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Applied Microbiology Edited by ALLEN I. LASKIN Exxon Research and Engineering Company Annandale, New Jersey
VOLUME 30
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1984
ACADEMIC PRESS, INC. (Harcourt Brace Jovanovich, Publishers) Orlando San Diego New York
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COPYRIGHT @ 1984, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, m O U T PERMISSION IN WRITING FROM TXE PUBLISHER.
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CONTENTS CONTHIBUTOHS
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vii
Interactions of Bacteriophages with Lactic Streptococci
CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.
CARLE. CERNIGLIA, National Center f o r Toxicological Research, Food and Drug Administration, Jefferson, Arkansas 72079 (31) CHARLES P. GERBA,Departments of Microbiology and Immunology, and Nutrition and Food Science, University of Arizona, Tucson, Arizona 85721 (133) D. S. KAPLAN, Food and Drug Administration Center for Devices and Radiological Health, Rockville, Maryland 20857, and George WashzingtonUniversity, Washington, D. C. 20037 (197) TODD R. KLAENHAMMER,Department of Food Science, North Carolina State University, Raleigh, North Carolina 27695 (1)
L. MODELEVSKY,Molecular and Cell Biology Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Zndiana 46225 (169)
JOSEPH
LASLOA. NAGY,Environmental Analysis, Program in Social Ecology, University of Calz$ornia, Zrvine, Zrvine, Calqornia 9271 7 (73) BETTYH . OLSON,Environmental Analysis, Program in Social Ecolog y , University of Calfornia, Zrvine, Zrvine, Calqornia 9271 7 (73) G. L. PICCIOLO,Food and Drug Administration Center for Devices and Radiological Health, Rockville, Maryland 20857 (197)
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ADVANCES IN
Applied Microbiology VOLUME 30
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Interactions of Bacteriophages with Lactic Streptococci' TODDR. KLAENHAMMER Department of Food Science, North Carolina State University, Raleigh, North Carolina
I. Introduction 11. Phage-Host Interactions A. Lytic Development
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.............. C. Transduction A. Origin of Bacteriophages ....... B. Phage-Inhibitory Media and Concentrated Cultures . . . . C. Strain Rotation and Multiple-Strain Starters . . . . . . . . . . .
V. Conclusions
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I. Introduction Production of cheese and cultured dairy products has long relied on the fermentation of milk by group N streptococci. Members of this group, composed of Streptococcus lactis, Streptococcus cremoris, and Streptococcus lactis subsp. diacetylactis, are directly responsible for the acid development, flavor production, and, often, coagulum characteristics in mesophilic dairy fermentations. Because efficient milk fermentations are dependent on the growth and activity of the lactic streptococci, great care is exercised to prepare starter cultures that are highly active and uncontaminated with undesirable microorganisms or bacteriophages. However, the fermentation process itself is nonaseptic, occurring in open vats with a nonsterile medium, pasteurized milk. It is therefore highly susceptible to contamination with bacteriophages. For the majority of strains of lactic streptococci employed in commercial dairy fermentations, lytic bacteriophages capable of halting IPaper Number 9066 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27650. The use of trade names in the publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named. nor criticism of similar ones not mentioned.
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ADVANCES IN APPLIED MICROBIOLOFY, VOLUME 30
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growth and acid production can appear within 1-2 days after introducing the culture into the cheese plant (Zehren and Whitehead, 1954). Although bacteriophage contamination of numerous industrial fermentations has been observed (Ogata, 1980), the destructive role of bacteriophages in milk fermentations is without parallel in other fermentation processes (Lawrence and Thomas, 1979). Historically, milk fermentations relied on starter cultures composed of undefined mixtures of lactic streptococci propagated without knowledge of, or protection from, bacteriophages. Natural phage contamination in these cultures established an equilibrium of evolving bacteriophages and phageresistant variants (Reiter and Moller-Madsen, 1963; Stadhouders, 1975). These cultures were highly variable in day-to-day levels of acid production, but remained moderately active and could be used continuously in small fermentation factories. Over the past 20 years starter culture failures due to bacteriophage infection have become prevalent throughout the dairy industry (Sandine et al., 1972; Lawrence, 1978). Increasing demand for cultured milk products in recent years has necessitated increases in both production capacity and process efficiency such that larger volumes of milk are processed, cheese vats are filled repeatedly within a single day, and total processing time is shortened (Lawrence, 1978). This modernization of the industry concurrently increased the probability of phage contamination and further dictated the use of defined mixtures of lactic streptococci capable of uniform and rapid rates of acid production. With the selection of highly fermentative lactic streptococci and their propagation under aseptic conditions (in the absence of bacteriophages), the majority of cultures now used by the industry have become highly susceptible to bacteriophage attack upon introduction into the cheese factory. To cope with bacteriophage problems a number of successful methods have been developed to minimize phage action during commercial milk fermentations. Through the use of concentrated cultures (Gilliland and Speck, 1974; Lawrence et al., 1976; Chapman, 1978; Wigley, 1977, 1980; Sandine, 1977; Porubcan and Sellars, 1979), aseptic bulk starter vessels (Sandine, 1977), and phage-inhibitory media (Lawrence et aZ., 1976; Richardson et al., 1976; Sandine, 1977; Wright and Richardson, 1982; Hargrove et aZ., 1961), the starter culture can be protected from bacteriophage infection prior to vat inoculation. However, phage contamination cannot be prevented following entrance into the fermentation vat. Therefore, emphasis for protection of the culture shifts to minimizing prolific phage-host interactions through rotation of phage-unrelated strains (Whitehead, 1953)or use of phage-resistant mutants in multiple-strain starters (Thunell et al., 1981; Limsowtin et al., 1977). Although, in theory, strain rotation should minimize developing phage populations within the plant, in practice it has proved
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difficult to identify strains that demonstrate completely different patterns of phage sensitivity (Nichols and Hoyle, 1949; Collins, 1962; Czulak and Naylor, 1956; Henning et aZ., 1968a; Chopin et al., 1976). Estimates of the total number of different, phage-unrelated lactic streptococci approximate 25 strains worldwide (Collins, 1962; Henning et al., 1968a; Lawrence et al., 1976, 1978). Considering the small number of phage-unrelated strains available, the choice of strains for incorporation into rotation programs is severely limited (Chopin et al., 1976). Similarly, few phage-unrelated strains are available for construction of multiple-strain starters containing composites of 4-6 strains. A decade ago Sandine et al. (1972) emphasized the need to isolate new strains of lactic streptococci for use in the dairy industry. Foremost among the criteria for selection of these strains was resistance to existing bacteriophages. It is now recognized that some strains of lactic streptococci are not attacked by any phage when challenged with large collections of laboratory phage banks (Nichols and Hoyle, 1949; Henning et al., 1968a; Chopin et al., 1976; Sanders and Klaenhammer, 1983,1984) or when used on a continuous, long-term basis in commercial fermentations (Zehren and Whitehead, 1954; Czulak and Naylor, 1956; Lawrence et al., 1976; Limsowtin and Terzaghi, 1976; Heap and Lawrence, 1976; Limsowtin et al., 1977; Richardson et al., 1980; Thunell et aE., 1981).These reports demonstrate the existence of lactic streptococci that are not sensitive to bacteriophage attack, in spite of devastating phage pressure such as that which routinely occurs within the factory environment. However, to date, only a limited number of phage-insensitive strains have been identified and studied for mechanisms of phage resistance. Despite significant progress in starter culture technology and strain selection (Heap and Lawrence, 1976; Thunell et al., 1981),the limiting factor in control of bacteriophages remains the small supply of strains available that meet industrial criteria for fermentative capabilities and long-term phage resistance. Significant progress in the genetics of lactic streptococci (for reviews see McKay, 1982, 1983; Gasson, 1983; Davies and Gasson, 1981, 1983) has established the potential to genetically engineer desperately needed strains for the dairy industry. Application of current techniques in molecular biology will provide exciting opportunities for construction of tailored lactic streptococci with desirable fermentative and phage-resistant characteristics. Knowledge of phage-host interactions in the lactic streptococci thus becomes critical for the judicious selection of strains that can be incorporated into starter cultures, for control of bacteriophage development during fermentation, and for identification of resistance mechanisms that potentially could be genetically engineered to provide an adequate supply of phage-insensitive starter cultures.
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ti. Phage-Host interactions A. LYTICDEVELOPMENT 1. Morphology Bacteriophages attacking the lactic streptococci have been subjected to extensive morphological and serological characterization. Structural identification and measurement of phage heads, tails, collars, and baseplates by electron microscopy have demonstrated a diverse number of morphological types (Henning et al., 1968b; Keogh and Shimmin, 1974; Terzaghi, 1976; Tsaneva, 1976; Heap and Jarvis, 1980; Lembke et al., 1980; Chopin and Rousseau, 1983; Teuber and Lembke, 1983). With the exception of the tubular-head phages described by Chopin and Rousseau (1983),most phages are either prolate, small isometric, or large isometric (Henning et al., 1968b; Keogh and Shimmin, 1974; Terzaghi, 1976; Tsaneva, 1976; Lawrence et al., 1978; Sozzi et al., 1980). Although both prolate and isometric phages cause failures in milk fermentations (Teuber and Lembke, 1983), Heap and Jarvis (1980) reported that prolate phages generally attack more strains than do isometric phages. On the other hand, isometric types may be more common. In New Zealand isometric phages account for 90% of the phage types isolated. Morphological groupings of bacteriophages attacking the lactic streptococci correlate well with serology and DNA-DNA homology studies (Tsaneva, 1976; Heap and Jarvis, 1980;Jarvis, 1984a). Recently Jarvis (1984a) studied four groups of lactic bacteriophages on the basis of morphology, serology, and DNA homology. These groups included collared or noncollared small isometric phages (head diameter, 50 nm; tail length, 150 nm); isometric phages with short tails (length, 110 nm); prolate phages (head diameter, 41-63 nm; tail length, 76-105 nm); and large isometric phages (head diameter, 85 nm; tail length, 450-473 nm). The morphological groups assigned showed a complete lack of DNA homology. From these data Jarvis (1984a) suggested that these phage groups do not have a common phage ancestor nor are different phage species derived by mutation between groups. Within morphological groups, distinct phage types demonstrating baseplates, collars, etc., can be subgrouped further by serotyping (Lawrence et d., 1978; Lembke and Teuber, 1981). However, bacteriophage groups assigned by morphology, serology, or DNA homology show no correlation with groupings based on host range (Keogh and Shimmin, 1974; Heap and Jarvis, 1980; Jarvis, 1978, 1984a).
2. Host Range Since the discovery of bacteriophage activity against the lactic streptococci (Whitehead and Cox, 1936), host-range studies of bacteriophage action have
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demonstrated a wide spectrum of interactions ranging from highly specific to indiscriminate. Early reports suggested that phages attacking S . cremoris strains were, for the most part, strain or group specific, while S. lactis phages were less restricted to strain or group boundaries (Hunter, 1946; Whitehead, 1953). In crosses of 60 phage races against 100 strains of lactic streptococci, Henning et al. (1968a)reported that with S . lactis, S . cremoris, and S . lactis subsp. diacetylactis, species boundaries were generally maintained; but they also noted a number of phages that attacked all three species. The most extensive study of phage-host crosses for lactic streptococci was conducted by Chopin et al. (1976)with 132 phages used to challenge 291 strains. The majority of phages (57.5%, group “g3”)acted on 2 or 3 species, with some phages active against a large number of strains within these species. Of the strains tested 68.8% were sensitive to the “g3” group phages. From these data it is apparent that the majority of lactic streptococcal strains are susceptible to broad host-range bacteriophages that respect neither species nor strain boundaries. The high degree of diversity in host range for lactic bacteriophages was far greater than was originally suspected. In recent years, numerous low-eEciency, heterologous phage crosses have been reported (Oram and Reiter, 1968; Terzaghi and Terzaghi, 1978; Pearce, 1978; Boussemaer et al., 1980; Sanders and Klaenhammer, 1980; Daly and Fitzgerald, 1982).The results of Pearce (1978) were most dramatic in this regard. Using a single phage ($643.ML3), plaquing ability was demonstrated on 23 different hosts of S . lactis and S . cremoris with efficiency of plaque formation ranging from 1 to 10- lo. Similarly, Boussemaer et al. (1980) demonstrated plaquing ability in 77% of 36 phage-host crosses using all combinations between four strains of S . lactis and two strains of S . cremoris and their homologous phages. Detection of low-efficiency phage replication has been facilitated by use of hightiter phage preparations (Reiter and Moller-Madsen, 1963; Lawrence et al., 1976; Heap and Lawrence, 1976)and, more importantly, through the development and use of buffered media for plaque assays. Buffered media developed for plaque assays include M16 (Lowrie and Pearce, 1971), tryptone yeast extract agar (Keogh, 1980), and M17 (Terzaghi and Sandine, 1975). Although specific phage-host responses may vary (Keogh, 1980), buffered media generally enhance strain growth and promote better plaque development than nonbuffered media. Use of M17 buffered with P-glycerophosphate has been widely accepted for plaque assays and undoubtedly enhances detection of heterologous phage development in the lactic streptococci. Bacteria and bacteriophages are constantly evolving entities which exhibit considerable genotypic and phenotypic variability. Changes in the resistance of lactic streptococci to bacteriophages have been routinely observed (Collins, 1958, 1962; Limsowtin and Terzaghi, 1977; Limsowtin et d . , 1978;
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Sinha, 1980; Sanders and Klaenhammer, 1981, 1983).Similarly, changes in bacteriophages due to mutation (Jarvis, 1978)or host-controlled modification (Collins, 1956; Potter, 1970; Keogh, 1973; Sinha, 1980; Sanders and Klaenhammer, 1980; Daly and Fitzgerald, 1982) can dramatically alter host range. Considering the dynamic state of phage-host interactions during milk fermentations (Lawrence and Thomas, 1979), it is apparent that host-range activities are subject to constant change throughout the lactic streptococci. 3. Adsorption
Initiation of the bacteriophage lytic cycle begins with phage adsorption. This interaction between the bacterium and phage is highly specific and dependent on the presence of reactive phage receptors localized near the cell surface. The only extensive study characterizing phage receptors in the group N streptococci was conducted by Oram and Reiter (1968) and Oram (1971). Cell walls isolated from six S. crerrwris strains readily adsorbed reactive phages suggesting that phage receptors were localized in the cell walls of these strains. On the other hand, phage receptors for S . lactis ML3 were positioned in the plasma membrane (Oram, 1971). It was proposed that access of the phage to this membrane occurs through cone-shaped holes present in the cell wall of S. Zactis ML3 (Hirst and Stubbs, 1969). Subsequent characterization of the M U phage receptors demonstrated a lipoprotein moiety with polypeptide components (Oram, 1971). Further evidence for proteinaceous phage receptors in lactic streptococci was reported by Keogh and Pettingill (1983). Treatment of S. cremoris EB7 with trypsin, pepsin, or rennet significantly reduced or eliminated adsorption of phage eb7. In addition, L-rhamnose, D-galactosamine, and mglucosamine inactivated phage eb7. From these data it was suggested that the receptor confers serological specificity to the phage. The degree of phage adsorption can be affected dramatically by the availability of mono- or divalent cations (Cherry and Watson, 194913). In general, cations enhance adsorptive interactions between the phage and bacterium. These reactions are not specific for calcium or any other cation (Cherry and Watson, 194913; Potter and Nelson, 1952; Reiter and Moller-Madsen, 1963; Oram and Reiter, 1968). Additional extrinsic factors that may affect phage adsorption to the lactic streptococci include changes in pH (Cherry and Watson, 1949a; Keogh and Pettingill, 1983) and temperature (Keogh, 1973; Sanders and Klaenhammer, 1984). Although adsorption interactions between a phage and bacterium are highly specific, efficient phage adsorption, or lack thereof, may have little effect on lytic development of the phage. Generally phages adsorb well (>SO%) to their homologous hosts and lytic development follows. But nu-
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merous heterologous phage-host crosses that demonstrate efficient adsorption show no lytic development (Oram and Reiter, 1968), or limited lytic development at low plaquing efficiencies (Sanders and Klaenhammer, 1980, 1983). It is likely that this response is due to the presence of restrictionlmodification systems (Pearce, 1978; Boussemaer et al., 1980; Sanders and Klaenhammer, 1980; Daly and Fitzgerald, 1982), other undefined defense mechanisms (Sanders and Klaenhammer, 1983, 1984), or simply failure of the adsorbing phage to replicate within the host. In contrast, inefficient phage adsorption, as determined within the limits of the detection methods, does not necessarily deter phages from initiating high efficiency infections (Oram and Reiter, 1968; Limsowtin and Terzaghi, 1977; Sanders and Klaenhammer, 1983, 1984). In the lactic streptococci, adsorption reactions are subject to considerable variation due to changes in either the bacterium or phage. Alterations in the adsorptive specificity of lactic bacteriophages that are caused by mutation (Jarvis, 1978) or host-induced modification (Keogh, 1973) can elicit changes in host range. In the latter case, the ability of the phage to adsorb to a new host is dependent on the previous host on which the phage was propagated. This nonclassical, host-induced modification of the phage is phenotypic and conferred by a protein modification of the phage particle (Kruger et al., 1980; Kruger and Bickle, 1983). Changes in the capacity of the cell to adsorb phages can accompany the transition of lactic streptococci from phage sensitive to phage resistant. Numerous studies have reported resistant mutants that do not adsorb phages which are capable of adsorption to, and lysis of, the phage-sensitive parent (Collins, 1958; Oram and Reiter, 1968; Limsowtin and Terzaghi, 1976; King et al., 1983). In this regard, it has been shown that changes in the adsorption properties of lactic streptococci may, in some cases, involve acquisition or loss of plasmid DNA (Sanders and Klaenhammer, 1983; de Vos et al., 1984). With the loss of a 30-MDa plasmid in the prototype phage-insensitive strain S . lactis ME2, adsorption of four heterologous phages increased significantly (Sanders and Klaenhammer, 1983). For one of these phages, a 99% enhancement of phage adsorption in the plasmid-cured variant was accompanied by a 2 log cycle increase in plaquing efficiency. Similarly de Vos et al. (1984) reported that adsorption of a phage to S. cremoris S K l l increased from 5 to 90% upon loss of a 34-MDa plasmid. These studies have provided evidence for novel phage-defense mechanisms in the lactic streptococci that are encoded by plasmid DNA elements. Furthermore, plasmid involvement in the adsorption specificity of the streptococcal cell could account for a portion of the highly variable and unstable phage-host interactions routinely observed throughout the lactic streptococci.
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4 . Lytic Phage Replication
Following adsorption, phage DNA is injected into the cell and proceeds to direct the DNA replication, transcription, and translation systems of the host bacterium to produce progeny phage particles. Propagation of lytic phages can be a rapid process when latent periods are short and large numbers of phages are released when the cell bursts. Latent periods reported for phagehost crosses in the lactic streptococci range from 9 to 139 minutes (Zehren and Whitehead, 1954; Keogh, 1973; Lawrence et al., 1976), with a majority occurring within 40 to 50 minutes at 30°C (Keogh, 1973). Burst sizes are highly variable throughout the lactic streptococci and range from 9 to 105 particles released per infected cell (Keogh, 1973). At higher temperatures (37°C) latent periods are generally reduced, but burst sizes may increase, decrease, or remain unchanged (Zehren and Whitehead, 1954; Keogh, 1973). Zehren and Whitehead (1954) recognized that short latent periods or high burst size, or both, are characteristic of phage races that develop quickly and achieve high populations in cheese whey. These phages are considered to have high “multiplicationfactors” (Pearce et al., 1970), as assigned through a starter culture activity test, and they can cause complete failure of the starter culture even when present in low concentrations at the start of cheesemaking (Lawrence et al., 1976). Phage multiplication in the lactic streptococci is dependent on a number of nutritional factors. Foremost among these is a requirement for electrolytes that are active in promoting phage replication and cell lysis. Cherry and Watson (1949b)reported that potassium phosphate, sodium and calcium chloride, magnesium sulfate, and sodium acetate could enhance lysis of S . lactis by bacteriophages, but the effect correlated closely with increased phage adsorption. Alternatively, removal of tryptone from the growth medium reduced burst sizes threefold without affecting the adsorption efficiency of the phage. Subsequent work on electrolyte stimulation of phage multiplication has emphasized requirements for calcium. In numerous studies calcium-dependent phage replication has been demonstrated (Collins et al., 1950; Potter and Nelson, 1952; Oram and Reiter, 1968; Sozzi et al., 1980). These calcium requirements are generally independent of adsorption interactions, but the actual mechanisms that utilize calcium during phage replication have not been determined (Lawrence et al., 1976). Calcium requirements may vary between different phage-host interactions (Potter and Nelson, 1952)with optimum concentrations ranging from 2.7 x 10-4 to M . Despite a general requirement for calcium during lytic 1.7 x maturation of lactic streptococcal bacteriophages, reports demonstrating calcium-independent phage growth (Collins et al., 1950; Sozzi et al., 1980) indicate that calcium is not an absolute requirement for all phages. Signifi-
BACTERIOPHAGES AND LACTIC STREPTOCOCCI
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cant improvement of phage multiplication in the acid-producing lactic streptococci was achieved by incorporating 6-glycerophosphate into the growth medium. This buffering agent does not chelate calcium (Terzaghi and Sandine, 1975). In M17 medium the availability of calcium and maintenance of the pH above 5.7 enhance both cell growth and phage multiplication in lactic streptococci. Beyond specific p H and calcium effects, marked differences in phage multiplication, burst size, and adsorption efficiency have also been observed in raw milk (Hull and Brooke, 1982), sterile reconstituted milk, and sterile skim milk (Pearce et al., 1970; Keogh, 1973). Among those factors which affect the lytic development of phage in lactic streptococci, temperature has generated the most varied responses. As expected, most bacteriophages exhibit optimum replication at temperatures that are optimum for growth of the host bacterium, typically 30-32°C (Sozzi et al., 1978; Whitehead and Cox, 1936). Although optimum conditions for the phage and bacterium are usually similar, Hunter (1943) first observed that phage replication and bacterial growth may not always exhibit identical temperature dependencies. Mullan et al. (1981) in examining the interactions of 23 bacteriophages on lactic streptococci at 30, 38, and 40°C observed two basic types of temperature responses: (1) growth of the phage paralleled strain growth at all three temperatures; and (2) the phage did not replicate at higher temperatures (38 or 40°C) at which bacterial growth was uninhibited. These observations encouraged Mullan et al. (1981) to propose that starter strains which are innately less susceptible to phage attack do not replicate phage at cooking temperatures. Inhibition of phage replication at higher temperatures, that permitted bacterial growth, was also observed by Hunter (1943), Keogh (1973), and Zehren and Whitehead (1954). Enhanced phage lytic development at elevated temperatures (37-40°C) is a third temperature response exhibited routinely for phage-host interactions in the lactic streptococci. Phages that are unable to proliferate at normal growth temperatures (i.e., 22-32°C) show an increase in the efficiency of plating or accelerated replication at higher temperatures (Hunter, 1943; Pearce, 1978; Sanders and Klaenhammer, 1980; Daniel1 and Sandine, 1981; Hull and Brooke, 1982). Stimulation of plaquing efficiency in low-efficiency heterologous phage crosses by growth at elevated temperatures (Pearce, 1978) or by heat shock (Sanders and Klaenhammer, 1980) appears to result from phenotypic inactivation of restriction/modification systems. This temporary loss of restrictive activity may allow phage replication in normally restrictive hosts. Additional evidence now suggests that growth at elevated temperatures may allow immediate and full-scale lytic development of phages that are incapable of maturation at lower temperatures in selected strains of lactic streptococci (Sanders and Klaenhammer, 1984; McKay and Baldwin, 1984). These studies suggest that lactic streptococci carry phage defense
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mechanisms, independent of classical restriction and modification systems, that are rapidly disarmed during growth at elevated temperatures. The importance of elevated temperatures in the emergence of lytic bacteriophages against lactic streptococci was recognized by Heap and Lawrence (1976). They developed a laboratory starter culture activity test that mimicked the temperature profile encountered by starter cultures during cheesemaking. Strains surviving repeated cycles of the test in the presence of diverse phages at high titers performed well over extended time periods in New Zealand cheese factories without failure due to bacteriophage contamination. Use of a 38°C incubation period for 160 minutes was an important factor that contributed to phage detection in the starter activity test. Similarly, Hull and Brooke (1982) have noted enhanced replication of “raw milk” phages in activity tests conducted at 35”C, whereas little phage development occurred at 30°C. Undoubtedly, elevated temperatures encountered during cheesemaking may allow phage replication in some strains that prevent, or restrict, lytic development at the lower temperatures. Therefore, strains which do not allow heterologous phage replication at elevated temperatures (Mullan et al., 1981) may, in fact, be the most desirable for incorporation into starter cultures.
5. Host-Dependent Phage Replication Once phage DNA has entered the cell, the bacterium may either replicate the invading DNA leading to phage propagation, abort the infection because the phage is not compatible with the host’s replication systems, destroy the phage DNA by restriction enzymes, or, at low efficiency, chemically modify the DNA so that it is unrecognized by the host’s restriction enzymes and remains intact to direct lytic phage maturation (Kruger and Bickle, 1983). Adaptation of heterologous phages by host-controlled modification allows unrestricted lytic development in the new host; but such modifications are reversible and are dictated by the last host in which the phage was propagated. Therefore, while restriction and modification systems provide a major defense mechanism against infection by heterologous (unmodified) bacteriophages, the associated modification enzymes provide the opportunity for adaptation of the phage to new hosts. The opcration of restriction and modification systems in the lactic streptococci is now well established (Collins, 1956; Pearce, 1978; Limsowtin et al., 1978; Sanders and Klaenhammer, 1980; Boussemaer et al., 1980; Daly and Fitzgerald, 1982) and has received increased attention in recent years (see Davies and Gasson, 1981, 1983; McKay, 1983; Teuber and Lembke, 1983). Restricted phage crosses reported throughout the lactic streptococci range in efficiency of plaquing from high yet restricted efficiency values of lo-’ to extremely low efficiency values of (Collins, 1956; Pearce,
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1978; Boussemaer et al., 1980; Sanders and Klaenhammer, 1980). In 30 heterologous phage crosses on 6 different strains of S . lactis and S. cremoris, Boussemaer et al. (1980) detected low-efficiency plaque formation in 23 reactions. From these data it was suggested that restriction and modification systems are distributed throughout the lactic streptococci. Based on a mathematical model it was further projected that multiple restriction and modification systems may exist within single strains. Although low-efficiency plaque development suggests operation of restriction and modification systems, numerous unrelated factors, such as poor adsorption or heterogeneity in the host population (Limsowtin et al., 1978; Sanders and Klaenhammer, 1983), may contribute to low plaquing efficiencies. Therefore, the operational definition of restriction/modification systems requires demonstration of host-dependent modification of the phage. In the 23 restrictive crosses reported by Boussemaer et al. (1980), host-dependent replication was demonstrated in only two cases. However, experimental support for the projections of Boussemaer et al. (1980) has accumulated from a number of studies demonstrating host-dependent replication of phages isolated from low-efficiency crosses (Collins, 1956; Limsowtin et al., 1978; Pearce, 1978;Terzaghi and Terzaghi, 1978; Sanders and Klaenhammer, 1980; Daly and Fitzgerald, 1982). Based on these reports it is apparent that restriction/modification systems are common to the lactic streptococci. Distribution of restrictionImodification systems throughout the lactic streptococci may be accounted for in light of evidence suggesting linkage to plasmid DNA elements (Sanders and Klaenhammer, 1981). In S . cremoris KH, a 10-MDa plasmid was correlated with restriction and modification activity. Variants which had lost the plasmid replicated c2 phage more efficiently, with a 1.8-2.9 log cycle increase in the efficiency of plaquing. Modification activity was lost simultaneously in the restriction-deficient mutants demonstrating that the plasmid coded for both restriction and modification activities. However, despite the reduction in restriction and modification activities, the mutants cured of the 10-MDa plasmid retained some restrictive ability against c2 phage (efficiency of plaquing = 5 X 10W4).These data provided evidence for the operation of additional restriction/ modification systems in S . cremoris KH that are independent of the plasmid-linked system. Expression of multiple restriction/modification systems within single strains of lactic streptococci provides a strong barrier that protects the strain from attack by heterologous bacteriophages. Acquisition or loss of plasmid-linked restriction/modification systems accordingly would strengthen or weaken this barrier. Considering the inherent instability of plasmid DNA, variants deficient in restrictive capacity can readily accumulate upon subculture (Limsowtin et al., 1978; Sanders and Klaenhammer, 1981) and provide a host reservoir for replication of heterologous bacteriophages.
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Performance of restriction/modification systems in the lactic streptococci can be retarded when the optimum growth or assay conditions are altered. When stationary phase cells of S. cremoris AM1 were challenged with a lowefficiency, heterologous phage from S . cremoris HI, plaquing efficiency increased considerably over values obtained with logarithmic phage cells (Lowrie, 1974). Terzaghi and Terzaghi (1978)further reported that aged cells grown on high levels of lactose, but not glucose or galactose, demonstrate a reduced ability to restrict growth of heterologous phages. This effect was partially attributed to the low pH levels achieved during the growth of cultures at unlimiting lactose concentrations. Of more practical significance were observations by Pearce (1978) that exposure of starved cells of S. crem r i s KH to elevated temperatures enhanced the efficiency of plaquing of the restricted phage m13. Using the m13 phage to further challenge 23 hosts of S. lactis and S. cremoris, efficiency of plaquing was increased at least 2 log cycles on 5 different strains when the incubation temperature was raised from 30 to 37°C. These data strongly suggest that elevated temperatures can retard the restrictive capacity of lactic streptococci during attack by heterologous bacteriophages. Similar observations were made in our laboratory for restricted, low-efficiency phage crosses in S . cremoris KH and S. cremoris 799 (Sanders and Klaenhammer, 1980). Following heat challenge of S. cremoris KH and 799, modified progeny phages were produced that were capable of full lytic development on the formerly restrictive hosts (Sanders and Klaenhammer, 1980). These data indicate that in the presence of a lowefficiency, heterologous phage, temporary loss of the bacterium’s restrictive abilities at elevated temperatures can lead to the appearance of a modified phage population with full lytic capacity on the previously restrictive host. In this regard, the elevated temperatures used in cheese manufacture may contribute directly to the appearance of new lytic phage populations by enhancement of heterologous phage replication in heat-shocked starter cultures. Restrictiodmodification systems are undoubtedly widespread in the lactic streptococci and are of crucial importance in their effect, positive or negative, on host-range interactions of heterologous bacteriophages. However, evidence for restriction and modification activities has been based primarily on operational data, with one notable exception. Of seven strains examined, Fitzgerald et al. (1982) were able to demonstrate a type 11, sequence-specific, restriction endonuclease from S . cremoris F. The enzyme, designated ScrFI, required Mg2+ or Mn2+, did not require S-adenosylmethionine or ATP, and recognized the sequence 5’-CC NGG-3‘. Although S. cremoris F expresses restriction and host-dependent modification of phages (Daly and Fitzgerald, 1982), it is not known whether ScrFI operates in oivo as part of the cell’s restrictiont’modification system. Unfortunately, additional re-
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13
striction enzymes were not detected in other strains exhibiting operationally defined restriction/modification systems (Fitzgerald et at., 1982). However, the presence of type I or type 111 enzymes was not investigated. These elegant studies have provided the first biochemical evidence for restriction enzymes in the lactic streptococci. Further studies are needed and it is hoped that they will elucidate the biochemical and molecular basis of restriction and modification in the lactic streptococci.
B. LYSOGENIC AND PHAGE-CARRYING CULTURES Lysogeny throughout the lactic streptococci has been well established (for reviews see Lawrence et al., 1976; Davies and Gasson, 1981, 1983; Teuber and Lembke, 1983). A majority of lactic streptococcal strains harbor temperate phages that can be readily induced by ultraviolet light, mitomycin C, or heat and, in some cases, induction can occur spontaneously (Reiter, 1949; Keogh and Shimmin, 1969; Kozak et al., 1973; McKay and Baldwin, 1973; Lowrie, 1974; Park and McKay, 1975; Huggins and Sandine, 1977; Meister and Ledford, 1979; Terzaghi and Sandine, 1981; Reyrolle et al., 1982). For most of these studies, lysis of the culture was observed following induction, and temperate phages were identified by lysis of the appropriate indicator strain or, more often, by detection of phage particles using electron rnicroscopy. Formal demonstration of classical lysogeny in S. Zactis and S. cremoris was accomplished by Gasson and Davies (1980). Prophage-cured derivatives were isolated and shown to be susceptible indicators for their own temperate phages. The cured derivatives could be relysogenized, thus completing the temperate cycle to the prophage state. Similar observations were made by Georghiou et al. (1981) who also demonstrated resistance of the prophage state to antiserum active against the temperate bacteriophage. The accumulated evidence clearly demonstrates the ubiquity of temperate bacteriophages which are carried as lysogens throughout the lactic streptococci. The attention focused on the lysogenic nature of the lactic streptococci is based on the possibility that lytic phages appearing in cheese plants originate from temperate bacteriophages harbored by the starter cultures in use (Lawrence et al., 1976). Evidence supporting this mechanism has been circumstantial and is limited to similarities in the morphology of the temperate and lytic bacteriophages (Lawrence, 1978; Lawrence and Thomas, 1979; Heap and Jarvis, 1980). However, Shimizu-Kadota et al. (1983)have demonstrated that the virulent phage appearing during milk fermentations with Lactobacillus casei S-1 originates from the temperate phage carried by this strain as a lysogen. The temperate and lytic phages were indistinguishable by serology, morphology, and restriction digests of DNA from both phages. Furthermore, use of L. casei derivatives cured of the prophage did not
14
TODD R. KLAENHAMMEH
produce the virulent phage (Shimizu-Kadota and Sakurai, 1982). The practically universal incidence of lysogeny in the lactic streptococci supports a similar mechanism and indicates that lysogenic strains may serve as a “reservoir” of phages that are potentially able to attack strains in mixed-strain starter cultures (Huggins and Sandine, 1977). In this regard, care has been exercised not to mix strains in starter cultures that are sensitive indicators to temperate phages which can be induced from other component strains of the culture (Lawrence et al., 1978; Daniel1 and Sandine, 1981). Despite the logical connection that can be made between temperate bacteriophages and the potential for lytic phage development, no definitive evidence has been provided to link the temperate and lytic cycles as a source of lytic phage in cheese plants. This fact, coupled with a conspicuous lack of known indicator strains that support the lytic development of temperate phages (Lawrence et al., 1976), might indicate that temperate phages are of minor importance and do not contribute to the appearance of lytic bacteriophages during cheese manufacture (Teuber and Lembke, 1983). Recent evidence strongly supporting this view was provided by Jarvis (1984b). In DNA homology studies between temperate phages induced from 3 strains of S. cremoris and 25 lytic phages isolated from New Zealand cheese factories, no significant genetic relatedness was detected. From these data Jarvis (1984b) concluded that temperate phages carried as lysogens in starter cultures are not a predominant source of lytic phage in cheese plants. Alternatively, some evidence continues to support the possibility that temperate phages may, in some instances, be responsible for lytic attack of starter cultures. Reyrolle et d.(1982) reported that 25% of 113 strains examined were sensitive indicators for temperate phages induced from the lactic streptococci. The lytic spectrum of the temperate phages was similar to virulent phage groups described earlier (Chopin et al., 1976). This high percentage of indicator strains and a close correlation between temperate and lytic phage activity spectra reemphasized the potential of temperate phages to provide a source of lytic phages (Reyrolle et al., 1982).The latter authors further noted that the transition from temperate to virulent activity may accompany a mutation of the phage. Basic studies which examine conversion of phages from temperate to lytic cycles, induced by mutation or genetic rearrangements, are desperately needed. Considering the dynamic genetic state of the lactic streptococci (Davies and Gasson, 1981, 1983; McKay, 1983), genetic alterations in the temperate phages carried as lysogens in these organisms may be equally dynamic, thus providing a complex array of temperate and lytic phage-host interactions. Perpetuation of bacteriophages by lactic streptococci can occur by lytic maturation, lysogeny, or establishment of a phage-carrier state. Pseudolysogeny (or the phage-carrier xtate) is established upon the persistant but in-
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15
complete lytic association between the phage and host bacterium (Barksdale and Arden, 1974). Pseudolysogeny can be readily differentiated from true lysogeny by treatment of the phage-carrying culture with antisera reactive against the phage, thereby eliminating the phage. Hunter (1947) first described pseudolysogeny in the lactic streptococci and reported that the phagecarrying state could be eliminated by repeated single colony isolation. In the lactic streptococci phage-carrying cultures appear to be composed of subpopulations that exhibit varied sensitivities to the carried phage (Graham et al., 1952; Limsowtin and Terzaghi, 1977).These data imply that the phage is allowed only limited development by a continuous balance of phage-sensitive and phage-resistant variants in the population. However, mechanisms by which lactic streptococci perpetuate phages in a pseudolysogenic state have not been investigated in detail and are poorly understood. Of major practical significance was the observation that phage-carrying cultures are protected from attack by other phages which can attack the corresponding phage-free cultures (Hunter, 1947, 1949). This phenomenon may represent a powerful mechanism of phage defense for the lactic streptococci, but remains uninvestigated. Detailed studies on the phage-carrying state of the lactic streptococci are needed to resolve the specific mechanisms involved and the significance of this phage-host interaction.
C. TRANSDUCTION Transfer of genetic information by temperate, and occasionally virulent, phages of the lactic streptococci has been demonstrated in S. lactis, S. cremoris, and S . Zactis subsp. diacetylactis. Transduction processes in the lactic streptococci have been extensively reviewed and therefore will not be reexamined in detail here (for reviews see Lawrence et al., 1976; Davies and Gasson, 1981, 1983; Gasson, 1983; Teuber and Lembke, 1983). It is pertinent, however, to note that phage-mediated transfer in the lactic streptococci can involve both chromosomal and plasmid markers including those which determine lactose, mannose, and maltose metabolism (McKay et al., 1973, 1976; Snook et al., 1981; Davies and Gasson, 1983), proteinase activity (McKay and Baldwin, 1974; McKay et al., 1976), streptomycin resistance (Allen et al., 1963; McKay et al., 1980), erythromycin resistance (Gasson, 1983), and tryptophan independence (Sandine et aZ., 1962). Transduction provides a mechanism for distribution of genetic information during naturally occurring phage-host interactions of the lactic streptococci. Further, with the recent emphasis on genetic engineering prospects for dairy starter cultures (Davies and Gasson, 1981, 1983; McKay, 1983), transductional processes may contribute to the construction of improved strains. In this regard, stabilization of lactose and proteinase genes in the chromosome of S. lactis
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TODD R. KLAENEIAMMER
C2 by transduction was a most significant accomplishment (McKay and Baldwin, 1978). Ill. Control of Bacteriophages
A. ORIGINOF BACTERIOPHAGES The origin of bacteriophages during milk fermentations remains an unresolved question for which there is no simple, or single, solution. Phages may enter the environment by airborne contamination or through the milk supply itself. Raw milk has been shown to harbor bacteriophages (Whitehead, 1953; Hull and Brook, 1982) which can survive pasteurization and spray drying (Chopin, 1980). Therefore the fermentation substrate is naturally contaminated with phages, albeit at low levels, that could serve as predecessors to developing lytic phage populations. In addition, use of lysogenic starter cultures may contribute to the contaminating phage population (Lawrence et al., 1976; Huggins and Sandine, 1977). Whatever external sources exist, it is apparent that phages will be present in the cheesemaking environment. Whether or not these phages, of either temperate or lytic origin, develop into lytic populations will depend on a variety of independent variables that affect the phage or host, or both. These can include, but are not limited to, an array of mutational events affecting the phage or host (Jarvis, 1978; Sinha, 1980), genotypic and phenotypic alterations in the host’s susceptibility to phages (Collins, 1958; Limsowtin et al., 1978; Pearce, 1978; Sanders and Klaenhammer, 1980, 1981, 1983), host-controlled modification of phages (Collins, 1956; Potter, 1970; Pearce, 1978; Sanders and Klaenhammer, 1980; Daly and Fitzgerald, 1982), or simply, chance selection of a starter strain that supports full lytic capability of a contaminating bacteriophage. The cheesemaking environment provides a highly dynamic situation for interactions between the lactic streptococci and their bacteriophages. When both bacteria and phages are present, which is unavoidable in the milk fermentation process, genotypic or phenotypic alteration of either entity could trigger a series of complex events leading to the appearance of a dominant lytic phage population. Consequently, the origin of lytic phages in the milk fermentation process cannot be targeted to a definitive mechanism or routine series of events.
B. PHAGE-INHIBITORY MEDIA AND CONCENTRATED CuLTU RE s
Prior to inoculation of the cheese vat, prevention of lytic phage replication during the preparation of the starter culture is essential. Control of bacterio-
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17
phages at this stage can be accomplished by aseptic methods, which eliminate external routes for phage contamination, and use of phage-inhibitory media to restrain development of lytic phages that might occur in the bulk starter culture. Bulk starter vessels which prevent airborne phage contamination of the culture were first constructed and used in New Zealand cheese factories (see Whitehead, 1953; Sandine, 1977). These vessels are designed to maintain aseptic conditions and can be used without reliance on phage-inhibitory bulk starter media (Lawrence et al., 1976; Sandine, 1977). Although most bulk culture tanks are routinely designed as closed systems (Wigley, 1980) they do not meet stringent aseptic requirements and may, therefore, be subject to phage contamination. Consequently, a common practice in the United States is to prepare bulk cultures in phage-inhibitory media. These media are supplemented with phosphates in order to chelate divalent cations, particularly calcium (Hargrove et d., 1961; Zottola and Marth, 1966). When calcium is not available, proliferation of most, but not all, bacteriophages is inhibited (Collins et al., 1950; Reiter and Moller-Madsen, 1963).The effectiveness of phage-inhibitory media is dependent on the type and concentration of phosphate (Hargrove et aZ., 1961; Zottola and Marth, 1966), the p H of the medium (Hargrove et al., 1961; Ausavanodom et al., 1977), and the specific requirements of the phage-host interaction (Sozzi et al., 1980). Further, commercial phage-inhibitory media can vary widely in composition and effectiveness (Sandine, 1977). Two major developments have occurred recently in the use of phage-inhibitory media for propagation of bulk starter cultures. Using recycled whey, whey-based medium containing phosphates has been formulated and used in conjunction with an externally pH controlled bulk starter system (Ausavanodom et al., 1977; Chen and Richardson, 1977; Richardson et al., 1976; Wright and Richardson, 1982). Using external pH control, phosphate levels could be reduced without affecting phage inhibition or starter culture activity (Ausavanodom et al., 1977). Furthermore, use of recycled whey in the formulation reduced the cost of bulk starter preparation. In a second major development, Sandine and Ayres (1981) have described a bulk starter medium containing water-insoluble phosphates, This medium does not require external p H control and exhibits excellent phage inhibition while stimulating culture growth and activity. Starter culture concentrates, which can be directly inoculated into the cheese vat, provide an alternative to bulk culture preparation and use of phage-inhibitory media. Because these concentrates are prepared aseptically and shipped to the plant, there is no possibility for phage contamination of the starter prior to inoculation of the fermentation vat. At present, culture concentrates designed for direct vat inoculation are frozen and stored at ultracold temperatures ranging from -70 to - 196°C (Porubcan and Sell-
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T O D D R. KLAENHAMMEH
ars, 1979). Frozen culture concentrates are highly active, but the expense required for distribution and storage at ultracold temperatures restricts their widespread use in the dairy industry. Alternatively, dried culture concentrates are less expensive to distribute and store, but these cultures are less active and usually require a series of intermediate transfers to regain full fermentative activity (Porubcan and Sellars, 1979; Yang and Sandine, 1979). Propagating culture concentrates through intermediate or bulk cultures in the cheese plant reestablishes the potential for phage contamination of the starter culture.
c. STRAIN ROTATION A N D MULTIPLE-STRAIN STARTERS Once the starter culture enters the cheese vat it can no longer be protected from bacteriophage contamination or proliferation. Whitehead (1953), realizing that phage exposure cannot be entirely eliminated in the cheesemaking process, attempted to reduce phage levels in the plant by thorough sanitation practices and use of a strain rotation system. From “hundreds” of strains examined, I0 strains were selected for the rotation that were fermentatively active and unrelated in phage sensitivity. Strains were paired and used in a 4-day cycle of rotation. It was reasoned that one strain of the pair could continue acid production if the other was attacked by bacteriophages. Use of each pair only every 4 days eliminated daily proliferation of phages that were specific for the strains of that pair. Therefore, phages generated over 1 day of cheese manufacture would be reduced in concentration during the use of the other phage-unrelated pairs over the next 3 days of rotation. Description of this starter culture program by Whitehead (1953) provided the basis for defined mixed-strain starters and the strain rotation programs that are still in use today. The objective of culture rotation, as described by Whitehead (1953), was to reduce levels of lytic phages in the plant. Later work suggested that residual phage levels were not reduced sufficiently over the rotation period to prevent slow acid production by strains susceptible to lytic phages present in the cheese plant (Collins, 19S8, 1962). Collins (1962) argued that pcrsistence of ltacteriuphages in the plant call be avoided by replacing susceptible strains in the rotation immediately upon the appearance of bacteriophages. The success of this program relied on continuous monitoring of the whey samples for phages that were active against any strain in the rotation. Upon detection of phages, strain combinatioiis were reformulated to eliminate sensitive hosts. Cultures used in the rotations were composed of 3-4 strains and selected from a collection of 20 compatible and phageunrelated strains. Collins (1962) recognized that there are, in fact, few phage-unrelated strains of lactic streptococci available for use in this type of
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19
culture program. Consequently, the effectiveness and longevity of traditional starter rotation and strain replacement programs are severely limited (Collins, 1955b; Chopin et al., 1976; Lawrence et al., 1976). Because phage-unrelated strains are limited in number and difficult to recognize (Lawrence, 1978), rotation programs inevitably use strains that are phage related. Under these circumstances susceptible hosts are present throughout the rotation cycle and phage populations can be maintained at high levels. Once present in high titers in the plant the host range of phages could be expanded through phage mutation or host-controlled modification (Jarvis, 1978; Heap and Lawrence, 1976; Sanders and Klaenhammer, 1980, 1981). Therefore, the practice of rotating large numbers of strains in mixedstrain starters may offer little protection from bacteriophages and could, in fact, promote phage proliferation and the appearance of different phage types (Thunell et al., 1981). In recent years, the use of multiple-strain starters has provided an alternative to traditional rotation programs (Limsowtin et al., 1977; Richardson et al., 1980; Daniel1 and Sandine, 1981; Thunell et al., 1981). Multiple-strain starters are composed of three to six selected strains of lactic streptococci that are used continuously in the plant as part of the phage-monitoring program. Upon detection of lytic phages for any strain in the composite, the susceptible strain is replaced either with a new strain (Limsowtin et al., 1977) or with a phage-resistant mutant (Richardson et al., 1980; Thunell et al., 1981). The initial success of the multiple-strain starter is dependent upon selection of phage-unrelated strains that resist attack by phages present in plants targeted for use of the culture. Over long-term use, strains must also be selected that will withstand attack by phages that have the potential to develop in the cheesemaking environment. The starter activity test described by Heap and Lawrence (1976) predicts such strain longevity in a factory environment. Strains under consideration are subjected to repeated growth cycles in the presence of phage composites containing high-titer phage preparations and bulk whey samples collected from New Zealand factories. The growth cycle includes a temperature profile that mimics timetemperature relationships encountered in cheesemaking. In their study Heap and Lawrence (1976) found five strains that survived six growth cycles through the activity test in the presence of the high-titer phage composite. After 8 months of continuous use in New Zealand factories, lytic phages did not appear for three of these strains and were detected at only low titers in one factory for the remaining two strains. This study emphasized the importance of temperature, phage types, and phage levels in the appearance of lytic bacteriophages in commercial cheesemaking. But of even more significance, the laboratory activity test provided a means to predict whether or
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not a phage will rapidly appear against a strain introduced into a cheese plant. Subsequently, Limsowtin et al. (1977) formulated a multiple starter composed of six strains, each selected by the laboratory activity test for phage insensitivity. The multiple starter was used continuously from 2 to 8 months without failure. Although activity was not affected, phages did appear against four of the six strains in the multiple starter. Consequently, Limsowtin et al. (1977) recommended that strains be replaced when phages are detected. The multiple-starter concept is effective only when strains can be selected that resist attack by bacteriophages long enough to make their use worthwhile (Heap and Lawrence, 1976; Limsowtin et al., 1977). These strains are obviously limited in number and, therefore, selection of strains as initial components of multiple starters, or as replacement strains, can be difficult. Once the multiple starter is formulated, an alternative to new strain substitution is replacement of strains with phage-resistant mutants derived from those original strains for which a phage has developed (Richardson et al., 1980; Thunell et al., 1981). These programs have overcome the typical problems encountered during selection and use of phage-resistant mutants, particularly selection of fast acid-producing derivatives (see below). However, maintenance of multiple-starter programs that rely on continual selection of phage-resistant mutants as replacement strains requires a program tailored on a daily basis to individual cheese plants. This level of commitment in a service rather than in a production program may ultimately be impractical to the major culture industries.
