Progress 09/01/09 to 08/31/12
Outputs
OUTPUTS: A transgenic switchgrass line expressing Arabidopsis transcription factor gene AtLov1 was generated. The transgenic switchgrass plants have erect leaf and increased lignin content (higher hydrocarbon content) of the cell wall biomass. Over300 new microsatellite molecular markers on a switchgrass mapping population were also screened. A draft genetic linkage map that will allow us to tag putative switchgrass genes that may associate with the hydrocarbon contents of switchgrass biomass was also screened. Following the last year's greenhouse trial, we conducted a field trial to compare the long term effects of poultry litter and wood biochar on soil properties and yields. These field trials were conducted at two field sites on organic farms growing peppers. An application of 4 tons biochar/acre is equivalent to approximately 0.2% biochar by weight in the greenhouse trial discussed previously, so well below where salt toxicity occurs. Six treatments were applied to the field sites: 1) Poultry litter biochar at 2 tons/acre; 2) Poultry litter biochar at 2 tons/acre plus compost addition for nitrogen; 3) Poultry litter biochar at 4 tons/acre; 4) Poultry litter biochar at 4 tons/acre plus compost addition for nitrogen; 5 (Wood biochar at 4 tons/acre plus compost addition for nitrogen and 6) Unfertilized control with no char or compost. Results indicated that due to highly variable yields, there was little effect of biochar addition on yield. Initially salts were elevated where poultry litter biochar was applied, but these leached harmlessly with rainfall and time. One significant finding was that the phosphorus in the biochar was plant available, as shown by the Mehlich 3 and Olsen soil tests. Earlier characterization of the biochar showed that although it contained 5% P by weight, this was not soluble when water was leached through the biochar. However, the pH of the biochar was 9.3 which favors the development of poorly soluble calcium phosphate, which probably dissolved slowly after application to the slightly acidic soils at the field sites. Application of poultry litter or wood biochar also increased the cation exchange capacity of the soils by 0.5 to 1.0 cmol/kg. This is a very significant benefit to soils and crop production, as soils with higher cation exchange capacities also have better nutrient use efficiencies, which leads to more efficient and profitable crop production. These results show that biochar produced from either poultry litter or wood has the potential to increase the quality and productivity of soils, especially poor soils. Several undergraduate and graduate students participated in the project and several presentations were made at various national and international meetings. A patent disclosure application on the low lignin-content switchgrass was filed. PARTICIPANTS: Bingyu Zhao, John Fike, Percival Zhang and Zhiyou Wen TARGET AUDIENCES: Biofuel industry, researchers and government agencies in charge of bioenergy. PROJECT MODIFICATIONS: No specific modifications were made to the project.
Impacts
Increasing the hydrocarbon of biomass feedstock could significantly improve the bio-oil production through thermal conversion process such as pyrolysis. Improved plant canopy structure with smaller leaf angles could increases shade-avoidance and thereby maximize plant biomass yield in a dense field population. A new plant gene, AtLov1, was identified that regulates the lignin content and also causes erect leaf in transgenic switchgrass plants. The cell wall biomass of the AtLov1 transgenic plants have 7 to 10 percent more lignin content than the wild type controls. Higher lignin content means higher hydrocarbon content, which is desirable for the thermal conversion. We expect the biomass feedstock of the new switchgrass line will be more efficient for bio-oil production in pyrolysis. Therefore, it will can lower the cost of bio-oil production and make the bio-refinery to be more profitable. The AtLov1 transgenic plants have dramatically smaller leaf angles resulting erect leaf. Therefore, farmers can grow switchgrass plants in higher planting density, which has the potential to produce more biomass and generate more income with limited land supply. It also could contribute to alleviating the public concern regarding use of food crops for bioenergy.
Publications
- Johnson M, Wen Z. 2010. Development of an attached microalgal growth system for biofuel production. Applied Microbiology and Biotechnology. 85 (3): 525-534.
- Johnson M, Wen Z. 2009. Production of biodiesel fuel from the microalga Schizochytrium limacinum by direct transesterification of algal biomass. Energy and Fuels. 23 (10): 5179-5183.
- Ethier S, Woisard K, Vaughan D, Wen Z. 2011. Continuous culture of the microalgae Schizochytrium limacinum on biodiesel-derived crude glycerol for producing docosahexaenoic acid. Bioresource Technology. 102(1): 88-93.
- Zhu ZG, Wang YR, Minteer SD, Zhang Y-HP. 2011. Maltodextrin-powered enzymatic fuel cell through a non-natural enzymatic pathway. Journal of Power Sources 196:7505-7509.
