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13C12C isotope fractionation of aromatic hydrocarbons during microbial degradation


Environmental Microbiology (1999) 1(5), 409–414

C/ 12C isotope fractionation of aromatic hydrocarbons during microbial degradation
Rainer U. Meckenstock,1* Barbara Morasch,1 Rolf Warthmann,1 Bernhard Schink,1 Eva Annweiler,2 Walter Michaelis2 and Hans H. Richnow2 1 University of Konstanz, Department of Microbial ¨ Ecology, Universitatsstr. 10, D-78457 Konstanz, Germany. 2 University of Hamburg, Institute of Biogeochemistry and Marine Chemistry, Bundesstra?e 55, D-20146 Hamburg, Germany. Summary The in?uence of microbial degradation on the 13C/ 12C isotope composition of aromatic hydrocarbons is presented using toluene as a model compound. Four different toluene-degrading bacterial strains grown in batch culture with oxygen, nitrate, ferric iron or sulphate as electron acceptors were studied as representatives of different environmental redox conditions potentially prevailing in contaminated aquifers. The biological degradation induced isotope shifts in the residual, non-degraded toluene fraction and the kinetic isotope fractionation factors C for toluene degradation by Pseudomonas putida (1.0026 0.00017), Thauera aromatica (1.0017 0.00015), Geobacter metallireducens (1.0018 0.00029) and the sulphate-reducing strain TRM1 (1.0017 0.00016) were in the same range for all four species, although they use at least two different degradation pathways. A similar 13C/ 12C isotope fractionation factor ( C ? 1.0015 0.00015) was observed in situ in a non-sterile soil column in which toluene was degraded under sulphate-reducing conditions. No carbon isotope shifts resulting from soil–hydrocarbon interactions were observed in a non-degrading soil column control with aquifer material under the same conditions. The results imply that microbial degradation of toluene can produce a 13C/ 12C isotope fractionation in the residual hydrocarbon fraction under different environmental conditions. Introduction A reliable characterization of contaminated aquifers and a
Received 25 February, 1999; accepted 12 May, 1999. *For correspondence. E-mail rainer.meckenstock@uni-konstanz.de; Tel. (?49) 7531 88 4541; Fax (?49) 7531 88 2966.
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prediction of the behaviour of hazardous plumes is possible only if the microbial degradation of contaminants can be quanti?ed. However, no method is available so far that could quantify the in situ degradation in the aquifer directly. Apart from laboratory investigations with soil columns or radioactive tracers, changes in contaminant concentrations can be monitored in situ , but a decrease in the course of the plume can result from manifold reasons, such as dilution, adsorption to soil constituents or microbial transformation (Albrechtsen and Christensen, 1994; Head, 1998). Many biological reactions produce 13C/ 12C isotope fractionation of the substrate. Usually, lighter 12C isotopes are used preferentially, and the naturally occurring 13C (1.11% abundance) is discriminated as, for example, in CO2 ?xation by photosynthetic organisms (O’Leary, 1984). Also, in methane formation from CO2 , the lighter 12CO2 isotope is preferred, resulting in a depletion of 13C in the generated methane up to ?30 to ?80 13C (Games et al ., 1978; Whiticar and Faber, 1985; Krzycki et al ., 1987; Blair and Aller, 1995; Summons et al ., 1998). Consequently, the 13C content in the non-used, residual substrate fraction increases, as observed for degradation of acetate by acetoclastic methanogens (Blair and Carter, 1992; Gelwicks et al ., 1994) and for methane oxidation by methanotrophic bacteria (Lebedew et al ., 1969; Coleman et al ., 1981; Blair and Aller, 1995; Botz et al ., 1996). Preliminary observations indicated that microbial degradation might also imply 13C/ 12C isotope fractionation with higher molecular mass compounds such as alkanes or chlorinated hydrocarbons (Stahl, 1980; Ertl et al ., 1996). Here, we show with toluene as a model substance that microbial degradation of aromatic hydrocarbons can induce a 13C/ 12C isotope fractionation in the remaining substrate fraction in pure cultures and in situ in a non-sterile soil column. Results and discussion Contaminated aquifers often produce a redox gradient with various growth conditions for the microbial community. The degradation processes in different redox zones potentially prevailing in a polluted aquifer were simulated using the aerobic bacterium Pseudomonas putida strain mt-2 (Worsey and Williams, 1975; Ramos et al ., 1997), the denitri?er Thauera aromatica strain K172 (Tschech and Fuchs, 1987), the iron-reducer Geobacter metallireducens (Lovley et al ., 1993) and a sulphate-reducing isolate strain TRM1

