**4. Methanogenic archaeal population in gastro-intestinal tract of ruminants**

The rumen was the initial environment of *Archaea* which is comprehensively investigated and studied. Hungate [15] reported that about 23 bacterial species played prominent role in ruminal metabolism whereas in 1996 the number increased up to 200 [16]. The culture based techniques had serious limitations as they failed to differentiate between two phylogenetic diverse species along with the dire need of maintaining anaerobic environment to culture and isolate bacteria. The 16S rRNA sequencing technology has been far and wide used to explore the methanogens residing inside rumen and to characterize and validate their community structure and taxonomic composition in evolutionary timeline. The methanogenic group in gastrointestinal tract of ovine, caprine and bovine using rRNA targeted oligonucleotide probes were identified and *Methanobacteriales* were reported to be the abundant methanogens in bovine and caprine rumen whereas *Methanomicrobiales* was found to be predominant in ovine rumen [17]. In 2000, the population of methanogens among rumen microbial diversity of sheep in Japan was reported using 16S rDNA cloning and fluorescence in situ hybridization (FISH) technique and most of the clones were found associated with *Methanomicrobium mobile, Methanobrevibacter ruminantium* and *Methanobrevibacter smithii*. The total methanogens accounted for 3.6% from the total microorganisms present in rumen and population of *M. mobile* among methanogens was found to be 54% [18]. A year later the archaeal libraries generated from the rumen of dairy Holstein cows from Japan revealed two groups of sequences produced from two different sets of archaeal primers. The library generated from primers-D30 and D33, revealed 21% of clones related to *M. mobile* and 79% of clones were anaerobic digester associated archaeal sequences with close identity to *Thermoplasma*. The second library generated from 0025e and 1492 primers showed 56% of the clones related to *M. mobile*, 20% related to the *Thermoplasma* associated sequences and 16% related to *Methanobrevibacter* spp. and 2 sequences were related to the unidentified rumen *Archaea* [19].

Similarly in bovine rumen, 41 cloned sequences were identified in 3 clusters. The largest cluster contained 24 clones with 2 distinct sub clusters with sequences affiliated with *Mbb. ruminantium*. The sub cluster Mbr I contained nine 16S rDNA sequences that had 98.5–98.8% sequence identity to *Mbb. ruminantium* whereas the sub cluster Mbr II contained 15 cloned sequences that had 97.2–97.7% similarity to *Mbb. ruminantium* whereas the second cluster contained 11 cloned sequences having similarity values of 96.1–97.5% to *Methanosphaera stadtmanae*, an organism first time recognized in rumen. The third cluster was found containing 6 cloned sequences that were 89% similar to *Methanosarcina* sp. str. WH1 and *Methanosarcina thermophila* indicating it to be comprised of a novel group of rumen methanogens [20]. In Japan, clones were deduced from bovine rumen that was 83.9–88.3% identical to *Mbb. ruminantium* [21]. In 2004, the archaeal populations from three fractions of rumen-rumen fluid, rumen solid and rumen epithelium from Korean Hanwoo cattle was constructed using 16S rDNA gene clone libraries. Species belonging to the family *Methanomicrobiaceae* were found dominant in fractions of fluid and epithelium in rumen while *Methanobacteriaceae* was abundant in solid fraction of rumen [22]. The *Methanomicrobium* phylotype was the most abundant phylotype among methanogenic population in rumen of Murrah buffaloes from India as revealed by constructing 16S rDNA gene library. A total of 15 phylotypes out of 17 were affiliated to *M. mobile*; one sequence was identical to *T. acidophilum* and one sequence with *Methanocorpusculum bavaricum* [23]. *Methanobacteriales* was

*Extremophilic Microbes and Metabolites - Diversity, Bioprospecting and Biotechnological...*

and *Euryarchaeota* [5].

**gases**

**2. Major rumen microbes**

further divided into 10 families and 31 genera [7–9].

nutrition and feed management strategies.

techniques led to the discovery of a third microbial kingdom, the *Archaebacteria*, which includes methanogens [3, 4]. Further the target specific sequence analysis of 16S rRNA gene in 1970's had redefined taxonomy of all living organisms into three main domains. Methanogens belong to the 3rd domain of life-*Archaea*, other two being—*Eucarya* and *Bacteria*. *Archaea* is further divided into phylums *Crenarchaeota*

At any time there are billions of any species of anaerobic bacteria and facultative anaerobic bacteria residing in rumen along with a mixed population of various anaerobic protozoa, anaerobic fungi and flagellates making it a diverse microbial consortium in nature. The bacteria along with protozoa make most of the microbial mass (nearly 80%) inside rumen. The bacteria present in specialized niches are a very small fraction that cannot be recovered by cultural methods and even among cultivable bacteria true number of diversity is now revealed only by molecular techniques [6]. The bacteria can further be cellulolytic (fiber digesting), amylolytic (starch and sugar digesting) and lactate utilizing bacteria. The role of symbiotic microbial ecosystem consisting of bacteria, protozoa and fungi is of great significance in ruminants. Phylum Euryarchaeota within domain *Archaea* includes 7 orders—*Methanobacteriales*, *Methanomicrobiales*, *Methanococcales*, *Methanopyrales*, *Methanocellales*, *Methanosarcinales* and *Methanomassiliicoccales*. The orders are

**3. Methane production in ruminants and its contribution to greenhouse** 

Methane is a main byproduct of digestion in ruminants produced by the microbial fermentation of plant biomass. Methanogens ferment the ingested feed into short chain fatty acids which consists of 70% of the total metabolizable energy source for ruminants. The methane is specifically produced by methanogens (*Archaea*) that resides symbiotically in the gut of ruminants by using hydrogen produced by bacteria, fungi and protozoa and reducing CO2 to methane. It is not used by ruminants and is lost in environment through eructation resulting in a loss of 2–12% of metabolic energy intake to the host [10, 11]. Among agricultural sources, enteric fermentation along with natural and man-made wetlands, animal wastes; paddy fields contribute to the release of major amount of methane in environment. Methane gas has a major global warming impact [12]. According to the fifth assessment report of Intergovernmental Panel on Climate Change (IPCC) published in 2014, global release of greenhouse gases from enteric fermentation grew from 1.4 to 2.1 GtCO2eq/yr between 1961 and 2010. The largest methane emission was by cattle (75% of the total) followed by goat, sheep and other ruminants during the year 2000–2010 [13]. The enteric fermentation in ruminants is a significant cause of methane emission in environment. It is an inevitable outcome of their normal digestive process [14], which is not used by them and is lost in environment. Since, methane is a potent greenhouse gas, to reduce the activity and number of methane producing *Archaea*, it is desirable to have knowledge about the community structure of methanogens and their feed conversion energy mechanism. In order to control various ruminal disorders the insight into microbial ecology will help to develop

**114**

a dominant order identified from the rumen of Surti buffaloes in India by cloning and sequencing of *mcr*A gene while in an another study on Murrah buffaloes 100% sequence similarity was reported by two isolates to *Mbb. smithii* and 100% sequence similarity by one isolate to *M. mobile* based on 16S rRNA [24, 25].
