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Fermentative toluene degradation in anaerobic defined syntrophic cocultures
FEMS Microbiology Letters 177 (1999) 67^73
Fermentative toluene degradation in anaerobic de¢ned syntrophic cocultures Rainer U. Meckenstock * ë kologie, Universita«tsstr. 10, 78457 Konstanz, Germany Universita«t Konstanz, Lehrstuhl fu«r Mikrobielle O Received 22 March 1999; received in revised form 28 May 1999; accepted 28 May 1999
Abstract A syntrophic coculture of a new sulfate-reducing isolate, strain TRM1, with Wolinella succinogenes degraded toluene with either fumarate or NO3 3 as the terminal electron acceptor. Neither strain TRM1 nor W. succinogenes could metabolise toluene under these conditions in pure culture. Syntrophic degradation was 2^3 times slower than toluene utilisation by strain TRM1 in pure culture with sulfate as electron acceptor. The culture did not produce benzoate or fatty acids like acetate or propionate in detectable amounts. An increase in biomass of the syntrophic toluene-degrading culture was shown in a growth curve with nitrate as the terminal electron acceptor. Both partner organisms were detected microscopically at the end of the growth experiment. Syntrophic degradation of toluene with W. succinogenes and fumarate as the terminal electron acceptor was also demonstrated with the iron reducer Geobacter metallireducens. The results provide the first example of a fermentative oxidation of an aromatic hydrocarbon in a defined coculture. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Biodegradation; BTEX contaminant; Bioremediation; Syntrophy; Methanogenesis
1. Introduction Aromatic hydrocarbons like benzene, toluene, ethylbenzene, and xylene (BTEX) or polyaromatic hydrocarbons (PAH) are among the most prominent groundwater contaminants. BTEX compounds and low molecular mass PAH are biodegradable in the presence of oxygen by bacteria or fungi whereas anaerobic degradation of aromatic hydrocarbons was found only recently [1^3]. E¡orts have been undertaken to isolate pure bacterial strains capable
* Tel.: +49 (7531) 88 4541; Fax: +49 (7531) 88 2966; E-mail: [email protected]
of anaerobic oxidation of aromatic hydrocarbons, but only a few pure cultures exist today. Apart from denitrifying and sulfate-reducing strains which can oxidise ethylbenzene or trimethylbenzene [4,5], most of the existing pure cultures can grow with toluene or xylene and either nitrate, sulfate, iron or manganese as electron acceptor [6^8]. Toluene appears to be the aromatic hydrocarbon which is most easily degraded by anaerobic bacteria, and the degradation pathway has been investigated in detail for the denitrifyer Thauera aromatica and the sulfate-reducing strain PRTOL1 [9^12]. Fermentative degradation of toluene and even benzene could be observed in microcosm studies, but the responsible organisms could neither be en-
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 2 9 0 - 6
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riched nor isolated [13,14]. Toluene cannot be degraded by disproportionation to organic acids and hydrogen under standard conditions because the reaction is endergonic [15,16]. Thus, fermentative degradation of toluene needs a hydrogen-accepting partner organism which reduces the hydrogen partial pressure to values that allow a negative Gibbs free energy vG for the ¢rst oxidation reaction. Such syntrophic cooperation has been shown in methanogenic degradation of compounds such as acetate, propionate or benzoate which cannot be degraded by fermenting bacteria in pure culture [16]. We report here on the fermentative degradation of toluene by de¢ned syntrophic cocultures. It is shown that toluene can be used as the sole carbon and energy source by a sulfate-reducing bacterium strain TRM1 or by Geobacter metallireducens, in cooperation with Wolinella succinogenes as electron-accepting partner organism. To our knowledge, this is the ¢rst report on a de¢ned fermenting coculture degrading an aromatic hydrocarbon.
2. Materials and methods 2.1. Isolation of strain TRM1 The sulfate-reducing strain TRM1 was enriched from a soil percolation column ¢lled with material from a BTEX-contaminated aquifer near Stuttgart, Germany. The column was run for 8 months with carbonate-bu¡ered freshwater medium, pH 7.2^7.4 [17], supplemented with 3 mM FeCl2 as reducing agent, benzene, toluene, and xylene as carbon source (100 WM each), and 3 mM sulfate as electron acceptor. After toluene degradation has started, 1 ml samples were taken from the column as an inoculum for further enrichments with the same medium supplemented with 0.5 mM toluene. Strain TRM1 was puri¢ed by serial agar dilution according to standard procedures [18]. 2.2. Organisms and growth conditions W. succinogenes (DSM 1740) and G. metallireducens GS-15 (DSM 7210) were obtained from the Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany. All strains were grown at
