Research ArticleBioreactor Scale-Up and Kinetic Modeling of Lactic Acid andBiomass Production byEnterococcus faecalis SLT13 duringBatchCulture on Hydrolyzed Cheese Whey

Manel Ziadi 1 Sana MrsquoHir 1 Abdelkarim Aydi2 and Moktar Hamdi1

1Laboratory of Microbial Ecology and Technology (LETMi) e National Institute of Applied Sciences and Technology (INSAT)Carthage University 2 Boulevard de la Terre BP 676 1080 Tunis Tunisia2Laboratory of Materials Molecules and Applications Preparatory Institute for Scientific and Technical StudiesCarthage University BP 51 La Marsa 2075 Tunis Tunisia

Correspondence should be addressed to Manel Ziadi manel_z52yahoofr and Sana MrsquoHir sana2617yahoofr

Received 26 June 2019 Accepted 15 October 2019 Published 9 March 2020

Guest Editor Abdullah Al Loman

Copyright copy 2020 Manel Ziadi et al 0is is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Kinetic modeling of biomass and lactic acid production by Enterococcus faecalis SLT13 have been developed during batch culturein M17 and Hydrolyzed Cheese Whey (HCW) in 2 L and 20 L bioreactors 0e specific growth rate μmax was higher in 20 Lbioreactor (109 hminus 1) however the maximum specific lactic acid production rate qpmax and maximum specific sugar utilizationrate qsmax were higher in 2 L bioreactor Biomass and sugar utilization were affected by lactic acid inhibition in HCW No effectsof substrate inhibition have been observed Substrate limitation of biomass has been observed on HCW in 20 L bioreactor thesubstrate limitation constant for biomass Ksx was 4229 gL Substrate limitation of sugar consumption has been observed on M17in 2 L bioreactor the substrate limitation constant for sugar consumption Kss was 273 gL Compared to experimental data themodel provided good predictions for biomass sugar consumption and lactic acid production

1 Introduction

Actually the lactic acid and its derivative polylactic acid(PLA)market appears among the most important markets ofthe chemical industry 0is market is in clear progress andit is predictable to attain USD 382 billion and 516 billionon 2020 for the lactic acid and the PLA respectively 0isgrowth is due to several reasons the increasing request forthe eco-friendly products the increasing prices of productsderived from the petrochemistry and the limited reserves offossil energy [1]

0e lactic acid presents several applications essentiallythe food industries (preservative and acidulant) textile andpharmaceutical industries Moreover the expansion of PLAmarket is the main reason for lactic acid market risingIndeed the lactic acid is used as monomer for PLA pro-duction [2] For its bioavailability and biodegradability thePLA is used not only in the food industries for packaging ofsensitive foods (ie eggs) and tablets in the pharmaceutical

industry but also in electronics and textile which can besubstituted for synthetic polymers derived from petrochemistry

Two processes are used to produce industrially lacticacid chemical synthesis and microbial fermentation How-ever microbial fermentation presents numerous advantagescompared to chemical synthesis these advantages are es-sentially environmental fermentation is a clean processalso the lactic acid obtained by fermentation is opticallypure (L (+)-lactic acid) compared to the racemic mixture(50 L-lactic acid and 50 D-lactic acid) obtainedchemically [3] Indeed PLA with L (+)-lactic acid contenthigher than 90 is a crystalline polymer and is preferred forcommercial uses than the amorphous polymer with highcontent of the D-isomer [4]

For advantageous commercial process choosing anappropriate carbon source for lactic acid production iscrucial Indeed a promising substrate must be of low costdoes not contain contaminants and allows fast growth ofthe lactic acid producer microorganism as well as high

HindawiJournal of ChemistryVolume 2020 Article ID 1236784 9 pageshttpsdoiorg10115520201236784

recovery in lactic acid [5] 0us several substrates havebeen used for the production of lactic acid through mi-crobial fermentation Among them cheese whey a majorbiowaste of the dairy industry is a good candidate forthis purpose 0is byproduct contains 5ndash8 (ww) of drymatter in which lactose is approximately 60ndash80 proteins10ndash20 minerals vitamins fat lactic acid and trace el-ements [6] Cheese whey has been widely used for variousproductions including organic acids single-cell proteinsenzymes and ethanol [7]

Many microorganisms are involved in lactic acid pro-duction from varied feedstock including Lactic Acid Bacteria(LAB) yeasts and fungi LAB species used for lactic acidproduction include Lactobacillus Streptococcus and En-terococcus producing L(+)-lactic acid while Leuconostoc andLactobacillus bulgaricus produce D(minus )-lactic acid [8] Ge-netically modified Saccharomyces cerevisiae was used for theproduction of lactic acid in continuous fermentation [9]Rhizopus is the major fungi used for lactic acid productionthe species R oryzae and R microsporus have been previ-ously used and high amounts of lactic acid were formed [10]Others bacterial strains Bacillus and Escherichia coli werealso used for the production of both lactate isomers [11 12]

Among lactic acid bacteria involved in lactic acid pro-duction Enterococcus faecalis has been described in manystudies as lactic acid producer Enterococcus faecalis RKY1 isable to produce lactic acid from agricultural feedstock suchas wheat barley corn hydrol soybean curd residues maltand from single and mixed sugars [13ndash15] 0e same strainwas used by Nandsana and Kumar [16] to produce lactic acidfrom molasses Enterococcus faecium No 78 has been usedfor lactic acid production from liquefied sago starch [17]Enterococcus faecalis QU11 is able to produce lactic acidfrom glycerol fermentation [18]

With increasing interest in the industrial application oflactic acid and PLA different kinetic models have beendeveloped in order to study the growth and lactic acidproduction by different species of LAB Indeed kineticmodeling is an essential step to optimize a fermentationprocess as though models help in process control reducingprocess costs and time and improving product quality [19]

Earlier studies have described lactic acid production byLactobacillus sp using LuedekingndashPiret model 0is kind ofmodel relates cell growth and lactic acid production by in-cluding two coefficients growth-associated coefficient (α) and anon-growth-associated coefficient (β) Unstructured kineticmodels such as the Gompertz equation have been also used[20] Further developments have proposed models whichincluded Monod equation to describe the relation betweena limiting substrate and biomass growth and then productformation [21 22] Monod equation relates the specificgrowth rate (micro) to the concentration of a single growth-limiting substrate (S) via two parameters the maximumspecific growth rate (micromax) and the substrate affinityconstant (KS) Other authors have proposed the inhibitoryeffects of high initial lactose concentrations lactose lim-itation and lactate inhibition [23 24] while others de-veloped models showing the inhibition effects of bothassociated and dissociated forms of lactic acid [25 26]

0is study is conducted to find themost suitablemodel thatdescribes biomass and lactic acid production by Enterococcusfaecalis SLT13 in batch cultures in a medium prepared withhydrolyzed cheese whey and in a synthetic medium (M17)0eeffects of culture media and scale-up from 2 to 20L bioreactorson the kinetic parameters were studied

2 Materials and Methods

21 Bacterial Strain and Culture Conditions 0e strainEnterococcus faecalis SLT13 used in this work was isolatedfrom Tunisian traditional fermented milk ldquoLebenrdquo [27]Stock cultures of this strain were stored in M17 brothcontaining 20 glycerol (Merck) at minus 80degC

Enterococcus faecalis cells were grown in M17 broth(Merck) containing glucose as main carbon source or inpapaın Hydrolyzed Cheese Whey (HCW) Cheese wheypowder (BHA Belgium SA) was rehydrated in distilledwater (6 wv) All fermentations were supplementedwith (L) 2 g yeast extract (YE) 05 g NH4Cl 2 g K2HPO4and 25mg MnSO4 Papaın was provided by the WalloonCenter of Industrial Biology (CWBI Belgium) and had anactivity of 22 000 IU 0e enzyme substrate ratio was 05 100 (ww) 0e whey hydrolysis was carried out at pH 50and 60degC 0e enzyme was inactivated by thermal treatment(90degC for 5min) and cooled in an ice bath Hydrolysis timewas 30min After hydrolysis the pH was adjusted to 70

Batch cultures using M17 or HCWmedia were carried outin 2L bioreactor Biostat B (B Braun Biotech InternationalMelsungen Germany) A working volume of 15 L was usedwhich comprised 14 L growth medium and 100mL of inoc-ulum Inoculum was prepared by adding 100 μL of the pre-served strain in 250mL Erlenmeyer flask containing 100mLof M17 broth (approx 1times 107 CFUmL) and then incubatedat 30degC 0e media used were sterilized in the bioreactor at121degC for 15min 0e set point of pH was 70plusmn 02 tem-perature 30degC and stirrer speed 100 rpm 0e pH was ad-justed with 1N HCl or 1N NaOH0e fermentation was runfor 10 h in M17 and 24 h in HCW Samples were withdrawnevery 2ndash4 h for determination of glucose lactose and lacticacid concentrations and viable cells count (CFUmL)

Batch culture in 20 L bioreactor was conducted as fol-lows 100 μL of the preserved strain was inoculated into20mL vial containing 10mL M17 broth and then incubatedat 30degC for 12 h and transferred again in 250mL Erlenmeyerflask containing 200mL M17 broth (preculture P1) that wasincubated at 30degC for 16 h 0e preculture P1 from the ex-ponential growth phase was inoculated into 1 L Erlenmeyerflask containing 800mL M17 broth (preculture P2) Batchculture was performed in 20 L stirred bioreactor (BiolafitteFrance) in HCW 0e media used were sterilized in thebioreactor at 121degC for 15min 0e preculture P2 in theexponential growth phase prepared in 1 L Erlenmeyerflask was added to the culture medium 0e temperatureof the bioreactor was maintained at 30degC and the pH wascontrolled at 7plusmn 02 by the addition of NaOH (3N) Sampleswere withdrawn every 2ndash4 h for determination of lactose andlactic acid concentrations and viable cells count (CFUmL)All experiments were conducted in triplicate

2 Journal of Chemistry

22 Biomass Determination For biomass determination acalibration curve relating dry cell weight with the cell con-centration (estimated by viable cells counting method) wasused For cells counting 01mL of diluted sample was spreadover M17-agar surface 0e plates were then incubated for onenight at 30degC 0e number of colonies was counted andexpressed in CFUmL (Colony-Forming UnitsmL)

23 Analytical Analysis Glucose lactose and lactic acidconcentrations in the fermentation broth were measured byHPLC (Agilent Technologies) using an ion-exclusion column(SupelCo Gel C-610H) operated at 35degC Components wereelutedwith 01 phosphoric acid at a flow rate of 05mLmiddotminminus 1Detection was accomplished by a refractive index detector

24 Kinetic Model

241 Model Development 0emodel used in this work is theone developed by Nandasana and Kumar [16] for lactic acidproduction by Enterococcus faecalis RKY1 and is as follows

Specific growth rate

μ μmaxSKiX

KSX + S( 1113857 KiX + S( 1113857e

minus PKpX( 1113857 (1)

0e biomass production ratedX

dt μ minus K d( 1113857X (2)

0e substrate consumption ratedS

dt minus qsmax

SKiS

KSS + S( 1113857 KiS + S( 1113857e

minus PKpS( 1113857X (3)

0e product production ratedP

dt α

dX

dt+ qpmax

SKiP

KSP + S( 1113857 KiP + S( 1113857e

minus PKpp( 1113857X (4)

For (3) the usual assumption proposed by Nandasanaand Kumar [16] was not used due to the nature of theprocess a minus sign was included According to the sameauthors these assumptions are taken in consideration for thesimplification of the modeling step

(i) Kss Ksp low lactose concentration affects in thesame way lactose uptake and lactic acid production

(ii) Kis Kip high lactose concentration inhibits in thesame way lactose uptake and lactic acid production

(iii) Kps Kpp lactate concentration affects in the sameway lactose uptake and lactic acid production

0e method used to solve model differential equationsfor a given set of kinetic parameters was ODE113s solver ofMATLAB [28] in order to obtain X S and P values 0einitial conditions were the experimental values of X S and P

at time zero (t t0)

242 Model Integration In order to estimate kinetic pa-rameters a search of these parameters values for which

predicted values of X S and P are closer to the experimentalone needs to be applied For this process an optimizationmethod was used 0e nonlinear optimization method chosenfor this problemwas the interior-point approach to constrainedminimization using the ldquofminconrdquo function in MATLAB[29 30] 0is function is a local search algorithm that startswith an initial guess and finds a constrained minimum

0e objective function (OF) used in this minimizationwas in the form of

OFX 1113936

ni1 Xi minus XiEXP1113872 1113873

2

1113936ni1 Xi minus Xi( 1113857

2 middot 100

OFS 1113936

ni1 Si minus SiEXP1113872 1113873

2

1113936ni1 Si minus Si( 1113857

2 middot 100

OFP 1113936

ni1 Pi minus PiEXP1113872 1113873

2

1113936ni1 Pi minus Pi( 1113857

2 middot 100

OF OFX + OFS + OFP

3

(5)

0us it was considered in the estimation of parame-ters that all variables are equally important and given equalweight to the approximation error for each one of them0e procedure to find the parameters was repeated at least30 times with random initial guesses in order to assure agood solution

3 Results and Discussion

31 Effect of Culture Media and Scale-Up on Lactic Acid andBiomass Productivity 0is study is conducted to evaluate theeffect of scale-up on growth and lactic acid production byEnterococcus faecalis SLT13 0e fermentation was first carriedout at laboratory scale in 2L bioreactor than at semipilot scalein 20L bioreactor All the studied fermentations were con-ducted in Stirred Tank Reactor (STR) Moreover two distinctculture media were analyzed including the synthetic mediumM17 and the papaın Hydrolyzed Cheese Whey (HCW) inorder to characterize the growth kinetics of Enterococcusfaecalis SLT13 2 L bioreactor cultures were conducted in M17and HCW under controlled conditions while only HCW wasused for 20L bioreactor the same parameters (pH tempera-ture and stirrer) were maintained

0e key kinetic parameters during the culture of E faecalisin the conditions mentioned below are estimated experi-mentally and summarized in Table 1

