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.

One of the main barriers to the enzymatic hydrolysis of cellulose results from its highly crystalline structure. Pretreating biomass with ionic liquids (IL) increases enzyme accessibility and cellulose recovery through precipitation with an anti-solvent. For an industrially feasible pretreatment and hydrolysis process, it is necessary to develop cellulases that are stable and active in the presence of small amounts of ILs co-precipitated with recovered cellulose. However, a significant decrease in cellulase activity in the presence of trace amounts of ILs has been reported in the literature, necessitating extensive processing to remove residual ILs from the regenerated cellulose. Towards that end, we have investigated the stability of hyperthermophilic enzymes in the presence of the IL 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) and compared it to the industrial benchmark Trichoderma viride (T. viride) cellulase. The endoglucanase from a hyperthermophilic bacterium, Thermatoga maritima, and a hyperthermophilic archaeon, Pyrococcus horikoshii, were over expressed in E. coli and purified to homogeneity. Under their optimum conditions, both hyperthermophilic enzymes showed significantly higher [C2mim][OAc] tolerance than T. viride cellulase. Using differential scanning calorimetry we determined the effect of [C2mim][OAc] on protein stability and our data indicates that higher concentrations of IL correlated with lowered protein stability. Both hyperthermophilic enzymes were active on [C2mim][OAc] pretreated Avicel and corn stover. Furthermore, these enzymes can be recovered with little loss in activity after exposure to 15% [C2mim][OAc] for 15 h. These results demonstrate the potential of using IL-tolerant extremophilic cellulases for hydrolysis of IL-pretreated lignocellulosic biomass, for biofuel production.

Generation of biofuels from sugars in lignocellulosic biomass is a promising alternative to liquid fossil fuels, but efficient and inexpensive bioprocessing configurations must be developed to make this technology commercially viable. One of the major barriers to commercialization is the recalcitrance of plant cell wall polysaccharides to enzymatic hydrolysis. Biomass pretreatment with ionic liquids (ILs) enables efficient saccharification of biomass, but residual ILs inhibit both saccharification and microbial fuel production, requiring extensive washing after IL pretreatment. Pretreatment itself can also produce biomass-derived inhibitory compounds that reduce microbial fuel production. Therefore, there are multiple points in the process from biomass to biofuel production that must be interrogated and optimized to maximize fuel production. Here, we report the development of an IL-tolerant cellulase cocktail by combining thermophilic bacterial glycoside hydrolases produced by a mixed consortia with recombinant glycoside hydrolases. This enzymatic cocktail saccharifies IL-pretreated biomass at higher temperatures and in the presence of much higher IL concentrations than commercial fungal cocktails. Sugars obtained from saccharification of IL-pretreated switchgrass using this cocktail can be converted into biodiesel (fatty acid ethyl-esters or FAEEs) by a metabolically engineered strain of E. coli. During these studies, we found that this biodiesel-producing E. coli strain was sensitive to ILs and inhibitors released by saccharification. This cocktail will enable the development of novel biomass to biofuel bioprocessing configurations that may overcome some of the barriers to production of inexpensive cellulosic biofuels.