A diverse bacterial community in an anoxic quinoline-degrading bioreactor determined by using pyrosequencing and clone library analysis

There is a concern of whether the structure and diversity of a microbial community can be effectively revealed by short-length pyrosequencing reads. In this study, we performed a microbial community analysis on a sample from a high-efficiency denitrifying quinoline-degrading bioreactor and compared the results generated by pyrosequencing with those generated by clone library technology. By both technologies, 16S rRNA gene analysis indicated that the bacteria in the sample were closely related to, for example, Proteobacteria, Actinobacteria, and Bacteroidetes. The sequences belonging to Rhodococcus were the most predominant, and Pseudomonas, Sphingomonas, Acidovorax, and Zoogloea were also abundant. Both methods revealed a similar overall bacterial community structure. However, the 622 pyrosequencing reads of the hypervariable V3 region of the 16S rRNA gene revealed much higher bacterial diversity than the 130 sequences from the full-length 16S rRNA gene clone library. The 92 operational taxonomic unit (OTUs) detected using pyrosequencing belonged to 45 families, whereas the 37 OTUs found in the clone library belonged to 25 families. Most sequences obtained from the clone library had equivalents in the pyrosequencing reads. However, 64 OTUs detected by pyrosequencing were not represented in the clone library. Our results demonstrate that pyrosequencing of the V3 region of the 16S rRNA gene is not only a powerful tool for discovering low-abundance bacterial populations but is also reliable for dissecting the bacterial community structure in a wastewater environment.     

Analyses of bacterial communities in meju,a Korean traditional fermented soybean bricks,by cultivation-based and pyrosequencing methods

Despite the importance of meju as a raw material used to make Korean soy sauce (ganjang) and soybean paste (doenjang), little is known about the bacterial diversity of Korean meju. In this study, the bacterial communities in meju were examined using both culture-dependent and independent methods in order to evaluate the diversity of the bacterial population. Analyses of the 16S rRNA gene sequences of the bacterial strains isolated from meju samples showed that the dominant species were related to members of the genera Bacillus, Enterococcus, and Pediococcus. The community DNAs extracted from nine different meju samples were analyzed by barcoded pyrosequencing method targeting of the V1 to V3 hypervariable regions of the 16S rRNA gene. In total, 132,374 sequences, with an average read length of 468 bp, were assigned to several phyla, with Firmicutes (93.6%) representing the predominant phylum, followed by Proteobacteria (4.5%) and Bacteroidetes (0.8%). Other phyla accounted for less than 1% of the total bacterial sequences. Most of the Firmicutes were Bacillus and lactic acid bacteria, mainly represented by members of the genera Enterococcus, Lactococcus, and Leuconostoc, whose ratio varied among different samples. In conclusion, this study indicated that the bacterial communities in meju were very diverse and a complex microbial consortium containing various microorganisms got involved in meju fermentation than we expected before.     

Biodiversity in Oscypek, a traditional Polish cheese, determined by culture-dependent and -independent approaches

Oscypek is a traditional Polish scalded-smoked cheese, with a protected-designation-of-origin (PDO) status, manufactured from raw sheep's milk without starter cultures in the Tatra Mountains region of Poland. This study was undertaken in order to gain insight into the microbiota that develops and evolves during the manufacture and ripening stages of Oscypek. To this end, we made use of both culturing and the culture-independent methods of PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing of 16S rRNA gene amplicons. The culture-dependent technique and PCR-DGGE fingerprinting detected the predominant microorganisms in traditional Oscypek, whereas the next-generation sequencing technique (454 pyrosequencing) revealed greater bacterial diversity. Besides members of the most abundant bacterial genera in dairy products, e.g., Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, and Enterococcus, identified by all three methods, other, subdominant bacteria belonging to the families Bifidobacteriaceae and Moraxellaceae (mostly Enhydrobacter), as well as various minor bacteria, were identified by pyrosequencing. The presence of bifidobacterial sequences in a cheese system is reported for the first time. In addition to bacteria, a great diversity of yeast species was demonstrated in Oscypek by the PCR-DGGE method. Culturing methods enabled the determination of a number of viable microorganisms from different microbial groups and their isolation for potential future applications in specific cheese starter cultures.     