D. PHAGE-RESISTANT MUTANTS With the discovery of bacteriophages in 1936 by Whitehead and Cox, it was immediately realized that a simple solution might be the isolation and use of phage-resistant mutants. Although “immune” forms could be readily isolated from secondary cultures growing out of lysed milk cultures, these mutant strains were subject to attack by new phage or gradually lost resistance to the original lytic phage. Since this initial report numerous investigators have attempted to isolate resistant mutants for phages that appear in dairy fermentations. Consistently, these mutants either revert to phage sensitivity, are attacked by a new phage, or show retarded acid-producing ability (Whitehead, 1953; Collins, 1955a; Czulak and Naylor, 1956; Limsowtin and Terzaghi, 1976; Marshall and Berridge, 1976; Jarvis, 1981; King et al., 1983). Moreover, for some phage-host combinations resistant mutants could not be isolated (Limsowtin and Terzaghi, 1976;Jarvis, 1981).Therefore, when phage-resistant mutants can be isolated, their use usually provides only short-term protection from bacteriophage attack of the starter culture (White-
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21
head and Cox, 1936). However, in rare cases, phage-resistant mutants have been shown to endure continuous use over several months without a recurrent phage attack (Limsowtin and Terzaghi, 1976;Jarvis, 1981; Thunell et al., 1981). One approach to preparing starter cultures that are not susceptible to phage attack during milk fermentations is to deliberately contaminate these cultures with phages. During daily propagation of the starter, usually of undefined composition, fresh whey samples are added to continually expose the culture to phages appearing in the plant (Thomas and Lowrie, 1975; Hull, 1977; Czulak et al., 1979). In the Netherlands, phage contamination of the starter occurred naturally by starter propagation and buildup in the plant where aseptic precautions were not taken to exclude phages (Galesloot et al., 1977; Stadhouders, 1975). Under these circumstances, constant selection of phage-resistant mutants occurs naturally in the starter. Consequently, these phage-derived starters can be used continuously without threat of failure due to bacteriophage attack. However, on a daily basis these cultures vary considerably in rates of lactic acid production due to the accumulation of slow variants in the population (Stadhouders, 1975; Thomas and Lowrie, 1975). With present-day manufacturing requirements and schedules, this fluctuation in acid-producing ability of the starter culture is often unacceptable (Lawrence, 1978). The routine appearance of slow acid-producing variants following selection of phage-resistant mutants has prompted speculation that phage resistance and fermentation ability are interrelated (Lawrence et al., 1976; Lawrence, 1978). Alternatively, Marshall and Berridge (1976) concluded that a correlation does not exist between phage resistance and slow acid production. They reasoned that the emergence of slow acid-producing variants was the direct result of the method used to isolate phage-resistant mutants. In broth or milk cultures which have been completely lysed by phages, development of secondary, phage-resistant cultures is a slow process and may favor outgrowth of debilitated mutants that are unsuitable for cheesemaking. Recent studies by King et aZ. (1983) have demonstrated that mutations to phage resistance and slow acid-producing ability are genetically independent events. Although mutation rates varied considerably between strains, spontaneous mutations to phage resistance were three to five orders of magnitude below the corresponding rates of mutation to slow acid production. These data strongly suggest that the appearance of slow acid-producing variants during selection of phage-resistant mutants is merely a coincidence rather than a direct cause and effect relationship. Nevertheless, phage-resistant mutants that are slow acid producers are routinely encountered during phage challenge. Consequently, efforts to isolate and use phage-resistant mutants in starter culture programs must include methods to differentiate
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TODD R. KLAENHAMMER
between fast and slow variants. In multiple-starter systems using phageresistant mutants as replacement strains, fast acid producers are detected quickly by plating on either fast-slow differential agar (Huggins and Sandine, 1979; Thunell et al., 1981) or milk-citrate agar (Richardson et al., 1980). Development of these differential media has made possible the routine selection of fast acid-producing, phage-resistant mutants for replacement of phage-sensitive strains in multiple-strain starters, thereby negating the need to substitute new phage-unrelated strains which are of limited availability.
E. PHAGE-INSENSITIVE STHAINS Phage-resistant mutants arise spontaneously through cell alterations that prevent adsorption, infection, or replication of a specific phage or a related group of phages. In contrast, there are reports of strains of lactic streptococci that persistently resist attack by all known phages and maintain this resistance for considerable periods under the most devastating commercial manufacturing conditions (Zehren and Whitehead, 1954; Chopin et al., 1976; Heap and Lawrence, 1977; Daniel1 and Sandine, 1981). The longevity of these strains is undoubtedly influenced by the specific phage complement and environmental conditions encountered in any particular cheese plant. However to some extent survival must also be dictated by innate and powerful mechanisms of phage resistance exhibited by the strain itself. In the search for lactic streptococci suitable for cheesemaking, strains exhibiting superior fermentative activity and long-term phage resistance have been routinely isolated from commercial mixed-strain cultures (Lawrence et al., 1976, 1978). Historically these mixed-strain starters were propagated continuously by traditional cheesemakers without protection from phage contamination. Lawrence (1978) reasoned that component strains of these starters are descendants from strains which originally contaminated these cultures over 60 years ago. Because the environment created by the cheesemaker was susceptible to constant phage pressure, it would be expected that through natural selection strains harboring dynamic mechanisms of phage resistance would evolve. In recent years evidence has accumulated suggesting that a higher order of phage resistance exists among select strains of lactic streptococci. Major barriers against phage attack have been defined in multiple restriction and modification systems (Boussemaer et al., 1980; Sanders and Klaenhammer, 1981) and in plasmid-encoded mechanisms that block phage adsorption or lytic phage maturation (Sanders and Klaenhammer, 1983, 1984; de Vos et al., 1984; McKay and Baldwin, 1984). These systems provide a first line of phage defense for the lactic streptococci, but as independent mechanisms they may be too easily penetrated by phages. However, in a prototype phage-insen-
BACTERIOPHAGES AND LACTIC STREPTOCOCCI
23
sitive strain of S . Zuctis, all three independent mechanisms appeared to function cooperatively to confer an impermeable phage-defense system (Sanders and Klaenhammer, 1984).Therefore, collective expression of multiple phage-defense mechanisms may be the basis of the insensitive state of the lactic streptococci. Because these strains are rarely isolated from natural sources, the availability of phage-insensitive strains for the dairy industry may ultimately depend on genetic engineering efforts to combine multiple phagedefense mechanisms within single strains of lactic streptococci.
IV. Roles of Plasmid DNA Within the lactic streptococci it has been well established that numerous fermentative and metabolic characteristics are genetically encoded by plasmid DNA elements. Plasmid DNA involvement has been demonstrated, or implicated, for carbohydrate metabolism (lactose, galactose, sucrose), proteolytic activity, citrate utilization, bacteriocin production (nisin, diplococcin), and resistance to inorganic salts and the lactoperoxidase-thiocyanatehydrogen peroxide system (for reviews see McKay, 1982, 1983; Davies and Gasson, 1981; Kempler and McKay, 1981). The lactic streptococci harbor an unusually large and diverse complement of plasmid DNA elements. Although the function of the majority of plasmid species remains unknown, it is apparent that select plasmids encode properties that positively affect the ability of the lactic streptococci to compete in a milk environment. Moreover, some of these plasmid-linked characteristics are crucial to the performance of strains responsible for successful dairy fermentations (McKay,
1983). It is believed that the lactic streptococci have recently evolved from green plant material to raw milk as a natural habitat (Hirsch, 1952; Sandine et aZ., 1972). Considering that plasmid DNA elements are genetic agents of rapid strain evolution and adaptation (Reanney, 1976), McKay (1982) reasoned that highly specialized strains of lactic streptococci would emerge in milk through acquisition of plasmid DNA elements. The accumulated information on plasmid linkages and the genetics of the lactic streptococci strongly supports this view. In this light it is interesting to note that the most probable source of phage-insensitive lactic streptococci is commercial mixed-strain starters propagated over decades without protection from bacteriophages (Lawrence et uZ., 1976; Lawrence, 1978). Adaptation to this environment may have similarly been facilitated by acquisition of plasmid DNA elements carrying determinants for phage-resistance mechanisms. The potential involvement of plasmid DNA in phage resistance of the lactic streptococci has been subject to study only in recent years. However, in early studies Collins (1958) recognized that phage resistance may be an
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TODD R. KLAENHAMMER
unstable characteristic. Loss of phage resistance in three cultures was observed after 4 months of repeated transfers in milk. Collins (1958) further noticed that changes in the bacteriophage sensitivity of the lactic streptococci occurred gradually and were less frequent if culture transfers were reduced. Although unrecognized at that time, instability of a plasmid-linked mechanism of phage resistance was probably responsible for the appearance of phage-sensitive variants in these cultures. Similarly, Limsowtin et al. (1978) reported that cultures of lactic streptococci subcultured in M 17 broth were highly heterogeneous and composed of a variety of phage-resistant and phage-sensitive variants. Some of the variants which were characterized exhibited different levels of bacteriophage restriction and modification activity. Noting the rapid appearance of phage-sensitive variants during sub-
FIG. 1. Agarose gel electrophoresis of plasmid DNA from Streptococcus cremoris K H and Streptococcus crenioris M12R. Each culture was plated onto M17 agar plates (Terzaghi and Sandine, 1975) for isolation of single colonies. Colonies were picked at random, propagated in MI7 broth, and examined for plasmid composition (Sanders and Klaenhammer, 1983). (A) Wells A - 6 , S . cremoris KH isolates. Well H. Escherichiu coli V517. (Plasmid molecular weights of 35.8, 4.8, 3.7, 3.4, 2.6, 2.0, 1.8, and 1.4 X IF.) (B) Wells A-C, E-C, S . cremoris M12R isolates. Well C, E . coli V517. The migration patterns revealed that individiial isolates from S. cremoris cultures can be highly heterogeneous in plasmid D N A composition. Observations of plasmid heterogeneity in single colony isolates of S. cremoris KH were first made by Sanders (1983). These data were kindly provided by R. B. Sanozky and L. R. Steenson, North Carolina State University.
BACTERIOPHAGES AND LACTIC STREPTOCOCCI
25
culturing, Limsowtin et al. (1978) suggested that plasmid DNA elements might be involved. Subsequently, correlative evidence for plasmid linkage of a restriction/modification system in S . cremoris KH was reported (Sanders and Klaenhammer, 1981). Recent work has further defined plasmid-linked mechanisms that block adsorption of phage (Sanders and Klaenhammer, 1983; de Vos et al., 1984) and has identified a phage-resistance mechanism that is heat sensitive and linked to a conjugative plasmid in S . Zuctis subsp. diacetylactis (McKay and Baldwin, 1984). From these data it is apparent that phage-defense mechanisms in the lactic streptococci can be encoded by plasmid DNA elements. However, the distribution, nature, and genetic determinants of phage-resistance mechanisms in the lactic streptococci have yet to be studied in detail. Their extrachromosomal location makes plasmid DNA elements and their associated traits inherently unstable. Therefore, plasmid linkage of phageresistant characteristics in the lactic streptococci establishes a genetic mechanism for the rapid appearance of phage-sensitive variants within starter cultures (Sanders and Klaenhammer, 1981, 1983). Lawrence (1978) emphasized that phage problems in the dairy industry may result from accumulation of phage-sensitive variants within starter cultures. Considering the large complement of plasmids carried by the lactic streptococci, genetic and phenotypic heterogeneity within single strain cultures can be common phenomena induced by simple subculturing. It is not unusual that pure strain cultures of lactic streptococci exhibit extensive heterogeneity in plasmid composition when single colony isolates from the culture are examined (Fig. 1). Therefore it is imperative that culture-handling practices in the dairy industry minimize the potential for genetic heterogeneity within starter cultures, especially when plasmid involvement in phage resistance is considered.
V. Conclusions The appearnce of bacteriophages in milk fermentations continues to be the major problem faced by the cultured dairy products industries. With the development of aseptic starter culture preparation and handling procedures, bacteriophage proliferation in the starter culture can be controlled prior to inoculation of the fermentation vat. Use of these methods will continue to be an integral part of any successful starter culture program using defined mixtures of lactic streptococci. However, starter culture failures due to bacteriophage attack will persist as long as phages appear and, more importantly, are allowed to replicate in the fermentation vat. A permanent solution to the bacteriophage problem may never be found. The nature of the fermentation process and the dynamic state of phage-host
26
TODD R. KLAENHAMMER
interactions in the lactic streptococci indicate that regardless of the strains used or precautions taken, it is probable that phages will appear and eventually attack starter culture strains. However, the frequency of such infections could potentially be reduced to an insignificant level. Toward this goal, it is imperative that phage-sensitive variants do not accumulate in starter cultures; that phage-defense mechanisms of the lactic streptococci remain active under the growth and temperature conditions of the cheesemaking process; and that phage-unrelated strains be employed that are, ideally, phage insensitive. In the latter case, such strains are of limited availability to the dairy industry and are not readily accessible from natural sources. However, progress in the genetics of lactic streptococci (see McKay, 1982, 1983; Davies and Gasson, 1981, 1983; Gasson, 1983) has established the potential for genetic engineering of phage-resistance mechanisms. As our knowledge of the genetics of lactic streptococci and their phage-defense mechanisms increases, application of recombinant DNA technology eventually may provide phage-insensitive starter cultures for the dairy fermentation industries. REFERENCES Allen, I>. K., Sandine, W. E., and Elliker, P. R. (1963).J. Dairy Sci. 30, 351-357. Ausavanodorn, N . , White, R. S., Young, G., and Richardson, G. H. (1977).J . Dnirrj Sci. 60, 1245-1251. Barksdale, L., and Arden, S. B. (1974). Anrtu. Reo. Microbiol. 28, 265-299. Boussemaer, J. P., Schrauwen, P. P., Sourrouille, J . L., and Guy, P. (1980).J. Dairy Res. 47, 401-409. Chapman, H. R. (1978).J . Soc. Dairy Technd. 31, 99-101. Chen, Y. L., and Richardson, C . H.(1977). J . Duiry Sci. 60,1252-125s. Cherry, W. B., and Watson, D. W. (1949a). J. Bacteriol. 58, 601-610. Cherry, W. B., and Watson, D. W. (1949b).1. Bacterial. 58, 611-620. Chopin, M.-C. (1980).J. Dairy Res. 47, 131-139. Chopin, M.-C., and Rousseau, M. (1983).Appl. Enoiron. Microbiol. 45,294-296, Chopin, M.-C., Chopin, A., and Roux, C. (1976). Appl. Enuiron. Microbiol. 32, 741-746. Collins, E. B. (1955a).Appl. Microbiol. 3, 137-140. Collins, E. B. (195511). Appl. Microbid. 3, 145-148. Collins, E. B. (1956). Virology 2, 261-271. Collins, E. B. (1958).J. Dairy Sci. 41, 41-48. Collins, E. B. (1962).J . Dairy Sci. 45, 552-558. Collins, E. B., Nelson, F. E., and Parmelee, C. E. (1950).J . Bacteriol. 60, 53-542. Cznlak, J., and Naylor, J. (1956).J . Dairy Res. 23, 120-125. Cziilak, J., Bant, D. J., Blythe, S. C., and Grace, J. B. (1979). Dairy Ind. Int. 44, 17-19. Daly, C., and Fitzgerald, G. F. (1982).In “Microbiology 1982” (D. Schlessinger, ed.), pp. 213216. American Society for Microbiology, Washington, D.C. Daniell, S. D., and Sandine, W. E. (1981).J . Dairy Sci. 64, 407-415. Davies, F. L., and Gasson, M. J. (1981).J . Dairy Res. 48, 363-376. Davies, F. L., and Gasson, M. J . (1983). Zr. J . Food Sci. Technol. 7, 49-60.
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de Vos, W. M., Underwood, H. M., and Davies, F. L. (1984). FEMS Microbwl. k t t e r s . In press. Fitzgerald, G . F., Daly, C., Brown, L. R., and Gingeras, T. R. (1982). Nucl. Acids Res. 10, 8171-8179. Galesloot, TH. E., Hassing, F., and Stadhouders, J. (1966). Znt. Dairy Congr. Proc. 17th, 491498. Gasson, M. J. (1983). Antonie oan Leeuwenhoek 49, 275-282. Gasson, M. J., and Davies, F. L. (1980). Appl. Enoiron. Microbiol. 40, 964-966. Georghiou, D., Phua, S. H., and Terzaghi, E. (1981). J . Gen. Microbiol. 122, 295-303. Gilliland, S. E., and Speck, M. L. (1974). J. Milk Food Technol. 37, 107-111. Graham, D. M., Parmelee, C. E., and Nelson, F. E. (1952).J. Dairy Sci. 35, 813-822. Hargrove, R. E., McDonough, F. E., and Tittsler, R. P. (1961).J . Dairy Sci. 44, 1799-1810. Heap, H. A., and Jarvis, A. W. (1980). N . 2.1. Dairy Sci. Technol. 15, 75-81. Heap, H. A., and Lawrence, R. C. (1976). N . Z. J. Dairy Sci. Technol. 11, 16-20. Henning, D. R., Black, C. H., Sandine, W. E., and Elliker, P. R. (1968a).J. Dairy Sci. 51,1621. Henning, D. R., Sandine, W. E., and Elliker, P. R. (1968b).J. Dairy Sci. 51, 345-351. Hirsch, A. (1952). J. Dairy Res. 19, 290-293. Hirst, A., and Stubbs, J. M. (1969). J. Bacteriol. 97, 1466-1479. Huggins, A. R., and Sandine, W. E. (1977). Appl. Enuiron. Microbiol. 33, 184-191. Huggins, A. R., and Sandine, W. E. (1979). J . Dairy Sci. (Suppl.) 62, 70. Hull, R. R. (1977). Aust. J. Dairy Technol. 32, 65-66. Hull, R. R., and Brooke, A. R. (1982). Aust. J . D a i y Technol. 32, 143-146. Hunter, G. J. E. (1943). J . Dairy Res. 13, 136-145. Hunter, G. J. E. (1946). J. Hyg. 44, 264-270. Hunter, G. J. E. (1947). J. Hyg. 45, 307-312. Hunter, G. J. E. (1949). J . Dairy Res. 16, 374-376. Jarvis, A. W. (1978). A p p l . Enoiron. Microbiol. 36, 785-789. Jarvis, A. W. (1981). N . Z. J. D a i y Sci. Technol. 51, 25-31. Jarvis, A. W. (1984a). A p p l . Enoiron. Microbiol. 47, 343-349. Jarvis, A. W. (1984b). A p p l . Enuiron. Microbiol. 47, 1031-1038. Kempler, G. M., and McKay, L. L. (1981). J . Dairy Sci. 64, 1527-1539. Keogh, 8. P. (1973).J . Dairy Res. 40, 303-309. Keogh, B. P. (1980). A p p l . Enoiron. Microbiol. 40, 798-802. Keogh, B. P., and Pettingill, G. (1983). A p p l . Enoiron. Microbiol. 45,1946-1948. Keogh, B. P., and Shimmin, P. D. (1969).J . D a i y . Res. 36, 87-93. Keogh, B. P., and Shimmin, P. D. (1974). Appl. Microbiol. 27, 411-415. King, W. R., Collins, E. B., and Barrett, E. L. (1983). A p p l . Enoiron. Microbiol. 45, 14811485. Kozak, W., Rajchert-Trzpil, M., Zajdel, J., and Dobrzanski, W. T. (1973). A p p l . Microbiol. 25, 305-308. Kruger, D. H., and Bickle, T. A. (1983). Microbiol. Reu. 47, 345-360. Kruger, D. H., Hasen, S., and Schroeder, C. (1980). Virology 102, 444-446. Lawrence, R. C. (1978). N . 2.1. Dairy Sci. Technol. 13, 129-136. Lawrence, R. C., and Thomas, T. D. (1979). In “Microbial Technology: Current State, Future Prospects” (A. T. Bull, D. C. Ellwood, and R. Ratledge, eds.), Vol. 29, pp. 187-219. Cambridge University Press, Cambridge. Lawrence, R. C., Thomas, T. D., and Terzaghi, B. E. (1976). J . Dairy Res. 43, 141-193. Lawrence, R. C., Heap, H. A., Limsowtin, G., and Jarvis, A. W. (1978).J . Dairy Sci. 61,11811191.
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Lemhke, I . , and Teuber, M. (1981). Milchwissenschaft 36, 10-12. Lemhke, J. Krusch, U., Lompe, A., andTeuber, M. (1980). Zentralbl. Bakteriol. Ilyg. I . Abt. Orig. C1, 79-91. Limsowtin, G. K. Y., and Terzaghi, B. E. (1976). N. Z. J. Dairy Sci. Technol. 11, 251-266. Limsowtin, G . K. Y., and Terzaghi, B. E. (1977). N . Z . 1. Dairy Sci. Technol. 12, 22-28. Limsowtin, G. K. Y., Heap, H. A., and Lawrence, R. C. (1977).N. Z. J. Dairy Sci. Teclinol. 12, 101-106. Limsowtin. G . K. Y., Heap, H. A., and Lawrence, R. C. (1978). N. Z. J . Dairy Sci. Techno/. 13, 1-8. Lowrie, R. J. (1974).Appl. Microbiol. 27, 210-217. Lowrie, R. J., and Pearce, L. E. (1971). N. Z. J . Dairy Sci. Technol. 6 , 166-171. McKay, L. L. (1982). In “Developments in Food Microbiology”(R. Uavies, ed.), pp. 153-182. Applied Science, London. McKay, L. L. (1983). Antonie van Leeuwenhoek 49, 259-274. McKay, L. L., and Baldwin, K. A. (1973). Appl. Microbiol. 25, 682-684. McKay, L. L., and Baldwin, K. A. (1974).A p p l . Microbiol. 28, 342-346. McKay, L. L., and Baldwin, K. A. (1978).Appl. Environ. Microbiol. 36, 360-367. McKay, L. L., and Baldwin, K. A. (1984).A p p l . Enoiron. Microbiol. 47, 68-74. McKay, L. L., Cords, B. R., and Baldwin, K. A. (1973).J. Bacteriol. 115, 810-813. McKay, L. L., Baldwin, K. A., and Efstathiou, J. D. (1976). Appl. Enuiron. Microbiol. 32, 4552. McKay, L. L., Baldwin, K. A., and Walsh, P. M. (1980).Appl. Enuiron. Microbiol. 40,84-91. Marshall, R. J., and Berridge, N. J. (1976).J. Dairy Res. 43, 449-458. Meister, K. A., and Ledford, R. A. (1979).J. Food Prot. 42,396-400. Miillan, M. A., Daly, C., and Fox, P. (1981). J. Dairy Res. 48, 465-471. Nichols, A. A., and Hoyle, M. (1949).J. Dairy Res. 16, 167-208. Ogata, S. (1980). Biotechnol. Bioeng. 22, 177-193. Oram, J. D. (1971).J. Gen. Virol. 13, 59-71. Oram, J. D.,and Reiter, B. (1968).J. Gen. Virol. 3, 103-119. Park, C., and McKay, L. L. (1975).J . Milk Food Technol. 38, 594-597. Pearce, L. E. (1978). N. 2. J. Dairy Sci. Technol. 13, 166-171. Pearce, L. E., Limsowtin, G. K. Y., and Crawford, A.M. (1970). N. Z . /. Dairy Sci. Technol. 5, 145-149. Poruhcdn, R. S., and S e h s , R. L. (1979). In “Microbial Technology” (H. J. Peppler and D. Perlman, eds.), 2nd ed., Vol. 1, pp. 59-92. Academic Press, New York. Potter, N. (1970). J . Dairy Sci. 53, 1358-1362. Potter, N. N., and Nelson, F. E. (1952). J, Bacteriol. 64, 113-119. Reanney, D. (1976). Bacteriol. Reo. 40, 552-596. Reiter, 8. (1949). Nature (London)164, 667-668. Reiter, B., and Moller-Madsen, A . (1963). J . Dairy Res. 30, 419-455. Reyrolle, J. Chopin, M.-C., Letellier, F., and Novel, G . (1982).Appl. Environ. Microbiol. 43, 359-356. Richardson, G. H., Cheng, C. T., and Young, R. (1976).J. Dairy Sci. 60, 378-386. Richardson, G . H.,Hong, G. L., and Ernstrom, C. A. (1980).J . Dairy Sci. 63, 1981-1986. Sanders, M. E., and Klaenhammer, T. R. (1980). Appl. Enuiron. Microbiol. 40, 500-506. Sanders, M. E., and Klaenhammer, T. R. (1981). Appl. Enuiron. Microbiol. 42, 944-950. Sanders, M. E.,and Klaenhammer, T. R. (1983). AppZ. Environ. Microbid. 46, 1125-1133. Sanders, M. E., and Klaenhammer, T. R. (1984). A p p i . Enuiron. Microbiol. 47, 979-985. Sandine, W. E. (1977). j . Dairy Sci. 60, 822-827. Sandine, W. E., and Ayres, J. W. (1981). U.S. Patent No. 4,282,255.
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Sandine, W. E., Elliker, P. R., Allen, L. K., and Brown, W. C. (1962).J . Dairy Sci. 45,12661271. Sandine, W. E., Radich, P. C., and Elliker, P. R. (1972). J . Milk Food Technol. 35, 176-185. Shimizu-Kadota, M., and Sakurai, T. (1982). Appl. Enuiron. Microbiol. 43, 1284-1287. Shimizu-Kadota, M., Sakurai, T., and Tsuchida, N. (1983). Appl. Enoiron. Microbiol. 45,669674. Sinha, R. P. (1980). A p p l . Enuiron. Microbiol. 40, 326-332. Snook, R. J., McKay, L. L., and Ahlstrand, G . G. (1981). Appl. Enoiron. Microbiol. 42, 897903. Sozzi, T., Poulin, J. M., and Maret, R. (1978).J. Dairy Res. 45, 259-265. Sozzi, T., Bauer, H., Maret, R., and Dentan, E. (1980). Milchwissenschaft 35, 17-20. Stadhouders, J. (1975). Neth. Milk Dairy J . 29, 104-126. Terzaghi, B. E. (1976). N. Z. J . Dairy Sci. Technol. 11, 155-163. Terzaghi, B. E., and Sandine, W. E. (1975). Appl. Microbiol. 29, 807-813. Terzaghi, B. E., and Sandine, W. E. (1981).J. Gen. Microbiol. 122, 305-311. Terzaghi, E. A., and Terzaghi, B. E. (1978). Appl. Enuiron. Microbiol. 35, 471-478. Teuber, M., and Lembke, J. (1983). Antonie oan Leeuwenhoek 49, 283-295. Thomas, T. D., and Lowrie, R. J. (1975).J. Milk Food Technol. 38, 275-278. Thunell, R. K., Sandine, W. E., and Bodyfelt, F. W. (1981). J . Dairy Sci. 64, 2270-2277. Tsaneva, K. P. (1976). A p p l . Enuiron. Microbiol. 31, 590-601. Whitehead, H. R. (1953). Bacteriol. Reo. 17, 109-123. Whitehead, H. R., and Cox, G. A. (1936).J . Dairy Res. 7, 55-62. Wigley, R. C. (1977).J. SOC.Dairy Technol. 30, 45-51. Wigley, R. C. (1980). J . Soc. Dairy Technol. 33, 24-30. Wright, S. L., and Richardson, G. H. (1982).J. Dairy Sci. 65, 1882-1889. Yang, N. L., and Sandine, W. E. (1979).J. Dairy Sci. 62, 908-915. Zehren, V. L., and Whitehead, H. R. (1954). J . Dairy Sci. 37, 209-219. Zottola, E. A., and Marth, E. H. (1966). J . Dairy Sci. 49, 1343.
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Microbial Metabolism of Polycyclic Aromatic Hydrocarbons CARLE. CERNIGLIA National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas
I. Introduction
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31
11. General Pathways of Mammalian Polycyclic Aromatic
Hydrocarbon Metabolism
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33
111. General Pathways of Microbial Polycyclic Aromatic
Hydrocarbon Metabolism
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IV.
V. VI. VII. VIII. Alkyl-Substituted Benz[a]anthracene A. Mono- and Dimethylbenz[a]anthr B. 3-Methylcholanthrene IX. Nitro-Substituted Polycycli X. Nature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... ....... XI. Conclusions and Comments . . . . . References ...........................................
62 64 65
1. Introduction During the last three centuries, a relationship between the higher incidence of cancer in urban and industrial areas than in rural areas and the exposure of humans to polycyclic aromatic hydrocarbons (PAHs) has prompted considerable research on the sources, occurrence, bioaccumulation, metabolism, and disposition of these pollutants in aquatic and terrestrial ecosystems. Historically, physician John Hill in 1761 recognized this relationship and documented the high incidence of nasal cancer as a consequence of excessive use of tobacco snuff (Redmond, 1970). Percival Pott (1775) similarly noted such a relationship in his report on the high rate of scrota1 skin cancer in chimney sweeps which was due to compounds contained in the soot. About 140 years later, Yamagiwa and Ichikawa (1915) induced tumors on the ears of rabbits by repeated application of coal tar. Studies by Kennaway and Hieger (1930), Cook et al. (1933), and Kennaway (1955)established that the carcinogenic fractions of coal tar contained PAHs. PAHs are compounds containing carbon and hydrogen with fused benzene rings in linear, angular, and cluster arrangements (Fig. 1).PAHs may 31 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 30 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-002630-9
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CARL E. CERNIGLIA
7 6 & & : 5
6 7@:
5
4
Naphthalene (inactive)
10
4
Anthracene (inactive)
8:
6o 7
8
0 1 0 9
Phenanthrene (inactive)
8
7
6
Benz[a]anthracene (inactive) 7
3-Methylcholanthrene (active)
FIG
7
6
5
Benzo [alpyrene (active)
CH3
7-Methylbenz[a]anthracene (active)
7,12-Dirnethylben z [a] ant h rac e ne (active)
The structures of polycyclic aromatic hydrocarbons and their bic.Jgical activity.
also contain alkyl and nitro substituents or can be considered heterocyclic molecules with the substitution of an aromatic ring carbon with nitrogen, oxygen, or sulfur. PAHs are universal products of the combustion of organic matter. They are formed during the combustion of a wide variety of materials such as fossil fuels, saturated and unsaturated hydrocarbons, peptides, and carbohydrates. Several theories on PAH pyrosynthesis have been proposed and extensive reviews on this subject are available (Blumer, 1976; Badger et al., 1958, 1964). PAHs and their alkyl homologs are widely distributed in soils and aquatic environments (Andelman and Snodgrass, 1974; Harrison et al., 1976). It has been estimated that 1300 tons of benzo[a]pyrene is emitted into the United States' atmosphere each year (National Academy of Science Reports USA, 1972). Occurrence of PAHs in the environment is due to both natural and anthropogenic processes (Blumer, 1976). Some major sources of PAHs are forest and prairie grass fires, volcanic ash, heat and power generation, refuse burning, motor vehicle emissions,
POLYCYCLIC AROMATIC HYDROCARBONS
33
industrial processes, petroleum leakage and spills, fallout from urban air pollution, coal liquefaction and gasification processes, and cigarette smoke. This review deals with the microbial transformation of PAHs. The similarities and differences between the microbial and mammalian metabolism of these ubiquitous environmental carcinogens are described. The structures of some of the PAHs discussed in this article are shown in Fig. 1. Various aspects of PAH metabolism by microorganisms have appeared in earlier reviews (Dagley, 1971, 1975; Chapman, 1972, 1979; Gibson, 1971, 1977; Fewson, 1981; Cerniglia, 1981; Hou, 1982).
11. General Pathways of Mammalian Polycyclic Aromatic Hydrocarbon Metabolism The metabolism of PAHs in mammals by microsomal enzymes has been demonstrated in many tissues such as liver, lung, kidney, placenta, intestinal tract, and skin. The highest activity is usually found in hepatic endoplasmic reticulum preparations which convert lipophilic compounds to metabolites that are water soluble and thus easily excreted from the body (Gillette, 1967; Conney, 1967). The level of enzyme activity may be affected by numerous factors such as the age, sex, and hormonal status of the experimental animal. As a result of the pioneering research of Elizabeth C. and James A. Miller at the University of Wisconsin, it has been generally accepted that many carcinogens including PAHs are in fact inactive per se and must be metabolically activated by mammalian microsomal enzymes to elicit their mutagenic, genotoxic, and carcinogenic properties (Miller and Miller, 1973, 1976). In the case of PAHs, initial monooxygenation of the aromatic nucleus to form an arene oxide is the key step in the activation of these compounds (Jerina and Daly, 1974). This oxidative reaction is catalyzed by the membrane-bound cytochrome P-450-containing monooxygenase enzyme system. This xenobiotic-metabolizing enzyme exists in multiple molecular forms with different, but in some cases overlapping, substrate specificities (Lu, 1979). The arene oxide which is formed can undergo further metabolism, such as enzyme-catalyzed hydration by epoxide hydrolase to yield trans-dihydrodiols (Oesch, 1973). These non-K-region transdihydrodiols can be further oxygenated via the cytochrome P-450 enzyme system to yield dihydrodiol epoxides (Sims and Grover, 1981). The toxicological significance of this metabolic pathway is illustrated below for the mammalian metabolism of benzo[a]pyrene. The arene oxide can also undergo nonenzymatic rearrangement to phenols by a mechanism termed the “NIH shift”; the migration of deuterium, tritium, or aryl substitutents from the site of hydroxylation to an adjacent carbon atom has also been termed the “NIH shift” (Daly et al., 1972, Guroff et al., 1967). These oxygenated inter-
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reductare
Glutathlona transferare
’
covalent bindins to DNA. R N A
Q-Sulfates
-O-Glucuronides
FIG.2. Major pathways of mammalian metabolism of polycyclic aromatic hydrocarbons.
mediates when conjugated with glutathione, sulfate, or glucuronic acid also form important pathways in the disposition of PAHs in mammals (Aitio, 1978). Reduction of the arene oxide back to the parent hydrocarbon has also been demonstrated. Besides the cytochrome P-450 monooxygenase enzyme system, other enzymes of PAH metabolism include epoxide reductase, epoxide hydrolase, glutathione transferase, UDP-glucuronosyltransferase, and sulfotransferase. Many of the enzymatic reactions that occur in the liver convert hydrophobic substrates to more polar oxygenated products, facilitating excretion from the cell and thus detoxification of these lipophilic compounds. However, some of the metabolites are very electrophilic and bind to nucleophilic sites in proteins and nucleic acids. This covalent binding to informational macromolecules is thought to be a necessary requirement for the initiation of malignant transformations. A scheme illustrating the activation and detoxification of aromatic hydrocarbons is shown in Fig. 2. This balance between activation and detoxification enzymatic pathways is important in determining how much of the reactive metabolites will be formed and will be available for binding to cellular macromolecules. A number of comprehensive reviews on the mechanisms of chemical carcinogenesis have been written (Heidelberger, 1975; Gillette et al., 1975; Miller and Miller, 1976).
111. General Pathways of Microbial Polycyclic Aromatic Hydrocarbon Metabolism Bacteria, fungi, and algae play an important role in the metabolism of PAHs in terrestrial and aquatic environments (Tables 1-111). Until recently
POLYCYCLIC AROMATIC HYDROCARBONS
35
TABLE I OXIDATION OF AROMATIC HYDROCARBONS BY DIFFERENT SPECIES O F BACTERIA Organism Pseudomonas putida P . putida (391D) P . putida (biotype B) P . putida (arvilla) mt-2 P. putida (119) P . aeruginosa
Reference Jeffrey et al. (1975); Axcell and Geary (1975) Gibson et al. (1970a,b) Jeffrey et al. (1975) Worsey and Williams (1975) Jeffrey et al. (1975) Kitagawa (1956); Marr and Stone (1961); Wu and Wong (1981) Jeffrey et al. (1975) Trecanni et al. (1954) Marr and Stone (1961) Nozaka and Kusunose (1968, 1969) Jeffrey et al. (1975) Trecanni et al. (1954) Trecanni et al. (1954) Kiyohara and Nagao (1978) Hogn and Jaenicke (1972) Gibson et al. (1975); Akhtar et al. (1975) Colla et al. (1959) Claus and Walker (1964) Trecanni et al. (1954) Dua and Meera (1981)
it was thought that bacteria initially oxidize aromatic hydrocarbons to transdihydrodiols (Walker and Wiltshire, 1953; Treccani et al., 1954; Marr and Stone, 1961). The assignment of a trans configuration for the dihydrodiols was based on their similarities to metabolites formed in previous studies of aromatic hydrocarbon metabolism in mammals. In 1968, Gibson and coworkers reported the isolation of a strain of Pseudomonas putida which could utilize ethylbenzene as the sole carbon and energy source. This organism could also grow on benzene and toluene. Subsequent experiments by Gibson et al. (1970a) showed that P. putida 39/D, a mutant strain of the wildtype benzene-oxidizing organism, when grown on glucose in the presence of benzene, accumulated cis-l,2-dihydroxy-l,2-dihydrobenzene. Oxygen-18 experiments showed that both oxygen atoms of the cis-benzene dihydrodiol were derived from molecular oxygen. The precise determination of a cis configuration for the benzene dihydrodiol and the careful measurement of 180, incorporation by David T. Gibson and colleagues laid the foundation for some general principles in the oxidative metabolism of aromatic hydrocarbons. First, bacteria initially oxidize aromatic hydrocarbons that range in size from benzene to benzo[a]pyrene to cis-dihydrodiols (Table IV). Second,
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CARL E. CERNIGLIA
TABLE I1 OXIDATION OF
AROMATIC HYPROCAHBONS BY DIFFERENT
Mastigomycota Chytridomycetes Phlyctochytrium reinboldtae (43-3) Rhizoph1yctf.s rosea (63-la) R . harderi (ATCC 63-2) Oornycetes Saprokgniu purasitica (ATCC 22284) Phytophthora cinnamoni (ATCC 16984) Thraustochytrium sp. (70-2E) Hyphochytrium cutenoides (75-l8b) Zygomycota Zygwnycetes Cunningham& ebgans (ATCC 36112) C. ebgans (NRRL 1392) C. ekguns (NRRL 1393) C. elegans (ATCC 9245) C. echinulata C . echinuluta (ATCC 9244) C. echinulata (NRRL 3655) C.echinulata (NRRL 1386) C. japonica C . blakesleeana (ATCC 8688a) C . blakesleeana (NRRL 1369) C. bainieri (ATCC 9244) C . bainieri Syncephalastrum sp. (UT-70) S. racemosum (UT 211a) Mucor sp. M. heimalis (UT 70-5) Cilbertella persicaria (NRRL 2357) Absidia sp. A. glauca (UT 5a) A. glauca (NRRL 1324) A. ranwsa (NRRL 1332) A. spinosa (NRHL 1347) A. pseudocylindrospora (NRRL 2770) Zygorhynchus nwelleri (UT 193) Cokeromyces poitrassi (NSF 402) Choanephora cumpincta (UT 66-54) Phycomyces blakesbeanus (UT 71-101) Circincllu sp. (UT 70-45) Thamnidium anamoltrm (UT 66-34)
SPECIES OF
FUNGI*
Rhizopus arrhizus R. stolonijer (NRRL 1477) Basidiobolus ranarum (ATCC 14449) Conidiobolus goninwdes (ATCC 14445) Tricomycetes Smittium culisetae (COC-18-3) S. simulii (JAP-51-1) S. culicis (WYO-51-11) Ascomycota Sacchurornyces cerevisiae Emericellopsis Neurospora crassa Sordaria jlmicola Clauiceps paspali Candida lipolytica (37-1) C. lipolytica (78-003) C . multosa (R-42) C . tropicalis (CBS 6947) C . guilliennondii (78-006) Deba yomyces hansenii (CBS 767) Basidiomycota Psilocybe strictipes P. subaeruginascens P. cubensis P. stuntzii Panaeolus subbalteatus P . canabodginensis Deuteromycota Aspergillus niger A. niger (ATCC 9142) A . niger (UTX 172) A. niger (NRRL 599) A . ochraceous (ATCC 1008) A. ochraceous (TS) Penicillium notatum P. chrysogenum P. ochro-chloron (ATCC 36110) Curnularia lunata Gliocladium sp. Epicoccum nigrum Pestalotia sp. Helicostylum pirifonne (QM 6945)
Cerniglia et al. (1978); Cerniglia and Crow (1981); Dodge et al. (1979);Smith and Rosazza (1974); Smith et al. (1980); Schwartz et al. (1980); Woods and Wiseman (1979, 1980); Lin and Kapoor (1979); Wong et al. (1983); Ghosh et al. (1983); Wu arid Wong (1981).
POLYCYCLIC AROMATIC HYDROCARBONS
37
TABLE I11 OXIDATION OF AROMATIC HYDROCARBONS DIFFERENT SPECIESOF CYANOBACTERIA AND MICROALGAE~
cis-dihydrodiols are formed by the incorporation of both atoms of molecular oxygen into the aromatic nucleus, The dioxygenase that catalyzes these initial reactions is a multicomponent enzyme system; the terminal oxygenase is an iron-sulfur protein (Axcell and Geary, 1975; Yeh et al., 1977; Crutcher and Geary, 1979; Subramanian et al., 1979, 1981; Ensley et al., 1982; Ensley and Gibson, 1983). Third, cis-dihydrodiols are rearomatized through a cis-dihydrodiol dehydrogenase to yield a dihydroxylated derivative (Pate1 and Gibson, 1974). Fourth, further oxidation of cis-dihydrodiols leads to the formation of catechols that are substrates for other dioxygenases that bring about enzymatic cleavage of the aromatic ring (Fig. 3). Catechol can be oxidized via the ortho pathway, which involves cleavage of the bond between carbon atoms of the two hydroxyl groups to yield &,cis-muconic acid,
Gibson et al. (1975) Dodge and Gibson (1980); Cerniglia et al. (1980a); Fu et al. (1983) Gibson et al. (1975) Dodge and Gibson (1980); Cerniglia et al. (1980a); Fu et al. (1983) Gibson et al. (1975)
FIG. 3. The pathways utilized by prokaryotic and eukaryotic microorganisms for the oxidation of polycyclic aromatic hydrocarbons.
or via the meta pathway, which involves cleavage of the bond between a carbon atom with a hydroxyl group and the adjacent carbon atom with a hydroxyl group. These ring fission pathways have been elegantly reviewed by Dagley (1971) and Chapman (1972). In contrast to bacteria, fungi oxidize PAHs via cytochrome P-450monooxygenase and epoxide hydrolase-catalyzed reactions to truns-dihydrodiols (Cerniglia, 1981) (Table IV). These reactions appear to be similar to those reported for mammalian enzyme systems (Ferris et d.,1973; Smith and Rosazza, 1974; Rosazza and Smith, 1979) (Fig. 3). Although there have not been any reports on the ability of fungi to utilize aromatic hydrocarbons as the sole source of carbon and energy, several studies have shown that a wide taxonomic and phylogenetic spectrum of fungi (Table 11) has the enzymatic capacity to oxidize PAHs when grown on an alternative carbon source. The products are nontoxic metabolites as well as compounds that have been implicated as biologically active forms of PAHs in higher organisms. It seems that fungi hydroxylate aromatic hydrocarbons as a prelude to detoxification, whereas bacteria oxidize aromatic hydrocarbons to dihydroxylated compounds as a prelude to ring fission and assimilation (Dagley, 1981). Recent studies with prokaryotic and eukaryotic algae have indicated that these pho-
POLYCYCLIC AROMATIC HYDROCARBONS
41
tosynthetic microorganisms can hydroxylate aromatic compounds such as naphthalene, methylnaphthalene, phenanthrene, biphenyl, and aniline (Table 111).Cyanobacteria (blue-green algae) are generally viewed as the first group of organisms to have developed the capacity for an oxygen-evolving photosynthesis (Schopf, 1975). This suggests that the first oxygen-requiring enzymes capable of the hydroxylation of an aromatic ring evolved in an ancestral cyanobacterium and that cyanobacteria may play an important role in the degradation of aromatic hydrocarbons in aquatic environments. These general principles are examined in some detail in this review of the microbial metabolism of naphthalene, phenanthrene, anthracene, benzo[a]pyrene, benz[a]anthracene, and related methyl- and nitro-substituted PAHs.
