Title: Western Region Hazardous Substance Research Center Project 2-OSU-05
Aerobic Cometabolism of Chlorinated Ethenes by Microorganisms that Grow on Organic Acids and Alcohols

Investigators: P. J. Bottomley, D.J. Arp, M. Dolan, L. Semprini, Oregon State University

Institution: Oregon State University

Research Category: Cometabolism, ethenes, volatile organic compounds

Project Period: 2004-2007


Part 1: Aerobic cometabolism of chlorinated aliphatic hydrocarbon compounds with butane-grown microorganisms.

Investigators: Peter Bottomley and Dan Arp

Goal: The proposal aimed to evaluate how to maximize the chloroethene degrading potential of individual strains of hydrocarbon degrading bacteria. Specific subobjectives included identifying conditions (a) that maximize reductant flow to cometabolism, (b) that promote maximum expression of monooxygenase genes and enzyme activity, and (c) that sustain enzyme activity with minimal cell damage.

Rationale: Studies conducted under laboratory and field conditions have shown that hydrocarbon-oxidizing bacteria cometabolize a wide range of chloroethenes. Nonetheless, there is considerable variability in the properties of cometabolism shown by different types of bacteria both in terms of the range of chloroethenes degraded and in their transformation capacities. More research is needed to better understand the microbiological reasons for the range of efficiencies observed, and to use this information to improve the biotechnology of bioremediation under cometabolism conditions.

Experimental Approaches:

(a) Throughout our studies with P. butanovora, it became clear that despite fast initial rates of CE oxidation it was difficult to sustain maximum rates of CE degradation with propionate and butyrate as electron donors, and this was particularly true for a poor substrate like 1,2 trans DCE (Doughty et al. 2005). We continued to study the activity of BMO in P. butanovora in the presence of organic acids (Doughty et al. 2006; Doughty et al. 2007). Incubation of alkane-grown P. butanovora with butyrate or propionate led to irreversible, time-, and O2-dependent loss of BMO activity. In contrast, BMO activity was unaffected by incubation with lactate or acetate. Chloramphenicol inhibited the synthesis of new BMO, but did not change the kinetics of propionate-dependent BMO inactivation, suggesting that the propionate effect was not simply due to it acting as a repressor of BMO transcription. BMO was protected from propionate-dependent inactivation by the presence of its natural substrate, butane.Although both the time and O2 dependency of propionate inactivation of BMO infer that it might be a suicide substrate, no evidence was obtained for BMO-dependent propionate consumption, or 14C labeling of BMO polypeptides by [2-14C] propionate during inactivation. We have also examined the BMO mutant strains mentioned in project 1 of 1-OSU-02 (Halsey et al. 2006) to determine if propionate sensitivity of BMO had been changed. The effects of propionate on BMO differed among the mutant strains. For example, in the case of the G113N mutant, in which a glycine was replaced by an asparagine residue in a region adjacent to, or contributing to the active site, BMO activity was not inactivated by propionate. In contrast, mutant strain T148C, in which a threonine in the active site region was replaced by a cysteine, showed a significant increase in propionate-dependent inactivation of BMO relative to wild type. We screened other well-studied monooxygenase enzymes for inactivation by fatty acids. In vivo studies showed that the diiron methane monooxygenases (sMMO) of M. capsulatus Bath, and M. trichosporium OB3b, and the toluene o-monooxygenase of Burkholderia cepacia G4 were not inactivated by propionate. In contrast, the toluene 4-monooxygenase (T4MO) of the excellent TCE degrader, Pseudomonas mendocina KR1, was inactivated following a10 min incubation with 10 mM propionate or butyrate by ~ 60 and 94% respectively. In contrast acetate or lactate did not inactivate T4MO activity, indicating that T4MO was sensitive to the same range of organic acids as BMO. Furthermore, T4MO activity was protected from propionate-dependent inactivation by toluene, the physiological substrate of T4MO. Our data certainly showed that oxygenase containing strains with bioremediatory potential must be screened to determine if potential electron donating organic acids might have negative effects on enzyme activity prior to considering them for use as bioremediatory agents of CEs.

