Basic Biotechnology 2 ed

BaSK Biotechnology Biotechnology impinges on everyone-'s lives. It is one oftlle maJor rechnologies oftbe twenty..first century. les huge, wide-r.mging, multhlisdplinaTY activities ¡nelude recombiIlanr ONA: techniques, doning 3ud geoetics, and the application ofmicrobiology to the production ofgoods as prosak as bread, beer, cheese and antibiotics. Jt continue$ tO revolutionise 1:realnlents ofmany diseases, and is used lo provide clea.n tedlnologie5 and todeal with environmental probJems. Basic Blo:t'chtwlogyis a textbook. thal gives aJuU accouDtofthecu.r rentstate of biotechnology, providing!he ~ader with iruight, inspiratlon and instruction. The fundamental aspects lhat Uflderpin biotechnoiogy are explained through examples fiom me pbarmaceutical, food and environmental industries. Olapters on the public pen::eption ofbiot.echnology and the business aud economics of the subject are <"l150 Included. The book is esse.Drial reading' fue all students and teachen ofbiotechnology and applied microbiology, and forreseuchers in the many blotl!Chnology industries.

-'

Basic Biotechnology

SECOND EDITION

eó'ed by

Calin Ratledge and Bj0rn Kristiansen Univefsiry a(Hul!, UK

Europeon Siorechoo/ogists, Norwoy

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www.k-t- dra .com

HCAMBRIDGE ~ UNIVERS.I TY l'RESS

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The Edlnburgb Building, Cambridg-t'CB2 2RU, UK -'10 West 20tb Strert. NewYork. NY l 00II-4 211. USA 10 Stamford Road, Oakl eigh. VlC3166, Australia Ru1zdeAlan::6n 13, 28014 Madrid. SpaiD Dock House, nle W:tlerfront, Cape TO\\.'D 8001. SOulhAfric~ brtp :lfWww·cambridge. org

o Cambridge University ('ress 2001 Thi¡; bwk is In copyrigbt.Subjec Lte .t.1tulOry eJIcc ption iltId to the provtsloru: ohelevant coll&ffi>e li~ns¡ng agreemenlt . no teproducti o!l of any part m ay (ake place withoul Ihe writtfn pe~ion ofCambridge Univ=;ity Press. f'irsl pub lis hed 2001 Re-prinled2001 Printcd in th/!; Uniled KingdOlIl al the Unil'!'ulty PI"eI'5. Cambridge

ISBN O 52\ no7:/¡ 2 hardba.ck ISBN O 521 779170 p;lpe1'back

Contents l.ist 01 c()f)trlburors ""fa"

page xi

'v

Part I Fundamenab and principies Publlc perceptfon ofblotec:hnology JOHN E. SMITl-t 1.1 1.2 1.3 lA

Introductlon Public il.warenc$S oC genetic engineering Regulatary requirl!mencs - saretyof ge.netically ellgineered fuods Labelling-howfarsho\Jldilgo? 1.5 Policy maldng 1.6 Areas ofsignificantpublic concem 1.7 Condusions 1.8 Further reading

2

Biochemistry and physiolo¡y 01 crowth and metaboli5m COLlN RATLEDGE 2.1 Introducrion 2.2 Metabolisrn 2.3 Catabolic pathways 2'< Gluconeogenesis 2.5 Energy production in aerobic m.iCJ'OoOrg;¡.nisros 2.6 Anaerobicmetabolism 2.7 Biosyntbesis 2.8 Control of m etabolic processes 29 Efficiency of microbial growtb

2.10 Further readJng

1

Stoichiometry and kinetics of mlcroblaJ growth from a thermodynamk perspective I IHEIJNEN 3.1 3.2 33 3.4 3.5

•

Nomendature lntroduction Stoichiometty ca1colations s ro lchiomell:)l pred.ictions based on Gibbs enl!l"8Y dissipation Growth kinetics froma tbermodynamic point(lfview Further readlng

Genome management and anatysis: prokaryotes COUN R. HARWOOD and ANIL W IPAT 4.1 4~

... '.3

'S

H

'.7

.., 4.8

4.10 4.11

lntroduction Bactmal chromosomes and natural gene transrer What i5 geoeru: eogineering and what is it used fuI'? The: bask tool5 of genetic engineering Coning vectors and librarie5 Analysis of genomeslproteomes Analysis ofgeoe expression 8ngineering genes and optimising productll Production ofheterologous prodUClS In rllko analysis orbacterial genomes Purther reading

3 3 4 7 10 11

12 16 16 17 17

18 21

,.

2. 32

35

..

38

43 45

45

"•• 52 56

" "

59 5.

65

•• 76 81

83 87

89 91

93

,;

-1 CONTENTS

5 Genetlc engineering: yea.st5 and filamentOU5 fungi 95 DA\IlD B. ARa-tER. DONAlD A MACKENZIEand DAVID J. JEENES 5.1 5.2 5.3 SA 5.5 5.6

5.7

6

Glossary lnrroduction Introducing DNA intO fungi (fungal uansformiltion) Gene c10ning Cene structure.organisation;wd expression SpedaJ m etbodologies Biotechno logical applications offungi Furtber reading

Mlcrobial proce!lS kinetics

jEN5 N IELSEN

Nomendature 6.1 6.2

6.3 6.4

7

Bloructor design 7.1 7:1. 7.3 704 7.5 7.6 7.7 7.8

a

9.4

9.5 9.6 9.7

9,8 9.9 9.10

10

lO'

116 119 120

126 127 127 128 130

141 14'

151 152 153

,,,

158

162 164

"8 171

HENKJ. NOORMAN

173

Nomeodatlire Introduction

173

The man transfur srep!

175 177 183 185 180

174

Mass lransfer equatioru Determining the volumerrk In3SS tr.msfercoeffidents The efl'ect o(scale on mass tr.IQ SreT Further reading

Downstream processlng In blotechnolosy RAJNI HATTI·ICAUL and BO MATIIA550N !U 9.2 9.3

100

YUSUF CHISTI and 11URRAY MOO-YOUNG 151

Nomecdature IntroductioD Bioreactorconfigurations BioreactordHign teatures Desigo fo r steril e opera tion Photobioreacton He3t transfer Shear effects in culture Purrher readios

Mass transfer 8.1 8.2 8.3 8.<4 8.5 8.6

9

lntrodU CtiOD

Kine tic modelling ofcell growth Mass bala nces fur ideal bioreilctors FUj"[her re¡¡diog

95 97

18'

In troductlon

187

Downstrea.m prol:esslng: a muldstage Oper.ldon SoHd-liquid separation

187

Rele;ueofinuaccllularcomponents ConcMuation ofbiologiciI] products PulificatiorL by chromatogra phy PnxIUCI funuuladoll Monitoring ofdownstream processing Process in tegradon Further reading

Measurement and control NomencJature 10.1 lntroduction

A LÜBBERT ancI R. SIMVTlS

188 193

195 204 207 209

210 211

213 2'13 214

CONTENTS

10.2 10.3 10.4 10.5 10.6 10.7 10..8 10.9

I1

Process economlcs BJ0 RN KRIsnANSENRAJNI tu Introduction 11.2 Tbe startingpoint 11.3 Cost e.1:imóltH n.4 Process design 11.5 DesigTI exercise. 11 .6 Cap¡tal costs estimates 11.7 Oper.lting cosu estimllte5 11.8 The costs case - ro build or not ro build 11.9 Further reading

Part 11 12

StructuJ'e OrpnX"eS5 models Kinetic Tate expreulons AdV3nc~ mode.lliugcollliideratiollS I'rocen supemsion and co ntrol Open·loop con trol Closed·Joop con trol Condusioo FUl1her read ing

Practical 8Jlpllcations

The business 01 blotechnology WI LLlAM BAlNS and CHRJS EVAN$ l2.1 Introductlon 12.2 What i5 biorechnoloQ' used for? 12.3 Biotechnology companies. tb eir care and nurturing 12.4 ln~onent in biotechnology 12.5 WhD needs management? 12.6 Patento; and biorechnology 12.7 Condusion: jumping the feoce 12.8 Punherreading

1) Amino acids L EGGEUNG, W PFEFFERLE and H. SAHM 13.1 13.2 13.3 13.4 13.5 13.6 13.7 13.8 13..9 13.10 13.11 13.12

14

Introduction CommericaJ l15eofatuinoadds Prodllction methods and 10015 L-Glutamate l-Lyslne L-1hrronine

215 216 222

"a 230 231 23.

"" 239 23' 240 240

242 2....

247 247 250

252 253 255

255 255 26()

26S 275 278

279

279 281 281

282

28'

285 289 2'"

L·PhenylaJaJ.1ine L'Tryptophan

296

L·AlIpartare

300

Outlook. Acknowledgements Furtherreading

302

Organic adds CHRISTIAN P. KUBICEK 14.1 lntroduction 14.2 CitrJc add 14.3 Gluconic acid 14.4 Lactic acld 14..5 Other acids 14.6 Furtber reading

29' 302 302 305 305

OO.

315

317 319 32<

I

viii

CONTENTS

15

Microbial polyhydroxyalkanoates.pofytaccharldesand lipids AUSTAIRJ. ANDERSON andJAMESP.WYNN 15.1 Introduction 15.2 Microb¡al polyhydroxyalhnoates 15.3 Microbial polysaccharides 15.4 Microbl.1..llipids 15.5 Further readiog

16

Antibiotics 16.1

16.2 16.3 16.4 16.5 16.6 16.7

16.8 16.9 16.10

16.11 16.12 16.13 16.14 16.15 16.16

17

Baker's yeast SVEN·OLOF ENfORS 17.1 17.1 17.3 17.4

17.5 17.6 17.7

18

19

DAVIDALOWE Jntroduction Biosynthesis 5traln improw:ment Genetic mgineering AnaIysis Culture preservarlon and aseptic.propagatioo 5cale-up Fermentadon hnicillim Cephalosporins New fJ-Jactam technologies Aminoglycoside.s Macrolides Economics Good Ma.nufac[uring Practices Furtherreading Nomenclature lntroduction Mediurnfor baker'syeastproduction Aerobicethanol formationandconsumption Ihe fed-batch technique used ro control ethanoJ proc:luction Industrial process control PrOCe5S outline Furtberre
Production of enqmes DAVID A LOWE 18.1 Introduction 18.2 Enzymes from
325 325 325 333 339 348 349 349

35 1 351 35<1

354 354 355 356 361

367 368 370

373 373 37' 375 377 377

378 380 381

384 386 387 389

391 391 393 395 396

39'

400 40' 403

'0' 40. 407 408

409

.12

CONTENTS

19.3 19.4 19.5 19.6 19.7

10

21

CJ¡jr;¡J building blocl3 for synthesis

<1,

Reductions and oxidCLtiOns Use of enzymes in sugar chemistry Use ofenzymes to make aminoacids and peptides Further reading

419 422 426 428

Recombinant proteins o( high value GEORG-B. KRESSE 20.1 Applications ofhigh-value proteins 20.2 Analytical enzymes 20.3 Therapeutic protei..n.s 20.4 Regulatory a.spectS aftherapeutic proteiru: 20.5 Outlook to the future ofprotein therapies 20,6 Funherread!ng

Mammallan cen culture

N. VRJEZEN. J. P. VA N DIJKEN

429

429 430 436 446

446 447

449

"'" L HAGGSTROM 21.1 21.2 21.3 21.4 21.S 21.6 21.7 21.8 21.9

22

lntroductioll Mammalian teU Ilnes and their characteristia COInmerical products Proteinglycosyliltion Media fot [he cultivation ofmammalian cells Mel.4lbolism Large-scale cultivatíon ofmammalian cells Genetic engineerlng ofmammalian (!!lis Further readiu.g

Blotransformatlons JOAQU1M M.S. CABRAl lntrodunion 22.2 Biocatalyst selection 22.3 Bioca.talyst irnmobilisatioll and performancr 22.4 [mmobilised enzyme reactDrs 22.5 Bioca.talysis in non
2J

14

449

4SO 452

453

'SS 458

46' 468 470 ' 7J

'71 473 475

'"'91 500 SOl

Immunochemic::al applications M1KE CLARK Glossary 23.1 lntroduction 23.2 Ann'body S!:n.lcture :md functions 23.3 Antibody prorein fragments 23.4 Autibodyaffinity 23.5 Antibody spec:i.ficity 2.3.6 lmmu.nlsation and productioo ofpol~ onal :rntisen 23.7 Monoclonal antibodies 23.8 Anrlbodyengineecing 23.9 Combinatorial and phage display librarles :D.IO In vUm uses ofrecombioant3nd monoclODaJ antibodies 23_11 rn viw usesofrecombiuant and mOnodonal antibodies 23. l2 Furrher read.ing

S03 503 506 506

Environmental applications and Wll1.Y VERSTRAETE 24..1 lntroduction 24..2 Tteatment ofwaste water

S31

PHIUPPE VANDEVIVERE

5 10

512 513 SI. 517 518 520 522 526 529

531

532

be

)(

CONTENTS

24.3 24.4 24,S 24.6

Digestion of org;¡.nicslun ies TreallIlent ofsolid wastes Treatmen! ofwastegases Soil rcmediiltion 24.7 Treatmenlofgl'oundwarer 24.8 Furtherreading

540 54' 545

1""'"

559

549

554 557

Contributors

ALASTAI!!. J. ANDERSON

L. EGGElING

Department of Bio Logica1 Sciences UniversiLy ofHull HuI!

¡nni tut fiir Biotechnologie

HU67RX, UK

0-52425 Jülich Germ:my

TH ORLElf ANTHONSEN

ForschungszentrumJülich CmbH PO Bax1913

Kjemisk InstituU

SVEN-OLOf ENFOR S

l
~partment of Biochemistr'y and Siotechnology RoyallDstitute ofTechno1ogy 5-100 +4 Stockholm

7034 Trondheim Norway DAVID B. ARCHER

School ofLife and Environmenral

Sweden

Sdences

C HRIS EVANS

UniVt'l'Sity of Notringham UniversilY Park Noningbam NG7ZRD.UK

Merlin Biosciences LuI 12 StJames's Square loodon SVV1Y
Wl l llAM SAINS MuLin Biosciences Ltd

L. H AGGST RÓM

12 5tJames's Square Lendon SW1Y4RB.UK

IOAQUIM M. S. CAUAl

laboratorio de Eugi!nharia Bioquimica Instituto Superior Te<;hn \CO 1000 Lisboa

Portugal

Department ofBiochemistry and Biotet!hnology Royallnstitute ofTedmology 5-100 44StDCkholm Sweden COLIN P.. HAP.WOOD o.-p;'!.rtm~ t

ofMicrobiology

The Medica! S¡;: hool University ofNewcastle Frarnlingron Place N~tle

YUSUf CHIST!

lnstitllte ofTedlnology and EngiDeering Massey Universily Private Bagl1 222 Palmerston Nortb NewZealand HIKE CLARK

Immunology nivision Dep3rtruentoFPathology UniversityofCambridge CAmbridge CB21Q,P.UK

upon Tyne NEZ4.HH.UK

II.AJNI HATTI-KAUl

Deparonenr of Biotechno logy Centre for Chemistry md Chemic3.1

Engineerlng LUlld Universlty PO Box 124 5-2.21 00 Lund

Sweden

J. J. HEIJNE N Delft: University ofTechnoLogy

Departme.nt ofSiochemical Engineering julianaLaall 67 Nl-2628 BC Delft TheNetherlands

xli

lIST OF CONlRIBUTORS

DAVID J . JEENES

MURRAY MOO-YO UNG

InstitUlc ofFood Resc.urll Norwich Research Park Colney. Norwich

NR47UA. UK

DepartmerttofChemical Engineering Unlversity ofWaterloo Waterloo. Ootario N2L 3Gl Canada

GEORG-B. ICRESSE

JENS NIELSEN

R& D Biotechnology

Institutc fur Biotecbnology Danish University ofTechnolog)'

Boehringer-Mannheim GmbH Werk Penzberg Nonnenwald 2 1>-82372 Penzberg Germany

DK 2800Lyngby

Denmark HENK] . NOORMAN

Gist-brocó1c\es NV

8J 0RN KRIHIANSEN

POBoxl

European Biotf.'Ch.nologist5 Gluppeveien 15 N-t6H. Fredrlkstad

2600 MA Delft lbe Netberlilllds

Norway CHRISTIAN P. ICUBICEK

Depanment ofMicrobial Biochemistry lnstitute ofBiochem.ical Technology Get reidemarkt9/l72 \Tienna a-1060 Austria

W. PFEfFERLE

Biotedmologie Deguna-Hüls AG PFllt2 0-33788 Halle

Germany COLlN R.ATlEDGE

Department ofBiological Sciences UruVl!nity ofHull

DAVID A. LOWE

HuU

Brlstol·Myen Co. Industrial Division

HU67RX, UK

PO Bax4755

Syracuse NY 13221, USA A . LÜBBERT

lnstiWt ffir Bioverfahreostechnik und Reaktionstechnik Martin Luther UniV{:rsital Halle Wirtenberg o.06099lialle{Saalc Gl!Tmany

H. SAHM

Insrirut für Biotechnologie Forscbung5zentrumJütich GmbH PO Bollt913 1).52-425 jülich GcrOlany R. SIMUTlS

Control Departrnent Kaunas Technical UniVl'fiity Kaunas lilhuania

DONALD A. MA CKENZ IE

Institute ofFood Research Norwich Research Park Colney. Norwich NR47UA,UK

JOHN E. SM ITH

Applle
BO MATTlASSON

Depanment ofBiotechnology CentIY (or Chemistry and Otcmical Engíneer:ing Lund Unirersity PO Boxl24 S-221 00 Lund Sweden

PHILlPPE VANDE V I VERE

OWS DokNoord4 B90DOGenl BeJgium

UST OF CONTRISUTORS

J. P. VAN OIJK EN Uepartment ofMicrobiOlogy and Enzymology De.lft Univers.ity ofTech nology Julian alaan 67A. NI. 2628 se Dclft The Nethedands

ANI l W IPAT

DeparonenrofMicrobiology The Medica! School Uruversiry of Newcastle Fra.mlingtolLPlace Newcastle upon 1'yne NE24HH, UK

WtLlYV ER STRAETE

Labora toyYofMicrobiaJEcology UnivemtyofGen t Gen t Belgium N. VR1E ZEN

CentnrorB. V. PO Box 251

NL 2300AG Leiden The Ne th erlan ds

JA MES P. WY NN

Departmem or BioJogical Sciellces UniversityofHull Hull HU67RX.UK

xiI

Preface Ir is sorne 14 years slnce the firstedition ofthis bookappeared. Mucil has happened ro biotcchnology in these illtCrvening years. Recombinant

DNA technology which wasjustbeginniog in rhe mid-1980s is now Olle ofthe major cornerstoncs ofmodern biotechnology. Deve.lopments in this area have radically a1tered our cancepes ofhealth-care with che arrival of numerous products that were untJünk.able 20 years ago. Such is me pace ofbiotcchnology tbat ¡l can be antidpated in the Da! 14 years that evt'n greater developmenrs will oecur thanks [Q such programmes as the Human Genome Project which will open up opportunities fortreatnlentof dIseases atthe individuallevel. AH such advances lhough relyon rhe 31'plication ofbasic koowledge and tbe appredation ofhow ro trans-Iate thatknowJedge into produces matcan be produced safely and as cheapJy as possible. The funda.w.emals ofbiotechnology remain. as always. pmduction ofgoods and scrvkestbatan" needed and can be provided with safety and reasonabte costo Biotechnology is notjust ahouLrecombinant DNA, of cloning and geneties: ir is equally aOOm producing more prosaic materials. Uke citl'ic aeid. beer. wine. bread. fermented foods such as cheese and yoghurts. antibiotics and the like. Ir i~ also ¡¡OOut providing de;;)n techo nolo&)' fol' a new miUennium; ofproviding means ofwaste disposal. of dealing: with rnvironmental probLems. It is, in short. one of me lWO major I.eChnologies ofthe tweOl)'-firstTcrttury mal will sustain gt'ówth and dcvelopmf.'llt in countries throughout the world IUL' several decades to come. Ir will continue to improve the standard ofal1 our Uves, from improved merucal treatments, through.its effects on foods and food supply and into rhe environment. No aspect of our lives will be unat: fected by biotechnology. This book has been writtcn to provide an ove.rview ofmany of me fundamental OlSpects lhal underpin a D biotechnology and to provide eXOlmples ofhow tbese principies are pUlintD operatioll: from the srarting substrateorfeedstock rnrough to thelinal producto Because biotech· nology is 1l0W such a huge. ttlulti·evcrything activity we have not been able ro include every single topi<.:. eVéL'y single product 01' process: for thm an encyclopedia would havc bccn neederl. lnstead we have attempted to provide a ma.instream acco\lnt of the curre.nt state of biotechnology that. we hope, wiLI provide the reader with insighl inspiratloD and instructioD inthe skills a nd arts ofthe subject, Sinte the ficst edition ofthis book. wesadlyhavc ro record me death ofour colleagueand friend,Johu Bu'Lock, whose perspicacity had led ro the firse erlition ofthis book be.ingwrittf.'ll_John. at the time ofhis death in 1996, was already beginning to plan this semncl edition and it has beena privilege for us tohave been able to continue in his footsteps tosee it through ¡mo print.John was an il1spiring figure in biorechnology fur mallyofus and ir is ro tbe memoryofa finescientist, dcdicatecl bioternnologist allrl a remarkabLe man (hat we dedicate this book to JOB.

Part I Fundamentals and principies

Chapter I

Public perception of biotechnology John E. Smith lntroducliOll PubLic awareness of genetic engineering Itfgulatory requiremenrs - safetyofgenetically engioeered foeds Labelliog - how f;rrshould it g01 Policy lIliIking Areas ofsígnlficant public concern Condusions Furthec reading

1.1

I

Introduction

Biotech.nology can be viewed as a grou p of useful, enabling technologies with wide and divene applications in industry, comroerce and the environment. Historic:aUy, biotechnology evolved as an artisanal skiU rather than a science, exemplified in tbe manufacture ofbeers. wines. cheeses Ctc. where the techniques ofrnanufacture were we1 1understood but the molecular mechanisms went unknown. in more recent times. with the advances in tbe understanding DE microbíology and biochemistry, all of these empirically derived processcs bave become better understood and as él result improved. The traditional biotechnology products h3ve now been added to with antibiotics. vaccines, monodonal antibodies and many others. me production ofwhich has been optimised by improved fermentation procedures and novel downstream processing. Tt is clearthat biotechnologyhas its roots in tbedistantpastand has large, highly profitable, modern industrial outlets of great value ro society. e.g. the fermentation. biopharmaceutical and food industries. Why then. has thcre been SUcll public awareness and mncem of this subjecl in reCt~n( years? The main reasons must be associated with rhe rapid advances in moLecular biology. in particular, recombinant ONA (rONA) technology. which is now giving biosdentists a remarkable undersranding and control over bioJogicaJ processes. By these techruques ir is increasing)y possible to dil:ectIy manipulate the heritable material of cells between different lypt'.s of organisms. creating new

"

SMITH

combinationsofcharacters and funcuons not previously aeruevable by traditional breeding methods. Itis moS[ probable that this rDNA teehnoLogyorgenetieengineeTIng will be the most revolutionary tedtnology in the firstpaTtortbe twe.Dtyfirst century. Genetic engineering will be increasingly viewed as a branch ofmodern menee which wiU bave profound impacts OD medicine, eontributing to tbe diagnosis and cure of hereditary defects and serious ruseases. the development ofnew biopharmaceutical drugs and vaccines tor human and animal use, tbe modification of microorganisms. plants and farmed animals for improved and tailored food production and to increased opportunities forenvironmental remediation and proteetion. In plant and animal breeding, the new teehnologies are much raster and have loweT costs than the traditional metbods ofselcctive breeding. Furthermore. tbe desired modifications caIl be achieved in fewer generatioos. llllprovements to plant yields for nutritional content eould result in significant inerease in om ability ro feed an ever·increasing world population (8 billion before the year 2025) at.a reduce
1.2 I Public awareness 01 genetic engineering However, genetic engineering is sw-prisingly being subjected to a massivc leve! of criticism frem a deeply suspicious publico While [he American public seem to have agreater acceptance ofme potentials of genetic engineering. in Emope the technology appears to amuse deep unease among many consumers. Consumers demonstrate coneern abour 'unknowo' health risks, possible deleterious effects on the envirooment and lhe ' ullllaturalness' oftransfening genes between uorelated spedes. Also for many people there is an inereasingconcern about me ever.growing influence of technology in their lives ando in sorne instances. an unjustified mistrustofscientists. While genetic engineering is aD irnmensely compLicated subject, not easily explained in lay terms, that does not mean that it must remain. in decision·making terms. only in tbe control of the scientist. indusrrialist or politician. A Royal Society report in 1985 on 'Public Understanding ofSdence·. finished with the following stateme.nt: 'Our moS[ direct and useful message must be to the scientists themselves -

PUSUC PERCEPTlON OF BIOTECHNOlOGY

learn to ~ommunicate with the publi ~. be willing to do so and consider ic your duty to do so!' Thel'e is no doubt tbat many ofthe public 01' consumers are interest.ed in the science of genetic engineering but are uoable ro understand me complexity of this subject. Furthermore. genetic engineering and its myriad ofimplic.ations musrnot be beyond debate. Public attitudes to genetic engineering will infiuence i15 evolu· tion and marketplact': applicatioDs.ltis importantfor public confidence for everyone to recognise (induding scientisu) thar a1l sd eoce is fallible - esperially complex biological sdences. All too often press aod TV repom on genetíc engineering present tbe dlscoverie!i as absolute ce..· tainties when this is rarely me case. Whatthen mustbedone toadvance public understandingofgenetic engineering in the cont.ext of biotechnology? What does the public oeed ro know aod how can (his be achieved to emure that rhe many undoubted benefits thar trus technology can bring to manlcind do not suffer the same fate as (he food irradiatíon debacle in the OK in rhe early 1990s? While garnma·irradiation offoods was demonstrated to be a safc and effldent method [O kill patbogenic bacteria, itwas not accepted by tbe lay publjc following the Chernobyl disaster, sincemostwere unable to differentiate: het'Neen me pTOCess of irradlatíon and radioactivity. Effcctive communication about tbe benefits and risks ofgenetic engineering will depend on understandjng the underJying concems of (he public together with any foreseeable technical risks. Over recentyeaI"S tbe..e bave beco many efforts made ro gauge the public awareness ofbiotecbnology by questionna.ires. Eurobarometers and Consensus Conferences. Early studies rughlighted public artitudes to!he application ofgenetic manipuladon to a wide range ofscenarlos (rabie 1.1). While medical applications were more generally a.cceprable others sucb as the manipulation of animal aod hmnan genomes were: highly unacceptable. Eurobarometer surveys revealed a broad spectrum of opinions t bat were influenced by na.t ionality, religion, knowledgeofthe subjec( aod how the technology will be applied (Box 1.1). A major contriburory factor is the plurality ofbeliefs and viewpoints thar are held explicitly or implicitly abau!. the moral and religious status of Natu ..e and what out relationshjp with it sbould be. Do we view Nature. in the. context of man's dependency on plants and animals, as perfect and complete derived by natural means ofreproduction and rherefore not to be taropered with by 'unnatura l' methods. or do we set': it as a source of raw material fu .. tbe benefit of mankind? For centuñes now man has beeo indirectly mampulating tbe genomes of plants and animals by guided matings primarily to enhance desired characteristics. In this way, food plants and animals bear little resemblance ro their predecessors. In essence, such cbanges have been driven by the needs a od demands of tbepublic or consumer, and have beeo readily accepted bythem. 1n the tradiriooal methods that have been used. tbe changes are made at tbe level of tbt': whole organism, selection is made for a desired phenotype and tbe genetic cbanges are often poorly characterised and QCcur together witb othe[: possibly undesired gent':tic changes. The oew

S

6

SMITH

Microbial production af bio-plastics Cell fusian ta improve crops Culing diseases such as cancer Extensron of shelf life oftomatoes Oeaning up ait slicks Oetoxifying Irtdu$trial waste Anti-b!ood-dotting enzymes produced by rats Medical research Making medicines Making crops to grow in the Third World Mastitis-resistant Ct::/INS by genetic modification of CO'HS Producing disease-resistant CIUps Chymosin production by micro-organisms Improving crop yields Using viruses to attack crop pests Improving milI< yields Cloning prize cattJe O1anging human physical appearance Producing hybrid animals Biological warfare

Comfortable

Neutral

Uncomfortable

91 81 71 71 65 65 65 59 57 54 52 46 43 39 23 22 72 45 4.5 1.9

6 10 17 11 20 10 14 13 26 25 16 29 30 31 26 30 18 9.5 12 2.7

3 10 9.5 19 13 13 11 15 13 19 31 23 17 29 49 47 72 84

82 95

PUBlIC PERCEPTION OF BIOTECHNOLOGY

m ethods. in contrasto enable genetic material (O be modified ilt the celo lular and m olecular level, are more precise and accurntc. and consequently produce beUer charactetistics and more predicrable results while still rctaining (be aims ofthe cJassical breeder. A great many sucb. cbanges can and will be done within species giving better and faster results tbanby traditional breedingmethods.

1.3

Regulatory requirements - safety of genetically engineered foods

Much debate is now taking place on rIle safety aod emital aspects of geneticalIy moditied organisms (GMOs) and their products destined for public consumption. Can such products with 'unnatural' gene changes lead to unforescen problems roc present and future gener.ations? The safetyofthehuman foorl su pply is ofcritical importance to most nations and all foods mould be fit forconsumption Le. not injurious to health or contaminated. When foods oc food ingredients are derived from GMOs tbey mustbe seen to be as safe as. orsafer tban, their tradi· rional counterparts. The concept oC subrtantial equivalence is widely applied in the detennination ofsafety by comparison with analogous CODVl!ntional roed products rogetherwitb intended use and exposure. \Vhen substantial equivalence can be shown then norrnaJly no further safety consideratiOIlS are necessary. \-Vhen substantial equivalence is DOl dearly established tbe points of differcnce murt be subjected lo fu.n:her scrutiny. When sucb novel produc{s aTe moving ioto the marl-..-etplace tbe con· sumer must be assul'ed oftheir qllality aod safety. Thus tbere must be tnxicological alld Jlutritiona l guidance in the cvolution ofnovel food s and ingredients to bighlight any potential risks whic:h can tben be dea lt: with appropriately. Safery assessmcnt of novel foods and food ingredients must satisf}r!he producer, the manufucturel'. Che legislator ana Che consumeroThe approach should b e in line witb accepted scientiJk considerations, the r:esuJts of the safet)' assessment muse be reprodu· cible a nd acceptable to the responsíble health authorities and tbc outcome mllstsarisfy una convince thc consuffier! A comprehensive regulatory framework is now in p lace within tbe EU with the aim to pr'otet:t human health and the environrnent from adverse activities involving GMOs. 'l'here a re two Directives providing horizontal controls i.e. (1) contained use and (2) delibera te release of GMOs. Thecontained use ofGMOs isregulated under theHealtb and Safety al Work Acr through the Genetically Modified Organisms (Contained Use) Regulations which a re administered by the Hea lth and Safety Executive (HSE) in the UK. The HSE receives advice from the Advisory Com.mittee 011 Genetic Modification. These Regulations, wbich implemen t Dh'eCtive 90/219/EEC. covet tbe use ofaU GMOs in contai llment and will incorporate GMOs used to produce food addirives oc processing aids. AH programmes must carry out detailed lisk assessmenrs witb

7

8

SM1TH

spedal emphasis 00 the organism thar is being modified aud the effect ofthe modificatioo. Anydeliberate release otGMOs into theenvironment is regulated io the UK by the Genetically Modified Organisms (Deliberate Release) Regulations, which are made under the Environmental Profection Aa and implement EC Directive 90{220IEC. Such regulations will cover the release into me environment ofGMOs foc experimental purposes (Le. field trials) and the marketing of GMOs. Current examples could inelude the growingofGM food crop planes orthe marketingofGM soya beans for food processing. AU ex:perimentall'elease trials must havegovernment apPfO\r.lI and the applicant must províde detailed assessment of the risk of harro ro human he.alth andjor the environrnent. AH applications and tbe risk assessmentsare scrotinised by the AdvisoryCommiU~ on Releases ioto the Environment which is largcly m .. de up otindependent experts-who tbeo advise tbe Ministers . The Ee Novel Foods Regulation (258/97) carne inro effect in May ]997 and reprcsents a mandatory EC-wide pre-market approval process forall novel foods. TIIe reguJatioll defines a nowl foad as one that has llot previously been coruumed ro a signific:mt degree within the EU. A partof their regulations will include foad containing or consistingofGMOs as defined in Directive 90/220and food produced byGMOs but notcontaining GMOs in the final producto In tbeUK the safetyof all novel foods including genetically modified foods is assessed by t be independent Advisory Committee on Novel Foods and Processes (ACNFP) wbkh has largely followed the approach developed by the WHO and OEDe in assessing the safety ofnovel food s. The ACNFP has encouraged openness in 311 ofits dcalings. pubUshing agendas. reports oi assessments and annual reports. a Newsle.tter and saoo a ComnlÍtree Website. By such means it llopes ro dispe1 any misgivings mat may be harboured by memben of the publk. The ultimare decisions are nor influenced by industrial pressurc and are based entirely on safety factors. Thereis undoubtedly going ro be a steady increase in the range ofGM foods coming to tbe market in the US and in Europe (rabIe 1.2). A comprehensive HU regulatory framework covering GMOs is now firrnly established and the specific legislarion now in force will ensure the safety ofCM foods. In aU ofthe foregoing. the risk assessments ofGMO products ere. have beeo made by experts and judged on the basis of safety to the consumer. However, ir must be recogni5ed thar subject experts define risk in a narrow technical way. whereas the publicor consumerwitboutsufficientknowledgegeneraUy displays a wider, more complex view ofrisk tbatincorporates vaJue-ladeo considerntions 5uch as unfamiliarity, cato astrophic patential and controllability. Furtbermore, the public, in general. will almost atways ove.restimate risks associated with technologkal hazards such as genetic engineering and underestimate risks associated with 'lifestyle' hazards such as driving cars. smoking. drinking, fatty foods etc. Ir is puz:.r:ling ro note that food-related technoJogies

PUBlIC PERCEPTION OF BIOTECHNOLOGY

C"'p

Trait

Company

Tomato Soya beans Tomato Potato Tomato

Modirted ripening Glyphosate tolerance Modified ripening Insect resistance Modifled ripening

Zeneca Plant Science Monsanto Ca, Monsanto·Co. Monsanto Ca. DNA Plant Technology

Cotton Tomato Squash Corton Oilseed rape

Bromoxynil tolerance Delayed ripening Vir1Js resistance Insect resistance Glyphosate tolerance

Calgene Calgene

Asgrow Seed Co.

Cotton Maize Oilseed rape Maize Oilseed rape

Glyphosphate tolerance Insect I"esistance High laurate - oil Glufosinate tolerance Glufosinate tolerance

Monsanto Co. Ciba Geigy Corp. Calgene Agrfvo Ine. Agrf vo Ine.

Maize Oílseed rape Maize Petato Maize

Male sterile Male sterileJfertility restorer Insed re5istance Insed resistance Insect resistance

Plant Genetic Systems Plarrt Genetic Systems Northrup King Monsanto Ca. Monsanto Ca.

Maize Cotton Maize Tomato Soy,

Insed resistance. glyphosate tolerance Sulpnonyiurea tolerance Glufosinate tolerance Modified ripening High aleic acid content of oH

Monsanto Ca. Du Pont Dekalb Genetic; Corp. Agritope Ine. Du Pont

Maize Maize

Herbicide tolerance Herbicidetolerance and ¡nsed resistance Herbicide tolerance and ¡nsed resistance Male sterile

Monsanta Ca. Monsanto Ca. Calgene BejoZaden

Cenon Chicory

rend to be perceived as high i.n risk relative to benefitwhen compare
Monsarlto Co. Monsanto Co.

9

10

SMfTH

1.4 I Labelling - how far should it gol Perbaps the most maten tiou! issue related to foods derived fro m genetic engineering is to what extent should chey be labelled. nlC' purpose oflabellinga food product is lO providesufficientinfonnation and advice. accurately and clearly, to allow consumers to select praducrs according ro their needs, to store and prepare them carreed)' and la consume tbeOl wiUl safety. With respeet to the principie oflabclling. infonnation should be accurate. t ruthful. suffidentlydctaiJed. not m isleading and above all understandable. Labelling ofa productwill only be relevane iftbe con sumer is ablt' to understand (he information printed 011 the labels. The Food a nd Drug Administration of tbe USA considers thar labelling should n othe based on the way a particular pl'oduct is obtained. Thisis, orshould be, a part ofno rmal approval for agricultural practice orindustrial processes. and j f approved. then JabeUing should be unnecessary. whkh i5 the COmIDon pTactice for rnost food products. lt c.lDbe argued th
PU8lIC PERCEPTION O F BIOTECHNOLOGY

necessicated by the nero to produce food fOT an ever increasing world population . Let U5 nor delude ourselves. Without the add ition of this technology te tbearmouryofthe plant breedermere wlll be serious and indeed calamitous food shorrages especially in the developing world. Ir aH aspects of genetic modificaDon must be recognised and recorded ir can only lead ro unacceptably complex labelting criteria. Consumer rights. now recognised by all member s{ares in the EU. involve a righr to information and irs corollary. a duty [o infonn. As a consequence.labeIling should be meaningfu1 bU( appropriare.

1.5 I Policy making Policy making on generic engineering throughout lbe industrial wodd ísstrongIy influeDced by thevaried interests ofg!M!mments. indusoy. academics and environmental groups. After almosr two decades of discussions, the dominant issue still is wbether government regulations should depe.nd on the characteristics ofthe products modified by rDNA lechnique or on tbe tet::hnique of rDNA. For rhose who support tbe product-based rcgulations. the new tedmiques couId be considered as an extension and refinement ofmoreconvenUonal or tradicional breroing appro ach~ . As a result they would expec[ [he new products to be treated in a way similar to products created by conventional technoJogy. In contrast, t here are those who profess tbar tbe regulation of me genetic modiflcation process could be viewed as a new biotechnology and as an undeveloped scienee abour which there is little knowledge and working expericnce. In this way tbe process is sufHciendy different and distinct from conventional techniques and should demand unique regu latory rules. As with other techniques. the genetic engineering debatecould also preve ro bea critica1 testingground for effoI1s te insert into geverrunenmI policies socio-economic and sociocultu1'a.l measures - the so-called 'fourth diterion '.!he advocates ofthis approach consider that measures ofefficacy, quality and safety are. alone, iruufficient ro judge the polentiall'isk associated witb sucb new techniques and tbeir prodUC1S and to these rheywould addsocial and moral considerations. These new approach~ are h aving a significan! impact on the pace of agricultural and environmental applieatiolls of genetic engineering. In contrast, biomedical applications have progressed relatively rapidly. MilIions of people throughout the world have accepted and benefited from diagnostics. Drugs provided by the new biotechnology companies ioduae the GM produc[s erythropoietin. for kidneydialysis patieOt5. aod lnsulin.for diabetics. whilediagnostics developed by geneticengineering in particular keep dangerous pathogens. such as l:UV and hepatitis viruses . out ofthe blood supplies. Those activists who oppose gcnetically engineered products have studiouslykept clear ofthese successful areas ofapplication.ln agriculture there has been conceru..'() opposition by the activists against CM bovine somatotropin (BST) bu[ almost total silence 00 che enginee.red chymosin enl.yOle used to dot milk in cheese

1I

12

SMJTH

production, which now dairns abaut 40-15% ofthe US market. MiUions of calves are now no Jonger required for trus process.lndeed. tbe success ofCM ehymosin has been applauded by animalrights activisu. How far the fourth critedon will infIuence saenee policy a nd, in particuln. genedc engineering in the malcing or agricultural and environmental poliaes are now at important cross·roads. While there is ~at need ro mercase the seienee Iiteracy of the pubUc in general. a wellinformed public must stiU rely heavily on independent experts foc the evaluadon ofsophisticared technieal i¡¡sues.

1.6 I Areas of signiflcant public concern 1.6.1 Antibiotic resistance marker genes Antibiotie 'marker' genes are used to identify and select cells wbkh have been suecessfully modified following a genetie modiflcation process. By the use of such genes, cclls which have been successfully modifled ean growin the presenceofthe particular antibiatic. The rnost commonly used antibiarle resistance m.a.rker genes in GM plants conter resiSl:ance to kanamycin or hygromycin while for GM bacteria the ampi· cillin resistanee market gene is more ofren used. Could such antibiotic resistanee genes be transferred from a consumed CM plant or micro· organism into the human gut microflora and so ¡ncrease antibiotic resistance in rhe human population? Bacterial resistance to commonly used antibiotics can nowbe found throughout the world and ir ismost probable thar this incidence is the result of rhe transfer of resistance genes bet:ween bacteria followed by che selective pressures imposed by the use ofantibiotics. To date it has not been possibJe to demonstrat.e the transfer of antibiotic resistance from a marker gene in a consumed GM plant ro micro-organisms nonn ally present in the gut ofhumans and other animals. HOwe~T, a potential does exist and should llOt be ¡gnored. TIleAdvisoryCornminl'Con Novel Foodsand Processes (ACNFP) in the UK has recomm.endec! [hat antibíotic resistance marlrers should be eliminatec! from all GM foods and micro-organisms that will be con· sumed Uve. Researchers developing GMOs for food should develop and use alternatives to antibiotic resistance markers andjor employ metbods to jettison tbose used in developing the early transgenic celIs. By foUowing cbis adviee. publk canceen over this facet of genetic engineering should be eliminated. The recent and heated conuoversy over the Ciba.ccigy transgt'nic com revolved around the presence ofa non· functional IHactamase (an enzyme that can inaetivate penicillin) sequence. While the risk oftransfer of ,8-laetamase DNA to gut microflon..-wouJd indeed be vanishingly small, it has been a massive publicity winner for those opposed ro GMOs. How easily it couId have been avoided wjrh a Hule foresight by the company.

1.6.2 Transfer of allergies Food allergies arise when the irnrnune systemresponds ro spedHc al1ergens which are usually g1yeoproteins in the food. 'l'his is now a major

PUBLlC PERCEPTION OF SlOTECHNOLOGY

ooncem espcdaliywith respect ro the peanuland othe. nuts and severe anaphylactic reactions are not uncommon. Consequently, labelling of fuod products with re:spect to tbe presence of peanut, is now widcly practised. Thus. itbecomes essential with GM foods to ensu.re that transo fer of allergens does llot occur from donor species to recipient spedes. Oearly, this is a complex process and one which 3ll producers ofGMOs are nowfu1Jy alerted ro and due comideration is beinggiven.Therehave been no recorded examples of new allergies by the process of recombinanl DNA technology. Many databases mat can identify proteins that could be problematic if inserted inoo food materials a re now increas· ingIy available.

1.6.3 Pollen transfer from GM plantS Ihe possibility of gene transfer fram transgenic crop plants lO compatible wild relatives has been given seriaus examination. Could pest or berbicide resistance incorporared inm transgenic plants be transferred meo otber dosely related plants and incl'ease their ' weediness'7 Under normal conditions, gene tr.msfer by way of pollen even berween close reJ.atives is exceprionally rare and there is litrle hard evidence that this will be different with transgenic plants. HOWeYer, released cransgenic plants will be routinely monitored to continue me already extensive validation that has Qccurred. The possible presence of toxk recombi· nane proleins in honey (which may cantain about 2% pallen) has been sbown to havc no relevance.

1.6.4 Social. moral and ethicaJ issues associated with GMOs lnitial concerns about- GMOs were. perceived basically on safety issues but more rccently social, moral and ethical issues have become part of me deosion'maJdng process. The control ofGM crop plants and their seeds by multinational agrodlemical companies and tbeir nced to rerover the high mvesonent costscould imply thatonly high technologyfarmers will be able ro meet tbe full costs. CM seeds are normaUy made rterile to prevent further propagation by farmen. However, tbis is normal practice by se.ed como panies where (he seeds used commercially are F¡ hybrids which do nat bree
1)

14

SMITH

engineered bovlne growth hormone (BST) may wcll cause severe difficulty for small farmers in USAand the EU. While tbis developmenrbas gane tbrough quite successt'ully in the USA there is a moratorium currently in operation in the EU. It must be expecred tha[ many aspects of this new genetic tec.hnology. partirularly when applied to agriculture and food production, could welllead to decreases ín e.mploymentwith an ensuing increa.st> in poverty in deveJoping economies. Different value judgements come into operation to re:concile theadvantages to society against the disadvantage5. The deve10ped nations muS[ endeavour to assist me developing oations botb technically a nd financially [O be par[ of this agriculture revolutioo. To what extenttbis is nowhappening is variable and questionable.lt is sad thatpublic awareness ofruch issues is seldom voiced by the western activists against genetic engineering. Ir is in the aTea of animal transgenics tbar publie awareness and roncero is most regularly expressed. Transgenic animals are those incorpol'
PUBLlC PERCEPTION OF BIOTECHNOLOGY

;¡ge of hurna n organs for essential transplants. Th e maill CODcerns have been possi blc trarnfer ofpigviruses to humans in the Iight o(the BSE sean:, Also the qut$tion ofbreeding pígs for this purpose as opposed to rhe accepted current practice for eating purposes does genera te ethical concenlS for some.l (5) Biomarkers for detecting environmentalpollution using trans· genic nematodes would appearto be a wortbwhile activity. (6) Tl-ansgenic animals can be used as models for human genetic diseases with the ultimare aim to develop new drugs or gene-the.rapy O"eatments. While many people. see Che aims oftbese studjes to be ofsignlficant value to mankind otben express genuine coocerns for animal we1fare, and wheCher wc havc the right to indu1ge in modifying ao animal genome ro human advantage. Central (O pubHc cOt\cem is a strongfecling of 'unnaturalness' in aansferringhumangenes inta animals with the newtransgenic animal containing copies of [he original human gene. Ir is difficult for the avuage layperson to comprehend thatwhile the transgene has hUUlan origin and structure ir.. is no[ its immedíate sourcc. In the pTOCess of genetic manipulation, gcncs canDot be directly transporred froro the donorto the redpientbllt rnllst progress through a complicated series of in \litrD c1onings. lo Chis manner a series of amplification steps are GlJ"ried out in which the original gene is copied manytimes during dIe wbole process such fhat the original genetic material is dilutcd 10". !bus the original DNA is not directly uscd. but rather. simila r DNA is synthesised ;utificiaUy. Secause the transgenic organ does not contain me actual human gene bu! on1y ao artificial created copy afthe gene, molecuJarblologists widely accept t he status ofChe transgene to be tbat i!IO'>y of thenew org:m.ism. It is tbe ir view that genes fuLfi] thcir biologial role only by the ir activitywithin the environment ofthe ccll and an organismo lbeissueofanimal rights is highlyeontentious - do they have intrinsic rights or nor? Sorne wou ld assign equal moral worth to sentient ;;u:rlmals as to humans. but wbere do you draw che line? A sub-committee of the Advisory Committec on Novel Foods and ?rocesses (ACNFP) considered sorne ofthe ma in ethical conceras arising from Che food use ofcertain transgenic organs and prepared the followfugexclus ions: (1) Transfer of genes from animals whose fLesh is forbidden for use Z5 food by certain religiou s groups to animals which they normally mnsume (e.g. pig genes into sheep)v.rould offend mostJews and l!cslims. (2) While transfer ofhuman genes to foad animals (e.g. transfer of die buman gene fur Factor IX, a protein involyed in blood cJotting, into sbeep) is acceptable for pharmaceuDcal and medical purposeS. the Zilimals should not, U poll slaughter, e nte r the food main. (31 Transfer of animal genes into food pla nts (e.g. for vaccine proé!ction) is acceptable forphannaceutical and medkal purposes but pbnr remains should n ot tben enter the animal a nd hwnan foad

15

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::< ¡,¡

~

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D

O ~

lO

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.'

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16

SMITH

ehain as theywould be anathema tovegetacians and especially vegans.

(4) Use oforganisms containing human genes as animal feed (e.g. micro-organismsmodifled ro produce pharmaceutical human proteins ruch as insulin). Clase eonsultation wjth a wide range ofre1igious fuims strongly suggested lhat there were no overwheJming objections to necessitate an absolute ban on tbe use of food prodUClS containing copy genes oC human origino(Ibis was particularly notlceable when the conce pt of the copy gene was understood.)The report, however. strongly advocared tha( the.insertion ofethicallysensitiwgenes into food organis ms bediscouraged espedally when alternative approaches were available. lf transgenic organisms conraining copy genes that are unacceptable to specific groups of che papulatioo rubject (O religious dierary restrictions were lO be presenr in sorne foods, ir should be rompulsory (hat rum foods were c1early labelled .

1.7 I Conclusions The safety and impact ofgenetically modified organisms continues ro be addressed by scientific research. Baste research into the natwe of genes, how Chey work and how they can be ttansferred between organisms has served to underpin the developm ent of rhe rechnology of genetic modificabon, [n tbis way. basic. informarion about the behaviOUT of genes and of GMOs will be builr up and used to address th.e concr:rns about the overall safety of GMOs and thciT impact on the environment.

1.8 I Further reading Frewer, L1. & ShqJherd, R. (1995). Ethical concems and risk perceptions associated wi t h different appllcationJ orgenetic engineering: intcTrelationship with the. pe~ved need for regulalion orlhe technology.Agric. Hu m. V
The Royal Socicty Statement (1998~ Genetitally MQdlfird Plan ts.for Food U~. Pp. 1-16.

I

~

,

I

¡.

Chapter2

Biochemistry and physiology of growth and metabolism Cotin Ratledge lnnoduction Metabolism Catabolic pathways Cluconeogenesis Energy .production in aerobic micrlXlrganisms Anaerobic merabolism Biosynthesis

Control ofmetabolic processes Efficiency ofmicrobial growth Further rcading

2.1

I Introduction

lhe FirstLaw ofBiology (iflhere was one) could be: The purpoSt ofa microOrganism fs to make another micro-orgonísm, In sorne cases biotechnologists. who seek to exploit che ollCl"Oorganismo may wish lhis to happen as frequently and as quick.ly as possible; in other words tbey wish to have as many micro-organism s ovailable at che end afthe process as possible.ln othercases. where (he product is not tbe organjsm i tse Lf. the biotechnologisr must manipu· latcit in such a wayrhar the primary goal ofrhe microbe is diverted.A5 lhe micro-organism [hen smves to OVf'rcome these restraints on in reproductive capacity. ir produces the product which Che biotechnologist. desires. The growth ofthe organism and its various products are tberefore intimately linked byvirtue ofits metabolismo In writing this chapter. 1have notattempted ro explain the strucrure ofthe maia microbial cells: t he bacteria, theyeasts, the fungi and t he microalgae. These are avai labJe in most biology textbooks and tlJese mould be consu1ted if there a re uncertainties a bout cell structures. However, biology textOOüks rarely explain the chemistry that goes on in the living cell (Le. their biochemistry) in simple cenos but. as rhe biochemistry oftbe cell is fundamental to the exploitation afthe organism, ir is impartanr ro be acquainted with rhe basic systems thar microbial cells use ro achieve th~ ir multiplication.

18

RATLEDGE

,,)

lb) CARBON NEWCELLS

SUBSTRATE

CARBON SUBSTRATE

NEWCEllS

Anabo lism Anaboli sm

Catabc lism

CSlabolism

Reduced end products

~

C
Carbcn

t

Reducing powar

Reducing power

~TH'O

'ENERGY

ENERGY

Pro eMSeS of caubolism (dez!'3datioo) and anabclism (blo~yntt.e:sis) linkcd 10 . nergy proóuctlon 1M provl,joo 01 reckKln¡ power. (a) AMeroblc metl.boIbm; o"phof =oxldativtl phOfPhorylation (see SKtlon 2.5); (b) Anlltl"Obtc metabol>sm.

The bjochernístry of the eell is therefore described as an aecount of the chemical changes which oecur within él eell as itgrows and multiplies to beoome two eclls. The physiology of [he eell. however. goes beyond the biochemistr}' of the tell as this term extends che simple aecount ofthe f10w ofcarbon. and the changes which cernrra other elemcnts. by describing how (hese processes relate te the whole growth process itself. The biochcmical changes therefore are to be seen ocrurring in the three
2.2

I Metabolism

2.2.1 Sorne definltions Metabolism is a matrh!: of two c10sely iDte rünked but divergent activo ities (see Fig. 2.1). Anabolic pr0ce5ses are c.oncerned with the buildingupofceU m aterials. not only the major cell ronstituents (protcin. nudeic acids. Iipids. carbohydr.ates. etc.) but also the intermediate prerursors ofthese materials- amino acids. purine :md pyrimidioes. fatty adds. various sugars and sugar phosphates. Anabolism concems processes which are endothermk overall (they 'require energy'). They 31so invariably r equire a source of rcducing power which rnust come by t he degradation of the substrate (or feedstock). The compensating exothermicity is provided by various catabolic ('energy·yielding·) processes. The degradation of carbohydrates, s ueh as

GROWTH ANO METABOU$M

sucrose or gl ucose. uJtimarely to give COl and water, is the principal eXIr tbermic process whereby 'energy generation' is accomplisbed. During tbis process reducing power nceded ror the subsequent anabolic pn> cesses is also generated. The same cODsideratiofls. however, apply to aU substances that a.re use
me eell. We can also distinguish between organisms which ca.rry out their metabolism aerobkally. using O] from the airo and those tbatare able to dothis anaerobically. thatis. witbourO r Theoverall reaction ofreduced carbon compounds witb 2 , to givewater and COl' is a highlyexothermic process: an aerobic organism can therefore balance a relatively smaller useofits substrates for catabolism ro sustain a gjven leve.! ofanaboLismo that ¡s, ofgrowth (see Fig. 2.1 a). Substrate tlOmsformatio ns for anaerobic organisms are essentiaUy disproport:i.onations, with a relatively low 'energy yield'. so tbat a larger proportion ofthe substrate has to be used eatabolicaJly to sustain a given level ofanabolism (Fig. 2.1b). The difference can be illustrated with an organism 5uch as yeast. Saccharomyces cereviSiae , wbicb is a facultative anaerobe - that is, ir can exist either aerobicaUy or anaerobically. Transforming glucase at the same rate, aerobic yeast gives COl' watfi and a relatively high yield of new yeast. whercas the yeaS! grown anaerobically has lawer yield of energy and reducing powet. Consequently. fewercells caDbe made tban under aerobic conditions. A1so it is nat possible (or the eeUs. in the abseneeof0l' (O ox.idise aIl the reducing power that is generated during catabolism oConsequentIy, sutplus earbon intermediares (in the case of . yeast it is pyruvic acid) are redueed in arder to recyde the reductants '(see Fig. 2.2) and, in thecast oryeast. cthanol is the product. Overall. this process can be described by the simple reaction:

°

X+ NAOH -+Xl4 + NAD't'

Reduced carbon metabolite o r H20

I C.. '~n liubstrntc ICmHnOpNQ) Nitroge" source

INH3)

K'!PO:-IMgl'I

SOl·tetc. 020r surplus carbon metabolites

HU

Thermodyl'wnk IMbnce

19

20

RATLEDGE

wb~re X ¡S3

metaboliteand NADH is tbereductant, and NAD·· is its oxid¡sed form (see Fig. 2.3a, bJ. NAO stands for nicotillamide ade.nine di· nucleotide; NADH is therefore reduced NAD. There is abo the phosphorylated form of NAD+, NAD phos:phate designated as NADP+. TltiJ; can aIso be reduced lO NADPH and it can also functiou as a reduc· tant but u sually in anabolic reactions of me cell, wbereas NADH is usually involved in the degradative reactLoos. All four forros (NAD+, NAOP". NADH and NAOPH) occur in both aerobic as well as anaerobic ceUs: in tbe former (ells re-oxidation of NADH can ocror with 0 2' but mis cannor take place in the a naerobe, hence the need for the altemative re-oxidation scrategy (see Section 2.6). A ce:U that grows obviouslyuses carbon but manyother elemen ts are needed to make up the final composition ofthe cell. These will inelude nitrogen, oxygen - wbich may come from the air if the organism is growing aerobically (otberwise the 0 2 musí come from a rearrangement oflhe molecules in which the organism is growing, or e.ven water ilSclf) - togetherwitb other elemellls sucb as r. Mg H , S {as SOr). Pías PO! - ) and an array of minor ions S"uch as Fe H , Zn H , Mn1-t , etc. The: dynamics ofthe sysrem are set out in Fig. 2.2.

2.2.2 Catabolism and energy

(a) NAD '· /NADP+ (olddlsed): (b) NADH / NADPH (rrduc:e d). ln NAD ~ ~nd NADH, R = H: In NADf>'" and NAOPH,

R=POI-·

The necessary linkage between catabolism and anabolism depends upon making t he catabolic processes 'drive' the syn.t hesis of reactive reagents, few in numbC!r, whicb in tum are used to 'drive' the full range of anabolic reactions. These key intermediates, of which the most important is adenosine triphospbare, ATP (Fig. 2.4), have what biologists t/!'TIU a 'high-energy bond': inATP itis the anhydride linkage in tbe pyrophospbate residue. Directly or indirectly the potential energy released by splitting chis bond is used fur the bond.forming steps in ana.bolic 5yn· t heses. Molecules such as ATP then provide me 'energycurrency' ofme cell . Wben ATP is used in a biosynthetic reaction ¡tgenerates ADP{adenosine diphosphare) or occasionally AMP (adenosine monophosphate) as the hydro1ysis product: A+B +ATP~AB +AD P + Pt

or A+B+ATP -+ AB+AMP+PP¡

(where A and B are botll carbon metabolites of the ceH and Pi'" morganic pnosphate, and pPJ "" inorganic P}'rophosphate). ADP, whlch still possesses a 'hjgh-energy bond', can aIso be used to produce ATP by the adenylate kinase reaction: ADP + ADP-+ ATP + AMP Phosphorylation reactions. which are very (ommon in living ceUs, usually occur through the mediation of ATP:

o 11

- e -OH + Al'P -+-C - O-P - OH + ADP

I

I

I OH

GROWTH ANO METABOUSM

The phosphorylated product is usual1y more reactive (in arre of sevual ways) than the original compourrd.

2.3 I Catabolic pathways 2.3.1 General considerations of glucose degradation

,l

, ,~

f ¡ t

The purpose of breaking down a substr.lte is to provide the microOIganiSDlS with: • building units fur the synthesis of new cells: • energy. prinopaUy in the forro ofATE, by which to syntbesise new bon4,s,and newcompounds: • reducingpower, which is main1y as reduced NAD (i.e. NADH) or reduce
donatlng encr¡)'. the ")'-bond Is hydrolyHd alld the :ava!lab~ enereY 1, used tO maleo a new bond In I molceule. Ade005¡ne dlphosphate (AOp) b wlthout the Iuc photpho group Ind adenoslne iUOIIOphospNte Is witl"lout the I&st fWO pho$pho

21

21

RATlEDGE

-.

~-."- --'-----------------~-

DNA)

RNA -

ATP ) ____

Nucleotides.

daoxynudootides,

¡

con .... .,/"'otc.

""''''''0''.''- Polysac:charide$ ¿"

---p",to,,~'P'­

histldine

Phenylalanlno. Iyrosine,

lipids

~-- S";nÓ- --'G"do,, , :-Cysteine. mothionine

NAD. etc. -tryptophan, p-hydroxyoonroate

quinooes

.

