RDX was biodegraded in serum bottles incubated under methanogenic conditions (Figure 2). In these bottles, three transient appearing peaks were observed in the chromatograms during HPLC analysis of the aqueous samples (Figure 2). These compounds were identified as mononitroso-RDX, dinitroso-RDX, and trinitroso-RDX by comparing their HPLC retention times with authentic standards (data not shown). After approximately 27 days, the compounds were no longer detected in the serum bottles. The purity of the standards used to identify the intermediates were unknown, therefore researchers were unable to quantitate them. However, Figure 2 shows the relative area of the peak on the chromatogram corresponding to the compound of interest. Although no other intermediates were observed in the HPLC chromatograms, the biodegradation of RDX was hypothesized to proceed in a manner similar to that proposed by McCormick, Cornell, and Kaplan (1981), who reported that the RDX molecule is destabilized and spontaneously fragments when the nitro groups are reduced to the hydroxylamino level. Others (Kitts, Cunningham, and Unkefer 1994) have also reported ring cleavage of the RDX molecule.
Figure 2. RDX biodegradation in serum bottles incubated under methanogenic conditions. Three transient appearing peaks were identified as the mono-, di-, and trinitroso-RDX intermediates. The integrator units of the peak corresponding to the nitroso-intermediate is shown.
Table 1. Methane production in serum bottles containing wastewater from an explosives manufacturing wastewater treatment plant. The bottles were amended with RDX to approximately 80 _M.
Sample |
Methane (_moles) Initial |
Final |
Methane (_moles) Formed |
Sterile Control |
0 |
0 |
0 |
Unamended Control |
0 |
40 |
40 |
RDX Amended |
0 |
3 |
3 |
The presence of RDX inhibited methane production (Table 1). For example, only 3 _moles methane was produced in bottles amended with RDX, compared to 40 _moles in RDX unamended controls. Inhibition of methane production by RDX was not surprising since RDX is known to be toxic to aquatic microorganisms (Drzyzga et al. 1995). Furthermore, nitroaromatic compounds can lyse methanogenic bacteria and inhibit methane formation in anaerobic sewage sludge (Gorontzy, Kuver, and Blotevogel 1993). TNT is also known to be mutagenic and toxic to microorganisms (Roberts, Ahmad, and Pendharkar 1996). Although previous reports suggest that the toxicity of RDX may be responsible for the inhibition on methane production, subsequent studies (described below) suggest that the inhibition is due to something other than the toxicity.
The initial RDX degradation rate in bottles incubated under methanogenic conditions in the screening studies was 9 _M day-1. Attempts were made to enrich for this activity by re-amending the bottles with RDX when it was no longer detected in the aqueous phase. Despite these efforts, the RDX biodegradation activity decreased over time. Ethanol and butyrate were added to the bottles in an attempt to sustain the activity. The RDX degradation rates in bottles amended with butyrate and water were about 2 _M day-1 and decreasing, while the degradation rate in the ethanol amended culture had stabilized at 4 _M day-1 (data not shown). The contents of the serum bottle was periodically transferred to fresh basal salts medium (10 to 20 percent by volume) and were amended with ethanol and RDX when they were depleted. After several transfers, the contents of the serum bottle was transferred to a 1-L bottle. Ethanol, RDX, and basal salts medium were periodically added. This enrichment culture was used in all subsequent studies.
Figure 3. RDX biodegradation by the methanogenic enrich-ment culture when amended with acetate, ethanol, pryuvate, or glucose. The electron donors were added to a concentration of 1 mM.
RDX was biodegraded by the enrichment culture when ethanol, pyruvate, or glucose were supplied as electron donors (Figure 3). Glucose and ethanol appeared to be better cosubstrates than pyruvate for supporting RDX biodegradation. After 7 days, RDX was no longer detected in serum bottles amended with glucose or ethanol, while in bottles amended with pyruvate >70 percent of the RDX remained. In the latter case, RDX was eventually depleted after 16 days. Interestingly, acetate did not support RDX biodegradation (Figure 3). There was no loss of RDX in cosubstrate unamended and sterile controls throughout the study period.
Several electron donors supported methane production when added to the enrichment culture, but the addition of RDX stopped methane production. For example, 5.2 _moles methane were produced when ethanol was added to the enrichment culture, but only 0.2 _moles were produced when RDX was added along with the ethanol (Table 2), for a 96 percent decrease in the amount of methane produced. RDX had a similar effect on methane production when glucose was the cosubstrate. Bottles amended with RDX showed a 91 percent decrease in methane production compared to the RDX unamended controls (Table 2). Acetate and pyruvate did not support methane production (Table 2). Methane production in these bottles was similar to the cosubstrate unamended control. Less than 25 percent of the methane predicted from the complete mineralization of ethanol to CH4 and CO2 was observed in RDX unamended controls (Table 3). However, acetate, an intermediate produced during the mineralization of ethanol to CH4 and CO2, did not support methane production by the enrichment culture (Table 2).
Table 2. Inhibition of methane production in the enrichment culture by RDX. The bottles were amended to a concentration of 1 mM with the respective electron donor and 25 _M RDX. Reported methane values (in _moles) were taken after 19 days incubation.