16S rRNA基因在微生物生态学中的应用

16S rRNA(Small subunit ribosomal RNA)基因是对原核微生物进行系统进化分类研究时最常用的分子标志物(Biomarker),广泛应用于微生物生态学研究中。近些年来随着高通量测序技术及数据分析方法等的不断进步,大量基于16S rRNA基因的研究使得微生物生态学得到了快速发展,然而使用16S rRNA基因作为分子标志物时也存在诸多问题,比如水平基因转移、多拷贝的异质性、基因扩增效率的差异、数据分析方法的选择等,这些问题影响了微生物群落组成和多样性分析时的准确性。对当前使用16S rRNA基因分析微生物群落组成和多样性的进展情况做一总结,重点讨论当前存在的主要问题以及各种分析方法的发展,尤其是与高通量测序技术有关的实验和数据处理问题。     

Influence of DNA Extraction Method, 16S rRNA Targeted Hypervariable Regions,and Sample Origin on Microbial Diversity Detected by 454 Pyrosequencing in Marine Chemosynthetic Ecosystems

Next-generation sequencing (NGS) opens up exciting possibilities for improving our knowledge of environmental microbial diversity, allowing rapid and cost-effective identification of both cultivated and uncultivated microorganisms. However, library preparation, sequencing, and analysis of the results can provide inaccurate representations of the studied community compositions. Therefore, all these steps need to be taken into account carefully. Here we evaluated the effects of DNA extraction methods, targeted 16S rRNA hypervariable regions, and sample origins on the diverse microbes detected by 454 pyrosequencing in marine cold seep and hydrothermal vent sediments. To assign the reads with enough taxonomic precision, we built a database with about 2,500 sequences from Archaea and Bacteria from deep-sea marine sediments, affiliated according to reference publications in the field. Thanks to statistical and diversity analyses as well as inference of operational taxonomic unit (OTU) networks, we show that (i) while DNA extraction methods do not seem to affect the results for some samples, they can lead to dramatic changes for others; and (ii) the choice of amplification and sequencing primers also considerably affects the microbial community detected in the samples. Thereby, very different proportions of pyrosequencing reads were obtained for some microbial lineages, such as the archaeal ANME-1, ANME-2c, and MBG-D and deltaproteobacterial subgroups. This work clearly indicates that the results from sequencing-based analyses, such as pyrosequencing, should be interpreted very carefully. Therefore, the combination of NGS with complementary approaches, such as fluorescence in situ hybridization (FISH)/catalyzed reporter deposition (CARD)-FISH or quantitative PCR (Q-PCR), would be desirable to gain a more comprehensive picture of environmental microbial communities.     

Generation of multimillion-sequence 16S rRNA gene libraries from complex microbial communities by assembling paired-end illumina reads

Microbial communities host unparalleled taxonomic diversity. Adequate characterization of environmental and host-associated samples remains a challenge for microbiologists, despite the advent of 16S rRNA gene sequencing. In order to increase the depth of sampling for diverse bacterial communities, we developed a method for sequencing and assembling millions of paired-end reads from the 16S rRNA gene (spanning the V3 region; ～200 nucleotides) by using an Illumina genome analyzer. To confirm reproducibility and to identify a suitable computational pipeline for data analysis, sequence libraries were prepared in duplicate for both a defined mixture of DNAs from known cultured bacterial isolates (>1 million postassembly sequences) and an Arctic tundra soil sample (>6 million postassembly sequences). The Illumina 16S rRNA gene libraries represent a substantial increase in number of sequences over all extant next-generation sequencing approaches (e.g., 454 pyrosequencing), while the assembly of paired-end 125-base reads offers a methodological advantage by incorporating an initial quality control step for each 16S rRNA gene sequence. This method incorporates indexed primers to enable the characterization of multiple microbial communities in a single flow cell lane, may be modified readily to target other variable regions or genes, and demonstrates unprecedented and economical access to DNAs from organisms that exist at low relative abundances.     

Characterization of the fecal bacteria communities of forage-fed horses by pyrosequencing of 16S rRNA V4 gene amplicons

The diversity of the equine fecal bacterial community was evaluated using pyrosequencing of 16S rRNA gene amplicons. Fecal samples were obtained from horses fed cool-season grass hay. Fecal bacteria were characterized by amplifying the V4 region of bacterial 16S rRNA gene. Of 5898 mean unique sequences, a mean of 1510 operational taxonomic units were identified in the four fecal samples. Equine fecal bacterial richness was higher than that reported in humans, but lower than that reported in either cattle feces or soil. Bacterial classified sequences were assigned to 16 phyla, of which 10 were present in all samples. The largest number of reads belonged to Firmicutes (43.7% of total bacterial sequences), Verrucomicrobia (4.1%), Proteobacteria (3.8%), and Bacteroidetes (3.7%). The less abundant Actinobacteria, Cyanobacteria, and TM7 phyla presented here have not been previously described in the gut contents or feces of horses. Unclassified sequences represented 38.1% of total bacterial sequences; therefore, the equine fecal microbiome diversity is likely greater than that described. This is the first study to characterize the fecal bacterial community in horses by the use of 16S rRNA gene amplicon pyrosequencing, expanding our knowledge of the fecal microbiota of forage-fed horses.     

