The direct conversion of xylan to lactic acid by Lactobacillus brevis transformed with a xylanase gene

Article abstract of DOI:10.1039/c1gc15169j

A xylanase gene (xynR8), obtained from the DNA of a pool of uncultured rumen microbes, was introduced via a plasmid into Lactobacillus brevis. The recombinant xylanase, with an estimated molecular weight of 32 KDa, was expressed in the transformants and showed obvious xylanase activity (0.412 U ml^-1) against oat-spelt xylan in broth when compared to the wild-type Lactobacillus brevis ATCC367. The transformants all shared a similar ability to utilize and metabolize xylooligosaccharides. When a selected transformant was inoculated into modified MRS medium containing xylan as the main carbon source, the cell density reached 2.20 × 10⁹ CFU ml^-1 on day 4, while the wild-type strain without the plasmid containing the recombinant xylanase did not grow at all under the same conditions. After fermentation, 1.70 g l^-1 of lactic acid and 0.44 g l^-1 of ethanol were present in the culture supernatant of the strain containing the recombinant xylanase. These results indicate that Lactobacillus brevis containing the xylanase gene is capable of directly saccharifying and fermenting xylan to produce lactic acid in one step. This strain will enable the development of a feasible and economical approach to the production of lactic acid directly from xylan.

Full text of DOI:10.1039/c1gc15169j

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PAPER
The direct conversion of xylan to lactic acid by Lactobacillus brevis
transformed with a xylanase gene
Chun-Yi Hu,a Da-Jun Chi,b Shih-Syuan Chenb and Yo-Chia Chen*b
Received 14th February 2011, Accepted 3rd May 2011
DOI: 10.1039/c1gc15169j
A xylanase gene (xynR8), obtained from the DNA of a pool of uncultured rumen microbes, was
introduced via a plasmid into Lactobacillus brevis. The recombinant xylanase, with an estimated
molecular weight of 32 KDa, was expressed in the transformants and showed obvious xylanase
activity (0.412 U ml-1) against oat-spelt xylan in broth when compared to the wild-type
Lactobacillus brevis ATCC367. The transformants all shared a similar ability to utilize and
metabolize xylooligosaccharides. When a selected transformant was inoculated into modified
MRS medium containing xylan as the main carbon source, the cell density reached 2.20 ¥ 109
CFU ml-1 on day 4, while the wild-type strain without the plasmid containing the recombinant
xylanase did not grow at all under the same conditions. After fermentation, 1.70 g l-1 of lactic acid
and 0.44 g l-1 of ethanol were present in the culture supernatant of the strain containing the
recombinant xylanase. These results indicate that Lactobacillus brevis containing the xylanase
gene is capable of directly saccharifying and fermenting xylan to produce lactic acid in one step.
This strain will enable the development of a feasible and economical approach to the production
of lactic acid directly from xylan.
Direct fermentation to produce lactic acid from starch has
Introduction
been achieved using various Lactobacillus strains that possess
Lactic acid is a valuable and multi-functional organic acid,
extracellular amylase activity.2,5 However, it is clear that the
and it has been widely used in many industries, including cos-
conversion of starchy feedstocks to lactic acid would compete
metic, food, pharmaceutical and chemical, due to its attractive
with food for humans and feed for livestock, and that this
properties.1,2 It also has been shown to have potential promise
is an important limitation. Excessive conversion of starchy
when used to manufacture the biodegradable and biocompatible
feedstock like corn grain into fuel has already increased the
polymer polylactic acid (PLA), which is an environmentally
prices of cereal and dairy products,6,7 and the situation would be
friendly alternative to non-degradable plastics derived from the
worse if extra starchy feedstock was used for the production
planet’s limited petrochemical resources.1,3 Intensive screening
of biodegradable plastic due to limited availability. In this
for various potential and economical feedstock substrates for the
context, the use of abundant but inedible material would be
production of lactic acid is currently under way to meet future
a better feedstock when using fermentation for lactic acid
demand. Lactic acid can be produced by chemical synthesis or
production.
