FEMS MicrobiologyLetters 90 ( 199212111-2114
c~
~) 1992 Federalionof European MicrobiologicalSocieties11378-1(197/92/$115.1111
Published by Elsevier
FEMSLE 04751
Dehalogenation of trichlorofluoromethane (CFC-11)
by Methanosarcina barkeri
U t e E. K r o n e a n d R u d o l f K. T h a u e r
Lahoratorium filr Mikrobh~h~gie der Phi~ipps-Unicersitiit. Karl-con-Frisch-Str,,ts.~e. 3550 Marburg. F.R.G.
Received t~sOctober 1901
Accepted28 October 1991
Key words: Halomethane degradation; Reductive dehalogenation: Methanogenic bacteria;
Anaerobic archaebacteria; Methanohacterium barkeri
i. S U M M A R Y
Methanobacterium barkeri was found to cat-
alyze the reductive dehalogenation of trich!~3rofluoromethane ( C F C - I I ) , also known as
F R E O N 11. Products detected were CHFCI,,
CH2FCI, CO and fluoride.
2. INTRODUCTION
Trichlorofluoromethane (CFC-II), also known
as F R E O N 11, is a volatile compound that has
been widely used as a foam-blowing agent, as an
aerosol propellant, and as a refrigerant. Because
of its high stability it has accumulated in the
atmosphere, with adverse effects on the ozone
layer [1] and on the climate [2]. It persists in the
atmosphere for as many as 40-45 years [3]. The
Correspondence to: R. Thauer, Laborato~ium fiir Mikrobiolo-
gie der Phillips-Universir;.it, Karl-von-Frisch-Strasse.3550
Marburg, F.R.G.
only major known decomposition of CFC-II appears to be its photolysis in the lower stratosphere [3,4]. We rcport here that CFC-II is
biodegradable by reductive dehalogenation.
It has already been known that many anaerobic bacteria can catalyze the reductive dehalogenation of CCI 4 to CHCI 3, CH2CI 2, and CH3CI
[5-10]. Evidence is available that corrinoids are
involved in the catalytic mechanism [11,12]. This
evidence is based on the two observations that (i)
the ability of bacteria to dehalogenate CCI 4 correlates with the presence of high concentrations
of corrinoids in the microorganisms; and (ii) corrineids such as aquocobalamin catalyze the reduction of CCI 4 to CHCI~, CH2CI 2, CH3CI, and
CO with titanium(liD citrate or with dithiothreitol as electron donor [11,13].
Recently it was found that corrinoids can also
catalyze the reductive dehalogenation of CFC-I 1
[13]. Products detectcd were CO and fluoride in
an 1 to 1 stoichiomctry, and CHFCI 2, CH2FCI,
CH3F, C2F2CI 2, C2F2CI4, and formate in minor
amounts. The product pattern indicates that the
corrinoid-mediated reduction of the chlorofluoro-
2(12
carbon involves the intermediacy of dihalocarbones.
CFCI 3 + 2 [ H ] --, CFCI + 2HCI
( 1)
CFCI + H , O - , C O + H F + HCI
(2)
The above mentioned findings promp!,:d an
investigation to determine w h e t h c r anaerobic
bacteria that can mediate the reductive dehalogenation of CCI 4 can also degrade CFC-I 1. For
our studies we selected Methanosarchta barkeri.
This organism is a strictly anaerobic m e t h a n o g e n i c
archaebacterium which is very a b u n d a n t in anaer-
A
7.0
O
o 6.0
E
7i
,o
obic e n v i r o n m e n t s [14}. Methanosarchm species
are known to have a relatively high dchalogenation capacity [10,1215-17] and to contain high
concentrations of corrinoids [18].
