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.     

Structure of planktonic microbial food web in a brackish stratified Siberian lake

The distribution of primary components of the microbial community (autotrophic pico- and nanoplankton, phototrophic bacteria, heterotrophic bacteria, microscopic fungi, heterotrophic flagellates, ciliates and heliozoa) in the water column of Lake Shira, a steppe brackish-water, stratified lake in Khakasia, Siberia (Russia), were assessed in midsummer. Bacterioplankton was the main component of the planktonic microbial community, accounting for 65.3 to 75.7% of the total microbial biomass. The maximum concentration of heterotrophic bacteria were recorded in the monimolimnion of the lake. Autotrophic microorganisms contributed more significantly to the total microbial biomass in the pelagic zone (20.2–26.5%) than in the littoral zone of the lake (8.7–14.9%). First of all, it is caused by development of phototrophic sulphur bacteria at the oxic-anoxic boundary. The concentrations of most aerobic phototrophic and heterotrophic microorganisms were maximal in the upper mixolimnion. Heterotrophic flagellates dominated the protozoan populations. Ciliates were minor component of the planktonic microbial community of the lake. Heterotrophic flagellates were the most diverse group of planktonic eucaryotes in the lake, which represented by 36 species. Facultative and obligate anaerobic flagellates were revealed in the monimolimnion. There were four species of Heliozoa and only three of ciliates in the lake.     

High resolution measurements of sediment resuspension above an accumulation bottom in a stratified lake

A detailed record of suspended particulate matter (SPM) concentrations in the benthic boundary layer (BBL) 1.5 m above an accumulation bottom and 13.5 m below the surface was obtained from frequent (30 min interval) beam attenuation measurements made with a Sea Tech transmissometer in the main basin of Lake Erken, a moderately deep (mean depth 9 m, maximum depth 21 m) dimictic lake in central Sweden. Concentrations of SPM (g m^–3) were not as strongly correlated to the beam attenuation coefficient (c, [m^–1]), as were concentrations of the inorganic SPM fraction. Apparently, this was caused by large optically inactive organic particles which significantly affected the measurements of SPM, but had little effect on the attenuation of light.When the water column was thermally stratified, SPM concentrations in the BBL showed a seasonal increase which was related to an increase in the thermocline depth. As the epilimnion deepened, there was also a marked increase in the occurrence of rapid and large changes in SPM concentration. After the loss of stratification, the amount of SPM and the temporal variability in its concentration was reduced. Since surface waves could not influence sediment resuspension at the depth of measurement, these data show the importance of internal waves in promoting sediment resuspension in areas of sediment accumulation. The relatively short period in each summer, when the thermocline reaches a sufficient depth to allow for resuspension over accumulation bottoms, can have important consequences for both the redistribution of lake sediments and the internal loading of phosphorus.     

Distribution and diversity of archaea corresponding to the limnological cycle of a hypersaline stratified lake (Solar lake, Sinai, Egypt)

The vertical and seasonal distribution and diversity of archaeal sequences was investigated in a hypersaline, stratified, monomictic lake, Solar Lake, Sinai, Egypt, during the limnological development of stratification and mixing. Archaeal sequences were studied via phylogenetic analysis of 16S rDNA sequences as well as denaturing gradient gel electrophoresis analysis. The 165 clones studied were grouped into four phylogenetically different clusters. Most of the clones isolated from both the aerobic epilimnion and the sulfide-rich hypolimnion were defined as cluster I, belonging to the Halobacteriaceae family. The three additional clusters were all isolated from the anaerobic hypolimnion. Cluster II is phylogenetically located between the genera Methanobacterium and Methanococcus. Clusters III and IV relate to two previously documented groups of uncultured euryarchaeota, remotely related to the genus Thermoplasma. No crenarchaeota were found in the water column of the Solar Lake. The archaeal community in the Solar Lake under both stratified and mixed conditions was dominated by halobacteria in salinities higher than 10%. During stratification, additional clusters, some of which may possibly relate to uncultured halophilic methanogens, were found in the sulfide- and methane-rich hypolimnion.     