by biotechnological processes.4 The chemical synthesis of lactic
Hemicellulose, which is mainly composed of xylan, is the
acid uses petroleum as the raw material and is less friendly to the
second most abundant carbohydrate after cellulose in lig-
environment.1 The production of lactic acid by biotechnological
nocellulosic biomass. It is also an important waste product
processes is becoming more and more preferred due to issues
from agriculture and food processing.8 Lactic acid bacteria
of environmental pollution, the reducing reserves of petroleum
are generally regarded as potentially good candidates for the
and the increased requirement for lactic acid worldwide.
production of lactic acid, but most lactobacilli lack the ability
to hydrolyze lignocelluloses.9 Consequently, a variety of pre-
treatment methods have been developed that allow the fermen-
aDepartment of Nutrition and Health Science, Fooyin University,
Kaohsiung, 83161, Taiwan, Republic of China
tation of lignocellulose to produce lactic acid. Enzymatic pre-
treatment, and simultaneous saccharification and fermentation
(SSF) are the two most prevalent procedures used to convert
lignocellulosic materials into lactic acid.3,10,11 The raw material
is depolymerized into its constituent monosaccharides or simple
bGraduate Institute of Biotechnology, National Pingtung University of
Science and Technology, No. 1 Shuehfu Road, NeiPu, Pingtung 91201,
Pingtung, Taiwan, Republic of China. E-mail: ox@mail.npust.edu.tw;
Fax: +886 8-7740550; Tel: +886 8-7703202 ext. 5181
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sugars, which can then be utilized by the lactic acid bacteria.
This is done by adding the hydrolysate to media, which is
then fermented by lactic acid-producing microorganisms. The
two most common pre-treatment methods for the hydrolysis of
lignocellulose to simple sugars are acid hydrolysis and enzymatic
hydrolysis. Enzymatic treatment is more economic because it
uses less energy, has relatively mild reaction conditions that avoid
the use of toxic and corrosive chemicals, and does not produce
inhibitors that affect the microbial fermentation.10 Nonetheless,
enzyme treatment is overall a more costly approach when used
for lactic acid production. In these circumstances, the integration
of enzyme production and microbial fermentation would be
a feasible and cost-effective way to produce lactic acid from
lignocellulose.
Lactobacillus brevis is a heterofermentative microorganism
found in milk, cheese, sauerkraut, sourdough, silage and the
gastrointestinal tracts of animals.12 It can simultaneously con-
sume a wide variety of carbon sources, including xylose and
glucose, for growth.13 It also has been shown to have b-xylosidase
activity when wheat straw hydrolysate is used as the starting
material for lactic acid production.10 However, Lactobacillus
brevis is unable to grow using xylan as its sole carbon source
since no xylanase gene is present in its genome.14 It seems highly
probable that Lactobacillus brevis would be able to directly utilize
xylan for growth if a xylanase gene was incorporated into its
genome. In this study, a xylanase gene from a metagenomic
sample of rumen fungi was introduced to Lactobacillus brevis
(Scheme 1), and the expression of the xylanase and the ability
of the transformed Lactobacillus brevis to produce lactic acid
using xylan as the main carbon source (Scheme 2) were
examined.
Experimental
Microorganisms and media
Escherichia coli DH5a (Invitrogen Corporation, Carlsbad, CA)
was used as the host when carrying out recombinant DNA exper-
iments and was grown at 37 ◦C on Luria-Bertani (LB) medium.15
Lactobacillus brevis ATCC 367, which was purchased from
the Bioresource Collection and Research Center (Hsingchu,
Taiwan), was selected as the candidate for transformation with
the xylanase gene. The Lactobacillus strain was maintained on de
Man, Rogosa and Sharpe (MRS) medium.10,16 MRS agar plates
containing chloramphenicol (10 mg l-1) was used to screen for
transformants containing the xylanase gene plasmid. MRS-X
medium, which was modified from MRS medium by replacing
the glucose with oat-spelt xylan (20 g l-1), was used for lactic
acid production. MRS-XOS medium was used to test sugar
utilization. The components of the MRS-XOS medium were
the same as for the MRS medium, except that glucose was
replaced with a 2% (w/v) xylooligosaccharide mixture that
contained 1.41% xylose, 29.11% xylobiose, 31.31% xylotriose,
15.06% xylopentaose, 6.73% xylotetraose, 4.39% xylohexaose,
8.55 xyloheptaose, 0.73% glucose and 0.75% arabinose (Sweet
Town Biotech. Crop, Taiwan). Lactobacillus strains were grown
at 30 ◦C without shaking.
