Cooperation of Three Denitrifying Bacteria in Nitrate Removal of Acidic Nitrate- and Uranium-Contaminated Groundwater

Abstract

In uranium-contaminated aquifers co-contaminated with nitrate, denitrifiers play a critical role in bioremediation. Six strains of denitrifying bacteria belonging to Rhizobium, Pseudomonas, and Castellaniella were isolated from the Oak Ridge Integrated Field Research Challenge Site (OR-IFRC), where biostimulation of acidic (pH 3.5–6.5), nitrate-contaminated (up to 140 mM) groundwater occurred. Three isolates were characterized in regards to nitrite tolerance, denitrification kinetic parameters, and growth on different denitrification intermediates. Kinetic and growth experiments showed that Pseudomonas str. GN33#1 reduced NO⁻ ₃ most rapidly (V_max = 15.8 μmol e⁻·min⁻¹·mg protein⁻¹) and had the fastest generation time (gt) on NO⁻ ₃ (2.6 h). Castellaniella str. 4.5A2 was the most low pH and NO⁻ ₂ tolerant and grew rapidly on NO⁻ ₂ (gt = 4.0 h). Rhizobium str. GN32#2 was also tolerant of low pH values and reduced NO⁻ ₂ rapidly (V_max = 10.6 μmol e⁻·min⁻¹·mg protein⁻¹) but was far less NO⁻ ₂ tolerant than Castellaniella str. 4.5A2. Growth of and denitrification by these three strains incubated together and individually were measured in OR-IFRC groundwater at pHs 5 and 7 to determine whether they cooperate or compete during denitrification. Mixed assemblages reduced NO⁻ ₃ more rapidly and more completely than any individual isolate over the course of the experiment. The results described in this article demonstrate 1) that this synthetic assemblage comprised of three physiologically distinct denitrifying bacterial isolates cooperate to achieve more complete levels of denitrification and 2) the importance of pH- and nitrite-tolerant bacteria such as Castellaniella str. 4.5A2 in minimizing NO⁻ ₂ accumulation in high-NO⁻ ₃ groundwater during bioremediation. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental files.

Keywords:

• bioremediation
• denitrification
• Castellaniella
• tri-culture
• nitrite

Acknowledgments

We thank Dave Watson for providing OR-IFRC site groundwater used in this study, Dr. John Senko for isolating Rhizobium and Pseudomonas strains, John Frink for assisting with experiments, and Yiran Dong and Elizabeth Butler for assisting in nonlinear regression estimates of Michaelis-Menten kinetic parameters. This work was supported by the Office of Biological and Environmental Research (OBER) of the Office of Science, U.S. Department of Energy (DOE), Environmental Remediation Sciences Program (ERSP) (FG03-02ER63443, DE-FC02-96ER62278, and FG02-00ER62986).