The Microbes of Moria: Characterization of active microbial members in the subsurface environment of the Witwatersrand Basin

Abstract

The subsurface borehole water of Beatrix mine in the Witwatersrand Basin in South Africa was sampled and tested for the presence of an active microbial community for the first time at the region. Gene diversity of 16S ribosomal DNA (rDNA) and 16S ribosomal RNA (rRNA), which represents respectively the total microbial community and the active microbial community, was characterized. If a microorganism is metabolically active, transcription by ribosomes must be taking place, and the rRNA concentrations within it will be far greater than that of microorganisms that are moribund or senescent. Analyzing rRNA is one of the well-accepted approaches to characterize the active community. Analysis for both the active and total communities was done to characterize both bacteria and archaea. Analysis was also done on selected functional genes in the active community in order to gain a greater understanding of what metabolic processes are actively taking place in the subsurface. Taxonomic affiliations of the 16S rRNA and 16S rDNA sequences from this study were assigned by comparing them to a public database and phylogenetic analysis. Alpha and Betaproteobacteria were prevalent in both the active and total bacterial community, while uncultured euryarchaotes and Methanobacteria were prevalent in both the active and total archaeal communities. The Firmicutes species Ca. D. audaxviator, which has been found in deep fractures across the entire Witwatersrand Basin, was detected in the 16S rRNA gene library of the DNA, but was not detected in the 16S rRNA library. The nifH gene sequence belonging to Ca. D. audaxviator was detected in the RNA indicating that the organism may actively fix N2 in the studied fracture.