Microscopic Examination of Distribution and Phenotypic Properties of Phylogenetically Diverse Chloroflexaceae-Related Bacteria in Hot Spring Microbial Mats†

We investigated the diversity, distribution, and phenotypes of uncultivated Chloroflexaceae-related bacteria in photosynthetic microbial mats of an alkaline hot spring (Mushroom Spring, Yellowstone National Park). By applying a directed PCR approach, molecular cloning, and sequence analysis of 16S rRNA genes, an unexpectedly large phylogenetic diversity among these bacteria was detected. Oligonucleotide probes were designed to target 16S rRNAs from organisms affiliated with the genus Chloroflexus or with the type C cluster, a group of previously discovered Chloroflexaceae relatives of this mat community. The application of peroxidase-labeled probes in conjunction with tyramide signal amplification enabled the identification of these organisms within the microbial mats by fluorescence in situ hybridization (FISH) and the investigation of their morphology, abundance, and small-scale distribution. FISH was combined with oxygen microelectrode measurements, microscope spectrometry, and microautoradiography to examine their microenvironment, pigmentation, and carbon source usage. Abundant type C-related, filamentous bacteria were found to flourish within the cyanobacterium-dominated, highly oxygenated top layers and to predominate numerically in deeper orange-colored zones of the investigated microbial mats, correlating with the distribution of bacteriochlorophyll a. Chloroflexus sp. filaments were rare at 60°C but were more abundant at 70°C, where they were confined to the upper millimeter of the mat. Both type C organisms and Chloroflexus spp. were observed to assimilate radiolabeled acetate under in situ conditions.     

Filamentous "Epsilonproteobacteria" dominate microbial mats from sulfidic cave springs

Hydrogen sulfide-rich groundwater discharges from springs into Lower Kane Cave, Wyoming, where microbial mats dominated by filamentous morphotypes are found. The full-cycle rRNA approach, including 16S rRNA gene retrieval and fluorescence in situ hybridization (FISH), was used to identify these filaments. The majority of the obtained 16S rRNA gene clones from the mats were affiliated with the "Epsilonproteobacteria" and formed two distinct clusters, designated LKC group I and LKC group II, within this class. Group I was closely related to uncultured environmental clones from petroleum-contaminated groundwater, sulfidic springs, and sulfidic caves (97 to 99% sequence similarity), while group II formed a novel clade moderately related to deep-sea hydrothermal vent symbionts (90 to 94% sequence similarity). FISH with newly designed probes for both groups specifically stained filamentous bacteria within the mats. FISH-based quantification of the two filament groups in six different microbial mat samples from Lower Kane Cave showed that LKC group II dominated five of the six mat communities. This study further expands our perceptions of the diversity and geographic distribution of "Epsilonproteobacteria" in extreme environments and demonstrates their biogeochemical importance in subterranean ecosystems.     

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%.     

Influence of sulfide and temperature on species composition and community structure of hot spring microbial mats

In solfataric fields in southwestern Iceland, neutral and sulfide-rich hot springs are characterized by thick bacterial mats at 60 to 80 degrees C that are white or yellow from precipitated sulfur (sulfur mats). In low-sulfide hot springs in the same area, grey or pink streamers are formed at 80 to 90 degrees C, and a Chloroflexus mat is formed at 65 to 70 degrees C. We have studied the microbial diversity of one sulfur mat (high-sulfide) hot spring and one Chloroflexus mat (low-sulfide) hot spring by cloning and sequencing of small-subunit rRNA genes obtained by PCR amplification from mat DNA. Using 98% sequence identity as a cutoff value, a total of 14 bacterial operational taxonomic units (OTUs) and 5 archaeal OTUs were detected in the sulfur mat; 18 bacterial OTUs were detected in the Chloroflexus mat. Although representatives of novel divisions were found, the majority of the sequences were >95% related to currently known sequences. The molecular diversity analysis showed that Chloroflexus was the dominant mat organism in the low-sulfide spring (1 mg liter(-1)) below 70 degrees C, whereas Aquificales were dominant in the high-sulfide spring (12 mg liter(-1)) at the same temperature. Comparison of the present data to published data indicated that there is a relationship between mat type and composition of Aquificales on the one hand and temperature and sulfide concentration on the other hand.     

