This book contains information obtained from authentic and highly regarded sources Reasonable efforts have been made to publish reliable data and information,
In this third edition of his popular undergraduate-level textbook, Desmond Nicholl recognises that a sound grasp of basic principles is vital
This book covers a wide range of current biotechnology methods developed and mutations and genetic engineering 2–11 The general principles of PCR start
The book contributes chapters on the basics of genetic engineering, on applications of the technology to attempt to solve problems of greater importance to
Principles of gene manipulation and genomics / S B Primrose and R M Twyman —7th ed another book, Principles of Genome Analysis, whose
DNA and Genetic Engineering—The Beginning of Modern Biotechnology The science of genetics was transformed by the discovery of DNA (deoxyribonucleic
You may search for incredible novel by the title of An Introduction to Genetic Engineering Desmond S T Nicholl Currentlyyou could easily check out each book
In contrast, recombinant DNA techniques, popularly termed 'gene cloning' or 'genetic engineering', offer potentially unlimited opportunities for creating new
No part of this Book may be reproduced in any form by mimeograph or any other means without Unit - 20 Biotechnology and Genetics Engineering in Human
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Geneticsandbiotechnology
3.1Introduction
Inessence,allpropertiesoforganismsdependonthesumoftheirgenes.There aretwobroadcategoriesofgenes:structuralandregulatory.Structuralgenes encodeforaminoacidsequencesofproteinswhich,asenzymes,determine thebiochemicalcapabilitiesoftheorganismbycatalysingparticularsynthetic orcatabolicreactionsor,alternatively,playmorestaticrolesascomponents ofcellularstructures.Incontrast,theregulatorygenescontroltheexpression ofthestructuralgenesbydeterminingtherateofproductionoftheirprotein productsinresponsetointra-orextracellularsignals.Thederivationofthese principleshasbeenachievedusingwell-knowngenetictechniqueswhichwill notbeconsideredfurtherhere. TheseminalstudiesofWatsonandCrickandothersintheearly1950s ledtotheconstructionofthedouble-helixmodeldepictingthemolecular structureofDNAandsubsequenthypothesesonitsimplicationsforthe understandingofgenereplication.Sincethentherehasbeenaspectacular unravellingofthecomplexinteractionsrequiredtoexpressthecodedchemical informationoftheDNAmoleculeintocellularandorganismalexpression. ChangesintheDNAmoleculemakingupthegeneticcomplementofan organismisthemeansbywhichorganismsevolveandadaptthemselvesto newenvironments.Innature,changesintheDNAofanorganismcanoccur intwoways: (1)Bymutation,whichisachemicaldeletionoradditionofoneormoreof thechemicalpartsoftheDNAmolecule.
34Geneticsandbiotechnology
(2)BytheinterchangeofgeneticinformationorDNAbetweenlikeorgan- ismsnormallybysexualreproductionandbyhorizontaltransferinbacteria. Ineukaryotes,sexualreproductionisachievedbyaprocessofconju- gationinwhichthereisadonor,calledÔmaleÕ,andarecipient,called ÔfemaleÕ.Often,thesearedeterminedphysiologicallyandnotmorpholog- ically.BacterialconjugationinvolvesthetransferofDNAfromadonorto arecipientcell.ThetransferredDNA(normallyplasmidDNA)isalways inasingle-strandedformandthecomplementarystrandissynthesisedin therecipient.TransductionisthetransferofDNAmediatedbyabacterial virus(bacteriophageorphage),andcellsthathavereceivedtransducing DNAarereferredtoasÔtransductantsÕ.Transformationinvolvestheuptake ofisolatedDNA,orDNApresentintheorganismÕsenvironment,intoa recipientcellwhichisthenreferredtoasaÔtransformantÕ.Genetictrans- ferbythiswayinbacteriaisanaturalcharacteristicofawidevarietyof bacterialgenerasuchaCampylobacter,NeisseriaandStreptomyces.Strains ofbacteriathatarenotnaturallytransformablecanbeinducedtotakeup isolatedDNAbychemicaltreatmentorbyelectroporation. Classicalgeneticswas,untilrecently,theonlywayinwhichhereditycould bestudiedandmanipulated.However,inrecentyears,newtechniqueshave permittedunprecedentedalterationsinthegeneticmake-upoforganisms, evenallowingexchangeinthelaboratoryofDNAbetweenunlikeorganisms. Themanipulationofthegeneticmaterialinorganismscannowbeachieved inthreeclearlydeÞnableways:organismal,cellularandmolecular.
Organismalmanipulation
Geneticmanipulationofwholeorganismshasbeenhappeningnaturallyby sexualreproductionsincethebeginningoftime.Theevolutionaryprogress ofalmostalllivingcreatureshasinvolvedactiveinteractionbetweentheir genomesandtheenvironment.Activecontrolofsexualreproductionhas beenpractisedinagriculturefordecadesÐevencenturies.Inmorerecent timesithasbeenusedwithseveralindustrialmicroorganisms,e.g.yeasts.It involvesselection,mutation,sexualcrosses,hybridisation,etc.However,itis averyrandomprocessandcantakealongtimetoachievedesiredresultsÐ ifatallinsomecases.Inagriculture,thebeneÞtshavebeenimmense withmuchimprovedplantsandanimals,whileinthebiotechnological industriestherehasbeengreatlyimprovedproductivity,e.g.antibioticsand enzymes.
3.2Industrialgenetics35
Cellularmanipulation
CellularmanipulationsofDNAhavebeenusedforovertwodecades,and involveeithercellfusionorthecultureofcellsandtheregenerationofwhole plantsfromthesecells(Chapter10).Thisisasemi-randomordirectedprocess incontrasttoorganismalmanipulations,andthechangescanbemoreread- ilyidentiÞed.Successfulbiotechnologicalexamplesofthesemethodsinclude monoclonalantibodiesandthecloningofmanyimportantplantspecies.
Molecularmanipulation
MolecularmanipulationsofDNAandRNAÞrstoccurredovertwodecades agoandheraldedaneweraofgeneticmanipulationsenablingÐfortheÞrst timeinbiologicalhistoryÐadirectedcontrolofthechanges.Thisisthemuch publicisedareaofgeneticengineeringorrecombinantDNAtechnology,which isnowbringingdramaticchangestobiotechnology.Inthesetechniquesthe experimenterisabletoknowmuchmoreaboutthegeneticchangesbeing made.ItisnowpossibletoaddordeletepartsoftheDNAmoleculewith ahighdegreeofprecision,andtheproductcanbeeasilyidentiÞed.Current industrialventuresareconcernedwiththeproductionofnewtypesoforgan- ismandnumerouscompoundsrangingfrompharmaceuticalstocommodity chemicals,andarediscussedinmoredetailinlaterchapters.
3.2Industrialgenetics
Biotechnologyhassofarbeenconsideredasaninterplaybetweentwocom- ponents,oneofwhichistheselectionofthebestbiocatalystforaparticular process,whiletheotheristheconstructionandoperationofthebestenviron- mentforthecatalysttoachieveoptimumoperation. Themosteffective,stableandconvenientformforthebiocatalystisawhole organism;inmostcasesitissometypeofmicrobe,e.g.abacterium,yeastor mould,althoughmammaliancellculturesand(toalesserextent)plantcell culturesareÞndingever-increasingusesinbiotechnology. Mostmicroorganismsusedincurrentbiotechnologicalprocesseswereorig- inallyisolatedfromthenaturalenvironment,andhavesubsequentlybeen modiÞedbytheindustrialgeneticistintosuperiororganismsforspeciÞcpro- ductivity.Thesuccessofstrainselectionandimprovementprogrammesprac- tisedbyallbiologicallybasedindustries(e.g.brewing,antibiotics,etc.)isa
36Geneticsandbiotechnology
directresultoftheclosecooperationbetweenthetechnologistandthegeneti- cist.Inthefuture,thisrelationshipwillbeevenmorenecessaryinformulating thespeciÞcphysiologicalandbiochemicalcharacteristicsthataresoughtin neworganismsinordertogivethefullestrangeofbiologicalactivitiesto biotechnology. Inbiotechnologicalprocesses,theaimisprimarilytooptimisetheparticular characteristicssoughtinanorganism,e.g.speciÞcenzymeproductionorby- productformation.GeneticmodiÞcationtoimproveproductivityhasbeen widelypractised.Thetaskofimprovingyieldsofsomeprimarymetabolites andmacromolecules(e.g.enzymes)issimplerthantryingtoimprovetheyields ofcomplexproductssuchasantibiotics.Advanceshavebeenachievedinthis areabyusingscreeningandselectiontechniquestoobtainbetterorganisms. Inaselectionsystem,allrareornovelstrainsgrowwhiletherestdonot;in ascreeningsystem,allstrainsgrowbutcertainstrainsorculturesarechosen becausetheyshowthedesiredqualitiesrequiredbytheindustryinquestion. InmostindustrialgeneticsthebasisforchangingtheorganismÕsgenome hasbeenbymutationusingX-raysandmutagenicchemicals.However,such methodsnormallyleadonlytothelossofundesiredcharactersorincreased productionduetolossofcontrolfunctions.Ithasrarelyledtotheappearance ofanewfunctionorproperty.Thus,anorganismwithadesiredfeaturewill beselectedfromthenaturalenvironment,propagatedandsubjectedtoa mutationalprogramme,thenscreenedtoselectthebestprogeny. Unfortunately,manyofthemicroorganismsthathavegainedindustrial importancedonothaveaclearlydeÞnedsexualcycle.Inparticular,thishas beenthecaseinantibiotic-producingmicroorganisms;thishasmeantthatthe onlywaytochangethegenome,withaviewtoenhancingproductivity,has beentoindulgeinmassivemutationalprogrammesfollowedbyscreeningand selectiontodetectthenewvariantsthatmightarise. Onceahigh-producingstrainhasbeenfound,greatcareisrequiredinmain- tainingthestrain.Undesiredspontaneousmutationscansometimesoccurat ahighrate,givingrisetodegenerationofthestrainÕsindustrialimportance. Straininstabilityisaconstantprobleminindustrialutilisationofmicroor- ganisms.Industryhasalwaysplacedgreatemphasisonstrainviabilityand productivitypotentialofthepreservedbiologicalmaterial.Mostindustrially importantmicroorganismscanbestoredforlongperiods,forexampleinliq- uidnitrogen,bylyophilisation(freeze-drying)orunderoil,andstillretain theirdesiredbiologicalproperties. However,despiteelaboratepreservationandpropagationmethods,astrain hasgenerallytobegrowninalarge-productionbioreactorinwhichthechances ofgeneticchangesthroughspontaneousmutationandselectionareveryhigh.
