Photochemical changes during rehydration of an intertidal cyanobacterial mat exposed to variations in salinity and light intensity.

This study focuses on a Lyngbya cf. aestuarii dominated mat community from the intertidal zone of the Laguna Ojo de Liebre, Baja California Sur. In this environment, the mat is desiccated for several days between spring tides. While the mats were desiccated, photosynthetic activity was absent but recovered rapidly (~3 h) upon rehydration. It has been shown previously that the rate of photosynthetic recovery is dependent on both light intensity and salinity. In the current study, photosynthetic recovery was measured based on chlorophyll a fluorescence using pulse amplitude modulated (PAM) fluorometry. Upon the addition of water, photosystem II (PSII) complexes recovered the capacity for reaction centre excitation. However, these functional centres were initially closed. Respiratory activity early in recovery probably reduced the plastoquinone pool through the shared use of part of the photosynthetic transport chain, thus temporarily blocking electron transport downstream of PSII. The time that PSII complexes remained closed increased with light intensities above saturation. This condition is potentially damaging to the cyanobacteria since the exposure of closed PSII centres to high light intensities can lead to the production of singlet oxygen. After this initial lag period, PSII centres opened rapidly indicating an increase in the flow of electrons from PSII to PSI. The rate of photosynthetic recovery appeared to be limited primarily by the relatively slow return of functional PSII. Photosynthetic recovery rates were slower in salinities greater than those that naturally occur in the intertidal zone.

The Abundance and Diversity of Fungi in a Hypersaline Microbial Mat from Guerrero Negro, Baja California, México.

The abundance and diversity of fungi were evaluated in a hypersaline microbial mat from Guerrero Negro, México, using a combination of quantitative polymerase chain reaction (qPCR) amplification of domain-specific primers, and metagenomic sequencing. Seven different layers were analyzed in the mat (Layers 1-7) at single millimeter resolution (from the surface to 7 mm in depth). The number of copies of the 18S rRNA gene of fungi ranged between 106 and 107 copies per g mat, being two logarithmic units lower than of the 16S rRNA gene of bacteria. The abundance of 18S rRNA genes of fungi varied significantly among the layers with layers 2-5 mm from surface contained the highest numbers of copies. Fifty-six fungal taxa were identified by metagenomic sequencing, classified into three different phyla: Ascomycota, Basidiomycota and Microsporidia. The prevalent genera of fungi were Thermothelomyces, Pyricularia, Fusarium, Colletotrichum, Aspergillus, Botrytis, Candida and Neurospora. Genera of fungi identified in the mat were closely related to genera known to have saprotrophic and parasitic lifestyles, as well as genera related to human and plant pathogens and fungi able to perform denitrification. This research suggests that fungi in the mat may participate in nutrient recycling, modification of community composition through parasitic activities, and denitrification.

Reference-guided metagenomics reveals genome-level evidence of potential microbial transmission from the ISS environment to an astronaut's microbiome.

Monitoring microbial communities aboard the International Space Station (ISS) is essential to maintaining astronaut health and the integrity of life-support systems. Using assembled genomes of ISS-derived microbial isolates as references, recruiting metagenomic reads from an astronaut's nasal microbiome revealed no recruitment to a Staphylococcus aureus isolate from samples before launch, yet systematic recruitment across the genome when sampled after 3 months aboard the ISS, with a median percent identity of 100%. This suggests that either a highly similar S. aureus population colonized the astronaut's nasal microbiome while the astronaut was aboard the ISS or that it may have been below detection before spaceflight, instead supporting a shift in community composition. This work highlights the value in generating genomic libraries of microbes from built-environments such as the ISS and demonstrates one way such data can be integrated with metagenomics to facilitate the tracking and monitoring of astronaut microbiomes and health.

Quantifying potential N turnover rates in hypersaline microbial mats by 15N tracer techniques.