- Wang YR, Huang WD, Sathisuksanoh N, Zhu ZG, Zhang Y-HP. 2011. Biohydrogenation from biomass sugar mediated by cell-free synthetic pathway biotransformation. Chemistry and Biology: 18:372-380.Huang WD, Zhang Y-HP. 2011. Analysis of biofuels production from sugar based on three criteria: Thermodynamics, bioenergetics, and product separation. Energy and Environmental Science 4:784-792.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: The computational method of identifying hexanucleotide pairs was applied to the C. acetobutylicum genome and additional parameters were included to improve predictions. In the promoter region of the cellulosome in C. acetobutylicum, two sigma factor binding sites were identified. The strong sigma A site that is strongly associated with solventogenesis was present along with the sigma F/G site, which is strongly associated with sporulation. Flanking each of these binding sites were two unknown protein-binding regulatory sites. This method was applied to the genomes of C. thermocellum and C. cellulolyticum. The sigma factor and unidentified regulatory binding sites were found in the promoters of these cellulsomes as well. This suggests that repression of the C. acetobutylicum may be more complex than simple transcriptional repression. A major effort was undertaken in this project to apply a previously-published gene reporter system for C. acetobutylicum using the pHT3 plasmid. The blunt-end cloning methods yielded poor results when it was applied to cloning cellulosomal promoter fragments. This blunt-end cloning method was replaced by a TA-cloning method; however,it was found that this method does not provide the precise quantitative measurements needed for analyzing the effects of clostridial promoter fragments. In response, a new gene reporter system for C. acetobutylicum was developed. This system contains a newly-available anaerobic GFP reporter gene and makes use of In-Fusion (Clontech) cloning technology. This method of cloning was found highly efficient for clostridial plasmids and will greatly facilitate metabolic engineering of clostridia. It is not yet known if the unidentified regulatory site in the proximity of the sigma A binding site is causing inhibition, but theexperiments that will be run in the coming months will provide answers. However, this project has already shown transcriptional repression of the C. acetobutylicum cellulosome is in the presence of an active sigma A binding site. This is a novel development and will significantly impact the field of clostridial biology. In a greenhouse experiment biochar was mixed with two soils, one a silt loam, and the other a sandy loam, at rates of 0% to 100% biochar by weight. Lettuce seeds were germinated to evaluate the benefits or toxicity of biochar, and following the germination the soils were analyzed for a broad range of characteristics. Adding biochar up to 1% by weight had a slightly beneficial effect or no effect on lettuce germination in either soil. However, above 2.5% by weight, lettuce germination dropped off dramatically. Testing of the soil showed that this was due to salt toxicity, due to its high concentration in the biochar. A simple wash of the biochar with water is capable of removing these salts. We evaluated the water holding capacity of the soils used in the lettuce germination study and poultry litter biochar increased the water holding capacity of both soils. Results suggest that biochar may have a greater potential to improve poor quality soils than good quality soils. Several undergraduate and graduate students were trained in the laboratory. PARTICIPANTS: John Fike, Ryan Senger, TARGET AUDIENCES: Industries and government agencies responsible for promoting alternative sources of energy and conservation of natural resources. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Discovering the mechanisms of repression of the C. acetobutylicum cellulosome is of the utmost importance for eliminating them. With an active cellulosome, C. acetobutylicum will be the first organism capable of operating in a consolidated bioprocess to produce the readily drop-in biofuel butanol. Multiple researchers have shown that a consolidated bioprocess is the most economically feasible means of producing biofuels from lignocellulosic plant materials. As an unintended consequence in this research, several molecular biology tools for clostridia were developed. A new gene reporter system containing an anaerobic functioning GFP resulted from this research. This will allow, for the first time, the continuous anaerobic monitoring of gene products and metabolism in the clostridia. This will have profound impacts on understanding clostridial metabolism and engineering biofuels production and lignocellulose degradation. A combinatorial method for sampling and analyzing the function of a gene promoter was developed in this research. This will lead to more effective studies of prokaryotic gene regulation and will help elucidate the many regulatory mechanisms. Relatively few of these are known, and the combination of the computational and experimental approach of this research will ultimately lead to the design of new cellular systems that can carry-out desired functions related to: (i) the production of biofuels, (ii) the production of commodity chemicals, (iii) remediation of toxins, and (iv) biomedical applications. The disposal of poultry litter is becoming a major problem for the poultry industry in the USA because of environmental concerns. The traditional disposal methods such as land application, composting, combustion for power and heat generation, are no longer suitable. Thus, there is a need to develop novel technologies to address these waste disposal problems, especially for small-scale growers and other growers in EPA non-attainment areas. The overall goal of this project is to develop suitable applications of biochar to preserve its fertilizer value and dispose of nutrients with minimal environmental footprint. These preliminary results show that biochar produced from either poultry litter or wood has the potential to increase the quality and productivity of soils, especially poor soils.
Publications
- Reed JL, Senger RS, Antoniewicz MR, Young JD. 2010. Computational approaches in metabolic engineering. Journal of biomedicine & biotechnology 2010:207414.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: During the first year of the project we conducted research experiments to improve the quality of the biomass feedstock for efficient processing by transforming switchgrass to express endoglucanase enzymes. The major achievements are: 1) Development of an efficient switchgrass tissue culture and genetic transformation protocol has been established. 2) 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. 3) More than 20 independent switchgrass transgenic lines have been obtained. The presence of endoglucanase gene was confirmed by PCR amplification with gene specific primers. 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. The transgenic plants will be further characterized by crossing to non-transgenic plants and test if the transgene will be stably inherited in the progenies. The transgenic plants will also be used to extract the endoglucasenase enzyme and then the residual biomass will be converted to biofuels by pyrolysis. We just completed the first year of the project. Research is also being conducted on characterizing poultry litter biochar, and engineering cellulose utilization by the biobutanol-producer bacteria. Results will be disseminated through publication of the results and presentations at several conferences. At least three graduate students have been recruited to work on various objectives of the project. PARTICIPANTS: Bingyu Zhao and Foster Agblevor from Virginia Tech contributed to the objectives reported here. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Natural biomass feedstocks are composed of lignin, cellulose, hemicelluloses and extractives. Although these polymeric constituents are potential raw materials that could be converted to various types of biofuels, their natural structures 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. Further, because biofuels are commodity chemicals and have low energy densities, they are currently not competitive with energy dense fossil fuels. 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 could be extracted first, and then the residual biomass can be converted into biofuels. Because the pharmaceutical proteins have very high value, they will make the entire process economically viable and competitive with fossil fuel production.
Publications
- No publications reported this period
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