Role of methanogenic and sulfate-reducing bacteria in the reductive dechlorination of tetrachloroethylene in mixed culture

Full text of DOI:10.1007/s001289900119

Bull. Environ. Contam. Toxicol. (1996) 56:817-824
1996 Springer-Verlag New York Inc.
©
Role of Methanogenic and Sulfate-Reducing Bacteria in
the Reductive Dechlorination of Tetrachloroethylene in
Mixed Culture
1
1
1
2
2
N. Ca b iro l, J. Pe rrie r, F. Ja c ob , B. Fo uille t, P. C ha m b o n
1
Laboratoire de Microbiologie Appliquée et Industrielle, Université Claude
Bernard-Lyon I, Bât. 405, 43, Bd. du 11 Novembre 1918, 69100 Villeurbanne,
France
2
Laboratoire de Toxicologie et d’Hygiène Industrielle, Faculté de Pharmacie, 8,
Av. Rockefeller, 69008 Lyon, France
Received: 24 May 1995/Accepted: 16 November 1995
Tetrachloroethylene (perchloroethylene, PCE) is widely used in many industries and particularly
as a degreasing and dry-cleaning solvent. It is commonly found as a groundwater contaminant
and because of its carcinogenic properties is considered a pollutant. which must be eliminated by
proper treatment (Fawell and Hunt, 1988).
Several reports have demonstrated biotransformation of tetrachloroethylene at low concentrations
under strict anaerobic conditions by sequential reductive dechlorination (Fathepure and Boyd,
1
988a; Freedman and Gosset, 1989). During this biodegradation, trichloroethylene (TCE), 1,1-
dichloroethylene, vinylidene chloride (DCE), and vinyl chloride (VC) are the intermediate
products ; ethene or ethane are the end products (De Bruin et al, 1992; Di Stefano et al, 1992).
Tetrachloroethylene dechlorination was achieved by using methanogenic-enriched cultures
(
Freedman and Gossett, 1989) and in methanogen pure cultures (Fathepure and Boyd, 1988b).
Some authors have also pointed out the ability of sulfate-reducing bacteria to degrade chlorinated
compounds (ie, 3-chlorobenzoate, carbon tetrachloride, 1,2-dichloroethane, tetrachloroethylene)
(Bagley and Gossett, 1990: Egli et al, 1987; Mohn and Tiedje, 1991).
The mixed culture used in this work was isolated from the sludge of an urban waste water
treatment plant. It contains methanogenic, sulfate-reducing, and acetogenic bacteria which have
an interdependant metabolism. Our research deals with the role of the mixed culture in PCE
dechlorination at high concentration, from an ecological point of view. In this study, we
investigated the respective role of sulfate-reducing and methanogenic bacteria in
tetrachloroethylene dechlorination.
MATERIALS AND METHODS
A methanogenic and sulfate-reducing mixed culture was isolated from anaerobic digested sludge
from the Bourg-en-Bresse (France) waste water treatment unit. After several subcultures, the
enrichment was conducted using basal medium modified from Zehnder et al (1980) : KH
.3 g, K H P O 0.3 g, NH Cl 0.3 g, NaCl 0.3 g, CaCl -2H O 0.11 g, MgCl - 6H O 0.10 g,
2
P O
4
0
2
4
4
2
2
2
2
Correspondence to: N. Cabirol
8
17

