High Improvement in Lactic Acid Productivity by New Alkaliphilic …downloads.hindawi.com/journals/bmri/2019/7212870.pdf · 2019-07-30 · lactic-acid-and-poly-lactic-acid-market)

Research ArticleHigh Improvement in Lactic Acid Productivity by NewAlkaliphilic Bacterium Using Repeated Batch FermentationIntegrated with Increased Substrate Concentration

Mohamed Ali Abdel-Rahman 1 Saad El-Din Hassan1 Mohamed Salah Azab1

Abdullah-Al- Mahin2 and Mahmoud Ali Gaber1

1Botany and Microbiology Department Faculty of Science (Boys) Al-Azhar University PN11884 Nasr City Cairo Egypt2Microbiology and Industrial Irradiation Division (MIID) Institute of Food and Radiation Biology (IFRB)Atomic Energy Research Establishment (AERE) Ganakbari Savar Dhaka-1349 Bangladesh

Correspondence should be addressed to Mohamed Ali Abdel-Rahman mohamedalikyudaijp

Received 18 September 2018 Revised 22 November 2018 Accepted 6 January 2019 Published 17 January 2019

Academic Editor Fabiano J Contesini

Copyright copy 2019 Mohamed Ali Abdel-Rahman et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Optically pure lactic acid (LA) is an important chemical platform that has a wide range of industrial and biotechnologicalapplications Improved parameters for cost effective LA production are of great interest for industrial developments In the presentstudy an alkaliphilic lactic acid bacterium BoM 1-2 was selected among 369 newly obtained bacterial isolates It was characterizedusing API 50 CHL kit and identified as Enterococcus hirae BoM 1-2 by 16S rRNA gene sequence analysis Efficient polymer-gradeL-lactic acid production was achieved at pH 90 and 40∘C In batch fermentation strategy using 20 g Lminus1 glucose 196 g Lminus1 lacticacid was obtained with volumetric productivity of 218 g Lminus1 hminus1 While using 100 g Lminus1 glucose 960 g Lminus1 lactic acid was obtainedwith volumetric productivity of 107 g Lminus1 hminus1 The highest lactic acid concentration of 1806 g Lminus1 was achieved in multipulse fedbatch strategy with volumetric productivity of 065 g Lminus1 hminus1 To achieve higher productivity repeated fermentation processes wereapplied using the two different strategies In the first strategy the lactic acid productivity was increased from 197 g Lminus1 hminus1 to 448 gLminus1 hminus1 when the total of 10 repeated runs were carried out using 60 g Lminus1 glucose but lactic acid productivity decreased to 295 gLminus1 hminus1 using 100 g Lminus1 glucose In second strategy repeated fermentation coupled with gradual increase in glucose concentrationfrom 40 to 100 g Lminus1 was conducted for 24 runs A dramatic increase in LA productivity up to 399 g Lminus1 hminus1 (18-fold compared tofirst run) was achieved using 40 g Lminus1 glucose while volumetric productivity ranging between 248 and 299 g Lminus1 hminus1 was achievedusing 60ndash100 g Lminus1 glucose

1 Introduction

Lactic acid (LA) is an industrially important platformchemical that is utilized as pH regulator and preservativesfor food and beverages sector and for cosmetics textilespharmaceuticals and chemical industries It has receivedmore interest in recent years as a raw material for pro-duction of biodegradable plastics (poly-lactic acid PLA)[1] Global LA demands were estimated to be 19472kilo tons in 2018 and are expected to grow annuallyby 162 from 2019 to 2025 as a result of increasingsales of medicines and perfumes in different countries

(httpwwwgrandviewresearchcompress-releaseglobal-lactic-acid-and-poly-lactic-acid-market)

Lactic acid has two optical isomers [l- and d-lactic acid]which only can be produced via microbial fermentationas chemical synthesis produces racemic mixture [dl-LA]Biotechnological LA production is mostly driven by wildtypes or genetically engineered lactic acid bacteria (LAB)that can produce LA with high concentration yield andproductivity either through homofermentative or heterofer-mentative pattern based on the fermentation end product [2]Homofermentative LAB are of interest for commercial scalelactic acid production that produces lactic acid as the major

HindawiBioMed Research InternationalVolume 2019 Article ID 7212870 13 pageshttpsdoiorg10115520197212870

2 BioMed Research International

end productwithout by-productsHowevermost industriallyapplicable LAB are mesophilic (optimal temperature le 37∘C)andor neutrophilic (optimal pH 70) facilitating contam-ination risk with unwanted microbes that might decreasefermentation efficiency and affect the end product quality [1]Therefore utilization of thermotolerant LAB strains mightreduce this risk Besides utilization of alkaliphilic strainsis advantageous not only for minimizing contaminationproblems due to their tolerance to high salt levels butalso for reducing the neutralizing agents required duringLA fermentation [3] Few alkaliphilic LA producer strainshave been reported including Halolactibacillus halophilusExiguobacterium sp Enterococcus spp and Bacillus spp butachieving low LA productivity [4ndash7]

The efficiency of lactic acid fermentation ismainly depen-dent on the producer strain and fermentation strategy Batchfermentation strategy is the simplest process but it suffersfrom low cell biomass and low LA productivity owing toexhausted nutrients substrate andor product inhibition andelongated fermentation time [8] Fed batch fermentationstrategy might overcome substrate inhibition and results inhigh LA concentration but also resulted in low LA produc-tivity [9] On the other hand repeated fermentation strategythat involves repeated cycles by inoculating a part or all of thefree or immobilized cells from a previous run into the nextrun has reported several advantages in saving labor and seedpreparation time and achieving high LA productivity [10]This fermentation mode is usually conducted using the samesubstrate concentrations in several runs that might result inincreased LA productivity or attained the same productivityas initial run

This study focused on the challenge to increase LAconcentration and LA productivity from high glucose con-centration using repeated batch fermentation Additionallynew isolated alkaliphilic LAB were used in order to avoidthe risk of contamination with other microbes that mightreduce fermentation efficiencyWe also aimed to optimize theLA production by investigating the physical and nutritionalrequirements as well as fermentation strategies Thereforeestablishment of new strategy for high improvement of LAproductivity via investigating the effect of different sugarconcentrations coupled with repeated fermentation was con-ducted for commercial application purposes

2 Materials and Methods

21 Isolation and Screening of Alkaliphilic LAB Different soiland water samples as well dairy products were collected andused as isolation sources One gram or mL was mixed with100mL of saline solution (085 NaCl) and then 5mL of thesuspensions were added to 100mL-Erlenmeyer flask contain-ing 50mL of enrichment MRSmedium (MRS) and incubatedat 37∘C for 48 h MRS media consisted of 20 g Lminus1 glucose10 g Lminus1 peptone 10 g Lminus1 beef extract 4 g Lminus1 yeast extract5 g Lminus1 sodium acetate 2 g Lminus1 ammonium citrate 2 g Lminus1K2HPO4 01 g Lminus1MgSO

4 05 g Lminus1MnSO

4 and 1mL Tween

80 All chemicals are purchased from Sigma unless otherwisementioned The obtained bacterial colonies were grown onMRS agar medium supplemented with 04 g Lminus1 bromocresol

green to indicate acid production Positive acid-producingisolates were tested for catalase activity using 3 H

2O2for

primary selection of lactic acid bacteria All bacterial isolateswere kept at minus80∘C in MRS media containing 15 glycerolCatalase positive isolates were grown onMRS broth mediumwithoutwith supplementation of 50 and 100 g Lminus1 glucose or10 and 20 g Lminus1 of sodium acetate separately and incubatedat 37∘C for 30 h for quantitative determination of lactic acid

22 Characterization and Identification of Isolate BoM1-2Culture characteristics of the highest LA producer iso-late BoM1-2 were determined on MRS agar plates Gramreaction cell shape and arrangements were investigatedusing Gram stain Biochemical and physiological char-acteristics were determined using API 50 CHL test kit(bioMerieux Marcy lrsquo Etoile France) for sugar assay growthat different temperatures and growth at different concen-trations of sodium chloride Various enzymatic activitieswere assayed as previously described [11] Genomic DNAwas extracted according to the method of Miller et al[12] Bacterial 16S rRNA genes were amplified using thegenomic DNA as template and bacterial universal primersof 27 f (5-GAGTTTGATCACTGGCTCAG-3) and 1492r (5-TACGGCTACCTTGTTACGACTT-3) [13] The PCRmixture contained 1x PCR buffer 05mM MgCl

2 25 U

Taq DNA polymerase (QIAGEN Germantown MD 20874USA) 025mM dNTP 05 120583M of each primer and 1 120583Lof extracted genomic DNA The PCR was performed in aDNA Engine Thermal Cycler (PTC-200 BIO-RAD USA)with 94∘C for 3min followed by 30 cycles of 94∘C for30 s 55∘C for 30 s and 72∘C for 1min followed by afinal extension performed at 72∘C for 10min The PCRproducts were checked for the expected size on 1 agarosegel and purified using GeneJET PCR Purification Kit(Thermo K0701) The PCR products were sequenced onGATC Company by use ABI 3730xl DNA sequencer byusing forward and reverse primers The sequences werecompared against the GenBank database using the NCBIBLAST research Multiple sequence alignment was doneusing ClustalX 18 software package (httpwww-igbmcu-strasbgfrBioInfoclustalx) and a phylogenetic analysis wasconstructed by the maximum likelihood method based onthe Kimura two-parameter model using MEGA (Version 61)software with confidence tested by bootstrap analysis (1000repeats)

