A novel cohabitation between two diazotrophic cyanobacteria in the oligotrophic ocean

The cyanobacterial genus Trichodesmium is biogeochemically significant because of its dual role in nitrogen and carbon fixation in the oligotrophic ocean. Trichodesmium species form colonies that can be easily enriched from the water column and used for shipboard rate measurements to estimate their contribution to oceanic carbon and nitrogen budgets. During a July 2010 cruise near the Hawaiian Islands in the oligotrophic North Pacific Subtropical Gyre, a specific morphology of Trichodesmium puff-form colonies were examined under epifluorescent microscopy and found to harbor a colonial endobiont, morphologically identified as the heterocystous diazotrophic cyanobacterium Calothrix. Using unialgal enrichments obtained from this cruise, we show that these Calothrix-like heterocystous cyanobionts (hetDA for ‘Trichodesmium-associated heterocystous diazotroph'') fix nitrogen on a diurnal cycle (maximally in the middle of the light cycle with a detectable minimum in the dark). Gene sequencing of nifH from the enrichments revealed that this genus was likely not quantified using currently described quantitative PCR (qPCR) primers. Guided by the sequence from the isolate, new hetDA-specific primers were designed and subsequent qPCR of environmental samples detected this diazotroph from surface water to a depth of 150 m, reaching densities up to ∼9 × 10³ l⁻¹. Based on phylogenetic relatedness of nifH and 16S rRNA gene sequences, it is predicted that the distribution of this cyanobiont is not limited to subtropical North Pacific but likely reaches to the South Pacific and Atlantic Oceans. Therefore, this previously unrecognized cohabitation, if it reaches beyond the oligotrophic North Pacific, could potentially influence Trichodesmium-derived nitrogen fixation budgets in the world ocean.     

Connecting biodiversity and potential functional role in modern euxinic environments by microbial metagenomics

Stratified sulfurous lakes are appropriate environments for studying the links between composition and functionality in microbial communities and are potentially modern analogs of anoxic conditions prevailing in the ancient ocean. We explored these aspects in the Lake Banyoles karstic area (NE Spain) through metagenomics and in silico reconstruction of carbon, nitrogen and sulfur metabolic pathways that were tightly coupled through a few bacterial groups. The potential for nitrogen fixation and denitrification was detected in both autotrophs and heterotrophs, with a major role for nitrogen and carbon fixations in Chlorobiaceae. Campylobacterales accounted for a large percentage of denitrification genes, while Gallionellales were putatively involved in denitrification, iron oxidation and carbon fixation and may have a major role in the biogeochemistry of the iron cycle. Bacteroidales were also abundant and showed potential for dissimilatory nitrate reduction to ammonium. The very low abundance of genes for nitrification, the minor presence of anammox genes, the high potential for nitrogen fixation and mineralization and the potential for chemotrophic CO₂ fixation and CO oxidation all provide potential clues on the anoxic zones functioning. We observed higher gene abundance of ammonia-oxidizing bacteria than ammonia-oxidizing archaea that may have a geochemical and evolutionary link related to the dominance of Fe in these environments. Overall, these results offer a more detailed perspective on the microbial ecology of anoxic environments and may help to develop new geochemical proxies to infer biology and chemistry interactions in ancient ecosystems.     

Pseudomonas stutzeri Nitrite Reductase Gene Abundance in Environmental Samples Measured by Real-Time PCR

We used real-time PCR to quantify the denitrifying nitrite reductase gene (nirS), a functional gene of biogeochemical significance. The assay was tested in vitro and applied to environmental samples. The primer-probe set selected was specific for nirS sequences that corresponded approximately to the Pseudomonas stutzeri species. The assay was linear from 1 to 10⁶ gene copies (r² = 0.999). Variability at low gene concentrations did not allow detection of twofold differences in gene copy number at less than 100 copies. DNA spiking and cell-addition experiments gave predicted results, suggesting that this assay provides an accurate measure of P. stutzeri nirS abundance in environmental samples. Although P. stutzeri abundance was high in lake sediment and groundwater samples, we detected low or no abundance of this species in marine sediment samples from Puget Sound (Wash.) and from the Washington ocean margin. These results suggest that P. stutzeri may not be a dominant marine denitrifier.     

