Growth kinetic models of five species of Lactobacilli and lactose consumption in batch submerged culture

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Biotechnology

and

Industrial

Microbiology

Growth

kinetic

models

of

five

species

of

Lactobacilli

and

lactose

consumption

in

batch

submerged

culture

Fazlollah

Rezvani

_,

_Fatemeh

_Ardestani

_,

_Ghasem

_Najafpour

c_{BabolNooshirvaniUniversityofTechnology,FacultyofChemicalEngineering,BiotechnologyResearchLab.,Babol,Iran}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received7June2015 Accepted20September2016 Availableonline3January2017 AssociateEditor:MaricêNogueira deOliveira

Keywords: Bacterialgrowth

Exponentialkineticmodel Contoismodel

Lactobacilli

a

b

s

t

r

a

c

t

KineticbehaviorsoffiveLactobacillusstrainswereinvestigatedwithContoisandExponential models.Awarenessofkineticbehaviorofmicroorganismsisessentialfortheirindustrial processdesignandscaleup.Theconsistencyofexperimentaldatawasevaluatedusing Excelsoftware.L.bulgaricuswasintroducedasthemostefficientstrainwiththehighest biomassandlacticacidyieldof0.119and0.602gg−1consumedlactose,respectively.The biomassandcarbohydrateyieldofL.fermentumandL.lactiswereslightlylessandclosetoL. bulgaricus.Biomassandlacticacidproductionyieldof0.117and0.358forL.fermentumand 0.114and0.437gg−1forL.actobacilluslactiswereobtained.L.caseiandL.delbrueckiihadthe lessbiomassyield,nearly11.8and22.7%lessthanL.bulgaricus,respectively.L.bulgaricus (R2_{=0.9500and0.9156)andL.casei(R}2_{=0.9552and0.8401)showedacceptableconsistency} withbothmodels.Theinvestigationrevealedthattheabovementionedmodelsarenot suitabletodescribethekineticbehaviorofL.fermentum(R2_{=0.9367and0.6991),L.delbrueckii} (R2_{=0.9493and0.7724)andL.lactis(R}2_{=0.8730and0.6451).Contoisrateequationisasuitable} modeltodescribethekineticofLactobacilli.SpecificcellgrowthrateforL.bulgaricus,L.casei, L.fermentum,L.delbrueckiiandL.lactiswithContoismodelinorder3.2,3.9,67.6,10.4and 9.8-foldofExponentialmodel.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Kinetic models are useful tools in design and control of biotechnological processes to obtain improved knowl-edgeabout microbial growth behavior using mathematical

∗ _{Correspondingauthor.}

E-mails:f.ardestani@qaemiau.ac.ir,Ardestanifatemeh@yahoo.com(F.Ardestani).

models along with specifically accurate and repeatable detailedexperiments.Cellgrowthtime-coursesareinvolved individual growth phases: lag phase, exponential growth phase,stationaryphaseandthephasewithexponentialdecay. Nonlinear mathematical models used to identify growth parameters.1

http://dx.doi.org/10.1016/j.bjm.2016.12.007

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Lactobacillusisagenusofgram-positivefacultative anaer-obic bacteria also known as one of the main groups of probioticorganismsinvolvedinfunctionalfoods.2–5 _A com-mon featureof these microorganisms istheir antagonistic activity against some pathogens such as salmonella spp., Escherichia coli, Listeria monocytogenes, Clostridium perfringens and Helicobacterhepaticus.6–11 _{Thecapability ofL.plantarum} and L.reuteriin phyticacid hydrolysisis valuableinbread processingtechnology.12_{Inoculatinghetero-fermentative} lac-tic acid bacteria to alfalfa silages cause to increase the production of lactic and acetic acids, hence decrease pH, reducethe number ofyeastsand molds,and inhibit Enter-obacteriumandKlebsiellapneumoniae.13_{Lactobacilliarewidely} appliedinindustrialenzymeproductionprocesses,for exam-pleglucose-formingamylase14_andlactase.15_{Differentstrains} ofLactobacilli are the main fermentative lactic acid and ␥-aminobutyricacidproducersusedincommercialprocesses aswellasstartersindairyproducts.16–18

