Productia de acid lactic cu tulpini de La.plantarum si L.brevis.pdf

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Lactic acid production from biomass-derived sugars via co-fermentation of Lactobacillus brevis and Lactobacillus plantarum

Yixing Zhang1 and Praveen V Vadlani12

Bioprocessing and Renewable Energy Laboratory Department of Grain Science and Industry Kansas State University Manhattan KS 66506 USA 1 and Department of Chemical

Engineering Kansas State University Manhattan KS 66506 USA2

Received 23 June 2014 accepted 29 October 2014

Available online xxx

Lignocellulosic biomass is an attractive alternative resource for producing chemicals and fuels Xylose is the domi-nating sugar after hydrolysis of hemicellulose in the biomass but most microorganisms either cannot ferment xylose or have a hierarchical sugar utilization pattern in which glucose is consumed 1047297rst To overcome this barrier Lactobacillusbrevis ATCC 367 was selected to produce lactic acid This strain possesses a relaxed carbon catabolite repressionmechanism that can use glucose and xylose simultaneously however lactic acid yield was only 052 g g L1 from amixture of glucose and xylose and 51 g L L1 of acetic acid and 83 g L L1 of ethanol were also formed during productionof lactic acid The yield was signi1047297cantly increased and ethanol production was signi1047297cantly reduced if L brevis was co-cultivated with Lactobacillus plantarum ATCC 21028 L plantarum outcompeted L brevis in glucose consumptionmeaning that L brevis was focused on converting xylose to lactic acid and the by-product ethanol was reduced due toless NADH generated in the fermentation system Sequential co-fermentation of L brevis and L plantarum increasedlactic acid yield to 080 g g L1 from poplar hydrolyzate and increased yield to 078 g lactic acid per g of biomass fromalkali-treated corn stover with minimum by-product formation Ef 1047297cient utilization of both cellulose and hemicellulosecomponents of the biomass will improve overall lactic acid production and enable an economical process to producebiodegradable plastics

2014 The Society for Biotechnology Japan All rights reserved

[Key words Lactic acid Poplar hydrolyzate Co-culture Corn stover Lactobacillus brevis Lactobacillus plantarum]

Lactic acid is a versatile chemical with a long history of appli-

cations in the food cosmetic and pharmaceutical industries (1)

and it has been listed as a platform chemical derived from biomass

by the US Department of Energy since 2004 (2) The estimated

world demand for lactic acid will be 600000 tons by 2020 (3) and is

expected to keep increasing because of their use in the develop-

ment of poly-lactic acid (PLA) and lactate solvents (4)

Biomass-based fermentation products have gained intensive

attention recently due to their potential as fossil fuel substitutes

More than 90 of global production of plant biomass is lignocel-

lulose which is mainly composed of cellulose hemicellulose and

lignin (5) Total cellulose and hemicellulose content is higher in

hardwood (788) than in softwood (703) but lignin content is

opposite (6) Various pretreatment methods have been developedsuch as alkali treatment and ammonia explosion to convert

structural carbohydrates to monomer sugars (7) Xylose is the

dominant sugar released from hemicellulose in hardwoods and

agricultural residues (8) Ef 1047297cient utilization of all sugars derived

from biomass has the potential to reduce the production cost of

chemicals by about 25 (9) Most homofermentative lactic acid

bacteria including Lactobacillus delbrueckii (1011) L paracasei

(12) and L lactis (13) cannot convert xylose to lactic acid In

contrast some heterofermentative lactic acid bacteria such as

Lactobacillus brevis CHCC 2097 and Lactobacillus pentosus CHCC

2355 have been used to produce lactic acid from xylose released

from wheat straw (14) These heterofermentative strains also

produce considerable amounts of byproducts such as acetic acid

and ethanol which increase product cost and decrease produc-

tivity (15) A third group of lactic acid bacteria known as the

facultative heterofermentative for example Lactobacillus planta-

rum use glucose through the Embden-Meyerhof pathway (EMP)

to produce lactic acid while they may also possess an inducible

phosphoketolase pathway (PK) with pentose acting as inducers

(16)

Vadlani et al (17) reported using L plantarum ATCC 21028 in the

1047297rst stage of fermentation to produce lactate from cheese whey aproduct yield of 098 g lactate per g of lactose was obtained Fu et al

(18) also reported the kinetic model of lactic acid production by

L plantarum ATCC 21028 and found out that lactic acid fermenta-

tion with this bacterium is homolactic and primary growth asso-

ciated L brevis a well-known heterofermentative strain was

reported to use xylose simultaneously with glucose (19) which is

highly desirable because the strain does not possess carbon

catabolite repression (CCR) especially when operating under

simultaneous sacchari1047297cation and fermentation (SSF) conditions

(20) In SSF the glucose consumption rate needs to be higher than

the release rate to ensure that no glucose remains in the medium

otherwise xylose will not be used by microorganisms that have a

hierarchical sugar utilization pattern

Corresponding author at Department of Grain Science and Industry Kansas

State University Manhattan KS 66506 USA Tel thorn1 785 532 5012 fax thorn1785 532

7193

E-mail address vadlaniksuedu (PV Vadlani)

wwwelseviercomlocatejbiosc

Journal of Bioscience and BioengineeringVOL xx No xx 1e6 2014

1389-1723$ e see front matter 2014 The Society for Biotechnology Japan All rights reservedhttpdxdoiorg101016jjbiosc201410027

Please cite this article in press as Zhang Y and Vadlani P V Lactic acid production from biomass-derived sugars via co-fermentation of Lactobacillus brevis and Lactobacillus plantarum J Biosci Bioeng (2014) httpdxdoiorg101016jjbiosc201410027