Whereas the final biomass is higher in synthetic media(M17) lactic acid concentration is more important whenE faecalis was grown on HCW Lactic acid yield on lactose(Yps) increases significantly in HCW (from 0314 to 098 gg)Lactic acid yield obtained for the strain E faecalis RKY1based on sugar consumption varies from 090 to 099 fordifferent initial sugar concentrations [2]

0e ability of Enterococcus faecalis SLT13 to conductlactose-to-lactic acid production is enhanced in hydrolyzed

Journal of Chemistry 3

whey0is suggests that the carbonnitrogen ratio in this kindof culture media is more favorable to lactic acid productionthan biomass growth0is fact is confirmed by the decrease ofbiomass yield on lactose (Yxs) which suggests that the carbonsource available in the medium is rather directed towardslactic acid production Values of Yxs are in the same range ofmagnitude than those reported for others species EnterococcusfaecalisCBRDO1 cultivated in LA5medium supplementedwithvarious concentration of glucose (5ndash20 gmiddotLminus 1) presentedYxs from 003 to 006 gg [31] Enterococcus faecalis RKY1grown on cane-sugar molasses had 007ndash019 gg [2] Ziadiet al [21] presented values varying between 018 and020 gg for two strains of Lactococcus lactis SLT6 andSLT10 cultured on hydrolyzed cheese whey

0e volumetric lactic acid productivities are higher inHCW and reach 133 gLmiddoth in 2 L bioreactor 0e ability ofE faecalis to produce L(+)- lactic acid from glucose orlactose by homolactic fermentation was earlier demon-strated in the literature Lactate is generated through py-ruvate reduction and two enzymes are involved in thisreaction the cytosolic NAD+ -dependent L-(+)-lactatedehydrogenases ldh-1 and ldh-2 [32]

Enterococcus faecalis is able to produce lactic acid fromdifferent substrates Oh et al [15] reported productivitieshigher than 08 gLmiddoth for E faecalis RKY1 using barley andwheat as nutrient source

While biomass is enhanced by scale-up lactic acidproduction is negatively affected although the scale-up ratio1 10 is respected 0is decrease can be explained by manyreasons During industrial fermentation scale-up causeschanges in the geometry and consequently hydrodynamicof the bioreactor especially for the STR Two importantchanges occurred and can affect microbial survival con-ducting to a decrease of productivities the mass transfer andthe shear stress 0e changed geometric conditions affectmixing behavior and consequently mass transfer whichlead to a less availability of nutriments for the cells 0eincreasing shear stress during scale-up affects intrinsicresistance of the lactic-acid-producer microorganism [33]

Indeed in mechanical agitated bioreactors and inde-pendently from microorganism type the stirrer is the maindispersing tool allowing contact between both phasesbiotic and abiotic of the system For an optimal processwith optimal kinetic parameters mass transfer betweencells and culture media is crucial 0us choosing the ap-propriate stirrer speed and design is essential [34] Severalstirrer designs are available the most used on microbialfermentation is the flat blade turbine type Rushton (con-taining 4 6 or 8 blades) assuring a radial flow 0is type of

turbine is adapted to the agitation of Newtonian fluids suchas culturemedia However shear stress caused by this type ofturbine is important particularly in the case of fragile mi-croorganisms Knowing that the impeller used in both 2 Land 20 L bioreactors consists of 2 and 3 turbine type Rushtonrespectively the decrease in lactic acid productivity duringscale-up can be attributed to the high shear stress caused bythis kind of turbine 0e effect of shear stress on LAB wasearlier demonstrated when studying the cell metabolism ofLactobacillus delbrueckii subsp bulgaricus cells cultivated at72 Pa were affected by shearing forces however whencultivated at 36 Pa metabolism was improved [35]

0us a good comprehension of kinetic behavior duringbioreactor scale-up of lactic acid production is of crucialimportance

32 Estimation of Parameters 0e experimental data ob-tained from batch fermentations of Enterococcus faecalis SLT13on M17 and Hydrolyzed Cheese Whey in 2L and 20L bio-reactors were used for developing the kinetic models and es-timation of kinetic parameters Results are presented in Table 2

Differences in the maximum specific growth rate (μmax)values obtained from the parameters estimation are observedHigher growth rate is achieved when the strain is cultivated inHCW in 20L bioreactor (109hminus 1) Similar values have beenreported for Enterococcus faecalis RKY1 grown on cane-sugarmolasses 16 hminus 1 [16] 0e low growth rate in M17 was foundpreviously for Enterococcus faecalis EF37 grown on the samemedium [20] Enterococcus faecalis CBRDO1 grown on LA5medium supplemented with various concentration of glucose(5ndash20 gmiddotLminus 1) showed μmax values of 059ndash064hminus 1 [31]

0e substrate limitation constants Ksx Kss and Ksp(Monod constants) for biomass production sugar utilizationand lactic acid production respectively are estimated Al-though no effects of substrate limitation are observed forfermentation in HCW in 2 L bioreactor substrate limitationof biomass is observed when Enterococcus faecalis SLT13 wascultured on HCW in 20 L bioreactor (the substrate limitationconstant for growth of biomass Ksx is 4229 gL) Substratelimitation of sugar consumption is observed when the strain iscultured on M17 in 2 L bioreactor (the substrate limitationconstant for sugar consumption Kss was 273 gL)

In this study substrate inhibition is not observed thekey constants for substrate inhibition (Kix Kis and Kip) arege100 gL (Table 2) Meanwhile initial substrate concentra-tion does not exceed 45 gL

0e product inhibition key constants (Kpx Kps and Kpp)are also estimated and listed in Table 2 Although no sub-strate inhibition is observed lactate inhibition of biomassgrowth occurs in HCW since the values of Kpx are 377 and500 gL for 2 L and 20 L bioreactors respectively 0e valuesof Kpx Kpp and Kps reported for Enterococcus faecalis RKY1cultured in molasses were 17074 and 291664 gL respec-tively [16] 0e inhibition by lactic acid can be caused byboth undissociated and dissociated forms Seen that lacticacid is completely dissociated at pHge 6 and the fermenta-tions occurs at pH 7 product inhibition is mainly due tocompletely dissociated form

Table 1 Main experimental data from the different batch culturesof Enterococcus faecalis SLT13

Parameters 2 L M17 2 L HW 20 L HWBiomass (gL) 316 257 267Lactic acid (LA) (gL) 604 3223 228Yps (gg) 0314 098 072Yxs (gg) 0142 0069 0074Volumetric productivityLA (gLmiddoth) 060 133 095

4 Journal of Chemistry

05

1

15

2

25

3

35

Biom

ass c

once

ntra

tion

(gL

)

0 1 32 54 76 98 10Time (h)

(a)

0

2

4

6

8

10

12

14

16

18

20

Glu

cose

conc

entr

atio

n (g

L)

1 2 3 4 5 6 7 8 9 100Time (h)

(b)

0

1

2

3

4

5

6

7

Lact

ic ac

id co

ncen

trat

ion

(gL

)

1 2 3 4 5 6 7 8 9 100Time (h)

(c)

Figure 1 Experimental data (points) and simulation (lines) of biomass (a) glucose (b) and lactic acid (c) concentrations of 2 L bioreactorbatch culture of Enterococcus faecalis SLT13 on M17 medium

Table 2 Best-fitting values of kinetic parameters obtained by modeling Enterococcus faecalis SLT13 growth sugar utilization and lactic acidproduction in M17 and HCW in 2 L and 20 L bioreactors

Kinetic parameters 2 L M17 2 L HCW 20 L HCWBiomass production modelμmax (hminus 1) 034 099 109Kix (gg) 11406 39975 39420Ksx (gg) 0023 00023 4229Kpx (gg) 1811 377 5001Kd (hminus 1) 0013 00001 00001Sugar utilization modelKis (gg) 29013 39999 143391Kss (gg) 273 001 015Kps (gg) 1044 1116 2007qsmax (ggmiddoth) 492 499 416Lactic acid production modelKip (gg) 13589 10000 37389Kpp (gg) 531 4499 4283Ksp (gg) 0025 001 0065qpmax (ggmiddoth) 1027 204 1863α (gg) 0052 001 0017R2 9434 8881 9787

Journal of Chemistry 5

For sugar consumption the higher maximum sugaruptake rate (qsmax) is estimated to be 499 g(gmiddoth) for HCWin 2 L bioreactor in spite of the values are close for the threefermentations 0e maximum specific lactic acid productionrate (qpmax) is higher for HCW and reach 204 g(gmiddoth) in 2 Lbioreactor

0e growth-associated term (α) is very low for all thefermentations In spite of that lactic acid production is bothgrowth-associated and non-growth-associated and seen thevalue of α the production of lactic acid is essentially oc-curred in the stationary phase Higher growth-associatedparameter (α) was reported by Nandasana and Kumar [16]for Enterococcus faecalis molasses and was 026 gg Lacto-coccus lactisNZ133 grown on lactose showed a highest value036 gg [36]

Evaluation of the cell death coefficient or endogenousdecay constant (Kd) of Enterococcus faecalis is conducted forthe three fermentations 0e estimated decay constants arevery low and equal to 00001 hminus 1 on HCW Neverthelessdecay constant is relatively important for the fermentation

in M17 and is equal to 0013 hminus 1 Death coefficient is straindependent and different Kd values were reported in theliterature Lactococcus lactis SLT6 and SLT10 grown in thesame media used in this study (HCW) presented values of0093ndash026 hminus 1respectively [21] Enterococcus faecalis RKY1grown on cane-sugar molasses had Kd value of 000318 hminus 1

[16] 0e lower Kd value for HCW can be ascribed to theprotective effect of cheese whey proteins Indeed wheyproteins are commonly used as protective media during thespray-drying and storage of lactic acid bacteria [37]

33 Comparison of Predicted and Experimental Parameters0e model used in this study and developed by Nandasanaand Kumar [16] reflects closely the kinetic behavior ofEnterococcus faecalis SLT13 0e comparisonof experimental data and predicted one is shown inFigures 1ndash3

0e value of correlation coefficient (R2) is presentedin Table 2 and is used to determine if the model fits well

0

05

1

15

2

25

3Bi

omas

s con

cent

ratio

n (g

L)

20 25151050Time (h)

(a)

0

5

10

15

20

25

30

35

40

45

50

Lact

ose c

once

ntra

tion

(gL

)

5 10 15 20 250Time (h)

(b)

0

10

20

30

40

50

60

Lact

ic ac

id co

ncen

trat

ion

(gL

)

5 10 15 20 250Time (h)

(c)

Figure 2 Experimental data (points) and simulation (lines) of biomass (a) lactose (b) and lactic acid (c) concentrations of 2 L bioreactorbatch culture of Enterococcus faecalis SLT13 on hydrolyzed cheese whey

6 Journal of Chemistry

the experimental data A good linearity between model andexperimental data is observed especially for the fermenta-tions occurring in 2 L M17 and 20 L HCW

For all the fermentations no lag phase is observedproving the ability of enterococci to adapt to differentgrowth conditions Exponential growth phase lasts 6 hwhen the strain was cultured in M17 followed by a shortstationary phase When cultured in HCW exponentialgrowth phase lasts only 5 h while the stationary phase isrelatively long Glucose is totally consumed after 6 h offermentation which confirmed that in M17 medium themain factor affecting sugar utilization is substrate limi-tation (the substrate limitation constant for sugar con-sumption Kss was 273 gL) In HCW while few quantitiesof lactose remain at the end of fermentation in 2 L bio-reactor it is almost totally consumed in 20 L bioreactorafter about 10 h of fermentation (14 gL) which confirmsthat the growth of biomass is limited by lactose con-centration since Ksx is 4229 gL Furthermore the initialconcentration of lactose is higher in 2 L bioreactor

Indeed in cheese whey powder concentration of lactosecan vary from batch to batch

In all fermentations lactate production began earlierin the exponential growth phase and continues during thestationary phase Indeed lactic acid production is growth-associated as well as non-growth-associated Lactic acidproduction remains constant after 7 h and 10h of fermentationin M17 and in HCW in 20 L bioreactor respectivelyHowever on HCW in 2 L bioreactor lactate concen-tration continues to increase with the availability of thesubstrate

4 Conclusions

0e overall results obtained in our study show thatcheese whey is suitable for lactic acid production vol-umetric productivities are higher than the one obtainedin M17 0e mathematical modeling is useful and allowscharacterizing growth and lactic acid production ofEnterococcus faecalis SLT13 in two different media and

0

05

1

15

2

25

3Bi

omas

s con

cent

ratio

n (g

L)

5 10 15 20 250Time (h)

(a)

ndash5

0

5

10

15

20

25

30

35

Lact

ose c

once

ntra

tion

(gL

)

5 10 15 20 250Time (h)

(b)

0

5

10

15

20

25

Lact

ic ac

id co

ncen

trat

ion

(gL

)

5 10 15 20 250Time (h)

(c)

Figure 3 Experimental data (points) and simulation (lines) of biomass (a) lactose (b) and lactic acid (c) concentrations of 20 L bioreactorbatch culture of Enterococcus faecalis SLT13 on hydrolyzed cheese whey

Journal of Chemistry 7

two different volume bioreactors 0e scale-up affectslactic acid productivity but enhances biomass growthSubstrate inhibition did not impact significantly themaximum lactic acid productivities attainable in theboth 2 L and 20 L bioreactors However product inhi-bition seems to play a major role in the two studiedmedia

Abbreviations

Kd Death coefficient (hminus 1)Kip Substrate inhibition constant for lactic acid

production (gL)Kis Substrate inhibition constant for sugar consumption

(gL)Kix Substrate inhibition constant for growth of biomass

(gL)Kpp Product inhibition constant for lactic acid

production (gL)Kps Product inhibition constant for sugar consumption

(gL)Kpx Product inhibition constant for growth of biomass

(gL)Ksp Substrate limitation constant for lactic acid

production (gL)Kss Substrate limitation constant for sugar consumption

(gL)Ksx Substrate limitation constant for growth of biomass

(gL)P Lactic acid concentration (gL)qpmax Maximum specific lactic acid production rate (g(g h))qsmax Maximum specific sugar utilization rate (g(g h))S Sugar concentration (gL)X Biomass concentration (gL)Ypx Lactic acid yield on growth of biomass (gg)Yps Lactic acid yield on sugar consumption (gg)Yxs Biomass yield on sugar consumption (gg)

