Phylogenetically and physiologically diverse methanogenic archaea inhabit the Indian hot spring environments.

Mesophilic and thermophilic methanogens belonging to the hydrogenotrophic, methylotrophic, and acetotrophic groups were isolated from Indian hot spring environments using BY and BCYT growth media. Following initial Hinf I-based PCR-RFLP screening, 70 methanogens were sequenced to ascertain their identity. These methanogens were phylogenetically and physiologically diverse and represented different taxa distributed across three physiological groups, i.e., hydrogenotrophs (53), methylotrophs (14) and acetotrophs (3). Overall, methanogens representing three families, five genera, and ten species, including two putative novel species, were recognized. The highest number and diversity of methanogens was observed at 40 â„ƒ, dominated by Methanobacterium (10; 3 species), Methanosarcina (9; 3 species), Methanothermobacter (7; 2 species), Methanomethylovorans (5; 1 species) and Methanoculleus (3; 1 species). Both putative novel methanogen species were isolated at 40 â„ƒ and belonged to the genera Methanosarcina and Methanobacterium. At 55 â„ƒ, limited diversity was observed, and resulted in the isolation of only two genera of methanogens, i.e., Methanothermobacter (28; 2 species) and Methanosarcina (4; 1 species). At 70 â„ƒ, only members of the genus Methanothermobacter (5; 2 species) were isolated, whereas no methanogen could be cultured at 85 â„ƒ. Ours is the first study that documents the extensive range of cultivable methanogenic archaea inhabiting hot springs across various geothermal provinces of India.

Pseudodesulfovibrio thermohalotolerans sp. nov., a novel obligately anaerobic, halotolerant, thermotolerant, and sulfate-reducing bacterium isolated from a western offshore hydrocarbon reservoir in India.

OBJECTIVE: Characterization and documentation of strain MCM B-1480T, a novel sulfate-reducing bacterium isolated from produced water of India's western offshore hydrocarbon reservoir. METHOD: Strain MCM B-1480T was unequivocally identified using a polyphasic approach routinely followed in bacterial systematics. The morphological and biochemical characterization of strain MCM B-1480T was carried out using standard microbiological techniques. RESULTS: MCM B-1480T was a Gram-stain-negative, motile, non-spore-forming, curved-rod-shaped bacterium. MCM B-1480T could grow at temperatures between 20 and 60 °C (optimum 37 °C), pH 6-8 (optimum 7), and required 1-6% NaCl (optimum 3%) for growth. Strain MCM B-1480T was reducing sulfate to produce hydrogen sulfide during growth. This strain used lactate and pyruvate as prominent electron donors, whereas sulfate, sulfite, thiosulfate, and nitrate served as electron acceptors. MCM B-1480T shared maximum 16S rRNA gene sequence homology of 98.65% with the members of the genus Pseudodesulfovibrio. The G + C content of the 3.87 Mb MCM B-1480T genome was 60.39%. Digital DDH (27.7%) and average nucleotide identity (ANI 84%) with the closest phylogenetic affiliate (less than 70% and 95%, respectively) reaffirmed its distinctiveness. The major cellular fatty acids components, namely iso-C15:0, anteiso-C15:0, C16:0, and anteiso-C17:0, differentiated strain MCM B-1480T from other species of Pseudodesulfovibrio. Genome annotation revealed the presence of genes encoding dissimilatory sulfate reduction and nitrate reduction in strain MCM B-1480T. CONCLUSION: The polyphasic studies, including SSU rRNA gene sequencing, average nucleotide identity, Digital DNA-DNA hybridization, cell wall fatty acids analysis, etc., identified strain MCM B-1480T as a novel taxon and Pseudodesulfovibrio thermohalotolerans sp. nov. was proposed (= JCM 39269T = MCC 4711T).

Description of Sporanaerobium hydrogeniformans gen. nov., sp. nov., an obligately anaerobic, hydrogen-producing bacterium isolated from Aravali hot spring in India.