Salinity and Bacterial Diversity: To What Extent Does the Concentration of Salt Affect the Bacterial Community in a Saline Soil?

In this study, the evaluation of soil characteristics was coupled with a pyrosequencing analysis of the V2-V3 16S rRNA gene region in order to investigate the bacterial community structure and diversity in the A horizon of a natural saline soil located in Sicily (Italy). The main aim of the research was to assess the organisation and diversity of microbial taxa using a spatial scale that revealed physical and chemical heterogeneity of the habitat under investigation. The results provided information on the type of distribution of different bacterial groups as a function of spatial gradients of soil salinity and pH. The analysis of bacterial 16S rRNA showed differences in bacterial composition and diversity due to a variable salt concentration in the soil. The bacterial community showed a statistically significant spatial variability. Some bacterial phyla appeared spread in the whole area, whatever the salinity gradient. It emerged therefore that a patchy saline soil can not contain just a single microbial community selected to withstand extreme osmotic phenomena, but many communities that can be variously correlated to one or more environmental parameters. Sequences have been deposited to the SRA database and can be accessed on ID Project PRJNA241061.     

Endophyte microbiome diversity in micropropagated Atriplex canescens and Atriplex torreyi var griffithsii

Microbial diversity associated with micropropagated Atriplex species was assessed using microscopy, isolate culturing, and sequencing. Light, electron, and confocal microscopy revealed microbial cells in aseptically regenerated leaves and roots. Clone libraries and tag-encoded FLX amplicon pyrosequencing (TEFAP) analysis amplified sequences from callus homologous to diverse fungal and bacterial taxa. Culturing isolated some seed borne endophyte taxa which could be readily propagated apart from the host. Microbial cells were observed within biofilm-like residues associated with plant cell surfaces and intercellular spaces. Various universal primers amplified both plant and microbial sequences, with different primers revealing different patterns of fungal diversity. Bacterial and fungal TEFAP followed by alignment with sequences from curated databases revealed 7 bacterial and 17 ascomycete taxa in A. canescens, and 5 bacterial taxa in A. torreyi. Additional diversity was observed among isolates and clone libraries. Micropropagated Atriplex retains a complex, intimately associated microbiome which includes diverse strains well poised to interact in manners that influence host physiology. Microbiome analysis was facilitated by high throughput sequencing methods, but primer biases continue to limit recovery of diverse sequences from even moderately complex communities.     

Pyrosequencing Reveals Diverse and Distinct Sponge-Specific Microbial Communities in Sponges from a Single Geographical Location in Irish Waters

Marine sponges are host to numerically vast and phylogenetically diverse bacterial communities, with 26 major phyla to date having been found in close association with sponge species worldwide. Analyses of these microbial communities have revealed many sponge-specific novel genera and species. These endosymbiotic microbes are believed to play significant roles in sponge physiology including the production of an array of bioactive secondary metabolites. Here, we report on the use of culture-based and culture-independent (pyrosequencing) techniques to elucidate the bacterial community profiles associated with the marine sponges Raspailia ramosa and Stelligera stuposa sampled from a single geographical location in Irish waters and with ambient seawater. To date, little is known about the microbial ecology of sponges of these genera. Culture isolation grossly underestimated sponge-associated bacterial diversity. Four bacterial phyla (Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria) were represented amongst ~200 isolates, compared with ten phyla found using pyrosequencing. Long average read lengths of ~430 bp (V1-V3 region of 16S rRNA gene) allowed for robust resolution of sequences to genus level. Bacterial OTUs (2,109 total), at 95% sequence similarity, from ten bacterial phyla were recovered from R. ramosa, 349 OTUs were identified in S. stuposa representing eight phyla, while 533 OTUs from six phyla were found in surrounding seawater. Bacterial communities differed significantly between sponge species and the seawater. Analysis of the data for sponge-specific taxa revealed that 2.8% of classified reads from the sponge R. ramosa can be defined as sponge-specific, while 26% of S. stuposa sequences represent sponge-specific bacteria. Novel sponge-specific clusters were identified, whereas the majority of previously reported sponge-specific clusters (e.g. Poribacteria) were absent from these sponge species. This deep and robust analysis provides further evidence that the microbial communities associated with marine sponge species are highly diverse and divergent from one another and appear to be host-selected through as yet unknown processes.     

