ANNALSofFacultyEngineeringHunedoara

InternationalJournalofEngineering

TomeXII[2014]Fascicule3[August]
ISSN:15842673[CDRom,online]
afreeaccessmultidisciplinarypublication
oftheFacultyofEngineeringHunedoara
AndrewAMENAGHAWON,2.SamuelOGBEIDE,3.CharityOKIEIMEN

1.

MODELLINGANDSIMULATIONOFETHANOL
PRODUCTIONVIAALCOHOLFERMENTATIONOF
DILUTEACIDHYDROLYSEDCORNSTOVERUSING
SACCHAROMYCESCEREVISIAE

DepartmentofChemicalEngineering,FacultyofEngineering,UniversityofBenin,BeninCity,NIGERIA

Abstract:Efficientdesign,controllabilityandoperabilityofbiologicalsystemsrequireanunderstandingof
howtheprocessfunctions.Thisisoftenachievedviamathematicalmodellingoftheprocess.Inthiswork,
the modelling of ethanol production during fermentation of corn stover hydrolysate was carried out. A
dynamicmathematicalmodelwasdevelopedandvalidatedviaestimationofmodelparameters.Thegrowth
of microbial cells was simulated using a low order kinetic model. The results obtained showed that the
modelwasabletoreplicatetoahighlevelofconfidence,theconcentrationsofsubstrate,microbialcellsand
ethanol as obtained experimentally indicating validity of the model. Also, the parameters were estimated
with very low confidence intervals and standard deviation values indicating that the model gave a good
representationoftheexperimentaldata.Furthermore,implementationofthevalidatedmodelviasimulation
using an equation oriented modelling software indicated that important fermentation variables such as
microbialcellyield,specificmicrobialgrowthrate,ethanolyield,andspecificethanolproductionratewere
affectedbyethanolconcentration.Theinhibitoryeffectofethanolonthesevariableswasshowntobeasa
resultofdecreasingmicrobialcellyield.
Keywords:Ethanol,Specificgrowthrate,Biomass,Hinshelwoodmodel,Fermentation
13.

1.INTRODUCTION
In trying to understand the dynamic behaviour of process systems, it is imperative to formulate
dynamicmodelsofsuchprocessesinordertostudytheircharacteristics[1].Modelsformulatedfor
aprocesscanbeutilizedinanalysingthebehaviouroftheprocessunderconsideration,simulation
oftheprocess,provisionofinsightsintothemechanismsthatdrivetheprocess,designofentirely
newprocessesanddesignofcontrollers[2].Forbiologicalprocesses,kineticmodellingisnotonly
important in describing the behaviour of the process, but it can also reduce the number of
experimentsneededtoeliminateextremepossibilitiesandprovidemathematicalexpressionsthat
canquantitativelydescribethemechanismoftheprocessasrequiredforoptimisationandcontrol
[3,4].Althoughseveralkineticmodelshavebeendevelopedforsimulatingthegrowthofcellsin
bothbatchandcontinuousprocesses,unstructuredmodelsstillgivethemostbasicunderstanding
of microbiological processes [5][7]. These unstructured models provide a fair approximation of
thedynamicbehaviouroftheseprocessesfornonsteadystatecases[8].
The growth of microorganisms during bioconversion is a complex process. TheMonod equation
has been traditionally used to relate the specific growth rate to the concentration of the limiting
substrate. The equation can be used to simulate the growth of cells for fermentation processes
where growth is not inhibited by toxic substances. However, it is well known that microbial
activity during ethanol fermentation is affected by certain factors namely, cell death, substrate
limitation,substrateinhibitionandproductinhibition.Kineticmodelsavailableintheliteraturedo
copyrightFacultyofEngineeringHunedoara,UniversityPOLITEHNICATimisoara

