Progress 10/01/04 to 09/30/05
Outputs
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? Number of broilers produced in Kentucky increased 142% from 1997 to 2002 (Kentucky Agricultural Statistics, 2002). Since the majority of production is occurring in the western third of the state, this presents challenges with respect to environmentally responsible management of litter in a restricted geographical region. These challenges are important to the poultry and other livestock producers, community stakeholders, and regulatory and conservation agencies. Utilization of poultry litter to provide optimum crop quality and yield while reducing potential soil phosphorous, copper, and zinc accumulations (accumulations relating to greater probability of water and soil pollution). Problems related to excess phosphorus (P) in soils are being addressed using a plant-based bioremediation
(phytoremediation) technology. The problem is remediation of phosphates in poultry waste. Research also was conducted on the possibility of using normal poultry litter microflora to absorb and maintain high intracellular levels of phosphate thereby reducing the amount of phosphate available for immediate run-off into local watersheds. Produce useful products from poultry litter by studying the combustion behavior of co-firing poultry litter with coal, and investigating multi- utilization and treatment technology of poultry litter for production of energy. New research projects are ongoing related to the animal waste management such as; production of useful products from poultry litter by studying the combustion behavior of co-firing poultry litter with coal, and also investigating multi-utilization and treatment technology of poultry litter for production of energy such as utilizing poultry litter to generate activated carbon, which could be used as sorbents to capture the mercury
produced in the coal combustion process, by testing several thermal chemical processes. In another experiment, research is providing new information on the environmental contamination of cattle housing areas with Mycobacterium avium paratuberculosis (MAP) an organism that is shed in the feces and causes chronic untreatable diarrhea in cattle and other species. There is speculation that there is a relationship between MAP and Crohns disease in humans. These problems directly relate to Manure and Byproduct Utilization (NP 206). 2. List the milestones (indicators of progress) from your Project Plan. a) Applied fertility treatments to plots; collected and analyzed soil and forage samples; presented findings at national/regional meetings; preparing paper for submission to Agronomy Journal; and beginning to quantify best management practices; b) Identified plants suitable for high P accumulation; characterized P accumulator plants under different conditions, such as pH, mode of planting,
etc.; prepared and analyzed a cDNA library and a mRNA subtraction library representing the phosphorus sufficient and deficient gulf ryegrass; isolated a partial clone of a high affinity phosphate transporter induced under phosphate deficiency in gulf ryegrass; c) Co-firing poultry litter with coal in a lab-scale fluidized bed combustion and a bench-scale circulating fluidized bed combustion system to identify the optimal combustion conditions. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Completed litter rate studies in tall fescue and corn (grain). Milestone Substantially Met 2. Training and education of several agricultural undergraduate students in field research using poultry litter application to forage crops. Milestone Substantially Met 3. A lab-scale fluidized bed combustor (FBC) was set up for testing co- firing of poultry litter and coal.
Milestone Substantially Met 4. Compared an ELISA test against a new FCT test for diagnosis of MAP using bovine serum. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? 2006: a) Collect background soil samples, establish research plots, apply broiler litter and inorganic fertilizer; b) Finish all experiments on the lab-scale fluidized bed combustor, and start to test on the bench-scale circulating fluidized bed combustor; complete the test on two screw reactors; establish the fluidized bed reactor for char activation, optimize the fluidized bed for activated carbon and RDF gasification; c) Continue screening plants for higher P accumulation, establishment of tissue cultures and development of a repeatable regeneration protocol for alfalfa Isolate, analyze and catalog sets of phosphate-induced genes from gulf ryegrass