23 Optimization of Fermentation Conditions To determinethe optimal pH for LA production by strain E hirae BoM1-2 batch fermentation processes were conducted at 37∘C inthe 250mL-Erlenmeyer flasks containing 100mL workingvolume of MRS medium containing 20 g Lminus1 glucose Theflasks were inoculated at 10 from preculture incubated at37∘C for 30 hThe pHwas adjusted at 50 60 70 80 85 90100 and 110 using intermitted pH control with 10N NaOHSamples were taken periodically every 3ndash6 h to analyzecell growth (OD

600nm) glucose and LA concentrations Todetermine the best nitrogen sources for LA productiondifferent concentrations of yeast extract (0 25 50 75 100 g

BioMed Research International 3

Lminus1) peptone (0 50 100 150 200 250 g Lminus1) and beefextract (0 50 100 150 200 250 g Lminus1) were supplementedseparately in 250mL-Erlenmeyer flasks containing 100mL ofthe media described above at 37∘C pH controlled at 90 for30 h Samples were withdrawn periodically for the analysis ofgrowth and fermentation products To determine the optimaltemperature media were prepared and inoculated at 10 ofpreculture broth Fermentation processes were conducted at25 30 37 40 45 50 55 and 60∘C

24 Fed Batch and Repeated Batch Fermentation Multipulsefed batch fermentation was conducted in a 10-l jar fermentorwith a working volume of 300mL of optimizedMRSmediumusing E hirae BoM1-2 Fermentation was initiated by cellinoculation at 10 (vv) pH was controlled at 90 by 10 Nof NaOH at 40∘C Samples were taken every 3-6 h for theanalysis of cell growth (OD

600nm) glucose and LA concen-trations Intermitted feeding using glucose (30 or 40 g Lminus1)and yeast extract (1 g Lminus1) was added to the fermentor whenthe residual glucose concentration reached 10 g Lminus1 Feedingwas supplemented after 36 h (40 g Lminus1) 69 h (40 g Lminus1) 138 h(30 g Lminus1) and 204h (30 g Lminus1) of fermentation Repeatedbatch fermentation processes were performed using 300mLin 1L jar fermentor supplemented with the desired glucoseconcentrations at 40∘C pHwas controlled at 90 usingNaOHAt the end of fermentation processes fermentation brothwas centrifuged (6000 rpm for 10min at 5∘C) and the cellswere resuspended in new fresh media to perform next batch(run) Two fermentation strategies were conducted In thefirst strategy fermentation processes were conducted foreleven runs using optimized MRS medium supplementedwith 60 g Lminus1 glucose in the first 10 runs and 100 g Lminus1glucose in the last run In the second strategy fermentationprocesses were conducted for 24 runs At the first 22 runsoptimizedMRSmediawere usedwith different initial glucoseconcentrations Runs 1ndash13 were supplemented with 40 g Lminus1

glucose runs 14ndash16 were supplemented with 60 g Lminus1 glucoseruns 17ndash19 were supplemented with 80 g Lminus1 glucose runs20-22 were supplemented with 100 g Lminus1 glucose Runs 23-24were supplemented with 100 g Lminus1 glucose but exclusion ofpeptone from medium composition was carried out in 23rdrun while peptone and beef extract were excluded in 24thrun

25 Analytical Methods and Fermentation Parameters Cellgrowth was estimated based on OD

600nm measurementsusing a visible spectrophotometer Fermentation sampleswere centrifuged at 6000 rpm (4427xg) for 10min at 4∘Cand the supernatants were analyzed for glucose and LAdetermination Residual glucose in the broth was estimatedby using 3 5-dinitrosalicylic acid reagent (DNSmethod) [14]LA was measured by the modified Barker and Summersonmethod [15] and confirmed with HPLC analysis as describedpreviously [10] The concentrations of residual glucose andLA were calculated using calibration curves obtained usingthe standard solutions The yield of LA (Y g gminus1) was definedas the ratio of lactic acid produced (g Lminus1) to amount ofglucose consumed (g Lminus1) LA productivity (g Lminus1 hminus1) was

calculated as the ratio of lactic acid concentration (g Lminus1)to the fermentation time (h) at which the maximum lacticacid concentration was obtained Maximum productivity wascalculated between each sampling period within exponentialgrowth phase All experiments were conducted in triplicateand data were statistically analyzed by SPSS v17 (SPSS IncChicago IL USA) One-way analysis of variance (ANOVA)test was used for sample comparison when normality andhomogeneity of variance were satisfied and followed bymultiple comparison using Tukeyrsquos range tests at Plt005

3 Results

31 Isolation and Characterization of Alkaliphilic Lactic AcidBacteria A total of 369 purified bacterial isolates wereobtained from the 27 different sources collected from EgyptAmong them 80 bacterial isolates were primarily consideredas lactic acid bacteria that showed catalase negative activityand yellow color around the colonies on MRS plates contain-ing bromocresol green These isolates were cultured in brothmedia for quantitative determination of LAOut of 34 isolatesthat produced LA gt 2 g Lminus1 only 17 isolates were selectedbecause these isolates produced LA higher than 5 g Lminus1 withhigh yield gt 080 g gminus1-consumed sugars as shown in Fig S1(see supplementary data) These isolates also exhibited bettertolerance to high glucose concentration (up to 100 g Lminus1) andto acetic acid (up to 20 g Lminus1) [data not shown] Among themthe isolate BoM1-2 which was obtained from milk samplecollected from Beheira governorate at Egypt was describedas the highest lactic acid producerThis isolate produced 91 gLminus1 of LAwith highLAyield (095 g gminus1 of glucose consumed)and showed the highest growth at OD

600nm of 0887The selected isolate BoM1-2 was characterized by

biochemical and molecular methods As indicated in TableS1 (see supplementary data) isolate BoM1-2 is Gram positiveof coccus shape catalase negative and facultative anaerobicBoM1-2 produced l-lactic acid homofermentatively at anoptical purity of 997 The colonies are white in color andconvex on agar plates This isolate showed high growth ata wide range of temperature 25ndash60∘C and had the abilityto grow at broad spectrum of pH values 50ndash100 On theother hand this isolate could not utilize citrate urea pectinstarch cellulose or gelatine Moreover isolate BoM1-2showed no blood haemolysis on agar plates which showedthe safety of this isolate BoM1-2 can grow under highconcentration of sodium chloride up to 75 The isolatewas also characterized by bacterial identification kit of API-50 CHL which tested the acid formation from varioussugars and sugar alcohol As shown in Table S2 the sugarfermentation pattern of strain BoM1-2 excluding utilizationof l-arabinose d-mannitol and amygdaline showed thehighest similarity to those of Enterococcus hirae amongthose of enterococcal species described by Manero andBlanch [16] The molecular identification of 16S rRNA genefragments of this isolate showed that it was identified asEnterococcus hirae with 99 identity to Enterococcus hiraestrain Egypt H (GenBank accession number of KU847975)and Enterococcus hirae strain 080204 (GenBank accessionnumber of KU353616) Phylogenetic analysis based on 16S


100KU353616 Enterococcus hirae strain 080204

KU847975 Enterococcus hirae strain Egypt HEnterococcus hirae strain BoM1-2

NR 115763 Enterococcus avium strain ATCCNR 108137 Enterococcus rotai strain CCM 4630

DQ411815 Enterococcus sulfureus strain ATCC 49903KJ865598 Bacillus cereus strain LCw-22LC034566 Bacillus thuringiensis strain TBTK040311B

KC867306 Bacillus sp strain SO67HM461171 Bacillus sp clone HSL48A

KF646689 Bacillus methylotrophicus strain DH-17

KF583692 Paenibacillus barengoltzii strain 1-39KF278964 Brevibacillus agri strain Ysn

KF600772 Brevibacillus agri strain SMR24

JN935016 Bacillus amyloliquefaciens strain SWFU2928

100

100

100

100

002

87

87

97

92

77

63

Figure 1 Phylogenetic tree based on 16S rRNA gene sequences The evolutionary history was inferred by using the maximum likelihoodmethod based on the Kimura two-parameter model The percentage of replicate trees in which the associated taxa clustered together in thebootstrap test (1000 replicates) is shown next to the branches Black squares indicate the organisms isolated in this study GenBank accessionnumbers of reference sequences are indicated Bar 002 nucleotide substitutions per position

rRNA gene sequencing and alignment revealed that isolateBoM1-2 was clustered with Enterococcus hirae strains withsupported bootstrap value of 96 (Figure 1) Enterococcus hiraestrain BoM 1-2 16S ribosomal RNA gene has been depositedin NCBI GenBank under the accession number MK026806(httpswwwncbinlmnihgovsearchallterm=mk026806)

32 Optimization of Fermentation Conditions

321 Effect of Various pH Values and Nitrogen Sources onLactic Acid Production To investigate the influence of pHon lactic acid production by E hirae BoM1-2 fermenta-tion processes were conducted at various initial pH values(50ndash110) using the modifiedMRSmedia supplemented with20 g Lminus1 glucose (Table 1) Strain BoM1-2 showed comparablegrowth at pH 50ndash100 with OD

600nm range of 0930ndash104while decreased growth of 0620 was obtained at pH 110 withdrastic decrease in glucose consumption (36 g Lminus1) and LAproduction (30 g Lminus1) The strain exhibited homofermenta-tive LA production at all tested pH values with LA yieldsof 083ndash090 g gminus1-consumed glucose The highest glucoseconsumption of (154ndash156 g Lminus1) LA production (139 g Lminus1)andmaximumLAproductivities (19 g Lminus1 hminus1) were obtainedat pH 85-90 Higher yield at 090 g gminus1-consumed sugar wasobtained at pH 90 and therefore was selected as the optimalfermentation condition for further experiments

