S156: Targeted enzyme discovery in thermophilic feedstock-adapted microbial communities for high temperature saccharification of ionic-liquid pre-treated biomass

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. To select for thermophilic biomass-degrading enzymes, microbial communities derived from compost were adapted to switchgrass at 60°C. Phylogenetic profiling of these communities show that they consist of simple mixed consortia with representatives of the Firmicutes, Bacteriodetes, Thermi, Gemmatimonadetes and Chloroflexi phyla. Metagenomic sequencing of these consortia has allowed us to reconstruct the genomes of the important members of these microbial communities, resolve strain populations and assign functions to individual consortial members. Secretomes of these microbial communities were screened for glycoside hydrolase activity and were used to hydrolyze ionic-liquid pre-treated switchgrass at 70ºC and 80ºC. 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 secretomes were identified by a combination of mass-spectrometry-based proteomics, using both a shotgun approach and in-gel digests of SDS-PAGE bands chosen through zymography. Synergistic effects on biomass hydrolysis have also been demonstrated by adding exogenous thermophilic cellulases to the bacterial secretomes.