Evidence for phylogenetically and catabolically diverse active diazotrophs in deep-sea sediment.
ABSTRACT: Diazotrophic microorganisms regulate marine productivity by alleviating nitrogen limitation. However, we know little about the identity and activity of diazotrophs in deep-sea sediments, a habitat covering nearly two-thirds of the planet. Here, we identify candidate diazotrophs from Pacific Ocean sediments collected at 2893?m water depth using 15N-DNA stable isotope probing and a novel pipeline for nifH sequence analysis. Together, these approaches detect an unexpectedly diverse assemblage of active diazotrophs, including members of the Acidobacteria, Firmicutes, Nitrospirae, Gammaproteobacteria, and Deltaproteobacteria. Deltaproteobacteria, predominately members of the Desulfobacterales and Desulfuromonadales, are the most abundant diazotrophs detected, and display the most microdiversity of associated nifH sequences. Some of the detected lineages, including those within the Acidobacteria, have not previously been shown to fix nitrogen. The diazotrophs appear catabolically diverse, with the potential for using oxygen, nitrogen, iron, sulfur, and carbon as terminal electron acceptors. Therefore, benthic diazotrophy may persist throughout a range of geochemical conditions and provide a stable source of fixed nitrogen over geologic timescales. Our results suggest that nitrogen-fixing communities in deep-sea sediments are phylogenetically and catabolically diverse, and open a new line of inquiry into the ecology and biogeochemical impacts of deep-sea microorganisms.
Project description:Ecological evidence suggests that heterotrophic diazotrophs fueled by organic carbon respiration in sediments play an important role in marine nitrogen fixation. However, fundamental knowledge about the identities, abundance, diversity, biogeography, and controlling environmental factors of nitrogen-fixing communities in open ocean sediments is still elusive. Surprisingly, little is known also about nitrogen-fixing communities in sediments of the more research-accessible marginal seas. Here we report on an investigation of the environmental geochemistry and putative diazotrophic microbiota in the sediments of Bohai Sea, an eutrophic marginal sea of the western Pacific Ocean. Diverse and abundant nifH gene sequences were identified and sulfate-reducing bacteria (SRB) were found to be the dominant putative nitrogen-fixing microbes. Community statistical analyses suggested bottom water temperature, bottom water chlorophyll a content (or the covarying turbidity) and sediment porewater Eh (or the covarying pH) as the most significant environmental factors controlling the structure and spatial distribution of the putative diazotrophic communities, while sediment Hg content, sulfide content, and porewater [Formula: see text]-Si content were identified as the key environmental factors correlated positively with the nifH gene abundance in Bohai Sea sediments. Comparative analyses between the Bohai Sea and the northern South China Sea (nSCS) identified a significant composition difference of the putative diazotrophic communities in sediments between the shallow-water (estuarine and nearshore) and deep-water (offshore and deep-sea) environments, and sediment porewater dissolved oxygen content, water depth and in situ temperature as the key environmental factors tentatively controlling the species composition, community structure, and spatial distribution of the marginal sea sediment nifH-harboring microbiota. This confirms the ecophysiological specialization and niche differentiation between the shallow-water and deep-water sediment diazotrophic communities and suggests that the in situ physical and geochemical conditions play a more important role than geographical contiguity in determining the community similarity of the diazotrophic microbiota in marginal sea sediments.
