Agreement between theory and measurement in quantification of ammonia-oxidizing bacteria

Autotrophic ammonia-oxidizing bacteria (AOB) are of vital importance to wastewater treatment plants (WWTP), as well as being an intriguing group of microorganisms in their own right. To date, corroboration of quantitative measurements of AOB by fluorescence in situ hybridization (FISH) has relied on assessment of the ammonia oxidation rate per cell, relative to published values for cultured AOB. Validation of cell counts on the basis of substrate transformation rates is problematic, however, because published cell-specific ammonia oxidation rates vary by over two orders of magnitude. We present a method that uses FISH in conjunction with confocal scanning laser microscopy to quantify AOB in WWTP, where AOB are typically observed as microcolonies. The method is comparatively simple, requiring neither detailed cell counts or image analysis, and yet it can give estimates of either cell numbers or biomass. Microcolony volume and diameter were found to have a log-normal distribution. We were able to show that virtually all (>96%) of the AOB biomass occurred as microcolonies. Counts of microcolony abundance and measurement of their diameter coupled with a calibration of microcolony dimensions against cell numbers or AOB biomass were used to determine AOB cell numbers and biomass in WWTP. Cell-specific ammonia oxidation rates varied between plants by over three orders of magnitude, suggesting that cell-specific ammonia oxidation is an important process variable. Moreover, when measured AOB biomass was compared with process-based estimates of AOB biomass, the two values were in agreement.     

Analysis of size distribution and areal cell density of ammonia-oxidizing bacterial microcolonies in relation to substrate microprofiles in biofilms

A fine-scale in situ spatial organization of ammonia-oxidizing bacteria (AOB) in biofilms was investigated by combining molecular techniques (i.e., fluorescence in situ hybridization (FISH) and 16S rDNA-cloning analysis) and microelectrode measurements. Important parameters of AOB microcolonies such as size distribution and areal cell density of the microcolonies were determined and correlated with substrate microprofiles in the biofilms. In situ hybridization with a nested 16S rRNA-targeted oligonucleotide probe set revealed two different populations of AOB, Nitrosomonas europaea-lineage and Nitrosospira multiformis-lineage, coexisting in an autotrophic nitrifying biofilm. Nitrosospira formed looser microcolonies, with an areal cell density of 0.51 cells microm(-2), which was half of the cell density of Nitrosomonas (1.12 cells microm(-2)). It is speculated that the formation of looser microcolonies facilitates substrate diffusion into the microcolonies, which might be a survival strategy to low O(2) and NH(4) (+) conditions in the biofilm. A long-term experiment (4-week cultivation at different substrate C/N ratios) revealed that the size distribution of AOB microcolonies was strongly affected by better substrate supply due to shorter distance from the surface and the presence of organic carbon. The microcolony size was relatively constant throughout the autotrophic nitrifying biofilm, while the size increased by approximately 80% toward the depth of the biofilm cultured at the substrate C/N = 1. A short-term ( approximately 3 h) organic carbon addition experiment showed that the addition of organic carbon created interspecies competition for O(2) between AOB and heterotrophic bacteria, which dramatically decreased the in situ NH(4) (+)-uptake activity of AOB in the surface of the biofilms. This result might explain the spatial distribution of AOB microcolony size in the biofilms cultured at the substrate C/N = 1. These experimental results suggest O(2) and organic carbon were the main factors controlling the spatial organization and activity of AOB in biofilms. These findings are significantly important to further improve mathematical models used to describe how the slow-growing AOB develop their niches in biofilms and how that configuration affects nitrification performance in the biofilm.     

Analysis of mammalian viable cell biomass based on cellular ATP

Analysis of cellular ATP as a means of measuring viable biomass loading was investigated in hybridoma cell culture. ATP analysis by the luciferin-luciferase assay was compared with trypan blue-stained hemocytometer counts. The cell-specific ATP content varied between 2 and 6 fmol per viable cell over a batch culture. ATP levels were highest during exponential growth, and decreased during the stationary and decline phases. Electronic counting and volume measurements were performed to assay the viable cell biomass. Cell sorting, using fluorescein diacetate, was used to separate viable and nonviable cells in cultures with between 35% and 90% viable cells. Viable cells contained over 2 orders of magnitude greater cell-specific ATP than nonviable cells. Cell-specific ATP correlated directly with the viable cell volume rather than viable cell numbers. Over the range of batch culture conditions, ATP analysis should provide a more accurate measurement of hybridoma viable biomass than hemocytometer counts.     

