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:
This project was initiated in July of 2016. The following report is for the second full year of research on this project (June, 2017 to June, 2018). Fourteen manuscripts detailing work associated with this project were published over this time. For Sub-objective 1A, a greenhouse study was completed and the soil metabolome analyzed using a refined soil extraction method where the volatile salt 0.25 M ammonium bicarbonate was included to enhance extraction of metabolites. This refined extraction procedure was used to successfully test the following hypotheses: (1) bulk soil metabolomic profiles are affected by soil type, (2) rhizosphere soil metabolomic profiles are affected by soil type, and (3) rhizosphere soil metabolomic profiles are impacted by crop species. This greenhouse study was also used to demonstrate that this refined extraction method, incorporating the volatile salt in the procedure, could be used for both metabolomic and microbial structure analysis of soil samples. A paper detailing this work is being finalized for submission to Applied Soil Ecology. For Sub-objective 1B we successfully completed sampling and data analysis regarding the impact of cropping systems diversity on the soil microbial community structure. The resulting dataset (not yet publicly available) is one of the most extensive assessments of fungal and bacterial rRNA to date within replicated field plots where cropping system impacts are compared. Two manuscripts are being prepared that describe this work. In work associated with Sub-objective 1B, and in cooperation with scientists at the Chinese Academy of Agricultural Sciences, Beijing, pot studies were completed demonstrating the impact of the phosphate-solubilizing fungus Aspergillus aculeatus P93 on soil available phosphate and promotion of maize growth in different soils. DGGE-based analysis of maize rhizosphere demonstrated alterations in microbial community structure due to the introduction of isolate P93. This study highlighted the impact of plant-beneficial microbes on the soil microbial community and the possibility that this altered soil microbial community may contribute to maize growth promotion. A paper was published detailing this work. A researcher on this project is co-PI on the funded NSF research coordination network grant (award number 1714276) RCN: AgMicrobiomes: An Interdisciplinary Research Network to Advance Microbiome Science in Agriculture. The goal of this network is to bring together researchers from around the world who conduct microbial research within agricultural systems. Progress was made on research under Objectives 2 and 3 but slowed due to the lack of production of manure solids and separated manure by the BARC dairy. We determined the influence of amendments and amendment ratios on drying rates of a biosolids product under composting conditions. We also evaluated a relatively low-cost anaerobic digestion system that has significant potential for use on small farms. Results indicated that anaerobic digestion using this system design removed 70% of monensin, an ionophore antibiotic used widely in animal husbandry. Levels of monensin in residues did not affect the anaerobic digestion process. However, methane production was reduced by 74% at 10 ppm monensin. This level of monensin is not typically found in dairy manure. Anaerobic metabolism was unaltered. For Sub-objective 4A, a refined multiresidue extraction and analytical method for 27 of the original proposed 53 compounds was developed. This refined method was tested across six wastewater treatment plants to determine the effectiveness of the waste treatment processes at these plants. Sixteen compounds were detected at least once at these waste water treatment plants demonstrating the effectiveness of this refined method. A manuscript detailing this work is under review at the journal. Also for Sub-objective 4A, the new combined thermal hydrolysis/anaerobic digester system (CAMBI) was compared with lime stabilization methods for degradation of residues using 2 bacteriostatic compounds and 4 phthalate plasticizers which are common in treated biosolids. Results were mixed using the newer, improved CAMBI system. The CAMBI system improved biosolid quality but resulted in increased concentrations of one of the bacteriostats and all the platicizers. Calculations of predicted environmental concentrations for soil receiving these biosolids suggested that concentrations were well below any level where toxicity might occur. For Sub-objective 4B, Task 1, some progress was made. Extraction and detection methods for artificial sweeteners were improved. Detection limits were lowered and results among replicates more consistent than with the old method. For Objective 4B, Task 2, the MESA-based age-dating method was compared with the old USGS dating method using the Bucks branch watershed in Delaware. Results indicated that the MESA-based age-dating method was comparable to the established USGS method for age-dating groundwater. This MESA-based method will also allow point-source tracking in streams, which is currently not possible.
