D-(-)-Lactic acid production from cellobiose and cellulose by Lactobacillus lactis mutant RM2-24

Article abstract of DOI:10.1039/b925975a

Lactobacillus lactis mutant RM2-24 utilizes cellobiose efficiently, converting it into d-(-)-lactic acid. Cellobiose-degrading enzyme activities were determined for whole cells, cell extracts and disrupted cells. Aryl-β-glucosidase activity was detected in whole cells and disrupted cells, suggesting that these activities are confined to the cells. The mutant produced 80 g l^-1 of lactic acid from 100 g l^-1 of cellobiose with 1.66 g l^-1 h^-1 productivity. Production of d-lactic acid from different cellulose samples was also studied. The cellulose samples at high concentration (10%) were hydrolyzed by cellulase enzyme preparation (10 FPU g^-1 cellulose) derived from Penicillium janthinellum mutant EU1 generated in our own laboratory. We obtained a maximum 72% hydrolysis, yielding glucose and cellobiose as the main end products. Lactic acid was produced from these cellulose samples by simultaneous saccharification and fermentation (SSF) in a media containing a cellulase enzyme preparation derived from Penicillium janthinellum mutant EU1 and cellobiose utilizing Lactobacillus lactis mutant RM2-24. A maximum lactic acid concentration of 73 g l^-1 was produced from a concentration of 100 g l^-1 of bagasse-derived cellulose, the highest productivity and yield being 1.52 g l^-1 h^-1 and 0.73 g g^-1, respectively. Considering that bagasse is a waste material available in abundance, we propose to use this biomass to produce cellulose and then sugars, which can be fermented to valuable products such as ethanol and lactic acid.

Full text of DOI:10.1039/b925975a

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D-(-)-Lactic acid production from cellobiose and cellulose by Lactobacillus
lactis mutant RM2-2†4
Mamta Singhvi,a Dipti Joshi,a Mukund Adsul,a Anjani Varmab and Digambar Gokhale*a
Received 9th December 2009, Accepted 1st April 2010
First published as an Advance Article on the web 14th May 2010
DOI: 10.1039/b925975a
Lactobacillus lactis mutant RM2-24 utilizes cellobiose efficiently, converting it into D-(-)-lactic
acid. Cellobiose-degrading enzyme activities were determined for whole cells, cell extracts and
disrupted cells. Aryl-b-glucosidase activity was detected in whole cells and disrupted cells,
suggesting that these activities are confined to the cells. The mutant produced 80 g l-1 of lactic acid
from 100 g l-1 of cellobiose with 1.66 g l-1 h-1 productivity. Production of D-lactic acid from
different cellulose samples was also studied. The cellulose samples at high concentration (10%)
were hydrolyzed by cellulase enzyme preparation (10 FPU g-1 cellulose) derived from Penicillium
janthinellum mutant EU1 generated in our own laboratory. We obtained a maximum 72%
hydrolysis, yielding glucose and cellobiose as the main end products. Lactic acid was produced
from these cellulose samples by simultaneous saccharification and fermentation (SSF) in a media
containing a cellulase enzyme preparation derived from Penicillium janthinellum mutant EU1 and
cellobiose utilizing Lactobacillus lactis mutant RM2-24. A maximum lactic acid concentration of
73 g l-1 was produced from a concentration of 100 g l-1 of bagasse-derived cellulose, the highest
productivity and yield being 1.52 g l-1 h-1 and 0.73 g g-1, respectively. Considering that bagasse is a
waste material available in abundance, we propose to use this biomass to produce cellulose and
then sugars, which can be fermented to valuable products such as ethanol and lactic acid.
around 230 ◦C.2 This finding has attracted more attention to the
Introduction
production of D-lactic acid.