Greek Symbols

α Growth-associated constant in LuedekingndashPiretmodel (gg)

μmax Maximum specific growth rate (hminus 1) of lactic acidμ Specific growth rate (hminus 1)

Data Availability

All the numerical data used to support the findings of thisstudy are available from the corresponding author uponreasonable request

Additional Points

Kinetic modeling of lactic acid and biomass productionby Enterococcus faecalis SLT13 was realized on syntheticmedia and Hydrolyzed Cheese Whey Hydrolyzed CheeseWhey enhances lactic acid production Scale-up from 2 Lbioreactor to 20 L bioreactor increases biomass but notlactic acid production Biomass growth and sugar

utilization are affected by lactic acid inhibition in Hy-drolyzed Cheese Whey

Conflicts of Interest

0e authors declare no financial or commercial conflicts ofinterest regarding the publication of this paper

Acknowledgments

0is work was supported by the Ministry of Higher Educationand Scientific Research (Tunisia) and the Walloon Region(Belgium) during the project Technological Platform for theDevelopment of Bioindustries

Supplementary Materials

Graphical abstract representing lactic acid production by En-terococcus faecalis SLT13 during batch culture on HydrolyzedCheese Whey in 2 L and 20 L bioreactors (SupplementaryMaterials)

References

[1] Markets and Markets ldquoLactic acid market by application(biodegradable polymer food amp beverage personal care amppharmaceutical) amp polylactic acid market by application(packaging agriculture automobile electronics textile) amp bygeography-global trends amp forecasts to 2020rdquo Report CodeFB 2647 Markets and Markets Hadapsar Pune 2015

[2] B Gupta N Revagade and J Hilborn ldquoPoly(lactic acid) fiberan overviewrdquo Progress in Polymer Science vol 32 no 4pp 455ndash482 2007

[3] N Narayanan P K Roychoudhury and A Srivastava ldquoL(+)lactic acid fermentation and its product polymerizationrdquoElectronic Journal of Biotechnology vol 7 no 2 2004

[4] R Mehta V Kumar H Bhunia and S N UpadhyayldquoSynthesis of poly(lactic acid) a reviewrdquo Journal of Macro-molecular Science Part C Polymer Reviews vol 45 no 4pp 325ndash349 2005

[5] M A Randhawa A Ahmed and K Akram ldquoOptimization oflactic acid production from cheap raw material sugarcanemolassesrdquo Pakistan Journal of Botany vol 44 pp 333ndash3382012

[6] A-N Vamvakaki I Kandarakis S KaminaridesM Komaitis and S Papanikolaou ldquoCheese whey as a re-newable substrate for microbial lipid and biomass productionby zygomycetesrdquo Engineering in Life Sciences vol 10 no 4pp 348ndash360 2010

[7] M I G Gonzalez ldquo0e biotechnological utilization of cheesewhey a reviewrdquo Bioresource Technology vol 57 pp 1ndash111996

[8] Y H Park K M Cho H W Kim and C Kim ldquoMethod forproducing lactic acid with high concentration and high yieldusing lactic acid bacteriardquo CJ CheilJedang Corporation SeoulRepublic of Korea US Patent 7682814 B2 2010

[9] T Mimitsuka K Sawai K Kobayashi et al ldquoProduction ofd-lactic acid in a continuous membrane integrated fermen-tation reactor by genetically modified Saccharomyces cer-evisiae enhancement in d-lactic acid carbon yieldrdquo Journal ofBioscience and Bioengineering vol 119 no 1 pp 65ndash71 2015

[10] V Kitpreechavanich T Maneeboon Y Kayano and K SakaildquoComparative characterization of l-lactic acid-producing

8 Journal of Chemistry

22 Biomass Determination For biomass determination acalibration curve relating dry cell weight with the cell con-centration (estimated by viable cells counting method) wasused For cells counting 01mL of diluted sample was spreadover M17-agar surface 0e plates were then incubated for onenight at 30degC 0e number of colonies was counted andexpressed in CFUmL (Colony-Forming UnitsmL)

23 Analytical Analysis Glucose lactose and lactic acidconcentrations in the fermentation broth were measured byHPLC (Agilent Technologies) using an ion-exclusion column(SupelCo Gel C-610H) operated at 35degC Components wereelutedwith 01 phosphoric acid at a flow rate of 05mLmiddotminminus 1Detection was accomplished by a refractive index detector

24 Kinetic Model

241 Model Development 0emodel used in this work is theone developed by Nandasana and Kumar [16] for lactic acidproduction by Enterococcus faecalis RKY1 and is as follows

Specific growth rate

μ μmaxSKiX

KSX + S( 1113857 KiX + S( 1113857e

minus PKpX( 1113857 (1)

0e biomass production ratedX

dt μ minus K d( 1113857X (2)

0e substrate consumption ratedS

dt minus qsmax

SKiS

KSS + S( 1113857 KiS + S( 1113857e

minus PKpS( 1113857X (3)

0e product production ratedP

dt α

dX

dt+ qpmax

SKiP

KSP + S( 1113857 KiP + S( 1113857e

minus PKpp( 1113857X (4)

For (3) the usual assumption proposed by Nandasanaand Kumar [16] was not used due to the nature of theprocess a minus sign was included According to the sameauthors these assumptions are taken in consideration for thesimplification of the modeling step

(i) Kss Ksp low lactose concentration affects in thesame way lactose uptake and lactic acid production

(ii) Kis Kip high lactose concentration inhibits in thesame way lactose uptake and lactic acid production

(iii) Kps Kpp lactate concentration affects in the sameway lactose uptake and lactic acid production

0e method used to solve model differential equationsfor a given set of kinetic parameters was ODE113s solver ofMATLAB [28] in order to obtain X S and P values 0einitial conditions were the experimental values of X S and P

at time zero (t t0)

242 Model Integration In order to estimate kinetic pa-rameters a search of these parameters values for which

predicted values of X S and P are closer to the experimentalone needs to be applied For this process an optimizationmethod was used 0e nonlinear optimization method chosenfor this problemwas the interior-point approach to constrainedminimization using the ldquofminconrdquo function in MATLAB[29 30] 0is function is a local search algorithm that startswith an initial guess and finds a constrained minimum

0e objective function (OF) used in this minimizationwas in the form of

OFX 1113936

ni1 Xi minus XiEXP1113872 1113873

2

1113936ni1 Xi minus Xi( 1113857

2 middot 100

OFS 1113936

ni1 Si minus SiEXP1113872 1113873

2

1113936ni1 Si minus Si( 1113857

2 middot 100

OFP 1113936

ni1 Pi minus PiEXP1113872 1113873

2

1113936ni1 Pi minus Pi( 1113857

2 middot 100

OF OFX + OFS + OFP

3

(5)

0us it was considered in the estimation of parame-ters that all variables are equally important and given equalweight to the approximation error for each one of them0e procedure to find the parameters was repeated at least30 times with random initial guesses in order to assure agood solution

3 Results and Discussion

31 Effect of Culture Media and Scale-Up on Lactic Acid andBiomass Productivity 0is study is conducted to evaluate theeffect of scale-up on growth and lactic acid production byEnterococcus faecalis SLT13 0e fermentation was first carriedout at laboratory scale in 2L bioreactor than at semipilot scalein 20L bioreactor All the studied fermentations were con-ducted in Stirred Tank Reactor (STR) Moreover two distinctculture media were analyzed including the synthetic mediumM17 and the papaın Hydrolyzed Cheese Whey (HCW) inorder to characterize the growth kinetics of Enterococcusfaecalis SLT13 2 L bioreactor cultures were conducted in M17and HCW under controlled conditions while only HCW wasused for 20L bioreactor the same parameters (pH tempera-ture and stirrer) were maintained

0e key kinetic parameters during the culture of E faecalisin the conditions mentioned below are estimated experi-mentally and summarized in Table 1

Whereas the final biomass is higher in synthetic media(M17) lactic acid concentration is more important whenE faecalis was grown on HCW Lactic acid yield on lactose(Yps) increases significantly in HCW (from 0314 to 098 gg)Lactic acid yield obtained for the strain E faecalis RKY1based on sugar consumption varies from 090 to 099 fordifferent initial sugar concentrations [2]

0e ability of Enterococcus faecalis SLT13 to conductlactose-to-lactic acid production is enhanced in hydrolyzed

Journal of Chemistry 3

whey0is suggests that the carbonnitrogen ratio in this kindof culture media is more favorable to lactic acid productionthan biomass growth0is fact is confirmed by the decrease ofbiomass yield on lactose (Yxs) which suggests that the carbonsource available in the medium is rather directed towardslactic acid production Values of Yxs are in the same range ofmagnitude than those reported for others species EnterococcusfaecalisCBRDO1 cultivated in LA5medium supplementedwithvarious concentration of glucose (5ndash20 gmiddotLminus 1) presentedYxs from 003 to 006 gg [31] Enterococcus faecalis RKY1grown on cane-sugar molasses had 007ndash019 gg [2] Ziadiet al [21] presented values varying between 018 and020 gg for two strains of Lactococcus lactis SLT6 andSLT10 cultured on hydrolyzed cheese whey

0e volumetric lactic acid productivities are higher inHCW and reach 133 gLmiddoth in 2 L bioreactor 0e ability ofE faecalis to produce L(+)- lactic acid from glucose orlactose by homolactic fermentation was earlier demon-strated in the literature Lactate is generated through py-ruvate reduction and two enzymes are involved in thisreaction the cytosolic NAD+ -dependent L-(+)-lactatedehydrogenases ldh-1 and ldh-2 [32]

Enterococcus faecalis is able to produce lactic acid fromdifferent substrates Oh et al [15] reported productivitieshigher than 08 gLmiddoth for E faecalis RKY1 using barley andwheat as nutrient source

While biomass is enhanced by scale-up lactic acidproduction is negatively affected although the scale-up ratio1 10 is respected 0is decrease can be explained by manyreasons During industrial fermentation scale-up causeschanges in the geometry and consequently hydrodynamicof the bioreactor especially for the STR Two importantchanges occurred and can affect microbial survival con-ducting to a decrease of productivities the mass transfer andthe shear stress 0e changed geometric conditions affectmixing behavior and consequently mass transfer whichlead to a less availability of nutriments for the cells 0eincreasing shear stress during scale-up affects intrinsicresistance of the lactic-acid-producer microorganism [33]

Indeed in mechanical agitated bioreactors and inde-pendently from microorganism type the stirrer is the maindispersing tool allowing contact between both phasesbiotic and abiotic of the system For an optimal processwith optimal kinetic parameters mass transfer betweencells and culture media is crucial 0us choosing the ap-propriate stirrer speed and design is essential [34] Severalstirrer designs are available the most used on microbialfermentation is the flat blade turbine type Rushton (con-taining 4 6 or 8 blades) assuring a radial flow 0is type of

turbine is adapted to the agitation of Newtonian fluids suchas culturemedia However shear stress caused by this type ofturbine is important particularly in the case of fragile mi-croorganisms Knowing that the impeller used in both 2 Land 20 L bioreactors consists of 2 and 3 turbine type Rushtonrespectively the decrease in lactic acid productivity duringscale-up can be attributed to the high shear stress caused bythis kind of turbine 0e effect of shear stress on LAB wasearlier demonstrated when studying the cell metabolism ofLactobacillus delbrueckii subsp bulgaricus cells cultivated at72 Pa were affected by shearing forces however whencultivated at 36 Pa metabolism was improved [35]

0us a good comprehension of kinetic behavior duringbioreactor scale-up of lactic acid production is of crucialimportance

32 Estimation of Parameters 0e experimental data ob-tained from batch fermentations of Enterococcus faecalis SLT13on M17 and Hydrolyzed Cheese Whey in 2L and 20L bio-reactors were used for developing the kinetic models and es-timation of kinetic parameters Results are presented in Table 2

Differences in the maximum specific growth rate (μmax)values obtained from the parameters estimation are observedHigher growth rate is achieved when the strain is cultivated inHCW in 20L bioreactor (109hminus 1) Similar values have beenreported for Enterococcus faecalis RKY1 grown on cane-sugarmolasses 16 hminus 1 [16] 0e low growth rate in M17 was foundpreviously for Enterococcus faecalis EF37 grown on the samemedium [20] Enterococcus faecalis CBRDO1 grown on LA5medium supplemented with various concentration of glucose(5ndash20 gmiddotLminus 1) showed μmax values of 059ndash064hminus 1 [31]

0e substrate limitation constants Ksx Kss and Ksp(Monod constants) for biomass production sugar utilizationand lactic acid production respectively are estimated Al-though no effects of substrate limitation are observed forfermentation in HCW in 2 L bioreactor substrate limitationof biomass is observed when Enterococcus faecalis SLT13 wascultured on HCW in 20 L bioreactor (the substrate limitationconstant for growth of biomass Ksx is 4229 gL) Substratelimitation of sugar consumption is observed when the strain iscultured on M17 in 2 L bioreactor (the substrate limitationconstant for sugar consumption Kss was 273 gL)

In this study substrate inhibition is not observed thekey constants for substrate inhibition (Kix Kis and Kip) arege100 gL (Table 2) Meanwhile initial substrate concentra-tion does not exceed 45 gL

0e product inhibition key constants (Kpx Kps and Kpp)are also estimated and listed in Table 2 Although no sub-strate inhibition is observed lactate inhibition of biomassgrowth occurs in HCW since the values of Kpx are 377 and500 gL for 2 L and 20 L bioreactors respectively 0e valuesof Kpx Kpp and Kps reported for Enterococcus faecalis RKY1cultured in molasses were 17074 and 291664 gL respec-tively [16] 0e inhibition by lactic acid can be caused byboth undissociated and dissociated forms Seen that lacticacid is completely dissociated at pHge 6 and the fermenta-tions occurs at pH 7 product inhibition is mainly due tocompletely dissociated form

