348731 The Aerobic Cometabolic Transformation of Chlorinated Ethenes By the Mycobacterium ELW1 Grown on Isobutene

Monday, November 4, 2013
Grand Ballroom B (Hilton)
Stephanie Rich, Chemical, Biological, and Environmental Engineering, Oregon State University, Corvallis, OR

The Aerobic Cometabolic Transformation of Chlorinated Ethenes by the Mycobacterium ELW-1 Grown on Isobutene

AUTHORS: Stephanie Rich1, Mohammad Azizian1, Michael Hyman2, Lewis Semprini1

1School of Chemical, Biological, and Environmental Engineering, Oregon State University;  2 Department of Microbiology, North Carolina State University.

            ELW1 is a recently isolated aerobic bacterium, in the mycobacterium family, that was discovered to have cometabolic capabilities, such as the ability to transform chlorinated ethenes into less toxic substances. The focus of this research was to determine the rates of transformation when this organism was exposed to high contaminant concentrations. This will help establish the practicality of using ELW1 for bioremediation techniques in the field. The chlorinated ethenes ELW1 was found to transform are notorious carcinogens, groundwater pollutants, and volatile organic compounds whose remediation has been a worldwide problem for over 40 years. Transformation rates for three different types of chlorinated ethenes were determined, (TCE), cis-dichloroethene (c-DCE), and vinyl chloride (VC).

            The ELW1 culture was grown and sustained in both batch culture and steady-state chemostat environments with isobutene as a growth substrate on a minimal growth media containing trace minerals, such as cobalt and a phosphate buffer. The batch-grown cells for these experiments were cultured in 600mL glass bottles with Teflon-lined septa, filled with 300mL of growth media. Batch cells were then harvested after a four-day incubation period at an optical density (OD600) of approximately of 0.34, while chemostat-grown cells were harvested at an optical density (OD600) of 1.3-1.5. Cells that were used for experiments were concentrated by centrifugation, washed with excess media and placed in a new media solution containing high concentrations of the compound to be tested. Initial rates of transformation were determined using gas chromatography to analyze 100L samples of headspace gas over a period of one to three days to determine the rate of compound loss.

            The rates of utilization of a range of isobutene concentrations using batch-grown cultures were determined in order to estimate values of the half-substrate coefficient, Ks and maximum utilization rate, kmax. A Ks value of 5.6 mol/L and kmax value of 38 mol/day/mg protein was estimated for isobutene, which is in the range of values previously determined at North Carolina State University. Isobutene rate tests were performed using the same procedure as contaminant rate tests, over a range of isobutene concentrations.

The rates at which ELW1 can utilize isobutene were compared with rates of chlorinated ethene transformation, which overall quantifies the practicality of using this organism in the field. The results of this type of comparison analysis are displayed in Table 1.

Table 1 Transformation rates determined for ELW-1 grown on isobutene

Cell Growth

Compound

Initial Liquid Concentration(umol/L)

Rate of Transformation

(umol/day/mg TSS)

Batch

VC

421

5.1

Chemostat

VC

382

3.2

Batch

c-DCE

1330

4.1

Chemostat

c-DCE

1400

3.5

Batch

TCE

198

0.2

Batch

Isobutene

118

15.9

Chemostat

Isobutene

129

11.2

            The values in Table 1 show that the ELW-1 culture can transform VC and c-DCE much faster than TCE. The rates measured using batch-grown cultures are consistently faster than the estimated rates obtained with the chemostat grown cells. By comparing the rate of TCE transformation with that of isobutene, it is not certain whether the ELW-1 culture would be effective for the bioremediation of TCE contamination.  c-DCE and VC, however, would be effectively treated using ELW-1.


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