KineticbehaviorofLactobacilliwasstudiedbysome pre-viousresearchers. Ghalyetal.,19 _{reportedthat highlactose} concentrationshadaninhibitoryeffectonL.helveticusgrowth rate. They also emphasized that adding yeast extract to culturemediumand using micro aerationcould caused to increase the specificgrowth rateand lactose consumption ofLactobacilli.19_{Vasudha andHari}20_{investigatedGompertz} andLogistickineticmodelsforL.plantarumNCDC414.The viablecellcountsincreasedfrom4×105_to7×1010_CFUmL−1 at24h.20_{CockandStouvenel}21_{studiedlacticacidproduction} byL.lactissubslactis and showedthat upto35gL−1 lactic acidwasobtainedinfermentationwithusing60gL−1ofinitial glucose.21_Amrane22_{evaluatedthegrowthkineticofL.} helveti-cusonwheypermeate.Hecharacterizedanddescribed five separatephasesduringL.helveticusgrowth.22_{L.rhamnosuscell} dryweightwasobtained;whichwasequalto23gL−1after18h incubationatappointedbioreactorconditions.23_Guptaetal.,24 foundthatL.plantarumcellgrowthratewasimprovedwith anincreaseinagitationspeed.24_{Itwasalsofoundthatmalic} acidascarbonsourceenhancedthespecificgrowthrateofL. plantarumfrom0.2to0.34h−1.25_{Thestudyofcellgrowthand} substrateutilizationkineticofL.caseiandL.rhamnosusshowed asstrongexponentiallydependentonproductinhibitionat lowlactic acid concentrations.26 _{In ourbest ofknowledge,} thereisnotanydocumentedreportonLactobacillikineticswith ContoisandExponentialkineticmodels.

Inthisarticle,kineticbehavior,cellgrowthandsubstrate consumptiontrendsoffivedifferentstrainsofLactobacilliwere investigated.BasedonContoisandExponentialkinetic mod-elsinabatchsubmergedcheesewheyasthemainnutrientsof themediumwasused.Ineachcase,influentialkinetic param-etersweredetermined.

Materials

and

methods

Microorganismsandinoculumpreparation

Lactobacillus species: L. casei subsp. casei PTCC1608, L. delbrueckiisubsp. delbrueckiiPTCC1333, L.delbrueckiisubsp. bulgaricusPTCC1737,L.fermentumPTCC1744andL.delbrueckii subsp.lactisPTCC1743werepreparedfromIranianResearch OrganizationforScienceandTechnology.

Thestockcultureofeachstrainwasseparatelyprepared onMRSmedium.Inoculatedcultureswereincubatedat37◦C for48handthenstoredinarefrigeratorat4◦C.Batch sub-merged fermentation process was performed in separate laboratory shake flasks contained of deproteinized sterile wheyasthemainsubstrate.Theculturemediawasenriched byaddingsomegrowthfactorsincluding(gL−1):lactose,50; yeastextract, 10;sodium acetate,5;KH2PO4, 2;MgSO4,0.2; MnSO4,0.05;FeSO4,0.03;peptone,10.

Culturepreparation

100mLofdeproteinizedandenrichedsterilewheywasadded toa250mLshakeflask.Beforeautoclaving,pHwasadjustedto 6.5bya2MNaOHsolutionofor2NHCl.Topreventundesired reactions, deproteinized whey and the enrichment media wereseparatelyautoclavedat121◦Cfor15minandthenwere mixedtogetherinsterileconditionstoobtained100mLfinal culturemediumineachshakeflask.

Batchsubmergedfermentation

Inoculationwasperformedbyadding2.5mLofLactobacillus stockculturetoeachshakeflask.Then,theflaskswere incu-batedat37◦Cmixedwithagitationspeedof50rpmfor50h.At thisperiod,sampleswereremovedatproper5hintervalsto assaylacticacid,lactoseandcelldryweightconcentrations.

