Progress 04/01/16 to 03/31/20

Outputs
Target Audience:Our target audiences during the finalno-cost extended reporting period consisted of the following: 1)USDA-ARS scientists ofthe Pasture Systems and Watershed Management unitat University Park, PA.During the final one-year extendedperiod of this project, the Dairy Cropping System (DCS)experiment (previously funded by USDA-NESARE for nine years) transitioned to becoming one of the long-term experimental sites in the USDA-ARS Long-Term Agricultural Research (LTAR) network. Since soils of the DCS experimental plots have been the subject of our soil microbiology research, the LTAR group represents a new target audience. 2) Approximately 60 atendees of a Cover Crop Field Day at the Penn State Agronomy Farm, Summer 2019. Attendees learned about effects of cover crop management on potential differences in N2O and other GHG emissions. 3) Undergraduate and graduate students (22) enrolled in SOILS 412W in Fall 2019, who visited DCS plots and learned about effects of tillage, cover cropping, and manure management practices on GHG emissions from soils. Target audiences reached during the 2016-2019 period of the project were as follows: 1) Research and extension collaborators and students at Penn State who work on the Sustainable Dairy Cropping Systems project funded by Northeast Sustainable Agriculture Research and Education (NE-SARE). Our team communicated with the SDCS team on a regular basis to explain the objectives of the NIFA project and to coordinate plans for soil sampling. 2) Dairy farmers identified by Penn State Extension, as well as farmers and a farm consultant in the NESARE SDC Advisory Panel. We explained project objectives, asked for their feedback, and requested manure samples from their operations. 3) Soil scientists and agronomists attending symposia at two international conferences: American Society of Agronomy Symposium--Environmental Quality: A One Health Perspective, Nov 6, 2016, Phoenix, AZ; and American Geophysical Union Symposium--Coupling the Nitrogen and Carbon Cycles of Terrestrial Ecosystems: Understanding the Nexus between Land Management and GHG Mitigation Strategies I, San Francisco 12-16 December, 2016, San Francisco, CA. At two oral presentations, PD Bruns introduced the idea that manure application enriches soils with bacteria capable of converting nitrate to ammonium and that nitrate's alternative fate in agricultural soils could affect climate change through soil N dynamics. 4) USDA Project Directors at USDA-NIFA Agroclimatology Meeting, Dec 17-18, San Francisco, CA. PD Bruns and Postdoc Bhowmik attended the meeeting to explain the objectives of this project to other NIFA PDs working on climate change mitigation/adaptation in agriculture. 5) Undergraduate students in Environmental Resource Management and graduate students in Soil Science, Biogeochemistry, and Ecology who learn about nitrate ammonification as an understudied step in the nitrogen cycle during coursework and discussions during poster presentations and various research meetings on campus. 6) Policy makers, professionals and other individuals who attended our symposium, Soil Health, Microbiomes, and Climate- Adaptive Agriculture at the National Council for Science and the Environment, Washington, DC, Jan 24, 2017. Changes/Problems:The major change was in tasks carried out by personnel due to the departure of postdoc Arnab Bhowmik, who left Penn State in the summer of 2018 to take a faculty position. Bhowmik's samples were analyzed by graduate student Mara Cloutier and technician Tiffanie Alcaide. Minor changes are noted here regarding changes in the journals for reported publications: Although the following paper was initially submitted to Frontiers in Microbiology in 2018, it was resubmitted to Agricultural and Environmental Letters and published in 2019: Cloutier, M.L., Bhowmik, A., Bell, T.H., and Bruns, M.A. 2019. Innovative Technologies Can Improve Understanding of Microbial Nitrogen Dynamics in Agricultural Soils. Agricultural & Environmental Letters. 4:190032 doi:10.2134/ael2019.08.0032 2. Although the following paper was initially submitted to ISME Jin 2018, it was resubmitted to Scientific Reports and published in 2019: Cloutier, M.L., Murrell, E., Kaye, J., Barbercheck, M., Finney, D., Gonzalez, I.G., Bruns, M.A., Fungal assemblages and functions shaped by brassicaceous crops and soil texture in a multi-species cover crop experiment. Scientific Reports (2020) doi: 10.1038/s41598-020-63173-7 3.The following paper was initially submitted to Global Change Biology in April 2019, but was returned. It was resubmitted to Ecological Applications in July 2020 Saha, D., Kaye, J.P., Bhowmik, A., Bruns, M.A., Wallace, J.M., and Kemanian, A.R. Organic fertility inputs synergistically increase denitrification-derived nitrous oxide emissions in agroecosystems. Submitted July 13, 2020 to Ecological Applications in review. What opportunities for training and professional development has the project provided?Graduate student Mara Cloutier, who was supported on this grant, conducted research to determine field practices and conditions with potential to affect N2O emissions from denitrification. To that end, she assessed the relationships between field practices and soil conditions to the numbers of nrfA gene copies in soils, because the nrfA gene is a molecular marker for bacteria capable of converting nitrate to ammonium, As such, Cloutier sampled soils from two field experiments: a long-term tillage (LTT) experiment and the sustainable dairy cropping systems (SDCS) experiment led by coPI Karsten. In addition to analyzing soils from the field experiments, Cloutier, with the help of technician Tiffanie Alcaide, completed the work of postdoctoral associate Arnab Bhowmik to analyze nrfA Illumina amplicon sequencing data from Bhowmik's incubation experiment. Bhowmik left Penn State in August 2018 to become an assistant professor at North Carolina State A&M University. How have the results been disseminated to communities of interest?Results have been disseminated at the annual meetings of the TriSocieties (ASA-CSSA-SSSA) in the past three years. The potential effects of management practices on denitrification and N2O emissions have been communicated to the dairy farmer-cooperators in the SDCS (Sustainable Dairy Cropping Systems) Advisory Panel and to the three dairy farmers who provided access for sampling raw and digested manure for the incubation study. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? SDCS Experiment. Results were obtained to determine whether nrfA gene abundances were impacted by harvesting of a perennial alfalfa-orchardgrass (AO) mixture, which is a part of a no-till, six-year rotation, and to evaluate how nrfA gene abundances changed with termination timing. Harvest of the AO aboveground biomass occurs several times during the second year of growth. In the SDCS rotations, AO is either chemically terminated in the fall of the second year or in the spring of the third year. Rhizosphere soil samples were taken before and two, five, and eight days after harvest. In 2018 samples from two harvest events were collected and samples from three harvests were collected in 2019. Samples for comparing termination timing were collected before fall termination from both the treatments (i.e. fall or spring termination) and collected before spring termination. All samples were collected by taking 3, 10X10 cm soil cores from areas with AO aboveground biomass in each plot and brought to the lab. Roots were removed from the core and excess soil was removed from the roots. Roots were then washed; aliquots were transferred to microcentrifuge tubes and soil was pelleted. Composite samples were created from the three soil cores collected from each plot. Soil genomic DNA was extracted from the composite samples and qPCR was performed on the extracts to quantify nrfA gene abundances.Results from the linear mixed effect models from the harvests indicated a significant interaction between the sampling time and the harvest event. Comparisons between the before samples and two, five or eight