Purines,

pyrimldinE

Porphyrim tnc.

p"amlnobenzoale,

Aespiratory .........

slora~

Stori\ge lipida

Glyeerol - ~Membrane

ADP

Folle acld _

..............

/

= =

= =::AJanlne

Va line, ¡eucine

= =: : Fatty &Cids. lipids, PHB, polyketldes

Mey¡¡lonato, !taroid!, carotenoids

Glut8mate, glutamine- Argini prolin,

l

Folie acid

Anabalic P'othway, (synmesls) and lhe centnl atJ.bo~c ~th-rs. Only me main DIo5)'flrhu15 rouUt~, and tlIelr maln COOlllCDOf\t with o.aboVc

Pilthways:are $hown, aY 11'1 h\gJIly slmplifled ~ions. ConnKtJgns through 'energy' (ATP) ilnd 'redox' (NAO·, NADP ') metaboVsm a!"ld throu¡h die metabo~.\Ill cA nitrogen, etc., are al omltted. (PHB

=pcty.,8-r.yd,.~j)(ybutyra~; p= phos.pooVOUp). The nIM principal pr«ursors ~ in the lihade
and tetrose (Col) phosphates. This is the pentase pbosphate patb sometirnes referred te as a 'shunf or as the hexost' monophosp pathway (see fig. 2.7). Thepurpose ofthis pathway is rwo-fold: to PI"( C~ and C4 unirs for biosynthesis (see Hg. 2.5) a nd .1.lso to provide NA furbiosyntheris. Although the EMP pathway and the pentose phosphare (PP) patl botb use glucose 6-phosphate. the extent to which each mute ope. depends Lugelyon what the cell is doing. During tlle most active ! ofceD growth. botb patbways operate in the approximare ratio 2: tbe EMP pathway over tbe PP pathway. Howev&, as growth sJows d the biosynthetic capacity of the cel1 also slQV.Is down and less N,Il and C5 and C4 sugarphospbat"es are needed so tbat tbe ratio betwee pathways nowmoves ro 10:1 or even to 20:1. Ir is therefore apparent mat metabolic pathways are control systems capable ofconsiderable refinement to meet tbe eh anging 1 ofthe eell. '!ltis is discussed lacu (see Section 2.8). Although lhe EMP and PP pathways are found in mon n organisms, a few bacteria have an alternative pathway to the fe patbway. This is the Enrner-Doudoroff pathway (see Fig. 2.8) ... occurs in pseudomonads and re1ated bacteria. Tbe pentose phos· pathway though soll operares in these bacteria as the En Doudoroffpathway does notgenerate C, and C4 pbosphat.es.

GROWTH ANO METABOUSM

2.3.2 The tricarboxylic acid cycle lbe. degradation of glucose. by whatever route or routes. invariably leads to the formation ofpyruvic acid: CH3 .CO.COOH. The fate ofpyruvate is diffe:rent in aerobic organisms aud anaerobic ones. ln aerobic sys~ms. pyruvate is decarboxylated (i.e. loses C0 2J and i5 simultaneously activated in r.he chemical sense. to acetylcocnzymeA (abbreviated as acetyl-CoA) in a complex reaction also involving NAD+: pyruvate+ CoA + NAD+ ---t acetyl-CoA +C0 2 + NADH This reaction is cataIysed bYPYf1lvate ddtydrogtttast'. (lbe fate ofpyruvate

in anaerobic cells is described latero) Acel:yl-CoA. by virrue ofit being a thioester. is highIy reactive. It is capable oC generating a large number of intermediates but iu principal though Dot sale tate is to be progressively oxidised through a cyelic series ofrcactions knOWD as the citrJc acid cyde. TIris is also known as the tricarboxyUc acid cyde or me Krebs cycle after its discoverer. The reacrions ofthe amc acid cyde are shown in Fig. 2.9. This cyele fulfils two essentiaJ functions: • it provides key intennediates fur biosynthetic reactions (sec Fig. 2.5), principal ofwhich are 2-oxoglutarate (to make glutamate and r.henceglutamine, arginine and Qroline), succinate (to make porphyrins) and QXaloacetl'l.te (to make aspartate and the aspartate famUy of amino acids - see Chapter 13): • to produce energy from me complete oxidationofaceryl-CoA to COz and Hp. (1bis process is desmbed in detall in Scction 2.5.) However tbe citric acid cycle canDOt fulfil either funcD oll exdusively: ifintermediates are removed fur biosynthesis. then sorne energy production must be sacrificed: if all the acelyl-COA is oxidised to CO 2 and RzO there wUl be no intermediates left for biosynr.hesis. Consequently. the cycle runs as a balance between the two objectives. Pyruvate, commg from glucose, provides the input and the cyele p~ vides [he output in fue way of energy and biosynthetic precursors (see Fig. 2.10). In meeting its twin objectives. the cyc1e cannot entirely replenish the ¡uitial oxaloacetate [hat is needed as a priming reactant tO makecitrate as sorne ofthe inrermediates must inevitably be used for biosynthetic purposes.. (lftheywerenotsoused. there would be no poillt in the cydejust producingenergy as this could nor then be used in any

Glucose

ATP

(i)

f= AOP

Glucosa &·phosph8te

®! Fructo68 6·phosphate

®

ATP f= AOP

Fructose 1.6-bisphosphate

~

1

1

®

OHA-P...- G3-P

P'yNAD ®f--NADH l,3-dlphospho-glycerate

F=

ADP

(!)

' - -ATP

3-pllosphog Ivcerate

® .

The

Embden--MlI)'erhof-Pa~ p;ithw~y

of IIy~ysb. O~nll:

2·phosphoglycerate

Glueose + 2 NAD + + 2 ADP + 2P, ..... 2 p)'T1JVlte + 2 NADH + lATP TIlo reacrions In! catalysed by:

(1) hexokinase, (2) gIucosM~bate iromerze, (3) pho$phofructokln:ue. (-4) aldolale. (5) ulose phosphate l5omerue, (6) gtrceraldehyde--J-phOspllate dehyd~ •. (7) 3-phc~glycerate kio
®! PhO'PhO'"f=o IPY'O:::: @ Pyruvate

ATP

2

24

RATlfOGE

Al?

NADP~

AOP

GIllCOse-"""-)~,

01'00"

NAOPH

5-P U6-P

NAOP' NAOPH

.,",on.,,~ RIb"o," '-P

(1)

EPi:'~'1 \ om."w Ribose 5-P

Xylulose 5-P

T'' ' ' '/{

Glycor¡"ldehyde 3-P

Sedoheptulose 7-P

T"n"/dO/'~ Fructose6-P

'--------c,

e,

Erythrose 4-P

TrlJnsketofaso

fructose 6-P

Glycereldehyde 3-P

Ttw. pentoH pho~te cyde (toexose monophosphue slwnt). Th~ number.d emymes are; (1) &lucose.fo-pho5phate dehyG'osenas., (lJ pl'losphacluconaa dehyclrogenue.lr'I$ft; summar)' ~.nx stoldliometry wnen fruCWs. 6-pho~le b recyded to IkicO$e 6-phosphale by:llll,~; glyeeraldehyde l-pnosphale an Ño be reqclcd by revur'Se glycolysis (Fl¡. 16). WIth MI r~fin(the pathway function5 as a llenM'UOf"of NAOPH. bUt!he ,~nsaJdolaH and tnnIb!olue roctions abo permlt sugat Illten:onvel'$im'15 whlch are us~ in o!her ways.

Ne' reodkwl: glucose+ATP+ lo NAQP+ .... gtycenldehyde 3-P+ADP+ 6 NAOPH

(Afrt ",maYal of~ and C. ,usarE lar bIosyntheril wllI dimJnhh me nqdiflg?fOCHJ and thus!he yield oif NAOPH wiJI 6ecntase..) sensible manner as tbere can be no biosynthesis witbout precursors.) It is therefore essenti ... 1for t here to be a seoond pathway by which oxaloacetate can be formed a nd this arises principally by fue carboxylation of pyruvate: pyruvate+ COl + ATP -+ oxaloacetate + ADP+ PI 'Ibis reaction is carne
J

25

GROWTH AND HETABOUSM

fono citrate). tbe concentration of acetyI-CoA will faH; pyruvate carboxylase will tben slaw down but. as pyruvate dehydrogenase still operares as befure. more acetyl-CoA will be produeed. In this way nol only will time add synthesis always eontinue, but [he two reaetions leading [o me precursors of cirrate will always be balaneed (see Fig. 2.11).lbis type oC reamon catalysed by pyruvate carboxylase is rekrred to as an aBapletoticreaction. meaning 'replenishing'_

2.3.3 The glyoxylace by-pass lor growth on C, compounds Ifan organism grows on a c:" compound, OE on a farty acid. hydrocarbon m any substrate fuat is degraded primarily into e 2 units (5ee Section 23-4), the tricarboxylic acid cycle i5 insufficientto accountfor It5 metaboIism. Acetyl-CoA can be generated directly from aeetate, ifthis is being as:ed as carbon source, or from a el compound more reduced than ~te, Le. acetaldehyde or ethanol: NAO'"

NADH

NAO'"

NADH ATP

ADP + PI

~HsOH~ CI\CHO~CH3COO-~ aceryl-CoA ethanol

acetaldehyde (ethanal)

acetic acid

CoA

1he manner in which acetate units are converted to C4 compounds 5!i::nawn as the glyoxylate by-pass (see Fig. 2.12) fur which tw'O enzymes

aiditional ro tbose ofthe tricarboxylic acid cycle are needed: isocitrate: f!est and ma!ate synthast. The former enzyme cleaves isocitrate mto succli:!are and glyoxylate. The latter enzyme rhen uses a second acetyl-COA if)zdd te the gIyoxylate to give malate. 80th theseenzymesare ' induced' (chal is they are synthesised ooly wben the spedfic signal is given - see Sel:rion 2.8.4) when micro-organisms are grown on el compounds . The a::rivity ofborb e nzymesincreases by sorne 20 to 50 times under such growth conditions. The glyoxylate by-pass docs nor supplant the operatil:moftbe tricarboxyLic acid cyde; fer example 2-oxoglutarate will stUI iEre ro be produced (from isocitrate) in arder to supply glutamate for ~ syntbesis etc. Succin;¡¡te. the oeber producr from isocitl'ate Iyase. __:JI be metabolised as before toyield maJa te, and tbence oxaJoacetate. l31!3 tbrough the Eeactions oftbe glyoxaJate cyde, the C4 compounds Gil now be produced frem C units and are tben available fur synthesis l fIÉ an ceU metabolites (see Fig. 2.5). Their convenion into sugars is *'t:;¡¡jJed in Section 2.4 .

2..3.-4 Carbon sources other than glucose ~compound

that is used by a micro-organism and can feed ioto any afme intermediates of glycolysis. 01' even the citric acid cyde. can be Simdled by the orgaaism witb its existing complement of enzymes. 6I:M~ a great many other substrares can be handled by micro~ms . 1n other words. aU natural compounds are capable ofdegra.eation and me majority of this degradative capacity is found in iiii!f::robia1 systems. The application ofmicro-org.misms as 'waste dispos;¡! units' is therefore paramounr aDd this activity forms aD intrlnsic

Glucose

f==m ADP

Glucose 6-phos phale

f==NAD P

NADPH 6-phosphogluconate

G)f'-H~ 2-tet0-3-deo)fy-6-phDSl)l1ogluconale

n

Gr

l____...... Pyluvate Entner--Doudoroff

pathwly. Thls rometimes repbcl$ ttH! Embd~n--Meyerhof--Paml$

pathway (see Rg. 2.6) in sorne psaudomonads an d re!¡ued baaeri.l. Numbered enzyrna 11,..: (1) pllruphoglucon:lte ~dntDlR. (2) 11 speclflC .!doIase. Gtycenldehyde J-pho:\'~ (G3p) b c.onverted to pyruvate by ÜMI re:levant enzymes- gfven In Al. 2.6.

o(

Z

O

..:

O-

()

<

¡.'¡

'"O

O

-' :¡

~

e o• ¡¡, ~

'"z

""

!-< ~

...¡ ~ ~

~

)

26

RATUDGE

---'--------------------------------

~~."-

AcetyI-CoA

f'-

--C0"'''-\--.--'~--_Citrate CoA ® IV H,o

Q)CaloélC&tate

NAOH NAO'

~

Maluta

cis·A,co nilrate

H,o --1®

®r- H,o

Fumarate

tsocitrate

FAOH =l (j) FAO

yf= 0)

Ceh

NAO' NADH

2·QxogtUUlrate

Succinate

CoA GTP

NAOH Suee¡nv~CoA

TIre trkarboxyUc "lcld cyc:le.. (ATP I AOP rmy replace GTP I GOP In rcacdon 7. The 0VI!r;1I ~don \s: Icef)'l·CoA + 3 NAO+ +fAO.¡. GDP(AOp) ~ 2 C01+coenzymeA + 3 NAOH + FADH' + GTP(ATP) Tñe n~ered stepsarg catalysed by: (1) drrate symhas e. (2. J) aconitasl!. (4) Is oc:itr.lte dehydl'QgellaSll. (5) 2-oxogll,lC;QmuI delTtdl"\'ltl!r>Ue, (6) weclnatl! thiolmase, (7) succinate dehydn),ena$(!, (8) fumara se. (9) rmbte dehyd~nue.

3AOP + 3P,

3ATP

(oKphos)

Oiagr;mmaric preunutioll 01 1M du.al role ~ el IIIfI U"h:.lrt>o~ytic add cyde: to prod!.Jtl! Intl!rmedllte~ lnd Me!X)' (ATP).

Citrie aeid cyele

GROWTH ANO METABOclSH

pan of cnvironmeo ral biotedtnology which is {'xpJaineu in detall in OJ.apter24 . To illustrate this diversity, the exa.mple of microbial degradation of fanyacids will be collsidered. The ability ofmicro.arg;misms to growon oils and fats is widespread. The difference betweeo 3D Di! and a fue is wherher one is liquid or salid at ambient temperatures: mey are both chemically the same, that is chey are fatty acyl triesters of glyceroL:

CH,OH I CHOH I CH,OH

CH,O.OC{CH,I.-CH, I

giycerol

triacylglycerol

wbere o. m and p are rypically 14 or 16: me long alkyl chain maybe saturated as indicated ormay b.aw ooeor more double bonds givingunsat· unted. ur polyun satttrated . fauy aeyl groups. TIle oils, when added to microbiaJ cull'ures, are initially hydrolysed by aUpase enzyme into ils constituent f.J.tty acids and glycerol. The latter is then metaboLised byconversioo lO gIycel'3Jde hyde 3·phosphate (ree Fig_ 2.6). The fany acids are raken iota the ceU aod immediately converted into their coenzyme A thioesters. The fatty acyl-CoA esters are degraded in a cyclic sequcnce of reactious (sce Fig. 2.13) in which the

Acetyl-CoA

($Ce A¡. 2. 10), are ( 1) isocltr.ate

¡

trasO! and (2) malatB synthue. ~

Isocitral6

~S""i"," 1

Glyoxylate

'umm"

Malate ~

¡

Oxaloacelate _ _ ATP

--J.

0

Hcw ceH ensures equal wpplies of onIoacetaU (OAA) and acetyl-CoA (AcCoA) for citric acld biosynt~esls . Tht activily of pyruvate urboxytUt (2) Is nimubttd by acetyl-CoA fOl'med by ~1"UY2[e ¿dYydrogen:ue ( 1)

The ¡Iyoxylate by-pm.. Th e acklitlonal reactloos. beyond tho~ 01 me trlarboxrl;( acl¿ cyde

Citrato

~j®

y

• Acetyl-CoA

tne

I Cf!,O.OCíCH,I,-CH,

I

~

Oxaloacetatll

Citrato

CHO.OC-{ CH2)m-C~

1Acotyl·CoA

Pyru vate

ca,

Aop .-/l - Phospnonnolpyruvate

~

_ _ ...J

~cheme ¡ llO shows oow che bypass functions lO permit rugar formatlon from acetyl-CoA. wim lhe ad~ ruetlo n (1) pOO~hoeno lpyruvat.e

arboxykl nne. folowed by nlVemd IIyc~s (d. Fi,. 2. 1'1).

27

28- 1_ RATlfOGE " ._ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __

IRCH,.CH¡.CODH)

,8-0xldaticll\ cyde of

f8try acid

"., K"~:oA "~r\.. En:r:yom.s are:

<,)

huy K)'I-CoA I)'nthetaw. (2) fauy ~·CoA oxidase (In yeast ~nd ~II) Wnkl d 'Q tbvirl. I;)r' htt)' K)'l-CoA dehydrogenaJ.ln ~t.erit ~nked to FA[); (3) 1.1. W1OrJ-CoA hy~QSIII (;¡Isa km>wn ZJ crotona¡e): (1) 3.hydroxyac:y\-

ATP= t m H-S-COA

G)

AOP

+

HR<14

1---- RCH';~~:: lo, ti"",,) t ' - FAOH, {or flavin-I·M

CoA~le;(5)3·

oxaacyl-CoA thio'ue. The new

R.CH;CH.CQ·S-CoA

0r H~

htty K)'1-CoA rheo re
A.CHOH.CH1.C0-5·CoA

(4)V-

NAD

'

t'-NAOH

H.S,COA-J

R.CO.CH l"CO-S·CoA

I

® t ' - CH3.CO-S.CoA

i_________ R.Ca-S-CoA new fatty 8cyr·CoA

fatty acyl chain is prOgTesriveJy shortened by 10$.5 oftwo e atoros. Thes 3toms are loS{ as acetyI.coA units. Thi5 is known as the /J-ox.idaUo cyde as the in¡tia1 attack on tbe fatty aeyl chaio is at the p. (or 3-) PO! tion. Each turo of the cycle produces a shorter fany acyl-COA ester whic then repeats the seque.nce ofthe four reactions until, finaUy. a C.. fat aeyl group C... (butyTyl-CoA) is produced which tben gives rise to ho acetyJ.coA units. For unsaturaled fatty acids some adjuscment in the position of c:: double bond may be needed [O ensure tbat itis in the right position al configuration for it to be attaclced bytbe second enzyme ofthe cyde, t hydratase(see Fig. 2.13). The degradation offatty acids ¡mera tes the energy as heat ratl chan metabolically us.ableenergy in the form of ATP. lltis is bycoupli the reoxidation ofFADH1(see fig . 2.13) to 0 1 which produces H ~O~. T is thendeaved by an enzyme, catalase, ro give HP and !-201 wiili the eration of considerable beato Thus micro-organisms growing on f. acids and related matmals, sucb as long chain alkanes, invariably f erateconsiderable amountsofheat.

GROWTH ANO METABOUSM

2.4

I Gluconeogenesis

When an organisrn grows on a C; or Cl compound, or a material whose metabolism win produce su ch compounds, at or below the metabolic ~I of pyrl1vate (fur example. aceta te, ethanol,lactate or fatry acids), it is necessary for the organism to synthesise various sugars to fuJfll its metabolic needs. This is tenned gluconcogenesis. Though mostofthe reactions in the glycolytic parhways (se.!: Figs. 2.5 and 2.6) are reversible. mase caralysed by pyruvate kinase and phosphofructokinase are not and it 1; necessary for me ceIJ to orcumvenl them. As phosphoenolpyruvate cannot be formed from pyruvate (rhere ~ though, a fcw exceptions). oxaloacetate is used as the precursor: oxaIoacetate + ATP --+ phosphoenolpyruvate + C0 2 + ATP 1lris reaction is catalysed by phosphoenolpyru\'ate carboxyhnase which is ii!e key enzym e of gluconeogenesis. (The formation of oxaJoacetate has ~dy been rusc\lssed in relarion [O acetare metabolism, in SectiOD :!..3.3.) For growth on lactate, or pyruvate ¡tself. rhe lactate would be midised to pynlvale: ~CH(OH).COOH +

NAD + --+ CH1.CO.COOH + .NADH

~

pyruvate will be carboxylated to oxaloacetate using pyruvate carlxJxylase: ~.CO.COOH

+ C0',l + ATP --+ COOH.CI·l:t.CQ,COOH + ADP + p¡

lile oxaloacetate is then conYel'tffl lo phosphoenolpyruvate (see abOYe). The net reaction for growtb inlactate is therefore:

lact:are+ NA01- + ZATP ~ phosphoenolpyruvate + NADH + 2ADP + >P, The irreversibility of the second gIycolytic enzyme. phosphofTuclO' (producing frucrose 1.6-bisphosphate). is circumvented by t he aaioD of¡ructose btsphosphatQ.n:: Üi!ia5e

fructose 1.6-bisphl>sphate + El:t0 ~ fructose 6-phosphate + Pi ñom'this point. hexose sugars can be forroed by the reversal ofglyand <; and C" s ugars can now be formed vi.. [he pentose phospiir¡:s:E: pathway (Hg, 2.7). Gtucose itself is not aD end-product of ~neogenesis' bU{ glucose &phosphate is used for the synthesis of ~

~

waU constituents and a large variet:y of extracellular and storage Fig. 2.1-4).

~arides (see

1.5

I Energy production in aerobic micro-organisms

• Stas a1ready been ex:plained how. iD the metabolism oC glucose (Figs 2.6md 2.7)and in.the tricarboxylic acid cyde (Fig, 2.9). oxidation ofthe ~ merabolic intermediates is linked to rbe red uction ofa limited

29

30

RATlEOGE

-." ,- --~--------------------Acetyl-CoA I

1(1)

¡ O,,-aloacetale

ATP ~ (2¡

ADP-~-CCh Phosphoeno lpyruvllte r

i

(3)

¡ FruC10~

l ,6-bisphosphate

p,~(4) Fructose 6-phosphate (6)

Glucosa 6-phosphate r

~ntose

1

phosphat8 cyc\tI

(es Bfld C6 sugars) pofys8CCharides etc.

G\uconeogel\l!Sls lequence. from a SUbslr.!.le suc.hu ac:eqt-CoA mis Is convcrted ( 1) by me reac:tlollJ of me glyoxylate by1"Ut (see Al- 2. 12) to = 'oac:et;lle:illd th
NADPH + 3ADP + 3P¡ + ~z -. NADP~' + 3ATP + H;eQ

NADH + 3ADP + 3Pi + ~Ol -. NAO I + 3ATP + H20 FADH+2ADP+2P¡+ YzO~-t FAD + 2ATP + HP

w

phosphoenolpyruY:ite by

phosphoenl>lpyruva« car-boxykinase (2). nls i$ ~rted by che rtvened glycolytic sequence ef erlZ)'me< (l)

¡ntofructD~ 1.6-blsphos phate (see

abo F"1g. 2.6) vffljdl is therJ hydroly,ed -...fth me n!leas. of

lno rganic ph05pN te (P¡) by fructes",-I .6-bisphospha~1 e

number of ro-factors (NAO+, NADP-¡'. FAD) produdng the correspon ing reduced forms (NADH. NADPH and PADHz). The reducing power I these products is released by a complex reaction seque.nce which, i aerobic systems, is linleed eventually to reduction of aonospheric e This pro<:e~s is Icnown as oxidative phosphorylation and me sequen! of carrie.rs that are used to convey the hydrogen ious and e Lectron eventuaUy ro bc.. coupled te 0 1 to form H20. is referred ro as tbe ele toon transpon chain. The function of the electron-O'ansport-coupl;¡ phosphorylation (ETP) is the synthesis of ATP_Ue elecll'on transpo chain. together witb A1P syntht'rase, forros n multi
("l,

Th e produce is thero homeri~ to

glucose 6-phosphate lS) whi ch aIl

men be red loto me reac:rJom mme peritos. phosphate padlway (Rg. 2.7) o( usedferthe bIosynthlSisof c:~' envelope poIysaccharido!s.

One can tberefore describe Lhe ATP yie.ld, ineach case:, as being 3 and 2, respect'i"...cly. These are sometimes referred to as the: P/O rati wruch is lhe amounl of ATP gained frorn the reductiOIl of IhO~ to ti; and involves the trallSport oftwo electrons. The yields of ATP per mole of glucose m etabolised by t Emlxlen-Meyel'hofpathway (Fig. 2.6) and from the resulting pyruva metaboli.sed by rhe reactions of tbe tricarboxylic acid cycle: (Hg. 2.9) ; summarised in TabJe:2.1 . As can be seen. the vastméÚorityof Anisg. erated with the reactions oftbe electron transport chain coupled to I reactions ofthe citric arid cyc.le.

GROWTH ANO HETABOUSM

Succ1nate

¡

Succinate dehydrogenasa (FADjFADHt)

ADP

¡

* F,"?( F,-· x ",e

ADP

ADP

¡

¡

¡

""""~OX. F'.S;(R'd""d:X: l~

NADH d h d e y rogenijse

Ublquinone

Red. Fe-S

Oxidised

¡

Ubiqulnonecytochrome ¡: redu¡:tase

Cytochrome

h oc romas

s, +8:) Fe'·

Fe l -

t

ATP

Cyt

e

~

ATP

~Ot

ATP

Succinal8

ICADH~ Ox

!~

¡

r

Succinale dehydrogenase (FADjFAOH2)

AOP

S

Fe-

;<:

¡ Reduced

NAOH dehydrogenase

. Menaqulnone

Red. Fe·S

Oxldlsed

¡

ATP

ATP

l Cy1ochmm , b556

~

ATP

cyt:~~ome bS58

ATP

I - - - CYloch¡Ome

°

ATP - - cyt1:me d

!

¡

,

ATP

ATP

ElecTJ"Oll-t" .,1$por¡.coup led phmphorylnlon (m) Jynem of: (a) miwcoond,;a 11"" lb) Er:oIi. Notallltw! electr"on ~n arriel"$. ln

lhe E. col! JyUllm, me eliKuor> tnlMpO(t chain diviCc and elllctrom;wd protons cm iIow tllroulh bom me deSCTbed. The $¡teS of phospho.-yladon 01 AOP to ATP ar. onIy Imlications ~ the actual fonna!ion of ATP is carl"ied out

~u

~ATP $)'fltha~es that are driven by

Outslde

( / __ nH'

\

'- -3"··~

me physlcal mO'lt!menc of prot.lm thrgtJ&h the membrwte (see ~ho Fig. 2.161.

Membrano

Inside

Electron tran spon chain AOP+ PI

Thecouplng m.charlÍsm of ET? The elec.lrOn transportcaml!l"s (tee Fil. 2. 1S)

arll locatlld wlthln a Il1l!mbr.me. As thll redllcW1t (AHJ Is oxidited, mis seu upa fTJO"VefTIeIlt 01 protonli throllgh the. membranll. The$e protoN are then pUfT'olXjd back acro" ene membr¡¡ne driving the ATP sym~ 1! Ima cauplin,ADP with PI [O JÍ""t! ATP.

31

'1

32

RATLEDGE

MolesATP prodLJced per

mole hexose

Glycolysis (gluco$e (O pyruYare): Net yield ofATP =2 mol NADH=2molx3 PyI1N(l(C

2" 6

ro oce!yl-ú:>A: NADH= I mol x3(x2 for 2pyruvate)

6

TricorboxyJic add cyc/e: NADH = 3 mol )( 3 (x 2 for '2 acetyl-CoA) FADH2 = I mol x 2 (x 2 fo r 2 acetyl·CoA) ATP= I mol (x2foracetyl-CoA}

Total

18 4 '2 38

r.'0lIt: • Under ~n.:u.rob;c c<>n
phosphurylation'. $ee SKt1nn 2.6.1).

2.6 I Anaerobic mecabolism 2.6.1 General concepts Under anaerobk conditions, the process of oxidative phospborylation cannot occor, and the ceH is deprived ofiu principal way of generating energy. Under such circumstances energy must be provided from the very process of degrnding the original substcate. TIlls process, kncwn as mbstrate Level phospborylation, yields only abour 8% or the energy that can be produce
X + NADH -+ XHl + NAO" Golya Httle ofthe reducing power per se ¡s needed for !.ynthesis ofnew ceUs. Gonst'quently. tht' reducing equivalents react with tbe accumulated carbon intermediates to reduce them in tbeir tum (Fig. 2.1b). The only enetgy that is then available to the cell is that whicb comes from ATP fonued during the anaerobic degradation nf the sübstTate. Examples nf substra.te-Ievel phosphOIylation are gi~n in Table 2.2. This process of anaerobic catabolism occurs not only In microorganisms, but m ay occur in higher animals too: an example would be

Emyme

Reaction cataJysed

OCCUITeOCe

l . Phosphoglycerol kinase

I ,3-bisphosphoglycerate + ADP ~ 3.phosphogly<erate + ATP

Wldespread, 5ee Fig. 26

2 Pyruvate kioase

phosphoenolpyruvate + AOP -Jo pyruvate + ATP

Widespread. see Flg. 2.6

3. Acetate kjnase

acetyl phosphate + ADP ~ acetate + ATP

Widespread

4. Butyrate kinase

but)ryt phosphate + ADP ~ butyrate + ATP

E.g. ef1terobacteria on allantoin

5. Carbamate kinase

carbarnoyl phosphate + ADP -Jo carbamate + ATP

E.g. dostridia en arginine

6. Formyl-tetr.Jhydrofolate synthetase

N,o.rormyl-H 4 folate + ADP + p¡ -Jo formate + H4 rolate + ATP

E.g. clostridia on xanthine

RATLEDGE '.._34_.L...... _ _ __ __ __

_

_ __

_

_

_ __

_

the accumul ation of.lactic acid i n musde fusue during byper-activity of an athlete. In micro-organisms we can see a whole range of redueed carOOn compounds being accumulated. by organisms growing allacrot>. ically. Examples may abo inelude lactie acid itself(produced by laetic bacteria), but would ¡nelude short ehain fatty acids such as butyric or propioruc acids, and aleohoIs such as butano!, propano! 3nd ethano! (see Fig. 2.17).Some orga.rnsms, such as me methanogenic.bacteria, may go even further and produce, as the comple:tely reduced end-product. methane (notshown in Fig. 2.17). It is important ro poinr out that pyruvate. produced by che glycolytíc pathway (Fig. 2.6). will still enter the tricarboxylic acid cyclewhich muse continue. aC lean in part ro provideessential precursors furbiosynthesiso pnnopally 2-oxoglutarate. and oxaloacerate. but not to p roduce energy. The NADH produced in the cyde cannor be converted inro ATP as the cells have no supply ofO~ to drive oxidative phosphorylarion; however in certain bacteria there are terminal electron acceptors other than 0 2 which are capable of being eou pled m to the E1l' system (Fig. 2.15) and which will allow the fOl'mat'ion of ATP ro take place. This indudes microbes tbat can use nitrate (which is reduced to nirrite). nitrite (rccluced to NH~ or in sorne cases N 2 in a proeessJcnown as denitriftcation), COl (reduced to merhane by metbanogens) or su1phate (reduced to HlS by rulphare reducing bacteria) as alternatives [O 01. ln all cases. although theyield of A'IPis less than occurs in aerobic systems. ir is muc.h greater than obtained by substrate-Ievel phosphorylation alonc.

2.6.2 Products of anaerobic metabolism Figure 2_17 summarises sorne ofthe maln reactioos leading to the for-

mation ofreduccd end-products in anaerobic micro-organisms. The major products are: • glycerol. produced byyeasts when the conversion ofpyruvate to ethanol is bloeked; • lactic add, forrned by lactic acid bacteria; • formic acid, formelnd sometimes purines. Metbane(notshown in Fig. 2.17) is pcrhaps me ultimare redueed ca.l·bon compound and is produeed by highIy specialised An:hatMcteria by eleavage ofacelate to ca} and CH~ or in sorne cases by red.uction ofCO, .

GRDWTH AND METABOLlSM

2Py'+"'HE-_L_ _""''''''''''''' .-'!-

OKaloacetate _ _ _

IR

~1t~r-Su CcinYI-COA

¡x, Acetoacetyl-CoA

P(O PjOL,-CoA

- - --1 R

" '- - - CoA, CO~ Butyryl·COA

/

~";~""a:.""'i!1il""j,, " ""~·

jR

1!íiU~1

memanal (CH30H). etbana l ( C~CH 10H) 01" fonnic acid {HCOOH) a1l in me presence of~ gas.

2.7

I Biosynthesis

The provision of energy (AIP). re ducing power (NADH and NADPH) and ;a variety of monomeric precursors (see Fig. 2.5) from the degradation of .2 substrate provides the cell with lhe neeessary means oC regenerating iiself. The cel! undertakes roe biosynthesis oCtbe macromolerules of me cell~ nudeic acids (DNA and RNA). proteins (for enzymes and other functions).lípids for membr.mes and polysaccbarides as eomponents of me ce11 envclope. from fuese simple buiJding blocks. As rnanyofthe bio-syntbetic patbways are eOYere<:! elsewhere in this book (e.g. Chapten; 13. 'K and 15) tbeseneed Dot be detailed bere. However, a distinetion needs m be drawn between primary metabotisrn and secondary metabolism as [bese have considerable imporrance fur formation of bioteclmologi· al products.

2.7.1 Primary r:netabolism Prlmary metabolism occurs during baJanced growth , sometimes mown as the tropophase, of tbe organism in which all nutrients needed by the ceH are provided in excess in the medium (see Eig. 2.18). Undef such conditions the celli grow at an exponential rate in keeping witb theirmodeofreproduction. The ceJl s wiU have optimum contents al all the vadous macromolecules of [he cel! - DNA, RNA. proteins. ipids etc. - but tbeir proportions willchange as growth progresses and dlen sJows down. Evenroaliy. though. tbe ceO must run out of sorne nutrient even if ¡bis is only 01.' and eoruequently me growtb rate slows and eventually

Produ cn gl anaerobic metabolllm in vario u$ micro. o r&aolsms. ReaC'tiofl$ le;Jdinl 10 th! fec)'dng of NADH l /'l! indiqlte
glYen in the wded bolees. mar 0«11' sklt ly o, in ¡roups acc:ordln, (O the orpnism.

35

36

RATlEDGE

ceases. However, metabolism does notcease. lbe onIy time that rnetabolism completcly ceases is when the ceU dies: thus as 10flg as tbe cell retams viabiJity it is able 10 carry out sorne metabollc processes ando converscly and most imponantly, ¡fthe cell wishes to remain alive it rnust carry out a modicum ofmetabolism.

2.7.2 Secondary metabolism The need for the cell to keep a flux (or flow) of carbon going through it when active multiplication has ceased requires that tbe cell diverls its core metabolites into products other than the primary ones which arl! OO! needed in the S3meabundance. Sorne maintenaoce ofvital ccmponents. though, mus! be car[ied out: k.ey proteins rnunbe replaced as all proteios undergo tumover; ONA must be repaired. RNA maintainedetc. Consequently, sorne primary metabolism must continue but the cell nowswitches into a secondary mode ofmetabolism (sce Fig. 2.18). These secondary products then begin to arise sometimes as storage products within the ceH (e.g. poly-,&hydroxybueyrate or triacylglycerols - sce Chapter 15). sometirnes as increased amounts ofprimary metabolites (5uch as organic acids. see Chapter 14), but sometimes llew products are synthesised which are llOt normaUy present in any great abundance during the baJanced phase-ofgrowth. These secondary metabolites can be ofconsiderable biotechnologi. cal importance as many ofthem are biologically active notonlywithin the producing cell but also in othercel1s and therefoTe some may act as autibiotics. As the rangeofsecondary metabolites vacies almostwith the species ofoTganisrn being studied, this phase ofunbalanced metabolism has beco referred ro as lhe idiophase in cont:r.tsttD fue tropophase phase of growth. The secondary metabolites are usually synthesised from unwanted tnonomers lbat are still produce
Whatever is the reason for the profusion ofsecondary metabolites. their properties make them sorne of the most exploited biotechIlologicaJ products ofaU time.

I I

e

,Q

1i8 ~

e " "' ~

"' -

MicrubAlgrowth: prlm;¡ryand 5K01ldary mt'tJboIic

I

tropoPhase,~·-tI~·~--i dloPha se ----;o­

...... , ..

I

,,

Iomass

I

I

, ~

I I

limit ing'\ nutrient ...

"

B~ .ot

,

E

...-

I \

I

\

I

."

.../--

,

......

............

phases. ln the initii!1 pha~e (babnced growili=~) all nutrMnU are kl e~ When one nutrMAt (not arlJon) G consumed (- - ) cell gvwth ( - -) s~ down ~d J«ondilry mr.aboite(5) (- - - - _o) are fol'm4td in the idiophue.

./ secondar y ./ metabolite

.../ "

\ I !' '.1 /" '. ./

t ime

Pyruvate

Formadon of !.eConebry metabolil$! from ¡cet}i-

~co,

CoA. The .$eCOIIcDry mnabclites are showo tri lhe slladed ttxts.

There can be considl!rab!. onl"latlon In s uu~ of sorne of mese mllltabollte5.

x3

Mevalonate (Cd

~co, Isopren e units (es)

l~

----1ij~. Mi~ ií·!i~

38

RAnEDGE

2.8 I Control of metabolic processes 2,8.1 Metabolic flux

NewDNA

Rapl~tlVI1

DNA

Tmnscnpt"on ma g ene Mosse oger RNA (mA NA)

Atlach ea 10 11

ric.osome mRNA .. rlbo.ome

Tra"s/a¡IOtI 01 mfl.NA

(ccde. for ,m¡!'Io acids 10 be .clded logetlteo- In conlKl

sequ~)

PnMein I IduHsed db.gn.m .$howing how DNA C\n elther be

repliuted to "", nI!'" DNA (for new ce l! synthesls) or be ¡ran¡tribed Into meUflnxer P.NA (mRNA) chal is decoelld (or tTatlw.ted) by It becomllll 3.ttached ro a ribotome whlch moo ITlilkes ¡¡ proteln rnolecule by 1.qoentiat .addition or amino aelc:fs. Thu$ lbe original sequen« oí b,u as ¡ long

the ONA tlhe gene)" flrst conven:ed to a correspcndlng sequence ofbau~, (mRNA) mat ¡¡:/YQS r!se f.O

a I'\(!W proteln; see also

Ch;¡pter 4, Flg. 4.1.

The concept of metabolic flux (or flow) has been developed th¡ attempts to describe iIunatbematical (ertnS the1
2.8,2 Nucrienc uptake

Control of cell metabolism begins by th e cel! regulating its uptake ( nutrients. MeS! nutrients, apart" from oxygen ¡¡nd a very few carho compounds, are {ak.eo up by specific:transport mechanisms so that tht maybeeoneen tra ted within the cell from dUute sol utionsoutside. Suc 'active' transport systems require an input ofenergy. The processes al controllable so that o nce the amount of nutnent tak.en into the cel] he reached a given concentratioD. further unnecessary (or even demmel tal) uptake can be stopped. (This is abo discussed in Sectioo 2.8.5 o catabalite repression.) In sorne cases the rate atwhich a earoon sourel

GROWTH ANO METABOUSM

such as glucose.is takenup inta the cell may be the limiting process for growth oftbewhole cell and themore should receive particular attenrion when evaluating potential bottle-necks to increased productivity ofa bioprocess.

2.8.3 Compartmentalism A simple form of metabolic control is the use of compartments, or

organelles, within the ccll wherein separate pools ofmetabolites can be roaintained. An obvious example is the mitochondrion of tbe eukaryodc ce.U which separates (amongstothers) the tricarbaxyl:ic acid cycle reactions from reactians in the cytoplasm. Anothe.r would be the biosynthesis of tatty acids which occurs in tbe cyroplasm of eukaryotic cells whereas the degradanon offatty acids (see Fig. 2. l3) ocrurs in the peroxisome organeUe. Separating [be two sets ofenzymes prevents any (aroman interDlediate being recyded in a futile manner. Qther organeUes (vacuoles, the nudeus, peroxisomes, etc.) are simHarly used to control other reacrions ofthe cen. Bacteria, bowever. do Dot have such compartments witbin tbeir celIs and therefore must rely on other means of metabolk control.

2_8A Control of enzyme synthesis Many enzymes within 3 ceU are present constitutively; that is. they are there under al1 growth conditioI1.'l. Other enzymes only 'appear' when nl'eded : e.g. isocitrate lyase aftbe glyoxylate by-pass (see Fig. 2.12) wben the cell grows on a C; substrate. This is termoo induction of enzyme synthesis. Conversely enzymes can 'disappear' w hen they are no looger requieed: for example, enzymes for histidine biosyntltesis stop being produced ifthere is sufflciem externa] histidine avaiJable lO satisfy the needs ofthe ccl1. 'Ihis is (emted reprt'ssion: when thegraruüous supplyofthe compound has gone, the enzymes for syntbesis of the material 'reappear' ; their synlhesis is de-repressed. The key to both induction and repressian is that the genes codingfor the synthesis ofthe proteíns bythe processes of transcription (see Fig. 2.20) are either switched on (indu etion) or off(repl'ession) according to the metabolite~ present (or absent) in the cell. These processes are shown diagrammatically in Fig. 2.21.

2.8.5 Catabolic repression This type ofmetabolic control is an extensioo ofthe ideas already set out with respect to enzyme ¡nduchon and repression, being brought ahaut by external nutnenlS added (Q tbe microbial culture. 'Ole term catabolite repression refers 10 seveJ.;:¡ ] general phenomena seen, for example. wben a micro-organism is able to select, from MO or more different carbon sources simulraneously presented to it, tbat substrate which it (Irefers lO utilise. For example, a micro-organism presented with both glucose and lactose may ignore the lactase until it has con· sumed all the glucose. This scquential utilisation oftwo substrates is referred ro as diauxic growtb. Simila r selection may cernr ror the choice ofa nitrogen source ifmorc than one is availablc. The advantage ro tbe cell is tbat it can use the compound which provides it with

)9

RATLEDGE

10

¡a)

DNA

~

~

2.

Transcriptlon

o· 00 • •

DNA

lb)

e

2. • e

J\JV' mRNA

~

Transcription

o· ¡¡D

JV\I" mRNA

!

~

Translatton

¡TmM"t'"" ~

$

2inding

•

~

O

z

O ~ w ~ O

~

• O

Proteio

~

••

•, o

¡;

"2 lE

1ha attached protei"

~tiv e

No binding 10 ONA. possible

StrucUlrlll genes IlOt exprossad as signal from the operator geno Is not given dUlI 10 the presence of

Oparator

'Switch 00' signal

Z

O ~ w ~ O

••e •

~

" , ~

. ~j

J\f\J' m"NA, J\f\J' mRNA ~ JV\F" mANAl

1

Proteína produced

lE

Con1rol of enzylT\ol! s)'TlI:htik thl'Ol.lgh re,gul;atlon of DNA I!Kpression. (a> Represslon: In the absence of any Indudng molecuhl the mes$t1'lser RNA (mRNA) from the reJUlatory gelMl produces a pl'O'te lo tN.t binds te an 'oper.Itor' ¡ _ funher down dl4t

ONA mo!ecllle. As a resultofthis binding. tM

openuor gene ¡~

Inact/vated ~nd no signal is given te allow the strUcwl'lll genes (tlut

WCluld makllcdve enzymes) te be eXprased. (b) InductiO!1: in me presellCe

ot ~n Induclrlg molecule.

tlw. protein arlslllffrom me "'g"blo')' , _ 1, now no 'on&er able ce bllld te> d'M>~r g~. Consequently, me openlotor 'switche,' Qfl the 1171.JCt:UraJ cenes and active prou:ins (enI)'mes) are

now made.

the most useful substrate ror production of energy aod provision of metabolites. The mechanisms by which catabolite repression is acl1ieved varies from organism to organismoA simple caseis with E. colt where control is exerted via 3n effector moLecule, cyclic AMP (cAMP). (lo cAMP (he single phospho groupofAMP-see Fig. 2,4 - bridges across IToro the 3' -hydroxy group ofnoose to tbe 5'·hydroxy group, tbereby forming acydicdiphosphoester.) cAMP interacts with a specific protein. catabolite activator protein(CAP;! (also known as the CRP "" catabolite receptorprotcin), and the cAMP-CAP comple>: binds to DNA causing the genes that follow afier (ordownstream of) the binding site to be tl'anscribed (see Ag. 2.22). Tbese genes may tben be used to syntbesise new proteins for uptake and metabolism ofthe next substrate (e.g. lactose if [he cells aregrowing on a glucoseflactose mixture).1his positive system ofgenetic conl:l'ol is the reverseofthe negativecoDl:I'ol systern described in Fig. 2.21. The key molerule is Lherefore cAMP. As long as gluoose or its catabolites are present, cAMP is not formed as ilS syntbesising enzyrne (adeny· 1a1'e cydase) is inhibited by these catabolites and thtlS Jactase uptake and metabolism cannotoccur. The catabolites therefore repress the syn· tbesis ofnewenzymes. Thc repression is removed when the catabolites disappear- i.e. a11 the gtucose has been consumcd.

2.8.6 Modification o( enzyme activiey Once an enzyme has been syntbesised, its activity can be modulated by a variety ofmeans.

GROWTH AND HETABOlISM

ONA

Catabo!lI:. rep!'Msion.

•e • D

CAP mRNA

j

~

Inhlblted by gJucose m81aboJites

CAP

e

~ cAMP --- ATP

AM:M~ ~

~

_~" _ v_V_V

"" mRNA') Proteins

~ mRNA, -~mRNAa

Th. stn.I(tur;a1 gen6anl therefore 'swI~

off' (I.e .• ~mud);u Ion¡ as ~a o r lu c:ar:aboliles anI pruefll. Sevenl operoru. may respond 10 the cAt1P-CAP slf:naL

) .

Th. mech;r,nism lhown ís medj¡ted by cyOlC AMP (cAMP). An operon Is <:ontTOl1ed by the oparatorxene beln, 3ctiv¡¡ted by a complex fol.llld batw..n a protein (tIM cataboJi~ acdvator proteln, CAP) and cyc.Jlc AMP (cAHP). cAMPbOflty formed wheo gIucose 15 ablento

produeed

Post-transcriptional mod16cations This process is so-called becauseit occurs afterthe enzyme has be:en syntbesised. ¡,e. after its fonnation bytranscription (see Fig. 2,20). Enzymes maybe moclified from one fono to anotber, oneform being active and rhe.other inactive or less active: E¡.rnWI H EjlJlao:ttw)

This process ofactivatingor inactivating an cnzyIDe is camed outby an entirely separate enzyme which has nothing to do wilh ca talysing rhe reaction thatthe original en:zyme will be ¡nvolved with. ~ common way ofachieving this converslon is by phosphorylation of the enzyme using a new enzyme - a proteill kina~. These protcin kinases. of wh.ich rhere can be maoy. usually react with only ene enzyme and thus are highly specific, They add a phospho group (from ATP) to a specific hydroxyl group (normalIy a serioe rcsidue) on the enzymc, Tbe activared enzyme may be:either the phospborylate(l form or its de-phosphorylated formo The de-phosphorylation will be carned out by a specific phosphatase enzyme. The activities of the protein mase and the phosphatase will be obviously controlled by other (acron within the cell aud will work according lO the metabolic status ofthe ce1 1. 'There are otber mechanisms of altering the activities ofspedfic proteins by tbe attachmenr (or removal) of a simple molecule ro a particular amino add residue in an enzyme. but the addition of a phospho group isby far the mosrcommon,

41

42

RATLEDGE

Aetion of effect.ors The second way in which an enzyme's activitycan be controlled is by its response to vanous effectors. (Effe<:tors can .lct positively. Le. are promoters, or neg.ltively, Le. are inhibitors.) An example is che process known as feedback inhibition. HeTe. in a sequence ofbiosyothesis A~B~C-+D~E

•

I

the end product. E, may be able to inhibit the 6rst enzyme of the sequence {converting Ato BJ. Tbis will only occur wheo sufficient E has beeo produeed bythecell for itsimmediate requirements and therefore no further carbon need be channelled down this pathway. AJ¡, the ceTI contlnues to grow itwiU consume the accumulated E and thus diminish theamountofitin circuladon. ThU$, as Eis withdrawo forthe ccll's own needs. t he inhibitoryeffect will be withdrawn and the conversion of Ato B will recommence with the further symhesis ofE then oerurring [O match the ce1l's requirements. Th1S process will .lIso occur ifthe end-product, E. is added ro m e growth medium ofthe organismo Here, as the product is now supplied gratuitously, the cell h as no nero to 'waste' ies resources syntbesising E so the pathway is now inhibited.ln addition to che feedback inhibition. a high concemr.ation of the endproduct can also lcad to the repression of tbe enzymes for the entire pathway; mus mere is a 'qukk' response mode to ahigh concentration of the end-product .1lising: [he initiaJ enzyme of the pathway is inhibited and there is no flux ofcarbon along che patbway; and then mere is a longer-term response whereby all the enzymes ueeded fur the pathway stop being syntbesised by repression (see above) ar tbe DNA level as they are surplus to requiremffit and theircontinued synthesis wouJd be a waste ofvaluable amino acid precursors. This process can. of course, be quite complicated should the pathway not be linear as depicted above bu[ be a branchiQg pathway witb multi· ple-end products. This is ofparticular importante in the biosynthesis of amino acids several ofwhich (such as phenylalanine, tyrosine aDd tryptophan) share a comlllon iDirial patbway. This is disrussed in greater detaiJ in Chapter 13.

2,8. 7 Degradation of enzyrnes Enzymes are not particularly stable molecules and may be quicldy and irreve.rsibly destto~. Their half-lives are veryvanable; they may be as shon as a few minutes or as long as several days. Although the syntbe· ses ofenzymes can be regulated at the genetic level (see Section 2.8.41, once an en2yme has been synthesised it can rem.lm functioDal foc some time. If the envllonme.nral conditions change abruptIy, it may not sufflce forthe syntbesis oftheenzyme to be 'switched off', i.e. repressed: the cel! may need ro inactivate the enzyme so as to avoid needless. or even pe.rhaps deJeterious. metabolic activity. This may be by feedback inhibition (Section 2.8.6). Additionally, under nitrogen-limited eond" tions, when a ceU becomes deplered of nitrogen and tben ceases tu

GROWTH AND METABOUSM

Organism

-

G ¡=ul

Methylomonos sp. Methylomonas sp. Candido utilis Klebsiella pneumonioe

Gu:ose

Escherichio coli'

tfefr-.a.ne Hefr-odlOl

Molar growth yield (g organism dry 'Nt. per g-mol substrate)

1.46 1.38

17.s 16.6 3 1.2 50.4

1.40

95.0 25.8

1.32 0.36

90 21 81

1.26 0.29 1. 13

KJebsiella pneumonioe Pseuoomonas sp. Camida uti/is

173 52.2 23.5 21.6

120 0.87 0.98

Yarrowia (Condida) lipolytiCCl

203

1.06

aerobic anaerobic Socchoromyces cerevisioe: aerobic anaerobic Penici/lium d1rysogenum KJebsiel/a pneumonioe

~

proteases. that are proteolytic enzymes, may be activated to copies of enZ)'lnes so that the amino acids therein can Ée.sa'reDged and used for the biosynthesis ofnew enzymes [hat may be 53111 essential. Thus, enzymes may be 'turned over' mote rapidly than Sil2j'0CCllr by simple denaturation.

~e surplus

2.9

Carbon conversion coefficient (g organism dry wt pero g suhstr
I Efficiency of microbial growth

"!iirre-owerall efficiency of microbial growth is discussed in serict thermo~c temlS in Chapter 3. Tt is usually expressed in terms aftbe yield &É a!ls fonned per unit weight of carbon suhstrate consurned. The ~ growth yield, Ys. is the cell yield (dry weight) per mole ofsubSi::Gre, while the carban conversion coeffirient, which allows more ..."n";inngful comparisons berween substrates of different molecular si:as.is the cell yield per gram ofsubstrate carbono A particular feature in Table 2.3 is the lower growth yields attained 'iIiiIen facultative organisms are rransferred from aerobic to anaerobic amditions. a phenomenon which is obviously connected with ea:reased enecgy production under these conditions. Empirical1y, the growth yield of a micro--organism wiU depend on ;;:;;;arry factors: (t) The namee ofthe cacbon source.

1.30

0.90

43

+4

RATlEDGE

(2) The pathways of substrate rntabolism. (3) Any supplementaryprovisionofcomplex substrares (obviating the nero for some anabolic pathways ro operare). (4) Energy requirements for assimilating othernutrients especially nitrogeo. (S) Varyin.g efficiencies of ATP-generatiog reaclions. (6) Presence ofinbibitory substrates, advene ionic balance. or other medium components imposing extra demands 00 transpon systems. (7) Tbe physiological nace ofthe organism: nearly al1 microorganisms modify their developrnent according to the external environment. and me different processes (e.g. primal)' aod secondary metabolism) will enrail different mass and energybalances. In conrinuous culture systems, in which the growth rate and nutritional status of the ceLls are controUed (see Chaprers 3 and 6), furtber facrors can be identified : (8) The nature ofthe limiting substrate: carbon·1imited growth is afien more 'effident' than. ror example, nitrogen-limited growth, in which catabolism of excess carben substrate may follow mutes which are energetically 'wasreful' (bowever useful they may be ro the biotechnologist!). (9) The pennirted growth tate: wbereas thegrowth rate is decreased , tbe propartion ofrhe substrate going towards maintaining the celIs increases, thereby diminishing the amountofsubrtrate that can go to other producrs. As a final faclOr governing aU aspects of mlcrobial performances, one might usefully add: (10) The competence ofehe microbiologist'.

2. 10

I Further reading

Hames, B. D. and Hooper, N. M. (ZOOO). Instan! No/es: Biochemistry. 2nd edn. Bios: Scientific Publishe:rs, Oxford. Lengeler,J. W.. Drews, G. and Schlegel. H., eds (1999). Biologyo{tht Prokat)lltes.

Blackwell Science, Oxford. Nicklin,J .. Graeme-Cook, K.. Paget, T. and KiUington, R.(1999).lnstantNolts: Microbiology. Bio5 Scientific Publuhers, Oxford. Madigan, M. T_ Ma:rtinJco.J. M. and parker.J.(2000J. BiologyofMimHlrE'Jnisms, 9th Editlon. Prentice-HaU InternadonaL(UIQ Ltd, London.

Chapter 3

Stoichiometryand kinetics of microbial growth from a thermodynamic perspective J.J. Heijnen Nomenclamre

[ntroduction Stoichiomeuy cakuJations Stoichiometl'Y predictions based 011 Gibbs energy dissipilDon

Growth kinetics from a Ihermodynamic point ofview Furth~Teading

No me nclatu re maximal growth yield ofbiomass (X) on C-mol X per Cmol S substrate(S) or electron donor (O) maintenance coefficient of rompound j Cmo! i per C-mol Xh maximum specific growth rafe h-' mol¡-\ affinicyconstant yleJd ofbiomass (X) on compound i C-mol X per mol i reactor specific convenion rnte of mol i m - 1 per h compoundi el concentradoD molim- l V liquid volume ofreactor m' JI. specific growth rate h- ' tili¡ stand.m! ('m balpyofformarion kJ mol - \ aC¡ standard Gibbs energy offormation kJmoJ - 1 - ó.C""r Gibbs energy ofcatabolism ~r Cmol kJ per (q mol organic elcct:ron donor OT per mol of inorganíc e lectron donor biomass speciftc convenion rafe of mol j pee Cmol Xh compound i y degsuofreduction X biomass GmoJ y:u

3.1

Introduction

Quantieative infoemation on microbia J growth is needed in many fec· meneation a nd biological waste treatmefltprocesses. Typic.ally. growth

46

HEIJNEN

Mi'

Growth represented as

i coupled anabo~smlcaabclism.

CATABOLISM

ANABOllSM

Electron donor (O)

BiomU9 (X) CHUlOo.sNo,

•

electron 8cceptor IAI Glbbs

energy

Oxldised dono r

• reduced accepto r

C-sourco, N·source H2D, HCOi". H'

i..s quantified usingwell known parameters such as ma"imum biomass yield on substrate S (also caBed electron donar D) (Y~iI.'( or Y~). maintenance requirementsfor substrate S or electton donor D (nls or mol, Mmu' K, and a threshold concentration of the electron donor. A practical problem is that the valueS for these parameters vary by one ro two orders ofmagnitude, depending on the growth systems being consi dered. Su ch growth systems are generally characterised (Fig. 3.1) by their electron donor and electron acreptor. their C-source and N-source. In additionin eachgrowth system HCO;, HP and H" are involved.A practical point i5 that rnany micro-organi5m5 have similar elemental compositions. as illustrated in Fig. 3.1_Th.is allows the use of a nandard biornass cornposition, in case tbis informationi5 notavaib..ble. However it is always preferable ro detennine tbis, using elemenral an.a.lysis. For practical purposes it is important to k:now the complete sooichiomerry of growth. ln fennentation processes DotOnJy is che biomass yield from the substrate (Ysx orYex ) importam. bu[so are theoxygen requirement. COl production and beat production in order lO design an optim.al process. TherefOre. me.thods of stoichlomerry calrulation are offundamental value. ln addition, the estima tion ofgrowth stoichiometry for arbitrary growth systems is relevant, for e.xample in biological wasre treatment. ln tbe past decades. many metbods have beel} proposed to estimaregrowth parame{f'rs for arbitr.u:y growth systems.

3.2 I Stoichiometry calculations 3.2. 1 Definition of the growth system A roicrobial growth system is convenilmtly represented as an overall reaction equarjon lFig. 3.2) where 1 C-mol of biornass is fonned and which takes ¡noo account the role ofN--source. HP, HCO;-. Hi". electron dona r and electron acceptor (ouple. In addition it is indicated that heat and Gibbs energy are also involved . Ofie C mol ofbiomass is the.amount wbich contains l2g' of carbon which usually amounts to about 25g of dry matter. knowing that the biomass carbon content is about45%. For microbial growth energy must be genera{f'd 00 enable the CODstruction of the complex biomass molecules frOID simple cacbon compounds. This energy is generated in a redox reacdon between t he electroD donor and electron acceptor. The pro per measure of energy spent in growtb

STOICHIOMETRY AND KINETICS OF GROWTH

_ -'- electroll donor - _'_ t1lectrOIl acceptor + 1C - mol biomass + .1... 1tJ haat YI)¡(

+.]

' o,

YAl(

y~

I C-moIQf biomau. YI)(Is dIe yiekl I C-mol X on I C-rnol of

U Gibbs energy + (, .. ) N·souree + (... ) HlO + ( •.. ) HCOi + (... ) H'

o(

compo~ i; ( .... ) ~ ~speclf1td

processes is DOC the: released heat but Gibbs energy. beca use Gibbs energy (~G) combines [he heat relate
3.2.2 Measuring yields It: is stressed that stoicbiometric yield coeffidents ;¡re ratios of conver·

sionrates (rx is given as e·mol X m- 3 reactor per h. ti in mol i m - 3 peth).