3. M A T E R I A L S A N D M E T H O D S
Methanosarcbta barkeri (strain Fusaro) (DSM
804) was from the Deutsche Sammlung von
Mikroorganismen (Braunschweig, F.R.G.). The
archaebacterium was grown on methanol and
harvested at the end of the exponential phase as
I
O
~
j
1.8
with N2i
I
B
1.2
CHFCl2
g o.6
5.0
-~
c~
~e
0
--~A~t--"-~"
j_CO A
4.0
r
0
20
40
60
0
Time (min)
i
i
,
i
40
20
,
,
60
Time (min)
D
6.0
4.0
CHFCI2
S'
~o 3.0
E
C,HFCI "2
-~
~
a_
4.0
3
/.,"
m 2.0
o
/~Fluoride
/"
/
"
1.0
,~
Fluoride
~
2.0
V
0
0
6
12
Protein ( m g / m l )
6
12
CFC- 11 (/~mol/vial)
Fig. 1. Reductive dehalogenation of CFC-I I by cell suspensions of Me,ham~sarcina barkeri. (A) Rate of CFC-I I ¢ons,~mption: (B)
rate of products formation: (C) protein dependence: (D) CFC-I 1 dependence. For assay conditions see MATERIAI,SAND METI-tODS.
0
2~)3
described [19]. Cell suspensions (5 mg p r o t e i n / m l
or as indicated) were anaerobically prepared in
25 mM imidazole/phosphate pH 7.0 containing 5
mM dithiothreitol, 2 mM MgCI_,, 411 mM NaCI
and 2/.tM resazqrin.
The dehalogenation assays were performed at
37°C in the dark in 120-ml serum vials containing
10 ml cell suspension and 11)0% H e (or 1110% N_,
where indicated) at 1.5 x 105 Pa as gas phase.
The reaction, which was n:n under continuous
shaking at 250 rpm, was started by the addition of
6.7 t~mol CFC-II in methanolic solution (or of
the amounts indicated). At 15-rain time intervals
0.3-ml samples of the head space were withdrawn
and analyzed by gas chromatography [13].
4. RESULTS AND DISCUSSION
Cell suspensions of M. barked grown on
methanol were found to degrade CFC-II in the
presence of H 2 (Fig. IA), which the organism can
use as electron donor via an active hydrogenase
[20,21]. CHFCI z, fluoride, and CO were found as
products (Fig. IB). CH_,FCI was detected in
minute amounts (not shown). The rate of product
formation increased linearly with the protein concentration (Fig. IC) and hyperbolically with the
CFC-11 concentration (Fig. ID). From Fig. ID an
apparent K m for CFC-I1 of approximately 10l~
ppm in the gas phase (6 p.mol per vial) was
estimated. The specific rates (apparent V~,,~)were
100 nmol CFC-11 consumed per h per mg protein, and 66 nmol CHFCI 2, 16 nmol fluoride, and
3 nmol CO formed per h per mg protein.
Carbon monoxide, which is converted to CO2
and H 2 by the organism via an active CO dehydrogenase [22], could substitute for H 2 as electron donor in CFC-I 1 reduction (data not shown).
CFC-11 v, as net degraded in the absence of either H., or CO (Fig. 1A). Cells denatured by
incubation at 100°C for 20 rain were unable to
catalyze the reduction of CFC-I 1 with H 2 or CO.
Th~ specific rate of CFC-II degradation was
half that of CCI 4 reduction to CHCI 3 and twice
that of CHC! 3 reduction to CHzCI 2 by M. barked. Degradation of CFC-12 (diehlorodifluoro-
methane) occurred at less than 5% the rate at
which CFC- 1 I was dehalogenated.
CFC-II completely inhibited mcthanogenesis
in M barked at the concentrations tested in the
dehalogenation experiments. An apparent K, of
51) l~pm in the gas phase was determined. It is
clear that CFC-I I is highly toxic for the organism.
However, at the concentrations prevailing in the
atmosphere (approx. 0.2 ppb) [23], no inhibition
of methanogenesis was observed. Based on the
apparent K m of 1000 ppm the rate of CFC-II
dehalogenation will be very low at these concer,trations. It has yet to be determined whether this
low rate is of biogeochemical significance
Methanogenie soils and sediments have to be
considered as possible sinks. Because of the long
tropospheric lifetime, even minor dehalogenation
rates could have a long-term i~,pact on the
amount of CFC-I 1 that reaches the.stratosphere.
