Brief Summary of Minutes of Annual Meeting

Correction to Nov, 2003 meeting minutes: · "K. Muthukumarappan from SDSU volunteered for secretary position and was elected" was incorrect. It should read as, K. Muthukumarappan from SDSU volunteered to create and lead the new listserver for S1007. · It also should include, Sundaram Gunasekaran of University of Wisconsin-Madison was elected secretary with suggestion that the secretary will then serve as vice president and then president in the following two years. Wednesday, September 29 · The second annual meeting of S-1007 was held at the NREL Headquarters in Golden, CO with a tour of the research laboratories on Wed, Sept 29, 2004. Thursday, September 30 7:30 am · Registration. 8:05 am · Milford Hanna opened meeting with introductory remarks. He reminded the Station representatives about the annual report. Please send an electronic version of a brief summary to Milford as soon as possible. 8:10 am · Roland Mote, S1007 Administrative Advisor, made brief remarks. He stressed the importance of the annual report and encouraged including any positive impacts of research in progress if it were to be successful. The current term of the group, which started in 2001, ends in September 2007. 8:20 am · Hongda Chen, USDA CSEERS Advisor, spoke. He indicated that a proposal for renewal should be submitted by December 2007 and encouraged forming a steering committee to oversee this and a writing committee to start working on the rewrite of the proposal by next year. 8:25 am · Carmel Bailey, National Program Leader, Agricultural Materials Program, spoke about the possibility of using the groups expertise to review proposal for the Biomass Grants Program. · The BBCC group is internal to USDA in which 14 agencies are involved which allows coordination with other federal, public, and private agencies. At the stakeholders workshop (held in 1999 and 2004) recommendations for future directions were obtained. More information is available at: www.usda.gov/bbcc 9:00 am · Hongda Chen introduced Susan Sun (Kansas State U.) who served as the Panel manager for 71.2 Bio-based Products Program of the National Research Initiative Competitive Grant Program of the USDA/CSREES. · Susan Sun described the NRI proposal review process from last year. Last year was the first time the bio-based products program was administered separately. The total funding allocated was $3 million. Out of 144 proposals only 11 received funding. This year the total funding is $6 million with the funding size ranging from $150k to $500k. · Hongda Chen indicated that the NRI budget for 2004 was $164 million. The request for 2005 is $180-183 million. The deadlines for the value-added products research program is December 3; and for bio-based products research program it is January 14. · Hatch proposals submitted for S-1007 group should have project objectives identical top the groups objectives. Specific sub-objectives can be listed under. · 9:15 am · Milford Hanna asked the group to think about the committee to organize the rewrite. He reviewed agenda for the station reports in the morning and in the afternoon. The reports will be scheduled alphabetically starting with Arkansas. Reports from station representatives leaving early will be accommodated. Any information submitted can be posted in the groups website at: www.egr.msu.edu.bio 9:20 am to 12:00 noon · The following station reports were made by the respective representatives. Arkansas; California; South Carolina; Florida; Illinois; Indiana; Kansan; Kentucky; Louisiana; Washington; Michigan; Maryland; Minnesota; North Dakota; Oklahoma; and Oregon. 1:00 pm · Carmela Bailey discussed the proposal for the S1007 group members to be part of the review team for USDA/DOE Biomass Initiative research projects. There are 15 funded projects which are ready fro site visits and review. Milford Hanna (and University of Nebraska) will coordinate the site review by interested members of the S-1007 group. · A list of these funded projects was distributed and the members were asked to indicate their preference to serve as site visit review team. The team will consists of 2 to 3 team members plus 1 USDA person. 1:45 pm  3:30 pm · Five Breakout sessions were held with the following serving as the team leaders: Sue Nokes  Feed Stock; Julie Carrier Biofuels; Dwayne Johnson  Biomaterials; Bernie Tao  Biorefinery; and Mark Worden Education and Training. · Following the presentation of the breakout session summaries a second round of small group discussions were held. The following team leaders were assigned the task of identifying a potential title for a project proposal: Ray Huhnke  Biomass handling; Julie Maupin Furlow  Microbial conversion; Dwayne Johnson  Biomaterials; and Bernie Tao  Thermochemical Conversion. Friday, October 1 8 am · Dr. Chen introduced Mike Harrington  Western Association of Experimental Station Director. · Five projects were introduced: Project 1: Research cooperation  faculty on DOE advisory board are invited for review panels, Project 2: Extension and Outreach  deployment, 2A greater energy efficiency in homes, 2B alternative energy services; Project 3: Youth education in science/technology; Project 4: Engage research capacity; Project 5: Gap analysis  enhance cooperation between federal depts. 