Literature on the production of lactic acid through co-fermen-

tation systems is limited (1421e23) Cui et al (21) reported co-

cultivation of Lactobacillus rhamnosus and L brevis to produce lactic

acid from corn stover with a yield of 07 g g1 To the best of our

knowledge no report discusses co-cultivation of L brevis with

L plantarum from lignocellulosic biomass We hypothesized that

L plantarum converts most glucose to lactic acid using the EMP

whereas L brevis converts xylose and a small portion of glucose tolactic acid and acetic acid via the phosphoketolase pathway (Fig 1)

In this case both glucose and xylose derived from lignocellulosic

biomass can be used ef 1047297ciently and L brevis can focus on con-

verting xylose to lactic acid only because available glucose is

limited the by-product ethanol which is produced mainly when

glucose concentration is high can be reduced due to glucose

depletion by L plantarum The objective of this study was to eval-

uate the performance of mixed cultures of L plantarum and L brevis

and to utilize poplar hydrolyzate and corn stover two promising

biomasses representing hardwood and agriculture residues

respectively as feedstocks to produce lactic acid

MATERIALS AND METHODS

Microorganism and culture conditions L brevis ATCC 367 and L plantarum

ATCC 21028 obtained from the American Type Culture Collection (Manassas VA

USA)were used in this work L brevis and L plantarum inocula were grown in 50 mL

liquid MRS medium (MRS broth Difco Laboratories Detroit MI USA) and incubated

at 37C for 15 h at an agitation rate of 150 rpm in a temperature-controlled shaker

(Innova 4300 New Brunswick Scienti1047297c NJ USA) N2 was sparged into the bottle to

create anaerobic growing conditions The initial cell density of L brevis and

L plantarum were measured using colony forming unit (CFU) counting method the

overnight cultures were diluted to different concentrations plated on MRS agar and

incubated at 37C for 24 h Colonies were counted using a colony counter (Lab-Aids

Inc NY USA) Initial cell concentrations of L plantarum and L brevis were 3 109

and 12 1010 CFUmL respectively which were adjusted to approximately 109 CFU

mL for inoculation in all fermentation experiments

Poplar hydrolyzate was obtained from Technology Holding LLC (Salt Lake City

UT USA) which contained 415 g L 1 of glucose 132 g L 1 of xylose and 13 g L 1 of

cellobiose The initial pH of poplar hydrolyzate was 26 which was adjusted to 65using 10 mol L 1 of NaOH

Corn stover was harvested from the Kansas State University Agronomy Farm in

Manhattan and Tribune Kansas USA Corn stover was treated with 1 NaOH (wv)

using the method described by Guragain et al (24) The alkali-treated corn stover

was dried at 60C overnight and ground to particlesize of lt1 mm usinga laboratory

mill (3303 Perten Instruments Spring1047297eld IL USA)

Fermentation Shake 1047298ask fermentation was performed in 100 mL Wheaton

serum bottles containing 50 mL of modi1047297ed MRS medium or poplar hydrolyzate The

modi1047297ed MRS medium consisted of glucose and xylose in a 31 ratio and supple-

mentedwith10g L 1 ofpeptone5 g L 1 ofyeast extract2 g L 1 of ammonium citrate

2 g L 1 of sodium acetate 2 g L 1 of K2HPO4 01 g L 1 of MgSO4$7H2O 005 g L 1 of

MnSO4$4H2O and 1 g L 1 of Tween 80 Poplar hydrolyzate was diluted and supple-

mented with all the components (except glucose and xylose) of the modi1047297ed MRS

medium pHof themediawas adjusted to65 using10 mol L 1 NaOH and3 (wv) of

calciumcarbonatewas added to control thepH Temperaturewas maintainedat 37C

for both L plantarum and L brevis Agitation was maintained at 150 rpm

In the simultaneous co-culture fermentation experiment L plantarum and

L brevis were inoculated at the same time at the beginning of fermentation with 5

(vv) inoculum for each strain at a 11 ratio In the sequential fermentation test 5(vv) L plantarum was added 1047297rst then 5 (vv) of L brevis was inoculated when

glucose concentration reached around 5 g L 1

Batch fermentationwas performed in a 7-L fermentor with a working volume of

5 L (Bio1047298o 110 New Brunswick Scienti1047297c Inc) The fermentation broth consisted of

1 L diluted poplar hydrolyzate (190 g L 1 glucose 72 g L 1 of xylose and 6 g L 1 of

cellobiose) 2 g L 1 of ammonium citrate 2 g L 1 of sodium acetate 2 g L 1 of

K2HPO4 01 g L 1 of MgSO4$7H2O 005 g L 1 of MnSO4$4H2O and 1 g L 1 of Tween

80 During fermentation the temperature was maintained at 37C agitation speed

at 100 rpm and pH at 65 by adding 10 mol L 1 NaOH N2 was sparged at 06 vvm

through the vessel to maintain anaerobic conditions

Simultaneous sacchari1047297cation and fermentation (SSF) was conducted in 100-mL

serum bottles Pretreated corn stover (4 wv) was suspended in 50 mL of

005 mol L 1 sodium citrate buffer supplemented with all the components except

sugarsof themodi1047297ed MRS medium Cellic CTec2 (CTec2)obtained fromNovozymes

Inc (Franklinton NC USA) was added at 8 FPUg of biomass Temperature was

maintained at 37C and the agitation rate was maintained at 150 rpm

Analytical methods Glucose xylose and lactic acid were measured ac-

cording to the method described by Zhang et al (11) Acetic acid and ethanol were

measured using high-performance liquid chromatography (HPLC Shimadzu

Scienti1047297c Instruments Inc Columbia MD USA) equipped with a Rezex ROA

organic acid column (150 78 mm Phenomenex Inc Torrance CA USA) and a

refractive index (RI) detector (RID-10A) 0005 N H2SO4 was used as the mobile

phase at a 1047298ow rate of 06 mL min1 Temperatures of the column and detector

were maintained at 83C and 40C respectively

Statistical methods SAS software version 91 (SAS Inc Cary NC USA) was

used to analyze experimental data by applying PROC GLM

RESULTS AND DISCUSSION

Lactic acid production from a mixture of glucose and

xylose The theoretical L plantarum yield of lactic acid from

glucose via the EMP pathway is 1 (g per g of glucose) Fig 2A shows

fermentation pro1047297le of L plantarum from a mixture of glucose andxylose L plantarum consumed glucose rapidly only 44 g L 1

glucose was left at 12 h and it was completely consumed within

24 h of fermentation Lactic acid (243 g L 1) was obtained from

255 g L 1 of glucose with a DL lactic acid molar ratio of 094

(485 of optical purity)

