Anaerobic Oxidation of [1,2-¹⁴C]Dichloroethene under Mn(IV)-Reducing Conditions

Abstract

Anaerobic oxidation of [1,2-¹⁴C]dichloroethene to ¹⁴CO₂ under Mn(IV)-reducing conditions was demonstrated. The results indicate that oxidative degradation of partially chlorinated solvents like dichloroethene can be significant even under anoxic conditions and demonstrate the potential importance of Mn(IV) reduction for remediation of chlorinated groundwater contaminants.

In chlorinated-ethene-contaminated groundwater systems, production of the Environmental Protection Agency priority pollutant vinyl chloride (VC) by anaerobic biodegradation of dichloroethene (DCE) is well documented (1, 2, 5, 7, 8, 10, 15, 17–19) and typically drives environmental concerns (2, 15). Aerobic oxidation of both compounds without accumulation of reduced intermediates is well-known but of limited relevance because DCE and VC are produced in situ under anaerobic conditions (1, 5, 7, 8, 10, 17–19). It is important, therefore, to determine which of the commonly occurring anaerobic terminal electron acceptors may be energetically sufficient to oxidize compounds like DCE and VC. Anaerobic microbial oxidation of VC with CO₂ as the product has been demonstrated in environmental samples under Fe(III)-reducing conditions (3, 4). In contrast, even though low but significant mineralization of DCE under anaerobic conditions has been reported (4, 16), the fact that mineralization involved significant accumulation of VC (4, 16) and was not enhanced by Fe(III) amendment (4) indicates that the initial step was a reduction and that Fe(III) reduction was not sufficient to oxidize DCE directly. These observations suggest that anaerobic oxidation of DCE requires a terminal-electron-accepting process that is more energetically favorable than Fe(III) reduction.

Mn(IV) oxides are common in alluvial and glacial aquifer sediments, and Mn(IV) reduction is more energetically favorable than Fe(III) reduction (9, 11, 14). The natural abundance of Mn(IV) (11, 14), the greater availability for microbial reduction of Mn(IV) oxides [relative to Fe(III) oxides] (14), and the more favorable energetics of Mn(IV) reduction (2, 11, 14) suggest that Mn(IV) reduction may support oxidation of DCE to CO₂ under anaerobic conditions without the accumulation of VC. To test this hypothesis, a microcosm study was initiated by using aquifer (shallow and intermediate) and surface sediments collected from a site at the Naval Undersea Warfare Center, Keyport, Washington. Aquifer material was characterized by fine to medium sands with some gravel and by an Mn(IV) oxide content of 0.05 to 0.1% (dry weight). Dissolved-Mn(II) concentrations at the site ranged from 0.04 to 5.7 mg/liter. Water chemistry analyses, field measurements of dissolved-H₂ concentrations, and laboratory incubations indicated that Mn(IV) reduction was the predominant terminal-electron-accepting process in the shallow and intermediate aquifers at this site. The surface fill layer was characterized by silt to silty sand and consisted of a mixture of natural material, including glacial till. Water chemistry and H₂ concentration data were not available for the surface fill material. However, based on the shallow sample collection depth, periodic fluctuations in the water table with resultant exposure to air, spatial variability in Mn(IV) oxide availability, and Fe(III) oxide staining on the fill material, the predominant terminal-electron-accepting processes in the zone of the surface fill sample appear to vary among O₂, Mn(IV), and Fe(III) reduction.

DCE mineralization was evaluated with a neat mixture of [1,2-¹⁴C]DCE (29% trans and 71% cis isomers; radiochemical purity ≥ 99.9%; Moravek Biochemicals, Inc., Brea, Calif.). Microcosm preparation, incubation conditions, sampling procedures, and methods for verification of ¹⁴CO₂ have been described previously (3, 4, 6). In brief, 20-ml microcosms containing 15 g of saturated material each were created with headspaces of air (aerobic treatments) or 100% helium (anaerobic treatments). Some were amended with 1.0 ml of anoxic, sterile distilled water (aerobic and unamended anaerobic treatments), and the rest were amended with 1.0 ml (approximately 0.05 g) of an anoxic, sterile slurry of poorly crystalline MnO₂ (12, 13) or Fe(OH)₃ (13). Killed controls were autoclaved twice for 1 h each time at 15 lb/in² and 121°C. The microcosms were preincubated for 5 days and were then spiked with 40,000 dpm of [1,2-¹⁴C]DCE (specific activity = 0.6 mCi/mmol). ¹⁴CO₂ was collected in 3 M KOH and quantified by liquid scintillation counting (3, 4, 6). Recovery of ¹⁴CO₂ was confirmed in select microcosms as described previously (3, 4, 6).