Viability of Indigenous Soil Bacteria Assayed by Respiratory Activity and Growth

The bacterial population in barley field soil was estimated by determining the numbers of (i) cells reducing the artificial electron acceptor 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) to CTC-formazan (respiratory activity), (ii) cells dividing a limited number of times (microcolony formation) on nutrient-poor media, (iii) cells dividing many times (colony formation) on nutrient-poor agar media, and (iv) cells stained with acridine orange (total counts). The CTC reduction assay was used for the first time for populations of indigenous soil bacteria and was further developed for use in this environment. The number of viable cells was highest when estimated by the number of microcolonies developing during 2 months of incubation on filters placed on the surface of nutrient-poor media. The number of bacteria reducing CTC to formazan was slightly lower than the number of bacteria forming microcolonies. Traditional plate counts of CFU (culturable cells) yielded the lowest estimate of viable cell numbers. The microcolony assay gave an estimate of both (i) cells forming true microcolonies (in which growth ceases after a few cell divisions) representing viable but nonculturable cells and (ii) cells forming larger microcolonies (in which growth continues) representing viable, culturable cells. The microcolony assay, allowing single-cell observations, thus seemed to be best suited for estimation of viable cell numbers in soil. The effect on viable and culturable cell numbers of a temperature increase from 4 to 17°C for 5 days was investigated in combination with drying or wetting of the soil. Drying or wetting prior to the temperature increase, rather than the temperature increase per se, affected both the viable and culturable numbers of bacteria; both numbers were reduced in predried soil, while they increased slightly in the prewetted soil.     

Application of Most-Probable-Number Statistics to Direct Enumeration of Microorganisms

A novel method for rapid determination of total microbial cell numbers was investigated. The method involves the application of most-probable-number estimation statistics to direct microscopic counting of microbial cells by using a particle sizing graticule. Its accuracy and reliability were tested with computer simulations of bacterial cell distributions encountered in ecological studies. Good estimates of cell numbers were obtained when the cell density varied from 3 to 6,000 cells per field, i.e., over 3 orders of magnitude. Low levels of contagion did not markedly influence cell estimates, although high levels, corresponding to discrete scattered microcolonies, did. However, these could be recognized visually. Estimates of cell numbers in Breed smears confirmed its speed and good correlation with the standard quadrat counting technique under real experimental conditions.     

Abundance and diversity of ammonia-oxidizing archaea in a biological aerated filter process

Ammonia-oxidizing archaea (AOA) represent an important group of ammonia-oxidizing microorganisms that are able to convert ammonia to nitrite, a function which is crucial for the removal of nitrogen from wastewater. In this study, we investigated the abundance and diversity of AOA in a full-scale wastewater treatment plant (WWTP) which used a biological aerated filter (BAF) as the main processing mode. According to the quantitative PCR results, AOA clearly outnumbered ammonia-oxidizing bacteria (AOB) during the whole process. The abundance of AOA amoA genes in the filter layer of BAF was highest with the value varied from 6.32 × 10³ to 3.8 × 10⁴ copies/ng DNA. The highest abundance of AOB amoA genes was 1.32 × 10² copies/ng DNA, recorded in the effluent of the ACTIFLO® settling tank. The ratios of AOA/AOB in the WWTP were maintained at two or three orders of magnitude. Most AOA obtained from the WWTP fell within the Nitrosopumilus cluster. The abundance of AOA and AOB was significantly correlated with ammonium nitrogen concentrations and pH value. The community structure of AOA was significantly influenced by dissolved oxygen concentrations, pH value and chemical oxygen demand.     

In situ distribution and activity of nitrifying bacteria in freshwater sediment

Nitrification was investigated in a model freshwater sediment by the combined use of microsensors and fluorescence in situ hybridization with rRNA-targeted oligonucleotide probes. In situ nitrification activity was restricted mainly to the upper 2 mm of the sediment and coincided with the maximum abundance of nitrifying bacteria, i.e. 1.5 x 107 cells cm-3 for ammonia-oxidizing Beta-proteobacteria (AOB) and 8.6 x 107 cells cm-3 for Nitrospira-like nitrite-oxidizing bacteria (NOB). Cell numbers of AOB decreased more rapidly with depth than numbers of NOB. For the first time, Nitrospira-like bacteria could be quantified and correlated with in situ nitrite oxidation rates in a sediment. Estimated cell-specific nitrite oxidation rates were 1.2-2.7 fmol NO2- cell-1 h-1.     

