Glycoside Hydrolase Activities of Thermophilic Bacterial Consortia Adapted to Switchgrass

Abstract

Industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. Thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. Compost-derived microbial consortia were adapted to switchgrass at 60°C to develop thermophilic biomass-degrading consortia for detailed studies. Microbial community analysis using small-subunit rRNA gene amplicon pyrosequencing and short-read metagenomic sequencing demonstrated that thermophilic adaptation to switchgrass resulted in low-diversity bacterial consortia with a high abundance of bacteria related to thermophilic paenibacilli, Rhodothermus marinus, and Thermus thermophilus. At lower abundance, thermophilic Chloroflexi and an uncultivated lineage of the Gemmatimonadetes phylum were observed. Supernatants isolated from these consortia had high levels of xylanase and endoglucanase activities. Compared to commercial enzyme preparations, the endoglucanase enzymes had a higher thermotolerance and were more stable in the presence of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid used for biomass pretreatment. The supernatants were used to saccharify [C2mim][OAc]-pretreated switchgrass at elevated temperatures (up to 80°C), demonstrating that these consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to more extreme reaction conditions.

Metagenomics of microbial and viral life in terrestrial geothermal environments

“…Gladden and collaborators adapted samples from composts to grow on switchgrass by multiple passages at 60°C. The enrichments produced a reduction in microbiome diversity showing by 16S rRNA the presence of thermophilic Gram-positive bacteria Firmicutes, Bacteriodetes, Chloroflexi, and, remarkably, an uncultivated lineage related to the Gemmatimonadetes phylum (Gladden et al 2011). In addition, supernatants of the enriched cultures showed endoglucanase and xylanase activities more stable than those commercially available and exploited in biorefineries.…”

Microbial Community Structures Differentiated in a Single- Chamber Air-Cathode Microbial Fuel Cell Fueled with Rice Straw Hydrolysate

“…One remaining issue with this technology that needs to be addressed to maximize efficiency and reduce capital costs is the incompatibility of ILs with cellulase cocktails derived from filamentous fungi. These enzyme cocktails can be strongly inhibited by certain ILs, such as 1-ethyl-3 methylimidazolium acetate [C2mim][OAc], necessitating expensive and inefficient washing steps to remove residual IL from the biomass prior to addition of enzymes [8,[12][13][14]. One solution to this issue is to develop enzyme cocktails that are tolerant to ILs.…”

“…One solution to this issue is to develop enzyme cocktails that are tolerant to ILs. Fortunately, it has been shown that certain thermophilic bacterial cellulase enzymes can tolerate high levels of the IL [C2mim] [OA]], and in fact these enzymes have been used to develop an IL-tolerant cellulase cocktail called JTherm [13][14][15][16][17]. It has been further demonstrated that JTherm can be used in a one-pot IL pretreatment and saccharification bioprocessing scheme that eliminates the need to wash the pretreated biomass with water, significantly reducing the number of process steps [18].…”

“…This concept drove recent work where complex compostderived microbial communities were cultivated on switchgrass under thermophilic conditions to enrich for organisms that produce mixtures of IL-tolerant cellulases and xylanases [14]. The community was composed of several abundant bacterial populations related to Thermus thermophilus, Rhodothermus marinus, Paenibacillus, Thermobacillus and an uncultivated lineage in the Gemmatimonadetes phylum [19].…”

“…Therefore, these communities provide a rich reservoir of potential enzyme targets to develop thermophilic and IL-tolerant cellulase cocktails to be used in lignocellulosic biofuel production platforms. To discover the genes that encode these IL-and thermo-tolerant enzymes, metagenomic and proteomic analysis was conducted on the community [14,19]. The analysis identified a variety of genes encoding potential cellulose and hemicellulosedegrading enzymes, a subset of which were assembled into complete open reading frames (ORFs) from the metagenome.…”