Nitrous Oxide Reductase Gene Research Articles

Diversity and Abundance of Denitrifying Bacteria in the Sediment of a Eutrophic Estuary

The relationships between denitrifying microbial communities and their controlling factors are largely unknown in eutrophic estuaries sediment. This work showed that in Liaohe Estuary, nirS-type denitrifiers were consistently more abundant and diverse than nirK and nosZ-type denitrifiers, which probably means that they play an important role in nitrogen removal, particularly around nearshore stations. The dominant genera of nirK, nirS and nosZ-type denitrifiers were Sinorhizobium, Pseudomonas, and Azospirillum. Salinity, nitrogen levels, and sediment grain size were the main factors affecting the denitrification process in this eutrophic estuary. These results provide more information about the dynamics of denitrifying microbiota in marine sediments. Summary: The relationships between denitrifying microbial communities and abundance in estuaries sediment and their controlling factors are largely unknown, especially in eutrophic estuaries. In this study, nitrite reductase genes (nirS, nirK) and nitrous oxide reductase genes (nosZ) were used as molecular markers, qPCR and illumina Miseq high-throughput sequencing technology were used to study the relative abundance of key functional microflora groups and major environmental impact factors in Liaohe Estuary. The results showed that nirS-type denitrifiers were consistently more abundant and diverse than nirK and nosZ-type denitrifiers, which suggested that nirS-type denitrifiers probably play an important role in nitrogen removal in Liaohe Estuary, particularly around nearshore stations. The dominant genera of the bacterial containing nirK, nirS and nosZ genes were Sinorhizobium, Pseudomonas, and Azospirillum, in which Sinorhizobium and Azospirillum were nitrogen-fixing bacteria, while Pseudomonas was denitrogenation bacteria. The different dominant denitrifiers indicated that sedimentary denitrification was accomplished by cooperation of different denitrifying species rather than a single species. Salinity, NH4 +, NO3 -, NO2 - and sediment grain size were regarded as determinants for the denitrification process in the sediment of the estuary. Overall, the results of this study suggest that a comprehensive analysis of different denitrifying functional genes may provide more information about the dynamics of denitrifying microbiota in marine sediments.

Flocs are the main source of nitrous oxide in a high-rate anammox granular sludge reactor: insights from metagenomics and fed-batch experiments

Nitrous oxide (N2O) emissions from anammox-based processes are well documented but insight into source of the N2O emission in high-rate anammox granular sludge reactors (AGSR) is limited. In this study, metagenomics and fed-batch experiments were applied to investigate the relative contributions of anammox granules and flocs to N2O production in a high-rate AGSR. Flocs, which constitute only ~10% of total biomass contributed about 60% of the total N2O production. Granules, the main contributor of nitrogen removal (~95%), were responsible for the remaining ~40% of N2O production. This result is inconsistent with reads-based analysis that found the gene encoding clade II type nitrous oxide reductase (nosZII) had similar abundances in both granules and flocs. Another notable trend observed was the relatively higher abundance of the gene for NO-producing nitrite reductase (nir) in comparison to the gene for the nitric oxide reductase gene (nor) in both granules and flocs, indicating nitric oxide (NO) may accumulate in the AGSR. This is significant since NO and N2O pulse assays demonstrated that NO could lead to N2O production from both granules and flocs. However, since anammox bacteria, which were shown to be in higher abundance in granules than in flocs, have the capacity to scavenge NO this provides a mechanism by which its inhibitory effects can be mitigated, limiting N2O release from the granules, consistent with experimental observation. These results demonstrate flocs are the main source of N2O emission in AGSR and provide lab-scale evidence that NO-dependent anammox can mitigate N2O emission.

