Sulfur cycling connects microbiomes and biogeochemistry in deep-sea hydrothermal plumes.

In globally distributed deep-sea hydrothermal vent plumes, microbiomes are shaped by the redox energy landscapes created by reduced hydrothermal vent fluids mixing with oxidized seawater. Plumes can disperse over thousands of kilometers and their characteristics are determined by geochemical sources from vents, e.g., hydrothermal inputs, nutrients, and trace metals. However, the impacts of plume biogeochemistry on the oceans are poorly constrained due to a lack of integrated understanding of microbiomes, population genetics, and geochemistry. Here, we use microbial genomes to understand links between biogeography, evolution, and metabolic connectivity, and elucidate their impacts on biogeochemical cycling in the deep sea. Using data from 36 diverse plume samples from seven ocean basins, we show that sulfur metabolism defines the core microbiome of plumes and drives metabolic connectivity in the microbial community. Sulfur-dominated geochemistry influences energy landscapes and promotes microbial growth, while other energy sources influence local energy landscapes. We further demonstrated the consistency of links among geochemistry, function, and taxonomy. Amongst all microbial metabolisms, sulfur transformations had the highest MW-score, a measure of metabolic connectivity in microbial communities. Additionally, plume microbial populations have low diversity, short migration history, and gene-specific sweep patterns after migrating from background seawater. Selected functions include nutrient uptake, aerobic oxidation, sulfur oxidation for higher energy yields, and stress responses for adaptation. Our findings provide the ecological and evolutionary bases of change in sulfur-driven microbial communities and their population genetics in adaptation to changing geochemical gradients in the oceans.

Globally-distributed microbial eukaryotes exhibit endemism at deep-sea hydrothermal vents.

Single-celled microbial eukaryotes inhabit deep-sea hydrothermal vent environments and play critical ecological roles in the vent-associated microbial food web. 18S rRNA amplicon sequencing of diffuse venting fluids from four geographically- and geochemically-distinct hydrothermal vent fields was applied to investigate community diversity patterns among protistan assemblages. The four vent fields include Axial Seamount at the Juan de Fuca Ridge, Sea Cliff and Apollo at the Gorda Ridge, all in the NE Pacific Ocean, and Piccard and Von Damm at the Mid-Cayman Rise in the Caribbean Sea. We describe species diversity patterns with respect to hydrothermal vent field and sample type, identify putative vent endemic microbial eukaryotes, and test how vent fluid geochemistry may influence microbial community diversity. At a semi-global scale, microbial eukaryotic communities at deep-sea vents were composed of similar proportions of dinoflagellates, ciliates, Rhizaria, and stramenopiles. Individual vent fields supported distinct and highly diverse assemblages of protists that included potentially endemic or novel vent-associated strains. These findings represent a census of deep-sea hydrothermal vent protistan communities. Protistan diversity, which is shaped by the hydrothermal vent environment at a local scale, ultimately influences the vent-associated microbial food web and the broader deep-sea carbon cycle.

Seafloor Incubation Experiment with Deep-Sea Hydrothermal Vent Fluid Reveals Effect of Pressure and Lag Time on Autotrophic Microbial Communities.

Depressurization and sample processing delays may impact the outcome of shipboard microbial incubations of samples collected from the deep sea. To address this knowledge gap, we developed a remotely operated vehicle (ROV)-powered incubator instrument to carry out and compare results from in situ and shipboard RNA stable isotope probing (RNA-SIP) experiments to identify the key chemolithoautotrophic microbes and metabolisms in diffuse, low-temperature venting fluids from Axial Seamount. All the incubations showed microbial uptake of labeled bicarbonate primarily by thermophilic autotrophic Epsilonbacteraeota that oxidized hydrogen coupled with nitrate reduction. However, the in situ seafloor incubations showed higher abundances of transcripts annotated for aerobic processes, suggesting that oxygen was lost from the hydrothermal fluid samples prior to shipboard analysis. Furthermore, transcripts for thermal stress proteins such as heat shock chaperones and proteases were significantly more abundant in the shipboard incubations, suggesting that depressurization induced thermal stress in the metabolically active microbes in these incubations. Together, the results indicate that while the autotrophic microbial communities in the shipboard and seafloor experiments behaved similarly, there were distinct differences that provide new insight into the activities of natural microbial assemblages under nearly native conditions in the ocean.IMPORTANCE Diverse microbial communities drive biogeochemical cycles in Earth's ocean, yet studying these organisms and processes is often limited by technological capabilities, especially in the deep ocean. In this study, we used a novel marine microbial incubator instrument capable of in situ experimentation to investigate microbial primary producers at deep-sea hydrothermal vents. We carried out identical stable isotope probing experiments coupled to RNA sequencing both on the seafloor and on the ship to examine thermophilic, microbial autotrophs in venting fluids from an active submarine volcano. Our results indicate that microbial communities were significantly impacted by the effects of depressurization and sample processing delays, with shipboard microbial communities being more stressed than seafloor incubations. Differences in metabolism were also apparent and are likely linked to the chemistry of the fluid at the beginning of the experiment. Microbial experimentation in the natural habitat provides new insights into understanding microbial activities in the ocean.

