Sep 18, 1989 - injected onto a Porapak Q (50/80 mesh) column (1.83 m by. 0.64 cm [inside ..... tion of coenzyme M (2-mercaptoethanesulfonic acid) by meth-.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1990, p. 1198-1201 0099-2240190/041198-04$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 56, No. 4
Dechlorination of Chloroform by Methanosarcina Strains MARK D. MIKESELLt AND STEPHEN A. BOYD*
Department of Crop and Soil Sciences, Michigan State University, East Lansing, Michigan 48824-1325 Received 18 September 1989/Accepted 18 January 1990
Dehalogenation of carbon tetrachloride, chloroform, and bromoform in pure cultures of Methanosarcina sp. strain DCM and Methanosarcina mazei S6 was demonstrated. The initial dechlorination product of chloroform was methylene chloride (dichloromethane), which accumulated transiently to about 70% of the added chloroform; trace amounts of chloromethane were also detected. The amount of chloroform dechlorinated per mole of methane produced was approximately 10 times greater than the ratio observed previously for tetrachloroethene dechlorination by these strains. The production of 14CO2 from [14C]chloroform and the absence of 14CH4 imply that processes in addition to reductive dechlorination operate. The fate of halogenated aliphatic compounds in methanogenic environments has received considerable attention because of the importance of methanogenic habitats such as landfills, waste treatment systems, aquatic sediments, and some aquifers. The most widely reported transformation of chlorinated one- and two-carbon compounds under methanogenic conditions is reductive dechlorination (2, 4, 5, 9, 16, 17). This occurs in a sequential fashion with progressively slower rates as the number of Cl atoms per molecule decreases (17). There is also evidence for the conversion of these compounds to CO2 under anaerobic conditions (2-4, 16, 17). Although haloaliphatic compounds are clearly metabolized in anaerobic habitats, relatively few reports have appeared on the anaerobic metabolism of these compounds in pure cultures. Belay and Daniels (1) found that both reductive dehalogenation and dehydrohalogenation (elimination of hydrogen halides) had occurred in Methanobacterium and Methanococcus cultures incubated with haloethanes and -ethenes. Egli et al. (5, 6) reported dechlorination of carbon tetrachloride (tetrachloromethane) by Desulfobacterium sp., Methanobacterium sp., and Acetobacterium woodii and proved that the latter organism produced [14C]acetate and 14CO2 from [14C]carbon tetrachloride. In previous experiments with Methanosarcina sp. strain DCM and Methanosarcina mazei S6, Fathepure et al. (10) and Fathepure and Boyd (8, 9) found that both strains were able to reductively dechlorinate tetrachloroethene to trichloroethene. In the present work, we used the same organisms to examine reductive dehalogenation of halomethanes. The procedure used to isolate Methanosarcina sp. strain DCM and aspects of its characterization and taxonomy have been reported previously (9). M. mazei S6 was obtained from R. A. Mah (11). Both strains were maintained on methanol or acetate (25 mM) in modified PREM medium (7) without yeast extract and with additional calcium and magnesium salts (25 mg of CaCl2 per liter and 15 mg of MgCl2 per liter). Incubations were performed in anaerobic culture tubes (27 ml containing 10 ml of growth medium) or, in experiments with 14CHCl3, serum bottles (160 ml containing 50 ml of medium) which were prepared by using standard anaerobic culture techniques (13) and sealed with butyl rubber stoppers and aluminum crimp seals. In all cases, the vessels * Corresponding author. t Present address: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-0620.
1198
flushed with N2-CO2 (4:1) by using a Hungate appara(13). Cultures were incubated statically in the dark at 37°C, and growth was monitored by measurement of CH4 in a sample of headspace gas. Haloforms were added either with the inoculum (growth phase 1) or with a second addition of the primary substrate (growth phase 2). For experiments in growth phase 2, when the primary substrate was consumed and CH4 production leveled off (10 days), the headspace was flushed, the culture was refed with methanol (25 mM), and the halomethane was added with a microliter syringe. For experiments in growth phase 1, chloroform and methanol (25 mM) were added to each serum tube or bottle just before inoculation (2%, vol/vol) with a logarithmicphase culture. Killed controls consisted of autoclaved time zero cultures for each set (phases 1 and 2) of experiments; the concentration of carbon tetrachloride and chloroform decreased by 5 to 7% in these controls (93 to 95% recovery), which contained the same time zero cell densities as the experimental bottles. After addition of halomethane, regardless of the culture growth phase, the vessels were sealed with autoclaved Teflon-lined rubber stoppers (The West Co., Phoenixville, Pa.) and aluminum crimps. A series of replicate vessels were incubated along with each experiment so that CH4 production could be monitored without piercing the were
tus
Teflon septa of the experimental bottles. Triplicate bottles were sacrificed for each time point and each treatment because subsamples could not be taken without subsequent loss of halomethane through the pierced septum. In all of the experiments reported here, the standard deviation of the reported mean values was less than 10% unless otherwise indicated. The Teflon-coated stoppers were impermeable to 02, as shown by the maintenance of reduced conditions (decolorized resazurin) in uninoculated vessels for 3 weeks
or more.