L brevis can use both glucose and xylose via the PK pathway and

produces a mixture of lactic acid acetic acid and ethanol The

theoretical yield of lactic acid fromglucose and xylose is 05 (g per g

FIG 1 Simpli1047297ed pathways for lactic acid production from a mixture of glucose and xylose by L plantarum and L brevis Enzymes 1 acetate kinase 2 lactate dehydrogenase 3

alcohol dehydrogenase Solid lines indicate the homofermentative pathway in L plantarum and dashed lines indicate the heterofermentative pathway in L brevis

2 ZHANG AND VADLANI J BIOSCI BIOENG




of glucose) and 06 (g per g of xylose) respectively As shown in

Fig 2B L brevis barely consumed glucose and xylose in the 1047297rst 8 h

of fermentation after which glucose was consumed much faster

than xylose all glucose was consumed after 24 h and xylose wascompletely used after 48 h Final lactic acid acetic acid and ethanol

concentration was 172 51 and 83 g L 1 respectively The DL

molar ratio of lactic acid was037 (270 of optical purity) Kim et al

(19) investigated the proteome of L brevis grown in glucose xylose

and a glucosexylose mixture The relative expression of a putative

acetate kinase was expressed at a much higher level when cells

were grown in xylose as single carbon source which resulted in a

different end-product pro1047297le L brevis in our study also showed a

different end-product pro1047297le when a different carbon source was

used The acetateethanol molar ratio was 01 when glucose was the

sole carbon source but the molar ratio changed to 25 when xylose

was used as the only carbon source The ratio of acetateethanol

depends on the oxidation reduction potential (NADHNADthorn ratio)

of the fermentation system (25) NADH is required for ethanol and

lactic acid production More NADH is generated from glucose

catabolism than from xylose metabolism therefore acetaldehyde

is converted to ethanol coupled with the regeneration of NADthorn

from NADH (Fig 1)

Unlike a single culture of L brevis simultaneous fermentation of

L brevis and L plantarum did not exhibit a sugar consumption lag

phase in the 1047297rst 8 h of fermentation Glucose was consumed

within 24 h and xylose was consumed within 48 h ( Fig 2C) Final

lactic acid concentration increased to 283 g L 1 with a DL molar

ratio of 061 (378 of optical purity) and ethanol decreased to

21 g L 1 (Table 1) The maximum glucose consumption rate of

L plantarum was almost 5 times greater than that of L brevis which

suggests that L plantarum outcompetes L brevis for glucose con-

sumption when these two strains grow together Furthermore the

ethanol production by L brevis was inhibited due to glucose

depletion by L plantarum

The fermentation pro1047297le of sequential co-fermentation of

L brevis with L plantarum is shown in Fig 2D Different inoculation

times of L brevis were tested no ethanol was produced when

L brevis was inoculated at glucose concentration around 5 g L 1

305 g L 1 of lactic acid was obtained by the end of fermentation

with a DL molar ratio of 065 (394 of optical purity) If all glucose

entered the EMP pathway and if all xylose entered the PK

pathway the theoretical production of lactic acid was

lactic acid ethg=L THORN frac14 ethglucoseethgTHORNTHORN xyloseethgTHORN 06=volumeethL THORN

Simultaneous and sequential fermentation of L plantarum and

L brevis increased lactic acid production ef 1047297ciency to 89 and 95

of the theoretical maximum production respectively

Taniguchi et al (23) also reported highest concentration of lactic

acid (95 g L 1) with a mixed culture system of Lactobacillus casei

and Enterococcus casseli 1047298avus from a mixture of glucose (100 g L 1)

and xylose(50g L 1) while simultaneous inoculation of L casei and

E casseli 1047298avus did not increase the lactic acid production sequen-

tially inoculated E casseli 1047298avus after 40 h allowed complete con-

sumption of xylose and enhanced 1047297nal lactic acid concentration at

the expense of lactic acid productivity (049 g L 1 h1) Compared

with the two-stage system reported by Taniguchi the co-cultiva-

tion system in this study has higher lactic acid productivity

(059 g1 L 1 h1) and higher lactic acid yield (085 g g1)

Lactic acid production from poplar hydrolyzate The poplar

hydrolyzate was detoxi1047297ed by the company and delivered to us

hence no inhibition of cell growth was observed in our experi-

ments Table 2 summarizes the fermentation results L plantarum

produced 256 g L 1 of lactic acid from 297 g L 1 glucose The D L

lactic acid molar ratio was 098 (495 of optical purity) which is

very close to that obtained from synthetic sugars L brevis

produced 188 g L 1 lactic acid from 296 g L 1 of glucose and

94 g L 1 of xylose with a DL molar ratio of 12 (545 of optical

purity) Acetic acid (45 g L 1) and ethanol (115 g L 1) were also

A

Time (h)

0 5 10 15 20

C o n c e

n t r a t i o n ( g L - 1 )

0

5

10

15

20

25

30

B

Time (h)

0 10 20 30 40

C o n c e

n t r a t i o n ( g L - 1 )

0

5

10

15

20

25

30

C

Time (h)

0 10 20 30 40

C o n c e n t r a t i o n ( g L - 1 )

0

5

10

15

20

25

30

35

D

Time (h)

0 10 20 30 40 50 60


0

5

10

15

20

25

30

35

FIG 2 Lactic acid production from a mixture of glucose and xylose by (A) L plantarum (B) L brevis (C) simultaneous fermentation of L plantarum and L brevis and (D) sequential

fermentation of L plantarum and L brevis Symbols 1047297lled circles xylose open circles glucose 1047297lled triangles lactic acid open triangles acetic acid 1047297lled squares ethanol

