INTRODUCTION

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Contamination of groundwater with vinyl chloride (VC) occurs primarily via anaerobic reductive dechlorination of tetrachloroethene, trichloroethene, and 1,1,1-trichloroethane (45). The maximum contaminant level for VC in drinking water is 2 µg/liter, which is lower than for any other volatile organic compound (34). This is consistent with the fact that VC is a known human carcinogen. Reductive dechlorination of VC to ethene (11, 16) and anaerobic oxidation of VC under iron-reducing and methanogenic conditions (4, 6) often occur at relatively low rates. The potential for persistence of VC has long been a concern with the exclusive reliance on anaerobic dechlorination as a method for groundwater remediation.

In contrast, it is generally accepted that VC is readily biodegradable under aerobic conditions. Cometabolism of VC has been demonstrated with numerous primary substrates, including ethene (17, 28), ethane (17), methane (8, 12), propane (30, 32), propylene (14), isoprene (15), 3-chloropropanol (8), and ammonia (37, 44). Under such conditions, cometabolism of VC occurs faster and with less apparent toxicity than cometabolism of more chlorinated alkenes. Aerobic biodegradation of VC during microcosm studies has also been widely reported, although it is not clear if the presence of organic matter other than VC served the role of a primary substrate (5, 9, 13). In spite of its apparent aerobic biodegradability, only a few organisms with the ability to use VC as a sole substrate capable of supporting growth have been isolated (23-25). These isolates have proven to be unstable in their sustained use of VC. The absence of VC for even short periods (i.e., less than 1 day) results in a complete loss of their ability to resume VC biodegradation (25). We hypothesized that other isolates which are capable of more robust use of VC as a sole organic substrate exist.

The isolate we obtained is capable of using VC as a growth substrate under aerobic conditions and resumes use of VC after periods of at least 24 days when none is present. In addition, we report on the kinetics of VC utilization (yield, maximum specific utilization rate, growth rate, and Monod half-saturation coefficient), growth of the isolate on other substrates (including ethene and acetate), the effect of oxygen deprivation on VC utilization, and the likely involvement of a monooxygenase in initiating VC biodegradation.

(Some preliminary results of this study were presented at the 99th Annual Meeting of the American Society for Microbiology, Chicago, Ill., 1999.)

	MATERIALS AND METHODS

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Chemicals and medium. VC gas (99.5%, containing <0.5% phenol to inhibit polymerization) was obtained from Aldrich; ethene (99.9%) and ethane (99.9%) were obtained from Matheson. All other chemicals used were of reagent grade. VC-epoxide was synthesized by reacting VC with 3-chloroperoxybenzoic acid dissolved in chloroform-d (27). The epoxide was identified by gas chromatographic analysis of headspace samples (see below) and comparison of peaks in vials with and without VC.

Analytical methods. Experiments involving VC, ethene, and ethane were performed with 70- and 160-ml serum bottles (Wheaton) sealed with slotted gray butyl rubber septa (diameter, 20 mm; Wheaton) and aluminum crimp caps. Autoclaved controls (121°C for 15 min) were used to evaluate abiotic losses, including adsorption and diffusion through the septa.

(1)

Culture maintenance, isolation, and identification. Routine maintenance of all cultures included purging the headspace of bottles with pure oxygen between feedings and adjusting the pH to 7.1 with 8 M NaOH. An isolate capable of growth on VC was obtained from an enrichment culture by streaking it onto trypticase soy agar plates (BBL) incubated at room temperature (23°C). Creamy-white colonies that grew after 24 h were placed into sterile liquid medium and fed VC. After the liquid cultures consumed several additions of VC and exhibited a substantial increase in optical density, additional transfers into fresh medium were made with VC as the only carbon source.

Chromosomal-DNA preparation, PCR, and sequencing applications. For chromosomal-DNA preparations, a 1.5-ml sample of strain MF1 grown in the presence of VC as the sole carbon source was pelleted with a microcentrifuge (model 5415C; Eppendorf) at 14,000 rpm for 10 min, and DNA was extracted using a Wizard genomic DNA purification kit (catalog no. A1120; Promega). Preparations were analyzed on a 1% agarose gel containing 0.5 µg of ethidium bromide per ml. The 16S rRNA gene was amplified by PCR using the forward (5'-TGGAGAGTTTGATCCTGGCTCAGATTGAACGCT-3') and reverse (5'-TACGGCTACCTTGTTACGACTTCACCCCAGTCA-3') primers (20 pmol/µl) corresponding to Escherichia coli positions 005 and 1540, respectively. Target DNA (25 ng) was cycled once on a Tetrad thermocycler (model PTC-225; MJ Research) at 98°C for 3 min and then for 30 times at 98°C for 2 min, 50°C for 2 min, and 72°C for 2 min, followed by a 10-min extension at 72°C. Samples were analyzed on a 1.2% agarose gel containing 0.5 µg of ethidium bromide per µl. Reaction mixtures were purified to remove excess buffer, nucleotides, and enzyme by using a PCR purification kit (catalog no. 28104; Qiagen).

