Environ. Sci. Technol. 1997, 31, 1489-1495
Bacterial Oxidation of Methyl Bromide in Mono Lake, California TRACY L. CONNELL,† SAMANTHA B. JOYE,‡ LAURENCE G. MILLER,† AND R O N A L D S . O R E M L A N D * ,† U.S. Geological Survey, 345 Middlefield Park, Menlo Park, California 94025, and Texas A&M University, College Station, Texas 77843
The oxidation of methyl bromide (MeBr) in the water column of Mono Lake, CA, was studied by measuring the formation of H14CO3 from [14C]MeBr. Potential oxidation was detected throughout the water column, with highest rates occurring in the epilimnion (5-12 m depth). The oxidation of MeBr was eliminated by filter-sterilization, thereby demonstrating the involvement of bacteria. Vertical profiles of MeBr activity differed from those obtained for nitrification and methane oxidation, indicating that MeBr oxidation is not simply a co-oxidation process by either nitrifiers or methanotrophs. Furthermore, specific inhibitors of methane oxidation and/or nitrification (e.g., methyl fluoride, acetylene, allyl sulfide) had no effect upon the rate of MeBr oxidation in live samples. Of a variety of potential electron donors added to Mono Lake water, only trimethylamine resulted in the stimulation of MeBr oxidation. Cumulatively, these results suggest that the oxidation of MeBr in Mono Lake waters is attributable to trimethylaminedegrading methylotrophs. Neither methyl chloride nor methanol inhibited the oxidation of [14C]MeBr in live samples, indicating that these bacteria directly oxidized MeBr rather than the products of MeBr nucleophilic substitution reactions.
Introduction Methyl bromide (MeBr) is a trace gas that is linked to stratospheric ozone destruction (1-3). The atmospheric residence time of MeBr is 2 min) or filter-sterilized (0.2 µm) cell suspensions. Subsamples (1 mL) were extruded during the incubation into microfuge tubes as outlined above, differing only in that that 1.0 N NaOH (0.02 ml) was added to each 1-mL sample aliquot in the tube before the addition of SrCl2‚6H2O in order to elevate the pH and prevent loss of counts as 14CO2. Formation of 14CO32- was nearly linear for a 3-h time course (data not shown), at which time 50% of the added [14C]MeBr was recovered as 14CO32-, while no oxidation was detected in the cell-free controls. An experiment was also conducted with cells of M. capsulatus that were resuspended in Mono Lake water. Cells were treated as above with the exception that resuspension was done in previously filter-sterilized (0.2 µm) Mono Lake surface water (final cell density ) ∼0.11 mg dry weight/mL). Due to the elevated pH of Mono Lake water and the fact that M. capsulatus grows only at circumneutral pH, only a small amount (3%) of the added [14C]MeBr was recovered as 14CO32- and only after a prolonged (68 h) incubation. Although the oxidation rate was ∼200-fold slower in Mono Lake water than in cells incubated in neutral pH mineral salts, the activity was readily discernible and nearly linear over the time course (data not shown). No activity was detected in killed controls. Washed cell suspensions of Nitrosomononas europaea treated as outlined above at circumneutral pH also oxidized [14C]MeBr to 14CO32-, but at a slower rate than M. capsulatus (data not shown). These preliminary experiments illustrated that the
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technique was applicable to study the oxidation of MeBr by bacterial assemblages in natural waters. Effect of Electron Donors, Potential Intermediates, and Inhibitors upon MeBr Oxidation. To determine whether methane oxidation, nitrification, or methylotrophy are linked to CH3Br oxidation in Mono Lake water, laboratory experiments were conducted using Mono Lake water and additions of various substrates, intermediates, or inhibitors of these processes. Samples were collected from a 5-m depth (4/95, 7/95, and 6/96) and processed in the laboratory within 2 months, and in some cases processing was done within 2 weeks of collection. Preliminary experiments indicate that cell counts