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It is frequently observed in aromatic hydrocarbons such as benzene, toluene, ethylbenzene, xylene (BTEX)-contaminated aquifers that toluene degrades faster than xylenes and benzene. In sediment column experiments which were run with a mixture of BTEX compounds toluene degradation started after a lag period of several weeks. When we omitted toluene from the culture medium o-xylene degradation started. Xylene degradation could be inhibited by adding toluene back to the medium and could be recovered when toluene was omitted again. This was observed repeatedly when toluene concentrations higher than 20 microM were added. Two sulphate-reducing bacterial species, isolated from the column material, were used to investigate the degradation behaviour in detail. Strain TRM1 degraded exclusively toluene, strain OX39 degraded preferentially o-xylene and toluene only after an adaptation period of more than 90 days when added as the sole substrate. Growth and o-xylene degradation of strain OX39 were inhibited by toluene concentrations as low as 40 microM, whereas, in contrast, toluene degradation by strain TRM1 was not inhibited by o-xylene concentrations up to 0.5 mM. Both the column data and the batch experiments indicated that two organisms were responsible for the toluene/xylene degradation in the sediment column. One strain degraded only toluene and was not effected by xylene and the second degraded xylene and was inhibited by toluene. Our findings offer an explanation that the observed differential degradation of BTEX compounds in contaminated aquifers could originate from a partial metabolic inhibition of xylene-degrading organisms by toluene.

To evaluate the intrinsic bioremediation potential in an anoxic tar oil-contaminated aquifer at a former gasworks site, groundwater samples were qualitatively and quantitatively analyzed by compound-specific isotope analysis (CSIA) and signature metabolites analysis (SMA). 13C/12C fractionation data revealed conclusive evidence for in situ biodegradation of benzene, toluene, o-xylene, m/p-xylene, naphthalene, and 1-methylnaphthalene. In laboratory growth studies, 13C/12C isotope enrichment factors for anaerobic degradation of naphthalene (epsilon = -1.1 +/- 0.4) and 2-methylnaphthalene (epsilon = -0.9 +/- 0.1) were determined with the sulfate-reducing enrichment culture N47, which was isolated from the investigated test site. On the basis of these and other laboratory-derived enrichment factors from the literature, in situ biodegradation could be quantified for toluene, o-xylene, m/p-xylene, and naphthalene. Stable carbon isotope fractionation in the field was also observed for ethylbenzene, 2-methylnaphthalene, and benzothiophene but without providing conclusive results. Further evidence for the in situ turnover of individual BTEX compounds was provided by the presence of acetophenone, o-toluic acid, and p-toluic acid, three intermediates in the anaerobic degradation of ethylbenzene, o-xylene, and p-xylene, respectively. A number of groundwater samples also contained naphthyl-2-methylsuccinic acid, a metabolite that is highly specific for the anaerobic degradation of 2-methylnaphthalene. Additional metabolites that provided evidence on the anaerobic in situ degradation of naphthalenes were 1-naphthoic acid, 2-naphthoic acid, 1,2,3,4-tetrahydronaphthoic acid, and 5,6,7,8-tetrahydronaphthoic acid. 2-Carboxybenzothiophene, 5-carboxybenzothiophene, a putative further carboxybenzothiophene isomer, and the reduced derivative dihydrocarboxybenzothiophene indicated the anaerobic conversion of the heterocyclic aromatic hydrocarbon benzothiophene. The combined application of CSIA and SMA, as two reliable and independent tools to collect direct evidence on intrinsic bioremediation, leads to a substantially improved evaluation of natural attenuation in situ.

