Microbial Physiology Genetics and Ecology

MICROBIAL PHYSIOLOGY GENETICS AND ECOLOGY

Dr. Jyotsna Rathi

M MANGLAM PUBLICATIONS DELHI -110053 (INDIA)

Published by :

MANGLAM PUBLICATIONS L-21/1, Street No. 5, Shivaji Marg, Near Kali Mandir, J.P. Nagar, Kartar Nagar, West Ghonda, Delhi-53 Phone: 011-22945677, 9968367559,9868572512 E-mail: [email protected] manglam. [email protected]

Microbial Physiology Genetics and Ecology

©Reserved First Edition 2009 ISBN 978-81-907127-6-7

All rights reserved no part ofthis work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the Publisher.

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Published by D.P. Yadav for Manglam Publications, Delhi. Laser Typeset by Amandeep (Graphic Era) Delhi-92. Printed at Sachin Printers, Maujpur, Delhi-53

PREFACE The present title "Microbial Physiology. Genetics and Ecology" is a fast expanding branch of science, and it is impossible to present all of it in a book of this size. Author has therefore tried to present selected portions of microbial physiology in sufficient detail that the student can understand them through reading the book. The present title covers the various physiological processes of microorganisms, the way of characters are transmitted and expressed, and the influence of climate conditi6ns on them. The aim in writing this book was to bring together the relevant aspects of the biology of microorganisms. Thus it offers a comprehensive and uptodate information on microbial science. Author has covered the vast information available on the subject in a concise form so as to cater to the needs of both undergraduate and postgraduate students. It will be equally useful as a reference book to the researchers and teachers as well. The author expresses her thanks to all those friends, colleagues, and research scholars whose continuous inspirations have initiated her to bring this title. The author wishes to thank the M/s. Manglam Publications, Delhi for bringing out this book. Constructive criticisms and suggestions for improvement of the book will be thankfully acknowledged. Author

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Contents 1. Introduction ........................................................ 1 1.1

1.2

Elements of Epidemiology ......................................... 1 1.1.1 Some Definitions ....................................... 1 1.1.2 Chain of Infection ...................................... 2 Pathogens and Parasites Found in Domestic Wastewater ............................................................... 8 1.2.1 Bacterial Pathogens .................................... 8 1.2.2 Viral Pathogens ........................................ 22 1.2.3 Protozoan Parasites .................................. 28 1.2.4 Helminth Parasites ................................... 39 1.2.5 Other Problem· Causing Microorganisms .... 40

2. Microbial Metabolism and Growth ................. 42 2.1

Metabolic Diversity of Freshwater Bacteria ............... 42 Key Metabolic Parameters ........................ 42 . 2.1.1 2.1.2 CO 2 Fixation ............................................ 43 2.1.3 Breakdown of Organic Matter in Aerobic and Anaerobic Environments .................... 44 2.1.4 Bacterial Adaptations to Low-nutrient Environments .......................................... 49 2.2 Photosynthetic Bacteria ............................................ 51 2.2.1 General Characteristics ............................. 52 2.2.2 Motility .................................................... 52 2.2.3 Ecology ................................................... 53 (i)

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2.3

2.4

2.5

2.6

2.7

Bacteria and Inorganic Cycles ... ,.............................. 54 2.3.1 Bacterial Metabolism and the Sulphur Cycle ....................................................... 54 Bacterial Populations ................................................ 56 2.4.1 Techniques for Counting Bacterial Populations .............................................. 57 2.4.3 Biological Significance of Total and Viable Counts .................................................... 58 Bacterial Productivity ............................................... 59 2.5.1 Measurement of Productivity .................... 59 2.5.2 Regulation of Bacterial Populations and Biomass ................................................... 60 2.5.3 Primary and Secondary Productivity: Correlation Between Bacterial and Algal Populations .............................................. 61 2.5.4 Role of Dissolved Organic Carbon ........... 64 2.5.5 Bacterial Productivity and Aqua tic Food Webs ....................................................... 65 Microbial Growth .................................................... 66 Algal Blooms and Eutrophication .............. 66 2.6.1 2.6.2 Formation of colonial blue-green algal blooms .................................................... 68 2.6.3 Environn'ental Effects of Blue-green Blooms .................................................... 71 Metabolism of Starter Cultures ................................ 74 2.7.1 Carbohydrate Utilization by Lactic Acid Bacteria ................................................... 75 2.7.2 Protein Metabolism .................................. 89 2.7.3 Citrate Metabolism ................................... 96 2.7.4 Metabolism of Propioni Bacteria ............... 99 2.7.5 Metabolism of Molds and other Flavorcontributing Microorganisms .................. 100 2.7.6 Metabolic Engineering ........................... 102

3. Fermentation ................................................... 103 3.1

Introduction .......................................................... 103 3.1.1 Defined and Characterized ..................... 104 3.1.2 Lactic Acid Bacteria ............................... 105

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3.1.3

3.2

3.3

3.4 3.5 3.6 3.7 3.8

3.9

3.10

Metabolic Pathways and Molar Growth Yields .................................................... 109 Dairy Products ...................................................... 110 3.2.1 Milk Biota .............................................. 110 3.2.2 Starter Cultures, Products ...................... 112 3.2.3 Cheeses ................................................. 116 Apparent Health Benefits of Fermented Milks ........ 118 3.3.1 Lactose Intolerance ................................ 118 3.3.2 Cholesterol ............................................ 120 3.3.3 Anticancer Effects .................................. 121 3.3.4 Probiotics .............................................. 121 Diseases Caused by Lactic Acid Bacteria ................. 122 Fermented Fruit and Vegetable Products ............... 122 Fresh and Frozen Vegetables ................................. 123 3.6.1 Spoilage ............................................... 124 Spoilage of Fruits .................................................. 129 Fresh-cut Produce ................................................. 130 3.8.1 Microbial Load ...................................... 130 3.8.2 Pathogens .............................................. 131 Fermented Products .............................................. 133 3.9.1 Breads ................................................... 133 3.9.2 Olives, Pickles, and Sauerkraut .............. 134 3.9.3 Beer, Ale, Wines, Cider, and Distilled Spirits Beer andAle ............................... 138 3.9.4 Cider ..................................................... 142 Miscellaneous Fermented Products ........................ 144

4. Microbial Genetics ......................................... 150 4.1 4.2 4.3 4.4 4.5

Genetics ................................................................ DNA Replication ................................................... 4.2.1 Chromosome Connection ...................... Protein Synthesis ................................................... 4.3.1 Genotype and Phenotype ....................... Controlling Genes ................................................. 4.4.1 Operon Model ............................................. Mutations .............................................................. 4.5.1 Mutation Rate ........................................

150 151 152 152 154 154 155 155 157

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4.6

4.7

Genetic Interactions .............................................. 157 4.6.1 Genetic Diversity .................................... 158 4.6.2 Mechanisms for Gene Transfer in Freshwater Systems ............................... 162 4.6.3 Evidence for Gene Transfer in the Aquatic Environment .............................. 166 Recombinant DNA Technology .............................. 169 4.7.1 Genetic Engineering: Designer Genes ..... 169 4.7.2 Gene Therapy: Makes You Feel Better .. , 171 4.7.3 DNA Fingerprinting ............................... 172 4.7.4 Recombinant DNA Technology and Society .................................................. 173

5. Microbial Ecology .......................................... 175 5.1

5.2

5.3

5.4

General Introduction ............................................. 175 5.1.1 Aquatic Existence ................................... 175 5.1.2 Global Water Supply - Limnology and Oceanography ....................................... 176 5.1.3 Freshwater Systems: Some Terms and Definitions ............................................. 177 5.1.4 Biology of Freshwater Microorganisms .. , 179 Biodiversity of Microorganisms .............................. 179 5.2.1 Domains of Life ..................................... 179 5.2.2 Size Range ............................................ 180 5.2.3 Autotrophs and Heterotrophs ................. 182 5.2.4 Planktonic and Benthic Microorganisms ... 184 5.2.5 Metabolically Active and Inactive States .. 186 5.2.6 Evolutionary Strategies: r-selected and Kselected Organisms ................................ 187 Biodiversity in Ecosystems, Communities, and Species Populations ............................................... 190 5.3.1 Main Ecosystems ................................... 191 5.3.2 DiversitywithinSubsidiaryCommunities .. 191 5.3.3 Biodiversity within Single-species Populations ............................................ 192 Bjofilm Community: A Small-scale Freshwater Ecosystem ............................................................. 193 5.4.1 Interactions between Microorganisms ..... 194

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5.4.2 5.4.3

5.5

5.6

5.7 5.8

Biomass Formation and Transfer ............ 196 Maintenance of the Internal Environment .......................................... 196 5.4.4 Interactions with the External Environment .......................................... 197 Pelagic Ecosystem ................................................. 197 5.5.1 Interactions between Organisms ............. 198 5.5.2 Trophic Connections and Biomass Transfer ................................................. 199 5.5.3 Maintenance of the Internal Environment .......................................... 205 5.5.4 Interactions with the External Environment .......................................... 205 Homeostasis and Ecosystem Stability ..................... 207 5.6.1 Stress Factors ........................................ 207 5.6.2 General Theoretical Predictions: Community Response ............................ 208 5.6.3 Observed Stress Responses: From Molecules to Communities ..................... 209 5.6.4 Assessment of Ecosystem Stability .......... 210 5.6.5 Ecosystem Stability and Community Structure ............................................... 211 5.6.6 Biological Response Signatures ............... 213 Pelagic Food Webs ................................................ 213 Communities and Food Webs of Running Waters ... 214 5.8.1 Allochthonous Carbon ............................ 214 5.8.2 Pelagic and Benthic Communities ........... 216 5.8.3 Microbial Food Web .............................. 219

6. Light as Abiotic Factor .................................. 222 6.1

Light Environment ................................................ 6.1.1 Physical Properties of Light: Terms and Units of Measurement ............................ 6.1.2 Light Thresholds for Biological Activities ................................................ 6.1.3 Light Under Water: Refraction, Absorption, and Scattering .....................

223 223 224 225

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6.1.4

6.2

6.3

.,

6.4

6.5

6.6

6.7

Light Energy Conversion: From Water Surface to Algal Biomass ........................ 228 Photosynthetic Processes in the Freshwater Environment ......................................................... 229 6.2.1 Light and Dark Reactions ...................... 229 6.2.2 Photosynthetic Microorganisms .............. 230 6.2.3 Measurement of Photosynthesis .............. 2.31 6.2.4 Photosynthetic Response to Varying Light Intensity ....................................... 232 Light as a Growth Resource .................................. 234 6.3.1 Strategies for Light Uptake and Utilization .............................................. 235 6.3.2 Light-Photosynthetic Response in DifferentAlgae ....................................... 235 6.3.3 Conservation of Energy .......................... 237 6.3.4 Diversity in Small Molecular Weight Solutes and Osmoregulation .................. 238 Algal Growth and Productivity ............................... 240 6.4.1 Primary Production: Concepts and Terms .................................................... 240 6.4.2 Primary Production and Algal Biomass ... 240 6.4.3 Field Measurements of Primary Productivity ........................................... 241 Photosynthetic Bacteria .......................................... 244 6.5.1 Major Groups ......... ~ .............................. 244 6.5.2 Photosynthetic Pigments ........................ 244 6.5.3 Bacterial Primary Productivity ................ 246 Photoadaptation: Responses of Aquatic Algae to Limited Supplies of Light Energy ....................... 246 6.6.1 Different Aspects of Light Limitation ...... 247 6.6.2 Variable Light Intensity: Light-Responsive Gene Expression .................................... 248 6.6.3 Photosynthetic Responses to Low Light Intensity ................................................ 250 6.6.4 Spectral Composition of Light: Changes in Pigment Composition ......................... 255 Carbon Uptake and Excretion by Algal Cells ........... 256 Changes in Environmental CO 2 and pH ... 256 6.7.1

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CONTENTS

6.7.2

6.8

6.9

6.10

Excretion of Dissolved Organic Carbon by Phytoplankton Cells ............................... 258 Competition for Light and Carbon Dioxide between Algae and Higher Plants ........................................ 262 6.8.1 Balance between Algae and Macrophytes in Different Aquatic Environments .......... 262 6.8.2 Physiological and Environmental Adaptations in the Competition between Algae and Macrophytes .......................................... 263 Damaging Effects of High Levels of Solar Radiation: Photoinhibition ...................................................... 267 6.9.1 Specific Mechanisms of Photoinhibition .... 268 6.9.2 General Effects of Photoinhibition .......... 270 6.9.3 Strategies for the Avoidance of Photoinhibition ...................................... 271 6.9.4 Photoinhibition and Cell Size .................. 273 6.9.5 Lack of Photo inhibition in Benthic Communities ......................................... 276 6.9.6 Photoinhibition in Extreme High-light Environments ........................................ 277 Periodic Effects of Light on Seasonal and Diurnal Activities of Freshwater Biota ................................. 279 6. 10.1 Seasonal Periodicity ............................... 279 6.10.2 Diurnal Changes .................................... 280 6.10.3 Circadian Rhythms in Blue-green Algae .. 281 6.10.4 Circadian Rhythms in Dinoflagellates ...... 283

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1 Introduction 1.1 ELEMENTS OF EPIDEMIOLOGY 1.1.1 Some Definitions Epidemiology is the study of the spread of infectious diseases in populations. Infectious diseases are those that can be spread from one host to another. Epidemiologists play an important role in the control of these diseases. Incidence of a disease is the number of individuals with the disease in a population, whereas prevalence is the percentage of individuals with the disease at a given time. A disease is epidemic when the incidence is high and endemic when the incidence is low. Pandemic refers to the spread of the disease across continents. Infection is the invasion of a host by an infectious microorganism. It involves the entry (e.g., through the gastrointestinal and respiratory tracts, skin) of the pathogen into the host and its multiplication and establishment inside the host. Inapparent infection (or covert infection) is a subclinical infection with no apparent symptoms (i.e., the host reaction is not clinically detectable). It does not cause disease symptoms but confers the same degree of immunity as an overt infection. For example, most enteric viruses cause inapparent infections. A person with inapparent infection is called a healthy carrier. Carriers constitute, however, a potential source of infection for others in the community. Nosocomial infections are hospitalacquired infections, which affect approximately 2.5 mi1lion patients annually in the United States. This represents approximately 5 percent of patients with a documented infection acquired in a hospital.

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2

Intensive care unit patients represent about 20 percent of hospitalacquired infections. In developing countries, the overall rates vary between 3 and 13.5 percent. The patients most at risk for nosocomial infections are elderly patients, patients with intravenous devices or urinary catheters, those on parenteral nutrition or antimicrobial chemotherapy, and HIV and cancer patients. Pathogenicity is the ability of an infectious agent to cause disease and injure the host. Pathogenic microorganisms may infect susceptible hosts, leading sometimes to overt disease, which results in the development of clinical symptoms that are easily detectable. The development of the 'disease depends on various factors, including infectious dose, pathogenicity, and host and environmental factors. Some organisms, however, are opportunistic pathogens that cause disease only in compromised individuals.

1.1:2 Chain of Infection The potential for a biological agent to cause infection in a susceptible host depends on the various factors described in the following.

1.1.2.1 Type of infectious agent Several infectious organisms may cause diseases in humans. These agents include bacteria, fungi, protozoa, metazoa (helminths), rickettsiae, and viruses. Evaluation of infectious agents is based on their virulence or their potential for causing diseases in humans. Virulence is related to the dose of infectious agent necessary for infecting the host and causing disease. The potential for causing illness also depends on the stability of the infectious agent in the environment. The minimal BACTERIA

VIRUSES

PROTOZOA

HELMINTHS

e.g. Salmonella Shigella Vibrio cholera

e.g. HAV Norwalk agent

e.g., Giardia Cryptosporidlum

e.g., Ascaris Taema

HUMAN

L -_ _ __

I

Figure 1.1 Categories of organism'> of public health significance.

INTRODUCTION

3

infective dose (MID) varies widely with the type of pathogen or parasite. For example, for Salmonella typhi or enteropathogenic E. coli, thousands to millions of organisms are necessary to establish infection, whereas the MID for Shigella can be as low as 10 cells. A few protozoan cysts or helminth eggs may be sufficient to establish infection. For some viruses, only one or a few particles are sufficient for infecting individuals. For example, 17 infectious particles of echovirus 12 are sufficient for establishing infection. Table 1.1 Minimal infective doses for some pathogens and parasites

Organism Salmonella spp. Shigella spp. Escherichia coli Escherichia coli 01 57: H 7 Vibrio cholerae Campylobacter jejuni Giardia lamblia Cryptosporidium Entamoeba coli Ascaris Hepatitis A virus

Minimal infective dose 10 1-102 106-108 <100 10; about 500 10 1-10 2 cysts 10 1 cysts 10 1 cysts 1-10 eggs 1-10 PFU

1.1.2.2 Reservoir of tlte infectious agent A reservoir is a living or nonliving source of the infectious agent and allows the pathogen to survive and multiply. The human body is the reservoir for numerous pathogens; person-to-person contact is necessary for maintaining the disease cycle. Domestic and wild animals also may serve as reservoirs for several diseases (e.g., rabies, brucellosis, turbeculosis, anthrax, leptospirosis, toxoplasmosis) called zoonoses, that can be transmitted from animals to humans. Nonliving reservoirs such as water, wastewater, food, or soils can also harbor infectious agents.

1.1.2.3 Mode of transmission Transmission involves the transport of an infectious agent from the reservoir to the host. It is the most important link in the chain of infection. Pathogens can be transmitted from the reservoir to a susceptible host by various routes.

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MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

1.1.2.3.1 Person-to-person transmission The most common route of transmission of infectious agents is from person to person. The best examples of direct contact transmission are the sexually transmitted diseases such as syphilis, gonorrhea, herpes, or acquired immunodeficiency syndrome (AIDS). Coughing and sneezing discharge very small droplets containing pathogens within a few feet of the host (droplet infection). Transmission by these infectious droplets is sometimes considered as an example of direct contact transmission. 1.1.2.3.2 Waterborne transmission The waterborne transmission of cholera was established in 1854 by 10hn Snow, an English physician who noted a relationship between a cholera epidemics and consumption of water from the Broad Street well in London. The waterborne route is not, however, as important as the person-to-person contact route for the transmission of fecally transmitted diseases. The World Health Organization (WHO) reported that diarrhoeal diseases contracted worldwide mainly by contaminated water or food, killed 3.1 million people, most of them children. In the United States, waterborne disease outbreaks are reported to the U.S. Environmental Proteotion Agency and the Centers for Disease Control and Prevention (CDC) by local epidemiologists and health authorities; the system was started in the 1920s. During the period 1971-1985, 502 waterborne outbreaks and 111,228 cases were reported. Gastrointestinal illnesses of unidentified etiology and giardiasis are the most common waterborne diseases for groundwater and surface water systems. The outbreak rate (expressed as the number of outbreaks/1000 water systems) and the illness rate (expressed as numbers of cases/million-person year) decrease as the raw water is filtered and disinfected. Foodborne transmission Food may serve as a vehicle for the transmission of numerous infectious diseases caused by bacteria, viruses, protozoa, and helminth parasites. The World Health Organization estimates that accidental food poisoning kills up to 1.5 million people per year. In the United States, it is estimated that foodborne illnesses affect some 6 to 80 million persons/ year, leading to approximately 9000 deaths. Food contamination results from unsanitary practices during production or preparation. Several pathogens and parasites have been detected

INTRODUCTION

5

Table 1.2 Etiology of waterborne outbreaks for groundwater and surface water systems Illness Gastroenteritis, undefined Giardiasis Chemical poisoning Shigellosis Hepatitis A Gastroenteritis, viral Campylobacterosis Salmonellosis Typhoid Yersiniosis Gastroenteritis, toxigenic E. coli Cryptosporidiosis Cholera Dermatitis Amoebiasis Total

No. of Outbreaks

Cases of Illness

251 92 50 33 23 20 11 10 5 2

61,478 24,365 3,774 5,783 737 6,254 4,983 2,300 282 103 I,COO 117 17 31 4 111,228

502

in risky foodstuffs such as shellfish, vegetables, raw milk, runny eggs or pink chicken, turkey, ground beef and ground pork, alfalfa sprouts, and unpasteurized apple juice/cider. Their presence is of public health significance, particularly for foods that are eaten raw (e.g., shellfish, fresh produce). There is also an increased risk among the elderly and immunocompromised people (HIV and leukemia patients, and those taking immunosuppressive drugs such as steroids, cyclosporine, and radiation therapy). Vegetables contaminated with wastewater effluents are also responsible for disease outbreaks (e.g., typhoid fever, salmonellosis, amebiasis, ascariasis, viral hepatitis, gastroenteritis). Raw vegetables and fruit become contaminated as a result of being handled by an infected person during processing, storage, distribution or final preparation, or following irrigation with fecally contaminated water. Vomitus (estimation of 20 to 30 million virus particles released during vomiting) from infected food handlers can also contaminate exposed food and surfaces via production of bioaerosols. In England and

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MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

Wales, viruses accounted for 4.3 percent of all foodborne outbreaks for the period 1992-1999, with Norwalk-like viruses (NLVs) being the most commonly found agents. Outbreaks of hepatitis were associated with fresh produce (e.g., salads, iceberg lettuce, diced tomatoes, frozen raspberries). Shellfish (e.g., oysters, clams, mussels) are significant vectors of human diseases of bacterial, viral, and protozoan origin. Several surveys have beell carried out worldwide to show the presence of pathogens in shellfish samples. The use of molecular techniques (RTpeR) has helped in the detection of enteric viruses (hepatitis A virus, Norwalk-like virus, enterovirus, rotavirus, and astrovirus) in oyster and mussel samples in France. In Switzerland, 8 of 87 imported oyster samples were positive for Norwalk-like viruses. Moreover, infectious oocysts of Cryptosporidium were detected in mussels and cockles in Spain. Enteric viruses (enteroviruses, Norwalk-like viruses, and adenoviruses) were detected in 50-60 percent of mussel samples in two sites in the west coast of Sweden. Of 36 mussel samples from the Adriatic Sea, 13 were contaminated with hepatitis A virus and 5 samples with enteroviruses. In several surveys, E. coli was not found to be a good indicator of virus presence in mussels. Shellfish are important in disease transmission for the following reasons: 1. They live in estuarine environments, which are often contaminated by domestic wastewater effluents. 2. As filter-feeders, they concentrate pathogens and parasites by pumping large quantities of estuarine water (4-20 Llh). The accumulation occurs mainly in the digestive tissues (mostly in the digestive gland as demonstrated for male-specific phage accumulation in mussels). The bioaccumulation of enteric bacteria and viruses by bivalve mollusks vary with the species of shellfish, type of microorganism, environmental conditions, and season. In oysters (Crassostrea virgil1ica) , the average accumulation factors for E. coli and P coliphage were 4.4 and 19.0 (range from < 1 to 99), respectively. 3. They are often eaten raw or insufficiently cooked. It has been estimated that only one-third of shellfish consumed every year in France are sufficiently cooked. In the United States, this habit has led to 2100 cases in the period 1991-1998. Sixty percent of the cases were caused by enteric viruses, particularly

INTRODUCTION

7

Norwalk-like viruses (NLV). Shellfish steaming or cooking do not seem to prevent viral-associated gastroenteritis. A temperature of about 90°C is necessary to inactivate hepatitis A virus in shellfish. 4. Depuration of shellfish, while effective for bacterial contaminants, is not successful for enteric viruses such as hepatitis A virus. 5. Other health hazards associated with shellfish consumption result from the ability of these mollusks to concentrate dinoflagellate toxins, heavy metals, hydrocarbons, pesticides, and radionuclides. 1.1.2.3.3 Airborne transmission Some diseases (e.g., Q fever. some fungal diseases) can be spread by airborne transmission. This route is important in the transmission of biological aerosols generated by wastewater treatment plants or spray irrigation with wastewater effluents. 1.1.2.3.4 Vector-borne tral1smission The most common vectors for disease transmission are arthropods (e.g., fleas, insects) or vertebrates (e.g., rodents, dogs, and cats). The pathogen mayor may not multiply inside the arthropod vector. Some vector-borne diseases are malaria (caused by Plasmodium), yellow fever, or encephalitis (both due to arboviruses) and rabies (from virus transmitted by the bite of rabid dogs or cats). 1.1.2.3.5 Fomites Some pathogens may be transmitted by nonliving objects or fomites (e.g., clothes, utensils, toys, environmental surfaces).

1.1.2.4 Portal of entry Pathogenic microorganisms call gaill access to the host mainly through the gastrointestinal tract (e.g., enteric viruses and bacteria), the respiratory tract (e.g., Klebsiella pneumOl1ae, Legionella, myxoviruses) or the skin (e.g., Aero111onas, Clostridium tetani, Clostridium perjril1gel1s). Although the skin is a formidable barrier against pathogens, wounds or abrasions may facilitate their penetration into the host.

1.1.2.5 Host susceptibility Both the immune system and nonspecific factors playa role in the resistance of the host to infectious agents. Immunity to an infectious agent may be natural or acquired. Natural immunity is genetically specified and varies with species, race, age (the young

MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

8

and the elderly are more susceptible to infection), hormonal status, and the physical and mental health of the host. People in poor health and the elderly are mOl e susceptible to infectious agents than healthy adults. Acquired immunity develops as a result of exposure of the host to an infectious agent. Acquired immunity can be passive (e.g., fetus acquiring the mother's antibodies) or active (e.g., active production of antibodies through contact with the infectious agent). The nonspecific factors include physiological barriers at the portal of entry (e.g., unfavorable pH, bile salts, production of digestive enzymes and other chemicals with antimicrobial properties, competition with the natural rnicroflora in the colon) and destruction of the invaders by phagocytosis.

1.2 PATHOGENS AND PARASITES FOUND IN DOMESTIC WASTEWATER Several pathogenic microorganisms and parasites are commonly found in domestic waste-water as well as in effluents from wastewater treatment plants. The three categories of pathogens encountered in the environment are: 1. Bacterial pathoge11s. Some of these pathogens (e.g., Salmonella, Shigella) are enteric bacteria. Others (e.g., Legionella, Mycobacterium (lviwl1, Aeromol1as) are indigenous aquatic bacteria. 2. Viral pathogens. These are also released into aquatic environments but are unable to multiply outside their host cells. Their infective dose is generally lower than for bacterial pathogens. 3. Prf/tozoall parasites. Thest are released into aquatic environments as cysts or oocysts, which are quite resistant to environmental stress and to disinfection, and do not multiply outside their hosts. 1.2.1 Bacterial Pathogens Fecal matter contains up to 10 12 bacteria per gram. The bacterial content of feces represents approximately 9 percent by wet weight. 16S rRNA-gene-targeted group-specific primers were used to detect and identify the predominant bacteria in human feces. Of 300 bacterial isolates from feces of six healthy individuals, 74 percent belonged to the Bacteroides fi'agilis group, Bifidobacteriul11 , Clostridium cocco ides group.
INTRODUCTION

9

2. Gram-negative aerobic bacteria: e.g., Pseudomonas, Alcaligenes, Flavobacterium, Acinetobacter. 3. Gram-positive spore forming bacteria: e.g., Bacillus spp. 4. Nonspore-forming gram-positive bacteria: e.g., Arthrobacter, Corynebacterium, Rhodococcus.

1.2.1.1 Salmonella Salmonellae are enterobacteriaceae and are widely distributed in the environment and include more than 2000 serotypes. The Salmo11ella numbers in wastewater range from a few to 8000 organisms/ 100 mL, are the most predominant pathogenic bacteria in wastewater, and cause typhoid and paratyphoid fever, and gastroenteritis. Salmonella numbers in wastewater in the United States range from 10 2 to 104 organisms/ 100 mL, but much higher concentrations (up to 1oq/ 100 mL) have been reported in developing countries. It is estimated that two to four million human Salmonella infections occur each year in the United States. An estimated 0.1 percent of the population excretes Salmonella at any given time. In the United States, salmonellosis is primarily due to food contamination, but its transmission through drinking water is still of great concern. Salmonella typhi is the etiological agent for typhoid fever, a deadly disease that was brought under control as a result of the development of adequate water treatment processes (e.g., chlorination, filtration). This pathogen produces an endotoxin that causes fever, nausea, and diarrhea and may be fatal if not properly treated by antibiotics. Species implicated in food contamination are S. enteriditis and S. typhimurium. These species can grow readily in contaminated foods and cause food poisoning, leading to diarrhea and abdominal cramps. Salmonella enteriditis, through consumption of contaminated poultry and eggs, emerged as a public health problem in the 1980s in Europe and in the United States. Of 371 outbreaks of S. el1leriditis that occurred in the United States, 80 percent were associated with consumption of insufficiently cooked eggs. Salmonella enteriditis is transmitted directly from breeding flocks to egg-laying hens. In Israel, about 30 percent of the 35 flocks of laying hens examined were contaminated with S. enteriditis. This has led to programs to control this pathogen.

1.2.1.2 Shigella Shigella is the causative agent of bacillary dysentery or shigellosis, an infection of the large bowel that leads to cramps, diarrhea, and fever. This disease produces bloody stools as a result of inflammation

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MICROBIAL PHYSIOLOGY. GENETICS AND E,COLOGY

(induction and release of proinflammatory cytokines) and ulceration of the intestinal mucosa. Globally, this pathogen is responsible for approximately one million deaths and 163 million cases of bacillary dysentery annually, with 99 percent of the cases occurring in developing countries. There are four pathogenic species of Shigella: S. dysenteriae (13 serotypes), S. flexneri 15 serotypes), S. boydii (18 serotypes) and S. s0I111ci. (1 serotype). S. d\'sel1teriae, serotype 1, produces a potent toxin called the Shiga toxin. Infection with bacteria producing this toxin may lead to hemolytic uremic syndrome which results in kidney failure. No current vaccine is available for protection against Shigella. This pathogen is transmitted by direct contact with an infected individual, who may excrete up to 10 9 shigellae per gram of feces. The infectious dose for Shigella is relatively small and can be as low as 10 organisms. Although person-to-person contact is the main mode of transmission of this pathogen, food-borne (via salads and raw vegetables) and waterborne transmissions have also been documented. At least three large epidemics caused by Shigella dysenteriae type 1 have occurred in Bangladesh between 1972 and 1994, and the pathogen was isolated from surface waters, using genetic probes and culturing followed by biochemical tests. The environmental isolates shared some virulence genes with clinical isolates and were resistant to one or more antibiotics. Groundwater was found to be responsible for a shigellosis outbreak in Florida that involved 1200 people. However, Shigella persists less in the environment than fecal coliforms. Few quantitative data are available on its occurrence and removal in water and wastewater treatment plants.

1.2.1.3 Vibrio cholerae Vibrio cholera is a gram-negative curved rod bacterium that is an autochtonous member of the aquatic microbial community. This bacterium is the causative agent of cholera. In 1854, John Snow first demonstrated that contaminated drinking water was the cause of cholera. This pathogen releases an enterotoxin that causes mild to profuse diarrhea, vomiting, and a very rapid loss of fluids, which may result in death in a relatively short period of time. The infectious dose for V. cholera is between 10 4 and 106 cells. Among about 200 known serogroups of Vibrio cholerae, only two (01 and 0139) are known to cause disease and can be detected using serum agglutination assays or monoclonal antibodies. Although rare in the

INTRODUCTION

11

United States and Europe, this disease appears to be endemic in various areas throughout Asia, particularly in Bangladesh. This pathogen is found in wastewater, and levels of 10-104 organisms/ 100 mL during a cholera epidemic have been reported. Explosive epidemics of cholera and typhoid fever have been documented in Peru and Chile and have been associated with the consumption of sewage-contaminated vegetables. Vibrio cholerae can be detected in environmental samples, using immunological or molecular methods. Although the nucleic acid-based methods are rapid and relatively easy, they do not allow a differentiation between viable and nonviable cells. Vibrios are naturally present in many aquatic environments and survive by attaching to solids, including zooplankton (e.g., copepods), cyanobacteria (e.g., Anabaena), and phytoplankton (e.g., Volvox) cells. These plankton-associated bacteria may occur in the viable but nonculturable state (VBNC) and can be observed under the microscope by means of the fluorescent-monoclonal antibody technique.

1.2.1.4 Escherichia coli Several strains of E. coli, many of which are harmless, are found in the gastrointestinal tract of humans and warm-blooded animals. There are several categories of E. coli strains, however, that bear virulence factors and cause diarrhea. There are enterotoxigenic (ETEC), enteropathogenic (EPEC) , enterohemorrhagic (EHEC), enteroinvasive (EIEC), and enteroaggregative (EAggEC) types of E. coli. Enterotoxigenic E. coli causes gastroenteritis with profuse watery diarrhea accompanied with nausea, abdominal cramps, and vomiting. Approximately 2-8 percent of the E. coli present in water were found to be enteropathogenic E. coli, which causes Traveler's diarrhea. Food and water are important in the transmission of this pathogen. However, the infective dose for this pathogen is relatively high, within the range of 106-109 organisms. Distinct features of enteroaggregative E. coli are its adherence to Hep2 cells in tissue culture in an aggregative pattern and its ability to cause persistent diarrhea. Some of these diarrhea genic strains of E. coli have been detected in treated water with genetic probes, and they can represent a health risk to consumers. During a 1989-1990 survey of waterborne disease outbreaks in the United States, the etiologic agent in one out of 26 outbreaks was enterohemorrhagic E. coli 0157:H7, an agent that

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MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

produces shiga-like toxins (SLT) I and/or II, has a relatively low infectious dose « 100 organisms) and causes bloody diarrhea, particularly among the very young and very old members of the community. Infections, if left untreated, may lead to hemolytic uremic syndrome, a leading cause of kidney damage and possible failure in children, with a 3-5 percent death rate in patients. In the United States, it is estimated that E. coli 0157:H7 causes more than 20,000 infections and as many as 250 deaths each year. Twenty outbreaks of E. coli 0157:H7 infections reported in the United States between 1982 and 1993 led to 1557 cases, 358 hospitalizations, and 19 deaths. Food (e.g., fresh or undercooked ground meats) appears to be the primary source of infections. Other sources include cider, raw milk, lettuce, and contaminated waters. Enterohemorrhagic E. coli was also isolated from a water reservoir in Philadelphia, PA, untreated and treated drinking water, but surprisingly was not detected in primary and secondary wastewater effluents. Several outbreaks of E. coli 0157:H7 were shown to be associated with waterborne transmission from sources such as groundwater, recreational waters, and municipal water systems. One outbreak occurred in Cabool, Missouri, in the winter of 1990 after disturbances in the water distribution network; it resulted in 243 documented cases of diarrhea and four deaths among elderly citizens. A more recent outbreak occurred in May 2000 in Canada and resulted in 2000 infections and six deaths. A rapid method has been developed for detecting respiring E. coli 0157:H7 in water. This membrane filtration method is based on the use of a specific fluorescent antibody combined with cyanoditolyl tetrazolium chloride (CTC) to assess the respiratory activity of the cells. In food, such as hamburger meat, this pathogen can be detected by combining immunomagnetic separation with a sandwich enzyme-linked immunosorbent assay (ELISA). A new proposed methodology detects E. coli 0157:H7 by using green fluorescent-labeled PP01 specific bacteriophage, which, following adsorption to the host cells, leads to the visualization of the cells under a fluorescence microscope. This method detects E. coli 0157:H7 in both the culturable and the culturable but not viable (VBNC) states.

1.2.1.5 Yersinia Yersinia enterocolitica is responsible for acute gastroenteritis with invasion of the terminal ileum. Swine are the major animal

INTRODUCTION

13

reservoir, but many domestic and wild animals can also serve as reservoirs for this pathogen. Food-borne (e.g., milk, tofu) outbreaks of yersiniosis have also been documented in the United States. The role of water is uncertain, but there are instances in which this pathogen was suspected to be the cause of waterborne transmission of gastroenteritis. This psychrotrophic organism thrives at temperatures as low as 4°C, is mostly isolated during the cold months, but is poorly correlated with traditional bacterial indicators. This organism has been isolated from wastewater effluents, river water, and from drinking water.

1.2.1.6 Campylobacter This pathogen (e.g., c. fetus and C. jejuni) is known to infect humans as well as wild and domestic animals. It is ubiquitous in domestic waste-water and in effluents from abattoirs and poultry processing plants. In Lancaster, United Kingdom, the occurrence of Campylobacters in wastewater showed a seasonal trend similar to that of the incidence of infections in humans. Its relatively low infectious dose of approximately 500 organisms (for C. jejuni) makes it the leading cause of food-borne infections, with approximately 4 million C. jejuni infections/year in the United States. Campylobacter is the most frequent cause of diarrhea in the United States and United Kingdom. These infections may lead in one of 1000 patients to Guillain-Barre syndrome, which is an acute paralytic illness. It is also a common cause of acute gastroenteritis (fever, nausea, abdominal pains, bloody diarrhea, vomiting) and is transmitted to humans through contaminated food, mainly undercooked poultry, unpasteurized milk, contaminated drinking water, and water from mountain streams. This pathogen has been the cause of several outbreaks of gastroenteritis in the United States and worldwide. The first outbreak in the United States was reported in 1978 in Vermont, where 2000 out of a population of 10,000 were affected. The seasonal occurrence of Campy/obaeter in surface waters has been documented. Campylobaeter has been detected in surface waters, potable water, and wastewater, but no organisms have been recovered from digested sludge. Recovery from surface waters was highest in the fall (55 percent of samples positive) and winter (39 percent of samples positive). Numbers of Campylobaeter did nut display any correlation with heterotrophic plate counts, total and fecal coliforms, or fecal streptococci. Owing to their sensitivity to oxygen and inability to grow at temperatures below 30°C (optimum

14

MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

temperature is 42°C), C. jejuni survives but does not grow in the environment. Campylobacters can be detec'ted in contaminated natural waters by using selective growth agar media or molecular methods following an enrichment step in a selective broth medium. 1.2.1.7 Leptospira

Leptospira is a small spirochete that can gain access to the host through abrasions of the skin or through mucous membranes. It causes leptospirosis, which is characterized by the dissemination of the pathogen in the patient's blood and the subsequent infection of the kidneys and the central nervous system. The disease can be transmitted from animals (rodents, domestic pets, and wildlife) to humans coming into contact (e.g., bathing) with waters polluted with animal wastes. This zoonotic disease may strike sewage workers. This pathogen is not of major concern because it does not appear to survive well in wastewater. 1.2.1.8 Legionella pneumophila

This bacterial pathogen is the etiological agent of Legionnaires' disease, first described in a 1976 outbreak in Philadelphia, PA. It is estima ted that there are 10,000-25,000 cases of Legionnaires' disease/year in the United States. This disease is a type of acute pneumonia with a relatively high fatality rate; it may also involve the gastrointestinal and urinary tracts as well as the nervous system. Legionella pneumophila causes pneumonia as a result of its ability to multiply within alveolar macrophages. Pontiac fever is a milder nonfatal form of Legionnaires' disease associated with Legionella infection. People manifesting this syndrome have fever, headaches, and muscle aches, but may recover without any treatment. Aquatic environments and soils can act as natural reservoirs for pathogenic species of Legionellae. This organism is transmitted mainly by aerosolization of contaminated water or soil. 1.2.1.S.1 Aerosolization Outbreaks of legionnaire's disease are associated with exposure to L. pneumophila aerosols from cooling towers, evaporative condensers, humidifiers, shower heads, air conditioning systems, whirlpools, mist machines in produce departments of grocery stores, mechanical aerosolization of soil particles during gardening, or dental equipment. A study of public showers in Bologna, Italy, showed

INTRODUCTION

J5

that 22 of 48 samples were positive for L. pneumophila. Natural draft cooling towers are used to cool the hot water generated by power-generating plants. These towers generate microbial aerosols, including Legionella at the top of the structure. It is postulated that the source of Legionella is the water drawn from nearby surface waters or the potable water supply to replace the moisture lost during the cooling cycle. Perhaps the world largest epidemics of Legionnaire's disease occurred in Murcia, Spain, in 2001, with 449 confirmed cases out of 800 suspected cases. The outbreak was due to contamination from the hospital cooling towers. 1.2.1.8.2 Ingestion Legionella pneumophila serogroup 1 has been detected in drinking water systems but, so far, no outbreak has been attributed to consumption of contaminated drinking water.

Table 1.3 Frequency of isolation of Legionella spp. from shower waters Number of positive samples (11 = 48)

Legiol1ella Legiol1ella pneumophila Legionella pl1eumophila Legionella pneumoph ila Legiol1eIla pl1eumophila Legionella pl1eumophila Legiollella bozemanii LegiolleIla dumofii LegioneIla gorl7lallii Legiollella micdadei All the species

(SG 1) (SG3) (SG4) (SG5) (SG6)

6 5 2 8

7 4 3 4 27

Range (CFU/L)

20-8,700 10-650 1530 15,150-15,440 20-19,250 100-6,000 20-510 600-4,000 30-1,800 10-25,250

Surveys have shown that hospitals are the setting for many outbreaks of Legionella infections. The sources of nosocomial Legionnaire's disease can be traced back to hospital cooling towers and to potable water distribution system in hospitals. The number of cases increase after a pressure drop in the distribution system, which probably causes the release of Legionella cells associated with biofilms growing in the distribution pipes. Biofilms appear to provide a protective environment to Legionella in distribution systems. However, the number of cases dropped after hyperchlorination (>2 mg/L free residual chlorine). This bacterium appears to be ubiquitous in the environment and has been isolated from waste-water, soil,

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MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

and natural aquatic environments including tropical waters. Its presence in wastewater has been linked, on one occasion at least, with increased levels of antibodies among wastewater irrigation workers. However, the epidemiological significance of this finding remains unclear. In the natural environment, this pathogen can thrive in association with other bacteria which may provide the L-cysteine required by Legionella, green and blue-green algae, amoeba (e.g., Acanthamoeba, Naegleria), and other protozoa (e.g., Hartmanella, Tetrahymena), or ciliates. Conversely, 16-32 percent of heterotrophic plate count bacteria isolated from chlorinated drinking water were found to inhibit Legionella species. Legionella association with protozoa provides increased resistance to biocides such as chlorine, low pH, and high temperatures. Protozoa are able to sustain the intracellular growth of L. pneumophila and have an effect on its virulence to mammalian cells. It was found that the same virulence genes are required to infect both protozoa and mammalian cells. Control of Legionella in water distribution systems includes thermal treatment (e.g., increase of water temperature to 60-70 C followed by flushing), treatment with bactericidal agents (e.g., copper, silver), and hyperchlorination up to 50 mg/L. However, because biofilms protect bacterial pathogens from inactivation by free chlorine, treatment with monochloramine provides a better control of Legionella in water distribution lines. An epidemiological study showed that 10 times fewer outbreaks of Legionnaires' disease occurred in hospitals having monochloramine as disinfectant residual than those using free chlorine as a residual. D

1.2.1.9 Bacteroides fragiis Bacteroides species are a major part of the microorganisms in the human colon and account for approximately 25 percent of all colonic isolates. This pathogen has been found in wastewater at levels ranging from 6.2 x 104 to 1.1 x 105 colony forming units/ mL, 9.3 percent of which were enterotoxigenic. Enterotoxinproducing strains of this anaerobic bacterium may be involved in causing diarrhea in humans.

1.2.1.10 Opportunistic bacterial pathogens This group includes heterotrophic gram-negative bacteria belonging to the following genera: Pseudomonas, Aeromonas, Klebsiella, Flavobacterium, Ellterobacter, Citrobacter, Serratia, Acinetobacter, Proteus and Prol'idencia, and nontubercular

INTRODUCTION

17

mycobacteria. Segments of the population particularly at risk of infection with opportunistic pathogens are newborn babies, and elderly and sick people. These organisms may occur in high numbers in institutional (e.g., hospital) drinking water and attach to water distribution pipes, and some of them may grow in finished drinking water. However, their public health significance with regard to the population at large is not well known. Pseudomonas aerugil10sa is ubiquitous in the environment and is frequently found in water, wastewater, soils and plants. Although it poses no risks in drinking water, it is responsible for 10-20 percent of nosocomial (i.e., hospital-acquired) infections. Other opportunistic pathogens are the nontubercular mycobacteria, which cause pulmonary infections and other diseases. The most frequently isolated nontubercular mycobacteria belong to the species of Mycobacterium avium complex (MAC) (i.e., M. avium and M. intracellulare), which infect humans (mostly AIDS and other immunocompromised patients) and animals (e.g., pigs). Another member of the MAC complex is M. avium subspecies paratuberculosis (MAP), which causes inflammation. There is now evidence that infection with MAP is one of the causes of Crohn' s disease, a chronic inflammation of the intestine affecting animals and humans. A new generation of antibiotics (rifabutin, clarithromycin, azithromycin) has been proposed to control or reduce MAP activity in the gastrointestinal tract. Mycobacteria are ubiquitous and are found in environmental waters, including drinking water, ice, and hospital hot water systems, soils, plants, air-water interface of aquatic environments, biofilms in water distribution systems, medical instruments, and aerosols. Owing to the hydrophobic nature of the cell surface of mycobacteria, a major route for their transmission is via aerosolization. This also explains their resistance to commonly used disinfectants and to antibiotics. Mycobacteria persist well and grow under environmental conditions. Potable water, particularly hospital water supplies, can support the growth of these bacteria, which may be linked to nosocomial infections. Their growth in water distribution systems was correlated with assimilable organic carbon and biodegradable organic carbon levels. Mycobacterium avium and M. il1tracellulare can be detected using cultural, biochemical, and molecular-based methods (commercially available DNA probes and PCR amplification).

MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

18

1.2.1.11 Helicobacter pylori Helicobacter pylori is a bacterial agent that is responsible for peptic ulcers (chronic gastritis), stomach cancer, lymphoma, and adenocarcinoma. In the United States alone, over 5 million people are diagnosed annually with peptic ulcers, and some 40,000 of them undergo surgery, sometimes leading to death in complicated cases. There are indications of person· to-person as well as waterborne and food-borne transmissions of this pathogen. It has been suggested that H. pylori may be transmitted via four routes: the fecal-oral route, oral-oral route (person-to-person transmission via saliva), gastric-oral route (e.g., contaminated vomit in children), and by endoscopic procedures in the hospital setting. This pathogen has been associated with an increased risk of gastric cancer among sewage workers. Helicobacter pylori infection is generally treated via administration of antibiotics such metronidazole, tetracycline, amoxycillin, clarithromycin, and azithromycin. The treatment is complicated by the appearance of antibiotic resistance in this pathogen. Helicobacter pylori has been detected in wastewater, seawater, and drinking water. When exposed to environmental conditions, H. pylori enters a viable but nonculturable (VBNC) state that allows its persistence in aquatic environments. Although Johnson and colleagues 5 4

• H. pylori

a

0

E. coli

3

c: 0

"" -52 ()

~

8'

~

1

0 -1 0.00

0.05

010

0.15

0.20

0.25

030

035

Chlorine (mg/L)

Figure 1.2 Effect of chlorine on Helicobacter pylori and Escherichia coh.

INTRODUCTION

19

(1997) reported that H. pylori was readily inactivated by free chlorine, it is, however, more resistant than E. coli to chorine. This higher resistance to disinfectants may allow this pathogen to persist in water distribution systems. Helicobacter pylori was detected in environmental samples, using culture-based methods as well as immunological, autoradiography, and molecular-based methods. Although no standard method is available for H. pylori detection, this pathogen was recently isolated from wastewater using fluorescent in situ hybridization (FISH), a combination of immunomagnetic separation OMS) and culturing techniques, and identified using PCR-based 16S rRNA sequences. Helicobacter DNA was also isolated from biofilms in municipal water distribution systems.

1.2.1.12 Antibiotic-resistant bacteria Antibiotics act on microorganisms by inhibiting peptidoglycan synthesis, protein synthesis, and nucleic acid synthesis (interruption of nucleotide metabolism, inhibitition of RNA polymerase or DNA gyrase) or by affecting the cell membrane integrity. Increased use of antibiotics is often associated with an increased resistance of bacteria to these chemicals, especially in the hospital setting. A global rise in antibiotic resistance has been reported. A comparison of preantibiotic era strains of E. coli and Salmonella enterica to contemporary strains showed that the former were susceptible to antibiotics, whereas 20 percent of the latter displayed resistance to at least one of the antibiotics. In the United States, 46 percent of Streptococcus pneumoniae isolates are now resistant to penicillin, and methicillin-resistant Staphylococcus aureus accounts for 30 percent of nosocomial infections with this pathogen. An investigation of the antibiotic resistance pattern of E. coli strains in a wastewater treatment plant in Austria showed that these strains were resistant to 16 of 24 tested antibiotics. Antibiotic-resistant strains numbers increase when the influent to the wastewater treatment plant is from a hospital source. Resistance to vancomycin has also been documented. Vancomycin-resistant enterococci (VRE) were isolated from 60 percent of raw wastewater and 36 percent of wastewater effluents. As regards resistance to vancomycin, the minimum inhibitory concentrations (MIC) of VRE from hospital wastewaters were found to be much higher than those from residential wastewaters. Using a real-time PCR assay, the antibiotic resistance genes vanA of VRE, and ampC (resistance gene for the synthesis

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MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

of ,B-Iactamase) of Enterobacteriaceae were detected in 20 and 78 percent of wastewater samples, respectively. Patients receiving antibiotic therapy harbor a large number of antibiotic-resistant bacteria in their intestinal tract. These bacteria are excreted in large numbers in feces and eventually reach the community wastewater treatment plant. The genes coding for antibiotic resistance are often located on plasmids (R factors) and, under appropriate conditions, can be transferred to other bacteria through conjugation that requires cell-to-cell contact, or through other. modes of recombination. If the recipient bacteria are potential pathogens, they may be of public health concern as a result of their acquisition of antibiotic resistance. Drug-resistant microorganisms produce nosocomial and community-acquired human infections, which can lead to increased morbidity, mortality, and disease incidence. Resistance to antibiotics, including quinolones (e.g., nalidixic acid, ciprofloxacin), can in turn complicate and increase the cost of therapy based on administration of antibiotics to patients exposed to pathogens of environmental origin. Patients infected with antibiotic-resistant bacterial strains are likely to require hospitalization, sometimes for long periods. Bacterial resistance to antibiotics has been demonstrated in terrestrial and aquatic environments, particularly those contaminated with wastes from hospitals. Gene transfer between microorganisms is known to occur in natural environments as well as in engineered systems such as wastewater treatment plants. Investigators have used survival chambers to demonstrate the transfer of R plasmids among bacteria in domestic wastewater. The mean transfer frequency in wastewater varied between 4.9 X 10--5 and 7.5 X 10-5• The highest transfer frequency (2.7 x 10-4 ) was observed between Salmonella enteritidis and E. coli. Nonconjugative plasmids (e.g., pBR plasmids) can also be transferred and this necessitates the presence of a mobilizing bacterial strain to mediate the transfer. Several indigenous mobilizing strains have been isolated from raw wastewater. Each of these strains is capable of aiding in the transfer of the plasmid pBR325 to a recipient E. coli strain. Under laboratory conditions, plasmid mobilization from genetically engineered bacteria to environmental strains was also demonstrated under low temperature and low nutrient conditions in drinking water. The occurrence of multiple-antibiotic resistant (MAR) indicator and pathogenic (e.g., Salmonella) bacteria in water and wastewater

21

INTRODUCTION

Total coliform

Total MAR colllform

5 4

3 2

6/84 Date Figure 1.3 Multiple-antibiotic resistant (MAR) bacleria in dOlllestic wastewater.

treatment plants has been documented. In untreated wastewater, the percentage of multiple-antibiotic resistant coliforms varies between less than 1 to about 5 percent of the total coliforms. Chlorination appears to select for resistance to antibiotics in wastewater treatment plants. However, others observed that chlorination increased the bacterial resistance to some antibiotics (e.g., ampicillin, tetracycline) but not to others (e.g., chloramphenicol, gentamicin). The proportion of bacteria carrying R factors seems to increase after water and wastewater treatment. For example, in one study, MAR was expressed by 18.6 percent of heterotrophic plate count bacteria in untreated water as compared to 67.8 percent for bacteria in the distribution system. Similarly, in a water treatment plant in Oregon, the percentage of MAR bacteria rose from 15.8 percent in untreated (river) water to 57.1 percent in treated water. Multiple-antibiotic resistance is furthermore associated with resistance to heavy metals (e.g., Cu 2 +, Pb 2 +, Zn2+). This phenomenon was observed both in drinking water and wastewater. The public health significance of this phenomenon deserves further study. Strategies for tackling the serious problem of drug resistance include the reduced use of antibiotics in humans and animals, preventive measures for the transmission of infectious diseases, and increased efforts by the scientific community to better understand the mechanisms of drug resistance in microorganisms.

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MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

1.2.2 Viral Pathogens Water and wastewater may become contaminated by approximately 140 types of enteric viruses. These viruses enter into the human body orally, multiply in the gastrointestinal tract, and are excreted in large numbers in the feces of infected individuals. Many of the enteric viruses cause non apparent infections that are difficult to detect. They are responsible for a broad spectrum of diseases ranging from skin rash, fever, respiratory infections, and conjunctivitis to gastroenteritis and paralysis. It was estimated that nonpolio enteroviruses cause 10-15 million symptomatic infections/ year in the United States. Virus presence in the community wastewater reflects virus infections among the population. Enteric viruses are present in relatively small numbers in water and wastewater. Therefore, environmental samples of 10-1000 L must be concentrated in order to detect these pathogens. An ideal method should fulfil the following criteria: applicability to a wide range of viruses, processing of large sample volumes with small-volume concentrates, high recovery rates, reproducibility, rapidity, and low cost. A number of approaches have been considered for accomplishing this task. The most widely used approach is based on the adsorption of viruses to electronegative and electropositive microporous filters of various compositions (e.g., nitrocellulose, fiber-glass, chargemodified cellulose, epoxy-fiberglass, cellulose + glass fibers, positively charged nylon membranes). This step is followed by elution of the adsorbed viruses from the filter surface. Further concentration of the sample can be obtained by membrane filtration, organic flocculation (using heef extract or casein) or aluminum hydroxide hydroextraction. The concentrate is then assayed using animal tissue cultures, immunological or genetic probes. Other adsorbents considered for virus concentration include glass powder, glass wool, bituminous coal, bentonite, iron oxide, modified diatomaceolls earth or pig erythrocyte membranes. From an epidemiological standpoint, enteric viruses are mainly transmitted via person-to-person contacts. However, they may also be communicated by water transmission either directly (drinking water, swimming, aerosols) or indirectly via contaminated food (e.g., shellfish, vegetables). Some enteric viruses (e.g., hepatitis A virus) persist on environmental surfaces, which may serve as vehicles for the spread of viral infections in day-care centers or hospital wards.

INTRODUCTION

23

Table lA Some human enteric viruses Virus Group A. Enteroviruses Poliovirus Coxsackievirus A

B

Echovirus

Enteroviruses (68-71) Hepatitis A virus (HAy) Hepatitis E virus (HEY) B. Reoviruses C. Rotaviruses D. Adenoviruses

Serotypes 3 23

6

34

4

3 4 41

E. Norwalk agent (calicivirus) E Astroviruses

5

Some diseases caused Paralysis Aseptic meningitis Herpangia Aseptic meningitis Respiratory illness Paralysis Fever Pleurodynia Aseptic meningitis Pericarditis Myocarditis Congenital heart Anomalies Nephritis Fever Respiratory infection Aseptic meningitis Diarrhea Pericarditis Myocarditis Fever, rash Meningitis Respiratory illness Infectious hepatitis Hepatitis Respiratory disease Gastroenteritis Respiratory disease Acute conjunctivitis Gastroenteritis Gastroenteritis Gastroenteritis

The infection process depends on the minimal infectious dose (MID) and on host susceptibility, which involves host factors (e.g., specific immunity, sex, age) and environmental factors (e.g., socioeconomic level, diet, hygienic conditions, temperature, humidity) factors.

24

MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY Excreta from humans and animals

•

Wastewater

Figure 1.4 Waterborne transmission of enteric viruses.

Although the MID for viruses is controversial, it is generally relatively low as compared with bacterial pathogens. Experiments with human volunteers have shown an MID of 17 PFU (plaque-forming units) for echovirus 12. Several epidemiological surveys have shown that enteric viruses are responsible for 4.7-11.8 percent of waterborne epidemics. Epidemiological investigations have definitely proved the waterborne and food-borne transmission of viral diseases such as hepatitis and gastroenteritis.

1.2.2.1 Hepatitis Hepatitis is caused mainly by the following viruses: 1. Infectious hepatitis is caused by hepatitis A virus (HAV) , a 27 nm RNA enterovirus (enterovirus type 72 belonging to the family picornaviridae) with a relatively short incubation period (2-6 weeks) and displaying a fecal-oral transmission route. Although it can be replicated on primary and continuous human or animal tissue cultures, it is hard to detect because it does not always display a cytopathic effect. Other means of detection of HAV include genetic probes, use of peR, and immunological methods. 2. Serum hepatitis is caused by hepatitis B virus (HBV), a 42 nm DNA virus displaying a relatively long incubation time (4-12 weeks). This virus is transmitted by contact with infected blood or by sexual contact. The mortality rate (1-4 percent) is higher

INTRODUCTION

25

than for infectious hepatitis «0.5 percent). Hepatitis B virus is responsible for approximately 60 percent of the 434,000 cases of liver cancer worldwide. 3. Non-A, non-B infectious hepatitis is caused by hepatitis E virus (HEy). Hepatitis A virus causes liver damage with necrosis and inflammation. After the onset of infection, the incubation period may last up to 6 weeks. One of the most characteristic symptoms is jaundice. In the United States, approximately 140,000 persons are infected annually with HAY. Hepatitis A is transmitted via the fecal-oral route either by person-to-person direct contact, waterborne, or food-borne transmission. Concentration of HAV in feces can reach 107 to 109/ g. This disease is distributed worldwide and the prevalence of HAV antibodies is higher among lower socioeconomic groups and increases with the age of the infected individuals. Direct contact transmission has been documented mainly in nurseries (especially among infants wearing diapers), mental institutions, prisons, or military camps. Waterborne transmission of infectious hepatitis has been conclusively demonstrated and documented worldwide on several occasions. It has been estimated that 4 percent of hepatitis cases observed during the period 1975-1979 in the United States were transmitted through the waterborne route. The hepatitis cases are due to consumption of improperly treated water or contaminated well water. Hepatitis A outbreaks were also associated with swimming in lakes or public pools. Food-borne transmission of HAV appears to be more important than waterborne transmission. Consumption of shellfish grown in wastewater-contaminated waters accounts for numerous hepatitis and gastroenteritis outbreaks documented worldwide. Passive immunization by means of pooled immunoglobulin is used for the prevention of infectious hepatitis. Vaccines against hepatitis A are available around the world. Hepatitis E virus (HEy) is a single-stranded RNA virus, the classification of which is not known. It is believed that this virus may be a calicivirus. This virus is not well characterized due to the lack of a tissue culture cell line for its assay. Unlike HAY, it is mainly transmitted via fecally contaminated water, while the personto-person transmission is very low. Hepatitis E virus epidemics generally involve thousands of cases. A notorious non-A, non-B hepatitis (now recognized as hepatitis E

26

MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

virus) epidemic broke out in 1956 in New Delhi, India, and resulted in approximately 30,000 cases. A more recent outbreak involved approximately 79,000 people in I
1.2.2.2 Viral gastroenteritis Gastroenteritis is probably the most frequent waterborne illness; it is caused by protozoan parasites, and by bacterial and viral pathogens (e.g., rotaviruses, Norwalk-like agents, adenoviruses, astroviruses). In this section we will examine rotaviruses, caliciviruses genetically related to Norwalk-type virus, and enteric adenoviruses as causal agents of gastroenteritis. 1.2.2.2.1 Rotaviruses Rotaviruses, belonging to the family Reoviridae, are 70 nm particles containing double-stranded RNA surrounded with a doubleshelled capsid. Rotaviruses are the major cause of infantile acute gastroenteritis in children less than two years of age. This disease largely contributes to childhood mortality in developing countries, and is responsible for millions of childhood deaths per year in Africa, Asia, and Latin America. It is also responsible for outbreaks among adult populations (e.g., the elderly) and is a major cause of travelers' diarrhea. Up to 1011 rotavirus particles can be detected in patient stools. The virus is spread mainly by the fecal-oral route, but a respiratory route has also been suggested. There have been several outbreaks of gastroenteritis where rotaviruses originating from wastewater have been implicated. Detection of rotaviruses in wastewater, drinking water, and other environmental samples is accomplished by using electron microscopy, enzyme-linked immunosorbent assays, reverse transcription-PCR method (RT-PCR), which helped identify types 1, 2, and 3 in wastewater, or tissue cultures (a popular cell line is MA-I04, which is derived from fetal rhesus monkey kidney). Detection in cell cultures include methods such as plaque assay, cytopathic effect (CPE), or immunofluorescence. Information on the fate of rotaviruses in the environment is available mostly for simian rotaviruses (e.g., strain SA-Ii) and little is known concerning the four known human rotavirus serotypes. A recent molecular epidemiological survey for

INTRODUCTION

27

rotaviruses in wastewater showed that the environmental rotavirus isolates from wastewater effluents displayed profiles similar to human rotaviruses isolated from fecal samples. Using RT-PCR, rotavirus RNA was detected in drinking water in homes with children suffering from rotaviral acute gastroenteritis, but the sequences found in drinking water were different from those found in the patients' feces.

1.2.2.2.2 Human caliciviruses Gastroenteritis is often caused by small round-structured viruses that have been characterized as caliciviruses genetically related to the Norwalk-like virus (NLV). The latter is a small 27 nm virus, first discovered in 1968 in Norwalk, Ohio, and is a major cause of waterborne disease and is also implicated in food-borne outbreaks, especially those associated with shellfish consumption. The virus causes diarrhea and vomiting and appears to attack the proximal small intestine, but the mechanism of pathogenesis is poorly understood as the exact site of virus replication has not been identified. The Norwalk virus plays a major role in waterborne gastroenteritis but also appears to playa role in travelers' diarrhea. There is a lack of long-term immunity to NLVs. These viruses are stable under environment?l conditions and appear to have a low infectious dose (estimated at 10-100 virus particles). Although personto-person transmission is the prevalent mode of transmission of NLVs, food-borne (e.g., via shellfish consumption, contaminated fruits and vegetables, food handlers) and waterborne transmission (e.g., tapwater, ice, well water, bottled water) have been well documented around the world. Approximately half of the reported outbreaks of gastroenteritis in the US, Japan and Europe are caused by Norwalklike viruses (NLV). Because NLV cannot be propagated in tissue cultures, the tools mostly used for their detection in clinical samples, immune electron microscopy and radioimmunoassay techniques, are not sensitive enough for environmental monitoring. New methodology includes immunomagnetic separation followed by RT-PCR or an RT-PCRDNA enzyme immunoassay for detection of NLV in stools and shellfish but more work is needed for the development of rapid diagnostic assays for environmental monitoring. 1.2.2.2.3 Enteric adenoviruses Enteric adenoviruses belong to subgroup F adenoviruses, which comprises 2 serotypes types 40 and 41, and are commonly found in stools « 1011 viruses/g feces) of children with gastroenteritis.

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MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

These nonenveloped double-stranded DNA viruses have an 80-nm diameter. The most frequent symptoms of infection by these viruses are diarrhea and vomiting. Enteric adenoviruses can be detected using a commercial monoclonal ELISA method or PCR technique. They are found in raw wastewater and effluents, and are suspected to also be transmitted by the water route. They are quite resistant to UV irradiation. 1.2.2.2.4 Astroviruses Astroviruses are 27-34 nm spherical nonenveloped singlestranded RNA viruses with a characteristic starlike appearance. Seven human serotypes have been identified to date. Astroviruses mostly affect children and immunocompromised adults and are transmitted by the fecal-oral route, spreading via person-to-person contacts and via contaminated food or water. Astroviruses are the second leading cause of viral gastroenteritis in children and adults. The mild, watery diarrhea lasts for 3-4 days, but can be long-lasting in immunocompromised patients. Astroviruses are traditionally detected with immune electron microscopy but molecular probes (e.g., RT-PCR, RNA probes) and immunoassays (e.g., monoclonal antibodies) are now available for their diagnosis. After treatment with trypsin, astroviruses can grow in human embryonic kidney cultures or in Caco cell cultures. Infectious astroviruses can be detected in environmental samples through growth on tissue cultures followed by hybridization with a specific cDNA probe. 1.2.3 Protozoan Parasites Most of the protozoan parasites produce cysts, which are able to survive outside their host under adverse environmental conditions. Encystment is triggered by factors such as lack of nutrients, accumulation of toxic metabolites, or host immune response. Under appropriate conditions, a new trophozoite is released from the cyst. This process is called excystment. The major waterborne pathogenic protozoa affecting humans are presented in the following subsections.

1.2.3.1 Giardia This flagellated protozoan parasite has a pear-shaped trophozoite (9-21 JLm long) and an ovoid cyst stage (8-12 JLm long and 7-10 JLm wide). An infected individual may shed up to 1-5 X 106 cysts/ g feces. Domestic wastewater is a significant source of Giardia, and wild and domestic animals act as important reservoirs of Giardia

29

INTRODUCTION

cysts. This parasite is endemic in mountainous areas in the United States and infects both humans and domestic and wild animals (e.g., beavers, muskrats, dogs, cats). Infection is caused by ingestion of the cysts found in water. Passage through the stomach appears to promote the release of trophozoites, which attach to the epithelial cells of the upper small intestine and reproduce by binary fission. They may coat the intestinal epithelium and interfere with absorption of fats and other nutrients. They encyst as they travel through the intestines and reach the large intestine. In humans, infections may last months to years. The infectious dose for Mongolian gerbils is more than 100 cysts, and is generally between 25 and 100 in human volunteers, but may be as low as 10 cysts. Giardia has an incubation period of 1-8 weeks and causes diarrhea, abdominal pains, nausea, fatigue, and weight loss. However, giardiasis is rarely fatal. Although its usual mode of transmission is the person-to-person or food routes, Giardia is recognized as one of the most important etiological agents in waterborne disease outbreaks. The first major documented outbreak of giardiasis in the United States occurred in 1974 in Rome, New York, and was associated with the presence of Giardia in the water supply. It affected approximately 5000 people (10 percent of the town's population). The outbreak occurred as a result of consumption of water that has been chlorinated but not filtered. Other outbreaks have been reported in Colorado, New Hampshire, Pennsylvania, South Dakota, Tennessee, Utah, Vermont, and Washington. In the United States, during the 1971-1985 period, more than 50 percent

Nucleus _ _....t"::f'll

G. lamblia trophozoite

=;~~r4_ Nucleus

G. lamblia cyst

Figure 1.5 Giardia lal1lblia trophozoite and cyst.

MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

30

of the outbreaks resulting from the use of surface waters were caused by Giardia. Approximately 80 giardiasis outbreaks were recorded in the United States from 1965 to 1983. In the United States, Giardia was the etiologic agent responsible for 27 percent of the outbreaks reported in 1989-1990, and for 16.6 percent of the outbreaks reported in 1993-1994. A survey of giardiasis cases in the United States between 1992 and 1997 indicated 0.9-42.3 cases of giardiasis per 100,000 population, with a national average of 9.5 cases per 100,000 population. It is estimated that as many as 2.5 million cases of giardiasis occur annually in the United States. It is estimated that Giardia is responsible for 100 million mild cases and one million severe cases per year, worldwide. Many animals may serve as reservoirs in the environment

Cysts in environmental waters

Cyst wall (0.3 11m thick)

Axonemes Median body

7-10 11m

(J)' J ! r

Cysts excreted in feces (up to 1DB/day)

Ingestion bya

1

suitaYle host (e.g. human)

Gall bladder

/

iI*

Tropozoites in upper .....-4-- small intestine

Figure 1.6 Life cycle of Giardia.

Non-infective trophozoites excreted in feces

INTRODUCTION

31

Most of the giardiasis outbreaks are associated with the consumption of untreated or unsuitably treated water (e.g., water chlorinated but not filtered, interruption of disinfection) and with recreational activities during the summer season. Amendments of the Safe Drinking Water Act (Surface Water Treatment Rule) in the United States mandates the U.S. EPA to require filtration and disinfection for all surface waters and groundwater under the direct influence of surface water to control the transmission of Giardia spp. and enteric viruses. However, exceptions (e.g., effective disinfection) to this requirement have been considered. Faulty design or construction of filters may lead to breakthrough of Giardia lamblia and subsequent contamination of drinking water. Traditional bacterial indicators are not suitable as surrogates for the presence of Giardia cysts in water and other environmental samples. A good correlation has been reported between the removal of Giardia cysts, Cryptosporidium oocysts and bacterial pathogens, and some traditional parameters of water quality such as turbidity. Microbiological quality can be significantly improved at a turbidity below 0.2 NTU. Oocyst-sized polystyrene microspheres also appear to be reliable surrogates for C. parvum oocyst removal by filtration. Giardia cysts generally occur in low numbers in aquatic environments and must be concentrated from water and wastewater, by means of ultrafiltration cassettes, vortex flow filtration, or adsorption to polypropylene or yarn wound cartridge filters. Because Giardia lamblia cannot be cultured in the laboratory, the detection of the cysts necessitates other approaches such as immunofluorescence, using polyclonal or monoclonal antibodies, or by phasecontrast microscopy. Moreover, cysts exposed to chlorine concentration from 1 to 11 mg/L, although fluorescing, could not be confirmed by phase contrast microscopy because they lost their internal structures. Cysts can also be selectively concentrated from waters samples by an antibody-magnetite procedure. Cysts, following exposure to a mouse anti-Giardia antibody, are allowed to react with an anti-mouse antibody-coated magnetite particles and then concentrated by high-gradient magnetic separation. U.S. EPA methods 1622 and 1623 consist of filtration of the sample through a pleated membrane capsule (1 /-Lm) or a compressed foam filter, followed by elution, purification by immunomagnetic separation (lMS) of the cysts, and observation under a florescence microscope following staining with fluorescein isothiocyanate (FITC)

32

MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

conjugated monoclonal antibody (FAb) and counterstaining with DAPI. Cyst recovery is significantly improved by heating at 80°C for 10 min prior to DAPI staining. In vivo infectivity assays, using gerbils, give information on both the viability and infectivity of the cysts. Alternative methods include the use of cell cultures (e.g., caco-2 cells), in vitro excystation, and fluorogenic dyes. The latter include a propidium iodide (PI) in combination with fluorescein diacetate (FDA) or DAPI. Fluorescein diacetate uptake and degradation releases fluorescein, which renders the cysts fluorescent. Although cysts' response to FDA sometimes correlates well with infectivity to animals, this stain may overestimate their viability. Conversely, there is a negative correlation between cyst staining with propidium iodide and infectivity, indicating that this stain can be used to determine the number of nonviable cysts. Alternative vital dyes include SYrO-9 and SYfO-59 flu orogenic dyes. It was proposed to combine the use of these fluorogenic dyes with Nomarski differential interference contrast microscopy for examination of morphological features of the cysts. Molecular-based methods have also been applied for the detection of cysts and oocyts in water and wastewater. A cDNA probe was constructed for the detection of Giardia cysts in water and wastewater concentrates. However, this method does not provide information on cyst viability. Amplification of the giardin gene by PCR has been used to detect Giardia, and several primer pairs have been developed to detect this parasite at the genus or species level. Distinction of live from dead cysts was made possible by measuring the amount of RNA before and after excystation. Viable Giardia cysts can also be detected by PCR amplification of heat-shock-induced mRNA that codes for heat shock proteins. Several substances (e.g., humic acids) that are present in environmental samples interfere with pathogen and parasite detection by PCR technology. However, several methods have been proposed to remove this interference. A survey of raw wastewater from several states in the United States showed that Giardia cyst& numbers varied from hundreds to thousands of cysts per liter, but cyst concentration may be as high as 10'/L. In Italy, Giardia cysts were found in raw wastewater throughout the year at concentrations ranging from 2.1 x 103 to 4.2 x 10 4 cysts/L. In Arizuna, Giardia was detected at concentrations of 48 cysts/40 I of activated sludge effluent. This

INTRODUCTION

33

concentration decreased to 0.3 cysts/40 L after sand filtration. It was suggested that wastewater examination for Giardia cysts may serve as a means for determining the prevalence of giardiasis in a given community. A survey of a wastewater treatment plant in Puerto Rico showed that 94-98 percent of these parasites are removed following passage through the plant. This parasite is more resistant to chlorine than bacteria, Furthermore, Giardia cysts have been detected in 16 percent of potable water supplies (lakes, reservoirs, rivers, springs, groundwater) in the United States at an average concentration of 3 cysts/ 100 L. Another survey of surface water supplies in the United States and Canada showed that cysts occurred in 81 percent of the samples. A survey of drinking water plants in Canada indicated that 18 percent of the treated water samples were positive for Giardia cysts, but viable cysts were found in only 3 percent of the samples. A total of 80 percent of the plants treated their water solely by chlorination without any filtration. In Japan, Giardia was detected in 12 percent of drinking water samples with a mean concentration of 0.8 cyts/lOOO L. 1.2.3.2 Cryptosporidium The coccidian protozoan parasite Cryptosporidium was first described at the beginning of the twentieth century. It was known to infect mostly animal species (calves, lambs, chicken, turkeys, mice, pigs, dogs, cats), but infection of humans was reported only during the 1970s in an immunocompetent child. Cryptosporidium parvum is the major species responsible for infections in humans and animals. The infective stage of this protozoan is a thick-walled oocyst (5-6 JLm in size), which readily persists under environmental conditions. An infected individual may release up to 109 oocysts per day. Following ingestion by a suitable host, the oocysts undergo excystation and release infective sporozoites, which parasitize epithelial cells mainly in the host's gastrointestinal tract. Animal models showed that as few as one to ten oocysts may initiate infection, while a study with 29 healthy human volunteers showed a minimum infective dose of 30 oocysts and a median infective dose of 132 C. parvum oocysts. The parasite causes a profuse and watery diarrhea that typically lasts for 10 to 14 days in immunocompetent hosts and is often associated with weight loss and sometimes nausea, vomiting, and low-grade fever. The duration of the symptoms and the outcome depend on the immunological status of the patient. The diarrhea

34

MICROBIAL PHYSIOLOGY. GE.NETICS AND ECOLOGY

generally lasts 1-10 days in immunocompetent patients, but may persist for longer periods (more than 1 month) in immunodeficient patients (e.g, AIDS patients, cancer patients undergoing chemotherapy). Drug therapy to control this parasite is not yet available. Examination of thousands of human fecal samples in the United States, Canada, and Europe has shown that the prevalence of human cryptosporidiosis is within a range of 1-5 percent. It is estimated that C. parvum is responsible for 250-500 million infections per year in developing countries. Person-to-person, waterborne, food-borne, and zoonotic routes are all involved in the transmission of Cryptosporidium. Person-to-person transmission is the major route, especially in day-care centers. The zoonotic route, the transmission of the pathogen from infected animals to humans, is suspected to be greater for Cryptosporidium than for Giardia. Molecular analysis of isolates from human and animal sources showed that there are two Cryptosporidium genotypes, with genotype 1 found in humans, and genotype 2 found in both animals and humans. This supports the existence of two separate transmission cycles of this parasite. Studies have been conducted on the prevalence of this protozoan parasite in the environment since some of the outbreaks are waterborne. Ten of the 30 drinking-water-associated outbreaks reported in 1993-1994 in the United States were attributed to Cryptosporidium or Giardia. Cryptosporidiosis outbreaks occurred in Georgia, Minnesota, Nevada, Texas, Washington, and Wisconsin. The outbreak in Carrollton, Georgia, affected approximately 13,000 people and was epidemiologically associated with consumption of drinking water from a water treatment plant where rapid sand filtration was part of the treatment processes. Problems discovered in the plant included ineffective flocculation and restarting of the sand filter without backwashing. Cl)'ptosporidium was identified in 39 percent of the stools of patients during the outbreak and in samples of treated water, while no other traditional indicator was identified in the samples. The largest documented waterborne disease outbreak occurred in Milwaukee, Wisconsin, where 403,000 people became ill, of whom 4400 were hospitalized and 54 died. A retrospective cost-of-illness analysis showed that the total cost of that outbreak was $96.2 million in medical costs and productivity losses. There are also reports of swimming-associated cases of cryptosporidiosis.

INTRODUCTION

35

Cryptosporidium is not efficiently removed or inactivated by traditional water treatment processes such as sand filtration or chlorination, although lime treatment for water softening can partially inactivate Cryptosporidium oocysts, and short-term (15 s) pasteurization at 71. rc is able to destroy the infectivity of C. parvum oocysts. Compliance with U.S. EPA standards does not guarantee protection from infection with Cryptosporidium. Concentration techniques have been developed for the recovery of this parasite but they are still at the developmental stage. The methods used involve, for example, the retention of oocysts on polycarbonate filters, polypropylene cartridge filters, vortex flow filtration, hollow fiber ultrafiltion, or concentration via passage through a membrane filter that is dissolved in acetone and then centrifuged to pelletize the oocysts. Although the recovery efficiency of these concentration techniques is relatively low, methodology improvements have been reported. The oocysts are detected in the concentrates using polyclonal or monoclonal antibodies combined with epifluorescence microscopy, flow cytometry, genetic probes in combination with PCR, or electronic imaging of the fluorescent oocysts, using cooled charge couple devices (CCD). The method currently used by the U.S. EPA is the immullomagnetic separation OMS) fluorescent antibody (FA) detection technique. Method 1623 and cell culture-PCR gave similar recovery efficiencies as regards the detection of Cryptosporidium in water. The comparison between the two methods revealed that about 37 percent of the Cryptosporidium oocysts detected by Method 1623 are infectious. The recovery efficiency of the method can be also be improved by using a polysulfone hollow-fiber single-use filter instead of the pleated polyethersulfone filter used in the EPA method. Immunomagnetic separation was also combined with PCR for oocyst detection. Viable oocysts can be detected with IMS followed by RT-PCR, which targets the hsp70 heat-shock-induced mRNA. The assay did not give any signal following heat treatment (20 min at 95°C) of the oocysts, confirming that the method detects only viable oocysts. Oocyst viability and infectivity is generally determined by in vitro excystation, mouse infectivity assay, in vitro cell culture infectivity assays, or staining with fluorogenic vital dyes such as DAPI (4,6diamino-2-phenylindoleL propidium iodide, SYfO-9 or SYfO-59. However, the vital-dye-based assays and in vitro excystation were found to overestimate oocyst viability. Although the mouse infectivity assay is considered the method of choice for assessing oocyst

36

MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

infectivity, the in vitro cell culture assay should be considered as a practical and accurate alternative, A PCR method, based on the amplification of an 873 bp gene fragment, detects the excysted sporozoite, and therefore allows a distinction between live and dead oocysts, An immunomagnetic capture PCR method was proposed to detect viable Cryptosporidium parvum in environmental samples. The procedure consists of capturing Cryptosporidium oocysts on IgG-coated magnetite particles followed by excystation, PCR amplification, and identification of the PCR products. Oocyst infectivity can also be determined by combining infectivity assays on cell cultures with RT-PCR, which targets a heat shock protein 70 (hsp 70) gene. It was also suggested that the Asian benthic freshwater clam (Corbicula fluminea) or the marine mussel (Mytilus edulis) , be used as biomonitors for the presence of Cryptosporidium oocysts in water and wastewater. Immunofluoresence microscopy showed that ,he clam concentrates the oocysts in the hemolymph, where they are phagocytosed by the hemocytes. Oocysts uptake and phagocytosis by hemocytes was also demonstrated for the Eastern oyster, Crassostrea virgil1ica. These methodologies allow the detection of this hardy parasite in wastewater, surface, and drinking water and show that oocysts occur in raw wastewater at levels varying between 850 and 13,700 oocysts/L. The range of oocyst concentrations in wastewater effluents varies between 4 and 3960 cysts/L. A survey of potable water supplies in the United States showed that oocysts were present in 55 percent of the samples at an average concentration of 43 oocysts/ 100 L. Other surveys indicated the presence of oocysts in up to 87 percent of surface water samples, 8.7 percent of coastal waters, and 16.7 percent of cistern waters. Analysis of river water in the western United States showed Cryptosporidiu111 oocysts in each of the 11 rivers examined, at concentrations ranging from 2 to 112 oocysts/ L. In Japan, a survey of 18 rivers that serve as sources of water supply, showed that 47 percent of samples were positive for C. parvul11 oocysts. Another study showed that 13 of 13 samples of source water in Japan were positive for Cryptosporidium oocysts. This parasite has also been detected in finished drinking water. 1.2.3.3 Cyclospora

Cyclospora cayetanensis is another emerging diarrhea -causing coccidian parasite, which was first reported in 1986, and is often

INTRODUCTION

37

mentioned in the literature as a "cyanobacterium-like body".

Cyclospora oocysts are spheroidal with an 8-10 JLm diameter and contain two sporocysts, with two sporozoites per sporocyst. Cyclosporiasis has an incubation period of approximately one week, and clinical symptoms include 10ng~lasting watery diarrhea, sometimes alternating with constipation, abdominal cramps, nausea, weight loss, sometimes vomiting, anorexia" and fatigue. Infections may last up to four months in AIDS patients. This parasite infects epithelial cells of the duodenum and jejunum. Diagnosis of infection is based on microscopic examination of stool specimens. The treatment of cyclosporiasis necessitates the use of trimethropim sulfamethoxazole (TMX-SMX). Cyclospora outbreaks have been reported in developing countries, and the parasite is endemic in certain countries such as Nepal, Haiti, and Peru. Some water-associated cases have been documented in the United States. Even after chlorination, drinking water appears to be implicated in outbreaks of diarrhea associated with Cyclospora. In the United States, most infections are associated with the consumption of contaminated fruits and vegetables. An outbreak of cyclosporiasis in 1996 in the United States and Canada resulted in 1465 cases, 67 percent of which were confirmed by various laboratories. The outbreak was associated with consumption of raspberries imported from Guatemala. Other outbreaks have been linked to the consumption of contaminated basil. Cyclospora oocysts are concentrated in environmental samples by filtration methQds similar to those employed for Cryptosporidium. Microscopic examination is used to detect oocysts in sample concentrates. A distinct feature of Cyclospora oocysts is their autofluorescence, as they appear as blue circles when examined under a fluorescence microscope (365 nm excitation filter). Polymerase chain reaction can detect less than 40 oocysts per 100 g of raspberries or basil. Fluorogenic probes, in conjunction with real-time PCR, have been used to detect Cyclospora oocysts. A method based on PCRrestriction fragment length polymorphism (PCR-RFLP) allows the differentiation of Cyclospora cayetanensis from other Cyclospora species, as well as other coccidian parasites such as Eimeria. Unfortunately, there is a lack of in vivo or in vitro culture assays to assess the viability of Cyclospora oocysts. No data are available on the removal/inactivation of this parasite following water and wastewater treatment.

38

MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

1.2.3.4 Microsporidia Microsporidia are obligate intracellular protozoan parasites that cause infections in humans, especially AIDS patients. Prevalence in patients with chronic diarrhea varies between 10 and 50 percent. Individuals ingesting the small (1-5 pm) resistant spores experience chronic diarrhea, dehydration, and significant weight loss. Some microsporidia species may be transmitted by the water route.

1.2.3.5 Elttamoeba histolytica Entamoeba histolytica forms infective cysts (10-15 }.Lm in diameter) that are shed for relatively long periods by asymptomatic carriers; it persists well in water and wastewater and may be subsequently ingested by new hosts. Level of cysts in raw wastewater may be as high as 5000 cysts/L. This protozoan parasite is transmitted to humans mainly via contaminated water and food. It causes amoebiasis or amoebic dysentery, which is a disease of the large intestine. Symptoms vary from diarrhea alternating with constipation to acute dysentery. It may cause ulceration of the intestinal mucosa, resulting in diarrhea and cramps. It is a cause of morbidity and mortality mostly in developing countries and is acquired mostly via consumption of contaminated drinking water in tropical and subtropical areas. Waterborne transmission of this protozoan parasite is, however, rare in the United States.

1.2.3.6 Naegleria Naegleria are free-living protozoa that have been isolated from wastewater, surface waters, swimming pools, soils; domestic water supplies, thermal spring waters, and thermally polluted effluents. Naegleria fowleri is the causative agent for primary amoebic meningoencephalitis (PAME), first reported in Australia in 1965. It is most often fatal, 4-5 days after entry of the amoeba into the body. The protozoan enters the body via the mucous membranes of the nasal cavity and migrates to the central nervous system. The disease has been associated with swimming and diving mostly in warm lakes in southern states of the United States. Using an animal model, it was estimated that PAME risk to humans, as a function of N. fOIVleri concentration in water, is 8.5 x 10-8 at a concentration of 10 amoebae per liter. Another concern is the fact that Naegleria may harbor Legionella pneumophila and other pathogenic microorganisms. The implications of this association with regard to human health are not well known.

INTRODUCTION

39

There are now rapid identification techniques (e.g., cytometry, API ,ZYM System, which is based on detection of enzyme activity), monoclonal antibodies, DNA probes, and PCR methods that can distinguish Naegleria 'fowleri from other free-living amoebas in the environment. A recent method for detecting N. fowleri is solidphase cytometry, which uses fluorescent labeling of microorganisms on a membrane filter followed by an automated counting system.

1.2.3.7 Toxoplasma gondii Toxoplasma gondii is a coccidian parasite that uses cats as a host. It also causes parasitic infections in humans worldwide. Many infections are congenitally acquired and cause ocular disease in children. Others are postnatally acquired and lead to enlargement of the lymph nodes. This parasite causes most damage among AIDS patients and other immunosuppressed individuals. Humans become infected following ingestion of uncooked or undercooked meat or water contaminated with T. gOl1dii oocysts. An outbreak of toxoplasmosis was linked to the consumption of water from a reservoir in Canada. Preventive measures are directed mostly to pregnant women, who should avoid contact with cats as well as contaminated meat. Following concentration and purification (immunomagnetic separation can be used) of environmental samples, T. gondii oocysts can be detected using light or fluorescence microscopy or PCRbased methods. The molecular methods are hampered by the multi layered nature of the oocyst walls, which cause difficulties in DNA extraction. Oocyst viability is assessed via mouse bioassay.

1.2.4 Helminth Parasites Although helminth parasites are not generally studied by microbiologists, their presence in wastewater, along with bacterial and viral pathogens and protozoan parasites, is nonetheless of great concern as regards human health. It is estimated that about 63 percent of the Chinese population is infected with one or more helminth parasites, particularly with Ascaris lumbricoides, Trichuris trichiura, and hookworms Ancylostoma duodenl.lle and Necator americanus. Most of these infections are acquired by the foodborne route. The ova (eggs) constitute the infective stage of parasitic helminths; they are excreted in feces and spread via wastewater, soil, or food. The ova are very resistant to environmental stresses

40

MICROBIAL PHYSIOlOGY. GENETICS AND ECOLOGY

and to chlorination in wastewater treatment plants. There are seasonal fluctuations in the number of helminth eggs in wastewater. Egg concentration in raw wastewater from Marrakech, Morocco, varied between 0 and 120 eggs/L with an annual mean of 32 eggs/L. The most important helminth parasites are presented in the following subsections.

1.2.4.1 Taenia spp. Taenia saginata (beef tapeworm) and Taenia solium (pig tapeworm) are now relatively rare in the United States. These parasites develop in an intermediate host to reach a larval stage called cysticercus and may finally reach humans, which serve as final hosts. Cattle ingest the infective ova while grazing, and serve as intermediate hosts for Taenia saginata, pigs being the intermediate hosts for Taenia solium. The cysticerci invade muscles, eyes, and brain. These parasites cause enteric disturbances, abdominal pains, and weight loss.

1.2.4.2 Ascaris lumbricoides The life cycle of this helminth includes a phase in which the larvae migrate through the lungs and cause pneumonitis (Loeffler' s syndrome). This disease can be acquired through ingestion of only a few infective eggs. Infected individuals excrete a large quantity of eggs and each female Ascaris can produce approximately 200,000 eggs/day. The eggs are dense and are well removed via sedimentation in wastewater treatment plants. Although they are effectively removed by the activated sludge treatment, they are quite resistant to chlorine action.

1.2.4.3 Toxocara canis This parasite infects mainly children with habits of eating dirt. In addition to causing intestinal disturbances, the larvae of this parasite can migrate into the eyes, causing severe ocular damage, sometimes resulting in loss of the eye.

1.2.4.4 Trichuris trichiura Trichuris trichiura causes whipworm infections in humans. The eggs are dense and settle quite well in sedimentation tanks.

1.2.5 Other Problem-Causing Microorganisms Surface waters feeding water treatment plants may harbor large concentrations of blue-green algae (cyanobacteria) such as Anabaena flos aquae, Microcystis aerugillosa, and Schi::.othrix calcicola. These

41

INTRODUCTION

--------] Hands carry Infective ova from soli contaminated with human txcreta. v~getable, dust ete

--

-~-

Ul~ae penetrate muc;;:'--

;0;;;;1

Iymphatics and vehules - 10 right

heart and lu ngs - brea k out Into alveOli - moult twice - ascend respiraloty tree - descend oesophagus to mature In the intestine.

Figure 1.7 Life cycle of Ascaris !umbricoids.

algae produce exotoxins (peptides and alkaloids) as well as endotoxins (lypopolysaccarides) that may be responsible for syndromes such as gastroenteritis. Presently, studies are being undertaken to gain knowledge about the occurrence and removal potential of these toxins in water and waste-water treatment plants. Their health risks have not yet been fully evaluated.

2 Microbial Metabolism' and Growth 2.1 METABOLIC DIVERSITY OF FRESHWATER BACTERIA Variations in the metabolic activities of bacteria are an important aspect of their diversity within the freshwater environment, and reflect their different roles and locations within the ecosystem. 'In this section metabolic diversity is considered in relation to key metabolic parameters, vadations in CO 2 fixation, aerobic and anaerobic decomposition'of organic substrates and the metabolic transition which occurs from high- toloV(-nutrif'nt availability: 2.1.1 Key Metabolic Parameters Four key features define the metabolic status of individual freshwater bacteria. 1. The source of energy. Either from light (phototrophs) by photosynthesis or from chemical energy (chemotrophs). Chemotrophs use energy obtained from energy-yielding (exergonic) reactions to oxidize organic matter. 2. The source of electrons for growth (electron donor). These are obtained either from organic (organotrophs), or from chemical compounds (lithotrophs) such as sulphide, hydrogen, andwater. 3. The source of carbon. required for synthesis of bacterial biomass. This is obtained either by reducing CO2 during photosynthesis (autotrophs), or from complex organic compounds (heterotrophs) . 42

MIC~OBIAL

43

METABOLISM AND GROWTH

4. The .terminal electron acceptor. The final electron acceptor in the process of respiration involves either oxygen (aerobic respiration) or other molecules (e.g., sulphate, nitrate) in anaerobic respiration. The main role of bacteria in the freshwater environment is the breakdown of organic biomass and the recycling of various key elements (nitrogen, phosphorus, sulphur) which are present within the various organic compounds. In line with this, the majority of freshwater bacteria are heterotrophic, living on organic carbon compounds present in the aquatic medium or in the sediments. Using the above terminology, the typical bacterium in the water column or substratum of a freshwater system is a chemo-organoheterotroph, while the typical algal cell is a photolitho-autotroph. Classification of freshwater bacteria in relation to their metabolic activities provides a set of ecologically relevant groups, some of which (chemosynthetic autotrophs, most non-photosynthetic heterotrophs) occur in aerobic environments, while others (e.g., photosynthetic autotrophs, photosynthetic heterotrophs, fermentation bacteria) are present in anaerobic hypolimnia, sediments and other anoxic sites. ENERGY - Light

ALGAE

ELECTRON DONOR

f+.! 0

CARBON -C02 SOURCE ELECTRON - 02 ACCEPTOR

I

Aerobic

Photo-Inho-autotrop~

:~:::RYON 1

DONOR

Organic carbon compounds

CARBON SOURCE ELECTRON - 02 or range ACCEPTOR of compounds Aerobic or anaerobic chemo-organo-heterotrophs

Figure 2.1 General metabolic comparison of freshwater algae and bacteria: algae are normally simply referred to as autotrophs and bacteria as heterotrophs.

2.1.2 CO 2 Fixation Although CO 2 fixation in freshwater environments is usually associated with the photosynthetic requirements of algae and macrophytes, some bacteria also have this activity. Bacteria whichare able to assimilate CO 2 directly are of three types, as follows. 1. Photosynthetic bacteria (photoautotrophs). Using light energy to mediate CO 2 uptake. 2. Chemosynthetic bacteria (chemoautotrophs). These organisms typically occur within the water column at the boundary layer

44

MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

between aerobic and anaerobic zones, using reduced inorganic compounds as energy substrates. The reduced compounds are largely derived by decomposition of organic matter within the anaerobic hypolimnion. Chemosynthetic fixation of CO 2 is particularly high in conditions of steep redox potential gradient which occur at the top of the anaerobic zone, but is low in other parts of the water column. The requirement for an anaerobic hypolimnion means that chemosynthesis tends to be prominent in eutrophic rather than oligotrophic lakes, and in the stable long-term redox gradients that develop in meromictic lakes. 3. Heterotrophic bacteria. Most non-photosynthetic fixation of CO 2 is carried out by heterotrophic bacteria. This dark CO 2 fixation provides a useful parameter for measuring heterotrophic productivity. 2.1.3 Breakdown of Organic Matter in Aerobic and Anaerobic Environments The breakdown of biological material by bacteria ultimately involves an oxidation/reduction reaction, with the transfer of electrons from the organic substrate (oxidation) to an electron acceptor (reduction). This may be summarized:

(Substrate + e) + Acceptor ~ Substrate ... (1) + (Acceptor + e) + free energy Bacteria act as catalysts in mediating this reaction, which can only take place in conditions which are thermodynamically suitable. The energy that is released by this process can be quantified as the free energy per mole of substrate (AG6)' Only a fraction of this energy (1-50 per cent) becomes directly available to the bacterial cell, and is used for catabolic and anabolic (synthetic) processes, both of which promote bacterial productivity and population increase. The free energy that is released in the oxidation process varies with substrate and electron acceptor, which depend on environmental conditions - including the presence (aerobic environment) and absence (anaerobic environment) of oxygen.

2.1.3.1 Aerobic conditions Aerobic conditions may be defined as environments where oxygen is freely available, and is used in the oxidation of organic (and some inorganic) substrates. Aerobic environments contain obligate aerobic microorganisms (which are restricted to oxygen as the secondary

45

MICROBIAL METABOLISM AND GROWTH

electron acceptor) and facultative anaerobes (which are able to use other secondary acceptors in addition to oxygen). In a lake, the concentration of dissolved oxygen (DO) ranges from supersaturation (lake surface) to very low levels in the hypolimnion and sediments. This range of oxygen concentration correlates with a range of oxidizing ability or oxidation/reduction potential (redox potential). Redox potential (E ,,) can be measured in reference to a standard electrode, and is normally expressed as millivolts (mY). In well-oxygenated environments, the redox potential is normally in excess of +360 mY, falling to --500 mY in highly reducing conditions. Because of the connection between free energy released during substrate oxidation and bacterial growth, bacteria that are involved in high-energy reactions have the potential for higher growth rates and can out-compete those that mediate lower-energy reactions. In aerobic environments, oxygen which generates most free energy, will become the main electron acceptor and bacteria that use this to oxidize their substrates will predominate. The surface waters of rivers and lakes are thus populated by obligate aerobes which use complex organic compounds as a source of energy, carbon, and electrons. The oxidation-reduction reaction can be summarized: Organic biomass I

I

(~H20)106(NH3)16(H3P04) + 13802-~

106C02 + 16HN0 3 + H 3 P04 + 122H 20

... (2)

Free energy (AGOl) = -3190 kJmorl The organic compounds which are degraded by bacteria in lakes include material from dead biota (particularly algae that have sedimented out of the epilimnion) and extracellular products (dissolved organic carbon, DOC). In oligotrophic lakes, both of these sources of carbon are minimal, limiting the activity of aerobic heterotrophs and the removal of oxygen, so that the whole water column remains aerobic. In eutrophic lakes, high substrate levels lead to much higher oxygen uptake, resulting in anaerobic conditions. This is particularly acute in the hypolimnion, where there is greatest accumulation of dead organic matter and no photosynthetic generation of oxygen by algae. In streams and rivers, where the entire water column is typically welloxygenated, much aerobic degradation by bacteria occurs on the sediments. Although most bacteria in aerobic environments are involved in the oxidation of organic material, some are able to use inorganic compounds. Such chemolithotrophic bacteria are active in

46

MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

many aerobic situations and are responsible for the oxidation of a range of inorganic substrates including methane, hydrogen sulphide, ammonium ions, nitrite ions, and ferro~s ions. The maximum amount of free energy (6G OI) released per mole of reactants ranges from4 to -810 kJ in these reactions, and is considerable less than the value of -3190 kJmol- 1 generated by oxidation of biomass. Many of the bacteria involved in these reactions are present in the oxygenated microzone of mud-water interfaces, where there is a ready supply of substrate by diffusion from the lower. sediments (internal loading).

2.1.3.2 Anaerobic decomposition of organic matter Anaerobic environments are those where the concentration of oxygen is too low for it to be used as an electron acceptor. These environments may be divided into two main groups - low oxygen environments, and anoxic environments, where oxygen is completely absent.

2.1.3.2.1 Low oxygen environments Although oxygen is the electron acceptor in fully-oxygenated environments, removal of DO by metabolic processes may reduce the availability to such an extent that other electron acceptors (Mn 4 +, N0 3-, Fe3 +, and SO/-) become used instead. The free oxidation energy released per molecule of organic matter via each of these acceptors varie~ considerably, from -380 to 3050 kJ mol-I. Since oxidation of organic matter fO,llows a sequence in which acceptors generating most energy take precedence, each of these acceptors will be used in tum until depleted levels lead on to the next one. This process continues until all oxidizable substrates or all electron acceptors are removed from the system. This chemical sequence is paralleled by an ecological succession, in which whole communities of bacteria change with the chemical envi(,onment. Bacteria that use Mn4 +, NO_-, Fe 3 + are facultative anaerobes, able to use either oxygen or an in~rganic electron acceptor - depending on prevailing conditions.

:i 1.3.i2 Anoxic environments These environments, where oxygen is compietely absent, contain populations of obligate aQaerobic bacteria. Oxidation/reduction reactions using inorganic electron acceptors are severely restricted, though sulphate-reducing bacteria are able to use SO/- when. oxygen is completely exhausted (Ell <200 mY). Most breakdown of organic material occurs by fermentation processes.

47

MICROBIAL METABOLISM AND GROWTH

In anaerobic conditions, organic matter is metabolized by a variety of heterotrophic bacteria, which obtain energy by substrate phosphorylations. In this situation, where oxygen no longer acts as the universal hydrogen acceptor, the situation becomes complex, with a range of organic and inorganic compounds taking Dver this role. In some cases, the same compound can act as hydrogen acceptor or donor, depending on environmental conditions. Large quantities of organic detritus are degraded under the anaerobic conditions which occur in the hypoli)11nion and sediments of eutrophic lakes, and in the sediments of ponds, rivers, and wastetreatment plants (septic tankS, anaerobic lagoons). The process of anaerobic degradation converts biomass to COl' CH 4 , and NH3 and can be summarized: Org"lnic ,biomass i

i

(CB20)106(NH3)16(H3P04) ~ 53C0 2 + 53CH 4 + 16NH3 + H 3P0 4

... (3)

Free energy (,~GOI) = -350 kJmol- 1 Comparison with the oxidation process (using oxygen as electron acceptor) shows that bacteria in the oxygenated part of the water column obtain much more energy from aerobic breakdown of organic material compar~d with those carrying out anaerobic fermentation on tlW se.diments. The process of fermentation occurs as two distinct stages as follows. 1. Initial hydrolysis and fermentation. Hydrolytic and fermentative conversion of proteins, carbohydrates, and fats to a range of breakdown products (primarily fatty acids) is carried out by a heterogeneous group of facultative and obligate anaerobic bacteria. These bacteria generate large amounts of organic acids and are collectively referred to as acid formers. The degradative activity of these organisms results in the formation of CO 2 and various reduced end products, including H 2; H 2S, acetic, , proprlonic, lactic, and butyric acids, ethanol, and amines. These compounds would accumulate in the anaerobic environment if they were not metabolized in various ways. 2. Removal of breakdow11 products. Removal of oxidizable intermediate and end products is carried out by obligate anaerobes which are able to LIse a raJ1ge of hydrogen acceptors such as sulphates, nitrates, and CO 2 , The different groups of

48

MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

r-~==~~IH~Y_dr_OI~%~ls~I______~I=Fe;~ __ en_ta~t=io=nl~__~--~--~-------'C02 Complex carbohydrates. proteins. and lipids

---.

Amino-acids. Fatty acids. simple sugars. - - - . alcohols fallyaclds H2. CO

ACid-forming bacteria

'--------+ CH. Methanogenesis

Figure 2.2 Stages of anaerobic metabolism of complex organic compounds.

organisms that carry out this terminal oxidation include the following. (a) Denitrifying and sulphate-reducing bacteria which use nitrate and sulphate as the ultimate electron acceptor. The activity of these organisms is limited by sulphate and nitrate availability, which has to diffuse from the epilimnion into the hypolimnion in eutrophic lakes. In aerobic sediments, bacteria such as Thioploca obtain their nitrate at the surface, then migrate into anaerobic regions to use the oxygen in nitrate for sulphur oxidation. (b) Methane-producing bacteria, operate under strict anaerobic conditions, and include two rod-shaped (Methanobacterium, Methal1obacillus) and two coccoid (Methanococcus, Methanosarcina) genera. Methane is generated by one of two processes. In the first situation, bacteria use CO 2 as the acceptor of hydrogen derived from the organic acids: CO 2 + 8H -7 CH 4 + 2H1 0 ... (4) In the second process, acetic acid is directly converted to CO 2 and methane: CH 3 COOH -7 CH 4 + CO 2 ... (5) The methane generated by reduction of CO 2 escapes to aerobic regions where it is readily oxidized. Below the top 1-2 mm, deep (profundal) sediments of stratified lakes are permanently anoxic, and methane production is a major activity as a key terminal process in the anaerobic decomposition of organic matter. Fermentation becomes the dominant breakdown process in such conditions because the only alternative, sulphate reduction, is limited in most freshwater lakes by low sulphate availability. In North American lakes, more than half of the total

MICROBIAL METABOLISM AND GROWTH

49

carbon output to the sediment was found to be converted to CH 4 , with H/C0 2 and acetate as the immediate substrates. Studies on Lake Kinneret (Israel) demonstrated a direct coupling in deep sediments between the oxidation of acetate and the generation of methane.

2.1.3.2.3 Photosynthetic bacteria . Reduced substrates such as H 2 S are released from decaying biomass as part of the fermentation process. Subsequent oxidation of reduced substrates by photosynthetic bacteria involves the removal of hydrogen and electrons to drive the reduction of CO 2 in the process of photophosphorylation. The uptake of CO 2 by these organisms as part of an assimilatory sulphate-reduction system is thus important for both carbon compound formation and the disposal of excess hydrogen and electrons from the reduced substrates. Other end products of anaerobic fermentations, including H 2, CH 4 , H 2 S, and N2 can also be metabolized by photosynthetic bacteria, which are able to use hydrogen as an electron acceptor simultaneously with the sulphate assimilatory reduction system. Unlike the other two major groups of anaerobic bacteria involved in the terminal degradation of biomass, photosynthetic bacteria are located at th.:? top of the anaerobic hypolimnion rather than in the lower regions and sediments. 2.1.4 Bacterial Adaptations to Low-nutrient Environments Many bacterial species have evolved special mechanisms to survive the adverse environmental conditions of low-nutrient availability. Gram-positive bacteria tend to form dormant spores, while Gram-negative bacteria have molecular and physiological mechanisms which enable them to persist at low metabolic (but not dormant) activity until adequate nutrient levels return; they then exploit the improved growth conditions and undergo a burst of synthetic activity and population increase. The ability of bacteria to live through conditions of acute nutrient deprivation is referred to as 'starvation-survival' and may be defined as 'the process of survival in the absence of energy-yielding substrates'.

2.1.4.1 Low-nutrient aquatic environments Starvation survival has been investigated particularly in relation to the planktonic bacteria of marine environments, where they may be carried passively within water masses for many years under conditions of extremely low organic carbon availability. Although

50

MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

many of the environmental starvation adaptations shown by aquatic bacteria have been studied specifically in marine organisms, the lownutrient responses of freshwater organisms are expected to be closely similar. Organic nutrients are normally available to freshwater bacteria from exogenous sources or from algae via the microbial loop. Carbon limitation of bacterial populations become particularly acute where there is limited exogenous supply and where algal populations are low due to a lack of available inorganic nutrients (e.g., oligotrophic standing waters) or absence of light.

2.1.4.2 Starvation response The starvation response in aquatic bacteria is a classic example of environmentally-induced molecular activity, and involves the activation of a cohort of 'starvation genes' followed by associated changes in biochemical activity, cell size and shape, and bacterial populations. 2.1.4.2.1 Molecular activation Transition from high- to low-nutrient environment promotes a fundamental change in cell physiology, with a switch from growth to maintenance. Energy reserves need to be mobilized and the cell has to survive in the absence of multiplication. These changes result from the activities of a large set of genes which are induced at the onset of starvation. This induction is the result of the initial activation of the rpoS gene and the formation of a starvation-specific transcription factor (RpoS or (}") which confers new promoter recognition sites on the RNA polymerase. The role of RpoS in the induction of starvation-induced dormancy parallels its role in the NUTRIENT LIMITATION

/'

"'\ Increased cAMP and ppG pp

I

transcription rpeS translation.

t

/' -+

Starvation [RpoS] ----.. RpoS protein ' \ . genes stability

Decreased UDP-glucose

'figure 2.3 Molecular response of Gram-negative bacteria to nutrient limitation.

MICROBIAL METABOLISM AND GROWTH

51

'transition of bacterial populations to a stationary phase during batch culture, and the induction of stationary phase characteristics during quorum sensing in biofilms. Regulation of RpoS by environmental changes in nutrient concentration is mediated by changes in the concentration of internal metabolites, including cAMP and ppGpp (which promote transcription) and UDP-glucose (which represses translation). Factor RpoS is also regulated by post-transcriptional control of its molecular stability.

2.1.4.2.2 Activity of RpoS-controlled genes About 30-50 proteins are thought to be induced via the RpoScontrolled genes, including enzymes which are involved in hydrolysis, protein and carbohydrate synthesis, and protection of DNA. Biochemical changes include a decrease in surface membrane (with changes in biochemical composition), decreases in ATP content, utilization of internal storage compounds and also conversion of internal non-storage molecules for energy formation. Decrease in the surface membrane, with utilization of cell contents for energy formation, results in a decrease in size and a change to spherical shape (for elongate bacteria). The small size of bacteria in the starvation state is one of the most obvious features of this metabolic condition, and may result in the formation of very tiny 'ultramicrocells'. Reduction in biomass formation and cell division leads to a marked decrease in the bacterial population. Amy and Morita (1983) noted four distinct patterns of change during the starvation survival process, the most common of which involved an initial increase in total count followed by a prolonged decline. Viable counts also showed sharp decline, with reductions of over 99 per cent over a 4-week starvation period being recorded for some aquatic bacteria. Resulting populations include a high proportion of dead cells, cells with minimal metabolic activity (surviving but unable to divide in the short-term), and cells with low metabolic activity that are able to respond rapidly to high nutrient and form colonies on nutrient agar. 2.2 PHOTOSYNTHETIC BACTERIA As noted earlier, photosynthetic bacteria can be divided into three major groups - the green sulphur bacteria (Chlorobacteriaceae), purple sulphur bacteria (Thiorhodaceae), and the purple non-sulphur bacteria (Athiorhodaceae).

52

MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

2.2.1 General Characteristics Although these groups are defined primarily in terms of colour and metabolic substrate, they also show differences in terms of cell size, fine structure, photosynthetic activities, involvement in the sulphur cycle, general ecology, and cell motility. Within each group, cell shape varies greatly between species, including spherical, elliptical, rod-shaped, and vibrioid forms. Differences in size occur between species and also between major groups. The overall size of green sulphur bacteria tends to be less than purple sulphur bacteria, which reach maximum diameters of 5-10 p.m. The location of photosynthetic pigments in green sulphur bacteria within distinctive 'chlorobium vesicles' distinguishes the fine structure of these organisms from the other two major groups. Gas vacuoles are found in those organisms (purple and green sulphur bacteria) which form discrete layers in stratified lakes, but not the less ecologically-defined purple non-sulphur bacteria. This distinction highlights the importance of gas vacuoles in the depth-regulation of photosynthetic bacteria. 2.2.2 Motility Motility of photosynthetic bacteria has been investigated particularly in laboratory cultures, where there is considerable variation within species and within major taxonomic groups. Individual photosynthetic bacteria, like algae, come in one of three categories .- non-motile, actively motile (by flagella), and passively motile (by gas vacuoles). Unlike algae, these characteristics are to some extent interchangeable, with environmental factors having an important effect. Cultures of Thiorhodaceae exposed to high sulphide concentrations and light intensity undergo a conversion of all motile to non- motile cells, which sink to the bottom of the vessel and produce copious slime. The ecological significance of active and passive motility is particularly apparent in situations such as the lake water column, where both processes are important in the exact positioning that occurs at the top of the hypolimnion. Many of these organisms are killed by exposure to oxygen in the presence of light, so it is important that they do not stray up into the aerated waters of the epilimniol1. Migration of purple sulphur and green sulphur bacteria has been ob~erved during summer stratification in holomictic lakes, where upward movement of bacterial populations follows the extension of the anaelObic high-sulphide zone that accompanies the rise in

MICROBIAL METABOLISM AND GROWTH

53

the thermocline. Flagellate forms are able to migrate by their own flagellar activity. In vitro studies have suggested that vertical movements of these organisms occur in response to gravity (negatively geotactic) and oxygen (negatively aerotactic). The response to light is non-directional in terms of the stimulus and involves a reversal in direction of flagellar movements followed by a reversal in the direction of cell movement. Gas-vacuolate photosynthetic bactel ia include green sulphur (e.g., Pelodictyol1) and purple sulphur (e.g., Amoebobacter) organisms, which may use their gas vacuoles il1 a similar way to blue-green algae for depth regulation. Observatiqns by Eichler and Pfennig (1990) showed that populations of gas-vacuolate purple bacteria overwintered in flocs of organic material in the sediments of Schleinsee. At stratification, these bacteria disappeared from the sediments, becoming dispersed throughout the hypolimnion and later forming a discrete layer at the top of the hypolimnion. At autumn overturn the bacteria developed deposits of oxidized irOli and manganese which acted as ballast, reducing buoyancy and depositing the cells on the sediments prior to the next over-wintering phase.

2.2.3 Ecology The metabolic activities and requirements of photosynthetic bacteria determine their ecological niche in the aquatic environment. These organisms are often found in narrowly-defined microenvironments within relatively heterogeneous aquatic systems including ponds, ditches, marshes, estuarine mud flats, rivers, and lakes. In contrast to the Athiorhodaceae, which occur widely but never at high population density, the Thiorhodaceae and Chlorobiaceae are often present at high abundance - appearing as red or greenish layers on mud or forming substantial blooms in lakes. Hidden blooms of photosynthetic ~ulphur bacteria are often found below the surface of well-stratified productive lakes, where they are restricted to the top of the hypolimnion (immediately below the thermocline) as a discrete layer. Within this layer, green sulphur bacteria are often localized below an overlaying population of purple sulphur bacteria, in accordance with the higher H,S tolerance of the Chlorobacteriaceae. At this interface, photosynthetic bacteria are just within the euphotic zone (light level typically < 1 per cent lake surface value), with adequate supplies of substrate (reduced sulphur compounds)

54

MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

and under anaerobic conditions. In lakes which completely mix during the annual cycle (holomictic), the bacterial bloom is limited to the period of summer stratification when the thermocline has risen to its highest point and light intensity is maximal. Lakes which do not completely mix (meromictic) have a thermocline (with an associated anaerobic high- sulphide zone) which may be stable over many years and have associated bacterial blooms which are constant over long periods of time. pfenllig et a!. (1966), for example, report the longterm presence of a pink bacterial layer 19.5m below the surface of meromictic lake Blankvann in Norway, from which they were able to isolate a range of purple and green sulphur bacteria. Although photosynthetic bacteria tend to accumulate at the thermocline boundary, they are not restricted to this part of the lake and studies on European lakes have shown that purple and green sulphur bacteria can be found throughout the hypolimnion to depths of 35 m. As noted previously, purple non-sulphur bacteria differ from other photosynthetic bacteria in not building up bloom populations under natural conditions. This is even true for conditions which might be expected to suit their mixotrophic metabolism, such as the presence of organic wastes (e.g., sewage ponds). This lack of bloom formation can be attributed to the presence of sulphate, which is normally present at high levels in such environments and is converted by sulphate reducing bacteria to H,S, directly inhibiting growth of purple non -sulphur organisms. Green and purple sulphur bacteria become dominant under such conditions, eventually reducing the level of sulphide to a point at which purple non-sulphur bacteria are able to co-exist. 2.3 BACTERIA AND INORGANIC CYCLES Freshwater bacteria have a key role in geochemical transitions within the aquatic environment, and are important in the cycling of metabolically-important elements such as nitrogen, Fe and sulphur. The cycling of other elements, such as silicon, may also involve bacterial activity. Studies by Patrick and Holding (1985) have shown that solubilization of diatom frustules (regenerating silicic acid) is increased in the presence of natural populations of freshwater bacteria. 2.3.t Bacterial Metabolism and the Sulphur Cycle The cycling of sulphur within the freshwater environment involves cllternate phases of anabolic and catabolic activity. Sulphate ions are taken up by all lake biota and converted to sulphydryl (-SH) groups

55

MICROBIAL METABOLISM AND GROWTH Lake biota organic-5H Degradation by heterotrophs (1)

Uptake and /' protein / synthesis I

/

~ "-i 504

~::~~~~~~~

_:::::

(2)

.... , . . .

Oxidation

(3)

~ \,

\:

I.H

_........

.' J ......

.: (4) ~ :

2S

#

OXidation (3)

~:~

Aerobic· - -- •• -Anaerobic---

Figure 2.4 Sulphur cycle in a Eutrophic lake: (I) Pseudomonas liquefaciells. Bacterium delicatutll. (2) Desulfovibrio. Desulfotomaculutll. (3) Clzlorobiul1l. Clzromatiutll. (4) Beggiatoa (H 2 S to S). Tlziobacillus (S to S04)'

in the synthesis of proteins. Breakdown of proteins on death of the organism results in the release and conversion of simple sulphur compounds, leading ultimately to the regeneration of sulphate ions. Four main types of microbial metabolic activity are involved in the sulphur cycle - protein decomposition, sulphate reduction, aerobic and anaerobic sulphide oxidation. Microbial interactions involved in the cycling of sulphur within the aquatic environment are clearly localised in eutrophic lakes, where the occurrence of distinct aerobic and anaerobic zones within the water column leads to clear separation of microbial activities. The aerobic epilimnion is the main region of incorporation of inorganic sulphur compounds into lake biomass (trophogenic zone), while the anaerobic hypolimnion and sediments are the primary sites of conversion from organic to inorganic sulphur (tropholytic zones). Dissolved inorganic sulphur occurs primarily as sulphate ions (SO/-) within the oxygenated lake water of the epilimnion. These ions are taken up by algae and other biota and are subsequently reduced to sulphydryl (-SH) groups during protein synthesis. Death and sedimentation of lake biota leads to cell breakdown in the hypolimnion and lake sediment, with further reduction of sulphydryl groups to H2S during the process of protein decomposition. This anaerobic process is carried out by a wide range of bacteria, including Pseudomonas liquelaciens and Bacterium delicatum. Protein

56

MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

decomposition occurs mainly in the surface sediments, where bacterial population counts are up to three times greater than in lake water. Reduction of sulphate generated from mineral sources also occurs in the sediment. In these anaerobic conditions, sulphate provides a source of oxygen for the oxidation of molecular hydrogen or carbon compounds by bacteria such as Desulfovibrio and Desulfotomaculum. H 2S0 4 + 4H2 ~ 4H 2 0 + H 2 S [M;OI = -14kJ mol-I] ... (6) H 2 S0 4 + 2[CHP] ~ 2C0 2 + 2Hp + H 2 S ... (7) H1S, generated in the sediments by protein decomposition and reduction of sulphate, diffuses vertically through the hypolimnion and is oxidized immediately prior to entry, or during entry, into aerobic conditions. Anaerobic sulphur oxidizing bacteria occur at the top of the hypolimnion, and can be divided into two main groups - the green sulphur bacteria and purple sulphur bacteria. In both cases, sulphide is oxidized to sulphur or sulphate as part of a light-mediated reaction. CO 2 + 2H 2 S

light

)[CH 2 0] + H 2 0 + S

... (8)

2C02 +2H 2 0+H2 S light )2[CH2 0]+H2 S04 ... (9) The other major group of sulphur-oxidizing bacteria are colourless chemosynthetic microbes which are mostly aerobic. These organisms oxidize sulphide to sulphate via elemental sulphur, which is deposited either inside (Beggiatoa and Thiothrix) or outside the cell (Thiobacillus) as an intermediate. Sulphur deposition in Beggiatoa and Thiothrix, two organisms typical of sites where H1S is being produced, continues for as long as sulphide is available: H 2S + t02 ~ S + H20[6Gb

= -lOkJ mol-I]

... (10)

Depletion of sulphide ultimately results in metabolism of internal stores of sulphur, with release of sulphate into the surrounding water. S + 1Yl0 2 + H 20 ~ H2S04[6Gb

= -28 kJ mol-I]

... (11)

2.4 BACTERIAL POPULATIONS In most freshwater environments bacteria form the largest population of all free-living biota, and are only exceeded by viruses in terms of total organisms present. The population ecology of freshwater bacteria is thus characterized by high cell counts (both planktonic and biofilm communities) and the capacity for rapid rates of reproduction. Bacterial populations tend to show marked

MICROBIAL METABOLISM AND GROWTH

57

fluctuation in response to environmental factors that promote (e.g., inorganic and organic poilu tants, increase DOC levels from phytoplankton blooms) or deplete (e.g., increased levels of zooplankton or protozoon grazing) the increase in biomass. 2.4.1 Techniques for Counting Bacterial Populations Populations of bacteria in suspension can be enumerated either as total or viable counts. Direct counting of bacteria (total counts) is regarded as the most reliable method for evaluation of community dynamics since all cells are included. Measurements of bacterial dimensions can be used to convert counts of cell numbers to estimates of total bacterial biomass, which in turn can be combined with measures of bacterial productivity to provide useful information on the dynamics of bacterial populations.

2.4.1.1 Total counts Although direct counts" of aquatic bacteria in environmental samples are routinely carried out by light microscopy, the small size (normally 0.2-5 p,m diameter) of these organisms may create problems since they are close to the resolution of the light microscope and are also within the size range of organiclinorganic particulate material typically present in lakes and rivers. Both of these problems may be largely overcome using fluorescent stains which enhance the detection of bacteria and do not normally label particulate nonliving material. Various stains are available. Acridine orange forms green and red fluorescing complexes with DNA and RNA respectively when excited with light (wavelengths 436 or 490 nm), while DAPI (4"6- diamidino-2-phenylindole) forms a blue fluorescing complex with DNA at or above 390 nm. The procedure for direct counting involves addition of the stain to the water sample, filtration of a known volume through a polycarbonate filter then examination and enumeration using epifluorescence microscopy. Preparations from lakes and rivers typically reveal a very high population of bacteria, with individual cells ranging in size and shape. Total counts can also be made under the scanning electron microscope after passing a known volume of chemically-fixed water sample through a 0.2p,m (pore diameter) filter membrane. 2.4.1.2 Viable counts Counts of viable (metabolically-active) heterotrophic bacteria can be readily carried out by plating water samples onto nutrient agar

58

MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

plates and counting the number of colonies that develop. Bacterial counts, expressed as colony forming units (CFU) ml-I, record those organisms that are able to grow and multiply on the nutrient medium. Although this approach can potentially give information on the total number of metabolically active heterotrophic bacteria (total heterotrophic viable count) present in the sample, there are a number of problems. Most importantly, all plating media are highly selective, and many viable organisms with complex nutrient requirements (fastidious organisms) will be excluded. Bacteria requiring specific physicochemical parameters (e.g., particular conditions of pH or oxygen concentration) may also be excluded from the count. 2.4.3 Biological Significance of Total and Viable Counts Total bacterial counts from freshwater sites are invariably higher than viable counts. For example, total bacterial counts in two freshwater lakes were 106-10 7 cells ml- I , compared with viable counts of 103 - 104 cells ml- I • In this situation, only 1 in 103 total bacteria were being recorded as viable cells. Differences between total and viable counts reflect the high degree of heterogeneity within natural microbial communities, and arise for two main reasons: 1. Most bacteria are metabolically inactive - although these organisms are able to become metabolically active when environmental conditions improve, they do not form colonies when plated out on nutrient agar and are referred to as 'non-viable'; 2. Many metabolically active bacteria require particular growth conditions which are not satisfied in routine laboratory cultures. Values for total and viable counts of planktonic bacteria vary with the trophic status of the water body and also the position within the water column. Comparison of mesotrophic Lake Windermere and eutrophic Esthwaite Water (UK) indicated higher levels of total bacteria (approaching 107 cells ml- I , and much higher levels of viable bacteria (over 10 4 cells ml- I ) under eutrophic conditions. In the nutrient-rich lake, total counts of bacteria are particularly high in the hypolimnion, where accumulation of algal detritus provides an organically-rich environment. The contrasting presence of highest viable counts in the epilimnion of the eutrophic lake partly reflects the high levels of dissolved organic carbon (DOC) released by the standing populations of phytoplankton, but may also reflect problems in culturing strictly anaerobic and fastidious organisms from the hypolimnion.

MICROBIAL METABOLISM AND GROWTH

59

2.5 BACTERIAL PRODUCTIVITY Bacterial productivity is a dynamic aspect of community function and an important component of biomass formation and transfer in freshwater microbial food webs. As with other lake biota, productivity refers to the intrinsic rate of increase in biomass and may be considered as 'net' and 'gross' terms, where: Net productivity = Gross productivity - Internal mass loss ... (12) Gross productivity of planktonic bacteria is the underlying rate of increase in biomass, and is the sum total of all the anabolic (synthetic) processes that promote growth. Internal mass loss includes catabolic (breakdown) processes such as respiration, and excretory/ secretory processes such as exoenzyme production: Net productivity gives a measure of the fundamental growth rate of the population before external factors such as grazing, parasitism, and sedimentation are taken into account. Net productivity and population increase are thus quite different concepts, though productivity is clearly an important factor in population increase. Although productivity is typically considered, and frequently measured, as the rate of increase in dry weight, these values are normally converted to carbon content and expressed as the increase in mass of carbon per unit volume of water per day (mg C m- 3 d- I ). 2.5.1 Measurement of Productivity Bacterial productivity can be determined with reference to a number of parameters, all of which are directly related to growth rate. These include: 1. Rate of cell division. Determination of the number of dividing cells within the population. 2. Assessment of heterotrophic activity. Determination of rates of decomposition of organic matter, count of viable heterotrophic bacteria. Non-light requiring fixation of CO) is carried out almost exclusively by heterotrophic bacteria and provides a useful index of heterotrophic activity. The level of dark CO) fixation is low in oligotrophic lakes, both in absolute ten115 anl as a proportion of photosynthetic CO 2 fixation. Mean 14 year values for oligotrophic Lawrence Lake, USA were 4.7 g C m-2 y-2 and 13.3 per cent respectively. Both of these parameters increase with increase in lake nutrient status. 3. Uptake of radioactive precursors. Including general metabolites such as CI-!-glucose and S35_S0 4 or specific precursorg such as

60

MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

H3-thymidine and H3-leucine (which are taken up into nascent DNA and proteins respectively). Measurement of bacterial productivity using any of the above parameters requires the subsequent use of appropriate conversion factors to express productivity as units of carbon increase. In practice, bacterial productivity is normally determined via uptake of radioactive precursors, measuring either DNA or protein synthesis. Measurement of DNA synthesis involves in situ incubation of bacterial suspensions with H3-thymidine over a brief period of time «45 minutes), with determination of the DNA increase from radioactive uptake. Measured changes in DNA are then converted to rates of bacterial productivity in carbon units. The application of H'-thymidine uptake is a particularly useful method to determine bacterial productivity, since the radioactive precursor is only taken up into newly-synthesized macro-molecules and is not involved in DNA turnover. Thymidine is rapidly transported across the bacterial membrane and converted to thymidine monophosphate by the enzyme thymidine kinase. Uptake of thymidine and incorporation into replicating DNA is much less rapid in blue-green algae, eukaryotic algae, and fungi, so the rapid incubation time used in this procedure effectively ensures that only bacterial productivity is being monitored.

2.5.2 Regulation of Bacterial Populations and Biomass A wide range of environmental factors affect bacterial biomass in freshwater systems, including predation (mainly protozoa and rotifers), parasitic (viral) attack, and nutrient (inorganic nutrients, dissolved organic carbon) availability. In terms of the microbial food web, these different factors operate by top-down or bottom-up control. In addition to food web factors, changes in the chemical environment (e.g., seasonal hypolimnetic oxygen depletion) also affect bacterial abundances. The relative importance of top-down and bottom- up control in determining planktonic bacterial populations has been a matter of some discussion. Analysis of ranges of aquatic systems by different workers have supported both sides of the debate. In some cases, such cross-system investigations have indicated that bacterial abundance relates primarily to predation or lysis, while in other cases the bacterial populations relate more to available nutrient resources. Sanders et aI., (1992) and Berninger et al. (1991 a), for example, have demonstrated a strong inverse correlation between bacterial abundance and predation by protozoa (heterotrophic nanoflagellates).

MICROBIAL METABOLISM AND GROWTH

61

I Bacteriophage I

Top-down control bacterial abundance relates to predation and/or lYSIS

i !

ID~e 1 Extra-cellular digestion

t

Marked spatial, diurnal and seasonal variation

Bottom-up control bacterial abundance relates to DOe and Inorganic nutrient availability

Particulate organic matter

Figure 2.5 Top-down and bottom-up control of bacterial biomass: DOe - dissolved organic carbon, continuous arrows indicate carbon flow.

In other studies, this coupling was not observed, possibly due to reduction of the protozoon populations by large zooplankton (particularly daphnids). The importance of bottom-up control is indicated by the increase in bacterial populations with progression from oligotrophic to eutrophic lakes, and by the clear correlation between algal (primary) and bacterial (secondary) productivity and the importance of dissolved organic carbon. As a general overview, it seems clear that both types of control may operate, but the balance between the two varies with time and ecological circumstances. Studies on both marine and freshwater systems suggest that control mechanisms vary temporally throughout the annual cycle. Marked spatial variations in predation can also occur within the water column of a freshwater lake, as demonstrated by Weinbauer and Hofle (1998).

2.5.3 Primary and Secondary Productivity: Correlation Between Bacterial and Algal Populations Comparison of data from a range of pelagic systems indicates a high degree of correlation between bacterial and phytoplankton

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MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

0.1 1.0 10 100 Chlorophyll-aconcentration (lJg r-1) Figure 2.6 Correlation between bacterioplankton and phytoplankton populations (chlorophyll-a concentrations) in freshwater system.

populations, with close quantitative similarities between freshwater and marine environments. Using acridine orange staining to enumerate total bacterial populations, Bird and Kalff (1984) demonstrated a close mathematical relationship between total bacterial count (Ct) and algal biomass (chi-a), where: ... (13) Ct = 5:867 + 0:776 log [Chi-a] The correlation between populations is also matched by a close coupling between primary and secondary productivity. This is seen particularly weIl in the photic zone of lakes and oceans, where bacterial production ranged from 004-150 J1.g Cl-I d- I and averaged 20 per cent of primary production. The contribution of bacterial production is even greater if regions below the photic zone are also taken into account, reaching about 30 per cent of primary production throughout the entire water column. Heterotrophic bacterial productivity is clearly a major component of secondary productivity in pelagic systems, and is roughly twice the level of macrozooplankton productivity at particular levels of primary production. The close coupling between algal and bacterial productivity is an overall property of the lake and does not extend to analyses within the water column. Depth measurements by Pace and Cole (1994) of primary and secondary productivity in a group of lownutrient lakes showed major vertical differences between primary and secondary productivity maxima at particular points in time.

MICROBIAL METABOLISM AND GROWTH

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Figure 2.7 Relationship between bacterial productivity and major food source in pelagic and benthic system: (a) bacterioplankton productivity and primary production. (b) benthic bacterial productivity and organic content of sediment.

2.5.3.1 Bent/de environments In benthic systems, bacterial productivity is expressed per unit dry weight of sediment, and ranges from about 0.1-50 (g DW) d -I, Most lake sediments are well below the photic zone and there is no resident photosynthetic algal population. In this situation, bacterial productivity is related more to the availability of degradable biomass rather than algal exudates. There is a general correlation between

64

MICROBIAL PHYSIOLOGY.

C;~NU'ICS

AND ECOLOGY

productivity, bacterial population, and sediment organic C content, though some systems with high rates of benthic primary production (e.g., coral reefs) have higher rates of bacterial production than expected, simply from the organic C content. 2.5.4 Role of Dissolved Organic Carbon The direct relationship between primary and secondary productivity in pelagic systems suggests that either the growth of bacteria and phytoplankton is a separate respOllse to common factors (e.g., inorganic nutrients, temperature) or that the growth of bacteria is directly related to particulate or soluble material derived from phytoplankton. Both of these are probably important, though the relationship between bacterial productivity and organic C content in sediments emphasizes the importance of organic substrates in promoting bacterial growth. As noted earlier the flow of organic carbon from algae to bacteria via algal exudates (m~robialloop) is an important part of the aquatic food web. The release of exudates as dissolved organic carbon (DOC) by algae involves processes of passive diffusion (photosynthetic products), active secretion (extracellular enzymes), and cell breakdown (lysis). In an actively growing and photosynthetic phytoplankton population, DOC production occurs mainly as the passive release of photosynthetic products, amounting to a maximum 10 per cent of primary productivity. This release of DOC provides a direct link between algal and bacterial productivity, with DOC occurring as a major substrate for bacterial growth. Indirect links also occur via the zooplankton population, where part of the carbon flow from algae is released back into the environment as excreted and faecal DOC. The ability of bacteria and other microheterotrophs to take up organic compounds released by algae was demonstrated in early studies on 14 CO, uptake. Kinetic tracer studies by Wiebe and Smith (1977) on estuarine water samples demonstrated steady-state release of photosynthetically-derived dissolved organic carbon (PDOC) of 0.13 mg C m- 3 h- I by algae, with a rate of incorporation of PDOC into heterotrophs of 0.10-0.12 mg C nr 3 h- I • In this system, there was clearly a rapid and almost complete heterotrophic uptake of PDOC within a short time of release by the autotrophic community. Increased bacterioplankton uptake of I4C-labelled extracellular phytoplankton products has been demonstrated during periods of intense algal bloom.

MICROBIAL METABOLISM AND GROWTH

65

In the case of blue-green algae. the trophic link with bacteria may be further enhanced by the activities of lytic bacteria such as Lysobacter, the abundance of which is closely correlated with the blue-green algal population. Laboratory studies by Fallowfield and Daft (1988) have shown that Lysubacter can specifically increase the release of DOC by blue-green, but not green algae, under conditions of active photosynthesis; this suggests that these organisms may have a very important role in promoting increased levels of carbon flow in the aquatic environment. 2.5.5 Bacterial Productivity and Aquatic Food Webs Quantitative determination of pelagic and benthic bacterial productivity can give various insights into the dynamics of aquatic food webs:

2.5.5.1 Carbon flux through bacteria The efficiency with which bacteria convert lake water DOC to bacterial biomass provides information on the total amount of carbon (carbon flux) which passes through the bacterial population. Within the photic zone of a eutrophic lake, bacterial production was about 20 per cent of primary production per unit volume of lake water. If the efficiency of conversion of substrate to bacterial biomass is 50 per cent, then approximately 40 per cent of primary production fluxes through bacteria in this part of the water column. Within the whole water column, bacterial production averaged about 30 per cent of primary production, giving a 60 per cent flux of primary production through bacteria. These values of 50 per cent growth efficiency are in line with the normally accepted levels of 40--60 per cent, based on observations of the uptake and efficiency of conversion of simple 14C-labelled organic molecules by bacteria. Bacterial growth efficiency (BGE) not only depends on conversion of simple to complex organic molecules (bacterial productivity, BP), but also on carbon loss due to bacterial respiration BR, where: ... (14) BGE = BP=(BR+BP) Recent studies have shown that bacterial respiration is generally high in aquatic environments, and that growth efficiencies of bacteria should be revised down to values of < 10-25 per cent in most aquatic systems. The amount of carbon flowing through the bacterial population is thus greater than originally estimated. In low-nutrient (oligotrophic) systems bacterial respiration may actually exceed

66

MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

phytoplankton net primary production. In this situation, where the total carbon processed by bacteria exceeds that fixed by phytoplankton, bacteria must also use other sources of carbon for respiration and the system is net heterotrophic.

