Progress 06/01/06 to 05/31/10

Outputs
OUTPUTS: Natural biomass feedstocks are composed of lignin, cellulose, hemicelluloses and extractives. However the natural structures of these polymeric constituents are such that they are very difficult to process efficiently to higher value products. The major challenges are the accessibility of the biopolymers to enzymes and other chemicals, multiplicity of products generated from the degradation of the biomass, toxicity of treated biomass to biocatalysts, and high oxygen content of the biomass which results in low energy density. One proposed method of improving profitability of the biomass is to genetically modify the biomass feedstock to produce active pharmaceutical proteins in addition to the biomass. These pharmaceutical proteins are extracted first, and then the residual biomass is converted into biofuels. Research experiments were conducted to improve the quality of the biomass feedstock for efficient processing by transforming switchgrass to express endoglucanase enzymes. An efficient switchgrass tissue culture and genetic transformation protocol has been established. A fungal belta-endoglucanase gene (from Hypocrea jecorina (AB003694)) was synthesized by Genscript. The codon optimized endoglucanase gene was fused to a chloroplast targeting signal of the rice RUBISCO small subunit gene. The targeting signal will drive the recombinant endoglucanase protein into chloroplast in the transgenic plants. The engloglucanse gene expression cassette was cloned into a special plant expression vector that allows us to transform switchgrass cultivar Alamo. More than 20 independent switchgrass transgenic lines have been obtained. The transgenic plants grow well in greenhouse condition, which suggests the possibility of expressing high value pharmaceutical protein, cell wall degrading enzymes, and other potential toxic proteins in switchgrass plants. Efforts were also made to recover phosphorus from liquid dairy manure through chemical precipitation using metal salts and as struvite. The use of alum and iron based salts in combination with polymers to remove phosphorus from dairy manure were tested. results showed that one can remove as up to 100% of P from the manure. The study on chemical phosphorus removal showed that use of chemical + polymers in large volumes of batch manure is feasible. The concept of designer manure was successfully demonstrated at the Virginia Tech dairy farm by chemically treating 2,000 cubic meters of manure in batch using a combination of aluminum chloride and a polymer with a trade name "Superfloc 4512". Recovery of phosphorus from liquid dairy manure is possible but it is a big challenge. For struvite recovery to be successful, pretreatment to increase the ratio of reactive phosphorus to total phosphorus is necessary. The project facilitated collaboration with industry, provided opportunities for several PhD students to receive training in the biofuels area and resulted in a large number of scholarly publications, outreach activities and conference presentations that aided in the dissemination of the results PARTICIPANTS: Several investigators worked on the project. Drs Zhiyou Wen and Jactone Aroge Ogejo were the faculty conducting nutrient removal and biogas studies. Drs Bigyu Zhao and Foster Agbelvor conducted the experiments on genetic engineering and biofuels studies. TARGET AUDIENCES: farmers, industry and citizens concerned with quality of water and protection of natural resources as well as sustainable energy production. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Because the pharmaceutical proteins have very high value, they will make the entire process economically viable and competitive with fossil fuel production. The processability of the biomass feedstock can be improved through plant breeding and genetic modification. Breeding and genetic modification can generate new plants with either high lignin or cellulose contents. Genetic tools can also be used to transform the biomass to improve accessibility to enzymes and chemicals. One such approach is the introduction of cellulose degrading enzymes into the plant in a dormant state and activating the enzymes to degrade the biomass after the harvest. These results provides a viable alternative to manure phosphorus management, to producers who have liquid manure handling systems and are in areas where soils may be saturated with phosphorus, have manure storage for 6 months and may be limited by land area to apply manure. Using chemicals to remove phosphorus can work well with dairies that use flush systems to handle manure. The recovered phosphorus, being small in volume but high in phosphorus content can then be transported out of the Shenandoah Valley, if the objective is to remove the excess phosphorus in the region. The project facilitated collaboration with industry, provided opportunities for several PhD students to receive training in the biofuels area and resulted in a large number of scholarly publications, outreach activites and conference presentations that aided in the dissemination of the results

Publications

• Debusk, J., J. Arogo Ogejo, K.F. Knowlton, and N.G. Love. 2008. Chemical phosphorus removal for separated flushed dairy manure. Applied Engineering in Agriculture 24(4): 499-506.
• Ogejo, J.A. and L. Li. 2010. Enhancing biomethane production from flush dairy manure with turkey processing wastewater. Applied Energy 87:3171-3177.