3.3Protoplastandcell-fusiontechnologies37
Thechanceofahighrateofspontaneousmutationisprobablygreaterwhenthe industrialstrainsinusehaveresultedfrommanyyearsofmutagentreatment. Greatsecrecysurroundstheuseofindustrialmicroorganismsandimmense careistakentoensurethattheydonotunwittinglypasstooutsideagencies (Section12.2). Thereisnowagrowingmovementawayfromtheextremeempiricism thatcharacterisedtheearlydaysofthefermentationindustries.Fundamen- talstudiesofthegeneticsofmicroorganismsnowprovideabackgroundof knowledgefortheexperimentalsolutionofindustrialproblemsandincreas- inglycontributetoprogressinindustrialstrainselection. Inrecentyears,industrialgeneticshascometodependincreasinglyon twonewwaysofmanipulatingDNA:Ð(1)protoplastandcellfusion,and (2)recombinantDNAtechnology(geneticengineering).Thesearenow importantadditionstothetechnicalrepertoireofthegeneticistsinvolved withbiotechnologicalindustries.Abriefexaminationofthesetechniques willattempttoshowtheirincreasinglyindispensablerelevancetomodern biotechnology.
3.3Protoplastandcell-fusiontechnologies
Plantsandmostmicrobialcellsarecharacterisedbyadistinctouterwall orexoskeletonwhichgivestheshapecharacteristictothecellororganism. Immediatelywithinthecellwallisthelivingmembrane,orplasmamem- brane,retainingallthecellularcomponentssuchasnuclei,mitochondria, vesicles,etc.Forsomeyearsnowithasbeenpossible,usingspecialtechniques (inparticular,hydrolyticenzymes),toremovethecellwall,releasingspher- icalmembrane-boundstructuresknownasprotoplasts.Theseprotoplastsare extremelyfragilebutcanbemaintainedinisolationforvariableperiodsof time.Isolatedprotoplastscannotpropagatethemselvesassuch,requiringÞrst theregenerationofacellwallbeforeregainingreproductivecapacity. Inpractice,itisthecellwallwhichlargelyhindersthesexualconjugation ofunlikeorganisms.Onlywithcompletelysexuallycompatiblestrainsdoes thewalldegenerate,allowingprotoplasmicinterchange.Thusnaturalsexual matingbarriersinmicroorganismsmay,inpart,beduetocell-walllimitations, andbyremovingthiscellwall,thelikelihoodofcellularfusionsmayincrease. Protoplastscanbeobtainedroutinelyfrommanyplantspecies,bacteria, yeastsandÞlamentousfungi.Protoplastsfromdifferentstrainscansometimes bepersuadedtofuseandsoovercomethenaturalsexualmatingbarriers. However,therangeofprotoplastfusionsisseverelylimitedbytheneedfor
38Geneticsandbiotechnology
DNAcompatibilitybetweenthestrainsconcerned.Fusionofprotoplastscan beenhancedbytreatmentwiththechemicalpolyethyleneglycol,which,under optimumconditions,canleadtoextremelyhighfrequenciesofrecombinant formationwhichcanbeincreasedstillfurtherbyultravioletirradiationofthe parentalprotoplastpreparations.Protoplastfusioncanalsooccurwithhuman oranimalcelltypes. Protoplastfusionhasobviousempiricalapplicationsinyieldimprovement ofantibioticsbycombiningyield-enhancingmutationsfromdifferentstrains orevenspecies.Protoplastswillalsobeanimportantpartofgeneticengineer- ing,infacilitatingrecombinantDNAtransfer.Fusionmayprovideamethod ofre-assortingwholegroupsofgenesbetweendifferentstrainsofmacro-and microorganisms. Oneofthemostexcitingandcommerciallyrewardingareasofbiotech- nologyinvolvesaformofmammaliancellfusionleadingtotheformation ofmonoclonalantibodies.Ithaslongbeenrecognisedthatcertaincells(- lymphocytes)withinthebodyofvertebrateshavetheabilitytosecreteanti- bodieswhichcaninactivatecontaminatingorforeignmolecules(theantigen) withintheanimalsystem.TheantibodyhasaY-shapedmolecularstructure andusesonepartofthisstructuretobindtheinvadingantigenandtheother parttotriggerthebodyÕsresponsetoeliminatetheantigen/antibodycomplex. Ithasbeencalculatedthatamammalianspeciescangenerateupto100mil- liondifferentantibodies,therebyensuringthatmostinvadingforeignantigens willbeboundbysomeantibody.AntibodieshavehighbindingafÞnitiesand speciÞcityagainstthechosenantigen.Forthemammaliansystemitisthe majordefenceagainstdisease-causingorganismsandothertoxicmolecules. Attemptstocultivatetheantibody-producingcellsinartiÞcialmediahave generallyprovedunsuccessful,withthecellseitherdyingorceasingtoproduce theantibodies.Itisnowknownthatindividual-lymphocytecellsproduce single-antibodytypes.However,in1975,GeorgesK¬ohlerandCesarMilstein successfullydemonstratedtheproductionofpureormonoclonalantibodies fromthefusionproduct(hybridoma)of-lymphocytes(antibody-producing cells)andmyelomatumourcells.In1984,theywereawardedtheNobelPrize forthisoutstandingscientiÞcachievement.Thecommercialimportanceof theirscientiÞcÞndingscanbejudgedfromtheestimatethat,inthelate1990s, thevalueoftherapeuticantibodiesalonewas$6billion. Themonoclonal-antibodytechniquechangesantibody-secretingcells(with limitedlifespan)intocellsthatarecapableofcontinuousgrowth(immor- talisation)whilemaintainingtheirspeciÞcantibody-secretingpotential.This immortalisationisachievedbyafusiontechnique,whereby-lymphocyte cellsarefusedtoÔimmortalÕcancerormyelomacellsinaone-to-oneratio, forminghybridsorhybridomasthatarecapableofcontinuousgrowthand
3.3Protoplastandcell-fusiontechnologies39
Fig.3.1Theformationofantibody-producinghybridomasbyfusiontechniques. Stage1:myelomacellsandantibody-producingcells(derivedfromimmunised animalorman)areincubatedinaspecialmediumcontainingpolyethyleneglycol, whichenhancesfusion.Stage2:themyelomaspleenhybridomacellsareselected outandculturedinclosedagardishes.Stage3:thespeciÞcantibody-producing hybridomaisselectedandpropagatedinculturevessels(invitro)orinanimal (invivo)andmonoclonalantibodiesareharvested. antibodysecretioninculture.Singlehybridcellscanthenbeselectedand grownasclonesorpureculturesofthehybridomas.Suchcellscontinueto secreteantibody,andtheantibodyisofoneparticularspeciÞcity,asopposed tothemixtureofantibodiesthatoccursinananimalÕsbloodstreamafter conventionalmethodsofimmunisation. Monoclonalantibodyformationisperformedbyinjectingamouseor rabbitwiththeantigen,laterremovingthespleen,andthenallowingfusionof individualspleencellswithindividualmyelomacells.Approximatelyl%ofthe spleencellsareantibody-secretingcellsandl0%oftheÞnalhybridomasconsist ofantibody-secretingcells(Fig.3.1).Techniquesareavailabletoidentifythe
40Geneticsandbiotechnology
correctantibody-secretinghybridomacell,cloningorpropagatingthatcell intolargepopulationswithsubsequentlargeformationofthedesiredantibody.
Thesecellsmaybefrozenandlaterre-used.
Monoclonalantibodieshavenowgainedwideapplicationinmanydiag- nostictechniqueswhichrequireahighdegreeofspeciÞcity.SpeciÞcmono- clonalantibodieshavebeencombinedintotestkitsfordiagnosticpurposes inhealthcare,inplantandanimalagriculture,andinfoodmanufacture. Monoclonalantibodiesmayalsobeusedinthefutureasantibodytherapyto carrycytotoxicdrugstothesiteofcancercells.Inthefermentationindustry theyarealreadywidelyusedasafÞnityligandstobindandpurifyexpensive products. SincethedevelopmentoftheÞrstmonoclonalantibodythemethodology hasdevelopedfromapurelyscientiÞctoolintooneofthefastestexpanding Þeldsofbiotechnology,whichhasrevolutionised,expandedanddiversiÞedthe diagnosticindustry.Themonoclonal-antibodymarketisexpectedtocontinue togrowataveryhighrateand,inhealthcarealone,theanticipatedannual worldmarketcouldbeseveralbillionUSdollarsoverthenextdecade.It isundoubtedlyoneofthemostcommerciallysuccessfulandusefulareasof modernbiotechnologyandwillbeexpandedoninseveralchapters.