V OL xx 2014 LACTIC ACID PRODUCTION VIA L BREVIS AND L PLANTARUM 3




obtained Compared with the simultaneous fermentation

experiment sequential fermentation of L plantarum and L brevis

increased lactic acid yield from 071 to 080 g g1 and increased

lactic acid production ef 1047297ciency from 79 to 88 of the

theoretical maximum production The DL lactic acid molar ratio

was 1 (500 of optical purity) for both simultaneous and

sequential co-fermentation Statistically sequential fermentation

gave the highest product concentration and yield at the expense

of relatively lower productivity Consequently sequential

fermentation was scaled up to a 7-L fermentor with 5-L working

volume using poplar hydrolyzate (Fig 3) Initial glucose and

xylose concentrations were 354 g L 1 and 143 g L 1respectively L brevis was added at 20 h when glucose

concentration was 52 g L 1 glucose was completely consumed

within 26 h Xylose (22 g L 1) was left after 96 h of fermentation

Final lactic acid concentration was 380 g L 1 with 72 g L 1 acetic

acid and no ethanol was detected The yield of lactic acid was

080 g g1 and productivity was 040 g L 1 h1

Garde et al (14) evaluated lactic acid production from hemi-

cellulose of wheat straw hydrolyzate by single or mixed culture of

L pentosus and L brevis The mixed culture system increased lactic

acid production ef 1047297ciency to 95 of the theoretical maximum yield

Nancib et al (22) also reported lactic acid production from date

juice extract by a mixed culture system of L casei and L lactis which

gave better lactic acid production and sugar utilizations All these

results corroborate the results we found that mixed cultures of

lactic acid bacteria are more ef 1047297cient than single culture regarding

lactic acid concentration and sugar utilizations

Lactic acid production from corn stover via SSF

process Corn stover is the most abundant agriculture residue

in USA with annual production of 105e117 million dry tons (26)

Alkali-treated corn stover consisted of around 54 (ww) glucan

29 (ww) xylan and a small amount of arabinan (24) The

theoretical sugar yields from 2 g of dried alkali-treated cornstover were 12 g glucose and 07 g xylose Temperature and pH

can be set at optimal conditions for either the enzyme or the

bacteria In this study optimal growth temperature of bacteria

was determined by measuring the optical density at 600 nm

under 30C 37C 40C and 45C Both culture grew best at 37C

The temperature was set to the optimum for the bacteria

because bacterial growth was signi1047297cantly reduced under the

optimal temperature range of CTec2 (45e50C) whereas the

CTec2 still remains 60 of its hydrolysis activity compared to

hydrolysis conducted under optimal conditions The initial pH

was set at 6 which between the optimal pH for enzymes and

bacteria and the relative performance of enzymes decreases only

10 under this pH according to the Novozymes application sheet

(Luna No 2010-01668-01) Fig 4A shows lactic acid production

from corn stover from a single culture of L plantarum Glucose

released from corn stover was consumed rapidly by L plantarum

the concentration of glucose was maintained at a low level

throughout the SSF process which suggests that hydrolysis was

the rate-limiting step L plantarum in this study cannot use

xylose but it was able to use arabinose at very slow rate and

produced lactic acid (yield of 038 g lactic acid per g of arabinose)

and trace amount of acetic acid (yield of 007 g acetic acid per g

of arabinose) Arabinose was not counted into lactic acid

production in this study due to the small amount of arabinose

(less than 15 g L 1) present in the corn stover hydrolyzate

Fig 4B shows L brevis performance for lactic acid production

from corn stover and L brevis consumed glucose faster than

xylose Glucose concentration reached a maximum level of

59 g L 1 at 12 h then rapidly decreased to 09 g L 1 in the next

12 h and no glucose was detected at 48 h Xylose increased to

TABLE 1 Lactic acid production by single or mixed culture of L brevis and L plantarum from a mixture of glucose and xylose

Glucose

(g L 1)

Xylose

(g L 1)

Lactic acid

(g L 1)

Acetic acid

(g L 1)

Ethanol

(g L 1)

Yielda

(g g1)

Productivity b

(g L 1 h1)

rsgluc

(g L 1 h1)

Optical purityd ()

L plantarum 255 11A 86 02A 243 04B 0B 0C 096 004A 101 002A 29 485

L brevis 250 05A 88 02A 172 05C 51 04B 83 01A 052 002C 036 001D 06 270

Simultaneous 264 02A 88 01A 283 02A 47 04B 21 00B 080 001B 059 000Be 378

Sequential 270 00A 90 00A 305 09A 49 04B 0C 085 002B 051 002Ce 394

Each mean is based on three replications ( p lt 005) Values with the same letters in the same column are not signi1047297cantly differenta Lactic acid yield was calculated by dividing the amount of lactic acid by the amount of sugar consumedb Productivity was de1047297ned as the amount of lactic acid produced per liter per hourc Maximum glucose consumption rate calculated based on the equation r sglu frac14 qsXd Optical purity (OP) calculated based on the equation OP frac14 100(D-lactic acid concentration)(total lactic acid concentration)

TABLE 2 Lactic acid production by a single or mixed culture of L brevis and L plantarum from poplar hydrolyzate

Glucose (g L 1) Xylose (g L 1) Lactic acid (g L 1) Acetic acid (g L 1) Ethanol (g L 1) Yiel da (g g1) Productivityb (g L 1 h1) Optical purityc ()

L plantarum 297 06A 94 02A 256 11C 0C 0C 087 003A 108 005A 495

L brevis 296 06A 94 03A 188 07D 45 03B 115 07A 048 002C 031 001D 545

Simultaneous 293 06A 102 03A 281 06B 53 05A 29 03B 071 001B 043 001C 500

Sequential 301 01A 101 02A 318 01A 56 02A 0C 080 001A 048 000B 500

Each mean is based on three replications ( p lt 005) Values with the same letters in the same column are not signi1047297cantly differenta Lactic acid yield was calculated by dividing the amount of lactic acid by the amount of sugar consumedb