Chloroform labeled with 14C was obtained from Dupont, NEN Research Products, Boston, Mass.; the specific activity of the material was 2.98 mCi/mmol (110 MBq/mmol). After growth of 50-ml cultures on 25 mM methanol, approximately 0.5 ,xmol (1.49 ,uCi) of ['4C]chloroform was added with a second addition of methanol (25 mM). The amount of radioactivity present as CO2 and CH4 was determined in duplicate bottles after completion of growth phase 2. Methane and CO2 were separated by gas chromatography and trapped in separate vials of scintillation cocktail (ScintiVerse II [Fisher Scientific Co., Pittsburgh, Pa.] and Carbon 14 Cocktail [R. J. Harvey Instruments Corp.] for CH4 and CO2, respectively). Recovery of 14CH4 and 14CO2 standards was
VOL. 56, 1990
NOTES
1199
TABLE 1. Effect of chloroform concentration on methanogenesis and dechlorination in growth cycles 1 and 2 of Methanosarcina sp. strain DCMa
0
0
E
0
0
h.
'a
Growth cycle and amt of chloroform added
CS
(nmol)
CH4 formed
(p.mol)
Concn of halomethane remaining (nmol) Methylene chloride Chloroform (dichloromethane)
0
c
0
0
S .0e
LI.
0
2
3
4
5
6
Incubation time, days FIG. 1. Time
course
for dechlorination of chloroform (CF) (10
,uM) and formation of methylene chloride (MC) by growth cycle 2 cultures of Methanosarcina sp. strain DCM.
85 to 90% by this method. Liquid scintillation counting was performed on a TriCarb 1500 liquid scintillation analyzer (Packard Instrument Co., Inc., Rockville, Md.) with external-standard quench correction. For quantification of CH4, a sample of headspace gas was injected onto a Porapak Q (50/80 mesh) column (1.83 m by 0.64 cm [inside diameter]); detection was by flame ionization, except for separation of CH4 and CO2 before scintillation counting, for which a nondestructive microthermistor detector was used. In each case, the column was kept at 35°C and the carrier gas (nitrogen) was set at 50 ml/min. The halomethanes were determined by purge and trap gas chromatography. After methane determination, a sample (1.0 ml) of liquid was removed from the culture vessel by using a glass syringe with a Teflon plunger. The sample was placed in a frit-type sparger and purged with helium (grade 5, >99.999% purity) for 12 min on a Tekmar Automatic Laboratory Sampler. The volatile compounds were trapped on a silica gel-Tenax trap contained in an LSC-2 liquid sample concentrator (Tracor Instruments, Austin, Tex.), and the trap was desorbed at 185°C. Separation of sample components occurred on a VOCOL column (60 m by 0.75 mm) (Supelco, Inc., Bellefonte, Pa.) in a Tracor 540 gas chromatograph. The oven temperature was maintained at 45°C for all of the compounds. Chloromethane was not retained by the silica gel-Tenax trap, so direct injection of a headspace sample (0.1 ml) onto the VOCOL column was required. The carrier was grade 5 helium at a flow rate of 10 ml/min. Compounds were detected by a Tracor 700A Hall electrolytic conductivity detector. Recovery of chloroform and methylene chloride from standard solutions prepared by using autoclaved cultures was 93% or greater. Reductive dechlorination of chloroform by growth cycle 2 cultures of Methanosarcina sp. strain DCM is illustrated in Fig. 1. The experiments summarized in Table 1 showed that chloroform was not dechlorinated and inhibited methanogenesis by Methanosarcina sp. strain DCM when added at concentrations of 0.1 to 10 p.M to freshly inoculated medium. However, when chloroform was added to grown cultures which were simultaneously refed with 25 mM methanol (growth cycle 2), chloroform did not inhibit methanogenesis (Table 1) and rapid dechlorination occurred (Fig. 1). Methylene chloride accumulated transiently in the culture, reaching a level of 65% (78% for M. mazei S6) of the added chloroform by day 3. That this was a biological reaction was established by the failure of autoclaved medium and cells to
Cycle 1b 100 10 1 0
Cycle 2c 100 10 1 0
0 5 16 149
93 9.0 1.0
0 0 0
128 123 121 130
5.0 0 0
19 6.0 0
a The cultures were incubated for 10 days for each cycle, and 0.25 mmol of methanol was provided as the primary carbon source at the beginning of each cycle (0 and 10 days). b Chloroform was added at time zero. c Chloroform was added at 10 days.
