Stable Isotope Probing with 15N2 Reveals Novel Noncultivated Diazotrophs in Soil

Biological nitrogen fixation is a fundamental component of the nitrogen cycle and is the dominant natural process through which fixed nitrogen is made available to the biosphere. While the process of nitrogen fixation has been studied extensively with a limited set of cultivated isolates, examinations of nifH gene diversity in natural systems reveal the existence of a wide range of noncultivated diazotrophs. These noncultivated diazotrophs remain uncharacterized, as do their contributions to nitrogen fixation in natural systems. We have employed a novel ¹⁵N₂-DNA stable isotope probing (⁵N₂-DNA-SIP) method to identify free-living diazotrophs in soil that are responsible for nitrogen fixation in situ. Analyses of 16S rRNA genes from ¹⁵N-labeled DNA provide evidence for nitrogen fixation by three microbial groups, one of which belongs to the Rhizobiales while the other two represent deeply divergent lineages of noncultivated bacteria within the Betaproteobacteria and Actinobacteria, respectively. Analysis of nifH genes from ¹⁵N-labeled DNA also revealed three microbial groups, one of which was associated with Alphaproteobacteria while the others were associated with two noncultivated groups that are deeply divergent within nifH cluster I. These results reveal that noncultivated free-living diazotrophs can mediate nitrogen fixation in soils and that ¹⁵N₂-DNA-SIP can be used to gain access to DNA from these organisms. In addition, this research provides the first evidence for nitrogen fixation by Actinobacteria outside of the order Actinomycetales.     

Coral-associated nitrogen fixation rates and diazotrophic diversity on a nutrient-replete equatorial reef

The role of diazotrophs in coral physiology and reef biogeochemistry remains poorly understood, in part because N₂ fixation rates and diazotrophic community composition have only been jointly analyzed in the tissue of one tropical coral species. We performed field-based ¹⁵N₂ tracer incubations during nutrient-replete conditions to measure diazotroph-derived nitrogen (DDN) assimilation into three species of scleractinian coral (Pocillopora acuta, Goniopora columna, Platygyra sinensis). Using multi-marker metabarcoding (16S rRNA, nifH, 18S rRNA), we analyzed DNA- and RNA-based communities in coral tissue and skeleton. Despite low N₂ fixation rates, DDN assimilation supplied up to 6% of the holobiont’s N demand. Active coral-associated diazotrophs were chiefly Cluster I (aerobes or facultative anaerobes), suggesting that oxygen may control coral-associated diazotrophy. Highest N₂ fixation rates were observed in the endolithic community (0.20 µg N cm⁻² per day). While the diazotrophic community was similar between the tissue and skeleton, RNA:DNA ratios indicate potential differences in relative diazotrophic activity between these compartments. In Pocillopora, DDN was found in endolithic, host, and symbiont compartments, while diazotrophic nifH sequences were only observed in the endolithic layer, suggesting a possible DDN exchange between the endolithic community and the overlying coral tissue. Our findings demonstrate that coral-associated diazotrophy is significant, even in nutrient-rich waters, and suggest that endolithic microbes are major contributors to coral nitrogen cycling on reefs.Subject terms: Microbial ecology, Biogeochemistry, Stable isotope analysis     

Nitrogen fixation and methanotrophy in forest mosses along a N deposition gradient