libraries; d) Find the most productive methods of sampling the environment for MAP organisms, do more environmental sampling of pasture, harvested forage and manure storage areas; e) Determine what environmental conditions influence expression of skatole synthesis. Skatole is a major odorant in manures, monitor indole producing organisms in in vitro experiments to determine which bacterial groups are most important in this process. 2007: a) Gather soil and forage samples, analyze soil and forage samples, present findings at national/regional meetings; b) Complete all experiments on the bench-scale circulating fluidized bed combustor, and develop a mobile apparatus for disposing poultry litter and test in the lab first. Integrate two screw reactors with fluidized bed reactor to set up the whole system for poultry litter multi-utilization, make the economic analysis and environmental impact for this technology; c) Clone some of the phosphate-induced genes (e.g. phosphate transporters,
phosphatase, phytase etc.) in to the binary vector containing AtPT2 promoter, infection and co-cultivation of plant parts/callus with Agrobacterium strains containing plasmids, regeneration of plantlets, optimize transformation of Alfalfa using both reporter genes and target genes identified above; d) evaluate the effect of anaerobic digestion of manure on the viability of the MAP organism; e) Isolation of skatole cultures from various manures and feces. This will allow, in part, for the determination of the microbial diversity responsible for this odor. 2008: a) Publication of peer-reviewed articles, field days/tours for target populations/stakeholders, develop a producer tour, begin to quantify best management practices; b) Test this mobile apparatus on site, perfect this unit, and transfer the technology to the poultry industry, put the technology into the practice in poultry industry; c) Standardization of high frequency regeneration of transformed plantlets, southern blot,
Northern blot and Western blot analyses of transformed alfalfa tissues/plantlets, transgenic plantlets will be assayed for phytase activity in rhizosphere, transgenics will be tested for use of phytate as a sole source of P in growth media.NB Based on the outcome of the above analyses in gulf ryegrass, we will choose 3 to 4 genes for further analysis to evaluate their roles in phosphate sequestration by plants. Analyze the expression of sets of phosphate starvation induced genes in phosphate efficient Gulf and Marshall rye grasses and in phosphate inefficient relatives of rye grasses; d) possibly look at vaccination as a control procedure for MAP; e) Isolation of the genes responsible for skatole synthesis. This will allow for the design of molecular probes to monitor this odorant. 4a What was the single most significant accomplishment this past year? a) Completed litter rate studies in tall fescue and corn(grain). Findings indicate no agronomic advantage to applying greater than 2
T/A litter on tall fescue or 4 T/A litter on corn; b) co-combustion of poultry litter and coal, set up the lab-scale fluidized combustions for studying the co-combustion of poultry litter and coal, understand mechanisms the dry/pyrolysis/activation of chicken waste; c) Characterization of P accumulations in annual ryegrass. Also, generation and analysis of molecular tools such cDNA and mRNA subtraction libraries; d) Demonstration that the phosphate binding capabilities of endogenous poultry litter microorganisms is increased by nutrient deprivation. 4b List other significant accomplishments, if any. Completed original orchardgrass and sorghum-sudangrass studies. Both of these studies are being modified for the coming year to better determine agronomically sustainable ways of removing excess soil nutrients; Construction of a phylogeny of tryptophanase genes which will allow for construction of molecular probes to monitor the potential for indole production. 4c List any significant
activities that support special target populations. Training and education of several Agricultural Undergraduate Students in field research using poultry litter application to forage crops. 4d Progress report. a) All milestones have been fully or substantially met. We are in the process of preparing a manuscript for peer-reviewed publication; b) Studies on physiological and growth parameters have confirmed the ability of Gulf and Marshall Grasses to sequester high levels of phosphorus in above ground parts. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. a) There were 2 field days for target populations. Another major accomplishment was finding that applying litter based on crop P requirements, and supplementing with inorganic N, produced forage yield and quality similar to what is produced with inorganic fertilizers or litter applied based on crop N requirements; b) Co-firing of poultry litter with coal could bring the
