Dilip R. Ranade

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 < 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.

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 ≈ 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., H2:CO2, 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.

Comparative Genome Analysis of Megasphaera sp. Reveals Niche Specialization and Its Potential Role in the Human Gut

With increasing number of novel bacteria being isolated from the human gut ecosystem, there is a greater need to study their role in the gut ecosystem and their effect on the host health. In the present study, we carried out in silico genome-wide analysis of two novel Megasphaera sp. isolates NM10 (DSM25563) and BL7 (DSM25562), isolated from feces of two healthy individuals and validated the key features by in vitro studies. The analysis revealed the general metabolic potential, adaptive features and the potential effects of these isolates on the host. The comparative genome analysis of the two human gut isolates NM10 and BL7 with ruminal isolate Megasphaera elsdenii (DSM20460) highlighted the differential adaptive features for their survival in human gut. The key findings include features like bile resistance, presence of various sensory and regulatory systems, stress response systems, membrane transporters and resistance to antibiotics. Comparison of the “glycobiome” based on the genomes of the ruminal isolate with the human gut isolates NM10 and BL revealed the presence of diverse and unique sets of Carbohydrate-Active enzymes (CAZymes) amongst these isolates, with a higher collection of CAZymes in the human gut isolates. This could be attributed to the difference in host diet and thereby the environment, consequently suggesting host specific adaptation in these isolates. In silico analysis of metabolic potential predicted the ability of these isolates to produce important metabolites like short chain fatty acids (butyrate, acetate, formate, and caproate), vitamins and essential amino acids, which was further validated by in vitro experiments. The ability of these isolates to produce important metabolites advocates for a potential healthy influence on the host. Further in vivo studies including transcriptomic and proteomic analysis will be required for better understanding the role and impact of these Megasphaera sp. isolates NM10 and BL7 on the human host.

Changes in human gut flora with age: an Indian familial study.

BackgroundThe gut micro flora plays vital role in health status of the host. The majority of microbes residing in the gut have a profound influence on human physiology and nutrition. Different human ethnic groups vary in genetic makeup as well as the environmental conditions they live in. The gut flora changes with genetic makeup and environmental factors and hence it is necessary to understand the composition of gut flora of different ethnic groups. Indian population is different in physiology from western population (YY paradox) and thus the gut flora in Indian population is likely to differ from the extensively studied gut flora in western population. In this study we have investigated the gut flora of two Indian families, each with three individuals belonging to successive generations and living under the same roof.ResultsDenaturation gradient gel electrophoresis analysis showed age-dependant variation in gut microflora amongst the individuals within a family. Different bacterial genera were dominant in the individual of varying age in clone library analysis. Obligate anaerobes isolated from individuals within a family showed age related differences in isolation pattern, with 27% (6 out of 22) of the isolates being potential novel species based on 16S rRNA gene sequence. In qPCR a consistent decrease in Firmicutes number and increase in Bacteroidetes number with increasing age was observed in our subjects, this pattern of change in Firmicutes / Bacteroidetes ratio with age is different than previously reported in European population.ConclusionThere is change in gut flora with age amongst the individuals within a family. The isolation of high percent of novel bacterial species and the pattern of change in Firmicutes /Bacteroidetes ratio with age suggests that the composition of gut flora in Indian individuals may be different than the western population. Thus, further extensive study is needed to define the gut flora in Indian population.

Evaluation of the use of sodium molybdate to inhibit sulphate reduction during anaerobic digestion of distillery waste

Biogas produced during anaerobic treatment of sulphate containing wastes, such as distillery waste, invariably contains around 1–3% (v/v) H2S. Being highly corrosive, H2S reduces the utility of biogas as a fuel for boilers and also makes the generation of electricity from biogas very problematic. Sodium molybdate is known to inhibit sulphate reduction and H2S production. To evaluate the efficacy of molybdate during anaerobic digestion of distillery waste, a single dose of 3 mM sodium molybdate was given to the well stabilized anaerobic digester. The digester was run at the loading rate of 1.33 kg COD m−3 d−1 on the distillery waste which had a sulphate content of 10 g l−1. A single dose of 3 mM molybdate inhibited production of H2S for 9 days. Continuous dosing of 3 mM molybdate inhibited H2S production for only 11 days after which H2S was again produced, while methane production declined from day 66. The inhibitor was bacteriostatic to both SRB and acetoclastic methanogens, but more toxic to SRB. It did not inhibit hydrolytic bacteria.

Upflow anaerobic filter for the degradation of adsorbable organic halides (AOX) from bleach composite wastewater of pulp and paper industry.