Growth experiments. Growth of the isolate on VC, ethene, and ethane was evaluated with 160-ml serum bottles that were modified by connecting a 1-cm-inside-diameter test tube at a right angle to the side of each bottle near the base, resulting in a final bottle volume of 173 ml. These modified serum bottles resemble culture flasks with a side arm (e.g., Bellco Biotechnology or Ace Glass), making it possible to monitor growth by determining optical density at 620 nm (Milton Roy Spec 20D spectrophotometer). Triplicate serum bottles containing 110 ml of sterile medium were used for all treatments, including controls.

Kinetic experiments. In order to provide a consistent source of culture for the kinetic experiments, the isolate was grown in a VC-fed batch reactor, consisting of a 2.3-liter glass bottle (Belco Biotechnology) capped with a gray butyl rubber septum (diameter, 30 mm), held in place with a screw cap. The bottle contained 1.5 liter of culture. Two septa were also placed in the side of the bottle. The reactor was operated in a semicontinuous mode, as follows: 100 ml of VC was added and consumed in approximately 14 days, 150 ml of culture was removed and replaced with fresh medium, the pH was adjusted, the headspace was purged with oxygen, and more VC was added. The process resulted in an average cell retention time of 140 days. Aseptic conditions were maintained during all manipulations. Between feedings, the reactor was stored at room temperature on a gyratory shaker table. After several months of operation in this mode, the biomass concentration in the reactor stabilized at approximately 162 mg of TSS/liter (138 mg of VSS/liter).

(2)

(3)

1

1

(4)

Nucleotide sequence accession number. The complete sequence (1,526 bases) of the 16S rRNA of strain MF1 has been deposited in the GenBank database under accession no. AF193514.

	RESULTS

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Enrichment culture and isolation. The culture used in this study originated from a mixed culture inoculated with activated sludge (Urbana, Illinois, Wastewater Treatment Plant) and enriched on ethane as the sole substrate, followed by several transfers into fresh medium (18). An aliquot of this enrichment culture was then fed VC as the sole substrate. Following a lag period of 80 days, it began consuming repeated additions of VC and has since been maintained through numerous transfers over a 4-year period. Duplicate subcultures of the enrichment were examined for their tolerance to VC. The amount fed was gradually increased to as much as 7.3 mM VC, with no apparent inhibition (data not shown).

Growth on VC and other substrates. Strain MF1 used VC (Fig. 1) and ethene (Fig. 2) as sole substrates, with average observed yields (Y_obs) reported in Table 1. The autoclave control results (Fig. 1a and 2a, insets) demonstrated that consumption of VC and ethene in the live bottles was a biotic process, and the gray butyl rubber septa were very effective in retaining VC and ethene. Over a 274-day period, there was no statistically significant loss of VC from the controls; loss of ethene averaged 20% over 75 days. Although gray butyl rubber septa are not appropriate for use with polychlorinated ethenes (data not shown), they are very effective at retaining VC and ethene.

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Use of VC as a growth substrate by strain MF1, indicated by repeated consumption of VC (a) (results from only one of three bottles are shown; results from the other two were similar), an increase in absorbance in the bottles fed VC (b), and a direct correlation between the amount of VC consumed per bottle and an increase in absorbance (c). Results with autoclave controls are shown in the inset, demonstrating no loss of VC through the gray butyl rubber septa.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Use of ethene as a growth substrate by strain MF1, indicated by repeated consumption of ethene (a) (results from only one of three bottles are shown; results from the other two were similar), an increase in absorbance in the bottles fed ethene (b), and a direct correlation between the amount of ethene consumed per bottle and an increase in absorbance (c). Results with autoclave controls are shown in the inset, demonstrating no major loss of ethene through the gray butyl rubber septa.

