nirS-type Denitrifiers Research Articles

Diversity and distribution of nirS-type denitrifiers as a biological indicator in response to environmental gradients in the eutrophied Haizhou Bay, China

ABSTRACTDenitrifying bacterial communities play an important role in nitrogen removal. The diversity, abundance and distribution of denitrifiers can be applied in eutrophication assessment for the bay ecosystems. In this study, the composition and variation of cytochrome cd1-type nitrite reductase gene (nirS)-type denitrifiers in the sediments of Haizhou Bay were investigated by using a PCR-based cloning approach. A total of 156 valid sequences were grouped into 76 OTUs based on 97% sequence similarity. The nirS sequences were identified with their closest-matched sequences from sediments of estuary and eutrophied bay, indicating similarity of the denitrifying bacterial communities in similar coastal environments. Canonical correspondence analysis indicated that nitrogen nutrient and dissolved oxygen were the major contributing parameters for the diversity and spatial distribution of denitrifying bacteria of Haizhou Bay.

Vegetation Effects on Bacteria and Denitrifier Abundance in the Soils of Two Tidal Freshwater Wetlands in Virginia

This study examined the abundance of bacteria and nirS-type denitrifiers associated with the rhizospheres of three emergent macrophyte species (Juncus effusus, Typha latifolia, and Peltandra virginica) to gain a greater understanding of plant-microbe interactions in wetland soils. Sampling of plant and soil properties was performed during the growing season (June) and following plant senescence (November) at two tidal freshwater wetlands. Quantitative polymerase chain reaction was used to determine the abundance of bacteria (16S rRNA) and nirS-type denitrifier genes from the rhizosphere and rhizoplane of each plant species and from nearby unvegetated soils. For bacteria, there was a positive rhizosphere effect that did not differ significantly across plant species. In contrast, significant differences in the abundance of nirS-type denitrifiers were observed across the plant species. Rhizosphere abundance was ∼2-fold greater in Peltandra virginica and 4-fold greater in Typha latifolia compared to Juncus effusus. For both bacteria and nirS-type denitrifiers, plant effects were greater during the growing season, and abundance was highly correlated with soil pH, moisture, and organic matter content. Overall, these results demonstrate plant effects on the rhizosphere microbial community can be species‐specific and that there is a synergistic relationship between plant species and environmental conditions.

Thiosulfate- and hydrogen-driven autotrophic denitrification by a microbial consortium enriched from groundwater of an oligotrophic limestone aquifer.

Despite its potentially high relevance for nitrate removal in freshwater environments limited in organic carbon, chemolithoautotrophic denitrification has rarely been studied in oligotrophic groundwater. Using thiosulfate and H2 as electron donors, we established a chemolithoautotrophic enrichment culture from groundwater of a carbonate-rock aquifer to get more insight into the metabolic repertoire, substrate turnover, and transcriptional activity of subsurface denitrifying consortia. The enriched consortium was dominated by representatives of the genus Thiobacillus along with denitrifiers related to Sulfuritalea hydrogenivorans, Sulfuricella denitrificans, Dechloromonas sp. and Hydrogenophaga sp., representing the consortium's capacity to use multiple inorganic electron donors. Microcosm experiments coupled with Raman gas spectroscopy demonstrated complete denitrification driven by reduced sulfur compounds and hydrogen without formation of N2O. The initial nitrate/thiosulfate ratio had a strong effect on nosZ transcriptional activity and on N2 formation, suggesting similar patterns of the regulation of gene expression as in heterotrophic denitrifiers. Sequence analysis targeting nirS and nosZ transcripts identified Thiobacillus denitrificans-related organisms as the dominant active nirS-type denitrifiers in the consortium. An additional assessment of the nirS-type denitrifier community in the groundwaterclearly confirmed the potential for sulfur- and hydrogen-dependent chemolithoautotrophic denitrification as important metabolic feature widely spread among subsurface denitrifiers at the Hainich Critical Zone Exploratory.

Abundance and diversity of anammox bacteria in a mainstream municipal wastewater treatment plant.