IV. Naphthalene Naphthalene and alkyl-substituted naphthalene are among the most toxic components in the water-soluble fraction of crude and fuel oils (Boylan and Tripp, 1971; Lee et al., 1974; Winters et al., 1976). Following the observations of Tattersfield (1927) and Tausson (1927), there have been numerous reports on the ability of bacteria to utilize naphthalene as the sole source of carbon (Gray and Thornton, 1928; Walker and Wiltshire, 1953; Trecanni et al., 1954; Davies and Evans, 1964). The metabolic sequence and enzymatic reactions leading to the degradation of naphthalene were first presented by Davies and Evans in 1964. More recent studies have indicated that bacteria initially oxidize naphthalene by incorporating both atoms of molecular oxygen into the aromatic molecule to form cis-1,2-dihydroxy-l,2-dihydronaphthalene (Catterall et al., 1971; Jerina et al., 1971) (Fig. 4). The absolute stereochemistry of the naphthalene cis-dihydrodiol formed by P . putida 119 was determined by Jeffrey et al. (1975) and established as (+)-(lR,2S).Ensley et al. (1982) and Ensley and Gibson (1983) have characterized the naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816. This enzyme system catalyzes the incorporation of both atoms of molecular oxygen into the aromatic nucleus to form (+)-cis-(lR,2S)-dihydroxy-1,2-dihydronaphthalene. Naphthalene dioxygenase consists of three protein components which are essential for naphthalene cis-dihydrodiol formation. This multicomponent property is similar to those reported for the benzene (Axcell and Geary, 1975; Crutcher and Geary, 1979) and toluene (Yeh et al., 1977; Subramanian et al., 1979, 1981) dioxygenase which consists of a flavoprotein, a two-iron, two-sulfur ferredoxin, and an iron-sulfur protein (Fig. 5). Naphthalene dioxygenase is very unstable, and rapid purification in the presence of dithiothreitol, 10% (v/v) ethanol, and 10%(v/v) glycerol in Trishydrochloride buffer is necessary for enzymatic activity (Ensley et al., 1982).
42
CARL E . CEHNIGLIA
a,:
6 7@:
5
4
Naphthalene
cis-1,2-Dhydroxy-l,2-dihydronaphthalene I
c&-o-Hydroxybenzalpyruvk
acid
coon
@OH
Salicylic acid
I @ noJ
OH
Catechoi
I
Ring Cleavage
FIG. 4. The pathway for the bacterial oxidation of naphthalene.
The terminal dioxygenase has a molecular weight of 158,000and is composed of two subunits which were established by SDS gel electrophoresis to be 55,000 and 20,000. The native and subunit molecular weights of the terminal oxygenase component are similar to those reported for the toluene dioxygenase (Subramanian et al., 1979). The subunit molecular weights are indicative of an azPzquaternary structure (Ensley and Gibson, 1983). The terminal naphthalene dioxygenase component is an iron-sulfur protein which contains 6 g-atoms of iron and 4 g-atoms of acid-labile sulfur per mole of the purified enzyme. In the presence of oxygen, NADH, and two other components of the naphthalene dioxygenase system, the terminal oxygenase forms
naphthalene-cis-dihydrodiol. A naphthalene oxygenase has also been purified from cells of Corynebacteriurnrenale grown on naphthalene as the sole source of carbon and energy (Dua and Meera, 1981). The enzyme has a molecular weight of approximately 99,000 and formed cis-1,2-dihydroxy-l,2-dihydronaphthalene as the predominant metabolite. The second step in the bacterial oxidation of naphthalene is the conversion to 1,2-dihydroxynaphthalene. of cis-1,2-dihydroxy-l,2-dihydronaphthalene This reaction is catalyzed by naphthalene-( +)-cis-dihydrodiol dehydrogenase and requires nicotinamide adenine dinucleotide as an electron acceptor (Patel and Gibson, 1974). The second enzyme in the naphthalene degradative pathway has a molecular weight of 102,000 and consists of four subunits of 25,500. Naphthalene-cis-dihydrodioldehydrogenase is highly stereoselective for the (+)-isomer of cis-l,2-dihydroxy-l,2-dihydronaphthaleneand (Patel and cannot metabolize trans-1,2-dihydroxy-1,2-dihydronaphthalene Gibson, 1974). Davies and Evans (1964) showed that 1,2-dihydroxynaphthalene was enzymatically cleaved by a dioxygenase from a Pseudomonus sp. to yield cis-2'-hydroxybenzalpyruvate which is then converted via a series of dioxygenases to salicylate and pyruvate. Salicylate is subsequently oxidized by salicylate hydroxylase to yield catechol, which can undergo either ortho or meta fission depending upon the bacterial species (Dagley, 1971). The proposed pathway for the bacterial oxidation of naphthalene is shown in Fig. 4. Evidence for a similar pathway for naphthalene metabolism by an Aeromonas sp. was reported by Kiyohara and Nagao (1978). Barnsley (1976) showed that Pseudomonas NCIB 9816 and other pseudomonads oxidized 1,2-dihydroxynaphthalene to 2-hydroxychromene-2-carboxylate which is enzymatically converted by an isomerase to cis-2'-hydroxybenzalpyruvate. Numerous investigations have indicated that the genes that code for naphthalene oxidation in pseudomonads are found on plasmids (Boronin et al., 1980; Cane and Williams, 1982; Connors and Barnsley, 1982; Yen and
44
CARL E. CERNIGLIA
6 7&: 5
4
Naphthalene
O2 &H
I
Cytochrome
P-450 H20
H
OH
Epoxide Hydrolase
Naphthalene- 1.2- oxide
9-Glucuronide
J.'
trans- 1,l-Oihydroxy- 1.2-dihydronaphthalene
OH
g-sulfate l-Naphthol (95%) 2-Naphthol (5%)
1
& H
OH
4-Hydroxy- 1-tetralone
FIG.6. The pathway for the fungal oxidation of naphthalene.
Gunsalus, 1982). The genes encoding the enzymes responsible for naphthalene degradation from P. putidu have recently been cloned and expressed in Escherichia coli (Schell, 1983; Ensley et al., 1983). Several studies have shown that a wide taxonomic and phylogenetic spectrum of fungi can metabolize naphthalene (Ferris et al., 1973; Smith and Rosazza, 1974; Cerniglia et al., 1978). Interestingly, these studies on the fungal metabolism of naphthalene have indicated different reactions in the enzymatic oxygenation of naphthalene from that reported for bacterial enzyme systems. In contrast to bacteria, fungi incorporate only one atom of molecular oxygen into naphthalene via a cytochrome-P-450 monooxygenase to form naphthalene-1,2-oxide (Fig. 6). This arene oxide is very unstable and can undergo other reactions such as (1) rearrangement to form l-naphthol (major) and 2-naphthol (minor) via the NIH shift mechanism and (2) enzymatic hydration catalyzed by cpoxide hydrolase to form (+)trans-(1S,2S)-
POLYCYCLIC AROMATIC HYDROCARBONS
45
dihydroxy-l,2-dihydronaphthalene(Cerniglia and Gibson, 1977, 1978; Cerniglia et al., 1983a). A trans-relative stereochemistry for the naphthalene dihydrodiol, occurrence of the NIH shift, oxygen-18 incorporation experiments, and the ratio of 1-naphthol to 2-naphthol suggested the prior formation of naphthalene-1,2-oxide as the initial oxidation product in the fungal metabolism of naphthalene (Cerniglia et al., 1983a). Microsomal preparations from the fungus Cunninghamella elegans oxidized naphthalene to trans-l,2-dihydroxy-1,2-dihydronaphthalene and 1-naphthol. From the observed requirements for oxygen and NADPH and from the detection of low levels of cytochrome P-450,results indicated a monooxygenase-catalyzed reaction (Cerniglia and Gibson, 1978). Although these reactions are similar to those reported for mammalian enzyme systems (Jerina et al., 1968, 1970; Oesch et al., 1971, 1972), the (+)-(1S,2S) absolute stereochemistry of the trans-l,2-dihydroxy-l,2-dihydronaphthaleneas the major enantiomer formed from naphthalene by C . elegans was opposite to the (-)-(lR,2R)dihydrodiol observed in mammalian systems (Jerina et al., 1970). This finding indicated differences in the stereoselectivity of the fungal cytochrome P-450 monooxygenase from similar enzymes purified from liver microsomes of rats, rabbits, pigs, and mice. In addition to the metabolites described above, 4-hydroxy-1-tetralone was produced from C . elegans cultures incubated with naphthalene and 1-naphthol (Cerniglia and Gibson, 1977) (Fig. 6). 4-Hydroxy-1-tetralone may be a common intermediate in the microbial oxidation of 1-naphthol since it has been isolated as an oxidation product of Inaphthol in transformation experiments with fungi (Cerniglia and Gibson, 1977; Cerniglia et al., 1978), bacteria (Bollag et al., 1975), and cyanobacteria (Cerniglia et al., 1980b,c). Fungi also have the ability to form glucuronide and sulfate conjugates of phenolic aromatic hydrocarbons. 1-Naphthyl glucuronic acid and 1-naphthyl sulfate were major water-soluble metabolites formed from the fungal metabolism of naphthalene (Cerniglia et al., 1982b). Since 1-naphthol has been shown to have toxic properties, the conjugation of I-naphthol with sulfate and glucuronic acid suggests that these reactions are important in the detoxification and elimination of xenobiotics in this fungus. A recent study by Wackett and Gibson (1982) has indicated that cell extracts of C . elegans contain UDP-glucuronosyltransferase activity. In contrast to mammalian UDP-glucuronosyltransferases, which are membrane bound, the fungal enzyme activity was located in the 100,000 g supernatant. Prokaryotic and eukaryotic algae grown photoautotrophically in the presence of naphthalene have the ability to oxidize naphthalene (Cerniglia et al., 1980b,c, 1982~).The cyanobacteria (blue-green algae) Agmenellum quadruplicatum strain PR-6and Oscillatoria sp. strain JCM oxidized naphthalene predominantly to 1-naphthol (Cerniglia et al., 1980~).An NIH shift mecha-
46
CARL E. CERNIGLIA
nism was recently implicated for the formation of l-naphthol from naphthalene in cyanobacteria (Narro et al., 1982). These data indicate that these prokaryotic organisms oxidize naphthalene to l-naphthol by a mechanism similar to that described previously for eukaryotic organisms. However, the isolation of cis-1,2-dihydroxy-l,2-dihydronaphthalenefrom cyanobacterial culture filtrates, which is a common intermediate of the bacterial metabolism of naphthalene, suggests multiple pathways for the metabolism of aromatic hydrocarbons in cyanobacteria (Cerniglia et al., 1980~).
V. Anthracene and Phenanthrene A considerable amount of information exists on the microbial metabolism of anthracene and phenanthrene. These tricyclic aromatic hydrocarbons are widely distributed throughout the environment as a result of pyrolytic processes and as minor contaminants in wastewater effluents from coal gasification and liquefaction processes (Blumer, 1976). Anthracene and phenanthrene and their metabolites are not acutely toxic, carcinogenic, or mutagenic; however, these compounds have been used as model substrates in studies on the environmental degradation of PAHs, since both structures are found in carcinogenic PAHs such as benzo[a]pyrene, benz[a]anthracene. and 3-methylcholanthrene (Fig. 1). Phenanthrene is the simplest aromatic hydrocarbon that contains a “bay-region” and a “K-region.” A bay-region occurs in a PAH when an angularly fused benzo ring is present (Jerina et al., 1977, 1978). The bay-region of phenanthrene is the sterically hindered area between carbon atoms 4 and 5 (Fig. 9). Based on quantum mechanical calculations, it has been predicted that dihydrodiol epoxides with the epoxy groups situated at this region are highly chemically and biologically reactive and are suspected to be ultimate carcinogens if they are formed metabolically (Jerina et al., 1978). The K-region of phenanthrene is the 9,lO double bond which is the most olefinic aromatic double bond with high electron density. According to the Schmidt-Pullman electronic theory, Kregion epoxides should be more carcinogenic than the parent hydrocarbon (Pullman and Pullman, 1955). However, recent studies have indicated that the biologically reactive metabolites of PAH might occur at sites other than the K-region. Phenanthrene is metabolized by rat hepatic microsomes and by highly purified cytochrome P-450 monooxygenase preparations to phenanthrene trans-l,&, -3,4-, and -9,lO-dihydrodiols as well as phenolic products (Boyland and Sims, 1962a,b; Sims, 1962, 1970; Chaturapit and Holder, 1978). Phenanthrene trans8,lO-dihydrodiol is the major metabolite. The lack of carcinogenicity and the low biological activity of phenanthrene are consistent with the observation that the trans-l,2-, -3,4-, and -9,lO-dihydrodiols
6 7&: 5
10
4
Anthracene
cis- 1,P-Dihydroxy- 1,2-dihydroanthracene
1.2-Dihydroxyanthracene
4 COOH
&-44
COOH
2’-Hydroxynaphth-3-y1)2-oxobut-enoic acid
COOH
2-Hydroxynaphthoic acid
2.3-Dihydrox ynaphthalane
@OH
COOH
Salicylic acid
Catechol
FIG.7. The pathway for the bacterial oxidation of anthracene.
48
CARL E. CERNIGLIA
as well as the bay region 1,2-dihydrodiol-3,4-epoxideformed are weakly mutagenic in Salmonella typhimurium strains TA 98 and 100 and in V79 Chinese hamster cells and inactive as tumor initiators on mouse skin (Bucker et al., 1979; Buening et al., 1979; Wood et al., 1979). Pure cultures of bacteria and microbial populations isolated from freshwater and marine environments have the ability to metabolize anthracene and phenanthrene as the sole source of carbon (Rogoff and Wender, 1957a,b; Colla et al., 1959; Evans et aZ., 1965; Akhtar et al., 1975; Jerina et al., 1976; Herbes and Schwall, 1978; Koreeda et al., 1978; Sherill and Sayler, 1980). Anthracene can be completely mineralized by soil pseudomonads with the initial oxygenated intermediate being a dihydrodiol (Evans et al., 1965; Akhtar et al., 1975; Jerina et al., 1976). Beijerinckia sp. B-836 and Pseudomonas putida 119, mutant strains deficient in dihydrodiol dehydrogenase activity, oxidize anthracene in the 1,2 positions to form (+)-cis-1R,2S-dihydroxy-1,2-dihydroanthracene (Akhtar et al., 1975; Jerina et al., 1976) (Fig. 7). Pseudomonas strains and a Nocardia strain oxidize (+)-cis-1,2-dihydroxy-1,2-dihydroanthracene via an NAD dependent dihydrodiol dehydrogenase to 1,2-dihydroxyanthracene (Pate1 and Gibson, 1974). Evans et al. (1965) have shown that cell extracts prepared from various Pseudomonas strains oxidized 1,Z-dihydroxyanthracene to the ring fission product cis-4-(2-hydroxynaphth-3-yl)-2-oxobut-3-enoic acid with subsequent conversion to 2-hydroxy-3-naphthoic acid (Fig. 7). This ring fission product is further metabolized to salicylate and catechol by a sequence similar to that described for the bacterial oxidation of naphthalene (Evans et al., 1965). Although there have been numerous studies on the bacterial oxidation of anthracene, very little is known about the metabolism of this compound in fungi. Cunninghamella elegans oxidized anthracene to trans-1,2-dihydroxy-1,2-dihydroanthraceneand l-anthryl sulfate (Cerniglia, 1982) (Fig. 8). The formation of a dihydrodiol with a transconfiguration and oxygen-18 experiments indicated a monooxygenase-catalyzed reaction and suggested the prior formation of anthracene-I,,%oxide (Fig. 8).These results are similar to those obtained in studies on the mammalian metabolism of anthracene, which showed initial oxygenation in the 1,2 positions to form a trans-dihydrodiol (Akhtar et al., 1975; Sims, 1970; Chaturapit and Holder, 1978). However, comparison of the circular dichroism spectrum of the fungal trans-1,2-dihydroxy-1,2-dihydroanthracene to that formed by rat liver microsomes indicated that the major enantiomer of the trans-1,2-dihydroxy-l,2-dihydroanthraceneformed by C . elegnns had a 1S,2S absolute stereochemistry, which is opposite to the predominantly 1R,2R dihydrodiol formed by rat liver niicrosomes (Cerniglia and Yang, 1984). Bacteria initially oxidize phenanthrene in the 1,2 and 3,4positions to form
+
POLYCYCLIC AROMATIC HYDROCARBONS
49
6 7 5 m 10 4 :
Anthracene
1-Anthrol
Sulfate
Anthracene- 1,2- oxide
@ I $J:
Conjugate
-
trans-l,2-Dihydroxy1,2-dlhydroanthracene
FIG. 8. The pathway for the fungal oxidation of anthracene.
optically pure (+)-cis-lR,2S-dihydroxy-l,2-dihydrophenanthrene and (+)-cis-3S,4R-dihydroxy-3,4-dihydrophenanthrene(Fig. 9). The phenanthrene-cis-3,4-dihydrodiol is the predominant isomer (Jerina et d., 1976; Koreeda et aZ., 1978). Pate1 and Gibson (1974) showed that cell extracts of Pseudomonas putida, Pseudomonas sp. NCIB 9816, and a Nocardia strain oxidized phenanthrene-cis-3,4-dihydrodiolto 3,4-dihydroxyphenanthrene. Evans and colleagues (1965) provided evidence that several pseudomonads further oxidized this dihydroxylated derivative of phenanthrene to cis-4-(1hydroxynaphth-2-yl)-2-oxobut-3-enoic acid (Fig. 9).This ring cleavage product is further metabolized to l-hydroxy-2-naphthoic acid, which is subsequently oxidatively decarboxylated to 1,2-dihydroxynaphthalene.This in turn is further metabolized through the naphthalene pathway (Fig. 4). Kiyohara and Nagao (1978)found that an Aeromonas strain as well as various fluorescent and nonfluorescent pseudomonads, vibrios, and unidentified bacteria utilize an alternative pathway for phenanthrene metabolism. They found that an Aeromonas strain converted phenanthrene to 1-hydroxy-2-naphthoic acid; however, none of the microorganisms could catalyze the decarboxylation of l-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene.Alternatively, they found that an Aeromonas strain converted l-hydroxy-2-naphthoate via an intradiol cleavage to form o-phthalic acid. This is then hydroxylated and decarboxylated to protocatechuate, which undergoes either ortho or meta
50
CARL E. CERNICLIA
Phmntkene
-
& & 00
cir-l.2-Dhy&oxy12-dihydrophenanthrene
dlhydrophenanthrene
00
&coo”
Wn &y-coo OH
OH
I&%OOHI
,2-DihydroXynapthalene
I
4
a::: a:”,”,:
2-Carboxybenzaldehyde I
flg-S-”ydroxybenzalpyruvic acid
L
I
&OH
q-Phthallc acid
I
cno
Salic ylaldehyde
&“ on
l-Hydroxy-2-nephtholc acid
I
Catechol
(a)
FIG.9. The different pathways for the bacterial oxidation of phenanthrene.
cleavage depending on the organism. The different pathways for the bacterial degradation of phenanthrene are illustrated in Fig. 9. Recent studies by Kiyohara et al. (1983) showed that the wild-type strain of Beijerenckia sp. contained a plasmid, pKGZ, which was responsible for the synthesis of enzymes involved in the oxidation of phenanthrene. The fungus C. elegans oxidized phenanthrene at the 1,2 and 3,4 positions to form phenanthrene trans-l,2- and truns-3,Cdihydrodiols (Cerniglia and Yang, 1984)(Fig. 10). There was no enzymatic attack at the 9,10 positions (Kregion) of phenanthrene; the K-region is a major site of metabolism for the mammalian oxidation of phenanthrene. Comparison of the circular dichroism spectra of the phenanthrene trans-l,&-and trans-3,4-dihydrodiols formed by C. elegans to those formed by mammalian enzymes indicated that the major enantiomers of each of the dihydrodiols formed by C. elegans had an S,S absolute configuration (Cerniglia and Yang, 1984).
51
POLYCYCLIC AROMATIC HYDROCARBONS
Bay Region
I Phennnthrene-9.10-Oxide)
Phenanthrene-trans9,lO-DhydrodT
(Phenanthrene- 1,2-0xide)
Phenanthrene-trans1,2-Dihydrodiol
\ Phenanthrene-trans3,4-Dihydrodiol
FIG. 10. The initial reactions in the fungal oxidation of phenanthrene.
VI. Benzo[a]pyrene Benzo[a]pyreneis a potent carcinogen when applied to mouse skin (Cook et
al., 1933). Benzo[a]pyrene is metabolized by mammals via a cytochrome P-450-dependent monooxygenase and an epoxide hydrolase to various primary and secondary metabolites including epoxides, phenols, trans-dihydrodiols, quinones, dihydrodiol epoxides, tetraols, and sulfate, glutathione, and glucuronide conjugates (Sims, 1970; Holder et al., 1974; Huberman et al., 1976; Yang et al., 1976; Thakker et al., 1977, 1978b). Metabolic activation of benzo[a]pyrene appears to proceed by the further metabolism of benzo[a]pyrene-7,8-oxideto an optically pure (-)-trans-7,8dihydroxy-7,8-dihydrobenzo[a]pyrene (Yang et al., 1977a,b). The (-)trans-7,8-dihydrodiol is further oxygenated highly stereoselectively at the 9,lO double bond to form mainly (+)-7P,8a-dihydroxy-9a, 1Oa-epoxy7,8,9,1O-tetrahydrobenzo[a]pyrene(benzo[a]pyrene 7,8-dihydrodiol-9,10epoxide 2) (anti), and a small amount of (-)-7P,8a-dihydroxy-9P, 10P-epoxy-7,8,9,lO-tetrahydrobenzo[a]pyrene (benzo[a]pyrene 7,8-dihydrodiol9,lO-epoxide 1) (syn) (Yang et al., 1976; Thakker et al., 1976, 1977). Both benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxides form C-10 carbonium ion intermediates that are responsible for their reactivity toward cellular nucleophiles (Yagi et al., 1977; Yang et al., 1977a,b). The benzo[a]pyrene dihydrodiol epoxides are very unstable and hydrolyze in aqueous media to tetraols or are reduced by NADPH or NADH to triols. The metabolic
52
CARL E. CERNIGLIA
Cytochrome
Cytochrome P-450
BP-Diol epoxide 2 (Anti)
DNA-BP-7.8-dlhydrodiol9,lO-epoxkfe adduct
FIG. 11. The mammalian metabolic activation of benzo[a]pyrene (BP).
activation of benzo[a]pyrene to bay-region 7,8-dihydrodiol-g, 10-epoxides and the binding to DNA are illustrated in Fig. 11. The diastereomeric benzo [ulpyrene 7,8-dihydrodiol-9,1O-epoxides are presumed to be the ultimate carcinogens ofbenzo[a]pyrene, since they are (1)highly carcinogenic for newborn mice (Kapitulnik et al., 1977), (2) mutagenic and cytotoxic for both mammalian and bacterial cells (Huberman et al., 1976; Wood et al., 1977; Levin et al., 1977; Slaga et al., 1976), and (3)chemically reactive in binding to DNA (Jeffrey et aZ., 1977; Osborne et al., 1978). Although bacteria with the ability to utilize di- and tricyclic aromatic hydrocarbons as their sole source of carbon and energy are readily isolated from water and soil samples, there has not been a published report on the ability of microorganisms to utilize aromatic hydrocarbons containing more than three aromatic rings as the sole source of carbon and energy. This may be due to the large size and extreme insolubility of such PAHs as benzo[a]pyrene or benz[a]anthracene (Wodzinski and Coyle, 1974). Microorganisms can, however, oxidize PAHs when grown on an alternative growth substrate (Lijinsky and Quastel, 1956; Poglazova et al., 1967; Gibson et al., 1975; Sisler and Zobell, 1947; Barnsley, 1975; Herbes and Schwall, 1978; Martinsen and Zachariah, 1978). Gibson and colleagues (1975) showed, using biotransformation techniques, that Beijerinckia sp. strain B-836growing on succinate in the presence of biphenyl, converted benzo[a]pyrene to cis-9,IO-dihydroxy-9,lO-dihydrobenzo[a]pyrene and cis-7,8-dihydroxy-7,8dihydrobenzo[a]pyrene (Fig. 12). The major isomer formed was cis-benzo[ alpyrene-9,lO-dihydrodiol. Various fungi have been reported to have the ability to oxidize ben-
53
POLYCYCLIC AROMATIC HYDROCARBONS
7
6
5
Benzo[a]pyrene
cis-9,1 O-Dihydroxy-9,1 O-dihydrobenzo[a]pyrene
-
cis-7,8-Dihydroxy 7,8-dihydrobenzo[a] pyrene
FIG. 12. Initial reactions in the oxidation of benzo[a]pyrene by Beijerinckia sp. B-836.
zo[a]pyrene by a mechanism similar to that observed in higher organisms. Saccharomyces cerevisiae, Neurospora crassu, Cunninghamella bainieri, Cunninghamella elegans, Aspergillus ochraceus TS, and various yeast strains have demonstrated benzo[a]pyrene hydroxylase activity (Cerniglia and Gibson, 1979; Wiseman and Woods, 1979; Ferris et al., 1976; Lin and Kapoor, 1979; Cerniglia and Crow, 1981; Ghosh et al., 1983). The filamentous fungus C. elegans oxidized benzo[a]pyrene to trans-9,lOdihydroxy-9,lO-dihydrobenzo[a]pyrene, trans-7-8-dihydroxy-7,8-dihydrobenzo[a]pyrene, benzo[a]pyrene-1,6- and 3,6-quinone, and 3- and 9-hydroxybenzo[a]pyrene (Cerniglia and Gibson, 1979). The pathway for the oxidation of benzo[a]pyrene by C. elegans is shown in Fig. 13. The major difference between the metabolism of benzo[u]pyrene by C . elegans and mammals was the lack of oxidative attack in the K-region of the benzo[a]pyrene molecule (4,5 positions). This suggests differences in regiospecificity of the fungal cytochrome P-450monooxygenase from that reported for mammalian hepatic microsomal and nuclear cytochrome P-450 preparations. Subsequent studies on the fungal oxidation of benzo[a]pyrene, (r)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, and (-+)-trans-g,10-dihydroxy-9,lO-dihydrobenzo[a]pyrene indicated the formation of benzo[a]pyrene dihydrodiol epoxides (Cerniglia and Gibson 1980a,b; Cerniglia et ul., 1980d) (Fig. 14). These results may be of medical, toxicological, and environmental significance since a typical soil fungus forms compounds that are highly tumorigenic and mutagenic and that appear to be the major ultimate carcinogens derived from benzo[u]pyrene. Microsomal preparations of C. bainieri (Ferris et al., 1976), S. cerevisiae
54
CARL E. CERNIGLIA
HO@&@ 9-OH-BP
/ I
3-0H-BP
000,
BP-9.10-Dihydrodiol
7
[7.8-EpoxideI
6
u)
5
I
- 3.s
Benzo [a] pyrene (BPI
0 0 BP-l,6-Ouinone
0 BP-3.6-Ouinone
FIG. 13. The pathways for the fungal oxidation of benzo[a]pyrene.
\
&AHQc]
000 __
-
Ho*a o.-'
.
ow
BP-9.10-Oxide BP9.10-Diol
\
nu
BP-7,8-Oxide BP-7.8-Diol
BPDiol Epoxide 2 --Tetra01 (Anti)
2
FIG. 14. The formation of benzo[a]pyrene (BP) dihydrodiol epoxides from benzo[a)pyrene, (+)truns-7,8-, and -Q,lO-benzo[a]pyrene dihydrodiols by Cunninghamella elegans. BP-diol epoxide 1 (syn), in which the epoxide oxygen and the Lenzylic hydroxyl groups are cis. BP-diol epoxide 1 (anti), in which the epoxide oxygen and the benzylic hydroxyl groups are trans.
POLYCYCLIC AROMATIC HYDROCARBONS
55
(Azari and Wiseman, 1982), and A . ochraceus (Ghosh et al., 1983)contain a cytochrome P-450 monooxygenase which catalyzes the hydroxylation of bena]zo[a]pyrene. The major metabolites are 7,8-dihydroxy-7,8-dihydrobenzo[ pyrene, 3-hydroxybenzo[a]pyrene, and 9-hydroxybenzo[a]pyrene.Woods and Wiseman (1979,1980) showed that the cytochrome P-45OlP-448 level and the benzo[a]pyrene hydroxylase activity were highest in S. cerevisiae grown aerobically at 20% glucose concentration. The high glucose concentration causes mitochondria1 repression, which lowers the level of cyclic AMP and increases the level of cytochrome P-4501P-448.
VII. Benz[a]anthracene Benz[a]anthracene is considered to be a weak carcinogen and has been reported to be a weak tumor initiator (Wislocki et al., 1979). Ben~[alanthraceneis metabolized by rat liver and lung microsomal preparations and by a purified cytochrome-P-448-dependent monooxygenase system to give the K-region trans-5,6-dihydrodiol and the non-K-region trans-8,9dihydrodiol as major metabolites. The trans-3,4-, -10,ll-, and -1,2-dihydrodiols were formed in minor amounts (Sims, 1970; Tierney et al., 1978; MacNicoll et al., 1980; Thakker et al., 1979). The optical purity and the absolute stereochemistry of the dihydrodiols formed from benz[alanthracene by rat liver microsomal fractions were determined by Thakker et al. (1979). The predominant enantiomers of the dihydrodiols were (-)-(3R,4R)-, (+)-(5R,6R)-, (-)-(8R,9R)-, and (-)-(lOR,llR)-dihydrodiols. Evidence obtained from mutagenicity studies indicated that the trans-3,4dihydrodiol is oxidized by the monooxygenase system to products that are 10 times more mutagenic in Salmonella typhimurium TA 100 than are metabolites of benz[a]anthracene or the other four dihydrodiols (Wood et al., 1976). Furthermore, the trans-3,4-dihydrodiol was 10-20 times more active in tumorigenicity experiments than was benz[a]anthracene (Wood et al., 1977). posIn addition, the benz[a]anthracene trans-3,4-dihydrodiol-l,2-epoxide sesses both mutagenic and carcinogenic activity (Wood et al., 1977). The mutagenicity, tumorigenicity, and carcinogenicity studies indicate that the 2-epoxides are the ultimate carcinogenic bay-region trans-3,4-dihydrodiol-l, metabolites of benz[a]anthracene (Fig. 15). However, it should be noted that the isolation of hydrocarbon-nucleoside adducts prepared from the DNA of mouse skin or hamster embryo cells which were treated with benz[alanthracene has shown that non-bay-region trans-8,9-dihydrodiol-l0,11epoxides may also be involved in the metabolic activation of benz[alanthracene (Vigny et al., 1980; Cooper et al., 1980a). The weak carcinogen benz[a]anthracene is metabolized by the bacterium Beijerinckia sp. strain B-836 to four dihydrodiols (Fig. 16). The major di-
FIG. 15. Structures of benz[a]anthracene trans-3,4-dihydrodiol and benz[a]anthracene krans-3,4-dihydrodiol-l,2-epoxide.
hydrodiol isomer formed was cis-1,2-dihydroxy-l,2-dihydrobenz[a]anthracene (Gibson et al., 1975). Fungi have also been reported to metabolize benz[a]anthracene to dihydrodiols but in contrast to bacteria, they are in a trans configuration. The fungus C.elcgans oxidized benz[a]anthracene to form truns-8,9-dihydroxy-8,9-dihydrobenz[u]anthracene,truns-lO,11-dihydroxy-l0,ll-dihydrobenz[u]anthracene,and a trace amount of trans-3,4dihydroxy-3,4-dihydrobenz[a]anthracene(Dodge and Gibson, 1980; Cerniglia et d . ,l980a). The structures of these metabolites are shown in Fig. 17. The major dihydrodiol formed was trans-8,9-dihydroxy-8,9-dihydrobenz[u]anthracene. The predominant enantiomer of trans-8,9-dihydroxy-8,9-dihydrobet~z[a]anthracene formed from benz[a]anthracene by C . eleguns had an S,S absolute configuration, which is opposite to the
&-10,1l-DhydrOxY-
10.1 1
-
dRydrobenz[a]anthracm
c&- 1,i-Oalydroxy- 1,2dihy&obenz[a]anthracene
(major)
~-5,6-Dhydroxy-5,6-
dhydrobenz [a]anthracene
FIG. 16. Initial reactions in the oxidation of benz[a]anthracerle by Reijerenckia sp. B-836.
E10,ll-Dihydroxy1 0 , l l -dihydrobenz [a] anthracene
trans-8,9-Dihydroxy 8.9-dihydro-
Y
benz [alanthracene (major)
Benz(a1anthracene-dihydrodiol epoxides
FIG. 17. The pathways for the fungal oxidation of henz[a]anthracene.
8R,SR-dihydrodiol formed from benz[a]anthracene by rat liver microsomes (Fu et al., 1983). As in the study on the fungal metabolism of benzo[a]pyrene, there was no evidence of enzymatic attack in the K-region (5,6 positions) of benz[a]anthracene. In addition, benz[a]anthracene 8,9-dihydrodiol-l0,11was detected epoxides or benz[a]anthracene 1O,ll-dihydrodiol-8,9-epoxides (Fig. 17). The formation of trans-3,4-dihydroxy-3,4-dihydrobenz[a]anthracene by C . elegans is of toxicological interest since this dihydrodiol has been reported to be highly mutagenic and tumorigenic in higher organisms and is thought to be a proximate carcinogen of benz[a]anthracene (Thakker et al., 1979).
VIII. Alkyl-Substituted Benz[a]anthracene A. MONO- AND
DIMETHYLBENZ[A]ANTHRACENE
Methylbenz[a]anthracenes are found in airborne particulates from cigarette smoke condensate, stack gases, roofing tar extracts, and industrial effluents (Thomas et al., 1978). The biotransformation of methylbenz[a]anthracenes in the environment is of considerable interest since certain isomers show moderate to high carcinogenic activity (Dunning and Curtis, 1960; Stevenson and Von Haam, 1965; Newman, 1976; Wislocki et al., 1982). Although benz[a]anthracene is a weak carcinogen, the substitution of a meth-
-
58
CARL E. CERNIGLIA
yl group at the 7 andlor 12 position converts the parent hydrocarbon to 7methylbenz[ alanthracene and 7,12-dimethylbenz[a]anthracene, both of which have been shown to be highly carcinogenic, mutagenic, and tumorigenic (Dipple, 1976; Chouroulinkov et al., 1977; Malaveille et al., 1977; Wislocki et al., 1981, 1982) (Fig. 1). Studies from several laboratories have suggested that the metabolism of methyl-substituted benz[a]anthracene to bay-region 3,4-dihydrodiol-l,2-epoxides(Fig. 15), strong electrophiles, is an important pathway for eliciting their biological activities (Jerina et al., 1977; Vigny et al., 1977; Yang et al., 1980). In contrast to the attention which has been given to the mammalian metabolism of mono- or disubstituted methylbenz[a]anthracenes, very little is known about the microbial metabolism of these compounds. Wu and Wong (1981)reported that both Penicillium notatum and Pseudomonas aeruginosa oxidized the methyl groups of 7,12-dimethylbenz[a]anthracene to 7-hydroxymethyl-12-methylbenz[a]anthracene and 12-hydroxymethyl-7-methylbenz[a]anthracene. Recently, these investigators (Wong et al., 1983) showed that C. elegans ATCC 9245 metabolized 7,12-dimethylbenz[u]anthraceneto trans-8,9- and 3,4-dihydrodiols of 7,12dimethylbenz[a]anthracene. In addition, the 7-hydroxymethyl and the 12hydroxymethyl derivatives of the trans-8,9- and 3,4-dihydrodiols were isolated. Cerniglia et al. (1982d) reported that C. elegans ATCC 36112 metabolized 7-methylbenz[a]anthracene primarily at the methyl group, followed by further metabolism at the 3,4 and 8,9 positions to form 7-hydroxymethylbenz[a]anthracene-trans-3,4-dihydrodioland 7-hydroxymethylbenz[a]anthracene-trans-8,9-dihydrodiol (Fig. 18). Incubation of 7-hydroxymethylbenz[a]anthracene with C. elegans gave similar results. Comparison of the metabolic patterns from the fungal and mammalian metabolism of 7-methylbenz[a]anthracene indicated that the regio- and stereoselectivity of the fungal enzymes have similarities and also some differences from those in the rat liver microsomes. Similar to previous findings on the fungal metabolism of PAHs (Cerniglia, 1981),there was a lack of metabolism in the K-region (5,6 positions) of 7-methylbenz[a]anthracene. However, the circular dichroism spectra are different for the trans-8,9-dihydrodiols formed from incubation of 7-methylbenz[a]anthracene and 7-hydroxymethylbenz[a]anthracene by C. elegans and rat liver microsomes. The major enantiomer of the trans-8,g-dihydrodiol formed from 7-methylbenz[a]anthracene and 7-hydroxymethylbenz[a]anthracene by C. elegans has an 8S,9S stereochemistry which is opposite to the 8R,SR-dihydrodiol formed by rat liver microsomes (Fu et al., 1983).In contrast, the circular dichroism spectrum ofthe trans-3,4dihydrodiol formed from the incubation of either 7-methylbenz[a]anthracene or 7-hydroxymethylbenz[a]anthracene with C. elegans indicated that both of the major enantiomers of the trans-3,4-dihydrodiol metabolites have 3R,4R absolute stereochemistries. These results indicate that the enzyme systems
FIG. 18. The pathways for the fungal oxidation of 7-methylbenz[a]anthracene
from rat liver microsomes and from the filamentous fungus C. elegans have similar stereoselective properties toward the 3,4 double bond, but different stereoselective properties toward the 8,9 double bonds of 7-methylbenz[a]anthracene and 7-hydroxymethylbenz[a]anthracene. Molecular oxygen-18 experiments and subsequent dehydration of the dihydrodiol metabolites indicated that the difference in stereoselective metabolism toward the 8,9 double bond of 7-methylbenz[a]anthraceneis due to the epoxidation by cytochrome P-450, not to the dehydration stage catalyzed by epoxide hydrolase. Cunninghamella elegans metabolized 4-methylbenz[a]anthracene to form trans-8,9-dihydroxy-8,9-dihydro-4-hydroxymethylbenz[a]anthracene and trans- 10,11-dihydroxy- 1O,ll-dihydro-4-hydroxymethylbenz[a]anthracene (Cerniglia et al., 1983b)(Fig. 19). There was no detectable trans-dihydrodiol formed at the methyl-substituted double bond (3,4 positions) or at the Kregion. The circular dichroism spectra of the trans-dihydrodiols formed from 4-methylbenz[a]anthracene by C. elegans indicated that the major enantiomers of the 4-hydroxymethylbenz[a]anthracene trans-8,9-dihydrodiol and trans-10, ll-dihydrodiol have S, S absolute stereochemistries which are opposite to the predominantely 8R,9R- and 10R, 11R-dihydrodiols formed by rat liver microsomes. The results of these studies indicate that the fungal monooxygenase-epoxide hydrolase enzyme systems are highly stereo- and regioselective in the metabolism of mono- and dimethylbenz[a]anthracenes. From an environmental and toxicological standpoint, the fungal transformation of methylbenz[a]anthracenes to potentially biologically active compounds (i.e., 3,4-dihydrodiols)as well as to detoxified products may be very important aspects of their biodegradation in natural habitats.
FIG. 19. The pathways for the fungal oxidation of 4-methylbenz[a]anthracene.
B.
3-METHYLCHOLANTHHENE
3-Methylcholanthrene has high biological activity in various in oitro and in oiuo test systems and it is one of the most potent carcinogenic polycyclic aromatic hydrocarbons. Most of the available evidence suggests that the metabolic activation of 3-methylcholanthrene involves the formation of one or both isomers of the bay-region 9,lO-dihydrodiol-7,8-epoxidesand possibly their hydroxylated derivatives (Wood et al., 1978; Thakker et al., 1978a; Tierney et al., 1979; Cooper et al., 1980a). D N A isolated from the skin of mice treated with 3-methycholanthrene indicated that the covalently bound DNA adducts derived from 3-methylcholanthrene occur via metabolism at the 7 , 8, 9, and 10 positions of the molecule (King et al., 1978; Vigny et al., 1980; Eastman and Bresnick, 1979; Cooper t al., 1980b). Little is known about the microbial metabolism of 3-methylcholanthrene. Beijerinckia sp. strain B-836 grown in the presence of 3-methylcholanthrene oxidized this alkyl-substituted derivative of benz[a]anthracene to cis-7,8-dihydroxy-7,8dihydro-3-methylcholanthrene (Kilbourn, 1980) (Fig. 20).Cerniglia et al. (1982a)reported that C. elegans oxidized 3-methylchulanthrene primarily at the methylene bridge to form 1-hydroxy- and 2-hydroxy-3-methylcholanthrene (Fig. 20). In addition, evidence was also reported for the formation of two diastereomeric 1-hydroxy-trans-9,lO-dihydrodiolsof 3methylcholanthrene. These compounds have been postulated to be proximate carcinogenic metabolites of 3-methylcholanthrene in higher organisms (Thakker et al., 1978a).
61
POLYCYCLIC AROMATIC HYDROCARBONS
on 1-Hydroxy-3-methylcholanthrene
on trans-9,lO-Dihydroxy9Kdlhydro-1-hydroxy3-methylcholanthrene
FIG. 20. The different pathways utilized by bacteria and fungi for the oxidation of 3methylcholanthrene.
IX. Nitro-Substituted Polycyclic Aromatic Hydrocarbons Nitro-PAHs are ubiquitous environmental pollutants which can be formed by nitration of PAHs and they have been found in diesel exhaust particulates, fly ash, cigarette smoke, photocopy toners, and in the products of various combustion processes. There has been concern regarding possible adverse human health effects due to exposure to nitrated PAHs since they have been shown to be extremely potent direct-acting mutagens in the Ames Salmonella mutagenesis assay. Furthermore, these compounds induce gene mutations, sister chromatid exchanges, unscheduled DNA synthesis in mammalian cells, and cancer in male rodents. It has been postulated that the biological activity of this class of compounds may be due to enzymatic reduction of the nitro group via nitroso intermediates to form aryl hydroxylamines. These aryl hydroxylamines either can undergo esterification to form highly electrophilic hydroxamic acid esters capable of reacting with DNA or can be direct-acting mutagens (Fig. 21). In addition to nitro reduction, ring hydroxylation of nitro-PAHs has recently been suggested as important in their metabolic activation. Both bacterial and mammalian nitroreductases have been implicated in the metabolic activation of nitro-PAHs. Details on the sources, metabolism, and biological activity of nitro-PAHs may be found in Mermelstein et al. (1982) and the references therein.
62
CARL E. CERNICLIA
@NO2
NilrosL
OH
Hrdroxylasins
Covalent Blndinn lo Prolains IndNucltic Acids
/
Rinp Hydroxylilion-Nllro. PIH
FIG. 21. The metabolic activation of nitro-substituted polycyclic aromatic hydrocarbons.
There have been several studies on the microbial metabolism of the mutagenic and carcinogenic nitro-PAH l-nitropyrene. Kinouchi et al. (1982) demonstrated a decreased mutagenicity of l-nitropyrene when this compound was mixed with the feces of a healthy individual or with various intestinal microflora. The decrease in mutagenicity was attributed to the enzymatic reduction of 1-nitropyrene to l-aminopyrene by intestinal microflora. Studies by Messier et aE. (1981)and Howard et aE. (1983)showed that anaerobic bacteria commonly associated with the intestinal tract of humans and animals are capable of metabolizing l-nitropyrene to 1-aminopyrene. The metabolism of l-nitropyrene in germ-free and conventional rats indicated that intestinal microflora are involved in the in uivo nitro reduction of l-nitropyrene (El-Bayoumy et al., 1983). Since the reduction of l-nitropyrene to 1-aminopyrene has been shown to be an important activation pathway, intestinal microflora could be important in the metabolism and subsequent toxicities elicited by nitro-PAHs. In contrast to the studies on the metabolism of nitro-PAHs by intestinal microflora, there are no published reports on the metabolism of these compounds by microbial populations isolated from soil or aquatic environments.
