Methanogenesis in a saltern in the Bretagne (France)

Abstract Salterns in the Bretagne (France), exhibiting different stages of salinity of 5% to 33% evolved up to 0.7 mmol methane per m² per day. Methane concentrations of up to 0.4 mM were found. High methane evolution rates and increased methane concetration were restricted to basins of up to 7% and more than 15% salinity, and to the upper 10 cm of the sediments, where high sulfate concentrations (50 to 100 mM) occurred as well. Basins of 10–15% salinity exhibited only low methane evolution rates (less than 0.05 mmol methane per m² per day) and low methane concentrations (less than 0.02 mM). Gas bubbles arising during times of increased photosynthetic activity from the microbial mats covering the sediments of the saltern basins contained up to 2% methane. Addition of methylated amines and methanol, but not of H₂/CO₂, formate or acetate, to sediments slurries from basins of up to 12% salinity resulted in a rapid enrichment of methanogenic populations. Enriched methanogenic bacteria did not grow at salinities exceeding 15% or temperatures exceeding 45°C, and showed characteristics similar to those documented for Methanococcus halophilus and strain SF1 (DSM 3243). No enrichments were obtained from basins of more than 20% salinity in spite of methane being produced and evolved from those basins.     

Spatial distribution of diatom and cyanobacterial mats in the Dead Sea is determined by response to rapid salinity fluctuations

Cyanobacteria and diatom mats are ubiquitous in hypersaline environments but have never been observed in the Dead Sea, one of the most hypersaline lakes on Earth. Here we report the discovery of phototrophic microbial mats at underwater freshwater seeps in the Dead Sea. These mats are either dominated by diatoms or unicellular cyanobacteria and are spatially separated. Using in situ and ex situ O₂ microsensor measurements we show that these organisms are photosynthetically active in their natural habitat. The diatoms, which are phylogenetically associated to the Navicula genus, grew in culture at salinities up to 40 % Dead Sea water (DSW) (14 % total dissolved salts, TDS). The unicellular cyanobacteria belong to the extremely halotolerant Euhalothece genus and grew at salinities up to 70 % DSW (24.5 % TDS). As suggested by a variable O₂ penetration depth measured in situ, the organisms are exposed to drastic salinity fluctuations ranging from brackish to DSW salinity within minutes to hours. We could demonstrate that both phototrophs are able to withstand such extreme short-term fluctuations. Nevertheless, while the diatoms recover better from rapid fluctuations, the cyanobacteria cope better with long-term exposure to DSW. We conclude that the main reason for the development of these microbial mats is a local dilution of the hypersaline Dead Sea to levels allowing growth. Their spatial distribution in the seeping areas is a result of different recovery rates from short or long-term fluctuation in salinity.     

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%.     

Structure of the phytoplankton communities in two lagoons of different salinity in the Sfax saltern (Tunisia)

The structure and ecophysiological characteristics of phytoplanktonassemblages were studied for 10 months in two lagoons of differentsalinity (40 and 90) in the saltern of Sfax (Tunisia), in relationto environmental factors. These assemblages were largely dominatedby diatoms and dinoflagellates, which accounted for >90%of total abundance. A principal components analysis clearlydifferentiated the observations made in the two lagoons, thedominant correlate being the salinity. Euryhaline species andparticularly diatoms developed preferentially in the less salinelagoon, and were largely replaced by stenohaline species representedby dinoflagellates, which were dominant in the more saline lagoon.Calculation of the pigment diversity index and the species diversityindex showed that the phytoplankton assemblages studied werepermanently in a juvenile stage, as the species restructuringrelated to environmental constraints did not allow them to reachthe climax stage at any given time in their development. Despitethe heavy constraint imposed by the salinity, it is evidentthat other environmental factors, e.g. temperature, play a rolein the regulation of the planktonic communities. Finally, thedifference in the size distribution of the total microbial biomass,estimated by the assay of particulate proteins, showed thatthere was a significant change in the community structure andthe planktonic trophic networks, in parallel with the increasein salinity.     