"

y . {)( ri

(3.1)

These tates are ca1culated fram measurements in experirnents which may be either batch, cantinuous or fed batch cultures. using correct mass balances. TIte mose frequentIy rncasured gtowth stoichiometric coeffident is the bíomass yield on substrate (or e1ectron donor ) Ysx (or Ypx)' In a constant volurne batch culture (O indicating tUne O), y ~ will be: (3.2a)

In a chemostat. where inputand o utflow cates are equa!, a similar equa· tion holds . wher-e ~ = Oand ~ is r-eplaced by the concentratioo ~ of the incoming substrilte. If volume variations Decur. more complex relations can be derived from rhe mass balances.Forbatch reactorswith variable volume V. WE':get:

(3.lb)

3.2.3 Maintenance effect lnitiaUy Ysr was inteoouced by Monod as a constant. However aftee the introduction of the chemostat, cultivanon of micro-organisrns undE':t diffe.rent growtb cates showed thar Ysx was dependent 00 the specific growth rate Jl.. The.proposed explanatían is basE':d on the maineenance concept (Herbert-Pict). ln this concept it is assumed that maintenaace of cellular functioIlS requires thE': expenditure of Gibbs E': nergy (ror restoring leaky gradienrs, protein degradation. erc). 1his Gibbs energy is produced by the catabolism ofa certain amountofelectron donor (= substrate). lfthis maintenance tate is ms or Irry e·mol substrate per C-mol X'h, the following equation holds 1 1 m,

- =~ +-

Ysx

Gene ... 1stoich lornetrlc

nlpresenQtion of t he fonn3 don of

ma.:

JI.

(3.3)

unknown stoichlometrlt compounds.

48

HEIJNEN

Dependtrn:e of biomass yleId Yu on sptelfic ¡rowth r.J.te (maintenance efl'ect), m,ls me

Y"

t

substnte maWltenill10Ct coefficlent.

r;-1s the mu.ima.1 biomus ylt!ld

0.5 Ys:;"

on sulmnote.

ExperimeoralJy y sx is rueasured in a chemostat under different speciflc growth mIes 1-/-. From the obtained Ysx and 1-/- values, one calrulates. llsingEqn(3.3). thf" modeJ parameters y~a:< and m,. Figure 3.3 shows how Y5X depends on Ik. Fot high valUe5 of JL. ysx approaches the value ofthe model paramf"tf"[ For low p. values Ysx drops signiticantly. becoming Y.! Y for p.=m.Y~. For mOSl conventiooal processes, itcan be shown that at nonual growth temperatures theeffectofmaintenanceonyield can be neglected ror 1-/- > O.OSh- ' . This means that in batch cultures ducing exponencial growth (where p.= JLmll<) YSJ("'Y~' However. in me fed-batch processes (whic.b are tbe nmm in most industrial processes). where I-/-
u

Yrx....

3.2.4 Conservatlon principies to calculate thefull stoichiometry of growth .Figure 3.2 shows [bar besides YSl( (or YDxl. there are many mOfe stoicbiometnc coe.fficients. Fortunately tbese nero 110t be detel'mined experimenralIy. !he applicatiol1 of conservation principIes often allows all otber coefflcients to be calculated if a single roefficient(Y sx) is measured.TItis calculation is most easUy explained in an example. Example: Use ol conservation principles in calcnlation of all stoichiometric coeffide.nts An aerobic micro-organismgroW5 on oxaJateusingNH¡ as N-source. The following overall reaction equatioo (according to Fig. 3.2) can be written based 00 1 vmol biomass bcing produce<'! witb a biomass yield on oxalate ofYDI"" t/5.815 e-mol biomass per mol oxalate -5.815

CP¡- + IlNH; + bll+ +cR20 + d0 2+ eHCO;- + lCH,.. Oo.,NI,1.2

There are five unknown stoichiometric coeffidents for which serva tion constcaints can be furrnlllated . C-conservation H-conservation Oronservation N·amservation Chacge
-11.63+e+l::0 4a+b+Zc+Je+ 1.8=0 -23.26 + c + 2d +3e+O.5=O

a+O.2=0 + 11.63 + a+b-e=O

Solving gives the full 5toichiometry.

fi~

con-

STOICHIOMfTRY ANO KINETICS O F GROWTH

-5 .815 C10!- + O.2NH: + O.8H'~ - 1.85701 -S.42Hp+ lCH.u0o,.,Na.z + lO.63HCO; Thus \Ve see that yNI: = 1/1_857 C-m ol XI mol 0 1 , Abo Yo:'" 1110.53 C-mol X/ mol HCO;. For mis overall equation o ne can . using AHr and AG values from rhennodynamic tables(Table3.1). also calculate tbe( - AHR)and (- dG Il). which then provide I ~HX aud l{Yex'

r

3_2.5 Balance of degree of reduction The applicaüon ofthe conservarion constraints is sn-aightforward. A usdu..l short cut of such calculations is to apply a degree of red uctíon balance (Roe1s, 19831_ The degrce of reduction tsymbol y) is defined for each compound and is a stoichiometric quantity, defined in sucb a way mat its value is zero for tbe referem.:e compounds H10. H~ . HCOl . 50:-_ NO;, Fe3 jo, N-source. The ')"value fol' each compound is found by calculating the redox ha lfreaction which eonverts the compound into the pl'evious defined rcfenmcechemicals anda numbel' ofelectrons. yfoJlowsas rhenumbel' of produced c1ectrons per Cmol fol' o rganic a nd per mol for inorganic compounds_ For e)(ample. thedegree ofreducrioDofOl fo l1ows from the redox ha lf re:'lction 02 + 4H+ -} 2H20 - 4e- , and -y = - 4. For glucose, the redox- half reactioll is C6H l~06 + 12H~0 --t 6HCO J + 30H+ + 2.-¡ c- and -y= 24/6=4. Using thc redoxhalf.reactions thc y-values fur atoms and eharge can also be calrulated (Table 3.2). For e..'lCample for the carbon atom one ohtains C + 3HP .....¡.HC0i' + SH"t- + 4e- and a value of ;, : 4 is found fol' carbono Similarly va lues for H, O. 5. N, + a ne! - charge are found (Table 3.2). Ir should be noted Lbauhe nitrogen atom in the biomassand in the N-source for growth has a degree of .reduction, whicb depend5 on the N-source used for gl'owth. This is needed ro ensure thar t he molecular degree ofreduction ofN-source becomes O. Forexample usingTable 3.2 the degree of reductíon for NH: follows as - 3 + 4 - 1 = O. 'Ihe degree of reduction ofa molerule (Table 3.1) represents the amount ofele<:trons becoming available from that molecule upon oxidation to the refe rence compounds. Fororganic molecules, its value i5 usua Uy normalised pe!' C-mole, fur inorganic molecules its value is per mole. Table 3.1 shows rhat for organic mol e<'U les the value range from Oro 8. For biomass (standard compasition) it follows tbat 'Y~ =4 .2 (1 X4 + 1.8 X 1 -0.5 X 2 0.2 X 3) forNtr; as a N-SOUl'ce and"x : 5.8 for NOJ as a N-somee. Because electrons are conserved, it Is possible to calculate the balance of degree ofredl.lcrion as shown in me following example. Example: Application oCthe balance oC degree o Creduction Consider the previous example of aerobic growth o n oxaIate. Calculationofthe degree ofreduction (using rabIe 3.2) ofl molecule oC oxalate (C.p!- ) gives y = 2 X 4 + 4 X(- 2)+ (+2) = 2.

49

SO

HEIJNEN

Compound name

CompositlOn

Biomass Water Btcarborlate

CH I e0o.sNcl

Proton

W 0,

ca, (g) 0, (g)

H,o HCO¡

ca,

0xalate 2 Carbon monoxide FormateGlyoxylateTartrate1-

c,ot

Malonate 1-

C]Hpl -

Fumarate 1Malate'-

C~H20¡-

Citrate1-

C,H~O, c,H,o¡ C~Hi O.. -

Pyruvate

Succinate 1GluconateFormaldehyde

""eme Dihydroxy acetone LaclateGlucose Mannrtol Glycerol ProptOnate -

Ethylene ~",oI

- 445. 18

C,H,O,

- 517,18 -91 7.22 - 9426 1 -488.52 -36 1.08 -33050 - 180 - 35263 -317 - 322 - 175.39 -1 8 1.75 - 175.8 1 + 60

C4H40~ .

C"Hn07-

C6H 11O" C6H I.O~

C)HaOJ C3Hs0 1_ C,H,O, C~H801

Propartechol Butanediol Methanol

C)Ha02 C4 H 1OO2 CH40

Ethanol

C,H,O

Propanol n-Alkane (1)

C)H80 CI~Hn

CJHa C,H, CH,

H,

Ammonium

NH.'¡'

N, (g)

N, NO;

Nitrite ion Nitrate ion Il"on ll Iron 111

°

S H,O]

CiH~Ol-

C. H]Ol

H,(g)

- 67 - 237,1 8 -586,85 - 394.359 - 39 ,87

CH,0 C,H,o,-

CO CHO; C,O,H-

Butyr.rt.e

Propane(g)

k! mo!-I

-674,04 -1 37. 15 - 335 - 468.6 -1 0 10 - 700 - 604.2 1 - 845,08 - 1168.34 -474.63 -690,2) - 1154 -1 3054 -369.4 1

Acetoine

Ethane (g) Methane (g)

~ql

N0 3-

Fe"

f e)-

l1If kj mol- I -9 1 -286 - 692 -394,1 O

°

- 82' - 111 - ' 10

-m -843 -1 5 15. -596

- 900 -486

- 687 - 1264 -676

- 535

- 32 89 -50.75

- 246 -288 - 33 1 - 439 - 104 - 85 -75

° - 37.2 °

° ° - 107

- 24

-79.37

- 11 1.34 -78.87 -4.6

- 133

-173 - 87

-.

Degr€e of reductlon

4.2 (N-source NH4"') O

°OO -4 +-1 +2 +2 +2 +2.5 + 2.67 +3 +-3 f-3

+-3.33 +350 +3.67 +. +4

+ 5.33 +. +4 +4,33 +4.67 +4.67 +5 +5 +5 + 5.33 + 5,50 +6

+6 +6 + 6.13 +6.66 +7 +8 +2 +8 +1 0 +2 O +1

°

STOICHIOHETRY ANO KINETICS OF GROWfH

Compound name

Composition

Sulphur Hydrogen sulphide (g) Sulphide ion Sulphate ion Thiosulphate ion Ammonium

S' H,S HSSO;-

S, 0 11 -

NH/

.ó. G,~1

kJ mol- I

O -3356 + 12.0S - 744.63 -5 13.2 - 79.37

O -20 -17 -909 - 608 - 133

H

1

O

-2

e s

N

+ I charge - I charge N In blomass and In N-$ource

J1H? kJ mol- 1

4 6

5 -1

+1 -3" O' 5'

N.~

.. 1o, < ~1;1.t" lO diff~renl N=tU'CH. be-!ns ¡a) l'i'H;-: ¡b) /'1,:1'" /'10;.

The 'Y value for biomass. (NH! as a N-source) foUows as 1 x 4 + 1.8 x 1 + 0.5 (-2) +0.2 (-3) = 4.2. Similarly. the degreeofreduction OfOl will be - 4. for me olher com· pounds y=O. e..g. for HCO:;- y = 1 x 1 + 1 x 4 + 3 X(- 2) + 1 =0 aud for NH: the valueof y = ~3 +4-1 =0. TIris gives foc tbe degree ofreduction balance: - 5.8.:15 X 2 ~ 4d+4 .2 =0_

lt.is seco that in mis balaDl_"'e oniy tbe smic:b.iometric coefficienrs ofsu!> strate (or eJectron donar). me electron acceptor and biomass occur. TIlis gives d = -1.857. being identical lo the full solution ofeonservatiOD eODstraims obtained before_ The other coefficients follow from appücation of (be regular conservauon constraints. i.c. the N-soutce coefficient fl"Om the N-balance. me HCO; from the C-balance etc. From me example severa! po¡nts mu!íI: be stressed: • the balance ofdegree of reduction specifies atways a linear relation between the stoichiometric coeffi.cients ofelectron donor, electron acceptorand biornass, making this relation extremclyuseful in prarnce; • the bala nce ofdegree ofreduction is not a newconstraint. it bjusta l>uirable combinatio," oftheC. H. N charge conservation constraints.

Degree of reduction

+6 +8 +8 O +8 +8

SI

Other useful applications of me conservatiotl constraims are outlined in tbe references and indude: • selection ofthe yield measuremenlS wbicb provide the least errors i.n tbe calculated otber yields (dlle ro e rror propagation in the meas' uremcuts); • improvementoftbe elTon in all yie1ds by measuring more than the minimal required yields (measurementredundnncy allowing data reconciliation); • use ofredundant measurements to investigate l he occurrence of systematicmeasure.ment errors 01" errors i.n the system definidon (e.g. a product has been forgatteo).

3.3

Stoichiometry predictions based on Gibbs energy dissipation

A number cf methods have previously been proposed to estimate biomass yields fl'ox) from corre.1atioDS. Aparticularly sitnple but useful and recent method has been the thermodynamically based approach using Gibbs energy dissipation per unit biomass (l(YGX) in kJ perC-mol X. This is a stoichiometric quantity which can ¡similar to che. biomass yie.ld YDX on e.lectron donor as in Eqn j3.3)1be written as

_1_ = _'_ + me Yex

y~x

(3.4)

¡J.

mG is the biomass specific rate of Gibbs energy dissipation fur maincenance purposes in kJ per C-mol X h and y~ is me maximal bioOlasS yield on Gibbs energy in Cmol X kJ- l. Eqn (3.4) show$ that the Gibbs eneTg)' dissipation contams a growth and a mainte.nance re.la ted termo Simple correlations have becn propased for 1{yGX' and for me (see Further reading. Section 3.5). Thcse correlations cover a wide IdIlge of microbiat growth systems and temperatures (heterotrophic. autotrophic. aerobic, anaerobic, den.itrifying growth systems on a wide rangeorC·sources. gTOwth systems with and withoutre.versed electron

transport - RE'I).

3.3. 1 Correlation for maintenance Gibbs energy The following corre1ati on has becn fouud to be valid for maintenance .

Gibbs cnergy Illc=4.sexp [

(! -.)_)]

690.00 8.314 T

(3.5) 298 This correlation holds for a temperature range of5 to 7S OC. for aerobic and anaerobic conditions. It does not depend an tbe C-source or electron donor OT acceptOr being applied and only shows a significaD[ ~m­ perature effec[. This see.ms IOglcal beca use maintenance only involves Gibbs enerID'. irrespective ofthe electron dOllorfacceptor combination which provides this Gibbs energy.

STOICHIOMETRY AND KINETICS OF GROWTH

3.3.2 Carrelatian far Gibbs energy needed far grawth For the growth-rela ted Gibbs energy requiremenr lfYGf. the following correlations can be used: Por her.erotrophic Ol" autotrophic growth witbour RET: 1 Yg';" = 200 + 18(6 - c)U + exp[({3.8 - 'Y.)2r l ~{3.6 + OAc)[

(3.6a)

Por autotropbic growth requiring reversed dectron transporl : 1 """iiii=3500 Ye<

(3 .6b)

Eqn (3.6a1 shows thar l fY(;'X" fur heterotrophic growth is mainly determined by lile carbon·source used. This C-source i5 characreri5ed by ils degree of reduction. 'Y.> and (he number of C-atoms {parameter el per mole ofe·saurce. Eqn (3.6a) shows that lfYr.x ranges between about 200 and 1000 kJ af Gibbs energy requirement per Cmol biomass dependenr on the e source use
5:

Example: Occurrence ofREf in autotrophic growth Consider the aUlotrophic aerobic microbial growtb using Fel+/FeJ + as electron donar. HCO:; is the C-source. This allows tbe foUowing biomass formation reaction te be drawn up where HCO:; is reduced using the donar elfftrons: HC03" + 4.2 Fe H

+ 0.2 NH,t + 5H+~ 1 CHUOO~II,j. +4.2 FeH + 2.5

H,D UsingTable3.1 one can calculare lhat AG~J = +454 kJ. Clearly. for lhe eJectron donor Fc~ -t-fFfi1-r REI is needed and Eqn (3.6b) applies.

3.3.3 Stolchiometry prediction using [he Gibbs cnergy correlations 111e correlations found ror m G and 1fY'G:' in Eqns (3.5. 3.6a, 3.Gb) Lan easily be used to estimare. fOT each microbial growth system, the como plete stoichiometry ofthe growth equation as a fimction of: • the C-source applied; • the electron donorJacceptor combination; • growth rate and teroperature. It has been shown thar for a wide rangeofmicrobial growth systems me estimation of YDX is possible in a l-ange of O.oJ ro 1 Gmol X per C-mol donar with a relative accuracyof ahout 10 to 15% (see Furtber reading. Section 3.5). 111e calculation of the complete stokhiomerry is best shown using un exarople. Example; Estimation of growth stoichiometry using the Gibbs eoergy correlations Consider the aerobic autotrophic growth oí a mkro-o.rganísm using Fel"" ro Fel+ as electron donar at SO gC. agrowth ra(e ofO.Olh- J , using NH; ' as N-source and growing al pH = 1.5. Ir is required ro calculate the complete growtb SloichiometIy. The folJowmg stoichiometric equation can be specified:

+aHCO; +bNH; + cHp+d02 +eFe H + 1 CH,.lPo.,No.z+JF¡:l-t- +gH'"

+ lfYal( Gibbs energy.

We can specifY six consewation constramts and one Gibbs energy balance ro calculare the seven (a to g) unknown stoichiometric coeffi· cients. Using Eqn(3.4) lfYcxfoUows from the correlarions (knowingthat RET is ¡nvolved, tbat p.=0.01 h- ¡ :l.nd thal r=323 K) as: . 38.84

1fYcx = 3500 + - - = 7384. kJ/C- molX

0.01

The- six conservanon constraints and the Gibbs energy balance (using 6G~"-va.lues froro Table 3.1) are as follows: C
a + l : : 1t O 3a+c+Zd+0.5=0 - 4d+e + 4.2=0

STOICHIOMETRY ANO KINETICS OF GROWTH

IrOI1·conselva tion N-conservation Charge-conservation

55

~+f"" O

b+O.2=O -a+b+2~+3f+g=0

Gibbs energy balance (- 586.85)11 + (- 79 .37)b +(- 237.18)c + (-78.87)e + ( -67) + (- 4.6lf+ (- 8.541g+ 7384 = O Iris noted thar forH t ~Cri s recalculated from pH =7(in Table3.1) te pH = 1.5 (ch ange from - 38 .87 to - S.54 kJ molH tl Also the balance of degree ofl'eduction has been used as a canstraint (:replacing theH-constraintl. After solving {he six equations ane obtains the complete stoichiomercy.

3.3.4 Algebraic relations to calculate stoichiometry Because al1 the stoichiomerric coeffidents are, through the conselVation conmaints, relatcd ro llYGX ' one can derive also algebraic relations, between lfY.x:and l/YC;X' For che biornass yield on electron donor yDX the following relatiOll is obtained as an cxample (see for additional relatioas Further reading, Section 3.5):

( - AG",,) 1 /Ycx + 1'~/"tn( 6.Gc..d

(3.7)

6.GCA1. is rhe Gibbs energy of the catabolic reaction of 1 C-mol organic electron donar or of 1 mol inorganic e1ectron donor in kJ per {C}-mol donar. 'Y~ and 1'u are che degree of reduction for biomass and e Lettron donar (per moL or C·mol). For the previous example the catabolic reaction of 1 mol dectron donor is Fe2 1• + y~ 0 1 + H+ ---t Fel+ + Y,¡ Hp. Using thevalues of6.Gru1in TabLeJ.l , and usingforH+ theaGfvalueatpH= 1.5 of -8.54 kJ/moL leads to 6.CCAT = - 35.78 kJ per mol FeZ' . In addirion 1" = 4.2. 'YI) = 1 and l JYc;x = 7384 kJ per C-mol X giving Yox = 0_0047 Cmol X

per mol fe2+. showing thar the stoichiome tric coe.fficiente in tbe previous example equals 215mol Fe~'" perCmoL X. Eqn (J.7) sbows that • YDX increases byperboLically with increasing Gibbs ene[gy productiOD in catabolism (- aG~T)' Thís explains why anaerobic growth systems (with low (- .ó.CC\:l'rvalues) have lower Yox values as aerobic systems . • Yox is higher for systems with lower lfYGX·values (as found for high specific growth cate p..lowtemperarure, favoulOlble C·source arrd the absence ofRET); • YDX depends byperbolically ODIL lsubstitute l{Ycx usingEqn (304)] due to maintenance effects in agreement with Eqn (3.3) ; • Yox has a theoretical maximallimit from me 2nd Jawoflhermodynamics (because according to tbe 2nd law 1{YGX has a theoretica.1 minimaJ value ofO kJ perC-mol X) of 1'r/ Yx'

3.3.5 Heat aspects Finally the aspect ofheat merits sorne thougbts. l /YHX (=kJ beat per C-mol X) follows by calculating (- aH:) of the

-'

.

~

¡.

e zo

... e

t.

G

¡.

'"10

e

D

•

E

,,

u

? 2

56

HEIJNEN

complete stoichiometricgrowth equation where 1 e-mol biomass is produced using MI¡'values from Table 3.1. Sorne relevant remarks: • For aerobicheterotrophic growth heat production is closely related to O[uptake. where 1 mol 02 =450 kJheat; • There are microbial growthsystems where hearuptake (not praduction) can be calculated to occm; • An example is the growth of methanogenic bacteria which split acerate to CH4 and CO 2 .

3.3.6 Limitation of the yield prediction using the thermodynamic approach The presented method provides Ysx-estimates where the biochemical details of metabolismo characteristic for eam micro-organism, are neglected. This is the attractive aspect of this method, beca use this knowledge is ofren not available. However one should always realise rhat differences in biochemistry are relevant. For example. ethanol fer· mentatioo from glucose 15 performed by Saccharomyccs cerevisiaewith Ysx measured [O be around 0.15 Cmol X per Cmo] glucose. A similar Ysx value ls also obtained using the above methad. However Zymomonas mobilis al50 performs the ethanoLfermentation butwith aYsx = 0.07 "Ole clifference is caused by a different biochemical pathway (glycolysis versus Entner-Doudoroff route, see Chapler 2). This example shows that, ifthevalue ofthe estimated Ysx deviates strongly from a measured Ysx ' o ne might expect that an unusual, perhaps new, biochemical pathwayis be.ingusf'd in catabolism (or anabolism). 0

3.4

Growth kinetics from a thermodynamic point ofview

Growth kinetics are generallycharacterised by the two parameters J.I.",n and K.o Iris known thatK, values can bevery different, dependent on the occurrence ofpassive diffusion, facilitated transporror active. transport for the transfer of electron donor (substrate) iuto me micrQ-Organism. A general thermodynamic correlation for K, is therefore not possible. Also for P-max a very wide range (0.001 to 1 h - I ) ofvalues is found, depending on the microorganism and cultivation conditionso However, itwould Seem reasonable to expect that a low maximal spf'cific rate of Gibbs energy production from catabolism leads to a lower rnaximal spe<:ific growth rateo Using thi5 concept of e.nergy limitation one can derive thf' following expressions for the maximal specific rate ofGibbs energy production q~ (kJ per Gmol X h). (3.8)

This relatioo is base
STOICHIOMETRY AND KINETlCS OFGROWTH

• A temperature effect 00 this rate accordiog ro anArrhenius relacon witb an energy of activation of690oo J per mol (equivalent to the tate doubling for every 10 "C inccease in temperaturel. R is tbe gas· constant (equal to 8.314 J per mol K): • The maximum rate ofC3rabolieGibbs energyproduction q~"" is then [he rate ofelectron transpoct multiplied by(~.6..Gc:.uIi'D) ' which 1S the cataoolk e nergy release pe.reLectron in tbe electron donor/acceptor reaction. Equating che maximal rate of catabolic Gibbs energy production (Eqn 3.8) to the Gibbs energy oeeded for growth under maximal growth rate coodition (being equal to the sum of IJ.J~;n. and maintt'nance beiog equal [04.5 x tempera ture coIT«tion,see Eqn (3.S))gives tben tbeP,mu' valne(io h - I ) according ro Eqn (3.9):

IJ( 1-'.......

6CCAT) I')'¡) ~ 4.5J f'X [ 1 fY~;l P

-

(!- ~~)l

69000 R T

298

(3.9)

Eqn (3.9) can be shown toprovide rea.~onab l e estimates of JLm ....·values for a wide varieeyofmicro-organisms (e.g. nitrifiers, methallogeos, heterotrophie aerobes). A final aspect to be discussed is the occurrenee ofso
3.5 I Further reading Baltley, E. H. (1987). Energttlcs ofMicrobial Growth.John Wiley and Sons, CJ:¡kht'.!ltu. He.ijnen.].j. and van DiJken.J. P. (1992). In search ora thf'l"lllodynamlc descrip-

tion ofbiomass yie1ds for the chemotrophic growth oflllicroorganism s. Blotechnol. Bioeng. 39. 833-858. Ht'ijnen,J.J.. van LDosdrecht. M. C. M. and Tijhuis. L. ('1992). A black bol! mathl!'"" matical.nlodel to caleulate aUID- and heterotrophic biomass yields based on

Gi bbs e.cergy dissipation. Biottrhnol. Bioeng. 40, 1139-1154. Roels.J. A. (1983). En<'Tgfficsand Kint'tio" in Biotahnology. Elsevier. NewYodt.. Tijhuis. L., vac Loo5drecht. M. C. M. ¡¡nd Heijnen.J. J . (1993). A [hermodynaml· cally based correlation formaintenam:e Gibbs energy requiremt'nts in aerobic and illlaerobic chemotrophic growth. Blokthnol. Bioeng. 42, 509-519.

Si

S8

HEIJNEN

van de r Heijden, R. T. J. M., Heijnen, J.]', Hellinga, c., Romcln. B. and lUybt>ll. K. Ch. A. M. (1994).linearconstraintTelatiamin biochemkalreartian systems: 1. Classif'iration ofthe calCU1.1bility and the balanceabilityof conver· sioll rateo .lI!o r~ch7l ol. Bloe-ng. 43. 3~ 10. van der Heijde n, R. T.J. M.. Romein, B.. Heijnen,J.J .. Remnga. C. and Luyben. K. CtL A. M. (199-4). Linearconstraint relations in biochemical reartion systems: Ji. Diagnosis and estimation of g~ errars. B!orechnoL Biorng. 43. 11-20. van der Hcijden, R. T. J. M.. Romein, B" Heijnen,j.J.. Rellinga. C. and Luyben. K. Ch. A. M. (1994). Linearconstraint reJatiOTU in biochemicall?lct:ion systems: 10. Sequen tial application of data reconciliation for sensitive defection ofsystematic enoTS. BiotertlTlol. Bioeng. 44. 7Bl-791. von Storkar, U. a nd MatisOD, 1, W. (19B9), The use of cal onmeny in biotechnol· ogy.t\d.... Blocru-m. E"8 . .lIlotechnol. 40, 93- 136. Westerboff. H. V. and va n Dam , K. (1987). MlJSaiC NOTl~'1uilibrium Thermodynam ics and rhc Control of8ioJoglcaJ Fm CTlergyTrUTlSdlóC!:wTI. El$evier. Amsterdam.

Chapter4

Genome management and analysis: prokaryotes Colin R. Harwood and Anil Wipat lntroduction

Bacterial c.hromosomes and natural gene transfer What is genetic engineering and what is ¡tused for? The bas ic OOols oC gene tic engineeriog Ooning vecto.rs and tibraries Analysis o f genomes/proteomes AnaJysis ofgene e..''{pression Engineering genes and optlmising products Proouction of hetl':rolagolls produC'(s In sl1ico analysis ofbacterial genomes Further rt'ading

4.1 I Introduction Gene manipu latioo is nowadays a eore technology u sed for a wide variety ofresearch and industrial applkations. In addition to representing an exttemely powerful analytical too1, it cm be used ro: (i) ¡ncrease the yield (ami quality) of existing products (proteins. me tabolites or even whole ceDs): (ii) improve che characteristics of exining products (e.g. proteio e ng ineering); (iü ) produce existing prodUCES by new routes (e.g. patbway en ginecring): and (iv) deve10p novel produces Dor previously found in Natuce. ln this chapte.r we assume a knowlcdge in the

reader of the basic structure and properties ofnudeic acids , m e organisatiOll of the genetic information into genes and operons amJ the mechanisms by which bacteria transcribe and translate this encoded infurmation f Ol" the synthesis ofproteim (see abo Chapter 2).

Bacterial chromosomes and natural gene transfer 4.2. 1 Bacterial chromosomes Chromosomes are tbe principal repositories of the ceU's genetic information, m e sire of gen e clq'ression and the vehide of inherita nce. The

60

HARWOOD ANO W!PAT

Genetic. element

Size range

Transposons Plasmids Prophages

800 bp te 30 kbp I kbp to ISOkbp

3 kbp

Bacteriophages

Bacterial c.hromosomes

''''' bp.

nlJcleolid~_ lx!sc

'o

300 I
pair(s): k. lhouu nd ; M. minian; !ll. nuclemide(S) .

teno cruomosome, meaning dark-straining body, was originally applied to the structures visualised in eukaryotie organisms by light mieroseopy. This tcnn has now been extended to deseribe me physical struetures thar enrode the genetic (hereditary) info[mation io aU ol'ganisms. lbe (erm genome is used in the more abstraet St'n5e to refer to the sum total of (he genetic informatian of an organismo The tenn nucleoid is applied ro a physical entity tha( can be isolated from a bacterial cell and mat contains the chromosomc in association with other eomponents including RNA and proteio. In addition to tbe main enromosome, other discrete types of replicating genetie material have been identified in bacterial cells, indudíng transposable genetk elements (transposons), plasmids and proviruses. The usual size ranges of me various genetic eJements found inside bacteria! cells are shown in Table 4..1_ The genetic material of bacteria consirts of double-stranded (ds) DNA. The nudeotide bases are usualJy unmodified excepting for the addition ofmetityl residues tbat fundion to: (i) identifY the 'oJd' (con· served) DNA strand foUowing replicarian; (n) protect the DNA from the acnon of specific nudcases; and (iül synchronise certain cell cycle events. Many viruses also use dsDNA as tite genetic inforrnation (e.g. T-phages and lambda). while others havc single-stranded (ss) DNA {e,g. <\lX174 and M13I, ssRNA (e.g. MS2) or dsRNA (e.g. rotoviruses). Microbial chromosomes rangc insize overseveral ol'ders of magnitude and vary in number, compositioll and ropology [rabIe 4.2). Ge:nome sizes tend (O refiel,..'t tbe organism's structural eomplcxity and JiCe style_ Obligare bacterial parasites, such as Myroplasma genítalium (S80 kbp), tend to have sOlall genomes, while bacteria witb complex Jife cydes, such as MyXOCOCCII5 xanthlls (9.45 Mbp). tend to have Iargc: genomes. The genomes of many eubacterial. archaeal and simple eukaryotic micro·mganisms have been completely scquenced. The chromosame af Escherilhla roli is typical ofmany eubacteria . It weighs S femtigrams (SX 10- 15 g), is 1100 ¡.un in length and its 4.6 Mbp ofDNA codes ror about 4400 proteins_ The chromosome has a single ser of genes (excepting for those eneoding ribosomaJ RNA). At least 90% or the DNA encodes proteins/polypeptides whilf' Lhe remaining 10% is use
GENETlC ENGINEERING: PROKARYOTES

Organism

Type

Number Size

MS2 <j> X 174

bacteriophage bacteriophage bacteriophage bacteriophage eubacterium eubacteril.m eubacterium eubacterium eubacterium euoocterium eubactenum archaea

1 1 1 1 1 1 1

lambda T4 M ycoplosmo geniwlium Borre/io burgdOt(eri

Campylobocter lelunJ Rhodobaclff sphaerOldes Bacillus sub61is Esch erichio wli

2

Myxococcus xonthus Methanococrus jonnaschii An:haeog/obus fUlgidus arcnaea Schizosoccharomyces pombe eukaryote

3

Socchoromyces cerevisioe

16

eul
3.6 knt S.4 knt 485 kbp 174 kbp S80kbp lA Mbp 1.7 Mbp 3.0 Mbp + 0.9 Mbp 4.2 t1bp 4.6 Mbp 9.45 Mbp

1.66 Mbp 1

Nucleicacid Topology ssRNA ssDNA dsDNA dsDNA dsO NA dsO NA dsD NA dsDNA dsDNA dsDNA dsDNA dsDNA dsONA

2.8 Mbp 3.5. 4.6 and 5.7 Mbp, dsDNA total I 8.8 Mbp 0.2 to 2.2 Mbp, total dsDNA 12.43 Mbp

""""

drcular linear linear linear circular linear circular 2 x circular circular circular ND

circular circular linear linear

ND. n"t dotnrn l!l€d: dsoHA. double-nrandw ONA; ."ONA. singJe.m:mded ONA; bp. nucJ~O\"td e biLSt ra.!r(s): M. million: nL

nud<'Otid~I):

t , thou!:md.

function . Genes of re.lared function are often. but nOl always. dustered rogemer 00 tlle chromosome. Proteio roding sequences cao be on either strand ofthe DNA. although there is a preference for an orienta· tion in the dire<:tion in wblch tbe DNA is replicated. Gene expression involves two distinct. highly co-ordinated processes. The ONA is firSlly transcribed by the enzyme.RNA polymerase into messe.nger (m)RNA, 3n lLDsrable molecular species withha1f.lives.li.e. the time taken for halfof the RNA ro be d egraded) thatare m,easured in minutes. Even as tbey are being transcribed. ribosomes Oarge nucleoprotein complexes) attnch ro spedfic sites on the mRNA, tbe ribosome binding sites , and translate the en coded inrOl'mation inro a linearpolypeptide. To enable bacterial cells (O regulate gene expTession, the DNA is organised into transcri p· rional unilS 01' operonswitb di.stinct control sequences and transcriptional and translational start and stop points (Fig. 4.1l. The E. roli chromosome repLicates by a bi-directional mode &om rile origin of replicaDon (oriq ro the terminus (teyq, primarily using lhe enzyme DNA polymerase m (Fig. 4.2). The rate ofreplieation (at 37 0q is abou ! 800 bases persecond. and consequeo dy ir takes approximately 40 minutes to replicare the entire chromosome. Sine!! E. cofj ean divide by binary fission into two similarly sized cells every 20 minutes when growing on highly nutritious culture media, tb e chromosome of a single cell mayhave multiple sires ofreplication .

61

62

HARWQOD ANO WIPAT

Schemaclc dia&r=' IHustnrJn& me. k.y c.omponents al ¡¡ bacterJaI ~ron. The ¡¡~tl'ator and reprenor blnding~¡te$ are cofltrol tites at whido!.he frequency of trnn5~ripllon Initi.ttion are concrolled. Altl\ough

SQrtalld

$lOP ~odon1.. ¡¡!"Id ribosome

bindlnt sites ClIn be r~01ed in the DNA ~u.mce, chey are only functlonal In themRNA

/

Promot8\

IJ" t ~

RNA polyrnoraso Repressor binding sita (operatorl

Transc:riptian tar7nmor

DNA~==Jlííí¡¡¡¡ll'Jl=liim~l=liilriiígl!!Ü= ! Activator

blndlng sila mRNA-

¡

••

~ • Start codon

Polypept ide A

¡

TRANSC RIPllON

¡

TRANSLATION

it

Polypeptide

B

a¡¡" Polypeplide

e

... Stop codon • Ribosome binding site

The bl...:llre ctkmal

,.,

replkatien oF!he oocrerial

c.hromosorne. Repfi~tIon is Inltlat.d at the erigin 01 .-.plication

(orlC) ~nd i5 completed Jt me

ta'"

u:rmlnlIs (terC). (:al In slow growll1g celh (doubling time:> 60 mm), each side of the c.hnJmCHOme has JUSI one repllu.tlon forle or slte of ONA synltletl:$. (h) In rapidIy ¡row'''l

Aepllcation fork

cell$ (doub~ng tilT"l'e - 20 mln). a n_ round ofrnplicadon Is iMllted befare che prev\ous Orle has (b)

rnadJed che ttrminus. Con~equendy.

ud! side<Jt" tI1.

chn:Jn1O$l)fIIe has

repliCillion fork.

mo~

INn ont

¡gr-:. - O - § O § § @- ao - - @ o @ @

4.2.2 Mechanisms of gene transfer lbe abi lity to engineer ebanges ro the cbaracrenstics of a bactenum dates back ro 1928 and lhe experiments ofGriffith. on the mode ofiofection ofStreptococcus pfleumoniM_Griffitb ohserved that ofthe two colonial morphologies exhibited by tbe:se bactcría on salid media, rougb and smooth, only the laner was ahle to cause: infection in mice. The rough and smoorh characterisrics (pbenotypes) were due to the

GENHIC ENGINEERlNG: PROKARYOTES

absence or presence. respectively. of a polysaccharide capsule that enables the bacrerium to avoid the bost's immune response, Griffith was able to show tb 150 kb) have been found in rcprcsentati\'cs of a numberofbacterial genera, induding Agro bacterfu m. Pseudomonas a nd Sn-epronlyces. Plasmids may account for between ">0.1 to about 4% of !:beir host'S genorype. although in race cases thismay be as rugh as 20%. Plasmids such as tbe F plasmid ofE colí tbatconferfertilil;Yon thcir hostcells are referred to as conjugative plasmids. Bacteria l conjugation involves (he tr:ansferofDNA from a donor to a

I

6J

64

HARWOOD ANO W IPAT

(,(

Sthematic ditgr.lm Dlustrar~

Plesmid ONA

me r-pHcation an
O"an
Con;ugatlve plasmid

conjvgatlon. (a) The<:ell onvelopes of donor 100 retip)lIrll , eal, make

oril; conjugal transfer origfn

c:onu« md thelr cytop lasms ar. Joined by ¡ tr¡nÚocatlon pare.

n.

c:onjugation-specific tr.Inmr orl¡in. orlT, inleracu wh:h mis port. (b) ~ pbsmld repliQw from ollTby a mode thndlrecu Orle oflfle orl¡inal itnmds of tts ONA lnto me redP; Mt, wherea compl ementar)' $(nnd is liynthesised. n.e omer ~ nnrod of thto! pb.lmld remall'\s lo dae donO!' whre It lOO has a complementar)' Slnmd syntl'ltilsed.

Q (b)

,~I-_ DNA

potvmerase

Donor

Re cipient ..

DNA polymerase

redpient 001 (Fig. 4.3). Usually this is plasmid DNA. although o<;cas1onal1y it is a pan of the donor's chrolUosomal ONA thaLis trausferred to the redpient. The machinery involved is almost exclusivcly encoded by the conjugative plasmid, tbe main exception being the enzymes involvoo in DNA transfer replicanoo. The transferred DNA is always in a single-stl.mded form and me complementary strand is synthesised in the recipienr. The tr.msfer ofhost chromosomaJ genes usually occurs at ~uencies that are very much lower than that of plasmids. although sorne plasmids are exceptional in bcing able to mobilise host chrOffiOS
GENETlC ENGINEERlNG: PROKARYOTES

the basis of cloning vectors. the mob genes are usually omined as a requirement ofeontainment regulations designed toavoid the dissemi· nation oftbe cloned genes to wild·type populations. Althougb transfer between bacteria! cells is rhe most common type of conjugation. conjugative transfer between bacteria and fungi 3Dd between bacteria and plants has .lIso becn demonstrated.ln the larrer case. strains of Agnlbacterlum tumefaciens with large (> 200 kbp) tUillour' inducing (Ti) plasmids can transfer part of their plasmid DNA - the socalle
4.3

What is genetic engineering and what is it used

for? Ihe al:lility ro manipulate and analyse DNA using genetic enginee.ring rechniques (recombinanr DNA technology) was foreseen in tbe mid 19605 and carne tofruition in the early 1970s. The technology. which is still developing rapidly, evolved from a series of basic studies in the interrelated disciplines of biochemistr)' and microbial genetics. Key amongst these was the elucidanoo ofme molecular basis ofbacreriaJ renrietioo and modificadon systems I:ly Wemer Arber that SUb5equently provided enzymes for cutting ONA ar precise locations (target sites). These restriction endonudeases (restriction enzymes) were quick:1yexploited for the anal}'llis and manipulation ofDNA molecules from a variety of sou.:rces. From these relatively modest beginnings . techniques fur manipulating and analysing both types ofnudeic acid (ONA and RNAI have become remarkably powerful arul sensitive ... ided by the developmentofkey technologies such as DNA sequencing, oligonucleotide synthesis and che polyme rase chain reaction fPCR). A( the sametime. the provisionofchemica!s. reagents and equipment ro facilitate this technology has become a mulri-million doUar industry. The advent ofrecombinant DNA (echnologies led to (he realisation thatDNAeould be analysed to aresolution thiltwas unimaginable only a few years before and consequently rhe genomes of almost any organ· ism. prokaryore. archaea 01' euk.uyote. could be manipulated to di:rect the synthesis ofbiological producrs thar were nonnaliy onIy produced

6S

66

HARWOOD AND WIPAT

~ ..

-

• • • • • • • Passenger DNA

Sc.htrnatic di3gnm

iUustratlng tIle buic concept of

genetlc enzinHr;"" using

I paullnlf!r DNA.

Digestie n el pa5S enger

l igatioo of YeCterlO pa!lSooger

I DNA wilh a restrldie n 1 endonucll.asa IhM , generale& overtulOgs

ONA

Uneliriaallon of vector DNA w;th the urne rllstric\¡on

&noon llcleue USQd 10 digest Ihe paue nger DNA

Recambina"! vecto r wilh clon&d pa5Setlger ONA

by othcrorganisrns. This technology, illusU"ated in Fig.4.4. has nor only facilita te
4.4 I The basic tools of genetic engineering lbe techniques for isolating. I.:utting and joining molecules of DNA, developed in the cady 19705. bave provided (he foundations of OUT current technology foT e ngineeling and analysing nucleic acids. Tbese. allow fragments ofONA from virtually any org;:¡nism to be doned..in a bacterium by inserting rhem into a vector (carl)'ing) molecule lhat is stably maintained in tbe bacteria] hos!.

4.4. 1 Isolation and purification of nudeic acids Biochemical techniq ues for preparing Iarge qua n tities of re lati\'ely pure nucleic acids from mkrobia] ce.lls are an essentiaJ prerequisite (or in vifmgene technology. The firsrstepin the ¡solarion of nucleic acids is!he mechanical Or e nzymatic disruptioD of the eeU ro release the intracellular components {bar indude the Ducleic acids. Once re1eased from the eell, the nudeic acids mustbe purified from o thercellular componen.r:s such as pl'oteins and polysaeeharides [O provide a substrate of appropriate purüy for nudeie aeid modifYing em:ymcs. '!be released nudeie adds are recovered using a combina non oftec.hniques induding centrifugation . electrophoresis. adsorption to inert insoluble substrare Ol" by precipitation with non-aqueous solvents.

GENETIC ENGINEERING: PROKARYOTES

4.4.2 Cutting DNA molecules Thc abiJity ca cut molecules oto NA. ei lher randomly or atspedfic target sites. is a requirement for Ulany reco mbinant DNA temnlques. DNA may be deaved using mechanical or enzymatic methods. Mechanical shearingresulr.s in thegene.rationofrandom DNAfl'agmenn which are onen lLsed for tbegeneration ofgenornic libraries (Section 4.5.5). When DNA molccules are mechanically sbeared ie is not possible to ¡solat,.. a specific fragment containing, for exalllpLe. a particular gene or operon. ll contrast, when che DNA is cutuslng restriction endonucleases whicb recognise and cleave sped.fic target base sequences in double-stlOmded (ds)DNA, specmcfragmems can be isolatcd. Restnction endonucleases cut the pbosphodiester bac:kbone ofboth strands ofthe DNA to generate 3'-OH and 5'-P0 4 [crrniDi. Several hUlldred restrictiOll endonudt.'ases have been isoLatcd frOID a wide vatiety of microbial spedes, and thf'ir numbf'rs continuc to grow. Different dasses of restriction codonucleases witb distinct biocbemical properties have beeo identifled. witb the type n enzyme:s being themain dass used for genetic engineering purposes. Resu'iction endonucJeases are named according to the species from which they were originaliy isolated; enzymes isolated from liaemopllilus f!lflucnzae are designated Hin, those fi:orn 1!aci1!us f!!!!)'IDlfq ue[adens, &1m etc. Whenmore tban one type of enzyme is isolated from a particular strain 01' species, tbe strain and isolation number(in roIDan nurncrals) are added to the name. Thus the three restrictioo endonudeases isolated from H. infiuenz.ae strain Rd are designated Hi1!dl, Hind ll and HindIII. The targetrecognition sequences (restriction.sites) oftype II restriction enzymes are usuallysbort. typically four lo six b
4..4,) Joining ONA f.-agments DNA molccuJes with either bLunt ends 01' with compatible (cohesive) ove.rlapping ends maybejoined logether il! \litro using specific 'joining e nzym es' called DNA ligases. These enz)'mes catalyse tbe fonnation of phO$phodiester bonds becwt'en 3'OH groups a.l the terminus of orre strand. Wilh the 5'PO" te.-minus of another mando The DNA ligase encoded by bacterioph age T4 is universaJly used for joining DNA

67

68

HARWOOO ANO WIPAT

ft.ewlc;don endonucleue d.,3Vlgot 01 moll'CuleJ of DNA It ~C target Jro:J te genlffilU!)' or 5' overhangs (QW!rl.tp~). or blunt

5'1llf4~3'

S'~ 3'

3,.gwMiS'- 3'1i"~"'''' ·'<''""''''s'

5'M.w.t~3'

3'~5'

...

ends.

EcoRI cuts asymmetrically leaving 5' overhangs

Pst I cuts asymmetrlcally leavlng 3' overhangs

s'at'í• • •b 3',,~5'

...

-

s'NA'II3' 3'.lls'

Eco RV cuts symmetrlcally leaving blunt ends

E"'Y"'e

Source

Recognition site

BamHI EcoRl EcoRU Ha€1II

No.

Badllus amy/oliquefadens H Escherichia coli RY I 3 Escherichia coli R245 Hoemophilus aegypfius Hoemophi/us in{luenzae Rd KJebsiella pneumomoe NOOJrdia o(Jtid;s-cUVlorum

Sou3A Smal

StaphylococOJS (JureU5 3A Serroua marr:escem

GJ,GATCC GJ,AATTC J,CC[rlA]GG GGJ,CC AJ,AGCTT GGTACJ,C GCJ,GGCCGC CTGCAJ,G J,GATC CCcJ.GGG

Hindlll

I
",,,

Providencia sruoroi

litUo:<; ~ wrillen in parcnlhelle..indinfe a.lrernative p¡'rmis~ibJ~ ba.\cs in !.he ~gnilinn sequt'.nce. St!<.lu~nc.~an Wriltl!n frnm S' In3' Qn Que !tr~ nd onJywilh!he poinlof c1NVOijie Indh:a(ed by 0111 arrow.

molecules with both blu nt-ended orcohesive ends. T4 DNA ligaseactivity requires ATP as a cofacoor to form an enzyme-AMP interm.ediary complex.. Ittben bimls to tbeexposed 3'OH and 5' P04 ends oftbeinteracting DNA molecules ro create the covale nt phospbodiesrer bond (Fig. 4.6).

Ligation reactioru usually involve joining a fragment ofpassenger DNA (that is [he piece ofnew DNA 00 be 'carned') to a vectormolecule

GENETIC ENGINEERING: PROKARYOTES

5' tnd

5'end

3' end

3' end

n

5' ene

3' end

)' end

~'

n

AMP

l iga. . . AMP l igase

::;~~ T40NA ligase 3' end

5' and

J' end

(Fig. 4.4). To increase [he probabilityofthevector attaching [O passenger DNA rather than to itlielf or another vector molecule, tbe molar ratio (Le. number rarher than mass of DNA) of passenger to vector DNA is usually about 10.An alternative strategy is to use a phosphatase (e.g. calf intestinal pbosphatase or ClP) to remove phosphate groups from the S'PO. ends ofthe linearised vector DNA. Since this phosphategroup is essentia l for joining the two ends oftbe vector togethec. redrcularisation is imposiiible. However, when passenger DNA is presento it can provide the S'P0 4 ends for ligation to the 3'OH ends ofthe vector. Thls generates a drruIar molecule with single gaps in each ofies nucleotide strands that are separated by the length of the passcnger ONA. This structure is stable enough te be transformed into a doning host where repan sysU"ms will seal the gaps.

4,4.4 The polyrnerase chain reaction (PCR) and its uses Since its introduction in the mid 19805. the polyrnerasechain I"eac.tion (PCR) has had a major impact on recombinanr DNA technology. PCR facilitares th.e: amplificabon of virtual1y any rragmenf af ONA from aboutO.2 (040 kbp insize.&cause the amplificabon reacdon iscyclical and (he concentradon ofDNA doubles al each eyde, rhe fOral amount of DNA in the reaction increases exponential1y; [he theoretical yield from each originaJ templare molerule. is abour 1~ mole.cu.les after 20 cycles. and abour 109 molecules alter 30 cyeles. PCR requires a thermostable DNA polymerase, template DNA, a pair of oligonueleotide primersand a complete setofdeaxynuc1eotide triphosphates (i.e. dATP. dcrP, dGTP and dTfP) substrates. Oligonudeotide primees for PCR are synrhesised chemically to be complementary to sequences whieh fiank the region to be amplified and are usually about 20 nucleotides in length. The primen are designed to bind (anneal) speci:fically to the opposite strands ofrhe template molecule. in sueh a way that their 3' ends faee tbe region f.O be amplified. It is the specifi.city oC the primer annealing reaetion which ensures thar the PCR amplifies the appropriare region of the template

end

T
phosphodil!!"Ster badlbone d tile ONAmd 1lAk.es a covaIent bond ~!he exposed ] 'OH VId S·PO.groUjK olll!!illm sideofthe

b...k

69

70

HARWOOD ANO WIPAT

DNA. Akeyfeat1l.ré ofthe PCR is thatthe entire DNAampli1kation reaction is carried out in n single tube containing enzyme, template, primers alld ~ubstrates. Each cycle of amplificaDon therefure involves annealiug. exteruion and denaturation reactions, eat"h brougbt about atdifferent temperarures (Hg. 4.7). Since the di.ssociation reaction may occur at temperatUTeS as bigb as 95·C. and mere may be as many as 35 cydes in a single PCR. a bigb1y thcl'mostable DNA po]ymerase is a basic requirement for PCR. Taq polymerase. isoLate
4.4.5 Transformation and other gene transfer methods The ability to introduce foreignDNAinto a bacterial cell host l¡es at the very heart ofrecombinant ONA technology. Transformation , in which the exogenous DNA is taken up by the host cell , is the most widely applied gene transfer tedmique fur doning purposes. While some bacteda posse.ss natutaJ transformation systems others, such as é. colí. require chernlcal pre-treatment to make tbem competen t for me uptake ofDNA. Although transformation is usuaUy efficient enough for most cloningpul'poses. there are sorne procedures. such as tbe generationof genomic librarles. for whic.h transformation is not efficient enough. ln [hese cases ili s posslble to cin.:umvent the transformation pl'ocedul'e by packaging Lbe recombinant DNA into virus particJes ín vitro {Section

GENETIC ENGINEERING: PROKARYOTES

The pol)'lTler.&se chaln tNetion. sl'Klwmgt;t,. cydiGI nawre of the annea~ng, i ynthesl$ and denaturadon react!on~ wh!ch a~ Cllrrled outalllDmaticaDy In a dedlcated thermoqding (PCR) machlne.

SI8p 3: DNA synthesls (Primer ext8nsion)

i

5'

•

•

IS'

,.,

~: !"o;L>.~.." i

¡¡

t:.~!""'!!i"6~·-·'·

, . .",;, . i.¡¡¡¡¡ Cyeras repealed 20-35 times leading 10 exponential doubling 01 the terget sequenee

71

72

I

HARWOOD AND WIPAT

4.5.2). More. recenrly ithas been discovered tbat bacteria are ableta ralee up DNA when given a high voltage pulse. In this process, called electroporation. mixtures of ce11s and exogenous DNA are subjected to a brief (typicallyofmillisecond duration) electric pulse ofup to 2500 volts. The high field strength induces pores to [onn in the ceH membrane, pennitting the entry of the negative1y charged DNA that is itself mobilised by the electncal gradient. In many cases electroporation is more effic.ient than transfonllation and sorne types of bacteria may only be transformed by this procedure.

4.4.6 Selection and screening of recombinants Mter most doning procedures it is necessary to scteen the resulring clones to ¡solate tbose carrying the required gene er fragmentofDNA. At the simplest leve! this may be done by selecting bacterial transformants tbar contain a copy ofthe vector. This is achieved by incorporating an antibiotic-resistance marker gene into tbe vector so that onIy transformed bacteria which have received a copy of the vector are able to grow on medi a containing the a ppropriate antibiotic. More advanced systems have been developed to allow the discrimination of t[ansiormants containing a vector with ar without a cloned inserto These systems indude the use of gene disrllption methods which result in the loss of a particular trait uponinsertion offoreign DNA (Section 4.5.]). Cones containing a specific gene or fragment can be identified directly by selection techniques 01" indirectly by restriction e.ndonuclease rnapping, PCR or hybridisation techniques. If the target gene is expressed. irs presence may be se.lected by compleme.ntation of a defeet in the cloning host (e.g. restoration ofthe ability to utilise a particular substra.te or to growin the absence of ill1 otherwise esse.ntiaJ nutrient). ln the case of restricrion mapping, plasmid DNA extracted from él. number of representative dones is digested with specific restriction endonucleases. Only dones containing the required gene ar DNA fragmentwill generate the correet pa ttern ofbands after agarose gel electro· phoresis. Restriction mapping is onIy feasible if the target clones are likely to occur at a high freqllency amongst the poplIlation to be screened. Diagnostic PCR, using oligonuc1eotide pLimers speciñc to the targetDNA sequence. may also be used ro identifYclones containing tbe required gene or DNA fragmento Since PCR may be used directly on unprocessed samples of colorues, it is feasible to test many more clones. Ifthe target DNAis likely to accur at a low frequencyin a population of clones, as would be the case with a genomic library (Section 4.5), a large number of clones need to be screened. In tbis case the methoq of choice is hybridisation of the bacteria colony that grows from a single cel! (ar in the case ofphage vectors. the. viral plaque). Colany ar plaque hybridisation makes use of1abelled nuc1eic add probes (DNA or RNA) that are able to detect the presence ofspecific DNA sequences within individual colonies oL.plaques. Biomass from individual transformant colorues or plaques is transfereed to a membrane ontowhichdenatured DNA, released by breaking the cells apen, will bind. The membrane is then e.xposed to a lahelled peohe (Section 4.4...7) which binds specifically

GENETIC ENGINEERING: PROKARYOTES

to the immobilised target DNA, revealing me idenlity of (alonies or plaques containingthe appropriate doned DNA.

4,4,7 Nucleic aeid probes and hybridisation Nucleie acid probes are osed to detect specific target DNA molecules. The soluble probe binds (Le. hybridises) to the targetDNA thatis immobilised anta a nylon or nitrocellu1ose membrane. Hybridisation is used for a variety ofbiotechnological applications induding lhe detection of cloned DNA (Section 4 .4.6), analysis of gelletic org-.misation and the diagnosis ofgenetic diseases. Althougb nucleicacid hybl'idisarion techo niques are used in a wide variety ofcontexts, the same basic principIes apply. Nudeic add hybridisatioD exploits the ability of single-stranded probe nudeic acid (DNA or RNAI to anneal lO complementary single· sll'anded target sequences lDNA or RNA) within a population of non· complementary nudeic aod molerules. The original technique, reterred to as Soutbern blotting afrer its inventor, Ed Southern, involved the size separation ofl'eSO'iction endonuclease digested fragments of DNA by gel electrophoresis. and their transfer by blotting onto nitroceUulose membranes. 111e probe nudeic acidjs applied as an aqueous solution ando under appropriate hybridisa· tion conditions. binds ro immobilised target ONA. lbe loeation of bOlmd probe nucleic acid on the membrane is indieated by tbe presence of a readily and sensitively detecte
73

74

HARWOODANDWIPAT

transcriptthat is complemenraryto the targetnucleic acid.As with the labelling ofONA. radiolabellcd nudeotides or nucleotide analogues are incorporated during the synthesis reaction.

4.4.8 DNA sequencing DNA sequencing is one ofthe mast important afthe techniques ¡¡vailable fortbe identification, analysis iI1ld dicected manipula nao ofDNA. Knowledge oftbe DNA sequences oftarger DNA molecules and doning vecmrs is fundamental to (be construction of advanced bactenal protein production sys tems. It aIso facilitates fue design of specific probes or primers and the productioll ofcompurergenerated tr.lllscription and restriction maps. The Maxam and Gilbcrt method for DNA sequcncing uses chemical reagents to bring abour the base-spccific deavage oC me DNA. Although still used for a limited number of applications, th.is che mically based technique has generally been.replaced by tbe elegant cbajnterminator rnethod developed by Sanget and colleagues in 1977. The Sanger pro<.'e dure exploits the ability ofa variantofE. roli ONA polymerase J (the so-called Klenow fragment) to synthesise a complementary strand of DNA from a single-stranded UNA template, incorporating both the naturaL deoxynudeotides and 2',3'-dideoxynudeDtide analogues. Dideoxynudeotides lack a hydroxyl group at the 3' position and are therefore Dot able to act as a substrare for further chaio elongarion. Their incorpomtion therefOre terminares me syntbesis of the DNA stl'and in question. A specific oligonudeotide primer, used fO initiate me chain elongatioll process, determines the start point ror aU of m e newly synthesised DNA molecules. DNA polymernse synrnesises DNA from a single-stranded template in the presence orall rOlLT deoxynucleotide triphosphate substrates (dATP, dCfP. dGTP, dTI'P), one of wruch is radiolabelled (e.g.la-l~l~TP).ldenrical reactions are carned out io fOUT rubes exceptingthateach rube also includes, at a lowerconcentration, one of rhe fOUT possible dideoxynucleotide ttiphosphate aoalogues (ddATP. ddCfP. ddG'IP, dd'ITP). When tbe appropriate relative concentratioilS ofnonnal and dideoxy nucleotides are used . newly synthesised DNA strands will be terminated 3t every possible base position and a ser of fragments of al1 possibJe le ngths will be generate
GENETIC ENGINEERING: PROKARYOTES

AA a\llOndlo~ of pare of l ONA nquentinggel ~rated by me Saoger cNm ttrmhmlon meehod. The I~ne$ l~ la"lIed acuordill¡ lO nud.otIdu al whlch mey l~ urmlnaeed. namely: A. aden!ne; C.

T G

A G A

me

e T

A

G G A

Directlon of elec1rophoresis

G

A

T

e e

T

A G

e

T

g~ 4.4.9 Site-directed mutagenesis Site-direcred muragenesis. the specific replacement ofnudeotides in a sequeoce afONA. is use
c)'ulllne; G. ¡uanlne; T. mymine. Tht sequen,e 5pedfied by die gel ¡, ~hown to the Ieft: of thc gel.

75

76

HAR-WOQO AND WIPAT

gene.lt is transformed iDtoE._~repair ofthe mismarches by the bost's mismatch repairsystemsis~byuseofa mutant(e.g. mutS) tha r is defective in this function. Aft:i5' ~ eac.h strand will forro a double-stranded molecu1e without mimla.lI:bes and mese will segl'egate into separate daughter cells. One molecuJewill oontain the mutations introduced by tbe oligonudeotides. whilst the other wm be ideDtieal to the original plasmid_Cells harbouring plasmids with the desired mUlation are selected byvirtue oftheir newly aequired 3mpidlIin resistance phenotype.