2.5.5.2 Pelagic and benthic bacteria in eutrophic and oligotrophic lakes Bacterial productivity is much greater in eutrophic compared with oligotrophic lakes, leading to the differences in population that were noted e::Jrlier. The total bacterial productivity of water column and sediments in eutrophic Lake Mendota (USA) was five times greater than oligotrophic Mirror Lake (USA). This difference is much greater than expected simply in terms of depth, and reflects greater substrate availability under eutrophic conditions. The transition from eutrophic to oligotrophic lakes may signal a ch~nge from net autotrophy (where most carbon uptake is via algal photosynthesis) to net heterotrophy (where most carbon uptake is into bacteria). This switch to bacterial dominance involves a transitioh in carbon availability changing from readily-assimilable autochthonous (algal) DOC to poorly-assimilable allochthonous (exogenous) carbon. Expressed on an area basis, bacterial productivity in the water column of a eutrophic lake such as Lake Mendota (USA) is much greater than in the sediments, implying that pelagic bacteria have a greater heterotrophic role than benthic ones in high nutrient conditions. The reverse appears to be true for oligotrophic lakes, where benthic organisms dominate bacterial secondary productivity. In a complex food web, where the same organic molecules may be consumed and recycled several times, secondary production by organisms such as bacteria could be nearly as large as primary production. 2.6 MICROBIAL GROWTH 2.6.1 Algal Blooms and Eutrophication Algal blooms are simply dense populations of planktonic algae which develop in aquatic systems. They may occur in a wide range of environments, including lakes and rivers, exposed mudflats, and snowpacks - and are part of the normal seasonal development in many ecosystems. In all of these environments, the development of algal blooms can be seen as a balance between the processes of population increase (high growth rate, ability to out-compete other algae) and population loss (effects of grazing, parasitic attack).

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67

Increased levels of inorganic nutrients lead to a general increase in primary productivity but may also promote algal blooms at different times of the year. In len tic environments, these blooms include the spring diatom bloom, late-spring blooms of green algae, and summer blooms of dinoflagellates and blue-green algae. Most of these blooms have no adverse effects on the environment, and the increased algal biomass is transferred to other lake biota via the normal food web. The major problems of eutrophication come with anthropogenic enrichment of the environment and the formation of dense blooms of toxic dinoflagellates (principally marine) and colonial blue-green algae (freshwater).

2.6.1.1 Toxic dinoflagellates These organisms are particularly characteristic of marine waters, and are thus largely outside the scope of this volume. They do occur in estuaries, however, and are occasionally seen in major rivers associated with estuaries - so do have some peripheral relevance to freshwater systems. Some of these dinoflagellates are important in the formation of neurotoxins, and are potential hazards in terms of human water contact and food consumption. Toxic dinoflagellates include Ga111bierdiscus toxicus (CTX toxin), GOl1yau/(L'( Alexandril1um (STX), Gymnodinium b,:eve (brevetoxins), Diophysis spp., and Pfiesteria piscicicia (unknown toxin). Pfiesteria has particular significance for freshwater systems since it has been implicated in eutrophication-related major fish kills in the Pocomoke and Chicamacomico Rivers which are part of the Chesapeake Bay complex. 2.6.1.2 Colonial blue-green algae Colonial blue-greens, not dinoflagellates, form the major nuisance-algae of freshwater systems and have the potential to cause deterioration in water quality and adverse environmental effects. In many eutrophic environments and mesotrophic lakes of lesser magnitude, quite dense blue-green blooms occur on an annual basis without any permanent environmental effects. It is only when these algae form very dense accumulations and totally out-compete other algae that their influence becomes severe. Four key questions arise in relation to the role of colonial bluegreen algae in the environmental effects of freshwater eutrophication. 1. What environmental factors lead to bloom formation? 2. Why is this particular group of algae able to out- compete other algae and form blooms?

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MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

3. What are the effects of blue-green algae on the freshwater environment? 4. How may these algae be controlled? 2.6.2 Formation of colonial blue-green algal blooms There has been much written in the past 20 years or so on the ability of blue-green algae to dominate freshwater environments and form blooms. These algae tend to become a prominent feature of northern temperate lakes in midsummer, when their dominance increases and bloom formation occurs.

2.6.2.1 General requirements for bloom formation Although the effects of blue-green blooms are often prolonged, their origin may appear to be rapid - with the sudden appearance of dense surface scums in eutrophic lakes and reservoirs. The origin of these blooms is partly due to massive growth (population increase) but also the ability to float to the water surface and form dense localized populations. The conditions necessary for the sudden development of surface blooms, and include high nutrient concentrations, physicochemical characteristics (high light, temperature, pH) of surface water that promote the dominance of blue-green over other algae, and conditions that lead to large-scale flotation of much of the algal biomass. The ability of blue-green algae to out-compete other members of the phytoplankton at a time of year when certain environmental aspects (light, temperature) are at an optimum, is a key feature of the success of these organisms in bloom formation. 2.6.2.2 Competition with other algae Various hypotheses have been put forward to explain the ability of blue-greens to out-compete other algae, including their optimum growth at high temperature, low-light tolerance, tolerance of low N/P nutrient ratios, depth regulation by buoyancy, resistance to zooplankton grazing, and tolerance of high pH/low CO 2 concentrations. Most of these features probably contribute to the success of blue-greens, without being individually of sole importance. The dominant success of colonial blue-greens probably results from the sum total of all these characteristics, with an overriding requirement for high nutrient input to achieve high biomass levels. 2.6.2.2.1 Optimum growth at high temperature Blue-green algae have higher growth optima than do green algae and diatoms. The midsummer increase in abundance of these algae

MICROBIAL METABOLISM AND GROWTH

69

in temperate lakes, and their success in tropical lakes, may be the direct result of their ability to grow well in warm water conditions. Chemos tat experiments of Tilman et al. (1986) have suggested that blue-greens isolated from Lake Michigan and Lake Superior (North America) have maximum growth ability at temperatures exceeding 20°C. Although temperature is important, it is secondary to nutrient requirements. Oligotrophic lakes in the same geographic area as eutrophic lakes, and with the same temperature regime, do not develop blue-green blooms. A survey of world lakes lead Robarts and Zohary (1987) to conclude that temperature was of subsidiary importance.

2.6.2.2.2 Tolerance of low-light conditions Various physiological studies have suggested that blue-green algae have lower light-energy requirements than green algae and diatoms. Although the ability to grow at low light may seem an unlikely advantage for algae present at the top of the water column in midsummer, the intense self-shading which occurs within turbid bloom populations make this a significant feature. Critical evidence is contradictory, however, with some studies showing that blue-greens are more dominant in turbid conditions, while others show the reverse. 2.6.2.2.3 Ability for growth at low NIP ratios The apparent ability of blue-greens to out-grow other algae at low nitrogen/phosphorus (N/P) ratios, has received much attention. General evidence in support of this is not conclusive, and it may be a feature of secondary importance. Analysis of 17 lakes by Smith (1983) concluded that although the blue-green contribution to the phytoplankton could be high in lakes with low TN/TP ratios «29/ 1), they only occurred at low level when ratios were higher. Other studies have been less supportive, and the general importance of low N/P ratios to blue- greens must be queried. 2.6.2.2.4 Depth regulation by buoyancy The ability of vacuolate blue-greens such as Anabaena and Oscillatoria to regulate their depth by buoyancy is probably important both in the early development of algal populations and in the final phase of dominance. In terms of population growth, it allows them to adopt an optimum position within the water column in relation to light and

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MICROBIAL PHYSIOLOGY, GF.NETICS AND ECOLOGY

CO 2 availability. Regulation of depth is also important in the diurnal migration of these algae to lower (high-nutrient) parts of the water column, allowing them to continue growth at a time of year when epilimnion nutrient levels have reached a low level. During bloom formation, the rapid flotation of these algae to the lake surface leads to changes in the water chemistry and light regime at the lake surface, depressing the growth of other phytoplankton. Depth regulation by buoyancy is particularly effective in a static water column and confers an advantage on these organisms in such stable conditions. In lakes where the water column remains stratified but without a high degree of stability, changes in the phytoplankton composition (with the emergence of blue-green dominance) still occur - suggesting that buoyancy is not absolutely essential for bloom formation in these algae.

2.6.2.2.5 Resistance to zooplankton feeding Filter-feeding zooplankton often appear to feed ineffectively, if at all, on blue-green algae. Blue-green dominance is further promoted by the elimination of competing green algae and diatoms from mixed phytoplankton populations during grazing activities. Even where bluegreen algae are ingested, the presence of a thick outer layer of mucilage often allows them to pass through the zooplankter alimentary canal without being digested. The relationship between blue-green algal dominance and 'cooplankton grazing is an important one. The notion that bluegreen algae may become dominant in the short term due to a failure of zooplankton grazing is suggested by several studies. These demonstrate that an abundance of filt'..:r-feeding grazers does promote dominance by blue- greens such as Aphanizomenon. In contrast to this shOl't· term effect, the effectiveness of biomanipulation as a control method depends on the long-term activities of zooplankton such as Daphnia magna for controlling blue-greens. Lakes which have acquired these zoop18nkton populations tend to show a shift from blue-green to green algal dominance. The overall relationship between blue-greens and zooplankton grazers at a particular site is thus highly variable, depending on zooplankton biomass, physiological state, species composition, and whether the interaction is being considered short-term or long-term. 2.6.2.2,6 Tolerance of high pH and low CO 2 cOllcentrations In conditions of high light intensity, elevated levels of photosynthesis within the epilimnion lead to pronounced CO 2 uptake

MICROBIAL METABOLISM AND GROWTH

71

resulting in strongly alkaline conditions with low CO 2 availability. It has been suggested that blue-green algae (but not other members of the phytoplankton) have the ability to tolerate these extreme environmental conditions - allowing them to continue active growth at a point when other algae are inhibited, thus out-competing other photosynthetic organisms. This concept is supported by the fact that blue- green dominance occurs in most lakes only when pH is high. Blue-green dominance is absent from lakes where pH does not rise during the summer including oligotrophic lakes (low algal biomass, low-photosynthesis) and lakes with CO 2 sources other than the atmosphere. The importance of CO 2 and pH to blue-green dominance is also supported by enclosure experiments. 2.6.3 Environmental Effects of Blue-green Blooms

2.6.3.1 General environmental changes The effects of dense blooms of blue-green algae are an extension of these features noted earlier for eutrophic lakes. Very high levels of algal biomass lead to extreme limitation of light penetration, preventing growth of other algae and completely suppressing growth of higher plants. Growth inhibition of other algae results in a dramatic loss of phytoplankton diversity within the water column. Oxygenation in the water column also reaches new extremes. Active photosynthesis at the water surface leads to high oxygen concentrations (often supersaturated) and high pH (frequently >pHlO). In the lower part of the water column, higher levels of algal detritus and heterotrophic decomposition result in extreme reducing conditions, with oxygen concentrations very low or nondetectable throughout most of the hypolimnion. These reducing conditions permit the accumulation of substantial levels of ammonia and nitrite in the water column, and lead to greater release of phosphate from the lake sediments adding to the already existing eutrophication problems. 2.6.3.2 Specific effects on water quality In addition to these rather general environmental effects, blooms of blue-green algae also cause more specific problems in relation to water quality and human use of the water body: 1. Production of high concentrations of small molecular weight toxins which are poisonous to a wide range of animals - these are discussed more fully in later section.

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MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

2. Fish kills: towards the end of an algal bloom, acute anoxia may develop throughout most of the water column due to largescale death of the algal cells, resulting in a decrease in oxygen evolution by photosynthesis and an increase in oxygen uptake by heterotrophic bacteria metabolizing the algal breakdown products. Under these sudden and extreme conditions of oxygen depletion, massive loss of fish populations may occur, with longterm effects on the ecology of the freshwater environment. 3. Problems with water extraction and treatment for domestic use: the filtration systems of water-treatment works can become blocked with algae, affecting the efficiency and maintenance costs of the extraction process. Mucopolysaccharides produced by algal breakdown can chelate the iron or aluminium coagulants added to the water during the treatment process, leading to an increase in the metal complexes entering the water supply. The presence of algae can also lead to unpleasant changes in the odour and taste of the water. Final problems come with the collapse of the bloom, which may lead to the accumulation of ammonia affecting the oxidation and disinfection capacity of chlorine and converting iron and manganese to soluble forms that can lead to discoloration of water. 4. Impacts on agriculture and fisheries: blue-green blooms can have adverse effects on agriculture through damage to livestock (toxin consumption), with an increased risk of flooding to farmland. In the case of fish farming, blue-green blooms can cause changes in fish species composition, and result in fish kills. 5. Loss of recreation amenity: development of intense algal blooms frequently leads to a ban on human access for swimming, sailing, and other recreational activities.

2_6.3.3 Production of toxins In the freshwater environment, toxins are produced and secreted in quantity by one major group of algae - the blue-greens. These toxins are part of a wide range of bioactive secondary metabolites produced by these organisms, including polyketones, alkaloids, and peptides. Toxins reach particularly high concentrations under conditions of bloom formation, when they contaminate the water supplies of wild and domestic animals and can also harm humans. Toxin production by other algae may occur in some situations. Toxic populations of the eukaryote alga Prymnesiu111 pan/um (Prymnesiophyceae), for example, may develop in the brackish waters

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73

of tidal or coastal freshwater systems causing dramatic changes in fish communities. In the marine environment, toxin production by dinoflagellates assumes greatest significance - contaminating sea water, but also causing shellfish to become poisonous due to ingestion and bioconcentration by these organisms. 2.6.3.3.1 General aspects of blue-green toxins Although blue-green toxins have harmful effects on cattle and other large vertebrates via their drinking supply, they appear to have evolved primarily as a defence against invertebrate predators reducing population loss by grazing. On the basis of their physiological effects on vertebrates, algal toxins have been divided into two main groups - neurotoxins (inducing neuromuscular dysfunction) and hepatotoxins (causing liver damage). Neurotoxins. These include anatoxin-a (a secondary amine alkaloid produced by some strains of Anabaena and other bluegreen algae), anatoxina(s) (an organophosphate produced by Anabaena and Oscillatoria), and saxitoxin/neosaxitoxin (produced by both freshwater blue-greens and marine dinoflagellates). These toxins act at neural synapses or neuromuscular junctions, causing loss of function by acting in various ways. Anatoxin-a is a neuromuscular blocking agent, mimicking the effect of the neurotransmitter acetylcholine and promoting prolonged muscular spasm. Anatoxin-a(s) achieves a similar effect by inhibiting acetylcholine esterase, thus preventing breakdown of the neurotransmitter. Saxitoxin and neosaxitoxin are both sodium channelblocking neurotoxins, preventing or delaying the normal post-synaptic processes. Hepatoxins. These toxins are of two main types - microcystins (cyclic heptapeptides) and nodularins (cyclic pentapeptides). They are produced by a wide range of freshwater blue-green algae, causing liver damage by promoting tumour development and specifically inhibiting protein phosphatases. Microcystin has been the most intensively-studied of all the bluegreen toxins, with information obtained on its molecular synthesis, structural diversity, and mode of action. The toxin is secreted by species belonging to the genera Anabaena, Oscillatoria, Nostoc, and Microcystis - of which Microcystis aeruginosa is the most widely distributed. In addition to microcystin, Micrucystis aeruginosa also synthesizes other peptides, including depsipeptides (cyanopeptolin,

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MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

micropeptin), tricyclic microviridins, and the linear microginins and aeruginosins. Most strains of Microcystis secrete at least one of these compounds, which act as protease inhibitors. The production and secretion of microcystin may be partially controlled by microcystin-related proteins (MrpA and MrpB), synthesis of which requires the presence of microcystin and is promoted by blue light. Close proximity of other Microcystis cells, detected by quorum sensing, may also be important and may lead to enhanced levels of toxin under bloom conditions. Field populations of individual species of blue- green algae are composed of a mixture of genotypes, not all of which encode the protein machinery for toxin production. In natural populations of Microcystis sp., for example, Kurmayer and Kutzenberger (2003) demonstrated that only 1-38 per cent of genotypes contained microcystin (mcy) genes. In these particular samples, the mean proportion of microcystin genotypes was stable from winter to summerwith no variation in relation to species population change. In addition to neurotoxins and hepatoxins, lipopolysaccharide (LPS) endotoxins are also produced by various blue-green algae. These resemble the LPS toxins produced by a range of Gramnegative bacteria such as Salmonella, causing fevers and inflammation. 2.7 METABOLISM OF STARTER CULTURES For most dairy fermentations, the role of starter culture bacteria is quite simple- they ferment lactose and produce lactic acid. As a result, the pH is reduced, and the ensuing low pH serves to preserve the product. In addition, lactic acid and low pH also are responsible for enhancing syneresis in cheese manufacture and for causing caseins to coagulate in yogurt, sour cream, and other cultured dairy products. However, lactic acid bacteria used as dairy starter cultures perform a number of other important functions in fermented milk products. They produce or generate several flavor compounds or flavor precursors, and they produce enzymes and other products that have profound effects on texture and body characteristics of cheese and cultured milk products. Not surprisingly, many functions performed by starter culture organisms are directly related to metabolic and physiological characteristics of those organisms. In this chapter, the specific means by which carbohydrates and proteins are metabolized and how endproducts are produced by lactic acid bacteria will be reviewed. The pathways for flavor production, not only by lactic

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MICROBIAL METABOLISM AND GROWTH

acid bacteria but also by non-lactic acid bacteria used as culture adjuncts and by fungi will also be described. 2.7.1 Carbohydrate Utilization by Lactic Acid Bacteria Lactic acid bacteria are classified as heterotrophic chemoorganotrophs, meaning that they require preformed organic carbon as a source of both carbon and energy. Lactic acid bacteria also glucose hexokinase

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MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

lack cytochrome or electron transport proteins, and therefore cannot derive energy via respiratory activity. Thus, substrate-level phosphorylation reactions that occur during glycolysis are the primary means by which ATP is obtained. There are, however, other means by which these organisms can conserve energy and save ATP that would ordinarily be used to perform necessary fUllctions, such as nutrient transport. Although there are some important differences between how various genera and species use and metabolize specific carbohydrates, lactic acid bacteria generally lack metabolic diversity and instead rely on two principal pathways for catabolism of carbohydrates. In the homofermentative pathway, hexoses are metabolized via enzymes of the Embden-Meyerhoff pathway, yielding 2 mol of pyruvate and 2 mol of ATP per mole of hexose. Pyruvate is subsequently reduced to lactate by lactate dehydrogenase, so that more than 90% of the starting material (i.e., glucose) is converted to lactic acid. The NADH formed via the glyceraldehyde-3-phosphate dehydrogenase reaction is also reoxidized (forming NAD+) by lactate dehydrogenase, thus maintaining the NADH/NAD+ balance. Among lactic acid bacteria used as dairy starter cultures, most are homofermentative, including Lactococcus iactis, Streptococcus thermophilus, Lactobacillus helveticus, and Lb. delbrueckii subsp. bulgaricus. In heterofermentative metabolism, hexoses are catabolized by the phosphoketolase pathway, wl'ich results in approximate equimolar production of lactate, acetate, ethanol, and COl' Only 1 mol of ATP is made per hexose. In actuality, however, product yields for both homo- and heterofermentative metabolism can vary, depending on the source and amount of available substrate, growth temperature, atmospheric conditions, and other factors. Under certain conditions, fur example, ·some homofermentative organisms can divert pyruvate away from lactate and toward other so-called "heterolactic" endproducts. Importantly, the pathway used by a particular strain or culture may have a profound effect on flavor, texture, and overall quality of fermented dairy products. Although several species of Lactobacillus are heterofermentative, Leuconostoc spp. are the only heterofermentative lactic acid bacteria used as starter cultures 111 dairy products.

2.7.1.1 Metabolism of lactose by lactic acid bacteria As described earlier, lactic acid bacteria generally rely on either the Embden-Meyerhoff or phosphoketolase pathway for metabolism

MICRORIAL METABOLISM AND GROWTH

77

g,uFcoseATP hexokinase ADP glucose-6-phosphate NAD glucose-6-phosphate dehydrogenase [ ; NADH + H 6-phosphogluconate 6-phosphogluconate dehydrogenase

k-:

NAD

~~~NADH+H

t

ribulose-5-phosphate ribulose-5-phosphate-3-epimerase

xyulose-5-phosphate

Ph~Pho~ glyceraldehyde-3-phosphate

acetyl phosphate

I

i i i

i

EM pathway

i i i

i

i

i

~COA

phosphotransacetylase ~ P acetyl-CoA

NAD acetaldehyde dehydrogenase [ ; NADH + H

'-V lactic acid

acetaldehyde ethanol dehydrogenase

~

J

NAD NADH + H

ethanol

Figure 2.9 The phosphoketolase pathway used by heterofermantative lactic acid bacteria.

of sugars. In fact, these catabolic pathways are only a part of the overall metabolic process used l~y these bacteria. The first, and

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MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

perhaps most important, step in carbohydrate metabolism involves transport of the sugar substrate across the cytoplasmic membrane and its 5ubsequent accumulation in the cytoplasm. This process of transport and accumulation is important for several reasons. First, active transport of sugars requires energy, and much of the energy gained by cells as a result of catabolism must then be used to transport more substrate. Second, the transport system used by a particular strain dictates, in part, the catabolic pathway used by that organism. The transport machinery also plays a regulatory role and can influence expression of alternative transport systems. Finally, the metabolic behavior of a particular strain and how that strain functions in fermented dairy foods may be influenced by the actual operation of the transport system itself.

2.7.1.2 Lactose pltospltotrallsferase system of Lc. lactis There are, in general, two different systems used by lactic acid bacteria to transport carbohydrates, and it is convenient to group lactic acid bacteria according to the system used to transport their primary substrate, lactose. The phosphoenolpyruvate (PEP)dependent phosphotransferase system (PTS) is used by most mesophilic, homofermentative lactic acid bacteria, especially lactococci used as starter cultures for cottage, Cheddar, Gouda, and other common cheese varieties. In contrast, other starter culture bacteria, such as S. ther1110philus and Lactobacillus spp. that are used for yogurt, Swiss, and mozzarella cheese production, transport lactose via a lactose permease. Dairy Leuco11ostoc bacteria also rely on a lactose permease for uptake of lactose. Some lactococci and lactobacilli have the ability to use both systems. Not only do these two systems differ in biochemical characteristics, but energy sources used to drive transport and accllmulated intracellular products differ as well. These differences have practical implications. The LactococCLlS lactose PTS, first described by McKay et al. (1969), consists of a cascade of cytoplasmic and membrane-associated proteins that transfer a high -energy phosphate group from its initial donor, PEp, to the final acceptor molecule, lactose. Phosphorylation of lactose occurs concurrent with the vectorial movement of lactose across the cytoplasmic membrane and results in intracellular accumulation of lactose phosphate. There are two cytoplasmic proteins, enzyme I and histidine-containing protein (HPr), that are nonspecific and function as the initial phosphorylating proteins for all PTS substrates. The substrate-specific PTS components comprise

MICROBIAL METABOLISM AND GROWTH

«-----

Glucose-6-P

PEPI pyruvate

Enzl

Xft~~P)8~

Enz I-P

-_

->

~

EIIB

HPr

AT? AD?

;;jt HPr Kmase ~I

IRPrI

l_~

-

~

--~

HPr

Ser-P

I

eRE

Figure 2.10 Signal transduction and the phosphotransferase system in gram-positive bacteria.

the enzyme II complex, which for the lactose PTS in Le. lactis, represents three protein domains (Enz rIAlae and Enz IIBClac). The phosphoryl group is transferred first from PEP to enzyme r, then to HPr, then to the cytoplasmic protein, Enz lIAlae, which then transfers it to the cytoplasmic domain of Enz IIBCloc. As lactose is translocated across the membrane by the integral membrane domain of Enz lIBClac, it becomes phosphorylated. The product of the lactose PTS, thus, is lactose-phosphate, or more specifically, glucose-[)-1,4-galactosyl-6-phosphate. Hydrolysis phospho-Il-galactosidase

lactose-6-P

> gala,tose-e-p

galactose-6-P isomerase

~

tagatose-GoP tagatose-6-P ki,)ase

~

+ glucose

tEM lactic acid

ATP

ADP

tagatose-1,6-di-P

~

glyceraldehyde-3' phosphate

tEM

\ dihydroxyacetone phosphate

lactic acid Figure 2.11 Tagatose pathway in lactococci.

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MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

of this compound occurs via phospho-l3-galactosidase, yielding glucose and galactose-6-phosphate. Glucose is phosphorylated by hexokinase (via an ATP) to glucose-6-phosphate, which then feeds directly into the Embden-Meyerhoff pathway, as described earlier. Galactose-6-phosphate, in contrast, takes a different route altogether, as it is first isomerized to tagatose-6-phosphate and then phosphorylated to form tagatose-l,6-diphosphate. The latter is then split by tagatose-l,6-diphosphate aldolase to form the triose phosphates, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, in a reaction analogous to the aldolase of the EmbdenMeyerhoff pathway. It is important to note that in Le. laetis, glucose and galactose moieties of lactose, despite taking parallel pathways, are fermented simultaneously.

2.7.1.3 Regulation oj the pllOspllOtransjerase system In Le. laetis, lactose fermentation is regulated at several levels. First, several glycolytic enzymes are allosteric, and their activities are therefore influenced by the intracellular concentration of specific glycolytic metabolites via feedback inhibition. During active lactose metabolism (i.e., when lactose is plentiful), the high intracellular concentration of fructose-l ,6-diphosphate (FDP) and low level of inorganic phosphate stimulate pyruvate kinase. Thus, much of the PEP made via glycolysis is used to drive ATP synthesis, which is consistent with a period of active cell growth. The activity of the NADH-dependent lactate dehydrogenase is also stimulated, which is important because reduced NAD+, formed via the glyceraldehyde3-phosphate dehydrogenase reaction, must be reoxidized to maintain the NAD+ /NADH balance. In contrast, when lactose is limiting, pyruvate kinase activity decreases causing PEP to accumulate, which forms a "bottleneck" in glycolysis. The concentration of triose phosphates subsequently increases, forming a pool of PEP equivalents. Thus, during a period when lactose is unavailable, a PEP "potential" exists, poising the cell for when lactose is available. A second and more effectual mechanism for controlling or regulating lactose metabolism is exerted at the level of the transport machinery itself. In particular, the phosphorylation state of HPr, the cytoplasmic PTS phosphocarrier protein, plays a major role in sugar metabolism. As noted earlier, HPr is phosphorylated by enzyme 1. This phosphorylation occurs specifically at the histidine-IS (His-IS) residue of HPr. However, HPr can also be phosphorylated at a serine residue (Ser-46) by an ATP-dependent HPr kinase, which

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81

is activated by fructose-I ,6-diphosphate (as would occur during active sugar metabolism). When BPr is in this state, that is, HPr (Ser46- P), phosphorylation at His-I5 is inhibited; thus, PTS activity is also inhibited and entry of other potential PTS substrates is prevented. Additional experimental evidence that HPr (Ser-46-P) can directly inhibit transport of sugars was provided by Saier and coworkers, who showed that HPr (Ser-46-P) can bind to or otherwise inactivate sugar permeases, a process known as inducer exclusion. Yet another means by which HPr (Ser-46-P) regulates sugar flux is via inducer expulsion. Presumably, this occurs when sugar phosphates have accumulated intracellularly beyond the rate at which metabolism can occur or when nonmetabolizable sugars have been taken up. Since these sugar phosphates could inhibit metabolism, they must first be dephosphorylated and then effluxed. In inducer expulsion, therefore, HPr (Ser-46-P) activates a sugar-specific phosphatase that dephosphorylates the sugar phosphates so that efflux of the free sugar can then occur. HPr not only exerts biochemical control on transport, but HPr (Ser-46-P) also plays an important role at the gene level through its interaction with the DNA-binding protein, CcpA, or catabolite control protein A. HPr (Ser-46-P) and CcpA (with the participation of fructose-l ,6-diphosphate) affect metabolism by blocking transcription of catabolic genes, including other PTS genes, a process called catabolite repression. CcpA or CcpA-like proteins appear to be widely distributed among gram-positive bacteria, including several species of lactic acid bacteria, and this mechanism of gene regulation, therefore, may be common. According to this model of carbon source-mediated gene regulation, HPr exists in one of two phosphorylation states, HPr (His-15- P) or HPr (Ser-46- P). The former accumulates when lactose (or another PTS sugar, such as glucose) is unavailable, since the enzyme II complex is without its substrate. In contrast, when lactose is available and the energy state of the cell is high, intracellular FOP levels increase and HPr kinase is activated, causing HPr (Ser-46- P) to accumulate. A complex is then formed between HPr (Ser-46-P) and CcpA. This complex, along with a glycolytic activator (fructose-I,6-diphosphate or glucose6-phosphate), binds to 14-base pair DNA regions called catabolite responsive elements (CREs) located near the transcription start sites of catabolic genes. When these CRE regions are occupied by the HPr (Ser-46-P)-CcpA complex, transcription by RNA polymerase is effectively blocked or reduced. In contrast, mutations in ccpA or

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MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

deletions of cre regions eliminate catabolite repression. Since eRE regions are found in the promoter regions of several catabolic genes, the phosphorylation status of HPr can have a profound effect on whether these genes are expressed. Identified gene clusters preceded by CRE regions in lactococci include genes coding for galactose (and thus lactose) and sucrose metabolism. For example, when Lc. lactis is grown on glucose, a PTS substrate, transcription of genes coding for galactose metabolism is repressed. Even the presence of galactose fails to induce expression of gal genes as long as glucose, the repressing sugar, is present. Not only does HPr have a negative regulatory role, but it was recently shown that HPr (Ser-46-P) and CcpA can also activate gene expression. Specifically, expression of the las operon, coding for lactate dehydrogenase, phosphofructokinase, and pyruvate kinase, is activated at high sugar conditions. The net effect, therefore, is that the phosphorylation state of HPr serves as a signal for activating expression of genes coding for glycolytic enzymes when the cell is actively metabolizing sugars. Recent genetic evidence indicates that HPr is also important in influencing sugar uptake by establishing a hierarchy for different sugars preferentially fermented by Lc. lactis. Finally, lactose metabolism is also genetically regulated via expression and repression of the lactose PTS genes. The lactose metabolism genes in Lc. lactis, like the genes coding for other important metabolic pathways, are often located on plasmids of varying size. Strains cured of the lactose plasmid, which encodes lactose metabolism genes, are unable to ferment lactose. In Lc. lactis MG 1820, the lac genes are organized as an 8-kb operon, consisting of eight genes in the order l£lcABCDFEGX. The first four genes, lacABCD, actually code fol' enzymes of the tagatose pathway and are necessary for galactose utilization. The lactosespecific genes, lacFEG, code for PTS proteins and phospho-13galactosidase. The operon is negatively regulated by LacR, a repressor pl'otein encoded by the lacR gene, which is located upstream of the lac promoter and which is divergently transcribed. In the presence of lactose, lacR expression is repressed, and transcription of the lac operon is induced. During growth on glucose or when lactose is unavailable (and cells are uninduced), LacR is expressed and transcription of the lac genes is repressed. A CRE site is also located nem the transcriptional start site of the lac operon. However, when lacR is inactivated, expression of lac genes becomes constitutive regardless of cmbon source (i.e., under conditions that presumably

MICROBIAL METABOLISM AND GROWTH

83

would activate CcpA-mediated repression). This implies that LacR, along with inducer expulsion-exclusion, have primary responsibility for regulating sugar metabolism, rather than CcpA, and that catabolite repression in lactococci is mediated mainly via the concentration of inducer. The lactose PTS, as described earlier for Lc. lactis, also exists in other dairy lactic acid bacteria, including Lb. casei. However, in Lb. casei, lac genes are chromosomally encoded and the nucleotide sequence and genetic organization are different from those in Lc. lactis. The Lb. casei lac cluster (lacTEGF) encodes, respectively, for a regulatory protein, two PTS proteins, and phospho-~­ galactosidase. Genes coding for galactose metabolism (lacABCD in Le. lactis) are absent in the Lb. casei lac cluster. Although expression of lac genes is repressed by a CcpA-mediated process, as in Le. lactis, an additional regulatory mechanism dependent on an antiterminator also exists in Lb. casei.

2.7.1.4 Lactose transport alld hydrolysis by S. thermophilus Although the PTS is widely distributed among lactic acid bacteria, several important dairy species rely on a lactose permease for transport and a ~-galactosidase for hydrolysis. Some species have both pathways for lactose, and some have a PTS for one sugar and a permease_ for another. The best example of the lactose permease/ ~-galactosidase system is that which occurs in S. therl11ophilus, Lb. helveticlls, and Lb. delbruecki subsp. bulgaricus. In these bacteria, lactose accumulates in an unmodified form via the LacS permease. A similar system also exists in some strains of Lc. lactis, but clearly it is not the primary system. The lactose permease in S. ther1710philus is dramatically different from other, well-studied lactose permeases, such as the LacY system in Escherichia coli. In E. coli, lactose transport is fueled by a proton motive force (PMF), and the permease binds and transports its substrate lactose in symport with a proton. In S. ther171ophilus, lactose transport can also be fueled by a PMF, but that is not the main way the permease can function. Instead, the transporter has exchange or antiporter activity, so that lactose uptake can be driven by efflux of galactose. That is, "uphill" lactose transport (uptake against a concentration gradient) occurs as a result of "downhill" galactose efflux. Since generation of a PMF requires ATP (or its equivalent), not having to llse the PMF for lactose uptake conserves energy. The lactose: galactose exchange

84

MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

reaction is actually quite remarkable, in that, as discussed below, galactose efflux, rather than galactose utilization, appears to be the preferred pathway for most strains of S. thermophilus. Why this phenomenon occurs and the important practical implications for this will be discussed later. Detailed analysis of the S. thermophilus LacS system has revealed that the permease protein itself is a hybrid consisting of two distinct regions or domains. The deduced amino acid sequence of the amino- terminal region is very similar to the melibiose permease of E. coli. However, the carboxy-terminal region is structurally similar to an E. coli PTS enzyme IIA domain. In fact, this enzyme IIA-like domain can be phosphorylated by HPr, reducing transport activity of LacS. It now appears that the permease region functions as the lactose carrier and the enzyme IIA-like domain functions as a regulatory unit. Hydrolysis of lactose in S. thermophilus occurs via a ~­ galactosidase that has modest amino acid homology to other LacZlike enzymes (30-50%). After hydrolysis, S. thermophilus rapidly ferments glucose to lactic acid by the Embden-Meyerhoff pathway, yet most strains, es[!ecially those used as dairy starter cultures, do not ferment the galactose moiety of lactose. Rather, galactose is effluxed and accumulates in the extracellular medium. In the manufacture of dairy products made with an S. thermophilllScontaining culture, such as yogurt or mozzarella cheese, galactose may appear in the finished product. With yogurt, accumulated galactose is of little consequence, but for mozzarella cheese, even a small amount of galactose can present problems. This is because of the nonenzymatic browning reaction that occurs when galactose, a reducing sugar, is heated in the presence of free amino acids. Since most mozzarella cheese is used for pizzas, high-temperature baking accelerates nonenzymatic browning reactions. Cheese containing galactose can brown excessively, a phenomenon considered as a defect by many pizza manufacturers. Therefore, mozzarella producers may be asked by their customers to satisfy specifications for "lowbrowning" or low-galactose cheese. Although some cheese manufacturers can rely on their cheese-making know-how and simply modify the production procedures to remove unfermented galactose, other manufacturers have chosen to use cultures that have lowbrowning potential, as described below. Why are most strains of S. thermophilus phenotypically galactose negative (Gal-) and unable to ferment either free or lactose-derived galactose? Evidence from several laboratories indicates that S.

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MICROBIAL METABOLISM AND GROWTH

thermophilus does contain genes necessary for galactose metabolism, but that these genes are not ordinarily expressed even under inducing conditions. Mutants have been isolated, however, that ferment free galactose, but when these strains are grown on lactose, galactose utilization is still repressed. Thus, it has been suggested that of the two routes that galactose can take, the efflux reaction is favored over the catabolic pathway.

2.7.1.5 Lactose metabolism by Lactobacillus and other lactic acid bacteria Most other lactic acid bacteria rely on one or the other of the two pathways described earlier. With the exception of Le. lactis and Lb. casei, however, most dairy lactic acid bacteria do not have a lactose PTS, and instead use a lactose permease/~-galactosidase system for metabolism of lactose. Some strains have more than one system; for example, Lc. lac/is and Lb. casei have both a lactose PTS and a lactose permease/~-galactosidase. It is important to note that not all strains or species that use non- PTS pathways for lactose metabolism excrete galactose into the medium, as described for S. thermophilus. Many of the lactobacilli and Leuconostoc spp. that transport and hydrolyze lactose by a permease and a ~-galactosidase, respectively, also ferment glucose and galactose simultaneously. This is important, because in almost all fermented dairy products made with a culture containing S. thermophilus, a galactose-fermenting Lactobacillus sp. is also present. For some products, such as Swissstyle cheeses, the galactose that is effluxed into the curd by S. thermophilus is subsequently fermented by Lb. helveticus. Otherwise, the free galactose could be fermented by other members of the microflora, resulting in heterofermentative end products that could contribute to off-flavors and other product defects. Table 2.1 Lactose transport and metabolic systems in dairy lactic acid bacteria Organism

Lactose transport system

Galactose pathway

Streptococcus thermophilus Lactococcus lactis Lactobacillus delbrueckii subsp. bulgaricus Lactobacillus helveticus Lactobacillus casei Leuconostoc lactis

Lac permease PTS Lac permeas'e

Leloir Leloir, tagatose Leloir

Lac permease PTS, Lac permease Lac permease

Leloir Leloir, tagatose Leloir

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MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

2.7.1.6 Galactose metabolism During growth in milk, lactic acid bacteria ordinarily encounter free galactose only after intracellular hydrolysis of lactose. For lactococci and those lactobacilli that transport lactose via the PTS, galactose-6-phosphate is the actual hydrolysis product (resulting from hydrolysis of lactose-phosphate by phospho-I)-galactosidase). Galactose-6-phosphate feeds directly into the tagatose pathway. However, as noted earlier, free galactose will appear and accumulate in fermented dairy products made with thermophilic starter cultures containing S. thermophilus, Lb. bulgaricus, or other galactosenonfermenting strains. Yogurt and mozzarella cheese, for example, can contain up to 2.5 and 0.8% galactose, respectively. Therefore, metabolism of free galactose may be of practical importance. For the lactococci and some lactobacilli, free galactose appears to be transported by either a galactose-specific PTS or by a galactose permease. The intracellular product of the galactose PTS (galactose6-phosphate) simply feeds into the tagatose pathway. When galactose accumulates via galactose permease. the intracellular product is free galactose. Subsequent metabolism occurs via the Leloir pathway, which phosphorylates galactose, and then converts galactose-lphosphate into glucose-6-phosphate. The latter then feeds into the glycolytic pathway. Interestingly, in Le. lactis, galactose permease may be the primary means for transporting galactose, since it has a much higher apparent affinity for galactose than the PTS transporter. galactose galactokinase

F

t

ATP ADP

galactose-l-P galactose-1-P

uridyl transferase

UDP glu

~UDP

galactose , ) eoimcrase

UDP gal

gJucose-1-P phosphoglucomutase

J

glucose-6-P EM

t.:

/'

"

PK ~

Figure 2.12 Leloir pathway in lactic acid bacteria.

MICROBIAL METABOLISM AND GROWTH

87

The Leloir pathway is used not only by lactococci, but it is also the pathway used by Lb. helveticus, Leuconostoc spp., and galactosefermenting strains of S. thermophilus. During growth on lactose, these bacteria rely on a lactose permease/~-galactosidase system and therefore generate free intracellular galactose. In some instances, they will also encounter free extracellular galactose, especially if they are grown in the presence of galactose-nonfermenting strains, as described earlier. Subsequent galactose fermentation by Lb. helveticus and Leuconostoc lactis occurs via the Leloir pathway. Transport is mediated by a permease, apparently driven by a PMF. A mutarotase (the product of the galM gene) may also be necessary to convert ~-D-galactose (the product of lactose hydrolysis) to its anomeric isomer, a-D-galactose, before it can be efficiently phosphorylated by galactokinase. Despite the inability of most strains of S. thermophilus to ferment galactose, genes coding for enzymes of the Leloir pathway appear to be present and functional. The S. thermophilus gal operon consists of four structural genes (gaIKTEM) and one divergently transcribed regulatory gene (gaLR). Transcription of these genes, however, does not occur in most wild-type strains, accounting for the galactose nonfermenting phenotype. Mutations in the gal promoter/regulatory region led to isolation of galactose-fermenting mutants that expressed gal genes and fermented galactose. Such efforts suggest that genetic modification of S. thermophilus may provide the basis for obtaining stable galactose-fermenting derivatives that would be of considerable value to the dairy industry. Although the gal genes in S. thermophilus, Leuc. lactis, Lc. lactis, Lb. casei, and Lb. helveticus share significant amino acid sequence homology and are chromosomally encoded, they are organized in a somewhat different order. All contain galK (galactokinase), gaIT(galactose-l-phosphate uridyl transferase), and galE (UDP-galactose-4-epimerase), and some clusters also contain the galM gene coding for mutarotase. In S. thermophilus, the gal genes are located immediately upstream of the lacS-IacZ cluster. There is also rather significant variation with respect to genetic structure even between strains of the same species. For example, a gaLA gene, thought to encode for a permease, is the first gene in the Lc. lactis MG 1363 gal cluster, but this gene does not appear in gal clusters from other organisms. The ability of these strains, especially lactobacilli, to ferment galactose can be quite variable, and strain selection is important.

MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

88

Galactose fermentation by lactobacilli has also been used as a basis for distinguishing between Lb. helveticus (Gal+) and Lb. delbrueckii subsp. bulgaricus (Gal-). As noted earlier, some culture suppliers promote "non browning" cultures for use in mozzarella cheese production; invariably, these cultures contain galactose-fermenting lactobacilli.

2.7.1.7 Altemate routes for pyruvate As described earlier, lactic acid bacteria are ordinarily considered as being either homofermentative or heterofermentative, with some species being able to metabolize sugars by both pathways. However, sugar metabolism, even by obligate homofermentative strains, can result in formation of endproducts other than lactic acid by a variety of pathways. In general, these alternative fermentation products are formed as a consequence of accumulation of excess pyruvate and the requirement of cells to maintain a balanced NADH/NAD+ ratio. That is, when the intracellular pyruvate concentration exceeds the rate at which lactate can be formed via lactate dehydrogenase, other pathways must be recruited not only to remove pyruvate but also to provide a means for oxidizing NADH. These alternative pathways may also provide cells with the means to make additional ATP. Under what conditions or environments would pyruvate accumulate? As noted earlier, when fermentation substrates are limiting, and the glycolytic activator, fructose-! ,6-diphosphate, is in short supply, activity of the allosteric enzyme, lactate dehydrogenase, is reduced

acetate

a-aceto/actate

CO 2

~tOI"t.t. a-as~enthase

/

pyruvate

pvruvate-::rmay IY'~

ethanol acetate formate

~eh~~~tate

~ogen.se lactate

Figure 2.13 Alternative routes of pyruvate metabolism

MICROBIAL METABOLISM AND GROWTH

89

and pyruvate accumulates. Low carbon flux may also occur during growth on galactose or other less preferred carbon sources, resulting in excess pyruvate. When the environment is highly aerobic, NADH that would normally reduce pyruvate is instead oxidized directly by molecular oxygen and is unavailable for the lactate dehydrogenase reaction. Several enzymes and pathways have been identified in lactococci and other lactic acid bacteria that are responsible for diverting pyruvate away from lactic acid and toward other products. In anaerobic conditions, and when carbohydrates are limiting and growth rates are low, a mixed-acid fermentation occurs, and ethanol, acetate, and formate are formed. Under these conditions, pyruvate-formate lyase is activated, and pyruvate is split to form formate and acetylCoA. Acetyl-CoA can be converted to ethanol and/or acetate. The latter also results in formation of an ATP via acetate kinase. If the environment is aerobic, pyruvate-formate lyase is inactive, and instead pyruvate is decarboxylated by pyruvate dehydrogenase to form acetate and COr Finally, excess pyruvate can be diverted to a-acetolactate via a-acetolactate synthase. This reaction has other important implications, since a-acetolactate is the precursor for diacetyl formation. Although these alternative pathways for pyruvate metabolism are influenced largely by environmental conditions, mutants unable to produce lactate dehydrogenase also must deal with excess pyruvate and, therefore, produce other endproducts. Under certain conditions, cells may divert excess pyruvate to highly desirable products, specifically the aroma compound diacetyl. Ordinarily diacetyl is made from citrate, but even citrate-nonfermenting cells will make diacetyl from lactose if appropriate conditions are established or if cells are genetically modified. For example, overexpression of NADH oxidase in Le. lactis decreases lactate formation from pyruvate, and instead a-acetolactate, the precursor for diacetyl, is formed. Enhancing diacetyl production by metabolic engineering will be discussed later.

2.7.2 Protein Metabolism Just as dairy lactic acid bacteria are well suited to utilize lactose as a source of energy and carbon, they are also well adapted to use casein as a source of nitrogen. Lactic acid bacteria cannot assimilate inorganic nitrogen and, therefore, they must be able to degrade proteins and peptides to satisfy their amino acid requirements. The absolute requirement for a system to degrade milk casein was first

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MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

demonstrated by McKay and Baldwin (1974), who showed that Le. lactis C2, cured of a plasmid containing the proteinase gene, was unable to grow to high cell density in milk. However, if milk was supplemented with hydrolyzed milk protein, the plasmid-cured strain grew like the parental strain. We now know that dairy lactic acid bacteria have evolved highly efficient systems for reducing large casein subunits to smaller pieces and for supplying cells with all of the amino acids necessary for growth in milk. The proteolytic system consists of three main components. The first involves the proteolysis of casein to form a large collection of peptides. In the second step, peptides are transported into cells by one of several transport systems. Once inside the cell, peptides are further hydrolyzed by a diverse group of peptidases to form free amino acids which are ultimately either metabolized or assimilated into protein.

2.7.2.1 Proteinase system Although lactic acid bacteria vary considerably in their ability to degrade 'milk protein, most organisms possess similar systems, as typified by the extensively studied proteolytic system of Lactococcus. For Le. lactis and other dairy lactic acid bacteria, casein is the primary source of amino acid nitrogen, since the non-protein nitrogen and fr,ee amino acids available in milk «300 mg/L) are quickly depleted. Because Le. lactis is a multiple amino acid auxotroph and requires as many as eight amino acids, casein hydrolysis is essential. Casein utilization by Le. lactis begins with elaboration of a cell envelope-associated serine 'proteinase. This proteinase, Prtp, is expressed as a large (>200 kD), inactive preproproteinase. The leader sequence, which is responsible for directing the protein across the cytoplasmic membrane, is removed, leaving the remaining protein anchored to the cell envelope. However, the propr6teinase is not active until it is further processed by the maturation protein, PrtM. The latter presumably acts by inducing an autocatalytic cleavage event that results in hydrolysis of the pro region of the enzyme, leaving a mature PrtP with a molecular mass of 180-190 kD. Although the proteinases among different strains of Lc. lactis are all genetically related and show only minor differences with respect to their amino acid sequence, the specific casein substrates and hydrolysis products of PrtP enzymes from lactococci can vary considerably. For example, proteinases belong to group A (formerly Pili-type) hydrolyze U SI -' 13-, and K-caseins, whereas group E proteinases (formerly PI-type) have a preference for l3-casein and

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relatively little activity for u s ,- and K-caseins. Still, the functional organization of the PrtP and PrtM system varies little among lactococci. Both are required for rapid growth in milk, and genes for both (prtP and prtM, respectively) are induced when cells are grown in low-peptide media (e.g., milk) and repressed in peptiderich media. Over 100 caseinolytic products result from action of PrtP on ~-casein. Most are large oligopeptides (4-30 amino acid residues) with a major fraction between 4 and 10 residues. Free amino acids, dipeptides, and tripeptides are not formed. The first and most abundant oligopeptides formed by PrtP are generated from the Cterminal end of ~-casein, and it now appears that initial hydrolysis events cause casein to unfold so that other cleavage sites are exposed.

2.7.2.2 Amino acid and peptide transport systems Although it was once believed that extracellular peptidases must be present to degrade further these peptides before transport, it is now well established that extracellular hydrolysis of peptides formed by PrtP does not occur, at least not by peptidases. Instead, lactococci and other lactic acid bacteria possess an array of amino acid and peptide transport systems able to transport substrates of varying size, polarity, and structure. Some of these are highly specific, whereas others have rather broad substrate specificity. They also vary as to energy Sources used to fuel active transport. As described earlier, the concentration of free amino acids in milk is too low to support growth of lactic acid bacteria. Still, lactococci possess at least 10 amino acid transporters, most of which are specific for structurally similar substrates. If the medium contains an adequate concentration of free amino acids, these transport systems can deliver enough amino acids to the cytoplasm to support growth. However, it has been suggested that the primary function of these transporters may be simply to excrete or efflux excess amino acids from the cytoplasm to maintain appropriate intracellular pool ratios. That is, if peptides are indeed the primary source of amino acids, then some amino acids, generated from intracellular peptidases, may accumulate faster than they can be assimilated. These free amino acids could then diffuse out of cells down their concentration gradient via the amino acid transporter operating in the reverse or efflux direction. If efflux of an amino acid is accompanied by a coupling ion (e.g., proton extrusion), then a net increase in the PMF is obtained. It may even be possible for amino acid efflux to provide

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enough energy to drive uptake of peptides. In contrast to the amino acid transporters, peptide transport is clearly necessary for lactic acid bacteria to grow in milk. Three groups of peptide transport systems have been identified. Two of these, DtpT and DtpP, transport dipeptides and tripeptides. DtpT is a large (463 amino acid residues) monomeric, PMF-dependent transporter that has affinity for hydrophilic peptides. Mutants with a deletion in the dtpT gene have been obtained and are unable to express DtpT and transport some peptides. In a defined medium, dtpT mutants grew poorly; however, growth of these mutants in milk was unaffected, indicating that DtpT is not essential in milk. DtpP, the other transport system in lactic acid bacteria that transports dipeptides and tripeptides, is an ATPdependent transporter that has high affinity for hydrophobic peptides. It also appears to be unnecessary for growth of lactococci in milk. The third and most important peptide transport system in lactic acid bacteria is the oligopeptid.e transport system (Opp). Since dipeptides and tripeptides are not released from casein, neither DtpT nor DtpP is required for growth in milk; lactococci instead rely on oligopeptides and Oppto satisfy all amino acid requirements. Indeed, mutants unable to express genes coding for the Opp system are unable to transport oligopeptides and ase unable to grow in milk. Although it was initially not known which oligopeptides were actually transported by Opp, many of the structural and genetic features of the Opp system in Le. lactis are .10W well defined. The Opp complex belongs to the ABC (ATP binding casette) family of transporters and consists of five subunits: two transmembrane proteins (OppB and OppC), two ATP binding proteins (OppD and OppF), and a membrane-linked substrate-binding protein (OppA). The five genes coding for Opp are organized as an operon in the order oppDFBCA. A gene coding for an oligopeptidase (pepO) is also located immediately downstream of oppA and is cotranscribed with the opp operon. The Opp system transports a diverse population of oligopeptides. Although PrtP releases over 100 peptides from p-casein, only 1014 pep tides apparently serve as substrates for Opp. All of these oligopeptides contain more than 4 and fewer than 11 amino acid residues. Detailed analysis revealed that they contain proportionally higher levels of valine, proline, and glutamate and moderate levels of alanine, leucine, isoleucine, lysine, and serine. Importantly, these oligopeptides provide all essential amino acids, with the exception of histidine, needed by lactococci for growth in milk.

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2.7.2.3 Peptidases The third and final step of protein catabolism involves peptidolytic cleavage of Opp accumulated peptides. Over 20 different peptidases have been identified and characterized, either biochemically and/or genetically, in lactococci and lactobacilli. Both endopeptidases (those that cleave internal peptide bonds) and exopeptidases (those that cleave at terminal peptide bonds) are widely distributed. Of the latter, only aminopeptidases have been reported; carboxypeptidases apparently are not produced. In general, concerted efforts of endopeptidases, aminopeptidases, dipeptidases, and tripeptidases are required fully to utilize peptides accumulated by the Opp system. Although there was once considerable debate on the location of these peptidases, it is now well accepted, based on genetic as well as physical evidence (e.g., lack of signal peptides and anchor sequences, cell fractionation, and immunogold labeling experiments), that they are intracellular enzymes. Substrate size and specificity and other properties of peptidases from lactic acid bacteria have been of considerable interest, not only because of their physiological importance but also because of the significant role peptidases play in cheese manufacture and ripening. 2.7.2.3.1 Endopeptidases Several endopeptidases have been described, including PepF and PepO in Lc. lactis and PepE, PepG, and PepO in Lb. helveticus. Most of these endopeptidases are metalloenzymes that contain sequences typical of zinc-binding domains and hydrolyze oligopeptides of varying lengths as substrates. It is interesting to note that some endopeptidases (e.g., PepF) have pH optima in an alkaline range (7.5-9.0) and that peptidase activity at pH levels typical of ripened cheese (e.g.,
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important functions. Several dipeptidases are also prolinases or prolidases and hydrolyze peptides having N - or C- terminal proline residues. For example, PepQ from Le. lactis and PepR from Lb. helveticus hydrolyze the dipeptides X-Pro and Pro-X, respectively. Another peptidase that hydrolyzes proline-containing dipeptides and tripeptides has also been described. The PepT tripeptidase from lactobacilli has preference for hydrophobic tripeptides.

2.7.2.3.3 Aminopeptidases Aminopeptidases, enzymes that hydrolyze N-terminal peptide bonds and release N -terminal amino acids, are the most widespread peptidases found in lactic acid bacteria. Mierau et al. (1997) classified aminopeptidases based on their specificity. The "general" or broadspecificity aminopeptidases, PepN and PepC, hydrolyze peptides ranging in size from 2 to 12 amino acids, and, in general, have little activity on proline-containing dipeptides. They are well conserved among lactococci and lactobacilli. Because p-casein is proline rich, many of the PrtP-generated oligopeptides contain proline. As noted above, proline-containing peptides are often poor substrates for general peptidases. "Specifictask" aminopeptidases (e.g., PepA, PepX, Pepp, PepR, and Pepl), in contrast, can hydrolyze these proline-containing peptides. Like other peptidases, these aminopeptidases vary as to substrate size and specificities. The substrates of PepP from Le. lactis, for example, are oligopeptides containing from 4 to 10 amino acids and having the sequence X-Pro-Pro-(X)n' In contrast, PepX from Le. lactis hydrolyzes similar oligopeptides but in addition can also act on tripeptides, as well as some non-proline-containing peptides. Both the general and specific aminopeptidases are especially important during cheese manufacture, since many oligopeptides contribute to bitter-flavored cheese if not degraded. The implications of prolinecontaining and other bitter pep tides in cheese and their effect on flavor is discussed later. Although it appears that no single peptidase is essential for cell growth, inactivation of multiple peptidases clearly is detrimental to growth in milk. Apparently, absence of a particular peptidase that degrades a particular peptide is not a very serious problem, since alternative pep tides and peptidases are readily available. However, if several peptidases are missing, the rate of peptide hydrolysis would be expected to decrease. Indeed, when cells containing multiple mutations in pepO, pepN, pepC, pepT, and pepX were grown in

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milk, growth rates were reduced more than lO-fold. That mutants reached final cell densities comparable to that of parent strains suggests that enough essential amino acids are eventually released by other peptidases.

2.7.2.4 Role of protein metabolism in cheese manufacture and cheese ripening Although the PrtP system and components of the peptide transport and hydrolysis steps are essential for starter culture growth and activity, they also have important implications during cheese manufacture. Recent identification and characterization of many of the genes involved in protein metabolism have made it possible to construct mutants defective in a single enzymatic or transport activity. Comparing such mutant strains with the isogenic parent has provided a clearer picture of the role of various proteinase components on cheese properties. Several studies have established that cheese made with strains deficient in proteinase activity lack flavor, have poor texture, and otherwise age poorly. Thus, products of starter culture proteinases, combined with products of residual coagulant and milk proteases, impart desirable cheese flavor, either directly or by serving as substrates for additional reactions. However, despite the necessary role of PrtP in developing desirable aged cheese flavor, casein hydrolysis by PrtP also releases several pep tides which are bitter. In general, bitter peptides are hydrophobic and their hydrolysis requires specific peptidases. Starter culture strains that possess the appropriate peptidases necessary to degrade these pep tides are often considered as being "nonbitter" strains, as opposed to "bitter" strains that lack those enzymes and produce bitter cheese. Several peptidases have been proposed to have debittering activity. Experiments using peptidase mutants have provided in vivo evidence for the debittering role of peptidases. Cheese made with PepN or PepX mutants was bitter and had lower organoleptic quality. Although it is clear that bitterness, or lack of bitterness, is an important determinant of cheese flavor and quality, other aspects of protein metabolism undoubtedly influence the properties of aged cheese. Free amino acids and small pep tides are thought to contribute to "nutty" and "sweet" flavor notes typical of Swiss, Parmesan, and other cheeses, whereas products of amino acid catabolism are primarily responsible for Cheddar cheese flavor. Among degradation products formed from amino acids, methanethiol and other sulfur-

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containing compounds are considered to be essential in many cheese varieties, especially those that are surface ripened, Most of these sulfur compounds evolve from methionine and, for Cheddar, are produced by starter as well as nonstarter bacteria. Catabolism of aromatic and other amino acids by lactic acid bacteria certainly results in a large number of volatile compounds, some of which may be desirable, but others may be considered as flavor defects. However, the specific means by which metabolism of amino acids occurs and how products of nitrogen metabolism contribute to cheese flavor and quality await further study.

2.7.2.5 Lactic acid bacteria as flavor adjuncts Once it was realized that peptidases from lactic acid bacteria could reduce bitterness and improve cheese flavor, several investigators began to identify suitable strains and to use them as culture adjuncts in cheese making. Species used as adjuncts include starter culture strains of Le. laetis as well as nonstarter strains of Lb. easei, Lb. hell'eticLls, and Lb. delbrueckii subsp. bulgaricus. In general, these strains have high peptidase activity. Since addition of such strains to cheese could also increase acid production, adjunct cultures are often prepared or used so that actual growth is minimized or prevented, while retaining their enzymatic activities. For example, using lactose-nonfermenting variants ensures that adjunct cells will not produce significant acid. Another way to deliver culture adjuncts is to heat- or freeze-shock the cells, treatments that cause cells to lose acid- forming ability, before addition to milk or curd or to lyse early in the ripening process. Cell extracts can also be added directly, and commercial products containing peptidase-rich extracts have been developed and are used for accelerated cheese-ripening programs. 2.7.3 Citrate Metabolism Although rapid fermentation of lactose and production' of lactic acid is a primary requirement for dairy lactic acid bacteria, the ability of selected strains to ferment citrate and form diacetyl is also an important property in many dairy products. Diacetyl contributes buttery aroma and flavor attributes in cultured butter, butter-milk, sour cream, and Gouda and Edam cheeses. Citrate fermentation also results in formation of CO 2 , which is responsible for eye development in Dutch-style cheeses. Despite the practical importance of this fermentation, however, only recently have the key biochemical and metabolic events been defined.

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2.7.3.1 Diacetyl synthesis Under ordinary conditions, citrate fermentation and diacetyl formation occur only in those strains of lactic acid bacteria that contain genes coding for transport and metabolism of citrate. Among the dairy lactic acid bacteria, citrate utilization is most often associated with Leuconostoc spp. and selected strains of Lactococcus sp. Accordingly, plasmids containing genes coding for citrate transport have been found in those strains that ferment citrate. In Lc. lactis subsp. lactis biovar diacetylactis, citrate fermentation is linked with an 8-kb plasmid, whereas in Leuconostoc, citrate genes are associated with plasmids as large as 22 kb. These plasmids contain a cluster of genes that encode citrate permease (CitP) in Lc. lactis subsp. lactis biovar diacetyiaclis and CitP ami citrate lyase in Leuc. paramesenteroides. How citrate-fermenting lactic acid bacteria actually form diacetyl has been the subject of considerable debate. Two pathways have been proposed. In both pathways, citrate is transported by the pHdependent CitP that has optimum activity between pH 5 and 6. Transport is mediated by a PMF; however, as described below, the

citrate

l

citrate lyase ~'---7 acetate

oxaloacetate

L.

oxaloacetate decarboxylase ~----:7 CO 2

pyruvate

TPP~

a-acetolactate

pyruvate decarboxylase COl

a-acetolactate

acetaldehyde-TPP

acetoin

2 , 3 -b ut ane

d" I 10

< Ireductase \ NAO

diacetyl

ace

NAOH +11

< reductase oln I '\

t'

NAD

Idiacetyl I

NAOH. H

Figure 2.14 Citrate fermentation pathway in lactic acid bacteria.

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MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

net bioenergetic effect of citrate metabolism may actually be an increase in the PMF. Intracellular citrate is then cleaved by citrate lyase to form acetate and oxaloacetate. Although acetate is ordinarily released into the medium, oxaloacetate is decarboxylated to pyruvate by oxaloacetate decarboxylase. Importantly, the evolved CO 2 can cause eye formation in some cheeses. Although lactic acid bacteria could conceivably reduce all excess pyruvate to lactate via lactate dehydrogenase, this does not normally occur. This is because pyruvate reduction requires NADH, which is made during glycolysis, but which is not formed in the citrate fermentation pathway. Using NADH to reduce citrate- generated pyruvate would quickly deprive cells of the NADH pool necessary to reduce pyruvate produced during glycolysis. Instead, excess pyruvate is decarboxylated by pyruvate decarboxylase in a thiamine pyrophosphate (TPP)-dependent reaction, and acetaldehyde-TPP is formed. Some researchers have proposed that an enzyme (diacetyl synthase) is responsible for converting acetaldehyde-TPP (in the presence of acetyl-CoA) directly to diacetyl. However, no evidence for the presence of diacetyl synthase currently exists. Instead, the accepted alternative pathway for diacetyl synthesis involves first a condensation reaction of acetaldehyde-TPP and pyruvate catalyzed by a-acetolactate synthase. This enzyme apparently has a low affinity for pyruvate in Le. lactis subsp. lactis biovar diacetylactis (Km = 50 mM); thus high concentrations of pyruvate are necessary to drive this reaction. The product, a-acetolactate, is unstable in the presence of oxygen and is next nonenzymatically decarboxylated to form diacetyl. This oxidative decarboxylation pathway is now supported by substantial biochemical, genetic, and nuclear magnetic resource evidence. Although utilization of citrate by lactic acid bacteria requires several enzymatic steps, it appears that citrate fermentation provides cells with no obvious benefits, as ATP-gf'nerating reactions are absent in this pathway and citrate consumption results only in excretion of organic endproducts and CO,. Why then do cells ferment citrate? As noted earlier, the driving-force for transport of citrate is the PMF, with divalent citrate transported in symport with a single proton. However, during the oxaloacetate decarboxylation reaction, a cytoplasmic proton is consumed, resulting in an increase in the cytoplasmic pH and an increase in the ~pH component of the PMF. In addition, when citrate-utilizing bacteria are grown in the presence of a fermentable sugar and lactate is produced, efflux of monovalent (anionic) lactate can drive uptake of divalent (anionic) citrate. Thus,

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CitP acts as a electrogenic precursor-product exchanger, with a net increase in the ~\jJ or electrical component of the PMF. Both of these mechanisms (electrogenic exchange and decarboxylation), therefore, result in an increase in the metabolic energy available to the cell.

2.7.3.2 Enhancing diacetyi formation in dairy products Even among citrate-fermenting lactic acid bacteria, the amount of diacetyl formed in dairy products is relatively low «2 mg/L) , and there is much interest in manipulating growth conditions and cultures in an effort to enhance diacetyl production in cheese and cultured milk products. Because citrate transport via CitP requires low pH, citrate-fermenting strains are usually combined with acidproducing strains during manufacture of cultured dairy products. Oxygen can also stimulate diacetyl formation by as much as 30fold. Presumably, high atmospheric oxygen can reduce activity of lactate dehydrogenase and accelerate the oxidative decarboxylation reaction responsible for diacetyl synthesis. In addition, oxygen can oxidize NADH, thereby slowing the rate at which diacetyl is reduced to acetoin or 2,3 -butanediol. Another strategy considered for enhancing diacetyl formation involves genetic modification of the cultures. Several metabolic steps have been identified at which mutations or blocks will lead to increased production of diacetyl. Inactivation of lactate dehydrogenase, for example, results in excess pyruvate, and such cells could theoretically produce more diacetyl than wild-type cells (even non-citrate-fermenting lactococci have been genetically manipulated to produce diacetyl). Enhanced expression of plasmid-borne copies of genes coding for a-acetolactate synthase or NADH oxidase in Lc. lactis also enhances diacetyl formation by increasing the concentration of a-acetolactate available for oxidative decarboxylation. Similarly, inactivation of the gene coding for aacetolactate decarboxylase, the enzyme that forms acetoin directly from a-acetolactate, also results in an increase in diacetyl production.

2.7.4 Metabolism of Propioni Bacteria Although not a lactic acid bacterium, Propionibacterium freudenreichii subsp. shermanii is an important part of the thermophilic starter culture used to manufacture Swiss-type cheeses. This organism is not only responsible for producing CO 2 that leads to eye or hole formation, but it also produces other compounds, including amino acids and their degradation products, that contribute to the characteristic flavor of these cheeses.

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During Swiss cheese manufacture, growth of Pro. freudenreichii subsp. shermanii does not occur until the primary lactic fermentation is completed and cheese is moved into a "warm room" held at 2025°C. Although propionibacteria can ferment lactose, essentially none is available at this time and instead lactate is the primary energy source for their growth in cheese. Fermentation of lactate yields propionate, acetate, and CO 2, with a theoretical molar ratio as: 3 lactate -; 2 propionate + 1 acetate + 1 CO 2 In cheese, the actual amount of CO 2 may vary either as a result of condensation reactions, cometabolism with amino acids, or strain variation. The propionate pathway consists of many reactions, and it requires several metal-containing enzymes and vitamin cofactors. Enzymes of the citric acid cycle are also required. One mole of ATP is generated per mole of lactate consumed. Although proteolysis of casein by Pro. shermanii is limited because of low proteinase activity, it does produce several peptidases. These peptidases are located intracellularly, and their substrates are the peptides released by starter culture proteinases and residual milk and coagulant proteinases. Although no information on peptide transport systems in propionibacteria is currently available, there is evidence that some peptidases could be released via autolysis. Several peptidases have activity on proline-containing peptides, accounting for high levels of proline that accumulate in Swiss-type cheeses. In addition, metabolism of the amino acids alanine and aspartate may contribute to CO 2 production.

2.7.5 Metabolism of Molds and other Flavor-contributing Microorganisms Despite their importance in several cheese types, much less is known about the metabolism of Pel1icilium spp. and brevibacteria used to make mold-ripened and surface-ripened cheeses. These organisms are not really starter cultures, since they do not contribute to acid development, but they are just as integral to the cheese making process as are the lactic starter cultures. Accordingly, their main role in cheese manufacture is to produce flavors and cause desirable changes in texture and appearance of the finished cheese.

2.7.5.1 Penicilium roqueforti The mold responsible for the well-kllOwn blue-veined appearance of Roquefort, Gorgonzola, and other blue cheese types is P. roqueforti. Although spores of P. roqueforti are added to milk or

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curds before the lactic fermentation, mold growth does not occur until after the lactic culture has fermented all or most of the available lactose to lactic acid. Lactic acid serves as an energy source for the mold. Importantly, consumption of lactic acid causes the pH to rise from about 4.6 to as high as 6.2. As P. roqueforti grows in cheese, substantial proteolysis occurs through elaboration of several extracellular proteinases, endopeptidases, and exopeptidases. Amino acids can be subsequently metabolized releasing amines, ammonia, and other possible flavor compounds (that also may raise the pH). However, the most characteristic blue cheese flavors are generated from lipid metabolism. As much as 20% of triglycerides in milk are hydrolyzed by lipases produced by P. roqueforti. Although free volatile fatty acids may themselves contribute to cheese flavor, their metabolism, via ~-oxidation pathways, results in formation of a variety of methylketones. It is this class of compounds that is responsible for the flavor of blue cheese.

2.7.5.2 P. camemberti Just as in blue-veined cheeses, growth of P. camemberti in the manufacture of Camembert and Brie cheeses occurs as a secondary fermentation, and again, lactic acid is used as an energy source. The subsequent rise in pH (from 4.6 to as high as pH 7.0 at the surface) because of lactate consumption and ammonia production provides opportunities for other organisms to grow, and the surface microflora of Camembert cheese can be quite diverse. The proteinases and peptidases produced by P. camemberti are similar to those produced by P. roqueforti. Although P. roqueforti grows throughout the cheese mass (because of deliberate aeration during cheese making), growth of P. camemberti is confined to the surface; therefore, protein breakdown in the interior of cheese is dependent on diffusion of excreted enzymes. Production of ammonia, methanethiol, and other sulfur compounds, presumably derived from amino acids, are also characteristic of Camembert cheese. Lipolysis of triglycerides and fatty acid metabolism by P. camemberti are just as important in surface-ripened cheese as in blue-veined cheese, and methylketones are abundant. 2.7.5.3 Brevibacterium linens Although B. linens is primarily used in the manufacture of Muenster, brick, and other surface-ripened cheeses, its potential use as a flavor adjunct has led to renewed interest in the metabolism of this organism. Most attention has focused on proteinases and

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peptidases produced by B. linens and subsequent formation of volatile flavor compounds from amino acid metabolism. Unlike lactic acid bacteria that produce a single proteinase (PrtP), B. linens produces several extracellular and intracellular proteinases and peptidases. Metabolism of released amino acids results in formation of many sulfur-containing compounds, including hydrogen sulfide, methanethiol, and other volatile flavors that are characteristic not only of surface-ripened cheese but which are also important in Cheddar cheese. The ability of B. linens to produce these flavor compounds. along with a high level of proteolytic activity, have led to the use of this organism as a flavor adjunct in Cheddar-type cheeses. 2.7.6 Metabolic Engineering Considerable information currently exists on many of the important genes and metabolic pathways that influence how lactic acid bacteria grow in yogurt, cheese, and other dairy products. Recently, the genome sequence of Lc. lactis was reported, and the genome sequence of Lb. acicloplzilus is expected to be completed soon. Efforts to use this information to improve or modify properties of lactic acid bacteria have already begun and are certain to be accelerated. As described earlier, metabolic engineering could be used in several ways to improve dairy fermentations. Diverting pyruvate from lactate to the flavor compound diacetyl can be accomplished by genetically disrupting genes coding for lactate dehydrogenase or a-acetolactate decarboxylase. Similarly, cheese ripening can be accelerated by either increasing expression of genes involved in proteolysis or by induced expression of genes coding for lytic enzymes. Increased synthesis of an exopolysaccharide by Lc. lactis subsp. cremoris was achieved by overexpressing the gene coding for fructose-bisphosphatase, an enzyme that makes more precursors available for polysaccharide synthesis. Finally, efforts are underway in several laboratories to engineer S. thermophilus so that galactose is fermented rather than released back into the curd or cheese.

3 Fermentation 3.1 INTRODUCTION Numerous food products owe their production and characteristics to the fermentative activities of microorganisms. Many foods such as ripened cheeses, pickles, sauerkraut, and fermented sausages are preserved products in that their shelf life is extended considerably over that of the raw materials from which they are made. In addition to being made more shelf stable, all fel mented foods have aroma and flavor characteristics that result directly or indirectly from the fermenting organisms. In some instances, the vitamin content of the fermented food is increased along with an increased digestibility of the raw materials. The fermentation process reduces the toxicity of some foods (for example, gari and peujeum), whereas others may become extremely toxic during fermentation (as in the case of bongkrek). From all indications, no other single group or category of foods or food products is as important as these are and have been relative to nutritional well-being throughout the world. The microbial ecology of food and related fermentations has been studied for many years in the case of ripened cheeses, sauerkraut, wines, and so on, and the activities of the fermenting organisms are dependent on the intrinsic and extrinsic parameters of growth. For example, when the natural raw materials are acidic and contain free sugars, yeasts grow readily, and the alcohol they produce restricts the activities of most other naturally contaminating organisms. If, on the other hand, the acidity of a plant product permits good bacterial growth and at the same time the product is high in simple sugars, lactic acid bacteria may be expected to grow, 103

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and the addition of low levels of NaCI will ensure their growth preferential to yeasts (as in sauerkraut fermentation). Products that contain polysaccharides but no significant levels of simple sugars are normally stable to the activities of yeasts and lactic acid bacteria due to the lack of amylase in most of these organisms. To effect fermentation, an exogenous source of saccharifying enzymes must be supplied. The use of barley malt in the brewing and distilling industries is an example of this. The fermentation of sugars to ethanol that results from malting is then carried out by yeasts. The use of koji in the fermentation of soybean products is another example of the way in which alcoholic and lactic acid fermentations may be carried out on products that have low levels of sugars but high levels of starches and proteins. Whereas the saccharifying enzymes of barley malt arise from germinating barley, the enzymes of koji are produced by Aspergillus oryzae growing on soaked or steamed rice or other cereals (the commercial product takadiastase is prepared by growing A. oryzae on wheat bran). The koji hydrolysates may be fermented by lactic acid bacteria and yeasts, as is the' case for soy sauce, or the koji enzymes may act directly on soybeans in the production of products such as Japanese miso. 3.1.1 Defined and Characterized The word fermentation has had many shades of meaning in the past. According to one dictionary definition, it is "a process of chemical change with effervescence . . . a state of agitation or unrest ... any of various transformations of organic substances." The word came into use before Pasteur's studies on wines. Prescott and Dunn and Doelle have discussed the history of the concept of fermentation, and the former authors note that in the broad sense in which the term is commonly used, it is "a process in which chemical changes are brought about in an organic substrate through the action of enzymes elaborated by microorganisms." It is in this broad context that the term is used in this chapter. In the brewing industry, a top fermentation refers to the use of a yeast strain that carries out its activity at the upper parts of a large vat, such as in the production of ale; a bottom fermentation requires the use of a yeast strain that will act in lower parts of the vat, such as in the production of lager beer . . Biochemically, fermentation is the metabolic process in which carbohydrates and related compounds are partially oxidized with the

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release of energy in the absence of any external electron acceptors. The final electron acceptors are organic compounds produced directly from the breakdown of the carbohydrates. Consequently, incomplete oxidation of the parent compound occurs, and only a sm~l amount of energy is released during the process. The products of fermentation consist of some organic compounds that are more reduced than others. 3.1.2 Lactic Acid Bacteria This group is composed of 12 genera of gram-positive bacteria at this time: Oenococcus Carnobacterium Pediococcus Enterococcus Streptococcus Lactococcus Tetragenococcus Lactobacillus Lactosphaera Vagococcus Leuconostoc Weissella With the enterococci and lactococci having been removed from the genus Streptococcus, the most important member of this genus of importance in foods is S. thermophilus. S. diacetilactis has been reclassified as a citrate-utilizing strain of Lactococcus lactis subsp. lactis. Related to the lactic acid bacteria but not considered to fit the group are genera such as Aerococcus, Microbacterium, and Propionibacterium, among others. The latter genus has been reduced by the transfer of some of its species to the new genus Propioniferax, which produces propionic acid as its principal carboxylic acid from glucose. The history of our knowledge of the lactic streptococci and their ecology has been reviewed by Sandine et al. These authors believe that plant matter is the natural habitat of this group, but they note the lack of proof of a plant origin for Lactococcus cremoris. It has been suggested that plant streptococci may be the ancestral pool from which other species and strains developed. Although the lactic acid group is loosely defined with no precise boundaries, all members share the property of producing lactic acid from hexoses. As fermenting organisms, they lack functional hemelinked electron transport systems or cytochromes, and they obtain their energy by substrate-level phosphorylation while oxidizing carbohydrates; they do not have a functional Krebs cycle.

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Kluyver divided the lactic acid bacteria into two groups based on end products of glucose metabolism. Those that produce lactic acid as the major or sole product of glucose fermentation are designated homofermentative. The homolactics are able to extract about twice as much energy from a given quantity of glucose as are the heterolactics. The homofermentative pattern is observed when glucose is metabolized but not necessarily when pentoses are metabolized, for some homolactics produce acetic and lactic acids when utilizing pentoses. Also the homofermentative character ,of homo lac tics may be shifted for some strains by altering growth conditions such a,s glucose concentration, pH, and nutrient limitation. Those lactics that produce equal molar amounts of lactate, carbon dioxide, and ethanol from hexoses are designated heterofermentative. All members of the genera Pediococcus, Streptococcus, Lactococcus, and Vagococcus are homofermenters, along with some of the lactobacilli. Heterofermenters consist of Leuconostoc, Oenococcus, Weissella, Carnobacterium, Lactosphaera. and some lactobacilli. The GLUCOSE

f- co.

I

2ATP4ATP

1 ATP --.

(A)

2ATP-4ATP

[-.2ATP Phosphokelolase

A

Hexose Isomerase

2

1

Lactate

Lactate

Ethanol

MethytmaJonyl-CoA - .

~

Oxalacetate

Proplonyl-CoA Succinate -'-+

I

(D) PYRUVATE

(C)

I

2H --.

Lacrate

I

~ Succinyt-CoA (Anaer~bICaIlY)

Propionate

Propionate, Acetate,

(E)}-+ Co. 2H-....

Etnanol (F)

1

Acetic Acid

(Gll co. + HP

CO;

Figure 3.1 Generalized pathways for the production of some fermentation products from glucose by various organisms. A-Homofermentative laclics; Bheterofermentative lacties; C and D-Propiollibacterium; E-Saccharomyces spp.; F-Acetobacter spp.; G-Acetobacter "overoxidizers."

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CITRATE

Acetate

Oxaloacetate

-COOH

Pyruvate

X2

TPP -+Acetyl-CoA -+-

Acetoin

Diacetyl

Figure 3.2 The general pathway by which acetoin and diacetyl are produced from citrate by group N lactococci and Leuconostoc spp.

heterolactics are more important than the homolactics in producing flavor and aroma components such as acetylaldehyde and diacetyl. The genus Lactobacillus has been subdivided classically into three subgenera: Betabacterium. Streptobacterium. and Thermobacterium. All of the heterolactic lactobacilli are betabacteria. The streptobacteria (for example, L. casei and L. plantarum) produce up to 1.5% lactic acid with an optimal growth temperature of 30°C, whereas the thermobacteria (such as L. acidophilus and L. bulgaricus) can produce up to 3% lactic acid and have an optimal temperature of 40°C.

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More recently, the genus Lactobacillus has been arranged into three groups based primarily on fermentative features. Group 1 includes obligate homofermentative species (L. acidophilus, L. bulgaricus, L. delbrueckii, etc.). These are the thermobacteria, and they do not ferment pentoses. Group 2 consists of facultative heterofermentative species (L. casei, L. plantarum. L. sake. etc.). Members of this group ferment pentoses. Group 3 consists of the obligate heterofermentative species, and it includes L. fer11lentum. L. brevis. L. reuteri. L. sanfrancisco. and others. They produce CO 2 from glucose. The lactobacilli can produce a pH of 4.0 in foods that contain a fermentable carbohydrate, and they can group up to a pH of about 7.1. In terms of their growth requirements, the lactic acid bacteria require preformed amino acids, B vitamins, and purine and pyrimidine bases-hence their use in microbiological assays for these compounds.Although they are mesophilic, some can grow below SOC and some as high as 4SoC. With respect to growth pH, some can grow as low as 3.2, some as high as 9.6, and most grow in the pH range 4.0-4.S. The lactic acid bacteria are only weakly proteolytic and lipolytic. The cell mucopeptides of lactics and other bacteria have been reviewed by Schleifer and Kandler. Although there appear to be wide variations within most of the lactic acid genera, the homofermentative lactobacilli 0; the subgenus Thermobacterium appear to be the most homogeneous in this regard in having Llysine in the peptidoglycan peptide chain and D-aspartic acid as the interbridge peptide. The lactococci have similar wall mucopeptides. Molecular genetics have been employed by McKay and coworkers to stabilize lactose fermentation by L. lactis. The genes responsible for lactose fermentation by some lactic cocci are plasmidborne, and loss of the plasmid results in the loss of lactose fermentation. In an effort to make lactose fermentation more stable, lac+ genes from L. lactis were cloned into a cloning vector, which was incorporated into a Streptococcus sanguis strain. Thus, the lac genes from L. lactis were transformed into S. sanguis via a vector plasmid, or transformation could be effected by use of appropriate fragments of DNA through which the genes were integrated into the chromosome of the host ,cells. In the latter state, lactose fermentation would be a more stable property than when the lac genes are plasmid-borne.

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3.1.3 Metabolic Pathways and Molar Growth Yields The end-product differences between homo-and heterofermenters when glucose is attacked are a result of basic genetic and physiological differences. The homolactics possess the enzymes aldolase and hexose isomerase but lack phosphoketolase. They use the Embden-Meyerhof-Parnas (EMP) pathway toward -their production of two lactates/glucose molecule. The heterolactics, on the other hand, have phosphoketolase but do not possess aldolase and hexose isomerase, and instead of the EMP pathway for glucose degradation, these organisms use the hexose monophosphate or pentose pathway. The measurement of molar growth yields provides information on fermenting organisms relative to their fermentation substrates and pathways. By this concept, the microgram dry weight of cells produced per micromole of substrate fermented is determined as the molar yield constant, indicated by Y. It is tacitly assumed that essentially none of the substrate carbon is used for cell biosynthesis, that oxygen does not serve as an electron or hydrogen acceptor, and that all of the energy derived from the metabolism of the substrate is coupled to cell biosynthesis. When the substrate is glucose, for example, the molar yield constant for glucose, YG' is determined by

_ y.G-

g dry weight of cells moles glucose fermented If the adenosine triphosphate (ATP) yield or moles of ATP produced per mole of substrate used is known for a given substrate, the amount of dry weight of cells produced per mole of ATP formed can be determined by

Y

_ g dry weight of cells/moles ATP formed moles substrate fermented A large number of fermenting organisms has been examined during growth and found to have YATP = 10.5 or close thereto. This value is assumed to be a constant, so that an organism that ferments glucose by the EMP pathway to produce 2 ATP/mole of glucose fermented should have YG = 21 (i.e., it should produce 21 g of cells dry weight/mole of glucose). This has been verified for E. faecalis, Saccharomyces cerevisiae, Saccharomyces rosei, and L. plantarum on glucose (all Yc = 21, YATP = 10.5, within experimental error). A study by Brown and Collins indicates that Yc ATP -

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and Y ATP values for Lactococcus lactis subsp. lactis biovar diacetylactis and Lactococcus lactis subsp. cremoris differ when cells are grown aerobically on a partially defined medium with low and higher levels of glucose, and further when grown on a complex medium. On a partially defined medium with low glucose levels (17 p,mol/mL), values for L. lac/is subsp. lac/is biovar diacetylactis were YG = 35:3 and YATP = 15.6, whereas for L. lactis subsp. cremoris, YG = 31.4 and YATP = 13.9. On the same medium with higher glucose levels (1-15 p,moI! mL), YG for L. lac/is subsp. Lactis biovar diacetylactis was 21, YATP values for these two organisms on the complex medium with glucose 2 p,moI!mL were 21.5 and 18.9 for L. lactis subsp. lactis biovar diacetylactis and L. lactis subsp. cremoris, respectively. Anaerobic molar growth yields for enterococcal species on low levels of glucose have been studied by Johnson and CoIlins. Zymomonas mobilis utilizes the Entner-Doudoroff pathway to produce only 1 ATP/mole of glucose fermented (YG = 8.3, YATP = 8.3). If and when the produced lactate is metabolized further, the molar growth yield would be higher. Bifidobacterium bifidum produces 2.5-3 ATP/mole of glucose fermented with YG = and YATP = 13. In addition to the use of molar growth yields to compare organisms on the same energy substrate, this concept can be applied to assess the metabolic routes used by various organisms in attacking a variety of carbohydrates.

3.2 DAIRY PRODUCTS 3.2.1 Milk Biota The microorganisms in raw cow's milk consist of those that may be present on the cow's udder and hide and on milking utensils or lines. Under proper handling and storage conditions, the predominant biota is gram positive. Raw milk held at refrigerator temperatures for several days invariably shows the presence of several or all bacteria of the following genera: Enterococcus, Lactococcus, Streptococcus, Leuconostoc. Lactobacillus, Microbacterium, Oerskovia. Propionibacterium, Micrococcus, Proteus, Pseudomonas, Bacillus, and Listeria, as well as members of at least one of the coliform genera. The biota that is unable to grow at the usual low temperature of holding tends to be present in very low numbers. Studies have revealed the presence of psychrotrophic spore formers and mycobacteria in raw milk. For example, psychrotrophic BaciLLus spp. were found in 25-36% of 97 raw milk samples in one study,"

FERMENTATION

III

and they were shown to grow at or below 7°C. Psychrotrophic clostridia were isolated from 4 or 48 raw milk samples in another study.' In another study, Mycobacterium and Nocardia spp. were isolated from about 69% of 51 raw milks." The pasteurization process eliminates all but thermoduric strains, primarily streptococci and lactobacilli, and spore formers of the genus Bacillus (and Clostridium, if present in raw milk). Milk is the vehicle for some diseases. Milkbome outbreaks generally involve the consumption of raw milk, including certified raw. Homemade ice cream containing fresh eggs, and dried and pasteurized milks contaminated after heat processing have been associated with foodbome outbreaks. Campylobacteriosis and salmonellosis are well established as illnesses that may be contracted from milk and milk products. Listeriosis and hemorrhagic colitis outbreaks have also been traced to milk. Questions' have been raised over the efficacy of milk pasteurization to destroy Mycobacterium paratuberculosis. The concern has to do with the fact that this bacterium causes Tohne's disease in cattle, and appears to playa role in Crohn's disease of humans. In one study, neither the high temperature short time (HTST) nor the low temperature long time (LTLT) method destroyed 103 -104 cfu/mL in all milk samples, but in another study, up to 106 cfu/mL were destroyed by HTST (72°C for 15 seconds). Crohn's disease is an inflammatory bowel disease (regional ileitis), a condition wherein the terminal ileum and sometimes the cecum and ascending colon are thickened and ulcerated. The lumen of the affected region is much narrowed, resulting in intestinal obstruction. The spoilage of pasteurized milk products has two common origins. First is the growth and metabolic activity of psychrotrophic organisms such as Pseudomonas, Alcaligenes, and Flavobacterium spp. These gram-negative rods, which are usually lipolytic and proteolytic, are postpasteurization contaminants. The proteolytic organisms are able to cause a destabilization of the casein micelles and cause a "sweet-curdling" of the milk. However, the predominant spoilage is manifest by bitter and fruity off-flavors. Second is the growth of heat-resistant organisms that are able to ferment lactose to lactic acid, and when the pH is reduced to about 4.6, the milk curdles. If mold spores are present, they may germinate and grow at the surface of the sour milk and elevate pH toward neutrality, thus allowing the more proteolytic bacteria such as Pseudomonas

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spp. to grow and bring about the liquefaction of the milk curd. In extended-shelf-life milk products, spoilage by psychrotrophic spore formers is a significant problem. Organisms such as Bacillus cereus can survive the UHT process, and because of the longer shelf life, can initiate growth and produce toxins as well as causing "sweetcurdling" of the products. The microbial spoilage of raw milk follows a pattern similar to the above, assuming the product is held under refrigeration. Ropiness is a condition sometimes seen in raw milk that is caused by Alcaligenes viscolactis. Its growth is favored by low-temperature holding of raw milk for several days. The rope consists of slimelayer material produced by the bacterial cells, and it gives the product a stringy consistency. 3.2.2 Starter Cultures, Products The products discussed in this subsection require the use of an appropriate starter culture. A lactic starter is a basic starter culture with widespread use in the dairy industry. For cheese making of all kinds, lactic acid production is essential, and the lactic starter is employed for this purpose. Lactic starters are also used for butter, cultured buttermilk, cottage cheese, and cultured sour cream and are often referred to by product (butter starter, buttermilk starter, and so on). Lactic starters always include bacteria that convert lactose to lactic acid, usually L. lactis subsp. lactis, L. lactis subsp. cremoris. orL. lactis subsp. lactis biovar diacetylactis. Where flavor and aroma compounds stich as diacetyl are desired, the lactic starter will include a heterolactic such as Leuconostoc mesenteroides subsp. cremoris, L. lactis subsp. lactis biovar diacetylactis, or Leuconostoc mesenteroides subsp. dextranicum. Starter cultures may consist of single or mixed strains. They may be produced in quantity and preserved by freezing in liquid nitrogen, or by freeze drying. The lactococci generally make up around 90% of a mixed dairy starter population, and a good starter culture can convert most of the lactose to lactic acid. The titratable acidity may increase to 0.81.0%, calculated as lactic acid, and the pH usually drops to 4.34.5. Butter, buttermilk. and sour cream are produced generally by inoculating pasteurized cream or milk with a lactic starter culture and holding until the desired amount of acidity is attained. In the case of butter, where cream is inoculated, the acidified cream is then churned to yield butter, which is washed, salted, and packaged.

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Butter-milk, as the name suggests, is the milk that remains after cream is churned for the production of butter. The commercial product is usually prepared by inoculating skim milk with a lactic or buttermilk starter culture and holding until souring occurs. The resulting curd is broken up into fine particles by agitation, and this product is termed cultured buttermilk. Cultured sour cream is produced generally by fermenting pasteurized and homogenized light cream with a lactic starter. These products owe their tart flavor to lactic acid and their buttery aroma and taste to diacetyl. Yogurt (yoghurt) is produced with a yogurt starter, which is a mixed culture of S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in a 1: 1 ratio. The coccus grows faster than the rod and is primarily responsible for initial acid production at a higher rate than that produced by either when growing alone, and more acetaldehyde (the chief volatile flavor component of yogurt) is produced by L. delbrueckii subsp. bulgaricus when growing in association with S. thermophilus. The coccus call produce about 0.5% lactic acid and the rod about 0.6-0.8% (pH of 4.2-4.5). However, if incubation is extended, pH can decrease to about 3.5 with lactic acid increasing to about 2%. The product is prepared either by reducing the water content of either whole or skim milk by at least one fourth (may be done in a vacuum pan following sterilization of milk), or by adding about 5% milk solids followed by water reduction (condensing). The concentrated milk is then heated. to 82°-93°C for 30-60 minutes and cooled to around 45°C. The yogurt starter is now added at a level of around 2% by volume and incubated at 45°C for 3-5 hours followed by cooling to 5°C. The titratable acidity of a good finished product is around 0.85-0.90%, and to get this amount of acidity the fermenting product should be removed from 45°C when the titratable acidity is around 0.65-0.70%.bo Good yogurt keeps well at 5°C for 1-2 weeks. The coccus grows first during the fermentation followed by the rod, so that after around 3 hours, the numbers of the two organisms should be approximately equal. Higher amounts of acidity, such as 4%, can be achieved by allowing the product to ferment longer, with the effect that the rods will exceed the cocci in number. The streptococci tend to be inhibited at pH values of 4.24.4, whereas the lactobacilli can tolerate pH values in the 3.5-3.8 range. The lactic acid of yogurt is produced more from the glucose moiety of lactose than the galactose moiety. Goodenough and Kleyn found only a trace of glucose throughout yogurt fermentation,

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PHYSIOLOGY, GENETICS AND ECOLOGY

whereas galactose increased from an initial trace to 1.2%. Samples of commercial yogurts showed only traces of glucose, but galactose varied from around 1.5% to 2.5%. Freshly produced yogurt typically contains around 109 organisms/ g, but during storage, numbers may decrease to 106 /g, especially when stored at 5°C for up to 60 days. The rod generally decreases more rapidly than the coccus. The addition of fruits to yogurt does not appear to affect the numbers of fermenting organisms. The International Dairy Federation norm for yogurt is 10 7/g or above. In a recent study, E. coli 0157:H7 did not survive in skim milk at pH 3.8, and the organism was inactivated in yogurt, sour cream, and buttermilk similarly. The antimicrobial qualities of yogurt, butter-milk, sour cream, and cottage cheese were examined by Goel et aI., who inoculated Enterobacter aerogenes and Escherichia coli separately into commercial products and studied the fate of these organisms when the products were stored at 7.2°C. A sharp decline of both coliforms was noted in yogurt and buttermilk after 24 hours. Neither could be found in yogurt generally beyond 3 days. Although the numbers of coli forms were reduced also in sour cream, they were not reduced as rapidly as in yogurt. Some cottage cheese samples actually supported an increase in coliform numbers, probably because the products had higher pH values. The initial pH ranges for the products studied by these workers were as follows: 3.65-4.40 for yogurts, 4.1-4.9 for buttermilks, 4.18-4.70 for sour creams, and 4.80-5.10 for cottage cheese samples. In another study, commercially produced yogurts in Ontario were found to contain the desired 1: 1 ratio of coccus to rod in only 15% of 152 products examined. Staphylococci were found in 27.6% and coliforms in around 14% of these yogurts. Twenty-six percent of the samples had yeast counts more than 1,000/g and almost 12% had psychrotroph counts more than 1,000/g. In his study of commercial unflavored yogurt in Great Britain, Davis found S. thermophilus and L. bulgaricus counts to range from a low of around 82 million to a high of over 1 billion/ g, and the final pH to range from 3.75 to 4.20. Kefir is prepared by the use of kefir grains, which contain L. lactis, L. bulgaricus, and a lactose-fermenting yeast held together by layers of coagulated protein. Acid production is controlled by the bacteria, and the yeast produces alcohol. The final concentration of lactic acid and alcohol may be as high as 1%. Kumiss is similar to

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115

kefir except that mare's milk is used, the culture organisms do riot form grains, and the alcohol content may reach 2%. Acidophilus milk is produced by the inoculation into sterile skim milk of an intestinal implantable strain of L. acidophilus. The inoculum of 1-2% is added, followed by holding of the product at 3 rc until a smooth curd develops. A popular variant of this product that is produced commercially in the United States consists of adding a concentrated implantable strain culture of L. acidophilus to a pasteurized and cold vat of whole milk (or skim or 2% milk), and it is bottled immediately. It has the pH of normal milk and is more palatable than the more acidic product. The numbers of L. acidophilus should be in the 107-lOB/mL range. Bulgarian buttermilk is produced in a similar manner by the use of L. bulgaricus as the inoculum or starter, but unlike L. acidophilus. L. bulgaricus is not implantable in the human intestines. Butter contains around 15% water, 81 % fat, and generally less than 0.5% carbohydrate and protein. Although it is not a highly perishable product, it does undergo spoilage by bacteria and molds. The main source of microorganisms for butter i!) cream, whether sweet or sour, pasteurized or nonpasteurized. The flora of whole milk may be expected to be found in cream because as the fat droplets rise to the surface of milk, they carry up microorganisms. The processing of both raw and pasteurized creams to yield butter brings about a reduction in the numbers of all microorganisms, with values for finished cream ranging from several hundred to over 100,000/g having been reported for finished salted butter. Salted butter may contain up to 2% salt, and this means that water droplets throughout may contain an effective level of about 10%, thus making this product even more inhibitory to bacterial spoilage. Bacteria cause two principal types of spoilage in butter. The first is a condition known as "surface taint" or putridity. This condition is caused by Pseudomonas putrefaciens as a result of its growth on the surface of finished butter. It develops at temperatures within the range 4-7°C and may become apparent within 7-10 days. The odor of this condition is apparently due to certain organic acids, especially isovaleric acid. The second most common bacterial spoilage condition of butter is rancidity. This condition is caused by the hydrolysis of butterfat with the liberation of free fatty acids. Lipase from sources other than microorganisms can cause the effect. The causative organism is Pseudomonas fragi. although P. jIuorescens is

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sometimes found. Bacteria may cause three other less common spoilage conditions in butter. Malty flavor is reported to be due to the growth of Lactococcus lac tis var. maltigenes. Skunklike odor is reported to be caused by Pseudomonas mephitica; black discolorations of butter have been reported to be caused by P nigrifaciens. Butter undergoes fungal spoilage rather commonly by species of Cladosporium. Alternaria, Aspergillus. Mucor. Rhizopus. Penicillium. and Geotrichum, especially G. candidum (Oospora lactis). These organisms can be seen growing on the surface of butter, where they produce colorations referable to their particular spore colors. Black yeasts of the genus Torula also have been reported to cause discolorations on butter. The microscopic examination of moldy butter reveals the presence of mold mycelia some distances from the visible growth. The generally high lipid content and low water content make butter more susceptible to spoilage by molds than by bacteria. Cottage cheese undergoes spoilage by bacteria, yeasts, and molds. The most common spoilage pattern displayed by bacteria is a condition known as slimy curd. Alcaligenes spp. have been reported to be among the most frequent causative organisms, although Pseudomonas. Proteus. Enterobacter, and Acinetobacter spp. have been implicated. Penicillium. Mucor, Alternaria, and Geotrichum all grow well on cottage cheese, to which they impart stale, musty, moldy, and yeasty flavors. The shelf life of commercially produced cottage cheese inAlberta, Canada, was found to be limited by yeasts and molds. Although 48% of fresh samples contained coli forms, these organisms did not increase upon storage in cottage cheese at 40°F for 16 days.

3.2.3 Cheeses Most but not all cheeses result from a lactic fermentation of milk. In general, the process of manufacture consists of two important steps: 1. Milk is prepared and inoculated with an appropriate lactic starter. The starter produces lactic acid, which, with added rennin, gives rise to curd formation. The starter for cheese production may differ depending on the amount of heat applied to the curds. S. thermophilus is employed for acid production in cooked curds because it is more heat tolerant than either of the other more commonly used lactic starters; or a combination of S.

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117

thermophilus and L. lactis subsp. lactis is employed for curds that receive an intermediate cook. 2. The curd is shrunk and pressed, followed by salting, and, in the case of ripened cheeses, allowed to ripen under conditions appropriate to the cheese in question. Although most ripened cheeses are the product of metabolic activities of the lactic acid bacteria, several well-known cheeses owe their particular character to other related organisms. In the case of Swiss cheese, a mixed culture of L. delbrueckii subsp. bulgaricus and S. thermophilus is usually employed along with a culture of Propionibacterium shermanii. which is added to function during the ripening process in flavor development and eye formation. These organisms have been reviewed extensively by Hettinga and Reinhold. For blue cheeses such as Roquefort, the curd is inoculated with spores of Penicillium roqueforti, which effect ripening and impart the blue-veined appearance characteristic of this type of cheese. In a similar fashion, either the milk or the surface of Camembert cheese is inoculated with spores of Penicillium camemberti. There are over 400 varieties of cheeses representing fewer than 20 distinct types, and these are grouped or classified according to texture or moisture content, whether ripened or unripened, and if ripened, whether by bacteria or molds. The three textural classes of cheeses are hard, semihard, and soft. Examples of hard cheeses are all cheddar, Provolone, Romano, and Edam. All hard cheeses are ripened by bacteria over periods ranging from 2 to 16 months. Semihard cheeses include Muenster and Gouda and are ripened by bacteria over periods of 1 to 8 months. Blue and Roquefort are two examples of semihard cheeses that are mold ripened for 2-12 months. Limburger is an example of a soft bacteria-ripened cheese, and Brie and Camembert are examples of soft mold-ripened cheeses. Among unripened cheeses are cottage, cream, and Neufchatel. The low moisture content of hard and semihard ripened cheeses makes them insusceptible to spoilage by most organisms, although molds can and do grow on these products as would be expected. Some ripened cheeses have sufficiently low oxidation-reduction (O/R) potentials to support the growth of anaerobes. It is not surprising to find that anaerobic bacteria sometimes cause the spoilage of these products when a, (water activity) permits growth to occur. Clostridium spp., especially C. pasteuriamlln. C. butyricu171, C. sporogel1es, and C. tyrobutyricUlI1 , have been reported to cause

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gassiness of cheeses, One of these (C, tyrobutyricum) is well established as the cause of a butyric acid fermentation or the lateblowing defect in cheeses such as Gouda, One aerobic sporeformer, Bacillus polymyxa, has been reported to cause gassiness. Gassiness results from the utilization of lactic acid with the production of CO 2 , Its activity can be delayed for several weeks by 0.5% of a food-grade long-chain polyphosphate, and possibly completely inhibited by 1.0%. For the years 1973-1992, there were 32 cheese-associated disease outbreaks in the United States with 1,700 cases and 58 deaths-52 of the latter caused by L. monocytogenes in the 1985 California outbreak. The most common vehicle was soft cheeses, and improper pasteurization was common.