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not all account for the effect of these factors at the same time. The models of Egamberdiev and
Jerusalimsky [9], Ghose and Tyagi [10], Hinshelwood [11], Holzberg et al. [12], Hoppe and
Hansford [13], Lee [14] and Aiba et al.[15] only account for the effect of product (ethanol)
inhibition.Inordertoaccuratelysimulatecellgrowthduringethanolfermentation,akineticmodel
musttakeintoconsiderationtheeffectofallfourfactors.However,thisisnotusuallythecasein
realscenariosaskineticmodelsmaynotbeabletoperfectlyrepresenttheprocess.
Thepurposeofthisstudywastomodelandsimulatethebatchfermentationofsugarsubstratesto
produce ethanol. A dynamic mathematical model capable of predicting the performance of the
fermentationprocesswasdevelopedfromfirstprinciples.Themodelwasvalidatedbyestimating
model parameters using experimental data and these were subsequently used for computer
simulationstopredicthowtheconcentrationofethanolaffectedimportantfermentationvariables
suchasmicrobialcellyield,microbialcellgrowthrate,ethanolyieldandethanolproductionrate.
2.MATERIALSANDMETHODS
MaterialsCollection,Culturemedium,InoculumandFermentation,SubstrateandPretreatment
CornstoverwasobtainedfromafarminamodelfarmintheFacultyofAgriculture,Universityof
Benin, Benin City, Nigeria. The yeast Saccharomyces cerevisiae ATCC 4126 which was used as
fermentingorganismwasobtainedfromthebiotechnologydepartmentoftheFederalInstituteof
IndustrialResearch(FIIRO),Oshodi,Lagos,Nigeria.
Thecomposition(g/L)ofthefermentationmediumusedforethanolproductionwas:Glucose,120;
Yeast extract, 8.5; NH4Cl, 1.32; MgSO4.7H2O, 0.11; CaCl2, 0.06; Antifoam, 0.01mL; Citric acid, 1.5;
Citrate,0.2;Water,1L.Thefermentationwascarriedoutin1LErlenmeyerflasks.Thefermenting
vesselwastightlycorkedtoensureanaerobicconditionprevailedduringthefermentation.
Thecollectedcornstoverwassundriedtoreduceitsmoisturecontent.Thedriedcornstoverwas
milledintosmallparticlestoincreaseitssurfaceareaandmakethecellulosereadilyavailablefor
hydrolysis. It was subsequently screened using standard sieves of known mesh sizes to obtain 2
mm particles. Dilute acid hydrolysis of the corn stover was carried out using an autoclave
accordingtotheoptimisedconditionsreportedbyAmenaghawonetal.[16]whichweresulphuric
acid concentration of 1.68 (%w/w), hydrolysis temperature of 168oC and hydrolysis time of 33
minutes. Each hydrolysate was subsequently neutralised with 1.0M Sodium Hydroxide solution
andthenallowedtocoolatroomtemperature.Theneutralisedhydrolysatewascentrifugedfor20
minutestoremoveanysuspendedsolids[17].Enzymatichydrolysiswassubsequentlycarriedout
accordingtothemethodreportedbyShietal.[18].
AnalyticalMethods
Liquidsamplesweretakenatintervalsof2hoursandanalysedtodeterminetheconcentrationof
fermentable sugars, microbial cells and ethanol. Microbial cell concentration was measured by
dispensing 5 mL of the fermentation broth into a tube and centrifuging it at 5000 rpm for 30
minutes. The optical density of the sample was measured spectrophotometrically at 600nm and
comparedtoastandardcurveofdryweightofyeastcells.Thetotalreducingsugarcontentofthe
finalhydrolysatewasdeterminedbythecolorimetricmethodusingglucoseasstandard[19].The
reducing sugars were treated with 3,5dinitrosalicylic acid (DNSA) which was reduced to 3
amino5nitrosalicylicacid.Thelatterwasquantifiedbymeasuringabsorbanceatawavelengthof
540nmusingaUVVisspectrophotometer(PGInstrumentsmodelT70).Theethanolconcentration
wasmeasuredbythedichromateoxidationmethod,whichisbasedonthecompleteoxidationof
ethanolbydichromateinthepresenceofsulphuricacid[20].
BatchFermenterModelDevelopmentMicrobialCellMaterialBalance
The rate at which the ethanol producing microorganisms in the fermenter are growing can be
representedbythefirstorderdifferentialequationshowninthefollowing.
dX
= X
dt

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(1)

ANNALS of Faculty Engineering Hunedoara International Journal of Engineering

Thisequationisvalidontheassumptionthatmicrobialgrowthisonlylimitedbytheavailability
oforganicsubstrateratherthanoxygenandalsothatendogenousdecayofcellsisnegligible.The
Hinshelwood kinetic model was adopted for describing cell growth during fermentation. This
model was chosen because it accounted for the effect of ethanol and the limiting substrate
concentrationonthegrowthofmicrobialcells[11].
= max

S
Ks + S

P
1

Pm

(2)

Where (h1) is the specific growth rate, max (h1) is the maximum specific growth rate of the
microbial cells, Ks (g/L) is the substrate affinity constant, and Pm (g/L) is the maximum
concentrationofethanolabovewhichcellgrowthceases.
SubstrateMaterialBalance
The equation describing the rate of consumption of fermentable sugars by microbial cells in the
courseoffermentationcanbedescribedasfollows.
dS

(3)
= q s X
dt
Thespecificsubstrateconsumptionrateqs(gsubstrate/gbiomass/h)isexpressedas:

qs =

Yx / s

(4)

EthanolMaterialBalance
Theequationdescribingtherateofethanolformationinthecourseoffermentationiswrittenas:
dP

(5)
= qp X
dt
Thespecificrateofethanolformationqp(gethanol/gbiomass/h)isgivenbytheLuedekingPiret
likeequation[21].