Productivity was de1047297

ned as the amount of lactic acid produced per liter per hourc Optical purity (OP) calculated based on the equation OP frac14 100(D-lactic acid concentration)(total lactic acid concentration)

Time (h)

0 20 40 60 80


0

10

20

30

40

FIG 3 Lactic acid production from poplar hydrolyzate by sequential fermentation

Symbols are the same as shown in Fig 2





44 g L 1 at 6 h then slowly decreased to 07 g L 1 during the

remaining time of fermentation The 1047297nal concentration of lactic

acid acetic acid and ethanol were 163 51 and 100 g L 1

respectively Fig 4C shows lactic acid production by simultaneous

fermentation of L plantarum and L brevis Glucose and xylose

accumulated to the maximum level of 16 g L 1 and 44 g L 1 at

6 h respectively Glucose concentration decreased to 02 g L 1 at

12 h and remained at zero during the duration of fermentation

Xylose concentration decreased to 19 g L 1 at 24 h and no

xylose was detected during the next 24 h Lactic acid (240 g L 1)

acetic acid (62 g L 1) and ethanol (12 g L 1) were obtained at

the end of fermentation In sequential fermentation as shown in

Fig 4D L brevis was added when xylose concentration reached

82 g L 1 at 24 h which was 61 of the theoretical hydrolysis

yield of xylose from 1 sodium hydroxide treated corn stover In

the 1047297rst stage glucose released from corn stover was quickly

consumed by L plantarum to produce lactic acid In the second

stage accumulated xylose was consumed by L brevis and

L plantarum kept consuming glucose Lactic acid increased to312 g L 1 and 63 g L 1 of acetic acid was obtained after 72 h of

fermentation

The performance of co-cultivation of L plantarum and L brevis

was better than the individual strain during fermentation (Table 3)

The highest lactic acid concentration (312 g L 1) and overall yield

(078 g g1) were obtained in sequential fermentation whereas the

highest productivity (050 g L h1) was obtained in simultaneous

fermentation with an overall yield of 057 g g1 In sequential

fermentation the overall yield was higher whereas productivity

was lower than that reported by Cui et al (21) which were

070 g g1 and 058 g L 1 h1 respectively in the fermentation of

alkali-treated corn stover with mixed cultures of L rhamnosus and

L brevis The lower productivity in our study is mainly attributed to

the lower enzyme dosage (8 FPUg) compared with that (25 FPUg)

used in Cuirsquos study consequently the total process time was

elongated

In conclusion the novel co-fermentation system in this study

took advantage of both lactobacillus strains and enabled optimum

utilization of sugars derived from lignocellulosic biomass This

mixed culture system showed better sugar utilization enhanced

lactic acid production and formed minimal by-products especially

when operated in SSF mode Metabolic 1047298ow of sugars in this co-

cultivation system need to be investigated in detail to further in-crease lactic acid yield and decrease by-product formation Because

the process is greatly simpli1047297ed by the similar cultivation condi-

tions of these two strains the co-cultivation system has enormous

potential for industrial applications In addition optimal conditions

A

Time (h)

0 10 20 30 40


0

5

10

15

20

25

B

Time (h)

0 10 20 30 40


0

5

10

15

20

C

Time (h)

0 10 20 30 40


0

5

10

15

20

25

30

D

Time (h)

0 20 40 60

C o n c

e n t r a t i o n ( g L - 1 )

0

5

10

15

20

25

30

35

FIG 4 Lactic acid production from pretreated corn stover via SSF process by (A) L plantarum (B) L brevis (C) simultaneous cultivation of L plantarum and L brevis (D) sequential

cultivation of L plantarum and L brevis Symbols are the same as shown in Fig 2

TABLE 3 Lactic acid production by a single or mixed culture of L brevis and L plantarum from NaOH-treated corn stover

Lactic acid (g L 1) Acetic acid (g L 1) Ethanol (g L 1) Overall yielda (g g1) Productivityb (g L 1 h1) Optical purityc ()

L plantarum 210 03C 0B 0C 050 003C 044 001B 422

L brevis 163 02D 51 02A 100 03A 039 001D 034 000C 397

Simultaneous 240 06B 62 01A 12 00B 057 001B 050 001A 471

Sequential 312 03A 63 06A 0C 078 008A 043 005B 432

Each mean is based on three replications ( p lt 005) Values with the same letters in the same column are not signi1047297cantly differenta

Lactic acid overall yield was calculated by dividing the amount of lactic acid produced by the amount of biomass consumedb Productivity was de1047297ned as the amount of lactic acid produced per liter per hourc Optical purity (OP) calculated based on the equation OP frac14 100(D-lactic acid concentration)(total lactic acid concentration)





such as inoculum size temperature and substrate concentration in

the SSF process can be found with the help of response surface

methodology

ACKNOWLEDGMENTS

This work was funded by the Consortium for Plant Biotech-

nology Research and the Development Initiative CompetitiveGrants Program (BRDI grant no 68-3A75-7-609) The authors are

grateful to Novozymes Inc for providing enzymes This article is

contribution number 14-402-J from the Kansas Agricultural

Experiment Station Kansas State University

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87e107 (2000)

5 Yang S Kataeva I Hamilton-Brehm S D Engle N L Tschaplinski T J

Doeppke C Davis M Westpheling J and Adams M W W Ef 1047297cient

degradation of lignocellulosic plant biomass without pretreatment by the

thermophilic anaerobe ldquo Anaerocellum thermophilumrdquo DSM 6725 Appl Environ

Microbiol 75 4762e4769 (2009)

6 Balat M Gasi1047297cation of biomass to produce gaseous products Energy Source

Part A 31 516e526 (2009)