9 am · Dr. Hanna suggested ideas for future meetings. Idea session on proposal development needed. Dr. Womack suggests 2-3 standard slides for future station presentations including objectives met and key impacts of work. Other suggestions included bringing a copy of the written reports and slide handouts to give to group beforehand to be more effective in creating collaboration, etc. · Terry Walker will be the group chair in 2005 with Sundaram Gunasekaran serving as vice chair/treasurer. Sundaram will then become chair in 2006. Kent Rauch was elected secretary for 2005, vice chair/treasurer in 2006 and chair in 2007. · Kent agreed to writing the terminal report and presiding over initiating continuation of S1007 for the next 5-year term if the group chooses to do so at the 2007 meeting. · The future meeting places were then determined: Knoxville/Oak Ridge with tours of the University of Tennessee and Oak Ridge National Lab with probable dates of Sept. 19-20, 2005; Minneapolis with tours of University of Minnesota and surrounding biomass industries was chosen for 2006; and Illinois/Peoria was chosen for 2007 meeting. 9:30 am · State section reports continued with Oregon  Dr. Penner: biosaccharification, cellulases, exoenzymes with tubular active site; Oklahoma State Univeristy  Dr. Huhnke: Grassohol project update; South Dakota  Dr. Gibbons: SFE, PHA project; Tennessee - Dr. Womak: pretreatment and harvesting processes  physical properties and stress analysis; Washington  Dr. Chen: algal bioprocessing and two-stage acid hydrolysis pretreatment; Utah  Dr. Taylor: cellulases and associated bioprocesses. 11:30 am · Dr. Hanna made closing statements and Dr. Chen expresses thanks to service of Dr. Hanna for this meeting and as the Chair of the group during the past year. · Meeting adjourned at noon.

Objective 1. Reduce the cost of harvesting, handling, storing and transporting biomass increasing the competitiveness of biomass as a feedstock for biofuels, biomaterials and biochemicals. Task 1. Feedstock quantification and characterization. (Mississippi) Miscanthus floridulus (giant miscanthus) and M. sinesis (Chinese maidengrass), Pennisetum lolium (Japanese foxtail) and P. purpurea (elephantgrass), Sorghum halpense (Johnsongrass) and S. vulgare (sorghum-sudangrass), Tripsacum dactyloides (Eastern gamagrass) Panicum virgatum (switchgrass), Andropogon gerardii (big bluestem), and Sorghastrum nutans (Indiangrass) were planted and yields indicated that 90-day harvest cycle probably would result in highest yields and sustainability. Greater harvest frequency caused decline in all species survivability, especially under rain-fed conditions. Ash was high (8%), and caloric potentials were similar at between 3500 and 3900 cal/g of material. The species that showed the greatest yield potential were giant miscanthus, elephantgrass, sorghum-sudangrass; and switchgrass. Switchgrass planted on a 3 ac plot yielded 7.2 tons/ac. The switchgrass can be pelletized but the expense is greater than cubing which will be investigated further. These species were subjected to sequential weathering to determine compositional changes in the standing but frost-killed crop. Yield loses varied by species with switchgrass and giant miscanthus retaining most of their yield (20% loss) after 30 days. Potassium loss was greatest during the first 30 days of weathering. Maximum yields were achieved with 150 lbs/A of nitrogen. Maximum yields are reached by mid-August, but a harvest at this date would compromise plant survival. Kansas State is researching grain sorghum as an economically viable crop for biobased products and bioenergy. Task 2. Harvest, process and handling. (UC Davis) Up to 50% dry matter loss occurred in unprotected rice straw bales in open storage, implying 8 to 12% overall loss for typical open stacks in California and higher costs compared to storage under permanent cover. Modeling and experimental efforts are underway to investigate chemical and biochemical mechanisms of spontaneous combustion in stored rice straw and other biomass materials. Work at the University of Kentucky showed that by collecting the fractions of the corn stover with the highest glucose-release potential (the cobs and 74% of the leaves and husks) and leaving the remaining fraction (26% of the leaves and husks, and the stalks) in the field for erosion control, the glucose potential of the collected biomass could increase by 21%. (Univ. of Tennessee) Switchgrass stem cross-sections failed with ultimate shearing stresses that were one-fifth the magnitude of ultimate tensile stresses, thereby indicating that shear-dominant size reduction equipment would be much more efficient than tensile-dominant size reduction processes. Mean shear strength was relatively unaffected by moisture content and elapsed time after harvest. Mean tensile strength increased as mean moisture content decreased from about 60 to 10 % wet basis, and tensile strength increased two-fold with a corresponding increase in elapsed time after harvest ranging from 2 to 386 h. This indicated that tensile-dominant size reduction should be conducted early in the harvest process and at a high moisture content to minimize energy consumption for grinding. A gradual decline in stover moisture, from an initial moisture content of about 50 to 70 % (w.b.) for the 25 % (w.b.) grain harvest, exhibited diurnal variation of about 8 % (w.b.). An occasional precipitation event typically resulted in stover moisture reaching original levels from as low as 15 % (w.b.). Combined stalks often had moisture 20 % (w.b.) less than mown stalks. Among the stover components, vertical stalks had the greatest wet mass followed by leaf and husk. Mass and moisture variation of above-ground plant components exhibited two moisture-reduction trends. The first trend was a period of rapid reduction. The second trend was a gradual reduction and stabilization of moisture. Stalk section below the typical ear generally had increased wet mass and dry matter. Wet mass and moisture content of stalks were not