Assessments

Lacticacidandlactoseconcentrationswereanalyzedbyhigh performanceliquidchromatography(HPLC,PerkinElmer200, Shimadzu,Japan)withAminexHEX-87Hcolumn.Themobile phaseconsistedof5mMsulfuricacidsolutionat40◦C and flowrateof0.6mLmin−1wasused.27

Cell dry weightwas assayed usinga spectrophotometer (Shimadzu,1601,Japan)atawavelengthof480nm.Standard dilutesolutionsofbacterialcellwerepreparedfromstationary phaseofcellgrowth.Todeterminecelldryweightcalibration curve,15mLofeachstandardsamplewaspassedthrougha celluloseacetatefilterwith0.45micronporesize.Filtersthen washedwithdistilledwateranddriedat100◦Cfor24h.Cell dryweightwascalculatedbasedondifferencesbetweenthe initialandthefinalfilterweights.ForeachLactobacillusspecies, aseparatestandardcurveofcelldryweightversusabsorption valuewasrecorded;whichwasappliedtodeterminecelldry weightinactualexperimentalsamples.

Kineticmodels

Contois(Eq.(1))isanun-structuredkineticmodelbasedon substrateandbiomassconcentrationandExponential(Eq.(2)) kineticmodelsisanotherun-structuredkineticmodelbased ononlysubstrateconcentration.

=max S ksx+S

(1)

60 50 40 30 20 10 0 6 5 4 3 2 1 0 0 10 20 30 Time (h)

Lactose con. Lactic acid con. Cell dry weight

Cell dr

y wight (g.L

–1

)

Lactose and lactic acid concentr

ation (g.L

–1

)

40 50 60

Fig.1–Celldryweightandlacticacidproductionaswellas

lactoseconsumptionprofileforLactobacillusdelbrueckii

subsp.bulgaricusPTCC1737inasubmergedbatchcultureof

deproteinizedwheyat37◦Cwith50rpmagitationspeedfor

52h.

whereandmaxisthespecificgrowthrateandthe maxi-mumspecificgrowth rateofbacterialstrainintermofh−1, respectively.Ks,S,S0,S1,XandXmisthesemi-saturated coef-ficient,thelimitingsubstrate (lactose)concentration,initial lactoseconcentration,requiredlactoseforinitialcellbiomass forming,celldryweightandthemaximumcelldryweightin termgL−1,respectively.

Results

FivedifferentLactobacillispecieswerestudiedforcellgrowth evaluationforincubationperiodof50h.Deproteinizedwhey ascarbon sourceswasused inasubmerged batchculture. Almost for all studied strains, a 6h lag phase period and 25–45h(dependonthestrain)exponentialgrowthphasewas observed.Table 1presentsbiomassproductionand lactose consumptiontrendsforfivedifferentstrainsofLactobacilli.

Fig.1presentscelldryweightandlacticacidproduction aswellaslactoseconsumptionprofileforL.delbrueckiisubsp. bulgaricusPTCC1737.Afterincubationperiodof(therealend oftheexponentialgrowthphase)maximumcelldryweight (5.1gL−1)wasobtained.Therateofbiomassproductivitywas calculated as 0.17gL−1h−1 for this strain. Maximum pro-ducedlacticacidbyL.delbrueckiisubsp.bulgaricusPTCC1737 wasdefinedto be26.6gL−1. For thisstrain, maximum lac-ticacidyieldandproductivitywasdeterminedof0.602gg−1 consumedlactose(after52hincubation)and 0.804gL−1h−1 (after24hincubation),respectively.Resultsshowedthatcell dryweightwasincreasedfrom0.9to5.1gL−1(morethan466% increase incell density) ata period of18h inexponential growthphase.