410 R. U. Meckenstock et al. (R. Meckenstock, unpublished), which were grown in batch cultures. With decreasing toluene concentration in the medium, the 13C/ 12C isotope composition in the remaining non-degraded toluene fraction shifted signi?cantly to higher values by 6–10‰, indicating an isotope fractionation (Fig. 1A–D). The isotope values were plotted on a graph based on the Rayleigh equation (Rayleigh, 1896; Hoefs, 1997) in order to calculate the individual 13C/ 12C kinetic isotope fractionation factors C, which were 1.0026 0.00017 for P. putida , 1.0017 0.00015 for T. aromatica , 1.0018 0.00029 for G. metallireducens and 1.0017 0.00016 for strain TRM1 (Fig. 2). Although the tested organisms use at least two different toluene degradation pathways (Biegert et al ., 1996; Beller and Spormann, 1997; Heider and Fuchs, 1997; Ramos et al ., 1997), the kinetic isotope fractionation factors were all in the same range. These fractionation factors were lower than those reported for CO2 ?xation by C3 plants (1.030) (O’Leary, 1984), CO2 reduction to CH4 (1.045) (Botz et al ., 1996), CH4 oxidation (1.004–1.020) (Coleman et al ., 1981; Whiticar and Faber, 1985) and acetate cleavage to CH4 and CO2 (1.032) (Gelwicks et al ., 1994), which might result from the different molecular masses of the substrates as well as the biophysical and biochemical characteristics of the enzymes and reactions involved. A non-inoculated ?ask served as a control for non-biological isotope effects that may be caused in a multiphasic

Fig. 1. Bacterial oxidation of toluene as sole carbon source in batch cultures and 13C/ 12C isotope fractionation in the residual, non-degraded substrate fraction. A. Pseudomonas putida mt-2. B. Thauera aromatica . C. Geobacter metallireducens . D. Strain TRM1. E. Sterile control.

1999 Blackwell Science Ltd, Environmental Microbiology, 1, 409–414

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C/ 12C isotope fractionation of aromatic hydrocarbons during microbial degradation 411
degradation generated a toluene gradient along the column that could be sampled at different ports (Fig. 3B). Analysis of the respective toluene isotope ratios revealed a signi?13 C in the residual toluene fraction cant increase of with decreasing concentrations (Fig. 3B), corresponding to an isotope fractionation factor C ? 1.0015 0.00015 for the microbial community present (Fig. 2). Non-biological isotope effects that might be induced by preferential binding of 13C or 12C toluene isotopes to the soil matrix were not observed in a control run with a second soil column ?lled with the same aquifer material in the absence of a terminal electron acceptor. This control experiment seemed to be appropriate because high-performance liquid chromatography (HPLC) with reversedphase columns has been reported to induce signi?cant isotope effects indicative of different isotope equilibria between the liquid eluent and the column matrix (Caimi and Brenna, 1997). If this were the case for toluene in the soil column as well, microbial degradation would induce an isotope shift as one isotope would be used preferentially because of its prevalence in the liquid phase. The substrate was added as a 50 ml medium pulse

Fig. 2. Toluene isotope fractionation by P. putida , T. aromatica , G. metallireducens , strain TRM1, an uninoculated control and a sulphidogenic soil percolation column. 13C isotope values and toluene concentrations were plotted according to eqn 4. The regression coef?cient (r ) for the slope of the regression curve and the error of the isotope fractionation factor C, which is calculated from the slope, are: P. putida (r ? ?0.9918; C ? 1.0026 0.00017); T. aromatica (r ? ?0.983; C ? 1.0017 0.00015); G. metallireducens (r ? ?0.939; C ? 1.0017 0.00037); strain TRM1 (r ? ?0.992; C ? 1.0017 0.00016); the sulphidogenic soil column (r ? ?0.998; C ? 1.0015 0.00015).

