Abstract

Microbial communities play a significant role in the treatment of inorganic constituents at mine sites, often through direct metabolic reduction. Soluble metal ions can act as the electron acceptor in microbial respiration with concurrent decreases in their solubility and toxicity, for example the reduction of aqueous selenate to precipitated elemental selenium. Targeted quantitative polymerase chain reaction (qPCR) allows quantitative monitoring of specific genes involved in chemical reduction processes, such as selenate reductases. Additionally, genetic colony identification and next generation sequencing (NGS) of 16S rRNA gene amplicons can be used to provide comprehensive microbial community profiles that are used to monitor important metabolic functions such as sulfate reduction, nitrate reduction, or groups that directly precipitate metals. In this study qPCR tests, NGS, and differential plating methods combined with genetic colony identification were used to detect and characterize microbial communities that reduced selenate (SeO₄^2-) to selenite (SeO₃^2-) or SeO₃^2- to elemental Selenium (Se). Pathways that produced selenophosphate (SePO₃^3-), an important precursor to biologically available organophosphates, were also explored.

Selenate metabolic pathways were used to develop qPCR tests specific to selenate reductase genes, including srdA and serA, which were used to quantify specialized selenium reducing microbial communities in samples of geologic material from a mine site. Metabolic pathways that facilitate the production of SePO₃^3- using a universally conservative selD gene were also investigated using specific qPCR tests. Other qPCR tests targeting sulfate reducing bacteria and nitrate reducing bacteria were used to quantify non-specialized selenium degraders. For the purposes of monitoring selenium metabolism through NGS, an open-source microbial functional database was modified to include a new functional category made up of 27 microbial taxa documented to be involved in selenium reduction. The database was then used to summarize the proportion of selenium metabolizers in the context of other metabolic functions. This provided rapid and easily interpretable outputs of complex data. The data indicated the presence of both specialized and non-specialized selenium reducers in the samples tested.

 In another approach, microbes from geologic material from a known selenium reducing site were plated and colonies of selenite reducing bacteria were grown and isolated. PCR amplification of the 16S rRNA gene combined with Sanger sequencing were used to identify the species of bacteria that reduced selenium based on the formation of characteristic red colonies on the selenite-amended agar plates. This is an example of using classical plate-based microbiology methods combined with molecular biology that provides a method for quickly isolating and identifying selenite reducing microbes that may be yet to be identified in the literature. The combination of molecular biological tools used in this study provided multiple lenses to view selenium metabolizing communities and can vastly increase our ability to understand how microbiology impacts metals treatment processes for selenium and other toxic metals.