Survival of heated or irradiated Chinese hamster ovary microcolonies that develop after an initial dose of heat

Asynchronous or synchronous G1 cells were heated initially and then heated or irradiated a second time when the multiplicity of viable cells in microcolonies that developed from cells surviving the first heat dose had increased to 6-30. The survival of these microcolonies was compared with the survival of single cells that were heated or irradiated after the microcolonies had been trypsinized and dispersed into single cells. The survival of the single cells was similar to the survival of the microcolonies and much higher than single cell survival calculated by correcting microcolony survival for multiplicity. However, when microcolonies developed from control unheated cells, the observed single cell survival corresponded to single cell survival calculated by correcting microcolony survival for multiplicity. Therefore, multiplicity corrections, which assume that cells within a microcolony survive independently from one another, are not valid when the microcolony has developed from a cell surviving an initial heat treatment.     

Ammonia oxidizing bacteria community dynamics in a pilot-scale wastewater treatment plant

Background

Chemoautotrophic ammonia oxidizing bacteria (AOB) have the metabolic ability to oxidize ammonia to nitrite aerobically. This metabolic feature has been widely used, in combination with denitrification, to remove nitrogen from wastewater in wastewater treatment plants (WWTPs). However, the relative influence of specific deterministic environmental factors to AOB community dynamics in WWTP is uncertain. The ecological principles underlying AOB community dynamics and nitrification stability and how they are related are also poorly understood.

Methodology/Principal Findings

The community dynamics of ammonia oxidizing bacteria (AOB) in a pilot-scale WWTP were monitored over a one-year period by Terminal Restriction Fragment Length Polymorphism (T-RFLP). During the study period, the effluent ammonia concentrations were almost below 2 mg/L, except for the first 60 days, indicting stable nitrification. T-RFLP results showed that, during the test period with stable nitrification, the AOB community structures were not stable, and the average change rate (every 15 days) of AOB community structures was 10%±8%. The correlations between T-RFLP profiles and 10 operational and environmental parameters were tested by Canonical Correlation Analysis (CCA) and Mantel test. The results indicated that the dynamics of AOB community correlated most strongly with Dissolved Oxygen (DO), effluent ammonia, effluent Biochemical Oxygen Demand (BOD) and temperature.

Conclusions/Significance

This study suggests that nitrification stability is not necessarily accompanied by a stable AOB community, and provides insight into parameters controlling the AOB community dynamics within bioreactors with stable nitrification.     

Dynamics of specific ammonia-oxidizing bacterial populations and nitrification in response to controlled shifts of ammonium concentrations in wastewater

Ammonia-oxidizing bacteria (AOB) are essential for the nitrification process in wastewater treatment. To retain these slow-growing bacteria in wastewater treatment plants (WWTPs), they are often grown as biofilms, e.g., on nitrifying trickling filters (NTFs) or on carriers in moving bed biofilm reactors (MBBRs). On NTFs, a decreasing ammonium gradient is formed because of the AOB activity, resulting in low ammonium concentrations at the bottom and reduced biomass with depth. To optimize the NTF process, different ammonium feed strategies may be designed. This, however, requires knowledge about AOB population dynamics. Using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy, we followed biomass changes during 6 months, of three AOB populations on biofilm carriers. These were immersed in aerated MBBR tanks in a pilot plant receiving full-scale wastewater. Tanks were arranged in series, forming a wastewater ammonium gradient mimicking an NTF ammonium gradient. The biomass of one of the dominating Nitrosomonas oligotropha-like populations increased after an ammonium upshift, reaching levels comparable to the high ammonium control in 28 days, whereas a Nitrosomonas europaea-like population increased relatively slowly. The MBBR results, together with competition studies in NTF systems fed with wastewater under controlled ammonium regimes, suggest a differentiation between the two N. oligotropha populations, which may be important for WWTP nitrification.     