Spatial Distribution of Nitrogen Metabolism Functional Genes of Eubacteria and Archaebacteria in Dianchi Lake

Nitrogen metabolism plays an important role in the nitrogen cycle and transformation in Dianchi Lake. Not only do eukaryotes participate in nitrogen transformation but prokaryotes, as the main drivers of the nitrogen cycle, also play an extremely important role in the nitrogen cycle. Based on 16S rDNA high-throughput sequencing technology, 13 sites in Caohai and Waihai of Dianchi Lake were monitored, and PICRUSt function analysis method was adopted to analyze the microbial community diversity and key genes of nitrogen metabolism in Dianchi Lake. Bacteria belonging to 35 phyla and 427 genera were found in Dianchi Lake water and mainly included Proteobacteria and Bacteroidetes. Archaea had 14 phyla and 61 genera and mainly belonged to Euryarchaeota. The overall bacterial richness index of Dianchi Lake was higher than that of archaea, and the bacterial diversity index of Caohai was higher than that of Waihai. Functional prediction showed functional richness of bacteria and archaea. There were 35 KO pathways involved in nitrogen metabolism in bacteria, including key genes such as nitrogenous nitrate-reducing gene nirB, nitric oxide reductase gene norB in denitrification, and nitroreductase gene nasK. There were 23 KO pathways involved in nitrogen metabolism in archaea, involving nifH, nifK, and nifD nitrogenase genes in nitrogen fixation. The copy number of nitrogenase genes was significantly higher than that of other nitrogenase genes. The copy number of nitrogen-fixing genes of archaea was higher than that of bacteria, the nitrogen metabolism capacity of archaea in Caohai was higher than that in Waihai, and the potential of nitrogen-fixation of archaea in Dianchi Lake water was higher than that of bacteria. From the perspective of community structure and function prediction of bacteria and archaea, this study discussed the differences of nitrogen cycle in bacteria and archaea in different areas of Dianchi Lake and provided a decision basis for water environment management in Dianchi Lake.

Crop residue carbon-to-nitrogen ratio regulates denitrifier N2O production post flooding

The response of nitrifier and denitrifier populations and associated N2O emissions to different carbon-to-nitrogen (C/N = 17 or 45) straw amendments was monitored under flooding-drying and non-flooding conditions. A 10-week laboratory mesocosm study was conducted in two soils, (i) a paddy soil with a long history of managed flooding-drying (CN), and (ii) a wheat cropping soil with no previous history of flooding (UK). We measured N2O fluxes and the abundances of ammonia-oxidizing archaea (AOA) and bacteria (AOB), nitrite reductase (nirK and nirS) genes, and nitrous oxide reductase (nosZI and nosZII) genes during flooding (4 weeks) and post-flooding (6 weeks). Straw addition enhanced N2O emissions, with higher fluxes apparent after incorporation of narrow C/N residues. Moreover, the impact of crop amendment on N2O emission was exacerbated when soil was under flooding-drying conditions. The abundances of nirS and nosZI genes in CN soil and AOA gene in UK soil were increased by straw amendment, with highest in narrow C/N straw amendments. Structural equation modeling showed that the impact of denitrifier gene abundance on the N2O flux was stronger than that of nitrifier gene abundance in the two soils, and significant correlations were observed between N2O fluxes and the consumption of DOC and NO3−, indicating that denitrification was the dominant N2O production pathway during the drying phase. The ratio of (nirS + nirK)/(nosZI + nosZII) in the narrow C/N amendment was greater than in the wide C/N treatment after flooding, suggesting that the straw C/N ratio had an effect on the capacity for N2O production via denitrification. We conclude that crop amendments with an appropriate C/N ratio could minimize N2O fluxes through regulating the denitrification process when soils are subjected to regular flooding and drying and also experiencing greater frequencies of flooding.

