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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Sustainable Agricultural Systems Laboratory » Research » Research Project #440891

Research Project: Soil, Crop, and Manure Biochemistry and Molecular Ecology: Bridging Knowledge Gaps in Microbiome Response to Management and Climate Change

Location: Sustainable Agricultural Systems Laboratory

2023 Annual Report

Objective 1: Determine the dispersal and activity patterns of fungi, bacteria and archaea with depth and across environmental gradients in agricultural systems and determine their impacts and influence on soil organic matter sequestration to inform better soil health management decisions. Objective 2: Develop a quantitative understanding of the impact of crop Genetics x Environmental context x Management strategies (G x E x M) on crop productivity as influenced by enhanced biological nitrogen fixation (BNF) and a fuller understanding of the soil and plant - microbiome symbiosis in leguminous cash and cover crop systems at local, long-term study sites and through LTAR collaborations. Sub-objective 2A: Study LTAR sites where legumes are grown in rotation with commodity crops to determine the factors that control regulation and efficiency of BNF and the net contribution of BNF nitrogen (N) to agroecosystems. Evaluations of the BAU and ASP cropping systems will be conducted. Sub-objective 2B: Establish fundamental understanding of BNF in the context of plant genotype by environment interactions in the commodity crop cowpea, Vigna unguiculata, and Soybean, Glycine max. Sub-objective 2C: Develop a standardized protocol for portable and low entry cost DNA sequencing platforms to evaluate critical sources of variability and error in analyses of biological transformations of soil carbon(C) and N. Objective 3: Assess thermal and anaerobic treatment processes of manure and in water resource recovery and treatment to reduce antibiotics in wastewater streams and develop effective approaches for treatment and monitoring materials of concern. Sub-objective 3A: Measure antibiotic removal during anaerobic processing of dairy manure and biosolids with small and large-scale processing methods. Sub-objective 3B: Develop protocols for anti-microbial gene detection in agricultural systems consistent with current recommendations from the EPA and One Health Initiative. Objective 4: Improve the ability to track the loading of nitrate from agricultural sources by using time dated metabolites of metolachlor to address N management strategies and to improve environmental and water quality. Sub-objective 4A: Redesign sampling and analysis protocols for metolachlor ethane sulfonic acid (MESA) to include metolachlor oxanilic acid (MOXA) for collection and analysis of stream water as a tool to track nitrate sources from groundwater. Sub-objective 4B: Determine isomer composition of both MESA and MOXA in watershed networks in order to describe groundwater nitrate loading from agriculture sources.

A molecular ecological approach will be taken to bridge gaps in understanding of biogeochemical stocks and flows in agroecosystems. Using classic chemistry, metabolomics, molecular biology, and plant physiology for analysis of samples from different cropping systems at the Farming Systems Project site critical issues in soil carbon sequestration and soil enzyme activity, and plant-microbiome interactions in relation to nitrogen fixation in legumes will be addressed. The Farming System Project in Beltsville, MD is part of the LTAR network and is a platform for comparison of long-term impacts of five cropping systems (conventional chisel till, conventional no-till, and three organic crop production rotations) commonly used in the Mid-Atlantic region of the US and elsewhere. New techniques will be developed to investigate how to improve manure anaerobic digestion systems for increased degradation of antibiotics and other compounds of concern in animal production waste streams to minimize the effect of their release into the environment. This research will also leverage the development of novel, passive sampling devices that detect breakdown products of the pesticide metolachlor as a surrogate for nitrate release from crop production fields. This improved technique will allow quantification of conservation practices directed towards reduction of agricultural waste in the nation’s water resources. In considering the connectivity and entirety and outcomes of the efforts of this project, this project will develop best management practices that improve water resources and soil quality in the Mid-Atlantic region helping to improve the sustainability of small, mid-sized, and large farms.