3.4Geneticengineering
Genesarethefundamentalbasisofalllife,determinethepropertiesofallliving formsoflife,andaredeÞnedsegmentsofDNA.BecausetheDNAstructure andcompositionofalllivingformsisessentiallythesame,anytechnology thatcanisolate,changeorreproduceageneislikelytohaveanimpacton almosteveryaspectofsociety. Geneticrecombination,asoccursduringnormalsexualreproduction,con- sistsofthebreakageandrejoiningofDNAmoleculesofthechromosomes, andisoffundamentalimportancetolivingorganismsforthereassortmentof geneticmaterial.Geneticmanipulationhasbeenperformedforcenturiesby selectivebreedingofplantsandanimalssuperimposedonnaturalvariation. Thepotentialforgeneticvariationhas,thus,beenlimitedtoclosetaxonomic relatives. Incontrast,recombinantDNAtechniques,popularlytermedÔgenecloningÕ orÔgeneticengineeringÕ,offerpotentiallyunlimitedopportunitiesforcreating newcombinationsofgeneswhich,atthemoment,donotexistundernatural conditions. GeneticengineeringhasbeendeÞnedastheformationofnewcombinations ofheritablematerialbytheinsertionofnucleicacidmoleculesÐproduced
3.4Geneticengineering41
bywhatevermeansoutsidethecellÐintoanyvirus,bacterialplasmidor othervectorsystemsoastoallowtheirincorporationintoahostorganismin whichtheydonotnaturallyoccurbutinwhichtheyarecapableofcontinued propagation.Inessence,genetechnologyisthemodiÞcationofthegenetic propertiesofanorganismbytheuseofrecombinantDNAtechnology.Genes maybeviewedasthebiologicalsoftwareandaretheprogramswhichdrive thegrowth,developmentandfunctioningofanorganism.Bychangingthe softwareinapreciseandcontrolledmanner,itbecomespossibletoproduce desiredchangesinthecharacteristicsoftheorganism. ThesetechniquesallowthesplicingofDNAmoleculesofquitediverse originand,whencombinedwithtechniquesofgenetictransformation,etc., facilitatetheintroductionofforeignDNAintootherorganisms.Theforeign DNAorgeneconstructisintroducedintothegenomeoftherecipientorgan- ismhostinsuchawaythatthetotalgenomeofthehostisunchangedexcept forthemanipulatedgene(s). ThusDNAcanbeisolatedfromcellsofplants,animalsormicroorgan- isms(thedonors)andcanbefragmentedintogroupsofoneormoregenes. SuchfragmentscanthenbecoupledtoanotherpieceofDNA(thevector) andthenpassedintothehostorrecipientcell,becomingpartofthegenetic complementofthenewhost.Thehostcellcanthenbepropagatedinmass toformnovelgeneticpropertiesandchemicalabilitiesthatwereunattainable byconventionalwaysofselectivebreedingormutation.Whiletraditional plantandanimalgeneticalbreedingtechniquesalsochangethegeneticcode, itisachievedinalessdirectandcontrolledmanner.Geneticengineeringwill nowenablethebreedertoselecttheparticulargenerequiredforadesired characteristicandmodifyonlythatgene. Althoughmuchworktodatehasinvolvedbacteria,thetechniquesare evolvingatanastonishingrateandwayshavebeendevelopedforintroducing DNAintootherorganismssuchasyeastsandplantandanimalcellcultures. Providedthatthegeneticmaterialtransferredinthismannercanreplicate andbeexpressedinthenewcelltype,therearevirtuallynolimitstothe rangeoforganismswithnewpropertieswhichcouldbeproducedbygenetic engineering.LifeformscontainingÔforeignÕDNAaretermedÔtransgenicÕand willbediscussedinmoredetailinchapter10. Thesemethodspotentiallyallowtotallynewfunctionstobeaddedtothe capabilitiesoforganisms,andopenupvistasforthegeneticengineeringof industrialmicroorganismsandagriculturalplantsandanimalswhicharequite breathtakingintheirscope.ThisisundoubtedlythemostsigniÞcantnewtech- nologyinmodernbioscienceandbiotechnology.Inindustrialmicrobiology itwillpermittheproductioninmicroorganismsofawiderangeofhitherto unachievableproductssuchashumanandanimalproteinsandenzymessuch
42Geneticsandbiotechnology
asinsulinandchymosin(rennet);inmedicinebettervaccines,hormonesand improvedtherapyofdiseases;inagricultureimprovedplantsandanimalsfor productivity,qualityofproducts,diseaseresistance,etc;infoodproduction improvedquality,ßavour,tasteandsafety;andinenvironmentalaspectsa widerangeofbeneÞtssuchaspollutioncontrolcanbeexpected.Itshouldbe notedthatgeneticengineeringisawayofdoingthingsratherthananendin itself.Geneticengineeringwilladdto,ratherthandisplace,traditionalways ofdevelopingproducts.However,therearemanywhoviewgeneticengineer- ingasatransgressionofnormallifeprocessesthatgoeswellbeyondnormal evolution.Theseconcernswillbediscussedinchapter14. Geneticengineeringholdsthepotentialtoextendtherangeandpower ofalmosteveryaspectofbiotechnology.Inmicrobialtechnologythesetech- niqueswillbewidelyusedtoimproveexistingmicrobialprocessesbyimprov- ingthestabilityofexistingculturesandeliminatingunwantedside-products. ItisconÞdentlyanticipatedthat,withinthisdecade,recombinantDNAtech- niqueswillformthebasisofnewstrainsofmicroorganismswithnewand unusualmetabolicproperties.Inthiswayfermentationsbasedonthesetech- nicaladvancescouldbecomecompetitivewithpetrochemicalsforproducinga wholerangeofchemicalcompounds,forexampleethyleneglycol(usedinthe plasticsindustry).Inthefoodindustry,improvedstrainsofbacteriaandfungi arenowinßuencingsuchtraditionalprocessesasbakingandcheese-making andbringinggreatercontrolandreproducibilityofßavourandtexture. AfullunderstandingoftheworkingconceptsofrecombinantDNAtech- nologyrequiresagoodknowledgeofmolecularbiology.Abriefexplanation willbeattemptedherebutreadersareadvisedtoconsultsomeofthemany excellenttextsthatareavailableinthisÞeld. Thebasicmoleculartechniquesfortheinvitrotransferandexpressionof foreignDNAinahostcell(genetransfertechnology)includeisolating,cutting andjoiningmoleculesofDNA,insertingintoavector(carrying)molecule thatcanbestablyretainedinthehostcell.
ThesetechniquesmaybedeÞnedthus:
IsolationandpuriÞcationofnucleicacids.Nucleicacidsfrommostorgan- ismscannowberoutinelyextractedandpuriÞedbymeansofarangeof biochemicaltechniques(Fig.3.2). CuttingandsplicingDNA.ThemostsigniÞcantadvancestowardsthecon- structionofhybridDNAmoleculesinvitrohavecomefromthediscov- erythatsite-speciÞcrestrictionendonucleaseenzymesproducespeciÞcDNA fragmentsthatcanbejoinedtoanysimilarlytreatedDNAmoleculeusing anotherenzyme,DNAligase.Restrictionenzymesarepresentinawide
3.4Geneticengineering43
Culture
Culture the bacteria.
Cell separation
Separate cells from media
by filtration or centrifugation.DNA isolation
Isolate DNA by
centrifugal fractionation, adsorption to a silica matrix for binding to magnetic beadsWashing
Wash DNA of all
salts and residual cellular contaminants.
Cell lysis
Lyse cells using enzyme,
detergent, pH or mechanical disruption.
Neutralisation
Neutralise lysis to prevent
dissociation of bacterial genomic DNA. Debris elimination
Eliminate cell wall, membrane,
lipids, carbohydrates, proteins and all other non-DNA particles by filtration, centrifugation, supernatant removal or wash steps.
Elution
Elute the purified DNA by
releasing it from matrix or beads or by pelleting the precipitated mass using centrifugation.
