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The anaerobic biodegradation of chlorinated aromatic compounds, such as chlorophenols, chlorobenzoates, and chlorinated phenylacetates, starts with reductive dechlorination (7, 9, 11, 15, 19). A few microorganisms that are able to conduct this step have been isolated in pure culture (1a, 3, 4, 5a, 8, 16a, 23). Among these, Desulfitobacterium dehalogenans, the first one isolated from freshwater sediment, is capable of reductively dehalogenating chlorophenols and chlorophenylacetates specifically at an ortho position to the hydroxyl group (22, 23). The potential to use these compounds as terminal electron acceptors has been indicated (6, 8, 14).

This paper reports on the contribution to both energy and growth of the reductive dechlorination of chlorophenols by D. dehalogenans. A comparison was made of the energy and growth yields for D. dehalogenans with 3-chloro-4-hydroxyphenylacetate (3-Cl-4-OHPA) as the chlorinated compound and with other nonchlorinated terminal electron acceptors. Competition experiments in which different electron acceptors were present in one culture provided insight on the preferences for their utilization by D. dehalogenans.

The premise for conducting these experiments was based on the assumption that the cell yield is proportional to the energy yield (1, 2, 16, 17, 18, 20). Thus, we calculated yields (X = dry weight biomass in g/liter) for substrate utilized (Y_X/S, where S is the substrate), products formed (Y_{X/reduced electron acceptor} and Y_{X/oxidized electron donor}), assumed ATP formation (Y_X/ATP), and two electrons transferred (Y_X/2e) (17). The values for Y_X/S, Y_{X/oxidized donor}, or Y_{X/reduced electron acceptor} were determined from the amount of substrate utilized or product formed while X amount of biomass was produced. Y_X/ATP was determined from the total theoretically assumed amount of ATP produced (Table 1). The yield per two electrons transferred, Y_X/2e, was determined from the biomass formed and the arithmetic average concentration of the oxidized electron donor and the reduced electron acceptor.

For each experiment, acclimated cells were grown in 1 liter of HEPES-based anaerobic minimal medium containing 0.1% (wt/vol) yeast extract and supplemented with 20 mM electron donor (pyruvate or formate) and 30 mM electron acceptor (fumarate, nitrate, or 3-Cl-4-OHPA), except in the case of sulfite (3 mM) as an electron acceptor, because higher concentrations were toxic to the cells (13, 23). The addition of yeast extract is required for growth (23). At four different time intervals, culture samples were taken for substrate and product analysis and dry weight biomass determination. Samples for sulfide determination were stored in antioxidant buffer (ATI Orion, Boston, Mass.). All organic acids were quantified by high-performance liquid chromatography with an Aminex ion exclusion HPX-87H column at 60°C, with a flow rate of 0.60 ml/min. The mobile phase consisted of a dilute sulfuric acid solution, and peak areas were calculated with an HP3395 integrator (Hewlett Packard, Palo Alto, Calif.). Nitrate and its reduced products were quantified with the Bran-Luebbe wet chemistry analysis package (Chemical Analysis Laboratory, University of Georgia). Sulfide determinations were made by titrating samples with 30 mM lead perchlorate by using a silver-sulfide ion-specific electrode (ATI Orion) as the endpoint indicator. Biomass determinations were made by filtering samples onto predried, preweighed 0.22-µm-pore-size filters (Micron Separations Inc., Westboro, Mass.). The filters were reweighed twice after a 24-h drying period at 80°C and a cooling period in a dessicator with calcium carbonate. In competition experiments, cells were inoculated in 1 liter of medium containing 60 mM pyruvate and each electron acceptor at 10 mM (fumarate, nitrate, and 3-Cl-4-OHPA) except for sulfite (3 mM). Additional competition experiments were conducted using only sulfite and 3-Cl-4-OHPA. At the conclusion of each experiment, the purity of the cultures was checked by microscopic examination. All experiments and measurements were performed in duplicate.

Under conditions where formate served as the electron donor and 3-Cl-4-OHPA served as the electron acceptor, the growth yields (Y_X/formate and Y_X/2e) conclusively showed that D. dehalogenans does conserve energy for growth through reductive dechlorination coupled to formate oxidation (Fig. 1). The electron-donating half-reaction (formate  CO₂ + 2e + 2H⁺) and the electron-accepting half-reaction [3-Cl-4-OHPA + 2e + 2H⁺  4-OHPA + HCl + (ATP)] yielded 3.2 to 11.3 g of Y_X/formate, Y_X/ATP, and Y_X/2e per mol. The Y_X/ATP range was calculated based on the 4-OHPA formed, assuming 1 mol of ATP was formed per mol of 3-Cl-4-OHPA reduced. The Y_X/2e range was calculated based on the average concentrations of 3-Cl-4-OHPA utilized and 4-OHPA formed. This number was divided by the number of two-electron transfers needed to complete the reduction. The range was determined by calculating the different yields for each set of data points in Fig. 1C. Growth was negligible or absent in control cultures consisting of minimal medium ± 0.1% yeast extract and supplemented with none or one of the substrates. The oxidation of formate does not support substrate-level phosphorylation; therefore, we hypothesize that energy conservation occurs during the two-electron reduction of the chlorinated compound via electron transport phosphorylation, resulting in a Y_X/ATP range of 3.2 to 11.3 g of cells per mol of ATP formed (5, 5b, 14, 21). This data was presented as a range because a nonlinear response between obtained dry weight and substrate utilized was observed for the different time points when the culture grew with formate. Although the reason for this remains unknown, we speculate that lower yields obtained during the late exponential phase were attributable to higher maintenance demands while greater yeast extract concentrations at the outset of the experiment promoted higher yields during early exponential growth. Mohn and Tiedje reported that Desulfomonile tiedjei was capable of conserving energy for growth by coupling the oxidation of formate to the reductive dechlorination of 3-chlorobenzoate, resulting in a molar growth yield of 2.8, expressed as the protein yield in grams per mole of Cl removed (14). Likewise, Holliger et al. reported that Dehalobacter restrictus is capable of conserving energy for growth by coupling formate oxidation to the reductive dechlorination of tetrachloroethene (PCE), forming cis-1,2-dichloroethene. The molar growth yield was 2.1 g of protein per mol of Cl removed (10, 11). Assuming that protein yield is 50% of the total dry weight biomass, then approximate molar growth yields of 5.6 and 4.2 g/mol, respectively, for these microorganisms fall within the growth yield range observed for D. dehalogenans.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Biomass formation (A), substrate utilization and product formation (B), and calculation of yields (C) for D. dehalogenans grown in the presence of formate plus 3-Cl-4-OHPA. Cultures were inoculated with cells from a pregrown, acclimated formate-3-Cl-4-OHPA culture. , biomass; , formate; , 3-Cl-4-OHPA; , 4-OHPA. Data shown are for a set of duplicates.