Progress Report
This report covers year two of this project. All 24-month milestones were met, and nine publications submitted. The recently hired project scientist was integrated into daily research activities as well as project stakeholder and collaborator groups. The new scientist completed a hiring search for a Physical Science Technician. The successful candidate has advanced experience in geochemical and agricultural systems, enhancing research capabilities. Project scientists served on the SciNet Scientific Advisory Committee and lead the Long-Term Agroecosystem Research (LTAR) Soils Working Group. The Soils Working Group created a common language data inventory of soils data from all LTAR sites and developed a web-based tool that enables users to harmonize datasets. This web-based tool is paired with a protocols and attributes tool that collects meta-data on protocols and procedures. Both tools will be utilized for the LTAR common experiment. Project scientists also provided leadership for the nationwide Soil Biology Network (SBGx). A project scientist served as scientific advisor to the NPS Microbiome Working Group and author on two budget initiatives submitted to REE. One initiative outlined an Agency microbiome research road map. The other initiative was in response to Executive Order 14081, Advancing Biotechnology and Biomanufacturing Innovation for Sustainable, Safe and Secure American Bioeconomy. For Objective 1, project scientists completed analysis of soil microbial and biochemical indicators of nitrogen fixation. Primers were designed to target genes contained within the nifH operon which is responsible for biological nitrogen fixation. In collaboration with the University of Maryland, relationships among common phylogenetic marker genes (16S and ITS rRNA) and specific functional genes involved in soil nitrogen and carbon cycling (nifH, nosZ, nirK, lacZ, lcc1) were explored. Predictable relationships among these genes were shown that differed with cropping system and the presence of the herbicide glyphosate. These relationships will be used to predict more accurately the impact of cropping systems on microbial soil nitrogen and carbon cycling. For Objective 2, improvement of protocols for cross-site comparison of soil biological attributes continued. Soils collected from Jornada, New Mexico, Pendleton, Oregon, Ft. Collins, Colorado, Columbus, Ohio, Ithaca, New York, and Beltsville, Maryland. were extracted and assessed for DNA quality and quantity using common protocols. These soils represent the regional diversity of LTAR Network agroecosystems. High-throughput, multiplexed-amplicon sequencing pipelines were developed to compare microbiomes based on relative abundance of assigned sequence variants (ASVs). This was achieved via creation of the app, Qseq, that performs statistical analysis and synthesis of multiplexed-amplicon-sequencing raw data and outputs formatted data for use in complementary downstream algorithms. This freely available app and other Scinet-based bioinformatic tools were used to complete two studies within the Farming System Project (FSP) LTAR site. One study showed that the herbicide Glyphosate does not impact the microbial community structure at plot scale but may impact some microbial functions, such as nitrogen fixation, under some conditions. The second study demonstrated that 26 years of farming systems management resulted in differences in microbial community structure and function corresponding to system scale differences at 0 to 15 cm soil depth. Below 15 cm depth soil type and physiochemical attributes were more predictive determinants of soil microbial community than farming system.The use of synchrotron µ-X-ray diffraction provided by the Brookhaven National Laboratory enabled project scientists to identify the distribution of minerals within manganese and iron hard concretions for the first time. These concretions are common in soils and accumulate substantial quantities of trace elements, including rare earth metals and plant nutrients, where they are removed from the bulk system. Furthermore, manganese is a powerful oxidizing agent in soils that can polymerize organic matter and alter contaminant and nutrient cycling. This study increased our understanding of natural forms of manganese and how it may impact critical geochemical cycles. For Sub-objective 3A, a new method to extract and quantitate antibiotics from various stages of processed and unprocessed manure was finalized. The resulting extract is subsequently cleaned and concentrated using solid phase extraction. The concentrated extract is then analyzed by use of this method has been validated for 10 antibiotics from four antibiotic classes. A manuscript detailing this method has been published. This new extraction method for antibiotics was applied to samples collected from a Bedding Recovery Unit (BRU) used to recover clean bedding from cow manure. Four manure types were sampled and extracted at sites along the BRU-processing line: unprocessed source material, a liquid fraction isolated by screw press separation, and the remaining solids from the screw press both before and after rotary drum heat treatment. Three antibiotics were detected in the manure samples: tetracycline, tulathromycin, and penicillin-G. Antibiotic concentrations ranged from 0.436 – 4.10 µg/kg. With animal-hospital farm manure containing incurred antibiotics, mass flow analysis of the sequential processing was determined by including corn kernels that followed the manure as it moved through this BRU device. Calculated mass flow rates indicated that 95% of the manure mass was fractioned with the separated liquid fraction. The remaining 5% of the manure mass was in the separated solid faction which contained 11% to 20% of tetracycline and tulathromycin antibiotics. No significant reduction of either antibiotic was found following BRU processing of the separated solids. Biochemical Methane Potential (BMP) testing was carried out at each BRU process sampling site to see if subsequent anerobic methane recovery caused destruction of antibiotics. Such methane recovery practices are popular on many dairy farms. Samples were spiked with oxytetracycline, ampicillin, and erythromycin and sampled and analyzed at five timed intervals up to 43 days. There was a significantly higher decrease in oxytetracycline with the heated versus the room-temperature incubations. Complete destruction of erythromycin and ampicillin occurred at room temperature and not under heated incubations. A lab-scale anaerobic digestion experiment was conducted to monitor antibiotic degradation within dairy manure based on temperature. Three antibiotics (oxytetracycline, erythromycin and ampicillin) were spiked into manure and monitored throughout a 43-day digestion under mesophilic (35°C) and thermophilic (55°C) conditions. Ampicillin was almost completely removed under both mesophilic and thermophilic conditions, while erythromycin was completely degraded (100%) under mesophilic conditions by day 36 and oxytetracycline had greater degradation under thermophilic conditions. Both conditions achieved > 30% degradation across all antibiotics throughout the digestion period. For Sub-objective 3B, there was a significant reduction in viable antibiotic-resistant bacteria in solids recovered from BRU-processed bedding material and complete elimination of pathogens. Solid, liquid separation prior to BRU heat processing, however, resulted in most of the mass staying with the separated liquid located in a storage lagoon; this material continuing to be a risk when released to the environment. Both mesophilic and thermophilic anaerobic treatment (BMP) of the final BRU solids were effective at eliminating spiked fecal indicator species (Enterococci and E. coli) by Day 9. Gene sequencing of the 16S ribosomal RNA gene is under way for comparative analysis of bacterial community diversity following the 43-day digestion of the final bedding material. Quantitative PCR (qPCR) was used to determine the relative gene abundance of eight antibiotic resistance genes (ARGs) and the 16S gene in samples from BRU and anaerobic digestion experiments. Genes included macrolide (ermB), beta-lactam (bla-2), sulfonamides (sul-1), tetracycline (tetX, tetM, tetW, tetQ), mobile genetic element (intl1), and 16S DNA for measuring bacterial populations. BRU processing resulted in significant reductions (>95%) of intl1, sul1, tetQ, tetX, and tetM while tetW, ermB and bla2 were unaffected. Thermophilic conditions had significantly lower bacterial populations compared to mesophilic conditions and both conditions reduced ARGs with specific gene reduction being temperature and condition dependent. There was evidence for some ARG enrichment throughout digestion under both conditions. For Sub-objective 4A, trials comparing approved MESA (metolachlor ethane sulfonic acid) extraction methods for the extraction of MOXA (metolachlor oxanilic acid) were completed and a methods paper published. MESA and MOXA were investigated as markers for monitoring nitrogen loss from agricultural fields. MESA and MOXA are dominant soil degradation products of the herbicide metolachlor. For Sub-objective 4B, chemical separation of MESA and MOXA was improved to allow separation of the four significant trans-isomers of both MOXA and MESA. Samples from the Choptank River Watershed were analyzed to quantitate and compare the four isomers of MESA and MOXA over three years (2007 to 2009). Lag-time calculations showed that MOXA is less persistent than MESA as a dating marker for groundwater transport of nitrogen. At the isomer-specific level, variations of the isomer pairs were closely linked to hydrologic variations which will allow development of predictive models describing flow paths for nitrogen based on soil properties. A manuscript on these results has been published.