3.3 APPARENT HEALTH BENEFITS OF FERMENTED MILKS The topic of health-promoting effects of certain fermented foods and/or the organisms of fermentation is beset by findings both for and against such effects. Some studies that appear to be well designed support health benefits; however, other equally well-designed studies do not. The three areas of concern are the possible benefits to lactose·-intolerant individuals, the lowering of serum cholesterol, and anticancer activity. 3.3.1 Lactose Intolerance Lactose intolerance (lactose malabsorption, intestinal hypolacternia) is the normal state for adult mammals, including most adult humans, and many more groups are intolerant to lactose than are tolerant. Among the relatively few groups that have a majority of adult5 who tolerate lactose are northern Europeans, white Americans, and members of two nomadic pastoral tribes in Africa. When lactose malabsorbers consume certain quanLities of milk or ice cream, they immediately experience flatulence and diarrhea. The condition is due to the absence or reduced amounts of intestinal lactase, and this allows the bacteria in the colon to utilize lactose with the production of gases. The breath hydrogen test for lactose intolerance is based on the increased levels of Hz produced by anaerobic and facultatively anaerobic bacteria utilizing the nonabsorbed lactose. A large number of investigators have found that lactose malabsorbers can consume certain fermented dairy products without

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harmful effects; other studies found no beneficial effects. When beneficial effects are found, they are attributed to the reduced level of lactose in the fermented product and to the production of 13galactosidase by the fermenting organisms following ingestion of the products. In one study, the lactose content of yogurt after storage for 11 days decreased about one-half (to about 2.3 g/ 100 g from 4.8 g/lOO g in nonfermented milk). During the same period, galactose increased from traces in milk to 1.3 g/ 100 g in yogurt, and similar results were found for acidophilus and bifid us milks. In a study employing rats, the animals were fed experimental diets containing yogurt, pasteurized yogurt, and simulated yogurt for 7 days. Those that received natural yogurt were able to absorb galactose more efficiently and also had higher levels of intestinal lactase. The yogurt bacteria remained viable in the gut for up to 3 hours. When eight lactose malabsorbers ingested yogurt or acidophilus milk, they did not experience any of the symptoms that resulted when low- fat milk was ingested. "Sweet" acidophilus milk has been reported by some to prevent symptoms of lactose intolerance, whereas others have found this product to be ineffective. Developed by M.L. Speck and co-workers, it consists of normal pasteurized milk to which is added large numbers of viable L. acidophilus cells as frozen concentrates. As long as the milk remains under refrigeration, the organisms do not grow, but when it is drunk, the consumer gets the benefit of viable L. acidophilus cells. It is "sweet" because it lacks the tartness of traditional acidophilus milk. When 18 lactase-deficient patients ingested unaltered milk for 1 week, followed by "sweet" acidophilus milk for an additional week, they were as intolerant to the latter product as to the unaltered milk. In a study with rats, the yogurt bacteria had little effect in preventing the malabsorption of lactose. The indigenous lactics in the gut tended to be suppressed by yogurt, and the rat lactobacillus flora changed from one that was predominantly heterofermentative to one that was predominantly homofermentative. It appears that several factors may be important in the contradictory findings noted: the strains of lactic acid bacteria employed, the basic differences between the digestive tracts of animals and humans, and the degree of lactose intolerance in test subjects. Overall, the amelioration of symptoms of lactose intolerance by lactic acid bacteria is well documented.

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3.'3.2 Cholesterol Impetus for studies on the effect of fermented milks on cholesterol came from a study of Masai tribesmen in Africa who, in spite of consuming substantial amounts of meat, have low serum cholesterol and a very low incidence of coronary diseases. This was associated with their common consumption of 4-5 Lid of fermented whole milk. Subsequent studies by a large number of groups leave unanswered the true effect of organisms of fermentation on serum cholesterol levels in humans, although the weight of evidence tends to support a positive effect. In a study by Mann using 26 human subjects, large dietary intakes of yogurt were found to lower cholesterolemia, and the findings suggested that yogurt contains a factor that inhibits the synthesis of cholesterol from acetate. This factor may be either 3hydroxy-3-methylglutaric acid and/or orotic acid plus thermophilus milk and methanol solubles of thermophilus milk on liver cholesterol, and the investigators found that both products significantly reduced liver cholesterol levels compared to controls. In another study with rats fed for 4 weeks with a stock diet plus 10% milk fermented by L. acidophilus, significantly lower serum cholesterol was found than when those rats were fed two other diets not containing fermented milk. Whereas in some studies the lowered cholesterol levels are believed to result from decreased synthesis, in others the bacteria were found to remove cholesterol or its precursors from the gastrointestinal tract. In a study by Gilliland et aI, two strains of L. acidophilus (recovered from swine) had the ability to grow in the presence of bile. One strain assimilated cholesterol from laboratory culture media in the presence of bile under anaerobic conditions and significantly inhibited increases in serum cholesterol levels in pigs that were fed a high-cholesterol diet. The other strain did not remove cholesterol from laboratory media and did not reduce serum cholesterol when fed to pigs. These investigators thus presented evidence that some strains of L. acidophilus reduce serum cholesterol by acting directly on cholesterol in the gastrointestinal tract. More . recently, cholesterol was shown to be reduced by 50% in a culture medium after 10-14 days of growth at 32°C by Propionibacterium freudenreichii. The organism did not degrade the compound because up to 70% could be recovered from washed cells. A total of 68 volunteers (ages 18 to 26) in groups of 10 or 13 were put on a regimen consisting of the following supplements: raw

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milk, whole milk, skim milk, yogurt, buttermilk, and "sweet" acidophilus milk. The regimen was maintained for 3 weeks, and the findings suggested that cultured buttermilk, yogurt, and acidophilus milk had no noticeable effect on serum cholesterol. From a study using rats fed for 4 weeks with chow plus skim milk fermented by S. thermophilus, L. delbrueckii subsp. bulgaricus, and L. acidophilus along with appropriate controls, no significant changes in plasma or whole-body cholesterol were found. After a 6-week feeding study of 58 healthy men of Danish descent, a statistically significant reduction of cholesterol was found in those fed a milk product (Gaio) fermented by Enterococcus faecium and two strains of Streptococcus thermophilus. The fermented product contained E. faecium at a level of about 2 x 10s/mL and S. thermophilus at about 7 x 10s/mL. 3.3.3 Anticancer Effects Apparently, the first observation of anticancer activity of lactic acid bacteria was that of I.G. Bogdanov and co-workers in the Soviet Union in 1962, who demonstrated an effect against a sarcoma and a carcinoma. Anticancer activities have been demonstrated in animal models by a large number of investigators who variously employed yogurt and yogurt extracts, L. acidophilus, L. delbrueckii subsp. bulgaricus, and L. casei in addition to extracts of these organisms. To study the effect of oral supplements of L. acidophilus on fecal bacterial enzyme activity, Goldin and Gorbach used 21 human subjects. The enzymes assayed were l3-glucuronidase, nitroreductase, and azoreductase because they can convert indirectly acting carcinogens to proximal carcinogens. The feeding regimen consisted of a 4-week control period followed by 4 we~ks of plain milk, 4 weeks of control, 4 weeks of milk containing 2 x 106 /mL of viable L. acidophilus, and 4 weeks of control. Reductions of twofold to fourfold in activities of the three fecal enzymes were observed in all subjects only during the period of lactobacillus feeding; fecal enzyme levels returned to normal during the final 4-week control period. Similar but more limited studies have been reported by others. Findings of the type noted may prove -to be significant in colon cancer where the body of evidence supports a role for diet. 3.3.4 Probiotics Although fermented foods such as yogurt contain viable organisms at the time of ingestion, their presence is not the ostensible

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reason why most individuals consume this product. Probiotics refers to the consumption of products that contain live organisms that are or are believed to be beneficial to the consumer. The objective here is the ingestion of the organisms, and they consist generally of various lactic acid bacteria and/or bifidobacteria.

3.4 DISEASES CAUSED BY LACTIC ACID BACTERIA Although the beneficial aspects of the lactic acid bacteria to human and animal health are unquestioned, some of these bacteria are associated with human illness. This subject has been reviewed by Aguirre and Collins, who noted that around 68 reports of involvement of lactobacilli in human clinical illness were made over about a 50-year period. Several species of the leuconostocs were implicated in about 27 reports in 7 years, the pediococci in 18 reports over 3 years, and the enterococci in numerous reports. The enterococci are the third leading cause of nosocomial (hospital acquired) infections, with E. faecalis and E. faecium being the two most common species. It appears that lactic acid bacteria are opportunists that are not capable of initiating infection in normal healthy individuals. To determine whether vancomycin-resistant enterococci (VRE) existed in ground beef and pork in Germany, 555 samples were examined for VRE, and overall their incidence in ground beef was too low to be a significant source in nosocomial infections. 3.5 FERMENTED FRUIT AND VEGETABLE PRODUCTS The microbial biota of land-grown vegetables may be expected to reflect that of the soils in which they are grown, although exceptions occur. The actinomycetes (gram-positive branching forms) are the most abundant bacteria in stable soils, yet they are rarely reported on vegetable products. On the other hand, the lactic acid bacteria are rarely found in soil per se, but they are significant parts of the bacterial biota of plants and plant products. The overall exposure of plant products to the environment provide many opportunities for contamination by microorganisms. The protective cover of many fruits and vegetables and the possession by some of pH values below which many organisms cannot grow are important factors in the microbiology of these products. Some attempt is made to treat fruits and vegetables separately even though this is difficult. In common usage, products such as tomatoes and cucumbers are called vegetables and yet from the botanical standpoint they are fruits. Lemons, oranges, and limes are

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fruits botanically as well as in common usage. By and large, the distinctions between fruits and vegetables are based on pH, irrespective of the lack of scientific merit.

3.6 FRESH AND FROZEN VEGETABLES The incidence of microorganisms in vegetables may be expected to reflect the sanitary quality of the processing steps and the microbiological condition of the raw product at the time of processing. In a study of green beans before blanching, Splittstoesser et al. showed that the total counts ranged from log 5.60 to over 6.00 in two production plants. After blanching, the total numbers were reduced to log 3.00-3.60/g. After passing through the various processing stages and packaging, the counts ranged from log 4.72 to S.94/g. In the case of french-style beans, one of the greatest buildups in numbers of organisms occurred immediately after slicing. This same general pattern was shown for peas and corn. Preblanched green peas from three factories showed total counts per gram between log 4.94 and 5.95. These numbers were reduced by blanching and again increased successively with each processing step. In the case of whole-kernel corn, the postblanch counts rose both after cutting and at the end of the conveyor belt to the washer. Whereas the immediate postblanch count was about log 3.48, the product had total counts of about log 5.94/g after packaging. Between 40% and 75% of the bacterial biota of peas, snap beans, and corn was shown to consist of leuconostocs and "streptococci," whereas many of the gram-positive, catalase-positive rods resembled corynebacteria. Lactic acid cocci have been associated with many raw and processed vegetables. These cocci have been shown to constitute from 41 % to 75% of the aerobic plate count (APC) biota of frozen peas, snap beans, and corn. It has been shown that fresh peas, green beans, and corn all contained coagulase-positive staphylococci after processing. Peas were found to have the highest count (log 0.86/g), whereas 64% of corn samples contained this organism. These authors found that a general buildup of staphylococci occurred as the vegetables underwent successive stages of processing, with the main source of organisms coming from the hands of employees. Although staphylococci may be found on vegetables during processing, they are generally unable to proliferate in the presence of the more normal lactic biota. Both coliforms (but not Escherichia coli) and enterococci have been found at most stages during raw vegetable

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processing, but they appear to present no public health hazard. In a study of the incidence of Clostridium botulinum in 100 commercially available frozen vacuum pouch-pack vegetables, the organism was not found in 50 samples of string beans, but types A and B spores were found in 6 of 50 samples of spinach. In a study of 575 packages of frozen vegetables processed by 24 factories in 12 states, Splittstoesser and Corlett found that peas yielded some of the lowest counts (mean of approximately log 1.93/ g), whereas chopped broccoli yielded the highest mean APCs-log 3.26/g. Using the three-class sampling plan of the International Commission on Microbiological Specifications for Foods (ICMSF), the acceptance rate for the 115 lots would have been 74% for the m specification of 10 5/g and 84% for M of 106 /g. In a study of 17 different frozen blanched vegetables, 63% were negative for fecal coliforms, and 33% of the 565 examined were acceptable when n = 5, c = 3, m = 10, and M = 103, and 70% were acceptable if n = 5, c = 3, m = 50, and M = 103 • In another study, the mean APC at 30°C for 1,556 frozen retail cauliflower samples was log 4.65/g; for 1,542 sample units of frozen corn, log 3.93/g; and for 1,564 units of frozen peas, log 3.83/g with 5/g or less of coliforms and <3/g of E. coli for all samples. Based on the APC, 97.2-99.6% of the latter foods were acceptable by ICMSF's sampling plan n = 5, c = 3, m = 105, and M = 106 • Microorganisms on fresh-cut or ready-touse vegetables are discussed further in the section below on freshcut produce. 3.6.1 Spoilage The average water content of vegetables is about 88%, with an average content of 8.6% carbohydrates, 1.9% proteins, 0.3% fat, and 0.84% ash. The total percentage composition of vitamins, nucleic acids, and other plant constituents is generally less than 1%. From the standpoint of nutrient content, vegetables are capable of supporting the growth of molds, yeasts, and bacteria and, consequently, of being spoiled by any or all of these organisms. The higher water content of vegetables favors the growth of spoilage bacteria, and the relatively low carbohydrate and fat contents suggest that much of this water is in available form. The pH range of most vegetables is within the growth range of a large number of bacteria, and it is not surprising, therefore, that bacteria are common agents of vegetable spoilage. The relatively high oxidation-reduction (O/R) potential of vegetables and their lack of high poising capacity suggest

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that the aerobic and facultative anaerobic types would be more important than the anaerobes. This is precisely the case; some of the most ubiquitous etiologic agents in the bacterial spoilage of vegetables are species of the genus Erwinia and are associated with plants and vegetables in their natural growth environment. The common spoilage pattern displayed by these organisms is referred to as bacterial soft rot.

3.6.1.1 Bacterial agents 3.6.1.1.1 Bacterial soft rot This type of spoilage is caused by Erwinia carotovora and pseudomonads such as Pseudomonas marginalis, with the former being the more important. Bacillus and Clostridium spp. have been implicated, but their roles are probably secondary. The causative organisms break down pectins, giving rise to a soft, mushy consistency, sometimes a bad odor, and water-soaked appearance. Some of the vegetables affected by this disease are asparagus, onions, garlic, beans (green, lima, and wax), carrots, parsnips, celery, parsley, beets, endives, globe artichokes, lettuce, rhubarb, spinach, potatoes, cabbage, Brussels sprouts, cauliflower, broccoli, radishes, rutabagas, turnips, tomatoes, cucumbers, cantaloupes, peppers, and watermelons. Although the precise manner in which Erwinia spp. bring about soft rot is not yet well understood, it is very likely that these organisms, present on the susceptible vegetables at the time of harvest, subsist on vegetable sap until the supply is exhausted. Plant roots are protected from invading microorganisms by their possession of hydrogen peroxide and superoxide, and invading microorganisms produce catalase and superoxide dismutase to overcome this defense. The Pseudomonas syringae group as well as erwiniae produce these enzymes. / The cementing substance of the vegetable body induces the formation of pectinases, which act by hydrolyzing pectin, thereby producing the mushy consistency. In potatoes, tissue maceration has been shown to be caused by an endopolygalacturonate transeliminase of Erwinia origin. Because of the early and relatively rapid growth of these organisms, molds, which tend to be crowded out, are of less consequence in the spoilage of vegetables that are susceptible to bacterial agents. Once the outer plant barrier has been destroyed by these pectinase producers, nonpectinase producers

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no doubt enter the plant tissues and help bring about fermentation of the simple carbohydrates that are present. The quantities of simple nitrogenous compounds present, the vitamins (especially the Bcomplex group), and minerals are adequate to sustain the growth of the invading organisms until the vegetables have been essentially consumed or destroyed. The malodors that are produced are probably the direct result of volatile compounds (such as NH 3, volatile acids, and the like) produced by the biota. When growing in acid media, microorganisms tend to decarboxylate amino acids, leaving amines that cause an elevation of pH toward the neutral range and beyond. Complex carbohydrates such as cellulose are generally the last to be degraded, and a varied biota consisting of molds and other soil organisms is usually responsible, as cellulose degradation by Erwinia spp. is doubtful. Arom~tic constituents and porphyrins are probably not attacked until late in the spoilage process, and again by a varied flora of soil types. The genes of E. carotovora subsp. carotovora that are involved in potato tuber maceration have been cloned. Plasmids containing cloned DNA mediated the production of endopectate lyases, exopectate lyase, endopolygalacturonase, and cellulases. The Escherichia coli strains that contained cloned plas'mids showed that endopectate lyases with endopolygalacturonase or exopectate lyase caused maceration of potato tuber slices. These enzymes, along with phosphatidase C and phospholipase A, are involved in soft rot by this organism. Carrots infected with Agrobacterium tumefaciens undergo senescence at a faster rate because of increased ethylene synthesis. In normal un infected plants, ethylene synthesis is regulated by auxins, but A. tumefaciens increases the synthesis of indoleacetic acid, which results in increased levels of ethylene. The genus Envinia belongs to the family Enterobacteriaceae. Its species are associated with plants where they are known to cause plant diseases of the rot and wilt types. These are gram- negative rods that are related to the genera Proteus, Serratia. Escherichia, Salmonella, and others. Erwil1ia spp. normally do not require organic nitrogen compounds for growth, and the relatively low levels of proteins in vegetables make them suitable for the task of destroying plant materials of this type. The pectinase produced by these organisms is actually a protopectinase, because the cementing substance of plants as it actually exists in the plant is protopectin. Many Erwinia spp. such as E. carotovora are capable of fermenting many of the sugars and alcohols that exist in certain vegetables

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such as rhamnose, cellobiose, arabinose, mannitol, and so forthcompounds that are not utilized by many of the more common bacteria. Although most Erwinia spp. grow well at about 3rC, most are also capable of good growth at refrigerator temperatures, with some strains reported to grow at lOC. 3.6.1.1.2 Other bacterial spoilage conditions E. carotovora pv. atroseptica. E. carotovora pv. carotovora. and E. chrysanthemi cause a rot of potatoes sometimes referred to as "black leg." In temperate regions, E. carotovora pv. atroseptica is usually involved with E. chrysanthemi to a lesser degree. Although direct contact with soil may be the source of normal forms of these organisms, L-phase variants may enter healthy tissues and later revert to classical forms. The genus Xanthomonas is undergoing reclassification, thus the species and pathovars are likely to be changed. A recent study revealed 20 DNA homology groups, each of which is considered a genomic species. X. campestris has been emended to include only pathovars from crucifers (cabbage, mustard, etc.), and X. axonopodis now includes 34 former X. campestris pathovars. Although the taxonomic status of the genus is in flux, it consists of some very important plant pathogens and spoilage organisms. Most form yellow mucoid and smooth colonies and produce the yellow-pigmented xanthomonadins. The mucoid colonies are due to xanthans, which are typical of the genus. Bacterial canker of stone fruits is caused by P. syringae pv. syringae, and this pathovar has been reported to cause disease in over 180 species of plants. '

3.6.1.2 Fungal agents Some of these spoilage conditions are initiated preharvest and others postharvest. Among the former, Botrytis invades the flower of strawberries to cause gray mold rot, Colletotrichum invades the epidermis of bananas to initiate banana anthracnose, and Gloeosporium invades the lenticels of apples to initiate lenticel rot. The largest number of market fruit and vegetable spoilage conditions occur after harvesting, and although the fungi most often invade bruised and damaged products, some enter specific areas. For example, Thielaviopsis invades the fruit stem of pineapples to cause black rot of this fruit, and Colletotrichum invades the crown cushion of bananas to cause banana crown rot. Black rot of sweet potatoes is caused by Ceratocystis, neck rot of onions by Botrytis aliii, and downey mildew of lettuce by Bremia spp.

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3.6.1.2.1 Gray mold rot This condition is caused by Botrytis cinerea, which produces a gray mycelium. This type of spoilage is favored by high humidity and warm temperatures. Among the vegetables affected are asparagus, onions, garlic, beans (green, lima, and wax), carrots, parsnips, celery, tomatoes, endives, globe artichokes, lettuce, rhubarb, cabbage, Brussels sprouts, cauliflower, broccoli, radishes, rutabagas, turnips, cucumbers, pumpkin, squash, peppers, and sweet potatoes. In this disease, the causal fungus grows on decayed areas in the form of a prominent gray mold. It can enter fruits and vegetables through the unbroken skin or through cuts and cracks. 3.6.1.2.2 Sour rot This condition of vegetables is caused by Geotrichum candidum and other organisms. Among the vegetables affected are asparagus, onions, garlic, beans (green, lima, and wax), carrots, parsnips, parsley, endives, globe artichokes, lettuce, cabbage, Brussels sprouts, cauliflower, broccoli, radishes, rutabagas, turnips, and tomatoes. The causal fungus is widely distributed in soils and on decaying fruits and vegetables. Drosophila melanogaster (fruit fly) carries spores and mycelial fragments on its body from decaying fruits and vegetables to growth cracks and wounds in healthy fruits and vegetables. Because the fungus cannot enter through the unbroken skin, infections usually start in openings of one type or another. 3.6.1.2.3 Rhizopus soft rot This condition is caused by Rhizopus stolonifer and other species that make vegetables soft and mushy. Cottony growth of the mold with small black dots of sporangia often covers the vegetables. Among those affected are beans (green, lima, and wax), carrots, sweet potatoes, potatoes, cabbage, Brussels sprouts, cauliflower, broccoli, radishes, rutabagas, turnips, cucumbers, cantaloupes, pumpkins, squash, watermelons, and tomatoes. This fungus is spread by D. melanogaster, which lays its eggs in the growth cracks on various fruits and vegetables. The fungus is widespread and is disseminated by other means also. Entry usually occurs through wounds and other skin breaks. 3.6.1.2.4 Phytophora rot This market condition, caused by Phytophora spp., occurs largely in the field as a blight and fruit rot of market vegetables. It appears to be more variable than some other market "diseases" and

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affects different plants in different ways. Among the vegetables affected are asparagus, onions, garlic, cantaloupes, watermelons, tomatoes, eggplants, and peppers. 3.6.1.2.5 Anthracnose This plant disease is characterized by spotting of leaves, fruit, or seed pods. It is caused by Colletotrichum coccodes and other species. These fungi are considered weak plant pathogens. They live from season to season on plant debris in the soil and on the seed of various plants such as the tomato. Their spread is favored by warm, wet weather. Among the vegetables affected are beans, cucumbers, watermelons, pumpkins, squash, tomatoes, and peppers.

3.7 SPOILAGE OF FRUITS The general composition of 18 common fruits shows that the average water content is about 85% and the average carbohydrate content is about 13%. The fruits differ from vegetables in having somewhat less water but more· carbohydrate. The mean protein, fat, and ash content of fruits are, respectively, 0.9%, 0.5%, and 0.5%-somewhat lower than vegetables except for ash content. Fruits contain vitamins and other organic compounds, just as vegetables do. On the basis of nutrient content, these products would appear to be capable of supporting the growth of bacteria, yeasts, and molds. However, the pH of fruits is below the level that generally favors bacterial growth. This one fact alone would seem to be sufficient to explain the general absence of bacteria in the incipient spoilage of fruits. The wider pH growth range of molds and yeasts suits them as spoilage agents of fruits. With the exception of pears, which sometimes undergo Erwinia rot, bacteria are of no known importance in the initiation of fruit spoilage. Just why pears with a reported pH range of 3.8 to 4.6 should undergo bacterial spoilage is not clear. It is conceivable that Erwinia initiates its growth on the surface of this fruit where the pH is presumably higher than on the inside. A variety of yeast genera can usually be found on fruits, and these organisms often bring about the spoilage of fruit products, especially in the field. Many yeasts are capable of attacking the sugars found in fruits and bringing about fermentation with the production of alcohol and carbon dioxide. Due to their generally faster growth rate than molds, they often precede the latter organisms in the spoilage process of fruits in certain circumstances. It is not clear whether some molds are dependent on the initial action of

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yeasts in the process of fruit and vegetable spoilage. The utilization or destruction of the high-molecular-weight constituents of fruits is brought about more by molds than yeasts. Many molds are capable of utilizing alcohols as sources of energy, and when these and other simple compounds have been depleted, these organisms proceed to destroy the remaining parts of fruits, such as the structural polysaccharides and rinds.

3.8 FRESH-CUT PRODUCE The production of precut packaged fruit and vegetable salads (minimally processed) has led to an explosion in the sale and consumption of these commodities during the past decade, and this trend shows signs of continuing. In essence, salad vegetables such as lettuce and carrots, and fruits such as cantaloupes and watermelons are cut, sliced, and packaged in see-through containers that are stored at chill temperatures such that they are ready to use (RTU) upon purchase. If packaged in high-oxygen permeable films, the primary concerns are product quality and enzymatic browning in the case of light-colored products. However, when low-02 permeable packaging is used with long-term storage, the possibility exists for the growth of microbial pathogens such as C. botulinum and L. monocytogenes. 3.8.1 Microbial Load Overall, RTU produce is by no means a microbe-free product. In its preparation, intact vegetables are washed, typically with water that contains chlorine from 50 to 200 ppm, followed by cutting and packaging. While washing reduces microbial numbers, the cutting operation has the potential to recontaminate. Also, the fresh- cut vegetables provide a higher level of moisture, more simple nutrients, and a higher surface area, all of which make the RTU product more susceptible to microbial growth than the original. The APCs of eight RTU vegetables in Ontario, Canada, recorded on day 0 and day 4 after storage at 4°C. It can be seen that the initial numbers ranged from 4.82 10g l ig to near 6.0 10glO/g on day 0, but after a 4-day storage, they ranged from 5.45 to > 7.0 10g lO/g. In an earlier study, the APC of RTU vegetables at harvest was around 105 _10 8 / g, and after storage at 7°C, the APC at time of sell-by date + 1 day for 12 vegetables ranged between 7.7 and 9.0 log I/g, a time when all products were organoleptically acceptable. In the latter study, coliforms ranged from 5.1 to 7.2 logl/g, but

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no type 1 E. coli strains were found i'he most predominant organisms were Pseudomonas and Pantoea. In a study of the types of organisms on RTU spinach that was stGfcd at 10°C for 12 days, mesophiles ranged between 10 7 and 10 to /g, psychrotrophs and pseudo monads between 106 and 10 to /g, and enteric bacteria between 104 and leg. 3.8.2 Pathogens The pathogen of greatest concern in RTU vegetables is C. botulinum and reasons for this concern are pointed up by several recent studies. In one, five RTU vegetables (butternut squash, mixed salad, rutabagas, romaine lettuce, and a stir-fry mix) were inoculated with a 10-strain cocktail-S each of proteolytic and nonproteolytic spores. The products were sealed in polystyrene trays with an oxygen transmission rate (OTR) of 2,100 mL and incubated at 5, 10, or 25°C. All 5 vegetables became toxic at some point during their storage. The time to toxin detection for non proteolytic strains in butternut squash was 7 days at 10°C with CO 2 at 27.8%; and for proteolytics in this product, 3 days at 25°C with 64.7% CO 2 , In butternut squash at 5°C with an inoculum of nonproteolytic strains of 10'/ g, toxin was detectable in 21 days. At the time of toxin detection in all samples, O 2 was < 1%. Although the packaging material was by no means of "zero" barrier quality, respiration of the products decreased 02 and increased CO 2 to the levels ~oted. It was the opinion of these investigators that the temperature of storage of RTU vegetables of the type noted is of critical importance to their safety. Most products were in states of detectable spoilage at the time of toxin detection. Another study employed cabbage and lettuce inoculated with about 102 spores/g of a 10-strain cocktail as above and packaged in film with an OTR of either 3,000 low OTR (LOTR) or 7,000 high OTR (HOTR) and stored at 4, 13, or 21°C for 21 or 28 days. Toxin was not detected under any conditions, and both vegetables were organoleptically spoiled before toxin could be produced. In the cabbage stored at 21°C for 10 days, the LOTR contained 69.4% and the HOTR 41.9% CO?, while in lettuce at 2l°C after 8 days, CO, was 41.9% and 9.0% i11 LOTR and HOTR. respectively. In contrast to the study by Austin et al. where 02 was < 100, both packaging materials allowed 0, ranging from 1.0% to 7.9%. In the fonner study, the packaging rl1aterial had an OTR of 2,100, while in the latter OTRs were 3,000 and 7,000. The more

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permeable film may have allowed the growth of more organisms that interfered with C. botulinum, A 10-strain cocktail of 7 proteolytics and 3 nonproteolytics was used in the study by Larson et al. in which five vegetables (broccoli, cabbage, carrots, lettuce, and green beans) were inoculated. Botulinal toxin was found in all grossly spoiled broccoli stored at 21°e, in one half of those grossly spoiled at 12°C, and in one third of the grossly spoiled lettuce stored at 21°C. No toxin was detected prior to spoilage, and no toxin was found in the other three vegetables. In contrast to the two studies noted above, the packaging material used in this study had varying OTRs ranging from 3,000 to 16,544, and the vegetables were sealed under vacuum with vacuum pulled. Interestingly, broccoli was packaged in material with OTRs of 13,013 to 16,544 while cabbage (which did not become toxic) was packaged in 3,000 to 8,000 OTR materials. The broccoli packs stored at 21°C for 7 days contained <2% 02 and about 12% CO 2 , while lettuce at 21°C for 6 days contained up to 40% eO r The APe of spoiled products was in the 108 to > 109 range. In a fourth study, romaine lettuce and shredded cabbage were each inoculated with a nine- strain cocktail of proteolytic and non proteolytic spores at a level of about 100 spores per gram, and the samples were stored in vented and nonvented plastic bags. The latter were vacuum packed but vacuum was not pulled. After 7 days at 21°e, the cabbage packaged in nonvented bags became toxic, but not when stored at 4.4 or 12.re for up to 28 days. Romaine lettuce became toxic after 14 days at 21°e in nonvented packs and in 21 days in vented packs. The toxic samples were organoleptically spoiled prior to toxin detection. A potential health hazard for RTU vegetables is pointed out by the above studies relative to botulinal toxin. However, these studies as well as others point to the importance of storage temperature in controlling not only this pathogen but others, including those below. Temperature and time of storage of RTU products are obviously critical to their safety. L. monocytogel1es has been demonstrated to grow on refrigerated vegetables, including lettuce, broccoli, cauliflower, and asparagus. Although it grew on raw tomatoes at 21°C, it did not at 10°C. Not only did this organism not grow on raw carrots, the numbers were actually reduced, with as little as 1% added to a broth base being effective. The antilisterial effect was destroyed when

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carrots were cooked. A study on the survival of Shigella sonnei in shredded cabbage revealed that numbers of this organism remained essentially unchanged for 1 to 3 days under three conditions of packaging-aerobic, vacuum, and in 30% N2 + 70% CO 2 • After 3 days, however, numbers decreased concomitant with decreasing pH. Thus, the organism could survive under refrigerator or room temperature conditions, but it did not grow. In an effort to control pathogens on raw fruits and vegetables, a spray containing 2,000 ppm of chlorine was shown capable of effecting a 2.3 loglo reduction following contact for 1 to 10 minutes. The products consisted of apples, tomatoes, and lettuce, and the pathogens studied were salmonellae, E. coli 0157:H7, and L. mOl1ocytogenes. To study the protective effect of a lactic organism, Lactobacillus casei and its culture permeate were tested on RTU salad vegetables at SoC. After 6 days of storage, 3% culture permeate reduced APC from 6 to 1 loglo cfu/g and suppressed coliforms, enterococci, and Aeromol1as hydrophila. Coliforms were reduced by about 2 logs and fecal coliforms by about 1 log by the use of 1% lactic acid. In a study of the effect of sodium hypochlorite on psychrotrophic organisms on minimally processed potato strips, the products that were treated with 100 or 300 ppm hypochlorite had higher numbers than controls after incubation at 2°C for 20 days in a modified atmosphere.

3.9 FERMENTED PRODUCTS 3.9.1 Breads San Francisco sourdough bread is similar to sourdough breads produced in various countries. Historically, the starter for sourdough breads consists of the natural biota of baker's barm (sour ferment or mother sponge, with a portion of each inoculated dough saved as starter for the next batch). The barm generally contains a mixture of yeasts and lactic acid bacteria. In the case of San Francisco sourdough bread, the yeast has been identified as Saccharomyces exiguus (Candida holmii) and the responsible bacteria are Lactobacillus sanfrallcisco, L. fermentu111, L. fructivorans, some L. brevis strains, and the recently named L. pOl1tis. The key bacterium is L. sanfrancisco, and it preferentially ferments maltose rather than glucose and it requires fresh yeast extractives and unsaturated fatty acids. The souring is caused by acids produced by

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these bacteria, and the yeast is responsible for the leavening action, although some CO 2 is produced by the bacterial biota. The pH of these sourdoughs ranges from 3.8 to 4.5. Both acetic and lactic acids are produced, with the former accounting for 20-30% of the total acidity. IdIi is a fermented bread-type product common in southern India. It is made from rice and black gram mungo (urd beans). These two ingredients are soaked in water separately for 3-10 hou:-s and then ground in varying proportions, mixed, and allowed to ferment overnight. The fermented and raised product is cooked by steaming and served hot. It is said to resemble a steamed, sourdough bread. During the fermentation, the initial pH of around 6.0 falls to values of 4.3-5.3. In a particular study, a batter pH of 4.70 after a 20-hour fermentation was associated with 2.5% lactic acid, based on dry grain weight. In their studies of idli, Steinkraus et al. found total bacterial counts of 108 _109 jg after 20-22 hours of fermentation. Most of the organisms consisted of gram-positive cocci or short rods, with L. mesenteroides being the single most abundant species, followed by E. [aecalis. The leavening action of idli is produced by L. mesenteroides. This is the only known instance of a lactic acid bacterium having this role in a naturally fermented bread. The latter authors confirmed the work of others in finding the urd beans to be a more important source of lactic acid bacteria than rice. L. mesenteroides reaches its peak at around 24 hours, with E. [aecalis becoming active only after about 20 hours. Other probable fermenters include L. delbrueckii, L. fermentum, and Bacillus spp. Only after idli has fermented for more than 30 hours does P. cerevisiae become active. The product is not fermented generally beyond 24 hours because maximum leavening action occurs at this time and decreases with longer incubations. When idli is allowed to ferment longer, more acidity is produced. It has been found that total acidity (expressed as grams of lactic acid per gram of dry grains) increased from 2.71 % after 24 hours to 3.70% after 71 hours, whereas the pH decreased from 4.55 to 4.10 over the same period.

3.9.2 Olives, Pickles, and Sauerkraut

3.9.2.1 Olives Olives to be fermented (Spanish, Greek, or Sicilian) are done so by the natural biota of green olives, which consists of a variety of bacteria, yeasts, and molds. The olive fermentation is quite similar

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to that of sauerkraut except that it is slower, involves a lye treatment, and may require the addition of starters. The lactic acid bacteria become prominent during the intermediate stage of fermentation. L. mesenteroides and P. cerevisiae are the first lac tics to become prominent, and these are followed by lactobacilli, with L. plantarum and L. brevis being the most important. The olive fermentation is preceded by a treatment of green olives with from 1.6 to 2.0% lye, depending on type of olive, at 21-2S0C for 4- 7 hours for the purpose of removing some of the bitter principal. Following the complete removal of lye by soaking and washing, the green olives are placed in oak barrels and brined so as to maintain a constant 28°-30° salinometer level. Inoculation with L. plantarum may be necessary because of destruction of organisms during the lye treatment. The fermentation may take as long as 6-10 months, and the final product has a pH of 3.8-4.0 following up to a 1% lactic acid production. Among the types of microbial spoilage that olives undergo, one of the most characteristic is zapatera spoilage. This condition, which sometimes occurs in brined olives, is characterized by a malodorous fermentation. The odor is due apparently to propionic acid, which is produced by certain species of Propionibacterium. A softening condition of Spanish-type green olives has been found to be caused by the yeasts Rhodotorula glutinis var. glutirzis. R. minuta var. minuta. and R. rubra. All of these organisms produce polygalacturonases, which effect olive tissue softening. Under appropriate cultural conditions, the organisms were shown to produce pectin methyl esterase, as well as polygalacturonase. A sloughing type of spoilage of California ripe olives was shown by Patel and Vaughn to be caused by Cellulomonas j1avigel1a. This organism showed high celluloytic activity, which was enhanced by the growth of other organisms such as Xanthomonas, El1terobacter. and Escherichia spp. 3.9.2.2 Pickles Pickles are fermentation products of fresh cucumbers, and as is the case of sauerkraut production, the starter culture normally consists of the normal mixed biota of cucumbers. In the natural production of pickles, the following lactic acid bacteria are involved in the process in order of increasing prevalence: L. mesel1teroides. E. [aecalis, P. cerevisiae, L. brevis. and L. plantarum. Of these the pediococci and L. plal1tarwn are the most involved. with L. brevis

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being undesirable because of its capacity to produce gas. L. plantarum is the most essential species in pickle production, as it is for sauerkraut. In the production of pickles, selected cucumbers are placed in wooden brine tanks with initial brine strengths as low as 5% NaCI (20 salinometer). Brine strength is increased gradually during the course of the 6- to 9-week fermentation, until it reaches around 60 salinometer (15.9% NaCI). In addition to exerting an inhibitory effect on the undesirable gram-negative bacteria, the salt extracts water and water-soluble constituents from the cucumbers, such as sugars, which are converted by the lactic acid bacteria to lactic acid. The product that results is a salt-stock pickle from which pickles such as sour, mixed sour, chowchow, and so forth may be made. The general technique of producing brine- cured pickles briefly outlined has been in use for many years, but it often leads to serious economic loss because of pickle spoilage from such conditions as bloaters, softness, off-colors, and so on. The controlled fermentation 0

0

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","Sugar

54

5.0 7

o-Actd A-pH a·Sugar

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05 0.4

.-Cells 3.8

0.3 0.2

3.4

0.2

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2

4

6

8

10

12

14

3.0

FERMENTATION TIME-days

Figure 3.3 Controlled fermcntation of cucumbers brincd in bulk.

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of cucumbers brined in bulk has been achieved, and this process not only reduces economic losses of the type noted but leads to a more uniform product over a shorter period of time. The controlled fermentation method employs a chlorinated brine of 25° salinometer, acidification with acetic acid, the addition of sodium acetate, and inoculation with P cerevisiae andL. plan/arum. or the latter alone. With a final pH of -4.0, pickles undergo spoilage by bacteria and molds. Pickle blackening may be caused by Bacillus nigrijicans. which produces a dark water-soluble pigment. Enterobacter spp., lactobaciIli, and pediococci have been implicated as causes of a condition known as "bloaters," produced by gas formation within the individual pickles. Pickle softening is caused by pectolytic organisms of the genera Bacillus. Fusarium. Penicillium. Phoma, Cladosporium. Alternaria, Mucor. Aspergillus. and others. The actual softening of pickles may be caused by anyone or several of these or related organisms. Pickle softening results from the production of pectinases, which break down the cementlike substance in the wall of the product.

3.9.2.3 Sauerkraut Sauerkraut is a fermentation product of fresh cabbage. The starter for sauerkraut production is usually the normal mixed flora of cabbage. The addition of 2.25-2.5% salt restricts the activities of gram-negative bacteria, while the lactic acid rods and cocci are favored. Leuconostoc mesenteroides andL. plantarum are the two most desirable lactic acid bacteria in sauerkraut product, with the former having the shorter generation time and the shorter life span. The activities of the coccus usually cease when the acid content increases to 0.7-1.0%. The final stages of kraut production are effected by L. plal1tarum and L. brevis. P. cerevisiae and E. faecalis may also contribute to product development. The final total acidity is generally 1.6-1.8%, with lactic acid at 1.0-1.3% and pH in the range 3.1-3.7. The microbial spoilage of sauerkraut generally falls into the following categories: soft kraut, slimy kraut, rotted kraut, and pink kraut. Soft kraut results when bacteria that normally do not initiate growth until the late stages of kraut production actually grow earlier. Slimy kraut is caused by the rapid growth of Lactobacillus cucumeris and L. plan/arum. especially at elevated temperatures. Rotted sauerkraut may be caused by bacteria, molds, and/or yeasts, whereas pink kraut is caused by the surface growth of Torula spp., especially

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T. glutinis. Due to the high acidity, finished kraut is generally spoiled by molds growing on the surface. The growth of these organisms effects an increase in pH to levels where a large number of bacteria can grow that were previously inhibited by conditions of high acidity. 3.9.3 Beer, Ale, Wines, Cider, and Distilled Spirits Beer and Ale Beer and ale are malt beverages produced by brewing. An essential step in the brewing process is the fermentation of carbohydrates to ethanol. Because most of the carbohydrates in grains used for brewing exist as starches, and because the fermenting yeasts do not produce amylases to degrade the starch, a necessary part of beer brewing includes a step whereby malt or other exogenous sources of amylase are provided for the hydrolysis of starches to sugars. The malt is first prepared by allowing barley grains to germinate. This serves as a source of amylases (fungal amylases may be used also). Both 13- and a-amylases are involved, with the latter acting to liquefy starch and the former to increase sugar formation. In brief, the brewing process begins with the mixing of malt, malt adjuncts, hops, and water. Malt adjuncts include certain grains, grain products, sugars, and other carbohydrate products to serve as fermentable substances. Hops are added as sources of pyrogallol and catechol tannins, resins, essential oils, and other constituents for the purpose of rrecipitating unstable proteins during the boiling of wort and to provide for biological stability, bitterness, and aroma. The process by which the malt and malt adjuncts are dissolved and heated and the starches digested is called mashing. The soluble part of the mashed materials is called wort (compare with koji). In some breweries, lactobacilli are introduced into the mash to lower the pH of wort through iactic acid production. The species generally used for this purpose is L. delbrueckii. Wort and hops are mixed and boiled for 1.5-2.5 hours for the purpose of enzyme inactivation, extraction of soluble hop substances, precipitation of coagulable proteins, concentration, and sterilization. Following the boiling of wort and hops, the wort is separated, cooled, and fermented. The fermentation of the sugar-laden wort is carried out by the inoculation of S. cerevisiae. Ale results from the activities of top-fermenting yeasts, which depress the pH to around 3.8, whereas bottom-fermenting yeasts (S. "carlsbergensis" strains) give rise to lager and other beers with pH values of 4.1 -4.2. A top fermentation is complete in 5- 7 days; a bottom fermentation requires

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7 -12 days. The freshly fermented product is aged and finished by the addition of CO 2 to a final content of 0.45-0.52% before it is ready for commerce. The pasteurization of beer at 140°F (60°C) or higher, may be carried out for the purpose of destroying spoilage organisms. When lactic acid bacteria are present in beers, the lactobacilli are found more commonly in top fermentations, whereas pediococci are found in bottom fermentations. The industrial spoilage of beers and ales is commonly referred to as beer infections. This condition is caused by yeasts and bacteria. The spoilage patterns of beers and ales may be classified into four groups: ropiness, sarcinae sickness, sourness, and turbidity. Ropiness is a condition in which the liquid becomes characteristically viscous and pours as an "oily" stream. It is caused by Acetobacter, Lactobacillus, Pediococcus cerevisiae, and Gluconobacter oxydans (formerly Acetomonas). Sarcinae sickness is caused by P cerevisiae, which produces a honey like odor. This characteristic odor is the result of diacetyl production by the spoilage organism in combination with the normal odor of beer. Sourness in beers is caused by Acetobacter spp. These organisms are capable of oxidizing ethanol to acetic acid, and the sourness that results is referable to increased levels of acetic acid. Turbidity and off-odors in beers are caused by Zymomonas anaerobia (formerly Achromobacter anaerobium) and several yeasts such as Saccharomyces spp. The growth of bacteria is possible in beers because of a normal pH range of 4-5 and a good content of utilizable nutrients. Some gram-negative obligately anaerobic bacteria have been isolated from spoiled beers and pitching yeasts, and the six species are represented by four genera:

Megasphaera cerevisiae Selenomonas lacticifex Pectinatus cerevisiiphilus Zymophilus paucivorans P. jrisingensis Z. raffinosivorans All but M cerevisiae produce acetic and propionic acids, and S. lacticifex also produces lactate. Although M. cerevisiae produces negligible to minor amounts of acetic and propionic acids, it produces large quantities of isovaleric acid in addition to H,S. P cerevisiiphilus was the first of these to be associated with spoiled beer when it was isolated from turbid and off- flavor beer in 1978. It has since been found in breweries not only in the United States but in several European countries and Japan. Among the unusual features of these organisms as beer spoilers is their Gram reaction and obligately

140

MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

anaerobic status. In the past the typical beer spoilers have been regarded as being either lactic acid bacteria or yeasts. Megasphaera and Selenomonas are best known as members of the rumen biota. In addition to the organic acids noted above, Pectil1atus spp. also produce H 2 S and acetoin. The beers most susceptible to their growth are those that contain <4.4% alcohol. With respect to spoiled packaged beer, one of the major contaminants found is Saccharomyces diastaticus. which is able to utilize dextrins that normal brewers' yeasts (S. "carlsbergel1sis" and S. cerevisiae) cannot." Pediococci, Flavobacterium proteus (formerly Obesumbacterium), and Brettal10myces are sometimes found in spoiled beer. 3.9.3.1 Wines

Wines are normal alcoholic fermentations of sound grapes followed by aging. A large number of other fruits such as peaches, pears, and so forth may be fermented for wines, but in these instances the wine is named by the fruit, such as peach wine, pear wine, and the like. Because fruits already contain fermentable sugars, the use of exogenous sources of amylases is not necessary, as it is when grains are used for beers or whiskeys. Wine making begins with the selection of suitable grapes, which are crushed and then treated with a sulfite such as potassium metabisulfite to retard the growth of acetic acid bacteria, wild yeasts, and molds. The pressed juice, called must, is inoculated with a suitable wine strain of S. "ellipsoideus." The fermentation is allowed to continue for 3 -5 days at temperatures between 70°F and gO°F (21°C and 32°C), and good yeast strains may produce up to 14-18% ethanol. Following fermentation, the wine is racked-that is, drawn off from the lees or sediment, which contains potassium bitartrate (cream of tartar). The clearing and development of flavor occur during the storage and aging process. Red wines are made by initially fermenting the crushed grape must "on the skins" during which pigment is extracted into the juice; white wines are prepared generally from the juice of white grapes. Champagne, a sparkling wine made by a secondary fermentation of wine, is produced by adding sugar, citric acid, and a champagne yeast starter to bottles of a previously prepared, selected table wine. The bottles are corked, clamped, and stored horizontally at suitable temperatures for about 6 months. They are then removed, agitated, and aged for an additional period of up to 4 years. The final sedimentation of yeast cells and tartrates is accelerated by

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reducing the temperature of the wine to around 25°C and holding for 1-2 weeks. Clarification of the champagne is brought about by working the sediment down the bottle onto the cork over a period of 2-6 weeks by frequent rotation of the bottle. Finally, the sediment is frozen and disgorged upon removal of the cork. Table wines undergo spoilage by bacteria and yeasts, Candida valida being the most important yeast. Growth of this organism occurs at the surface of wines, where a thin film is formed. The organisms attack alcohol and other constituents from this layer and create an appearance that is sometimes referred to as wine flowers. Among the bacteria that cause wine spoilage are members of the genus Acetobacter. which oxidize alcohol to acetic acid (produce vinegar). The most serious and the most common disease of table wines is referred to as tourne disease. Tourne disease is caused by a facultative anaerobe or an anaerobe that utilizes sugars and seems to prefer conditions of low alcohol content. This type of spoilage is characterized by an increased volatile acidity, a silky type of cloudiness, and later in the course of spoilage, a "mousy" odor and taste. Malo-lactic fermentation is a spoilage condition of great importance in wines. Malic and tartaric acids are two of tbe predominant organic acids in grape must and wine, and in the malolactic fermentation, contaminating bacteria degrade malic acid to lactic acid and CO 2: L( -) - Malic acid

"malo-lactic enzyme"

)

L( +) - Lactic acid + CO 2

L-Malic acid may be decarboxylated also to yield pyruvic acid. The effect of these conversions is to reduce the acid content and affect flavor. The malo-lactic fermentation (which may also occur in cider) can be carried out by many lactic acid bacteria, including 1puconostocs, pediococci, and lactobacilli. Although the function of the malolactic fermentation to the fermenting organism is not well understood, it has been shown that 0. oeni is actually stimulated by the process. The decomposition in wines of tartaric acid is undesirable also, and this process can be achieved by some strains of Lactobacillus plantarum in the following general manner: Tartaric acid ~ Lactic acid + Acetic acid + CO 2 The effect is to reduce the acidity of wine. Unlike the malo-lactic fermentation, few lactic acid bacteria break down tartaric acid. The bacterium Oel1ococcus oeni is an acidophile that can grow in grape must and wine at pH 3.5-3.8, and actually prefers an

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MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

initial growth pH of 4.8. It can grow in the presence of 10% ethanol but requires special growth factors found in grape or tomato juice.

3.9.4 Cider Cider, in the United States, is a product that represents a mild fermentation of apple juice by naturally occurring yeasts. In making apple cider, the fruits are selected, washed, and ground into a pulp. The pulp "cheeses" are pressed to release the juice. The juice is strained and placed in a storage tank, where sedimentation of particulate matter occurs, usually for 12-36 hours or several days if the temperature is kept at 40°F or below. The clarified juice is cider. If pasteurization is desired, this is accomplished by heating at 170°F for 10 minutes. The chemical preservative most often used is sodium sorbate at a level of 0.10%. Preservation may be effected also by chilling or freezing. The finished product contains small amounts of ethanol in addition to acetaldehyde. The holding of nonpasteurized or unpreserved cider at suitable temperatures invariably leads to the development of cider vinegar, which indicates the presence of acetic acid bacteria in these products. In their study of the ecology of the acetic acid bacteria in cider manufacture, Passmore and Carr found six species of Acetobacter and noted that those that display a preference for sugars tend to be found early in the cider process, whereas those that are more acid tolerant and capable of oxidizing alcohols appear after the yeasts have converted most of the sugars to ethanol. Zymomonas spp., gram-negative bacteria that ferment glucose to ethanol, have been isolated from ciders, but they are presumed to be present in low numbers. A recently discovered bacterium, Saccharobacter ferl71el1tatus. is similar to Zymomonas in that it ferments glucose to ethanol and COl' It was isolated from agave leaf juice, but its presence and possible role in spoiled ciders have yet to be determined. Other ethanol-producing bacteria are found in the genus Zymobacter.

3.9.4.1 Distilled spirits Distilled spirits are alcoholic products that result from the distillation of yeast fermentations of grain, grain products, molasses, or fruit or fruit products. Whiskeys, gin, vodka, rum, cordials, and liqueurs are examples of distilled spirits. Although the process for producing most products of these types is quite similar to that for beers. the content of alcohol in the final products is considerably higher than for beers. Rye and bourbon are examples of whiskeys.

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In the former, rye and rye malt, or rye and barley malt, are used in different ratios, but at least 51 % rye is required by law. Bourbon is made from corn, barley malt, or wheat malt, and usually another grain in different proportions, but at least 51 % corn is required by law. A sour wort is maintained to keep down undesirable organisms, the souring occurring naturally or by the addition of acid. The mash is generally soured by inoculating with a homolactic such as L. delbrueckii. which is capable of lowering the pH to around 3.S in 6-10 hours. The malt enzymes (diastases) convert the starches of the cooked grains to dextrins and sugars, and upon completion of diastatic action and lactic acid production, the mash is heated to destroy all microorganisms. It is then cooled to 75-S0°F (24-2JOC) and pitched (inoculated) with a suitable strain of S. cerevisiae for the production of ethanol. Upon completion of fermentation, the liquid is distilled to recover the alcohol and other volatiles, and these are handled and stored under special conditions relative to the type of product being made. Scotch whiskey is made primarily from barley and is produced from barley malt dried in kilns over peat fires. Rum is produced from the distillate of fermented sugar cane or molasses. Brandy is a product prepared by distilling grape or other fruit wines. Palm wine or Nigerian palm wine is an alcoholic beverage consumed throughout the tropics and is produced by a natural fermentation of palm sap. The sap is sweet and dirty brown in color, and it contains 10-12% sugar, mainly sucrose. The fermentation process results in the sap's becoming milky-white in appearance due to the presence of large numbers of fermenting bacteria and yeasts. This product is unique in that the microorganisms are alive when the wine is consumed. The fermentation has been reviewed and studied by Faparusi and Bassir and Okafor, who found the following genera of bacteria to be the most predominant in finished products: Micrococcus. Leuconostoc, "Streptococcus." Lactobacillus. andAcetobacter. The predominant yeasts found were Saccharomyces and Candida spp., with the former being the more common. The fermentation occurs over a 36- to 48-hour period, during which the pH of sap falls from 7.0 or 7.2 to less than 4.5. Fermentation products consist of organic acids in addition to ethanol. During the early phases of fermentation, Serratia and Ellterubacter spp. increase in numbers, followed by lactobacilli and leuconostocs. After a 48-hour fermentation, Acetobacter spp. begin to appear.

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MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

Sake is an alcoholic beverage commonly produced in Japan. The substrate is the starch from steamed rice, and its hydrolysis to sugars is carried out by A. oryzae to yield the koji. Fermentation is carried out by Saccharomyces sake over periods of 30-40 days, resulting in a product containing 12-15% alcohol and around 0.3% lactic acid. The latter is produced by hetero- and homolactic lactobacilli. 3.10 MISCELLANEOUS FERMENTED PRODUCTS Coffee beans, which develop as berries or cherries in their natural state, have an outer pulpy and mucilaginous envelope that must be removed before the beans can be dried and roasted. The wet method of removal of this layer seems to produce the most desirable product, and it consists of depulping and demucilaging followed by drying. Whereas depulping is done mechanically, demucilaging is accomplished by natural fermentation. The mucilage layer is composed largely of pectic substances, and pectinolytic microorganisms are important in their removal. Envinia dissolvens has been found to be the most important bacterium during the demucilaging fermentation in Hawaiian and Congo coffee cherries, although Pederson and Breed indicated that the fermentation of coffee berries from Mexico and Colombia was carried out by typical lactic acid bacteria (Ieuconostocs and lactobacilli). Agate and Bhat in their study of coffee cherries from the Mysore State of India found that the following pectinolytic yeasts predominated and played important roles in the loosening and removal of the mucilaginous layers: Saccharomyces marxianus, S. bayanus. S. "ellipsoideus, " and Schizosaccharomyces spp. Molds are common on green coffee beans, and in one study, 99.1 % of products from 31 countries contained these organisms, generally on the surface. Seven species of aspergilli dominated the biota, with A. ochraceus being the most frequently recovered from beans before surface disinfection, followed by A. niger and species of the A. glaucus group. The toxigenic molds, A. flavus and A. versicolor, were found, as were P cyclopium. P citrinum. and P expansum. but the penicillia were less frequently found than the aspergilli. Microorganisms do not contribute to the development of flavor and aroma in coffee beans as they do in cocoa beans. Cocoa beans (actually cacao beans-cocoa is the powder and chocolate is the manufactured product), from which chocolate is derived, are obtained from the fruits or pods of the cacao plant in

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parts of Africa, Asia, and South America. The beans are extracted from the fruits and fermented in piles, boxes, or tanks for 2-12 days, depending on the type and size of beans. During the fermentation, high temperatures (4S-S0°C) and large quantities of liquid develop. Following sun or air drying, during which the water content is reduced to less than 7.S%, the beans are roasted to develop the characteristic flavor and aroma of chocolate. The fermentation occurs in two phases. In the first, sugars from the acidic pulp (about pH 3.6) are converted to alcohol. The second phase consists of the alcohol being oxidized to acetic acid. In a study of Brazilian cocoa beans by Camargo et aI., the biota on the first day of fermentation at 21°C consisted of yeasts. On the third day, the temperature had risen to 49°C, and the yeast count had decreased to no more than 10% of the total biota. Over the 7 -day fermentation, the pH increased from 3.9 to 7.1. The cessation of yeast and bacterial activity around the third day is due in part to the unfavorable temperature, lack of fermentable sugar1>, and increase in alcohol. Although some decrease in acetic acid bacteria occurs because of high temperature, not all of these organisms are destroyed. The importance of lactic acid in the overall process was shown earlier. In a recent study, the cocoa fermentation was carried out with a defined microbial cocktail consisting of only five organisms rather than the SO or so that have been isolated from natural fermentations. The five consisted of Saccharomyces cerevisiae var. chevaliers, Lactobacillus plantarum. L. lactis. Acetobacter aceti. and Gluconobacter oxydans subsp. subo).}'Cians. The defined inoculum led to a product highly similar to that produced by natural fermentation. The key roles for the yeasts involved elevating pH from about 3.5 to 4.2, breaking down citric acid in pulp, producing ethanol, producing organic acids (oxalic, succinic, malic, etc.) that destroy bean cotyledons, producing volatile substances that may play a role in chocolate flavor, and reducing viscosity of pulp. S. cerevisiae was the most important organism in the above activities. Although yeasts play important roles in producing alcohol in cocoa bean fermentation, their presence appears even more essential to the development of the final, desirable chocolate flavor of roasted beans. Levanon and Rossetini found that the endoenzymes released by autolyzing yeasts are responsible for the development of chocolate precursor compounds. The acetic acid apparently makes the bean

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MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

tegument permeable to the yeast enzymes. It has been shown that chocolate aroma occurs only after cocoa beans are roasted and that the roasting of unfermented beans does not produce the characteristic aroma. Reducing sugars and free amino acids are in some way involved in the final chocolate aroma development. Soy sauce or shoyu is produced in a two-stage manner. The first stage, the koji (analogous to malting in the brewing industry), consists of inoculating either soybeans or a mixture of beans and wheat flour with A. oryzae or A. soyae and allowing them to stand for 3 days. This results in the production of large amounts of fermentable sugars, peptides, and amino acids. The second stage, the moromi, consists of adding the fungal-covered product to around 18% NaCl and incubating at room temperatures for at least a year. The liquid obtained at this time is soy sauce. During the incubation of the moromi, lactic acid bacteria, L. delbrueckii in particular, and yeasts such as Zygosaccharomyces rouxii carry out an anaerobic fermentation of the koji hydrolysate. Pure cultures of A. oryzae for the koji and L. delbrueckii and Z. rouxii for the moromi stages have been shown to produce good quality soy sauce. Tempeh is a fermented soybean product. Although there are many variations in its production, the general principle of the Indonesian method for tempeh consists of soaking soybeans overnight in order to remove the seed coats or hulls. Once seed coats are removed, the beans are cooked in boiling water for about 30 minutes and spread on a bamboo tray to cool and surface dry. Small pieces of tempeh from a previous fermentation are incorporated as starter followed by wrapping with banana leaves. The wrapped packages are kept at room temperature for 1 or 2 days during which mold growth occurs and binds the beans together as a cake-the tempeh. An excellent product can be made by storing in perforated plastic bags and tubes with fermentations completed in 24 hours at 31°C. The desirable organism in the fermentation is Rhizopus oligosporus, especially for wheat tempeh. Good soybean tempeh can be made with R. oryzae or R. arrhizus. During the fermentation, the pH of soybeans rises from around 5.0 to values as high as 7.5. Miso. a fermented soybean product common in Japan, is prepared by mixing or grinding steamed or cooked soybeans with koji and salt and allowing fermentation to take place usually over a 4- to 12-month period. White or sweet miso may be fermented for only a week, whereas the higher-quality dark brown product (mame)

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may ferment for 2 years. In Israel, Ilany-Feigenbaum et al. prepared miso-type products by using defatted soybean flakes instead of whole soybeans and fermenting for around 3 months. The koji for these products was made by growing A. oryzae on corn, wheat, barley, millet or oats, potatoes, sugar beets, or bananas, and the investigators found that the miso-type products compared favorably to Japaneseprepared miso. Because of the possibility that A. oryzae may produce toxic substances, koji was prepared by fermenting rice with Rhizopus oligosporus at 25°C for 90 days; the product was found to be an acceptable alternative to A. oryzae as a koji fungus. Ogi is a staple cereal of the Yorubas of Nigeria and is the first native food given to babies at weaning. It is produced generally by soaking corn grains in warm water for 2-3 days followed by wetmilling and sieving through a screen mesh. The sieved material is allowed to sediment and ferment and is marketed as wet cakes wrapped in leaves. Various food dishes are made from the fermented cakes or the ogi. During the steeping of corn, Corynebacterium spp. become prominent and appear to be responsible for the diastatic action necessary for the growth of yeasts and lactic acid bacteria. Along with the corynebacteria, S. cerevisiae and L. plantarum have been found to be prominent in the traditional ogi fermentation, as are Cephalosporium, Fusarium. Aspergillus. and Penicillium. Most of the acid produced is lactic, which depresses the pH of desirable products to around 3.8. The corynebacteria develop early, and their activities cease after the first day; those of the lactobacilli and yeasts continue beyond the first day of fermentation. A more recent process for making ogi has been developed, tested, and found to produce a product of better quality than the traditional process. By the new method, corn is dry-milled into whole corn and dehulled corn flour'. Upon the addition of water, the mixture is cooked, cooled, and then inoculated with a mixed culture (starter) of L. plantarum. L. lactis. and Z. rouxii. The inoculated preparation is incubated at 32°C for 28 hours, during which time the pH of the corn drops from 6.1 to 3.8. This process eliminates the need for starchhydrolyzing bacteria. In addition to the shorter fermentation time, there is also less chance for faulty fermentations. Gari is a staple food of West Africa prepared from the root of the cassava plant. Cassava roots contain cyanogenic glucosides, linamarin and lotaustra/il1. which make them poisonous if eaten fresh or raw. The roots can be detoxified by the addition of

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MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

linamarase, which acts on both. In practice the roots are rendered safe by a fermentation during which the toxic glucoside decomposes with the liberation of gaseous hydrocyanic acid. In the home preparation of gari, the outer peel and the thick cortex of the cassava roots are removed, followed by grinding or grating the remainder. The pulp is pressed to remove the remaining juice and placed in bags for 3 or 4 days to allow fermentation to occur. The organisms most responsible for the product include L. plantarum. E. faeciul11. and Leuconostoc mesenteroides. The fermented product is cooked by frying. Bongkrek is an· example of a fermented food product that in the past has led to a large number of deaths. Bongkrek or semaji is a coconut presscake product of central Indonesia, and it is the homemade product that may become toxic. The safe products fermented by R. oligosporus are finished cakes covered with and penetrated by the white fungus. In order to obtain the desirable fungal growth, it appears to be essential that conditions permit good growth within the first 1 or 2 days of incubation. If, however, bacterial growth is favored during this time and if the bacterium Burkholderia cocovenenans (formerly Pseudomonas cocovenenans) is present, it grows and produces two toxic substances-toxoflavin and bongkrekic acid. Both of these compounds show antifungal and antibacterial activity, are toxic for humans and animals, and are heat stable. Production of both is favored by growth of the organisms on coconut (toxoflavin can be produced in complex culture media). The structural formulas of the two antibiotics-toxoflavin, which acts as an electron carrier, and bongkrekic acid, which inhibits oxidative phosphorylation in mitochondria. CH.

Y:;:

o 1bxoflavin

N

/N HC • 0

Bongkrekic aCId

I

N

~

N

yiN

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Bongkrekic acid has been shown to be cidal to all 17 molds studied by Subik and Behun by preventing spore germination and mycelial outgrowth. The growth of B. cocovenenans in the preparation of bongkrek is not favored if the acidity of starting materials is kept at or below pH 5.5. It has been shown that 2% NaCI in combination with acetic acid to produce a pH of 4.5 will prevent the formation of the bongkrek toxin in tempeh. A fermented cornmeal product that is prepared in parts of China has been the cause of food poisoning by strains B. cocovenenans. The product is prepared by soaking corn in water at room temperature for 2-4 weeks, washing in water, and grinding the wet corn into flour for various uses. The toxic organisms apparently grow in the moist product during its storage at room temperature. The responsible organism produced both bongkrekic acid and toxoflavin, as do the strains of B. cocovenenans in bongkrek. Ontjom (oncom) is a somewhat similar but more popular fermented p·roduct of Indonesia made frolI1 peanut presscake, the material that remains after oil has been extracted from peanuts. The presscake is soaked in water for about 24 hours, steamed, and pressed into molds. The molds are covered with banana leaves and inoculated with Neurospora sitophila or R. oligosporus. The product is ready for consumption 1 or 2 days later.

4 Microbial Genetics "Who does she look like, mom or dad?" That's probably one of the first few questions everyone asks when hearing about a new arrival in the family. "Does she have Aunt Jane's eyes? Uncle Joe's nose?" "How about Gramps' sandy hair?" These are some of the many noticeable characteristics that can be passed down from family members. What is really being asked is what genetic traits did the current generation inherit from previous generations. Think of genetic traits as our computer program; it provides us with instructions on how to do everything needed to stay alive. Some instructions are passed along to the next generation while other instructions are not. _If microorganisms could speak, they might also ask the same questions as we do when a new offspring arrives, because microorganisms also pass along genetic traits to new generations of their species. Those traits preprogram new microorganisms on how to identify and process food, how to excrete waste products, and how to reproduce, as well as nearly everything the microorganism needs to know to survive. In this chapter you'll learn how microorganisms inherit genetic traits from previous generations of microorganisms. 4.1 GENETICS Genetics is the branch of science that studies heredity and how traits (expressed characteristics) are passed to new generations of species and between microorganisms. Scientists who study genetics are called geneticists and are interested in how traits are expressed within a cell and how traits determine the characteristics of an 150

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organism. Think of a trait as an instruction that tells an organism how do something, such as how to form a toe. Each instruction is contained in a gene. As you can imagine, there are thousands of genes (instructions) necessary for an organism to grow and flourish. This is why if a youngster looks and behaves like her mother, family members tend to say she has her mother's genes-that is, she has more genes (instructions) from her mom than from her dad. Genes are actually made up of segments or sections of deoxyribonuclear acid (DNA), or in the case of a virus, ribonucleic acid (RNA) molecules. These segments are placed in a specific sequence that code for a functional product.

4.2 DNA REPLICATION In 1868, Swiss biologist Friedrich Miescher carried out chemical studies on the nuclei of white blood cells in pus. His studies led to the discovery of DNA. DNA was not linked to hereditary information until 1943 when work performed by Oswald Avery, Colin MacLeod, and Maclyn McCarty at the Rockefeller Institute revealed that DNA contained genetic information. These studies also revealed that genetic information is passed from "parent cells" to "daughter cells," creating a pathway through which genetic information is passed to the next generation of an organism. Scientists were baffled about how the exchange of DNA occurred. The answer came in 1953 when American geneticist James Watson and English physicist Francis Crick discovered the double-helical structure of DNA at the University of Cambridge in England. Discovery of the double-helical structure was the key that enabled Watson and Crick to learn how DNA is replicated. In the late 1950's, Mathew Meselson and Franklin Stahl first described the DNA molecule and how DNA replicates in a process called semiconservative replication. DNA is replicated by taking one parent double-stranded DNA molecule, unzipping it and building two identical daughter molecules. Bases along the two strands of double-helical DNA complement each other. One strand of the pair acts as a template for the other. DNA is replication requires complex cellular proteins that direct the sequence of replication. Replication begins when the parent double-stranded DNA molecule unwinds; then the two strands separate. The DNA polymerase enzyme uses a strand as a template to make a new strand of DNA. The DNA polymerase enzyme examines the new DNA and removes bases that do not match and

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then continues DNA synthesis. The point at which the double-stranded DNA molecule unzips is called the replication fork. The two new strands of DNA each have a base sequence complimentary to the original strand. Each double-stranded DNA molecule contains one original and one new strand. In bacteria, each daughter receives a chromosome that is identical to the parent's chromosome. 4.2.1 Chromosome Connection Chromosomes are structures that contain DNA. DNA consists of two long chains of repeating nucleotides that twist around each other, forming a double helix. A nucleotide in a DNA chain consists of a nitrogenous base, a phosphate group. and deoxyribose (pentose sugar). The two DNA chains are held together by hydrogen bonds between their nitrogenous bases. There are two major types of nitrogenous bases. These are purines and pyrimidines. There are two types of purine bases: adenine (A) and guanine(G). There are also two types of pyrimidine bases: cytosine (C) and thymine (T). Purine and pyrimidine bases are found in both strands of the double helix. Base pairs are arrangements of nitrogenous bases according to their hydrogen bonding. Adenine pairs with thymine and cytosine pairs with guanine. Adenine is said to be complementary to thymine and cytosine is said to be complementary with guanine. This is known as complementary base pairing. Genetic information is encoded by the sequence of bases along a strand of DNA. This information determines how a nucleotide sequence is translated into an amino acid which is the basis of protein synthesis. The translation of genetic information from genes to specific proteins occurs in cells. Table 4.1 Complementary messenger RNA bases and DNA bases MessengerRNA Base Adenine (A) Guanine (G) Cytosine (C) Uricil (U)

Double-helical strand DNA molecule base Thymine (T) Cytosine (C) Guanine (G) Adenine (A)

4.3 PROTEIN SYNTHESIS Protein synthesis is the making of a protein and require5 ribonucleic acid (RNA), which is synthesized from nucleotides that

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contain the bases A, C, G, and U (uracil). There are three types of RNA. These are: 1. Ribosomal RNA (rRNA), which is the enzymatic part of ribosomes. 2. Trailsfer RNA (tRNA), which is needed to transport amino acids to the ribosomes in order to synthesize protein. 3. Messenger RNA (mRNA), which carries the genetic information from DNA into the cytoplasm to ribosomes where the proteins are made. An enzyme called RNA polymerase is required to make (synthesize) rRNA, tRNA, and mRNA. Protein synthesis begins with the transcription process, in which DNA sequences are replicated producing mRNA. The mRNA carries genetic information from the DNA to ribosomes. Ribosomes are organelles and the site of protein synthesis. Nucleotides contained in DNA are duplicated by enzymes before cell division, enabling genetic information to be carried between cells and from one generation to the next. This is referred to as gene expression and happens in RNA only. During transcription, the bases A, C, G, and uracil (U) pair with bases of the DNA strand that is being transcribed. The G base in the DNA template pairs with the C base in the mRNA. The C base in the DNA template pairs with the G base in the mRNA. The T base in the DNA template pairs with an A in the mRNA. An A base in the DNA template pairs with the U in the mRNA. This happens because RNA contains a U base instead of a T base. Transcription begins when the RNA polymerase binds to DNA at the promotor site. The DNA unwinds. One of the DNA strands, called a coding strand, serves as a template for RNA synthesis. RNA is synthesized by pairing free nucleotides of the RNA with nucleotide bases on the DNA template strand. The RNA polymerase moves along the DNA as the new RNA strand grows. This continues until the RNA polymerase reaches the terminator site on the DNA or is physically stopped by a section of RNA transcript. The new single-stranded mRNA and the RNA polymerase are released from the DNA. Here is what's happening: Information of the nucleic acid assembles a protein. The mRNA strand consists of several sections, one being the reading frame. The reading frame is made up of codons. These are AUG, MA, and GGC. Each codon contains

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information for a specific amino acid. The sequence of codons on the RNA determines the sequence that amino acids are used to synthesize proteins. Once the transcription process is completed, information of the mRNA is turned into protein in the translation process. The translation process is one in which genetic information encoded ill mRNA is translated into a specific sequence of amino acids that produce proteins. Appropriate amino acids are brought to the translation site in the ribosomes and are assembled into a growing chain. It is here that tRNA recognizes specific codons. Each tRNA molecule has an anticodon, which is a sequence of three bases that is complementary to the bases on the codon. These bases are then paired, and amino acids are brought to the chain. This process continues until a polypeptide is produced. The polypeptide is removed from the ribosome for further processing. The polypeptide may be stored in the Golgi body of a eukaryotic organism. The mRNA molecule degenerates and the nucleotides are returned to the nucleus. The tRNA molecule is returned to the cytoplasm and combines with new molecules of amino acids. 4.3.1 Genotype and Phenotype The genetic makeup of an organism is called a genotype and represents that organism's potential properties. Some properties may not have developed. Those that do develop are called an organism's phenotype. The phenotype represents expressed properties, such as blue eyes and curly hair. A genotype is the organism's DNA (a collection of genes). The phenotype is a collection of proteins. The majority of the cell's properties comes from the structures and functional properties of these proteins.

4.4 CONTROLLING GENES The process of making proteins (remember, polypeptides become proteins either after they are combined with other polypeptides or when they become biologically functional) begins with the copying of the genetic information found in DNA, into a complimentary strand of RNA. This copying is called transcription. Messenger RNA (mRNA) will carry the coded information or instructions for assembling the polypeptides from DNA to the ribosomes of the cell's endoplasmic reticulum where the polypeptides will be made.

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The actual building of polypeptides is called translation. Translation involves the deciphering of nucleic acid information and converting that information into a language that the proteins can understand. 4.4.1 Op·eron Model In 1961 Francois Jacob and Jacques Monod formulated the operon model that described how transcription of mRNA is regulated. Transcription of mRNA is regulated in two wayS. These are repression and induction. Repression inhibits gene expression and decreases the synthesis of enzymes. Proteins called repressors stop the ability of RNA polymerase to initiate transcription from repressed genes. Induction activates transcription by producing inducer, which is the chemical that induces transcription. Jacob and Monod identified genes in E. coli as structural gel1es, regulatory genes, and control regions. Collectively these form a functional unit called the operon. Certain carbohydrates can induce the presence of enzymes needed to digest those carbohydrates. For example when lactose is present, E. coli synthesize enzymes needed to breakdown lactose. Lactose is an inducer molecule. If lactose is absent, a regulator gene produces a repressor protein that binds to a control region caned the operator site, preventing the structural genes from encoding the enzyme for lactose digestion. Lactose binds to the repressor at the operator site when lactose is present, freeing the operator site. The structural genes are released and produce their lactose-digesting enzymes. 4.5 MUTATIONS A mutation is a permanent change in the DNA base sequence. Some mutations have no expressive effect while other mutations have an expressive effect. When a gene mutates, the enzyme encoded by the gene can become less active or inactive because the sequence ofthe enzyme amino acids may have changed. The change can be harmful or fatal to the cell, or it can be beneficial-especially if the mutation creates a new metabolic activity. The most common type of mutation is point mutation, which is also known as base substitution mutation. Point mutation occurs when an unexpected base is substituted for a normal base, causing alteration of the genetic code, which is then replicated.

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Table 4.2 Types of mutations Type of Mutation

Description

Also known as base substitution, this is the most common type of mutation and involves a single base pair in the DNA molecule. In point mutation, a different base is substituted for the original base, causing the genetic code to be altered. The substituted base pair is used when DNA is replicated. Missence mutation A mutation when a new amino acid is substituted in the final protein by the messenger RNA during transcription. Nonsense mutations A mutation when a terminator codon in the messenger RNA appears in the middle of a genetic message instead of at the end of the message, which causes premature termination of transcription. Frame shift mutation Pairs of nucleotides are either added or removed from a DNA molecule. Loss-of-function mutation This mutation causes a gene to malfunction. Spontaneous mutation Natt:rally occurring mutation that happens without the presence of a mutationcausing agent. Induced mutation Induced in a laboratory.

Point mutation

If the mutated gene is used for protein synthesis, the mRNA transcribed from the gene carries the incorrect base for that position. The mRNA may insert an incorrect amino acid in the protein. If this happens, the mutation is called a missence mutation. Mutations that change or destroy the genetic code are called nonsense mutations. If nucleotides are added or deleted from mRNA, the mutation is called a frame shift mutation. A mutation occurring in the laboratory is called an induced mutation; mutations occurring outside the laboratory are called spontaneous mutation. A spontaneous mutation occurs when a mutation -causing agent is present. Base substitution and frame shift mutations occur spontaneously. Agents in the environment or those introduced by industrial processing

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can directly or indirectly cause mutations. These agents are called mutagens. Any chemical or physical agent that reacts with DNA can potentially cause mutations. Certain mutations make microorganisms resistant to antibiotics or increase their pathogenicity. There are many naturally occurring mutagens, such as radiation from x-rays, gamma rays, and ultraviolet light. These rays break the covalent bonds between certain bases of DNA-producing fragments. Ultraviolet light binds together adjacent thymines in a DNA strand, forming thymine dimers that cannot function in protein synthesis. Unless repaired, these dimers cause damage or death to cells due to improper transcription or replication of DNA. Some bacteria can repair damage caused by ultraviolet radiation by employing light-repairing enzymes that separate the dimer into the original two thymines. This process is called photoreactivation.

4.5.1 Mutation Rate Mutations occur naturally and can be induced by mutationcausing agents in the environment. However, not all cells experience mutation even if they are exposed to mutation-causing agents. Scientists measure the impact that mutation has on an organism by determining the mutation rate. The mutation rate is the number of mutations per cell division. For example, suppose you observe the growth of 100 cells that began from a parent cell. If 90 of those cells replicate the parent cell and 10 cells are mutations, than the mutation rate is 10 percent. Measuring the mutation rate is a way to compare the number of mutations that occur naturally to the number of mutations that occur when a cell is exposed to a potential mutation-causing agent. First, scientists measure the mutation rate that occurs naturally when a cell is not exposed to a potential mutation-causing agent. Next, the mutation rate is calculated when a cell is not exposed to a potential mutation-causing agent. The results of these two observations are compared. If both mutation rates are relatively the same, then the substance being tested is not a mutation-causing agent. However, the substance is a mutation-causing agent if its mutation rate is appreciably higher than the natural mutation rate.

4.6 GENETIC INTERACTIONS The ecological significance of genetic activity in aquatic bacteria has already been emphasized in relation to biofilm communities,

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where physiological differences between biofilm and planktonic organisms relate to quorum sensing and the expression of stationaryphase genes. This section considers aspects of genetic diversity in aquatic bacteria, mechanisms of gene transfer and evidence for gene transfer in freshwater systems. 4.6.1 Genetic Diversity Genetic diversity involves variation in genetic composition and gene expression, and is a key factor in: 1. The variety of bacterial metabolic activities in the freshwater environment, and 2. Ecological adaptations of bacteria to particular habitats. The genetic composition of particular bacteria and bacterial groups within the microbial community may be considered in relation to the relative contributions of chromosomal and accessory DNA, the ecological importance of gene transfer in freshwater systems, and the complexity of the 'community genome.' Expression of individual genes is regulated by both external (e.g., specific substrate availability) and internal (e.g., stage of cell cycle) factors. Major environmental changes may induce the expression of a whole array of genes, which in prokaryotes (bacteria and bluegreen algae) may be triggered by the expression of a controlling transcription factor. In bacteria, this is seen in the response of biofilm communities to high population density and the response of bacterioplankton to acute nutrient deprivation (starvation response). In blue-green algae, multiple induction of genes via sigma factors has been demonstrated during exposure to physico-chemical stress (heat, salinity, nutrient deprivation) and in the diurnal oscillation of gene expressIOn.