Similar behavior, of course with lower efficiency was observedforL.caseisubsp. casei PTCC1608(Fig.2).For this strain, producedcell biomasshasreachedto 3.4gL−1 after 36hincubation.BiomassproductivityofL.caseisubsp.casei PTCC1608wascalculated0.119gL−1h−1.Maximumlacticacid concentrationof24.2gL−1wasobtained.Forthisstrain, max-imum lactic acid yield and productivity was determined as0.586gg−1 consumed lactose(48hafterincubation) and 0.696gL−1h−1(after 24hincubation),respectively. However,

60 50 40 30 20 10 0 5 4 3 2 1 0 0 10 20 30 Time (h)

Lactose con. Lactic acid con. Cell dry weight

Cell dr

y wight (g.L

–1)

Lactose and lactic acid concentr

ation (g.L

–1)

40 50 60

Fig.2–Celldryweightandlacticacidproductionaswellas

lactoseconsumptionprofileforLactobacilluscaseisubsp.

caseiPTCC1608inasubmergedbatchcultureof

deproteinizedwheyat37◦Cwith50rpmagitationspeedfor

52h.

celldryweightwasincreasedfrom0.8to4.3gL−1(morethan 437%increaseincelldensity)ataperiodof24hinexponential growthphase.

L.delbrueckiisubsp.lactisPTCC1743reachedtoitsendof exponentialgrowthphaseafter24hincubation.Atthistime, celldryweightwas3.1gL−1andthen,remainedconstantfora periodof18h.Butattheendofthestationaryphase(after48h incubation)suddenlyincreasedto3.9gL−1andthendecreased againto3.1gL−1after52hincubation(Fig.3).Biomass pro-ductivityofL.delbrueckiisubsp.lactisPTCC1743wasevaluated equalto0.081gL−1h−1after48h.Inexponentialgrowthphase, celldryweightwasincreasedfrom0.6to3.1gL−1(morethan 416%increaseincelldensity)ataperiodof12h.Maximum lac-ticacidconcentrationwasobtainedas14.7gL−1atafter42h incubation.Maximumlacticacidyieldandproductivitywas obtained0.437gg−1consumedlactose(after42hincubation) and0.47gL−1h−1(after30hincubation),respectively.

L.delbrueckiisubsp.delbrueckiiPTCC1333hadanextended exponentialgrowthphaseuntil42hafterincubation.Atthis time, cell dry weight reached to 3.2gL−1 (Fig. 4). Biomass productivityofL.delbrueckiisubsp.delbrueckiiPTCC1333was obtainedas0.076gL−1h−1 after52hincubation.Lactic acid production wasobserved both atexponentialand the sta-tionary growth phases. Maximumlactic acid concentration (13.2gL−1)wasobtainedafter52hincubation(endofthe sta-tionary phase).Maximumlactic acidyieldandproductivity wasobtained0.351gg−1consumedlactose(after52h incuba-tion)and0.317gL−1h−1(after36hincubation),respectively.

60 50 40 30 20 10 0 5 4 3 2 1 0 0 10 20 30 Time (h)

Lactose con. Lactic acid con. Cell dry weight

Cell dr

y wight (g.L

–1

)

Lactose and lactic acid concentr

ation (g.L

–1)

40 50 60

Fig.3–Celldryweightandlacticacidproductionaswellas

lactoseconsumptionprofileforLactobacillusdelbrueckii

subsp.lactisPTCC1743inasubmergedbatchcultureof

deproteinizedwheyat37◦Cwith50rpmagitationspeedfor

Table1–BiomassandlactoseconcentrationsandspecificgrowthrateforfivestudiedLactobacilliinasubmergedbatch culturemediumoflactosefortifiedwheyat37◦Cwith50rpmagitationspeedfor52h.