significantly influenced by the observed soil and environmental parameters. On an average, the bottom 1 to 4 stalk sections had 66±3% of total wet mass, and 61±3% of total dry matter. Task 3. (No report.) Objective 2. Expand the scientific knowledge leading to significant economic improvements in biofuel production processes. Task 1. Pretreatment for bioconversion processes. The University of Kentucky characterized the specific cellulase activity and thermostability derived from solid-state cultivation of thermophilic anaerobic bacteria. Direct measurement of cellulase from SSC cultures was not accomplished due to the strong binding of the cellulose binding domain. Unlike liquid cultivations, the substrate was still present for cellulase adsorption, rendering cellulase separation incomplete. Therefore, an indirect measurement of cellulase activity was conducted by measuring the saccharifying ability of the SSC cultures on new substrate. The maximum measured cellulase activities were 0.003 and 0.01 IU/ml at 37 and 60°C for Avicel; and 0.013 and 0.046 IU/ml at 37 and 60°C for paper sludge as compared to values of 0.012 to 0.1 reported in the literature for liquid production. Economic analyses of cellulase enzyme production costs indicated unit costs of $15.67/kg enzyme and $40.36/kg enzyme, for the SSC and SmF methods, respectively, compared to a market price of $36.00/kg. (Louisiana State Univ.) The effectiveness of AFEX pretreatment and the LSU process were tested. This LSU process is currently proprietary. The effectiveness of the treatment was monitored using the ability of the commercial enzyme cocktail to produce glucose from the treated materials. For enzyme test, sample loading was approximately 1% (w/v) in phosphate buffer solution. Crude cellulases from T. viride (Sigma Co., Cat. No. C-2274) were used. Currently, the LSU process appears to be a rapid, low temperature, economic method for pretreatment of biomass cellulosics. Within 40 min lignins are separated from cellulose giving a stream containing recoverable chemicals and a pre-swollen cellulosic pulp that easily digested with cellulases. With simultaneous fermentation, theoretical yields of ethanol were produced. A second approach was to modify the enzyme process to produce initially, only cellobiose, rather than glucose. Currently we are selecting cellulolytic organisms that produce low levels of ±-glucosidase. A cellobiose source will give the opportunity of sugar modification to other products. By middle of 2005 we plan to have an operational pilot system running to study technology and economics for bagasse to ethanol technology. Concurrently we will be working on alternative products from cellulosic biomass. Task 2. Biological conversion processes. University of Illinois researchers obtained corn, ground corn, beer, wet grains, syrup and DDGS samples from 9 dry grind plants. Concentrations of most elements were about 3 times those of corn, due to disappearance of starch during fermentation. Concentrations of Na, K and P were much higher in syrup than in wet grains. Corn protein content was 8.9 g/100 g. There were no effects of plant or week on corn protein content. Protein contents were 33.1 and 19.1 g/100 g for wet grains and syrup, respectively. Phosphorus levels were 3 times higher in DDGS than in corn. High phosphorus levels may become a limiting factor on demand and use of DDGS. Based on chemical characterization of 23 elements in various streams in several dry grind plants, we found no elements exceeded safe or recommended levels. Phosphorus was the only one of concern. Samples were taken at 23 locations in three plants and analyzed for total phosphorus content. Flow of phosphorus was simulated using a computer model for a 2,700 tonne/day plant. Steepwater, gluten and process water contained most of the phosphorus, with CGF steepwater having the most (5,380 g/kg). Ground corn was mixed with water and fermented for 72 h at 32°C to yield 37.4 ± 0.8 g ethanol/100 g corn by a gravimetric technique and 35.1 ± 3.1 g ethanol/100 g corn by an HPLC technique. To determine effects of corn flour particle size on sugar profile and ethanol yield, a 2L laboratory dry grind procedure was developed. Mash was subjected to addition of ±-amylase (0.24 to 2.4 mg/g corn) and glucoamylase (0.575 to 3.45 mg/g corn); liquefaction (80 to 110°C) and saccharification (50 to 65°C) temperatures; solids content of mash (20 to 35%) and yeast inoculum (5 to 60 ml). The optimized procedure was reproducible with low standard deviation. Hybrids were classified according to kernel density and evaluated for sugar profiles and ethanol yields, after saccharification and fermentation, respectively. True density varied from 1,118 to1,218 kg/m3. As hardness varied, ethanol yield varied by 1.89% (v/v) among hybrids. Sugars produced were found to be higher for hard endosperm corn. Presence of Aflatoxin B1 altered ethanol yields using the small scale lab dry grind procedure. (Mississippi) Clostridium ljungdahlii, C. acetobutilicum, and C. thermoaceticium cultures were evaluated for their ability to convert CO2, H2, and CO into acetic acid and ethanol. One culture had the ability to grow at a high rate with CO as it sole carbon source and produce ethanol. It is being investigated to determine the optimal nutrient composition for growth and ethanol production. Numerous syngas fermenters have and are being screened using a novel isolation approach. Using Winogradsky columns, organisms capable of converting CO2, H2, and CO into acetic acid or ethanol were allowed to establish themselves in a biofilm, and then samples were taken and used to inoculate microcosms for further enrichment of the