4.5 I Cloning vectors and libraries A c:loning vector is a molecule ofDNA into which passenger DNA can be cloned to allow it ro be replicated inside a bacteriaLhost cell. The vector and passenger ONA are covalently joined by ligation (Sel.-tion 4.4 .3). CJoning vectors have four basic characteristics: (i) they must be easily introduced into the hon bactenum by O'ansformation 01', after in vilro paekaging, by phage infection; (ii) chey must be able ro replicate in me host bacterium. prefel'd,bly so thar the number of copies oftbe vector \CQpy-number) exceeds that ofme host chromosome by between SO and 200; (jii) they shouId contain unique sites for the action of a varietyof .restriction endonucleases; and (iv) tbey should encode a means for selecting 01' screening host celis that contain a copy of the vector, Cloning vectors are derived from narurally oa:urring DNA moleculcs, such as plasmids and phages, which are capable ofrepLicating indepeIl"" dently of me host chromosome. A wide variety of cloning vectors llas been deveJoped for specific applicatloru and these are briefly descnbed below.

4.5.1 General purpose plasmid vectors General purpose plasmid vectors are designe
GENETlC ENGINEERING: PROKARYOTES

Polylinlter multiple --:::::::~¡¡;;_ ~/aC~/ Clonlng sita

pUC19 2686 bp

Multiple cloning site:

..,,,

Aval

Ec/13611

"".,

,~ ,

~"

~qt\lunc.G.\GL'1'CCG'l'.I.('Ct" GO" G ·,~~~~ ~",,~ ... t =t>¡~(C

EcaRI

Knpl

Apal

,01",,651

B~mHI

S~II

$phl

/11",,11

C-Sg,Ams..rs.. rP, aV~lArgPraA."<; luL""TM,S"",A '~Cv.AI . HlsLauS""P,aTh,lleM",-N

The generdl purpose plasmid doning vector pUCI9 i~ ~ member of the pUC ~erie~ of plasmid vectors. The pUC vectors haV'e been genernted h1 p;¡lrs that differ only with respect to thelr multlple donlng slte whlch are located In opposite orlentadon. allowlng the pusenger DNA to be located in aitherthe same dirllCtlon or opposed to me trnnscrfpdon ofthe /acZ'gene. The arrows on the 1ocZ', ampldllin reslstance (Ap) and repllcatio" protein (Ori) genes Indicate me dlrectlon of transc:ription. The sequence of ¡he multiple. doning site (capit:¡r,ls) and adjacent (Iower ClUe) sequences are

fJ-galactosidase. IfX-gal is induded in the selective agarplates, transformanteolonies are blue in the case oC a vector withno inserte
4.5.2 Bacteriophage and cosmid vectors Bactel'iophage lambda (A), which infects E. eoli. has provided the basis for the most mmmon1y used phage vectors. Lambda has been of greatest value for doning relativeJy large fragments (> 10 kbp) thar are nor easily doned by general-purpose plasmid vectoes. Lambda has a linear dsDNA genome of approximate1y 48.5 kbp in sizewith short 12-base pair singlestranded 5' projections at eam end to facilitate its circularisation in E. colí. The site generated bythe eirculansationreaction is known as the cos site (cohesive ends). In the deve10pment oflambda doning-vcetors, llonessential genes have been removed to provide space for the inserti.o n of DNA fragments ofup to 23 kbp in size. Additionally, because the phag!:' genome is only circularising upon infection afthe host. lambda vectors can be supplicd as separate 'left' alld 'right' arlllii. Each arm has an appropriare restriction endonudease site at one of its ends and the recombinant DNA is cloned between the anns (Fig. 4.10). The transformation oflambda molecules intoE. coli is relatively inefficient and this has led [O the development of in vitTo phage packaging systerns tor the efficicnt de1ivery ofrecombillant lambda genomes into their bacterial hosts. Cosmid vectol'S combine the advantages of cloning in a plasmid vector (e.g. ease of cloning and propagation) with the high effidency of delivery and cloning capacity of a phage vector. Cosmids are plasmids which have a copyofthe ros site llormally found 011 the lambda gellome.

~hown

together wlth restr1ction endonucle~se urget slte5 and transrated amlno acid,.

77

78

HARWOOO ANO WIPAT

I Simplifled map of b.lct4!lriophage lambda silOwing the left Jnd righl al'TllS 3M mi noneuenual ct!JIlr.l1 region thlt 15 c mltled rrom Iambd3_band tlcn.,g ve<:t.crs.

He"d

Ta il

genes

genes

DNA synthesis genes Non·essential genes B.g.lntegration Regulatory l ysi s genes genes alld immunity

Le'

cohesive end

"'" '''''

Right cohesive Righ1 a"" e nd

Thc prese:nceoftbecos siteel1ables [hese plasmids (O be: used in conjune= tion witb a lambda iJl \/itro pack
4.5.3 Bacterial artificial chromosomes Bacteria! artiñcial chromosomes (pBACs whcre 'p' refers tO plasm.id) have becn developed for c1ol1ing vt':ry large (> SO kbp) sequences ofDNA. BACvectors are usu allybased on che F plasm.id ofE. mil and are able to accept DNA inserrs as largeas 300 kbp. pBACs are maintained as single copy plasmids in E. eoli, excluding the reptication of more dlan one pBAC in tbesame hostcell. Ord~ pBAClibrariesofbactcrialgenomes maybeconstrtlcted in wruch theentiregenome sequence is represented by a series of dones with overlapping ¡nserl!.

4.5.4 Spedal purpose vectors In add itioll to the vectors described aboYe. a range ofspedal purpose vcctors have been developed and are described briefly below. E:c:press Loh vettOTS Expression vectors are designed to achieve high level , controlled expreso sion ofa targetgene with resultingproductionofa protein productat concentl'ations as high as 40% total cellularprotein. Expressioll vectors often incorporate a system that .adds an. affinity tag to the protein to facilitate ies purification by affinity chromatograpby. .Expression vectors are mostly plasmid-based and ofien use the tight1y controlled and highly efficientphage 1'7 RNA po1yrnerase gene expression sysrelll. The targetgene is doned downstream ofthe transcriptional (promoter) a nd rranslational (ribasome binding site) control signili derived from T7. The ve<:fOr is transformed into an E. rol! host with a chromosomany located gene encoding tbe17 RNApolymerase. Switching the polymerase gene on leads ro high·level synthesis ofthe target gent'_ Ifthe urget gene is not fused lo an affinity tag. the protein m ust be purified l1sing expensive me thodologies. However. a numberof affioity tag systems have been developed in recen[ years tbat provide for highly speciflc purification prorocols. A varicty oftags have been used, inel uding a rag of six histidjlle residucs (6 X His) that binds ro nickeL TIte

GENETlC ENGINEEKING: PROKARYOTES

CIontld proteln Add ceU lyS8.1111C GIU101hlone Sepharo.!le

C~~)

CIeave rusIcm p!CI~

wíth slte-<sped"'- Pfol6Me {11wtwnbin Ot Factor)(.al

Analyse by SOS·PAGE lo check purlty

---

-

-

'5il. lha of ¡Iucuhione S-tnamferue (GST)

Callad eluale

¡u dfinlo/tlIlI for rhe purificatlon of protein~ The targer protein is syntheslsed u an N·termilUl fwion w GST. The producer ceh are Iysed and me Clrget prntelnlGST fU$lonll"3pped on i Gkn.othione Sepharos. columr\ After elltem lve wuhlng, the target pro!t'lln 1$ r~ased fmm the column by addlng i proteiSe th~t cluve~ tite targe( prottln from m. GST molet)'.

,In

ligand is attached lO an insoluble resin and the tOlgged fusian protein recovered by passing tbrough a cbrornatographic column containing (he resin·bollnd ligand (Fig. 4.11). The boulld fusion proreinis e1uted as a virtually pOfe protein and me affiniry tag removed by trcatrnent with a chemica l or by digestion with a prorease.

79

80

HARWOOD AND WIPAT

Propertlel or ~ se<:retiQn "..ctor. (a) Str v(nre of ..

typic.J.1 EcoII slp (Clr leader) peptide requlr.d tcl target a proteln acrou the cytopl3$nlK: membraNl.. (b) Organi$;ltio" of a se
¡mmedj~n,ty

slgl\alloquence. (e} Secreti(1Il vector ..... ith m. ONA leqUef'oCe of

lal Signal paptide sirueture (-25 rasldues);

N "eH."i!:h~= l!il"''''¡¡¡;·¡¡·'f''!IIIIIII!IIl!!III....IIIil.IIII!!1i11ii1iI!llIlll• •__ e N·d ornillO

---

Potilil/&Iy·t:hllrlled IImino 8ckls (erginine er Iysine). (2- '6 <",s¡dues)

H.
C..:IomHln

Hydrophoblc amioo acid! forming lI·he/leal strul:"l.ure (>8 residuul

LeS!! hydrophobie. wilh . 19nal peptid ase re.::ogni,ion site ( ~ 8 rlsl tloosJ

lb) Socretion vector: lnéuc ibh'l promoter

che tilrgetproteio1 fu~ed ....fram. dowNlream el ,lgnal

---Ciimm;,';-i_ _llIIlHe.-

me

/

l equence.

(c)

\

Signal

Clerning

Anllbiatic

$&Quunce

!lite

."lst' nc8gl'lnll

-

Rtlpllcatiern o rigin

Secretion lIac1or wi\h insert: -Yi¡i IlIl1 WVUZUII

e

largal gene MC¡uence fus ed in·frame with thlt s ign ai sequence

Secretion Veclors Currently most systems for the production of TeCombinant protcins lead to the in tracdluLar acrumulation ofthe productoHoweyec, mm· cellular accumulation can ¡ead to lower production levels, protein aggregation, proteoJy5is and perm¡¡nent 105s or biologicaJ activity (Section 4.9.41. T.his can sometimes be oven:ome by 5ecrcting the target protein diIeclly into the cuJture medium since secrete
GENmC ENGINEERING: PROKARYOTES

Single-str.mded phage and phagemid vectors It is sometimes necessary to generate single-stranded DNA, partirularly for DNA sequencing and oligonucleotide-directed mutagenesis. Mcssingdeveloped a series ofvectors based on bacteriophage M13 ,
4.5.5 Genomic and gene libraries Agenomic übrary is a collecrion ofrecombinant dones containing, at a tbeoretical level, representatives of aU of the genes encoded by (he genome of a particular organismo ln practice, the best tbat can be achieved is a library wirh a high p robability (usually >95%) rhat a par· ticular gene will be represented. Libraries are produced by 'shotgun d orung' randomly generated DNA fragments into a suitable c10ning vector. These fragmenls may be generated by mechanical shearing or by enzymatic digestion. Librarie!l may also be genera ted using copy DNA which has been synthesised from the mRNA ofa partirular tissue or organism using the enzyme revel"Se transcriptase.

4.6 I Analysis 01 genomes/proteomes The genetic infOI:mation oC a bacterial cell is physical1y located on i[5 chromosome{s). The physical organisalion ofbacterial cbromosomes has recendy been shown [O be more variable (han was previousJy supposed. a nd both linear andcircularchl"omosomes have ~nrecognised as a remlt oCthe a.pplication of physical mapping techniques such as pulsed-field ge1 clectrophore.sis (PFGE).

111_1(l rlg ln

w-~

Tite phag~mld 15 a p1asmid ~ecr(>r u5ed for lhe generation of sl ngl,.. slrlInded DNA m ole~ules. C.ns contil(ning tlle ph~g¡¡mld:ml Infected wtth an fl 'helper· phag. tnat stim ulales the fl replkation orIgg, 10 gener;ue nO NA mal Í$ us.embled ¡nto phage partide'l and reluse r:l from me b;Jttl'!rium. Two venlons of p8!uewipt. me (-t-) or (- ) derivat.lves. ;¡JSow for rq¡lleadon of elmer the po$iti-..e o r nep~ nnnd;& required. T3 and T7 promolen either ~de ofth e pBlue~cript

IT'IU~

doning sÍle allow me

tJn¡l~" !n.nded

DNA 10 be ll'ied as

a substr¡m~ from In v/tro RNA syn~sls

utlng tl1e cognate RNA pol)'l11erase and NTP rubstntes. An am plcillin resistil nce gene (A¡.) Is used to selecl for and 10 rrntintil;n me phag.mld In E.ccH.

81

ANDWIPAT .•' _82_LHARWOOD -_ _ __ __ __ __

_

_ _ _ __ _ __

4.6. 1 DNA fingerprintinglphysical mappinglpulsed-field gel

electrophoresis Priortowhole.genome sequencing, various physical mapping methods were developed to determine tbe physical structure of bat.1:eriaJ genom es and detennIDe relatedness between individual baClerial str".uns. The latter is usefu1 fur strain identificat"ion and epidem..iologieal studies. Physical maps ofthe cllromosome can be COllstrueted by useofPFGE, a method developed specifkally to resolve very large (30 to 2000 kbp) fragments ofDNA To avoid mechanical sllearing, the DNAis extracred dlrectly from bacterial r.:ells in agarose blocks and, whcn required, digested fn sltu with restriction endonuc1eases thar cleave rhe genome sequenees infrequently (e.g. 10 to 30 times) . Tbe agarose block i..s tben incorporated into a slab ofagarose and subjccted to 3n oscillating eleerrie field. Separation is based on the time taken for rhe individ ual molecu1es of ONA to re-oricmate in the modulatillg electric field; larger molecules taking looger than smaller molecules. The various fragmClUS are aligned using ti vadety ofstrategies that ¡nelude digesting the ONA with ty,.'o rarc-cuttingenzymes, hybridisation between fragments isolared from separare digests and transformatian af pudfied tragm en ts into mutams wilh specific lesioos. Genome fingerprinting methods have been developed for examino illg tbe relat.ionships between strains for epidern.iological studics (e.g. monitoring· outbreaks oí" disease) or heterogeneity in naturdl populatioos. Tbe bandingpatternsgcucrated by restnctio n endonudeases (e..g. rcstricrion Ji usi ng s pcd6c or random o Ligonudeotide primees (e.g. r.rndom omplified polymorphic DNA OT RAPD)cau citber be used diagnostically Oi fur rcvealing relationships benveeD strains.

4.6.2 Analysis of the proteome The tenn proteome has been
GENETIC ENGINeERlNG: PROKARYOTES

proteil1s are ~aled by reacting (probing) with an antiserum wokh ¡s theo detected using asecondary antibody or pro be. The currcnl system ofchoice for analysing che proteome invoIves a combination of two-dimensiona.l polyacrylamide gel electrophoresis (2-DPGE) and polypeptide micro-sequencillg or mass spectTometry techo niques. 2-0PCE fadlimtes the separation ofhundreds ofpolypeptides extracted fr0111 whole <ells. Polypeptides are initiaUy sepaTated in the firsrdimension on irnmobilised pHgradientgels on the basis ofthe-irpL These gels are tben separatcd in the second direcdon by SOS-polyacrylamide gel electrophoresis in which tbe rateof migration is pdmaríly based on tbeir size. The rates ofmigration of polypeptides in the (wo dimensiollS are extrcmcly reproducible. Individual JXllypeptides caD be detecte
4.7

1

Analysis of ge ne expression

Promoten influence the frequenc:y of transcriptioD iniba.tion rathe.r than me rue oftranscription. and strongpromoters bave a high affinity for RNA polymerase binding. A comparison of a number of E. coH promoters has loo ro the recognJtion of a consensus promoter seqll~ce: S' -TATAAT-J ' .cenrred around 10nudeotides up.streamof(prior to) the transcriptiol1 ¡niriarlon site (- lO regian) aDd 5 '-TI'GACA-3', located about 35 nudeotides upstream (- 35 region). The strongcstpromoters are mese which show tite closest identities ro [his sequence. Additionally. the spaang between tbe - 10 and -35 regions is impor· tant the optima! being 17 nucleotides. The ability te analyse gene expression is an important prerequisite for optimising the biotechno· logieal potcntial ofbacreria and many highly sensitive and precise techo niques have been developed ror this purposc.

4.7. 1 Analysis o( messenger (m)RNA transcripts 1hree methods are used for the analysis of mRNA u-aflScripts: Nortbem

blotting. S1-nudease mapping and p.ri.mer extension analysis. The lauer two techniques have tbe potential to identif}r the transcription ¡nidaDon sitcs. Northern blotting involves lhe separation ofrnRNA species by cleclrOpboresis ttlrough agarose Ol" polyacrylamide. Formam.ide. urea or other dcnatur.mts are indudOO (O avoid the sjngle-stranded molecuIes fonning secoDdary strucrures (e.g. duplexes. loops) that might affect Lheirmobility. The separated mRNA species are transferred to activated

83

84

I

HARWOOD ANO WIPAT

Promoter

l'1ipf>lng 01 tl"al'lstrlptlon Inltlarlnn poinl$ u51ng

SI

noclUSI.

mRNA Í!5 hybrid~d lO

a dflMWred ONA rr'alrnent th;¡.t has been bbelled at iu 5'...end. The ONA fra¡rrterlt is cho~en 50 mat la S' ·.,.d b Intel'nal to the t:lrxet mRNA. whUfI th,l' -eod extenm beyond da putativa mRNA St3rt

DNAII mRNA

~....

TIE

Gene 2

Gene 1

9

.....

'i

I!

Hybridlse extracted mRNA

transcripts 10 a deneluroo, 5'-laba lled, ONA Irll9mont

5'·labelled

1

;;... _e:

'M

polnf. Thll RNAJDNA hybrid moleal~

__o

hu sift&le-stnnded

UQnsiOlU Úlatal"O!. d~raded by

1M sln¡le-smond-:;pecific anivity of

S I nuc!e¡¡s-e. TheJ'-endofthe DNA l~lIlnt is ootennlned by r unning II o n .. denaturing gel ¡pinst a DNA sequencll8 of me ongml fngmem geoer.J.ted by me Mu;¡¡m;aml Gilbert chernial d~'Bge

method.

,, , ,,, ,,

,,, ,,,

I

ElectrophoreS8

,. product on a denaturing

,

T polyaerylamlde gel

GG

ee

1

-j

nylon membranes by blotting and theu covalentlycross-linked. Specific mRNA species are detected by hybridisation (Section 4.4.7), using labelled oligonucleotide. DNA or RNA pro bes. The use ofmarkers wilh different mo!eLwar sizes allows the sizes af specific transcripts to be estimated whkh provides ciLles as ro the organisation ofthe transcripriona! unit from which rhe transcriprwas syntbesisro. Sl-nudease and primer extension analyses facilitate the identificarion ofthe 5' -prime eods ofmRNA transcripts or the processed products of primary transcripts, In rhe case of Sl-mapping (Fig. 4.14), mRNA is hybridised [O a speciflc spedes of ssDNA that overlaps tbe start afthe targct !r.lnscl"Ípt. Tbe resu!ting RNAfDNA hybrid malecule has an overll'lpofDNAat theJ'-end thatisdigested bythe singl~strand specific Sl-nuc1ease. The size ofilie processed ssDNA molecule, which is labelled al ¡u unmodified 5'-end, is determined by denaturing polyacrylamide gel elecrropboresis using a ONA sequence ladder as molecular size markcr. ID the case of primer extension analysis (Fig. 4.15). a 5' -end la belled oligonudeotide, hybridising about 60-100 nucleatides downstream ol' the predicted tr.:mscription initiation rite. is used to prime the synth esis of a ONA copy of the mRNA tr.mscript, using the enzyme reve rse tl"atlscriprase. Syntbesis ofthe complementary DNA strand terminales at me 5'-end of the transcriPl, ro genera te a prodUL1: of defined length. Again t.b.is can be sized using a ONA sequence ladder. geneI
GENEl1C ENGINEERING: PROKARYOTES

PrDm0l9r

ONAI I

5

Gene 1

Gene 2 Riiid

mRNA

, t

1/ __ J

....

¡¡

&O

Reverse transcrlptase S'-Iebelled primer

,""o:

Primer extensiOll an.alysls ola specifK: mRNA transcrlpt. An oIigonucleotide prilTlflr, radlobbelled I t iu 5 ' -ell d, ~

ilnnt!aled to extracted mRNA

about 60-100 nudeoddes down stntam ofthe putative trallKript sun polnt. Revene ~Klipase (1111 RNA-dependent ONA poIym~) alld d~oxyribonlldeotide uipho:sphaU!

(dNTP) subnn~es are added, anrl copy (cl ONA 5Ynthesis Í'litiated.

cONA synthesls termblates at the 5 ' IInd olthe rnRNA transcripr aOO tI-H! ,tu of tlle run-off cO NA prodUCt Is determined o n a sequenclng gel (Ianes I ar.d 2 o n

T

G A

GA fe

12

e G T A

e

T G

_ _

j

Denaturing polyacrylamide

9"

e

me &el) uslog 11 DNA sequence iadder cenerl ted with the same primer as I molerulu me nurker.

Tllls IUOWStÍle pretise nucJeotide II whk:h

me trans<:Tllt wu

Inltiate-d 10 be iden'tified. Prlmet-

A

extenslon anal)'$1s Í$ semI-

G

q ullltltative. lnd the ~trength of ¡ignal lrom each pri!Ml" extefl$ion reaWOIl ~ecu the amount o fthespedfoc; mRNA. The reutlons can also bol UHd to c;ompare lhe Stre1'1¡th of adjlcent

T

4.7.2 Gene fusion rechnology One of the most powerful and widely used techniques for analysing gene express ion is reporter gene technology. Reportee genes are generally used when tbe gene oroperon under investigation does nor encode an easily assayed prodUCL Fusion to 11 reportergene lhereforeallows the fuctors controlling gene expression to be identified. In recenr years, gene fusion technology h as been developed to facilitate tbe study of target gene expression a r the singte-cclllevel, aUowing visualisation of popu lation heterogenei ty, sites ofsynthesis and che location ofspecific protein s within the cell. Theuseofrepocrergene recbnology iovotvcs three main variables:(i) tbe type offusion comtructed (j.e. transcriptionat oc translational); (ii) the type ofreporter gene used; and (iii) the methods of detection (e.g. enzymc. immuno- or cytor.:hemical assay). Types ofgene fusion Reportee genes caD- be fused as either transcriptional oc tr.mslational fu sions (Fig. 4.16). In the case of a tr-anscriptiooal fu sion. tbe reportee gene is clone
me

promoters.

86

HARWOODANDWIPAT

Consuuction á reporter gene fuslons. Ina cranscriptional fw.ion me reponer gene Is transcribed from tf¡e tólrget gerMI prometer, buc is tr.ms lated from fu own ribosome bindrng sin:. In ~ translatlonal fu~. the reporcer ~ne lada iu own rlbosome bindlng sJte and Is fUSl::d In fi-ame Wfth the targetgene ~ne 1). Tht: product or chis fuslon event 15;¡ UU!Cued protein I fU$ed tO a complete ropy ofthe fu:;ion gene proceln. TTE, Innscrlpcion termlnatkm elemeru.

Target operon: Promoter ONA mRNA

Gene 2

Gene 1

LI

...,.

b i

9#+ rbs2

rbs1

TIE

0_

=.

Protoin 2

Protein 1 Transcriptional fusion: Reporter gene

UI

I

I ••u !

Truncated protein 1

Reportar protein

Protein 2

T¡anslotionol fuslon:

Protaln 1/reporter prom!n fusion

Pro tein 2

In the case of a trans..lational fusion (Fig. 4.16). the reportff gene is fused in Che same. codoo .reading frame as me large[ gene so tbat when me [argel gene is transcribed and translatcd, tbe pcoduct is a hybrid protein consin ing. fo.r example. of a portion of the target protein at me N·tenninus and tbe emire reporter at tbe C-tennmus. The length of the target protein depends on the type. of analysis. lf roe purpose is simply (O understand mechanisms controlling synthesis. me portion of target may be as little as 10 amino acid residues. However, if the purpose is to identi1Y the ce1lular location of the target protein. virtually all oft:he target proteln may be requiTed lo be fused to the Teporter pt"Olein. Reporter genes A variety of reporter genes have been developed tOí particular applicalions and most can be adapted for use in a wide. range of bacterial

spedes. These inelude ch.romoge.nk reporters (e.g.E. coli lacZ. encoding ~alactosidilse) that are detecte
GENETIC ENGINEERING: PROKARYOTES

(e.g. JuxAB. encodlng luciferase which catalyses a light-emitting reaetion 01' gfp, encoding a grcen fluorescent protein) that are detecte<1 by spectrometry, luminometry 01' videolphoto-microscopy. Hybridlsation array technology The genes being transcribed by a bacteritun at any particular time are referre
4.8

I Engineering genes and optimising product5

Many cornmercially important biotechnoJogical processes have beeu developed usmg micro-orgarusms found in the environment. These include. traditional p_roce5ses 5Uth as the producLion of milk products (e.g. yoghurt production by Lacrococcuscasei). the synthesisoforganic solvents (e.g. acetone production byOIlS!ridium Qcetobllfylirum), and [he production of enzymes for domestic and industrial C3.t
4.8.1 Protein and pathway engineering A well studied example of enzyme o ptimisaLion is that ofsubtilisin. an albline protease ¡solatOO from Rsubnlis and c10serelatives fe.g . Rlichenifonnis, B. stearotherlflopl.i1l1s) and used asa stain remover in the detergent industry, Subtili5in i5 used ln95% ofwashing detergent fonnulatiolls a.nd allows prorein stains to be re moved mote effective1y and a tlower temperatures dlan are usually needed for laundry processing. The ideal requirements far such an cnzymc are stability up to 70 ~ C and wi thin the pH range 8- 11. resistance te non-ioruc detergenrs and oxidising reagents such as hydrogcll peroxide. and the absence o( metal ion require ments. Based on an exte.ns ive knowledge of its catalytic activity a nd Lhree-di01ensional structure, subtilisin was engineereerby

Si

:!8

J.lARWOOD ANO WIPAT

site-directcd mu tagenesis (Section 4.4.9) to produce variant enzymeS with combmations ofthese improved charactenstics. An alternative approach bas beco used to improve tbe enzymatic characteristics ofBaciUu5 a-amylases. In this case natural recombina tion was used to generate fun ctionaI hybrid amylases from genes encodlng c10sely related enzymes from B. arnylolfquejacic!lts, B. licheniformis and B. stearothermophilus. Tbe genes ror tbese enzymeswece doned in pair-wise combinations and then aUowed toundergoroundsofreciproca.l recom· bination. TIlis gene.ca.ted a population of cells witb a. large numbcr of hybrid a-amyJases, which were then screened for ceUs produdng amyJases witb improved catalytic oc structucal characteristics. More recently, k.nowledge: ofthe tJu-ee.dimenslonal structure of tbese amylases. togetherwith information on (he relationship between structure and fundional cha.racteristics such as thermostabiliry, has enabJed more directed approoches to be used. These have induded [he use oC PCR gene splidng techniques ror the construction of spcci.tic hybrid a-amyJases. Comparative DNA and proteinsequence studies have demonstra ted the importance of recombination of blocks of sequence rather than point mutation alone in m e evolueon of protein structure. These studies have loo to the development ofmolecular techniques such as DNA shuffiing or sexual PCR 10 facilitate the rapid evolution of proteins_ The principIe involves mixing randomly fragmented DNA encoding dosely reJated genes and then using PCR (Section 4.4.4) to reassembJe tbem into fulllength fragments, witb the individual fragments acting as primen. The PCR produces are used to generate a tibrary (Section 4.5) of chimaeric. genes foc lhe se.lection of proteins with modified or improved characterisr-ics.Ihis system hasbeen used to generate a variant of an E (oli ,B-galactosidase with a 6()"fold increase in specitic activity for sugar that is normally a poor substrate fo. this enzyme and a variant of the green fluorescent protein (Section 4.7.2) with a 45-foId increase in f1uore scent signa!. Bacteria produce a number of compounds mat, if syntherised at suitabl e concentrations, represent commercially viable products. Traditional1y. bacteria tbat make a significant amount ofa potentially commercial product can bedirected to synthesise larger amounts. This may be acrueved by randomly mutageuising a population of the target organismo and screening fo. mut:mts producing higbe.r concentrations ol the product, or by doning the synthetíc genes together and placing them under the l'Ontrol of efficient transcription and transIation signals. While there are examples chat testifY to the success of such approaches (e.g. antibiotic production. syuthesis of amino acids), recentJy more ratioual approaches have becn made by engineering merabolic pathways_ either to increase. productivicy or to direct syntbesis towards specjfic products.

GENETIC ENGINEERING: PROKARYOTES

4.9 I Production of heterologous products Traditionally, genetic techniques have been applied by indllstry to increase the.production ofnaturaJ products such as enzy:mes, antibiotics and vitamins. Dnly a limited numberofprotein prodllcts wereproduced commercia1ly. and these were produced using existing technologies frorn their natural hosts (e.g. proteases frorn Badnus species j. Specific genes can now be ¡sola red from.virtua Uy any biological material and c10ned lnto a bacteri um or other host system. However, cloning a gene does not, pe.r. se, eru;ure its expression, nor does expression ensure the biological activity ofits producto Many factors need to be considered to ensure commercially viable leve1s of prodllction and biological activity. FoI example, the choice ofhosr/vector systems determines both the strategy used fur thedoning and expression, and these in tUIn can affect the quantity and fidelity ofthe product.ln sorne cases itis nor possible lo use a bacterial system to produce a biologicallyactive product or one that is acceptable for phannaceuticaJ purposes.lnstead hostfvector systerru based on higher organisms. fuI example mammalian or insect cell culture systems. may be used.

4.9.1 Host systems and their relative advantages Prior tú tbe 1970s, the only methods fur obtaining proteiru or polypeptides for analysis or fm: therapeutic purposes were to ¡solate them frOID natural sources or, in a limited number of cases (e.g. bioactive peptides). to synthesise the-m chemically. Recombinant DNA technology opened up the possibility rodone the gene responsible fur a particular product and to produce it in unlimited amounts in a bacterium sllch as E. eolio Initially, the only eukaryotic genes that couId be doned were those encoding products that were already available in re1atively large amounts and that hao very sensitive assays (e.g. insulin, human growth honnone. inteñeron). This was because it was necessary to have extensive information about theiI amino acid seqllence. and proteinsequenc· ing techniques available al that time required relatively large amounts oftbe purified protein. Current teehnical improvements permitalmost any characterised protein to be doned, either directlyvia copy DNA syn' tbesis from mRNA extracted from biological material, or indirectly by gene synthesis. Although recombinant rechnology was developed in bacteria! systems. it has been increasingly expanded into a range of eukaryotic organisms, using a wide variety ofinteresting and novel technologies (see Chapter S). However, from an ~onomic point ofview. bacteria are still the organisms of choice because ofthe case with which theycan be genetically manipulated, their rapid growth rate and relatively simple nutritional requirements. Proteins derived from recombinant technology are expected to mee! the same exacting standards as conventionally produced drugs; particularly with respect ro product potency, purity and identity. Sensitive analytical techniques are used ID cbaIacterise the products,

8~

90

HARWOOD AND WIPAT

with particular attentiOll being paid to undesirable biologiC'al activities such as adverse immunogenic and allergenic react.ions. It is therefore me exacting requirements of regulatory authorities, sucb as tbe US Food and Drug Administr.ition (FDA), for increasingly .mthenti c phormacological produc.:ts that has led producers to switch from ba<:terial to eukaryotic systems ror the production of certain products. These requirements haY(' been driven by the increasing sensitivity ofanalyti· cal teclmiques suro as high perfonnance liquid chromatogr
4.9.2 Transcription The c10ning ofa DNA fragment encoding a protein ofinlerest is not in itself sufficient to ensure its expression. lnstead, lbe cJouing strategy has to include me inclusion oí seqllences designed 10 express the ONA atan appropriate timeand level in the host bacterium. Transcription is facilitated by cloning thl! targct gene. downsn'e
GENETlC ENGINEERING: PROKARYOm

4.9.3 Transl.tion The effldent translation ofmRNA n.mscripts requires the incorporaoon of an efficient ribosome binding sire (RBS), located about 5 bp upstream of (prior to) the translational start codon. The structure of Che RBS tend5 to vary frorn bacterium to bacterium acccrding to the sequ ences at che 3'OE{ end cfthe 16S ribosomal (r)RNA that interact wito the mRNA The genetic code is degenerate. that is many amino acids are specified by more than one codon (triplet ef nucleotide bases). Codeus t hat sped1y the same amino acid are said to be synenyroous but are not nocessarily used with similar ftequencies. ln facr. mon bacterial spedes exhibit preferences in their use ef codons, partic ularly for highJy expressed genes. Variations in CodOD usage are, in partoa refJection of the %GC oontent of the organism's DNA. with favoured codous correspondiog to che organism's most abundant transfer (t)RNA spec.ies. Since most codous a re recognised. by specific aminoacyl tRNA molerules. the use of non-favoured codons results in a reduction in me rate of translaoon and an increase in the mis-incorporation of amino acids above that of me normal error rate oí aboue 1 in 3000 amino acids. Codon bias can be determined by calculating the Relative Synonymous Codon Usage (RSCU) ofincj.ividual codens: RSCU "" ohserved number orrimes a particular codon is used expccted number ifall codons are used with equa J freqllency therefore: ifRSCU equals 1. fue codon is used without bias ifRSCU is Jess [han 1, the CodOD is 'non-favoured ' ifRSCU is lno/l: chan 1 thecodon is ' favoured '. Gene syn tbesis technology alIows genes to be conrtructed so as te optimise the codon usage ofilie host producer strain. The signiflcance of codon usage was dernQnstrated in srudies on the production of interleukin-2 (lL-2) by E. coH. When (he native IL·2 gene (399 hp) was analysed for 115 coden u sage only 43%ofthe codons were .favoured' byE. mil. When an altern.ative copy ofme [L·2 gene was generated by gene synthesis , it was possible to adjust the codon usage such that 85% ofthecodons coro responded to those faVOllred by E. col!. When the twOvel'5iOllS were d oned and ex:pressed in E. coli on identical vectOTS. despite tbeir producing identical amounts ofmRNA, e.ight times more biologicaUy active 11-2 was produced.from tbe syn thetic gene as compa.red wi th thenative gene.

4.9.4 Formation of indusion bodies Many .recombinam proteins. particuJarly when produced al high conccn(rations, are unable te fold properly witrun lhe producingcell and instead associate with each other te fel'm large protein aggregates referred to as indusion bodies. lnc1usion bodies are particu1arJy oomm on in bacteria expressing mamrnalian proteins. The proteins in inclusion bodies can vary frOID a native-like state mat are easilydissociatoo. to completeJy misfolded molecules t bat are dissociated onIyunder

9I

n

HARWOOO ANO WIPAT

highly denaturing conditions. 111e size, stat.e and aggregation deDsity of indus ions are affected by the characteristics of the recombirunt protein itself and factors lhat affect ccll physiology (e.g. growth rateo temperature. culture medium etc.). In sorne cases aggregation can be prevente
4. 10

I In si/ico analysis of bacterial genomes

The availability of e.mire genome sequences fuI' a significant num ber of micro-organisms apeos up new approaches Cal the anaJysis oCbacreria. and the rapidly expanding field ofbioinformatics has the potential to reveal relevant and novel insights on bacterial evolution and gene tunetion, lt has the potential te provide a nswers ro long-standing questions celari ng toevolutionary mecbanisms and (O the relatiollships bel:Ween gene order a nd function . One of the mast significant advances in metbods for studying and analysingmicro-org;rnisms has come about through the availability of powcrful personal computers which, togetherwith the developmentof the inrernet, provides researchers with access to powerful bioinformatkal mols, Bioinformatics, often retened to as jn silfco analysis, nicely complements In Vivo and in vitro methodologi es. An ultimare goal ís to model rhe bebaviour ofwhole organisms, including asptttsof theirevo-lutíon. Although nor currently a subsotute for in vivo and in \litro experimenration, bioinformatics has already demonstrated jts potential [O direct rhe focu s of more tradicional approaches. Severa l rypes ofcomputerprogram are avaUable for analysing bacterial genome sequences. These ¡nelude prognms thata ttempt to identify protein-encoding genes. sequence signals slIch as ribosomc binding sites, promoters and protein. binding sites, a nd rclatioruhips ro previously sequeneed DNA oC whatever somec. Programs thar attempt ro identifY protein-e.ncoding genes translate t he consensus DNA sequence in a1l sixreadingframes and then anaJyse tbe resultingdata forthe presE'nce oflongstretches ofamina acid-e.ncodingcodons, uninterrupted by tcnnination codons. These so-called open t'eading fuunes (ORF) are usuaUy at least 60 aminoacids long, burmay be several thousand amina adds in lengtb. The more advanced prOgr.'lms for predicting protein roding genes are ab!e te search for the presence oC ribosome binding

GENETIC ENGINEERING: PROKARYOTES

sites locared immediately upstl'e3m ofa purative srarr codon and even to idenrify potential DNA sequencing errors that generare frame-shift mutations. Once purative prateins have been identified. orher bioinformarical t001s can be used to derermine their relationships to previousJy identified proteins OT putative proteins. A prerequisire for this type of analysis is rhe availability of data libraries which act as repositories of l."11tJ'eDtly available DNA and prorein sequem;es. The databases can be routinely accessed via the internet. using programs such as FASTA and BLASf that provide a list of DNA sequence.s and proteins. respective.ly. sbowing bomology to alt oc part oftlte query sequence. The rnternetis a1so a souree of molecular biological tools tltat facilitate a wide r.mge of analyses including the identificatioll of putative transmembrane domains. secondarystructures and the signa! peptides of secretory pl'Oteins.

4.1 1

I

Further reading

Davies.J. E. and ~ain. A. L (1999). Manual o/Industrial Mirrobi%gy amI

Hlotichnology. 2nd Editíon. American Society foe Microbiology, Washington OC. Glazer. A N. and Nikaldo. H. {1995). MiCTObill! Hiotechnology: r'llndamentuls oJ Jl.l'p!itd Mlcrobl%gy. W. H. Freeman and Company. NcwYQrk. Lewin. B. (2000). Gt'It~ VJ1. Oxford University Press. Ox:ford. Old. R. W. and Prlmrose. S.a. (1994 ). Pr1nc1pl~ ofGcnt Manipuladon: AA lruroductian toGcnetic E"gincmng. 5th I:dition. 8lackwcl1 Scientific

Publicatiolls. Oxford .

Snyder. L. and Champoess. w'(1997). Mo!rcularCeuma oJj'Bacwria. American Society fur Microbiology. Washi ngton Oc.

93

Chapter 5

Genetic engineering: yeasts and filamentous fungi David B. Archer, Donald A. MacKenzie and David J. Jeenes Glossary IntroductiOll

Introduong ONAimu fungi (fungal transform:uiun) Gcnedoni ng Gene SI1'UCIUrt", organisatioll alld e.xpres.s ion Spccial llle[hOdolugies Biotechnologit:al apptications offungi rurther rt"ading

1

Glossary

Auxotrophic lllufauon A mutation in a gene t.hat confkr.; the requiremcnt fur a g rowth factor lO bt! ru pplied ratber than syntheslsed by the organism./\ gene that complemears this auxotr0IJltic mutation is o ne that can ¡'etu ro lbe o rg
lactarl· cONA Single-stralldcd DNA with oomplemf'nt.uy sequence to mes.senger RNA (mRNA). synthesiscd ift vHm. DoubJe-strandcd cDNA can then be madc. dJNA libraries containdouble'sm nded cONA molecul!!!;, e¡¡eh ofwhich forms partofa. vector. The cOllection of cDNA molet:ules. eac.h in a separate ~ctor. fuTl11S rhe cDNA Iibrary. Cbaperone A protein wruch aJlSlsb th e folding ofanothcr protein. ChromarLn A highly organised Lum plex of proteio and DNA Otromosome A discrele unit ofDNA (containing many gt!DeS) and prote.in. Differe ntspecies ha"e diffcreDt nu mbers ofchmillosom cs (See Table 5.1). Complem~lalJon Theabilityofa gene w<:on\lel' a mUGlDI ph.enotype [O wild-type. Cosmid Pl:JSmlcJ containing sequences (phage lambda (OS sites) which permit packaging (}f the plasmid into tb e pruteinaceous phagC' coat. Cross-o\-er SC''' homologous recolllbination. Dimorpllism Ability to exlst as rwo structuro.llly distinctforms. Endoplasruic rctiru1um (ER) Inn·aceUuln.r memhrane stnlcum' in eukaryotes forming (he carJy par! oftlIe protcin secretory p
96

ARCHER, MACKENZIE AND JEENES

Expression A Il"rm used 10 descdbe the process ofproteln syntbesis from a

&='. Expression cassette AnarnngememorDNA which pennlls m e transcription ora gt!n<." for protein production. Le. ind udd 3 prom otu. tbeopco reading frame and a tr.msaiptionaJ termloalor. Gene A reg:i01l ofDNA wllicb is [ranscribeOratlon ofthe entire plasmid ¡uto a chro lllosome. A d oubJe cross-over employs a linea.riscc\ ONA lDolecule and leads ro t h e replacement ofa chromos,omal gen e by a gClle wim sequecce similarity to the chromosomal gene atthe cross-over regioos. Intron A segment ofRNA which is eJidsed before the mRNA is translate
Open readlngframe(ORF) St:retc::h orrodoos uninterrupted by stop cooon3. Origlo (tln) The site oflniliation ofDNArcplica.lion. Phmo~ Pro~ rties le.g. biochemicalor phy¡;ioLogical) ofan organism wh.ich are de termincd by the genotype. Plasm.1d DNA rno!cculc mar replicates independentlyofthe chrornosomes. Plotdy The n\.lmbc.r ofcolDple te sen ofchrarnosom es in a cell. e.g. haploid (1). diploid (2), polyploid (> 2). Polymerase chaiu reaction{pcR) A procedore for =pont!lltlal ampllficadon of DN .... fragmenn (see Fig. 4.7). P.romoler TIle region ofDNA upstream (Le. S') ofa gene which rontains signa!s fOf initiating and regulating tr.Illsoiptiou afthe gene. Protoplasts CeUs frOID which the cell wall has becn removed by the action of carbohydl'llse enzymes. These ceUs are bounded by me plasma membrane and are osmoticaJly fragile. Recombination The exchan¡;e ofDN.... beN."eeD two DNAmolecules a r the incorpol'lldon of one DNA molecuJe inm anotber. e.g. be~1l [he ch romosome and introduced DNA. Rl!sllictlon enzyme Enzyme which deaves ar or oear a specific, sbort ONA seguence (restrictiol1 site/_ Shuttle ftCtOr A vector tbatcan replicate iodependently in mo re than one type of organism. e.g. can 'shutlle' betweeo a bac[eflum and a ~ast.

GENETIC ENGINEERING: FUNGI

Sonthern blotting DNA fragments ilre separated according ro size bye1ectrophoresis. tr.msrerred to a membrane and probed with a labelle
Transform ..tion (genetlc) The uprakl"! and stable inrorporatioo ofexogenous DNA..illto 3 cell. Vector A DNA molecule (usually .. plasmid) used for transferring DNA Intll 30 org:mism. WOd-rype Strain of 3n organism notdeUberately mut:alf'd or modified by genetic milnipulation. The tenn can also ~ uscd to describe the phenot:ype ohuch a strain.

5. 1 I Introduction Fungi are eukaryotes cJassified as eirher yeasts orfilamentous fungi pri· marily by lheir predominant form oC growth in culture. Those spedes which are normally unicellular are theyeasts 3nd thus su perficia lly distillguished from their filiLmeotous rclativ~. This distinctioQ, is Dar always adhered to by the organisms themselves as several are able to grow in both forms and are called dirnorphic{Fig_ 5.1). More discriminatory genotypíc methods are nowenabling a more soundly based classifi.. cation of the fungi but rhe substantial similarities between yeasts and filamentDus fungí enable us to treat them [ogether in one chapter. Despíte the sim.il arities. their differences.have given rise toa wide diver.. sity oCbiotechnological appti cations which is increasing as the number ofdifferent spccies examined rises and as theirexploitation is extended by genetic engineering. In tb..is dlapter. we focus 00 rhe molecular biology involved in genetic engineering offungi and describe appllcations ofthe fungi as hosts for the production of proteins e.ncoded by introduced foreign genes. Many oftheexperimentaJ approaches adopted in [bis workhave been described in the preceding chapter (Chapter 4) with prokaryores and are essentially the same-with fungi. Their desc.riptions are Dot repeated here except where differences between bacteria 3nd fungi requireaiterations to me prorocols. Differences arise primarily be:cause fungi are larg~ in both size and~nome rhan bact~ta and the organts· ing principies for many of tbeir cellular functions are typical oC complex bigber organisms rather than simple bacterial cells. ThU5. fungi have defined membrane-OOund nudei containing severa! chromosomes and other membrane-bound organel1es including a membranous intra-ceUular system called rhe endoplasmic reticulum

97

98

ARCHER. MACKENZIE ANO JEENES

Plw.1I (OIltr.ut micnJgraph 01 (a) lhe yt::as t

SacchQromyces c~ (b) t he yeut (aboYe r i3ht) and hyphal (balow right) lorou of Yarmwoo ¡;poiytlca, (,) brighl field mlcrogrijlh ol lhe fibme:noou. fungul Asperginus nIget grown on a cllllophane sht.et, (d) differelltlal imenerence contras! micro¡raph of prntoplnt formatian from AsPetE¡:fuslliduJans.

Notll!he brantNng ofhyph)fl. Bar markers '" IO¡un. Cs,

~

onidiO$por«

(nexual): H. hyph;t:

P. protoplut. Micrographs a tD c

are courtesy ci Linda andJame!. Barnl!tt.

(ERJ ioto which protcins are transported tor targetingeither to theccll exterior or to other sub-cellular organel.lcs. Fungal ceU w,¡¡jls do not contain peptidoglycan which is found only in bacteria. Rather, their waUs are compose
GENEl'IC ENGINEER1NG: FUNGl

Organism Soccharomyces cerevisiae Schaosoccharomyces pombe Condida orbiccms

Numberof Genome chromosomes size (MW web sit~ 16

12. 1

3

14

8

16

Neurosporo crossa

7

43

Aspergillus nidulans

8

JI

http://genome-www.stanford.edulsaccharomyces http://www.sanger.ac.uk/ProjectslS-pombel http://aJces.med,umn.edulCandida.html http://sequence-vvww.stanford.edu/gtoup/ candida/lndex.html http://w#w.unm.edu/-n?PI http://gene.genetJcs.ugaedu

Notn

• Ilapl <>id &Cll r¡m~ oompkm"nl ",
in fungi is muffilarger tban thatin prokaryotes (c.f. 4.7 Mb - tbat is 4,' million nucleotide bases in length - for EschericMa coli). This is due to fungi having more genes and more DNA which does not code for pro· teins, eitherwithin genes (as introns) or as 'spacer' DNA between genes. Bacteria economise on non
99

100

ARCHER, MACKENZIE AND jEENES

5.2

Introducing DNA into fungí (fungal transformation)

5.2.1 Background The lirst fungal transformation experiments in the enrIy 1970s were perlormed by traruferring chromosomal ONA fragmen ts frem wild· type strains in to mutan lhosts whieh had a specifie growth requiremenc for an amino acid or nudeotlde base (examples of auxotropbic mutants), Successful transformad on relied on converting che fu ngaJ cells into protoplasu (Fig. S.ld) bydigesting away m e cell wall(a major barrier to ONA uptake) with carbobydrase enzyme mi.xtu.res before introducing lhe transforming DNA. Transfonnants (Quid then be selected by th.eir ability to growwithoul supplcmentation for (he auxotrophie requirement due to the activity of the wild-type gene, a process Itnown as genetic complementation. The fate ofthe transformin g DNA in thecell could noc be studied in grear detail until the ¡¡d~n [ ofmore sophisticated molecular techniques in the late 19705 and early 19805 when it was subsequently shown that the DNA in these early experj· ments had imegrated by genetic recomblllation into tbe fungaJ chromosomes. SubsequentJy, transformarlon me.thods were developed for theyeast Sacchal"omycescerevislae usingsbuttle vecton wh.ich could be propagated as intact pJasmids, without chrom050mal integration, in botb E. coH and yeast, Manipulation ofthese plasmids and 'bulking up' the amount of ve<:tor DNA were more effectively performed in E. coli prior ro their introdllction into yeast. Similar ve.ctO[$ have been designed fu r the uansfo[mation offilamentous fungi but in most cases the introduced DNA integrates jnto tbe fungal chromosomes rathcr than replieating as ti plasmid. Transformarion was accomplished flrs[ in genetically well· characterised organisms such as S. cerevfslae, Ncurospora crassa and AspngirlllS nldulans because [hese fungi. had been studied extensi~ly in che laboratory and a numbetofsuitable auxotrophic mutants and their correspondingwild·type genes were avaiJable. Methorls are now continually being modified or arlapted far more biotechnologicalIy importanr fungi in which genetie systems may be less we1\ defined. [n cases wherc auxotrophic complementation of industrial strains is 1l0t fcas ible. othe.r sclection markers such as resistance to an autibiotic can be used in me t:rausformation.

5.2.2 Transformation protocols For the transformation of many filam entous fungi, the method of ehojee still relies on conveniog the fungal myceliwn into protoplasts which are prepare
GENETIC ENGINEERING: FUNGI

Typical transformatlon protocol U!;ing protoplasts dll!l'Í\led from a filamentou~ fungus.. FoUowing harverting, me filtered mycelium is resuspended in a buffer containlng an ~motic ~tab~i~er ,uch as :¡orbital or KCI ro pr"event protoplans fmm burning. The ,electlveagar can either be a

Inoculate wilh fungal spores

IncuIJate tor 16-24 h

minimal medium (lacking che

Remove cell walls with carbohydrase emyme for 1-2 h

requlred growth suppl\lment which 15 now supplied by me activlt)' of me Introduced gene) or it can contaln an antibiotic. For anclbioti<: se lectioo, che tr.msfOl"mants are nonnally allowed to regener.ate

Wash protoplasls wilh buffer containing osmotic stabilser

their cell waJls and

e)(p~

antiblotlc-resi~nce

me

prole]n befo,,"

being challenged with tlle antibiotic. PEG , pol)'elhylene

glycol. Add plasmid DNA, Ca 2• ions and PEG

Plate on to selective ager containing osmotic stabiliser Allow cell wall regeneration then pick off indi v idual transforman! colonies

Control (minus ONA)

¡

+

¡

DNA

¡

0000

Thus. mere is a need ro have effective selection methods ro obtain thase eells eontaining the introduced DNA Transformation frequencies. in teI:lI1S of the number of transfunnants obtained per microgram of vector DNA added, can val}' significantly depending on the organism and ttansformation protocol used, Since the first reports of fungal transformation, various modifications to this basic method have been described wrnch improve ttansformation frequencies by orders of magnitude. Alrl'Tnative methods whieh obviate the need far protop1ast fonnation. such as the lithiuDl acetatefyeastwhole eell method, electroporarion of germinating spores or biolistic transformation of fungal mycetia, have had varied success but these suffer from the limitations of suitable host range and the need fuI' specialised equipment in sorne cases.Alist ofrransformation methods is given in Table 5.2.

5.2.3 Transfonnation vectors Transformation veetors can be designed to introduce DNA which either integra tes inro the recipient organism's genome (integrative transformation) orean be maintained as a plasmid.lntegrative transformation is med toinsertDNAeither atsite(s) in the chromosome which showsignifieant sequence similarity ro a regian on the plasmid (integratian by

101

H¡n~~~:~ai.ff#i~~t~~~m?K~H~b~fmmW~mImj¡mHHnfmi~~mgg1¡;~!mmmHm~g!¡m~m~im¡HH~mmHmUnIm~mgmH¡m¡Hfili Method 0 1' trea1ment

Examples of fungi transformed

TransfonT1ation frequency ~

Remarks

Socchamrnyr:es cereviSioe, PichiQ poswris

1Q2- 1 ~

Most widely used method but frequellcies genera11y lowerwith fl lamentous fungi

Aspergillus ntdulans. A. niger. T,ichodermo reesei, MULGrorOnel'oides Protoplasts Electroporation< S. cerevlsioe, A. rliger, r rcesei Whole cells Electroporation S. cerevtsiae, A. niger. A oryzae, Neurospcra crasso W hole cells liAcd/PEG S. cerevisioe, Yorrowio ¡ipolyuca

lOO_ ID)

Protoplasts PEGb/Ca~

10"-10'

Can be as effident as the PEG/CaCl1 method

IOO- IOl

Forfijamentous fungi, best resutts- are obtained using germinating conidiospores witt1 weakened cell walJs

10'- 10'

Only applicable to a few yeast species and

not

effective with flIalllentous fungi

Whole cells Biolistics" Protoplasts Agroborterium 0'

tumefaciens(

S. cerevisine. A nidulans, N.crossa, Trkhoderma h[Jrzjorlum S. cerevrsrae, Asp€rgillus owamori, T.reesei. N.crossa

lOO-l O)

Most effective with Intact yeast cells or conidiospores but myceJium can also be used

102-1 ()'Ig

Equally effectNe with protoplasts or conidiospores bU! transformation frequency is specieHIependent

""""'ole cells ¡'¡!neJ;

• b;pkS$l!
high vollage el«lric fi~ld. • UrbJum Uf'ta.!t'. , Shr>ol!ng DNA-
GENEnC ENGINEERING: RJNGI

A

Stylls.d yeoad-f. coli ,hume expresslol'l vector $Ilowin¡ origi~s orONA

yeas! ori

• E. (;011

orl

homologous recombination) or randomly al one oc more locations (ecropic intcgl'dtion). Yeast shuttle vectors contain genes whicb aHow tor their selection in botb bacterial and yeast cells. The:y also comain bacterial ,mel yeast origins of replication (orI). sequE'nces which are essential for the iniriation ofplasmid DNA replication in borh organiffilS. A styüsed yeast shuttle vector which is capable of replication witbout intcgratioo inm the ycast chromosorncs (exU'achromosornal vceroe) or which can be speci.flcal ly targeted iuto a region of a chromosorne (intcgrativevecroe) is iIIustcated in Fig. 5.3. In order to flnd those cclJs which are 'rransfurmed ' , ¡,e. contain tbe introduced DNA, the transformation vector is designed to comain a gene which confers a selec::tabJe characteristic on the transformed cells. Thesc'selection markc!rs' faU into three maingroups. Pirst, a numberof genes from ....1Id·type fungi bave been doned which comptement auxotrophic growth rcquirements. as already explained. In many cases. genes hom one fungus are able [O complcment tbe appropriate muta· tions in a diffcrent fungus (het~rologous transformation). Howeve:r. for sorne fungi , only theie own genes are able to complement mutations (homologolls transformation). If the required auxotrophic mutant is not available. a second C]
replicatlon (on) eiVl~r

in ye;¡st or E. eoIi. selectIon muirers and clonlng ~ite A for IIlsenll1g the gene to be el<prened. For t¡rgeted Integrarlon or the pla~m¡d In lo ~ cnromosome. a DNA sequence from !he rlbosom31 RNA-encod!ng reglan (rDNA) could be: tndude.:! in me vector. Slte El In thl~ rDNA seq~erJc e 15 ~ ,..striction ~¡te requl re d to convart the Ye(;tor from,¡ drcula<" molecule w a lineu one which increues the effic!tllC)' of ON" hnegrnrion by homolOjOUs r.combinauol'l inlO -me rONA region af me chromosome. which function

103

I (}4

ARCHER., MACKENZIE ANO JEENES

carbon or nitrogen sources which the host strain would no! normal1y be able ro use. A good example is the acetamidase gene (amdS) ofA. "td",lans which aUows growth of the red pient strain on acetamide or acrylam ¡de as sale nirrogensource. This marker has been introduced inm a number of.Aspergtllus and Trfchoderma spp. and is particularly useful in generar· ing ttansronnants which contain multiple copies of the integrated vector. lnyeast, p lasmid vectors are maintained witbin tbecell provided fue transfonnants are grown under selective conditions, e.g. in me presence of an antibiotic. When me se1ective pressute is removed. plasmids can be lost relatively rapidly from the eelli because there is no advan· tage to the eells in maintainlng a plasmid. Al! plasmids ñlUstcontain an on' which can be derived eirher from naturally occurring plasmids or from ehromosomal DNA sequences. Vectors with al] ori from one yeast can normaUy function in a variety oC different yeast hosts, albeir nor always wim the same degl'ee oC efficiency. Plasmid vectors can be presenr al up to 200 copies per ceH and rhus provide a simple system for increasing tbe number of copies of me introduced genes. This ofien leads to higher yields of rhe prorein encoded by tbe introduced gene. The disadvantage is thalselecrtve pressure is normally required lO avoid significant plasmid 1055, i.e. maintaining me desired characreristic can be a problem. particularly in S. rerevislae. Conversely, in otber yeasts, suro as KIuyveromyces ¡actú, sorne extrachTOmo50mal vectOI"S are campar· atively stable.without rhe need rorcontinuous selection. lnregralion oC the plasmid roto the yeasr genome brings enhanced stabilitybut lower numbers ofthe intrOduced gene. One wayofachiev· ing trus in S. cerevtstae has been ro target tbe plasmid ro ribosomal DNA sequences{rONAI (which can be presentar about 150 tandemlyrepeated copies per genome). (ncorpor.!tion ofr.DNAsequences into a vector (Fig. 5.3) enhances integration ofthar plasmid into tlle chromosomal rONA regíon by recombination of homologous sequences, especialIy when lhe vector has been linearised in tbe rDNA regian. Tbe number ofgene copies can be increased by placingthe gene used Cor selection under the transcriptional control of weak, or deLiberate.ly weakened. promotecs. This approach encourag-es tbe sclection of multiple gene copies through selection pressure for a critica! leve] of gene producto This approach is used togemer with rUNA targering to obtain high numbers onnregra,ted gene copies. Vectol's thar integrate inlO tbe chromosomes have severalimportant uses in the molecular manipulation offungi. In addition ro increasing lhe number of copies of a particular gene in a chromosome, they can also be used lo disruptor replace il desired gene. In S. cerevístae, the use oC'replacemenr cassette' vecton has permitted rhe deletian of each oC the.6000 or so genes identified by the Yeast Genome SequencingProject lO test {he functionofeachgene in thecell. Eachcassette. which consists of a kanamycin {G418) selec:tion gene tlanked at each errd by $hoct regíons oC gene-spedficsequence, is released from me vector bycuning with a restriction enzyme. Homologous recombination between the ends oftbe cassette and me target gene in tbe chromosome leads to the

GENEnC ENGtNEERING: RJNGI

gene A chromosome

replacement cassette

¡

1

1;

le p

doubJe cross-over

Ka.