30³C in half-¢lled 100 ml serum bottles sealed with viton rubber stoppers (Maag Technik, Du«bendorf, Switzerland). For W. succinogenes the carbonatebu¡ered freshwater medium, pH 7.2^7.4 [17], was reduced with sul¢de (1 mM) and supplemented with the electron acceptors fumarate (40 mM) or NaNO3 (10 mM). Strain TRM1 was supplied with 10 mM Na2 SO4 as electron acceptor and 3 mM FeCl2 to scavenge produced sul¢de. G. metallireducens was grown in the same freshwater medium, pH 7.2^7.4, supplemented with 50 mM ferric citrate as electron acceptor. Toluene was added as a carbon source for strain TRM1 and G. metallireducens to 0.5 mM ¢nal concentration and W. succinogenes was grown with 20 mM formate. 2.3. Syntrophic toluene degradation For syntrophic toluene degradation, strain TRM1 or G. metallireducens was grown in 200 ml volumes with toluene as the sole carbon and energy source (see Section 2.2) and harvested in the stationary growth phase by centrifugation (30 min, 10 000Ug). Cell pellets from 200 ml volume were resuspended in 10 ml freshwater medium, pH 7.2, without electron acceptor, and 2 ml aliquots of the dense cell suspension were added to a 100 ml serum bottle with 25 ml freshwater medium, pH 7.2, without electron acceptor which was reduced with 1 mM Na2 S and 3 mM FeCl2 . A stationary phase culture of W. succinogenes (25 ml) and the electron acceptors fumarate (40 mM) or NaNO3 (2 mM) were added, the bottle was £ushed with CO2 /N2 (20/80), and closed with a viton stopper. Toluene was added from a 2 mM stock solution in the same medium without electron acceptor to a ¢nal concentration of 70^100 WM, and cells were incubated at 30³C. In controls where one of the two partner organisms was omitted, the respective amount of medium was added to the culture. Growth of a coculture of strain TRM1 and W. succinogenes was monitored in 20 ml tubes. In order to prevent high toluene concentrations which might be toxic to W. succinogenes the cells were grown in the presence of Amberlite XAD-7 (Fluka, Buchs, Switzerland). XAD-7 was carefully washed ¢ve times with ethanol p.a. and ¢ve times with distilled water, and dried at 90³C for 5 days. 0.1 gram XAD-7 was
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added before autoclaving the tubes. The tubes were ¢lled with 10 ml carbonate-bu¡ered freshwater medium, pH 7.2, which was reduced with sodium sul¢de (1 mM) and FeCl2 (3 mM). Five mM NaNO3 were added as electron acceptor and the tubes £ushed with N2 /CO2 (80/20). Two Wl toluene were added with a syringe through the stopper and the system was allowed to equilibrate for 3^4 days. For the inoculum strain TRM1 was grown in a preculture (see above) with an excess of toluene (1 mM) and a limiting amount of Na2 SO4 (1 mM). One ml of the sulfate-depleted culture of strain TRM1 or 1 ml W. succinogenes culture pregrown with 20 mM formate, 1 mM acetate, and 10 mM NaNO3 was inoculated to the respective tubes. 2.4. Analytical procedures For toluene quanti¢cation, 250 Wl samples were withdrawn from the medium with a syringe through the stopper and mixed with 1 ml of 99.8% ethanol.
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After centrifugation (5 min, 14 000Ug) the samples were analysed on a Beckman HPLC system Gold with a C18 reversed phase column and isocratic acetonitril/ammonium phosphate bu¡er (50 mM, pH 3.5) (70/30) eluent by monitoring UV absorption at 206 nm. Samples for nitrate quanti¢cation were diluted 10-fold with water and analysed via UV absorption at 220 nm on the same HPLC system equipped with a Grom-Sil anion exchange column that was run with methanol/30 mM KCl (70/30). Fatty acids were analysed from undiluted samples on a BioRad Aminex HPX-87H column with a refraction index detector (Erma, Tokyo, Japan) and 5 mM sulfuric acid as eluent. Bacterial growth was monitored either by optical density measurement at 578 nm in 20 ml test tubes with a Bausch and Lomb spectrophotometer or substrate turnover by quanti¢cation of produced sul¢de or ferrous iron respectively [19,20].
3. Results 3.1. Growth of strain TRM1 and G. metallireducens with toluene A sulfate-reducing isolate, strain TRM1, grew with toluene as sole carbon and energy source optimally at 30³C, pH 7.4, with a doubling time of 36 h. The electron balance showed 95% recovery of electrons from toluene in the produced sul¢de, indicating complete oxidation of toluene to CO2 (Fig. 1A). Fatty acids or benzoate were not produced and the residual toluene concentration was below the detection limit of our HPLC analysis (1 WM). G. metallireducens grew with toluene and ferric iron with an electron recovery of 87% in the produced Fe(II) (Fig. 1B) [8]. 3.2. Toluene degradation by syntrophic cocultures
Fig. 1. Anaerobic toluene degradation by (A) strain TRM1 with sulfate and (B) by G. metallireducens with ferric citrate as electron acceptor.
