Community Structures and Distribution of Anaerobic Ammonium Oxidizing and nirS-Encoding Nitrite-Reducing Bacteria in Surface Sediments of the South China Sea

Anaerobic ammonium oxidation (anammox) and denitrification are two important processes responsible for nitrogen loss; monitoring of microbial communities carrying out these two processes offers a unique opportunity to understand the microbial nitrogen cycle. The aim of the current study was to characterize community structures and distribution of anammox and nirS-encoding nitrite-reducing bacteria in surface sediments of the northern South China Sea (SCS). The consistent phylogenetic results of three biomarkers of anammox bacteria, including 16S rRNA, hzo, and Scalindua-nirS genes, showed that Scalindua-like bacteria were the only anammox group presenting in surface sediments of the SCS. However, a relatively high micro-diversity was found within this group, including several SCS habitat-specific phylotypes, Candidatus “Scalindua zhenghei”. Comparing to 16S rRNA gene, hzo and Scalindua-nirS genes provided a relatively higher resolution to elucidate anammox bacteria. For the nirS-encoding nitrite-reducing bacteria, the detected nirS gene sequences were closely related to various marine nirS denitrifiers, especially those which originated from coastal and estuarine sediments with a much higher diversity than anammox bacteria. Anammox bacterial communities shifted along with the seawater depth, while nirS-encoding nitrite-reducing bacteria did not. Although nirS-encoding nitrite-reducing bacteria have a much higher abundance and diversity than anammox bacteria, they showed similar abundance variation patterns in research sites, suggesting the two microbial groups might be affected by the similar environmental factors. The significant correlations among the abundance of the two microbial groups with the molar ratio of NH₄ ⁺ to (NO₂ ^??+?NO₃ ^?), pH, and organic matters of sediments strongly supported this hypothesis.     

Residence of Habitat-Specific Anammox Bacteria in the Deep-Sea Subsurface Sediments of the South China Sea: Analyses of Marker Gene Abundance with Physical Chemical Parameters

Anaerobic ammonium oxidation (anammox) has been recognized as an important process for the global nitrogen cycle. In this study, the occurrence and diversity of anammox bacteria in the deep-sea subsurface sediments of the South China Sea (SCS) were investigated. Results indicated that the anammox bacterial sequences recovered from this habitat by amplifying both 16S rRNA gene and hydrazine oxidoreductase encoding hzo gene were all closely related to the Candidatus Scalindua genus. A total of 96 16S rRNA gene sequences from 346 clones were grouped into five subclusters: two subclusters affiliated with the brodae and arabica species, while three new subclusters named zhenghei-I, -II, and -III showed ≤97.4% nucleic acid sequence identity with other known Candidatus Scalindua species. Meanwhile, 88 hzo gene sequences from the sediments also formed five distant subclusters within hzo cluster 1c. Through fluorescent real-time PCR analysis, the abundance of anammox bacteria in deep-sea subsurface sediment was quantified by hzo genes, which ranged from 1.19 × 10⁴ to 7.17 × 10⁴ copies per gram of dry sediments. Combining all the information from this study, diverse Candidatus Scalindua anammox bacteria were found in the deep-sea subsurface sediments of the SCS, and they could be involved in the nitrogen loss from the fixed inventory in the habitat.     

Mimicking the oxygen minimum zones: stimulating interaction of aerobic archaeal and anaerobic bacterial ammonia oxidizers in a laboratory‐scale model system

In marine oxygen minimum zones (OMZs), ammonia‐oxidizing archaea (AOA) rather than marine ammonia‐oxidizing bacteria (AOB) may provide nitrite to anaerobic ammonium‐oxidizing (anammox) bacteria. Here we demonstrate the cooperation between marine anammox bacteria and nitrifiers in a laboratory‐scale model system under oxygen limitation. A bioreactor containing ‘Candidatus Scalindua profunda’ marine anammox bacteria was supplemented with AOA (Nitrosopumilus maritimus strain SCM1) cells and limited amounts of oxygen. In this way a stable mixed culture of AOA, and anammox bacteria was established within 200 days while also a substantial amount of endogenous AOB were enriched. ‘Ca. Scalindua profunda’ and putative AOB and AOA morphologies were visualized by transmission electron microscopy and a C₁₈ anammox [3]‐ladderane fatty acid was highly abundant in the oxygen‐limited culture. The rapid oxygen consumption by AOA and AOB ensured that anammox activity was not affected. High expression of AOA, AOB and anammox genes encoding for ammonium transport proteins was observed, likely caused by the increased competition for ammonium. The competition between AOA and AOB was found to be strongly related to the residual ammonium concentration based on amoA gene copy numbers. The abundance of archaeal amoA copy numbers increased markedly when the ammonium concentration was below 30 μM finally resulting in almost equal abundance of AOA and AOB amoA copy numbers. Massive parallel sequencing of mRNA and activity analyses further corroborated equal abundance of AOA and AOB. PTIO addition, inhibiting AOA activity, was employed to determine the relative contribution of AOB versus AOA to ammonium oxidation. The present study provides the first direct evidence for cooperation of archaeal ammonia oxidation with anammox bacteria by provision of nitrite and consumption of oxygen.     