of live bacteria are roughly comparable for freshly collected samples (∼5 × 106 cells/ml) as opposed to those stored at 4 °C for 2 months after retrieval from the field (∼3 × 106 live cells/mL as determined by acridine orange direct counts; 30). Filter-sterilized controls were run for each experiment, and in addition, heat-killed controls (boiled for 5 min then cooled to 20 °C) were also run in a preliminary experiment. Incubations were conducted in 10-ml plastic syringes as above, except in the case of the sodium sulfide experiment where the water was bubbled with N2 for 10 min before sampling to lower dissolved oxygen (DO) and incubations were carried out in glass syringes. 14CH3Br (final concentration ) 2.5 µmol/L; 0.06 µCi/mL) was added, and samples were incubated at room temperature (∼20 °C) in the dark for several days. The effects of the following substances upon the rate of MeBr oxidation were tested (concentrations given in the figure legends): CH4, NH4Cl, sodium sulfide, methane thiol (MeSH), trimethylamine (TMA), dimethylamine (DMA), glucose, methanol (MeOH), methyl chloride (MeCl), dimethyl sulfide (DMS), and dimethylsulfonopropionate (DMSP). Inhibitor additions were 50 µM N-(N-butyl)thiophosphoric triamide (NBPT), 50 µM allyl sulfide, 54 µM dicyandiamide (DCD), 4 mM acetylene, and 4 mM CH3F. These substances inhibit CH4 oxidation and/or nitrification (27, 28, 31, 32). Gaseous inhibitors and substrates were added from saturated solutions prepared with deionized water. Chemical Degradation of MeBr in Mono Lake Water. The degradation of MeBr by hydrolysis to MeOH and by chloride substitution to form MeCl was investigated with filtersterilized Mono Lake water. In this experiment, we employed high levels (mM) of unlabeled MeBr in order to be able to detect products by analytical chemical means. Lake water (5 mL) was drawn into a 10-mL glass syringe and capped as outlined above. A saturated stock solution of [12C]MeBr was prepared by bubbling MeBr through a glass frit into de-ionized water for 5 min. A subsample (0.25 mL) was injected into the syringe to achieve an initial dissolved MeBr concentration of 13.5 mM, and the syringe was incubated in a water bath (20 °C) for 3 days, during which time subsamples were withdrawn for the determination of MeBr, MeCl, and MeOH concentrations. Upon subsampling, the syringe was hand-shaken and 5 µL was withdrawn for analysis via flame ionization gas chromatography using direct aqueous injection (HewlettPackard Model 5730 A; column ) 3 m × 0.32 cm krytox; He carrier flow ) 30 mL/min; oven temperature ) 100 °C). The injection port was packed with glass wool and held at 200 °C in order to vaporize the samples prior to their reaching the column. The retention times for MeOH, MeCl, and MeBr were 1.7, 1.9, and 2.5 min, respectively. For comparative purposes, we ran an identical experiment with filtered seawater collected from Santa Cruz, CA.
Results Field Studies. In April 1995 the lake had undergone winter turnover, and water temperatures ranged from 6 °C at the surface to as low as 1.5 °C at the bottom (Figure 1B). The oxycline began at 9-10 m and the waters below 15 m were anoxic. The lake was also chemically stratified, with a chemocline evident at 12-14 m (28). Highest levels of
FIGURE 1. (A) Rate constants for methane oxidation (0), nitrification ×100 ((), and MeBr oxidation (b) vs depth. (B) Dissolved oxygen (() and temperature (O).
FIGURE 2. Time course of [14C]MeBr oxidation experiments in April 1995. Symbols represent the mean of three samples, and bars indicate (1 SD. Absence of bars indicates the error was smaller than the symbols. Open symbols, live samples; closed symbols, filtered sterilized controls. methane (∼2 µM) and ammonia (25 µM) detected in the hypolimnion exceeded those in the epilimnion, but sulfide levels were comparable (1-5 µM). The dissolved concentrations of these reduced substances, however, are orders of magnitude below the concentrations detected during Mono Lake’s episode of meromixis in the mid-1980s (20, 21).
