Environ. Sci. Technol. 2000, 34, 2525-2527
Microbial Transhalogenation: A Complicating Factor in Determination of Atmospheric Chloro- and Bromomethane Budgets D A V I D B . H A R P E R , * ,† R O B E R T M . K A L I N , ‡ MICHAEL J. LARKIN,§ JOHN T. G. HAMILTON,| AND C A T H E R I N E C O U L T E R †,§ Microbial Biochemistry Section, School of Agriculture and Food Science, The Queen’s University of Belfast, Belfast BT9 5PX, U.K., Environmental Engineering Research Centre, School of Civil Engineering, The Queen’s University of Belfast, Belfast BT9 5AG, U.K., QUESTOR Centre, The Queen’s University of Belfast, Belfast BT9 5AG, U.K., and Department of Agriculture for Northern Ireland, Belfast BT9 5PX, U.K.
The sources and sinks of the ozone-depleting halocarbons, chloromethane (CH3Cl) and bromomethane (CH3Br), have been the subject of recent controversy. Considerable uncertainty surrounds the relative contributions of oceanic and terrestrial sources of CH3Cl and natural versus anthropogenic fluxes of CH3Br. Halogen stable isotope ratios in atmospheric halomethanes could provide a valuable tool in estimating relative magnitudes of sources, particularly those of CH3Cl. However, the reliability of such techniques is critically dependent on the conservative nature of the halogens within these atmospheric halomethanes. Here we demonstrate that intact cells of the soil bacterium strain CC495 under anaerobic or microaerophilic conditions rapidly exchange 37Clwith organically bound chlorine in CH3Cl. Since Cl- occurs ubiquitously and such bacteria appear to be widespread, any chlorine isotope fractionation during biological or abiotic CH3Cl production may therefore not be apparent in atmospheric CH3Cl. Cells of strain CC495 also catalyzed transhalogenation of CH3Br to CH3Cl, suggesting that this transformation may represent a significant sink for atmospheric CH3Br in soil.
Introduction With an average tropospheric mixing ratio of about 600 pptv and a global atmospheric burden of 4-5 million t, CH3Cl is the most abundant volatile halocarbon in the atmosphere (1-3). Indeed between 15 and 20% of chlorine-catalyzed ozone destruction in the stratosphere can be attributed to CH3Cl (4). Important sources identified to date include biomass burning (5), oceanic emissions (2), and production by wood-rotting fungi (2, 6-8). Additionally higher plants may release significant quantities of CH3Cl (9-12). However, * Corresponding author telephone: 44-2890-255343; fax: 44-2890669551; e-mail:
[email protected]. † School of Agriculture and Food Science, The Queen’s University of Belfast. ‡ School of Civil Engineering, The Queen’s University of Belfast. § QUESTOR Centre, The Queen’s University of Belfast. | Department of Agriculture for Northern Ireland. 10.1021/es991329r CCC: $19.00 Published on Web 05/13/2000
2000 American Chemical Society
there is uncertainty regarding the relative magnitudes of the various sources of the halocarbon. A recent downward revision of the oceanic contribution from 3 million to 440 000 t/yr (2) and a significant discrepancy emerging between the estimates of fluxes from the known sources and the modeled sinks have fueled further debate. Possible explanations of this anomaly include a substantial underestimation of one or more of the emission fluxes or a major unidentified source. One approach to refining source terms is to compare the chlorine stable isotope composition of atmospheric CH3Cl with that of CH3Cl from possible sources as it seems likely that biologically produced CH3Cl (i.e., from fungal, plant, and possibly oceanic sources) may show significant isotopic fractionation as compared with that produced by abiotic processes (13). The potential of using δ37Cl to provide information on sources and transformation of chlorocarbons in the environment has been the subject of several recent investigations (14-16). The technique has been proposed as a means of quantifying the contribution of natural CH3Cl to the stratospheric inorganic chlorine budget (17). The validity of such a technique is dependent on the organically bound chlorine of atmospheric CH3Cl behaving conservatively, i.e., not exchanging once the halocarbon is produced. Chemical considerations would suggest that this assumption is justified as, even at the high concentrations of Cl- present in seawater, exchange