Compound-specific stable isotope analysis (CSIA) using gas chromatography-isotope ratio mass spectrometry (GC/IRMS) has developed into a mature analytical method in many application areas over the last decade. This is in particular true for carbon isotope analysis, whereas measurements of the other elements amenable to CSIA (hydrogen, nitrogen, oxygen) are much less routine. In environmental sciences, successful applications to date include (i) the allocation of contaminant sources on a local, regional, and global scale, (ii) the identification and quantification of (bio)transformation reactions on scales ranging from batch experiments to contaminated field sites, and (iii) the characterization of elementary reaction mechanisms that govern product formation. These three application areas are discussed in detail. The investigated spectrum of compounds comprises mainly n-alkanes, monoaromatics such as benzene and toluene, methyl tert-butyl ether (MTBE), polycyclic aromatic hydrocarbons (PAHs), and chlorinated hydrocarbons such as tetrachloromethane, trichloroethylene, and polychlorinated biphenyls (PCBs). Future research directions are primarily set by the state of the art in analytical instrumentation and method development. Approaches to utilize HPLC separation in CSIA, the enhancement of sensitivity of CSIA to allow field investigations in the microg L(-1) range, and the development of methods for CSIA of other elements are reviewed. Furthermore, an alternative scheme to evaluate isotope data is outlined that would enable estimates of position-specific kinetic isotope effects and, thus, allow one to extract mechanistic chemical and biochemical information.

We present an approach for characterizing in situ microbial degradation using the 13C/12C isotope fractionation of contaminants as an indicator of biodegradation. The 13C/12C isotope fractionation of aromatic hydrocarbons was studied in anoxic laboratory soil percolation columns with toluene or o-xylene as the sole carbon and electron source, and sulfate as electron acceptor. After approximately 2 months' of incubation, the soil microbial community degraded 32 mg toluene l(-1) and 44 mg o-xylene l(-1) to less than 0.05 mg l(-1), generating a stable concentration gradient in the column. The 13C/12C isotope ratio in the residual non-degraded fraction of toluene and o-xylene increased significantly, corresponding to isotope fractionation factors (alphaC) of 1.0015 and 1.0011, respectively. When the extent of biodegradation in the soil column was calculated based on the measured isotope ratios (R(t)) and an isotope fractionation factor (alphaC=1.0017) obtained from a sulfate-reducing batch culture the theoretical residual substrate concentrations (C(t)) matched the measured toluene concentrations in the column. This indicated that a calculation of biodegradation based on isotope fractionation could work in systems like soil columns. In a field study, a polluted, anoxic aquifer was analyzed for BTEX and PAH contaminants. These compounds were found to exhibit a significant concentration gradient along an 800-m groundwater flow path downstream of the source of contamination. A distinct increase in the carbon isotope ratio (delta13C) was observed for the residual non-degraded toluene (7.2 per thousand ), o-xylene (8.1 per thousand ) and naphthalene fractions (1.2 per thousand ). Based on the isotope values and the laboratory-derived isotope fractionation factors for toluene and o-xylene, the extent to which the residual substrate fraction in the monitoring wells had been degraded by microorganisms was calculated. The results revealed significant biodegradation along the groundwater flow path. In the wells at the end of the plume, the bioavailable toluene and o-xylene fractions had been almost completely reduced by in situ microbial degradation. Although indane and indene showed decreasing concentrations downstream of the groundwater flow path, suggesting microbial degradation, their carbon isotope ratios remained constant. As the physical properties of these compounds are similar to those of BTEX compounds, the constant isotope values of indane and indene indicated that microbial degradation did not lead to isotope fractionation of all aromatic hydrocarbons. In addition, physical interaction with the aquifer material during the groundwater passage did not significantly alter the carbon isotope composition of aromatic hydrocarbons.

Stable carbon isotope analysis of tetrachloroethene (PCE) and trichloroethene (TCE) was applied to evaluatenatural attenuation processes in the upper Quaternary and lower Tertiary aquifer in the area of a former dry-cleaning plant located in Leipzig, Germany. Groundwater samples were taken during one monitoring campaign in 2001. The 13C enrichment in contaminants along the water flow path suggested that both, PCE and TCE were degraded in the Quaternary aquifer. The enrichment of 13C in the residual PCE fraction and an isotope fractionation factor from laboratory experiments were used to calculate the extent of biodegradation in the Quatemary aquifer. These calculations indicated that a major portion of PCE was biodegraded in the course of the plume. In the Tertiary aquifer the carbon isotope ratios of PCE and TCE indicated that the decreasing concentrations of these contaminants were probably not caused by microbial processes.