Among the factors that obstruct the application of anammox-based technology for nitrogen removal from mainstream municipal wastewater is the water's high organic loads. We hypothesized that some anammox species can adapt and grow in mainstream wastewater in which a minimal temperature of 13-15°C is maintained. Using the AMX368F and AMX820R PCR-primers, anammox bacteria were detected in influent wastewater (COD/N ratio > 13) and in the anaerobic, anoxic, and aerobic chambers of a full-scale municipal wastewater treatment plant, reaching 107 copies/g VSS of the16S rRNA gene. Furthermore, anammox activity was demonstrated by 15N-isotopic tracing. The DNA sequences of clones randomly selected from a clone library were mainly clustered with Candidatus Brocadia flugida in addition to Ca. Brocadia sinica, Ca. Jettenia asiatica, and Ca. Anammoxoglobus propionicus. However, Ca. Brocadia was the only genus detected by high-throughput next-generation sequencing and denaturing gradient gel electrophoresis. The nitrite producers, ammonia-oxidizing archaea and bacteria, were both detected in the influent wastewater and the other chambers, while the nitrite consumers, Nitrospira nitrite oxidizers and the nirS-type denitrifiers, dominated all chambers. The results indicate the occurrence and potential activity of anammox bacteria in mainstream wastewater under certain conditions (proper temperature). The dominance of Brocadia flugida and Anammoxoglobus propionicus suggests a role for volatile fatty acids in selecting the anammox community in wastewater.

Differences in microbial community structure and nitrogen cycling in natural and drained tropical peatland soils

Tropical peatlands, which play a crucial role in the maintenance of different ecosystem services, are increasingly drained for agriculture, forestry, peat extraction and human settlement purposes. The present study investigated the differences between natural and drained sites of a tropical peatland in the community structure of soil bacteria and archaea and their potential to perform nitrogen transformation processes. The results indicate significant dissimilarities in the structure of soil bacterial and archaeal communities as well as nirK, nirS, nosZ, nifH and archaeal amoA gene-possessing microbial communities. The reduced denitrification and N2-fixing potential was detected in the drained tropical peatland soil. In undisturbed peatland soil, the N2O emission was primarily related to nirS-type denitrifiers and dissimilatory nitrate reduction to ammonium, while the conversion of N2O to N2 was controlled by microbes possessing nosZ clade I genes. The denitrifying microbial community of the drained site differed significantly from the natural site community. The main reducers of N2O were microbes harbouring nosZ clade II genes in the drained site. Additionally, the importance of DNRA process as one of the controlling mechanisms of N2O fluxes in the natural peatlands of the tropics revealed from the results of the study.

Cause and effect of N/P ratio decline with eutrophication aggravation in shallow lakes

To explore the relationship and cause and effect between eutrophication and the nitrogen (N)/phosphorus (P) ratio, samples from 38 lakes in Wuhan City, China, with differing degrees of eutrophication, were collected for nutrient levels and extracellular enzyme activities (EEA) in the water column from July 2011 to November 2011. The phosphorus fraction, abundance and potential denitrification rate (PDR) as well as community composition of nirS-type denitrifier in sediments of five typical lakes were further analyzed. A higher trophic level index (TSI) corresponded to a lower N/P ratio, which can be attributed to a loss of N and an increase in P. Specifically, in more eutrophic lakes, the enrichment of total organic carbon and all forms of P in sediments could fuel PDR by shaping community composition and increasing the abundance of nirS-type denitrifier as evidenced by correlation and redundancy analysis, ultimately resulting in a loss of N. Meanwhile, iron-bound phosphorus release induced by anoxia and the hydrolysis of organic P accounted for the observed increase of P in the water column. The lower N/P ratio facilitated the production of leucine aminopeptidase, which was unexpectedly induced by high P but not by low N. Similarly, alkaline phosphatase was induced by high N but not by low P. These findings indicate a mutual coupling and interplay between N and P cycling and confirm our hypothesis that P accumulation accelerates N loss in the process of eutrophication.

