P.R. Pandya

Metagenomic analysis of Surti buffalo (Bubalus bubalis) rumen: a preliminary study

The complex microbiome of the rumen functions as an effective system for the conversion of plant cell wall biomass to microbial proteins, short chain fatty acids and gases. In this study, metagenomic approaches were used to study the microbial populations and metabolic potential of the microbial community. DNA was extracted from Surti Buffalo rumen samples (four treatments diet) and sequenced separately using a 454 GS FLX Titanium system. We used comparative metagenomics to examine metabolic potential and phylogenetic composition from pyrosequence data generated in four samples, considering phylogenetic composition and metabolic potentials in the rumen may remarkably be different with respect to nutrient utilization. Assignment of metagenomic sequences to SEED categories of the Metagenome Rapid Annotation using Subsystem Technology (MG-RAST) server revealed a genetic profile characteristic of fermentation of carbohydrates in a high roughage diet. The distribution of phylotypes and environmental gene tags (EGTs) detected within each rumen sample were dominated by Bacteroidetes/Chlorobi, Firmicutes and Proteobacteria in all the samples. The results of this study could help to determine the role of rumen microbes and their enzymes in plant polysaccharide breakdown is fundamental to understanding digestion and maximising productivity in ruminant animals.

Microbial and Carbohydrate Active Enzyme profile of buffalo rumen metagenome and their alteration in response to variation in the diet

Rumen microbiome represents rich source of enzymes degrading complex plant polysaccharides. We describe here analysis of Carbohydrate Active Enzymes (CAZymes) from 3.5 gigabase sequences of metagenomic data from rumen samples of Mehsani buffaloes fed on different proportions of green or dry roughages to concentrate ration. A total of 2597 contigs encoding putative CAZymes were identified by CAZyme Analysis Toolkit (CAT). The phylogenetic analysis of these contigs by MG-RAST revealed predominance of Bacteroidetes, followed by Firmicutes, Proteobacteria, and Actinobacteria phyla. Moreover, a higher abundance of oligosaccharide degrading and debranching enzymes in buffalo rumen metagenome and that of cellulases and hemicellulases in termite hindgut was observed when we compared glycoside hydrolase (GH) profile of buffalo rumen metagenome with cow rumen, termite hindgut and chicken caecum metagenome. Further, comparison of microbial profile of green or dry roughage fed animals showed significantly higher abundance (p-value<0.05) of various polysaccharide degrading bacterial genera like Fibrobacter, Prevotella, Bacteroides, Clostridium and Ruminococcus in green roughage fed animals. In addition, we found a significantly higher abundance (p-value<0.05) of enzymes associated with pectin digestion such as pectin lyase (PL) 1, PL10 and GH28 in green roughage fed animals. Our study outlines CAZyme profile of buffalo rumen metagenome and provides a scope to study the role of abundant enzyme families (oligosaccharide degrading and debranching enzymes) in digestion of coarse feed.

Methanogen diversity in the rumen of Indian Surti buffalo (Bubalus bubalis), assessed by 16S rDNA analysis.

The methanogenic communities in buffalo rumen were characterized using a culture-independent approach of a pooled sample of rumen fluid from three adult Surti buffaloes. Buffalo rumen is likely to include species of various methanogens, so 16S rDNA sequences were amplified and cloned from the sample. A total of 171 clones were sequenced to examine 16S rDNA sequence similarity. About 52.63% sequences (90 clones) had ≥ 90% similarity, whereas, 46.78% of the sequences (81 clones) were 75-89% similar to 16S rDNA database sequences, respectively. Phylogenetic analyses were also used to infer the makeup of methanogenic communities in the rumen of Surti buffalo. As a result, we distinguished 23 operational taxonomic units (OTUs) based on unique 16S rDNA sequences: 12 OTUs (52.17%) affiliated to Methanomicrobiales order, 10 OTUs (43.47%) of the order Methanobacteriales and one OTU (4.34%) of Methanosarcina barkeri like clone, respectively. In addition, the population of Methanomicrobiales and Methabacteriales orders were also observed, accounting 4% and 2.17% of total archea. This study has revealed the largest assortment of hydrogenotrophic methanogens phylotypes ever identified from rumen of Surti buffaloes.

Molecular identification of methanogenic archaea from surti buffaloes (bubalus bubalis), reveals more hydrogenotrophic methanogens phylotypes.

Methane emissions from ruminant livestock are considered to be one of the more potent forms of greenhouses gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, a thorough knowledge of the diversity of these microbes in breeds of buffaloes, as well as in response to geographical location and different diets, is required. Therefore, molecular diversity of rumen methanogens in Surti buffaloes was investigated using 16S rRNA gene libraries prepared from pooled rumen contents from three Surti buffaloes. A total of 171 clones were identified revealing 23 different sequences (phylotypes). Of these 23 sequences, twelve sequences (12 OTUs, 83 clones) and 10 sequences (10 OTUs, 83 clones) were similar to methanogens belonging to the orders Methanomicrobiales and Methanobacteriales, and the remaining 1 phylotype (5 clones) were similar to Methanosarcina barkeri. These unique sequences clustered within a distinct and strongly supported phylogenetic group. Further studies and effective strategies can be made to inhibit the growth of Methanomicrobiales and Methanobacteriales phylotypes to reduce the methane emission from rumen and thus help in preventing global warming.

