The lesser chlorinated ethenes, cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC), tend to accumulate at chloroethene-contaminated sites under anaerobic conditions, limiting the application of natural attenuation and enhanced reductive anaerobic in-situ treatment technologies. Aerobic degradation of lesser-chlorinated ethenes has been demonstrated; however, present understanding of the transformation potentials of cDCE and VC is limited, thus limiting the reliability of and confidence in natural and enhanced biological alternatives for site remediation.
This project determined the distribution and metabolic capabilities of microorganisms able to mineralize cDCE and/or VC in aerobic subsurface environments.
Thirty-seven sample-types were screened from 22 installations. Microcosms were prepared from all sample-types, and enrichment on cDCE and/or VC was attempted. Microcosms demonstrating cDCE or VC degradation were diluted into minimal salts medium (MSM), and subsequent enrichment on the chlorinated compound as sole carbon and energy source in MSM was attempted to isolate and characterize the degrading organisms.
Only two samples displayed presence of aerobic cDCE degraders; one aerobic bacterium capable of growth on cDCE as a sole carbon and energy source was isolated by enrichment culture. The 16S ribosomal DNA sequence of the isolate (strain JS666) had 97.9% identity to the sequence from Polaromonas vacuolata. At 20°C, strain JS666 grew on cDCE with a minimum doubling time of 73 h and a growth yield of 6.1 g of protein/mol of cDCE. Chloride analysis indicated that complete dechlorination of cDCE occurred during growth. The half-velocity constant (Ks) for cDCE transformation was 1.6 μM, and the maximum specific substrate utilization rate ranged from 12.6 to 16.8 nmol/min/mg of protein. Resting cells grown on cDCE could transform cDCE, ethene, VC, trans-dichloroethene, trichloroethene, and 1,2- dichloroethane. Epoxyethane was produced from ethene by cDCE-grown cells, suggesting that an epoxidation reaction is the first step in degradation.
Twelve different bacteria (11 Mycobacterium strains and 1 Nocardioides strain) capable of growth on VC as sole carbon source were isolated, and five representative strains were examined. All the isolates grew on ethene in addition to VC and contained VC-inducible ethene monooxygenase activity. The Mycobacterium strains all had similar growth yields (5.4 to 6.6 g of protein/mol), maximum specific growth rates (0.17 to 0.23 day-1), and maximum specific substrate utilization rates (9 to 16 nmol/min/mg of protein) with VC. The Nocardioides strain had a higher growth yield (10.3 g of protein/mol), growth rate (0.71 day-1), and substrate utilization rate (43 nmol/min/mg of protein) with VC but was much more sensitive to VC starvation. Ks values for VC were between 0.5 and 3.2 μM, while for oxygen they ranged from 0.03 to 0.3 mg/liter.
The isolation and preliminary characterization of a novel, cDCE-degrading aerobe is significant — it is the first report of such an organism. The ability of this bacterium to subsist on cDCE as sole carbon and energy source makes it a good candidate for use as a bioaugmentation agent at chlorinated ethene-contaminated sites where degradation has stalled and cDCE has migrated to aerobic zones. The VC results indicate that aerobic VC-degrading microorganisms are widely distributed at sites contaminated with chlorinated solvents and are likely to be responsible for the natural attenuation of VC. (Project Completed – 2004)