Abundance Of Denitrifying Genes Research Articles

Coupled molecular and isotopic evidence for denitrifier controls over terrestrial nitrogen availability.

Denitrification removes ecologically available nitrogen (N) from the biosphere and influences both the pace and magnitude of global climate change. Disagreements exist over the degree to which this microbial process influences N-availability patterns across Earth's ecosystems. We combine natural stable isotope methods with qPCR to investigate how denitrifier gene abundance is related to variations in nitrate (NO3-) pool sizes across diverse terrestrial biomes and conditions. We analyze NO3- isotope composition (15N/14N, 18O/16O) and denitrifier gene nirS in 52 soil samples from different California ecosystems, spanning desert, chaparral, oak-woodland/savanna and forest. δ15N-NO3- correlates positively with δ18O-NO3- (P⩽0.03) and nirS abundance (P=0.00002) across sites, revealing the widespread importance of isotopic discrimination by soil denitrifiers. Furthermore, NO3- concentrations correlate negatively to nirS (P=0.002) and δ15N-NO3- (P=0.003) across sites. We also observe these spatial relationships in short-term (7-day), in situ soil-incubation experiments; NO3--depletion strongly corresponds with increased nirS, nirS/16 rRNA, and enrichment of heavy NO3- isotopes over time. Overall, these findings suggest that microbial denitrification can consume plant-available NO3- to low levels at multiple time scales, contributing to N-limitation patterns across sites, particularly in moist, carbon-rich soils. Furthermore, our study provides a new approach for understanding the relationships between microbial gene abundance and terrestrial ecosystem functioning.

Temporal Changes Rather than Long-Term Repeated Burning Predominately Control the Shift in the Abundance of Soil Denitrifying Community in an Australian Sclerophyll Forest.

To understand the temporal dynamics of soil bacterial denitrifying community in response to long-term prescribed burning and its resilience and recovery following a fire, a wet sclerophyll forest study site under two treatments (2 yearly burning (2YB) and no burning (NB)) and with 40-year-old burning history was used. Similar temporal patterns in the abundance of total (16S rRNA) and denitrifying (narG, nirK, nirS, nosZ) bacteria between two burning treatments revealed strong temporal influences. The magnitude of burning impacts on the abundance of 16S rRNA and denitrification genes was smaller compared with the impact of sampling time, but significant burning and temporal impacts were recorded for all (P < 0.001)-except for the nirS gene. Impacts of prescribed fire on the abundance of soil denitrifying community could be observed immediately after fire, and this impact diminished over a 24-month period prior to the next prescribed burning event. In conclusion, temporal changes govern the fluctuations of the abundance of soil denitrifying genes over the sampling period and the denitrifying community can recover after fire, suggesting that this community is resilient to the effects of prescribed burning. A combination of biotic and abiotic factors may account for the different temporal dynamics of denitrification gene abundance.

Optimization of ozonation combined with integrated fixed-film activated sludge (IFAS) in the treatment of oil sands process-affected water (OSPW)

Effects of hydraulic retention time (HRT) and the chemical oxygen demand/nitrogen (COD/N) ratio on the microbial community composition of integrated fixed-film activated sludge (IFAS) and its ability to treat raw and ozonated oil sands process-affected water (OSPW) were evaluated. After 11 months of HRT and ammonium optimization, 54.56% of the COD and 30.20% of the acid extractable fraction (AEF) were removed in raw OSPW IFAS, and 56.83% of the COD and 51.51% of the AEF were removed in ozonated OSPW IFAS. Extension of the HRT in the IFAS had no significant effect on the removal of COD and nitrogen, whereas a lower COD/N ratio increased the removal of organics and total nitrogen. The quantitative polymerase chain reaction (q-PCR) indicated that the abundance of nitrifier and denitrifier genes decreased during HRT optimization, and increased significantly after ammonium optimization. Proteobacteria, Nitrospirae, Acidobacteria, and Bacteroidetes were the dominant phyla in IFAS flocs and biofilms according to 454 sequencing. Proteobacteria showed significant decreases in flocs and biofilms after HRT and ammonium IFAS optimization. Nitrospirae and Acidobacteria showed significant upward trends in all biomass forms in optimized IFAS systems compared to unoptimized IFAS systems.