Lactic acid and its derivatives have been widely used in food,
Cellulosic substances are abundantly available resources of
pharmaceutical, cosmetic and industrial applications.1 It has
renewable biopolymer which can be utilized as a feedstock for
a potential to become a commodity chemical for produc-
producing a number of bulk chemicals like ethanol or lactic
tion of biodegradable polymers, oxygenated chemicals, plant
acid through fermentation processes. The production of such
growth regulators and special chemical intermediates. It has
value-added products from renewable feedstocks is a present
been receiving great attention as a feedstock for manufacture
need, and there is a demand to make it an economically and
of polylactic acid (PLA), a biodegradable polymer used as
environmentally feasible process. Such bioprocesses involving
environmentally friendly biodegradable plastic. Lactic acid is
cellulosic residues not only provide alternative substrates but
manufactured either chemically or by microbial fermentation.
also help solve their disposal problems. With the advent of
Chemical synthesis always results in racemic mixture of lactic
biotechnological innovations, mainly in the area of enzyme
acid, which is a major disadvantage. Microbial fermentation
technology and fermentation technology, many new avenues
offers the advantage in both utilization of renewable carbohy-
have opened up for their exploitation as value-added products.
drates and production of pure L- or D-lactic acid depending on
Currently, optically pure lactic acid is produced mainly from
the strain selected. The physical properties of PLA depend on
corn starch. However, the use of agro-waste materials for lactic
the isomeric composition of lactic acid. Poly(L-lactide) (PLLA)
acid production appears to be more attractive because they do
obtained by polymerization of L-lactic acid or L-lactide has a
not then have any impact on the human food chain.
melting temperature of 175 ◦C. The melting point of this polymer
Unfortunately, the process for converting cellulosic material
can be increased by blending with poly (D-lactide) (PDLA) in a
into lactic acid is not yet feasible due to the high cost of cellulase
solvent. Recently, it was found that the polymer blend of PLLA
enzymes involved in cellulose hydrolysis3,4 and also to the use of
and PDLA produces a stereo-complex with melting temperature
fastidious organisms.5 The utilization of cellulosic wastes such as
cardboard and corn cobs as substrates for lactic acid fermenta-
tion, simultaneous saccharification and fermentation (SSF) has
been considered to be a promising approach.6 However, there
have been many technical problems – for instance, the enzymes
of cellulose hydrolysis are inhibited by the intermediate products
such as cellobiose, and lactic acid biosynthesis is inhibited by
lactic acid.7 Many investigations have been carried out to relieve
such inhibitions, for example, in situ product removal technology
aNCIM Resource Center, National Chemical laboratory, Dr Homi
Bhabha Road, Pune, 411008, India. E-mail: dv.gokhale@ncl.res.in;
Fax: 91-20-25902671; Tel: 91-20-25902670
bPolymer Science and Engineering, National Chemical laboratory, Pune,
411008, India
† Electronic supplementary information (ESI) available: Additional
experimental information. See DOI: 10.1039/b925975a
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has been applied to the SSF process, but this needs large electric
energy or high-level equipment.8,9 Cellulase inhibition by glucose
and cellobiose during the hydrolysis of cellulosic material by
cellulases is the main bottleneck, which significantly slows down
the rate of hydrolysis. The addition of b-glucosidase at the
beginning of SSF is recommended for the removal of cellobiose
inhibition, but sometimes it does not work because of rapid
deactivation of the enzyme.10 Therefore, it is advantageous to
use a lactic acid producing strain that has the ability to utilize
both glucose and cellobiose efficiently.5 It is known that some
Lactobacillus strains utilize cellobiose as a carbon source,11 but
very little information is available about lactic acid production
from cellobiose.
were carried out using Penicillium janthinellum mutant EU1
cellulase, as per the methodologies reported earlier.12
Enzyme assay
b-Glucosidase (b-D-glucosideglucohydrolase; E.C. 3.2.1.21) ac-
tivity was estimated according to the method described earlier13
using pNPG as substrate. The total of assay mixture (1 ml)
consisting of 0.9 ml of substrate and 0.1 ml of suitably diluted
◦
enzyme was incubated at 50 C for 30 min. The p-nitrophenol
liberated was measured at 410 nm after developing the color with
2 ml of sodium carbonate (2%). One unit of enzyme activity is
equivalent to one mmol of p-nitrophenol generated per minute.
