Isolation and characterization of Methylophaga sulfidovorans sp. nov.: an obligately methylotrophic, aerobic, dimethylsulfide oxidizing bacterium from a microbial mat

Abstract: Sediment from a microbial mat from the South-West coast of the Netherlands consumed dimethylsulfide (DMS) under oxic and anoxic conditions. From this sediment, a Gram-negative, oval DMS oxidizing bacterium, strain RB-1, was isolated. Its substrate range is typical of an obligately methylotrophic organism. Enzyme analysis revealed the presence of the ribulose monophosphate pathway for carbon assimilation, and the ability to use the linear dissimilatory pathway via formate to carbon dioxide, as well as the cyclic pathway via the ribulose monophosphate route for carbon dissimilation. 16S rRNA sequence analysis showed high similarity with species belonging to the genus Methylophaga . Because of the specific dimethylsulfide and hydrogen sulfide oxidizing capacity, the new isolate was named Methylophaga sulfidovorans .     

Dimethyl sulfide emissions from a sedimental microbial ecosystem subject to diel variations of oxic and anoxic conditions: a simple mathematical model

This modelling study relates dimethylsulfide emission from a microbial mat to the flux of dimethylsulfoniopropionate that is exuded into the interstitial space of the mat by phototrophs. Dimethylsulfoniopropionate may be either cleaved or demethylated. Only cleavage results in the production of dimethylsulfide, which itself is further oxidized or escapes from the mat. The fate of dimethylsulfoniopropionate depends on the functional group composition of the mat, the physiological characteristics of these groups, and the eco-physiological conditions oxic/anoxic and light/dark, which both vary in a diel cycle. These three factors are accounted for in a mathematical model of a microbial mat typical of the Wadden Islands of The Netherlands and Germany. Model simulations quantify increased dimethylsulfide production under alkaline stress as well as additional dimethylsulfoniopropionate loads.     

Analysis of diurnal and vertical microbial diversity of a hypersaline microbial mat

Microbial mats are prokaryotic communities that provide model systems to analyze microbial diversity and ecophysiological interactions. Community diversity of microbial mat samples was assessed at 8:00 a.m. and 3:00 p.m. in a combined analysis consisting of 16S rRNA-denaturing gradient gel electrophoresis (DGGE) and phospholipid fatty acid (PLFA) profiles. The divergence index determined from PLFA and DGGE data showed that depth-related differences have a greater influence on diversity than temporal variations. Shannon and Simpson indices yielded similar values in all samples, which suggested the stable maintenance of a structurally diverse microbial community. The increased diversity observed at 3:00 p.m. between 2.5 and 4 mm can be explained mainly by diversification of anaerobic microorganisms, especially sulfate-reducing bacteria. In the afternoon sampling, the diversity index reflected a higher diversity between 4 and 5.5 mm depth, which suggested an increase in the diversity of strict anaerobes and fermenters. The results are consistent with the conclusion that hypersaline microbial mats are characterized by high degree of diversity that shifts in response to the photobiological adaptations and metabolic status of the microbial community. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Dedicated to the memory of David C. White.     

Sulfide fluxes in a microbial mat from the Ebro Delta, Spain

The sulfur cycle of Ebro Delta microbial mats was studied in order to determine sulfide production and sulfide consumption. Vertical distribution of two major functional groups involved in the sulfur cycle, anoxygenic phototrophic bacteria and dissimilatory sulfate-reducing bacteria (SRB), was also studied. The former reached up to 2.2×10⁸ cfu cm^–3 sediment in the purple layer, and the latter reached about 1.8×10⁵ SRB cm^–3 sediment in the black layer. From the changes in sulfide concentrations under light-dark cycles it can be inferred that the rate of H₂S production was 6.2 μmol H₂S cm^–3 day^–1 at 2.6 mm, and 7.6 μmol H₂S cm^–3 day^–1 at 6 mm. Furthermore, sulfide consumption was also assessed, determining rates of 0.04, 0.13 and 0.005 mmol l^–1 of sulfide oxidized at depths of 2.6, 3 and 6 mm, respectively. Electronic Publication     

