Denitrification in human dental plaque.

BACKGROUND: Microbial denitrification is not considered important in human-associated microbial communities. Accordingly, metabolic investigations of the microbial biofilm communities of human dental plaque have focused on aerobic respiration and acid fermentation of carbohydrates, even though it is known that the oral habitat is constantly exposed to nitrate (NO3-) concentrations in the millimolar range and that dental plaque houses bacteria that can reduce this NO3- to nitrite (NO2-). RESULTS: We show that dental plaque mediates denitrification of NO3- to nitric oxide (NO), nitrous oxide (N2O), and dinitrogen (N2) using microsensor measurements, 15N isotopic labelling and molecular detection of denitrification genes. In vivo N2O accumulation rates in the mouth depended on the presence of dental plaque and on salivary NO3- concentrations. NO and N2O production by denitrification occurred under aerobic conditions and was regulated by plaque pH. CONCLUSIONS: Increases of NO concentrations were in the range of effective concentrations for NO signalling to human host cells and, thus, may locally affect blood flow, signalling between nerves and inflammatory processes in the gum. This is specifically significant for the understanding of periodontal diseases, where NO has been shown to play a key role, but where gingival cells are believed to be the only source of NO. More generally, this study establishes denitrification by human-associated microbial communities as a significant metabolic pathway which, due to concurrent NO formation, provides a basis for symbiotic interactions.

Real-time microsensor measurement of local metabolic activities in ex vivo dental biofilms exposed to sucrose and treated with chlorhexidine.

Dental biofilms are characterized by structural and functional heterogeneity. Due to bacterial metabolism, gradients develop and diverse ecological microniches exist. The aims of this study were (i) to determine the metabolic activity of microorganisms in naturally grown dental biofilms ex vivo by measuring dissolved oxygen (DO) and pH profiles with microelectrodes with high spatial resolution and (ii) to analyze the impact of an antimicrobial chlorhexidine (CHX) treatment on microbial physiology during stimulation by sucrose in real time. Biofilms were cultivated on standardized human enamel surfaces in vivo. DO and pH profiles were measured in a flow cell system in sterile human saliva, after sucrose addition (10%), again after alternative treatment of the sucrose exposed biofilms with CHX (0.2%) for 1 or 10 min or after being killed with paraformaldehyde (4%). Biofilm structure was visualized by vitality staining with confocal microscopy. With saliva as the sole nutrient source oxygen consumption was high within the superficial biofilm layers rendering deeper layers (>220 mum) anoxic. Sucrose addition induced the thickness of the anaerobic zone to increase with a concurrent decrease in pH (7.1 to 4.4). CHX exposure reduced metabolic activity and microbial viability at the biofilm surface and drove metabolic activity deeper into the biofilm. CHX treatment led to a reduced viability at the biofilm surface with minor influence on overall biofilm physiology after 1 min; even after 10 min there was measurable respiration and fermentation inside the biofilm. However, the local microenvironment was more aerated, less acidogenic, and presumably less pathogenic.

Fluorescence "in situ" hybridization for the detection of biofilm in the middle ear and upper respiratory tract mucosa.

Most chronic bacterial infections are associated with biofilm formation wherein the bacteria attach to mucosal surfaces, wound tissue, or medical device surfaces in the human body via the formation of an extracellular matrix. Biofilms assume complex three-dimensional structures dependent on the species, the strain, and the prevailing environmental conditions and are composed of both the bacteria and the extracellular slime-like matrices, which surround the bacteria. Bacteria deep in the biofilm live under anaerobic conditions and must use alternatives to O(2) as a terminal electron acceptor. Thus, the metabolic rates of these deep bacteria are greatly reduced, which renders them extremely resistant to antibiotic treatment, and for reasons not clearly understood, it is often very difficult to culture biofilm bacteria using traditional microbiologic techniques. To directly identify and visualize biofilm bacteria in a species-specific manner, we developed a confocal laser scanning microscopy (CLSM)-based 16S rRNA fluorescence in situ hybridization (FISH) protocol, to find biofilm bacteria in middle ear and upper respiratory tract mucosa, which preserves the three-dimensional structure of the biofilm and avoids the use of traditional culture techniques.

