Host and diet specific differences

Even though the general archaeal community composition was similar in all samples, we did find that the archaeal communities of the different ruminant species tested (cattle, sheep, and red deer) were distinctive, regardless of diet, indicating host- specific community structures. We also found that, within ruminant species, archaeal communities in cattle were different when different diets were fed, but that this was not as strong with red deer, and not detectable in sheep. While the major clades of archaea detected were similar in all samples, the differences were in minor parts of the communities, and subgroups within major clades. The amount of variation in the archaeal communities was less than in the bacterial communities of the same animals,

153 and there were fewer detectable bands on DGGE gels of archaeal 16S rRNA genes than of bacterial 16S rRNA genes.

Archaeal communities in the rumens of red deer had structures similar to those in cattle and sheep, with some minor differences. The dominant bands present in cattle and sheep samples were also common to red deer. However, compared to cattle and sheep, the individual variation among red deer rumen methanogen communities was greater. When they were fed with summer pasture, a Methanosarcina sp. was found as one of the dominant bands in all of the red deer. In addition, the samples from silage-fed red deer contained a Methanosphaera sp. of a different sequence type that resulted in a DGGE band distinct from those formed from other Methanosphaera spp. These two distinct bands were largely absent when these same red deer were fed winter pasture. It has been found that red deer exhibit strong seasonal cycles of rumen digestive function, with increased rumen retention time in summer (Freudenberger et al., 1994).

Methanosarcina spp. grow relatively slowly and have higher Km values for H2 than

other methanogens (Zinder, 1993) and they would be washed out of the rumen unless the flow through the rumen is slowed. The unusual digestive physiology of red deer may also partly be the reason for the high variation in their banding patterns compared to cattle and sheep. To our knowledge, there have been no published studies on the rumen methanogens of red deer. Sundset et al. (2009a, 2009b) have reported studies on the diversity of rumen methanogens in Norwegian and Svalbard reindeer. They found the methanogens present in the reindeer were similar to the methanogens found in domesticated ruminants (cattle and sheep). Our study shows that the same is true of domesticated red deer.

Several studies have found host-species differences on the bacterial community of the rumen (Shi et al., 2008; Sundset et al., 2007). Effects of diet on changes on the diversity of bacterial species in the rumen have also been reported (Kocherginskaya et al., 2001; Tajima et al., 2001a). Different microbes act on the ingested feed during feed fermentation, and the species composition can be expected to be strongly influenced by the properties of the feed. Therefore, it is expected that there should be differences in the bacterial communities in rumens when animals are fed different diets. We found the bacterial community to be more complex than the archaeal community. We also found that the amount of variation between bacterial communities of animals fed different

154 diets, and between animals on the same diet, was significantly greater than variations between the archaeal communities. Most rumen methanogens grow with the H2

produced during the fermentation of the ingested feed. The rumen environment (T, pH, CO2, H2 and salts) is relatively constant over time. This is probably the reason why the

diversity of methanogens in the rumen of different ruminant species fed with different diets is relatively similar. The effect of diet was strongest in cattle, less so in red deer, and not detectable in sheep. It is interesting to speculate that the observation of effects of diet on cattle methanogen community relative to sheep and red deer is due to the fact that cattle rumens are much larger than those in either sheep or red deer. Cattle rumens may represent a more stable environment leading to less variability in methanogen community structure between individuals. Variation between individual sheep and red deer may be greater, masking the subtle diet effects.

All rumens sampled in this study contained remarkably similar archaeal communities, suggesting a common core of ruminal methanogen species. This is significant for efforts to develop methane mitigation strategies, as it limits the number of methanogen groups that need to be targeted to control the majority of methane producers. It is unclear, however, if the minor components of the methanogen community are specialists occupying narrow niches with limited capacity to contribute to total methane production, or if they will increase in abundance if the dominant groups are eliminated.

Note

A shorter version of this chapter has been published in FEMS Microbiology Ecology. This chapter therefore has more extensive methods and discussion sections than the other chapters.

