A novel, multi-layered methanotrophic microbial mat system growing on the sediment of the Black Sea.

A novel microbially diverse type of 1- to 5-cm-thick mat performing anaerobic oxidation of methane (AOM) and covering several square metres of the seafloor was discovered in the Black Sea at 180 m water depth. Contrary to other AOM-mat systems of the Black Sea these floating mats are not associated to free gas and are not stabilized by authigenic carbonates. However, supply of methane is ensured by the horizontal orientation of the mats acting as a cover of methane enriched fluids ascending from the underlying sediments. Thorough investigation of their community composition by molecular microbiology and lipid biomarkers, metabolic activities and elemental composition showed that the mats provide a clearly structured system with extracellular polymeric substances (EPS) building the framework of the mats. The top black zone, showing high rates of AOM (15 mumol g(dw) (-1) day(-1)), was dominated by ANME-2, while the following equally active pink layer was dominated by ANME-1 Archaea. The lowest AOM activity (2 mumol g(dw) (-1) day(-1)) and cell numbers were found in the greyish middle part delimited towards the sediment by a second pink, ANME-1-dominated and sometimes a black outer layer (ANME-2). Our work clearly shows that the different microbial populations are established along defined chemical gradients such as methane, sulfate or sulfide.

Seasonal and diel distributions of denitrifying and bacterial communities in a hypersaline microbial mat (Camargue, France).

Changes in spatio-temporal distribution of bacterial and denitrifying communities were qualitatively studied in a microbial mat from Camargue (France). During a diel and a seasonal cycle, patterns of 16S rRNA and nitrite reductase genes (nirS and nirK) were compared by denaturing gradient gel electrophoresis (DGGE). Statistical analysis of DGGE profiles showed a significant seasonal shift in the community structure of the nirS-containing bacteria with a winter superficial population that extended in summer, whereas the nirK-containing bacteria seemed more affected by vertical gradients rather than by month-to month-changes. Denitrifying activities remained stable during these sampling times. The bacterial community at the surface of the mat also changed according to season, but appeared stable over a day. Finally, during a diel cycle nirK populations were localized in zones with large fluctuations of environmental parameters (oxygen, pH, and sulfur levels) while nirS populations seemed more restricted to the permanent anoxic layer of the microbial mat.

Regulation of photosynthesis and oxygen consumption in a hypersaline cyanobacterial mat (Camargue, France) by irradiance, temperature and salinity.

Short-term effects of irradiance (0-1560 micromol photons m(-2) s(-1)), temperature (10-25 degrees C), and salinity (40-160) on oxygenic photosynthesis and oxygen consumption in a hypersaline mat (Salin-de-Giraud, France) were investigated with microsensors under controlled laboratory conditions. Dark O(2) consumption rates were mainly regulated by the mass transfer limitations imposed by the diffusive boundary layer. Areal rates of net photosynthesis increased with irradiance and saturated at irradiances >400 micromol photons m(-2) s(-1). At low irradiances, oxygen consumption increased more strongly with temperature than photosynthesis, whereas the opposite was observed at saturating irradiances. Net photosynthesis vs. irradiance curves were almost unaffected by decreasing salinity (100 to 40), whereas increasing salinities (100 to 160) led to a decrease of net photosynthesis at each irradiance. Dark O(2) consumption rates, maximal gross and net photosynthesis at light saturation were relatively constant over a broad salinity range (60-100) and decreased at salinities above the in situ salinity of 100. Within the range of natural variation, temperature was more important than salinity in regulating photosynthesis and oxygen consumption. At higher salinities the inhibitory impact of salinity on these processes and therefore the importance of salinity as a regulating environmental parameter increased, indicating that in more hypersaline systems, salinity has a stronger limiting effect on microbial activity.

Characterization of functional bacterial groups in a hypersaline microbial mat community (Salins-de-Giraud, Camargue, France).

A photosynthetic microbial mat was investigated in a large pond of a Mediterranean saltern (Salins-de-Giraud, Camargue, France) having water salinity from 70 per thousand to 150 per thousand (w/v). Analysis of characteristic biomarkers (e.g., major microbial fatty acids, hydrocarbons, alcohols and alkenones) revealed that cyanobacteria were the major component of the pond, in addition to diatoms and other algae. Functional bacterial groups involved in the sulfur cycle could be correlated to these biomarkers, i.e. sulfate-reducing, sulfur-oxidizing and anoxygenic phototrophic bacteria. In the first 0.5 mm of the mat, a high rate of photosynthesis showed the activity of oxygenic phototrophs in the surface layer. Ten different cyanobacterial populations were detected with confocal laser scanning microscopy: six filamentous species, with Microcoleus chthonoplastes and Halomicronema excentricum as dominant (73% of total counts); and four unicellular types affiliated to Microcystis, Chroococcus, Gloeocapsa, and Synechocystis (27% of total counts). Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments confirmed the presence of Microcoleus, Oscillatoria, and Leptolyngbya strains (Halomicronema was not detected here) and revealed additional presence of Phormidium, Pleurocapsa and Calotrix types. Spectral scalar irradiance measurements did not reveal a particular zonation of cyanobacteria, purple or green bacteria in the first millimeter of the mat. Terminal-restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA gene fragments of bacteria depicted the community composition and a fine-scale depth-distribution of at least five different populations of anoxygenic phototrophs and at least three types of sulfate-reducing bacteria along the microgradients of oxygen and light inside the microbial mat.

