L-(+)-Lactic acid production by co-fermentation of cellobiose and xylose without carbon catabolite repression using Enterococcus mundtii QU 25

Article abstract of DOI:10.1039/c4ra02764g

The use of lignocellulosic biomass for the production of optically pure lactic acid remains challenging because it requires efficient utilisation of mixed sugars without carbon catabolite repression (CCR). Enterococcus mundtii QU 25, a novel l-lactic acid-producing strain, was used in this study to ferment mixed sugars. This strain exhibited apparent CCR in a glucose-xylose mixture; however, replacement of glucose by cellobiose (cellobiose-xylose mixture) led to simultaneous consumption of both sugars without CCR. The production of lactic acid and activity of enzymes related to xylose metabolism were also investigated. Xylose isomerase and xylulokinase specific activity in cellobiose-xylose grown cells was three times higher than that in glucose-xylose grown cells. The addition of yeast extract and ammonium hydroxide effectively improved sugar utilisation and cell growth. Under the optimal conditions with simulated lignocellulosic hydrolysates, a high l-lactic acid concentration (up to 163 g L^-1) was produced with a yield of 0.870 g g^-1 and maximum productivity of 7.21 g L^-1 h^-1 without CCR in the fed-batch fermentation. Thus, we could establish rapid and simultaneous consumption of hexose and pentose sugars by using a lactic acid bacterium strain, which significantly increased production of high-purity l-lactic acid. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra02764g

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L-(+)-Lactic acid production by co-fermentation of
cellobiose and xylose without carbon catabolite
repression using Enterococcus mundtii QU 25
Cite this: RSC Adv., 2014, 4, 22013
Ying Wang,a Mohamed Ali Abdel-Rahman,ab Yukihiro Tashiro,cd Yaotian Xiao,a
a
c
ae
*
Takeshi Zendo, Kenji Sakai and Kenji Sonomoto
The use of lignocellulosic biomass for the production of optically pure lactic acid remains challenging
because it requires efficient utilisation of mixed sugars without carbon catabolite repression (CCR).
Enterococcus mundtii QU 25, a novel L-lactic acid-producing strain, was used in this study to ferment
mixed sugars. This strain exhibited apparent CCR in a glucose–xylose mixture; however, replacement of
glucose by cellobiose (cellobiose–xylose mixture) led to simultaneous consumption of both sugars
without CCR. The production of lactic acid and activity of enzymes related to xylose metabolism were
also investigated. Xylose isomerase and xylulokinase specific activity in cellobiose–xylose grown cells
was three times higher than that in glucose–xylose grown cells. The addition of yeast extract and
ammonium hydroxide effectively improved sugar utilisation and cell growth. Under the optimal
conditions with simulated lignocellulosic hydrolysates, a high L-lactic acid concentration (up to 163 g
Received 28th March 2014
Accepted 2nd May 2014
L
À1) was produced with a yield of 0.870 g gÀ1 and maximum productivity of 7.21 g LÀ1 hÀ1 without CCR
in the fed-batch fermentation. Thus, we could establish rapid and simultaneous consumption of hexose
and pentose sugars by using a lactic acid bacterium strain, which significantly increased production of
high-purity L-lactic acid.
DOI: 10.1039/c4ra02764g
www.rsc.org/advances
the focus of intense research interest as an environmentally
friendly method of lactic acid production because of the low
Introduction
temperature and energy requirements, as well as the high
optical purity of the produced lactic acid.2,3
Lactic acid (2-hydroxypropionic acid, CH3CHOHCOOH) is one
of the most widely used organic acids in the food, pharmaceu-
tical, cosmetic, and chemical industries. Lactic acid production
is currently a subject of active research and development
because it serves as feedstock in the synthesis of polylactic acid,
which can be used for manufacturing of biodegradable mate-
rials.1 Lactic acid can be produced by either chemical synthesis
or microbial fermentation. Recently, lactic acid fermentation
from non-food feedstock, such as renewable biomass, has been
Among the variety of potential renewable biomass sources,
lignocellulosic biomass is available in large quantities with
widespread distribution and comparatively low prices.2 The
major components of lignocellulose are cellulose (insoluble
bres of b-1,4-glucan), hemicellulose (polysaccharides such as
xylans, mannans, and glucans other than b-1,4-glucan), and
lignin (amorphous phenylpropanoid polymer).2,3 The hydroly-
sates of lignocellulosic biomass aer pretreatment and
saccharication are mainly composed of mixed sugars,
including hexoses (such as glucose and cellobiose) and
pentoses (such as xylose and arabinose). Furthermore, the
composition of mixed sugars differs depending not only on the
type of lignocellulosic biomass but also on the method of
pretreatment and saccharication.1,3 Efficient fermentation of
mixed sugars derived from lignocellulosic biomass to lactic acid
by lactic acid bacteria (LAB) is hampered by the following
problems: (1) few LAB can utilise xylose, (2) most of the xylose-
utilising LAB produce by-products such as acetic acid and
ethanol, and (3) LAB, as many other bacteria, tend to utilise a
preferred (rapidly metabolisable) sugar (such as glucose) rst,
while inhibiting catabolism of sugars other than that preferred
(such as xylose); a phenomenon well known as carbon
aLaboratory of Microbial Technology, Division of Applied Molecular Microbiology and
Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of
Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku,
Fukuoka 812-8581, Japan. E-mail: sonomoto@agr.kyushu-u.ac.jp; Fax: +81-(0)92-
642-3019; Tel: +81-(0)92-642-3019
bBotany and Microbiology Department, Faculty of Science (Boys), Al-Azhar University,
PN:11884, Nasr City, Cairo, Egypt
cLaboratory of Soil Microbiology, Division of Applied Molecular Microbiology and
Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of
Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku,
Fukuoka 812-8581, Japan
dInstitute of Advanced Study, Kyushu University, 6-10-1 Hakozaki, Higashi-ku,
Fukuoka 812-8581, Japan
eLaboratory of Functional Food Design, Department of Functional Metabolic Design,
Bio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka
812-8581, Japan
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catabolite repression (CCR).4,5 As a result of CCR, the fermen- pre-culture was performed by transferring 4 mL of the refreshed
tation of xylose in sugar mixtures is inhibited by glucose, which culture to a 100 mL ask with 36 mL mMRS medium and
ꢀ
becomes a major obstacle to efficient utilisation of all sugars incubating at 43 C for 8 h. All main cultures, except for those
derived from lignocellulosic biomass.2
used in investigation of nitrogen sources, were grown at 43 ꢀC in
In regard to lactic acid fermentation using LAB, various a 1 L jar fermenter (Biott; Tokyo, Japan) containing 0.4 L mMRS
studies have reported the approaches to overcome CCR caused medium or in a test tube with 15 mL mMRS medium including
by glucose and improve fermentation of mixed sugars to lactic 10% (v/v) of pre-culture grown under different conditions, as
acid, such as (1) usage of two LAB strains specic for xylose and described for each experiment. Samples were taken at different
glucose,6 (2) establishment of a fermentation process based on time intervals and analysed for cell growth and composition of
controlling glucose concentration at lower than the CCR sugars and fermentation products.