Functional diversity and abundance of nitrogen cycle-related genes in paddy soil

The nitrogen cycle and the associated microbes play an important role in natural ecosystems, including terrestrial habitats; they also have a major effect on climate change. The aim of this study was to explore microbial communities in rice paddy soil by detecting and quantifying some key functional genes involved in the nitrogen cycle using molecular techniques such as conventional polymerase chain reaction (PCR), clone library construction, sequencing, phylogenetic analysis, and real-time PCR. The genes analyzed were as follows: nitrogenase reductase gene (nifH), hydrazine synthase gene (hzsA), nitrous oxide reductase gene (nosZ), copper-containing (nirK) and cytochrome cd1-containing (nirS) nitrite reductase genes, nitrite oxidoreductase gene (nxrB), and ammonium monooxygenase gene (amoA). The sequence assessment using the clone library targeting these genes revealed high diversity and dominance of bacterial communities. Furthermore, real-time PCR using SYBR green dye and some primers specific for each gene revealed the high abundance of nxrB (4.1 × 109 ± 0.4 × 109 copies g−1 soil) and low abundance of hzsA (4.0 × 105 ± 1.1 × 105 copies g−1 soil). The findings of our study will be useful to explore microbial communities in terrestrial habitats, such as agricultural paddy fields.

Wheat rhizodeposition stimulates soil nitrous oxide emission and denitrifiers harboring the nosZ clade I gene

Stimulatory effects of growing plants on nitrous oxide (N2O) emissions have been widely reported in terrestrial ecosystems, but the potential mechanisms responsible for these effects remain unclear. This study revealed that wheat can induce a 3.5–9.2-fold increase in N2O emissions under different soil fertility levels, and that this “plant” source of N2O occurs in the rhizosphere. Moreover, plants induced soil niche differentiation between denitrifiers harboring the nitrous oxide reductase genes nosZI and nosZII. Pulse labeling of wheat demonstrated that 67% of 13C-labeled nosZI-type denitrifiers, but no nosZII-type denitrifiers, were more abundant in the rhizosphere than in bulk soil. Furthermore, a higher percentage of bacterial genomes containing nitrite reductase genes was found within plant-associated nosZI-type denitrifiers than nosZII-type denitrifiers, favoring NO2− to N2O conversion. Overall, this study revealed a strong selective stimulating effect of wheat on soil denitrifiers through root-derived carbon and a key role of the nosZI-type community in rhizosphere denitrification.

The responses of bacterial community and N2O emission to nitrogen input in lake sediment: Estrogen as a co-pollutant

Excessive nitrogen (N) input is one of the most important causative factors of lake eutrophication, which has aroused increasing public attention in past decades. Estrogen contamination is also an increasing environmental problem in aquatic systems around the world. Although both substances usually co-exist in aquatic ecosystems, many researches have only investigated the influences of either N or estrogen individually on sediment bacterial community and nitrous oxide (N2O) emission. Knowledge regarding the combined effects of N and estrogen is still very limited. In this study, a 30-day laboratory incubation experiment was performed to examine the impacts of different N sources (ammonium and nitrate) combined with 17β-estradiol (E2) on sediment bacterial community. High-throughput 16S rRNA gene sequencing technique was used and N2O emission was measured. The results revealed that the relative abundances of Proteobacteria and Bacteroidetes were higher in nitrate treatment than ammonium treatment. Compared to N treatments, N and E2 combined treatments showed higher relative abundances of Proteobacteria, Bacteroidetes, and Firmicutes, but lower relative abundances of Chloroflexi, Acidobacteria, and Actinobacteria over entire incubation period. At the genus level, the relative abundances of genera Flavobacterium, Pseudomonas, Arenimonas, Novosphingobium, Massilia, Aquabacterium, and Bacillus were enhanced by N treatments and especially N and E2 combined treatments, compared to sediment without addition of N and E2. However, the relative abundances of Sporacetigenium, Gaiella, Desulfatiglans, Nitrospira, and Haliangium were decreased in N treatments. Apart from the changes in bacterial community structure, N2O emission was also influenced by different treatments. Nitrate exerted a more significant positive effect on N2O emission than ammonium, and the cumulative emission of N2O was highest in nitrate and E2 combined treatment. Very low abundances of ammonia monooxygenase (amoA) gene and hydroxylamine oxidase (hao) gene were observed in sediments compared to other genes involved in N cycles (such as nitrate reductase (narG and napA) genes, nitrite reductase (nirB, nirK, and nrfA) genes, and nitric oxide reductase (norB) gene), implying that denitrification rather than nitrification played an important role in sediments. The abundances of napA, nirK, and norB were higher in N and E2 combined treatments, indicating that E2 might provide a carbon source for denitrifiers. Moreover, decrease in the abundance of nitrous oxide reductase (nosZ) gene during the denitrifying process in N and E2 combined treatment might be an important reason for increases of N2O emission. These results indicated that alterations of the bacterial community structure due to the co-existence of N and E2 could also change the abundances of genes involved in N cycle.