Microarray analysis and barcoded pyrosequencing provide consistent microbial profiles depending on the source of human intestinal samples

Large-scale and in-depth characterization of the intestinal microbiota necessitates application of high-throughput 16S rRNA gene-based technologies, such as barcoded pyrosequencing and phylogenetic microarray analysis. In this study, the two techniques were compared and contrasted for analysis of the bacterial composition in three fecal and three small intestinal samples from human individuals. As PCR remains a crucial step in sample preparation for both techniques, different forward primers were used for amplification to assess their impact on microbial profiling results. An average of 7,944 pyrosequences, spanning the V1 and V2 region of 16S rRNA genes, was obtained per sample. Although primer choice in barcoded pyrosequencing did not affect species richness and diversity estimates, detection of Actinobacteria strongly depended on the selected primer. Microbial profiles obtained by pyrosequencing and phylogenetic microarray analysis (HITChip) correlated strongly for fecal and ileal lumen samples but were less concordant for ileostomy effluent. Quantitative PCR was employed to investigate the deviations in profiling between pyrosequencing and HITChip analysis. Since cloning and sequencing of random 16S rRNA genes from ileostomy effluent confirmed the presence of novel intestinal phylotypes detected by pyrosequencing, especially those belonging to the Veillonella group, the divergence between pyrosequencing and the HITChip is likely due to the relatively low number of available 16S rRNA gene sequences of small intestinal origin in the DNA databases that were used for HITChip probe design. Overall, this study demonstrated that equivalent biological conclusions are obtained by high-throughput profiling of microbial communities, independent of technology or primer choice.     

Microbial diversity in degraded and non-degraded petroleum samples and comparison across oil reservoirs at local and global scales

Microorganisms have shown their ability to colonize extreme environments including deep subsurface petroleum reservoirs. Physicochemical parameters may vary greatly among petroleum reservoirs worldwide and so do the microbial communities inhabiting these different environments. The present work aimed at the characterization of the microbiota in biodegraded and non-degraded petroleum samples from three Brazilian reservoirs and the comparison of microbial community diversity across oil reservoirs at local and global scales using 16S rRNA clone libraries. The analysis of 620 16S rRNA bacterial and archaeal sequences obtained from Brazilian oil samples revealed 42 bacterial OTUs and 21 archaeal OTUs. The bacterial community from the degraded oil was more diverse than the non-degraded samples. Non-degraded oil samples were overwhelmingly dominated by gammaproteobacterial sequences with a predominance of the genera Marinobacter and Marinobacterium. Comparisons of microbial diversity among oil reservoirs worldwide suggested an apparent correlation of prokaryotic communities with reservoir temperature and depth and no influence of geographic distance among reservoirs. The detailed analysis of the phylogenetic diversity across reservoirs allowed us to define a core microbiome encompassing three bacterial classes (Gammaproteobacteria, Clostridia, and Bacteroidia) and one archaeal class (Methanomicrobia) ubiquitous in petroleum reservoirs and presumably owning the abilities to sustain life in these environments.

     

Members of the Candidate Division OP10 are spread in a variety of environments

Our planet holds a huge bacterial diversity. Most of these bacteria have only been detected by their 16S rRNA gene sequences remaining to be cultured. Many are classified within Candidate Divisions. One them is the Candidate Division OP10. Analysis of environmental 16S rRNA gene sequences available in public repositories revealed the existence of numerous sequences clustering within the Candidate Division OP10 but currently unclassified or assigned to other bacterial phyla. Newly proposed 16S rRNA sequences multiply several fold the reported sequences for the Candidate Division OP10. This study showed that the Candidate Division OP10 is a diverse and broadly distributed bacterial phylum and represents a stable microbial component in different natural environments.     