The other substrates used were pNPC and pNPgal.
We have previously reported the production of L-lactic acid
from sugarcane bagasse cellulose with high productivity and
yield using a Lactobacillus mutant created in our laboratory.12
In this paper, we describe the efficient utilization of cellobiose
and cellulosic materials by Lactobacillus lactis mutant RM2-24
for lactic acid production. The mutant was isolated following
UV mutagenesis and selected on the basis of a greater zone
of acid formation on a sucrose-based medium. We also report
the aryl-b-glucosidase activity (involved in cellobiose utilization)
that this mutant displays.
Analytical methods
Cell growth was measured by spectrophotometrically using
Spectrometer-117 (Systronics, Mumbai, India) at a wavelength
of 660 nm. Reducing sugar concentration was determined by
DNS method. The amounts of glucose and lactic acid were
determined using a high-performance liquid chromatography
(HPLC) system (Dionex India Ltd.) equipped with UV- or
RI-detectors.15 An ion exclusion column (Aminex, HPX-87H,
Biorad, Hercules, CA) was used at a temperature of 30 ◦C with
0.008 N H2SO4 as a mobile phase at flow rate of 0.6 ml min-1.
An injection volume of the sample was 50 ml. The presence of
L-(+)-lactic acid was analyzed by an L-lactate oxidase enzyme kit
(Randox Laboratories, UK), and the D-lactic acid content was
calculated by subtracting L-lactic acid values from total lactic
acid estimated by HPLC.14
Experimental
Chemicals
p-Nitrophenyl-b-D-glucopyranoside (pNPG), p-nitrophenyl-b-
D-cellobioside (pNPC), p-nitrophenyl-b-D-galactopyranoside
(pNPgal), 3,5-dinitrosalicylic acid and Sigma cellulose were
obtained from Sigma-Aldrich Co., St Louis, MO, USA. Avicel
PH-101 was obtained from Fluka Chemie GmbH. Solka Floc
SW44 was purchased from Brown Co., Berlin, NH. The a-
cellulose with 0.18% lignin and 98% cellulose was prepared from
sugarcane bagasse in our laboratory.12
Results and discussion
The profile of growth (optical density) and lactic acid production
from cellobiose is shown in Fig. 1. The maximum amount of
lactic acid was produced within 48 h of fermentation, with
an increase in optical density from 0.6 to 11 and a decrease
in pH from 6.5 to 4.8. The maximum (80 g l-1) amount of
lactic acid was produced from 100 g l-1 of cellobiose with
1.66 g l-1 h-1 productivity and 0.8 g g-1 yield. These are the
highest productivity and efficiency values reported so far for the
production of lactic acid from cellobiose. This strain, therefore,
Strain information and cellulase production
Mutant EU1 was isolated by exposing conidia of Penicillium
janthinellum NCIM 1171 to UV irradiation. The procedure
of generation of mutant EU1 and its crude enzyme prepa-
ration have already been reported earlier.13 The mutant was
maintained on potato dextrose agar (PDA) and sub-cultured
once every three months. PDA contained (per liter) extract
from 200 g potatoes, glucose (20.0 g), yeast extract (1.0 g),
and agar (20.0 g). Enzyme production was carried out in a
250 ml Erlenmeyer flask, with 70 ml of production medium
containing 1% (w/v) cellulose-123 powder and 2.5% wheat
bran.13 Lactobacillus lactis mutant RM2-24, producing lactic
acid with high productivity, was isolated by UV mutagenesis.14 It
was maintained in liquid MRS medium supplemented with 0.1%
CaCO3.
