Progress 04/15/09 to 04/14/13
Outputs
Target Audience: The target audience was scientists and managers who use published results. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? We have trained three graduate and two undergraduate students. In addition, three research technicians participated in this project. -Bongkeun Song (UNCW) is a lead PI of this project with overall responsibility. -Craig Tobias (UCONN) is a co-PI responsible for stable isotope analysis and rate measurement of anammox and denitrification -David Cady (UCONN) is a research technician who developed N2O isotope methods to help better characterize the role of denitrification and co-denitrification concurrent with anammox -Joshua Heitman (NCSU) is a co-PI responsible for sampling site selection and coordination, and soil property analysis. -Andrew Long (UNCW) is a graduate student seeking a MS degree in Biology. He has worked on molecular and stable isotope analyses of anammox, codenitrification and denitrification in the soil samples. -Kelly Fridey (UNCW) is an undergraduate student who has worked on molecular analysis of denitrifying communities. -Amy Zemsta (UNCW) is an undergraduate student who has worked on molecular analysis of denitrifying communities. -Kimberly Duernberger (UNCW) is a research technician who developed a new stable isotope method to measure anammox activity in soil samples. -Scott King (NCSU) is a research associate who collected and analyzed samples from NC and IN. -Jennifer Etheridge (NCSU) is undergraduate/graduate researcher responsible for soil property analysis. -Nicole Chang (UCONN) is a graduate student working on anammox. She assisted with N2O isotope analyses. -Veronica Rollinson (UCONN) is a research technician in the IRMS lab. How have the results been disseminated to communities of interest? New methods of stable isotope and molecular analyses were developed to differential microbial activities involved in anammox, denitrification and codenitrification. Implications of anammox in the soil nitrogen cycle were discussed in the review article “Microbes and sustainable production of biofuel crops: A nitrogen perspective” published in Biofuels. After careful investigation of anammox activities and bacterial abundance in soil, fungal codenitrification was found to be an important nitrogen removal process in arable soils, rather than anammox and denitrification. This finding was published in Applied and Environmental Microbiology. The initial findings on soil anammox were presented at the annual meeting of the American Society of Agronomy and Soil Science Society of America in 2010, and the importance of fungal codenitrification was presented at the meeting of the American Society for Microbiology in 2012. In addition, the PD collaborated with Dr. Rebecca Philips at USDA-Agriculture Research Service to examine soil denitrification in disturbed and undisturbed soils. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Our project started with soil core samples collected from six different arable fields in the United States. Three sites were located in North Carolina while the other three sites were located in Indiana, Kansas and Iowa. Only mineral fertilizers were applied to the selected sites for at least five years preceding this study. Molecular detection of anammox bacteria was conducted by targeting the genes encoding hydrazine oxidoreductase (hzo), which is a central enzyme in the anammox pathway. Based on the detected hzo genes, anammox bacteria in soils were found to be “Candidatus Jettenia spp.”. Abundance of anammox bacteria was quantified using Q-PCR of hzo genes in the soil samples. The hzo gene abundance ranged from 4.99 X 103 to 1.57 X 104 copies per g of soils. Abundance of denitrifying bacteria in the soil samples was also measured by targeting the genes encoding N2O reductase (nosZ). The nosZ gene abundance was from 3.21 X 106 to 7.88 X 106 copies g-1 of soils. Based on the abundances of both genes anammox bacteria accounted for less than 0.2 % of total N2 producing bacterial communities (%anammox abundance). Soil slurry experiments were conducted with 15N isotope pairing techniques (IPT) to measure potential rates of anammox and denitrification in the soils. The anammox rates were substantially higher than expected based