Electron |
Methane Produced | |
Donor |
RDX Amended |
RDX Unamended |
none |
aND |
0.4 |
ethanol |
0.2 |
5.2 |
glucose |
0.2 |
2.2 |
acetate |
0.2 |
0.6 |
pyruvate |
0.1 |
0.3 |
anot done | ||
Table 3. Methane recovery from 1 mM ethanol when added to the enrichment culture.
Ethanol
|
_moles
|
Expected Methane
|
Observed Methane
|
%
|
0 |
0.0 |
0.0 |
0.7 |
aNA |
1 |
17.3 |
b25.9 |
6.0 |
23 |
1 |
17.3 |
c8.6 |
6.0 |
70 |
a Not applicable
| ||||
Furthermore, subsequent studies demonstrated that acetate accumulates in the medium and is apparently not used by the enrichment culture (data not shown). When acetate's contribution to methane production is subtracted, 70 percent of the expected amount of methane from ethanol was observed (Table 3).
However, the loss of methane production when the enrichment culture was fed RDX along with ethanol (Table 2) is still unexplained. Since the enrichment culture could not use acetate, the proposed reactions for ethanol utilization by the enrichment culture are:
Reaction |
Equation |
_Go' (kJ) |
|||
|
ethanol + H2O |
_ |
Acetate- + H+ + 2 H2 |
+ 9.6 |
Eq 1 | |
4 H2 + HCO3- + H+ |
_ |
CH4 + 3 H2O |
- 135.6 |
Eq 2 | |
Net: |
2 ethanol + HCO3- |
_ |
2 acetate- + CH4 + H2O + H+ |
-116.4 |
Eq 3 |
This pathway is consistent with known reactions occurring under methanogenic conditions with a consortium of bacteria (McInerney 1986). RDX is hypothesized to serve as a hydrogen sink, diverting hydrogen away from methane production (Eq 2). In other words, H2 produced during the metabolism of ethanol was used to reduce RDX, not CO2, thus suppressing methane production. To test this hypothesis, increasing concentrations of ethanol (10 mM) were added to the enrichment culture to ensure that more H2 was produced than needed for RDX biodegradation. As predicted, no methane was produced in the presence of RDX, but after its depletion, methane production started (Figure 4). It appears that, when RDX is no longer present, the H2 produced by the bacteria (Eq 1) becomes available to the methanogens and methane production resumes (Eq 2). There was no lag in the methane production in RDX unamended controls. These observations support the hypothesis that RDX serves as an H2 sink, diverting H2 away from methane production. Boopathy and Kulpa have suggested a similar phenomenon with sulfate-reducing bacteria and nitroaromatic compounds (Boopathy and Kulpa 1993). They reported nitroaromatic compounds may substitute for sulfate as catabolic electron acceptors for Desulfovibrio sp. (B strain).
Other evidence supports our contention that RDX is serving as a terminal electron acceptor in methanogenic environments. For example, the metabolism of ethanol to acetate and H2 is not thermodynamically favorable (+9.6 kJ), as shown in Eq 1. Ethanol metabolism only proceeds if one of the products is removed, resulting in a thermodynamically favorable reaction. Typically the methanogens remove H2 (Eq 2), pulling Eq 1 forward. The sum of the two reactions results in an overall thermodynamically favorable reaction for the metabolism of ethanol to acetate and CH4 (Eq 3). Methane production is inhibited when bromoethanesulfonic acid (BESA), an inhibitor of methanogenic bacteria, is added to the serum bottles (Figure 5). The partial pressure of H2 increases and ethanol degradation stops, as predicted by Eq 1. RDX biodegradation should therefore also cease. Interestingly, this did not occur. Figure 6 demonstrates that BESA had no effect on RDX biodegradation. The metabolism of ethanol continued, supplying H2 for the reduction of RDX. Furthermore, greater than 50 percent of the predicted amount of methane from ethanol was observed, compared to less than 5 percent in the presence of BESA or RDX (Table 4). RDX appears to be replacing CO2 as an H2 sink during ethanol degradation since the metabolism of ethanol to acetate without H2-using methanogens is not thermodynamically feasible (McInerney 1986).
Figure 4. Inhibition of methane formation by RDX in serum bottles amended with 10 mM ethanol. The RDX concentrations are also shown.
Figure 5. Methane formation by the enrichment culture in serum bottles amended with RDX and BESA containing 2 mM ethanol.
Figure 6. Biodegradation of RDX by the methanogenic enrichment culture in serum bottles amended with BESA and 2 mM ethanol. The RDX concentrations in the sterile and ethanol unamended controls are also shown.
Sample |
_moles
|
aStoichiometry
|
CH4 (_moles) Expected |
CH4 (_moles) Formed |
% CH4
|
No ethanol |
0 |
- |
0 |
<1 |
NA |
No BESA |
30 |
0.5 |
15 |
8 |
>50 |
+ BESA |
40 |
0.5 |
20 |
<1 |
<5 |
+ RDX |
40 |
0.5 |
20 |
<1 |
<5 |
acalculated according to the following equation: 2 ethanol + HCO3- --- >2 acetate + CH4 + H2O + H+ | |||||