Increasing emissions of greenhouse gases from the transportation sector is recognized as one of the powerful contributors to global warming and climate change. In addition to these environmental problems, dwindling fossil fuel resources make the search for renewable energy resources more urgent than ever. In the short term, ethanol has been trusted as an environmentally sound motor fuel alternative that can be used either in blend with gasoline or alone. Although ethanol production from starch and sugar crops, such as corn, wheat, sugar cane, sugar beet, etc., is a mature biotechnology, the high feedstock costs, accounting for about 50% of the ethanol production costs, and the relatively limited contractibility of starch and sugars, pose major barriers to a large-scale implementation of ethanol as the alternative transportation fuel. Widely available and renewable lignocellulosic waste streams from agriculture and food industry, paper and wood industry, etc., as well as specially grown energy crops are the plentiful and low-cost feed stocks for fuel ethanol large-scale production. Such an approach is strengthened further by the fact that cellulosic ethanol exhibits a net energy content three times higher than corn ethanol, and emits a low, net level of greenhouse gases. The primary impediment to widespread production of ethanol from recalcitrant lignocellulosic materials is the general absence of a low cost technology. In particular, the state-of-the-art, multi-stage biotechnological cycles involving discrete process steps for (i) production of lignocellulose hydrolyzing enzymes, (ii) hydrolysis of cellulose and hemicellulose, and (iii) fermentation of the hydrolysis products (6 and 5 carbon sugars) to ethanol do not provide price-performance at the industrial scale. Therefore, development of a consolidated bioprocessing strategy (CBP) that offers accomplishing cellulose and hemicellulose hydrolysis and fermentation of both cellulose and hemicellulose hydrolysis products in a single-stage process, excluding a dedicated, high-expense procedure for enzyme production is considered of great potential for making fuel ethanol cost competitive with gasoline. However, no single microbial culture with combined cellulose utilization and ethanol formation abilities is known presently. Development of microorganisms for CBP may be pursued using two approaches: (i) a native cellulolytic strategy that involves development of a syntrophic microbial consortium comprising of naturally occurring hypercellulolytic and saccharolytic microorganisms with improved ethanol productivity and high tolerance to fermentation products and inhibitors, or (ii) combining recombinant DNA technology and metabolic engineering with the aim to genetically modify an outstanding cellulase-producer that makes ethanol, acetate, and lactate in a way that mainly ethanol is produced. The main impediment to the latter studies at this time is the absence of a well established gene transfer system for cellulolytic thermophiles. Anaerobic, thermophilic, cellulolytic, and saccharolytic bacteria of the genus Clostridia are recognized as the most suitable microorganisms for achieving CBP and therefore, screening of novel hypercellulolytic and saccharolytic strains with improved ethanol productivity and decreased acids production with high tolerance to fermentation end products is one of the main R&D focus in fuel ethanol biotechnology.
Proposed project is focused on a Consolidated Bioprocessing (CBP) strategy, and aims to substantially contribute to the development of an economically sound and environmentally safe biotechnology process for fuel ethanol production from lignocellulosic materials. The overall goal of the project is the development of a novel bioprocess for cost effective production of ethanol from herbaceous lignocellulosic wastes, such as corn stalks and wheat straw. The main objective of the project is the development of a syntrophic microbial consortium for ethanol production in a single-stage process. Specific approaches proposed for project realization involve (i) collection and characterization of thermophilic, cellulolytic, and saccharolytic anaerobic bacteria in the genus Clostridia endemic in more than 12 soil/climate zones of Georgia; (ii) screening of hypercellulolytic and C5 and C6 sugars fermenting strains with improved ethanol productivity and decreased acids production; (iii) identification of industrially relevant, robust microorganisms with extended tolerance to high substrate, ethanol, and fermentation inhibitors; (iv) development of a stable, syntrophic microbial consortium, which will directly convert herbaceous lignocellulosic wastes, such as corn stalks and wheat straw to fuel ethanol; (v) testing the efficiency of a CO2-based freeze explosion pretreatment method for increasing digestibility of herbaceous biomass; (vi) optimization of ethanol production in batch and continuous fermentations with recycled cells; and (vii) testing a stable, syntrophic microbial consortium for herbaceous biomass direct conversion to ethanol at a 3-30-liter scale. Considering that the Clostridium-based fermentation by-products, hydrogen and volatile fatty acids, are highly valuable commercial products that may be used for energy production on-site, an optimized scheme for their use will be developed. Finally, cost benefit and mass and energy balance analyses for ethanol fermentation using a syntrophic microbial consortium and use of fermentation by-products will be conducted. Cooperation of highly qualified Georgian researchers and engineers with advanced foreign scientists will guarantee successful realization of the project. Considering the rapidly increasing worldwide demand for environmentally sound and low-cost automotive fuel, specific project results, such as unique collections of endemic anaerobic, thermophylic cellulose-degrading and ethanol-producing clostridia; novel strains of hipercellulolytic and ethanol-tolerant bacteria; C5 and C6 sugars fermenting strains with improved ethanol productivity and decreased acid production; strains capable to simultaneously ferment C5 and C6 sugars; syntrophic microbial consortia for direct conversion of herbaceous biomass to ethanol have extremely high potential for commercialization. For this purpose, substantial effort will be made to establish business contacts with research teams and companies involved in cellulosics-based fuel ethanol R&D and production.
