Thermophilic lignocellulose deconstructing microbial consortium: Mining of Cellulolytic glycoside hydrolases for saccharification of agro residues

Abstract

Plant cell wall lignocellulose is recognized as one of the most abundant source of fennentable sugars with potential use in the production of different value added products like biofuel and other chemicals. Deconstruction of the complex lignocellulose into useable monomers by microbial enzymes has been long adapted; however a major bottleneck in efficient bioconversion of cellwall polysaccharide by enzymes is that plant cell walls have evolved to resist enzymatic deconstruction - a factor collectively known as "biomass recalcitrance". Using a broader suite of enzymes for hydrolysis of cellulose and hemicelluloses to fennentable sugars would be a better approach to combat biomass recalcitrance. Ln nature also, lignocellulosic biomass is degraded with the cooperation of many microorganisms producing a variety of cellulolytic and hemicellulolytic enzymes under aerobic and anaerobic conditions. The biodegradation of cellulosic biomass through the use of microbial co-cultures or complex communities has been proposed as a highly efficient approach for biotechnological application, since it avoids the problems of feedback regulation and metabolite repression posed by isolated single strains. In this study microbial communities of lignocellulosic habitat of forest soil (FS) and goat rumen contents (GR) were adapted to grow and survive at higher temperatures in destarched wheat bran /rice straw as sole carbon source to generate four thennophilic microbial consortia amongst which the consortium developed from goat rumen contents bred on rice straw (GR~ RS) was selected for further analysis due to its better hydrolytic potential on the basis of enzyme assays and in-gel analysis. Characterisation of cellulolytic and xylanolytic potential of GR-RS revealed both the cellulolytic and xylanolytic potential to be thennophilic and thermostable over a broad range of pH. The enzymes were tolerant to and activated in the presence of many metal ions such as Magnesium, Calcium, Barium, Sodium etc. whereas it was greatly reduced to 10% by Hg2+. Comparative Taxonomic profiling of GR-RS-T (developed at 60°C) and GR-RS-M (developed at 37°C) by meta.genomic analysis revealed GR-RS-M had a majority of Proteobacteria (48.67%), Finnicutes (30.34%), Verrucomicrobia (5.45%), Bacteroidetes (5. 12%), Planctomyecetes (3.13%), wheras GR-RS-T exhibited majority of bacteria belonging to phyla Proteobacteria (34.65%), Bacteroidetes (31 %) and Finnicutes (30.83%). Analysis of differential transcriptional expression of cellulolytic and xylanolytic enzymes in GR-RR-T revealed that expression of cellulases and xylanases were mostly from Paenibacillus , Thermobacillus, Pseudoclostridium, Geobacillus, Clostridium etc in case of GR-RS-T whereas its expression were mostly from Klebsiella, Cellulomonas and Pseudoxanthomonas in case of GR-RS-M. The analysis of transcriptome for distribution of different classes of CAZymes revealed that GR-RS-T had GH (34%), GT (38%), CBM (17%), AA (I%), CE (9%) and PL (1 %) whereas GR-RS-M had GH (29%), GT (43%), CBM (16%), AA (3%), CE (8%) and PL (I%). It was also revealed that most families of GH, PL and AA had higher abundance in GRRS- M as compared to GR-RS-T based on the number of reads detected. The study highlights the significance of sub-culturing the lignocellulolytic population from goat rumen contents at 60°C which selects and maintains the therrnophiJic members for production of thermophilic GHs as also evidenced by in vitro analysis. Comparison of saccharification potential of Conso,tiurn Enzyme Preparation (CEP) from GR-RS-T with commercial cellulase blend (CCB) on biologically pretreated rice straw (Spent rice straw) revealed a much higher release by CCB as compared to CEP indicating the supreme potential of the former which has been designed and developed for commercial applications. SEM image of SRS control showed prominent holes and cracks on its surface as compared to the intact strucrure of URS control indicating the delignification of rice straw and loosening of its structure during the growth of P!eurotus sp on rice straw. The structure of URS and SRS after saccharification showed prominently higher level of disorganisation when CCB was used as compared to CEP which cotTelated with the release of reducing sugars from the substrates. The release of reducing sugars in case of SRS-CEP ( 175 mg/gm) was comparable to, in fact slightly higher than URS-CCB (158 mg/gm) indicating that the saccharification potential of CEP could be made comparable to CCB if biologically pretreated rice straw was used. The outcome of this research contributes to the growing interest in lignocellulose deconstruction by enzymes, and highlights the potential of biological pretreatment in enhancing sacbharification by GHs.