of Cl- with chlorine in CH3Cl by nucleophilic attack is relatively slow with a calculated half-life of 3-4 months at 20 °C (18). However the findings presented in this paper now indicate that certain microorganisms may rapidly catalyze such exchange. In our investigations, we used a facultative methylotrophic bacterium (strain CC495) that had been isolated from woodland soil (19). This microorganism is closely related to Rhizobium and capable of growing on CH3Cl as the sole carbon and energy source. Initial dehalogenation of CH3Cl by this bacterium has been shown to be mediated by a novel corrinoid enzyme, halomethane: bisulfide/halide ion methyltransferase (19). In the presence of HS-, the purified methyltransferase converted CH3Cl, CH3Br, or CH3I to CH3SH. It is postulated that, in whole cells under aerobic conditions, HS- present intracellularly at low concentrations is metabolized to CH3SH. The latter compound is then oxidized via formaldehyde with the concomitant release of HS-, which is recycled to act as the endogenous acceptor ion for further CH3Cl degradation. Formaldehyde is then further oxidized through formate to CO2. Surprisingly, in the presence of the halide ions (Cl-, Br-, and I-) enzyme preparations in vitro catalyzed displacement of organically bound halogen in halomethane with halide ion. We here report experiments designed to examine the possibility that intact cells of strain CC495 may mediate the transhalogenation of halomethanes in environments where significant concentrations of inorganic halide ion are present and oxygen supply is restricted, preventing recycling of HS-.
Experimental Section Cultures of bacteria strain CC495 were grown on minimal medium (pH 7.2, 500 mL) in sealed 2-L conical flasks containing 0.4 g of CH3Cl as the sole carbon source as previously described (19). Cells were harvested in the late exponential phase after 3 days growth by centrifugation (3000g, 90 min), washed twice with 50 mM phosphate buffer, pH 7.2 and suspended in this buffer. For experiments on incorporation of 37Cl- into CH3Cl by cell suspensions of strain CC495, washed cells (25 mg wet wt) were incubated at 25 °C in duplicate 20-mL vials (crimp-capped sealed with a PTFEVOL. 34, NO. 12, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Incorporation of 37Cl- from 50 mM Na37Cl into CH3Cl (400 µg) by cell suspensions of bacterial strain CC495 under N2 (O) and pure halomethane:bisulfide/halide ion methyltransferase in air (9). Each value is the mean of duplicates (ranges are contained within each of the data points). lined butyl rubber septum) in 50 mM phosphate buffer, pH 7.2, containing 50 mM Na37Cl (Sigma Aldrich, purity 95% 37Cl) and 400 µg of CH Cl (total volume, 1 mL) under 1 atm 3 of either N2, 1% O2, 5% O2, or air. For experiments on incorporation of 37Cl- into CH3Cl by halomethane:bisulfide/ halide ion methyltransferase, the pure enzyme (0.1 mg of protein, specific activity 983 µkat kg-1), obtained from cell extracts of strain CC495 as previously described (19) was incubated under similar conditions in air. The proportion of CH335Cl to CH337Cl was determined using a Hewlett-Packard 5890 series II gas chromatograph linked to an HP5971 mass selective detector. The gas chromatograph was fitted with a Poraplot Q capillary column (10 m × 0.32 mm), and helium (1 mL min-1) was used as the carrier gas (19). Headspace (50 µL) was injected onto the column at a split ratio of 50:1, and the oven temperature was held at 30 °C for 1 min and then programmed at 10 °C min-1 to 150 °C. Ion currents at m/z 35, 37, 50, and 52 were monitored. The ratio of ion currents at m/z 50 and 52 was used to calculate the proportion of CH335Cl to CH337Cl in the sample. For experiments on transhalogenation of CH3Br, washed cells (100 mg wet wt) of strain CC495 were incubated in duplicate 20-mL vials in 50 mM phosphate buffer, pH 7.2, containing 50 mM NaCl and 325 µg of CH3Br (total volume, 1 mL) under 1 atm of either N2, 1% O2, 5% O2, or air at 25 °C. CH3Cl and CH3Br concentrations in samples of headspace (0.2 mL) were determined by gas chromatography using a column packed with Tenax TA 60-80 and a flame ionization detector (19). Calibration was against samples of the headspace above standard solutions equilibrated at 25 °C. No detectable transhalogenation was observed in the absence of cells.