Distribution of cultivated and uncultivated cyanobacteria and Chloroflexus-like bacteria in hot spring microbial mats.

Oligodeoxynucleotide hybridization probes were developed to complement specific regions of the small subunit (SSU) rRNA sequences of cultivated and uncultivated cyanobacteria and Chloroflexus-like bacteria, which inhabit hot spring microbial mats. The probes were used to investigate the natural distribution of SSU rRNAs from these species in mats of Yellowstone hot springs of different temperatures and pHs as well as changes in SSU rRNA distribution resulting from 1-week in situ shifts in temperature, pH, and light intensity. Synechococcus lividus Y-7c-s SSU rRNA was detected only in the mat of a slightly acid spring, from which it may have been initially isolated, or when samples from a more alkaline spring were incubated in the more acid spring. Chloroflexus aurantiacus Y-400-fl SSU rRNA was detected only in a high-temperature mat sample from the alkaline Octopus Spring or when lower-temperature samples from this mat were incubated at the high-temperature site. SSU rRNAs of uncultivated species were more widely distributed. Temperature distributions and responses to in situ temperature shifts suggested that some of the uncultivated cyanobacteria might be adapted to high-, moderate-, and low-temperature ranges whereas an uncultivated Chloroflexus-like bacterium appears to have broad temperature tolerance. SSU rRNAs of all uncultivated species inhabiting a 48 to 51 degrees C Octopus Spring mat site were most abundant in the upper 1 mm and were not detected below a 2.5-to 3.5-mm depth, a finding consistent with their possible phototrophic nature. However, the effects of light intensity reduction on these SSU rRNAs were variable, indicating the difficulty of demonstrating a phototrophic phenotype in light reduction experiments.     

Distribution of cultivated and uncultivated cyanobacteria and Chloroflexus-like bacteria in hot spring microbial mats

Oligodeoxynucleotide hybridization probes were developed to complement specific regions of the small subunit (SSU) rRNA sequences of cultivated and uncultivated cyanobacteria and Chloroflexus-like bacteria, which inhabit hot spring microbial mats. The probes were used to investigate the natural distribution of SSU rRNAs from these species in mats of Yellowstone hot springs of different temperatures and pHs as well as changes in SSU rRNA distribution resulting from 1-week in situ shifts in temperature, pH, and light intensity. Synechococcus lividus Y-7c-s SSU rRNA was detected only in the mat of a slightly acid spring, from which it may have been initially isolated, or when samples from a more alkaline spring were incubated in the more acid spring. Chloroflexus aurantiacus Y-400-fl SSU rRNA was detected only in a high-temperature mat sample from the alkaline Octopus Spring or when lower-temperature samples from this mat were incubated at the high-temperature site. SSU rRNAs of uncultivated species were more widely distributed. Temperature distributions and responses to in situ temperature shifts suggested that some of the uncultivated cyanobacteria might be adapted to high-, moderate-, and low-temperature ranges whereas an uncultivated Chloroflexus-like bacterium appears to have broad temperature tolerance. SSU rRNAs of all uncultivated species inhabiting a 48 to 51 degrees C Octopus Spring mat site were most abundant in the upper 1 mm and were not detected below a 2.5-to 3.5-mm depth, a finding consistent with their possible phototrophic nature. However, the effects of light intensity reduction on these SSU rRNAs were variable, indicating the difficulty of demonstrating a phototrophic phenotype in light reduction experiments.     

Diversity of phototrophic bacteria in microbial mats from Arctic hot springs (Greenland)

We investigated the genotypic diversity of oxygenic and anoxygenic phototrophic microorganisms in microbial mat samples collected from three hot spring localities on the east coast of Greenland. These hot springs harbour unique Arctic microbial ecosystems that have never been studied in detail before. Specific oligonucleotide primers for cyanobacteria, purple sulfur bacteria, green sulfur bacteria and Choroflexus/Roseiflexus-like green non-sulfur bacteria were used for the selective amplification of 16S rRNA gene fragments. Amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced. In addition, several cyanobacteria were isolated from the mat samples, and classified morphologically and by 16S rRNA-based methods. The cyanobacterial 16S rRNA sequences obtained from DGGE represented a diverse, polyphyletic collection of cyanobacteria. The microbial mat communities were dominated by heterocystous and non-heterocystous filamentous cyanobacteria. Our results indicate that the cyanobacterial community composition in the samples were different for each sampling site. Different layers of the same heterogeneous mat often contained distinct and different communities of cyanobacteria. We observed a relationship between the cyanobacterial community composition and the in situ temperatures of different mat parts. The Greenland mats exhibited a low diversity of anoxygenic phototrophs as compared with other hot spring mats which is possibly related to the photochemical conditions within the mats resulting from the Arctic light regime.     

