Microbial diversity of extant stromatolites in the hypersaline marine environment of Shark Bay, Australia

Stromatolites have been present on Earth, at various levels of distribution and diversity, for more than 3 billion years. Today, the best examples of stromatolites forming in hypersaline marine environments are in Hamelin Pool at Shark Bay, Western Australia. Despite their evolutionary significance, little is known about their associated microbial communities. Using a polyphasic approach of culture-dependent and culture-independent methods, we report the discovery of a wide range of microorganisms associated with these biosedimentary structures. There are no comparable reports combining these methodologies in the survey of cyanobacteria, bacteria, and archaea in marine stromatolites. The community was characterized by organisms of the cyanobacterial genera Synechococcus, Xenococcus, Microcoleus, Leptolyngbya, Plectonema, Symploca, Cyanothece, Pleurocapsa and Nostoc. We also report the discovery of potentially free-living Prochloron. The other eubacterial isolates and clones clustered into seven phylogenetic groups: OP9, OP10, Marine A group, Proteobacteria, Low G+C Gram-positive, Planctomycetes and Acidobacteria. We also demonstrate the presence of sequences corresponding to members of halophilic archaea of the divisions Euryarchaeota and Crenarchaeota and methanogenic archaea of the order Methanosarcinales. This is the first report of such archaeal diversity from this environment. This study provides a better understanding of the microbial community associated with these living rocks.     

Microbial diversity in modern marine stromatolites, Highborne Cay, Bahamas

Living marine stromatolites at Highborne Cay, Bahamas, are formed by microbial mat communities that facilitate precipitation of calcium carbonate and bind and trap small carbonate sand grains. This process results in a laminated structure similar to the layering observed in ancient stromatolites. In the modern marine system at Highborne Cay, lamination, lithification and stromatolite formation are associated with cycling between three types of microbial communities at the stromatolite surface (Types 1, 2 and 3, which range from a leathery microbial mat to microbially fused sediment). Examination of 923 universal small-subunit rRNA gene sequences from these communities reveals that taxonomic richness increases during transition from Type 1 to Type 3 communities, supporting a previous model that proposed that the three communities represent different stages of mat development. The phylogenetic composition also changes significantly between these community types and these community changes occur in concert with variation in biogeochemical rates. The dominant bacterial groups detected in the stromatolites include Alphaproteobacteria , Planctomycetes , Cyanobacteria and Bacteroidetes . In addition, the stromatolite communities were found to contain novel cyanobacteria that may be uniquely associated with modern marine stromatolites. The implications of these findings are discussed in the context of current models for stromatolite formation.     

Carbon isotope discrepancy between precambrian stromatolites and their modern analogs: Inferences from hypersaline microbial mats of the sinai coast

The isotopic composition of organic carbon from extant stromatolite-type microbial ecosystems is commonly slanted toward heavy ¹³ C values as compared to respective compositions of average organic matter (including that from Precambrian stromatolites). This seems the more enigmatic as the bulk of primary producers from benthic microbial communities are known to fix carbon via the C3 pathway normally entailing the sizable fractionations of the RuBP carboxylase reaction.There is reason to believe that the small fractionations displayed by aquatic microorganisms result from the limitations of a diffusion-controlled assimilatory pathway in which the isotope effect of the enzymatic reaction is largely suppressed. Apart from the diffusion-control exercised by the aqueous environment, transport of CO₂ to the photosynthetically active sites will be further impeded by the protective slime (polysaccharide) coatings commonly covering microbial mats in which gas diffusivities are extremely low. Ineffective discrimination against¹³C becomes, however, most pronounced in hypersaline environments where substantially reduced CO₂ solubilities tend to push carbon into the role of a limiting nutrient (brine habitats constitute preferential sanctuaries of mat-forming microbenthos since the emergence of Metazoan grazers 0.7 Ga ago). As the same microbial communities had been free to colonize normal marine environments during the Precambrian, the CO₂ concentration effect was irrelevant to the carbon-fixing pathway of these ancient forms. Therefore, it might not surprise that organic matter from Precambrian stromatolites displays the large fractionations commonly associated with C3 photosynthesis. Increased mixing ratios of CO₂ in the Precambrian atmosphere may have additionally contributed to the elimination of the diffusion barrier in the carbon-fixing pathways of ancient mat-forming microbiota.     

Microbial community composition and dolomite formation in the hypersaline microbial mats of the Khor Al-Adaid sabkhas,Qatar

Extremophiles - The Khor Al-Adaid sabkha in Qatar is among the rare extreme environments on Earth where it is possible to study the formation of dolomite—a carbonate mineral whose origin...     

