Changes in Diversity and Functional Gene Abundances of Microbial Communities Involved in Nitrogen Fixation, Nitrification, and Denitrification in a Tidal Wetland versus Paddy Soils Cultivated for Different Time Periods

In many areas of China, tidal wetlands have been converted into agricultural land for rice cultivation. However, the consequences of land use changes for soil microbial communities are poorly understood. Therefore, we investigated bacterial and archaeal communities involved in inorganic nitrogen turnover (nitrogen fixation, nitrification, and denitrification) based on abundances and relative species richness of the corresponding functional genes along a soil chronosequence ranging between 50 and 2,000 years of paddy soil management compared to findings for a tidal wetland. Changes in abundance and diversity of the functional groups could be observed, reflecting the different chemical and physical properties of the soils, which changed in terms of soil development. The tidal wetland was characterized by a low microbial biomass and relatively high abundances of ammonia-oxidizing microbes. Conversion of the tidal wetlands into paddy soils was followed by a significant increase in microbial biomass. Fifty years of paddy management resulted in a higher abundance of nitrogen-fixing microbes than was found in the tidal wetland, whereas dominant genes of nitrification and denitrification in the paddy soils showed no differences. With ongoing rice cultivation, copy numbers of archaeal ammonia oxidizers did not change, while that of their bacterial counterparts declined. The nirK gene, coding for nitrite reductase, increased with rice cultivation time and dominated its functionally redundant counterpart, nirS, at all sites under investigation. Relative species richness showed significant differences between all soils with the exception of the archaeal ammonia oxidizers in the paddy soils cultivated for 100 and 300 years. In general, changes in diversity patterns were more pronounced than those in functional gene abundances.     

Nitrogen-Cycling Genes in Epilithic Biofilms of Oligotrophic High-Altitude Lakes (Central Pyrenees,Spain)

Microbial biofilms in oligotrophic environments are the most reactive component of the ecosystem. In high-altitude lakes, exposed bedrock, boulders, gravel, and sand in contact with highly oxygenated water and where a very thin epilithic biofilm develops usually dominate the littoral zone. Traditionally, these surfaces have been considered unsuitable for denitrification, but recent investigations have shown higher biological diversity than expected, including diverse anaerobic microorganisms. In this study, we explored the presence of microbial N-cycling nirS and nirK (denitrification through the conversion of NO₂ ^? to NO), nifH (N₂ fixation), anammox (anaerobic ammonium oxidation), and amoA (aerobic ammonia oxidation, both bacterial and archaeal) genes in epilithic biofilms of a set of high-altitude oligotrophic lakes in the Pyrenees. The concentrations of denitrifying genes determined by quantitative PCR were two orders of magnitude higher than those of ammonia-oxidizing genes. Both types of genes were significantly correlated, suggesting a potential tight coupling nitrification-denitrification in these biofilms that deserves further confirmation. The nifH gene was detected after nested PCR, and no signal was detected for the anammox-specific genes used. The taxonomic composition of denitrifying and nitrogen-fixing genes was further explored by cloning and sequencing. Interestingly, both microbial functional groups were richer and more genetically diverse than expected. The nirK gene, mostly related to Alphaproteobacteria (Bradyrhizobiaceae), dominated the denitrifying gene pool as expected for oxygen-exposed habitats, whereas Deltaproteobacteria (Geobacter like) and Cyanobacteria were the most abundant among nitrogen fixers. Overall, these results suggest an epilithic community more metabolically diverse than previously thought and with the potential to carry out an active role in the biogeochemical nitrogen cycling of high-altitude ecosystems. Measurements of activity rates should be however carried out to substantiate and further explore these findings.     

Changes in Bacterial and Archaeal Community Structure and Functional Diversity along a Geochemically Variable Soil Profile

Spatial heterogeneity in physical, chemical, and biological properties of soils allows for the proliferation of diverse microbial communities. Factors influencing the structuring of microbial communities, including availability of nutrients and water, pH, and soil texture, can vary considerably with soil depth and within soil aggregates. Here we investigated changes in the microbial and functional communities within soil aggregates obtained along a soil profile spanning the surface, vadose zone, and saturated soil environments. The composition and diversity of microbial communities and specific functional groups involved in key pathways in the geochemical cycling of nitrogen, Fe, and sulfur were characterized using a coupled approach involving cultivation-independent analysis of both 16S rRNA (bacterial and archaeal) and functional genes (amoA and dsrAB) as well as cultivation-based analysis of Fe(III)-reducing organisms. Here we found that the microbial communities and putative ammonia-oxidizing and Fe(III)-reducing communities varied greatly along the soil profile, likely reflecting differences in carbon availability, water content, and pH. In particular, the Crenarchaeota 16S rRNA sequences are largely unique to each horizon, sharing a distribution and diversity similar to those of the putative (amoA-based) ammonia-oxidizing archaeal community. Anaerobic microenvironments within soil aggregates also appear to allow for both anaerobic- and aerobic-based metabolisms, further highlighting the complexity and spatial heterogeneity impacting microbial community structure and metabolic potential within soils.     