The physiological potential of anammox bacteria as revealed by their core genome structure

We present here the second complete genome of anaerobic ammonium oxidation (anammox) bacterium, Candidatus (Ca.) Brocadia pituitae, along with those of a nitrite oxidizer and two incomplete denitrifiers from the anammox bacterial community (ABC) metagenome. Although NO2− reduction to NO is considered to be the first step in anammox, Ca. B. pituitae lacks nitrite reductase genes (nirK and nirS) responsible for this reaction. Comparative genomics of Ca. B. pituitae with Ca. Kuenenia stuttgartiensis and six other anammox bacteria with nearly complete genomes revealed that their core genome structure contains 1,152 syntenic orthologues. But nitrite reductase genes were absent from the core, whereas two other Brocadia species possess nirK and these genes were horizontally acquired from multiple lineages. In contrast, at least five paralogous hydroxylamine oxidoreductase genes containing candidate ones (hao2 and hao3) encoding another nitrite reductase were observed in the core. Indeed, these two genes were also significantly expressed in Ca. B. pituitae as in other anammox bacteria. Because many nirS and nirK genes have been detected in the ABC metagenome, Ca. B. pituitae presumably utilises not only NO supplied by the ABC members but also NO and/or NH₂OH by self-production for anammox metabolism.     

Identification of Denitrifying Bacteria Diversity in an Activated Sludge System by using Nitrite Reductase Genes

Nitrite reduction is the key step in the denitrification reaction with two predominant types of nitrite reductase genes: nirS and nirK. The diversity of denitrifying bacteria in a municipal wastewater treatment plant is described by using both these genes. Of the cultured colonies, 22.5% contained the NirS gene and 12.5% the nirK gene. These nitrite reductase-containing colonies could be further divided into five different types by using both restriction fragment length polymorphism and denaturing gradient gel electrophoresis analysis. Phylogenetic analysis showed that these five types of denitrifying bacteria were phylogenetically diverse. Finally, one nirS gene was obtained and compared with the published sequences.     

牛场肥水灌溉对土壤nirK、nirS型反硝化微生物群落结构的影响

以徐水县梁家营长期定位施肥试验田为研究对象,利用末端限制性片段长度多态性(T-RFLP)分析和克隆文库构建,研究了5种施肥处理(清水灌溉CK、无机肥灌溉CF、牛场肥水不同浓度、不同次数灌溉T4、T5和T11)下土壤中nirK、nirS型反硝化细菌群落多样性及其群落结构的演变。结果表明,不同施肥处理下nirK、nirS型反硝化细菌群落多样性无显著差异,但群落结构却有明显变化:nirK型反硝化细菌群落结构既受施肥种类又受施肥量影响,优势种群尤其对施肥种类和施肥量响应显著;nirS型反硝化细菌则主要受施肥种类影响,施肥量影响微弱。牛场肥水处理和无机肥处理分别促进和抑制不同的nirS型反硝化细菌,群落主成分受无机肥促进、牛场肥水抑制。系统发育分析结果表明,土壤中nirK型反硝化细菌主要与假单胞菌属(Pseudomonas)、产碱杆菌属(Alcaligenes)和根瘤菌属(Rhizobium)的反硝化细菌具有较近的亲缘关系;nirS型反硝化细菌主要与劳尔氏菌(Ralstonia)和红长命菌属(Rubrivivax)有较近的亲缘关系。试验土壤中反硝化微生物多与目前已报道的好氧反硝化细菌亲缘关系较近,这可能与微生物分析取自表层土有关。     

