Transition from cometabolic to growth-linked biodegradation of vinyl chloride by a Pseudomonas sp. isolated on ethene.

Pseudomonas aeruginosa strain DL1 was isolated on ethene as a sole carbon and energy source. When ethene-grown DL1 was first exposed to vinyl chloride (VC), the rate of VC consumption was very rapid and then declined sharply, indicative of a cometabolic process. A lack of growth and significant release of soluble products during this interval also indicates that the initial activity on VC was cometabolic. Following the rapid initial rate of VC cometabolism, a slow rate of VC utilization continued. After an extended period of incubation (>40 days), a transition occurred that allowed DL1 to begin using VC as a primary growth substrate, with an observed yield, maximum growth rate, and Monod half saturation coefficient of 0.21 mg of total suspended solids/mg VC, 0.046 d(-1), and 1.17 microM VC, respectively, at 22 degrees C. Acetylene inhibits consumption of ethene and VC by ethene-grown cells, suggesting a monooxygenase is responsible for initiating metabolism of these alkenes. Resting cells grown on ethene cometabolized VC with an observed transformation capacity of 9.1 micromol VC/mg total suspended solids and a transformation yield of 0.22 mol VC/mol ethene. The presence of 40 microM ethene increased the rate and amount of VC cometabolized. However, consumption of higher concentrations of ethene decreased the total amount of VC consumed, and VC inhibited ethene utilization. A kinetic model was developed that describes substrate interactions during batch depletion of ethene and VC for a range of initial concentrations. The results suggest that ethene may stimulate in situ biodegradation of VC either by functioning as a primary substrate to support cometabolism of VC or by selecting for organisms that can utilize VC as a primary substrate.

Substrate interactions during aerobic biodegradation of methane, ethene, vinyl chloride and 1,2-dichloroethenes.

Intrinsic biodegradation of trichloroethene and 1,1,1-trichloroethane in groundwater at a Superfund site in California has been observed. An anaerobic zone exists in the area closest to the source location, yielding the expected complement of reductive dechlorination daughter products, including cis-1,2-dichloroethene (cis-DCE) and vinyl chloride (VC). Significant levels of methane and ethene were also generated in the anaerobic zone. The groundwater returns to aerobic conditions downgradient of the source, with methane, ethene, VC, and several other compounds still present. Attenuation of VC in the aerobic zone suggests that it is being biodegraded. In this study microcosms were used to evaluate the role of methane and ethene as primary substrates for aerobic biodegradation of VC. Biodegradation of VC was fastest in the bottles containing ethene, with 40 mumol of VC consumed over a 150 day period, compared to approximately 15-20 mumol with methane or a mixture of methane and ethene. VC did not noticeably inhibit ethene biodegradation but did slow the rate of methane use. Methane inhibited ethene metabolism, which apparently caused a reduction in VC biodegradation when methane was present with ethene. These results suggest that ethene plays an important role during in situ natural attenuation of VC under aerobic conditions. Microcosms were also set up with VC alone. Following a 75 day lag period. VC consumption began and subsequent additions were consumed without a lag, suggesting the presence of organisms capable of using VC as a growth substrate. After providing VC alone for nearly 400 days, aliquots of the enrichment culture were used to evaluate its ability to biodegrade cis- and trans-DCE. Both compounds were readily consumed, although addition of VC as the primary substrate was needed to sustain biodegradation of repeated additions. This result suggests that organisms capable of using VC as a sole substrate may play an active role in aerobic natural attenuation of DCEs.

Characterization of an isolate that uses vinyl chloride as a growth substrate under aerobic conditions.