An obligately anaerobic bacterium XHS1971T, capable of degrading cellulose and xylan, was isolated from a sediment sample of Aravali hot spring, Ratnagiri, India. Cells of strain XHS1971T were Gram-stain-negative, spore-forming, motile, long-rods. Growth was observed at temperatures 30-50 °C (optimum 40-45 °C), pH 5.0-10.0 (optimum pH 8.0) and NaCl concentrations 0-0.5% (optimum 0%). Generation time of strain XHS1971T was 5 h under optimised growth conditions. Strain XHS1971T showed the ability to metabolise different complex and simple sugars constituting lignocellulosic biomass. Glucose was fermented majorly into hydrogen, formic acid, acetic acid, and ethanol, whereas carbon dioxide, butyric acid, lactic acid and succinic acid were produced in traces. 16S rRNA gene analysis of strain XHS1971T revealed < 94.5% homology with Cellulosilyticum lentocellum DSM5427T followed by Cellulosilyticum ruminicola JCM14822T, identifying strain as a distinct member of family Lachnospiraceae. The major cellular fatty acids (> 5%) were C14:0, C16:0, C18:0, and C16:1 ω7c. The genome size of the strain was 3.74 Mb with 35.3 mol% G + C content, and genes were annotated to carbohydrate metabolism, including genes involved in the degradation of cellulose and xylan and the production of hydrogen, ethanol and acetate. The uniqueness of strain was further validated by digital DNA-DNA hybridisation (dDDH), Average Nucleotide Identity (ANI), and Average Amino Acid Identity (AAI) values of 22%, 80%, and 63%, respectively, with nearest phylogenetic affiliates. Based on the detailed analyses, we propose a new genus and species, Sporanaerobium hydrogeniformans gen. nov., sp. nov., for strain XHS1971T (= MCC3498T = KCTC15729T = JCM32657T) within family Lachnospiraceae.

Patterns and determinants of the global herbivorous mycobiome.

Despite their role in host nutrition, the anaerobic gut fungal (AGF) component of the herbivorous gut microbiome remains poorly characterized. Here, to examine global patterns and determinants of AGF diversity, we generate and analyze an amplicon dataset from 661 fecal samples from 34 mammalian species, 9 families, and 6 continents. We identify 56 novel genera, greatly expanding AGF diversity beyond current estimates (31 genera and candidate genera). Community structure analysis indicates that host phylogenetic affiliation, not domestication status and biogeography, shapes the community rather than. Fungal-host associations are stronger and more specific in hindgut fermenters than in foregut fermenters. Transcriptomics-enabled phylogenomic and molecular clock analyses of 52 strains from 14 genera indicate that most genera with preferences for hindgut hosts evolved earlier (44-58 Mya) than those with preferences for foregut hosts (22-32 Mya). Our results greatly expand the documented scope of AGF diversity and provide an ecologically and evolutionary-grounded model to explain the observed patterns of AGF diversity in extant animal hosts.

Actinomyces ruminis sp. nov., an obligately anaerobic bacterium isolated from the rumen of cattle.

An obligately anaerobic, rod-shaped, Gram-stain-positive, non-spore-forming, non-motile bacterial strain; designated as CtC72T was isolated from the rumen of cattle. The 16S rRNA gene sequence similarity of less than 98.65% revealed the strain as a member of the genus Actinomyces, nearest to but distinct from Actinomyces qiguomingii DSM 106201T, Actinomyces ruminicola DSM 27982T, Actinomyces procaprae JCM 33484T, Actinomyces succiniciruminis TISTR 2317, Actinomyces glycerinitolerans TISTR 2318. The low values of digital DNA-DNA hybridization (< 70%) and average nucleotide identity (< 95%) further highlighted the distinctive nature of strain CtC72T from its closest relatives. The strain CtC72T could grow at temperatures between 30 and 50 °C (optimum 40 °C), pH between 6.0 and 9.0 (optimum 7.5-8.0), and NaCl between 0 and 1.5% (optimum 0%). The strain hydrolysed cellulose and xylan and utilised a range of mono-, di-, and oligo-saccharides as a source of carbon and energy. Glucose fermentation resulted in acetic acid and formic acid as major metabolic products, while propionic acid, lactic acid, and ethanol as minor products along with CO2 production. The DNA G + C content of strain CtC72T was 68.40 (mol%, Tm) and 68.05 (%, digital). Major cellular fatty acids (> 10%) were C16:0, C18:1 ω9c, and C18:1 ω9c DMA. Based on these data, we propose that strain CtC72T be classified as a novel species, Actinomyces ruminis sp. nov., under the genus Actinomyces. The type strain is CtC72T (= KCTC 15726T = JCM 32641T = MCC 3500T).