Concentrations and isotopic compositions (13C/12C) of aromatic hydrocarbons were determined in eight samples obtained from the strongly anoxic part of the leachate plume downgradient from the Vejen Landfill (Denmark), where methanogenic, sulfate-reducing and iron-reducing conditions were observed. Despite the heterogeneous distribution of the compounds in the plume, the isotope fractionation proved that ethylbenzene and m/p-xylene were subject to significant biodegradation within the strongly anoxic plume. The isotope fractionation factors (alphaC) for the degradation of the m/p-xylene (1.0015) and ethylbenzene (1.0021) obtained from the field observations were similar to factors previously determined for the anaerobic degradation of toluene and o-xylene in laboratory experiments, and suggest that in situ biodegradation is one major process controlling the fate of these contaminants in this aquifer. The isotope fractionation determined for 1,2,4-trimethylbenzene and 2-ethyltoluene suggested in situ biodegradation; however, the isotopic composition did not correlate well with the respective concentration as expressed by the Rayleigh equation. Some other compounds (1,2,3-trimethylbenzene, o-xylene, naphthalene and fenchone) did not show significant enrichments in delta13C values along the flow path. The compound concentrations were too low for accurate isotope analyses of benzene, toluene, 1- and 2-methylnaphthalene, while interferences in the chromatography made it impossible to evaluate the isotopic composition for 4-ethyltoluene, 1,3,5-trimethylbenzene and camphor. In addition to demonstrating the potential of assessing isotopic fractionation as a means for documenting the in situ biodegradation of complex mixtures of aromatic hydrocarbons in leachate plumes, this study also illustrates the difficulties for data interpretation in complex plumes and high analytical uncertainties for isotope analysis of organic compounds in low concentration ranges.

13C/(12)C and D/H stable isotope fractionation during aerobic degradation was determined for Pseudomonas putida strain mt-2, Pseudomonas putida strain F1, Ralstonia pickettii strain PKO1, and Pseudomonas putida strain NCIB 9816 grown with toluene, xylenes, and naphthalene. Different types of initial reactions used by the respective bacterial strains could be linked with certain extents of stable isotope fractionation during substrate degradation.

A liquid chromatography (LC) method followed by electrospray ionization (ESI) and tandem mass spectrometry (MS-MS) was developed for the quantification of acidic naphthalene derivatives in the concentration range 0.1 to 100 microg/l without excessive sample preparation. For optimal sensitivity the LC-MS-MS measurements were performed recording mass fragmentation by collision induced dissociation in the multiple reaction mode. The collision energy was optimized for every analyte. The matrix effects of the sample were investigated by spiking standards of 1-naphthoic acid with humic acid (HA) and with calcium chloride. While HA decreased the signal intensity an increase was observed in the presence of calcium chloride. For the investigated groundwater samples of a tar oil contaminated site a complete separation of the analytes from the sample matrix by reversed-phase separation could be obtained. The absence of matrix effects on quantification results was confirmed by comparison of results based on external calibration with those based on standard addition of the analytes to a groundwater sample. In four groundwater samples of the contaminated site naphthalene derivatives like 1-naphthoic acid, 2-naphthoic acid, 1-naphthylacetic acid, 2-naphthylacetic acid, 1-hydroxy-2-naphthoic acid, 2-hydroxy-3-naphthoic acid, and naphthyl-2-methylenesuccinic acid have been detected.

A concept to assess in situ biodegradation of organic contaminants in aquifers is presented. The alteration of the carbon isotope composition of contaminants along the groundwater flow path indicates microbial degradation processes and can be used as an indicator for in situ biodegradation. The Rayleigh equation was applied to calculate the percentage of the in situ biodegradation (B[%]) using the change in the isotopic composition of contaminants (Rt/R0) along the ground water flow path and a kinetic carbon isotope fractionation factor (alphaC) derived from defined biodegradation experiments in the laboratory. When the groundwater hydrology is known and a representative source concentration (C0) for a groundwater flow path can be determined, the extent of in situ biodegradation can be quantified.