(6)
q p = Yp / x
Thebatchfermentermodelwasdescribedbythesystemofequations(1)(6)whichisasetofnon
linear differential and algebraic equations (DAEs) and described the dynamic behaviour of the
concentration of all species present in the fermentation vessel. The summary of the model
equationsisgiveninTable1.
Table1.Summaryofmodelequations,variablesandcorrespondingassumptions
Numberof
New
Numberof
Equations
Assumptions
Equations Variables NewVariables
dX
Perfectmixingofthe
= X
1
X,
2
fermentercontent
dt
Perfectmixingofthe
dS
= q s X
1
S,qs
2
fermentercontent
dt
Perfectmixingofthe
dP
= qp X
1
P,qp
2
fermentercontent
dt

S
P
Fermentablesugaristhe

1
= max
1
N/A
0
onlylimitingsubstrate
Ks + S
Pm
qs =

Yx / s

q p = Yp / x
Total

N/A

N/A

Fermentablesugaristhe
onlylimitingsubstrate
Fermentablesugaristhe
onlylimitingsubstrate

Degreeoffreedomanalysis
Thedegreeoffreedomanalysisisusedtodeterminethecompletenessofamodel.Thenumberof
degreesoffreedomgivesanindicationoftheextravariablesorequationsthatmustbespecifiedin
order to have a complete model. In common chemical engineering terminology, the degree of
freedomisdefinedas[22,23].
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Number of model Number of model
=

variables

independent equations

Number of degrees

of freedom

(7)

Forthebatchfermentermodelunderconsideration,wehavethefollowing:
NumberofModelVariables:

6
NumberofModelIndependentEquations:

6
FromEquation(7),thenumberofdegreesoffreedomisgivenas:DOF=(6)(6)=0
Since the number of degrees of freedom is 0, it means the model is complete and no further
specificationisneededwithrespecttotheequationsorvariables.
3.RESULTSANDDISCUSSION
ParameterEstimation(ModelValidation)
The formulated fermentation model was validated against experimental data by estimating the
model parameters. Table 2 shows the estimated parameters, max, Ks and Pm, optimal estimated
value, 95% confidence interval, 95% tvalue, standard deviation, weighted residual and the 2
value. For all the parameters estimated, the 95% tvalue ranged from 0.011 to 0.021. The 95% t
value shows the percentage accuracy of the estimated parameters with respect to the 95%
confidence interval. For parameter accuracy, the 95% tvalues should be smaller than each
individualconfidenceintervalvalues.ThiswasindeedthecasehereasshowninTable2.The95%
confidenceintervalvaluesrangedfrom0.031to1.125.The95%confidenceintervalindicatesthat
thereisa95%probabilitythattheestimatedparameterwillbewithinthestatedinterval.Anarrow
intervalusuallyindicatesahighaccuracyofestimatedparameters.Thestandarddeviationofthe
estimatedparametersvariedbetween0.031and1.125.Thelowmagnitudeofthesevaluesindicates
thattherewasverylittledeviationofthevaluesoftheestimatedparametersfromthemean.The
weightedresidualand2valuearestatisticaltoolsusedtotesthowwellamodelfitsexperimental
data. A good fit results if the weighted residual is less than the 2value. For this study, the 2
valuewas71.32whiletheweightedresidualwas35.Sincetheweightedresidualwaslessthanthe
2value, it indicates that the model results showed a good fit and proper correlation with the
experimental results. max represents the maximum specific growth rate which indicates the
greatestrateofgrowthofthemicrobialcells.Thehalfsaturationconstant(Ks)indicatestheaffinity
ofthemicroorganismsforthesubstratewhilePmindicatesthemaximumconcentrationofethanol
beyondwhichthegrowthofmicrobialcellsceases.
Table2.Statisticalinformationfortheestimationofmodelparameter
Model
Optimal
Parameter
Estimate
max(1/h)
0.36
Ks(1/h)
5.42
Pm(g/L)
30.13
Yx/s(g/g)
0.20
Yp/x(g/g)
3.74
Weightedresidual

