Sumit Singh Dagar

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.

D1/D2 Domain of Large-Subunit Ribosomal DNA for Differentiation of Orpinomyces spp.

ABSTRACT This study presents the suitability of D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA) for differentiation of Orpinomyces joyonii and Orpinomyces intercalaris based on PCR-restriction fragment length polymorphism (RFLP). A variation of G/T in O. intercalaris created an additional restriction site for AluI, which was used as an RFLP marker. The results demonstrate adequate heterogeneity in the LSU rDNA for species-level differentiation.

Isolation and characterization of methanogens from rumen of Murrah buffalo

Methanogens were isolated from the rumen of Murrah buffaloes (Bubalus bubalis). These isolates (BRM-1, -2 and -3) were found to utilize CO2 + H2 mixture, formate and acetate as substrate, but failed to grow on ethanol and methanol. Their physiological analysis showed that they could tolerate NaCl and bile salts up to 1.0% but 2.0% bile salt inhibited their growth. Based on 16S rRNA/mcrA gene sequence analysis, the isolates showed their phylogenetic relation with genus Methanobrevibacter and Methanomicrobium. BRM-1 and -3 showed 100% similarity with Methanobrevibacter smithii, while BRM-2 showed 100% similarity with Methanomicrobium mobile. The mcrA protein-based phylogeny also showed similar results to the mcrA gene, suggesting no apparent difference in the phylogeny between DNA and amino acid sequences of these isolates.

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.

Role of live microbial feed supplements with reference to anaerobic fungi in ruminant productivity: A review

Abstract To keep the concept of a safe food supply to the consumers, animal feed industries world over are showing an increasing interest in the direct-fed microbials (DFM) for improved animal performance in terms of growth or productivity. This becomes all the more essential in a situation, where a number of the residues of antibiotics and/or other growth stimulants reach in milk and meat with a number of associated potential risks for the consumers. Hence, in the absence of growth stimulants, a positive manipulation of the rumen microbial ecosystem to enhance the feedstuff utilization for improved production efficiency by ruminants has become of much interest to the researchers and entrepreneurs. A few genera of live microbes (i.e., bacteria, fungi and yeasts in different types of formulations from paste to powder) are infrequently used as DFM for the domestic ruminants. These DFM products are live microbial feed supplements containing naturally occurring microbes in the rumen. Among different DFM possibilities, anaerobic rumen fungi (ARF) based additives have been found to improve ruminant productivity consistently during feeding trials. Administration of ARF during the few trials conducted, led to the increased weight gain, milk production, and total tract digestibility of feed components in ruminants. Anaerobic fungi in the rumen display very strong cell-wall degrading cellulolytic and xylanolytic activities through rhizoid development, resulting in the physical disruption of feed structure paving the way for bacterial action. Significant improvements in the fiber digestibility were found to coincide with increases in ARF in the rumen indicating their role. Most of the researches based on DFM have indicated a positive response in nutrient digestion and methane reducing potential during in vivo and/or in vitro supplementation of ARF as DFM. Therefore, DFM especially ARF will gain popularity but it is necessary that all the strains are thoroughly studied for their beneficial properties to have a confirmed ‘generally regarded as safe’ status for ruminants.

A new anaerobic fungus (Oontomyces anksri gen. nov., sp. nov.) from the digestive tract of the Indian camel (Camelus dromedarius)

Two cultures of anaerobic fungi were isolated from the forestomach of an Indian camel (Camelus dromedarius). Phylogenetic analysis using both the internal transcribed spacer (ITS) and large-subunit (LSU) regions of the rRNA locus demonstrated that these isolates were identical and formed a distinct clade within the anaerobic fungi (phylum Neocallimastigomycota). Morphological examination showed that these fungi formed monocentric thalli with filamentous rhizoids and uniflagellate zoospores, broadly similar to members of the genus Piromyces. However, distinctive morphological features were observed, notably the pinching of the cytoplasm in the sporangiophore and the formation of intercalary rhizoidal swellings. Since genetic analyses demonstrated this fungus was only distantly related to Piromyces spp. and closer to the polycentric Anaeromyces clade, we have assigned it to a new genus and species Oontomyces anksri gen. nov., sp. nov. Interrogation of the GenBank database identified several closely related ITS sequences, which were all environmental sequences obtained from camels, raising the possibility that this fungus may be specific to camelids.

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.

β-Glucosidase Activity of Lactobacilli for Biotransformation of Soy Isoflavones

Thirty isolates of lactobacilli were screened for β-glucosidase production and isoflavones biotransformation. The isolates exhibited enzyme activities in the range of 0.14–3.31 IU. Five highest enzyme producers were assessed for isoflavones biotransformation potential in soymilk that varied greatly among the isolates with an increase of 2–3 fold in genistein and 6–14 fold in daidzein. The biochemical and molecular identification classified the isolates as Lactobacillus rhamnosus. Among isolates, L. rhamnosus D13 was the highest enzyme producer, while L. rhamnosus D7 was the highest isoflavone aglycone producer. Our results are distinct because of no proportional relation between enzyme activity and isoflavones biotransformation. The study further confirms that the degree of biotransformation of isoflavones is characteristic of an individual strain.

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.

Prospective use of bacteriocinogenic Pediococcus pentosaceus as direct-fed microbial having methane reducing potential

Abstract Direct-fed microbials (DFM), generally regarded as safe status, are successfully used in improving rumen ecology, gastro-intestinal health, feed efficiency, milk production and growth rate in ruminants. On the other hand, methanogenesis in rumen, which accounts for a significant loss of ruminant energy and increased greenhouse gas in environment, is of great concern, therefore, use of DFM for improving productivity without compromising the animal health and ecological sustainability is encouraged. The present study was conducted to investigate the methane reducing potential of bacteriocinogenic strain Pediococcus pentosaceus-34. Since, the culture showed no hemolysis on blood agar and DNase activity, hence, it was considered to be avirulent in nature, a prerequisite for any DFM. The culture also showed tolerance to pH 5.0 for 24 h with 0.5% organic acid mixture, whereas when given a shock for 2 h at different pH and organic acids concentrations, it showed growth at pH 3.0 and 4.0 with 0.1 and 1.0% organic acids, respectively, as having good animal probiotics attributes. The total gas production was significantly (P