This SBIR Phase I project will genetically engineer a thermophilic bacterial strain capable of expressing and secreting all the enzymes necessary for biomass hydrolysis and that can grow on minimal media in aerobic and anaerobic conditions. The National Energy Plan Renewable Fuel Standard mandates the production of 21 billion gallons of advanced biofuels from non-food based crops by 2022. In order to achieve this goal, an economically feasible process to hydrolyze biomass must be developed. Production of cellulosic ethanol currently involves pretreating biomass with steam, acid or base to make it more accessible to enzymatic digestion. Enzymes, namely those possessing cellulase, xylanase, â-xylosidase and â-glucosidase activities, will be added after pretreatment to hydrolyze the biomass to its constituent five and six carbon sugars that can be fermented to produce fuel.
The broader/commercial impact of the project will be low cost bio-fuel made from local biomass. This could reduce and stabilize energy prices, strengthen the national energy security, reduce the fiscal deficit, create American jobs and promote economic development.
The National Energy Plan Renewable Fuel Standard mandates the production of 21 billion gallons of advanced biofuels from non-food based crops by 2022. In order to achieve this goal, an economically feasible process to hydrolyze biomass must be developed. Production of cellulosic ethanol currently involves pretreating biomass with steam, acid or base to make it more accessible to enzymatic digestion. Current cellulosic ethanol processes are hindered by the high cost and low specific activities of the required enzymes. The goal of this project is to produce a cocktail of biomass-degrading enzymes directly from a thermophilic Geobacillus strain. The following critical milestones were set for Phase I of this Research Project. 1. Production of a concentrated Geobacillus culture broth containing b-glucosidase, b-xylosidase, xylanase, α-L-arabinofuranosidase, feruloyl esterase and xylan esterase activities. 2. Conversion of 80% of the xylan in hemicellulose in 0.6% pretreated corn stover to free xylose in 80 hours using a mixture of Clostridium thermocellum cellulases and Geobacillus concentrated culture broth. 3. Conversion of 80% of the cellulose in 0.6% pretreated corn stover to glucose in 80 hours using a mixture of Cl. thermocellum cellulases and Geobacillus concentrated culture broth. Milestone #1 involving the production of a Geobacillus culture broth containing b-glucosidase, b-xylosidase, xylanase, α-L-arabinofuranosidase, feruloyl esterase and xylan esterase activities has been achieved. Milestone #2 seeking the conversion of 80% of the xylan in hemicellulose in pretreated corn stover to free xylose not only has been achieved, but it has been surpassed. The Geobacillus cocktail, in the presence of four additional carbohydrases, catalyzed the conversion to xylose of 100 % of the xylan in pretreated corn stover in 24 hours. Milestone #3 seeking the conversion of 80% of the cellulose in pretreated corn stover has been completed. The original plan involved using a mixture of Cl. thermocellum cellulases to convert 80 % of the cellulose in pretreated corn stover to glucose within 80 hours. Experiments revealed that high concentrations of fifteen Cl. thermocellum cellulases and accessory enzymes were unable to convert 20 % of the cellulose in Avicel to glucose in 72 hours. Based on these data, research was redirected to identifying and producing more effective cellulases for conversion assays. In response to these data, two proprietary enzymes cocktails were developed that were capable of achieving significantly higher cellulose conversion rates. C5-6 Cel#1 cocktail was shown to convert 82% of the cellulose in filter paper to glucose in 258 hours, with 68% conversion being achieved after 90 hours. In addition, the distinct C5-6 Cel#2 proprietary enzyme cocktail has been shown to convert 70% of the cellulose in pretreated corn stover to glucose within 70 hours. Proposed Phase II engineering of Geobacillus was accelerated in order to obtain the cellulases necessary for phase I work. The gene encoding an important cellulase was fused to a targeting signal and Geobacillus promoter. This construct was cloned into the Geobacillus-E. coli expression vector pC56-1 and transformed into Geobacillus. Geobacillus not only produced this enzyme, but successfully secreted it into the media. Previous attempts to produce this enzyme in E. coli, Bacillus subtilis and Lactobacillus were unsuccessful. These data not only provide a means to produce the cellulase required to complete milestone#3, but also validates the Phase II plan to engineer Geobacillus to secrete a potent biomass degrading cocktail.