A mixed cell suspension of the sulfate-reducing strain TRM1 with W. succinogenes was tested for syntrophic toluene utilisation with fumarate as electron acceptor (Fig. 2A). Toluene degradation started without a lag phase and the toluene concentration decreased below the detection limit (1 WM) within 8 days. Microscopic examination of the coculture
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after 8 days showed that both cell types were present and W. succinogenes was highly motile. Substrate depletion was 2^3 times slower than the positive control with the same amount of TRM1 cells in the presence of 10 mM Na2 SO4 as electron acceptor, in the absence of W. succinogenes. Neither the negative control with strain TRM1 plus fumarate but no W. succinogenes cells added nor that with W. succinogenes alone with fumarate showed any toluene degradation. A mixed cell suspension of strain TRM1 and W. succinogenes was also able to degrade toluene syntrophically with NO3 3 as electron acceptor (Fig. 2B). The toluene utilisation rate was comparable to that of the mixture of strain TRM1 and W. succinogenes with fumarate as electron acceptor and increased after a second toluene addition. W. succinogenes cells were still motile after 8 days of growth, and the toluene concentration decreased below the detection limit. With strain TRM1 and NO3 3 in the absence of W. succinogenes, the toluene concentration remained constant. An electron balance for syntrophic toluene degradation by the mixed cell suspension of strain TRM1 and W. succinogenes was performed with nitrate as the terminal electron acceptor. Toluene was completely oxidised and 121% of the electrons were recovered in reduced nitrate and 98% in produced ammonia respectively. Mixed cell suspensions of G. metallireducens and W. succinogenes degraded toluene syntrophically with fumarate as electron acceptor, too (Fig. 2C). Toluene depletion by this coculture was as fast as by the positive control with ferric citrate as electron acceptor. No toluene utilisation was observed with G. metallireducens and fumarate alone. Fig. 2. Syntrophic degradation of toluene by cell suspensions of A: (F), strain TRM1 in syntrophic cooperation with W. succinogenes and nitrate as electron acceptor ; (8), strain TRM1 with nitrate and no partner organism ; and (R), positive control with strain TRM1 with sulfate. B: (F), strain TRM1 together with W. succinogenes and fumarate as electron acceptor ; (8), strain TRM1 with fumarate and no partner organism. C: (F), G. metallireducens in syntrophic cooperation with W. succinogenes and fumarate as electron acceptor ; (8), G. metallireducens with fumarate and no partner organism ; (b), W. succinogenes with fumarate only; and (R), G. metallireducens with ferric citrate.
3.3. Growth of the syntrophic coculture Formation of cell material was analysed in a 10 ml growth experiment inoculated with 1 ml washed cell suspension of strain TRM1 and W. succinogenes respectively and nitrate as electron acceptor (Fig. 3). During the ¢rst 3 days, the optical density decreased due to precipitation of Fe(II), most likely in the form of FeCO3 and FeS. After a lag phase of 15 days signi¢cant increase in optical density was observed in the coculture with nitrate as electron acceptor. The controls with only one of the two organisms
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Fig. 3. Toluene-dependent growth of strain TRM1 in syntrophic cooperation with W. succinogenes with nitrate as terminal electron acceptor. (F), strain TRM1 with W. succinogenes plus toluene and nitrate; (8), strain TRM1 with toluene and nitrate only; and (b), W. succinogenes with toluene and nitrate only.
plus toluene and nitrate did not show an increase in optical density. After the cells of the coculture entered the stationary phase both types of bacteria could be detected microscopically. W. succinogenes could be detected only in very small numbers because the organism tends to stick to iron minerals and was precipitated. Nevertheless, some W. succinogenes cells were free swimming and highly motile. The coculture could be transferred several times indicating cells growth.
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most common electron shuttle system in anoxic environments [16]. Hydrogen serves as a growth substrate for W. succinogenes and TRM1 and we could measure hydrogenase activity in crude cell extracts of all three organisms [21] (data not shown). Nevertheless, it has been shown for G. metallireducens and W. succinogenes that syntrophic acetate degradation was stimulated by addition of the humic acid analogue anthraquinone-2,6-disulfonate (AQDS) indicating an electron shuttle function [22]. Other authors reported on a possible electron shuttle function of extracellular cytochromes [23,24]. Toluene conversion to CO2 and H2 is an endergonic reaction under standard conditions (Eq. 1) but the reaction becomes exergonic if coupled to fumarate (Eq. 2) or nitrate reduction (Eq. 3). C7 H8 14H2 O ! 7CO2 18H2 v G0 0 504:1 kJ mol31
1
C7 H8 14H2 O 18fumarate ! 7CO2 18succinate v G0 0 31044:2 kJ mol31
2
C7 H8 0:5H2 O 4:5NO3 3 9H ! 0 31 7CO2 4:5NH 4 v G0 32195:1 kJ mol
3 4. Discussion Here we report on fermentative degradation of toluene by de¢ned bacterial cultures. Syntrophic cocultures of a new sulfate-reducing bacterium, strain TRM1, or G. metallireducens with W. succinogenes and either fumarate or nitrate as terminal electron acceptor were able to degrade toluene. Neither strain TRM1 nor G. metallireducens degraded toluene in the absence of W. succinogenes because they could not use the added electron acceptor (nitrate or fumarate for strain TRM1, and fumarate for G. metallireducens). Thus, the two toluene-oxidising strains shuttle electrons from toluene to W. succinogenes which can reduce the added electron acceptor. This could be proven by the closed electron balance for toluene oxidation and nitrate reduction by the mixed cell suspension of strain TRM1 and W. succinogenes. The electron carrier could be hydrogen which is the
