Sunil Kumar Sirohi

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.

Diversity analysis of methanogens in rumen of Bubalus bubalis by 16S riboprinting and sequence analysis

The molecular diversity of rumen methanogens was investigated by 16S rDNA gene library prepared from the rumen contents obtained from Murrah buffaloes in India. Genomic DNA was isolated from adult male fistulated buffaloes and PCR conditions were set up using specific primers. Amplified product was cloned into a suitable vector, and the positive clones were selected assuming based on blue-white screening and sequenced. Positive clones were reamplified and the resulting PCR products were further subjected to Amplified Ribosomal DNA Restriction Analysis (ARDRA) by using HaeIII enzyme. A total of 108 clones were examined, and the analysis revealed 16 phylotypes. Out of sixteen phylotypes, nine phylotypes belong to the uncultured group of methanogens, and the rest of seven phylotypes belong to the order Methanomicrobiales, Methanococcales and Methanobacteriales. Out of the 108 rDNA clones, 66 clones which constitute 61.1% of the total clone representing 9 phylotypes, show less than 97% sequence similarity with any of the cultured strain of methanogens. The second largest group of clones (24 clones) represented by four phylotypes show a sequence similarity ranging from 91% to 99% with Methanomicrobium mobile strain of methanogens. The third group of 16S rDNA clones clustered along with M. burtonii strain of methanogens. This group consists of 6 clones and constitutes about 5.5% of the total clones and represented by only single phylotype. Fourth and fifth clusters of 16S rDNA clones consist of 5 and 7 clones respectively, and these were matched with Methanobrevibacter gottschalkii and Methanobrevibacter rumanatium strain of methanogens and constitute about 4.6% and 6.4% of the total clones.

Rumen methanogens: a review

The Methanogens are a diverse group of organisms found in anaerobic environments such as anaerobic sludge digester, wet wood of trees, sewage, rumen, black mud, black sea sediments, etc which utilize carbon dioxide and hydrogen and produce methane. They are nutritionally fastidious anaerobes with the redox potential below −300 mV and usually grow at pH range of 6.0–8.0 [1]. Substrates utilized for growth and methane production include hydrogen, formate, methanol, methylamine, acetate, etc. They metabolize only restricted range of substrates and are poorly characterized with respect to other metabolic, biochemical and molecular properties.

The 16S rRNA and mcrA gene based comparative diversity of methanogens in cattle fed on high fibre based diet.

In the present study, the diversity of rumen methanogens in crossbred Karan Fries cattle was determined by constructing 16S rRNA and mcrA (methyl coenzyme-M reductase α subunit) gene libraries using specific primers. All thirteen OTUs or phylotypes from 16S rRNA library clustered with order Methanobacteriales, twelve of which aligned with Methanobrevibacter spp., whereas one OTU resemble with Methanosphaera stadtmanae. Out of eighteen OTUs identified from mcrA gene library, fifteen clustered with order Methanobacteriales, two resemble with Methanomicrobiales and remaining one grouped with Methanosarcinales. These results revealed that Methanobrevibacter phylotype was predominantly present in Karan Fries crossbred cattle fed on high fibrous diet containing wheat straw. Compared to 16S rRNA gene, mcrA gene OTUs clustered in three orders providing better insights of rumen methanogens diversity in cattle.

Performance of crossbred calves with dietary supplementation of garlic extract.

Twelve crossbred calves (Holstein cross) in their pre-ruminant stage were used to study the effect of garlic extract feeding on their performance and they were randomly allotted into treatment and control groups in equal number. Performance was evaluated by measuring average body weight (BW) gain, feed intake [dry matter (DM); total digestible nutrient (TDN) and crude protein (CP)], feed conversion efficiency (DM, TDN and CP), fecal score and fecal coliform count. Diets were same for both groups. In addition, treatment group received garlic extract supplementation at 250 mg/kg BW/day/calf. BW measured weekly, feed intake measured twice daily, proximate analysis of feeds and fodders analysed weekly, fecal scores monitored daily and fecal coliform count done weekly. There was a significant (p < 0.01) increase in mean BW gain and feed intake and a significant (p < 0.01) decrease in severity of scours as measured by fecal score in the treatment group compared to the control group. The results suggest that garlic extract can be supplemented to the calves for better performance.

Structure modeling and inhibitor prediction ofNADP oxidoreductase enzyme from Methanobrevibacter smithii.

The F420-dependent NADP oxidoreductase enzyme from Methanobrevibacter smithii catalyzes the important electron transfer step during methanogenesis. Therefore, it may act as potential target for blocking the process of methane formation. Its protein sequence is available in GenBank (accession number: ABQ86254.1) however no report has been found about its 3D protein structure. In this work, we first time claim 3D model structure of F420-dependent NADP oxidoreductase enzyme from Methanobrevibacter smithii by comparative homology modeling method. Swiss model and ESyPred3d (via Modeller 6v2) softwares were generated the 3D model by detecting 1JAX (A) as template along with sequence identities of 34.272% and 35.40%. Furthermore, PROCHECK with Ramachandran plot and ProSA analysis revealed that swiss model produced better model than Modeller6v2 with 98.90% of residues in favored and additional allowed regions (RM plot) as well as with ProSA Z score of -7.26. In addition, we investigated that the substrate F420 bound at the cavity of the model. Subsequently, inhibitor prediction study revealed that Lovastatin (-22.07 Kcal/mol) and Compactin (Mevastatin) (-21.91 Kcal/mol) produced more affinity for model structure of NADP oxidoreducatse as compared to F420 (-14.40 Kcal/mol). It indicates that the Lovastatin and Compactin (Mevastatin) compounds (Negative regulator) may act as potential inhibitor of F420 dependent NADP oxidoreducatse protein.