organisms. The rate of CO2 and H2 consumption resulted in a vacuum being created within the vessels and resulted in a loss of anaerobic conditions and subsequent loss of CO2 and H2 consumption. This was overcome through recent improvements in anaerobic cultivation techniques that have resulted in the prevention of air and more importantly O2 entering the system and providing the microorganisms with 26 times more CO2 and H2. To date, 15 Winogradsky columns have been prepared using a variety of sources of microorganisms ranging from deeply anaerobic sediments to animal manures. Additionally, a novel dynamically charged column assembly has been designed and will be tested. This new system should provide the ecological conditions to culture improved isolates with better chemical production capabilities. The hyperthermophiles continue to show great promise but require more research to optimize growth and ethanol production. Isolation of syngas fermenters has been improved significantly with improvements to culturing techniques and gas management techniques. Researchers at the University of Florida have developed an integrated approach for the metabolic engineering of new biocatalysts for the conversion of renewable biomass to fuels and other value added chemicals. Bacterial strains have been engineered for the homogeneous production of acetic acid, L-lactic acid, D-lactic acid, and pyruvic acid in mineral salts medium at 80 to 90 % of theoretical yield (50 g per liter). To enhance microbial tolerance to the high-level formation of these products, levels of cellular osmoprotectants have been enhanced via genetic engineering and medium supplementation. Modifications and improvements continue to be made on ethanologenic strains of Escherichia coli and Klebsiela oxytoca to enhance the conversion of xylan and cellulose derived oligosaccharides to ethanol in the absence of enzyme supplementation. In addition, a new strain of Bacillus sp. (strain 17C5) has been isolated that grows readily at pH 5.0, 50°C. Under these conditions, the strain ferments sugar cane bagasse hemicellulose, hydrolyzed by dilute H2SO4 and supplemented with mineral salts and 0.5% corn steep liquor, to L(+)-lactic acid at 89% theoretical yield with an optical purity of 99.5%. A genetic exchange system has been developed for this strain with the goal of inserting metabolic genes to channel the intracellular pools of pyruvate to ethanol and other useful fermentation products. With this same goal, a synthetic Gram-positive ethanol operon has been constructed that enables high-level production and activity of Sarcina ventriculi pyruvate decarboxylase and Geobacillus stearothermophilus alcohol dehydrogenase enzymes in diverse Gram-positive bacteria including species of Bacillus and Corynebacterium. Production of recombinant pyruvate decarboxylase activity also has been engineered into extreme haloarchaea which grow at 2  5 M salt. Through the cloning and characterization of several new bacterial pyruvate decarboxylase genes, a novel subfamily of Gram-positive bacterial genes are anticipated to facilitate decarboxylation and carboligation reactions required for production of value added chemicals including flavor additives and (R)- phenyacetylcarbinol, a central intermediate of pharmaceutical ephedrine and pseudoephedrine production. In addition, a large cluster of Gram-positive genes have been cloned and sequenced that are involved in the metabolism of xylans and xyloglucuronic acid. Task 3. Development of improved thermochemical processes for biofuel production. (Mississippi) A Community Power Corp. down-draft gasifier was used to convert soft and hardwood chips to syngas. The gasifier ran consistently and control of the fire-front was at the appropriate level. The output temperature is a key control parameter for the gasification system and this temperature was consistent throughout several test trials. Syngas quality was consistent after the unit has reached the warmed-up condition. Changes in flow rate had minimal effect on the output. CO2 levels were 21 to 25 mol percent, methane levels were from 3 to 4 mol percent, and H2 levels 16 to 18 mol percent. O2 levels can run as high as 18 mol percent, but commonly run in the 13 to 14 percent range. (UC Davis) Mineral phase relationships were explored for wood and straw ash separately and in fuel blends over temperatures from 500 to 1300°C. Addition of rice straw to a predominantly wood based fuel caused a marked freezing point depression in the liquidus temperature from above 2000°C to below 1260°C. However, fuel blends containing more than 30% ash from rice straw exhibited enhanced retention of potassium in solid slag, reducing the volatile fraction potentially contributing to heat exchanger fouling. Agglomeration of bed media in fluidized beds was restricted for fuel blends containing less than 8% rice straw, allowing use in commercial systems without further fuel pretreatment such as leaching. Studies were initiated on coupling an anaerobic digester to a thermal gasification system for syngas treatment and fuel gas enhancement through syngas and biogas blending. Experiments were designed to investigate the impacts of tar and producer gas on digester function and operation. Phytoremediation techniques are being developed for producing biomass for bioenergy and bioproducts from agricultural lands that are drainage impaired and have high salt contents. Properties of this biomass are being investigated, as are thermo- and biochemical conversion and manufacturing of particle boards and other products. Statewide assessments of biomass in California were completed including surveys of bioenergy producers in the