Structure and assembly cues for rhizospheric nirK- and nirS-type denitrifier communities in long-term fertilized soils

The stimulatory effect of plants on soil denitrification activity has been widely reported, and root-derived carbon (C) is considered a factor contributing to enhanced denitrification activity in the rhizosphere. However, the mechanisms through which root-derived C shapes the rhizospheric denitrifying community structure remains unclear, especially under different soil fertility levels. Here, DNA-based stable isotope probing (DNA-SIP) and Illumina MiSeq sequencing were employed to characterize root-associated denitrifier communities containing nitrite reductase genes (nirS and nirK) in the rhizosphere of wheat grown in soils with three distinct long-term (32-year) fertilization regimes. Fertilization showed a significant impact on the composition of denitrifying communities that actively utilized photosynthetically fixed C in the wheat rhizosphere. In soils amended with inorganic fertilizers, wheat root-derived 13C was mainly assimilated by nirS-type denitrifiers affiliated to Alcaligenaceae and nirK-type denitrifiers affiliated to Phyllobacteriaceae. In contrast, organic fertilization resulted in larger diversity of 13C-labeled denitrifier communities where nirS-type denitrifiers such as Rhodobacteraceae and unclassified Burkholderiales and some unknown nirK-type denitrifiers were more abundant. The nirS-type denitrifier community was found to be more sensitive to the rhizosphere effect than the nirK-type community. Approximately 31% of the 13C-labeled nirS-type denitrifiers were more abundant in the rhizosphere than in the bulk soil, but only 2% of the 13C-labeled nirK-type denitrifiers showed increased abundance in the rhizosphere. The results of this study present direct evidence that root exudates can act as inducible C sources for heterotrophic denitrifying bacteria, but this induction pattern differs between nirS- and nirK-type communities and is dependent on soil fertility level.

Water Quality and Diversity of Denitrifier Community Structure of Typical Scenic Water Bodies in Xi'an

Six micro-polluted landscape water bodies were selected to explore the relationship between water quality and the nirS-Type denitrifier community structure and diversity with an Illumina high-throughput sequencing technique. The results of a physico-chemical analysis of the water bodies showed that the Fengqing Park (FQ) and Laodong Park (LD) water bodies were classified as inferior Ⅴ water quality, the Qujiang Park (QJ), Mutasi Park (MTS), and Xinjiyuan Park (XJY) water bodies were classified as Ⅴ water quality, and the Yongyang Park (YY) water quality was classified as Ⅳ. The TN values varied from 1.21 mg·L-1 to 6.50 mg·L-1, with the lowest value found in YY and the highest in FQ (P<0.05). TP was significantly higher in LD (0.10 mg·L-1) and significantly lower in QJ[0.02 mg·L-1 (P<0.05)]. The NH4+-N in LD was 4.44 times higher than that in QJ and FQ (P<0.01). Illumina high-throughput sequencing revealed that the denitrifier community composition was significantly different among the six water bodies, and Paracoccus sp., Pseudomonas sp., and Rubrivivax sp. were the dominant genus species. A principle component analysis (PCA) indicated that the nirS-Type denitrifier communities of XJY and MTS were mainly regulated by NH4+-N and the permanganate index, LD and FQ were significantly influenced by NO3--N, NO2--N, TN, TP, and DO, and YY was significantly influenced by pH value. Our results showed that nirS-Type denitrifier communities were regulated by different water quality indicators.

Enhancing Nitrate Removal from Freshwater Pond by Regulating Carbon/Nitrogen Ratio.