The structure and biogeochemical activity of the phototrophic communities from the Bol'sherechenskii alkaline hot spring

Microbial communities growing in the bed of the alkaline, sulfide hot spring Bol'sherechenskii (the Baikal rift area) were studied over many years (1986-2001). The effluent water temperature ranged from 72 to 74 degrees C, pH was from 9.25 to 9.8, and sulfide content was from 12 to 13.4 mg/ml. Simultaneous effects of several extreme factors restrict the spread of phototrophic microorganisms. Visible microbial fouling appears with a decrease in the temperature to 62 degrees C and in the sulfide content to 5.9 mg/l. Cyanobacteria predominated in all biological zones of the microbial mat. The filamentous cyanobacteria of the genus Phormidium are the major mat-forming organisms, whereas unicellular cyanobacteria and the filamentous green bacterium Chloroflexus aurantiacus are minor components of the phototrophic communities. No cyanobacteria of the species Mastigocladus laminosus, typical of neutral and subacid springs, were identified. Seventeen species of both anoxygenic phototrophic bacteria and cyanobacteria were isolated from the microbial mats, most of which exhibited optimum growth at 20 to 45 degrees C. The anoxygenic phototrophs were neutrophiles with pH optimum at about 7. The cyanobacteria were the most adapted to the alkaline conditions in the spring. Their optimum growth was observed at pH 8.5-9.0. As determined by the in situ radioisotope method, the optimal growth and decomposition rates were observed at 40-32 degrees C, which is 10 to 15 degrees C lower than the same parameter in the sulfide-deficient Octopus Spring (Yellowstone, United States). The maximum chlorophyll a concentration was 555 mg/m2 at 40 degrees C. Total rate of photosynthesis in the mats reached 1.3 g C/m2 per day. The maximum rate of dark fixation of carbon dioxide in the microbial mats was 0.806 g C/m2 per day. The maximum rate of sulfate reduction comprised 0.367 g S/m2 per day at 40 degrees C. The rate of methanogenesis did not exceed 1.188 micrograms C/m2 per day. The role of methanogenesis in the terminal decomposition of the organic matter was insignificant. Methane formation consumed 100 times less organic matter than sulfate reduction.     

Denaturing gradient gel electrophoresis used to monitor the enrichment culture of aerobic chemoorganotrophic bacteria from a hot spring cyanobacterial mat.

Previous studies investigating microbial diversity in the Octopus Spring cyanobacterial mat community (Yellowstone National Park) have shown a discrepancy between bacterial populations observed by molecular retrieval and cultivation techniques. To investigate how selective enrichment culture techniques affect species composition, we used denaturing gradient gel electrophoresis (DGGE) separation of PCR-amplified 16S rRNA gene fragments to monitor the populations contained within enrichment cultures of aerobic chemoorganotrophic bacteria from the ca. 50 degrees C region of the mat community. By varying the degree of dilution of the inoculum, medium composition, and enrichment conditions and duration and by analyzing the cultures by DGGE, we detected 14 unique 16S rRNA sequence types. These corresponded to alpha-, beta-, gamma-, and delta-proteobacteria, Thermus relatives, and gram-positive bacteria with high G + C ratio and, at the highest inoculum dilutions, Chloroflexus aurantiacus relatives, which were estimated to still be approximately 300 times less abundant than cells of the mat primary producer, Synechococcus spp. Only three of these populations were previously cultivated on solidified medium after similar enrichment. Only two of these population have 16S rRNA sequences which were previously cloned directly from the mat. These results reveal a diversity of bacterial populations in enrichment culture which were not detected by either molecular retrieval or strain purification techniques.     

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.     

Phylogenetic diversity of bacteria associated with the marine sponge Rhopaloeides odorabile

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the beta- and gamma-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.     