Competition between anoxygenic phototrophic bacteria and colorless sulfur bacteria in a microbial mat

Abstract The populations of chemolithoautotrophic (colorless) sulfur bacteria and anoxygenic phototrophic bacteria were enumerated in a marine microbial mat. The highest population densities were found in the 0–5 mm layer of the mat: 2.0 × 10⁹ cells cm⁻³ sediment, and 4.0 × 10⁷ cells cm⁻³ sediment for the colorless sulfur bacteria and phototrophs, respectively. Kinetic parameters for thiosulfate-limited growth were assessed for Thiobacillus thioparus T5 and Thiocapsa roseopersicina M1, both isolated from microbial mats. For Thiobacillus T5, growing at a constant oxygen concentration of 43 μmol l⁻¹, μ_max was 0.336 h⁻¹ and K _s 0.8 μmol l⁻¹. Phototrophically grown Thiocapsa strain M1 displayed a μ_max of 0.080 h⁻¹ and a K _s of 8 μmol l⁻¹ when anoxically grown under thiosulfate limitation. In a competition experiment with thiosulfate as electron donor, Thiocapsa became dominant during a 10-h oxic/14-h anoxic regimen at continuous illumination, despite the higher affinity for thiosulfate of Thiobacillus .     

Structure and development of a benthic marine microbial mat

Abstract Vertically stratified microbial communities of phototrophic bacteria in the upper intertidal zones of the North Sea island of Mellum were investigated. Growth and population dynamics of the cyanobacterial mat were followed over three successive years. It was concluded that the initial colonization of the sandy sediments was by the cyanobacterium Oscillatoria . In well-established mats, however, the dominant organism was Microcoleus chthonoplastes . The observed succession of cyanobacteria during mat development is correlated with nitrogen fixation. Nitrogen fixation is necessary in this low-nutrient environment to ensure colonization by mat-constructing cyanobacteria. Under certain conditions, a red layer of purple sulfur bacteria developed underneath the cyanobacterial mat in which Chromatium and Thiocapsa spp. dominated, but Thiopedia and Ectothiorhodospira spp. have also been observed. Measurements of light penetrating the cyanobacterial mat indicated that sufficient light is available for the photosynthetic growth of purple sulfur bacteria. Profiles of oxygen, sulfide and redox potential within the microbial mat were measured using microelectrodes. Maximum oxygen concentrations, measured at a depth of 0.7 mm, reached levels more than twice the normal air saturation. Dissolved sulfide was not detected by the microelectrodes. Determination of acid-distilled sulfide, however, revealed appreciable amounts of bound sulfide in the mat. Redox profiles measured in the mat led to the conclusion that the upper 10 mm of the sedimentary sequence is in a relatively oxidized state.     

Sulfide oxidation under oxygen limitation by a thiobacillus thioparus isolated from a marine microbial mat

Abstract The colorless sulfur bacterium Thiobacillus thioparus T5, isolated from a marine microbial mat, was grown in continuous culture under conditions ranging from sulfide limitation to oxygen limitation. Under sulfide-limiting conditions, sulfide was virtually completely oxidized to sulfate. Under oxygen-limiting conditions, sulfide was partially oxidized to zerovalent sulfur (75%) and thiosulfate (17%). In addition, low concentrations of tetrathionate and polysulfide were detected. The finding of in vivo thiosulfate formation supports the discredited observations of thiosulfate formation in cell free extracts in the early sixties. In a microbial mat most sulfide oxidation was shown to take place under oxygen-limiting conditions. It is suggested that zerovalent sulfur formation by thiobacilli is a major process resulting in polysulfide accumulation. Implications for the competition between colorless sulfur bacteria and purple sulfur bacteria are discussed.     

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.     

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.     