Pelagic Sargassum community metabolism: Carbon and nitrogen

The production, nitrogen fixation, and release rates and fate of dissolved organic matter of a pelagic Sargassum community have been investigated at eight stations in the Gulf Stream and the Sargasso Sea. Net production and gross nitrogen fixation rates of Sargassum and epiphytes varied significantly between stations, 328 ± 114μg C (g dry wt)^?1h^?1 and 18 ± 7.4μg N g^?1h^?1, respectively. The net release rates of dissolved organic carbon (287 ± 150μg DOC g^?1h^?1) also showed the same variability between stations. On the other hand, the community carbon and nitrogen content, 268 ± 4.8 and 16.9 ± 2.4 mg g dry wt^?1, respectively, remained constant at all stations. The results of chemical measurements indicate that ≈ 0–50 % of the gross production was lost as a result of photosynthate release. From ¹⁴C-tracer experiments it was found that the planktonic and epiphytic heterotrophs mineralized 50–70 % of the photosynthate released by Sargassum and epiphytic algae. Based on the community gross production and fixation rates, carbon and nitrogen content, the amount of nitrogen required for the observed production rates, the Sargassum community appears to obtain a substantial part (40%) of its nitrogen from nitrogen fixation.     

Effects of available P and N:P ratios on non-symbiotic dinitrogen fixation in tallgrass prairie soils

Summary Prescribed burning is a major control over element cycles in Tallgrass prairie (Eastern Kansas, USA). In this paper we report potential effects of fire on nonsymbiotic nitrogen fixation. Fire resulted in additions of available P in ash, which may stimulate nitrogen fixation by terrestrial cyanobacteria. Cyanobacterial nitrogenase activity and biomass responded positively to additions of ash or P in laboratory assays using soil. Further assays in soil showed that cyanobacteria responded to changes in available N:available P ratio (aN:P) across a range of concentrations. Nitrogen fixation rate could be related empirically to aN:P via a log-linear relationship. Extrapolation of laboratory results to the field yielded a maximal estimate of 21 kg N ha^-1 y^-1. Results support arguments from the marine and terrestrial literature that P availability is central to regulation of ecosystem N budgets.     

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.     

Modeling the Dynamic Regulation of Nitrogen Fixation in the Cyanobacterium Trichodesmium sp.

A physiological, unbalanced model is presented that explicitly describes growth of the marine cyanobacterium Trichodesmium sp. at the expense of N₂ (diazotrophy). The model involves the dynamics of intracellular reserves of carbon and nitrogen and allows the uncoupling of the metabolism of these elements. The results show the transient dynamics of N₂ fixation when combined nitrogen (NO₃⁻, NH₄⁺) is available and the increased rate of N₂ fixation when combined nitrogen is insufficient to cover the demand. The daily N₂ fixation pattern that emerges from the model agrees with measurements of rates of nitrogenase activity in laboratory cultures of Trichodesmium sp. Model simulations explored the influence of irradiance levels and the length of the light period on fixation activity and cellular carbon and nitrogen stoichiometry. Changes in the cellular C/N ratio resulted from allocations of carbon to different cell compartments as demanded by the growth of the organism. The model shows that carbon availability is a simple and efficient mechanism to regulate the balance of carbon and nitrogen fixed (C/N ratio) in filaments of cells. The lowest C/N ratios were obtained when the light regime closely matched nitrogenase dynamics.     

Elevated temperature alters proteomic responses of individual organisms within a biofilm community

Microbial communities that underpin global biogeochemical cycles will likely be influenced by elevated temperature associated with environmental change. Here, we test an approach to measure how elevated temperature impacts the physiology of individual microbial groups in a community context, using a model microbial-based ecosystem. The study is the first application of tandem mass tag (TMT)-based proteomics to a microbial community. We accurately, precisely and reproducibly quantified thousands of proteins in biofilms growing at 40, 43 and 46 °C. Elevated temperature led to upregulation of proteins involved in amino-acid metabolism at the level of individual organisms and the entire community. Proteins from related organisms differed in their relative abundance and functional responses to temperature. Elevated temperature repressed carbon fixation proteins from two Leptospirillum genotypes, whereas carbon fixation proteins were significantly upregulated at higher temperature by a third member of this genus. Leptospirillum group III bacteria may have been subject to viral stress at elevated temperature, which could lead to greater carbon turnover in the microbial food web through the release of viral lysate. Overall, these findings highlight the utility of proteomics-enabled community-based physiology studies, and provide a methodological framework for possible extension to additional mixed culture and environmental sample analyses.     

An assessment of nitrogen fixation as a source of nitrogen to the North Atlantic Ocean

The role of nitrogen fixation in the nitrogen cycle of the North Atlantic basin was re-evaluated because recent estimates had indicated a far higher rate than previous reports. Examination of the available data on nitrogen fixation rates and abundance ofTrichodesmium, the major nitrogen fixing organism, leads to the conclusion that rates might be as high as 1.09 × 10¹² mol N yr^–1. Several geochemical arguments are reviewed that each require a large nitrogen source that is consistent with nitrogen fixation, but the current data, although limited, do not support a sufficiently high rate. However, recent measurements of the fixation rates per colony are higher than the historical average, suggesting that improved methodology may require a re-evaluation through further measurements. The paucity of temporally resolved data on both rates and abundance for the major areal extent of the tropical Atlantic, where aeolian inputs of iron may foster high fixation rates, represents another major gap.     

