Department of Biotechnology
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Item Open Access Studies on cellulase-producing bacteria isolated from a vermicompost-derived consortium and to evaluate their synergism in depolymerization of agricultural residues(University of North Bengal, 2023) Basak, Arijita; Ghosh, ShilpiThe microorganisms growing in lignocellulose-degrading environment produce multiple enzyme systems that act synergistically to break down the polysaccharide constituents of JjgnocelluLosic material, namely cellulose, and bemicellulose. Vennicompost has taxonomically distinct microorganisms that could be used for the production of biocatalysts for the decomposition of industrial agroresidues. In this study, the microbial communities present in vermicompost were gradually adapted to grow at higher temperature of 60 °C while utilizing chopped rice straw as carbon source. The lignocellulolytic consortium RSV so developed exhibited the ability to degrade cellulose and hemicellulose. This was backed by quantitative estimation whereby RSV produced glycoside hydrolases belonging to cellulases such as exoglucanase, endoglucanase and ~-glucosidase with maximum activity of 5.8 ± 0.05, 19.56 ± 0.07 and 5.8 ± 0.03 ill/mg, respectively; and xylanases such as endoxylanase, ~xylosidase and a -L-arabinofuranosidase with highest activity 54 ± 15. 76, 5.2 ± 0.04, and 4.8 ± 0.03 IU/mg, respectively, on Day 2 of growth at pH 6 and 60 °C. The extracellular enzyme concentrate of RSV showed the ability to saccharify rice straw pretreated by glacial acetic acid, hydrogen peroxide, acid-peroxide combinations and heat The saccharification yield of reducing sugar equivalents was significantly greater from acid-peroxide combinations of 1: l (460 mg/g of rice straw), 2:1 (360 mg/g ofR and 4:1 (290 mg/g of rice straw on Day 5). The total genomic DNA of RSV was isolated, sequenced and the raw reads were trimmed and assembled into a metagenome for taxonomic and CAZyme profiling. The RSV consortium was comprised of a majority of bacteria from the phyla Finnicutes (56.91 %), Proteobacteria (28.32 %) and Bacteroidetes ( 12.22 %) and included members of the genera Pseudoc/osrridium (21.29 %), Chelatococcus ( I 6. l 3 %), Thermoanaerobacrerium (14.8 %), Algoriphagus (10.45 %), Bacillus (3.95 %), Pseudoxanthomonas (3.88 %), Geobacillus (3.57 %), Halomonas (3.29 %), Aeribacillus (3 .26 %) and Syrnbiobacterium (1.77 %) as the most abundant bacteria. Apart from producing GHs, RSV also produced enzymes belonging to CAZymes groups GH, CE and AA and auxiliary active enzymes such as multi copper oxidase, catalase/peroxidase, glycolate oxidase, GMC oxidoreductase and quinone oxidoreductase. Lignocellulolytic bacteria were obtained from RSV by dilution plating technique. Eight of them were capable of cellulose degradation while one was predominantly ligninolytic. Phylogenetic analysis of the isolates based on l 6S rRNA gene sequence identified these bacteria as belonging to genera Geobacillus, Parageobacillus, Aeribacil/us and Micrococcus. Among the bacterial isolates two cellulolytic isolates exhibiting significantly higher cellulase activity, Parageobacil!us thermoglucosidasius NBCB 1 (23.94±1 .34 TU/mg on Avicel, Day3), and Aeribacillus composti XLN l (22. 75± 1.50 ill/mg on A vicel, Day3 ), and the ligninolytic isolate Micrococcus yunnanensis B4 with laccase ()40.81±10.3 l μIU/mg on Day 3), lignin peroxidase (191. I 9±26.66 μTU/mg on Day 3) and manganese peroxidase (3239.73±177.11 μIU/mg oa Day 3) activities, were selected for the development of synthetic triculture consortium. The synthetic triculture consortium was further assessed for cellulase production and activity. Inoculation of the three cultures simultaneously to the celluJase production medium MSM-RS gave 53.24±0.96 TU/mg of cellulase production on Day 3 with the highest degree of synergy among all synthetic consortia (1.26±0.04). The cellulase production of the triculture was further optimized by OFAT (94.00±4.52 ill/mg), RSM-CCRD (115.68±0.01 ill/mg) and ANN-GA (139.00±0.00 IU/mg). Of all of them, ANN was found to give better process precision with optimized culture parameters of 3.33 days of incubation, medium pH of 6.06, 0.89 % sorbitol and 1.09 % peptone. The enzyme preparation obtained from triculture on the optimized MSM-RS medium was then utilized in the saccbarification of alkali, peroxy acetic acid, moist heat pretreated or untreated rice straw which gave reducing sugar equivalent yields of l 70.40±2.13 mglg of pretreated rice straw (Day 5), 156.00± l.25 mg/g (Day 5), 113.54±2.49 mg/g (Day 5) and 82.07±0.00 mg/g (Day 5), respectively, which were ~53.47, ~58.01 , ~65.87 and - 137.10 % of the yields from commercial cellulase Celluclast. The cellulase produced by Parageobacillus thermoglucosidasius NBCB 1 was purified to homogeneity, characterized and the encoding gene was cloned and sequenced. Before purifying, the enzyme production by the bacterium was enhanced to 78.47±2.23 IU/mg through ANN-GA. The cellulase enzyme, designated as PtCell , was purified through ammoniwn sulphate precipitation, gel filtration and DEAE-Sephacel anion exchange chromatography with 4 1.95-fold purification and final y ield 21 .52 %. The specific activity of PtCel 1 was 184 TU/mg on CMC and 305 IU/mg on A vicel thereby qualifying it as a processive endoglucanase. It was functional within a broad range of pH (4.5-8.5) and temperature (4-80 °C) with optimum activity at pH 5.5 and 60 °C. PtCel 1 showed enhanced activity in presence of Zn2+ (~369.14 %), Mg2+ (~245.15 %), Ca2 i (~261.10 %), Na+ (~ 179.77 %), Sn2+ (~ 150.80 %), SDS (- 132.8 %) and P-ME (~ 186.81 %). It was resistant to and retained activity in presence of galactose (~85.53 %), xylose (~74.28 %), cellobiose (~63.37 %), EDTA (- 62.18 %) and H202 (~33.33 %). PtCell had Km and Ymax at 0.363 mg/ml and 308.64 IU/mg, respectively. The putative gene encoding PtCell it was cloned and sequenced. It was characterized to be a Zn•dependeut endoglucanase/metallopeptidase from the M42 family.