Bacterially mediated precipitation in marine stromatolites

Stromatolites are laminated, lithified (CaCO₃) sedimentary deposits formed by precipitation and/or sediment accretion by cyanobacterial–bacterial mat communities. Stromatolites have been associated with these communities as far back as the Precambrian era some 2+ billion years ago. The means by which microbial communities mediate the precipitation processes have remained unclear, and are the subject of considerable debate and speculation. Two alternative explanations for microbially mediated precipitation include: (i) cyanobacterial photosynthesis increases pH in a system supersaturated in respect of CaCO₃, resulting in CaCO₃ precipitation and then laminated lithification, and (ii) decomposition of cyanobacterial extracellular organic matter (e.g. sheaths, mucilage and organic acids) by microheterotrophs leads to release of organic-bound Ca²⁺ ions and CaCO₃ precipitation. We evaluated these explanations by examining metabolically active, lithifying stromatolitic mat communities from Highborne Cay, Bahamas, using microautoradiography. Microautoradiographic detection of ¹⁴CO₂ fixation and ³H organic matter ( d -glucose and an amino acid mixture) utilization by photosynthetically active cyanobacteria and microheterotrophs, combined with community-level uptake experiments, indicate that bacteria, rather than cyanobacteria are the dominant sites of CaCO₃ deposition. In the oligotrophic waters in which stromatolites exist, microheterotrophs are reliant on the photosynthetic community as a main source of organic matter. Therefore, autotrophic production indirectly controls microbially mediated precipitation and stromatolite formation in these shallow marine environments.     

Metabolic shifts in hypersaline microbial mats upon addition of organic substrates

The responses of hypersaline microbial mats to the addition of acetate, glycolate or glucose were investigated using oxygen, pH and sulphide microsensors. Changes in community structure were investigated with molecular techniques. Acetate addition inhibited respiration in the photic zone, stimulated respiration in the aphotic zone and had no effect on gross photosynthesis. Glycolate addition strongly increased both respiration and gross photosynthesis in the photic zone. Thus, glycolate and acetate were probably consumed in those regions of the mat where these substrates are usually formed. Moreover, photosynthesis was only stimulated by increased respiration and concomitant CO2 production in the photic zone which indicates that the photosynthetic and respiratory populations must be present in close proximity to each other. Glucose addition had an unexpected negative effect on the microbial population, strongly inhibiting both respiration and gross photosynthesis within hours. After four days, oxygen profiles in the light were equal to those measured in the dark. After replacing the water phase with unamended water, photosynthesis and respiration recovered within a week. None of the physiological changes were accompanied by detectable shifts in the cyanobacterial or the overall microbial community. The mechanism of inhibition of photosynthesis by glucose requires further investigation.     

Effect of oxygen concentration on photosynthesis and respiration in two hypersaline microbial mats

The effects of oxygen concentration on photosynthesis and respiration in two hypersaline cyanobacterial mats were investigated. Experiments were carried out on mats from Eilat, Israel, with moderate photosynthetic activity, and mats from Mallorca, Spain, with high photosynthetic activity. The oxygen concentration in the overlying water above the mats was increased stepwise from 0% to 100% O2. Subsequent changes in oxygen concentration, gross photosynthetic rates, and pH values inside the mats were measured with microelectrodes. According to published reports on the regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key enzyme in the CO2-fixation pathway of phototrophs, we expected photosynthetic activity to decrease with increasing oxygen concentration. Gross photosynthetic and total respiration rates in both mats were highest when the O2 concentration was at 0% in the overlying water. Net oxygen production rates under these conditions were the same as under air saturation (21% O2), while gross photosynthetic and respiration rates were lowest at air saturation. In both mats, gross photosynthetic and respiration rates increased upon gradually increasing the oxygen concentration in the overlying water from 21% to 100%. These results contradict the expectation that photosynthesis decreases with increasing oxygen concentration. Increased photosynthetic rates at oxygen concentrations above 21% were probably caused by enhanced oxidation of organic matter and concomitant CO2 production due to the increased oxygen availability. The cause of the high respiration rates at 0% O2 in the overlying water was presumably the enhanced excretion of photosynthetic products during increased photosynthesis. We conclude that the effect of the O2/CO2 concentration ratio on the activity of Rubisco as demonstrated in vitro on enzyme extracts cannot be extrapolated to the situation in intact microbial mats, because the close coupling of the activity of primary producers and heterotrophic bacteria plays a major role in this ecosystem.     