An aerobic enrichment culture was developed by using vinyl chloride (VC) as the sole organic carbon and electron donor source. VC concentrations as high as 7.3 mM were biodegraded without apparent inhibition. VC use did not occur when nitrate was provided as the electron acceptor. A gram-negative, rod-shaped, motile isolate was obtained from the enrichment culture and identified based on biochemical characteristics and the sequence of its 16S rRNA gene as Pseudomonas aeruginosa, designated strain MF1. The observed yield of MF1 when it was grown on VC was 0.20 mg of total suspended solids (TSS)/mg of VC. Ethene, acetate, glyoxylate, and glycolate also served as growth substrates, while ethane, chloroacetate, glycolaldehyde, and phenol did not. Stoichiometric release of chloride and minimal accumulation of soluble metabolites following VC consumption indicated that the predominant fate for VC is mineralization and incorporation into cell material. MF1 resumed consumption of VC after at least 24 days when none was provided, unlike various mycobacteria that lost their VC-degrading ability after brief periods in the absence of VC. When deprived of oxygen for 2.5 days, MF1 did not regain the ability to grow on VC, and a portion of the VC was transformed into VC-epoxide. Acetylene inhibited VC consumption by MF1, suggesting the involvement of a monooxygenase in the initial step of VC metabolism. The maximum specific VC utilization rate for MF1 was 0.41 micromol of VC/mg of TSS/day, the maximum specific growth rate was 0.0048/day, and the Monod half-saturation coefficient was 0.26 microM. A higher yield and faster kinetics occurred when MF1 grew on ethene. When grown on ethene, MF1 was able to switch to VC as a substrate without a lag. It therefore appears feasible to grow MF1 on a nontoxic substrate and then apply it to environments that do not exhibit a capacity for aerobic biodegradation of VC.

Modeling the kinetics of vinyl chloride cometabolism by an ethane-grown Pseudomonas sp.

Pseudomonas sp strain EA1 was isolated under aerobic conditions using ethane as the sole organic carbon and electron donor source, with an observed yield of 0.99 mg total suspended solids/mg ethane (0.85 mg volatile suspended solids / mg ethane) and a maximum specific growth rate of 0.015 d(-1). When grown on ethane, EA1 cometabolizes vinyl chloride (VC) at a maximum rate of 0.350 micromol/mg volatile suspended solids/d and with a half saturation constant of 0.62 microM VC. The rate of VC use by EA1 is twice as high when ethane is also provided, even though consumption of ethane is almost completely inhibited until VC is consumed. The presence of ethane also reduces the total amount of VC cometabolized. A model was developed that adequately describes the batch kinetics of VC cometabolism in the presence and absence of ethane, as well as ethane metabolism in the presence and absence of VC. Terms are included that increase the initial rate of VC use in the presence of ethane (according to the ratio of initial ethane concentration to the half saturation coefficient) but decrease the total amount of VC cometabolized. Parameter estimates for the model were obtained using a step-wise experimental approach, with varying initial concentrations of VC and ethane. Strain EA1 completely dechlorinates VC in the presence and absence of ethane. Measurements of soluble chemical oxygen demand indicate that approximately 50% of the VC consumed is mineralized, with the balance released as soluble, nonchlorinated products. Ethene is not used as a substrate by EA1 but it does inhibit ethane metabolism and VC cometabolism. In mixtures containing all three compounds, more VC is degraded and at a faster rate compared to VC plus ethene. The results suggest that ethane-enhanced biodegradation of VC may contribute to VC removal at the aerobic fringe of groundwater plumes undergoing reductive dechlorination.

Physical map and gene survey of the Ochrobactrum anthropi genome using bacterial artificial chromosome contigs.

Bacterial artificial chromosome (BAC) clones are effective mapping and sequencing reagents for use with a wide variety of small and large genomes. This report describes research aimed at determining the genome structure of Ochrobactrum anthropi, an opportunistic human pathogen that has potential applications in biodegradation of hazardous organic compounds. A BAC library for O. anthropi was constructed that provides a 70-fold genome coverage based on an estimated genome size of 4.8 Mb. The library contains 3072 clones with an average insert size of 112 kb. High-density colony filters of the library were made, and a physical map of the genome was constructed using a hybridization without replacement strategy. In addition, 1536 BAC clones were fingerprinted with HindIII and analyzed using IMAGE and Fingerprint Contig software (FPC, Sanger Centre, U.K.). The FPC results supported the hybridization data, resulting in the formation of two major contigs representing the two major replicons of the O. anthropi genome. After determining a reduced tiling path, 138 BAC ends from the reduced tile were sequenced for a preliminary gene survey. A search of the public databases with the BLASTX algorithm resulted in 77 strong hits (E-value < 0.001), of which 89% showed similarity to a wide variety of prokaryotic genes. These results provide a contig-based physical map to assist the cloning of important genomic regions and the potential sequencing of the O. anthropi genome.