~ cbromosome

lacking gene A

e

m p

Kan

Citone deletion in S.cereviOOe wing liltl kal1anl)'d" «(118) 'n!placement auette', The assene (01\11:1:ts of rI'>I'llwlamyd n (G 418)-resistance gene (Kan) under the control of a runla! prornotet (P) and short !link;"g sequences ofonly about 40 bp (O) whlch corrtipClnd 10 !he ends oftMl:ene lO be deleted. A doubl. o:roJ~r by homologou1retOlTlblnation between tht..SOl endsand me chrorTlOSOIl"OfI ~d:s 10 lhe

deletlon of d-..gene. At th e tron·OYer 11tU, cJvumo$OlNl ONA molKule$al""ll broken en¡ymlc.1l/y ¡nd DNA $tr;lnd, ei'!d\arJ&ed with mat ofthe ¡ncomin¡ cassette ONA viii DNA repair mechani$m whlch re-joins me DNA molecules_ BecaU5e many genes I I""Il essentiaJ ror surviYlll, gene deleooo is flrst arded OUt in dlploid ceh wheNl on/yone of lile [WO copies o/ th.gene is deleted. Y~diploid tr.ansformoonu COI"Iulni ng Ihe dele'".ed

gene ue seletttd on moolum contailllnll the ;votiblotic G418, which is more effective
specific deletion of that gene (Fig. 5.4). In these srudies, individual genes. or groups of genes. were deleted from the yeast and c.hanges in phenotype were thenlooked for.ln this way, a particular gene might be associated with a particularfunctionalthough, in practice. thede1etion ofagene does notalways give.a detectable phenotype. Tbe majorityofvectors used for tr.a.nsforming filameotous fungi rely on random, integranon events which occur at a rclative.lylowfrequency in mosteases_Qne approach (O increase tr.msformation &equendes has been theuseofRestriction lillzyme-Mediated Integrarion (REMI). ID th~ method, the plasmid DNA is added to ,he ce.lls alongwith a restriction en2yme (see Cbaprer 4) which cuts once in the vector. Under rbese con· ditions. the DNA is rnrgNed to the corresponding restriction sires in rhe genome. Wirh standard integrati\'e transformation in filam entous fungi . the vector is fOlUld predominan Uy as multiple copies at one or more sites in the genomebur with REM1 the proportion ofsillgle copies ar severa) different sires is increased (Ag. 5.5). Tbe extent ro whicb an introduced gene is able to lead to production ofthe protejn ir encodes is affected by its site ofinregrationin the hosr's genome. Therefore. methods have been developed to target genes to

105

106

ARCHER. MACKENZIEAND JEENES

(.1

(bl

chromosollle

cbromosome E

single cross~ove r

x

O

E

o

gene

circular plasmid

gene

circular plasmid

+ restriction cnzyme cutting at sile E

t

OT more copies orthe vector integrated per sHe

=

predomioantly 1 copy orIbe vector integrated per site E

E

Random pl:umid nr.esration inlO dW!I chrornoscm., offlbmenmus fungl. (a) Random (<II by cunIng al the same restriction lite a!lcws imegrarion of t he entire pblmid ilt Sllvtl~ chrom
GENETIC ENGINEERING: FUNGI

1')

Targeted integr;¡don al

di, Pf1G Iocus (eocoding orQtidine-

chromosome

S' -pllO$JlM,.!! deorboxylne ) In DWIlmOrl. The hozt m-;¡In of A. awumQri contl.;ns il mugnt (non_functiooal) ~rG gene c:onferringa growth requlrement fo r urldine. The P"iln~format\on ~tor cont;lins il norHunttional pyrGwith a mutatlOl1 in a differem: pan: ofme gEne. By a Sin,1e homologous recombln;¡¡tiOll ~ent (::ro l ingle CI"OSJl-Q'fer) (a). the emlre 1/OCtOf" is inle,... wd al the pyIC Iocus aOO " functional pyrC gene is r:;r-ealed (b). The cron-9Yer.!!Yeflt Involves brel!k¡,g\! and re-jolnJlg oí DNA mofecules both in the yector and chromo~ome by a DNA rep~rr !TIe<:han-l:!5m similar to that O~lllned in Fig. 5.4. FlrIgaI transtQrmants are 1el.!!aed by their ablliq (O grow wimout the need lor add~d urldin.!! In me gmwth medium. As~$

vector

lb) py", •

l""i'f?üffl#

double mlJtation (Inactlve)

p,,",

E'

wHd-type gellE! (fUl1ctional)

spccific chromosomaJ locatiol\S thar ensure good express ion. Que me thod for gene targeting. developed in AspergiUus spp., celies on transfonniog a strain which has oue muration in a particular gene with an integrative plasmirl which c.:ontaios t]le same gene with a diff'erel1t mUnltion. A functiona] gene will only be fonne
5.3

I Gene cloning

TIte success with wbich Fungal genes are isolared and charaeterised is still beavily dependent on the species being studied. Seve.ral different approacbes bave been a.dopted lo ove:rcome problems presented by genetically poor1y characterised. butbiorechnologkaLly useful, fungi. The use of mutant stra.ins has been the cornerstone of fungal gene doning.ln this approach, mutant strains are transformed to the wildtype phenotype by introduction ofONA and, when that DNA contains only one gene, ir identiJies the function ofthe gene. Other options are 1l0W available which exploit the rapidJy expanding amount of gene sequence infonnation available in gene darabases aJthough a funetional assessment of ;¡ clone
107

108

ARCHER. MACKENZtE ANO JEENES

1,1

S'

::>-

chromosO!TM'

lir1Nrised vector

Ibl ch r olTlO'JOfl'lt'

gene X

t

X

J'

-c:::::;:::,

X

::>-~

S'

are B

3'

"::>-

"gB

J'

•

:::::o

Spetlfit gen!! deletlon In f~~ment~ funll. Upstreml (5 ' ) and down5tr'Um (l') reglons of a~ le¡¡5t I kb In Jlze, Immedlalfllyadjacllnt te thII chromowmal ¡en. to be deleted (gene X). Incorponl\e
¡re

enE)'me ¡nvolved in allinlne blolynthesis. The plasmld whldl is normaHya drrulu molecule of DNA 1, conven.ed lO alinear OM; ti)' C).¡tting witll a rero1ction eN)'me tlu.t (un the plllnld once. Thl$line¡¡rl!ied .-ec10l" ISlnen InI/lSlom'l6d ¡nto 111 «¡B mutan! stl"illn. A double homol0g0u5 rec;ombination IM!nt (doublll tnn,s-over. as oudinad In Flg. S.") in the S' and 3' regions (iI) repbteS gt!~e X;n medlromo5Ol'ne with me OIJa ,electioo mlrlc~ (b). FunpJ tr;vufOrmlnl.S are selected lYt melr abiHtyto ero« wIttIout me n~ ror added ll'linine In lhe &"lWlh med¡u",.

gen~.

'expression donlng' is a convenient and efficient approach mar

is based on the introduction into ycast of a new metabolk artivity detfX':table by a simple plate assay. We describe [he basic principies involved in these and other strategies below.

5.3. 1 Mutant ¡soladon The complementarion of defined rnutants remajos [he most effective means for isolating genes ofknown funcrion although me generation ofsuch mutants remains a problem formany fungi ofinteresC. Phys¡c~ chemical mutagens. e.g. UV or nitrosoguanidine. are soH che most commonmeans forgenerating fuogal mutantswhich mayo on occasion, be charactensed quicldy when simple growth tests are avai1able. Because mutatioos i.n othergenes in a biosynthetic pathway can confcr the same growth requircmcnt. the mutaot 1S tested for its ability to metabolise different sl1bstrates to confirm the precise location of the mutation. A disadvantage of physicO
GENETIC ENGINEERING: RJNGt

M.utlnt cell

•

Plasrnid with wild-type gene

+

, Compkoment:'lclon of 3 muumpne 'a· in I cell wfth liIl eOfTMpOl\diog wlld.typt gene 'A' on a pbsrnid. ~ represt!IlG me ml.!t:.lntgelle ','Ind c::::J repreleflU wIId-type gene 'A'. The prese nee oIpne. JI, withln the

me

Ph, notype:

..

DO growtb

,ell, either n 3 plnmid or llltegr3ted [nto th. chrornosome. alJows growth, ebromMfl'" ,1

lra~bromo»
pla3.m1d

• Pbenotype:

inttj¡rtIolion

OA growth

•

A f-

growch

methods where the nccd for a defined genetic background is paramauot, Fragrnents of DNA. tcrmed ttansposons. with the ability to move from ane site to another within the ban genome occur natura.lly in many spec:ies and can be used for chis purpose_ The transposon Ty is used in tbis way ¡nS. cerevisiae a.nd similar elements have been identified in Schiwsaccharomyces pom.bt, Candfdo albicans, YorroWÍa lipolytica and Pichia membranaefaciens although these bave not yet been.furthcr developed as g~tic teols, Otber insertional io.activation systems are a.vaila ble and ¡nelude re.striction enzyme-mediated integradon (REMI). as alreadydescribed in Section 5.2.J, Ofnecessity, mutants must provide an easily assayed phenotype SO that large numbers of colonies can be quickly tened. Screening for me productionofextracellularproducts provides a simple and rapid means to do this, Agar plates containing substrates which. fur example. allow ll\e detection ofdearing zones orcolour changes are frequendy used to ¡solare genes encodingtbose extracellular enzymes associated witb the saprophytic lifestyle oí many filamentous fungi , TIlus, agar pIares coo.taining starch as the sole carbo n source can be used [O testfor the production ofamylases . Oftcn.specific strategies can be employed lO ¡solare particular dasses of mutant. For example. the location of a protein within a c:t'llcan beexploi[ed:ifan essential protein is normaUy located in the cytoplasm buthas been geneticaliy engineered to contain signals which will divert ittbrough the secretory pathway [O [he outside ofthe eell, then mutants in (he secretory patbway which prevent this occurríng can be selccted for,

5,3.2 Mutant compl ementatíon DNAfragmencs which represent the emire genome ofan organism can be doned iuto a vector to produce a papuJadon of ONA mo]ecuJes

109

110

ARCHER. MACKENZIEAN D JEENES

.

Ilolatlon of ¡

,en<¡ by

Motllnt (';('11

eo mplemcntatlo n of a definecl mL!~t

•

In a f~:lIT1ernQoJ$ fungU$.

~ repre~cnu me rnutant gene 'a' .lId c:::::l rep~~Cf1u the wlld·f)'lIe gene 'A' fOUl'ld I~ genomk ONA ( ), repr~enu me f",n&ll ori¡in _ of replbdOfl (01'1) Ofl lhe plumid. Recombin,ulon between me ~nornle DNA and!he pbumid rewlts In an Iloslllble In!ter¡llIn(t! of

1'llIsmid

+ intcgnrtioll

.~A~ 1

__

c:J rttombinatioa

a

+

A~ A

~

the wlld·type: gene 'A' which causes seaored (01011)' morphology. I,e. growth o(cu r$ Ylhere the pbsmid 11 present b UI 1, preventod wh~n puu. ofthe (oIony lose ~he pbsmid. The plismld cOI,g,ining gene 'A' COIn be recOVli!red kom lhe calonjes shawlng secta red gr'OWlh.

:1

Colony morp hoJogy: eveo growth

settored growth

termed u library. ComplementatioD ofdefineia¡: andA . nidulalls. wbich can be nansformed at high frcquem.y (Fig. 5.8). Al ternatively. sorne gene produces are sufficiently welJ conselVed across specics boundaries tbat [hey wiU function in a bacterial background. Thus. a number of fuoga l genes have beco isolated Vla complemen tation of E. co¡'¡ mutantS although transformation offungal bosts rernains thc nonu. The surest route ro assess gene function rcl icson tbein tegtation ofa singLecopyoflhe genc in qucstion iOlo [he fimgal host DNA. Many yearu. such as S. cert'V'isiae, a nd sorne fitamenrous fungi al10w thc introduction ofa gene on a circu· lar DNA molecule (plasmid) which can bE' pre.sent al several copies ¡>er cel!.ln sorne cases. th..is allows closelyrelated (but not identica 1) genes to comple.men [ the muration and can therefore provide a misleading pictul'e ofthe gene's function. Plasmids which replicate extrachromosornally are much less comm.on in filamentous fungi t han in yeam. Despitc tbis. sucb ve<:tOI'S can sti1l be exploited to idt!Iltify funga l genes wruch are able lo complement specific lllu tations (Fig. 5,9). For example. ifanA 11idulans mutant is transformed with a mixture oCa plasmid. containingonly a bacteria! se1et.1Íon marker and a funga l orl, aud linear genomic DNA isolated from tbe fungus undel' study, two distinctdasses ofuansformant a re obtained. TIle first shows a stable. wild-type phcllotype on plates resulting fram direct integl'iltion ofrhe complementing DNA mm meA. 11¡du· 1ans chromosomes. The second dass of transformant sh ows uneven growth wlthin colonies on pIares wh ere ccrtain scctors of eam colony appeill' wild-typc whilst other sectol'$ display lhe mUlam phcnotype. Th.is 'sectorcd' morpboJogy results from recom bination betwee.n the two source DNAs produdng .plasmids which carry the complementing

GENETrC ENGINEERrNG: FUNGr

geuebutwltich can be lost during the process ofcell division. Extractio n of tbe total DNA from su ch Ullstable A nidul,ms colonies, and transforrnationintof. eoli, pennj ts the isolation aftbe plasmicl s containüIg the rungal DNA fragment which complem ented che original A nidulans mutation. Advantages ofthc.method are that itjs rapid and it is nOl necessary to construct a library of individual genes from m e total DNA of tbe fungoso

5_3.3 Gene isoladon by the polymerase chain reaction Extensive use of tbe po lymerase chain rearnon (PCR. (see Fig. 4.7) is now mOlde to isolate many specific genes. This approach requires profetnS with rhe same function ro have been identified in other orgOlrnsms and for the gene sequent.:es ro be available in a DNA sequellCE' data.base (e.g. 0 0 the intemet)_ Alignment of tbe protein sequences eucodecl by these genes Olg<¡instone anotber can then be med to identifY highly con· serve
III

112

ARCHER, MACKENZlE AND JEENES

'Nested primer'· PCR. Redundant primer mike~ (00 and
(j)

==--------- 3' ~

Genomic DNA template

5' 3'

...,..-- 5'

® 1_Primary peA reaction

@

~

Prlmary peA reaction product

1

S' - - - - - - - - - - 3 ' 3'

5' ~

@

gene.

2. Secondary peA reaction

1

5'====3'

Secondary peR reaction product

3'

5'

1

Probe library

Pcssible mechanlsm for

me down-~gulation of proteln

p

I

productio n by 'antlsense ' RNA tr.msc ripu. Open bO)(es represent me urget gene X cloned In elther oftwo o rlentiltlons md flan~d by promoter (P) and terminater (T) sequences. Transcription provides seme and ¡¡ntlsense mRNA molecules (both with polyA talls) depending on me orlent:atlon of me gene ~btive to me promot.el'. TI>ese two complemenW'y RNA moIecules blnd together ro form iI.

-GeneX-

IT

p

I---Genax-I

T

J} s'

3'

AMA Sensa mRNA

S'

MM3'

S'

+

~

3'

........ ··.. ··..... AAAA

Antisense mRNA 3'

---MM

Duple x RNA

S'

duplex RNA Formation of duplex RNA in the nudeus reduces me amount of m3tun- mRNA available IOr translatJon in che cytoplasm.

which is espedally useful when tbe gene in question is essential fOI" growth. is to decrease the amount ofa protein produced within cells_ TIüs uses a process known as 'antisense' in which a messenger KNA (mRNA) sequence complementary to the sense roRNA interferes with [he production afthe protein encoded by the targetgene fFig. 5.11). This. approach is becoming more commonIy used fol' the functi:onal assess· ment of daned genes inyeasts and filamentous fungi altbough it has not a1ways proved successful.

GENETIC ENGINEERING: FUNGI

Fungal transformllnta

• • • •

! Grow mycallll

1.

from p~rlfled funpi tranifol"rnllnl colonIas on agar pl.it.s

a,..

iOCKuI.ued ¡nlO gTOWth medium Wl

mlerowe plates. Mtr 16-24 Il growm. funpl mycella aJ'& v~sfl!ml!d from lile orltlnal microtitnl pbte toa n_ platll PUrifyIO..-.g Ia~_

~ lnCIc\II3Ie miGrotitre ~"1.

0000 00 000000 000000000000 000000000000 000000000000 000000000000 000000000000 000000000000 00000 00 00000

1 Cen wall digestion

Screenil:g fun,gal

UIDsrormanu by PCR. Sporu

3. Tf1ifl5I" myaIIi. ¡rIlO T....." ,,*_p/III" 4 ....lId ceII ..... -
00 0000000000 000000000000 00000 00000 00 00000 0000000 000000000000 0000 0000 0000 0000000 0 0000 000 000 000000

!

So Heat 111 95 'C 11. Ncot .safl"l9i11 to SIIIlIe en ice

7.

u.. wpetna.tanI as temDIIl'.. IoI I'CR

peR frllgmenb

5.3.4 PCR and lungi In addition [O its use i.n the ¡solation of genes, PCR is also cornrnonly used in so·eening for the outcome of specific DNA manipulations in many systems, For sorne filamentous fun gi. howcver, the cell wall has proved a substantial barrier to obtaining DNA ofsufficient quantity or purity for la rge seale screening by PCR.. A prococol has now been deve1oped in which fungal colonies a re gro\'ffi in Liquid culture inmicrotitre plates and their cell walls removed by em;ymic digestion to release profOpla.sts. The 'hot' dcnaturing Sfcp ofthe PCR cyde Iyses the protopla.sts providing DNA w hich can be used to screen for the presence oC speciflc DNA fragments (Hg. 5.12). Other protocols, which use mycelia , spores or celli and incorporate a short heuting step to burst thc cells prior lO PCRampli6cation. work weIl with many fungal species.

conulnlnca KC!lcitnu buffer and iIll enzyrne whicll di;geus _y lhe funga! all....-¿lIls adele!! ro COfl'I\!f"t l Ome: orme celb 10 protoplun.. After I h n37· C. 5-IO¡LIcl

illptlmatólnt 1$ u.ken ~d che DNA

whkh ir ~ber.lted from me re1ultl", protopbru by hutins: am u 3 ternpbte In th. PCR.

113

11...

ARCHER. MACKfNZIE ANO JEENES

5.3.5 Heterologous gene probes A third s1l
5.3.6 Database and linkage-based methods for gene ¡solado n Finally. two otber strategies may be u sed for gene isolarion. Th~ firse. termed synteny, relies upon rhe fact that even wbere there are significant c1ifferences between Che ONA sequences of genes from different species. gene linkages may be physicallymainrainoo. Thus. ifin arganism X. genes A ¡¡nd B are neighboul's 011 che same chromosome then. if the same case pertains in me fungal system. screening for gene B may allow one ro isolategene A. The main requirementfol'sucb a method to work is a detan~d genome map ofthe model ol'garusm (X). A number of genomes are currently being sequenced and maps established which should provide a basis for this approach (rabIe 5.1). The S. cerevisiae genome sequence is already complete, that of S. pombe is expected to be finished shortly. aud those ofe. ulbicans. N. crassa.Aspt'Tgmus ful11¡gatu.~ and A. /lídu/ans are aH ullder way. An alrernative approach employs partial complementary DNA (cDNA) libraries which can be made from fungal RNA samples using readily available cDNA sYllthesis kits. These cDNAs are termed Bxpressed Sequence Tags (ESTs) since thcycomprise the ends of rran~ scribed sequences. '#hen allied to
5.3.7 Expression cloning The life style of fungi means mat they secrete l1igb concentralions of protein. many of which have applicarion in a variety of industries. A quid: and effective method. tcl:med expression doning. has recently been developed ro isolate genes e ncoding extr.1cellular enzymes (Fig. 5.13). In this method. thefungus isgrown underconditionsexpected to induce expression oftbe GUget gene(s). and the mRNA is extracted and used te construct a cDNA library in R eoli. This Library is tbeo usc:d to

GENEnC ENGtNEERING: RJNGI

Expres$jon doN"g. from c~lwres seClllting a range ri PrQtelns are dOl"lfld ¡nto a yeaft expruslon vector Clpabl e (J f ~li1:.ltingln E. cok DNA molec:~lel I'rom E. cm tr.ltlSfonnants a.re pooll!d belO<"e ~ DNAs

Fungal culture

' . ANA ~

2. cONAsyntl"l6!till

cONA

~

, . """' " !

4 . E. cailr.lnltormtlion

E. coJllibmry

11s.

1

P!asmld DNA ex!tllctlcn

6. Yeas! trans/otm¡¡11on

7. RepIca platng

Veast screenlng

1

8. P!ur
Cloned fungal cONA

transform S. Ct'm'tsiat and the tr.msformants are grown on media cbal provide a simple assay for the new1y acquired enzyrne activity. Otber yeasthosts such as Y. lIpolytica. K. !acns, Plchfa ungusta (formerly Ranse!1111a polymorpha) and S. pambe may be prefen'ed rOL" the u-dllsfonnalion ¡¡tep. The effectiveness of this system has bcen demonstrated by its use ro done genes encoding over 150 fungal enzymes induding arabinanases. endoglucanases. galactan
trilndo rmlll8 yeast (5.cete
an;J:)'$ for the re levant enzymic anlvl\ies..

115

116

ARCHER. MACKENZlE ANO JEENES

5.4 I Gene structure, organisation and expression In general, transcriptional signals Ut genes (rom higher organisms are

more compLex (han those found in bacteria! syste ms. Within fungi, gene organisation shares many common reaturesacross a wide number of genera. The three rn3in organisationa! units in fungal genes can be split up imo tbose signals which (a) control the switching on OT offoe gene transcription (promoters), (b)concroJ tbe termination oftranscripdon (terminaton) and (c)control che necessary excision ofintrons fram

mRNA UnJike their bacrerial councerparts, ñmgal promoters can extend a substantial distance (> 1 kb) upstream (Le. 5' ) of che transcriptional start point (rsp). EarJy cloningexperiments in S. certVÍ5iae suggesred tbat native S, cerellfsiae promoten: were required for the expression offoreign genes a!though s\lbsequent experiments have shown that sorne promoten from otheryeasts. e.g. K. lactis. can also ftmction in S. cert"visiae. ln filarnentous fungi. promoters tend to function '\'Iell within their own genus but are less predictable in more distantlyrelated species. Promoters can generaUy be defined as either constitutive, Le. they are switched on permanently. orinducible. i.e. contain elements which allow them ro be swhcbed on or offin a regulated fashioD.ln both types oi promoter, one 01' more tsp can existo Seqnences in the promoter region define both the tsp and the binding sites for regulatory proteins. TA-rich regions, known as TAIA-bo~s. are often involved in Mtermining the tsp and abo ma.intaining a basallevcl oftransCliption.An illustration of their function is provided by the S. cerevisiae JUS4 promoter where transcnption can occur either witb 01' withont a TATA-box (the HIS4 gene encodes h.istiruno! dehydrogenase involvcd in histidine biosynthesis); high tr;mscription levels are only observed from rhe lliS4 promoter containing a TATA-bol{. However, thel'e are alJio strong yeast promoters whkh lack a TATA-box. Since many yeast and filamentous fungal promoters do not contain this sequence, other motifs, such as the pyrimidine-rich tr.acts (Cf-boxes) found in fiLamentOlls fungal prOmoteTS, ean perfarm me same function. These sequences can be used to desl."¡bewbatis essentiaUy a 'core' promoter.AthiJ:d sequence, CUAT, is also aften associated with eore promoter function. Rowever. mast (> 95%) S. cen:risiae genes appear not ro require CCAAT-boxes for function althoughthis motifisknown tofunction in A.1'IiduJolls. [tshould be stressed that in the majority of fungal promotet'S mat have been ¡solated, the funcrlonal significance DI sequences identified in rhem has uot been detennined and therefore remains uuclear. With a constitutive promoter, the basal level of transcription is determined by me binding to the 'core promoter' ofa proteio cornplex which eontains RNA polymerase and tbe so
GENETIC ENGINEERING: FUNGI

Allcaline pH ====,..=== Plflsma me mbrana

¡

Slgnal transdoction pathwav

¡ PACC Inactive

Active

Activation o f 'alkllllne' genes represslon of 'aeid' genes Rqulatlon aftnn.Kriptioll by pH In flbmentous ftr,gi. The externa! pH b Jonsed by ceUs aOO leadsi ro cm.nges in lenr expreulon . In fungi, the tn.nKnpdoo of genes whleh e ncode proteln~ chat ~ re necenary for survivaJ lt aJka~r.e pH IJ mtodlate d by ~ tran5Crlptlon factor (PAC e). Alkalinu pH IJ 5en$ed and luds, through a sign...J tran'lducVon pathwzy. te Úle deavage ofan In~ctlve form of PACC to produce the active formoThe :KI:rv::ued PACC :Stimulates the cranserlptlon of a;enes lead ing te unI)'mes such as ab!¡lle phmph;nase matare ac:tive at akallne pH (·a1kalint!' genes) and repreu es the tliln)("riptioo of ·xid' genes. T he uunc:r.ted I'oITll of PACe ICtiv.:rms by bindltl¡ t.o ia tar¡el promoters nw nquence S'-GCCARG·)' (where R=G or A).

a-

sequen ces (UAS or URS). These UASfURS sequences bind regulatory proteins which are theught to stabilise (er Mstabilise), either directly or indirectly, t he transcriptional complex bound ro tbe cOte promoter thus elevatingjdecreas ing the r.ate. oftranscriptional initiation. Regulatory sequences whkh control expression of an inducible prÜ" moter m.ayoften be faund in promoten afseveral genes thaL encade prÜ" teins of unlinked function . all of whic.h are controlled by a single regularory protein. Such a network ofco-regulilted genes may respond to physiological parameters affecting che cell such as pIi , carbon or nitrogen sou rce. For example, in the filamentous fungus A. nidull1llS, a protein (termed PACqwhich binds ro a specific sequence in (he promoten of genes encoding pH-respousive proteins has been identified (Flg. 5.14). When the ambíent pH is allcaline, PACe is activared by a protease ro a form whic.h per.mits expression of a wide. range of alkalineexpressedgenes le.g. isopenicillin N synthase) and represses many genes normaUy expressed under acid condirions (e.g. acid phosphatases). Similarly. i.n S. crrevisw and sorne f..1uyvm¡myces spp., Lhe DNA-binding protei.n . MIGlp, binds in a glucose-depe.ndent fushion te GC·1ich regioos (GGboxes) of many promote.rS ofgenes llvolved in carbon $Ourc:e utilisa· {ion. MIGl p forms a complex with orbe r proteins lhar represses tr.mscnption of genes required for me catabolism of carbon sources whic.h are less efficient in producing energy than glucose and related sugars, a process known as carhan catabolite repression.ln filamentous fungi, suc:h asA. nidulalls and Trichodmna rcesei. a similarfunction.is perfonned bythe CREA snd CRE·1 proteins, respectively. whic:h bind to GCboleeS in the promoten¡ ofgenes involved in carbon catabolismo Another Iletwork of genes, w hose control is mediated by the re:gulatory protein

11;

11 8

ARCHER. MACKENZlE ANO JEENES

AREAinA. nldulalls(NIT2 iaN. ct'assa).is that involved in the synthesis of enzymes for nítrogen catabolismo In the presence oC the preferred simpLe nitrogen sources, arumonium and glutamine. genes responsible for breaking down complex nitroge.n sources are switched off. In addition to tbese regulatory systemsofbroad specificitycovering networks of genes, th el'e are aJso control mecbanisms spedftc tO a particular metabolic pathway. Thus. the positive regulatory protein, co-ordinates !:be expression ofar leaS[ 25 differentgenes responsibte for tbe synthesis afthe funga l torin. aOatoxin. AlI these genes exarnined so fM contain a specific sequence in their promoters to which the AFlR protein binds and activates transcription.Although in this instance the genes are dustered on a smaU regioo of Dne chromosome, thel'e is no known requ.!rement for genes which are cOtability is increased. Most yeast genes lack tbe sequence AATAAA associated with polyadenylation in higher cuhryotes although two otber motifs bave been associated with tenninator function:a TTITIATmotifwhich functions oruyiDoneorientation, and a tripartite signal based 00 a TAG ..rr·rich).:rA(T}G1'..{AT-rich)..TIT sequence which can function in eitherorientation. rtseems likelythata number ofsignaIs are used totcnnínate transcription in S. cenvisiae. The genes oí higher organisms also differ from thei.r bacteria] cou ntcrparts through the presence of introns which mus( be exased from the rnRNA before ir is translated into protein; a pl'ocess known as spUdng. S. cerevisiae and filamentous fungal genes sbow considerable differences with regard to the presence and nature oftheil'introns. The majority of S. cerevi.siae genes lack introns altogcther and those thar do contain introns often contain only one. Genes from filamentous fungi. and those of S. pombe. afien have several inttons. usually betwffn 50- 100 bp in size. The fuer that the introduction offilamenrous fungal genes into S. cerevisiae results in incorrect splicing suggests tIlat the splicing me
"A.R.

GENETIC ENGINE~R1NG: FUNG I

(a)

}--------------------dSONA (b)

1!5.~~~----------------- dsONA (a) Yoast two-hybrid srstem base!! Oll me GAl4p tnnsa1ption rnctol'". Thls proteln tont;1ins regions fQr DNA-bJnding (D B) ¡nd fer tn.nscripdoo acdvadon (A D) Ind 15 IllvolVld In ~w, u:hing on gal ~c tQSe utHlsatkm in S.cerevisiae. P. GAL4p-lctivltlld run promour: Te, cran~criptio n cornplex including the RNA

polrmer;u~;

DB, che GA l 4p

ONA.blndln¡ .-.gJon fl,lSlId rn S, the 'b;tit' protein of known nmctioo; PR, the 'prey' proteln undllf.nudy er ThfI prnducu of a cONA &bnu')' tu sed to AD. the GAl4p acdvatlon rt¡jO!'\; UAS. ups~m actlva tiOll ~eqUerK:e requJred rOl' inltiadon of

traI'IKrlptIon lt che prcmot.e r; dsDNA, dovble·m'anded DNA. Ó:prenJon of the reporu!r g_, e.l. me lDcZ 1_ encod iog ¡3-gala(twidase, is $wltrned onlf ch e AO and OB rqlQfl1 of!he GAI..:4p transcrlption actívator a", brought togetll4lr thrO\.lgh Im.eracuon ofthe 'Míe' and ' prey' protl!'w,s. (b) Ye~t one-hybrid synem whlch is liso bued on che GAl 4p tr.lIIlcripoon mctOr. TE, target.elernent (DNA se(;juence), norrmly COll$U'UcteóU al lean mrH reput:ed copies: AD.De, me GAl4p acdvatioo ~glon flAed lO ONA-bindlll8 proleln of IntI!res1: or to the producu from a cONA Wbrary: !Xhll!' bbel~ogu In (1). Expnmlon of die reponer cene (e.g. 1ocZ) is switdloo on If the pu~tiY'll

me

OS re&Jon binds [O che TEs.

5.5 I Special methodologies 5.5.1 Yeast !Wo-hybr¡d system This genetic-based assay was developed in S. íerevfsiar: ro rest whether protcins ofinterestinteract within the cell (Fig, 5.1 5a). The method can be used either ro study two proteins whose genes have already been ¡solated or, more impot:tantly, to identifY genes from a cDNA librarywhose gene products wiil interactwith a known protein ofinterest, termed the 'bait' protcin. The two-hybrid assay reHes on t he faer tbat most eukar· yotie transcription factors are single proteins which contain rcgiollS involved either in promotcl' ONA·binding or traDscription initiation. A common. system used is based on the GAl.4p transcription activator protein which regulates ga lactose utilisatioD in S. rerr:'IIsiae. Typica11y, the 'reportee' gene, whose expression is measured in the yeast transfor· ma..nts, is tbe loa gene which encocles ¡3-galactosidase, an easily measured enzyme activiry. Positive interactiODS between the proteins under

119

120

ARCHER, MACKENZIE ANO JEENES

srudy result in an Increased production of ,&galacrosidase compared wilh nega tive controls, This Oexible syslem cao be used to investigate interactions between proteins frolO any organism but once a positive result has been obrained, me interaction in quesdon mustbe verified biochemicaUy, nte two-hybrid system is now commerdalIy available io a number ofdifferent kits.

5.5,2 Yeast one-hybrid system !he one-hybrid system was developed from the two-hybrid version to identify genes which encode protelns recognising Icnown spedflcDNA sequences, such as trnnscription fac:tors which regulate gene expression or proteins which bind to other 5equences such as DNA replication origins(Fig.5.15b). Positiveclones whic:h show increased 'reporter' gene activity are analysed by searches through DNA databases for sequence identity to k:nown DNA-binding proteins and tbeir functional activity verified by prorein-ONA binding assays_

5.5.3 Cosmids and artificial chromosomes W¡th the development ofvectors which can carry large fragments oí c:bromosomaJ DNA. up to 50 kb in size.itis now possible to clone entice fungal pathways, provided all the genes are clustered in one region of

the genome, and to transfer these ioto another organism, Cosmid vedors have been ronstructed which can be propagatedinE. cali oc in filamentous fungi. like other vectors. these DNA molecules can be designed ro either integrare ioto me chromosome or replicate extrachromosomally when introduced into the fungus. Cosmids with overlapping inserts have been used ro clone tbe entUe pamway fm aflaroxin/slerigmatocystin biosynthesis. a total of at least 25 coregulated genes. from a number of Asprrgillus spp. In another case, a rosmid containing tbe tbree structural genes for penicillin biosynthesis from Penidllium dl1ysogenutn has been integrated inlo me chromosornes of Aspugilll.ls nigcr aud N, crasia, both of which then gained the ability to syntbesise penicillin_ Yeast artificial chromosomes (YACs) are now abo available for cloning largeDNAfragments. These are large linear molecule5 up t0620 kb in size and can be considered to behave as smalI chromosomes . Problems ofYACs[ability have loo to thedevdopmentofalternative bac· terial artificial chromosome systems (BAes) but YACs still prove useful in severaJ applications. They bave been used ro analyse foreign gene expression in marnmalian ceD lines and also in whole animals. YAes have also been used to study chromosome damage in higber ceUs, to map ESTs in the genome and ro clone large DNA fragments for genome sequencingprojeds.

5.6

I Biotechnological applications offungi

Genetic engineeri.ng of yeasts and flJamentous fungi is now commonly used roc investigating aspects of tbeir biological functioll and also fOf

GENETlC ENGINEERING: FUNGI

Product

Uses

Yema

Filamentous fungusa

Biornass

Foods

Sacchoromyc:e.s cerevisiae

Aganrus bisporus Fusonl.Jm I'enenotum

Ethanol

Beer. wine

Saccharomyces cereYisioe

CO,

Bread, wine

Soccharomyces cerevisiae

Sulphite

Preservative (beer)

Sacchoromyces cerevisioe

F1avours (e.g. lactone s. Foods. beverages peptides. terpenoids)

Socchoromyces cerevisioe Pichio gui/liermondii Sporobolomyces odorus

Po lyunS
Foods

Crypwcoccus curvotus

Organic acids (e.g. crtric, gluconic, ltaconic)

Yorrowio IipoJytica Preservatives. food ingredients. ehemieal synthesis

Antibiotics (e.g, penicillin.

Health

Aspergillus niger Aspergillus terreus

PenidlJium chrysogenum. Aeremonium dwysogenum Pen¡ólUum griseo{Ulvum Aspergil/us tamorii

Homologous e nzymes Fo od processing. (e .g. amyiases. paper production. cellulases, proteases) detergents

....

Moroereila alpioo MUCO(GndneUo~

cephalospom, po lyketides)

Heterologous proteÍlls

Trit:hoderma viride Gibberello (ujikuroi Mueor dra"ne/loides Phycomyces bkJr.esleeanus

Foods. therapeutics

KJuyveromyces loais

Socchoromyces ceretisioe Kluyyeromyces loeUs P!chIO paston·s Pichio ongtJSto Yorrowia lipolytico

' Main ~ocly

constructing strains for particular biorechnological a pplications. Altho ugh genetic engineering is well developed. foc on1y a few species, the technology can normalIy be deve.loped [oc m ost species provided suffident effort is expended . Sorne of the commercial1y important products and species are shown.in Table 5.3. Each producr is a target for improved production using genetical1y modified str.rins: this is most advanced in the production ofenzymes and antibiotics. The use ofyeastS" and filamentous fungi to produce heterologous proteins. I.e. proteins .not naturalIy produced from that species and encoded by a gene derived from another organism, serves as a convenie.nt ta pie roc a comparative discwsion. Th.is is because both yeasts and filamentous fungi are used as hosts foc heterologous proteio production. the tec.hnology has advanced sufficie.ntly foc com.m.ercial use aud

Aspergi/lus spp. Rhizopus spp. Trichodenno spp. Aspergillus niger Aspergillus oryzoe Aspergillus nidu/ans Tridloderma reesei

121

122

ARCHER, MACKENZIE ANO JEENES

researc.h is still active in order to improve the systems . To be effective. a production system must deliver sufficient yields (whether it be for commercial viability or ro provide research material) oftbe rarget protein and the protein must be.authentic in its properties, te. the same as, or dose ro, thoseofthe protein from its natural source.

5.6. 1 Protein production: che importance of secretion Most of tbe commerciaUy available enzymes are secreted from their soun:e organisms although sorne important enzymes are extracted front cell biomass. lbe main advant
5.6.2 Heterologous proteíns from yeasts Sacmaromyces cert'V1siae is widely used in the produetion of bread and alcohol. and is regarded as safe. Gene transfer and gene regulationl expression have been extensively studied in S. certvisiae and its widespread familiarity makes it a superficiaUy attractive host organism for

heteroJogous protein production. A largc number of different h eterologous proteins have be en prodm:e
a hydrophobic core) of the signal sequence. Sever.il beterologous, homologous and syntheticsignal sequeoces baw been cxamined with many working effectively. It has become more common tO ¡nelude a short 'pro-sequence' after the signal sequence aud before the N'lerminus ofthe target protein. Pro--sequences are naturallypresent in many secreted homologous proteins and can aid folding. In S. rerevísiae. the signal sequence .md pro-sequence ofthe secreted a-mating factor prorein i.s ofren employed. This pro-sequence ends in a dibasic pai.r of amino adds.lysine-argin..ine. aud an endopeptidase that is loc3red in the Golgi body (an orgauelJe in [he secre lDry pathway) c1eaves after the Iysine-arginine ro release matllrc Urget protein with its correct N·terminal sequence. TIlls endopeptidasE" is caUed KEX2 in S. ctm'islae and equivaleD[ enzymes are found in o ther yeasts and flJamentous fungi. Many heterologous protcins are produced at yields which are [00 low (ror commercial viabillty oc for experimental purposes) or are struc· tural ly and functionalJydifferenr from theauthenticprorein. Yeasts are no differenr in this regard ro other expression systems and modifica· tions ro the standard procedures have been pursued in. order to overo come tbe difficulties. The use ofvectors which replicate at high copy number can titrate o ut tbe ne<essary mmscription facrors so that tbey be<:ome limiting. This OCCllrs in S. rLrtVisiae with the galactose-inducible GALl promoter. lncreasing the exp.ression ofthe assodated regulatory protein (GAL4p) overcomes tbis titration effect. Manyofrhe lirnitations leading to lowsecreted yields are posHranslational and relate ro the secretot}' pathway oc to proteolytic degrada· tion. Thus, protease-deficient mutants have l>een examined and o conversely, enhancemen t of the specific. proteolytic activities of [he signal peptidase and KEX2 protease havc aIso becn examined. Each approac.h has shown sorne promise witbout wholly ~rcoming !:he bottle-necks to highyields. Folding ofproteins duringsecretion occurs within the ER and is assisted by residentchaperone proteins. Other proteins, tenned foldases, aOO cataJyse folding by the focmation of disul· phide bonds within tbe ER. Up-regulated express io n of foldases and chaperones.has increased the secreted yield ofsome. hut not aH, heterologaus proteins from S. cerevisiae. Mutagenesis oC strains has been used ro increase secreted protein yields and in a few cases mutations have been locaJised to panicular genes. Mutanons thar afIeet aU aspeclS, induding transcription. proteoIysis. secretion and glycosylation, have bee.n recorded. Although mutagenesiswillcontinue to be used as a tool ror improvingyields, manyrnutations are recessive and no[easily incorporated ifito polyploid commercial strains which have multiple copies ofeaeh chromosome. Thus targeted gene manipulations provide a como plementary and valuable approach.

5.6.3 Hetero logous proteins from filamentous fungi Typical fullgal e.xpression vectors are not disslmilar to those descnbed already for yeast. As discussed in Section 5.2.3. the main dif· ference i5 thatautonomous replication is Ilotnormally an option with

GENfTlC ENGINEERING: FUNGI

commercial fil.amentol1S fungi and a U YeCto rs , with the exception of sorne used fue reseaTch porposes. are designed ro in tegrate iota tbe fungal genome. As with yeast inregration vectors. genomic integranon of the transform.ing DNA brings added. though not nec:essarily complete. stability but sorne uncertainty about the levels of gcne expressioll expecred a nd a limit to the number of gene copies that can be integrated. From a pracncal standpoint. a fungaJ transformation produces transformed strains which differ in their level ofheterologous protein produce
125

(a} En1.y o, na8cant protein in10 t ha ER iumen BiP: ehapa ra ll e Irat"lsioc:on ) ribosomll eytos o l

lumcn

(b ) An'sted protein 'olding in t he ER lum en p< o-sequenclI

Cr-í~ COOH~

=

G ~

he~u5

~

prole;n

C.

(e) Endopeptld... el....eg. In tIMo I.t. PC•• tory pathll\lIlY

LLI O

(001,,0 .nd .'!aa.. o,

O

ca,rla. p.otl!Ó n and /"utterolollO'ls prou!n 10

...

tha c" l osteril>l'"

O

lQ

cK~T C?OOH~ •

FoIdlng and proCeJ$ln&

of ffcre10ry l'ullon pIO{eÍll$In flIunenlOU$ '~ (a)

Entry of

JUs(ent polypepdde Imo the tumen oIthe e~opWm!c reticulum lE"R).

n. sI¡nal s.qllel"lCt whkh d lreca erTll'y of!h. polypepdde is r.mo-v.d by slgnal pcpcld.ase so th:.Ic ma emar¡ln¡ poIypepudc wlthln malllmen IlW me slpl SllqUiIn". SIP Is m ~bI.r1d~l1t chaperone wlchil me IUll1en whtch 11 nroclated wich nrly prot.eln fQldtn, e-.n:nt$ , Other chaperones

an d foldues (lee tElxt) are also preient. (b) Fokl ing of!he fuI!· length fu$ion proce~, within the ER. (e) The rus Ion proceln is

clea~.d

withln til . Golgl body by a lpeclfl(

peptldan (KEX2I n S.cereviJioe) to release me hetw-otogous prote;n tO m e ceUextMlo r fOIIowl"g tnnsport of me prot eln by membra.r.e·boun d ycsJ.t:1e5 (el).

126

ARCHER. MACKENZIEANO JEENES

a1ready c1ear tb3t. as wirh ycast expression, severa} factors can conspire to present a bottle-neck and l llat their relative importance depends on the heterologous protein. Foreign genes which use CodOllS Ilotcommon in fungi , the presence of sec¡uences which destabilise mRNA, differences in the protein folding/secretOlY pathway and the abuodance of protcases aO contribttte ro me observed bottlenecks. lo addition, although hyperglycosylation of heterologous proteins Is not such a problem with1i.1amentous fungi as it is with S. cerevisiae, it can still be a difficulty. In addioon. [he patterns of gIycosylation diffcr from those seen in mammalian ceJls wh ich could be importa nt for therapeutic protein production. TIte glycan su-uctures in fungaJ glycoproteillS are being analysed aod the genes tbat enrode enzytDes respons ible fur gIyc3n assembly are being doncd, providing the possibilil)' in che fuorre of manipuladng glycan synthesis. The essential details ofthe secretory pathway in filamentous fun gi appearto be qualjtativelyverysimilar ro those in theyeastsystem whích has becn studied more exrensively. Sorne of the genes that encode chaperones aud foldases have -been d oncd, as have genes that encocle proteins lnvolved in vesicular traosport.Although successful manipulation o( the protein sec.retory pathwayusrng these genes has notyetbeen reported, the nccessary rools to do so are becoming available.

5.7 I Further reading Atuubel. F. M_ Brent. R., Kingston. R. E.. Mool'e, D. D.. Seidman.J. e .• Smith,J_A. and Sttuhl. K. (1995). CllrrmtE'roI(l(.l,¡'S in Molmslnr f:liology. John W iJey, New

York, Broda, P" Oliver, S, G. aud Sinu, P_ F. G. (1.993). The cuknl)'tlllC Cfflcttw:Organisution ¡HIt! Rlgulatioll. Cambridge University Press. Cambridge. Gel lissen, e, and HoUcubcrg, C. P. (1997). Application of ycasts in gene expresslcn studies: a comparison ofS[J[charomycts cercvisiac, HanuIJu la polymorpha and f(!uyveromym: lactis - a review. Gm~ 190. 87- 97. Gow, N.A. R. and Gadd. G.M. (eds.) (1995). The Growing Funsus. Olapman and Hall, London . Kinghorn,j.R. and Tumer, G. (eds.) (1992). J\pplied Molf'CU larCenalCJ 01 FilatlU'ntollsFungi. Blackie Academic!lr Profusional. GlilsgtJW. I.uban.J. 31ld GoEr, S.I'. (1995). The.ycasl lwo-bybrid s~te m for studying proteinprorein inter.1roolls. Cun: apill. Biotl'dlnol. 6, 59- 64. Oliver, R. P. and Scb weizer, M. (eds.). (1999). MoIcculur FlIngtll Diolug)'. Cambridge

Universiry Press. Cambridge. Wolf. 1<.. (ed.J (1996). NmH:ollW'T!tiornd Yearo 111 BiotooutOlogy. Sprini,oer-Verlag, Berlin.

Chapter 6

Microbial process kinetics Jens Nielsen NomenclatUN Introduction Kinetic mode1ling of cell growth

Mass balances for ideal bioreactors Further reading

I

Nomenclature

a

COllstant in de:fined by Eqn (6.62)

A.

constallt

B e " ' "O c... ' .., D Dcdt f.e~-

E8 F FOUI

aGd ~

K,. Kl

K, m, p.

r¡ r" r, T 14 V

cOllStant

collcentration (g \- , or moles 1- 1 ) COllcelltration of a substrate essential fur grCJWth (g ¡- 1 or moles ¡-1) ¡nitial concentratian afthe limiting substrate (g 1- 1 or moles 1 ~'1) concentration ofa growth enhancing 5Ubstrate (g 1- 1 or moles ¡-1) conccntration (g 1- 1 Dr molesl-') dilution rate (h -1) critical dilution Tate (h- ') activt! funn afthe ellzyme inactive forms oftheenzyme activation energyofthe growth process (kJ mole-I) flowI
flow rate o,u t ofthe bioreactor (1 h- ') change in free energy (XJ moJe-') inhibitlon constant (g g ' l) dissociatlon <:onstant dissociation '-"Orutant saturation coeffident (g 1. 1or moles ¡-I) maintenance coefficient (g g ¡'h) productivity (g 1- 1·hor moles 1-1.h) specific rates (g g-' -h or moles g-l'l1 j specific productionrare (g g-l'h or moles g-I'h) spedfic substrate.uptakerate (g g-lf¡ or moles g-I·n) tempenture (K) time(h) doubling time (h) volume (1)

128

NlELSEN

bio.m ass co[lc~trnLion (g ¡- I). yif!ld L'Ol'.fIlcientspecifylng the 3mount ofl produce yield cocJ:ficicntspecifying the 3mollnt ofproduct fonned per unit bioma5s formed (g g- ') Y...ur yield roeffl.cie.n l specifying the. a mountof All' consume(!r unit biomass formed (g g- I)

JI

Y,.

SubscripLS t growthcnhancingcompound f.th SUbStT3te o r produce e.ssential growth compound o initia] conditions substra lc x biomass p product Supe.rn:riprs

r

fero

Greek letlers a.f3 cOó!fficients in equalion 6.11 6.G,¡

free enerxY change spttifir gl'owlh mteofthe total biomass (g g- "lLorsimply b- 1) I'inu maximum specljir growth rote of[he rotal biomns (gg-l'b 01' simply b- I) p.

6.1

Introduction

Quantitative desc:ription of cellular processes is an indispensable tool in the design offennentation proc:esses . The cwo most importantquantitative design parameters. yield and productivity, are quantitative measures that specify how the cells convert the substrates to the producto The yield specmes the amount of product obtained from the substrate, and it is ofpartirularimportance when the rawmaterial costs make up a large fraction ofthe total costs, as exemplifled in the produc:· tion of solvents. antibiotics . alcohol. and other primary rnetabolites, The productivity specifies the rate af product formation, and is partic· ularlyimportant when the capital investments play an important role. such as in a growmg market where there is an increasing demand for producing the product by a given capadty(or factory). These two design parameters can easily be derived from experimental data but, what ts more difficult to predlct. is how theychange with che operating condi· dons. e.g. if the mediurn c:omposition c:hanges or the temperature changes. To do this it is necl!:Ssaryto set up a rnathematical model.

MICROBIAl PROCESS KJNETlCS

A modells a setofre1ationships between the variables in rhe system being studied. These relationships ate normally expressed in the form of mathematical equations. but they may also be specmed as logic expressioos lar c3use/eff'ect relationships) which are used in che opera· bon ofa process . The variables ¡nelude any property mat are ofimportance fol' the process, suc.h as me agitaban mte, [he feed rateo pH. temperature. concentrations of substrates. metabolic produces and biomass, and the stateorthe biomass - ofien represented by {he caneentration of a set ofkey intracellular compounds. To set up a malhematical mode! it is necessary to specüy a control volume wherein aJl the variables ofinterest are taken ro be uniformo For fermentadon processes the control votume is typic:ally the whoLe bioreactor, but fu, large bioreactors the medium may be nonhomogeneous due to mixing problems and hereitis necessary to divide fue bioreactor into several control volumes. When me control volume is tbe whole bioreactor ir may either be of constant volume or ir may changewith time depending on the operation ofthe bioprDCess. When fue controL volume has been defined . a set ofbalance equatioos can be spedfied for the valiables of interest. These balance equa· tions speci:fy how material is flowing in amI out of the control volume and.how materiaL is converted withjn the control volume. Rate equa· tions (or kinetic expressions) specify the convenion ofmarerialwithin the control volume. They may be anything from a simple empirical corretation mar specifies rhe product formatlon rate as a function of me medium composition to a complex model mat accounts for all the major cellular reactioos involved in the conversion ofthesubsuates to rhe producto Independentafthe model structure. the process ofdefining a quantirative description of a fennentation process involves a number of steps.assbown in Fig_6.1. A key aspect in setting up a model is to specify the model complex¡ty_ This depends on what rhe model is going to be used rOl' (see Secrion 6.2.1)_ Specificatioo of the model complexity invoLves defining the numhe,ofreactions tobeconsidered io che modelo and specification oí' tbesroichiomerry for these reactions . When the mode! complexity has been specified. mtes afrhe cellular reactions considered in the model are described with.mathematical expressions, Le. the rates are specified as functions af the variables: namely rhe concentrarion of me substrates (and in sorne cases the metabolic praducts). These functions are normally referred to as kinetic expressions. since rhey specify the kioetics ofthe reactions considered in che model. !bis is an important step in the overall modellingcyde ando in many cases. differentkinetic expressions have to be examined befare a satisfaclory model is obtained. The next step in tbe modelling process is ro combine the kinetics of the cellular reactions wim a model for the reactor in whicb the ceUular process occurs. Such a model specifies how the concentradons of substrates. biomass, and metabolic products ch.ange wirh time. and what fiows in and out ofme bioreactor. These bioreactor models are

Specify mooel complexily Sel uptkioellQ

I

~

&~'~IPlinces mass

Rtdenne model compkxity

llii

..timol'

~-"J

¡

Simu[a1.~ ferm~nlllliDn proces ~

Dlffenlllt ueps In fermematlon prcx:esse5.

12~

1]0

NIa5EN

nonnally represented in terms of simple mass balances over the whole reactor, bu! more detalle« reactor models may also be applied, if inbomogeneityofthe medium is likely toplay él role. Thecombination of the kinetic and the reactor model makes up a complete matbematical description ofthe fermentation process and this model can be used la simulate the profile ofthedifferentvanables ofthe process, e.g. the sul> strate and producr concentrations. However, before this can be done ir is necessary to assign values to the par.uneters of tbe mnde!' Sorne of these pararneters are operaring par.uneters. which are dependent on bow me process is operated. e.g. the volumetric flow in and out oftlle bioreactor. whereas otbers are kinetic parameters which are 3ssooated with !he cellular sysrem . To assign values tu these parametcu ir is necessary to compare model simulations witb experimental data and herebyestiman.' a parameter setthatgives the bestfltofthe model tothe experimental data. This is referred to as paramete.r estimation. The evaluation oftbe fitofthe modellO the experimental data can be done by simple visuaJ inspection ofthe fit. but gener.illy it is preferential to use a more rationaJ procedure, such as minimising the s um ofsquared eITOl'S between the fiodel and the experime ntal data. ln the fullowing wewill consider tbe two different elements o.eeded for setting up a bioprocess modeJ , na melykinet ic modelling and mass balances. This wilIlead to a description ofdifferenr types ofbioreactor operation. and bereby simple design probtems can be illustrated. Even thOllgh parameter estimation is an imporrantstep in theoverall modelling cycle. we will notconsider tbis, since the tools available for this are extensively described elsewhere.

6.2

I

Kinetic modelling 01 cell growth

All researcbets in life saences use madels when resuhs froro individual experiments are in lerpreted aod when results froro several different expe.riments are compared wü.,h the aim ofsetting up a rondel m a r may explain the differeot observations. During rhe last 10 years: rhere has been a revolution in experimental tecbniques applied in life sciences. and this has made possible (ar more detailed modellingofcellularproct'sses. Furthermore. theavailabilityofpowerful computers h as made it possible to salve complex: nume rical problems with a reasonable computational time; l'Ven complex mar.hcmaticalmodels for biological pro· cesses can be handled and experimentallyverified. However, often such detailed (or mechanistic) models are oflittle use in tbedesign ofa binprocess. whereas they mainly serve a purpose in fundamental research ofbiological phenomena.ln t bis presentation we wiII fucus on models which are usefuJ fordesign ofbiop rocesses, bur in order to givean ove.cview ofthe different mathematicaJ models applied to describe biologj· cal processes we start the presentation of kinetic models witb a discussion of model comptexity.

MICROBIAL PROCESS KINETICS

StructurillO al !he eell 'aval

Unstructured No n-segre9ll t ed

Slructured Non·segrngated

Unstructurad Segregated

Structured Seg¡egaled

6.2. 1 Model structure and model complexity BioJogical processes are per se ex:tremely complex. CeIL growth and metabolite formation are the remlt ofa very large- number of ce!lular reactiOllS ¡¡nd cvcnts like gene express ion, translation of m.RNA iuto functional protejns. further processing of proteins into functional enzyrnes 01" structural proteins. and !K'Cjuencesofbiochemical reactions leading to building blocks needed for synthesis ofcellularcomponenbi (see Chapre.r 2). lt is cJear that a t:Ompleu" descriptioll of 311 these reactions and events cannor possibly be incJudcdin a mathemalical model.ln fennen· tation processcs, wheTe there is a large populanoo of cells. nonhomogeneity of the cells with respect to activity and function may add funher to the complcxity. In setting up fe rmentation models lumping ofcelJular reactions and e...-ents is therefore always done but the detall leveI considered in che model. i.e. the degree of lumping, dcpends on the aim ofthc mode.lling. Fermentation mode1s ca n roughly be divided' iota fout groups depending on the detaillevel induded in the mode l. see Fig. 6.2. The simplest description is the so-
Oiffl!l'1!flll}'PeS of model eomple.IClty. wim Increa1ng compll!J!.ity going from me upper leh comer l
1)

132

NlELSEN

Metaboliles

Genenl representatkm of celluiar llrowth and prnduct formalioo. Viaa luge number al Jou,¡ce.ularbiochemical reJCDons. $\Ibstn~ J rE: <:oowen:OO Inro meubolic prodUC:l$. ag. emano!. 3CeUte, I~ct¡)te, orpe.nic.lllln (and other $E:co;md;vy rrw!l.lIoolites), exCr-¡celk.Jlar macromolf!Cules, •. g. I ,ecreted ~~. a heterologous protein. or a polysaccharide, an
.¡

Sobsltates- .....

Inlr.lceUular biuchcmj(aJ reaclions

}-_. . Extracellular macromorecult:~

Biomass As simple as pússible bm not simpler, This TuJe impües that the basic mecho

anisms always should be induded and that the madel strtlctuTe depc.nds Qn the airo afthe modeUing C'XerclSe.

6.2.2 Definitions of rates and yield coefficienrs Befare we [um [O describing different unStnlClured models, a few defi.. nilions are needed. Figure 6.3 is a representatioR ofthe overall conversion ofsubstrates into metabolic products and biamass components (oI' (Otal biomass). Ihe r.rtes ofsubstrate cons umption can be determined d~ring a fermentatian process by measuring the concentration ofthese substrates in memedium. Similarly. the tates afformationofmetabol ic products and biomass can be deletmined from measutements of [he corresponding concentrations. It is th erefore possible to detennine what flows into the total pool of cells and whatflows out ofthis pool. The inflow ofa substrate is nannally referred to as the substrate uptakerate and tbe outOow ofa metabolic product is normaUy referred to as the p,r oduct fonnatioo rateoProm me directtueasuJ'ements ofthe concen1:rations. ane ahtains Sa. Often it is convellient lO noI'malise che rates witb respect lO the amount ofbiomass presento since the rates hereby easily can be compared between fermentation experiments. even when the amountofbiamass changes. Such normal¡sed rates are referred to as specific tates, and t hese are often represented as r¡. where che subscript indicates whether it is a substrate (s) oI' a metabolic product (P). The specificgrowth rateofthe total biomass ís also a very important variable. and it is generatly designated /L. The specific growt.h l'ate is relaree! ro the doubling time ttl (b) oftbe biomass through:

In 2

"

,~ ­

,

(6.1)

The doubling time td is equal (O the generation time rar a cell . ¡.e. [he lengthofa cell cyde for u¡úcellular organisms. which is frequentlyused by life scientists to quantify the rate ofccll growt.h. The speciftcrates. ar the flow in and out ofthe cell. are very impor· tant design paramcters since they are related to the productivity of the celL Thus , the specific productivity of a given met.abolite directIy indicates the capacity of tile cells ro synthesise uds metabalite. FuI'thennore. iftbesped.fic raCe is multiplied by lhe biomass concentra· tion in tbe bioreactor one obtains the volu.meni.c productivity. or the capacity of tbe biomass papulation per reactor volume. In simple IUnetic models the speci1ic rates are specified as runctions oC the vari-

MICROBIAL PROCESS KINET1CS

ables in tbe system, e.g. the 5ubstrate coneentratioo!!. In more complex models where tbe rates ofthe intracellular reactiODs are spedfied as functions ofthe variables in the ~ys(em, me substrate uptake rates aad productformation rates are given as funcnons ofthe intraeeUular reaetion mtes. Another dass oC very important design parameren; are the yield coefficients, which qu:mtify the amount of substr.lte re<:overed in biornass and the merabotic products. The yield coefficients are ghre n as ratios ofthe specific rates, e.g. fm the yicld ofbiomass on a substrate: y

.,.