X. Biodegradation of Polycyclic Aromatic Hydrocarbons in Nature The fate of PAHs in the environment is influenced by a number of factors which determine the degradation rate and extent of metabolism. Such fac-
POLYCYCLIC AROMATIC HYDROCARBONS
63
tors include the physicochemical properties of the PAH such as molecular size, water solubility, lipophilicity, volatility, concentration, and the presence of various substituents and functional groups. Environmental factors include temperature, pH, oxygen concentration, salinity, season, light intensity, sediment type, presence of cosubstrates, and nutrient status. Microbial factors include the types, population, and distribution of microorganisms present as well as their predators and other microbiota and the bioavailability of the PAH. There are several review articles that include' aspects on factors which can influence the persistence and biodegradation of xenobiotics in the environment (Alexander, 1974, 1979a,b). Although much is known concerning the degradation of PAHs by pure cultures of microorganisms or by enzyme systems, far less is known about the transformation of these compounds in natural habitats. Herbes (1981) investigated the biodegradation of naphthalene, anthracene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene in stream water collected in the vicinity of a coal-coking treated wastewater discharge. These six PAHs were incubated with sediment and water samples. Mean rate constants in sediments collected downstream from the effluent outfall were 7.8 X 10-2/hour (naphthalene), 1.6 X 10 - 2/hour (anthracene), and 3.3 X 10 - 3/hour (ben~[alanthracene).Benzo[a]pyrene and dibenz[a,h]anthracene were not degraded. Only naphthalene and anthracene were observed to be degraded in water samples, with rate constants 5- to 20-fold lower, respectively, than in sediment samples. These results are similar to those of an earlier study by Herbes and Schwa11 (1978) which indicated a consistent decrease in the biodegradation rate with the increasing number of fused benzene rings and higher biodegradation rates in PAH-contaminated sediments than in pristine sediments. Sherrill and Sayler (1980) showed that microorganisms in surface waters from three diverse reservoir systems degraded approximately 80% of the phenanthrene added to the sample. The rate of phenanthrene biodegradation was directly related to incubation temperatures, history of pollution of the sampling sites, and time of incubation. In addition, phenanthrene was degraded to a greater extent than pyrene. Khesina et al. (1969) showed as much as 50% degradation of benzo[a]pyrene in soil from oil contaminated soil over a 3-month period. Hinga et al. (1980) reported that after 230 days, 29% of the initial benz[a]anthracene added to a microcosm which contained water and sediments from Narragansett Bay had been respired to CO,. They estimated that if the CO, production rate were to remain the same as during the last 100 days of the experiment, then all the benz[a]anthracene would be converted to CO, within 3.5 years. Due to the different approaches and experimental designs to simulate the environment, it is difficult to extrapolate results on the biodegradation of
64
CARL E. CERNIGLIA
PAHs in nature. In general, there are major rate differences between the decomposition of PAHs in soil and in aquatic systems with rates slower in aquatic systems, and PAHs become more recalcitrant to microbial degradation as the number of benzene rings in the compound is increased.
XI. Conclusions and Comments Bacteria, filamentous fungi, yeasts, cyanobacteria, diatoms, and other eukaryotic algae have the enzymatic capacity to oxidize PAHs that range in size from naphthalene to benzo[a]pyrene. Di- and tricyclic aromatic hydrocarbons are more susceptible to microbial degradation than are higher molecular weight PAHs. The hydroxylation of PAHs always involves the incorporation of molecular oxygen; however, there are diiferences in the mechanism of hydroxylation of PAHs by prokaryotic and eukaryotic microorganisms (Fig. 3). Bacteria oxygenate PAHs to form a dihydrodiol with a cis configuration (Table IV). In this reaction both atoms of molecular oxygen are incorporated into the PAH via a dioxygenase. cis-Dihydrodiols can undergo further metabolism via a pyridine nucleotide-dependent dehydrogenation reaction to yield catechols, which can act as substrates for ring cleavage enzymes with complete mineralization of the PAH (Fig. 3). The genes for the initial oxidation of PAHs are localized on plasmids. In contrast to bacteria, fungi oxidize PAHs via a cytochrome P-450 monooxygenase to form arene oxides (Fig. 3) which can isomerize to phenols or undergo enzymatic hydration to yield trans-dihydrodiols (Table IV). These reactions appear to be similar to those reported for mammalian enzyme systems except that there is a lack of metabolism at the K-region of the PAH molecule, which is a major site of enzymatic attack in mammals. In addition, the absolute stereochemistries of the fungal dihydrodiols in most cases are opposite to those observed in mammalian systems. These findings suggest differences in the regio- and stereoselectivity of the fungal cytochrome P-450 monooxygenases from similar enzymes purified from hepatic and nuclear microsomal cytochrome P-450 preparations. The fungal degradation of PAHs is of toxicological and environmental significance since some of the metabolic products have been implicated as biologically active forms of PAHs in higher organisms. In addition, the capacity of fungi to form glucuronide and sulfate conjugates of phenolic PAHs suggests that these reactions may be important in the detoxification and elimination of PAHs. Multiple oxidative pathways may be involved in the cyanobacterial metabolism of PAHs. Virtually nothing is known about the nature of the enzymes and cofactors involved in these reactions. Further insight into the algal oxidation of aromatic hydrocarbons requires much more understanding of
POLYCYCLIC AROMATIC HYDROCARBONS
65
the mechanisms of oxidation than is now available; this area warrants further investigation. Much is known concerning the microbial metabolism of PAHs using both pure cultures and purified enzymes. Such studies have given us greater insight into the metabolic potential of microorganisms and the mechanisms by which they metabolize these compounds. There is a relative paucity of reports, however, on the decomposition of PAHs under environmental conditions. Research is needed to establish rates of degradation in natural habitats and to determine what contribution prokaryotic and eukaryotic microorganisms make to the overall biodegradation of PAHs. Additional studies designed to compare the predictions based on pure or mixed culture studies to “real world” systems are necessary to determine how accurately data can be extrapolated from experimental results obtained in uitro to aquatic and terrestrial ecosystems. It is also apparent that relatively little is known concerning the identification of the ring cleavage products and the exact reaction sequence for the bacterial oxidation of PAHs which contain more than three aromatic rings. Little attention has been paid to the transport processes for these hydrophobic compounds and the site of aromatic hydrocarbon hydroxylation in microbial cells. Studies on PAH metabolism are entering a new era; biochemical genetic techniques such as gene cloning and transposon mutagenesis will provide new insight into the biochemistry and regulation of PAH degradative pathways. The genetic manipulation of the biodegradative activities of bacteria should also be useful in enhancing the removal of PAHs in the environment. REFERENCES Aitio, A. (1978). In “Conjugation Reactions in Drug Biotransformation,” p. 529. Elsevier, Amsterdam. Akhtar, M. N., Boyd, D. R., Thompson, N. J., Koreeda, M . , Gibson, D. T., Mahadevan, V., and Jerina, D. M. (1975).J . Chem. SOC. 2056. Alexander, M. (1974). Ado. Appl. Microbiol. 18, 1. Alexander, M. (1979a).In “Microbial Ecology: A Conceptual Approach” 0. M. Lynch and N. J. Poole, eds.), p. 246. Blackwell, Oxford. Alexander, M . (1979b). In “Microbial Degradation of Pollutants in Marine Environments” (A. W. Bourquin and P. H. Pritchard, eds.), p. 67. EPA, Gulf Breeze. Andelman, J. B., and Snodgrass, J. E. (1974). CRC Crit. Rev. Enuiron. Control 5, 69. Axcell, B. C., and Geary, P. J. (1975). Biochem. J . 146, 173. Azari, M. R., and Wiseman, A. (1982). Anal. Biochem. 122, 129. Badger, G. M., Buttery, R. G., Kimber, R. W. L., Lewis, G. E., Meritz, A. C., and Napier, I. M. (1958).J . Chem. Soc. 2249. Badger, G. M., Kimber, R. W. L., and Novotny. J. (1964). Aust. J. Chem. 17, 778. Barnsley, E. A. (1975). Can. J . Microbiol. 21, 1004. Barnsley, E. A. (1976). Biuchem. Biophys. Res. Commun. 72, 1116.
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Microbiology of Potable Water BETTYH. OLSONAND LASLOA. NAGY Environmental Analysis, Program in Social Ecology, University of CaZifornia, Irvine, Iroine, CaZ$ornia
I. Introduction 11. Source Water 111. Treated Water
1. Introduction The topic of the microbial quality of water and wastewater is one of continuing concern and general interest. In the mid-MOs, the association of waterborne diseases with massive loss of human life in continental Europe and England focused attention on the importance of public health aspects of water quality. As better sanitation methods were employed and water treatment processes were initiated the incidence of waterborne diseases decreased. Perhaps most significant was the introduction of chlorination in water supplies to reduce the number of microbial pathogens. As can be seen in Fig. 1, there was a decrease in the number of waterborne disease outbreaks in the United States toward the middle of the twentieth century. This 73 ADVANCES IN APPLIED MICROBIOLOGY. VOLUME 30 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-002630-9
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YEARS
Fic. 1. Number of outbreaks of waterborne disease in the United States, 1920-1980.
trend was then reversed by a small but steady increase in waterborne disease outbreaks continuing to the present. Two explanations for this have been developed. One invokes better reporting efforts through the cooperation of the Center for Disease Control, the United States Environmental Protection Agency (USEPA), and state agencies. The other points to increased pressure on water sources through urbanization and extended use of remote areas for recreation. Most likely, both factors are important in explaining the increase. The number of waterborne disease cases in the United States approximates 20,000 per year. The importance of this figure is difficult to assess because of the uncertainty associated with this reported value. The general sentiment is that 20,000 cases per year may underestimate the actual number by as much as 30%. Given the size of the population of the United States and the expectation of high water quality, many instances of waterborne disease could be attributed to other sources such as food, or could go unrecognized, since symptoms are often transient. Nevertheless, waterborne disease is still a recognizable phenomenon in this country. In developing countries, waterborne diarrheal diseases are often the leading cause of infant and childhood morbidity and mortality, as exemplified by Central and South America (PanAmerican Health Organization, 1982).The lack of basic sanitation and the lack of access to safe water supplies constitute the major problems for decreasing the incidence of waterborne diseases in these countries. Thus, the majority of the population in the world is still concerned with waterborne diseases in the manner that Europe and North America were in the middle and late nineteenth century. Today, in both Europe and the United States, waterborne diseases are viewed as a minor health concern.
MICROBIOLOGY OF POTABLE WATER
75
The questions that remain unanswered in the United States involve the persistence of pathogens in aquatic environments, their level of pathogenicity after environmental exposure, their ability to survive treatment barriers, and how to predict the possibility of a disease outbreak if those barriers fail. Microbiologists and sanitary engineers grapple with this persistent threat to public health in order to ensure microbially safe water supplies. This requires an approach which underscores the behavior of pathogenic microorganisms, opportunistic pathogens, and nonpathogenic microorganisms, as well as the behavior of indicator organisms throughout the system, from source water to consumer. An ecological approach is needed to understand the microbiology of potable water from its origin to the consumer’s tap. Until recently, few studies in the literature adopted this perspective. Instead the literature has assumed an engineering approach which is designed to look at removal, survival, etc., under controlled conditions or to report observational data collected in the field. Findings in recent years regarding disinfectant efficiency, ability of injured pathogens to produce disease, and the persistence of unexpected organisms in distribution systems have certainly pointed out the shortcomings of a laboratory strategy for defining what actually occurs in the system (Ridgway and Olson, 1982; R. R. Colwell. 1983, personal communication). This review describes the microbiology of potable water from a historical as well as an ecological perspective. The review does not focus directly on methodology, pathogens, or coliform or other indicator organisms in potable water as these subjects have been reviewed recently (Bitton, 1980; Dutka, 1981; Hendricks, 1979; James and Evison, 1979; Mitchell, 1972; Pipes, 1982; Sobsey and Olson, 1983). The historical perspective reacquaints the reader with the insights and endeavors of investigators who preceded us in the quest for protection and understanding of the microbial quality of water. The ecological perspective is not used in this review as it applies to theoretical ecology, but rather in the sense of applied ecology, which attempts to explain why and how organisms move from one ecosystem to another and the mechanisms which prevent or enhance such movement. “Ecosystem” is defined here as each unique environment, i.e., source waters, treatment plants, or distribution systems. Once this view is adopted, the subject of potable water microbiology becomes a series of discrete yet intimately linked parts. One can imagine the source water, treatment plants, and the distribution system as separate ecosystems linked together by the continuous flow of water from its origin to the consumer. This review tries to identify the critical linkages and how they Eunction in each of these systems. It endeavors to report not only the state of knowledge of microbiology, but also the importance of chemistry and engineering aspects, in each compartment of the potable water system.
76
B E ” Y H . OLSON A N D LASLO A. NAGY
EARLYHISTORY The first microbiological study of drinking water can be traced back to the middle of the nineteenth century. It was conducted on the water supply of London by Hassell, who in 1850 published his findings in a report entitled “A Microscopic Examination of the Water Supplied to the Inhabitants of London and the Suburban Districts” (Rafter, 1892; Whipple et al., 1927). Hassell stressed the importance of microbiological examination of drinking water and outlined a relationship between sanitary quality and microbiological activity. At about the same time, similar conclusions were being reached by Ferdinand Cohn, who in 1853 published a treatise entitled “Living Organisms in Drinking Water” (Whipple et al., 1927).These works were preceded by studies on the waterborne nature of cholera (Snow, 1855) and typhoid fever (Budd, 1857). The initial observations of Hassell and Cohn were confirmed by other investigators during the second half of the nineteenth century (Rafter, 1892; Whipple et al., 1927). The idea of specific microbial indicators evolved around the 1880s with the work of von Fritsch and Escherich on fecal coliforms, and Miquel on plate count microorganisms as indicators of sanitary quality (Prescott and Winslow, 1904). The first book dealing with the subject of drinking water microbiology was MacDonalds 1875 “Guide to the Microscopical Examination of Drinking Water.’’ This was followed by a book by Fox (1878) on the sanitary examination of water, a book by Rafter (1892)called “The Microscopical Examination of Potable Water,” and a book by Whipple (1899) entitled “The Microscopy of Drinking Water.” Whipple’s book, with the title unchanged, was reedited in 1905, 1914, and 1927. The last edition was actually completed by Whipple’s associates after his death and has become “Whipple et al.” (1927). In 1904 Prescott and Winslow compiled “Elements of Water Bacteriology,” a book that was similarly reedited and expanded over the years. The first edition of “Standard Methods for the Examination of Water and Wastewater,” appearing in 1904, was largely based on the previously formed set of procedures and recommendations of a committee appointed in 1897 by the American Public Health Association (APHA).
II. Source Water Historically, efforts to designate the quality of source water to be used as a potable supply resulted in two divergent viewpoints. One argues that the best available source in terms of initial quality must be obtained, while the other proposes heavy reliance on treatment processes to improve a water source of initially poor quality. Regardless of the position taken, increasing urbanization and the increased use of recreational facilities have placed ex-
77
MICROBIOLOGY OF POTABLE WATER
treme pressure on the microbiological quality of both groundwater and remote surface water sources. Further, the location of large populations far from water sources has led to the development of water transport systems that carry water hundreds of miles from its source. These transport systems and storage facilities en route to the customer become the major foci of public health and management concerns. Therefore, no matter what the philosophical background, in reality more reliance is being placed on water treatment to produce finished water of acceptable microbiological quality, regardless of source quality. The importance of source water quality and our increasing reliance on treatment process is demonstrated by data collected in the United States from 1971 to 1977 which showed that 67% of the largest waterborne disease outbreaks occurred due to source water contamination where treatment was either inadequate or nonexistent (Sobsey and Olson, 1983). Microbiological quality of source water has been assessed through the use of indicator organisms. In the United States, most often total coliforms have been used to determine the level of treatment required for a source water. Table I shows treatment strategies recommended by the United States Public Health Service in relation to source water quality. As can be seen, the level of treatment increases as microbial quality decreases. In recent years, TABLE I RECOMMENDED SANITARY REQUIREMENTS FOR WATERTREATMENT SYSTEMS OF THE SOURCE WATER" RELATEDTO THE QUALITY
Designation Group1 Group I1 Group 111
Group IV
Treatment required None
Type of water
Protected groundwater Ground and surface Chlorination water Complete Water requiring filtration for turbidity; waters polluted by sewage Complete plus auxilia- Polluted ry treatment to rapid sand filtration with continuous postchlorination
From Public Health Reports (1927).
Level of sanitary contamination
Coliform count (per 100 ml per month)
None
51
Low
550
Medium
~ ~ 5 0 and 0 0 20% of samples exceeding 5000
High
25000 in more than 20% of the samples and 220,000 in not more than 5% of samples
78
BETTY H. OLSON AND LASLO A. NAGY
the validity of coliforms as an adequate indicator of source water microbial quality has been increasingly compromised. Newly identified pathogens such as Legionella, or pathogens only recently recognized as causing waterborne disease, e.g., Yersinia, have the ability under certain circumstance to grow in water. Other pathogenic agents have resistant life stages, such as Giardia. These produce a cyst stage that survives for much longer periods in the environment than Escherichia coli or other fecal coliforms. Such factors limit our ability to relate pathogen occurrence to fecal contamination by traditional indicators. Increasing evidence, mostly in the form of unpublished reports in the United States, suggests that coliforms can regrow under a variety of conditions. E. coli has been shown to survive and grow in a warm (28.5-38°C) monomictic reservoir which receives thermal effluent (Gordon and Fliermans, 1978). Blooms of E. coli 08 have been reported in Lake Burragorang, the raw water source for Sydney, Australia (MacKay and Ridley, 1983). E. coli blooms occurred at several locations simultaneously in this 2.1 X lo6 M1 lake after spring rainfall and algal blooms. The same serotype of E. coli was found in several areas of the lake, which suggested regrowth as opposed to direct fecal contamination. The authors concluded that E. co2i was able to grow on the organics released from decaying algae. In the summer of 1983, a persistent coliform bloom was observed in an open finished water reservoir in Southern California. The bloom of organisms, identified as Etiterobacter cloacae, lasted for several weeks. The coliform was found to be associated with a concurrent algal bloom and a resident frog population in the reservoir. Addition of chlorine to the reservoir eliminated the problem (E. G. Means, personal communication, 1983). Thus, interpreting the meaning of the presence of coliform bacteria in relation to fecal contamination of a source water continues to be difficult for the water industry. One might wonder why these indicators, which have been relied upon with great success, are suddenly less valid. There are, of course, many possible explanations. In the United States, the Safe Drinking Water Act of 1974 focused attention and research dollars on the microbial qualtity of drinking water and is a probable link. One has only to look at a comparison of the work produced in Europe and the United States over the last several decades to realize that drinking water microbiology has had a significant resurgence in the United States in the 1970s. This research focused on finished water microbial quality and has led to renewed interest in source water quality and indicator organisms in general. Further, difficulty in understanding temporal relationships between source water and resultant problems in the distribution system caused these two systems to be viewed as separate and unrelated entities. Focus on isolating the causative agents of waterborne diseases has reinforced the link-
MICROBIOLOGY OF POTABLE WATER
79
age not only between the source water and the distribution system, but also between treatment practices and efficiency. This linkage is becoming increasingly important as protection comes to rely more implicitly on treatment. One such example of the linkage between systems is the deposition of silt in distribution lines from a surface water source which received only chlorination and fluoridation as treatment. This deposition of sediment in the distribution system gave rise to colonization of the mains by the coliform Klebsiella (Ireland et al., 1983). The problem of how to deal with a water supply in which coliforms are regrowing is faced by an increasing number of water utilities with increasing frequency. For detailed information on source protection the reader is directed to Pojasek (1977).
Ill. Treated Water Water treatment is the most important and direct means of controlling the microbial quality of drinking water. Treatment schemes consisting of chemical coagulation and flocculation, filtration, and disinfection are usually used for surface waters, although high quality surface waters are sometimes treated by direct filtration and disinfection or just disinfection. Groundwaters, being generally lower in such contaminants as microbial agents, particulates, and organics, often receive no treatment or only disinfection. A number of laboratory and field studies have shown that under optimum conditions, these treatment processes can substantially reduce the levels of microbial agents and other contaminants in water. A variety of treatment schemes are shown in Fig. 2, depicting storage, pretreatment, coagulation, filtration, and disinfection. Storage is one of the oldest means of treating water. Today, storage of water in reservoirs usually serves to regulate the supply of water available on a short- or long-term basis. However, it is also of value as a form of microbial treatment. Storage serves to reduce not only dissolved and suspended organic matter, but also the numbers of bacteria and pathogenic agents, such as viruses and pathogenic protozoa (Poynter and Stevens, 1975). Several factors act to reduce the numbers of microorganisms and these include flocculation, sedimentation, ultraviolet light, production of bactericidal agents by certain organisms, predation, and competition for nutrients (Hutchinson and Ridgway, 1977). Storage can also result in the degradation of water quality by pollution from water fowl or sea gulls, and by algal growth which can promote growth of coliform and other bacteria. Products released by decaying algal blooms can promote actinomycete growth, which can result in taste and odor problems. (Geldreich, 1966; Williams and Richards, 1976). The processes shown in Fig. 2 are the major treatment schemes used today, although specific types may be more common in one country than
Borehole abstraction
2
Storage on protected catchments
:Chlorination
Storage
Micro-st raining
Direct abstraction
-
filtration
>-
Storage
\
Slow sand
*
Micro-straining/ Rapid sand filtration
Mrect abstraction
Biological Mrect _ IPretreatmentabstraction (rapid sand/ sediment at i o n )
Prechlorination-
Chlorination -Supply
bChlorination
Coagulat Ion Rapid sand + -filtration Clarification
FIG.2. TyTical wmbinations of watcr treatment pwcsses used in the Unitrri Kingdom (from Hutchinsari and Fiidgway, 1977).
Supply
MICROBIOLOGY OF POTABLE WATER
81
another. Flash chlorination prior to treatment is commonly used in the United States where trihalomethanes are not a problem. This reduces the microbial load and also oxidizes organic matter. It should be noted that enteric viruses and protozoan cysts are less effectively reduced by certain treatment processes than are enteric bacteria. These findings have raised concerns about the possibility of producing drinking water that meets current bacteriological standards but still contains sufficient viral and protozoan pathogens to pose a health risk to consumers. Such situations are most likely to occur when treatment is minimal or only marginally effective and raw water pathogen levels are high. At the present time there is inadequate epidemiological information available to show that this is a significant or widespread problem. A. VIRUSES
The topic of virus removal and inactivation by different drinking water treatment processes was first reviewed by Clark and Chang (1959). Since that time a number of subsequent review articles have covered this expanding research field (Committee Report, 1979; Hutchinson and Ridgway, 1977; LAWPRC Study Group on Water Virology, 1983; Leong, 1983; Report to Congress, 1978; Roebeck et al., 1962; Sobsey, 1975; Sobsey and Olson, 1983; Taylor, 1974). It is generally accepted that conventional water treatment practices (composed of coagulation, flocculation, sedimentation, filtration, and disinfection) can reduce viral levels by 6-8 logs in the finished water (Committee Report, 1979; LAWPRC Study group on Water Virology 1983; Leong, 1983; Report to Congress, 1978; Sobsey, 1975). Coagulation, flocculation, and sedimentation remove approximately 99% (a 2 log reduction) of the viral plaque forming units (PFUs) found in raw water (Clarke and Chang, 1959; Committee Report, 1979; Leong, 1983; Sobsey, 1975; Sobsey and Olson, 1983);however, as would be expected, the type of coagulant, virus, and water can produce wide deviations from this value (Clarke and Chang, 1959; Leong, 1983; Sobsey and Olson, 1983). Filtration is relatively ineffective in terms of virus removal, especially if the viruses are not associated with large particles (Leong, 1983; Sobsey, 1975; Sobsey and Olson, 1983). However, under correct operating practices and without floc breakthrough, sand filtration can produce a 1-2 log reduction in viral numbers (Clarke and Chang, 1959; Leong 1983; Sobsey, 1975; Sobsey and Olson, 1983). Disinfection, usually in the form of chlorination, has been the main method of virus inactivation in drinking water, generally resulting in a 4 log reduction in viral levels (Clarke and Chang, 1959; Committee Report, 1979; IAWPRC Study Group on Water Virology, 1983; Leong, 1983; Report to
82
BETTY H. OLSON AND LASLO A. NAGY
Congress, 1978; Roebeck et al., 1962; Sobsey, 1975; Sobsey and Olson, 1983; Taylor, 1974). The many laboratory, pilot, and field experiments on the impact of chlorination on a number of different viruses have been recently reviewed by a number of authors (IAWPRC Study Group on Water Viology, 1983; Leong, 1983; Sobsey and Olson, 1983), and are not individually discussed here. It appears from the information currently available that viruses are considerably more resistant to chlorination than are coliforms, and consequently they require higher chlorine residuals and longer contact times than coliforms (IAWPRC Study Group on Water Virology, 1983; Leong, 1983; Sobsey and Olson, 1981). Ozonation, at least from bench tests, appears to be a relatively good viricidal treatment; however, treatment plant studies with ozone have often recorded viruses in the finished water (Leong, 1983; McDermott, 1974; Taylor, 1974). The use of chloramines for virus disinfection has indicated that in comparison to chlorine, chloramines require higher doses and much longer contact times for satisfactory inactivation (Wolfe et al., 1984).
B. BACTERIA There are few reports in the literature from the 1920s to the present on the effectiveness of removal of various bacterial pathogens or indicators by treatment processes. 1 . Pretreatment
Generally, three types of pretreatment are used: microstrainers, roughing filters, and biological sedimentation. Microstrainers are not commonly used in the United States. In actuality, microstraining does little to remove bacteria because the smallest mesh size is 23 pm (Boucher, 1967). It does, however, remove larger particles and certain types of algae. Microstrainers can develop bacterial slimes or biofilms which reduce thier efficiency. Therefore, routine maintenance practices often require control of the biofilm by ultraviolet light or disinfection. Roughing filtration utilizes large diameter sand particles to remove larger suspended solids or filamentous algae. Its main benefits are the passage of large volumes of water through the filter in a short period of time and reduction of the need for backwashing during filtration. Biological sedimentation is sometimes used in Europe, but is not often found in the United States as a pretreatment process. Water is run through horizontal and upflow tanks in a manner which keeps the biofloc (river sand, sand, or alum floc) in suspension. The large surface area of these flocs is excellent for removing ammonia (Millner et al., 1972).
MICROBIOLOGY OF POTABLE WATER
83
2 . Slow Sand Filtration Slow sand filtration is not widely practiced in Europe today, but has a long history beginning in the early 1800s. In the United States, rapid sand filtration has always been the favored form of water treatment. Slow sand filtration is the classical form of biological treatment and it was responsible for the identification of water treatment as a means to reduce waterborne outbreaks of typhoid and cholera (Baker, 1949). Water passes through the filter at a slow rate (0.1 mph). The filter is composed of fine sand (0.2-0.4 mm in diameter). A biofilm (Schmutzdecke) is formed on the surface, which removes not only bacteria, protozoa, and viruses, but also organics and nitrogen. Further, the mechanical process of filtration occurs as the water moves through the sand bed, increasing removal of various chemical constituents as well as microorganisms. As with all treatment processes this is not free from problems, including seeding with sporeformers which can result in spores passing through the filter.
3. Coagulation and Filtration In the United States, coagulation and sedimentation are frequently used to treat surface water sources. Flocculants include ferrous or aluminum sulfate which form positively charged flocs of the respective hydroxide. Treatment plants are designed so that slow mixing enhances floc formation, and water movement down channels allows settling of the floc, which is then discharged to the sewer or to sludge-drying beds. Coagulation and sedimentation are usually followed by rapid sand filtration. Rapid sand filtration as opposed to slow sand filtration removes the organisms throughout the filter depth, not just at the surface. Rapid sand filtration is purely a physicochemical process, but the filters can become contaminated with microorganisms (Hutchinson and Ridgway, 1977). This condition usually results from ineffective backwashing or the formation of mud balls in stagnant areas of the filter. Dual or mixed media filters are preferred. Table I1 shows reduction of indicator organisms by coagulation and filtration. Removal rates for E . coli and coliforms by aluminum sulfate range from 75 to 99.4%and closely parallel reductions in turbidity. Slow sand filtration examples shown in Table I1 removed from 41 to 99.5%of the bacteria. These studies do not designate the numbers of bacteria in the source water, but generally a 2-3 log removal appears to be the norm. Thus, disinfection is a critical barrier to bacteria entering the distribution systems if the source water carries greater than 103 bacterialml.
84
B E m H . OLSON A N D LASLO A. NACY
TABLE I1
INUICATOHBACTEHIAREDUCTIONBY CIIEMICAI. COAGULATION A N D FILTHATION"
Agent
Type of water
Dose (mgfliter)
Total coliforin
River
12.6
Total coliform
River
20
Total coliform E. coli E. coli
River River Lake
25 10.5 12.1
Coagulationb Turbidity removal
("/.I
T I C
Bacterial removal
is)
14
96
97
8
40
74
9699.6 90 72
99.4 83 76
140-255
168 40
Reference Cummins and Nash (1978) Mallman and Kahler (1948) Chang et al. (1959) Streeter (1927) Streeter (1927)
Filtration
Agent
Type of filter
Aerial loading rate
Total coliform Total coliform
Slow sand Slow sand
5 m/day Unknown
Unknown Unknown
Total coliform Total coliform
Slow sand Slow sand
Unknown Unknown
Unknown Unknown
E. coli
Slow sand
Unknown
Unknown
0
Initial concentration
Removal (%)
Reference
70-98 96.S99.5
Hoekstra (1978) Poynter and Slade (1977) 88 (low temp) Burman (1962) SO Robeck et nl. (1962) 41 (low temp) Burman (1962)
From Sobsey and Olson (1983). Using Al2(S0&. Initial turbidity units.
4. Disinfection In Table 111examples of disinfection efficiency for inorganic chloramines, chlorine, and chlorine dioxide are shown. Chlorine dioxide is the most efficient disinfectant for bacteria, having both the lowest effective dose and the shortest contact time. Unfortunately, human health problems can arise with its use in certain populations (Bercz et d., 1982; Bull, 1982; Lubbers et al., 1982). All reactions listed in Table I11 are pH and temperature dependent. At higher pH values (> 7.0) chlorine becomes less effective because hypochlorite ion is formed; increased pH levels (> 7.5) also shift dichloramines to monochloramines, which are less bactericidal. However, increasing pH increases the bactericidal activity of chlorine dioxide (Hoff and Geldreich, 1981). interestingly dichloramines are less efficient viricides than monochloramines (Esposito, 1974; Dorn, 1974). Increasing the temperature increases the inactivation rate of bacteria for these three disinfectants. For
TABLE I11 INDICATOR AND PATHOGEN REDUCTION BY DISINFECTION^ _
Agent
Disinfectant
Escherichia coli E . coli E . coZi E . coli E . coli E . coli (ATCC 11229) E . coli E . coli E . coli E . coli E . coli E . coli E . coli (ATCC 11229) Salmonella typhi Pseudonwnas pyocyaneo Pseudomonas pyocyanea Legwnella pneumophh Legwnella pneumophih Campylobacter jejuni Campylohacter jejuni Campylohacter jejuni
HOCl
4
b
Concentration (mgliter)
oc1-
0.1 1.0 1.0 1.0 1.2 1.0 0.3
OCI ClOZ ClOZ ClOZ ClOZ
0.25 0.25 0.25 0.25
oc1NHzCl NHzCl NlizCl NHClz
uv2
OCI OCI -
oc1-
HOCl HOCl HOCl HOCl HOCl
From Sobsey and Olson (1983). Calculated, in many instances. NA, Not applicable. NR, Not reported
Contact time (minutes) pH
0.4
6.0
("/.I
Reference
5
99
Scarpino et nl. (1974) Scarpino et d. (1974: Siders et al. (1973) Siders el al. (1973) Siders et d . (1973) Esposito (1974) Butterfield (1948) Butterfield (1948) Cronier et d. (1978) Cronier et al. (1978) Cronier et al. (1978) Cronier et d. (1978) Rice and Hoff (1981) Butterfield (1948) Butterfield (1M8) Butterfield (1948) Skaliy et d. (1980) Skaliy et ul. (1980) Wang et al. (1982) {Vng et ul. (1982) Wang et 01. (1982)
0.63
30
NR
5.0
1
NR
0.4
3 x lo3 W-s/cmZ
0.3 0.75 0.4 3.3 1.W1.5 2.5
m.5
~
Reductionb
5.5 LO 10 1.8 1.3 0.68 0.27 NAC 10 10 10 <1 19 1
64
_
Temp. ("C)
10.0 5 9.0 5 15 9.0 9.0 25 4.5 15 10.0 20-25 10.0 20-25 5 6.5 10 6.5 6.5 20 32 6.5 7.0 20 10.7 20-25 10.7 2&25 10.0 20-25 NRd 25 NR NR NR NR
0.92 175
_
NR NR
99
99 99 9Y 99 100 100 99 99 99 99 99.9 100 100
100 99.9 99 100 100 100
86
BETTY H. OLSON AND LASLO A. NAGY
more detail the reader is referred to the National Research Council (NHC) Report (1980) on disinfection of drinking water.
C. ACTINOMYCETES The impact of water treatment practices on actinomycete numbers has been reviewed by Burman (1973). This review, and a number of other papers, indicated that filtration results in a comparatively small reduction (approximately 50-90%) in actinomycete colony forming units (CFU) (Bays et al., 1970; Niemi et al., 1982); this is attributed to the presence of actinomycetes in the natural flora of the filters (Burman, 1973). Actinomycetes in the vegetative or the spore form are considerably more resistant to chlorination than are coliform bacteria, such that under commonly employed chlorination practices, actinomycete CFU/ml may be reduced by only 2090% (Bays et al., 1970; Burman, 1973). Chloramines are even less effective against actinomycetes, and may even be used to enhance their recovery (Burman, 1973).
D. FILAMENTOUS FUNGI The influence of different water treatment practices on filamentous fungi has not been investigated in detail. However, it appears that water treatment generally results in a 2 log reduction in filamentous fungal CFUs (Bays et al., 1970; Niemi et al., 1982). As stated in Standard Methods for the Examination of Water and Wastewater (APHA, 1980), the chlorine resistance of filamentous fungi is relatively unknown. However, a number of recent papers have provided some information on this topic (Ah0 and Hirn, 1981; McLaughlin et al., 1983; Rosenzweig et aZ., 1983) and it appears that filamentous fungi are morc chlorine tolerant than are coliforms. Similar results have been observed with respect to ozone (Brewer and Carmichael, 1979; Haufele and Sprockhoff, 1973).
E. YEASTS Although the passage of yeasts through wastewater treatment plants has received considerable attention (Cooke, 1970), corresponding studies have not been conducted for water treatment plants. Several studies have compared the chlorine and ozone resistance of yeasts with that of coliforms (Haufele and Sprockhoff, 1973; Jones and Schmitt, 1978; Rosenzweig et al., 1983; Farooq and Akhlayue, 1983) and have found that yeasts are generally more resistant to these two disinfectants than are coliforms.
MICROBIOLOGY OF POTABLE WATER
87
F. ALGAE The occurrence of algae in drinking water treatment plants has been reviewed for different sections of the United States by Palmer (1958, 1960, 1961) and by Palmer and Poston (1958). These articles provide a detailed review of the historical literature from the last century on algae in drinking water treatment plants as well as an analysis of more recent information. Algae are important in surface water treatment because they frequently clog both slow and rapid sand filters (Palmer, 1958). Consequently they can reduce the duration of filter runs from 100 to 5 hours (Palmer, 1961). These problems are usually highly seasonal, with the greatest clogging in the late summer months (Baylis, 1922). Diatoms are frequently problem organisms, mainly because their silica shells clog filters even though the protoplasm inside has been inactivated. Algae are also important because they can cause taste and odor problems (Izaguirre et al., 1982; Palmer 1958, 1960, 1961; Palmer and Poston, 1958; Smith, 1972; Task Group Report, 1966),as well as an increase in trihalomethane (THM) precursors (Briley et al., 1978; Oliver and Shindler, 1980; Veenstra and Schnoor, 1980). The chlorination of these precursors results in chlorinated organics that may be carcinogenic (Jolley, 1978; Jolley et al., 1978, 1980), and consequently are limited to 100 pg/liter by the USEPA. Although coagulation and filtration, depending upon operating conditions, will remove varying levels of algae from raw water (Palmer, 1958, 1960, 1961; Palmer and Poston, 1958), copper sulfate (Joint Discussion, 1954; Flentje, 1952; Muchmore, 1978), chlorine (Joint Discussion, 1954; Kay et al., 1980), and chlorine dioxide (Ringer and Campbell, 1955) have been relatively effective in controlling algal levels in raw and in finished waters. Unfortunately, the chlorination of large numbers of algal blooms in finished drinking water increases THM levels and releases nutrients which may be utilized by aftergrowth bacteria. G . PROTOZOA
Over the years, several investigators have tried to determine the impact of different water treatment practices on protozoa numbers in finished drinking water (Sobsey and Olson, 1983). Initially, work centered on removal or inactivation of Entamoeba, whereas more recently investigations of Giardia have become more frequent (Logsdon et al., 1981). When properly performed, coagulation, flocculation, sedimentation, and filtration can remove 90-99% of protozoan cysts in water (Baylis et al., 1936; Jakubowski and Hoff, 1979; Logsdon et al., 1981; Sobsey and Olson, 1983). Outbreaks of waterborne illness caused by protozoa have frequently been attributed to improper operation of treatment plants (Kirner et al., 1978; Lippy, 1978; Shaw
88
B E m H. O L S O N A N D LASLO A. NAGY
et aZ., 1977). Chlorination can further reduce levels of protozoa by an additional 90-99% (Chang and Fair, 1941; Cooper and Bowen, 1983; Jarroll et al., 1981; Rice et al., 1982). Other disinfectants such as ozone and chloramines exhibit a similar degree of inactivation (Newton and Jones, 1949; Wolfe et al., 1984). Currently, small utilities are being advised by the USEPA to use slow sand filtration to remove Giardia cysts.
IV. Distribution Systems Sobsey and Olson (1983) reported that 27% of the waterborne disease outbreaks between 1971 and 1977 occurred due to contamination of the distribution system. This fact indicates that there is a substantial need to better understand the functioning of distribution systems in order to protect the microbiological quality of the water after it leaves the treatment plant. The possibility of microbial aftergrowth in distribution systems was first suggested in the published literature about 50-60 years ago (Baylis, 1930; Committee on Water Supply, 1930; Powell, 1921; Schaut, 1929; Whipple et al., 1977), but the topic has received relatively little attention until the early 1970s. At that time, certain principles from aquatic and terrestrial microbial ecology were adapted and applied to drinking water distribution systems. This microbial ecosystem approach has provided valuable explanations and raised some important public health and system management questions.
A. VIRUSES There is some controversy about the presence of viruses in drinking water distribution systems. Most investigators agree that viruses can be isolated from drinking water that has been improperly treated or subjected to contamination (Cookson, 1974; Dennis, 1959; Hejkal et al., 1982; IAWPRC Study Group on Water Virology, 1983; Mahdy, 1979; Rao et al., 1981; Roy and Tittlebaum, 1982; Roy et al., 1981b; Sobsey, 1975; Sobsey and Olson, 1983), but there is disagreement about the presence of viruses in properly treated and maintained distribution system waters. Although other microbial groups are discussed in terms of their presence in drinking water and on distribution system wall/pipe surfaces, this separation is impractical for viruses, as all the work thus far has involved possible isolations from the actual drinking water.
I. Viruses in Drinking Water Several French investigators reported in 1966 and 1967 the presence of viruses in 8-9% of drinking water samples analyzed from Nancy and Paris (Taylor, 1974). Partly as a result of these findings, ozone levels were in-
MICROBIOLOGY OF POTABLE WATER
89
creased in France, and subsequent surveys have not been able to recover viruses from drinking water (McDermott, 1974; Taylor, 1974). In 1972 the USEPA reported the results of a preliminary survey of viruses in two Massachusetts communities, in which 9% of the samples were positive for viruses (McDermott, 1974). Another USEPA-funded study reported poliovirus in effluent from a supposedly functioning water treatment plant (Hoehn et al., 1977). Partly as a result of the above findings, the USEPA conducted a more detailed survey in which all possible external sources of viral contamination were reduced or controlled. This study examined 255 drinking water samples from 56 communities and found no viruses in any of these waters (Clarke et al., 1975; Committee Report, 1979; Report to Congress, 1978). However, more recently, investigators have isolated viruses from apparently well-functioning treatment plants (Payment, 1981; ShaEer et al., 1980; Sekla et al., 1980)These studies suggest that naturally occurring viruses have a significantly lower chlorine inactivation rate than their laboratory cultured counterparts (Payment, 1981; ShaRer et al., 1980). It may not be possible to provide definite answers about the occurrence of viruses in drinking water until some standardized sampling techniques are developed. Several researchers have investigated viral recovery techniques (Berman et al., 1980; Farrah et al., 1976; Fenters and Reed, 1977; GuttmanBass and Armon, 1983; Rao et al., 1968, 1981; Sobsey and Glass, 1980; Sobsey et aE., 1981). The fourteenth edition of Standard Methods for the Examination of Water and Wastewater (APHA, 1976) contained a tentative prodedure. This procedure has been evaluated (Sobsey et al., 1980a) and some additional changes have been suggested (Sobsey et al., 1980b) as a result of the evaluation. 2 . Importance of Viruses in Drinking Water Environments
The public health importance of viruses in drinking water has been reviewed by a number of authors (Cookson, 1974; Hutchinson and Ridgway, 1977; Sobsey, 1975; Sobsey and Olson, 1983). Where viruses are present in drinking water due to improper treatment or distribution, serious outbreaks can result (Clarke and Chang, 1959; Dennis, 1959; Hejkal et al., 1982; Mayr, 1980). The inactivation of viruses by various disinfectants was recently reviewed by Leong (1983) and by Sobsey and Olson (1983). A considerable amount of research has been conducted on this topic and apart from investigating the impact of chlorine, researchers have tried to assess the viricidal efficiency of a number of other disinfectants, including chlorine dioxide (Alvarez and O’Brien, 1982), bromine chloride (Keswick et al., 1981), chloramine (Gowda et al., 1981), ammonia (Craemer et al., 1983), iodine (Alvarez and O’Brien,
90
BETTY H. OLSON A N D LASLO A. NAGY
1982), sodium fluoride (Eubanks and Farrah, 1981), and ozone (Roy et al., 1981b, 1982). Although results are influenced by disinfectant concentration, water quality, pH, and a variety of other factors, it appears that viruses generally are slightly more resistant to disinfection than are coliform bacteria. With the currently available information, it is not possible to accurately judge the ecological or system management importance of viruses in drinking water distribution systems. However, unlike the other groups of microorganisms discussed in this review, it is highly improbable that in a wellhnctioning distribution system virus levels would increase as the water moved from treatment plant to the consumer.