Different concentrations of nitrogen sources includingyeast extract (0ndash10 g Lminus1) peptone (0ndash25 g Lminus1) and beefextract (0ndash25 g Lminus1) were supplemented to modified MRSmedia to determine the best medium composition for BoM1-2 using 20 g Lminus1 glucose at pH 90 and 37∘C The highestOD600nm (113) sugar consumption (188 g Lminus1) LA con-

centration (170 g Lminus1) LA productivity (057 g Lminus1 hminus1) andmaximum LA productivity (29 g Lminus1 hminus1) were obtained in

medium contained 75 g Lminus1 yeast extract 20 g Lminus1 peptoneand 20 g Lminus1 of beef extract (data not shown) that is 22increment in LA concentration compared to control Inaddition supplementation of inorganic nitrogen sources didnot exhibited effect on increment of LA production

322 Effect of Different Temperatures on Lactic Acid Produc-tion Theeffect of temperature on the growth and productionof lactic acid by Enterococcus hirae was studied in therange of 25-60∘C and pH 90 for 30 h (Table 2) StrainBoM1-2 exhibited comparable growth pattern at all testedtemperatures that record high OD

600nm ranging from 097 to105 with the highest value at 40ndash45∘C Comparable glucoseconsumption was also achieved ranging between 187 and197 g Lminus1 at wide range of temperature (25ndash55∘C) with themaximum value of 197 g Lminus1 at 40∘C On the other handdecreased glucose consumption of 152 g Lminus1 was obtainedat 60∘C Similar pattern was exhibited for LA productionwithin the range 171ndash184 g Lminus1 at 25ndash55∘C with the highestvalue of 184 g Lminus1 at 40∘C Decreased LA production of 132 gLminus1 was obtained at 60∘C Maximum LA yield of 093 g gminus1-glucose consumed was obtained at 40∘Cwith amaximum LAproductivity of 31 g Lminus1hminus1 and total productivity of 061 gLminus1hminus1 so that 40∘C was selected as the optimal temperaturefor further studies

323 Effect of Different Glucose Concentrations on Lactic AcidProduction To investigate the effect of glucose concentrationon lactic acid production fermentation processes were con-ducted with different initial glucose concentrations (20ndash150 gLminus1) at pH controlled at 90 (Table 3 Figure 2) Strain BoM1-2 exhibited short lag phase at all tested concentrations withfinal biomass increment from OD

600nm of 199 at initial 20 gLminus1 glucose up to OD

600nm of 972 at initial 150 g Lminus1 glucose


Table 1 Effect of different pH values on the growth and kinetic parameters of lactic acid production from glucose by Enterococcus hiraeBoM1-2

pH value OD600 nma Consumed glucose (g L-1) LA conc (g L-1)b 119884LA (g g-1)c 119875LA (g L-1 h-1)d Max 119875LA (g L-1 h-1)e

50 0930plusmn005 680plusmn01 610plusmn01e 090 020e 080(6 h)60 0970plusmn003 850plusmn03 760plusmn02d 089 025d 090(6 h)70 101plusmn001 980plusmn01 880plusmn01c 090 029c 160 (3 h)80 101plusmn001 121plusmn02 109plusmn02b 090 036b 180 (3 h)85 103plusmn001 156plusmn02 139plusmn01a 089 046a 190 (3 h)90 104plusmn006 154plusmn03 139plusmn01a 090 046a 19 (3 h)100 0980plusmn005 101plusmn01 90plusmn00c 089 030c 080(3 h)110 0620plusmn002 360plusmn01 30plusmn00f 083 010f 020(6 h)aOD maximum optical density b Maximum lactic acid concentration after 30 h c Lactic acid yield d Lactic acid productivity at the end of fermentation timee Maximum lactic acid productivity at indicated time Different letters between columns denote that mean values are significantly different (ple005) by Tukeytest values are means plusmn SE (n=3)

Table 2 Effect of different temperatures on the growth and kinetic parameters of lactic acid production from glucose by Enterococcus hiraeBoM1-2

Temperature (∘C) OD600 nma Consumed glucose (g L-1) LA conc (g L-1)b 119884LA (g g-1)c 119875LA (g L-1 h-1)d Max119875LA (g L-1 h-1)e

25 101plusmn0002 187plusmn03 168plusmn01a 089 057a 2630 102plusmn0003 186plusmn01 169plusmn01a 091 057a 2837 104plusmn0004 189plusmn05 171plusmn03a 091 057a 2940 105plusmn0003 197plusmn00 184plusmn00a 093 061a 3145 105plusmn0004 196plusmn01 182plusmn01a 093 061a 3050 104plusmn0006 194plusmn02 179plusmn02a 092 060a 2855 101plusmn0006 195plusmn02 178plusmn02a 091 059a 2460 097plusmn0008 152plusmn03 132plusmn03b 087 044b 13aOD maximum optical density bMaximum lactic acid concentration after 30 h cLactic acid yield dLactic acid productivity at the end of fermentation timeeMaximum lactic acid productivity at indicated time Different letters between columns denote that mean values are significantly different (ple005) by Tukeytest values are means plusmn SE (n=3)

Table 3 Effect of different glucose concentrations on growth and kinetic parameters of lactic acid production by Enterococcus hirae BoM1-2

Conc ofglucose (gL-1)

OD600 nma LA conc (g

L-1)b 119884LA (g g-1)c 119875LA (g L-1 h-1)d Residual sugar(g L-1)

Fermentationtime (h)

Max 119875LA (g L-1

h-1)e

20 199plusmn0009 196plusmn01f 098 218a 01plusmn01 9 32 (3h)40 419plusmn0021 373plusmn05e 093 207a 00plusmn00 18 33 (3h)60 625plusmn0120 579plusmn01d 097 175b 00plusmn00 33 34 (3h)80 769plusmn0074 774plusmn00c 097 123c 05plusmn01 63 31 (3h)100 919plusmn0160 960plusmn01b 096 107d 00plusmn00 90 33 (3h)150 972plusmn0064 1099plusmn04a 096 102d 357plusmn04 108 26 (3h)aOD maximum optical density bMaximum lactic acid concentration cLactic acid yield dLactic acid productivity at the end of fermentation time eMaximumlactic acid productivity at indicated time Different letters between columns denote that mean values are significantly different (ple005) by Tukey test valuesare means plusmn SE (n=3)

(Figure 2(a)) The strain exhibited long stationary phase at80ndash150 g Lminus1 glucose while glucose consumption and LApro-duction was achieved Glucose was completely consumed atan initial glucose concentration 20ndash100 g Lminus1with productionof 196ndash960 g Lminus1 lactic acid at high yield ranging from 093 to096 g gminus1-consumed glucose (Figures 2(b) and 2(c)) On theother hand 359 g Lminus1 of glucose remained in fermentationmedium after 108 h in fermentation processes with 150 g Lminus1

glucose with maximum LA production of 1099 g Lminus1 LAproductivities were decreased with an increase of glucoseconcentration achieving the highest value of 218 g Lminus1hminus1

in fermentation processes using 20 g Lminus1 glucose and 102 gLminus1hminus1 in fermentation processes using 150 g Lminus1 glucosewhile themaximal LAproductivities were almost comparableat fermentation processes with 20ndash100 g Lminus1 glucose withinthe range 31ndash34 g Lminus1 hminus1 The highest value for maximalLA productivity of 34 g Lminus1 hminus1 was obtained from 60 g Lminus1glucose (Table 3)

33 Fed Batch Fermentation for Lactic Acid Production In atrial to increase LA production and productivity multipulsefed batch strategy was conducted with an initial glucose


20 gl 40gl 60 gl80 gl 100 gl 150 gl

20 40 60 80 100 1200Fermentation Time (h)

0123456789

10

Gro

wth

(OD

600n

m)

(a)

20 gl 40gl 60 gl80 gl 100 gl 150 gl


020406080

100120140160

Residu

al glucose

(gL)

(b)

Lactic

acid concentration

20 gl 40gl 60 gl80 gl 100 gl 150 gl

0

20

40

60

80

100

120

(gL)

24 48 72 96 1200Fermentation Time (h)

(c)

Figure 2 Profiles of lactic acid fermentation using different glucose concentrations by E hirae BoM1-2 (a) Growth [OD600nm] (b) Residual

glucose concentration [g Lminus1] (c) Lactic acid concentration [g Lminus1]

concentration of 60 g Lminus1 and feed with 40 g Lminus1 of glucoseand 1 g Lminus1 of YE when the glucose concentration reached toabout 10 g Lminus1 until the total added glucose reached 140 g Lminus1after that 30 g Lminus1 of glucose and 1 g Lminus1 of YE were addeduntil the glucose concentration reached 200 g Lminus1 As shownin Figure 3 the growth of the BoM1-2 strain was increasedwith time achieving maximum OD

600nm of 2203 at 72 hbecame almost stable for another 90h and then decreasedgradually to OD

600nm of 156 at the end of fermentation(276 h) Glucose consumption rate was gradually decreasedafter 72 h of fermentation that is in accordance with LAproduction rate A final LA concentration of 1806 g Lminus1 wasobtained with a yield of 098 g gminus1-consumed glucose at LAproductivity of 065 g Lminus1 hminus1 and residual glucose concentra-tion of 156 g Lminus1Therefore fed batch fermentation increasedfermentation parameters in terms of sugar consumption andLA production compared to batch mode while resulting indecreased LA productivity

