Microbial organisms use a wide variety of metabolic processes to generate energy and maintain cellular growth. These processes involve the transfer of electrons from an electron donor (food source) to an electron acceptor. Three categories of metabolic processes are involved in the biological degradation of chlorinated ethenes. Reductive dechlorination is an anaerobic process in which chlorinated ethenes are used as electron acceptors (McCarty, 1994; Montgomery and others, 1994). Cometabolism is an aerobic process in which chlorinated ethenes are degraded as a result of fortuitous biochemical interactions which yield no benefit to the bacteria (Alvarez-Cohen and McCarty, 1991; Hanson and Brusseau, 1994; Ely, Williamson, and others, 1995). Direct oxidation is an aerobic or mildly anaerobic (iron reducing) process in which sparsely chlorinated ethenes are used as electron donors (McCarty and Semprini, 1994; Bradley and Chapelle, 1996). One or all of these processes could be occurring at a given site, depending on environmental conditions.
Generally, organic molecules with abundant carbon-hydrogen bonds are good food sources (electron donors) because they contain high-energy electrons. Highly chlorinated solvents such as tetrachloroethylene (PCE) and TCE, however, are electron poor because they have chlorine-carbon bonds. These compounds are likely to be used by bacteria as final electron acceptors instead of electron donors (Wiedemeier and others, 1998). During reductive dechlorination, chlorine atoms are replaced by electrons coupled to hydrogen atoms, resulting in sequential dechlorination from PCE to TCE to dichloroethylene (DCE) to vinyl chloride (VC) to ethene (fig. 1). During reductive dechlorination, cis-1,2-dichloroethylene (cDCE) is the most commonly formed isomer of DCE.
Soil and ground-water bacteria use a variety of natural electron acceptors. The use of these final electron acceptors is not arbitrary but is based on energy-transfer efficiency and availability (Montgomery and others, 1994). The most common inorganic electron acceptor in ground water is oxygen. Once the oxygen has been depleted, bacteria will preferentially use the next most efficient electron acceptor; usually this is nitrate (NO3-) or insoluble manganese (Mn4+). After NO3- and Mn4+ have been depleted, the bacteria will use insoluble ferric iron (Fe3+), followed by sulfate (SO42-), and carbon dioxide (CO2), respectively. The byproducts resulting from transfer of electrons to various electron acceptors are shown in table 1.
Dechlorination of PCE and TCE to DCE can occur under mildly reducing conditions such as NO3- or Fe3+ reduction (fig. 2); however, the dechlorination of DCE to VC, and VC to ethene requires the stronger reducing conditions of SO42--reduction or methanogensis (Vogel and others, 1987). The effectiveness of chlorinated solvents to serve as electron acceptors is proportional to the number of chlorine molecules attached (Vogel and McCarty, 1985). For example, PCE is more likely to serve as an electron acceptor and lose a chlorine atom through reductive dechlorination than is TCE, DCE, or VC.
Wilson and Wilson (1985) first reported that TCE was degraded under aerobic conditions by methanotrophic bacteria in a soil enriched with CH4 and oxygen (O2). Further studies revealed that the methane monooxygenase (MMO) enzyme was responsible for catalyzing the oxidation of TCE (Alvarez-Cohen and McCarty, 1991; Henry and Grbic´-Galic´, 1994). Other oxygenase enzymes such as ammonia monooxygenase (AMO) (Arciero and others, 1989; Rasche and others, 1991) and toluene dioxygenase (Nelson and others, 1988; Hopkins and others, 1993; Heald and Jenkins, 1994) also have been shown to oxidize certain chlorinated solvents. This oxidation reaction is called cometabolism because the reaction uses metabolic enzymes, but does not contribute any energy in return.
These oxidative enzymes catalyze a reaction that incorporates O2 into the target substrates (fig. 3). This oxidation reaction requires an energy molecule such as nicotinamide adenine dinucleotide (NADH) to incorporate the O2. The enzymes lack the ability to efficiently distinguish CH4, NH3, or toluene from certain chlorinated solvents. This lack of substrate specificity results in a chemical reaction in which oxygen is incorporated into the solvent molecule forming an unstable molecule such as TCE epoxide during MMO cometabolism (Alvarez-Cohen and McCarty, 1991). The unstable molecule will spontaneously degrade to one of several chloroacetic acids, such as dichloroacetic acid. These chloroacetic acids are soluble in water and will slowly degrade to CO2, chloride, and water (fig. 3).
Cometabolism is limited to chlorinated solvents that have at least one hydrogen atom attached to the carbon (fig. 1). No studies have found a cometabolic pathway capable of degrading PCE (Henry and Grbic´-Galic´, 1994). Cometabolism tends to be an unsustainable process under stagnant conditions because of substrate competition and enzyme inhibition and inactivation (Ely and others, 1997). Competition occurs between the natural substrates, such as CH4, NH3, or toluene, and chlorinated solvents for binding on the active site of the nonspecific oxygenase enzyme (Semprini and others, 1991; Broholm and others, 1992). Competitive inhibition occurs when enzymes cometabolize chlorinated solvents to the exclusion of natural substrates, ultimately depleting the bacteria of energy molecules (Chang and Alvarez-Cohen, 1995) (NADH in fig. 3). The TCE oxidation byproducts such as TCE epoxide may result in the inactivation of the oxygenase activity caused by damage to the enzymes (Ely, Hyman, and others, 1995). Inhibition and inactivation may be overcome by additional natural substrates (Alvarez-Cohen and McCarty, 1991; Ely and others, 1997).
Recent studies have reported that chlorinated solvents with only one or two chlorine atoms (the least oxidized compounds) can serve as electron donors by bacteria. Several studies have shown that VC and 1,2-dichloroethane (DCA) can serve as food under aerobic conditions (McCarty and Semprini, 1994). Iron-reducing bacteria also can mineralize VC (Bradley and Chapelle, 1996) and DCE (Bradley and Chapelle, 1997) as a food source under aerobic conditions. Direct oxidation is limited to degrading lightly chlorinated solvents such as DCA, DCE, and VC; however, direct oxidation may serve a vital role in the sequential steps of chlorinated-solvent biodegradation. Aerobic or iron-reducing zones are commonly found downgradient of methanogenic or sulfate-reducing zones. Thus, partially dechlorinated byproducts (DCE and VC) produced by reductive dechlorination in the methanogenic or sulfate-reducing zones may be consumed in the more oxidized zones downgradient.
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