95%Confidence
Interval
0.031
0.503
1.125
0.022
0.201
2value(95%)

35.00

71.32

95%tvalue

StandardDeviation

0.014
0.021
0.011
0.021
0.012

0.031
0.512
1.125
0.001
0.021

Inference
Weightedresiduallessthan
2valueimpliesgoodfit

The estimated parameters were used to generate time profiles for substrate, microbial cell and
product (ethanol) concentrations using the model. Figure 1 shows the comparison between the
experimental and model predicted results for substrate, microbial cell and product (ethanol)
concentrations. The results presented in Figure 1 shows that the developed model was able to
replicatetheconcentrationsofethanol,substrate,andmicrobialcellsasobtainedfromexperiment.
This is evident in the high level of correlation between the experimental and model predicted
resultsindicatingthatthemodelexhibitsagoodfitwiththeexperimentaldata.
The variation of fermentable sugar concentration with time for ethanol fermentation by
SaccharomycescerevisiaerisalsoshowninFigure1.Itwasobservedthattherewasagradualand
progressivedecreaseintheconcentrationofsubstrate(fermentablesugars)withtime.Theamount
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of fermentable sugars was observed to reduce from an initial value of 120 g/L at the start of
fermentationtoabout38g/Lattheendoffermentation(18hours).Thisindicatedthatthesubstrate
wasbeingmetabolisedbythefermentingmicroorganismtoproduceethanol.Theseresultsarein
agreement with those reported by Ocloo and Ayernor [24] for the production of ethanol from
cassava flour hydrolysate. According to them, there was an observed decrease in the substrate
concentrationwhichtheyattributedtothemetabolicactivityofthefermentingmicroorganismin
producingethanol.SimilarresultswerealsoreportedbyBaeietal.[25].
Theconcentrationofmicrobialcellswasobservedtoincreaseinthecourseoffermentation.Thisis
indicative of the growth of microbial cells as a result of the presence of sufficient amount of
substrate in the fermenter. Stanbury [26] reported that if a microorganism is introduced into a
nutrientmediumthatsupportsitsgrowth,thepopulationofmicrobialcellswillincreaseasaresult
of the favourable conditions presented. Generally, microbial growth proceeds in four distinct
phases,namely,thelag,exponential,stationaryanddeathphases.However,Figure1onlyshows3
phases,alimitedlagphase,theexponentialandtheonsetofthestationaryphase.Thedeathphase
did not come into play possibly because the limiting substrate was still available for the
microorganisms to metabolise. The exponential phase was observed within the period of 2 to 16
hoursoffermentation.Thisphasewascharacterisedbyagradualincreaseintheconcentrationof
microbialcells,indicatinggrowthandcelldivisionwithinthisphaseofgrowth.Theincreaseinthe
concentration of microbial cells was accompanied by a concomitant decrease in the fermentable
sugarconcentration.Themaximumgrowthrateofmicrobialcellsduringthefermentationprocess
was recorded during the exponential phase
andwasestimatedtobe0.36h1.Themaximum
specific growth rate of Saccharomyces
cerevisiae has been reported to be 0.42, 0.33,
and0.51h1byAibaetal.[15],Monod[27]and
Aiba and Shoda [28] respectively. From these
values, it can be seen that the value of the
maximum
specific
growth
rate
of
Saccharomycescerevisiaeobtainedinthiswork
iscomparabletothosereportedinliterature.