Genomic architecture of three newly isolated unclassified Butyrivibrio species elucidate their potential role in the rumen ecosystem.

One cellulose-degrading strain CB08 and two xylan-degrading strains XB500-5 and X503 were isolated from buffalo rumen. All the strains were designated as putative novel species of Butyrivibrio based on phylogeny, phylogenomy, digital DNA-DNA hybridization, and average nucleotide identity with their closest type strains. The draft genome length of CB08 was ~3.54 Mb, while X503 and XB500-5 genome sizes were ~3.24 Mb and ~3.27 Mb, respectively. Only 68.28% of total orthologous clusters were shared among three genomes, and 40-44% of genes were identified as hypothetical proteins. The presence of genes encoding diverse carbohydrate-active enzymes (CAZymes) exhibited the lignocellulolytic potential of these strains. Further, the genome annotations revealed the metabolic pathways for monosaccharide fermentation to acetate, butyrate, lactate, ethanol, and hydrogen. The presence of genes for chemotaxis, antibiotic resistance, antimicrobial activity, synthesis of vitamins, and essential fatty acid suggested the versatile metabolic nature of these Butyrivibrio strains in the rumen environment.

Genomics and simulated laboratory studies reveal Thermococcus sp. 101C5 as a novel hyperthermophilic archaeon possessing a specialized metabolic arsenal for enhanced oil recovery.

Laboratory evaluation of hyperthermophiles with the potential for Enhanced Oil Recovery (EOR) is often hampered by the difficulties in replicating the in situ growth conditions in the laboratory. In the present investigation, genome analysis was used to gain insights into the metabolic potential of a hyperthermophile to mobilize the residual oil from depleting high-temperature oil reservoirs. Here, we report the 1.9 Mb draft genome sequence of a hyperthermophilic anaerobic archaeon, Thermococcus sp. 101C5, with a GC content of 44%, isolated from a high-temperature oil reservoir of Gujarat, India. 101C5 possessed the genetic arsenal required for adaptation to harsh oil reservoir conditions, such as various heat shock proteins for thermo-adaptation, Trk potassium uptake system proteins for osmo-adaptation, and superoxide reductases against oxidative stress. Microbial Enhanced Oil Recovery (MEOR) potential of the strain was established by ascertaining the presence of genes encoding enzymes involved in the production of the metabolites such as hydrogen, bio-emulsifier, acetate, exopolysaccharide, etc. Production of these metabolites which pressurize the reservoir, emulsify the crude oil, lower the viscosity and reduce the drag, thus facilitating mobilization of the residual oil was experimentally confirmed. Also, the presence of crude oil degradative genes highlighted the ability of the strain to mobilize heavy residual oil, which was confirmed under simulated conditions in sand-pack studies. The obtained results demonstrated additional oil recoveries of 42.1% and 56.5% at 96 °C and 101 °C, respectively, by the strain 101C5, illustrating its potential for application in high-temperature oil reservoirs. To our best knowledge, this is the first report of genome analysis of any microbe assessed for its suitability for MEOR from the high-temperature oil reservoir.

Genome sequencing and analysis of a psychrotrophic methanogen Methanosarcina sp. nov. MSH10X1 cultured from methane hydrate deposits of Krishna Godavari Basin of India.

Methanosarcina sp. strain MSH10X1, a psychrotrophic methanogen, was isolated from sub-seafloor methane hydrate deposits of Krishna Godavari Basin on India's east coast. The strain could grow from 5 to 40 °C following all three i.e. methylotrophic, acetoclastic, and hydrogenotrophic modes of methanogenesis utilizing different substrates like methanol, trimethylamine, H2/CO2 (80/20), acetate, valerate, isobutyrate, isopropanol, and isobutanol. The genome sequencing and analysis of this strain revealed a circular chromosome of 3,557,383 bp length having 42.47 mol% G + C content, which consisted of 3110 coding genes, 58 tRNA genes, and 3 rRNA operons. The KEGG analysis highlighted the presence of genes responsible for all three modes of methanogenesis. The presence of genes like mtaB, mtaC, and mttB in the genome provided evidence for possible adaptation of strain MSH10X1 in the deep sea's low-temperature conditions.