Microbial mats, due to stratification of the redox zones, have a potential to include a complete N cycle, however an attempt to evaluate a complete N cycle in these ecosystems has not been yet made. In this study, occurrence and rates of major N cycle processes were evaluated in intact microbial mats from Elkhorn Slough, Monterey Bay, CA, USA, and Baja California Sur, Mexico under oxic and anoxic conditions using 15N-labeling techniques. All of the major N transformation pathways, with the exception of anammox, were detected in both microbial mats. Nitrification rates were found to be low at both sites for both seasons investigated. The highest rates of ammonium assimilation were measured in Elkhorn Slough mats in April and corresponded to high in situ ammonium concentration in the overlying water. Baja mats featured higher ammonification than ammonium assimilation rates and this, along with their higher affinity for nitrate compared to ammonium and low dissimilatory nitrate reduction to ammonium rates, characterized their differences from Elkhorn Slough mats. Nitrogen fixation rates in Elkhorn Slough microbial mats were found to be low implying that other processes such as recycling and assimilation from water are main sources of N for these mats at the times sampled. Denitrification in all of the mats was incomplete with nitrous oxide as end product and not dinitrogen. Our findings highlight N cycling features not previously quantified in microbial mats and indicate a need of further investigations in these microbial ecosystems.Importance: Nitrogen is essential for life. The nitrogen cycle on Earth is mediated by microbial activity and has had a profound impact on both the atmosphere and the biosphere throughout geologic time. Microbial mats, present in many modern environments, have been regarded as living records of the organisms, genes, and phylogenies of microbes, as they are one of the most ancient ecosystems on Earth. While rates of major nitrogen metabolic pathways have been evaluated in a number of ecosystems, it remains elusive in microbial mats. In particular it is unclear what factors affect nitrogen cycling in these ecosystems and how morphological differences between mats impact nitrogen transformations. In this study we investigate nitrogen cycling in two microbial mats having morphological differences. Our findings provide insight for further understanding of biogeochemistry and microbial ecology of microbial mats.

Greenhouse gases and biogeochemical diel fluctuations in a high-altitude wetland.

The landscapes of high-altitude wetland ecosystems are characterized by different kinds of aquatic sites, including ponds holding conspicuous microbial life. Here, we examined a representative pond of the wetland landscape for dynamics of greenhouse gases, and their association with other relevant biogeochemical conditions including diel shifts of microbial communities' structure and activity over two consecutive days. Satellite image analysis indicates that the area of ponds cover 238 of 381.3 Ha (i.e., 62.4%), representing a significant landscape in this wetland. Solar radiation, wind velocity and temperature varied daily and between the days sampled, influencing the biogeochemical dynamics in the pond, shifting the pond reservoir of inorganic versus dissolved organic nitrogen/phosphorus bioavailability, between day 1 and day 2. Day 2 was characterized by high dissolved organic nitrogen/phosphorus and N2O accumulation. CH4 presented a positive excess showing maxima at hours of high radiation during both days. The microbial community in the sediment was diverse and enriched in keystone active groups potentially related with GHG recycling including bacteria and archaea, such as Cyanobacteria, Verrucomicrobia, Rhodobacterales and Nanoarchaeaota (Woesearchaeia). Archaea account for the microbial community composition changes between both days and for the secondary productivity in the water measured during day 2. The results indicate that an intense recycling of organic matter occurs in the pond systems and that the activity of the microbial community is correlated with the availability of nutrients. Together, the above results indicate a net sink of CO2 and N2O, which has also been reported for other natural and artificial ponds. Overall, our two-day fluctuation study in a representative pond of a high-altitude wetland aquatic landscape indicates the need to explore in more detail the short-term besides the long-term biogeochemical variability in arid ecosystems of the Andes plateau, where wetlands are hotspots of life currently under high anthropogenic pressure.

Shotgun metagenome data of a defined mock community using Oxford Nanopore, PacBio and Illumina technologies.