Spatial and temporal variability in a stratified hypersaline microbial mat community

Hypersaline microbial mat communities have recently been shown to be more diverse than once thought. The variability in community composition of hypersaline mats, both in terms of spatial and temporal dimensions, is still poorly understood. Because this information is essential to understanding the complex biotic and abiotic interactions within these communities, terminal restriction fragment analysis and 16S rRNA gene sequencing were used to characterize the near-surface community of a hypersaline microbial mat in Guerrero Negro, Mexico. Core samples were analyzed to assay community variability over large regional scales (centimeter to kilometer) and to track depth-related changes in population distribution at 250-μm intervals over a diel period. Significant changes in total species diversity were observed at increasing distances across the mat surface; however, key species (e.g. Microcoleus sp.) were identified throughout the mat. The vertical position and abundance of >50% of the 60 peaks detected varied dramatically over a diel cycle, including Beggiatoa sp., cyanobacteria, Chloroflexus sp., Halochromatium sp., Bacteroidetes sp. and several as-yet-identified bacteria. Many of these migrations correlated strongly with diel changes in redox conditions within the mat, contributing to strong day–night community structure differences.     

Effects of salinity and light on organic carbon and nitrogen uptake in a hypersaline microbial mat

Utilization of dissolved organic matter (DOM) is thought to be the purview of heterotrophic microorganisms, but photoautotrophs can take up dissolved organic nitrogen (DON) and dissolved organic carbon (DOC). This study investigated DOC and DON uptake in a laminated cyanobacterial mat community from hypersaline Salt Pond (San Salvador, Bahamas). The total community uptake of (3)H-labeled substrates was measured in the light and in the dark and under conditions of high and low salinity. Salinity was the primary control of DOM uptake, with increased uptake occurring under low-salinity, 'freshened' conditions. DOC uptake was also enhanced in the light as compared with the dark and in samples incubated with the photosystem II inhibitor 3(3,4-dichlorophenyl)-1, 1-dimethylurea, suggesting a positive association between photosynthetic activity and DOC uptake. Microautoradiography revealed that some DOM uptake was attributed to cyanobacteria. Cyanobacteria DOM uptake was negatively correlated with that of smaller filamentous microorganisms, and DOM uptake by individual coccoid cells was negatively correlated with uptake by colonial coccoids. These patterns of activity suggest that Salt Pond microorganisms are engaged in resource partitioning, and DOM utilization may provide a metabolic boost to both heterotrophs and photoautrophs during periods of lowered salinity.     

Characteristics and turnover of exopolymeric substances in a hypersaline microbial mat

The properties and microbial turnover of exopolymeric substances (EPS) were measured in a hypersaline nonlithifying microbial mat (Eleuthera, Bahamas) to investigate their potential role in calcium carbonate (CaCO₃) precipitation. Depth profiles of EPS abundance and enzyme activities indicated that c . 80% of the EPS were turned over in the upper 15–20 mm. Oxic and anoxic mat homogenates amended with low-molecular-weight (LMW) organic carbon, sugar monomers, and different types of EPS revealed rapid consumption of all substrates. When comparing the consumption of EPS with that of other substrates, only marginally longer lag times and lower rates were observed. EPS (5–8%) were readily consumed during the conversion of labile to refractory EPS. This coincided with a decrease in glucosidase activity and a decrease in the number of acidic functional groups on the EPS. Approximately half of the calcium bound to the EPS remained after 10 dialyses steps. This tightly bound calcium was readily available to precipitate as CaCO₃. We present a conceptual model in which LMW organic carbon complexed with the tightly bound calcium is released upon enzyme activity. This increases alkalinity and creates binding sites for carbonate and allows CaCO₃ to precipitate. Therefore, this model explains interactions between EPS and CaCO₃ precipitation, and underscores the critical role of aerobic and anaerobic microorganisms in early diagenesis and lithification processes.     