Impact of Long-Term Fertilization on the Composition of Denitrifier Communities Based on Nitrite Reductase Analyses in a Paddy Soil

The effect of long-term fertilization on soil-denitrifying communities was determined by measuring the abundance and diversity of the nitrite reductase genes nirK and nirS. Soil samples were collected from plots of a long-term fertilization experiment started in 1990, located in Taoyuan (110°72″ E, 28°52″ N), China. The treatments were no fertilizer (NF), urea (UR), balanced mineral fertilizers (BM), and BM combined with rice straw (BMR). The abundance, diversity, and composition of the soil-denitrifying bacteria were determined by using real-time quantitative PCR, terminal restriction fragment length polymorphism (T-RFLP), and cloning and sequencing of nirK and nirS genes. There was a pronounced difference in the community composition and diversity of nirK-containing denitrifiers responding to the long-term fertilization regimes; however, less variation was observed in communities of nirS-containing denitrifiers, indicating that denitrifiers possessing nirK were more sensitive to the fertilization practices than those with nirS. In contrast, fertilization regimes had similar effects on the copy numbers of nirK and nirS genes. The BMR treatment had the highest copy numbers of nirK and nirS, followed by the two mineral fertilization regimes (UR and BM), and the lowest was in the NF treatment. Of the measured soil parameters, the differences in the community composition of nirK and the abundance of nir denitrifiers were highly correlated with the soil carbon content. Therefore, long-term fertilization resulted in a strong impact on the community structure of nirK populations only, and total organic carbon was the dominant factor in relation to the variations of nir community sizes.     

The Impact of Using Mature Compost on Nitrous Oxide Emission and the Denitrifier Community in the Cattle Manure Composting Process

The diversity and dynamics of the denitrifying genes (nirS, nirK, and nosZ) encoding nitrite reductase and nitrous oxide (N₂O) reductase in the dairy cattle manure composting process were investigated. A mixture of dried grass with a cattle manure compost pile and a mature compost-added pile were used, and denaturing gradient gel electrophoresis was used for denitrifier community analysis. The diversity of nirK and nosZ genes significantly changed in the initial stage of composting. These variations might have been induced by the high temperature. The diversity of nirK was constant after the initial variation. On the other hand, the diversity of nosZ changed in the latter half of the process, a change which might have been induced by the accumulation of nitrate and nitrite. The nirS gene fragments could not be detected. The use of mature compost that contains nitrate and nitrite promoted the N₂O emission and significantly affected the variation of nosZ diversity in the initial stage of composting, but did not affect the variation of nirK diversity. Many Pseudomonas-like nirK and nosZ gene fragments were detected in the stage in which N₂O was actively emitted.     

Diversity of Nitrite Reductase (nirK and nirS) Gene Fragments in Forested Upland and Wetland Soils

The genetic heterogeneity of nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in forested upland and marsh soil was investigated using molecular methods. nirK gene fragments could be amplified from both soils, whereas nirS gene fragments could be amplified only from the marsh soil. PCR products were cloned and screened by restriction fragment length polymorphism (RFLP), and representative fragments were sequenced. The diversity of nirK clones was lower than the diversity of nirS clones. Among the 54 distinct nirK RFLP patterns identified in the two soils, only one pattern was found in both soils and in each soil two dominant groups comprised >35% of all clones. No dominance and few redundant patterns were seen among the nirS clones. Phylogenetic analysis of deduced amino acids grouped the nirK sequences into five major clusters, with one cluster encompassing most marsh clones and all upland clones. Only a few of the nirK clone sequences branched with those of known denitrifying bacteria. The nirS clones formed two major clusters with several subclusters, but all nirS clones showed less than 80% identity to nirS sequences from known denitrifying bacteria. Overall, the data indicated that the denitrifying communities in the two soils have many members and that the soils have a high richness of different nir genes, especially of the nirS gene, most of which have not yet been found in cultivated denitrifiers.     

Effect of Sulfadiazine on Abundance and Diversity of Denitrifying Bacteria by Determining nirK and nirS Genes in Two Arable Soils

Sulfadiazine (SDZ) is an antibiotic frequently used in agricultural husbandry. Via manuring of excrements of medicated animals, the drug reaches the soil and might impair important biochemical transformation processes performed by microbes, e.g., the nitrogen turnover. We studied the effect of pig manure and SDZ-spiked pig manure on denitrifying bacteria by quantifying nirK and nirS nitrite reductase genes in two arable soils. Addition of manure entailed mainly an increase of nirK-harboring denitrifiers in both soils, whereas in the SDZ-amended treatments, primarily the nirS denitrifiers increased in abundance after the bioavailable SDZ had declined. However, the community composition of nirS nitrite reducers investigated by denaturing gradient gel electrophoresis did not change despite the observed alterations in abundance.     