Incubations conducted at various depths in the water column demonstrated biological oxidation of [14C]MeBr in live samples but not in controls. Results of time course experiments for a complete depth profile are shown in Figure 2. No activity was detected in any of the filter-sterilized controls, and only relatively small differences were noted
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between the activites sustained at the various depths, with the highest rate of oxidation occurring at 11 m. Rates of oxidation declined after the first day of sampling for all the depths assayed (Figure 2). Therefore, rate constants for all the sampling periods were determined from the first incubation period (∼28 h). The potential rates of MeBr oxidation at ambient light in 5-m samples (10.8 ( 1.7 nmol L-1 day-1) were comparable to those incubated in the dark (9.6 ( 2.5) and at low DO levels (13.8 ( 1.3) (mean of three samples ( 1 SD; data not shown). The depth profiles of rate constants for the oxidation of MeBr, methane, and ammonia are shown in Figure 1A. The highest rate constants for MeBr oxidation were generally observed in the epilimnion, with maxima evident between 9 and 13 m depth. Although maxima were also evident for methane oxidation and nitrification, these did not coincide in depth with those for MeBr oxidation. Methane oxidation rates were highest in the anoxic hypolimnion, which is consistent with previous observations made in Mono Lake as well as in chemically similar Big Soda Lake (18, 22). Rate constants for nitrification were greatest in the epilimnion, but did not correlate with those for MeBr oxidation. There was no significant correlation between the rate constants for MeBr oxidation vs methane oxidation (r2 ) 0.18) or nitrification (r2 ) 0.15). Turnover times for MeBr, based only on its oxidation to CO32-, ranged from 0.25 to 0.63 years. Laboratory Studies. Experiments with Electron Donors and Inhibitors. Water from 5 m depth demonstrated a capacity for biological oxidation of MeBr in all the samples tested. In a 21-h experiment, live samples produced 0.190 ( 0.010 nCi/ml of 14CO32-, while heat-killed and sterile-filtered controls formed 0.097 ( 0.003 and 0.097 ( 0.005, respectively (mean of 3 samples ( 1 SD). Addition of TMA to samples resulted in a clear stimulation of MeBr oxidation (Figure 3A). Samples containing the largest additions of TMA (100 µM) were able to sustain the initial rate of MeBr oxidation for ∼7 days, while rates in unamended samples leveled off after only 2 days. No MeBr oxidation was observed in filtered controls incubated with or without TMA. In contrast, neither DMA nor glucose additions had any effect upon the rate of MeBr oxidation (Figure 3B). The effects of sulfur compounds were followed in similar time course experiments, and the results are summarized in Table 1. No enhancement or inhibition of MeBr oxidation was observed after addition of the alkylated sulfur compounds DMS, MeSH, and DMSP, although a clear enhancement of oxidation occurred with the addition of 50 µM sulfide. The effects of methyl chloride and methanol, both products of MeBr’s nucleophilic reactivity with Mono Lake water, are shown in Figure 4. Neither substance had any effect upon MeBr oxidation. A slight stimulation of MeBr oxidation was noted with addition of 10 µM ammonium, but higher concentrations resulted in a clear inhibition. Thus, 50 µM ammonium inhibited oxidation by ∼60%, and complete inhibition was achieved with 500 µM ammonium (Figure 5A). Methane additions had no significant effect on MeBr oxidation (Figure 5B). None of the inhibitors of nitrification or methane oxidation had any effect upon the rate of MeBr oxidation (Table 2). Chemical Degradation of MeBr in Mono Lake Water. The results of MeBr chemical degradation experiments are shown in Figure 6. For Mono Lake water (Figure 6A), removal of MeBr exhibited a nearly exponential decline, which indicated first-order kinetics (correlation coefficient r2 ) 0.96) and an apparent rate constant for loss of 1.4 day-1. The major product detected was MeOH, which accounted for ∼33% of the observed MeBr degradation after 3 days incubation. Methyl chloride accounted for only 4% of MeBr degradation. Therefore, ∼63% of the MeBr loss from lake water could not be accounted for as recovered products from filtered lake water. In contrast, the degradation of MeBr in seawater was
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FIGURE 3. Laboratory incubations of 5 m of lake water. (A) Water was collected during May 1996 and processed within 2 weeks of collection. Symbols: no additions (4), 10 µM TMA (3), 50 µM TMA (O), 100 µM TMA ()), filtered (+), filtered with 50 µM TMA (0). (B) Water from July 1995. Symbols: no additions (4), 10 µM glucose (O), 50 µM glucose (3), filtered with 50 µM glucose ()), 10 µM DMA (0), 50 µM DMA (+), filtered with 50 µM DMA ((). Results represent the mean of three samples, and bars indicate (1 SD. Absence of bars indicates the error was smaller than the symbols.