days after harvest showed a decrease in nrfA gene copy numbers five days and two days after the first and second harvest collected from in 2018, respectively. In 2019, nrfA gene abundances were lower eight days after harvest from the first harvest collected, but higher at two days and eight days after the second harvest in 2019. Comparing nrfA gene abundances by termination timing also indicated a significant interaction between the treatment (i.e. fall termination and spring termination, hereby referred to as FT and ST) and date (i.e. fall versus spring sampling). In the fall, nrfA gene abundances were similar between the plots that would be terminated in the fall and spring, which meant that the starting nrfA gene copy numbers were not different between the treatments. The spring sampled FT plots had a 10-100 million increase in nrfA gene copy numbers when compared to the fall sampled FT plots and the spring sampled ST plots. There were no differences observed between the ST plots sampled in the fall compared to the spring.Results from this analysis indicated that nrfA gene abundances are sensitive to management practices like harvest and termination timing in these AO perennial systems. Fall termination resulted in increased nrfA abundances in the spring sampled rhizospheres of AO, which may be due to availability of carbon over the winter/early spring from the decomposing AO roots. Further exploration needs to be performed to determine how these practices are impacting NH4+ and NO3- cycling of these AO systems but there is evidence of changes to nrfA gene copy numbers due to management practices. Tillage Experiment. Tillage treatments in the LTT experiment included no-till (NT), moldboard plow (MP), and chisel-disk (CD), which had been maintained for the previous 40 years prior to sampling. The experimental set up is a randomized complete block design with four blocks and has a corn-soybean-wheat/cover crop rotation.Soil sampling and analysis were conducted to address the following objectives: 1) identify whether tillage impacts gene abundances of nrfA; 2) determine if nrfA gene abundances are different in rhizosphere and bulk soils; and 3) evaluate nrfA gene abundances at different growth stages of corn and soybean. Rhizosphere and bulk soil samples were taken in each of the tillage treatments from the volunteer wheat in the spring of 2018, from corn at growth stage V3/4 (6/14/2018), V5/6 (7/3/2018), and V8/9 (7/13/2018) and on soybean at growth stages V2 (7/7/2019), V5 (7/23/2019), and R1 (7/31/2019). Approximately 12 samples from each sample type (i.e. rhizosphere or bulk) were taken from each plot and homogenized to make 1 composite sample for each block of each tillage treatment. DNA was extracted from each composited sample and quantitative polymerase chain reaction (qPCR) was performed to quantify nrfA gene abundances. Results from the linear mixed effect models indicated a significant interaction between tillage treatment and type of sample (p = 0.0238), and between type of sample (rhizosphere or bulk) and sample date (p < 0.0001). Analysis of pairwise comparisons between tillage*type revealed a significantly higher number of gene copies in the NT bulk soils compared to the MP bulk soils, but no differences were observed between sample type within tillage treatments (e.g. NT bulk versus NT rhizosphere). Analysis of pairwise comparisons between sample type*date indicated nrfA gene copy numbers were lower in the rhizosphere at corn V3/4 compared to bulk, but were higher in the rhizosphere at corn V5/6. Additionally, nrfA genes were higher in bulk samples during soybean at R1 compared to rhizosphere samples. Analyses comparing samples taken from corn and soybean indicated a higher abundance of nrfA sequences in the corn rhizosphere and bulk soils compared to the soybean rhizosphere and bulk soils. Raw and Digested Manure-Amended Soil Incubation experiment Treatments within the SDCS plots were sampled to include soils taken from the soils that were in a corn-soybean rotation and were either amended previously with synthetic fertilizer (FERT) or broadcasted manure (BM). Samples were taken across all four blocks and homogenized to create composite samples for each soil type. Objectives were 1) to evaluate differences in the nrfA communities between the two soil types and two amendments prior to the incubations, 2) determine whether nrfA communities were impacted by soil type, amendments, water filled pore space (WFPS) or a combination of these variables, 3) assess nrfA community compositions between day 2 and day 16 of the incubation; and 4) identify potential correlations between NO3-, NH4+, and nrfA gene copy numbers from day 2 and day 16 samples. Incubations in the lab were carried out with FERT and BM to include amendments with either manure digestate (DIG) or raw manure (RAW). An additional treatment that was included was water filled pore space (WFPS) that was held constant at either 60 or 95%. Control samples for the FERT and BM samples were incubated that did not receive an amendment. Samples for DNA extraction were taken from the FERT and BM soils and from the DIG and RAW amendments prior to incubations. Additional samples for DNA extractions were taken at day 2 and day 16 during the incubations. Incubations that were sampled at day 2 and day 16 were also analyzed for NO3-, NH4+, and nrfA qPCR.Results from this incubation experiment indicate that the nrfA communities from the soils (FERT and BM) and amendments (DIG and RAW) were different and those differences in communities were still noticeable into day 16 of the incubation, with Bacteroidia still a dominant Class in the RAW soils. Amendment explained a significant portion of the phylogenetic community dissimilarity across the samples. Community dissimilarities were highly correlated with chances in NH4+ and nrfA gene abundances observed in the Day 2 and Day 16 samples. These results demonstrated the importance of amendment and type of amendment in structuring nrfA communities and possibly impacting the cycling of inorganic nitrogen species in agricultural soils.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Bhowmik, A., Cloutier, M., Bell, T., Karsten, H., Dell, C., and Bruns, MA. 2019. Impact of Soil and Manure Management on Nitrate Ammonifying Bacteria and Greenhouse Gas Emissions. Soil Science Society of America (SSSA) International Meetings, San Antonio, TX.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Cloutier, M.L., Murrell, E., Kaye, J., Barbercheck, M., Finney, D., Gonzalez, I.G., Bruns, M.A., Fungal assemblages and functions shaped by brassicaceous crops and soil texture in a multi-species cover crop experiment. Scientific Reports (2020) doi: 10.1038/s41598-020-63173-7
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Cloutier, M.L., Chatterjee, D., Elango, D., Cui, J., Bruns, M.A. Chopra, S. Sorghum root flavonoid chemistry, cultivar, and frost stress effects on rhizosphere bacteria and fungi. Phytobiomes Journal (2020) doi: 10.1094/PBIOMES-01-20-0013-FI
• Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Saha, D, Kemanian, A.R., Bhowmik, A., Bruns, M.A., Wallace J. M., and Kaye, J.P. Controls on nitrous oxide emissions from organic agriculture. Submitted to Global Change Biology, April 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Bruns, M.A., Cloutier, M.L, Karsten, H.D. Soil Biogeochemical Responses to Legume Crop Termination, oral presentation 120062. Soil Science Society of America International Meetings, San Antonio, TX.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Cloutier, M., Karsten, H.D., Alcaide, T., Bruns, M.A. Above Ground Disturbances Impact below Ground Nitrogen Cycling in Rhizospheres of Alfalfa and Orchard Grass, Presentation 120231, Soil Science Society of America International Meetings, San Antonio, TX.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Cloutier, M., Duiker, S., Bruns, M.A. Microbial N Dynamics Affected by Tillage and Root Exudation. Oral presentation at Environmental Chemistry and Microbiology Student Symposium, Penn State University, University Park, PA, April 2019.