Conversion of monoculture cropland and open grassland to agroforestry alters the abundance of soil bacteria, fungi and soil-N-cycling genes.

Integration of trees in agroforestry systems can increase the system sustainability compared to monocultures. The resulting increase in system complexity is likely to affect soil-N cycling by altering soil microbial community structure and functions. Our study aimed to assess the abundance of genes encoding enzymes involved in soil-N cycling in paired monoculture and agroforestry cropland in a Phaeozem soil, and paired open grassland and agroforestry grassland in Histosol and Anthrosol soils. The soil fungi-to-bacteria ratio was greater in the tree row than in the crop or grass rows of the monoculture cropland and open grassland in all soil types, possibly due to increased input of tree residues and the absence of tillage in the Phaeozem (cropland) soil. In the Phaeozem (cropland) soil, gene abundances of amoA indicated a niche differentiation between archaeal and bacterial ammonia oxidizers that distinctly separated the influence of the tree row from the crop row and monoculture system. Abundances of nitrate (napA and narG), nitrite (nirK and nirS) and nitrous oxide reductase genes (nosZ clade I) were largely influenced by soil type rather than management system. The soil types’ effects were associated with their differences in soil organic C, total N and pH. Our findings show that in temperate regions, conversion of monoculture cropland and open grassland to agroforestry systems can alter the abundance of soil bacteria and fungi and soil-N-cycling genes, particularly genes involved in ammonium oxidation.

Variable responses of nitrification and denitrification in a paddy soil to long-term biochar amendment and short-term biochar addition

Biochar-mediated change in soil N transformations has gained much attention. Biochar properties undergo changes through ageing process that may impact variably on N cycling, yet the degree to which soil N transformations may be impacted by biochar ageing process on long-time scale remains unclear. Here, we compared the effects on nitrification, denitrification and N2/N2O gas flux of short-term fresh biochar addition with long-term biochar amendment over six consecutive years in a paddy soil under identical amounts of soil organic C contents. We found that short-term treatments elicited a stronger effect on nitrification than long-term treatments, due to more positive effects on soil pH and ammonia monooxygenase gene (amoA) abundance of ammonia oxidizing bacterial (AOB). However, there were contrasting effects on soil denitrification and total N gas loss for two types of biochar addition, where the long-term treatments limited availability of C and N, and decreased net NO3− reduction and total N gas (N2O + N2) losses by up to 55.5% and 25%. Short-term biochar addition increased dissolved organic C, nutrient content and soil pH, enhanced net NO3− reduction by 88.7%, also reduced the ratio of nitrite reductase genes (nirS and nirK) and nitrous oxide reductase gene (nosZ) and N2O emissions, but increased total N gas loss by 0.2–1.2 times. Our study quantified the differences in nitrification and denitrification capacity in response to short-term biochar addition and long-term biochar amendment and bridged the knowledge-gaps of fresh and aged biochar-mediated N transformation.

Performance and microbial consortium structure in simultaneous removal of sulfur and nitrogen compounds under micro-oxygenated condition