Metagenomic sequence data from defined mock communities is crucial for the assessment of sequencing platform performance and downstream analyses, including assembly, binning and taxonomic assignment. We report a comparison of shotgun metagenome sequencing and assembly metrics of a defined microbial mock community using the Oxford Nanopore Technologies (ONT) MinION, PacBio and Illumina sequencing platforms. Our synthetic microbial community BMock12 consists of 12 bacterial strains with genome sizes spanning 3.2-7.2 Mbp, 40-73% GC content, and 1.5-7.3% repeats. Size selection of both PacBio and ONT sequencing libraries prior to sequencing was essential to yield comparable relative abundances of organisms among all sequencing technologies. While the Illumina-based metagenome assembly yielded good coverage with few misassemblies, contiguity was greatly improved by both, Illumina + ONT and Illumina + PacBio hybrid assemblies but increased misassemblies, most notably in genomes with high sequence similarity to each other. Our resulting datasets allow evaluation and benchmarking of bioinformatics software on Illumina, PacBio and ONT platforms in parallel.

Morphological and isotopic changes of heterocystous cyanobacteria in response to N2 partial pressure.

Earth's atmospheric composition has changed significantly over geologic time. Many redox active atmospheric constituents have left evidence of their presence, while inert constituents such as dinitrogen gas (N2 ) are more elusive. In this study, we examine two potential biological indicators of atmospheric N2 : the morphological and isotopic signatures of heterocystous cyanobacteria. Biological nitrogen fixation constitutes the primary source of fixed nitrogen to the global biosphere and is catalyzed by the oxygen-sensitive enzyme nitrogenase. To protect this enzyme, some filamentous cyanobacteria restrict nitrogen fixation to microoxic cells (heterocysts) while carrying out oxygenic photosynthesis in vegetative cells. Heterocysts terminally differentiate in a pattern that is maintained as the filaments grow, and nitrogen fixation imparts a measurable isotope effect, creating two biosignatures that have previously been interrogated under modern N2 partial pressure (pN2 ) conditions. Here, we examine the effect of variable pN2 on these biosignatures for two species of the filamentous cyanobacterium Anabaena. We provide the first in vivo estimate of the intrinsic isotope fractionation factor of Mo-nitrogenase (εfix  = -2.71 ± 0.09‰) and show that, with decreasing pN2 , the net nitrogen isotope fractionation decreases for both species, while the heterocyst spacing decreases for Anabaena cylindrica and remains unchanged for Anabaena variabilis. These results are consistent with the nitrogen fixation mechanisms available in the two species. Application of these quantifiable effects to the geologic record may lead to new paleobarometric measurements for pN2 , ultimately contributing to a better understanding of Earth's atmospheric evolution.

Mechanisms of nitrogen attenuation from seawater by two microbial mats.

Microbial mats, due to their high microbial diversity, have the potential to express most biogeochemical cycling processes, highlighting their prospective use in bioremediation of various environmental contaminants. In this study the mechanisms of nitrogen attenuation were investigated in naturally occurring microbial mats from Elkhorn Slough, Monterey Bay, CA, USA, and Baja California Sur, Mexico. Key processes responsible for this removal were evaluated using quantification of functional genes related to nitrification, denitrification, and nitrogen fixation. Both microbial mats were capable of removing high (up to 2 mM) concentrations of ammonium and nitrate. Ammonium assimilation rates measured for Elkhorn Slough mats showed that this process was responsible for most of the ammonium uptake in these mats. While Elkhorn Slough mats did not show any evidence of nitrogen removal pathways other than microbial assimilation, Baja mats exhibited the potential for nitrification, denitrification, and DNRA as well as assimilation. The results of this study demonstrate the potential of microbial mats for bioremediation of nitrogenous pollutants independent of the mechanisms responsible for their removal.

Metagenomics reveals niche partitioning within the phototrophic zone of a microbial mat.