Anaerobic degradation of naphthalene, 2-methylnaphthalene, and tetralin (1,2,3,4-tetrahydronaphthalene) was investigated with a sulfate-reducing enrichment culture obtained from a contaminated aquifer. Degradation studies with tetralin revealed 5,6,7,8-tetrahydro-2-naphthoic acid as a major metabolite indicating activation by addition of a C(1) unit to tetralin, comparable to the formation of 2-naphthoic acid in anaerobic naphthalene degradation. The activation reaction was specific for the aromatic ring of tetralin; 1,2,3,4-tetrahydro-2-naphthoic acid was not detected. The reduced 2-naphthoic acid derivatives tetrahydro-, octahydro-, and decahydro-2-naphthoic acid were identified consistently in supernatants of cultures grown with either naphthalene, 2-methylnaphthalene, or tetralin. In addition, two common ring cleavage products were identified. Gas chromatography-mass spectrometry (GC-MS) and high-resolution GC-MS analyses revealed a compound with a cyclohexane ring and two carboxylic acid side chains as one of the first ring cleavage products. The elemental composition was C(11)H(16)O(4) (C(11)H(16)O(4)-diacid), indicating that all carbon atoms of the precursor 2-naphthoic acid structure were preserved in this ring cleavage product. According to the mass spectrum, the side chains could be either an acetic acid and a propenic acid, or a carboxy group and a butenic acid side chain. A further ring cleavage product was identified as 2-carboxycyclohexylacetic acid and was assumed to be formed by beta-oxidation of one of the side chains of the C(11)H(16)O(4)-diacid. Stable isotope-labeling growth experiments with either (13)C-labeled naphthalene, per-deuterated naphthalene-d(8), or a (13)C-bicarbonate-buffered medium showed that the ring cleavage products derived from the introduced carbon source naphthalene. The series of identified metabolites suggests that anaerobic degradation of naphthalenes proceeds via reduction of the aromatic ring system of 2-naphthoic acid to initiate ring cleavage in analogy to the benzoyl-coenzyme A pathway for monoaromatic hydrocarbons. Our findings provide strong indications that further degradation goes through saturated compounds with a cyclohexane ring structure and not through monoaromatic compounds. A metabolic pathway for anaerobic degradation of bicyclic aromatic hydrocarbons with 2-naphthoic acid as the central intermediate is proposed.

Anaerobic cometabolic conversion of benzothiophene was studied with a sulfate-reducing enrichment culture growing with naphthalene as the sole source of carbon and energy. The sulfate-reducing bacteria were not able to grow with benzothiophene as the primary substrate. Metabolite analysis was performed with culture supernatants obtained by cometabolization experiments and revealed the formation of three isomeric carboxybenzothiophenes. Two isomers were identified as 2-carboxybenzothiophene and 5-carboxybenzothiophene. In some experiments, further reduced dihydrocarboxybenzothiophene was identified. No other products of benzothiophene degradation could be determined. In isotope-labeling experiments with a [(13)C]bicarbonate-buffered culture medium, carboxybenzothiophenes which were significantly enriched in the (13)C content of the carboxyl group were formed, indicating the addition of a C(1) unit from bicarbonate to benzothiophene as the initial activation reaction. This finding was consistent with the results of earlier studies on anaerobic naphthalene degradation with the same culture, and we therefore propose that benzothiophene was cometabolically converted by the same enzyme system. Groundwater analyses of the tar-oil-contaminated aquifer from which the naphthalene-degrading enrichment culture was isolated exhibited the same carboxybenzothiophene isomers as the culture supernatants. In addition, the benzothiophene degradation products, in particular, dihydrocarboxybenzothiophene, were significantly enriched in the contaminated groundwater to concentrations almost the same as those of the parent compound, benzothiophene. The identification of identical metabolites of benzothiophene conversion in the sulfate-reducing enrichment culture and in the contaminated aquifer indicated that the same enzymatic reactions were responsible for the conversion of benzothiophene in situ.