34 Different Repeated Batch Fermentation Strategies Re-peated batch fermentation processes using all recycled cellswere conducted with an initial glucose concentration of 60 gLminus1 for the first 10 runs and 100 g Lminus1 glucose for the last

run (11th run) (Figures 4(a) and 4(b)) The growth of BoM1-2 strain was increased with subsequent runs achieving amaximum OD

600nm of 38 in first run and OD600nm of 137 at

the 10th run Fermentation time was greatly decreased from30h at the 1st run to 13 h at 9th and 10th runs with comparableLA concentrations (588-600 g Lminus1) and LA yields (097-099 g gminus1-consumed glucose) LA productivity was increasedfrom 195 g Lminus1 hminus1 at the 1st run up to 448 g Lminus1hminus1 at 10thrun (Table 4) Increasing glucose concentration to 100 g Lminus1at 11th run has resulted in long fermentation time (33h) withproduction of 972 g Lminus1 of LA at yield of 097 g gminus1-consumedglucose and LA productivity of 295 g Lminus1hminus1 (Table 4)

In a rational trial for improved LA productivity repeatedbatch fermentation processes were conducted with initialglucose concentration of 40 g Lminus1 for the first 13 runsand then glucose concentration was gradually increased insubsequent runs [60 g Lminus1 at runs 14th-16th 80 g Lminus1 atruns 17thndash19th and 100 g Lminus1 at runs 20thndash24th] (Table 5Figure 5) The data recorded in Table 5 summarized thekinetic parameters of all runs Strain BoM1-2 showed anincreased growth with repeated cycles from OD600nm 31 atthe 1st run up to 212 at 13th runs At these runs glucose wascompletely consumed with comparable LA production that


0

20

40

60

80

100

120

140

160

180

200

Glucose

and

lactic

acid (g

L)

0

4

8

12

16

20

24

28

Cell gro

wth

(OD

nm

)

40 80 120 160 200 240 2800Fermentation time (h)

Figure 3 Fed batch fermentation for lactic acid production by BoM1-2 using medium containing initial 60 g Lminus1 glucose at 40∘C Symbols998771 residual glucose (g Lminus1) LA (g Lminus1) e optical density at 600 nm

Table 4 Lactic acid production in repeated batch fermentation by Enterococcus hirae BoM1-2 using initial 60 g Lminus1 of glucose for the first 10runs and 100 g Lminus1 glucose for run 11th

BatchNumber

Initial glucose (gLminus1) OD

600 nma LA conc (g

Lminus1)bFermentation

time (h) 119884LA (g gminus1)c 119875LA (g Lminus1 hminus1)d Max 119875LA (g Lminus1hminus1)e

1 60 380plusmn0024 592plusmn04 30 099 197 352 60 470plusmn0560 590plusmn03 24 098 246 383 60 670plusmn0063 591plusmn02 22 099 269 394 60 840plusmn0038 578plusmn02 21 098 275 395 60 106plusmn0093 588plusmn03 20 098 294 46 60 123plusmn0570 581plusmn09 18 097 323 427 60 125plusmn0102 588plusmn05 16 098 368 458 60 129plusmn0097 581plusmn06 14 098 415 549 60 133plusmn0109 581plusmn04 13 097 447 5110 60 137plusmn0048 583plusmn07 13 097 448 511 100 150plusmn0102 972plusmn09 33 097 295 39aOD maximum optical density bMaximum lactic acid concentration after 30 h cLactic acid yield dLactic acid productivity at the end of fermentation timeeMaximum lactic acid productivity at indicated time

ranged from 396 to 399 g Lminus1 at LA yield of 099ndash10 g gminus1-consumed sugar In the 1st run fermentation lasted after 18 hafter that the fermentation time was significantly decreasedat the subsequent runs to achieve 18-fold decrease (only 1 h)at 12th and 13th run Significant increase in LA productivity(18-fold) was obtained which achieved 399 g Lminus1hminus1 at the13th run compared to 22 g Lminus1 hminus1 at the 1st run

To increase the final lactic acid concentration 14th-16thruns were conducted in the same MRS media supplementedwith 60 g Lminus1 glucose At the 14th run the growth wasfurther increased to achieve OD

600nm of 223 and completeconsumption of glucose was achieved with production of LAat 597 g Lminus1 at LA yield of 10 g gminus1-glucose consumed Onthe other hand fermentation timewas increased to 3 h which

achieved lower LA productivity of 199 g Lminus1 hminus1 compared toprevious runs Similar lactic acid concentration (594ndash598 gLminus1) and LA yield (099-10 g gminus1 of glucose consumed) wereobtained in the 15th and 16th run while shorter fermentationtime (2 h) and higher LA productivity (297 g Lminus1hminus1) wereobtained Almost comparable results were obtained in the16th run as 15th run with little increase in OD

600nm (247)(Table 5)

In a further attempt to increase the lactic acid concen-tration with the same components of MRS media 17thndash19thruns were conducted with higher concentration of 80 g Lminus1glucose As shown in Figure 5 OD

600nm was increased withinthe range 255ndash274 in these runs with complete consump-tion of glucose LA concentration was increased to achieve


Average OD

0

2

4

6

8

10

12

14

16

Cell gro

wth

(OD

nm

)

50 100 150 200 2500Fermentation time (h)

(a)

Residual glucose (gL) Lactic acid (gL)

0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)

20 40 60 80 100 120 140 160 180 200 220 2400Fermentation time (h)

(b)

Figure 4 Lactic acid production by E hirae BoM 1-2 in repeated batch fermentation (1ndash11 runs) with initial glucose concentration of 60 gLminus1 at the first 10 runs and 100 g Lminus1 at run 11 pH 90 at 40∘C (a) Cell growth (b) Residual glucose and lactic acid concentration Symbols 998771OD ◼ residual sugar ◻ lactic acid

794ndash796 g Lminus1 with high LA yield that ranged from 099to 10 g gminus1 On the other hand decreased LA productivitywas obtained at 17th run (198 g Lminus1hminus1) compared to theprevious 16th run (299 g Lminus1hminus1) while it became increasedin subsequent runs to achieve 2647ndash2653 g Lminus1hminus1 in run18th and 19th with short fermentation time of only 3 h

To further increase the LA concentration the 20thndash22ndruns were conducted using 100 g Lminus1 of glucose with thesame media components Growth and LA concentrationwere increased to record maximal value of 281 and 991 gLminus1 respectively after 5 h of fermentation time while LAproductivity decreased to 198 g Lminus1hminus1 compared to theprevious 19th run (265 g Lminus1 hminus1) On the subsequent run(21st) the increasing growth (OD

600nm of 289) resulted inincreasing LA productivity to 248 g Lminus1 hminus1 with almost thesame LA concentration Similar results were obtained at 22ndrunwith little increase of growth recordingmaximalOD

600nmof 296 with fermentation time of only 4 h

To decrease the production cost for LA fermentationby BoM1-2 strain reducing of nitrogen source in mediawas achieved through excluding the peptone from MRSmedia in 23rd run Growth LA concentration and LA yieldwere almost similar to previous run at 297 988 g Lminus1 and099 g gminus1 of glucose consumed respectively compared to22nd (containing peptone) at 296 992 g Lminus1 and 099 ggminus1 of glucose consumed respectively On the other handfermentation proceeded for longer time (6 h) than 4 h in22nd run which resulted in decrease of LA productivityto 1647 g Lminus1hminus1 compared to 248 g Lminus1hminus1 achieved inthe previous run Exclusion of peptone and beef extractin 24th run [medium contained only 75 g Lminus1 of YE asthe sole nitrogen source] resulted in a decrease in LA acidconcentration with only 920 g Lminus1 (compared to 988 g Lminus1 ofprevious run) and LAproductivity of 115 g Lminus1hminus1 comparedto 165 g Lminus1hminus1 was obtained On the other hand almostno changes in the cell growth and LA yield at 298 and


Table 5 Lactic acid production by repeated batch fermentation coupled with gradual increase in glucose concentrations from 40 to 100 gLminus1 by Enterococcus hirae BoM1-2 at pH 90 and 40∘C

BatchNumber

Initial glucose(g L-1) OD600 nm

a LA conc (gL-1)b 119884LA (g g-1)c 119875LA (g L-1 h-1)d Fermentation

time (h)Max 119875LA (g L-1

h-1)e

1 40 310plusmn0196 396plusmn02 099 220 18 322 40 540plusmn0030 398plusmn02 10 249 16 373 40 710plusmn0016 397plusmn02 099 284 14 454 40 880plusmn0001 397plusmn01 099 331 12 455 40 101plusmn0012 399plusmn03 10 399 10 486 40 114plusmn0013 396plusmn05 099 495 8 637 40 123plusmn0020 397plusmn02 099 662 6 708 40 139plusmn0016 396plusmn03 099 990 4 1159 40 149plusmn00217 397plusmn03 099 132 3 13910 40 167plusmn0013 399plusmn04 10 199 2 22711 40 179plusmn0019 399plusmn05 10 266 15 28212 40 200plusmn0114 397plusmn02 099 397 1 47313 40 212plusmn0013 399plusmn02 10 399 1 49214 60 223plusmn0576 597plusmn02 10 199 3 24515 60 234plusmn0032 594plusmn04 099 297 2 34716 60 247plusmn0418 598plusmn03 10 299 2 36217 80 255plusmn0016 795plusmn03 099 199 4 22618 80 268plusmn0131 794plusmn05 099 265 3 29519 80 274plusmn0521 796plusmn03 10 265 3 31020 100 281plusmn0070 991plusmn03 099 198 5 23421 100 289plusmn0340 992plusmn05 099 248 4 27822 100 296plusmn0253 992plusmn03 099 248 4 28323 100 297plusmn0079 988plusmn05 099 165 6 21524 100 298plusmn0435 920plusmn04 099 115 8 142aOD maximum optical density bMaximum lactic acid concentration after 30 h cLactic acid yield dLactic acid productivity at the end of fermentation timeeMaximum lactic acid productivity at indicated time