Hypersaline photosynthetic microbial mats are stratified microbial communities known for their taxonomic and metabolic diversity and strong light-driven day-night environmental gradients. In this study of the upper photosynthetic zone of hypersaline microbial mats of Elkhorn Slough, California (USA), we show how metagenome sequencing can be used to meaningfully assess microbial ecology and genetic partitioning in these complex microbial systems. Mapping of metagenome reads to the dominant Cyanobacteria observed in the system, Coleofasciculus (Microcoleus) chthonoplastes, was used to examine strain variants within these metagenomes. Highly conserved gene subsystems indicated a core genome for the species, and a number of variant genes and subsystems suggested strain level differentiation, especially for nutrient utilization and stress response. Metagenome sequence coverage binning was used to assess ecosystem partitioning of remaining microbes to both reconstruct the model organisms in silico and identify their ecosystem functions as well as to identify novel clades and propose their role in the biogeochemical cycling of mats. Functional gene annotation of these bins (primarily of Proteobacteria, Bacteroidetes, and Cyanobacteria) recapitulated the known biogeochemical functions in microbial mats using a genetic basis, and revealed significant diversity in the Bacteroidetes, presumably in heterotrophic cycling. This analysis also revealed evidence of putative phototrophs within the Gemmatimonadetes and Gammaproteobacteria residing in microbial mats. This study shows that metagenomic analysis can produce insights into the systems biology of microbial ecosystems from a genetic perspective and to suggest further studies of novel microbes.

Bacterial and archaeal profiling of hypersaline microbial mats and endoevaporites, under natural conditions and methanogenic microcosm experiments.

Bacterial and archaeal community structure of five microbial communities, developing at different salinities in Baja California Sur, Mexico, were characterized by 16S rRNA sequencing. The response of the microbial community to artificial changes in salinity-sulfate concentrations and to addition of trimethylamine was also evaluated in microcosm experiments. Ordination analyses of the microbial community structure showed that microbial composition was distinctive for each hypersaline site. Members of bacteria were dominated by Bacteroidetes and Proteobacteria phyla, while Halobacteria of the Euryarchaeota phylum was the most represented class of archaea for all the environmental samples. At a higher phylogenetic resolution, methanogenic communities were dominated by members of the Methanosarcinales, Methanobacteriales and Methanococcales orders. Incubation experiments showed that putative hydrogenotrophic methanogens of the Methanomicrobiales increased in abundance only under lowest salinity and sulfate concentrations. Trimethylamine addition effectively increased the abundance of methylotrophic members from the Methanosarcinales, but also increased the relative abundance of the Thermoplasmata class, suggesting the potential capability of these microorganisms to use trimethylamine in hypersaline environments. These results contribute to the knowledge of microbial diversity in hypersaline environments from Baja California Sur, Mexico, and expand upon the available information for uncultured methanogenic archaea in these ecosystems.

Metagenomic analysis of intertidal hypersaline microbial mats from Elkhorn Slough, California, grown with and without molybdate.

Cyanobacterial mats are laminated microbial ecosystems which occur in highly diverse environments and which may provide a possible model for early life on Earth. Their ability to produce hydrogen also makes them of interest from a biotechnological and bioenergy perspective. Samples of an intertidal microbial mat from the Elkhorn Slough estuary in Monterey Bay, California, were transplanted to a greenhouse at NASA Ames Research Center to study a 24-h diel cycle, in the presence or absence of molybdate (which inhibits biohydrogen consumption by sulfate reducers). Here, we present metagenomic analyses of four samples that will be used as references for future metatranscriptomic analyses of this diel time series.

Enhanced performance of the microalga Chlorella sorokiniana remotely induced by the plant growth-promoting bacteria Azospirillum brasilense and Bacillus pumilus.