threshold level,7 and (3) genetic engineering of LAB strains by
For fermentation experiments, sugar mixtures containing
introduction of the genes related to xylose metabolism.8,9 100 g LÀ1 glucose and 60 g LÀ1 xylose (G100X60) or 100 g LÀ1
However, in those studies, wild type LAB exhibited not only cellobiose and 60 g LÀ1 xylose (C100X60) were used in mMRS
simultaneous consumption of mixed sugars but also heterolactic medium. Batch fermentation was carried out at agitation of 200
acid fermentation with production of by-products, such as acetic rpm with an automatic pH control at 7.0 by 10 M NaOH addition.
acid and ethanol, resulting from xylose utilisation of hetero-
To investigate the effects of nitrogen sources, C100X60-con-
fermentative LAB.2 To date, there are no reports on production of taining mMRS medium was supplemented with yeast extract,
lactic acid by wild type LAB through homolactic fermentation of peptone, ammonium sulphate, or urea (all at 6 g LÀ1). Batch
mixed sugars derived from lignocellulosic biomass.
fermentation was performed in test tubes containing 15 mL
We recently reported a unique fermentation strategy for mMRS medium supplemented with 100 g LÀ1 CaCO3 as a
butanol production by using wild type clostridial bacterium neutralizing agent.
based on replacement of glucose by cellobiose without CCR of
To test lactic acid production using simulated energy cane
xylose consumption.10 On the other hand, Enterococcus mundtii hydrolysates,13 mMRS medium containing 10 g LÀ1 glucose, 80
QU 25, isolated and characterised in our laboratory, is a g LÀ1 cellobiose, and 40 g LÀ1 xylose (G10C80X40) was supple-
homofermentative LAB that can utilise xylose11 and cellobiose12 mented with 6 g LÀ1 yeast extract. Batch fermentation was
as a sole substrate to produce optically pure L-lactic acid. No carried out at agitation of 200 rpm and the pH was maintained
studies on lactic acid fermentation using mixtures of cellobiose at 7.0 by addition of 10 M NaOH or NH4OH.
and xylose have yet been reported. Thus, the aim of the present
To test lactic acid production in the fed-batch fermentation,
work was to establish an efficient L-lactic acid production mMRS media (G10C80X40) was supplemented with 6 g LÀ1
process without CCR by using wild type E. mundtii QU 25 and yeast extract. The fed-batch fermentation was performed with
sugar mixtures containing cellobiose and xylose derived from agitation at 200 rpm and the pH was maintained at 7.0 with 10
lignocellulosic biomass.
M NH4OH. A mixture of 5 g LÀ1 glucose, 40 g LÀ1 cellobiose, and
20 g LÀ1 xylose (G5C40X20) with 1 g LÀ1 yeast extract was added
to the fermentation broth aer 36 h cultivation.
Experimental
Microorganism and media
Fermentation parameters
E. mundtii QU 25 was used throughout this study. The stock
culture was stored at À80 ꢀC in vials containing 15% (v/v)
glycerol until use.
The fermentation parameters evaluated in this study were as
follows: maximum sugar consumption rate (g LÀ1 hÀ1) was
calculated as the ratio of consumed sugar concentration (g LÀ1
)
Unless otherwise stated, cell growth, inoculum preparation,
and fermentation was conducted using a modied Man, Rogosa,
and Sharpe (mMRS) medium containing the following compo-
nents (LÀ1): 10 g peptone (Becton, Dickinson and Company;
Sparks, MD, USA), 8 g beef extract, 4 g yeast extract, 2 g K2HPO4, 5
to each sampling period (h). The yield of lactic acid based on
substrate consumed (g gÀ1) was dened as the ratio of lactic
acid produced (g LÀ1) to amount of sugar consumed (g LÀ1).
Lactic acid productivity (g LÀ1 hÀ1) was calculated as the ratio of
the highest lactic acid concentration (g LÀ1) to the indicated
fermentation time (h). Maximum lactic acid productivity (g LÀ1
hÀ1) was calculated between each sampling period within
exponential growth phase.
g
CH3COONa$3H2O, 2 g tri-ammonium citrate, 0.2 g
MgSO4$7H2O, 0.05 g MnSO4$4H2O, 1 mL Tween 80 (all from
Nacalai Tesque; Kyoto, Japan). As indicated in each experimental
description, glucose, xylose (both from Nacalai Tesque), and
cellobiose (Carbosynth; Berkshire, UK) were added at various
concentrations as carbon sources. The medium pH was adjusted
to 7.0 by 10 M NaOH prior to sterilisation at 115 ꢀC for 20 min.