白洋淀湖滨湿地岸边带氨氧化古菌与氨氧化细菌的分布特性

摘要：本研究通过分子生物学分析方法,以amoA基因为标记,考察了氨氧化古菌（Ammonia-Oxidizing Archaea, AOA）和氨氧化细菌（Ammonia-Oxidizing Bacteria,AOB）在华北平原的白洋淀这一典型湖泊的湖滨湿地岸边带系统中的生物多样性和丰度分布。在前人的研究中,氨氧化古菌在海洋、原生态土壤和人为干扰土壤等环境中主导氨氧化过程的完成。但本研究发现,在湿地岸边带系统中氨氧化过程并不是完全由氨氧化古菌主导完成,即氨氧化古菌和氨氧化细菌在不同区域分别占据主导地位。根据主导微生物的不同,可以将湿地岸边带区域划分为陆相区、中间区和湖相区。在湿地岸边带陆相区,氨氧化古菌主导氨氧化过程,氨氧化古菌的amoA基因丰度是氨氧化细菌的526倍（AOA：1.23?108每克干土;AOB：2.34?105每克干土）;在岸边带湖相区,氨氧化细菌主导氨氧化过程,氨氧化古菌的amoA基因丰度只有氨氧化细菌的1/50倍（AOA：3.17?106每克干土;AOB：1.39?108每克干土）;在岸边带中间区,两种微生物对氨氧化过程的贡献相当,二者的amoA基因丰度也相当 (AOA：9.83?106, AOB：4.08?106)。研究还发现,湿地中间区的微生物生物多样性高于陆相区和湖相区。在湿地中间区,氨氧化古菌和氨氧化细菌的生物多样性都最高,分别有5和7个操作分类单元（OTUs）;相比之下,岸边带陆相区和湖相区的多样性依次降低,陆相区的氨氧化古菌和氨氧化细菌分别有3和6个操作分类单元,湖相区的氨氧化古菌和氨氧化细菌分别有2和6个分类单元。本研究的两个结论进一步反映了湿地岸边带极强的空间异质性。     

Application of cation-exchange membranes for characterisation and imaging ammonia-oxidising bacteria in soils

A new approach, in which ammonia-oxidizing bacteria (AOB) are entrapped from soil onto cation-exchange membranes, was applied to identify terrestrial AOB by fluorescence in situ hybridization (FISH). An experimental hot spot of ammonia oxidation was developed by establishing a gradient of ammonium substrate (200 to <20 mg NH4+-N l(-1)) diffused through the cation-exchange membranes incubated in soil for 6 months. By this approach we were able to characterise and image indigenous AOB populations growing in heavily oil-polluted soil using FISH and sequence analysis of PCR-amplified 16S rRNA genes, respectively. The FISH results revealed that Nitrosospira-like AOB were dominant on the ammonium-enriched membranes incubated in the soil. Fourteen unique Nitrosospira-like 16S rRNA gene sequences belonging to clusters 2 and 3 were recovered from the soil-incubated membranes and from the soil, suggesting the importance of Nitrosospira-like AOB in the oil-polluted landfarming soil.     

Failure of the ammonia oxidation process in two pharmaceutical wastewater treatment plants is linked to shifts in the bacterial communities

AIMS: To investigate whether two different wastewater treatment plants (WWTPs) -- treating the same pharmaceutical influent -- select for a different bacterial and/or ammonia oxidizing bacterial (AOB) community. METHODS AND RESULTS: Molecular fingerprinting demonstrated that each WWTP had its own total bacterial and AOB community structure, but Nitrosomonas eutropha and N. europea were dominant in both WWTP A and B. The DNA and RNA analysis of the AOB communities revealed different patterns; so the most abundant species may not necessarily be the most active ones. Nitritation failures, monitored by chemical parameter analysis, were reflected as AOB community shifts and visualized by denaturing gradient gel electrophoresis (DGGE)-based moving window analysis. CONCLUSIONS: This research demonstrated the link between functional performance (nitritation parameters) and the presence and activity of a specific microbial ecology (AOB). Clustering and moving window analysis based on DGGE showed to be valuable to monitor community shifts in both WWTPs. SIGNIFICANCE AND IMPACT OF THE STUDY: This study of specific community shifts together with functional parameter analysis has potential as a tool for relating functional instability (such as operational failures) to specific-bacterial community shifts.     