Extent of VC biodegradation. The extent of VC biodegradation was determined based on the stoichiometry of chloride ion release and accumulation of soluble COD. Chloride measurements were taken at the beginning and end of the growth experiment (Fig. 1), with VC serving as the sole substrate. The net increase in chloride at the end of the growth experiment accounted for 97% of the VC consumed, indicating nearly complete dechlorination (Table 2).

Starvation experiments. The ability of MF1 to resume biodegradation of VC after various periods of VC starvation was examined. Three sets of duplicate cultures were fed VC (25-ml culture in 70-ml serum bottle). Once the VC was consumed below the detectable level, none was added for 1, 3, or 7 days, although oxygen was always present in the bottles. When VC was added again, all of the bottles resumed use of VC, although the longer the starvation period, the lower the rate of biodegradation following resumption of VC feeding. Results for the 7-day starvation period are shown in Fig. 3.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Recovery of VC utilization by strain MF1 after a 7-day period when VC was below the detectable limit. The results shown are for a single serum bottle; similar results were obtained for a duplicate bottle.

View larger version (26K): [in this window] [in a new window]   FIG. 4.   Effect of oxygen deprivation for 2.5 days on VC utilization by strain MF1. The results shown are for a single serum bottle; similar results were obtained for a duplicate bottle.

Kinetic experiments. The kinetics of VC utilization of strain MF1 were evaluated using batch depletion data (Fig. 5). Initial VC concentrations were set high enough to demonstrate a maximum rate of utilization (i.e., zero order) but low enough to avoid significant growth. For the highest initial concentration, the increase in biomass was estimated to be only 2% (based on an initial biomass concentration of 205 mg of TSS/liter and the Y_obs reported in Table 1). The frequency of sampling was increased with time as the rate of depletion slowed, in order to adequately characterize the nonlinear portion of the curve. Simultaneously fitting all of the depletion data to equation 2 resulted in the k and K_s values shown in Table 1, along with the calculated value for µ_max using equation 4. Similar experiments were conducted with ethene as the growth substrate, with the resulting kinetic parameters shown in Table 1. Numerically computed sensitivity functions for k and K_s were not multiples of one another (data not shown), indicating that both parameters were uniquely identifiable from the experimental data (39).

View larger version (23K): [in this window] [in a new window]   FIG. 5.   VC batch depletion results used to determine k and Ks for strain MF1. Symbols represent data from six separate cultures fed various initial concentrations of VC; lines represent a simultaneous fit of equation 2 to the entire data set. The inset shows depletion at low VC concentrations for two of the bottles.

Effect of acetylene. The presence of monooxygenase activity in MF1 was examined using acetylene as an inhibitor. Duplicate cultures without acetylene added consumed a single addition of VC within 6 days, while another set of duplicates fed the same amount of VC plus 5% acetylene consumed less than 10% of the VC after 13 days (Fig. 6). In order to evaluate potential effects of acetylene on metabolic processes other than monooxygenase activity, two sets of duplicate cultures were fed acetate (20 mM) as the sole substrate, one of which also received 5% acetylene. After 1.5 days of incubation, the absorbance (620 nm) in both sets of bottles was 0.45 (±0.016), indicating no apparent inhibitory effect of acetylene during growth on acetate.

View larger version (20K): [in this window] [in a new window]   FIG. 6.   Effect of acetylene on biodegradation of VC by strain MF1.

	DISCUSSION

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Strain MF1 appears to be the first Pseudomonas sp. reported that can use VC as a growth substrate. Castro et al. (8) demonstrated biodegradation of VC by a Pseudomonas sp., but the culture first had to be grown on 3-chloroproponol as the primary substrate. Cometabolism of VC began with a direct hydroxylation of the C-Cl bond to produce acetaldehyde. This finding is in contrast to the likely involvement of a monooxygenase in the initial step of VC metabolism by MF1.

M. aurum L1 is the best characterized of previously described isolates that use VC as a growth substrate (24). A comparison of MF1 and L1 reveals several differences and similarities (Table 1). Both organisms are able to grow on VC and ethene, with comparable yields on each substrate. However, the µ_max for L1 grown on VC is several orders of magnitude higher, while the K_s for MF1 is approximately an order of magnitude lower. From this perspective, L1 may be viewed as a "µ_max strategist" (40), having a competitive advantage at higher VC concentrations. MF1 may be viewed as a "K_s strategist" (40), because its lower K_s allows it to utilize lower concentrations of VC. Similar comparisons have been made between other organisms that use 1,2-dichloroethane (43) and dichloromethane (19) as growth substrates.