Meta-omics based analyses of microbiome involved in biomethanation of rice straw in a thermophilic anaerobic bioreactor under optimized conditions.

Biomethanation of rice straw was performed at 55 °C without thermochemical pretreatment using cattle dung supplemented with Methanothermobacter thermautotrophicus strains. Methane yield of 323 ml g-1 VS obtained under optimized conditions such as particle size (1 mm), carbon to nitrogen ratio (15:1), substrate to inoculum ratio (1:1), organic loading rate (7.5% w/v) and hydraulic retention time (20 days), was one of the highest ever reported from rice straw. Metagenome analysis revealed several putative novel taxa among resident microbes. The genomes of Clostridium, Hungateiclostridium, Alkaliphilus, Anaerocolumna, Olsenella, Paenibacillus, Pseudoclostridium, Tepidanaerobacter and Turicibacter were recovered as metagenome assisted genomes. Clostridium spp. and M. thermautotrophicus were the dominant hydrolytic and methanogenic microbes, respectively. Syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis was found to be the major pathway for methane production. Efficient thermophilic biomethanation of rice straw without thermochemical pretreatment using cattle dung supplemented with M. thermautotrophicus is reported for the first time.

Liebetanzomycespolymorphus gen. et sp. nov., a new anaerobic fungus (Neocallimastigomycota) isolated from the rumen of a goat.

An extended incubation strategy to culture slow growing members of anaerobic fungi resulted in the isolation of a novel anaerobic fungus from the rumen of a goat after 15 days. The novel genus, represented by type strain G1SC, showed filamentous monocentric thallus development and produced uniflagellate zoospores, hence, showing morphological similarity to the genera Piromyces, Buwchfawromyces, Oontomyces and Pecoramyces. However, strain G1SC showed genetic similarity to the genus Anaeromyces, which, though produces uniflagellate zoospore, also exhibits polycentric thallus development. Moreover, unlike Anaeromyces, strain G1SC did not show hyphal constrictions, instead produced a branched, determinate and anucleate rhizoidal system. This fungus also displayed extensive sporangial variations, both exogenous and endogenous type of development, short and long sporangiophores and produced septate sporangia. G1SC utilised various complex and simple substrates, including rice straw and wheat straw and produced H2, CO2, formate, acetate, lactate, succinate and ethanol. Phylogenetic analysis, using internal transcribed spacer 1 (ITS1) and D1/D2 domain of large-subunit (LSU) rRNA locus, clearly showed a separate lineage for this strain, near Anaeromyces. The ITS1 based geographical distribution studies indicated detection of environmental sequences similar (93-96%) to this strain from cattle faeces. Based on morphological and molecular characterisation results of strain G1SC, we propose a novel anaerobic fungus Liebetanzomycespolymorphus gen. et sp. nov., in the phylum Neocallimastigomycota.

Comparative diversity analysis of ruminal methanogens in Murrah buffaloes (Bubalus bubalis) in four states of North India.

We compared the community structure of methanogens in Murrah breed of buffaloes of four states of north India using 16S rRNA gene clone library method. The results revealed the dominance of methanogens related to Methanobrevibacter in three states, while Methanomicrobium-related methanogens were abundant in one state.

Cultivation of multiple genera of hydrogenotrophic methanogens from different environmental niches.

Six genera of hydrogenotrophic methanogens, namely Methanobrevibacter, Methanobacterium, Methanocorpusculum, Methanothermobacter, Methanoculleus, and Methanospirillum were cultivated from diverse environmental niches like rumen, feces, gut, and sediments using BY medium. We also report a putative novel genus and two novel species of methanogens isolated from termite, Indian star tortoise, and green iguana.

Comparative evaluation of lignocellulolytic activities of filamentous cultures of monocentric and polycentric anaerobic fungi.