dechlorinate chloroform. Identical experiments with M. mazei S6 gave essentially the same result as with strain DCM. The absence of dechlorination in freshly inoculated medium of each strain and reduced methanogenesis indicate the toxicity of chloroform to methanogens when the initial ratio of chloroform to cells is high. Strain DCM was more susceptible to chloroform inhibition of methanogenesis during growth phase 1, which was significant at 0.1, 1.0, and 10 ,M chloroform (Table 1); strain S6 showed no effect at 0.1 ,M chloroform and comparatively less at 1.0 and 10 ,uM chloroform (data not shown). No inhibition occurred in growth phase 2 for either strain (Table 1), when the ratio of chloroform to cells was low. This result may be attributed in part to the fact that growth cycle 2 cells are gathered into large clumps that are characteristic of the genus Methanosarcina and may provide some protection. By analysis of the time course shown in Fig. 1, the CH4-normalized dechlorination rate for strain DCM was ca. 0.71 ,umol of chloroform dechlorinated per mmol of CH4 produced (at day 6). This normalized rate is more than an order of magnitude greater than that reported previously for dechlorination of tetrachloroethene by strain DCM; the CH4-normalized rate was 0.05 ,umol of tetrachloroethene dechlorinated per mmol of CH4 produced (9). The product of chloroform dechlorination, methylene chloride, was also degraded, but at a slower rate. The product of methylene chloride dechlorination, chloromethane, was detected consistently at trace levels in these experiments but could not be accurately quantified by our analytical method. Degradation of methylene chloride by mechanisms other than reductive dechlorination (e.g., oxidation through phosgene, COCl2, or alcohols) has been suggested previously, even for methanogenic environments (16). For comparative purposes, strain DCM was incubated with bromoform and carbon tetrachloride in addition to chloroform (Fig. 2). The brominated compound was expected to be dehalogenated at a higher rate on the basis of earlier results obtained with brominated aromatic compounds (12, 14) and on the basis of the lower bond dissociation energies of C-Br than C-Cl bonds. This was not the case, however, and because the amount of CH4 produced by
APPL. ENVIRON. MICROBIOL.
NOTES
1200
TABLE 2. Methanogenesis and chloroform dechlorination by Methanosarcina sp. strain DCM growing on different primary substratesa
CCC14
Mean (SD) halomethane concn remaining (nmol)
Mean (SD) CH4 concn
(SDmol)
0
6
Substrateb
Chloroform
dechlori-
Methylene nated/Lmol
Actual
Chloro- chloride form (dichloro-
Expected
of CH4
(nmol)
methane)
0
44-
None
c
0
0
1
2
3
4
5
6
7
8
Incubation time, days FIG. 2. Biodegradation of carbon tetrachloride, chloroform, and bromoform by Methanosarcina sp. strain DCM.
zero.