1. Characterizing soil nematode biology. Communities of soil nematodes impact ecosystem functions and vital processes in agriculture, including plant growth, nutrient cycling, and soil health. Understanding nematode communities through identification and characterization has been hampered by the need to physically isolate nematodes and subsequently use a microscope to identify nematode species by eye and expert opinion. We developed a DNA meta-barcoding technique that utilizes indicator gene sequences to rapidly identify and characterize nematode communities without bias. This DNA meta-barcoding technique allows molecular ecologists, who do not have expertise in nematology, to use standard high throughput sequencing techniques to assess the community structure of this critical group of soil dwelling organisms.
2. Extraction method for soil microbial community analyses. Characterization of the collection of chemicals (metabolites) in soil contributes to our understanding of how the functions of soil microorganism communities are impacted by agricultural management practices. This information is central for sustainable agriculture. ARS scientists in Beltsville, Maryland, optimized a metabolite extraction method to increase the number of metabolites pool extracted from soil. Modification of the extraction method also allowed for the isolation and analysis of distinct biologically important lipids called phospholipid fatty acids that can be used to characterize the microorganism community makeup in the soil. This work has provided scientists an improved method with increased effectiveness for metabolite analysis in soil that saves time, reduces analysis cost and enables the characterization of soil microorganism communities.
Kim, E.J., Lee, S.H., Jo, H.S., Jeong, J.H., Mulbry III, W.W., Rhaman, S., Ahn, H.K. 2018. The effect of moisture content on solid-state anaerobic digestion of dairy manure from a sawdust-bedded pack barn. Energies. 11(3):484.
Lozano, N., Rice, C., Ramirez, M., Torrents, A. 2018. Fate of triclocarban in agricultural soils after biosolid applications. Environmental Science and Pollution Research. 25:222-223.
Mulbry III, W.W., Selmer, K.J., Lansing, S. 2017. Influence of liquid surface area on hydrogen sulfide oxidation during micro-aeration in dairy manure digesters. PLoS One. 12(10):e0185738. https://doi.org/10.1371/journal.phon.0185738.
Armstrong, D.L., Lozano, N., Rice, C., Ramirez, M., Torrents, A. 2018. Degradation of triclosan and triclocarban and formation of transformation products in activated sludge using benchtop bioreactors. Environmental Research. 161:17-25.
Arikan, O., Mulbry III, W.W., Rice, C., Lansing, S. 2018. The fate and effect of monensin during anaerobic digestion of dairy manure under mesophilic conditions. PLoS One. 13(2):e0192080. https://doi.org/10.1371/journal.pone.0192080
Kangas, P., Mulbry III, W.W., Klavon, P., Laughinghouse, D., Adey, W. 2017. High diversity within the periphyton community of an algal turf scrubber on the Susquehanna River. Ecological Engineering. 108:564-572.
Treonis, A.M., Unangst, S., Kepler, R.M., Buyer, J.S., Cavigelli, M.A., Mirsky, S.B., Maul, J.E. 2018. Characterization of soil nematode communities in three cropping systems through morphological and DNA metabarcoding approaches. Scientific Reports. 8:2004. https://doi.org/10.1038/s41598-018-20366-5.
Davis, B.W., Mirsky, S.B., Needelman, B.A., Cavigelli, M.A., Yarwood, S.A., Maul, J.E., Bagley, G.A. 2016. A novel approach to estimating nitrous oxide emissions during wetting events from single-timepoint flux measurements. Journal of Environmental Quality. 46:247-254.
Yin, Z., Fan, B., Roberts, D.P., Chen, S., Shi, F., Buyer, J.S., Jiang, H. 2017. Enhancement of maize growth and alteration of the rhizosphere community by phosphate-solubilizing fungus Aspergillus aculeatus P93. Journal of Agriculture Biotechnology. https://dx.doi.org/10.20936/JAB/170201.