Biogeochemistry and community composition of iron- and sulfur-precipitating microbial mats at the Chefren mud volcano (Nile Deep Sea Fan, Eastern Mediterranean)

In this study we determined the composition and biogeochemistry of novel, brightly colored, white and orange microbial mats at the surface of a brine seep at the outer rim of the Chefren mud volcano. These mats were interspersed with one another, but their underlying sediment biogeochemistries differed considerably. Microscopy revealed that the white mats were granules composed of elemental S filaments, similar to those produced by the sulfide-oxidizing epsilonproteobacterium "Candidatus Arcobacter sulfidicus." Fluorescence in situ hybridization indicated that microorganisms targeted by a "Ca. Arcobacter sulfidicus"-specific oligonucleotide probe constituted up to 24% of the total the cells within these mats. Several 16S rRNA gene sequences from organisms closely related to "Ca. Arcobacter sulfidicus" were identified. In contrast, the orange mat consisted mostly of bright orange flakes composed of empty Fe(III) (hydr)oxide-coated microbial sheaths, similar to those produced by the neutrophilic Fe(II)-oxidizing betaproteobacterium Leptothrix ochracea. None of the 16S rRNA gene sequences obtained from these samples were closely related to sequences of known neutrophilic aerobic Fe(II)-oxidizing bacteria. The sediments below both types of mats showed relatively high sulfate reduction rates (300 nmol x cm(-3) x day(-1)) partially fueled by the anaerobic oxidation of methane (10 to 20 nmol x cm(-3) x day(-1)). Free sulfide produced below the white mat was depleted by sulfide oxidation within the mat itself. Below the orange mat free Fe(II) reached the surface layer and was depleted in part by microbial Fe(II) oxidation. Both mats and the sediments underneath them hosted very diverse microbial communities and contained mineral precipitates, most likely due to differences in fluid flow patterns.     

Novel uncultured Epsilonproteobacteria dominate a filamentous sulphur mat from the 13 degrees N hydrothermal vent field, East Pacific Rise

Rapid growth of microbial sulphur mats have repeatedly been observed during oceanographic cruises to various deep-sea hydrothermal vent sites. The microorganisms involved in the mat formation have not been phylogenetically characterized, although the production of morphologically similar sulphur filaments by a Arcobacter strain coastal marine has been documented. An in situ collector deployed for 5 days at the 13 degrees N deep-sea hydrothermal vent site on the East Pacific Rise (EPR) was rapidly colonized by a filamentous microbial mat. Microscopic and chemical analyses revealed that the mat consisted of a network of microorganisms embedded in a mucous sulphur-rich matrix. Molecular surveys based on 16S rRNA gene and aclB genes placed all the environmental clone sequences within the Epsilonproteobacteria. Although few 16S rRNA gene sequences were affiliated with that of cultured organisms, the majority was related to uncultured representatives of the Arcobacter group (< or = 95% sequence similarity). A probe designed to target all of the identified lineages hybridized with more than 95% of the mat community. Simultaneous hybridizations with the latter probe and a probe specific to Arcobacter spp. confirmed the numerical dominance of Arcobacter-like bacteria. This study provides the first example of the prevalence and ecological significance of free-living Arcobacter at deep-sea hydrothermal vents.     

Diversity and function of Chloroflexus-like bacteria in a hypersaline microbial mat: phylogenetic characterization and impact on aerobic respiration

We studied the diversity of Chloroflexus-like bacteria (CLB) in a hypersaline phototrophic microbial mat and assayed their near-infrared (NIR) light-dependent oxygen respiration rates. PCR with primers that were reported to specifically target the 16S rRNA gene from members of the phylum Chloroflexi resulted in the recovery of 49 sequences and 16 phylotypes (sequences of the same phylotype share more than 96% similarity), and 10 of the sequences (four phylotypes) appeared to be related to filamentous anoxygenic phototrophic members of the family Chloroflexaceae. Photopigment analysis revealed the presence of bacteriochlorophyll c (BChlc), BChld, and gamma-carotene, pigments known to be produced by phototrophic CLB. Oxygen microsensor measurements for intact mats revealed a NIR (710 to 770 nm) light-dependent decrease in aerobic respiration, a phenomenon that we also observed in an axenic culture of Chloroflexus aurantiacus. The metabolic ability of phototrophic CLB to switch from anoxygenic photosynthesis under NIR illumination to aerobic respiration under non-NIR illumination was further used to estimate the contribution of these organisms to mat community respiration. Steady-state oxygen profiles under dark conditions and in the presence of visible (VIS) light (400 to 700 nm), NIR light (710 to 770 nm), and VIS light plus NIR light were compared. NIR light illumination led to a substantial increase in the oxygen concentration in the mat. The observed impact on oxygen dynamics shows that CLB play a significant role in the cycling of carbon in this hypersaline microbial mat ecosystem. This study further demonstrates that the method applied, a combination of microsensor techniques and VIS and NIR illumination, allows rapid establishment of the presence and significance of CLB in environmental samples.     