Impact of nitrate on the structure and function of bacterial biofilm communities in pipelines used for injection of seawater into oil fields.

We studied the impact of NO(3)(-) on the bacterial community composition, diversity, and function in in situ industrial, anaerobic biofilms by combining microsensor profiling, (15)N and (35)S labeling, and 16S rRNA gene-based fingerprinting. Biofilms were grown on carbon steel coupons within a system designed to treat seawater for injection into an oil field for pressurized oil recovery. NO(3)(-) was added to the seawater in an attempt to prevent bacterial H(2)S generation and microbially influenced corrosion in the field. Microprofiling of nitrogen compounds and redox potential inside the biofilms showed that the zone of highest metabolic activity was located close to the metal surface, correlating with a high bacterial abundance in this zone. Upon addition, NO(3)(-) was mainly reduced to NO(2)(-). In biofilms grown in the absence of NO(3)(-), redox potentials of <-450 mV at the metal surface suggested the release of Fe(2+). NO(3)(-) addition to previously untreated biofilms induced a decline (65%) in bacterial species richness, with Methylophaga- and Colwellia-related sequences having the highest number of obtained clones in the clone library. In contrast, no changes in community composition and potential NO(3)(-) reduction occurred upon subsequent withdrawal of NO(3)(-). Active sulfate reduction was below detection levels in all biofilms, but S isotope fractionation analysis of sulfide deposits suggested that it must have occurred either at low rates or episodically. Scanning electron microscopy revealed that pitting corrosion occurred on all coupons, independent of the treatment. However, uniform corrosion was clearly mitigated by NO(3)(-) addition.

Nitrosomonas Nm143-like ammonia oxidizers and Nitrospira marina-like nitrite oxidizers dominate the nitrifier community in a marine aquaculture biofilm.

Zero-discharge marine aquaculture systems are an environmentally friendly alternative to conventional aquaculture. In these systems, water is purified and recycled via microbial biofilters. Here, quantitative data on nitrifier community structure of a trickling filter biofilm associated with a recirculating marine aquaculture system are presented. Repeated rounds of the full-cycle rRNA approach were necessary to optimize DNA extraction and the probe set for FISH to obtain a reliable and comprehensive picture of the ammonia-oxidizing community. Analysis of the ammonia monooxygenase gene (amoA) confirmed the results. The most abundant ammonia-oxidizing bacteria (AOB) were members of the Nitrosomonas sp. Nm143-lineage (6.7% of the bacterial biovolume), followed by Nitrosomonas marina-like AOB (2.2% of the bacterial biovolume). Both were outnumbered by nitrite-oxidizing bacteria of the Nitrospira marina-lineage (15.7% of the bacterial biovolume). Although more than eight other nitrifying populations were detected, including Crenarchaeota closely related to the ammonia-oxidizer 'Nitrosopumilus maritimus', their collective abundance was below 1% of the total biofilm volume; their contribution to nitrification in the biofilter is therefore likely to be negligible.

Biofilm plaque and hydrodynamic effects on mass transfer, fluoride delivery and caries.

BACKGROUND: The biofilm concept of dental plaque now is widely accepted in the dental clinic, particularly with respect to its importance to oral hygiene. A number of reviews have focused on the microbial ecology of biofilm with regard to oral health; however, there has been less focus on how the interaction of biofilms and hydrodynamics with mass transfer (the movement of molecules and particulates) and physiological processes may relate to caries. TYPES OF STUDIES REVIEWED: The authors reviewed reports in the microbiology and dental literature addressing microbiological, engineering and clinical aspects of biofilms with respect to mass transport and microbial physiology, with an emphasis on fluoride ions (F(-)). CONCLUSIONS: and Practical Implications. These data illustrate how dental plaque biofilms may affect the delivery of cariogenic agents, such as sucrose, or anticariogenic agents, such as F(-), into and out of the biofilm, with subsequent consequences for the development of physio-chemical microenvironments at the tooth surface. Increasing the flow rate in an overlying fluid (such as saliva or mouthrinse) increases transport from the fluid into and through biofilms. Increasing the delivery of anticariogenic agents such as F(-) into the plaque biofilm, by generating strong fluid flows, may be a useful strategy for enhancing the anticaries effects of F(-) in areas of the mouth where complete biofilm removal is not possible with routine daily cleaning techniques.

