|Abstract or Summary
- Reductive dechlorination of chlorinated ethenes is dependent upon suitable
substrates promoting microbial activity and creating anaerobic conditions. At the
periphery of active reductive dechlorinating zones combinations of lesser
chlorinated ethenes should exist along with end products of the anaerobic
metabolism that is driving reductive dechlorination. Potential end-products of
anaerobic metabolism were investigated for their ability to stimulate oxidative
cometabolism of dichloro ethenes (DCEs) by the butane-degrading bacterium,
Pseudomonas butanovora. Organic acids that supported butane-monooxygenase
(BMO) activity were acetate, propionate, lactate, and butyrate. Lactate
consistently supported and sustained greater rates of cooxidation than did the other
organic acids. When propane replaced butane as the growth substrate, lactate
remained the superior electron donor, while the ability of butyrate and acetate to
support BMO activity decreased. In contrast, propionate-supported cooxidation
was only observed in propane-grown cells.
Lactate supported the degradation of 1,2-trans dichloroethylene (1,2-trans
DCE), 1,2-cis dichloroethylene (1,2-cis DCE) and 1,1-dichloroethylene (1,1-DCE)
in butane-grown P. butanovora. 78 nmoles (25 μM) of 1,2-cis DCE were
completely degraded by butane-grown P. butanovora. In contrast, smaller
amounts of 1,1-DCE and 1,2-trans DCE were degraded over the twenty minute
time course. Decreasing rates of cooxidation over time were observed for of all
three DCEs, and 50% of BMO activity was irreversibly lost after 15 min, 6 min,
and 0.5 min exposures to 1,2-cis DCE, 1,2 trans-DCE, and 1,1-DCE respectively.
Cell viability decreased by over 90, 95, and 99.95% during the transformation of
25 nmoles/mg protein of 1,2-cis DCE, 1,2-trans DCE and 1,1-DCE. These results
indicate that cellular viability was more sensitive to cooxidation of 1,2-cis DCE
and 1,2-trans DCE than was BMO.
1,2-cis DCE and 1,2-trans DCE induced BMO activity to 25 and 45% of
the butane control, respectively. Induction by 1,2-trans DCE was observed at a
threshold of about 20 μM and higher concentrations did not increase BMO
activity. Fusion of lacZ to the BMO catabolic promoter, with consequent knock
out of BMO activity, provided the opportunity to assess substrate induction
without the confounding effects of enzyme inactivation and product induction.
While BMO substrates, butane, 1,2-cis DCE, and ethylene, were unable to induce
lacZ activity the BMO products, 1-butanol, and ethylene oxide, effectively induced
lacZ activity. 1,2-trans DCE was unique among the BMO substrates tested in it's
ability to induce expression of lacZ, 2-fold above background, in the reporter strain. A wide range of concentrations induced lacZ activities (10 to 100 μM), and
low levels of 1,2-trans DCE achieved high levels of induction after 4 hrs.
However, lacZ activities were limited to an induction of about four-fold above
background and this limit allowed lower concentrations of 1,2-trans DCE to
eventually produce equal levels of beta-galactosidease. These data provide proof-of-
concept that BMO-dependent cometabolism can occur independently of butane
as an inducer and electron donor for BMO gene expression and activity.