Plasmid construction and transformation
The xylanase R8 gene (accession no. AY941119) was obtained
from the metagenomic DNA sample of a water buffalo by
PCR (polymerase chain reaction) amplification, as published
previously.17 The PCR fragment encoding xynR8 was digested
with XhoI and PstI, and ligated into similarly digested pNZ3004
to generate pNZR8. Next, pNZR8 was transformed into compe-
tent Lactobacillus brevis by electrophoration.18 The electrophor-
ated cells were spread onto MRS plates containing 10 mg ml-1
chloramphenicol and incubated at 30 ◦C until transformants
appeared. The transformants were selected and transferred to
another MRS plate containing 0.5% oat-spelt xylan. The plate
was stained with Congo red solution after 2 d of incubation.19
Zymogram and enzyme activity assays
Culture supernatants were collected to determine xylanase
activity, and zymogram analysis was also performed.20 For
image documentation, the stained gels were scanned at a
resolution of 600 dpi using a V200 Photo Scanner (Epson
Corporation, Nagano, Japan), and Adobe Photoshop software,
version 6.0 (Adobe Systems Incorporated, San Jose, CA, USA).
Scheme 1 Construction of a xylanase-producing transformant of
Lactobacillus brevis.
Scheme 2 Direct conversion of xylan to lactic acid by a Lactobacillus brevis transformant bearing a xylanase gene.
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The xylanase activity was detected by measuring the quantity of
reducing sugar produced by enzymatic hydrolysis. The reducing
sugar was determined by the DNS (dinitrosalicylic acid) method.
One unit of xylanase activity was defined as 1 mmol of reducing
sugar equivalents released from oat-spelt xylan per minute under
assay conditions.20
Lactic acid fermentation from xylooligosaccharides and xylan
Fig. 1 Clear zones and orange-colored zones formed by (a) Lactobacil-
lus brevis R8 and (b) Lactobacillus brevis ATCC 367. Both strains were
inoculated on an MRS plate containing 0.5% (w/v) xylan. The plates
were incubated at 30 ◦C for 3 d and stained with 0.5% (w/v) Congo red
solution.
The wild-type Lactobacillus brevis and the Lactobacillus brevis
strain containing the xylanase gene plasmid were cultured in
MRS-X and MRS-XOS media to assess the fermentation of
lactic acid. The lactic acid production experiments were carried
out as batch fermentations under aerobic conditions. The strains
were grown in a 250 ml Erlenmeyer flask containing 100 ml
MRS-X broth, with the Lactobacillus brevis strains inoculated
with 5% (v/v) of a bacterial suspension obtained after 15–20 h
growth. The inoculated broth was then incubated at 30 ◦C for 5 d
without shaking. The pH values of the broth were measured by
a pH meter. The total reducing sugars in the fermentation broth
were determined by the DNS colorimetric assay, as described
by Cheng et al.20 To determine viable cell counts (CFU (Colony
Forming Units) ml-1), serial dilutions of the samples were spread
on MRS plates and incubated for 48 h at 30 ◦C. The Lactobacillus
brevis CFU ml-1 in the broth were transformed to logarithmic
values before statistical analysis. The reported bacterial count
values are the mean values of triplicate determinations the
standard deviations (SD).
product of approximately 900 bp was amplified and was then
detected by agarose gel electrophoresis. The sequence of the
xylanase gene incorporated in the plasmid from Lactobacillus
brevis R8 was confirmed by DNA sequencing, and the sequence
was found to be identical to the sequence of the xylanase R8
gene, as previously described.
Analysis of the broth by zymogram after Lactobacillus brevis
R8 had been grown in it revealed that xylanase activity was
present and that the protein involved was a single clear band
with an apparent molecular weight of 32 kDa (Fig. 2). The size
of this expressed xylanase is close to the predicted molecular
weight of xylanase R8. These results indicate that he xylanase
R8 gene was successfully transformed, and is expressed and
secreted by Lactobacillus brevis R8.