The removal of AOX from bleach plant effluent of pulp and paper industry was studied using upflow anaerobic filter. In this paper biodegradation of AOX at different concentrations and effect of electron donors like acetate and glucose thereon in an upflow anaerobic filter at 20 d HRT is described. Results showed significant improvement in AOX degradation when electron donors such as acetate and glucose were supplemented to the influent. AOX degradation was 88% at 28 mg AOX L(-1) and 28% at 42 mg AOX L(-1). The percent degradation efficiency was enhanced to 90.7, 90.2, and 93.0 at 28 mg AOX L(-1) when the influent was supplemented with glucose, acetate and both glucose and acetate, respectively. Similarly, the efficiency was 57, 56.6 and 79.6 at 42 mg AOX L(-1) when the influent was supplemented with glucose, acetate and both glucose and acetate, respectively. The GC-MS analysis data indicated that supplementation of the influent with electron donor increased the biodegradability of number of chlorinated organic compounds.

Selenomonas lipolytica sp. nov., an obligately anaerobic bacterium possessing lipolytic activity

A novel, oligately anaerobic bacterium capable of hydrolysing lipids was isolated from a tropical anaerobic lagoon receiving waste water from an edible oil mill. The isolate had many characteristics similar to those of members of the genus Selenomonas. The isolate showed lipolytic activity on tributyrin, triolein and groundnut oil in qualitative plate clearance assays, which has not been reported for the type strain of the genus Selenomonas. It did not require n-valerate supplementation for growth on glucose. Acetate and propionate were the only volatile fatty acids produced from glucose fermentation with propionate as the major end product. The isolate could grow optimally at pH 6.8 and at a temperature of 40 degrees C. It could tolerate NaCl concentrations of up to 40 g l-1. The G&C content of the DNA was 40 mol% as determined by thermal denaturation analysis. Comparison of partial 165 rRNA gene sequences revealed that the isolate was most closely related to genus Selenomonas with 91% sequence similarity (250 bp compared) to Selenomonas ruminantium strain GA 192. On the basis of the results obtained in the present investigation, it is suggested that a new species of Selenomonas should be created for this novel isolate and the name Selenomonas lipolytica is proposed for this new species. The type strain is strain CF1BT (= MCMB 505T).

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.

Megasphaera indica sp. nov., an obligate anaerobic bacteria isolated from human faeces

Two coccoid, non-motile, obligately anaerobic, Gram-stain-negative bacteria, occurring singly or in pairs, or as short chains, with a mean size of 1.4-2.5 µm were isolated from the faeces of two healthy human volunteers, aged 26 and 56 years, and were designated NMBHI-10(T) and BLPYG-7, respectively. Both the strains were affiliated to the sub-branch Sporomusa of the class Clostridia as revealed by 16S rRNA gene sequence analysis. The isolates NMBHI-10(T) and BLPYG-7 showed 99.1 and 99.2% 16S rRNA gene sequence similarity, respectively, with Megasphaera elsdenii JCM 1772(T). DNA-DNA hybridization and phenotypic analysis showed that both the strains were distinct from their closest relative, M. elsdenii JCM 1772(T) (42 and 53% DNA-DNA relatedness with NMBHI-10(T) and BLPYG-7, respectively), but belong to the same species (DNA-DNA relatedness of 80.9 % between the isolates). According to DNA-DNA hybridization results, the coccoid strains belong to the same genospecies, and neither is related to any of the recognized species of the genus Megasphaera. Strains NMBHI-10(T) and BLPYG-7 grew in PYG broth at temperatures of between 15 and 40 °C (optimum 37 °C), but not at 45 °C. The strains utilized a range of carbohydrates as sources of carbon and energy including glucose, lactose, cellobiose, rhamnose, galactose and sucrose. Glucose fermentation resulted in the formation of volatile fatty acids, mainly caproic acid and organic acids such as succinic acid. Phylogenetic analysis, specific phenotypic characteristics and/or DNA G+C content also differentiated the strains from each other and from their closest relatives. The DNA G+C contents of strains NMBHI-10(T) and BLPYG-7 are 57.7 and 54.9 mol%, respectively. The major fatty acids were 12 : 0 FAME and 17 : 0 CYC FAME. On the basis of these data, we conclude that strains NMBHI-10(T) and BLPYG-7 should be classified as representing a novel species of the genus Megasphaera, for which the name Megsphaera indica sp. nov. is proposed. The type strain is NMBHI-10(T) ( = DSM 25563(T) = MCC 2481(T)).

Isolation of propionate degrading bacterium in co-culture with a methanogen from a cattle dung biogas plant.

Various anaerobic hydrolytic and methanogenic bacteria active in cattle dung biogas plants are reported in the literature. Anaerobic bacteria with ability to use volatile fatty acids constitute a vital bridge between hydrolytic bacteria and methanogenic bacteria. The present paper describes the isolation ofSyntrophobacter wolinii a propionate degrading bacterium in co-culture with a hydrogen utilizing methanogenviz.,Methanobacterium formicicum from the fermenting slurry of cattle dung biogas plant. Earlier studies on propionate and butyrate degradation indicatedMethanospirillum hungatei as the hydrogen utilizing partner of the co-culture whereas in the present studies this was not the case. Temperature 35° C, pH 7.5 and 20 mM of propionate were found optimal for growth and activity of co-culture.