Progress 06/01/08 to 05/31/09

Outputs
OUTPUTS: The faculty at the Center continued their investigation on several projects related to a) Optimizing biogas production potential and nutrient recovery from dairy manure and b)development of a biodegradable litter amendment product for removing odor from manure. Regarding the biogas production the focus was made on 1)development of a microwave-based pretreatment method to treat dairy manure, with the purpose of enhancing the biogas production during the anaerobic digestion process, and improving nutrient (particularly phosphorus, P) recovery efficiency; and 2) studying the fate of pathogens during different manure treatment processes.we used microwave heating combined with different chemicals (NaOH, CaO, H2SO4, or HCl) to successfully enhance solubilization of manure and degradation of glucan/xylan in dairy manure. The pretreatments also released 20-40% soluble phosphorus and 9-14% ammonium. Microwave heating, combined with NaOH or HCl, led to a high anaerobic digestibility and phosphorus recovery. Using these two chemicals, the performance of microwave- and conventional-heating in thermochemical pretreatment was further compared and the results show that the microwave heating resulted in a better performance in terms of COD solubilization, glucan/xylan reduction, phosphorus solubilization and anaerobic digestibility. A DNA-based method was developed to identify and quantify E. coil in manure samples. We adopted a quantitative PCR method, in which gadA and gadB genes of E. coil were the targets. An E. coli mutant strain lacking gadA and gadB genes was constructed and was added to each sample prior to processing as an internal control to quantify the DNA extraction efficiency during the Q-PCR analyses The results indicated that DNA-based methods could detect and quantify pathogen more precisely than culture based method. We have developed a biodegradable litter amendment material (Amosoak) that is capable of removing 98% of the ammonia present in air streams; it can also reduce odor and VOCs. The material is produced from agricultural residues such as corn cob, cotton gin waste, peanut hulls, corn stover, wood waste and other agricultural residues. It is a dry acidic material, which can be produce as either coarse particles or fibrous structure. When Amosoak is applied as a litter amendment, the litter has a pleasant fruity smell depending on the quantity of Amosoak added to the litter. Amosoak acts by reacting with ammonia and VOCs forming organic salts and esters that are retained in the coarse particles or fibrous structure of the material. The nitrogen content of the litter is therefore enhanced and this improves on its nutrient value. The pH of Amosoak ranges from 2.5 to 4 and therefore it is not harmful to birds when applied to litter. It is also not harmful to birds when eaten. We have started screening other agricultural materials such as soybean straw, corn fodder, and sawdust as material for producing Amosoak. This will diversify our raw material sources and make the process more flexible. The projects results were disseminated at various national and international meetings. Several graduate students were trained on the technologies. PARTICIPANTS: Virginia Tech College of Agriculuture and Life Sciences faculty worked with an industrial partner, Novozymes Biological, on this project. Several PhD students, post-doctoral reaserach associates and visiting scholars participated in these projects. We also involved several undergraduates in research related to the Center's activities. Dr. Saied Mostaghimi, professor and Head of the Department of Biological Systems Engineering at Virginia Tech served as the Interim Director of the Center. TARGET AUDIENCES: Farmers, industry and citizens concerned about the impact of manure on downstream water bodies and odor and air quality problems PROJECT MODIFICATIONS: No modifications was made to the project's goal during the reporting period.

Impacts
The results of the investigations on biogas production will impact the animal industry by providing a novel pretreatment of animal manure, which can be implemented into anaerobic digestion processes. This pretreatment technology can improve the biogas production and nutrient recovery, simultaneously. The improved biogas yield and the potential for developing other value-added products from nutrient recovery provide incentives for farmers to install an onsite digester on their farms. The results of the study could lead to development of a protocol to rapidly and accurately identify and quantify the pathogen organisms in animal manure samples and evaluate the effectiveness of different manure treatment process on the pathogen reduction. These results will impact the U.S. agriculture industry by providing methods for better assessing the fate of pathogenic microbes in dairy waste, and understanding the source, fate and the transport of pathogens in soil, surface and ground water. The US poultry industry produces several billon tons of poultry litter per year. Poultry litter has basic pH and undergoes microbial degradation that produces ammonia, odor, and volatile organic acids. These poultry litter degradation products are harmful to the health of birds and poultry workers and they also have negative impact on plants and aquatic ecosystems. Ammonia and odor are currently controlled using non-biodegradable inorganic acidic materials such as alum, zeolite, poultry guard, and poultry litter treatment.We are currently applying the Amosoak on a pilot scale pyrolysis project in Dayton VA. The addition of Amosoak to poultry litter not only removes odor during processing of the litter, but it also increases the bio-oil yield. Thus, we are currently adding 10 wt% of Amosoak to poultry litter before the pyrolysis. The patent application on the process has been reviewed by the US Patent office and recommendations made for the patent to be split into four patent applications instead of one.

Publications

• Jin Y, Hu Z, Wen Z. (2009). Enhancing anaerobic digestibility and phosphorus recovery of dairy manure through microwave-based thermochemical pretreatment. Water Research. 43 (14): 3493-3502.