Draft Genome Sequence of an Ammonia-Oxidizing Archaeon, “Candidatus Nitrosopumilus koreensis” AR1, from Marine Sediment

Ammonia-oxidizing archaea (AOA) are ubiquitous in various marine environments and play important roles in the global nitrogen and carbon cycles. We here present a high-quality draft genome sequence of an ammonia-oxidizing archaeon, “Candidatus Nitrosopumilus koreensis” AR1, which was found to dominate an ammonia-oxidizing enrichment culture in marine sediment off Svalbard, the Arctic Circle. Despite a significant number of nonoverlapping genes (ca. 30%), similarities of this strain to “Candidatus Nitrosopumilus maritimus” were revealed by core genes for archaeal ammonia oxidation and carbon fixation, G+C content, and extensive synteny conservation.     

Soil microbes of an urban remnant riparian zone have greater potential for N removal than a degraded riparian zone

Soils in the riparian zone, the interface between terrestrial and aquatic ecosystems, may decrease anthropogenic nitrogen (N) loads to streams through microbial transformations (e.g., denitrification). However, the ecological functioning of riparian zones is often compromised due to degraded conditions (e.g., vegetation clearing). Here we compare the efficacy of an urban remnant and a cleared riparian zone for supporting a putative denitrifying microbial community using 16S rRNA sequencing and quantitative polymerase chain reaction of archaeal and bacterial nitrogen cycling genes. Although we had no direct measure of denitrification rates, we found clear patterns in the microbial communities between the sites. Greater abundance of N-cycling genes was predicted by greater soil ammonium (N-NH₄), organic phosphorus, and C:N. At the remnant site, we found positive correlations between microbial community composition, which was dominated by putative N oxidisers (Nitrosomonadaceae, Nitrospiraceae and Nitrosotaleaceae), and abundance of ammonia-oxidizing archaea (AOA), nirS, nirK and nosZ, whereas the cleared site had lower abundance of N-oxidisers and N cycling genes. These results were especially profound for the remnant riparian fringe, which suggests that this region maintains suitable soil conditions (via diverse vegetation structure and periodic saturation) to support putative N cyclers, which could amount to higher potential for N removal.     

Abundance of Microbes Involved in Nitrogen Transformation in the Rhizosphere of Leucanthemopsis alpina (L.) Heywood Grown in Soils from Different Sites of the Damma Glacier Forefield

Glacier forefields are an ideal playground to investigate the role of development stages of soils on the formation of plant–microbe interactions as within the last decades, many alpine glaciers retreated, whereby releasing and exposing parent material for soil development. Especially the status of macronutrients like nitrogen differs between soils of different development stages in these environments and may influence plant growth significantly. Thus, in this study, we reconstructed major parts of the nitrogen cycle in the rhizosphere soil/root system of Leucanthemopsis alpina (L.) Heywood as well as the corresponding bulk soil by quantifying functional genes of nitrogen fixation (nifH), nitrogen mineralisation (chiA, aprA), nitrification (amoA AOB, amoA AOA) and denitrification (nirS, nirK and nosZ) in a 10-year and a 120-year ice-free soil of the Damma glacier forefield. We linked the results to the ammonium and nitrate concentrations of the soils as well as to the nitrogen and carbon status of the plants. The experiment was performed in a greenhouse simulating the climatic conditions of the glacier forefield. Samples were taken after 7 and 13 weeks of plant growth. Highest nifH gene abundance in connection with lowest nitrogen content of L. alpina was observed in the 10-year soil after 7 weeks of plant growth, demonstrating the important role of associative nitrogen fixation for plant development in this soil. In contrast, in the 120-year soil copy numbers of genes involved in denitrification, mainly nosZ were increased after 13 weeks of plant growth, indicating an overall increased microbial activity status as well as higher concentrations of nitrate in this soil.     