H. BACTERIA 1. Bacteria in Drinking W a t e r By the early 1920s it was well established that bacterial levels in drinking water increase due to passage through a distribution system (Powell, 1921). This was further substantiated by Schaut (1929), Baylis (1930), and the Committee on Water Supply (1930). Powell (1921) in a survey of 32 municipal water plants indicated that 92% experienced aftergrowth in their treated water. Schaut (1929) found higher bacterial levels in winter months when chlorination was less effective. Baylis (1930, 1938) pointed out that under certain conditions aftergrowths occur in distribution systems, probably when chlorine residuals are lost, or when a chlorine-free zone develops in pipe sediments or at the pipe surface. Furthermore, he demonstrated a log increase in bacterial numbers as a result of passage through a distribution system, and he demonstrated a log increase in bacterial numbers as a result of passage through the distribution system, and he showed a possible association between bacterial and coliform numbers in distribution pipe sediments. Bushwell (1938) described various food sources that microorganisms in drinking water may be utilizing, and indicated that bacteria may be deriving their energy from the oxidation of ammonia. At about the same time, Howard (1940) observed that there is a greater tendency for bacterial aftergrowth in the summer months. This was the opposite of what had been observed by Schaut (1929),but Howard suggested that higher summer numbers would be due to faster bacterial multiplication, more frequent loss of a chlorine residual, or greater introduction of bacteria from the pipe surface into the water as a result of greater variations in water pressure. He also stated that the possibility of properly treated water degrading to unsafe levels as a result of bacterial aftergrowth was extremely unlikely, and thus
MICROBIOLOGY OF POTABLE WATER
91
ruled out any public health importance assignable to microorganisms in drinking water distribution systems. The above-mentioned assumption was soon questioned by some of the findings of Shannon and Wallace (1941, 1944). These researchers were the first to carry out a comprehensive bacteriological analysis of drinking water consisting of isolation, enumeration, and identification techniques (Shannon and Wallace, 1944). They examined drinking water from various dead ends in the Detroit distribution system and observed numerous coliforms as well as members of the genus Salmonella. Shannon and Wallace (1941, 1944) also isolated several other genera including Flavobacterium, Achromobacter, Pseudomonas, Alcaligenes and Proteus. They were also the first to use lower incubation temperatures (20 instead of 37°C) and extended incubation times (96 instead of 48 hours). Howard’s (1940)assumption about the lack of public health importance of microorganisms in properly treated water was further questioned by investigators who found coliforms surviving chlorination, or reinfecting distribution systems once chlorine levels decreased (Charlton, 1933; Levine et al., 1939, 1942; Mellman and Fontes, 1955). The research generated since the early 1970s can be divided by environment, into investigations of bacteria isolated from drinking water and those isolated from drinking water distribution system wall/pipe surfaces. It is generally accepted that bacterial aftergrowth occurs on surfaces; however, as a result of problems of ready access to such surfaces and the lack of established techniques of sampling them, the majority of studies have examined bacteria in the actual water. This approach is not entirely unjustified as microbial aftergrowths on pipe surfaces generally would only become a problem once they reenter the passing water. Since the early 1970s a large number of studies have observed, enumerated, and in some cases identified bacteria in drinking water; and a number have reviewed the general topic [Allen, 1978, 1979, 1980, 1981, 1982, Anon, 1981; Bonde, 1977; Englebrecht, 1983; Geldreich et d.,1972; Greenberg, 1983; Herman, 1978; Hutchinson and Ridgway, 1977; National Academy of Sciences-National Research Council (NAS-NRC) 1982; NATO, 1982; Olivieri, 1983; Pipes, 1983; Reasoner, 1983; Seidler and Evans, 1983; Sobsey and Olson, 19831. Studies conducted in the 1970s were based on a better understanding of the need to integrate physical, chemical, and engineering data into the evaluation process. Therefore, more complete information was gathered on treatment of the water prior to entering the system and on the chlorine residuals maintained than in earlier published reports. A survey of the literature also indicates that most studies conducted outside of Europe (specifically Germany) contain little or no information on the chemical properties of the associated water. The importance of chemical information is well docu-
92
BETTY H. OLSON A N D LASLO A. NAGY
TABLE IV
RELATIONSIIIPBETWEEN TOTALORGANIC CARBON (TOC) I N A WATERSOURCEA N D BACTERIAL NUMBEMU
Source
(mdliter)
Geometric mean (CFU/ml)
Well River Moorland reservoir Impounding reservoir Lowland impounding reservoir Stored river water Direct river abstraction
0.2-0.3 1.7 1.0-1.1 1.0 4.6 2.4-2.9 3.13.8
32 18 100 347 4075 4OOO51,OOo
TOC
1 4
Dissolved oxygen (% change in distribution system)
0 to -2 0
0 to -5 -9
-5 -9 to -11 -15 to -25
Adapted from Ainsworth et al. (1980)
mented in corrosion and disinfection field studies, but has generally obtained less visability in studies of the microbial quality of distribution systems. The role of pH, total organic matter, and dissolved oxygen in promoting bacterial growth is frequently not addressed in field studies and when addressed appears to make little sense. The data in Table IV suggest a direct relationship between the amount of total organic carbon (TOC) in a water supply and the colony forming units of bacteria recovered per milliliter. Further, dissolved oxygen in the distribution system of waters generally decreases as organic content and bacterial numbers increase. It appears that bacterial numbers have a greater effect on reducing oxygen than does TOC. A decrease in dissolved oxygen also suggests an increase in chlorine demand, making it more difficult to maintain a chlorine residual. Data in Table V have been extracted from a number of studies to provide information on the types of bacteria that occur in water supply systems. Table VI shows the numbers of microorganisms likely to be present in various treated water as that water moves from the source to the consumer. None of the studies reported here followed the same sample of water as it moved through the system, but rather, in the best cases, relied upon numerous samplings to provide representative data at the two points within the system. In Tables V and VI four types ofwater are denoted: river, aqueduct, lake, and well/ground. Investigators now make a distinction between samples collected at the well and those collected directly from a groundwater aquifer. Because of the difficulty in sampling aquifers, the literature contains few reports on bacteria in these waters. In the southwestern United States water is often transported hundreds of miles from its source through open or
TABLE V OCCURRENCE OF BACTERIAL GENERA IN WATER SUPPLY SYSTEMS
Cincinnati, dead end Dis. S Y S . ~ . hydrant. hydrant dis. ys.
+ + + +
-
Well Lake hf ichigan, dis. sys.. hydrants
Artificial
-
-
-
-
+
+
Rhine. di5. sys.
laked
Treated dis. sys.
+ -
-
-
-
-
-
-
+ +
+
+
+
+
-
-
+
+ 4 + +
-
-
+ +
-
-
-
-
35°C 48 hours MSPC SP Olson and Hanami
35°C
SPC MF Nash and Geldreich
(1980)
(1980)
48 hours
-
W C 3 days
v MF Dott and Schoenen (1981)
-
WC 20°C 3G72 hours 55 days TGE NSM YF SP Shannon and Dott and Wallace (1944) Tampisch
+
NR
(1981.
SPC NR Reilly and Kippin
1983)
(19831
Wellell” tap
Welld
-
-
+ +
+ +
+ +
+ +
-
-
-
-
-
-
-
Dis. sys. Customer hydrantd tap
+
.t
-
+
-
f
I I
-
-
38C 35°C 35°C 48 hours 48 hours 48 hours 48 hours NR MSPC MSPC MSPC SP NR SP SP Olson Olson Lamke el Olwn and and and d. (1980) Hanami Hanami Hanami ilW@
Treated, imless otherwise indicated. Chlorination only. L‘ DistrihutioIh systetn. Untreated. e Category not used. I Observed via scanning eledron microscope studies. g RT, Room temperature; NR, not reported. h MA, Low nutrient [Reasoner and Geldrich, 1984); MSPC, bl standard plate count (Taylor and Geldrich, 1 W 1 : SPC, standard plate count (Difco); V, various media; TGE, tq-ptose glucose extract (Difco); NSM, nonselective media; NR not reported. SP, Spread plate: MF, membrane filtration; NR, not reported. 2’
TABLE VI BACTERIAL NUMBERSRECOVEREDFROM SOURCEA N D DISTRIBUTION SYSTEM LOCAT~ONS OF VARIOUSWATERS
Source water
Ground Ground Ground WelUspring Rhine River Rhine River River ~
O7
Aqueduct Aqueduct Aqueduct Aqueduct Aqueduct
water water water water water
Aqueduct water
System Well Well Distrib. system Distrib. system Finished water reservoir Finished water clearwell Distrib. system Trunkline Distrib. system Trunkline Distrib. fiystem Open finished water reservoir
Lake
Open finished water reservoir Open finished water reservoir Distrib. system
Lake, reservoir
Uistritr. systetn
Aqueduct water
Location Polyamid pipe Well water Hydrant Customer tap Surface water column Wall
CFU/mP 1-50 60-8 X 102 40-4 X 103 5-1.9 X lob 106 103 2.2 x 109
Schoener~and Dott (1982) Olson et al. (1980) Olson et al. (1950) Lamka et al. (1980) Schmnen and Dott (1982)
C,Fl,S,F,Cld C, F1,S,F,CI C,Fl,S, F,CI C1 CI c,H,S, F, c1
NR 0.5-0.1) 0.28-0.85
Nash and Geldreich (1980)
0
Olson et al. (1980) Olson et al. (1980) McCoy et al. ( 1 9 s ) McCoy et d.(1983) Olson et al. (1980)
103-106 0-350b
C,F1, S, F,CI
0
Silverman et al. (1983)
104
C, Fl,S, F,CI
0
Ward et al. (1982)
5-48 X 18 (37"C)b, 40-80 x 1 8 (U)"C)b <3->500
C1
0.15-0.33
Shannon and Wallace (1944)
C,FI, S, F,CI
0->I.O
Reilly and Kippin (198.3)
102-104 70
Middle and dead
Reference
0.15 0 0 NRC 0.15 0.15 0.3
Hydrant Hydrant Pressure vdve Customer tap TOP Middle Bottom Water column
sudace Dead end hydrant
Clz residual (mgAiter)
None None None None? Complete Complete CompIete?
Dead end hydrant
5 meters below
Treatment
3.2 X 102 35-170 102- 103 1@~-1W
1.32 1.0
Scboenen and Dott (1977)
105
end
Mean values. Forty-eight-hour incubation. NH, Not reported. C, Coagulation; GI, chlorination; S, settling: F, filtration; FI, flocculation.
96
BETIT H . OLSON AND LASLO A. NAGY
closed cement-lined canals to where it is treated and delivered. The long distance in transport sufficiently changes the characteristics of the water to reflect those of the transport system, necessitating the source of these waters to be classified as aqueduct and not as the site of origin. Of the 12 studies reported on in Table V, Acinetobacter and Alcakgenes were isolated from 73% of the systems, while Flauobacterium and Pseudomonas occurred in YO and 100% of the systems, respectively. Grampositive or gram-variable genera were found to occur less frequently, but were present in 27 and 36% of the systems sampled, respectively. Coliforins isolated from nonselective media were frequently identified in 58 and 83%of the systems examined, and were represented respectively by Klebsiella spp. and Entwobacter spp. Stuph ylococcus saprophyticus was found in several supplies (Nash and Geldreich, 1980) and Staphylococcus aureus has been reported by Lamka et al. (1980) and LeChevallier and Seidler (1980). Aeromonos spp. were isolated from all groundwater systems and one river supply system. Generally, these data indicate a remarkable concurrence in the types of organisms isolated even though a variety of incubation temperatures, durations, media, and isolation techniques were employed. Identification schemes also employed various techniques, with the German contributions using the most sophisticated of these (numerical taxonomy) (Dott and Schoenen, 1981). The data in Table VI indicate that bacterial numbers are highly variable at both source and distribution locations regardless of the nature of the water supply. Groundwater counts ranged from 1 to lo3 CFU/ml. Generally, numbers were lower if a chlorine residual was reported. Surface waters (river, aquaduct, and lake) contained greater numbers of organisms even in the presence of a chlorine residual. The highest reported value, lo9 CFU/ml, came from the surface of a clear well (Schoenen et al., 1979). As will be discussed later, surface colonization of distribution systems or source water supply facilities can lead to the introduction of organisms into the system in a unpredictable manner. Even at free residual chlorine levels of greater than 1mg/liter bacterial numbers can exceed lo2 CFU/ml. However, in general, highest values are reported for supplies with low chlorine residuals.
2. Bacteria on Drinking Water Distribution System WalllPipe Surfaces By the early 1970s aftergrowth in distribution systems was thought to be largely a result of microbial growth on pipe surfaces in the distribution system and its subsequent reentry into the passing water (Baylis, 1938; Ewing and Hopkins, 1930; Howard, 1940; Larson et al., 1960; Shannon and Wallace, 1944). The ability of various microorganisms, and in some cases
MICROBIOLOGY OF POTABLE WATER
97
macroorganisms, to grow and survive on the surface of drinking water pipes was relatively well established by the turn of the century (Whipple et al., 1927). As early as 1876, Hartwig Petersen described minute animals inhabiting the water pipes of the city of Hamburg (Whipple et al., 1927). These findings were substantiated around the turn of the century by other investigators in several other water distribution systems (E. G. Smith, 1903; 0. T. Smith, 1904; Whipple, 1899; Whipple et al., 1927). During the next 60-70 years, the ability of pipe surface microorganisms to influence water quality was most clearly demonstrated by iron and maganese bacteria. Numerous investigators observed these organisms in the water andlor on cast iron pipe surfaces (Alexander, 1974; Berry, 1932; Brown, 1934; Clark et al., 1967; Grainge and Lund, 1969; Hasselbarth and Ludemann, 1972; Leuschow and Mackenthun, 1962; McMillan and Stout, 1977; Tenny, 1939; Myers, 1961; Wilson, 1945), resulting in considerable corrosion and water coloration. These growths are usually not a public health hazard, however, colored water invariably reduces consumers’ confidence in their water utility, and may encourage the use of aesthetically more pleasing but microbiologically inferior alternatives. Although iron and manganese bacteria provided the best example of a relationship between events at the pipe surface and subsequent water quality, in the early 1970s it was strongly believed that other microorganisms could also survive and multiply at the pipe surface, or within sediment at the bottom of such pipes, and at a later time reenter the water column (Baylis, 1930, 1938; Ewing and Hopkins, 1930; Howard, 1940; Larson et al., 1960; Shannon and Wallace, 1944; Shindala and Chisholm, 1970; Victoreen, 1969). The resurgence of activity in drinking water microbiology in the 1970s was largely a result of increased availability of research tools such as scanning electron microscopes, vital stains, and a wider range of media. Also, investigators found that coliforms and total count bacteria were surviving chlorination, or were protected once within the distribution system. The complexity of the issues facing researchers caused them to place greater emphasis on understanding the microbial ecology of distribution systems. In addition, increased interest in drinking water quality came about as a result of the United States Safe Drinking Water Act of 1974. Two critical papers of this period were by Victoreen (1969) and Geldreich et al. (1972). The former presented the pipe-water interface as a highly heterogeneous microbial ecosystem, often removed from the direct influence of disinfectants, whereas the latter paper reaffirmed the public health and management significance of bacterial aftergrowth in drinking water distribution systems. Several investigators have observed the occurrence of coliforms on the surface of reservoirs (Ellgas and Lee, 1980; Seidler et al., 1977), on pipe surfaces from dead ends (Earnhardt, 1980), and in finished water reservoir
98
BElTY H. OLSON AND LASLO A. NAGY
sediments (Olson et al., 1980). The growth of coliforms has been found to be stimulated by tubercle deposits from drinking water pipes (Victoreen, 1980), and Salmonella species have been isolated from such pipe sediments (Muller, 1979). Burman and Colbourne (1977) and Colbourne and Brown (1979) have developed extensive procedures to determine the abilities of various materials used in drinking water systems to support microbiological growth. Similar research has been conducted for materials used in finished water reservoirs (Dott et al., 1979; Schoenen, 1980; Schoenen and Dott, 1979, 1982; Schoenen and Hotter, 1981; Schoenen and Thofern, 1981a,b; Schoenen et al., 1978; Thofern et al., 1978). Drinking water pipe surfaces have been examined by a number of investigators using a variety of microbiological media (Bigham and Tuovinen, 1983; Lee et al., 1980; Olson et al., 1981; Tuovinen and Hsu, 1982; Tuovinen et a l p ,1980) as well as scanning electron microscopy (Allen et al., 1980; Olson et al., 1981; Ridgway and Olson, 1982; Ridgway et al., 1981; Tuovinen et al., 1980). It is difficult to arrive at a comparison of bacterial numbers on the surface of walls and reservoirs, since results have been expressed in terms of CFUlmilliliter of biofilm (Dott et al., 1979;Thofern et al., 19781, CFWlmilliliter of sediment (Olson et al., 1981), or CFU/gram of sediment (Tuovinen and Hsu, 1982). When recalculated in terms of surface area, bacterial levels are usually in the loo-lo6 CFU/cm2 range on drinking water wall/pipe surfaces. Although relatively few investigations have identified heterotrophic bacteria on drinking water surfaces (Dott et al., 1979; Nagy, 1984; Olson et al., 1981), the organisms isolated have been found to be basically the same as those isolated from the actual water (Table V). 3. lmportance of Bacteria in Drinking Water Environments a . Public Health. Investigators have also isolated from well-functioning drinking water systems bacteria of definite public health importance such as Legionella (Brown et al., 1982; Tison and Seidler, 1983; Yee and Wadowsky, 1982; Wadowsky et al., 1982), Klebsiella pneumoniae (Ptak et al., 1973; Reilly and Kippin, 1983; Seidler et al., 1977), Yersinia enterocolitica (Weber et al., 1981a,b), S. aureus (Lamka et al., 1980; LeChevallier and Seidler, 1980), and Salmonella (Mendis et al., 1976; Muller, 1979; Schubert and Scheiber, 1979; Sinegre et al., 1975). In addition to these organisms a number of bacterial genera (see Table V) which are classified as opportunistic pathogens have been routinely encountered. These include Pseudomonas, Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium, Klebsiella, Enterobacter, Serratia, Proteus, Providencia, Citrobacter, and Acinetobacter. These organisms usually only produce infections in indi-
MICROBIOLOGY OF POTABLE WATER
99
viduals who are in some manner compromised (postoperative or immunosuppressed patients, the elderly, and newborn babies). The fact that many of these organisms are found in water supplies, and that they are capable of growth in water (as in the case of Pseudomonas aeruginosa) (Favero et al., 1971; Carson et al., 1972) indicate a need to understand their pathogenicity for high risk groups in the population. This is especially true for certain institutions such as convalescent and nursing homes, and acute care hospitals. Routine analysis of new hospital water supplies has revealed as many as 3000 to 4000 CFU/ml (Eichorn et al., 1977).
b. Disinfection in Relation to Coli&orms. A number of studies have identified and/or enumerated coliforms in drinking waters, usually with respect to chlorine levels (Allen et al., 1976;Armstrong et al., 1981; Clark and Pagel, 1977; Clark et al., 1982; Earnhardt, 1980; Goshko et al., 1981, 1983; Haas et al., 1983; LeChevallier et al., 1980; Lister, 1979; Martin et al., 1982; Mossel et al., 1977; Ptak et al., 1973; Seidler et al., 1977; Talbot et al., 1979; Tracy et al., 1966; Victoreen, 1980), or high recoveries (Evans et al., 1981a,b,c; Feng and Hartman, 1982; Fujioka and Narikawa, 1982; Hsu and Williams, 1982; LeChevallier et al., 1983a,b; McDaniels and Bordner, 1983; McFeters et al., 1982; Nash and Geldreich, 1980; Seidler et al., 1981; Standridge and Delfino, 1982). The most frequently isolated coliforms appear to be Enterobacter (Clark and Pagel, 1977; Clark et al., 1982; LeChevallier et al., 1983a) and Citrobacter (Evans et al., 1981b). As pointed out by Seidler et al. (1981), the isolation of these two genera is just as important from the public health and system management viewpoint as is the isolation of Escherichia or Klebsiella species. c. Systems Management. Microorganisms not only represent potential health threats, but they also serve as excellent tools to evaluate how well a system is functioning. Recent investigations have illuminated the inadequacies of the coliform. test. The standard plate count (SPC) (APHA, 1980) will be modified in the sixteenth edition of Standard Methods. This modification changes the name of SPC to the heterotrophic plate count (HPC), because several new methods have been introduced. These methods should allow utilities the option to select a medium and enumeration technique suitable to the individualities of each system. Although bacterial numbers fluctuate seasonally, a pattern is soon established for a system if samples are processed on a regular basis and are representative of the system. As opposed to the coliform test which is mandated by law, the HPC is currently proposed as a guideline. The object of this latter method is not to achieve zero or any particular value but rather to be able to follow trends throughout a system. If values suddenly deviate in an upward manner from the norm,
100
BETTY H. OLSON A N D LASLO A. NAGY
this can provide an important clue as to the location and magnitude of the problem. Although most outbreaks occur due to major problems, i.e., crossconnections, a number are not easily explained, especially in the distribution system. Again, an understanding the system as a whole is necessary; this is best achieved by an ecological approach.
C. ACTINOMYCETES Actinomycetes are frequently found in aquatic environments (Cross, 1981) and consequently in drinking water distribution systems (Burman, 1973; Dott and Waschko-Dransmann, 1981). As in the section on bacteria, the discussion below encompasses the occurrence of actinomycetes in drinking water, and on drinking water distribution system walllpipe surfaces; followed by a description of their importance in drinking water environments.
1. Actinomycetes in Drinking Water Numerous studies examining the occurrence of bacteria in drinking water have reported the isolation of actinomycetes, even though no special isolation techniques were used for the latter group (Armstrong et al., 1981; Lamka et al., 1980; Olson and Hanami, 1980; Reilly and Kippin, 1983). Other studies have attempted to isolate and enumerate actinomycetes specifically, usually using membrane filtration and selective media (Bays et al., 1970; Burman, 1965; Burman et al., 1969; Donlan, 1983; Hsu and Lockwood, 1975; Niemi et al., 1982). Media employed include chitin agar (Bays et al., 1970; Burman, 1965; Burman et at., 1969; Donlan, 1983), nutrient and actinomycete isolation agar (Niemi et al., 1982), and starch-casein agar (Donlan, 1983); incubation temperatures and durations have usually been 20-22°C and 7-14 days, respectively. Donlan (1983) found that starchcasein agar recovered higher levels of actinomycete CFU than chitin agar, with no significant difference between 7 and 14 days incubation. Standard Methods for the Examination of Water and Wastewater (fifteenth edition, APHA, 1980) suggests the use of starch-casein agar with a pour overlay technique, although on this medium, membrane filtration may be superior to both the pour overlay and spread plate procedure (Donlan, 1983). Levels of actinomycetes in drinking water are usually in the 1O0-1@ CFU/100 ml range (Bays et al., 1970; Burman, 1965, 1973; Niemi et al., 1982) although numbers as high as 103 CFUllOO ml have been reported (Bays et al., 1970). Representatives of the genus Streptomyces are probably the most frequently isolated actinomycetes from drinking water (Burman, 1973) although Nocardia and Micronwnospora species have also been reported (Bays et al., 1970; Burman, 1965, 1973).
MICROBIOLOGY OF POTABLE WATER
101
2 . Actinomycetes on Drinking Water Distribution System WalllPipe Surfaces Actinomycetes have been observed microscopically in biofilm on the surface of a drinking water reservoir (Dott et al., 1979), as well as in drinking water pipe encrustations in several different systems (Allen et aZ., 1980). Furthermore, they have been isolated from reservoir biofilm (Schoenen et al., 1979) and from rubber joints used to seal drinking water pipes (Hookey et aZ., 1980; Leeflang, 1963, 1968). No quantitative assessments are available to indicate the possible levels of actinomycetes per given area of surface, and it is doubtful if such assessments will be feasible; however, both Streptomyces (Hookey et al., 1980; Leeflang, 1963, 1968) and Nocardia (Hookey et al., 1980; Schoenen et al., 1979) have been isolated from drinking water surfaces. As indicated earlier, these genera are also the frequently found actinomycete genera in the actual drinking water.
3. lmportance of Actinomycetes in Drinking Water Environments As far as can be ascertained, actinomycetes in drinking water systems are of no public health importance. However, in terms of distribution system ecology and management they require considerable attention. Actinomycetes are the microorganisms primarily responsible for taste and odor problems in drinking water (Bays et al., 1970; Larson, 1966; Lewis, 1966; Silvey and Roach, 1953, 1959). Most of these actinomycete taste and odor problems are attributable to certain members of the Streptomyces (Bays et al., 1970; Lewis, 1966; Raschke et al., 1975; Rosen et al., 1970; Silvey, 1966), although some Micromonospora (Morris, 1962) have also been implicated. In addition to contributing to a degradation in water quality, actinomycetes in drinking water distribution systems may also contribute to the degradation of the physical system. Because they are able to grow on or in rubber used for sealing pipe joints (Leeflang, 1963, 1968) they may produce economic problems due to earlier replacement and higher water loss through faulty pipe joints.
D. FILAMENTOUS FUNGI Studies of filamentous fungi in drinking water distribution systems have been considerably less numerous than those on bacteria. However, even the limited currently available information indicates that filamentous fungi can be isolated from most drinking water environments. As in the previous section, studies on these fungi can be divided into those addressing occurrence in drinking water or on drinking water distribution system surfaces.
102
BETIT H . OLSON A N D LASLO A . NAGY
1 . Filamentous Fungi in Drinking Water A number of studies have made reference to the occurrence of filamentous fungi in drinking waters (Burman et al., 1969; de Malignon, 1976; Mackenthun and Keup, 1970; O’Connor et al., 1975; Rae, 1981). In most of these instances filamentous fungi were observed in hydrant flushings or water taken from dead ends. Several investigations have also enumerated, and in some cases identified, filamentous fungi from drinking water (Bays et al., 1970; Burman, 1965; Nagy and Olson, 1982; Niemi et aZ., 1982). These enumerations have generally used membrane filtration (Qureshi and Dutka, 1976; Sherry and Quereshi, 1981) and subsequent incubation at room temperature for 7-10 days on media such as Martin’s (Burman, 1965), Czapek (Nagy and Olson, 1982) or malt extract (Niemi et al., 1982). Standard Methods for the Examination of Water and Wastewater (APHA, 1980) recommends either neopeptone-glucose (similar to Martin’s), Czapek, or yeast extract-malt extract agar for the isolation of filamentous fungi from water. Results of fungal enumerations are usually expressed in CFU1100 ml of water, with typical levels in the loo-102 CFU1100 ml range. Filamentous fungi isolated from drinking water generally belong to the Deuteromycotina, or imperfect fungi, with genera such as Ceyhalosporium, Verticillium, T r i c h o d e m , Penicillium, Sporocybe, Acremonium, Fusarium, Alternark, and Epicoccum commonly observed (Bays et al., 1970; Burman et aE., 1969; Nagy and Olson, 1982). With respect to filamentous fungi, great care should be taken in assigning relative ecological importance to different genera based upon the levels of spores or CFU recovered. There is considerable variation in the spore-forming ability of different genera, and the number of spores enumerated may not be an accurate representation of that organism’s true occurrence and environmental importance in drinking water distribution systems (Bays et al., 1970). 2 . Filawntous Fungi on Drinking Water Distribution System WallIPipe Surjiaces
Filamentous fungi have been noted on, or isolated from, the surface of drinking water reservoirs (Dott et al., 1979; Dott and Thofern, 1980; Schoenen and Dott, 1977; Schoenen et al., 1979, 1981; Thofern et al., 1978), drinking water tanks (Seidler et al., 1977), drinking water pipes (Nagy and Olson, 1984), and pipe joints (Roesch and Leong, 1983). Some of these studies have also enumerated and identified these organisms, using media such as Sabouraud dextrose, Czapek, and nutrient agars. Results have been expressed as CFU/milliliter of slime (Thofern et al., 1978), CFU/gram of slime (Schoenen et al., 1979), or CFU/100 cm2 of surface (Nagy and Olson,
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1984). Levels of filamentous fungi in reservoir biofilm or slime have been in the l@-105 CFU/g range (Schoenen et al., 1979), whereas on pipe surfaces the observed numbers were 10°-104 CFU/100 om2 (Nagy and Olson, 1984). Genera identified have usually included members of the Deuteromycotina such as Penicillium, Cladosporium, Cephalosporium, Verticillium, Tric h o d e m , Fusarium, Alternaria, and Epicoccum (Nagy and Olson, 1984; Schoenen et al., 1979; Thofern et al., 1978). These genera are the same as those isolated from drinking water (Bays et al., 1970; Burman, 1965; Nagy and Olson, 1982) and this result substantiates a similar relationship as noted for bacteria in regard to the presence on surfaces and in the distribution water itself. 3. Importance of Filamentous Fungi in Drinking Water Environments The possible public health importance of filamentous fungi in drinking water has been indicated by the isolation of a pathogenic fungus, Petriellidium boydii, from drinking water pipe joints (Roesch and Leong, 1983), and by the observation that some fungi in aquatic environments produce humic substances (Day and Felbeck, 1974) which may subsequently act in the presence of chlorine as precursors for trihalomethanes (THM). THMs are formed in the presence of chlorine, and because many of this class of compounds are suspected human carcinogens their levels in drinking water are limited to 100 pglliter in the United States. Furthermore, a number of filmentous fungi isolated from drinking water have been found to be positive for mycotoxin production, at least under laboratory conditions (Rosenzweig et al., 1983). As stated in Standard Methods for the Examination of Water and Wastewater (APHA, 1980), the “amount of chlorine or other disinfectant required for fungal control is essentially unknown.” However, several recent studies have indicated that filamentous fungi, in both the vegetative and spore form, are somewhat more resistant to chlorination than are coliforms (Ah0 and Hirn, 1981; Rosenzweig et al., 1983). Fungi growing in pipe joints may be particularly well protected from the effects of chorination, as a 350-400 ppm initial dose over a 3-day contact period was insufficient to inactivate the previously mentioned pathogenic fungus in core samples taken from caulk in pipe joints (Roesch and Leong, 1983). Consequently, the pipeline had to be abandoned. From the limited information available, the resistance of filamentous fungi to ozone appears to be similar to that of bacteria such as E . coli and P . aeruginosa (Haufele and Sprockhoff, 1973). With the currently available information, it is not possible to accurately judge the ecological and management importance of filamentous fungi in drinking water distribution systems. However, it is clear that some filamen-
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tous fungi can grow actively in such environments, as indicated by observations of fungal mycelia in biofilm on the surface of drinking water reservoirs (Schoenen and Dott, 1977; Schoenen et al., 1981), drinking water storage tanks (Seidler et al., 1977), and drinking water pipe joints (Roesch and Leong, 1983). Filamentous fungi have also been found to increase in number as water moves from the treatment plant to the consumer (Bays et al., 1970), contribute to taste and odor problems (Burman, 1965), and frequently appear in hydrant washings from dead ends (OConnor et al., 1975; Rae, 1981).
E. YEASTS Yeasts, or nonfilamentous fungi, have received even less attention in drinking water systems than their filamentous counterparts. Although a number of studies have observed yeasts in drinking water systems, very few have identified and even fewer have enumerated these organisms in the water or on surfaces. 1. Yeusts in Drinking Wuter
Yeasts have been isolated from hydrant washings (O’Connor et al., 1975), tap washings (Reilly and Kippin, 1983), and from regular drinking water (LeChevallier et al., 1980). They were found to number 2.3%of the bacterial population (LeChevallier et al., 1980) on media formulated for bacterial isolation and enumeration. Rosenzweig et ul. (1983) isolated and identified three species from drinking water (Cryptococcus luurentii, Rhodotorulu glutinis, and Rhodotorula rubra), which they subsequently used in chlorine resistance experiments. Stantlard Methods for the Examination of Water and Wastewater suggests the use of Diamalt agar or quantitative enrichment agar for isolating yeast from water, although Sabouraud dextrose (supplemented with 33.3 mg/ml rose bengal and 80 mg/ml streptomycin) has also been used effectively (Rosenzweig et al., 1983). Membrane filtration on a suitable medium would probably provide the most acceptable results (Buck and Bubucis, 1978). The accurate enumeration of yeasts in drinking water is complicated by their nonfilamentous growth pattern, which makes their colonies difficult to distinguish from the usually more numerous but similar looking bacterial colonies. Only microscopic examination for cell size (at x400 or X1000) allows differentiation of the two groups.
2 . Yeasts on Drinking Water Distribution System WalllPipe Surfaces Only one investigation so far has reported on the occurrence of yeasts on the surface of drinking water pipes (Nagy and Olson, 1984). In this study
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yeasts were enumerated on Czapek, nutrient, and Sabouraud dextrose agars (using microscopy to differentiate between yeast and bacterial colonies) and then identified using the AP1-2OE system. Frequently occurring yeasts were Cryptococcus albidus, C . laurentii, Rhodotorula minuta, R . glutinis, R. rubra, and Sporobolomyces salmonicolor, some of which have also been isolated from drinking water (Rosenzwieg et al., 1983). 3. lmportance of Yeasts in Drinking Water Environments
It is difficult to evaluate the public health or ecological/management importance of yeasts in drinking water distribution systems. There is relatively little information from drinking water environments on this group of microorganisms; however, the topic would probably be a promising area of research, especially since one yeast, Candida albicans, has been suggested as a possible indicator organism in water quality analysis (Buck and Bubucis, 1978; Sherry et al., 1979; Svorcova, 1982). Yeasts thus far isolated and identified from drinking water are of some public health importance, but are not actual pathogens. The chlorine resistance of yeasts from drinking water is slightly higher than that of bacteria (Rosenzweig et al., 1983) but their ozone resistance is approximately the same (Farooq and Akhlaque, 1983; Haufele and Sprockhoff, 1973).
F. ALGAE Algae can be observed in most aquatic environments, and consequently are present in drinking water environments, particularly if a light source is present. In a survey of water supplies (Mackenthun and Keup, 1970), algae were the organisms creating the greatest number of reported problems, affecting about three times as many supplies as iron bacteria, the next highest category. (For the purposes of this discussion blue-green algae will be regarded as part of the algae group.) 1 . Algae in Drinking Water Numerous studies and reports have pointed out the widespread occurrence of algae in drinking water (Baylis, 1930; Berry, 1932; Committee Report 1953, 1983; Committee on Water Supply 1930; Kay et al., 1980; Mackenthun and Keup 1970; Palmer, 1960, 1961; Rae, 1981; Silverman et al., 1983; Task Group Report, 1966). Most of this widespread occurrence results from the use of uncovered finished water reservoirs within the distribution system (Baylis, 1930; Committee Report, 1983; Committee on Water Supply, 1930; Palmer, 1960, 1961; Silverman et al., 1983). Several investigators have attempted to enumerate and in some cases identify algae in such finished drinking water reservoirs (Kay et al., 1980). These enumera-
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tion procedures rely on light microscopy, with a number of different techniques available in Standard Methods for the Examination of Water and Wastewater (APHA, 1980). These techniques usually express their results in terms of algae cellslmilliliter with average numbers for finished drinking water reservoirs in the 10'-10$ cells/ml range (Kay et at., 1980; Silverman et al., 1983; Sykora et al., 1980). The water entering these reservoirs is usually of relatively high quality with very low leyels of algae. However, during the time period of detention, water quality decreases and algal numbers increase (Committee Report, 1953, 1983; Committee on Water Supply, 1930). This increase can be relatively dramatic (Kay et al., 1980; Silverman et al., 1983) and usually occurs in the warmer months. One of the main parameters influencing algae numbers in such finished drinking water reservoirs is temperature, however reservoir flow-through rate may also be critical (Kay et al., 1980; Silverman et al., 1983). Frequently observed algal genera are green algae such as Chlorella, Ankistrodesmus, and Scenedesmus; the bluegreen, Schizothrix, and the diatom, Glenodinium (Kay et al., 1980). Of these, Chlorella appears to be particularly widely distributed and adapted to finished drinking water reservoirs (Kay et al., 1980; Silverman et al., 1983). Water from reservoirs frequently carries with it an algal load, and despite chlorination practices, the algae frequently survive passage through the distribution system (Kay et al., 1980).
2. Algae on Drinking Water Distribution System WalllPipe Surfaces Algae, usually diatoms, have often been observed in encrustations on the surface of drinking water pipes (Allen et al., 1980). Organisms associated with these walls are usually of the same genera as those found in high numbers in the water (Kay et al., 1980; Smith, 1972) once again reinforcing the previously noted similarities between microorganisms in the water and those attached to surfaces. 3. Importance of Algae in Drinking Water Environments
Although algae themselves have minimal public health significance in drinking water systems, their presence in such systems could result in some secondary public health concerns. The tastes and odors that algae may add to water could cause a loss of public confidence in the water supplier (Palmer, 1960) and the utilization of alternative, perhaps less wholesome, sources. Furthermore, the chlorination of water leaving finished drinking water reservoirs could substantially increase the THM content of those waters (Hoehn et al., 1980; Kay et al., 1980; Veenstra and Schnoor, 1980; Oliver and Schindler, 1980). As stated above, THMs are suspected human carcinogens, and their levels are strictly regulated by the USEPA. With the exception of copper in reservoirs, the impact of various disinfec-
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tants upon the different algal populations in drinking water is relatively uninvestigated. Chlorine appears to be highly algicidal for Chlorella (Kay et al., 1980)and it is probably also relatively effective against other green algae. Chlorination can be a useful control measure for reducing algal blooms in finished drinking reservoirs although copper is probably more widely utilized (Committee Report, 1953, 1983; Committee on Water Supply, 1930; Flentje, 1952; Muchmore, 1978; Palmer, 1961). Most of the ecological or management problems caused by algae in drinking water systems originate in uncovered finished water reservoirs and would be dramatically reduced if the reservoirs were covered. This solution has been long obvious to the drinking water industry (Committee Water Supply, 1930); however, its implementation has been considerably more difficult.
G . PROTOZOA Apart from a few pathogenic species, protozoa have not been investigated in detail in drinking water distribution systems. However, several reports have pointed out their frequent occurrence in such systems (Chang, 1960, 1961; Cooper and Brown, 1983; Ingram and Bartsch, 1960) and since most protozoa feed on detritus and bacteria, their presence in drinking water is not unexpected.
1 . Protozoa in Drinking Water Protozoa in both the ciliate and amoeboid form are commonly observed in uncovered finished water reservoirs (Ingram and Bartsch, 1960). Amoebae have also been observed, during regular monitoring, in uncontaminated distribution systems (Chang, 1960; Cooper and Bowen, 1983). A number of different isolation and enumeration techniques, usually developed for pathogenic protozoa, are available (APHA, 1980). Approximately half of the treated potable water samples from one distribution system were positive for free-living amoebae (Cooper and Bowen, 1983). Numbers of amoeba ranged from <1 to 17 per 1000 ml with older parts and dead ends of the distribution system representing a disproportionately greater share (Cooper and Bowen 1983). The most frequently isolated genus in treated waters is Hartmannella (Chang, 1960; Cooper and Bowen, 1983), although Naegleria, Echinarnoeba, and Acanthamoeba have also been recorded (Cooper and Bowen, 1983).
2. Protozoa on Drinking Water Distribution System WalllPipe Surfaces Protozoa have been reported in slime or biofilm on the surface of finished water reservoirs (Dott et al., 1979; Schoenen and Dott, 1977; Schoenen et
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al., 1981; Thofern et al., 1978). Ciliate as well as flagellate forms have been observed (Dott et al., 1979; Schoenen et al., 1981), with numbers in one instance in the range of 105/ml of biofilm (Schoenen et al., 1981). The genera Bodo, Vorticella, Uronemu, and Euplotes have been observed in such environments (Schoenen et al., 1981).
3. Importance of Protozoa in Drinking Water Environments Most protozoa have little or no public health importance in drinking water environments. However, Giardia lamblia, Entamoeba histolytica, Balantidium coli, Naegleria floweri, and the genus Acanthamoeba are regarded as protozoan pathogens that can be acquired from known drinking water supplies (Sobsey and Olson, 1983). However, these pathogens usually are present only as a result of some gross contamination, or negligence. Relatively little is known about the impact of various disinfectants on the different protozoan groups. However with some pathogenic protozoa, such as Giardia, the cyst form is apparently more resistant to chlorine than are coliforms or viruses (Jarroll et al., 1981; Rice and Hoff, 1981; Rice et al., 1982). Additionally, cyst-forming amoebae are generally resistant to chlorine residuals greater than 0.3 mg/liter (Cooper and Bowen, 1983). The ecological or system management importance of protozoa in drinking water environments is difficult to evaluate. High protozoan numbers probably indicate high levels of bacteria or soluble organics in the system. The presence of non-cyst-formers signifies low to absent chlorine residuals. But both of these parameters (bacterial levels and free chlorine residuals) could be assessed directly, without a need to determine protozoan levels. Extremely high or sudden increases in protozoan levels should act as a warning to system operators, however, and the cause of the increase should be investigated in detail.
H. OTHERORGANISMS Although this review is specifically concerned with microorganisms, it should be mentioned that numerous other larger organisms can also occur in drinking water distribution systems (APHA, 1980; Ingram and Bartsch, 1960; Mackenthun and Keup, 1970). These include sponges (Ingram and Bartsch, 1960), water lice (Phillips, 1968), cyclops (Crabill, 1956), nematodes (Chang, 1960, 1961; Chang et al., 1959, 1960; Schonen et al., 1981;Thofern et al., 1978), and bloodworms (Silvey, 1956), as well as snails, clams, and insects (Ingram, 1956, Ingram and Bartsch, 1960).
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V. Distribution System Dynamics A. ECOLOCXCAL PROCESSES
A solid object such as a pipe surface in contact with flowing water will undergo a series of physical-chemical and biological modifications at the solid-liquid interface (Characklis, 1973a,b, 1981; Marshall, 1976). These modifications consist of four main stages (Characklis, 1981) which are summarized as follows: (1)absorption of organic molecules onto solid surface; (2) adhesion of microorganisms to this “conditioned” surface: (3)continued adhesion and/or multiplication of microorganisms forming biofilm at the solidliquid interface; and (4) dynamic equilibrium of biofilm with film growth and film destruction in relative balance. The overall process can be regarded as a microbiological succession starting with a conditioned surface that supports a primary, then a secondary, and finally a climax community of microorganisms (Alexander, 1971; Hudson, 1968; Webster, 1970). A distribution system pipe surface can thus be viewed as a microbial ecosystem increasing in complexity over time until the development of the dynamic equilibrium/climax state. From investigations of microbial successions at solid-liquid interfaces it appears that the primary colonizers are invariably bacteria with certain physiological characteristics. These characteristics allow the bacteria to produce sticky extracellular slime covers, generally composed of polysaccharides and water (Characklis, 1973a). Such slime coats would have obvious ecological advantages in drinking water systems as they would facilitate adhesion to surfaces and afford better protection against chlorine (Reilly and Kippin, 1983). Scanning electron microscope studies have in fact revealed the presence of bacteria with possible extracellular slime layers on distribution pipe surfaces (Ridgway and Olson, 1981). Once slime-producing bacteria colonize a pipe surface, inorganic and organic particles as well as cells of other microorganisms can become entrapped in the matrix (Characklis 1973a, 1981). The new arrivals can be bacteria or other microorganisms and they may grow and become part of the biofilm, or die and together with other dead cells add to the biochemical/nutritional diversity of the matrix. Microbial successions range in duration from a few months (Harper and Webster, 1964; Nagy and Harrower, 1979, 1980) to a number of decades (Rayner, 1977a,b). Generally, their duration is inversely proportional to the range and concentration of available nutrients in the particular ecosystem. In drinking water distribution systems the time required for the microbial succession to reach dynamic equilibrium is unknown, however, the relatively low levels of nutrients probably serve to extend the duration.
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A number of papers have suggested rate-limiting factors for microbial successions on distribution system pipe surfaces. These include physical, chemical, and biological parameters such as pH, temperature, redox potential, TOC, chlorine residual, hardness, mineral content, etc. (Geldreich et al., 1972; Goshko et al., 1981; Hutchinson and Ridgway, 1977; LeChevallier et al., 1981; Lee et al., 1980; NAD-NRC, 1982; Olson et al., 1981; Reilly and Kippin, 1983; Shindala and Chisholm, 1970; Tuovinen et al., 1980; Victoreen, 1974). There is widespread agreement that free residual chlorine is a critical rate-limiting factor for the microbial succession, as is total organic carbon (Ainsworth et al., 1980). There is also general agreement concerning the idea of microbial detachment from pipe surfaces. It has been suggested that the flow of water within the pipes enhances the removal particles and attached microorganisms from surfaces (Baylis, 1930, 1938; Geldreich et al., 1972; Goshko et al., 1981; Howard, 1940; Olson et al., 1981; Shannon and Wallace, 1944; Shindala and Chisholm, 1970; Tuovinen et al., 1980), which then reenter the water distribution system and are delivered to consumers. Depending upon free chlorine residuals and water utilization, these resuspended microorganisms may or may not be of public health importance. Although it is generally accepted that drinking water pipes act as sites for microbiological successions, that free residual chlorine is important in controlling both attached and detached microbial aftergrowths, and that microorganisms from the pipe surface can reenter the passing water, few studies have evaluated these ideas directly under field conditions.
B. SYSTEMSMODEL As suggested by previous authors (Anon., 1982; Armstrong et al., 1981; Baylis, 1930, 1938; Ewing and Hopkins, 1930; Goshko et al., 1981; Howard, 1940; Larson et al., 1960; Olson et al., 1981; Shannon and Wallace, 1944; Shindala and Chisholm, 1970; Tuovinen et aZ., 1980), flow rates influence bacterial levels in drinking water at a single site, such that as flow rates increase bacterial levels also increase per unit volume of water (Nagy, 1984). This has important monitoring consequences, as most distribution systems experience considerable flow fluctuations in any 24-hour period (Fig. 3; Walker, 1978). The correlation between flow rates and bacterial levels indicates that the microbiology of distribution systems is highly dynamic. Monitoring efforts often fail because the importance of flow changes on microbial levels is not considered. Disregarding an important component of sample variability may produce confounding results, unless samples are taken at the same time of day and site conditions remain very similar. An increase in the microbiological levels of drinking water as a result of
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passage through the distribution system is often but not always observed (Baylis, 1930, 1938; Geldreich et al., 1972; Hutchinson and Ridgway, 1977; MacKenthun and Keup, 1970; NATO, 1982; Victoreen, 1969, 1974). Hutchinson and Ridgway (1977) showed a decrease in average free residual chlorine levels (from 0.21 to 0.02 mg/liter) as the water moved from the intermediate to the end sections of the distribution system and a general increase in bacterial numbers per milliliter. Similarly, Reasoner and Geldreich (1979)found a 2 log increase in heterotrophic bacteria in drinking water as a result of passage through 42 km of pipes. However, Nagy (1984) observed a decrease in bacterial levels with distance traveled, as did Haas et al., (1983). A model to explain these contradictory results is presented in Fig. 4. This descriptive model relies on two factors which act as opposite forces on bacterial numbers. These are free chlorine residual (contact time) and detachment of bacteria from pipe surfaces. Most chlorine disinfection analyses indicate that after a contact time of 5-10 minutes very little additional change in bacterial numbers is observed (Safe Drinking Water Committee, 1980) at free residual levels of approximately 1 mg/liter. However, most disinfection studies have been conducted under laboratory conditions and it is highly probable that bacteria under field conditions would exhibit higher survival if not actual resistance (Safe Drinking Water Committee, 1980). There is evidence to indicate that heterotrophic bacteria recovered from chlorinated drinking water systems have a higher tolerance to chlorine than those isolated from unchlorinated systems (Ridgway and Olson, 1982). High levels of bacteria have on numerous occasions been isolated from drinking water with free residual chlorine levels in the range of 0.4-0.7 mg/liter (Olson et al., 1981). Laboratory studies for both bacteria and viruses
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have suggested increased tolerance after repeated exposure to this disinfectant (Leyval et al., 1983; Schatter et al., 1980). Field studies have shown that Legionella pneumophila, Flavobacterium sp., and nontuberculous mycobacteria lose their tolerance to chlorine after subculturing under laboratory conditions (Wolfe et al., 1984; Carson and Favero, 1984; Kutcha et al.,
1984). At relatively short contact times (0-30 minutes) considerable decreases are observed in bacteria levels due to inactivation. This is followed by a period (probably 1-6 hours), when the more resistant bacteria in the water are gradually inactivated. These bacteria may be biochemically or physically less susceptible to the biocidal effects of chlorination, due to such characteristics as clumping (Reilly and Kippin, 1983) or attachment to particulate materials (Ridgway and Olson, 1981). At these middle contact times, the contribution provided by detachment begins to increase gradually, such that a point is reached where the reduction produced by chlorine is the same as the increase from the pipe surface, as shown in Fig. 4. Beyond this contact time (probably over 8 hours), bacterial levels increase in drinking water as a result of passage down a distribution system, because the contribution from the pipe surface is greater than the reduction due to chlorination. This descriptive model (Fig. 4) brings together the observations of disin-
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fection studies using short contact times and usually high chlorine levels with field experiments that test water with a range of contact times and bacterial levels. Laboratory studies (Butterfield et al., 1943; Haas and Englebrecht, 1980a,b; Kruse et al., 1970; Morris, 1966; Safe Drinking Water Committee, 1980; Scarpino et al., 1974; Skaliy et al., 1980; Wang et al., 1982) have found dramatic decreases in bacterial levels over time in a given body of water, and because these experiments were carried out under controlled conditions, they could ensure that (1) the same water was resampled over time, (2) the chlorine demand remained stable, and (3)bacteria tested were washed and disaggregated. Field experiments on the other hand have usually, but not always, found a negative correlation between free residual chlorine and bacterial levels, although the same body of water was not resampled (Goshko et al., 1983; Haas et ul., 1983; Hutchinson and Ridgway, 1977; LeChevallier et al., 1980; Olson et al., 1981; Reasoner and Geldreich, 1979; Shannon and Wallace, 1944). Recent experiments have allowed resampling of the same body of water in the field and have also measured contact times (Nagy, 1984). Interestingly, the descriptive model predicts that free residual chlorine and bacterial levels will positively correlate in the early sections, not correlate over the middle sections, and show negative correlations in the later sections of the distribution system. This explains the observed results of Nagy (1984), where in field resampling (flow) experiments, there was a simultaneous decrease in both free residual chlorine and bacterial levels in an early section of a distribution system. The model also explains some unusual findings by other investigators (Goshko et ul., 1983) who have noticed significant positive correlations between chlorine levels and coliforms in some, and significant negative correlations in other distribution systems. According to the above model such results are possible if one system has a short and the other a long contact time before water reaches the consumer or the sampling site.