Analytical methods
Cell growth was monitored by optical density at 562 nm (OD562
)
using an ultraviolet (UV)-1600 visible spectrophotometer (Bio-
Spec; Shimadzu; Tokyo, Japan). One unit of OD562 corresponded
to 0.218 g dry cell weight (DCW) per L.12 Cellobiose, xylose,
Fermentation process
For inoculum preparation, 1 mL of glycerol stock was inocu- glucose, and fermentation products were detected by high-
lated into 9 mL mMRS medium containing 15 g LÀ1 cellobiose performance liquid chromatography (HPLC; US HPLC-1210;
and 15 g LÀ1 xylose and refreshed for 24 h at 43 C. Then, the Jasco, Tokyo, Japan) equipped with a SUGAR SH-1011 column
ꢀ
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(Shodex; Tokyo, Japan). Samples taken during fermentation consisting of 0.9 mL of substrate and 0.1 mL of diluted whole
were centrifuged at 2000 Â g at 4 ꢀC for 10 min to remove solids, cell suspension was incubated at 43 ꢀC for 30 min. The release
and supernatants were ltered through a membrane lter of p-nitrophenol was measured at 410 nm and compared with
(Dismic-13HP, 0.45 mm; Advantec; Tokyo, Japan). HPLC analysis the p-nitrophenol standard. One unit of the enzyme activity was
ꢀ
was performed at the column temperature of 50 C with 3 mM equivalent to 1 mmol of p-nitrophenol generated per min.
HClO4 as a mobile phase at a ow rate of 1.0 mL minÀ1 using an
Transketolase (EC 2.2. 1.1) activity in the pentose phosphate/
injection volume of 20 mL. Protein concentration in crude glycolytic pathway was measured according to the method of
enzyme extracts was determined by using the BCA Protein Assay Tanaka et al.17 and Kochetov.18 The reaction mixture contained
Kit™ (Thermo Scientic; Rockford, IL, USA).
citrate-phosphate buffer (pH 7.0), 100 mM; MgCl2, 1.5 mM;
triose phosphate isomerase, 0.5 U; a-glycerophosphate dehy-
drogenase, 0.5 U; thiamine pyrophosphate, 0.1 mM; NADH,
0.15 mM; potassium ribose 5-phosphate, 1.2 mM; potassium
xylulose 5-phosphate, 0.9 mM; and crude enzyme extract. One
unit of the enzyme activity was dened as the amount of the
enzyme catalysing the formation of 1 mmol glyceraldehyde 3-
phosphate per min.
Preparation of crude enzyme extracts
For the preparation of crude enzyme extracts to measure the
activity of xylose isomerase and xylulokinase, cells were grown
in mMRS medium with G100X60 and C100X60 for 14 h when
glucose and cellobiose were still present in the fermentation
broth and collected by centrifugation at 8000 Â g for 15 min at 4
ꢀC. The harvested cells were washed twice with 0.85% (w/v)
NaCl, suspended in 10 mL of 50 mM potassium phosphate
buffer (pH 7.0), pretreated with 15 g LÀ1 lysozyme, and dis-
rupted by using a French press. Cell debꢀris was removed by
centrifugation at 7190 Â g for 10 min at 4 C, and the claried
supernatants were used as crude extracts for the enzymatic
assays described below.
Results and discussion
Batch fermentation using mixtures of glucose–xylose and
cellobiose–xylose
In our previous study, it was shown that E. mundtii QU 25 effi-
ciently produced lactic acid using high initial concentrations of
glucose (100 g LÀ1),19 xylose (103 g LÀ1),11 and cellobiose (151 g
To investigate the localisation of b-glucosidase, cell super-
natants were ultracentrifuged at 75 000 Â g for 40 min at 4 ꢀC,
and the enzymatic activity was measured in the intracellular
and cell membrane-bound fractions.
LÀ1
)
as the sole carbon sources. However, there were no
12
reports on the fermentative production of lactic acid using high
concentration of mixed sugars derived from lignocellulosic
biomass. In this study, we initially performed batch fermenta-
tions with E. mundtii QU 25 using a mixture of glucose (100 g
LÀ1) and xylose (60 g LÀ1) (G100X60). During the early phase (up
Enzyme assays
Assays were performed using a Shimadzu UV-160A recording to 24 h), E. mundtii QU 25 grew rapidly, reaching the maximum
spectrophotometer and 1 mL quartz cuvettes at 43 ꢀC. All DCW of 3.19 g LÀ1 mainly by using glucose at a much higher
chemicals were of analytical grade. Enzymes and other maximum consumption rate (6.19 g LÀ1 hÀ1) than xylose (0.748
biochemical reagents were purchased from Sigma-Aldrich Co. g LÀ1 hÀ1) (Fig. 1A and Table 1). Xylose consumption was not
(St Louis, MO, USA). All experiments of each enzyme were stimulated even aer 24 h of cultivation when glucose concen-
carried out in triplicate.
tration decreased to less than 20 g LÀ1, and the amount of
Xylose isomerase (EC 5.3.1.5) activity was determined in a utilised xylose was only 25.6 g LÀ1 with a residual concentration
reaction mixture containing Tris–HCl buffer (pH 7.0), 100 mM; of 36.7 g LÀ1 aer 96 h. As a result, E. mundtii QU 25 growing on
MgCl2, 10 mM; NADH, 0.15 mM; sorbitol dehydrogenase, 2 U; G100X60 produced 71.2 g LÀ1 lactic acid with a yield of 0.603 g
and crude enzyme extracts. The reaction was initiated by addi- gÀ1. Thus, E. mundtii QU 25 growing on this sugar mixture
tion of 500 mM xylose as described by Kuyper et al.14 The apparently exhibited CCR of xylose utilisation, resulting in an
decrease in the absorbance at 340 nm, indicative of NADH insignicant decrease of xylose even aer almost complete
oxidation, was monitored for 5 min. One unit of the enzyme consumption of glucose.
activity was dened as the amount of enzyme catalysing the
formation of 1 mmol xylulose per min.