Selection Is a Significant Driver of Gene Gain and Loss in the Pangenome of the Bacterial Genus Sulfurovum in Geographically Distinct Deep-Sea Hydrothermal Vents.

Microbial genomes have highly variable gene content, and the evolutionary history of microbial populations is shaped by gene gain and loss mediated by horizontal gene transfer and selection. To evaluate the influence of selection on gene content variation in hydrothermal vent microbial populations, we examined 22 metagenome-assembled genomes (MAGs) (70 to 97% complete) from the ubiquitous vent Epsilonbacteraeota genus Sulfurovum that were recovered from two deep-sea hydrothermal vent regions, Axial Seamount in the northeastern Pacific Ocean (13 MAGs) and the Mid-Cayman Rise in the Caribbean Sea (9 MAGs). Genes involved in housekeeping functions were highly conserved across Sulfurovum lineages. However, genes involved in environment-specific functions, and in particular phosphate regulation, were found mostly in Sulfurovum genomes from the Mid-Cayman Rise in the low-phosphate Atlantic Ocean environment, suggesting that nutrient limitation is an important selective pressure for these bacteria. Furthermore, genes that were rare within the pangenome were more likely to undergo positive selection than genes that were highly conserved in the pangenome, and they also appeared to have experienced gene-specific sweeps. Our results suggest that selection is a significant driver of gene gain and loss for dominant microbial lineages in hydrothermal vents and highlight the importance of factors like nutrient limitation in driving microbial adaptation and evolution.IMPORTANCE Microbes can alter their gene content through the gain and loss of genes. However, there is some debate as to whether natural selection or neutral processes play a stronger role in molding the gene content of microbial genomes. In this study, we examined variation in gene content for the Epsilonbacteraeota genus Sulfurovum from deep-sea hydrothermal vents, which are dynamic habitats known for extensive horizontal gene transfer within microbial populations. Our results show that natural selection is a strong driver of Sulfurovum gene content and that nutrient limitation in particular has shaped the Sulfurovum genome, leading to differences in gene content between ocean basins. Our results also suggest that recently acquired genes undergo stronger selection than genes that were acquired in the more distant past. Overall, our results highlight the importance of natural selection in driving the evolution of microbial populations in these dynamic habitats.

Fluid geochemistry, local hydrology, and metabolic activity define methanogen community size and composition in deep-sea hydrothermal vents.

The size and biogeochemical impact of the subseafloor biosphere in oceanic crust remain largely unknown due to sampling limitations. We used reactive transport modeling to estimate the size of the subseafloor methanogen population, volume of crust occupied, fluid residence time, and nature of the subsurface mixing zone for two low-temperature hydrothermal vents at Axial Seamount. Monod CH4 production kinetics based on chemostat H2 availability and batch-culture Arrhenius growth kinetics for the hyperthermophile Methanocaldococcus jannaschii and thermophile Methanothermococcus thermolithotrophicus were used to develop and parameterize a reactive transport model, which was constrained by field measurements of H2, CH4, and metagenome methanogen concentration estimates in 20-40 °C hydrothermal fluids. Model results showed that hyperthermophilic methanogens dominate in systems where a narrow flow path geometry is maintained, while thermophilic methanogens dominate in systems where the flow geometry expands. At Axial Seamount, the residence time of fluid below the surface was 29-33 h. Only 1011 methanogenic cells occupying 1.8-18 m3 of ocean crust per m2 of vent seafloor area were needed to produce the observed CH4 anomalies. We show that variations in local geology at diffuse vents can create fluid flow paths that are stable over space and time, harboring persistent and distinct microbial communities.

Spatially distinct, temporally stable microbial populations mediate biogeochemical cycling at and below the seafloor in hydrothermal vent fluids.