Nitrogen accumulation is a serious environmental problem in freshwater ponds, which can lead to massive death of fish and shrimps as well as the eutrophication. The removal of nitrate by regulating the carbon to nitrogen (C/N) ratio and the underlying mechanisms were investigated. The nitrate removal system comprised 530-mL medium containing 5 mg/L -N and 0–66.6 mg/L COD (i.e., C/N ratio of 0–13.3) and 20 g ponds sediments. When the C/N ratio was higher than 8, the nitrate removal efficiency nearly reached 100% during the incubation period and the accumulation of nitrite was negligible. When the C/N ratio was below 8, the nitrate removal efficiency was lower and significant nitrite accumulation occurred. The nitrate removal rate increased with the C/N ratio increased, which was ascribed to the increase in the absolute abundance of denitrifiers (nirS, nirK, and nosZ). Although both nirS-type and nirK-type denitrifiers were found in the sediments of freshwater pond, nirS-type denitrifiers were predominant. Dechloromonas was the major nirS-type denitrifier for nitrate removal in nirS-type with the C/N ratios above 5.33, while the majority of the nirK-type denitrifiers were unclassified. Thus, this study implied that the appropriate C/N ratio played an important role on the removal of excess nitrate from freshwater ponds.

Micro-electrolysis/retinervus luffae-based simultaneous autotrophic and heterotrophic denitrification for low C/N wastewater treatment.

Nitrogen bioremediation in organic insufficient wastewater generally requires an extra carbon source. In this study, nitrate-contaminated wastewater was treated effectively through simultaneous autotrophic and heterotrophic denitrification based on micro-electrolysis carriers (MECs) and retinervus luffae fructus (RLF), respectively. The average nitrate and total nitrogen removal rates reached 96.3 and 94.0% in the MECs/RLF-based autotrophic and heterotrophic denitrification (MRAHD) system without ammonia and nitrite accumulation. The performance of MRAHD was better than that of MEC-based autotrophic denitrification for the wastewater treatment with low carbon nitrogen (COD/N) ratio. Real-time quantitative polymerase chain reaction (qPCR) revealed that the relative abundance of nirS-type denitrifiers attached to MECs (4.9%) and RLF (5.0%) was similar. Illumina sequencing suggested that the dominant genera were Thiobacillus (7.0%) and Denitratisoma (5.7%), which attached to MECs and RLF, respectively. Sulfuritalea was discovered as the dominant genus in the middle of the reactor. The synergistic interaction between autotrophic and heterotrophic denitrifiers played a vital role in the mixotrophic substrate environment.

Distinct distribution patterns of proteobacterial nirK- and nirS-type denitrifiers in the Yellow River estuary, China.

Denitrification is considered to be the critical process in removing reactive nitrogen in estuarine ecosystems. In the present study, the abundance, diversity, and community structure of nirK- and nirS-type denitrifiers were compared in sediments from the Yellow River estuary. Quantitative polymerase chain reaction showed that the 2 types of denitrifiers exhibited different distribution patterns among the samples, indicating their distinct habitat preference. Phylogenetic analysis revealed that most of the sequences from clusters I, III, IV, and V for nirK-type denitrifiers were dominant and were distributed at sites where dissolved oxygen (DO) was lower, and the sequences in the other clusters were dominant at sites with higher DO. However, there was no spatially heterogeneous distribution for the nirS-type denitrifier community. Canonical correlation analysis and correlation analysis demonstrated that the community structure of nirK was more responsive to environmental factors than was that of nirS. Inversely, the abundance and α-diversity targeting nirS gene could be more easily influenced by environmental parameters. These findings can extend our current knowledge about the distribution patterns of denitrifying bacteria and provide a basic theoretical reference for the dynamics of denitrifying communities in estuarine ecosystem of China.

Attached and Suspended Denitrifier Communities in Pristine Limestone Aquifers Harbor High Fractions of Potential Autotrophs Oxidizing Reduced Iron and Sulfur Compounds

