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

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

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

similarity by one isolate to *M. mobile* based on 16S rRNA [24, 25].

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

SM9, M6, and NT7 [27].

the structure of ruminal methanogens [31].

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

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

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

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.

**116**

Tymensen and McAllister [43] reported the archaeal spp. linked with ruminal protozoa in cattle and obtained 276 final sequences generated from clone libraries using five diverse universal archaeal primer pairs and found that the three genera/taxa viz. *Methanobrevibacter*, Rumen Cluster C (RCC) and *Methanomicrobium* accounted for 94–100% of the sequences in each library. Metatranscriptomics approach-Illumina deep-sequencing with overlapping read paired-end technology revealed that *Bacteria* and *Eukaryotes* contributed to the majority of ribotags (approximately 50%) whereas *Archaea* contributed only 1% of ribotags mainly comprised of the order *Methanobacteriales* (*Methanobrevibacter* and *Methanosphaera*) and *RCC Thermoplasmata*. The *RCC Thermoplasmata* lowered down considerably on rape seed oil (RSO) supplementation whereas *Methanobacteriales* did not show any decrease. A notable decrease in the mcrA and mcrB transcripts of RCC on change in was noticed suggesting the reduced CH4 emissions [44].

The abundance of two archeal orders-*Methanobacteriales* and *Methanomassiliicoccales* in rumen of sheep and cattle from New Zealand were studied. From the order *Methanobacteriales*, sequences were assigned to only four species—*Mbb. gottschalkii*, *Mbb. ruminantium*, *Methanosphaera* sp. ISO3-F5 and *Methanosphaera* sp. group5. The members of the order *Methanomassiliicoccales* contributed 10.4% of the total relative abundance of the methanogenic archaeal community, *Methanobacteriales* (89.6%) being dominant [45]. The methanogenic *Archaea* in yak from China grazing on natural pastures exhibited the species of the family *Methanobacteriaceae* to be predominant in yak rumen followed by members from the family *Methanomassiliicoccaceae* and *Methanosarcinaceae* [46].

The archaeal methanogenic community from rumen of two indigenous ruminant species-yak and Tibetan sheep and two introduced species-cattle and crossbred sheep in Qinghai-Tibetan plateau from China under similar diet of oaten hay and barley and environmental conditions revealed the more archaeal diversity in indigenous species than in introduced species. *Methanomassiliicoccaceae* was predominant family representing most of the sequences while *Methanobacteriaceae* was second most dominant archaeal family. Among *Methanobrevibacter* genus, *Mbb. gottschalkii* and *Mbb. ruminantium* were the most observed species. Interestingly, *Mbb. woesei* and *Mbb.* sp. RT were only found associated with yak rumen [47]. Salgado-Flores et al. [48] reported archaeal methanogenic density by quantitative real-time PCR and diversity from rumen and cecum samples of Norwegian reindeer fed on standard pellets and lichens by 454 pyrosequencing of 16S rRNA genes. The population density of archaeal methanogens remained almost constant for both the diets in rumen and cecum samples. In rumen samples, *Methanobrevibacter* was found to be main genus and strain *Mbb. thaueri* CW to be predominant in both groups fed different diets. *Mbb. wolinii* SH was second most abundant species found in group fed pellet based diet whereas constituted only 1.5% of the total sequences in group fed lichens. The second most prevalent species was *Mbb. ruminantium* strain M1 in reindeers fed lichens but accounted only 4.2% of the total sequences in pellet fed group of reindeers. In cecum samples also, genus *Methanobrevibacter* was detected predominantly in both the groups. *Mbb. millerae* strain ZA-10 was found to be most abundant in reindeer group fed with pellet but had less than 97% similarity with this archaeal methanogen whereas strain *Mbb. thaueri* CW was main species in lichen fed group with 98% similarity. Franzolin and Wright reported that the density of archaeal methanogens was very low as compared to bacterial counterparts in grazing and feedlot group of buffaloes from Brazil. The density of methanogens as compared to bacteria in reticulum was more as compared to rumen [49].

The rumen methanogenic structure in three Indian cattle and buffaloes which were fed on wheat straws based diet using RT-PCR revealed most abundant orders

**119**

**Figure 1.**

*downloaded from NCBI Genbank database [58].*

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

of *Methanomicrobiales* and *Methanobacteriales* along with total bacteria and that it remained constant for two animals using a particular diet [50]. Similarly, the ruminal diversity in Indian Murrah buffaloes by using amplified ribosomal DNA restriction analysis (ARDRA) maintained under standard diet of wheat straws revealed a total of 108 clones that were classified into 16 phylotypes. The 9 phylotypes showed less than 97% sequence similarity to any of the cultivated methanogen strain and represented a novel uncultured group of methanogens. The second group comprised of 4 phylotypes that showed 92–99% sequence similarity with *M. mobile*. The third group consisted of a single phylotype clustered with *M. burtonii*, reported for

*A phylogenetic tree based on 16S rRNA sequences obtained from camel foregut and reference sequences* 

*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*

of *Methanomicrobiales* and *Methanobacteriales* along with total bacteria and that it remained constant for two animals using a particular diet [50]. Similarly, the ruminal diversity in Indian Murrah buffaloes by using amplified ribosomal DNA restriction analysis (ARDRA) maintained under standard diet of wheat straws revealed a total of 108 clones that were classified into 16 phylotypes. The 9 phylotypes showed less than 97% sequence similarity to any of the cultivated methanogen strain and represented a novel uncultured group of methanogens. The second group comprised of 4 phylotypes that showed 92–99% sequence similarity with *M. mobile*. The third group consisted of a single phylotype clustered with *M. burtonii*, reported for