1. Soil depth is a more significant determinate of soil microbial community than farming system below 15 cm. The soil microbial community is important in agriculture as it functions in transformation and storage of soil carbon and plant nutrients. Models of stocks and flows of carbon and availability of nutrients currently use outdated estimates of soil metabolic kinetics; these estimates do not consider how farming systems regulate and impact transformation of carbon and nutrients. USDA-ARS scientists in Beltsville, Maryland, extracted and analyzed DNA and RNA from fungal, bacterial, and archaeal communities in 1-meter-deep soil cores from three different farming systems at the 26-year-old Farming Systems Project in Beltsville, Maryland. Results showed differences in soil microbial community at 0 to 15 cm among the three different farming systems. Below 15 cm depth, soil type and physiochemical attributes were more predictable determinants of soil microbial community than farming system. Results from this study are important to scientists and will be used to improve models of soil carbon dynamics which are needed to improve soil carbon sequestration and plant nutrient availability assessments.

2. Glyphosate application does not significantly alter microbial community structure but can impact certain traits. The impacts of the herbicide glyphosate on the plant and soil microbial community structure are unclear and there are concerns regarding non-target ecosystem effects. A variety of farming systems styles using Round-up Ready® corn and soybean at three USDA-ARS locations nationally were analyzed for microbial community structure and function to resolve these issues. Glyphosate impacted nitrogen fixing metabolism, resulting in increased biological nitrogen fixation in some cropping systems and decreased nitrogen fixation in others. This indicates that herbicides can have non-target impacts on the soil microbial community involved important agroecological functions such as nitrogen fixation. Other findings, reported in a series of seven papers, were that in modern corn and soybean farming operations use of glyphosate does not significantly change crop metabolic profiles or populations of pathogenic and endophytic fungi associated with the crop.

Review Publications
Fischel, M.H., Clarke, C.E., Sparks, D.L. 2023. Synchrotron resolved microscale and bulk mineralogy in manganese-rich soils and associated pedogenic concretions. Geoderma. 430: Article e116305.
Porell, M., Cushman, M., Fischel, J.S., Fischel, M.H., Sparks, D.L., Grayburn, R. 2023. Scanning x-ray fluorescence spectroscopy and micro-x-ray absorption near-edge structure analysis as a guiding tool for the conservation treatment of two eighteenth-century Philadelphian portraits. X-Ray Spectrometry. Article e3345.