Methyl coenzyme M reductase (mcrA) gene based phylogenetic analysis of methanogens population in Murrah buffaloes (Bubalus bubalis)

The aim of the present study was to decipher the diversity of methanogens in rumen of Murrah buffaloes so that effective strategies can be made in order to mitigate methane emission from these methanogens. In the present study diversity of rumen methanogens in Murrah buffaloes (Bubalus bubalis) from North India was evaluated by using mcr-A gene library obtained from the pooled PCR product from four animals and by using MEGA4 software. A total of 104 clones were examined, revealing 26 different mcr-A gene sequences or phylotypes. Of the 26 phylotypes, 16 (64 of 104 clones) were less than 97% similar to any of the cultured strain of methanogens. Seven clone sequences were clustered with Methanomicrobium mobile and three clone sequences were clustered with Methanobrevibacter gottschalkii during the phylogenetic analysis. Uncultured group of methanogens comes out to be the major component of the methanogens community structure in Murrah buffaloes. Methanomicrobium phylotype comes out to be major phylotype among cultured methanogens followed by Methanobrevibacter phylotype. These results help in making effective strategies to check the growth of dominant communities in the rumen of this animal which in turn help in the reduction of methane emission in the environment and ultimately helps us in fighting with the problem of global warming.

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.

Effect of urea supplemented and urea treated straw based diet on milk urea concentration in crossbred Karan-Fries cows

Abstract The study was undertaken to evaluate the effect of urea supplemented and urea treated straw based diet on milk urea concentration. Six multiparous crossbred Karan-Fries (Holstein Friesian × Tharparkar) cows were blocked into three groups of nearly equal body weight, DIM, milk yield and milk fat content and were randomized into a 3 × 3 Latin square design with 3-week period. Three experimental diets were fed to the animals. Composition of these diets were: Diet 1) green maize, wheat straw and concentrate mixture; Diet 2) green maize, wheat straw, concentrate mixture (urea supplemented) and molasses; Diet 3) green maize (4 % of total DM), 4 % urea treated wheat straw and concentrate mixture. Intake of DM and CP did not vary across the diets. Intake of digestible crude protein (DCP) was found significantly higher in diet 2, while ME and NEl intakes were found significantly lower in diet 3 but did not differ between diets 1 and 2. Average milk and plasma urea concentrations (mg dl-1) were found 29.2 ± 2.6, 45.3 ± 0.9, 34.5 ± 2.3 and 28.9 ± 2.4, 36.6 ± 1.4, 33.9 ± 2.2, respectively in diet 1, diet 2 and diet 3. Urea concentrations in morning milk samples were found significantly lower than noon or evening samples in all the three diets. Concentrations of urea in milk and plasma were found closely correlated (r = 0.94) and the regression equation developed was, plasma urea = 8.90 (.89) + .79 (.02) milk urea. Intake (g) of DCP than CP, per unit (MCal) of ME was found more closely associated with milk urea concentration. The study revealed that urea supplementation and urea treated straw based diet increased urea concentration significantly in milk and plasma. Morning milk urea values that estimated at a time gap of 15 hr since last major feeding may be considered as the lowest level and can be used for interpretation to monitor feeding adequacy or reproductive performances in dairy cows.

Influence of Albizia lebbeck Saponin and Its Fractions on In Vitro Gas Production Kinetics, Rumen Methanogenesis, and Rumen Fermentation Characteristics

The present study was undertaken to investigate the effect of crude seed powder (CSP) and gross saponins extract (GSE) of seeds of Albizia lebbeck on antimicrobial activity by taking two Gram-positive (Staphylococcus aureus and Bacillus cereus), two Gram-negative (Escherichia coli and Salmonella Typhi) bacteria, and two fungi species (Aspergillus niger and Candida butyric) were taken at 25, 50, 100, 250, and 500 µg levels using agar well diffusion method. Zone of inhibition was increased with increasing of concentration of CSP and saponins which indicates that Gram-negative bacteria (E. coli), Gram-positive bacteria (B. cereus), and A. niger were significantly susceptible to inhibition. Another experiment was conducted to study the effect of GSE and saponins fraction A and B of A. lebbeck supplementation at 6% on DM basis on methane production and other rumen fermentation parameters using in vitro gas production test, by taking three different type diets, that is, high fiber diet (D1, 60R : 40C), medium fiber diet (D2, 50R : 50C), and low fiber diet (D3, 40R : 60C). Significant (P ≤ 0.05) increase was seen in IVDMD, methane production; however ammonia nitrogen concentration decreased as compared to control. The methane production was reduced in a range between 12 and 49% by saponin supplemented diets except in case of GSE in D2. Sap A showed the highest methane reduction per 200 mg of truly digested substrate (TDS) than other treatment groups. Results in relation with quantification of methanogens and protozoa by qPCR indicated the decreasing trend with saponins of A. lebbek in comparison with control except total methanogen quantified using mcr-A based primer.