Transient development of filamentous Thiothrix species in a marine sulfide oxidizing, denitrifying fluidized bed reactor.

In this study, microscopic and molecular microbial analyses were integrated to characterize rapidly developing white filamentous tufts in a fluidized bed reactor used for nitrate removal from a marine recirculating fish culture system. Formation and rapid elongation of the tufts (often exceeding 50 mm day (-1)) was strongly correlated to transient elevated sulfide concentrations (>50 microM) in the reactor. The dominant bacterial constituents of these tufts were filamentous gram-negative bacteria with densely packed intracellular sulfur granules. Using 16S rRNA gene analysis and fluorescence in situ hybridization it was found that these filamentous bacteria represented a novel Thiothrix phylotype closely related (97% sequence identity) to a previously identified Thiothrix strain endogenous to the marine crustacean Urothoe poseidonis. In addition to filamentous morphotypes, rosette-shaped morphotypes of Thiothrix were also detectable within the tufts.

Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media.

CONTEXT: Chronic otitis media (OM) is a common pediatric infectious disease. Previous studies demonstrating that metabolically active bacteria exist in culture-negative pediatric middle-ear effusions and that experimental infection with Haemophilus influenzae in the chinchilla model of otitis media results in the formation of adherent mucosal biofilms suggest that chronic OM may result from a mucosal biofilm infection. OBJECTIVE: To test the hypothesis that chronic OM in humans is biofilm-related. DESIGN, SETTING, AND PATIENTS: Middle-ear mucosa (MEM) biopsy specimens were obtained from 26 children (mean age, 2.5 [range, 0.5-14] years) undergoing tympanostomy tube placement for treatment of otitis media with effusion (OME) and recurrent OM and were analyzed using microbiological culture, polymerase chain reaction (PCR)-based diagnostics, direct microscopic examination, fluorescence in situ hybridization, and immunostaining. Uninfected (control) MEM specimens were obtained from 3 children and 5 adults undergoing cochlear implantation. Patients were enrolled between February 2004 and April 2005 from a single US tertiary referral otolaryngology practice. MAIN OUTCOME MEASURES: Confocal laser scanning microscopic (CLSM) images were obtained from MEM biopsy specimens and were evaluated for biofilm morphology using generic stains and species-specific probes for H influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. Effusions, when present, were evaluated by PCR and culture for evidence of pathogen-specific nucleic acid sequences and bacterial growth, respectively. RESULTS: Of the 26 children undergoing tympanostomy tube placement, 13 (50%) had OME, 20 (77%) had recurrent OM, and 7 (27%) had both diagnoses; 27 of 52 (52%) of the ears had effusions, 24 of 24 effusions were PCR-positive for at least 1 OM pathogen, and 6 (22%) of 27 effusions were culture-positive for any pathogen. Mucosal biofilms were visualized by CLSM on 46 (92%) of 50 MEM specimens from children with OME and recurrent OM using generic and pathogen-specific probes. Biofilms were not observed on 8 control MEM specimens obtained from the patients undergoing cochlear implantation. CONCLUSION: Direct detection of biofilms on MEM biopsy specimens from children with OME and recurrent OM supports the hypothesis that these chronic middle-ear disorders are biofilm-related.

Effect of nitrate on sulfur transformations in sulfidogenic sludge of a marine aquaculture biofilter.

The effect of NO(3)(-) addition on dissimilatory SO(4)(2-) reduction and sulfide conversion in organic-rich sludge from the digestion basin of a recirculating marine aquaculture system was studied. SO(4)(2-) reduction could only explain a minor fraction (up to 4-9%) of the observed total sulfide production (up to 35 mmol L(-1) day(-1)), indicating that the main source of sulfide in the sludge was not SO(4)(2-) reduction, but desulfuration during the decomposition of organic matter. Although NO(3)(-) inhibited SO(4)(2-) reduction, but not desulfuration, the primary NO(3)(-) mitigation effect was the onset of NO(3)(-)-mediated sulfide oxidation (up to 75 mmol L(-1) day(-1)), partially to elemental sulfur (S(0)). Above NO(3)(-) concentrations of 0.6 mM in the bulk water, the net sulfide production and oxidation zones were moved deeper into flocs and sludge cores, which effectively prevented sulfide from entering the water column. However, the sulfide efflux from the sludge instantly recovered after NO(3)(-) depletion. Thus, the NO(3)(-) level in the water column controls the zonation and magnitude of sulfur transformations in the sludge. The effect of NO(3)(-) relies therefore on its sustained presence in the water column, which in turn depends on a well-functioning nitrification in the mariculture system.

