Rapid Microbial Production of Filamentous Sulfur Mats at Hydrothermal Vents

During recent oceanographic cruises to Pacific hydrothermal vent sites (9°N and the Guaymas Basin), the rapid microbial formation of filamentous sulfur mats by a new chemoautotrophic, hydrogen sulfide-oxidizing bacterium was documented in both in situ and shipboard experiments. Observations suggest that formation of these sulfur mats may be a factor in the initial colonization of hydrothermal surfaces by macrofaunal Alvinella worms. This novel metabolic capability, previously shown to be carried out by a coastal strain in H₂S continuous-flow reactors, may be an important, heretofore unconsidered, source of microbial organic matter production at deep-sea hydrothermal vents.     

Speciation of sulfur from filamentous microbial mats from sulfidic cave springs using X-ray absorption near-edge spectroscopy

Most transformations within the sulfur cycle are controlled by the biosphere, and deciphering the abiotic and biotic nature and turnover of sulfur is critical to understand the geochemical and ecological changes that have occurred throughout the Earth's history. Here, synchrotron radiation-based sulfur K-edge X-ray absorption near-edge structure (XANES) spectroscopy is used to examine sulfur speciation in natural microbial mats from two aphotic (cave) settings. Habitat geochemistry, microbial community compositions, and sulfur isotope systematics were also evaluated. Microorganisms associated with sulfur metabolism dominated the mats, including members of the Epsilonproteobacteria and Gammaproteobacteria. These groups have not been examined previously by sulfur K-edge XANES. All of the mats consisted of elemental sulfur, with greater contributions of cyclo-octasulfur (S8) compared with polymeric sulfur (Smicro). While this could be a biological fingerprint for some bacteria, the signature may also indicate preferential oxidation of Smicro and S8 accumulation. Higher sulfate content correlated to less S8 in the presence of Epsilonproteobacteria. Sulfur isotope compositions confirmed that sulfur content and sulfur speciation may not correlate to microbial metabolic processes in natural samples, thereby complicating the interpretation of modern and ancient sulfur records.     

Bacterial sulfur cycling shapes microbial communities in surface sediments of an ultramafic hydrothermal vent field

The ultramafic-hosted Logatchev hydrothermal field (LHF) is characterized by vent fluids, which are enriched in dissolved hydrogen and methane compared with fluids from basalt-hosted systems. Thick sediment layers in LHF are partly covered by characteristic white mats. In this study, these sediments were investigated in order to determine biogeochemical processes and key organisms relevant for primary production. Temperature profiling at two mat-covered sites showed a conductive heating of the sediments. Elemental sulfur was detected in the overlying mat and metal-sulfides in the upper sediment layer. Microprofiles revealed an intensive hydrogen sulfide flux from deeper sediment layers. Fluorescence in situ hybridization showed that filamentous and vibrioid, Arcobacter-related Epsilonproteobacteria dominated the overlying mats. This is in contrast to sulfidic sediments in basalt-hosted fields where mats of similar appearance are composed of large sulfur-oxidizing Gammaproteobacteria. Epsilonproteobacteria (7-21%) and Deltaproteobacteria (20-21%) were highly abundant in the surface sediment layer. The physiology of the closest cultivated relatives, revealed by comparative 16S rRNA sequence analysis, was characterized by the capability to metabolize sulfur components. High sulfate reduction rates as well as sulfide depleted in (34)S further confirmed the importance of the biogeochemical sulfur cycle. In contrast, methane was found to be of minor relevance for microbial life in mat-covered surface sediments. Our data indicate that in conductively heated surface sediments microbial sulfur cycling is the driving force for bacterial biomass production although ultramafic-hosted systems are characterized by fluids with high levels of dissolved methane and hydrogen.     

