Development of Novel Technology for In-Situ Saccharification and Biomass Conversion

The biological conversion of agricultural, forestry, and municipal waste biomass into value-added chemicals offers great promise for increasing industrial sustainability but technological limitations still exist. Enzymes are an important component of this growing technology, but in many cases the cost of enzymes is prohibitively expensive when compared to more traditional methods. Our proposal links (1) a new, valueadded use for under-utilized biomass resources with (2) a novel method for supplying enzymes for biofuel and biochemical production. This process should result in a lessexpensive method for producing such chemicals and lessen the nation's dependence on foreign energy supplies. The results are transferable to other processing industries that use enzymes, such as biochemicals, animal feeds, paper processing, beverage alcohols, and detergent additives. Many industrial processes do not need purified enzymes, but do require concentrated, organism-free enzyme preparations. sse typically produces concentrated enzymes, but there are no economically practical methods for deactivating the organism without denaturing the enzyme. Our choice of organisms provides a solution: many enzymes produced by thermophilic, anaerobic organisms typically function in mesophilic and/or aerobic environments, but the organisms do not. Therefore, unprocessed enzyme activities can be added to a bioconversion process without fear of unwanted microbial activity. These advantages indicate that sse of anaerobic thermophiles has great potential for supplying enzymes for biomass conversion, potentially at a significant cost savings. The overall goal of this project is to develop the technology for using anaerobic, thermophilic sse cultures directly in the enzymatic saccharification of insoluble substrates under aerobic and/or mesophilic conditions for ethanol production. This goal is based on the preliminary data which show that sse of anaerobic, thermophilic bacteria produce low-cost thermostable enzyme complexes (cellulases, xylanases, and amylases) from agricultural, forestry, and municipal waste residues. We will investigate two model configurations in the following specific aims: A) Develop a two step process where the cellulose undergoes enzymatic conversion to glucose using these novel enzymes followed by a subsequent yeast fermentation step and B) Develop simultaneous saccharification of the cellulose by these novel enzymes and fermentation by yeast. Experiment A will allow us to quantify bacterial metabolism in the enzyme saccharification step and then subsequently determine whether bacterial metabolism occurs in the yeast fermentation due to a more nutrient-rich fermentation environment. Experiment B will allow us to observe enzyme catalysis in an environment where glucose concentrations are less likely to inhibit enzyme performance. Preliminary Data: Clostridium thermocellum 27405 were incubated with waste paper pulp and avicel, respectively, in both liquid and solid-state cultivation. Samples were periodically taken over a 10 day (liquid) and 25 day period (SSe) and fermentation endproducts measurements were used as a measure of microbial activity. Both organisms were able to form end products from the insoluble substrates. In addition, the accumulation of reducing sugars in the solid-state cultures and disappearance of 50% of the cellulose indicated that microbial enzymes were depolymerizing the insoluble substrates. The results clearly show that the bacteria are able to survive and metabolize at 70% moisture content. It is also clear that hydrolytic enzyme activities must be present because microbial metabolism continued for the entire 25 days in sse.