Characterization of the rumen microbiota of pre-ruminant calves using metagenomic tools

The temporal sequence of microbial establishment in the rumen of the neonatal ruminant has important ecological and pathophysiological implications. In this study, we characterized the rumen microbiota of pre-ruminant calves fed milk replacer using two approaches, pyrosequencing of hypervariable V3-V5 regions of the 16S rRNA gene and whole-genome shotgun approach. Fifteen bacterial phyla were identified in the microbiota of pre-ruminant calves. Bacteroidetes was the predominant phylum in the rumen microbiota of 42-day-old calves, representing 74.8% of the 16S sequences, followed by Firmicutes (12.0%), Proteobacteria (10.4%), Verrucomicrobia (1.2%) and Synergistetes (1.1%). However, the phylum-level composition of 14-day-old calves was distinctly different. A total of 170 bacterial genera were identified while the core microbiome of pre-ruminant calves included 45 genera. Rumen development seemingly had a significant impact on microbial diversity. The dazzling functional diversity of the rumen microbiota was reflected by identification of 8298 Pfam and 3670 COG protein families. The rumen microbiota of pre-ruminant calves displayed a considerable compositional heterogeneity during early development. This is evidenced by a profound difference in rumen microbial composition between the two age groups. However, all functional classes between the two age groups had a remarkably similar assignment, suggesting that rumen microbial communities of pre-ruminant calves maintained a stable function and metabolic potentials while their phylogenetic composition fluctuated greatly. The presence of all major types of rumen microorganisms suggests that the rumen of pre-ruminant calves may not be rudimentary. Our results provide insight into rumen microbiota dynamics and will facilitate efforts in formulating optimal early-weaning strategies.     

Pyrosequencing analysis of bacterial diversity in 14 wastewater treatment systems in china

To determine if there is a core microbial community in the microbial populations of different wastewater treatment plants (WWTPs) and to investigate the effects of wastewater characteristics, operational parameters, and geographic locations on microbial communities, activated sludge samples were collected from 14 wastewater treatment systems located in 4 cities in China. High-throughput pyrosequencing was used to examine the 16S rRNA genes of bacteria in the wastewater treatment systems. Our results showed that there were 60 genera of bacterial populations commonly shared by all 14 samples, including Ferruginibacter, Prosthecobacter, Zoogloea, Subdivision 3 genera incertae sedis, Gp4, Gp6, etc., indicating that there is a core microbial community in the microbial populations of WWTPs at different geographic locations. The canonical correspondence analysis (CCA) results showed that the bacterial community variance correlated most strongly with water temperature, conductivity, pH, and dissolved oxygen (DO) content. Variance partitioning analyses suggested that wastewater characteristics had the greatest contribution to the bacterial community variance, explaining 25.7% of the variance of bacterial communities independently, followed by operational parameters (23.9%) and geographic location (14.7%). Results of this study provided insights into the bacterial community structure and diversity in geographically distributed WWTPs and discerned the relationships between bacterial community and environmental variables in WWTPs.     

Alkane Biodegradation Genes from Chronically Polluted Subantarctic Coastal Sediments and Their Shifts in Response to Oil Exposure

Although sediments are the natural hydrocarbon sink in the marine environment, the ecology of hydrocarbon-degrading bacteria in sediments is poorly understood, especially in cold regions. We studied the diversity of alkane-degrading bacterial populations and their response to oil exposure in sediments of a chronically polluted Subantarctic coastal environment, by analyzing alkane monooxygenase (alkB) gene libraries. Sequences from the sediment clone libraries were affiliated with genes described in Proteobacteria and Actinobacteria, with 67?% amino acid identity in average to sequences from isolated microorganisms. The majority of the sequences were most closely related to uncultured microorganisms from cold marine sediments or soils from high latitude regions, highlighting the role of temperature in the structuring of this bacterial guild. The distribution of alkB sequences among samples of different sites and years, and selection after experimental oil exposure allowed us to identify ecologically relevant alkB genes in Subantarctic sediments, which could be used as biomarkers for alkane biodegradation in this environment. 16?S rRNA amplicon pyrosequencing indicated the abundance of several genera for which no alkB genes have yet been described (Oleispira, Thalassospira) or that have not been previously associated with oil biodegradation (Spongiibacter-formerly Melitea-, Maribius, Robiginitomaculum, Bizionia and Gillisia). These genera constitute candidates for future work involving identification of hydrocarbon biodegradation pathway genes.     