Nevertheless, in a syntrophic coculture both partner organisms have to gain energy from the reaction to be able to promote growth (Gibbs free energy demand vG for one ATP = 370 kJ mol31 ). The minimal amount of energy which can be used for ATP formation is equivalent to the energy required for the transfer of one single ion across the charged cytoplasmic membrane, corresponding to one third of an ATP (vG = minus;20 kJ mol31 ) [16]. The energy gain for the syntrophic coculture of strain TRM1 and W. succinogenes with nitrate as the terminal electron acceptor is high enough to promote growth which was measured as an increase in optical density. In addition, the coculture could be transferred repeatedly. These results indicate that both organisms can conserve energy from the reaction for generation of cell material. Obviously, W. succinogenes can grow with hydrogen and nitrate,
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but microscopic examination showed signi¢cant growth of strain TRM1 in the syntrophic coculture too indicating that it could gain energy from this process. Syntrophic cooperation has been reported mostly for purely fermenting bacteria that release electrons in the form of hydrogen. Fermentative degradation of aromatic compounds by de¢ned cultures has been demonstrated in a few examples leading to acetate, CO2 and hydrogen as fermentation products [25^27]. Consumption of produced hydrogen by the syntrophic partner organism can make an otherwise endergonic reaction possible as e.g. acetate oxidation by Geobacter sulfureducens in coculture with W. succinogenes or by Clostridium ultunense in coculture with a hydrogenotrophic methanogenic bacterium [22,23,28,29]. However, some sulfate-reducing bacteria such as Desulfovibrio vulgaris, which grows with ethanol and sulfate, can grow in syntrophic coculture with an hydrogen-oxidising methanogenic partner organism in the absence of sulfate [30]. Our ¢nding of syntrophic fermentative oxidation of an aromatic hydrocarbon by sulfate- or iron-reducing organisms expands this concept to more complex substrates. One attempt to identify the prevailing electron-accepting process in contaminated aquifers is the in situ quanti¢cation of bacteria representing metabolic types. Taking aside the well-known di¤culties in obtaining accurate counts of microorganisms, our ¢ndings demonstrate that a certain number of tolueneoxidising bacteria that have been cultivated with sulfate or iron as electron acceptor must not necessarily imply that they reduce the respective electron acceptor in the environment. Rather the organism might cooperate with an hydrogen consuming organism that can use other electron acceptors like nitrate or even CO2 which the toluene-oxidising bacterium could not use itself.
Acknowledgements The author would like to thank Prof. Bernhard Schink for support. This work was ¢nanced partly by the Deutsche Forschungsgemeinschaft and represents publication no. 71 of the priority program 546 `geochemical processes with long-term e¡ects in
anthropogenically a¡ected seepage- and groundwater'.
References [1] Heider, J. and Fuchs, G. (1997) Anaerobic metabolism of aromatic compounds. Eur. J. Biochem. 243, 577^596. [2] Lovley, D.R., Coates, J.D., Woodward, J.C. and Phillips, E.J.P. (1995) Benzene oxidation coupled to sulfate reduction. Appl. Environ. Microbiol. 61, 953^958. [3] Zhang, X. and Young, L.Y. (1997) Carboxylation as an initial reaction in the anaerobic metabolism of naphthalene and phenanthrene by sul¢dogenic consortia. Appl. Environ. Microbiol. 63, 4759^4764. [4] Ball, H.A., Johnson, H.A., Reinhard, M. and Spormann, A.M. (1996) Initial reactions in anaerobic ethylbenzene oxidation by a denitrifying bacterium, strain EB1. J. Bacteriol. 178, 5755^5761. [5] Ha«ner, A., Ho«hener, P. and Zeyer, J. (1997) Degradation of trimethylbenzene isomers by an enrichment culture under N2 O-reducing conditions. Appl. Environ. Microbiol. 63, 1171^1174. [6] Evans, P.J., Mang, D.T., Kim, K.S. and Young, L.Y. (1991) Anaerobic degradation of toluene by a denitrifying bacterium. Appl. Environ. Microbiol. 57, 1139^1145. [7] Rabus, R., Nordhaus, R., Ludwig, W. and Widdel, F. (1993) Complete oxidation of toluene under strictly anoxic conditions by a new sulfate-reducing bacterium. Appl. Environ. Microbiol. 59, 1444^1451. [8] Lovley, D.R. and Lonergan, D.J. (1990) Anaerobic oxidation of toluene, phenol, and p-cresol by the dissimilatory iron-reducing organism, GS-15. Appl. Environ. Microbiol. 56, 1858^ 1864. [9] Seyfried, B., Glod, G., Schocher, R., Tschech, A. and Zeyer, J. (1994) Initial reactions in the anaerobic oxidation of toluene and m-xylene by denitrifying bacteria. Appl. Environ. Microbiol. 60, 4047^4052. [10] Biegert, T., Fuchs, G. and Heider, J. (1996) Evidence that anaerobic oxidation of toluene in the denitrifying bacterium Thauera aromatica is initiated by formation of benzylsuccinate from toluene and fumarate. Eur. J. Biochem. 238, 661^668. [11] Chee-Sanford, J.C., Frost, J.W., Fries, M.R., Zhou, J. and Tiedje, J.M. (1996) Evidence for acetyl coenzyme A and cinnamoyl coenzyme A in the anaerobic toluene mineralization pathway in Azoarcus tolulyticus Tol-4. Appl. Environ. Microbiol. 62, 964^973. [12] Beller, H.R. and Spormann, A.M. (1997) Benzylsuccinate formation as a means of anaerobic toluene activation by sulfatereducing strain PRTOL1. Appl. Environ. Microbiol. 63, 3729^ 3731. [13] Grbic-Galic, D. and Vogel, T.M. (1987) Transformation of toluene and benzene by mixed methanogenic cultures. Appl. Environ. Microbiol. 53, 254^260. [14] Weiner, J.M. and Lovley, D.R. (1998) Rapid benzene degra-