Sulfur isotope's signal of nanopyrites enclosed in 2.7 Ga stromatolitic organic remains reveal microbial sulfate reduction

Microbial sulfate reduction (MSR) is thought to have operated very early on Earth and is often invoked to explain the occurrence of sedimentary sulfides in the rock record. Sedimentary sulfides can also form from sulfides produced abiotically during late diagenesis or metamorphism. As both biotic and abiotic processes contribute to the bulk of sedimentary sulfides, tracing back the original microbial signature from the earliest Earth record is challenging. We present in situ sulfur isotope data from nanopyrites occurring in carbonaceous remains lining the domical shape of stromatolite knobs of the 2.7‐Gyr‐old Tumbiana Formation (Western Australia). The analyzed nanopyrites show a large range of δ³⁴S values of about 84‰ (from ?33.7‰ to +50.4‰). The recognition that a large δ³⁴S range of 80‰ is found in individual carbonaceous‐rich layers support the interpretation that the nanopyrites were formed in microbial mats through MSR by a Rayleigh distillation process during early diagenesis. An active microbial cycling of sulfur during formation of the stromatolite may have facilitated the mixing of different sulfur pools (atmospheric and hydrothermal) and explain the weak mass independent signature (MIF‐S) recorded in the Tumbiana Formation. These results confirm that MSR participated actively to the biogeochemical cycling of sulfur during the Neoarchean and support previous models suggesting anaerobic oxidation of methane using sulfate in the Tumbiana environment.     

Characterization of sulfide-oxidizing microbial mats developed inside a full-scale anaerobic digester employing biological desulfurization

The microbial mats responsible for biological desulfurization from biogas in a full-scale anaerobic digester were characterized in terms of their structure, as well as their chemical and microbial properties. Filament-shaped elemental sulfur 100–500 μm in length was shown to cover the mats, which cover the entire headspace of the digester. This is the first report on filamentous sulfur production in a non-marine environment. The results of the analysis of the mats suggest that the key players in the sulfide oxidation and sulfur production in the bio-desulfurization in the headspace of the digester were likely to be two sulfide-oxidizing bacteria (SOB) species related to Halothiobacillus neapolitanus and Sulfurimonas denitrificans, and that the microbial community, cell density, activity for sulfide oxidation varied according to the environmental conditions at the various locations of the mats. Since the water and nutrients necessary for the SOB were provided by the digested sludge droplets deposited on the mats, and our results show that a higher rate of sulfide oxidation occurred with more frequent digested sludge deposition, the habitat of the SOB needs to be made in the lower part of the headspace near the liquid level of the digested sludge to maintain optimal conditions.     

Biogeochemical processes in the saline meromictic Lake Kaiike, Japan: implications from molecular isotopic evidences of photosynthetic pigments

Stable carbon and nitrogen isotopic compositions were determined for individual photosynthetic pigments isolated and purified from the saline meromictic Lake Kaiike, Japan, to investigate species-independent biogeochemical processes of photoautotrophs in the natural environment. In the anoxic monimolimnion and benthic microbial mats, the carbon isotopic compositions of BChls e and isorenieratene related to brown-coloured strains of green sulfur bacteria are substantially ( approximately 10 per thousand) depleted in (13)C relative to those found in the chemocline. In conjunction with 16S rDNA evidence reported previously, it strongly suggests that Pelodyctyon luteolum inhabited and photosynthesized in the anoxic monimolimnion and benthic microbial mats by using (13)C-depleted regenerated CO(2). By contrast, both Chl a and BChl a in the monimolimnion and microbial mats have similar isotopic compositions as they do in the chemocline, implying that the source organisms live only in the chemocline. In the chemocline, the nitrogen isotopic compositions of BChl e homologues ranges from -7.7 to-6.5 per thousand, whereas that of BChl a is -2.1 per thousand. These isotopic compositions suggest that green sulfur bacteria Chlorobium phaeovibrioides would conduct nitrogen fixation in the chemocline, whereas purple sulfur bacteria Halochromatium sp. and cyanobacteria Synechococcus sp. may assimilate nitrite.     

