Aerobic bacterial degradation of the water pollutant N-nitrosodimethylamine (NDMA)

The water contaminant N-nitrosodimethylamine (NDMA) is a probable human carcinogen whose appearance in the environment is related to the release of rocket fuel and to certain forms of water and wastewater treatment. Although this compound has been shown to be biodegradable, there is minimal information about the organisms capable of performing degradation, and little is understood of the mechanisms or biochemistry involved. The goal of this dissertation is to gain a better mechanistic understanding of the bacterial oxidation of NDMA in order to assess the potential for biological attenuation of this compound.; Organisms were identified that could cometabolically degrade NDMA under aerobic conditions. Results demonstrate that bacteria expressing certain monooxygenase enzymes functionally similar to those demonstrated to degrade NDMA in eukaryotes have the capability to degrade NDMA. Specifically, induction of the soluble methane monooxygenase (sMMO) expressed by Methylosinus trichosporium OB3b, the propane monooxygenase (PMO) enzyme of Mycobacterium vaccae JOB-5, and the toluene 4-monooxygenases found in Ralstonia pickettii PKO1 and Pseudomonas mendocina KR1, resulted in NDMA degradation by these strains. In each of these cases, brief exposure to acetylene gas, a suicide substrate for certain monooxygenases, inhibited the degradation of NDMA. Further, Escherichia coli TG1/pBS(Kan) containing recombinant plasmids derived from the toluene monooxygenases found in strains PKO1 and KR1 mimicked the behavior of the parent strains. In contrast, M. trichosporium OB3b expressing the particulate form of MMO, Burkholderia cepacia G4 expressing the toluene 2-monooxygenase and Pseudomonas putida mt-2 expressing the toluene side chain monooxygenase, were not capable of NDMA degradation. In addition, bacteria expressing aromatic dioxygenases were not capable of NDMA degradation. Finally, Rhodococcus sp. RR1 exhibited the ability to degrade NDMA by an unidentified, constitutively-expressed enzyme that was not inhibited by acetylene exposure.; It was determined that cellular growth on propane led to the fastest NDMA transformation rates and a kinetic analysis of cometabolic NDMA degradation by two propanotrophs was conducted. Growth of Rhodococcus sp. RR1 on propane resulted in a maximum transformation rate, numax,n , of 44 +/- 5 mug NDMA (mg protein)-1 hr -1, while similarly cultured bacteria of Mycobacterium vaccae Job-5 exhibited a maximum rate of 28 +/- 3 mug NDMA (mg protein) -1 hr-1. The half saturation constant, Kn, for each strain was 36 +/- 10 mug NDMA L-1 and 2200 +/- 1000 mug NDMA L-1 respectively. While strain Job-5 did not exhibit the ability to transform NDMA after growth on soy broth, strain RR1 exhibited a numax,n of 0.14 +/- 0.01 mug NDMA (mg protein)-1 hr-1 and a Kn of 45 +/- 10 mug NDMA L-1 when grown in this way. Both strains were capable of NDMA degradation at trace levels resulting in final concentrations below the detection threshold of 0.02 mug NDMA L-1. The co-presence of propane had a stronger inhibitory effect on NDMA degradation in strain JOB-5 than in strain RR1 as expected from the differences in propane half saturation constants for the two strains (Kp of 6200 +/- 400 mug propane L-1 for strain RR1 and 790 +/- 400 mug propane L-1 for strain JOB-5).; None of the tested bacteria were capable of growth using NDMA as a sole energy and carbon source. Measurements of the evolution of 15N 2-labeled molecular nitrogen during the metabolism of doubly 15N-labeled NDMA indicated that 19 +/- 2 of the NDMA was metabolized by alpha-hydroxylation. In addition, conversion of NDMA to nitrite accounted for 13 +/- 1% transformation while approximately 50% of the carbon from NDMA was converted to formaldehyde prior to further respiration by the cells.; A comparison of NDMA degradation by soil enrichments demonstrated that cells enriched on pyruvate can degrade NDMA; however degradation was strongly enhanced by enrichment with pro...