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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.

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(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

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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

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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

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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

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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.

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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(95Ð98
C)andseparatesintotwocomplementarysinglestrands. InStep2(60
C),thesyntheticoligonucleotideprimers(chemicallysynthe- sisedshort-chainnucleotides)Ðshortsequencesofnucleotides(usuallyabout

20nucleotidebasepairslong)Ðareaddedandbindtothesinglestrandsin

placeswherethestrandÕsDNAcomplementstheirown.InStep3(37
C),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