Table 1 Main experimental data from the different batch culturesof Enterococcus faecalis SLT13

Parameters 2 L M17 2 L HW 20 L HWBiomass (gL) 316 257 267Lactic acid (LA) (gL) 604 3223 228Yps (gg) 0314 098 072Yxs (gg) 0142 0069 0074Volumetric productivityLA (gLmiddoth) 060 133 095

4 Journal of Chemistry

05

1

15

2

25

3

35

Biom

ass c

once

ntra

tion

(gL

)

0 1 32 54 76 98 10Time (h)

(a)

0

2

4

6

8

10

12

14

16

18

20

Glu

cose

conc

entr

atio

n (g

L)

1 2 3 4 5 6 7 8 9 100Time (h)

(b)

0

1

2

3

4

5

6

7

Lact

ic ac

id co

ncen

trat

ion

(gL

)

1 2 3 4 5 6 7 8 9 100Time (h)

(c)

Figure 1 Experimental data (points) and simulation (lines) of biomass (a) glucose (b) and lactic acid (c) concentrations of 2 L bioreactorbatch culture of Enterococcus faecalis SLT13 on M17 medium

Table 2 Best-fitting values of kinetic parameters obtained by modeling Enterococcus faecalis SLT13 growth sugar utilization and lactic acidproduction in M17 and HCW in 2 L and 20 L bioreactors

Kinetic parameters 2 L M17 2 L HCW 20 L HCWBiomass production modelμmax (hminus 1) 034 099 109Kix (gg) 11406 39975 39420Ksx (gg) 0023 00023 4229Kpx (gg) 1811 377 5001Kd (hminus 1) 0013 00001 00001Sugar utilization modelKis (gg) 29013 39999 143391Kss (gg) 273 001 015Kps (gg) 1044 1116 2007qsmax (ggmiddoth) 492 499 416Lactic acid production modelKip (gg) 13589 10000 37389Kpp (gg) 531 4499 4283Ksp (gg) 0025 001 0065qpmax (ggmiddoth) 1027 204 1863α (gg) 0052 001 0017R2 9434 8881 9787

Journal of Chemistry 5

For sugar consumption the higher maximum sugaruptake rate (qsmax) is estimated to be 499 g(gmiddoth) for HCWin 2 L bioreactor in spite of the values are close for the threefermentations 0e maximum specific lactic acid productionrate (qpmax) is higher for HCW and reach 204 g(gmiddoth) in 2 Lbioreactor

0e growth-associated term (α) is very low for all thefermentations In spite of that lactic acid production is bothgrowth-associated and non-growth-associated and seen thevalue of α the production of lactic acid is essentially oc-curred in the stationary phase Higher growth-associatedparameter (α) was reported by Nandasana and Kumar [16]for Enterococcus faecalis molasses and was 026 gg Lacto-coccus lactisNZ133 grown on lactose showed a highest value036 gg [36]

Evaluation of the cell death coefficient or endogenousdecay constant (Kd) of Enterococcus faecalis is conducted forthe three fermentations 0e estimated decay constants arevery low and equal to 00001 hminus 1 on HCW Neverthelessdecay constant is relatively important for the fermentation

in M17 and is equal to 0013 hminus 1 Death coefficient is straindependent and different Kd values were reported in theliterature Lactococcus lactis SLT6 and SLT10 grown in thesame media used in this study (HCW) presented values of0093ndash026 hminus 1respectively [21] Enterococcus faecalis RKY1grown on cane-sugar molasses had Kd value of 000318 hminus 1

[16] 0e lower Kd value for HCW can be ascribed to theprotective effect of cheese whey proteins Indeed wheyproteins are commonly used as protective media during thespray-drying and storage of lactic acid bacteria [37]

33 Comparison of Predicted and Experimental Parameters0e model used in this study and developed by Nandasanaand Kumar [16] reflects closely the kinetic behavior ofEnterococcus faecalis SLT13 0e comparisonof experimental data and predicted one is shown inFigures 1ndash3

0e value of correlation coefficient (R2) is presentedin Table 2 and is used to determine if the model fits well

0

05

1

15

2

25

3Bi

omas

s con

cent

ratio

n (g

L)

20 25151050Time (h)

(a)

0

5

10

15

20

25

30

35

40

45

50

Lact

ose c

once

ntra

tion

(gL

)

5 10 15 20 250Time (h)

(b)

0

10

20

30

40

50

60

Lact

ic ac

id co

ncen

trat

ion

(gL

)

5 10 15 20 250Time (h)

(c)

Figure 2 Experimental data (points) and simulation (lines) of biomass (a) lactose (b) and lactic acid (c) concentrations of 2 L bioreactorbatch culture of Enterococcus faecalis SLT13 on hydrolyzed cheese whey

6 Journal of Chemistry

the experimental data A good linearity between model andexperimental data is observed especially for the fermenta-tions occurring in 2 L M17 and 20 L HCW

For all the fermentations no lag phase is observedproving the ability of enterococci to adapt to differentgrowth conditions Exponential growth phase lasts 6 hwhen the strain was cultured in M17 followed by a shortstationary phase When cultured in HCW exponentialgrowth phase lasts only 5 h while the stationary phase isrelatively long Glucose is totally consumed after 6 h offermentation which confirmed that in M17 medium themain factor affecting sugar utilization is substrate limi-tation (the substrate limitation constant for sugar con-sumption Kss was 273 gL) In HCW while few quantitiesof lactose remain at the end of fermentation in 2 L bio-reactor it is almost totally consumed in 20 L bioreactorafter about 10 h of fermentation (14 gL) which confirmsthat the growth of biomass is limited by lactose con-centration since Ksx is 4229 gL Furthermore the initialconcentration of lactose is higher in 2 L bioreactor

Indeed in cheese whey powder concentration of lactosecan vary from batch to batch

In all fermentations lactate production began earlierin the exponential growth phase and continues during thestationary phase Indeed lactic acid production is growth-associated as well as non-growth-associated Lactic acidproduction remains constant after 7 h and 10h of fermentationin M17 and in HCW in 20 L bioreactor respectivelyHowever on HCW in 2 L bioreactor lactate concen-tration continues to increase with the availability of thesubstrate

4 Conclusions

0e overall results obtained in our study show thatcheese whey is suitable for lactic acid production vol-umetric productivities are higher than the one obtainedin M17 0e mathematical modeling is useful and allowscharacterizing growth and lactic acid production ofEnterococcus faecalis SLT13 in two different media and

0

05

1

15

2

25

3Bi

omas

s con

cent

ratio

n (g

L)

5 10 15 20 250Time (h)

(a)

ndash5

0

5

10

15

20

25

30

35

Lact

ose c

once

ntra

tion

(gL

)

5 10 15 20 250Time (h)

(b)

0

5

10

15

20

25

Lact

ic ac

id co

ncen

trat

ion

(gL

)

5 10 15 20 250Time (h)

(c)

Figure 3 Experimental data (points) and simulation (lines) of biomass (a) lactose (b) and lactic acid (c) concentrations of 20 L bioreactorbatch culture of Enterococcus faecalis SLT13 on hydrolyzed cheese whey

Journal of Chemistry 7

two different volume bioreactors 0e scale-up affectslactic acid productivity but enhances biomass growthSubstrate inhibition did not impact significantly themaximum lactic acid productivities attainable in theboth 2 L and 20 L bioreactors However product inhi-bition seems to play a major role in the two studiedmedia

Abbreviations

Kd Death coefficient (hminus 1)Kip Substrate inhibition constant for lactic acid

production (gL)Kis Substrate inhibition constant for sugar consumption

(gL)Kix Substrate inhibition constant for growth of biomass

(gL)Kpp Product inhibition constant for lactic acid

production (gL)Kps Product inhibition constant for sugar consumption

(gL)Kpx Product inhibition constant for growth of biomass

(gL)Ksp Substrate limitation constant for lactic acid

production (gL)Kss Substrate limitation constant for sugar consumption

(gL)Ksx Substrate limitation constant for growth of biomass

(gL)P Lactic acid concentration (gL)qpmax Maximum specific lactic acid production rate (g(g h))qsmax Maximum specific sugar utilization rate (g(g h))S Sugar concentration (gL)X Biomass concentration (gL)Ypx Lactic acid yield on growth of biomass (gg)Yps Lactic acid yield on sugar consumption (gg)Yxs Biomass yield on sugar consumption (gg)

Greek Symbols

α Growth-associated constant in LuedekingndashPiretmodel (gg)

μmax Maximum specific growth rate (hminus 1) of lactic acidμ Specific growth rate (hminus 1)

Data Availability

All the numerical data used to support the findings of thisstudy are available from the corresponding author uponreasonable request

Additional Points

Kinetic modeling of lactic acid and biomass productionby Enterococcus faecalis SLT13 was realized on syntheticmedia and Hydrolyzed Cheese Whey Hydrolyzed CheeseWhey enhances lactic acid production Scale-up from 2 Lbioreactor to 20 L bioreactor increases biomass but notlactic acid production Biomass growth and sugar

utilization are affected by lactic acid inhibition in Hy-drolyzed Cheese Whey

Conflicts of Interest

0e authors declare no financial or commercial conflicts ofinterest regarding the publication of this paper

Acknowledgments

0is work was supported by the Ministry of Higher Educationand Scientific Research (Tunisia) and the Walloon Region(Belgium) during the project Technological Platform for theDevelopment of Bioindustries

Supplementary Materials

Graphical abstract representing lactic acid production by En-terococcus faecalis SLT13 during batch culture on HydrolyzedCheese Whey in 2 L and 20 L bioreactors (SupplementaryMaterials)

References

[1] Markets and Markets ldquoLactic acid market by application(biodegradable polymer food amp beverage personal care amppharmaceutical) amp polylactic acid market by application(packaging agriculture automobile electronics textile) amp bygeography-global trends amp forecasts to 2020rdquo Report CodeFB 2647 Markets and Markets Hadapsar Pune 2015

[2] B Gupta N Revagade and J Hilborn ldquoPoly(lactic acid) fiberan overviewrdquo Progress in Polymer Science vol 32 no 4pp 455ndash482 2007

[3] N Narayanan P K Roychoudhury and A Srivastava ldquoL(+)lactic acid fermentation and its product polymerizationrdquoElectronic Journal of Biotechnology vol 7 no 2 2004

[4] R Mehta V Kumar H Bhunia and S N UpadhyayldquoSynthesis of poly(lactic acid) a reviewrdquo Journal of Macro-molecular Science Part C Polymer Reviews vol 45 no 4pp 325ndash349 2005

[5] M A Randhawa A Ahmed and K Akram ldquoOptimization oflactic acid production from cheap raw material sugarcanemolassesrdquo Pakistan Journal of Botany vol 44 pp 333ndash3382012

[6] A-N Vamvakaki I Kandarakis S KaminaridesM Komaitis and S Papanikolaou ldquoCheese whey as a re-newable substrate for microbial lipid and biomass productionby zygomycetesrdquo Engineering in Life Sciences vol 10 no 4pp 348ndash360 2010

[7] M I G Gonzalez ldquo0e biotechnological utilization of cheesewhey a reviewrdquo Bioresource Technology vol 57 pp 1ndash111996

[8] Y H Park K M Cho H W Kim and C Kim ldquoMethod forproducing lactic acid with high concentration and high yieldusing lactic acid bacteriardquo CJ CheilJedang Corporation SeoulRepublic of Korea US Patent 7682814 B2 2010

[9] T Mimitsuka K Sawai K Kobayashi et al ldquoProduction ofd-lactic acid in a continuous membrane integrated fermen-tation reactor by genetically modified Saccharomyces cer-evisiae enhancement in d-lactic acid carbon yieldrdquo Journal ofBioscience and Bioengineering vol 119 no 1 pp 65ndash71 2015

[10] V Kitpreechavanich T Maneeboon Y Kayano and K SakaildquoComparative characterization of l-lactic acid-producing

8 Journal of Chemistry

whey0is suggests that the carbonnitrogen ratio in this kindof culture media is more favorable to lactic acid productionthan biomass growth0is fact is confirmed by the decrease ofbiomass yield on lactose (Yxs) which suggests that the carbonsource available in the medium is rather directed towardslactic acid production Values of Yxs are in the same range ofmagnitude than those reported for others species EnterococcusfaecalisCBRDO1 cultivated in LA5medium supplementedwithvarious concentration of glucose (5ndash20 gmiddotLminus 1) presentedYxs from 003 to 006 gg [31] Enterococcus faecalis RKY1grown on cane-sugar molasses had 007ndash019 gg [2] Ziadiet al [21] presented values varying between 018 and020 gg for two strains of Lactococcus lactis SLT6 andSLT10 cultured on hydrolyzed cheese whey

0e volumetric lactic acid productivities are higher inHCW and reach 133 gLmiddoth in 2 L bioreactor 0e ability ofE faecalis to produce L(+)- lactic acid from glucose orlactose by homolactic fermentation was earlier demon-strated in the literature Lactate is generated through py-ruvate reduction and two enzymes are involved in thisreaction the cytosolic NAD+ -dependent L-(+)-lactatedehydrogenases ldh-1 and ldh-2 [32]

Enterococcus faecalis is able to produce lactic acid fromdifferent substrates Oh et al [15] reported productivitieshigher than 08 gLmiddoth for E faecalis RKY1 using barley andwheat as nutrient source

While biomass is enhanced by scale-up lactic acidproduction is negatively affected although the scale-up ratio1 10 is respected 0is decrease can be explained by manyreasons During industrial fermentation scale-up causeschanges in the geometry and consequently hydrodynamicof the bioreactor especially for the STR Two importantchanges occurred and can affect microbial survival con-ducting to a decrease of productivities the mass transfer andthe shear stress 0e changed geometric conditions affectmixing behavior and consequently mass transfer whichlead to a less availability of nutriments for the cells 0eincreasing shear stress during scale-up affects intrinsicresistance of the lactic-acid-producer microorganism [33]