Oxygen and nitrate availability as well as the presence of suitable organic or inorganic electron donors are strong drivers of denitrification; however, the factors influencing denitrifier abundance and community composition in pristine aquifers are not well understood. We explored the denitrifier community structure of suspended and attached groundwater microorganisms in two superimposed limestone aquifer assemblages with contrasting oxygen regime in the Hainich Critical Zone Exploratory (Germany). Attached communities were retrieved from freshly crushed parent rock material which had been exposed for colonization in two groundwater wells (12.7 and 48m depth). Quantitative PCR and amplicon pyrosequencing of nirK and nirS genes encoding copper-containing or cytochrome cd1 heme-type nitrite reductase, respectively, and of bacterial 16S ribosomal RNA genes showed a numerical predominance of nirS-type denitrifiers in both attached and suspended groundwater communities and a dominance of nirS-type denitrifiers closely related to the autotrophic thiosulfate- and hydrogen-oxidizing Sulfuritalea hydrogenivorans and the iron- and sulfide-oxidizing Sideroxydans lithotrophicus ES-1. Potential rates of nitrate reduction in association with exposed crushed rock material were higher with an inorganic electron donor (thiosulfate) compared to an organic electron donor (fumarate/acetate) in the upper aquifer assemblage but similar in the lower, oxic aquifer. Our results have clearly demonstrated that groundwater from pristine limestone aquifers harbors diverse denitrifier communities which appear to selectively attach to rock surfaces and harbor a high potential for nitrate reduction. Our findings suggest that the availability of suitable inorganic versus organic electron donors rather than oxygen availability shapes denitrifier communities and their potential activity in these limestone aquifers.

NirS- and nirK-type denitrifier communities are differentially affected by soil type, rice cultivar and water management

Denitrification is one of the main biological sources of N2O, a greenhouse gas. In paddies, water flooding provides anaerobic soil conditions that can boost denitrification. In this work, the influences of rice cultivar, water management and soil type on the abundance and community structure of denitrifying bacteria were evaluated; nirS and nirK-type denitrifiers were considered separately. Two microcosm experiments were performed, and the abundance and community structure for both nitrite reductase genes were assessed using qPCR and T-RFLP, respectively. In the first experiment, two rice cultivars and two water managements were evaluated; in the second experiment, two soils, which mainly differed in their organic matter content, and two flooding times were evaluated. The results show that sampling time was the main factor driving nirS and nirK abundances. The nirK:nirS ratio, recently proposed as an indicator of the soil N2O emission potential, was higher in samples taken at the flower primordium stage of rice in both experiments, suggesting that this stage could be a hot moment for N2O emissions. Soil type was the main factor influencing the nirS community structure, while it had no effect on nirK communities. Although the nirK community structure was not affected by cultivar or soil type, statistical analysis showed that some T-RFs were associated with each cultivar or soil type, indicating that these factors exerted effects on certain subsets of the denitrifying population rather than the whole community. The community structure of nirS-type denitrifiers was more sensitive to the analyzed factors than the nirK-type, and soil type was the main factor that influenced this community structure.

NirS-Encoding denitrifier community composition, distribution, and abundance along the coastal wetlands of China.

For the past few decades, human activities have intensively increased the reactive nitrogen enrichment in China's coastal wetlands. Although denitrification is a critical pathway of nitrogen removal, the understanding of denitrifier community dynamics driving denitrification remains limited in the coastal wetlands. In this study, the diversity, abundance, and community composition of nirS-encoding denitrifiers were analyzed to reveal their variations in China's coastal wetlands. Diverse nirS sequences were obtained and more than 98% of them shared considerable phylogenetic similarity with sequences obtained from aquatic systems (marine/estuarine/coastal sediments and hypoxia sea water). Clone library analysis revealed that the distribution and composition of nirS-harboring denitrifiers had a significant latitudinal differentiation, but without a seasonal shift. Canonical correspondence analysis showed that the community structure of nirS-encoding denitrifiers was significantly related to temperature and ammonium concentration. The nirS gene abundance ranged from 4.3×10(5) to 3.7×10(7) copies g(-1) dry sediment, with a significant spatial heterogeneity. Among all detected environmental factors, temperature was a key factor affecting not only the nirS gene abundance but also the community structure of nirS-type denitrifiers. Overall, this study significantly enhances our understanding of the structure and dynamics of denitrifying communities in the coastal wetlands of China.