Molecular and morphological characterization of the association between bacterial endosymbionts and the marine nematode Astomonema sp. from the Bahamas.

Marine nematode worms without a mouth or functional gut are found worldwide in intertidal sandflats, deep-sea muds and methane-rich pock marks, and morphological studies show that they are associated with endosymbiotic bacteria. While it has been hypothesized that the symbionts are chemoautotrophic sulfur oxidizers, to date nothing is known about the phylogeny or function of endosymbionts from marine nematodes. In this study, we characterized the association between bacterial endosymbionts and the marine nematode Astomonema sp. from coral reef sediments in the Bahamas. Phylogenetic analysis of the host based on its 18S rRNA gene showed that Astomonema sp. is most closely related to non-symbiotic nematodes of the families Linhomoeidae and Axonolaimidae and is not closely related to marine stilbonematinid nematodes with ectosymbiotic sulfur-oxidizing bacteria. In contrast, phylogenetic analyses of the symbionts of Astomonema sp. using comparative 16S rRNA gene sequence analysis revealed that these are closely related to the stilbonematinid ectosymbionts (95-96% sequence similarity) as well as to the sulfur-oxidizing endosymbionts from gutless marine oligochaetes. The closest free-living relatives of these gammaproteobacterial symbionts are sulfur-oxidizing bacteria from the family Chromatiaceae. Transmission electron microscopy and fluorescence in situ hybridization showed that the bacterial symbionts completely fill the gut lumen of Astomonema sp., suggesting that these are their main source of nutrition. The close phylogenetic relationship of the Astomonema sp. symbionts to known sulfur-oxidizing bacteria as well as the presence of the aprA gene, typically found in sulfur-oxidizing bacteria, indicates that the Astomonema sp. symbionts use reduced sulfur compounds as an energy source to provide their hosts with nutrition.

Consumption of methane and CO2 by methanotrophic microbial mats from gas seeps of the anoxic Black Sea.

The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH(4) and CO(2) assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO(2) reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average delta(13)C carbon isotopic signature of -67.1 per thousand, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (-66.4 per thousand +/- 3.9 per thousand [mean +/- standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (-72.9 per thousand +/- 2.2 per thousand; n = 7). Incorporation of (14)C from radiolabeled CH(4) or CO(2) revealed one hot spot for methanotrophy and CO(2) fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with (14)CH(4) or (14)CO(2) revealed that there was interconversion of CH(4) and CO(2.) The level of CO(2) reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis.

Nitrification in a biofilm at low pH values: role of in situ microenvironments and acid tolerance.

The sensitivity of nitrifying bacteria to acidic conditions is a well-known phenomenon and generally attributed to the lack and/or toxicity of substrates (NH3 and HNO2) with decreasing pHs. In contrast, we observed strong nitrification at a pH around 4 in biofilms grown on chalk particles and investigated the following hypotheses: the presence of less acidic microenvironments and/or the existence of acid-tolerant nitrifiers. Microelectrode measurements (in situ and under various experimental conditions) showed no evidence of a neutral microenvironment, either within the highly active biofilm colonizing the chalk surface or within a control biofilm grown on a nonbuffering (i.e., sintered glass) surface under acidic pH. A 16S rRNA approach (clone libraries and fluorescence in situ hybridizations) did not reveal uncommon nitrifying (potentially acid-tolerant) strains. Instead, we found a strongly acidic microenvironment, evidence for a clear adaptation to the low pH in situ, and the presence of nitrifying populations related to subgroups with low Km s for ammonia (Nitrosopira spp., Nitrosomonas oligotropha, and Nitrospira spp.). Acid-consuming (chalk dissolution) and acid-producing (ammonia oxidation) processes are equilibrated on a low-pH steady state that is controlled by mass transfer limitation through the biofilm. Strong affinity to ammonia and possibly the expression of additional functions, e.g., ammonium transporters, are adaptations that allow nitrifiers to cope with acidic conditions in biofilms and other habitats.

