Location: Sustainable Agricultural Systems Laboratory
2021 Annual Report
Objectives
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.
Approach
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.
Progress Report
This is the final report for the project. Two scientists on this project retired during the project cycle, and one support scientist left ARS. One vacant scientist position and the vacant support scientist position were abolished. A candidate has accepted the informal job offer for the remaining vacant scientist position and is expected to start in mid-September 2021.
For Objective 1, a new method to simultaneously extract phospholipids and metabolites from soil was developed. With this method phospholipid fatty acids were measured by gas chromatography while metabolites were measured by gas chromatography-mass spectrometry. The method was tested in a greenhouse experiment using three soil types and three crop species, and the work describing this method was published in Applied Soil Ecology. In collaboration with ARS scientists in Stoneville, Mississippi, and Urbana, Illinois, we determined that applications of glyphosate on Round-up Ready® corn and soybean did not increase the abundance or risk of Fusarium Root Rot. Metabolic profiling of the grain showed that the glyphosate application did not change the amino acid profile. Results from this study were published in Pest Management Science and Applied and Environmental Microbiology. Also, for Objective 1, two manuscripts were published (Diversity, Soil Biology, and Biochemistry) on work performed with USDA-ARS collaborators in Beltsville, Maryland, and Stuttgart, Arkansas. This work was directed at determining the impact of rice cultivars on the associated soil microbial community. In the first study, the rice rhizosphere microbial community composition of nine recombinant inbred lines (RILs) and their parents was determined using metagenomic sequencing to examine how rice biomass influences the rhizosphere community. Species that correlated with increases in either root or shoot biomass were widely present in soil and included those involved in nitrogen cycling (Anaeromyxobacter spp.) and methane production (Methanocella avoryzae), as well as known endophytes (Bradyrhizobium spp.). In a second study with these collaborators, we sequenced the soil metagenomes associated with two rice RILs and their parents displaying a range of low to high methane emitting phenotypes. Methane emissions were low at booting and increased during heading and grain fill stages, where peak methane emissions were observed and returned to basal levels at maturity. In response to genotype and plant developmental stage, we observed changes in rhizosphere microbial community structure in several methanogenic archaea and bacterial methane oxidizers and sulfur cyclers. Rice genotype played a larger role in influencing the soil microbial community structure during the reproductive phases of booting and heading compared to the ripening phases (grain fill and maturity). This indicated that the reduced methane emissions trait was associated with small changes in the composition of methanogens rather than wholesale community shifts. This finding suggests that future plant breeding efforts can reduce methane emissions during high methane emitting phases (i.e., reproductive phases) by selecting rice genotypes with lower methanogen and higher methanotroph populations during these developmental stages. A manuscript is also in preparation on the impact of a Trichoderma mycoparasite applied in a biological fertilizer on the soil microbial community of the field crop oilseed rape. This work was performed in collaboration with scientists at the Oil Crops Research Institute in Wuhan, China.
In collaboration with scientists at the University of Maryland, we isolated and genotyped a novel dark-septate fungus associated with grasses and imparted varying degrees of salt tolerance, depending on host/endophyte genotypes. For this work, various marsh grasses were grown in native soils and subjected to a salinity gradient. From this process, we were able to use the plant's growth characteristics and associated endophytic fungi to identify fungal/plant pairs that resulted in improved salt tolerance by the plant. This work was published in The ISME Journal.
Over the course of this project plan, novel and innovative techniques were developed that enabled analysis of difficult to analyze soil samples. This resulted in one of us becoming Co-Principal Investigator on a National Science Foundation (NSF) research coordination network that developed and published standards and technical bulletins for soil health and soil microbiology measurements (NRCS No. 450-03). This work is ongoing and evolving with support from the National Program Staff for building research networks such as the Soil Biology Group, Soil BiologyX, and the ARS-LTAR network.
Objectives 2 and 3 addressed the growing concern about residual antibiotics and feed additives in the manure of farm animals. Progress on Objectives 2 and 3 was slowed due to the retirement of the scientist working on this aspect of the Project, but there was progress with the treatment of monensin, the most widely used ionophore coccidiostat in the U.S. A relatively low-cost anaerobic digestion system that has significant potential for use on small farms in the Mid-Atlantic and Northeast Regions of the U.S. was evaluated. Results from experiments with these anaerobic digestion systems showed that small farms have an option for on-farm anaerobic digestion, which is an effective treatment for reducing, but not eliminating, monensin in dairy manure. New studies were begun to study the fate of other antibiotics in dairy manure. Clean manure spiked with fourteen likely veterinary drugs was used to optimize a screening method designed from published methods. This optimized screening method was used to study the occurrence and fate of these antibiotics using dairy manure from medicated cows. This dairy manure was treated with a commercial bedding recovery system (BRU) that employed heated anaerobic digestion to reduce pathogens and limit antibiotic carryover. After the operation of the BRU for 24 hours, a significant loss of two beta-lactam antibiotics (ampicillin, penicillin) was observed, but no other significant degradations occurred for three other antibiotics (tetracycline, epitetracycline, tulathromycin) detected in the manure. Tests are underway to examine existence and persistence of antibiotic resistance genes within the microbial community under different digestion conditions, using PCR to target key antimicrobial genes for ampicillin and tetracycline resistance.