Primary features of hydrogen and carbon isotope fractionation during toluene degradation were studied to evaluate if analysis of isotope signatures can be used as a tool to monitor biodegradation in contaminated aquifers. D/H hydrogen isotope fractionation during microbial degradation of toluene was measured by gas chromatography. Per-deuterated toluene-d(8) and nonlabeled toluene were supplied in equal amounts as growth substrates, and kinetic isotope fractionation was calculated from the shift of the molar ratios of toluene-d(8) and nondeuterated toluene. The D/H isotope fractionation varied slightly for sulfate-reducing strain TRM1 (slope of curve [b] = -1.219), Desulfobacterium cetonicum (b = -1.196), Thauera aromatica (b = -0.816), and Geobacter metallireducens (b = -1.004) and was greater for the aerobic bacterium Pseudomonas putida mt-2 (b = -2.667). The D/H isotope fractionation was 3 orders of magnitude greater than the (13)C/(12)C carbon isotope fractionation reported previously. Hydrogen isotope fractionation with nonlabeled toluene was 1.7 and 6 times less than isotope fractionation with per-deuterated toluene-d(8) and nonlabeled toluene for sulfate-reducing strain TRM1 (b = -0.728) and D. cetonicum (b = -0.198), respectively. Carbon and hydrogen isotope fractionation during toluene degradation by D. cetonicum remained constant over a growth temperature range of 15 to 37 degrees C but varied slightly during degradation by P. putida mt-2, which showed maximum hydrogen isotope fractionation at 20 degrees C (b = -4.086) and minimum fractionation at 35 degrees C (b = -2.138). D/H isotope fractionation was observed only if the deuterium label was located at the methyl group of the toluene molecule which is the site of the initial enzymatic attack on the substrate by the bacterial strains investigated in this study. Use of ring-labeled toluene-d(5) in combination with nondeuterated toluene did not lead to significant D/H isotope fractionation. The activity of the first enzyme in the anaerobic toluene degradation pathway, benzylsuccinate synthase, was measured in cell extracts of D. cetonicum with an initial activity of 3.63 mU (mg of protein)(-1). The D/H isotope fractionation (b = -1.580) was 30% greater than that in growth experiments with D. cetonicum. Mass spectroscopic analysis of the product benzylsuccinate showed that H atoms abstracted from the toluene molecules by the enzyme were retained in the same molecules after the product was released. Our findings revealed that the use of deuterium-labeled toluene was appropriate for studying basic features of D/H isotope fractionation. Similar D/H fractionation factors for toluene degradation by anaerobic bacteria, the lack of significant temperature dependence, and the strong fractionation suggest that analysis of D/H fractionation can be used as a sensitive tool to assess degradation activities. Identification of the first enzyme reaction in the pathway as the major fractionating step provides a basis for linking observed isotope fractionation to biochemical reactions.

Anaerobic sulfate-reducing bacteria were enriched from contaminated aquifer samples with naphthalene, o-, and m-xylene as sole carbon and energy source in the presence of Amberlite-XAD7, a solid adsorber resin. XAD7 served as a substrate reservoir maintaining a constantly low substrate concentration in the culture medium. In equilibration experiments with XAD7, the aromatic hydrocarbons needed up to 5 days to achieve equilibrium between the water and the XAD7 phase. The equilibrium concentration was directly correlated with the amount of added substrate and XAD7. In the enrichments presented here, XAD7 and aromatic hydrocarbons were adjusted to maintain substrate concentrations of 100 microM m-, or o-xylene, or 50 microM naphthalene. After five subsequent transfers, the three cultures were able to grow with higher substrate concentrations in the absence of XAD7 although they grew best with lower hydrocarbon concentrations. Two new xylene-degrading cultures were obtained that could not utilise toluene as carbon source. O-xylene was degraded anaerobically by a culture, which could also oxidise m-xylene but not p-xylene. Eighty-three percent of the electrons from o-xylene oxidation were recovered in the produced sulfide, indicating a complete oxidation to CO2. Another sulfate-reducing enrichment culture oxidised m-xylene completely to CO2 but not o-, or p-xylene. A naphthalene-degrading sulfate-reducing enrichment culture oxidised naphthalene completely to CO2. Metabolites of naphthalene degradation were recovered from the XAD7 phase and subjected to GC/MS analysis. Besides the metabolites 2-naphthoic acid and decahydro-2-naphthoic acid which were identified by the mass spectrum and coelution with chemically synthesised reference compounds, the reduced 2-naphthoic acid derivatives 5,6,7,8-tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. Cultivation of bacterial cultures in the presence of XAD7 and subsequent derivatisation and extraction of metabolites directly from the solid XAD7 resin provides a new method for the isolation of sensitive bacteria and identification of metabolites.