Jeyanathan J, Kirs M, Ronimus RS, Hoskin SO & Janssen PH (2011) Methanogen community structure in the rumens of farmed sheep, cattle and red deer fed different diets. FEMS Microbiol Ecol, no. doi: 10.1111/j.1574-6941.2011.01056.

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Chapter 5

Effects of rumen-administered chloroform on the abundance and diversity of mcrA

and 16S rRNA genes of rumen archaea indicate the presence of as-yet uncultured methanogens

5.1 Introduction

Methanogenic archaea living in the rumen of ruminant animals produce methane (CH4) as an end product of ruminal feed fermentation, primarily from the hydrogen (H2)

produced during the fermentation of feed by bacteria, fungi and protozoa. The environmental and economic concerns of methane production from ruminant animals have lead to research to develop means of mitigating CH4 production emanating from

the rumen and to channel the carbon and energy to more volatile fatty acids (VFAs) and microbial biomass (McAllister & Newbold, 2008). Research has focused on reducing the amount of CH4 formed in the rumen by feed manipulation (Fievez et al., 2003; Guo

et al., 2008), broad-range inhibitors (Božic et al., 2009: Hamilton et al., 2010), animal selection (Chagunda et al., 2009), interfering with H2 transfer to the methanogens

(Joblin, 1999), or by specifically inhibiting the methanogens themselves using vaccines and small-molecule inhibitors (Wedlock et al., 2010; Williams et al., 2009). Many of these techniques have limitations and met with only limited success (McAllister & Newbold, 2008). Vaccines and small-molecule inhibitors which target methanogens could be effective technologies to reduce methane emissions from New Zealand farmed ruminants, as they are relatively readily applied to pasture-grazed animals. To produce targeted vaccines and inhibitor-based technologies, identification of targets found only in rumen methanogens is essential. This requires knowledge of rumen methanogens.

A meta-analysis of rumen archaea suggested that most are known methanogens, but some archaeal lineages of unknown physiology were also present (Janssen & Kirs, 2008). The largest of these, designated Rumen Cluster C (RCC), made up about 16% of the global dataset (Janssen & Kirs, 2008), and up to 81% of the archaeal 16S rRNA genes detected in some rumen samples (Wright et al., 2006). Unlike other known rumen methanogens, the contribution of RCC group to the total archaeal pool in various rumen samples has varied widely from highly dominant to completely absent (Table 1.2). In

156 New Zealand ruminants, RCC was one of the dominant groups of archaea and qPCR analysis of rumen samples obtained from sheep fed a range of diets indicated that, RCC made up an average of 26.5% of the archaeal pool (Chapter 4). However, the role of RCC in the rumen is not known yet.

Metabolic inhibitors are very useful to study microbial processes in a given environment. Chloroform (CHCl3) is an inhibitor that selectively inhibits methanogens

at low concentrations. Chloroform been used experimentally at low concentrations to specifically inhibit methanogens without changing the remaining overall microbial community structure (Achtnich et al., 1995; Chidthaisong & Conrad, 2000; Chin & Conrad, 1995; De Graaf et al., 1996). The inhibitory action of CHCl3 on rumen

methanogens was accidently found by Bauchop in 1967. Low concentrations of CHCl3

inhibit cobamide dependent methyl-transfer reactions (Wood et al., 1968: Kenealy & Zeikus, 1981) that normally lead to the formation of methane by methanogens. However, it appears that CHCl3 also inhibits the methyl coenzyme M reductase, an

enzyme diagnostic of methanogens that catalyzes the formation of CH4 from methyl-

coenzyme M (Gunsalus & Wolfe, 1978). Thus, there may be multiple modes of inhibition by CHCl3. Upon the CHCl3 treatment, archaea that are methanogens should

be inhibited and be eliminated from the rumen, while non-methanogenic archaea may be resistant.

There is no strong evidence to suggest that RCC are methanogens thus far. In this study CHCl3 was added to the rumens of cows and the responses of the archaeal

community were studied by monitoring the abundance and diversity of genes encoding for the 16S rRNA, and for the subunit of the methyl-coenzyme M reductase.

5.2 Materials and Methods