Project description:The South China Sea (SCS), the largest marginal sea in the Western Pacific Ocean, is a huge oligotrophic water body with very limited influx of nitrogenous nutrients. This suggests that sediment microbial N(2) fixation plays an important role in the production of bioavailable nitrogen. To test the molecular underpinning of this hypothesis, the diversity, abundance, biogeographical distribution, and community structure of the sediment diazotrophic microbiota were investigated at 12 sampling sites, including estuarine, coastal, offshore, deep-sea, and methane hydrate reservoirs or their prospective areas by targeting nifH and some other functional biomarker genes. Diverse and novel nifH sequences were obtained, significantly extending the evolutionary complexity of extant nifH genes. Statistical analyses indicate that sediment in situ temperature is the most significant environmental factor influencing the abundance, community structure, and spatial distribution of the sediment nifH-harboring microbial assemblages in the northern SCS (nSCS). The significantly positive correlation of the sediment pore water NH(4)(+) concentration with the nifH gene abundance suggests that the nSCS sediment nifH-harboring microbiota is active in N(2) fixation and NH(4)(+) production. Several other environmental factors, including sediment pore water PO(4)(3-) concentration, sediment organic carbon, nitrogen and phosphorus levels, etc., are also important in influencing the community structure, spatial distribution, or abundance of the nifH-harboring microbial assemblages. We also confirmed that the nifH genes encoded by archaeal diazotrophs in the ANME-2c subgroup occur exclusively in the deep-sea methane seep areas, providing for the possibility to develop ANME-2c nifH genes as a diagnostic tool for deep-sea methane hydrate reservoir discovery.
Project description:The Eurasian basin of the Central Arctic Ocean is nitrogen limited, but little is known about the presence and role of nitrogen-fixing bacteria. Recent studies have indicated the occurrence of diazotrophs in Arctic coastal waters potentially of riverine origin. Here, we investigated the presence of diazotrophs in ice and surface waters of the Central Arctic Ocean in the summer of 2012. We identified diverse communities of putative diazotrophs through targeted analysis of the nifH gene, which encodes the iron protein of the nitrogenase enzyme. We amplified 529 nifH sequences from 26 samples of Arctic melt ponds, sea ice and surface waters. These sequences resolved into 43 clusters at 92% amino acid sequence identity, most of which were non-cyanobacterial phylotypes from sea ice and water samples. One cyanobacterial phylotype related to Nodularia sp. was retrieved from sea ice, suggesting that this important functional group is rare in the Central Arctic Ocean. The diazotrophic community in sea-ice environments appear distinct from other cold-adapted diazotrophic communities, such as those present in the coastal Canadian Arctic, the Arctic tundra and glacial Antarctic lakes. Molecular fingerprinting of nifH and the intergenic spacer region of the rRNA operon revealed differences between the communities from river-influenced Laptev Sea waters and those from ice-related environments pointing toward a marine origin for sea-ice diazotrophs. Our results provide the first record of diazotrophs in the Central Arctic and suggest that microbial nitrogen fixation may occur north of 77°N. To assess the significance of nitrogen fixation for the nitrogen budget of the Arctic Ocean and to identify the active nitrogen fixers, further biogeochemical and molecular biological studies are needed.
Project description:Diverse nifH and nifH-like gene sequences were obtained from the deep-sea surface sediments of the methane hydrate-bearing Okhotsk Sea. Some sequences formed novel families of the NifH or NifH-like proteins, of currently unresolved bacterial or archaeal origin. Comparison with other marine environments indicates environmental specificity of some of the sequences, either unique to the methane seep sediments of the Okhotsk Sea or to the general deep-sea methane seep sedimentary environments.
Project description:Nitrogen (N) cycling microbial communities in marine sediments are extremely diverse, and it is unknown whether this diversity reflects extensive functional redundancy. Sedimentary denitrifiers remove significant amounts of N from the coastal ocean and diazotrophs are typically regarded as inconsequential. Recently, N fixation has been shown to be a potentially important source of N in estuarine and continental shelf sediments. Analysis of expressed genes for nitrite reductase (nirS) and a nitrogenase subunit (nifH) was used to identify the likely active denitrifiers and nitrogen fixers in surface sediments from different seasons in Narragansett Bay (Rhode Island, USA). The overall diversity of diazotrophs expressing nifH decreased along the estuarine gradient from the estuarine head to an offshore continental shelf site. Two groups of sequences related to anaerobic sulphur/iron reducers and sulphate reducers dominated libraries of expressed nifH genes. Quantitative polymerase chain reaction (qPCR) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) data shows the highest abundance of both groups at a mid bay site, and the highest nifH expression at the head of the estuary, regardless of season. Several potential environmental factors, including water temperature, oxygen concentration and metal contamination, may influence the abundance and nifH expression of these two bacterial groups.