An experiment with ['4C]chloroform was performed to observe the formation of 14C-labeled CH4 or CO2 in cultures of the two strains. In repeated experiments, no 14CH4 was detected, although the cultures were actively methanogenic. However, 14CO2 was detected in headspace samples from both strains at about 7% of the radioactivity added. The metabolism of halogenated aliphatic compounds to CO2 in methanogenic systems has already been reported. Vogel and McCarty (17) found that 24% of the [14C]tetrachloroethene they added to a continuous-flow fixed-film methanogenic column was recovered as 14CO2. Bouwer and McCarty (2) reported production of CO2 from carbon tetrachloride and
0
94 (5)
Trc
Methanol 0.10 mmol 0.25 mmol
88 (7) 157 (11)
75 188
55 (4) 30 (2)
28 (2) 64 (4)
0.51 0.60
Methylamine
139 (10)
188
22 (3)
70 (6)
0.56
Dimethylamine 324 (15)
375
11 (1)
78 (7)
0.27
Trimethylamine 472 (18)
562
a
the cultures was about the same regardless of which halomethane was present (data not shown), the data cannot be attributed to inhibition effects. Nor can the relatively slow rate of bromoform debromination be attributed to the reduction potentials for the reaction, since the values reported (16) for dehalogenation are 0.67, 0.56, and 0.61 V, respectively, for carbon tetrachloride, chloroform, and bromoform. The relatively larger size of bromine than chlorine may be manifested in a failure of the compound to mesh effectively with the catalytic apparatus because of steric constraints. The more rapid dechlorination of carbon tetrachloride than chloroform is consistent with the general observation that more highly chlorinated structures are dechlorinated more rapidly, corresponding to their reduction potentials (14). The effects on the chloroform dechlorination rate of (i) differing primary substrate concentrations and (ii) substrates which differ in CH4 yield and energy yield per mole of CH4 (Table 2) were also examined to establish the dependence of dechlorination on CH4 production. The extent of dechlorination was proportional to the amount of CH4 produced, and when normalized for CH4 production, the values were relatively consistent, ca. 0.5 to 0.6 ,umol of chloroform dechlorinated per mmol of CH4. That strain DCM can dechlorinate chloroform (this work) and tetrachloroethene (8, 9) while generating CH4 from a variety of substrates (methanol, methylamines, and acetate) suggests that dechlorination proceeds in nature regardless of the methanogenic substrate used. The values for di- and trimethylamines were somewhat lower (0.27 and 0.21 ,umol dechlorinated per mmol of CH4, respectively), which may reflect a limitation imposed by the chloroform concentration, which was low or
11
0
70 (4) 0.21 The data presented are means of triplicate samples. The data were
collected after 10 days of incubation of strain DCM in growth cycle 2. At the start of the experiment, 10-ml cultures were flushed and refed with substrate, and 100 nmol of chloroform was added. b Methylamine, dimethylamine, and trimethylamine were added at 0.25 mmol per 10 ml. c Tr, Trace.
chloroform in a similar methanogenic column. In neither of these cases nor in any other of which we are aware has 14CH4 been recovered from 14C-labeled halomethanes. The biochemical factors responsible for reductive dehalogenation of halomethanes in methanogenic bacteria have yet to be elucidated. Figure 3 schematically illustrates a possible interpretation of our results; while this scheme is intended merely as a proposal, it is reasonable in light of the results presented here, as well as consistent with data published previously. It is well known that methanol is initially bound by a corrinoid protein in M. barkeri (15), and corrinoid enzymes have been shown to catalyze dehalogenation of halomethanes (18). A similar mechanism has been invoked by Egli et al. (6) to explain the dechlorination of carbon tetrachloride by a number of anaerobic bacteria, notably A. woodii. Presumably, formation of 14CO2 from [14C]chloroform follows the pathway by which CO2 is formed from the disproportionation of methanol.
CH31H4MPT
[Coll MT,-CH3-O - DECHLORINATION
C
-
PRODUCT CHOH, H-M H3H MT2 or [Co'J-MT1co |CHLOROMETH NES1 CH3-S-CoM
O,>CH4
FIG. 3. Speculated pathway for metabolism of chloromethanes to yield dechlorinated products and CO2 in Methanosarcina sp. growing on methanol. Abbreviations: MT1, methanol-5-hydroxybenzimidazolylcobamide methyltransferase; MT2, Co-methylcobamide: HS-CoM methyltransferase; H4MPT, tetrahydromethanopterin; MR, methyl reductase; HS-CoM, coenzyme M (2-mercaptoethanesulfonic acid); CH3-S-CoM, methyl coenzyme M.