Glycine betaine is the main organic osmotic solute in a stratified microbial community in a hypersaline evaporitic gypsum crust

We have examined the organic osmotic solutes content within the stratified microbial communities in an evaporitic gypsum crust found in an evaporation pond (~194 g/l total dissolved salts) of the salterns of the Israel Salt Company, Eilat. We extracted intracellular solutes from the upper three pigmented layers of the crust: a yellow-orange layer dominated by unicellular cyanobacteria, a green layer with filamentous cyanobacteria, and a layer colored red-purple by purple sulfur bacteria; dense communities of heterotrophic bacteria were present in all layers. The solutes were analyzed by Raman spectroscopy, ¹H and ¹³C nuclear magnetic resonance, and HPLC. All layers contained glycine betaine as the only detectable osmotic solute; ectoine and other solutes known to be produced by many halophilic and halotolerant prokaryotes were not found. In this first attempt to assess the osmotic solute content within complex natural communities of halophilic microorganisms, the predominant role of glycine betaine as an osmolyte was established. Most heterotrophic bacteria cannot produce glycine betaine but preferentially use it when it is supplied. Presence of glycine betaine produced by the photoautotrophic members of the community, therefore, may relieve the heterotrophs from the need to synthesize other compounds at a high-energy cost.     

Community composition of a hypersaline endoevaporitic microbial mat

A hypersaline, endoevaporitic microbial community in Eilat, Israel, was studied by microscopy and by PCR amplification of genes for 16S rRNA from different layers. In terms of biomass, the oxygenic layers of the community were dominated by Cyanobacteria of the Halothece, Spirulina, and Phormidium types, but cell counts (based on 4',6'-diamidino-2-phenylindole staining) and molecular surveys (clone libraries of PCR-amplified genes for 16S rRNA) showed that oxygenic phototrophs were outnumbered by the other constituents of the community, including chemotrophs and anoxygenic phototrophs. Bacterial clone libraries were dominated by phylotypes affiliated with the Bacteroidetes group and both photo- and chemotrophic groups of alpha-proteobacteria. Green filaments related to the Chloroflexi were less abundant than reported from hypersaline microbial mats growing at lower salinities and were only detected in the deepest part of the anoxygenic phototrophic zone. Also detected were nonphototrophic gamma- and delta-proteobacteria, Planctomycetes, the TM6 group, Firmicutes, and Spirochetes. Several of the phylotypes showed a distinct vertical distribution in the crust, suggesting specific adaptations to the presence or absence of oxygen and light. Archaea were less abundant than Bacteria, their diversity was lower, and the community was less stratified. Detected archaeal groups included organisms affiliated with the Methanosarcinales, the Halobacteriales, and uncultured groups of Euryarchaeota.     

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.     

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.     

Intensities of microbial production and oxidation of methane in bottom sediments and water mass of the Black Sea

Intensities of biogeochemical (microbial) processes of methane production and methane oxidation were determined in bottom sediments and water column of the Black Sea. Aerobic bacterial oxidation of methane is confined to the upper 20-30 cm of Holocene bottom sediments of the shelf (0.7-259 ng C/(dm3 day)) and oxygenated waters (0.2-45 ng C/(dm3 day)). In reduced sediments of the deep-sea zone and in the hydrogen sulfide-containing water column, considerable intensities of anaerobic methane oxidation were recorded, comparable to or exceeding the intensities of methane oxidation in oxygenated layers. From one fourth to one half of the methane formed in bottom sediments was oxidized immediately therein. The major part of the remaining methane was oxidized in the water column, and a smaller portion arrived in the atmosphere.     

Dynamics of microbial oxidation of methane in the water of stratified lakes

Fluctuations of methane (CH₄) concentration and the dynamics of microbial methane oxidation (MO) were investigated in the water column of freshwater stratified lakes of different trophicity levels during various seasonal periods and throughout the diurnal cycle. Characteristics of vertical CH₄ distribution and ranges of methane transformation rates were determined and found to depend upon the lake productivity as well as seasonal and daily fluctuations of hydrological and hydrochemical parameters. The highest rate of MO was registered in highly eutrophic lakes during summer stagnation under conditions of formation of a distinct metalimnial water layer with MO up to 0.4–1.2 ml CH₄/(l day). Under the same conditions, a maximum amount of bacterioplankton (6–13 × 10⁶ cells/ml) was detected and CO₂ bacterial dark assimilation (DA) reached 50–72 μg C/(l day). In the metalimnion layer, a strong correlation (R = 0.74) was revealed between diurnal fluctuation dynamics of MO and DA.     