VI. Future Research As stated in Section I the need to understand how the organisms of interest function in potable water is of great importance. This is likely to be achieved only if an ecological approach is adopted. An incomplete understanding of systems and processes has led to anecdotal and contradictory results. The most obvious of these contradictions are found in the disinfection literature. The vast majority of disinfection work has focused on the use of laboratory or environmental pure culture organisms which are grown under optimal conditions, harvested, washed to remove chlorine demand, disaggregated, and then subjected to the disinfectant in sterile, demand-free
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water. Although such a system is excellent in allowing the development of models of disinfection, it is difficult to see how these studies relate to natural environments where bacteria are usually in a static state, aggregated, covered with extracellular polysaccharides, and in a water milieu containing one or more interfering compounds. All of these factors will influence disinfection efficiency. As the model in Fig. 4 indicates, a holistic view of a system often leads to sensible explanations for previously divergent views (in this instance disinfection efficiency) and does not require complete knowledge of all variables. This points to the need to develop methods which are concerned with estimating the survival of microorganisms under natural conditions. The recent work of Ward et al. (1982)indicated that mixed natural populations of microorganisms had distinctly different die-off curves than pure cultures in the presence of chloramines and free residual chlorine, reflecting differences in susceptibility to each disinfectant. Further, the concept of environmental strains being more “hardy” than culture collection organisms may be inaccurate. Ward et al. (1984) have shown that the phenomenon is not one of environmental versus culture collection, but rather one of strain differences in susceptibility. In the future it is hoped that greater emphasis will be placed on understanding the many competing and complex factors which exist under field conditions and that more techniques will be developed to assess disinfection efficiency under in situ conditions. Another area of growing concern (and of increasing appreciation of its complexity) is the meaning and validity of the coliform as an indicator organism. This subject seems to be discussed by scientists approximately every 20 years only to reconfirm the conclusion that the best available indicator is the coliform. Yet, with the methodology that exists today, it should be possible for microbiologists interested in potable water to develop more sensitive and specific measures of water quality. The relative conservatism of the water community in demanding foolproof solutions, which have as yet never been attained, inadvertently decreases the possibility for investigators to introduce innovation and perhaps to move the field forward. It strikes us as interesting that since the introduction of the basic coliform test in the late 1800s, little has changed except for the development of membrane filtration and a few modifications in the ingredients of the media. One could, of course, argue that the method was so good that it could not be improved upon, but all that is necessary to dispel this idea is to ask microbiologists what they think of the testing procedure and its relationship to public health. Unfortunately, microbiologists are told that alternative methods are too expensive or too difficult for water treatment personnel to handle. We believe that individuals involved in protecting the safety of drinking water should be required to be well trained and that testing should not be limited to tests which are inexpensive but only partially accurate.
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The relationship between indicators and incidences of waterborne disease outbreaks has resulted in various models. Figures 5 and 6 show the relationship between coliforms or fecal coliforms, respectively, and the risk of illness of viruses and SaZmonelZa. However, according to a compilation of data presented by Sobsey and Olson (1983), this relationship seldom seems to hold in actual outbreaks. The numbers of coliforms are usually exceedingly low if present at all and frequently no records exist on coliform levels. Even in the best documented case for coliform occurrence, the number of coliforms detected was far below the level which would have predicted disease according to these models. This points to the importance in understanding pathogen-indicator relationships and emphasizes, in the absence of such knowledge, the need for small systems to frequently monitor for indicators. Both microbiologists and engineers need to have a greater understanding of the interactions of the physical, chemical, and microbiological components of water treatment and distribution. The recent results of Wadowsky
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60
70
4
30 40 50 COLIFORM (10’ MPN1100rnl) 1
1
20 25 30 F E C A L COLIFORM (lo3 M P N I I O O ~ I )
10
1
12
a
I
L
10
1
I
35
40
FIG. 6. Relationship between disease risk and salmonella coliforins, and fecal coliforms (after Mechalas et al., 1972).
and Lee (1983) showing that LegionelZn’s presence in distribution systems is due to symbiotic relationships with the natural flora in pipe sediment have both public health as well as management implications. However, it is doubtful that such an understanding would have arisen without an applied ecological approach. Too often a report in the literature ignores the chemistry of a water supply or does not adequately describe the physical system. This lack of description leads to an incomplete understanding of the major question being asked, which in turn produces controversial results. In reality these contradictions can often be explained by simple differences of environment. Investigators with an incomplete understanding of an environment or an approach which is solution oriented may resolve a problem for the moment, but too often it recurs with no available explanation. This has produced a field of anecdotal works which contribute only in a minor way to the understanding of broad concepts and solutions toward which the field strives. Hence, as seen in this review, investigators in the 1970s were asking basically the same questions as those in the 1940s. If a clearer understanding of the ecological parameters which promote or decrease disease transmission in water systems is achieved, better design and operation of facilities will be possible which in turn will result in better protection of public health. The value of microbiology in understanding water treatment management and distribution maintenance has only recently been appreciated. The po-
MICROBIOLOGY OF POTABLE WATER
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tential now exists for drinking water microbiology, with its ecological approach, to move the field from a science concerned only with public health hazards into a broader based discipline concerned also with system management through the use of microorganisms. But here again, it is necessary to view the system as an integrated whole. Bacteria may play an important role in one phase of a process, such as corrosion, but they are often ignored in the examination of the problem because historically the process has been viewed to be only chemical or physical. In reality, it is generally accepted that few multicompartment systems function in this manner. The task ahead for potable water microbiologists is to extend their knowledge and training in the fields of chemistry, engineering, and ecology. If this is done many questions currently left unanswered will fall into place as the ecology of microorganisms in the treatment process or the distribution system is finally understood. REFERENCES Aho, R., and Hirn, J. (1981). A survey of fungi and some indicator bacteria in chlorinated water of indoor public swimming pools. 761. Zentralbl. Bakteriol. Hyg., Abt. 1 , Orig. Reihe B 173, 242-249. Ainsworth, R. G., Oliphant, R., and Ridgway, J. (1980). The Introduction of new water supplied into old water systems. Water Res. Center TR 146, 36. Alexander, M. (1971). “Microbial Ecology.” Wiley, New York. Alexander, L. (1974). Control of iron and sulfur organisms by super-chlorination and de-chlorination. Am. Water Works Assoc. 36, 1349-1355. Allen, M. J. (1978). Microbiology of ground waters. J. Water Pollut. Control Fed. 50, 13421344. Allen, M. J. (1979). Microbiology of potable water. J. Water Pollut. Control Fed. 51, 17471751. Allen, M. J. (1980). Microbiology of potable water. J. Water Pollut. Control Fed. 52, 18071812. Allen, M. J. (1981). Microbiology of potable water. J. Water Pollut. Control Fed. 53, 11091112. Allen, M. J. (1982). Microbiology of potable water. J. Water Pollut. Control Fed. 54,943-946. Allen, M. J., Taylor, R. H., and Geldreich, E. E. (1980). The occurrence of microorganisms in water main encrustations. J . Am. Water Works Assoc. 7 2 , 614-625. Allen, M. J., Taylor, R. H., and Geldreich E. E. (1976). The impact of excessive bacterial populations on coliforrn methodology. Proc. Am. Water Works Assoc. Water Qual. Technol. Conf., Denver pp. 3B-4, 1-7. Alverez, M. A,, and O’Brien, R. T. (1982). Mechanisms of inactivation of poliovirus by chlorine dioxide and iodine. Appl. Enwiron. Microbiol. 44, 1064-1071. American Public Health Association (1976). “Standard Methods for the Examination of Water and Wastewater,” 14th ed. American Public Health Association, Washington, D.C. American Public Health Association (1980). “Standard Methods for the Examination of Water and Wastewater,” 15th ed. American Public Health Association, Washington, D.C. Anon. (1981). Biological quality of water in the distribution system. In “Drinking Water and
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Sobsey, M. D., and Glass, J. S. (1980). Poliovirus concentration from tap water electropositive adsorbent filters. Appl. Enoiron. Microbiol. 40, 201-210. Sobsey, M. D., and Olson, B. H. (1983). Microbial agents of waterborne disease. In “Assessment of Microbiology and Turbidity Standards for Drinking Water (P. S. Berger and Y. Argaman, eds.). EPA, Washington, D.C. Sobsey, M. D., Glass, J. S., Carrick, R. J., Jacobs, R. R., and Ratula, W. A. (1980a). Evaluation of the tentative standard method for enteric virus concentration from large volumes of tap water. J . Am. Water Works Assoc. 72, 292-299. Sobsey, M. D., Glass, J. S., Jacobs, R. R., and Ratula, W. A. (1980b). Modification of the tentiative standard method for improved virus recovery efficiency. J . Am. Water Works ASSOC.72, 350-355. Sobsey, M. D., Moore, R. S., and Glass, J. S. (1981). Evaluating absorbent filter performance for enteric virus concentrations in tap water. J . Am. Water Works Assoc. 73, 542-548. Standridge, J. H., and Delfino, J. J. (1982). Underestimation of total-coliform counts by the membrane filter verification procedure. Appl. Enoiron. Microbiol. 44, 1001-1003. Skeeter, H. W. (1927). Studies of the effect of water purification processes. Publ. Health Bull. 172. Svorcova, L. (1982). Yeasts in spa establishments. Zentralbl. Bakteriol. Nyg., Abt. 1 , Orig., Reihe B 176, 167-175. Sykora, J. L., Keleti, G., Roche, R., Volk, D. R., Kay, G. P., Burgess, R. A., Shapiro, M. A., and Lippy, E. C. (1980). Endotoxins, algae and Limulus amoebocyte lysate test in drinking water. Water Res. 14, 829-839. Talbot, H. W., Jr.. Morrow, J. E., and Seidler R. J, (1979). Control of coliform bacteria in finished drinking water stored in redwood tanks. J. Am. Water Works Assoc. 71,349-353. Task Group Report (1966).Nutrient-associated problems in water quality. J . Am. Water Works ASSOC., 58, 1337-1355. Taylor, F. B. (1974).Viruses-What is their significance in water supplies. J . Am. Water Works ASSOC. 66, 306-311. Tenny, M. K. (1939). Des Moines iron problem. J . Am. Water Works Assoc. 31, 96-104. Thofern, E., Schoenen, D., and Schoenen, R. (1978). Microbial settlement of paint- and building-materials in the sphere of drinking-water 1. Information: Long time observation of a bitumen paint in a drinking-water-reservoir. Zentralbl. Bakteriol. Hyg., Abt. 1 , Orig., Reihe B 167, 306313, Tison, D. L., and Seidler, R. J. (1983). Legionella incidence and density in potable drinking water supplies. Appl. Environ. Microbiol. 45, 337-339. Tracy, H. W., Camarena, V. M., and Wing, F. (1966). Coliform persistence in highly chlorinated waters. J . Am. Water Works Assoc. 58, 1151-1159. Tuovinen, 0. H., and Hsu, J. C. (1982). Aerobic and anaerobic microorganisms tubercles of the Columbus, Ohio water distribution system. Appl. Environ. Microbiol. 44, 761-764. Tuovinen, 0. H., Buttons, K. S., Vuorinen, A,, Carlson, L., Mair, D. M., and Yut, L. A. (1980). Bacterial, chemical and mineralogical characteristics of tubercules in distribution pipelines. J . Am. Water Works Assoc. 72, 626-635. Veenstra, J. N . , and Schnodor, J. A. (1980). Seasonal variations in trihalomethane levels in an Iowa River water supply. J . Am. Water Works Assoc. 72, 583-540. Victoreen, H. T. (1969).Soil bacterial and color problem in distribution systems. J . Am. Water Works Assoc. 61, 529-431. Victoreen, H. T. (1974). Control ofwater quality in transmission and distribution mains. J . Am. Water Works Assoc. 66, 369-320. Victoreen, H. T. (1980). The stimulation of coliform growth by hard and soft water main deposits. Proc. Am. Water Works Assoc. Water Qual. Technol. Conf:, Denver. pp. 111122.
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Wadowsky, H. M., and Yee, R. B. (1983). Satellite growth of Legionella pnevmophilu with an environniental isolate Flaovbucterium breve. Appl. Enoiron. Microbiol. 46, 1447-1449. Wadowsky, R. M., Yee, R. B., Mazmer, L., Wing, E. J., and Dowling, J. N. (1982). Hot water systems as a source of Legionella pneumophila in hospital and nonhospital plumbing fixtures. A p p l . Envirvn. Microbiol. 43, 1104-1110. Walker, R. (1978). “Water Supply, Treatment a d Distribution” Prentice-Hall, New York. Wang, W. L. L., Powers, B. W., Blaser, M. J., and Leuchtfeld, N. W. (1982). Laboratory studies of disinfectants against Campylobacter jejoni. Annzr. Meet. Am. Soc. Microbiol. C159. Ward, N. R., Wolfe, R. L., Means, E. G., and Olson, B. H. (1982). The inactivaton of total couut and selected gram-negative bacteria by inorganic monochloramines. Proc. Am. Water Works Assoc. Water Qual. Techno/. Conf. Denver. pp. 81-97. Ward, N. R., Wolfe, R. L., and Olson, B. H. (1984). Disinfectant activity of inorganic chloramines with pure culture bacteria: Effect of pH, application technique, and chlorine to nitrogen ratio. Appl. Enuiron. Microbiol. Weber, G . , Stanek, G., Massiczek, N., and Klenner M.-F. (1981a). First isolation of Yersinia enterocoliticu from drinking-water in Austria. Zentralbl. Bakteriol. Hyg., Abt. I , Orig., Reihe B 173, 207-208. Weber, G . , Stanek, G., Massiczek, N., and Klenner M.-F. (1981b). Yersinia enterocolitica in drinking-water. Zentralbl. Bukteriol. Hyg. Abt. I, Orig., Reihe B 173, 209-216. Webster, J. (1970). Coprophilous fungi. Trans. Br. Mycol. SOC. 54, 161-180. Whipple, G. C. (1897). Some observations on the growth of organisms in water pipes. J . N . Engl. Water Works Assvc. 12, 1-19. Whipple, G . C. (1899). “The Microscopy of Drinking Water.” Wiley, New York. Whipple, G. C., Fair, G . M., and Whipple, M. C. (1927). “The Microscopy of Drinking Water,” 4th Ed. Wiley, New York. Williams, B. M., and Richards, D. W. (1976). Salmonella infection in the herring gull (Larns argentatus). Veterinurian 51, 978. Wilson, C. (1945). Bacteriology of water pipes. J . Am. Water Works Assoc. 37, 52-58. Wolfe, R. L., Ward, N. R., and Olson, B. H. (1984a). Chloramines as drinking water disinfectants: A review. 1. Am. Water Works Assoc. 76, 74-88. Wolfe, R. L., Ward, N. R., and Olson, B. H. (1984b). Inactivation of natural aquatic bacterial populations by chlorine and chloramines. Wuter Res. In press. Yee, R. B., and Wadowsky, R. M. (1982). Multiplication of Legionella pneuniophila is unsterilized tapwater. Appl. Enuiron. Microbiol. 43, 1330-1334.
Applied and Theoretical Aspects of Virus Adsorption to Surfaces CHARLES P. GERBA Departments of Microbiology and Immunology, and Nutrition and Food Science, University of Arizona, Tucson, Arizona
.............................. 11. Mechanisms of Virus Adsorption . . . . . . . . . . . . . . . . . . . . . . . . . A. Theoretical Aspects .......................... B. Variables Affecting Virus Adsorption . . . . . . . . . . . . 111. Protective Effects of Virus Adsorption .................... A. Aquatic and Soil Environments ...................... B. Disinfection ....................................... IV. Inactivation of Viruses on Solid Surfaces . . . . . . . . . . . . . . . . . . A. Aquatic Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Metal Surfaces C. Soils . . . . . . . . . V. Applied Aspects of Virus Adsorption A. Wastewater Tre B. Advanced Wastewater and Drinking Water Treatment . . C. Land Application of Wastes ......................... D. Detection of Viruses in the Environment . . . E. Implications.. ..................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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149 149 151 153 153
157 160 161 163 163
1. Introduction
The response of viruses to their environment (i.e., inactivation) and to changes in their environment (pH, temperature, ionic strength, etc.) has, in the past, largely been characterized on the basis of the virus as a separate entity. Today it is realized that viruses in the environment are often associated with particulate matter or other surfaces and that this has a major effect on their persistence and transport in the environment. A major emphasis of previous studies has been on the role of solids in the transmission of waterborne human enteric pathogenic viruses. Members of this group include hepatitis A virus (enterovirus type 72), rotavirus, and Nonvalk virus, all of which are known to have caused numerous outbreaks of waterborne disease (Gerba et al., 1984). There are over 100 additional enteric viruses which are excreted in the feces of man and these could potentially be transmitted by water. It has become increasingly evident in the last decade that virus interactions with surfaces such as fecal material, clays, soils, biological and chemical flocs, etc. play a major role in determining their removal by sewage 133 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 30 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-002630-9
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and drinking water treatment processes, the effectiveness of disinfectants, contamination of groundwater, hydrotransportation in the marine environment, and persistence in the environment. Viral adsorption phenomena are also important in many methods currently in use for virus concentration and virus detection in the environment. This article emphasizes how the current understanding of the mechanisms and factors influencing virus adsorption can be used to interpret and control virus behavior in the environment.
II. Mechanisms of Virus Adsorption A. THEORETICAL ASPECTS
The types of interactions possible between an adsorbed molecule and a solid surface range from weak nonpolar van der Waals forces to strong chemical bonding. The latter, chemisorption, is characterized by a high specificity of interaction and by heats of adsorption in the range of 10-100 kcal/mol. It is physical adsorption which is of most interest in the consideration of virus adsorption to solid surfaces. The relative importance of the various forces ,which can be involved in adsorption will depend greatly on the nature of the adsorbent and virus, as well as on the associated environment. Virus particles are of a colloidal nature, and it is therefore expected that theories describing colloidal behavior are applicable to viruses in a dispersion medium. Dispersed particles have been observed to carry an electrical charge, yet a colloidal system as a whole remains electrically neutral (Venvey and Overbeek, 1948). This phenomenon is explained by the double-layer theory, developed in its original form by Gouy (1910, 1917), Chapman (1913), and Debye and Hiickel(1923). Accordingly, ions on particle surfaces attract ions of opposite charge (counterions) from the dispersion medium into a compact layer around the particle. This layer is called the Stern layer (Fig. l), and ions within it partially neutralize the change on the colloid (Stern, 1924). Other counterions are held further away, yet still in the vicinity, in a diffuse layer called the Gouy layer. The distance the diffuse second layer extends into the bulk solution determines the force and distance over which particles repel each other. Forces acting between particles due to their interacting double layers are not the only electrical forces of importance in colloidal systems. London (1930) described an additional type of force, always attractive, between atoms and molecules. This force is known as van der Waals attraction, an electrical force between instantaneous dipole moments within the different molecules. As calculated by London, the potential energy of the attractive forces between two atoms is inversely proportional to the sixth power of the distance between them (Fig. 1).
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VIRUS ADSORPTION TO SURFACES
I STERN LAYER
SOLID €2
-0
HYDROPHOBIC QROUPS \
H-0 H
FIG. 1. Diagrammatic representation of double layers and hydrophobic groups about a virus and solid. The presentation shows ionizing residues and surface hydroxyl groups thought responsible for charge development as explained by the electrical double-layer theory of colloidal chemistry; the Stern layer of bound cations and anions, and the Gouy layer with cation in+) excesses and anion (n-) deficits. €1, €2, and €3 are the complex dielectric susceptibility functions important in controlling the differences in magnitudes of van der Waals interactions with different materials. Hydrophobic groups on virus and solid may interact by exclusion of water molecules.
Interactions between two particles in a dispersion are thought to result from a balance between repulsive double-layer interactions and attractive van der Waals forces, best described by the DLVO (Derjaguin-LandauVenvey-Overbeek) theory of colloid stability (Verwey and Overbeek, 1948). A particle, such as a virus, immersed in an aqueous solution develops a surface charge by adsorbing ions on its surface. A fixed layer of oppositely charged ions develops around the surface of the adsorbent. To maintain the electrically neutral system, there is a diffused layer containing a sufficient number of counterions extending for some distance into the solution. If the bulk solution of counterions increases by addition of cationic salts or increasing pH, the thickness of this layer decreases because less volume is required to contain enough counterions to neutralize the surface charge. The reduction of the thickness of this layer facilitates the approach of the two surfaces, allowing van der Waals forces to have an effect. Murray (1978) showed the applicability of the DLVO theory to poliovirus adsorption to metal surfaces. He examined adsorption from the standpoint of equilibrium thermodynamics, and suggested that the overall free energy of adsorption resulted from contributions by various energy components (po-
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CHARLES P. GERBA
tentials). All of these potentials were said to arise from the interaction of electromagnetic fields, and might include contributions from double-layer, van der Waals, and covalent-ionic forces. When the contributions of these components, plus other possible bonding mechanisms (induced image forces, hydrogen bonding, and hydrophobic interactions) were evaluated, only double-layer and van der Waals contributions were found to be significant. The extent of adsorption of virus from suspension onto an adsorbent surface may be thought of as defining a distribution of virus between the surface and the liquid phase. This distribution reflects the position of equilibrium in the adsorption process. The extent of adsorption can be measured only as a net adsorption rate, yet the process is a dynamic one of adsorption and subsequent desorption of virus particles. Equilibrium is achieved when the rate of adsorption equals the tate of desorption. During adsorption, the amount of solute adsorbed per gram of solid depends upon several conditions, including the concentration of the solute, the temperature, and the nature of the particles involved. Several types of isothermal relationships have been described. The most common, and the one of particular interest with respect to virus adsorption, is obtained from systems in which adsorption is limited to the deposition of only a single layer of solute molecules on the surface of the solid. The Langmuir model is one such treatment, valid only for single-layer adsorption (Langmuir, 1918). The model is based upon three assumptions: (1) that maximum adsorption corresponds to a saturated monolayer of solute molecules on the adsorbent surface; (2) the energy of adsorption is uniform across the adsorbent surface, i.e., there is a homogeneity of active sites for adsorption; and (3) there is no interaction between adsorbed molecules on the surface. According to Langmuir, the rate of adsorption can be expressed as HateAds = L,C(X,-X) where kA = rate constant for adsorption, C = concentration of solute in , = total number of adsorption sites on the surface, X = dispersion, X number of sites occupied (amount of solute adsorbed), and X,,-X = number of available adsorption sites. The Langmuir isotherm is expected to describe adsorption of viruses to surfaces. Since adsorption is a displacement phenomenon, i.e., a molecule of liquid will be displaced by virus on the solid surface, the process would not be expected to continue beyond the formation of one layer. Moore et al. (1981)constructed isotherms for poliovirus type 2 adsorption to minerals and soils. Their data conformed well to the Langmuir equation and, from the degree of surface coverage of sand by the virus, they determined that at maximum adsorption under conditions of pH 7.5, only 1%of the total surface of the sand was covered.
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An isothermal relationship more commonly applied to virus adsorption is the Freundlich model (Freundlich, 1926). This treatment also assumes monolayer coverage of a surface by adsorbate, but makes no assumption for homogeneity among active sites for adsorption. The theoretical basis for the model is not as significant as for the Langmuir representation (Weber and Morris, 1964), yet it has satisfactorily described poliovirus and echovirus 1 adsorption to estuarine sediments (LaBelle and Gerba, 1979), bacteriophage R17 adsorption to allophane (Taylor et al., 1980), +X-174 adsorption to soil (Burge and Enkiri, 1978a), MS-2 adsorption to bentonite (Stagg, 1976), and poliovirus 2 to minerals and soil (Moore et al., 1981). The isotherm is expressed as X = KCn where, X = the amount of adsorbed viruslweight of adsorbent, C = the virus in suspension at equilibrium, and n and K are constants. The linear form of this equation is log X = log K + n log C and a plot of log X against log C gives a line having slope and intercept log K . Kinetic studies have been applied to the adsorption of virus particles to surfaces. Valentine and Allison (1959) studied the adsorption of latex particles, as models of virus particles, on metallic surfaces in static and agitated systems. Their studies also included vaccinia and fowl-plague viruses, and their data analysis led them to conclude that the process of adsorption was diffusion limited, i.e., dependent upon the rate of virus movement through suspending medium based upon Brownian motion of the particles. The boundary conditions of their theory called for the existence of a static layer of the suspending medium in contact with the adsorbent surface. This layer would remain static regardless of whether or not the system was agitated mechanically, and virus present in the layer would contact the adsorbent at a rate determined by a diffusion constant. The amount of virus adsorbed was found to be a linear function of the square root of time. Cookson (1967) studied the kinetics of adsorption of bacterial viruses on activated carbon. H e also found virus adsorption to be diffusion limited, and reported that the process may follow second-order kinetics. Filmer et al. (1971) studied adsorption of albumin molecules on silica particles as a model for adsorption of viruses to soils. They suggested that adsorption was a two-phase process involving movement of virus into the vicinity of the solid surface (by diffusion, flow velocity, or other mechanical means), and attachment of the virus to the adsorbent. The transport phase, being a slower process, would limit the actual amounts of adsorption. Experimental evidence suggested a diffusion-limited process when the fraction of adsorbed virus was plotted against the square root of time. Data conformed to the Freundlich isotherm.
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Stagg (1976) studied the adsorption rate of bacteriophage MS-2 on bentonite. The adsorption rate was first order with respect to unadsorbed virus, and the variation of rate constant with stirring speed indicated a diffisionlimited process. In contrast to earlier reports, Burge and Enkiri (1978a)concluded that the actual rate-determining process may not be diffusion. Rather, the number of approaches to or impacts on the adsorption surface before a virus hits an adsorption site would limit the process. They suggested that virus charge is therefore implicated. Their findings were in agreement with earlier observations by Krueger (1931). He had studied bacteriophage adsorption to host cells, and found that doubling the viscosity of the suspending medium by adding glycerol did not affect adsorption rate. His results were inconsistent with a diffusion-related process, which would be expected to be very sensitive to viscosity changes. Recent studies by Farrah and co-workers (Farrah et al., 1981a; Farrah, 1982; Shields and Farrah, 1983) have suggested that hydrophobic effects may play a significant role in virus-surface interactions. The results obtained in these studies with viruses, and in other studies with proteins (Hatefi and Hanstein, 1969) suggest that chaotropic salts and un-ionized compounds such as Tween 80, urea, and ethanol disrupt hydrophobic interactions, whereas antichaotropic salts promote such interactions. Ions which disrupt the structure of water and thereby enhance the accommodation of hydrophobic groups are called “chaotropic.’’ Conversely, ions which increase the ordering of water molecules and promote the sequestering of hydrophobic entities are called “antichaotropic.” It has been proposed that hydrophobic interactions are a result of the unfavorable interaction of apolar groups with water rather than of attraction between different polar groups (Ben-Naim, 1980). Consistent with this hypothesis, chaotropic agents are viewed as disordering the structure of water and thus reducing the thermodynamic barrier to the introduction of apolar groups to the aqueous environment (Hatefi and Hanstein, 1969). Solutions of chaotropic ions have been found to solubilize membrane proteins and organic compounds such as riboflavin and adenine and to disrupt antigen-antibody complexes (Dandiker et al., 1967; Farrah, 1982; Hatefi and Hanstein, 1969). Chaotropic ions are relatively large, singly charged ions such as trichloroacetate, thiocyanate, and iodide (Hatefi and Hanstein, 1974). In contrast, antichaotropic ions are generally small, singly charged ions such as fluoride or multivalent ions such as citrate, nitrate, calcium, or magnesium. These antichaotropic ions have been found to promote hydrophobic interactions. Presumably by increasing water structure, the ability of solutions to accommodate hydrophobic groups is reduced and hydrophobic interactions between apolar groups in the solution are increased. These antichaotropic salts have been shown to counteract the
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VIRUS ADSORPTION TO SURFACES
ability of chaotropic salts to disrupt hydrophobic interactions (Farrah, 1982; Farrah et al., 1981a). Chaotropic salts promote elution of viruses adsorbed to membrane filters used for virus concentration from water (Farrah et al., 1981a)and estuarine sediments (Wait and Sobsey, 1983). Un-ionized detergents such as Tween 80 and urea have also been used to elute viruses adsorbed to membrane filters, estuarine and freshwater sediments, and wastewater sludges (Farrah et al., 1981b; Bitton et al., 1982; Shields and Farrah, 1983). Solutions containing antichaotropic ions such as citrate, EDTA, and magnesium have been found either to promote virus adsorption to membrane filters or to be relatively ineffectual in eluting viruses adsorbed to filters (Fig. 2) (Farrah, 1982;Wallis and Melnick, 1967~). Shields and Farrah (1983) have hypothesized that at different pH values, the relative importance of electrostatic and hydrophobic interactions varies. During virus interactions with cellulose nitrate filters at high pH values (pH HYDROPHOBIC QROUPS
FILTER SURFACE
FIG. 2. Effect of chaotropic and antichaotropic agents on virus interaction with filter surfaces. In the top figure virus elution from the filter surface is promoted by chaotropic ions which disrupt hydrophobic interactions. In the bottom figure virus adsorption is promoted by antichaotropic ions which promote hydrophobic interactions (Shields and Farrah, 1983).
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CHARLES P. GERRA
9-11), both virus and filter surfaces are negatively charged (Mandel, 1971; Sobsey and Jones, 1979). Solutions having minimal effects on hydrophobic interactions (glycine buffer) or solutions capable of disrupting hydrophobic interactions (chaotropic salts and detergents) promote efficient virus elution (Fig. 2). In contrast, antichaotropic ions have no effect on elution. This suggests that at high pH, hydrophobic interactions may be the major factor maintaining virus-filter adsorption (Farrah et al., 19Sla). At low pH (pH 4), solutions which disrupt only electrostatic interactions or solutions which disrupt hydrophohic interactions alone do not elute appreciable amounts of virus. For example, for efficient elution of virus at low pH, both a detergent (Tween 80) and an antichaotropic salt (e.g., CaCl, or citrate) are necessary. These results can be explained by assuming that both electrostatic and hydrophobic interactions are crucial in maintaining virusfilter association at low pH. When a neutral detergent disrupts hydrophobic interactions, addition of a charged species or increase in pH is able to disrupt electrostatic interactions and bring about elution of the virus (Fig. 3). Alternatively, the charge can be incorporated as part of the detergent (e.g.,
HYDROPnOBlC
FILTER SURFACL
DETERGENT
FIG.3. Effect of antichaotropic agents, cations, and pH on virus interaction with filter surfaces. Figure at left illustrates virus adsorbed to filter at low pH in the presence of the nonionic detergent Tween 80. Electrostatic interaction prevents virus elution. The addition of a cation or increase in pH is able to disrupt electrostatic interactions and bring about elution of virus (Shields and Farrah, 1983).
VIRUS ADSORPTION TO SURFACES
141
cetyltrimethylammonium chloride), and elution’ is also a result (Shields and Farrah, 1983).
B. VARIABLESAFFECTING VIRUS ADSORPTION 1 . Nature of the Virus Virus adsorptive behavior depends greatly upon the virus being studied (Gerba et al., 1980). It has been suggested that differences in virion surface charge or hydrophobicity play a significant role in virus adsorption to solids (Bitton, 1975; Shields and Farrrah, 1983). Most viruses have coats composed of protein polypeptides that contain amino acids such as glutamic acid, aspartic acid, histidine, and tyrosine. These amino acids contain weakly acidic and basic groups (i.e., carboxyl and amino groups), which upon ionization give the viral capsid an electrical charge (Fig. 1). Each ionizing group in the polypeptide has a characteristic dissociation constant. The variation of dissociation constants among the various polypeptides ensures that most viruses have net charges that vary continuously with pH. At a characteristic pH, defined as the isoelectric point (PI), ionization is such that the virion exists in a state of zero net charge. At this pH, localized pockets of positive and negative charge will exist across the viron surface, depending upon the charge state of individual component amino acids (Mix, 1974). However, pZ data provide only an identification of overall virus charge under given pH conditions and cannot yield data concerning local areas of charge over the virion surface. Viruses will be positively charged below their isoelectric point and negatively charged above it. Isoelectric points of several viruses have been reported in the literature, and some of these data are summarized in Table I. The isoelectric point of a virus may vary not only by the type of virus but also by the strain (Zerda, 1982; Mandel, 1971). The variation in virus adsorption observed with soils, sludge, and estuarine sediments could be attributed to differences in their isoelectric points (Gerba et al., 1980). In a study of adsorption of various types and strains of viruses to various soils, Gerba et at. (1981)observed that by using factor analysis viruses could be grouped into two general categories (Table 11). For group I, the most important factors affecting adsorption were pH, organic matter, and exchangeable iron content of the soil. No studied soil characteristic was found to be significantly associated with adsorption of group I1 viruses. The implication is that adsorption-elution of group I viruses is more sensitive to certain soil characteristics than group 11. The coliphage f2 was placed in a third group as it adsorbed poorly to almost all the soil studied. An examination of the known isoelectric points would sug-
? Column I E F Horizontal IEF Microelectrophoresis Horizontal IEF Microelectrophoresis Moving boundary Moving boundary Moving boundaq Gel Moving boundary
Reference Floyd and Sharp (1978) Korant et nl. (1975) La Colla et al. (1972) Floyd and Sharp (1978) Ward (1978) Mandel (1971) Zerda (1982) Murray and Parks (1980) Zerda (1982) Zerda (1982) Zerda (1982) Zerda (1982) Zerda (1982) Murray and Parks (1980) Murray and Parks (1980) Salo and Mayor (1978) Mouillot and Netter (1977) Douglas and Williams (1969) Mouillot and Netter (1977) Douglas and Williams (1969) Miller et al. (1944) Beard and Wyckoff (1938) Sharp ct al. (1946) Childs and Birnhoim (1975) Zerda (1982)
IEF, Isoelectric focusing.
gest that group I viruses generally have lower isoelectric points than group I1 (Table I). The exception would appear to be T2,but this may be due to the fact that it is a tailed phage whose tail fiber configurational charges (at various pH values) may greatly affect attachment mechanisms (Cookson, 1970). Results of several studies indicate that knowledge of the pZ of a virus makes it possible to predict the likelihood of its adsorption to a charged surface, as long as the suspending conditions are known (Zerda, 1982; Fuhs and Taylor, 1982). Hydrophobic interactions can be expected to play a major role in the adsorption of lipid-containingviruses and their adsorptive behavior could be expected to be quite different than the more hydrophilic coliphages and enteroviruses. This is perhaps best illustrated by the studies of Stotzky
143
VIRUS ADSORPTION TO SURFACES
TABLE I1 VIRUSGROUPING BY ADSORPIWE BEHAVIOR TO .SOIL= Group 1 Coxsackie B4 (V216)b Coxsackie B4 (V240)
From Gerba et al. (1980). Codes in parentheses refer to virus strain.
(1980) and Stotzky et al. (1981)who observed that the adsorption of the lipidmembraned virus herpes hominis type 1to clays was very different from that of coliphages T1 and T7, and reovirus type 3. The polypeptides of the nonlipid-coated viruses contain hydrophobic regions. In general, proteins in an aqueous environment fold in such a way as to allow hydrophilic side chains to associate with the surrounding water molecules, while shielding hydrophobic side chains. In the case of membrane-bound proteins, hydrophilic side chains are exposed on the surface while hydrophobic side chains are located within the lipid bilayer. Evaluation of amino acid sequences can be used to predict whether particular segments of a protein are hydrophobic, and therefore likely to reside within the interior of a membrane. The complete amino acid sequences for the coat protein of MS-2 and for poliovirus protein VP4 have been reported (Weber and Konigsberg, 1975; Kitamura et al., 1981). The sequences were recently analyzed (Zerda, 1982) using a computer program designed to evaluate the hydrophobicity and hydrophilicity of a protein along its amino acid sequence. The program is highly indicative of internal and external portions of proteins whose structures have been studied (Zerda, 1982). A computer evaluation of the MS-2 coat protein sequence indicated that most of the spans of amino acids along the sequence are hydrophobic, separated by four to five interspersed hydrophilic runs (Zerda, 1982). Presumably, the hydrophilic spans reside on the surface of the virion, but since the complete removal of hydrophobic side chains of proteins from contact with water is not generally possible (Tanford, 1979), some hydrophobic portions are also exposed. Such spans would be expected to act as sites for association with other hydrophobic moieties. Because of the possible significant role of hydrophobic forces in the ad-
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CHARLES P. GERRA
sorption of enteric viruses to at least some surfaces, an understanding of the relative degree of hydrophobicity may be useful in explaining and predicting the degree of adsorption of different viruses to surfaces.
2 . Nature of the Surface The role of adsorbent charge in virus adsorption has been considered in
detail for soils, filters used to concentrate viruses from water, and metal surfaces. Knowing the net charge on the virus and adsorbent has been seen as an aid in predicting and controlling virus adsorption to surfaces. The actual charges on the virus and surface will vary depending upon the type and concentration of ions in solution and pH but these can be predicted from experimental data. The isoelectric points of substances that have been used in virus adsorption studies arc shown in Table 111. As with viruses, the isoelectric point is defined to be that pH at which the solid has a net electrical potential of zero. Below the isoelectric point the solid has a positive charge and above it, it has a negative charge. When the electrostatic components of adsorption are considered by themselves, solids that have high isoelectric points are better virus adsorbents TABLE 111 ISUELECTRIC POINTS OF VAHIOUS NATURALA N D AHTIFICIAI.S O l ~ i D S IMPORTANT I N V l H U S ADSORPTION Solid Soil and mineral surliaces Quartz a-SiOe Corundum a-Al203 Albite NaA1Si30s Hematite cu-Fe203 Pyrolusite a-MnOa Birnessite 6-Mn02 Tenorite CuO Kaolinite AI(Si40,0)(OH)8 Montmorillonite Allophane Filters Cellulose nitrate, cellulose acetate Epoxy fiberglass (Filterite) Fiberglass-ashestos (Cox) Zeta-plus (50s) Zeta-plu, (1MDS)
PI
Reference
6.5
Murray and Parks (1980) Murray and Parks (1980) Murray and Parks (1980) Murray and Parks (1980) Murray and Parks (1980) Murray and Parks (1980) Murray and Parks (1980) Murray and Parks (1980) Murray and Parks (1980) Fuhs and Taylor (1982)
1.5-2.0 1.0-1.5 3.0 5.2 6.5 3.5
Keswick and Wagner (1978) Sobsey and Jones (1979) Sobsey and Jones (1979) Sohsey and Jones (1979) Hou et nl. (1980) Hou at al. (1980)
2-3.5 5-9.2 2.0 4.2-9.3 7.3 1.5
9.5 <2-4.6 52.5
VIRUS ADSORFITON TO SURFACES
145
than those with low values. For example, A1,0, could be considered a better adsorbent than SiO, on this basis alone (Murray and Parks, 1980). High values for the isoelectric point may correspond with a greater probability of the solids having a net positive zeta potential at the pH of a natural water system. If the virus has a net negative charge under these conditions, electrostatic forces would be attractive, leading to a greater adsorption compared to a solid that would have a negative zeta potential under these same conditions. An understanding of the significance of net charges on the virus and adsorbent has led to its application in the development of microporous filters for virus concentration from water. Thus, Sobsey and Jones (1979) and Hou et al. (1980) demonstrated that microporous filters modified to have a net positive charge were better adsorbents of viruses and bacteria from tap water than filters with a net negative charge. It is important to point out that virus behavior toward a charged surface will vary also with the type of virus because of differences in the net charges between types and strains and even among populations (Burge and Enkiri, 1978b). The possible interactions between virus and adsorbent are best illustrated by the work of Zerda (1982). In this study adsorbent particles of defined surface chemistry were prepared by reacting colloidal silica with organosilanes under controlled conditions and selectively adding residues containing primary amine, quaternary amine, or carboxyl groups to silica surface hydroxyls. The amine-modified silica was positively charged over a pH range of 4.0-8.5 while the carboxyl-modified silica was negatively charged under the same conditions. Adsorption of MS-2, T2, and reovirus type 1 (all with isoelectric points near pH 4.0) and poliovirus strains LSc and Brunhilde with higher isoelectric points of 6.6 and 7.1 to these solids at various pH values was studied. It was found that all viruses adsorbed exclusively to negatively charged silica at p H values below their isoelectric points, i.e., under conditions favoring a positive surface charge on the virions. Conversely, all of the viruses adsorbed to positively charged silica at pH values above their isoelectric points, i.e., where virus surface charge was negative, Viruses in near isoelectric state adsorbed to a small degree to all types of modified silica. But, because of the differences in charge between surfaces and viruses, optimal pH conditions for maximum adsorption varied greatly among the viruses. For example, at pH 4.0 no significant adsorption of MS-2 occurred to the carboxyl treated surface, while over 99% of the poliovirus adsorbed. In contrast, at the same pH, 90% of the MS-2 adsorbed to the amine-treated surface while no poliovirus adsorbed to the same surface. This phenomenon can be used as a tool in developing methods for virus concentration and separation, as well as in explaining differences in virus behavior during water and wastewater treatment.
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CHARLES P. GERBA
Clays and other naturally occurring solids may have surfaces that have a very heterogeneous distribution of charges at their surfaces. For example, a greater concentration of positive charge is associated with the crystalline edges of kaolinite, although some adsorption occurs to the outer surfaces (Stotzky et al., 1981). Viruses with different isoelectric points may also be expected to adsorb to different regions of the same clay. Thus, reoviruses have been reported to adsorb primarily to negatively charged sites on clays, while T1 and T7 coliphages to positively charged sties (Schiffenbauer and Stotzky, 1982; Lipson and Stotzky, 1983). Competition between virus groups for adsorption sites has also been observed for clays (Stotzky et al., 1981). The difference in the ability of various materials to generate large van der Waals potentials is probably the most important factor in many cases according to Murray and Parks (1980). The Lifshitz theory, which can be used to quantify van der Waals interactions (Murray and Parks, 1980), predicts the following general series of adsorbent effectiveness on the basis of the dielectric properties of the materials: Metals (strong) > Sulfides > Transition Metal Oxides > SiOz > Organics (weak)
A comparison of Lifshitz-van der Waals potentials for various materials, as reported by Murray and Parks (1980), is shown in Table IV. The extent of virus interaction with the various materials listed in Table IV appears to correspond closely to that observed in laboratory studies, i.e., weak adsorption of viruses to organic surfaces like polystyrene and SiO, and strong adsorption to aluminum metal (Murray, 1980).Murray and Parks (1980) also believe that since the magnitudes of the van der Waals potentials are larger than double-layer interactions at ionic strengths found in most natural waters (O.O2), Lifshitz predictions may well be more important than electrokinetic TABLE IV LIFSIIITFVAN DEH WAALSPOTENTIALS FOR VARIOUS SOLIDS~ uvdw
considerations in adsorption of viruses to different materials in natural waters. This theory predicts that most organics would be poor adsorbents of virus except at low pH and high ionic strength. However, the predicted weak effectiveness of organics is appropriate for characterizing only general, nonspecific types of interactions, and cannot be applied to other types of interactions, A rule of hydrophobic interactions involving various surfaces and virus adsorption has not yet been formulated, but this matter is certainly deserving of future study.
