2.5: Example analysis

To provide an example of a microbial community analysis using 16S rRNA gene sequencing, we can consider some of the findings of Kirk et al. (2015). Their study examined variation in the chemistry and microbiology of water and natural gas samples collected from coal-bearing strata in the Cherokee basin in southeastern Kansas, USA. One research question they considered was: how did methane in the coalbed gas form? Some Archaea can form methane by a few different pathways (see Section 5.1.4), but methane can also form abiotically through thermal degradation of organic matter.

The researchers’ geochemical analysis indicated that methanogenic microorganisms formed the methane by oxidizing dihydrogen \(\left(\mathrm{H}_{2}\right)\) and reducing carbon dioxide \(\left(\mathrm{CO}_{2}\right)\). Consistent with that result, their analysis of 16S rRNA genes in their samples indicated that most of the archaeal sequences were closely related to groups of methanogens that form methane by reducing carbon dioxide. To illustrate, Figure \(2.5\) shows the relative abundances of sequences that classified within classes of Archaea. The relative abundance of a microbial group (e.g., a genus, a family, or etc.) is the number of sequences that classified within that group divided by the total number sequences obtained for the sample. This value is typically expressed as a percentage. Of the classes identified, only those within Methanosarcinales are thought to be capable of producing methane by pathways other than carbon dioxide reduction (Thauer et al., 2008), and these sequences accounted for only a small portion of the sequences overall. Thus, the results from the geochemical and microbial analyses provided strong evidence for understanding how methane formed in the coalbeds.

Figure \(2.5\): Results of 16S rRNA gene analysis by Kirk et al. (2015). Results are only shown for sequences that classified within orders of Archaea, and only classes with average relative abundance greater than 2% are specifically identified.

In addition to helping reveal the main pathway of methanogenesis in the coalbeds, their analysis also provided insight into potential environmental controls on microbial methane production. They observed that the relative abundance of Methanosarcinales sequences, though small overall, increased significantly with the salinity of the formation water. This result is also consistent with their analysis of gas and water geochemistry. Thus, they concluded that methane formed primarily by carbon dioxide reduction but that the contribution of other pathways increased with the salinity of the formation water. This result indicates that formation water salinity is an environmental control on the overall function of the microbial community in the coalbeds.

For this type of analysis, care should be taken in interpreting the functional capabilities of microorganisms based on their taxonomy. Microbial species can often use more than one type of reaction as a source of energy and just because one species is closely related to another, it does not mean that they would both use the same reaction. Therefore, it is helpful to combine microbial community analyses with other results that provide constraints on community function, such as results that show how the community is affecting the chemistry of the environment.