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Research Article
19 December 2013

Co-Occurring Anammox, Denitrification, and Codenitrification in Agricultural Soils


Anammox and denitrification mediated by bacteria are known to be the major microbial processes converting fixed N to N2 gas in various ecosystems. Codenitrification and denitrification by fungi are additional pathways producing N2 in soils. However, fungal codenitrification and denitrification have not been well investigated in agricultural soils. To evaluate bacterial and fungal processes contributing to N2 production, molecular and 15N isotope analyses were conducted with soil samples collected at six different agricultural fields in the United States. Denitrifying and anammox bacterial abundances were measured based on quantitative PCR (qPCR) of nitrous oxide reductase (nosZ) and hydrazine oxidase (hzo) genes, respectively, while the internal transcribed spacer (ITS) of Fusarium oxysporum was quantified to estimate the abundance of codenitrifying and denitrifying fungi. 15N tracer incubation experiments with 15NO3 or 15NH4 + addition were conducted to measure the N2 production rates from anammox, denitrification, and codenitrification. Soil incubation experiments with antibiotic treatments were also used to differentiate between fungal and bacterial N2 production rates in soil samples. Denitrifying bacteria were found to be the most abundant, followed by F. oxysporum based on the qPCR assays. The potential denitrification rates by bacteria and fungi ranged from 4.118 to 42.121 nmol N2-N g−1 day−1, while the combined potential rates of anammox and codenitrification ranged from 2.796 to 147.711 nmol N2-N g−1 day−1. Soil incubation experiments with antibiotics indicated that fungal codenitrification was the primary process contributing to N2 production in the North Carolina soil. This study clearly demonstrates the importance of fungal processes in the agricultural N cycle.


The application of inorganic nitrogen fertilizers in agricultural fields causes various environmental problems, including eutrophication and habitat degradation. Accurate quantification of the processes removing fixed nitrogen is increasingly important to gain a better understanding of the fate of nitrogen in agricultural soils. Three microbial processes, denitrification, codenitrification, and anaerobic ammonium oxidation (anammox), are involved in the removal of nitrogen from soils through the production of nitrous oxide (N2O) or dinitrogen gas (N2) (1, 2). Denitrification is a microbial process in which nitrate (NO3) and nitrite (NO2) are converted to N2O and N2 in aerobic and anaerobic soils. It is a well-studied process in bacteria and has recently been found to occur in archaea and fungi (3). Codenitrification produces N2O and N2 through the reduction of nitrite (NO2) by other nitrogen compounds, including azide, ammonium (NH4+), salicylhydroxamic acid, and hydroxylamine (4, 5). Codenitrification can occur in both fungi (e.g., Fusarium oxysporum) and bacteria (e.g., Streptomyces antibioticus) (5, 6) and has been measured in grassland and agricultural soils (4, 7). Fungal codenitrification has been estimated to contribute up to 92% of the N2 produced in grassland soils (7).
Anammox produces N2 by oxidizing NH4+ with NO2 reduction (8, 9). Anammox has been detected in a number of aquatic ecosystems, including marine sediments (10, 11, 12), oxygen-minimum zones (13, 14, 15, 16), freshwater marshes (3, 17), rivers (18), meromictic lakes (19), and river estuaries (20, 21, 22). Anammox bacteria have also been found in various soil types, including permafrost soils (3), reductisol, agricultural soils (17), peat soils (23), and rice paddy soils (24). However, the importance of anammox in soil N cycling has not been fully explored.
The potential rates of both anammox and denitrification can be calculated using 15N isotope-pairing techniques (25). Under anoxic incubation conditions utilizing 15NO3 and ample 14NH4+, anammox and denitrification produce 29N2 and 30N2, respectively. The relative importance of anammox can be calculated as the percentage of the total N2 gas produced by anammox (%anammox). The %anammox has been shown to vary across aquatic environments, from being essentially absent to being the dominant pathway, with up to 79% of the N removed by anammox in various marine sediments (26). However, estimating %anammox in soils becomes difficult because codenitrification can also generate 29N2 by reducing 47N2O that is produced from the utilization of 14NH4+ and 15NO3/15NO2 in 15N isotope-pairing experiments. The contribution of anammox and codenitrification to N2 production can be presented as the percentage of N2 produced as 29N2 (%29N2). In addition, both bacteria and fungi can generate 30N2 as an end product of anaerobic denitrification. In order to gain a better understanding of soil microbial N cycling, it is important to determine the biological source of N2 production in soils. The fungal and bacterial contributions to N2 production may be differentiated using antibiotic inhibition experiments (7). Using antibiotic inhibition coupled with 15N isotope-pairing techniques, the contributions of anammox, codenitrification, and denitrification to total N2 production in soil samples may be determined.
Molecular methods can be used to determine the genetic potential of organisms involved in N2 production in soils. Anammox bacteria in the environment can be detected and quantified based on 16S rRNA genes, as well as the functional genes encoding dissimilatory nitrite reductase (nirS), hydrazine oxidase (hzo), and hydrazine synthase (hzs) (19, 27, 28, 29, 30, 31). Nitrous oxide reductase genes (nosZ) have been used to quantify N2-producing denitrifying bacteria in soils and sediments (32, 33, 34). However, genetic markers for codenitrifying and denitrifying fungi have yet to be developed. Alternatively, rRNA genes in selected fungal species can be targeted to estimate, as a proxy, the abundance of fungi capable of codenitrification and denitrification in environmental samples. F. oxysporum is the best-characterized fungus capable of codenitrification and denitrification (5). The detection and quantification of F. oxysporum have been demonstrated in inoculated soil samples by targeting the internal transcribed spacer (ITS) region of F. oxysporum rRNA genes (35).
By combining molecular quantification and 15N isotope-pairing techniques with antibiotic inhibition, the contributions of anammox, anaerobic denitrification, and codenitrification to total N2 production might be assessed in soils. We aimed to (i) identify the abundances of microbes capable of anammox, codenitrification, and denitrification and (ii) quantify the contributions of fungi and bacteria to total N2 production from agricultural soils.


