Chemical markers for rumen methanogens and methanogenesis

Abstract

The targeting of mcrA or 16S rRNA genes by quantitative PCR (qPCR) has become the dominant method for quantifying methanogens in rumen. There are considerable discrepancies between estimates based on different primer sets, and the literature is equivocal about the relationship with methane production. There are a number of problems with qPCR, including low primer specificity, multiple copies of genes and multiple genomes per cell. Accordingly, we have investigated alternative markers for methanogens, on the basis of the distinctive ether lipids of archaeal cell membranes. The membranes of Archaea contain dialkyl glycerol ethers such as 2,3-diphytanayl-O-sn-glycerol (archaeol), and glycerol dialkyl glycerol tetraethers (GDGTs) such as caldarchaeol (GDGT-0) in different proportions. The relationships between estimates of methanogen abundance using qPCR and archaeol measurements varied across primers. Studies in other ecosystems have identified environmental effects on the profile of ether lipids in Archaea. There is a long history of analysing easily accessible samples, such as faeces, urine and milk, to provide information about digestion and metabolism in livestock without the need for intrusive procedures. Purine derivatives in urine and odd-chain fatty acids in milk have been used to study rumen function. The association between volatile fatty acid proportions and methane production is probably the basis for empirical relationships between milk fatty acid profiles and methane production. However, these studies have not yet identified consistent predictors. We have evaluated the relationship between faecal archaeol concentration and methane production across a range of diets in studies on beef and dairy cattle. Faecal archaeol is diagnostic for ruminant faeces being below the limit of detection in faeces from non-ruminant herbivores. The relationship between faecal archaeol and methane production was significant when comparing treatment means across diets, but appears to be subject to considerable between-animal variation. This variation was also evident in the weak relationship between archaeol concentrations in rumen digesta and faeces. We speculate that variation in the distribution and kinetics of methanogens in the rumen may affect the survival and functioning of Archaea in the rumen and therefore contribute to genetic variation in methane production. Indeed, variation in the relationship between the numbers of micro-organisms present in the rumen and those leaving the rumen may explain variation in relationships between methane production and both milk fatty acid profiles and faecal archaeol. As a result, microbial markers in the faeces and milk are unlikely to relate well back to methanogenesis in the rumen. This work has also highlighted the need to describe methanogen abundance in all rumen fractions and this may explain the difficulty interpreting results on the basis of samples taken using stomach tubes or rumenocentesis.

Effects of supplementing a mixed diet with echium (<i>Echium plantagineum</i>) oil on methanogenesis in a rumen simulation system

“…Several studies (Hook et al, 2011;Lillis et al, 2011) have reported higher numbers of methanogens in rumen solids than in rumen liquid regardless of diet. However, diet may affect the distribution of methanogens between the solid-and liquid-phases of the rumen, with important implications for methanogenesis (McCartney et al, 2013). In the present study, despite not being significant, when compared to control, EO4 increased the proportion of methanogens in the effluents and decreased the proportion of methanogens in residues, suggesting a change in the microbial communities between liquid and solid phases.…”

Methyl-coenzyme M reductase A as an indicator to estimate methane production from dairy cows

“…Whereas the Mlas and Mlas-mod primers designed by Steinberg and Regan (2009) were primarily developed for clone library construction and qPCR analyses in oligotrophic fen and anaerobic digester sludge, the qmcrA primer set has been widely used to quantify methanogens specifically from the ruminant gastrointestinal tract, which harbors a different methanogen population (Denman et al, 2007). First attempts were made to relate rumen methanogens as quantified by qmcrA primers with whole-animal methane production in small ruminants, but the results show some discrepancies (McCartney et al, 2013). Briefly, Ding et al (2012) reported a highly positive correlation between mcrA DNA abundance and methane yield measured in 12 Tibetan sheep, whereas Morgavi et al (2012) failed to demonstrate a significant relationship for 6 Texel wethers, which could have been due to defaunation applied in the latter study.…”

Microbial trophic interactions and mcrA gene expression in monitoring of anaerobic digesters

“…Recently biochemical markers using archaeol (2,3-diphytanyl- O -sn-glycerol) also have been developed (McCartney et al, 2013a). In the case of molecular analysis, various methanogen specific primers targeting 16S rRNA gene have been developed (Castro et al, 2004; Yu et al, 2005; Zhou et al, 2011).…”