Diversity and distribution in hypersaline microbial mats of bacteria related to Chloroflexus spp

Filamentous bacteria containing bacteriochlorophylls c and a were enriched from hypersaline microbial mats. Based on phylogenetic analyses of 16S rRNA gene sequences, these organisms form a previously undescribed lineage distantly related to Chloroflexus spp. We developed and tested a set of PCR primers for the specific amplification of 16S rRNA genes from filamentous phototrophic bacteria within the kingdom of "green nonsulfur bacteria." PCR products recovered from microbial mats in a saltern in Guerrero Negro, Mexico, were subjected to cloning or denaturing gradient gel electrophoresis and then sequenced. We found evidence of a high diversity of bacteria related to Chloroflexus which exhibit different distributions along a gradient of salinity from 5.5 to 16%.     

Chemolithotrophic sulfur bacteria in sediments,mats, and stromatolites of western Australian saline lakes

Samples of stromatolites, microbial mats, and sediments from four saline lakes (approximate seasonal salinity ranges 20–220%o) in Western Australia were used to establish enrichments for elective cultures of aerobic and anaerobic denitrifying chemolithoautotrophs that could grow with thiosulfate as sole energy source. Organisms of these types were obtained from all sources tested. Twenty‐four pure cultures were isolated, all of which were gram‐negative, rod‐shaped bacteria exhibiting a considerable diversity of metabolic capability. Isolation of these obligate and facultative sulfur‐oxidizing chemolithotrophs from the stromatolite and mat habitats indicates the possibility that these rod‐shaped bacteria contribute to the oxidative phase of the sulfur cycle in these habitats, in addition to oxidation by phototrophs or Beggiatoa. Only four of the pure cultures could grow without salt, but all 24 showed significant halophily, some tolerating 3 M NaCl. Three novel isolates of NaCl‐dependent, thiosulfate‐oxidizing, aerobic and denitrifying obligate chemolithotrophs are described. In addition, a facultatively heterotrophic halophilic strain growing either methylotrophically on methylamine or chemolithotrophically on thiosulfate aerobically or with anaerobic denitrification was found.     

Attached vermiform gastropods in Carboniferous marginal marine stromatolites and biostromes

Tubiform fossils conventionally referred to Serpula cf. advena Salter and species of Spirorbis Lamarck from the British Lower Limestone Shales and Border Group (Lower Carboniferous) are re-examined. They occur in peritidal carbonate environments of schizohaline aspect. These fossils superficially resemble calcareous polychaete tubes but have skeletal characters, including molluscan wall structure, numerous internal septa, and protoconch, which indicate that they represent a new group of substrate-attached, disjunctly coiled gastropods. They resemble archaeogastropods in internal morphology of the skeleton but show parallels in external form and occurrence with the extant Vermetidae. There are two principal modes of occurrence: (1) erect tubes forming intertidal biostromes associated with non-skeletal algal laminites, and (2) prostrate discoidal tubes encrusting subtidal skeletal stromatolites or occasionally forming larger irregular bioherms. These biostromes and bioherms are comparable in structure to Recent vermetid reef developments.     

Microbial mats for multiple applications in aquaculture and bioremediation

Microbial mats occur in nature as stratified communities of cyanobacteria and bacteria, but they can be cultured on large-scale and manipulated for a variety of functions. They are complex systems, but require few external inputs. The functional uses of mats broadly cover the areas of aquaculture and bioremediation. Preliminary research also points to promising uses in agriculture and energy production. Regarding aquaculture, mats were shown to produce protein, via nitrogen fixation, and were capable of supplying nutrition to tilapia (Oreochromis niloticus). Current research is examining the role of mats in the nitrification of nutrient-enriched effluents from aquaculture. Most research has addressed bioremediation, within which two majors categories of contaminants were examined: metals and radionuclides, and organic contaminants. Mats sequester or precipitate metals/radionuclides by surface absorption or by conditioning the surrounding chemical environment, thus bioconcentrating the metal/radionuclide in a small volume. Organic contaminants are degraded and may be completely mineralized. For agriculture mats hold promise as a soil amendment and nitrogen fertilizer. The use of mats in biohydrogen production has been verified, but is in a preliminary phase of development. We propose a comprehensive closed system based on microbial mats for aquaculture and waste management.     

Microbial mats and the early evolution of life

Microbial mats have descended from perhaps the oldest and most widespread biological communities known. Mats harbor microbes that are crucial for studies of bacterial phylogeny and physiology. They illustrate how several oxygen-sensitive biochemical processes have adapted to oxygen, and they show how life adapted to dry land long before the rise of plants. The search for the earliest grazing protists and metazoa in stromatolites is aided by observations of mats: in them, organic compounds characteristic of ancient photosynthetic protists can be identified. Recent mat studies suggest that the 13C/12C increase observed over geological time in stromatolitic organic matter was driven at least in part by a long-term decline in atmospheric carbon dioxide levels.     