Sample collection.

In 2009, surface soil samples (30-cm depth increments) were collected in triplicate with a core sampler (10.16-cm diameter) in six different agricultural fields in the United States: Pasquotank County, NC (36°07′30.249″N, 76°10′10.776″W); Beaufort County, NC (35°27.681′N, 076°55.0926′W), Currituck County, NC (36°23′09.77″N, 76°07′18.82″W); Tippecanoe County, IN (40°29′20.027″N, 87°00′07.256″W); Jewell County, KS (39°56.1008′N, 098°2.1027′W); and Boone County, IA (41°55.186′N, 93°44.891′W). All the fields had a history (>5 years) of maize, soybean, and/or wheat production following typical regional management practices. The selected sites had been treated with inorganic fertilizers only in at least the 5 years preceding the study. Soil samples from each core were homogenized separately for subsampling. Two grams of each homogenized soil sample was transferred to 2-ml microcentrifuge tubes and stored in a −80°C freezer for DNA analysis, while the rest was stored in a 4°C cold room in sealed mason jars for rate measurements.

Physical and chemical analyses of soil properties.

Additional bulk samples from the same depths and locations were used for soil characterization (Table 1). The soil texture was measured using a hydrometer (36). A 1:1 ratio of soil to water slurry was used to measure the pH (37). Organic matter was measured from the loss on ignition at 360°C (38). The inorganic N (NH4+ and NO3) was measured using the methods of Dahnke (39). Phosphorus was measured through the use of the Mehlich III soil test (40). The soil texture and nutrient profiles are reported in Table 1.
Table 1
Table 1 Physical and chemical characteristics of soil samples
Sample siteSoil seriesUSDAa texturepHOrganic matter (%)Content (mg/kg)
Beaufort, NCPortsmouthSandy loam5.,08013969230.2719230.891.321,625
Pasquotank, NCChapanokeLoam6.32.2410.23.4204161,095276299290.6331503.826.111,007
Currituck, NCRoanokeSilt loam5.92.318.95.993131,063274131290.32350131.63.39632
Boone, IANicolletteLoam5.92.915.43.2793,315530119440.65173212.180.75773
Tippecanoe, INChalmersSilty clay loam7.23.535.25.520143,112975192430.76114253.511.92821
Jewell, KSGibbonLoam7.11.748.14.17172,340414275750.67126791.931.19611
USDA, U.S. Department of Agriculture.

DNA extraction from soil samples.

Soil DNA extraction was performed with a ZR Soil Microbe DNA kit (Zymo Research Corporation, Orange, CA) using the manufacturer's instructions. The soil DNA concentration was measured with a Quant-It PicoGreen double-stranded DNA (dsDNA) assay kit according to the manufacturer's protocol (Invitrogen, Carlsbad, CA).

qPCR of hzo and nosZ genes and F. oxysporum.