3. Salts and pH The tendency of viruses to adsorb strongly to various materials at high ionic strength is well known and consistent with electrostatic double-layer interactions. Carlson et al. (1968)found that adsorption of phage T2 (with a low isoelectric point) on montmorillonite, kaolinite, and illite at p H 7 increased with increasing concentrations of NaCl and CaC1,; and CaC1, was as effective as NaCl at one-tenth the concentration. When electrophoretic mobilities of the clays in the electrolytes were determined, virus adsorption in CaCl, increased with decreasing clay mobility. Again, since colloids are negatively charged, divalent Ca2 would be particularly effective in reducing surface potentials and consequently promoting adsorption. However, the clays did not display mobility changes as NaCl concentration was increased, even though adsorption was enhanced. This suggests that doublelayer interactions were of secondary importance. Since NaCl is an antichaotropic salt the increased NaCl concentrations could have encouraged the enhanced adsorption by strengthening hydrophobic interactions, as suggested by Farrah et al. (1981a). Studies of virus adsorption to microporous filters and soils also indicate that divalent and trivalent salts are more effective than monovalent salts in promoting virus adsorption (Wallis et aZ., 1972; Lance and Gerba, 1984). The effective concentrations of trivalent salts are 1% those of divalent salts. Mix (1974) has proposed that the effect of electrolytes is more than a simple neutralization of charges. Rather, multivalent ions can link virus and adsorbents of like charge by forming salt bridges between them. Keswick and Wagner (1978) described this phenomenon as a cross-complexation of the cation with groups on the two surfaces simultaneously. Divalent cations such as Mg2+ would not be expected to complex as effectively as trivalent cations such as A13+ ; and monovalent ions would be incapable of crosscomplexation. The enhanced adsorption of virus to negatively charged surfaces by cations as described by electrostatic interactions also accounts for decreased adsorption of viruses at high cation concentrations when both surfaces become +
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CHAHLES P. GERBA
positively charged (Murray and Parks, 1980). This effect is also more pronounced on surfaces possessing a net positive charge. Thus, Hou et al. (1980) observed that increasing concentrations of cations decreased adsorption of virus and negatively charged polystyrene beads to positively charged filters. As discussed in the previous sections, salts such as magnesium sulfate can promote virus adsorption to membrane filters by strengthening hydrophobic interactions (Farrah, 1982). Anions also affect hydrophobic interactions and have been shown to affect virus adsorption to filters and soils (Farrah, 1982; Lance and Gerba, 1984). The dependence of virus adsorption to surfaces upon pH has been demonstrated many times and has been assumed to indicate the effects of surfacecharge development (Fuhs and Taylor, 1982). Zerda (1982) studied in detail the effects of pH and charge development on virus adsorption (see Section 11, B, 1). In general, high pH favors free virus and low pH favors adsorbed virus, although isoelectric points of both the virus and surface may play roles in this interaction.
4 . Organic Mutter Dissolved organic matter tends to compete with virus adsorption sites and thereby reduce virus adsorption. Wastewater organics (Lo and Sproul, 1977; Bitton et al., 1976a), humic and fulvic acids (Scheuerman et ul., 1979; Bixby and O’Brien, 1979), proteins, polypeptides, and amino acids (Carlson et al., 1968; Goyal and Gerba, 1982; Katzenelson et ul., 1976; Farrah et al., 19811)) interfere with virus adsorption to surfaces and can be used to elute previously adsorbed viruses. Considering the low dielectric nature of organics and the predominantly negative charge of humic substances at the pH of most natural waters, we can expect them to affect the double-layer or van der Waals contributions to adsorption (Murray and Parks, 1980). Lipidmembraned viruses though may respond differently. For example, Stotzky et al. (1981) found that preadsorption of clays with bovine serum albumin (which inhibits adsorption of the nonlipid coliphages and enteroviruses) actually increases the amount of herpesvirus hominis type 1 adsorption. The enhancement probably occurs because of the strong hydrophobic interactions between hydrophobic groups on the albumin and herpesvirus. DeSilva and Toth (1964) studied the pH-dependent electrokinetic mobility of humic materials in the free state and when adsorbed on kaolinite and bentonite. In all cases the humic matter had an isoelectric point below pH 3 with continuing negative charge development with increasing pH. These charge properties were conferred to the clay minerals after adsorption of the organic matter. Naturally occurring humic substance can have important effects on virus adsorption. Bixby and O’Brien (1979) found bacteriophage adsorption to be
VIRUS ADSORPTION TO SURFACES
149
reduced in the presence of fulvic acid. Humic matter also reduces poliovirus adsorption to magnetite (Bitton et al., 1976b). Muck and other soils high in organic matter are poor adsorbers of virus (Fuhs and Taylor, 1982). Soluble humic matter isolated from cypress dome water with a molecular weight of less than 50,000 interferes with virus adsorption to soil (Scheuerman et al.,
1979). The effectiveness of any particular organic eluant is related to its chemical nature. Studies have shown that poliovirus adsorbed to epoxy-fiberglass membrane filters could be eluted by solutions of basic amino acids, but that acidic amino acids were poor eluants (Farrah and Bitton, 1978). The molecular weight of eluant compounds was not found to be a significant factor in elution efficiency, but these investigators later reported that the functional groups on a compound may influence elution (Farrah and Bitton, 1979). Basic groups such as amino and guanidino groups favored elution from the filters, while carboxyl groups hindered elution.
111. Protective Effects of Virus Adsorption A. AQUATICA N D
SOIL E N V I R O N M E N T S
Potential adsorbents of viruses in natural waters have been found to include sand, pure clays (e.g., montmorillonite, illite, kaolinite, bentonite, and allophane), bacterial cells, naturally occurring suspended colloids, and estuarine silts and sediments (Gerba and Schaiberger, 1975; Mitchell and Jannasch, 1969; Taylor et al., 1980). In addition, viruses may be disharged into natural waters already associated with solids (Gerba et at., 1978; Hejkal
et al., 1981). Adsorption of coliphages to pure clays has been shown to greatly reduce inactivation rates in natural and artificial seawater (Bitton and Mitchell, 1974; Gerba and Schaiberger, 1975) and in freshwater (Babich and Stotzky, 1980). Differences may exist in the degree of protection afforded by various minerals. For example, attapulgite and vermiculite were observed to have a greater protective effect than montmorillonite and kaolinite on the rate inactivation of a bacteriophage (Babich and Stotzky, 1980). In a similar study the survival of coliphage T7 at 4°C was extended from 5 to 31 weeks in the presence of clay material, while survival at 24°C was enhanced from 1 to 9 weeks with montmorillonite and to 7 weeks with kaolinite (Stotzky et al., 1981). In this same study coliphage T1 and T7 were found to adsorb poorly to a wide variety of bacterial species and it was concluded that adsorption to microbial cells did not play a major role in their survival in natural waters. Estuarine sediments have been demonstrated to prolong survival of enteroviruses and rotaviruses (Smith et d.,1978; LaBelle and Gerba, 1980).
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CHARLES P. GERBA
Enteric bacteria are also known to survive longer in sediments than in the overlying water (Gerba and McLeod, 1976). DeFlora et aZ. (1975)observed that under laboratory conditions poliovirus type 1 was inactivated 45 times faster in seawater than in marine sediments. Under field conditions in which poliovirus type 1 was suspended in seawater and sediment and placed in survival chambers, survival was 4-100 times greater in the sediment than in the seawater (LaBelle and Gerba, 1980). These findings suggest that accumulation of viruses will be favored by their longer survival in sediments. This is probably why high concentrations of enteric viruses are often observed in marine sediments (DeFlora et al.,
1975). Virus association with soils is also known to prolong virus survival. Green
(1976) observed that at room temperature the inactivation rate of poliovirus adsorbed to soil was lower than nonadsorbed virus. In a study of factors which control virus survival in the soil matrix, Hurst et d . (1980) examined the persistence of various enteric viruses and coliphages in nine different soils. Overall, temperature and the degree of virus adsorption to the soil were found to be the most important predictors of virus die-off. Both the nature of the soil and the virus were reflected in the relative survival of viruses. For example, viruses which inherently adsorb poorly to soils exhibit shorter survival times (Gerba et al., 1981). Similar soil-protective effects were reported by Sobsey et al. (1980a) who found extended survival of poliovirus to be associated with adsorption to certain soils. The time required for a 99% reduction of virus adsorbed to Cecil soils was 167 days compared to 86 days for unadsorbed virus. These investigators noted, however, that not all soils offered this protection. Ponzer soils, which were poor adsorbers under test conditions, apparently accelerated the inactivation time to only 18 days. Apparently, factors associated with this soil type not only inhibited viral adsorption, but enhanced viral inactivation by some unknown mechanism. Virus adsorption to containers used to hold liquids in viral survival experiments may also affect virus survival. Akin et al. (1976)in studying poliovirus type 1 survival in seawater observed that the virus appeared to survive longer in those containers which permitted adsorption to their walls. Poliovirus readily adsorbs to glass surfaces, but not to polycarbonate or polyethylene surfaces. Thus, the container in which a viral survival experiment is conducted could conceivably influence the rate of viral decline. The protective effect of virus association with particulate matter or other surfaces includes protection from proteolytic enzymes or other substances which inactivate viruses, increased stability of the viral capsid, prevention of aggregate formation, and blocking of ultraviolet radiation. Clays such as
VIRUS ADSORPTION TO SURFACES
151
kaolinite are capable of sorbing a greater variety of inorganic and organic substances that could inactivate viruses. As an example, bentonite clays have been shown to protect viruses against inactivation by ribonuclease by the adsorption of this enzyme by the clay (Singer and Fraenkel-Conrat, 1961). The protective effects of clays were also shown by their ability to protect coliphages T1 and T7 against inactivation by lysozyme and reovirus against chymotrypsin (Stotzky, 1980). Particulate matter in water may offer some protection against photoinactivation. Bitton (1980b) observed a protective effect of three different clays toward phage T7 subjected to ultraviolet (UV) irradiation but found no detectable viable virus in control samples after 30 seconds of exposure to UV light. In another study, the time for a 90% reduction in poliovirus type 1 titer in a tank containing groundwater exposed to solar radiation was 75 minutes and 163 minutes in groundwater containing a clay (Bitton et al., 1979b). Protection of viruses against thermoinactivation has been suggested from the results of Bitton et al. (1979b), Liew and Gerba (1980), and Stotzky et a!. (1981). Inactivation of poliovirus type 1and echovirus type 1is prolonged in the presence of marine sediments at 24, 37, and 55°C but not at 4°C (Liew and Gerba, 1980). Supernatant fluids of seawater and sediment mixtures lack the protective effect against thermoinactivation, suggesting that prolonged virus survival in the presence of sediments is due to adsorption to particulates. It has been demonstrated that poliovirus particles are ruptured during heat treatment and both the nucleic acid and one structural polypeptide (VP4) are released (Breindl, 1971; Gebhard, 1960).The released nucleic acid retains its infectivity. It may be possible that adsorption of the virus particles acts to stabilize the virus against disruption by steric effects or that the apparent increase in stability of the virus is due to adsorption of released nucleic acid by the sediment particles which would be expected to retard degradation of the nucleic acid. B. DISINFECTION As mentioned previously, solid-associated viruses are present in domestic wastewater. The quantity of solid-associated viruses being discharged by a sewage treatment facility is important because it has been shown to affect both the resistance of viruses to disinfection and survival in natural waters. Wellings et al. (1976) first reported a greater recovery of solid-associated enteric viruses after chlorination. The relative concentrations of freely suspended and solid-associated enteric viruses and coliphages change significantly after wastewater chlorination (Stagg et al., 1978; Hejkal et al., 1981),
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CHARLES P. GERBA
apparently because of the greater sensitivity of freely suspended viruses to inactivation by chlorine. The nature of the particulate matter with which the virus is associated and the virus type may play a role in the degree of protection afforded to a virus. Boardman and Sproul (1977) reported that inorganic particles including alum, calcium carbonate, and kaolin did not interfere with tailed coliphage "7 inactivation by chlorine. Hoff (1978) also reported no protection for poliovirus l adsorbed to bentonite or AIPO,. These authors suggest that protection from disinfection would occur only after total encapsulation of the virus with the particulate matter. Hydrated aluminum oxide floc and bentonite clay have also been reported to afford little or no protection to poliovirus l or coxsackievirus A9 (Sproul et d., 1979). In contrast to these studies, coliphage f2 adsorbed to bentonite clay was inactivated at a slower rate in the presence of ozone (Sproul et al., 1979);and coliphage MS-2 was also more resistant when adsorbed to bentonite in the presence of chlorine (Stagg et al., 1977). Kaolin clay has been reported to protect poliovirus 1 against inactivation by ozone (Kaneko and Igarashi, 1983). Although results on the possible protection afforded to solid-associated viruses from chlorine and ozone by inorganic particulates have not been consistent, all the current evidence indicates that protection is clearly afforded by organic particulates. Protection of enteroviruses against inactivation by chlorine (Hoff, 1978), chlorine dioxide (Moffa and Smith, 1974), and ozone (Sproul et al., 1979; Foster et al., 1980) has been demonstrated with tissue culture cell debris. Of course, these are artificial conditions and may not reflect "real w o r l d conditions. But studies using fecal homogenates also indicate that protection is afforded by organic matter (Foster et al., 1980; Hejkal et al., 1979) more approaching what would be expected in nature. Recent laboratory experiments have also shown that poliovirus 1 is af€orded significant protection against inactivation by chlorine when associated with activated sludge particulates, which was significantly greater than that afforded by kaolin (Kaneko and Igarashi, 1983). The actual degree of virus protection from disinfection afforded by virus adsorption to solids is difficult to determine. For example, the greater protective effect observed with organic particulates may be due to inclusion of the virus within the particulate or viral aggregation rather than protection afforded by similar adsorption of the virus to the surface of the particle. Evidence indicates that occluded viruses are protected to a greater degree than surface-associated viruses (Hejkal et d.,1979). Also, experimental conditions are often difficult to control and experimental design is critical in the interpretation of results. It would appear though that the degree of protection is greatly dependent on the type of virus and the nature of the particulate material.
VIRUS ADSORPTION TO SURFACES
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IV. Inactivation of Viruses on Solid Surfaces A. AQUATIC ENVIRONMENT Although virus association with solids in natural waters has generally been observed to be protective, some may actually enhance virus inactivation. Gerba and Schaiberger (1975) using differential filtration and centrifugation were able to demonstrate that particles of a certain size and density in seawater were detrimental to virus survival. Since the particulates did not lose their activity after autoclaving, it was presumed that the observed activity was not due to interaction with viable bacteria which are known to possess antiviral activity (Gundersen et al., 1967) or due to a solid-associated enzyme. It was suggested that such inactivation could be caused by irreversible adsorption of the virus to sites on bacteria or other particulate matter not influenced by autoclaving.
B. METAL SURFACES While heavy metals in solution have been known for some time to be toxic for both bacteria and viruses (Sterritt and Lester, 1980; Allan, 1973; Edebo et al., 1967), only recently have studies been done on the fate of viruses associated with metal and metal oxide surfaces. Murray and Laband (1979) studied the interaction of poliovirus 1 with five metal oxides, i.e., SiO,, Fe,O,, Al,O,, MnO,, and CuO. Poliovirus readily adsorbed to the metal oxide surfaces. Alteration of the specific infectivity of radioactively labeled viral nucleic acid and protein indicated that degradation of the virus was occurring at the surface of MnO, and CuO. All of the virus adsorbed on the other surfaces could be recovered by elution with a mixture of sera, tryptone, and detergent. Virus was most easily eluted from the SiO,, but multiple elutions were required to recover virus from the other metal oxides. Further analysis indicated that not only was the virus being inactivated at the surface of the CuO but that degradation of the virus was taking place. Sedimentation analysis of the eluted virus indicated that the RNA was being released from the virai capsid and that the RNA and protein were being broken down into small fragments (Fig. 4). Recovery data indicated that virus is rapidly inactivated at first and proceeds more slowly after 2 hours. Degradation on aluminum metal was found to be even more dramatic than on the metal oxides (Murray, 1980). To determine if aluminum metal could be applied to disinfection a bench-scale process experiment was performed with poliovirus 1 seeded into unchlorinated secondary effluent that was continuously passed through a 1-cm column of coarse aluminum powder.
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CHARLES P. GERBA
-
$
RELEASE OF INTACT NUCLEIC ACID
\ <3
3
MICROORGANISMS
#(
DEGRADATION
OF NUCLEIC ACID
RELEASE OF DEGRADED NUCLEIC ACID
4
FIG.4. Mechanisms of virus inactivation on a solid surface. (1) Poliovirus eluted from CuO and aluminum metal is broken down into small RNA and protein fragments (Murray and Laband, 1979).(2) Reovirus is inactivated on soils but remains intact &er elution (Moore et al., 1982a). (3) Under drying conditions in sterile soil poliovirus RNA is released from the capsids with the virus capsid remaining irreversibly bound to the soil (Yeager and O’Brien, 1979). (4) Under moist conditions poliovirus RNA is damaged before dissociation from the capsid and under nonsterile conditions biological activity degrades the HNA once it is released.
Substantial virus removal was observed even after passage of 300 bed volumes. Although the mode of action of the metal on virus degradation was not determined, Yamamoto et u2. (1964) found that the RNA coliphages MS-2 and f2 are inactivated in fluids which have been in contact with aluminum, zinc, or magnesium. They believed that the inactivation was due to traces of Cu2+ and electrolytically formed H,O,. The phages were protected by adding either catalase or EDTA, which they suggested as support for the mechanism of inactivation. Iron oxides of hematite or magnetite have been known to have a strong affinity for viruses (Larkin and Gahlmore, 1971; Rao et al., 1968; Bitton and Mitchell, 1974; Bitton et al., 1976b). Magnetite also appears to have potential for wastewater treatment as it is also capable of removing a large proportion of the dissolved and suspended matter present (Atherton and Bell, 1983). Degradation of coliphage MS-2 into small fragments after elution from magnetite at pH 10 has been observed (Atherton and Bell, 1983).
VIRUS ADSORPTION TO SURFACES
155
C. SOILS Inactivation of viruses can also occur during association with soils. Work
by Yeager and O’Brien (1979) suggests that poliovirus inactivation in moist and drying soils is by different mechanisms. Under drying conditions in sterile soil the viral ribonucleic acid (RNA) was released from the capsids with the poliovirus capsids apparently remaining irreversibly bound to the soil (Fig. 4). In moist soils the viral RNA was damaged before dissociation from the capsid. Since events in the viral inactivation were identical under sterile and nonsterile conditions, biological activity was not believed responsible for the mode of inactivation (Fig. 4). The irreversible binding of soil occurred only when the viruses were dried in the presence of soil. The mechanisms of viral inactivation in soils are similar to those observed in aerosols (Dimmock, 1967) and during desiccation (Ward and Ashley, 1977) and probably represent general mechanisms of viral inactivation. While these studies provide information on the general mechanisms of viral inactivation in soils, it does not follow that such an association is detrimental to virus survival. Some recent studies have indicated that inactivation can occur because of the physical association of the virus with soils and clays. Taylor et al. (1980) found that reovirus eluted from clay with sodium hexametaphosphate was not disintegrated but was of lower infectivity. However, aggregates of the virus may have formed, accounting for the result. Moore et al. (1982a) also suggested that reovirus was inactivated during contact with the surface of various soils and minerals. As the sedimentation behavior of the recovered virus was indistinguishable from that of the input virus, loss of infectivity was not attributable to the formation of virus aggregates or disruption of virions (Fig. 4).Only the presence of humic organic matter in the soil protected and prolonged virus survival. Since the infectivity of the virus was stable in the presence of exopolymer-coated substrates, the authors concluded that the observed loss of infectivity was the consequence of physical adsorption on the mineral and soil surfaces. In a similar study with poliovirus no inactivation was observed during its association with the same soils indicating that such inactivation may be specific to certain types of viruses (Moore et al., 1981).
V. Applied Aspects of Virus Adsorption A. WASTE WATER TREATMENT Domestic sewage contains solids of biological and mineral origin (Rickert and Hunter, 1971). During biological wastewater treatment more solids are
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CHARLES P. GERBA
produced by microbial growth during the conversion of soluble organic substances to cellular material. The size and nature of the solids present in raw domestic wastewater also change dramatically during treatment (Rickert and Hunter, 1972). Although many of the viruses present in raw domestic sewage are believed to be adsorbed or embedded in solids, few quantitative studies have been done to assess the relative solid-associated versus freely suspended viruses. In a study of the raw sewage received by two sewage treatment plants, Wellings et al. (1976) observed that the amount of solidassociated virus as determined by centrifugation ranged from 23 to 81%. Duff (1970) found that 25-70% of the total viruses were associated with solids. Much of the remaining information on virus association with solids in raw wastewater rests on observation of the reduction of virus which occurs during the settling of solids (Berg, 1973). Removal of virus during primary settling has been reported to range from 0 to 65%, with a probable average removal under normal wastewater treatment practices of 50% (Gerba, 1981). Considering that much of the sewage discharged into the world’s aquatic environment is untreated, additional information on the degree of virus association with raw sewage could be most useful in understanding the fate of viruses after discharge into the environment. Although differences were small, it would appear that enteroviruses and adenoviruses may be more effectively removed during primary settling of raw sewage than reoviruses (Irving and Smith, 1981). The three most widely used biological processes for secondary sewage treatment are trickling filtration, activated sludge, and oxidation ponds. All three generate large amounts of microbial biomass to which viruses readily adsorb. In fact, the degree of virus removal which occurs during these processes appears to largely depend on virus adsorption to the solids (Berg, 1973; Sobsey and Cooper, 1973; Balluz and Butler, 1979), although biological antagonism may also be a major factor in the eventual inactivation of the virus (Berg, 1973; Sobsey and Cooper, 1973; Ward, 1982). Studies on the detection of enteric viruses in activated sludge aeration basins indicate that 83-99% of the indigenous enteroviruses may be solids associated (Moore et al., 1978). Generally, trickling filtration is less effective than activated sludge because of the lower biomass available for viral adsorption, and the relatively short contact time between the wastewater and the biological growth on the filter medium (Sorber, 1983). Laboratory studies using bench-scale reactors also confirm that 90% or greater removal of enterovirus can be expected during activated sludge treatment (Malina, 1976). Results of several studies indicated that the eEciency of a treatment plant is closely related to the concentration of mixed liquor-solids and its capacity to remove them (Balluz et al., 1977). Clarke et al. (1961) showed that coxsackievirus A9 was removed much more efficiently
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157
than poliovirus 1, although removal of both viruses was always greater than 80%. B a h z et al. (1978) found that there was a distinct difference between the behavior of poliovirus 1 and coliphage f2 in a laboratory-scale model, activated sludge, sewage treatment plant, which was directly related to differences in the adsorption of virus to solids in the aeration tank. The most striking contrast was in the distribution of the two viruses between the liquid and solid fractions of the mixed liquor. Approximately 84% of the f2 remained in the liquid fraction with 16% in the solid fraction, while for poliovirus it was virtually the opposite. Another dissimilarity was the percentage of virus removal across the plant, which for the phage was 79.6%and for poliovirus 99.96%. Laboratory-grown viruses can be demonstrated to readily adsorb to activated sludge flocs (Farrah et d . , 1978). Major differences between the degree of poliovirus type 1 and rotavirus adsorption to preformed activated sludge flocs were observed by Farrah et al. (1978). Human and simian SA-11 rotavirus adsorbed significantly less to the flocs than did the poliovirus. In a later study, poliovirus, echoviruses 7 and 29, and coxsackievirus B3 were found to be more readily adsorbed to sludge flocs than strains of coxsackievirus B4 and echovirus 1 (Gerba et al., 1980). Removal of indigenous virus during activated sludge treatment is dependent on type. Irving and Smith (1981) reported that removal of enteroviruses, adenoviruses, and reoviruses was 93, 87, and 30%, respectively. Studies on the concentrations of enteroviruses and rotaviruses in effluents of activated sludge plants also indicate that rotaviruses are less effectively removed than enteroviruses (Smith and Gerba, 1982). In a bench-scale, extracted sludge test system, poliovirus and coxsackie viruses B1 and B5 were slightly more efficiently removed than echovirus 7 (Vasl, 1981).
B. ADVANCEDWASTEWATER AND DRINKING WATER TREATMENT
1. Coagulation Tertiary treatment of wastewater and modern drinking water treatment involves a series of physicochemical processes, like coagulation with alum, lime, iron salts, or polyelectrolyte followed by filtration and passage through activated carbon or resins to remove residual organics. Most often the first step involved in water treatment is coagulation. Most studies have not separated the virus-removal effectiveness of the coagulation and filtration processes. This is particularly true when the studies have been conducted on indigenous viruses at naturally occurring levels (Sorber, 1983)
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because, often, sufficiently high viral levels were not observed to permit this degree of discrimination. Laboratory studies have shown the coagulation process to be quite effective for the removal of enteroviruses and laboratory strains of bacteriophages (Leong, 1983; Sorber, 1983). Lime coagulation is highly effective (Leong, 1983; Gerba, 1981), with 90-99% or more of the virus removed from the water by this treatment. Application of lime during this process results in a final treated water pH of 9-12 which probably is a major factor in the effectiveness of this process, as many viruses are readily inactivated at these pH levels. Alum coagulation is often performed during drinking water treatment, and is highly effective in enterovirus removal, removals of 90% or greater usually being achieved (Leong, 1983). In a comparative study Chang et al. (1958)found that while 95-98% of coxsackievirus A2 could be removed from river water by alum coagulation only 40% of coxsackievirus A3 was removed under similar conditions. Farrah et a2. (1978) also found significantly less adsorption of simian rotavirus SA-11 to preformed alum flocs than poliovirus 1. However, because preformed floc was used and the coagulation and flocculation processes are dynamic, the results may not necessarily represent actual removal of the two viruses during a treatment process. Steinmann and Havemeister (1982) also observed lower removal of rotavirus SA-11 than poliovirus 1 with flocs formed in situ in treated sewage by ferric chloride or aluminum chloride with and without the addition of polyelectrolytes, although the differences were small in most cases. It is now accepted that the coagulation process involves an interaction between negatively charged hydrophobic or hydrophilic colloidal particles (i.e., viruses) and positively charged hydrolysis products of aluminum, calcium, or iron (Bitton, 1980a). Aluminum hydroxide apparently retains viruses more efficiently than do the other precipitable salts (Bitton, 1980b). Cookson (1974) suggested that adsorption of viruses to Al(OH), flocs could result from coordinated hydroxyl groups or from electrostatic attraction between negatively charged viruses and positively charged Al(OH),. The latter mechanism is highly dependent on p H and salt concentration. Aluminum phosphate does not have the charge benefit that aluminum hydroxide has and does not have the large number of OH- ion bridges to aid virus adsorption. Thus, the number of possible mechanisms for virus attachment to AIPO, is less than that for aluminum hydroxide. Wallis and Melnick (1967a,b) demonstrated this by finding that AIPO, would adsorb only the acid-sensitive viruses (pox, herpes, myxo, arbo, rhino, rubella, influenza, and measles). Cookson (1974) thus suggested that the ability of AlPO, to adsorb viruses appears to depend on the presence of phospholipids in the viral membrane. Adsorption of these viruses onto aluminum phosphate could involve hydrogen bonding of the phosphate to positively charged groups on the phospholipids, notably amines.
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Preformed precipitates of calcium phosphate were found to be effective virus adsorbents, except for adenovirus and reovirus (Wallis and Melnick, 1967a).The precipitate formed by calcium phosphate is Ca, [OH(PO,),] and while it adsorbs more types of viruses than AIPO,, it is not as efficient an adsorbent because of its lower degree of hydrolysis and lower positive charge (Cookson, 1974). It is important to note that reovirus failed to adsorb to aluminum and calcium precipitates. Coagulant aids, such as polyelectrolytes, which are polymers of synthetic origin are often added to treatment processes to improve floc size which leads to faster settling times. Certain polyelectrolytes have also been used to concentrate viruses from water (Wallis et al., 1970, 1971). Under laboratory conditions polyelectrolytes are able to interact directly with viruses. Cationic polyelectrolytes generally display a higher adsorptive capacity than nonionic or anionic polyelectrolytes (Bitton, 1980a; Foliguet and Doncoeur, 1975). Negatively charged viruses are probably attracted to positively charged amino groups on the surface of the cationic polyelectrolyte (Thorup et al., 1970). Cookson (1974) proposed that the virus-polyelectrolyte interaction may be due to hydrogen bonding between the virus and chemical groups (carboxylor amino groups) on the polymer surface and to electrostatic forces that depend on pH and salt concentration.
2. Filtration and Adsorption Sand filtration is often practiced in advanced wastewater and drinking water treatment to remove suspended matter. Without addition of a coagulant this process is inefficient and the amount of virus removed is erratic. Removal of viruses may occur by direct adsorption of the virus to sand, coal particles, or floc. Viruses generally have a low affinity for sand, and the degree of removal is dependent on flow rates, pH, concentration of divalent cations, and presence of organic matter (Bitton, 1980b). The high flow rates are probably a major reason for the poor adsorption and probably account for the large range of reported performances (0-99%) (Leong, 1983). Optimal use of coagulants preceding filtration can bring about greater than 99% removal of the viruses. Diatomaceous earth is also used as a filtration medium in some treatment processes, but it also is a poor adsorbent of viruses (Amirhor and Engelbrecht, 1975). Adsorption of the virus can be enhanced by treatment of the diatomite by coating with a cationic polyelectrolyte or with ferric or aluminum hydrates (Amirhor and Engelbrecht, 1975). Activated carbon can be used in advanced wastewater treatment to remove additional organics and in drinking water treatment to remove odor and taste. Virus adsorption has been studied by a number of investigators, and generally the carbon was found to be a poor adsorbent of viruses, especially in the presence of organics naturally present in water and waste-
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water effluents (Cookson, 1967, 1970; Gerba et al., 1975a; Sproul et al., 1969). Cookson (1969) performed the most detailed studies on virus adsorption to activated carbon. He suggested that adsorption of T4 was due to electrostatic attraction between carboxyl groups on the activated carbon and amino groups on the virus surface. Following esterification of the carboxyl groups with acid alcohol, he found that the attachment of T4 to activated carbon was suppressed almost completely. Dual media filtration involving sand and coal is sometimes practiced during water and wastewater treatment. Virus adsorption apparently involves hydrogen bonding (Oza and Chaudhuri, 1976), as adsorption to acetyl chloride-treated coal is greatly reduced. Acetyl chloride converts the hydroxyl groups on coal to acetate or anhydride groups which would, in turn, result in reduced hydrogen-containing hnctional groups on coal. The adsorption interaction is also evident from differences in virus adsorption capabilities of various coals with different hydrogen content (Oza and Chaudhuri, 1977). It also appears that coliphages MS-2 and T4 compete for the same adsorption sites on the coal. Viruses adsorb only to the outer surfaces of the coal and are excluded from the small pores which give coal and activated carbon their large surface area for adsorption of chemical pollutants. Surface coverage of coal is only 0.1% (Oza and Chaudhuri, 1975). c . LANDAPPLICATION OF
WASTES
With almost half of the waterborne disease outbreaks in the United States caused by contaminated groundwater there is a major interest in the fate of human pathogenic viruses in groundwater (Keswick and Gerba, 1980).Over 800 billion gallons of raw sewage from septic tanks and cesspools leach into the ground on an annual basis, with an additional 250 billion gallons of raw and treated sewage reaching the soil each year from leaking municipal sewage systems (Keely, 1977). In addition to these major sources, intentional land application of wastes to land as a treatment method is also practiced and has gained widespread acceptance in recent years. Intentional land application of wastes includes rapid infiltration of wastes for groundwater recharge or renovation, irrigation of crops, and deep-well injection. Much of the work discussed in the previous sections has related to virus adsorption to soils. Virus removal which occurs during the land application of wastes is primarily by adsorption and processes which govern adsorption are paramount in predicting safe distances between the location of wells used for domestic drinking water and sites of sewage discharge. Salt concentration, pH, the presence of organic matter, soil composition, nature of the virus, and flow rates can affect the degree of retention of
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viruses by soil particles (Gerba et al., 1975b). Retention of viruses by soil particles does not result in their permanent immobilization from the liquid phase, and changes in water quality can result in their elution and further subsurface travel (Lance et al., 1976; Duboise et al., 1976; Bitton et al., 1976a; Landry et a l . , 1979). Simulated cycles of rainfall and sewage effluent applications, resulting in ionic gradients, have been shown to result in the migration of poliovirus through soil columns in the laboratory (Duboise et al., 1976; Landry et al., 1979). In addition, viruses adsorbed to soil in tap water could be eluted with water high in humic acids (Bitton et al., 1976a). If the columns are of sufficient length, readsorption of eluted virus is observed (Lance et al., 1976; Landry et al., 1979). Thus, through adsorption and elution events viruses could migrate for long distances in the subsurface by a chromatographic-type effect. This type of behavior could result in a phenomenon in which viruses are concentrated near the soil surface by adsorption during the infiltration of wastewater and then after a drying period are eluted in a concentrated band if rainfall occurs. The reported possible association of enteroviruses in groundwater beneath wastewater land treatment sites after periods of heavy rainfall indicates that such events occur under field conditions (Wellings et al., 1975). Thus, consideration must be given to environmental conditions in attempts to detect viruses in groundwater. The extent of virus adsorption and degree of virus elution is type and strain dependent and appears to be governed by the overall electronegativity of the virus (Goyal and Gerba, 1979; Gerba et al., 1981; Landry et al., 1979; Burge and Enkiri, 1978b). In addition, viruses within a given population may vary in their adsorption to soil (Burge and Enkiri, 1978b). Strain dependence of virus elution was studied in detail by Landry et at. (1979), who observed that the extent of elution from soil cores, by either rainwater or sewage effluent rinses, varied with virus strain. From their observations the authors cautioned against over-extrapolation from data derived from the study of a few virus types. Vaughn et al. (1978) suggested that all polioviruses may be poorly eluted, since members of the group were the least encountered during a field study of virus-contaminated aquifers. D. DETECTIONOF VIRUSES
IN THE
ENVIRONMENT
Methods for detection of viruses in water must be capable of concentrating them from 10 to 2000 liters of water of varying quality. The most successful method for accomplishing this task has been the microporous filter adsorpIn tion-elution process originally developed by Wallis and Melnick (1967~). this procedure the water being sampled is passed through a microporous filter; viruses retained on the filters are then eluted by passage of a small
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volume of eluent. The eluent is usually a proteinaceous solution adjusted to pH 8-11 (Goyal and Gerba, 1982). When large-diameter or pleated filters are used the eluent is further concentrated by a flocculation or precipitation step. A wide variety of filters have been used for concentration of viruses from water including nitrocellulose, cellulose acetate, asbestos, epoxy-fiberglass, charge-modified inorganic filter aids, and borosilicate glass. A good deal of study on the mechanisms of virus adsorption and elution has been conducted in attempts to optimize virus recovery by this method. Farrah and co-workers (Farrah et al., 1981a; Farrah, 1982; Shields and Farrah, 1983)and Sobsey and Jones (1979) have suggested that electrostatic and hydrophobic interactions play a significant role in virus adsorption to filters. Cellulose and fiberglass filters possess a net negative charge at the pH of most natural, drinking, and waste waters; and for optimal virus adsorption, the water being sampled is first adjusted to pH 3-4 and then aluminum or magnesium salts are added (Gerba et al., 1978). To reduce the need for pH adjustment and salts, Sobsey and Jones (1979) reported the application of positively charged filters for virus concentration from tap water. These electropositive filters retain a net positive charge at the pH of most waters and readily adsorb the negatively charged viruses. The efficiency of virus concentration also varies greatly among virus types and may be both a reflection of the virus isoelectric point and its sensitivity to the high pH often used to elute adsorbed virus (Sobsey et al., 1977). Efficiency of reconcentration by bioflocculation or the use of other flocculents may also be virus dependent (Morris and Waite, 1980; Bitton et al., 1979a). For example, Morris and Waite (1980) reported large differences in the efficiency of virus concentration using the beef-extract organic flocculation method of Katzenelson et al. (1976). The efficiency of concentration was as follows: coxsackievirus B4, 9%; echovirus 1, 7%; coxsackievirus B3, 98%; and poliovirus 1, 40%. Bitton et aE. (1979a), using nonfat dry milk as an organic flocculant for virus reconcentration from filter eluants, also observed that the efficiency of concentration of poliovirus 1 and coxsackievirus B3 was almost 10-fold greater than that of echovirus 1. It would appear from a general review of the literature of methods for virus concentration that viruses with low isolectric points have a lower efficiency of concentration with the adsorption-elution methodology commonly employed today. As previously discussed, recent studies have indicated that hydrophobic interactions may play a significant role in virus adsorption to filter surfaces and other types of solids (Farrah et al., 1981a). Sobsey and co-workers reported the enhanced recovery of enteric viruses from estuarine sediments and electropositive filters using a combination of a chaotropic agent and beef extract (Moore et al., 1982b; Wait and Sobsey, 1983).
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An understanding of virus-adsorption mechanisms in recent years has clearly contributed to improved methods for their isolation from the environment. Further application of this knowledge should result in continued improvements.
E. IMPLICATIONS This article has sought to demonstrate the importance of virus association with solids and their fate in the environment. In addition, an understanding of factors controlling the interaction has already led to new and improved methods for the concentration of viruses from water, and their isolation from the environment. Further studies on mechanisms involved in virus adsorption could lead to improved methods for their removal during sewage and drinking water processes. For example, it is now known that viruses are readily inactivated by contact with certain metal surfaces. A further understanding of this phenomenon could lead to new and simple methods of viral disinfection. It is also evident that not all viruses behave alike toward a solid under identical conditions. Under most natural conditions viruses with a low isoelectric point appear to be more poorly adsorbed to most solid surfaces. This must now be taken into consideration when evaluating concentration or treatment systems which involve adsorption. This phenomenon is also important in determining the transport of viruses in the environment. Certain enteric viruses appear to adsorb less readily to soils and aquatic sediments than others. Thus their potential transport to groundwater may be greater and they may be less likely to settle in surface waters. To take into consideration these differences in virus behavior, it is perhaps best to use viruses with widely varying isoelectric points or marked differences in hydrophobicity to evaluate the extremes in virus interaction with a given surface which could occur. REFERENCES Akin, E. W., Hill, W. F., Cline, G. B., and Benton, W. H. (1976). Water Res. 10, 59-63. Allan, W. H. (1973). Vet. Record 93, 448. Amirhor, P., and Engelbrecht, R. S. (1975). J . Am. Water Works Assoc. 67, 187-192. Atherton, J. G . , and Bell, S. S. (1983). Water Res. 17, 943-948. Babich, H . , and Stotzky, G. (1978). A p p l . Enoiron. Microbiol. 36, 906-914. Babich, H . , and Stotzky, G. (1980). Water Res. 14, 185-187. Balluz, S. A., and Butler, M . (1979). J . Hyg. 82, 285-291. Balluz, S. A., Jones, H. H., and Butler, M . (1977). J . Hyg. 78, 165-173. Balluz, S. A., Butler, M . , and Jones, H. H. (1978). J . Hyg. 80, 237-242. Beard, J. W., and Wyckoff, R. W. G . (1938). J . Biol. Chenz. 123, 461-470.
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SaIo, R. J., and Mayor, H. D. (1978). Interuirology, 10, 87-93. Scheuerman, P. R., Bitton, G., Overman, A. R., and Gifford, G. E. (1979)./. Enuiron. Eng. Diu. Am. SOC. Ciu. Eng. 15, 629-640. Schiffenbauer, M., and Stotzky, G. (1982). Appl. Enuiron. Microbiol. 43, 590-596. Sharp, D. G., Hook, A. E., Taylor, A. R., Beard, D., and Beard, J. W. (1946)./. Biol. Chem. 165, 259-270. Shields, P. A., and Farrah, S. R. (1983). Appl. Enuiron. Microbiol. 45, 526-531. Singer, B., and Fraenkel-Conrat, H. (1961). Virology 14, 59-61. Smith, E. M., and Gerba, C. P. (1982). Appl. Enuiron. Microbiol. 43, 1440-1450. Smith, E. M., Gerba, C. P., and Melnick, J. L. (1978).Appl. Enuiron. Microbiol. 35,685-689. Sobsey, M. D., and Cooper, R. C. (1973). Water Res. 1, 669-685. Sobsey, M. D., and Jones, B. L. (1979). Appl. Enuiron. Microbiol. 37, 588-595. Sobsey, M. D., Gerba, C. P., Wallis, C., and Melnick, J. L. (1977).Can. J . Microbiol. 23,770778. Sobsey, M. D., Dean, C. H., Knuckles, M. E., and Wagner, R. A. (1980a). Appl. Enuiron. Microbiol. 40, 92-101. Sobsey, M. D., Glass, J. S., Carrick, R. J., Jacobs, R. R., and Rutula, W. A. (1980b).J . Am. Water Works Assoc. 72, 350-355. Sorber, C. A. (1983). I n “Viral Pollution of the Environment” (G. Berg, ed.), pp. 39-75. CRC Press, Boca Raton, Florida. Sproul, 0. J., Warner, M., LaRochelle, L. R., and Brunner, D. R. (1969). In “Advances in Water Pollution Research” (S. H. Jenkins, ed.), pp. 541-554. Pergamon, Oxford. Sproul, 0. J., Buck, C. E., Emerson, M. A., Boyce, D., Walsh, D., and Howser, D. (1979). “Effect of Particulates on Ozone Disinfection of Bacteria and Viruses in Water.” EPA Report EPA-60012-089, U.S. Environmental Protection Agency, Cincinnati, Ohio. Stagg, C. H. (1976). Ph.D. Dissertation, Rice University, Houston, Texas. Stagg, C. H., Wallis, C., and Ward, C. H. (1977). Appl. Enoiron. Microbiol. 33, 385-391. Stagg, C. H . , Wallis, C., Ward, C. H., and Gerba. C. P. (1978). Prog. Water Technol. 10,381387. Steinmann, J., and Havemeister, G . (1982). Zentralbl. Bakteriol. I . Abt. Orig. B176, 546. Stern, 0. (1924). Z. Elektrochem. 30, 506-516. Sterritt, R. M., and Lester, J. N. (1980). Sci. Total Enuiron. 14, 5-17. Stotzky, G. (1980). In “Microbial Adhesion to Surfaces” (R. C. W. Berkeley, J. M. Lynch, J. Melling, R. R. Rutter, and B. Vincent, eds.), pp. 231-247. Ellis Horwood, Chichester, England. Stotzky, G., Schiffenbauer, M., Lipson, S . M., and Yu, B. H. (1981). In “Viruses and Wastewater Treatment” (M. Goddard, and M. Butler, eds.), pp. 199-209. Pergarnon, Oxford. Taylor, D. H., Bellamy, A. R., and Wilson, A. T. (1980). Water Res. 14, 339-346. Tanford, C. (1979). “The Hydrophobic Effect: Formation of Micelles and Biological Membranes.” Wiley, New York. Tborup, R. R., Nixon, F. P., Wentworth, D. F., and Sproul, 0. J. (1970)./, Am. Water Works Assoc. 62, 97-101. Valentine, R. C., and Allison, H. C. (1959). Biochim. Biophys. Acta 34, 10-23. Vasl, R. (1981). Ph.D. Dissertation, Technion, Hafia, Israel. Vaughn, J. M., and Landry, E. F. (1983). In “Viral Pollution of the Environment” (G. Berg, ed.), pp. 163-210. CRC Press, Boca Raton, Florida. Vaughn, J. M . , Landry, E. F., Baranosky, L. I., Beckwith, C. A,, Dahl, M. C., and Delihas, N. C. (1978). Appl. Enuiron. Microbiol. 36, 47-51. Venvey, E. J. W., and Overbeek, J. G. (1948). “Theory of the Stability of Lyophobic Colloids.” Elsevier, Amsterdam. Wait, D. A., and Sobsey, M. D. (1983). Appl. Enuiron. Microbiol. 46, 379-385.
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Wallis, C., and Melnick, J. L. (1967a).Am. J . Epidemiol. 85, 459-468. Wallis, C., and Melnick, J. L. (196%). In “Transmission of Viruses by the Water Route” ( C . Berg, ed.), pp. 129-141. Wiley, New York. Wallis, C., and Melnick, J. L. (1967~). J . Virol. 1, 472-477. Wallis, C., Melnick, J . L., and Fields, J. E. (1970). Water Res. 4, 787-796. Wallis, C., Melnick, J. L., and Fields, J. E. (1971). Appl. Microbid. 21, 703-709. Wallis, C., Henderson, M., and Melnick, J. L. (1972). Appl. Enuiron. Microbid. 23,476-480. Ward, R. L. (1978).J . Virol. 26, 299-305. Ward, R. L. (1982). A p p l . Enoiron. Microbiol. 43, 1221-1224. Ward, R. L., and Ashley, C. S. (1977). Appl. Enoiron. Microbiol. 34, 564-570. Weber, K . , and Koniysberg, W. (1975).I n “RNA Phages,” p. 51. Cold Spring Harbor Laborator y , Cold Spring Harbor, New York. Weber, W. J., and Morris, J . C. (1964). J . Sun. Eng. Diu. Am. Soc. Clu. Eng. 90, 79-107. Wellings, F. M., Lewis, A. L., Mountain, C. W., and Pierce, L. V. (1975).Appl. Microbiol. 29, 751-757. Wellings, F. M., Lewis, A. L., and Mountain, C. W. (1976). Appl. Enuiron. Microbiol. 31, 354-358. Yamamoto, N., Hiatt, C. W., and Haller, W. (1964). Biochim. Biophys. Acta 91, 257-261. Yeager, J. G., and O’Brien, R. T. (1979). Appl. Enoiron. Microbid. 38, 702-709. Zerda, K. S. (1982). Ph.D. Dissertation, Baylor College of Medicine, Houston, Texas.
Computer Applications in Applied Genetic Engineering JOSEPH
L. MODELEVSKY
Molecular and Cell Biology Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana
During the last few years it has become increasingly obvious that the computer is a vital tool for scientists, including the genetic engineer. Proper application of computers can provide labor savings, increase productivity, and permit instantaneous acquisition, analysis, and exchange of information. Computers can assist scientists in the modeling of test systems and in process monitoring and control; and they can be used as interactive storehouses of expert information. In many cases, a computer can carry out operations which are virtually impossible for humans to carry out. Through a desk top workstation as an entry port to a computer, a laboratory can access a system which provides text editors, file sharing, analytical programs, databases and database managers, project managers, electronic mail and bulletin boards, and even electronic journals and bibliographic services. A concerted effort at building knowledge and databases into such a system can put the combined knowledge of a group of scientists at the fingertips of everyone accessing that system.