At deep-sea hydrothermal vents, microbial communities thrive across geochemical gradients above, at, and below the seafloor. In this study, we determined the gene content and transcription patterns of microbial communities and specific populations to understand the taxonomy and metabolism both spatially and temporally across geochemically different diffuse fluid hydrothermal vents. Vent fluids were examined via metagenomic, metatranscriptomic, genomic binning, and geochemical analyses from Axial Seamount, an active submarine volcano on the Juan de Fuca Ridge in the NE Pacific Ocean, from 2013 to 2015 at three different vents: Anemone, Marker 33, and Marker 113. Results showed that individual vent sites maintained microbial communities and specific populations over time, but with spatially distinct taxonomic, metabolic potential, and gene transcription profiles. The geochemistry and physical structure of each vent both played important roles in shaping the dominant organisms and metabolisms present at each site. Genomic binning identified key populations of SUP05, Aquificales and methanogenic archaea carrying out important transformations of carbon, sulfur, hydrogen, and nitrogen, with groups that appear unique to individual sites. This work highlights the connection between microbial metabolic processes, fluid chemistry, and microbial population dynamics at and below the seafloor and increases understanding of the role of hydrothermal vent microbial communities in deep ocean biogeochemical cycles.

Factors affecting the bacterial community composition and heterotrophic production of Columbia River estuarine turbidity maxima.

Estuarine turbidity maxima (ETM) function as hotspots of microbial activity and diversity in estuaries, yet, little is known about the temporal and spatial variability in ETM bacterial community composition. To determine which environmental factors affect ETM bacterial populations in the Columbia River estuary, we analyzed ETM bacterial community composition (Sanger sequencing and amplicon pyrosequencing of 16S rRNA gene) and bulk heterotrophic production (3 H-leucine incorporation rates). We collected water 20 times to cover five ETM events and obtained 42 samples characterized by different salinities, turbidities, seasons, coastal regimes (upwelling vs. downwelling), locations, and particle size. Spring and summer populations were distinct. All May samples had similar bacterial community composition despite having different salinities (1-24 PSU), but summer non-ETM bacteria separated into marine, freshwater, and brackish assemblages. Summer ETM bacterial communities varied depending on coastal upwelling or downwelling conditions and on the sampling site location with respect to tidal intrusion during the previous neap tide. In contrast to ETM, whole (>0.2 µm) and free-living (0.2-3 µm) assemblages of non-ETM waters were similar to each other, indicating that particle-attached (>3 µm) non-ETM bacteria do not develop a distinct community. Brackish water type (ETM or non-ETM) is thus a major factor affecting particle-attached bacterial communities. Heterotrophic production was higher in particle-attached than free-living fractions in all brackish waters collected throughout the water column during the rise to decline of turbidity through an ETM event (i.e., ETM-impacted waters). However, free-living communities showed higher productivity prior to or after an ETM event (i.e., non-ETM-impacted waters). This study has thus found that Columbia River ETM bacterial communities vary based on seasons, salinity, sampling location, and particle size, with the existence of three particle types characterized by different bacterial communities in ETM, ETM-impacted, and non-ETM-impacted brackish waters. Taxonomic analysis suggests that ETM key biological function is to remineralize organic matter.

Bacterial Biogeography across the Amazon River-Ocean Continuum.

Spatial and temporal patterns in microbial biodiversity across the Amazon river-ocean continuum were investigated along ∼675 km of the lower Amazon River mainstem, in the Tapajós River tributary, and in the plume and coastal ocean during low and high river discharge using amplicon sequencing of 16S rRNA genes in whole water and size-fractionated samples (0.2-2.0 µm and >2.0 µm). River communities varied among tributaries, but mainstem communities were spatially homogeneous and tracked seasonal changes in river discharge and co-varying factors. Co-occurrence network analysis identified strongly interconnected river assemblages during high (May) and low (December) discharge periods, and weakly interconnected transitional assemblages in September, suggesting that this system supports two seasonal microbial communities linked to river discharge. In contrast, plume communities showed little seasonal differences and instead varied spatially tracking salinity. However, salinity explained only a small fraction of community variability, and plume communities in blooms of diatom-diazotroph assemblages were strikingly different than those in other high salinity plume samples. This suggests that while salinity physically structures plumes through buoyancy and mixing, the composition of plume-specific communities is controlled by other factors including nutrients, phytoplankton community composition, and dissolved organic matter chemistry. Co-occurrence networks identified interconnected assemblages associated with the highly productive low salinity near-shore region, diatom-diazotroph blooms, and the plume edge region, and weakly interconnected assemblages in high salinity regions. This suggests that the plume supports a transitional community influenced by immigration of ocean bacteria from the plume edge, and by species sorting as these communities adapt to local environmental conditions. Few studies have explored patterns of microbial diversity in tropical rivers and coastal oceans. Comparison of Amazon continuum microbial communities to those from temperate and arctic systems suggest that river discharge and salinity are master variables structuring a range of environmental conditions that control bacterial communities across the river-ocean continuum.