The DNA samples extracted from the soil samples were utilized for quantitative-PCR (qPCR) analysis. The abundances of anammox and denitrifying bacteria were quantified using primers targeting the hzo gene and nosZ gene, respectively. The qPCR primers (HZOQPCR1F, 5′-AAGACNTGYCAYTGGGGWAAA-3′, and HZOQPCR1R, 5′-GACATACCCATACTKGTRTANACNGT-3′) of hzo genes were designed to target highly conserved regions of hzo gene cluster 1 after comparing the hzo gene cluster 1 sequences available in the NCBI database. qPCR of hzo genes generated 224-bp amplicons, which were confirmed to be hzo genes belonging to cluster 1 based on cloning and sequence analysis (data not shown). The detection limit of hzo gene qPCR was determined to be 10 copies per sample based on a serial dilution of plasmid standards carrying an hzo gene (see Fig. S1 in the supplemental material). qPCR of the nosZ gene was conducted with nosZ2F and nosZ2R primers as described previously (33). The abundance of the codenitrifying and denitrifying fungus F. oxysporum was measured by targeting the ITS region using the primers FOF1 and FOR1 designed by Mishra et al. (41). The qPCR standards were generated by serial dilution of the plasmids carrying the respective gene targets. All qPCR utilized GoTaq qPCR Master Mix Green (Promega, Madison, WI) and a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA). The PCR cycling for hzo genes included an initial denaturation step for 10 min at 95°C, followed by 50 cycles of 95°C for 45 s, 53°C for 45 s, and 72°C for 35 s and a measurement step for 35 s at 75°C. The PCR cycling for nosZ genes started with an initial denaturation step for 10 min at 95°C, followed by 50 cycles of 95°C for 45 s, 55°C for 45 s, and 72°C for 35 s and a measurement step for 35 s at 80°C. The PCR cycling for the F. oxysporum ITS region began with an initial denaturation step for 2 min at 95°C, followed by 40 cycles of 95°C for 1 min, 65°C for 30 s, and 72°C for 30 s and a measurement step for 10 s at 79°C. PCR specificity and primer dimer formation were monitored by analysis of dissociation curves. All qPCRs were performed in triplicate. The R2 values for the standard curves were 0.998, 0.996, and 0.997 for the hzo qPCR, nosZ pPCR, and F. oxysporum ITS qPCR, respectively. The efficiency and detection limit of the hzo qPCR are shown in Fig. S1 in the supplemental material.

Examination of anammox community structures in soils.

PCR of hzo gene cluster 1 was conducted to examine community structures of soil anammox bacteria using the primers hzocl1F1 and hzocl1R2, following the method of Schmid et al. (42) with some modifications. The PCR mixture was a 25-μl-volume reaction mixture containing 12.5 μl GoTaq Green Master Mix (Promega, Madison, WI), 1 μl of each primer (10 μM), and 1 μl of DNA as the template (10 to 100 ng). The PCR cycle began with a 5-min, 95°C denaturation step, followed by 40 cycles of denaturation at 94°C for 45 s and a primer-annealing step for 1 min at 50°C, and concluding with a 1-min extension step at 72°C. Gel electrophoresis on a 1.0% agarose gel was used to examine the PCR products, which were subsequently purified using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI) according to the manufacturer's instructions. The purified amplicons were cloned using the Perfect PCR Cloning Kit (5Prime, Gaithersburg, MD). Clone libraries for the following sites were constructed: Beaufort, NC (BS), Currituck, NC (CS), Pasquotank, NC (PS), Jewell, KS (JS), Tippecanoe, IN (IS), and Boone, IA (OS) (see Table S1 in the supplemental material). The clones were sequenced using BigDye Terminator (Applied Biosystems, Foster City, CA) and an ABI 3130xl automated genetic analyzer (Applied Biosystems, Foster City, CA). NCBI BLAST (http:/ was used to find closely related sequences. The sequences, along with closely related reference sequences, were aligned using ClustalW (http:/ MEGA version 4.0 was utilized to create neighbor-joining trees with bootstrapping of 16S rRNA gene sequences (43). Protein sequences were deduced from hzo sequences, and MEGA was utilized to create a Dayhoff model tree with bootstrapping. Similarities were calculated using EBI EMBOSS ( DOTUR was used to compare the diversity of hzo genes detected in six sites based on the Shannon and chao1 indices (44).

Soil slurry incubation experiments utilizing 15NO3 or 15NH4+ substrates.