Time(h) 0 6 12 18 24 30 36 42 48 52 L.bulgaricus X(gL−1) 0 0.2 0.9 2.5 4.2 5.1 5.1 5 5.1 5.2 S(gL−1) 50 47.9 40.1 26.6 13.5 7.3 7 6.7 6.1 5.8 (h−1) – 0 0.250 0.210 0.169 0.135 0.108 0.089 0.077 0.071 L.casei X(gL−1_{) 0 0.2 0.8 2.6 3.9 4.2 4.3 4.3 4.2 3.8} S(gL−1_{) 50 48.2 42.1 31.9 17.8 10.5 9.2 9.1 8.7 8} (h−1) – 0 0.231 0.214 0.165 0.127 0.102 0.085 0.072 0.064 L.lactis X(gL−1) 0 0.2 0.6 1.9 3.1 3.2 3.1 3.4 3.9 3.1 S(gL−1) 50 49.2 38.8 27.3 21.4 19.6 18.2 16.4 15.8 13.2 (h−1) – 0 0.183 0.188 0.152 0.115 0.091 0.079 0.071 0.059 L.delbrueckii X(gL−1) 0 0.2 0.6 1.7 2.4 2.6 2.9 3.2 3.1 3 S(gL−1) 50 48.1 36.4 25.3 22.1 18.2 16.4 15.1 13.6 12.4 (h−1) – 0 0.183 0.178 0.138 0.107 0.089 0.077 0.065 0.058 L.fermentum X(gL−1_{) 0 0.1 0.7 2.1 2.5 2.7 2.7 2.9 3.3 3.5} S(gL−1) 50 49.3 44.2 35.6 29.1 26.8 25.3 23.2 22.6 20.1 (h−1) – 0 0.324 0.254 0.179 0.137 0.110 0.093 0.083 0.077 60 50 40 30 20 10 0 5 4 3 2 1 0 0 10 20 30 Time (h)

Lactose con. Lactic acid con. Cell dry weight

Cell dr

y wight (g.L

–1

)

Lactose and lactic acid concentr

ation (g.L

–1)

40 50 60

Fig.4–Celldryweightandlacticacidproductionaswellas lactoseconsumptionprofileforLactobacillusdelbrueckii

subsp.delbrueckiiPTCC1333inasubmergedbatchculture ofdeproteinizedwheyat37◦Cwith50rpmagitationspeed for52h.

L.fermentumPTCC1744hadthelongestexponentialgrowth phase;after52hincubationperiod,maximumcelldryweight of 3.5gL−1 was obtained (Fig. 5). In addition, its biomass productivity was0.067gL−1h−1.Lactic acid productionwas observed at both exponential and the stationary growth phases. Maximumlactic acid concentration(10.7gL−1)was obtainedattheendofthestationaryphase.Maximumlactic

60 50 40 30 20 10 0 5 4 3 2 1 0 0 10 20 30 Time (h)

Lactose con. Lactic acid con. Cell dry weight

Cell dr

y wight (g.L

–1

)

Lactose and lactic acid concentr

ation (g.L

–1)

40 50 60

Fig.5–Celldryweightandlacticacidproductionaswellas

lactoseconsumptionprofileforLactobacillusfermentum

PTCC1744inasubmergedbatchcultureofdeproteinized

wheyat37◦Cwith50rpmagitationspeedfor52h.

acid yield and productivity was defined as0.358gg−1 con-sumedlactose(after52hincubation)and0.29gL−1h−1(after 30hincubation),respectively.

Contoiskinetic

Inordertoevaluatetheconsistencyoffivestudiedstrainswith Contoiskineticmodel,experimentaldataoflactose consump-tion and cell dry weightproduction inexponential growth phasewereused(Table1).Fig.6representsthelinearfitted experimentaldatausingContoiskineticmodelforfive inves-tigatedLactobacillistrains.

Malthuslawexplainstheexponentialgrowthphaseof Lac-tobacillusspeciesinabatchculture(Eq.(3)).IntegrationofEq.

(3)usingsuitableinitialcondition(X=X0att=t0)resultedin Eq.(4)thatdemonstratesspecificcellgrowthrate().The val-uesforinitialbiomassconcentrationandlagphasetimedelay (X0andt0)wereconsidered0.2gL−1and6h,respectively.

dX dt =X (3) =ln





Specific cell growth rate valueswere calculated accord-ing tothecelldryweightasbiomassconcentration(X)and averagelactoseconcentrationaslimiting substrate concen-tration(Save)fortheexponentialgrowthphaseusingEq.(4). Kineticconstantcoefficients(max,Ks)werecalculatedusing thecurvefittingmethod.