We discovered that BMO expression in P. butanovora is induced by the products of its activity i.e. alcohols, aldehydes, and epoxides, by the xenobiotic substrate, 1,2-trans DCE, but not by its natural alkane substrates (Doughty et al. 2005; Sayavedra-Soto et al. 2005). Furthermore, we showed that when butane and propane or pentane were present simultaneously, that BMO expression was repressed. This was subsequently attributed to the accumulation of propionate when cells were exposed to odd chain length alkanes and the inability of cells grown on even chain length alkanes to process propionate (Doughty et al. 2006). Although it is recognized that aerobic transformation of lesser chlorinated ethenes might occur at the aerobic/anaerobic interface of contaminant plumes undergoing reductive dechlorination, it is unclear to what extent monooxygenases like BMO can be induced under the low concentrations of O2 found at the fringes of contaminated zones. Data were obtained to show that induction of BMO by alcohols or 1,2-trans DCE could occur at low O2 concentrations (<1%), however, the combination of low concentrations of Cu (<1µM) and low O2 levels (<2%) repressed induction of BMO by alcohols (Doughty et al. 2007, in review). Furthermore, a combination of a low concentration of Cu (0.5 µM), and the reducing agent Na ascorbate was an effective repressor of alcohol dependent induction of BMO under fully oxic conditions. This result was intriguingly similar to that observed in methanotrophs in which the synthesis of soluble methane monooxygenase is repressed upon exposure to low concentrations of Cu (>1µM). Again, the results indicate that care must be taken to screen strains for bioremediatory purposes that are in possession of monooxygenase genes whose induction occurs at low O2 and is not sensitive to the presence of Cu.


Part 2: Aerobic metabolism and cometabolism of vinyl chloride and fluoroethene on microorganisms that grow on ethene

Investigators: Lewis Semprini, Peter Bottomley and Mark Dolan

Considerable attention has been given to bacteria that degrade VC aerobically because it is often a persistent product of reductive dechlorination and can move out of the anoxic contaminated plume into the adjacent oxic zone. It is difficult to estimate rates of aerobic VC transformation in situ because the mineralization of VC yields CO2 and Cl-; neither of which can be tied solely to VC transformation. Fluoroethene (FE) is a stable molecule in aqueous solution and its aerobic degradation yields fluoride (F-), which is a unique signature in most aquifers.Work with cytochrome P450-dependent monooxygenases suggests that FE is aerobically degraded in a manner similar to VC; an epoxide is formed from the initial oxidation. FE-epoxide is unstable and is expected to yield spontaneous degradation products analogous to VC-epoxide. Laboratory experiments were carried out with various alkene monooxygenase-containing bacteria that either cometabolically or catabolically metabolize VC, to evaluate if (i) rates of FE transformation are similar to those of VC transformation, (ii) VC and FE have similar affinities for the monooxygenase that mediates the initial transformation, (iii) a competitive inhibition kinetic model accurately simulates concurrent FE and VC degradation, and (iv) the rate of F- accumulation can be correlated with that of VC utilization. In addition the potential for bacteria to use FE as a carbon and energy source was evaluated (Taylor et al. 2007, in press). Despite the fact that the three VC-degrading isolates responded differently to Eth, VC and FE as growth substrates, there were no differences between the Ks/c or kmax values for FE and VC of any individual isolate, and there was little difference between the three isolates in their rates of transformation or affinity for the halogenated substrates. Additionally, rates of maximum VC and FE transformation or utilization were similar for all three isolates, indicating that the presence of the smaller F atom did not affect the alkene monooxygenase’s ability to accept FE as a substrate. In a separate experiment we also determined if it was possible to monitor VC transformation by modeling the rates of F- accumulation. During cotransformation, the initial rates of halide release matched that of substrate transformed for each VC-degrading isolate, and in separate experiments where the degradation of individual substrates was followed, halide release ceased as soon as substrate transformation was complete. This demonstrated that there was no further halide release from halogenated products that might have been formed during cometabolism. For VC, mass balance of the substrate transformed and halide released were nearly stoichiometric regardless of whether it was a growth or nongrowth substrate and averaged 0.98 (±0.11) on a mole fraction basis. There was a trend for stoichiometric release of F- during transformation of FE by JS614 (1.0 ±0.16 mole percent), while F- released during cometabolic transformation of FE by EE13a and JS60 averaged 0.84 (±0.04) mole percent. Competitive inhibition between substrates, halide release rates equivalent to substrate transformation rates, a known mole fraction of halide released, and previously determined X, kmax and Ks/c values were incorporated into the model. This model accurately estimated the accumulation of halide in the batch reactors using heuristically fit Ki values. For the three aerobic VC-degrading isolates studied, both the rates of FE transformation and F- accumulation could be correlated with the rate of aerobic degradation of VC. FE therefore has the potential to be used as a surrogate reactive tracer for estimating rates of VC degradation in situ. In a VC contaminated aquifer, for example, in single-well push-pull tests, FE addition and subsequent cotransformation could be utilized as an indicator of in situ VC transformation rates.