The rates of 29N2 and 30N2 production were measured and calculated using a modification of the method of Dalsgaard and Thamdrup (10). Approximately 2 g of soil was transferred to 12-ml Exetainer tubes (Labco, High Wycombe, United Kingdom) and mixed with 2 ml of Milli-Q water to generate saturated soil slurries. The tubes were sealed with gas-tight septa and flushed with He gas. The tubes with soil slurries were incubated overnight at room temperature to reduce the background concentrations of NO3 and NO2 (NOx). The remaining background NOx levels were measured using reduction by vanadium(III) and chemiluminescent detection with an Antec model 7020 nitric oxide analyzer (Antek Instruments, Houston, TX). After the initial overnight incubation, the tubes were vacuumed and flushed with He gas three times. A final concentration of 1 mM Na15NO3 (99.5 atom%; Cambridge Isotope Laboratory, Andover, MA) was added to each tube. 14NH4+ was not added, as sufficient quantities (>5 mg/kg) were present in the surface soils. Time course incubations were carried out in duplicate (time points 0, 1, 2, 3, and 5 h) at room temperature. A saturated ZnCl2 solution was added at each time point during the incubation in order to stop microbial activity. The N2 gas in the headspace of each sample was measured on a continuous-flow isotope ratio mass spectrometer (Thermo Finnigan Delta V; Thermo Scientific, Waltham, MA) in line with an automated gas bench interface (Thermo Gas Bench II). All samples from a single site were measured on the same day. 29N2 and 30N2 production rates were calculated from the samples amended with 15NO3. The background nitrate levels, based on the nitrate and nitrite reduction measurements, were taken into account in the rates of 29N2 and 30N2 production, along with tracer dilutio, as described by Dalsgaard and Thamdrup (10).
The presence of anammox and codenitrification were confirmed by conducting additional incubation experiments utilizing 15NH4+ substrate additions. These conditions produce 29N2 only if anammox or codenitrification occurs in the soil samples. Approximately 5 g of soil was transferred to 30-ml Wheaton serum bottles (Sigma-Aldrich, St. Louis, MO) and mixed with 5 ml of Milli-Q water to produce saturated soil slurries. The bottles were sealed with gas-tight butyl rubber stoppers and flushed with He gas. After an overnight incubation, the headspace of each serum bottle was vacuumed and flushed with He gas. The serum bottles were injected with He-flushed stock solutions of (15NH4)2SO4 (99.2 atom%; Cambridge Isotope Laboratory, Andover, MA) to give a final concentration of 1 mM 15NH4+. The headspace gas in the serum bottles (5 ml) was sampled at the beginning (0 h) and end (24 h) of incubation and transferred to a He-filled 12-ml Exetainer tube (Labco, High Wycombe, United Kingdom) using a gas-tight syringe (Hamilton Company, Reno, NV). The collected gas samples were measured on a continuous-flow isotope ratio mass spectrometer (Thermo Delta V; Thermo Scientific, Waltham, MA). The rate of 29N2 production was calculated without considering 15NH4+ tracer dilution.

Soil slurry incubation experiments with antibiotic treatments.

Additional soil samples were collected in 2010 from the same agricultural field at Beaufort, NC, following the same sampling procedures, to conduct soil slurry incubation experiments with addition of selective antibiotics of bacteria and fungi. Streptomycin was used to inhibit bacterial activity, while cycloheximide was used to inhibit fungal activity, as described by Laughlin and Stevens (7). The contribution of bacteria and fungi to the production of 29N2, 30N2, and N2O was measured using the same incubation conditions described for the 15NO3 addition experiments with the following modifications. Approximately 2 g of soil was transferred to 12-ml Exetainer tubes (Labco, High Wycombe, United Kingdom) and mixed with 2 ml of Milli-Q water to generate saturated soil slurries. Cycloheximide was added to a series of soil slurries at a final concentration of 15 mg/g, while streptomycin was added to another series of soil slurries at a final concentration of 3 mg/g. A series of soil slurries with no antibiotic additions was used to calculate an activity baseline for 29N2, 30N2, and N2O production and to act as a positive control. Another series of soil slurries contained both antibiotics in the concentrations listed above as a negative control. The initial preincubation was extended from overnight to 48 h in order to allow the antibiotics to inhibit microbial activity. After the initial preincubation, the tubes were vacuumed and flushed with He gas three times. A final concentration of 1 mM Na15NO3 (99.5 atom%; Cambridge Isotope Laboratory, Andover, MA) was added to each tube. Time course incubations were carried out in duplicate (time points 0, 3, and 6 h). A saturated ZnCl2 solution was added at each time point during the incubation in order to stop microbial activity. The N2 gas in the headspace of each sample was measured on a continuous-flow isotope ratio mass spectrometer (Thermo Finnigan Delta V; Thermo Scientific, Waltham, MA) in line with an automated gas bench interface (Thermo Gas Bench II). The fungal and bacterial 29N2 and 30N2 rates in these incubation experiments were calculated using the methods of Dalsgaard and Thamdrup (10). Separate incubation experiments were set up for N2O measurements using the same incubation conditions. Gas samples stored in the Exetainer tubes were analyzed for N2O using a Varian Model 3800 Gas Chromatograph with a Combi-Pal autosampler. In this system, the sample is autoinjected into a 1-ml sample loop and then loaded into columns and routed through a 63Ni electron capture detector (ultrapure 95% argon-5% CH4 carrier gas). The gas chromatograph was calibrated with commercial blends of N2O balanced in N2 (Scott Specialty Gases, Philadelphia, PA) following verification of stated concentrations with standards from the National Institute of Standards and Technology. The precision of analysis, expressed as a coefficient of variation for 10 replicate injections of low and high concentration standards, was consistently <2%. The minimum detectable concentration change was 7 nl N2O liter−1. The time-linear change in the headspace N2O molar concentration was used to calculate production rates of N2O (45, 46). Percent inhibition by antibiotic treatments was calculated by dividing the rates of N2O, 29N2, and 30N2 production in three different antibiotic treatments by those measured in the control.