ACKNOWLEDGEMENTS
This work was supported by the Deutsche
Forschungsgemeinschaft and by the Fonds der
Chemischen industrie. We thank Dr. H.C. Friedmann, University of Chicago, and Dr. H.P.C.
Hogenkamp, University of Minnesota, Minneapolis, for helpful suggestions in preparing the
manuscript.
REFERENCES
[1] Anderson, J.G.. Toohey, D.W. and Bruno, W.H. (1991)
Science 251, 39-46.
[2] Fisher, D.A., times, C.It., Wang, W.-C.. Ko, M.K.W.and
Sze, N.D. (1990) Nature 344. 513-516.
[3] Singh, H.B., Sahs, LJ., Shigeishi. tl. and Scribner, E.
(1979) Science 203. 899-903.
[4] Prather. M.J. and Watson, R.T. (19911)Nature 344, 729734.
[5] Bouwcr, E.J. and McCarly, P.L. (19"~3)Appl. Environm.
Microbiol. 8.5. 1286-1294.
[6] Egli,C., Scholtz,R., Cook. A.M, and Leisinger.T (1987)
FEMS Microbiol. Left. 43, 257-261.
[71 Egli, C., Tschan, T., Scholtz, R., Cook. A.M. and
Leisinger, T. (1988) Appl. Envmmm. Microbiol. 54,
28!0-2824.
2(14
[8] Egli, C., Stromeyer, S.. Cook. A.M. and Leisinger, T.
(19t~)) FEMS Microbiol. Left. 68, 2117-212.
[9] Criddlc, C.S,, DeWitt, J.T. and McCarty, P.L. (19901
Appl. Environm. Microbiol. 56, 3247-3254.
[ll}] Mikesell, M.D. and Boyd. S.A. (1990) Appl. Environm.
Microbiol. 56, 1198-12(11.
[11] Krone, U.E., Thauer, R.K. and Hogenkamp, H.P.C.
(It,~89) Biochemistp~ 28, 49118-4914.
[12] Krone, U.E., Laufer, K., Thauer, R.K. and Hegenkamp,
H.P.C. (19891 Biochemistry 28, 111061-101165.
[13] Krone, U.E., Thauer, R.K., Hogenkamp, H.P.C. and
Steinbach, K. (19911 Biochemistry 30, 2713-2719.
[14] Balch, W.E., Fox, G.E., Magrum, L.J., Woese, C.R. and
Wolfe, R.S. (19791 Microbiol. Rev. 43, 260-296.
[15] Fathepure, B.Z., Nengu, J.P. and Boyd, S.A. (19871 Appl.
Environm. Microbiol. 53, 2671-2674.
[16] Fathepure, B.Z. and Boyd, S.A. (19881 FEMS Microbiol.
Left. 49, 149-156.
[17] Fathepure, B.Z. and Boyd, S.A. (198~) .,",ppl. Environm.
Microbiol. 54, 2976-2981).
[18] Gorris, L.G.M., van der Drift, C. and Vogels, G.D. (1988)
J. Microbiol. Methods 8, 175-190.
[19] Karrasch, M., Boll, M. and Thauer, R.K. (1989) Arch.
Microbiol. 151, 137-142.
[20] Fiebig, K. and Friedrieh, B. (1989) Eur. J. Biochem. 184,
79-88.
[21] Schw6rer, B. and Thauer, R.K. (1991) Arch. Microbiol.
155, 459-465.
[22] Bott, M. and Thauer, R.K. (19891 Eur. J. Biochem. 179,
469-472.
[23] Fisher, D.A., Hales, C.H., Filkin, D.L., Ko, M.K.W., Sze,
N.D., Connelt, P.S., Wuebbles, D.J., Isaksen, I.S.A. and
Stordal, F. (19901 Nature 344, 508-512.