4.6.1.1 Chromosomal and accessory DNA Genetic diversity in freshwater bacteria, as with bacteria generally, is characterized by the presence of two separate genetic systems, composed of chromosomal and extra-chromosomal DNA. Chromosomal DNA carries the main gene bank of the bacterial cell, contains all the genes that are essential for growth and division of the bacterium, and replicates in strict synchrony with the timing of the cell cycle. Extra-chromosomal DNA is present in the bacterial cell as separate, relatively short-sequence fragments, and includes phages, plasmids, transposons, and insertion sequences. This DNA can be regarded as accessory to chromosomal DNA, since it typically

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encodes for non-essential characteristics such as Uye resistance, catabolism of unusual carbon sources, resistance to antibiotics and heavy metals, and metabolites involved in secondary metabolism. These accessory elements are present in variable amounts and are able to replicate independently of the cell cycle. Genetic diversity in bacteria is promoted by the active transfer of genes between organisms, which involves the transport and incorporation of accessory elements (containing the transferring gene) from one cell to another. Transferred genes may have long-term and short-term effects as follows. 1. Many of the transferred genes are retained on the transferred DNA (e.g., plasmid) and are expressed in the host bacterial cell. Plasmid-encoded genes often confer short-term environmental advantage, such as resistance to heavy metals in a polluted habitat and resistance to ultra-violet (UYe) damage to DNA under conditions of high irradiation. Many bacteria (e.g., Escherichia coli) are able to repair DNA damage caused by uve irradiation via a chromosomally encoded Uye-inducible, mutagenic repair system, which is mediated via a key macromolecule - RecA protein. The common freshwater bacterium Pseudomonas aeruginosa has little resistance to the damaging effects of Uye radiation since it does not contain this system, but it does contain RecA protein. Many naturallyoccurring plasmids of Pseudomonas species encode for Uye resistance and are found abundantly in aquatic habitats. Introduction of such plasmids into RecA + strains of Pseudomonas aeruginosa allowed Uye-inducible repair, allowing these organisms to grow and survive in exposed environments. The Uye-repair system is also mutagenic, so introduction of Uye plasmids also resulted in an increased mutation rate. 2. Accessory elements may also carry and introduce fragments of DNA which become incorporated into the main chromosome. The chromosomal alterations are of three main types recombination, mutation, and transposition. Recombination involves replacement of a region of chromosomal DNA by homologous DNA introduced into the bacterial cell, and is an important aspect of DNA repair. Direct insertion of the introduced DNA into the bacterial chromosome can cause loss or alteration of existing gene function (mutation) or expression of the gene in a new region of the chromosome (transposition). Alteration of gene expression on the chromosome has long-term

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significance, since the modified chromosomal DNA replicates and passes to daughter cells in a stable manner. The incorporation and expression of plasmid genes in different bacteria is important for phenotypic diversity within the bacterial community. This includes the expression of novel genes introduced via human activity as well as natural mutations arising within the ecosystem.

4.6.1.2 Ecological importance of gene transfer m freshwater systems The potential for gene transfer between bacteria in aquatic systems has been the subject of much research and is of interest for two main reasons - introduction and spread of novel genes arising from human activities, and the natural process of bacterial evolution in freshwater environments. 4.6.1.2.1 Introduction of novel genes This has received much attention in recent years because of potential problems with: I. The discharge of bacteria with plasmids containing antibiotic resistance genes (originating from clinical and agricultural sources) from sewage and waste-treatment facilities. The possible transfer of these plasmids to the indigenous microbial flora and to human pathogens imposes a potential threat to human health; 2. The potential release of genetically-engineered microorganisms (GEMs) into the terrestrial environment, with aquatic contamination and transfer of the novel genes to other freshwater biota. Genetically engineered bacteria which might be released into the environment include a wide range of organisms that have been modified for agricultural purposes - other possibilities include the use of GEMs which have been modified for the detoxification of polluted soil, water, and land-fill sites by the manipulation of normal catabolic activities. In both cases, transfer and spread of the novel genes within the aquatic environment has potential implications for human health. Introduction of novel genes may also be important within the natural ecological framework, affecting the short-term survival and longterm evolution of bacterial species. 4.6.1.2.2 Gene transfer within the ecosystem The transfer and spread of genes (including natural mutations) within aquatic bacterial populations is important for a number of

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key processes that affect the ability of bacteria to adapt to changes in the freshwater environment and compete with other organisms. 1. Gene transfer speeds up the process of bacterial evolution by bringing together new genes that originally arose as advantageous mutations in different bacterial lines. The origin of these mutations can thus be exploited by the entire bacterial community, allowing these organisms to adapt more rapidly to new environments. 2. Gene transfer gives accessory genetic elements a major role in contributing to genetic diversity. As noted previously, these elements are of various types and code for phenotypic characteristics such as antibiotic resistance, heavy metal resistance, and degradation of complex organic compounds - all of which may give short-term ecological advantage. The role of accessory elements as agents for rapid transfer across bacterial lineages is indicated by the fact that the genetic control of conjugation is plasmid -mediated. 3. The introduction of new DNA into the bacterial cell, particularly into the bacterial chromosome, not only generates genetic novelty but is also important for maintaining the status quo. Gene transfer by transformation is thought to be particularly important in DNA repair, replacing DNA that has become non-functional due to adverse mutation restoring the original gene function.

4.6.1.3 Total genetic diversity: the 'community genome' Total genetic diversity can be estimated from the molecular complexity of the collective DNA sample that is isolated from the entire community. The DNA complexity can be determined by measuring the re-annealing (reassociation) rate for single-stranded DNA in solution under defined conditions. If the microbial community can be regarded for the sake of the calculation as a 'single species', the reassociation data provide an estimate of the total genome size of this species. The total number of putative species can then be derived by dividing this value by the standard genome size of a 'typical species' such as Escherichia coli. As an example of this, Ovreas et al. (2003) quote values for the total DNA complexities of bacterial communities in pristine sediments in the range 2.7 x 10 10 to 4.8 X 10 10 bp. Taking the genome size of E. coli as 4.1 x 106 bp gives a DNA complexity within the sediments that is equivalent to 6500--11500 genomes.

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The number of bacterial species that actually contribute to this diversity must be a matter of speculation. 4.6.2 Mechanisms for Gene Transfer in Freshwater Systems Three main mechanisms for bacterial gene transfer have been demonstrated in the freshwater environment- transformation (uptake of naked DNA), transduction (mediated by bacteriophages), and conjugation (requiring cell-to-cell contact). The transferred genes may either by carried on intact plasmids (frequently the case with transduction and conjugation) or on other types of DNA fragment (transformation). Once initial transfer has occurred, long-term survival of the introduced gene will depend either on persistence of the DNA fragment within the bacterial population or on transposition of the gene to permanent genomic or extra-chromosomal DNA within the bacterial cell. Movement of genes within bacterial cells includes the activities of insertion elements and transposons. Insertion elements are DNA sequences which encode the movement of the pNA sequence from one location to another. These elements carry no selectable markers and are typically less than 1 kb in size. Transposons, in comparison, are larger and carry other genes, often encoding antibiotic resistance, flanked by insertion sequences. Insertion of transposons into the bacteriql genome has a variety of effects including inactivation of the gene or operon into which they transpose, delivery of novel genes contained within their sequence, and rearrangement of other DNA sequences within the bacterial DNA. The transfer of transposons from host to host can be mediated by any of the three transfer mechanisms noted below~

4.6.2.1 Transformation: uptake of exogenous DNA The ability of planktonic or attached (e.g., biofilm) bacteria to take up external (exogenous) DNA from the water medium and incorporate it into their genome is referred to as transformation. In this process, genes present on exogenous DNA originally derived from a donor bacterium are transferred to a new cellular environment, where they may become active as novel genes or replace existing genes (DNA repair). 4.6.2.1.1 Requirements for transformation The two major requirements for transformation ill the aquatic environment are free availability of extra-cellular DNA and the physiological capability of recipient cells (referred to as 'transformable'

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or 'competent'). The conversion of non-competent to competent bacterial cells is prompted by environmental factors and is considered in the following section. Exogenous DNA appears to be universally available in freshwater systems, occurring over a wide range of concentrations (1-200 ng ml-'). This compares with a standard level of approximately 27 ng ml-' in sea water. Although this DNA occurs as insoluble macromolecular fragments within the aquatic medium, it is technically part of the dissolved organic material (DOM) or dissolved organic carbon (DOC) since it passes through a 0.2 p.m filter membrane. Unlike most of the DOC derived from lake biota, which is secreted by living algal cells, exogenous DNA is produced by breakdown or lysis which results from cell death. Within the freshwater environment, the exogenous DNA is heterogeneous both in terms of origin (derived from bacteria, algae, invertebrates) and state - comprising both single- stranded (ssDNA) and double-stranded (dsDNA) fragments. This DNA is in a continuous dynamic flux and has a rapid turnover, with persistence times in the range 4-24 hours. DNA is continuously being released from lysed cells into the freshwater system, and is continuously being removed by processes of adsorption (to cell surfaLes and other particulate matter) and enzymatic breakdown (by exogenous nuclease enzymes). Bacterial transformation only involves homologous (i.e., from the same strain or species) dsDNA. DNA from other organisms, ssDNA, and RNA are not taken up and may actually interfere with the transformation process by competing for adsorption sites on the recipient bacterial surface.

4.6.2.1.2 Transformation process The natural process of transformation can be thought of as a sequence of three distinct events - (1) development of competence, (2) DNA binding, and (3) DNA uptake and incorporation into the host cell. Development of competence. Competence is a regulated state in transformable bacteria. In Gram-negative organisms, this is internally controlled and appears to be 'correlated with a state of limited or unbalanced growth. Competence may be promoted where environments are inherently nutrient-limited or where high bacterial densities in niche habitats (e.g., biofilms) have a localized growthlimiting effect. In Gram-positive bacteria, the induction of competence

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depends on the external accumulation of secreted polypeptide competence factors to a critical level, and is largely determined by population density. The development of bacterial competence involves the induction of various competence genes, resulting in the synthesis of surface DNA receptors and other key proteins. DNA binding. The binding of exogenous DNA to the surface of competent bacterial cells is closely dependent on the chemical composition of the water medium - including concentration of homologous DNA, presence of heterologous DNA, concentration of nuclease enzymes, and ionic composition. The binding process involves a sequence of events, with initial bacterial/DNA contact, followed by loose (electrostatic) attachment to the protein receptor then firmer (covalent) association. The aquatic concentration of homologous dsDNA is clearly important for initial contact and binding;" and in some bacteria the presence of specific nucleotide recognition sequences are also necessary. Any factors which reduce the level of homologous dsDNA in the environment (e.g., presence of nuclease enzymes) or interfere with the binding process (e.g., presence of heterologous DNAs) will inhibit transformation. In addition to these effects, loose binding of DNA is antagonized by high cation levels, and at each stage of binding the surface DNA is vulnerable to attack by nuclease enzymes. The opposing effects of ion antagonism, interference by other nucleic acids, and enzyme degradation on bacterial surface receptor binding can be viewed as a complex framework of competitive interactions in the freshwater environment. DNA uptake and incorporation into the bacterial cell. DNA uptake and incorporation represents the final stage of bacterial transformation. The dsDNA attached to the surface receptor is nicked by a surface nuclease, which sequentially degrades one of the strands allowing the other strand to enter in isolation. The entry of a singlestranded intermediate into the cytoplasm allows the key bacterial recombination protein (recA protein) to bind to the transformed strand. This protein is then activated for binding to the doublestranded chromosomal DNA, subsequently positioning and inserting the transformed strand at the homologous region of the main chromosome (genetic recombination). More detailed information on the molecular events taking place during transformation can be found in standard texts on microbiology.

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4.6.2.1.3 Significance of bacterial transformation in the aquatic environment Although the extent to which transformation occurs in aquatic environments is not known, external DNA availability and the presence of favourable environmental (nutrient limitation, localized cell concentrations) conditions suggest that it may be significant. In terms of environmental significance, however, it is important to distinguish between gene transfer at .the interspecific (between species) and intraspecific (within species) levels. Transformation is generally considered to have only limited ecological potential for gene transfer at the interspecific level, since: 1. Only a limited number of bacterial species are known to be transformable, so there is a limit on host bacteria, 2. Base-sequence homology is often required for stable maintenance of introduced DNA, so there is a limit on donor DNA, 3. There are restrictions to the size and type of DNA that can be successfully taken up by transformable cells, placing further limitations on the range of DNA uptake. These limitations are reduced when considering the uptake of small fragments of homologous DNA within species, and transformation may have more significance for DNA repair than gene transfer. The relevance of transformation to DNA repair is emphasized by the fact that most transformable bacteria use their primary recombination (recA) system for the final step in the transformation process. 4.6.2.2 Transduction The potential for transductive gene transfer within aquatic bacterial communities has been shown by experimental studies on Pseudomonas aeruginosa model systems. These have demonstrated ,/ that stable phage-mediated transfer of both chromosomal and plasmid genes can occur between strains of the same species at significant frequencies within freshwater habitats. Although the potential for this process is clearly present in aquatic environments, the general importance of transduction for gene transfer may be limited, since: 1. Bacteriophages often have restricted host ranges, reducing the variety of species available for gene"~change, 2. There is a limit to the size of bacterial DNA that can be packaged inside phage heads.

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4.6.2.3 Transfer of plasmid DNA by direct cell contact Gene transfer between bacterial cells by conjugation typically involves the transfer of plasmids and is probably the genetic exchange system of greatest importance within freshwater bacterial communities. Plasmids (and other extra-chromosomal elements) have evolved to achieve maximum gene expression within bacterial populations, and have developed capabilities for: 1. Optimal transfer between conjugating bacterial cells, 2. Post-conjugation replication (i.e., survival) in the new (transconjugant) host cells - plasmids typically have 'wide-range' replication functions, 3. Post-conjugation gene expression in the new host cells. The process of conjugation which leads to plasmid transfer is itself determined almost entirely by plasmid rather than chromosomal DNA. This encodes specific transfer functions allowing the DNA to pass from one bacterial cell to another. These conjugative plasmids, which promote their own transfer during conjugation, are also able to promote the transfer of ~on-conjugative plasmids. This is referred to as mobilization, and requires the presence of a mob site on the non-conjugative plasmid. Numerous genetically distinct and noninteracting systems have evolved to effect plasmid transfer. Some systems (broad range) have the capacity to encode plasmid transfer to a wide range of phyllogenetically-unrelated organisms, while other (narrow-range) systems only permit plasmid transfer to a small group of closely-related bacteria. The importance of conjugative gene transfer in the freshwater environment is underscored by the role of plasmids in this process. Much of the evidence for gene transfer in the aquatic environment comes from studies on plasmids and their movement within bacterial populations. 4.6.3 Evidence for Gene Transfer in the Aquatic Environment Evidence for gene transfer between bacteria in the freshwater environment comes from three major sources - retrospective analysis, laboratory (in vitro), and field (in situ) experimental studies.

4.6.3.1 Retrospecth'e analysis Epidemiological analysis of plasmid populations in natural communities of bacteria has provided much useful information on gene transfer. Retrospective analysi~ of plasmid transfer involves three stages:

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1. Isolation of specific groups of bacteria such as pseudomonads, antibiotic-resistant bacteria, and mercury-resistant bacteria from the environment, 2. Size and molecular analysis of the plasmids, 3. Inference of transfer events from molecular similarities found between plasmids isolated from related and unrelated bacteria. Various published reports provide evidence for gene transfer in different aquatic environments, including rivers, estuarine and river sediments and lakes.

4.6.3.2 Laboratory (in vitro) studies on plasmid transfer The basis of laboratory experiments on plasmid transfer in bacteria is to mix donor cells (plasmid-containing strains) with plasmid-free recipient cells, promote mating between the bacteria, then determine the characteristics and frequency of transconjugant formation. Laboratory (in vitro) studies on freshwater bacteria have the advantage over field (in situ) studies in that gene transfer can be studied between defined strains of organisms under controlled physical, chemical, and biological conditions. In order to match the field situation, however, laboratory studies normally involve the use of bacteria that have originally been isolated from the freshwater environment, with mating being carried out in conditions which closely approximate to the river or lake situation.

4.6.3.3 Field (in situ) studies on bacterial gene transfer Early experiments on gene transfer focussed on R-factor transmission between Escherichia coli and other coliform bacteria and were laboratory-orientated. These studies then developed a more environmental approach, attempting to create lake or river conditions in terms of the organisms involved (field isolates) and the laboratory microenvironment for plasmid transfer. This approach was extended into the field, with enclosed systems (dialysis tubing, Teflon bags, diffusion chambers) containing mating mixtures of bacteria being placed directly into the freshwater environment. In their studies on gene transfer between strains of Pseudomonas aeruginosa, O'Morchoe et al. (1988) also carried out test matings within enclosed chambers placed in a reservoir environment. A significant number of transconjugants was recovered from these field trials, demonstrating gene transfer under semi-natural conditions, but the frequency of detectable transfers was considerably lower than had been obtained from the laboratory matings involving

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pure cultures. The presence of other microorganisms in the field probably has the same effects as the addition of natural microbial populations to laboratory systems. The use of enclosed systems may be further extended to studies on unenclosed systems. Bale et al. (1988) studied plasmid transfer between pseudomonad species in river epilithon by attaching filters containing donor and recipient cells to the surface of stones and submerging them in river water. In such situations, transfer of marker plasmids can be investigated between donor and recipient bacteria (within the filter) or between donor bacteria and the indigenous epilithic bacteria which can be isolated as a mixed natural suspension from the stone surface. Plasmid transfer studies carried out in a variety of running- and standing-water environments, show that gene transfer frequency is highly variable, ranging from high (10-1) to almost undetectable (10-9 ) transconjugants per donor cell. In all reported studies, however, some plasmid transfer was detectable, and was typically in the range 10-3 to 10-<> transconjugants per donor cell. These studies indicate that plasmid transfer between bacteria is widely significant within the freshwater environment.

4.6.3.3.1 Relative importance of environmental conditions on gene transfer Relatively little direct information is available on the importance of environmental factors in gene transfer, and much of the speculation on this is derived from laboratory studies. In general, this process would be expected to be stimulated by conditions which promote: 1. Enhanced survival and activity of the host cells (favourable temperatures, high nutrients), and 2. The specific processes of transformation, transduction, or conjugation. Environmental factors influencing the different processes of gene transfer are highly varied and are clearly important in maintaining genetic diversity within the bacterial community. These include aspects such as concentrations of soluble DNA, exogenous nuclease and cations (transformation), nutrient availability (induction of competence in transformation), presence of bacteriophage virions (transduction), and proximity for cell contact (conjugation, transformation). The relative importance of the three processes of gene transfer may also vary between microenvironments, with conjugation possibly

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predominating in high population density biofilms - while transformation and transduction may be relatively more important in dispersed planktonic populations. 4.7 RECOMBINANT DNA TECHNOLOGY Our genes are a strong determining factor of who we are and what we are going to be because genes program our bodies to express specific characteristics. Some of those characteristics enable us to carry out basic life functions, such as converting food to energy, while others make us stand out in a crowd, such as being a seven-foot professional basketball player. The same concept holds true with microorganisms. A microorganism's genes determine characteristics expressed by that microorganism. Genetic information is encoded in our DNA by the linkage of nucleic acids in a specific sequence, which you learned about in the previous chapter. Think of what would happen if you could change the sequence. You could reprogram genes to express desirable characteristics and to repress undesirable characteristics, such as those that cause diseases. Reordering genetic information is called genetic engineering. In this chapter, you'll learn about genetic engineering and how to use recombinant DNA technology to alter the genetic program of an organism. 4.7.1 Genetic Engineering: DesigQer Genes The modification of an organism's genetic information by changing its nucleic acid genome is called genetic engineering and is accomplished by methods known as recombinant DNA technology. Recombinant DNA technology opens up totally new areas in research and applied biology and is an important part of biotechnology, a field that is increasingly growing. Biotechnology is the term used for processes in which organisms are manipulated at the genetic level to form products for medicine, agriculture, and industry. Recombinant DNA is DNA with a new sequence formed by joining fragments from different sources. One of the first breakthroughs leading to recombinant DNA, or rDNA, technology was the discovery of microbial enzymes that make cuts into the double-stranded DNA. These were discovered by Werner Arber, Hamilton Smith, and Dan Nathans in the late 1960s. These enzymes recognize and cleave specific sequences of four to eight base pairs and are known as restriction enzymes. These enzymes recognize specific sequences in DNA and then cut the DNA to produce

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fragments called restriction fragments. The enzymes cut the bonds of the DNA backbone at a point along the exterior of the DNA strands. There are three types of restriction enzymes. Types I and III cleave DNA away from recognition sites. Type II restriction endonucleases cleave DNA at specific recognition sites. The type II enzymes can be used to prepare DNA fragments containing specific genes or portions of genes. A gene can be defined as a segment of DNA (a segment is a sequence of nucleotides) that codes for a functional product. ECORI cleaves the DNA between guanine (G) and adenine (A) in the base sequence GMTTC. In the double-stranded condition, the base sequence GMTTC will base pair with a sequence, which runs in the opposite direction. ECORI cleaves both DNA strands between the G and the A. When the two DNA fragments separate they contain single-stranded complementary ends called sticky ends. In 1972, David Jackson, Robert Symons, and Paul Berg generated recombinant DNA molecules. They allowed the sticky ends of the fragments to base pair with each other and covalently joined the fragments with the enzyme DNA ligase. The enzyme DNA ligase links the two sticky ends of the DNA molecules at the point of union. In 1973, Stanley Cohen and Herbert Boyer constructed the first recombinant plasmid capable of being replicated within a bacterial host. A'plasmid is a circular DNA molecule that a bacterium can replicate without a chromosome. In 1975, Edwin M. Southern developed procedures for detecting specific DNA fragments so that a particular gene could be isolated from a complex DNA mixture. This technique is called the Southern blotting technique. DNA fragments are separated by size with agarose gel electrophoresis. Gel electrophoresis takes advantage of the chemical and physical properties of DNA to separate the fragments. The phosphate groups in the backbone of DNA are negatively charged. This makes the DNA molecules attracted to anything that is positively charged. In gel electrophoresis the DNA molecules are placed in an electric field so that they migrate towards the positive charge. The DNA is placed in agarose, a semi solid gelatin, and placed in a tank of buffer. When electrical current is applied, the DNA molecules migrate through the agarose gel, separate, and travel toward the positive poles of the electric fields. The entire DNA

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fragments migrates through the gel. The larger DNA fragments have a harder time moving than the smaller ones, so the small fragments travel farther through the gel.

4.7.1.1 Artificial DNA: putti,tg together the pieces Oligonucleotides, from the Greek word oligo meaning "few," are short pieces of DNA or RNA that are 2 to 30 nucleotides long. The ability to synthesize DNA oligonucleotides of a known sequence is incredibly important and useful. A DNA probe is used to analyze fragments of DNA. A DNA probe is a single- stranded fragment of DNA that recognizes and binds to a complementary section of DNA in a mixture of DNA molecules. DNA probes can be synthesized and DNA fragments can be prepared for use in molecular techniques such as polymerase chain reaction (PCR). Poly~erase chain reaction is a technique that was developed by Kary Mullis in 1985. It produces large quantities of a DNA fragment without needing a living cell. Starting with one small piece of DNA, PCR can make billions of copies in a few hours. These large quantities of DNA can be easily analyzed. PCR and DNA probes have been of great value to the areas of molecular biology, medicine, and biotechnology. Using these tools, scientists can detect the DNA associated with HIV (the virus that causes AIDS), Lyme disease, chlamydia, tuberculosis, hepatitis, HPV (human papilloma virus), cystic fibrosis, muscular di strophy, and Huntington's disease.

4.7.2 Gene Therapy: Makes You Feel Better Gene therapy is a recombinant DNA process, in which cells are taken from the patient, altered by adding genes, and returned to the patient. A type of genetic surgery called somatic gene therapy may be possible. Cells of a person with a genetic disease could be removed, cultured, and transformed with cloned DNA containing a normal copy of the defective gene. They could be reintroduced into the individual. If these cells become established, the expression of the normal genes may be able to cure the patient. In the early 1990s, gene therapy of this type was used to correct a deficiency of the enzyme adenosine deaminase (ADA). An immune deficiency disease patient lacking the enzyme adenosine deaminase, an enzyme that destroys toxic metabolic byproducts, had been treated. Some of the patient's lymphocytes were removed. Lymphocytes are a type of white blood cell that fights infection. The lymphocytes were given the adenosine deaminase gene with the use of a modified

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retrovirus-which served as a vector-and placed back into the patient's body. Once established in the body, the cells with altered genes began to make the enzyme adenosine deaminase (ADA) and alleviated the deficiency. 4.7.3 DNA Fingerprinting DNA fingerprinting is an area of molecular biology that involves analyzing genetic material. It involves the use of restriction enzymes, which cut DNA molecules into pieces. When DNA samples obtained from different individuals are cut with the same restriction enzyme, the number and size of restriction fragments produced may be different. This difference provides the basis for DNA fingerprinting. The use of DNA fingerprinting depends upon the presence of repeating base sequences. These sequences are called restriction fragment length polymorphosis, or the RFLP pattern, which is unique for every individual. This is a sort of molecular signature or fingerprint. In order to perform DNA fingerprinting, DNA must be taken from an individual. Samples can be taken from hair, blood, skin, cheek cells, or other tissue. The DNA is taken from the cells and is broken down with enzymes. The fragments are separated with electrophoresis. The DNA fragments are then analyzed for RFLPs using DNA probes. An evaluation enables crime lab scientists (forensic pathologists) to compare a person's DNA with the DNA taken from a scene of a crime. This technique has a 99 percent degree of certainty that a suspect was at a crime scene.

4.7.3.1 Industrial application Industrial applications of recombinant DNA technology inc.\ude manufacturing protein products by the use of bacteria, fungi, and cultured mammal cells. The pharmaceutical industry is producing several medically important polypeptides using biotechnology. An example is bacteria that metabolize petroleum and other toxic materials. These bacteria are constructed by assembling catabolic genes on a single plasmid and then transforming the appropriate organism. Another example is vaccines. The hepatitis B vaccine is made up of viral protein manufactured by yeast cells, which have been recombined with viral particles. 4.7.3.2 Agricultural applications: crops and cows Recombinant DNA and biotechnology have been used to increase plant growth by increasing the efficiency of the plant's ability to fix

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nitrogen. Scientists take genes for nitrogen fixation from bacteria and place the genes into plant cells. Because of this, plants can obtain nitrogen directly from the atmosphere. The plants can produce their own proteins without the need for bacteria. Another way to insert genes into plants is with a recombinant tumor-inducing plasmid Ti plqsmid. This is obtained from the bacterium Agrobacteriul11 tUl11ejaciens. This bacteria invades plant cells and its plasmids insert chromosomes that carry the genes for tumor induction. An example of recombinant DNA with livestock is the recombinant bovine growth hormone that has been used to increase milk production in cows by 10 percent. U.S. farmers grow substantial amounts of genetically modified crops. About one-third of the corn and one-half of the soybean and cotton crops are genetically modified. Cotton and corn have become resistant to herbicides and insects. Soybeans have herbicide resistance and lower saturated fat content. Having herbicidal-resistant plants is important because many crop plants suffer stress when treated with herbicides. Resistant crops are not stressed by the chemicals that are used to control weeds. 4.7.4 Recombinant DNA Technology and Society GeneticalIy altering an organism raises scientific and philosophical questions. Recombinant DNA technology has had a positive impact on society, although there may be associated dangers with rDNA. There have been concerns raised by the scientific community that genetically engineered microorganisms carrying dangerous genes might be released into the environment and cause widespread infection. Due to these worries, the federal government has established guidelines to regulate and limit the locations and types of experiments that are potentially dangerbus. Biomedical rDNA research has been regulated by the Recombinant DNA Advisory Committee (RAe) of the Natural Institutes of Health. The Food and Drug Administration (FDA) has principal responsibility in overseeing gene therapy research. The Environmental Protection Agency (EPA) and state governments have jurisdiction over field experiments in agriculture. One of the biggest efforts in biotechnology has been the human genome project, which began in 1990 and formally ended in 200l. The goal of this project has been to determine the sequences of all human chromosomes. Advances like this in biotechnology will make genetic screening incredibly effective. Physicians will one day be

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capable of detecting genetic flaws in DNA long before the disease becomes manifested in a patient. Another area of controversy is agriculture. Some scientists state that the release of recombinant organisms without risk assessment may disrupt the ecosystem. Viral nucleic acids, inserted into plants to make them resistant to viruses, might combine with the genome of an invading virus to make the virus even stronger. Genetically modified food might even trigger an allergic response in people or animals that consume them. As of this writing, obvious health or ecological events have not been observed. However, due to the consensus of the public, many food producers have stopped using genetically modified crops.

5 Microbial Ecology S.t GENERAL INTRODUCTION This chapter explores the diversity, interactions and activities of microbes (microorganisms) within freshwater environments. These form an important part of the biosphere, which also includes oceans, terrestrial environments and the earth's atmosphere. S.1.t Aquatic Existence It is now generally accepted that life originated between 3.5 and 4 billion years ago in the aquatic environment, initially as selfreplicating molecules. The subsequent evolution of prokaryotes, followed by eukaryotes, led to the existence of microorganisms which are highly adapted to aquatic systems. Life in the aquatic environment (freshwater and marine) has numerous potential advantages over terrestrial existence. These include physical support (buoyancy), accessibility of three-dimensional space, passive movement by water currents, dispersal of motile gametes in a liquid medium, minimal loss of water (freshwater systems), lower extremes of temperature and solar radiation, and ready availability of soluble organic and inorganic nutrients. Potential disadvantages of aquatic environments include osmotic differences between the organism and the surrounding aquatic medium (leading to endosmosis or exosmosis) and a high degree of physical disturbance in many aquatic systems. In undisturbed aquatic systems such as lakes, photosynthetic organisms have to maintain their position at the top of the water column for light availability. In many water bodies (e.g., lake water column), physical and chemical parameters show a continuum - with few distinct microhabitats. In 175

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these situations, species compete in relation to different growth and reproductive strategies rather than specific adaptations to localized environmental conditions. 5.1.2 Global Water Supply - Limnology and Oceanography Water covers seven tenths of the Earth's surface and occupies an estimated total volume of 1.38 x 109 km;. Most of this water occurs between continents, where it is present as oceans (96.1 per cent of global water) plus a major part of the atmospheric water. The remaining 3.9 per cent of water, present- within continental boundaries (including polar ice-caps), occurs mainly as polar ice and ground water. The latter is present as freely exchangeable (i.e., not chemically-bound) water in subterranean regions such as aquifers at varying depths within the Earth's crust. Non-polar surface freshwaters, including soil water, lakes, rivers and streams occupy approximately 0.0013 per cent of the global water, or 0.37 per cent of water occurring within continental boundaries. The volume of saline lake-water approximately equals that of freshwater lakes. The largest uncertainty is the estimation of ground water volume. Annual inputs by precipitation are estimated at 5:2 x 108 km 3 , with a resulting flow from continental (freshwater) systems to oceans of about 38600 km 3 • The distinction between oceans and continental water bodies leads to the two main disciplines of aquatic biology - oceanography and limnology. 1. Oceanography is the study of aquatic systems between continents. It mainly involves saltwater, with major impact on global parameters such as temperature change, the carbon cycle and water circulation. 2. Limnology is the study of aquatic systems contained within continental boundaries, including freshwater and saltwater sites. The study of freshwater biology is thus part of limnology. Although freshwater systems do not have the global impact of oceans, they are of major importance to biology. They are important ecological features within continental boundaries, have distinctive groups of organisms, and show close links with terrestrial ecosystems. The two main sites (over 99 per cent by volume) of continental water - the polar ice-caps and exchangeable ground water - are extreme environments which have received relatively little microbiological attention until recent years. Although 1110st Iimnological

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studies have been carried out on lakes, rivers, and wetlands the importance of other water bodies - particularly the vast frozen environments of the polar regions - should not be overlooked. Although there are many differences between Iimnological (inland) and oceanic (intercontinental) systems, there are also some close similarities. The biology of planktonic organisms in lakes, for example, shows many similarities to that of oceans - and much of our understanding of freshwater biota (e.g., the biology of aquatic viruses) comes from studies on marine systems. 5.1.3 Freshwater Systems: Some Terms and Definitions

5.1.3.1 Freshwater microorganisms Microorganisms may be defined as those organisms that are not readily visible to the naked eye, requiring a microscope for detailed observation. These biota have a size range (maximum linear dimension) up to 200 p,m, and vary from viruses, through bacteria and archea, to micro-algae, fungi and protozo
5.1.3.2 Freshwater ellvirollmellts: water ill the liquid and frozen state Freshwater environments are considered to include all those sites where freshwater occurs as the main external medium, either in the liquid or frozen state. Although frozen aquatic environments have long been thought of as microbiological deserts, recent studies have shown this not to be the case. The Antarctic sub-continent, for example, is now known to be rich in microorganisms, with protozoa, fungi, bacteria, and microalgae often locally abundant and interacting to form highly-structured communities. New microorganisms, including freeze-tolerant phototrophs and heterotrophs, have been discovered and include a number of endemic organisms. New biotic environments have also been discovered within this apparently hostile environment - which includes extensive snow-fields, tidal lakes, iceshelf pools, rock crystal pools, hypersaline soils, fell-field microhabitats, and glacial melt-water streams. Many of these polar environments are saline, and the aquatic microbiology of these regions is considered here mainly in terms of freshwater snow-fields in relation

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to extreme aquatic environments and the cryophilic adaptations of micro-algae. This chapter deals with aquatic systems where water is present in the liquid state for at least part of the year. In most situations (temperate lakes, rivers, and wetlands) water is frozen for only a limited time, but in polar regions the reverse is true. Some regions of the ice-caps are permanently frozen, but other areas have occasional or periodic melting. In many snow-fields, the short-term presence of water in the liquid state during the annual melt results in a burst of metabolic activity and is important for the limited growth and dispersal of snow microorganisms and for the completion of microbial life cycles.

5.1.3.3 Freshwater and saline environments Within inland waters, aquatic sites show a gradation from water with a low ionic content (freshwater) to environments with a high ionic content (saline) - typically dominated by sodium and chlorine ions. Saline waters include estuaries, saline lakes and extensive regions of the polar ice-caps. The high ionic concentrations of these sites can also be recorded in terms of high electrical conductivity (specific conductance) and high osmotic potential. The physiological demands of saline and freshwater conditions are so different that aquatic organisms are normally adapted to one set of conditions but not the other, so they occur in either saline or freshwater conditions. The importance of salinity in determining the species composition of the aquatic microbial community was demonstrated in a recent survey of Australian saline lakes, where distinct assemblages of diatoms were present in low salinity (oligosaline) and high salinity (hypersaline) waters. Some diatom species, however, were present over the whole range of saline conditions, indicating the ability of some microorganisms to be completely independent of salt concentration and ionic ratios. Longterm adaptability to different saline conditions is also indicated by the ability of some organisms to migrate from saltwater to freshwater sites. and establish themselves in their new conditions. This appears to be the case for various littoral red algae of freshwater lakes, which were originally derived from marine environments. Differences between freshwater/saltwater environments and their microbial communities. are particularly significant in global terms, where the dominance of saline conditions is clear in terms of area coverage, total biomass, and overall contribution to carbon cycling.

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5.1.3.4 Lelltic alld lotic freshwater systems Freshwater environments show wide variations in terms of their physical and chemical characteristics, and the influence these parameters have on the microbial communities they contain. These aspects are considered further, but one important distinction needs to be made at this stage - between lentic and lotic systems. Freshwater environments can be grouped into standing waters (Ientic systems - including ponds, lakes, marshes and other enclosed water bodies) and flowing waters (lotic systems - rivers, estuaries and canals). The distinction between len tic and lotic systems is not absolute, and almost all water bodies have some element of throughflow. 5.1.4 Biology of Freshwater Microorganisms The biology of freshwater microorganisms is considered from five major aspects: 1. Microbial diversity and interactions within ecosystems; these interactions include temporal changes in succession and feeding (trophic) interactions. 2. Variations between different environmental systems, including lakes, rivers, and wetlands. Each system has its own mixture of microbial communities, and its own set of physical and biological , characteristics. 3. Characteristics and activities of the five major groups of microbial organisms - algae, bacteria, viruses, fungi, and protozoa. 4. The requirement of freshwater microorganisms for two major environmental resources - light and inorganic nutrients. These are considered immediately after the section on algae, since these organisms are the major consumers of both commodities. 5. The microbial response to eutrophication. Environmental problems asss>ciated with nutrient increase are of increasing importance and reflect both a microbial response to environmental change and a microbial effect on environmental conditions. 5.2 BIODIVERSITY OF MICROORGANISMS 5.2.1 Domains of Life With the exception of viruses (which constitute a distinct group of non-free-living organisms) the 1110St fundamental element of taxonomic diversity within the freshwater environment lies in the

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separation of biota into three major domains - the Bacteria, Archaea, and Eukarya. Organisms within these domains can be distinguished in terms of a number of key fine-structural, biochemical, and physiological characteristics. Cell organization is a key feature, with the absence of a nuclear membrane defining the Bacteria and Archaea as prokaryotes. These prokaryote domains also lack complex systems of membrane-enclosed organelles, have 70s ribosomes and have genetic systems which include plasmids and function by operons. Prokaryote features also include a unicellular or colonial (but not multicellular) organization, and a small cell size « 5 p,m diameter). The domain Bacteria is widely represented in all freshwater environments and contains a single kingdom, the Eubacteria, which includes bacteria, actinomycetes and blue-green algae. In contrast to this, members of the domain Archaea (single kingdom Archaebacteria) tend to be restricted within freshwater environments to extreme situations. The domain Eukarya takes its name from the fundamental eukaryote organization of its cells and may be conveniently divided into four kingdoms - the Protista (unicellular and colonial organisms), Plantae (multicellular photosynthetic eukaryotes), Fungi and Animalia (multicellular heterotrophic eukaryotes). The Protista include two important groups of freshwater microorganisms - the micro-algae and protozoa.

5.2.2 Size Range Size is an important parameter for all freshwater microorganisms, affecting their location within the freshwater environment, their biological activities, and their removal by predators. The importance of size and shape in planktonic algae is considered later, and includes aspects such as predation by other organisms, sinking rates, and potential growth rates. In the case of free-floating (planktonic) organisms, the maximum linear dimension ranges from <0.2 p,m-> 200 p,m, with separation of the biota into five major size categories, from femtoplankton to macroplankton. 5.2.2.1 Femtopiallktolt «0.2 p,m)

The distinction between particulate (insoluble) and non-particulate (soluble) material in freshwater systems is usually defined in terms of retention by a 0.2 p,m pore-size filter membrane. On this basis, the smallest size group, the Femtoplankton «0.2 p,111) falls within

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the non-particulate category and the constituent viruses and small bacteria are strictly part of the dissolved organic material (DOM) or dissolved organic carbo11 (DOC) of the freshwater environment.

5.2.2.2 Picoplanktoll (0.2-2 JLm) This group is almost entirely composed of prokaryotes (bacteria and blue-green algae) with potentially rapid growth rates and the ability to carry out rapid colonization of freshwater environments. These organisms have negligible sinking rates, and are subject to significant predation by small rotifers, protozoa, and filter-feeding crustaceans. Some large linear viruses (family 1l1oviridae) also fit into this size categor and have been linked to the infection of bacterial populations. 5.2.2.3 Nanoplankton (2-20 JLm) Typically eukaryote flagellated unicellular organisms, this group includes fungal zoospores, algae, and protozoa. These organisms are the principal food of micro- and macro-zooplankton, and have low sinking rates and high potential growth rates. Within this assemblage, unicellular algae, along with picocyanobacteria, are particularly important in the short-term development of algal blooms which may occur during brief growing seasons or at various points in a more prolonged seasonal sequence. 5.2.2.4 Microplankton (20-200 JL11l) Larger microplankton are retained by traditional -70 JL111 mesh size phytoplankton nets, and are highly prone to sinking in the absence of buoyancy aids. These organisms are consumed by larger crustacea, and are also the principal food of pelagic and benthic omnivorous fish. Growth rates are moderate to low. 5.2.2.5 Macroplankton (>200 JL11l) These have similar biological features to the larger microplar..kton, and are characterized by the colonial blue-green algae and by the multicellular zooplankton (rotifers and crustacea). The biology of mesotrophic and eutrophic lakes in temperate climates are typically dominated by these size categories over the summer growth period, with separate population peaks of colonial nlgae (diatoms, blue-greens) and zooplankton (crustacea) at different times of year. Although macroplanktonic organisms are chara~·teristically slow-growing, they typically make the greatest contributioll to biumass' under conditions of adequate nutrient supply. Differences betw,~en picoplankton and

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macroplankton in terms of growth rate, sl'''rt-term colonization and long-term domination of freshwater enviro. lents reflect fundamental differences in evolutionary selection strategy and the way these biota are adapted to different environmental conditions. The distinction between small size (r-strategist) and large size (K-strategist) organisms is considered more fully below.

5.2.3 Autotrophs and Heterotrophs Freshwater microorganisms may be divided according to their feeding activity (trophic status) into two major groups: 1. Autotroplzs - synthesize their complex carbon compounds from external CO,. Most also obtain their supplies of nutrient (e.g., nitrogen and phosphorus) from simple inorganic compounds. These phototrophic microorganisms include microalgae and photosynthetic bacteria, and are the main creators of biomass (primary producers) in many freshwater ecosystems. This is not always the case, however, since photosynthetic microorganisms are out-competed by larger algae and macrophytes in some aquatic systems, particularly wetland communities. 2. Heterotrophs - use complex organic compounds as a source of carbon. By far the majority of freshwater microorganisms (most bacteria, protozoa, fungi) are heterotrophic. Even within the algae, various groups have evolved the capacity for heterotrophic nutrition and many organisms currently included in the protozoon assemblage have probably evolved from photosynthetic ancestors. Hetrrotrophic nutrition involves a wide diversity of activities with microorganisms obtaining their organic material in three main ways - saplOtrophy, predation, and in association with living organisms. Saprotrophic organisms obtain their nutrients from non-living material. This may be assimilated in three main ways: direct uptake as soluble compounds (chiefly bacteria), indirect uptake by secretion of exterllal enzymes (exoenzymes) followed by absorption of the hydrolytic products (bacteria and fungi), and ingestion of particulate matter by phagocytosis (protozoa). Predation is carried out by protozoa, and involves capture, ingestion, and internal digestion of other living organisms such as bacteria and algae. Protozoa can capture their prey either by active motility or, as sedentary organisms, by the use of filter feeding processes. The third major aspect of heterotrophy involves associations with living organisms and includes parasitism and symbiosis. Parasitism almost invariably involves a strict dependence of the parasite on the host organism as part of the

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parasitic life cycle, though in some cases the benefits to the parasite are not entirely clear. The common presence of Opalinid protozoa in the gut of adult amphibia. for example, is thought to involve nourishment from mucous secretions and gut fluids, but there is no penetration of host tissues or any apparent adverse effects on the host. As with other freshwater parasites, this organism has a welldefined life cycle that fits in with the aquatic biology of the host. Commensal microorganisms such as epiphytic bacteria, protozoa, and algae derive no direct nutrient from their associated host, though there may be indirect nutritive benefits. These are seen in the phycosphere community that occurs in association with mucilaginous colonial algae, where a close coupling occurs between the local growth of bacterial populations and the trophic activities of bacterivorous protozoa. Within the nutritive diversity, particular heterotrophic activities stand out as having special ecological significance. These include the uptake of DOC by planktonic bacteria, breakdown of detritus by benthic organisms (bacteria, fungi, and protozoa) and limitation of phytoplankton populations by parasitic activity (viruses and fungi). The importance of heterotrophic nutrition in the freshwater environment runs as a theme throughout the book, with specific examples discussed in appropriate sections.

5.2.3.1 Autotrophic and heterotrophic actiJ7ities within communities The overall balance between autotrophic and heterotrophic activities within freshwater communities is an important aspect of ecosystem function. This determines the net exchange of carbon with the surrounding atmosphere (which influences global warming) and can be expressed as the net ecos)'stem production (NEP). where NEP = P - R and P = primary productivity (carbon uptake during photosynthesis) R = community respiration (carbon loss by respiratory breakdown). NEP will have a positive value in ecosystems that are net autotrophic (P > R), and a negative value where the community is net heterotrophic (P < R). The balance between P and R is determined by the relative metabolic contributions of autotrophic and heterotrophic organisms, which in turn relates to environmental parameters such as light availability (promoting photo trophy) and availability of externallyderived (allochthonous) carbon-promoting heterotrophy.

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Benthic communities vary from net heterotrophy (e.g., profundal sediments, dominated by anaerobic heterotrophic bacteria) to net autotrophy (e.g., shallow lake sediments, dominated by benthic algae). Pelagic communities typically have greater light exposure than benthic systems, and (in lentic water bodies) often have a greater autotrophic contribution. The balance of available nutrients is important, and low nutrient (oligotrophic) lakes are typically net heterotrophic while high nutrient (eutrophic) lakes show net autotrophy. This differ.ence arises because oligotrophic conditions support only limited carbon uptake by phototrophy (low inorganic nutrient availability) but significant heterotrophy can still occur due to input of allochthonous carbon. In eutrophic lakes, both modes of nutrition are increased, but the increased availability of soluble inorganic nutrients promotes autotrophy to a greater extent. The pelagic communities of lotic systems may also show great variation in net ecosystem production. This is seen particularly well in some estuaries, where the value for NEP shows marked seasonal fluctuation with river inflow. 5.2.4 Planktonic and Benthic Microorganisms Freshwater organisms can be divided into two main groups, according to where they spend the major part of their growth phase - pelagic organisms (present in the main body of water) and benthic organisms (associated with the sediments). Pelagic biota can be further subdivided into nekton (strongly swimming organisms such as fish) and plankton (free-floating). The latter tend to drift within the water body, though they may have limited motility and achieve vertical movement. Pelagic microorganisms are essentially plankton, so the distinction within this assemblage is between planktonic and benthic states. Although freshwater organisms can be categorized as planktonic or benthic, most species have both planktonic and benthic phases within their life cycle. Biofilm bacteria, for example, have a dynamic equilibrium between attached and planktonic states, and freshwater algae also show clear planktonic/benthic interactions in lake and stream environments. Planktonic and benthic phases of the same species typically show considerable differences in metabolic activity. This is particularly the case for freshwater bacteria, where quorumsensing mechanisms lead to the induction of stationary-phase physiology i~ biofilm communities.

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5.2.4.1 Planktonic microorganisms These biota frequently have specialized mechanisms to migrate or maintain their position vertically within the water column, and are particularly well represented by micro-algae and bacteria. Planktonic organisms can be further divided into holoplanktonic forms (where the organism is present in the water column for a major part of the annual cycle) and meroplanktonic organisms (where the planktonic phase is restricted over time) biota. The distinction between holoplanktonic and meroplanktonic species is particularly well shown by the algae, where the meroplanktonic state can beviewed as an adaptation for short-term competition. 5.2.4.2 Benthic microorganisms Present at the surface and within sediments, these are dominated by biota such as fungi, protozoa, and bacteria that are able to break down sedimented organic debris. The diversity of benthic life forms is particularly well represented by the protozoa, which include both attached and freely-motile organisms, with a variety of feeding mechanisms. A number of key terms have been used to define particular groups of benthic organisms, including one major group - the periphyton. This term is used to describe all the 'plant-like' microorganisms (microflora) present on substrata, including microscopic algae, bacteria and fungi. This term excludes 'animal-like' organisms, such as micro-invertebrates and protozoa, but includes filamentous algae (e.g., Cladophora, Spirogyra, Chara, and Vaucheria). Differences occur within the periphyton in terms of the nature of the substrate, which may be living (e.g., plant surfaces) or nonliving (organic or inorganic, with different particle sizes). 1. Epiphytic organisms are associated with the surfaces of higher plants and macroalgae. The substratum in this case is often metabolically active, and the epiphytic association may be important in terms of competition for light, metabolic exchange and nutrient availability. 2. Epilithic, epipsammic and epipelic microorganisms grow on non-living substrates which differ in particle size. Epilithic biota occur on hard, relatively inert substrata such as gravel, pebbles and large rocks. Decrease in particle size leads to epipsammic organisms (present on sand) and epipelic organisms (present on fine sediments such as mud). Relatively few of the larger microorganisms, such as algae, live in sand, since particles are

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too unstable and may crush them. Quite large algae are present on fine sediments, however, including large motile diatoms, motile filamentous algae and large motile flagellates such as Euglena. A further group of plant-like benthic organisms includes the metaphyton, which are loosely associated (but not attached) to the substratum. These are characterized by clouds of filamentous green algae such as Spirogyra, Mougeotia or Zygnema, which become loosely aggregated and accumulate in regions of substratum that are free from currents and waves. Metaphyton usually arises from other substrata, such as higher plant surfaces (epiphyton). 5.2.5 Metabolically Active and Inactive States Although freshwater microorganisms are often thought of as dynamic, metabolically active biota, this is not always the case. All species have inactive or inert periods for at least part of their life cycle; these may occur as temporary dormant phases (surviving adverse conditions) or terminal phases of senescence, leading to death.

5.2.5.1 Dormant phases In temperate climates, particularly, many microorganisms overwinter on the sediments in a dormant state. The formation of resistant spores typically occurs as environmental conditions deteriorate (overcrowding, reduced nutrient and light availability, accumulation of toxic metabolites, reduced temperature), and may be preceded by sexual reproduction. In some cases, dormancy appears to relate to a specific environmental change, such as oxygen concentration. In the water column of lakes, obligate anaerobes such as photosynthetic purple bacteria are metabolically active in the anaerobic hypolimnion during stratification, but overwinter in flocs of organic material once the lake becomes oxygenated at autumn overturn. In bacteria and viruses, metabolic inactivity is also nutrientrelated. Comparisons of total and viable bacterial counts suggest that only a small fraction of aquatic populations are metabolicallyactive. Under certain environmental conditions, bacteria remain inert until they encounter appropriate growth conditions - particularly in relation to nutrient supply. Viruses are also metabolically inactive within the water medium, and are only activated when they encounter a healthy host cell. In the inert state, they occur as free particles (virions) within the water medium, and are vulnerable to inactivation and loss processes.

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5.2.5.2 Senescence Senescence and cell death are just as much a part of population development as growth processes, and play an important role in the dynamics of microbial communities. In some situations, death is induced by specific environmental factors such as nutrient limitation, solar irradiation and parasitism. In other situations, senescence may occur due to internal changes, particularly after a long sequence of divisions and probably due to programmed cell death. The occurrence of growth and senescence within a single-species population is taken from a stationary phase in a laboratory monoculture of the green alga Micrasterias. Mature cells (M) of this placoderm desmid have two halves (semi-cells) of equal size, each with a single chloroplast. Cells that are undergoing senescence (S) show condensation of the chloroplasts to the centre of the cell, leading to degeneration and the formation of colourless dead cells that just have the remains of the cell wall. Actively growing cells that have recently divided (inset) have one normal sized semicell (b) derived from the mother cell, plus a new semicell (a) that will attain full size on completion of the growth cycle. Although cell differences in growth cycle and the onset of senescence are particularly clear in populations of this organism, they are also characteristic of other populations where they are not so easily observed. Witl1in phytoplankton populations at the top of the water column, for example, senescence and cell death occurring in a wide range of species results in a continuous rain of mixed organisms to the lower part of the water column and the sediments. These can be collected in sediment traps suspended in the water column and subsequently analy~ed.

5.2.6 Evolutionary Strategies: r-selected and K-selected Organisms In the population of cells, regeneration (formation of new organisms) and death continuously occur. The growth of populations that include continuous cycles of regeneration and death is best described by 'models of continuous growth', and leads to a consideration of the importance of competition in population increase and the designation of two opposing evolutionary strategies - adopted by r-selected and K-selected organisms.

5.2.6.1 Exponential and sigmoidal models of continuous growth The speed with which a single-species population (N) increases with time will be denoted by dN Ielt. The increase in size of the

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whole population is the sum of the growth contribution of all individuals, so the rate of increase per individual (r) will be:

r= dN[~] dt N or

rN= dN dt The parameter r is also referred to as the 'per capita rate of increase' and describes the fundamental ability of individual organisms to grow and reproduce. The above equation describes population growth in conditions of unlimited resources and generates an exponential increase in population with time. In practice, intra-specific competition for resources (particularly nutrients and space) must also be taken into account, and the population growth curve then becomes sigmoidal. In this situation, the population (N) rises to a maximum value (K), which is the maximum value (the carrying capacity) that the environment can support. The growth equation which describes the sigmoidal curve is referred to as the 'logistic equation' and has the form:

dN =rN[K-N] dt K This sigmoidal curve is well-known to microbiologists, where the growth of microorganisms in batch culture follows a sequence of lag, logarithmic and stationary phases. In the lower part of the curve (lag phase), when population is sparse, the population is beginning to colonize the new environment and relatively little competition occurs between cells. As the population approaches the carrying capacity (K), resources (nutrients, space) become limited and high levels of competition occur between cells.

5.2.6.2 r-selected and K-selected organisms The conditions seen in the single-species growth curves of organisms cultured under laboratory conditions can also be applied to freshwater environments, where mixed populations occur. Some environments have low population densities, and are dominated by organisms that are adapted for high rates of growth and rapid colonization, while others have high population densities and are dominated by organisms that are adapted to survive in these highly competitive conditions.

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This fundamental distinction between two major types of adaptation was first described by MacArthur and Wilson (1967) in relation to differences in selection pressure at different phases in the colonization of oceanic islands. These authors separated organisms into r-strategists, which were the initial colonizers of the island environment, and K-strategists - adapted to the more crowded, later conditions. In uncrowded environments, organisms able to grow and reproduce rapidly, with high productivity, will be most suited to dominate the environment. They are subsequently replaced by organisms which are more suited to a crowded community that is approaching its population limit and are referred to as K-strategists. The terms rand K are derived from the logistic equation for population growth, which specifies that the per capita rate of increase (r) is maximized under sparse conditions. With increasing crowding, a decline in the per capita rate of increase occurs until the population density equilibrates to its upper level or carrying capacity (K). The adaptation of r- and K-strategists to environments of differing population density has a range of biological implications, including differences in biological diversity, competition, accumulation of metabolites, resource availability, and liability to parasitic attack. The incidence of ecological stress can be high or low in either situation, depending on the particular circumstances. The adaptation of r-strategists to uncrowded conditions also makes them suitable to unstable environments, where growth is limited to short periods of time and high population levels cannot become established. Under such conditions the ability of these organisms to grow rapidly and exploit growth opportunities as they become available gives them a competitive advantage. In line with this, r-strategists are typically small, with a short life cycle and high growth rate. In contrast, Kstrategists are adapted to stable environmental conditions, where dense populations develop and rapid growth is not an advantage. All groups of freshwater microorganisms contain r- and K-selected species, and all aquatic environments have situations (during colonization, and at different times in the mature community) when r- and K-strategists are respectively adapted to the prevailing situation. Planktonic bacteria are particularly good examples of r-strategists, existing in a metabolically inert form for much of their life cycle, but able to grow and multiply within a short space of time when nutrients become available. Phytoplankton contain well-adapted examples of both types of organism, with changes in dominance of r- and Kselected algae during the seasonal progression. One particular group

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of algae, the dinoflagellates, are perhaps the best example of all microbial K-strategists. 5.3 BIODIVERSITY IN ECOSYSTEMS, COMMUNITIES, AND SPECIES POPULATIONS Individual microorganisms are part of larger groups or communities, and any consideration of biodiversity must take into account the wider community and the environment in which it occurs. A community is a naturally occurring group of organisms living in a particular location, such as a lake or stream. The occurrence and interactions of organisms living within a discrete environment constitutes a functional unit, referred to by Tansley (1935) as an ecological system or 'ecosystem'. This was considered by Tansley to consist of two major components - the biome or ecosystem community (the entire group of organisms) and the habitat (physical environment). Each particular ecosystem can be regarded to some extent as self-contained and as a basic unit of ecology. Ecosystems are themselves part of a larger geographic or global unit, the biosphere, which is the sum of all ecosystems within a particular zone. ECOLOGICAL UNIT

l\lAIN ECOSYSTEM le g. J.. kC. nver. v.·ell.and)

SUBSIDIARY COl\lMUNITIES 'c.g .• hh,lill11. pdaglc communlly)

SINGLE SPECIES POPULATION te g.• I,lIlglc !tlgal 'p~t'I(!'" waler column)

III

HlOLOGICAL DIVERSITY MIxture uflubsidiary ecosystems, each with il.l' ()WII c01llmunity

Single cOll/IlI/mity, with: • Different types of organism • VlIrietyof species

iJil't'rsity within 6pecies - TIIaleeular, chemical, and pizysiait)gil"al variation

Figure 5.1 Diversity wIthin species, communities, and groups of communities at different levels of organization in freshwater systems.

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The nature and evaluation of community diversity partly depends on environmental scale and can be considered as a hierachy of three levels - main ecosystems, subsidiary communities, and species populations. 5.3.1 Main Ecosystems Major aquatic environments such as lakes, rivers and wetlands form a discrete ecological unit, with their own characteristic community of organisms; they can be referred to as main ecosystems. Each of these main ecosystems contains a diverse array of distinctive groups of organisms (subsidiary communities), each in their own particular environment, forming subsidiary ecosystems. As an example of this, a typical temperate lake (main ecosystem) can be divided into two major regions - the peripheral shoreline (littoral) zone and the central zone, each with its own subsidiary communities and ecosystems. The central zone can be separated into pelagic and benthic groups of organisms, with further resolution within the pelagic zone of neuston, phycosphere and photosynthetic bacterial communities. These occur in discrete small-scale environments, and are entirely composed of microorganisms. The littoral zone of the lake is dominated by attached organisms, which in eutrophic lakes can be divided into three major groups macrophyte, upper periphyton and lower periphyton communities. Within these, various distinctive micro-communities occur including epiphytic communities on macrophyte leaves, and algal and bacterial biofilms on exposed rock surfaces. Although these various groups of organisms have a degree of autonomy, they all interrelate within the main ecosystem. The pelagic and benthic communities, for example, may appear very distinct during the summer growth phase of eutrophic lakes but the benthic community depends on continuous biomass input from the pelagic zone by sedimentation, and populations of planktonic organisms arise by recruitment from the benthic zone. 5.3.2 Diversity within Subsidiary Communities Different communities have their own distinctive pattern of organisms with their own level of biodiversity. Within these communities, this diversity can be measured in various ways including variation in size of organisms, presence of mucilaginous and non-mucilaginous types, attached and free-floating biota and proportions of autotrophic and heterotrophic species. In practice,

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diversity is often determined simply in terms of species content. The variety of algae within phytoplankton is considered in terms of indices of species diversity. Significant changes occur in these indices during seasonal development of phytoplankton, indicating fundamental changes in community diversity throughout the growth season.

5.3.3 Biodiversity within Single-species Populations Phenotypic and genetic diversity within the ecosystem populations of individual species represents an important but relatively unexplored area of biological diversity in freshwater systems. One of the problems in studying variability at this level is that classical biochemical (testtube) techniques are difficult to apply to single species within mixed populations. The use of analytical microscopical techniques, however, has gone some way to overcoming this problem since these have sufficient spatial resolution to determine the chemical and molecular composition of single microorganisms within mixed populations. Some of these techniques are discussed in relation to algal populations and include the use of light microscope infrared spectroscopy to study the vibrational states of different molecular groups and the use of scanning electron microscope X-ray micro-analaysis to determine variations in elemental composition. Both of these approaches have demonstrated considerable variability within the single-species populations that make up the mixed phytoplankton assemblage, indicating that intra-specific variation in planktonic algae is an important feature of the pelagic environment. Molecular techniques also have considerable potential for looking at biodiversity within single species populations, and have been used to study various aspects of genetical variation within natural populations. These include sub-species (strain) variation in blue-green algae and variations in plasmid-borne resistance in aquatic bacteria. Differences between planktonic and attached (biofilm) bacteria in terms of the expression of stationary phase genes also constitute an important an important intraspecific variable within benthic systems. Ecosystems vary in size and composition, and contain a wide range of organisms which interact with each other and with the environment. Individual ecosystems have a number of important properties: (i) a distinct pattern of interactions between organisms, (ii) defined routes of biomass formation and transfer, (iii) maintenance of the internal environment, and (iv) interactions with the external environment.

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The range of organisms present in aquatic communities define and characterize the system concerned, and are involved in the generation and transfer of biomass. They have distinct roles and interactions within the ecosystem, occupying particular trophic levels and forming an interconnected system of feeding relationships (the food web). The balance of individual species within the food web is determined primarily by resource (light, nutrient availability) and competition. This in turn affects variety in the range and proportions of the different organisms (biodiversity), with important implications for the overall functioning of the system. Community structure is closely related to ecosystem stability and physical stress levels in the environment. Interactions between ecosystems and their surrounding environment occur in various ways. One example of this is the net exchange of carbon between the aquatic ecosystem and the adjacent atmosphere, which can be quantified in relation to net ecosystem production (NEP). 5.4 BIOFILM COMMUNITY: A SMALL-SCALE FRESHWATER ECOSYSTEM This section considers the dynamic properties of freshwater communities within two ecosystems where microorganisms have a key role - the microbial biofilm and the pelagic ecosystem of lakes. The microbial biofilm is a small-scale ecosystem composed entirely of microorganisms, while the pelagic ecosystem is a large-scale functional unit containing a wide range of biota. Contrasts between the diagrams to some extent reflect diversity in research approach rather than fundamental ecosystem differences. In the case of biofilms, for example, a lot of information has been obtained on genetic interactions, but relatively little is known about food webs. The reverse is true for pelagic ecosystems. Microbial biofilms provide a useful model system for considering fundamental aspects of community interactions and ecosystem function. Their small scale makes them amenable to laboratory as well as environmental experimentation, and the close proximity of organisms within the biofilm leads to high levels of biological interaction. Within the context of this book. the exclusively microbial composition of biofilms also makes them particularly appropriate for consideration. Microbial biofilms occur as discrete communities within a gelatinous matrix, and are present as a surface layer on rocks and stones in lakes and rivers. The biological composition of biofilms

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varies with environmental conditions, including factors such as ambient light intensity, water flow rate and prior colonization history. In some cases they are entirely bacterial, while in others they initiate with the settlement of diatoms and develop into larger scale periphyton communities. The biofilm is a mature mixed biofilm, such as might occur on a river substratum under conditions of limited light. and consists of a balanced community composed mainly of bacteria, with algae, protozoa, and fungi also present. The organisms are largely present within a gelatinous matrix, which defines the extent of the ecosystem. The matrix typically has a columnar appearance, with channels or pores between the columns through which water percolates. The microbial biofilm shares with other small-scale microbial (e.g., neuston and phycosphere) ecosystems and with larger ecosystems the four key aspects of ecosystem function listed above - interactions between organisms, biomass transfer, homoestasis and interactions with the external environment.

5.4.1 Interactions between Microorganisms Microbial interactions occur both within the main population of bacteria and in relation to other microorganisms. Bacterial interactions determine the structure and diversity of the biofilm and include gene transfer, quorum sensing and specific adhesion processes. Trophic (feeding) interactions occur between different groups of biota and can be considered in terms of food webs.

5.4.1.1 Gene transfer Microbial biofilms are particularly important in relation to gene transfer between bacterial cells, since they are a part of the aquatic environment where the transfer process is optimized due to the close proximity of the organisms concerned. Gene transfer between bacterial cells has been studied under both laboratory and environmental conditions. 5.4.1.2 Quorum sensing The physiological activities of bacteria vary considerably in relation to population density (number of cells per unit volume of medium). The pattern of gene activity, in particular, differs markedly within a single bacterial species between the sparse planktonic populations that occur in the general water medium and the dense community of cells present in the microbial biofilm. The mechanism underlying this difference in gene expression is referred to as 'quorum sensing'

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and depends (in Gram-negative bacteria) on the release of the signal molecule acyl homoserine lactone (AHL) into the water medium. At low population density, release of AHL results in the formation of low signal concentrations in the water medium, and no quorum response. At high population density, the concentration of AHL in the water reaches a critical level, activating DNA transcription factors (by binding to them) and triggering the activation of AHL-responsive genes. These AHL-responsive genes have been shown to be present in a wide range of biofilm bacteria (including Pseudomonas aeruginosa) and to operate in environmental (stream) biofilms. They encode a variety of cell functions, including population density regulation, pseudomonad virulence factors and stationaryphase characteristics. The induction of stationary-phase physiology is an important feature of biofilms, distinguishing these organisms from their planktonic counterparts. In Pseudomonas aeruginosa, the stationary phase state is caused by induction of a stationary phase sigma (transcription) factor known as RpoS, which activates a wide spectrum of stationary phase characteristics. These include greater antibiotic resistance, decrease in protease secretion, reduced motility and higher levels of extracellular polysaccharide (EPS) synthesis. These changes promote the formation of the biofilm (EPS) matrix, and the retention of cells within the surface film (reduced motility), and are thus crucial in forming and maintaining the biofilm ecosystem.

5.4.1.3 Specific adhesion processes Specific adhesion mechanisms are important in the secondary colonization and maturation of bacterial biofilms. During biofilm development, different species of bacteria enter the community at different times, and there is a need for specific recognition systems to maintain the necessary sequence and hierarchy of association. This is achieved by specific co-aggregation, which involves receptormediated recognition and specific binding between different species of bacteria. 5.4.1.4 Trophic interactions Ingestion of biofilm bacteria, diatoms and blue-green algae is carried out by protozoa that move over exposed submerged surfaces. These protozoa include hypotrich ciliates, hypos tome ciliates (with ventral mouths) plus bodonid and euglenoid flagellates. Ingestion of biofilm microorganisms is the first stage in the sequence of biomass

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transfers that forms the food web of the biofilm ecosystem, but also connects with the food web of the wider lake or river ecosystem. In addition to protozoa, surface biofilms are also grazed by larger invertebrates such as snails and larvae of Ephemeroptera and Trichoptera. These larger organisms are extraneous to the biofilm community. 5.4.2 Biomass Formation and Transfer Relatively little is known about biomass formation and transfer in aquatic biofilms. The presence of autotrophic algae such as diaroms and blue-green algae generates fixed carbon by /' photosynthesis, and some release of dissolved organic carbon (DOC) would be expected. Biomass transfer occurs via ingestion of cellular and other particulate material by protozoa and invertebrates (see above), and also by assimilation of secreted organic material such as DOC and matrix by bacteria and fungi. Extraneous organic material may also enter biofilms and become part of the biomass transfer. 5.4.3 Maintenance of the Internal Environment The microbial biofilm is a mature film that has arisen from a sequence of colonization processes. This has resulted in a stable and balanced internal environment which has two major components: 1. The gelatinous matrix, secreted by the bacteria, and forming a complex architecture that includes internal spaces (pores and channels) with a water circulation connecting to the outside medium. 2. Populations of different microorganisms that are in a state of balanced equilibrium. Once the biofilm has become established, the internal environment will be maintained and controlled by the resident microorganisms. This involves the following processes. (a) Continued production of gelatinous matrix. Continued secretion of the extracellular polysaccharide (EPS) matrix by bacterial cells tends to balance the loss caused by detachment of pieces of biofilm at the water surface and entrainment in the current. (b) Controlled population growth. Growth rates of bacteria within the biofilm are controlled by the quorum sensing system that acts as a negative feedback process. High population densities trigger the induction of stationary phase characteristics, including reduced rates of cell division. If the population of bacteria

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becomes depleted (e.g., due to ingestion) the quorum control will cease to operate, and division will increase. (c) Balance of mixed populations. The balance of different organisms will be determined by differential growth rates, stable food webs and specific adhesion mechanisms. The latter is particularly important during biofilm development and maturation, when the changing pattern of species composition that accompanies the colonization process is determined by differential recognition and adhesion characteristics. These features may also control the entry of bacteria into the mature biofilm and their retention within the community. 5.4.4 Interactions with the Exter.nal Environment Although microbial biofilms operate as a functional (and to some extent self-contained) unit, they are not separate from either their physicochemical or biological surrounding environment. Important physicochemical characteristics include light (required for photosynthesis), inorganic nutrients, soluble organic nutrients and dissolved oxygen. The entry and exit of soluble components into and out of the biofilm is promoted by internal water circulation, and leads to concentration gradients within the gelatinous matrix. These concentration gradients may be important determinants of local biofilm physiology and microstructure. The potential importance of nutrients is indicated by laboratory studies on monospecies biofilms, which have suggested that biofilm structure is greatly influenced by the concentration and quality of nutrients. The external biotic environment also has direct influences on biofilms. These are exerted through invertebrate grazing activities, and also entry and loss of resident microbes at the water interface. The entry of particulate (by sedimentation) or soluble (water flow) organic materials into the biofilm is a further biotic effect that provides important substrates for heterotrophic nutrition. 5.5 PELAGIC ECOSYSTEM Pelagic ecosystems occupy the main water body of the lake and contain the largest subsidiary community within the main lake ecosystem, encompassing all the free floating (planktonic) and strongly swimming (nektonic) organisms. Pelagic ecosystems of lakes occupy the largest volume of all freshwater environments (excluding snow and ice systems), and show close similarities to the marine pelagic ecosystems of seas and oceans.

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5.5.1 Interactions between Organisms A range of defined interactions occur between microorganisms in pelagic lake ecosystems, including competition for resources, antagonism, trophic interactions and epiphytic associations. Competition for resources such as light and inorganic nutrients (silicate, phosphate, nitrate) are an important contributory factor in determining the relative ability of different species populations to grow and dominate the pelagic environment. In cases where competition for more than one nutrient is involved, a change in algal dominance may occur with shift in nutrient balance. Antagonistic interactions may also occur as part of the competition for resources, allowing the population of the successful antagonist to access the resources and also generating nutrients from the target organism. Freshwater bacteria in particular, which are known to produce a range of anti-algal metabolites, may be important as antagonists in the termination of algal blooms, and are also potentially useful as biological control agents. Dominance of bluegreen algae in bloom conditions may also be regarded as antagonistic activity, since the low CO/ high pH micro-environment created by these organisms inhibits the growth of eukaryote algae in the top part of the water column. Trophic interactions in the pelagic zone are often very specific, and may result in close coupling of particular microbial populations. This is the case for heterotrophic bacteria and phytoplankton, which are linked by algal DOC production. A trophic link also occurs between bacteria and predatory heterotrophic nanoflagellate (HNF) protozoa, with a buildup of HNF populations in some lakes at times of high bacterial count. Parasitic viral and fungal infections of bke bacteria and phytoplankton provide examples of highly spec ific microbial interactions and are ecologically important in limiting host population growth and productivity. Epiphytic associations are common in the pelagic environment, with many unicellular organisms (bacteria, protozoa and algae) occurring on (or within) the surface of larger biota such as colonial algae and zooplankton. With the exception of blue-green algal heterocyst bacteria, little is known about the possible exchange of nutrients between epiphyte and host. Whether this occurs or not, epiphytic sites such as colonial blue-green algae are trophically significant in providing microcosms of microbial activity, each with its own localized food ·'\·eb. Epiphytic sites also provide a point of

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attachment in a part of the lake that is otherwise devoid of substratum, and allow planktonic organisms the opportunity to exist in equilibrium with a sedentary (non-planktonic) phase.

5.5.1.1 Seasonal changes in a temperate lake The importance of microbial interactions in the pelagic ecosystem of temperate lakes is seen in the seasonal progression of biomass and populations that occurs during the growth season. This sequence is considered in the section on algae, since it is driven primarily by the response of phytoplankton populations to environmental alteration, and all other lake biota show correlated changes. The role of other lake biota within the seasonal transition is important and includes: 1. Control of phytoplankton spring and autumn blooms by fungal and viral epidemics; 2. Control of phytoplankton populations by protozoon and zooplankton grazing, with the occurrence of a clear-water pha<;l'; 3. Peaks in total bacterial count that correlate (slightly out of phase) with phytoplankton blooms, in relation to DOC production and accumulation. 5.5.2 Trophic Connections and Biomass Transfer Biomass is created by the photosynthetic activity of autotfcrhc organisms and is subsequently consumed by heterotrophs, pa~~;ng from one group to another in a defined sequence of t101;!;':c progression. The interconnections and transfer processes rLat are involved in this sequence can be considered at three main lcveis of complexity: 1. The food chain - a linear sequence describing the rrcgl t:ssion in terms of major groups of organisms; 2. The ecological pyramid - a linear sequence, in Wl:!C:l l,rganisms are grouped in terms of ecological role, and dY:13tliics of biomass transfer are quantified - diagrammatic rep"f'~LI;.z:tlOns have a pyramidal shape, hence the name; 3. The food web - showing detailed interaction~ bdv:een particulm' groups and species, often including details of l>ieJ!11aSS or energy transfer within the network.

5.5.2.1 Pelagic food chain In pelagic environments, interactions between Olganisms can be considered in relation to two major routes of bio\l1a~s transfer - the major trophic sequence and the microbial loop.

