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Exercise 11. Batch Growth of Escherichia coli

Objectives
Knowledge of the growth characteristics of an organism is an essential part of
biotechnologyinordertodesignbioreactors,achievereproducibletransformationefficiencies
andforobtainingreproducibleplasmidandrecombinantproteinyields.Therateofgrowthofan
organismdeterminesthebatchfermentationtimeandthemaximumdilutionratethatcanbe
employedincontinuousfermentation.Inthisexperimentwewillexaminethekineticsofgrowth
ofthebacteriumEscherichiacoli.
PrelabQuestionsforExercise11:
1. SketchagrowthcurveforbatchgrowthofE.coliwithxaxisasgrowthtimeandyaxisas
thelog(#ofcells).Labelthephasesofgrowth.
2. WhatistheaveragedoublingtimeofE.coli?Whatfactorscontributetochangesinthis
value?
3. Whatfactorscausethecellstodieduringthecelldeathexponentialphase?
Phases of the Batch Growth-Cycle
Whenmicrobialcellsareinoculatedintoabatchreactorcontainingfreshculturemedium
and their increase in concentration is monitored, several distinct phases of growth can be
observed.Thereisaninitiallagphase,whichisofvariableduration.Thisisthenfollowedbythe
exponentialgrowthphase,wherecellnumber(anddryweight)increasesexponentially.Thisis
alsoreferredtoasthelogarithmicphase,thenamearisingfromthecommonmethodofplotting
thelogarithmofcellnumberagainsttime.Followingthisisashortphaseofdeclininggrowth,
and then the stationary phase. Here the cell numbers are highest. Finally the cell numbers
declineduringthedeathphase.
The lag phase results from several factors. When cells are placed in fresh medium,
intracellularlevelsofcofactors(e.g.,vitamins),aminoacidsandions(e.g.,Mg 2+,Ca2+ etc.)
may be transported across the cell membrane and thus their concentration may decrease
appreciably. If intermediates in metabolic pathways are required for enzyme activity, this
dilutionmayreducetherateatwhichvariouspathwaysoperate.Cellsmustthenmetabolizethe
available carbon sources to replenish the intracellular pools prior to initiating cell division.
Similarly,iftheinoculumisgrowninamediumcontainingadifferentcarbonsourcefromthat
ofthenewmedium,newenzymesmayneedtobeinducedtocatabolizethenewsubstrateand
thiswillalsocontributetoalag.Thepointinthegrowthcyclefromwhichtheinoculumwas
derived is also important. Cells taken from the exponential phase and used as an inoculum
generally showashorterlagphasethanthosetakenfromlaterphases.Theseexponentially

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derivedinoculawillhaveadequateconcentrationsofintermediatesandwillnotsufferfromthe
dilutioneffect.Ifaninoculumisplacedinarichmedium,onecontainingaminoacidsandother
complex carbon and nitrogen sources, a shorter lag phase results as the intermediates of
metabolismarealreadyprovided.
Whencellsareplacedinamediumwhichcontainsseveralcarbonsources,severallag
phasesmayresult.Thisisknownasdiauxicgrowth.Cellspreferentiallyuseonecarbonsource
prior to consuming the second, due to catabolite repression of the enzymes required to
metabolize the second carbon source. For example, when E. coli is placed in a medium
containingbothglucoseandlactose,glucoseisconsumedfirstandalagphasefollowsascells
synthesizetheenzymegalactosidase,whichisrequiredforlactoseutilization.Duringgrowth
onglucose,theformationofthisenzymeisundercataboliterepressionbycyclicAMP,andthus
indirectlybyglucose.Itsconcentrationinthecellisthusverylow.Thecellsconsumeglucose
(withnoadditionalmetabolicenergyexpenditure)priortolactose,whichrequiresthesynthesis
ofthisenzyme.
Celldivisionoccursintheexponentialphase.Therateofincreaseofcellnumber(N)is
proportionaltothenumberofcells.Cellsincreaseinageometricprogression2 0,21,22,..2m
aftermdivisions.Forexample,iftheinitialcellnumberwasN o,thenumberaftermgenerations
is2mNo.
Insteadofcellnumber,itisoftenmoreconvenienttousedrycellweightpervolumeXas
ameasureofcellconcentration.Duringtheexponentialphaseinabatchreactorwecanwrite
dX
X
dt
whereisthespecificgrowthrateofthecells.Theaboveequationcanbeintegratedfromthe
endofthelagphase(X=Xo,t=tlag)toanypointintheexponentialphase(X,t)
X X oe

( t tlag )