Carbon amendment and soil depth affect the distribution and abundance of denitrifiers in agricultural soils.

The nitrite reductase (nirS and nirK) and nitrous oxide reductase-encoding (nosZ) genes of denitrifying populations present in an agricultural grassland soil were quantified using real-time polymerase chain reaction (PCR) assays. Samples from three separate pedological depths at the chosen site were investigated: horizon A (0-10cm), horizon B (45-55cm), and horizon C (120-130cm). The effect of carbon addition (treatment 1, control; treatment 2, glucose-C; treatment 3, dissolved organic carbon (DOC)) on denitrifier gene abundance and N2O and N2 fluxes was determined. In general, denitrifier abundance correlated well with flux measurements; nirS was positively correlated with N2O, and nosZ was positively correlated with N2 (P < 0.03). Denitrifier gene copy concentrations per gram of soil (GCC) varied in response to carbon type amendment (P < 0.01). Denitrifier GCCs were high (ca. 10(7)) and the bac:nirK, bac:nirS, bac:nir (T) , and bac:nosZ ratios were low (ca. 10(-1)/10) in horizon A in all three respective treatments. Glucose-C amendment favored partial denitrification, resulting in higher nir abundance and higher N2O fluxes compared to the control. DOC amendment, by contrast, resulted in relatively higher nosZ abundance and N2 emissions, thus favoring complete denitrification. We also noted soil depth directly affected bacterial, archaeal, and denitrifier abundance, possibly due to changes in soil carbon availability with depth.

Rhizospheric Denitrification Potential and Related Microbial Characteristics Affected by Secondary Salinization in a Riparian Soil

A pot experiment with simulated saltwater incursion was conducted to study the effects of secondary salinization on the denitrification potential and related microbiological activities and size of the denitrifier community by real-time PCR quantification of denitrifying genes in a rhizospheric soil vegetated with different riparian plants. The results showed that saltwater incursion significantly inhibited denitrification potential in the riparian soil, with the average denitrification rate decreased by 34.9%. The inhibitory effects differed with the vegetation types. The rhizosphere soils of Acorus calamus and Phragmites australis showed a significantly lower inhibitory percent of the denitrification rate compared with the corresponding monoculture. Secondary salinization resulted in a decrease in the number of N-cycling microorganisms and in the activities of soil N-transforming enzymes, especially the number of denitrifiers and nitrite reductase activity, which exhibited declines of 51.5% and 43.5%, respectively. Real-time PCR quantification showed that the saltwater incursion significantly changed the abundance and composition of denitrifying genes in the riparian soil despite the vegetation types. The community size of narG was the most sensitive, while nosZ was the least affected by secondary salinization. There were significant differences in the responses of N-transforming microbiological activities and denitrifying gene abundance to secondary salinization in riparian soils with different vegetation types. The inhibition percents of N transforming-related enzyme activities and the abundances of denitrifying genes in the rhizospheric soil of A. calamus and P. australis were significantly lower compared with the pure plant community, indicating that mixed vegetation provided a significant alleviation of the inhibition of microbiological parameters and denitrification processes due to saltwater incursion in riparian soil. Principal component analysis (PCA) analysis indicated that the denitrification rate correlated significantly with the activities of N transform-related enzymes and the abundance of narG, nirK and nirS rather than nosZ.

Molecular techniques and stable isotope ratios at natural abundance give complementary inferences about N2O production pathways in an agricultural soil following a rainfall event