099 g gminus1 of glucose consumed respectively were obtainedas compared to previous runs From the above resultsrepeated batch fermentation by E hirae BoM1-2 coupledwith increased glucose concentration strain achieved gradualincrease in cell growth and greatly improved LA productivityas compared to fermentation in batchmodes using respectivesugar concentrations

4 Discussion

Lactic acid (LA) and its derivatives are among the mostimportant products having applications in many industries[9] LA can be produced by various microorganisms includ-ing bacteria fungi andmicroalgae however bacterial strainsare preferred over other microorganisms because of theirfast growth and high productivities [8] For commercialapplications robust microbes that can meet the industrialneeds for high LA titre productivity yield and high opticalpurity are required In addition overcoming some produc-tion challenges such as contamination risk by neutrophilicmicroorganisms and reduction of neutralizing agents is ofgreat importance [1] Consequently new microbial isolateswith enhanced LA production capabilities are needed to

reduce the production and purification costs Therefore thepresent study aimed to isolate and characterize potential alka-liphilic LAB and improve the production and productivity byusing different modes of fermentation

In this study 369 bacterial isolates were obtained fromdifferent environmental samples collected from differentlocalities in Egypt among those 80 isolates produced acidon agar medium and showed no catalase activity that pre-liminary considered as LAB Quantitative determination ofLA investigation of high glucose tolerance and high aceticacid tolerance indicated that isolate BoM 1-2 is considered asthe most potent LAB producer based on LA production LAyield and homofermentative LA production pattern Mor-phological and biochemical characterization revealed thatthis isolate was similar to Enterococcus spp (Tables S1 and S2see supplementary data) Molecular identification dependingon 16S rRNA gene fragments amplifications and sequencingconfirmed that this isolate was identified as Enterococcushirae BoM1-2 Natural sources are always the most powerfulmeans for obtaining useful and genetically stable strainsfor industrially important products [17] Several enterococcistrains have been isolated from natural resources such asthermophilic E faecium QU 50 [17] and E faecalis CBRD01


05

1015202530

Cell gro

wth

(OD

nm

)


(a)


0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)

(b)

Figure 5 Repeated batch fermentation (1ndash24 runs) for lactic acid production byBoM1-2 usingmediumcontaining initial 40ndash100 g Lminus1 glucoseat 40∘C pH controlled at 90 (a) Cell growth profile (b)Glucose consumption and lactic acid production Symbols998771 average optical densityOD at 600nm ◼ glucose concentration ◻ produced lactic acid

[18] that were isolated from soil sample and E mundtii QU25 that was isolated from bovine fecal samples [19]

To improve LA production in E hirae BoM 1-2 fer-mentation parameters including pH medium compositiontemperature and initial sugar concentrations were variedand modified E hirae BoM1-2 strain showed better growthsugar consumption LA production LA productivity and LAyield at alkaline pH values of 85ndash90 On the other handlow pH (less than 70) and highly alkaline pH (more than100) had negative effect on the growth of strain BoM1-2 which eventually reflected in terms of less metabolitesproduction This might be due to the enzyme denatura-tion at these conditions These results indicated that strainBoM 1-2 is alkaliphilic that is more advantageous for LAproduction than commonly used LAB Alkaliphilic strainscan tolerate high levels of salts and consequently reduce therisk of contamination and decrease the amount of requiredneutralizing agents [4] This is the first study to reporton alkaliphilic production of LA using E hirae Previouslyreported Enterococcus strains achieved LA fermentation atneutral or slightly acidic conditions Abdel-Rahman et al [10]reported that the optimal pH for LA production from glucosein batch fermentation by E mundtii QU 25 was 70 Value of65 was considered as the optimal pH for LA production by Efaeciumusing xylose [17] or starch [20] in batch fermentation

LAB bacteria are fastidious organisms which need com-plex nutrients as amino acids and vitamins for cell growth[21] therefore it is necessary to determine the optimal mediacomposition for the selected strains Different concentrations

of nitrogen sources were tested at pH 90 The optimal con-centrations of yeast extract peptone and beef extract were75 g Lminus1 20 g Lminus1 and 20 g Lminus1 respectively which achievedthe highest fermentation efficiency Yeast extract peptoneand beef extract supplement contain critical amounts ofvitamins and trace elements are essential for growth andLA biosynthesis [22] Higher concentrations led to decreaseof all fermentation parameters indicating that these highconcentrationsmight be toxic [22] Also the absence of any ofthese nitrogen sources decreases the fermentation efficiencyand leads to weak growth due to nutritional limitations anddeficiency in peptide sources (amino acids) or in growthfactors Dumbrepatil et al [23] reported that YE at 10 g Lminus1 isthe optimum concentration for LA production from corncobmolasses Chiarini et al [24] reported that a maximumLA concentration by Lb helveticus using whey permeateprovided with 15ndash25 g Lminus1 yeast extract Also Wee et al [25]found that the optimal concentration of YE that achieved thehighest productivity and dry cell weight for E faecalis RKY1was 20 g Lminus1 while Kotzamanidis et al [22] reported that the50 g Lminus1 YE was the optimum concentration of YE for allfermentation parameters by L delbrueckiiNCIMB 8130 usingbeet molasses as the sole carbon source

It is important to maintain the operating temperatureat the optimal level because it affects the rate of growthenzyme activities biochemical reactions as well as substrateconsumption rate and LA production efficiency [2 17 19]In this study E hirae BoM1-2 produced LA at a wide range


of temperature 25ndash55∘C with the optimal fermentation at40∘CHigher temperature over 55∘Cadversely affected the LAfermentationThismight be due to the decreased biochemicalreactions rates generation time substrate conversion rateand the enzymatic activities catalysing LA production [17]Most of the lactic acid-producing enterococci described inthe literature show maximum production of lactic acid inthe temperature range of 30-43∘C Sun et al [26] foundthat 40∘C was the optimal temperature for LA productionby E faecium Nandasana and Kumar [27] reported that38∘C was the optimal temperature for LA production by EfaecalisRKY1 while Abdel-Rahman et al [10 19 28] reportedthat maximum LA production from cellobiose glucose andxylose occurred at 43∘C by E mundtii QU 25 Shibata et al[20] found that 30∘C was the optimal temperature for theproduction of lactic acid from sago starch by E faecalis Incontrast Abdel-Rahman et al [17] found that 50∘C was theoptimal temperature for the production of lactic acid fromxylose by E faecium QU 50

For commercial production of LA high LA concentra-tion yield and productivity are important key fermentationparameters to reduce the production cost The high initialsubstrate tolerance is very important for getting high LAproduction to reduce the downstream processing costs [18] Interestingly fermentation by BoM1-2 strain under pHcontrol at 90 showed increased biomass with an increase ofinitial glucose concentration from 199OD

600nm at initial 20 gLminus1 of glucose up to 972 OD

600nm at 150 g Lminus1 glucose withhomofermentative production of LA at yield range of 090-098 g gminus1 of glucose consumed This indicates that strainBoM1-2 exhibited osmotolerance at high glucose concen-trations which offers potential advantages in fermentativeLA production enabling high production and facilitatingdownstream processing This also indicates that substrateinhibition may be negligible because it showed a short initiallag phase followed by a rapid growth phase and the growth isnot suppressed at all tested concentrations Stationary phaseappears to be long at high initial concentration of glucose(80-150 g Lminus1) after the cell mass reached the maximum valueas a result of accumulation of LA andor nutrient exhaus-tion On the other hand LA was still produced as glucosemetabolized but led to decreased LA productivity at highglucose concentration due to the increase of fermentationtime The maximum LA productivity of 218 g Lminus1hminus1 wasachieved at 20 g Lminus1 glucose and decreased by increasingglucose concentration Moreover the residual sugar wasalso increased as a result of LA accumulation in the mediathat might indicate end product inhibition LA fermentationefficiency by strain BoM1-2 using glucose was mostly compa-rable to the LA concentration LA yield and LA productivityobtained using cellobiose (898 g L 0913 g gminus1 and 125 gLminus1hminus1 respectively) and xylose (866 g Lminus1 083 g gminus1 and0902g Lminus1 hminus1 respectively) [19 28] from 100 g Lminus1 of eachsugar in batch fermentation Our results are in agreementwith other reports showing increased LA concentration withthe increase of initial glucose concentrations while veryhigh concentrations of sugar resulted in increasing osmoticpressure and decreasing LA productivity [10 11]