Thetimerequiredforthecellnumbersordryweighttodouble,thedoublingtimetd,isrelatedto
thespecificgrowthrateby
td

ln 2 0.69

Occasionallyitisfoundthatthedoublingtimesforcellnumberandcelldryweightmaydiffer,
asaresultofanonconstantcellmasspercellduringtheexponentialphase.Therefore,wedefine
thecellnumberspecificgrowthrate(hr1)separatelyfromthespecificgrowthrate(hr1)as
follows:

1 dN
N dt

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1 dX
X dt

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When and areequal,growthisreferredtoas balanced.Inbalancedgrowth,thereisan

adequatesupplyofallnonlimitingnutrients,sothatthecompositionofthecellisconstanteven
thoughtheconcentrationsofallothernutrientsmaybedecreasing.Ontheotherhand,when
growthisunbalanced,variationsincellcomposition(e.g.,proteincontent)mayoccur.Although
thecellnumbergrowthratemaybeconstant,thecellmassgrowthratewillvary.
Atlownutrientconcentrations,itisfoundthatthespecificgrowthratedependsonthe
nutrient concentration. At high concentrations, the specific growth rate reaches a maximum
value, set by the intrinsic kinetics of intracellular reactions, which are related to DNA
transcription and translation. The end of the exponential phase arises when some essential
nutrient,forexamplethecarbonornitrogensource,isdepletedorwhensometoxicmetabolite
accumulatestoasufficientlevel.Evenifveryhighconcentrationsofnutrientsareemployed,the
accumulationoftoxicmetabolites(e.g.,aceticacidinthecaseof E.coli growingonglucose)
willlimittheconcentrationofcellsthatcanbeattainedintheexponentialphaseinabatch
reactor. This limitation can be overcome by retaining cells by filtration, while supplying a
continuousflowofnutrientsandremovingproducts.
Following the exponential growth phase, the rate of exponential growth decreases
(declininggrowthphase)andisfollowedbythestationaryphase.Thedurationofthestationary
phasemayvarywithcelltype,previousgrowthconditionsetc.Somecellsmaylyse,releasing
nutrientsthatcanbeconsumedbyothercells,andthusmaintainthecellpopulation.Following
thisisthe deathphase. Duringthedeathphase,itisthoughtthatcelllysisoccursandthe
population decreases. Intracellular metabolites are scavenged by different enzyme systems
withinthecellandtoxicmetabolitesmayaccumulate.Therateofdeclineisalsoexponential,and
isrepresentedduringthedeathphaseas
dX
k d X
dt

We shall now turn our attention to models which relate the specific growth rate to
substrateconcentrationsandotherexternalvariables.Thesimplestofthesedonotconsiderthe
variousphasesofthegrowthcycle,butpredictonlytherateofgrowthintheexponentialphase.
Weshallthenconsidermorecomplexmodels.
Unstructured Growth Models
Thesimplestrelationshipsdescribingexponentialgrowthareunstructuredmodels.These
modelsviewthecellasasinglespeciesinsolutionandattempttodescribethekineticsofcell
growthbasedoncellandnutrientconcentrationprofiles.Themodelsthatwerefirstdeveloped
forcellgrowthdidnotaccountforthedependencyoftheexponentialgrowthrateonnutrient
concentration; they were devised to have a maximum achievable population built into the
constitutive expressions employed. Such models find applicability today when the growth
limitingsubstratecannotbeidentified.ThesimplestmodelisthatofMalthus:
rX X

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whererX isthevolumetricrateofincreaseindrycellweight(whichweshallabbreviateas
DCW)(e.g.,gmDCW/literhr)and (hr1)isconstant.Thismodelpredictsunlimitedgrowth
withtime("Mathusian"growth).Toprovideameanstolimitgrowth,Verlhulst(1844)andlater
PearlandReed(1920)proposedtheadditionofaninhibitiontermwhichwascellconcentration
dependent:
rX kX (1 X )
whichforabatchsystembecomes
dX
rX
dt

Xo e kt
1 X o (1 e kt )

where X=Xo at t=0. This result is known as the logistic equation. The maximum cell
concentration attained at large times is 1/, and the initial rate of growth is approximately
exponential,asisusuallyconsiderablylessthanunity.
The Monod Model
Oneofthesimplestmodelswhichincludestheeffectofnutrientconcentrationisthe
modeldevelopedbyJacquesMonod1basedonobservationsofthegrowthofE.coliatvarious
glucoseconcentrations.Itisassumedthatonlyonesubstrate(thegrowthlimitingsubstrate,S)is
importantindeterminingtherateofcellproliferation.TheformoftheMonodequationissimilar
tothatofMichaelisMentenenzymekinetics;infactifsubstratetransporttothecellislimitedby
theactivityofapermease,cellgrowthmightwellbeexpectedtofollowtheMichaelisMenten
formgivenbelow:

max S
KS S

Thusforbatchgrowthatconstantvolume:
dX max SX

dt
KS S

wheremaxisthemaximumspecificgrowthrateofthecells,andK Sisthevalueofthelimiting
nutrientconcentrationwhichresultsinagrowthrateofhalfthemaximumvalue.Thisequation
hastwolimitingforms.Athighsubstrateconcentrations,S>>KS,andtheequationreducestoa
zerothorderdependenceonsubstrateconcentration.Atlowsubstrateconcentrations,S<<K S
andafirstorderdependenceresults.