Microbial communities in iron-silica-rich microbial mats at deep-sea hydrothermal fields of the Southern Mariana Trough

The abundance, diversity and composition of bacterial and archaeal communities in the microbial mats at deep-sea hydrothermal fields were investigated, using culture-independent 16S rRNA and functional gene analyses combined with mineralogical analysis. Microbial mats were collected at two hydrothermal areas on the ridge of the back-arc spreading centre in the Southern Mariana Trough. Scanning electron microscope and energy dispersive X-ray spectroscopic (SEM-EDS) analyses revealed that the mats were mainly composed of amorphous silica and contained numerous filamentous structures of iron hydroxides. Direct cell counting with SYBR Green I staining showed that the prokaryotic cell densities were more than 10⁸ cells g⁻¹. Quantitative polymerase chain reaction (Q-PCR) analysis revealed that Bacteria are more abundant than Archaea in the microbial communities. Furthermore, zetaproteobacterial cells accounted for 6% and 22% of the prokaryotic cells in each mat estimated by Q-PCR with newly designed primers and TaqMan probe. Phylotypes related to iron-oxidizers, methanotrophs/methylotrophs, ammonia-oxidizers and sulfate-reducers were found in the 16S rRNA gene clone libraries constructed from each mat sample. A variety of unique archaeal 16S rRNA gene phylotypes, several pmoA , dsrAB and archaeal amoA gene phylotypes were also recovered from the microbial mats. Our results provide insights into the diversity and abundance of microbial communities within microbial mats in deep-sea hydrothermal fields.     

Phylogenetic Evidence for the Existence of Novel Thermophilic Bacteria in Hot Spring Sulfur-Turf Microbial Mats in Japan

So-called sulfur-turf microbial mats, which are macroscopic white filaments or bundles consisting of large sausage-shaped bacteria and elemental sulfur particles, occur in sulfide-containing hot springs in Japan. However, no thermophiles from sulfur-turf mats have yet been isolated as cultivable strains. This study was undertaken to determine the phylogenetic positions of the sausage-shaped bacteria in sulfur-turf mats by direct cloning and sequencing of 16S rRNA genes amplified from the bulk DNAs of the mats. Common clones with 16S rDNA sequences with similarity levels of 94.8 to 99% were isolated from sulfur-turf mat samples from two geographically remote hot springs. Phylogenetic analysis showed that the phylotypes of the common clones formed a major cluster with members of the Aquifex-Hydrogenobacter complex, which represents the most deeply branching lineage of the domain bacteria. Furthermore, the bacteria of the sulfur-turf mat phylotypes formed a clade distinguishable from that of other members of the Aquifex-Hydrogenobacter complex at the order or subclass level. In situ hybridization with clone-specific probes for 16S rRNA revealed that the common phylotype of sulfur-turf mat bacteria is that of the predominant sausage-shaped bacteria.Microbial mats develop in a wide variety of aquatic environments, including geothermal hot springs and hydrothermal vents. There are several types of thermophilic microbial mats, e.g., those of cyanobacteria, anoxygenic phototrophic bacteria, and chemotrophic sulfur bacteria, which differ according to the physical and chemical conditions they favor and other environmental factors (10, 38). These microbial mats in thermal habitats have been studied extensively as a peculiar microbial community of the ecosystem, in relation to the phylogeny and evolution of thermophilic prokaryotes, or as a source of new functional enzymes.So-called sulfur-turf microbial mats are macroscopic bundles of white filaments consisting of colorless sulfur bacteria and elemental sulfur particles that form in shallow streams of sulfide-containing high-temperature hot springs. Since first reported by Miyoshi in 1897 (33), this kind of microbial mat has been recorded for several geographically remote hot springs in Japan, although there have been only scattered reports of sulfur-turf microbial mats or chemotrophic sulfur streamers in geothermal springs in other countries (9, 13, 14). The sulfur-turf mats generally develop within a temperature range of 45 to 73°C, within a pH range of 6 to 9, and at discrete sulfide-oxygen interfaces in geothermal springs. These characteristics suggest that the major constituents of the sulfur-turf prokaryotic community are (hyper)thermophilic, neutrophilic, microaerophilic, and chemolithotrophic bacteria. Early studies of these sulfur-turf mats distinguished microscopically three morphotypes of bacteria, two of which were tentatively named Thiovibrio miyoshi and Thiothrix miyoshi (15). Moreover, in situ ecophysiological and microscopic studies have shown that one of these bacteria, the large sausage-shaped “Thiovibrio miyoshi,” predominates in sulfur-turf mats and oxidizes environmental sulfide to elemental sulfur and then to sulfate via thiosulfate (27–31). So far, however, it has not been possible to isolate and cultivate any thermophilic prokaryotes from the sulfur-turf mats predominated by these sausage-shaped bacteria with artificial media, and no attempt has been made to clarify their taxonomic and phylogenetic positions.Determination of 16S rRNA genes is a useful research strategy for identifying uncultivated prokaryotes and is now commonly performed in ecological studies. This technique, involving PCR amplification of 16S rRNA genes or synthesis of cDNAs from bulk 16S rRNAs of natural mixed microbial populations, has been used successfully for the phylogenetic characterization of prokaryotes in hydrothermal environments (6, 7, 34, 40, 41, 47, 48). In the present study, this approach was applied to characterize the sausage-shaped bacteria in sulfur-turf mats without isolating and cultivating them. Here we report that sulfur-turf mats contain novel thermophilic bacteria belonging to the earliest-branching lineage of the domain bacteria.     