Exponentialmodel

Theconsistencyofthepracticaldataforthecellgrowthand lactoseconsumptionoffivestudiedLactobacillusspecieswith

8 7 6 5 4 3 2 1 0 0 0.2 0.4 (X/S)ave

Lactobacillis bulgaricus Lactobacillus casei

Lactobacillus delbrueckii Lactobacillus fermentum Lactobacillus lactis 1/ µ (h) 0.6 0.8 16 14 12 10 8 6 4 2 0 0 0.1 0.2 (X/S)_ave 1/ µ (h) 0.3 0.4 12 10 8 6 4 2 0 0 0.1 0.2 (X/S)ave 1/ µ (h) 0.4 0.3 0.5 12 14 10 8 6 4 2 0 0 0.05 0.1 (X/S)ave 1/ µ (h) 0.2 0.15 0.25 12 14 10 8 6 4 2 0 0 0.05 0.1 (X/S)ave y=72 687x+0.3859 R2_=0.9367 y=42.17x+3.2158 R2_=0.9493 y=38.21x+2.9994 R2=0.873 y=5.0736x+3.7738 R2_=0.95 y=11.965x+3.3463 R2=0.9552 1/ µ (h) 0.2 0.15

Fig.6–ThelinearplottofittingtheexperimentaldataonsubstrateutilizationandcellgrowthtoContoiskineticmodelfor

fivestudiedLactobacilliinasubmergedbatchculturemediumoflactosefortifiedwhey.

Exponential kinetic model is presented in Fig. 7. F(S) was definedasEq.(5). F(S)=





Discussion

Theanalysisofobtainedresults(presentedinTable1)showed that L. delbrueckii subsp. bulgaricus PTCC1737, L. fermentum PTCC1744 and L.delbrueckii subsp. lactis PTCC1743had the highestbiomassyieldof0.119,0.117and0.114gg−1of con-sumed lactose, respectively. L. casei subsp. casei PTCC1608

30 25 20 15 10 5 0 0 10 20 Time (h) f (S) 30 40 25 20 15 10 5 0 0 10 20 Time (h) f (S) 30 40 50 60 70 50 40 30 20 10 0 0 20 40 Time (h) f (S) 60 60 70 50 40 30 20 10 0 0 10 20 Time (h) Lactobacillus fermentum

Lactobacillus lactis Lactobacillus delbrueckii

Lactobacillus casei Lactobacillis bulgaricus f (S) 30 40 50 60 35 30 40 25 20 15 10 5 0 0 10 20 Time (h) f (S) 30 40 y=2.3216e0.0824x R2_=0.9156 y=11 788e0.0339x R2_=0.6451 y=6.6256e0.03x R2_=0.7724 y=8.2789e0.0383x R2_=0.6991 y=2284e0.0757x R2=0.8401

Fig.7–TheexponentialplottofittingtheexperimentaldataonsubstrateutilizationandcellgrowthtoExponentialkinetic

modelforfivestudiedLactobacilliinasubmergedbatchculturemediumoflactosefortifiedwhey.

and L.delbrueckiisubsp. delbrueckiiPTCC1333showed lower biomassproductionyield,relatively11.8and22.7%lessthan L.delbrueckiisubsp.bulgaricus PTCC1737, respectively.Fig. 1

represents that lactic acid production rate in exponential growthphasewashigherthanthestationaryphase.Biomass productivityofL.caseisubsp.caseiPTCC1608wascalculated 30%lessthanL.delbrueckiisubsp.bulgaricusPTCC1737. Max-imumlacticacidconcentrationwas9%lessthanL.delbrueckii subsp.bulgaricusPTCC1737.Forthisstrain,maximumlactic acidyieldandproductivity wasinorder2.65 and8.7%less thanL.delbrueckiisubsp.bulgaricusPTCC1737. Biomass pro-ductivityofL.delbrueckiisubsp. lactisPTCC1743was 52.35% lessthanL.delbrueckiisubsp.bulgaricusPTCC1737.Maximum