On the other hand, when glucose was replaced by cellobiose
(C100X60), the DCW was increased to 3.54 g LÀ1 (Table 1).
Xylulokinase (EC 2.7.1.17) activity was measured as described C100X60-grown cultures also exhibited simultaneous consump-
previously15 in a reaction mixture containing glycylglycine, 250 tion of both cellobiose and xylose signicantly increasing the
mM (pH 7.0); MgSO4, 5 mM; NADH, 0.15 mM; pyruvate kinase, 1 maximal consumption rate of xylose from 0.748 g LÀ1 hÀ1 in
U; lactate dehydrogenase, 3 U; phosphoenolpyruvate, 1.5 mM; D- G100X60 to 1.78 g LÀ1 hÀ1 in C100X60, and demonstrating high
xylulose, 5 mM; and ATP, 1 mM. The reaction was initiated by total consumption (56.2 g LÀ1) and low residual concentration
adding crude enzyme extracts, and the decrease in the absor- (3.78 g LÀ1) of xylose (Fig. 1B and Table 1). These results suggest
bance at 340 nm during the reaction was monitored for 5 min. that replacement of glucose by cellobiose could counteract CCR
One unit of activity was dened as the amount of the enzyme of xylose consumption. Furthermore, C100X60-grown cultures
phosphorylating 1 mmol of xylulose per min.
demonstrated a much higher lactic acid concentration of 90.2 g
b-Glucosidase (EC 3.2.1.21) activity was estimated according LÀ1 and yield of 0.756 g gÀ1 compared to those in G100X60
to a previously described method12,16 using p-nitrophenyl-b-D- cultures (Table 1). Interestingly, only small amounts of by-
glucopyranoside as substrate. The assay mixture (1 mL) product (0.369 g LÀ1 acetic acid) were detected. This study
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LAB have been poorly characterised, the activity of enzymes
involved in sugar metabolism has been considered to be
responsible for CCR in some LAB such as Lactobacillus brevis20
and Lactobacillus plantarum.8 However, knowledge of the related
mechanisms in the Enterococcus genus is quite limited. In order
to clarify CCR of xylose consumption by glucose and to under-
stand the mechanisms underlying CCR absence in E. mundtii
QU 25 grown on cellobiose and xylose mixtures, the activity of
key enzymes in xylose metabolism and cellobiose hydrolysis
were analysed in cells grown in C100X60 and G100X60 for 14 h
(Table 2).
In the C100X60-grown cells, the enzymes initiating xylose
catabolism, xylose isomerase, and xylulokinase, showed specic
activities of more than three-fold higher (0.511 Æ 0.057 and
0.633 Æ 0.088 U per mg protein, respectively) than those in
G100X60-grown cells (0.153 Æ 0.011 and 0.183 Æ 0.015 U per mg
protein, respectively), which led to high xylose consumption in
cellobiose-cultured cells. Therefore, in E. mundtii QU 25,
glucose-induced CCR of xylose consumption might result from
a low activity of xylose isomerase and xylulokinase, as observed
in L. brevis20 and L. plantarum.8 On the other hand, the activity of
transketolase, an intermediate enzyme in xylose catabolic
pathway, was similar in C100X60 and G100X60 cultures (0.598
Æ 0.160 and 0.530 Æ 0.087 U per mg protein, respectively).
Furthermore, the activity of b-glucosidase, which catalyses the
hydrolysis of cellobiose to glucose, was much higher in C100X60
than in G100X60 whole cells suspensions (24.2 Æ 1.8 and 0.266
Æ 0.024 U per mg DCW, respectively) (Table 2). This value was
almost equal to 25.7 U per mg DCW exhibited by E. mundtii QU
25 grown on cellobiose as a sole carbon source.12 Furthermore,
b-glucosidase was localised in the cytoplasmic membrane
fraction (Table 2). Consequently, in E. mundtii QU 25 using the
cellobiose and xylose mixture for lactic acid fermentation, CCR
of xylose consumption could be avoided because of the high
activity of the enzymes initiating xylose catabolism and simul-
taneous cellobiose consumption from the high activity of b-
glucosidase.
In Firmicutes bacteria, several mechanisms of CCR have been
considered, including genetic repression of metabolic
enzymes.4,21 In general, two proteins are thought to regulate
genetic expression, phospho-carrier protein (HPr) and catabo-
lite control protein A (CcpA). Serine-phosphorylated HPr
couples with CcpA in the presence of the high intracellular
concentration of glucose 6-phosphate and fructose 1,6-
bisphosphate derived from glucose (favourable sugar), thereby
repressing transcription of the genes or operons encoding
metabolic enzymes for non-favourable sugars. However, we did
not detect glucose in the fermentation broth of C100X60-grown
E. mundtii QU 25 (Fig. 1B), and the cellobiose consumption rate
(2.58 g LÀ1 hÀ1) with C100X60 was much lower than the glucose
consumption rate (6.19 g LÀ1 hÀ1) with G100X60. Although it is
not known whether glucose originated from the extracellular or
intracellular hydrolysis of cellobiose, these results indicate that
the hydrolysis of cellobiose by b-glucosidase is a rate-limiting
Fig. 1 Profile of lactic acid production from sugar mixtures G100X60
(A), C100X60 (B), and C100X60 supplemented with yeast extract (C). E.
mundtii QU 25 was cultured in a 1 L jar fermenter containing 0.4 L
mMRS medium at 43 ꢀC with agitation at 200 rpm and pH 7.0
controlled with 10 M NaOH. Symbols: open circles, glucose concen-
tration; closed circles, lactate concentration; open triangles, xylose
concentration; closed triangles, dry cell weight; open squares, cello-
biose concentration. Data points represent the mean values from
three independent experiments.
succeeded in establishing conditions for homolactic fermenta-
tion of sugar mixtures by wild type LAB without CCR, which have
not been previously reported. Other fermentation studies also
showed that replacement of glucose by cellobiose prevented CCR
of xylose consumption in production of ethanol and butanol
using genetically engineered Saccharomyces cerevisiae13 and wild
type Clostridium saccharoperbutylacetonicum,10 respectively.