on the bacterial abundance. Anammox contribution to total N2 production (%anammox) was estimated to be 32 to 78%, which was dramatically different from %anammox abundance. These discrepancies in activities and abundance of anammox bacteria led us to look for alternative microbial processes producing N2 in soil ecosystems. Fungal codenitrification was found to produce 29N2 and could be mimicked with anammox in soils because both processes can utilize the same N substrates. Fungal codenitrification was measured using the Substrate Induced Respiration-Inhibition (SIRIN) method, which utilizes cycloheximide and streptomycin as selective antibiotics for fungi and bacteria, respectively. We combined the SIRIN method and 15N IPT (15N-SIRIN method) to differentiate N2 production from anammox, codentirification and denitrification in a NC soil sample. Highest inhibition was observed in 29N2 production when the soil was treated with cycloheximide (fungal antibiotic). This indicates that fungal codenitrification is the more important N2 producing pathway, although anammox and codenitrification are co-occurring in soils. A modified 15N-SIRIN experiment was conducted with four different treatments to differentiate N2 production in anammox and codenitrification. Soil core samples were collected from the Beaufort and Currituck sites of NC in 2011. Cyclohexaminde and streptomycin were used as described above. A cocktail of cycloheximide, oxacillin and ampicillin was added as a modification and was expected to inhibit the growth and activities of codenitrifying and denitrifying microorganisms while anammox bacteria were not susceptible to this treatment. Cycloheximide treatments inhibited more than 50% of 29N2 and 30N2 productions in the Beaufort soil while 77% and 100% inhibition of 29N2 and 30N2 production, respectively, were observed in the Currituck soils. This indicates that fungi were major N2 producing microorganisms in both soil communities. Since fungal codenitrification was inhibited by cycloheximide, 29N2 was produced by anammox and codenitrifying bacteria. Complete inhibition of N2 production in bacterial denitrification was observed in the soil samples treated with streptomycin. There was no 30N2 production in both soil communities although 29N2 production was detected. Fungal codenitrification was responsible for 29N2 production since both anammox and bacterial codenitrifiation were inhibited by streptomycin. It should be noted that fungi are able to conduct codenitrification and denitrification simultaneously. However, the end product of fungal denitrification is N2O rather than N2 since fungi do not carry N2O reductase. Lack of 30N2 production in streptomycin treatment assures that fungal codenitrification is an important pathway of producing N2 in soil ecosystems. The soil samples treated with a cocktail of three antibiotics showed 29N2 production only, which could be from anammox activities. Based on the rate calculations, anammox accounted for 4.1% and 8.4% of total N2 production in the Beaufort and Currituck soils, respectively. Overall, codenitrification was found to be a major pathway of N2 production in arable soils. A modified 15N-SIRIN experiment was also conducted to differentiate N2O production between codenitrification and denitrification. A continuous flow isotope ratio mass spectrometer (IRMS) was used to measure isotopic compositions of 44,45,46N2O produced from the soil communities. Higher production of 45N2O than 46N2O was observed under different antibiotic treatments. Based on the N substrates utilized in the pathways of codenitrification and denitrification, 45N2O was generated from codenitrification while denitrification produced 46N2O. Codenitrification accounted for 90% of N2O production in the soils without antibiotic treatments. Streptomycin repressed 35% of N2O production, while cycloheximide treatments inhibited 85% of N2O production from fungal codenitrification and denitrification. Bacterial codenitrification had higher contribution of N2O production than bacterial denitrification. Thus, we were able to identify fungal codenitrification as a major pathway of N2 and N2O production in arable soils. Molecular analysis of bacterial and fungal communities was conducted to quantify the abundance of microbes involved