Effects of COD/SO42− and SO42−/NH4+ ratios on simultaneous removal of sulfate- and ammonium-rich synthetic wastewaters were investigated under micro-oxygenated condition (dissolved oxygen concentration at 0.10–0.15 mg/L). Lactate was served as carbon source to generate COD/SO42− ratios of 2.0, 2.5, 3.0 3.5 and 4.0. The batch experimental results indicated that the highest sulfate (72.1%) and ammonium (62.8%) removal efficiencies could be reached at COD/SO42− ratio of 4.0. The main metabolic products were elemental sulfur (0.63 g S0/g SO42−–Sadded) and nitrogen gas (0.57 g N2/g NH4+–Nadded). Subsequently, various SO42−/NH4+ ratios (0.5, 1.0, 1.5, 2.0 and 2.5) were performed at controlled COD/SO42− ratio of 4.0. The highest SO42−/NH4+ ratio of 2.5 provided 76.6 and 72.8% sulfate and ammonium removal efficiencies, respectively, and also reached the highest yield of elemental sulfur and nitrogen gas of 0.68 g S0/g SO42−–Sadded and 0.66 g N2/g NH4+–Nadded, respectively. Microbial consortium structure providing the highest removal efficiencies was consequently analyzed using Illumina sequencing and polymerase chain reaction-denaturing gradient gel electrophoresis approaches. Taxonomic assignments demonstrated that Proteobacteria (46%), Firmicutes (15%), and Bacteroidetes (14%) were the most abundant phyla. Almost core genera analysis with two distinguished approaches demonstrated similar results. Aside from microbial community analysis, quantitative real-time polymerase chain reaction was used to validate existing abundance of aforementioned seven dominant microorganisms. The nitrous oxide reductase gene was shown the most abundance (~ 108 copies/µL) which plays a crucial role for simultaneous removal of sulfur and nitrogen compounds.

New Breeding Techniques for Greenhouse Gas (GHG) Mitigation: Plants May Express Nitrous Oxide Reductase

Nitrous oxide (N2O) is a potent greenhouse gas (GHG). Although it comprises only 0.03% of total GHGs produced, N2O makes a marked contribution to global warming. Much of the N2O in the atmosphere issues from incomplete bacterial denitrification processes acting on high levels of nitrogen (N) in the soil due to fertilizer usage. Using less fertilizer is the obvious solution for denitrification mitigation, but there is a significant drawback (especially where not enough N is available for the crop via N deposition, irrigation water, mineral soil N, or mineralization of organic matter): some crops require high-N fertilizer to produce the yields necessary to help feed the world’s increasing population. Alternatives for denitrification have considerable caveats. The long-standing promise of genetic modification for N fixation may be expanded now to enhance dissimilatory denitrification via genetic engineering. Biotechnology may solve what is thought to be a pivotal environmental challenge of the 21st century, reducing GHGs. Current approaches towards N2O mitigation are examined here, revealing an innovative solution for producing staple crops that can ‘crack’ N2O. The transfer of the bacterial nitrous oxide reductase gene (nosZ) into plants may herald the development of plants that express the nitrous oxide reductase enzyme (N2OR). This tactic would parallel the precedents of using the molecular toolkit innately offered by the soil microflora to reduce the environmental footprint of agriculture.

Seasonal N2O emissions respond differently to environmental and microbial factors after fertilization in wheat–maize agroecosystem

Biogeochemical processes regulating cropland soil nitrous oxide (N2O) emissions are complex, and the controlling factors need to be better understood, especially for seasonal variation after fertilization. Seasonal patterns of N2O emissions and abundances of archaeal ammonia monooxygenase (amoA), bacterial amoA, nitrate reductase (narG), nitrite reductase (nirS/nirK), and nitrous oxide reductase (nosZ) genes in long-term fertilized wheat–maize soils have been studied to understand the roles of microbes in N2O emissions. The results showed that fertilization greatly stimulated N2O emission with higher values in pig manure-treated soil (OM, 2.88 kg N ha−1 year−1) than in straw-returned (CRNPK, 0.79 kg N ha−1 year−1) and mineral fertilizer-treated (NPK, 0.90 kg N ha−1 year−1) soils. Most (52.2–88.9%) cumulative N2O emissions occurred within 3 weeks after fertilization. Meanwhile, N2O emissions within 3 weeks after fertilization showed a positive correlation with narG gene copy number and a negative correlation with soil NO3− contents. The abundances of narG and nosZ genes had larger direct effects (1.06) than ammonium oxidizers (0.42) on N2O emissions according to partial least squares path modeling. Stepwise multiple regression also showed that log narG was a predictor variable for N2O emissions. This study suggested that denitrification was the major process responsible for N2O emissions within 3 weeks after fertilization. During the remaining period of crop growth, insufficient N substrate and low temperature became the primary limiting factors for N2O emission according to the results of the regression models.