Lactic acid production in shake flasks
Lactic acid production from cellobiose in shake flasks was
performed as reported earlier.14 The saccharification of cellulosic
substrates and simultaneous saccharification and fermentation
Fig. 1 Profile of lactic acid production (᭹), residual sugar (ꢀ) and
growth (ꢀ) during fermentation by Lactobacillus lactis mutant RM2-24
using cellobiose (100 g l-1).
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Table 1 Detection of aryl-b-glucosidase activity from Lactobacillus
lactis mutant RM2-24 grown in different sugar substratesa
grown cells) were detected in sonicated cells (Table 1), suggesting
that the enzymes could be bound to the cell wall/membrane. The
activity levels were low compared to those of the intact cells,
which could be due to the inactivation of the enzymes during
sonication. It is noteworthy that aryl-glucosidase activities were
higher in the Lactobacillus lactis mutant RM2-24 compared
to those observed in Lactobacillus delbrueckii mutant Uc-3
reported earlier.16
Experiments were performed to evaluate the hydrolysis of
various cellulose samples using cellulase enzyme preparations
derived from Penicillium janthinellum EU1. Hydrolysis of the
most crystalline substrate, Avicel, was always lower than that
of other cellulose samples due to its microcrystalline structure
which prevents easy access to the enzyme. However, 71%
hydrolysis was obtained in case of bagasse-derived cellulose,
indicating its greater accessibility towards cellulase enzyme
(Fig. 2). Solka-Floc and Sigma cellulose were hydrolyzed to
approximately 50%, probably due to the more amorphous nature
of these two substrates.
Enzyme activity (U g-1 of cells) grown in:
Hydrolyzed
Enzyme source Substrate Glucose Lactose sucrose
Cellobiose
Cells
pNPG
pNPgal 5.18
2.88
7.56
6.6
1.215 ND
4.02
5.01
0.81
2.7
5.1
23.4
4.41
6.075
10.5
2.48
3.79
pNPC
Sonicated cells pNPG
ND
ND
ND
2
ND
pNPgal 2.1
pNPC ND
a The cells were harvested at the onset of stationary phase for the
determination of enzyme activity. ND, not detected.
is proven to be highly efficient for the conversion of cellobiose
to lactic acid, and could be exploited at a commercial level.
Previously, we reported the production of L-(+)-lactic acid from
cellobiose by Lactobacillus delbrueckii mutant Uc-3.16
This mutant strain, RM2-24, was observed to utilize cel-
lobiose, indicating the presence of cellobiose-degrading en-
zymes. Therefore, we attempted to detect the cellobiose-
degrading enzymes by using pNPG, pNPC and pNPgal as
substrates. The cells were grown in different sugars at 42 ◦C and
harvested at the late-exponential phase by centrifugation. After
centrifugation, the supernatant and cells were used for an analy-
sis of aryl-b-glucosidase and aryl-b-galactosidase activities. We
could not detect any enzyme activity in the supernatant. The cells
were washed three times with phosphate buffer (50 mM, pH 7.0)
and resuspended in the same buffer. The suspension was used for
analyzing cell-bound glucosidase and galactosidase activities.
Table 1 presents the data for all the enzyme activities in the
mutant strain grown in liquid medium with different sugars. All
activities were detected in lactose and cellobiose grown cells.
However, a higher level of aryl-b-glucosidase was observed in
cellobiose-grown cells. Other than cellobiose, lactose-grown cells
also exhibited all activities. When cells were grown in glucose
and a hydrolyzed sucrose-based medium, aryl-b-glucosidase and
aryl-b-galactosidase activities were detected in small amounts.
There was no activity detected using pNPC as substrate. These
results show that the aryl-b-glucosidase or aryl-b-galactosidase
gene could be constitutively expressed, and enhancement in
activity was observed when the mutant was grown in either cel-
lobiose or lactose, respectively. Activity with pNPC as substrate
was detected only when the culture was grown in cellobiose or
lactose. However, the induction in lactose-grown cells is less than
that in cellobiose-grown cells.