Analysis of sugars and fermentation products
Samples taken during the fermentation were centrifuged (4 ◦C,
10 000 g, 30 min) on a Sigma 2K15 Centrifuge (Sigma Ltd.,
Osterode, Germany). The supernatant was filtered through a
0.45 mm filter (Pall Corporation, East Hills, NY, USA) and
stored at 4 ◦C. The sugars and fatty acids of the filtrate were
analyzed by high performance liquid chromatography (HPLC)
using a Hitachi D7100 (Merck–Hitachi, Tokyo, Japan) with an
ICSep COREGEL 87H3 column (Transgenomic Inc., Omaha,
NE, USA) coupled to a UV detector and an RI detector; 0.01 M
sulfuric acid (J. T. Baker Inc., Phillipsburg, NJ, USA) was used
as the mobile phase at a flow rate of 1.0 ml min-1. All chemicals
in this study were purchased from Sigma-Aldrich (St. Louis,
MO, USA) unless otherwise stated.
Fig. 2 Zymogram analysis of xylanase activity in crude extracts from
Lactobacillus brevis R8 (lane 1) and Lactobacillus brevis ATCC 367
(lane 2). The crude extracts were separated on a 10% acrylamide gel
supplemented with 1% oat-spelt xylan. Following electrophoresis, the
gels were incubated in the presence of a citrate buffer (50 mM, pH 6)
containing 50 mM dithiothreitol and stained with Congo red solution.
Results
Constructing the Lactobacillus brevis containing the xylanase
gene
The transformants of Lactobacillus brevis harboring the xy-
lanase gene plasmid were constructed and screened. The trans-
formant shown in Fig. 1 shows xylanolytic ability on an MRS
plate containing xylan. A yellow area around the colonies is
visible after Congo red staining, and no such region of xy-
lanolytic activity was found when a similar plate was inoculated
with the wild-type untransformed strain. The transformant with
xylanolytic ability was designated as Lactobacillus brevis R8.
The plasmid present in Lactobacillus brevis R8 was isolated and
checked for the presence of the xylanase gene by PCR. A single
Lactic acid fermentation from xylooligosaccharides
To investigate the ability of Lactobacillus brevis R8 to uti-
lize xylooligosaccharides, the glucose in the MRS medium
was replaced with xylooligosaccharides. Lactobacillus brevis
ATCC367, the wild-type strain, and Lactobacillus brevis R8
were inoculated individually into the modified medium, and
the bacterial cell density, consumption of xylooligosaccharides
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and accumulation of fermentation products were analysed.
Lactobacillus brevis ATCC367 and the transformant both grew
quickly and reached 109.30 CFU ml-1 and 109.18 CFU ml-1,
respectively, during which time the xylooligosaccharides were
utilized (Fig. 3). The quantitative disappearance of xylotriose,
xylobiose and xylose can be detected using HPLC, as shown
in Fig. 3. Of these three sugars, which were present in the
broth initially, xylobiose and xylotriose were quickly consumed,
but xylose slightly increased after 1 d of incubation by both
Lactobacillus brevis ATCC367 and Lactobacillus brevis R8.
Xylotriose was totally exhausted after fermentation. In contrast,
xylose and xylobiose were not completely depleted and a
small amount remained in the broth after fermentation. The
consumption of these substrates during fermentation indicates
that both Lactobacillus brevis wild-type and R8 are able to
utilize the various sugars present in the medium, including
xylose, xylobiose and xylotriose. The concentration of lactic acid
reached 6.81 0.17 and 7.10 0.53 g l-1 in the culture supernatant
after 3 d of fermentation with Lactobacillus brevis ATCC367 and
Lactobacillus brevis R8, respectively. The amount of acetic acid
also increased to 0.76 0.06 and 0.71 0.04 g l-1, respectively.
Ethanol could not be found after fermentation when either
strain was grown in MRS-XOS medium. These results show
that Lactobacillus brevis ATCC367 and Lactobacillus brevis R8
are both capable of utilizing xylooligosaccharides as a carbon
source for growth, and seem to possess a similar growth rate and
similar metabolic pathways for xylooligosaccharide utilization.