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.     

Iron deficiency increases growth and nitrogen-fixation rates of phosphorus-deficient marine cyanobacteria

Marine dinitrogen (N₂)-fixing cyanobacteria have large impacts on global biogeochemistry as they fix carbon dioxide (CO₂) and fertilize oligotrophic ocean waters with new nitrogen. Iron (Fe) and phosphorus (P) are the two most important limiting nutrients for marine biological N₂ fixation, and their availabilities vary between major ocean basins and regions. A long-standing question concerns the ability of two globally dominant N₂-fixing cyanobacteria, unicellular Crocosphaera and filamentous Trichodesmium, to maintain relatively high N₂-fixation rates in these regimes where both Fe and P are typically scarce. We show that under P-deficient conditions, cultures of these two cyanobacteria are able to grow and fix N₂ faster when Fe deficient than when Fe replete. In addition, growth affinities relative to P increase while minimum concentrations of P that support growth decrease at low Fe concentrations. In Crocosphaera, this effect is accompanied by a reduction in cell sizes and elemental quotas. Relatively high growth rates of these two biogeochemically critical cyanobacteria in low-P, low-Fe environments such as those that characterize much of the oligotrophic ocean challenge the common assumption that low Fe levels can have only negative effects on marine primary producers. The closely interdependent influence of Fe and P on N₂-fixing cyanobacteria suggests that even subtle shifts in their supply ratio in the past, present and future oceans could have large consequences for global carbon and nitrogen cycles.     

Nitrogen metabolism and seed composition as influenced by foliar boron application in soybean

The physiological effects of foliar boron application (FB) on nitrogen metabolism and seed composition have not been well established in soybean [(Glycine max(L.)Merr.)]. Therefore, the effect of FB on nitrogen metabolism and seed composition was investigated. Nitrate assimilation was evaluated by measuring nitrate reductase activity (NRA) and nitrogen fixation was evaluated by measuring nitrogenase activity and natural abundance of ¹⁵N/¹⁴N. NRA were significantly (P?≤?0.05) higher in plants that received FB than the control plants. Higher rate of FB (One application of four times of commercial rate) inhibited nitrogen fixation as measured by natural abundance of ¹⁵N/¹⁴N ratio, but increased NRA. The higher activities of NR and nitrogenase by FB were accompanied with a higher B concentration in leaves. The significant (P?<?0.0001) enrichment of ¹⁵N/¹⁴N, accompanied with a higher rate of FB, suggested a possible mechanism where nitrate assimilation may compensate for the decrease in nitrogen fixation. FB increased seed protein by 13.7% and oleic acid by 30.9% compared to the control plants. This alteration was accompanied by a higher B concentration in leaves and seed. The results suggest that FB affects nitrogen metabolism and alters seed compositions, especially protein and unsaturated fatty acids.     

Molecular Fingerprint and Dominant Environmental Factors of Nitrite-Dependent Anaerobic Methane-Oxidizing Bacteria in Sediments from the Yellow River Estuary,China

Nitrite-dependent anaerobic methane oxidation (n-damo) is performed by “Candidatus Methylomirabilis oxyfera” (M. oxyfera), which connects the carbon and nitrogen global nutrient cycles. In the present study, M. oxyfera-like bacteria sequences were successfully recovered from Yellow River Estuary sediments using specific primers for 16S rRNA and pmoA genes. A M. oxyfera-like sequences analysis based on the 16S rRNA gene revealed greater diversity compared with the pmoA gene; the 16S rRNA gene sequences retrieved from the Yellow River Estuary sediments belong to groups A as well as B and were mainly found in freshwater habitats. Quantitative PCR showed that 16S rRNA gene abundance varied from 9.28±0.11×10³ to 2.10±0.13×10⁵ copies g^-1 (dry weight), and the pmoA gene abundance ranged from 8.63±0.50×10³ to 1.83±0.18×10⁵ copies g^-1 (dry weight). A correlation analysis showed that the total organic carbon (TOC) and ammonium (NH₄ ⁺) as well as the ratio of total phosphorus to total nitrogen (TP/TN) influenced the M. oxyfera-like bacteria distribution in the Yellow River Estuary sediments. These findings will aid in understanding the n-damo bacterial distribution pattern as well as their correlation with surrounding environmental factors in temperate estuarine ecosystems.     

