55NISR Research GRANT
2010 Young Investigator Research Grant

Construction of a microbial community for effective hydrolysisof the cellulosic and lignocellulosic biomass
Keiko WATANABE
Department of Environmental Engineering for Symbiosis, Faculty of Engineering, Soka University(Present address: Soka Senior High School)

Research aims
In recent years, bioethanol has become an importantproduct. Cellulosic and lignocellulosic biomass has highpotential as feedstock for bioethanol, and has been thefocus of active research and development. Wood wastes,rice straws and rice husks, etc. are suitable biomass materials for bioethanol production because use of these materials
for ethanol production does not compete with food production. However, they are complex mixtures of cellulose,
hemicellulose (including xylan) and lignin, and cannot be
decomposed easily. Microorganisms have been found to decompose cellulose and lignin with high efficiency previously1). However, microorganisms which can decompose
xylan in the same environment have not been reported.
During our analysis of microbial community compositionin the composting reactor, we detected cellulose-degrading
bacteria Thermobifida fusca, lignin-degrading bacteria Ureibacillus thermosphaericus, and xylan-degrading bacteria
Thermobacillus composti in the same compost sample.
Therefore, these three bacteria may be possible symbiotic
microorganisms. In this study, to construct the microbialcommunity for effective hydrolysis of the cellulosic and lignocellulosic biomass, we carried out the following three investigations: 1) searching for optimum conditions for coculturing T. fusca, U. thermosphaericus and T. composti inthe same reactor, 2) screening of microorganisms, from thesame compost sample, that enhance the hydrolysis efficiency and improve the stability of microbial community
composition, and 3) characterization of xylanase derived
from T. composti.
Methods
Optimum conditions for culturing three microorganisms
in the same reactor
T. fusca (DSM43792), U. thermosphaericus (DSM10633)
and T. composti (DSM18247) were obtained from DSMZ(The German Resource Centre for Biological Material). T.
composti is the most difficult to grow among these bacterial
species. Therefore, according to the optimal growth conditions for T. composti, the modified Brock’s basal salts(MBS)2) supplemented with 0.5% yeast extract was used asa growth medium. And the growth potentials of the threebacterial species in the medium with various initial pH

were determined at 50°C. U. thermosphaericus and T. composti were cultured in the liquid medium. Their growth wasmonitored by the optical cell density at 600 nm by using aspectrophotometer. T. fusca is an actinomycetous bacterium. T. fusca forms aggregates, not dispersed uniformly
in the liquid medium. Since its optical cell density could
not be measured by using spectrophotometer, the optimum
growth conditions for T. fusca were determined by counting
colonies on solid medium.
Cellulosic and lignocellulosic biomass glycosylation
Rice husks were used as cellulosic and lignocellulosic
biomass. They were milled by using a household use
kitchen mixer. After size fractionation (1 mm, 1 mm–0.1 mm, 0.1 mm) by using sieve, milled rice husks wereautoclaved twice. MBS (pH 8.0) supplemented with 0.5%
rice husks or MBS (pH 8.0) supplemented with 0.5% ricehusks and 0.5% yeast extract was used as medium. Microorganisms were inoculated into the medium and static
cultivation was performed at 50°C for 2 weeks. Experiments were conducted under the following eight conditions:
growth medium without inoculation (blank), cultures inoculated individually with single species, mixed cultures inoculated with two species (T. fusca and U. thermosphaericus,
T. fusca and T. composti, U. thermosphaericus and T. composti), and mixed cultures inoculated with all the threespecies. Released reducing sugar was measured by using
DNS method3). To confirm the presence of the three speciesof bacteria in cultures, RFLP analysis was conducted as follows: genomic DNA mixture was extracted from bacterial
cells grown in liquid culture and used as template DNA forpolymerase chain reactions (PCR). 16S rDNAs were amplified by PCR. PCR products were digested with restriction
enzymes SacI and PstI.
Screening of microorganisms from compost sample
The composting sample was added into MBS (pH 8) supplemented with 0.5% yeast extract, and incubated at 50°C
for 24 hours. This enrichment culture was plated onto solid
medium for isolation of microorganisms. After single
colony isolations, purified isolates were analyzed by 16S
rDNA sequencing.

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Characterization of xylanase derived from Thermobacillus composti
T. composti was cultured in MBS (pH 9) supplemented
with 0.5% beechwood xylan, with shaking at 150 rpm at50°C, for 3 days. The culture was separated into the supernatant and bacterial cells by centrifugation. Proteins in thesupernatant were precipitated by ammonium sulfate precipitation. After centrifugation, the proteins were resuspendedin buffer. A crude enzyme solution was obtained after desalting treatment. For analysis of xylanase activity, thecrude enzyme solution was added to a buffer solution containing 0.5% beechwood xylan. After incubation for 1 hourat various temperatures, the amount of produced xylose wasquantitated by the DNS method.
Results
The optimum growth pH for T. fusca, U. thermosphaericus, and T. composti were 8.0, 7.5–8.0, and 8.0–9.0, respectively. These results showed that pH 8.0 is a suitable growth
pH for co-culturing these three species in the same
medium. Therefore, for effective hydrolysis of the cellulosic and lignocellulosic biomass, the mixed cultures withvarious combinations of the three species were incubated atpH 8.0. Examination of biomass hydrolysis efficiency withthe each culture showed that the mixed culture containing
all of the three bacterial species released a larger amountsof reducing sugars (total of C6 and C5 sugars) than any
other mixed cultures, regardless of presence of yeast extract, at the end of experiments (after 2 weeks). When ricehusks with a particle size of 0.1 mm were used, hydrolysis
efficiency was about 65% (reducing sugars calculated asglucose). The hydrolysis efficiency observed with themixed culture containing all of the three bacterial species
was about 1.5-times higher than that of the culture containing only T. fusca, and about 1.1–1.8 times higher than thoseobserved with the mixed cultures containing two bacterial
species out of the three species. These results showed thatcombination of the tree different bacterial species could enhance hydrolysis efficiency of cellulosic and lignocellulosic
biomass.
The 16S rDNA sequencing analysis showed that the microorganisms isolated from the same compost sample belonged to Geobacillus thermoglucosidasius and Geobacillus toebii. It was reported that certain G. thermoglucosidasius strain can ferment major C6 and C5 sugars in cellulosic biomass and can tolerate high concentrations ofethanol4). From cellulosic and lignocellulosic biomass, themicrobial community constructed in this study was found
to release both C6 and C5 sugars. Therefore, there is a possibility that inclusion of G. thermoglucosidasius in themixed microbial community containing the three bacterial
species might lead to more efficient production of ethanolfrom cellulosic and lignocellulosic biomass. In addition, theoptimum temperature of xylanase derived from T. composti
was estimated at 55–60°C.
Conclusion
In this study, a microbial community capable of effectivehydrolysis of the cellulosic and lignocellulosic biomass wassuccessfully constructed. In addition, the optimum temperature of xylanase derived from T. composti was estimated.
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