Quantitative PCR Analysis of Functional Genes in Iron-Rich Microbial Mats at an Active Hydrothermal Vent System (Lō'ihi Seamount,Hawai'i)

The chemolithotrophic Zetaproteobacteria represent a novel class of Proteobacteria which oxidize Fe(II) to Fe(III) and are the dominant bacterial population in iron-rich microbial mats. Zetaproteobacteria were first discovered at Lō''ihi Seamount, located 35 km southeast off the big island of Hawai''i, which is characterized by low-temperature diffuse hydrothermal venting. Novel nondegenerate quantitative PCR (qPCR) assays for genes associated with microbial nitrogen fixation, denitrification, arsenic detoxification, Calvin-Benson-Bassham (CBB), and reductive tricarboxylic acid (rTCA) cycles were developed using selected microbial mat community-derived metagenomes. Nitrogen fixation genes were not detected, but all other functional genes were present. This suggests that arsenic detoxification and denitrification processes are likely cooccurring in addition to two modes of carbon fixation. Two groups of microbial mat community types were identified by terminal restriction fragment length polymorphism (T-RFLP) and were further described based on qPCR data for zetaproteobacterial abundance and carbon fixation mode preference. qPCR variance was associated with mat morphology but not with temperature or sample site. Geochemistry data were significantly associated with sample site and mat morphology. Together, these qPCR assays constitute a functional gene signature for iron microbial mat communities across a broad array of temperatures, mat types, chemistries, and sampling sites at Lō''ihi Seamount.     

Microbial Metabolic Potential for Carbon Degradation and Nutrient (Nitrogen and Phosphorus) Acquisition in an Ombrotrophic Peatland

This study integrated metagenomic and nuclear magnetic resonance (NMR) spectroscopic approaches to investigate microbial metabolic potential for organic matter decomposition and nitrogen (N) and phosphorus (P) acquisition in soils of an ombrotrophic peatland in the Marcell Experimental Forest (MEF), Minnesota, USA. This analysis revealed vertical stratification in key enzymatic pathways and taxa containing these pathways. Metagenomic analyses revealed that genes encoding laccases and dioxygenases, involved in aromatic compound degradation, declined in relative abundance with depth, while the relative abundance of genes encoding metabolism of amino sugars and all four saccharide groups increased with depth in parallel with a 50% reduction in carbohydrate content. Most Cu-oxidases were closely related to genes from Proteobacteria and Acidobacteria, and type 4 laccase-like Cu-oxidase genes were >8 times more abundant than type 3 genes, suggesting an important and overlooked role for type 4 Cu-oxidase in phenolic compound degradation. Genes associated with sulfate reduction and methanogenesis were the most abundant anaerobic respiration genes in these systems, with low levels of detection observed for genes of denitrification and Fe(III) reduction. Fermentation genes increased in relative abundance with depth and were largely affiliated with Syntrophobacter. Methylocystaceae-like small-subunit (SSU) rRNA genes, pmoA, and mmoX genes were more abundant among methanotrophs. Genes encoding N₂ fixation, P uptake, and P regulons were significantly enriched in the surface peat and in comparison to other ecosystems, indicating N and P limitation. Persistence of inorganic orthophosphate throughout the peat profile in this P-limiting environment indicates that P may be bound to recalcitrant organic compounds, thus limiting P bioavailability in the subsurface. Comparative metagenomic analysis revealed a high metabolic potential for P transport and starvation, N₂ fixation, and oligosaccharide degradation at MEF relative to other wetland and soil environments, consistent with the nutrient-poor and carbohydrate-rich conditions found in this Sphagnum-dominated boreal peatland.     

Strain-level genomic variation in natural populations of Lebetimonas from an erupting deep-sea volcano