Results and Discussion Figure 1 shows that the exchange of 37Cl with organically bound chlorine of CH3Cl observed in vitro with pure halomethane:bisulfide/halide ion methyltransferase also occurred rapidly when cell suspensions of strain CC495 were incubated anaerobically under 1 atm of N2. The ratio of 37Cl/ 35Cl in CH Cl rose from its natural abundance of 24.6/75.4 3 to 63/37 in 80 min and to approximately 80/20 in 240 min. When cell suspensions were incubated under microaerophilic conditions (i.e., 1% or 5% O2 atmosphere), much of the CH3Cl present was oxidized to CO2; nevertheless, significant isotope exchange occurred (data not shown). Thus, the proportion of 37Cl in CH3Cl increased in 80 min to 53.4% under 1% O2 and to 41.9% under 5% O2 while the CH3Cl concentration in the aqueous phase fell from 0.25 to 0.18 and 0.04 mM, respectively. However, when the experiment was conducted under 1 atm of air, only 0.01 mM CH3Cl remained after 80 min and 37Cl in CH3Cl did not exceed 26%. 2526
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FIGURE 2. Transhalogenation of CH3Br (0, 9) to CH3Cl (O, b) under N2 (0, O) or 5% O2 (9, b) by cell suspensions of bacterial strain CC495 in the presence of 50 mM NaCl. Each value is the mean of duplicates (bars show ranges). There is phylogenetic and molecular genetic evidence that two other CH3Cl/CH3Br-degrading bacterial strains recently identified, strain IMB-I isolated from agricultural soil in California (20) and Methylobacterium strain CM4 isolated from a Russian soil (21), possess corrinoid methyltransferase pathways for the degradation of halomethanes. Thus, bacteria containing Cl-/halomethane exchange capability are probably widespread in the environment. In conditions where Cl- concentrations are high, and particularly where oxygen tension fluctuates sharply leading to intermittent periods of restricted oxygen supply (e.g., salt marsh sediments or soils subject to episodes of waterlogging by brackish water), the presence of such bacteria will lead to the rapid exchange of Cl- in solution with atmospheric CH3Cl. However, ample documentation in the literature shows that, even in normal aerobic soils, the microaerophilic and anaerobic microniches necessary for halogen exchange occur within the active microbial biofilms that surround soil particles (22). It is also pertinent in this context that an estimated 340 million ha of agricultural land worldwide has salinity sufficiently high to cause osmotic stress to plants. It is therefore likely that any potential isotopic fractionation in the pool of atmospheric CH3Cl due to chlorine isotope effects in biological production or degradation of CH3Cl is likely to be significantly reduced, perhaps even eliminated. A rapid biological transhalogenating capability also has implications for the atmospheric CH3Br budget. Atmospheric CH3Br, although present at a mixing ratio of only about 10 pptv, is responsible for between 10 and 15% of halogencatalyzed ozone depletion in the stratosphere as bromine is approximately 50-fold more effective than chlorine in ozone destruction (23). As yet no scientific consensus exists as to the magnitudes of many of the main sources of CH3Br (biomass burning, oceanic emissions, terrestrial vegetation, anthropogenic release due to CH3Br use as a fumigant, and during combustion of leaded petrol) and the sinks (destruction by hydroxyl radical in the atmosphere, uptake by oceans and soils) (11, 23-31). Our findings are particularly relevant to the latter. Figure 2 shows that cell suspensions of strain CC495 in the presence of Cl- rapidly catalyze the conversion of CH3Br to CH3Cl under anaerobic and microaerophilic conditions. Thus, CH3Br (0.52 mM in the aqueous phase) is transformed with a 70% yield to CH3Cl within 40 min in the presence of 50 mM NaCl under N2. The ratio of CH3Cl formed to CH3Br consumed after this period was 0.92:1. The lack of a completely stoichiometric relationship can be attributed to some oxidation of halomethane occurring due to traces of O2 introduced during injection of CH3Br into the vial. Under a 5% oxygen atmosphere, significant oxidation of halomethane occurred, as observed in the 37Cl- exchange experiment. Nevertheless a substantial proportion (21%) of
CH3Cl. As the population of such organisms is likely to be enhanced in environments where biological release of CH3Cl occurs (e.g., rotting wood, forest soils, salt marshes, and wetlands), these habitats deserve special attention as possible sinks for atmospheric CH3Br either by microbial oxidation to CO2 or transhalogenation to CH3Cl.