Two-dimensional mapping of photopigment distribution and activity of Chloroflexus-like bacteria in a hypersaline microbial mat

Pigment analysis in an intact hypersaline microbial mat by hyperspectral imaging revealed very patchy and spatially uncorrelated distributions of photopigments Chl a and BChl a/c, which are characteristic photopigments for oxygenic (diatoms and cyanobacteria) and anoxygenic phototrophs (Chloroflexaceae). This finding is in contrast to the expectation that these biomarker pigments should be spatially correlated, as oxygenic phototrophs are thought to supply the Chloroflexaceae members with organic substrates for growth. We suggest that the heterogeneous occurrence is possibly due to sulfide, whose production by sulfate-reducing bacteria may be spatially heterogeneous in the partially oxic photic zone of the mat. We furthermore mapped the near-infra-red-light controlled respiration of Chloroflexaceae under light and dark conditions and found that Chloroflexaceae are responsible for a major part of oxygen consumption at the lower part of the oxic zone in the mat. The presence of Chloroflexaceae was further confirmed by FISH probe and 16S rRNA gene clone library analysis. We assume that species related to the genera Oscillochloris and 'Candidatus Chlorothrix', in contrast to those related to Chloroflexus and Roseiflexus, depend less on excreted photosynthates but more on the presence of free sulfide, which may explain their presence in deeper parts of the mat.     

Growth of mixed cultures on mixtures of substitutable substrates: the operating diagram for a structured model

The growth of mixed microbial cultures on mixtures of substrates is a problem of fundamental biological interest. In the last two decades, several unstructured models of mixed-substrate growth have been studied. It is well known, however, that the growth patterns in mixed-substrate environments are dictated by the enzymes that catalyse the transport of substrates into the cell. We have shown previously that a model taking due account of transport enzymes captures and explains all the observed patterns of growth of a single species on two substitutable substrates (J. Theor. Biol. 190 (1998) 241). Here, we extend the model to study the steady states of growth of two species on two substitutable substrates. The model is analysed to determine the conditions for existence and stability of the various steady states. Simulations are performed to determine the flow rates and feed concentrations at which both species coexist. We show that if the interaction between the two species is purely competitive, then at any given flow rate, coexistence is possible only if the ratio of the two feed concentrations lies within a certain interval; excessive supply of either one of the two substrates leads to annihilation of one of the species. This result simplifies the construction of the operating diagram for purely competing species. This is because the two-dimensional surface that bounds the flow rates and feed concentrations at which both species coexist has a particularly simple geometry: It is completely determined by only two coordinates, the flow rate and the ratio of the two feed concentrations. We also study commensalistic interactions between the two species by assuming that one of the species excretes a product that can support the growth of the other species. We show that such interactions enhance the coexistence region.     

Effect of flooding-drawdown cycle on vegetation in a system of floating peat mat and pond

The relationship between vegetation and seasonal changes in water level was examined in a system of terrestrializing floating peat mat and pond in a warm temperate zone. The duration of flooding, or drawdown period, is related to vegetation in the mat. The first process of terrestrialization would be the attenuation of floating-sinking movement of the mat and the consequent stabilization of water level. Water level at the time when the movement of the mat was attenuated determined the subsequent vegetation change at the site. Two series of succession according to terrestrialization were recognized, and started from initial conditions of low and high water levels respectively. Habitats in low initial water levels experienced short flooding and long drawdown periods. Species changes in the successional series were:Menyanthes trifoliata-Rhynchospora fauriei-Eriocaulon sikokianum-Sphagnum cuspidatum. Submerged peat was experimentally emerged, and the crowded community ofRhynchospora fauriei andEriocaulon sikokianum established within one or two years. Appearance of these species was controlled by the water level of the habitats in winter. Another series of species changes in terrestrialization process was:Menyanthes trifoliata-Phragmites australis-Carex thunbergii orIsachne globosa. Habitats of these types of vegetation were in the area with relatively high initial water level (long flooding and short drawdown) when the floating-sinking movement of the mat attenuated. After the temporal paludification, the water level was lowered by the accumulation of peat in the process of terrestrialization.     