in codenitrification and denitrification in the croplands of NC. Five soil cores were collected from two transects of the Beaufort and Currituck fields. Q-PCR targeting internal transcribed spacer (ITS) region of fungal rRNA genes was used to measure abundance of fungi in the soil samples. Bacterial abundance was estimated based on Q-PCR results of bacterial 16S rRNA genes. The ratio of fungal ITS:16S rRNA genes was from 0.1 to 0.3 throughout the soil samples in two transects. Pyrosequencing of fungal ITS regions was conducted using the 454 GS Junior instrument. A total of 28 fungal genera were identified in the Currituck field while the Beaufort fields contained 37 fungal known genera. Among the identified taxa, Aspergillus, Chaetomidium, Fusarium, Hypocrea, Nectria, Penicillium and Trichoderma were present as potential codenitrifying/denitrifying fungi in the NC soils. Relative abundance of Fusarium spp. ranged 2 to 12% in the soil fungal pyrosequences. Since Fusarium spp. was a dominant codenitrifying/denitrifying fungus, abundance of Fusarium spp. was measured using Q-PCR of Fusarium ITS region and used as a quantitative indicator of codenitrifying/denitrifying fungi. The Beaufort field contained 1.77 X 105 to 4.44 X 106 Fusarium cells g-1 of soils while Fusarium spp. were counted for 1.78 X 106 to 3.76 X 106 cells g-1 of soils in the Currituck field (Long, 2011). Soil slurry incubations with 15N-IPT were conducted to measure the rates of 29N2 and 30N2 production from the transect soils. Principal Coordinate Analysis was conducted to find a correlation between abundance of N2 producing organisms and the rates of N2 productions. Relative abundance of Fusarium spp. in fungal communities showed positive correlations with both 29N2 and 30N2 productions in both field transects. Thus, we found the importance of codenitrifying/denitrifying fungus such as F. oxysporum in agricultural soil N cycle.
Publications
- Type:
Theses/Dissertations
Status:
Other
Year Published:
2011
Citation:
Long, A., 2011. The importance of anammox and codenitrification in agricultural soil. UNCW, Masters thesis.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2012
Citation:
Long, A., Heitman, J., Tobias, C. Song, B., 2012. Resolving the activity of codenitrification from denitrification and anammox in agricultural soils through antibiotic inhibition. ASM meeting abstract.
- Type:
Journal Articles
Status:
Published
Year Published:
2010
Citation:
Germaine, K.J., Chhabra, S., Song, B., Brazil, D, Dowling, D.N., 2010. Microbes and sustainable production of biofuel crops: a Nitrogen perspective. Biofuels 1: 877-888.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2010
Citation:
Long, A., Heitman, J., Tobias, C., Song, B., 2010. Importance of anaerobic ammonium oxidation (anammox) in agricultural soils. ASA-SSSA meeting abstract.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Long, A., Heitman, J., Tobias, C., Philips, R., Song, B., 2013. Co-occurring anammox, denitrification, and codenitrification in agricultural soils. Applied and Environmental Microbiology 79, 16876.
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Progress 04/15/10 to 04/14/11
Outputs
OUTPUTS: One graduate student and one undergraduate student were trained for molecular and stable isotope analyses of soil microbial communities. A new method was developed to measure anammox activity only by adding 15NH4+ in soil slurry incubation experiments. The initial findings of soil anammox were presented at the annual meeting of the American Society of Agronomy and Soil Science Society of America in 2010. The PD was invited to write a review article "microbes and sustainable production of biofuel crops: a nitrogen perspective" and described the implication of anammox in soil nitrogen cycle. The PD also collaborated with Dr. Rebecca Philips at USDA-Agriculture Research Service and provided an experimental protocol of stable isotope incubation to measure anammox and denitrification in the undisturbed soil communities from agricultural activities. PARTICIPANTS: -Bongkeun Song (UNCW) is a lead PI of this project with overall responsibility. -Craig Tobias (UCONN) is a co-PI responsible for stable isotope analysis and rate measurement of anammox and denitrification -David Cady (UCONN) is a research