system by transitions between the liquid and the gas phase (Fig. 1E). As biodegradation takes place only in the liquid phase, artifacts in the degradation experiments could possibly be caused by isotope fractionation upon diffusion of toluene from the gas phase into the liquid (Henry coef?cient for toluene H (20 C) ? 0.26) or from different 13C/ 12C equilibrium isotope constants of toluene in the liquid and the gas phases. For example, gaseous CO2 and aqueous carbonate are known to differ by ?7.9‰ 13C in isotope equilibrium (O’Leary, 1984). No non-biological isotope effects other than those caused by microbial degradation could be observed in the batch experiments (Fig. 2). A non-sterile percolation column was operated as a microcosm in order to simulate a natural aquifer with anaerobic sulphate-dependent toluene degradation. The column was ?lled with aquifer material from a PAH-contaminated site near Stuttgart, Germany, and was run for several months with a mineral medium containing 350 M toluene and sulphate as electron acceptor. After a lag phase of 20 days, a toluene-degrading microbial community became established, which decreased the concentration at the outlet below the detection limit (1 M) (Fig. 3a). Microbial
1999 Blackwell Science Ltd, Environmental Microbiology, 1, 409–414

Fig. 3. Toluene degradation under sulphate-reducing conditions in a soil percolation column. A. Microbial toluene degradation during the course of the experiment. C/Co depicts the toluene concentration at the outlet divided by the inlet concentration. B. Toluene concentration along the column pro?le and 12C/ 13C toluene isotope fractionation.

412 R. U. Meckenstock et al. (1 mM toluene) at the inlet of the column, followed by continuous operation with medium without substrate, and the isotope signature of toluene at the outlet was monitored. Although toluene was signi?cantly retarded by the soil matrix and migrated four times slower than the carrier water phase (Fig. 4A), no differences between the 13C/ 12C isotope composition at the beginning and the end of the toluene peak eluting from the column could be observed (Fig. 4B). The same result could be shown for different aromatic hydrocarbons, such as naphthalene, benzene and m ,p -xylene (Fig. 4A and B). Thus, isotope fractionation based on liquid–solid phase interactions of the substrate, similar to effects observed in reversed phase chromatography, are probably not relevant in the soil column. The data presented show that microbial degradation is the reason for a shift in the 13C/ 12C isotope composition of the remaining toluene in our laboratory systems. No other variables, such as degradation rates or substrate concentrations, which changed during the course of the batch experiments as a result of cell growth, in?uenced the results (Fig. 2). As in a closed system, isotope fractionation can be described mathematically by the Rayleigh eqn 4, microbial toluene degradation can be calculated in the batch culture or soil column at different time points or distances from the inlet, respectively, based on isotope values, the isotope fractionation factor C and the initial substrate concentration. In a more complex environmental system with less de?ned conditions, such as an aquifer, more parameters, for example dilution with pristine groundwater, diffusion, adsorption, evaporation and temperature, could play an important role. Whereas dilution is neutral to isotope effects in this case, adsorption to soil material could possibly affect the isotope composition over longer migration distances in the plume, which might not be adequately simulated by our short soil columns. Evaporation of BTEX compounds from the water phase did not show kinetic or equilibrium isotope effects in laboratory studies (Slater et al ., 1999), but temperature is certainly in?uencing the isotope fractionation of chemical or enzymatic reactions. However, it has yet to be demonstrated whether 13C/ 12C toluene isotope fractionation resulting from microbial degradation is also taking place in the environment and if a fractionation factor C is similar to the laboratory studies considering temperature and other factors. Nevertheless, there are indications that the carbon or chlorine isotope signatures of some groundwater contaminants change in the course of a plume (Kelley et al., 1997; Sturchio et al ., 1998). Experimental procedures
Geobacter metallireducens GS-15 was obtained from the Deutsche Sammlung von Mikroorganismen (DSMZ). Pseudomonas putida mt-2 (pWWO) was a kind gift from J. R. van der ¨ Meer, Dubendorf, Switzerland, and Thauera aromatica a gift from G. Fuchs, Freiburg, Germany. Strain TRM1 was isolated from a soil column using standard techniques (Widdel and Bak, 1992) under the growth conditions mentioned below. P. putida mt-2 was grown at 30 C on minimal medium M9 (Sambrook et al ., 1989) in half-?lled 120 ml serum bottles sealed with Viton stoppers. The three anaerobic strains were cultivated in carbonate-buffered freshwater medium (Widdel and Bak, 1992) with a 20% CO2 /N2 gas phase and supplemented with either 10 mM NO3?, 50 mM Fe(III) citrate or 3 mM FeSO4 as electron acceptor. After the addition of 0.5 mM toluene, the cultures were allowed to equilibrate overnight before the ?rst sample was taken. Growth of P. putida and T. aromatica was measured as an increase in OD 578 and of G. metallireducens and strain TRM1 by quanti?cation of reduced electron acceptor (Cline, 1969; Stookey, 1970). Decreasing toluene concentrations in the control experiment were generated by repeatedly removing 50% of the medium every 1.5 h and immediate addition of the same volume of toluene-free medium without changing the gas phase. Toluene concentrations were measured by isocratic HPLC on a C18 reversed-phase column after ?vefold dilution of the samples with ethanol. The eluent was 70:30 (v/v) acetonitrile/50 mM ammonium phosphate buffer, pH 2.6. 13C/ 12C isotope ratios were determined by isotope ratio monitoring–gas
1999 Blackwell Science Ltd, Environmental Microbiology, 1, 409–414

