2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Both scientists and policymakers view as inevitable the transition of industrial economies from those based on petroleum to those based on renewable materials. Biomass is the only sustainable source of fuels and chemicals available to humanity, but biomass production for the purpose of conversion to fuels and industrial chemicals in the U.S. and other industrialized nations is based almost exclusively on grains, particularly corn. This represents a diversion of these grains from the food supply, and the high inputs and ecological consequences of grain-based fermentations at industrial scale have led many to question their long-term sustainability. Forages have many advantages in agricultural production systems, including high biomass yields, ability to be cultivated on marginal lands, low input requirements, and ecological sustainability. However, forage acreage is declining in the U.S., largely because the low digestibility of forage cell walls limits their use in livestock feeding, and because alternative end uses for forage fiber are not available. The objective of this project is to identify and develop novel value-added products from alfalfa and other forages. Two basic process configurations are being explored. The first uses dry fractionation to separate alfalfa herbage into a high-value, leaf fraction and a low-value stem fraction. The second process configuration uses wet fractionation to produce a high-value juice fraction rich in protein and other value-added biochemicals (e.g., phytase and other recombinant enzymes), and a low-value fiber fraction. Our intent is to use the fiber directly as a biofiltering agent (e.g., in heavy metal removal from water) or to upgrade the fiber by microbial fermentation to produce ethanol (a liquid fuel) or lactic acid (a food acidulent). During these fermentations a substantial portion of the fiber is lignified resists fermentation, resulting in a fermentation residue that also contains bacterial cells and a sticky "glycocalyx" required by the bacteria to attach to the fiber. We intend to use the residue as a biological adhesive competitive with soy protein. 2.List the milestones (indicators of progress) from your Project Plan. Objective 1. Harvesting, processing and storage technologies. i) Establish stands of alfalfa, switchgrass and reed canarygrass for harvesting research. ii) Determine effects of cutting height and winter harvest on yield, fermentability, and persistence of alfalfa, switchgrass and reed canarygrass. iii) Develop field-scale fractionation technology. iv) Evaluate low-moisture storage technology. Objective 2: Biomass screening. i) Compare in vitro gas production and SSF methods. ii) Screen gama grass and bluestem samples. iii) Screen switchgrass and bermuda grass samples. iv) Screen alfalfa samples. v) Develop expert system for predicting fermentability. Objective 3a. Selection of switchgrass for vigor, lodging resistance, and disease resistance. i) Complete cycle 2 of selection, produce maternal seed. ii) Complete cycle 3 of selection. iii) Field trials for C0, C1, C2 and C3 evaluation. Objective 3b. Switchgrass hybrids for yield and energy conversion. i) Transplant crossing blocks. ii) Harvest hybrid seed (upland x lowland and lowland x upland). iii) Field trials of hybrids and parents. iv) Lab work on samples from field trials and screening for fermentability. Objective 4a. Consolidated bioprocessing. i) Isolate and characterize microbial strains. ii) Medium minimization. iii) Mass balance determinations. iv) Optimization of yield and product concentration. Objective 4b: Bio-based adhesives. i) Structural characterization of glycocalyces. ii) Large-scale production of fermentation residues; iii) Optimization of adhesive formulations and applications. iv) Technology transfer to manufacturers. 4a.What was the single most significant accomplishment this past year? Gene expression in ethanol-producing bacteria. Four genes involved in cellulose degradation by Clostridium thermocellum, a bacterium that grows at high temperature in the absence of oxygen, were shown to be regulated in a manner that varied in response to the conditions under which the bacterium is grown. The process of cellulose degradation (a key process in producing ethanol from cellulosic biomass) is poorly understood at the level of gene expression. This research -- the most complete study to date in any organism of the relationship between cell growth rate and gene expression -- provides new information on the regulation of cellulose degradation by an organism that shows particular promise for converting cellulosic biomass to both ethanol and a fermentation residue having desirable properties as a wood adhesive. The results provide new strategies for improving cellulose degradation and ethanol production by this bacterium. 4b.List other significant accomplishments, if any. 1. Biomass screening. Fermentability data have been determined for Eastern gamagrass, bluestem, and switchgrass varieties grown at a variety of locations east of the 100th meridian, and NIR prediction equations have been developed for in vitro fermentability of these forages. 