Environmental Fate and Transport of Solvent-Stabilizer Compounds - Environmental Investigation and Remediation - page 165

characteristics of the bacterium are unlike the other species identii ed in the Pseudonocardia genus. To
identify the strain, the researchers extracted genomic DNA from CB1190 T to perform polymerase
chain reaction (PCR) amplii cation of the 16S rRNA gene to obtain gene sequences for comparison
with the GenBank database for phylogenetic analyses. Genotypic and phenotypic data support the
formal recognition of Pseudonocardia dioxanivorans as a unique species.
CB1190 T was grown aerobically in ammonium BSM at 30°C with 5 mM 1,4-dioxane. Cell
growth was determined by measuring total protein and by assaying the enzyme activities of soluble
monooxygenases. The observed maximum growth rate over 1 h was 90 g/L, and the maximum

1,4-dioxane degradation rate, 0.1 mg/mg protein/h, is described by Monod kinetics. Cell yield was
0.09

μ

0.002 g of protein per gram of 1,4-dioxane. Mahendra and Alvarez-Cohen also investigated
the ability of CB1190 T to grow on 1,4-dioxane under anaerobic conditions; however, their data did
not show anaerobic growth.
Twenty bacterial isolates, 13 of which were capable of transforming 1,4-dioxane, were evaluated
by Mahendra and Alvarez-Cohen (2006). 1,4-Dioxane served as a growth substrate for Pseudono-
cardia dioxanivorans CB1190 and Pseudonocardia benzenivorans B5; yields were 0.09 g of protein
per gram of 1,4-dioxane and 0.03 g of protein per gram of benzene, respectively. The researchers
observed cometabolic transformation of 1,4-dioxane for monooxygenase-expressing bacterial
strains induced with methane, propane, THF, and toluene, including Methylosinus trichosporium
OB3b, Mycobacterium vaccae JOB5, Pseudonocardia K1, Pseudomonas mendocina KR1, Ralstonia
pickettii PKO1, Burkholderia cepacia G4, and Rhodococcus RR1. Many of the cometabolic reac-
tions resulted in incomplete degradation of 1,4-dioxane because of product toxicity. Subjecting the
bacteria to brief exposure to acetylene, a known monooxygenase inhibitor, prevented oxidation of
1,4-dioxane and toluene in all cases, which supported the hypothesis that monooxygenase enzymes
expressed by these strains are the active agent for degrading 1,4-dioxane. The researchers also used
a colorimetric assay of naphthol production to rapidly detect monooxygenase activity. The ability of
the strains to oxidize naphthalene to naphthol independently coni rms the strains' ability to express
(i.e., utilize) monooxygenases and that dioxane degradation was positively correlated with monoox-
ygenase activity. Bacterial monooxygenases are similar in structure, function, and reaction mecha-
nisms to mammalian P450 enzymes, which have been documented in the metabolism of 1,4-dioxane
during toxicological studies using rats (Woo et al., 1977).
Most of the cultures that acted on 1,4-dioxane cometabolically degraded dioxane incompletely,
which suggests that their transformation capacity for dioxane is limited. The primary substrate (e.g.,
THF) was removed to prevent competition with dioxane during the cometabolic degradation reactions.
Incomplete degradation of 1,4-dioxane could be caused by a limited reductant (NADH) * supply to fuel
the monooxygenase reaction, by toxicity due to dioxane, and/or by toxicity due to dioxane metabolites.
1,4-Dioxane is not likely to exert direct toxicity; CB1190 is able to degrade 1,4-dioxane at concentra-
tions above 1,000,000

±

μ g/L and Strains JOB5, OB3b, and RR1 all tolerate 1,4-dioxane concentrations

as high as 500,000 g/L. Metabolite toxicity is more likely to explain incomplete 1,4-dioxane degrada-

tion: the major dioxane metabolite in mammalian cells, 1,4-dioxane-2-one, is more toxic than dioxane.
In rats, the lethal dose of 1,4-dioxane-2-one is about one-tenth that of 1,4-dioxane (Woo et al., 1977).
Mahendra and Alvarez-Cohen (2006) found that two of the bacterial strains studied were
capable of sustained growth on 1,4-dioxane as the sole carbon and energy source: Pseudonocardia
dioxanivorans CB1190 and Pseudonocardia benzenivorans B5. Rates of oxidation of 1,4-dioxane at
50,000

μ

μ g/L were

• 0.01-0.19 mg/h for the metabolic processes

• 0.1-0.38 mg/h for cometabolism by the monooxygenase-induced strains

• 0.17-0.60 mg/h for the recombinant strains per milligram of bacterial protein present

* NADH is nicotinamide adenine dinucleotide, an important coenzyme found in cells that serve as a carrier of electrons
and a participant in metabolic redox reactions.