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dation in methanogenic sediments from a petroleum-contaminated aquifer. Appl. Environ. Microbiol. 64, 1937^1939. Thauer, R.K., Jungermann, K. and Decker, K. (1977) Energy conservation in chemotrophic anaerobic bacteria. Bacteriol. Rev. 41, 100^180. Schink, B. (1997) Energetics of syntrophic cooperation in methanogenic degradation. Microbiol. Mol. Biol. Rev. 61, 262^280. Widdel, F., Kohring, G.W. and Mayer, F. (1983) Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids. III. Characterization of the ¢lamentous gliding Desulfonema limicola gen. nov., and Desulfonema magnum sp. nov.. Arch. Microbiol. 134, 286^294. Widdel, F. and Bak, F. (1992) Gram-negative mesophilic sulfate-reducing bacteria. In: The Prokaryotes (Balows, A., Tru«per, H.G., Dworkin, M., Harder, W. and Schleifer, K.H., Eds.), Vol. 4, pp. 3352^3378. Springer, New York. Cline, J.D. (1969) Spectrophotometric determination of hydrogen sul¢de in natural waters. Limnol. Oceanogr. 14, 454^ 458. Stookey, L.L. (1970) Ferrozine - a new spectrophotometric reagent for iron. Anal. Chem. 42, 779^781. Gorby, A.Y. and Lovley, D.R. (1991) Electron transport in the dissimilatory iron reducer, GS-15. Appl. Environ. Microbiol. 57, 867^870. Lovley, D.R., Fraga, J.L., Coates, J.D. and Blunt-Harris, E.L. (1999) Humics as an electron donor for anaerobic respiration. Environ. Microbiol. 1, 89^98. Cord-Ruwisch, R., Lovley, D.R. and Schink, B. (1998) Growth of Geobacter sulfurreducens with acetate in syntrophic
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cooperation with hydrogen-oxidizing anaerobic partners. Appl. Environ. Microbiol. 64, 2232^2236. Seeliger, S., Cord-Ruwisch, R. and Schink, B. (1998) A periplasmic and extracellular c-type cytochrome of Geobacter sulfurreducens acts as a ferric iron reductase and as an electron carrier to other acceptors or to partner bacteria. J. Bacteriol. 180, 3686^3691. Mountfort, D.O. and Bryant, M.P. (1982) Isolation and characterization of an anaerobic syntrophic benzoate-degrading bacterium from sewage sludge. Arch. Microbiol. 133, 249^256. Hopkins, B.T., McInerney, M. and Warikoo, V. (1995) Evidence for an anaerobic syntrophic benzoate degradation threshold and isolation of the syntrophic benzoate degrader. Appl. Environ. Microbiol. 61, 526^530. Wallrabenstein, C., Gorny, N., Springer, N., Ludwig, W. and Schink, B. (1995) Pure culture of Syntrophus buswellii, de¢nition of its phylogenetic status, and description of Syntrophus gentianae sp. nov.. Syst. Appl. Microbiol. 18, 62^66. Schnu«rer, A., Houwen, F.P. and Svensson, B.H. (1994) Mesophilic syntrophic acetate oxidation during methane formation by a triculture at high ammonium concentration. Arch. Microbiol. 162, 70^74. Schnu«rer, A., Schink, B. and Svensson, B.H. (1996) Clostridium ultunense sp. nov., a mesophilic bacterium oxidising acetate in syntrophic association with a hydrogenotrophic methanogenic bacterium. Int. J. Syst. Bacteriol. 46, 1145^1152. Bryant, M.P., Campbell, L.L., Reddy, C.A. and Crabill, M.R. (1977) Growth of Desulfovibrio in lactate or ethanol media low in sulfate in association with H2 -utilizing methanogenic bacteria. Appl. Environ. Microbiol. 33, 1162^1169.
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Fermentative toluene degradation in anaerobic de¢ned syntrophic cocultures Rainer U. Meckenstock * ë kologie, Universita«tsstr. 10, 78457 Konstanz, Germany Universita«t Konstanz, Lehrstuhl fu«r Mikrobielle O Received 22 March 1999; received in revised form 28 May 1999; accepted 28 May 1999
Abstract A syntrophic coculture of a new sulfate-reducing isolate, strain TRM1, with Wolinella succinogenes degraded toluene with either fumarate or NO3 3 as the terminal electron acceptor. Neither strain TRM1 nor W. succinogenes could metabolise toluene under these conditions in pure culture. Syntrophic degradation was 2^3 times slower than toluene utilisation by strain TRM1 in pure culture with sulfate as electron acceptor. The culture did not produce benzoate or fatty acids like acetate or propionate in detectable amounts. An increase in biomass of the syntrophic toluene-degrading culture was shown in a growth curve with nitrate as the terminal electron acceptor. Both partner organisms were detected microscopically at the end of the growth experiment. Syntrophic degradation of toluene with W. succinogenes and fumarate as the terminal electron acceptor was also demonstrated with the iron reducer Geobacter metallireducens. The results provide the first example of a fermentative oxidation of an aromatic hydrocarbon in a defined coculture. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Biodegradation; BTEX contaminant; Bioremediation; Syntrophy; Methanogenesis
1. Introduction Aromatic hydrocarbons like benzene, toluene, ethylbenzene, and xylene (BTEX) or polyaromatic hydrocarbons (PAH) are among the most prominent groundwater contaminants. BTEX compounds and low molecular mass PAH are biodegradable in the presence of oxygen by bacteria or fungi whereas anaerobic degradation of aromatic hydrocarbons was found only recently [1^3]. E¡orts have been undertaken to isolate pure bacterial strains capable
* Tel.: +49 (7531) 88 4541; Fax: +49 (7531) 88 2966; E-mail: [email protected]