The abatement of anthropogenic nitrous oxide (N2O) emissions depends on sound management of manure nitrogen (N) and an ability to track and predict manure-derived microbial N2O production in soils. The objective of this study was to investigate the utility of applying a novel combination of micrometeorological, stable isotope, and molecular methods to determine the short-term dynamics of N2O production processes in soil. Nitrous oxide emissions were continuously monitored in two drought-stressed agricultural fields treated with liquid dairy manure applied either in the fall or spring over an N2O emission event triggered by a heavy rainfall. In situ δ15N–N2O and δ18O–N2O measurements were used in conjunction with abundance (DNA) and potential activity (cDNA) measurements of key microbial N cycling gene and transcript (mRNA) targets to evaluate if these two techniques provided similar inferences about N2O soil processes occurring over the emission event. Soil gas was sampled at 4 depths along a profile from 10 to 50 cm below the surface and a multi-layer diffusion model was used to calculate the vertical N2O fluxes, the change in storage, and the net production of N2O in each layer.The rainfall event triggered N2O production in both fields at all depths, but the uppermost soil layer was the main source of N2O emissions throughout. Soil concentrations and surface emissions of N2O increased rapidly reaching a maximum 4 d following rainfall and were accompanied by a significant increase in the abundance of both nitrifier and denitrifier genes and total bacterial and archaeal genes and transcripts. A similar increase in the transcription of functional genes was not detected, but relatively high levels of archaeal ammonia monooxygenase (crenamoA) and bacterial nitrite reductase (nirS) transcripts were present in both fields throughout the study, indicating nitrification and denitrification were occurring concomitantly. High levels of H2O–NOx oxygen exchange at our study site precluded the use of δ18O–N2O values to separate different sources. Prior to the rain, both fields had very small amounts of soil ammonium and the stable nitrogen isotope characterization of N2O in the dry soil implicated denitrification as the main source of N2O rather than nitrifier-denitrification or hydroxylamine oxidation. Following the rain event, the δ15N–N2O values showed a change in production process as nitrifier-denitrification became a more dominant N2O source. By pairing stable isotope measurements with molecular analyses we were able to verify that these two approaches provide similar inferences about soil N2O production processes during an emission event in manure-applied fields. Although significantly higher N2O flux and N2O accumulation in the soil profile were observed for the field receiving manure in the spring, differences in isotope signal and molecular analysis between fields were not observed. To our knowledge this is the first time that isotope and molecular techniques have been used to study processes resulting in N2O production in manure-amended soils in situ. Whereas stable isotopes were useful for directly tracking the pathways of N2O production, molecular analyses revealed the status of the N cycling communities before, during and after the emission event. This information helped explain the observed differences in emissions between fields, and it helped to support our isotope-based conclusions.

Relationships between denitrification gene expression, dissimilatory nitrate reduction to ammonium and nitrous oxide and dinitrogen production in montane grassland soils

The montane grassland soils of Europe store significant amounts of nitrogen (N), and climate change might drive their volatilization due to the stimulation of gaseous nitrous oxide (N2O) and dinitrogen (N2) losses. Hence, a thorough, mechanistic understanding of the processes responsible for N loss and retention such as denitrification and dissimilatory nitrate reduction to ammonium (DNRA) in these soils is urgently needed. Here we aimed to explore the relationships between denitrifier gene abundance and expression with N2 and N2O production and the importance of DNRA versus denitrification in nitrate consumption and N2O production for typical montane grassland soils of Southern Germany. In a laboratory incubation experiment with glucose and nitrate addition, we combined direct measurements of N2O and N2 production with a molecular analysis of the denitrifier communities involved in nitrite, nitric oxide (NO) and N2O reduction and with the quantification of DNRA. The soils originated from a space-for-time climate change experiment, where intact plant-soil mesocosms were exposed for three years either to ambient conditions at a high elevation site (“HE” control treatment) or to predicted climate change conditions (warming, reduced summer precipitation and reduced winter snow cover) by translocation to lower elevation (“LE” climate change treatment).The abundance (DNA) of cnorB genes was significantly reduced in LE soils, whereas the abundance of nosZ genes did not differ between the HE and LE soils. However, the decreased abundance of cnorB genes unexpectedly resulted in slightly increased rather than decreased potential N2O emissions. This effect could be explained by the increased levels of cnorB mRNA and, therefore, the higher physiological activity of the NO reducers in the LE soils. In contrast with the DNA levels, the dynamics of the cnorB mRNA levels followed N2O emission patterns, whereas the nosZ expression was strongly correlated with the N2 emission (R2 = 0.83). The potential rates of DNRA were approximately one-third of the rates of denitrification, and DNRA was not a source for N2O.We conclude that DNRA significantly competes with denitrification in these soils, thus contributing to N conservation. This work demonstrates that the molecular analysis of nosZ gene expression has great potential to contribute to solving the enigmatic problem of understanding N2 loss from soil.

Effects of sulfamethazine on denitrification and the associated N2O release in estuarine and coastal sediments.