Indeed in mechanical agitated bioreactors and inde-pendently from microorganism type the stirrer is the maindispersing tool allowing contact between both phasesbiotic and abiotic of the system For an optimal processwith optimal kinetic parameters mass transfer betweencells and culture media is crucial 0us choosing the ap-propriate stirrer speed and design is essential [34] Severalstirrer designs are available the most used on microbialfermentation is the flat blade turbine type Rushton (con-taining 4 6 or 8 blades) assuring a radial flow 0is type of

turbine is adapted to the agitation of Newtonian fluids suchas culturemedia However shear stress caused by this type ofturbine is important particularly in the case of fragile mi-croorganisms Knowing that the impeller used in both 2 Land 20 L bioreactors consists of 2 and 3 turbine type Rushtonrespectively the decrease in lactic acid productivity duringscale-up can be attributed to the high shear stress caused bythis kind of turbine 0e effect of shear stress on LAB wasearlier demonstrated when studying the cell metabolism ofLactobacillus delbrueckii subsp bulgaricus cells cultivated at72 Pa were affected by shearing forces however whencultivated at 36 Pa metabolism was improved [35]

0us a good comprehension of kinetic behavior duringbioreactor scale-up of lactic acid production is of crucialimportance

32 Estimation of Parameters 0e experimental data ob-tained from batch fermentations of Enterococcus faecalis SLT13on M17 and Hydrolyzed Cheese Whey in 2L and 20L bio-reactors were used for developing the kinetic models and es-timation of kinetic parameters Results are presented in Table 2

Differences in the maximum specific growth rate (μmax)values obtained from the parameters estimation are observedHigher growth rate is achieved when the strain is cultivated inHCW in 20L bioreactor (109hminus 1) Similar values have beenreported for Enterococcus faecalis RKY1 grown on cane-sugarmolasses 16 hminus 1 [16] 0e low growth rate in M17 was foundpreviously for Enterococcus faecalis EF37 grown on the samemedium [20] Enterococcus faecalis CBRDO1 grown on LA5medium supplemented with various concentration of glucose(5ndash20 gmiddotLminus 1) showed μmax values of 059ndash064hminus 1 [31]

0e substrate limitation constants Ksx Kss and Ksp(Monod constants) for biomass production sugar utilizationand lactic acid production respectively are estimated Al-though no effects of substrate limitation are observed forfermentation in HCW in 2 L bioreactor substrate limitationof biomass is observed when Enterococcus faecalis SLT13 wascultured on HCW in 20 L bioreactor (the substrate limitationconstant for growth of biomass Ksx is 4229 gL) Substratelimitation of sugar consumption is observed when the strain iscultured on M17 in 2 L bioreactor (the substrate limitationconstant for sugar consumption Kss was 273 gL)

In this study substrate inhibition is not observed thekey constants for substrate inhibition (Kix Kis and Kip) arege100 gL (Table 2) Meanwhile initial substrate concentra-tion does not exceed 45 gL

0e product inhibition key constants (Kpx Kps and Kpp)are also estimated and listed in Table 2 Although no sub-strate inhibition is observed lactate inhibition of biomassgrowth occurs in HCW since the values of Kpx are 377 and500 gL for 2 L and 20 L bioreactors respectively 0e valuesof Kpx Kpp and Kps reported for Enterococcus faecalis RKY1cultured in molasses were 17074 and 291664 gL respec-tively [16] 0e inhibition by lactic acid can be caused byboth undissociated and dissociated forms Seen that lacticacid is completely dissociated at pHge 6 and the fermenta-tions occurs at pH 7 product inhibition is mainly due tocompletely dissociated form

Table 1 Main experimental data from the different batch culturesof Enterococcus faecalis SLT13

Parameters 2 L M17 2 L HW 20 L HWBiomass (gL) 316 257 267Lactic acid (LA) (gL) 604 3223 228Yps (gg) 0314 098 072Yxs (gg) 0142 0069 0074Volumetric productivityLA (gLmiddoth) 060 133 095

4 Journal of Chemistry

05

1

15

2

25

3

35

Biom

ass c

once

ntra

tion

(gL

)

0 1 32 54 76 98 10Time (h)

(a)

0

2

4

6

8

10

12

14

16

18

20

Glu

cose

conc

entr

atio

n (g

L)

1 2 3 4 5 6 7 8 9 100Time (h)

(b)

0

1

2

3

4

5

6

7

Lact

ic ac

id co

ncen

trat

ion

(gL

)

1 2 3 4 5 6 7 8 9 100Time (h)

(c)

Figure 1 Experimental data (points) and simulation (lines) of biomass (a) glucose (b) and lactic acid (c) concentrations of 2 L bioreactorbatch culture of Enterococcus faecalis SLT13 on M17 medium

Table 2 Best-fitting values of kinetic parameters obtained by modeling Enterococcus faecalis SLT13 growth sugar utilization and lactic acidproduction in M17 and HCW in 2 L and 20 L bioreactors

Kinetic parameters 2 L M17 2 L HCW 20 L HCWBiomass production modelμmax (hminus 1) 034 099 109Kix (gg) 11406 39975 39420Ksx (gg) 0023 00023 4229Kpx (gg) 1811 377 5001Kd (hminus 1) 0013 00001 00001Sugar utilization modelKis (gg) 29013 39999 143391Kss (gg) 273 001 015Kps (gg) 1044 1116 2007qsmax (ggmiddoth) 492 499 416Lactic acid production modelKip (gg) 13589 10000 37389Kpp (gg) 531 4499 4283Ksp (gg) 0025 001 0065qpmax (ggmiddoth) 1027 204 1863α (gg) 0052 001 0017R2 9434 8881 9787

Journal of Chemistry 5

For sugar consumption the higher maximum sugaruptake rate (qsmax) is estimated to be 499 g(gmiddoth) for HCWin 2 L bioreactor in spite of the values are close for the threefermentations 0e maximum specific lactic acid productionrate (qpmax) is higher for HCW and reach 204 g(gmiddoth) in 2 Lbioreactor

0e growth-associated term (α) is very low for all thefermentations In spite of that lactic acid production is bothgrowth-associated and non-growth-associated and seen thevalue of α the production of lactic acid is essentially oc-curred in the stationary phase Higher growth-associatedparameter (α) was reported by Nandasana and Kumar [16]for Enterococcus faecalis molasses and was 026 gg Lacto-coccus lactisNZ133 grown on lactose showed a highest value036 gg [36]

Evaluation of the cell death coefficient or endogenousdecay constant (Kd) of Enterococcus faecalis is conducted forthe three fermentations 0e estimated decay constants arevery low and equal to 00001 hminus 1 on HCW Neverthelessdecay constant is relatively important for the fermentation

in M17 and is equal to 0013 hminus 1 Death coefficient is straindependent and different Kd values were reported in theliterature Lactococcus lactis SLT6 and SLT10 grown in thesame media used in this study (HCW) presented values of0093ndash026 hminus 1respectively [21] Enterococcus faecalis RKY1grown on cane-sugar molasses had Kd value of 000318 hminus 1

[16] 0e lower Kd value for HCW can be ascribed to theprotective effect of cheese whey proteins Indeed wheyproteins are commonly used as protective media during thespray-drying and storage of lactic acid bacteria [37]

33 Comparison of Predicted and Experimental Parameters0e model used in this study and developed by Nandasanaand Kumar [16] reflects closely the kinetic behavior ofEnterococcus faecalis SLT13 0e comparisonof experimental data and predicted one is shown inFigures 1ndash3

0e value of correlation coefficient (R2) is presentedin Table 2 and is used to determine if the model fits well

0

05

1

15

2

25

3Bi

omas

s con

cent

ratio

n (g

L)

20 25151050Time (h)

(a)

0

5

10

15

20

25

30

35

40

45

50

Lact

ose c

once

ntra

tion

(gL

)

5 10 15 20 250Time (h)

(b)

0

10

20

30

40

50

60

Lact

ic ac

id co

ncen

trat

ion

(gL

)

5 10 15 20 250Time (h)

(c)

Figure 2 Experimental data (points) and simulation (lines) of biomass (a) lactose (b) and lactic acid (c) concentrations of 2 L bioreactorbatch culture of Enterococcus faecalis SLT13 on hydrolyzed cheese whey

6 Journal of Chemistry

the experimental data A good linearity between model andexperimental data is observed especially for the fermenta-tions occurring in 2 L M17 and 20 L HCW

For all the fermentations no lag phase is observedproving the ability of enterococci to adapt to differentgrowth conditions Exponential growth phase lasts 6 hwhen the strain was cultured in M17 followed by a shortstationary phase When cultured in HCW exponentialgrowth phase lasts only 5 h while the stationary phase isrelatively long Glucose is totally consumed after 6 h offermentation which confirmed that in M17 medium themain factor affecting sugar utilization is substrate limi-tation (the substrate limitation constant for sugar con-sumption Kss was 273 gL) In HCW while few quantitiesof lactose remain at the end of fermentation in 2 L bio-reactor it is almost totally consumed in 20 L bioreactorafter about 10 h of fermentation (14 gL) which confirmsthat the growth of biomass is limited by lactose con-centration since Ksx is 4229 gL Furthermore the initialconcentration of lactose is higher in 2 L bioreactor

Indeed in cheese whey powder concentration of lactosecan vary from batch to batch

In all fermentations lactate production began earlierin the exponential growth phase and continues during thestationary phase Indeed lactic acid production is growth-associated as well as non-growth-associated Lactic acidproduction remains constant after 7 h and 10h of fermentationin M17 and in HCW in 20 L bioreactor respectivelyHowever on HCW in 2 L bioreactor lactate concen-tration continues to increase with the availability of thesubstrate

4 Conclusions

0e overall results obtained in our study show thatcheese whey is suitable for lactic acid production vol-umetric productivities are higher than the one obtainedin M17 0e mathematical modeling is useful and allowscharacterizing growth and lactic acid production ofEnterococcus faecalis SLT13 in two different media and

0

05

1

15

2

25

3Bi

omas

s con

cent

ratio

n (g

L)

5 10 15 20 250Time (h)

(a)

ndash5

0

5

10

15

20

25

30

35

Lact

ose c

once

ntra

tion

(gL

)

5 10 15 20 250Time (h)

(b)

0

5

10

15

20

25

Lact

ic ac

id co

ncen

trat

ion

(gL

)

5 10 15 20 250Time (h)

(c)

Figure 3 Experimental data (points) and simulation (lines) of biomass (a) lactose (b) and lactic acid (c) concentrations of 20 L bioreactorbatch culture of Enterococcus faecalis SLT13 on hydrolyzed cheese whey

Journal of Chemistry 7

two different volume bioreactors 0e scale-up affectslactic acid productivity but enhances biomass growthSubstrate inhibition did not impact significantly themaximum lactic acid productivities attainable in theboth 2 L and 20 L bioreactors However product inhi-bition seems to play a major role in the two studiedmedia

Abbreviations

Kd Death coefficient (hminus 1)Kip Substrate inhibition constant for lactic acid

production (gL)Kis Substrate inhibition constant for sugar consumption

(gL)Kix Substrate inhibition constant for growth of biomass

(gL)Kpp Product inhibition constant for lactic acid

production (gL)Kps Product inhibition constant for sugar consumption

(gL)Kpx Product inhibition constant for growth of biomass

(gL)Ksp Substrate limitation constant for lactic acid

production (gL)Kss Substrate limitation constant for sugar consumption

(gL)Ksx Substrate limitation constant for growth of biomass

(gL)P Lactic acid concentration (gL)qpmax Maximum specific lactic acid production rate (g(g h))qsmax Maximum specific sugar utilization rate (g(g h))S Sugar concentration (gL)X Biomass concentration (gL)Ypx Lactic acid yield on growth of biomass (gg)Yps Lactic acid yield on sugar consumption (gg)Yxs Biomass yield on sugar consumption (gg)

Greek Symbols

α Growth-associated constant in LuedekingndashPiretmodel (gg)

μmax Maximum specific growth rate (hminus 1) of lactic acidμ Specific growth rate (hminus 1)

Data Availability

All the numerical data used to support the findings of thisstudy are available from the corresponding author uponreasonable request

Additional Points

Kinetic modeling of lactic acid and biomass productionby Enterococcus faecalis SLT13 was realized on syntheticmedia and Hydrolyzed Cheese Whey Hydrolyzed CheeseWhey enhances lactic acid production Scale-up from 2 Lbioreactor to 20 L bioreactor increases biomass but notlactic acid production Biomass growth and sugar

utilization are affected by lactic acid inhibition in Hy-drolyzed Cheese Whey

Conflicts of Interest

0e authors declare no financial or commercial conflicts ofinterest regarding the publication of this paper

Acknowledgments

0is work was supported by the Ministry of Higher Educationand Scientific Research (Tunisia) and the Walloon Region(Belgium) during the project Technological Platform for theDevelopment of Bioindustries

Supplementary Materials

Graphical abstract representing lactic acid production by En-terococcus faecalis SLT13 during batch culture on HydrolyzedCheese Whey in 2 L and 20 L bioreactors (SupplementaryMaterials)

References

[1] Markets and Markets ldquoLactic acid market by application(biodegradable polymer food amp beverage personal care amppharmaceutical) amp polylactic acid market by application(packaging agriculture automobile electronics textile) amp bygeography-global trends amp forecasts to 2020rdquo Report CodeFB 2647 Markets and Markets Hadapsar Pune 2015

[2] B Gupta N Revagade and J Hilborn ldquoPoly(lactic acid) fiberan overviewrdquo Progress in Polymer Science vol 32 no 4pp 455ndash482 2007

[3] N Narayanan P K Roychoudhury and A Srivastava ldquoL(+)lactic acid fermentation and its product polymerizationrdquoElectronic Journal of Biotechnology vol 7 no 2 2004

[4] R Mehta V Kumar H Bhunia and S N UpadhyayldquoSynthesis of poly(lactic acid) a reviewrdquo Journal of Macro-molecular Science Part C Polymer Reviews vol 45 no 4pp 325ndash349 2005