Project description:Seagrass colonization changes the chemistry and biogeochemical cycles mediated by microbes in coastal sediments. In this study, we molecularly characterized the diazotrophic assemblages and entire bacterial community in surface sediments of a Zostera marina-colonized coastal lagoon in northern China. Higher nitrogenase gene (nifH) copy numbers were detected in the sediments from the vegetated region than in the sediments from the unvegetated region nearby. The nifH phylotypes detected were mostly affiliated with the Geobacteraceae, Desulfobulbus, Desulfocapsa, and Pseudomonas. Redundancy analysis based on terminal restriction fragment length polymorphism analysis showed that the distribution of nifH genotypes was mostly shaped by the ratio of total organic carbon to total organic nitrogen, the concentration of cadmium in the sediments, and the pH of the overlying water. High-throughput sequencing and phylogenetic analyses of bacterial 16S rRNA genes also indicated the presence of Geobacteraceae and Desulfobulbaceae phylotypes in these samples. A comparison of these results with those of previous studies suggests the prevalence and predominance of iron(III)-reducing Geobacteraceae and sulfate-reducing Desulfobulbaceae diazotrophs in coastal sedimentary environments. Although the entire bacterial community structure was not significantly different between these two niches, Desulfococcus (Deltaproteobacteria) and Anaerolineae (Chloroflexi) presented with much higher proportions in the vegetated sediments, and Flavobacteriaceae (Bacteroidetes) occurred more frequently in the bare sediments. These data suggest that the high bioavailability of organic matter (indicated by relatively lower carbon-to-nitrogen ratios) and the less-reducing anaerobic condition in vegetated sediments may favor Desulfococcus and Anaerolineae lineages, which are potentially important populations in benthic carbon and sulfur cycling in the highly productive seagrass ecosystem.
Project description:The Baltic Sea receives large nitrogen inputs by diazotrophic (N2-fixing) heterocystous cyanobacteria but the significance of heterotrophic N2 fixation has not been studied. Here, the diversity, abundance and transcription of the nifH fragment of the nitrogenase enzyme in two basins of the Baltic Sea proper was examined. N2 fixation was measured at the surface (5?m) and in anoxic water (200?m). Vertical sampling profiles of >10 and <10??m size fractions were collected in 2007, 2008 and 2011 at the Gotland Deep and in 2011 in the Bornholm Basin. Both of these stations are characterized by permanently anoxic bottom water. The 454-pyrosequencing nifH analysis revealed a diverse assemblage of nifH genes related to alpha-, beta- and gammaproteobacteria (nifH cluster I) and anaerobic bacteria (nifH cluster III) at and below the chemocline. Abundances of genes and transcripts of seven diazotrophic phylotypes were investigated using quantitative polymerase chain reaction revealing abundances of heterotrophic nifH phylotypes of up to 2.1 × 10(7) nifH copies?l(-1). Abundant nifH transcripts (up to 3.2 × 10(4) transcripts?l(-1)) within nifH cluster III and co-occurring N2 fixation (0.44±0.26?nmol?l(-1)?day(-1)) in deep water suggests that heterotrophic diazotrophs are fixing N2 in anoxic ammonium-rich waters. Our results reveal that N2 fixation in the Baltic Sea is not limited to illuminated N-deplete surface waters and suggest that N2 fixation could also be of importance in other suboxic regions of the world's oceans.