Progress 06/01/07 to 05/31/08

Outputs
OUTPUTS: Seed funding was provided by the Biodesign and Bioprocessing Research Center to: a) develop processes for producing high Value Polymers from poultry and dairy processing in Virginia, b) Optimize biogas and acid production potential and nutrient recovery from dairy manure, and c) conduct a proof-of-concept study to produce high-yield hydrogen from polysaccharides and water through a novel enzymatic method. Uses of agricultural by-product proteins from poultry and dairy processing as commodity plastics were investigated. These proteins do not have much feed value and cannot be converted into fuels and are usually disposed. Dr. J. Barone's group has been investigating ways to toughen the protein by eliminating the diffusible glycerol component and to stabilize the protein against biodegradation for longer life. They have discovered that choice of the correct protein structure through processing can stabilize the protein to microbial attack and more efficiently tailor product life. Laboratory studies are being conducted to recover nutrients (P and N) from liquid dairy manure by Dr. Ogejo's research group. This study focuses on P recovery using chemical precipitation and N conservation through nitrification. The studies also determined the optimum manure total solids (TS) content for chemical P removal. Use of polymer in combination with chemicals decreased P in the supernatant by more than 80% and produced a more compact sludge. Hydrogen is a clean energy carrier. The hypothetical hydrogen economy will provide a promising future energy vision. A novel enzymatic method producing high-yield hydrogen from polysaccharides and water was proposed in collaboration with the researchers from the Oak Ridge national laboratory. A proof-of-concept experiment is being conducted to validate the theory. Dr. Percival Zhang's research group used 13 enzymes from different species to construct a synthetic enzymatic pathway, which released the energy in the sugar step by step. In order to address the remaining challenges of slow reaction rates and unstable enzymes, researchers plan to clone and express a number of thermophilic recombinant enzymes to replace costly Sigma mesophilic enzymes and accelerating hydrogen production rate at higher temperatures. In June 2008, Dr. Zhang and his group produced high-yield hydrogen from cellulosic materials. In addition, they have increased the hydrogen production rate by 10x fold through optimization. The next 10-fold increase in reaction rate will greatly enhance the chances for commercialization of the technology. The results of these investigations have been disseminated at numerous national and international conferences throughout the World. The Center has provided opportunities for training of a large number of graduate students in an effort to produced skilled work force for the bi-industry of the future. PARTICIPANTS: Partner Organizations: Virginia Tech, College of Agriculture and Life Sciences Novozymes Biological, Inc, Salem Virginia TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Plastics represent an important class of products totaling 175 billion pounds in the U.S. each year. The rise in petroleum prices has meant that prices for gasoline are two times higher than 2 years ago while for plastics they are three times higher. Finding new bio-based sources for plastics would help solve this problem. Since most plastics are processed at factories powered also by fossil fuels, finding low energy "green" processing methods is advantageous. The work being conducted at the Center aims to find higher value uses for protein by-products from poultry and dairy processing in Virginia. While biogas production from animal manure is not a new idea, the economic feasibility of this technology has been a challenge in Virginia. Anaerobic digesters are mostly sited at dairies milking herd sizes well over 250 head. Virginia diaries have an average herd size of 100. Though small in size, we contend that the focus of this study, combining organic residue streams from adjacent farms and other industries producing organic residues with dairy manure may provide a creative, cost-effective, and practical way to generate biogas from smaller dairies and strengthening rural economies. Managing manure phosphorus to produce a fertilizer with a nitrogen to phosphorus ratio that matches crop fertilizer needs will be helpful to farmers who use animal manure as fertilizer and are restricted by phosphorus based manure application regulations. Recovering some of the phosphorus from manure will also help protect and or improve the quality of the water caused by phosphorus in runoff water due to excess application of manure to crop lands. Nitrogen conservation through nitrification increases the fertilizer value of manure and also improves air quality through reduced ammonia volatilization to the atmosphere resulting in: reduction of the consequences of emitting ammonia to the atmosphere. The experimental results on hydrogen production validated the theory by using a novel synthetic enzymatic pathway for hydrogen production. The unique features of this biocatalysis, such as complete conversion, high hydrogen yields, low temperature temperatures (30oC), etc, provide a great potential to mobile and stationary applications. This technology promises to solve several problems of the hydrogen economy, such as, cheap hydrogen production, safe and efficient hydrogen storage, and hydrogen distribution infrastructure.

