1a. Objectives (from AD-416):
Objective 1: Analyze the effects of agricultural management systems on soil biodiversity and functions. Sub-objective 1.A: Validate combined soil PLFA/metabolomics method for analysis of microbial community structure and function. Sub-objective 1.B: Analyze soil microbial community structure, diversity, metabolomic profile, and functional diversity in different cropping systems. Objective 2: Develop improved management practices to reduce emissions of ammonia and greenhouse gases, and reduce pathogens during manure composting. Objective 3: Develop technologies and practices that improve anaerobic digestion of animal manures and other feedstocks, contributing to improved system economics, recovery of nutrients, and water quality. Objective 4: Improve the ability to quantify organic contaminants released during the process of water resource recovery, treatment, and reuse of resulting biosolids. Sub-objective 4.A. Use bioreactors as a model system to measure xenobiotic degradation losses using a newly implemented CAMBI system. Sub-objective 4.B. Study two specific soluble source tracers, artificial sweeteners for urban sources and metolachlor ethane sulfonic acid for agriculture sources, in order to model nitrogen loading to impacted watersheds.
1b. Approach (from AD-416):
We are developing a new method to simultaneously extract phospholipids and metabolites from soil. Phospholipid fatty acids are measured by gas chromatography while metabolites are measured by gas chromatography-mass spectrometry. Our methods will be tested in a greenhouse experiment using 3 soil types and 3 crop species. We will then analyze soil microbes and their activity using DNA sequencing, transcriptomics, metabolomics, and PLFA analysis in 3 different replicated field trials: the Beltsville Farming Systems Project (FSP), the Beltsville Cover Crop Systems Project (CCSP), and a cover crop field experiment at the ARS lab in Brookings, South Dakota (SD). Composting studies will be conducted at the BARC composting facility. Initial experiments will be conducted outdoors using replicate pilot-scale compost piles composed of manure and bedding from the BARC dairy. Subsequent large-scale experiments will be conducted using extended static piles and windrows of the manure/bedding. Experimental variables include aeration and compost covers. Gases and compost pile temperatures will be monitored. Results will be validated using large-scale extended static piles. Six identical pilot-scale anaerobic digesters will be operated using solids-separated manure effluent from the BARC dairy. For H2S removal experiments, duplicate digesters will be randomly assigned to one of three treatments: no air injection; air injection, low rate; air injection, high rate. Depending on the results, additional experiments may be conducted to evaluate other aeration rates or recirculation of biogas or manure in order to optimize H2S removal. Experiments during the second year of operation will evaluate manure pre-heating as a means to maintain digester temperature and improve overall energy use. The first task will be to establish analytical methods using liquid chromatography-mass spectrometry for up to 53 pollutants. Incubation studies of samples obtained at different stages in the CAMBI process will then be carried out. Based on these results, compounds that appear to be degraded will be singled out for separate individual incubation studies. The goal will be to better describe their degradation rates and formation of metabolites in the CAMBI system. An artificial sweetener will be used as a tool to track urban pollution from wastewater treatment plants while MESA will be used to track agricultural pollutants for rural sources. A liquid chromatography-mass spectrometry method for assessment of both MESA and the most easily detected sweetener will be developed. The second step will be to test the method by analyzing selected real samples. The final step will be to apply the method to base-flow fed streams in the Choptank and Bucks Branch watersheds in Delaware in order to measure groundwater residence times.
3. Progress Report:
With regard to Objective 1, progress was made on both sub-objectives. Under sub-objective 1A, greenhouse experiments were completed in order to validate a new method for combined phospholipid fatty acid and metabolite analysis. Analysis of the greenhouse samples is ongoing. Numerous chemical standards were analyzed in order to improve our ability to identify metabolites, and detection limits were improved. Several different experiments were carried out under sub-objective 1B. We are looking at the impact of plant-beneficial microorganisms on the indigenous soil microflora and plants in collaboration with scientists at the Chinese Academy of Agricultural Sciences (CAAS), Beijing and the CAAS, Wuhan. Work regarding the phosphate-solubilizing fungus, Penicillium oxalicum P8 was continued. This work showed that this fungus increased soil available phosphorus and maize fresh weight in several soils in greenhouse pot experiments. A gene was cloned from isolate P8 into Escherichia coli that enabled this bacterium to solubilize inorganic phosphorus in vitro. Follow up pot study experiments were conducted with a Trichoderma isolate, which altered soil enzyme activity in field trials, to look at the impact of this Trichoderma isolate on the soil microbial community. Additional research contributing to sub-objective 1B focused on analysis of the soil and root associated microbial community in conventional and organic small grain farming systems after exposure to glyphosate herbicide. We have found that plant associated microbial communities differ significantly by location (Beltsville, MD, Urbana, IL or Stoneville, MS). The historic legacy of farming system practices have more influence on the root and soil microbes than the application of glyphosate. In a related study, researchers on our team conducted phytopathological assessment of sweet corn in production systems with and without glyphosate. A separate study was focused on the fungal group Hypocreales which plays critical roles in both plant disease and plant health. Many of these fungi enhance plant uptake of soil nutrients such as phosphorus and nitrogen but understanding how their phylogeny relates to their functionality in the ecosystem remains difficult to discern. Using recent Hypocreales genomic data we determined that a more accurate arrangement of phylogeny could be created with multiple gene targets within the Hypocreales genomes. We suggested a new way to classify related Hypocreales fungi based on the combination of this multi-locus genomic information and functional phenotype. Progress was made under Objective 2 which is focused on reducing unwanted gas emissions during composting. Work on this project was delayed because biogas hydrogen sulfide treatment experiments took much longer than expected to complete. In addition, the BARC compost facility was not available for experiments because of needed site repair. Initial composting experiments using pilot-scale piles were initiated in June 2017. Progress was also made under Objective 3 which is focused on improving our basic understanding of anaerobic digestion. Under that objective, we successfully completed a study of a low cost method (micro-aeration) to reduce levels of hydrogen sulfide in biogas from anaerobic digesters. Progress was made on all sub-objectives under Objective 4. With regard to sub-objective 4A, after considerable study on 53 proposed compounds for extraction and analysis by LC/MS-MS methods, follow-up studies were carried out on subsets involving triclosan and triclocarban and their metabolites; and also studies on 4 phthalate plasticizers, diisononyl phthalate (DiNP), diisodecyl phthalate (DiDP), bis(2-ethylhexyl) phthalate (DEHP), and benzyl butyl phthalate (BBP). These studies compared the CAMBI process with the prior waste treatment process involving lime stabilization. Under sub-objective 4B, method development is underway and a revised method has been tested on field collected samples. Samples have been analyzed from two different field sites, Bucks Branch and Choptank, and the findings appear to be correlated with drainage types in the watershed.
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Kepler, R., Maul, J.E., Rehner, S.A. 2017. Managing the plant microbiome for biocontrol fungi: Examples from Hypocreales. Current Opinion in Microbiology. 37(1):48-53. doi:10.1016/j.mib.2017.03.006.
Armstrong, D., Rice, C., Ramirez, M., Torrents, A. 2017. Influence of thermal hydrolysis-anaerobic digestion treatment of wastewater solids on concentrations of Triclosan, Triclocarban, and their transformation products in biosolids. Chemosphere. 171:609-616.
Chang, C., Slavecz, K., Buyer, J.S. 2016. Species identities, not functional groups, explain the effects of earthworms on litter carbon-derived soil respiration. Soil Biology and Biochemistry. 100:129-139.