Chemolithoautotrophic Epsilonproteobacteria are ubiquitous in sulfidic, oxygen-poor habitats, including hydrothermal vents, marine oxygen minimum zones, marine sediments and sulfidic caves and have a significant role in cycling carbon, hydrogen, nitrogen and sulfur in these environments. The isolation of diverse strains of Epsilonproteobacteria and the sequencing of their genomes have revealed that this group has the metabolic potential to occupy a wide range of niches, particularly at dynamic deep-sea hydrothermal vents. We expand on this body of work by examining the population genomics of six strains of Lebetimonas, a vent-endemic, thermophilic, hydrogen-oxidizing Epsilonproteobacterium, from a single seamount in the Mariana Arc. Using Lebetimonas as a model for anaerobic, moderately thermophilic organisms in the warm, anoxic subseafloor environment, we show that genomic content is highly conserved and that recombination is limited between closely related strains. The Lebetimonas genomes are shaped by mobile genetic elements and gene loss as well as the acquisition of novel functional genes by horizontal gene transfer, which provide the potential for adaptation and microbial speciation in the deep sea. In addition, these Lebetimonas genomes contain two operons of nitrogenase genes with different evolutionary origins. Lebetimonas expressed nifH during growth with nitrogen gas as the sole nitrogen source, thus providing the first evidence of nitrogen fixation in any Epsilonproteobacteria from deep-sea hydrothermal vents. In this study, we provide a comparative overview of the genomic potential within the Nautiliaceae as well as among more distantly related hydrothermal vent Epsilonproteobacteria to broaden our understanding of microbial adaptation and diversity in the deep sea.     

The Different Potential of Sponge Bacterial Symbionts in N2 Release Indicated by the Phylogenetic Diversity and Abundance Analyses of Denitrification Genes,nirK and nosZ

Nitrogen cycle is a critical biogeochemical process of the oceans. The nitrogen fixation by sponge cyanobacteria was early observed. Until recently, sponges were found to be able to release nitrogen gas. However the gene-level evidence for the role of bacterial symbionts from different species sponges in nitrogen gas release is limited. And meanwhile, the quanitative analysis of nitrogen cycle-related genes of sponge microbial symbionts is relatively lacking. The nirK gene encoding nitrite reductase which catalyzes soluble nitrite into gas NO and nosZ gene encoding nitrous oxide reductase which catalyzes N₂O into N₂ are two key functional genes in the complete denitrification pathway. In this study, using nirK and nosZ genes as markers, the potential of bacterial symbionts in six species of sponges in the release of N₂ was investigated by phylogenetic analysis and real-time qPCR. As a result, totally, 2 OTUs of nirK and 5 OTUs of nosZ genes were detected by gene library-based saturated sequencing. Difference phylogenetic diversity of nirK and nosZ genes were observed at OTU level in sponges. Meanwhile, real-time qPCR analysis showed that Xestospongia testudinaria had the highest abundance of nosZ gene, while Cinachyrella sp. had the greatest abundance of nirK gene. Phylogenetic analysis showed that the nirK and nosZ genes were probably of Alpha-, Beta-, and Gammaproteobacteria origin. The results from this study suggest that the denitrification potential of bacteria varies among sponges because of the different phylogenetic diversity and relative abundance of nosZ and nirK genes in sponges. Totally, both the qualitative and quantitative analyses of nirK and nosZ genes indicated the different potential of sponge bacterial symbionts in the release of nitrogen gas.     

Identification of a Thiomicrospira denitrificans-Like Epsilonproteobacterium as a Catalyst for Autotrophic Denitrification in the Central Baltic Sea

Identification and functional analysis of key members of bacterial communities in marine and estuarine environments are major challenges for obtaining a mechanistic understanding of biogeochemical processes. In the Baltic Sea basins, as in many other marine environments with anoxic bodies of water, the oxic-anoxic interface is considered a layer of high bacterial turnover of sulfur, nitrogen, and carbon compounds that has a great impact on matter balances in the whole ecosystem. We focused on autotrophic denitrification by oxidation of reduced sulfur compounds as a biogeochemically important process mediating concomitant turnover of sulfur, nitrogen, and carbon. We used a newly developed approach consisting of molecular analyses in stimulation experiments and in situ abundance. The molecular approach was based on single-strand conformational polymorphism (SSCP) analysis of the bacterial community RNA, which allowed identification of potential denitrifiers based on the sequences of enhanced SSCP bands and monitoring of the overall bacterial community during the experiments. Sequences of the SSCP bands of interest were used to design highly specific primers that enabled (i) generation of almost complete 16S rRNA gene sequences using experimental and environmental DNA as templates and (ii) quantification of the bacteria of interest by real-time PCR. By using this approach we identified the bacteria responsible for autotrophic denitrification as a single taxon, an epsilonproteobacterium related to the autotrophic denitrifier Thiomicrospira denitrificans. This finding was confirmed by material balances in the experiments that were consistent with those obtained with continuous cultures of T. denitrificans. The presence and activity of a bacterium that is phylogenetically and physiologically closely related to T. denitrificans could be relevant for the carbon budget of the central Baltic Sea because T. denitrificans exhibits only one-half the efficiency for carbon dioxide fixation per mol of sulfide oxidized and mol of nitrate reduced of Thiobacillus denitrificans hypothesized previously for this function.     