7 Kumar P Barrett D M Delwiche M J and Stroeve P Methods for pre-

treatment of lignocellulosic biomass for ef 1047297cient hydrolysis and biofuel pro-

duction Ind Eng Chem Res 48 3713e3729 (2009)

8 Taherzadeh M J and Karimi K Pretreatment of lignocellulosic wastes to

improve ethanol and biogas production a review Int J Mol Sci 9 1621e1651

(2008)9 Hinman N Wright J Hoagland W and Wyman C Xylose fermentation e

an economic-analysis Appl Biochem Biotechnol 2021 391e401 (1989)

10 Fukushima K Sogo K Miura S and Kimura Y Production of D-lactic acid

by bacterial fermentation of rice starch Macromol Biosci 4 1021e1027

(2004)

11 Zhang Y and Vadlani P D-lactic acid biosynthesis from biomass-derived

sugars via Lactobacillus delbrueckii fermentation Bioprocess Biosyst Eng 36

1897e1904 (2013)

12 Moon S Wee Y and Choi G A novel lactic acid bacterium for the pro-

duction of high purity L -lactic acid Lactobacillus paracasei subsp paracasei

CHB2121 J Biosci Bioeng 114 155e159 (2012)

13 Kosugi A Tanaka R Magara K Murata Y Arai T Sulaiman O

Hashim R Hamid Z A A Yahya M K A Yusof M N M Ibrahim W A

and Mori Y Ethanol and lactic acid production using sap squeezed from oldoil palm trunks felled for replanting J Biosci Bioeng 110 322e325 (2010)

14 Garde A Jonsson G Schmidt A S and Ahring B K Lactic acid production

from wheat straw hemicellulose hydrolysate by Lactobacillus pentosus and

Lactobacillus brevis Bioresour Technol 81 217e223 (2002)

15 Abdel-Rahman M A Tashiro Y and Sonomoto K Lactic acid production

from lignocellulose-derived sugars using lactic acid bacteria overview and

limits J Biotechnol 156 286e301 (2011)

16 Fugelsang K C and Edwards C G Lactic acid bacteria pp 36e38 in

Fugelsang K C (Ed) Wine microbiology Practical applications and pro-

cedures Springer New York (2007)

17 Vadlani P V Mathews A P and Karr G S Low-cost propionate salt as road

deicer evaluation of cheese whey and other media constituents World J

Microbiol Biotechnol 24 825e832 (2008)

18 Fu W and Mathews A P Lactic acid production from lactose by Lactobacillus

plantarum kinetic model and effects of pH substrate and oxygen Biochem

Eng J 3 163e170 (1999)

19 Kim J Shoemaker S P and Mills D A Relaxed control of sugar utilization

in Lactobacillus brevis Microbiology 155 1351e

1359 (2009)20 Kim J Block D E and Mills D A Simultaneous consumption of pentose

and hexose sugars an optimal microbial phenotype for ef 1047297cient fermentation

of lignocellulosic biomass Appl Microbiol Biotechnol 88 1077e1085 (2010)

21 Cui F Li Y and Wan C Lactic acid production from corn stover using mixed

cultures of Lactobacillus rhamnosus and Lactobacillus brevis Bioresour Technol

102 1831e1836 (2011)

22 Nancib A Nancib N and Boudrant J Production of lactic acid from date

juice extract with free cells of single and mixed cultures of Lactobacillus casei

and Lactococcus lactis World J Microbiol Biotechnol 25 1423e1429 (2009)

23 Taniguchi M Tokunaga T Horiuchi K Hoshino K Sakai K and

Tanaka T Production of L -lactic acid from a mixture of xylose and glucose by

co-cultivation of lactic acid bacteria Appl Microbiol Biotechnol 66 160e165

(2004)

24 Guragain Y N Wilson J Staggenborg S McKinney L Wang D and

Vadlani P V Evaluation of pelleting as a pre-processing step for effective

biomass deconstruction and fermentation Biochem Eng J 77 198e207

(2013)25 Kandler O Carbohydrate metabolism in lactic acid bacteria Antonie Van

Leeuwenhoek 49 209e224 (1983)

26 Graham R L Nelson R Sheehan J Perlack R D and Wright L L Current

and potential US corn stover supplies Agron J 99 1e11 (2007)


Please cite this article in press as Zhang Y and Vadlani P V Lactic acid production from biomass-derived sugars via co-fermentation of b ill b i d b ill l i i i ( ) h d d i j jbi



Literature on the production of lactic acid through co-fermen-

tation systems is limited (1421e23) Cui et al (21) reported co-

cultivation of Lactobacillus rhamnosus and L brevis to produce lactic

acid from corn stover with a yield of 07 g g1 To the best of our

knowledge no report discusses co-cultivation of L brevis with

L plantarum from lignocellulosic biomass We hypothesized that

L plantarum converts most glucose to lactic acid using the EMP

whereas L brevis converts xylose and a small portion of glucose tolactic acid and acetic acid via the phosphoketolase pathway (Fig 1)

In this case both glucose and xylose derived from lignocellulosic

biomass can be used ef 1047297ciently and L brevis can focus on con-

verting xylose to lactic acid only because available glucose is

limited the by-product ethanol which is produced mainly when

glucose concentration is high can be reduced due to glucose

depletion by L plantarum The objective of this study was to eval-

uate the performance of mixed cultures of L plantarum and L brevis

and to utilize poplar hydrolyzate and corn stover two promising

biomasses representing hardwood and agriculture residues

respectively as feedstocks to produce lactic acid

MATERIALS AND METHODS

Microorganism and culture conditions L brevis ATCC 367 and L plantarum

ATCC 21028 obtained from the American Type Culture Collection (Manassas VA

USA)were used in this work L brevis and L plantarum inocula were grown in 50 mL

liquid MRS medium (MRS broth Difco Laboratories Detroit MI USA) and incubated

at 37C for 15 h at an agitation rate of 150 rpm in a temperature-controlled shaker