Journal articles

Doughty, D.M., L.A., Sayavedra-Soto, D.J. Arp and P.J. Bottomley (2006). Product repression of butane monooxygenase expression in ‘Pseudomonas butanovora.J. Bacteriol. 188: 2586-2592.

Doughty, D. M., K. H. Halsey, L. A. Sayavedra-Soto, D. J. Arp, and P. J. Bottomley (2007). Alkane monooxygenase inactivation by propionate in Pseudomonas butanovora; physiological and biochemical implications. Microbiology, 153: 3722-3729.

Doughty, D.M., Sayavedra-Soto, L.A., D.J. Arp, and P.J. Bottomley (2007). Evidence for copper ions and redox involvement in regulation of butane monooxygenase in Pseudomonas butanovora. J. Bacteriol. (In review).

Sayavedra-Soto, L.A., D.M. Doughty, E. Kurth, P.J. Bottomley, and D.J. Arp (2005). Inducer and inducer –independent induction of butane oxidation in Pseudomonas butanovora. FEMS Microbiology Letters, 250:111-116.

Taylor, A.E., M. Dolan, P.J. Bottomley, and L. Semprini (2007). Use of fluoroethene as a surrogate for aerobic vinyl chloride transformation. Environ. Sci. Technol. 41: 6378-6383.


Abstracts and Posters

Doughty, D. M., K. H. Halsey, C. Vievelle, L. A. Sayavedra-Soto, D. J. Arp and P. J. Bottomley (2006). Propionate-dependent Inactivation of Butane Monooxygenase. Molecular Basis for Microbial One-carbon Metabolism, Gordon Research Conference at Magdalen College, Oxford, UK.

Halsey, K. H., D. M. Doughty, L. A. Sayavedra-Soto, P. J. Bottomley and D.J. Arp. (2006). Investigating the Basis of Substrate Specificity of Butane Monooxygenase and Chlorinated Ethene Toxicity in Pseudomonas butanovora. Molecular Basis for Microbial One-carbon Metabolism, Gordon Research Conference at Magdalen College, Oxford, UK.

Halsey, K. H., D. M. Doughty, L. A. Sayavedra-Soto, P. J. Bottomley and D.J. Arp (2006). Investigating the basis of substrate specificity of butane monooxygenase and chlorinated ethene toxicity in Pseudomonas butanovora. Subsurface Biosphere Initiative Workshop/IGERT Retreat, Newport, OR.

Taylor, A.E., L. Semprini, P.J. Bottomley, M. Dolan (2005). Evaluation of fluoroethene as an analogue for aerobic vinyl chloride degradation. The Joint International Symposia for Subsurface Microbiology and Environmental Biogeochemistry. Jackson Hole, Wyoming.



Blatchford, C. (2005). Aerobic Degradation of Chlorinated Ethenes by Mycobacterium Strain JS60 in the Presence of Organic Acids. M.S., Oregon State University.

Doughty, D.M. (2008). Control of alkane monooxygenase activity and expression in Pseudomonas Butanavora. Ph.D. Department of Microbiology, Oregon State University.


Supplemental Keywords: biotransformation; characterization; VOCs; chlorinated solvents; bioremediation; in situ