The microorganisms indigenous to all three sample materials were capable of significant DCE mineralization to CO₂ under unamended anaerobic conditions (Fig. 1). For aquifer microcosms, mineralization ranged from 14 to 38% over three days. For the surface fill material, approximately 30% mineralization of [1,2-¹⁴C]DCE was observed after 17 days. In all cases, DCE mineralization was attributable to biological activity because the final recovery of ¹⁴CO₂ in killed control microcosms was less than 4%. Mn(IV) reduction was the predominant terminal-electron-accepting process under unamended, anaerobic conditions, as indicated by the lack of significant dissolved O₂ ([O₂] < 3 μM), NO₃ ([NO₃] < 0.2 μM), and SO₄ ([SO₄] < 20 μM); the lack of significant production of CH₄ (not detected, i.e., [CH₄] < 1 μmol/liter of headspace), dissolved sulfide (not detected, i.e., [HS] < 0.2 μM), and dissolved Fe(II) [2 ± 2 nmol of Fe(II) produced (Table 1)]; and the significant accumulation of Mn(II) (9 ± 1 nmol produced [Table 1]) within these experimental microcosms.

FIG. 1.

Percentage mineralization of [1,2-¹⁴C]DCE to ¹⁴CO₂ in microcosms containing material collected from the surface fill (depth, 1 m), the shallow aquifer (depth, 3 to 5 m) and the intermediate aquifer (depth, 8 to 10 m). Experimental data are means ± standard deviations for duplicate microcosms, and the control data are from a single sterile control microcosm. For each material studied, superscript letters indicate statistically significantly different final mean ¹⁴CO₂ recoveries according to the Kruskal-Wallis one-way analysis of variance on ranks (P < 0.05).

TABLE 1.

Mn(IV) and Fe(III) reduction during oxidation of [1,2-¹⁴C]DCE in intermediate aquifer microcosms^a

Microcosm	Amt (nmol) of productb
	Mn(II)		Fe(II)
	Exptl	Control	Exptl	Control
Anaerobic	9 ± 1	1	NS	NS
Fe(OH)3 amended	NS	NS	35 ± 11	3
MnO2 amended	47 ± 6	NS	NS	NS
Aerobic	NS	NS	NS	NS

^aMicrocosms were vortex mixed and 1.0 ml of sediment supernatant was removed, filtered (0.2-μm pore size), and analyzed colorimetrically with commercially available test kits (Hach Chemical Co.). 

^bThe amount of Mn(II) or Fe(II) produced is presented as a qualitative indicator of Mn(IV) or Fe(III) reduction because the Mn(II) or Fe(II) found in the dissolved phase is a small fraction (less than 10%) (14) of the total Mn(II) or Fe(II) present in the system. Experimental data are means ± standard deviations from duplicate microcosms, and control data are from a single sterile control. NS, no significant change. 

The bioavailability of Mn(IV) was a major factor affecting anaerobic DCE mineralization for all three sample materials (Fig. 1). Amendment of anaerobic microcosms with poorly crystalline MnO₂ resulted in a fivefold increase in Mn(IV) reduction (Table 1). The stimulation of Mn(IV) reduction by MnO₂ amendment was associated with increased DCE mineralization and ¹⁴CO₂ recoveries similar to those under aerobic conditions (Fig. 1). The combined results from the unamended and MnO₂-amended microcosm studies demonstrate that, under anaerobic conditions, both Mn(IV) reduction and DCE mineralization were limited by the bioavailability of Mn(IV) and indicate that DCE mineralization was coupled to Mn(IV) reduction.