Preparation for sequencing
Chronology of steps
varies with protocol Fig.3.2DiagramofatypicalseriesofsamplepreparationstepsrequiredforDNA puriÞcationfrombacterialcells(fromWellsandHerron,2002). rangeofbacteriaandcandistinguishbetweenDNAfromtheirowncells andforeignDNAbyrecognisingcertainsequenceofnucleotides.There aretechniquesavailableforbreakingopenalengthofDNAintoshorter fragmentswhichcontainanumberofgenesdeterminedbytheenzyme used.SuchDNAfragmentscanthenbeseparatedfromeachotheron thebasisofdifferingmolecularweights,andcansubsequentlybejoined togetherinanumberofways,providedthattheendsarecomplementary. ThesourcesofDNAcanbequitedifferent,givinganopportunityto replicatetheDNAbiologicallybyinsertingitintoothercells. ThecompositemoleculesintowhichDNAhasbeeninsertedhavealso beentermedÔDNAchimerasÕbecauseoftheanalogywiththeChimera ofmythologyÐacreaturewiththeheadofalion,thebodyofagoatand thetailofaserpent. Thevectororcarriersystem.Twobroadcategoriesofvectormoleculeshave beendevelopedasvehiclesforgenetransfer,namelyplasmids(smallunits ofDNAdistinctfromchromosomes)andbacteriophages(orbacterial viruses).Vectormoleculeswillnormallyexistwithinacellinaninde- pendentorextra-chromosomalform,notbecomingpartofthechro- mosomalsystemoftheorganism.Vectormoleculesshouldbecapable ofenteringthehostcellandreplicatingwithinit.Ideally,thevector
44Geneticsandbiotechnology
shouldbesmall,easilypreparedandmustcontainatleastonesitewhere integrationofforeignDNAwillnotdestroyanessentialfunction.Plas- midswillundoubtedlyofferthegreatestpotentialinbiotechnologyand havebeenfoundinanincreasinglywiderangeoforganisms,e.g.bacteria, yeastsandmouldfungi;theyhavebeenmostlystudiedingram-negative bacteria. IntroductionofvectorDNArecombinants.ThenewrecombinantDNAcan nowbeintroducedintothehostcellbytransformations(thedirectuptake ofDNAbyacellfromitsenvironment)ortransductions(DNAtrans- ferredfromoneorganismtoanotherbywayofacarrierorvectorsystem) and,ifacceptable,thenewDNAwillbeclonedwiththepropagationof thehostcell. NovelmethodsofensuringDNAuptakeintocellsincludeelectropo- rationandmechanicalparticledeliveryorbiolistics.Electroporationisa processofcreatingtransientporesinthecellmembranebyapplication ofapulsedelectricÞeld.Creationofsuchporesinamembraneallows theintroductionofforeignmoleculessuchasDNA,RNA,antibodies, drugs,etc.,intothecellcytoplasm.Developmentofthistechnologyhas arisenfromsynergyofbiophysics,bioengineeringandcellandmolecu- larbiology.Whilethetechniqueisnowwidelyusedtocreatetransgenic microorganisms,plantsandanimals,itisalsobeingincreasinglyusedfor theapplicationoftherapeuticsandgenetherapy.Themechanicalparticle deliveryorÔgenegunÕmethodsdeliverDNAonmicroscopicparticlesinto targettissueorcells.Thisprocessisincreasinglyusedtointroducenew genesintoarangeofbacterial,fungal,plantandmammalianspeciesand hasbecomeamainmethodofchoiceforgeneticengineeringofmany plantspeciesincludingrice,corn,wheat,cottonandsoybean. Thestrategiesinvolvedingeneticengineeringaresummarisedin
Table3.1andFig.3.3.
Althoughthetheoryunderlyingtheexchangeofgeneticinformation betweenunrelatedorganismsandtheirpropagationisbecomingbetterunder- stood,difÞcultiesstillpersistatthelevelofsomeapplications.Furtherresearch isrequiredbeforesuchexchangesbecomecommonplaceandthehostorgan- ismsarepropagatedinlargequantities. Earlystudiesongeneticengineeringweremainlycarriedoutwiththebac- teriumEscherichiacolibut,increasingly,otherbacteria,yeastandÞlamentous fungihavebeenused.Mammaliansystemshavebeenincreasinglydevel- opedusingthesimianvirus(SV40)andoncogenes(genesthatcausecancer), whileseveralsuccessfulmethodsareavailableforplantcells,inparticularthe
3.4Geneticengineering45
Table3.1.Strategiesinvolvedingeneticengineering
StrategyMethod
FormationofDNA
fragmentsExtractedDNAcanbecutintosmallsequencesby speciÞcenzymesÐrestrictionendonucleasesfoundin manyspeciesofbacteria.
SplicingofDNAinto
vectorsThesmallsequencesofDNAcanbejoinedorspliced intothevectorDNAmoleculesbyanenzymeDNA ligase,creatinganartiÞcialDNAmolecule.
Introductionofvectors
intohostcellsThevectorsareeithervirusesorplasmids,andare repliconsandcanexistinanextra-chromosomalstate; theycanbetransferrednormallybytransductionor transformation.
Selectionofnewly
acquiredDNASelectionandultimatecharacterisationofthe recombinantclone. Fig.3.3RecombinantDNA:thetechniqueofrecombininggenesfromonespecies withthoseofanother. Agrobacteriumsystem(Chapter10).Thus,inthelastfourdecades,molecular biologyhasformulatedevidencefortheunityofgeneticsystemstogetherwith thebasicmechanismsthatregulatecellfunction.Geneticengineeringhascon- Þrmedtheunityofthelivingworld,demonstratingthatalllivingcreatures arebuiltofmoleculesthataremoreorlessidentical.Thus,thediversityof lifeformsonthisplanetderivesfromsmallchangesintheregulatorysystems thatcontroltheexpressionofgenes.
46Geneticsandbiotechnology
3.5ThepolymerasechainreactionandDNAsequencing
Twomolecularbiologytechniquesinrecentyearshaverevolutionisedthe availabilityofDNAdata,namelythepolymerasechainreaction(PCR)andthe developmentofautomatedDNAsequencing.APCRisbasicallyatechnique whichallowstheselectiveampliÞcationofanyfragmentofDNAprovided thattheDNAsequencesßankingthefragmentareknownÐdescribedasa techniquewhichÞndsaneedleinahaystackandthenproducesahaystackof needlesbyspeciÞcampliÞcation!TheinventorofPCR,KaryMullis,shared theNobelPrizeinChemistryin1993. ThePCRprocessreliesonthesequenceofÔbasepairsÕalongthelengthof thetwostrandsthatmakethecompleteDNAmolecule.InDNAthereare fourdeoxynucleotidesderivedfromthefourbases,adenine(A),thymine (T),guanine(G)andcytosine(C).Thestrandsorpolymersthatcom- prisetheDNAmoleculeareheldtoeachotherbyhydrogenbondsbetween thebasepairs.Inthisarrangement,AonlybindstoTwhileGonlybindstoC, andthisuniquesystemfoldstheentiremoleculeintothenowwell-recognised double-helixstructure. PCRinvolvesthreeprocessingsteps:denaturation,annealingandthenexten- sionbyDNApolymerase(Fig.3.4a,b).InStep1,thedouble-strandedDNA isheated(95Ð98C)andseparatesintotwocomplementarysinglestrands. InStep2(60C),thesyntheticoligonucleotideprimers(chemicallysynthe- sisedshort-chainnucleotides)Ðshortsequencesofnucleotides(usuallyabout
20nucleotidebasepairslong)Ðareaddedandbindtothesinglestrandsin
placeswherethestrandÕsDNAcomplementstheirown.InStep3(37C),the primersareextendedbyDNApolymeraseinthepresenceofallfourdeoxynu- cleosidetriphosphates,resultinginthesynthesisofnewDNAstrandsthatare complementarytothetemplatestrands.Thecompletionofthethreesteps comprisesacycleandtherealpowerofPCRisthat,with25Ð30cycles,this experimentalsynthesisleadstomassiveampliÞcationofDNAwhichcanthen beusedforanalyticalpurposes.Amajorrecentadvancehasbeenthedevelop- mentofautomatedthermalcyclers(PCRmachines),whichallowtheentire
PCRtobeperformedautomaticallyinseveralhours.
PCRwasÞrstpatentedinl987andthencommercialisedbytheAmerican CetusCorporationin1988.However,in1991,HoffmanLaRocheandPerkin ElmerpurchasedthefulloperatingrightsofPCRfor$300million.Theappli- cationsofPCRincreasealmostdailyandinclude:molecularbiology/genetic engineering,infectiousandparasiticdiseasediagnosis,humangeneticdisease diagnosis,forensicvalidation,plantandanimalbreeding,andenvironmental;
3.5Thepolymerasechainreaction47
Fig.3.4(a)Thepolymerasechainreaction.Thedouble-strandedDNAisheatedand separatesintotwosinglestrands.Thesyntheticoligonucleotideprimersthenbindto theircomplementarysequenceandareextendedinthedirectionofthearrows, givinganewstrandofDNAidenticaltothetemplateÕsoriginalpartner; (b)PCRtemperaturecyclingproÞle(seeGraham,1994).
48Geneticsandbiotechnology
Forensic sampleDNA is extracted from the
sample, and amplified by the PCR if necessaryThe DNA is cut into fragments by a restriction endonuclease
The fragments are separated
by size by electrophoresis on an agarose gel
The X-ray film is
developed to reveal a pattern of bands which is known as a
DNA FINGERPRINT
32P labelled cDNA
or RNA probe
A sheet of X-ray
film is placed on the membrane to detect the radioactive pattern
The probe binds to
specific sequences of DNA on the membrane
The pattern of
DNA bands is
transferred to a nylon membrane ('Southern blotting') Fig.3.5DNAÞngerprinting(fromGraingerandMadden,1993). monitoring.PCRhasbeenextensivelyusedinthewell-knownprocedureof geneticorDNAÞngerprinting,thefallibilityofwhichisnowbeingchallenged incourtsoflaw(Fig.3.5). WhilePCRisÞndingconsiderableanduniqueuseinarchaeology,itis doubtfulwhetherwewilleverbeabletoresurrectwoollymammothsand dinosaursfromancientanimalremains,asrecentlyepitomisedinMichael
CrightonÕsJurassicPark.
Genomesofallorganismsconsistofmillionsofrepetitionsofthefour nucleotidesÐC,G,AandT.Inhumans,thereareover3000million nucleotides.Analysingthesequenceofthenucleotides(DNAsequencing) hasbecomeacriticallyusefultechniquefortheidentiÞcation,analysisand directedmanipulationofgenomicDNA.Originally,methodsofseparation andidentiÞcationreliedupongelelectrophoresisandautoradiography.How- ever,recentdevelopmentsinsequencingtechnologyhaveallowedtheprocess tobeautomatedandgreatlyspeededup.Fluorescentdye-labelledsubstrates areused,whichallowtheuseofalaser-inducedßuorescentdetectionsys- tem.Inmanyapplicationsautomatedsequencerscanproduceover1000base pairsofsequencesfromovernightoperations.Therearenowpubliclyavailable databasessuchasGenBank,whichprovidenumerousonlineservicesforiden- tifying,aligningandcomparingsequences.Individualchromosomescontain
3.6Genomicsandproteomics49
manythousandsofsequences,someofwhichareorganisedintogeneswhile othersappeartobemerelyßankingorspacerregions.
