Revisiting N2 fixation in Guerrero Negro intertidal microbial mats with a functional single-cell approach.

Photosynthetic microbial mats are complex, stratified ecosystems in which high rates of primary production create a demand for nitrogen, met partially by N2 fixation. Dinitrogenase reductase (nifH) genes and transcripts from Cyanobacteria and heterotrophic bacteria (for example, Deltaproteobacteria) were detected in these mats, yet their contribution to N2 fixation is poorly understood. We used a combined approach of manipulation experiments with inhibitors, nifH sequencing and single-cell isotope analysis to investigate the active diazotrophic community in intertidal microbial mats at Laguna Ojo de Liebre near Guerrero Negro, Mexico. Acetylene reduction assays with specific metabolic inhibitors suggested that both sulfate reducers and members of the Cyanobacteria contributed to N2 fixation, whereas (15)N2 tracer experiments at the bulk level only supported a contribution of Cyanobacteria. Cyanobacterial and nifH Cluster III (including deltaproteobacterial sulfate reducers) sequences dominated the nifH gene pool, whereas the nifH transcript pool was dominated by sequences related to Lyngbya spp. Single-cell isotope analysis of (15)N2-incubated mat samples via high-resolution secondary ion mass spectrometry (NanoSIMS) revealed that Cyanobacteria were enriched in (15)N, with the highest enrichment being detected in Lyngbya spp. filaments (on average 4.4 at% (15)N), whereas the Deltaproteobacteria (identified by CARD-FISH) were not significantly enriched. We investigated the potential dilution effect from CARD-FISH on the isotopic composition and concluded that the dilution bias was not substantial enough to influence our conclusions. Our combined data provide evidence that members of the Cyanobacteria, especially Lyngbya spp., actively contributed to N2 fixation in the intertidal mats, whereas support for significant N2 fixation activity of the targeted deltaproteobacterial sulfate reducers could not be found.

Competition for inorganic carbon between oxygenic and anoxygenic phototrophs in a hypersaline microbial mat, Guerrero Negro, Mexico.

While most oxygenic phototrophs harvest light only in the visible range (400-700 nm, VIS), anoxygenic phototrophs can harvest near infrared light (> 700 nm, NIR). To study interactions between the photosynthetic guilds we used microsensors to measure oxygen and gross oxygenic photosynthesis (gOP) in a hypersaline microbial mat under full (VIS + NIR) and VIS illumination. Under normal dissolved inorganic carbon (DIC) concentrations (2 mM), volumetric rates of gOP were reduced up to 65% and areal rates by 16-31% at full compared with VIS illumination. This effect was enhanced (reduction up to 100% in volumetric, 50% in areal rates of gOP) when DIC was lowered to 1 mM, but diminished at 10 mM DIC or lowered pH. In conclusion, under full-light illumination anoxygenic phototrophs are able to reduce the activity of oxygenic phototrophs by efficiently competing for inorganic carbon within the highly oxygenated layer. Anoxygenic photosynthesis, calculated from the difference in gOP under full and VIS illumination, represented between 10% and 40% of the C-fixation. The DIC depletion in the euphotic zone as well as the significant C-fixation by anoxygenic phototrophs in the oxic layer influences the carbon isotopic composition of the mat, which needs to be taken into account when interpreting isotopic biosignals in geological records.