GeoChip-based analysis of the functional gene diversity and metabolic potential of soil microbial communities of mangroves

Mangroves are unique and highly productive ecosystems and harbor very special microbial communities. Although the phylogenetic diversity of sediment microbial communities of mangrove habitats has been examined extensively, little is known regarding their functional gene diversity and metabolic potential. In this study, a high-throughput functional gene array (GeoChip 4.0) was used to analyze the functional diversity, composition, structure, and metabolic potential of microbial communities in mangrove habitats from mangrove national nature reserves in China. GeoChip data indicated that these microbial communities were functionally diverse as measured by the number of genes detected, unique genes, and various diversity indices. Almost all key functional gene categories targeted by GeoChip 4.0 were detected in the mangrove microbial communities, including carbon (C) fixation, C degradation, methane generation, nitrogen (N) fixation, nitrification, denitrification, ammonification, N reduction, sulfur (S) metabolism, metal resistance, antibiotic resistance, and organic contaminant degradation. Detrended correspondence analysis (DCA) of all detected genes showed that Spartina alterniflora (HH), an invasive species, did not harbor significantly different microbial communities from Aegiceras corniculatum (THY), a native species, but did differ from other species, Kenaelia candel (QQ), Aricennia marina (BGR), and mangrove-free mud flat (GT). Canonical correspondence analysis (CCA) results indicated the microbial community structure was largely shaped by surrounding environmental variables, such as total nitrogen (TN), total carbon (TC), pH, C/N ratio, and especially salinity. This study presents a comprehensive survey of functional gene diversity of soil microbial communities from different mangrove habitats/species and provides new insights into our understanding of the functional potential of microbial communities in mangrove ecosystems.     

Abundance and Diversity of Bacterial Nitrifiers and Denitrifiers and Their Functional Genes in Tannery Wastewater Treatment Plants Revealed by High-Throughput Sequencing

Biological nitrification/denitrification is frequently used to remove nitrogen from tannery wastewater containing high concentrations of ammonia. However, information is limited about the bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants (WWTPs) due to the low-throughput of the previously used methods. In this study, 454 pyrosequencing and Illumina high-throughput sequencing, combined with molecular methods, were used to comprehensively characterize structures and functions of nitrification and denitrification bacterial communities in aerobic and anaerobic sludge of two full-scale tannery WWTPs. Pyrosequencing of 16S rRNA genes showed that Proteobacteria and Synergistetes dominated in the aerobic and anaerobic sludge, respectively. Ammonia-oxidizing bacteria (AOB) amoA gene cloning revealed that Nitrosomonas europaea dominated the ammonia-oxidizing community in the WWTPs. Metagenomic analysis showed that the denitrifiers mainly included the genera of Thauera, Paracoccus, Hyphomicrobium, Comamonas and Azoarcus, which may greatly contribute to the nitrogen removal in the two WWTPs. It is interesting that AOB and ammonia-oxidizing archaea had low abundance although both WWTPs demonstrated high ammonium removal efficiency. Good correlation between the qPCR and metagenomic analysis is observed for the quantification of functional genes amoA, nirK, nirS and nosZ, indicating that the metagenomic approach may be a promising method used to comprehensively investigate the abundance of functional genes of nitrifiers and denitrifiers in the environment.     

Response of the Abundance of Key Soil Microbial Nitrogen-Cycling Genes to Multi-Factorial Global Changes

Multiple co-occurring environmental changes are affecting soil nitrogen cycling processes, which are mainly mediated by microbes. While it is likely that various nitrogen-cycling functional groups will respond differently to such environmental changes, very little is known about their relative responsiveness. Here we conducted four long-term experiments in a steppe ecosystem by removing plant functional groups, mowing, adding nitrogen, adding phosphorus, watering, warming, and manipulating some of their combinations. We quantified the abundance of seven nitrogen-cycling genes, including those for fixation (nifH), mineralization (chiA), nitrification (amoA of ammonia-oxidizing bacteria (AOB) or archaea (AOA)), and denitrification (nirS, nirK and nosZ). First, for each gene, we compared its sensitivities to different environmental changes and found that the abundances of various genes were sensitive to distinct and different factors. Overall, the abundances of nearly all genes were sensitive to nitrogen enrichment. In addition, the abundances of the chiA and nosZ genes were sensitive to plant functional group removal, the AOB-amoA gene abundance to phosphorus enrichment when nitrogen was added simultaneously, and the nirS and nirK gene abundances responded to watering. Second, for each single- or multi-factorial environmental change, we compared the sensitivities of the abundances of different genes and found that different environmental changes primarily affected different gene abundances. Overall, AOB-amoA gene abundance was most responsive, followed by the two denitrifying genes nosZ and nirS, while the other genes were less sensitive. These results provide, for the first time, systematic insights into how the abundance of each type of nitrogen-cycling gene and the equilibrium state of all these nitrogen-cycling gene abundances would shift under each single- or multi-factorial global change.     