technician who developed N2O isotope methods to help better characterize the role of denitrification and co-denitrification concurrent with anammox -Joshua Heitman (NCSU) is a co-PI responsible for sampling and soil property analysis. -Andrew Long (UNCW) is a graduate student seeking a MS degree in Biology. He has worked on molecular and stable isotope analyses of anammox, codenitrification and denitrification in the soil samples. -Kelly Fridey (UNCW) is an undergraduate student who has worked on molecular analysis of denitrifying communities. -Kimberly Duernberger (UNCW) is a research technician who developed a new stable isotope method to measure anammox activity in soil samples. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Soil slurry incubation experiments with 15N isotope were able to measure anammox rates in various soil samples collected from 8 sites in NC, IA, IN and KS. The anammox contribution to the total N2 production was estimated to be from 32.1 to 77.9%, which are much higher than the percent anammox reported in aquatic sediments. We considered codenitrification as another N2 production pathway in soil communities. Codenitrification may produce 29N2, which is the same isotope composition of N2 gas produced by anammox in the soil experiments. We are currently developing a new stable isotope method to differentiate anammox and codenitrification. Alternatively, we determined genetic potentials of anammox, denitrification and codenitrification in soils by measuring the abundance of anammox and denitrifying bacteria as well as Fusarium oxysporum, which is a known fungal species with codenitrification capability. Quantitative PCR (qPCR) of nosZ and hzo genes were used to quantify the abundance of denitrifying and anammox bacteria, respectively while the ITS region of rRNA operon in F. oxysporum was targeted to quantify the number of potential codenitrifying microorganisms. Anammox bacterial abundance was estimated to be 1,220 to 7,850 while denitrifiers were accounted to be 23,300 to 7,880,000 cells per gram of soil. The presence of F. oxysporum was detected in some soil samples and its abundance was estimated to be 49,000 to 3,120,000 cells per gram of soil. We also conducted transect samplings at two agricultural fields in North Carolina to gain better understanding of the contribution of anammox, codenitrification and denitrification to total N2 production. Soil core samples were collected from the top 30 cm of soils across two transects located at either Beaufort or Currituck, North Carolina. Both qPCR and soil slurry incubation experiments were conducted to measure the abundance and activities of anammox, codenitrifying and denitrifiying communities. Abundance of denitrifiers in the transect at the Beaufort field ranged from 13,600 to 17,800,000, while anammox bacteria ranged from 680 to 63,000, and. F. oxysporum ranged from 15,800 to 6,920,000 cells of g of soil. The transect at the Currituck field had a range of 1,570,000 to 28,000,000 of denitrifiers, 376 to 16,300 of anammox bacteria, and 10,800 to 376,000 of F. oxysporum per g of soil. The potential denitrification rates in the Beaufort transect ranged from 0.2 to 30.5 nmoles N2 g-1h-1 while the rates of combined anammox and codenitrification were 0.05 to 2.6 nmoles N2 g-1h-1. The Currituck transect had a range of 0.6 to 35.2 nmoles N2 g-1h-1 for denitrification and 0.04 to 9.6 nmoles N2 g-1h-1 for anammox and codenitrification. Overall, denitrification is the dominant N2 production pathway across the two transects. However, the contribution of anammox and codenitrification significantly varied across the two transects with at least one sampling location where their contribution to the N2 production exceeds denitrification. This suggests that anammox and codenitrification may have substantial roles in N removal in agricultural soils.
Publications
- Germaine, K. J., Chhabra, S., Song, B., Brazil, D and D. N. Dowling (2010) Microbes and sustainable production of biofuel crops: a Nitrogen perspective. Biofuels 1: 877-888.
- Published Abstract: Long, A. M., Heitman, J., Tobias, C. R., Song, B. (2010) Importance of anaerobic ammonium oxidation (anammox) in agricultural soil. The annual meeting of the American Society of Agronomy and Soil Science Society of America.