Literature Cited FIGURE 3. Transformations catalyzed by cell suspensions of bacterium (strain CC495) under aerobic and anaerobic/microaerophilic conditions. CH3Br originally present was converted to CH3Cl within 40 min. Under a 1% oxygen atmosphere this proportion was even higher at 50%. However under fully aerobic conditions (air atmosphere), oxidation of CH3Br was the preferred metabolic pathway, and 97% of the CH3Br originally present was lost by oxidation after 40 min with only trace quantities of CH3Cl (ca. 0.4% conversion) being detected (data not shown). Soils, in particular those of temperate forest and woodland, have been identified as significant sinks for CH3Br. Uptake by aerobic bacterial activity appears to be sufficiently fast at ambient CH3Br atmospheric mixing ratios to account for 25% of the global annual loss of atmospheric CH3Br (25, 26). Populations of CH3Cl-degrading bacteria such as strain CC495 are likely to be higher in woodland soils where emission of CH3Cl by wood-rotting fungi will locally enhance CH3Cl concentrations in the soil (8). Apparent CH3Br uptake in these environments could be due to the use of CH3Br rather than CH3Cl as carbon and energy source by such bacteria. Alternatively their transhalogenating capability may result in the previously unforeseen conversion of CH3Br to CH3Cl in the microaerophilic and anaerobic microniches within the biofilm surrounding soil particles mentioned above (Figure 3). As leaf litter contains relatively high concentrations of Cl-, ca. 400 ppm on a dry weight basis (5), such a transformation may partly explain the observation by Shorter et al. (25) that the litter layer of a temperate forest soil possessed the highest CH3Br uptake of all the soil types examined by these workers. In the marine environment, biotranshalogenation could also have an important impact as an additional CH3Br sink. Chloride substitution is currently regarded as the chief abiotic removal process for CH3Br in seawater with a calculated loss rate of about 8% day-1 at 20 °C (23). However, investigations by King and Saltzman (28) indicate that bacterial uptake of CH3Br in subtropical waters may be of the same order with loss rates of 4-10% day-1 measured at 21 °C. Indeed it has been postulated that in polar waters where the chemical loss rate is very slow (less than 1% day-1) biological degradation may dominate CH3Br removal (28, 29). This hypothesis would explain the observed undersaturation of such waters with CH3Br as compared with model predictions (30). While such biological utilization under aerobic conditions may be attributable to the direct oxidation of CH3Br to CO2 by bacteria, the high concentration of Cl- in seawater renders enzymic transhalogenation to CH3Cl another likely biological sink for CH3Br in anaerobic and microaerophilic estuarine and littoral environments, perhaps rivaling the chemical process in significance. In conclusion, microbial biotranshalogenation greatly reduces the value of δ37Cl as a potential indicator of the origin of the atmospheric CH3Cl. It is also clear that a comprehensive evaluation of the biological sinks of CH3Br in both terrestrial and marine environments will require a consideration of the biochemistry and ecology of the organisms responsible for the utilization of the much more abundant atmospheric gas,
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Received for review December 1, 1999. Revised manuscript received March 21, 2000. Accepted March 22, 2000. ES991329R VOL. 34, NO. 12, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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