VOL. 56, 1990
These results demonstrate that axenic cultures of Methanosarcina spp. reductively dehalogenate chloroform, carbon tetrachloride, and bromoform. The reductive dehalogenation of haloforms observed in nature may result in part from the dehalogenating activity of these methanogens. This work was supported by the U.S. Geological Survey, Department of the Interior, under award 14-08-0001-G1290; the Michigan Agricultural Experiment Station; and the Michigan State University Center for Environmental Toxicology. We are grateful to Babu Fathepure for reviewing the manuscript. LITERATURE CITED 1. Belay, N., and L. Daniels. 1987. Production of ethane, ethylene, and acetylene from halogenated hydrocarbons by methanogenic bacteria. Appl. Environ. Microbiol. 53:1604-1610. 2. Bouwer, E. J., and P. L. McCarty. 1983. Transformations of 1and 2-carbon halogenated organic compounds under methanogenic conditions. Appl. Environ. Microbiol. 45:1286-1294. 3. Bouwer, E. J., and P. L. McCarty. 1983. Transformations of halogenated organic compounds under denitrification conditions. Appl. Environ. Microbiol. 45:1295-1299. 4. Bouwer, E. J., B. E. Rittman, and P. L. McCarty. 1981. Anaerobic degradation of halogenated 1- and 2-carbon organic compounds. Environ. Sci. Technol. 15:596-599. 5. Egli, C., R. Scholtz, A. M. Cook, and T. Leisinger. 1987. Anaerobic dechlorination of tetrachloromethane and 1,2-dichloroethane to degradable products by pure cultures of Desulfobacterium sp. and Methanobacterium sp. FEMS Microbiol. Lett. 43:257-261. 6. Egli, C., T. Tschan, R. Scholtz, A. M. Cook, and T. Leisinger. 1988. Transformation of tetrachloromethane to dichloromethane and carbon dioxide by Acetobacterium woodii. Appl. Environ. Microbiol. 54:2819-2824. 7. Fathepure, B. Z. 1987. Factors affecting the methanogenic activity of Methanothrix soehngenii VNBF. Appl. Environ. Microbiol. 53:2978-2982. 8. Fathepure, F. Z., and S. A. Boyd. 1988. Reductive dechlorination of perchloroethylene and the role of methanogens. FEMS
NOTES
1201
Microbiol. Lett. 49:149-156. 9. Fathepure, B. Z., and S. A. Boyd. 1988. Dependence of tetrachloroethylene dechlorination on methanogenic substrate consumption by Methanosarcina sp. strain DCM. Appl. Environ. Microbiol. 54:2976-2980. 10. Fathepure, B. Z., J. P. Nengu, and S. A. Boyd. 1987. Anaerobic bacteria that dechlorinate perchloroethene. Appl. Environ. Microbiol. 53:2671-2674. 11. Mah, R. A., and D. A. Kuhn. 1984. Transfer of the type species of the genus Methanococcus to the genus Methanosarcina, naming it Methanosarcina mazei (Barker 1936) comb. nov. et emend. and conservation of the genus Methanococcus (Approved Lists 1980) with Methanococcus vannielii (Approved Lists 1980) as the type species. Int. J. Syst. Bacteriol. 34: 263-265. 12. Mikesell, M. D., and S. A. Boyd. 1986. Complete reductive dechlorination and mineralization of pentachlorophenol by anaerobic microorganisms. Appl. Environ. Microbiol. 52:861-865. 13. Miller, T. L., and M. J. Wolin. 1974. A serum bottle modification of the Hungate technique for cultivating obligate anaerobes. Appl. Microbiol. 27:985-987. 14. Tiedje, J. M., S. A. Boyd, and B. Z. Fathepure. 1987. Anaerobic degradation of chlorinated aromatic hydrocarbons. Dev. Ind. Microbiol. 27:117-127. 15. Van der Meijden, P., L. P. J. M. Jansen, C. van der Drift, and G. D. Vogels. 1983. Involvement of corrinoids in the methylation of coenzyme M (2-mercaptoethanesulfonic acid) by methanol and enzymes from Methanosarcina barkeri. FEMS Microbiol. Lett. 19:247-251. 16. Vogel, T. M., C. S. Criddle, and P. L. McCarty. 1987. Transformations of halogenated aliphatic compounds. Environ. Sci. Technol. 21:722-736. 17. Vogel, T. M., and P. L. McCarty. 1985. Biotransformation of tetrachloroethylene to trichloroethylene, dichloroethylene, vinyl chloride, and carbon dioxide under methanogenic conditions. Appl. Environ. Microbiol. 49:1080-1083. 18. Wood, J. M., F. S. Kennedy, and R. S. Wolfe. 1968. The reaction of multihalogenated hydrocarbons with free and bound reduced vitamin B12. Biochemistry 7:1707-1713.