Potential virus-mediated nitrogen cycling in oxygen-depleted oceanic waters

Viruses play an important role in the ecology and biogeochemistry of marine ecosystems. Beyond mortality and gene transfer, viruses can reprogram microbial metabolism during infection by expressing auxiliary metabolic genes (AMGs) involved in photosynthesis, central carbon metabolism, and nutrient cycling. While previous studies have focused on AMG diversity in the sunlit and dark ocean, less is known about the role of viruses in shaping metabolic networks along redox gradients associated with marine oxygen minimum zones (OMZs). Here, we analyzed relatively quantitative viral metagenomic datasets that profiled the oxygen gradient across Eastern Tropical South Pacific (ETSP) OMZ waters, assessing whether OMZ viruses might impact nitrogen (N) cycling via AMGs. Identified viral genomes encoded six N-cycle AMGs associated with denitrification, nitrification, assimilatory nitrate reduction, and nitrite transport. The majority of these AMGs (80%) were identified in T4-like Myoviridae phages, predicted to infect Cyanobacteria and Proteobacteria, or in unclassified archaeal viruses predicted to infect Thaumarchaeota. Four AMGs were exclusive to anoxic waters and had distributions that paralleled homologous microbial genes. Together, these findings suggest viruses modulate N-cycling processes within the ETSP OMZ and may contribute to nitrogen loss throughout the global oceans thus providing a baseline for their inclusion in the ecosystem and geochemical models.Subject terms: Virus-host interactions, Biogeochemistry, Microbial biooceanography, Microbial ecology     

Characterization of microbial community in an aerobic moving bed biofilm reactor applied for simultaneous nitrification and denitrification

A continuous-flow moving bed biofilm reactor (MBBR) under aerobic conditions was established for simultaneous nitrification and denitrification (SND), and microbial communities were investigated by a combination of denaturing gel gradient electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). DGGE analysis has revealed more similar microbial community structures formed in the biofilms with more similar carbon nitrogen (C/N) ratios. FISH analysis shows that the dominance of both Betaproteobacteria ammonia-oxidizing bacteria and Nitrospira-like nitrite-oxidizing bacteria were negatively correlated to C/N ratios. Sequence analysis of DGGE bands has indicated the presence of anoxic denitrifying bacteria Agrobacterium tumefaciens and Rhizobium sp., suggesting that the oxygen gradient inside the biofilm may be responsible for the mechanism of SND in aerobic MBBRs. The study confirms that appropriate control of microbial community structure resulting from optimal C/N ratio is beneficial in improving SND, thus optimizing nitrogen removal in aerobic MBBR. The established SND-based MBBR can save operation space and time in comparison to the traditional nitrogen removal process, and might be very attractive for future practical applications.     

Organic carbon availability limiting microbial denitrification in the deep vadose zone

Microbes in the deep vadose zone play an essential role in the mitigation of nitrate leaching; however, limited information is available on the mechanisms of microbial denitrification due to sampling difficulties. We experimentally studied the factors that affect denitrification in soils collected down to 10.5 meters deep along the soil profile. After an anoxic pre‐incubation, denitrification rates moderately increased and the N₂O/(N₂O + N₂) ratios declined while the microbial abundance and diversity did not change significantly in most of the layers. Denitrification rate was significantly enhanced and the abundance of the denitrification genes was simultaneously elevated by the increased availability of organic carbon in all studied layers, to a greater extent in the subsurface layers than in the surface layers, suggesting the severe scarcity of carbon in the deep vadose zone. The genera Pseudomonas and Bacillus, which are made up of a number of species that have been previously identified as denitrifiers in soil, were the major taxa that respond to carbon addition. Overall, our results suggested that the limited denitrification in the deep vadose zone is not because of the lack of denitrifiers, but due to the low abundance of denitrifiers which is caused by low carbon availability.