Variability in benthic diazotrophy and cyanobacterial diversity in a tropical intertidal lagoon

Benthic nitrogen fixation has been estimated to contribute 15 Tg N year(-1) to the marine nitrogen budget. With benthic marine nitrogen fixation being largely overlooked in more recent surveys, a refocus on benthic diazotrophy was considered important. Variations in nitrogenase activity (acetylene reduction-gas chromatography) in a tropical lagoon in the western Indian Ocean (Zanzibar, Tanzania) were monitored over a 3-year period (2003-2005) and related to cyanobacterial and diazotrophic microbial diversity using a polyphasic approach. Different nitrogenase activity patterns were discerned, with the predominant pattern being high daytime activities combined with low nighttime activities. Analyses of the morphological and 16S rRNA gene diversity among cyanobacteria revealed filamentous nonheterocystous (Oscillatoriales) and unicellular (Chroococcales) representatives to be predominant. Analyses of the nifH gene diversity showed that the major phylotypes belonged to noncyanobacterial prokaryotes. However, as shown by cyanobacterial selective nifH-denaturing gradient gel electrophoresis analysis, cyanobacterial nifH gene sequences were present at all sites. Several nifH and 16S rRNA gene phylotypes were related to uncultured cyanobacteria or bacteria of geographically distant habitats, stressing the widespread occurrence of still poorly characterized microorganisms in tropical benthic marine communities.     

Nitrogen fixation in denitrified marine waters

Nitrogen fixation is an essential process that biologically transforms atmospheric dinitrogen gas to ammonia, therefore compensating for nitrogen losses occurring via denitrification and anammox. Currently, inputs and losses of nitrogen to the ocean resulting from these processes are thought to be spatially separated: nitrogen fixation takes place primarily in open ocean environments (mainly through diazotrophic cyanobacteria), whereas nitrogen losses occur in oxygen-depleted intermediate waters and sediments (mostly via denitrifying and anammox bacteria). Here we report on rates of nitrogen fixation obtained during two oceanographic cruises in 2005 and 2007 in the eastern tropical South Pacific (ETSP), a region characterized by the presence of coastal upwelling and a major permanent oxygen minimum zone (OMZ). Our results show significant rates of nitrogen fixation in the water column; however, integrated rates from the surface down to 120 m varied by ～30 fold between cruises (7.5±4.6 versus 190±82.3 μmol m(-2) d(-1)). Moreover, rates were measured down to 400 m depth in 2007, indicating that the contribution to the integrated rates of the subsurface oxygen-deficient layer was ～5 times higher (574±294 μmol m(-2) d(-1)) than the oxic euphotic layer (48±68 μmol m(-2) d(-1)). Concurrent molecular measurements detected the dinitrogenase reductase gene nifH in surface and subsurface waters. Phylogenetic analysis of the nifH sequences showed the presence of a diverse diazotrophic community at the time of the highest measured nitrogen fixation rates. Our results thus demonstrate the occurrence of nitrogen fixation in nutrient-rich coastal upwelling systems and, importantly, within the underlying OMZ. They also suggest that nitrogen fixation is a widespread process that can sporadically provide a supplementary source of fixed nitrogen in these regions.     

Biological nitrogen fixation in acidic high-temperature geothermal springs in Yellowstone National Park, Wyoming

The near ubiquitous distribution of nifH genes in sediments sampled from 14 high-temperature (48.0-89.0°C) and acidic (pH 1.90-5.02) geothermal springs in Yellowstone National Park suggested a role for the biological reduction of dinitrogen (N(2)) to ammonia (NH(3)) (e.g. nitrogen fixation or diazotrophy) in these environments. nifH genes from these environments formed three unique phylotypes that were distantly related to acidiphilic, mesophilic diazotrophs. Acetylene reduction assays and (15) N(2) tracer studies in microcosms containing sediments sampled from acidic and high-temperature environments where nifH genes were detected confirmed the potential for biological N(2) reduction in these environments. Rates of acetylene reduction by sediment-associated populations were positively correlated with the concentration of NH(4)(+), suggesting a potential relationship between NH(4)(+) consumption and N(2) fixation activity. Amendment of microcosms with NH(4)(+) resulted in increased lag times in acetylene reduction assays. Manipulation of incubation temperature and pH in acetylene reduction assays indicated that diazotrophic populations are specifically adapted to local conditions. Incubation of sediments in the presence of a N(2) headspace yielded a highly enriched culture containing a single nifH phylotype. This phylotype was detected in all 14 geothermal spring sediments examined and its abundance ranged from ≈ 780 to ≈ 6800 copies (g dry weight sediment)(-1), suggesting that this organism may contribute N to the ecosystems. Collectively, these results for the first time demonstrate thermoacidiphilic N(2) fixation in the natural environment and extend the upper temperature for biological N(2) fixation in terrestrial systems.     