Physiology of purple sulfur bacteria forming macroscopic aggregates in Great Sippewissett Salt Marsh, Massachusetts

Abstract Purple bacterial aggregates found in tidal pools of Great Sippewissett Salt Marsh (Falmouth, Cape Cod, MA) were investigated in order to elucidate the ecological significance of cell aggregation. Purple sulfur bacteria were the dominant microorganisms in the aggregates which also contained diatoms and a high number of small rod-shaped bacteria. Urea in concentrations of ≥ 1 M caused disintegration of the aggregates while proteolytic enzymes, surfactants or chaotropic agents did not exhibit this effect. This suggests that polysaccharides in the embedding slime matrix stabilize the aggregate structure. In addition cell surface hydrophobicity is involved in aggregate formation. The concentration of dissolved oxygen decreased rapidly below the surface of aggregates while sulfide was not detected. The apparent respiration rate in the aggregates was high when the purple sulfur bacteria contained intracellular sulfur globules. In the presence of DCMU, respiration remained light-inhibited. Light inhibition disappeared in the presence of KCN. These results demonstrated that respiration in the aggregates is due mainly to purple sulfur bacteria. The concentration of bacteriochlorophyll (Bchl) a in the aggregates (0.205 mg Bchl a cm⁻³) was much higher than in the pool sediments but comparable to concentrations in microbial mats of adjacent sand flats. Purple aggregates may therefore originate in the microbial mats rather than in the pools themselves. Rapid sedimentation and high respiration rates of Chromatiaceae in the aggregates would prevent the inhibition of Bchl synthesis if aggregates were lifted off the sediment and up into the oxic pool water by tidal currents.     

Geomicrobiological exploration and characterization of a novel deep-sea hydrothermal system at the TOTO caldera in the Mariana Volcanic Arc