N a
(
0
2
S e O
FeSO -7H
.05 g. A1K(SO
8
0.03 g, NaHCO
O 0.3 g, H B O
0.05 g, CoCl
3
3.2 g, CH
0.05 g, ZnCl
-6H O 0.05 g. NiCl
3
OH 2.6 g, Yeast extract 1 g, Mineral solution 10 mL
0.05 g, CuCl 0.03 g, MnCl 0.5 g, Na M o O -2H
0.05 g. distilled deionized water qsp 1 L),
4
2
3
3
2
2
2
2
4
2
O
4
)
2
2
2
2
Vitamin solution 10 mL, L-cystein 1.2 g, resazurin, distilled deionized water qsp 1L ; pH 7.5.
Laboratory reactor was a 1-liter semicontinuous anaerobic digester operated at 37°C.
All experiments were conducted in batch conditions with 20 ml-serum vials. Each serum vial
was amended with 50 µM tetrachloroethylene and 5 mL sterile basal medium. and inoculated
with 5 mL of an actively growing mixed culture. The vials were sealed with PTFE-lined rubber
septa and aluminium crimp caps. Incubation was conducted in the dark at 37°C. Each series of
experiments with inoculated vials was accompanied by triplicate sterile basal medium controls
(
10 mL of sterile medium plus the chlorinated compound). At different incubation periods,
triplicates of active and control vials were sacrificed and stored at -20°C for analysis: chlorinated
compounds, biogas production. methanol degradation and biomass production. Biomass
production was determined by the Lowry method for protein quantification.
Both gentamicin and 2-bromoethane sulfonic acid (BESA) inhibitors were separately applied to
the mixed culture to investigate the roles of sulfate-reducing bacteria (SRB) and methanogens in
PCE dechlorination. A series of experiments, prepared as above (with inoculated vials), was
supplemented with 10 mM BESA. BESA-induced inhibition is specific to methanogens because
of the BESA structural similarity to coenzyme M; this latter is unique to methanogens (Daniels et
-
1
al, 1984). Another series of experiments was supplemented with 50 mg
L gentamicin. A
preliminary study was conducted to determine the best inhibitor and its active concentration to
inhibit sulfate-reducing bacteria without affecting methanogens, under our experimental
conditions. Tanimoto et al (1989) screened growth inhibitors of sulfate-reducing bacteria which
did not affect methanogenesis. Some antibiotics were found to be effective and specific to SRB. In
our study, bacitracin and gentamicin were screened at several concentrations. Gentamicin at a
-
1
concentration of 50 mg L was the more suitable inhibitor (data not shown). This antibiotic
binds to eubacterial ribosomes at multiple sites and specifically inhibits the translocation process
(
Gale et al, 1981).
For each measurement of the three series, enumerations of SRB were carried out by the most
probable-number technique (three tubes) in anaerobic conditions. The anaerobic liquid medium
contained the following substrates : Yeast extract 1 g. Sodium lactate 4 g, Ammonium chloride
0
.5 g, K
2
H P O
4
1 g, MgSO
4
0.2 g, CaCl
2
0.1 g, FeSO
4
0.1 g, Na
2
S O 0.5 g, distilled deionized
4
water qsp 1 L plus iron thread (pH 7.2 - 7.4) (Gerhardt et al, 1994). A tube was positive only
when sulfide production could be shown by a black precipitate.
The amounts of PCE, TCE, 1.1 DCE, 1.2 DCE, and VC were determined by direct injection of
headspace samples into a Perkin Elmer model Sigma 2000 gas chromatograph equipped with a
HS 100 autosampler, an electron capture detector and a flame ionization detector. Gas
chromatographic separation was achieved with a fused-silica capillary column, CP Sil 8B
methylsilicone (50 m X 0.23 mm i.d.. 1.2 µm film thickness). The time for thermostated vials in
the headspace analyzer was 60 min at a temperature of 60°C (state of equilibration). The column
gradient temperature was : 60°C (10 min) ; 5°C/min to 120°C ; 120°C (0 min); run-time 22 min;
detectors temperature 300°C; carrier flow (He) 2 mL/min.
The amounts of CH
4
, C
2
H
2
, and CO were determined by a Girdel 330 gas chromatographic
2
analysis of a 1 mL-headspace sample, injected with a gas-tight syringe. into a stainless-steel
8
18