Nitrogen deposition has decreased the plant-associated nitrogen (N₂) fixation when measured using the indirect acetylene reduction assay (ARA). However, nitrogen deposition can also lead to changes in the diversity of moss symbionts, e.g. affect methanotrophic N₂ fixation, which is not measured by ARA. To test this hypothesis we compared ARA with the direct stable isotope method (¹⁵N₂ incorporation) and studied methanotrophy in two mosses, Hylocomium splendens and Pleurozium schreberi, collected from seven forest sites along a boreal latitudinal N deposition transect. We recognized that the two independent N₂ fixation measures gave corresponding results with the conversion factor of 3.3, but the ¹⁵N₂ method was more sensitive for finding a signal of low N₂ fixation activity. Methane carbon fixation associated with mosses was under the detection limit (<2 nmol C g⁻¹ h⁻¹). N₂ fixation rates were more pronounced in the mosses with higher C/N ratio, and in the green upper parts of the shoot than in the lower brownish parts. Sequencing of nifH genes revealed that dominating diazotrophs were affiliated to cyanobacterial genera Nostoc and Nodularia, but methanotrophic diazotrophs were not found in the nifH libraries. We conclude that the suppression of N₂ fixation along the deposition gradient was consistent regardless of the measurement technique, and microbial community changes toward methanotrophic or otherwise acetylene-sensitive N₂ fixation could not explain this trend.     

Facets of diazotrophy in the oxygen minimum zone waters off Peru

Nitrogen fixation, the biological reduction of dinitrogen gas (N₂) to ammonium (NH₄⁺), is quantitatively the most important external source of new nitrogen (N) to the open ocean. Classically, the ecological niche of oceanic N₂ fixers (diazotrophs) is ascribed to tropical oligotrophic surface waters, often depleted in fixed N, with a diazotrophic community dominated by cyanobacteria. Although this applies for large areas of the ocean, biogeochemical models and phylogenetic studies suggest that the oceanic diazotrophic niche may be much broader than previously considered, resulting in major implications for the global N-budget. Here, we report on the composition, distribution and abundance of nifH, the functional gene marker for N₂ fixation. Our results show the presence of eight clades of diazotrophs in the oxygen minimum zone (OMZ) off Peru. Although proteobacterial clades dominated overall, two clusters affiliated to spirochaeta and archaea were identified. N₂ fixation was detected within OMZ waters and was stimulated by the addition of organic carbon sources supporting the view that non-phototrophic diazotrophs were actively fixing dinitrogen. The observed co-occurrence of key functional genes for N₂ fixation, nitrification, anammox and denitrification suggests that a close spatial coupling of N-input and N-loss processes exists in the OMZ off Peru. The wide distribution of diazotrophs throughout the water column adds to the emerging view that the habitat of marine diazotrophs can be extended to low oxygen/high nitrate areas. Furthermore, our statistical analysis suggests that NO₂⁻ and PO₄³⁻ are the major factors affecting diazotrophic distribution throughout the OMZ. In view of the predicted increase in ocean deoxygenation resulting from global warming, our findings indicate that the importance of OMZs as niches for N₂ fixation may increase in the future.     

Environment-Dependent Distribution of the Sediment nifH-Harboring Microbiota in the Northern South China Sea

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 N₂ 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 NH₄⁺ concentration with the nifH gene abundance suggests that the nSCS sediment nifH-harboring microbiota is active in N₂ fixation and NH₄⁺ production. Several other environmental factors, including sediment pore water PO₄³⁻ 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.     

Abundance and distribution of major groups of diazotrophic cyanobacteria and their potential contribution to N2 fixation in the tropical Atlantic Ocean

The abundances of six N₂‐fixing cyanobacterial phylotypes were profiled at 22 stations across the tropical Atlantic Ocean during June 2006, and used to model the contribution of the diazotrophs to N₂ fixation. Diazotroph abundances were measured by targeting the nifH gene of Trichodesmium, unicellular groups A, B, C (UCYN‐A, UCYN‐B and UCYN‐C), and diatom‐cyanobiont symbioses Hemiaulus–Richelia, Rhizosolenia–Richelia and Chaetoceros–Calothrix. West to east gradients in temperature, salinity and nutrients [NO₃^‐ + NO₂^‐, PO₄^3?, Si(OH)₄] showed the influence of the Amazon River plume and its effect on the distributions of the diazotrophs. Trichodesmium accounted for more than 93% of all nifH genes detected, dominated the warmer waters of the western Atlantic, and was the only diazotroph detected at the equatorial upwelling station. UCYN‐A was the next most abundant (> 5% of all nifH genes) and dominated the cooler waters of the eastern Atlantic near the Cape Verde Islands. UCYN‐C was found at a single depth (200 m) of high salinity and low temperature and nutrients, whereas UCYN‐B cells were widespread but in very low abundance (6.1 × 10¹ ± 4.6 × 10² gene copies l^?1). The diatom‐cyanobionts were observed primarily in the western Atlantic within or near the high Si(OH)₄ input of the Amazon River plume. Overall, highest diazotroph abundances were observed at the surface and declined with depth, except for some subsurface peaks in Trichodesmium, UCYN‐B and UCYN‐A. Modelled contributions of Trichodesmium, UCYN‐B and UCYN‐A to total N₂ fixation suggested that Trichodesmium had the largest input, except for the potential of UCYN‐A at the Cape Verde Islands.     