Microbial players and processes involved in phytoplankton bloom utilization in the water column of a fast-flowing, river-dominated estuary.

Fueled by seasonal phytoplankton blooms, the Columbia River estuary is a natural bioreactor for organic matter transformations. Prior metagenome analyses indicated high abundances of diverse Bacteroidetes taxa in estuarine samples containing phytoplankton. To examine the hypothesis that Bacteroidetes taxa have important roles in phytoplankton turnover, we further analyzed metagenomes from water collected along a salinity gradient at 0, 5, 15, 25, and 33 PSU during bloom events. Size fractions were obtained by using a 3-µm prefilter and 0.2-µm collection filter. Although this approach targeted bacteria by removing comparatively large eukaryotic cells, the metagenome from the ES-5 sample (5 PSU) nevertheless contained an abundance of diatom DNA. Biogeochemical measurements and prior studies indicated that this finding resulted from the leakage of cellular material due to freshwater diatom lysis at low salinity. Relative to the other metagenomes, the bacterial fraction of ES-5 was dramatically depleted of genes annotated as Bacteroidetes and lysogenic bacteriophages, but was overrepresented in DNA of protists and Myxococcales bacterivores. We suggest the following equally plausible scenarios for the microbial response to phytoplankton lysis: (1) Bacteroidetes depletion in the free-living fraction may at least in part be caused by their attachment to fluvial diatoms as the latter are lysed upon contact with low-salinity estuarine waters; (2) diatom particle colonization is likely followed by rapid bacterial growth and lytic phage infection, resulting in depletion of lysogenic bacteriophages and host bacteria; and (3) the subsequent availability of labile organic matter attracted both grazers and predators to feed in this estuarine biogeochemical "hotspot," which may have additionally depleted Bacteroidetes populations. These results represent the first detailed molecular analysis of the microbial response to phytoplankton lysis at the freshwater-brackish water interface in the fast-flowing Columbia River estuary.

Coupled RNA-SIP and metatranscriptomics of active chemolithoautotrophic communities at a deep-sea hydrothermal vent.

The chemolithoautotrophic microbial community of the rocky subseafloor potentially provides a large amount of organic carbon to the deep ocean, yet our understanding of the activity and metabolic complexity of subseafloor organisms remains poorly described. A combination of metagenomic, metatranscriptomic, and RNA stable isotope probing (RNA-SIP) analyses were used to identify the metabolic potential, expression patterns, and active autotrophic bacteria and archaea and their pathways present in low-temperature hydrothermal fluids from Axial Seamount, an active submarine volcano. Metagenomic and metatranscriptomic results showed the presence of genes and transcripts for sulfur, hydrogen, and ammonium oxidation, oxygen respiration, denitrification, and methanogenesis, as well as multiple carbon fixation pathways. In RNA-SIP experiments across a range of temperatures under reducing conditions, the enriched (13)C fractions showed differences in taxonomic and functional diversity. At 30 °C and 55 °C, Epsilonproteobacteria were dominant, oxidizing hydrogen and primarily reducing nitrate. Methanogenic archaea were also present at 55 °C, and were the only autotrophs present at 80 °C. Correspondingly, the predominant CO2 fixation pathways changed from the reductive tricarboxylic acid (rTCA) cycle to the reductive acetyl-CoA pathway with increasing temperature. By coupling RNA-SIP with meta-omics, this study demonstrates the presence and activity of distinct chemolithoautotrophic communities across a thermal gradient of a deep-sea hydrothermal vent.

Microbial Gene Abundance and Expression Patterns across a River to Ocean Salinity Gradient.