2. Improved switchgrass. Using conventional plant breeding methods, new experimental varieties crosses of switchgrass have been selected for improved performance in northern latitudes, for subsequent additional experimental evaluation in the coming year. 4c.List any significant activities that support special target populations. None. 4d.Progress report. The effect of growth conditions on gene expression in the cellulose-fermenting, ethanol-producing bacterium Clostridium. thermocellum ATCC 27405 was studied, using cells grown in continuous culture under cellobiose or cellulose limitation over approximately a tenfold range of growth rates. Fermentation product distribution displayed similar patterns in cellobiose- or cellulose-grown cultures, including substantial shifts in the proportion of ethanol and acetic acid with changes in growth rate. Expression of seventeen genes involved or potentially involved in cellulose degradation, intracellular phosphorylation, catabolite repression, and fermentation end-product formation was quantified by real-time PCR, with normalization to two calibrator genes (recA and 16S rDNA) to determine relative expression. Thirteen genes displayed modest (fivefold or less) differences in expression with growth rate or substrate type: sdbA (cellulosomal scaffoldin-dockerin binding protein), cdp (cellodextrin phosphorylase), cbp (cellobiose phosphorylase), hydA (hydrogenase), ldh (lactate dehydrogenase), ack (acetate kinase), one putative type IV alcohol dehydrogenase, two putative cAMP binding proteins, three putative Hpr-like proteins, and a putative Hpr serine kinase. By contrast, four genes displayed > tenfold reduced levels of expression when grown on cellobiose at dilution rates > 0.05 h-1: cipA (cellulosomal scaffolding protein), celS (exoglucanase), manA (mannanase) and a second type IV alcohol dehydrogenase. The data suggest that at least some cellulosomal components are transcriptionally regulated, but that differences in expression with growth rate or among substrates do not directly account for observed changes in fermentation end-product distribution. These results suggest targets for genetic manipulation of this organism for enhanced cellulose degradation and ethanol production. In addition, this work represents the most complete study to date of the effect of growth rate on gene expression in any bacterial species. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. Strategies for processing alfalfa have been developed that include wet fractionation to produce high-protein juice and a fibrous solids material. The juice fraction can be treated by ultrafiltration or by pH adjustment to recover the protein, and has been shown to have high nutritional value as a protein supplement in foods that may be particularly useful in third-world countries whose populations eat protein-deficient diets. The solids remaining from wet fractionation of alfalfa have been shown to be fermentable to ethanol by Ruminococcus albus, although yields are currently low. A collaboration with the USDA Forest Service, Forest Products Laboratory has demonstrated that the fermentation residues (containing undegraded fiber, bacterial cells, and extracellular polymers produced by the bacteria) have adhesive properties and can partially replace petroleum-derived phenol-formaldehyde resins used in plywood manufacture. Acceptable shear strength and wood failure values were not obtained with rehydrated pure fermentation residues, but were obtained if the adhesive mixture was combined in a proportion of 30% fermentation residue plus 70% phenol-formaldehyde resin (dry weight basis). An improved means of establishing strictly anaerobic conditions for cultivation of our fermentative cultures has been developed that is broadly applicable to other anaerobic microorganisms. A continuous flow, packed-bed reactor system has been constructed for cultivating the anaerobic bacterium Ruminococcus albus in a manner that produces more fermentation residue for bioadhesive testing. A novel strain of fungus has been isolated that can produce ethanol from cellulose under anaerobic, non-growing conditions in a completely mineral medium, and progress was made toward developing a genetic system for this strain. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Technologies for wet fractionation of herbage were transferred under a Cooperative Research and Development Agreement (CRADA) to a major plant biotechnology company. Parents of protein-deficient children were taught to make food supplements from green plants via fractionation. Technology for intensive conditioning of forage crops to accelerate drying and to increase digestibility was transferred under a CRADA to a major farm equipment manufacturer. A U.S. patent application was filed 05/05/2004 covering the use of cellulosic fermentation residues as bioadhesive materials. We have already been contacted by one company regarding the licensing of this technology. Review Publications Weimer, P.J., Dien, B.S., Springer, T.L., Vogel, K.P. 2005. In vitro gas production as a surrogate measurement of the fermentability of cellulosic biomass. Applied Microbiology Biotechnology. 67:52-58. Casler, M.D. 2005. Ecotypic variation among switchgrass population from the northern USA. Crop Science. 45:388-398. Contreras-Govea, F.E., Muck, R.E., Filya, I., Mertens, D.R., Weimer, P.J. 2005. In vitro gas production and bacterial biomass estimation for lucerne silage inoculated with one of three lactic acid bacterial inoculants. In: Park, R.S., Stronge, M.D., editors. Silage production and utilisation, XIVth International Silage Conference, July 3-6, 2005, Belfast, Northern Ireland. p. 207. Martin, N.P., Mertens, D.R., Weimer, P.J. 2004. Alfalfa: hay, haylage, baleage and other novel products. In: Proceedings of the Idaho Alfalfa and Forage Conference, February 24-25, 2004, Twin Falls, Idaho. p. 9-18.