Molecular Existence and Diversity of Nitrite-Dependent Anaerobic Methane Oxidizing (n-Damo) Bacteria in the Lakes of Badain of the Gobi Desert

Nitrite-dependent anaerobic methane oxidation (n-damo) process, mediated by Candidatus Methylomirabilis oxyfera of the candidate phylum NC10, was discovered recently which plays an important role in coupling the global nitrogen and carbon cycles. However, the distribution and diversity of this new anaerobic methane-oxidizing microorganism have not been investigated in desert lakes yet. The present study successfully retrieved n-damo bacterial 16S rRNA and pmoA gene sequences using PCR technique from lakes in Badain Jaran Desert of China. Phylogenetic analyses showed that n-damo bacteria widely occurred in brine and freshwater lakes on the desert with high diversity, including both sediment and water samples. The results of quantitative PCR indicated that the abundance of the 16S rRNA gene in lake sediments varied from 1.12?±?0.68?×?10⁵ to 1.64?±?0.70?×?10⁵ copies g^?1 (dry weight), while that in water samples per milliliter was generally one order of magnitude lower than sediments. Correlation analyses suggested that n-damo bacterial abundance and diversity strongly depended on salinity. In lake sediments, the distribution, abundance, and diversity of n-damo bacteria were significantly associated with depth due to the concentration gradient of the NO_x^- and ammonium. This study provided new insights into both the n-damo community patterns and its interaction with ambient environmental factors in the desert lake ecosystem.     

No stimulation of nitrogen fixation by non‐filamentous diazotrophs under elevated CO2 in the South Pacific

Nitrogen fixation by diazotrophic cyanobacteria is a critical source of new nitrogen to the oligotrophic surface ocean. Research to date indicates that some diazotroph groups may increase nitrogen fixation under elevated pCO₂. To test this in natural plankton communities, four manipulation experiments were carried out during two voyages in the South Pacific (30–35^oS). High CO₂ treatments, produced using 750 ppmv CO₂ to adjust pH to 0.2 below ambient, and ‘Greenhouse’ treatments (0.2 below ambient pH and ambient temperature +3 °C), were compared with Controls in trace metal clean deckboard incubations in triplicate. No significant change was observed in nitrogen fixation in either the High CO₂ or Greenhouse treatments over 5 day incubations. qPCR measurements and optical microscopy determined that the diazotroph community was dominated by Group A unicellular cyanobacteria (UCYN‐A), which may account for the difference in response of nitrogen fixation under elevated CO₂ to that reported previously for Trichodesmium. This may reflect physiological differences, in that the greater cell surface area:volume of UCYN‐A and its lack of metabolic pathways involved in carbon fixation may confer no benefit under elevated CO₂. However, multiple environmental controls may also be a factor, with the low dissolved iron concentrations in oligotrophic surface waters limiting the response to elevated CO₂. If nitrogen fixation by UCYN‐A is not stimulated by elevated pCO₂, then future increases in CO₂ and warming may alter the regional distribution and dominance of different diazotroph groups, with implications for dissolved iron availability and new nitrogen supply in oligotrophic regions.     

Levels of Daily Light Doses Under Changed Day-Night Cycles Regulate Temporal Segregation of Photosynthesis and N2 Fixation in the Cyanobacterium Trichodesmium erythraeum IMS101

While the diazotrophic cyanobacterium Trichodesmium is known to display inverse diurnal performances of photosynthesis and N₂ fixation, such a phenomenon has not been well documented under different day-night (L-D) cycles and different levels of light dose exposed to the cells. Here, we show differences in growth, N₂ fixation and photosynthetic carbon fixation as well as photochemical performances of Trichodesmium IMS101 grown under 12L:12D, 8L:16D and 16L:8D L-D cycles at 70 μmol photons m^-2 s^-1 PAR (LL) and 350 μmol photons m^-2 s^-1 PAR (HL). The specific growth rate was the highest under LL and the lowest under HL under 16L:8D, and it increased under LL and decreased under HL with increased levels of daytime light doses exposed under the different light regimes, respectively. N₂ fixation and photosynthetic carbon fixation were affected differentially by changes in the day-night regimes, with the former increasing directly under LL with increased daytime light doses and decreased under HL over growth-saturating light levels. Temporal segregation of N₂ fixation from photosynthetic carbon fixation was evidenced under all day-night regimes, showing a time lag between the peak in N₂ fixation and dip in carbon fixation. Elongation of light period led to higher N₂ fixation rate under LL than under HL, while shortening the light exposure to 8 h delayed the N₂ fixation peaking time (at the end of light period) and extended it to night period. Photosynthetic carbon fixation rates and transfer of light photons were always higher under HL than LL, regardless of the day-night cycles. Conclusively, diel performance of N₂ fixation possesses functional plasticity, which was regulated by levels of light energy supplies either via changing light levels or length of light exposure.