Culture-based and culture-independent approach for the study of the methanogens and obligate anaerobes from different landfill sites.

The landfill is a cheap way of solid waste management in developing countries. The majority of landfills are non-sanitary and work as open garbage dumping sites and pose threats to public and environmental health. Therefore, an in-depth understanding of the chemistry and microbiology of landfills is imperative to develop the right policies for landfill management. In the current study, we investigated the chemistry and microbiology of three Indian landfill sites using culture-based and culture-independent molecular approaches. Our data indicate that the nature of landfills varies from site to site in terms of chemistry, pollutants, and pathogens. We also enriched and cultivated three methanogens using an optimized medium and constructed two high-quality draft genomes from enriched microbiomes using metagenome-assembled genome approaches. The phylogenomic study of one draft genome showed the highest 93% sequence similarity with members of Methanomassiliicoccaceae and was always enriched with Acholoplasma and Anaerohalosphaera lusitana. Despite all the efforts, we did not isolate it in pure culture and hypothesized that for the cultivation of some not-yet-cultured methanogen, the presence of other organisms plays an important role, and their syntrophic interaction must be discerned for its successful cultivation in the future. Co-cultivation of amino acid-degrading organisms indicates that their co-culture can assist in boosting the growth of methanogens. In addition, our data indicated that landfill leachate contains a heavy load of pollutants and treatment is a must before discharge in nature or use in irrigation or biofertilizer.

A culture-based and culture-independent approach to the study of landfill leachate bacterial and archaeal communities.

The landfill is a convenient and affordable method of municipal solid waste (MSW) management. Landfill leachate contains a heavy load of pollutants and pathogens. Discharge of untreated leachate is the leading cause of surface and groundwater contamination and a threat to public and environmental health. To develop an efficient leachate treatment technology, an in-depth understanding of landfill chemistry and microbiology is essential. In the present manuscript, we conducted a comparative study of three different landfill leachate samples using cultivation-based and culture-independent molecular studies. We cultivated 85 species of aerobic, anaerobic bacteria and archaea from leachate represented by a total of 200 strains using extensive culturomics approaches. Twelve out of 200 cultivated strains of bacteria showed very low 16S rRNA gene sequence similarity (84-98.6%) with their closest relatives and could be the potential novel taxa, the first time cultivated from leachate. Members of the six genera only have 2-5 representatives from past studies from other habitats but first time cultivated from leachate. In addition to bacteria, we also cultivated and characterized different groups of methanogenic archaea. Our chemistry data indicate that leachate is a highly stressed ecosystem with an assemblage of many toxic wastes like sulfur, zinc, mercury, chromium, etc. 16S rRNA gene-based amplicon analysis showed the dominance of (30-55%) methanogens and haloarachaea. Our data suggest that archaea are the significant regulators of leachate ecology, and more in-depth studies with multiple leachate samples are required to understand their role in leachate nutrient cycling and the development of effective leachate treatment technology.

Assessment of the Role of Wastewater Treatment Plant in Spread of Antibiotic Resistance and Bacterial Pathogens.

In current study, we performed a comparative study on bacterial load, total coliform counts and type of organisms present in pre- and post-treated wastewater samples from municipal wastewater treatment plant of Pune, India. In addition, we also studied the antibiotic resistance profiling and role of the selected treatment plant in spread of antibiotic resistance in the environment. Data showed that total 30 different bacterial species from 18-different genera were present in untreated wastewater while only 9 species from 6-different genera were present in post-treated effluent. Furthermore, pre-treated wastewater sample contains wide range of organisms with high levels of antibiotic resistance while bacterial load reduced drastically and pathogens were absent from post-treated effluent.

Enhanced n-butanol production by Clostridium beijerinckii MCMB 581 in presence of selected surfactant.

Extractive butanol fermentation with non-ionic surfactant, a recently explored area, has shown promising results with several advantages but is relatively less investigated. This work reports the extractive fermentation with selected non-ionic surfactants (L62 and L62D) to enhance butanol production using a high-butanol producing strain (Clostridium beijerinckii MCMB 581). Biocompatibility studies with both the surfactants showed growth. Higher concentrations of surfactant (>5%) affected the cell count. 15.3 g L-1 of butanol and 21 g L-1 of total solvents were obtained with 3% (v/v) L62 which was respectively, 43% (w/w) and 55% (w/w), higher than control. It was found that surfactant addition at 9th h doubled the productivity (from 0.13 to 0.31 g L-1 h-1 and 0.17 to 0.39 g L-1 h-1, respectively for butanol and total solvent). Butanol productivity obtained was 2-3 times higher than similar studies on extractive fermentation with non-ionic surfactants. Interestingly, mixing did not improve butanol production.

