SÍNTESIS DEL CASO

Frozen cultures of methanogens were warmed quickly to 39°C and inoculated (0.5 and 1 ml) into 10 ml of appropriate media (BY, BY+ or RM02). These inoculated tubes were pressurised with H2/CO2 and incubated at 39°C with shaking.

74 Clones of Escherichia coli were regenerated by streaking them onto pre-warmed (37°C) LB plate or adding small amount of clones into pre-warmed (37°C) LB broth media. The LB plate/LB broth was then kept at 37°C overnight.

76

Chapter 3

Selection of primers for the molecular-based studies of rumen methanogens

3.1 Introduction

The diversity of the microbial community in the rumen environment has become a great interest in the last few years as the methane produced in the rumen is a potent greenhouse gas. Methane contributed 30.3% to New Zealand‟s antrophogenic greenhouse gas total emission in 2008 (Ministry for the Environment, 2009). Ninety six percentage (96%) of this methane was generated by enteric fermentation, nearly all of this from the rumens of farmed ruminants. The methanogens, which account for a minor proportion (0.5-3.0%) of the total rumen microbes, produce this methane as a part of their energy metabolism (Hedderich & Whitman, 2006; Wolin et al., 1997). Formation of methane is the major way to eliminate the H2, which is the by-product of feed

fermentation in the rumen. Thus, methanogens in the rumen play a major part in improving the efficiency of the conversion of complex carbohydrates to fermentable sugars which is in part due to the effective disposal of H2, and they also contribute

substantially to the GHG emission.

Cultivation methods to study the rumen methanogens are not always possible as this group of microbes are nutritionally and culturably fastidious (Chaban et al., 2006; Garcia et al., 2000). Due to this constraint, to explore the diversity of methanogens in the rumen, culture-independent molecular approaches have become indispensable. Molecular techniques use the nucleic acids extracted directly from the environment. Subsequently, the marker genes, such as 16S rRNA and mcrA genes are used to determine the methanogen diversity in that environment. The 16S rRNA gene has functional regions that are highly conserved as well as regions that are semi-conserved at the phylum level or highly diverse even among closely related genera which help to determine the phylogenetic relatedness of organisms (Ludwig & Schleifer, 1994; Woese, 1987). To study the methanogens in the environmental samples, archaeal 16S rRNA gene phylogenetic markers are usually used. This is because designing 16S rRNA gene markers that are specific to methanogens is quite difficult due to their high phylogenetic diversity (Banning et al., 2005).

77 The other important target for the methanogens, the mcrA gene, is the gene encoding α subunit of methyl-coenzyme M reductase (MCR) (Hales et al., 1996; Luton

et al., 2002; Springer et al., 1995). Methyl-coenzyme M reductase is one of the key enzymes of methanogenesis, which catalyzes the final step of methane formation and is unique to methanogens (Friedrich, 2005), except for methane oxidising Archaea (Hallam et al., 2003). Studies conducted on methanogen diversity in landfills using 16S rRNA and mcrA gene fragments have confirmed that both phylogenies are similar and that the mcrA gene can be used as a phylogenetic marker to study methanogens (Luton

et al., 2002). The 16S rRNA gene markers used in the study of rumen methanogens are common to all Archaea, but the mcrA gene marker is specific to methanogens.

One potential concern in using these targets for PCR-based molecular techniques is the possibility of PCR bias. One factor due to the primers themselves stands out as being very important for faithful amplification of the original populations. PCR bias can lead to the preferential amplification of certain templates (PCR selection) (Polz & Cavanaugh, 1998; von Wintzingerode et al., 1997), the lack of amplification of certain groups (Skillman et al., 2006; Pei et al., 2008) and different amplification efficiencies for different environmental samples (Juottonen et al., 2006). There have been a number of archaeal 16S rRNA and methanogen mcrA gene primers designed to target different regions of these genes to study rumen methanogens (Table 1.2). However, it‟s not sure whether the resultant PCR products by these primers represent the original samples. A few studies have compared different primers targeting the 16S rRNA and mcrA genes for the suitability to represent the methanogen diversity of selected environment (Friedrich, 2005; Juottonen et al., 2006; Pei et al., 2008; Skillman et al., 2006; Tajima

et al., 2001). Despite this progress, more studies are still required to check the primer suitability for environmental samples to study the methanogen diversity.

In this thesis, methanogen diversity of New Zealand farmed-ruminants was determined using molecular techniques, and then cultivation techniques used to isolate previously uncultured rumen methanogens. Although there are other factors (e.g. DNA extraction methods, PCR inhibitors) that can also affect the determination of methanogen diversity in environmental samples, the selection of suitable primers was considered to be an important first step in this thesis. There were a number of 16S rRNA and mcrA gene primers published which targeted different regions of the

78 respective genes. However, in this study only a few 16S rRNA and mcrA gene primers were selected and checked for their suitability to use on the rumen sample. Small clone libraries were constructed using a single rumen sample with selected primers, and the different groups of methanogens identified and their relative proportions were considered in choosing suitable primers for molecular ecological studies of rumen methanogens to use in this thesis.