Effects of modified nutrient concentrations and ratios on the structure and function of the native phytoplankton community in the Neuse River Estuary, North Carolina, USA

A variety of analyses were used to assess the structure (community composition) and function (assimilation number, nitrogen fixation) of phytoplankton in the Neuse River Estuary (NRE), NC under ambient and modified nutrient concentrations. Dilution bioassays were employed to reduce the concentration of nitrogen (N) or both N and phosphorus (P) and thus compare varied DIN:DIP ratios. Experimental manipulations created conditions that may result from mandated N load reductions to the estuary. We hypothesized that unilateral reduction of N loading to the NRE would increase the activity, abundance and diversity of N₂ fixing cyanobacteria. Changes in phytoplankton primary productivity, N₂ fixation (nitrogenase activity), genetic potential for N₂ fixation (presence of nifH), phytoplankton taxonomic composition (diagnostic photopigment concentration) and abundances of N₂ fixing cyanobacteria (microscopy) were determined. Decreasing ambient DIN:DIP ratios in NRE samples resulted in increased rates of N₂ fixation when seed populations were present and environmental conditions were amenable. Decreasing the DIN:DIP ratio did not lead to an increase in the abundance or diversity of N₂ fixing cyanobacteria. Because N₂ fixing cyanobacteria were only actively fixing nitrogen during periods of low riverine N discharge (summer and early autumn), lowering nutrient ratios may not have a major impact on the NRE. However, the maximum potential amount of N from N₂ fixation was calculated using rates from this study and was found to be approximately 3% of total riverine loading of N to the NRE. Because N₂ fixation occurs farther downstream and later in the year than riverine N loading to the NRE, there is potential for N₂ fixation to modify N dynamics. Analyses of the phytoplankton community as a whole in these relatively short term experiments indicated that reduced DIN:DIP may not have a major impact on their structure and function.     

Nitrogenase gene expression in the Chesapeake Bay Estuary

Like many estuaries, the Chesapeake Bay has pronounced gradients in salinity and nutrients. Previous studies have shown that there is a high diversity of nitrogenase (nifH) genes in the estuary, and that there are specific distributions of individual nifH phylotypes. In contrast to previous work that revealed the remarkable diversity of nifH phylotypes in the Chesapeake estuary, in this study of nifH expression we only detected two phylotypes, and both were phylogenetically related to cyanobacterial nifH genes. One of the phylotypes was closely related to a nifH sequence from the filamentous, heterocystous cyanobacterium Anabaena cylindrica, and was found at the head of the estuary. The other phylotype was found in a sample collected near the mouth of the estuary and was closely related to nifH sequences from Group A unicellular cyanobacteria, which has previously been reported in oceanic waters only. These nifH phylotypes had distinct patterns of expression that were restricted to different regions of the Chesapeake Bay. This study provides the first evidence of nifH expression in the Chesapeake Bay, and suggests that diazotrophic unicellular cyanobacteria have a broader distribution and activity than previously recognized.     

Composition and diversity of nifH genes of nitrogen-fixing cyanobacteria associated with boreal forest feather mosses

Recent studies have revealed that nitrogen fixation by cyanobacteria living in association with feather mosses is a major input of nitrogen to boreal forests. We characterized the community composition and diversity of cyanobacterial nifH phylotypes associated with each of two feather moss species (Pleurozium schreberi and Hylocomium splendens) on each of 30 lake islands varying in ecosystem properties in northern Sweden. Nitrogen fixation was measured using acetylene reduction, and nifH sequences were amplified using general and cyanobacterial selective primers, separated and analyzed using density gradient gel electrophoresis (DGGE) or cloning, and further sequenced for phylogenetic analyses. Analyses of DGGE fingerprinting patterns revealed two host-specific clusters (one for each moss species), and sequence analysis showed five clusters of nifH phylotypes originating from heterocystous cyanobacteria. For H. splendens only, N(2) fixation was related to both nifH composition and diversity among islands. We demonstrated that the cyanobacterial communities associated with feather mosses show a high degree of host specificity. However, phylotype composition and diversity, and nitrogen fixation, did not differ among groups of islands that varied greatly in their availability of resources. These results suggest that moss species identity, but not extrinsic environmental conditions, serves as the primary determinant of nitrogen-fixing cyanobacterial communities that inhabit mosses.     

