Molybdenum Availability Is Key to Nitrate Removal in Contaminated Groundwater Environments

Abstract

The concentrations of molybdenum (Mo) and 25 other metals were measured in groundwater samples from 80 wells on the Oak Ridge Reservation (ORR) (Oak Ridge, TN), many of which are contaminated with nitrate, as well as uranium and various other metals. The concentrations of nitrate and uranium were in the ranges of 0.1 μM to 230 mM and <0.2 nM to 580 μM, respectively. Almost all metals examined had significantly greater median concentrations in a subset of wells that were highly contaminated with uranium (≥126 nM). They included cadmium, manganese, and cobalt, which were 1,300- to 2,700-fold higher. A notable exception, however, was Mo, which had a lower median concentration in the uranium-contaminated wells. This is significant, because Mo is essential in the dissimilatory nitrate reduction branch of the global nitrogen cycle. It is required at the catalytic site of nitrate reductase, the enzyme that reduces nitrate to nitrite. Moreover, more than 85% of the groundwater samples contained less than 10 nM Mo, whereas concentrations of 10 to 100 nM Mo were required for efficient growth by nitrate reduction for two Pseudomonas strains isolated from ORR wells and by a model denitrifier, Pseudomonas stutzeri RCH2. Higher concentrations of Mo tended to inhibit the growth of these strains due to the accumulation of toxic concentrations of nitrite, and this effect was exacerbated at high nitrate concentrations. The relevance of these results to a Mo-based nitrate removal strategy and the potential community-driving role that Mo plays in contaminated environments are discussed.

FIG 1

Model of the effects of NO₃⁻, Mo, and W on the growth of Pseudomonas isolates under denitrifying conditions. The first enzyme in the denitrification pathway, nitrate reductase, requires a molybdopterin guanine dinucleotide cofactor for activity. The activity of nitrate reductase can be increased during growth of a Pseudomonas isolate by supplementing the growth medium with Mo or NO₃⁻. This effect is beneficial to growth up to a point; however, oversupplementation with Mo and NO₃⁻ can result in an inhibitory buildup of nitrite, decreasing growth. Supplementation of the growth medium with W results in antagonistic interactions with Mo that decrease nitrate reductase activity.

FIG 2

Metals in pristine and contaminated wells at the ORR. (A) Concentrations of metals and nitrate (inset) measured in the soluble fractions of groundwater samples from pristine well FW-300 (black) and contaminated well FW-126 (gray). Measurements at or below detection limits are indicated with asterisks. The error bars indicate standard deviations. (B) The groundwater samples were divided into two subsets based on the soluble U concentration: those below (black) and those above (gray) the maximum amount (126 nM) allowed in drinking water by the EPA. For each subset of groundwater samples, the low, median, and high concentrations are represented. Metals whose median concentrations are higher in U-contaminated than in noncontaminated wells are represented by circles, and those whose median concentrations are lower are represented by triangles.

FIG 3

Nitrate and Mo concentrations in groundwater wells at the ORR. Each point represents a groundwater sample taken from a well at the ORR. A total of 93 groundwater samples were taken from 80 wells. Duplicate samples from the same well were taken either on different dates or at different well depths. The concentrations of nitrate and Mo are plotted on logarithmic scales. Wells containing U above (blue) or below (green) a concentration of 126 nM are indicated. The red dashed line crosses the Mo concentration axis at 10 nM, and the purple dashed line crosses at the limit of detection for Mo. The nitrate data were collected as part of a previous study (9).

FIG 4

Effect of high concentrations of Mo on the growth of Pseudomonas strains in the presence of high concentrations of nitrate. The three strains (RCH2, N2A2, and N2E2) were grown anaerobically on basal medium with 60 mM nitrate (black circles) or 120 mM nitrate (gray squares) at various Mo concentrations (expressed on a logarithmic scale). Growth is represented as a percentage of the maximum growth measured for the strain. The error bars indicate standard deviations.