S127: Metagenomic and Proteomic Analysis of Thermophilic Feedstock-Adapted Microbial Communities

Plant biomass is an enormous potential resource for generating renewable biofuels, however it is a recalcitrant substrate that requires pre-treatment and enzymatic hydrolysis to access the carbohydrates that will be transformed to biofuels. The enzyme cocktails used for saccharification of pre-treated biomass need to be robust and able to withstand inhibitors produced during pre-treatment. Enzymes secreted by filamentous fungi have traditionally been used for saccharification, however bacterial enzymes may be more suitable for saccharification under the harsh conditions encountered in an industrial process. To identify these enzymes, we have focused on adapting thermophilic bacterial microbial communities to specific feedstocks and pre-treatment conditions, followed by microbial community analysis and functional characterization of secreted glycosyl hydrolases. Metagenomic sequencing of these consortia has allowed us to reconstruct the genomes of important members of these microbial communities and assign functions to individual consortial members. Supernatants of these microbial communities were used to hydrolyze ionic-liquid pre-treated switchgrass at 80 ºC. These assays indicated that the complement of glycosyl hydrolases in the secretomes could hydrolyze a broad range of polysaccharide linkages and were more active than commercial fungal enzyme cocktails at elevated temperature. Individual enzymes in the supernatants were identified by mass-spectrometry-based proteomics, using both a shotgun approach and in-gel digests of SDS-PAGE bands chosen through zymography. This approach has been used to compare thermophilic bacterial communities on enriched on different pre-treated feedstocks and to compare thermophilic bacterial cocktails with supernatnats generated from growth of thermophilic fungi on pre-treated feedstocks.