3.6Genomicsandproteomics
Thegeneticheritablematerialoflivingcellsresideswiththenucleicacids ofthechromosomesandistermedtheÔgenomeÕ.Arisingfromthepreviously describedtechniques,itwaspossiblein1995todeterminetheÞrstcom- pletegenomeorDNAsequenceofafree-livingorganism,thebacterium Haemophilusinßuenzae.Sincethen,aconsiderablenumberofprokaryotes, theyeastSaccharomycescerevisiae,thefruit-ßyDrosophilamelanogasterandthe plantArabidopsisthalianahavebeensequenced.However,themajorevent inmoleculargeneticswastheelucidationofthehumangenomesequencein
2001.Theacademicandcommercialdrivetodecipherthehumangenome
waslargelydrivenbyabeliefthatmajormedicaldevelopmentswouldunfold. Consequently,manybillionsofdollarshavebeenspenttoachievethismomen- touslevelofgenomicknowledge.Whiletherehasbeenmuchhypeconcerning theethicalandcommercialimplicationsofthesediscoveries,thisisonlythe beginningoftheunderstandingoftherealfunctionalactivitywithincells, intissuesandinwholeorganisms.Throughoutthislastdecadeofgenomic researchtherehasbeeninsufÞcientemphasisonotheraspectsofcellularorgan- isationandmuchill-judgedscientiÞcbeliefthattheenigmaofcellfunction inhealthanddiseasecouldbeunderstoodsolelythroughknowledgeofgenes alone. Biochemicalstudiesovermanydecadeshaveshownthatcellularactivity isachievedthroughavastarrayofsignallingandregulatoryandmetabolic pathways,eachinvolvingmanyspeciÞcmolecules.Therestillexistsavastgulf betweenourunderstandingofindividualmolecularmechanismsandpathways andhowtheyareintegratedintoanorderlyhomeostaticsystem. Majormolecularbiologyattentionhasnowmoveddramaticallytothe studyoftheproteomeÐthecollectivebodyofproteinsmadewithinanorgan- ismÕscellsandtissues.Whilethegenomesuppliestherecipesformakingthe cellÕsproteins,itistheproteomethatrepresentsthebricksandmortarofthe cellsandcarriesoutthecellularfunctions.TheproteomeisinÞnitelymore complicatedthanthegenome.Whileacellwillhaveonlyonegenome,it canhavemanyproteomes.TheDNAalphabetiscomposedoffourchained bases,whileproteins,incontrast,areconstructedfromapproximately20 aminoacids.Whilethegenesthroughtranscriptiondeterminethesequence ofaminoacidsinaprotein,itisnottotallyclearwhattheproteindoesandhow
50Geneticsandbiotechnology
itinteractswithotherproteins.Unlikegeneswhicharelinear,proteinsfold intothree-dimensionalstructureswhicharedifÞculttopredict.Theproteome isextremelydynamic,andminoralterationsintheexternalorinternalenvi- ronmentcanmodifyproteomefunction.Understandingproteomicsshould giveabetterholisticviewofcellularmetabolism. Thedominantbiochemicalapproachtoproteomicscombinestwo- dimensionalpolyacrylamidegelelectrophoresis(2D-PAGE),whichseparates, mapsandquanitiÞesproteins,withmassspectrometry(MS)-basedsequenc- ingtechniqueswhichidentifyboththeaminoacidsequencesofproteinsand thepost-translationarymolecularadditions.Proteomicswillrelatetogenomic databasestoassistproteinidentiÞcationandconsequentlywillindicatewhich geneswithinthedatabaseareimportantinspeciÞcconditions.Thetwoareas ofgenomicsandproteomicsmusthaveastrongsynergisticrelationship.The potentialofproteomicstoidentifyandcomparecomplexproteinproÞlesis nowgeneratinghighlyaccuratebutsensitivemolecularÞngerprintsofpro- teinspresentinhumanbodyßuidsatagiventime.Thesemaywellofferearly markersofdiseasedstatusinthehumansystem.Suchmolecularmedicine couldwellbeoneofthemostremarkableachievementsofbiotechnologyof thiscentury. TheabilitytocloneDNAormanipulategenesandtoobtainsuccessful expressioninanorganismisnowadaysacoretechnologyofquiteunparalleled importanceinmodernbioscienceandbiotechnology.Theexpressionand acceptanceofgeneticengineeringinthecontextofbiotechnology,where novelgenepoolscanbecreatedandexpressedinlargequantities,willoffer outstandingopportunitiesforthewell-beingofhumanity.
3.7Potentiallaboratorybiohazardsof
geneticengineering Theearlystudiesongenemanipulationprovokedwidediscussionandcon- siderableconcernatthepossiblerisksthatcouldarisewithcertaintypesof experiment.Thusitwasbelievedbysomethattheconstructionofrecombi- nantDNAmoleculesandtheirinsertionintomicroorganismscouldcreate novelorganismswhichmightinadvertentlybereleasedfromthelaboratory andbecomeabiohazardtohumansortheenvironment.Incontrast,oth- ersconsideredthatnewlysynthesisedorganismswiththeiradditionalgenetic materialwouldnotbeabletocompetewiththenormalstrainspresentin nature.Thepresentviewsofgenemanipulationstudiesarebecomingmore moderateasexperimentshaveshownthatthisworkcanproceedwithinastrict
3.7Potentiallaboratorybiohazards51
safetycodewhenrequired,involvingphysicalandbiologicalcontainmentof theorganism. Thestandardsofcontainmentenforcedintheearlyyearsofrecombinant DNAstudieswereunnecessarilyrestrictiveandtherehasbeenasteadyrelax- ationoftheregulationsgoverningmuchoftheroutinegeneticengineering activities.However,formanytypesofstudyÐparticularlywithpathogenic microorganismsÐthestandardswillremainstringent.Thus,forstrictphysi- calcontainment,laboratoriesinvolvedinthistypeofstudymusthavehighly skilledpersonnelandcorrectphysicalcontainmentequipment,e.g.negative pressurelaboratories,autoclavesandsafetycabinets. Biologicalcontainmentcanbeachievedorenhancedbyselectingnon- pathogenicorganismsasthecloningagentsofforeignDNAorbythedelib- erategeneticmanipulationofamicroorganismtoreducetheprobabilityof survivalandpropagationintheenvironment.Escherichiacoli,abacterium whichisextremelyprevalentintheintestinaltractsofwarm-bloodedand cold-bloodedanimalsaswellasinhumans,isthemostwidelyusedcloning agent.Tooffsettheriskofthiscloningagentbecomingadangerintheenviron- ment,aspecialstrainofE.colihasbeenconstructedbygeneticmanipulation whichincorporatesmanyfail-safefeatures.Thisstraincanonlygrowunder speciallaboratoryconditionsandthereisnopossibilitythatitcanconstitute abiohazardifitescapesoutofthelaboratory. Thegovernment-controlledHealthandSafetyExecutivecontrolsandmon- itorsrecombinantDNAworkwithintheUK.Thiscommitteeseeksadvice fromtheGeneticManipulationAdvisoryGroup(GMAG),whoformulate realisticproceduralguidelineswhich,ingeneral,haveprovedwidelyaccept- abletotheexperimentingscientiÞccommunity.MostotheradvancedscientiÞc nationsinvolvedinrecombinantDNAstudieshavesetupsimilaradvisory committees.Thedeliberatereleasingofgeneticallymanipulatedorganismsto theenvironmentisdiscussedinChapter14. 4
Bioprocess/fermentation
technology
4.1Introduction
Bioprocessorfermentationtechnologyisanimportantcomponentofmost ÔoldÕandÔnewÕbiotechnologyprocessesandwillnormallyinvolvecomplete livingcells(microbe,mammalianorplant),organellesorenzymesasthebio- catalystandwillaimtobringaboutspeciÞcchemicaland/orphysicalchanges inorganicmaterials(themedium).InordertobeviableinanyspeciÞcindus- trialcontext,bioprocessingmustpossessadvantagesovercompetingmethods ofproductionsuchaschemicaltechnology.Inpractice,manybioprocessing techniqueswillbeusedindustriallybecausetheyaretheonlypracticalwayin whichaspeciÞcproductcanbemade(e.g.vaccines,antibiotics). Theverybeginningsoffermentationtechnology,orasitisnowbetterrecog- nised,ÔbioprocesstechnologyÕ,werederivedinpartfromtheuseofmicroor- ganismsfortheproductionoffoodssuchascheeses,yoghurts,sauerkraut, fermentedpicklesandsausages,soysauce,andotherOrientalproducts,and beveragessuchasbeers,winesandderivedspirits(Table4.1).Inmanycases, thepresent-dayproductionprocessesforsuchproductsarestillremarkably similar.Theseformsofbioprocessingwerelongviewedasartsorcraftsbutare nowincreasinglysubjectedtothefullarrayofmodernscienceandtechnol- ogy.ParallelingtheseusefulproductformationswastheidentiÞcationofthe rolesthatmicroorganismscouldplayinremovingobnoxiousandunhealthful wastes,whichhasresultedinworldwideserviceindustriesinvolvedinwater puriÞcation,efßuenttreatmentandsolidwastemanagement(Chapter9). Bioprocessinginitsmanyformsinvolvesamultitudeofcomplexenzyme- catalysedreactionswithinspeciÞccellularsystems,andthesereactionsare
4.1Introduction53
Table4.1.Fermentationproductsaccordingtoindustrialsectors
SectorProducts/activities
Chemicals
Organic(bulk)Ethanol,acetone,butanol
Organicacids(citric,itaconic)
Organic(Þne)Enzymes
Perfumeries
Polymers(mainlypolysaccharides)
InorganicMetalbeneÞciation,bioaccumulationandleaching(Cu,U)
PharmaceuticalsAntibiotics
Diagnosticagents(enzymes,monoclonalantibodies)
Enzymeinhibitors
Steroids
Vaccines
EnergyEthanol(gasohol)
Methane(biogas)
Biomass
FoodDairyproducts(cheeses,yoghurts,Þshandmeatproducts)
Beverages(alcoholic,teaandcoffee)
BakerÕsyeast
Foodadditives(antioxidants,colours,ßavours,stabilisers)
Novelfoods(soysauce,tempeh,miso)
Mushroomproducts
Aminoacids,vitamins
Starchproducts
Glucoseandhigh-fructosesyrups
FunctionalmodiÞcationsofproteins,pectins
AgricultureAnimalfeedstuffs(SCP)
Veterinaryvaccines
Ensilageandcompostingprocesses
Microbialpesticides
RhizobiumandotherN-Þxingbacterialinoculants
Mycorrhizalinoculants
Plantcellandtissueculture(vegetativepropagation,embryo production,geneticimprovement)
AdaptedfromBulletal.(1982).