Matching molecular diversity and ecophysiology of benthic cyanobacteria and diatoms in communities along a salinity gradient

The phylogenetic diversity of oxygenic phototrophic microorganisms in hypersaline microbial mats and their distribution along a salinity gradient were investigated and compared with the halotolerances of closely related cultivated strains. Segments of 16S rRNA genes from cyanobacteria and diatom plastids were retrieved from mat samples by DNA extraction and polymerase chain reaction (PCR), and subsequently analysed by denaturing gradient gel electrophoresis (DGGE). Sequence analyses of DNA from individual DGGE bands suggested that the majority of these organisms was related to cultivated strains at levels that had previously been demonstrated to correlate with characteristic salinity responses. Proportional abundances of amplified 16S rRNA gene segments from phylogenetic groupings of cyanobacteria and diatoms were estimated by image analysis of DGGE gels and were generally found to correspond to abundances of the respective morphotypes determined by microscopic analyses. The results indicated that diatoms accounted for low proportions of cells throughout, that the cyanobacterium Microcoleus chthonoplastes and close relatives dominated the communities up to a salinity of 11% and that, at a salinity of 14%, the most abundant cyanobacteria were related to highly halotolerant cultivated cyanobacteria, such as the recently established phylogenetic clusters of Euhalothece and Halospirulina . Although these organisms in cultures had previously demonstrated their ability to grow with close to optimal rates over a wide range of salinities, their occurrence in the field was restricted to the highest salinities investigated.     

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%.     

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.     

Salinity control of the distribution of diatoms in lakes of the Bunger Hills, East Antarctica

The biogeography of Antarctic lacustrine diatoms is incompletely known due in part to the absence of information from some important ice-free regions. In the first detailed study of the diatoms of the Bunger Hills, 29 species were identified in near-edge microbial mats of lakes ranging in salinity from fresh to 77‰. Most of the species present had previously been recorded in other East Antarctic coastal oases. The distribution and relative abundance of diatoms within the Bunger Hills were strongly influenced by salinity, but other factors, notably the characteristics of lakes in the immediate area of a particular lake, also played important roles.     

Variation of environmental features and microbial populations with salt concentrations in a multi-pond saltern

A multi-pond saltern that creates a gradient of salt concentrations has been studied with respect to some characteristics of the resulting environments and their microbial populations. The increase in salt concentration was correlated with increase in diurnal temperature and biomass present and with decrease in oxygen concentrations. Many types of organisms below 15% (w/v) total salts, were found, many of them normal inhabitants of seawater and even freshwater. Most organisms over 15% salts were halophilic. The salt concentrations comprised two ranges, each characterized by different microbial populations. First, between 15 and 30% salts, the populations ofDunaliella increased, reaching large numbers; moderately halophilic eubacteria and some fast-growing halobacteria predominated as heterotrophic microorganisms and, among the first, thePseudomonas-Alteromonas-Alcaligenes group andVibrio were the more abundant taxonomic groups; and gram-positive cocci appeared mainly over 25% salts. Phototrophic bacteria, both oxygenic and anoxygenic, were also found in this range, and among the anoxygenic type,Chromatium species andRodospirillum salexigens were probably predominant. Second, over 30% salts the diversity decreased greatly, all organisms found at the lower salt concentrations disappeared, and instead large populations of halobacteria developed. Over 50% salts, only three species of halobacteria were found.     

Virioplankton community structure along a salinity gradient in a solar saltern

The virioplankton community structure along a salinity gradient from near seawater (40 per thousand ) to saturated sodium chloride brine (370 per thousand ) in a solar saltern was investigated by pulsed-field gel electrophoresis. Viral populations with genome sizes varying from 10 kb to 533 kb were detected. The viral community structure changed along the salinity gradient. Cluster analysis of the viral genome-banding pattern resulted in two main clusters. The virioplankton diversity within the samples with salinity from 40 per thousand to 150 per thousand was on the same cluster of a cladogram. The other group consisted of virioplankton from samples with salinity above 220 per thousand. The virioplankton diversity in the different samples was calculated using the Shannon index. The diversity index demonstrated an increase in diversity in the samples along the gradient from 40 per thousand to 150 per thousand salinity, followed by a decrease in the diversity index along the rest of the salinity gradient. These results demonstrate how viral diversity changes from habitats that are considered one of the most common (seawater) to habitats that are extreme in salt concentrations (saturated sodium brine). The diversity index was highest in the environments that lie in between the most extreme and the most common.     