Statistical analysis.

The rates calculated from the soil slurry incubation experiments; the abundances of anammox, denitrifying, and codenitrifying organisms; and the physical and chemical characteristics of soil samples were used for principal-component analysis (PCA) and Pearson correlation values using the Canoco program (version 4.5; Microcomputer Power, Ithaca, NY) and Microsoft (Redmond, WA) Excel, respectively. R2 and P values were calculated from linear regression analyses using Microsoft Excel. Due to the extraordinarily high rate of 29N2 production from the Beaufort soils compared to the other sites, the data from the site were excluded from the statistical analysis.

Nucleotide sequence accession numbers.

The hzo gene sequences were deposited in the NCBI database with accession numbers ranging from JQ314231 to JQ314341.


Physical and chemical properties of soils collected from agricultural fields.

The measurements of organic matter, NH4+, NO3, and other chemical parameters in soil samples from six agricultural fields are reported in Table 1. High concentrations of NH4+ and NO3 were characteristic of all soil samples, ranging from 1.1 to 10.2 mg/kg for NH4+ and from 1.3 to 5.9 mg/kg for NO3. Soil pH varied considerably from more acidic in North Carolina (ranging from 5.2 to 6.3) to near neutral or basic in Midwestern states (ranging from 5.9 to 8.2). Soil textures ranged from sandy loam in Beaufort to silt loam in Currituck to clay loam in Pasquotank.

Detection and identification of anammox bacteria in agricultural soils.

PCR with primers hzocl1F1 and hzocl1R2 generated amplicons with a length of 470 bp from all the soil samples. Cloning and sequencing of the amplicons confirmed the detection of only hzo genes associated with hzo gene cluster 1 (42). Based on the hzo gene detection, anammox bacteria were found to be ubiquitous across the six sample sites in four states. The hzo gene sequences were translated into amino acid sequences, which were used to select representative sequences based on sequence identity. Phylogenetic analysis of the representative HZO sequences showed that soil anammox bacteria were closely related to “Candidatus Jettenia,” sharing 94.1 to 100% sequence similarity (Fig. 1). None of the sequences were closely associated with the HZO sequences found in “Candidatus Scalindua spp.,” “Candidatus Anammoxoglobus spp.,” “Candidatus Kuenenia spp.,” or “Candidatus Brocadia spp.” There was no clear segregation of anammox bacterial communities associated with the soils sampled from the different locations. However, higher diversity of hzo genes was found in the Currituck soil community than in other soil samples (see Table S1 in the supplemental material).
Fig 1
Fig 1 Phylogenetic tree of representative HZO sequences deduced from the hzo genes detected from agricultural soils. Neighbor joining with the Dayoff model was used for tree construction. The HAO sequence from the planctomycete KSU-1 was selected as an outgroup. Abbreviations for sample sites are as follows: BS (Beaufort surface layer), CS (Currituck surface layer), IS (Tippecanoe surface layer), JS (Jewell surface layer), OS (Boone surface layer), and PS (Pasquotank surface layer). The bootstrap numbers are percentages of 1,000 iterations.

Abundances of hzo and nosZ genes and the F. oxysporum ITS.

Quantitative PCR was performed on DNA samples from the top 30 cm of all the sample sites using primers specific for hzo and nosZ genes for anammox and denitrifying bacteria, respectively. The abundances of hzo, nosZ, and the F. oxysporum ITS are reported in Table 2.
Table 2
Table 2 Abundances of denitrifying and anammox bacteria, as well as F. oxysporum, in agricultural soils
Sample siteNo. of copies g−1
hzonosZF. oxysporum ITS
Beaufort, NC1.24 × 104 ± 5.30 × 1037.88 × 106 ± 1.40 × 1061.92 × 105 ± 2.10 × 104
Pasquotank, NC5.53 × 103 ± 1.80 × 1033.67 × 106 ± 7.10 × 1051.19 × 105 ± 7.50 × 103
Currituck, NC9.04 × 103 ± 6.10 × 1033.65 × 106 ± 8.40 × 1053.12 × 105 ± 4.50 × 104
Boone, IA1.57 × 104 ± 5.20 × 1033.21 × 106 ± 7.10 × 105NDa
Tippecanoe, IN4.99 × 103 ± 8.80 × 1025.15 × 106 ± 6.50 × 1042.38 × 105 ± 1.80 × 104
Jewell, KS1.15 × 104 ± 1.20 × 1033.33 × 106 ± 4.50 × 105ND
ND, not detected.
The highest hzo gene abundance was found in the Boone soils, while the nosZ gene abundance was highest in Beaufort soils. The highest abundance of F. oxysporum was recorded in the Currituck soils, but F. oxysporum was not detected in soil samples collected from Boone and Jewell.