J. Monod, Ann. Inst. Pasteur, Paris, 79, 390 (1950).

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Glucose (GO) Assay Method Sigma Diagnostics (From Kit Instructions)

WewillbeusingadiagnostickitfromSigmathatusesenzymaticreactionstoassay
glucoseconcentrationinthegrowthmedium.Thereactionsinvolvedareasfollows:
Glucose+H2O+O2

GlucoseOxidase

GluconicAcid+H2O2

H2SO4

H2O2+oDianisidineOxidizedoDianisidine
(brown)
(pink)

Glucoseisfirstoxidizedtogluconicacidandhydrogenperoxideinthereactioncatalyzedby
glucoseoxidase. Thehydrogenperoxideformedreactsinthepresenceofperoxidasewitho
dianisidinetoformacoloredproduct,withanabsorbancemaximumat540nm.Theintensityof
thecolorproducedisdirectlyproportionaltotheglucoseconcentrationinthesample.

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Procedure
Normally,thisprocedurewouldbeconductedusingafermentor(bioreactor);however,here,we
willbeusingshakeflaskstodeterminegrowthandsubstrateutilizationkinetics.Inafermentor,
the contents of the vessel are constantly stirred by an impeller to achieve a wellmixed
environment, and air is sparged through the liquid medium to supply oxygen to the
microorganisms.Inshakeflasks,mixingisachievedbyrapidagitationonanorbitingplatform
andoxygencannotbecontinuallysuppliedtotheculture.Wewillassumethatoxygentransport
isnotlimitinggrowthandthatthereissufficientairintheflasktosustainthegrowingculture.
Thesearegenerallygoodassumptionsinshakeflaskculturesifthesurfaceareaoftheliquidair
interfaceislargerelativetothedepthoftheliquid,andtheliquidvolumeisnotgreaterthan20%
ofthetotalflaskvolume.
Note: Aseptic technique must be used in this Exercise. Review yournotes from previous
Exercisestorefamiliarizeyourselfwithasepticandsteriletechnique.
Culture Sampling
1. E.colicultureswillbegrownin1litershakeflaskswith200mlM9minimalmedium.The
initialglucoseconcentrationwillbe0.1%(1g/L).Theflaskswillbeinoculatedwithculture
grownovernightinM9priortothestartofthelabperiod.
2. Youwillbesamplingfromyourgroupsflaskevery20minutes.Ateachsampletime,you
willneedtomeasureandrecordtheopticaldensityofthecultureat600nm.Youwillalso
needtodeterminetheglucoseconcentrationintheculturemedium.Besuretoproperlylabel
allsamplesthatyoutake.
3. Whenyouarereadytotakeasample,carefullyremoveyourflaskfromthemetalclampin
theshakingincubator.Donottaketoomuchtime;youwanttominimizethetimethatthe
incubatorisopentotheairandthattheplatformisnotshaking.
4. Usingasepticetechnique, remove1mlofculturefromtheflaskasfollows. Lightan
alcohollamp.Removethemetalcapfromthemouthoftheflaskandimmediatelyflamethe
opening.Quicklyremove1mlofculturewithouttouchingtheinsideofthecontainerwith
thePipetman.(YoumayneedtouseapipetinsteadofaPipetmantomakesurethatyoucan
reachtheliquidinthebottomoftheflask.Sincethewholepipetissterilewhentakenfrom
thewrapping,youonlyneedtomakesurethatthebulbdoesnttouchtheinside.)Flamethe
openingoncemore,recaptheflask,andreturntheflasktotheincubator.Becarefulnotto
usetoomuchforceplacingtheflasksbackinthemetalclampsintheincubator.Theycan
andwillbreak.DONTFORGETTORECORDTHESAMPLETIME.
5. TransfertheculturetoasmallcuvetteandmeasuretheODat600nm.Youwillrelatethis
ODtothedrycellweightofthesample.(RememberyourcalibrationcurvefromExercise