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Progress 04/15/09 to 04/14/10
Outputs
OUTPUTS: Two graduate students and one undergraduate student have been trained for molecular and stable isotope analyses of soil microbial communities. Molecular approaches of detecting anammox bacteria were presented at the American Society for Microbiology meeting, the Gordon Conference (Applied and Environmental Microbiology) and the 1st International Conference on Nitrification in 2009. The PD communicated with colleagues at France and the Northlands regarding the detection method of anammox bacteria in soils. PARTICIPANTS: 1.Bongkeun Song (UNCW) is a lead PI/PD of this project with overall responsibility. 2.Craig Tobias (UNCW) is a co-PI responsible for stable isotope analysis and rate measurement of anammox and denitrification 3.Joshua Heitman (NCSU) is a co-PI responsible for sampling and soil property analysis. 4.Scott King (NCSU) is a research associate who has assisted in soil sampling and analysis for Kansas, Iowa, Indiana, and North Carolina. 5.Jessica Lisa (UNCW) is a graduate student seeking a MS in Marine Biology. She has involved in the method development of molecular detection and 15N tracer incubation experiments. 6.Andrew Long (UNCW) is a graduate student seeking a MS degree in Biology. He has worked on molecular detection of anammox bacteria based on hydrazine oxidase genes. He also conducted 15N tracer incubation experiments to measure anammox and denitrification rates from the soil samples. 7.Adam Howard (NCSU) is a graduate student seeking a MS in Soil Science. He has assisted in soil sampling and analysis for North Carolina. 8.James Tyler (UNCW) is an undergraduate student who has worked on DNA extraction from soil samples and PCR screen of anammox bacteria. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Anaerobic ammonium oxidation (ANAMMOX) is a recently described process of N2 production, coupling ammonium oxidation and nitrite reduction under anoxic conditions. Initially anammox was discovered in a wastewater treatment facility. Since then, it has been found in numerous marine environments as a key pathway of the global nitrogen cycle. Until very recently, there was limited evidence for the existence of anammox bacteria in terrestrial environments. Detection, diversity and activity of anammox bacteria in the soils have yet to be examined. Therefore, we investigated various soil communities to detect anammox bacteria and to measure anammox rates and its contribution to total N2 production. A total of 24 soil core samples were collected from representative row-crop agricultural fields in North Carolina, Kansas, Indiana, and Iowa. Each core sample was subdivided into surface and subsurface layers based upon the depth of the water table and frequency of saturation. This yielded a total of 54 samples to be examined for anammox detection. By targeting hydrazine oxidase genes (hzoA/hzoB) as a genetic marker, anammox bacteria were detected from 16 out of 54 samples. Anammox bacteria were mostly found at depths shallower than 30 cm, where complete soil saturation is infrequent. Soil samples have been analyzed for macro- and micro-nutrient content, organic matter (C content), pH, and physical properties (particle size distribution, density, porosity and water retention characteristics). These properties will be correlated to presence of anammox bacteria in order to target further soil sample collection and characterize soil environments for anammox bacteria. Based on phylogenetic analysis of the detected hzoA/hzo B genes, soil anammox bacteria were closely associated with either Candidatus Jettenia spp. or Candidatus Anammoxoglobus spp. The highest diversity was observed in the soil communities from North Carolina compared with the samples collected from other states. Soil slurry incubation experiments with 15N tracer were conducted with the samples that were positive for hzoA/hzoB gene detection. The rates of anammox and denitrification were simultaneously measured using isotope ratio mass spectrometry. Preliminary data suggested that anammox contributes substantially to total N2 production in the soils. More than 50% of N2 production was mediated by anammox in North Carolina soil samples. We found that anammox bacteria are not ubiquitous in soil ecosystems. However, they may play an important role in removing nitrogen via N2 production in agricultural fields if they are present. Therefore, anammox should be considered an integral pathway in the soil nitrogen cycle.
Publications
- Published Abstract 1.Song, B., M. Hirsch and J. Lisa 2009. New genetic marker (hzoAB gene) to elucidate the diversity and ecology of anammox bacteria in sediments, soils and animal waste treatments. Gordon Research Conferences for Applied and Environmental Microbiology. 2.Lisa, J. A. and B. Song 2009. Molecular detection of anaerobic ammonium oxidizing (ANAMMOX) bacteria in agricultural soil and animal manure storage. 109th American Society for Microbiology General Meeting, Philadelphia, PA.
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