Impact of protozoan grazing on nitrification and the ammonia- and nitrite-oxidizing bacterial communities in activated sludge

In activated sludge, protozoa feed on free-swimming bacteria and suspended particles, inducing flocculation and increasing the turnover rate of nutrients. In this study, the effect of protozoan grazing on nitrification rates under various conditions in municipal activated sludge batch reactors was examined, as was the spatial distribution of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) within the activated sludge. The reactors were monitored for ammonia, nitrite, nitrate, and total nitrogen concentrations, and bacterial numbers in the presence and absence of cycloheximide (a protozoan inhibitor), allylthiourea (an inhibitor of ammonia oxidation), and EDTA (a deflocculating agent). The accumulations of nitrate, nitrite, and ammonia were lower in batches without than with protozoa grazing. Inhibition of ammonia oxidation also decreased the amount of nitrite and nitrate accumulation. Inhibiting protozoan grazing along with ammonia oxidation further decreased the amounts of nitrite and nitrate accumulated. Induction of deflocculation led to high nitrate accumulation, indicating high levels of nitrification; this effect was lessened in the absence of protozoan grazing. Using fluorescent in situ hybridization and confocal laser scanning microscopy, AOB and NOB were found clustered within the floc, and inhibiting the protozoa, inhibiting ammonia oxidation, or inducing flocculation did not appear to lower the number of AOB and NOB present or affect their position within the floc. These results suggest that the AOB and NOB are present but less active in the absence of protozoa.     

Architecture of a nascent Sphingomonas sp. biofilm under varied hydrodynamic conditions

The architecture of a Sphingomonas biofilm was studied during early phases of its formation, using strain L138, a gfp-tagged derivative of Sphingomonas sp. strain LB126, as a model organism and flow cells and confocal laser scanning microscopy as experimental tools. Spatial and temporal distribution of cells and exopolymer secretions (EPS) within the biofilm, development of microcolonies under flow conditions representing varied Reynolds numbers, and changes in diffusion length with reference to EPS production were studied by sequential sacrificing of biofilms grown in multichannel flow cells and by time-lapse confocal imaging. The area of biofilm in terms of microscopic images required to ensure representative sampling varied by an order of magnitude when area of cell coverage (2 x 10(5) microm(2)) or microcolony size (1 x 10(6) microm(2)) was the biofilm parameter under investigation. Hence, it is necessary to establish the inherent variability of any biofilm metric one is attempting to quantify. Sphingomonas sp. strain L138 biofilm architecture consisted of microcolonies and extensive water channels. Biomass and EPS distribution were maximal at 8 to 9 mum above the substratum, with a high void fraction near the substratum. Time-lapse confocal imaging and digital image analysis showed that growth of the microcolonies was not uniform: adjacently located colonies registered significant growth or no growth at all. Microcolonies in the biofilm had the ability to move across the attachment surface as a unit, irrespective of fluid flow direction, indicating that movement of microcolonies is an inherent property of the biofilm. Width of water channels decreased as EPS production increased, resulting in increased diffusion distances in the biofilm. Changing hydrodynamic conditions (Reynolds numbers of 0.07, 52, and 87) had no discernible influence on the characteristics of microcolonies (size, shape, or orientation with respect to flow) during the first 24 h of biofilm development. Inherent factors appear to have overriding influence, vis-a-vis environmental factors, on early stages of microcolony development under these laminar flow conditions.     

Identification of bacteria responsible for ammonia oxidation in freshwater aquaria.

Culture enrichments and culture-independent molecular methods were employed to identify and confirm the presence of novel ammonia-oxidizing bacteria (AOB) in nitrifying freshwater aquaria. Reactors were seeded with biomass from freshwater nitrifying systems and enriched for AOB under various conditions of ammonia concentration. Surveys of cloned rRNA genes from the enrichments revealed four major strains of AOB which were phylogenetically related to the Nitrosomonas marina cluster, the Nitrosospira cluster, or the Nitrosomonas europaea-Nitrosococcus mobilis cluster of the beta subdivision of the class Proteobacteria. Ammonia concentration in the reactors determined which AOB strain dominated in an enrichment. Oligonucleotide probes and PCR primer sets specific for the four AOB strains were developed and used to confirm the presence of the AOB strains in the enrichments. Enrichments of the AOB strains were added to newly established aquaria to determine their ability to accelerate the establishment of ammonia oxidation. Enrichments containing the Nitrosomonas marina-like AOB strain were most efficient at accelerating ammonia oxidation in newly established aquaria. Furthermore, if the Nitrosomonas marina-like AOB strain was present in the original enrichment, even one with other AOB, only the Nitrosomonas marina-like AOB strain was present in aquaria after nitrification was established. Nitrosomonas marina-like AOB were 2% or less of the cells detected by fluorescence in situ hybridization analysis in aquaria in which nitrification was well established.     