Deciphering Community Structure of Methanotrophs Dwelling in Rice Rhizospheres of an Indian Rice Field Using Cultivation and Cultivation-Independent Approaches.

Methanotrophs play a crucial role in filtering out methane from habitats, such as flooded rice fields. India has the largest area under rice cultivation in the world; however, to the best of our knowledge, methanotrophs have not been isolated and characterized from Indian rice fields. A cultivation strategy composing of a modified medium, longer incubation time, and serial dilutions in microtiter plates was used to cultivate methanotrophs from a rice rhizosphere sample from a flooded rice field in Western India. We compared the cultured members with the uncultured community as revealed by three culture-independent methods. A novel type Ia methanotroph (Sn10-6), at the rank of a genus, and a putative novel species of a type II methanotroph (Sn-Cys) were cultivated from the terminal positive dilution (10(-6)). From lower dilution (10(-4)), a strain of Methylomonas spp. was cultivated. All the three culture-independent analyses, i.e., pmoA clone library, terminal restriction fragment length polymorphism (T-RFLP), and metagenomics approach, revealed the dominance of type I methanotrophs. Only metagenomic analysis showed significant presence of type II methanotrophs, albeit in lower proportion (37 %). All the three isolates showed relevance to the methanotrophic community as depicted by uncultured methods; however, the cultivated members might not be the most dominant ones. In conclusion, a combined cultivation and cultivation-independent strategy yielded us a broader picture of the methanotrophic community from rice rhizospheres of a flooded rice field in India.

A pmoA-based study reveals dominance of yet uncultured Type I methanotrophs in rhizospheres of an organically fertilized rice field in India.

Rice fields are one of the important sources of anthropogenic methane. Methanotrophs can oxidize up to 30 % of the produced methane and thus have a pivotal environmental role in methane mitigation. India occupies the largest region under rice cultivation; however, most of the studies done on methanotrophic communities have focused on the Northern region. We studied methanotrophic community of a flooded, organically fertilized rice field using pmoA clone library approach. Organic rice fields impose a more serious threat as they produce more methane. pmoA gene is the main functional gene which is primarily used for taxonomical analysis of methanotrophs. Our results showed that the pmoA clone libraries from two growth stages of rice were dominated by pmoA sequences which were very distant from cultivated Type Ia methanotrophic genera (80-82 % nucleotide similarity) indicative of the presence of a putatively novel genus. We designated this group of clones as 'rice field clones' as this also includes many pmoA sequences originating from other rice fields. Thus, our current knowledge of methanotroph diversity from Indian rice fields has been expanded revealing that a substantial portion of methanotrophic diversity is unexplored.

Draft genome sequence of Clostridium sulfidigenes 113A isolated from sub-seafloor sediments associated with methane hydrate deposits.

Clostridium sulfidigenes 113A is a strictly anaerobic, rod shaped, gram positive bacterium isolated from sub-seafloor sediments associated with methane hydrates. Here, we report the first draft genome of C. sulfidigenes strain 113A, which comprises 3,717,420 bp in 96 contigs with the G+C content of 30.1%. A total of 3148 protein coding sequences were predicted. The genome annotation revealed that 113A could play an important role in biogeochemical cycles and have potential biotechnological applications such as production of organic acids and butanol.

Draft genome sequence of Clostridium celerecrescens 152B isolated from sub-seafloor methane hydrate deposits.

Clostridium celerecrescens 152B is an obligate anaerobic, Gram positive rod shaped bacterium isolated from sub-seafloor methane hydrate sediments of Krishna Godavari basin, India. Here, we report the first draft genome sequence of C. celerecrescens 152B, which comprises 5,050,495bp in 92 contigs with the G+C content of 43.5%. The whole genome of C. celerecrescens 152B was sequenced for further biotechnological exploitation of its genome features especially regarding the production of secondary metabolites as well as for environmental bioremediation and production of industrially valuable enzymes.

Genome sequencing of multidrug resistant novel Clostridium sp. BL8 reveals its potential for pathogenicity.