Influence of Supplementing Solid State Fermented Biomass on Digestibility, Microbial Nitrogen Supply and Enteric Methane Emissions in Cattle

Present study was carried out to evaluate the effect of supplementing solid state fermented (SSF) biomass on nutrient intake, digestibility, rumen fermentation patterns, microbial nitrogen supply and methane emissions in crossbred cattle. Six non-lactating crossbred cattle (445.6 kg BW) were used in a replicated 3 × 3 latin square design. Animals in control group (T1) were fed total mixed ration (TMR) containing concentrates and wheat straw in 40: 60 ratio. Animals in group T2 were fed TMR containing concentrates, wheat straw and groundnut straw in 40: 30: 30 ratio whereas the animals in group T3 were fed TMR containing concentrates, wheat straw, groundnut straw and SSF biomass in a ratio of 40: 25: 30: 5. The intakes of nutrients and rumen parameters were not affected by the treatments. Digestibility coefficient of DM, OM, CF, NDF and cellulose improved by 8.9 (P<0.05), 9.3 (P<0.05), 13.0 (P<0.01), 11.0 (P<0.01) and 10.1% (P<0.01) respectively in group T3 as compared to group T1. Intestinal flow of microbial nitrogen supply improved (P<0.01) by 13.3 and 15.6% whereas enteric methane emissions (g/d) reduced (P<0.01) by 7.8 and 11.7% in groups T2 and T3. respectively as compared to group T1. Similarly, energy loss as methane (% of gross energy intake) was lower (P<0.01) by 10.6 and 15.1%, respectively in groups T2 and T3 than group T1. Hence, inclusion of legume (groundnut) straw alone or with SSF biomass in whest straw based diet helped in improving nutrient digestibility and reducing enteric methane emissions in crossbred cattle.

Influence of Incorporating Different Levels of Solid State Fermented Biomass in Total Mixed Rations on In Vitro Digestibility and Methane Production

The aim of this study was to evaluate the effect of incorporating different levels of solid state fermented (SSF) biomass on in vitro digestibility and methane (CH4) production. The total mixed rations (TMRs) were prepared using concentrates (40%), wheat straw (30%), groundnut straw (30%) and were used as substrate for in vitro studies. The SSF biomass was prepared using Aspergillus oryzae and Trichoderm areesei spp. of fungi. The SSF biomass having enzyme carboxymethyl cellulase having activity of 680 IU/g was incorporated in TMRs @ 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% by replacing wheat straw on part basis. The in vitro apparent and true dry matter digestibility (DMD) of TMR increased with increasing levels of SSF biomass in the TMRs. The highest in vitro apparent and true DMD was found at inclusion of 5.0% SSF biomass which increased significantly (P<0.01) by 14.36 and 12.92%, respectively. Digestibility of OM increased significantly by 13.94% (P<0.01) as compared to control group. With increasing level of SSF biomass, in vitro CH4 production reduced as compared to control TMR and maximum reduction was observed at an inclusion level of 5.0% SSF level. In vitro CH4 production (mL/100 mg DM) reduced (P<0.01) by 16.91% than the value observed in control group. Methane production in terms of mL/100 mg apparent and true DMD reduced by 27.55 and 26.80%, respectively as compared to control group. Hence, solid state fermentation biomass improved digestibility and reduced methane production under in vitro conditions, however, in vivo studies are required for such confirmation.

In Vitro Digestibility and Methane Production as Affected by Incorporation of Different Levels of Solid State Fermented Biomass in Total Mixed Rations

The aim of this study was to evaluate the effect of incorporating different levels of solid state fermented (SSF) biomass on in vitro digestibility and methane (CH4) production. The total mixed rations (TMRs) were prepared using concentrates (40%), wheat straw (30%), groundnut straw (30%) and used as substrate for in vitro studies. The SSF biomass was prepared using Aspergillus oryzae and Trichoderma reesei spp. of fungi. The enzyme carboxymethyl cellulose having activity of 680 IU/g was incorporated in TMRs @ 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% by replacing wheat straw on part basis. The in vitro apparent and true dry matter digestibility (DMD) of TMR increased with increasing levels of SSF biomass in the TMR. The highest in vitro apparent and true DMD was found at inclusion of 5% SSF biomass which increased (P<0.01) by 14.36 and 12.92%, respectively. Digestibility of OM increased (P<0.01) by 13.94% as compared to control group. With increasing levels of SSF biomass, in vitro CH4 production reduced as compared to control TMR and the maximum reduction was observed at inclusion level of 5% SSF level. In vitro CH4 production (mL/100 mg DM) reduced (P<0.01) by 16.91% than the value observed in control group. Methane production in terms of mL/100 mg apparent and true DMD reduced (P<0.01) by 27.55 and 26.80%, respectively as compared to control group. Therefore, inclusion of SSF biomass in the TMRs at 5% level has the potential for improving digestibility and reducing CH4 production in ruminants, however, in vivo studies need to be conducted for substantiation of in vitro results.