-~ T,

(6.21

and similarly fortheyie1d ofa metabolicproduct on a substrate: (6.3)

The yie..ld cocl'fidents are clearly determined by how the carron in tbe substrate is distributed among the different eellular pathways Cow.m1s me end products ofthe catabolkandanabolic mutes. TIlese parameters can be considered as 3n overall determination ofmetabolicfluxes, a key aspeet in modern physiologica.l studies where methods te quantify intraeellular, metabolic Ouxes have become 3n important tool in defining tbe activity of the different pathways within the complete. metabolicnetwork. ln me production oflow-value added products, e.g. etbanol, antibiotics, amino acids and baker's ye;J.st, ir is generally of utmost importance to optimise tbe yield oC product on the substrate and tbe targ('( is lheruore to direct as much carban as possible tawards the product and minimíse tbe earbon Oow to by-products (including biomass in metabolite production processes). [n this process tbe yield coefficient is the most important design parameler, baro for chatacterising different mutants and fol' characterising different fermentation schemes . Far aerobie processes the yield of CO2 from 0 %is ofien used to charaeterise rhe metabolism ofthe cells. This yield coefficient is refer.red tO as [he respiratory quotient (RQ). with complete respiration me RQis clase te I whereas ifa metabolite is focrned it dcviates fraro 1 (see also Section 6.2.4). Theyield coeffidents arealways given with a double index !hat indicates (he direction of!he. eonversion. i.e. the yie.ld for the conversion of substrate to biornass (s~~) has the index sx. Thus. the yield coeffideut y In spedfies the amountofsubstrate oonverted pe.r unit biomass foerned and , sinlilarly, tbe yield coefticient. YIIp' specifies che amountofproduet forme
6.2.3 Black box model, The simplest mathematieal present3tion oC ceU growth is the so-called black box. mode!, wbere al! the celluJar reacuons are lumped into a single overall reactiolL This implies thut theyield ofbiomass on !he substtate (as well as tbe. yield of 0111 otber compounds consumed and

133

134

I

NIELSEN

. ' ¡l-. n

41

Thupe<:lfkgrowtnrnte.

funcrlon

¡;

oltl1. ~oncltfltr.ltlon

of the IImltln¡ Wlm:ntll, s. when the MMod modells applltd.

1.0 0.8 -

-SO' _ 0.6 Q

.g 0.4 .~ 0.2 ID

ooC----CsL----c,~ O ----~'~ S ----~2·0 Subsllate concenlratlon

produced by theceJls) is constant. Consequently the specific ~ubstrate uptake rate can b(' specified as a funetian afthe ~eciflc growth rate of the biomass, simply by rewritingEqn (6.2)! (6.4)

Similarly, thespedfic uptakerolte ofother substrates, such as O2 , and the formation rate of metabolic prodllCts are proportional to t he specific growth rate. In tbe blackbox modelothe lOnetics reduces to a descrip.. tion of the specific growth rate a.s a functiOll of tlle varjables in tbe system . ln the most simplemodel description. ir is assumed that tbe re is ooly one Iimiting substrate, typicaUy che (arban souree. and the specifkgrowth rate is therefore specified as a ñ.lllction ofthe concentradon oftros substrate only. A ve.rygeneral observation for ceUgrowth on a single limitingsubmate is thatatlow substrate concentrations (c,1the specifk growth rate, p, is pl'Opol'tional with e" but roc increasing concentrations thcrc is an upper value for the specific gtowth rateoThis verbal presentation can be descl'ibed witb many diffel'cnt mathematical models, but the Olost often applied is the Monod model. which sta tes that: (6.5)

K. is the substrate concentration at which the specific growth rate is 0.5 and is sometimes interprete
HICROBlAl PROCES5 KINETICS

K, (mg 1- 1)

Species

Subru-ate

Aspergillus oryzae Escherichia col! KJebsjello pneumonioe Aerobocter oerogenes KJebsiello oxytoco

Penicilll-um chrysogenum Socchoromyces cef'evisioe

Glucose Glucose Glucose Glycerol Glucose Arabinose Frudose Glucase Glucose

Name

Kinetic expres"¡on

5 4 9 9

10 50 10 4 180

Tessier Moser Contois

e,

Blaclrm3n

J.I.=

l' ~' ( s 2K rr,al<2J(," ,

{ t"'"rm>( "

e, ~2K¡

Logistic law

Contais ldnetics, an influence of the biomass eoncentration, x, is induded. i,e. at high biomass concenttations there i.s an inhibition of cell growth.1t is unlikely that the biomass concentration as such inhibits cell growth but there may well be 3n indireeteffect, e.g. the tormation of an inhibitory compound by the biomass or high biamass concentI-ations may glve a very viscous medium that results in mass trarufer problems. Similarly the Logistic Law expresses a negative inf1uenee ofthe biomass concentration on the specificgrowth rateo These different expressions clear1y demonstrate tbe empirical nature of these kinetic models. and it is therefore futile to discuss which model is to be preferred. sinee. they are 3U simply data fitters. and Qne shauld simply choose the madel that gives the best deseriptian afthe system beiug studied. All tbe kinetic eXQressians presented in Table 6.2 assume that there is anly ane limiting substrate. but afien more than one subsrrate

135

136

NIELSEN

concentratioh lnOueuces rhe specific growth rateo In these situations, complex interactioos can occur which are difficulc to model wirh unstructured models unless many adjustable parameters are included. SeveraJ different multiparameter. unstructured models fOf growth on multiple substrates have been proposed where it is often difficulr te dis· ringuish between whether a second substrate is growth enhancing 01' limiting growth. A general metic expression thar accounes for both types ofsubstrates is: • =

(1 + L.: ~ -~~j,~-;-) rr JA.u,.x,J ' •.1 ¡ c¡i.e + K~j

! c•. j

+ K..j

(6.6)

The presence of growth-enhancing substrates increases tbe specific growth rate whel'eas the essential substrates are necessaryfor growth to take place. A special case ofEqn (6.6) is the growth in tbe presence oftwo essential substrates. el,! and e•.2:

.-

J.LUI:ax.I IJ-IllU. :.l C.,lCs.:.!

(C,.I + KI~c•.2 + K,)

(6.7)

Jfbotb substrates are ar concenrrations where tbe specific growth rate for eaeh substrate reaches 90% ofits maximum value. Le. ' ..,=0 .9 K,. then tbe total rate ofgrowth is limited to 81%ofthe maximum possible vaJue. TItis is hal'dly pra<:tical and several altematives [O Eqn (6.7) have therefore been proposed. and one ofthese is:

" . (,... <",)

Jl-mu = mm "-1 + KI 'c..2 + K1

(6.8)

Growth on two 01' more substrates chat may substitute each other, e.g. glucoSf' and laClose. cannot be describe
Po""",c~ f14 + c. + X.

and forinhibition by a rnetabolic product:

(6.9)

MICROBIAL PROCESS KINETICS

-

~ ---'+ plKt

P - ~DWtc.+X. 1

(6.10)

Equilrions (6.9) and (6.10) may be a useful way ofinduding product or substrate inhibition in a simple model . Extension ofthe Monod rnodel with additional terms or factors should. howevcr. be done witb sorne resrr.aintsince the result may be a modeJ with a large number ofpararn· eters hut oflittle value outside the range in which rhe experiments were made.

6.2.4 Linear rate equations In the black box modelaUthe yield coefficients are taken to be constanL This implies that all the cellular reactions are lumped jnto a single, QVerall growth reaction where subsO"ate is conver~ ro biomass. A requiremencfor this assumption is thar the re is a constant distnouoon of (luxes through al! the differenr cellular pathW
sperific substr
The marntenance coefficients qu antity tbe rate of substr.a te consumptioll for cellu}ar maintenance, and it is normally given as a constant. In principIe, this gives cise ro a contlict since this may resu lt in substrate con· sumption even when the substratí'. concentr
wruch shows tbat Y,,,, decreases at Low specific growth rates where an increasing fraction of the suhstrate is used ro meet the maintenance

13

:J

NIB.SEN

Organism

$ubstrate

Y'~

• ') (g,,

m, (g,,"h)

AspergilJlJ.5 awamori

Glucose

1.92 1.67 2.00 2.27 2.27 21 7 1.85

0.0 16 0.020 0.031 0.057 0.063 0.02 1 0.0 15 0.OB9

AspergiJ/us nidu!ans Candida uti!is Escherlchia coli KJebsiello aerogenes Penicillium r::hrysogenurn SocdJQromyr::es cerev!siae Aerobacter oeragenes Boci!lus megatarium KJebsielJa aerogenes

1.79 1.67

Gltcerol

}.1 3

0.074

requirements ofthe cen. For la.rge specific growth rates tbeyield coefficient approaches the reciprocal ofY:"". ie. Y", becomes equal to Y~. This cOn'esponds to!he situation where the maintenance substrateconsumption betomes negligible compared with the substr'tion fur biomass growth, and Eqn (6.12.) can be approximated with Eqn (6.4 ). !>espite its simple structure che linear GIre equation (6.12) o[Pict is found to hold for many difIerent species, and Thble 6 .3 compiles true yield coeftidents and maintenance coefficients for various microbial spooes. !he empicically derived, linear coITelations are veryuseful to correlate growth data, especially in steady smb" continuolls cultures where linear correlations similar to Eqn (6_U) are found for mos! of the important specific rates. The remarkabLe robustness and general validity of the Linear oorrelations indicares tbat they have a fundamemal basis and t his basis is likely to be the continuous supply and consumption of ATP, sincc these two pTOCCSSCS are tightly coupled in all ce.lls. Thus. m e role oC the energy producing substrate is to provide ATP lo drive both the biosyntbetic and polymerisation react:ions of tbe cell and the different mamtenance processes accO[ding ro the linear re1ationship: (6.14)

which is a formal a nalogue to the linear correlation ofPict. and states [bal (he ATP being produced is balanced by its oonsumption for growth and ror maintcnancc. lftheATP yield on theenergy-prodllcing sllbstrate is coostant, Le, r...TI' is proportional to r" ir is quite obvious thatEqn (6.14) can be used lo derive rhe litlCar correlation Eqn (6.12), y .....TP used in Eqn (6.14) is <1 true yicLd coeffident but it is normally specified without the superscript 'true', Witb the linear rate equations che cellular reactions can be 5truc-

MICROBlAl PROCESS KINETICS

tured ¡nto severnI individual reactions. ll1is cOllcept can. in principIe. be extended tocons:ider individual reactions fordifferentceUular pathways, as i1Justrated in me SonnJeimer alld Kappcli mode1 ror bakeú yeast (see
there may be a mixed m e taboli5nl with both respiranon an.d fermentation being active. At ltigh glucose uptake rates there is a limitation in the respirarory pathway which results in an overflow metabolism towards ethanol. The point atwhich the glllcose lIprake rate initiates ferrnentative metabolism is often referred to as the critical glucose uptake rateo and this is dependent on the oxygen concentration in the bioreactor. Thus. at low dissolvcd oxygen concentrarions [he critical glucose uptakf rate is Jowc.r than at high dissolved oxygen concentrations {and cJearly :¡{ anaerobic conditions there is only ferruentative mctabolism corresponding to the critical g1ucose uptake rate being zero).A model for lbis fcnnentation process can be found in Chapter 17.

6,2_5 Effect of temperature and pH The reaction temperature and the pH afthe growth mcdium ill:e other process condhions with a bealing on the growth kinetics. Ir is normally desued to keep both ofthese variables constanr (and at rhcir optimal values) throughouttbe cultivation process, hCllce lhey areoften called cuJture paramelers ro distinguish them from other variables s uch as reacrant concenrrntions, stiTri ng rareooxygen su pply rate etc. which c.ro change dralllaticaUy fram tbe start to fue end ofa cultivation. The influence oftemperature and pH on individual 001 processes can be very difrerent. and since the growth process is tbe result ofmany enzymatic proc:esses the influence ofbotb variables (or culture paramerers) on the overaU bioreacOon is quite complex. The influence oftemperarure on the maximum specific growth rate of a micrÜ"Organism is similar to thut observed for the activity of an enzyme: un ¡ncrease wl th in(reasing temperature up to a cl!rtam point where protrln de.naturation starts, and a tupid decreasc bcyond this temperature. FottempC'ídtures belowtbe onsetof protein denaturatian me ma..'l'imum spedOc growth rate increases in ruuch Ute same way as foc
~m.~ =Aexp(-~)

(6.15)

Assuming tbar tbe proteins a re temp~ture denatured by a reversible cbemical reaclian witb free energy change .ó.Gd. and thar denatured proteins are i.nal-tlve one may propase (Roels. 1983) an expression for ~nl'u:

A exp( - E, /Rll J.'=x '"

1 + B exp( - AGd IRT)

(6.16)

Figure 6.5 is a typical AIrbenius plor (reciprocalofrbe absolure remperature on the absassa and log J.l. on tbe ordinate) for Ii. eoli. The lin ear portian of lbe curve betw~n approximately 294 and 300_5 K is accurateJy represented by Bqn {6.27) while the shacp bend and rapid deo:ease

139

110

NlELSEN

"-

The Influel\Ct 01 temperature Oll the m3xlmum Jpeclfic VOwth race of EKherk;hfa ,g¡¡ SIr. {el Growth on a glLKO$erich medlum: (11) ¡rowr.h on ~

10

~

~"'"

•e ~

gNCO$e-mlnlrml medlum. The IInes

~

u

are ealculated \U1n8 the mod~ In Eqr¡ (6.18} with che pu;ameters: ~ - SSk,lmole- '. AG. '"" 550 kj

~

&

I

0.1 '.1

~

mole-',A - I Olllh- ' ,B= ].O 10'10,

~ ,., ,. ,..

'.2

1000/T t1/KI The ¡"nueoce a r pH on th tnaximum specilic growth rate of 1M r~;unentous r~us Aspet¡olllus Otyt
,,-

O.,

"S,

O.,

•o.

~

"1

o

u

~

0.2 0.1

'g o

f\

O

~

2

,

4

5 pH

•

I

7

8

9

oí JJ.. forT > 312K ("" 39 "C) sbows lhe influence afthe denominatorterm inEqn (6.16).

The influence of pH on [he cellular activity is determined bythe seositiviry of the individual enzymes to changes in tbe pR. Enzymes are nonnaliy onIy active within a c..'ettain pH range, and the toml enzyme activity ofthe ceU is therefore a complex functlon ofrhe environmental pH. As 3D example. we sball consider the influence of pH on a single enzyme which is taken to represent the ceH activity. The enzyme is assumed to exi.st in th)'ec forms: (6.17)

where e- is taJeen to be the active form of the enzyme while the rvro otber fOl'ms
l

cwr

1 + [H +IfK, + .!(2!IH+ )

(6.18)

and rhe enzyme activity is taken to bek = k~t'- . Ifthe cel! activity is determined by the activity ofthe enzyme considered above the maxJmum sped.fk growth tate will be: 1

(6.19)

Although the dependence of cen activity 00 pH cannot possibly be explained by thissimple modeLit is. however.found thatEqn 16.19) gives an adequate: fit for m;¡ny m.kro-orgarusrns. and Fig_ 6.6 shows fitofthe modeJ for sorne data ofthe .fiJamentous fungus~rgillusoryzat.

MICROBIALPROCESS KINrnCS

F,--
General rep.ttS«lt.1.tIon of ¡ bloreactor wtth ;r,ddluon crffresh, nerllll m&dium and removal of sp<ellt medlum . el, Js the concenuadon of me rm compoUlld (typkaH)' ¡¡ sulntl"i.tt!) ¡n [1If¡ f"d and dls che cOt1cenuadon ofthe rth compound in me spent medium. ne b1Or'eactcr Is ;usumed tO be verywell mlxed (or ideal), whereby the concenll"i.tion of t¡¡eh compound In tIle spent medKlm betome~ IdllntiCil I to iu concentr2t1on In me blortactor. ln sm~1I voIume blOrtKtorS « 50 1) (intluding ~hake f1asks) this can 1_1'.I1y be achi_d throu¡h aer:!ldon and ¡glCldon. ln Ilrger bloreactOl~ there mi)'. howeY
6.3 I Mass balances fo r ideal bio reacto rs The Jast step in modelJing offermentation processes is to combine the kinetic modeJ witha model forthc bioreactor. A bioreac:tormodcl isnormal1y represented by a set ofdynamic mass balances foc the substrates, me metabolic prod ucts and the biomass, whicb describes the change in time ofme concentration ofthese stal:e: variables. lbe bioreac[Qr may be any type of device ranging from a test tube or a shake tlask to a wellinstrumented bioreactoc. Figure 6.7 is a general representatioo of a bioreactor. The feed is normally assumed tO be ste.rile.. Le. me biomass concenll"auon in me feed is zero. The bioreactoc m ay be operated in three dlfferent modes: • batch, w O, i.e. (be volume is constant; • fed-batdl (or semi-batch), where F> Oand FQU ( = O. Le. me volume ¡ncreases. The mass balances for the different bioreactor modes can all be c1erived from a set of general mass balances, and we therefore start to consider tbese general balances.

6.3.1 General mass balance equations The basis for derivation of the general dynamic mass balances is [he mass balanceequation: Accumulated=Nct fonnation rate + ln - Out

(6.20)

The term AccumuJated specifies the rate of change of a compound in the. bioreactor, such as the..rate ofincre.ase in me biomass ooncentratioll

141

1-42

NlELSEN

du ring J. batch fermenlaDon . For subsrrates. tbe tenn Net formatlon r.ate is given by a substrate uptake rate (tha.t js regarded as neg-dtive being tbe withdrnwal ofcarbon from tbe system), whereas for metabolic products and biomass tlús tenn is given by the fonnation rate ofthese variables , TIIe tenn In represents the flowofthe compoundinro t he bio· reactor and the te.rm Out tbe fl ow of tbe compound out from the bio· reactor. For the ith substráte. which is added to the bioreactorvia the feed and is consu med by the ceOs presen t in the bioreactor, the mass baJancc is: d{coJV ) - - - ""' -'r xV+Fcf-F

dr.

IJ

.J

In"r

_ 'J

(6.21)

The first term in Eqn (6.21) is the accumulation term, the second term accounts foc su bstrate cOll5umption (or net farm ation), the (hird tClm accounts foc the ¡nlet, and the last rerm aCCOlmts for rhe oudet. Rearrangement ofEqn (6.21) givcs; dc,j

F ,

(Fout 1~

Tt=-r"ix+V'·j- V .+Vdt¡'· j

(6.22)

Since for a fed-batch reaLtor: dV dr

F--

(623)

and PO Dl = O the term within tbe parentheses becomes equal to the socalled dilUtiO D rate given by: F

D~­

V

(6 .24)

Fo!' both a continllOUS and a batch reactor, che volume is constant. i.e. dV¡dt=O. and F=F"u,' and also foI' mese bioreactor m odes tbe term witbin tbe parentheses becomes equal to che diLucion cate. Eqn (6.24) thetefore reduces to Lbe mass balance (6.25) foI' any type of operatioo . dco.l _ _ f d~ - T~x + Dfc'J - 'I~)

(6,25)

The first tenn on the righthand sideofEqn (6,25) is thevolumetric mte ofsubstrate collSumpdon, whicll is given as the product ofthe specmc tate of substrate consumptioll and the biomass concentration. The second term acrounts for the addition..and re moval of substr.1te from the bioreacror. Oynamic mass balanl'es for the metabolic products are derived in analogy with those fo! me substrate5 and ralces tbe form:

~ = -T .x +D(cf -e ) dt '.' p.! p.!

(6.26)

where the first term on the right hand side is thl! volumetric formation r.Ltf oftbe ¡th metabolic pl·oduct. Normally the meraboLic products are Dot present in thesterile feed (O the biorcactor and c:¡is therefore ofien zero.

MICROBIAL PROCESS KlNmcs

With sterile feed me man balance for the total biomass is:

dlxV)

-- =~V-F

dt

16.27)

:Ji

0111'"

which in analogy with thesubstrate balance can be rewritten as: dx - :(,,- D)x dI

(6.28)

6.3.2 The batch reactor This is the classical operation ofthe bioreactor thac is use
dx

-=IJ..)C~t= {)) = ;( dt' II

(6.29)

d~

-de =- rlt'c( r=o)=c•.0 r' •

(6.30)

According ro these mass balances the biomass concentranon will increase and the substrare concentration will decrease until its coneentradon reOlches 'lero and_growth stops_ Assuming Manod kinetics, tlle mass balances for biamass and rhe lirniting substrate can be rearrangcd into one first-ordel' differentiaJ equation in [he bio1ll3SS concentration a.nd an algebraic equation relating the substr.Jte concentratioD to tlle biomass concentl'arion. The algebraic equanon is given by: (6.31)

and the saludan to the dlffe.rential equationfor [he biomass concentration i5 given by: P. n=r

f;=

- (1 + c"o +K.Y.,.}Io) In (X) XII

(X,-X)

- - K,- -In 1 +- Cl,lI

+ Y~,X()

Y",

c"o

16.Ja)

Using these equations [he profLles oftbe biomass and the glucosc con· centrations dllring a typkal batch culture are easily derived. as shown in Fig, 6,8. Since the substraJe cOn<:entration is zero at the end ofthe culo tivation the overall yield ofbiomass on the substrate can be fOllnd from: {6.33) C.,O

1"3

14..

NIElSEN

Simulatioo of the bloman and g!U(o~" conClll1traoon durlng a batch cultur". The slmuiadon ha, becn camed out U'Qlg the MOIlod modei wjth /ot.",. .. 0.5 h- J• K, = 0.05 ,1- 1• and Yu; =

o.so.

,0

'0

B

:L

1.S' 6

!

4

'"

2

"',

O O

E!

•• •E

0.1 o ¡¡; 0.01

5

10

Time th)

NormaUy xI) <::x linaJ '
6.3.3 The chemoscat A typlcal operationofthe continuous bioreactor is tlle scx:alled chernastat, where the added ll1ediurn is designed such that there is a single lim.iting substrate. This allows for controlled variarion in the specific growth rate ofthe biomass. Byvaryingthe feed flow rate to the bioreactor, the environmental conditions can be varied and thereby valuable infonnation concemmg tbeinfluence afme environrne ncaJ conditions on tlle cellular phYslologycan be obtained. For industrial applicadons. rhe continuous bioreactor is atO'active since the pl"Oductivity may be higb . Howeve r, often me titre. l.e. the product concentration, is lower than what can be obtained in the fed-batcb reactor. Furthermorc. it is ra.rely used in industrial processessince ir is sensitive to contaminarian. e_g. via the feed stream, and to tbe appear.mceofspontaneously formed mutants that may out-compete the production ¡¡train. Oth~examples ofcontinuous oper.ltion besides the chemostat are the pH·stat, where tbe feed flow Is adj usted to maintain fuE' pH constant in the bioTc3ctar,

HICI\OBlAl PROCESS KINETICS

and tbe turbidostat where tbe.feed flow is adjusred ro maintain (he biomass conce.ntration at aconstantlevel. Frem tbe biornass mass balance (6.40). ir iseasilyseen that in a steady state continuous reactor tbe specific growth mte equals the diluoon rate: p,=D

16.34)

Thus, byvarying tltedilution rate(ortbe feed fiow .rate) in aconnnuous culture differe nl speciftc growth rafes can be obtained. TIris a1lows derailed physiologic
which uponcombinatioo wilh Eqn (6.34)and rhedefinition oftheyield coefficient directly gives: [6.36)

Thus, theyield coefficientcanbe determined from mearurementofthe biomass aod the substrate.concentrutions in the bioreactor.From measurements of the substrate concentration aud the biornass concentra· non al steady rute the specific glucose uptake rute can easily be calculated using Eqn (6.35). and similarly th e specific rates ofproduct formation can be determine
16.37)

0'

'.

DK, Pm",, -D

(6.38)

lbus, [he coucentration of [he limiting substrate ¡ncrea.ses wilh the dilution rateoWhen substrate concentration becomes equal ro che substra te concentration in tbe feed the dilution rate attaios in maximum value, which is often called [he critical dilution rate:

O

-

c:ri, -

~

Pm..~cr + ~

(6.39)

When the dilution tate becomes equa! to 01' larger thao this va tue the biolUass i5 washed out of the bioreaclor. Equation (6.38) c1early shows thar the steady slate c.hemostat is well suited to study the influence of che substrate concentranon on the cenuJar function , e.g. produce formation, si nce by changing tbe dilution tate it is possible to change the substrate concentration as the on1y variable. Furtbennore, it i5 possible to study the influence of differe.nt limiting substratts on lhe ceHular physiology, e.g. glucose and arnmonia.

1<15

1-16

NIELSEN

Besides quantification ofthe Monod parameters the chem ostat is weU suitcd to determine tne maintenance coefflcienc. Since the dilution rate equals che speciflc growth rateo combination ofEqn (6.13) and (6.36) gives : D

,'( - YV'i'"D + m.,(ci- c,)

(6.40)

which shows ehat che biomass concentration decreases at low speci.fic growth rates, where the subsu-ate consumption for maintenanc:e is significant compared with ehar for growth. At high specific growth rates (high dilution rates) Ulaintenance is negligible and the yield coefficien.t becomes equal to the truc yield coeffident, see Fig. 6.10. Since IL =D ar steady state, Eqn (6.12) expresses that there is a linear re Jatio n between the specific substrate uptake l'ate and the dilution rateo In this linea r relationship the true yield coefficient and th e mamrenance coeffidenr caneasily be estimated using linear regression. For proouction ofbiomass. e.g. baker's yeast or single ceU protein. and growth-related products the chemostar is verywell suited since ir is possible ro maintain a high prOd\1Ctivity over very long periods o( oper' arion. The productivity ofbiomass is given by:

P=Dx

•

(6.41)

ando in Fig. 6.9, (he productivity is shown ilS a function ofche dihltion rare.By inserting the expression fur the biomass conCentraDOn (6.40) in Eqll {6.41 1, with Eqn (6.38) il15erted fuI' the substra(e concentration. it is possible to calcnlate che dilution rates which give che maximum productivity. lf chere is no maintena nce, ¡.e. m~ =0, the opümal dilution [ate is giwn by: D"I'" =

~mn(1 - ~G; KJ

(6.42)

is imponant to emphasise thar this optirnum ooly holds for Monod kinetics without mamtenance. Wben maintenance is induded finding tbe optimum dilutioo rate will involve solving iI third-degree polynomial. This polynomiaL will have one saludon in me possible range of diludon Tates. However. instead ofsolvingihe tbird-degree polynomiaJ it is generally e:asie:r to find m e solution numerically. [t

6.3.4 The fed -batch reac.tor l1lis operaDou is probably the most cerumon operariou in industrial proce5ses. sínce it aJlows for control ofthe environmental conditions, e.g. mainraining tbe glucose concentration at a certain level; it also enables formatiOIl of very higb titre5 (up to several hundred gr.uns per litre ofsomemecabolites). whicb is ofimporcance forsubsequentdownstre
,---

10 -

MICROBIAl PROCESS KINETlCS

.

Oilution rate (h -\) GrowU\ of KJcbslda pMa/morllof (AerobCicter C1t!/Qfent$) in a chemmt3t wIth glycllrol as rlMollmlting ~ubs'tl'ale. TIle blomau concentratlon (.) decrus~ at low rli luti on r:l1:ftl; ¿ue tO ~ m::o.lnterl3nCe met:lbollsm, and when Eh .. dilullo n r:ate3ppro3cnes the crtdcal vJlue me bklmul concentr~t¡OIl decreale~ r.tpid ly. Thellycerol concentntion (.A) increases dowly al low dilu tlon rates. but when the dllutlon rate ~pproathe$!he critical ~alue it incruseJ mpldly. Th e IIne$ are modelsimul3tioru U$ing!he Monod model wI'th malntenance, and wltil pan.meter ~lue l: e: '" 10 &1- 1: /l""" = 1.0 h- I; ". =-0.0 1 It 1 1'0 ; m," 0.08 ,,-l. h; Y.":,, "" 1.70 11- ' . The broken Wne i~ the produt.tMty accorrli ng to

me

Eqn (6.41 ).

tbefeed is a very concentTllted solution , and the fee
V d'

(6.43)

and ifDis lo be keptconstant there needs tO bean exponencially inacasing ~ed fl ow ro the bioreactor. Iftbeyield coefficient is CODstant combination ofthe mass balances for the biomass and the substrate gives: (6.44)

orsincep.=Y.. r.

di' -

y.(<( - ~»

dI

(6.45)

Th.rough combination witb Eqn (6.43) this differendal equalion can easily be solve
(6.46)

where xo' eJ.o and Vodefine the biomass conce.ntration , the substrate con· centration and tbe reactorvolume at the startofthe fed-batch process. As mentioned aboYe the substrate conc~tration in tbe feed e~ is normally very high and much higher than both the iniria1 substrate

147

148

NIB..5EN

concentration and the substrate concentratíon during tbe process (C.), Furthermore, a very high c! means that Y is larger than [be biomass concentration, both initiaUy and during the proeess. Consequently the ¡nerease in volume can be kept low even when tbere is a very largc increase in the biomass concentration. If there is an exponential feed f10\\' to che bioreaetor there will be substantial biomass growth a nd, SLnce the biomass concentration ¡ncreases, this may lead to limitations in Che O}supply. The feed Oow is therefore t:ypically increased untillimitations in tbe O} supply set in and thereafter tbe feed flow 1s kept constant. lhis wiU give a decreasing specific growth rateo However, since the biomass concentration nor~ mally will.increase. the volumetric uptake rdtc ofsubstrates (induding oxygen) may be kept approxirnately constant. From the above it 15 dear {bat mere may be many different feeding strategies in a fed·batch process and optimisation ofthe operaDon is a complex problem tbat is difficult te solve empiricaUy; and, even when a very good process Olodel i5 available. calculation of the optimal feeding strategy i5 a complex optimisation problem, In an emprncal search fOT rhe oprimal feeding policy the two most obvious criteria are : (1) keep the concentration of {he liruitingsub5trateconstant. and (2) kcep the volumetric growth rate oftbe biomass (or uptakeof a given substrate) constant. A constant volumetric growth rate (or uprake of a given subsn-ate) is applied if there are limitanoos in the supply of oxygeD or in heat removal. A COD5tant concentration of the timiting substrate is often applied ifthe substrate inhibits producr formation, and tbe chosen concentration therefore depends on tbedegree ofinhibition and thedesire te maintain a certaingrowth ofthe cells. The required feeding profile to maintam a constan t 5ubstrate concentration c•.ocorrespondlng to a constant specific growth rate iJ.o is quite simple to derive:. From Eqn (6.27)

uc!'

withFOU/ = O,

d(xV)

(6.47)

Tt=J.I.oXV oc

(6.48)

Since me: substrate cont:t!Iltration is constant the 5ubstrate balance gives: (6,49) 0,

(6.50)

Finally, the biomass concentration I'(t) is obtained from Eqn (6046) with.

c. = (,.o:

:!lE "" Xo

e¡z[X

1

QX(I

+ axoei'tlI

(6.51)

MICI\OBtAl PROCESS KINEnCS

~

..

0.3

lOO

,

~.s 80 t<. e o

,E

-Bu-

''= "O 60 O,

(.). me bloreaetOl'

0.2

yolume (_ ). ilnd me l'eed tIowl'iltt (A ) rOl" iI fed·batch ructor opel'ilted wim iI constant wbstratll concencl'ildon. The yleld

0.1

speelflc growth I'iItI! ~Q;5 0.0211 - 1• ¡lid the !ubstraU! concentrarlon I.n

e o

coefflclent Y" Is 0.5, mI! cOl1st:¡nt

..

o ' 40 oo ~ o.~ .~

the~ ,: ls400kg

E o 20 o •

~

The blorTQSS

~on

O O

, 20

40

60

m- l. Tlle

$ubstn~ concew.ndon 15 assurned

80

tO be much leS! !han é,. The Inltlal biomass concentntion; O\I'Id the Inltlill bloreactor voJume are alcen

O lOO 120

Time (h)

to b!!! 10 kgm-1andSOm1• respectively

wbere (6.51 )

The bioreactor volume is given by: V ....... =l - QX +ax t"Ot V o

o

o

(6.53)

Figure 6.10 illustrates typical profiJes for the biomass concentration. the bioreactor volume and the feed tJow rafe during a fed-batch process with constant substrate concentration. Fed-batch processing is applied to pl'Ocesses where control ofculrure condítioos is required. m
6.4 I Further readi ng Herbert. D. (1959). Sorne principies of cantinuou s culture. RtCCTÚ- PTOg. MkTobfo!. 7. 381 - 396. Monod. J.(194l). R«hm:htS$&Ir la Crolssana lks Cultures B!lcurlmf1C5. Hennallll er Cie, Pzris. Monad. J .. Wyman.j. and Changeux.j.-P.(1965).). MuL8 tol.12:88-118 Nielsen,J. and VilladseD.). {1994). BioTC'amon Engtllt mn.1; Principle•. Plenum Preu, NewYork. Pirt, S.J. (1965). The malntena.nce energy ofb:u::teria in growing cUltureS. Proc. Rvyql SoCo I.ondon. Sm

a B 163. 224-231.

RoeJs. j. A. (1983). fnergctfa ond Kinmcrill Biotechllulogy, Elrevier Biomed.ical Press, Amsterdam. Sonnleirnl!l", B. and Klippeli, O . (1986). Growtb ofSIU(lu¡rum)Us reTtvbit\c.> js con· trolled by its lintited I"C!spiratory capaciry. Biortch nal, mOt"n,g. 28.927-937. Nil"lsen.J . a.nd Aristodou. A. (1998). Mem!:Jollc Eng intering. Srephanopoulos. Pr1l1clpl~s und MethodoJogla. Academic Press. San Diego.

e..

149

Chapter 7

I

Bioreactor design Yusuf Chist i and Murr ay Moo -Young Nomendacure 1ntroduction

Bioreactor configurations Biort-ilctor de~ign tea tu res Design fOI sterile operation

Photobioreactors Heat trailifer Shear effects in culture Further reading

I

Nomenclature

Roman lener.! cross"5fftional area oftht' downcomer (m') AH .uea for heat transfer (m") A, [[oss-section;¡! area afilie mer (m") a paramctef in equaüon (7.8) (-) A~

ClP clean-in-place

specific heat capacity ofthe broth tI kg-!."C) dimensionJess constant (- ) cilaracteristic Jength dimension (m) di diameter ortbe impeller (m) d p partide dlameter (m) dr d iameter ofbubble COlUffill DI tan1:: (m) d", rermenterwall thic:kness (m) R energy dissipation rate per unit mass offl uid Ukg 1) Gr Grashofnumber(-) gravitational acceleration (m s ' 2) K jacket side fuul ing film heat trausfer coefficien tO rn - 2 ."Q film heat transfer coefficient fur the cooling water fil m o n thejacket side Uro - o."C) hL height of gas·free liquid (ro) 110 broth film heat transfur coefficient m-1."q lo: paramelM"inequation (7.8) (m-1) Impeller-dependenrconstant(- ) lc.r thermal conductivityofthe cullure brothO m-I."e¡ 1.:", thermal conductivity ofthe fermenter.waU U m-I.0C)

e,

,

,

a

"

mean length afilie energy dissipating Huid eddy (m) N rotadonal speed oftbe Impeller (S-I) Nu Nu!sel t"Ilumber(-) 11' Ilaw behaviour indexofa 8uid (- ) f' powerinputingas.freestateffi Po power input in presenceofgas01 Po pawernumber(- ) l'r Pralldtl number (- ) Q volum(o: flow [".lte ofg¡¡s tmJ s-') <4. heat.transfer r;¡te Os 1)

RE

Reynolds number(- )

impeUer Reynolds nUlnber (- ) SG sigbr glaS3 R ~,

4T temperature difference (oC) superficial gas velocity based on che total cross-sectional area ofthe vesseJ {ms 1) Ue , rruperfidal velodty ofgas i.n ruer (m S- I)

U..

UU orer.dl beaTtr::losfer coefficientO m- l.oq Vl ~"Uperficial UC)uld ~locity{rus- I I Vt volumeofliquidin there.u:tor(m 3)

Greek lerten

fJ

roefficient ofvolumetric expansiOll (ro' kg-I ."q average shear tate (s-') Y"""", maxiInum sbear fati! (S- I) et volume franion ofliquid (- ) j,Lt vis¡:osityofliquid (kgm - 1·s)

l'

J.L.., IJ,....

Pt

.,

viscosityofwarer{kgm-1 ·s) viscosity ofliquid at wall temperaturc (kg m- l·s) de.nsity of]iquid orslurry (kg m l )

shearstress (N m-2 )

7. 1

I

Introducti on

BioreacwtS, or [ennenten, are the core of any biotechnology-based produrnon process be it for vaccines. proteins. organic acids. amino acids, anttl>iotics. enzymatic or microbiaJ biotransformatioDs, bioremediation and biodegradation, and microbial inoculantsfor use as biofertilisers . Biocatalysts. micrQ-(Jrganisms. anima l 01" plant celIs. are produced iIlld maincained in bioreactors. A production faci Jity typically bas a train ofbioreactors ranging fram 20 litres to 250 m 3 _ Stilllarger ~ssels are encounrered in cerra¡o processes. In a grea t majority ofprocesses, the reactors are operare
BIOREACTOR DESIGN

sterility considerations, vessel design and surfuce finishes, clean-inplace issues, and aspects ofbioreactor performance are ofien coromon to bioreactoI'll_ The bioreactor types used extensively are the airlift, stirred tankand bubble column bioreactor_

7.2 I Bioreactor configurations 7.2. 1 Stirred tank reactors

D--CJ

Stirred tank bioreactors consist of a cyJindric-dl vessel with a motordriven central sbaft thatsupports one or more agitators. The shaftmay enter through the top oc the botlorn of the Teactor vessel. A typical stirred tankreactoris showninFig. 7.1. Microbial culturevessels are generally provided with four baffles projecting into the vessel from tbe walli to prevent swirling and vortexing afthe tluid. The baffle width is Ylo oc Yu of the tank diameter. The aspect ratio (Le. height-to-diameter ratio) of the vessel is 3-5, except in animal ceU culture applications where aspect ratios do not normally exceed 2. OCten. the animal cell culture vessels are unbaffled (especially small-scale reactocs) to reduce turbulen<.Oe thar may damage the cells. The number of impeUers depends on che aspect ratio. The bottom impeller is located at a distance abont % afthe tank diameter aboye the bottom of the tank. Additional impelleI'll are spaced approximately 1.2 impeller diameter distance aparto The impeller diameter is about ~ ofthe vessel diameter fue gas dispersion impellers sueh as Rnshton dise turbines and concave bladed impellers (Fig. 7.2). Larger bydrofoil impellers (Eig. 7.2)with diametecs oí 0.5 to 0.6 times the tank diameter are especially effective bulk mixers (,)

8affle

o--!-c>-1r i(npaller sparger-\t::::tlt--.Y

L ..

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bloreaaor.

(o)

Sorne commonly usad imP<"!hm: (al Rusllron disc wrbine;

(b) a.co n(:i\ve bladed wrbine: (e) 3 hydrofoil impeller; and (d) prop0ller.

(d)

a marine

153

5i

CHISTI ANO MOO· 'rOUNG

and are used in fennente.rs fur highly viscous mycelial broth~. Animal cell culture ve~sels typically cmploy a single,large diameter. low-shear impellcr sucb as a marine propeller (Hg. 7.2). Gas is sparged into the reactor Uquid belm" tbe. bottom impeller using a perforated pipe ring sparger with a ring diameter that is sUghtly smalle r tha.n that of the. impcller. Alternatively. a single hole spargermay be used. in animal or plant cell culture applications. the impeller speed generally does nor exceed about 120 rpm in vessels larger than abour 50 litreS. Higher stirring rates are employed in microbial culture. except with rnycelial and filamentous cultures where tbe impeller tip spe.ed (ie. 3.143 X impellerdiameter X speed ofrotation) does notin general exceed 7.6 m S- l. Eve.n lower speeds have been documented to damage certain mycelialfungi. The superficiaLaeration velodty(i.e. the volume flmvrate ofgas divided by the cross·sectional area ofthe vcssel) instirred vessels must remain below tbe value needed to f100d !he impeller. (An impeller is flooded when it receives more gas [bao ir can effective1y disperse.) A floocled impeller is a. poor mixer. Superficial aetation velocities do not generally exceed 0_05 m S- l.

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A bubble column bioreac.tor is shown 00 Pig. 7.3. Usually. the column is cylindrical with an aspecr ratio of 4- 6 (height-tcxliameter). Gas is sparged at tbe base ofthe columo through perforated pipes. perforatcd plates. 01' sin te red glass 01' metal micro-porous spargers. 02 transfer. mixing and other performance factors are iniluenced munIy bythe gas Oow rate and the rheoLogical properties of the fluid. Internal devices sud as horizontal p('rforated plates. vertical baffles and corrugated sheet packi.ngs may be placed in the vessel to improve mass transfer and rnodify the basic.:design. The colum n diameterdoes not affect its behaviourso long as the diameterexreeds 0.1 m. Oue exception is the axial mixing performance. Por a given gas tlow rateo the Illixing improves witll increasingvessel diameter. Mass and heat transfer and the prevailing shear rale ¡ncrease as gas fiow rate is increa.sed.. (n bubbLe colurnos the maximum aetation vclocity does not usually exceed 0.1 m S- l. The liquid flow cate does not iniluence tbe gas-liquid mass transfer coeffidentso long as the superficialliquid veloc.ityremaim below 0.1 m S-l.

7.2.3 Airlift bioreactors In airlitt bioreactors. the fluid volume of the vessel is divided into two interconnccted rones by means ofa baffle or draft tube as shown inFig. 7.4. Onty one of [he two zones is sparged with air or other gas. The sparged ZOlle is knowTl as tbe riser; the zone that receives no gasis the downcomer. The bulk density of the gas-liquid dispersion in the gassparged J:iser tends to be lower than the oulk density in the downcomer. consequen tly me dispcnion f10ws up in lhe riser ZQne and down.flow occurs in che downcomer. Sometimes the riser and the downcomer are 1WO separate vertical pipes (hat are interconnected..at the top and the botrom to form an externa} circulation loop. Far optimal gas-liquid mass transfer performance. the Liser-rooowncomer CToss-sectional

BIOREACTOR OESIGN

(e)

155

A1r1lh blorUCtOI'1:

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area ralio should be between 1.8 and 4.3. External-loop airlift reactors are less common in commercial processes compa.red te the interna.lloop designs. TIte imernalloop configuration may be eitber a concentrie dr.út-rube device or a split cylinder. Airlift bioreactors are highly energy-efficient relative to stin'ed fermenten, yet t he productivitics of both types are comparable. Being espedaI1y suited to shear-sensirive cultures. airlift devices are afien e mployed in largc-scalc manufacture of biophannaceutkal proteins obtained from fragUe animal ceUs. Heat and ma$s transfer capabilitie.s of airlift reactors are at least as good as those of otber systems. and airlift reactors are more effective in ruspending solids than are bubble columns. AJI performance characteristics ofairlift bioreactors are linked ultima tely ro r.be gas injetuon rate and the resulting rare ofliquid circulation. In genera.!. the Jale ofliqui d circulation increases wíth the square root of tbe heiglH of the airlift device. Consequently. the reactors are designed with high aspcct ranos. Because the liquid circulanoo is drive n by [he gas hold-up difl'ere ol..1i': be.tween the riser and the clowncomer. ci.rculatiou is en11allced if tbere is little or no gas in the down· com er. Al] the gas in the downcomer comes fram being entrained in with che liquid as it ftows into the downcomer from the riser near the top of the reaCf.Or. Various designs of gas-liquid separ.!tors are sorne{imes used in m e head zone ro reduce oreliminate the gas carry-overto m e downcomer. Re1ative lo a reactOr without a gas-liquid separ.Jtor. illstallation ofa suitably designed separ.Jmrwilla lways enhance liquid circulatiOD. i.e. the merease
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th;;m compensate for any additional resistance to flow due to the separator.

7.2.4 Fluidised beds F1uidised bed bioreactors are suited to reactions involving: a fluidsuspended particulate biocataLyst such as me hnmobilised enzyme and ceU partides or microbial fiocs.An up-Oowing stream oftiquid is used to suspend or 'f1uidise' che solids as in Fig. 7.5. Geometrically, che reactor is similar [o a bubble column except that the top section is expandec1 to reduce che superficial velocity of the Ouidising liquid ro a level below titat nceded [O keep tbe solids in suspe.nsioll. COllsequently, the solids sedimentin the expanded zone and drop back into (he narrower reactor column beJow; hence. the solids are retained in the reactor whereas the liquid flows out. A liquid fluidised hed may be spa:rged with air orsome O[her gas to produce a gas-liquid-solid Huid bed. lftbe solid partides are too light. tbey may have to be artificially weighted. for example by embedding stainless steel balls in aD otherwise Iig ht solid matrix. A high density of solids improves solid- liquid mass tt"ansfer by increasing the re[ative velocity belWCen the phases. Denser solids are also easier [O sediment

BlOREACTOR DESIGN

TI

but me densi(}' should not be too high relative to that ofme Iiquid . or fluidisation wilJ be difficu)t. Liquid Ouidised beds tend ro be fuirly quiescem but introduction of a gas subsrantially e nhances turbuJence and agitanan. Even withrclatively Iight partides. the superfidal liquid velocity needE'd to suspend mE' soJids may be so high that the liquid lE'avE's tbE' reactor much too quickly, i.E'. thesoLid- liquid contact time is insufficient for tbe reaction. ln mis case. me Iiquid may have to be recycLed to eruure a suffidently longcumularive contact time with the biocatalyst. The minimumfluid¡sacian velocity - t.e. the superficial liquid velocity needed to just suspend the solids from a settled state - depends on severa! factors. induding the density difference between (he phases. the dia.meter of the particles. and theviscosity ofthc liquido

pad<¡ng

7.2.5 Packed bed columns A bed of solid parricles, usually with conflning walls, constitut.es a packed be
matrix ofsolids tharrnay be porous or a homogeneous non-porcus gel. The solids may be particles of compressible polymeric Or more rigid materiaL A fluid conraining nutrienr.s flows continuously through the bed to provide the needs of the immobilised bioca.talyst. Metabolites and products are released into the fluid and removed in the outflow. The flow may be upward or rlownward . bur downflow under graviry is the norm. Ifthe fluid Oows up the bed. the maximum flowvelocity is limited because the velocity cannot exceed the minimum fiuidisation velocity orthe bed will fluidise. The depth of the bed is limited by several (actors. including the density and fue compressibility of the solids, rhe need {O mainrain a certaifl minimal leve! of a critical nutrient. such as O~, through the entire depth. and the fI.ow rate mal is needed Cor a given -pressuredrop. For a given void volume (Le. solids-fi·cc volume fraction of che bed) che gravity-driven.flow rate tbrough the bed dedmes as thedepth ofthe bed toereases. Nutrienrs and substrates are depLeted as lhe fluid moves down the bed. Conversely. cODcentraUODS of metabolites and products iucrease. Thus. tbe environmem of a packe
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¡ biore.:lctor.

157

158

CHt5TI AND MOO-YOUNG

7.3 I Biore.ctor design fe.tures

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A typical b ioreaetC>r.

( 1) reactO r vessel; (2) jacket;

(J) insubtlon; (-4) ~roud: (S) itloa.olum COmfleOon; (6) polU for pH, temperawreand dlnc>lved oxygM senSOrl; (T} aglt>ltor; (8) gas sp~rger: (9) mer.hanlal ~a!s; (10) n1d ucitlg gurbole: (11 ) mexor; (12) harvest nozzle; (13) Jadoot connecdoM 5;; ( 14) u mple ,.., I~ wlth ltl!am

co nnecrion; (IS) 51g1lt glass: ( 16) connecliom ror a<:id, ¡11eaI¡ and antifoam chtmkals; ( 17) air ¡nlec (18) remcwa.ble top; (19) medium o r leed oouJe: (20) ,Ir exm.ust nouJe; (2 1)

~mTument

poru;

(several); (22) foam breal(U; (2.1) sigile glm With nght (OOt shown) an d Stllam connccllon:

(24) rupl~ dlsc nonte ,

lrrespective of the speci6c bioreactor configuranon use
The reactor vessel is invariably j3.cketcd. In the absence of especial requirements. the jacket is designed ro the same speci fica tions as the vesscl. !he jacketis covered with chloride-free fibreglass insulacion lhat i5 fu lly cnclose
7.4 I Design for sterHe operation Mast oommercia! fermentation processes are mouo-cultures. To establish and maintain aseptic conditions are vital for the success ofthese processes. Hence, a bioreactormustbe sterilised priortoinoculation and contaruÍnation during operation must be prevented. Contamination during culture i5 a common cause of process failuIe.

7A.I Sterilisation-in-place A bioreactor iatended fOT in sih.l sterilisation requires a complex arrangement ofpipework. valves. aod filters to e.nable iniual sterilisation aod maintenance ofsterility. A typicaJ arrangentent for in situ sterilisarion is shown in Pig. 7.8. Recause almost all biopharmaceutical production processes involve aeration. the figure ¡neludes aerarion and exhauSl groups that must also be sterilised. 111e air ¡olet and exhaust Unes have in sítu steam·sterilisable gas filters. Typic:a1ly. hydrophobic membrane cartridge filtcrs are used. These filters are rated for rcmoving partides down to 0.45 ¡.¡.m or E"ven 0.1 ¡..Lm. Often lhe gas screams have two filler cartridges in series; with the first seIVing to prorect the final filter. A good system is designed so that tbe different sections can be stcrilised mdependently of any of rhe others. thus steriHsation of any section durmg fermentation can be carned out i.f. and when. requrred. Saturated dean steam (1.1-1.4 bargauge) is lIsed for sterilisaUon. The air inlet and exh:mst groups are sterilised first, and then. in a second step, tbe bioreactor. The system is designed so that che filters. valvcs and the assodated pipe-work reach sterili$ation temperature (- 121 °C) very quic.:kly (-1 minute). and are held at the temperature Cor the required time l25-30 minutes). Apartfrom theharvestvalve, allothervalvessbown in Fig. 7.8sho uld eithe.r be diaphragm orpinch valves.The harvestvah-e is usually a pistan valve witb a metal beLlows se<:lJed stcm. The valve doses fiush with the internal sucfuce of the bioreactor and tbere js an unobstructed flow

60

I

CHISn ANO MOO-YOUNG

A biore:actor with alr

inlet and ewustgroups arranged

elean steam

_ ~" Fllter Exhaust

for In-place sterillsadon wlth

steam.

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path through thevalve body. The valves maybt': operated manually, but pneumatic operation under automatic control is more efficient aud reproducible. '!be bioreactor is sterilised either filled with the mffiium orwitbout.Empty sterilisatiou is the norro in cell culture applications where the media are invariably heat sensiti~. In this case. filter sterilisation is osed to srerilise themedium. Proper closing aud opening sequence ofthe various valves is important for attaining sterility aud I?Teventing recontaminatiOIl from the adjacent non-sterile areas. Once the sterilising steam supply is shutoff, the bioreador is immediately pressurised with sterile ah' through tbe air inletfilter so that any leakage from tbe outside to the sterile vessel is prevented. In bioreactors with stirrer 01' foam breaker shaft penetrations. the shaft seals require. suitable. piping and valves for steam ste.rilisation and maintenance of a sterile barrier fluid betwee.n the·('ontents of the fennenter and the outside.

7.4.2 Clean-in-place considerations Industrialbioreactors and much ofthe other processing equipment are deaned in-p11ce using automated methods. Automation ensures consistency of c1eaning and reduces down-time (Le. unproductive time of a machine). Attaining an acceptable state of cleanliness is essential to prevent contamination and IToss-contamination of biophannaceuticals and tbod products. An effective and trouble-free cleaning capability requires atlention to design ofthe bioreactors and the dean-iD-place (ClP) systems.At any given time a plant may have. several bioreactors at differentstages ofprocessing and some emptyreactors which need ro be cleaned along with any associated transfer piping. The CIP devices and procedures must be matched to the speci.fic configuranon ofthe biareactor and to the ferme.ntation process to emure satisfactory cleaning. GeneraUy, a bioreactorwhich has processed hybridoma or othe1' animal ceU cu.lture broth is far easier to dean than one whic.h has processed broths oí Srreptomyces or rnycelial fungi such as PenidUium.

SIOREACTOR DESIGN

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Design aspects To ensute removal of solid particles and avoid sedimentation. the minimum fiowvelocity through piping sh ould be 1.5 rn 5- 1. but ahigh~ value of2.0 m 5 - 1 ¡s preferred. ln addition , the piping should be free of dead spaces as much as possible; ifunavoidable, the depth ofthe dead zone must be less than twopipe diam eters te ensure adequate deaning using CIP tcch.niqucs. OnJy valves with a metal·bel1ows-sealed mm . or diaphragm and pinch valves are recommended as all othervalves carry a significant risk of contaminating reactors with accumulated debris during the final rinse cycle. For adequatecleaning. the ClPsolutions are sprayed into the reactor through one or more removable. static or dynamic.spray baBs. ordynamic spray nozzles (see Fig. 7.9). In addition. the piping fo!" air exhaust. which 15 upstream oftbe exhal1st gas fiI{er, and the air ínlet piping. shouLd also receive the deaning 5OJutions. For deaning with jet spray. pressuI't$ of 308 ro 377 kPa (absolure) are optimal Permanently installed spray heads are no( reconunended for bioreactors because oi potential difficulties with steriJisation, These devices must be inserted into the reactor through one of [be ports on the head plate. Ofien. the spray heads are designed to spray the upper one-third ofthe cankand the falling liquid film inigates the remaining sw:face. For bioreactors for parente!"al (inject:able) products and othcr biopharmaceuticals. potable quality deionised water i8 recomlnended for all pre-rinsing and detergent fonnulations. Pre-rinse should be on a once-through basis witbout reciI"culation. A five minute pre-rinse is usually sufficient for bacterial, yeast and animal ceH culture reactors. Following pre-rinse. 1% (wfv) NaOH ar 75-80 ~C should be circulared through the equipmentso tharall productcontactsurfaccs are exposed ro this solution for 15-20 minutes. TIte alkali should be discaroed afterwaros. Dilution. contarnmation withsoil and microbial spores that can survive for long periods aud los5 of quality definition of che starting material far the Dcxt deaning. are sorne ofthe arguments against l'e-use of c1caning chemicals. A deionised or reverse osmosis water rinse al 25- 35 oC is used to remove all alkalifrom the system. Process equipment

DeI..... ry 01 me dean-In(ClP) Ilqulds. [O tN bioreactor. The fIow of CIP rolutions is '~nted throu¡h the tnnsfer in
16 1

161

CHISTIANO MOO-YOUNG

fur ptoducts mar are injected into the body mUS! nndergo a final wash with hot water-for-injection grade water. This erunres that 0111 residual water compl1es with rhe requisite quality standards. In mechanically agitated bioreactors. the spr.Jyofd eaning solutions may be unable to achieve proper deaning of m e agüators, magnetic couplings. mecha nical seals and the lower partions of baffLes. Therefore, filIing afthe vessel ro at (east above thelevel ofthe lowerrnost hnpeHer and agitation at impeller Reynolds numbers (see Se¡;tion 7.6) of lOL IQ8$ is recommended during pre-rinse. alkaJi recirculalion and the final rinse. Agitation for 2- 3 minutes is. sufficient ro dislodgc adhe.ring dirt or sollo These recommcndations assurne thar re'H.:tDrs are being deaned in·place sooo afte.r use and caking ofdirt b.ts nor occurrcd.