The growth of Lactobacillus brevis R8 in xylan medium
Both the wild-type and Lactobacillus brevis R8 were subcultured
in MRS-X broth to examine their growth and to determine
whether there was heterologous xylanase expression. Xylanase
activity increased, reaching a level of 0.412 U ml-1 after 5 d
of incubation, when Lactobacillus brevis R8 was used for
the fermentation, whereas no detectable xylanase activity was
found when the wild-type strain was cultured under the same
conditions (Fig. 4). The accumulation of reducing sugars in
the fermentation depended on the production of xylanase by
Lactobacillus brevis R8. As shown in Fig. 4, the level of reducing
sugars (equivalent to xylose) in the broth continuously increased
during the Lactobacillus brevis R8 fermentation on the MRS-X
medium and reached 2.62 g l-1 after 5 d of incubation. However,
there was no detectable increase in reducing sugar concentration
when the wild strain was cultured under the same conditions.
These results show that reducing sugars were released from the
oat-spelt xylan, and that this was very likely to be due to the
action of the expressed xylanase in Lactobacillus brevis R8.
The growth curves of the wild-type and Lactobacillus brevis
R8 are presented in Fig. 4. Lactobacillus brevis R8 can be seen to
grow continuously on MRS-X medium and reached a maximum
cell density of 2.20 ¥ 109 CFU ml-1 after 4 d of incubation; the
bacterial counts then decreased slightly to 1.46 ¥ 109 CFU ml-1
on day 5. Although Lactobacillus brevis ATCC367 without the
xylanase gene showed a similar level of growth at the beginning
of the incubation period, the cell density did not rise noticeably
after the second day of fermentation. These results support the
idea that the presence of the xylanase activity in Lactobacillus
brevis R8 allows growth without pre-treatment on 0.5% oat-
spelt xylan and that the xylan is utilized as its main carbon
source. This does not occur with the wild-type Lactobacillus
brevis because it lacks the plasmid encoding the xylanase gene.
Lactic acid fermentation from xylan
Fig. 3 The (a) growth and trend of xylooligosaccharide consumption
in MRS-XOS broth inoculated with (b) Lactobacillus brevis R8 and
(c) Lactobacillus brevis ATCC 367 during 5 d of fermentation. The
peak areas of refractive index chromatograms were obtained from
HPLC analysis using an 87H3 column. The samples were examined as
described in the Experimental section. Data points represent the mean
and standard deviations from triplicate experiments.
The ability of the Lactobacillus brevis strain containing the
plasmid encoding the xylanase gene to directly ferment xylan
to lactic acid was then assessed, together with an analysis of
the intermediate products and the pH of the broth (Fig. 5).
During fermentation, the pH was found to fall from an initial
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Fig. 4 Changes in (a) growth, (b) xylanase activity and (c) reducing
sugar (equivalent to xylose) accumulation in the broth of MRS-
X inoculated with Lactobacillus brevis R8 and Lactobacillus brevis
ATCC367 during 5 d of fermentation. The number of Lactobacillus
brevis was counted colony-forming units (CFU). The bacteria in the
broth were spread on an MRS medium plate after dilution and incubated
for 1 d at 37 ◦C. The xylanase activity was detected by measuring the
quantity of reducing sugar produced by enzymatic hydrolysis from oat-
spelt xylan for 30 min at 30 ◦C. The reducing sugar was determined by
the DNS (dinitrosalicylic acid) method. One unit of xylanase activity is
defined as 1 mmol of reducing sugar equivalents released from substrate
per minute. Data points represent mean and standard deviations from
triplicate experiments.
Fig. 5 Changes in (a) pH, (b) lactic acid production and (c) acetic acid
(circle symbols), and the ethanol (triangle symbols) concentration in
MRS-X broth inoculated with Lactobacillus brevis R8 (solid symbols)
and Lactobacillus brevis ATCC367 (open symbols) during 5 d of
fermentation. The samples were examined as described in the section of
Experimental. Data points represent the mean and standard deviations
from triplicate experiments.
of Lactobacillus brevis R8 and reached 0.46 g l-1, whereas no
significant change in either lactic acid or ethanol was detected
with the wild-type strain. For both strains, the concentration
of acetic acid changed only negligibly during the fermentation.