Novel hydrothermal activities accompanying effluent white smokers and elemental sulfur chimney structures at the north-east lava dome of the TOTO caldera depression in the Mariana Volcanic Arc have been explored and characterized by geochemical and microbiological surveys. White smoker hydrothermal fluids were observed in the potential hydrothermal activity centre of the field and represented the maximal temperature of 170 degrees C and the lowest pH of 1.6. The chimney structures, all consisting of elemental sulfur (sulfur chimney), were also unique to the TOTO caldera hydrothermal field. Microbial community structures in a sulfur chimney and its formation hydrothermal fluid with a high concentration of hydrogen sulfide (15 mM) have been investigated by culture-dependent and -independent analyses. 16S rRNA gene clone analysis and fluorescence in situ hybridization (FISH) analysis revealed that epsilon-Proteobacteria dominated the microbial communities in the sulfur chimney structure and formed a dense microbial mat covering the sulfur chimney surface. Archaeal phylotypes were consistently minor components in the communities and related to the genera Thermococcus, Pyrodictium, Aeropyrum, and the uncultivated archaeal group of 'deep-sea hydrothermal vent euryarchaeotal group'. Cultivation analysis suggested that the chemolithoautotrophs might play a significant ecological role as primary producers utilizing gas and sulfur compounds provided from hydrothermal fluids.     

Formation and Taphonomy of Human Footprints in Microbial Mats of Present-Day Tidal-flat Environments: Implications for the Study of Fossil Footprints

This study concerns the formation, taphonomy, and preservation of human footprints in microbial mats of present-day tidal-flat environments. Due to differences in water content and nature of the microbial mats and the underlying sediment, a wide range of footprint morphologies was produced by the same trackmaker. Most true tracks are subjected to modification due to taphonomic processes, leading to modified true tracks. In addition to formation of biolaminites, microbial mats play a major role in the preservation of footprints on tidal flats. A footprint may be consolidated by desiccation or lithification of the mat, or by ongoing growth of the mat. The latter process may lead to the formation of overtracks. Among consolidated or (partially) lithified footprints found on present-day tidal flats, poorly defined true tracks, modified true tracks, and overtracks were most frequently encountered while unmodified and well-defined true tracks are rather rare. We suggest that modified true tracks and overtracks make up an important percentage of fossil footprints and that they may be as common as undertracks. However, making unambiguous distinctions between poorly defined true tracks, modified true tracks, undertracks, and overtracks in the fossil record will remain a difficult task, which necessitates systematic excavation of footprints combined with careful analysis of the encasing sediment.     

Mathematical simulation of the diel O, S, and C biogeochemistry of a hypersaline microbial mat

The creation of a mathematical simulation model of photosynthetic microbial mats is important to our understanding of key biogeochemical cycles that may have altered the atmospheres and lithospheres of early Earth. A model is presented here as a tool to integrate empirical results from research on hypersaline mats from Baja California Sur (BCS), Mexico into a computational system that can be used to simulate biospheric inputs of trace gases to the atmosphere. The first version of our model, presented here, calculates fluxes and cycling of O(2), sulfide, and dissolved inorganic carbon (DIC) via abiotic components and via four major microbial guilds: cyanobacteria (CYA), sulfate reducing bacteria (SRB), purple sulfur bacteria (PSB) and colorless sulfur bacteria (CSB). We used generalized Monod-type equations that incorporate substrate and energy limits upon maximum rates of metabolic processes such as photosynthesis and sulfate reduction. We ran a simulation using temperature and irradiance inputs from data collected from a microbial mat in Guerrero Negro in BCS (Mexico). Model O(2), sulfide, and DIC concentration profiles and fluxes compared well with data collected in the field mats. There were some model-predicted features of biogeochemical cycling not observed in our actual measurements. For instance, large influxes and effluxes of DIC across the MBGC mat boundary may reveal previously unrecognized, but real, in situ limits on rates of biogeochemical processes. Some of the short-term variation in field-collected mat O(2) was not predicted by MBGC. This suggests a need both for more model sensitivity to small environmental fluctuations for the incorporation of a photorespiration function into the model.     

