| dc.description.abstract | The world is now facing one of the greatest difficulties of modern time. As she is
running out of energy, scientists need to think of a new and alternative way to produce
sustainable energy source. Luckily bio-ethanol production from lignocellulosic waste can
solve this problem completely. However the structure of lignocellulose poses substantial
recalcitrance to the path of ethanol production. In order to produce bioethanol two basic
steps are involved; first one is the pretreatment of lignocellulosic waste such as rice
straw, maize straw, corn stover, barley straw etc. and the hydrolysis step which can be
achieved by enzymatic manipulation using different microbes. Many articles have
already reviewed different pretreatment methods and hydrolysis mechanisms in order to
conquer the recalcitrant structure of cellulose. However no significant advancement in
this criteria has still been achieved as various obstacles such as cellulose crystallinity,
degree of polymerization, lignin residues, and hemicellulose content keep getting in the
way of our success. The use of bioconsortium in order to break the recalcitrant structure
of cellulose has not been done very briefly and appears to promise more than the
conventional biological treatments of lignocellulose. Bio-consortium can be the
breakthrough that scientists are looking for. As one species of bacteria can’t break
through the recalcitrant cellulose, two or three different bacterial strains together in the
right environment can produce significant results. In this article a total of two cellulase
producing bacterial strains (HSTU-2 and HSTU-3) have been isolated from cow dung
and identified at molecular level using 16s rRNA gene sequencing. Both the strains
(HSTU-2 and HSTU-3) have been identified as Bacillus sp. and have been submitted to
gene bank in National Centre for Biotechnology Information (NCBI). Bacillus sp.
HSTU-2 (accession no. MK659878) and Bacillus sp. HSTU-3 (accession no.
MG582599.1) have also been run through different bioinformatics software and online
tools for their sequence similarity including AT-GC content determination and
evolutionary analysis using ‘MEGA X’. Phylogenetic tree has also been described for all
the six strains with maximum likelihood method and proper node and branch lengths has
been maintained to identify all of these strains. Four more bacterial strains have also
been used (HSTU-6, HSTU-7, HSTU-9 and HSTU-10) in this study which have been
studied previously in the Molecular biology lab of Hajee Mohammad Danesh Science
and Technology University, Dinajpur as part of the bioethanol production research.
HSTU-6 (MG582600.1) and HSTU-7 (MG582601.1) are pectinase producers isolated
from cow rumen content while HSTU-9 (MG582602.1) and HSTU-10 (MG582603.1)
are amylase producing strains which have been isolated from vermicompost.
Phylogenetic trees have also been described for all the six strains (HSTU-2, HSTU-3,
HSTU-6, HSTU-7, HSTU-9 and HSTU-10) with maximum likelihood method and
proper node and branch lengths has been maintained to identify all of these strains.
Various biochemical tests have been done using Bacillus sp. HSTU-2 and Bacillus sp.
HSTU-3 to identify their metabolic habits. Both of them (Bacillus sp. HSTU-2 and
Bacillus sp. HSTU-3) are gram positive bacteria as indicated by gram’s staining test,
citrate test and different selective media growth (Gelatin mannitol salt agar, MacConkey agar, Salmonella shigella agar etc) analysis. Both the strains produced acetyl-methyl
carbinol from pyruvate as indicated by Voges-Proskauer test. These strains are able to
utilize complex sugars such as lactose, maltose and sucrose as depicted by the
differential sugar fermentation tests. Bacillus sp. HSTU-2 has been found to be catalase
positive while Bacillus sp. HSTU-3 has been detected to utilize cytochrome C. oxidase.
Bacillus sp. HSTU-2 and Bacillus sp. HSTU-3 are efficient cellulose degrading bacteria
since both strains performed great in the Congo-red agar media with holo zone diameter
of 8 mm and 6 mm respectively. In order to prove the hypothesis that these strains can
effectively make the perfect bio consortium that can reduce the lignin-hemicellulose
content as well as the crystallinity and degree of polymerization, these six strains
(HSTU-2, HSTU-3, HSTU-6, HSTU-7, HSTU-9 and HSTU-10) have been used to
directly treat four types of fibers (cotton, areca, banana and coir fiber). The strains have
been used in pair (amylase, pectinase and cellulase producing pairs) to make several bio
consortiums by changing the concentration of bacteria and manipulating the temperature
and time of the treatment. In order to determine the structural deformities of cellulose
after treatment with the different bio consortiums, the fibers (cotton, areca, coir and
banana) have been analyzed by FTIR analysis and found significant improvement of
band transmittance at particular wavelengths of cellulose structure indicating band
sharpening. The curves clearly indicate the removal of impurities such as lignin,
hemicellulose, pectin, waxes etc. Also the XRD analysis have been performed for the
treated cotton and areca fiber for determining the crystallinity index (CrI) of cellulose.
Astoundingly, after treating the cotton and areca fiber with Bacillus sp. HSTU-2 and
Bacillus sp. HSTU-3, the CrI dropped from 31.5% to a staggering 13.69%, which proves
that these strains under optimum temperature can overcome the recalcitrance structure of
cellulose. Bacillus sp. HSTU-2 and Bacillus sp. HSTU-3 have also been used to treat
maize straw in order to see their combined effectiveness in reducing sugar yield as this is
the primary concern of the experiment. Both of the strains (Bacillus sp. HSTU-2 and
Bacillus sp. HSTU-3) performed splendidly as with a combined dose of HSTU-2 and
HSTU-3 the reducing sugar yield have been found 27%, 35%, 43%, 60% and 63% in
20h, 30h, 40h, 50h and 72h timeframe respectively. All of these evidence points out to
the fact that the use of these strains in making bio consortium will not only benefit in
bioethanol production of lignocellulose but also will be able to overcome the cellulose
recalcitrant challenge. | en_US |