Dominance of Candidatus Scalindua species in anammox community revealed in soils with different duration of rice paddy cultivation in Northeast China

The anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the oxygen-limited zone for nitrogen cycling, but their roles in agricultural ecosystems are still poorly understood. In this study, soil samples were taken from the rhizosphere and non-rhizosphere and from surface (0–5 cm) and subsurface (20–25 cm) layers with 1, 4, and 9 years of rice cultivation history on the typical albic soil of Northeast China to examine the diversity and distribution of anammox bacteria based on 16S rRNA gene and hydrazine oxidoreductase encoding gene (hzo). By comparing these soil samples, no obvious difference was observed in community composition between the rhizosphere and non-rhizosphere or the surface and subsurface layers. Surprisingly, anammox bacterial communities of these rice paddy soils were consisted of mainly Candidatus Scalindua species, which are best known to be dominant in marine and pristine environments. The highest diversity was revealed in the 4-year paddy soil based on clone library analysis. Phylogenetic analysis of 16S rRNA gene and deduced HZO from the corresponding encoding gene showed that most of the obtained clones are grouped together with Candidatus Scalindua sorokinii, Candidatus Scalindua brodae, and Candidatus Scalindua spp. of seawater. The obtained clone sequences from all samples are distributed in two subclusters that contain sequences from environmental samples only. Tentative new species were also discovered in this paddy soil. This study provides the first evidence on the existence of anammox bacteria with limited diversity in agricultural ecosystems in Northern China.     

New PCR primers targeting hydrazine synthase and cytochrome c biogenesis proteins in anammox bacteria

PCR primers targeting genes encoding the two proteins of anammox bacteria, hydrazine synthase and cytochrome c biogenesis protein, were designed and tested in this study. Three different ecotypes of samples, namely ocean sediments, coastal wetland sediments, and wastewater treatment plant (WWTP) samples, were used to assess the primer efficiency and the community structures of anammox bacteria retrieved by 16S ribosomal RNA (rRNA) and the functional genes. Abundances of hzsB gene of anammox bacteria in South China Sea (SCS) samples were significantly correlated with 16S rRNA gene by qPCR method. And hzsB and hzsC gene primer pair hzsB364f-hzsB640r and hzsC745f-hzsC862r in combination with anammox bacterial 16S rRNA gene primers were recommended for quantifying anammox bacteria. Congruent with 16S rRNA gene-based community study, functional gene hzsB could also delineate the coastal-ocean distributing pattern, and seawater depth was positively associated with the diversity and abundance of anammox bacteria from shallow- to deep-sea. Both hzsC and ccsA genes could differentiate marine samples between deep and shallow groups of the Scalindua sp. clades. As for WWTP samples, non-Scalindua anammox bacteria reflected by hzsB, hzsC, ccsA, and ccsB gene-based libraries showed a similar distribution pattern with that by 16S rRNA gene. NH₄ ⁺ and NH₄ ⁺/Σ(NO₃ ⁻ + NO₂ ⁻) positively correlated with anammox bacteria gene diversity, but organic matter contents correlated negatively with anammox bacteria gene diversity in SCS. Salinity was positively associated with diversity indices of hzsC and ccsB gene-harboring anammox bacteria communities and could potentially differentiate the distribution patterns between shallow- and deep-sea sediment samples. SCS surface sediments harbored considerably diverse community of Scalindua. A new Mai Po clade representing coastal estuary wetland anammox bacteria group based on 16S rRNA gene phylogeny is proposed. Existence of anammox bacteria within wider coverage of genera in Mai Po wetland indicates this unique niche is very complex, and species of anammox bacteria are niche-specific with different physiological properties towards substrates competing and chemical tolerance capability.