following benefits: First, SO2 emission could be reduced due to very low sulfur content in poultry litter. Second, co-firing poultry litter with coal could abate NOx emission level effectively due to NH3 released from poultry litter. Finally, mercury content in poultry litter is very low, but chlorine content in poultry litter is much higher than that in coal. These characteristics could be helpful to capture mercury emission from the coal combustion process; c) Generation of transgenic plants efficient in sequestering phosphate in above ground parts should also aid in phytoremediation of excess phosphate in soils. Isolation and analysis of genes induced during phosphate deficiency in Gulf rye grass may lead to identification of molecular targets for improving phosphate uptake and efficiency in plants. 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? a) Lower litter rates and supplement with inorganic N. There is no agronomic advantage to applying more than 2 T/A litter on tall fescue or 4 T/A litter on corn. These findings have been passed on to producers and other researchers; b) A phytoremediation strategy to deal with the excess soil P problem will be the outcome of this research after a few years; c) A presentation at the Kentucky Dairymans conference provided information to over 100 dairy industry persons about MAP and how we are dealing with the disease and its eradication. We have also made several farm visits to discuss with farmers their manure management issues. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Gilfillen, R.A., Willian, W.T, Henderson, H. 2004. Soil Nutrient
Accumulations in Corn Soil as Influenced by Poultry Litter Application Rate (4241). Proc. American Society of Agronomy Annual Meeting. Nov. 6-10. Seattle, WA. Henderson, H., Gilfillen, R.A., Sleugh, B.B., Willian, W.T. 2004. Available N and P in Orchardgrass-Alfalfa soils following poultry litter application. Proc. American Society of Agronomy Annual Meeting. Nov. 6-10. Seattle, WA. Sleugh, B.B., Gilfillen, R.A., Willian, W.T., Henderson, H., Embrey, D., Simmons, J. 2004. Nutrient Uptake and Forage Quality of SorghumSudangrass Under Different Poultry Litter Fertility Programs. Proc. American Society of Agronomy Annual Meeting. November 6-10. Seattle, WA. Willian, W.T., Gilfillen, R.A., Sleugh, B.B, Sistani, K.R., Henderson, H. D. 2004. Soil nutrient accumulation and field corn yield as influenced by poultry litter application rate. Proc. American Society of Agronomy Annual Meeting. Nov. 6-10. Seattle, WA. Sleugh, B.B., Gilfillen, R.A., Willian, W.T., Henderson, H.D. 2005. Poultry
Litter Rate Study in Tall Fescue. Proc. American Forage and Grassland Council Annual Meeting. June 11-15. Bloomington, IL. Whitely, N. R., Ozao, R., Wu, C.H., Chen, D., Pan, W.P. Solving the Chicken Litter Problem: Development of Novel Practices for Chicken Litter Management and Disposal, Proceedings, 35th Mississippi Water Resources Conference and 2nd Symposium on Safe Management and Utilization of Animal Waste. Jackson, MS. April 26-27, 2005. Whitely, N.R., Ozao, R., Wu, C.H., Chen, D., Pan, W.P. Combustion Behavior of Chicken-litter Coal Blends, March 15-17. Prepr. Pap.-Am. Chem. Soc. Div. Fuel Chem. 2005. 50(1):249. Sharma, N. C., Sahi, S.V. Strategy for phosphate phytoremediation. Proceedings of the 4th International Phosphorus Workshop (Eds. Chardon, W. J. and Koopmans, G.F. Wageningen. The Netherlands. 2004. Britt J, Pike A, Cook K. The Western Kentucky University Johnes Eradication Program, Proceedings Kentucky Dairymans Conference. March 2005. P 25-32. Mason, B. P., Vadari,
Y., Doerner, K.C. 2005. Characterization of Phosphate Hyper-Accumulating Staphylococcus sp. Isolated from Poultry Litter. In Abstracts of the 105th General Meeting of the American Society for Microbiology. American Society for Microbiology. Washington D.C. Groves, C, Bolster, C., Meiman, J. Spatial and Temporal Variations in Epikarst Storage and Flow in South Central Kentuckys Pennyroyal Plateau Sinkhole Plain. USGS Karts Interest Group Conference Proceedings. 2005. Accepted 7/2005. Ozao, R., Okabe, T., Arii, T., Nishimoto, Y., Cao, Y., Whitely, N., Pan, W.P. TGA/DTA/GC-MS Study of Odorless Woodceramics from Chicken Wastes. Journal of Thermal Analysis and Colorimetry. 2005. 80:489-493. Sharma, N. C., Sahi, S.V. Characterization of phosphate accumulation in Lolium multiflorum for remediation of phosphorus-enriched soils. Environmental Science and Technology. 39:5475-5480. 2005. Doerner, K.C., Mason, B.P. 2005. Nutritional Stress Increases Intracellular Phosphate and Polyphosphate in
Poultry Litter Microflora. [Accepted for publication 25 June 2005] Lett. Appl. Microbiol. (LAM 2005- 0506.R1)
Impacts
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Progress 10/01/03 to 09/30/04
Outputs
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? Utilization of poultry litter to provide optimum crop quality and yield while reducing potential soil phosphorous, copper, and zinc accumulations (accumulations relating to greater probability of water and soil pollution). Establishment of field studies to assess the impact of various levels of nutrient input on soil nutrient accumulation, forage nutrient uptake and forage quality. Impact of annual poultry litter application on crop yield and soil nutrient content in a corn monoculture. Problems related to excess phosphorus (P) in soils are being addressed using a plant-based bioremediation (phytoremediation) technology. The problem is remediation of phosphates in poultry waste. Research also was conducted on the possibility of using normal poultry litter microflora to absorb and maintain high