Publications

• Barone, J.R., Arikan, O. 2007. Composting and biodegradation of thermally-processed feather keratin polymer. Pol. Deg. Stab. 92:859-867.
• Barone, J.R., Medynets, M. 2007. Thermally processed levan polymers. Carb. Pol. 69:554-561.
• Barone, J.R., Dangaran, K.L., Schmidt, W.F. 2007. Protein-transition metal ion networks. J. Appl. Pol. Sci. 106:518-1525.
• Hu Z, Wen Z. 2008. Enhancing enzymatic digestibility of switchgrass by microwave-assisted alkali pretreatment. Biochemical Engineering Journal. 38: 369-378.
• Hu Z, Wang YF, Wen Z. 2008. Alkali (NaOH) pretreatment of switchgrass by radio frequency-based dielectric heating. Applied Biochemistry and Biotechnology. 148: 71-81.
• Hong J, Ye X, Wang Y, Zhang Y-HP. 2008. Bioseparation of recombinant cellulose binding module-protein by affinity adsorption on an ultra-high-capacity cellulosic adsorbent. Analytica Chimica Acta 621:193-199.
• Hong J, Wang Y, Ye X, Zhang Y-HP. 2008. Simple protein purification through affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage. Journal of Chromatography A. 1194(2): 150-154.
• Zhang Y-HP. 2008. Reviving the carbohydrate economy via multi-product biorefineries. Journal of Industrial Microbiology and Biotechnology 35 (5): 367-375.
• Hong J, Ye X-H, Zhang Y-HP. 2007. Quantitative determination of cellulose accessibility to cellulase based on adsorption of a non-hydrolytic fusion protein containing CBM and GFP with its applications. Langmuir 23 (25): 12535-12540.

Progress 06/01/06 to 05/31/07

Outputs
During the first year of the project the Center was engaged in several projects related to utilization of waste and other biological materials. We investigated methods for safe and environmentally benign utilization of poultry litter. The traditional methods of land application and feeding to cattle are no longer acceptable because of safety and environmental concerns. We have developed a new technology that will safely dispose of the poultry and yet preserve all the nutrient value of the litter. The poultry litter nutrient is encrusted in the char after treatment at high temperatures. Because of the high temperature treatment all potential pathogens are destroyed in the process. The new product, "black manure", has no smell, has no pathogens but contains most of the original nutrients in the poultry litter. Above all, the nutrient release rate into the soil from black manure is considerably lower than that of the raw poultry litter fertilizer. Phosphorous release is about 10 times less than that of raw poultry litter and potassium and calcium releases are about three times less than that from the raw litter fertilizer. The black manure can be potentially sold as organic fertilizer and it can substitute for poultry litter fertilizer. Because it is odorless, it is a much better product than current organic fertilizer on the market. Hydrogen is a clean energy carrier. The hypothetical hydrogen economy will provide a promising future energy vision. We have proposed a novel enzymatic method - producing high-yield hydrogen from polysaccharides and water. A proof-of-concept experiment was conducted to validate the theory. The idea of new hydrogen production method is to use the energy stored in polysaccharide to split water and release all chemical energy in the form of hydrogen. We used 13 enzymes from different species to construct a synthetic enzymatic pathway, which released the energy in the sugar step by step. This process is like sugar catabolism, but here water is used as an oxidant rather than oxygen in sugar oxidation. The overall chemical reaction is C6H10O5 + 7 H2O = 12 H2 + 6 CO2. The reaction is a spontaneous process and conducted at 30 degrees C and atmospheric pressure. This process can produce 12 molecules of hydrogen from 1 molecule of glucose unit, three times of the theoretical yield of the natural process - anaerobic fermentation. We wish to develop some super hydrogen-producing microorganisms sometimes.

Impacts
Improved water and air quality through reduced application of animal wastes and ammonia volatilization to the atmosphere would result in reduction of surface and groundwater pollution and reduce the consequences of emitting ammonia to the atmosphere, and thus the deposition of nitrogen in nitrogen sensitive ecosystems through ammonia deposition. Removing some of the phosphorus in manure to conform manure to a fertilizer with a nitrogen to phosphorus ratio that conforms or matches crop fertilizer needs will be helpful to farmers who use animal manure as fertilizer and are restricted by phosphorus based manure application regulations. The biocatalysis process we have developed for production of hydrogen from biomass has unique features such as complete conversion, high hydrogen yields, and low temperature temperatures (30 degrees C) provide a great potential to mobile and stationary applications. This technology promises to solve several problems of the hydrogen economy, such as, cheap hydrogen production, safe and efficient hydrogen storage, and hydrogen distribution infrastructure. With further development and integration of processes, it is anticipated that we can run vehicles on sugar in the future. If the technology is successful, the US could significantly reduce its dependence on foreign oil import, greatly promote farmer income, and decrease the future greenhouse gas emission by up to 50 percent.

Publications

• Zhang Y.-H.P., Mielenz J.R., Evans B.R., Hopkins R.C., Adams M.W.W. 2007. High-yield hydrogen production from starch and water by synthetic enzymatic pathway. PLOS ONE 2(5): e456. http://dx.doi.org/10.1371/journal.pone.0000456.