54Bioprocess/fermentationtechnology
criticallydependentonthephysicalandchemicalconditionsthatexistintheir immediateenvironment.Successfulbioprocessingwillonlyoccurwhenallthe essentialfactorsarebroughttogether. Althoughthetraditionalformsofbioprocesstechnologyrelatedtofoods andbeveragesstillrepresentthemajorcommercialbioproducts,newproducts areincreasinglybeingderivedfrommicrobialandmammalianfermentations, namely: (1)intheoverproductionofessentialprimarymetabolites,e.g.aceticand lacticacids,glycerol,acetone,butylalcohol,organicacids,aminoacids, vitaminsandpolysaccharides; (2)intheproductionofsecondarymetabolites(metabolitesthatdonotappear tohaveanobviousroleinthemetabolismoftheproducerorganism),e.g. penicillin,streptomycin,cephalosporin,gibberellins; (3)intheproductionofmanyformsofindustriallyusefulenzymes,e.g.exo- cellularenzymessuchasamylases,pectinasesandproteasesandintracellu- larenzymessuchasinvertase,asparaginaseandrestrictionendonucleases; (4)intheproductionofmonoclonalantibodies,vaccinesandnovelrecom- binantproducts,e.g.therapeuticproteins. Alloftheseproductsnowcommandlargeindustrialmarketsandareessen- tialtomodernsociety(Table4.1). Morerecently,bioprocesstechnologyisincreasinglyusingcellsderived fromhigherplantsandanimalstoproducemanyimportantproducts.Plant cellcultureislargelyaimedatsecondaryproductformationssuchasßavours, perfumesanddrugs,whilemammaliancellculturehasbeenconcernedwith vaccineandantibodyformationandtherecombinantproductionofprotein moleculessuchasinterferon,interleukinsanderythropoietin. Thefuturemarketgrowthofthesebioproductsislargelyassuredbecause, withlimitedexceptions,mostcannotbeproducedeconomicallybyother chemicalprocesses.Itwillalsobepossibletomakefurthereconomiesin productionbygeneticallyengineeringorganismstohigheroruniqueproduc- tivitiesandutilisingnewtechnologicaladvancesinprocessing.Theadvantages ofproducingorganicproductsbybiological,asopposedtopurelychemical, methodsarelistedinTable4.2. Theproductformationstagesinbioprocesstechnologyareessentiallyvery similarregardlessoftheorganismselected,themediumusedandtheprod- uctformed.Inallexamples,largenumbersofcellsaregrownunderdeÞned controlledconditions.Theorganismsmustbecultivatedandmotivatedto formthedesiredproductsbymeansofaphysical/technicalcontainmentsys- tem(bioreactor)andthecorrectmediumcompositionandenvironmental
4.1Introduction55
Table4.2.Advantagesanddisadvantagesofproducingorganiccompounds bybiologicalratherthanchemicalmeans
AdvantagesDisadvantages
Complexmoleculessuchasproteinsand
antibodiescannotbeproducedby chemicalmeans.Theproductcanbeeasilycontaminated withforeignunwantedmicroorganisms, etc. Bioconversionsgivehigheryields.Thedesiredproductwillusuallybe presentinacomplexproductmixture requiringseparation.
Biologicalsystemsoperateatlower
temperatures,nearneutralpH,etc.Thereisaneedtoprovide,handleand disposeoflargevolumesofwater.
ThereismuchgreaterspeciÞcityof
catalyticreaction.Bioprocessesareusuallyextremelyslow whencomparedwithconventional chemicalprocesses.
Exclusiveproductionofanisomeric
compoundcanbeachieved.
SubstrateIntracellular
biochemical reactionsBiomass
Metabolites
Extracellular macromolecules
Fig.4.1Thebiotechnologyprocess.
growth-regulatingparameterssuchastemperatureandaeration.Optimisa- tionofthebioprocessspansboththebio-andthetechnicalsystems.The properexploitationofanorganismÕspotentialtoformdistinctproductsof deÞnedqualityandinlargeamountsrequiresadetailedknowledgeofthe biochemicalmechanismsofproductformation. Bioprocessinginitsmanyformsiscatalysedwitheachrespectivecellular systembyalargenumberofintracellularbiochemicalreactions.Substrates derivedfromthemediumareconvertedintoprimaryandsecondaryproducts, intra-andextracellularmacromolecules,andbiomasscomponentssuchas
DNA,RNA,proteinsandcarbohydrates(Fig.4.1).
Thesereactionswillbedependentonthephysicalandchemicalparameters thatexistintheirimmediateenvironments. ThesameapparatuswithmodiÞcationscanbeusedtoproduceanenzyme, anantibiotic,anaminoacidorasinglecellprotein.Initssimplestform,the
56Bioprocess/fermentationtechnology
Table4.3.Examplesofproductsindifferentcategoriesinbiotechnological industries
CategoryExample
CellmassaBakerÕsyeast,SCP
Cellcomponents
bIntracellularproteins
Biosyntheticproducts
bAntibiotics,vitamins,aminoandorganicacids
Catabolicproducts
aEthanol,methane,lacticacid
Bioconversion
aHigh-fructosecornsyrup,6-aminopenicillanicacid
WastetreatmentActivatedsludge,anaerobicdigestion
aTypically,conversionoffeedstockcost-intensiveprocesses. bTypically,recoverycost-intensiveprocess. bioprocesscanbeviewedmerelybymixingthemicroorganismswithanutrient brothandallowingthecomponentstoreact,e.g.mixingyeastcellswithasugar solutiontogivealcohol.Moreadvancedandsophisticatedprocessesoperating onalargescaleneedtocontroltheentiresystemsothatthebioprocesscan proceedefÞcientlyandbereadilyandexactlyrepeatedwiththesameamounts ofrawmaterialsandinoculum(theparticularorganism)toproduceprecisely thesameamountofproduct. Allbiotechnologicalprocessesareessentiallyperformedwithincontain- mentsystemsorbioreactors.Largenumbersofcellsareinvariablyinvolved intheseprocessesandthebioreactorensurestheircloseinvolvementwiththe correctmediumandconditionsforgrowthandproductformation.Italso shouldrestrictthereleaseofthecellsintotheenvironment.Amainfunction ofabioreactoristominimisethecostofproducingaproductorservice.Exam- plesofthediverseproductcategoriesproducedindustriallyinbioreactorsare giveninTable4.3.