A Microbial Mat of a Large Sulfur Bacterium Preserved in a Miocene Methane-Seep Limestone

A Miocene methane-seep limestone from the Romagna Apennine (Pietralunga, Italy) was found to contain an extraordinarily well-preserved microbial mat consisting of filamentous fossils. Individual filaments of the lithified Pietralunga mat are 50 to 80 μ m in diameter and resemble the sulfide-oxidizing bacterium Beggiatoa. Mats of sulfur bacteria are common around modern methane-seeps, but have not yet been reported from ancient seep limestones. This is thought to be related to the conditions prevailing in metabolically active mats of sulfur bacteria that do not favor carbonate formation. The preservation of the Pietralunga mat was most likely caused by a sudden change from oxidizing to anoxic conditions, leading to the rapid carbonate precipitation induced by anaerobic oxidation of methane. Lipid biomarkers specific for archaea and sulfate-reducing bacteria linked with the anaerobic oxidation of methane co-occur with compounds derived from methanotrophic bacteria and ciliates. These findings confirm a close proximity of oxic and anoxic conditions, as required for the growth of sulfide-oxidizing bacteria in the methane-based ecosystem. The lack of earlier reports on fossilized thiotrophic mats in seep limestones is most likely related to the rarity of environmental changes rapid enough to preserve the filaments rather than to a lower frequency of thiotrophic mats around methane-seeps in the geological past.     

Metagenomic resolution of microbial functions in deep-sea hydrothermal plumes across the Eastern Lau Spreading Center

Microbial processes within deep-sea hydrothermal plumes affect ocean biogeochemistry on global scales. In rising hydrothermal plumes, a combination of microbial metabolism and particle formation processes initiate the transformation of reduced chemicals like hydrogen sulfide, hydrogen, methane, iron, manganese and ammonia that are abundant in hydrothermal vent fluids. Despite the biogeochemical importance of this rising portion of plumes, it is understudied in comparison to neutrally buoyant plumes. Here we use metagenomics and bioenergetic modeling to describe the abundance and genetic potential of microorganisms in relation to available electron donors in five different hydrothermal plumes and three associated background deep-sea waters from the Eastern Lau Spreading Center located in the Western Pacific Ocean. Three hundred and thirty one distinct genomic ‘bins'' were identified, comprising an estimated 951 genomes of archaea, bacteria, eukarya and viruses. A significant proportion of these genomes is from novel microorganisms and thus reveals insights into the energy metabolism of heretofore unknown microbial groups. Community-wide analyses of genes encoding enzymes that oxidize inorganic energy sources showed that sulfur oxidation was the most abundant and diverse chemolithotrophic microbial metabolism in the community. Genes for sulfur oxidation were commonly present in genomic bins that also contained genes for oxidation of hydrogen and methane, suggesting metabolic versatility in these microbial groups. The relative diversity and abundance of genes encoding hydrogen oxidation was moderate, whereas that of genes for methane and ammonia oxidation was low in comparison to sulfur oxidation. Bioenergetic-thermodynamic modeling supports the metagenomic analyses, showing that oxidation of elemental sulfur with oxygen is the most dominant catabolic reaction in the hydrothermal plumes. We conclude that the energy metabolism of microbial communities inhabiting rising hydrothermal plumes is dictated by the underlying plume chemistry, with a dominant role for sulfur-based chemolithoautotrophy.     

Microbial Mat Boundaries between Chemolithotrophs and Phototrophs in Geothermal Hot Spring Effluents

Among the various microbial mats that develop in geothermal hot springs in solfataric fields, colorless sulfur-turf (ST)—macroscopic white bundles consisting of large sickle-shaped bacteria belonging to Aquificales and elemental sulfur particles–develops in a limited environment of geothermal effluent containing hydrogen sulfide with neutral pH and low in oxygen. Photosynthetic cyanobacterial mat (CY) often grow just downstream of chemolithotrophic ST, or they coexist with ST where the temperature is slightly lower. Knowledge of the environmental regimes of these microbial mats will lead to better understanding of the distribution of thermophilic microorganisms on the Earth and provide clues about evolutionary processes in the microbial ecosystems of the Precambrian era. We studied the environmental parameters of the boundary zone and examined the distribution of these types of mats and measured the in situ growth rates of the microorganisms composing them. In situ examination revealed that temperature and Eh constrain the development of the microbial mats. At the boundary between ST and CY, temperature and Eh ranged between 51.1°C and 63.2°C and between ?112 mV and ?25 mV, respectively. These environmental parameters were not significantly different among Japanese, Yellowstone (North American), and Icelandic hot spring effluents with genetically similar thermal sulfur oxidizers. Sickle-shaped bacteria rarely coexist with cyanobacteria, although they can potentially grow in some CY environments. This suggests that the boundary between ST and CY might be partly determined by exclusive ecological competition.     