Biodiversity and monitoring of colorless filamentous bacteria in sulfide aquatic systems of North Caucasus region

Bacterial mats in sulfide aquatic systems of North Caucasus are basically composed by the species of genera Thiothrix and Sphaerotilus. Additionally, several non-filamentous sulfur-oxidizing bacteria were isolated from the mats and several minor 16S rRNA phylotypes were found in clone libraries from these mats. The minor components were affiliated with Proteobacteria, Chlorobia, Cyanobacteria and Firmicutes. Even in an individual mat population heterogeneity of Thiothrix spp. was revealed by analysis of 16S rRNA gene and RAPD-PCR. Five Thiothrix isolates were described as new species Thiothrix caldifontis sp. nov. and Thiothrix lacustris sp. nov. In the Thiothrix-Sphaerotilus type of bacterial mat the proportion of dominant organisms might be influenced by sulfide concentration in the spring water. The higher sulfide concentration (more than 10 mg/l) in the spring water is more favorable for the development of bacterial mats with dominant Thiothrix organisms than for Thiothrix-Sphaerotilus type of sulfur mat.     

Metabolically active eukaryotic communities in extremely acidic mine drainage

Acid mine drainage (AMD) microbial communities contain microbial eukaryotes (both fungi and protists) that confer a biofilm structure and impact the abundance of bacteria and archaea and the community composition via grazing and other mechanisms. Since prokaryotes impact iron oxidation rates and thus regulate AMD generation rates, it is important to analyze the fungal and protistan populations. We utilized 18S rRNA and beta-tubulin gene phylogenies and fluorescent rRNA-specific probes to characterize the eukaryotic diversity and distribution in extremely acidic (pHs 0.8 to 1.38), warm (30 to 50 degrees C), metal-rich (up to 269 mM Fe(2+), 16.8 mM Zn, 8.5 mM As, and 4.1 mM Cu) AMD solutions from the Richmond Mine at Iron Mountain, Calif. A Rhodophyta (red algae) lineage and organisms from the Vahlkampfiidae family were identified. The fungal 18S rRNA and tubulin gene sequences formed two distinct phylogenetic groups associated with the classes Dothideomycetes and Eurotiomycetes. Three fungal isolates that were closely related to the Dothideomycetes clones were obtained. We suggest the name "Acidomyces richmondensis" for these isolates. Since these ascomycete fungi were morphologically indistinguishable, rRNA-specific oligonucleotide probes were designed to target the Dothideomycetes and Eurotiomycetes via fluorescent in situ hybridization (FISH). FISH analyses indicated that Eurotiomycetes are generally more abundant than Dothideomycetes in all of the seven locations studied within the Richmond Mine system. This is the first study to combine the culture-independent detection of fungi with in situ detection and a demonstration of activity in an acidic environment. The results expand our understanding of the subsurface AMD microbial community structure.     

Phylogenetic diversity and activity of aerobic heterotrophic bacteria from a hypersaline oil-polluted microbial mat