of anaerobic oxidation of aromatic hydrocarbons, but only a few pure cultures exist today. Apart from denitrifying and sulfate-reducing strains which can oxidise ethylbenzene or trimethylbenzene [4,5], most of the existing pure cultures can grow with toluene or xylene and either nitrate, sulfate, iron or manganese as electron acceptor [6^8]. Toluene appears to be the aromatic hydrocarbon which is most easily degraded by anaerobic bacteria, and the degradation pathway has been investigated in detail for the denitrifyer Thauera aromatica and the sulfate-reducing strain PRTOL1 [9^12]. Fermentative degradation of toluene and even benzene could be observed in microcosm studies, but the responsible organisms could neither be en-
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 2 9 0 - 6
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riched nor isolated [13,14]. Toluene cannot be degraded by disproportionation to organic acids and hydrogen under standard conditions because the reaction is endergonic [15,16]. Thus, fermentative degradation of toluene needs a hydrogen-accepting partner organism which reduces the hydrogen partial pressure to values that allow a negative Gibbs free energy vG for the ¢rst oxidation reaction. Such syntrophic cooperation has been shown in methanogenic degradation of compounds such as acetate, propionate or benzoate which cannot be degraded by fermenting bacteria in pure culture [16]. We report here on the fermentative degradation of toluene by de¢ned syntrophic cocultures. It is shown that toluene can be used as the sole carbon and energy source by a sulfate-reducing bacterium strain TRM1 or by Geobacter metallireducens, in cooperation with Wolinella succinogenes as electron-accepting partner organism. To our knowledge, this is the ¢rst report on a de¢ned fermenting coculture degrading an aromatic hydrocarbon.
2. Materials and methods 2.1. Isolation of strain TRM1 The sulfate-reducing strain TRM1 was enriched from a soil percolation column ¢lled with material from a BTEX-contaminated aquifer near Stuttgart, Germany. The column was run for 8 months with carbonate-bu¡ered freshwater medium, pH 7.2^7.4 [17], supplemented with 3 mM FeCl2 as reducing agent, benzene, toluene, and xylene as carbon source (100 WM each), and 3 mM sulfate as electron acceptor. After toluene degradation has started, 1 ml samples were taken from the column as an inoculum for further enrichments with the same medium supplemented with 0.5 mM toluene. Strain TRM1 was puri¢ed by serial agar dilution according to standard procedures [18]. 2.2. Organisms and growth conditions W. succinogenes (DSM 1740) and G. metallireducens GS-15 (DSM 7210) were obtained from the Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany. All strains were grown at
30³C in half-¢lled 100 ml serum bottles sealed with viton rubber stoppers (Maag Technik, Du«bendorf, Switzerland). For W. succinogenes the carbonatebu¡ered freshwater medium, pH 7.2^7.4 [17], was reduced with sul¢de (1 mM) and supplemented with the electron acceptors fumarate (40 mM) or NaNO3 (10 mM). Strain TRM1 was supplied with 10 mM Na2 SO4 as electron acceptor and 3 mM FeCl2 to scavenge produced sul¢de. G. metallireducens was grown in the same freshwater medium, pH 7.2^7.4, supplemented with 50 mM ferric citrate as electron acceptor. Toluene was added as a carbon source for strain TRM1 and G. metallireducens to 0.5 mM ¢nal concentration and W. succinogenes was grown with 20 mM formate. 2.3. Syntrophic toluene degradation For syntrophic toluene degradation, strain TRM1 or G. metallireducens was grown in 200 ml volumes with toluene as the sole carbon and energy source (see Section 2.2) and harvested in the stationary growth phase by centrifugation (30 min, 10 000Ug). Cell pellets from 200 ml volume were resuspended in 10 ml freshwater medium, pH 7.2, without electron acceptor, and 2 ml aliquots of the dense cell suspension were added to a 100 ml serum bottle with 25 ml freshwater medium, pH 7.2, without electron acceptor which was reduced with 1 mM Na2 S and 3 mM FeCl2 . A stationary phase culture of W. succinogenes (25 ml) and the electron acceptors fumarate (40 mM) or NaNO3 (2 mM) were added, the bottle was £ushed with CO2 /N2 (20/80), and closed with a viton stopper. Toluene was added from a 2 mM stock solution in the same medium without electron acceptor to a ¢nal concentration of 70^100 WM, and cells were incubated at 30³C. In controls where one of the two partner organisms was omitted, the respective amount of medium was added to the culture. Growth of a coculture of strain TRM1 and W. succinogenes was monitored in 20 ml tubes. In order to prevent high toluene concentrations which might be toxic to W. succinogenes the cells were grown in the presence of Amberlite XAD-7 (Fluka, Buchs, Switzerland). XAD-7 was carefully washed ¢ve times with ethanol p.a. and ¢ve times with distilled water, and dried at 90³C for 5 days. 0.1 gram XAD-7 was
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added before autoclaving the tubes. The tubes were ¢lled with 10 ml carbonate-bu¡ered freshwater medium, pH 7.2, which was reduced with sodium sul¢de (1 mM) and FeCl2 (3 mM). Five mM NaNO3 were added as electron acceptor and the tubes £ushed with N2 /CO2 (80/20). Two Wl toluene were added with a syringe through the stopper and the system was allowed to equilibrate for 3^4 days. For the inoculum strain TRM1 was grown in a preculture (see above) with an excess of toluene (1 mM) and a limiting amount of Na2 SO4 (1 mM). One ml of the sulfate-depleted culture of strain TRM1 or 1 ml W. succinogenes culture pregrown with 20 mM formate, 1 mM acetate, and 10 mM NaNO3 was inoculated to the respective tubes. 2.4. Analytical procedures For toluene quanti¢cation, 250 Wl samples were withdrawn from the medium with a syringe through the stopper and mixed with 1 ml of 99.8% ethanol.