Remote effects (occurring without physical contact) of two plant growth-promoting bacteria (PGPB) Azospirillum brasilense Cd and Bacilus pumilus ES4 on growth of the green microalga Chlorella sorokiniana UTEX 2714 were studied. The two PGPB remotely enhanced the growth of the microalga, up to six-fold, and its cell volume by about three-fold. In addition to phenotypic changes, both bacteria remotely induced increases in the amounts of total lipids, total carbohydrates, and chlorophyll a in the cells of the microalga, indicating an alteration of the microalga's physiology. The two bacteria produced large amounts of volatile compounds, including CO2, and the known plant growth-promoting volatile 2,3-butanediol and acetoin. Several other volatiles having biological functions in other organisms, as well as numerous volatile compounds with undefined biological roles, were detected. Together, these bacteria-derived volatiles can positively affect growth and metabolic parameters in green microalgae without physical attachment of the bacteria to the microalgae. This is a new paradigm on how PGPB promote growth of microalgae which may serve to improve performance of Chlorella spp. for biotechnological applications.

A radiative transfer modeling approach for accurate interpretation of PAM fluorometry experiments in suspended algal cultures.

The results of a numerical study on the simulation of pulse amplitude modulated (PAM) fluorometry within dense suspensions of photosynthetic microorganisms are presented. The Monte Carlo method was used to solve the radiative transfer equation in an algae-filled cuvette, taking into account absorption, anisotropic scattering, and fluorescence, as well as Fresnel reflections at interfaces. This method was used to simulate the transport of excitation and fluorescence light in a common laboratory fluorometer. In this fluorometer, detected fluorescence originates from a multitude of locations within the algal suspension, which can be exposed to very different fluence rates. The fluorescence-weighted fluence rate is reported, which is the local fluence rate of actinic light, averaged over all locations from which detected fluorescence originated. A methodology is reported for recovering the fluorescence-weighted fluence rate as a function of the transmittance of measuring light and actinic light through the sample, which are easily measured with common laboratory fluorometers. The fluorescence-weighted fluence rate can in turn be used as a correction factor for recovering intrinsic physiological parameters, such as the functional cross section of Photosystem II, from apparent (experimental) values. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1601-1615, 2016.

Permanent draft genome of strain ESFC-1: ecological genomics of a newly discovered lineage of filamentous diazotrophic cyanobacteria.

The nonheterocystous filamentous cyanobacterium, strain ESFC-1, is a recently described member of the order Oscillatoriales within the Cyanobacteria. ESFC-1 has been shown to be a major diazotroph in the intertidal microbial mat system at Elkhorn Slough, CA, USA. Based on phylogenetic analyses of the 16S RNA gene, ESFC-1 appears to belong to a unique, genus-level divergence; the draft genome sequence of this strain has now been determined. Here we report features of this genome as they relate to the ecological functions and capabilities of strain ESFC-1. The 5,632,035 bp genome sequence encodes 4914 protein-coding genes and 92 RNA genes. One striking feature of this cyanobacterium is the apparent lack of either uptake or bi-directional hydrogenases typically expected within a diazotroph. Additionally, a large genomic island is found that contains numerous low GC-content genes and genes related to extracellular polysaccharide production and cell wall synthesis and maintenance.

Light Regimes Shape Utilization of Extracellular Organic C and N in a Cyanobacterial Biofilm.