Vertical Distribution of Nitrogen-Fixing Phylotypes in a Meromictic,Hypersaline Lake

We investigated the diversity of nitrogenase genes in the alkaline, moderately hypersaline Mono Lake, California to determine (1) whether nitrogen-fixing (diazotrophic) populations were similar to those in other aquatic environments and (2) if there was a pattern of distribution of phylotypes that reflected redox conditions, as well as (3) to identify populations that could be important in N dynamics in this nitrogen-limited lake. Mono Lake has been meromictic for almost a decade and has steep gradients in oxygen and reduced compounds that provide a wide range of aerobic and anaerobic habitats. We amplified a fragment of the nitrogenase gene (nifH) from planktonic DNA samples collected at three depths representing oxygenated surface waters, the oxycline, and anoxic, ammonium-rich deep waters. Forty-three percent of the 90 sequences grouped in nifH Cluster I. The majority of clones (57%) grouped in Cluster III, which contains many known anaerobic bacteria. Cluster I and Cluster III sequences were retrieved at every depth indicating little vertical zonation in sequence types related to the prominent gradients in oxygen and ammonia. One group in Cluster I was found most often at every depth and accounted for 29% of all the clones. These sequences formed a subcluster that contained other environmental clones, but no cultivated representatives. No significant nitrogen fixation was detected by the ¹⁵N₂ method after 48 h of incubation of surface, oxycline, or deep waters, suggesting that pelagic diazotrophs were contributing little to nitrogen fluxes in the lake. The failure to measure any significant nitrogen fixation, despite the detection of diverse and novel nitrogenase genes throughout the water column, raises interesting questions about the ecological controls on diazotrophy in Mono Lake and the distribution of functional genes in the environment.     

Aphotic N2 Fixation in the Eastern Tropical South Pacific Ocean

We examined rates of N₂ fixation from the surface to 2000 m depth in the Eastern Tropical South Pacific (ETSP) during El Niño (2010) and La Niña (2011). Replicated vertical profiles performed under oxygen-free conditions show that N₂ fixation takes place both in euphotic and aphotic waters, with rates reaching 155 to 509 µmol N m⁻² d⁻¹ in 2010 and 24±14 to 118±87 µmol N m⁻² d⁻¹ in 2011. In the aphotic layers, volumetric N₂ fixation rates were relatively low (<1.00 nmol N L⁻¹ d⁻¹), but when integrated over the whole aphotic layer, they accounted for 87–90% of total rates (euphotic+aphotic) for the two cruises. Phylogenetic studies performed in microcosms experiments confirm the presence of diazotrophs in the deep waters of the Oxygen Minimum Zone (OMZ), which were comprised of non-cyanobacterial diazotrophs affiliated with nifH clusters 1K (predominantly comprised of α-proteobacteria), 1G (predominantly comprised of γ-proteobacteria), and 3 (sulfate reducing genera of the δ-proteobacteria and Clostridium spp., Vibrio spp.). Organic and inorganic nutrient addition bioassays revealed that amino acids significantly stimulated N₂ fixation in the core of the OMZ at all stations tested and as did simple carbohydrates at stations located nearest the coast of Peru/Chile. The episodic supply of these substrates from upper layers are hypothesized to explain the observed variability of N₂ fixation in the ETSP.     