The concentration of ethanol produced during

Figure1.Comparisonbetweenexperimentaldataand
fermentation was observed to increase with
themodelpredicteddataforsubstrate,biomassand
time.Theincreaseinethanolconcentrationcan
productconcentrations
beseentomirrorthegrowthofSaccharomyces
cerevisiaecellsasshowninFigure1.Theincreaseintheconcentrationofethanolcanbeattributed
totherapidgrowthoftheSaccharomycescerevisiaecellsasaresultoftheabundanceofsubstrate
and nutrients required for cell maintenance and growth [29]. The growth of the Saccharomyces
cerevisiaecellsresultedinthedecreaseobservedintheconcentrationoffermentablesugarsasthe
sugarsservedassubstratefortheSaccharomycescerevisiaecellsandtherebyusingthemasenergy
sourceandforgrowth,resultingintheproductionofethanolandcarbondioxideasendandby
productsrespectively.Prescottetal.[30]reportedthatethanolisusuallyclassifiedastheprimary
fermentation product as it is formed during the exponential growth phase. The onset of the
stationaryphaseobservedcouldbeattributedtothelimitationofsubstrateasaresultofcontinued
consumptionbythemicrobialcellsandtheaccumulationofethanolinthefermentationvessel.A
high ethanol level has been reported to be toxic and hence inhibitive to the activity of microbial
cells[31].
EffectofEthanolConcentrationonFermentationVariables
Theaccumulationofethanolinthefermentationvesselhasbeenreportedtoinhibitthegrowthof
microorganisms during fermentation as the cells begin to die due to the toxicity of ethanol. The
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effect of ethanol concentration on key fermentation variables was analysed in the following
sections.
MicrobialCellYield
TheeffectofethanolconcentrationontheyieldofmicrobialcellsisshowninFigure2.TheFigure
wasobtainedbyusingtheparametersestimated(Table2)tosimulatethegrowthofmicrobialcells
in a batch system. The yield of microbial cells during fermentation decreased gradually but
progressivelyastheconcentrationofethanolincreased.Thedecreaseobservedmaybeattributed
to the accumulation of ethanol in the fermentation broth which inhibits the growth of the cells.
SimilarobservationswerereportedbyWarrenetal.[32]andTayloretal.[31].

Figure2.Effectofethanolconcentrationontheyield
ofmicrobialcells

Figure3.Effectofethanolconcentrationonthe
specificgrowthrateofmicrobialcells

SpecificGrowthRateofMicrobialCells
The effect of ethanol on the specific growth rate of the microbial cells is shown in Figure 3. The
relativegrowthrateofthemicrobialcellswasexpressedastheratiooftheirspecificgrowthrateto
itsmaximumvalue(max).TheFigureshowsthattherewasanalmostlinearrelationshipbetween
the relative specific growth rate and ethanol concentration. A steady decrease in the specific
growth rate of fermenting organism relative to its maximum value was observed as the
concentration of ethanol was increased. The almost linear behaviour observed is similar to those
obtainedbyAibaetal.[15]andHolzbergetal.[12].Theseresearchersreportedthattheinhibitory
effectofethanolonthespecificgrowthrateisprincipallyduetodecreasingbiomassyieldjustlike
thatshowninFigure2.
EthanolYield
TheeffectofethanolconcentrationontheyieldofethanolduringfermentationisshowninFigure
4.Itwasobservedthatastheconcentrationofethanolincreased,theyieldofethanolalsoincreased
uptoapointafterwhichitbecameconstant.Thetrendobservedcanreadilyexplainedbythefact
thatastheconcentrationofethanolinthefermentationvesselincreases,theyieldwillalsoincrease
as the yield is an indication of the amount of a ethanol produced. The constant trend observed
towards the end of the plot could be attributed to inhibition which might have resulted in the
inabilityofthecellstoproducemoreethanol.Warrenetal.[32]alsoreportedasimilartrend.They
obtainedacharacteristicplotofethanolyieldasafunctionofethanolconcentrationsimilartothat
presentedinFigure4.
SpecificEthanolProductionRate
Theeffectofethanolconcentrationonthespecificrateofethanolproductionduringfermentation
isshowninFigure5.Alinearrelationshipwasobservedbetweenthespecificethanolproduction
rateandethanolconcentration.Thetrendobservedshowthatthespecificethanolproductionrate
decreased linearly with ethanol concentration. The trend is similar to that presented in Figure 3
suggesting that the primary effect of ethanol inhibition is the reduction in microbial cell yield
which in turn affects the production of ethanol. These results are similar to those obtained by
DaugulisandSwaine[33].
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Figure4.Effectofethanolconcentrationonthe
yieldofethanol

Figure5.Effectofethanolconcentrationonthe
specificethanolproductionrate

4.CONCLUSIONS
Ethanol production from corn stover hydrolysate obtained from dilute acid hydrolysis was
modelledandsimulatedusinganequationorientedmodellingplatform.Avalidatedmathematical
model developed for the process was able to replicate to a high level of confidence, the
experimental concentration of substrate (fermentable sugars), microbial cells and ethanol. The
parameterswereestimatedwithhighaccuracyasseenfromthelowvaluesofstandarddeviation
and confidence intervals. The concentration of ethanol affected fermentation variables such as
specific growth rate, microbial cell yield, ethanol yield and specific ethanol production rate. The
primaryeffectofethanolconcentrationisontheyieldofmicrobialcellswhichinturnaffectsother
fermentationvariables.
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