29N2 and 30N2 production from soil slurry incubation experiments.

Potential anammox, codenitrification, and denitrification rates in soil samples were measured using two different 15N substrates (15NH4+ or 15NO3) (Table 3). The 29N2 production from 15NH4+ tracer incubations is considered an indication of the presence of anammox and codenitrification in the soils samples, while the 29N2 and 30N2 production rates from 15NO3 tracer incubations were used to calculate the combined potential rates of anammox and codenitrification and the potential rate of denitrification, respectively (Fig. 2).
Table 3
Table 3 N2 production rates calculated from 15N isotope-pairing experiments
Sample site15NH4+ (nmol N2-N g−1 day−1) 29N215NO3 + 14NH4+ (nmol N2-N g−1 day−1)%29N2Residual NO3/NO2 (mM)15NO3 enrichment (%)
Beaufort, NC0.011 ± 0.006147.711 ± 1.84842.121 ± 3.16877.90.25174.9
Pasquotank, NC0.024 ± 0.01719.212 ± 6.37140.811 ± 4.81232.00.14185.9
Currituck, NC0.018 ± 0.02421.611 ± 3.65133.611 ± 3.81639.10.36163.9
Boone, IA0.181 ± 0.0972.796 ± 0.4084.188 ± 0.28840.00.18281.8
Tippecanoe, IN0.353 ± 0.2668.172 ± 0.52811.291 ± 0.16842.00.05194.9
Jewell, KS0.040 ± 0.01913.212 ± 2.89212.011 ± 2.05252.40.21178.9
Fig 2
Fig 2 29N2 and 30N2 production from 15NO3 addition incubation experiments. (A) Beaufort. (B) Currituck. (C) Pasquotank. (D) Boone. (E) Jewell. (F) Tippecanoe. The error bars indicate standard deviations.
The 29N2 production rates from anammox and codenitrification varied from 2.796 to 147.711 nmol N2-N g−1 day−1, while the denitrification rates ranged from 4.118 to 42.121 nmol N2-N g−1 day−1. The %29N2 production ranged from 32.1 to 77.9% of total N2 production. Both of the lowest potential 29N2 production and denitrification rates were from the Boone soils, while both of the highest potential 29N2 production and denitrification rates were from the Beaufort soils. The highest %29N2 production was found in Beaufort, while the lowest was in Pasquotank. The overall trend was higher potential 29N2 production and denitrification rates in North Carolina than in the other states in the sample set.
The presence of anammox and codenitrification in soil samples was confirmed with the incubation experiments with 15NH4+ addition. The production of 29N2 was observed from the incubation conditions, and the potential anammox and codenitrification rates were calculated to be from 0.011 to 0.353 nmol N2-N g−1 day−1 based on the 29N2 production (Table 3). The lowest potential rate of 29N2 production was in the Beaufort soils, and the highest potential rate was in the Tippecanoe soils. Overall, the potential 29N2 production rates from North Carolina were lower than those from the other states.

Correlation analysis of rate measurements, gene abundance, and soil properties.

Weighted and normalized PCAs were conducted to determine correlations of rate measurements with the soil characteristics and the abundance of anammox and denitrifying bacteria and F. oxysporum (Fig. 3). Principal component 1 (PC1) explained 97.1% of the variability, while principal component 2 (PC2) explained 2.9% of the variability. The potential 30N2 production rates showed a significant and positive correlation with the abundance of F. oxysporum in the soils (r = 0.748, r2 = 0.560, and P = 0.032). The denitrification rates showed no other statistically significant correlations but exhibited a strong correlation with NO3 (r = 0.895; r2 = 0.801). The potential 29N2 production rates calculated from the 15NO3 incubation experiments showed strong correlations with the levels of NO3 (r = 0.905; r2 = 0.819) and the abundance of F. oxysporum (r = 0.799; r2 = 0.638).
Fig 3
Fig 3 PCA plot comparing 29N2 and 30N2 production rates, qPCR data on microbial abundance, and soil characteristics.

Comparison of bacterial and fungal activities in N2 and N2O production.