BACKGROUND: The human gut microbiome is important for maintaining the health status of the host. Clostridia are key members of the human gut microbiome, carrying out several important functions in the gut environment. Hence understanding the role of different Clostridium species isolated from human gut is essential. The present study was aimed at investigating the role of novel Clostridium sp. isolate BL8 in human gut using genome sequencing as a tool. FINDINGS: The genome analysis of Clostridium sp. BL8 showed the presence of several adaptive features like bile resistance, presence of sensory and regulatory systems, presence of oxidative stress managing systems and presence of membrane transport systems. The genome of Clostridium sp. BL8 consists of a wide variety of virulence factors like phospholipase C (alpha toxin), hemolysin, aureolysin and exfoliative toxin A, as well as adhesion factors, proteases, Type IV secretion system and antibiotic resistance genes. In vitro antibiotic sensitivity testing showed that Clostridium sp. BL8 was resistant to 11 different tested antibiotics belonging to 6 different classes. The cell cytotoxicity assay confirmed the cytotoxic effect of Clostridium sp. BL8 cells, which killed 40% of the Vero cells after 4 hrs of incubation. CONCLUSIONS: Clostridium sp. BL8 has adapted for survival in human gut environment, with presence of different adaptive features. The presence of several virulence factors and cell cytotoxic activity indicate that Clostridium sp. BL8 has a potential to cause infections in humans, however further in vivo studies are necessary to ascertain this fact.

Development of a microbial process for the recovery of petroleum oil from depleted reservoirs at 91-96°C.

A consortium of bacteria growing at 91°C and above (optimally at 96°C) was developed for the recovery of crude oil from declining/depleted oil reservoirs having temperature of more than 91°C. PCR-DGGE-Sequencing analysis of 16S rRNA gene fragments of NJS-4 consortium revealed the presence of four strains identified as members of the genus Clostridium. The metabolites produced by NJS-4 consortium included volatile fatty acids, organic acids, surfactants, exopolysaccarides and CO2, which reduced viscosity, emulsified crude oil and increased the pressure that facilitated displacement of emulsified oil towards the surface. NJS-4 enhanced oil recovery by 26.7% and 10.1% in sand pack trials and core flood studies respectively in optimized nutrient medium comprised of sucrose and sodium acetate as carbon/energy source and urea as nitrogen source (pH 7-9, 96°C, and 4% salinity). Nutrient medium for MEOR was constituted using commercial grade cheap nutrients to improve the economic viability of MEOR process.

Molecular analysis of gut microbiota in obesity among Indian individuals.

Obesity is a consequence of a complex interplay between the host genome and the prevalent obesogenic factors among the modern communities. The role of gut microbiota in the pathogenesis of the disorder was recently discovered; however, 16S-rRNA-based surveys revealed compelling but community-specific data. Considering this, despite unique diets, dietary habits and an uprising trend in obesity, the Indian counterparts are poorly studied. Here, we report a comparative analysis and quantification of dominant gut microbiota of lean, normal, obese and surgically treated obese individuals of Indian origin. Representative gut microbial diversity was assessed by sequencing fecal 16S rRNA libraries for each group (n=5) with a total of over 3000 sequences. We detected no evident trend in the distribution of the predominant bacterial phyla, Bacteroidetes and Firmicutes. At the genus level, the bacteria of genus Bacteroides were prominent among the obese individuals, which was further confirmed by qPCR (P less than 0.05). In addition, a remarkably high archaeal density with elevated fecal SCFA levels was also noted in the obese group. On the contrary, the treated-obese individuals exhibited comparatively reduced Bacteroides and archaeal counts along with reduced fecal SCFAs. In conclusion, the study successfully identified a representative microbial diversity in the Indian subjects and demonstrated the prominence of certain bacterial groups in obese individuals; nevertheless, further studies are essential to understand their role in obesity.

Novel methanotroph diversity evidenced by molecular characterization of particulate methane monooxygenase A (pmoA) genes in a biogas reactor.

The present study for the first time investigated the diversity of methanotrophs in a biogas reactor running on cattle dung by sequence analysis of the "functional" gene, particulate methane monooxygenase A (pmoA). Community structure was determined by operational taxonomic unit (OTU) phylogenetic analyses of 396 clones belonging to pmoA library. On the basis of 95% peptide identity, 396 inferred PmoA peptide sequences were clustered in 22 phylotypes. These OTUS were phylogenetically affiliated to alpha-proteobacteria (5.30% clones) and gamma-proteobacteria (94.7% clones) and were only distantly related to those of known methanotrophs, indicating the existence of unknown methanotrophs involved in aerobic methanotrophy inside the biogas plant. Good's coverage indicated that the present library covered 96.21% of the dominant species that could be cloned from the biogas reactor. The molecular approaches used in this study provided useful description of the microbial community involved in aerobic oxidation of methane.