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After centrifugation (5 min, 14 000Ug) the samples were analysed on a Beckman HPLC system Gold with a C18 reversed phase column and isocratic acetonitril/ammonium phosphate bu¡er (50 mM, pH 3.5) (70/30) eluent by monitoring UV absorption at 206 nm. Samples for nitrate quanti¢cation were diluted 10-fold with water and analysed via UV absorption at 220 nm on the same HPLC system equipped with a Grom-Sil anion exchange column that was run with methanol/30 mM KCl (70/30). Fatty acids were analysed from undiluted samples on a BioRad Aminex HPX-87H column with a refraction index detector (Erma, Tokyo, Japan) and 5 mM sulfuric acid as eluent. Bacterial growth was monitored either by optical density measurement at 578 nm in 20 ml test tubes with a Bausch and Lomb spectrophotometer or substrate turnover by quanti¢cation of produced sul¢de or ferrous iron respectively [19,20].
3. Results 3.1. Growth of strain TRM1 and G. metallireducens with toluene A sulfate-reducing isolate, strain TRM1, grew with toluene as sole carbon and energy source optimally at 30³C, pH 7.4, with a doubling time of 36 h. The electron balance showed 95% recovery of electrons from toluene in the produced sul¢de, indicating complete oxidation of toluene to CO2 (Fig. 1A). Fatty acids or benzoate were not produced and the residual toluene concentration was below the detection limit of our HPLC analysis (1 WM). G. metallireducens grew with toluene and ferric iron with an electron recovery of 87% in the produced Fe(II) (Fig. 1B) [8]. 3.2. Toluene degradation by syntrophic cocultures
Fig. 1. Anaerobic toluene degradation by (A) strain TRM1 with sulfate and (B) by G. metallireducens with ferric citrate as electron acceptor.
A mixed cell suspension of the sulfate-reducing strain TRM1 with W. succinogenes was tested for syntrophic toluene utilisation with fumarate as electron acceptor (Fig. 2A). Toluene degradation started without a lag phase and the toluene concentration decreased below the detection limit (1 WM) within 8 days. Microscopic examination of the coculture
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after 8 days showed that both cell types were present and W. succinogenes was highly motile. Substrate depletion was 2^3 times slower than the positive control with the same amount of TRM1 cells in the presence of 10 mM Na2 SO4 as electron acceptor, in the absence of W. succinogenes. Neither the negative control with strain TRM1 plus fumarate but no W. succinogenes cells added nor that with W. succinogenes alone with fumarate showed any toluene degradation. A mixed cell suspension of strain TRM1 and W. succinogenes was also able to degrade toluene syntrophically with NO3 3 as electron acceptor (Fig. 2B). The toluene utilisation rate was comparable to that of the mixture of strain TRM1 and W. succinogenes with fumarate as electron acceptor and increased after a second toluene addition. W. succinogenes cells were still motile after 8 days of growth, and the toluene concentration decreased below the detection limit. With strain TRM1 and NO3 3 in the absence of W. succinogenes, the toluene concentration remained constant. An electron balance for syntrophic toluene degradation by the mixed cell suspension of strain TRM1 and W. succinogenes was performed with nitrate as the terminal electron acceptor. Toluene was completely oxidised and 121% of the electrons were recovered in reduced nitrate and 98% in produced ammonia respectively. Mixed cell suspensions of G. metallireducens and W. succinogenes degraded toluene syntrophically with fumarate as electron acceptor, too (Fig. 2C). Toluene depletion by this coculture was as fast as by the positive control with ferric citrate as electron acceptor. No toluene utilisation was observed with G. metallireducens and fumarate alone. Fig. 2. Syntrophic degradation of toluene by cell suspensions of A: (F), strain TRM1 in syntrophic cooperation with W. succinogenes and nitrate as electron acceptor ; (8), strain TRM1 with nitrate and no partner organism ; and (R), positive control with strain TRM1 with sulfate. B: (F), strain TRM1 together with W. succinogenes and fumarate as electron acceptor ; (8), strain TRM1 with fumarate and no partner organism. C: (F), G. metallireducens in syntrophic cooperation with W. succinogenes and fumarate as electron acceptor ; (8), G. metallireducens with fumarate and no partner organism ; (b), W. succinogenes with fumarate only; and (R), G. metallireducens with ferric citrate.