Community survey of ammonia-oxidizing bacteria in full-scale activated sludge processes with different solids retention time

AIMS: To study the effects of different solids retention time (SRT) on the nitrification activity and community composition of ammonia-oxidizing bacteria (AOB) in two full-scale activated sludge processes during a 5-month period. METHODS AND RESULTS: The AOB community composition was analysed using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE), and the identified populations were enumerated by quantitative FISH. Potential nitrification rates were determined in batch tests and the in situ rates were calculated from mass balances of nitrogen in the plants. Increased SRT reduced the nitrification activity, but neither the number per mixed liquor suspended solids nor community composition of AOB were affected. Two dominant AOB populations related to Nitrosomonas europaea and Nitrosomonas oligotropha were identified by FISH, whereas only the latter could be detected by DGGE. CONCLUSIONS: The effect of a longer SRT on the activity was probably because of physiological changes in the AOB community rather than a change in community composition. SIGNIFICANCE AND IMPACT OF THE STUDY: Physiological alterations of a stable AOB community are possible and may stabilize activated sludge processes. The commonly used FISH probes designed to target all beta-proteobacterial AOB does not detect certain Nitrosomonas oligotropha populations, leading to an underestimation of AOB if a wider set of probes is not used.     

Microcolony epifluorescence microscopy for selective enumeration of injured bacteria in frozen and heat-treated foods.

A rapid (less than 6 h) method for selectively enumerating coliforms, pseudomonads, and staphylococci has been developed which involves counting microcolonies grown on the surface of polycarbonate membranes under selective conditions. The method was not directly applicable to foods containing injured bacteria due to the poor formation of or an inability to form microcolonies under selective conditions. However, the introduction of a 3- to 5-h resuscitation step in tryptone soya broth allowed the method to give reliable estimates of these organisms in a variety of frozen and heat-processed foods. Under nonselective conditions, i.e., for total counts, the microcolony method enabled a rapid count to be made of viable bacteria in heat-treated foods, but these results were also made more consistent by the introduction of a resuscitation step. This method makes results from these foods available far faster than conventional enumeration methods.     

Microcolony epifluorescence microscopy for selective enumeration of injured bacteria in frozen and heat-treated foods

A rapid (less than 6 h) method for selectively enumerating coliforms, pseudomonads, and staphylococci has been developed which involves counting microcolonies grown on the surface of polycarbonate membranes under selective conditions. The method was not directly applicable to foods containing injured bacteria due to the poor formation of or an inability to form microcolonies under selective conditions. However, the introduction of a 3- to 5-h resuscitation step in tryptone soya broth allowed the method to give reliable estimates of these organisms in a variety of frozen and heat-processed foods. Under nonselective conditions, i.e., for total counts, the microcolony method enabled a rapid count to be made of viable bacteria in heat-treated foods, but these results were also made more consistent by the introduction of a resuscitation step. This method makes results from these foods available far faster than conventional enumeration methods.     

Activity,Distribution, and Diversity of Sulfate Reducers and Other Bacteria in Sediments above Gas Hydrate (Cascadia Margin,Oregon)

Cold seep environments such as sediments above outcropping hydrate at Hydrate Ridge (Cascadia margin off Oregon) are characterized by methane venting, high sulfide fluxes caused by the anaerobic oxidation of methane, and the presence of chemosynthetic communities. Recent investigations showed that another characteristic feature of cold seeps is the occurrence of methanotrophic archaea, which can be identified by specific biomarker lipids and 16S rDNA analysis. This investigation deals with the diversity and distribution of sulfate-reducing bacteria, some of which are directly involved in the anaerobic oxidation of methane as syntrophic partners of the methanotrophic archaea. The composition and activity of the microbial communities at methane vented and nonvented sediments are compared by quantitative methods including total cell counts, fluorescence in situ hybridization (FISH), bacterial production, enzyme activity, and sulfate reduction rates. Bacteria involved in the degradation of particulate organic carbon (POC) are as active and diverse as at other productive margin sites of similar water depths. The availability of methane supports a two orders of magnitude higher microbial biomass (up to 9.6 2 10 10 cells cm m 3 ) and sulfate reduction rates (up to 8 w mol cm m 3 d m 1 ) in hydrate-bearing sediments, as well as a high bacterial diversity, especially in the group of i -proteobacteria including members of the branches Desulfosarcina/Desulfococcus , Desulforhopalus , Desulfobulbus , and Desulfocapsa . Most of the diversity of sulfate-reducing bacteria in hydrate-bearing sediments comprises seep-endemic clades, which share only low similarities with previously cultured bacteria.