lactic acid concentration was approximate 45% less than L. delbrueckii subsp. bulgaricus PTCC1737. Maximum lactic acid yieldand productivity was inorder of27.4 and 41.5% lessthanL.delbrueckiisubsp.bulgaricusPTCC1737.Cockand Stouvenel21found35gL−1lacticacidproductionsbyL.lactis subs lactis.In the presentwork, maximumlactic acid con-centrationwasobtainedas26.6gL−1byL.delbrueckiisubsp. bulgaricus PTCC1737. Biomass productivity of L. delbrueckii subsp.delbrueckiiPTCC1333was55.3%lessthanL.delbrueckii subsp. bulgaricus PTCC1737 and approximately near to L. delbrueckiisubsp.lactisPTCC1743.Lacticacidproductionwas observed atexponential andthe stationarygrowth phases. Maximum lacticacidconcentrationwasobtainedafter52h

Table2–AcomparisonofContoisandExponentialkineticconstantsforfivedifferentspeciesofLactobacilli.

Strain R2 _

max(h−1) Ks(gL−1)

Contois Exponential Contois Exponential Contois

L.delbrueckiisubsp.bulgaricusPTCC1737 0.9500 0.9156 0.265 0.0824 1.34

L.caseisubsp.caseiPTCC1608 0.9552 0.8401 0.299 0.0757 3.58

L.delbrueckiisubsp.lactisPTCC1743 0.8730 0.6451 0.333 0.0339 12.72

L.delbrueckiisubsp.delbrueckiiPTCC1333 0.9493 0.7724 0.311 0.03 13.11

L.fermentumPTCC1744 0.9367 0.6991 2.591 0.0383 188.33

incubation(endofthestationaryphase),halfofL.delbrueckii

subsp.bulgaricus PTCC1737.Maximum lacticacid yieldand productivitywasinorder41.7and60.6%lessthanL.delbrueckii

subsp.bulgaricusPTCC1737. L.fermentumPTCC1744hadthe longestexponentialgrowth phase.Itsbiomassproductivity was60.6%lessthanL.delbrueckiisubsp.bulgaricusPTCC1737. Thisstrainwasthesameaspreviousmentionedstrain,lactic acid productionwasobserved atbothexponentialand the stationarygrowth phases. Maximum lactic acid concentra-tionwasabout 60%lessthan L.delbrueckiisubsp.bulgaricus

PTCC1737.Maximumlactic acidyieldand productivitywas in order of 40.5 and 63.9% less than L. delbrueckii subsp.

bulgaricusPTCC1737.Polak-Bereckaetal.,23 _reported23gL−1 of dry cell weight after 18h for L.rhamnosus, much more thanourstudiedstrains.Thisindicatethatstraintypeand culture composition have considerable impact on biomass production.23_{ReporteddatabyBerryetal.,}28_{onL.rhamnosus} ATCC10863growthcharacteristicsanditslacticacid produc-tioninbatchcultureofadefinedmediumshowedayieldof 0.84glacticacidg−1ofconsumedsubstrate.28_Bustosetal.,29 studiedlacticacidproductionbyL.pentosusATCC8041from vine-trimmingwastes.They reporteda productionyieldof 0.77(glactic acidg−1 consumed substrate)that isrelatively lessthanourobtaineddata.29_{Itwasfoundthatthequalityof} substrateandtypeoforganismspeciesarekeyparametersin productyield.Themainreasonmightbeduetoexistenceof highmineralconcentrationinthewhey.Kimetal.,30reported 13.7gL−1 lactic acid concentrations ina 48h fermentation usingL.lactisssp.lactisthatismuchlowerthanourobtained datausingglucoseasthemainsubstrate.30