state. Large undeveloped resource exists within the agricultural, forestry, and urban sectors. Biogasification of various organic solid wastes studies using an APS-Digester at thermophilic temperatures yielded methane production potentials of 292, 284, 431, and 138 L/kgVS fed for animal bedding, green waste, food waste, and dairy manure, respectively, and mixed waste yielded 282 L/kgVS fed. Biogas yield of onion waste was 340 - 470 L / kg VS fed, with methane yields ranging from 50 - 80%. The APS-Digester is being scaled up to a pilot scale facility of 3 tons per day capacity and a commercial scale of 25 tons per day. A two-stage anaerobic fermentation process is being developed to produce hydrogen and methane gases from food processing wastes. Cheese permeate and fruit processing wastes are being studied as feedstocks. Batch experiments showed that the anaerobic digester sludge was a good innoculum for both feedstocks. The hydrogen yield and organic acid production from each feedstock in the first stage fermentation have been determined. (Oklahoma State Univ.) The effect of biomass moisture content (mc) on gasification operating parameters and hence producer gas composition, was studied. Switchgrass was harvested at approximately 35% mc (w.b). Bales were dried and gasified in the fluidized bed gasifier at lower mc. Elevated biomass mc decreased the operating temperature by as much as 100oC for a 20% change in mc. The decreased operating temperature altered the composition of the producer gas, i.e. CO and H2 concentrations were lowered by 30-40% when mc was increased from 9% to 29%. Changes in the quantity of ash, tar, and water produced did not seem to show specific patterns with changes in mc. In an effort to understand and optimize the operation of the fluidized bed gasifier, a full-scale plexiglass model of the gasifier was built and used to study the fluidization process. Feedstocks studied were a switchgrass, bermudagrass, and corn fermentation waste. Baffles were added to the reactor bed in an effort to simulate the added electric heating coils being used for pyrolysis and steam gasification. Use of gasifier-generated producer gas in fermentation studies also has continued. Producer gas is being cleaned, cooled, and compressed into storage tanks. It is then used in a bioreactor during fermentation to determine how well the microorganisms can grow and produce ethanol and other value-added products. Four-liter bioreactor studies on syngas for the fluidized bed gasifier did not kill the cells but cell growth stopped. The cell decline observed in the bioreactor agreed with a model suggesting no growth and loss of non-growing cells from the exit stream of the bioreactor. These results demonstrate that cell recycle is necessary. Ethanol production increased following exposure to producer gas, whereas hydrogen consumption stopped immediately. Preliminary results suggest that nitric oxide may be the inhibitorperhaps directly interacting with the hydrogenase enzyme. This result demonstrates that nitric oxide, if the inhibitor, must be removed from the producer gas prior to entering the bioreactor. Acetylene, oxygen, ethylene, and methane at the concentrations found in the producer gas did not interfere with cell growth. These results suggest that gas cleanup following gasification may not need to be applied extensively - just some minor cleanup such as removing nitric oxide. Objective 3. Develop, evaluate, and optimize integrated processes to convert biomass resources into biomaterials with commercial applications. Task 1. Raw feedstock evaluation. (Louisiana State University) The thermal characteristics of (TGA) for cellulose, lignin, raw bagasse fiber, and bagasse fibers were determined by thermogravimetry and derivative thermogravimetry profiles. Higher content of cellulose and lower content of lignin resulted in higher onset degradation temperature, higher peak rate of decomposition, and lower residual weight. The onset decomposition temperature and peak rate of weight loss indicated that higher content of cellulose occurred in the bagasse fiber treated with 1.5 to 2N NaOH for 1.5 to 2h. The Univ. of Minnesotas Center for Biorefining has a number of projects related to converting biomass to fuels and materials. The Center also is discussing a possible collaboration on microwave pyrolysis/liquefaction with researchers in Norway. The University of Nebraska is continuing its efforts to improve the starch acetylation process and to characterize acetylated starch blended with native starch, fibers, other biodegradable polymers and synthetic polymers. Some of our most recent results include preparing starch acetate nanocomposite foams with four organoclays (Cloisite 30B, 10A, 25A, and 20A) by melt-intercalation methods. XRD results indicated the extents of intercalation depended on the types of organoclay and were exhibited in the sequence of Cloisite 30B>10A>25A>20A. SEM results indicated a decrease in cell size in the starch acetate foam matrix with the addition of nanoclay. Glass transition temperature and onset temperatures of thermal degradation increased with the addition of organoclay into starch acetate matrix. The incorporation of organoclays decreased significantly the compressibilities of starch acetate nanocomposites and did not substantially affect their spring indices. Task 2. Methodologies for producing biomaterials. (University of Illinois) Modified dry grind processes (QG, QGQF and E-Mill) increased ethanol concentration by 8 to 27%, reduced fiber content of DDGS 24 to 81% and increased protein content of DDGS 26 to 105%, relative to the conventional dry grind process. Elutriation (air separation) and