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#1?) DontforgettodilutethesamplewithcarbonfreeM9mediumiftheODisbeyond
thelinearrangeofthecalibrationcurve.Ifthespecisbeingused,placethesampleonicein
a closed container. This will slow the growthsubstantially (eventually stopping growth
completely)sothatyourmeasurementscanmoreaccuratelyreflectthesampletime.Blank
withcarbonfreeM9medium.
6. Transfer0.5mlofthesampletoa1.5mlmicrocentrifugetube. Centrifuge2minutesat
12,000RPM. Remove200ulofthesupernatant,whichwillbeusedtomeasureglucose
concentration(procedurebelow).
7. Continuetotakesamplesuntilyouseenoincrease(oryouseeadecrease)intheODreadings
for3successivesamples.
Glucose Assay (Sigma GO Kit)
Thisassayisextremelysimpleandstraightforward;however,you can ruinyourresultsifyou
dontpayattention!
8. Pipet130uLH2Ointoacuvette.Add10lculturesupernatantandmixbygentleinversion
orwithpipette.Fortheblank,add10uLofwater.Besuretolabelyourcuvettes.
9. Next,pipet280uLofGlucoseReagentintoeachcuvette(includingBlank).
10. Incubatethesample(andBlank)forexactly30minutesat37C.Stopthereactionbyadding
280uLof12NH2SO4intoeachcuvette(includingBlank)andmixingthouroughly.
11. Measuretheabsorbanceofeachtubeagainstthereagentblankat540nm.
12. After you have assayed all of your samples, prepare a onepoint calibration curve by
performingtheGOglucoseassayasdescribedabovewiththeglucosestandardprovided
(0.1%). Your calibration curve will have two points; one at {0,0} and one at {OD of
standard,0.1%}.

Guidelines for Analysis & Conclusions Section

(Remember,thesearepointsyoushouldconsiderandincludeinyouranalysis. Thissection,
however,neednotbelimitedtothesespecificguidelines.)
1. Makea semilog graph,plotting drycell weight pervolume (X)(recall that youhave a
calibrationcurvefromthefirstexperiment)andglucoseconcentration(S)asafunctionof
time.CommentontheXvs.tcurvewithrespecttotheexpectedphasesofbatchgrowth,i.e.
whatphasesdoyousee,whatistheirduration,etc.Findyourobservedmaxfromtheslope
ofthelinearexponentialgrowthregion. Theglucoseconcentrationshoulddecreasewith
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time,with=maxforlargevaluesofSand=0whenthesubstrateisdepleted.Isthis
observationconfirmedbyyourgraph?
2. MakeanappropriatefitoftheXvs.tdata(datafromthelagphaseshouldbeneglected).
Usingthisfit,generatevaluesofdX/dtforeachvalueofX.Plot(dX/dt)/Xvs.Stofindas
afunctionofS.Dotheseresultsmeetyourexpectations?Ifthetrendisnotwhatyouexpect
youmaytryapproximatingdX/dtastheslopebetweentwopoints(thiswouldbethevalueat
themidpointofthetwopointsused).
3. Plot1/ vs.1/SusingaLineweaverBurketypeplot. Alsoplot vs. /susinganEadie
Hofsteetypeplot.Finally,fittheMonodequationtothevs.Sdata.Fromeachofthese
three analyses, findKs and max. Compare yourresults. How doyourvalues of max
comparewiththatfoundfromtheXvs.tgraph?
4.PlotXvs.S.Fromthisgraph,findtheyieldcoefficientYx/s.
5. Calculatetheglucoseconsumptionrate(dS/dt)andthespecificglucoseconsumptionrate
(dS/dt/X).Therelationshipbetweenthespecificglucoseconsumptionrateandthespecific
growthrategivesyoutheyieldcoefficient. How? Howdoesthisvaluecomparetothat
obtainedin(4)?Whichdoyouconsidermorereliable?

EQUIPMENT AND REAGENTS

E.coliW3110
1Lshakeflasks
M9MinimalMedium,with0.1%Glucose(1liter):
786.5mlDeionizedH2O(autoclavesterilized)
200ml5xM9salts(autoclavesterilized)
1.0ml1MMgSO4(filtersterilized)
10ml0.01MCaCl2(autoclavesterilized)
2.5ml40%Glucose(autoclavesterilized)
5xM9salts:Dissolvethefollowingsaltsindeionizedwater,bringingthefinalvolumeto
1.0liter.
30gNa2HPO4
15gKH2PO4
2.5gNaCl
5.0gNH4Cl
Spectrophotometer
Disposablecuvettes,small(~1.5ml)andlarge(~3.5ml)
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1.5mlmicrocentrifugetubes
Alcohollamps
Icebucketwithice
Sterilepipets
Glucose(Trinder)ReagentfromSigma

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