Progress 04/01/18 to 03/31/19

Outputs
Target Audience:Our immediate target audience has been the advisory panel of five dairy famers on the Dairy Cropping Systems project at Rock Spring and three dairy famers in the region who own and operate manure anaerobic digestion facilities. We have interacted with other growers in the Pennsylvania No-Till Alliance and in the Pennsylvania Association for Sustainable Agriculture. We have provided research findings to Penn State faculty and extenstion educators on the effects of management practices on nitrogen cycling and nitrous oxide emissions from agricultural soils. We collaborate with USDA-ARS scientists at the Watershed Management and Pasture Lab who also conduct experiments at Rock Spring and with faculty in Plant Science, Dairy and Animal Science, Plant Pathology and Environmental Microbiology, and Entomology. Other target audiences are undergraduate students in Soil Ecology (SOILS 412W) and graduate students in other programs who are mentored by the CoPIs of the project. To reach scientists in the disciplines of Agronomy, Soil Science, and Soil Microbiology, we have published in the peer reviewed literature and presented our work at conferences. Changes/Problems:Postdoctoral Fellow Arnab Bhowmik left the project to take a faculty position at North Carolina A&T University. Prior to Arnab's departure, Arnab trained a wage-payroll technician, Tiffanie Alcaide, to carry out quantitative PCR tests on soil samples. When Tiffanie became responsible for the laboratory work conducted by Arnab, she demonstrated proficiency which warranted her being hired as a Research Technologist. We hired new wage-payroll undergraduate students to assist in both lab and field work. We will request a one-year, no-cost extension to enable completion of the project, which will have a new termination date of 3/31/2020. What opportunities for training and professional development has the project provided?Graduate student and postdoc participated in April 4 Penn State Sustainable Agriculture Conference, 2018 Graduate student and postdocs participated in Student Field Orientation at Agronomy Farm, May 24, 2018 Attended Energy Days on May 31 and sessions on anaerobic digestion Assigned Arnab weeklong teaching modules for two of my courses, Soil Ecology 412W and Environmental Soil Microbiology, SOILS 512 with student evaluations Discussed teaching materials and provided Arnab with my lecture notes for Environmental Soil Microbiology for him to review and evaluate Reviewed Arnab Bhowmik's teaching and research statements for faculty positions and provided advice for improvement. Listened to Arnab's oral presentations prior to his interviews and coached him on flow and confidence. Arnab started a tenure-track position at North Carolina State A&T University, Greensboro, NC, in August 2018 Assistant Professor in Soil Science/Soil Microbiology, Dept. of Natural Resources & Environmental Design How have the results been disseminated to communities of interest?Oral Presentations Bruns, Mary Ann. 2018. Role of nitrate ammonification in mitigating greenhouse gases. USDA Climate and Agroecology Project Directors" Meeting, Dec 6-7, Washington, DC. Bruns, Mary Ann. 2018. How might soil properties constrain phytobiome manipulation? Manipulating Wild and Tamed Phytobiomes: 21st Annual Plant Biology Symposium, June 19, University Park, PA. Poster Presentations Bruns, Mary Ann, Xin Peng, Ryan Trexler, and Terrence Bell. 2018. Provision of agroecosystem services by soil-surface microbial consortia. Penn State Microbiome Center Networking Event, Oct. 2, University Park, PA. Mara Cloutier, Sjoerd William Duiker, Mary Ann Bruns. Microbial N Dynamics Affected By Tillage and Root Exudation. Poster Presentation at Soil Science Society of America Meetings, January 8, 2019, San Diego, CA. Arnab Bhowmik, Terrence Bell, Heather Karsten, Curtis Dell, Mary Ann Bruns. 2019. Can Bacteria in Manures Help Reduce Nitrogen Losses from Agricultural Soils?. Poster Presentation at Soil Science Society of America Meetings, January 8, 2019, San Diego, CA. What do you plan to do during the next reporting period to accomplish the goals?Graduate student Emily Ball plans to graduate with MS in Soil Science in May 2019 We are requesting a one-year, no-cost extension to enable completion of data analysis for manuscripts in preparation.

Impacts
What was accomplished under these goals? Research: Four dairy farmer-cooperators with anaerobic manure digestorsreceived nutrient content results of their raw and digested manure after we obtained samples for nutrient and microbial analyses in our USDA-NIFA project on application of manures and digestate liquids to farmland. Northeast Sustainable Agriculture, Research and Education (NESARE) Fellowship; Francis E. Clark Soil Biology Scholarship, Soil Science Society of America

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Cloutier, M.L., Bhowmik, A., Bell, T.H., and Bruns, M.A. 2019. Innovative Technologies Can Improve Understanding of Microbial Nitrogen Dynamics in Agricultural Soils. Agricultural & Environmental Letters. 4:190032 doi:10.2134/ael2019.08.0032
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Peng, X., & Bruns, M. A. V. 2019. Development of a nitrogen-fixing cyanobacterial consortium for surface stabilization of agricultural soils. Journal of Applied Phycology. http://doi.org/10.1007
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Bell, T.H., Hockett, K.L., Alcala-Briseno, R.I., Barbercheck, M., Beattie, G.A., Bruns, M.A., Carlson, J.E., Chug, T., Collins, A., Emmett, B., Esker, P., Garrett, K.A., Glenna, L., Gugino, B.K., Jimenez-Gasco, M., Kinkel, L., Kovac, J., Kowalksi, K.P., Kuldau, G. Leveau, J.H.J., Michalska-Smith, M.J., Myrick, J., Peter, K., Vivanco Salazar, M.F., Shade, A., Stopnisek, N., Tan, X., Welty, A.T., Wickings, K., Yergeau, E. 2019. Manipulating wild and tamed phytobiomes: challenges and opportunities. Phytobiomes: Published Online:13 Mar 2019, https://doi.org/10.1094/PBIOMES-01-19-0006-W
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Chen, L, ML Hile, EE Fabian-Wheeler, Z Xu, MA Bruns, V Brown. 2018. Iron oxide to mitigate hydrogen sulfide gas release from gypsum-bedded dairy manure storages. Transactions of the American Society of Agricultural and Biological Engineers 61:1101-1112.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Cloutier ML, E Murrell, M Barbercheck, I Garcia, J Kaye, MA Bruns. 2017. What actually controls fungal community structure in agroecosystems? An analysis of cover crop treatments and soil physiochemical parameters. Poster presentation at 8th Argonne Soil Metagenomics Meeting, Nov. 1-3, 2017, Argonne, IL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Cloutier ML, E Murrell, M Barbercheck, I Garcia, J Kaye, MA Bruns. 2017. Fungal communities associated with cover cropping in a tilled agroecosystem. Poster presentation at Northeast Cover Crop Council Educational Meeting, Cornell University, Nov. 8, 2017.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Peng, X., and Bruns, M.A. 2019. Cyanobacterial soil surface consortia mediate N cycle processes in agroecosystems. Frontiers in Environmental Science Volume 6: Article 156.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Kreider, A. N., Fernandez Pulido, C., Bruns, M.A., and Brennan, R.A. 2019. Duckweed as an agricultural amendment: nitrogen amendment, leaching, and sorghum uptake. J. Environ. Qual. 48:469-475.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Ball E, Bruns MA, Karsten H, Dell C. 2017. Soil redox potential and carbon fractions in manured and cover-cropped soils under reduced tillage. Poster no. 313-1233 at the 2017 International Annual Meeting of the Soil Science Society of America, Tampa, FL, Oct. 22-25, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Saha D, A Bhowmik, AR Kemanian, JP Kaye, MAV Bruns. 2017. Assessing in-situ sources of soil N2O emissions in response to manure and residue management in organic systems. Poster 1106 at the 2017 International Annual Meeting of the Soil Science Society of America, Tampa, FL, Oct. 22-25, 2017

Progress 04/01/17 to 03/31/18

Outputs