UNLABELLED: Although it is becoming clear that many microbial primary producers can also play a role as organic consumers, we know very little about the metabolic regulation of photoautotroph organic matter consumption. Cyanobacteria in phototrophic biofilms can reuse extracellular organic carbon, but the metabolic drivers of extracellular processes are surprisingly complex. We investigated the metabolic foundations of organic matter reuse by comparing exoproteome composition and incorporation of (13)C-labeled and (15)N-labeled cyanobacterial extracellular organic matter (EOM) in a unicyanobacterial biofilm incubated using different light regimes. In the light and the dark, cyanobacterial direct organic C assimilation accounted for 32% and 43%, respectively, of all organic C assimilation in the community. Under photosynthesis conditions, we measured increased excretion of extracellular polymeric substances (EPS) and proteins involved in micronutrient transport, suggesting that requirements for micronutrients may drive EOM assimilation during daylight hours. This interpretation was supported by photosynthesis inhibition experiments, in which cyanobacteria incorporated N-rich EOM-derived material. In contrast, under dark, C-starved conditions, cyanobacteria incorporated C-rich EOM-derived organic matter, decreased excretion of EPS, and showed an increased abundance of degradative exoproteins, demonstrating the use of the extracellular domain for C storage. Sequence-structure modeling of one of these exoproteins predicted a specific hydrolytic activity that was subsequently detected, confirming increased EOM degradation in the dark. Associated heterotrophic bacteria increased in abundance and upregulated transport proteins under dark relative to light conditions. Taken together, our results indicate that biofilm cyanobacteria are successful competitors for organic C and N and that cyanobacterial nutrient and energy requirements control the use of EOM. IMPORTANCE: Cyanobacteria are globally distributed primary producers, and the fate of their fixed C influences microbial biogeochemical cycling. This fate is complicated by cyanobacterial degradation and assimilation of organic matter, but because cyanobacteria are assumed to be poor competitors for organic matter consumption, regulation of this process is not well tested. In mats and biofilms, this is especially relevant because cyanobacteria produce an extensive organic extracellular matrix, providing the community with a rich source of nutrients. Light is a well-known regulator of cyanobacterial metabolism, so we characterized the effects of light availability on the incorporation of organic matter. Using stable isotope tracing at the single-cell level, we quantified photoautotroph assimilation under different metabolic conditions and integrated the results with proteomics to elucidate metabolic status. We found that cyanobacteria effectively compete for organic matter in the light and the dark and that nutrient requirements and community interactions contribute to cycling of extracellular organic matter.

An Unusual Inverted Saline Microbial Mat Community in an Interdune Sabkha in the Rub' al Khali (the Empty Quarter), United Arab Emirates.

Salt flats (sabkha) are a recognized habitat for microbial life in desert environments and as analogs of habitats for possible life on Mars. Here we report on the physical setting and microbiology of interdune sabkhas among the large dunes in the Rub' al Khali (the Empty Quarter) in Liwa Oasis, United Arab Emirates. The salt flats, composed of gypsum and halite, are moistened by relatively fresh ground water. The result is a salinity gradient that is inverted compared to most salt flat communities with the hypersaline layer at the top and freshwater layers below. We describe and characterize a rich photosynthetically-based microbial ecosystem that is protected from the arid outside environment by a translucent salt crust. Gases collected from sediments under shallow ponds in the sabkha contain methane in concentrations as high as 3400 ppm. The salt crust could preserve biomarkers and other evidence for life in the salt after it dries out. Chloride-filled depressions have been identified on Mars and although surface flow of water is unlikely on Mars today, ground water is possible. Such a near surface system with modern groundwater flowing under ancient salt deposits could be present on Mars and could be accessed by surface rovers.

Cyanobacterial reuse of extracellular organic carbon in microbial mats.

Cyanobacterial organic matter excretion is crucial to carbon cycling in many microbial communities, but the nature and bioavailability of this C depend on unknown physiological functions. Cyanobacteria-dominated hypersaline laminated mats are a useful model ecosystem for the study of C flow in complex communities, as they use photosynthesis to sustain a more or less closed system. Although such mats have a large C reservoir in the extracellular polymeric substances (EPSs), the production and degradation of organic carbon is not well defined. To identify extracellular processes in cyanobacterial mats, we examined mats collected from Elkhorn Slough (ES) at Monterey Bay, California, for glycosyl and protein composition of the EPS. We found a prevalence of simple glucose polysaccharides containing either α or ß (1,4) linkages, indicating distinct sources of glucose with differing enzymatic accessibility. Using proteomics, we identified cyanobacterial extracellular enzymes, and also detected activities that indicate a capacity for EPS degradation. In a less complex system, we characterized the EPS of a cyanobacterial isolate from ES, ESFC-1, and found the extracellular composition of biofilms produced by this unicyanobacterial culture were similar to that of natural mats. By tracing isotopically labeled EPS into single cells of ESFC-1, we demonstrated rapid incorporation of extracellular-derived carbon. Taken together, these results indicate cyanobacteria reuse excess organic carbon, constituting a dynamic pool of extracellular resources in these mats.