Temporal shifts of nitrite reducing communities in a rice field soil in Ibague (Colombia)

Denitrification and nitrification are microbial processes that regulate the cycle of nitrogen and nitrous oxide, which is considered an important greenhouse gas. Rice field soils have been known to have strong denitrifying activities; however, the microorganism structure that is responsible for denitrification and the temporal variation of these communities in the agricultural soils of Ibague (Colombia) is not well known. In this study, the denitrifying community composition was compared between a rice field soil and an uncultivated soil at three different times during the year using a terminal restriction fragment length polymorphism analysis of the nirS functional gene, which codes the enzyme that reduces nitrite, one of the key steps in the denitrification process. The results showed changes in the richness, relative abundance and diversity of the operational taxonomic units between the soils and sampling times. The canonical correspondence analysis indicated that the moisture and the pH were the environmental factors that explained the observed changes in the nirS-type denitrifiers' community composition in the studied soils.

Long-term organic and inorganic fertilization alters temperature sensitivity of potential N2O emissions and associated microbes

Emissions of the greenhouse gas nitrous oxide (N2O) from soil are sensitive to many factors including temperature, nutrient status, pH and bulk-density. Interactions among these are complex, particularly in agricultural systems where fertilizer-addition has interactive influences across many soil properties. Under laboratory conditions, the temperature sensitivity of N2O emissions, as measured by Q10 values (fractional change in rate with a 10 °C increase in temperature), was higher in soils receiving long-term fertilizer addition compared with control. Different fertilization regimes significantly influenced the emission pathways. Application of manure increased the proportion of potential N2O derived from denitrification although the incubation condition used in the present study might not be favorable for anaerobic denitrification. The abundance of archaeal amoA gene copies increased under all fertilizer treatments, but that of bacterial amoA only increased under mineral (NPK) fertilization. Meanwhile, abundance of nirS, nirK and nosZ only increased under OM and MNPK fertilization. T-RFLP analyses showed that both ammonia oxidizing and denitrifier community structures were altered by fertilization, but only the nirS community structure was sensitive to temperature change. Furthermore, a strong correlation was observed between nirS gene abundance and potential N2O emissions. Relationships between AOA, nirK gene abundances and potential N2O emission were significant but relatively weak. PLS path model revealed that besides direct effect, potential N2O emission was also indirectly influenced by temperature through mediation of NH4+ concentration and nirS-type denitrifier. Our work suggests that warming-induced elevation of potential N2O emission could be strengthened by long-term application of fertilizers, especially organic manure, via shifting community abundance and structure of nirS-type denitrifier.

Impact of Land Use Management and Soil Properties on Denitrifier Communities of Namibian Savannas.

We studied potential denitrification activity and the underlying denitrifier communities in soils from a semiarid savanna ecosystem of the Kavango region in NE Namibia to help in predicting future changes in N(2)O emissions due to continuing changes of land use in this region. Soil type and land use (pristine, fallow, and cultivated soils) influenced physicochemical characteristics of the soils that are relevant to denitrification activity and N(2)O fluxes from soils and affected potential denitrification activity. Potential denitrification activity was assessed by using the denitrifier enzyme activity (DEA) assay as a proxy for denitrification activity in the soil. Soil type and land use influenced C and N contents of the soils. Pristine soils that had never been cultivated had a particularly high C content. Cultivation reduced soil C content and the abundance of denitrifiers and changed the composition of the denitrifier communities. DEA was strongly and positively correlated with soil C content and was higher in pristine than in fallow or recently cultivated soils. Soil type and the composition of both the nirK- and nirS-type denitrifier communities also influenced DEA. In contrast, other soil characteristics like N content, C:N ratio, and pH did not predict DEA. These findings suggest that due to greater availability of soil organic matter, and hence a more effective N cycling, the natural semiarid grasslands emit more N(2)O than managed lands in Namibia.

Diversity, Abundance, and Distribution of nirS-Harboring Denitrifiers in Intertidal Sediments of the Yangtze Estuary.