Denitrification is an important pathway of nitrogen removal and nitrous oxide (N2O) production in estuarine and coastal ecosystems, and plays a significant role in counteracting aquatic eutrophication induced by excessive nitrogen loads. Estuarine and coastal environments also suffer from increasing antibiotic contamination because of the growing production and usage of antibiotics. In this study, sediment slurry incubation experiments were conducted to determine the influence of sulfamethazine (SMT, a sulphonamide antibiotic) on denitrification and the associated N2O production. Genes important for denitrification and antibiotic resistance were quantified to investigate the microbial physiological mechanisms underlying SMT's effects on denitrification. SMT was observed to significantly inhibit denitrification rates, but increasing concentrations of SMT enhanced N2O release rates. The negative exponential relationships between denitrifying gene abundances and SMT concentrations showed that SMT reduced denitrification rates by restricting the growth of denitrifying bacteria, although the presence of the antibiotic resistance gene was detected during the incubation period. These results imply that the wide occurrence of residual antibiotics in estuarine and coastal ecosystems may influence eutrophication control, greenhouse effects, and atmospheric ozone depletion by inhibiting denitrification and stimulating the release of N2O.

Emissions of nitrous oxide from casts of tropical earthworms belonging to different ecological categories

Abstract While earthworms’ feeding behavior has been shown to be a key driver of greenhouse gas like nitrous oxide (N 2 O) emissions for temperate species, this assessment remains to be demonstrated for tropical ones. This study constitutes a comparative assessment of the impact of different feeding strategies of tropical earthworms on their cast emissions of N 2 O and the characterization of microbial communities associated with these emission dynamics. N 2 O flux was determined in vitro from casts of 2 epigeic ( Dichogaster annae and Eisenia andrei ) and 4 endogeic/epiendogeic ( Metaphire houlleti , Octolasion tyrtaeum , Pontoscolex corethrurus and Amynthas corticis ) earthworm species in the presence or absence of acetylene. Casts of epigeic earthworms, fed on fresh coffee pulp, emitted about 392-fold higher amounts of N 2 O than those of endogeic ones, fed on a tropical soil. Acetylene exposure significantly increased the cast emission rates indicating that (i) apart from N 2 O, dinitrogen (N 2 ) is also emitted by earthworm casts, particularly by the epigeic guild (45%) and (ii) denitrification is the major microbial process responsible for these N-gas emissions from casts. Total bacteria were 10-fold higher in epigeic casts than endogeic but the relative abundance of nitrifier (AOB) and denitrifiers (nirK and nirS) was many times higher in endogeic casts than epigeic ones. The relative gene density of nosZ to (nirK + nirS) was 4-fold higher in epigeic casts than endogeic ones. Moreover, bacterial amoA gene abundance was higher in casts of all species than for their archaeal counterparts. Cast N 2 O emission was positively correlated with nitrifier as well as denitrifier gene abundance, while it was positively correlated with epigeic nitrate and negatively with endogeic nitrate contents. Ammonium contents did not show any relationship with cast emissions. Mean ammonium and nitrate contents were many times higher in casts than food substrates except nitrate in endogeic casts which was 4-fold less than parent food substrate.

Effect of nitrogen and waterlogging on denitrifier gene abundance, community structure and activity in the rhizosphere of wheat

Microbial denitrification plays a key role in determining the availability of soil nitrogen (N) to plants. However, factors influencing the structure and function of denitrifier communities in the rhizosphere remain unclear. Waterlogging can result in root anoxia and increased denitrification, leading to significant N loss from soil and potential nitrous oxide (N(2)O) emissions. This study investigated denitrifier gene abundance, community structure and activity in the rhizosphere of wheat in response to anoxia and N limitation. Denitrifier community structure in the rhizosphere differed from that in bulk soil, and denitrifier gene copy numbers (nirS, nirK, nosZ) and potential denitrification activity were greater in the rhizosphere. Anoxia and N limitation, and in particular a combination of both, reduced the magnitude of this effect on gene abundance (in particular nirS) and activity, with N limitation having greater impact than waterlogging in rhizosphere soil, in contrast to bulk soil where the impact of waterlogging was greater. Increased N supply to anoxic plants improved plant health and increased rhizosphere soil pH, which resulted in enhanced reduction of N(2)O. Both anoxia and N limitation significantly influenced the structure and function of denitrifier communities in the rhizosphere, with reduced root-derived carbon postulated to play an important role.