Target Audience:Our target audiences during this reporting period:: 1) Research faculty, students, and farm management staff at Penn State and the USDA-ARS Pasture Lab who work on the Sustainable Dairy Cropping Systems (SDCS) project funded by Northeast Sustainable Agriculture Research and Education (NE-SARE). Our group interacts regularly with other members of the SDCS team to coordinate soil sampling and pursue the objectives of the NIFA project. Participating departments include Plant Science, Dairy and Animal Science, Ecosystem Science and Management, and Entomology. Penn State Extension collaborators include Ron Hoover (Agronomy),Kristy Borrelli (Sustainable Agriculture), and Sjoerd Duiker (Soil Management). 2) Dairy farmers and managers who sit on the NESARE SDCS advisory panel (including Joel Myers, Dean Patches, Gerard Troisi), In the past year we also worked with five dairy farmers who operate anaerobic digestors for converting dairy manure to methane for energy generation. We explained project objectives, asked for their feedback, obtained samples, and reported results of N, C, and microbial analyses. 3) Soil scientists, agronomists, and students attending Soil Biology and Biochemistry session (organized by Bruns and Bhowmik) on Understanding the Biology of High Carbon and Low Disturbance Soils at the Soil Science Society of America Annual Meeting in Tampa in October 2017. During two oral presentations, Bruns and Bhowmik presented data on soil microbial N dynamics. 4) USDA-NIFA Project Directors. Bruns presented an overview of nine NIFA projects on soil microbial response to climate change and adaptive management at USDA-NIFA meeting in Tampa, FL, in October 2017. 5) Undergraduate audiences in Soil Ecology course (SOILS 412W) and graduate students in Environmental Soil Microbiology course (SOILS 512). discussions during poster presentations and various research meetings on campus. Changes/Problems:One problem we encountered was damage to the datalogger-recorder for redox measurements in October, when it was overturned inadvertently by field equipment. We have not been able to repair the datalogger nor get a response from the consultant who sold us these materials, so we are unable to collect redox data in 2018. Another potential problem may be difficulty in finding a suitable postdoc to replace Arnab Bhowmik. If there is a delay in being able to find a postdoc, we may need to request a no-cost extension. Fortunately, a new postdoc will have funding from the next USDA grant that started in April 2018. What opportunities for training and professional development has the project provided?Major accomplishments included the training of postdoctoral scientist Arnab Bhowmik who gained more expertise in molecular and biochemical techniques (including qPCR, reverse transcriptase (RT)-PCR, microbial community analysis by high-throughput sequencing and nitrous oxide isotopomer analysis). Bhowmik's professional development during his project has been very successful because he has been hired by North Carolina A&T State University, Greensboro, NC, as an assistant professor of soil microbiology. Arnab will leave Penn State in August 2018, to start the next phase of his research and teaching career. Other professional development activities for Bhowmik included co-chairing two sessions on "Understanding the Biology of High Carbon and Low Disturbance Soils: A Key to Soil Health and Sustainable Intensification" for the Soil Biology and Biochemistry Division along with delivering two presentations (oral and poster) at the Soil Science Society of America (SSSA) annual meetings held in Tampa, Florida October 22-25 2017. During that meeting, Arnab served as an 'ACS Golden opportunity mentor' for Mr. Alec Martin Weber (ACS Golden Opportunity Scholar) who is a senior Agronomy major at South Dakota State University. Arnab is now the student mentoring program chair for Soil and Water Management and Conservation Division, SSSA. Bhowmik continued gaining mentoring experience through his work withf three graduate and two undergraduate students in designing soil microcosm experiments, DNA extraction, electrophoresis gel run, PCR, quantitative PCR, greenhouse gas measurements and analysis of soil biochemical properties. Project funds supported mentoring and training of two graduate students, who are making excellent progress toward their degrees. Soil Science MS student Emily Ball presented a poster (Ball E, Bruns MA, Karsten H, Dell C. 2017. Soil redox potential and carbon fractions in manured and cover-cropped soils under reduced tillage. Poster no. 313-1233) at the 2017 International SSSA Annual Meeting, Tampa, Oct 2017. She also received an award to participate in the Graduate Student Leadership Conference at the same Annual Meeting. Soil Science PhD student Mara Cloutier received an award in 2017 from the PSU Dual Title Program in Biogeochemistry at PSU. She also successfully applied for the Clark Soil Biology Scholarship fom SSSA and will be recognized for the award at the SSSA Annual Meeting in January 2019. Two undergraduate students, Ana Turosky and Haley Stauffer, contributed to this project in 2017 by assisting graduate students Mara Cloutier and Emily Ball and postdoc Bhowmik with lab and field work. In addition, Bruns mentored Ana in preparing a successful Erickson Discovery Grant from the University, "How does glyphosate affect beneficial microbes?" Ana continued to work on our project after graduating in December and before taking a position in March as Soil Conservation Technician for Capital Resource Conservation and Development Area Council in York County, PA. Bruns also mentored Haley Stauffer and wrote a reference letter for Haley to receive a World Food Prize Student Scholarship to attend the Borlaug Dialog in Des Moines, IA, in October 2017. Bruns also helped Haley with a successful proposal to the College of Agricultural Sciences Undergraduate Research Program to conduct a research project entitled, Comprehensive Literature Review of the History of Industrial Hemp in Pennsylvania, Current Marketability Aspects, and Future Product Potential, with Dr. Greg Roth, Dept of Plant Science, as advisor. Haley also received an Undergraduate Research Fellowship to work at the University of Maine's Bioproducts Research Institute in summer of 2018. Bruns also assisted in mentoring Janelle Thompson, an African American undergraduate working in the lab of Dr. Surinder Chopra by facilitating Janelle's application for travel awards to attend and present research at the Annual Biomedical Research Conference for Minority Students (ABRCMS 2017) in Phoenix. Bruns also participates in the USDA REEU project led by Dr. Tim McNellis in the Dept of Plant Pathology and Environmental Microbiology. This project partners with Alcorn State University (1890 Land Grant institution); California State University-Monterey (Hispanic serving institution); University of Puerto Rico-Mayaguez; Morgan State University (Historically Black University); Virginia State University (1890 Land Grant institution). How have the results been disseminated to communities of interest?During their contacts and visits with the owners of five dairy operations in the region, Bruns and Bhowmik explained their research on how the anaerobic digestion process could the numbes and activities of manure microbes. The cooperators received results of carbon and analysis tests run on their manure samples, with additional follow-up on microbial tests planned for the coming year. Bruns, Bhowmik and graduate students have presented research results to advisory board members for the NESARE SDCS project. Two of our cooperators have provided manure samples for testing, and all have expressed interest in learning about nitrate ammonification that may be occurring in their soils. PIs Karsten and Bruns participate in a NRCS Regional Conservation Partnership Program (RCPP) led by the Chesapeake Bay Foundation and coordinated at Penn State by Extension Educator Kristy Borelli, This project focuses on agricultural management for water quality and soil health in Clinton, Center, and Lycoming Counties. Bruns has provided workshop materials and slides for presentations by Kristy at RCPP meetings. In the Spring/Summer 2017 issue of Penn State Ag Science Magazine, our research was featured in a section on protecting water quality and restoring soils in the article, "Microbiome: Our Invisible World," by PSU writer Sarah LaJeunesse. Information on this USDA research project is also available on our website, http://sites.psu.edu/soilmicrobiology/ What do you plan to do during the next reporting period to accomplish the goals?Postdoc Arnab Bhowmik will leave PSU on July 31, 2018, to become an assistant professor at North Carolina Ag and Technical State