Microbial communities mediate the biogeochemical cycles that drive ecosystems, and it is important to understand how these communities are affected by changing environmental conditions, especially in complex coastal zones. As fresh and marine waters mix in estuaries and river plumes, the salinity, temperature, and nutrient gradients that are generated strongly influence bacterioplankton community structure, yet, a parallel change in functional diversity has not been described. Metagenomic and metatranscriptomic analyses were conducted on five water samples spanning the salinity gradient of the Columbia River coastal margin, including river, estuary, plume, and ocean, in August 2010. Samples were pre-filtered through 3 µm filters and collected on 0.2 µm filters, thus results were focused on changes among free-living microbial communities. Results from metagenomic 16S rRNA sequences showed taxonomically distinct bacterial communities in river, estuary, and coastal ocean. Despite the strong salinity gradient observed over sampling locations (0 to 33), the functional gene profiles in the metagenomes were very similar from river to ocean with an average similarity of 82%. The metatranscriptomes, however, had an average similarity of 31%. Although differences were few among the metagenomes, we observed a change from river to ocean in the abundance of genes encoding for catabolic pathways, osmoregulators, and metal transporters. Additionally, genes specifying both bacterial oxygenic and anoxygenic photosynthesis were abundant and expressed in the estuary and plume. Denitrification genes were found throughout the Columbia River coastal margin, and most highly expressed in the estuary. Across a river to ocean gradient, the free-living microbial community followed three different patterns of diversity: 1) the taxonomy of the community changed strongly with salinity, 2) metabolic potential was highly similar across samples, with few differences in functional gene abundance from river to ocean, and 3) gene expression was highly variable and generally was independent of changes in salinity.

Metagenomic and metatranscriptomic inventories of the lower Amazon River, May 2011.

BACKGROUND: The Amazon River runs nearly 6500 km across the South American continent before emptying into the western tropical North Atlantic Ocean. In terms of both volume and watershed area, it is the world's largest riverine system, affecting elemental cycling on a global scale. RESULTS: A quantitative inventory of genes and transcripts benchmarked with internal standards was obtained at five stations in the lower Amazon River during May 2011. At each station, metagenomes and metatranscriptomes were obtained in duplicate for two microbial size fractions (free-living, 0.2 to 2.0 µm; particle-associated, 2.0 to 297 µm) using 150 × 150 paired-end Illumina sequencing. Forty eight sample datasets were obtained, averaging 15 × 10(6) potential protein-encoding reads each (730 × 10(6) total). Prokaryotic metagenomes and metatranscriptomes were dominated by members of the phyla Actinobacteria, Planctomycetes, Betaproteobacteria, Verrucomicrobia, Nitrospirae, and Acidobacteria. The actinobacterium SCGC AAA027-L06 reference genome recruited the greatest number of reads overall, with this single bin contributing an average of 50 billion genes and 500 million transcripts per liter of river water. Several dominant taxa were unevenly distributed between the free-living and particle-associated size fractions, such as a particle-associated bias for reads binning to planctomycete Schlesneria paludicola and a free-living bias for actinobacterium SCGC AAA027-L06. Gene expression ratios (transcripts to gene copy ratio) increased downstream from Óbidos to Macapá and Belém, indicating higher per cell activity of Amazon River bacteria and archaea as river water approached the ocean. CONCLUSION: This inventory of riverine microbial genes and transcripts, benchmarked with internal standards for full quantitation, provides an unparalleled window into microbial taxa and functions in the globally important Amazon River ecosystem.

Determining indicator taxa across spatial and seasonal gradients in the Columbia River coastal margin.

Bacterioplankton communities are deeply diverse and highly variable across space and time, but several recent studies demonstrate repeatable and predictable patterns in this diversity. We expanded on previous studies by determining patterns of variability in both individual taxa and bacterial communities across coastal environmental gradients. We surveyed bacterioplankton diversity across the Columbia River coastal margin, USA, using amplicon pyrosequencing of 16S rRNA genes from 596 water samples collected from 2007 to 2010. Our results showed seasonal shifts and annual reassembly of bacterioplankton communities in the freshwater-influenced Columbia River, estuary, and plume, and identified indicator taxa, including species from freshwater SAR11, Oceanospirillales, and Flavobacteria groups, that characterize the changing seasonal conditions in these environments. In the river and estuary, Actinobacteria and Betaproteobacteria indicator taxa correlated strongly with seasonal fluctuations in particulate organic carbon (ρ=-0.664) and residence time (ρ=0.512), respectively. In contrast, seasonal change in communities was not detected in the coastal ocean and varied more with the spatial variability of environmental factors including temperature and dissolved oxygen. Indicator taxa of coastal ocean environments included SAR406 and SUP05 taxa from the deep ocean, and Prochlorococcus and SAR11 taxa from the upper water column. We found that in the Columbia River coastal margin, freshwater-influenced environments were consistent and predictable, whereas coastal ocean community variability was difficult to interpret due to complex physical conditions. This study moves beyond beta-diversity patterns to focus on the occurrence of specific taxa and lends insight into the potential ecological roles these taxa have in coastal ocean environments.