Prokaryotic diversity of a Tunisian multipond solar saltern

16S rRNA gene clone libraries were separately constructed from three ponds with different salt concentrations, M2 (15%), TS38 (25%) and S5 (32%), located within a multipond solar saltern of Sfax. The 16S rRNA genes from 216 bacterial clones and 156 archaeal clones were sequenced and phylogenetically analyzed. 44 operational taxonomic units (OTUs) were generated for Bacteria and 67 for Archaea. Phylogenetic groups within the bacterial domain were restricted to Bacteroidetes and Proteobacteria, with the exception that one cyanobacterial OTU was found in the TS38 pond. 85.7, 26.6 and 25.0% of the bacterial OTUs from M2, TS38 and S5 ponds, respectively, are novel. All archaeal 16S rRNA gene sequences were exclusively affiliated with Euryarchaeota. 75.0, 60.0 and 66.7% of the OTUs from, respectively, M2, TS38 and S5 ponds are novel. The result showed that the Tunisian multipond solar saltern harbored novel prokaryotic diversity that has never been reported before for solar salterns. In addition, diversity measurement indicated a decrease of bacterial diversity and an increase of archaeal diversity with rising salinity gradient, which was in agreement with the previous observation for thalassohaline systems. Comparative analysis showed that prokaryotic diversity of Tunisian saltern was higher than that of other salterns previously studied. A. Sghir and E. Ammar have equally contributed to this work.     

Prokaryotic genetic diversity throughout the salinity gradient of a coastal solar saltern

Bacterial and archaeal assemblages have been studied in a multipond solar saltern using a range of microbial ecology techniques by four laboratories simultaneously. These include 16S rDNA sequencing from both denaturing gradient gel electrophoresis (DGGE) and clone libraries, and culturing methods. Water samples from eight ponds were analysed, covering a salinity range from near sea water (4% salt) to saturated sodium chloride (37% salt; ponds called crystallizers). Clone libraries focused on ponds with salinity of 8%, 22% and 32%. Although different cloning strategies were able to retrieve the same type of dominant sequences, there were differing degrees of success with less abundant sequences. Thus, the use of two sets of primers recovered a higher number of phylotypes. Bacterial and archaeal isolates were, however, different from any of the retrieved environmental sequences. For Bacteria, most sequences in the 8% salt pond were related to organisms of marine origin. Thus, representatives of the alpha-, beta-, gamma- and epsilon-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides group (CFB), high-G+C Gram-positive bacteria and cyanobacteria were found. In the 22% salt pond, alpha- and gamma-Proteobacteria, cyanobacteria and CFB were the only groups found, and most of them were related to specialized halophilic bacteria. From the 32% salt pond, only CFB were found, and most of the sequences retrieved clustered with Salinibacter ruber, an extremely halophilic bacterium. A decrease in the richness of bacterial genera was therefore apparent along the gradient. Archaea behaved quite similarly. In the lowest salinity ponds, sequences were related to environmental clones of Marine Archaea Group III (Thermoplasmales relatives) and to unclassified branches of Euryarchaeaota. In the 8%, 22% and 32% ponds, most of the clones were related to different cultured strains of Halobacteriaceae. Finally, most sequences from the crystallizers clustered with the uncultured square archaeon SPhT. Crenarchaeaota were not detected. Despite the fact that higher prokaryotic richness was apparent in the lower salinity ponds than in the crystallizers, the diversity index from clone libraries calculated according to Shannon and Weaver did not show this trend. This was because diversity in the crystallizers can be considered as 'microdiversity', the co-existence of several closely related clones of Bacteria (the S. ruber cluster) and Archaea (the SPhT cluster). Regardless of the changes in abundance, both Bacteria and Archaea showed the same pattern; as salinity increased, the number of different clusters decreased, and only one cluster became dominant. Both clusters, however, showed a considerable degree of microdiversity. The meaning of such microdiversity remains to be determined.     