Bulk soil bacterial community structure and function respond to long-term organic and conventional agricultural management.

Understanding how soil microbiomes respond to management is essential to maximizing soil health. We contrasted microbiomes in bulk soil under long-term organic and conventional management in a grain production setting. Management category significantly impacted the relative abundances of 17% of the most abundant taxa. Both conventional and organic management favored particular taxa, but these effects were not reflected in summary richness and diversity indices. Management systems also lead to differences in soil edaphic properties, including pH and nutrient status; this may have been the mechanism by which change in the prokaryote community was enacted. Community change between years of sampling was less pronounced, with only 6 taxa differentially abundant among years. Management category also impacted the abundance of functional genes related to the production and consumption of greenhouse gases. Particulate methane monooxygenase genes were more frequent in soil under organic management, while soluble methane monooxygenase genes were more frequent in soil under conventional management in 1 of 2 years. Nitrous oxide reductase genes were significantly less abundant in soils under second-year alfalfa than in soils under corn. This work highlights the ability of agricultural management to enact broad rearrangements to the structure of bulk soil bacterial communities.

Denitrifier communities impacted by heavy metal contamination in freshwater sediment

Heavy metals are widely detected in natural environments, however their impacts on denitrifier community in freshwater ecosystem remain unclear. The present study investigated the changes of denitrifier communities (based on nosZ (nitrous oxide reductase) gene) in a freshwater reservoir contaminated by a severe accidental spill of heavy metals. The abundance of nosZ-denitrifiers drastically decreased, and their community richness, diversity and structure also showed considerable variations. The mainly detected denitrifying bacteria included Pseudogulbenkiania, Pseudomonas and two unknown groups. These major nosZ-denitrifier groups responded in different ways to heavy metal pollution. Metal contamination could exert a profound influence on denitrifier community in freshwater sediment. This work could provide some new insights to the impact of metal pollution on nitrogen cycling.

High pyrolysis temperature biochars reduce nitrogen availability and nitrous oxide emissions from an acid soil

AbstractBiochar–bioenergy coproduction from biomass pyrolysis has the potential to contribute to climate change mitigation. Biochar produced at various pyrolysis temperatures (<600°C) has been widely studied. However, the effect of biochars, produced at high pyrolysis temperature (≥600°C), on soil nitrogen (N) dynamics and nitrous oxide (N2O) emission is largely unknown. A pot trial was performed to examine the effect of high pyrolysis temperature (600, 700, 850 and 950°C) woody biochars on soil N dynamics, microbial gene abundance and N2O emissions with (+N) and without N (−N) fertilization from an acid soil. Results showed that all biochar treatments significantly lowered the N2O emissions in both fertilized and unfertilized regimes. However, the suppressive effect on N2O emission among different high pyrolysis temperatures was not statistically different. Biochar amendment significantly decreased the concentration of soil NH4+, and lower levels of soil NO3− were observed at the later stage of experiment. Under −N, plant biomass and N uptake were significantly lowered in all biochar treatments. Under +N, biochar addition significantly increased plant biomass, while only the 700°C biochar significantly increased N uptake. This suggests that single application of biochar could limit soil mineral N bioavailability and further decrease plant growth and N uptake in the plant–soil system. Biochar amendments tended to increase nitrous oxide reductase (nosZ) gene abundance, but this effect was only significant for biochar produced at 950°C under +N. In conclusion, high pyrolysis temperature biochars can be effectively used to reduce N2O emission, while increases in nosZ gene abundance and decreases in NH4+ and NO3− concentrations in the acid soil are likely to be responsible for the reduction in N2O emission. Thus, woody biochars as a by‐product produced at high pyrolysis temperature have the potential to mitigate soil N2O emission via modifying N transformation and further affect climate change.