A glimpse under the rim – the composition of microbial biofilm communities in domestic toilets

Aim: To determine the microbial composition of biofilms in domestic toilets by molecular means. Methods and Results: Genomic DNA was extracted from six biofilm samples originating from households around Düsseldorf, Germany. While no archaeal 16S rRNA or fungal ITS genes were detected by PCR, fingerprinting of bacterial 16S rRNA genes revealed a diverse community in all samples. These communities also differed considerably between the six biofilms. Using the Ribosomal Database Project (RDP) classifier tool, 275 cloned 16S rRNA gene sequences were assigned to 11 bacterial phyla and 104 bacterial genera. Only 15 genera (representing 121 sequences affiliated with Acidobacteria, Actinobacteria, Bacteroidetes, Planctomycetes and Proteobacteria) occurred in at least half of the samples or contributed at least 10% of the sequences in a single biofilm. These sequences were defined as ‘typical’ for toilet biofilms, and they were examined in more detail. On a 97% sequence similarity level, these sequences represented 56 species. Twelve of these were closely related to well‐described bacterial species, and only two of them were categorized as belonging to risk group 2. No 16S rRNA genes of typical faecal bacteria were detected in any sample. Virtually all ‘typical’ clones were found to be closely related to bacteria or to sequences obtained from environmental sources, implicating that the flushing water is the main source of recruitment. Conclusion: In view of the great diversity of mostly yet‐uncultured bacteria and the considerable differences between individual toilets, very general strategies appear to be most suited for the removal and prevention of toilet biofilms. Significance and Impact of the Study: For the first time, a molecular fingerprinting and cloning approach was used to monitor the species composition in biofilm samples taken from domestic toilets. Knowledge about the microbial composition of biofilms in domestic toilets is a prerequisite for developing and evaluating strategies for their removal and prevention.     

High through put 16S rRNA gene-based pyrosequencing analysis of the fecal microbiota of high FCR and low FCR broiler growers

The performance of birds appears to vary among the flock of growing broilers which may in part be due to variation in their gut microbiota. In the view of poultry industry, it is desirable to minimise such variation. We investigated metagenomic profile of fecal bacteria in birds with high and low feed conversion ratio (FCR) to identify microbial community linked to low and high FCR by employing high throughput pyrosequencing of 16S rRNA genomic targets. Therefore feeding trial was investigated in order to identify fecal bacteria consistently linked with better feed conversion ratio in bird performance as measured by body weight gain. High-throughput 16S rRNA gene based pyrosequencing was used to provide a comparative analysis of fecal microbial diversity. The fecal microbial community of birds was predominated by Proteobacteria (48.04?% in high FCR and 49.98?% in low FCR), Firmicutes (26.17?% in high FCR and 36.23?% in low FCR), Bacteroidetes (18.62?% in high FCR and 11.66?% in low FCR), as well as unclassified bacteria (15.77?% in high FCR and 14.29?% in low FCR), suggesting that a large portion of fecal microbiota is novel and could be involved in currently unknown functions. The most prevalent bacterial classes in high FCR and low FCR were Gammaproteobacteria, Clostridia and Bacteroidia. However in low FCR birds Phascolarctobacterium, Faecalibacterium and Clostridium predominated among the Clostridia. In FCR comparison of fecal bacteria, about 36 genera were differentially abundant between high and low FCR birds. This information could be used to formulate effective strategies to improve feed efficiency and feed formulation for optimal gut health.     

Microbial diversity in sediments associated with a shallow methane seep in the tropical Timor Sea of Australia reveals a novel aerobic methanotroph diversity

This study examined the diversity of Bacteria, Archaea and in particular aerobic methanotrophs associated with a shallow (84 m) methane seep in the tropical Timor Sea, Australia. Seepage of thermogenic methane was associated with a large carbonate hardground covered in coarse carbonate-rich sediments and various benthic organisms such as solitary corals. The diversity of Bacteria and Archaea was studied by analysis of cloned 16S rRNA genes, while aerobic methanotrophic bacteria were quantified using real-time PCR targeting the α-subunit of particulate methane monooxygenase ( pmoA ) genes and diversity was studied by analysis of cloned pmoA genes. Phylogenetic analysis of bacterial and archaeal 16S rRNA genes revealed diverse and mostly novel phylotypes related to sequences previously recovered from marine sediments. A small number of bacterial 16S rRNA gene sequences were related to aerobic methanotrophs distantly related to the genera Methylococcus and Methylocaldum . Real-time PCR targeting pmoA genes showed that the highest numbers of methanotrophs were present in surface sediments associated with the seep area. Phylogenetic analysis of pmoA sequences revealed that all phylotypes were novel and fell into two large clusters comprised of only marine sequences distantly related to the genera Methylococcus and Methylocaldum that were clearly divergent from terrestrial phylotypes. This study provides evidence for the existence of a novel microbial diversity and diverse aerobic methanotrophs that appear to constitute marine specialized lineages.