(Innova 4300 New Brunswick Scienti1047297c NJ USA) N2 was sparged into the bottle to

create anaerobic growing conditions The initial cell density of L brevis and

L plantarum were measured using colony forming unit (CFU) counting method the

overnight cultures were diluted to different concentrations plated on MRS agar and

incubated at 37C for 24 h Colonies were counted using a colony counter (Lab-Aids

Inc NY USA) Initial cell concentrations of L plantarum and L brevis were 3 109

and 12 1010 CFUmL respectively which were adjusted to approximately 109 CFU

mL for inoculation in all fermentation experiments

Poplar hydrolyzate was obtained from Technology Holding LLC (Salt Lake City

UT USA) which contained 415 g L 1 of glucose 132 g L 1 of xylose and 13 g L 1 of

cellobiose The initial pH of poplar hydrolyzate was 26 which was adjusted to 65using 10 mol L 1 of NaOH

Corn stover was harvested from the Kansas State University Agronomy Farm in

Manhattan and Tribune Kansas USA Corn stover was treated with 1 NaOH (wv)

using the method described by Guragain et al (24) The alkali-treated corn stover

was dried at 60C overnight and ground to particlesize of lt1 mm usinga laboratory

mill (3303 Perten Instruments Spring1047297eld IL USA)

Fermentation Shake 1047298ask fermentation was performed in 100 mL Wheaton

serum bottles containing 50 mL of modi1047297ed MRS medium or poplar hydrolyzate The

modi1047297ed MRS medium consisted of glucose and xylose in a 31 ratio and supple-

mentedwith10g L 1 ofpeptone5 g L 1 ofyeast extract2 g L 1 of ammonium citrate

2 g L 1 of sodium acetate 2 g L 1 of K2HPO4 01 g L 1 of MgSO4$7H2O 005 g L 1 of

MnSO4$4H2O and 1 g L 1 of Tween 80 Poplar hydrolyzate was diluted and supple-

mented with all the components (except glucose and xylose) of the modi1047297ed MRS

medium pHof themediawas adjusted to65 using10 mol L 1 NaOH and3 (wv) of

calciumcarbonatewas added to control thepH Temperaturewas maintainedat 37C

for both L plantarum and L brevis Agitation was maintained at 150 rpm

In the simultaneous co-culture fermentation experiment L plantarum and

L brevis were inoculated at the same time at the beginning of fermentation with 5

(vv) inoculum for each strain at a 11 ratio In the sequential fermentation test 5(vv) L plantarum was added 1047297rst then 5 (vv) of L brevis was inoculated when

glucose concentration reached around 5 g L 1

Batch fermentationwas performed in a 7-L fermentor with a working volume of

5 L (Bio1047298o 110 New Brunswick Scienti1047297c Inc) The fermentation broth consisted of

1 L diluted poplar hydrolyzate (190 g L 1 glucose 72 g L 1 of xylose and 6 g L 1 of

cellobiose) 2 g L 1 of ammonium citrate 2 g L 1 of sodium acetate 2 g L 1 of

K2HPO4 01 g L 1 of MgSO4$7H2O 005 g L 1 of MnSO4$4H2O and 1 g L 1 of Tween

80 During fermentation the temperature was maintained at 37C agitation speed

at 100 rpm and pH at 65 by adding 10 mol L 1 NaOH N2 was sparged at 06 vvm

through the vessel to maintain anaerobic conditions

Simultaneous sacchari1047297cation and fermentation (SSF) was conducted in 100-mL

serum bottles Pretreated corn stover (4 wv) was suspended in 50 mL of

005 mol L 1 sodium citrate buffer supplemented with all the components except

sugarsof themodi1047297ed MRS medium Cellic CTec2 (CTec2)obtained fromNovozymes

Inc (Franklinton NC USA) was added at 8 FPUg of biomass Temperature was

maintained at 37C and the agitation rate was maintained at 150 rpm

Analytical methods Glucose xylose and lactic acid were measured ac-

cording to the method described by Zhang et al (11) Acetic acid and ethanol were

measured using high-performance liquid chromatography (HPLC Shimadzu

Scienti1047297c Instruments Inc Columbia MD USA) equipped with a Rezex ROA

organic acid column (150 78 mm Phenomenex Inc Torrance CA USA) and a

refractive index (RI) detector (RID-10A) 0005 N H2SO4 was used as the mobile

phase at a 1047298ow rate of 06 mL min1 Temperatures of the column and detector

were maintained at 83C and 40C respectively

Statistical methods SAS software version 91 (SAS Inc Cary NC USA) was

used to analyze experimental data by applying PROC GLM

RESULTS AND DISCUSSION

Lactic acid production from a mixture of glucose and

xylose The theoretical L plantarum yield of lactic acid from

glucose via the EMP pathway is 1 (g per g of glucose) Fig 2A shows

fermentation pro1047297le of L plantarum from a mixture of glucose andxylose L plantarum consumed glucose rapidly only 44 g L 1

glucose was left at 12 h and it was completely consumed within

24 h of fermentation Lactic acid (243 g L 1) was obtained from

255 g L 1 of glucose with a DL lactic acid molar ratio of 094

(485 of optical purity)

L brevis can use both glucose and xylose via the PK pathway and

produces a mixture of lactic acid acetic acid and ethanol The

theoretical yield of lactic acid fromglucose and xylose is 05 (g per g

FIG 1 Simpli1047297ed pathways for lactic acid production from a mixture of glucose and xylose by L plantarum and L brevis Enzymes 1 acetate kinase 2 lactate dehydrogenase 3

alcohol dehydrogenase Solid lines indicate the homofermentative pathway in L plantarum and dashed lines indicate the heterofermentative pathway in L brevis

























from NADH (Fig 1)














































A

Time (h)

0 5 10 15 20

C o n c e

n t r a t i o n ( g L - 1 )

0

5

10

15

20

25

30

B

Time (h)

0 10 20 30 40

C o n c e

n t r a t i o n ( g L - 1 )