Distribution, localization, and phylogeny of abundant populations of Crenarchaeota in anaerobic granular sludge.

Eight anaerobic granular sludges were surveyed for Crenarchaeota using rRNA gene cloning. Microbial arrangement and substrate uptake patterns were elucidated by fluorescent in situ hybridization and beta imaging. Group 1.3 Crenarchaeota represented up to 50% of Archaea and 25% of the total microbiota in five sludges. Crenarchaeota were localized in close association with methanogenic Archaea.

In situ substrate conversion and assimilation by nitrifying bacteria in a model biofilm.

Local nitrification and carbon assimilation activities were studied in situ in a model biofilm to investigate carbon yields and contribution of distinct populations to these activities. Immobilized microcolonies (related to Nitrosomonas europaea/eutropha, Nitrosomonas oligotropha, Nitrospira sp., and to other Bacteria) were incubated with [14C]-bicarbonate under different experimental conditions. Nitrifying activity was measured concomitantly with microsensors (oxygen, ammonium, nitrite, nitrate). Biofilm thin sections were subjected to fluorescence in situ hybridization (FISH), microautoradiography (MAR), and local quantification of [14C]-bicarbonate uptake (beta microimaging). Nitrifying activity and tracer assimilation were restricted to a surface layer of different thickness in the various experiments (substrate or oxygen limitation). Excess oxygen uptake under all conditions revealed heterotrophic activity fuelled by decay or excretion products during active nitrification. Depth limits and intensity of tracer incorporation profiles were in agreement with ammonia-oxidation activity (measured with microsensors), and distribution of incorporated tracer (detected with MAR). Microautoradiography revealed a sharp individual response of distinct populations in terms of in-/activity depending on the (local) environmental conditions within the biofilm. Net in situ carbon yields on N, expressed as e- equivalent ratios, varied between 0.005 and 0.018, and, thus, were in the lower range of data reported for pure cultures of nitrifiers.

Identification of bacteria potentially responsible for oxic and anoxic sulfide oxidation in biofilters of a recirculating mariculture system.

Bacteria presumably involved in oxygen- or nitrate-dependent sulfide oxidation in the biofilters of a recirculating marine aquaculture system were identified using a new application of reverse transcription-PCR denaturing gradient gel electrophoresis (DGGE) analysis termed differential-transcription (DT)-DGGE. Biofilter samples were incubated in various concentrations of sulfide or thiosulfate (0 to 5 mM) with either oxygen or nitrate as the sole electron acceptor. Before and after short-term incubations (10 to 20 h), total DNA and RNA were extracted, and a 550-bp fragment of the 16S rRNA genes was PCR amplified either directly or after reverse transcription. DGGE analysis of DNA showed no significant change of the original microbial consortia upon incubation. In contrast, DGGE of cDNA revealed several phylotypes whose relative band intensities markedly increased or decreased in response to certain incubation conditions, indicating enhanced or suppressed rRNA transcription and thus implying metabolic activity under these conditions. Specifically, species of the gammaproteobacterial genus Thiomicrospira and phylotypes related to symbiotic sulfide oxidizers could be linked to oxygen-dependent sulfide oxidation, while members of the Rhodobacteraceae (genera Roseobacter, Rhodobacter, and Rhodobium) were putatively active in anoxic, nitrate-dependent sulfide oxidation. For all these organisms, the physiology of their closest cultured relatives matches their DT-DGGE-inferred function. In addition, higher band intensities following exposure to 5 mM sulfide and nitrate were observed for Thauera-, Hydrogenophaga-, and Dethiosulfovibrio-like phylotypes. For these genera, nitrate-dependent sulfide oxidation has not been documented previously and therefore DT-DGGE might indicate a higher relative tolerance to high sulfide concentrations than that of other community members. We anticipate that DT-DGGE will be of general use in tracing functionally equivalent yet phylogenetically diverse microbial populations in nature.

Sulfide-oxidizing activity and bacterial community structure in a fluidized bed reactor from a zero-discharge mariculture system.

