**5. Effect of diet on diversity of rumen methanogens**

The rumen is a dynamic system therefore the microbes must change qualitatively and quantitatively in response to the changes in the chemical composition of diet of animal rather than geographical location in general. Wang et al [26] reported members of the order Rumen Cluster C (RCC) to be most abundant ruminal methanogen present in cattle from China fed agricultural residues like corn stover, rapeseed and cottonseed meals followed by the order *Methanobacteriales*. By constructing a gene clone library of *mcrA* gene, they found that by increasing the agricultural residues in diet of cattle, the methanogen community structure did not change however methane production was increased*.* The effect of diet on rumen methanogen population has also been studied in Western Australia where sheep were fed different diets. Analysis revealed that archaeal diversity in sheep from grazing pasture was more as compared to sheep fed forage diets-oaten hay or lucerne hay. The maximum numbers of clones identified were from *Methanobrevibacter* strains SM9, M6, and NT7 [27].

A corn and cottonseed diet of cattle from Jinnan region of China also reported members of *Methanobrevibacter, Methanobacterium*, *Methanosphaera, Methanomicrobium* and unidentified Euryarchaeota. Overall, *Methanobrevibacter* spp. appeared to be predominant in all three rumen fractions [28]. Similarly, methanogenic population in dairy cattle from Canada was estimated that were fed diets supplemented with enzyme additive by PCR-DGGE and quantitative real-time PCR (qRT-PCR) analysis. The PCR-DGGE profiles were made up of 26 different bands, with two bands affiliated to Methanogenic archaeon CH1270 and one band to *Mbb. gottschalkii* strain HO. Three bands similar to Methanogenic archaeon CH1270 or *Mbb. smithii* ATCC 35061 appeared after enzyme was supplemented [29]. The diversity of rumen methanogens present in Mediterranean water buffaloes from Brazil which were maintained on three different diets-corn silage (library 1), pasture grazing (library 2) and sugar cane (library 3) revealed all three 16S rRNA clone libraries to be consisted of *Methanobrevibacter*-related sequences. The abundance of *Methanobrevibacter* like sequences in water buffaloes was in contrast to previous reports that showed *M. mobile* like methanogens to be predominant *Archaea* isolated from water buffaloes of Murrah and Surti breeds from India [30]. The taxonomy and structure of methanogens in Swedish dairy cattle fed two different diets through clone library consisted by terminal restriction fragment length polymorphism (T-RFLP) showed the genus *Methanobrevibacter* to be dominant in rumen and that the diet may not be an obvious factor affecting the community composition of methanogenic population inside rumen but may give an insight to the structure of ruminal methanogens [31].

Another study on sheep in Queensland, Australia in 2006 revealed 78 clones of 26 different methanogen related sequences were obtained. Eight sequences consisted of 15 clones were found 95–100% similar to the orders *Methanobacteriales* and *Methanomicrobiales*, and rest 18 sequences consisted of 63 clones were 72–75% affiliated to *Thermoplasma acidophilum* (*T. acidophilum*) and *Thermoplasma volcanium* (*T. volcanium*) [32]. The structure of archaeal diversity in feedlot cattle (starch based diet) from two different provinces of Canada-Ontario and Prince Edward Island, were deduced by constructing a clone library of 241 sequences.

**117**

leaf library [36].

**conditions of diet**

*Methanogenic Diversity and Taxonomy in the Gastro Intestinal Tract of Ruminants*

Eleven phylotypes (38 clones) in cattle from Ontario region (corn-based diet) were unique to this group as they were not found in cattle from Prince Edward Island. Similarly, 7 phylotypes (42 clones) from Prince Edward Island cattle (potato by-products) were found only in this group whereas 5 sequences representing 161 clones were found common in both herds. Out of 23 different sequences obtained, 10 sequences consisting of 136 clones were 89.8–100% affiliated to the species of the orders *Methanobacteriales*, *Methanomicrobiales* and *Methanosarcinales* and remaining 13 sequences consisting of 105 clones showed 74.1–75.8% sequence similarity to the species *T. volcanium* and *T. acidophilum* [33]. The dominance of total rumen *Archaea* from different ruminant species around the world in a global data set report surveying nine studies assessed that genus *Methanobrevibacter* (61.6%), *Methanomicrobium* (14.9%) and uncultured species from Rumen Cluster C (15.8%) constituted 92.3% of total rumen *Archaea* [34]. Another study from Venezuela indicated *Methanobrevibacter* phylotype to be the most abundant genera in 14 different 16S rRNA gene sequences or phylotypes from 104 clone library constructed in sheep [35]. The rumen of Sika deer fed oak leaf diet and corn stalk diet from China revealed thirty six OTUs assigned to 146 unique sequences and in both the diet group, genus *Methanobrevibacter* was detected as a predominant methanogen. Among the species, *Mbb. millerae* was most abundant in both groups but accounted for a slightly higher population (69.5%) in corn stalk library than in oak leaf library (51.4%). Clones with similarity to *Mbb. smithii* like clones and *Mbb. ruminantium* like clones were present in corn stalk library but were absent in oak