Microbial diversity and activity in hypersaline high Arctic spring channels

Lost Hammer (LH) spring is a unique hypersaline, subzero, perennial high Arctic spring arising through thick permafrost. In the present study, the microbial and geochemical characteristics of the LH outflow channels, which remain unfrozen at ≥−18°C and are more aerobic/less reducing than the spring source were examined and compared to the previously characterized spring source environment. LH channel sediments contained greater microbial biomass (~100-fold) and greater microbial diversity reflected by the 16S rRNA clone libraries. Phylotypes related to methanogenesis, methanotrophy, sulfur reduction and oxidation were detected in the bacterial clone libraries while the archaeal community was dominated by phylotypes most closely related to THE ammonia-oxidizing Thaumarchaeota. The cumulative percent recovery of ¹⁴C-acetate mineralization in channel sediment microcosms exceeded ~30% and ~10% at 5 and −5°C, respectively, but sharply decreased at −10°C (≤1%). Most bacterial isolates (Marinobacter, Planococcus, and Nesterenkonia spp.) were psychrotrophic, halotolerant, and capable of growth at −5°C. Overall, the hypersaline, subzero LH spring channel has higher microbial diversity and activity than the source, and supports a variety of niches reflecting the more dynamic and heterogeneous channel environment.     

Proterozoic stromatolites: The first marine evolutionary biota

A biometric analysis of the morphology of a late Pliocene planktic diatom lineage, Rhizosolenia praebergonii Mukhina indicates that it appeared abruptly in the fossil record in the Indian Ocean at 2.9 Ma after which it remained virtually unchanged. The observed pattern can be explained by “local”; evolution from the ancestral form Rhizosolenia bergonii, Peragallo or by migration from thecentral Pacific Ocean, where it originated gradually, in conjunction with an accelerated rate of evolution. At present time, it is not possible to conclude which one of the two hypotheses is more likely.     

Microbial eukaryotes in the hypersaline anoxic L'Atalante deep-sea basin

The frontiers of eukaryote life in nature are still unidentified. In this study, we analysed protistan communities in the hypersaline (up to 365 g l⁻¹ NaCl) anoxic L'Atalante deep-sea basin located in the eastern Mediterranean Sea. Targeting 18S ribosomal RNA retrieved from the basin's lower halocline (3501 m depth) we detected 279 protistan sequences that grouped into 42 unique phylotypes (99% sequence similarity). Statistical analyses revealed that these phylotypes account only for a proportion of the protists inhabiting this harsh environment with as much as 50% missed by this survey. Most phylotypes were affiliated with ciliates (45%), dinoflagellates (21%), choanoflagelates (10%) and uncultured marine alveolates (6%). Sequences from other taxonomic groups like stramenopiles, Polycystinea , Acantharea and Euglenozoa , all of which are typically found in non-hypersaline deep-sea systems, are either missing or very rare in our cDNA clone library. Although many DHAB sequences fell within previously identified environmental clades, a large number branched relatively deeply. Phylotype richness, community membership and community structure differ significantly from a deep seawater reference community (3499 m depth). Also, the protistan community in the L'Atalante basin is distinctively different from any previously described hypersaline community. In conclusion, we hypothesize that extreme environments may exert a high selection pressure possibly resulting in the evolution of an exceptional and distinctive assemblage of protists. The deep hypersaline anoxic basins in the Mediterranean Sea provide an ideal platform to test for this hypothesis and are promising targets for the discovery of undescribed protists with unknown physiological capabilities.     

Archaeal populations in hypersaline sediments underlying orange microbial mats in the Napoli mud volcano

Microbial mats in marine cold seeps are known to be associated with ascending sulfide- and methane-rich fluids. Hence, they could be visible indicators of anaerobic oxidation of methane (AOM) and methane cycling processes in underlying sediments. The Napoli mud volcano is situated in the Olimpi Area that lies on saline deposits; from there, brine fluids migrate upward to the seafloor. Sediments associated with a brine pool and microbial orange mats of the Napoli mud volcano were recovered during the Medeco cruise. Based on analysis of RNA-derived sequences, the "active" archaeal community was composed of many uncultured lineages, such as rice cluster V or marine benthic group D. Function methyl coenzyme M reductase (mcrA) genes were affiliated with the anaerobic methanotrophic Archaea (ANME) of the ANME-1, ANME-2a, and ANME-2c groups, suggesting that AOM occurred in these sediment layers. Enrichment cultures showed the presence of viable marine methylotrophic Methanococcoides in shallow sediment layers. Thus, the archaeal community diversity seems to show that active methane cycling took place in the hypersaline microbial mat-associated sediments of the Napoli mud volcano.