The fungal and bacterial contributions to 29N2, 30N2, and N2O production rates were evaluated using the slurry incubation experiments with antibiotic additions. Cycloheximide inhibits both fungal codenitrification and denitrification, while bacterial denitrification and anammox are repressed by streptomycin. Based on the rates calculated from control conditions without antibiotic treatment, the Beaufort soil communities produced larger amounts of N2O than of N2. The N2O production rate was 24.14 nmol N2O n g−1 day−1, while the total N2 production rate was estimated to be 7.27 nmol N2-N g−1 day−1 by combining the 29N2 and 30N2 production rates (see Table S2 in the supplemental material). Based on the comparison of N2O and N2 production rates from the control and antibiotic treatments, cycloheximide was shown to inhibit 65% of the N2O production and 85% of the total N2 production (Table 4). Cycloheximide inhibited both 29N2 and 30N2 production by 85%. Streptomycin inhibited 45% of N2O production and 62% of total N2 production. Streptomycin inhibited 29N2 and 30N2 production by 60% and 68%, respectively. Higher inhibition was observed in both bacterial and fungal N2 production than N2O production. The incubations with both antibiotics added nearly completely inhibited N2 production, while 71% of N2O production was repressed. Among N2 production rates measured from the antibiotic treatments, the highest inhibition (62.6%) was found in 29N2 production with cycloheximide addition (Table 4). Streptomycin treatment inhibited 44.1% of total N2 production, which may account for anammox and bacterial codenitrification.
Table 4
Table 4 Percent inhibition of N2O and N2 production with antibiotic treatments
Antibiotic treatment% Inhibition
Streptomycin + cycloheximide70.699.972.526.4


Detection and identification of anammox bacteria in agricultural soils.

The presence of anammox bacteria in agricultural soils was determined based on the detection of hzo genes in this study. This is the first report of finding the hzo genes in agricultural soils. Phylogenetic analysis showed that all the detected anammox bacteria are closely associated with “Ca. Jettenia spp.” with low diversity. This was in contrast to the findings of Humbert et al. (17), which showed high diversity of anammox bacteria across terrestrial ecosystems with “Ca. Jettenia,” “Ca. Brocadia,” and “Ca. Kuenenia.” A number of selective pressures may have contributed to the lack of diversity in anammox bacteria in the agricultural soils. Paramount in these factors is tillage, which introduces air into the soil and disrupts the structural integrity and horizons of the soil profile. Tillage is known to have a detrimental effect on the abundance of denitrifiers in the soil, with up to 60% less denitrifiers in tilled versus untilled soils (47). Another selective pressure in agricultural fields that may have an effect on soil anammox diversity is the long-term application of inorganic nitrogen fertilizers. The application of NH4+- and NO3-based fertilizers can alter the composition of denitrifier community structures (48). The selective pressures in agricultural soils may have allowed “Ca. Jettenia” to be enriched and to predominate in the examined soils. Dominance of “Candidatus Jettenia asiatca” was reported in the anammox bacterial enrichment study with peat soils after feeding an increased concentration of NO2 and NH4+ (23).

Abundance of N2-producing microorganisms in agricultural soils.

Denitrifying bacteria were found to be the most abundant N2-producing microorganisms in agricultural soils, based on the qPCR assays. The nosZ gene abundance was at least 200 times higher than that of the hzo genes (Table 2) and fell into the range of nosZ gene copy numbers measured in other soils (105 to 107 gene copies g−1) (33). qPCR of hzo genes was used to estimate anammox bacterial abundance in the different soil samples. The hzo gene abundance in soils was much lower than those reported in Jiaozhou Bay sediments (27). Lower anammox bacterial abundance in soils might be related to aeration and the physical disruption of soil communities in agricultural fields. Anammox bacteria may be better suited to stratified anoxic sediments than to agricultural soils.
Based on qPCR of the ITS, F. oxysporum was detected and quantified in four of the six agricultural soil samples. The abundance of F. oxysporum in the soils was greater than in the soil measured by Jimmenez-Fernandez et al. (35), who inoculated soil samples. with F. oxysporum. This is the first report to quantify F. oxysporum using qPCR in agricultural soils. Sequence analysis of the amplicons showed 100% sequence identity to the ITS region of F. oxysporum (data not shown). F. oxysporum is just one of the codenitrifying and denitrifying fungi (4). Since the qPCR assay of the F. oxysporum ITS region is highly specific for F. oxysporum species, the abundance of codenitrifying and denitrifying fungi in soils is underestimated in this study. It is necessary to develop new molecular methods to estimate the abundance of codenitrifying and denitrifying fungi in soil ecosystems.

Microbial interaction of N2 production in agricultural soils.