Project description:BACKGROUND:Holobionts comprising nitrogen-fixing diazotrophs and phytoplankton or zooplankton are ubiquitous in the pelagic sea. However, neither the community structure of plankton-associated diazotrophs (PADs) nor their nitrogenase transcriptional activity are well-understood. In this study, we used nifH gene Illumina sequencing and quantitative PCR to characterize the community composition and nifH expression profile of PADs with >?100??m size fraction in the euphotic zone of the northern South China Sea. RESULTS:The results of DNA- and RNA-derived nifH gene revealed a higher alpha-diversity in the active than in the total community. Moreover, the compositional resemblance among different sites was less for active than for total communities of PADs. We characterized the 20 most abundant OTUs by ranking the sum of sequence reads across 9 sampling stations for individual OTUs in both nifH DNA and RNA libraries, and then assessed their phylogenetic relatedness. Eight of the 20 abundant OTUs were phylogenetically affiliated with Trichodesmium and occurred in approximately equal proportion in both the DNA and RNA libraries. The analysis of nifH gene expression level showed uneven attribute of the abundance and nitrogenase activities by the remaining 12 OTUs. Taxa belonging to cluster III and Betaproteobacteria were present at moderate abundance but exhibited negligible nitrogenase transcription activity. Whereas, the abundances of Richelia, Deltaproteobacteria and Gammaproteobacteria were low but the contribution of these groups to nitrogenase transcription was disproportionately high. CONCLUSIONS:The substantial variation in community structure among active dizatrophic fractions compared to the total communities suggests that the former are better indicators of biological response to environmental changes. Altogether, our study highlights the importance of rare PADs groups in nitrogen fixation in plankton holobionts, evidenced by their high level of nitrogenase transcription.
Project description:Sinking particles transport carbon and nutrients from the surface ocean into the deep sea and are considered hot spots for bacterial diversity and activity. In the oligotrophic oceans, nitrogen (N2)-fixing organisms (diazotrophs) are an important source of new N but the extent to which these organisms are present and exported on sinking particles is not well known. Sinking particles were collected every 6?h over a 2-day period using net traps deployed at 150?m in the North Pacific Subtropical Gyre. The bacterial community and composition of diazotrophs associated with individual and bulk sinking particles was assessed using 16S rRNA and nifH gene amplicon sequencing. The bacterial community composition in bulk particles remained remarkably consistent throughout time and space while large variations of individually picked particles were observed. This difference suggests that unique biogeochemical conditions within individual particles may offer distinct ecological niches for specialized bacterial taxa. Compared to surrounding seawater, particle samples were enriched in different size classes of globally significant N2-fixing cyanobacteria including Trichodesmium, symbionts of diatoms, and the unicellular cyanobacteria Crocosphaera and UCYN-A. The particles also contained nifH gene sequences of diverse non-cyanobacterial diazotrophs suggesting that particles could be loci for N2 fixation by heterotrophic bacteria. The results demonstrate that diverse diazotrophs were present on particles and that new N may thereby be directly exported from surface waters on sinking particles.
Project description:The coral holobiont often resides in oligotrophic waters; both coral cells and their symbiotic dinoflagellates possess ammonium assimilation enzymes and potentially benefit from the nitrogen fixation of coral-associated diazotrophs. However, the seasonal dynamics of coral-associated diazotrophs are not well characterized. Here, the seasonal variations of diazotrophic communities associated with three corals, Galaxea astreata, Pavona decussata, and Porites lutea, were studied using nifH gene amplicon pyrosequencing techniques. Our results revealed a great diversity of coral-associated diazotrophs. nifH sequences related to Alphaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria were ubiquitous and dominant in all corals in two seasons. In contrast with the coral P. decussata, both G. astreata and P. lutea showed significant seasonal changes in the diazotrophic communities and nifH gene abundance. Variable diazotroph groups accounted for a range from 11 to 49% within individual coral samples. Most of the variable diazotrophic groups from P. decussata were species-specific, however, the majority of overlapping variable groups in G. astreata and P. lutea showed the same seasonal variation characteristics. Rhodopseudomonas palustris- and Gluconacetobacter diazotrophicus-affiliated sequences were relatively abundant in the summer, whereas a nifH sequence related to Halorhodospira halophila was relatively abundant in spring G. astreata and P. lutea. The seasonal variations of all diazotrophic communities were significantly correlated with the seasonal shifts of ammonium and nitrate, suggesting that diazotrophs play an important role in the nitrogen cycle of the coral holobiont.