Activity of enzymes involved in xylose metabolism in cultures
with and without CCR
Here, we described a unique strategy to co-ferment hexose and step in lactic acid production, and that the intracellular
pentose sugars simultaneously for production of lactic acid concentration of glucose, glucose 6-phosphate, and fructose
without glucose-induced CCR. Although CCR mechanisms in 1,6-bisphosphate in C100X60-grown cells is too low to cause
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CCR. These considerations and the enzymatic activity results
suggest that fermentation with C100X60 should not repress
transcription of the genes encoding xylose isomerase and
xylulokinase. More studies are needed to further conrm our
hypothesis such as metabolome and transcriptome analyses
and investigation of the transport systems for cellobiose and
xylose.
Effects of nitrogen sources on lactic acid production
The addition of nitrogen sources to bacteria growth media has
been shown to stimulate not only sugar utilisation but also
lactic acid production.22 In normal MRS media, the C : N ratio
was extremely high because of the high concentration of carbon
sources in C100X60, which led to 37.3 g LÀ1 residual cellobiose
even aer 192 h fermentation (Fig. 1B). In this study, the effects
of several organic and inorganic nitrogen sources on sugar
utilisation and lactic acid production were investigated in order
to improve sugar consumption by E. mundtii QU 25. In a
preliminary experiment, batch fermentations in C100X60-con-
taining mMRS medium supplemented with 6 g LÀ1 of yeast
extract, peptone, ammonium sulphate, or urea was performed.
Among the nitrogen sources, yeast extract showed the best
improvement in the consumption of both cellobiose and xylose
as well as in the production of lactic acid (concentration and
yield) (data not shown).
The consumption of cellobiose and xylose was enhanced by
the addition of yeast extract (Fig 1B and C and Table 1). The
maximal DCW was also signicantly increased from 3.54 to 5.43
g LÀ1 following the addition of yeast extract (Table 1). Further-
more, simultaneous utilisation of cellobiose and xylose was
achieved (Fig. 1C). As a result, the addition of yeast extract could
improve lactic acid production (concentration, 122 g LÀ1
maximum productivity, 5.58 g LÀ1 hÀ1, and yield, 0.766 g gÀ1
,
)
compared to the corresponding parameters (90.2 g LÀ1, 3.23 g
LÀ1 hÀ1, and 0.756 g gÀ1, respectively) in the culture without any
additions. Increases in sugar utilisation, cell growth, and lactic
acid production by supplementation with yeast extract have
been also reported for other LAB including L. delbrueckii23 and L.
coryniformis subsp. torquens.24 In summary, the addition of yeast
extract signicantly improved both the utilisation of mixed
sugars and the production of lactic acid by E. mundtii QU 25.
Lactic acid production from simulated energy cane
hydrolysate
Energy cane is a hybrid of commercial and wild sugarcanes and
a source of inedible biomass, which has a much higher bre
content (including cellulose and hemicellulose) than commer-
cial sugarcane. There is growing interest in using energy cane
for production of valuable chemicals and fuel.25 Recently,
ethanol production from simulated energy cane hydrolysates
with simultaneous utilisation of mixed sugars by genetically
engineered S. cerevisiae (10 g LÀ1 glucose, 80 g LÀ1 cellobiose,
and 40 g LÀ1 xylose) has been reported.13 Therefore, to investi-
gate the feasibility of lactic acid production from energy cane
hydrolysates, batch fermentations were carried out in
G10C80X40-containing mMRS medium supplemented with 6 g
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Table 2 Enzymatic activities for cells grown in media containing glucose–xylose or cellobiose–xylose mixturea
Xylose isomerase Xylulokinase Transketolase
b-Glucosidase
(U per mg of DCW) (U per mg protein) (U per mg protein) (U per mg protein)
Carbon
sourceb
(U per mg
protein)
(U per mg
protein)
(U per mg
protein)
[whole cell]
[cell-bound]
[intracellular]
[extracellular]
G100X60 0.153 Æ 0.011
C100X60 0.511 Æ 0.057
0.183 Æ 0.015 0.530 Æ 0.087 0.266 Æ 0.024
0.633 Æ 0.088 0.598 Æ 0.160 24.2 Æ 1.8
NDc
NDc
NDc
NDc
NDc
2.39 Æ 0.62
a
b
Averages with standard deviations are based on three independent fermentations. G100X60, glucose 100 g LÀ1 and xylose 60 g LÀ1; C100X60,
cellobiose 100 g LÀ1 and xylose 60 g LÀ1
.
Not detected.
c
LÀ1 yeast extract with pH controlled at 7.0 by 10 M NaOH or C100X60 (due to a lower initial sugar concentration), the lactic
NH4OH as neutralisers.
acid yield (0.798 g gÀ1) and the maximal lactic acid productivity
The batch fermentation with G10C80X40 using NaOH resul- (5.96 g LÀ1 hÀ1) obtained with G10C80X40 were comparable or
ted not only in simultaneous sugar utilisation without CCR but higher than those obtained with C100X60 (0.766 g gÀ1 and 5.58 g
also in a high DCW (5.33 g LÀ1) (Fig. 2A) similar to that obtained LÀ1 hÀ1, respectively). Thus, these results support the feasibility
with C100X60 (5.43 g LÀ1) (Fig. 1C). This suggested that low of efficient lactic acid production from energy cane hydrolysates
glucose concentration in hydrolysates derived from lignocellu- by E. mundtii QU 25 without CCR.
losic biomass should not affect the ability of E. mundtii QU 25 to
To investigate the effect of pH neutraliser on lactic acid
simultaneously ferment hexose and pentose sugars into lactic production from mixed sugars, the batch fermentation was
acid. Although the 106 g LÀ1 lactic acid concentration obtained performed in G10C80X40-grown cultures at pH 7.0 controlled
with G10C80X40 was lower than the 122 g LÀ1 obtained with with 10 M NH4OH. As shown in Fig. 2B, in NaH4OH-controlled
cultures, the maximal DCW increased from 5.33 to 6.67 g LÀ1
.