Nitrogen removal and metabolic profiling of a cold-adaptive and biofilm producing paddy soil bacterium Cupriavidus sp. PDN31

ABSTRACTTo assess the physiology and low temperature adaptability of the key players of nitrification and denitrification, denitrifying bacteria were isolated and characterized from the selected paddy fields. Bacterial strains belonging to Cupriavidus and Ochrobactrum sp. were explored through the selective screening of heterotrophic nitrifying and aerobic denitrifying bacteria. The direct implication of nitrate removal in the natural sample was estimated by taking the nitrate supplemented soil as well as the enriched culture. A more prominent cold-adaptive bacterium was identified as Cupriavidus sp. PDN31. The utilization of ammonium, nitrate, and nitrite and the presence of nitrous oxide reductase (nosZ) gene, catalyses the first step of the denitrification conferred its heterotrophic nitrification and aerobic denitrification ability. The ammonium, nitrate, and nitrite removal efficiency of PDN31 was found to be 92.1%, 93.5%, and 99.8%, respectively. The functional traits, evaluated from metabolizing various nitrogen substrates (Biolog) suggested its ability to utilize some sources as L-arginine, L-asparagine, L-cysteine, L-glutamic Acid, L-glutamine, L-histidine, L-citrulline and N-acetyl-L glutamic acid. The adaptive behaviour of PDN31 with its ability to remove nitrogen and induced biofilm production under low temperature regime makes it a suitable candidate among the plethora of microorganism resided in any agriculture environment.

Absence of the Nitrous Oxide Reductase Gene Cluster in Commercial Alfalfa Inoculants Is Probably Due to the Extensive Loss of Genes During Rhizobial Domestication.

As other legume crops, alfalfa cultivation increases the emission of the greenhouse gas nitrous oxide (N2O). Since legume-symbiotic nitrogen-fixing bacteria play a crucial role in this emission, it is important to understand the possible impacts of rhizobial domestication on the evolution of denitrification genes. In comparison with the genomes of non-commercial strains, those of commercial alfalfa inoculants exhibit low total genome size, low number of ORFs and high numbers of both frameshifted genes and pseudogenes, suggesting a dramatic loss of genes during bacterial domestication. Genomic analysis focused on denitrification genes revealed that commercial strains have perfectly conserved the nitrate (NAP), nitrite (NIR) and nitric (NOR) reductase clusters related to the production of N2O from nitrate but completely lost the nitrous oxide (NOS) reductase cluster (nosRZDFYLX genes) associated with the reduction of N2O to gas nitrogen. Based on these results, we propose future screenings for alfalfa-nodulating isolates containing both nitrogen fixation and N2O reductase genes for environmental sustainability of alfalfa production.

Quantitative analyses of denitrifying bacterial diversity from a seasonally hypoxic monsoon governed tropical coastal region

Microbial community mediated denitrification leads to nitrate reduction and, its eventual removal from marine ecosystems. Therefore, it is necessary to understand the functional diversity in terms of the abundance and types of nitrate reductases in the microbial communities extant in the hypoxic/suboxic waters where organic matter oxidation is predominantly nitrate- reduction mediated. For this study, water samples from the surface, mid-depth and close to bottom were collected from three coastal locations along the west coast of India during Summer monsoon (SuM), Fall intermonsoon (FIM) and, Spring intermonsoon (SIM). The abundance of nitrate (narG), nitrite (nirS) and nitrous oxide (nosZ) reductase genes from these water samples were quantified by qPCR method. Besides the distinct spatio-temporal differences, the overall ranges of these three functional genes across the sampling sites ranged from 1.9 x 107 to 7.9 x 107 copies L-1 (narG), from 0.001 x 106 to 0.30 x 106 copies L-1 (nirS) and from 0.30 x 106 to 2.9 x 106 copies L-1 (nosZ). Functional gene sequencing of bacterial communities possessing these reductases were diverse and, dominated by members of Alpha-, Beta- and Gamma-proteobacteria, including taxonomic groups containing well known denitrifiers such as Pseudogulbenkiania, Kocuria, Pseudomonas, Herbaspirillum, Achromobacter, Rhodoferax, Mesorhizobium, Sinorhizobium, Shinella, Bradyrhizobiaceae, Rhodopseudomonas, Dinoroseobacter, Paracoccus, Rhodobacter, Ruegeria and Azospirillum sp. Assemblages of bacteria possessing narG, nirS, and nosZ indicated that complete denitrification could occur, particularly in the samples collected close to the bottom.