intracellular levels of phosphate thereby reducing the amount of phosphate available for immediate run-off into local watersheds. Number of broilers produced in Kentucky increased 142% from 1997 to 2002 (Kentucky Agricultural Statistics, 2002). Since the majority of production is occurring in the western third of the state, this presents challenges with respect to environmentally responsible management of litter in a restricted geographical region. These challenges are important to the producers, community stakeholders, and regulatory and conservation agencies. Long-term application of chicken litter and other animal manures in soils has accelerated P movement leading to a large-scale eutrophication of water bodies. Recent emergence of Pfiesteria spp. from P-enriched water has caused a serious human health concern. Furthermore, deterioration in water quality seriously impacts fish and other aquatic life forms. Various methods using different soil amendments are being tried to
control P loss from soil via runoff and leaching, but these methods are not free from drawbacks. Thus, plant-assisted remediation of excess P was researched in this project. The problem directly relates to the National Program for Natural Resources and Sustainable Agricultural Systems, and Manure and Byproduct Utilization (206). 2. List the milestones (indicators of progress) from your Project Plan. a) Two manuscripts in the area of poultry litter nutrient uptake were submitted for publications, b) Lachat and NIR instruments are in place, which strengthen the research laboratory capability for chemical analysis of soil, plant, and manure analyses, c) annual ryegrass cultivars marshall and gulf were grown in P-enriched soils in greenhouse under different cultural conditions. Alterations in culture conditions included: soil pH, atmospheric temperature, mode of planting (seed sowing vs. transplantation), micorrhizal inoculation and number of clippings, d) The capacity of poultry litter
microflora to increase phosphate up-take and increase intracellular phosphate stores in response to nutritional stresses was studied. 3. Milestones: Six presentations and three publications were made from this research project. 4. What were the most significant accomplishments this past year? A. Orchardgrass Study - High rates of poultry litter, inorganic fertilizer or lower rates of poultry litter supplemented with inorganic N seem to have similar yields. Addition of supplemental inorganic fertilizer has not consistently improved forage yield. After 4 consecutive years of high poultry litter application rates, significant soil accumulations of P, Cu, and Zn have occurred in comparison to low poultry litter application rates. B. Sorghum-Sudangrass Study - Low rates of poultry litter had the lowest concentration of Cu and Zn in plant tissue. High rates of poultry litter had similar plant Cu concentrations to inorganic fertilizers and low rates of poultry litter supplemented with
inorganic N. Low rates of poultry litter application resulted in lower crude protein concentrations. After 3 consecutive years of poultry litter application, low poultry litter application rates result in less soil accumulations of P, Cu, and Zn compared to high rates of application. However, accumulations are much lower in this study in comparison to the orchardgrass study due to lower rates of application and annual tillage. C. Carbon Sequestration Study - After 3 consecutive years of application, fertility treatments have resulted in statistically equivalent grain yields. Soil accumulations of P and Zn have been numerically highest in the full manure rate but not statistically different from the half manure/half inorganic fertilizer treatment. Full manure rates of application have resulted in significantly higher P, K, and Zn than plots treated with inorganic fertilizer. D. Litter Rate Study (Corn) - Broiler litter applied at greater than or equal to 9 Mg/ha: (a) produced grain
yield equivalent to plots receiving inorganic fertilizer and (b) resulted in higher available soil P, K and PO4-P than other treatments. E. Phytoremediation uses hyperaccumulator plants or those accumulating substantial amounts in their aerial parts. Thus the screened plant: annual ryegrass cultivars marshall and gulf were grown in P-enriched soils in greenhouse under different cultural conditions. Alterations in culture conditions included: soil pH, atmospheric temperature, mode of planting (seed sowing v/s transplantation), micorrhizal inoculation and number of clippings. Under certain conditions, these grasses accumulate P approximately 1% shoot dry weight with productivity of satisfactory biomass. It is believed these crops can accumulate >1% shoot P under optimal conditions. Also, genomic analysis of Gulf ryegrass, subtraction cDNA library (under P sufficiency and deficiency) of gulf ryegrass was prepared. The technique of subtractive hybridization was employed to obtain clones
