Fixed nitrogen (N) often limits the growth of organisms in terrestrial and aquatic biomes, and N availability has been important in controlling the CO2 balance of modern and ancient oceans. The fixation of atmospheric dinitrogen gas (N2) to ammonia is catalysed by nitrogenase and provides a fixed N for N-limited environments. The filamentous cyanobacterium Trichodesmium has been assumed to be the predominant oceanic N2-fixing microorganism since the discovery of N2 fixation in Trichodesmium in 1961 (ref. 6). Attention has recently focused on oceanic N2 fixation because nitrogen availability is generally limiting in many oceans, and attempts to constrain the global atmosphere-ocean fluxes of CO2 are based on basin-scale N balances. Biogeochemical studies and models have suggested that total N2-fixation rates may be substantially greater than previously believed but cannot be reconciled with observed Trichodesmium abundances. It is curious that there are so few known N2-fixing microorganisms in oligotrophic oceans when it is clearly ecologically advantageous. Here we show that there are unicellular cyanobacteria in the open ocean that are expressing nitrogenase, and are abundant enough to potentially have a significant role in N dynamics.

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In this study we determined the composition and biogeochemistry of novel, brightly colored, white and orange microbial mats at the surface of a brine seep at the outer rim of the Chefren mud volcano. These mats were interspersed with one another, but their underlying sediment biogeochemistries differed considerably. Microscopy revealed that the white mats were granules composed of elemental S filaments, similar to those produced by the sulfide-oxidizing epsilonproteobacterium "Candidatus Arcobacter sulfidicus." Fluorescence in situ hybridization indicated that microorganisms targeted by a "Ca. Arcobacter sulfidicus"-specific oligonucleotide probe constituted up to 24% of the total the cells within these mats. Several 16S rRNA gene sequences from organisms closely related to "Ca. Arcobacter sulfidicus" were identified. In contrast, the orange mat consisted mostly of bright orange flakes composed of empty Fe(III) (hydr)oxide-coated microbial sheaths, similar to those produced by the neutrophilic Fe(II)-oxidizing betaproteobacterium Leptothrix ochracea. None of the 16S rRNA gene sequences obtained from these samples were closely related to sequences of known neutrophilic aerobic Fe(II)-oxidizing bacteria. The sediments below both types of mats showed relatively high sulfate reduction rates (300 nmol ⅐ cm ؊3 ⅐ day ؊1 ) partially fueled by the anaerobic oxidation of methane (10 to 20 nmol ⅐ cm ؊3 ⅐ day ؊1 ). Free sulfide produced below the white mat was depleted by sulfide oxidation within the mat itself. Below the orange mat free Fe(II) reached the surface layer and was depleted in part by microbial Fe(II) oxidation. Both mats and the sediments underneath them hosted very diverse microbial communities and contained mineral precipitates, most likely due to differences in fluid flow patterns.

The nitrogenase activity and phylogenetic diversity of nitrogen fixing microorganisms in several different cyanobacterial mat types from Guerrero Negro, Baja California, Mexico were investigated by acetylene reduction assay, and by amplification and sequencing of the nitrogenase nifH gene. Acetylene reduction assays performed on a Lyngbya sp. and two Microcoleus chthonoplastes dominated microbial mats showed a typical diel pattern of nitrogenase activity in these mats. The highest rates of activity were found at night, with 40 and 37 micromol C(2)H(4) m(-2) h(-1) measured in the Microcoleus mats, and 9 micromol C(2)H(4) m(-2) h(-1) in the Lyngbya mat. Nitrogenase sequences were obtained that clustered with sequences from cyanobacteria, gamma-Proteobacteria, and cluster 3 of nifH. In addition, novel and divergent sequences were also recovered. The composition of nifH sequence types recovered differed between the Lyngbya and Microcoleus mats. Interestingly, nifH sequences belonging to filamentous cyanobacteria were absent in most mat samples even though both mats were dominated by filamentous cyanobacteria. nifH sequences clustering with those of unicellular cyanobacteria were found, some of which were virtually identical to the nifH sequence from Halothece sp. MPI96P605, which had previously been isolated from the mat. In manipulation experiments, the Lyngbya and Microcoleus mats were allowed to re-colonize a cleared surface. In these developing mats, nifH sequences not previously observed in the mats were discovered. Our results showed that organisms capable of N(2) fixation were present in N(2) fixing mats, that the composition of the N(2) fixing communities differs between mats, and that filamentous cyanobacterial diazotrophs may not have a large role in the early stages of mat development.