TABLE 1. Effect of Sulfide and Alkylated Sulfide Compounds upon Oxidation of [14C]MeBra in 5 m of Mono Lake Waterb addition
14CO 23
(nCi/mL)
Inorganic Sulfide Experiment (Water Collected April 1995) none 0.91 ( 0.03 sulfide (1.0 µM) 1.09 ( 0.12 sulfide (10 µM) 0.93 ( 0.16 sulfide (50 µM) 2.06 ( 0.17 filtered + 10 µM sulfide 0.06 ( 0.01 Alkylated Sulfide Experiment (Water Collected July, 1995) none 0.70 ( 0.40 DMS (50 µM) 0.69 ( 0.02 filtered + DMS 0.05 ( 0.01 DMSP (50 µM) 0.62 ( 0.40 filtered + DMSP 0.06 ( 0.01 MeSH (50 µM) 0.54 ( 0.10 filtered + MeSH 0.05 ( 0.05 a Data represent mean of three water samples (1 SD after 50-51-h incubation; [14C]MeBr added ) 66 nCi/mL (1.4 µM) for sulfide experiments and 41 nCi/mL for alkylated sulfides (2.2 µM). b Samples incubated with 10 µM alkylated sulfur compounds did not affect rates as compared to uninhibited samples (data not shown).
much slower (Figure 6B), with an apparent rate constant for loss of 0.27 day-1. After 3 days, about 50% of the MeBr added was removed, with a recovery of 8% of this loss as MeCl. Significant production of MeOH was not noted during the time course of this experiment, and levels remained near the limit of analytical detection. Therefore, we were unable to account for 92% of the observed MeBr loss in the seawater experiments.
TABLE 2. Effect of Nitrification and Methane-Oxidation Inhibitors upon Oxidation of [14C]MeBra in 5 m of Mono Lake Water Collected in May 1996 addition none NBPT (50 µM) DCD (50 µM) allyl sulfide (50 µM) in ethanol ethanol solvent (550 µM) methyl fluoride acetylene filtered
14CO 23
(nCi/mL)
0.74 ( 0.04 0.80 ( 0.03 0.73 ( 0.04 0.75 ( 0.05 0.79 ( 0.04 0.77 ( 0.06 0.68 ( 0.05 0.11 ( 0.01
a Results represent the mean ( 1 SD of 3 water samples incubated for 47 h with [14C]MeBr (final concentration ) 63 nCi/mL; 2 µM).
FIGURE 4. Effect of methanol and methyl chloride on MeBr oxidation in 5 m of lake water collected during May 1996. Symbols: no additions (4), 10 µM methanol (3), 50 µM methanol (O), 10 µM methyl chloride ()), 50 µM methyl chloride (0), filtered with 50 µM methanol (+), filtered with 50 µM methyl chloride ((). Symbols represent the mean of three water samples, and bars indicate (1 SD. Absence of bars indicates the error was smaller than the symbols.
FIGURE 5. Effect of ammonium and methane upon MeBr oxidation in 5 m of lake water collected in April 1995. (A) No additions (4), 10 µM NH4Cl (O), 50 µM NH4Cl (3), 500 µM NH4Cl ()), and filtered + 50 µM NH4Cl ((). (B) No additions (4), 1 µM methane (O), 10 µM methane (3), 50 µM methane (0), filtered + 50 µM methane ((). Symbols represented the mean of three samples, and bars indicate (1 SD. Absence of bars indicates the error was smaller than the symbols. Detection limits for MeCl and MeOH were both e0.1 mM.