Fig. 4. Elution pro?le of a non-degrading control soil column after the addition of a pulse of different aromatic hydrocarbons. A. Closed symbols represent the aromatic hydrocarbon concentrations. B. Open symbols represent the respective 13C/ 12C isotope values.

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C/ 12C isotope fractionation of aromatic hydrocarbons during microbial degradation 413
formation as a means of anaerobic toluene activation by sulfate-reducing strain PRTOL1. Appl Environ Microbiol 63: 3729–3731. 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. Blair, N.E., and Aller, R.C. (1995) Anaerobic methane oxidation on the Amazon shelf. Geochim Cosmochim Acta 59: 3707–3715. Blair, N.E., and Carter, W.D. (1992) The carbon isotope biogeochemistry of acetate from a methanogenic marine sediment. Geochim Cosmochim Acta 56: 1247–1258. Botz, R., Pokojski, H.-D., Schmitt, M., and Thomm, M. (1996) Carbon isotope fractionation during bacterial methanogenesis by CO2 reduction. Org Geochem 25: 255–262. Caimi, R.J., and Brenna, J.T. (1997) Quantitative evaluation of carbon isotopic fractionation during reversed-phase high-performance liquid chromatography. J Chromatogr A 757: 307–310. Cline, J.D. (1969) Spectrophotometric determination of hydrogen sul?de in natural waters. Limnol Oceanogr 14: 454– 458. Coleman, D.D., Risatti, B., and Schoell, M. (1981) Fractionation of carbon and hydrogen isotopes by methane-oxidizing bacteria. Geochim Cosmochim Acta 45: 1033–1037. Ertl, S., Seibel, F., Eichinger, L., Frimmel, F.H., and Kettrup, A. (1996) Determination of the 13C/ 12C isotope ratio of organic compounds for the biological degradation of tetrachloroethene (PCE) and trichloroethene (TCE). Acta Hydrochim Hydrobiol 24: 16–21. Games, L.M., Hayes, J.M., and Gunsalus, R.P. (1978) Methane producing bacteria: natural fractionation of stable carbon isotopes. Geochim Cosmochim Acta 42: 1295–1297. Gelwicks, J.T., Risatti, J.B., and Hayes, J.M. (1994) Carbon isotope effects associated with acetoclastic methanogenesis. Appl Environ Microbiol 60: 467–472. Hayes, J.M., Freeman, K.H., Po, B.N., and Hoham, C.H. (1990) Compound-speci?c isotope analysis. A novel tool for reconstruction of ancient biogeochemical processes. Org Geochem 16: 1115–1128. Head, I.M. (1998) Bioremediation: towards a credible technology. Microbiology 144: 599–608. Heider, J., and Fuchs, G. (1997) Anaerobic metabolism of aromatic compounds. Eur J Biochem 243: 577–596. Hoefs, J. (1997) Stable Isotope Geochemistry. Berlin: Springer-Verlag. Kelley, C.A., Hammer, B.T., and Cof?n, R.B. (1997) Concentrations and stable isotope values of BTEX in gasolinecontaminated groundwater. Environ Sci Technol 31: 2469–2472. Krzycki, J.A., Kenealy, W.R., DeNiro, M.J., and Zeikus, J.G. (1987) Stable isotope fractionation by Methanosarcina barkeri during methanogenesis from acetate, methanol, or carbon dioxide-hydrogen. Appl Environ Microbiol 53: 2597–2599. Lebedew, W.C., Owsjannikow, W.M., Mogilewskij, G.A., and Bogdanow, W.M. (1969) Fraktionierung der Kohlenstof?sotope durch mikrobiologische Prozesse in der biochemischen Zone. Angew Geol 15: 621–624.