University, where the administrators have agreed for him to devote the first semester to research and writing (no teaching), which will enable him to complete project-associated tasks this fall. To provide continuity for qPCR and other molecular analyses, Bhowmik has been training Tiffany Alcaide, whom Bruns hired in June to work on this project as a part-time research technologist. Two new undergraduate students, Josephine Beck and Tyler Bailey, have also been hired to assist the graduate students with lab and field work. PIs Bruns and Karsten plan new outputs, products, and research progress from the SDCS experiment as a result of an additional USDA NIFA award. This award is for two years starting in April 2018 to conduct additional research at SDCS, "Nitrous oxide consumption in soils under adaptive management to climate change." The new project will test for effects of management practices on non-denitrifier microbial populations that do not produce N2O yet can reduce N2O to the inert gas N2, thereby lowering overall green house gas impacts. We will assess these microbial populations in the SDCS experiment, which is now becoming a part of the Long-Term Agricultural Research (LTAR) site system being developed by USDA-ARS. Through our collaboration with the Watershed Management and Pasture Systems Lab, the new project will enable sampling and analysis to be extended to additional LTAR treatments and sites. Soil Science MS student Emily Ball expects to graduate in December 2018 and is interested in working as an extension educator after graduation. PhD student Mara Cloutier has submitted a NESARE graduate student proposal and is preparing to submit a pre-doctoral fellowship proposal to USDA in July 2018. Bruns and Karsten will continue to make presentations and provide materials and advice for the project, Soil Health: Improving Land, Water, and Producer Profitability, USDA NRCS RCPP # 17-S-PA-1645 -- Regional Conservation Partnership Program (RCPP), led by the Chesapeake Bay Foundation. Bhowmik and Bruns will continue a relevant collaboration in the coming year with an agroecosystem modelling group that has established a USDA certified organic systems experiment adjacent to the SDCS plots at Penn State's Agronomy Research Farm. This experiment demonstrated the challenges encountered when adding organic amendments to reduced-tillage soils. Co-locating manure and fresh legume residues may create a brief temporal window of N availability in excess of plant uptake and increase N2O emissions, a potent greenhouse gas, thereby offsetting the environmental benefits of organic agriculture. In 2017, a micro-plot study was conducted to investigate the effects of manure and/or cover crop exclusions on the magnitude and microbial sources of N2O emissions in a corn-soybean-winter grain rotation.A manuscript is in preparation on results indicating that cover crop biomass removal (+M-C) caused 60% reduction in cumulative N2O as compared to the +M+C treatment, while having no significant effect on subsequent corn yield (P<0.05). We also observed higher N2O emissions associated with the incorporation of cover crop than with manure. We also can report on N2O isotopomer analysis which indicated that denitrification was the major source of N2O production irrespective of manure and cover crop treatment. These results indicate that organic management that controls the input of leguminous cover crop and relies more on manure as the source of N may mitigate N2O emissions. Bhowmik.will be presenting two oral talks at the next annual SSSA meetings to be held at in 2019 in San Diego, California.

Impacts
What was accomplished under these goals? Accomplishments will be summarized under each of three goals: GOAL 1 (ORGANISMS) Manures contain nitrate-ammonifying (NA) bacteria (including E. coli) that have potential to improve N retention in soils amended with manures. We undertook molecular and culturing methods to evaluate presence of NA in manures and manure-amended soils. To test the hypothesis that soils after manure application would have greater abundance of NA following manure amendment, we collected soils before and after broadcasting of manure to soils in plots of the SDCS experiment. Raw manure being applied to these plots was also sampled, Microbial community DNA was extracted from composite soil samples for quantitative polymerase chain reaction (qPCR) analysis of NA bacteria via nrfA genes. Standard curves were obtained by amplifying five serial dilutions of Escherichia coli DNA. The qPCR results indicated that nrfA gene copy numbers in the manures varied from 3.5×10^3 to 1.5×10^4 per gram of fresh pellet (range of 2.6 x 10^6 to 1.1 x 10^7 nrfA genes per gallon of liquid manure). With a manure application rate of 8000 gal per ha, these gene copy densities would be not sufficiently high to be detected in soils immediately after sampling to a depth of 15 cm. Gene copy numbers in soils before and after manure application were in the range of 2-5×10^3 nrfA per gram dry soil. Since these soils had received 8000 gal of manure annually in the previous six years, results indicated that NA bacterial populations were not building up or persisting in the soil, a finding which does not support our hypothesis that manured soils contain higher numbers of nrfA-containing bacteria. Although attempts were made to culture several hundred isolates from manures and manure-amended soils using nitrate-rich growth medium, none of these cultures tested positive for nrfA genes using PCR. Another objective under this goal was to determine whether NA organisms differ in raw and anaerobically digested manures. Manure samples were collected before (raw) and after anaerbic digestion (digestate) from 5 different dairy manure digestor facilities in Pennsylvania during 2016-2017. DNA was extracted from manure, and real-time qPCR and amplicon sequencing with Illumina MiSeq were conducted to evaluatenrfAgene abundance and diversity. Results indicated that anaerobic digestion lowered the carbon: nitrogen (C: N) ratio by 36-56 % in digestate as compared to raw manure. ThenrfAgene abundance ranged from 102-105gene copy numbers ml-1manure, with similar results for all five dairies. Results showed a community shift from Enterobacteraceae NA in raw manure to Verrucomicrobiaceae in digestate. Higher diversity of nrfA was observed in raw manure compared to the digestate. Our results indicate that the type of manure applied to soil can regulate nrfA gene structure in soil, thereby affecting net N2O emissions. GOAL 2 (FIELD PROCESSES) Soil redox values (Eh) were studied to gain insights into an important soil factor that affects the pathways by which microbes carry out anaerobic respiration using nitrate in soil (denitrification and NA). Seed funding from the PSU College of Agricultural Sciences supported a training visit by Michel Vorenhout of MVH Consult to help us start using redox probes and a semi-continuous datalogger and multi-recorder. Probes were deployed in several locations of the SDCS plots in August-September of 2017 to obtain readings every 15 min for 7-day periods at 4-cm and 10-cm depths. Very high variation in redox recordings within an individual plot and between two depths of one probe were observed. The main findings were that 1) soil redox values varied diurnally often, but not always, and by as much as 500 mV; 2) average redox values tended to be higher at the 4-cm depth than at 10-cm depth. and 3) redox values were lower and more variable under alfalfa than under canola, where redox values did not go below 260 mV (which is the approximate redox value at which denitrification is induced). Lowest redox measured under alfalfa was -100 mV, which indicates that soils in these locations could favor NA over denitrification. Data are being analyzed from additional redox measurements made in long-term conventionally tilled and no-till plots. The multirecorder was damaged beyond repair during field operations in October, so further measurements cannot be made. These observations suggest that soils under deeper rooting and perennial crops like alfalfa could favor NA over denitrification. N2O fluxes from soils were measured between 5 May and 5 August 2017 in conjunction with soil sampling and measurement for moisture, temperature, pH, carbon fractions, and inorganic N. Measurments were made with a Gasmet FTIR gas analyzer and custom-made chambers in SDCS plots planted with corn and receiving broadcast manure