Furthermore, sugar concentration and consumption were quite
similar between NH4OH- and NaOH-controlled cultures (Table
1 and Fig. 2). Surprisingly, despite the similar sugar consump-
tion, lactic acid production (concentration, 115
g ;
LÀ1
maximum productivity, 7.33 g LÀ1 hÀ1; yield, 0.863 g gÀ1) was
markedly improved by using NH4OH compared to NaOH (106 g
LÀ1, 5.96 g LÀ1 hÀ1, and 0.798 g gÀ1, respectively). Inhibition of
Na+ on LAB growth has been reported.26 In addition, using
NH4OH as a neutraliser enhanced cell growth of several LAB,
including L. delbrueckii subsp. lactis, Lactobacillus pentosus, L.
+
brevis, and Leuconostoc sp.,27,28 because NH4 is considered to
serve as a nitrogen source for LAB,29 and lactic acid production
by LAB is known to be associated with cell growth.30 These
results indicate that NH4OH is a better neutraliser for pH
control instead of NaOH, which can also serve as a nitrogen
source to stimulate growth of E. mundtii QU 25, thereby pre-
venting inhibition by Na+.
To achieve homolactic acid production process without CCR
from various lignocellulosic biomasses, the sugar mixture
compositions of hydrolysates suggested to be signicant, in
particular concentrations of xylose and glucose. While initial
xylose concentration of more than 40 g LÀ1 exhibited homolactic
acid production (Table 1 and Fig. 2), heterolactic acid produc-
tion was observed from low initial xylose concentration (<ca. 10 g
LÀ1) using glucose–xylose mixture (unpublished data). Although
CCR of xylose consumption was considered to occur under high
glucose concentration of 25 g LÀ1 and over in glucose–xylose
mixture (unpublished data), even high concentration of cello-
Fig. 2 Production of lactic acid by E. mundtii QU 25 from simulated
energy cane hydrolysate (G10C80X40) in the batch fermentation using
different neutralising agents, (A) NaOH and (B) NH4OH. Symbols: open
circles, glucose concentration; closed circles, lactate concentration;
open triangles, xylose concentration; closed triangles, dry cell weight
(DCW); open squares, cellobiose concentration. Data points represent
the mean values from three independent experiments.
biose (80 g LÀ1) in sugar mixture with low glucose (10 g LÀ1
)
should not result in CCR (Fig. 2). These considerations
suggested the accomplishment of homolactic acid
fermentation without CCR by E. mundtii QU 25 from
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lignocellulosic biomass-derived hydrolysates containing mainly fermentation (Fig. 2B). In an attempt to improve lactic acid
xylose ($10 g LÀ1) and cellobiose with less glucose (<ca. 25 g production, a single-pulse fed-batch fermentation was per-
LÀ1). Therefore, it is desirable for hydrolysates to be obtained formed: the culture started in G10C80X40-containing mMRS
from lignocellulosic biomasses containing high proportion of medium supplemented with 6 g LÀ1 yeast extract and was
hemicellulose such as leaves2 and grasses2 by b-glucosidase-free additionally fed with G5C40X20 and 1 g LÀ1 yeast extract aer
cellulases to suppress the formation of glucose.31
36 h (pH was controlled at 7.0 with 10 M NH4OH) (Fig. 3 and
Table 1). Aer additional feeding, E. mundtii QU 25 fermented
all the sugars (glucose, cellobiose, and xylose) simultaneously to
lactic acid (Fig. 3). As a result, the fed-batch fermentation
signicantly improved sugar consumption (cellobiose, 113 g
LÀ1; xylose, 58.7 g LÀ1) and L-lactic acid production (163 g LÀ1) at
an optical purity of $99.7% compared to those values observed
Improved lactic acid production in fed-batch fermentation
mode
All sugars in the mixture were completely consumed when the
simulated energy cane hydrolysate was used in the batch
in the batch fermentation (83.7 g LÀ1, 39.1 g LÀ1, and 115 g LÀ1
,
respectively) (Table 1). In addition, the maximum consumption
rates of cellobiose (4.51 g LÀ1 hÀ1) and xylose (2.50 g LÀ1 hÀ1),
and the maximum lactic acid productivity (7.21 g LÀ1 hÀ1) and
yield (0.870 g gÀ1) in the fed-batch fermentation were quite
comparable to those in the batch fermentation (4.23 g LÀ1 hÀ1
,
2.44 g LÀ1 hÀ1, 7.33 g LÀ1 hÀ1, and 0.863 g gÀ1, respectively).
These results indicate that the fed-batch fermentation method
is a better approach than batch fermentation in regard to
improvement of mixed sugar consumption and lactic acid
production without CCR.
There is currently great interest in efficiently simultaneous
utilizations of pentoses and hexoses in hydrolysates derived
from lignocellulosic biomasses in co-fermentation for lactic
acid production.2 Table 3 shows the results of recent studies on
lactic acid co-fermentation by several microorganisms such as
fungi,32 genetically engineered bacteria (Escherichia coli33 and L.
plantarum8), and L. brevis20 using several sugar mixtures derived
from lignocellulosic biomass. Basically, most of these studies
mainly investigated mixtures of glucose and xylose in the batch
fermentations. A few authors reported production of lactic acid
without CCR by using genetically engineered strains.8,33 The
Fig. 3 Production of lactic acid by E. mundtii QU 25 from simulated
energy cane hydrolysate by single-pulse fed-batch fermentation using
NH4OH as a neutralising agent. Fermentation was conducted in a 1 L
jar fermenter containing 0.4
L of mMRS medium with initial
G10C80X40 sugar mixture and 6 g LÀ1 yeast extract. G5C40X20
mixture and 1 g LÀ1 yeast extract were added at 36 h of cultivation.