[5] M A Randhawa A Ahmed and K Akram ldquoOptimization oflactic acid production from cheap raw material sugarcanemolassesrdquo Pakistan Journal of Botany vol 44 pp 333ndash3382012

[6] A-N Vamvakaki I Kandarakis S KaminaridesM Komaitis and S Papanikolaou ldquoCheese whey as a re-newable substrate for microbial lipid and biomass productionby zygomycetesrdquo Engineering in Life Sciences vol 10 no 4pp 348ndash360 2010

[7] M I G Gonzalez ldquo0e biotechnological utilization of cheesewhey a reviewrdquo Bioresource Technology vol 57 pp 1ndash111996

[8] Y H Park K M Cho H W Kim and C Kim ldquoMethod forproducing lactic acid with high concentration and high yieldusing lactic acid bacteriardquo CJ CheilJedang Corporation SeoulRepublic of Korea US Patent 7682814 B2 2010

[9] T Mimitsuka K Sawai K Kobayashi et al ldquoProduction ofd-lactic acid in a continuous membrane integrated fermen-tation reactor by genetically modified Saccharomyces cer-evisiae enhancement in d-lactic acid carbon yieldrdquo Journal ofBioscience and Bioengineering vol 119 no 1 pp 65ndash71 2015

[10] V Kitpreechavanich T Maneeboon Y Kayano and K SakaildquoComparative characterization of l-lactic acid-producing

8 Journal of Chemistry

05

1

15

2

25

3

35

Biom

ass c

once

ntra

tion

(gL

)

0 1 32 54 76 98 10Time (h)

(a)

0

2

4

6

8

10

12

14

16

18

20

Glu

cose

conc

entr

atio

n (g

L)

1 2 3 4 5 6 7 8 9 100Time (h)

(b)

0

1

2

3

4

5

6

7

Lact

ic ac

id co

ncen

trat

ion

(gL

)

1 2 3 4 5 6 7 8 9 100Time (h)

(c)

Figure 1 Experimental data (points) and simulation (lines) of biomass (a) glucose (b) and lactic acid (c) concentrations of 2 L bioreactorbatch culture of Enterococcus faecalis SLT13 on M17 medium

Table 2 Best-fitting values of kinetic parameters obtained by modeling Enterococcus faecalis SLT13 growth sugar utilization and lactic acidproduction in M17 and HCW in 2 L and 20 L bioreactors

Kinetic parameters 2 L M17 2 L HCW 20 L HCWBiomass production modelμmax (hminus 1) 034 099 109Kix (gg) 11406 39975 39420Ksx (gg) 0023 00023 4229Kpx (gg) 1811 377 5001Kd (hminus 1) 0013 00001 00001Sugar utilization modelKis (gg) 29013 39999 143391Kss (gg) 273 001 015Kps (gg) 1044 1116 2007qsmax (ggmiddoth) 492 499 416Lactic acid production modelKip (gg) 13589 10000 37389Kpp (gg) 531 4499 4283Ksp (gg) 0025 001 0065qpmax (ggmiddoth) 1027 204 1863α (gg) 0052 001 0017R2 9434 8881 9787

Journal of Chemistry 5

For sugar consumption the higher maximum sugaruptake rate (qsmax) is estimated to be 499 g(gmiddoth) for HCWin 2 L bioreactor in spite of the values are close for the threefermentations 0e maximum specific lactic acid productionrate (qpmax) is higher for HCW and reach 204 g(gmiddoth) in 2 Lbioreactor

0e growth-associated term (α) is very low for all thefermentations In spite of that lactic acid production is bothgrowth-associated and non-growth-associated and seen thevalue of α the production of lactic acid is essentially oc-curred in the stationary phase Higher growth-associatedparameter (α) was reported by Nandasana and Kumar [16]for Enterococcus faecalis molasses and was 026 gg Lacto-coccus lactisNZ133 grown on lactose showed a highest value036 gg [36]

Evaluation of the cell death coefficient or endogenousdecay constant (Kd) of Enterococcus faecalis is conducted forthe three fermentations 0e estimated decay constants arevery low and equal to 00001 hminus 1 on HCW Neverthelessdecay constant is relatively important for the fermentation

in M17 and is equal to 0013 hminus 1 Death coefficient is straindependent and different Kd values were reported in theliterature Lactococcus lactis SLT6 and SLT10 grown in thesame media used in this study (HCW) presented values of0093ndash026 hminus 1respectively [21] Enterococcus faecalis RKY1grown on cane-sugar molasses had Kd value of 000318 hminus 1

[16] 0e lower Kd value for HCW can be ascribed to theprotective effect of cheese whey proteins Indeed wheyproteins are commonly used as protective media during thespray-drying and storage of lactic acid bacteria [37]

33 Comparison of Predicted and Experimental Parameters0e model used in this study and developed by Nandasanaand Kumar [16] reflects closely the kinetic behavior ofEnterococcus faecalis SLT13 0e comparisonof experimental data and predicted one is shown inFigures 1ndash3

0e value of correlation coefficient (R2) is presentedin Table 2 and is used to determine if the model fits well

0

05

1

15

2

25

3Bi

omas

s con

cent

ratio

n (g

L)

20 25151050Time (h)

(a)

0

5

10

15

20

25

30

35

40

45

50

Lact

ose c

once

ntra

tion

(gL

)

5 10 15 20 250Time (h)

(b)

0

10

20

30

40

50

60

Lact

ic ac

id co

ncen

trat

ion

(gL

)

5 10 15 20 250Time (h)

(c)

Figure 2 Experimental data (points) and simulation (lines) of biomass (a) lactose (b) and lactic acid (c) concentrations of 2 L bioreactorbatch culture of Enterococcus faecalis SLT13 on hydrolyzed cheese whey

6 Journal of Chemistry

the experimental data A good linearity between model andexperimental data is observed especially for the fermenta-tions occurring in 2 L M17 and 20 L HCW

For all the fermentations no lag phase is observedproving the ability of enterococci to adapt to differentgrowth conditions Exponential growth phase lasts 6 hwhen the strain was cultured in M17 followed by a shortstationary phase When cultured in HCW exponentialgrowth phase lasts only 5 h while the stationary phase isrelatively long Glucose is totally consumed after 6 h offermentation which confirmed that in M17 medium themain factor affecting sugar utilization is substrate limi-tation (the substrate limitation constant for sugar con-sumption Kss was 273 gL) In HCW while few quantitiesof lactose remain at the end of fermentation in 2 L bio-reactor it is almost totally consumed in 20 L bioreactorafter about 10 h of fermentation (14 gL) which confirmsthat the growth of biomass is limited by lactose con-centration since Ksx is 4229 gL Furthermore the initialconcentration of lactose is higher in 2 L bioreactor

Indeed in cheese whey powder concentration of lactosecan vary from batch to batch

In all fermentations lactate production began earlierin the exponential growth phase and continues during thestationary phase Indeed lactic acid production is growth-associated as well as non-growth-associated Lactic acidproduction remains constant after 7 h and 10h of fermentationin M17 and in HCW in 20 L bioreactor respectivelyHowever on HCW in 2 L bioreactor lactate concen-tration continues to increase with the availability of thesubstrate

4 Conclusions

0e overall results obtained in our study show thatcheese whey is suitable for lactic acid production vol-umetric productivities are higher than the one obtainedin M17 0e mathematical modeling is useful and allowscharacterizing growth and lactic acid production ofEnterococcus faecalis SLT13 in two different media and

0

05

1

15

2

25

3Bi

omas

s con

cent

ratio

n (g

L)

5 10 15 20 250Time (h)

(a)

ndash5

0

5

10

15

20

25

30

35

Lact

ose c

once

ntra

tion

(gL

)

5 10 15 20 250Time (h)

(b)

0

5

10

15

20

25

Lact

ic ac

id co

ncen

trat

ion

(gL

)

5 10 15 20 250Time (h)

(c)

Figure 3 Experimental data (points) and simulation (lines) of biomass (a) lactose (b) and lactic acid (c) concentrations of 20 L bioreactorbatch culture of Enterococcus faecalis SLT13 on hydrolyzed cheese whey

Journal of Chemistry 7

two different volume bioreactors 0e scale-up affectslactic acid productivity but enhances biomass growthSubstrate inhibition did not impact significantly themaximum lactic acid productivities attainable in theboth 2 L and 20 L bioreactors However product inhi-bition seems to play a major role in the two studiedmedia

Abbreviations

Kd Death coefficient (hminus 1)Kip Substrate inhibition constant for lactic acid

production (gL)Kis Substrate inhibition constant for sugar consumption

(gL)Kix Substrate inhibition constant for growth of biomass

(gL)Kpp Product inhibition constant for lactic acid

production (gL)Kps Product inhibition constant for sugar consumption

(gL)Kpx Product inhibition constant for growth of biomass

(gL)Ksp Substrate limitation constant for lactic acid

production (gL)Kss Substrate limitation constant for sugar consumption

(gL)Ksx Substrate limitation constant for growth of biomass

(gL)P Lactic acid concentration (gL)qpmax Maximum specific lactic acid production rate (g(g h))qsmax Maximum specific sugar utilization rate (g(g h))S Sugar concentration (gL)X Biomass concentration (gL)Ypx Lactic acid yield on growth of biomass (gg)Yps Lactic acid yield on sugar consumption (gg)Yxs Biomass yield on sugar consumption (gg)

Greek Symbols

α Growth-associated constant in LuedekingndashPiretmodel (gg)

μmax Maximum specific growth rate (hminus 1) of lactic acidμ Specific growth rate (hminus 1)

Data Availability

All the numerical data used to support the findings of thisstudy are available from the corresponding author uponreasonable request

Additional Points

Kinetic modeling of lactic acid and biomass productionby Enterococcus faecalis SLT13 was realized on syntheticmedia and Hydrolyzed Cheese Whey Hydrolyzed CheeseWhey enhances lactic acid production Scale-up from 2 Lbioreactor to 20 L bioreactor increases biomass but notlactic acid production Biomass growth and sugar

utilization are affected by lactic acid inhibition in Hy-drolyzed Cheese Whey

Conflicts of Interest

0e authors declare no financial or commercial conflicts ofinterest regarding the publication of this paper

Acknowledgments

0is work was supported by the Ministry of Higher Educationand Scientific Research (Tunisia) and the Walloon Region(Belgium) during the project Technological Platform for theDevelopment of Bioindustries

Supplementary Materials

Graphical abstract representing lactic acid production by En-terococcus faecalis SLT13 during batch culture on HydrolyzedCheese Whey in 2 L and 20 L bioreactors (SupplementaryMaterials)

References

[1] Markets and Markets ldquoLactic acid market by application(biodegradable polymer food amp beverage personal care amppharmaceutical) amp polylactic acid market by application(packaging agriculture automobile electronics textile) amp bygeography-global trends amp forecasts to 2020rdquo Report CodeFB 2647 Markets and Markets Hadapsar Pune 2015

[2] B Gupta N Revagade and J Hilborn ldquoPoly(lactic acid) fiberan overviewrdquo Progress in Polymer Science vol 32 no 4pp 455ndash482 2007

[3] N Narayanan P K Roychoudhury and A Srivastava ldquoL(+)lactic acid fermentation and its product polymerizationrdquoElectronic Journal of Biotechnology vol 7 no 2 2004

[4] R Mehta V Kumar H Bhunia and S N UpadhyayldquoSynthesis of poly(lactic acid) a reviewrdquo Journal of Macro-molecular Science Part C Polymer Reviews vol 45 no 4pp 325ndash349 2005

[5] M A Randhawa A Ahmed and K Akram ldquoOptimization oflactic acid production from cheap raw material sugarcanemolassesrdquo Pakistan Journal of Botany vol 44 pp 333ndash3382012

[6] A-N Vamvakaki I Kandarakis S KaminaridesM Komaitis and S Papanikolaou ldquoCheese whey as a re-newable substrate for microbial lipid and biomass productionby zygomycetesrdquo Engineering in Life Sciences vol 10 no 4pp 348ndash360 2010

[7] M I G Gonzalez ldquo0e biotechnological utilization of cheesewhey a reviewrdquo Bioresource Technology vol 57 pp 1ndash111996

[8] Y H Park K M Cho H W Kim and C Kim ldquoMethod forproducing lactic acid with high concentration and high yieldusing lactic acid bacteriardquo CJ CheilJedang Corporation SeoulRepublic of Korea US Patent 7682814 B2 2010

[9] T Mimitsuka K Sawai K Kobayashi et al ldquoProduction ofd-lactic acid in a continuous membrane integrated fermen-tation reactor by genetically modified Saccharomyces cer-evisiae enhancement in d-lactic acid carbon yieldrdquo Journal ofBioscience and Bioengineering vol 119 no 1 pp 65ndash71 2015

[10] V Kitpreechavanich T Maneeboon Y Kayano and K SakaildquoComparative characterization of l-lactic acid-producing

8 Journal of Chemistry

For sugar consumption the higher maximum sugaruptake rate (qsmax) is estimated to be 499 g(gmiddoth) for HCWin 2 L bioreactor in spite of the values are close for the threefermentations 0e maximum specific lactic acid productionrate (qpmax) is higher for HCW and reach 204 g(gmiddoth) in 2 Lbioreactor

0e growth-associated term (α) is very low for all thefermentations In spite of that lactic acid production is bothgrowth-associated and non-growth-associated and seen thevalue of α the production of lactic acid is essentially oc-curred in the stationary phase Higher growth-associatedparameter (α) was reported by Nandasana and Kumar [16]for Enterococcus faecalis molasses and was 026 gg Lacto-coccus lactisNZ133 grown on lactose showed a highest value036 gg [36]

Evaluation of the cell death coefficient or endogenousdecay constant (Kd) of Enterococcus faecalis is conducted forthe three fermentations 0e estimated decay constants arevery low and equal to 00001 hminus 1 on HCW Neverthelessdecay constant is relatively important for the fermentation

in M17 and is equal to 0013 hminus 1 Death coefficient is straindependent and different Kd values were reported in theliterature Lactococcus lactis SLT6 and SLT10 grown in thesame media used in this study (HCW) presented values of0093ndash026 hminus 1respectively [21] Enterococcus faecalis RKY1grown on cane-sugar molasses had Kd value of 000318 hminus 1