None of these activities were detected in fermented broth,
suggesting the intracellular location of these enzymes. To check
the intracellular location of these enzymes, cells were subjected
to sonication (SONICS Vibra cell; model VC 130) in phosphate
buffer (pH 7.0, 50 mM) containing 0.5 mM EDTA and 1 mM
dithiothreitol. The sonication was performed at 60% amplitude
(125 mm) for 3 min by using a 2 mm probe under cold conditions.
Almost 90% of the cells were disrupted by this method. The
supernatant and sonicated cell debris were analyzed for all
above-mentioned enzyme activities. No activities were detected
in the supernatant, showing that enzymes are not located in the
cytoplasm. However, all activities (for lactose- and cellobiose-
Fig. 2 Profile of hydrolysis of Avicel (ꢁ), baggasse cellulose (ꢀ),
Solka-Floc (ꢀ) and Sigma cellulose (ꢀ) by mutant (EU1) enzyme
preparations. The hydrolysis was carried out using 10% substrate
concentrations with mutant enzyme preparations (10 FPU g-1).
SSF experiments were performed under the selected con-
ditions (42 ◦C, pH 6.5) because the organism used in this
fermentation is a mutant of Lactobacillus lactis (RM2-24) and
◦
cannot grow at temperatures more than 42 C. We carried out
the SSF at pH 6.5, at which the cellulases used were active
with retention of more than 50% activity. SSF experiments
were performed in production media containing cellulases
(10 FPU g-1 of substrate). The pH of the fermentation broth
dropped to 5.4 within 24 h (Fig. 3), which is the pH at
Table 2 Lactic acid production by Lactobacillus lactis mutant RM2-24
using different cellulosic substrates
Yield (g l-1)
24 h 48 h
Productivity (g l-1 h-1)
Substrates
72 h
24 h
48 h
72 h
a-Cellulose
47.92 73.31 73.1
Sigma cellulose 17.78 17.94 20.8
1.99
0.74
1.52
0.37
0.64
0.35
1.01
0.28
0.45
0.24
Solka-Floc
Avicel PH-101
30.96 31.13 32.62 1.29
15.88 17.11 17.33 0.66
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Table 3 Comparison of recent data with the present work on SSF production of D-(-)-lactic acid from cellulosic substrates
Fermentation Maximum lactic acid Yield
time (h)
Productivity
Substrate (g l-1)
Filter paper (33)
Microorganism
Enzyme (FPU g-1)
concentration (g l-1)
(% g g-1) (g l-1 h-1)
Ref.
L. coryniformis
ATCC 25600
L. delbrueckii IFO Cellulase-Y-NC
3202
L. coryniformis
ATCC 25600
Celluclast and
Novozyme (28)
48
25
28
23
73
75
28
56
73
0.5
4
Defatted rice
bran (100)
Pretreated
cardboard (41)
a-Cellulose (100) L. lactis RM2-24
36
0.77
0.49
1.52
8
Celluclast and
Novozyme (22.8)
48
17
P. janthinellum EU1 (10) 48
This work
which enzymes are most active. No cellobiose accumulation
was observed during the fermentation at any time. Cellobiose
was either converted to glucose by b-glucosidase present in the
cellulase preparations or utilized by the mutant strain to produce
lactic acid. We obtained 73 g l-1 of lactic acid from 100 g l-1 of
a-cellulose. The yield (g g-1) and productivity (g l-1 h-1) of lactic
acid were 0.73 and 1.52, respectively (Table 2). This is, to our
knowledge, the highest yield and productivity of D-lactic acid
reported in the literature so far from cellulose (Table 3).
cellulosic materials in SSF. Bottlenecks like feedback inhibition
by glucose and cellobiose were removed by using this strain,
leading to the complete conversion of cellulosic substrates to
the value-added products like lactic acid. This strain utilized
cellobiose effectively and produced lactic acid in a homo-
fermentative manner. These studies show the potential of such
a strain for producing value-added products from renewable
biomass.