Vertical migration of phototrophic bacterial populations in a hypersaline microbial mat from Salins-de-Giraud (Camargue, France)

The spatio-temporal distribution of phototrophic communities of the hypersaline photosynthetic Camarguc microbial mat (Salins-de-Giraud, France) was investigated over a diel cycle by combining microscopic and molecular approaches. Microcoleus chthonoplastes and Halomicronema excentricum, the dominant cyanobacteria of this oxyphotrophic community, were observed with confocal laser scanning microscopy to determine their biomass profiles. Both bacteria have similar vertical distributions, varying from a homogenous distribution through the mat during the night, to a specific localization in the upper oxic zone of 1.5 mm during the day. Terminal restriction fragment length polymorphism of PCR-amplified pufM gene fragments revealed three groups of anoxyphototrophic populations, which varied according to the two opposite periods of the diel cycle under study. They were either specifically detected in only one period, or homogenously distributed through the mat in all periods, or located in specific zones of the mat depending on the period considered. Oxygen concentrations, pH and biomass of the major filamentous cyanobacteria were the determinative factors in the distribution of these anoxyphototrophs across the mat. Thus, vertical migration, cell-cell aggregate formation and metabolic switches were the most evident defence of the photosynthetic populations against the adverse effects of sulfide and oxygen fluxes during a diel cycle.     

Effects of microbial mats on germination and seedling survival of typical dune slack species in the Netherlands

The role of microbial mats in wet dune slack succession is often discussed. We tested if presence of microbial mats may retard dune slack succession by lowering the germination and seeding survival of successor species. This hypothesis was tested on a set of typical dune slack species of the Frisian Islands in two climate chamber experiments. The species were separated into early-, intermediate- and late successional species. There were large differences in germination rates between species (2% – >200% compared to the reference), but within a species the high germination rates were mostly found on sand without a microbial mat. Only the germination of Agrostis stolonifera appeared to be stimulated by the presence of a well-developed microbial mat, they were even higher than in the reference. Seedling survival also did not show different responses between successional stages. Seedlings placed on top of a microbial mat showed for most species lower survival rates compared to seedlings that were planted or placed on top of the sand. Growth was the only measured variable that differed between successional groups. Species of the early- and intermediate successional stages grew significantly better if a microbial mat was present whereas late successional species were not stimulated. Early and intermediate successional species seem to be favored by the presence of a microbial mat. An explanation for this may be that they can profit from the enhanced nitrogen availability caused by N₂-fixation by cyanobacteria in the microbial mat. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of a deep-sea microbial mat from an active cold seep at the Milano mud volcano in the Eastern Mediterranean Sea

A white, filamentous microbial mat at the Milano mud volcano in the Eastern Mediterranean Sea was sampled during the Medinaut cruise of the R/V Nadir in 1998. The composition of the mat community was characterized using a combination of phylogenetic and lipid biomarker methods. The mat sample was filtered through 0.2 and 5-microm filters to coarsely separate unicellular and filamentous bacteria. Analyses of 16S rRNA gene sequences amplified from the total community DNA from these fractions showed that similar archaeal populations were present in both fractions. However, the bacterial populations in the fractions differed from one another, and were more diverse than the archaeal ones. Lipid analysis showed that bacteria were the dominant members of the mat microbial community and the relatively low delta(13)C carbon isotope values of bulk bacterial lipids suggested the occurrence of methane- and sulfide-based chemo(auto)trophy. Consistent with this, the bacterial populations in the fractions were related to Alpha-, Gamma- and Epsilonproteobacteria, most of which were chemoautotrophic bacteria that utilize hydrogen sulfide (or reduced sulfur compounds) and/or methane. The most common archaeal 16S rRNA gene sequences were related to those of previously identified Archaea capable of anaerobic methane oxidation. Although the filamentous organisms observed in the mat were not conclusively identified, our results indicated that the Eastern Mediterranean deep-sea microbial mat community might be sustained on a combination of methane- and sulfide-driven chemotrophy.