Spatial variability overwhelms seasonal patterns in bacterioplankton communities across a river to ocean gradient.

Few studies of microbial biogeography address variability across both multiple habitats and multiple seasons. Here we examine the spatial and temporal variability of bacterioplankton community composition of the Columbia River coastal margin using 16S amplicon pyrosequencing of 300 water samples collected in 2007 and 2008. Communities separated into seven groups (ANOSIM, P<0.001): river, estuary, plume, epipelagic, mesopelagic, shelf bottom (depth<350 m) and slope bottom (depth>850 m). The ordination of these samples was correlated with salinity (ρ=-0.83) and depth (ρ=-0.62). Temporal patterns were obscured by spatial variability among the coastal environments, and could only be detected within individual groups. Thus, structuring environmental factors (for example, salinity, depth) dominate over seasonal changes in determining community composition. Seasonal variability was detected across an annual cycle in the river, estuary and plume where communities separated into two groups, early year (April-July) and late year (August-Nov), demonstrating annual reassembly of communities over time. Determining both the spatial and temporal variability of bacterioplankton communities provides a framework for modeling these communities across environmental gradients from river to deep ocean.

Bacterioplankton community variation across river to ocean environmental gradients.

Coastal zones encompass a complex spectrum of environmental gradients that each impact the composition of bacterioplankton communities. Few studies have attempted to address these gradients comprehensively. We generated a synoptic, 16S rRNA gene-based bacterioplankton community profile of a coastal zone by applying the fingerprinting technique denaturing gradient gel electrophoresis to water samples collected from the Columbia River, estuary, and plume, and along coastal transects covering 360 km of the Oregon and Washington coasts and extending to the deep ocean (>2,000 m). Communities were found to cluster into five distinct groups based on location in the system (ANOSIM, p < 0.003): estuary, plume, epipelagic, shelf bottom (depth < 150 m), and slope bottom (depth > 650 m). Across all environments, abiotic factors (salinity, temperature, depth) explained most of the community variability (ρ = 0.734). But within each coastal environment, biotic factors explained most of the variability. Thus, structuring physical factors in coastal zones, such as salinity and temperature, define the boundaries of many distinct microbial habitats, but within these habitats variability in microbial communities is explained by biological gradients in primary and secondary productivity.

Nitrifier and denitrifier molecular operational taxonomic unit compositions from sites of a freshwater estuary of Chesapeake Bay.

Temporal and spatial changes in the molecular operational taxonomic unit (OTU) compositions of bacteria harboring genes for nitrification and denitrification were assessed using denaturing gradient gel electrophoresis (DGGE), clone-based DNA sequencing of selected PCR products, and analyses of ammonium and organic matter concentrations. Sediment, overlying water, and pore-water samples were taken from different vegetated sites of Jug Bay National Estuarine Research Reserve, Maryland, during spring, summer, and fall 2006. OTU richness and the diversities of nitrifiers and denitrifiers were assessed by the presence of bands on DGGE gels, both ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were seasonally dependent. AOB OTU richness was highest in the summer when NOB richness was decreased, whereas NOB richness was highest in the spring when AOB richness was decreased. The OTU diversities of nitrifiers did not correlate with ammonium concentrations, organic matter concentrations, or the presence of vegetation. The OTU diversities of denitrifiers possessing either the nirK or nosZ genes were not seasonally dependent but were positively correlated with organic matter content (p = 0.0015, r2 = 0.27; p < 0.0001, r2 = 0.39, respectively). Additionally, the presence of vegetation significantly enhanced nosZ species richness (Wilcoxon/Kruskal-Wallis test, p < 0.008), but this trend was not seen for nirK OTU richness. Banding patterns for nirK OTUs were more similar within sites for each season compared with any of the other genes. Over all seasons, nirK OTU richness was highest and AOB and nosZ OTU richness were lowest (Wilcoxon/Kruskal-Wallis test, p < 0.0001). High levels of sequence divergence among cloned nirK PCR products indicate a broad diversity of nirK homologs in this freshwater estuary.