Microbial N2 production in soils can be mediated by anammox, denitrification, and codenitrification. Both anammox and codenitrification can generate 29N2 gas from 15N isotope-pairing experiments with 15NH4+ or 15NO3 substrates. The N2 production rates from 15NH4+ treatments were significantly lower than the rates measured from the 15NO3 treatments, which may have been due to dilution of the 15NH4+ tracer with unlabeled 14NH4+ continuously generated from remineralization of organic nitrogen during the incubation. Lower 29N2 production rates of 15NH4+ treatments observed in the North Carolina soils could be explained by higher remineralization coupled with denitrification.
The 29N2 production from the 15NO3 treatments ranged from 2.8 to 147.71 nmol N2-N g−1 day−1, and the %29N2 production ranged from 32.1 to 77.9% of the total N2 produced. Both 29N2 production rates and %29N2 production were higher than those reported in rice paddy soils (24). The potential denitrification rates based on the 30N2 production fall into the lower range of N2 gas fluxes from denitrification measured in other agricultural soils (49). There was no significant correlation between the abundance of hzo and nosZ genes and the production rates of 29N2 and 30N2 gases. This may indicate that anammox and bacterial denitrification are not major N2-producing pathways in the agricultural soils. Alternatively, there might be a detection limitation of the nosZ gene primers used for qPCR analyses. The diversity of N2O-respiring bacteria in the examined soils might be greater than those reported by Henry et al. (33).
The presence of F. oxysporum in the soils suggests that codenitrification and denitrification by fungi may play important roles in 29N2 and 30N2 production. PCA showed a strong correlation between the abundance of F. oxysporum and the production rates of 29N2 and 30N2 (Fig. 3). The results from soil slurry incubation with antibiotics confirmed that fungal codenitrification and denitrification had a greater contribution to the total N2 production in the examined soil samples than did anammox and bacterial denitrification (Table 4; see Table S2 in the supplemental material). Greater inhibition of 29N2 production by fungus was also observed in the antibiotic inhibition experiments. Interestingly, the sum of percent inhibition by either cycloheximide or streptomycin in N2 production is larger than the percent inhibition of both antibiotic treatments. This may due to an unknown mechanism of inhibition by either cycloheximide or streptomycin on bacteria or fungus, respectively. The highest inhibition of N2 production by cycloheximide indicates that fungal codenitrification is a major N2-producing process in the examined soil communities. This, along with the higher overall 29N2 production rates compared to the 30N2 production rates, suggests that fungal codenitrification is the dominant pathway of N2 production in the Beaufort soils. Anammox and bacterial codenitrification were also found to make significant contributions to the total N2 production compared to that of denitrification.
Future studies must continue to address the fungal contribution to the production of N2 and N2O in agricultural soils with the development of new methods. Current denitrification, codenitrification, and anammox calculations based on the production of labeled N2 cannot fully differentiate the 29N2 produced by bacterial codenitrification and anammox (7, 25, 50). Due to the characteristics of anammox bacteria, which, like other planctomycetes, lack cell walls, antibiotics targeting cell wall synthesis, coupled with fungal inhibitors, may inhibit both bacterial and fungal codenitrification, thereby allowing anammox to be measured separately from bacterial codenitrification (51). Characterization and development of molecular probes targeting genes in the codenitrification pathway may prove useful in gaining further understanding of the abundance of codenitrifiers in soils.

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Published In

cover image Applied and Environmental Microbiology
Applied and Environmental Microbiology
Volume 79Number 11 January 2013
Pages: 168 - 176
PubMed: 23087029


Received: 14 August 2012
Accepted: 10 October 2012
Published online: 19 December 2013


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Andrew Long
Department of Biology and Marine Biology, University of North Carolina, Wilmington, North Carolina, USA
Present address: Bongkeun Song, Virginia Institute of Marine Science, Department of Biological Sciences, Gloucester Point, Virginia, USA; Andrew Long, University of Toronto, Department of Ecology and Evolutionary Biology, Toronto, Ontario, Canada.
Joshua Heitman
Department of Soil Science, North Carolina State University, Raleigh, North Carolina, USA
Craig Tobias
Department of Marine Sciences, University of Connecticut, Storrs, Connecticut, USA
Rebecca Philips
USDA-ARS Northern Great Plains Research Laboratory, Mandan, North Dakota, USA
Bongkeun Song
Department of Biology and Marine Biology, University of North Carolina, Wilmington, North Carolina, USA
Present address: Bongkeun Song, Virginia Institute of Marine Science, Department of Biological Sciences, Gloucester Point, Virginia, USA; Andrew Long, University of Toronto, Department of Ecology and Evolutionary Biology, Toronto, Ontario, Canada.


Address correspondence to Bongkeun Song, [email protected].

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