0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Tetracycline treatment nmoles N 2 O2N g 21 h2 1 0 20 40 60 80 100 Tetracycline treatment nmoles N 2 1N g 11 h1 1 Control 0.1 mg/kg 0.5 mg/kg 1 mg/Kg 10 mg/Kg 0 50 100 150 200 250 300 0 1 5 9 15 22 30 μmol N 2 O)N m )2 hr )1 Day a0er treatment applica7on Control Tetracycline Manure Manure + Tetracycline Methods Results Acknowledgements This research is funded by the AFRI program of National Institute of Food and Agriculture. Figure 2. Rates of N 2 (A) and N 2 O (B) productions measured in soil slurry incubations with the samples collected from a North Dakota grassland. Different concentrations of tetracycline were used to test antibiotic inhibition on N 2 and N 2 O production. Water was added to the controls. Columns represent mean ± SE. Figure 5. Percent inhibition of cycloheximide (fungal inhibitor) on N 2 production in the soil samples collected at the end of the mesocosm experiment . % inhibition =[( N 2 production without cycloheximide – N 2 production with cycloheximide)/ N 2 production without cycloheximide] x 100. N 2 or N 2 O Anammox/ Codenitrifica4on Figure 1. Revised soil nitrogen cycle A North Dakota Soil Sampling (16 cores) Soil Laboratory Experiments (1 week) An?bio?c Treatments Tetracycline 0.1 – 1000 mg Kg 1 soil N 2 and N 2 O poten?al rates (soil slurry incuba?ons with 15 NO 3 ) Soil Mesocosm Experiment (1 month) No an?bio?c N 2 O fluxes measurements N 2 poten?al rates (soil slurry incuba?ons with 15 NO 3 ) Tetracycline Manure Manure + tetracycline B 0.0 2.0 4.0 6.0 8.0 Tetracycline treatment nmoles N 2 O2N g 21 h2 1 Control 1000 mg/Kg Figure 3. N 2 O fluxes measured in the soil mesocosm experiments with manure and antibiotic treatments. Tetracycline (2 mg Kg -1 ) was applied for the antibiotic treatment. Markers represent mean ± SE. Figure 4. Rates of N 2 production in soil slurry incubations with the samples collected at the end of the mesocosm experiment. Columns represent mean ± SE. Summary 1. Antibiotic inhibition of soil N 2 production was dose- dependent, reaching 25 and 80% inhibition in the samples treated with 0.5 mg Kg -1 and 1,000 mg Kg -1 of tetracycline, respectively. 2. N 2 O production was enhanced 8 times in the soils treated with high concentration of tetracycline, but no effect on N 2 O production was observed at lower doses of tetracycline. 3. Higher N 2 O fluxes were generally measured in the soil mesocosms treated with manure plus tetracycline until day 15. However, N 2 O fluxes in each mesocosm decreased during the incubation period. 4. Inhibition of N 2 production was only observed in the soil mesocosms treated with tetracycline but not with manure. 5. Higher inhibition of fungal N 2 production was found in the soil mesocosms treated with either tetracycline or manure. Impact 1. Agricultural producers, industry advisors, and government program officials will be advised of the potential consequences of antibiotic carryover from livestock manures to field soils. 2. This will encourage development and enable selection of appropriate livestock production and nutrient management planning schemes to minimize agricultural N 2 O emission. Relevance Environmental impacts of nitrogen (N) fertilization are well documented, including contributions to the increasing concentration of atmospheric nitrous oxide (N 2 O), a powerful greenhouse gas. While denitrification and nitrification are the primary pathways leading to N 2 O emission in the soils, there is uncertainty regarding the organisms responsible for N 2 O production. Previously, bacteria were considered the only microbial N 2 O source. Current studies, however, indicate that fungi also produce N 2 O by denitrification. While bacteria can produce N 2 O or N 2 as an end product of denitrification, fungal denitrification produces only N 2 O. Higher N 2 O emissions are likely to occur when most of the denitrification is due to fungal rather than bacterial activity. One potential factor influencing soil N 2 O emissions is the application of animal manures to agricultural fields. Antibiotics targeting mostly bacteria can pass through to animal manure as a result of antibiotic use in the livestock industry. Antibiotic contaminated manures may have significant impacts on soil communities, which could lead to higher contributions of fungi to N 2 O emissions. Thus, we investigate the importance and contribution of fungal denitrification to N 2 O production under variable fertilizer practices. We expect to identify the primary microbial N 2 O sources in grasslands fertilized with manure. This information will yield data to support potential N 2 O mitigation strategies. Objectives 1. Examine the effects of antibiotic on microbial communities responsible for N 2 and N 2 O production in grassland soils 2. Determine the effects of organic fertilization on bacterial and fungal N 2 and N 2 O in grassland soil communities. 3. Examine the effects of organic fertilization and antibiotic on N 2 O emission in grassland fields. 4. Identify the major microbial pathway producing N 2 O and N 2 under different scenario of fertilization and antibiotic at grasslands. Unveiling fungal contributions to agricultural soil nitrogen cycling following application of organic and inorganic fertilizers Miguel Semedo 1* , Bongkeun Song 1 , Tavis Sparrer 1 , Carl Crozier 2 , Craig Tobias 3 , and Rebecca Phillips 4 1 Department of Biological Sciences, Virginia Institute of Marine Science, College of William & Mary 2 Department of Soil Science, North Carolina State University 3 Department of Marine Sciences, University of Connecticut, 4 Ecological Insights Corporation Miguel Semedo [email protected] 0 20 40 60 80 Tetracycline treatment nmoles N 2 1N g 11 h1 1 Control 1000 mg/Kg 0 5 10 15 20 25 30 35 Mesocosm treatment nmoles N 2 /N g /1 h/ 1 Control Tetra Manure Manure + Tetra 0% 10% 20% 30% 40% 50% Mesocosm treatment % inhibi0on of N 2 produc0on Control Tetra Manure Manure + Tetra July 29, 2015