Characterization of a sulfide-oxidizing biofilm developed in a packed-column reactor.

The potential of microbial mats to develop sulfide-oxidizing biofims was explored. A bioreactor specially designed for the treatment of sulfide-containing effluents was inoculated with a microbial-mat sample, and a complex microbial biofilm with sulfide-oxidation activity developed. The microbial composition of the biofilm was studied by pigment, microscopy, and 16S rRNA gene analyses. Purple sulfur bacteria and diatoms were observed by microscopy, chlorophyll a and bacteriochlorophyll a were detected in the pigment analysis, and high genetic diversity was found in the 16S rRNA gene library. Specialized anaerobic sulfur oxidizers (i.e., phototrophic purple and green sulfur bacteria) dominated the library. Aerobic phototrophs (diatoms) also developed and the oxygen produced allowed the growth of aerobic sulfide oxidizers, such as Thiomicrospira-like spp. Cyanobacteria, which are significant organisms in natural microbial mats, did not develop in the reactor but unexpected uncultured members from the Epsilonproteobacteria developed profusely. Moreover, a variety of more minor organisms, such as members of the Cytophaga-Flavobacterium-Bacteroides (CFB) and purple non-sulfur bacteria (Roseospirillum sp.), were also present. The results showed that a complex community with high genetic and metabolic diversity, including many uncultured organisms, can develop in a laboratory-scale reactor.     

Use of specific PCR primers for the study of sulfate-reducing bacteria diversity in microbial mats of Ebro Delta,Spain

Microbial mats are prokaryotic communities that provide model systems to analyze microbial diversity and ecophysiological interactions. Sulfate-reducing bacteria (SRB) play a key role in sulfur and nutrient recycling in these ecosystems. In this work, specific primers for 16S rRNA encoding gene, previously described, were used to study the diversity of SRB in microbial mats of the Ebro Delta. We confirm that this method is reliable to identify the diversity of SRB in these ecosystems. However, some mismatches in obtained sequences had been observed in our system and must be taken under consideration. Various genera of SRB in Ebro Delta microbial mats were identified, such as Desulfonema, Desulfatitalea, Desulfosalsimonas, Desulfoccocus, and Desulfovibrio. The diversity observed in our samples is very similar to previously reported in other microbial mats communities.     

Diversity of anoxygenic phototrophic sulfur bacteria in the microbial mats of the Ebro Delta: a combined morphological and molecular approach

The diversity of purple and green sulfur bacteria in the multilayered sediments of the Ebro Delta was investigated. Specific oligonucleotide primers for these groups were used for the selective amplification of 16S rRNA gene sequences. Subsequently, amplification products were separated by denaturing gradient gel electrophoresis and sequenced, which yielded a total of 32 sequences. Six of the sequences were related to different cultivated members of the green sulfur bacteria assemblage, whereas seven fell into the cluster of marine or halophilic Chromatiaceae. Six sequences were clustered with the family Ectothiorhodospiraceae, three of the six being closely related to chemotrophic bacteria grouped together with Halorhodospira genus, and the other three forming a group related to the genus Ectothiorhodospira. The last thirteen sequences constituted a cluster where no molecular isolate from microbial mats has so far been reported. Our results indicate that the natural diversity in the ecosystem studied has been significantly underestimated in the past and point out the presence of novel species not related to all known purple sulfur bacteria. Furthermore, the detection of green sulfur bacteria, after only an initial step of enrichment, suggests that -- with the appropriate methodology -- several genera, such as Prosthecochloris, could be established as regular members of marine microbial mats.