Symbols: open circles, glucose concentration; closed circles, lactate
concentration; open triangles, xylose concentration; closed triangles,
dry cell weight (DCW); open squares, cellobiose concentration. Data
points represent the mean values from three independent
experiments.
Table 3 Comparison on recent data with present work using various sugar mixtures derived from lignocellulosic biomass
b
c
d
e
Fermentation CLA
mode
YLA
PLA
Max. PLA
Isomer (optical
purity, %)
Substratea
Microorganism
(g LÀ1
)
(g gÀ1
)
(g LÀ1 hÀ1
)
(g LÀ1 hÀ1
)
pH neutraliser Ref.
G75X25
G40X40
Rhizopus oryzae RQ4015 Batch
83
64.3
0.83
0.77
1.38
0.45
2.1
—
L (À)
L (À)
Na2CO3
32
33
Escherichia coli
FBR19 DptsG
Lactobacillus plantarum
DldhL1::PxylAB-xpk1::tkt-
Dxpk2::PxylAB
Batch
Batch
2.5 M KOH +
2.5 M NaOH
10 M NH4OH
G50X25A5
G75X25
61.2
74.2
0.80
0.78
1.7
4.9
5.6
D (99.5)
8
8
Lactobacillus plantarum
DldhL1::PxylAB-xpk1::tkt-
Dxpk2::PxylAB
Batch
2.85
D (99.5)
10 M NH4OH
G10X10
Lactobacillus
brevis ATCC 14869
Enterococcus
Batch
12.5
122
0.579
0.766
0.870
0.568
0.635
0.679
—
—
No control
20
C100X60
Batch
5.58
7.21
L (99.7)
L (99.7)
10 M NaOH
10 M NH4OH
This work
This work
mundtii QU 25
G10C80X40 Enterococcus
mundtii QU 25
Fed-batch
163
a
G75X25, glucose 75 g LÀ1 and xylose 25 g LÀ1; G40X40, glucose 40 g LÀ1 and xylose 40 g LÀ1; G50X25A5, glucose 50 g LÀ1, xylose 25 g LÀ1 and
arabinose 5 g LÀ1; G10X10, glucose 10 g LÀ1 and xylose 10 g LÀ1; C100X60, cellobiose 100 g LÀ1 and xylose 60 g LÀ1; G10C80X40, glucose 10 g
b
c
d
L
À1, cellobiose 80 g LÀ1 and xylose 40 g LÀ1
.
CLA, maximum lactic acid concentration. YLA, lactic acid yield. PLA, overall lactic acid
productivity. e Max. PLA, maximum lactic acid productivity. —, not described.
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present study is the rst to investigate lactic acid fermentation
from glucose–cellobiose–xylose mixture and to overcome CCR
of xylose consumption even in the presence of low glucose by
using cellobiose, which resulted in a high yield of lactic acid and
fewer by-products. In addition, we rst establish a fed-batch
fermentation process for lactic acid production using sugar
mixtures and could produce approximately 2 times higher lactic
3 A. Pessione, M. Zapponi, G. Mandili, P. Fattori,
E. Mangiapane, R. Mazzoli and E. Pessione, J. Biotechnol.,
2014, 173, 31–40.
¨
¨
4 B. Gorke and J. Stulke, Nat. Rev. Microbiol., 2008, 6, 613–624.
5 J. Kim, D. E. Block and D. A. Mills, Appl. Microbiol.
Biotechnol., 2010, 88, 1077–1085.
6 M. Taniguchi, T. Tokunaga, K. Horiuchi, K. Hoshino,
K. Sakai and T. Tanaka, Appl. Microbiol. Biotechnol., 2004,
66, 160–165.
7 A. Thani, L. Laopaiboon, S. Vichitphan, V. Leelavatcharamas
and P. Laopaiboon, Thai. J. Biotechnol., 2006, 7, 1–5.
8 S. Yoshida, K. Okano, T. Tanaka, C. Ogino and A. Kondo,
Appl. Microbiol. Biotechnol., 2011, 92, 67–76.
9 K. Okano, S. Yoshida, R. Yamada, T. Tanaka, C. Ogino,
H. Fukuda and A. Kondo, Appl. Environ. Microbiol., 2009,
75, 7858–7861.
acid (163 g LÀ1) than the maximum reported value (83 g LÀ1
)
previously.32 Compared with low lactic acid concentrations in
the literatures, the obtained high concentration of lactic acid
should be separated and puried from the fermentation broth
more easily by several downstream technologies such as elec-
trodialysis34 and crystallization.35 Moreover, because a high
concentration of b-glucosidase is always required for complete
the hydrolysis of cellulose to glucose during the pretreatment of
lignocellulosic biomass,36 our fermentation approach should
reduce the cost of this process. Furthermore, to the best of our 10 T. Noguchi, Y. Tashiro, T. Yoshida, J. Zheng, K. Sakai and
knowledge, we achieved the highest L-lactic acid concentration K. Sonomoto, J. Biosci. Bioeng., 2013, 116, 716–721.
(163 g LÀ1), maximum lactic acid productivity (7.21 g LÀ1 hÀ1), 11 M. A. Abdel-Rahman, Y. Tashiro, T. Zendo, K. Hanada,
and yield (0.870 g gÀ1) to date at $99.7% of optical purity in the
fed-batch fermentation. Therefore, this study demonstrates
K. Shibata and K. Sonomoto, Appl. Environ. Microbiol.,
2011, 77, 1892–1895.
potential of using low-cost feedstock of lignocellulosic biomass 12 M. A. Abdel-Rahman, Y. Tashiro, T. Zendo, K. Shibata and
for the production of valuable compounds.