B. COMPUTER SUPPORTFOR GENETICENGINEERING Many genetic engineering operations lend themselves to computer support. Genetic engineers must routinely carry out a variety of nucleic acid and 169 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 30 Copyright 6 1984 by Academic Press, Inc. All nghts of reproduction in any form reserved. ISBN 0-12-002630-9
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amino acid sequence analyses. Many such analyses may, conceivably, be carried out by hand, but they are often based upon complex algorithms which require tremendous reiterative skills, extensive and rapidly accessed memory, and the ability to recognize patterns over great lengths of sequence. Few humans have these skills and abilities, but most computers do. In the past few years, a variety of computer programs have been developed which address genetic engineering applications. Such programs can simulate many genetic engineering operations and can perform a large number of analyses useful to recombinant DNA research. Programs of general utility for support of genetic engineering laboratories have also been developed. These programs carry out routine calculations, plot data, and generally replace written calculation and record keeping. This large body of independently developed software is beginning to coalesce into complete systems designed to provide broad computational support for genetic engineering. Rather than providing a disjointed set of programs which answer a few specific questions, these systems try to meet all the computational needs of a genetic engineering laboratory. The programs fit and flow together, with conserved operational conventions, eliminating the need to do such things as interconvert data from one program to fit another. In this article I shall discuss the design and application of computational support systems for genetic engineering. This article is not a thorough review of all programs available in the field. For such a review, I refer you to the computer applications issues of Nucleic Acids Research, 1982 and 1984. It is extremely difficult, if not impossible, to evaluate the usefulness of programs and systems without actually applying them. Therefore, I shall describe and provide examples of the components of one such system which has proven its utility for support of genetic engineering in the Eli Lilly and Company DNA Computing Environment (DNACE). II. Computational Support Systems
A. THE COMPUTING ENVIRONMENT A computing environment is a computer system in which all the computational supports (tools) demanded by the end users are readily accessible through a single port of entry (for example, a desk-top terminal). The tools contained in the computing environment are logically integrated and transparently flow together; i.e., the inner workings of the system are invisible to the user. The formation of a computing environment establishes a community of users with common interests in the development and application of the tools
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provided. In DNACE, this community consists of three contributing groups. The architects of the environment are information-filled users, knowledgeable in the field of application, familiar with scientific computing, and able to recognize and design applications that computers can address. The builders of the environment are systems experts who are able to write the required programs to address the applications. The third and most important group is the end user group. The end user group operates within the environment and applies the programs to real problems. This group determines if the applications programs are functional, useful, friendly, and error free. These three groups constantly interact and are responsible for the continued development of the computing environment. The users of DNACE are located in several buildings. These users are connected together via electronic communications (bulletin boards, electronic mail; see Fig. 1) provided in the computing environment. Much of the information shared electronically could not be as easily nor as quickly shared in the absence of DNACE. The ability to exchange text and displays (such as restriction maps) is a distinct advantage over telephone communications. Electronic project management can occur: a group of physically separated laboratories working on a common project can readily exchange data without meeting to do so. A major factor in the development of any computing environment is the hardware support provided. The architecture and operating system of the host computer will determine the structure of the programs and files, the package of system-provided utilities, the availability of compilers and other software, and the amount of computation which may be carried out. The more than 100 DNACE-specific executable images (what the machine works with when a program is “run”) range from one to hundreds of blocks each in size (1 block = 512 bytes = 0.5K).The sequence data bases fill more than 30,000 blocks. Add to this the sizes of all the supporting command files, data
8 mail H A I L > send To: j o e S u b j : sample m a i l E n t e r your message b e l o w . P r e s s CTRL/Z when c o m p l e t e , C T R W C t o q u i t : This i s a test. Had t h i s been a r e a l message, I c o u l d haue s i m u l t a n e o u s l y s e n t t h i s message t o s e v e r a l i n d i v i d u a l s o r a l a r g e , p r e - s e l e c t e d group. The r e c i p i e n t s of such a message may r e a d i t and respond t o i t a t t h e i r leisure. T h e r e i s no need t o s y n c h r o n i z e c o m n u n i c a t i o n .
Mojo ^Z New m a i l from JOE MIL>
FIG. 1. An electronic mail message.
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files, etc., and the need for a large machine becomes obvious. DNACE is hosted by a cluster of VAX 11/780s. Different computational applications demand different input and output types. The computing environment must provide the necessary devices to support such demands. Lilly, like most large industrial research organizations, owns a heterogeneous supply of terminals, modems, printers, and plotters. DNACE has had to accommodate this wide variety of devices from many different manufacturers. For this reason, it has been of great value to design and implement applications software which is device independent. To communicate with DNACE, most laboratories have at least one terminal [CRT (video terminal) or personal computer]. Provisions have also been made for input from tape, disk, voice, video, digitizing tablet, or instruments. Output is generated on a variety of alphanumeric and graphics CRTs, printers, and plotters. All of this hardware is expensive. In many cases, specialized devices may be accessed by everyone in the environment, whether the user is at the same location as the device or not. The ability to share such devices provides considerable cost efficiency.
B. A GENETICENGINEERING COMPUTING ENVIRONMENT In this section, I describe the overall design, the interactive style, and some of the applications addressed in DNACE. It is important to note that DNACE is itself part of a larger Lilly Ineractive Drug Design System within the Lilly Research Laboratories Research Computing Environment. Gateways are available to other environments which address activities such as molecular mechanics and design. The discussion below is limited solely to what is within DNACE. Figure 2 represents an overview of the major portions of DNACE. The programs of DNACE are organized into program libraries which address various applications. Programs are available to assist activities such as routine experimental calculation, simulation of plasmid or sequence construction, nucleic acid and amino acid sequence analysis, and evolutionary studies. DNACE maintenance programs automate much of the upkeep of DNACE. Experience has shown that computer programs are virutally useless if their operation is a completely foreign task to the end user. In DNACE, the programs are designed to be interactive and friendly: the dialogue between the user and the computer is in the vernacular of the molecular biologist. Several different types of menus are available to guide the user to the
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COMPUTER APPLICATIONS IN GENETIC ENGINEERING
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programs required to solve his problem. The user may enter DNACE through a totally menu-driven pathway which displays options as molecular biological operations or through a menu-driven pathway which provides program descriptions; or the user may run any of the programs in DNACE from the command interpreter level (Fig. 3). On-line help is available as a complete, selectively accessed help library and is nested in the program headers and interactive prompts (Fig. 4). The documentation is designed to let the user know where he is, what he is doing, and what his current options are. The programs in DNACE are constructed from modular subroutines. These subroutines carry out all of the basic character-string manipulations
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A W Canputinp Enuironment W Canputinp Environment
1 Access W H E L P l i b r a r y . 2 Examine o v r r v i e w o f W C E
3 S r l e c t CURRENT MOLECULE o p t i o n . 4 Run p r o g r a m s d i r r c t l y by nun*. 5 Work w i t h NUCLEIC ACID s e q u r n c e s . 6 Work w i t h W I N O ACID sequences. 7 Access e l e c t r o n i c DATA BASES. 8 Use LAEOWITORY ASSISTWCE programs. 9 Use CALENMWSCHEDULER p r o g r u n . 18 Use W PHONE u t i l i t y . 1 1 UCQ W M I L u t i l i t y . 12 SELECT a n o t h e r menu. 13 RETURN t o W C E m a i n menu. 1 4 EXIT fran W C E . E n t e r t h e number o f y o u r c h o i c e : m I n f o r m a t i o n auai I a b l e : CMPUTE MENUS Top i c ?
DATABCISES WL
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WERVIEW T h i s s e c t i o n , WEWIEW, w i l l p r o v i d e you w i t h an o u r r v i r w o f W C E . The OVERVIEW s u b t o p i c , O I A G W W . w i l l d i s p l a y a d i a g r a m o f t h e a r e a s o f canputational support a u a i l a b l r . The DIAGRM s u b t o p i c s w i l l d i s p l a y a d i a g r a m Of each c a n p o n r n t of t h e WERVIEW DIAGIWn. Additional
information available:
D1A G M
B C4W CMPLITING ENVIRONIENT R E C M B I M W PROORMS T h i s mrnu p u i d e r you i n t o t h e W C E s y s t r m o f r e c a n b i n a n t DNII programs. pranptr You have s r u w a l c h o i c e s of what t o r e p l y t o t h e CD (carriagr return) HELP or ? DESCRIBE BRIEF REGULAR VERBOSE BIT p r o g r a m name
e
Advances you t o t h e n e x t s e g n r n t o f t h e menu. D i s p l a y s t h i s h r l p message. G i v e s t h e v e r b o s e d r s c r i p t i o n o f a propram. P r o m p t s f o r t h e namr o f t h e p r o g r a m . S w i t c h e s t o t h e menu w i t h p r o g r a m names only. S w i t c h e s t o t h r mrnu w i t h o n r - l i n e d e s c r i p t i o n s of t h e p r o g r a m s . T h i s i s t h e s t a r t - u p menu. S w i t c h r r t o t h e mrnu w i t h d e t a i l e d d e s c r i p t i o n s o f the programs. E x i t s f r o m t h e menu. E x e c u t o r named program. You can r n t e r t h e name o f a n y program, w h e t h e r i t i s d i s p l a y e d on t h e c u r r e n t menu c r g n e n t o r n o t .
Comnand: d e s c r i b e Program: ycma Y W P - G r n e r a e s l i n e a r r e s t r i c t i o n maps on t h e l i n e p r i n t r r . Calculatrs W sequence i s i n p u t f r o m f r a g n e n t s i z e and s o r t s f r a g n e n t s by s i z e . a f i l e a n d i s c u t w i t h all c u m n e r c i a l l y a v a l l a b l r r e s t r i c t i o n e n z m r s or any o t h e r i n p u t r e s t r i c t i o n enzyme f i l e .
FIG.3. (A) DNACE entry menu for operation-description menu-driven port of entry. Sample of DNAHELP library access is presented. User input in this and all following figures is enclosed in boxes. (B) DNACE program-description menu-driven port of entry.
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Restrict ion Mapping Program Library Restriction Mapping Program Library 1 Access the W H E L P library. 2 Cut primary sequence with one e n z m e at a time. 3 Cut plasmid map with restriction e n z m e s . 4 Print out single-I inr/enzyme restriction map. 5 Print out fragment-sizr map for each enzyme. 6 Predict restriction cuts from a protein srquence. 7 Store, retrirue, edit and display circular maps (PLA-P). 8 NEW OPTION: Display a plasmap +ile as a LINEAR map. 9 SELECT another menu. 18 RETUW to W C E main menu. 1 1 M I T frun W C E .
Enter the number of your c h o i c e : D There is no default choi for this menu. Do YOU want to continue? Y Enter the number of your c oice: @ To restriction cut a sequence, one enznne at a time, WQ will use thr program, E M I N When we enter E M I N , W > R E A D in the srquencc to be cut, then W > E Z to carry out the restriction.
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required for nucleic acid and amino acid sequence analysis, and are addressable from a variety of programs. Building large programs from modular subroutines provides a great deal of flexibility and economy in programming. To meet a new application, the subroutines often may be quickly reassembled. Applications in information storage and management, routine computation, sequence manipulation and analysis, and display of genetic information will be examined below.
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1. lnformation Storage and Management The use of the computer as a laboratory information storage and management tool is expanding. Computer systems are repositories for laboratory notes, reagent recipes, technical protocols and protocol aids, databases, and even electronic journals. The storage of such information in a computer provides all the benefits of electronic information management, for example, file editing and instantaneous, asynchronous communication of data. This information may be retrieved on-line in the laboratory. To facilitate information input, DNACE users can access text editors and word processing utilities. Database management systems (DBMS) are accessible; DNACE users can custom design their own databases. The DBMSs are being used to record experimental data, purchases, chemical inventories, equipment inventories, bibliographies and abstracts, and more. The ability to rapidly sequence DNA has created a virtual explosion in available gene sequence information. The quantity of protein sequence information is also growing rapidly. The need to manage all this data is being addressed by several electronic sequence database projects. The dominant available databases are the GenBank Genetic Sequence Data Bank (established by Bolt, Beranek and Newman, Inc., Cambridge, MA, and Los Alamos National Laboratory under a contract with several departments of the National Institutes of Health and other government agencies), the European Molecular Biology Laboratory’s Nucleotide Sequence Data Library (Rindone et al., 1983, and Nucleic Acids Research, 1984b), and the Protein and Nucleic Acid Data Banks of the National Biomedical Research Foundation (Orcutt et al., 1982). Each database is available in a variety of media. These databases are setting the conventions for sequence database file structure, format, and information content. They contain a wealth of supporting information for the sequences (locus descriptions, references, features tables). In the absence of an adopted convention for sequence database structure, interconversion programs that interface DNACE programs with the databases had to be developed. To make efficient use of the databases in these early stages of development, a variety of sequence database management, searching, and display programs were also developed for DNACE (Fig. 5). In a genetic engineering environment, the kinds of sequence database searches carried out include searches for entries containing a protein or its gene sequence, for sequence-supporting references, for authors, for statistical information, and whole database homology searches against input experimental sequences. For such searches, it is desirable to examine the most complete collection of information available; for example, a single database constructed from the merging of all the available databases. The sample
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D a t a Base L i b r a r y . D a t a Base L i b r a r y . 1 Access the W H E L P l i b r a r y . 2 Check f o r the presence o f a I o c u s i n t h e d a t a b a s e s . 3 Check t o 5ee i f a sequence was added t o GenBanK t h i s month. 4 Search GenBank f o r a p i u e n l o c u s and s u p p o r t i n g i n f o . 5 Who’s Who: Author s e a r c h . 6 Examine the WYHOFF PROTEIN d a t a b a s e . 7 Examine t h e WIYHOFF NUCLElC ACID d a t a base. 8 Examine s y n t h e t i c OLIOONUCLEOTIDE d a t a base. 9 Copy a sequence from a WYHOFF W T & BASE 10 R E F O W T a n o n - W C E sequence f i l e . I 1 SELECT a n o t h e r menu. 12 RETURN t o W C E main menu. 13 B I T fram W C E . E n t e r the number o f your c h o i c e :
FIG.5. Menu for the electronic DATA BASE PROGRAM LIBRARY. GenBank is automatically integrated into DNACE on a monthly basis. The National Biomedical Research Foundation (DAYHOFF) database is accessed selectively.
search provided below demonstrates the power of database searching tools and demonstrates the kinds of records one may generate with such tools (Fig. 6).
2 . Routine Computation The computer can provide routine computational support for the laboratory (Fig. 7 ) . Complex or repeated calculations can become simple tasks if a computer carries them out. Computer programs can provide a reproducible calculatory method for all users to follow. All the variables can be clearly defined, and can be requested in a logical order, documenting all the essential factors in the calculation. As a result, all such calculations may be standardized and, thereby, have the same meaning from laboratory to laboratory. The program represented in Fig. 8 calculates the efficiency of cDNA synthesis reactions and displays many of these desirable program attributes. Such a program can save considerable time during repeated analytical runs.
3. Sequence Manipulations and Analysis Sequence manipulating programs can simulate most of the operations the genetic engineer may carry out in the test tube. The main sequence editor in DNACE can carry out 31 common operations involving basic sequence manipulations, statistics, and record keeping (Fig. 9). The ability to manipulate sequence information and, thereby, simulate genetic engineering experiments, provides a number of advantages. Simulations allow the genetic engineer to predict or confirm experimental results (e.g., comparison of the predicted restriction pattern of a proper construct with observed patterns). The simulation of sequence rearrangements can sometimes confirm what rearrangements might have occurred. Simulated constructions can be used
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JOSEPH L. MODELEVSKY D a t a Base L i b r a r y . D a t a Base L i b r a r y .
I Access the W H E L P l i b r a r y . Check f o r t h e p r e s e n c e o f a l o c u s i n t h e d a t a b a s e s .
2 3 4 5 6
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18 11 12 13
Check t o 8.. i f a sequence was added t o GQnBank t h i s month. S e a r c h GenBank f o r a p i u e n l o c u s and s u p p o r t i n g i n f o . Who’s Who: A u t h o r s e a r c h . Examine t h e DAYHOFF PROTEIN d a t a b a r e . Examine t h e DAYHOFF NUCLEIC ACID d a t a base. Examine s y n t h e t i c OLIGONUCLEOTIDE d a t a b a r e . Copy a sequence fran a DAYHOFF DATA BASE R E F O W T a n o n - W C E sequence f i l e . SELECT a n o t h e r menu. RETUFW t o W C E m a i n menu. EXIT frm W C E .
E n t e r t h e number o f y o u r c h o i c e : a w o u l d you l i k e a d e s c r i p t i o n ? ( Y / N ) : ~ THIS I S FINDALL. You a r e i n FINDALL. T h i s d e s c r i p t i o n c o n t a i n s a d i s c u s s i o n of PURPOSE, STRATEW FOR APPLICATION, M D INTERACTIONS of FINDALL. PURPOSE I FINDALL a l l o w s you t o a s e l e c t e d requence. and r e t r i e v e t h e code p r o v i d e s you w i t h t h e i n f o r m a t i o n ONLY.
s e a r c h t h r o u g h t h e GenBank d a t a base f o r t h e p r e s e n c e o f By u s i n g k e y words, you can s e a r c h t h e l o c i d e f i n i t i o n s name f o r each s e a r c h e d - f o r sequence. T h i s program GenBank CODE W E S o r d e s c r i p t i v e l o c u s - s u p p o r t i n g
I f YOU want t o s t o p t h e s c r o l l i n g s c r e e n , p r e s s CONTROL S ( h o l d down To s t a r t t h e c o n t r o l k e y and t h e S k e y a t t h e same t i m e ) . s c r o l l i n g a g a i n , p r e s s CONTROL 0. If You n e e d h e l p t y p e HELP.
What k e y w o r d s aha11 1 s e a r c h w i t h ? ( e . p . , h u m a n , u i r u s ) : S h a l l I s e a r c h f o r t i t l e c o d e s o n l y ( T > o r p r o v i d e d e t a i l e d l i s t i n g s (D): h e l p T h i s p r o p r a n w i l l allow YOU t o s e a r c h f o r e i t h e r 1 O C U 8 t i t i e s ( c o d e names) o f It sequence f i l e s o r f o r d e t a i l e d l i s t i n g s o f t h e s u p p o r t i n g i n f o f o r a l o c u s . i s b e s t t o f i r s t ~ e n e r a t ea l i s t o f l o c u s t i t l e s , a n d t h e n p e t d e t a i l e d l i s t i n g s on s e l e c t e d l o c i i n t h e l i s t . Detailed l i s t i n p r contain s t a t i s t i c a l i n f o r m a t i o n . a u t h o r s , r e f e r e n c e s , comnentr, e t c . I f YOU want t o s t o p t h e s c r o l l i n g s c r e e n , p r e s s CONTROL S ( h o l d down To s t a r t t h e c o n t r o l k e y and t h e S k e y a t t h e came t i m e ) . s c r o l l i n p a g a i n , p r e s s CONTROL Q.
(D>BB
S h a l l I s e a r c h f o r t i t l e c o d e s on ly ( T ) o r p r o v i d e d e t a i l e d ~ i s t i n p s WKAIAT MONKEY(BFIBOON) ALPW-I+NTITRYPSIN MIM. I~~BBP mKATII1BFI MONKEY(BAB0ON) M I T H R M B I N 1 1 1 GENE M W (PARTIAL). 120BP MONKEY (BABOON) HIGHLY REPETITIVE DIW SEQUENCE. 343BP WKRS BABOON ENDOGENOUS TYPE C VIRUS (MEW M7) W 5’ END. 132BP BAWGCIOM7 BABOON ENDOGENOUS VIRUS M7 (PRWIRAL); LTR, GAG AND POL GENE. BANGCIGP MBOON ENDOGENOUS UIRUS(M7)PARTIAL GAG GENE. 268BP BAEUGAGP3B BABOON ENDOGENOUS VIRUS, P38 REGION OF GCIG GENE. 92BP WNLTR MBOON ENDOGENOUS WlRUS(BAELJ) L W O T E M I M L REPEAT W . 554BP Do YOU want a d e t a i l e d l i r t l n p f o r ny o f t h e t i t l e s I f o u n d (YiN):@ WCYJT TO DO ANOTHER SEARCH? ( Y / N > I n BEFORE YOU GO, w o u l d YOU l i k e t o r e v ew p r o p r a m d e s c r i p t i o n ? : m
ww5
9
FIG.6. Output from program which searches the databases and retrieves informatioll using user-selected key word(s).
COMPUTER APPLICATIONS IN GENETIC ENGINEERING
179
L a b o r a t o r y A s s i s t a n c e Program L i b r a r y Laboratory Assistance Program L i b r a r y 1 2 3 4 5 6
7 8
9
ie
4ccess the W H E L P l i b r a r y . F o r m u l a t e CsCl g r a d i e n t s . C a l c u l a t e 32P d e c a y a n d volum e C o r r e c t i o n s . E xa mi ne r e s t r i c t i o n enzyme b u f f e r r e c i p e s . Manufacture micro-inJection needles. Make up t r i s b u f f e r s . E xami ne c a l c u l a t i o n p r o g r a m menu. SELECT a n o t h e r menu. RETURN t o DNACE m a i n menu. EXIT f r a W C E .
E n t e r t h e number of Y o u r c h o i c e :
Computation and C a l c u l a t i o n L i b r a r y Computation and C a l c u l a t r o n L i b r a r y
I A c c e s s t h e DNAHELP l i b r a r y . 2 C a l c u l a t e cDNA s y n t h e s i s e f f i c i e n c y . 3 Calculate terminal transferase t a i l i n g results. 4 Account f o r temperature e f f e c t s i n t r i s buffer p r e p a r a t i o n . 5 C a l c u l a t e mol. wt. from m i g r a t i o n d i s t a n c e i n a g e l . 6 SELECT a n o t h e r menu. 7 RETURN t o DNACE m a i n menu. 8 E X I T fran DNACE. E n t e r t h e num ber o f y o u r c h o i c e :
FIG. 7 . Entry menus for LABORATORY ASSISTANCE and COMPUTATION PROGRAM LIBRARIES.
T H I S IS A PROGRAM WHICH W ILL DETERMINE LABELLING EF F IC IEN C IES CVJD IS USED TO CALCULATE cDNA SYNTHESIS EFFICIENCIES. YOU MUST USE M P I T A L LETTERS FOR T H I S PROGRAM.
uwul
R = TEMPLATE CaJCENTRATION I N D = dpm LABELLED dJTP INCORPORATED / u l H = LABELLED W P CONCENTRATION I N u C i / u l C = TOTAL CaJCENTRATIW OF NUCLEOTIDE LABELLEO+CARRIER I N uMOLAR ENTER R, D , H, CYJD C--BE CERTAIN OF W I T S ! ! ENTERED WLU E S WILL BE FIXED N I L C W G E D
*!&&
ENTER R t u g / u l ) ’ ENTER D ? & YOUR SYNTHESIS
DO IT AGAIN
EFFICIENCY
counting e f f i c i e n c y (decimal f r a c t i o n
less
WAS 37.9383 PERCENT
ON A DIFFERENT WPLE
OF WE
RX t 1 1 x 3 9 a
ARE YOU FINISHED~~Q
BYE-BYE,
I HOPE YOU ’SEE‘
DNA’
FIG.8. Output from program which calculates cDNA synthesis efficiency. Interaction demonstrates desirable attributes of calculation programs.
180
JOSEPH L. MODELEVSKY
n lw w l
F un c i o n Action Comnand<s) _--___--_-_ -_--__-__-_--_--__ _--_-_---__ -- 1-3 o r 3-1 Afr c o d e s < 1 3 , 3 1 > -- Change e u e r y o c c u r . -- C o n u c r t D W t o aa -- C a n p u t c 9 c c m p . -- Change f r o m f i l e -- Change
MCWERT ALLCWGE WINO ACIDS BASE C W P CF CWGE CUIPLEMENT DELETE DISPLAY ENZYME F 1ND INERT I uc INPUT INSERT I F
LIST PALINDROMES PRINT WYJDUIIZE READ RDJUlBER REVERSE W E SEARCH S H IF T STATISTICS STOP SYMBOLS TRWSFORM VJWE WRITE
---
----
-------
--
--
--
--
------
----
----
C a n p l e m e n t DNA reg. (CW,COMP> D e l e t e a segm ent Compact s e q d i s p l a y R e s t r i c t i o n enzyme <EZ> F i n d a sequence I n v e r t DW s t r i n g I n u e r t dr c o m p l e m e n t < IC,I U C > C r e a t e a new seq. I n s e r t a 5equence (IS> I n s e r t fran f i l e < INSF > L i s t a sequence (LI) Find palindranes L i s t t o l i n e p r i n t . Random ize s e q u e n c e s (RAN> Read f r m f i l e (RE> S e t s t a r t i n g num ber (R”) Reverse a sequence T e m p o r a r y s a v e seq. <%> Character searching.<SE> S h i f t s e q . r t o r I t <SH> Sum nar y s t a t i s t i c s <ST> E x i t t h r program <W> To see t h e DNA symb.<SYMB> C o n v e r t DNA <-> RNA
Get saved sequence <W,WS> W r i t e t o per m . f i l e <WR>
FIG.9. Help message from EDMAIN, the main sequence editor of DNACE. Message describes the operations available in EDMAIN.
to determine if experimental problems may develop as a project continues; for example, one optional construction may yield a situation where a cloned gene or fragment cannot be distinguished from a vector fragment when digested with a selected set of restriction enzymes. Computer-generated restriction maps can trap such errors. The analysis of expression units is a common computer application in genetic engineering. The construction of an in-house database of expression units and their components (promoters, ribosome binding sites, linkers, adapters, structural genes, terminators, vectors) is, in itself, valuable. Beyond this, the ability to electronically manipulate this data into new simulated constructions can permit the evaluation of potential constructs. One may, for example, examine the restriction map of a simulated expression unit or, as shown in Fig. 10, the folding of the messenger RNA (Zuker and Stiegler, 1981). The computer analysis of proteins can involve hydropathicity predictions to determine solvent accessibility or antigenic target sites (Kyte and Doolittle, 1982; Hopp and Wood, 1983), predictions of secondary structures, or prediction of protein stability (Fig. 11). The initial cloning of a gene presents many analytical problems. Often, the target gene product is poorly described; assays for the gene product may
COMPUTER APPLICATIONS IN GENETIC ENGINEERING
181
You have j u s t e n t e r e d W F O L D . W F O L D I S an i n t e r a c t i u e u e r s 1 o n o f a p r o g r a m d e v e l o p e d hy M i c h a e l Z u k e r , C a n a d i a n R e s e a r c h C o u n c i l , w h i c h w i l l f o l d n u c l e i c a c i d s e q u e n c e s i n t o a s i n g l e m l nl m um e n e r g y c o n f o r m a t i o n . y o u may g e n e r a t e a p r e t t i e r d i s p l a y u s i n g CI c r u d e d i s p l a y i s p r o v i d e d ; R. F e l h a n n ' s p r o g r a m , NUCSHO, i f y o u e l e c t t o g e n e r a t e a CT f i l e d u r i n g the interaction. ENTER ENERGY F l L E NAME
LENERGU37)
ENTER C FOR A CONTINWTION RUN,
S FOR W E (FUTURE CCNTINWTION)
M D FOR A REGULAR RUN. ENTER SEQUENCE F I L E W
ITESTFOLD.DNA
2
E
a
E n t e r two id's: 7 2 NUCLEDTIDES READ OUTPUT TO T E R H I M L 3 Y OR N
i 3 CT F l L E
GENEWITION3 Y OR N
I3 ENTER ENDPOINTS 11,72'1 **I
OF FRAGMENT TO BE FOLDED.
TABLE OF C W G E A B L E PARAMETERS
AUXILIARY CONSTRAINT DEFAULTS: I ) FORCED BASE PAIRS 2 ) PROHlBlTED BASE PAIRING
------
-
(DEFAULT
=
1,N)
***
SET TO 'NO' SET TO 'NO'
LOOP PARAMETER DEFAULTS: 1 ) 0-U BASE PAIRING ONLY ALLOWED I N THE INTERIOR OF STACKING REGIONS 2) WIMW BULGE OR N I TERO IR LOOP SIZES SET TO 38 3) W I M U I N O . LNWIIRED BASES O N BOTH SIDES OF BULGE OR LOOP SET TO 313 FOLDING PARAMETER DEFAULTS: 1) M I N I M W HPIRPIN LOOP S I Z E SET TO 3 (NEVER < 3) 2 ) W X I M u 1 DISTW CE BETWEEN W S E P6IRED NUCLEDTIDES SET TO 1 6 e e e . PLEASE
I3
ENTER ONE OF THE FOLLW ING: TO BEGIN FOLDING: TO QUIT THE PRDGWYl TO SET W Y OF THE DEFAULT P A M E T E R S
FOLDING BASES 1 TO -16.9 ENERGY =
7 2 OF
ie 28 38 CGGWTATGCTCG AGT TAC ACA CTWTACWTTGC T TGT WCTTTATGGTTMCG G WGTWCCA --G TAC 78 68 58 48 ENTER: T T E R H I M T E , NS NEW SEQUENCE, NF NEW FRAGMENT, 0 OLTPUT PARAMETER 1 Fy.(O 72. D E F IN IT ION , OR THE ENDPOINTS OF A SUBFWIMENT BETWEEN E D WITH 1 TO FORCE ENDS TO BASEPAIR.
_---
4
FORTRPN STOP
S h a l l we d o t h i s a g a i n ? ( Y / N ) : h
You a r e n w l e a v i n g RNAFOLD.
FIG. 10. Output from RNAFOLD, a nucleic acid folding program. The original program was generously provided by its author, Michael Zuker, Canadian Research Council, and has been integrated into DNACE.
182
JOSEPH L. MODELEVSKY
You h a v e j u s t e n t e r e d HYDROPATH. D o y o u w a n t a d e s c r i p t i o n of
t h e program?
You h a v e j u s t e n t e r e d HYDROPATH. HYDROPATH p r o g r e s s i v e l y e v a l u a t e s t h e h y d r o p h i l i c i t Y a n d h y d r o p h o b i c i t y o f a p r o t e i n a l o n g i t s amino a c i d sequence. HYDROPATH UECE a m o v i n g s e g n e n t a p p r o a c h t o c o n t i n u o u s l y d e t e r m i n e t h e a u e r a g e HYDROPATHy w i t h i n a s e g n e n t a s i t a d v a n c e s t h r o u g h t h e segm ent a n d c o m p a r e s t h e s e a v e r a g e d s e g n e n t t o t h e w h o l e p r o t e i n HYDROPATHy. I n t e r i o r segments o f t h e p r o t e i n s h o u l d E x t e r i o r segnrntc c o r r e s p o n d t o t h e h y d r p h o b i c s i d e of t h e o u t p u t r a n g e . s h o u l d c o r r e s p o n d t o t h e h y d r o p h y l l i c s i d e of t h e o u t p u t r a n g e . ( J . KYTE a n d R . DOOLITTLE, J.Mol. E i o l . 157, 185-132, 1982). You MUST USE ONE-LETTER a m i n o a c i d c o d e s . Code c o n v e r s i o n i s p e r m i t t e d i n t h e p r o g r a m . The r i g h t s i d e of t h r p l o t i s HYDROPHOBIC. The l e f t s i d e o f t h e p l o t i s HYDROPHILIC. F o r an m i n o a c i d f i l e E n t e r t h e f i l e name: )-it
(i.e
SAnPLE.W),
1s t h e a a f i l e i n 1 o r 3 l e t t e r c o d e s 7 ( e n t e r E n t e r t w o c(A id’s:
1 o r 3):m
i n p u t l e n g t h of hydropath se e n t over which to average, (n u mbe r o f aa r e s i d u e s ) : Want a P l o t ? Want a p r e d i c t i o n of s e c o n d a r y s t r u c t u r e ? Llr) You h a w J U S ~e n t e r e d a v e r s i o n o f t h e Fasman A n a l y s i s . T h i s u e r r i o n p r e d i c t s s e c o n d a r y s t r u c t u r e c h a r a c t e r i s t i c s of t h e a m i n o a c i d sequence i n p u t i n HYDROPATH. The c a l c u l a t i o n s a r e b a s e d u p o n c o n f o r m a t i o n a l p a r a m e t e r s e s t a b l i s h e d f o r 29 r e f e r e n c e p r o t e i n s : Reference Set I
-
Chou a n d Fasman, B i o c h e m 13, p.211- 245 1974 a n d A d v a n c e s i n E n z y m o l o g y , 1978; R e f e r e n c e S e t 2 = FEBS L e t t e r s 93, 19-24, 1978.
FIG. 11. Output from program HYDROPATH. The output contains a hydropathicity analysis of the amino terminus of the OmpF protein of Escherichia coli
exist, but there may be limited data on the protein itself. The target gene product may be associated with a cloned segment of DNA, but the resolution of the DNA into hnctional domains is not a simple problem to resolve without more data. The computer can assist in the analysis of newly cloned DNA. Once a clone has been isolated, the cloned DNA sequence is readily determinable. Some useful computational tools which can be applied to analyze such DNA sequences include searches for open reading frames, determinations of coding probability for those open reading frames (Fickett, 1982), and searches for functional elements (promoters, ribosome-binding sites, terminators).
183
COMPUTER APPLICATIONS IN GENETIC ENGINEERING
12)
E n t e r r e f e r e n c e s e t number ( I o r 2 ) : T r a n s l a t i n g I l e t t e r codes t o 3 l e t t e r s . 368 codes. E n t e r P r o t e i n Name i n c a p i t a l
l e t t e r s (e.g.
l i m i t 20 l e t t e r s : [ E . c o l i mpF]
W E ) ,
T r a n s l a t i n g 3 l e t t e r codes t o I l e t t e r . 368 codes ( 1888 c h a r a c t e r s ) .
These analytical results can suggest the necessary experiments to confirm assignments of functional domains. 4. Display of Genetic Z n f o m t i o n All of the analyses described above create output which must be interpreted by the researcher. The information generated by these analyses must somehow be displayed for review. Computerized display of genetic information has been addressed in a number of ways. Simple data tables containing a list of information (e.g., strain designations and their genetic markers) may be used. Restriction maps may be displayed linearly across a screen, with cut sites delineated by labeled or unlabeled tick marks, or as circular maps. For the genetic engineer, the circular restriction map is the
184
JOSEPH L. MODELEVSKY
predominant format for display of plasmid genetic information. Circular restriction maps frequently contain a large amount of information, including dozens of restriction sites, genetic markers, and statistical and historical information; however, such maps do not readily lend themselves to computerized display. Often, sites of interest overlap or are congested into small display areas; circles themselves require relatively high graphic resolution for display. Recently, storage, retrieval, and display of circular restriction maps have been implemented in DNACE using the program PLASMAP (Stone et al., 1984). PLASMAP forms the basis for the graphic display of color-coded genetic information which is superimposed upon conventional line displays. Using this approach, a variety of analytical results and constraints can be displayed on a single screen of information (Fig. 12).
PSEUDO p B R 3 2 2 D e r i v a t i v e Clal Hincl I Rrul
Pvul
, Xmal I I Nrul
1000
Sonple PLASYAP doto flle ond dlsploy. A alnple olpho-nummrlc toble Is convmrtmd lnto o grophfc dlrploy of o convmntlonol. tnformotlon-dmn#e, cfrculor rertrlctlon m a p . Thfs mop lncluder locatlonr o f reverol known restrtctlon enrym recognltlon sequmncer occurlng two or fewer timer ln pBR322DNA. The poaltlona of the tetrocycllnm ond omploillfn rmrtrtoncm goner: romplms of overlopplng Iondmorkr and o pmeudo cloned erprmsrlon unlt (Px. pronotrr X; RBS. rlboronm blndlng slte: Gene X ) orm olro Indlcatmd. Plotted on HewImttPcokord 7220 plotter. Rmstrlctlon rltes from Sutcllffe. J.G.. Cold Spring Horbor Symposlum (1979) 43. 77-90.
FIG. 12. Sample display from PLASMAP, the storage, edit, and retrieval system for circular restriction maps. Color-coded information may be superimposed on graphic display.
COMPUTER APPLICATIONS I N GENETIC ENGINEERING
185
C . A SAMPLEAPPLICATION Thus far, I have dealt mainly with the computational side of computer applications in genetic engineering. Next, to describe how genetic engineers might operate, I wish to specifically demonstrate the applications described above via a model genetic engineering project. The target gene in this model is the hypothetical human INSIGHT gene. Assume that the INSIGHT gene product is expressed in the pituitary at barely detectable levels and is believed to have a molecular weight of approximately 9000. The demonstration begins with the only known sequence, 21 amino acids at the carboxyl terminus of the protein (Fig. 13). The decision has been made to clone INSIGHT cDNA. To begin, RNA is extracted from human pituitaries, and from this RNA preparation, a fraction of poly(A) (messenger) RNA is isolated. This poly(A) RNA will be the template for cDNA synthesis. To increase the specificity of the synthesized cDNA population, a cDNA synthesis primer specific for INSIGHT messenger RNA will be used in lieu of an oligo(dT) primer. To design this DNA primer, the computer will reverse-translate the known INSIGHT amino acid sequence and select the coding region of minimal degeneracy (i.e., the region coded for by a minimum number of possible DNA sequences). Then a set of synthetic oligonucleotides (DNA pieces) complementary to this coding region will be made; these oligonucletides will be used as primers for cDNA synthesis. Since the known amino acid sequence is at the carboxyl terminus of INSIGHT protein (i.e., coded from the 3’ end of the message), using a primer for this region should provide highly discrete, nearly full-length, cDNAs for INSIGHT protein. As shown‘ in Fig. 14, there is a region of the INSIGHT amino acid sequence (beginning at amino acid number 9 of the known 21) that may be coded for by 8 different 12-mers (12 base long oligonucleotides) or 16 different 14-mers. A 12-mer should be capable of priming cDNA synthesis, should be highly discriminatory in the RNA population, and may be used as a colony hybridization probe to screen resultant clones for the presence of INSIGHT cDNA. (Colony hybridization is a technique which may identify bacterial colonies which contain “probed-for’’ DNA sequences.) To use these oligonucleotides as probes in a colony hybridization screen-
Carboxy t e r m i n u s of hypothetical I N S I G H T protein.
-_---h i n o
a c i d sequence
S t a r t i n g point
1.
I L E T R P MET GLY -- A S P ARG PHE PRO ARG _-_-__-_- -__ THR -_____ __---- --- -- --- -__-____-__GLY I L E MET ARG PRO -- THR __---_ --_ -_- -_- _- --_
I V A L GLY ALA THR SER
_ _ -___
16
Frc. 13. The carboxyl terminus of the hypothetical INSIGHT protein.
186
JOSEPH L. MODELEVSKY i**I+.*l****C*I******f+************************~*********ff*
APROF You h a v e j u s t e n t e r e d APROF: APROF s c a n s s u b s e q u e n c e s i n an M s t r i n g a n d g e n e r a t e s a s e t o f DNA s e g m e n t s w h i c h c o u l d code f o r t h o s e subsequenes. The D M s e q u e n c e s w i t h t h e h i g h e s t number o f f i x e d b a s e s o r GC c o n t e n t a n d minimum number of DNA c h a i n s r e q u i r e d t o c o d e f o r t h a t M subsequence i s d e t e r m i n e d a n d p r i n t e d . Useful i n the design o f s p e c i f i c DNCI p r o b e s +or unknown gene s e q u e n c e s b a s e d u p o n known M sequences.
READ t o r e a d an aa f i l e SCAN t o compute f i x e d b a s e s a n d p o s s i b l e sequences DNA t o d i s p l a y t h e c o d o n s f o r a segment Ll ST d i s p l a y s t h e a m i n o a c i d sequence ENTER i n p u t an a m i n o a c i d sequence f r m the teletype. PRINT e n a b l e s o u t p u t t o t h e l i n e p r i n t e r NOPF.INT d i s a b l e s l i n e p r i n t e r o u t p u t
+a
F o r an a m i n o a c i d f i l e E n t e r t h e f i l e name: i n s i h t f . a a a E n t e r t w o PA id‘s: Translate 1 l e t t e r t o l e t t e r codes’ T r a n s l a t e 3 l e t t e r t o I l e t t e r codes’
&-
The p e p t i d e i s The pseudo-dna
QOCJ
R
21 a m i n o a c i d s l o n g . f r o m bases 1 to
63.
F u l l listing’ O p t i m i z e C h a i n s , F i x e d Bases, o r GC c o n t e n t 7 C C F G I : m L e n g t h ( i n c o d o n s ) o f amina a c i d s e g n e n t t o be c o n s i d e r e d : E n t e r l o w c o d o n , h i g h codon:
I 2 6 7 The The The The
Fixed = Fixrd = Fixrd = Fixed =
segnent maximum maximum minimum
6 6 7 8
GC GC GC GC
=
=
6.42993 8.37837
E n t e r t h e l o w amino a c i d l o c a t i o n , Fixed =
19
GC
Chains = Chains = Chains = Chains =
64 64 12 3
3 8 6.72222 3
length is: number o f f i x e d b a s e s i s : gc c o n t e n t i s : number o f c h a i n s i s :
m
5
= 8.72222 = 8.72222
a
length of s e g m e n t : m
= 8.51667
Chains
=
27648
4 THR SER THR I L E TRP MET GLY ASP ARG PHE PRO ARC
Fic;. 14. Computer-assisted INSIGHT primerlprobe design. Minimally redundant coding region is enclosed in box.
187
COMPUTER APPLICATIONS IN GENETIC ENGINEERING
D o you want t o do a h m o l o g y ( h y b r i d i z a t i o n ) t e s t o r a s i m p l e s t r i n g match’ Answer H o r H : F o r t h e s t r i n g YOU want t o s e a r c h , t h e base CNA f i l e E n t e r t h e f i l e name. p b r 3 2 2 dna E n t e r two DNCI i d , s : ’ F ’ 1 to The DNA c o n t a i n s 4363 b a s e s ( l o c a t i o n F o r t h e s t r i n g YOU want t o s e a r c h w i t h , t h e M S K f i l e , E n t e r t h e f i l e name:lprobes.dna\ E n t e r two 1.5’5: hasps 1 to The M S K c o n t a i n s E n t e r minimum l e n g t h and p e r c e n t : Output t o the l i n e p r i n t e r ’ 4363 The W s t r i n g goes from l o c a t i o n 1 to E n t e r low and h i g h l o c a t i o n s f o r DNA s e g 1 : 1 to 28 The M S K s t r i n g goes f r o m l o c a t i o n E n t e r l o w a n d h i g h l o c a t i o n s f o r M S K s e g 2: L i s t the input s t r i n g s ?
-
m28 :m
4363)
28)
:m
Beginning search.
I n d i c e s are
From 443 From 1 ATCGCCWWTC PTCNCCCATC
to to
454. 18.
From 888 From 1 GTCCCGCCACCA PTCNCCCATCCA
to to
899.
Fran 3523 3 From CGCCTCCATCCA CNCCCATCCA
1
4363
28
1
Len
12.
12.
P c t 9.83333 Len
12.
to to
3534. 12.
P c t 8.83333 Len
12.
to to
542. 28.
P c t 9.83333 Len
12.
P c t 8.83333
I@: From 531 Fran 17 TGGCCGGGGWC TGGATGGGNGAQ
3875 t o 3887. From From 17 t o 28. P c t 8 . 8 4 6 1 5 Len TGTATGCGGCGAC TGWTGGWGAQ 28: 5 homologies found. There were DNA c o n t a i n s 4363 b a s e s ( l o c a t i o n The The MASK c o n t a i n s 28 b a s e s ( l o c a t i o n
13.
1 to I to
4363)
28)
FIG. 15. Homology test between cloning vector pBR322 and INSIGHT prirner/probes.
ing process, there can be no homology between the probes and pBR322, the selected cloning vector. The results of a homology analysis reveal that there is no significant homology between the primer-probes and either strand of the vector (Fig. 15).Therefore, the eight 12-mer primers will be synthesized for use in the project. The oligonucleotide database is checked for the availability of precursors; none is found, so an automated synthesis machine is set up according to the formulations programmed into the synthesis management program library by the chemists (Fig. 16). Next, the cDNA is chemically synthesized. The progress of the synthesis
188
JOSEPH L. MODELEVSKY
T H I S PROGRAM W I L L CALCULATE THE AMOUNTS OF NLICLEOTIDE MONOMERS AND CONDENSING REAGENT TO DO A SAM RUN, THE SEQUENCE W I L L BE STORED I N ‘WWILOG.LIS BY RUNNING 0DD.EXE.
AND CAN BE ACCESSED
ENTER THE SEQUENCE TO BE SYNTHESIZED I N THE 5‘
TO 3‘
I F THERE ARE ANY M I X E D S I T E S , USE ‘P‘ ’N’ FOR A/C/G/T.
‘12‘ FOR A/G,
TYPE I N THE I D E N T I F Y I N G NCIME, USE, W D SYNTHESIS METHOD
NNWNNNNM‘I
e.g.
SEQUENCE’
PROBE
GGH-I
FOR T/C,
REQUESTOR,
SMITP
DIRECTION. AND
NOTEBOOK REFERENCE,
im-e-em
SAM
IGT CTCCCATCCA?
OTHER lNFDRMATION7 DO YOU WANT T H I S SEQUENCE WRITTEN I N ‘DNALOG’? W I L L SFIM BE DOING T H I S SYNTHESIS7 Y ’ N ’ D CCTACCCTCTG T H I S SEQUENCE IS A
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