Diazotrophic diversity and distribution in the tropical and subtropical Atlantic Ocean

To understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean, the molecular diversity of the nifH gene was studied by nested PCR amplification using degenerate primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental DNA samples collected during three cruises (November-December 2000, March 2002, and October-November 2002) covering an area between 0 to 28.3 degrees N and 56.6 to 18.5 degrees W. A total of 170 unique sequences were recovered from 18 stations and 23 depths. Samples from the November-December 2000 cruise contained both unicellular and filamentous cyanobacterial nifH phylotypes, as well as gamma-proteobacterial and cluster III sequences, so far only reported in the Pacific Ocean. In contrast, samples from the March 2002 cruise contained only phylotypes related to the uncultured group A unicellular cyanobacteria. The October-November 2002 cruise contained both filamentous and unicellular cyanobacterial and gamma-proteobacterial sequences. Several sequences were identical at the nucleotide level to previously described environmental sequences from the Pacific Ocean, including group A sequences. The data suggest a community shift from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters. With one exception, filamentous cyanobacterial nifH sequences were present within temperatures ranging between 26.5 and 30 degrees C and where nitrate was undetectable. In contrast, nonfilamentous nifH sequences were found throughout a broader temperature range, 15 to 30 degrees C, more often in waters with temperature of <26 degrees C, and were sometimes recovered from waters with detectable nitrate concentrations.     

Diversity and distribution of diazotrophic communities in the South China Sea deep basin with mesoscale cyclonic eddy perturbations

The South China Sea (SCS) is an oligotrophic subtropical marginal ocean with a deep basin and a permanently stratified central gyre. Upwelling and nitrogen fixation provide new nitrogen for primary production in the SCS. This study was aimed at an investigation of phylogenetic diversity and quantification of the diazotroph community in the SCS deep basin, which is characterized by frequent mesoscale eddies. The diazotroph community had a relatively low diversity but a distinct spatial heterogeneity of diversity in the SCS deep basin. The potential for nitrogen fixation consistently occurred during cyclonic eddies, although upwelling of nutrient-replete deep water might have alleviated nitrogen limitation in the SCS. However, diazotrophic proteobacteria were dominant, but neither Trichodesmium nor heterocystous cyanobacterial diatom symbionts. Quantitative PCR analysis using probe-primer sets developed in this study revealed that the nif?H gene of the two dominant alpha- and gammaproteobacterial groups was at the highest abundance (up to 10(4) to 10(5) copies?L(-1) ). Trichodesmium thiebautii was detected with an average density of 10(2) trichomes?L(-1) in the euphotic waters, while Richelia intracellularis was observed sporadically under the microscope. The unicellular cyanobacterial groups A and B were not detected in our libraries. Our results suggested that diazotrophic proteobacteria were significant components potentially contributing to nitrogen fixation in this oligotrophic marginal ocean ecosystem.     

Mesopelagic N2 Fixation Related to Organic Matter Composition in the Solomon and Bismarck Seas (Southwest Pacific)

Dinitrogen (N₂) fixation was investigated together with organic matter composition in the mesopelagic zone of the Bismarck (Transect 1) and Solomon (Transect 2) Seas (Southwest Pacific). Transparent exopolymer particles (TEP) and the presence of compounds sharing molecular formulae with saturated fatty acids and sugars, as well as dissolved organic matter (DOM) compounds containing nitrogen (N) and phosphorus (P) were higher on Transect 1 than on Transect 2, while oxygen concentrations showed an opposite pattern. N₂ fixation rates (up to ~1 nmol N L^-1 d^-1) were higher in Transect 1 than in Transect 2, and correlated positively with TEP, suggesting a dependence of diazotroph activity on organic matter. The scores of the multivariate ordination of DOM molecular formulae and their relative abundance correlated negatively with bacterial abundances and positively with N₂ fixation rates, suggesting an active bacterial exploitation of DOM and its use to sustain diazotrophic activity. Sequences of the nifH gene clustered with Alpha-, Beta-, Gamma- and Deltaproteobacteria, and included representatives from Clusters I, III and IV. A third of the clone library included sequences close to the potentially anaerobic Cluster III, suggesting that N₂ fixation was partially supported by presumably particle-attached diazotrophs. Quantitative polymerase chain reaction (qPCR) primer-probe sets were designed for three phylotypes and showed low abundances, with a phylotype within Cluster III at up to 10³ nifH gene copies L^-1. These results provide new insights into the ecology of non-cyanobacterial diazotrophs and suggest that organic matter sustains their activity in the mesopelagic ocean.     

Characterization of nifH gene expression, modification and rearrangement in Nodularia spumigena strain AV1

The annually reoccurring blooms that characterize the surface waters of the Baltic Sea are dominated by filamentous, heterocystous cyanobacteria such as Nodularia spumigena. In a previous study, we have demonstrated that N. spumigena strain AV1 differentiates heterocysts in the absence of detectable nitrogen fixation activity, an unusual physiological trait that is clearly distinct from other well-studied cyanobacteria. To further analyze the uncoupling between these two processes, we analyzed the gene expression and modification of the nitrogenase enzyme (the enzyme responsible for nitrogen fixation) in N. spumigena AV1, as well as in several other N. spumigena strains. Here, we demonstrate the occurrence of two nifH gene copies in N. spumigena strain AV1, only one of which is located in a complete nifHDK cluster and several NifH protein forms. Furthermore, we demonstrate the occurrence of a DNA rearrangement mechanism acting within the nifH gene copy located in the nifHDK cluster and present only in the strains exhibiting the previously reported uncoupling between heterocyst differentiation and nitrogen fixation processes. These data stress the existence of a distinct and complex regulatory circuit related to nitrogen fixation in this ecologically significant bloom-forming cyanobacterium.     