0

5

10

15

20

25

30

C

Time (h)

0 10 20 30 40


0

5

10

15

20

25

30

35

D

Time (h)

0 10 20 30 40 50 60


0

5

10

15

20

25

30

35






































































Glucose

(g L 1)

Xylose

(g L 1)

Lactic acid

(g L 1)

Acetic acid

(g L 1)

Ethanol

(g L 1)

Yielda

(g g1)

Productivity b

(g L 1 h1)

rsgluc

(g L 1 h1)

Optical purityd ()


L brevis 250 05A 88 02A 172 05C 51 04B 83 01A 052 002C 036 001D 06 270







L brevis 296 06A 94 03A 188 07D 45 03B 115 07A 048 002C 031 001D 545






Time (h)

0 20 40 60 80


0

10

20

30

40


























fermentation














elongated











A

Time (h)

0 10 20 30 40


0

5

10

15

20

25

B

Time (h)

0 10 20 30 40


0

5

10

15

20

C

Time (h)

0 10 20 30 40


0

5

10

15

20

25

30

D

Time (h)

0 20 40 60

C o n c

e n t r a t i o n ( g L - 1 )

0

5

10

15

20

25

30

35






L brevis 163 02D 51 02A 100 03A 039 001D 034 000C 397











methodology

ACKNOWLEDGMENTS






References



(2011)










87e107 (2000)





Microbiol 75 4762e4769 (2009)


Part A 31 516e526 (2009)










(2004)



1897e1904 (2013)





















Eng J 3 163e170 (1999)








102 1831e1836 (2011)







(2004)
































from NADH (Fig 1)














































A

Time (h)

0 5 10 15 20

C o n c e

n t r a t i o n ( g L - 1 )

0

5

10

15

20

25

30

B

Time (h)

0 10 20 30 40

C o n c e

n t r a t i o n ( g L - 1 )

0

5

10

15

20

25

30

C

Time (h)

0 10 20 30 40


0

5

10

15

20

25

30

35

D

Time (h)

0 10 20 30 40 50 60


0

5

10

15

20

25

30

35






































































Glucose

(g L 1)

Xylose

(g L 1)

Lactic acid

(g L 1)

Acetic acid

(g L 1)

Ethanol

(g L 1)

Yielda

(g g1)

Productivity b

(g L 1 h1)

rsgluc

(g L 1 h1)

Optical purityd ()


L brevis 250 05A 88 02A 172 05C 51 04B 83 01A 052 002C 036 001D 06 270







L brevis 296 06A 94 03A 188 07D 45 03B 115 07A 048 002C 031 001D 545






Time (h)

0 20 40 60 80


0

10

20

30

40


























fermentation














elongated











A

Time (h)

0 10 20 30 40


0

5

10

15

20

25

B

Time (h)

0 10 20 30 40


0

5

10

15

20

C

Time (h)

0 10 20 30 40


0

5

10

15

20

25

30

D

Time (h)

0 20 40 60

C o n c

e n t r a t i o n ( g L - 1 )

0

5

10

15

20

25

30

35






L brevis 163 02D 51 02A 100 03A 039 001D 034 000C 397











methodology

ACKNOWLEDGMENTS






References



(2011)










87e107 (2000)





Microbiol 75 4762e4769 (2009)


Part A 31 516e526 (2009)










(2004)



1897e1904 (2013)





















Eng J 3 163e170 (1999)








102 1831e1836 (2011)







(2004)











































































Glucose

(g L 1)

Xylose

(g L 1)

Lactic acid

(g L 1)

Acetic acid

(g L 1)

Ethanol

(g L 1)

Yielda

(g g1)

Productivity b

(g L 1 h1)

rsgluc

(g L 1 h1)

Optical purityd ()


L brevis 250 05A 88 02A 172 05C 51 04B 83 01A 052 002C 036 001D 06 270







L brevis 296 06A 94 03A 188 07D 45 03B 115 07A 048 002C 031 001D 545






Time (h)

0 20 40 60 80


0

10

20

30

40


























fermentation














elongated











A

Time (h)

0 10 20 30 40


0

5

10

15

20

25

B

Time (h)

0 10 20 30 40


0

5

10

15

20

C

Time (h)

0 10 20 30 40


0

5

10

15

20

25

30

D

Time (h)

0 20 40 60

C o n c

e n t r a t i o n ( g L - 1 )

0

5

10

15

20

25

30

35






L brevis 163 02D 51 02A 100 03A 039 001D 034 000C 397











methodology

ACKNOWLEDGMENTS






References



(2011)










87e107 (2000)





Microbiol 75 4762e4769 (2009)


Part A 31 516e526 (2009)










(2004)



1897e1904 (2013)





















Eng J 3 163e170 (1999)








102 1831e1836 (2011)







(2004)































fermentation














elongated











A

Time (h)

0 10 20 30 40


0

5

10

15

20

25

B

Time (h)

0 10 20 30 40


0

5

10

15

20

C

Time (h)

0 10 20 30 40


0

5

10

15

20

25

30

D

Time (h)

0 20 40 60

C o n c

e n t r a t i o n ( g L - 1 )

0

5

10

15

20

25

30

35






L brevis 163 02D 51 02A 100 03A 039 001D 034 000C 397











methodology

ACKNOWLEDGMENTS






References



(2011)










87e107 (2000)





Microbiol 75 4762e4769 (2009)


Part A 31 516e526 (2009)










(2004)



1897e1904 (2013)





















Eng J 3 163e170 (1999)








102 1831e1836 (2011)







(2004)














methodology

ACKNOWLEDGMENTS






References



(2011)










87e107 (2000)





Microbiol 75 4762e4769 (2009)


Part A 31 516e526 (2009)










(2004)



1897e1904 (2013)





















Eng J 3 163e170 (1999)








102 1831e1836 (2011)







(2004)










Productia de acid lactic cu tulpini de La.plantarum si L.brevis.pdf

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