Organic geochemical studies of modern microbial mats from Shark Bay: Part I: Influence of depth and salinity on lipid biomarkers and their isotopic signatures

The present study investigated the influence of abiotic conditions on microbial mat communities from Shark Bay, a World Heritage area well known for a diverse range of extant mats presenting structural similarities with ancient stromatolites. The distributions and stable carbon isotopic values of lipid biomarkers [aliphatic hydrocarbons and polar lipid fatty acids (PLFAs)] and bulk carbon and nitrogen isotope values of biomass were analysed in four different types of mats along a tidal flat gradient to characterize the microbial communities and systematically investigate the relationship of the above parameters with water depth. Cyanobacteria were dominant in all mats, as demonstrated by the presence of diagnostic hydrocarbons (e.g. n‐C₁₇ and n‐C_17:1). Several subtle but important differences in lipid composition across the littoral gradient were, however, evident. For instance, the shallower mats contained a higher diatom contribution, concordant with previous mat studies from other locations (e.g. Antarctica). Conversely, the organic matter (OM) of the deeper mats showed evidence for a higher seagrass contribution [high C/N, ¹³C‐depleted long‐chain n‐alkanes]. The morphological structure of the mats may have influenced CO₂ diffusion leading to more ¹³C‐enriched lipids in the shallow mats. Alternatively, changes in CO₂ fixation pathways, such as increase in the acetyl COA‐pathway by sulphate‐reducing bacteria, could have also caused the observed shifts in δ¹³C values of the mats. In addition, three smooth mats from different Shark Bay sites were analysed to investigate potential functional relationship of the microbial communities with differing salinity levels. The C_25:1 HBI was identified in the high salinity mat only and a lower abundance of PLFAs associated with diatoms was observed in the less saline mats, suggesting a higher abundance of diatoms at the most saline site. Furthermore, it appeared that the most and least saline mats were dominated by autotrophic biomass using different CO₂ fixation pathways.     

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.     

The Santa Pola saltern as a model for studying the microbiota of hypersaline environments

Multi-pond salterns constitute an excellent model for the study of the microbial diversity and ecology of hypersaline environments, showing a wide range of salt concentrations, from seawater to salt saturation. Accumulated studies on the Santa Pola (Alicante, Spain) multi-pond solar saltern during the last 35 years include culture-dependent and culture-independent molecular methods and metagenomics more recently. These approaches have permitted to determine in depth the microbial diversity of the ponds with intermediate salinities (from 10 % salts) up to salt saturation, with haloarchaea and bacteria as the two main dominant groups. In this review, we describe the main results obtained using the different methodologies, the most relevant contributions for understanding the ecology of these extreme environments and the future perspectives for such studies.     

Changes in archaeal,bacterial and eukaryal assemblages along a salinity gradient by comparison of genetic fingerprinting methods in a multipond solar saltern

Microbial communities inhabiting a multipond solar saltern were analysed and compared using SSU rRNA polymerase chain reaction (PCR)-based fingerprintings carried out in parallel by four laboratories. A salinity gradient from seawater (3.7%) to NaCl precipitation (37%) was studied for Bacteria, Archaea and Eukarya, and laboratories applied their own techniques and protocols on the same set of samples. Members of all three domains were retrieved from all salt concentrations. Three fingerprinting techniques were used: denaturing gradient gel electrophoresis (DGGE), ribosomal internal spacer analysis (RISA), and terminal-restriction fragments length polymorphism (T-RFLP). In addition, each laboratory used its own biomass collection method and DNA extraction protocols. Prokaryotes were addressed using DGGE and RISA with different 'domain-specific' primers sets. Eukaryotes were analysed by one laboratory using DGGE and T-RFLP, but targeting the same 18S rDNA site. Fingerprints were compared through cluster analysis and non-metric multidimensional scaling plots. This exercise allowed fast comparison of microbial assemblages and determined to what extent the picture provided by each laboratory was similar to those of others. Formation of two main, salinity-based groups of samples in prokaryotes (4-15% and 22-37% salinity) was consistent for all the laboratories. When other clusters appeared, this was a result of the particular technique and the protocol used in each case, but more affected by the primers set used. Eukaryotic microorganisms changed more from pond to pond; 4-5% and 8-37% salinity were but the two main groups detected. Archaea showed the lowest number of bands whereas Eukarya showed the highest number of operational taxonomic units (OTUs) in the initial ponds. Artefacts appeared in the DGGE from ponds with extremely low microbial richness. On the other hand, different 16S rDNA fragments with the same restriction or internal transcribed spacer (ITS) length were the main limitations for T-RFLP and RISA analyses, respectively, in ponds with the highest OTUs richness. However, although the particular taxonomic composition could vary among protocols, the general structure of the microbial assemblages was maintained.     