Discussion Methyl bromide was oxidized at all depths in the water column of Mono Lake (Figure 1). Because no significant increases in 14CO 2- were noted in the filter-sterilized controls as compared 3 with the live samples, we conclude that the oxidation detected at each depth was due to biological processes. This conclusion is reinforced by the fact that boiling effectively eliminated activity in lab-incubated samples, which rules out the possible involvement of some particle-based chemical phenomenon. The decline in rates after the first sampling period suggests that some substance became limiting or inhibitory to the reaction as time progressed (Figure 1). Only about 1% of the
FIGURE 6. Production of methanol (0) and methyl chloride ()) from 13.5 mM MeBr (O) added to filter-sterilized Mono Lake water (A) and from 10 mM MeBr added to filter-sterilized seawater (B). Symbols represent the mean of three water samples, and bars indicate (1 SD. Absence of bars indicates the error was smaller than the symbols. [14C]MeBr was oxidized in the samples after 24-h incubation, so rates did not decline because of depletion of the labeled substrate. No differences were noted between light and dark samples incubated in situ, which eliminates the involvement of photochemical or photobiological processes in the oxidation. Furthermore, significant activity was detected in the samples from the anoxic hypolimnion (Figures 1 and 2), which indicated that DO was not a controlling factor. Indeed, when we lowered the DO by purging with N2, we still obtained rates equivalent to the unperturbed samples. Cumulatively, these results suggest that some other factor, perhaps a co-metabolic substrate, limited rates of MeBr degradation by the resident microbial population.
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Neither methane oxidation nor nitrification appeared to be directly linked to MeBr oxidation, based on the depth profiles of rate constants for these processes (Figure 2), and correlation coefficients yielded insignificant values. These results are surprising because both nitrifiers and methanotrophs can oxidize MeBr (16, 17), and we detected significant oxidation of [14C]MeBr by cell suspensions of M. capsulatus and N. europaea in our preliminary experiments (see Methods). The potential for involvement of nitrifiers was suggested from the laboratory experiments with Mono Lake water in which additions of 10 µM ammonium slightly stimulated MeBr oxidation, while higher levels inhibited it (Figure 5A). This pattern is consistent with the involvement of nitrifiers since the provision of low levels of electron donor should enhance the activity of these organisms, which would encompass cooxidation of MeBr, while higher levels of ammonium would act as a competitive inhibitor of MeBr oxidation. Methane caused competitive inhibition of MeBr oxidation by M. capsulatus (16), but the addition of methane to lake water had no noticeable effect on MeBr oxidation by the resident bacterial assemblage (Figure 5B). Furthermore, inhibitors of methane oxidation and nitrification had no influence upon MeBr oxidation (Table 2). Hence, although a potential linkage of MeBr oxidation to nitrifying bacteria could sometimes be elicited in lake water, the preponderance of both field and laboratory data indicate that neither nitrification nor methane oxidation were the primary processes driving this reaction. There was, however, a clear enhancement of the extent of MeBr oxidation when TMA was added to lake water (Figure 3A). The effect was to sustain the initial rate of oxidation over a prolonged period rather than to increase the rate itself. Thus, TMA is likely to be the substance limiting the overall oxidation of MeBr observed in the field incubations (Figure 1). This indicates that the primary organisms involved in MeBr oxidation in this system were methylotrophs rather than methanotrophs or nitrifiers. Similar conclusions were drawn for the microbial oxidation of dibromomethane in seawater (33). The fact that no enhancement occurred with samples amended with glucose, dimethylamine, MeOH, or DMS (Figures 3B and 4; Table 1) argues not only for methylotrophs but also specifically for TMA-utilizing methylotrophs. Pure culture investigations with the marine methylotrophic bacterium BIS-6 (34) demonstrated that cooxidation of MeBr was achieved with TMA but not MeOH (35), which is consistent with our Mono Lake experiments. The lack of any inhibitory effect by either MeOH or MeCl upon MeBr oxidation (Figure 4) suggests that bacteria oxidize MeBr directly rather than a chemical product of its nucleophilic attack in this mileu (11-13). Should either MeOH or MeCl have been the direct focus of bacterial oxidation rather than MeBr, their addition would have resulted in a strong isotope dilution effect on the oxidation of [14C]MeBr, and the counts recovered as 14CO32- would have been much lower. The enhanced oxidation of MeBr achieved with sulfide was probably caused by its nucleophilic attack on MeBr, which results in the production of DMS (15). Bacterial populations capable of DMS as well as TMA degradation are common in marine waters since DMSP and glycine betaine are important osmolytes (36). Therefore, it is not surprising that hypersaline Mono Lake also harbors bacterial populations that are readily able to degrade these substances (37). The oxidation of MeBr in the anoxic hypolimnion (Figure 2) probably stemmed from its chemical conversion to DMS by reaction with the small levels of ambient sulfide (