classification techniques were developed to remove fiber from DDGS. At the University of Kentucky, an extractive process utilizing a heavy solvent to directly convert biomass, was used to convert red and white oak into heavy liquids (pitches), chemical feedstocks (phenols, cresols, and oxygenates), and carbon materials. With biomass conversion levels of 99%, the solvent loss was 36%. A decrease in solvent loss resulted in a lower conversion rate. Continuing research is focused on finding operating conditions where an economical balance between biomass conversion and solvent is achieved. In an effort to reduce the cost of producing polyhydroxyalkanoate (PHA) South Dakota State Univ. scientists conducted 3L bioreactor trials with Pseudomonas putida KT2442 and basal condensed corn solubles (CCS) medium, with agitation of 300-500 rpm and aeration of 2 L/L/min to maintain 30% of the maximum dissolved oxygen level during exponential phase. Variables include carbon sources (glucose, glycerol, corn oil, and soapstock), fed-batch additions of carbon sources, ammonia supplementation, and carbon:nitrogen ratios. Findings thus far indicate the 400 g/L level of CCS was optimal, and that glucose and organic acids (acetic, lactic) were utilized before glycerol. Corn oil, glycerol, and soapstock can be added effectively during stationary phase to boost PHA production. In these trials P. putida yielded over 30 g/L cell mass and 24% of the cell mass was PHA. Researchers previously reported cell yields of 173 g/L, consisting of 30% PHA, in high cell density fed-batch bioreactor trials with oleic acid as the carbon source. Future trials will focus on PO4 and NH4 supplementation feeding strategies, and cell recycling methods to obtain high cell density for PHA accumulation. Dual stage carbon feeding (e.g., glucose/soapstock) will be evaluated. PHA was extracted from the cell pellets using supercritical fluid extraction (SFE) with CO2, with 15% ethanol modifier at 60°C and 9000 psi. Task 3. Biomaterial applications. Kansas States research focus was on improving flexibility of biobased resins from poly(lactic acids) and starch for wide range applications; fundamental research on grain/plant polymers and materials, and applying the knowledge to biobased products for the future; and commercial feasibility testing of a soy adhesive. (Louisiana State University) Composite weight (planar density) and thickness influenced bagasse/PP, kenaf/PP composite acoustical performances (ASTM E 1050). Larger composite weight and thickness enhanced sound absorption. In general, the bagasse and ramie composites had very similar acoustic performances, and both were superior to the kenaf composite. With a greater concern for environmental protection, it is more and more important for automakers to improve recyclability of newly produced vehicles. Recycling auto interior parts is a major barrier to improving vehicle recyclability. Use of natural fibers for producing biobased nonwoven composites for auto interiors could remove this barrier. This research promotes utilization of U.S. agricultural fibrous residues and other fiber crops for automotive interior parts production and investigates new processing technologies that can be adopted to enhance the biobased material performance and to lower production cost. (Univ. of Nebraska) High quality textile fibers were extracted from corn stover. The traditional methods of fiber extraction hydrolyzed the corn stover, making the fibers too short and too weak for textile applications. A unique process was developed to extract fiber bundles from corn residues with the length, strength and elongation required for textile applications. Characterizations of fibers and textiles are on going. Cellulose fibers extracted from corn stalk were blended with starch acetate to make biodegradable packaging foams. Starch acetate was blended with treated fiber in concentrations of 0, 2, 6, 10 and 14 % (w/w) and extruded in a twin-screw extruder with 12 to 18 % ethanol content as a plasticizer and 5 % talc as a nucleating agent. The samples were extruded at 150 C and selected physical and mechanical properties were evaluated. Fiber incorporation at lower concentrations enhanced the physical properties of the extruded foams. Fiber contents greater than 10 % decreased expansion. Good compatibility between starch and corn fiber was observed. Objective 4. Expand the scientific knowledge for development of processes and systems for economical production of biobased speciality chemicals from agricultural feedstocks and residues. Task 1. Biochemical Identification Characterization and Separation from Biofeedstocks. University of Arkansas focus was on energy crops that contain valuable phytochemicals. The idea is to extract phytochemicals prior to or in combination with the dilute acid pretreatment step during enzymatic hydrolysis and fermentation to ethanol. HPLC and MS analyses of methanolic kudzu foliage extracts confirmed the presence of 0.65 % ± 0.16 robinin (kaempferol-3-O-robinoside-7-O-rhamnoside). One mg of FPLC fractionated robinin generated ORAC values of 5.15 ± 2.00 umol/mg Trolox, while one mg of pure robinin generated ORAC values of 12.34 ± 0.45 umol/mg Trolox. Compounds found in the methanol-soluble fraction of A. julibrissin foliage (mimosa) were an unknown quercetin derivative with mass of 610 daltons, hyperoside (quercetin-3-O-galactoside), and quercitrin (quercetin-3-O-rhamnoside). The quercetin glycosides accounted for 2.3% of total foliage mass and contributed 85% of total antioxidant activity of the A julibrissin foliage. South Dakota State Univ. focused on recovering high value phytochemicals from DDGS. We have