Addition of molasses and urea was tested as a means of stimulating microbial urea hydrolysis in the Eastern Snake River Plain Aquifer in Idaho. Ureolysis is an integral component of a novel remediation approach for divalent trace metal and radionuclide contaminants in groundwater and associated geomedia, where the contaminants are immobilized by coprecipitation in calcite. Generation of carbonate alkalinity from ureolysis promotes calcite precipitation. In calcite-saturated aquifers, this represents a potential long-term contaminant sequestration mechanism. In a single-well experiment, dilute molasses was injected three times over two weeks to promote overall microbial growth, followed by one urea injection. With molasses addition, total cell numbers in the groundwater increased 1-2 orders of magnitude. Estimated ureolysis rates in recovered groundwater samples increased from < 0.1 to > 25 nmol L(-1) hr(-1). A quantitative PCR assay for the bacterial ureC gene indicated that urease gene numbers increased up to 170 times above pre-injection levels. Following urea injection, calcite precipitates were recovered. Estimated values for an in situ first order ureolysis rate constant ranged from 0.016 to 0.057 d(-1). Although collateral impacts such as reduced permeability were observed, overall results indicated the viability of manipulating biogeochemical processes to promote contaminant sequestration.

Polymerase chain reaction primers based on the ureC gene are described for use in detecting diverse groundwater ureahydrolyzing bacteria. Six degenerate primers were designed and evaluated for their ability to detect the gene encoding the large catalytic subunit of urease, ureC. Five combinations of these primers were tested pair-wise and displayed an overlapping detection range for bacterial isolates. Pair L2F/L2R exhibited the greatest detection range for described bacterial species and for bacterial isolates from groundwater samples belonging to the bacterial divisions Firmicutes, Actinobacteria, and the α, β, and γ subdivisions of Proteobacteria. Primers L2F/L2R exhibited a greater detection range than previously described ureC-specific primers, and amplified novel ureC sequences from groundwater isolates in the genera Hydrogenophaga, Acidovorax, Janthinobacterium, and Arthrobacter. A comparative phylogenetic analysis of ureC and 16S rRNA genes was performed to determine the utility of groundwater ureC sequence information as a phylogenetic marker for ureolytic species. Our results were consistent with previous analyses of urease genes which demonstrated that the ureC gene has undergone lateral transfer and is not a robust phylogenetic marker. However, the ureC-specific primers, L2F/L2R, demonstrate a broad detection range for ureolytic species, and can serve to enhance functional diversity analyses of ureolytic bacteria.