column (2 m X 2 mm id.) packed with Porapak Q operated at 65°C, connected with a
methanation oven (Ni catalytic) and a flame ionization detector. The temperature of the injector
was 100°C. the temperature of the methanation oven 400°C, and the temperature of the detector
1
50°C; carrier pressure (H ) 1 bar. The amount of methanol was determined by the internal
2
standard method (ie mixed culture supernatant and ethanol [ internal standard ] vol/vol). A 1 µL-
mixture was injected into a Girdel 300 gas chromatograph equipped with a flame ionization
detector connected with a stainless-steel column (3 m X 2 mm id.) packed with Porapak Q
8
0/100 mesh operated at 170°C. The temperature of the injector was 200°C the temperature of
the detector 210°C. Carrier gas was N
2
.
Experimental data were statistically studied in order to assess the significant differences between
the three series. PCE dechlorination followed an exponential decrease. The incubation time is
linearly related to the logarithm (1n) of the PCE concentration, Then. the parameters of linear
regression were calculated for the three series : correlation coefficient. regression coefficient and
its 95 % confidence intervals (Table 1). A Student’s t-test was used to compare the methane
production of the three series.
The following compounds were obtained in neat liquid form : ICE (CPG, 99%, Prolabo, Vaulx
en Velin, France), TCE (CPG, 99%, Prolabo, Vaulx en Velin, France). 1,1-DCE (99 %, Aldrich,
Saint Quentin Fallavier, France), vinylidene chloride (98 %, mixture of isomers, Aldrich, Saint
-
1
Quentin Fallavier, France), VC (ampoule 200 µg · mL methanol, Supelco, Saint Germain en
Laye, France). CH , and CO were obtained as mixed gases in reference bottles (Air
4
C
2
H
2
2
Liquide, Lyon, France). PCE was added to cultures from a 5 mM solution in methanol (BP
Normapur, 99.8 %, Prolabo, Vaulx en Velin, France). 2-Bromoethane sulfonic acid (BESA,
Sodium salt) and Gentamicin Sulfate were obtained from Sigma Chemical (Saint Quentin
Fallavier, France).
RESULTS AND DISCUSSION
The PCE completely disappeared within 37 days in a mixed culture without inhibitors (Fig. 1).
Correlatively the formation of TCE was observed. During the first nine days of incubation, the
-
1
PCE concentration decreased at a rate of 3.3 µM . day . The TCE concentration increased at the
same rate. During the 37 days of incubation, the dechlorination rate was 5.2 nmol of degraded
-
1
-1
PCE · mg protein . day . Low levels of DCE and VC were also detected. The whole mass
balance of chlorine (0.43 µmol Cl) was conserved during the experiment
Our results suggest that this mixed culture has the potential to dechlorinate a high concentration
of PCE via reductive dechlorination, as has been previously observed at low concentrations of
PCE in other anaerobic environments (De Bruin et al. 1992; Fathepure and Boyd, 1988a;
Freedman and Gossett, 1989). PCE reductive dechlorination led essentially to TCE. In other
studies conducted by Freedman and Gossett (1989) and De Bruin et al (1992), PCE was
completely degraded into ethene or ethane. But the concentrations of PCE were about ten times
less than the concentration used in our study. High concentrations of PCE could inhibit
methanogenesis (Di Stefano et al, 1991). Then, PCE toxicity could reduce the capacity of
methanogens consortium to degrade TCE. In addition. the culture conditions (batch culture)
could carry limiting factors : De Bruin et al (1992) observed a much slower dechlorination of
PCE in batch cultures than in continuous flow, fixed-bed columns, Further studies could be
carried out to investigate the ability of this mixed culture to dechlorinate TCE.
8
19

Figure 1. PCE dechlorination by the mixed culture without inhibitor (Mean ±SD).
Figure 2. PCE dechlorination by the mixed
Figure 3. Methane production in mixed
culture supplemented with 10 mM BESA
cultures A= Without inhibitor. B= +BESA,
C= +Gentamicin (Mean ±SD).
(
Mean ±SD).
In the mixed culture supplemented with 10 mM BESA, the concentration of PCE appeared to
slightly decrease during the first four days and the dechlorination process stopped soon
afterwards (Fig. 2).
8
20