Results showed that L. delbrueckii subsp. bulgaricus PTCC1737andL.caseisubsp.caseiPTCC1608hadgood accept-ableconsistencyusingContoiskineticmodel.R-square,max andKsforthefirststrainwereinorderof0.95,0.265h−1and 1.34gL−1 andforthesecondonewere0.9552,0.299h−1and 3.85gL−1,respectively(Table2).L.delbrueckiisubsp.delbrueckii PTCC1333 also showed an acceptable fitting with Contois kinetic model. For this strain R-square, max and Ks were 0.9493, 0.311h−1 and 13.11gL−1, respectively. L. fermentum PTCC1744wasfittedtoContoismodelwithagoodR-square (0.9367)andthegreatestamountsofspecificcellgrowthrate (2.591h−1).ButitsContoissemi-saturatedcoefficient(Ks)was the greatest obtained value of 188.33gL−1. Therefore, it is perceivedthatContoiskineticmodelmaynotbedesiredto describethecellgrowthandsubstrateconsumptionbehavior ofL.fermentumPTCC1744.Resultsindicatedthatthe consis-tency ofL. delbrueckiisubsp. lactis PTCC 1743 with Contois kineticmodelisthelessthan allotherinvestigatedstrains. Forthisstrain,R-squarewasdeterminedtobeaslowas0.873.

Vasudha and Hari 20 _{studiedthe Gompertzand Logistic} kineticmodelsforL.plantarumNCDC414.Theyfoundthatthe viablecellcountsincreasedfrom4×105_to7×1010_CFUmL−1_, lacticacidconcentrationincreasedbyabout4.5foldsand44% w/vofsubstrateconsumptionwasoccurredatanincubation periodof24h.20 _{Inthiswork,significantlacticacid} produc-tion observedfortheexponentialandstationaryphases.In addition,thecell dryweightincreasedbyabout10–15folds (depend on thestrain) in24h. Alvarezet al.,26 _{studied the} kineticsofcellgrowth,lactic acidproductionandsubstrate utilization ofL.caseivar.rhamnosus.Theyreportedastrong exponentiallydependentproductinhibitionatlowlacticacid concentrations.Theirresultsindicatedthatlacticacid produc-tionratewaspartiallyassociatedwithbiomassgrowthasthis workdemonstrated.26

Based onthe calculated kineticparameters (Table 2),L. delbrueckii subsp. bulgaricus PTCC1737 and L. casei subsp. caseiPTCC1608hadgoodacceptableconsistencywithContois kineticmodeltoo.R-squareandmaxforthefirststrainwere inorder0.9156and0.0824h−1andforthesecondstrainwere 0.8401and0.0757h−1,respectively(Table2).

Itwas foundthatother studiedstrains didnothavean acceptableconsistencywithExponentialkineticmodel.Thus, Exponential kinetic model is not a well desired model to describethecellgrowth andsubstrateconsumption behav-iorofthesethreestrains. Theobtainedspecificgrowthrate forL.delbrueckiisubsp.bulgaricusPTCC1737withExponential kineticwasabout70%lessthantheobtainedvaluewith Con-toiskineticmodel.Also,forL.caseisubsp.caseiPTCC1608,75% declinewasdocumentedinthiswork.

Conclusion

Thisisthefirstreportonthecellgrowthandsubstrate utiliza-tionkineticoffivedifferentstrainsofLactobacilliwithrespect toContoisandExponentialkineticmodels.L.delbrueckiisubsp. bulgaricus PTCC1737 was introduced as the desired strain infields ofbiomassand lacticacid productionyield.L. del-brueckii subsp. bulgaricusPTCC1737and L.casei subsp. casei PTCC1608showedacceptableconsistencywithbothContois andExponentialkineticmodels.Wefound,Contoisisbetter thanExponentialmodeltodescribecellgrowth andlactose consumptionbehaviorofL.strains.

Conflicts

of

interest

Acknowledgments

TheauthorswishtothanktheOfficesofViceChancellorsin ResearchatIslamicAzadUniversity,QaemshahrBranchfor theirsupportofvaluedexperimentalandanalyticalwork con-ductedduringthecourseofpresentresearch.

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