[16] 0e lower Kd value for HCW can be ascribed to theprotective effect of cheese whey proteins Indeed wheyproteins are commonly used as protective media during thespray-drying and storage of lactic acid bacteria [37]

33 Comparison of Predicted and Experimental Parameters0e model used in this study and developed by Nandasanaand Kumar [16] reflects closely the kinetic behavior ofEnterococcus faecalis SLT13 0e comparisonof experimental data and predicted one is shown inFigures 1ndash3

0e value of correlation coefficient (R2) is presentedin Table 2 and is used to determine if the model fits well

0

05

1

15

2

25

3Bi

omas

s con

cent

ratio

n (g

L)

20 25151050Time (h)

(a)

0

5

10

15

20

25

30

35

40

45

50

Lact

ose c

once

ntra

tion

(gL

)

5 10 15 20 250Time (h)

(b)

0

10

20

30

40

50

60

Lact

ic ac

id co

ncen

trat

ion

(gL

)

5 10 15 20 250Time (h)

(c)

Figure 2 Experimental data (points) and simulation (lines) of biomass (a) lactose (b) and lactic acid (c) concentrations of 2 L bioreactorbatch culture of Enterococcus faecalis SLT13 on hydrolyzed cheese whey

6 Journal of Chemistry

the experimental data A good linearity between model andexperimental data is observed especially for the fermenta-tions occurring in 2 L M17 and 20 L HCW

For all the fermentations no lag phase is observedproving the ability of enterococci to adapt to differentgrowth conditions Exponential growth phase lasts 6 hwhen the strain was cultured in M17 followed by a shortstationary phase When cultured in HCW exponentialgrowth phase lasts only 5 h while the stationary phase isrelatively long Glucose is totally consumed after 6 h offermentation which confirmed that in M17 medium themain factor affecting sugar utilization is substrate limi-tation (the substrate limitation constant for sugar con-sumption Kss was 273 gL) In HCW while few quantitiesof lactose remain at the end of fermentation in 2 L bio-reactor it is almost totally consumed in 20 L bioreactorafter about 10 h of fermentation (14 gL) which confirmsthat the growth of biomass is limited by lactose con-centration since Ksx is 4229 gL Furthermore the initialconcentration of lactose is higher in 2 L bioreactor

Indeed in cheese whey powder concentration of lactosecan vary from batch to batch

In all fermentations lactate production began earlierin the exponential growth phase and continues during thestationary phase Indeed lactic acid production is growth-associated as well as non-growth-associated Lactic acidproduction remains constant after 7 h and 10h of fermentationin M17 and in HCW in 20 L bioreactor respectivelyHowever on HCW in 2 L bioreactor lactate concen-tration continues to increase with the availability of thesubstrate

4 Conclusions

0e overall results obtained in our study show thatcheese whey is suitable for lactic acid production vol-umetric productivities are higher than the one obtainedin M17 0e mathematical modeling is useful and allowscharacterizing growth and lactic acid production ofEnterococcus faecalis SLT13 in two different media and

0

05

1

15

2

25

3Bi

omas

s con

cent

ratio

n (g

L)

5 10 15 20 250Time (h)

(a)

ndash5

0

5

10

15

20

25

30

35

Lact

ose c

once

ntra

tion

(gL

)

5 10 15 20 250Time (h)

(b)

0

5

10

15

20

25

Lact

ic ac

id co

ncen

trat

ion

(gL

)

5 10 15 20 250Time (h)

(c)

Figure 3 Experimental data (points) and simulation (lines) of biomass (a) lactose (b) and lactic acid (c) concentrations of 20 L bioreactorbatch culture of Enterococcus faecalis SLT13 on hydrolyzed cheese whey

Journal of Chemistry 7

two different volume bioreactors 0e scale-up affectslactic acid productivity but enhances biomass growthSubstrate inhibition did not impact significantly themaximum lactic acid productivities attainable in theboth 2 L and 20 L bioreactors However product inhi-bition seems to play a major role in the two studiedmedia

Abbreviations

Kd Death coefficient (hminus 1)Kip Substrate inhibition constant for lactic acid

production (gL)Kis Substrate inhibition constant for sugar consumption

(gL)Kix Substrate inhibition constant for growth of biomass

(gL)Kpp Product inhibition constant for lactic acid

production (gL)Kps Product inhibition constant for sugar consumption

(gL)Kpx Product inhibition constant for growth of biomass

(gL)Ksp Substrate limitation constant for lactic acid

production (gL)Kss Substrate limitation constant for sugar consumption

(gL)Ksx Substrate limitation constant for growth of biomass

(gL)P Lactic acid concentration (gL)qpmax Maximum specific lactic acid production rate (g(g h))qsmax Maximum specific sugar utilization rate (g(g h))S Sugar concentration (gL)X Biomass concentration (gL)Ypx Lactic acid yield on growth of biomass (gg)Yps Lactic acid yield on sugar consumption (gg)Yxs Biomass yield on sugar consumption (gg)

Greek Symbols

α Growth-associated constant in LuedekingndashPiretmodel (gg)

μmax Maximum specific growth rate (hminus 1) of lactic acidμ Specific growth rate (hminus 1)

Data Availability

All the numerical data used to support the findings of thisstudy are available from the corresponding author uponreasonable request

Additional Points

Kinetic modeling of lactic acid and biomass productionby Enterococcus faecalis SLT13 was realized on syntheticmedia and Hydrolyzed Cheese Whey Hydrolyzed CheeseWhey enhances lactic acid production Scale-up from 2 Lbioreactor to 20 L bioreactor increases biomass but notlactic acid production Biomass growth and sugar

utilization are affected by lactic acid inhibition in Hy-drolyzed Cheese Whey

Conflicts of Interest

0e authors declare no financial or commercial conflicts ofinterest regarding the publication of this paper

Acknowledgments

0is work was supported by the Ministry of Higher Educationand Scientific Research (Tunisia) and the Walloon Region(Belgium) during the project Technological Platform for theDevelopment of Bioindustries

Supplementary Materials

Graphical abstract representing lactic acid production by En-terococcus faecalis SLT13 during batch culture on HydrolyzedCheese Whey in 2 L and 20 L bioreactors (SupplementaryMaterials)

References

[1] Markets and Markets ldquoLactic acid market by application(biodegradable polymer food amp beverage personal care amppharmaceutical) amp polylactic acid market by application(packaging agriculture automobile electronics textile) amp bygeography-global trends amp forecasts to 2020rdquo Report CodeFB 2647 Markets and Markets Hadapsar Pune 2015

[2] B Gupta N Revagade and J Hilborn ldquoPoly(lactic acid) fiberan overviewrdquo Progress in Polymer Science vol 32 no 4pp 455ndash482 2007

[3] N Narayanan P K Roychoudhury and A Srivastava ldquoL(+)lactic acid fermentation and its product polymerizationrdquoElectronic Journal of Biotechnology vol 7 no 2 2004

[4] R Mehta V Kumar H Bhunia and S N UpadhyayldquoSynthesis of poly(lactic acid) a reviewrdquo Journal of Macro-molecular Science Part C Polymer Reviews vol 45 no 4pp 325ndash349 2005

[5] M A Randhawa A Ahmed and K Akram ldquoOptimization oflactic acid production from cheap raw material sugarcanemolassesrdquo Pakistan Journal of Botany vol 44 pp 333ndash3382012

[6] A-N Vamvakaki I Kandarakis S KaminaridesM Komaitis and S Papanikolaou ldquoCheese whey as a re-newable substrate for microbial lipid and biomass productionby zygomycetesrdquo Engineering in Life Sciences vol 10 no 4pp 348ndash360 2010

[7] M I G Gonzalez ldquo0e biotechnological utilization of cheesewhey a reviewrdquo Bioresource Technology vol 57 pp 1ndash111996

[8] Y H Park K M Cho H W Kim and C Kim ldquoMethod forproducing lactic acid with high concentration and high yieldusing lactic acid bacteriardquo CJ CheilJedang Corporation SeoulRepublic of Korea US Patent 7682814 B2 2010

[9] T Mimitsuka K Sawai K Kobayashi et al ldquoProduction ofd-lactic acid in a continuous membrane integrated fermen-tation reactor by genetically modified Saccharomyces cer-evisiae enhancement in d-lactic acid carbon yieldrdquo Journal ofBioscience and Bioengineering vol 119 no 1 pp 65ndash71 2015

[10] V Kitpreechavanich T Maneeboon Y Kayano and K SakaildquoComparative characterization of l-lactic acid-producing

8 Journal of Chemistry

the experimental data A good linearity between model andexperimental data is observed especially for the fermenta-tions occurring in 2 L M17 and 20 L HCW

For all the fermentations no lag phase is observedproving the ability of enterococci to adapt to differentgrowth conditions Exponential growth phase lasts 6 hwhen the strain was cultured in M17 followed by a shortstationary phase When cultured in HCW exponentialgrowth phase lasts only 5 h while the stationary phase isrelatively long Glucose is totally consumed after 6 h offermentation which confirmed that in M17 medium themain factor affecting sugar utilization is substrate limi-tation (the substrate limitation constant for sugar con-sumption Kss was 273 gL) In HCW while few quantitiesof lactose remain at the end of fermentation in 2 L bio-reactor it is almost totally consumed in 20 L bioreactorafter about 10 h of fermentation (14 gL) which confirmsthat the growth of biomass is limited by lactose con-centration since Ksx is 4229 gL Furthermore the initialconcentration of lactose is higher in 2 L bioreactor

Indeed in cheese whey powder concentration of lactosecan vary from batch to batch

In all fermentations lactate production began earlierin the exponential growth phase and continues during thestationary phase Indeed lactic acid production is growth-associated as well as non-growth-associated Lactic acidproduction remains constant after 7 h and 10h of fermentationin M17 and in HCW in 20 L bioreactor respectivelyHowever on HCW in 2 L bioreactor lactate concen-tration continues to increase with the availability of thesubstrate

4 Conclusions

0e overall results obtained in our study show thatcheese whey is suitable for lactic acid production vol-umetric productivities are higher than the one obtainedin M17 0e mathematical modeling is useful and allowscharacterizing growth and lactic acid production ofEnterococcus faecalis SLT13 in two different media and

0

05

1

15

2

25

3Bi

omas

s con

cent

ratio

n (g

L)

5 10 15 20 250Time (h)

(a)

ndash5

0

5

10

15

20

25

30

35

Lact

ose c

once

ntra

tion

(gL

)

5 10 15 20 250Time (h)

(b)

0

5

10

15

20

25

Lact

ic ac

id co

ncen

trat

ion

(gL

)

5 10 15 20 250Time (h)

(c)

Figure 3 Experimental data (points) and simulation (lines) of biomass (a) lactose (b) and lactic acid (c) concentrations of 20 L bioreactorbatch culture of Enterococcus faecalis SLT13 on hydrolyzed cheese whey

Journal of Chemistry 7

two different volume bioreactors 0e scale-up affectslactic acid productivity but enhances biomass growthSubstrate inhibition did not impact significantly themaximum lactic acid productivities attainable in theboth 2 L and 20 L bioreactors However product inhi-bition seems to play a major role in the two studiedmedia

Abbreviations

Kd Death coefficient (hminus 1)Kip Substrate inhibition constant for lactic acid

production (gL)Kis Substrate inhibition constant for sugar consumption

(gL)Kix Substrate inhibition constant for growth of biomass

(gL)Kpp Product inhibition constant for lactic acid

production (gL)Kps Product inhibition constant for sugar consumption

(gL)Kpx Product inhibition constant for growth of biomass

(gL)Ksp Substrate limitation constant for lactic acid

production (gL)Kss Substrate limitation constant for sugar consumption

(gL)Ksx Substrate limitation constant for growth of biomass

(gL)P Lactic acid concentration (gL)qpmax Maximum specific lactic acid production rate (g(g h))qsmax Maximum specific sugar utilization rate (g(g h))S Sugar concentration (gL)X Biomass concentration (gL)Ypx Lactic acid yield on growth of biomass (gg)Yps Lactic acid yield on sugar consumption (gg)Yxs Biomass yield on sugar consumption (gg)

Greek Symbols

α Growth-associated constant in LuedekingndashPiretmodel (gg)

μmax Maximum specific growth rate (hminus 1) of lactic acidμ Specific growth rate (hminus 1)

Data Availability

All the numerical data used to support the findings of thisstudy are available from the corresponding author uponreasonable request

Additional Points

Kinetic modeling of lactic acid and biomass productionby Enterococcus faecalis SLT13 was realized on syntheticmedia and Hydrolyzed Cheese Whey Hydrolyzed CheeseWhey enhances lactic acid production Scale-up from 2 Lbioreactor to 20 L bioreactor increases biomass but notlactic acid production Biomass growth and sugar

utilization are affected by lactic acid inhibition in Hy-drolyzed Cheese Whey

Conflicts of Interest

0e authors declare no financial or commercial conflicts ofinterest regarding the publication of this paper

Acknowledgments

0is work was supported by the Ministry of Higher Educationand Scientific Research (Tunisia) and the Walloon Region(Belgium) during the project Technological Platform for theDevelopment of Bioindustries

Supplementary Materials

Graphical abstract representing lactic acid production by En-terococcus faecalis SLT13 during batch culture on HydrolyzedCheese Whey in 2 L and 20 L bioreactors (SupplementaryMaterials)

References

[1] Markets and Markets ldquoLactic acid market by application(biodegradable polymer food amp beverage personal care amppharmaceutical) amp polylactic acid market by application(packaging agriculture automobile electronics textile) amp bygeography-global trends amp forecasts to 2020rdquo Report CodeFB 2647 Markets and Markets Hadapsar Pune 2015

[2] B Gupta N Revagade and J Hilborn ldquoPoly(lactic acid) fiberan overviewrdquo Progress in Polymer Science vol 32 no 4pp 455ndash482 2007