Anaerobic degradation of 2-methylnaphthalene was investigated with a sulfate-reducing enrichment culture. Metabolite analyses revealed two groups of degradation products. The first group comprised two succinic acid adducts which were identified as naphthyl-2-methyl-succinic acid and naphthyl-2-methylene-succinic acid by comparison with chemically synthesized reference compounds. Naphthyl-2-methyl-succinic acid accumulated to 0.5 microM in culture supernatants. Production of naphthyl-2-methyl-succinic acid was analyzed in enzyme assays with dense cell suspensions. The conversion of 2-methylnaphthalene to naphthyl-2-methyl-succinic acid was detected at a specific activity of 0.020 +/- 0.003 nmol min(-1) mg of protein(-1) only in the presence of cells and fumarate. We conclude that under anaerobic conditions 2-methylnaphthalene is activated by fumarate addition to the methyl group, as is the case in anaerobic toluene degradation. The second group of metabolites comprised 2-naphthoic acid and reduced 2-naphthoic acid derivatives, including 5,6,7,8-tetrahydro-2-naphthoic acid, octahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. These compounds were also identified in an earlier study as products of anaerobic naphthalene degradation with the same enrichment culture. A pathway for anaerobic degradation of 2-methylnaphthalene analogous to that for anaerobic toluene degradation is proposed.

Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture was studied by substrate utilization tests and identification of metabolites by gas chromatography-mass spectrometry. In substrate utilization tests, the culture was able to oxidize naphthalene, 2-methylnaphthalene, 1- and 2-naphthoic acids, phenylacetic acid, benzoic acid, cyclohexanecarboxylic acid, and cyclohex-1-ene-carboxylic acid with sulfate as the electron acceptor. Neither hydroxylated 1- or 2-naphthoic acid derivatives and 1- or 2-naphthol nor the monoaromatic compounds ortho-phthalic acid, 2-carboxy-1-phenylacetic acid, and salicylic acid were utilized by the culture within 100 days. 2-Naphthoic acid accumulated in all naphthalene-grown cultures. Reduced 2-naphthoic acid derivatives could be identified by comparison of mass spectra and coelution with commercial reference compounds such as 1,2,3, 4-tetrahydro-2-naphthoic acid and chemically synthesized decahydro-2-naphthoic acid. 5,6,7,8-Tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. The metabolites identified suggest a stepwise reduction of the aromatic ring system before ring cleavage. In degradation experiments with [1-(13)C]naphthalene or deuterated D(8)-naphthalene, all metabolites mentioned derived from the introduced labeled naphthalene. When a [(13)C]bicarbonate-buffered growth medium was used in conjunction with unlabeled naphthalene, (13)C incorporation into the carboxylic group of 2-naphthoic acid was shown, indicating that activation of naphthalene by carboxylation was the initial degradation step. No ring fission products were identified.

The influence 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 alphaC 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 (alphaC = 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.

A syntrophic coculture of a new sulfate-reducing isolate, strain TRM1, with Wolinella succinogenes degraded toluene with either fumarate or NO3- 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.

Acetylene hydratase of Pelobacter acetylenicus is a tungsten iron-sulfur protein involved in the fermentation of acetylene to ethanol and acetate. Expression of the enzyme was increased 10-fold by feeding a 50-L batch culture continuously with 104 Pa acetylene at pH 6.8-7.0. Acetylene hydratase was purified to homogeneity by a three-step procedure in either the absence or presence of dioxygen. The enzyme was a monomer with a molecular mass of 73 kDa (SDS/PAGE) or 83 kDa (matrix-assisted laser-desorption ionization MS) and contained 0.5 +/- 0.1 W (inductively coupled plasma/MS) and 1.3 +/- 0.1 molybdopterin-guanine dinucleotide per mol. Selenium was absent. EPR spectra (enzyme as isolated, under air) showed a signal typical of a [3Fe-4S] cluster with gav = 2.01, at 10 K. In enzyme prepared under N2/H2, this signal was absent and reaction with dithionite led to a rhombic signal with gz = 2.048, gy = 1.939 and gx = 1.920 indicative of a low-potential ferredoxin-type [4Fe-4S] cluster. Upon oxidation with hexacyanoferrate(III), a new signal appeared with gx = 2.007, gy = 2.019 and gz = 2.048 (gav = 2.022), which disappeared after further oxidation. The signal was still visible at 150 K and was tentatively assigned to a W(V) center. The iron-sulfur center of acetylene hydratase (prepared under N2/H2) gave a midpoint redox potential of -410 +/- 20 mV in a spectrophotometric titration with dithionite. Enzyme activity depended on the redox potential of the solution, with 50% of maximum activity at -340 +/- 20 mV. The presence of a pterin-guanine dinucleotide cofactor differentiates acetylene hydratase from the aldehyde ferredoxin oxidoreductase-type enzymes which have a pterin mononucleotide cofactor.