Investigation of methanogen population structure in biogas reactor by molecular characterization of methyl-coenzyme M reductase A (mcrA) genes.

The methanogen community in biogas reactor running on cattle dung was investigated in two different seasons; summer (April, 36 degrees C) and winter (December, 24 degrees C), in the year 2004 by a culture-independent approach. Community structure was determined by phylogenetic analyses of 343 and 278 mcrA clones belonging to summer and winter month libraries, respectively. In summer month's library, 41.7% clones were affiliated to Methanomicrobiales, 30% to Methanosarcinales, 19% to Methanobacteriales, 5% to Methanococcales and a total of 4.3% clones belonged to unclassified euryarchaeotal lineages. In winter month's library, Methanomicrobiales encompassed 98.6% clones, and Methanobacteriales included 1.4% of total clone diversity. Biogas plant performance data collected during the winter month indicated significant reduction in daily biogas produced as compared to summer month because of lowering in ambient temperature and associated shift in microbial community. Results from this molecular study showed the existence of highly diverse and complex methanogens communities present in biogas plant.

Phylogenetic analysis of methanogenic enrichment cultures obtained from Lonar Lake in India: isolation of Methanocalculus sp. and Methanoculleus sp.

The diversity of methanogenic archaea in enrichment cultures established from the sediments of Lonar Lake (India), a soda lake having pH approximately 10, was investigated using 16S rDNA molecular phylogenetic approach. Methanogenic enrichment cultures were developed in a medium that simulated conditions of soda lake with three different substrates viz., H(2):CO(2), sodium acetate, and trimethylamine (TMA), at alkaline pH. Archaeal 16S rRNA clone libraries were generated from enrichment cultures and 13 RFLP groups were obtained. Representative sequence analysis of each RFLP group indicated that the majority of the 16S rRNA gene sequences were phylogenetically affiliated with uncultured Archaea. Some of the groups may belong to new archaeal genera or families. Three RFLP groups were related to Methanoculleus sp, while two related to Methanocalculus sp. 16S rRNA gene sequences found in Lonar Lake were different from sequences reported from other soda lakes and more similar to those of oil reservoirs, palm oil waste treatment digesters, and paddy fields. In culture-based studies, three isolates were obtained. Two of these were related to Methanoculleus sp. IIE1 and one to Methanocalculus sp. 01F97C. These results clearly show that the Lonar Lake ecosystem harbors unexplored methanogens.

Comparison of 16S rRNA gene sequences of genus Methanobrevibacter.

BACKGROUND: The phylogeny of the genus Methanobrevibacter was established almost 25 years ago on the basis of the similarities of the 16S rRNA oligonucleotide catalogs. Since then, many 16S rRNA gene sequences of newly isolated strains or clones representing the genus Methanobrevibacter have been deposited. We tried to reorganize the 16S rRNA gene sequences of this genus and revise the taxonomic affiliation of the isolates and clones representing the genus Methanobrevibacter. RESULTS: The phylogenetic analysis of the genus based on 786 bp aligned region from fifty-four representative sequences of the 120 available sequences for the genus revealed seven multi-member groups namely, Ruminantium, Smithii, Woesei, Curvatus, Arboriphilicus, Filiformis, and the Termite gut symbionts along with three separate lineages represented by Mbr. wolinii, Mbr. acididurans, and termite gut flagellate symbiont LHD12. The cophenetic correlation coefficient, a test for the ultrametric properties of the 16S rRNA gene sequences used for the tree was found to be 0.913 indicating the high degree of goodness of fit of the tree topology. A significant relationship was found between the 16S rRNA sequence similarity (S) and the extent of DNA hybridization (D) for the genus with the correlation coefficient (r) for logD and logS, and for [ln(-lnD) and ln(-lnS)] being 0.73 and 0.796 respectively. Our analysis revealed that for this genus, when S = 0.984, D would be <70% at least 99% of the times, and with 70% D as the species "cutoff", any 16S rRNA gene sequence showing <98% sequence similarity can be considered as a separate species. In addition, we deduced group specific signature positions that have remained conserved in evolution of the genus. CONCLUSIONS: A very significant relationship between D and S was found to exist for the genus Methanobrevibacter, implying that it is possible to predict D from S with a known precision for the genus. We propose to include the termite gut flagellate symbiont LHD12, the methanogenic endosymbionts of the ciliate Nyctotherus ovalis, and rat feces isolate RT reported earlier, as separate species of the genus Methanobrevibacter.