In other work, the influence of moisture content on the digestion of bedded pack dairy manure using solid-state anaerobic digestion (SS-AD) was investigated. Mixtures of sawdust bedding and dairy manure were prepared with moisture contents (M.C.s) of 70, 76, and 83% and digested at 37°C for 85 days. The performance of digesters containing manure at 83% MC was 1.3 to 1.4-fold higher than that of digesters containing 70% MC manure in terms of volatile solids reduction and biogas production. In terms of efficient management of farm odors and providing a renewable energy source for heating, SS-AD of bedded pack manure offers a potential alternative to the conventional composting systems currently in use.
For Objective 4, a multiresidue method to detect 27 preselected contaminants was developed and tested on biosolids from six different waste-water treatment plants (WWTPs) located in the Mid-Atlantic region of the U.S.; four WWTPs utilized anaerobic digestion for sludge treatment, and two utilized aerobic processes for treatment. This multi-residue method had great utility for screening as all contaminants were detected with recoveries at 50% or greater in experiments using samples spiked with the contaminants. Additionally, 16 of these contaminants were detected in non-spiked samples from all WWTPs. In other work under Objective 4, phthalate residues in the biosolid were found to degrade more rapidly under aerobic conditions while the CAMBI™ thermal hydrolysis/anerobic digestion, a process run at one WWTP, caused concentrations to increase due to solids alterations. In separate experiments, bioreactor vessels were operated using CAMBI™-treated solids and screened for the known metabolites triclosan (TCS) and triclocarban (TCC). Denitrifying conditions in the bioreactors using CAMBI™-treated biosolids did not alter TCC amounts but did improve TCS removal.
Metolachlor ethane sulfonic acid (MESA) and metolachlor oxanilic acid (MOXA) are breakdown products of the commonly used herbicide Metolachlor® and have been shown to co-elute from soil profiles with nitrate into waterways. We published evidence from an 11-year study in the LCB LTAR Choptank watershed showing close association of MESA and MOXA with nitrate transport in a first-order stream that discharged a precision agriculture site. Correlation analysis determined a 0.9 correlation for the flux of both MESA and MOXA with the flux of nitrate-N, both for base flow and storm flow. Improved analytical methods were necessary to increase the resolution of detection of Metolachlor. Stable isotopes of the source Metolachlor allowed us in collaboration with USDA-ARS researchers at Beltsville, Maryland, to separate isomers of MESA and MOXA isomerically enriched with the s-isomer of metolachor. This application allowed collaborating USDA-ARS modelers in Beltsville, Maryland, to date MESA movement from stored groundwater to streams to account for the residual flow of MESA and MOXA from previous history and mixing of sources in the sub-surface water table. Reanalyzing these eleven years of Choptank sub-watershed samples with the stable isotope approach allowed excellent dating results for this experimental site. Dating allows the determination if different farming practices, such as conservation tillage, reduce the amount of nitrate-N being released into watersheds from farmlands.
Accomplishments
1. Cold temperatures limit biological nitrogen fixation by winter legume cover crops. Winter annual legume cover crops can reduce the need for spring nitrogen fertilization via biological nitrogen fixation. However, cold fall temperatures in the northern regions of the U.S. can limit root growth and symbiotic bacterial establishment, thereby reducing the biological nitrogen fixation of these crops. In a pair of studies aimed to examine effects of cold temperatures on nodulation and nodule microbiome compositions in legume cover crops, researchers at the University of Minnesota and ARS researchers in Beltsville, Maryland, found that the lower limits of nitrogen fixation in three common cover crops [Hairy vetch (Vicia villosa), Austrian winter pea (Pisum sativa) and Crimson clover (Trifolium incarnatum)] were different and all species failed to establish symbiotic relationships with bacteria below an ambient temperature of 10°C. This is critical information to estimate annual winter cover crop nitrogen contributions and will be of interest to farmers, scientists, and policymakers considering nitrogen balance in cropping systems.
Review Publications
Roberts, D.P., Short, N.M., Jr., Sill, J., Lakshman, D.K., Hu, X., Buser, M.D. 2021. Precision agriculture and geospatial techniques for sustainable disease control. Indian Phytopathology. https://doi.org/10.1007/s42360-021-00334-2.
Fernandez-Baca, C.P., Rivers, A.R., Maul, J.E., Kim, W., McClung, A.M., Roberts, D.P., Reddy, V., Barnaby, J.Y. 2021. Rice plant-soil microbiome interactions driven by root and shoot biomass. Diversity. https://doi.org/10.3390/d13030125.
Fernandez-Baca, C.P., Rivers, A.R., Kim, W., McClung, A.M., Roberts, D.P., Reddy, V., Barnaby, J.Y. 2021. Changes in rhizosphere soil microbial communities across plant developmental stages of high and low methane emitting rice genotypes. Soil Biology and Biochemistry. http://doi.org/10.1016/j.soilbio.2021.108233.
Moore, V., Davis, B., Maul, J.E., Kucek, L.K., Mirsky, S.B. 2020. Phenotypic and nodule microbial diversity among crimson clover (Trifolium incarnatum L.) accessions. Agronomy. 10(9):1434. https://doi.org/10.3390/agronomy10091434.
Vega, F.E., Emche, S.E., Shao, J.Y., Simpkins, A., Summers, R., Mock, M., Ebert, D., Infante, F., Aoki, S., Maul, J.E. 2021. Cultivation and genome sequencing of bacteria isolated from the coffee berry borer (Hypothenemus hampei), with emphasis on the role of caffeine degradation. Frontiers in Microbiology. 12:644768.