recovered and characterized the oil products from DDGS. We have also established a procedure to refine these products for human consumption. In the future, we have plans to study the phytochemical profiles of these products and also the most economical means of recovering these oil products. (Univ. of Nebraska) Lipid material (a waxy fraction and an oily fraction) was recovered by hexane extraction of grain sorghum kernels, flour and dried distillers grains (DDG). Waxy fraction yields of 0.16-0.31% (w/w) were observed for 86 commercial grain sorghum hybrids from 1993-1997 seasons and have remained consistent since then. Waxy fraction yields from 1996 hybrids grown in Nebraska were lower than yields from other years and were attributable to weather conditions. Long-chained lipids (LCL) composition mg/100g,w/w,db in waxy fractions of unpolished grain sorghum, polished grain sorghum, brown rice, purple rice, whole wheat, flint maize were 223,36.6,32.9,61.2,9.6,9.7 respectively. As would be expected the LCL concentration in DDG was higher than in whole kernel due to the concentration effect of removing the starch. Long-chained alcohols or policosanols are a broad group of lipid plant compounds which are effective in limiting oxidation, lowering LDL cholesterol in blood and enhancing ergogenic (endurance) effects on muscle tissue. Approximately 1 kg of policosanols can be recovered from a tonne of DDG. Ongoing work expects to further understand the effects of extraction process parameters on the composition of hexane extract, to assess the levels of tocols (Viatamin E compounds) and phytosterols in the oily fractions, to further develop purification methods for lipid fractions and to screen parent lines of grain sorghum for variation in the constituent lipids. Task 2. Process Development. (UC Davis) Variables for Agrobacterium tumefaciens mediated transformation, including vacuum application, photoperiod, surfactant and cell density, were investigated for optimizing transient expression of recombinant protein in leaf tissue. The optimal produced 0.16% recombinant protein, which was 10 times greater than reported levels in transgenic corn seed produced commercially. Protein extraction methods from leaf tissue are being investigated. Transient recombinant protein expression using Agrobacterium tumefaciens mediated transformation appears to be a technology that can potentially be scaled up to produce high-value proteins. It provides safety and environmental advantages compared to other plant-based expression systems because no transgenic crop or vector is exposed to the environment and the possibility of animal or human consumption is eliminated. A new multidisciplinary research center, the Center for Nanostructured Biomimetic Interfaces (CNBI) was established at Michigan State University (MSU). The CNBI is combining biotechnology with nanotechnology to develop new enzyme- and cell-based processes for the biomedical and biobased-products industries. Research applications include development of biological fuel cells and reactors that use electricity to drive enzymatic reactions. A new 150 L bioreactor is being installed for process scale-up. Task 3. Product Application. Stability of grain sorghum waxy fraction or its resistance to oxidation of during storage needs to be established in order to be viable in certain industrial uses such as a polishing wax. Little change was observed in waxy fraction composition and melting point during 5 months of storage in dark at room temperature at the Univ. of Nebraska. Slight but insignificant oxidation of aldehydes to acids was observed during 5 months of storage in sunlight or in dark at elevated temperature. Objective 5. Identify needed educational materials, develop those materials in distance based delivery methods and develop a trained work force to support a biobased products industry. Task 1. (No report.) Task 2. Development of an Advisory Board for the National Resource Center. Univ. of Minnesota, Kansas State, South Dakota State and Iowa State, are forming an education consortium in biobased products and bioenergy area. At the University of Kentucky, work is being done to develop and distributevirtual tours of an ethanol and a biodiesel production facility in an effort to raise awareness about the need and advantages of renewable fuels. The target audience will be both the general public and state and local government officials. UK also is offering a graduate level class on biorenewables. A multi-state consortium including Idaho, Washington State University, and MSU began developing new curricular material related to biorefineries. This work is being sponsored by a USDA Higher Education Challenge Grant entitled, Biorefinery Process Analysis and Design. A white paper developed by the S-1007 Objective V subgroup entitled, Educational Initiatives for a Biobased Economy was sent to Dr. Jim Fischer and Dr. Ian Maw. The U of Minnesotas Initiative for Renewable Energy and Environment funded more than 35 projects in the past year. These projects ranged from biomass production to conversion technology, from genomics to increase biomass yield to environment and economy assessments. IREE and the (co) sponsored, and participated in: · Energy Alley presents Minnesota's Renewable Energy Research: Status & Opportunities (Nov. 14, 2003) · Biorefining Video Conference (June 17, 2004) · The Third Crop Conference · Research Symposium (Nov. 18, 2004) IREE also is coordinating a seminar by Chemrec, an European company developing technology to convert paper mills black liquors to transportation fuels.