Molecular ecology of nifH genes and transcripts in the eastern Mediterranean Sea

The eastern Mediterranean Sea is one of the most extreme oligotrophic oceanic regions on earth in terms of nutrient concentrations and primary productivity. Nitrogen fixation has been suggested to contribute to the high N : P molar ratios of approximately 28:1 found in this region. Surprisingly, no molecular biological work has been performed in situ to assess whether N(2) fixation genes actually occur in the eastern Mediterranean Sea, or to determine which organisms are responsible for this process. In this study, we examined the presence and expression of nitrogenase genes (nifH) in the upper water layer of the eastern Mediterranean. Clone libraries constructed from both DNA and reverse-transcribed PCR-amplified mRNA were examined and compared. We observed different nifH genes from diverse microbial groups, such as Cyanobacteria, Proteobacteria and methanogenic Archaea. Interestingly, numerous phylotypes were observed in coastal stations at the DNA level but none were active. However, in far offshore stations, the phylotypes observed at the DNA level were the ones that were actually active. Our preliminary study revealed diverse diazotrophs that possess and express nifH genes, which may support N(2) fixation in the eastern Mediterranean Sea.     

Proteomic responses to ocean acidification of the marine diazotroph Trichodesmium under iron-replete and iron-limited conditions

Photosynthesis Research - Growth and dinitrogen (N2) fixation of the globally important diazotrophic cyanobacteria Trichodesmium are often limited by iron (Fe) availability in surface seawaters. To...     

Is dinitrogen fixation significant in the Levantine Basin,East Mediterranean Sea?

We report N(2) fixation rates measured from two stations monitored monthly off the Mediterranean coast of Israel during 2006 and 2007, and along a transect from Israel to Crete in September 2008. Analyses of time-series data revealed expression of nifH genes from diazotrophs in nifH clusters I and II, including cyanobacterial bloom-formers Trichodesmium and diatom-Richelia intracellularis associations. However, nifH gene abundance and rates of N(2) fixation were very low in all size fractions measured (> 0.7 μm). Volumetric (15) N uptake ranged from below detection (～ 36% of > 300 samples) to a high of 0.3 nmol N l(-1) d(-1) and did not vary distinctly with depth or season. Areal N(2) fixation averaged ～ 1 to 4 μmol N m(-2) d(-1) and contributed only ～ 1% and 2% of new production and ～ 0.25% and 0.5% of primary production for the mixed (winter) and stratified (spring-fall) periods respectively. N(2) fixation rates along the 2008 east-west transect were also extremely low (0-0.04 nmol N l(-1) d(-1), integrated average 2.6 μmol N m(-2) d(-1) ) with 37% of samples below detection and no discernable difference between stations. We demonstrate that diazotrophy and N(2) fixation contribute only a minor amount of new N to the P impoverished eastern Mediterranean Sea.     

Facultative diazotrophy increases Cylindrospermopsis raciborskii competitiveness under fluctuating nitrogen availability

Relative fitness of three bloom-forming and potentially toxic cyanobacteria from the subtropical St. John's River, Florida was investigated under a range of nutrient conditions, during a bloom dominated by Cylindrospermopsis raciborskii. Nitrogen (N) was the primary nutrient limiting phytoplankton primary productivity and biomass. Phytoplankton biomass was also enhanced by phosphorus (P) added either alone or jointly with N, suggesting different components of the phytoplankton experienced distinct nutrient limitations. Based on quantitative PCR, the diazotrophic cyanobacteria Anabaena sp. and C.?raciborskii were responsible for the primary production response to P additions, while the nondiazotrophic Microcystis aeruginosa appeared to benefit from N released from the diazotrophs. Cylindrospermopsis raciborskii maintained high net growth rates under diazotrophic and nondiazotrophic conditions, while Anabaena sp. growth was significantly reduced under DIN enrichment. C.?raciborskii appears to be a generalist with regard to N source, a lifestyle traditionally not considered a viable ecological strategy among diazotrophs. Using facultative diazotrophy, C.?raciborskii gains a growth advantage under fluctuating DIN conditions, such as systems that are under the influence of anthropogenic N loading events. The described niche differentiation may be a key factor explaining the recent global expansion of C.?raciborskii.