Contribution of Chloroflexus respiration to oxygen cycling in a hypersaline microbial mat from Lake Chiprana, Spain

In dense stratified systems such as microbial mats, photosynthesis and respiration are coupled due to a tight spatial overlap between oxygen-producing and -consuming microorganisms. We combined microsensors and a membrane inlet mass spectrometer with two independent light sources emitting in the visible (VIS) and near infrared (NIR) regions to study this coupling in more detail. Using this novel approach, we separately quantified the activity of the major players in the oxygen cycle in a hypersaline microbial mat: gross photosynthesis of cyanobacteria, NIR light-dependent respiration of Chloroflexus-like bacteria (CLB) and respiration of aerobic heterotrophs. Illumination by VIS light induced oxygen production in the top approximately 1 mm of the mat. In this zone CLB were found responsible for all respiration, while the contribution of the aerobic heterotrophs was negligible. Additional illumination of the mat with saturating NIR light completely switched off CLB respiration, resulting in zero respiration in the photosynthetically active zone. We demonstrate that microsensor-based quantification of gross and net photosyntheses in dense stratified systems should carefully consider the NIR light-dependent behaviour of CLB and other anoxygenic phototrophic groups.     

Sedimentary anaerobic microbial biogeochemistry in the Gulf of Trieste, northern Adriatic Sea: Influences of bottom water oxygen depletion

Sediment samples from two locationsin the Gulf of Trieste (northern Adriatic Sea) werecollected during periods of maximum and minimumtemperatures for two years. Both sites were rich incarbonate material and inhabited by a diverse benthicinfaunal community. However, Site F exhibited adeeper dwelling faunal community, higher content ofcarbonate minerals, and larger grained sediments thanat site MA, which was closer to shore. Depth profilesof sulfate reduction and potential rates of iron andmanganese reduction were determined together withmeasurements of pore water and solid phase chemistry. Bottom waters at all sites were nearly saturated withoxygen for all of the dates sampled except forSeptember 1993 when bottom waters at site F were lessthan 50% saturated. Sulfate reduction rates were ashigh as 400 nmol ml^-1 day^-1 during latesummer and fall when temperatures were >20 °C,while rates during March (8 °C) were <30 nmolml^-1 day^-1. Potential rates of ironreduction, as determined by the accumulation of bothdissolved and acid-soluble reduced iron, were high insurficial sediments at each site except at site F whenbottom waters were partially depleted in oxygen. In the latter instance, sulfate reduction overwhelmedmetal reduction. Although the portion of metalreduction due directly to enzymatic use by bacteriawas not determined, the potential rate data suggestedthat Fe and perhaps Mn reduction were significantcomponents of anaerobic carbon degradation in thesesediments during much of the year. Both sitesappeared to support active metal-reducing bacterialcommunities. However, occasional depletion of oxygenin bottom waters appeared to cause a decrease inirrigation/reworking activity by infauna whichdepressed redox cycling of elements enhancing theimportance of sulfate reduction. A shift from metalreduction to sulfate reduction potentially exacerbatestoxic effects of oxygen depletion on fauna byincreasing the accumulation of toxic sulfide.     

Limnological studies on the Pretoria Salt Pan,a hypersaline maar lake

The Pretoria Salt Pan is shallow (maximum depth 2.85 m) and alkaline (surface water pH varied from 9.6 to 10.9) with pronounced mesothermy (38.2 °C in spring) at a depth of between 0.55 and 0.7 metres. Secchi disc transparencies ranged from 7 to 19 cm. A total ionic concentration gradient increasing from 59 500 mg · ?^?1 near the surface to 298 000 mg · ?^?1 at 2.75 metres stabilized the thermally inverted water column. Sodium was the major cation and nearly equal proportions of chloride plus carbonate and bicarbonate accounted for over 98% of the anions. The lake was meromictic with a steep chemocline, persistent thermally inverted temperature profile and complete anoxia at depths greater than 50 cm. The diel pattern of dissolved oxygen distribution involving a nocturnal deoxygenation and diurnal reoxygenation was unusual.     