**Figure 1.**

*A phylogenetic tree based on 16S rRNA sequences obtained from camel foregut and reference sequences downloaded from NCBI Genbank database [58].*

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

Tymensen and McAllister [43] reported the archaeal spp. linked with ruminal protozoa in cattle and obtained 276 final sequences generated from clone libraries using five diverse universal archaeal primer pairs and found that the three genera/taxa viz. *Methanobrevibacter*, Rumen Cluster C (RCC) and *Methanomicrobium* accounted for 94–100% of the sequences in each library. Metatranscriptomics approach-Illumina deep-sequencing with overlapping read paired-end technology revealed that *Bacteria* and *Eukaryotes* contributed to the majority of ribotags (approximately 50%) whereas *Archaea* contributed only 1% of ribotags mainly comprised of the order *Methanobacteriales* (*Methanobrevibacter* and *Methanosphaera*) and *RCC Thermoplasmata*. The *RCC Thermoplasmata* lowered down considerably on rape seed oil (RSO) supplementation whereas *Methanobacteriales* did not show any decrease. A notable decrease in the mcrA and mcrB transcripts of RCC on change in was noticed suggesting the reduced CH4

The abundance of two archeal orders-*Methanobacteriales* and

from the family *Methanomassiliicoccaceae* and *Methanosarcinaceae* [46].

to bacteria in reticulum was more as compared to rumen [49].

The rumen methanogenic structure in three Indian cattle and buffaloes which were fed on wheat straws based diet using RT-PCR revealed most abundant orders

The archaeal methanogenic community from rumen of two indigenous ruminant

species-yak and Tibetan sheep and two introduced species-cattle and crossbred sheep in Qinghai-Tibetan plateau from China under similar diet of oaten hay and barley and environmental conditions revealed the more archaeal diversity in indigenous species than in introduced species. *Methanomassiliicoccaceae* was predominant family representing most of the sequences while *Methanobacteriaceae* was second most dominant archaeal family. Among *Methanobrevibacter* genus, *Mbb. gottschalkii* and *Mbb. ruminantium* were the most observed species. Interestingly, *Mbb. woesei* and *Mbb.* sp. RT were only found associated with yak rumen [47]. Salgado-Flores et al. [48] reported archaeal methanogenic density by quantitative real-time PCR and diversity from rumen and cecum samples of Norwegian reindeer fed on standard pellets and lichens by 454 pyrosequencing of 16S rRNA genes. The population density of archaeal methanogens remained almost constant for both the diets in rumen and cecum samples. In rumen samples, *Methanobrevibacter* was found to be main genus and strain *Mbb. thaueri* CW to be predominant in both groups fed different diets. *Mbb. wolinii* SH was second most abundant species found in group fed pellet based diet whereas constituted only 1.5% of the total sequences in group fed lichens. The second most prevalent species was *Mbb. ruminantium* strain M1 in reindeers fed lichens but accounted only 4.2% of the total sequences in pellet fed group of reindeers. In cecum samples also, genus *Methanobrevibacter* was detected predominantly in both the groups. *Mbb. millerae* strain ZA-10 was found to be most abundant in reindeer group fed with pellet but had less than 97% similarity with this archaeal methanogen whereas strain *Mbb. thaueri* CW was main species in lichen fed group with 98% similarity. Franzolin and Wright reported that the density of archaeal methanogens was very low as compared to bacterial counterparts in grazing and feedlot group of buffaloes from Brazil. The density of methanogens as compared

*Methanomassiliicoccales* in rumen of sheep and cattle from New Zealand were studied. From the order *Methanobacteriales*, sequences were assigned to only four species—*Mbb. gottschalkii*, *Mbb. ruminantium*, *Methanosphaera* sp. ISO3-F5 and *Methanosphaera* sp. group5. The members of the order *Methanomassiliicoccales* contributed 10.4% of the total relative abundance of the methanogenic archaeal community, *Methanobacteriales* (89.6%) being dominant [45]. The methanogenic *Archaea* in yak from China grazing on natural pastures exhibited the species of the family *Methanobacteriaceae* to be predominant in yak rumen followed by members

**118**

emissions [44].

the first time in rumen. The fourth group was a single phylotype that showed 97% sequence identity with *Mbb. gottschalkii*. The last group of single phylotype showed a sequence similarity to *Mbb. ruminantium* [51].

Likewise, the comparative diversity analysis of methanogens using 16S rRNA and *mcr*A in cattle rumen fed on a high fiber diet reported 13 OTU's consisting of 102 clones from 16S rRNA gene based library. All OTU's were clustered with order *Methanobacteriales* and were further splitted into Cluster I that had 12 OTU's related to *Methanobrevibacter* spp. and Cluster II comprised of one OTU related to *M. stadtmanae* [52]. The Surti buffaloes that were fed wheat straw and compound concentrate mixture diet generated a total of 76 clones representing 21 sequences based on PCR-RFLP patterns. BLAST analysis revealed 13 OTU's (55 clones) that showed sequence identity with *Methanomicrobium* sp., 3 OTU's (15 clones) that showed sequence similarity with *Methanobrevibacter* sp. The remaining 5 OTU's (6 clones) were associated with uncultured *Archaea*. Overall, the methanogenic population inside rumen of buffaloes was from the order of *Methanomicrobiales* (18 OTUs) and *Methanobacteriales* (3 OTUs) [53]. The rumen metagenome of buffalo using q-PCR were compared with MG-RAST based annotation of the metagenomes sequences of 16S rDNA amplicons and high throughput shotgun sequencing and found *Methanomicrobiales* in lower number [54] (**Figure 1**).