The results further indicate that, under Mn(IV)-reducing conditions, DCE is efficiently oxidized to CO₂ without detectable accumulation of intermediates (Table 2). Following the 3-day incubation period, the headspace of intermediate aquifer microcosms was analyzed for the presence of the daughter products of DCE reductive dechlorination (VC, ethene, and ethane). No volatile organic compounds other than DCE were observed in this study. Moreover, the percent change in headspace DCE concentrations closely corresponded to the percent recoveries of ¹⁴CO₂ in unamended and MnO₂-amended anaerobic microcosms (Table 2). The stoichiometric conversion of DCE to CO₂, the lack of accumulation of volatile intermediates, the lack of a detectable lag in the production of ¹⁴CO₂ (Fig. 1), and the similar degrees of mineralization observed in aerobic and MnO₂-amended microcosms (Fig. 1) are consistent with direct oxidation of DCE with CO₂ as the end product.

TABLE 2.

Oxidation of [1,2-¹⁴C]DCE to ¹⁴CO₂ in anaerobic, intermediate aquifer microcosms^a

Microcosm	% of DCE lostb		% of 14CO2 recovered
	Exptl	Control	Exptl	Control
Anaerobic	37 ± 2	15	38 ± 1	3
MnO2 amended	42 ± 12	5	50 ± 0	1

^aExperimental data are means ± standard deviations for duplicate microcosms, and control data are from a single sterile microcosm. 

^bDCE concentrations were determined by flame ionization detection-gas chromatography, and DCE loss was estimated from the final headspace DCE concentration and expressed as a percentage of the initial headspace DCE concentration. All DCE loss was as cis-DCE. No significant change in the headspace concentration of trans-DCE was observed. 

The oxidation of DCE observed in this study under anaerobic conditions was not attributable to Fe(III) reduction (Table 3). Because others (12) have shown that Fe(III) reduction can proceed under Mn(IV)-reducing conditions without significant accumulation of dissolved Fe(II), we examined the possibility that anaerobic oxidation of DCE was associated with Fe(III) reduction by quantifying the mineralization of [1,2-¹⁴C]DCE in anaerobic microcosms amended with poorly crystalline Fe(III) oxide [provided as Fe(OH)₃]. Addition of Fe(OH)₃ resulted in significant Fe(III) reduction, as indicated by increased accumulation of dissolved Fe(II) (Table 1), but inhibited DCE mineralization (Table 3). The lack of stimulation of DCE mineralization under Fe(III)-amended conditions has been observed previously (4) and indicates that Fe(III)-reducing conditions are not sufficient to support DCE oxidation. Moreover, the apparent inhibitory effect of Fe(III) amendment on DCE oxidation (Table 3) provides additional, compelling evidence that the anaerobic oxidation of DCE observed in the present study was coupled to Mn(IV) reduction.

TABLE 3.

Effect of Fe(OH)₃ or MnO₂ amendment on the final recovery of ¹⁴CO₂

Microcosm	% of 14CO2 recovered from:
	Surface filla		Intermediate aquiferb
	Exptl	Control	Exptl	Control
Anaerobic	31 ± 2	1 ± 0	38 ± 1	3
Fe(OH)3 amended	21 ± 6	5 ± 1	28 ± 1	3
MnO2 amended	51 ± 5	6 ± 1	50 ± 0	1

^a17-day incubation period. Data are means ± standard deviations for triplicate experimental microcosms and duplicate control microcosms. 

^b3-day incubation period. Experimental data are means ± standard deviations for duplicate microcosms, and control data are from a single sterile control microcosm. 

This investigation is the first report of anaerobic oxidation of DCE under Mn(IV)-reducing conditions and, to our knowledge, the first report of anaerobic mineralization of DCE to CO₂ without the accumulation of reduced intermediates. Mn(IV) oxides are potentially powerful oxidants which are common in aquifer sediments and groundwater systems. Much of the risk associated with DCE contamination in groundwater is due to the potential production of VC. The present results demonstrate that DCE can be oxidized to CO₂ under Mn(IV)-reducing conditions without the environmental risk associated with VC accumulation.