Abstract
Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, ∼10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50°C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.
Highlights
Enzymatic hydrolysis is one of the most expensive steps in biofuel production from lignocellulosic biomass primarily due to the need for high enzyme loading caused by low catalytic efficiencies [1,2]
Carbon dioxide evolution (CER) and oxygen uptake rate (OUR) were calculated from continuous measurements of carbon dioxide and oxygen. Both Carbon dioxide evolution rates (CER) and OUR peaked after one day of composting corresponding to initial consumption of sugars [7] and again after eight days during the thermophilic phase (Figure 1B & 1C)
Respiration rates decreased by the end of the incubation, but microorganisms were still active indicating that substrate degradation continued
Summary
Enzymatic hydrolysis is one of the most expensive steps in biofuel production from lignocellulosic biomass primarily due to the need for high enzyme loading caused by low catalytic efficiencies [1,2]. Since a variety of biomass sources are envisioned for future biofuel production (e.g. switchgrass, miscanthus, poplar), a broad spectrum of lignocellulolytic enzymes (cellulases, hemicellulases, ligninases) is required to meet future demands. These enzymes are highly modular and usually classified by their domain structure [3]. Despite extensive efforts to engineer existing glycoside hydrolases to improve activity and stability, there is still a great need to expand the current enzyme repertoire as well as improve our understanding of how these enzymes function in complex environments [5]
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