In the present work we describe a comprehensive analysis of sulfide oxidation in a fluidized bed reactor (FBR) from an environmentally sustainable, zero-discharge mariculture system. The FBR received oxygen-depleted effluent from a digestion basin (DB) that is responsible for gasification of organic matter and nitrogen. The FBR is a crucial component in this recirculating system because it safeguards the fish from the toxic sulfide produced in the DB. Microscale sulfide oxidation potential and bacterial community composition within FBR biofilms were correlated to biofilter performance by integrating bulk chemical, microsensor (O2, pH, and H2S), and molecular microbial community analyses. The FBR consistently oxidized sulfide during two years of continuous operation, with an estimated average sulfide removal rate of 1.3 g of sulfide-S L(FBR)(-1) d(-1). Maximum sulfide oxidation rates within the FBR biofilms were 0.36 and 0.21 mg of sulfide-S cm(-3) h(-1) in the oxic and anoxic layers, respectively, indicating that both oxygen and nitrate serve as electron acceptors for sulfide oxidation. The estimated anoxic sulfide removal rate, as extrapolated from bench scale, autotrophic, nitrate-amended experiments, was 0.7 g of sulfide-S L(FBR)(-1) d(-1), which is approximately 50% of the total estimated sulfide removal in the FBR. Community composition analyses using denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rRNA gene fragments from FBR samples taken at six-month intervals revealed several sequences that were closely affiliated with sulfide-oxidizing bacteria. These included the denitrifying, sulfide-oxidizing bacteria Thiomicrospira denitrificans, members of the filamentous Thiothrix genus, and sulfide-oxidizing symbionts from the Gammaproteobacteria. In addition, marine Alphaproteobacteria and Bacteroidetes species were present in all of the DGGE profiles examined. DGGE analyses showed significant shifts in the bacterial community composition between profiles over two years of sampling, indicating the presence of a diverse and dynamic microbial community within the functionally stable FBR. The FBR's combined capacity for both oxic and anoxic sulfide oxidation, as indicated by bulk chemical, microsensor, and molecular microbial analyses, gives it significant functional elasticity, which is crucial for proper performance in the dynamic environment of this mariculture system.

Structure and activity of multiple nitrifying bacterial populations co-existing in a biofilm.

A biofilm from a nitrifying pilot-scale sequencing batch reactor was investigated for effects of varying process conditions on its microscale activity and structure. Microsensor measurements of oxygen, substrates and products of nitrification were applied under incubation at different ammonium and oxygen concentrations which reflected various situations during a treatment cycle. A high net N loss was observed under high ammonium (HA) concentrations in contrast to low ones. Additionally, results indicated inhibition of nitrite-oxidizing bacteria (NOB), but not of ammonia-oxidizing bacteria (AOB) by free ammonia under HA conditions. Diversity, spatial distribution, and abundance of nitrifying bacteria as analysed by fluorescence in situ hybridization (FISH) revealed six different nitrifying populations with heterogeneous distributions. Nitrosococcus mobilis formed conspicuous microcolonies locally surrounded by cells of the dominating N. europaea/eutropha-related AOB population. A third less abundant population was affiliated to N. oligotropha. Nitrite-oxidizing bacteria of the genera Nitrobacter and Nitrospira (with at least two distinct populations) showed a large scale heterogeneity in their distribution. Nitrospira spp. were also found in deeper inactive layers where they might persist rather than thrive, and act as seed population when detached. Results of functional and structural analyses are discussed with respect to specific niches of individual populations in this system.

Microbial reefs in the Black Sea fueled by anaerobic oxidation of methane.

Massive microbial mats covering up to 4-meter-high carbonate buildups prosper at methane seeps in anoxic waters of the northwestern Black Sea shelf. Strong 13C depletions indicate an incorporation of methane carbon into carbonates, bulk biomass, and specific lipids. The mats mainly consist of densely aggregated archaea (phylogenetic ANME-1 cluster) and sulfate-reducing bacteria (Desulfosarcina/Desulfococcus group). If incubated in vitro, these mats perform anaerobic oxidation of methane coupled to sulfate reduction. Obviously, anaerobic microbial consortia can generate both carbonate precipitation and substantial biomass accumulation, which has implications for our understanding of carbon cycling during earlier periods of Earth's history.