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5.5.2.1.1 Major trophic sequence The major trophic or grazing sequence is the principal system of mass transfer in the aquatic environment. It involves production of biomass by photosynthetic algae (primary producers) followed by a succession of grazing and ingestion. Ingestion of algae is carried out by herbivores (e.g., zooplankton), followed by a series of carnivores (e.g., zooplankivorous then piscivorous fish). NeklOIl

Ncklun

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I'rcdulory zooplankton

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~DOC Figure 5.2 Incorporation of microbial loop into the classical pelagic food chain: (a) classical pelagic food chain; (b) incorporation of various microbial components.

Until quite recently, trophic interactions within freshwater systems were considered almost entirely in relation to this classic sequence. involving organisms which could be caught in plankton nets or were clearly visible as planktonic or benthic biota. This has now changed. with the realization that: • in many pelagic systems, primary production is also carried out by algae which pass through phytoplankton nets and form major populations of actively photosynthetic picoplankton; • ill addition to algae, primary production i~ also carried out by photosynthetic bacteria and by some protozoa (containing symbionts); • many planktonic and benthic algae also have heterotrophic capabilities, able to assimilate complex carbon compounds 111 addition to their autotrophic activities;

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• primary productivity is also channelled into a second major trophic sequence which involves bacterial and protozoon populati.ons and is referred to as the microbial loop.

5.5.2.1.2 Microbial loop The existence of a second major trophic sequence, the microbial loop, was realized when it became clear that: 1. A substantial part of primary productivity does not enter the grazing sequence, but is released as dissolved organic carbon (DOC) into the environment; 2. Large populations of viruses, bacteria and protozoa are present in many aquatic systems and playa major role in the release and utilization of algal DOC. In the microbial loop, DOC released by microalgae is routed back to the grazing sequence via a succession of microbial organisms. DOC is metabolized by bacteria, which are then ingested in sequence by protozoa and smaller zooplankton, leading back into the main sequence. The microbial loop is particularly well seen in planktonic systems, where stable populations of protozoa, algae and bacteria may occur under balanced (steady-state) conditions. At other times, populations are not in equilibrium, and many studies have confirmed a temporal sequence of microorganisms in such circumstances, with bacteria succeeding phytoplankton blooms and protozoa succeeding the bacteria.

5.5.2.2 Ecological pyramid At any point in time, the biota contained within the pelagic zone of a lake may be considered to be in a state of equilibrium with a balance between major groups in terms of numbers, biomass and energy content. These broad interrelationships are typically illustrated as ecological pyramids, for the pelagic community of a temperate lake during winter and summer. The main conclusions that emerge from this concept are as follows. 1. Summer populations show a clear pyramid of biomass, with phytoplankton occupying the base of the pyramid (108 individuals 1-1) leading to herbivores, primary carnivores and secondary carnivores (10; individuals I-I , or one individual every 1000 I of lake water). 2. Winter conditions do not permit active growth of the primary producer, and the overall pyramidal structure is lost. At this time of year, the population of primary producer is down to

202

MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY Mas •• energy. nutritional valu•. kcnl m-2 year-I

Numb"r>. individuals per htre

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about 101 organisms I-I and herbivorous fish and zooplankton are the main population, The classical concept of the ecological pyramid is based on growth of the primary producer, with a succession of dependent groups of primary and secondary consumers. Although it provides a useful overview of population levels at different stages in the trophic sequence, there are a number of limitations. 1. Organisms are considered either as producers or consumers. Recent studies have shown that this is not the case with a number of algal groups - where organisms can exist either a" autotrophs or as heterotrophs.

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2. The classical ecological pyramid deals only with the main trophic sequence and ignores the microbial loop. A substantial amount of primary productivity is diverted into the production of dissolved organic carbon (DOC), and is then routed into the microbial community via the loop system, supporting a large population of heterotrophic bacteria and protozoa. 5.5.2.3 Food web Trophic interactions between individual species or particular groups of biota can be considered in more detail as a two· dimensional interconnected flow diagram (food web).

5.5.2.3.1 Generalized food web A generalized food web for the major groups of biota within the pelagic lake system is shown. This diagram includes the five major groups of microorganisms - viruses, fungi, bacteria, protozoa and algae - and again emphasizes the distinction between the main trophic sequence (ingestion) and the microbial loop Dissolved organic carbon (DOC) plays a key role in linking the productivity of phytoplankton and zooplankton to bacteria, and also linking the pelagic ecosystem to the external environment via exogenous DOC. The main trophic sequence involves input of energy (light), CO 2 and inorganic nutrients into phytoplankton, which can be separated into a gradation of subgroups. These are then differentially consumed by protozoa and different zooplankton groups, which are then consumed by zooplanktivorous fish - and ultimately piscivorous fish and other vertebrates as top predators. Trophic interactions between groups are indicated as carbon flow. This relatively simple diagram illustrates some of the complexities of the food web. These include multiple connections - picoalgae, for example, are consumed by protozoa, rotifers and copepods - and the fact that protozoa have a key role in both the main trophic sequence and the microbial loop. Interconnections between the different groups give the appearance of a cascade of organisms, and this generalized food web is sometimes referred to as the 'trophic cascade'. One other important aspect that emerges from this diagram is the question of control of phytoplankton and zooplankton productivity. Is this determined primarily by the supply of light and nutrients (bottom-up control) or by fish predation (top-down control)? The question of bottomup and top-down control is considered later in relation to particular microbial groups, and also in relation to the control of blue-green algal blooms resulting from eutrophication.

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5.5.2.3.2 Patterns of ingestion and biomass transfer Patterns of ingestion (i.e., which organisms are eating which) within aquatic food webs can be determined by gut content analysis, though this may fail where soft-bodied organisms are ingested or where advanced digestion has occurred. In most situations, food web interactions may also be inferred from known predator-prey relationships, particularly in relation to ingestion of defined size ranges by zooplankton. Other approaches may also be useful, such as the use of X-ray microanalysis to detect silicon in groups of zooplankton that are feeding on diatoms, and the use of trophic biomarker compounds and stable isotope analyses.

5.5.2.3.3 Trophic biomarker compounds The trophic biomarker concept is based on observations that particular dietary components pass from one organism to another, and are incorporated without any chemical change. This has been used particularly in relation to lipid composition, where fatty acid profiles of primary producers, herbivores and carnivores have revealed useful information Qn aquatic food web relationships.

5.5.2.3.4 Stable isotope analysis Uptake of stable carbon and nitrogen isotopes by primary consumers, and selective retention during biomass transfer, provides useful information on patterns of ingestion in aquatic food webs. Analysis of carbon and nitrogen stable isotopes in Lake Baikal (Russia), for example, has shown that the pelagic food web has an isotopically ordered structure, where the concentrations of o\3e and 015N within individual species both showed a well-defined relationship to trophic level. 015N levels in particular had a clear trend of stepwise enrichment with progression from primary producer to zooplankton, pelagic fish and freshwater seal. The technique 1.:an also be used to demonstrate changes in food web dynamics caused, for example, by eutrophication. The resulting transition in shallow Greenland lakes, from benthic to pelagic primary production, is matched by a change in the feeding habits of primary consumers. The use of carbon stable isotope analysis in this situation to detect changes in resource use is possible because phytoplankton discriminate against o\3e more than benthic algae (periphyton), and consumers conserve these differences. During the eutrophication process, the o\3e content of benthic grazers (amphipods, isopods and snails feeding on benthic algae) changes to that of benthic filter

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feeders (mussels and chironomids consuming phytoplankton). This transition in the ol3e signature of grazers results from a decrease in periphyton availability and an increase in phytoplankton biomass, which settles out on the sediments.

5.5.2.3.5 Biomass and energy j10w The flow of biological material within the food web involves a sequence of ingestion, breakdown and synthesis of new biomass. During the transition from one organism to another there are progressive changes in the chemical composition of biomass and also loss of potential energy (biomass) due to inefficiencies in the conversion process and respiratory breakdown of some of the ingested material. Incorporation of data on mass or energy content of different groups provides quantitative information on the flow of materials within the food web and the efficiency of energy conversion within the ecosystem. 5.5.3 Maintenance of the Internal Environment Maintenance of the internal environment (homeostasis) is an important property of all ecosystems and is particularly well seen in the pelagic environment. In practical terms, homeostasis involves the ability of the system to overcome short-term physical, chemical and biological perturbations which may be imposed from outside (e.g., severe climatic change, pollutant inflow), or arise internally (e.g., random increase in a microbial population). Homeostasis operates to restore the ecological balance that occurs between biota and to maintain the biodiversity of the ecosystem. In addition to its importance for natural ecological processes, homeostasis has a key role in the community response to humanmediated nutrient-enrichment (eutrophication) of the freshwater environment. The adverse environmental effects that result from eutrophication arise due to a breakdown in ecosystem homeostasis, and the recovery of freshwater systems requires the restoration of a balanced community and the return of homeostatic mechanisms. 5.5.4 Interactions with the External Environment Interactions with the external environment have a key effect on both parts of the pelagic ecosystem, influencing both the community of organisms and the habitat that they occupy. These interactions have particular importance for the lake pelagic ecosystem.

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1. A large surface area of the pelagic system is directly exposed to the atmosphere, and is thus rapidly affected by external physicochemical change. ' 2. The pelagic ecosystem is the major area in the lake for penetration of light and the generation of biomass through photosynthesis (primary productivity). The pelagic system acts as a source of biomass for other systems such as the deep benthic environment, and for littoral microsystems such as bacterial (non-mixed) biofilms. 3. External changes lead to seasonal stratification (and subsequent de-stratification) of the pelagic environment, with major impact on the biology of lake organisms. During daytime hours, penetration of light into the pelagic environment is continuously changing, with correlated changes in the response of phytoplankton cells. These changes include activation of light-responsive genes and alterations in the rate of photosynthesis. Longer-term changes in light level are also important, particularly in relation to seasonal changes and diurnal periodicity.

5.5.4.1 Seasonal challges The impact of external effects is seen particularly clearly in seasonal changes in lake biota, where alterations in surface light, temperature and wind trigger the onset of the spring diatom bloom, cause stratification in the water column, and promote a succession of changes in phytoplankton and other lake organisms.

5.5.4.2 Diurnal periodicity In addition to seasonal effects, external changes in light intensity also promote a diurnal periodicity in phytoplankton activities. These involve entrainment of phytoplankton dynamic activities (vertical migration), physiological processes (photosynthetic activity, nitrogen fixation) and gene transcription (expression of clock genes). Other biota such as zooplankton are also directly affected by daily changes in light intensity, and diurnal periodicity promotes the vertical migration of these organisms and their interactions with phytoplankton. These include the timing and position (within the water column) of zooplankton grazing of phytoplankton and the recycling of phosphorus from zooplankton to phytoplankton. The dynamics of lake hydrology may also exert an important external effect on the pelagic ecosystem, with inflow and outflow of aquatic biota, particulate matter and soluble nutrients. Towards the

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end of the growth season, for example, inflow may be particularly important in the supply of soluble inorganic nutrients to a nutrientdepleted system, while the outflow of biota such as planktonic algae may influence bloom formation. Flushing the pelagic system is an important management option with many freshwater systems in the control of late summer blue-green algal blooms. The loss and recruitment of biota may also occur via the surface of the lake. This does not appear to have been documented for lake microorganisms, but insects such as mosquitoes and chironomid flies enter the system as eggs, have an aquatic larval stage, then exit the ecosystem on metamorphosis to the imago stage.

5.6 HOMEOSTASIS AND ECOSYSTEM STABILITY All biological systems are subject to environmental changes that may impair function. These are referred to as 'stress factors' and can operate at the level of ecosystems, individual organisms (e.g., physiological influence of high light) and molecular systems. Prokaryotes respond to physicochemical stresses such as salinity, high temperature and acute nutrient deprivation by the induction of special transcription factors (sigma proteins) that lead to the expression of a range of stress genes. Ecosystems are frequently subject to external or internal environmental effects that tend to bring about changes in the biological community. These effects can be defined as 'environmental stress' when they impair the structure or function of food webs and other dynamic aspects of the biological system. In this section, the response of aquatic ecosystems (including microbial communities) to stress is considered in relation to general ecosystem theory, observed stress responses, assessment of ecosystem stability, changes in community structure and biological response signatures.

5.6.1 Stress Factors Examples of external environmental stresses which affect aquatic environments include major environmental change (floods, alteration of land usage), climatic change (droughts, temperature, wind disturbance), local physical parameters (rate of water flow) and chemical effects (toxic pollutants, nutrient enrichment). Biological perturbations, such as the introduction of new species, may also be important stress factors. The ecology of many lakes in the UK, for example, is affected by the introduction and spread of non-native plants such as the New Zealand pygmy-weed. Originally brought into the country for use as a garden pond plant, this readily colonizes

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other freshwater habitats -- spreading across water bodies, eliminating native plants and reducing habitat diversity.

5.6.1.1 Stress factors affecting microbial communities Various examples are given in this volume of the effects of external stress on aquatic microbial communities. The rate of water flow is an important parameter in lotic systems, for example, affecting the development of biofilm and epilithon communities. The flow rate of effluent into activated sludge (sewage) systems also provides a good example of external stress. At high flow rates the protozoon community is limited to rapidly reproducing species, with all the features typical of r-selected organisms. In lakes and other standing waters, inflow of inorganic nutrients also constitutes a major stress factor, leading to eutrophication and the breakdown of ecosystem homeostasis. Stress factors developing internally within aquatic environments may also be important. In some cases these internal stresses are secondary effects of external factors. such as the effects of bluegreen algal blooms resulting from eutrophication. Another example of an internal stress that may result from external factors is the induction of anoxic conditions by the bacterial bloom that develops as a response to organic pollution. This has an inlportant effect on the composition and activities of the emergent protozoon community. In other cases, internal stress factors are not directly linked to external perturbations. In lakes, they may occur as part of the seasonal cycle - and include the substantial increase in zooplankton grazing activity that occurs during the clear-water phase, which has a major influence on the phytoplankton population. Development of late summer intense blue-green algal blooms in natural eutrophic waters may also impose stress on the ecosystem, affecting the growth of other algae and in some cases limiting the development of zooplankton. 5.6.2 General Theoretical Predictions: Community Response Theoretical considerations suggest that a number of general responses may be expected in ecosystem communities in relation to external stress. The principles that underlie these responses apply equally to combinations of communities as well as single communities (e.g., biofilms, plankton populations) within complex ecosystems. These responses include changes in energetics, nutrient cycling, community structure and general system characteristics.

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5.6.2.1 Energetics Energy changes would be expected as an early response to external stress, with an increase in respiration (R) as organisms cope with the disorder caused by disturbance. Chemical energy within the system is directed from biomass production (productivity P) to respiration, the P/ R ratio becomes unbalanced and the R/ B (respiration/biomass) ratio increases. The drain of productive energy within the system means that auxiliary energy (from outside) becomes increasingly important for continued survival. Disturbance also results in less efficient use of primary production, with an increase in unused resources.

5.6.2.2 Nutrient cycling Increased nutrient turnover and decreased cycling frequently appear in stressed ecosystems. The decreased nutrient cycling results from changes in community structure, with increased horizontal transport but reduced vertical cycling. Together, these changes in turnover and cycling result in nutrient accumulation which, as with unused primary production, may be lost from the system.

5.6.2.3 Community structure Under stressed conditions, we would expect an increase in species that are able to grow rapidly and exploit temporary advantages. Such opportunistic (r-selected) organisms typically have a small size and decreased life spans. The competitive strategy of r-selected organisms and their adaptation to uncrowded and unstable envirohments has been considered above. Disturbance to the food web a:;o results ill shorter food chains and decreased species diversity as particular species attain temporary dominance.

5.6.2.4 Gelleral systcm-lel'el trends Under stress conditions, ecosystems tend to become more open and existing (autogenic) successional changes become reversed. The efficiency of resource use decreases due to unused primaty production and increased nutrient loss. As individual organisms become physiologically stressed, their susceptibility to parasitic infection increases.

5.6.3 Observed Stre~s Responses: From Molecules to Communities Observed responses to stress range from molecular c\ ,'fIts (e.g., light inactivation of proteins) to alterations in physiology, balance of populations (selective mortality), ecological dynamic~ (reduced

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recruitment) and community composition (reduced diversity). In terms of the aquatic community, many of the observed stress responses have been predicted on the basis of general ecosystem theory (previous section), which applies equally to aquatic and terrestrial systems. Aquatic ecosystems show many of the stress responses. Cattaneo et al. (1988), for example, demonstrated a general decrease in size in a range of lake biota (diatoms, thecamoebians and cladocerans) under chronic and progressive conditions of metal pollution. Havens and Carlson (1998), investigating plankton community structure in a range of lakes of different pH level (7.3 to 4.2), showed that reductions in pH correlated with reductions in food web complexity and species diversity. Schindler (1987), in a study of lake community responses to anthropogenic stress, demonstrated changes in the proportion of r-strategists, organism life spans, species diversity and the relative openness of the ecosystem. 5.6.4 Assessment of Ecosystem Stability The ability of ecosystems to resist stress and return to, or maintain, their biological integrity is an example of the general principle of homeostasis or maintenance of the internal environment. In ecosystems, the internal environment comprises the biological, chemical and physical characteristics of the system - with particular emphasis on the species composition and dynamic interactions of the constituent organisms. The extent to which an ecosystem is altered by any of the above stress factors is a measure of its stability - the greater the stability, the more able is the system to resist external change. Although concepts of ecosystem stability and the mechanisms that control it have been developed mainly in relation to terrestrial environments, many of the principles may also be applied to aquatic systems. The stability of ecosystems has been defined and discussed in relation to three interrelated aspects.

5.6.4.1 Species composition Stable systems show constancy in overall species numbers or relative proportions of individual species under stress. This has been defined as 'no-oscillation stability' and implies predictability and continuity of species composition within the ecosystem. 5.6.4.2 BiodilJersity alld complexity of the food web It is generally accepted that ecosystem stability is partly a function of trophic structure and the overall diversity of food webs. This

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diversity of trophic interconnections means that oscillations within the ecosystem are reduced and that there is increased resilience to outside influences. MacArthur (1955) 'has proposed a direct quantitative relationship between ecosystem stability and the number of links in the food web, and suggested that a large number of interactive feeding links allows a wide variety of adjustments to stress within the system and also provides alternative channels for energy flow. The ability of the system to maintain (or return to) its original state under external stress. This property has been referred to as 'stability resistance'. This property of environmental homeostasis is particularly relevant when considering the impact of blue-green algal blooms on the lake ecosystem. Some freshwater bodies have developed very stable ecosystems with high levels of biodiversity. This is particularly the case for ancient lakes such as Lake Baikal (Russia), where a diverse assemblage of species has evolved in a relatively stable environment over a long period of time.

5.6.5 Ecosystem Stability and Community Structure Although some freshwater systems have evolved relatively stable and complex communities, the concept that ecosystem stability relates primarily to biotic interactions and food web diversity applies particularly to established terrestrial systems-such as tropical rain forests. These typically conform to the community structure model developed by Hairston et al. (1960), where physical disturbance is of minimal importance. In this model, competition occurs mainly between primary producers (for space, nutrients, light) and between predators (competition for limited food resource). Populations of herbivores are unable to limit the growth of primary producers, and are kept below the theoretical carrying capacity of their available food supply by predation. Freshwater ecosystems frequently differ from terrestrial ones in having greater physical disturbance and greater physical heterogeneity. These systems relate more to the community structure model developed by Menge and Sutherland (1987), where varying level: of physical stress (disturbance) cause major alterations in competition and predation. Highest stress levels (continual disturbance) do not allow any species to establish, but as stress is reduced the community becomes more complex until, at low stress levels, it is equivalent to that envisaged by Hairston et al. (1960). Variations in stress level

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and community structure can be seen in both lotic and lentic aquatic systems.

5.6.5.1 Streams and rivers Turbulent water disturbance in fast-moving rivers provides a good example of a high-stress environment, corresponding to stages of the Menge-Sutherland model. In this situation, unattached organisms such as invertebrates and free-moving microorganisms are not able to establish themselves and are swept away by the current. Only attached microorganisms such as periphyton communities and biofilms are able to remain in such conditions, with varying degrees of competition between organisms colonizing exposed surfaces. Even these attached communities may be limited by environmental stress. In highly disturbed ecosystems such as head-water streams, algal succession may show little progression beyond diatom colonization of exposed surfaces.

5.6.5.2 Lakes The microbiology of lakes is influenced by stress factors in both natural and derived situations. Under natural conditions, variations in community structure and stress levels are seen during the seasonal cycle. High stress levels occur during the clear-water phase, where competition between herbivores (zooplankton) is high and phytoplankton populations are low and show rapid change. Only rapidly growing algae (r-selected organisms) are able to survive the adverse conditions at this time. In contrast to this, the summer bloom period is typically a phase of minimal stress levels, where zooplankton grazing pressure is reduced and high levels of competition occu'r between established populations of different phytoplankton species.

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An example of induced stress occurs where lakes are subject to high nutrient pollution (eutrophication) as a result of human activities. The growth of blue-green algae results in the suppression of both herbivores (zooplankton) and predators (fish), with strong competition within the phytoplankton community leading to blue-green dominance.

5.6.6 Biological Response Signatures Different types and degrees of stress result in different community responses. These responses can be used to evaluate the source, type and impact of stress on a particular aquatic system, and have been referred to as 'biological response signatures'. Biological response signatures may be defined as discernable patterns in the response of aquatic communities, allowing the investigator to discriminate between different types of stress. In their paper describing the selective effects of discrete environmental disturbances on rivers and streams in Ohio (USA), Yoder and Rankin (1995) were able to segregate various impacts into nine categories of disturbance. These comprised complex toxic release, conventional municipal discharge, sewer overflows, channelization, diffuse agricultural pollution, flow alteration, impoundment, combined sewer overflow with toxic discharge and livestock access. The use of biological signatures normally requires a broad assessment of the aquatic community rather than looking at individual 'indicator species.' This assessment involves the determination of community indices using multiple indicator groups, detailed taxonomic resolution and standardized sampling procedures. Most programmes of biological assessment use macro-scale indicators such as fish, invertebrates and higher plant communities. There is considerable scope, however, for using microbial communities - including benthic diatom assemblages to assess nutrient impacts on flowing waters and phytoplankton communities to evaluate eutrophication of standing waters. 5.7 PELAGIC FOOD WEBS The ecosystems of standing water (len tic) and running water (len tic) systems show a number of important differences. These reflect differences in the biota (e.g., relative proportions of pelagic and benthic organisms), the environment (e.g., absence and presence of water motion), and in the input of external (allochthonous) material into the aquatic system.

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The generalized pelagic food web was based on major groups of organisms, and becomes highly complex when individual species - each showing multiple connections to other species - are considered. For this reason investigations involving food webs tend to focus on particular aspects of the lake ecosystem, such as particular groups of biota (e.g., excluding the major microbial component), particular parts of the lake (e.g., the pelagic ecosystem), or particular time sequences (e.g., during a particular algal bloom). These different approaches will be illustrated by two case studies from lake ecosystems - the transient microbial food web associated with a spring algal bloom and a generalized annual food web present within the water column of an oligotrophic lake. Changes in the nutrient status of lakes can also influence interactions between pelagic and benthic food webs. Recent studies by Vadebonceur et al. (2003) on shallow oligotrophic lakes of Greenland have shown a shift in productivity from benthic to pelagic food webs with increased nutrient status, accompanied by a breakdown in the linkage between the two habitats in terms of food web interconnections. 5.8 COMMUNITIES AND FOOD WEBS OF RUNNING WATERS In standing water (lentic) systems, the ecology is dominated by phytoplankton and the entry of carbon into the food chain mainly occurs internally (autochthonous origin) via photosynthesis. In contrast to this, running waters are dominated by attached (benthic) organisms and the entry of carbon is principally from external sources (allochthonous origin) as complex organic compounds. As C\ result of these differences, len tic systems often have a net autotrophic metabolism, while lotic systems typically show net heterotrophy. At the microbial level, biofilm communities are particularly important in rivers and streams, and are dominated by either bacteria or algae (periphyton) - depending on stage of development and location. The lotic ecosystem will be considered in relation to the mainly allochthonous ()rigin of the organic carbon, the relative importance of pelagic and benthic communities and microbial food webs. 5.8.1 Allochthonous Carbon Although there is some production and release of autochthonous carbon by riverine phytoplankton, benthic algae, and higher plants, most of the carbon in streams comes from the catchment area or

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flood plain. The dependence of flowing waters on allochthonous sources of carbon involves -the entry of both dissolved organic carbon (DOC) and particulate carbon (POe) into the aquatic system, as reflected in the changes in concentration of these components during the seasonal river cycle.

5.B.l.l Dissolved organic carbon (DOC) The amount - and characteristics - of DOC that enters streams is controlled by both biotic (vegetation type, human activity) and physicochemical (geochemistry, hydrology) factors of the catchment area. Hydrological characteristics such as the flow path and residence time of water in soil horizons (differing in organic matter content and sorption capacities) are of particular importance. The molecular composition of DOC in streams is diverse, including both highly stable (refractory) and rapidly changing, unstable (labile) components. Only part of this can be broken down by bacteria, with relatively little degradation « 1 per cent) of the more refractory components such as hurnic acids and high molecular weight DOC but considerably more breakdown (>50 per cent) of the more labile low molecular weight DOC compounds, particularly those of human origin. Studies on the elemental composition of DOC in relation to bioavailability have suggested that the atomic ratios of Hie and ole in DOC can be used to predict the ability of bacteria to degrade these compounds. The Hie ratio in particular is directly proportional to readily degradable aliphatic compounds and inversely proportional to less degradable aromatic material. Because of the variation in DOC bioavailability, the amount of DOC entering the system is not a direct measure of DOC supporting the microbial food web.

5. B.1.2 Particulate organic carbon Particulate organic matter (diameter >0.2 JLm) ranges from finely dispersed material (including bacteria) to large particulate matter such as leaf litter and other plant debris. Much of this material is directly deposited into the flowing water from surrounding vegetation. Large particulate matter such as leaf litter is an important carbon source for the microbial food web, serving as a substrate for fungal and bacterial growth and as a source of DOC. On entering a stream or river, leaf litter releases an initial pulse of rapidly leachable, water soluble material, followed by a slow release of DOC due to microbial degradation. The breakdown and release of organic material from leaf litter is accelerated by the feeding activities of invertebrates.

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5.8.1.3 Changes in the concentrations of dissolved and particulate carbon during the seasonal cycle Concentrations of dissolved and particulate carbon show wide fluctuations in many streams and rivers during the annual cycle, with recorded DOC values generally in the range 1-10 mg 1-1, but reaching much higher levels in some rivers. In most cases, this annual variation reflects the allochthonous derivation of these compounds and the seasonal fluctuations of entry into the river system. The large rivers of Southern Asia, for example, have large changes in DOC concentration, with close correlations between seasonal flow patterns and the timing of DOC maxima and minima. The dissolved organic carbon levels in the Indus and GangesBrahmaputra Rivers reach a maximum near the end of rising water levels, due to overflow and entrainment from highly productive flood plains. The pulse of DOC then rapidly declines as water levels recede due to mixing, metabolic removal, and dilution. In the upper Mississippi River (USA), the very high autumn allochthonous DOC levels are derived not from soil, but from leaching of leaf litter. Although many rivers show elevated allochthonous DOC concentrations at times of flood and terrestrial runoff, wide differences in seasonal patterns can occur. The Shetucket River (USA), for example, is unusual in showing minimal DOC levels during high inflow winter months, but maximum levels during the low-flow summer period. The summer maximum was attributed to the generation of autochthonous carbon by secretion and senescence of benthic algae. In winter, the allochthonous DOC input was overridden and diluted by high discharge due to ice melt and heavy rain. Seasonal patterns in some rivers are at least in part driven by in situ planktonic primary production. In the Gambia River (West Africa), allochthonous DOC concentrations reach a maximum at the time of maximum discharge into the system, but accumulation of autochthonous DOC occurs during the low-flow period. This was linked to phytoplankton production and was associated with . elevated river water pH levels. 5.8.2 Pelagic and Benthic Communities Lotic systems differ considerably in the extent to which pelagic and benthic communities are able to develop. This depends particularly on size and flow, with a major distinction between large, slow-flowing rivers and small, turbulent streams.

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5.8.2.1 Large rivers: the development of a phytoplankton community In'most lotic systems, phytoplankton are simply displaced by the current, and are not able to form standing populations. Because of this continuous displacement, net production within a defined section can only occur when local growth rates exceed downstream losses. Phytoplankton growth in lotic systems tends to be limited by ambient light intensity (overhanging foliage), turbidity, and circulation within the water column (no stratification), while downstream loss is largely a function of current velocity. Phytoplankton production in riverine systems, and the development of a pelagic community, is thus largely regulated by light availability in combination with hydrological processes. Recent studies by Sellers and Bukaveckas (2003) have demonstrated that phytoplankton production may be significant in large rivers. Local biomass accumulation occurs particularly in shallow reaches during peaks of low discharge and turbidity, when phytoplankton experiences prolonged exposure to favourable light conditions. Observations on a large navigational pool in the Ohio River (USA), for example, showed that at times of high discharge, phytoplankton productivity within the pool was < 10 per cent of phytoplankton input from upstream and tributary sources. At times of low discharge, phytoplankton production in the pool exceeded external algal sources.

5.8.2.2 Small rivers and streams: development of a benthic community Most lotic systems are dominated by benthic communities, with little development of pelagic food webs. Solid surfaces are rapidly colonized by microbial organisms, leading to the development of bacterial and algal biofilms. These permanent microbial communities are an important aspect of the lotic environment, with algal biofilms making a significant contribution to primary productivity and both algal and bacterial biofilms forming part of the ecosystem food web. Bacterial biofilms typically produce copious amounts of extracellular matrix, creating a distinctive micro-environment that limits the extent of the community.

5.8.2.2.1 Benthic bacteria Bacteria present on the surface, and in subsurface regions, of stream-bed sediments are involved in a number of key ecosystem

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processes - including the breakdown (mineralization) of organic matter, assimilation of inorganic nutrients, and acting as a food source for consumer organisms. While quantitative aspects of the supply of organic matter clearly influence the abundance and productivity of sediment bacteria, qualitative aspects are also important. These include delivery, particle size distribution, and chemical composition - all of which affect the spatial and temporal composition of bacterial communities. Chemical composition is particularly important, and recent studies by Findlay et al. (2003) have emphasized differences between labile (easily assimilable) and recalcitrant (poorly assimilable) carbon sources in promoting bacterial community responses such as oxygen consumption, productivity, extracellular enzyme activity, and community composition. The growth of heterotrophic bacterial populations in benthic environments relates to both productivity (availability of organic carbon, inorganic nutrients, terminal acceptors) and loss processes such as grazing and viral infection.

5.8.2.2.2 Ecological pyramids and food webs The comparatively low level of phytoplankton and zooplankton in lotic systems means that ecological pyramids from rivers and streams look very different from those of standing waters. Food webs of lotic systems are also very different. The grazing (phytoplankton, zooplankton, fish) food web that dominates lakes is of much reduced importance, and the low level of internal (autochthonous) DOC production by phytoplankton and other photosynthetic organisms means that the pelagic microbial loop recycling carbon released from phytoplankton back into the macrobiota - has little application. The metabolic coupling and correlation in populations between planktonic algae and bacteria seen in lakes is not a feature of lotic systems. 5.8.2.3 Microbial loop of lotic systems Although the pelagic microbial loop has little relevance in most lotic systems, the benthic microbial community is important in cycling externally derived DOC into multicellular organisms within the stream environment. This benthic microbial loop is initiated by the arrival of externally-derived carbon into the river system as dissolved organic carbon (DOC) and particulate organic matter. The latter is broken down by invertebrates (conversion of coarse to fine particulate matter) then digested by fungal and bacterial exoenzymes to form DOC, with subsequent uptake by these two groups of organisms. Protozoa

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are involved in the direct uptake of particulate material and the ingestion of bacteria. Comparison between pelagic and benthic microbial loops emphasizes a number of key differences in addition to the internal or external origins of the carbon supply. Non-microbial biota have a greater impact on the benthic microbial loop, carrying out the initial shredding or breakdown of the coarse particulate organic (CPOM) matter and generating fine particulate material ahd DOC which are ingested by microorganisms. Connections with the overall food web also differ, with relatively good linkage between benthic and planktonic biota in lotic systems, but fewer biomass transfers between microorganisms and top carnivores within the river ecosystem.

5.8.3 Microbial Food Web The microbial loop of flowing water is completed by the assimilation of bacterial carbon, which then passes into multicellular biota. Bacteria form the main microbial biomass and are present mainly within biofilms and associated with organic debris such as leaf litter. They are ingested by three main groups of benthic organisms protozoa, insect larvae, and meiofauna. These differ in the efficiency with which they ingest bacteria, as determined by bacterial carbon uptake per unit weight of the organism.

5.8.3.1 Protozoa (flagellates and ciliates) This group of unicellular organisms are important grazers of biofilms. They have significant impact on bacterial populations, with assimilation rates of the order of 10- 1 to 10-2 p.g bacterial carbon 111g-1 protozoon biomass d- I •

5.8.3.2 Insect larvae Aquatic insect larvae ingest bacteria that are present mainly on organic debris such as leaf litter. They are both predators and competitors of microbial organisms, directly consuming bacteria and fungi and also digesting the organic detritus that is an important basis for the bacterial food chain. Some insect larvae (e.g., stoneflies and craneflies) ingest bacteria simply as part of the litter that they collect, resulting in relatively low bacterial assimilation rates (about 10-4 p.g mg- I d- I ). Other larvae have specialized bacterial collection strategies, including filtration (black fly) and surface scraping (mayfly), resulting in much higher bacterial assimilation rates.

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5.8.3.3 Meiofauna These are animals inhabiting the bottom of a lake or river that are just visible to the namost important ked eye and include copepods, nematodes and rotifers. These are generally regarded as the most important bacterial predators in lotic systems, and include both filter feeders and biofilm grazers. Their bacterial carbon assimilation rates are typically 1-4 orders of magnitude greater than that of other bacterial consumers. The microbial loop in running waters is part of a more complex food web that also involves photosynthetic carbon production by algae and higher plants, saprophytic and parasitic activities of fungi and viruses and important roles for the large invertebrate and vertebrate predators. Primary production by benthic algae (periphyton) varies in importance in different systems, depending partly on the depth of the water column and light penetration to the substratum. Light is also important in the water column in relation to degradation of non-assimilable (refractory) DOC. UV-irradiation, in particular, has been shown to have a major effect in converting humic acids to more labile forms of DOC. The ecological importance of bacteria, protozoa, and fungi in lotic food webs has been mentioned in relation to the microbial loop. Further information is subsequently given in relation to the breakdown of organic matter by benthic bacteria, saprophytic activities of benthic fungi, and the role of protozoa in the ingestion of both living and non-living particulate matter. The transfer of biomass from microbes to top carnivores does not have such a defined route as pelagic food-webs, and the domination of herbivorous activities in the water column by crustaceans (zooplankton) does not occur in the lotic food web. Herbivory in running waters occurs mainly at the sediment surface and involves the ingestion of either unattached or attached microorganisms. The consumption of microorganisms in lotic communities involves ingestion of: 1. Free-moving biota such as bacteria and protozoa, present as localized populations around organic debris, by meiofauna and protozoa; 2. Microorganisms, particularly fungi and bacteria, that are present within organic debris such as leaf litter - this is carried out by

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invertebrate shredders, gougers, and collector gatherers, prominent amongst which are insect larvae; 3. Biofilm and other microorganisms that are attached to solid substratum - this is carried out by shredding, scraping and rasping. The fragmentation and ingestion of leaf litter by invertebrates requires partial breakdown of this material by fungal activity. The substrate colonization, invasion, and macerating activity of these organisms thus promotes their own ingestion during the final stages of leaf processing.

6 Light as Abiotic Factor Sunlight (solar radiation) is important to aquatic microorganisms in four major and interrelated ways. 1. Determination of their physico-chemical environment. Light is a major determinant of the dynamics and structure of aquatic environments. Energy from the sun provides the heat that generates the Earth's wind patterns, resulting in mixing of the surface layers of lakes and oceans. Transformation of light to heat energy within surface waters results in a localized temperature increase which combines with wind energy to produce thermal stratification. This in turn limits the distribution of nutrients within the water column and thus has a secondary effect on aquatic chemistry. Light also has direct effects on chemical characteristics and is important, for example, in the degradation of humic acids in peaty lakes to more biologically available compounds. 2. Production of biomass. Light penetration of surface waters drives the photosynthetic activity of the primary producers. These include both planktonic and benthic organisms, and comprise three major groups - higher plants, algae, and photosynthetic bacteria. The photosynthetic reducing power which is generated by light is important for both carbon (C0 2 ) and nitrogen (NO" N0 2 , NH 4 ) assimilation. Nitrogen fixation (in colonial blue-green algae) and phosphorus metabolism are also closely linked to photosynthesis. but the cellular energy required for uptake and deposition of silicon in diatoms is derived solely from aerobic respiration without any involvement of photosynthetiC energy. 222

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3. Damage to cell processes. In extreme conditions, where general light intensity is high, or where there is a high level of ultraviolet radiation, damage to cell metabolic and genetic processes (photoinhibition) can seriously impair biological activities. 4. Induction of periodic seasonal and diurnal activities. Temporal changes in the occurrence and activities of freshwater biota are considerably influenced by the periodicity of light. This affects both seasonal activities (where day length is important) and diurnal activities (where alternation of light and dark entrains circadian rhythms). This chapter initially considers aspects of light intensity and wavelength within aquatic systems. The major part of the chapter then deals with light as a resource for the production of biomass, followed by a consideration of the damaging effects of light and the importance of light periodicity.

6.1 LIGHT ENVIRONMENT 6.1.1 Physical Properties of Li&.ht: Terms and Ur-its of Measurement Solar radiation can be considered either as a continuous wave of energy or as discrete packets (photons) of excitation. These two concepts give rise to two fundamental measurements of light: 1. Wavelength, which describes the quality of light and the type of effect it will have on living organisms, and 2. Photon intensity, which describes the quantity of light to which freshwater microorganisms are exposed. Solar energy has a wavelength range of about 100-3000 nm, with most of the energy being present between 300-2000 nm. The spectral range can be separated into three main regions - ultraviolet radiation (100--400 nm), visible light (approximately 400-700 nm), and infrared radiation (700-300 nm). These three groups comprise 3 per cent, 46 per cent and 51 per cent respectively of solar radiation arriving at the Earth's outer atmosphere. The visible radiation also coincidentally corresponds to the 'photosynthetically available radiation' (PAR) which drives the photosynthetic activities of most phototrophic organisms (algae and higher plants). Infrared radiation appears to have little specific metabolic effect on living organisms, although photosynthetic bacteria do absorb some of it for photosynthesis. Ultraviolet light is of major importance in causing damage to microorganisms.

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The measurement of light is based on the physical properties of electromagnetic radiation and is carried out using a 'quantum sensor'. Numbers of photons are usually expressed directly in moles, where 6:02 x 1023 photons equal 1 mol. Commonly used terms and units for different light parameters and include the number of incident photons (photon flux, photon flux density), energy content (irradiance), and wavelength (range of wavelength). Light intensity in both environmental and laboratory systems is typically expressed as photon flux density (PFD) and is an important parameter is assessing the light-response of living organisms in relation to photosynthesis, growth, and photoinhibition. The terms 'photosynthetic PFD' and 'photosynthetic irradiance' are also sometimes used to denote the amount of incident light within the restricted range of photosynthetically active radiation. 6.1.2 Light Thresholds for Biological Activities In the freshwater environment, photon flux density varies with time (seasonal, diurnal), ~ter quality (content of dissolved substances, presence of biota), and depth in the water column. Light intensity is quantitatively important for the photosynthesis and growth of autotrophic microorganisms, and is also qualitatively important in determining the presence or absence of particular biological activities. Threshold levels of light are particularly relevant to the activities of photosynthetic organisms. At the top end of the PFD scale, these include light saturation of photosynthesis in periphyton and phytoplankton. With decreasing light intensity levels, a sequence of thresholds operate for the growth and survival of macrophytes, algae, and photosynthetic bacteria.

6.1.2.1 Macrophytes The threshold PFD level for macrophyte survival (45-90 Ilmol III 1 S-I) is ecologically important and is directly linked to microbial activity, since in eutrophic waters shading by algal blooms reduces PFD below the critical level and causes eradication of the macrophyte flora. 6.1.2.2 Algae In temperate and polar lakes, critical levels of light intensity are required at the beginning of the growing season to trigger the onset of the spring diatom bloom. Studies on laboratory cultures indicate the onset of net photosynthesis and growth at PFD values of 0.1--1 Ilmol mIs-I.

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6.1.2.3 Photosynthetic bacteria These organisms are restricted to low-light anaerobic regions of the water column, and the onset of growth is triggered by very low-light levels « 1 /-Lmol m- I S-I). In addition to the direct effects of light on photosynthetic microorganisms, indirect effects on freshwater microbes also occur via the influence of light on zooplankton and fish. The onset of maximal vertical migration of Daphnia hyalinarequires threshold light levels of 10-3 /-Lmol m- I sI, and has implications for the grazing of microorganisms in surface waters. This light level is equivalent to the irradiance from a full moon, which also allows fish such as perch to feed visually on zooplankton in surface waters at night - indirectly influencing populations of algae and other microorganisms via the food web.

6.1.3 Light Under Water: Refraction, Absorption, and Scattering Both the overall intensity of light and the sp~ctral composition are modified by its passage through water, largely due the effects of refraction, absorption, and scattering. Refraction of light, with separation into different wavelengths, occurs as the radiation passes through regions of different refractive index - including the air/ water interface and strata of different density in the water column. Absorption (conversion of light to heat energy) and scattering (reflection) are mediated by water molecules, dissolved substances, and particulate matter.

6.1.3.1 Decrease in light intensity The intensity of light at the surface of a body of water does not normally exceed 2000 -mol photons m-2 S-I, equivalent to about 400 Wm-2 in energy units. Loss of light due to absorption and scattering results in an exponential decrease in intensity with depth. The fraction of light lost per metre of water is expressed mathematically as the extinction coefficient (eJ. For parallel beams of monochromatic (single wavelength) light, the intensity of light at a particular depth in the water column is given by the LambertBeer law, which can be expressed as: '" (1) I Z -- I oe -E).Z where: I z = light intensity at depth z (mol photon m- 2 S-I), 10 = light intensity penetrating the water surface (mol photon m- 2 S-I), e; = extinction coefficient for the particular wavelength (m- I ), and z = depth (m).

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The extinction coefficient (c) is very wavelength-dependent and can be separated into separate components which relate to light absorption by water molecules (cJ, dissolved substances (cd) and particulate matter (E/ For pure water, where Ed and c p are zero, EA = cw .

6.1.3.1.1 Light zonation of water column The top part of the water column has a distinct zonation in relation to light availability. The depth at which light intensity reaches the point where 02 evolution by photosynthesis equals 02 uptake by respiration is referred to as the compensation point. Light intensity at the compensation point, le' varies with environmental (e.g., temperature) and physiological characteristics, but is normally about 0.1-1 per cent of the value at the lake surface. The vertical distance between the water surface and compensation point is the photic zone, and is the region within which net anabolic (synthetic) processes occur in phototrophic organisms. Below this, in the aphotic zone, respiration exceeds photosynthesis and metabolism is predominantly katabolic. The depth of the photic zone varies widely between water bodies. In eutrophic lakes, light attenuation with depth is pronounced due to high phytoplankton biomass and the photic zone does not extend Incident light 10 (per cent)

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below the thermocline. In contrast, oligotrophic lakes have little particulate matter and the photic zone may extend below the thermocline. In the case of shallow lakes, rivers, and wetlands, the photic zone may extend down to the sediments or bed of the body of water. This promotes growth of microbenthic algae, which can become major primary producers within the particular ecosystem.

6.1.3.2 Changes in the wavelength of light The spectral composition of underwater radiation is important to lake biota in various ways, including photosynthesis, lightresponsive activities (phototaxis, diurnal variation), and radiationinduced damage to cells and molecules. Underwater separation of light into different wavelengths is complex, with the end result depending on the balance between the processes of refraction, absorption, and scattering. The bending of light by refractio'1 as the radiation passes through water of different densities is higher for short wavelengths (blue light) than for long wavelengths (red light). Absorption of light is also wavelengthselective, being most pronounced at both ends of the spectrum. As light passes through the water column, the ultraviolet and infrared ends of the spectrum are absorbed first, leaving a progressively narrower band of light, restricted to the blue-green part of the spectrum. Reflection of light by particulate or molecular material may also cause changes in spectral composition. The predominant .. ~ ~

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scattering of blue light by water molecules gives clear-water (oligotrophic lakes) a distinctive blue colour, and results in a clear shift of transmitted light towards the red end of the spectrum with depth. Changes in the spectral composition of light with depth differs considerably between lakes, varying particularly with the content of dissolved substances (e.g., humic acids), phytoplankton content, and the amount of nonliving organic and inorganic material. Depth changes in light intensity and composition are least in clear-water lakes, but may become very pronounced in high nutrient lakes with extensive phytoplankton populations. Photons in the blue and green portions of the spectrum are rapidly attenuated in turbid or coloured waters, while photons in the red part of the spectrum show least alteration under such conditions. 6.1.4 Light Energy Conversion: From Water Surface to Algal Biomass Only a small proportion of the light energy incident at the water surface is converted to chemical energy in the form of algal biomass. The loss of energy is partly due to depletion within the water medium and partly due to losses during light transformation within the algal cell. Factors affecting light depletion may be very different when comparing phytoplapkton and benthic algal communities.

6.1.4.1 Phytoplankton Approximately 1-2 per cent of incident light is lost by reflectance at the water surface (varying with the angle of incidence). Subsequent transmission within the water medium leads to a decrease in light intensity and changes in spectral composition, as described previously. The proportion of light that is captured by phytoplankton cells within the water column will depend on: 1. Light removal and alteration by physical parameters - particularly water molecules, dissolved matter, and non-living particulate matter; 2. Light removal and alteration by biological factors such as other phytoplankton cells (self shading) and macrophytes, and 3. How much algal biomass is available for light uptake. Calculations of the proportion of light quanta absorbed at the water surface which are subsequently captured by phytoplankton range from 4-80 per cent varying with chlorophylla concentration in individual water bodies. The effect of water turbidity is indicated by comparison of the clear-water of Lake Constance (70 per cent

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light capture at 30 mg chl-a m- 3) with the turbid water of Lough Neagh (50 per cent light capture at 92 mg chl-a m-3 ).

6.1.4.2 Benthic algae Although physical light absorption and alteration within the water column is also important for benthic algae, interception and absorption by other lake organisms may impose extreme conditions. Benthic algae may experience particularly severe light attenuation from the water surface due to: 1. Their existence in a physically compressed community (biofilms, algal mats) that promotes self-shading, 2. Light attenuation by the whole water column, including the entire mass of phytoplankton, 3. Shading by macrophytes - benthic algal communities can only occur in shallow water (due to the need for light penetration) which also favours the growth of macrophytes, 4. Benthic algae are fixed in position, while planktonic algae are able to migrate in the water column to optimal light conditions. After the removal of light by the environment, further losses are involved during the conversion of the light (captured by phytoplankton cells) to the chemical energy of the primary photosynthetic product (phosphoglyceric acid) and subsequently to proteins, lipids, and carbohydrates. These losses include inefficiencies during the electron transfer and carbon fixation parts of photosynthesis, and are calculated by Kirk (1994) to allow a maximum transformation of light energy to carbohydrate as 25 per cent. Subsequent conversion of carbohydrate to algal biomass involves further energy depletion, including loss of newly-synthesized material by respiration and leakage of small molecular weight dissolved organic carbon (DOC) into the environment. All of these factors result in an estimated maximum conversion of light to biomass in algal cells of about 18 per cent.

6.2 PHOTOSYNTHETIC PROCESSES IN THE FRESHWATER ENVIRONMENT Photosynthetic processes form the basis for phytoplankton productivity and the net assimilation of carbon in aquatic ecosystems.

6.2.1 Light and Dark Reactions Photosynthesis takes place in the chloroplasts of eukaryotic algae and within the entire cells of blue-green algae and photosynthetic bacteria, and can be separated into two processes: light-dependent reactions and dark reactions.

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6.2.1.1 Light reactions These occur on specialized photosynthetic (thylakoid) membranes and involve the capture of light energy by photosynthetic pigments and the transfer of this energy to adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate hydride (NADPH). In organisms that carry out oxygenic photosynthesis (eukaryotic and prokaryotic algae), water is split as part of the reaction, releasing oxygen and making available electrons which convert NADP to NADPH. Light reactions are temperature-independent since they are photochemical in nature and are not driven by enzymatic processes. Light reactions are of two main sorts - photosystem I (PS I) which is associated with the conversion of NADP to NADPH, and photosystem II (PS II) responsible for the ~plitting of water. Each system involves a specific macromolecular assembly, the lightharvesting complex, which is divided into two separate regions - a surface antennal complex and an internal reaction centre. Absorption of light t!nergy is carried out by surface pigments within the antennal complex, with subsequent funneling' of the energy to the reaction centre. This contains a single molecule of chlorophyll-a (P680) to mediate electron transfer to electron chains. Antennal pigments include chlorophyll-a, plus a range of accessory pigments such as chlorophyll-b, chlorophyll-c, carotenoids, phycocyanins, and phycoerythrins. Although both photosystems are required for complete photo-synthesis, they are physically distinct, have independent requirements for photons, and may have different types of antennal p;gments. In the red and blue-green algae, for example, chlorophylla is associated particularly with PS I, while phycoerythrins are present mainly in PS II harvesting complexes. 6.2.1.2 Dark reactions These. are associated with the fixation of CO 2 , and although they do not require the presence of light, they follow light reactions by only a few hundredths of a second. These reactions occur in the spaces between thylakoid membranes and utilize the energy and reducing power of ATP and NADPH to reduce CO, to hexose, ultimately generating a variety of sugars, amino- and fatty acids. 6.2.2 Photosynthetic Microorganisms Two distinct groups of photosynthetic microorganisms occur in the freshwater environment - algae (including blue-green algae) and photosynthetic bacteria.

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Algae are the most prominent of the two, occurring in almost all freshwater environments and typically having greater biomass and greater productivity. Algae are also more dispersed within individual environments, occurring both as pelagic (present throughout the water column) and benthic forms, whereas photosynthetic bacteria are typically restricted to limited microhabitats (anaerobic zones). Hnally, algae are more biologically diverse, present as both eukaryotes and prokaryotes, with a wide range of structural and physiological features. Most of the work on light and photosynthesis has been carried out in relation to algae, and much of the subsequent description relates specifically to them. Photosynthetic bacteria and their contribution to ecosystem productivity are considered separately. Light is important to photosynthetic organisms both as a general growth resource (generating ATP and carbohydrates) and also as a more specific factor in cell processes such as gamete differentiation. Light has a key role, for example, in promoting sexual reproduction (gamete formation) in green algae. In Scenedesmus, illumination is important at two critical periods in the laboratory induction of gamete formation - shortly after nutrient (N) withdrawal, and when somatic cells are becoming irreversibly committed to gamete differentiation. 6.2.3 Measurement of Photosynthesis Photosynthesis, involving the first two of the above processes (light capture to carbon fixation), is typically measured either a~ 02 evolution or CO 2 uptake. Measurement via 02 evolution (by chemical analysis, an oxygen electrode, or manometry) is normally carried out where photosynthesis is particularly active, as in many laboratory studies. For field measurements, where photosynthesis is often. at a low level, a more sensitive technique may be required and the process is normally determined as fixation of 14CO,. This is carried out by the addition of small amounts of [14C] :bicarbonate to bottles containing phytoplankton samples, then suspending the sealed bottles at points in the aquatic environment for a defined incubation period (normally a few hours) where photosynthesis is being determined. The algae are then deposited on a membrane filter, treated with acid, and assayed for the amount of radioactivity incorporated. Although measurement of photosynthesis by 14C02 uptake has been widely criticized, its strength lies in its sensitivity - not for slow rates but for low biomass concentrations.

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The rate of photosynthesis may be expressed either as gross photosynthesis Pg (where 02 uptake and CO2 evolution during respiration are not taken into account) or net photosynthesis Pn (where corrections are made for respiration). Measuring photosynthesis simply as overall CO 2 uptake would thus give a measure of net photosynthesis, to which CO, generated during respiration would have to be added to obtain a- gross rate of photosynthesis. The converse is true where photosynthesis is measured by 02 evolution. Although the terms gross and net photosynthesis are clear in terms of definition, it is not always apparent exactly what is being measured. The use of 14C0 2 fixation to measure CO 2 uptake introduces a complication in terms of the fate of the labelled carbon atom, and there is some controversy as to whether this method measures gross or net photosynthesis, or gives an intermediate value. In addition to gas exchange methods, in situ photosynthesis can also be determined by the use of active fluorescence. This involves estimating photosynthetic rates from light-stimulated changes in the quantum yield of chlorophyll fluorescence, and has the advantage that artefacts associated with isolating phytoplankton assemblages in bottles are avoided and that there are no problems in differentiating between net and gross procedures. Whatever procedure is used for measurement, the rate of photosynthesis can be expressed either in relation to environmental dimensions (per unit area or per unit volume of water) or per unit phytoplankton biomass.

6.2.4 Photosynthetic Response to Varying Light Intensity The relationship between rate of photosynthesis (P) and light intensity or irradiance (l) can be investigated in the laboratory or under environmental conditions. Bottles containing algae can be suspended at different points in the water column of a lake, for example, where the attenuation of light with depth provides the necessary range of irradiance values. Photosynthesis - irradiance curves of particular algae are measured for two main reasons: to evaluate ecophysiological responses to light, and to predict in situ photosynthesis over a range of irradiance conditions.

6.2.4.1 PhotosYllthesis - irradiallce curves In freshwater algae (and higher plants) the rate of photosynthesis typically varies in a nOll-lineal relationship with light intensity. As light intensity increases from zero, the photosynthetic response

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undergoes three main phases - light limited increase (zone A), light saturation (zone B), and light-inhibited decrease (zone C).

6.2.4.1.1 Phase 1: Light limitation In the dark, photosynthesis does not occur, so exchanges of 02 and CO 2 are solely due to respiration. As light intensity is increased from zero, there is an initial low rate of photosynthesis, which increases with light intensity until the gas exchange of photosynthesis exactly compensates for that of respiration. The irradiance value (I) at this point is the light compensation point. Further increase in light intensity results in net photosynthesis, with an initial linear increase in the rate of photosynthesis with light intensity. The slope of the graph during the linear phase (a) represents the highest rate of increase of photosynthesis with time, and has a value which is determined by the rate of the light-dependent reactions. At these low irradiances the rate of photosynthesis is limited primarily by the number of photons captured by photosynthetic pigments, and the value 'a' provides a measure of the efficiency with which this occurs. 6.2.4.1.2 Phase 2: Light saturation With further increase in irradiance the graph becomes nonlinear as light becomes saturating. Photosynthesis over this nonlinear part of the PI graph is limited primarily by dark reactions such as those controlled by ribulose 1,5-bisphosphate carboxylase (RUBISCO) activity. Saturation of photosynthesis might also occur with lightdependent reactions if the light-harvesting complexes of photosystem II were no longer able to accept all photons that are incident on the antenna complexes. The maximum rate of photosynthesis (Prn.) is reached when the system becomes fully saturated at light intensity I m• x and is sometimes referred to as the photosynthetic capacity. 6.2.4.1.3 Phase 3: Photoinhibitiol1 In addition to limitation of photosynthesis by light dependent (phase 1) and dark-dependent (phase 2) reactions, photoinhibition may also be important (phase 3). This is the damaging effect of light on a range of cell activities, including photosynthesis. The effects of photoinhibition are initially seen at mid-level irradiances, resulting in an extended non-linear part of the curve and higher values for I . Photosynthesis at irradiances beyond I mo , also shows a sharp d~~~ease, with the slope of the curve (p) providing a measure of the degree of photoinhibition under these conditions.

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6.2.4.2 Variation ill PI curves

The onset of light saturation (transition from straight line to curve) is an important measure of algal light response, but is difficult to define from the graph. In view of this, an arbitrary measure can be obtained from the intersection of the line (slope a) with the line at Pm..' The irradiance value (/) at this point is a marker for the onset of light saturated photosynthesis, and can be used as an index of species acclimation to different light levels. Microcystis r111lacoseira 6

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All photosynthetic rate parameters vary with cell size and morphology, taxonomy and environmental factors such as previous light regime, CO, concentration, and temperature. The light intensity required to re-ach light compensation point (l), saturate photosynthesis (/k) and achieve maximum photosynthesis level (Ion.) varies markedly from one algal species to another, as also does the degree of photoinhibition. 6.3 LIGHT AS A GROWTH RESOURCE Light is an environmental growth resource, leading to the synthesis of macromolecules, increased production of biomass, and potential increase in population. The role of light as a growth resource may be compared with that of other environmental resources such as inorganic nutrients (phosphates, nitrates, silicates) and CO 2 concentration. 1. The potential supply of light to algal cells (mol photons m-2 S-I) can be expressed in similar units to the potential supply of inorganic nutrients (mol nutrient m- 2 S-I).

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2. In both cases, acquisition of the resource occurs as a two-step process - physical uptake and metabolic assimilation. In the case of light, this involves absorption by the light-harvesting complex followed by primary photochemistry, while nutrient acquisition involves transport across the plasmalemma followed by primary assimilation via respective enzyme complexes. 3. At low resource levels, acquisition of light and nutrients both show limiting concentrations, below which net growth does not occur. These limiting concentrations are determined partly by the efficiency of resource collection (light absorption, nutrient uptake) at low levels, but also by the balance of metabolic activities. In the case of light, the limiting light intensity at the bottom of the euphotic zone has been defined as the point at which the energetic gains of photosynthesis are just balanced by respiratory losses. An analogous situation occurs with inorganic nutrients, where the initial uptake and assimilation may be counterbalanced by sub~equent leakage and regeneration at subsequent points in metabolism. 6.3.1 Strategies for Light Uptake and Utilization As with inorganic nutrient resources, competition between algae has lead to the evolution of strategies for maximum uptake under different environmental conditions and in the optimum use of growth materials derived from it. In the case of light, these strategies involve adaptations and evolutionary commitments within different algal groups at all levels of uptake and utilization: 1. Capture of light, by photosynthetic pigments within a light harvesting complex, 2. Conversion of kinetic (light) to potential (molecular) energy this involves electron transfer and carbon fixation, 3. Synthesis of low MW compounds for physiological activities (e.g., osmoregulation) and complex macromolecules for growth. These aspects have evolved in various ways within the different groups of algae, leading to clear distinctions in terms of their ability to grow at different minimum and maximum light levels, their energy requirements in relation to cell wall synthesis and motility and their use of different small MW compounds in osmoregulation. 6.3.2 Light-Photosynthetic Response in Different Algae Differences in light-response between different algae, and the importance of environmental conditions, can be seen by comparison

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of the photosynthesis - irradiance (P -l) curves for blue-green algae and diatoms at different temperatures. PI curves are shown for two commonly-occurring members of the lake phytoplankton community - Microcystis (blue-green alga) and Aulacoseira (diatom). Microcystis (and other blue-greens) showed a general increase in the rate of photosynthesis with temperature (up to 30°C), but the diatom did not respond to increases above 20°C. In both cases, the rate of photosynthesis increased with photon flux density up to levels of 210-550 /Lmol photons m-2 S-I (about 10-25 per cent full sunlight), above which photo inhibition was observed in Microcystis but not Aulacoseira. Although considerable variation exists in P-I curves between individual algae and in relation to environmental parameters, each major group of algae has its own characteristics. The advantage displayed by Microc),stis over Aulacoseira at high temperatures, for example, by having higher values for Pmax and a under these conditions, is typical of other members within the blue-greens and diatoms. The general response of different algal groups (blue-green algae, dinoflagellates, diatoms, and green algae) have been summarized by Horne and Goldman (1994) in relation to light requirements for minimum photosynthesis (I), maximum photosynthesis (lma) and the onset of photoinhibition (I). The data indicate that: p 1. The range of light intensities over which algae grow varies considerably; 2. For most algae, photosynthesis commences at light intensities of around 5-7 /Lmol m-2 S-I - the threshold for green algae is substantially higher at 21 /Lmol m-2 S-I; 3. The light intensity at which maximum photosynthesis occurs is also higher for green algae compared with blue-greens, dinoflagellates, and diatoms; 4. Photoinhibition occurs at much lower light levels in diatoms (86 /Lmol m-2 S-I) compared with other algal groups (>200/Lmol m- 2 s -I). The differences in light response exhibited by the different algal groups represent varying strategies in their adaptation to environmental light conditions. This is shown in the seasonal succession of algae, where periods of major dominance broadly coincide with appropriate light conditions. In the case of diatoms, for example, the range of light that is most readily used fits the spring bloom

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period where light levels are generally low due to circulation of water prior to stratification and to ambient seasonal conditions. In polymictic lakes of temperate regions, continued mixing and circulation of water may lead to reduced average light exposure and domination by diatoms over much of the year. Green algae tend to dominate in the summer, particularly during the clear-water phase, when light intensitit;s are high. Although these algae can use up to 211 jLmol m-2 s- 1, this exceeds the light intensity normally available in the epilimnion.

6.3.3 Conservation of Energy Algae show adaptations in terms of conservation or optimal use of the energy that has been transformed by photosynthesis. A major part of this energy is used in the synthesis of macromolecules for growth, of which cell wall materials represent a major investment. Comparison of different algae shows that the energy required for cell wall construction varies considerably. The silica cell wall of diatoms requires about 12 times less energy for construction compared with the cellulose and peptidoglycan cell walls of other algae, giving these organisms the potential to outcompete other algae on an energy basis. This may contribute to the evolutionary success of these organisms and the fact that diatoms are by far the most common algae in both fresh and saline waters. The saving of energy by diatoms does have a trade-off, however, in terms of silicon requirement and high mass. The need for silicon means that growth of diatom popuiations may become suddenly limited when supplies of the element become depleted. The high mass of diatom cell walls confers a high specific gravity and sedimentation rate, so that many species require turbulent water conditions to stay in suspension, and may be restricted to particular types of lake or particular times of year. Complete absence of a cell wall saves on energy of construction, but .the naked cell requires considerable expenditure of energy in using a contractile vacuole to maintain its osmotic balance so this strategy saves little energy in the freshwater environment. Optimal use of energy is also important in movement within the water column. Active swimming of motile organisms such as cryptophytes and dinoflagellates involves continuous expenditure of energy. Organisms such as blue-green algae that use buoyancy to regulate their position within the water column require energy to synthesize the gas vacuole proteins, but once these have become

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established regulation is by the deposition and removal of carbohydrate ballast. This is a normal part of the diurnal activities of these organisms, so the diurnal migration in the water column requires little extra energy. The formation of ballast at the lake surface under conditions of high light will also sink the cells out of the zone of photoinhibition, so depth regulation in relation to shortterm changes in light intensity will also require little energy. The lack of any buoyancy or active motility mechanism in diatoms means that they have no direct energy expenditure in terms of maintaining their position within the water column, but does impose reliance on water turbulence. In temperate lakes, the relative success of these different strategies during the annual cycle depends on the physico/chemical environment and on competition with other algae. Diatoms are clearly adapted to turbulent water conditions from autumn to spring, but tend to be replaced by organisms with buoyancy regulation or active motility during the more static conditions of the stratified water column in summer. Although actively motile organisms such as dinoflagellates require high energy expenditure to migrate in the water column, their ability for nocturnal movement from a nutrientdepleted epilimnion down to lower parts of the hypolimnion for nutrient uptake gives them a competitive advantage in late summer. Blue-green algae are also able to migrate into the hypolimnion, but their buoyancy mechanism requires active photosynthesis for ballast formation. This may become limited in autumn due to conditions of reduced light, leading to later dominance by dinoflagellates.

6.3.4 Diversity in Small Molecular Weight Solutes and Osmoregulation Photosynthesis is closely linked to the generation of low molecular weight organic solutes that are involved in osmoregulation. The formation of simple sugars, in particular, results in the formation of a range of osmotically-active compounds which are diagnostic for particular algal groups. These compounds are important in the osmotic balance within freshwater environments, where algal cells are surrounded by a hypotonic medium. In such conditions the tendency for water to enter by endosmosis is either counterbalanced by cell wall pressure, or (in naked algae) requires continuous expulsion of water by contractile vacuole activity. In conditions of increasing salinity, where the external medium becomes hypertonic, higher intracellular concentrations of osmotically-active solutes are

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required to balance the elevated external molarity. The importance of light in the osmoregulatory process is illustrated by the diatom Cyclindrotheca fusiformis, where photosynthesis is responsible for maintaining the free internal mannose concentration at an adequate level to balance outside osmolarity. In the dark, free mannose can be generated from its polymer, polymannose. Under light conditions, a decrease in external molarity (hypotonic conditions) results in a conversion of free mannose to polymannose. If the external molarity is raised, polymannose synthesis is inhibited and free mannose is synthesized directly through CO 2 fixation. Some algae such as Dunaliella are euryhaline, able to grow over a wide range of salt concentrations. This alga exists as two varieties able to 'grow at NaCl concentrations of >0.5 M (halotolerant cells) and >2 M (halophilic cells) respectively. The halotolerant strain has large red cells which contain high concentrations of glycerol, and is able to grow in environments of high salinity such as the Great Salt Lake of Utah, where it forms dense populations colouring the water red or green. Blue-green algae form a particularly interesting group in terms of osmoregulation, with different small MW molecules being involved in different environmental situations as follows. 1. In environments with little variation in salinity, blue-green algae respond to increases in salinity by the formation of glucosylglycerol. This is formed relatively slowly, but is adequate to adjust to small changes in salinity. 2. In estuarine environments, where salinity changes occur rapidly as the tide goes in and out, blue-green algae produce sucrose or trehalose as the major osmoregulant. This production occurs over a short time period, allowing a rapid response to environmental change. 3. In hypersaline environments, such as the Great Salt Lake of Utah (USA), blue-green algae make long-term adjustment to high salinity by the continuous production of osmotically-active quaternary ammonium compounds glycine, betaine, and glutamate betaine. In addition to the production of small MW organic compounds, internal molarity may also be controlled via the internal concentration of inorganic ions such as K+. This is particularly important in bluegreen algae, where diurnal fluctuations in photosynthetically-mediated K+ uptake result in fluctuations in internal pressure (turgor) and ., the periodic formation and loss of gas vesicles.

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6.4 ALGAL GROWTH AND PRODUCTIVITY The photosynthetic conversion of light (kinetic) energy into chemical (potential) energy, with the formation of reduced carbon compounds, is the key process which generates biomass within aquatic systems. The rate of synthesis of biological material (primary production) is important in relation to the net increase in biomass and in the development of algal populations. 6.4.1 Primary Production: Concepts and Terms Algal growth requires the synthesis of a wide range of cellular material and photosynthetic production of organic carbon compounds is clearly only part of this process. The rate of synthesis of algal biomass is an important parameter since it determines the increase in algal population, and is referred to as the 'gross primary production'. Although this term is widely used, there is no generally accepted definition. Different workers have variously defined gross primary production as follows. 1. The rate of conversion of light energy into chemical energy. This reflects the view held by theoretical ecologists and plant biophysicists, who consider primary production in terms of energy transformations during the initial steps of photosynthesis. 2. The rate of organic carbon production resulting from photosynthetic activity. This definition considers production in terms of carbon flow and reflects the viewpoint of the community ecologist and plant physiologist. 3. The rate of assimilation of inorganic carbon and nutrients into organic and inorganic matter by autotrophs. This definition considers primary production in terms of the overall production of biomass, and follows the approach of workers such as biogeochemists who are interested in the full range of nutrients within the environment. 6.4.2 Primary Production and Algal Biomass Gross primary production does not directly translate into algal biomass since the synthetic increase in biomaterials is countered by a range of loss process, including respiration. Net primary production is normally defined as gross primary production minus autotrophic respiration. Other loss processes are also important, including excretion of materials, cell lysis, and activities of other freshwater biota. In the case of phytoplankton, loss of dead cells from the euphotic zone by sedimentation, and exchange of living cells bet;veen the

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water column and sediments are also important. The growth of phytoplankton can be expressed mathematically: I:!J3 / f1t = (P - R - E)B - Gp - S ± BE ... (2) where: till/ b.t = increase in biomass with time, P = rate of gross primary production, R = rate of respiration, E = excretion and cell lysis, B = existing biomass, G p = loss by grazing or parasitism, S = loss by sedimentation of dead cells, and Be = exchange of biomass between sediments and water column.

6.4.2.1 Seasonal relationship between growth and photosynthesis Investigations of the factors that determine seasonal changes in phytoplankton populations often focus on photosynthetic and growth responses of phytoplankton. Because growth of a population of algal cells primarily depends on the increase in protein biomass, which directly relates to carbon assimilation and photosynthesis, the relationship between specific growth rate (p,) and environmental conditions (light, temperature) might be expected to reflect the photosynthetic rate P. The increase in protein and carbon biomass (growth rate) depends on a balance of factors, however, and although photosynthesis, growth rate, and population increase are often closely coupled, this is not always so. The relationship between photosynthesis and population increase may vary because of a range of imbalances in cellular processes as follows. 1. Respiration rate does not change in direct proportion to photosynthesis at different light and temperature levels. 2. Cell division rates may peak at lower temperatures than photosynthetic rates. 3. Extracellular release of DOC (loss of biomass) may be less than 5 per cent at optimum light and temperature but can increase to nearly 40 per cent under conditions of low light and high temperature. DOC release under conditions of high light (causing photoinhibition) may also be excessive. 4. Net photosynthesis and carbon assimilation are restricted to the illuminated part of the day, while protein synthesis may continue in the dark.

6.4.3 Field Measurements of Primary Productivity Most measurements of algal productivity involve determination of photosynthetic activity, either in terms of CO 2 fixation or 02 evolution. The Heo 2uptake method developed by Steemann Nielson

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(1952) has been extensively used to estima - primary production, which is expressed as fixed carbon mass per l.nit area per unit time. Productivity may also be considered in terms of long-term development of biomass (algal production). This may be expressed as units of chlorophyll-a per unit area (benthic algae) or per unit volume of water (phytoplankton). Measurements of algal production as biomass are particularly useful in complex aquatic environments where carbon fixation by different algal groups may be difficuh to determine. Assessment of primary productivity varies with complexity of the ecosystem, ranging from relatively simple planktonic populations in lakes to more complex communities in wetlands.

6.4.3.1 Primary productivity of lake phytoplankton Primary productivity varies considerably in relation to the nutrient status of the lake, environmental conditions and type pf algae present. In some cases, productivity occurs mainly via the picoplankton. Measurement of photosynthetic uptake of 14C-bicarbonate during the summer growth phase of Lake Baikal (Russia), for example, indicated total productivity values of 36 j.Lg C I-I day-I, with over 80 per cent being carried out by unicellular blue-green algal picoplankton and nanoplankton.

6.4.3.2 Primary productivity in wetland communities Measurement of algal productivity in wetland systems is complicated in two main ways. 1. The algae being monitored occur as a diverse assemblage of attached and free-floating forms, and can be divided into four main types on the basis of their life style and microhabitat epipeJic algae (present on mud surfaces), epiphytic algae (attached to higher plant and macroalgal surfaces), metaphyton (surface or benthic floating masses of filamentous green algae), and phytoplankton (free-floating cells and colonies entrained in the water column). 2. Because of their different microhabitats, measurements of biomass and carbon fIXation tend to use different units in terms of the environmental parameter, making comparability difficult. Thus the biomass of epipelic algae is typically expressed per unit area of wetland, epiphytic algae per unit area of attachment sub~trate (e.g., higher plant surface), and phytoplankton per unit volume of water.