The majority of sequences were related to genera *Methanobrevibacter* and *Methanosphaera* and a group of novel uncultured methanogens "uncultured marine bacteria" were identified in Moxoto breed goats from Brazil by constructing 16S rRNA gene clone libraries [37]. Likewise, the archaeal methanogen population inside rumen of lactating Jersey and Holstein cattle fed same diet from America revealed species level similarity to *Mbb. ruminantium* [38]. The community structure of methanogens inside rumen of farmed sheep, cattle and red deer which were fed different diets revealed diet and host based differences in framing community structure, but the presence of dominant archaeal species was uniform in all host animals. The dominant members were from following clades: RO clade-*Mbb. ruminantium* and *Mbb. olleyae*, SGMT clade-*Mbb. gottschalkii, Mbb. millerae and Mbb.* 

*thaueri* and species of the genus *Methanosphaera* [39].

and remainder of the OTU's were formed by *Methanosphaera*.

**6. Methanogen diversity inside rumen and/feces under similar** 

The sequences obtained from rumen and feces of local sheep from Xinjiang, China were divided into three groups based on their affiliation to the following genera: *Methanobrevibacter*, *Methanocorpusculum* and an unclassified methanogenlike group [40]. Order *Methanobacteriales* was found to be dominant in rumen of faunated and unfaunated Holstein cattle from Japan by constructing clone libraries from 16S rDNA gene and mcrA gene [41]. The methanogenic archaeal population in sheep of Scottish uplands were illustrated by Snelling et al. [42] by different methods-Sanger amplicon sequencing by constructing 16S rRNA gene libraries, 16S rRNA gene amplicon sequencing by Illumina, Illumina metagenome sequencing. All the methods revealed the order *Methanobacteriales* containing genera: *Methanobrevibacter*, *Methanosphaera* and *Methanobacteria* to be the most abundant. Among the *Methanobacteriales* order, *Mbb. millerae* comprised of ≥91% of OTU's

*DOI: http://dx.doi.org/10.5772/intechopen.82829*

#### *Methanogenic Diversity and Taxonomy in the Gastro Intestinal Tract of Ruminants DOI: http://dx.doi.org/10.5772/intechopen.82829*

Eleven phylotypes (38 clones) in cattle from Ontario region (corn-based diet) were unique to this group as they were not found in cattle from Prince Edward Island. Similarly, 7 phylotypes (42 clones) from Prince Edward Island cattle (potato by-products) were found only in this group whereas 5 sequences representing 161 clones were found common in both herds. Out of 23 different sequences obtained, 10 sequences consisting of 136 clones were 89.8–100% affiliated to the species of the orders *Methanobacteriales*, *Methanomicrobiales* and *Methanosarcinales* and remaining 13 sequences consisting of 105 clones showed 74.1–75.8% sequence similarity to the species *T. volcanium* and *T. acidophilum* [33]. The dominance of total rumen *Archaea* from different ruminant species around the world in a global data set report surveying nine studies assessed that genus *Methanobrevibacter* (61.6%), *Methanomicrobium* (14.9%) and uncultured species from Rumen Cluster C (15.8%) constituted 92.3% of total rumen *Archaea* [34]. Another study from Venezuela indicated *Methanobrevibacter* phylotype to be the most abundant genera in 14 different 16S rRNA gene sequences or phylotypes from 104 clone library constructed in sheep [35]. The rumen of Sika deer fed oak leaf diet and corn stalk diet from China revealed thirty six OTUs assigned to 146 unique sequences and in both the diet group, genus *Methanobrevibacter* was detected as a predominant methanogen. Among the species, *Mbb. millerae* was most abundant in both groups but accounted for a slightly higher population (69.5%) in corn stalk library than in oak leaf library (51.4%). Clones with similarity to *Mbb. smithii* like clones and *Mbb. ruminantium* like clones were present in corn stalk library but were absent in oak leaf library [36].

The majority of sequences were related to genera *Methanobrevibacter* and *Methanosphaera* and a group of novel uncultured methanogens "uncultured marine bacteria" were identified in Moxoto breed goats from Brazil by constructing 16S rRNA gene clone libraries [37]. Likewise, the archaeal methanogen population inside rumen of lactating Jersey and Holstein cattle fed same diet from America revealed species level similarity to *Mbb. ruminantium* [38]. The community structure of methanogens inside rumen of farmed sheep, cattle and red deer which were fed different diets revealed diet and host based differences in framing community structure, but the presence of dominant archaeal species was uniform in all host animals. The dominant members were from following clades: RO clade-*Mbb. ruminantium* and *Mbb. olleyae*, SGMT clade-*Mbb. gottschalkii, Mbb. millerae and Mbb. thaueri* and species of the genus *Methanosphaera* [39].