4.2Principlesofmicrobialgrowth
Thegrowthoforganismsmaybeseenastheincreaseofcellmaterialexpressed intermsofmassorcellnumberandresultsfromahighlycomplicatedand coordinatedseriesofenzymaticallycatalysedbiologicalsteps.Growthwillbe dependentbothontheavailabilityandtransportofnecessarynutrientsto thecellandsubsequentuptakeandonenvironmentalparameterssuchas temperature,pHandaerationbeingoptimallymaintained. ThequantityofbiomassorspeciÞccellularcomponentinabioreactorcan bedeterminedgravimetrically(bydryweight,wetweight,DNAorprotein)
4.2Principlesofmicrobialgrowth57
Table4.4.Approximatesizeofcells
usedinbiotechnologyprocesses
CelltypeSize(m)
Bacterialcells1×2
Yeastcells7×10
Mammaliancells40×40
Plantcells100×100
ornumericallyforunicellularsystems(bynumberofcells).Doublingtime referstotheperiodoftimerequiredforthedoublingintheweightofbiomass, whilegenerationtimerelatestotheperiodnecessaryforthedoublingofcell numbers.Averagedoublingtimesincreasewithincreasingcellsize(Table4.4) andcomplexity,e.g.doublingtimeforbacteriais0.25Ð1h;yeast1Ð2h;mould fungi2Ð6.5h;plantcells20Ð70h;andmammaliancells20Ð48h. Innormalpracticeanorganismwillseldomhavetotallyidealconditions forunlimitedgrowth;rather,growthwillbedependentonalimitingfactor, forexampleanessentialnutrient.Astheconcentrationofthisfactordrops, soalsowillthegrowthpotentialoftheorganismdecrease. Inbiotechnologicalprocessestherearethreemainwaysofgrowingmicroor- ganismsinthebioreactor:batch,semi-continuousorcontinuous.Withinthe bioreactor,reactionscanoccurwithstaticoragitatedcultures,inthepresence orabsenceofoxygen,andinliquidorlow-moistureconditions(e.g.onsolid substrates).Themicroorganismscanbefreeorcanbeattachedtosurfacesby immobilisationorbynaturaladherence. Inabatchculture,themicroorganismsareinoculatedintoaÞxedvolumeof mediumand,asgrowthtakesplace,nutrientsareconsumedandproductsof growth(biomass,metabolites)accumulate.Thenutrientenvironmentwithin thebioreactoriscontinuouslychangingand,thus,inturn,enforcingchanges tocellmetabolism.Eventually,cellmultiplicationceasesbecauseofexhaus- tionorlimitationofnutrient(s)andaccumulationoftoxicexcretedwaste products. ThecomplexnatureofbatchgrowthofmicroorganismsisshowninFig.4.2. Theinitiallagphaseisatimeofnoapparentgrowthbutactualbiochemical analysesshowmetabolicturnover,indicatingthatthecellsareinthepro- cessofadaptingtotheenvironmentalconditionsandthatnewgrowthwill eventuallybegin.Thereisthenatransientaccelerationphaseastheinoculum beginstogrow,whichisquicklyfollowedbytheexponentialphase.Inthe exponentialphasemicrobialgrowthproceedsatthemaximumpossiblerate forthatorganismwithnutrientsinexcess,idealenvironmentalparameters
58Bioprocess/fermentationtechnology
Fig.4.2Growthcharacteristicsinabatchcultureofamicroorganism.1,lagphase;
2,transientacceleration;3,exponentialphase;4,decelerationphase;5,stationary
phase;6,deathphase. andgrowthinhibitorsabsent.However,inbatchcultivationsexponential growthisoflimiteddurationand,asnutrientconditionschange,growthrate decreases,enteringthedecelerationphase,tobefollowedbythestationaryphase, whenoverallgrowthcannolongerbeobtainedowingtonutrientexhaustion. TheÞnalphaseofthecycleisthedeathphase,whengrowthratehasceased. Mostbiotechnologicalbatchprocessesarestoppedbeforethisstagebecause ofdecreasingmetabolismandcelllysis. Inindustrialusage,batchcultivationhasbeencarriedouttooptimiseorgan- ismorbiomassproductionandthentoallowtheorganismtoperformspeciÞc biochemicaltransformationssuchasend-productformation(e.g.aminoacids, enzymes)ordecompositionofsubstances(sewagetreatment,bioremediation). Manyimportantproductssuchasantibioticsareoptimallyformedduringthe stationaryphaseofthegrowthcycleinbatchcultivation. However,therearemeansofprolongingthelifeofabatchcultureandthus increasingtheyieldbyvarioussubstratefeedmethods: (1)bythegradualadditionofconcentratedcomponentsofthenutrient,e.g. carbohydrates,soincreasingthevolumeoftheculture(fedbatch)Ðused fortheindustrialproductionofbakerÕsyeast; (2)bytheadditionofmediumtotheculture(perfusion)andwithdrawalof anequalvolumeofusedcell-freemediumÐusedinmammaliancell cultivations. Incontrasttobatchconditions,thepracticeofcontinuouscultivationgives nearbalancedgrowthwithlittleßuctuationofnutrients,metabolitesorcell numbersorbiomass.Thispracticedependsonfreshmediumenteringabatch systemattheexponentialphaseofgrowthwithacorrespondingwithdrawal
4.2Principlesofmicrobialgrowth59
Culture vessel
Air ßow control
Rota meterRota
meter
Air Þlter
Gas analysers
Air Þlter
(air) Þlter
Hooded sampling
points Valve
Product receiver
Medium
Medium
Pump O2CO2 Fig.4.3Asimplelaboratoryfermenteroperatingonacontinuous-cultivationbasis. ofmediumpluscells.Continuousmethodsofcultivationwillpermitorgan- ismstogrowundersteadystate(unchanging)conditionsinwhichgrowth occursataconstantrateandinaconstantenvironment.Inacompletely mixedcontinuous-culturesystem,sterilemediumispassedintothebioreac- tor(Fig.4.3)atasteadyßowrateandculturebroth(medium,wasteproducts andorganisms)emergesfromitatthesamerate,keepingthevolumeofthe totalcultureinthebioreactorconstant.FactorssuchaspHandthecon- centrationsofnutrientsandmetabolicproducts,whichinevitablychange duringbatchcultivation,canbeheldnearconstantincontinuousculti- vations.Inindustrialpracticecontinuouslyoperatedsystemsareoflimited useandincludeonlysinglecellprotein(SCP)andethanolproductionsand someformsofwastewatertreatmentprocesses.However,formanyreasons (Table4.5)batchcultivationsystemsrepresentthedominantformofindustrial usage.Thefullrangeofcultivationmethodsformicroorganismsisshownin
Table4.6.
Microorganismsutilisedinindustrialbiotechnologyprocessesarenormally heldingreatsecrecybythecommercialcompanies.Theyhavebeenderived fromextensiveselectionprocessesandoptimisedbyculturedevelopmentfor optimumproductivity.Methodshavebeendevelopedforlong-termstorage tomaintainculturestabilityandproductivity.NationalandInternational CultureCollectionCentresconserveawiderangeofmicrobialcultures, whichprovideanorganismbaseforbiosystematicsandsupportbioscience andbiotechnologyresearchanddevelopment.
60Bioprocess/fermentationtechnology
Table4.5.Advantagesofbatchandfed-batchculturetechniquesinindustry (1)Theproductsmayberequiredonlyinrelativelysmallquantitiesatanygiventime. (2)Marketneedsmaybeintermittent. (3)Theshelf-lifeofcertainproductsisshort. (4)Highproductconcentrationisrequiredinbrothtooptimisedownstream processingoperations. (5)Somemetabolicproductsareproducedonlyduringthestationaryphaseofthe growthcycle. (6)Theinstabilityofsomeproductionstrainsrequirestheirregularrenewal. (7)ContinuousprocessescanoffermanytechnicaldifÞculties.
4.3Thebioreactor
Bioreactorsarethecontainmentvehiclesofanybiotechnology-basedproduc- tionprocess,beitforbrewing,organicoraminoacids,antibiotics,enzymesor vaccinesorforbioremediation.Foreachbiotechnologyprocessthemostsuit- ablecontainmentsystemmustbedesignedtogivethecorrectenvironmentfor optimisingthegrowthandmetabolicactivityofthebiocatalyst.Bioreactors rangefromsimplestirredornon-stirredopencontainerstocomplexasep- ticintegratedsystemsinvolvingvaryinglevelsofadvancedcomputercontrol (Fig.4.4). Bioreactorsoccurintwodistincttypes(Fig.4.4).IntheÞrstinstancethey areprimarilynon-asepticsystemswhereitisnotabsolutelyessentialtooperate withentirelypurecultures,e.g.brewing,efßuentdisposalsystems,whilein thesecondtypeasepticconditionsareaprerequisiteforsuccessfulproduct formation,e.g.antibiotics,vitamins,polysaccharides.Thistypeofprocess involvesconsiderablechallengesonthepartofengineeringconstructionand operation. Thephysicalformofmanyofthemostwidelyusedbioreactorshasnot alteredmuchoverthepast40years;however,inrecentyears,novelforms ofbioreactorshavebeendevelopedtosuittheneedsofspeciÞcbioprocesses, andsuchinnovationsareÞndingincreasinglyspecialisedrolesinbioprocess technology(Fig.4.4). Inallformsoffermentationtheultimateaimistoensurethatallparts ofthesystemaresubjecttothesameconditions.Withinthebioreactorthe microorganismsaresuspendedintheaqueousnutrientmediumcontaining thenecessarysubstratesforgrowthoftheorganismandrequiredproduct
4.3Thebioreactor61
Table4.6.Characteristicsofcultivationmethods
TypeofcultureOperationalcharacteristicsApplication
SolidSimple,cheapselectionof
coloniesfromsinglecell possible;processcontrol limitedMaintenanceofstrains, geneticstudies;productionof enzymes;composting
FilmVarioustypesofbioreactors;
tricklingÞlter,rotatingdisc, packedbed,spongereactor, rotatingtubeWaste-watertreatment, monolayerculture(animal cells);bacterialleaching; vinegarproduction
Submerged
homogeneous distributionof cells;batchÔSpontaneousÕreaction, varioustypesofreactor:stirred tankbioreactor,airlift,loop, deepshaft,etc;agitationby stirrers,air,liquidprocess controlforphysicalparameters possible;lessforchemicaland biologicalparametersStandardtypeofcultivation: antibiotics,solvents,acids,etc.
Fed-batchSimplemethodforcontrolof
regulatoryeffects,e.g.glucose repressionProductionofbakerÕsyeast
Continuous
one-stage homogeneousPropercontrolofreaction; excellentroleforkineticand regulatorystudies;higher costsforexperiment;problem ofasepticoperation,theneed forhighlytrainedoperatorsFewcasesofapplicationin industrialscale;productionof
SCP;wastewatertreatment
formation.Allnutrients,includingoxygen,mustbeprovidedtodiffuseinto eachcellandwasteproductssuchasheat,CO2andwastemetabolitesremoved. Theconcentrationofthenutrientsinthevicinityoftheorganismmustbe heldwithinadeÞniterangesincelowvalueswilllimittherateoforganism metabolismwhileexcessiveconcentrationscanbetoxic.Biologicalreactions runmostefÞcientlywithinoptimumrangesofenvironmentalparameters,and inbiotechnologicalprocessestheseconditionsmustbeprovidedonamicro- scalesothateachcellisequallyprovidedfor.Whenthelargescaleofmany bioreactorsystemsisconsidered,itwillberealisedhowdifÞcultitistoachieve
62Bioprocess/fermentationtechnology
(a) (c) (e) (b) (d)Motor
Stirrer gland
Foam breaker
Flat-bladed
impeller
Bafße
Air-sparger
AirAir
InßuentInletBafße
Air inlet
sample pointsTemperature indicatorClarifying tubeAttemporator jacketAir outlet
Outlet
EfßuentMixing
CH 4+CO2
AirAir
Air entrainment Fig.4.4Variousformsofbioreactor.(a)Stirredtankbioreactor;(b)towerreactor; (c)loop(recycle)bioreactor;(d)anaerobicdigesterorbioreactor;(e)activatedsludge bioreactor.(a)and(b)reproducedbypermissionfromKristiansenandChamberlain (1983).