     

Distribution of denitrifying bacterial communities in the stratified water column and sediment–water interface in two freshwater lakes and the Baltic Sea

We have studied the distribution and community composition of denitrifying bacteria in the stratified water column and at the sediment–water interface in lakes Plußsee and Schöhsee, and a near-shore site in the Baltic Sea in Germany. Although environmental changes induced by the stratification of the water column in marine environments are known to affect specific populations of denitrifying bacteria, little information is available for stratified freshwater lakes and brackish water. The aim of the present study was to fill this gap and to demonstrate specific distribution patterns of denitrifying bacteria in specific aquatic habitats using two functional markers for the nitrite reductase (nirK and nirS genes) as a proxy for the communities. The leading question to be answered was whether communities containing the genes nirK and nirS have similar, identical, or different distribution patterns, and occupy the same or different ecological niches. The genes nirK and nirS were analyzed by PCR amplification with specific primers followed by terminal restriction fragment length polymorphism (T-RFLP) and by cloning and sequence analysis. Overall, nirS-denitrifiers were more diverse than nirK-denitrifiers. Denitrifying communities in sediments were clearly different from those in the water column in all aquatic systems, regardless of the gene analyzed. A differential distribution of denitrifying assemblages was observed for each particular site. In the Baltic Sea and Lake Plußsee, nirK-denitrifiers were more diverse throughout the water column, while nirS-denitrifiers were more diverse in the sediment. In Lake Schöhsee, nirS-denitrifiers showed high diversity across the whole water body. Habitat-specific clusters of nirS sequences were observed for the freshwater lakes, while nirK sequences from both freshwater lakes and the Baltic Sea were found in common phylogenetic clusters. These results demonstrated differences in the distribution of bacteria containing nirS and those containing nirK indicating that both types of denitrifiers apparently occupy different ecological niches.     

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.     

Nitrite Reductase Genes (nirK and nirS) as Functional Markers To Investigate Diversity of Denitrifying Bacteria in Pacific Northwest Marine Sediment Communities

Genetic heterogeneity of denitrifying bacteria in sediment samples from Puget Sound and two sites on the Washington continental margin was studied by PCR approaches amplifying nirK and nirS genes. These structurally different but functionally equivalent single-copy genes coding for nitrite reductases, a key enzyme of the denitrification process, were used as a molecular marker for denitrifying bacteria. nirS sequences could be amplified from samples of both sampling sites, whereas nirK sequences were detected only in samples from the Washington margin. To assess the underlying nir gene structure, PCR products of both genes were cloned and screened by restriction fragment length polymorphism (RFLP). Rarefraction analysis revealed a high level of diversity especially for nirS clones from Puget Sound and a slightly lower level of diversity for nirK and nirS clones from the Washington margin. One group dominated within nirK clones, but no dominance and only a few redundant clones were seen between sediment samples for nirS clones in both habitats. Hybridization and sequencing confirmed that all but one of the 228 putative nirS clones were nirS with levels of nucleotide identities as low as 45.3%. Phylogenetic analysis grouped nirS clones into three distinct subclusters within the nirS gene tree which corresponded to the two habitats from which they were obtained. These sequences had little relationship to any strain with known nirS sequences or to isolates (mostly close relatives of Pseudomonas stutzeri) from the Washington margin sediment samples. nirK clones were more closely related to each other than were the nirS clones, with 78.6% and higher nucleotide identities; clones showing only weak hybridization signals were not related to known nirK sequences. All nirK clones were also grouped into a distinct cluster which could not be placed with any strain with known nirK sequences. These findings show a very high diversity of nir sequences within small samples and that these novel nir clusters, some very divergent from known sequences, are not known in cultivated denitrifiers.     

NirK and nirS Nitrite reductase genes from non-agricultural forest soil bacteria in Thailand

The genetic heterogeneity of the nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in a non-agricultural forest soil in Thailand was investigated using soil samples from the Plant Germplasm-Royal Initiation Project area in Kanchanaburi Province, Thailand. Soil bacteria were screened for denitrification activity and 13 (from 211) positive isolates were obtained and further evaluated for their ability to reduce nitrate and to accumulate or reduce nitrite. Three species with potentially previously unreported denitrifying activities were recorded. Analysis of the partial nirK and nirS sequences of these 13 strains revealed a diverse sequence heterogeneity in these two genes within the same environment and even potentially within the same host species, the potential existence of lateral gene transfer and the first record of both nirK and nirS homologues in one bacterial species. Finally, isolates of two species of bacteria (Corynebacterium propinquum and Micrococcus lylae) are recorded as denitrifiers for the first time.     