The challenge to increase LA concentration and LAproductivity from high glucose concentrations needs to beaddressed for commercial LA production We conductedmultipulse fed batch fermentation to reduce the osmoticstress of high initial sugar on microbial growth achievedin the batch culture condition that might combine withplasmolysis and result in a decrease in the rate of fermentationand sugar utilization A high increase in cell biomass achievedamaximum value of 2203 [OD600nm] at 72 h of fermentationA final LA concentration of 1806 g Lminus1 was obtained after276 h of fermentation This led to decreasing the total LAproductivity recording 065 g Lminus1hminus1 that is lower thanthat obtained by batch fermentation No by-products weredetected at all fermentation time with LA yield at 098 ggminus1-glucose consumed Results obtained in the current studyunder alkalophilic condition were superior to other LABstrains reported so far Yoshimune et al [29] achieved amaximum LA production of 149 g Lminus1 and 153 g Lminus1 from180 g Lminus1 glucose at pH 90 and 35∘C by E casseliflavusL-120 and E faecalis AY103 respectively A maximum LAconcentration of 103 g Lminus1 and LA yield of 08 g gminus1 were from129 g Lminus1 of glucose by E casseliflavus strain 79w3 at pH 80 inbatch fermentation [6] Exiguobacterium sp 8-11-1 produced125 g Lminus1 of LA at pH 85 and 37∘C in fed batch fermentation[5]

The repeated batch process involves repeated cycles offermentation by reinoculating a part or all of the cellsfrom one batch fermentation broth into the next batchfermentation medium [30] In comparison with batch orfed batch culture repeated batch operation has proved tohave several advantages in increasing LAproductivity besidessaving the time and labor work [30] BoM1-2 strain was firstlycultivated in repeated batch fermentation using 60 g Lminus1 ofglucose for 10 runs at controlled pH 90 Increased cell masswas obtained from OD of 38 at the 1st run up to 137 at 10thrun that consequently resulted in increase of LA productivityfrom 197 g Lminus1hminus1 at 1st run to 448 g Lminus1hminus1 at 10th run(227-fold) while the final LA concentration and LA yieldremain at similar valueThis improvement of LAproductivitywas attributed to increased cell biomass In the 11th runthe fermentation with 100 g Lminus1 of glucose resulted in littleincrease in biomass to OD of 150 with LA concentration of972 g Lminus1 at yield of 097 g gminus1 of glucose consumed whiledecrease in LA productivity of 295 g Lminus1hminus1 was obtained ascompared to 448 g Lminus1 hminus1 obtained in the previous run

In another strategy we conducted repeated batch fermen-tation processes coupled with gradual increase of glucoseconcentrations in the range 40-100 g Lminus1 as a rational trialfor cell adaption to higher sugar concentrations via 24-run fermentation High increase in cell biomass (OD

600nmof 212) was obtained at 13th run using 40 g Lminus1 glucoseThis resulted in a significant increase in the LA productivitycompared to previous experiment where LA productivityincreased by 18-fold from 22 g Lminus1hminus1 at 1st run to 399 gLminus1hminus1 at 13th run In the subsequent three runs using60 g Lminus1 glucose cell biomass was further increased result-ing in increased LA concentration (597 g Lminus1) with LA


productivity of 299 g Lminus1hminus1 compared to LA productivityof 175 obtained in normal batch fermentation (Table 3) Forfurther increase of LA concentration the three subsequentruns were conducted using initial 80 g Lminus1 of glucose andthen another three runs were performed with 100 g Lminus1 ofglucose Interestingly the strain showed the same behavioras that obtained with previous runs of increased cell biomasscomplete glucose consumption and increased LAproductionas expected LAproductivitywas 264 and 248 g Lminus1hminus1 using80 and 100 g Lminus1 of glucose respectively compared to 123and 107 g Lminus1hminus1 obtained in normal batch fermentation(Table 3) that is about 23- and 21-fold respectively Thissignificant increment of LA productivity of about 17-23 wasnot reported previously in literature for LA productionAlthough decreased cell growth was obtained with nitrogensource exclusion in subsequent runs (23th and 24th) LAproductivity was 115-165 g Lminus1 hminus1 which is also higher thanresults obtained in normal batch fermentation with completemedium (107 g Lminus1hminus1 Table 3) This superior improvementmight be due to the adaption of cultivated cells to higher sugarconcentrations and increasing the potency and efficiency ofmicrobial cells for substrate conversion and LA productionThis indicated that our new strategy is a significant findingfor improved green chemical productivity To the best of ourknowledge this study is not only the first study to reporton the LA production by repeated batch fermentation underalkaliphilic conditions but also the first study to reportvery high LA productivity (399 g Lminus1 hminus1) and to achieve24 runs for repeated batch in LA fermentation Thereforewe succeeded in establishing a highly productive system forobtaining LA at high concentration and high productivity inrepeated batch fermentation for at least 24 runs within 1405 hthat would greatly reduce the production cost of LA Thereare only a few reports that related LA production for usingrepeated batch fermentation by LAB Abdel-Rahman et al[10] achieved the highest LA productivity of 167 g Lminus1hminus1by E mundtii QU 25 Oh et al [21] achieved the highestLA productivity of 603ndash620 g Lminus1 by E faecalis RKY1 with100 g Lminus1 glucoseThe highest LA productivity of 40 g Lminus1hminus1was obtained by E faecalis RKY1 from wood hydrolyzatecontaining 50 g Lminus1 glucose [31] Kim et al [32] obtainedLA productivity at 634 g Lminus1hminus1 from 100 g Lminus1 lactose incheese whey by Lactobacillus sp RKY2 through the cell-recycle repeated batch fermentation

In conclusion efficient production of lactic acid fromalkaliphilic wild type E hirae BoM1-2 was obtained Its alka-liphilic characteristics could reduce the risk of contaminationduring fermentation process and using NaOH as neutralizingagent allowed for cleaner processing Batch fermentationexhibited limited lactic acid productivity of 218 g Lminus1 hminus1 asa maximum total LA productivity at pH 90 and 40∘C Lacticacid concentration was substantially enhanced by fed batchfermentation that reached 1806 g Lminus1 Repeated batch fer-mentation processes were performed for 24 runs without anyloss in the fermentation efficiency Lactic acid productivityandmaximal productivity reached the highest values in lacticacid fermentation technique 399 g Lminus1hminus1 and 497 g Lminus1hminus1

respectively which resulted in a considerable decrease in thefermentation time as a result of significant increase of culturebiomass Therefore we could establish a highly efficientsystem for the production of polymer-grade lactic acid usingE hirae BoM1-2 a strong candidate for industrial productionof lactic acid with superior LA productivity via couplingincreased sugar concentration during repeated fermentation

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Mohamed Ali Abdel-Rahman and Saad El-Din Hassan con-tributed equally to the manuscript

Acknowledgments

The authors are greatly thankful to Prof Dr Ayman FaragDardear DrMahmoud Esmael and the members of Fermen-tation Biotechnology and Applied Microbiology Centre Al-Azhar University for their great support technical assistanceand contributions throughout this workThe authors are alsoindebted to Dr Abdullah-Al-Mahin for his support contri-bution during study helpful comments on the organizationof the paper discussion and critical evaluation during thepreparation of this paper

Supplementary Materials

Table S1 morphological and biochemical characterizationof isolate BoM1-2 Table S2 sugar fermentation pattern ofisolate BoM1-2 by API 50 CHL and data for Enterococcushirae described by Manero and Blanch (1999) Fig S1lactic acid production and yield by the selected isolates inMSR medium containing 20 g Lminus1 glucose at 37∘C for 30 hSymbols ◼ lactic acid concentration g Lminus1 ◻ lactic acid yieldg gminus1 of glucose consumed (Supplementary Materials)

References

[1] M A Abdel-Rahman and K Sonomoto ldquoOpportunities toovercome the current limitations and challenges for efficientmicrobial production of optically pure lactic acidrdquo Journal ofBiotechnology vol 236 pp 176ndash192 2016

[2] Y Wang M A Abdel-Rahman Y Tashiro et al ldquol-(+)-Lactic acid production by co-fermentation of cellobiose andxylose without carbon catabolite repression using Enterococcusmundtii QU 25rdquo RSC Advances vol 4 no 42 pp 22013ndash220212014

[3] Y Meng Y Xue B Yu C Gao and Y Ma ldquoEfficient productionof l-lactic acid with high optical purity by alkaliphilic BacillusspWL-S20rdquo Bioresource Technology vol 116 pp 334ndash339 2012


[4] B P Calabia Y Tokiwa and S Aiba ldquoFermentative productionof l-(+)-lactic acid by an alkaliphilic marine microorganismrdquoBiotechnology Letters vol 33 no 7 pp 1429ndash1433 2011

[5] X Jiang Y Xue A Wang et al ldquoEfficient production ofpolymer-grade l-lactate by an alkaliphilic Exiguobacteriumsp strain under nonsterile open fermentation conditionsrdquoBioresource Technology vol 143 pp 665ndash668 2013

[6] H Yokaryo and Y Tokiwa ldquoIsolation of alkaliphilic bacteria forproduction ofhigh optically pure l-(+)-lactic acidrdquoe Journalof General and AppliedMicrobiology vol 60 no 6 pp 270ndash2752015

[7] N Assavasirijinda D Ge B Yu Y Xue and Y Ma ldquoEfficientfermentative production of polymer-grade d-lactate by anengineered alkaliphilic Bacillus sp strain under non-sterileconditionsrdquo Microbial Cell Factories vol 15 no 1 article no 32016

[8] M A Abdel-Rahman Y Tashiro and K Sonomoto ldquoRecentadvances in lactic acid production by microbial fermentationprocessesrdquo Biotechnology Advances vol 31 no 6 pp 877ndash9022013

[9] J Tan M A Abdel-Rahman and K Sonomoto ldquoBiorefinery-Based Lactic Acid Fermentation Microbial Production of PureMonomer Productrdquo inAdvances in Polymer Science vol 279 pp27ndash66 Springer New York LLC 2018