4.3Thebioreactor63
Table4.7.Standardsofmaterialsusedinsophisticatedfermenterdesign (1)Allmaterialscomingintocontactwiththesolutionsenteringthebioreactoror theactualorganismculturemustbecorrosionresistanttopreventtrace-metal contaminationoftheprocess. (2)Thematerialsmustbenon-toxicsothatslightdissolutionofthematerialor componentsdoesnotinhibitculturegrowth. (3)Thematerialsofthebioreactormustwithstandrepeatedsterilisationwith high-pressuresteam. (4)Thebioreactorstirrersystem,entryportsandendplatesmustbeeasily machinableandsufÞcientlyrigidsoasnottobedeformedorbrokenunder mechanicalstress. (5)Visualinspectionofthemediumandcultureisadvantageous;transparent materialsshouldbeusedwhereverpossible. theseconditionsinawholepopulation.Itisherethattheskillsoftheprocess orbiochemicalengineerandthemicrobiologistmustcometogether. Fermentationreactionsaremultiphase,involvingagasphase(containing N
2,O2andCO2),oneormoreliquidphases(aqueousmediumandliquid
substrate)andasolidmicrophase(themicroorganismsand,possibly,solid substrates).Allphasesmustbekeptinclosecontacttoachieverapidmassand heattransfer.Inaperfectlymixedbioreactor,allreactantsenteringthesystem mustbeimmediatelymixedanduniformlydistributedtoensurehomogeneity insidethereactor. Toachieveoptimisationofthebioreactorsystem,thefollowingoperating guidelinesmustbecloselyadheredto: (1)Thebioreactorshouldbedesignedtoexcludeentranceofcontaminating organismsaswellascontainingthedesiredorganisms; (2)Theculturevolumeshouldremainconstant,i.e.noleakageorevaporation; (3)Thedissolvedoxygenlevelmustbemaintainedabovecriticallevelsof aerationandcultureagitationforaerobicorganisms; (4)Environmentalparameterssuchastemperature,pH,etc.,mustbecon- trolled,andtheculturevolumemustbewellmixed. Thestandardsofmaterialsusedintheconstructionofsophisticatedfer- mentersisimportant(Table4.7). Fermentationtechnologistsseektoachieveamaximisationofculturepoten- tialbyaccuratecontrolofthebioreactorenvironment.Butstillthereisagreat lackoftrueunderstandingofjustwhatenvironmentalconditionswillproduce anoptimalyieldoforganismorproduct.
64Bioprocess/fermentationtechnology
SuccessfulbioprocessingwillonlyoccurwhenallthespeciÞcgrowth-related parametersarebroughttogetherandtheinformationusedtoimproveandopti- misetheprocess.Forsuccessfulcommercialoperationofthesebioprocesses, quantitativedescriptionofthecellularprocessesisanessentialprerequisite. Thetwomostrelevantaspects,yieldandproductivity,arequantitativemeasures thatwillindicatehowthecellsconvertthesubstrateintotheproduct.The yieldrepresentstheamountofproductobtainedfromthesubstratewhilethe productivityspeciÞestherateofproductformation. Tounderstandandcontrolafermentationprocessitisnecessarytoknowthe stateoftheprocessoverasmalltimeincrementand,further,toknowhowthe organismrespondstoasetofmeasurableenvironmentalconditions.Process optimisationrequiresaccurateandrapidfeedbackcontrol.Inthefuture,the computerwillbeanintegralpartofmostbioreactorsystems.However,there isalackofgoodsensorprobesthatwillallowon-lineanalysistobemadeon thechemicalcomponentsofthefermentationprocess. Alargeworldwidemarketexistsforthedevelopmentofnewrapidmeth- odsformonitoringthemanyreactionswithinabioreactor.Inparticular,the greatestneedisforinnovatorymicro-electronicdesigns. Whenendeavouringtoimproveexistingprocessoperationsordesigning, itisoftenadvisabletosetupmathematicalmodelsoftheoverallsystem. Amodelisasetofrelationshipsbetweenthevariablesinthesystembeing studied.Suchrelationshipsareusuallyexpressedintheformofmathematical equationsbutcanalsobespeciÞcascause/effectrelationshipswhichcanbe usedintheoperationofthespeciÞcprocesses.Theactualvariablesinvolved canbeextensivebutwillincludeanyparameterthatisofimportanceforthe process,andcanincludepH,temperature,substrateconcentration,agitation, feedrate,etc. BioreactorconÞgurationshavechangedconsiderablyoverthelastfew decades.Theoriginalfermentationsystemwasashallowtankthatwasagi- tatedorstirredbymanpower.Fromthishasdevelopedthebasicaerationtower systemwhichnowdominatesindustrialusage.Asfermentationsystemswere furtherdeveloped,twodesignsolutionstotheproblemsofaerationandagi- tationhavebeenimplemented.TheÞrstapproachusesmechanicalaeration andagitationdevices,withrelativelyhighpowerrequirements;thestandard exampleisthestirredtankbioreactor,whichiswidelyusedthroughoutcon- ventionallaboratoryandindustrialfermentations.Suchbioreactorsensure goodgas/liquidmasstransfer,havereasonableheattransfer,andensuregood mixingofthebioreactorcontents. Thesecondmainapproachtoaerobicbioreactordesignusesairdistribution (withlowpowerconsumption)tocreateforcedandcontrolledliquidßowina
4.3Thebioreactor65
recycleorloopbioreactor.Inthiswaythecontentsaresubjectedtoacontrolled recycleßow,eitherwithinthebioreactororinvolvinganexternalrecycleloop. Thusstirringhasbeenreplacedbypumping,whichmaybemechanicalor pneumatic,asinthecaseoftheairliftbioreactor. TheCSTRconsistsofacylindricalvesselwithamotor-drivencentralshaft thatsupportsoneorseveralagitators,withtheshaftenteringeitherthrough thetoporthebottomofthevessels.Theaspectratio(i.e.height-to-diameter ratio)ofthevesselis3:5formicrobialsystems,whileformammaliancell culturetheaspectratiosdonotnormallyexceed2.Sterileairisspargedinto thebioreactorliquidbelowthebottomimpellerbywayofaperforatedring sparger.Thespeedoftheimpellorswillberelatedtothedegreeoffragility ofthecells.Mammaliancellsareextremelyfragilewhencomparedwithmost microorganisms.Inagreatmanyofthehigh-valueprocesses,thebioreactors willbeoperatedinabatchmannerunderasepticmonoculture.Thebioreactors canrangefromabout20litrestoinexcessof250m3forparticularprocesses. Theinitialcultureexpansionofthemicroorganismswillcommenceinthe smallestbioreactor,andwhengrowthisoptimised,willthenbetransferredto alargerbioreactor,andsoforth,untiltheÞnal-operationbioreactor.Through- outsuchoperationsitisimperativetomaintainasepticconditionstoensure thesuccessoftheprocess.Bioreactorsarenormallysterilisedpriortoinocula- tion,andcontaminationmustbeavoidedduringallsubsequentoperations.If contaminationoccursduringthecultivationthiswillinvariablyleadtopro- cessfailuresince,moreoften,thecontaminantcanoutgrowtheparticipating monoculture. Largeamountsoforganicwastewatersfromdomesticandindustrialsources areroutinelytreatedinaerobicandanaerobicsystems.Activatedsludgepro- cessesarewidelyusedfortheoxidativetreatmentofsewageandotherliquid wastes(Fig.4.4d).Suchprocessesusebatchorcontinuouslyagitatedbioreac- torsystemstoincreasetheentrainmentofairtooptimiseoxidativebreakdown oftheorganicmaterial.Thesebioreactorsarelargeand,foroptimumfunc- tioning,willhaveseveralormanyagitatorunitstofacilitatemixingandoxygen uptake.Theyarewidelyusedinmostmunicipalsewagetreatmentplants. Anaerobicbioreactorsordigestorshavelongbeenusedtotreatsewage matter.Intheabsenceoffreeoxygen,certainmicrobialconsortiaareableto convertbiodegradableorganicmaterialtomethane,carbondioxideandnew microbialbiomass.Mostcommonanaerobicdigestersworkonacontinuous orsemi-continuousmanner. AnoutstandingexampleofmethanegenerationistheChinesebiogaspro- gramme,wheremillionsoffamily-sizeanaerobicbioreactorsareinoperation. Suchbioreactorsareusedforthetreatmentofmanure,humanexcreta,etc.,
66Bioprocess/fermentationtechnology
producingbiogasforcookingandlightingandthesanitisationofthewaste, whichthenbecomesanexcellentfertiliser. Inalmostallfermentationprocessesperformedinabioreactorthereisgen- erallyaneedtomeasurespeciÞcgrowth-relatedandenvironmentalparameters, recordthemandthenusetheinformationtoimproveandoptimisethepro- cess.Bioreactorcontrolmeasurement