or synthetic fertilizer. Prior crops before corn planting were rye, crimson clover, or none. Cumulative N2O flux was calculated from measured values and plotted with rainfall values collected from an on-site weather station. The data revealed large peaks in nitrous oxide flux that did not appear correlated with rainfall events or nitrogen application.Gas and soil measurements will be carried out in SDCS plots during the 2018 growing season. GOAL 3 (MICROCOSMS) We set up 30-day soil incubations to compare N2O emissions from soils amended with either raw or digested manures. For this study ten random soil cores (0-15 cm) were combined to make a composite sample from all four replicated plots of either synthetically fertilized or manure-amended soils. Sieved soil samples were packed to a bulk density of 1.2 g cc^-1 in separate microcosms containing soil equivalent of 50 g (dry weight). Manures were added at a rate of 180 kg N ha-1. Soil moisture was maintained at 60 or 95 % water filled pore space (WFPS). Each of the soil microcosms were placed in a 1 quart canning jar fitted with a butyl septum and incubated for 30 d at 25° C. Gas samples were collected on multiple days and analyzed for CO2, N2O and methane using the Varian CP3800 gas chromatograph. Destructive soil sampling was also done at multiple points during incubations to measure inorganic N, POX-C and nrfA gene copies. Manure digestate resulted in overall higher cumulative N2O emissions than raw manure. No significant differences were observed in cumulative N2O emissions in soils with different fertilization histories (manure or synthetic fertilizer). Ammonium-N, nitrate-N, N2O and CO2 all varied significantly (P<0.001). Overall the N2O emissions from soils amended with digestate were significantly higher that raw manure amended soils (P<0.01) which could be due to lower C and higher N availability in digestate. Our results indicate that benefits from manure digestion process may be associated with the tradeoff of increased greenhouse gas emissions.. To complement field measurements of soil redox values, we compared Eh with depth in microcosms consisting of intact soil cores and packed soil columns. The sieved soils for packed columns and intact cores were obtained from SDCS corn-soybean control plots that had been fertilized either with synthetic fertilizer or broadcast dairy manure. Eh was measured semi-continuously (every 15 minutes) in the laboratory over a seven-day incubation period. For both fertilizer treaments, soils in intact cores exhibited stratified redox potentials with depth. In packed cores, redox was higher (> 0 mV) in the upper one cm but dropped to below -100 mV at 3.5 cm and remained low to the deepest depth (9.5 cm). At depths of 3.5 to 9,5 cm, redox values of intact cores varied from -220 to +320 mV, while packed cores Eh ranged from -161 to -380 mV, Fertilizer history did not result in significant Eh differences. These results indicate that conditions affecting microbial process in packed soil columns may not be representative of conditions in the field.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Bhowmik A, Cloutier M, Ball E, Bruns MA. 2017. Underexplored microbial metabolisms for enhanced nutrient cycling in agricultural soils. AIMS Microbiology 3:826-845.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Cloutier, M., Bhowmik A. Bell, T., and Bruns, M.A. Synergies between high-throughput sequencing and other technologies advance the understanding of agroecosystem nitrogen cycling. Frontiers in Microbiology, Terrestrial Microbiology section, in review
• Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Cloutier ML, Murrell E, Barbercheck M, Kaye J, Finney D, Garcia-Gonzalez I, and Bruns MA. Fungal assemblages and functions shaped by brassicaceous crops and soil texture in a multi-species cover crop experiment. Submitted to ISME Journal.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Bruns MA (Invited Talk) 2017. Integrated management, underexplored microbial metabolisms, and nutrient flow in soils. Oral presentation 256-4 at the International Annual Meeting of the Soil Science Society of America, Tampa, FL, Oct. 22-25, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Bhowmik A, MA Bruns, and T Bell. 2017. Assessing the abundance and diversity of nrfA genes, molecular markers for nitrite ammonification, in manures. Oral presentation 406-4 at the International Annual Meeting of the Soil Science Society of America, Tampa, FL, Oct. 22-25, 2017
• Type: Other Status: Other Year Published: 2017 Citation: Bruns MA (Invited plenary talk). 2017. Importance of understanding and shaping microbial responses to climate-adaptive management. Plenary for USDA-NIFA AFRI Project Directors Meeting, October 21, 2017.

Progress 04/01/16 to 03/31/17

Outputs
Target Audience:Our target audience during this reporting period consisted of the following groups: 1)Research and extension collaborators and students at Penn State who work on the Sustainable Dairy Cropping Systems project funded by Northeast Sustainable Agriculture Research and Education (NE-SARE). Our team communicated with the SDCS team on a regular basis to explain the objectives of the NIFA project and to coordinate plans for soil sampling. 2)Dairy farmers identified by Penn State Extension, as well as farmers and a farm consultantin the NESARESDC Advisory Panel. We explained project objectives, asked for their feedback,and requested manure samples from their operations. 3)Soil scientists and agronomists attending symposia at two international conferences: American Society of Agronomy Symposium--Environmental Quality: A One Health Perspective, Nov 6, 2016, Phoenix, AZ; and American Geophysical Union Symposium--Coupling the Nitrogen and Carbon Cycles of Terrestrial Ecosystems: Understanding the Nexus between Land Management and GHG Mitigation Strategies I, San Francisco 12-16 December, 2016, San Francisco, CA. At two oral presentations, PD Bruns introduced the idea that manure application enriches soils with bacteria capable of converting nitrate to ammonium and that nitrate's alternative fate in agricultural soils could affect climate change through soil N dynamics. 4)USDA Project Directors at USDA-NIFA Agroclimatology Meeting, Dec 17-18, San Francisco, CA. PD Bruns and Postdoc Bhowmik attended the meeeting to explain the objectives of this project to other NIFA PDs working on climate change mitigation/adaptation in agriculture. 5)Undergraduate students in Environmental Resource Management and graduate students in Soil Science, Biogeochemistry, and Ecology who learn about nitrate ammonification as an understudied step in the nitrogen cycle during coursework and discussions during poster presentations and various research meetings on campus. 6)Policy makers, professionals and other individuals who attended our symposium, Soil Health, Microbiomes, and Climate-Adaptive Agriculture at the National Council for Science and the Environment, Washington, DC, Jan 24, 2017. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Main training accomplishments include the recruitment of a postdoctoral scientist, Dr. Arnab Bhowmik, who received his PhD in Soil Science from North Dakota State University in July 2016. Arnab's dissertation, "Greenhouse gas emissions and soil quality in long-term integrated and reduced tillage organic systems," is highly relevant to this NIFA project, on which he started work on August 15. Arnab's professional development activities include co-authoring presentations with PD Bruns at the American Geophysical Union annual meeting and the USDA Project Director's meeting, both held in San Francisco in November 2016. Arnab also received training at a Mentorship Workshop at Penn State, conducted by Dr. Carolee Bull on Sept 24. He also helped propose a symposium at the National Council for Science and the Environment on Jan 24, 2017, in Washington, DC, where he also gave an oral presentation. Another accomplishment is the enrollment of two Soil Science graduate students, who will train under the supervision of PD Bruns. PhD student Mara Cloutier's program is funded by NIFA, while MS student Emily Ball's program is funded largely by the Dept of Ecosystem Science and Management. Co-investigators Regan and Karsten and collaborator Curtis Dell will serve on advisory committees and guide thesis proposal development and research. PD Bruns has met with her team every week for the last six months. Two students who contributed