chromatography–mass spectrometry (IRM–GC–MS) (Hayes et al ., 1990; Merrit et al ., 1994) on a gas chromatograph interfaced to a Finnigan Mat combustion device with a water removal assembly. The combustion line was coupled to a Finnigan Mat 252 mass spectrometer. The IRM-GC-MS was controlled by the ISODAT software (Finnigan Mat). Toluene was extracted from water samples (1–5 ml) with 1 ml of pentane, and 1 l of the pentane phase was used for IRM-GCMS analysis. Microcosm experiments were performed with soil taken from a BTEX-contaminated aquifer near Stuttgart, Germany, at 8 m depth. Soil-?lled glass columns (5 × 45 cm) were run bottom to top with carbonate-buffered freshwater medium (Widdel and Bak, 1992) supplemented with 3 mM FeSO4 . A ?ow of 0.25 column volumes per day was adjusted with peristaltic pumps (Minipuls 3; Gilson), and toluene was added continuously in a mixing chamber with a SP 220i infusion pump (World Precision Instruments) to a ?nal concentration of 350 M. Chemicals were from Fluka.

Calculations
C values [‰ ] [Pee Dee Belemnite (PDB) standard] were calculated with eqn 1. R is the isotope ratio 13C/ 12C.
13 t C ?‰?
13

? ?13 C=12 C sample ?13 C=12 C standard?= ?13 C=12 C standard? × 1000 ? ?Rt =R Std ? 1? × 1000 ?1? ?2? ?3? ?4?

Rt =R0 ? ?

t

? 1000?=?

0

? 1000?

Rt =R0 ? ?Ct =C0 ??1=

C?1?

ln?Rt =R0 ? ? ?1= C ? 1? × ln?Ct =C0 ?

The kinetic isotope fractionation factors C were calculated using eqn 4, which is derived from the Rayleigh equation for a closed system (eqn 3) (Rayleigh, 1896; Hoefs, 1997). t is the 13C/ 12C isotope signature at time t ; 0 is the initial isotope signature of the substrate; and Ct / C 0 is the fraction of substrate remaining in the sample at time t . If ln(Rt / R 0 ) is plotted over ln (Ct / C 0 ) for the time intervals t , the slope of the linear regression curve gives the kinetic isotope fractionation factor C as (1/ C–1).

Acknowledgements
We are grateful to Dr Pierre Albrecht and Patrick Wehrung for providing facilities and technical assistance for the IRM-GCMS measurements. This paper represents publication no. 67 of the priority program 546 ‘Geochemical processes with long-term effects in anthropogenically affected seepage- and groundwater’ by the Deutsche Forschungsgemeinschaft, Bonn – Bad Godesberg.

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1999 Blackwell Science Ltd, Environmental Microbiology, 1, 409–414


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