Biogeochemistry of a gypsum-encrusted microbial ecosystem

Gypsum crusts containing multicolored stratified microbial populations grow in the evaporation ponds of a commercial saltern in Eilat, Israel. These crusts contain two prominent cyanobacterial layers, a bright purple layer of anoxygenic phototrophs, and a lower black layer with active sulphate reduction. We explored the diel dynamics of oxygen and sulphide within the crust using specially constructed microelectrodes, and further explored the crust biogeochemistry by measuring rates of sulphate reduction, stable sulphur isotope composition, and oxygen exchange rates across the crust–brine interface. We explored crusts from ponds with two different salinities, and found that the crust in the highest salinity was the less active. Overall, these crusts exhibited much lower rates of oxygen production than typical organic‐rich microbial mats. However, this was mainly due to much lower cell densities within the crusts. Surprisingly, on a per cell‐volume basis, rates of photosynthesis were similar to organic‐rich microbial mats. Due to relatively low rates of oxygen production and deep photic zones extending from 1.5 to 3 cm depth, a large percentage of the oxygen produced during the day accumulated into the crusts. Indeed, only between 16% to 34% of the O₂ produced in the crust escaped, and the remainder was internally recycled, used mainly in O₂ respiration. We view these crusts as potential homologs to ancient salt‐encrusted microbial ecosystems, and we compared them to the 3.45 billion‐year‐old quartz barite deposits from North Pole, Australia, which originally precipitated gypsum.     

The algal microflora of the salt works of Tarquinia (Italy)

The microflora and benthic microbial mats of the salt works of Tarquinia (Italy) are described. From 144 samples, 111 species and varieties of microalgae were identified. Diatoms prevailed up to 110. S. They were substituted by Cyanophyta at higher salinities. The microbial mats consisted of Cyanophyta, Beggiatoa spp., purple sulphur bacteria, and diatoms. Information on hypertonic resistance in some euryhaline species is given.     

Halocafeteria seosinensis gen. et sp. nov. (Bicosoecida), a halophilic bacterivorous nanoflagellate isolated from a solar saltern

Recently, heterotrophic nanoflagellates (HNF) have been reported to actively ingest prokaryotes in high salinity waters. We report the isolation and culture of an HNF from a Korean saltern pond of 300‰ salinity. The organism is biflagellated with an acronematic anterior flagellum and never glides on surfaces. The mitochondria have tubular cristae. Neither transitional helix nor spiral fiber were observed in the transition zones of the flagella. The cell has a cytostome supported by an arc of eight microtubules, suggesting that our isolate is a bicosoecid. Our isolate had neither mastigonemes, lorica, body scales, nor cytopharynx and thus could not be placed in any of the presently described bicosoecid genera. Phylogenetic analysis of 18S rRNA gene sequences from stramenopiles confirmed the bicosoecid affinities of our isolate, but did not place it within any established genus or family. Its closest relatives include Caecitellus and Cafeteria. The optimal range of growth temperature was 30–35°C. The isolated HNF grew optimally at 150‰ salinity and tolerated up to 363‰ salinity, but it failed to grow below 75‰ salinity, indicating that it could be a borderline extreme halophile. On the basis of its morphological features and position in 18S rRNA trees we propose a novel genus for our isolate; Halocafeteria, n. gen. The species name Halocafeteria seosinensis sp. nov. is proposed.