Long-term effects of contrasting tillage on soil organic carbon, nitrous oxide and ammonia emissions in a Mediterranean Vertisol under different crop sequences

This 2-year study aimed to verify whether the continuous application of no tillage (NT) for over 20years, in comparison with conventional tillage (CT), affects nitrous oxide (N2O) and ammonia (NH3) emissions from a Vertisol and, if so, whether such an effect varies with crop sequence (continuous wheat, WW and wheat after faba bean, FW). To shed light on the mechanisms involved in determining N-gas emissions, soil bulk density, water filled pore space (WFPS), some carbon (C) and nitrogen (N) pools, denitrifying enzyme activity (DEA), and nitrous oxide reductase gene abundance (nosZ gene) were also assessed at 0–15 and 15–30cm soil depth. Tillage system had no significant effect on total NH3 emissions. On average, total N2O emissions were higher under NT (2.45kgN2O-Nha−1) than CT (1.72kgN2O-Nha−1), being the differences between the two tillage systems greater in FW than WW. The higher N2O emissions in NT treatments were ascribed to the increased bulk density, WFPS, and extractable organic C under NT compared to CT, all factors that generally promote the production of N2O. Moreover, compared to CT, NT enhanced the potential DEA (114 vs 16μgNkg−1h−1) and nosZ gene abundance (116 vs 69 copy number mg−1 dry soil) in the topsoil. Finally, NT compared to CT led to an average annual increase in C stock of 0.70MgCha−1year−1. Though NT can increase the amount os soil organic matter so storing CO2 into soil, some criticisms related to the increase of N2O emission arise, thereby suggesting the need for defining management strategies to mitigate such a negative effect.

Responses of N2O reductase gene (nosZ)-denitrifier communities to long-term fertilization follow a depth pattern in calcareous purplish paddy soil

The effect of long-term fertilization on soil denitrifying communities was analysed by measuring the abundance and diversity of the nitrous oxide (N2O) reductase gene, nosZ. Soil samples were collected from plots of a long-term fertilization experiment established in 1982 in Suining City, China. The fertilizer treatments were no fertilizer (CK), three chemical fertilizer (CF) treatments (N, NP, NPK), manure (M) alone, and manure with chemical fertilizers (NM, NPM, NPKM). The abundance and diversity of the denitrifying bacteria were assessed by real-time quantitative PCR, terminal restriction fragment length polymorphism (T-RFLP), and cloning and sequencing of nosZ genes. The diversity and abundance of nosZ-denitrifiers was higher in soil amended with manure and chemical fertilizers (CFM) than in soil amended with CF alone, and the highest in topsoil (0–20 cm). The nosZ-denitrifier community composition was more complex in CFM soil than in CF soil. Specific species were detected only in the CFM soil. The abundance of nosZ-denitrifier in the NPKM treatment was approximately two times higher than that in the CK, N, and NPK treatments. Most of the cloned nosZ sequences were closely related to nosZ sequences from Bradyrhizobiaceae and Rhodospirillaceae in Alphaproteobacteria. Of the measured abiotic factors, soil organic matter correlated significantly with the abundance (P<0.01); available phosphorus correlated significantly with the topsoil community composition (P<0.01), whereas soil organic matter correlated significantly with the subsoil (20–90 cm) community composition (P<0.01). This study demonstrated that long-term CFM fertilization affected both the abundance and composition of the nosZ-denitrifier community.