[10] M A Abdel-Rahman Y Tashiro T Zendo and K SonomotoldquoImproved lactic acid productivity by an open repeated batchfermentation system using Enterococcus mundtii QU 25rdquo RSCAdvances vol 3 no 22 pp 8437ndash8445 2013

[11] M A Abdel-Rahman S E D Hassan M S Azab and M AGaber ldquoEffective production of lactic acid by a newly isolatedalkaliphilic Psychrobacter maritimus BoMAir 5 strainrdquo Journalof Applied Biotechnology Bioengineering vol 1 no 3 pp 68ndash762016

[12] D N Miller J E Bryant E L Madsen and C W GhiorseldquoEvaluation and optimization of DNA extraction and purifi-cation procedures for soil and sediment samplesrdquo Applied andEnvironmental Microbiology vol 65 no 11 pp 4715ndash4724 1999

[13] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic AcidsTechniques in Bacterial Systematics E Stackebrandt and MGoodfellow Eds pp 115-47 John Wiley amp Sons ChichesterUK 1991

[14] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959

[15] S B Barker and W H Summerson ldquoThe colorimetric deter-mination of lactic acid in biological materialrdquo e Journal ofBiological Chemistry vol 138 pp 535ndash554 1941

[16] AManero andA R Blanch ldquoIdentification ofEnterococcus sppwith a biochemical keyrdquo Applied and Environmental Microbiol-ogy vol 65 no 10 pp 4425ndash4430 1999

[17] M A Abdel-Rahman Y Tashiro T Zendo K Sakai and KSonomoto ldquoEnterococcus faeciumQU 50 A novel thermophiliclactic acid bacterium for high-yield l-lactic acid productionfrom xyloserdquo FEMSMicrobiology Letters vol 362 no 2 pp 1ndash72015

[18] M R Subramanian S Talluri and L P Christopher ldquoProduc-tion of lactic acid using a new homofermentative Enterococcusfaecalis isolaterdquo Microbial Biotechnology vol 8 no 2 pp 221ndash229 2015

[19] M A Abdel-Rahman Y Tashiro T Zendo K Shibata andK Sonomoto ldquoIsolation and characterisation of lactic acid

bacterium for effective fermentation of cellobiose into opticallypure homo l-(+)-lactic acidrdquoAppliedMicrobiology and Biotech-nology vol 89 no 4 pp 1039ndash1049 2011

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

[21] H Oh Y-J Wee J-S Yun and H-W Ryu ldquoLactic acid produc-tion through cell-recycle repeated-batch bioreactorrdquo AppliedBiochemistry and Biotechnology - Part A Enzyme Engineeringand Biotechnology vol 107 no 1-3 pp 603ndash614 2003

[22] C Kotzamanidis T Roukas and G Skaracis ldquoOptimizationof lactic acid production from beet molasses by Lactobacillusdelbrueckii NCIMB 8130rdquo World Journal of Microbiology andBiotechnology vol 18 no 5 pp 441ndash448 2002

[23] A Dumbrepatil M Adsul S Chaudhari J Khire and DGokhale ldquoUtilization of molasses sugar for lactic acid produc-tion by Lactobacillus delbrueckii subsp delbrueckiimutant Uc-3in batch fermentationrdquo Applied and Environmental Microbiol-ogy vol 74 no 1 pp 333ndash335 2008

[24] L Chiarini L Mara and S Tabacchioni ldquoInfluence of growthsupplements on lactic acid production in whey ultrafiltrate byLactobacillus helveticusrdquo Applied Microbiology and Biotechnol-ogy vol 36 no 4 pp 461ndash464 1992

[25] Y-J Wee J-N Kim J-S Yun and H-W Ryu ldquoUtilizationof sugar molasses for economical l(+)-lactic acid productionby batch fermentation of Enterococcus faecalisrdquo Enzyme andMicrobial Technology vol 35 no 6-7 pp 568ndash573 2004

[26] W Sun J Liu H Xu W Li and J Zhang ldquol-Lactic acidfermentation by Enterococcus faecium a new isolate frombovine rumenrdquo Biotechnology Letters vol 37 no 7 pp 1379ndash1383 2015

[27] A D Nandasana and S Kumar ldquoKinetic modeling of lactic acidproduction from molasses using Enterococcus faecalis RKY1rdquoBiochemical Engineering Journal vol 38 no 3 pp 277ndash2842008

[28] M A Abdel-Rahman Y Tashiro T Zendo K Hanada KShibata and K Sonomoto ldquoEfficient homofermentative l-(+)-Lactic acid production from xylose by a novel lactic acidbacterium Enterococcus mundtii QU 25rdquo Applied and Environ-mental Microbiology vol 77 no 5 pp 1892ndash1895 2011

[29] K Yoshimune M Yamamoto T Aoyagi and I Yumoto ldquoHighandRapid l-lactic Acid Production byAlkaliphilic Enterococcussp by Adding Wheat Bran Hydrolysaterdquo Fermentation Technol-ogy vol 06 p 138 2017

[30] J Tan M A Abdel-Rahman M Numaguchi et al ldquoTher-mophilic Enterococcus faecium QU 50 enabled open repeatedbatch fermentation for l-lactic acid production from mixedsugarswithout carbon catabolite repressionrdquoRSCAdvances vol7 no 39 pp 24233ndash24241 2017

[31] Y-JWee J-S Yun D Kim andH-W Ryu ldquoBatchand repeatedbatch production of l(+)-lactic acid by Enterococcus faecalisRKY1 using wood hydrolyzate and corn steep liquorrdquo Journalof Industrial Microbiology and Biotechnology vol 33 no 6 pp431ndash435 2006

[32] H O Kim Y J Wee J N Kim J S Yun and H WRyu ldquoProduction of lactic acid from cheese whey by batchand repeated batch cultures of Lactobacillus sp RKY2rdquo inProceedings of the Twenty-Seventh Symposium on Biotechnologyfor Fuels and Chemicals pp 694ndash704 Humana Press 2006

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018


Genetics Research International


Advances in

Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


end productwithout by-productsHowevermost industriallyapplicable LAB are mesophilic (optimal temperature le 37∘C)andor neutrophilic (optimal pH 70) facilitating contam-ination risk with unwanted microbes that might decreasefermentation efficiency and affect the end product quality [1]Therefore utilization of thermotolerant LAB strains mightreduce this risk Besides utilization of alkaliphilic strainsis advantageous not only for minimizing contaminationproblems due to their tolerance to high salt levels butalso for reducing the neutralizing agents required duringLA fermentation [3] Few alkaliphilic LA producer strainshave been reported including Halolactibacillus halophilusExiguobacterium sp Enterococcus spp and Bacillus spp butachieving low LA productivity [4ndash7]

The efficiency of lactic acid fermentation ismainly depen-dent on the producer strain and fermentation strategy Batchfermentation strategy is the simplest process but it suffersfrom low cell biomass and low LA productivity owing toexhausted nutrients substrate andor product inhibition andelongated fermentation time [8] Fed batch fermentationstrategy might overcome substrate inhibition and results inhigh LA concentration but also resulted in low LA produc-tivity [9] On the other hand repeated fermentation strategythat involves repeated cycles by inoculating a part or all of thefree or immobilized cells from a previous run into the nextrun has reported several advantages in saving labor and seedpreparation time and achieving high LA productivity [10]This fermentation mode is usually conducted using the samesubstrate concentrations in several runs that might result inincreased LA productivity or attained the same productivityas initial run

This study focused on the challenge to increase LAconcentration and LA productivity from high glucose con-centration using repeated batch fermentation Additionallynew isolated alkaliphilic LAB were used in order to avoidthe risk of contamination with other microbes that mightreduce fermentation efficiencyWe also aimed to optimize theLA production by investigating the physical and nutritionalrequirements as well as fermentation strategies Thereforeestablishment of new strategy for high improvement of LAproductivity via investigating the effect of different sugarconcentrations coupled with repeated fermentation was con-ducted for commercial application purposes

2 Materials and Methods

21 Isolation and Screening of Alkaliphilic LAB Different soiland water samples as well dairy products were collected andused as isolation sources One gram or mL was mixed with100mL of saline solution (085 NaCl) and then 5mL of thesuspensions were added to 100mL-Erlenmeyer flask contain-ing 50mL of enrichment MRSmedium (MRS) and incubatedat 37∘C for 48 h MRS media consisted of 20 g Lminus1 glucose10 g Lminus1 peptone 10 g Lminus1 beef extract 4 g Lminus1 yeast extract5 g Lminus1 sodium acetate 2 g Lminus1 ammonium citrate 2 g Lminus1K2HPO4 01 g Lminus1MgSO

4 05 g Lminus1MnSO

4 and 1mL Tween

80 All chemicals are purchased from Sigma unless otherwisementioned The obtained bacterial colonies were grown onMRS agar medium supplemented with 04 g Lminus1 bromocresol

green to indicate acid production Positive acid-producingisolates were tested for catalase activity using 3 H

2O2for

primary selection of lactic acid bacteria All bacterial isolateswere kept at minus80∘C in MRS media containing 15 glycerolCatalase positive isolates were grown onMRS broth mediumwithoutwith supplementation of 50 and 100 g Lminus1 glucose or10 and 20 g Lminus1 of sodium acetate separately and incubatedat 37∘C for 30 h for quantitative determination of lactic acid