The diversity and function of aerobic heterotrophic bacteria (AHB) in cyanobacterial mats have been largely overlooked. We used culture-dependent and molecular techniques to explore the species diversity, degradative capacities and functional guilds of AHB in the photic layer (2mm) of an oil-polluted microbial mat from Saudi Arabia. Enrichment isolation was carried out at different salinities (5% and 12%) and temperatures (28 and 45 degrees C) and on various substrates (acetate, glycolate, Spirulina extract and crude oils). Counts of most probable number showed a numerical abundance of AHB in the range of 1.15-8.13x10(6) cellsg(-1) and suggested the presence of halotolerant and thermotolerant populations. Most of the 16S rRNA sequences of the obtained clones and isolates were phylogenetically affiliated to the groups Gammaproteobacteria, Bacteriodetes and Alphaproteobacteria. Groups like Deltaproteobacteria, Verrucomicrobia, Planctomycetes, Spirochaetes, Acidobacteria and Deinococcus-Thermus were only detected by cloning. The strains isolated on acetate and glycolate belonged to the genera Marinobacter, Halomonas, Roseobacter and Rhodobacter whereas the strains enriched on crude oil belonged to Marinobacter and Alcanivorax. Members of the Bacteriodetes group were only enriched on Spirulina extract indicating their specialization in the degradation of cyanobacterial dead cells. The substrate spectra of representative strains showed the ability of all AHB to metabolize cyanobacterial photosynthetic and fermentation products. However, the unique in situ conditions of the mat apparently favored the enrichment of versatile strains that grew on both the cyanobacterial exudates and the hydrocarbons. We conclude that AHB in cyanobacterial mats represent a diverse community that plays an important role in carbon-cycling within microbial mats.     

Diazotrophic microbial community of coastal microbial mats of the southern North Sea

The diazotrophic community in microbial mats growing along the shore of the North Sea barrier island Schiermonnikoog (The Netherlands) was studied using microscopy, lipid biomarkers, stable carbon (δ(13) C(TOC) ) and nitrogen (δ(15) N) isotopes as well as by constructing and analyzing 16S rRNA gene libraries. Depending on their position on the littoral gradient, two types of mats were identified, which showed distinct differences regarding the structure, development and composition of the microbial community. Intertidal microbial mats showed a low species diversity with filamentous non-heterocystous Cyanobacteria providing the main mat structure. In contrast, supratidal microbial mats showed a distinct vertical zonation and a high degree of species diversity. Morphotypes of non-heterocystous Cyanobacteria were recognized as the main structural component in these mats. In addition, unicellular Cyanobacteria were frequently observed, whereas filamentous heterocystous Cyanobacteria occurred only in low numbers. Besides the apparent visual dominance of cyanobacterial morphotpyes, 16S rRNA gene libraries indicated that both microbial mat types also included members of the Proteobacteria and the Cytophaga-Flavobacterium-Bacteroides group as well as diatoms. Bulk δ(15) N isotopes of the microbial mats ranged from +6.1‰ in the lower intertidal to -1.2‰ in the supratidal zone, indicating a shift from predominantly nitrate utilization to nitrogen fixation along the littoral gradient. This conclusion was supported by the presence of heterocyst glycolipids, representing lipid biomarkers for nitrogen-fixing heterocystous Cyanobacteria, in supratidal but not in intertidal microbial mats. The availability of combined nitrogen species might thus be a key factor in controlling and regulating the distribution of the diazotrophic microbial community of Schiermonnikoog.     

Bacterial diversity of a cyanobacterial mat degrading petroleum compounds at elevated salinities and temperatures

Cyanobacterial mats of the Arabian Gulf coast of Saudi Arabia experience extreme conditions of temperature and salinity. Because they are exposed to continuous oil pollution, they form ideal models for biodegradation under extreme conditions. We investigated the bacterial diversity of these mats using denaturing gradient gel electrophoresis and 16S rRNA cloning, and tested their potential to degrade petroleum compounds at various salinities (fresh water to 16%) and temperatures (5 to 50 degrees C). Cloning revealed that c. 15% of the obtained sequences were related to unknown, possibly novel bacteria. Bacteria belonging to Beta-, Gamma- and Deltaproteobacteria, Cytophaga-Flavobacterium-Bacteroides group and Spirochetes, were detected. The biodegradation of petroleum compounds at different salinities by mat microorganisms showed that pristine and n-octadecane were optimally degraded at salinities between 5 and 12% (weight per volume NaCl) whereas the optimum degradation of phenanthrene and dibenzothiophene was at 3.5% salinity. The latter compounds were also degradable at 8% salinity. The same compounds were degraded at temperatures between 15 and 40 degrees C but not at 5 and 50 degrees C. The optimum temperature of degradation was 28-40 degrees C for both aliphatics and aromatics. We conclude that the studied microbial mats from Saudi Arabia are rich in novel halotolerant and thermotolerant microorganisms with the potential to degrade petroleum compounds at elevated salinities and temperatures.     

Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the β- and γ-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.