of genes that are expressed in one population (P+) but not in other (P-). A total of 513 clones of P+ and 495 clones of P- were obtained. A large number of P+ and P- clones showed significant homology with genes from the grass family (mainly Oryza sativa). These genes (P specific) will now be evaluated for expression analysis with the help of Oryza sativa EST. F. The capacity of poultry litter microflora to increase phosphate up- take and increase intracellular phosphate stores in response to nutritional stresses was studied. The results indicated deprivation of poultry litter microflora of a carbon and energy source increases total intracellular phosphate and polyphosphate by 38% and 50%, respectively. Nonpathogenic bacteria that occur naturally in poultry litter which hyper- accumulate phosphate were isolated. These Staphylococcus sp strains display roughly a 6-fold increase of total intracellular phosphate and a 4-5-fold increase in intracellular polyphosphate when exposed to
0.75 M potassium phosphate. We are currently conducting further studies for identification of these organisms as well as determination of conditions which yields maximum phosphate absorption. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Demonstrated the potential alternative management regime of utilizing poultry litter which may reduce nutrient buildup in soil but provide comparable crop yield and quality to higher poultry litter rates. Some of the potential plants to be applied for phytoremediation have been identified. They should accumulate increased concentrations of P in their aerial tissue. Thus, they are expected to control P loss through run off or leaching when grown in P-enriched soils. Suitable environmental conditions wherein the selected crops can accumulate and remove P from soil have been initially identified. Under determined optimal conditions, these plant agents are believed to function
efficiently. P+ and P- genes from Gulf ryegrass have been identified for the study of expression analysis. Efficiency of remediation technology can be improved with the knowledge gained from P+ gene expression pattern. 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? Field Day: Agronomy Tour for University of Kentucky Agronomy Personnel - August 13, 2004. A part of this technology may be transferable in a few years but the genetically engineered product will take longer time before it can be used by the end-users. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Presented the following papers at the American Society of Agronomy Meetings - Oct. 31-Nov. 4, 2003 -
Denver, CO: Nutrient Composition of Orchardgrass with Different Poultry Litter Fertility Treatments. Authors: B. Sleugh, D. Embrey, R. Gilfillen, W. Willian. Influence of Poultry Litter Application Rate on Field Corn Stover and Grain Yields. Authors: W.T. Willian, R.A. Gilfillen, B.B Sleugh, and K.R. Sistani. Poultry Litter Application Rates Influence on Soil Nutrient Management. Authors: R.A. Gilfillen, B.B. Sleugh, W.T. Willian, and D. Embrey. Presented the following papers at the Annual American Forage and Grassland Council Meeting - June 12-16, 2004 - Roanoke, VA: Nutrient Uptake and Yield of Sorghum-Sudangrass Fertilized with Poultry Litter Authors: D.L. Embrey, B.B. Sleugh, R.A. Gilfillen, and W.T. Willian. Utilizing Poultry Litter in a Fertility Program for Orchardgrass Hayfields. Authors: Byron Sleugh, D. Embrey, W.T. Willian, and R.A. Gilfillen. Baum, J. 2004. A whole lot of chicken.....whatever. The Western Scholar, Western Kentucky University Press, Vol. 4. Spring
Edition. Vadari, Y. and K.C. Doerner. 2004. Isolation of Phosphate Hyperaccumulating Bacteria from Poultry Litter. In Abstracts of the 104th General Meeting of the American Society for Microbiology. American Society for Microbiology, Washington D.C. Scientific Publications: B. Sleugh, D. Embrey, W.T. Willian, and R.A. Gilfillen. Utilizing Poultry Litter in a Fertility Program for Orchardgrass Hayfields. Proc. AFGC Meeting, 13, p. 285 289. June 12-16, 2004, 2004, Roanoke, VA. Sharma, N.C., Sahi, S.V., Jain, J.C., Raghothama, K.G. 2004. Enhanced accumulation of phosphate by Lolium multiflorum cultivars grown in phosphate-enriched medium. Environmental Science and technology, 38, 2443- 2448. Sharma, N.C., Jain, J., Sahi, S.V. 2003. Evaluation of phosphate phytoremediation potential of ryegrass. Proceedings of 2nd International. Agronomy Congress: Balancing food and environment security, New Delhi, India.
Impacts
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Publications
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