to but were not supported directly by the NIFA project, graduated during this period. International student Clauden Zanolli gained exposure to environmental and agricultural issues by auditing PD Bruns's Environmental Soil Microbiology course in the spring. Clauden did a laboratory rotation project on manure DNA extractions before receiving an MS in Biotechnology in August 2016. Hiroyuki Kashima received a PhD in Environmental Engineering in May 2016, with Co-investigator John Regan as major advisor and PD Bruns as graduate advisory committee member. Finally, the professional development of all NIFA project participants has been enriched through consultations and joint meetings. On March 21, 2017, our team participated in the meeting of the NESARE Dairy Cropping Systems Advisory Panel. This was an excellent opportunity for the postdocs and scientists to hear from farmer stakeholders and to incorporate their suggestions in research planning. How have the results been disseminated to communities of interest?TO POLICY MAKERS AND OTHER MEMBERS OF THE PUBLIC: PD Bruns and Postdoc Bhowmik co-organized a symposium entitled, "Soil Health, Microbiomes, and Climate-Adaptive Agriculture, at the 17th National Conference and Global Forum on Science, Policy, and the Environment: Integrating Environmental and Health, Jan 24-26, 2107, Washington, DC. The symposium and ensuing discussion were attended by 30 U.S. and international attendees. Symposium was moderated by PD Bruns. Speakers included Bianca Moebius-Clune, Director of the USDA-NRCS Division of Soil Health ("USDA-NRCS Division of Soil Health"); Terrence Bell, Assistant Professor of Phytobiomes, Penn State University ("The Agricultural Microbiome: Embracing Complexity to Enhance Plant and Soil Health"); and Arnab Bhowmik, NIFA Postdoctoral Researcher, Penn State University ("Greenhouse Gas Emissions and Soil Health in Organic Agroecosystems"). TO NESARE DAIRY CROPPING SYSTEMS ADVISORY PANEL (FARMER STAKEHOLDERS AND EXTENSION STAFF): Presentations by PD Bruns entitled, "Nitrite Ammonification in Manures and Soils under Adaptive Management for Climate Change," and by Postdoc Bhowmik, "Fertilizers and Microbial Processes Determine which Forms of N Predominate in Soil." During the NESARE meeting on March 21, 2017, we explained to the panel that we are assessing whether bacteria in SDCS soils are converting nitrate to ammonium, rather than to nitrous oxide or dinitrogen gas. We also explained how manure applications may be enriching soils with nitrate-ammonifying bacteria and that our project is an early step toward insights on managing manures and soil redox potential for N retention. The four farmer-advisors indicated interest in redox potential measurements in cover-cropped soils andwillingness to provide manure samples for ourtesting. After the meeting, Dr. Ron Hoover, PSU On-Farm Research Coordinator described our NIFA project to other collaboratorsin this way: "Dr. Bruns has begun some investigations into soil microbiology to increase our understanding of some of the many processes/cycles that we know little or nothing about, with the hope that we can use this information to help farm advisors and farmers do a better job as managers of our soil resource to enhance its productivity." TO UNIVERSITY RESEARCH AND EXTENSION FACULTY, STUDENTS, COLLABORATORS: ORAL PRESENTATIONS 1)Bruns, MA, CJ Dell, HD Karsten, A Bhowmik, JM Regan. Greenhouse gas trade-offs and N cycling in low-disturbance soils with long term manure additions. Agroclimatology USDA-NIFA Project Directors Meeting, Dec 17-18, San Francisco, CA. 2)Bruns, MA, CJ Dell, HD Karsten, A Bhowmik, JM Regan. Nitrogen Cycling Considerations for Low-Disturbance, High-Carbon Soil Management in Climate-Adaptive Agriculture . Oral Presentation B11H-03, Abstract ID: 198152, American Geophysical Union, San Francisco 12-16 December, 2016, San Francisco, CA. 3)Bruns, MA. Of Humans, Soil, and Microbes--Soil Microbial Responses to Anthropogenic Disturbances. Invited talk in American Society of Agronomy Symposium--Environmental Quality: A One Health Perspective, Nov 6, 2016, American Society of Agronomy Annual Meeting, Phoenix, AZ, Nov 6, 2016. POSTER PRESENTATIONS 1)Ball, E, A Bhowmik, MCloutier, H Karsten, and MA Bruns. Soil Redox Potential in Manured and Cover-Cropped Soils Under Reduced Tillage. Poster Presentation at 7th Annual PSU Sustainable Cropping Systems Symposium, Sustainable Cropping Systems for Water Quality, March 31, 2017, Penn State University, University Park. 2)Bhowmik, A, MA Bruns, H Karsten, and C Dell. Greenhouse Gas Trade-offs in Climate-Adaptive Agriculture: Fate of Nitrogen Cycling Associated with Soil Carbon Banking. Poster Presentation at 7th Annual PSU Sustainable Cropping Systems Symposium, Sustainable Cropping Systems for Water Quality, March 31, 2017, Penn State University, University Park. 3)Kashima, H, ABhowmik, M Cloutier, E Ball, J Regan, and MA Bruns. Metagenomic Analyses of Prokaryotic Functional Genes Increase Understanding of Nitrogen Cycling: the Case of nrfA in Soils and Bio-Wastes. Microbiome Initiative Networking Event, Oct. 17, 2016, Penn State University, University Park, PA. What do you plan to do during the next reporting period to accomplish the goals?OUTREACH. Based on interest generated at the NESARE SDC Advisory meeting, plans are underway for a field day this summer at the Myers Farm in Centre County, where the NIFA team plans a demonstration of soil redox potential measurement. The field day will be an outreach event for the Regional Conservation Partnership Program (RCPP) targeting Centre, Clinton, andLycoming Counties, which wasrecently funded by USDA NRCS and is being coordinated by Dr. Kristy Borelli, PSU Sustainable Ag Extension Educator. Co-investigators Karsten and Bruns are contributing time and effortto this RCPP. Additional PSU faculty are partnering with the Chesapeake Bay Foundation to develop another RCPP proposal for Lancaster, York, Cumberland, Adams, and Franklin Counties. The RCPP missions are producer education and outreachto promoteagricultural Best Management Practices for soil health and stream health. Program priorities include implementation of no till, cover crops, and riparian buffers through NRCS's Conservation Stewardship Program and Environmental Quality Incentives Program. SESSION AT ANNUAL MEETING OF SOIL SCIENCE SOCIETY OF AMERICA is another planned accomplishment. Drs. Bhowmik and Bruns are co-organizing oral and poster sessions for the Soil Biology and Biochemistry Division of SSSA inOctober 2017, "Understanding the Biology of High Carbon and Low Disturbance Soils: A Key to Soil Health and Sustainable Intensification." RESEARCH GOAL 1 (ORGANISMS). Culturable nitrate-ammonifying bacteria from dairy manures and manured soils will be isolated andclassified by 16S rRNA sequencing. Isolates which carry out NA in biochemical tests but which do notyield PCR amplicons with the nrfA primers of Welsh et al. (2014) will be evaluated for development of more comprehensive primer sets. Using quantitative PCR and primers of Welsh et al. (2014),we will determine whether culturable or non-culturable NA bacteria are more important NA drivers.Through a newcollaboration with Dr. Terrence Bell, PSU Dept of Plant Pathology and Environmental Microbiology, we willadd tothe high-throughput nrfA amplicon data generated last year by Dr. Kashima by obtaining sequences from manures and SDCS soils. We also are investigating the presence of nrfA genes in cultured members of the bacterial order Myxococcales. This group includes Anaeromyxobacter spp., whichare known NA bacteriafound in agricultural soils. RESEARCH GOAL 2 (FIELD PROCESSES) Field measurements in SDCS replicated plots will compare N2O flux, redox potential, and N cycling gene copies in soils managed with: synthetic fertilizer or broadcast manure (no additional carbon from cover crops) in the corn-soybean control system; broadcast manure (corn silage entry) in the manure management system with allfalfa/orchardgrass (different periods of cover cropgrowth over two seasons). RESEARCH GOAL 3 (MESOCOSM PROCESSES) Laboratory mesocosm design will be finalized and experiments conducted using selected SDCS soils. Extracts of DNA and RNA will be analyzed by qPCR and nfrA primers to evaluate effect of soil redox potential and carbon amendments on nitrate ammonification. The objective is to gain understanding of how soil physicochemical status influences nrfA gene expression.