It should be noted that the kinetic parameters reported in Table 1 were measured under extant conditions (21), i.e., the ratio of substrate to biomass (on an electron equivalent basis) was comparatively low. The physiological state of cells used in extant tests has an impact on the rate of substrate depletion during batch tests. It is likely that some of the differences in k and K_s shown in Table 1 are attributable to differences in how MF1 and L1 were grown prior to measurement of the parameters. Even this consideration, however, is unlikely to alter the conclusion that MF1 grows much more slowly than L1 and has a lower K_s with VC.

L1 and MF1 also differ significantly in their ability to resume VC biodegradation following an absence of the substrate. L1 completely lost its ability to metabolize VC when addition to a reactor was discontinued for as little as 9 h (25). When addition of VC resumed, VC-epoxide accumulated, presumably to a toxic level that prevented further growth (24). In contrast, MF1 resumed VC utilization after 7 days without VC and VC-epoxide did not accumulate when VC was added after the starvation period. Depriving MF1 of oxygen for several days had a much more severe impact on resumption of VC utilization and did result in an accumulation of VC-epoxide. The ability of an organism to withstand periods of electron donor or acceptor starvation is an important factor for environmental applications, including biofiltration and in situ bioremediation.

The pathway for VC metabolism by MF1 is not yet known. VC-epoxide formation, a lack of VC metabolism in the absence of oxygen, and inhibition of VC use by acetylene strongly suggest that a monooxygenase is involved in the first metabolic step. Acetylene inhibition of monooxygenases has been demonstrated with a number of other substrates (3, 35). It is less clear how VC-epoxide is then transformed, although tests with several possible products provides some insight. Rearrangement to chloroacetaldehyde and oxidation to chloroacetate (22, 33) seems unlikely, since MF1 does not grow on chloroacetate. Hydrolysis and dechlorination of VC-epoxide to glycolaldehyde has been observed during cometabolic transformation of VC by Methylosinus trichosporium Ob3b (7) but is unlikely with MF1, since MF1 does not grow on glycolaldehyde. Another possibility is direct conversion of VC-epoxide to acetyl coenzyme A. A similar transformation involving epoxyethane dehydrogenase has been demonstrated during growth of Mycobacterium sp. E20 on ethene (10). More studies are needed to clarify the metabolic pathway utilized by MF1 when it grows on VC.

The results shown in Table 1 for yields on VC and ethene are based on mass of cells per mass of substrate. Since VC is more oxidized than ethene, a more equitable comparison is based on electron equivalents. Expressed in this way, Y_obs for MF1 is 0.22 eq of biomass per eq of VC, versus 0.30 eq of biomass per eq of ethene. Even on this basis, the yield on VC remains lower than on ethene, although the difference is smaller. The reason for this is not yet known, but the results suggest that there is a substantial energetic cost associated with VC metabolism, perhaps due to repair of proteins that become alkylated by VC-epoxide (2, 22, 26). Furthermore, the lower yield on VC than on ethene suggests that neither MF1 nor L1 have a mechanism to conserve the substantial free energy available from breaking the carbon-chlorine bond.

Whether or not the enrichment process that led to isolation of MF1 was purposeful or fortuitous requires further investigation. The process began with ethane as the sole substrate in order to examine the potential for ethane-promoted cometabolism of VC (18). One of the treatments during these experiments involved addition of VC alone. These bottles later started consuming repeated additions of VC in the absence of any added ethane and became the source of enrichment culture from which MF1 was isolated on VC as the sole substrate. How MF1 was able to survive during the extended enrichment process on ethane is not yet clear, especially in light of the fact that MF1 does not grow on ethane. We previously reported the same phenomenon with a different ethane enrichment culture capable of using VC as a sole substrate, although in that study we did not pursue an isolate from the enrichment (17). The source of inoculum for both ethane enrichment cultures was the same activated sludge reactor, although the samples were taken 2 years apart. We are currently exploring the potential for obtaining additional isolates capable of growth on VC as a sole substrate by beginning with an ethene enrichment culture, as well as by using VC from the start.

The ability of MF1 to grow on ethene and then switch to VC has implications for possible use of this organism in bioaugmentation of contaminated groundwater or inoculation of biofilters. Given its toxicity, relatively high cost in neat form, and low Y_obs, VC is a poor substrate for growing an initial source of biomass. Ethene is less costly and far less toxic, and the apparent absence of a lag period when switching to VC makes it a preferential substrate.