Acknowledgements
The authors gratefully acknowledge the financial support from
TNBD Division, CSIR, New Delhi.
References
1 T. B. Vickroy, Comprehensive Biotechnology, ed. M. Moo-Young,
Pergamon Press, New York, 1985, vol. 3, pp. 761–776.
2 H. T. Tsuji and I. Fukui, Polymer, 2003, 44, 2891–2896.
3 C. E. Wyman, B. E. Dale, R. T. Elander, M. Holtzapple, M. R. Ladish
and Y. Y. Lee, Bioresour. Technol., 2005, 96, 1959–1966.
4 R. Yanez, A. B. Moldes, J. L. Alonso and J. C. Parajo, Biotechnol.
Lett., 2003, 25, 1161–1164.
5 A. B. Moldes, J. L. Alonso and J. C. Parajo, Bioprocess Eng., 2000,
22, 175–180.
6 B. Rivas, A. B. Moldes, J. M. Dominguez and J. C. Parajo, Enzyme
Microb. Technol., 2004, 34, 627–634.
7 Z. Lu, M. Lu, F. He and L. Yu, Bioresour. Technol., 2009, 100, 2026–
2031.
Fig. 3 Course of lactic acid production (ꢀ) and pH (ꢀ) during SSF
of a-cellulose (100 g l-1) using mutant enzyme preparation (EU1,
10 FPU g-1).
8 T. Tanaka, M. Hoshina, S. Tanabe, K. Sakai, S. Ohtsubo and M.
Tainguchi, Bioresour. Technol., 2006, 97, 211–217.
9 A. Romani, R. Yanez, G. Garrote and J. L. Alonso, Bioresour.
Technol., 2008, 99, 4247–4254.
Conclusion
10 K. Nakasaki and T. Adachi, Biotechnol. Bioeng., 2003, 82,
Lactobacillus lactis mutant RM2-24 is capable of utilizing high
concentrations of cellobiose, producing lactic acid with high
yield (0.8 g lactic acid per g cellobiose). Batch experiments
were conducted for conversion of cellulose to lactic acid by
simultaneous saccharification and fermentation (SSF) using
cellulase preparations derived from mutants of Penicillium
janthinellum (EU1) and Lactobacillus lactis (RM2-24). Batch
SSF yielded 73 g l-1 of lactic acid from 100 g l-1 of a-cellulose with
a yield of 0.73 g per g of cellulose substrate and a productivity
of 1.52 g l-1 h-1.
263–217.
11 F. J. Carr, D. Chill and N. Maida, Crit. Rev. Microbiol., 2002, 28,
281–370.
12 M. G. Adsul, A. J. Varma and D. V. Gokhale, Green Chem., 2007, 9,
58–62.
13 M. G. Adsul, K. B. Bastawade, A. J. Varma and D. V. Gokhale,
Bioresour. Technol., 2007, 98, 1467–1473.
14 D. S. Joshi, M. S. Singhvi, J. M. Khire and D. V. Gokhale, Biotechnol.
Lett., 2010, 32, 517–520.
15 S. R. Kadam, S. S. Patil, K. B. Bastawde, J. M. Khire and D. V.
Gokhale, Process Biochem., 2006, 41, 120–126.
16 M. G. Adsul, J.M. Khire, K. B. Bastawade and D. V. Gokhale, Appl.
Environ. Microbiol., 2007, 73, 5055–5057.
17 R. Yanez, J.L. Alonso and J. C. Parajo, J. Chem. Technol. Biotechnol.,
2005, 80, 76–84.
In conclusion, Lactobacillus lactis mutant RM2-24 is a
promising strain for the production of D-lactic acid from
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The Royal Society of Chemistry 2010
Green Chem., 2010, 12, 1106–1109 | 1109
©