Benthic nutrient fluxes in a eutrophic,polymictic lake

Sediment release rates of soluble reactive phosphorus (SRP) and ammonium (NH₄) were determined seasonally at three sites (water depth 7, 14 and 20 m) in Lake Rotorua using in situ benthic chamber incubations. Rates of release of SRP ranged from 2.2 to 85.6 mg P m⁻² d⁻¹ and were largely independent of dissolved oxygen (DO) concentration. Two phases of NH₄ release were observed in the chamber incubations; high initial rates of up to 2,200 mg N m⁻² d⁻¹ in the first 12 h of deployment followed by lower rates of up to 270 mg N m⁻² d⁻¹ in the remaining 36 h of deployment. Releases of SRP and NH₄ were highest in summer and at the deepest of the three sites. High organic matter supply rates to the sediments may be important for sustaining high rates of sediment nutrient release. A nutrient budget of Lake Rotorua indicates that internal nutrient sources derived from benthic fluxes are more important than external nutrient sources to the lake.     

DNA-based analysis of planktonic methanotrophs in a stratified lake

1. The assemblage of planktonic methanotrophs in a stratified freshwater lake was investigated. Vertical patterns were analysed by denaturing gradient gel electrophoresis, using the primer pair specific for 16S rRNA genes of type I methanotrophs.
2. The resulting banding patterns could be divided into three distinct groups, and sequenced bands were all related to the Methylobacter species. No amplicon was obtained with the primer pair specific for type II methanotrophs.
3. Cloning analysis of the pmoA gene was performed using samples from three water depths (epilimnion, metalimnion and hypolimnion). The compositions of the clone libraries from the three depths were distinct from each other but all three libraries were dominated by clones related to Methylobacter species.     

Prokaryotic diversity in a Tunisian hypersaline lake,Chott El Jerid

Prokaryotic diversity was investigated in a Tunisian salt lake, Chott El Jerid, by quantitative real-time PCR, denaturing gradient gel electrophoresis (DGGE) fingerprinting methods targeting the 16S rRNA gene and culture-dependent methods. Two different samples S1-10 and S2-10 were taken from under the salt crust of Chott El Jerid in the dry season. DGGE analysis revealed that bacterial sequences were related to Firmicutes, Proteobacteria, unclassified bacteria, and Deinococcus-Thermus phyla. Anaerobic fermentative and sulfate-reducing bacteria were also detected in this ecosystem. Within the domain archaea, all sequences were affiliated to Euryarchaeota phylum. Quantitative real-time PCR showed that 16S rRNA gene copy numbers of bacteria was 5 × 10⁶ DNA copies g^?1 whereas archaea varied between 5 × 10⁵ and 10⁶ DNA copies g^?1 in these samples. Eight anaerobic halophilic fermentative bacterial strains were isolated and affiliated with the species Halanaerobium alcaliphilum, Halanaerobium saccharolyticum, and Sporohalobacter salinus. These data showed an abundant and diverse microbial community detected in the hypersaline thalassohaline environment of Chott El Jerid.     

Limitation of oxygenic photosynthesis and oxygen consumption by phosphate and organic nitrogen in a hypersaline microbial mat: a microsensor study

Microbial mats are characterized by high primary production but low growth rates, pointing to a limitation of growth by the lack of nutrients or substrates. We identified compounds that instantaneously stimulated photosynthesis rates and oxygen consumption rates in a hypersaline microbial mat by following the short-term response (c. 6 h) of these processes to addition of nutrients, organic and inorganic carbon compounds, using microsensors. Net photosynthesis rates were not stimulated by compound additions. However, both gross photosynthesis and oxygen consumption were substantially stimulated (by a minimum of 25%) by alanine (1 mM) and glutamate (3.5 mM) as well as by phosphate (0.1 mM). A low concentration of ammonium (0.1 mM) did not affect photosynthesis and oxygen consumption, whereas a higher concentration (3.5 mM) decreased both process rates. High concentrations of glycolate (5 mM) and phosphate (1 mM) inhibited gross photosynthesis but not oxygen consumption, leading to a decrease of net photosynthesis. Photosynthesis was not stimulated by addition of inorganic carbon, nor was oxygen consumption stimulated by organic compounds like glycolate (5 mM) or glucose (5 mM), indicating that carbon was efficiently cycled within the mat. Photosynthesis and oxygen consumption were apparently tightly coupled, because stimulations always affected both processes to the same extent, which resulted in unchanged net photosynthesis rates. These findings illustrate that microsensor techniques, due to their ability to quantify all three processes, can clarify community responses to nutrient enrichment studies much better than techniques that solely monitor net fluxes.     

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.