Peer-reviewed journal articles: Belyea, R.L., K. D. Rausch and M.E. Tumbleson. 2004. Composition of corn and distillers dried grains with solubles from dry grind ethanol processing Biores. Technol. 94:293-298. Calvo, L.R., M. Otero, B.M. Jenkins, A. Moran and A.I. Garcia. 2004. Heating process characteristics and kinetics of rice straw in different atmospheres. Fuel Processing Technology 85:279-291. Chen, Y., O. Chiparus, L. Sun, I. Negulescu, D.V. Parikh and T.A. Calamari. 2004. Waste bagasse for production of nonwoven composites. International Sugar Journal. 106(2): 86-92. Crofcheck, C. L. and M. D. Montross. 2004. Effect of stover fraction on glucose production using enzymatic hydrolysis. Trans. ASAE. 47(3):841-844. Datar, R.P., R.M. Shenkman, B.G. Cateni, R.L. Huhnke, and R.S. Lewis. 2004. Fermentation of Biomass-Generated Producer Gas to Ethanol. Biotechnology and Bioengineering. 86(5):587-594. Dien, B.S., N. Nagel, V. Singh, R.A. Moreau, M.P. Tucker, N.N. Nichols, D.B. Johnston, M.A. Cotta, K.B. Hicks, N. Quang and R.J. Bothast. 2004. Fermentation of quick fiber produced from a modified corn milling process into ethanol and recovery of corn fiber oil. Appl. Biochem. Biotech. 115:937-949. Duan, L., D.J. Carrier and E. Clausen . 2004. Extraction of health beneficial compounds from milk thistle using hot/liquid water. Appl. Biochem. Biotech. 114:559-568. Ganjyal, G. and M.A. Hanna. 2004. Effects of extruder die nozzle dimensions on expansion and micrographic characteristics of acetylated starch. Starke. 56(3-4):108-117. Ganjyal, G.M., Reddy, N., Yang, Y. and Hanna M.A. 2004. Biodegradable packaging foams of starch acetate blended with corn stalk fibers. J. Appl. Polym. Sci. 93(6): 2627-2633. Guan, J., K. Eskridge and M.A. Hanna. 2004. Functional properties of extruded acetylated starch-cellulose foams. J. of Polymers and the Environment. 12(3): 113-121. Guan. J., Q. Fang and M.A. Hanna. 2004. Marcomolecular characteristics of starch acetate extruded with natural fibers. Trans. of ASAE. 47(1):205-212. Johnston, D.B. and V. Singh. 2004. Enzymatic milling of corn: optimization of soaking, grinding and enzyme incubation steps. Cereal Chem. 81:626-632. Kohli, N., P.R. Dvornic, S.N. Kaganove, R.M. Worden, and I. Lee. 2004. Nanostructured cross-linkable micropatterns via amphiphilic dendrimer stamping. Macromolecular Rapid Communications. 25:935941. Lau, S., D.J. Carrier, L. Howard, J. Lay, J. Archambault and E. Clausen . 2004. Extraction of antioxidant compounds from energy crops. Appl. Biochem. Biotech. 114:569-584. Maupin-Furlow, J. A., M. A. Gil, I. M. Karadzic, P. A. Kirkland, and C. J. Reuter. 2004. Proteasomes: perspectives from the archaea [update 2004]. Front Biosci. 9:1743-1758. Montross, M. D. and C. Crofcheck. 2004. Effect of stover fraction and storage method on glucose production during enzymatic hydrolysis. Bioresource Tech. 92:269-274. Patel, M., M. Ou, L.O. Ingram, and K.T. Shanmugam. 2004. Fermentation of sugar cane bagasse hemicelluose hydrolysate to L(+)-lactic acid by a thermotolerant acidophilic Bacillus sp. Biotechnol. Lett. 26:865-868. Rausch, K.D., C.I. Thompson, R.L. Belyea and M.E. Tumbleson. 2003. Characterization of light gluten and light steep water from a wet milling plant. Biores. Technol. 90:49-54. Singh, V. and D.B. Johnston. 2004. An enzymatic process for corn wet milling. Adv. Food and Nutr. Res. 48:151-171. Singh, V., D.B. Johnston, R.A. Moreau, K.B. Hicks, B.S. Dien, and R.J. Bothast. 2003. Pre-treatment of wet-milled corn fiber to improve recovery of corn fiber oil and phytosterols. Cereal Chem. 80:118-122. Shukla, V.B., S. Zhou, L.P. Yomano, K.T. Shanmugam, J.F. Preston, and L.O.Ingram. 2004. Production of D(-)-lactate from sucrose and molasses. Biotechnol. Lett. 26:689-693. Thorsell, S., F.M. Epplin, R.L. Huhnke and C.M. Taliaferro. 2004. Economics of a coordinated biorefinery feedstock harvest system: lignocellulosic biomass harvest cost. Biomass and Bioenergy. 27(4):327-337. Ulmer, J.D., R.L. Huhnke, D.D. Bellmer, and D.D. Cartmell. 2004. Acceptance of ethanol-blended gasoline in Oklahoma. Biomass and Bioenergy. 27(5):437-444. Underwood, S.A., M.L. Buszko, K.T. Shanmugam, and L.O. Ingram. 2004. Lack of protective osmolytes limits cell growth and volumetric productivity of ethanologenic Escherichia coli KO11 during xylose fermentation. Appl. Environ. Microbiol. 70:2734-2740. Other communications: Jenkins, B.M., V. Tiangco, Z. Zhang, G. Simons, G.C. Matteson, R.B. Williams, H.I. vonBernath, L. Yan and M. Gildart. 2004. Biomass energy and power development in California: resources, generation, and future potentials. Paper V8.50, Proceedings 2nd World Conference and Technology Exhibition on Biomass for Energy, Industry, and Climate Protection, 10-14 May 2004, Rome, Italy. Johnson, D. 2004. Canola: Applications in high performance lubricants. Encyclopedia of Plant and Crop Science. Ulmer, J., R. Huhnke, B. Parr, and D. Bellmer. 2004. Alternative Energy. Educational video. Oklahoma Cooperative Extension Service. Oklahoma State University. Theses and Dissertations: Yu, M. 2004. Ultimate strength characteristics of switchgrass stem cross-sections at representative processing conditions. Thesis - MS in Biosystems Engineering. Univ. of Tennessee. August 2004. Zhuang, J. 2004. Economic Analysis of Cellulase Production by Clostridium thermocellum in Solid State and Submerged Fermentation. Thesis. University of Kentucky. Patents: Patent disclosure filed on soy oil-based home heating fuel. Purdue University. 2003. Patent disclosure filed on urea fractionation of fatty acid methyl esters. Purdue University. 2003. Patent disclosure filed on soy-based jet aviation fuels. Purdue University. 2003. Patent disclosure filed on aqueous separation processing of lipids. Purdue University. 2003.