7.5 I Photobioreactors Cenain micro-a1gae and cyanobacteria provide imponant chemicals. such as as.talGlnthin and ,B
BIOREACTORDESIGN

,

I'hotoblcreactars lor (a) com:inuous run wbu!ar IoXlp: (b) a ~olar I'flcelver Ill3de of multiple paraJlel wbes: (e) helkal wound rubular loop: (d) fin panel configuration. Coofigo..ntion (a) and (b) mar be moomed vertically, or paraHel ro the ground. mono-cult~re:

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tb)

tdl

circuJated through thc solar receiver by a variety ofmethods. including centrifugaJ pumps. positive displacement mono pumps , Archimedean screws and airlift devices. Airlift pumps penorm well. have no mechanicalparts. are eaS}' to operate asepticaUy and are suited ro shcal' semi· uve apptications. Thc flow in a solar receiver [ube or panel sbould be turl>ulent enough ro aid periodk movement of cells fram the deeper peorly lit intenorto the regiolls-ncarcrthewalh_ GeneraUy. a minimum Reynolds number value of 104 is recommended. While turbulence is needed (O improve radiallllÍXing. too much turbuJence can be harmfuJ. Tbe veJoc· ity everywhere shouJd be sufficienI (O prE'Vt'm sedlmentation of cells. Typicallinear veJocities through receiver tubes [end ro be 0.3-0.5 m S- l. Bccause of the need tu m ainta.in adequate sun light penetratian. a tubular solar receiver canno[ be scaled up bysimply increasing [he tube diametel~ The diameter should not exceed 6 cm. although this constraim may be relaxed somewba t bydeploying specially designed sratic mixers rhatimprove radial mixinginside the tube. Without themixers, reducing !he tulle di<:Jmeter from 6 cm to 5 cm and further to 2 cm noticeably improves culture performanCE'. light penecration cfepends on biomass density, cellu)ac morpbology and pigmentation, and absorption chacacteristics ofthe ccl1-free culture medium. Photobioreactors a re normalIy operated in continuo us lllode. Micro' algae grow slowly; the doubling time ofChiort'Ua pyrtrloicloSt'l is approximatcly 9 hours. Conscquently, the dilution rare is kepr low; about one culture volume pe:r day. confined ro daylight hours . Although the biomass grows only during daylight, certain products are produced predomi nantly dunng' the dark hatirs. Biomass prodmTIvity of outdoor cultures is generaUy less than 2 g l ' l'day, ofien no more tball 1.5 g I- L.day. CuJture is generally carried out at 22-37 ac. During

163

16<1

CHISTI ANO MOO-YOUNG

dayUghthours. thesolarreceivertubes need ro becooled toprevenr temo perature [ise to damaging levels. A solar receiver is more amenable to scaJe-up by increasing che length of a contínuous run tube; bowever. tbe maximum permissible length should nor exceed 50-100 m because the pltorosynthetically generare
7.6 I Heattransfer All fermentations generate heat; in submerged cultures. lO- SO MJ m.- 3

ofthe heatoutput typically comes from microbial activity. Heat production is especially large when the biomass is growing rapidly in highdensity fermentations and when reduced c.lrbon sources suro as hydrocarbons and rnethanol are oseo as 5ubstrate. Themetabolic heat generation rate in kJ l-l·h 15 typicalIy about 12% ofthe 01 consumption tate expressed in mmol 0ll- l·h. Heat removal in large vesscls bccomes difficult as the heat generation rate approaches 20 MJ m - J. corresponding to a n 0l consumption rate orabout S kgm-3.h.ln addition [O metabolic heat mecharncal agitanon ofLhe broth produces up to 50 MJ m-l. In ait driven ferment~. a1l e:nergy input due to gassing is eventuaUy dissipated as heat. Consequently. a fermemer must be cooled to prevent temperatute rue and damage te culture.As the scaleofopt'r.ltion increases, heat transfer and no[ 01 mass transfer hecomes (be limiting process in bioreactors because the available surface area rOl" cooling decreases as Lhe fermenter volume increases. Thmperature is controlled by heating or cooling through external j ackees and internal coils. Lcss frequently. additional double walled baffles, draft tubes or heatexchangecs locared ¡nside the fermentation vcsse! are needcd to provide suEfkient heat transfer su:rfuce aTea. TIle rate ofheat removaJ. Q.¡, i5 related to (he surface area,J\•. avail· able for heatexchange and (he mean temperature difference. tJ.T. thus, (7.1)

The overall heat transfer coe.fficient, UH' is the inverse of!be overall resistance. to heat transfel". During cooIing, heat flows from the broth side to the roolin gwat~ in a jacketOl"coolingcoil. TIletransferring heat

BIOREACTOR DESIGN

t - - - Jacket

Cutlure

broth

--1--- Cooting water

1'-----11--- Water

film (1'hl l

A+i-----t--- deposils Fouli ng (1/h¡)

Broth film l1ih.,)

...-Cooting

water Direction ----'Lb~-f1fL. of heat tran sfer

---11--- - - - - --

Fermenter wa lt (dwfk..,)

eocounters several resistances in senes as illustrated in Fíg. 7.11 : the thin sta'goant film of broth on tbe [mide wall of tbe fennenter ; the ml."tal wal l ofthe fermenterorcooling coil; scaleor 'fouLing' de posits on the cooling water side; and a thin S[agnant fibn of the cooling fluid 00 the jacket side of the fennenter waU. These individuaJ resistances are related to t he overall resistance as folJows: 1

1

U.

h,

OYer311 resislanct"

broth lilm

resistance

1

+

+ vessel waJl n!Slst.am:e

h, foul1ng fl.lm resistanct"

+

(7.2) water film resistance

'Ihe film heat tTansfer coefficient is infIuenced by llumerous fac..:tors, inc1uding (he density and the viscosity of the Huid, tbel'mal conduc tivity and heat capacity, the velodty offlow a l' sorne otber rneasure of tu 1'bulence (e.g. power input. gas flow rateoete.). and the geornetry of the bioreactor. 111e manyvariables that affectheat transfer can be grouped into a few 'dimens ionless numbers' to. greatly simplifY the study and description ofthose effects. The groups relevant to heat transrer a nd the corresponding fluid dynamics (e.g. Ulrbulence) are as fo110W5: totaJ beat tr.msfer Nu (Nusselt number)= condu~üve bea[ tra.nsfer

Pr(Prandtlnumber) -

momenrum diffusivity h iffu' . termal d SlVlty

h.,d

k,-

(7.3)

(7.4)

inertia I force PlVId Re fReynolds number) : . , VlSCOUS orce ¡.LL

(7.5)

' ~a~ti~o~n force Gr (Grashof number) - -~gr~'7_"~'r VlSCOUS force

(7.6)

165

166

CHISTI ANO MOO-YOUNG

In these equations. d is a charaC'teristic length (e.g. diamerer oftube or impeller). The above noted dimcü5ionless groups expl'CSS (he relative significance of the various factors infiuencing a given situatÍon. The value ofthe Nusselr number rells us about the relative magnitudes oí total bcat transfer and that transferred by conduction alone. Ihe Grashofnumber is importanr in situations where 60w is produced by density differences thar may tbcmselves ~generared by thermal gmdientS (hence me!ll'f3 in [he Grashof Dumber). The Reynolds number is employed indcscribing fluid motion in situations where fol'ccd con Ve(:· tion [$ predominant. Correlatioru fur estimating thc film heat rransfe!' coefficient, h", are often briven in rerlllS ofthese dimensionless graups. Equations for quantifying the heat transfer resistances of tbe fouling films and films of heating and cooling fluids are disclISsed in readily availablc process engineering handbooks. Suitable corrclations fuI" cstimating the heat tcansfcrcoeffidem. h D• for m e film ofLiquid OI culture broth in various (:onfigurations ofbioreactors are summarisoo in Table 7. 1, Note thar me couelatíons given for mrred ve.55e1s utilise a Rcynolds number thar has becn deflned in terms of the tip speect ofrhe impelJer. lo somecases, the correlations in Table 7.1 require the rhermal conductivity. "-r. and the specific heat capacity. Cp ' ofthc ferrnentalion broth for estimation of the heat tró\nsfer cocffici.ent. For most bl'oths. the va lu e~ ofthase paramerers are close ro those ofwater. The film coefficielltgencrally illcreases with increasing turbuJence. gas flow ratc, 3nd the agitation power input. lbe roeffident typically declines with increasingviscosity ofthe culture broth. The geome nyof tbe bioreactor affccts the film heat trnnsfer cocfficient mainIy by illflu· cncing the degree of turbulence 01' related parameters such as the induced liquid cirrulation rate in airlift vessc1s. lo bubble columns the film coeffil.'ient i~ independent ofthe column diameter so long as che diamerer excecds abour 0.1 m. SimUarly, in bubble columns the ho value is nor affcc:ted by the height ofthe gas-free fluid . 111e value ot'h" mereases with increasing superficial gas velocity, 01: power input. bu t onJy up ro a veladt)' of abour 0.1 m s l. Furthennore. for jdenticaJ specificpowerinputs. bubble columru and stirred vessels providequite similarvalues ofthe hcat transfer coefficient. Litcratu.re on heat [rarufer in airlift reactors is sparse. Equations developed fu r bubble columns fTable 7.1) may be used to provide a low estimateofh" in aidiftvessels when the induced liquid circulatiOll ratcs are small. Undel' other conditions, the coefficient in airlift reactors can be more than two-fold greater rhall in a bubble column. When liquid flow velocity does Dot exceed abaut 0.015 m S- l . [he film hcat transfer coefficient is largely independent ofliquid velocity: however, for higher liquid velodties hn increases with Iiquid velocity as follows: (7.7)

A large amoune ofpublished datais available on heat rrarufer in verti· cal two-phase Oows. Sorne oi' this information may be applicable to airlift reactors provided tha[. the fluid properties, gas hold·up and rclative velocities ofthe two phases are idenrical in rhe airlift and the

. ·iiiiik;~;¿i'~w.áiaiá~aihlmb~~;f#~OO~~~t:i~~tiji.i!ñ~~¡~iW~:.@iis!~~rm!i!~~ . ..H~,lt~H#¡¿¡tH , .m ·'! · t~· .. ¡;"'liffl "r.¡'·" ... • ...... ....... "::·![""t··¡" ·h .• •• " ....... . ....... 'P In~ \ ...... . U ..... :::¡: .• • _.. .. :, ' . ·,:m.,:fH~~i¡Hgñi;.·imi,~m, •• • ..... ........ ¡~mnml~¡H¡mn , .. .. ¡

Bioreactor configuration

t. Stirred tanks (coiI5)

Ranges

Correlatlon

(",w)"" = 0,87 (dl NP )"" (Ck",), r

-ho
~

Jli.

2. Stin-ed tanks Uacketed)

h: (':::r ~036(d:1J r(1;)1

3. Bubble columns

ho = 939 I U~(::r3S

4. Bubble columm.

S. Airlift vessels

For cooling coils; Ne'Ntonian tluids

:::1'-

/J.i..

Forjacketed vessels: Newtonian fluids

Newtonian broths

10- 1
~~ O.I (U&.. (~)').¡

",-("UG h

o

I".g

Newtonian broths

kr

~ 87 IOU" n (A,)", (~)-'-' Gt

A.:t

Ne'vV!:onlan brcths

kT

~=

{draft-tube sparged)

0.78- 5.27 mPa s: Q,008 S UGr ::::; 0. 16 m S- I; 1.20. The hovaried frcm 600 to

O.25 $ A/Ad ~

1400W m-2. oC 6. Airlift vessels (annulus sparged)

A ) -'" Uo.m. h = 13340 ( 1 +2 " A, G

7. Fluidised beds (gas-liquid-solid)

k-r{I -Eu

h"dp6 L

Air----'"l,ldter: 0.0 1~ UG :S 0.04 m s- I;Ad/Ar = 0.241 and 0.452

O.044[ dpULPl C;¡J.4.]C.18 + lO (~1)C, ' 7 JJ.t.(I- F.L) k l

\,idp

A Hproperties are forthe Ilquid phase

168

CHISTI ANO MOO-YOUNG

vertical rwo-phase ftow device, Fungal mycelia-like solids may enhan,ce or reduce heat transfer depending 00 hydrodynamic moditions in the airlift device. Whereas in .microbial fermentations tbe temperature control tolerances are fairly narrow, animal cell cultures demalld even more dosely controlled temperature regimens. TypicaUy, cells are cultu red O1t37 == 0.2 °C.1he cells generate Httle heatand tbe heat produeed by agitation is aIso small. In addition, the almostwater-like consistency of eeU culture broths means that beat transfer is relatively easy; howeve.r. the temperature differences betweeo tbe heating/eooling sumce and the broth must remain small, aI: the cells maybe damaged .

7.7

I

Shear effects in culture

Shear rate is a measure ofspatial variation in local velocities in a fluid. Ceu dam01ge in a moving fluid is sometimes associ01ted with the magoitude afthe prevailingshear rateo But the shearrate in the re.latively turbulent environment afmOSI bioreactors is neither easily defined nor easUy measured. Mareover, the shear rate varies -with location within the vessel. Attempts have becn made to ch01racterise an average shear rale or a maximum shetlr rate in various types ofbioreactors.ln bubble columns an average sbear mte: has been defined as a function of the superfidalgas velodty as foll ows: (7.8)

where the parameter el equals 1.0 in mosteases, but the k value has been reported variously as 1000. 2800, SOOO m- I , e tc. Equation (7.8) has been appliedalso loairliftbioreactol's using tbesuperfidal gas velodty in the riser zone as a correlating paramete.r; howe.ver, thar usage is ineorrect. A more ruitable form ofthe equation fur airliftreactors is

kUCt

y; - -

(7.9)

1 + '" A,

Depending on lhe value of l: equations such as (7.8) illld (7.9) produce: wildlydifferenlvalues ofthe supposed shear rate. ln addition. the equatioos fail to take jnto account the momentum transfer capability, Le. the density and rhe viscosity, of tbe fluid . 80th of these will influence thesb~rate.

An average shear rate in stirred fermentcrs is given bythe equation:

( '"

).~n- ll

'Y=k - I 3n+ 1

N

(7.10)

whert" n, the flow index ofa fluid, equals 1.0 ror a Newtonian liquid such as water and thick glucose syrup. Sorne typical lc¡ values are: 11-13 ror 6-bladed disc turbines, 10-13 for paddJe impeUers, - 10 for propellers, and - 30 fur hetical ríbbon impellers. The maximum she:ar rate on a Rushton dise turbine in Newtonian fluidshas bee:n expressed as:

BIOREACTOR OESIGN

"Y

""'~

(pe)'"

= 3.3N u d -

i J.I.f..

(7.11)

Equation (?ll.applie.swhe:n 100 s (NtP¡pJ,uJ s 29000. Tt also applies to non-Newtonian fluids if 1\ js takenas the zero she:arviscosity. Theshear rate can be conve:rte:d to a paramete:r known as sbear stress T, wbere (7.12)

The: susce:ptibility ofsorne animal cells to she:ar stress levels has been characterised in wel1.ctefined laminar fiow environments. Another method ofdeciding whether the turbulence in a Hu id could potendally damage a suspended biocatal~[ is base:d on comparing the dime nsi ons ofthecell 01" the biocatalyst ftocwitb me Le:ngth scale aftbe fluid e
e =(:rtE-II~

(7.13)

In most cases. aH. the energy input te the fluid is dissipated in fluid eddies and Eequals the rate ofenergy input. Methods fur calculadngthe energyinput rate in the principal kinds ofbioreactors are noted in Sox ?.l. Equarion (?13) applies to isocropically turbulent f1ujd, Le. one in wh1ch the size ofthe pnrnary eddies generatect by the turbu]c Dce pro· ducing mechanismis a.thousand·fold ormore compared to the size of the enecgydissipating micro~ddies. The size ofthe micro-eddíes is cal· culate
16~

170

CHISTIANO MOO-YOUNG

BIOREACTOR DESIGN

smaU as 200 Jlffi in diameter a re suspended in me cul tu.re fluid to support adherent cells olllhe sunace oftbe carrier, inter-panicJe colli· sions are genETally infrequent under the conditions that are typicaUy employed. However, [be size ofthe fl uid eddies in micro<arrier culture systems may be similar to al' smaller than the dímensio ns o f the cal'" riers; henee, the adhering ceUs may experience cu rbule nce-re.La ted darnage. Freely suspended animal cells are generaUy roo smaU ro be damaged by fluid turbulent:e levels that are typically employcd in cell culture b ioreactors.ln micro-carri erCUlttlre. sh ear stress Jevels as low as 0.25 N m- 2 may interfere with the initial attachment ofcell s o n micrO"

carriers.

7.8

I

Further reading

Cbisti. Y. (1999). Shenr senSitivity. ln li'ncyclopeclia o!BIOpT'QC('SS 1'rchnology: Fennrntalion, BIOCGtalyrfs, uncl Ri()~epllrarion, Vol. 5 (M. C. Flickinger and S. W. DrL'W, eds.), pp. 2379- 2406.John Wi ley, NewYork, Cbisti, Y. (1999). SoUd SubsU'iHe fernumtati ons. enzyme production, fooot. In cncydopl!Ji¡¡ o!BflJpruce.~s TerhnoJogy: Ferm~ntatjOIl, Blocarlllysis, ¡¡na BI05epamtiou. Vol. S (M. C. Flickinger and S. W. Drew. eds.), pp. 2446-2462.

Wi\ey. New York. Chisti. Y. and Moo-Young. M.(1999). Fermentation technology, bioprOCí!'ssing.

scalt-Up and manufacture. In Dloleduwlogy: The Scimce IInd the Husiness.2nd Ed{ticm{V. Mases. R.l:. capo> and D. G. Springham, 005.). pp. 177- 222. Harwood Acaderuic Publ i1; tl(~rs. NC'W York. Doran, p, M. (1995). Bioprows Engillct'rlttg Prllldples. Academk Press. London. Grima, E. M., Fernández, F. G. A.. Camacho. F. G. ;lnd Chis[i. Y. (1999). Photobiore¡tctors: ligbt rr:gimt>, mas! trans(er, and sCilleup.]. Bioti!chnoL 70, 231 -247. LyderseTl. B. K.. D' Elia. N. A. and fllelsoll. K. L (ed s.)(l~). Hiopl'Oce.u Engineering: SyslrnlJ. F.quipmenr tina J-(¡cllitiu. John Wiley. Ncw York.

Van't Riel. K.and Tramper. j. (1991). Bane Bioro'actorlkstgn . Mareel Dekkff. New York.

Chapter8

Mass transfer HenkJ. Noorman No mendature Introductíon Tite mass tra nsfer steps Mass transfer equiltions Determining tbe vo]u metdc mass tr.1Rsfer coefficienls The effect ofscall' on mass ttansfer Furthe'r reading

- 1Nomenclature Rom= a inrertacial arca per unit ¡¡quid volume a' interfacial area per unit total rcardan valume e e,

e

(gas plus liquid) conte.ntration in liquid phase concenlr.ltion al ¡,quid side ofinterface sarur:l.tion ('" equi.l ibrium) concentratiol1 in liquld pbast' (= p/ffJ

CI d D D H

biomass concentrarion liqllid film lhicknes$ düfusion coeffici entor etTectivt' diffilSiviry impellerdiameter HC!nrycoeffldenl

H v liquJd hl1:lght

)

molar mass flux

J,

molar lllilSS fiux across gaJi film JI molar ma5S flux ilcroSS liquid film k mass transfer coeffident k, gas film mass transfer coefficient. "1 liquid film mass transfur coeffident kla volllmetricmass transfetcoefficient K OYel'all nl.:lSS transfu coeffident K

1

m m1 s ·1 m barm' mol ~ 1

m

mol m- 2,s mol m ~J ,s mol m- ~ · s ms- I ms- I ms- L 1S~1

ms- l

COllsistcncy index

power law index N impeUer rotational speed OTR OJ transfer r
moLm kgm'

,

.

mol m · ·s

174

NOORMAN

p Po

pressllre referlo'nce prcssure (= 1 bar) Pi pre!iSU1'e atgas sidc ofinterfdce P¡" inletg'"dS pressure Pool outletgas pressun~ l'

1', q

bar (N rn !) bu

"'"

power input power input by stirrer

bac bac W W

consumption T
molm l .:s

time ·l~.

ta.nk diametcr

.'< VII

distanct! supedicialgasvelocity

V

voluml'.

VI gasvolume VI liquid volume

m m

m, m' rn'

m'

G~k

'"

power law index

t

a~r.J.gt': !hcar rate

e

hQld-uporvoid franian

dynamicviscosity referl'.nce.dynamicviscosity A liquid phase densi ty

JI.

~\)

,-o kgm l·S kgm l·S kgm '

Dimenslonless Dumbers P" impellerpowerllumber Re Reynolds numbe r

8. 1 I Introduction In a bioreaction process. substJ:ates are consumed ¡¡nd products are formed by action of a micro-organism, 01' cata1ytic parts oforganisms,

[or example enzymes. Typical substrates for a living cell are tamon sources 5uch as sugar and oil . nitrogen soueces such as a mmonia and amino acids. and clectron acceptorssuch asOlOeroducrs can be all kinds of org-.mic compounds. biomass a nd COl' For an optimaJ rate of reaelion. rhe rnicro-organisrn. the academic researcher or the industrial process engineer should sel'. to it that transfer of suostrates te the enzyme or ceH surfacc (Oí the site of reaerlon ¡nside the ceU) and removal of products away from rhe enzyme or organism is as rapid as possible. and prefcrably notrate-limiting. Usuallythistransferinvolves a chain or mass transfer steps as shown in Fig. B.~ The slowest ofthese rteps wi U determine me o~r.l.ll mass transfer rateo and its value is lObe cornpared with Che slowest kinetic reaction step in order to find out if mass transfer will affect m e 0YeJ'31l process performance or noto ln this chapter. attenDon will be focused on reactions involviug whole ce lis. ln enzyrnatic biotransformations. cells are abSl'.nt and me number ofmass tramfer steps is decreased, but the same concepts cnn be applicd.

HASS TRANSFER

gas bubble or liquid droplat

3 bulk IIquk!

..group _amI""" • 01organlsms

so~d

particle ella,n of rN.n transfer stCps for a substrate or nutrltint from a PI bubble.

Ilquid droplet or 'iQlid partido towardi the lite of ruedon Inllde a cel : 1: Transfer (malnly by diffusion) 01 subslnteli from gall.llquid or lolld phale tO the 1~rf.Jce wlth the IIquld wat~r

phaslI; '2: Transport (mgs~ gnen by a cOl11binadon of diffuslon and convec:don) ao:mss a &:hIn. nU:Mr stagnanl.l ayer d wate r phale tht surrounds the rou bubble.llquld droplet ar saTld partK:I ~: 1 : Tran$pO!'t (usually by COIlveCtlon or turbulence) through die bullliquid phase ro a thln layer surroundirlg a single mh:ro-argilllbm ar a ~rtkle (dump.

canier) corltalnlng agroup af orpnlw1S:~: Transpart (dlffusive) mis !ayer 10 tlle cel l s~rbce; S: Trarn.part (paulve by dlfl'u,lan andlor lCtlve wlth a tran§JKlrt ~nzyme) overthe (ell enveIope tO a slte In,ide tlM:> (eU where me reaction [3kes place. NB: Products form!!d take ah!! roverse l"Q~te. pellet.

immobil~lion

.l CroSS

8.2 I The mass transfer steps 8.2. 1 Effects of transfer limitations lf one mass transfer step is slowcr than tbe key kinetic reaction step. 1t will limit the formation of a desired product from a se1ected substrate. As a resu lr. [W() effects may be observed. botb with freeJy suspended cells as well as organisms immobi lised inside cell aggregates 01' solid partic1es: • The ovemll J'~actjo" rute Is beluw 01(' thcon:tirol maximum. und the process output is slowcr titan desi~d. This is the case in the formation ofgluconicacid from glucose by the acrobic bacte.rium, Gluconobucter o.\yda ns. Hete, tbe overall reaction rnte is detennined by thc fate O]t which O~ is transferred to tbe Liquid phase. After reLiev· ingthe limitation. rhere is no irreversible effecton this particu lar micl'o-organism. Allothel' example 1S a limited supply of sugac lo immobilised ceJ Is due to slowdiffusion inside an immobilisatlon canier. lbe overaU rate ofproduction is ofren reversibly reduced. However. the:re: are a1so examples ofsystems where me biosynthetic capacity ofa ceH i5 ineversibly damaged afterimposingan 02

175

176

NOORMAN

transfer Iimitation (e,g, in penidUin fennentation). Such processes are very St!nsitive to mass transfer limitations . • !he selerrivityofthe reacrion is altered. Forexample. 0l serves as an ele<:tron acceptorln the formation ofbaker'syeast from glucose. ln theabsence ofOl the clectrons will bedirected to pyruvate resulting in the formation of ethanol and C01 instead of more yeasL BacUlus subri/is cultures produceacetoin and 2,3-butanediol when devaid of 0l' The ratio of the two products is great1y dependent on the disso lved 0l concentradon. and thus 0 0 the ratio Of02 transfer and 02 consumption.rares. Again. the damage can be either reversible or irreversible.

8.2.2 Transfer between phases 1'he transferofO'J from 3n air bubble ro themicro-organism in an aerobic bioprocess is a re1arively slow transport srep. Oxygen, and other sparingly soluble gases in aqueous solutions !such as bydrocarbons wirb up ro four carbon atoros), may become rapidly depleted when it is consumed. Jfnot replaced at the same high tate t he situation will be d eo-imencal for the micro-organism, Transfe.rofmatl!t:ial over a liquid-liqllid ar liquid-solid boundary is comparable with gas-liquid mass transfer. An example is the growth on higher hydrocarbons (> C61- lbe.oil phaseis present in [he form of smaU droplets and mass uansfe.r resistance is a e tbe side of tbe surroundíng water layer_ Also. the exrnaoge of material between a solid phase (substrate partic1es, parceles tbat coutaio microoTl:fdnisms) and the liquid phase obeys similar principies.

8.2.3 Transfer inside a single phase lnside a gas bubble. oroil dropler lhere is usuallyenough motion to guaraotee a quick transfer of molecules ro the interface with the water pbase. so the resistance is at [he water side ofthe. inrerface. lftbe d istances in tbe bu!k liquid phase to be bridged are rela tively large. a traos' port resistance can occu r in this pbase. $uch a sltuation is t'ncountered in large bioreactorswhere bulk liquid mixing is usually sll~ primal .ln industrial practice. ir is important to reaUse thar one has te live with this potentiaJ Iimitation . The.refore Its effectson the microbial reacrion system sbould be borne in mind during process developmentwotk. Mass tr.msfer Iimilatiolls imide a solid phase can QCcu r within biocaraJyst particles thar contain immobilised micro-organisms. either as a surface biofilm attached to a carricr, or dispersed throughout the carrier material. Alternatively, the micro-organism (usual1y mam en· tous) itselfmay be preseot as a c1ump 01' pellet. A substnlte cntering the particleor pelletmay be consumed so fast th;:¡t nothing enters the inner part of the particle. so that the efficiency of tbe caCalysc is below maximum. Also, che reacrion {llay be slowed down because a toxi<: or inhibiting product cannotmove away quicldy enough.

8.2.4 Transfer across the ce!! enve!ope The mia-u-organism itselfcan also be considered as a separate (solid or liquid) phase_ Transportacross!he ceU envelope (most1y a combinati on

HASS TRANSFER

aC1ive transport

facilitated diffusion

free diffusion

out

ATP~

1

in

of ce11 wall and c:y1:oplasmkmembrane) can be Limited, depending 00 me SLze and physical properties (hydrophobicity, electrical charge) of the molerule and whether me organism is equipped with a specific uanspor[mechanism or DOt. Gencrally (hree mechanisms can be distinguished (Fig. 8.2): • free diffusion; passive transportdown a concentrationgradient; • faci1itared diffusion: as above butspccded up by a ca[rier protein: • active transport: transport by a carrier proteio with inputoffree energy. The diamete:r ofthe microbial cell itselfis very small (arder afmagnitude 1-5 ¡.Lm) so that diffusioo ¡nside tbe cell is more rapid than transportacross tbe cell envelope.Addicionally, in eukaryot1ccells there are inu-acellular organs (vacuoles, mitochondria) which can prescor another tr.,msport barrier. Howeve:r, in quantirative temu this type of transport i5 much more rapid than the consumption rate ¡mide the ceU and will nonnally not limit the ovt!r.Jl1 rate in the chaln of u-ansport steps.

8.3 I Mass transfer equations 8.3, I Fundamental principIes The Pickequañon (8.1a)sta le5 thanhe mass ttansfer,) , ofa componcnt in single pbasewiU be proportional to theconcentrntiongradjent in the direction of tbe transporto The phenomenological expression fOf steady-state mass flux is: J~ -DdC¡dx

(8.1a)

Wbenmass transfer in a salid phasc is considered, O is the efJecNve diffusivity. a function of the diffusion coerficient, the porosiry of rhe solid and me shape oftbe channels inside the salid. For the geomeo-y ofa fiar sheetboundary layerwith thidrnessd in a stationaryfluid, the retationship between mass flux and concenu-ation difference, AC, becomes: J~Di!.Cld

(8.lb)

D/el is the mass transfer coefficient and the inverse, cljD, can be interprete
177

178

NOORMAN

. ..... p

I

bulk Iiq
I

p

- -:-.~

e,

I

"""""

e

"fwo·fllm lheory: mass

tranU'era
(8.2)

These fundamental , buttheoretical. equationscan be used to calculate mass transfer by a diffusion process. A prerequisire is that convective transport ís absent but (bis i5 rarely the case. More afien. a combination ofdiffusion .and convection witb phase transfer is enoountered.Now we h ave the additionaLproblem tbat the veJocity pattern ofthe üquid f10w is not known. Thus. for gas-liquid aud liquid- particle mass transfer in real bioreacto[S a more empirical approach is preferred. f'Or mass transfer between liquid and gas pbases ortiquid and salid phases, the well·known two-film theory (see any standard cheu'llcal cngi· ncering tex:tbook on mass transfcr) can be adopted. Mass flux in both phases must be scpararely described, whereas the over.ill transfer 1S determined bytwosteps in series across rhe film,.as sbown in Fig. 8.3 . Por gas- liquid transfer the mass flux is described by: gas filrn transpon:

J, = k¡:- (p - p¡)

(8.3)

liquid film transpon:

JI = k¡ (C.-q

(8 .4)

The CODcen crations at both sid~s ofthe interface PI and el are notide orical, but.rclated rhrough the Henry coefficient. H: p, =HC¡

(8.5)

In prarnce it is Dat possible to llleasure the interfacial valut's, so ir is bener ro eliminate mese fram Eqns (8.3). (8.4) ¡¡nd (8.5) and wrire the mass flow as a function ofthcconcentrations in both bu1.k phases: (8.6)

where C- (=¡I/Rl is t he saruration v-.llue in the liquid phase. Note titar Eqn (8.6) is oftbe same farol as Eqn (8.lb) . (e - e¡ is the OVt'raU driving force nnd the overaU transfer coefficient. K, resules from me sum ofthe transfer resistances: (8 .7)

Often this general equatian cnn be simpliHed as 1l(H kg)< l fkl(i .e. the gas phase film resistance is negligible compare
- q

(8.8)

where a is (he gas-liquid inte.nilcial area per unit liquid voLume. or aTea per unit gas/solid p lus liq uid vo[ume. 0 1" area per umt g ross vessel voJumein the bioreactor. Whendealingwith the transfer ofO¡ from gas to liquid, the l'roduct Ja is usualJy caBed the OTR, Ol" 02 transfer rate. For aIl accurate estimate ofk1d, assumptions must be made on the values of C- (or p) and C. For a labora tory scale bioreactor « 10 litres

MASSTAANSFER

Po~

fMl Ixmibfe curves of the changl!'l" in Ih~ pal-tla! ~!.Ure

P=Pin

reactor height

•• • ••

in the gas pitase H th~J.¡r bubbles tr.lvel up through J.l'f!acror YeS$~ .

¿,~:"¡'-I~-1r---- P == complex p

". Iinear P (-hydrostatic pressure)

p

I

I-_~_+--- p = lag mean p

.•

I

P = POllt Pin

partial pressure

operating volurnc) the bulk Iiquid pha!ie is assumed tO bt! well·m.ixed and henceCis constant throughout the liquid oHowever. in pilor planrs or prodUCtiOD scale vessels (> 100 lirres) this win nor be the case. and local concentration variations nee
179

180

NOORMAN

transfur. Detail~ on such texfbooks.

meones can be found iD mosr (bio)chem.ical

8.3.2 Gas-liquid mass transfer in real systems There has been a lotoffocus on 0 a transferfrom the gas phase into [he liquid phase in bioreactor processes. SiDce it is expcrimeDtallyl'ttY difficult ro estimate thevalues ofk1 and a separately, k¡a is ofien treated as a lumped parameter. ln bioprocess engineering literature, one can find a large number of expressions for chis [voluroetric mass transfer) ooeffi. ciento Here. a divisioI1 should be made between the doroinant types of reactors used: bubble columns, alr·lift reactors and $tirred tank reacto1'5 (sec Cbapter 7). In caro case the physical properties of che liquid mayinfluence the magnHudeofmass rransfer.Extremevalucs are given by,

• A llquid which greatly stimulates bubble roalesce nce, i .c. a coalescing liquidoAere mass transfer is pOOrest. • A llquid which suppresses roalescence to a large extent. ¡.e. a nonooatescillg liquido This gives the highest mass transfer rates . In a bubbLe colurno (sec Chapter 7, Section 7.2.2) the gas enters through the sparger orífices. lf the brotn is coalescenc and noo·viscous. e .g. distilled or tap water, the bubbles will rapidly take their equiJíbrium average diamNcrof ca. 6 mm. When the air f10w rate is high enough the vesseJ is operated in the hcterogeneous Oow regime and the hold-up is a funroon of the superficial gas ve10city (:= gas flow per unit crosssedional area of the reactor). corrected for pressure differences (PI! is a reference pressure ofl bar): (8.9)

For lhe mass transfer coef:ficient, the following correJation has beco experimentallyverified: (8.10)

In non-coalescing Ii'luids. e.g. lOl1ic SOIUtiOllS and sorne fermentatioll broths. the bubbles thatoriginate from the spargerwill rise 3nd not mil( with otber bubbles. provided tbat the size is smaller than ca. 6 mm. The interfacial area. and hence "la, will be higher chan when larger bubbles are preseut.lf the bubbles are larger [hey will disperse and take che same equilibrium valllc as in coaleScing liquids. It is noted that in a large bllbbLecoJumn (> 50 mll. the bubbles wiIl significantlyexpand as rhey rise through the reactor beca use ofthedecreasing hydr05taticpresSUTe. This will influencemasstransfer. In Oln air-Uft reactor{Chapter 7. Section 7.2.3). [here ¡53 riser section . in which the spargingofbubbles results in an upward liquid flOW,;1 tap section. where the bubbLes escape from rhe liquido aud a dOWTlcomer section, in which the liquid is .recirculated downward. Although [he riser resemb)es a bubble co)umn. che gas hold·up is lower than predicted by Eqn (8.9) due to the interaruoD with the Uquid f1ow. Correspondingly. "la will be lower, up to ane tbird ofthe bubble rolumn value. Aprecise quantification. howcver, cannot be easily made.

MASSTRANSFER

In a stirred tank reactor (Chapter 7. Section 7.2.1) the flow phenomena are determined by the balance between.aeration forces and agitation forces, and large local variations in combination with a number of tlow regime. transitions make a precise quantification ofmass transfer difficult. Sparged gas: is usually rapidly coLlecte
l;a = 0.026 (P JVl·4. (v¡(pofp)o.~

18.12)

non-coalescing: k¡a =0.002 (PJVI)O.1 (vgpJp) 0.2

(6.13)

coalesring:

In a coalesci.ng liquid the influence of aeration is larger than agitanon, while for a non-coalescing liquid the opposite is true. (Nore that me COI"relatioru are independenr ofthe agitator type.) The energy input by agiration is a vital variable in Eqns (8.11), (B.U) and (8.13). The amount of power dr.iWD by a stirrer of diameter D with a rotational speed N is lIsuallyexpressed as:

~

IB.14) The impeller power number is a function oftbe aerationrate. tbe impeller Reynolds number (= PI N IJl¡ ¡,.t) and the impeller type (see Fig. B.5). When a brotb is aerated, Pa generally falls due ro a growing size ofthe cavities behind the blades. A typical value fer a RushtOll impeller is a factor 0.5, while for a Scaba type (6SRGT] tbere is hardly any drop.

Prochem 10

1!Xl

kWm- 3, wegec:

Bubble column; Egn (8.10):

kl u= 0.05 s-J

Stirred tank;

kl t1=0.14s- 1

Eqn (8 .12):

Assuming (C'"-C)=O.59 mol m- l -0_l0 mol m- l =0.49 mol m- 3 , the bubble colllmn OTR is 0.024 mol m -l·S, whereas for the stirred tankit is

10000

Impelle r RevnQlds numoer, Re Som~

Example The o~ transfer perfonnance oC a stirred tank reactor is generally better than ofa bubble column witb similar geometry and aeration rateoFor a coalescillgliquid ina vessel ofl00 m J reacrion volume. with tankdiameter 3.5 m, aeration rate 1 vvm (or 1.67 Nrn 3 s-1 ), head-space pressure 2 bar, impeller diameteJ: 1.75 m. and powcrinpur per unir volume P/VI = 2

'QIXl

unaflrlIed

Impeller pOWt!r numbers, Po' as a

function of the Impener Reynolds number. Re. In me turbulent now reglme (Re>4000) in the absence of aeration the value of Po for a sClndard slx-bladed Rushoon turbine Is consumt al Q. 5. ror a Sa.ba 6SRGT I'''pene.- it l. 1;3. 1.7 in<:! for a Prochem agitator it is ca. I .O. ln thelamlnar fIow rflgimfl (Rfl < 1000), thel"flls an inversfl proportionalitywith Re (e.¡. ror a Rushton Impellflr Po" MIRe).

181

82

NOORMAN

:. IIluurulon of 0 1 tran5l'er In dWereM ru~ors and m~dla ~s a functlon of superfldal en velod!)' uslng Eqns (B. 10), (B. 12) and (B.13). Here It 15 usum,d thatk,O In a bubbl e ,olumn with non-co.\!escent medl¡

ls three times the value of coaJes.cem media.

0.30 0.25

••• • _ •••• -._. SlllTed tank,. non-coalescent

::- 0.20 ..--•• - .... ....... ~ 0.15 • •• ·--

•••••••

~ ::~ '~--_//-----0.00

0.02

0.04

._•• , Bubble col umn, non-coalescent StilTedtllnk,.coalescenl

Bubble column. coalescenl 0.06

vg p ,./p (m

o...

0.10

S _I )

O.070mol m-J·s. In spiteofthis, bubble columns are frequentlyuscd in bioprocesses. due toother advantages suro as simpleconstruction. eve.n distribution ofshear rate,lowerenergy input, etc. (see abo Fig. 8.6). When bigh concentrations of filamenrous micl'Ú"Organisms are used. the branchecl filaments ofthe mycclium interac.t with each other and fonu larger aggregates which decrease lhe free flow of liqwd. This rcsults in high viscosity and pselldoplastic or elastic brotb behaviour. Similar observations are made when a micro-organism excretes poIymerk subs1ances. such as xantban. The decrease oC maSS transfer ili partIy dlle. to the stimulatiol1.ofbubble coalescence. wbkb ¡eaves large bubbles in the broth. Abo the gas hold-up i5 reporred to be lowcr. As a consequence the interfacial area will be small. In large bioreactors uncler extreme conditions, bubbles of 1 metre diame ter can be obsCI\lcd. Sometlmes thiseffectLs p.1.rtlycompellsated by the simultaneo ous presence ofalarge Dumberofvery smaU bubbles (cliamerer< 1 mm) which llave a long residence time in the broth {15 min or mote). There is a lso ao effect ofvisrosily on kl' Thisis due to a decrease ofliquid velocity and nOl a dirc<:t coosequcncc of the viscóSity itselr. FUrlhermore, jll aerated broths lbe power inputmay be lowered because lbe size ofthe cavities behind me impeUer blades aTe Iarger, This will diminish tbe shear rates and bubble break·up. In rhe üterature, [be complex, combined effecrs are descl'ibed by an extension of Eqns (8.10), (8.U) and (8,13) wirh a factor p.- n. where n usually ranges from 0.5 to 0.9. Por stirred tanks and non-coalcscent media: (8.15)

provided [hOl p. > ¡"¡'o. where 1-1-0:= O.05Pa s. Ifthc broth behaves like a pseudopIastic fluid (viscosity isd ecreased at higher shear rates) or dilatant fluid (viscosityis incre3sed with higher shear rates), an average viscosity can be taken over the reactor:

¡.,¡.= K '5'"- 1

(8.16)

The average sbear ratecan beestimated from : y=lON

(8. 17 )

The parameters K and n in Che rheology model depend on the biomass concentratioo. Typically K is proportional [O C;. with the value of el' r.mging from 1.5 to4. Por pseudoplastic broths n < 1, fordilatant liquids n> 1. whereas for Newmruan media 11 =-1.

HASS TRANSFER

llxaOlple A pseudoplastie broth in a bioreactorhas the foUowing properties: e" "" 30 g 1- 1, K= 1, ti =0.4. The stir.rer speed is 3 revolutioru pers. Using Eqn (8.16), che apparent viseosity i5 0 .13 fu s. According to Eqn (8.15), k¡a is reduced to 51 %ofthe value in a lowviscosity broth. Whatwould happen ¡fthe biomass concentration o r [be impeUerspeed doublcd? (AnsWers: with d ouble biomass conC<.'ntration and a = 2, kfl is reduced to 19%: with double impeUer s peed . k.a is.reduced t069%.)

8.3.3 Uquid-solid mass transfer The description of mass transfe l' through a liquid film to orfrom a solid surface is much simpler tItan ror gas-liquid mass tr.msfer. k. is dependent on the liquid{solid propercies. and the value ofthe inlerfadal area can be detennined experi.mentally. Us ually, liquid-salid transfec applies to situaóons where mass transfer and reaction are interacting; a rubstrate is transported tbrough a liquid film to tite surface of a partide whece. micro-organisms are present to consume the substrate. Products formed are transported back th.rougb me ñlm to the bulk liquido These situations are often t"rcated in teITOs of apparent kinetics. Le. the observed rate of rcaetion js desc:ribed with standard kinetic exprcssions, but an efft!ctiwness factor(ranging from Oto 1) is introduced ro describe the penormance ofthe reaedon r.:ompared ro thesame reaction with no transport limitations.

8.3A Mass transfer ins ide a solid partide Wben tIt~ are micro-organisms active insidc apartide or c.:eUuJar aggregate, the tl:anspoTt (by diffusion) within che partides may prese.nt anotlter resistance. lhis situation is fo und in biocatalytic proce5SeS wh en dealing with cclls immobiLised in alginate or porous solid partides. A proper description of this phe nomenon is difficult because tbe kine tics of microbiaJ .reac rions ¡nside [he partide may greatly differ from those witlt free suspended ( d ls, and hence be lLDknown. 'Ibis is due ro altered physiological conditions for the cells. In addition to aD effectiveDess factor foI' external mass mnsfer, an overall effectiveness factor maybe use
8.4

Determining the volumetric mass transfer coefficients

Ir one is interested in approrimate kla values, or when measurelllents witb real bioreaction systems are impractical or ceon omi cally not feasibl e. such. as in large bioreactors, model f1 uids can be llsed. lnformatioll is provided from li terature, eitber more tbeoretic.a.l or mo re empíricaJ as described aboYe, 01' a lH.·W series ofexperimenrs can be designed. TIle use of model fluids . su ch as water oI' salt soJutíons, ir ne<:essary witb paper pulp or polymefS addcd to mimic.:vlscoUS broths. gives extremes ofwhat can be expected in real systems , and qllalilati ~ trcnds as a fllnc-[ion of changes. There are a few possibIe mcasuremenr methods:

183

184

NOORMAN

• Cht111lm /1?act1on method. In order to mimic: a microbial reaction, the transferred component ca n be consumed or produced in a chemiC411 reactíon. f'Or a l nansfer studíes. sulphite can be used, as it rapidly oxidises to sulphate in the presence OfOl and a catalyst. k¡a is found from Eqn (8.8) by measuring the rale ofsulphite conrumption,jal= dCrnlpt.nJdtj and C' (= p/H for 01)' for mostaccurate results, condítious should be st>lectM so thatC equals zero. Altematives for sulphite are glucose in combinanon with the glucose oxidase (01 is coruumed), or él mixtu re ofH l 0 2 and catatase (al is produced). Similarly NaOH solutions can be used to study ca] transfer (Cal rapidly reacts with OH-l. _ Ph}'!ica! replacement m~thod. Consider él gas·sparged liquid in steady sta te, so thal e equals C. The expenment is staned when che mole fraction Of02 in the gas phase is rapidly changed from me steady sta~ to anothervallle. Forexample, the Nl gas in a N2 sparged liquid is replaced by airo Tbenja in Eqn (8.8)equals dCldt and bycontinuollsly measllnng the 0 l concentration in·the liquid, k¡a can be evaluated from [his equation. Another possibitity is a slldden change in pressure ofthe componentto be transfen-ed. This method is usu a liy vt'I)' fast. and a rapidly enough respondingOl electrode is thercl'ore an absolute requiremenr. lfil is required to have real values fork¡a, theoríes. model systemso.t p ublished correlations shouJd uot be used.ln acdvely grO'oVing cultures . ~a can be experimentally determined using the.following metbods: • Suady stare bioreaction method. When using a microbial c:onsuming system in (pseudo) steady state, k¡a can be calculate
from che upwlrd ctlr"'e then the nlue ofkp can be-lCund,

Air off

I

tla '= (dC/dt - ql/( C· - C)

e q .. dCldt

~Iuat_d.

for example uliing ¡ Io¡lrithmlc plotof (~ -q

t

Alr on

n . ,lm.. Time

MASS TRANSFER

phase romposition will Dot be uniform afier sparging with N1(or [he gas hold-up must be built up again after shutdown ofthe ai,. flow). In any case, a rapid 0 2 electrode is a p,.erequisite for accurate results.

8.5

Large bioreat:lor

I The effect of scale on mass transfer

8.5.1 Scale-up In large-scale bioreac[ors. 0l transfer is usually better than in a labora[ory scale orpilotplant reactor. This is due to a larger contribution of the gas phase (higher superficial gas veloclty). and a larger driving force (high headspace pressure and hydrostatic head). Example Consider two geometricaJly similar. ideaUy mixed stirred tankreactors, one ofO.1 m l reaction volume. and one oflOO m 3 , 111e H.jTv ratio is 3,0, and the D{ry ratio 0.5. Scale-up is (arried out according to a constant power input, Prv;. of2kW m- 3 • aconstant relalive air flow rate ofl vvm, and a constantaverage pressure in the broth (determined by [he headspace pressure plus the hydrostatic head) of 2,45 bar. Assuming thar there ¡s no deplerion ofthe gas phase, the followingcomparison is made (coalescent brorh, C"= 0.2-4 mol m- J ar 1 bar, C= 0.10 mol m- J ): 0.1 ml : \1,

pJp= 0.007 ms- I "La = 0 ,046 S- l OTR = 0.022 mol m - l·s

100m3: v. vJp= 0.071 ms- L

lela

=0.145 S-

I

OTR ::: 0.071 mol m - J·s

What would be tbe OTR diffe.re ncc for a non-coalescing bl'Oth? (Answer: OTR= 0.07 orO.12 m ol m- 3 ,s, resp.) In a large bioreactor the OTRhas maximum limiLS due to rbe following restr;crlve conditiOn$, • Tbere may be mecha nical construcnon difficulties invery large ferme nten (> 300 01 3), Furthermore, liquid rransport and mixing will become very sJow compared to mass tr.msfer and reaction. and tbus rule tbe overall reac tion raleo Cooling limitations may become more significant. • The average power inputshould notexceed 5 kW m- ~.]fhigher, the mkro-organisms may be mechanicalIy damaged in areaswhere the value is locally much higher: in addition the energy costs and investmenr costs for rhe motorwill become excessively high. • !he pressure corrected superficial airvelocity should be beJow 0.10 m S- l. Compressor costs are restricting, and high gas bold-up will ¡ncrease ar the rost ofbrorhspace. • The head-space pressure has a maximum fue mechanicalreasons_ln addition CO 2 partial pressure will also ¡ncrease. and inbibit growth and production. • The gas pbdse canDor be consídered ídeally mixed. The O}partial presrure will faU as the bubbles travel up through tbe reactor and rhis reduces rhe driving force for mass transfer.

Low

o)(ygen

Air liquid now and DI tranger In a large biore:lctor. Most

ofthe 0 1 is transferTed In me region n...artlle impeller. In the circulation loop. Le. me path the brom travels Imm thft lmpeller; Out

into the body 01 th. reactor and back to the mpder. more 0lls consumid than cransferred and tite

0 1 conCf)mraúon wi!! decrease. In a ful.sale stlr~d t:ank reactor the l!quld circuladon loop can be as long as 10m. Wlth ~ IIquid 'ieloclty of r m 5- 1 che. me~n circulltion timI wiD be 10 seconds. As. a worst

cas.e Istimat. (no tr.msl.r at atI ouuide the impeller re¡ion).ltwlll Clke 10 s befOl'l'- the 0 1 wil become dep!eted In the /uo¡:>. lherefon=, theO l concent~tIon In me bottom Co:!mp;irtmellt sl'lould be JO tllgl'llhalloal OepIetlon. whlch can be 6elrlmentallorthe microbial state and produa.

fonnati<Jll ratl.'s~, No:!t. that Eqn (8.8) prMka thu tllfs wll r.d1lA cM mus trandu ratl blcaUK m. drMng fon:l (C"- q in che boaom part Is Iow as both Cand C will be h1;tl.

185

186

NOORMAN

lnreactoTs largertban ca. 10m3, the prcx:ess~oruquid transportand mass transfer become comparably slow. Mass transfer and liquid e~-ulatioll willinrerfere, and should be treated togelber.ln a stirred tankreactorwitb one si ngle impe1ler, most or the Oz ttansfer takes place in the impcl1er 1:One. A comp.nison of 0l rransrer in the bubble column and the stirred tank(see example in SectioD 8.3.2) revealed thatoutside the impellerzonf' the OTRmay be only one tbiId ofthc value around (he impeller.The im por· tance ofdrculation loops is described in Fig, 8.8 onprevious page.

8.4.2 Scale-down As illust:r.lted aboye, micro-organisms can experience a continuously ehanging environment when travclling around in a large bioreacLOr. This may giw undesired scale-up effcets. To avoid these problems the large scaJe should be taken as the poi nt of referenee, and the possible effects should be studied by simulado n ofthe large-scale val'iations in a sma U·scale experimental set-up (think big, ael smal1). Limitip.g factors 00 a large $Cale, such as mass transter, are thus sealed down and can be srudied and minimised in a practical and economic way. In reality, scale-down cannot be precise. beca use the large-scale con· ditions are difficult to detennine, and a lso too complex to be fully undcrstood. Several tools are available to find adequate SOIUtiOllS to down·scaling: • Two-compartmentreactor ser-ups ean be used, mirnicking (he two mosti mportantreaetor zones, and recirculation ofbroth between the zones (see Fig. 8.9). The size oftbe two eompartments and the circulation rate are e rioeal. but also the Oow type in e3eh companment, Le. rangingfrom well·mixed ro plug flow. • DeveJopment and (large-scale}verifieation ofsimple or more sophisticated m3thematical How models fuI' me large vesselcan be c3rried ou t, :m d then these used to design t he sealed-down experiment. • Wcll<.haraeterised rnicrobiaJ test systems can be used, in which th~ seDSitivi ty to selel-'ted variations is known. lo the litenlture these approaches have bee.n extensiveJy srudied fuI' tne improvementof01 transrer ñnd substr.lte(feed)mixingin large bioreaetors,

8.6 I Further reading

d,

[J- -El

NITrogen gas

BrOl h

lo- -El

,

Air

• An eJ(ample of a sma l¡" sale 2-compilrtme nt nlactor set· 1Ji! fer sa.lill:-down ma conditions larse biore3.CtOr. As an altemative, tila N 1 spargedvestel lfl3)' be In plugflowmode (wkh sorne dil; ~on) .

'" me:

B¡¡iley.J. E.. ;lDd OUis, D. F.(1986). BIoch~mklll Engfnurlng Ftmdam
Nielsen,J. and ViUadsen,j. (1994). 8/tmaction lí'ngillt'~rtng Prindpl<'5. Plcnum Pl'ess.NewYork. Nicuow, A. W. (199Oj. Agi.latl)[lj fOT myceli31 fermentations. Trrnds BiotwllloJ . 8, 224- 233.

'van t Rlct. K. aod Tr.r.mper, J. (1991). Dasie tlillrelUtm'Vnign. Marcld Dekker. New York

Chapter 9

Downstream processing in biotechnology Rajni Hatti-Kaul and Bo Mattiasson lntroduction Downstream processing: a multistage operation Sotid- Uquid separadon Release of Incracell ular compOllents Concentrating biological products Purification by chromatography Product formularion Monitoring of downstream processing Process integration Further reading

9.1

I Introduction

The ¡solatian aod purificatían of a biotec.hnological product to a form suitable forits ¡atended use is popular1ytermed 'downstream processing' (DSP). rn mosreases this means recovery ota product from él dilute aqueous solution , 'fhe complexity of downstream prlXessing is deterrnined by the required purüy afthe product, in tufn dete,rtnined by its application. The products ofbiotechnology incl\lde whole eeUs, org<mic adds, arnino acids, solvents. antibiotics, industrial e.nzymes, tberapeutic proteiru. vaccines, gums, !!tC.M the products vary greatlyin SlzC and nature, diffcrcnt sepax3tion principIes are required ror tbeir recovery and purificarlon. The lability 0 1· seruitivil}' ofmany of tbe bioproducts, particularly the proteins. (O the environrucntaJ conditions, places further demands on tbe dla ractcristics of the separation processcs usoo ror tbeir prodUcDOU , It is also i.mperative to mil1imise the number of steps and to mamrain hlgh yields in the differentsrages, since rhe ulti· mate result.will be poor ifscvcra11ow-yieJding steps are combined.

9.2

I

Downstream processing: • multistage oper.tion

Many ofthe stcps in DSP are traditional unitprocesses used excensively in chemical industry and have b!!en described lnchemical engineering

188

HATTl-KAUlAND MATIIASSON

liteeature. This chapter is devotro lO DSP processes especially adapted tor biotechnology. The downstream processÍng scheme normally employed for isolationand purificatioo o(biomolecu!escan be divided into the following stages: (1) solid-liquid separ.3tion or clarification (2) concentration (3) purification (4) fonnul ation. Sometimes, as in th~ case of industrial enzymes, the concentralion stage may provide purificatioo sufficieot for the final produCl quaUty, obviating the need foe a separate purifica non srage.A wide range of urot operations is available foreach stage, see Fig. 9.1. ('lbe eeader should be awa re tha! the industrial realityofDSPstarts upstream at the medium preparation stage. as rnany r.lW material s require pretreatmeo[ to remove impurities that would have increased the dcmands on tbe purificaban stages.)

9.3

I Solid-liquid separation

Solid-I¡quid separanon is a primary recovery opcration for the separanon ofwhole ceUs from the culture broth. removal ofceU debris, ooUection ofprotein precipitate. ooUection ofinclusion boclies. etc. The unit operations commonly used are centrifuganon and tiltrarlon.. Table 9 .1 lists [he cbaracteristics of sorne. of [he culture beoths used ror production ofbiomolecules. Yeasts and bacteria are usually homogeneous ly suspended in the fermentation broths. Some bacteria may form slime layers depending on tbe strain and fermentadon conditions and [his willlead ro separation problems . PiJamentous fungi are frequenrlycharacterised by a network ofintertwined ftlaments. producingviscous fermentation broths t:h.at may be difficult to dewater. Under certain conditiollS these fungi wiH form agglom erates caHed ·peUeu', whkh are eeJative.ly easy to recover owing lO theu- large size (100-4000 ¡.LfI1).

9.3.1 Filtradon A filter medium constitutes the separating agent. whichIetams che par-

tieles according ro sizewhile aUowing the passage ofrbe Hquid through !he filter. In cake. fUtration , the partieles are retained as a cake on rbe filter medium. Tbe tlow rhrough the filter layers is dependent on the area ofthe filterand flow resistance provided by the filter moo.i um and me cake. Provided the partidesdo not pene.trare me filter medium, the tlow resistance of tbe latler should remaio unchaoged; however tbe cake-Iayer, as ir grows thicker. will provide. increasing resistance. lo many cases ofbiomass separation, the filrer cakes obtained are compressibJe. and tbechanging effectivc pressure difference will influence rhe flow through the ftlter.Exarnples offilter media are perfora ted sintered metal, cloth. symhetic flbres, ceUulose. glass wool. ceramics and syntbetic membranes. Many types of filtratioh equipment are avai1able. Because of

OOWNSTREAM PROCESSING IN BIOTECHNOLOGY

Upstream proooss

OOWl'l5cream

procesJlrl(. Dlfferent s~ In the: Ilolatlol\ and ptWlflutlon of dl_

Productlon

produmofbl~rt·

Oownslream process Extra-

Intra-

callular

celluliH product

product

Chem ical Enzymatlc

Mechanical Physlcal

¡

Cantrifugation!sedimentation Extraction, fillration EVllporstion, ultrafiltration Adsorptlon, preclpltatlon Ch romatography

Formulé1lion

Solidstype

Size ( ~m)

Cell debnso Bacterial eells Yeast cells MammaJian eells Plant eells Fungal hyphae Microbial fl oes

O.2xO.2

CryS1allisation, fre ne drying, SprllY drying. stertlo flltradon

Ix2 7x lO 40x40

100xl00 IxI Ox(marted) IOOx l OO

Density dilference bet>Neen 50Iids and broth (kg m- ' )

0-120" 70 90 70 50 10

Nou:l: • Ce ll dE'bris dell$ity!h.·~nds on compolitinn. e.¡¡r. lipld co nTen to

simplicity of operation a nd low tosts, vacuum filters are frequE'ntly used for clarification oí" fermenta tion broths conta ining 10-40% solids by volume and partides wirll sizes ofO.5-10 ~m . The bestlcnown are rotary drum vacuum fllter and filter press. The former type is commonlyemployed for filtration offilame.ntous fungi and yeast cells . and

is scbematical1y presented in Fig. 9.2. Resides simplicity and effectiveness offiltration . its a t tractive features a re Iow power consumption and

Cost of recovery Highest

< < < < < Lowe"

18'3

190

HATTI-KAUL ANO MATTIASSON

Washing

A rm;u-y drum vacuum f~ter. The

filtralion eI!'!ment

compriSe5 <11 rootin¡ drtJm. malntalned UndN reduc.ed intem;¡J

i

pressur!'!. partlally immersed In, cank 01 brom. Rotating wlth a speed 010.2.5-5 rpm, t~é dn.tlll

plcks

~

th!'! bloman, me filtr.lte Is

drawn ami che u.ke Is depo$ited 011

Ca.ke

Feed - -- -,

removal

che drum sume.. The contlfJlJOUS rollltion al ehe dr um 3110ws subsequellt opentlons 0 1 dewJ.teñtg. wuhln¡. and dry;og tu be perlormed on mi r~t.er cake

prior te lu dls.chargl fronllhe

Filtr;lli on

drum sumce. Deplndlng on lOO propertIes. che blomM5 filt.er cake 15 di5char¡:ed by elcNr: knife-,

mili&-, bell_. or roll.r~l$elurge . The dnrms could 1M! ,11111&- or mlAú-comp3r tmlnt ¡¡nd 3rt! often c
2 te) 80 ml.

a (ontained operation. The filter pl'ess is built up of a sequen(c ofperforated plates alternating with hollow frames mountro on suitable supparts. TIle piares are covcred with a filrcr meruum (doth) to creare a series of c1otb·waUed chambers ioto which slurry can be forced under pressure. The sol ids are retamed within thechambers, while the filtrdte discharges iuto holloW$ on the pIare surface, and bence to drain poinlS_ At the end ofthe filtration cycle. the hydrauüc pressure is released and the cake manually removed from the doth. Typical filrer sizes
Cenuifuges.. (a) TIle tubular bowI typot" me simplen sedlll'l-l!notlon centri rugl! used In pilot planu, whlch un be oper.ated botll In ootch o r t.OnUnlJOllS modt, wIth nunt.131 d lKh;¡rge 01 tIt. soUeIs. Hi8~ cen~rilug;¡.l fleld an ba achieYed ow1nx to tu slender shape and small voIame.

(b) TIle multidDmber bowI type is 1. modiflc.atlon el che tubul;¡¡~ type, COlltamlne a numbftr of

cenclntric tubes connected to alto w ligug iIow ofdle p;m:ia.liate leed.. The cwO'lfugal force" illa-e
th, bowl wlm lne result mat the t-mllle$cp~rtide5 are collected in che outermostchamber. An imPOt'tlllnt3pplk.atlon ofthe~ centrifuga¡; i. in the Iractlorntion 01 human blood pruma.. (e) lile dl$c st;ac!(centrifup is u$<.ld widely in blotKhlXllogy. tr Ineorporate~ sep~race

numerous dises (typlcaBy between 30 ~ nd 200) n ~n ~e

0135-- 50'

~nd

Upt 0 .......2 mm aparto dlvldln¡ m!'! bowI.,to

seruing zones. The leed Inulrs tIle centrlfuge through a central feed pipe leadlog to me leed dmmbe r at th!'! bouom of (he

uack, SoWt:k $!'!ttle on che kJwer surlacI of IIiIcll disc an d mlgratl !.OW3rd che bowl perlphery. wlllle t~1!I clarified l!qul d moves Inward 3M upw3r d LO re
none end, h;rtiOJ;ll; a length-d¡.,m..,..,.. ... tio of 1 :-4.~nd fittedWith ~ dme-fiuing hellal1crew th.\~ rotaCIIS at a stighdy

dlft"erent spe-ed from me bowI. The 101lds dlpMd on meW311 al lbe bowt 3nd ill'l scr.\ped off h)I the s.crtW and d i«.h:!:rged lrom che Ilarrow efld al me bowl. TIH! ctlltrffuge can 1M! U$fId ror reeds wittl blomau conr.ent of 5-80% vlv.

(b)

(. )

Uquid di<;charge

~

Slurry m

Uquid

di ~ch a'1lc

.,1----

("

Súlids

--::=-~ di~harge

" ""

,., ,

~

-;-

.-- --- ~.

(d)

"

I I

I

I j

j

/ Liquids ¡$ioclwge

f

1. / j

/

/

f

I

1;:0 I

1 SI""" io

192

HAlTl-KAUlAND MAmASSON

9.3.2 Centrifugadon In centrifugation tbe removal of solids relies on the density difference

between the pMtides ro be separated and the surrounding medium. Batch centrifugation is commonly applied at a laboratoq¡ scale. Altbough pmviding higb centrifuga) rorces. low processing capacüy limits m e use ofsuch centrifuges on 3n industrial scale, where contin· uow flow centcifuges are the norm. Here. feed ing of tbe slurry and col· lection of the darified soludon is continuous, whilst the deposited soLids can eicher be continuously or intermittendy removed from the ccntl'ifuge, Sorne of che centrifuge cypes used in the isolation of bioproducrs are shown in Fig. 93. TraditionaLly, centrifugadon is used fur I"emoval of microbial cells and olherdiscrete large particles. Tbe super· natantobtained by centrifugarion is not freeofce lls (1()3-1OS ceUs ml- I ). and COSts of maintenance and power consumption are both high . Separatioll ofparticuJate debris from cel! homogenates is quite ¡neffi· den t by centrifugation .