K. Sonomoto, Appl. Microbiol. Biotechnol., 2011, 89, 1039–1049.
13 S. J. Ha, J. M. Galazka, S. R. Kim, J. H. Choi, X. Yang, J. H. Seo,
N. L. Glass, J. H. Cate and Y. S. Jin, Proc. Natl. Acad. Sci. U. S.
A., 2011, 108, 504–509.
Conclusion
14 M. Kuyper, H. R. Harhangi, A. K. Stave, A. A. Winkler,
M. S. Jetten, W. T. de Laat, J. J. den Ridder, H. J. Op den
Camp, J. P. Dijken and J. T. Pronk, FEMS Yeast Res., 2003,
4, 69–78.
15 C. F. B. Witteveen, R. Busink, P. van de Vondervoort,
C. Dijkema, K. Swart and J. Visser, J. Gen. Microbiol., 1989,
135, 2163–2171.
16 M. Singhvi, D. Joshi, M. Adsul, A. Varma and D. Gokhale,
Green Chem., 2010, 12, 1106–1109.
17 K. Tanaka, A. Komiyama, K. Sonomoto, A. Ishizaki, S. J. Hall
and P. F. Stanbury, Appl. Microbiol. Biotechnol., 2002, 60,
160–167.
We rst demonstrated a unique strategy for co-fermentation of
hexose and pentose sugars derived from lignocellulosic
biomass by E. mundtii QU 25 and for the production of L-lactic
acid by using cellobiose as an alternative substrate to glucose.
We found that CCR of xylose consumption by glucose may be
caused by low activity of the enzymes initiating the catabolism
of xylose, and that fermentation of cellobiose did not negatively
affect the activity of these enzymes. Furthermore, supplemen-
tation with yeast extract and the use of NH4OH as a pH neu-
traliser improved the sugar utilisation and production of high
purity lactic acid. Finally, an efficient system of L-lactic acid
production based on the fed-batch fermentation of mixed
sugars was successfully established.
18 G. A. Kochetov, Methods Enzymol., 1982, 90, 209–223.
19 M. A. Abdel-Rahman, Y. Tashiro, T. Zendo and K. Sonomoto,
RSC Adv., 2013, 3, 8437–8445.
Acknowledgements
20 J. H. Kim, S. P. Shoemaker and D. A. Mills, Microbiology,
2009, 155, 1351–1359.
21 J. Deutscher, Curr. Opin. Microbiol., 2008, 11, 87–93.
22 Z. H. Lu, F. He, Y. Shi, M. B. Lu and L. J. Yu, Bioresour.
Technol., 2010, 101, 3642–3648.
23 C. Kotzamanidis, T. Roukas and G. Skaracis, World J.
Microbiol. Biotechnol., 2002, 18, 441–448.
24 C. M. Nguyen, J. Kim, J. K. Song, G. J. Choi, Y. H. Choi,
K. S. Jang and J. Kim, Biotechnol. Lett., 2012, 34, 2235–2240.
25 M. Kim and D. F. Day, J. Ind. Microbiol. Biotechnol., 2011, 38,
803–807.
26 Y. Cai, S. Ohmomo, M. Ogawa and S. Kumai, J. Appl.
Microbiol., 1997, 83, 307–313.
27 Y. Tashiro, W. Kaneko, Y. Q. Sun, K. Shibata, K. Inokuma,
T. Zendo and K. Sonomoto, Appl. Microbiol. Biotechnol.,
2011, 89, 1741–1750.
This study has been supported by a Grant-in-Aid (P12088) for
JSPS Fellows to Mohamed Ali Abdel-Rahman from the Japan
Society for the Promotion of Science (JSPS), Japan. This research
was nancially supported in part by the Sumitomo Corporation
(Japan). Ying Wang gratefully acknowledges support by a
Chinese government scholarship offered by the China Schol-
arship Council during this study.
References
1 M. A. Abdel-Rahman, Y. Tashiro and K. Sonomoto,
Biotechnol. Adv., 2013, 31, 877–902.
2 M. A. Abdel-Rahman, Y. Tashiro and K. Sonomoto,
J. Biotechnol., 2011, 156, 286–301.
22020 | RSC Adv., 2014, 4, 22013–22021
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
28 E. Zannini, S. Santarelli, A. Osimani, L. Dellaquila and 32 P. Wang, J. Li, L. Wang, M. L. Tang, Z. L. Yu and Z. M. Zheng,
F. Clementi, Res. Microbiol., 2005, 55, 273–278. J. Ind. Microbiol. Biotechnol., 2009, 36, 1363–1368.
29 M. M. Peebles, S. E. Gilliland and M. L. Speck, Appl. 33 B. S. Dien, N. N. Nichols and R. J. Bothast, J. Ind. Microbiol.
Microbiol., 1969, 17, 805–810. Biotechnol., 2002, 29, 221–227.
30 C. Schiraldi, V. Adduci, V. Valli, C. Maresca, M. Giuliano, 34 X. L. Wang, Y. M. Wang, X. Zhang and T. W. Xu, Bioresour.
M. Lamberti, M. Carteni and M. De Rosa, Biotechnol.
Bioeng., 2003, 82, 213–222.
Technol., 2012, 125, 165–171.
35 K. Xu and P. Xu, Bioresour. Technol., 2014, 163, 33–39.
´
31 E. A. Znameroski, S. T. Coradetti, C. M. Roche, J. C. Tsai, 36 P. Andric, A. S. Meyer, P. A. Jensen and K. Dam-Johansen,
A. T. Iavarone, J. H. D. Cate and N. L. Glass, Proc. Natl.
Acad. Sci. U. S. A., 2012, 109, 6012–6017.
Biotechnol. Adv., 2010, 28, 308–324.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 22013–22021 | 22021