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Epipelon biomass can be measured in two main ways. Most studies of freshwater systems involve the collection of an algal sample by placement of lens paper onto the exposed sediment surface. Estimates of epipeIic algae by this method tend to give quite low biomass values « 10 mg m- 2 chlorophyll-a). These low values to some extent reflect the fact that growth of wetland epipelon is limited by shading from submerged and emergent macrophytes, their associated epiphytes, floating metaphyton mats, and phytoplankton blooms - all of which reduce light levels at the sediment surface over much of the growth season. Low biomass values may also reflect limited sample pick-up by this technique, which relies on the migration of algal cells into the lens paper. The other method of measuring epipelic biomass is from a core sample. This has the advantage that a1l epipelic algae are measured, but suffers from the drawback that degraded pigments from sedimented phytoplankton may also be present, resulting in an overestimate of living biomass in some situations. Chlorophyll-a analysis from core samples give values that are much higher and are more comparable to salt marsh epipelon. Higher epipelon biomass values might be expected in salt marshes, since the algae in these systems are part of a much more open community. Although salt marsh determinations are also taken from core samples, there is less contamination from phytoplankton in this situation. Epiphyton biomass is not usually expressed per unit area of wetland because of relating plant surface area to this. Data are normally based on algae that are attached to morphologically simple artificial substrata, with biomass values in freshwater marshes typically <5 p.,g cm-2 of chlorophyll-a. In those studies where epiphyton biomass has been estimated per unit wetland area, epiphyton biomass is equivalent to the level of algae present in other shallow waters. The relatively little quantitative information available for metaphyton is normally expressed in terms of dry weight, making comparability with other algae difficult. Biomass of metaphyton is generally < 10 mg cm- 2 , rising to much higher levels when metaphyton is undergoing rapid growth and forming extensive surface mats. Levels of phytoplankton in wetland systems are highly variable, but normally exceed 50 p.,g I-I chlorophyll-a. Assumi1)g a homogeneous distribution of phytoplankton within the water column and a depth of 1 111, this value is equivalent to 50 mg m-2 of

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chlorophyll-a per unit wetland area. Small eutrophic wetlands often have chlorophyll-a levels well in excess of this value (>200 mg m-2 chlorophyll-a). These wetland phytoplankton levels indicate biomass levels as high as those of epiphyton and metaphyton in less turbid areas, and are as high as phytoplankton levels that develop in eutrophic lakes.

6.5 PHOTOSYNTHETIC BACTERIA Although algae are normally the major microbial primary producers in freshwater environments in terms of overall biomass and productivity, photosynthetic bacteria are also frequently present and contribute to carbon fixation within the ecosystem. This section focuses on photosynthetic activities which differ from those of algae in lacking oxygen evolution and only occurring under anaerobic conditions. Bacterial photosynthesis requires external electron donors, such as reduced sulphur compounds or organic compounds in the photosynthetic reduction of COl' Assimilation of CO, via the Calvin Cycle occurs in all photosynthetic bacteria. Some straIns in the purple sulphur and non-sulphur bacteria are also able (mixotrophic) to use simple organic molecules such as fatty acids as their carbon source, separately or in combination with CO 2 uptake. 6.5.1 Major Groups Three major groups of photosynthetic bacteria have been recognized - the green sulphur bacteria (Chlorobacteriaceae), purple sulphur bacteria (Thiorhodaceae), and the purple and brown nonsulphur bacteria (Athiorhodaceae). The Chlorobacteriaceae are almost entirely obligate phototrophs, only able to grow under conditions of light and CO 2 availability. Thiorhodaceae are also predominantly photoautotrophs, but many species are potentially mixotrophic, able to photo-assimilate simple organic compounds. Athiorhodaceae photometabolize simple organic substances and are inhibited by H,S. Several species have the potential for aerobic heterotrophy. 6.5.2 Photosynthetic Pigments The absorption characteristics cf bacterial photosynthetic pigments have considerable ecological significance. These pigments are of two main types - bacteriochlorophylls and carotenoids - matching the light requirements of surface and deep water habitats respectively.

6.5.2.1 Bacteriochlorophylls Four major bacteriochlorophylls (a-d) have been isolated from photosynthetic bacteria, differing fr0111 chlorophyll-a (present in plants

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and algae) in details of various side groups. Although there is no simple correlation between taxonomic groups and type of bacteriochlorophyll, bacteriochlorophyll-a does tend to predominate in the purple sulphur and non-sulphur bacteria. Bacteriochlorophyllb appears to be restricted to these two groups, while bacteriochlorophylls-c and -d are typically found in the green sulphur bacteria. Bacteriochlorophylls differ from chlorophyll-a in having absorption maxima in the red and near infrared part of the spectrum. Since this part of the spectrum is strongly absorbed by water, these pigments will function primarily in exposed situations such as shallow ponds and estuarine mud flats.

6.5.2.2 Carotenoids Cultures of purple sulphur and non-sulphur bacteria have a variety of colours, ranging from peach to brown, pink, and purple red - varying with population density, sulphur content, and age of culture. These colours. are mainly due to the presence of carotenoids, which mask the bacteriochlorophylls and in the absence of which the purple bacteria would have the coloration of blue-green algae. More than 25 carotenoids have been characterized in photosynthetic bacteria. These have been divided into five major groups in terms of chemical structure and biosynthesis, with a diversity of carotenoids (Groups I-IV) being typical of purple bacteria, and Group V typical of the green sulphur bacteria. Taxonomic distinctions in terms of carotenoid content are shown by the presence of characteristic carotenoids for green sulphur bacteria (Chlorobactene), purple sulphur bacteria (Okenone), and purple nonsulphur bacteria (OH-Spheroidenone). Purple sulphur and nonsulphur bacteria show similarity in the mutual possession of Lycopene and Spirilloxanthin, but with none of the major carotenoids found in green sulphur bacteria. Carotenoids are physiologically and ecologically important in absorbing a different range of wavelengths compared with bacteriochlorophylls. Red and brown bacterial carotenoids selectively absorb light in the blue-green part of the spectrum, giving an absorption maximum at 450-550 mIL, which is the prominent wavelength in deep lakes. The presence of these pigments in deepwater purple and green bacteria (giving the latter a distinct brownish tinge) is clearly important for light absorption in this part of the water column.

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6.5.3 Bacterial Primary Productivity Photosynthetic bacteria are able to assimilate CO 2 in the presence of light and are thus primary producers. Although much is known about their physiology and occurrence in freshwater systems, relatively little is known about their contribution to overall productivity in aquatic environments. Studies on primary productivity of photosynthetic bacteria in lakes have tended to occur over a limited part of the annual cycle, at a time when bacterial populations were temporarily high. Work by Czeczuga (1968a,b) in Poland, for example, has demonstrated high rates of bacterial primary productivity during successive years of Chlorobium and Thiopedia blooms, reaching carbon fixation levels of 55 and 157 mg C m-2 day-I respectively. These values represent a substantial part of the overall primary productivity, in one case exceeding the algal contribution. In spite of temporary maxima in CO 2 fixation by photosynthetic bacteria, their contribution to prOductivity over the whole annual cycle is considered to be generally quite low. Exceptions to this may occur where algal production is depressed due to circumstances such as input of allochthonous materials. This is the case at Smith Hole Lake, USA where the mean annual productivity of photosynthetic bacteria was about half that of the algae.

6.6" PHOTOADAPTATION: RESPONSES OF AQUATIC ALGAE TO LIMITED SUPPLIES OF LIGHT ENERGY In most aquatic environments, the supply of light may vary , considerably (both spatially and temporally) and may become limited in relation to algal optimal photosynthetic requirements. In the water column of a typical temperate lake or river, for example. midday light intensities during the summer vary from high levels at the water surface (over 800 J,Lmolm-2 S-I, on a bright day) to zero, just below the euphotic zone. These light levels have potentially damaging effects on algal cells at the water surface (photoinhibition), while the provision of energy for photosynthesis becomes extremely limited in the lower part of the water column. The ability of algal cells to continue photosynthesis and to survive in environmental conditions of low light availability is referred to as photoadaptation. Both planktonic and benthic algae are able to adjust their molecular and physiological activities in relation to different levels of light availability.

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6.6.1 Different Aspects of Light Limitation During daylight hours, exposure of algal cells to the sun's energy may become limited in three main ways: 1. Temporal changes in light intensity, where alternating levels of high and low irradiance affect the continuity of physiological processes and the degree of .adaptation at anyone point in time. In the planktonic environment, variations in light intensity may be' caused by factors such as moving cloud cover and changes in shading by drifting populations of algae and zooplankton. Circulation of algae within the water column due to water turbulence also limits overall light availability, and is important during periods of seasonal non-stratification, in polymictic lakes, and in water bodies subject to artificial disturbance (hypolimnetic aeration). Benthic communities are also subject to similar time limitations. In forest streams, for example, sun beams (sunflecks) penetrating the le::}f canopy can contribute 10-85 per cent of total daily irradiance, but are very transient, moving across the streambed as the sun moves across the sky. 2. Continuous exposure to low light intensity, where algal cells have a long-term location within a particular light-limiting microenvironment. In the case of lake phytoplankton, this may involve the permanent location of populations within lower parts of the euphotic zone during the slimmer growth period. During winter, low ambient light levels combine with water mixing in unfrozen lakes to limit photosynthesis. Frozen Arctic and Antarctic lakes are particularly limited in relation to light penetration. Benthic algal communities (periphyton) frequently experience conditions of external and internal light limitation. External limitation involves a reduction in light levels reaching the community, while internal limitation occurs due to self-shading within the algal mat. External light limitation is important in both stream and lake environments. The shading effects of terrestrial vegetation are particularly pronounced in streams, where leaf canopies can intercept 95 per cent or more of incident sunlight, reducing maximum photon flux densities (PFDs) to less than 40 JLmol m-2 S-I. Benthic algae in lakes experience low light levels due to attenuation within the water column. This affects benthic algal communities at the edge of lakes (where light-mediated differences in community stt~ucture occur with depth) and also

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deep-water periphyton communities in oligotrophic clear-water lakes. In Lake Tahoe (USA), for example, 1 per cent of the light penetrates to sediments 60 m below the surface, allowing the development of a benthic community at the bottom of the water column. 3. Limited range of wavelengths. Both planktonic and benthic algae may become exposed to a limited range of wavelengths due to differential absorption of light above the water surface and within the water column. Differential absorption prior to water entry is particularly important in relation to forest streams, where the spectral composition of light is altered as it filters through terrestrial vegetation. Leaf absorption of red and blue wavelengths leads to a light environment which is weighted towards green wavelengths. Passage of light through the water column of lakes and streams results in progressive loss of wavelengths at both ends of the visible spectrum with depth, leading to a predominantly green/blue spectrum. The ability of algal cells to condition themselves to limiting light conditions is achieved by a range of molecular and physiological modifications. These vary in time from short-term changes in lightresponsive gene activity to long-term alterations in cell compartmentation and pigment content.

6.6.2 Variable Light Intensity: Light-Responsive Gene Expression Phytoplankton cells are able to react to transient increases in light intensity by activation of specific light-responsive genes. This activation has been investigated particularly in relation to blue-green algae and involves three main processes - stimulation of wavelengthspecific receptors, light-responsive signal transduction pathways, and activation of a range of genes.

6.6.2.1 Receptor-signal transduction pathways In addition to light. blue-green algae are also able to sense and respond to changes in nutrient and oxygen concentrations. Cyclic nucleotides, including cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), appear to playa key role in the signal transduction process. In Anabaena and other blue-green algae, cAMP concentration changes rapidly in response to environmental changes, with an increase in concentration on transition from light to dark and vice versa. These results suggest

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that cAMP acts as a second messenger of light signal transduction in blue-green algae, moderating light-responsive activity of a variety of processes. These include cell motility, as demonstrated in the unicellular blue-green alga Synechoc)'stis by Terauchi and Ohmori (1999). Mutants of this alga which lack the enzyme adenylate cyclase lose their motility. This enzyme is required for cAMP synthesis, and motility can be restored by exogenous addition of cAMP. Under standard growth conditions, the two second messengers cAMP and cGMP normally occur at similar levels in blue-green algae but changes in light, nutrients, and oxygen lead to changes in the level of both nucleotides. The intracellular levels of both molecules are regulated by cyclic nucleotide phosphodiesterases, which playa pivotal role in cAMP and cGMP signal transduction. Ochoa de AIda and Houmard (2000) have identified two putative phosphodiesterases by screening the complete genome sequence of the unicellular bluegreen alga Synechoc)'stis, using bioinformatic tools and applying protein sequence-function studies. These phosphodiesterase genes are surrounded by a cluster of regulatory genes, with all the genes being transcribed as a single unit. The regulatory genes encode polypeptides that act as two component systems, containing both signal transmitter (sensory histidine kinase) and receiver (response regulator) domains. Two of these genes contain signalling motifs known to regulate light-stimulated signalling pathways in other organisms, particularly involving blue light. In Synechocystis, blue light is known to induce changes in the intracellular level of cAMp, suggesting that the putative cAMP phosphodiesterase is part of the blue light signal transduction pathway.

6.6.2.2 Light-responsive genes Signal transduction of changes in light intensity allows cells to respond to environmental change by modulating the expression of light-responsive genes, thus producing an integrated response. Lightmodulated gene expression affects a wide range of metabolic activities in freshwater algae, various examples of which are considered in different sections. Details of light induction of microcystin-related proteins and gas vesicle formation are considered elsewhere, but the light-responsive formation of photosystem proteins and pigments is considered here since it is particularly relevant to photcadaptation. This light-modulated activity means that algae can make responses to light fluctuations within the highly variable aquatic environment, optimizing their

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photosynthetic activity over relatively short time periods. This occurs by promoting the synthesis of photosystem II centre proteins, chlorophyll-a binding proteins and phycobilin pigments (phycocyanin and phycerythrin). Wavelength-specificity for light-responsive gene expression varies considerably between genes, suggesting the involvement of multiple receptors and multiple transduction pathways. With some genes, the activation wavelength relates directly to the function of the end-product. The synthesis of phycocyanin in the blue-green alga Calothrix (cpcB2 operon), for example, is promoted by red light while the synthesis of phyocerythrin (cpe operon) is activated by green light. With some light-dependent physiological processes, Iightresponsive gene expression and protein induction are not sufficiently rapid to provide adequate response to short-term environmental changes. This is the case for nitrate assimilation, for example, where a more responsive reversible enzyme inhibition/activation system has also developed. 6.6.3 Photosynthetic Responses to Low Light Intensity Phytoplankton and benthic algal cells can adapt to different light levels by adjustments to their physiology, as indicated by changes in the characteristics of their photosynthesis - irradiance (P-I) curves. These changes reflect alterations in the balance of biochemical processes within photosynthesis and by adjustments in the allocation of photosynthetic products to different metabolic pools. Short-term changes in photosynthetic parameters are triggered by activation of the light-responsive genes discussed previously.

6.6.3.1 Physiological adaptations to low light levels: characteristics of P-I curves Quantitative parameters in the P-I relationship vary with ambient light conditions in both planktonic and benthic algae. Adaptations to low light intensities are usually characterized by: 1. Lower photosynthetic rates at saturation irradiances (P rna)' 2. A low compensation point (I), 3. Increased photosynthetic efficiency at low irradiances (increased a), and by 4. Decreased saturation parameters (lower Ik or Ima)' Relatively little information is available on (. levels, though some species of phytoplankton and benthic algae are known to have values less than <2 pmol m-2 S-I, and are clearly adapted to habitats where

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photons are scarce. Differences in photosynthetic efficiency and saturation levels have been demonstrated in samples from different light environments for both planktonic and algae.

Planktonic algae Physiological adaptations to low light levels have been demonstrated particularly clearly in cultures of planktonic algae. Cells of the green alga Scenedesmus, for example, showed markedly different P-I responses when cultured at high and low light levels. Continuous cultures of low-light grown cells saturated at much lower irradiance levels Urn.X> compared to high-light grown cells, with a lower maximum photosynthesis rate (P maX> per unit chlorophyll. 10

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One of the most extreme cases of light adaptation in the planktonic environment is provided by microalgae living under ice in Arctic and Antarctic lakes. These organisms exhibit light saturation at less than 10 ILmol m-2 S-I.

6.6.3.2 Bentltic algae Analyses of P-I curves from benthic algae provide an interesting contrast to the phytoplankton data discussed previously. These laboratory studies on intact stream periphyton communities obtained from shaded (maximum light 50 ILmol m 2 S-I) and open sites (maximum 1100 ILmol m- 2 S-I) demonstrated that P-I responses of benthic algae were not well related to the ambient light environment, but showed better correlation to algal biomass. The key difference between phytoplankton and periphyton in terms of P-/ response is that the phytoplankton population occurs in suspension, while

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periphyton occurs as dense mats - with self-shading and other aspects of development in the matrix affecting the light response. Even under bright light, due to the thickness of the periphyton mat, the overall community P-/ response may be more typical of low light conditions. This is because the P-/ curve is an integration of the responses of cells that are high-light adapted (upper layer) and shade-adapted (lower layer). For periphyton mats, biomass and related thickness are an important and, in some cases, dominant influence on the pI responses of the community. Despite the varied light regimes of benthic algae, most P-/ studies indicate /k values within a narrow range (100-400 JLmol m-2 s I). These saturation irradiances are generally much higher than ambient irradiance values occurring in situ. In heavily shaded forest streams, for example, photosynthesis by attached algae typically saturates at between 100-200 JLmol nr2 s I, yet maximum irradiances during summer (time of greatest shade) rarely exceed 30 JLmol m-2 S-I. This suggests that light saturation irradiance levels for benthic algae are not normally reached in the lotic environment, and that photosynthesis in streams is typically light-limited. The relatively high light saturation values for stream benthic algae is surprising and may reflect the fact that the photosynthetic machinery of these organisms is geared to harness the higher irradiances that occur infrequently (e.g., sunflecks) or seasonally (e.g., winter and spring maxima for streams shaded by deciduous trees).

6.6.3.3 Biochemical adaptations For phytoplankton populations, differences in P-/ responses reflect adaptations to low light at the biochemical level. These adaptations occur in relation to the machinery of photosynthesis (balance of chemical activities, chlorophyll concentration) and the allocation of photosynthetic products.

6.6.3.3.1 Balance of chemical activities Within photosynthesis, algal cells need to balance the processes of electron generation (by the light-harvesting complex), electron transfer, and carbon fIxation. Reduction in light intensity leads to an immediate reduction in the generation of electrons, and requires a rapid cell response. Transfer from high to low light intensity involves an up-regulation in the synthesis of pigments and light-harvesting complex proteins, and a down-regulation of electron transfer and carbon fIxation.

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Studies on the green alga Dunaliella tertiolecta have shown that the signal regulating these photoadaptative changes is provided by the redox state of the plastoquinone pool within the electron transfer chain of photosystem II. A shift from high to low light intensity results in a decrease in electron flow from the photosystem II light-harvesting complex, conversion of the plastoquinone pool to a highly oxidized state, and increased synthesis of mRNA encoding the light-harvesting complex proteins (cab mRNA). The rate of synthesis of these proteins is enhanced after a lag phase during which cab mRNA accumulates. A shift from low to high light intensity has the converse effects.

6.6.3.3.2 Chlorophyll concentration Up-regulation of pigment synthesis at lower light intensities, leading to increased pigment content, has been well-documented for both prokaryote and eukaryote algae. These changes in chlorophyll content can be directly measured in laboratory and also in environmental phytoplankton samples as an increase in the concentration of chlorophyll per unit cell biomass. This may be expressed as the chlorophyll-a/carbon ratio (8). The changes also lead to a decrease in the rate of carbon fixation per unit mass of chlorophyll (chlorophyll-specific photosynthesis rate (PChl), though the rate of carbon fixation per unit mass (pBlomass) may remain unchanged. Photoadaptation of phytoplankton under environmental conditions is demonstrated by physiological observations of depth samples and also by the occurrence of deep chlorophyll maxima as follows. 6.6.3.3.3 Physiology of depth samples Photoadaptation within the water column is well illustrated by the studies of Tilzer and Goldman (1978) on depth variations in algal physiology in Lake Tahoe. These authors sampled phytopiankton from three depths (surface, 50 m, and 105 m) and incubated these samples with 14CO, at a series of depths throughout the water column (samples re:suspended down to 105 m). The results show that photosynthetic rates expressed per unit chlorophyll (PChl) were much higher in samples originally derived from the middle and top of the water column. Values for carbon fixation per unit biomass (pBiomass) showed more similarity in the three samples, and showed clear variation with original sample depth. Phytoplankton originally sampled at the lake surface showed a peak of pBlomass at a much

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254

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Figure 6.5 Photoadaptation at different depths in a clear lake.

shallower level (about 20 m) compared with phytoplankton originally derived from 105 m, indicating clear algal adaptations to local light regimes within the water column. The growth rate of phytoplankton at different depths VJ.) can be related to the parameters pchl and 8, where: JLd = p Chl 8

6.6.3.3.4 Deep chlorophyll maxima The increased chlorophyll/biomass ratio that occurs during photoadaptation also provides an explanation for sub-surface chlorophyll maxima which are frequently observed in the clear waters of oligotrophic lakes. These chlorophyll maxima do not correspond to peaks in phytoplankton biomass, but are localized regions of high chlorophyll concentration (per unit carbon algal biomass) at the bottom of the euphotic zone, where there is an increased nutrient supply with depth and conditions are oxygenated. Studies by Fennel and Boss (2003) on algal distribution in the water column of oligotrophic Crater L'lke (USA) showed that biomass and chlorophyll maxima are generated by fundamentally different processes. Under steady-state conditions, maxima in algal biomass occur primarily in relation to positioning mechanisms and the local balance between growth increase and losses (respiration and grazing), while the vertical distribution of chlorophyll is determined mainly by photoadaptation.

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6.6.3.4 Allocation of photosynthetic resources Changes in the above balance of activities within photosynthesis occur as a short-term response (minutes to hours) to changes in light intensity. Continuation of high or low light conditions leads to more long-term (hours to days) changes in pigment content and in the fate of the photosynthetic products. Geider and MacIntyre (1996) have modelled these changes with a view to establishing long-term quantitative parameters of photoadaptation, and have identified three main destinations for fIXed carbon - light-harvesting apparatus, biosynthetic apparatus (for growth and division), and energy storage reserves. In their dynamic model of phytoplankton growth and light adaptation, the relative distribution of photosynthate to these different pools varied considerably with light intensity. At high irradiance, the proportion of fixed carbon being cycled back to the light-harvesting apparatus was minimal, with substantial allocation to energy storage reserves. At low levels of irradiance, the balance between these pools was reversed. At aU levels of irradiance, the relative flux of carbon to the biosynthetic apparatus was substantial, emphasizing the need for continued cell growth and population increase.

6.6.4 Spectral Composition of Light: Changes in Pigment Composition The spectral composition of light in the freshwater environment varies with depth and with water quality. These changes in the light spectrum impose limitations on light absorption by algal cells, which may respond by making compensatory adjustments to the balance of their photosynthetic pigments. The effect of changes in spectral composition have been investigated by Pick (1991), who examined the abundance and composition of small-ceUed blue-green algae (picocyanobacteria) in the surface waters of 38 freshwater lakes of varying trophic status and dissolved inorganic carbon (DIC) content. Two major groups of these organisms could be distinguished on the basis of the presence of the accessory pigments phycocyanin and phycoerythrin, with absorption peaks in the orange-red (620-650 nm) and green (peak 550 nm) parts of the spectrum respectively. One group of the algae (PE cells) had both pigments, while the other (non-PE cells) completely lacked phycoerythrin. The proportion of PE cells within the picocyanobacterial assemblage declined markedly in lakes which

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were nutrient enriched or were coloured by humic material. These lakes showed high light absorption and a shift in predominant spectral composition towards the yellow part of the spectrum. The change in algal composition indicated a change in pigmentation to optimize longer wavelength absorption under these conditions. Conversely, PE cells were particularly abundant in oligotrophic and mesotrophic lakes, where conditions of high light penetration and substantial levels of blue/green wavelengths prevail. 6.7 CARBON UPTAKE AND EXCRETION BY ALGAL CELLS Uptake of CO 2 by freshwater algae, the major microbial primary producer, has been considered so far in terms of energy input (solar radiation), carbon fixation, and growth of the organisms involved. Carbon incorporation and processing during photosynthesis also has major effects on the chemical composition of the aquatic environment, both in terms of CO 2 removal and excretion of dissolved organic substances. 6.7.1 Changes in Environmental CO 2 and pH Carbon dioxide is one of the major inorganic nutrient requirements for phototrophic organisms, occurring as the most oxidized and abundant form of inorganic carbon available in the biosphere. It is present in solution as dissolved CO 2 , carbonic acid (H 2 CO), and as bicarbonate (HC0 3-) and carbonate (CO/-) ions. Between pH 4 and pH 11, these species are linked by the following equations: CO 2 + Hp = H 2 C0 3 HS0 3 = H+ + HC0 3••• (4) HC0 3- = H++CO/The total inorganic carbon (C T) C T = lC0 2] + [H 2 C0 3] + [HC0 3-] + [CO/-] The relative proportions of these chemical species is governed by the equilibrium, the state of which depends to a large extent on pH. Free CO, is present in significant amounts between pH4 and pH7, decreasrng rapidly by pH8 and occurring only at about 0.003 per cent of the total carbon species by pH9. This environmental equilibrium is influenced by various factors, including the net removal of CO, by autotrophs (photosynthesis), CO, generation by heterotrophs- (respiration). and addition of lime part of management activities.

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6.7.1.1 Autotrophic activity Active photosynthesis of macrophytes and algae (phytoplaIlkton, benthic algae) results in the removal of free CO? from solution. This leads in turn to the reaction of bicarbonate ions with water to generate more CO? and CO_2- ions. The carbonate ions react with water to form hydroxyl (OH-) ions, giving rise to an increase in pH. The extent of the pH rise depends on the buffering capacity of the water, which has a pronounced maximum of pHS.1-S. 3 in fresh waters. Because of this, a small increase in pH brought about by photosynthesis results in a large fall in the concentration of free CO? Accumulations of algae (particularly blue-green algae) at the lake surface thus tend to increase pH and make CO? less available under conditions of bright light and resulting high rates of photosynthesis.

6.7.1.2 Heterotrophic activity Addition of CO 2 to lake and river water occurs from both external (atmospheric) and internal (heterotrophic) sources. Many aquatic systems are heterotrophic, or contain heterotrophic compartments, and are supersaturated with CO? This occurs particularly in anaerobic regions of high bacte-rial population, such as the hypolimnion and sediments of eutrophic lakes. Direct addition of any of the carbon species in Equation (4) will have an effect. increasing the total amount of inorganic carbon present and affecting the concentration of all components. Addition of bicarbonate for example will increase the absolute levels of CO 2 and H 2 CO,.

6.7.1.3 Addition of lime to aquatic systems Addition of elements other than carbon may also be important in affecting the total concentrations and balance of carbon species in lake water. This is particularly the case for calcium, which is often applied to commercial fishponds in the form of limestone (CaCn) or lime (a mixture of calcium hydroxide and calcium bicarbonate). Addition of lime to water of moderate to low alkalinity results in an increase in pH due to the removal of free CO? by the calcium hydroxide and the formation of soluble calcium bicarbonate. This rise in pH increases the buffering capacity of the water and accompanies a rise in the level of inorganic carbon pool in the aquatic system. These changes in water chemistry increase the primary productivity of both macrophytes and algae. ultimately resulting in increased levels of fish production.

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The total concentration of inorganic carbon in aquatic environments, and its distribution between the various chemical forms, is relatively constant in the surface waters of oceans, but varies considerably in freshwater systems. In freshwater bodies, pH can range from 1.0-11.0, with total inorganic carbon concentrations varying from 10 mmol m- 3 (acidic waters) to 100 mol m- 3 (bicarbonate-rich waters at high pH). The uptake of CO 2 by algae can be severely limited at the low end of this concentration spectrum, particularly as inorganic carbon species have low diffusion coefficients and low rates for conversion between chemical species at low pH values. Variation in algal species composition between waters of different pH can be partly attributed to the adaptations of different organisms to differences in CO 2 availability. 6.7.2 Excretion of Dissolved Organic Carbon by Phytoplan kton Cells Phytoplankton cells release a wide range of so.Juble organic compounds into the surrounding water medium. These mllke an important contribution to the overall dissolved material present in the ecosystem and are referred to as dissolved organic carbon (DOC) or dissolved organic matter (DOM). Many of these compounds are derived directly from the process of photosynthesis. DOC released by phytoplankton cells includes carbohydrates, polypeptides, free amino-acids, organic acids (particularly glycollic acid) and cyclic AMP. Considerable molecular diversity occurs within each of these major groups. Analysis of soluble carbohydrates in the Plussee (Germany), for example, involved the determination of 26 monosaccharides and six disaccharides. The major derivation 'of lake water concentrations of these organic compounds from phytoplankton cells is indicated by environmental observations of seasonal correlations of DOC with algal biomass, diurnal correlations with phytoplankton activities, and laboratory studies on photosynthesis and DOC release.

6.7.2.1 Range of activities involved ill DOC release Release of DOC occurs from both healthy and degenerating (lytic) algal cells. In healthy cells, DOC exudation takes place due to passive diffusion (leakage of photosynthetic products) and active secretion (e.g., production of extracellular enzymes, siderophores). The passive diffusion of these substances out of algal cells depends on their intracellular and extracellular concentrations, and the

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259 Curbuhydrutc and nucleic acid

0.8

Lipid

0.2

4000

2000

1000

Wavenumhcl1i (cm· I ) Figure 6.6 Molecular composition of

Doe

from a eutrophic lake.

membrane permeability of the cell and is directly related to the level of photosynthesis. Measurements of algal extracellular DOe in natural phytoplankton assemblages have generally indicated higher rates of release compared with those recorded in the laboratory using axenic algal cultures. Although algae are probably the major source of DOe in many standing freshwater systems, these compounds are also derived by secretion and lysis of other biota, including bacteria and zooplankton. Viruses, with sizes <0.2 pm, are also part of the defined soluble fraction. In addition to autochthonous DOe (derived from algae and other biota contained within the water body), DOe may also enter the system by inflow from external sources. Such allochthonous DOe includes humic acids and other material derived from the catchment area, and is particularly important in flowing (iotic) systems. The derivation of DOe from different sources is indicated by infra-red analysis of filtered water samples. Infra-red (FTIR) absorption spectra show clear bands of carbohydrate, polypeptide, and lipid material, consistent with origins via passive release, active secretion, and lytic activities of phytoplankton cells and other biota.

6.7.2.2 Ecological significance of Doe release by

pilytopialtkton Although the release of DOe represents only a small fraction (usually <5 per cent) of primary production, the dominance of phytoplankton over most of the Earth's surface makes this release a significant global carbon flow, with important implications for both

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marine and freshwater systems. DOC exudation is ecologically significant in a number of ways. 6. 7.2.2.1 Decrease in net primary productivity DOC release represents a loss of carbon which had been fixed by algal cells during photosynthesis, and thus decreases the net primary productivity. Most of this loss can be regarded as 'accidental' in the sense that the majority of these compounds are simply leaking out of the algal cells, and their release does not bring any apparent benefit to the algal cells. The loss of carbon is accompanied by a loss of nitrogen and phosphorus compounds.

6.7.2.2.2 Photosynthetic balance Although the loss of photosynthetic products is essentially a wasteful process, it may be important in enabling phytoplankton to dispose of excess carbon products accumulation of which may overwhelm the cell's ability to process fixed carbon.

6.7.2.2.3 Nutrient uptake and retrieval Some of the compounds released by algae are actively secreted and are important in nutrient uptake and retrieval. The enzyme alkaline phosphatase, for example, is excreted by many phytoplankton species under conditions of low phosphorus availability. This acts by releasing inorganic phosphate from organic phosphate complexes within the aquatic medium and is important in phosphorus recycling. Siderophores, released by blue-green algae, are important under conditions of iron limitation for the scavenging of Fe 3 + ions from the surrounding environment.

6.7.2.2.4 Microbial loop DOC represents a major carbon source for heterotrophic organisms, including bacteria, protozoa, and some algae. The release of DOC by algal cells is the main driving force for the microbial loop, promoting the growth of these microorganisms and the flux of carbon throughout the whole food web. It is a major aspect of the carbon cycle within the aquatic environment.

6.7.2.2.5 Indirect interactions between phytoplankton and bacteria The excretion of DOC by phytoplankton has direct effects on bacterial productivity, which may in turn have reverse adverse or beneficial effects back on the algal population. As an example of the former, competition between algae and bacteria for inorganic nutrients will be increased with greater bacterial populations, reducing

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the levels of nitrate and phosphate available to the algae. Increased DOC levels will also increase the population of antagonistic bacteria. Specific beneficial effects include the promotion of surface symbiotic bacteria, some of which are important in algal nitrogen fIxation. Murray (1995) has suggested that promotion of the microbial loop by phytoplankton is also an important defence against viruses. The increased bacterial population will result in increased levels of random algal virus adsorption to bacterial cells, with rapid destruction of non-infecting viruses at the bacterial surface.

6.7.2.3 Photosynthesis and DOC release Early studies on primary productivity in algae indicated that, during photosynthesis, algae release a substantial quantity of their newly-formed photosynthetic products into the surrounding medium as dissolved organic carbon. The quantitative relationship between carbon fixation during photosynthesis and release as DOC has been studied by supplying axenic algal cultures with radiolabelled carbon (14C) and following the course of transfer. The amount of 14C which is taken up into organic carbon in cells (primary productivity) and excreted into culture medium (DOC) can be determined by scintillation counting after removal of inorganic carbon and by making appropriate background corrections. Fallowfield and Daft (1988) used this approach to investigate the relationship between photosynthesis and DOC release in a range of blue-green and green algae, and to determine the effect of one major external factor - the presence of lytic bacteria - on this relationship. Under the conditions used in these experiments:. 1. All algae showed active photosynthesis, witfi 'higher rates of carbon fixation in blue-green (5-9 jLgC jLgChl~-1 h- I ) compared with green algae (1-5 jLgC jLgChl-a- 1 h- I ), 2. In all cases, active photosynthesis was associa;ted with the release of significant amounts of DOC. The rate of DOC release was related to the rate of carbon fIxation, The high carbon assimilation rates of blue-green algae were associated with particularly high rates (0.2-0.4 jLgC jLgChl-a- 1 h- I ) of DOC release in these organisms, In individual cultures, the linear increase in carbon fixation was matched by a linear increase in DOC production, indicating a close coupling between the two processes. In general, the loss of carbon as DOC ranged from 1-7 per cent of productivity,

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Inoculation of the cultures with the bacterium Lysobacter, an organism closely associated with bloom-forming blue-green algae, reduced the level of carbon fixation and increased the amount of DOC released by blue-green but not green algae. The promotion of increased blue-green algal DOC release by this organism suggests a specific uncoupling of this process from photosynthesis. The excretion of algal organic molecules has clear benefits for the bacterium, since LysobLlcter showed active growth in algal culture filtrates, taking up a range of amino-acids present within the DOC. This has potential environmental importance for increasing the availability of algal-derived DOC to associated bacteria such as Lysobacter, and to heterotrophic organisms generally within the plankton.

6.7.2.4 Location of DOC release and assimilation within the water column Conventional theory postulates that photosynthetically derived DOC is ~ecreted by algae in the euphotic zone, where it is locally recycled via the microbial loop. Soluble carbon is thus assimilated by heterotrophic bacteria in the top part of the water column, while particulate carbon sediments into the hypolimnion and is broken down in the lower water column and benthic zone. Although this model holds for some lakes, it does not apply to others. Recent studies by McManus et al. (2003) on Lake Superior (USA), for example, have shown a substantial amount of DOC passing intQ hypolimnion where it is broken down and contributes to deep lake oxygen consumption. Measured rates of oxygen consumption in this lake subst8l1tially exceed those that would occur simply from particulate carbon.

6.8 COMPETITION FOR LIGHT AND CARBON DIOXIDE BETWEEN ALGAE AND HIGHER PLANTS In most aquatic habitats, environmental conditions support the growth of higher plants (macrophytes) as well as algae. Both groups of biota are actively photosynthetic (primary producers) and compete for carbon dioxide, light, space, and inorganic nutrients. 6.8.1 Balance betwet:n Algae and Macrophytes in Different Aquatic Environments The balance between algae and macrophytes varies with the type of aquatic system and the water quality.

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6.8.1.1 Lakes In temperate climates, lake macrophytes are largely restricted to the edge of the lake (littoral zone) or other shallow regions, and there is relatively little overall competition for light with phytoplankton. The development of macrophytes in the littoral zone increases with progression from oligotrophic to eutrophic status, but very high nutrient levels lead to intense algal blooms and repression of higher plants. Tropical.' and subtropical lakes often show extensive development of floating macrophytes such as the water hyacinth (Eichornia crassipes) and water lettuce (Pistia stratoites). These may cover large areas of lake water and totally dominate primary production in the lake.

6.8.1.2 Wetlands Wetland sites such as shallow pools and water meadows typically have extensive developments of macrophytes, which can dominate primary production and out-compete algae. The interception of light by both rooted and floating macrophytes can be very high. During pedods of maximum Phragmites growth, up to 95 per cent of incident radiation can be removed by the developing canopy. Similarly, transmission of light through floating Lemna mats may be only 0.1 per cent of surface irradiance. Mac~ophyte dominance is particularly well seen in seasonal wetlands. In North West Australia, for example, the wet season brings high humidity, thunderstorms, and torrential rain, with extensive flooding of large tracts of land. These seasonal wetlands support a wide range of rapidly-growing hydrophytes, which die or become Qormant with the onset of the dry season. More permanent wetlands, such as those of the Trebon. Biosph'ere Reserve, also show marked seasonal variation and have been the subject of intensive research. 6.8.2 Physiological and Environmental Adaptations in the Competition between Algae and Macrophytes The high nutrient status of the Trebon fishponds favours algal domination, and macrophytes need to employ a range of ecological and physiological adaptative strategies to survive in such eutrophic and hypereutrophic conditions. These strategies include seasonal growth patterns, which are important in relation to light and CO)availability, and are part of a broad range of environmentid adaptations which affect competition between these biota.

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6.8.2.1 Assimilation of CO 2 and bicarbonate ions The ability of vascular plants and algae to assimilate bicarbonate ions in addition to free CO 2 is one of the most important adaptations to life in the aquatic environment. This physiological adaptation allows both sets of organisms to tolerate high pH and overcome frequent depletion of free CO 2 resulting from intense photosynthesis. In general, algae are more efficient than higher plants in the assimilation of bicarbonate ions, which gives them a competitive advantage when pH rises to high values. In addition to this, not all higher plants show this activity. The hydrophyte Ceratophyllum demersu111, for example, has light-adapted and shilde-adapted varieties which differ in bicarbonate uptake. The light-adapted varieties are able to exist in competition with algae under high light levels, and can overcome the marked fluctuations in pH by bicarbonate as well as free CO 2 assimilation. Shade-adapted varieties grow in water where the pH remains relatively low (close to neutral) and are only able to assimilate free CO 2 , Under extreme conditions, intense uptake of CO? by higher plants results in pH levels rising to values of 10-10.5. At this level, bicarbonate and carbonate ions are present but free CO? is absent, and only bicarbonate users such as Elodea canadensis, srnall species of Potamogeton, and the varieties of Ceratophyllum noted above are able to survive. Lack of free CO? is a major limitation for many higher plant species in eutrophic and hypereutrophic environments, and the photosynthesis of macrophytes that rely on free CO 2 ceases at pH 8.5-9.

6.8.2.2 Growtlt and life cycles The importance of rapid growth early in the season is an important aspect of algal - macrophyte competition and has been emphasized in the previous section. As well as timing of growth, algae and macrophytes are also able to concentrate their photosynthetic biomass in the top part of the water column, where light and CO 2 availability are optimal. Reduction in water transparency caused by algal growth results in the accumulation of vascular plant photosynthetic organs at or near the water surface, and enables them to compete with or even suppress phytoplankton. The evolutionary development of surfacefloating leaves or partial shoot emergence is the ultimate expression of this trend, and confers substantial advantage over algae in the competition for light and COl'

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6.8.2.3 Adaptations to anoxic sediments One important effect of intense algal blooms is the imposition , of a steep oxygen gradient within the water column, with high levels at the water surface and low levels at the bottom. This gradient arises due to high algal photosynthetic activity in the euphotic zone, coupled with bacterial breakdown of the increased algal biomass which sinks to the lake sediments. Anoxic sediments have no direct effect on phytoplankton, but they do cause physiological stress to the root and rhizome systems of attached macrophytes. These plants show a number of adaptations to the anoxic conditions, including the presence of high levels of reserve materials, allowing them to consume large amounts of carbohydrate in inefficient anaerobic metabolism. Macrophytes also typically possess air-conducting aerenchymatous tissues, allowing them to transfer oxygen into anaerobic regions. These adaptations enable vascular plants living in aquatic environments to survive long periods of anoxic stress, and are particularly well developed in emergent macrophytes (helophytes) which are rooted in low-oxygen sediments close to the water surface. 6.8.2.4 Mineral nutrient uptake Uptake of inorganic nutrients is discussed later in relation to nitrates and phosphates, and is considered here specifically in relation to competition between algae and higher plants. In oligotrophic and mesotrophic environments, shortage of mineral nutrients is the main limiting factor for both groups of organism. Althdugh oligotrophic water bodies tend to show little development of macrophytes, rooted vascular plants are present with increased nutrient levels and do have some advantages over both planktonic and attached filamentous algae in limiting nutrient conditions. These plants are able to take up scarce mineral nutrients via their root systems and recycle them internally, while algae have little chance to grow within the lownutrient water column. In eutrophic conditions, both sediments and the water column are rich in nutrients, allowing algae to grow abundantly and outcompete macrophytes. Algal growth in such conditions is accentuated by increased release of phosphorus from anoxic sediments, which is then transported upwards through the water column by thermal mixing during the night. Although this increased phosphorus supply enhances the growth of all aquatic plants, there is higher relative uptake by algae (compared to macrophytes) due to their greater

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surface area/volume ratio. Both algae and macrophytes are able to take up and store mineral nutrients at times of high availability, as noted elsewhere in relation to phosphorus. This luxury uptake effectively uncouples the organism from limiting external conditions, and is important in their competitive interactions.

6.S.2.5 Shading and competition for light Interception of light is an important aspect of competition between algae and higher plants. Excessive growth of filamentous algae at the. water surface can cause a dramatic decrease in the penetration of photosynthetically-active radiation (PAR), with 98 per cent being removed within the top 1 cm of the water column. Submerged macrophytes are shaded by both phytoplankton and periphyton, and can in turn deprive algae of light. Quite apart from competition between these organisms, dense growth of algae and higher plants may also result in self-shading. Self-shading by hydrophytes is one of the major factors in limiting their growth in eutrophic and hypereutrophic waters. Hydrophyte adaptations to avoid or minimize the effects of shading by planktonic and periphytic algae include the following. 1. Early growth prior to development of algal blooms. 2. Development of aerial foliage and floating leaves, both of which intercept light before it reaches algae. 3. Production of numerous growth tips early in the season, developing new shoot surface area at a higher rate than that of colonization by periphytic algae. In some vascular hydrophytes (e.g., Elodea), the smooth cuticle of this new growth reduces surface colonization by periphytic algae which can, however, form dense communities on older leaves and on the surfaces of other macrophytes. 4. Physiological adaptation to low irradiance. This is typical of submerged hydrophyte leaves, particularly those shaded by dense phytoplankton or periphyton. Photo-adapted leaves of hydrophytes have low photosynthesis saturation levels and low light compensation points, with respective values of 20-40 Wm-2 and 1-2 Wm-2 being reported by Pokorny et al. (2002b). In situations where phytoplankton levels are low, it is the periphyton which may take over as the major algal competitor for light. These biota have a lower CO, compensation point than macrophytes, and their higher efficiency of bicarbonate uptake

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means that they not only deprive the vascular plant of light but also COr Although ability to grow at low light levels is important, filamentous algae do not show any gradation in adaptation within the water column. The light compensation point for algae deep in the water column is the same as those at the surface. These organisms appear to rely simply on rapid growth rather than physiological adaptations to out-compete hydrophytes. The existence of hydrophytes in these conditions is variable, and although a wide range are able to survive algal competition in high nutrient conditions, others cannot. Pokorny et al. (2002b), for example, report that the water lily Nymphaea candida and other sensitive vascular plants are rapidly disappearing from the increasingly hyper-eutrophic fishponds of the Trebon wetlands. In addition to interactions within the water column, competition for light is also an important factor at the edge of ponds and lakes, where shading of littoral algae by helophytes is ecologically significant.

6.9 DAMAGING EFFECTS OF HIGH LEVELS OF SOLAR RADIATION: PHOTOINHIBITION Aquatic organisms are frequently exposed to high light intensities, with irradiance values at the top of the water column in lakes and other water bodies exceeding 800 JLmol m- 2 S-1 on bright summer days. Although these aquatic light levels are considerably less than values of up to 1800 JLmol m- 2 S-1 encountered in terrestrial environments the radiation may still have an adverse effect on the metabolism and activities of algae and other biota close to the water surface. High light intensity is an example of an environmental stress factor which can perturb life processes over a range of biological levels, from ecosystems to cell physiology and molecular control mechanisms. Photoinhibition may be defined as the destructive effects of light on cell processes leading to impairment of metabolic activities, reduced growth, and in some situations cell death. The most obvious examples of photoinhibition COme from depth measurements of photosynthetic activity close to the lake or river surface, where there is a clear depression in carbon fixation without any decrease in algal biomass, as measured by chlorophyll-a concentration. The adverse effects of light irradiation are particularly important in relation to phytoplankton and algal communities in extreme highlight environments. The biological implications of photoinhibition will

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be considered in relation to the mechanisms of metabolic destruction, algal strategies for avoiding or minimizing photoinhibition, and the importance of this process in different environments. 6.9.1 Specific Mechanisms of Photoinhibition The destructive effects of light are increased with a reduction in the wavelength of the irradiation. The inhibitory effects seen with photosynthetically active radiation (PAR 400-700 nm) are thus much enhanced with a shift to the ultraviolet end of the spectrum, comprising UV-A (315-400 nm) and UV- B (280-315 nm) radiation. Light incident at the water surface has a variable content of UV-B, depending on the solar angle and the thickness of the ozone layer. Penetration of the water column results in a rapid loss of UV radiation, with 99 per cent absorption of UV-A in clear-water occurring within 2-5 m of the surface and equivalent absorption of UV-B occurring within 0.5 m. In spite of the rapid attenuation of UV-B in the water column, this radiation will have major effects on microorganisms in surface waters and many workers have used this wavelength band in studies on the molecular mechanism of photoinhibition. Incident levels of UV-B, with associated microbiological effects, are expected to increase in line with predicted reductions in the stratospheric ozone layer. The destructive effects of visible and ultraviolet light are mediated both by direct action on molecular bonds and by the generation of active (superoxide) oxygen spec:~s, which leads to photooxidation of a range of molecular species. These effects are similar for both aquatic algae and higher plants and involve direct effects on macromolecular targets (DNA, photosynthetic machinery, photoreceptor, and motor organelle proteins) as well as indirect effects on a range of cellular processes. 6.9.1.1 DNA damage and repair Absorbance of UV-B irradiation by DNA triggers the dimerization of thymine pairs, with the formation largely of cyclobutane-pyrimidine dimers (CPDs). These DNA modifications are mutagenic. They also cause disruptions to cellular metabolism because of blockages in gene transcription and DNA replication arising from the inability of RNA- and DNA-polymerase to read through unrepaired dimers. Such blockages in protein synthesis and DNA replication inhibit cell growth and can limit increases in algal population. Repair of UV-B damaged DNA occurs via light-dependent photoreactivation, lIsing photolyase enzymes which restore the bases

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to their original state. Photolyases carry light-absorbing components (chromophores) which have absorption maxima between 350 and 400 nm and are activated primarily by UV-A, and to a lesser extent, blue light. Since UV-A and blue light invariably accompany UV-B irradiation under natural conditions, the photo reactive role of these specific wavelengths is environmentally highly effective. Lightdependent repair of CPDs has been demonstrated in both eukaryote and prokaryote algae, and is closely similar to the system operating in higher plants.

6.9.1.2 Photosynthetic machinery Although there is a clear advantage in terms of competition for light and promotion of photosynthesis for algae to be at the top of the euphotic zone, extensive residence (for more than a few hours) can expose these organisms to harmful radiation. Many investigators have linked impairment of photosynthesis to the high levels of light in surface waters. As noted previously, there is a sharp depression in phytoplankton carbon fixation close to the water surface, even though light intensity and chlorophyll-a concentration increase in this region. Mechanisms underlying this environmental effect can be investigated in laboratory cultures, where the damaging effect of light radiation is revealed by plotting photosynthesis - irradiation curves. As noted previously, these show a levelling off of photosynthesis, with a maximum value (P rna) at irra.diance (lrn,)' followed by a decline due to photoinhibition. Modelling of light saturation curves by Platt et al. (1980) indicates, however, that the process of photoinhibition commences at irradiation levels lower than Imax, before the net decrease in photosynthesis. Theoretical curves without photoinhibition reach a higher maximum rate of photosynthesis (P) at an irradiance value higher than 1m"" and there is no subsequent decline once the system has become saturated. Visible and UV light have a range of effects on the photosynthetic machinery, including bleaching of photosynthetic pigments, damage to photosynthetic (thylakoid) membranes and reduced activity of the enzyme ribulose bisphosphate carboxylase (Rubisco). Bleaching of photosynthetic pigments has been demonstrated by comparing the absorption spectra of intact algal cells, with and without exposure to solar radiation, and has demonstrated pigment loss specifically in the wavelength absorption range of the carotenoids and chlorophylls.

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In addition to these general effects, UV- B causes direct and specific damage to the PSII complex, which catalyses the transfer of electrons from water to plastoquinone. The degradation of PSII is maximal at 300 nm, with shorter wavelengths having much less effect. Within the highly-structured protein pigment complex, UV-B radiation causes rapid light-driven degradation of the central chlorophylla molecule (P680), two key proteins - Dl/D2 and the electron acceptors quinone A (OA) and quinone B (OB)' This leads to an accumulation of inactive PSI! complexes. Repair of the accessory proteins in PSI! complexes is thought to be mediated by rapid turnover of D 1 protein (driven by photosynthetically active radiation) and also combined turnover of D I-D2 proteins.

6.9.1.3 Photoreceptor and motor organelle proteins Orientation and diurnal migration of planktonic organisms within the water column involve both receptor activity to external stimuli (light, gravity) and motility. Recent laboratory studies have shown that high levels of solar radiation can impair both of these activities in a wide range of cultured algae (including euglenoids, cryptomonads, and dinoflagellates), acting by denaturation of photoreceptor and motor organelle proteins. These photoinhibitory effects lead to an inability of the organisms to adapt to constantly changing conditions in the environment, and may affect diurnal migration. Laboratory studies on photoinhibition of light and gravitational responses have been carried out mainly on dinoflagellates, which show pronounced diurnal migrations and active short-term responses in relation to these stimuli. Hader (1995) used continuous CCD camera recording and computer analysis of individual cell movements under controlled laboratory conditions to assess the directional precision of the dinoflagellate response and the general effects on motility. Control cultures of a test dinoflagellate (tentatively identified as Gymnodinium) showed a high precision of orientation under lateral illumination. Exposure to high levels of unfiltered solar radiation lead to a sharp decline in the directional precision of the photoresponse after 40 minutes. The percentage of motile cells also showed a marked decline after a threshold period of time, though in these experiments the average swimming motility of the motile fraction remained constant at about 250 JLm S-I. 6.9.2 General Effects of Photoinhibition The specific effects of photoinhibition on particular target molecules and particular processes lead to a wide range of secondary

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effects. UV- B irradiation in particular leads to general inhibition of cell division and growth, decreased levels of ATP, reduced rates of nutrient (ammonia, nitrate, phosphate) uptake, and loss of motility. Some of these general effects may be quite complex, varying with time, and also with radiation intensity and physiological state of the cells. Exposure of cells of Chlamydomonas to UV-B radiation, for example, leads to an initial increase in growth at low radiation levels (1.2 kJ nr1 ) with a subsequent decline. Marked inhibition of growth occurs throughout the time course at high radiation levels (16 kJ m-2). Stimulation and inhibition of growth in Chlamydomonas is closely correlated with phosphorus uptake and loss of flagella, demonstrating a close interlinking of the physiological effects of photoinhibition. The immediate effects of UV-B irradiation on cell processes, and the recovery of cells from this photoinhibition, vary with the physiological state of cells. In Chlamydomonas the degree of phosphorus limitation appears to be particularly important, with a higher recovery of flagella in P-limited cells (87 per cent within 72 hours after termination of radiation) compared with only 34 per cent of non P-limited cells. Different algae have varying susceptibilities to UV- B radiation. Larger species of diatoms are reported to be'more susceptible than smaller ones, and flagellated algae more susceptible than nonflagellated. These differential effects of photoinhibition will potentially lead to changes in community composition under natural conditions.

6.9.3 Strategies for the Avoidance of Photoinhibition Primary producers face the challenge that they depend on solar energy in the middle range (>400 nm) for photosynthesis, but are damaged in high intensity visible light and by UV irradiation. Of the two groups of primary producers, the potential risk of radiation damage is particularly high for algae (which are frequently found close to the water surface), but is not significant for photosynthetic bacteria which are limited to the lower part of the euphotic zone. Algae are able to minimize photoinhibition in two main ways by avoidance (migration) and by reducing the harmful effects of the radiation in situ.

6.9.3.1 Avoidance of radiation Algae are able to migrate from regions of high light intensity in both planktonic and benthic environments.

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6.9.3'.1.1 Planktonic environments In planktonic environments, avoidance by actively motile (flagellated) algae and by algae with a buoyancy mechanism, involves migration away from the water surface to lower regions of the water column. Such avoidance mechanisms are typically achieved by phototaxis, supported by other receptor processes. In the case of blue-green algae, sinking under conditions of high surface illumination may also depend on the formation of ballast by photosynthesis. Any interference with this process may lead to the colonies being stranded at the water surface, resulting in large-scale destruction of the algal population. 6.9.3.1.2 Benthic environments Various groups of benthic algae, including blue-green algae, diatoms, and desmids (green algae) are able to carry out lightrelated active movement on substratum. Blue-green algae. Many blue-green algae, including both singlecelled and filamentous forms, are capable of gliding movements. Single cells knd to make irregular jerky movements while filamentous forms such as Oscillatoria glide on substratum in a more controlled way, achieving speeds of 2-11 p,m S-I. Recent studies have suggested that movement is achieved by numerous microfibrils in the cell wall, which are spirally wound around the cell. Waves propagated in rapid succession along these fibrils are thought to produce a rotating forward movement of the whole organism, through friction between microfibrils and substratum. As the algae creep around like slugs, they leave behind them a trail of mucilage. The production of this mucilage was originally thought to be the means of locomotion, but is now considered to provide a solid substratum over which the organisms can move. The movement of benthic blue-green algae in response to high light intensity involves three types of response, each mediated by its own set of photoreceptor pigments and its own physiological mechanism: 1. Phototaxis, where the light source determines the direction of movement - blue-green algae exhibit positive phototaxis to dim light and negative phototaxis to bright light; 2. Photokinesis, where the speed rather than the direction of movement is influenced by light intensity; 3. Photophobic response, where a rapid reversal of movement is induced by a sudden increase or decrease in light intensity.

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As a result of these mechanisms, populations of benthic algae tend to accumulate in regions of moderate light intensity, avoiding high illumination. This may either involve lateral movement on benthic surfaces, or vertical movement into the substratum of environments such as mudflats where there are marked diurnal migrations. Diatoms. Some benthic diatoms, including various pennate and centric forms, are able to migrate over substrata by the extrusion of mucilage. In some cases, movement is influenced by light. Cell clumps of Nitzschia paiea, obtained from liquid culture, tend to disperse on transference to a glass slide in the presence of light but do not disperse in darkness.

6.9.3.2 Reduction of harmful effects High levels of solar radiation do not always lead to inhibition of photosynthesis. This is seen in such diverse freshwater environments as snowfields and lake pelagic systems, where high levels of irradiation can lead to protective physiological changes in the microalgae.

6.9.3.2.1 Photoprotection irz different freshwater envirol1ments In snow environments, physiological responses to harmful irradiation have been studied particularly in relation to green algae, such as Chlamydomonas niJlaiis. High levels of visible light and UV radiation induce the formation of photoprotectants, antioxidants, and photoreactive repair systems. Formation of the xanthophyll pigment astaxanthin (photoprotectant) is particularly intense under conditions of high light aQd low nutrient, leading to the formation of red blooms of snow algae referred to as 'red snow'. In lake pelagic environments, irradiation-induced physiological adaptations in phytoplankton have been inferred from studies such as those by Paerl et al. (1983) on the summer bloom of the blue-green alga Microc),slis aerugil1osa. The rate of carbon fixation in high-light adapted populations of this organism increased right up to the water surface, compared with surface inhibition in nonadapted populations seen at other times of the year. This ability to withstand high levels of solar radiation can be largely attributed to the production of Uvabsorbing molecules which essential1y block-out the radiation, limiting the damaging effects on other molecules in the cell.

6.9.3.2.2 Photoprotectiol1 in bille-green algae Blue-green algae are particularly effective in carrying this out. The development of such protection mechanisms in these organisms

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may relate to the fact that they arose in the early pre-Cambrianperiod, when UV levels were much higher than at present due to the absence of absorbing gases (0, and 0_) in the atmosphere. The evolutionary development of this- strategy' was also important in ecological terms, since fossil evidence has indicated that many of these pre- Cambrian algae were present in shallow pools where an avoidance mechanism would not have been relevant. Blue-green algae produce various compounds that absorb shortwavelength radiation, particularly UV light, and act as photoprotectants. These UV-absorbing compounds include mycosporinlike amino-acids (MMs), a blue-green algal-specific compound (scytonemin), and pigments that are associated with the photosynthetic machinery (carotenoids). MMs are generally present within bluegreen and other algae, fungi, aquatic invertebrates, and fish, and clearly have a widespread role for photoprotection within aquatic biota. Scytonemin is an inducible pigment which is specific to bluegreen algae. It is contained in the extracellular sheath and is synthesized in response to irradiation by wavelengths at the UV-blue end of the spectrum. Scytonemin absorbs strongly in the blue, UVA, UV-B and UV-C wavelengths and has been shown to protect metabolically active and inactive blue-green algal cells against the deleterious effects of UV radiation. The ability to synthesize scytonemin is known only in blue-green algae, and is found in all five subgroups of the phylum. The widespread occurrence of scytonemin in this phylum, particularly in different evolutionary lines that are believed to have diversified early in the history of the group, suggests that the protection mechanism arose early in the evolution of these algae at a time when UV radiation was an important stress factor. The strong absorption of UV-C radiation (190-280 nm) has little ecological relevance under present conditions. but would have been important in pre-Cambrian times. Reduction in the harmful effects of radiation can also be achieved by adjustments to photosynthetic pigment composition, increasing the proportion of carotenoid pigments that act in a similar way to scytonemin by absorbing the radiation and limiting its penetration to other parts of the cell. Increased carotenoid/chlorophyll-a ratios have been observed for developing bloom populations of Microcystis, leading to a high resistance to photoinhibition and greater photosynthetic efficiency.

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6.9.3.2.3 Role of carotenoids in photoprotection Carotenoids exhibit strong in vitro absorbance in the UV and near- UV parts of the spectrum, and increased proportions of these pigments increase light utilization in the low and middle ranges of the photosynthetically active radiation (PAR) as well as providing protection from UV damage. The protective effect of carotenoids involves direct removal of UV light by absorption and also alleviation of damage caused by the formation of highly reactive chlorophyll-a and oxygen species. Under UV irradiation, Chlorophyll-a can be converted to a reactive triplet state (ChI-aT), leading in turn to the formation of triplet oxygen (02 T ):

Chl- a ~ Chl- aT

... (5)

ChI-aT +0 2 ~Chl-a +oI oI can lead to photooxidative destruction of a range of organic compounds, including chlorophyll-a. Carotenoids act by transferring the triplet state from ChI-aT and oI in the following ways: Chl- aT + Carot. ~ Chl- a (groundstate) + CarotT Carot. T ~ Carot. (ground state) + heat T

... (6)

T

Carot. + O 2 ~ Carot. + O 2 (ground state) Carot.T ~ Carot. (ground state) + heat

... (7) Individual carotenoid pigments differ in their biochemical roles. Zeaxanthin is an important photoprotectant, with a limited role in photosynthesis, while ~-carotene is the converse.

6.9.4 Photoinhibition and Cell Size A variety of evidence in the literature suggests that cell size is a key factor in determining sensitivity to ultraviolet radiation. This evidence comes from theoretical analysis and modelling studies, experimental results from cultured algae, and studies on environmental assemblages. In theoretical terms, two related aspects suggest that small cells should be more sensitive than large ones. 1. Pigment-specific light absorption increases with decreasing cell size and decreasing internal molecular shading. This leads to higher UV exposure per unit pigment and per unit cell volume. 2. The protective efficiency of UV-absorbing molecules such as mycosporine-like amino-acids is a function of cell concentration,

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but also of cell size through its effect on molecular self-shading. Model calculations suggest that while nano- and microplankton cells contain sufficient UV-screening pigments to give protection, these are not sufficient within the small cell volume of picoplankton. Experimental and environmental evidence for the importance of cell size in relation)o UV damage comes mainly from oceanographic studies. Studies by I<arentz et aI., 1991, for example, showed that the UV sensitivity of 12 species of Antarctic diatoms was highest in smaller cells, which sustained more damage per unit DNA and were killed by a lower UV flux, compared with larger cells. Other studies have suggested that factors other than size may take precedence. In the sub-Arctic lake communities studies by Laurion and Vincent (1998), cell size was not a good index of UV sensitivity. The bluegreen algal dominated picoplankton in these lakes was less sensitive to the impact of UV irradiation than would have been predicted simply on the basis of cell size. Although cell size is important for UV sensitivity, picoplanktor. cells may have their own adaptations to minimize the effect. Bluegreen algal picoplankton in particular dominate high-UV freshwater and marine environments (tropical oceans) and have a wide range of effective defenses against UV damage. These include multiple copies of their genome, protective pigments and enzymes against reactive oxygen species, and repair mechanisms for UV-damaged DNA and photosystems.

6.9.5 Lack of Photoinhibition in Benthic Communities Benthic light environments are highly variable, with maximum light intensities to which algae are exposed varying from near zero in deep, turbid water~ to full sunlight in shallow, clear conditions. Laboratory studies on stream periphyton communities suggest that benthic algae taken from shaded sites are capable of showing photoinibition, while algae from exposed sites are not. These two groups have photosynthesis - irradiation (P-I) curves corresponding with laboratory irradiance levels up to 1200 ILmol m-2 S-I. Although shaded benthic algae are capable of showing photoinhibition, their low-light environment would not normally reach levels to cause this. Although laboratory P-I curves of exposed benthic algae do not give absolute proof of absence of photoinhibition under exposed environmental conditions, they do suggest that it is much less important in benthic than in planktonic organisms. Lack of

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photoinhibition in benthic algae may be partly attributed to the permanent development of protective pigments in these fixed communities. The typical occurrence of benthic algae in dense mats will also be important, since high irradiance will be rapidly attenuated due to self-shading. Even if photoinhibition occurs at the mat surface, this will be masked by lack of photo inhibition through the rest of the community. 6.9.6 Photoinhibition in Extreme High-light Environments The degree of exposure of phytoplankton to high-intensity visible and UV light varies with the type of water body and with climate, altitude, and water quality. In many situations, photoinhibition has only a limited effect on algal productivity. In temperate lakes, for example, although high levels of solar radiation occur on sunny days at the water surface, the algae are able to migrate down the water column to a position of optimum light. In other lakes, this avoidance of photoinhibition is not possible. Many polar and some alpine lakes are very shallow. These lakes also have very high light exposure with clear oligotrophic water, so that photoinhibition occurs throughout a major part of the water column. The overall decrease in pelagic productivity in these lakes is balanced by the development of a community of benthic algae and mosses, in some cases forming an extensive growth on the lake bed. The effects of UV irradiation (UVR) on aquatic systems in extreme environments is particularly relevant at the present time, since these systems are currently experiencing increased levels of underwater UVR due to stratospheric ozone depletion and to climate, related changes in spectral UVR attenuation in the water column. The effects of increased UVR on size may be particularly important, since any shift in size would have major implications for pelagic food web processes and for interactions with the benthos via sedimentation.

6.9.6.1 Antarctic lakes The periphery of the Antarctic continent is characterized by the presence of many lakes which are small, shallow, and are much influenced by the surrounding ocean (maritime lakes). Although these lakes do not have a high level of precipitation, they do not dry up because of the low temperature « 1Goe throughout the year) and high humidity. These lakes typically have a low nutrient input and a

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low productivity. Many of the algae present are washed in from surrounding terrain or from the bottom of the lake. In summer, at a time of long daylight hours and high insolation, photosynthetic rates are generally low - with minimum rates at midday and highest rates at midnight. This pattern arises due to severe limitation of photosynthesis for most of the day during Antarctic summer. In these lakes where photoinhibition is greatest at noon, photosynthesis is inversely related to light intensity. In autumn a more normal pattern of productivity occurs, since overall light levels are much less than summer and maximum productivity returns to the middle of the day. Benthic communities of blue-green algae dominate the bottoms of shallow lakes, providing a major habitat for Antarctic invertebrates such as protozoa, rotifers, crustacea, copepods, and Cladocera. These populations typically have little species diversity due to the isolation of Antarctica fr0111 the world's land masses, resulting in limited colonization of this remote region.

6.9.6.2 Alpine lakes These are found in most mountainous regions of the world. They are typically cold, deep lakes that are ice-free for most of the year. The light regime of these lakes at high altitude differs from lowland lakes in an increased level of solar radiation. rich in UV light. The oligotrophic nature of many of these lakes gives the water a high degree of clarity, enhancing UV penetration and increasing further the adverse effects of solar radiation. In many cases the low algal productivity is matched by a relatively simple ecosystem. Verderer Finstertaler See in Austria remains below 10c C over most of the SU11lmer, and has dinoflagellates as the only common group of phytoplankton. The zooplankton population is composed of two rotifers and one copepod. Although the planktonic flora and fauna are limited, extensive benthic flora develops on the lake beds where sufficient light penetrates. These plants provide a microenvironment for an extensive community of nematodes, oligochaetes, ostracods, and chironomid larvae. Light penetration and photo inhibition vary with the seasonal cycle. In winter months, populations of dinoflagellates accumulate under the ice, where they adapt to low light levels. Melting of the ice and increased insolation in spring lead to greater penetration of the water column, with downward migration of the algae to depths of 5-15 m to avoid photoinhibition.

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6.10 PERIODIC EFFECTS OF LIGHT ON SEASONAL AND DIURNAL ACTIVITIES OF FRESHWATER BIOTA Periodic changes in the intensity and quality of light have major effects on the biology of aquatic organisms. These effects are enhanced by the low light absorption (a) and scattering (b) coefficients of water which maximize the depths to which these fluctuations exert an influence. Periodic changes operate over two main time scales - seasonal and diurnal.

6.10.1 Seasonal Periodicity The importance of seasonal changes in light intensity on the annual succession of phytoplankton has been previously noted, and can be explained in terms of general environmental adaptations and competitive ability of different algal grotJps. In addition to this rather broad effect of light on microbial interactions, annual changes in light intensity, wavelength, alid daylength may also have a more specific effect on the succession and growth patterns of particular algae and other microorganisms. The filamentous green alga Ulothrix ZOl1ata provides a good example of such specific and direct effects of light on growth pattern. This alga is common in many northern lakes of the USA and in Canada, where it grows abundantly in spring and autumn in shallow waters. At these times of year the increase in biomass occurs due to high vegetative growth. The decline in growth rate and total biomass in late spring is due'to a switch from vegetative growth to asexual reproduction - with the formation of zoospores and the disruption of vegetative filaments. Experiments on laboratory cultures have shown that this transition is favoured by high temperatures (> lOoC), high light levels (about 520 J-LE m-2 S-I) and photoperiods of either short day (8: 16 light - dark) or long day (16:8 light - dark) cycles. Conversely, zoospore formation is minimal under conditions of low temperature « lOOC) , low light levels, and neutral day-lengths (12:12 light - dark). Seasonal changes in the growth pattern of Ulothrix can largely be explained in terms of these environmental parameters. In spring, the switch to zoospore production results from the combined effects of rising water temperatures and increasing light intensity and day-length. During summer, when conditions limit growth, Ulothr;', survives as short filaments on rocks or as epiphytes on other algae. Growth is resumed in autumn when temperatures and light levels decline, and day-length shifts towards neutral.

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6.10.2 Diurnal Changes The response of freshwater organisms to periodic changes in light becomes particularly marked in relation to the daily alternation of day and night. This well-defined environmental regime leads to biological periodicities that are typically both 'diel' (occurring at 24 hour intervals) and 'diurnal' (occurring every day, as a sequence of activity within a 24 hour time frame). 'Circadian rhythms', are an example of diurnal change, and are metabolic or behavioural cycles with a periodicity of about 24 hour. 6.10.2.1 General aspects Diurnal periodicities in freshwater biota include patterns of growth and death, metabolism (photosynthesis, nitrogen fixation) and behaviour (general motility and migration). Some of these responses (e.g., photosynthesis) are a direct physical result of the presence or absence of light, while others (e.g., vertical migration) relate more to an underlying, ultimately cellular, timing mechanism. This intracellular clock operates via diurnal variations in gene transcription and relate~ molecular activity. Organisms in the pelagic environment have greater light exposure compared to benthic biota, and show particularly strong periodic behaviour. This includes the diurnal migrations of phytoplankton and zooplankton, and the related movements of fish predators. These often show a similar light-regulated pattern to migrating zooplankton as they follow their prey. Benthic organisms on lake sediments and in streams also show diurnal activity. Epibenthic animals, living on the surface of sediments, avoid light and predators by hiding under stones during the day and only emerging at night. Crayfish and other predators also emerge from under rocks and plants at night to begin foraging, and deep water fish may move into shallower regions at dawn or dusk for their major feeding activities. 6.10.2.2 Circadian rhythms: endogenous actil1ity and

entrainment Although these diurnal activities relate to external changes in the environment, such as changes in light, temperature, oxygen concentration, and food availability, they may be driven by innate rhythms in the organism that continue irrespective of environmental alteration. Such daily or circadian rhythms operate as endogenous biological programs which time metabolic and/or behavioural events to occur at optimum phases of the daily cycle. These rhythms have three diagnostic characteristics:

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1. In constant conditions the programmes freerun with a period of approximately 24 hours duration; 2. In an environmental cycle involving periodic alternation of characteristics such as light--dark, or high-low temperature, the rhythm will take on the period of the environmental cycle this is referred to as entrainment; 3. The period of the free-running rhythm varies little with temperature. Within the physiological range circadian rhythms are temperature-compensated. The mechanisms that control circadian rhythms and their biological role in the freshwater environment have been investigated particularly in blue-green algae and dinoflagellates.

6.10.3 Circadian Rhythms in Blue-green Algae The occurrence of circadian rhythms in blue-green algae was first demonstrated by Grobbelaar et a1. (1986), who studied the rhythm of nitrogen fixation and amino-acid uptake in the unicellular freshwater blue-green alga SYl1echococcus. These authors were the first to report the isolation of mutants affecting these processes, establishing a genetical molecular basis for control of the diurnal cycle. Circadian rhythms are now known to be expressed in other genera of blue-green algae as well, including Anabaena, Cyal1othece, and Triochodesmium. Molecular studies on the control of the diurnal cycle in these algae were advanced considerably by the creation of a reporter strain of SYl1echococcus. This was achieved by inserting a DNA construct in which the luciferase gene set luxAB was expressed under the control of the promoter for a Synechococcus photosystem II gene, psbAI. Luminescence in this genetically-modified organism follows a circadian cycle, rising during the day and falling at night, and conforms to all three criteria for circadian rhythms noted previously. 6.10.3.1 Control sequence Further studies have now revealed a chain of molecular events controlling the circadian cycle, which can be considered in three parts - the central clock, light modulation, and control of circadian gene expression. 6.10.3.1.1 Central clock Central control of the circadian rhythm resides in a molecular oscillator (clock) composed of a cluster of three genes - kaiA, kaiB, and kaiC. The clock gene products interact with each other,

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forming' an autoregulatory feedback loop which has rhythmic periodicity, Deletion or inactivation of any of the kai genes causes disruption of the diurnal cycle, with changes in the cycle length, but has no effect on cell viability,

6.10.3.1.2 Light modulation Light/dark signals are considered to be the primary environmental events which set the phase of circadian clocks. Spectral analysis of circadian activation indicates that blue and red light are most effective in blue-green algae, while green and far-red light are ineffective. The pigments involved in light reception for circadian rhythms in blue-green algae have not yet been determined, and may be specific for this activity. They do not appear to include either phytochromes (phasing effect of red light not reversed by far red) or photosynthetic pigments (action spectrum for circadian entrainment does not coincide with that for photosynthesis). :1.10.3.1.3 Circadian gene expression Liu et al. (1995), investigated the extent of circadian control of gene expression in Synechococcus by inserting a luciferase gene set (luxAB) into the genome to achieve random insertion throughout the chromosome. Screening the luminescence patterns from the large number (-800) of clones indicated that circadian expression was widespread throughout the genome, and could be separated into two major categories, day-time and night-time expression. Expression of day-time genes builds up throughout the day, with a trough at dawn and a peak at dusk, and includes the psbAI gene for photosystem II. In contrast to this, night-time genes build up their expression during the night, with a minimum at dusk and a maximum transcription at dawn. Night-time genes include purF which encodes glutamine PRPP amidotransferase. Because of the large number of genes which are controlled by the circadian oscillator, it seems unlikely that each is controlled by a specific transcription factor. It seems most probable that global transcription (sigma) factors are involved, produced by genes that are activated by the oscillator (clock-controlled genes), and promoting expression of groups of circadian genes which encode specific nocturnal or daytime biological activities.

6.10.3.2 Circadian rhythms and biological processes The molecular control of circadian rhythms in blue-green algae is of prime importance to the biology of these organisms, where it

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is involved in the diurnal timing of processes such as photosynthesis, carbohydrate deposition, amino-acid uptake, and nitrogenase activity. These activities are seen particularly well in the process of nitrogen fixation by unicellular blue-greens such as Cyanothece, where the diurnal separation of photosynthesis and nitrogenase activity requires clockwork precision. Maintenance and timing of circadian rhythms may also be of general importance in competition between algae. Comparison of wild type and mutant cells (with different clock periods) suggests that the presence or absence of a fully operational 24 hour clock does not affect viability since growth of monocultures was not affected by mutation. Laboratory competition experiments, however, involving mixtures of strains showed that cycle duration is of key importance in cell interactions. Using light - dark regimens of different cycle length, Ouyang et al. (1998) showed that organisms whose endogenous cycle most closely matched the external regimen invariably out-competed those algae who were less well matched irrespective of whether they were wild type or mutant. These competition experiments are consistent with the idea that the circadian programme orders cellular processes to match environmental cycles optimally. When this correspondence is lost, fitness <md competitive ability are reduced. Circadian rhythms are clearly a major aspect of interactions between blue-green algae and their environment. The extent and importance of such rhythms in other freshwater biota remains to be seen.

6.10.4 Circadian Rhythms in Dinoflagellates Although nothing is yet known of the central clock in dinoflagellates, recent studies on the expression of clock-related genes indicates many similarities to blue-green algae. Okamoto and Hastings (2003) carried out a genome-wide study of circadian gene expression in Pyrocystis IUl1ula using microarray technology, and demonstrated that 3 per cent of the genes screened were circadiancontrolled. These included transcription factors, proteases, lightharvesting proteins, transporters, and metabolic enzymes. As with the blue-green algae, circadian regulation operated mainly at transcription level. with most gene expression occurring in early day and late night circadian time (CT).

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