Molecular Analysis of Diazotroph Diversity in the Rhizosphere of the Smooth Cordgrass, Spartina alterniflora

N₂ fixation by diazotrophic bacteria associated with the roots of the smooth cordgrass, Spartina alterniflora, is an important source of new nitrogen in many salt marsh ecosystems. However, the diversity and phylogenetic affiliations of these rhizosphere diazotrophs are unknown. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified nifH sequence segments was used in previous studies to examine the stability and dynamics of the Spartina rhizosphere diazotroph assemblages in the North Inlet salt marsh, near Georgetown, S.C. In this study, plugs were taken from gel bands from representative DGGE gels, the nifH amplimers were recovered and cloned, and their sequences were determined. A total of 59 sequences were recovered, and the amino acid sequences predicted from them were aligned with sequences from known and unknown diazotrophs in order to determine the types of organisms present in the Spartina rhizosphere. We recovered numerous sequences from diazotrophs in the γ subdivision of the division Proteobacteria (γ-Proteobacteria) and from various anaerobic diazotrophs. Diazotrophs in the α-Proteobacteria were poorly represented. None of the Spartina rhizosphere DGGE band sequences were identical to any known or previously recovered environmental nifH sequences. The Spartina rhizosphere diazotroph assemblage is very diverse and apparently consists mainly of unknown organisms.     

Molecular Characterization of Diazotrophic and Denitrifying Bacteria Associated with Mangrove Roots

An analysis of the molecular diversity of N₂ fixers and denitrifiers associated with mangrove roots was performed using terminal restriction length polymorphism (T-RFLP) of nifH (N₂ fixation) and nirS and nirK (denitrification), and the compositions and structures of these communities among three sites were compared. The number of operational taxonomic units (OTU) for nifH was higher than that for nirK or nirS at all three sites. Site 3, which had the highest organic matter and sand content in the rhizosphere sediment, as well as the lowest pore water oxygen concentration, had the highest nifH diversity. Principal component analysis of biogeochemical parameters identified soil texture, organic matter content, pore water oxygen concentration, and salinity as the main variables that differentiated the sites. Nonmetric multidimensional scaling (MDS) analyses of the T-RFLP data using the Bray-Curtis coefficient, group analyses, and pairwise comparisons between the sites clearly separated the OTU of site 3 from those of sites 1 and 2. For nirS, there were statistically significant differences in the composition of OTU among the sites, but the variability was less than for nifH. OTU defined on the basis of nirK were highly similar, and the three sites were not clearly separated on the basis of these sequences. The phylogenetic trees of nifH, nirK, and nirS showed that most of the cloned sequences were more similar to sequences from the rhizosphere isolates than to those from known strains or from other environments.     

Coliform Bacteria and Nitrogen Fixation in Pulp and Paper Mill Effluent Treatment Systems

The majority of pulp and paper mills now biotreat their combined effluents using activated sludge. On the assumption that their wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N₂ fixation in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase (nifH) gene probing, and bacterial isolations. In situ N₂ fixation was undetectable in all seven bioreactors but was present in six associated primary clarifiers. One primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained nifH, of which >90% were Klebsiella strains. All primary-clarifier coliform bacteria growing on MacConkey agar were identified as klebsiellas, and all those probed contained nifH. In contrast, analysis of 48 random coliform isolates from other mill water system locations showed that only 24 (50%) possessed the nifH gene, and only 13 (27%) showed inducible N₂-fixing activity. Thus, all the pulp and paper mill primary clarifiers tested appeared to be sites of active N₂ fixation (0.87 to 4.90 mg of N liter⁻¹ day⁻¹) and a microbial community strongly biased toward this activity. This may also explain why coliform bacteria, especially klebsiellas, are indigenous in pulp and paper mill water systems.