In many environments, biological nitrogen fixation can alleviate nitrogen limitation. The high rates of N 2 fixation often observed in cyanobacterial mats suggest that N 2 fixation may be an important source of N. In this study, organisms expressing nifH were identified in a Lyngbya sp.-and two Microcoleus chthonoplastes-dominated cyanobacterial mats. The pattern of nitrogenase activity was determined for the Lyngbya sp. mat and a Microcoleus chthonoplastes mat sampled directly in Guerrero Negro, Mexico. Their maximum rates were 23 and 15 mol of C 2 H 4 m ؊2 h ؊1 , respectively. The second Microcoleus mat, which was maintained in a greenhouse facility, had a maximum rate of 40 mol of C 2 H 4 m ؊2 h ؊1 . The overall diel pattern of nitrogenase activity in the three mats was similar, with the highest rates of activity occurring during the dark period. Analysis of nifH transcripts by reverse transcription-PCR revealed that several different organisms were expressing nifH during the dark period. nifH phylotypes recovered from these mats were similar to sequences from the unicellular cyanobacterial genera Halothece, Myxosarcina, and Synechocystis, the filamentous cyanobacterial genera Plectonema and Phormidium, and several bacterial nifH groups. The results of this study indicate that several different organisms, some of which were not previously known to fix nitrogen, are likely to be responsible for the observed dark-period nitrogenase activity in these cyanobacterial mats.

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Studies of the diversity of microorganisms in the environment have been facilitated by use of PCR and reverse transcription PCR (RT-PCR). Inhibition of the PCR by complex sample matrices and low abundance of some target microorganisms require the use of high-sensitivity amplification procedures, involving a large number of cycles or nested PCR methods. Using these methods, we frequently observed contamination of the amplification reagents, including polymerases, by genomic DNA containing nitrogenase (nifH) and rRNA genes. Contaminating genes were sequenced and found to belong to a variety of rRNA clades, but only three major nifH clades. These sequence types included a few nifH sequences reported in previous studies of the environment. Contamination could be reduced by restriction digestion and ultrafiltration of PCR reagents, but efficiency of amplification was also reduced. Our results suggest that studies relying on large numbers of PCR amplification cycles to assess environmental gene diversity should take precautions to assure that clone libaries generated from amplified PCR products are not the result of contaminated PCR reagents.

Microcosms containing sediment from an aquifer in Cambodia with naturally elevated levels of arsenic in the associated groundwater were used to evaluate the effectiveness of microbially mediated production of iron minerals for in situ As remediation. The microcosms were first incubated without amendments for 28 days, and the release of As and other geogenic chemicals from the sediments into the aqueous phase was monitored. Nitrate or a mixture of sulfate and lactate was then added to stimulate biological Fe(II) oxidation or sulfate reduction, respectively. Without treatment, soluble As concentrations reached 3.9 ± 0.9 μM at the end of the 143-day experiment. However, in the nitrate- and sulfate-plus-lactate-amended microcosms, soluble As levels decreased to 0.01 and 0.41 ± 0.13 μM, respectively, by the end of the experiment. Analyses using a range of biogeochemical and mineralogical tools indicated that sorption onto freshly formed hydrous ferric oxide (HFO) and iron sulfide mineral phases are the likely mechanisms for As removal in the respective treatments. Incorporation of the experimental results into a one-dimensional transport-reaction model suggests that, under conditions representative of the Cambodian aquifer, the in situ precipitation of HFO would be effective in bringing groundwater into compliance with the World Health Organization (WHO) provisional guideline value for As (10 ppb or 0.13 μM), although soluble Mn release accompanying microbial Fe(II) oxidation presents a potential health concern. In contrast, production of biogenic iron sulfide minerals would not remediate the groundwater As concentration below the recommended WHO limit.