D. dehalogenans is capable of utilizing different electron acceptors (Table 1). The yields for Y_X/acetate (Y_X/ATP) in experiments when fumarate, nitrate, sulfite, and 3-Cl-4-OHPA were used as electron acceptors (25.9, 23.8, 23.3, and 24.2 g/mol, respectively) are nearly twice as high as those obtained with pyruvate (13.4 g/mol) as the electron acceptor. Since no lactate was formed, all the supplemented electron acceptors apparently alleviated the need to use pyruvate as an electron acceptor. The oxidation of pyruvate yields acetate, CO₂, reducing equivalents, and ATP through substrate level phosphorylation. The additional energy, and hence additional biomass, is most likely obtained from electron transport phosphorylation involving the supplemented electron acceptor (Table 1).

In contrast to the situation in the original description of D. dehalogenans (23), nitrate was fully reduced to ammonia, suggesting that an assimilatory nitrate reductase may be involved. The extent to which nitrite is further reduced to ammonia depends on culture conditions, including the concentrations of electron donors present. This aspect is presently under study (12).

Competition experiments in which the different electron acceptors were all present in one culture (Fig. 2) indicated that D. dehalogenans utilizes nitrate and 3-Cl-4-OHPA simultaneously for growth. Nitrate was completely reduced to ammonia; no reduced intermediates were detected. Initially, fumarate utilization was low, but it increased significantly with depletion of 3-Cl-4-OHPA. Sulfide was not detected in the assay, but sulfite reduction was assumed because the amount of pyruvate utilized was not stoichiometric with the electron-accepting reactions.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Substrate utilization and product formation over time when D. dehalogenans was grown in minimal medium containing 60 mM pyruvate and 10 mM each electron acceptor, except for sulfite (3 mM), all in one culture. Pyruvate-acetate balance (A), nitrate-ammonia balance (B), fumarate-succinate balance (C), and 3-Cl-4-OHPA-4-OHPA balance (D) are shown. Cultures were inoculated with cells that were nonacclimated to any of the electron acceptors used. A drop line was drawn at 30 h for comparison. Sulfide was not detected in the assay and no intermediates were detected in the reduction of nitrate to ammonia. , pyruvate; , acetate; , nitrate; , ammonia; , fumarate; , succinate; , 3-Cl-4-OHPA; , 4-OHPA.

Additional experiments with sulfite and 3-Cl-4-OHPA were performed to clarify whether sulfite can be used competitively as an electron acceptor (Fig. 3). In both experiments, the formation of sulfide was observed (2.2 and 0.6 mM), but it was less prominent when the concentration of 3-Cl-4-OHPA was in excess. The formation of sulfide was not stoichiometric to the amount of pyruvate utilized, indicating that reduction of sulfite to sulfide was not always complete and may have resulted in undetectable intermediates. From these results we assume that sulfite reduction did occur in the previous competition experiments but that sulfite was not completely reduced to sulfide.

View larger version (26K): [in this window] [in a new window]   FIG. 3.   Representative example of substrate utilization and product formation when D. dehalogenans was grown in minimal medium containing 20 mM pyruvate and equimolar concentrations (3 mM) of 3-Cl-4-OHPA and sulfite (A) or at a 3-Cl-4-OHPA/sulfite ratio of 6:1 (18:3 mM) (B). Cultures were inoculated with cells nonacclimated to either sulfite or 3-Cl-4-OHPA. , pyruvate; , acetate; , 3-Cl-4-OHPA; , 4-OHPA; , sulfide.

For naturally occurring microorganisms like D. dehalogenans, the ability to use several different terminal electron acceptors simultaneously is quite advantageous, especially under conditions and in environments where the availability of electron acceptors is growth limiting. In summary, the use of 3-Cl-4-OHPA in the presence of other energy-yielding terminal electron acceptors strengthens the argument that chlorinated phenols can competitively serve as alternative electron acceptors for D. dehalogenans and that reductive dehalogenation of these compounds leads to energy formation, apparently through electron transport phosphorylation.