Evidence of novel phylogenetic lineages of methanogenic archaea from hypersaline microbial mats.

Methanogenesis in hypersaline and high-sulfate environments is typically dominated by methylotrophic methanogens because sulfate reduction is thermodynamically favored over hydrogenotrophic methanogenesis in these environments. We characterized the community composition of methanogenic archaea in both unmanipulated and incubated microbial mats from different hypersaline environments in Baja California Sur, Mexico. Clone libraries of methyl coenzyme-M reductase (mcrA) sequences and DGGE band patterns of 16S rRNA and mcrA sequences showed that the methanogen community in these microbial mats is dominated by methylotrophic methanogens of the genus Methanohalophilus. However, phylogenetic analyses of mcrA sequences from these mats also revealed two new lineages corresponding to putative hydrogenotrophic methanogens related with the strictly hydrogenotrophic order Methanomicrobiales. Stimulated methane production under decreased salinity and sulfate concentrations also suggested the presence of hydrogenotrophic methanogens in these samples. The relative abundance of mcrA gene and transcripts, estimated by SYBR green I qPCR assays, suggested the activity of different phylogenetic groups of methanogens, including the two novel clusters, in unmanipulated samples of hypersaline microbial mats. Using geochemical and molecular approaches, we show that substrate limitation and values of salinity and sulfate higher than 3 % and 25 mM (respectively) are potential environmental constraints for methanogenesis in these environments. Microcosm experiments with modifications of salinity and sulfate concentrations and TMA addition showed that upper salt and sulfate concentrations for occurrence of methylotrophic methanogenesis were 28 % and 263 mM, respectively. This study provides phylogenetic information about uncultivated and undescribed methanogenic archaea from hypersaline environments.

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.

Trimethylamine and Organic Matter Additions Reverse Substrate Limitation Effects on the δ13C Values of Methane Produced in Hypersaline Microbial Mats.

Methane production has been observed in a number of hypersaline environments, and it is generally thought that this methane is produced through the use of noncompetitive substrates, such as the methylamines, dimethylsulfide and methanol. Stable isotope measurements of the produced methane have also suggested that the methanogens are operating under conditions of substrate limitation. Here, substrate limitation in gypsum-hosted endoevaporite and soft-mat hypersaline environments was investigated by the addition of trimethylamine, a noncompetitive substrate for methanogenesis, and dried microbial mat, a source of natural organic matter. The δ(13)C values of the methane produced after amendments were compared to those in unamended control vials. At all hypersaline sites investigated, the δ(13)C values of the methane produced in the amended vials were statistically lower (by 10 to 71‰) than the unamended controls, supporting the hypothesis of substrate limitation at these sites. When substrates were added to the incubation vials, the methanogens within the vials fractionated carbon isotopes to a greater degree, resulting in the production of more (13)C-depleted methane. Trimethylamine-amended samples produced lower methane δ(13)C values than the mat-amended samples. This difference in the δ(13)C values between the two types of amendments could be due to differences in isotope fractionation associated with the dominant methane production pathway (or substrate used) within the vials, with trimethylamine being the main substrate used in the trimethylamine-amended vials. It is hypothesized that increased natural organic matter in the mat-amended vials would increase fermentation rates, leading to higher H2 concentrations and increased CO2/H2 methanogenesis.