9.3.3 Pretreatment of brotn to facilitate clarification Pretrea tmen t or cond itioning of th e broth by changing the biomass parriele size, the fermentation liquol" viscosity, and the interactions between che biomass partides are sometimes required to ensure effi· cient solid-liquid separation. This implies use ofa filter aid (body-feed) in the broth andfor fOl" l'recoating the filter medium. Filter aids are ¡ncompressible. di,screte particles of higb permeability with size ranging from 2-20 ¡A.m. Filter aids shouId be inert to the broth being treated: [he most frequentIy used are Diatomite (skeletal remains of aquatic plants), Perlite (volcanic rockprocessed ro give expanded struc(ure) and inactive carbon o Agglomeration of individual cells oc ce11-particles ineo large fl ocs. which can be ea.sily separated at low centrifugal forces, is done by the addition offlocculating agents such as polycations, either cellulosic or based on synthetic polymers, inorganic salts or mineral hydrocoUoids. Besides rhe flocculating agent itself, factors like tbe physiologicaJ state of tbe ceUs, the ionie environment, remperature, and naron! of the organism influence f1occulation. Interestingly, cationic filter a.ids also reduce the load of pyrogen, nucleic add and addie prote¡n, which normaJly foul chromatogt'aphy coIurnos.

9.3.4 Flotation In flmadon. panicles areadsorbed on gas bubbles, get trapped in a roarn

layerand can becollected. The gas mayeither be sparged into rhe particulate feed . orveryfine bubbles can ~ generated from dissolved gases by releasing lhe overpressure or by e1ectrolysis. Formation of stable roaro is supportfil by thepreseoceof'coIlector rubstances' such as long chain fatty acids or amines.

DOWNSTREAM PROCESSING IN BIOTECHNOlOGY

9.4 I Release 01 intracellular components The primarygoal for the.first step in a sequence fuc release ofintracellu· lar compounds is to liberare maximum amount of the praduct in a n active state. The inactivating eft"ec.ts ofshear. temperature and proteases need to be borne in mind at tlús stage. 111e choice ofthe disruption methodhas to be mad e emplrically. at the same time taking ÍDlO consid· eration rhe subsequent processing steps.

9.4. 1 Disruption ofmicrobial cells The different straregies m at may be used for rupture of microbial eells ¡ndude breaking the eeUs' ~tructure by mechanical forces. damaging preferentiallythecell wall e.g. bydrying ocenzymatic Iysis, oc lysing pri· marily the membranes e.g, by treatmenr with chemica!! (see Table 9 .2). Mechanical disro pdon of cells is the most common.means of releasing ¡ntcacellular products botb at rhe laboratory and indusuial scale. mtrasonlcation disruplS tbe ceLls by caviration, and is cornmonly uscd ae laboratory seale; removal ofthe heatgenented is difficulton alarger scale. Jndust:r:ial scale d isruprion of microbial cells is achieved by high· pressure homogenisation or by vigorous agitation with abrasives.ln the fonner case, the cell suspension is forced at high pressure tbrough an orifice of narraw intemal dlameter to emerge at acnospberic pressure. The sudden rclease ofpressure crea.tes a liqu.id shearcapableofdisruptingthecelli.Agitation with glass in bead milis ruprures tbeceUs by a combination of high shear and impact with tbe cetls. The size of the beads varies from 0.2-0.5 mm for bacteria to 0.4-0.7 mm for yeasts.

Me
Microbiol ce1ls Ultrasonication High-pressure homogenisation Agitation with abrasives

Non-mechanical

Drying Heat shock Osmotic shock Freeze-thaw Organic. so/vents Chaotropic agentsAlkali Detergents Enzymes

Animal rissue Homogenisation

Ptant tissue Mac.eratlon

Freezing and grinding

Enzym.,

193

19-4

HATTI-KAUlAND MATTIAS50N

Recently microfluidisation has beeninrroduced forcf"lJ disintegratiou_ Here, tbe (ell suspens.ion is fed underrngh press ure tllrough a cbamber where it is split into two streams, (O be rurected a( each other at h i~h ve.lodty befare emerging a( atmospheric pressure. Non-mechanica.l disruptioo of cells is possible by pbysil:;al, chemical or enzymatic means. Orying is a wide.ly applied method ofcell lysis. causiogchanges in tbe structure ofthe celJ \l/aU and making itpossible to subseque.nUy extraet tbe ceU contents in buffer or sale solution. Osmotie shock ruptures the cel l membrane and is particularlyeffective for animal ( ells thal lack a ceU wall. The presence of a ceU wall in the ase ofmicrobial cells may necessiC
9:4.2 H omogenisation of animal/plant tissue Animal cel1s are nonnal1y easy [O break because ofthe absence of eell walIs. A typical procedul1.' fuI' homogenising animal tissue is ro cut it ioto sma!! pleces. suspend in icc-<.:old.homogenisation buffer.and griod in a blender. Plant cells are more resistanr particularlywben they bave tough ceU walls. '[be extract of a fleshy or non-6brous p!ant tissue is prcpared by rapid homogenisation of the material suspended in a partiaUy frozen .suitable buffer in a Waring blendcr pre-cooled to - 20 ..C. The more fibrous material, which is difficull ro macera te, is frozen ó'lnd grouDd ro a dry powder before adding the extraction buffer for hornogenisation. Concentrated buffers with pH values 3round 6.5-7.2 are used io order to neutralise tbe addic materials inc1uding the phenols.

OOWNSTREAM PROCE551NG IN B10TECHNOLOGY

and also contain phcnol scavengers such as polyvinylpyrrolidone IPVP) and/o!" Am.b crlite. a hydropbobic poly5cyrene-b
9.5

I Concentration of biological products

Mer separanng rhe eeOs from the whoIe brom othe filtrate contains 85-98% ofwater. wirh the product forming oruy a minar eonstiruent Rcmaving Jarge arnounts ofwatcr is eostiy. re is done in differenr ways: evapoJ
Evaporation

~vapora tion

is a simple. but inmost cases. an energyerofstages {maximum 5even} using rhe vapouJ' of ·ne stage as a heating 50Ul'Ce for the nexr. In falling film evaporamrs, tbe liquld to be concentrated flows .own long tubes and distributes unifonnlyowr lile hearingsurface as thin film_ lbe vapours fiowing in rhe same direction ¡ncrease the .near velocity of the liquido rhcreby improving the heat transfer. .csidence times in the evapOl'ator are in rhe order of minutes. Tbese vaporaton are suited fol' concentrating viscous products (up to 200 lPa), and are frequently used in the fennentation indusrry_ Plateevapratol'S, where the hcaring surface is a pIate as oppo~d ro ;:¡ rube. have relatively large evaporation area in a sITIal! voJ ume, but the posslbility ftreatingvi5cous andsolids-containing fluid s is limited. For higher vis:lsities. forred filmevaporators with rnechanica11y driven liquid films re suitable. in sorne cases producing a dryproduct.1'he residence time

195

96

HAm-KAUL ANO MAITIASSON

ranges from a few seconds to a few minutes. CentriCugal forced..fUm evaporators pe.rmit a further redurnon ofthe residence time so that even the heat labile subscmces can be concentrated under gentIe conditions. EvapOl:ation takes place ona heated conical rurface or plates over which tbe transport of the liquid takes place through the centrifugal force produccd by the rotating bowL

9.5.2 liquid-liquid extraction Liquid- liquid extraction is applied on a large scale in biotechnology both for concentration and far purification. It involves [he transfer of solute from one liquid phase to another. The efficiency ofan extraction process is governed by the distribution ofsubstances between the two phases, defined by the p.aTtition coeffic:ient K (= concentration of sub-stance in extraet phasefconcentration of su bstance in raffinate phase). A partition coefficient well removed from unity is desirable. TIle physico-chcmical properties oftheproductinfiuence thedemands on a liquid-IiCjuirl extraction process. as illustrated in the following sectioos. Extractlon oflow molecuJar weight products Small lipophilic target molecules are extraeted using a.n organic solvent. whereas for hydrophilic compounds it may be difficult to design an efficient extraction process. Extraction in organicsolventcan be done in one of three ways: Physical extractlon: The compound distributes itself between the two phases according to its physical preference. Th.is applies [O nonionising compounds and the extraction isoptirnised by screening for [he solvems thar would lead to a high K value aDd also show a max:imal difference in K for the differentcomponents present in the crude mixture. Dissociative extractiOQ,: Difference in [he dissociation constant of the ionisable compone",S i5 exploi ted tO achieve separation. and these differences are onen large enough to overcome an adverse ratio of par tilion coefficients. Exuaction orpe.nic.ilJin 3nd some other antibiotics are typical for this type ofextraction principie. Reactiveextraction: A carrier, such as an aliphatic amine or a pbospiloroos compound. is added to the organic solvent which forms selective solvatioo bondsorsrokbiometric complexes thar are also insoluble in the aqueous pha5e. Thus, the compouud i5 carned from the aqueous to the orgaruc phase. This type of extraction is advantageou5 fur compounds thar have a high solubility in aqucous medium. e.g. organic 4

3ods. In most cases, cells and orher particulates ilre removed prior to

extraction to avoid the form arion ofemulsions ar the interface. After extraction, the productis recovered from the solventeithel' bydistilling off the product in case of a higb·boiling solvent or disrilling off the solvent when this is low boiling. lf the product is heat st'nsitive. ir is recovered by back-extraction into a new aqueous phase unde.r conditioos different from the firsrextraction. e.g. penicillin 1S extracted into butyl acetate or amyI acetate frem the fennentatioll medium ae pH 2.5-3.0 and back-extracted into aCjueous phosphate buffer atpH 5-7.5.

DOWNSTREAM PROCESSING IN BlOTECHNOLOGY

For hlgh extracLion yields. multi·step extraction in a countercurrent mode is used. which provides abo savings in both solvent and time. Oifferent kinds ofextraction equipment are availabte induding mixer-settlel'S, coIurnns and ceJltrifugal extractors. Tbe latter are often used in exCTaction ofantibiotics and steroids; representative examples beingthe Podbielniak extractor. Delaval contactor and westfalia extraetion-decanter. Superoitical fluid (SCF¡ extraction has beell consjdered as an alternative [echnique to convencional extractioll which has the disadvantageoftoxicity and Hammabilityororganie solvenrs. SCFs are materials thatexistas fluids aboye their critieal teOlperatureand pressnre. re."pec. tively. Many ofthe properties ofSCFs are intermedia te hetween those of gases and liquids; e.g. their diffusivir.y is bigher than those of liquids while viscosity is lower. The atttOlctive feature of SCFs as extraetants 1S that theirsolvent properties are highly sensitive toehanges in both temo peratureand pressure, which provides the opporrunity oftailoring me solvent strength lo a given application. Superc:ri tical COz is most eom· monIy llSed for extrac:tions because ofits r elatively low critieal tempero ature (31.3 oC) and pressure (72.9 bars). Supercritical extraetíon is still rather expensive. though a few large-seale applieatious in the area of fuod processing have demonsrrated the possibility of economicaUy viable operations. By adding a co-solvent such as ethanol. in small amounts. ir is possible to modífY the properties of the system and thereby influence the extraction behaviour. In a typical extraction precESs. the compressed SCF is con meted with che feedstoek to be e,,:tracted in an extraetíon column, and tbe loade
197

198

HATTI-KAULAND MATTIASSON

Mi'

Extnction in a'luaous two-pnas.e system. The components of die twO-pl'we synem are mblf!d dirKtly WldI the cruda hornoge/l3.te. EquiJibradon Is dorMI by ¡ende ml~"r1g alld is foll~d by phase sepoa ....t1on. The protlin prodyct partitioned ro th. top ptwe Is subsequer1tty re(o....red enner by a sec:o nd extr.Ktion nep. where it. i!l u-anderred i¡¡oo a new salt phase. or by d:rect adsorpticll'l O!'l tO a dlromnography matrix. Recyd"'i of TM ph3 Sf! componenu ¡, normally possible, mus rnil1lmlsln¡ th, matcrlll COSts.

CruOe homogenale

+ - - - - . PEG end salt - - -- - - -1

-

Equilibrium + phase separatlon

Cleaning & recycling

I ! \

PEG-riGh

LOp pnase (productl

S~lt·rich

! ! Clennlng & ! rocycling

! I

bottom phase (cell debris, prota ins,

~~_- _ n_,,";"";~''--i

PEG-dch

Salt-rich bottom phase

top ph8se

(productl

Desalling by utl rafiltllltion

I Recycling

! i i

Retenllte (product)

Permeate salt __ solution

J

homogenates having high viscosity and heterogeneous distribution of partide sizes. Otber aUractive features ofthe technique are bigh capacity(biomass tovolume ratio) and str.r.igh tforward scale-up. The process is easily adapted ro the extracrlon equipment used for water~rganic solllen[ systems. Figure 9.4 shows a schematic presentation of pl'olcin isolarion and partía] purificaríon in AUS . Por industrial scale separations. PECfsalt systellls are used beca use of their relative]y low costo lnexpensive and biodegradable po]ymers may aIso be considere
DOWNSTREAM PROCESSING IN BIOTECHNOLOGY

Type of membrane

Driving force

Applic.atlon

Microflltrauon

Hydrostatic pressure Ap 0.5-2 b.,.

Concentration of bacteria and viruses Harvesfmg of cell" O arincation orrermentation broth

Ultrafiltration

ü p2-I Obar

Fractionation of biomolecules

Desalting Production of enzymes Processing of whey Hyperllltration

.6.p 20- I00 bar

ConcentratJon of pharmaceuticals Production eflactose Part desalination of solutions

Bectrodialysis

Pervaporation

~El ectric

fi eld

llpdrtial vapour pressure

Purification of charged small molecules e.g. organic acid" SelectNe removal of solvents (ethanol. pressure acetone-butanol) during fermentation

Puriflcation of solvenls fmm azeotropic mí>..1.ures

F'erstraction

Partition

Extraction of SITIall molecules from aqueous/organic solutions

interactions between the protein surface and chargedsurfactam . and is thus dependenton pHand ionicstrength.

9.5.3 Membrane flltradon The use of me.mbrane technology for separadon of biomollX'llles aud ['anides and concentration of process fluids has expande
I'J'J

200

HATTI-KAUl ANO MATTlASSON

deposi tion of colloidal spedes, adsorption ofmacromolecuJar solutes, precipitanoo of small salutes, etc. on tbe surfat e and in the pores of (he filter leads to a deposition ofa cake which grows in thickness with time, reducing the flow through the filter. Cross-fiow or tangential How liltraCion has now completely replaeed the dead end filtration for harvesting cells on a large sede. Here. a flowofthe fL>ed stream is maintained parallel to the separation sut:fuce, with (he aim tO provide sufficient shear force clase to the membrane surface, thereby preventing particlllace matter from settling on, or within. the membrane scruc· ture. In pr.1ctice, the membranes in cross·flow filtratioo are also rubject (O fouling. but the cake thic:k:ness is Limited to a thin layer as rompared to the dead end mode. Although mast filtrarlon media are relatively merr. the formarlon of gcl layer is inevitable. The probIem of permeate flux reduction can be minimised by optimising the filter selection. operating pressure, Oow properties of feed . and frequent back·flusbing. Microfilrers and ultrafilters are avaiIable in materials sumas ceramies and steel tbat can be aggress ive:ly deaned and srerilised in place. Membranes composed ofpolymerie materials such as polyvinyldifiuoride ¡PVDF) and polyethersulphone (PES) are also used, but are more difficul t to clea n and may require chem icaLrather than steam sterilisati on. Membrane ruten are commonly pIate and frame systems. e mploying eartridge filten within which the membrane is present in a highly folded formal. This gives a large filtradon sumee area in a compact space with no dead spaces. Another fonn is lbe hoUow·fibre system, wh.ich eomprises a bundle ofhoUow capiUaries packed in a tube. TIlc liquid to be filtered is pumped through tlle central eore ofthe hoUow fibres. The permeate passing through (he capillarywalls can be drained as a pe:rmeate from one end of tbe caruidge. while tbe concentrated retentate.emerges from tbe othel' cnd. Membrane adsorbers New micro¡m.acroporous membrane matrices with ion exchange groups and affinity ligands. called membrane adsorbers . have been develaped which bind proteins from the c1arified feed pumped over them.Desorpdoo ofrhe protein is laterperformed using solutions as in chromatogl'aphy(see Section 9.6).A staekofmembranes provides a total surtace area for adsorption equivalent to chromatography gels, giviog similar high resolutioo separatioo as chromarograpic metbods. In membranes, liquid transport is by coovection as opposed to the ditTusional Oow in gels Isee Section 9.6), which increases the speed ofseparation ttemendously. Pervaporation Pcrvaporation is a membrane based process havingpotential for re<:ov· ery and concentratioo ofvolatile products. see Fig. 9.5. The majol' limi· tatioos of the tKhnique fur large-scale work are high en~rgy consumption. insufficient selectivity of the membrane and ditticult process design due te a temperature drop across the membrane.

DOWNSTREAM PROCESStNG IN BIOTECHNOlOGY

Relentale Uquid

Vacuum pump

Pamc:~le

Coolaol

Condcll!w:d pcnnclllC Pervapor.UjOfL The pt'OCeu allows H:p~tion el vobúle prodw:u by a combilRtion of penneation dlrou¡b a membrane and 8YapOI"alion ~cl!ieYe<1 by ¡ Iow preutre on me downsrream slde cf die tntillbrantl. The tnns-membran.e llull: of varlOU$ compooellts in ~ mixwre and lhelr separadon ¡¡ determ!ned by dlfferen(1l$1n thelr vapourp~tJI"e and bythe permubillry oIt/le memmne:; lhe I¡Uer beill' a function of (~) d;fl"u~ion oflhe ccmpooent to be separated throu¡h (he membr:ione Ind (b) membnne thick"eu. Pervaporllliofl membr.mel Cdn be consldered u homogeneotl$ $wolen poIymen, e,g. pol)'-{dmetfTytslloltar.e). Thtt solubilit)' 01 the«Jmponanl in the mernbrme material and the ell:tent to whlcl'llt Is lwollm determllle die dlffusloll ccodIitient. Selectivity ofthe membrln~ I1 iI.I1Odler factor Influencin( me periormance a flhe process.

Perstraction Perstraction is another soprusticated tl'chnique for product concentration and recovery that combines membrane processes and salven! extractian. By using che mem brane as a barrier between an aqueous feed and an organic solvent ir is possible to [ransfer molecules thar partition into the liquid filling the mcmbrane pores, This rcchnology has been used for recovery of hydropbobic substances dur:ing fermenrations. The membrane protects the cells from the toxic or inhibitoIY effects ofthE' extr.lction sotvenl. Depending on the direction of mass transfer. the membranes could be hydrophobic or hydrophilic. The hydrophobic membrnnes. which have pores filled wirh the organic phase. are used to exlr.lct [he non-polar product from the aqueous medium. 10 hydropni1ic membranes tbe pares are filled with a suitable aqueous buffer. which facilitates the removal ofrhe producr from rhe solvent.

9.5.4 Pl"ecipitation Precipitabon is an esta blished separation techn.ique in industryforconcentraban of proteins and polysaccharides. Often it constitu tes the onl y

201

202

HATIl·KAULANDMATTIASSON

Mode

Example

Addrtion or neutral 5alt

Comments Ino'&lSed hydrophobic interactions between neutral protein mo lecules; salt is removed priorto next purification step (except for hydrophobic interaction chromatography) by dialysis, ultrafiltration or gel f1Itration

Addition o f organic salvent

Acetone , ethanol

Reduced dielectric constant enhances e!ectro statlc interactions between protein molecules: Jow tem perature required fo r o peration

Addition of non-ionic: polymer

Polyethylene glyco l

Reduction in the effective qua.ntity ofwater available for proteln solvation: polyrner has often a stabilising effect o n p rote in ~

Addrdon o f charged polymer

Polyethyleneimine, polyacrylic aad

Complex formation between oppositely

charged mo lecules leads to charge neutralisation and precipltatJon

Increase in temperature

Increased hydrophobic interactions; used for precipitatio n of))eat sensmve proteins

Change in pH

Low solubility of protein al isoelectric po int, eldremes of pH denature and precipitate sensitive proteins

unit ope:ration after solid-liquid separ;Jtion in the recoveryofbulk pro. teins. Prccipiration can also be used to achieve the removal ofund esired by-products such as nudeic acids, pigments and other residual components from a crnde extracto Precipitation is usually carried out in a batch m ode in stirred tm.ks.Aggregates settle to che bottoro oftbe tank, the mother liguor is removl>d and me aggregate slurry is fed [O a centrifuge orafilter_ Precipitation ofproteiruis usually base<:! on a decrease in solubility induced byexternal factors (Table9.4). SaJtsand organic solvents are the precipiu.ting agents commonty employed in industry. TIlese precipitation processes ~re non-specifie in me sense that they exploit the ionie and hydrophobic ¡nte[;letioos, wllich are comrnon [O all proteins. There are a few examples of precipi tations chal are semi-specific, e.g. prorein-carbohydrnte complexes have beell. predpirated by meaos of borax: additions. Selectivity in precipiration has been introduced by use of affInity interactions. Creation of large complexes as a result of affinity interactions. as between antigen aod antibody, i5 one mode of afflnity predpltation. which is used in immunoprecipitation. This concept has becn generalised te sorne arene far selecti~ precipitation ofmultimeric proteins (having more than one binding site 101" a ligand) by using homobifunctional ligands (synthcsised by coupling two ligand moleculcs by a spacer). The modified ligand is ablc te bridge

DOWNSTREAH PROCESSING IN BlOTECHNOLOGY

differentprotcin molecules thereby forming aggregates. The precipitation afthe affinity complexoccurs only at a dcfinite ratio ofthe ligand and the protein. Transition metal ions are able toprccipirate proleins by bonding with m e surface histidine residues, the more tbe res¡dues the casier is the precipitation. With heterobifunctional ligands. whcre Qne functional ity is responsible for the Olffinity binding and thc other is exploitcd for the precipitation, irhecomes possíble to operale affillity precipitarion in a more general mode. The precipitatingcomponentofthe heterobifunc· donal ügand is a 'smart polymer' which responds to minor ehanges in an environmental parametel', e.g. pH. remperaturc. iome strength, etc. by a visi.ble c.bange in solubility. Examples ofsuc.h polymers are chit
Expanrled bed

9.5.5 Adsorption to chromatographic partieles Another sn-aregy often used fur concentrarlng a particular molecule from a erude extract is captunng on high-capaci[}' solid adsorhent par-ticles. Active marcoal was the first reponed adsorbent material for produelconcenn-ation.lon exchange resins have sinee been widely used for initial capturing of low molecular weight pl'oducts and proteins from erude extracts. because oftheirhigh billding Ld.paci[}', theirappli· cabiUly ro harsh c1eaning-in-place (GIP) pTOtocols and tbeir relatively low cost, Syntbl:'.tic adsorbenrs with hydrophobic adsorption rnar.lcterisoes bave also been dcveJoped whic.b can be use
t '.

Expande
adsorvtion dlromltDgt'aphy. The

bed of adiOrbent be3ds lo a colurm ls expanded by an Ilpw:!.rd flow of Hquld. A roIbte be
203

2~

HATTI-KAUL ANO MATTIAS$ON

9.6 I Pu rification by chromatography Chromatography is the technique used for high-resolution purificarían. !he components ro be separare
DOWNSTREAH PROCESSING IN BIOTECHNOLOGY

Chromatography

Separation principie

Size-exdusion (gel liltratlon) lon-exchange chromatography Chromatorocusing Hydrophobic interaction chromatography Affinity chromatography Immobilised metal-ion affinity chromatography

Size and shape Net charge Netcharge

C~ent chnornatography

Content of free - SH groups

Hydrophobkity Molecular recognition Metal ion binding

Matrix

Trade name

Cross-linked dextran Cellulose

Sephadex 'vVhatrnan TM. Cellurlne, 5ephacel. Cellex Sepharose, Sepharose Fast flow Sepharose, Ultrogel, Superase BioGel P

Agarose Cross-linked polyacrylamide Composite of polyacrylamide .lnd dextran Composite of agaroSe and dexlran Composite of agaro)e and polyacryjamide Composite of parcus kjeselguhr and agarose Hydroxylated acrylic polymers Hydroxyet hyl methacry!ate polymer Ethyleneglycol-methacr)'late co-polymer Polyacrylamide Porcus silica RJgid organlc polymers Polystyrene/divinyl benzene

a...

s.pnaoryt Supe<dex Ultrogel AcA

Macrosorn KA Trisacryl Spheron Fractogel TSK, Toyopearl

ELJpergn e Spherosil, Accell Mo nobeads, TSK-PW Poros

ueed ro be done by increasing the bed height, aud tbus the problem of bed compressiou ofsoft gels obrained in conventional downward flow ofeluant is ove«:orne.

9,6, I Size-exclusion chromatography This invoLves partitioning of proteins between che stationary liquid heId by pores of rhe gel partic1es and the mobile liquid in the void volume bctween the particles. The gel matrices used for size exclusion

chromatography haVE' .a defined pote size range Co allow small molerules imo the potes whíle rhe larget molecules are excluded and pass through rhe column with the mobile liquido Size-exclusion chromatography is effective only with small sampk volumes (equivalent ta 2-5% ofthe total bed volume), and is thus suitably use
I

205

206

HATII-KAULANDMATIIASSON

9.6.2 Adsorption chromatography Ln adsorption chroma tography. resolutioll af me macromolecu)es is a surfdcE'-mediated process ¡.e. there is differential adsorption of tbe

moleculcs ar the surrace of me m3trix (see r abie 9.5). The matrices empJoyed are de rivatiscd to con tain covalen tly attached functional groups, which ansorb and sepal:ilte protcins by diffecent mcchanisms.

Ion achange chromatography Ion exch ange d u omatograpby is by far the mostwidely used [echnique becausc ofüs general applicabiliry. good I'c solution and high capadty, Moreover, it is insensitivf.' ro sample volumes and is ohen used in t he initial phase or downstream processing to provide both product purifi· c.ation and volume rcduc.:Oon of the proccss Huid. Compounds are separated according lO the difference in tbeir surface chacges. Hence, pH of tb e medium is one ofthe Olost.impoltaot paramelers (or binding of the target molerule, as itdetermines the effective charge on both tbe (arget molecuJeand the ion exch anger. lonic buund molecules are eluted from the matrix, either by incrcasing the concentr,;¡ttoll ofsalr ions wb.idl compete fOl" tbe same binding sites on lhe ion exchanger, or by changing tbe pH of the eJuan t so thar the rno)ecules lose thcir charges. Displacement chromatography is an aI(ernative mede wherein a more heavily charged molecule is used (O 'displace' rhe bound matct"ial. 1011 excllangcrs are grouped inm anjon exehangel's with positively charged groups like diethylaminoethyl (DEAE) and qU3Cremary amino (Q). and cation exchangers which have negatively charged groups lil:e carboxymethyl (CM) and sulphonatc (S). Hydropbobic interaction e h roma tography Hydrophobic intecaction chromatography (Hlq i5 also a robusto high capacity method. It may be used early with dilure producl streams , lending to conccntratiOll and purification. me i5 analogous to n'verse pha.se chromatography (RPC) bur rc.1ies on comparative.ly weak hydrophobic interactions betwt!cn hydrophobic ligands. aHcyl oc aryl side chaios on the gel ma trix and the accessible bydrophobic ami no acids on protein surface_Differences in the content ofthese amiDa add residues can be used for separation of proteins. Binding is perfocm ed in a Illcdium favou ring hydl"ophobic i1ucr.lctions, e.g. a solution ofhigh salt concentraDon. flUtiOD ofbuund material is achievcd by reducing the hydrophobic interacDons, either by lowt!ring the salt conccntration, or the tempcrature, oc by decreasing the polarity oflhe medium {by indu· sion ofsolvents Jike ethylene glycol or cthanol in che buffer). Matrices with different hydrophobic groups like butyl--, octyl- and phenyl- are commercial1yavailable. Affini ty chromatogr-lpby

Molecular recognition rorms the basis ofadsorpnoD and separation by affinity chl'Omamgraphy. One of the react"ants in an 'affinity pair' . the Iigand. is irnmobilised on a salid móltrix aud is used nnder suitable conditions te fish out tbecomple.ment'lfY structure (the ligate). The llinding

Ligand type

Biospedf¡c Iigands Mono-speciflc Receptor Arltibody Hapten Substrate/substrate analogue, Inhibito~

(ofador

Protein 1ype

Ho rmane

Antigen Ant ibody Enzyme

Group-spedfte Cofactor Lectins Sugar derivatives Protein NG Heparin

Pseudobiospedfrc ligands Triazlne dyes Metal ions Hydrophobic groups

Enzymes Glycoproteins Lectins

Immunoglobulins Coagulation fadors, protein kJnases Dehydrogenases, kinases and other proteim Metal ion binding proteins

Various proteins

is reversible and can be broken by changing the buffer conditions. Potentially, sum methods possess very high resolving power. Table 9.7 shows that a variety ofligands , with specificity for one or a group of proteins, can be used for affinity chromatography. A trend in downstream processing has been to exploit the speeificity of affinity interaetions earlierin theseparation trainso as to reduce the numberofpurification steps; however this puts extra demands on the ligands with respect 10 chemical and biologicalstability. This has led to increased imerest in the use ofpseudobiospecific ligands like dyes and metal iOlls. The chemical coupling procedure for imrnobilisation of a ligand is chosen so as to provide sahsfactoryyields. strong linkage to minimise Ligand leakage during chromatographic operatian. and minimaJ nonspecific interactions with biomolecules. !be adsorbed protein is generallyeluted undeI' conditions that minimise its interactions with the ligand oe.g. by increasing the ionic strength OI' changing the pH ofthe buffer. o. by a free ligand m olecule. In the latter case, a subsequent step would be ro separate the protein from the free ligand.

9.7 I Product formulation The commerrial viability of a biotechnological product is dependcnt on the maintenance of its activity and stability during distribution and storage. Low molecular weight pwduds such as bulk solvcllts. bulk organic acids. etc. are formulated as concentrated solutions afier removing most of the water. 'Nhen high purity is required, smaU

208

HATII·KAUl AND MATTlASSON

Type o( dryer

Mode of heat transrer

Movement or the product

Belt dryer Fluidised bed dryer Spray·dryer Freeze-dryer Drum-dryer

Convection Convection ConvectJan ConLlct and radiation Contad Convection and corrtact

IntenSNe due to gas fiow Intensi\le due lo gas flow Intensive due to gas flO\o\l None or mechanical

Chamber dryer

Slight mechanical

Nene

molecules such as antibiotics, citri.c acid, sodium glutamate etc" are crystalliscd from salution by addition ofsa lts once tbey have reachee! the required degrec ofpurity. Proterns are particularly sensitive ro 1055 ofbioJogical activity during downstream processing and subsequently during storage. TIlis could be due 00 factan; 50ch as Olddatioll. temperature, preseoce of proteases elc. Proteio products are formulated as solutions, suspe:nsions ordry powders. A variety ofnabilising additives ar-e incJuded in me rormulaoons in order to prolong the product shelflife. The choice ola suitable stabiliscr ls done empirically. Among tbe non· specific chemical additives used regularly as stabilisers in proteio formulations are salts (arnmonium sulphate or sodium ch1oride). sugan (mcrose. lactose etc.). polyhydric aleohols (sorbitol, glyccrol etc_) or polymers (polyethylene glycol, bovine serum albumi n etc.). Bulk e nzymes are cornrnonJy sold as concentrared Iiquid ConnulaDom. Il is, howeve:r. often prefe.rred ro dry the product to decrease the volume as weU as the deoaturing reactions thar are enhanced in aqueoUll saludon. Bioproducrs, ofien being sensitive te heat, require gentle drying rnethods. rabIe 9.8 lists sorne of the commonly used dryers. Depending on the mechanjsm oC heat trarufer, these may be broadly classiflcd as contacto. con\lectioo-. and radiation-dryers. Batchwise drying in rnany contact drye rs is facili tated using mecbanically moved layen:. TIte advantage is [he unifono thennal stress exerted 00 the materia l beingdried. bigh throughpu[ and possibUity fur deveJ.. opment of conllnuous processes. A common feature of convertion dryers is thar the movemenr ofthe material to be dried is promoted by a flow of gas. Drying of large stre
Freeze-drying or lyophilisation l'epresents one of the least harsh methods ofprotein drying. IUs used for tbe drying ofpharmaceutical products, diagnostics, foodstuffs, viruses, bacteria etc. The drying principle is based on sublimation ofthe liquid from a frozen material The liquid containing the productisfrozen, ideally to a temperature below its glass tranSjtiOD temperature, and subjected to vacuum in a freezedryer. While maintaining the internal vial temperature still below fue glass transition value, the shelftemperature is increased to <1. temper
9.8

I

Monitoring 01 downstream processing

Therc may be severa! reasons for monitoring a DSP step. Gne such reason is a desire [O k.eep control over tbe presence and concentration ofthe target molecule which , if monitored directly e.g. by using sorne kiod ofa sensor. would enable one to take appropriate.rneasures quickly in tbe event oían unexpected performance. Fw-thermore, when the performance of a process is well established, the signal froro the sensormay be used for control purposes, thereby increasing the level of autornarion. Continuous monitoring of DSP is also desirable in processes dealing with the production ofpharmat:euticals, as a means to facilitate. establishing an improved level of documentation, and hence the approvaJ process. Furthennore, implementation of measuring and control in DSP will increase the reliability conceming reproducibility ofrepeated batches. Frem a practical point ofview, fraetion collection and other aspects of sampling and sample handling can be substantially facilitated by on-line manitoring cornbined with pre-set conditions for collectionjrejection ofthe stream. Sorne ofthe signals that are used for monitoring DSP-e~nts art' listed in Ta.ble 9.9.lrnportant fcatnres when selecting signals for Pl'OCesS monitoring are l'esponse-tinle, selectivity and seruitivity. In process monitoring itis important to carry out the analyses under sterile conditions so that infection ofthe bioproccss does not take plate. An often-nsed method is off-line analyses. Flow injection analysis (FlA) h.1S proven superior in this contextoA small tiquid sample froro the

210

HATTl-KAULAND MATIIASSON

Measun ng principie

Sensitivity

Selectivity

Response time

Comments

UV-absorbance

Medium

F,"

Commonty used

Conductivity

Low

Medium/fast

pH

Medium

Molecular síze

Low

No No No No

Enzyme activity

Mediumlhigh

Yes

Protein mo nitoring

H;gh

No

Fastlmedium

Needs addition of reagents

Biospecifk binding reaaions

High

High

Slow/medium/

Range of new blnding

F," F,,¡ < lOs

fust

l\leeds additio n of substrate

reactions

ruedium to be aoalysed is introduced into a continuous flow ofbuffer. The sample is transported over a biosensor Ot a reaction site and is theo analysed in a flow detector.

9.9 I Process integration Low productivity and the high cost of produdng biomolecuJes in general have bet!tllimiting factors in the devclopment ofbio rechnologka! processes. As the isolarion and purifica non Slage contlibutes substamialIy to this scenano. process integratioo has been suggested as a means to overcome tbese bottlenecks, wherein two or more different processingstages are integrated inro one step. The outcome wou ld be a dccrease in the aumber of necessary steps for the complete process, leading to lower COS[S and higher product reeovery. ¡ntegrating a produet isolation stagewith the fermenration proccss i5 possible for reeove.ry of both small molecules ¡¡nd protei ns. III sitll adsorption ofthe product is a working example. lntegration of e>.:trae· tion in organic solvents with fermentation for in situ reOOVC1'Y of low moleLular weight products has also been investig
DOWNSTREAM PROCESSIN G IN BlOTECHNOlOGY

inreracríons , into techlliques used for primary separaÚOD, e,g. as in affinity precipitatio!]. membraneaffinity filtr.Jtion etc.

9. 10

Further reading

AlbcrlSSOll. P. A. i 19861. /tlrtition ofCtU f'artidt's i.lnd Mlia(llllm«lIlt-s. 3m editfon. Wilcy In tel'Science. New York. Ascnjo. j. A.. ed. (1990). Separutitm Prounts jlJ lUoUChno.!ogy, Mareel Dekker. New York. IWher. P. A.. CU5sle .·. F.. L alld Hu. \,{·S. (19B8~ BioSé!p(lrilt!om: Downstn:am Procmingfor Hiotechno!oK)'.John wn~, Nt;w York. RUlldr. S.• Cofre. Il A.. Kessler. S. B.• O·Connor. J . L andZale, S.E. (1988). Mcmbrilue bilsed affinity technology for commerdal scale purifieiltiOlU. Biofli'dmoI6. 779-782. pieryan. M. (1986). UltrcjiltTatlon Handbook. TechnomkPubl. Laueastcr. USA. Deutscher. M. P.• ed. (1990). Methods in Rnzymology, Vol. 182. Gl.lide to Protcin I'lIrijicanon. Academic l'ress, San Diegu. Hatti·Ka.ul. R.• ed. (2000).Aq1i~OUS Tlw-Pltase Syst~ms. Mtthods dnd Protocols. Humana Press. New Jersey. Janson.Je. alld Ryd él1. 1... eds. (1998). Protein Purlfimtlnn. Ptindpln. Hlgh Resolution Methods, 11M Arrlicatio!ls.Johl1 Wiley. New York ](au!, R. ilIld Mattiasson, B. (1992). Secondary purlfkation. Bioscp..ltlltion 3. 1-26. Krijgsman,j. (1992). ProdUCf Ru(lVfry in R!O!Jrocess TtdllUllogy. BIOTOL Series Uenkins. R. O.. ed.). Butterworth Heinemann. O"Jord. Lo. T. c.. Baird. M. H. l alld Hanson. C. {1983). fiarnl book ofSolwnt Exlrllrtioo.John Wiley. New York. MaUUrnul'a, M. (1991 ). l'eTSlr.u: tion. I.n F.xlruclit.Oe' Biocllnwrsiolls(Mattiasson , B. and Holst, 0" eds.). 'PP. 9 1- 13 1. Mareel Dekker. N~ York. MaLl:Í.iIssoD. B.• ed. (1999). Expollded Rtd OIrtmUl lllgraph)'. Kluwer Al:ade mic PubUsbers. Donl rechl, NI ~ McCregol'. W. e.. ro. (1986). MemhTQrlt StpllTPfijM.~ in Bin/.t'chnology. MarceJ Dekker, New York. Schmidr·Kastner. G. and Gó lker. C. (1987). Product ret:OVéry In biotedmoLogy. In Fundamental! ofHiot«hnology (Prave.I'., Faust, U" Sittiy. W. and Sukatsch. n. A.. 005.), pp. 279-3Z1. VCH, Weinheim. SCO~5. R. ( 1994~ PYotein Purijication. Principia ami Practire, 3m F.ditltm. Spring~r­ Verlag, New York. Stephanopoulos. G" oo. (1993). RJotechnology Vol. 3. Rioprorming (Series ediw rs: Rebm. H-J.. Reed, G.. Pühler.A.and Stadler, P.). VCH. Weinheim. Strathman, H. ¡¡ud Gudematsch, W. (1991). Continuous removal ofethanol from bioreactor by pervaporation. In F.xtnlCtive Riflcoltversion.~ (Mattiasson. B. and Holst, O.. eas.), pp. 67-89. Maree] De'k'ker, New York. Street. e .• ed. (1994). HighLy Selecliv~ Separalioll$ in Riot.:chnoJogy. Blackie Academic & Professional. London. Verral! . .M. (1996).Downstrellm Proccssing ofNlltumJ Products.A PrnctlCill Handbook. Joh n Wi]ey & Sons. Cbichester. Walter, H. and johansson, G .• eds. í 1994). Melhods in F.nzymuWID~ ~. 22R. Aqurous fwo..i'hoseS)'stems. Academic Press, S¡Tl Diego.

2 11

Chapter 10

Measurement and control A. Lübbert and R. Simutis Nomeodature Inrroduction Structure of process models Kinetic rateexpressions Advanced modelling considerations Process supervi.sion and control Open-loop control Closed-loop control

Condusion Further reading

1

Nomenclature

ANN r e edil)

concentTation ofa metabolic by·product produced by microorganism s. de1ay time indiv:idual comp onenU ora blochemical reactioD system. (can be Ll!presented by mea ns o freaetion equatioM ~I V",, = O. where lhe component A¡ i5 usuaUy repre$ented by tu suro formula. and J'¡ are t he usual stoichiometriccoeffloenu) artificial neul
c¡;

concentrarían vector ofthe solution red to the reactor

A

a A¡

CER

COl evolution tate coefficient matrix ('Element<:omposition·Mattix') r. or F(t) the r.lte al which [he m..:Lterial is red to [he reactor a$ a function af time K-AfB amplirude1'3tio K.¡ inhibition constant fur the inhibitor A K< L'ú ntrollergain kLCI mass transferc()('fficient K, Monod constant KoJ the substrate inhibition constant m mainteniUlce rerm in Eqn 110.28) ni 31110unt afcomponent A¡

E

111

LÜSBERT ANO SIMUTIS

n pOl p02' OTR OWR RQ

SCR Po

'1", P RI R.

R S T

vectorofamour.ltS of compone.nts diS$Olved 0 1concentratioo in me medium thesolubilityofO¡ 01 trcmsfer rate o~

uptake rate re'"spiratioo coeffident

substnte cousumption rafe time deviatioo m;¡inten;¡nce tenn prodUCTconcentration conVEnioo t a teS absolute O~ t:(msumption rate wcLor ofvolumetric ["¡Ites ofchange afilie amounts oftbe componl"nts being created or consumed duting a process substrdte roncentration res~ns e

time

ue manipulable variable V, or Vit) \IOlume X biomass concentratian Y, yield roeffidentof component j on componentj L\tI.¡ amount of compcnent j synthesised or consume vector ofme r.ttes by which theamo unts ofrne components change as;) consequenceofthe maS$" transpon acl'OSS the boundarics ofme dornain u. spectfic acetatl' production rate Uf

JL 'TT T

Il

T[

", if

(T,,¡, w", w~

specific aU!late consumpoo n rate specific growth rolle spccifk productpn:xluaion 1'3te ~ coDStant fur derivative controllel' time corutant fur integral controller stoichiometrlc coeffident spccific substrate upul:e rate critica] speciflc subStrdte up take rale specific O2uptake rd.l e used for maintenallce speci1ic O2 uptake rale used for biom¡¡ss growth

10. 1 I Introduction In industrial production plants, process control is on everybody's agenda whe:n the cost[benefit.ratio ofa process must be improved. It is

de:sirable to guide the process aJong a path. whicb. guarantee.s the process to prod uce the prod uct in such a way thar ir meets predefined quality specifkations. This confumed. the aim is to produce this product at a m inimum of Ca$(. The detennmation of such optimal proct'SS patbs is the essential pan ofapen·loop control The two kt>y eJements of process monitoring and control are: (il measurements bywhich inforUlation aboutthecurrent process state is beillg acquired. and (B) models that dynamically ÍDte rrel.l te tbe various process variables, which are importantwith.respect to the task to be solved . Ofparticular importance are rhose variables by which the

stare afthe process ca(l be described unambiguausly.ll1ese variables. however. are not necessarily the most importam ones from [he practical point' ofview. Ofimmediate practical importance are the variabl es which describe the perfor:mance ofthe process. In arder to gel access to the performance, its relatio nships to the variables which can be measured dh'ectly and to the variables thal can be manipulated are of importance. Thus. modelling for process supetvision and control needs a quantitative definition ofme objectives afme process aud rhe particular task to be solved . For supervision and control applications in industrial environmenu. [be complexity of tbe models muS( be Jeept as low as possible to minimise me expenses ofmanpower needed te maintain them. I( only makes seose to implement complex precess conlTollers, arter it was made sure: that they will work significanlJy better than conventional simpler ones. It is the cost[beneflt-ratio thar is me final c.rirerion (or whether simpleT ormore complex contrallers are used and this must indude me cost ofprovidingthe relevan t man-

po_o lt is of advantage te formula te tbe mulo-dimensional problems of proces5 modellLng, 5upervision and control using a vector representation. TIüs nor only helps to keep tbings dear, buthelps to translate them into modern software mols which are mainly matrix based. The matrix notation \Ised in rhis artide was adapted to software products available on the maTket such as MATLAB OI SOLAB where tbe variable. x, i5 generilly assumed to be a matrix. Vectors and tbe Ul>1JaJ scalar Quantities are.considered matrices ofspecia l dimensions.

10.2

I Structure of process models

Process identification is tbe procedure of de\'cloping a process model

from prior knowledge and experimental data. The dassical approach to process modclling is the development ofa mamematical model in the forro of adynamic differe.ntial equation sysrem deriverl from rnechanistic considerations. The prior knowledge usually leads to the structure of a parameterised model. leaving me parameters assooatcd with tbis model structure to be estima ted from process data. However, suirable model structures for sorne parts of the process may not always be known. Then, 'blackbox' ideutification methods that make onJy minimal a.ssumptions about the strucn¡re of these sub-processes aTe al ternatives. The state of a biopTocess is mainly determined by tbe amount, n , (measured in mol) of irs key components. !be vectoT, n , may be composed oftbe amollnts ofthe substr:ate. bromass. productetc_ The basis of a bioproce5s model is a baJance eqllation (bar can describe tbe changes of n as a fllnction of time. Please uotc that bold cbaracters or abbrcviations such as n indicare mal che corresponding quantity is a ve<:tor-vaJued quantity. n "" Ibioouss X; SUbStl'3le S; product P; , .. 1

(10.1)

216

lOBBERT ANO SIMtmS

As it is more convenient [O formulate \bese balance equations in rerms ofconcentr.ations, c. which are related to theamounts , n,ofthecomponenes by c"" n{V, ourgeneral balancemass balance equation reads: dn d(cV) - - - -=RV+cb

dt

dt

(10.2)

¡P is the rate by which the vacious components are transported across the borders of the balance volume, Y, and RY me r:lte at whicb these component 5 are synthesised or consumed within V. In bioproces~es. the rates, lL. are usually non-linear funcci ons ofrhe variol1s components of norc. In the red·batch operational mode, wruch is the usual industrial production mode. the amount of substrate red to the reactor lead! to an increase in thetotal volume so that the volume iu el fis variable. Hence . an additional differentiaJ equation mu~a be speot Cor the (scalar) volume. Y_

dY

- -FI<) dI

(10.3)

After resolving the eql1ation fur the concentration vector. c. we obtain the more convernent form ofthe bal ance. de dt

tP V

dV dt

- =R+-+eor, with dc dt

(10.4)

CP=F~

F(t)

- =R + -(c - cl . V(t)

(10.5)

F

A sbart remark is required a baut the balance domain. In n on-lineal' systems, the f'SSential c rite rion for justifying ro represent the con ce otrafion of a particular compone nc. e.g_. m e substTate concentranoo_by a single value. S. is bomogeneily across che regioo over which the balance is drawn. It is possible 10 use the entire culturevolume, V. as the balance volume wh en the bioreactor can be considel'ed to be an ideal stir:red tank reactor. Wb en t his
10.3 I Kinetic rate expressions 10.3. 1 Considerations of scoichiomecry The first pan in Eqn (lO.S), R. describes tbe r.ates ofthe biochemical conversian of the componenrs i.n the system. As tbe products are synthesised from tbe substrates. the dilferent e1ements of c da Ilot change independently [rom each othe r. The interrelatianships are de termined by biochemical stoichiomeuy. The individual components.A r of a biochemical reaction system can be represente
MEASUREMENT ANO CONTROL

LIV¡AI"'O where tbe components A, are usually represenred by tbeir sum formula. and v¡ are the usual stoichiometric coefficienrs. As elemental analyses sbowed. the components, Al' can be considered lO be mainly composed of a few diffe.rent elements only. Often ir suffices to consider four elements le R o N] only. lfthis 15 a5rumed ro be sufficient, tben tbe individual componenlS can be identified by tbe.ir index VlXtOrs, e.g.: H

o

~1

Clucose - (O

t2

O,

O 3

O 2

o

2

t

O

2

01' 01' 11' 01' 01'

(e 1. 2.

- (O

3. Ammonia= 10 4. Water - (O

s.

CO,

- (t

Basic elements consfdtrrd Substmte

Ox""" Nitrogen source

(LO.61

Water Carbon dim:fdt

where l... J' mcaDS [be transposed oftbe row-vector,\ ... 1. Rere and in the following texto the vedor representatian by components is adopted Erom MA11AB. Problellls appear with tbe corresponding represent3tions of biomass and otber complex biapolymers. Here it is straigbtforward ro represent them as moleculeswitb tbe same relati ve composition as the originals, but with the C-index fixed ro 1. e.g. 6. Yeast""11

1.75 038 0.25]'

Biamass

With this assumption we can write down concrete reaction equations, e.g. ror aerobic yeast (Saccharnmyces cerevislac) production we get: JI,

C, HI2 0 , +

"n N~ + v.. 02~ ,,~C H,.~ 0 1).;18 No.lS + "wH,¡0 +

V,

COl (tO.7)

Using matrix representanon this equatian COlO be refonllulated into a homogeneous linearequañoD system: E ,,= 0

(10.8)

The coe.fficient matrix. E, in literature referred to as tbe 'E1emen[Composition-Matrix', is defined by the index vectors, llsing the ddlnitions in Eqn (10.6): E= r~ Glucose - 02 ~ Ammonia Yeast Waterroll

(10.91

The problem ofsalving rhe linear Eqn (lO.8l for the stoicbiometric c~ efficienlS vrequires five equations, since ane ofthe Jo'elements, e.g. that for gIucase, can be arbitrarily setta ane. lfthis substratc is chosen as me reference component. tben "s"" 1. Then we can transfonn tbe hornogeneous equation system (10.8) ioto a non-homogeneous one

Ee v= Glucose with the coefficient matrix Be =

I~0 2 -

Am.m.onia YeastWater C0 2l

and, according to Eqns (10.7) the five component yector:

(10.10)

217

218

LÜBBER.T AND SIMvnS

(JO.11) As Eqn (10.101 contains onIy four linear equiltioas and five unknowns,

we nee
R¡

(10.12)

II- = -~-

, tan, Re

whicb is equal to the ratio ofthe corresponding consumption or developmenr rates, RI' Far ¡nstaDee. R~ "" CER. tbe CO2 evolution rate and Ro "" OUR, tbe 01 uptake rate tbatare mea su red duringmanyfermenrations, can ofren be assumed ro be proportional. Tbe proportionaliry constant is referrcd to as the RQcoeffident. CER 1I~""RQp~ = OURJI~

(10.13)

Too can be reformulated by a scalar product [RQOOO -l1X 1-'= RQV X 11=0

(10.14)

wbich is a new li.near equation in 11. Henee, tbe row vector. RQV, de.fined in Eqn (lO.14). can be used lo extend tbe coefficienr matrix, Ec' ln the MAnAB representation we get: 110.15)

Correspondingly. we mustalso extend tiJ.e column vector on the right hand side ofEqn (10.10): b = [Substrate:O[

(10.16)

Nowwe have fivc linear equations for rhe flve unknown stoicbiometric coefficients. JI. (10.17)

Equalion (10.17) can now be solved directly lIsing linear algebra. This can be done wirh a single MATLABsta.tement v= ECJfb. From the solulÍon. 1-', we can dete rmine theyields. which are defined as:

y = all¡ ~ 6.na MW~ [kg] = v~MW~ ~b

ano

dnb MWb kg

[kg]

1"0 MW"b kg

(10.18)

Tbis rcquires the molecular weights MW. ofthe spedes ¡nvolved ro be known. The most important yields are the biomass per substrate yield telling how muro biomass is generate
HEASUREMENT AND CONTROL

Yxs =

~~: [=]

(10.19)

and the oxygen per biomass yield

Y~= ;:~: [:]

(10.20)

For measurement and control. s(oichiometric relationships are ofien used with adV
10.3.2 Conversion rates While stoichiomemc con sideratiOJl5 provide.i nformarion a bout tb ~ reJ· ative rates by which the different components are cOllsumed or produced, the basic cOl1vcrsion rate expressions, R, are a matter ofkinetics. Sino:, the performance oftbe mkro-organism is central in tbis respecto it is straightforward to discuss conversion Tates perbiomass cOnCellrr.l' tion. These quantities
[T=~ R

(10.21)

With increasing S. uapproaches (T~. At largcr S, q remains constanL Thus, the main idea behind the equation is todescribe the decreaseofu al low values of S, i.e. under substrate-limitingconditiol1s. The cell mannro the suhstrate into several metabolic. patbs. Mathematically, we may express this by tbe differential expression: 1 dS

1 rlS ax

1 OS ap

u =X dt =xax a¡+xap at +qm

(10.22)

. l aS l aS orWJ.th - = - aud - =~ Yg ax y~ ap ldS

po. 1T + - +q Yxs Y", m

u-- - - X dt

(10..23)

which is of high practicaJ value in modellíng for process supetVision alld controL The Monod cquation ofien d ~s not describe substrate degradation acctmltely enough. The n. further mechanisms lllust be taken imo account, e.g. suhrt1"a.tc inhibition and inhibition by undesirable

I

219

220

LUSBERT ANO SIMuns

by·producrs. A. TheSE' mecnanisms can be considered by attaching addi· tional factors to the original Monod equation for u: 0'=

Maximal specific uptake rate (capac.'ity)

u ......

s

x -K.+ S

K,¡

x- - -

KsI + S

ConventionaJ substrate limitation (erro

Substrate inb.ibition terro

K. lnllibi tion by by·product A +A

x - - .Ka;

110.24)

The parameters, u max ' K" K.¡ and Ka; must be determined from process data. The formation oE me by-productoA. neros an additiollal kinetic consideration. CeUs are assumed (O be .. ble to Clkeup more substrate than they can ful]y oxidise.ln terms ofthe specific substrate uptake rateS. (1', there is a critica] rateo " ( dI' above which all substrate taken up cannot beoxidised directly. In such situations, tbe cclJ produces el by-product, A, which usually, at leasr .. t higher conceDtrations. acts as an inhibitor. In R. coli this by·product is acerare. while in the yeast Sacdwromyres crrevisfae it is ethanol or in mammalian cells it may be lactate. In the example ofan E. coli model , we may assume that at v > U crlt ' aceta te, A. is generated with the specilic by-product generation rate, ag :

10.25) '

Wheo 0'< <<011' the acetate already fonned. can be consurned by the cells. This occurs when the cODcentration. S, ofthe main substrate drops belowa critical value. The specific by-producl consumption rate, "'c' can also be described by a Monod-like expression:

A

K..

a~:::: 0uw¡ - - - - --

Ka + A Ka¡+S

for 0' <

0', "1

and lt'c =Oelse

110.26)

where the repression ofthe by·product consumption bythe substrate. S, is described by the third termo The net speciñc by·product production rate a is obviously the difference

a=a, - oc' When we assume mat no further produce is being synthesised, the spedflc growth rateop., can be detennined, lo order to ke:ep the description as trnnsparent
In thesecond ase, atu> O'crit' tbe cells convertpartofthesubstrateinto biomass with a high biomass/substrate yield, Y:u' a nd use the restofth e

MEASUREMENT ANO CONTROL

*

40

l .~ ~

.3

Typlc.al result lar fed-

30 20

10 ·_"~"""'."H. ___ .. ..____ ._...

A

_...... _._·_H'-.-~-"H:

O

" ··......'H I

2

3

4

5 6 Time(h)

7

.~

8

9

10

15

- - Weight

!t

• E • J'

'1'1 the up~ I»'rt tlle computed ln¡ectOI'ln lor b jOfl'lUJ. Wbstraf •

O

1

1.

b:uch evldv¡don of modified E cnII.

-o-Biom3SS ......H. Substrote ~ Acatate )(10

.----------.-

'--'''- Feedrate )(10 .___ . . _...H . . . ._· . _. . . ..______.____. _. ...--' -----

10

~

5

OL-~~~--J-~~~--~--~~L-~

1

2

3

4

5 6 Tlme(h)

7

8

9

10

entire specific substrate cOllsumption Tate with a significantly lower yield, YKS~' Hence we obtain for

U > U cril

(10.28)

Finally, the specific product developmenl rate, "fr. must descrWed. It mostoften primarily depends 00 the sped6c biomass growth rate. lL. in sorne more or less complex relationship, The desired produce may be a recombinant protein. Then. a highly non-linear relationship. sucb as e.g.: w

(10.29)

l+ (~r

may be u sed in the simuJation. The sigmoidal form ofthis rate expression reflects the experience thar lhe cells need sorne growth to start protein produrnon and that there is a final specific production rate limit. 1l"uw' that is asymptotically approached at higner specific growth mtes. IL. The parameters of this expression must be deterntined from experimental data, in particular from p.(t) and 1T{t) estimations discllssed latero With these specific rate expressions, the absolutevolumetMe rate vector. R, can be detennined as:

R=Xlp.. -

fT,

a. '!TI

(10.30)

With R. the entire dynamic system is determined. Hence, when the required parameters, kinetic constants and yields can be supplied . we only need tO salve Eqn (10.5). This, once again, i5 very ea5)' when a modern software toollike MATLAB is used. Pigure 10.1 sbows a typjcal result oi a process simulation where

Il'1daC"'Ute. a~ eompaN!d.,.,.;th musur.G claa. clepi cce cl by tlt. symbolJ.ln me Iower p;irt the carre~ncllng feedrat.e (gluc:ose saludon) In.:! cultUN! _ight prom ...! are shown.

221

222

LÜBBERT ANO SIMlJT1S

t hese model components were used .The kineric bortle neck mechanism is of general use llot only for [he accompanying example of E. col! growth, bu( alsofor most other sysrems ofpractical interest.

10.4 I Advanced modelling considerations 10.4.1 Methods of lean modelling When a rnodcl is (O be used in process optimisation or for on-line process measurcmeut orcontrol , ir must be salvable quickly. ¡ inee [he model evaluation must be frcque ndy repeared. This requi res that the m ode! must be strictly resrricted (O rhose aSpccts thatdirectly influence tbe process performan ce, Rcstrictioo to these mostimportant variables i~ nor the only possibility to reduce the computing time, An important further meaSUl:C is ro identifYvariables that are importanr, but change with much smaller timeconstants in comparison with the keyvariables mbstrate, biomass oc produet concentration, These variables can be assumed [Q be atany time in an equilibrium state with the keyva riables. Hencc. their dyna mic changes nero Ilor be considered sepa.rately. In otherwords. they can be statically related ro cheother stare varjables. ln chis way che numbe.r of diffcrcntial equatioDs can be decreascd and thus the compudug time fo r the simulanoo, All important example is che dissolved 0 2l:onccnrradon . pOr on e of the most importanr variables in aerobic production processcs. Since p02 is irnmediateIy adapting ro rhe biomass gl'owth rate , its rate of change can be neglecred. The specific O) consumpnoo rateo f
(10.31 )

°

Thc absolule 1 upta ke rate, Ro=OUR = ", X, is balanced by tbe 02-transfer rate OTR, which 15 mostoften modelled by: OTR "" k¡p ( pO~ - pO¡)

(10.32)

Thus, we can combine Eqns (10.31 ) and (10.32) to ger:

p02 =

pO; - OUR,/(kLa);= pO; -

(1-'1'"" + wm}X/(k¡a)

(m.33)

The computadonal advantage (in tenns of the computing time) ofthis approach is flot alone due to the reduce
MEASUREMENT AND CONTROL

quasi-collstantleve1. Then. al] tbesubstratefed to tbereactoris ¡mmedi· ately consuml!d by (he cells.

10.4.2 Representations of the kinetic expressions by artificial neural networks In comparison with (he knowlt"dge. behind tbemass balance equéltions. tbe knowle