Sixteen strains of monocentric and polycentric anaerobic fungi were evaluated for cellulase, xylanase and esterase activities. Though strain level variations were observed among all genera, Neocallimastix and Orpinomyces strains exhibited the highest lignocellulolytic activities. The esterase activities of monocentric group of anaerobic fungi were better than the polycentric group.

New aspects and strategies for methane mitigation from ruminants.

The growing demand for sustainable animal production is compelling researchers to explore the potential approaches to reduce emissions of greenhouse gases from livestock that are mainly produced by enteric fermentation. Some potential solutions, for instance, the use of chemical inhibitors to reduce methanogenesis, are not feasible in routine use due to their toxicity to ruminants, inhibition of efficient rumen function or other transitory effects. Strategies, such as use of plant secondary metabolites and dietary manipulations have emerged to reduce the methane emission, but these still require extensive research before these can be recommended and deployed in the livestock industry sector. Furthermore, immunization vaccines for methanogens and phages are also under investigation for mitigation of enteric methanogenesis. The increasing knowledge of methanogenic diversity in rumen, DNA sequencing technologies and bioinformatics have paved the way for chemogenomic strategies by targeting methane producers. Chemogenomics will help in finding target enzymes and proteins, which will further assist in the screening of natural as well chemical inhibitors. The construction of a methanogenic gene catalogue through these approaches is an attainable objective. This will lead to understand the microbiome function, its relation with the host and feeds, and therefore, will form the basis of practically viable and eco-friendly methane mitigation approaches, while improving the ruminant productivity.

Changes in methane emission, rumen fermentation in response to diet and microbial interactions.

To evaluate relative contributions of different microbial groups in rumen, the mono-culture (i.e. bacteria, protozoa and fungi) and co-cultures (i.e. bacterial-protozoal, fungal-protozoal and bacterial-fungal) were tested in vitro using high and low roughage diets. Total gas and methane were higher in bacterial-fungal and bacterial-protozoal co-cultures, while lower in fungal-protozoal than controls (high and low roughage with complete rumen consortia; control 1 and 2, respectively). Digestibility and total volatile fatty acids were lower in bacterial-fungal co-culture with both high and low roughage diets. Methanogens decreased in bacterial-fungal co-culture with high roughage. With high roughage, counts were lower for bacteria with bacterial-protozoal, protozoa with fungal-protozoal, and fungi with the bacterial-fungal co-cultures. Total gas was higher in bacterial mono-culture with low roughage, but methane was not detected in any mono-culture. Digestibility and total volatile fatty acids were significantly lowered with protozoal mono-culture. Methanogens reduced significantly in mono-cultures with high roughage diet than control 1. Defaunation reduced methanogens without significantly affecting rumen fermentation.

Molecular tools for deciphering the microbial community structure and diversity in rumen ecosystem.

Rumen microbial community comprising of bacteria, archaea, fungi, and protozoa is characterized not only by the high population density but also by the remarkable diversity and the most complex microecological interactions existing in the biological world. This unprecedented biodiversity is quite far from full elucidation as only about 15-20 % of the rumen microbes are identified and characterized till date using conventional culturing and microscopy. However, the last two decades have witnessed a paradigm shift from cumbersome and time-consuming classical methods to nucleic acid-based molecular approaches for deciphering the rumen microbial community. These techniques are rapid, reproducible and allow both the qualitative and quantitative assessment of microbial diversity. This review describes the different molecular methods and their applications in elucidating the rumen microbial community.

Enumeration of methanogens with a focus on fluorescence in situ hybridization.

Methanogens, the members of domain Archaea are potent contributors in global warming. Being confined to the strict anaerobic environment, their direct cultivation as pure culture is quite difficult. Therefore, a range of culture-independent methods have been developed to investigate their numbers, substrate uptake patterns, and identification in complex microbial communities. Unlike other approaches, fluorescence in situ hybridization (FISH) is not only used for faster quantification and accurate identification but also to reveal the physiological properties and spatiotemporal dynamics of methanogens in their natural environment. Aside from the methodological aspects and application of FISH, this review also focuses on culture-dependent and -independent techniques employed in enumerating methanogens along with associated problems. In addition, the combination of FISH with micro-autoradiography that could also be an important tool in investigating the activities of methanogens is also discussed.