Figure 4. Hypothetical scheme for enzymatic complex involved in PCE dechlorination
BESA induced almost complete inhibition of methanogens, shown by the lack of methane
production (Fig. 3). On the other hand. sulfate-reducing bacteria were not inhibited by BESA
(Table 2) : their number was higher than the number in the standard mixed culture.
BESA inhibited methanogens because of its structural similarity to coenzyme M. Near total
inhibition of methanogens led to the absence of methane production. The reductive
dechlorination of PCE was nearly completely inhibited in this series of experiments as confirmed
by a regression coefficient near zero (Table 1), demonstrating that methanogens had a deciding
role in the dechlorination process. Fathepure and Boyd (1988a;1988b) proposed a hypothetical
scheme : electrons transferred during methanogenesis are diverted to PCE by an electron transfer
agent which is specific of methanogens and involved in methane formation. Holliger et al
(
1
1992a) observed that corrinoids or factor F430 were responsible for reductive dechlorination of
,2-dichloroethane by Methanosarcina
b a r k e r i . Coenzyme and factor F4 3 0 w e r e
M
indispensable for the methylcoenzyme M reductase activity. Also Holliger et al (1992b)
demonstrated that the methylcoenzyme M reductase of Methanobacterium thermoautotrophicum
DH catalyses the reductive dechlorination of 1.2-dichloroethane into ethene and chloroethane.
Table 1. Linear regression parameters used for modelling PCE dechlorination :
1
n[PCE] = (a X time) + b
Table 2. Enumeration of sulfate-reducing bacteria in mixed cultures with the 95% confidence
limits (Wardlaw, 1985) : Lower 95% confidence limit < Mean < Upper 95% confidence limit
8
21

-
1
Figure 5. PCE dechlorination by the mixed culture supplemented with 50 mg · L gentamicin
(Mean ±SD).
Other enzymatic complexes might also play a part in dechlorination, for instance the methyl
transferase complex which transferes the methyl-group from methanol to coenzyme M, via
corrinoid (Holliger et al, 1992b). The low rate of dechlorination observed might be due to this
methyl transferase complex. But the methyl coenzyme M reductase complex is responsible for the
largest production of ATP. Since the methyl coenzyme M reductase complex is inhibited by
BESA, the bacterial cells can not gain enough energy to support growth. The inhibition of BESA
is then effective (Fig. 4).
Sulfate-reducing bacteria were not inhibited by BESA. The absence of methanogens permitted a
better growth for sulfate-reducing bacteria, showing a competition between the two bacterial
groups. Methanogens were dominant compared to sulfate-reducing bacteria in the standard
mixed culture. The adapted culture medium was favorable to methanogens because of its lack of
sulfates,which are the last acceptor of electrons for sulfate-reducing bacteria.
-
1
PCE was completely degraded into TCE in the mixed culture with 50 mg · L gentamicin (Fig.
-
1
5
). However, during the first nine days, the dechlorination rate of PCE (1.7 µM · day ) was
-1
lower than that calculated in the standard mixed culture (3.3 µM · day ). The observed decrease
is noted too in Table 1 : the regression coefficient was lower than the standard regression
coefficient. For all that, PCE dechlorination was effective : during the 37 days of incubation, the
-
1
-1
dechlorination rate of PCE was 5.08 nmol of degraded PCE mg protein · day , which is
similar to the standard rate.
Inhibition of all sulfate-reducing bacteria was noted (Table 2) when the activity of methanogenic
bacteria was partially and temporarily inhibited (Fig. 3). After a lag time of nine days, the
production of methane was slightly lower with gentamicin than without any inhibitor. Also, the t-
8
22