Denitrification plays a critical role in nitrogen removal in estuarine and coastal ecosystems. In this study, the community composition, diversity, abundance, and distribution of cytochrome cd1-type nitrite reductase gene (nirS)-harboring denitrifiers in intertidal sediments of the Yangtze Estuary were analyzed using polymerase chain reaction (PCR)-based clone libraries and quantitative PCR techniques. Clone library analysis showed that the nirS-encoding bacterial biodiversity was significantly higher at the lower salinity sites than at the higher salinity sites. However, there was no significant seasonal difference in the nirS gene diversity between summer and winter. Phylogenetic analysis revealed that the nirS-harboring denitrifier communities at the study area had distinctive spatial heterogeneity along the estuary. At the lower salinity sites, the nirS-harboring bacterial community was co-dominated by clusters III and VII; while at the higher salinity sites, it was dominated by cluster I. Canonical correspondence analysis indicated that the community compositions of nirS-type denitrifiers were significantly correlated with salinity, ammonium, and nitrate. Quantitative PCR results showed that the nirS gene abundance was in the range of 1.01 × 10(6) to 9.00 × 10(7) copies per gram dry sediment, without significant seasonal variation. Among all the environmental factors, the nirS gene abundance was only significantly related to the change of salinity. These results can extend our current knowledge about the composition and dynamics of denitrification microbial community in the estuarine ecosystem.

Community composition of bacteria involved in fixed nitrogen loss in the water column of two major oxygen minimum zones in the ocean

The community composition of bacteria involved in nitrogen (N) transformations in the oxy- gen minimum zones (OMZ) may be related to the rates of fixed N loss in these systems. The abundance of both denitrifying and anammox bacteria and the assemblage composition of denitrifiers were investi- gated in the Eastern Tropical South Pacific and the Arabian Sea using assays based on bacterial molecu- lar markers. Quantitative PCR was used to investi- gate the abundance and distribution of genes encod- ing nitrite reductase (nirK and nirS) in denitrifying bacteria and hydrazine oxidase (hzo) and 16S rRNA genes in anammox bacteria. All of these genes had depth distributions with maxima associated with the secondary nitrite maximum in low-oxygen waters. NirS was much more abundant than nirK and much more abundant than the 16S rRNA gene from anam- mox bacteria. The ratio of hzo:16S rRNA was low and variable, implying greater unexplored diversity in the hzo gene. Assemblage composition of the abun- dant nirS-type denitrifiers was evaluated using a functional gene microarray. Of the nirS archetypes represented on the microarray, very few occurred specifically in one region or depth interval, but the assemblages varied significantly. Community com- position of denitrifiers based on microarray analysis of the nirS gene was most different between geo- graphical regions. Within each region, the surface layer and OMZ assemblages clustered distinctly. Thus, in addition to spatial and temporal variation in denitrification and anammox rates, microbial abun- dance and community composition also vary between OMZ regions and depths.

Response of an Arctic Sediment Nitrogen Cycling Community to Increased CO2

Ocean acidification influences sediment/water nitrogen fluxes, possibly by impacting on the microbial process of ammonia oxidation. To investigate this further, undisturbed sediment cores collected from Ny Alesund harbour (Svalbard) were incubated with seawater adjusted to CO2 concentrations of 380, 540, 760, 1,120 and 3,000 µatm. DNA and RNA were extracted from the sediment surface after 14 days' exposure and the abundance of bacterial and archaeal ammonia oxidising (amoA) genes and transcripts quantified using quantitative polymerase chain reaction. While there was no change to the abundance of bacterial amoA genes, an increase to 760 µatm pCO2 reduced the abundance of bacterial amoA transcripts by 65 %, and this was accompanied by a shift in the composition of the active community. In contrast, archaeal amoA gene and transcript abundance both doubled at 3,000 µatm, with an increase in species richness also apparent. This suggests that ammonia oxidising bacteria and archaea in marine sediments have different pH optima, and the impact of elevated CO2 on N cycling may be dependent on the relative abundances of these two major microbial groups. Further evidence of a shift in the balance of key N cycling groups was also evident: the abundance of nirS-type denitrifier transcripts decreased alongside bacterial amoA transcripts, indicating that NO3− produced by bacterial nitrification fuelled denitrification. An increase in the abundance of Planctomycete-specific 16S rRNA, the vast majority of which grouped with known anammox bacteria, was also apparent at 3,000 µatm pCO2. This could indicate a possible shift from coupled nitrification–denitrification to anammox activity at elevated CO2.