MICROBIAL COMMUNITY FOUND THRIVING IN VERY ALKALINE (PH 12-13) GROUNDWATER
Genetic identification using 16S rRNA PCR amplification with a universal bacterial primer yielded 140 unique clones, which fell into two distinct phylogenetic groups. At five of the sites, the sequences identified belonged dominantly to the Comamonadacea family of the β-Proteobacteria. Two sequences, Leptothrix mobilis and a closely related uncultured bacterium, respectively comprised 10% and 8% of the clones. Sequences from a sixth site most closely matched low G+C, gram positive Clostridium and Bacillus species, many of which have been identified in alkaline environments, such as at Mono Lake, California, tufa columns in Greenland, and cement-contaminated groundwater in a deep gold mine in South Africa. Other sequences were most closely related to thermophilic sulfate-reducing bacteria, or to species associated with chlorinated organic contaminants found at the site.
In free-drift microcosm experiments, pH decreased to below 9 as the result of the microbial conversion of the glucose nutrient to CO2. In microcosms buffered by contact with Ca(OH)2 pellets, initial activity appeared limited, with only a few rods observed under the microscope. After six months, microbial activity in microcosms containing slag and glucose had increased considerably, overcoming the buffer and lowering pH to 11.8. Sequencing showed the presence of another Comamonadacea β-Proteobacteria. The complete sequence of this bacterium most closely matches Hydrogenophaga pseudoflava, which is a hydrogen oxidizing bacterium. This suggests the bacteria in the microcosm are deriving energy from hydrogen produced by the corrosion of metallic iron in the slag. Raising the pH of a microcosm back to 12.33 with Ca(OH)2 caused activity to decrease; however, a subsample raised to pH 13.2 with NaOH showed only a slightly reduced population, but with a decreased number of motile bacteria.