test gave a hightly significant difference in methane production between this mixed culture and
the standard mixed culture during the first nine days (P = 0.01). The inhibiton of methanogens
was about 50 % after six incubation days and was reduced to 13 % after nine incubation days.
The inhibition of sulfate-reducing bacteria, induced by gentamicin. did not stop the reductive
dechlorination of PCE. So these bacteria did not have a major role in this biotransformation
under our experimental conditions. However, at the beginning of the experiment, the reductive
dechlorination rate was lower than the standard reductive dechlorination rate. In the same way.
methane production was lower than the standard methane production during the first nine days.
This lag time of methanogenesis seems to result from the momentary inhibition of methanogens
by gentamicin. At a high concentration, this antibiotic could be toxic for methanogens (ie, 100
-
1
mg · L ) (Tanimoto et al. 1989). This temporary inhibition would decrease the reductive
dechlorination rate of PCE.
Only the methanogenic bacteria dechlorinate PCE under our experimental conditions, where the
concentration of PCE is ten times higher than the most widely used one. The growth of sulfate-
reducing bacteria increased when methanogens were inhibited by BESA. But since other studies
have shown a positive role of sulfate-reducing and acetogenic bacteria in the dechlorination of
PCE (Bagley and Gossett. 1990: Stromeyer et al. 1992). it is essential to have a better
understanding of the relationships between sulfate-reducing and methanogenic bacteria during
the dechlorination of PCE. We are currently investigating the competitive distribution of the
reducing powers during dechlorination in our consortium. The knowledge of these intergroup
relationships is necessary to optimize the reductive dechlorination of PCE, a carcinogenic
product.
Acknowledgments. We would like to thank Mrs Verner and Mrs Delescluse for their outstanding
technical assistance.
REFERENCES
Bagley DM, Gossett JM (1990) Tetrachloroethene transformation to trichloroethene and cis 1,2
dichloroethene by sulfate-reducing enrichment cultures. Appl Environ Microbiol 56:2511-2516
Daniels L, Sparling R, Sprott GD (1984) The bioenergetics of methanogenesis. Biochim Biophys
Acta 768:113-163
De Bruin WP, Kotterman MJJ, Posthumus MA, Schraa G. Zehnder AJB (1992) Complete
biological reductive transformation of tetrachloroethene to ethane. Appl Environ Microbiol
5
8: 1996-2000
Di Stefano TD, Gossett JM, Zinder SH (1991) Reductive dechlorination of high concentration of
tetrachloroethene to ethene by an anaerobic enrichment culture in the absence of
methanogenesis. Appl Environ Microbiol 57:2287-2292
Di Stefano T, Gossett JM, Zinder SH (1992) Hydrogen as an electron donor for dechlorination of
tetrachloroethene by an anaerobic mixed culture. Appl Environ Microbiol 58:3622-3629
Egli C, Scholtz R, Cook AM, Leisinger T (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
Fathepure BZ, Boyd SA (1988a) Reductive dechlorination of perchloroethylene and the role of
methanogens. FEMS Microbiol Lett 49: 149-156
Fathepure BZ, Boyd SA (1988b) Dependance of tetrachloroethylene dechlorination on
methanogenic substrate consumption by Methanosarcina sp. Strain DCM Appl Environ
8
23

Microbiol 54:2976-2980
Fawell JK, Hunt S (1988) Tetrachloroethylene. In : Environmental toxicology : Organic
pollutants. Ellis Horwood Limited, Chichester
Freedman DL, Gossett JM (1989) Biological reductive dechlorination of tetrachloroethylene and
trichloroethylene to ethylene under methanogenic conditions. Appl Environ Microbiol 55:2144-
2
151
Gale EF, Cundliffe E. Reynolds MH, Richmond MH, Waring MJ (1981) The molecular basis of
antibiotic action. John Wiley&Sons, London
Gerhardt P, Murray RGE, Wood WA, Krieg NR (1994) Methods for general and molecular
bacteriology. American Society for Microbiology, Washington
Holliger C, Schraa G, Stupperich E, Stams AJM, Zehnder AJB (1992a) Evidence for the
involvement of corrinoids and factor F430 in the reductive dechlorination of 1,2 dichloroethane
by Methanosarcina barkeri. J Bacteriol 174:4427-4434
Holliger C, Kengen SWM, Schraa G, Stams AJM, Zehnder AJB (1992b) Methyl-coenzyme M
reductase of Methanobacterium thermoautotrophicum DH catalyses the reductive
dechlorination of 1,2 dichloroethane to ethylene and chloroethane. J. Bacteriol 174:4435-4444
Mohn WW, Tiedje JM (1991) Evidence for chemiosmotic coupling of reductive dechlorination
and ATP synthesis in Desulfomonile tiedje. Arch Microbiol 157: 1-6
Stromeyer SA, Stumpf K, Cook AM, Leisinger T (1992) Anaerobic degradation of
tetrachloromethane by Acetobacterium woodii : separation of dechlorinative activities in cell
extracts and roles for vitamin B12 and other factors. Biodeg 3: 113-123
Tanimoto Y, Tasaki M, Okamma K, Yamaguchi M, Minami K (1989) Screening growth
inhibitors of sulfate-reducing bacteria and their effects on methane fermentation. J Ferment
Bioeng 68:353-359
Wardlaw AC (1985), Practical statistics for experimental biologists. John Wiley&Sons. New
York, p 290
Zehnder AJB, Huser BA, Brock TD, Wurhman K (1980) Characterisation of an acetate
decarboxylating non hydrogen oxidizing methane bacterium. Arch Microbiol 124: 1-11
8
24