Impacts
What was accomplished under these goals? During spring and summer of 2016, preliminary research was conducted as part of a lab rotation project by MS student in Biotechnology, Clauden Zanolli. Dairy manure samples were obtained from Farm A, which is the local dairy farm source of all slurry applied to experimental plots of the Sustainable Dairy Cropping System (SDCS) during the last six years. It was determined that a centrifugation step was required for sufficient concentration of manure solids for the DNA extraction protocol deemed to give the most consistent DNA yields and quality. The extraction protocol also produced satisfactory yields of PCR-amplifiable DNA from SDCS soils before and after slurry application and will be used throughout this project. Additional research was conducted earlier in the year by PhD student Hiroyuki Kashima, advised by Co-Investigator Jay Regan. Before graduating in May 2016, Hiroyuki analyzed a large dataset of next-generation amplicons from activated sludge samples (fromvaried locations in a wastewater treatment plant) using primers for genes encoding nrfA (the respiratory enzyme that reduces nitrite to ammonium). Hiroyuki's research had been funded by a seed grant from Penn State's Institutes for Energy and the Environment to Regan and Bruns, who are Co-Investigators on the NIFA project. Hiroyuki's research confirmed the presence of nrfA genes in aerobic, suboxic, and anaerobic samples of the activated sludge. However, nrfA transcripts (obtained through extraction of RNA, rather than DNA) were detected only in suboxic and anaerobic samples and not in aerobic samples. These findings suggest that reduced O2 availability would also be an important condition for expression of nrfA genes in soils. These findings support our hypothesis that nitrate ammonification genes are expressed in soils having lower redox potentials, such as soils managed with reduced-tillage and more frequent additions of organic matter. Hiroyuki also observed that many nrfA sequences recovered from activatedsludges were highly similar to cloned sequences reported from soils. Postdoctoral scholar Dr. Arnab Bhowmik, began work on August 15. Soil Science graduate students Mara Cloutier (PhD) and Emily Ball (MS) also were recruited to work on the project. Overall, our NIFA project has three main goals, which we abbreviate as Organisms, Field Processes, and Mesocosm Processes. Accomplishments during this period are described for each goal: GOAL 1 (ORGANISMS). Characterize the nitrate-ammonifying bacteria present in manures and manured soils. Graduate student Mara Cloutier has begun purifying bacteria with diverse colonial morphologies from samples of Farm A manure as well as from SDCS soils before and after manure application. These isolates are being tested to confirm their ability to carry out "dissimilatory nitrate reduction to ammonium" (DNRA), which we refer to as "nitrate ammonification" (NA) for brevity. Genomic DNAs from these isolates also are being tested for the presence of nrfA genes using standard PCR with the primers developed by Welsh et al. (2014, Appl Environ Microbiol 80:2110). Welsh and coworkers report that these primers are capable of detecting nrfA genes from a broader array of bacterial phyla. Undergraduate student Ana Turosky contributes to the ongoing cultural and molecular work under this goal. Additional samples of dairy manures have been and will be obtained from other farms. Two farms (B and C) employ anaerobic co-digestion of dairy manures, and we have collected samples from barn floors and before, during, and after anaerobic digestion. At both farms, digested solids are used as bedding in dairy barns (typically mixed with sand or other materials), while the liquids are applied to soils as a source of N fertilizer. To assess the persistence of NA bacteria during anaerobic digestion of manures, DNA is being extracted from these samples for quantitative PCR using nrfA primers. Preliminary findings indicate that nrfA genes can be detected in digestion products. GOAL 2 (FIELD PROCESSES). Our first-year accomplishments for this goal include the development of a sampling plan for the 2017 growing season of the Sustainable Dairy Cropping System (SDCS) experiment at the Penn State Agronomy Farm; and development of methods to measure redox potential in unsaturated field soils. The SDCS project, which is entering its third three-year cycle of funding from NE-SARE, consists of three cropping systems, each designed to compare contrasting management practices: 1) nutrient management system (corn-soybean rotation, with no cover crops) comparing fertilization with synthetic fertilizer or dairy manure; 2) manure management system (two-year alfalfa/orchardgrass rotated with four-year series of cover-cropped silage) comparing broadcast and injected dairy manure; and 3) pest management system (two-year alfalfa/orchardgrass rotated with canola, soybean, silage and oats) comparing standard and reduced herbicide treatments. Treatments are replicated in four randomized blocks, and all crop years are represented in each block. For the NIFA project, we will focus on measuring redox potential, moisture and inorganic N content, N2O emissions, and nrfA gene copies in soils receiving a) synthetic fertilizer or dairy manure (no cover crops); b) soils receiving broadcast dairy manure witha green cover crop ofalfalfa/orchardgrass after recent establishment or after two seasons of growth; and 3) soils in cover crops receiving broadcast manure before and after moldboard plowing (one tillage eventper six years). Co-investigators Bruns and Karsten have held joint meetings nearly every month to discuss research progress and field operations. We have made progress in identifying the equipment and methodology for measuring soil redox potential. Because we are investigating soil conditions that promote different types of anaerobic bacterial respiration, we consider it critical to determine how carbon additions in the form of dairy manure and cover crop residues affect soil redox in reduced-tillage soils. We conducted a literature review and found that few research groups have published soil redox data for unsaturated agricultural soils. One relevant study comparing soil redox potential in plowed and no-till soils was published by Clay et al. (1990, Soil Sci Soc Am J 54:516) and was based on investigator-made platinum wire probes.Based on their study, whichshowed measurably lower soil redox potentials in no-till than in rototilled soils, we hypothesize that addition of organic amendments will result in even lower redox potentials that can influence respiratory pathways among resident bacterial communities. The body of information we obtained from our literature and internet searches indicated that redox probe readings are highly sensitive to temporal fluctuations and varied interfering factors. For those reasons we sought advice from MVHConsult, which offers custom probes and dataloggers. We have developed methods for deploying redox potential measurement equipment in the field and lab to evaluate effects of management (tillage, organic inputs, moisture). Collaborator Curtis Dell, from ARS, will supervise use of FTIR instrument by students to measure N2O emissions in the field. Other measurements will include carbon fractions, soil pH, EC, and inorganic N. Subsamples of soils will be frozen at -80C, and selected samples will be used for DNA/RNA extractions and quantitative PCR with primers for specific N cycling genes. GOAL 3 (MESOCOSM PROCESSES). Preliminary plans have been developed for soil mesocosm studies, which will be initiated as part of a dissertation research project next year. SDCS soils from different treatments will be incubated under controlled conditions for redox measurements and RNA extraction to assess expression of nrfA genes. Additional planning is underway for design and analysis of mesocosm experiments.

Publications

• Type: Book Chapters Status: Published Year Published: 2016 Citation: Bruns, MA. 2017. Bacteria, pp 175-183. In R. Lal (ed.), Encyclopedia of Soil Science, 3rd ed., Taylor & Francis DOI: 10.1081/E-ESS3-120053846.