These results show that Lactobacillus brevis R8 is able to directly
ferment xylan and generate lactic acid and ethanol.
value of 6.4 to 6.1 after 2 d, and then continue to fall, finally
reaching 5.4 on day 5. Lactic acid was found to be the primary
product of the fermentation by Lactobacillus brevis R8, and
the concentration of lactic acid increased continuously during
the 5 d of incubation, reaching a maximum of 1.70 g l-1 in the
culture supernatant after 4 d of fermentation. The amount of
lactic acid did not increase but decrease day-by-day when wilt-
type Lactobacillus brevis was cultured in the same conditions.
The levels of ethanol also increased during the fermentation
Discussion
It has been found that Lactobacillus brevis possesses a metabolic
preference for pentose sugars such as xylose and arabinose
over glucose, and xylose utilization in Lactobacillus brevis
is not stringently controlled, as has been seen in other
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xylose-utilizing lactobacilli such as Lactobacillus pentosus.13,21
In the present study, we found that xylotriose was quickly
exhausted, but that some xylobiose and xylose remained in the
broth after fermentation with Lactobacillus brevis R8 (Fig. 3).
In a previous study, it was also found that Lactobacillus brevis
ATCC 8287 prefers a xylooligosaccharide mixture to pure xylose
for growth.22 These results suggest that Lactobacillus brevis
might be better at utilizing xylotriose compared to xylose, sup-
porting the idea that Lactobacillus brevis is an excellent candidate
for the utilization and fermentation of xylan hydrolysate.
Obligately heterofermentative lactobacilli like Lactobacillus
brevis are able to use the pentose phosphoketolase (PK) pathway
for the metabolism of pentose, and the enzymes involved in the
PK pathway, except for pyruvate-formate lyase and pyruvate
decarboxylase, are also present in the genome of Lactobacillus
brevis ATCC 367. In these circumstances, lactic acid and ethanol
or acetate should be generated when the sugar is consumed,13–14
and there should be a direct correlation between the sugar
present and the production of ethanol or acetate. Acetate,
rather than ethanol, is quickly utilized at the beginning of
incubation, and increased amounts of acetate are only produced
by Lactobacillus brevis when the amount of sugar present is
high. The accumulation rate of acetate then slows down and
trace amounts of ethanol are produced when sugars are almost
depleted. This phenomenon has been found with several strains
of Lactobacillus brevis.13,23 The production of acetate rather than
ethanol allows the synthesis of additional ATP via acetate kinase
and leads to a higher specific growth rate. Protons are transferred
from a coenzyme like NADH, which is generated by the PK
pathway, to oxidative metabolites that allow faster growth.
However, NADH will accumulate quickly when the sugar is
metabolized on a large scale, and the accumulated NADH will
then retard the production of lactate from glyceraldehyde-3
phosphate and will also have an influence on the generation
of energy. Ethanol is made by recycling NADH, and this would
allow the regeneration of the coenzyme.13,23
In the present study, acetic acid and ethanol can only be
found in MRS-XOS and MRS-X, respectively (Fig. 5). The
sugar concentration in MRS-XOS was sufficiently high (20 g l-1
xylooligosaccharides) to allow the fast growth of Lactobacillus
brevis and the cell density reached a high level (Fig. 3). This was
accompanied by the generation of ATP via acetate kinase, and
as a result acetate accumulated significantly in the Lactobacillus
brevis broth. In contrast to the above scenario, reducing sugars
were released slowly from the xylan in MRS-X medium due to
hydrolysis by the xylanase expressed by Lactobacillus brevis R8,
and the available xylooligosaccharides were depleted as they
were produced; as a result, the sugar concentration (Fig. 4)
remained low (0.08~2.62 g l-1). Under these circumstances, the
production of ethanol was able to regenerate NADH, which
allowed lactate to be scavenged for ATP generation due to the
low levels of the available sugar.
transformed plasmid, is able to directly ferment xylan to lactic
acid in one step. This is an attractive approach to the production
of lactic acid from a xylan-containing material and is worthy of
further development in the future.
Acknowledgements
The financial support of the National Science Council of
Taiwan (Grant no. NSC 99-2313-B-020-006-MY3) is greatly
appreciated.
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Conclusion
In conclusion, our results clearly demonstrate that Lactobacillus
brevis R8, a lactobacillus expressing a xylanase carried on a
1734 | Green Chem., 2011, 13, 1729–1734
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