Genetic and Environmental Controls on Nitrous Oxide Accumulation in Lakes

We studied potential links between environmental factors, nitrous oxide (N₂O) accumulation, and genetic indicators of nitrite and N₂O reducing bacteria in 12 boreal lakes. Denitrifying bacteria were investigated by quantifying genes encoding nitrite and N₂O reductases (nirS/nirK and nosZ, respectively, including the two phylogenetically distinct clades nosZ _I and nosZ _II) in lake sediments. Summertime N₂O accumulation and hypolimnetic nitrate concentrations were positively correlated both at the inter-lake scale and within a depth transect of an individual lake (Lake Vanajavesi). The variability in the individual nirS, nirK, nosZ _I, and nosZ _II gene abundances was high (up to tenfold) among the lakes, which allowed us to study the expected links between the ecosystem’s nir-vs-nos gene inventories and N₂O accumulation. Inter-lake variation in N₂O accumulation was indeed connected to the relative abundance of nitrite versus N₂O reductase genes, i.e. the (nirS+nirK)/nosZ _I gene ratio. In addition, the ratios of (nirS+nirK)/nosZ _I at the inter-lake scale and (nirS+nirK)/nosZ _I+II within Lake Vanajavesi correlated positively with nitrate availability. The results suggest that ambient nitrate concentration can be an important modulator of the N₂O accumulation in lake ecosystems, either directly by increasing the overall rate of denitrification or indirectly by controlling the balance of nitrite versus N₂O reductase carrying organisms.     

The influence of intense chemical pollution on the community composition, diversity and abundance of anammox bacteria in the Jiaojiang Estuary (China)

Continuous chemical pollution is one of the most serious environmental problems in the Jiaojiang Estuary of the East Sea (China). This chemical pollution has significantly changed the estuarine environmental conditions and may have profoundly influenced the distribution of anammox bacterial communities in this estuary. Here, we investigated the influence of chemical pollution on the community composition, diversity and abundance of anammox bacteria in Jiaojiang estuarine sediments. Phylogenetic analysis of 16S rRNA genes showed that the majority of anammox bacterial sequences retrieved from the estuarine intertidal sediments were associated with Kuenenia. In contrast, different anammox communities composed of Brocadia, Kuenenia, Scalindua and Jettenia were found in the estuarine subtidal sediments. Redundancy analysis (RDA) indicated that the sediment nitrobenzene and organic content had significant impacts on the distribution of anammox communities in the intertidal sediments. Pearson correlation analysis showed that the diversity of anammox bacteria in the intertidal sediments was positively correlated with the organic content. In contrast, RDA results showed that the nitrobenzene content, NO(3)(-) concentration and salinity significantly influenced the distribution of anammox communities in the subtidal sediments. The diversity and relative abundance of anammox bacteria in the subtidal sediments were positively correlated with NO(3)(-) concentration.     

Isolation,genetic and functional characterization of novel soil nirK-type denitrifiers

Denitrification, the reduction of nitrogen oxides (NO₃⁻ and NO₂⁻) to N₂ via the intermediates NO and N₂O, is crucial for nitrogen turnover in soils. Cultivation-independent approaches that applied nitrite reductase genes (nirK/nirS) as marker genes to detect denitrifiers showed a predominance of genes presumably derived from as yet uncultured organisms. However, the phylogenetic affiliation of these organisms remains unresolved since the ability to denitrify is widespread among phylogenetically unrelated organisms. In this study, denitrifiers were cultured using a strategy to generally enrich soil microorganisms. Of 490 colonies screened, eight nirK-containing isolates were phylogenetically identified (16S rRNA genes) as members of the Rhizobiales. A nirK gene related to a large cluster of sequences from uncultured bacteria mainly retrieved from soil was found in three isolates classified as Bradyrhizobium sp. Additional isolates were classified as Bradyrhizobium japonicum and Bosea sp. that contained nirK genes also closely related to the nirK from these strains. These isolates denitrified, albeit with different efficiencies. In Devosia sp., nirK was the only denitrification gene detected. Two Mesorhizobium sp. isolates contained a nirK gene also related to nirK from cultured Mesorhizobia and uncultured soil bacteria but no gene encoding nitric oxide or nitrous oxide reductase. These isolates accumulated NO under nitrate-reducing conditions without growth, presumably due to the lethal effects of NO. This showed the presence of a functional nitrite reductase but lack of a nitric oxide reductase. In summary, similar nirK genotypes recurrently detected mainly in soils likely originated from Rhizobia, and functional differences were presumably strain-dependent.