Cyanobacterial ecotypes in the microbial mat community of Mushroom Spring (Yellowstone National Park, Wyoming) as species-like units linking microbial community composition, structure and function.

We have investigated microbial mats of alkaline siliceous hot springs in Yellowstone National Park as natural model communities to learn how microbial populations group into species-like fundamental units. Here, we bring together empirical patterns of the distribution of molecular variation in predominant mat cyanobacterial populations, theory-based modelling of how to demarcate phylogenetic clusters that correspond to ecological species and the dynamic patterns of the physical and chemical microenvironments these populations inhabit and towards which they have evolved adaptations. We show that putative ecotypes predicted by the theory-based model correspond well with distribution patterns, suggesting populations with distinct ecologies, as expected of ecological species. Further, we show that increased molecular resolution enhances our ability to detect ecotypes in this way, though yet higher molecular resolution is probably needed to detect all ecotypes in this microbial community.

Diel variations in carbon metabolism by green nonsulfur-like bacteria in alkaline siliceous hot spring microbial mats from Yellowstone National Park.

Green nonsulfur-like bacteria (GNSLB) in hot spring microbial mats are thought to be mainly photoheterotrophic, using cyanobacterial metabolites as carbon sources. However, the stable carbon isotopic composition of typical Chloroflexus and Roseiflexus lipids suggests photoautotrophic metabolism of GNSLB. One possible explanation for this apparent discrepancy might be that GNSLB fix inorganic carbon only during certain times of the day. In order to study temporal variability in carbon metabolism by GNSLB, labeling experiments with [13C]bicarbonate, [14C]bicarbonate, and [13C]acetate were performed during different times of the day. [14C]bicarbonate labeling indicated that during the morning, incorporation of label was light dependent and that both cyanobacteria and GNSLB were involved in bicarbonate uptake. 13C-labeling experiments indicated that during the morning, GNSLB incorporated labeled bicarbonate at least to the same degree as cyanobacteria. The incorporation of [13C]bicarbonate into specific lipids could be stimulated by the addition of sulfide or hydrogen, which both were present in the morning photic zone. The results suggest that GNSLB have the potential for photoautotrophic metabolism during low-light periods. In high-light periods, inorganic carbon was incorporated primarily into Cyanobacteria-specific lipids. The results of a pulse-labeling experiment were consistent with overnight transfer of label to GNSLB, which could be interrupted by the addition of unlabeled acetate and glycolate. In addition, we observed direct incorporation of [13C]acetate into GNSLB lipids in the morning. This suggests that GNSLB also have a potential for photoheterotrophy in situ.

Cyanobacterial ecotypes in different optical microenvironments of a 68 degrees C hot spring mat community revealed by 16S-23S rRNA internal transcribed spacer region variation.

We examined the population of unicellular cyanobacteria (Synechococcus) in the upper 3-mm vertical interval of a 68 degrees C region of a microbial mat in a hot spring effluent channel (Yellowstone National Park, Wyoming). Fluorescence microscopy and microsensor measurements of O(2) and oxygenic photosynthesis demonstrated the existence of physiologically distinct Synechococcus populations at different depths along a light gradient quantified by scalar irradiance microprobes. Molecular methods were used to evaluate whether physiologically distinct populations could be correlated with genetically distinct populations over the vertical interval. We were unable to identify patterns in genetic variation in Synechococcus 16S rRNA sequences that correlate with different vertically distributed populations. However, patterns of variation at the internal transcribed spacer locus separating 16S and 23S rRNA genes suggested the existence of closely related but genetically distinct populations corresponding to different functional populations occurring at different depths.

Microscopic examination of distribution and phenotypic properties of phylogenetically diverse Chloroflexaceae-related bacteria in hot spring microbial mats.

We investigated the diversity, distribution, and phenotypes of uncultivated Chloroflexaceae-related bacteria in photosynthetic microbial mats of an alkaline hot spring (Mushroom Spring, Yellowstone National Park). By applying a directed PCR approach, molecular cloning, and sequence analysis of 16S rRNA genes, an unexpectedly large phylogenetic diversity among these bacteria was detected. Oligonucleotide probes were designed to target 16S rRNAs from organisms affiliated with the genus Chloroflexus or with the type C cluster, a group of previously discovered Chloroflexaceae relatives of this mat community. The application of peroxidase-labeled probes in conjunction with tyramide signal amplification enabled the identification of these organisms within the microbial mats by fluorescence in situ hybridization (FISH) and the investigation of their morphology, abundance, and small-scale distribution. FISH was combined with oxygen microelectrode measurements, microscope spectrometry, and microautoradiography to examine their microenvironment, pigmentation, and carbon source usage. Abundant type C-related, filamentous bacteria were found to flourish within the cyanobacterium-dominated, highly oxygenated top layers and to predominate numerically in deeper orange-colored zones of the investigated microbial mats, correlating with the distribution of bacteriochlorophyll a. Chloroflexus sp. filaments were rare at 60 degrees C but were more abundant at 70 degrees C, where they were confined to the upper millimeter of the mat. Both type C organisms and Chloroflexus spp. were observed to assimilate radiolabeled acetate under in situ conditions.