3.3. Growth of the syntrophic coculture Formation of cell material was analysed in a 10 ml growth experiment inoculated with 1 ml washed cell suspension of strain TRM1 and W. succinogenes respectively and nitrate as electron acceptor (Fig. 3). During the ¢rst 3 days, the optical density decreased due to precipitation of Fe(II), most likely in the form of FeCO3 and FeS. After a lag phase of 15 days signi¢cant increase in optical density was observed in the coculture with nitrate as electron acceptor. The controls with only one of the two organisms
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Fig. 3. Toluene-dependent growth of strain TRM1 in syntrophic cooperation with W. succinogenes with nitrate as terminal electron acceptor. (F), strain TRM1 with W. succinogenes plus toluene and nitrate; (8), strain TRM1 with toluene and nitrate only; and (b), W. succinogenes with toluene and nitrate only.
plus toluene and nitrate did not show an increase in optical density. After the cells of the coculture entered the stationary phase both types of bacteria could be detected microscopically. W. succinogenes could be detected only in very small numbers because the organism tends to stick to iron minerals and was precipitated. Nevertheless, some W. succinogenes cells were free swimming and highly motile. The coculture could be transferred several times indicating cells growth.
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most common electron shuttle system in anoxic environments [16]. Hydrogen serves as a growth substrate for W. succinogenes and TRM1 and we could measure hydrogenase activity in crude cell extracts of all three organisms [21] (data not shown). Nevertheless, it has been shown for G. metallireducens and W. succinogenes that syntrophic acetate degradation was stimulated by addition of the humic acid analogue anthraquinone-2,6-disulfonate (AQDS) indicating an electron shuttle function [22]. Other authors reported on a possible electron shuttle function of extracellular cytochromes [23,24]. Toluene conversion to CO2 and H2 is an endergonic reaction under standard conditions (Eq. 1) but the reaction becomes exergonic if coupled to fumarate (Eq. 2) or nitrate reduction (Eq. 3). C7 H8 14H2 O ! 7CO2 18H2 v G0 0 504:1 kJ mol31
1
C7 H8 14H2 O 18fumarate ! 7CO2 18succinate v G0 0 31044:2 kJ mol31
2
C7 H8 0:5H2 O 4:5NO3 3 9H ! 0 31 7CO2 4:5NH 4 v G0 32195:1 kJ mol
3 4. Discussion Here we report on fermentative degradation of toluene by de¢ned bacterial cultures. Syntrophic cocultures of a new sulfate-reducing bacterium, strain TRM1, or G. metallireducens with W. succinogenes and either fumarate or nitrate as terminal electron acceptor were able to degrade toluene. Neither strain TRM1 nor G. metallireducens degraded toluene in the absence of W. succinogenes because they could not use the added electron acceptor (nitrate or fumarate for strain TRM1, and fumarate for G. metallireducens). Thus, the two toluene-oxidising strains shuttle electrons from toluene to W. succinogenes which can reduce the added electron acceptor. This could be proven by the closed electron balance for toluene oxidation and nitrate reduction by the mixed cell suspension of strain TRM1 and W. succinogenes. The electron carrier could be hydrogen which is the
Nevertheless, in a syntrophic coculture both partner organisms have to gain energy from the reaction to be able to promote growth (Gibbs free energy demand vG for one ATP = 370 kJ mol31 ). The minimal amount of energy which can be used for ATP formation is equivalent to the energy required for the transfer of one single ion across the charged cytoplasmic membrane, corresponding to one third of an ATP (vG = minus;20 kJ mol31 ) [16]. The energy gain for the syntrophic coculture of strain TRM1 and W. succinogenes with nitrate as the terminal electron acceptor is high enough to promote growth which was measured as an increase in optical density. In addition, the coculture could be transferred repeatedly. These results indicate that both organisms can conserve energy from the reaction for generation of cell material. Obviously, W. succinogenes can grow with hydrogen and nitrate,
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but microscopic examination showed signi¢cant growth of strain TRM1 in the syntrophic coculture too indicating that it could gain energy from this process. Syntrophic cooperation has been reported mostly for purely fermenting bacteria that release electrons in the form of hydrogen. Fermentative degradation of aromatic compounds by de¢ned cultures has been demonstrated in a few examples leading to acetate, CO2 and hydrogen as fermentation products [25^27]. Consumption of produced hydrogen by the syntrophic partner organism can make an otherwise endergonic reaction possible as e.g. acetate oxidation by Geobacter sulfureducens in coculture with W. succinogenes or by Clostridium ultunense in coculture with a hydrogenotrophic methanogenic bacterium [22,23,28,29]. However, some sulfate-reducing bacteria such as Desulfovibrio vulgaris, which grows with ethanol and sulfate, can grow in syntrophic coculture with an hydrogen-oxidising methanogenic partner organism in the absence of sulfate [30]. Our ¢nding of syntrophic fermentative oxidation of an aromatic hydrocarbon by sulfate- or iron-reducing organisms expands this concept to more complex substrates. One attempt to identify the prevailing electron-accepting process in contaminated aquifers is the in situ quanti¢cation of bacteria representing metabolic types. Taking aside the well-known di¤culties in obtaining accurate counts of microorganisms, our ¢ndings demonstrate that a certain number of tolueneoxidising bacteria that have been cultivated with sulfate or iron as electron acceptor must not necessarily imply that they reduce the respective electron acceptor in the environment. Rather the organism might cooperate with an hydrogen consuming organism that can use other electron acceptors like nitrate or even CO2 which the toluene-oxidising bacterium could not use itself.
Acknowledgements The author would like to thank Prof. Bernhard Schink for support. This work was ¢nanced partly by the Deutsche Forschungsgemeinschaft and represents publication no. 71 of the priority program 546 `geochemical processes with long-term e¡ects in
anthropogenically a¡ected seepage- and groundwater'.
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