22 Characterization and Identification of Isolate BoM1-2Culture characteristics of the highest LA producer iso-late BoM1-2 were determined on MRS agar plates Gramreaction cell shape and arrangements were investigatedusing Gram stain Biochemical and physiological char-acteristics were determined using API 50 CHL test kit(bioMerieux Marcy lrsquo Etoile France) for sugar assay growthat different temperatures and growth at different concen-trations of sodium chloride Various enzymatic activitieswere assayed as previously described [11] Genomic DNAwas extracted according to the method of Miller et al[12] Bacterial 16S rRNA genes were amplified using thegenomic DNA as template and bacterial universal primersof 27 f (5-GAGTTTGATCACTGGCTCAG-3) and 1492r (5-TACGGCTACCTTGTTACGACTT-3) [13] The PCRmixture contained 1x PCR buffer 05mM MgCl

2 25 U

Taq DNA polymerase (QIAGEN Germantown MD 20874USA) 025mM dNTP 05 120583M of each primer and 1 120583Lof extracted genomic DNA The PCR was performed in aDNA Engine Thermal Cycler (PTC-200 BIO-RAD USA)with 94∘C for 3min followed by 30 cycles of 94∘C for30 s 55∘C for 30 s and 72∘C for 1min followed by afinal extension performed at 72∘C for 10min The PCRproducts were checked for the expected size on 1 agarosegel and purified using GeneJET PCR Purification Kit(Thermo K0701) The PCR products were sequenced onGATC Company by use ABI 3730xl DNA sequencer byusing forward and reverse primers The sequences werecompared against the GenBank database using the NCBIBLAST research Multiple sequence alignment was doneusing ClustalX 18 software package (httpwww-igbmcu-strasbgfrBioInfoclustalx) and a phylogenetic analysis wasconstructed by the maximum likelihood method based onthe Kimura two-parameter model using MEGA (Version 61)software with confidence tested by bootstrap analysis (1000repeats)

23 Optimization of Fermentation Conditions To determinethe optimal pH for LA production by strain E hirae BoM1-2 batch fermentation processes were conducted at 37∘C inthe 250mL-Erlenmeyer flasks containing 100mL workingvolume of MRS medium containing 20 g Lminus1 glucose Theflasks were inoculated at 10 from preculture incubated at37∘C for 30 hThe pHwas adjusted at 50 60 70 80 85 90100 and 110 using intermitted pH control with 10N NaOHSamples were taken periodically every 3ndash6 h to analyzecell growth (OD

600nm) glucose and LA concentrations Todetermine the best nitrogen sources for LA productiondifferent concentrations of yeast extract (0 25 50 75 100 g









3 Results














100

100

100

100

002

87

87

97

92

77

63


























Max 119875LA (g L-1

h-1)e







20 gl 40gl 60 gl80 gl 100 gl 150 gl


0123456789

10

Gro

wth

(OD

600n

m)

(a)

20 gl 40gl 60 gl80 gl 100 gl 150 gl


020406080

100120140160

Residu

al glucose

(gL)

(b)

Lactic

acid concentration

20 gl 40gl 60 gl80 gl 100 gl 150 gl

0

20

40

60

80

100

120

(gL)


(c)












0

20

40

60

80

100

120

140

160

180

200

Glucose

and

lactic

acid (g

L)

0

4

8

12

16

20

24

28

Cell gro

wth

(OD

nm

)




BatchNumber


600 nma LA conc (g








600nm (247)(Table 5)




Average OD

0

2

4

6

8

10

12

14

16

Cell gro

wth

(OD

nm

)


(a)


0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)


(b)









BatchNumber




h-1)e



4 Discussion





05

1015202530

Cell gro

wth

(OD

nm

)


(a)


0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)

(b)




















Data Availability






Acknowledgments




References





































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018










3 Results














100

100

100

100

002

87

87

97

92

77

63


























Max 119875LA (g L-1

h-1)e







20 gl 40gl 60 gl80 gl 100 gl 150 gl


0123456789

10

Gro

wth

(OD

600n

m)

(a)

20 gl 40gl 60 gl80 gl 100 gl 150 gl


020406080

100120140160

Residu

al glucose

(gL)

(b)

Lactic

acid concentration

20 gl 40gl 60 gl80 gl 100 gl 150 gl

0

20

40

60

80

100

120

(gL)


(c)












0

20

40

60

80

100

120

140

160

180

200

Glucose

and

lactic

acid (g

L)

0

4

8

12

16

20

24

28

Cell gro

wth

(OD

nm

)




BatchNumber


600 nma LA conc (g








600nm (247)(Table 5)




Average OD

0

2

4

6

8

10

12

14

16

Cell gro

wth

(OD

nm

)


(a)


0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)


(b)









BatchNumber




h-1)e



4 Discussion





05

1015202530

Cell gro

wth

(OD

nm

)


(a)


0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)

(b)




















Data Availability






Acknowledgments




References





































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018












100

100

100

100

002

87

87

97

92

77

63


























Max 119875LA (g L-1

h-1)e







20 gl 40gl 60 gl80 gl 100 gl 150 gl


0123456789

10

Gro

wth

(OD

600n

m)

(a)

20 gl 40gl 60 gl80 gl 100 gl 150 gl


020406080

100120140160

Residu

al glucose

(gL)

(b)

Lactic

acid concentration

20 gl 40gl 60 gl80 gl 100 gl 150 gl

0

20

40

60

80

100

120

(gL)


(c)












0

20

40

60

80

100

120

140

160

180

200

Glucose

and

lactic

acid (g

L)

0

4

8

12

16

20

24

28

Cell gro

wth

(OD

nm

)




BatchNumber


600 nma LA conc (g








600nm (247)(Table 5)




Average OD

0

2

4

6

8

10

12

14

16

Cell gro

wth

(OD

nm

)


(a)


0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)


(b)









BatchNumber




h-1)e



4 Discussion





05

1015202530

Cell gro

wth

(OD

nm

)


(a)


0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)

(b)




















Data Availability






Acknowledgments




References





































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018














Max 119875LA (g L-1

h-1)e







20 gl 40gl 60 gl80 gl 100 gl 150 gl


0123456789

10

Gro

wth

(OD

600n

m)

(a)

20 gl 40gl 60 gl80 gl 100 gl 150 gl


020406080

100120140160

Residu

al glucose

(gL)

(b)

Lactic

acid concentration

20 gl 40gl 60 gl80 gl 100 gl 150 gl

0

20

40

60

80

100

120

(gL)


(c)












0

20

40

60

80

100

120

140

160

180

200

Glucose

and

lactic

acid (g

L)

0

4

8

12

16

20

24

28

Cell gro

wth

(OD

nm

)




BatchNumber


600 nma LA conc (g








600nm (247)(Table 5)




Average OD

0

2

4

6

8

10

12

14

16

Cell gro

wth

(OD

nm

)


(a)


0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)


(b)









BatchNumber




h-1)e



4 Discussion





05

1015202530

Cell gro

wth

(OD

nm

)


(a)


0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)

(b)




















Data Availability






Acknowledgments




References





































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



20 gl 40gl 60 gl80 gl 100 gl 150 gl


0123456789

10

Gro

wth

(OD

600n

m)

(a)

20 gl 40gl 60 gl80 gl 100 gl 150 gl


020406080

100120140160

Residu

al glucose

(gL)

(b)

Lactic

acid concentration

20 gl 40gl 60 gl80 gl 100 gl 150 gl

0

20

40

60

80

100

120

(gL)


(c)












0

20

40

60

80

100

120

140

160

180

200

Glucose

and

lactic

acid (g

L)

0

4

8

12

16

20

24

28

Cell gro

wth

(OD

nm

)




BatchNumber


600 nma LA conc (g








600nm (247)(Table 5)




Average OD

0

2

4

6

8

10

12

14

16

Cell gro

wth

(OD

nm

)


(a)


0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)


(b)









BatchNumber




h-1)e



4 Discussion





05

1015202530

Cell gro

wth

(OD

nm

)


(a)


0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)

(b)




















Data Availability






Acknowledgments




References





































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



0

20

40

60

80

100

120

140

160

180

200

Glucose

and

lactic

acid (g

L)

0

4

8

12

16

20

24

28

Cell gro

wth

(OD

nm

)




BatchNumber


600 nma LA conc (g








600nm (247)(Table 5)




Average OD

0

2

4

6

8

10

12

14

16

Cell gro

wth

(OD

nm

)


(a)


0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)


(b)









BatchNumber




h-1)e



4 Discussion





05

1015202530

Cell gro

wth

(OD

nm

)


(a)


0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)

(b)




















Data Availability






Acknowledgments




References





































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Average OD

0

2

4

6

8

10

12

14

16

Cell gro

wth

(OD

nm

)


(a)


0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)


(b)









BatchNumber




h-1)e



4 Discussion





05

1015202530

Cell gro

wth

(OD

nm

)


(a)


0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)

(b)




















Data Availability






Acknowledgments




References





































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




BatchNumber




h-1)e



4 Discussion





05

1015202530

Cell gro

wth

(OD

nm

)


(a)


0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)

(b)




















Data Availability






Acknowledgments




References





































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



05

1015202530

Cell gro

wth

(OD

nm

)


(a)


0

20

40

60

80

100

120

Glucose

and

lactic

acid (g

L)

(b)




















Data Availability






Acknowledgments




References





































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018















Data Availability






Acknowledgments




References





































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018






Data Availability






Acknowledgments




References





































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Documents

High Improvement in Lactic Acid Productivity by New Alkaliphilic …downloads.hindawi.com/journals/bmri/2019/7212870.pdf · 2019-07-30 · lactic-acid-and-poly-lactic-acid-market)