[3] N Narayanan P K Roychoudhury and A Srivastava ldquoL(+)lactic acid fermentation and its product polymerizationrdquoElectronic Journal of Biotechnology vol 7 no 2 2004

[4] R Mehta V Kumar H Bhunia and S N UpadhyayldquoSynthesis of poly(lactic acid) a reviewrdquo Journal of Macro-molecular Science Part C Polymer Reviews vol 45 no 4pp 325ndash349 2005

[5] M A Randhawa A Ahmed and K Akram ldquoOptimization oflactic acid production from cheap raw material sugarcanemolassesrdquo Pakistan Journal of Botany vol 44 pp 333ndash3382012

[6] A-N Vamvakaki I Kandarakis S KaminaridesM Komaitis and S Papanikolaou ldquoCheese whey as a re-newable substrate for microbial lipid and biomass productionby zygomycetesrdquo Engineering in Life Sciences vol 10 no 4pp 348ndash360 2010

[7] M I G Gonzalez ldquo0e biotechnological utilization of cheesewhey a reviewrdquo Bioresource Technology vol 57 pp 1ndash111996

[8] Y H Park K M Cho H W Kim and C Kim ldquoMethod forproducing lactic acid with high concentration and high yieldusing lactic acid bacteriardquo CJ CheilJedang Corporation SeoulRepublic of Korea US Patent 7682814 B2 2010

[9] T Mimitsuka K Sawai K Kobayashi et al ldquoProduction ofd-lactic acid in a continuous membrane integrated fermen-tation reactor by genetically modified Saccharomyces cer-evisiae enhancement in d-lactic acid carbon yieldrdquo Journal ofBioscience and Bioengineering vol 119 no 1 pp 65ndash71 2015

[10] V Kitpreechavanich T Maneeboon Y Kayano and K SakaildquoComparative characterization of l-lactic acid-producing

8 Journal of Chemistry

two different volume bioreactors 0e scale-up affectslactic acid productivity but enhances biomass growthSubstrate inhibition did not impact significantly themaximum lactic acid productivities attainable in theboth 2 L and 20 L bioreactors However product inhi-bition seems to play a major role in the two studiedmedia

Abbreviations

Kd Death coefficient (hminus 1)Kip Substrate inhibition constant for lactic acid

production (gL)Kis Substrate inhibition constant for sugar consumption

(gL)Kix Substrate inhibition constant for growth of biomass

(gL)Kpp Product inhibition constant for lactic acid

production (gL)Kps Product inhibition constant for sugar consumption

(gL)Kpx Product inhibition constant for growth of biomass

(gL)Ksp Substrate limitation constant for lactic acid

production (gL)Kss Substrate limitation constant for sugar consumption

(gL)Ksx Substrate limitation constant for growth of biomass

(gL)P Lactic acid concentration (gL)qpmax Maximum specific lactic acid production rate (g(g h))qsmax Maximum specific sugar utilization rate (g(g h))S Sugar concentration (gL)X Biomass concentration (gL)Ypx Lactic acid yield on growth of biomass (gg)Yps Lactic acid yield on sugar consumption (gg)Yxs Biomass yield on sugar consumption (gg)

Greek Symbols

α Growth-associated constant in LuedekingndashPiretmodel (gg)

μmax Maximum specific growth rate (hminus 1) of lactic acidμ Specific growth rate (hminus 1)

Data Availability

All the numerical data used to support the findings of thisstudy are available from the corresponding author uponreasonable request

Additional Points

Kinetic modeling of lactic acid and biomass productionby Enterococcus faecalis SLT13 was realized on syntheticmedia and Hydrolyzed Cheese Whey Hydrolyzed CheeseWhey enhances lactic acid production Scale-up from 2 Lbioreactor to 20 L bioreactor increases biomass but notlactic acid production Biomass growth and sugar

utilization are affected by lactic acid inhibition in Hy-drolyzed Cheese Whey

Conflicts of Interest

0e authors declare no financial or commercial conflicts ofinterest regarding the publication of this paper

Acknowledgments

0is work was supported by the Ministry of Higher Educationand Scientific Research (Tunisia) and the Walloon Region(Belgium) during the project Technological Platform for theDevelopment of Bioindustries

Supplementary Materials

Graphical abstract representing lactic acid production by En-terococcus faecalis SLT13 during batch culture on HydrolyzedCheese Whey in 2 L and 20 L bioreactors (SupplementaryMaterials)

References

[1] Markets and Markets ldquoLactic acid market by application(biodegradable polymer food amp beverage personal care amppharmaceutical) amp polylactic acid market by application(packaging agriculture automobile electronics textile) amp bygeography-global trends amp forecasts to 2020rdquo Report CodeFB 2647 Markets and Markets Hadapsar Pune 2015

[2] B Gupta N Revagade and J Hilborn ldquoPoly(lactic acid) fiberan overviewrdquo Progress in Polymer Science vol 32 no 4pp 455ndash482 2007

[3] N Narayanan P K Roychoudhury and A Srivastava ldquoL(+)lactic acid fermentation and its product polymerizationrdquoElectronic Journal of Biotechnology vol 7 no 2 2004

[4] R Mehta V Kumar H Bhunia and S N UpadhyayldquoSynthesis of poly(lactic acid) a reviewrdquo Journal of Macro-molecular Science Part C Polymer Reviews vol 45 no 4pp 325ndash349 2005

[5] M A Randhawa A Ahmed and K Akram ldquoOptimization oflactic acid production from cheap raw material sugarcanemolassesrdquo Pakistan Journal of Botany vol 44 pp 333ndash3382012

[6] A-N Vamvakaki I Kandarakis S KaminaridesM Komaitis and S Papanikolaou ldquoCheese whey as a re-newable substrate for microbial lipid and biomass productionby zygomycetesrdquo Engineering in Life Sciences vol 10 no 4pp 348ndash360 2010

[7] M I G Gonzalez ldquo0e biotechnological utilization of cheesewhey a reviewrdquo Bioresource Technology vol 57 pp 1ndash111996

[8] Y H Park K M Cho H W Kim and C Kim ldquoMethod forproducing lactic acid with high concentration and high yieldusing lactic acid bacteriardquo CJ CheilJedang Corporation SeoulRepublic of Korea US Patent 7682814 B2 2010

[9] T Mimitsuka K Sawai K Kobayashi et al ldquoProduction ofd-lactic acid in a continuous membrane integrated fermen-tation reactor by genetically modified Saccharomyces cer-evisiae enhancement in d-lactic acid carbon yieldrdquo Journal ofBioscience and Bioengineering vol 119 no 1 pp 65ndash71 2015

[10] V Kitpreechavanich T Maneeboon Y Kayano and K SakaildquoComparative characterization of l-lactic acid-producing

8 Journal of Chemistry

thermotolerant rhizopus fungirdquo Journal of Bioscience andBioengineering vol 106 no 6 pp 541ndash546 2008

[11] J Qin B Zhao X Wang et al ldquoNon-sterilized fermentativeproduction of polymer-grade L-lactic acid by a newly isolatedthermophilic strain Bacillus sp 2-6rdquo PLoS One vol 4 no 2Article ID e4359 2009

[12] S Zhou T B Causey A Hasona K T Shanmugam andL O Ingram ldquoProduction of optically pure D-lactic acid inmineral salts medium by metabolically engineered Escherichiacoli W3110rdquo Applied and Environmental Microbiologyvol 69 no 1 pp 399ndash407 2003

[13] L V Reddy Y M Kim J S Yun H W Ryu and Y J WeeldquoL-lactic acid production by combined utilization of agri-cultural bioresources as renewable and economical substratesthrough batch and repeated-batch fermentation of Entero-coccus faecalis RKY1rdquo Bioresource Technology vol 209pp 187ndash194 2015

[14] J Vijayakumar R Aravindan and T Viruthagiri ldquoRecenttrends in the production purification and application of lacticacidrdquo Chemical and Biochemical Engineering Quarterlyvol 22 pp 245ndash264 2008

[15] H Oh Y-J Wee J-S Yun S Ho Han S Jung andH-W Ryu ldquoLactic acid production from agricultural re-sources as cheap raw materialsrdquo Bioresource Technologyvol 96 no 13 pp 1492ndash1498 2005

[16] A D Nandasana and S Kumar ldquoKinetic modeling of lacticacid production from molasses using Enterococcus faecalisRKY1rdquo Biochemical Engineering Journal vol 38 no 3pp 277ndash284 2008

[17] K Shibata D M Flores G Kobayashi and K SonomotoldquoDirect l-lactic acid fermentation with sago starch by a novelamylolytic lactic acid bacterium Enterococcus faeciumrdquo Enzymeand Microbial Technology vol 41 no 1-2 pp 149ndash155 2007

[18] N Murakami M Oba M Iwamoto et al ldquoL-lactic acidproduction from glycerol coupled with acetic acid metabolismby Enterococcus faecalis without carbon lossrdquo Journal ofBioscience and Bioengineering vol 121 no 1 pp 89ndash95 2016

[19] J M Dodic D G Vucurovic S N Dodic J A GrahovacS D Popov and N M Nedeljkovic ldquoKinetic modeling ofbatch ethanol production from sugar beet raw juicerdquo AppliedEnergy vol 99 pp 192ndash197 2012

[20] F Gardini M Martuscelli M C Caruso et al ldquoEffects of pHtemperature and NaCl concentration on the growth kineticsproteolytic activity and biogenic amine production of En-terococcus faecalisrdquo International Journal of Food Microbi-ology vol 64 no 1-2 pp 105ndash117 2001

[21] M Ziadi F Rezouga H Bouallagui et al ldquoKinetic study ofLactococcus lactis strains (SLT6 and SLT10) growth on pa-pain-hydrolysed wheyrdquo World Journal of Microbiology andBiotechnology vol 26 no 12 pp 2223ndash2230 2010

[22] F Leroy and L De Vuyst ldquoGrowth of the bacteriocin-pro-ducing Lactobacillus sakei strain CTC 494 in MRS broth isstrongly reduced due to nutrient exhaustion a nutrient de-pletion model for the growth of lactic acid bacteriardquo Appliedand Environmental Microbiology vol 67 no 10 pp 4407ndash4413 2001

[23] A W Schepers J 0ibault and C Lacroix ldquoLactobacillushelveticus growth and lactic acid production during pH-controlled batch cultures in whey permeateyeast extractmedium Part II kinetic modeling and model validationrdquoEnzyme and Microbial Technology vol 30 pp 187ndash194 2002

[24] M M Alvarez E J Aguirre-Ezkauriatza A Ramırez-Medrano and A Rodrıguez-Sanchez ldquoKinetic analysis andmathematical modeling of growth and lactic acid production

of Lactobacillus casei var rhamnosus in milk wheyrdquo Journal ofDairy Science vol 93 no 12 pp 5552ndash5560 2010

[25] C Akerberg K Hofvendahl G Zacchi and B Hahn-Hagerdal ldquoModelling the influence of pH temperatureglucose and lactic acid concentrations on the kinetics of lacticacid production by Lactococcus lactis ssp lactis ATCC 19435in whole-wheat flourrdquo Applied Microbiology and Biotechnologyvol 49 no 6 pp 682ndash690 1998

[26] C J Gadgil and K V Venkatesh ldquoStructured model for batchculture growth of Lactobacillus bulgaricusrdquo Journal ofChemical Technology amp Biotechnology vol 68 no 1 pp 89ndash93 1997

[27] M Ziadi S Mhir R Dubois-Dauphin et al ldquoAnalysis ofvolatile compounds amino acid catabolism and some tech-nological properties of Enterococcus faecalis strain SLT13isolated from artisanal Tunisian fermented milkrdquo BritishMicrobiology Research Journal vol 14 no 1 pp 1ndash12 2016

[28] L F Shampine and M K Gordon Computer Solution ofOrdinary Differential Equations e Initial Value ProblemW H Freeman and Company San Francisco CA USA 1975

[29] R H Byrd J C Gilbert and J Nocedal ldquoA trust regionmethod based on interior point techniques for nonlinearprogrammingrdquo Mathematical Programming vol 89 no 1pp 149ndash185 2000

[30] R H Byrd M E Hribar and J Nocedal ldquoAn interior pointalgorithm for large-scale nonlinear programmingrdquo SIAMJournal on Optimization vol 9 no 4 pp 877ndash900 1999

[31] M R Subramanian S Talluri and L P Christopher ldquoPro-duction of lactic acid using a new homofermentative En-terococcus faecalis isolaterdquo Microbial Biotechnology vol 8no 2 pp 221ndash229 2015

[32] A Feldman-Salit S Hering H L Messiha et al ldquoRegulationof the activity of lactate dehydrogenases from four lactic acidbacteriardquo Journal of Biological Chemistry vol 288 no 29pp 21295ndash21306 2013

[33] F R Schmidt ldquoOptimization and scale up of industrial fer-mentation processesrdquo Applied Microbiology and Biotechnol-ogy vol 68 no 4 pp 425ndash435 2005

[34] F Garcia-Ochoa and E Gomez ldquoBioreactor scale-up andoxygen transfer rate in microbial processes an overviewrdquoBiotechnology Advances vol 27 no 2 pp 153ndash176 2009

[35] J P Arnaud C Lacroix C Foussereau and L ChoplinldquoShear stress effects on growth and activity of Lactobacillusdelbrueckii subsp bulgaricusrdquo Journal of Biotechnologyvol 29 no 1-2 pp 157ndash175 1993

[36] M Boonmee N Leksawasdi W Bridge and P L RogersldquoBatch and continuous culture of Lactococcus lactis NZ133experimental data and model developmentrdquo BiochemicalEngineering Journal vol 14 no 2 pp 127ndash135 2003

[37] A Ghandi I Powell M Broome and B Adhikari ldquoSurvivalfermentation activity and storage stability of spray driedLactococcus lactis produced via different atomization re-gimesrdquo Journal of Food Engineering vol 115 pp 83ndash90 2012

Journal of Chemistry 9