This work gives an overview of the recent achievements which have contributed to the understanding of the structure and function of molybdenum and tungsten enzymes. Known structures of molybdo-pterin cofactor-containing enzymes will be described briefly and the structural differences between representatives of the same and different families will be analyzed. This comparison will show that the molybdo-pterin cofactor-containing enzymes represent a very heterogeneous group with differences in overall enzyme structure, cofactor composition and stoichiometry, as well as differences in the immediate molybdenum environment. Two recently discovered molybdo-pterin cofactor-containing enzymes will be described with regard to molecular and EPR spectroscopic properties, pyrogallol-phloroglucinol transhydroxylase from Pelobacter acidigallici and acetylene hydratase from Pelobacter acetylenicus. On the basis of its amino acid sequence, transhydroxylase can be classified as a member of the dimethylsulfoxide reductase family, whereas classification of the tungsten/molybdenum-containing acetylene hydratase has to await the determination of its amino acid sequence.

The reaction center free light-harvesting core complex of Rp. marina was purified by DEAE 52 ion exchange chromatography in the presence of the detergent OG. The protein complex was crystallised by microdialysis yielding two-dimensional crystals with a diameter of up to 10 microns. The crystals were negatively stained with uranyl acetate or prepared in vitrified ice and electron micrographs were taken. They exhibited a hexagonal lattice with a lattice constant of 102 +/- 3 A. The optical diffraction pattern of the best ordered areas of electron micrographs showed spots up to a resolution of 29 A. Image processing revealed a six fold symmetry of the ring like B880-complex. The protein ring is hexagonal with one subunit in each corner of the hexagon and two subunits forming the connection site to the neighbouring B880-complex in the crystal. In freeze fracture preparations of whole cells the intra-cytoplasmic photosynthetic membranes are seen to be organised into large stacks that affect the organisation of the photosynthetic complexes. Most notably, the stacked membrane regions exhibit hexagonally packed photosynthetic complexes with a repeat of approximately 100 A, which is very similar to the lattice of the artificial B880-complex crystals. The same quasi-crystalline structure appeared in the cytoplasmic membrane of the contact sites with the intra-cytoplasmic membrane stack, but was absent from the end membrane of the stack. Thus, membrane stacking appears to induce the formation of the crystalline arrays, presumably through interactions between the cytoplasmic surface domains of the photosynthetic complexes. Tight packing of the photosynthetic particles is not sufficient to induce the crystalline order. The intra cytoplasmic membranes form a continuum with the cytoplasmic membrane via their origins at the round invagination sites.

The BChla-containing B880-complex (core-complex) of Rhodopseudomonas marina (Rhodospirillaceae) was isolated with a new purification method. The isolation of the B880-complex was performed by solubilisation of the photosynthetic membranes with the detergent LDAO and subsequent fractionated ammonium-sulfate precipitation with about 50% recovery. The B880-complex retained its original spectral properties as revealed with absorption, fluorescence and circular dichroism spectroscopy. Furthermore, we dissociated the B880-complex with the detergent n-octyl-beta-glucoside (OG) and purified the developed subcomplex by the method of Miller et al. [1], which showed an absorption maximum at 820 nm (B820). The alpha- to beta-polypeptide ratio and the alpha- or beta-polypeptide to BChla ratio, respectively, were estimated to be 1:1 in both complexes. The molecular weights of the B880 and the B820-complexes, determined by gel filtration chromatography, were 181 and 32 kDa, respectively. Thus, it appears that the B880-complex of Rp. marina consists of 24 polypeptides and the B820-complex of four polypeptides. Six B820-complexes or possible subunits could form the B880-complex. On the basis of these data we propose a model for the structure of BChla containing core-complexes.

Electron micrographs of photosynthetic membranes of the BChla-containing bacterium Rp. marina showed a quasi-crystalline structure. The photoreceptor units are arranged in a hexagonal lattice with a reaction center to reaction center distance of 102 +/- 3 A. Purified B880-complex was concentrated up to an OD880 of 60 which induced the formation of large protein vesicles. The protein complexes within these vesicles were highly ordered and showed a hexagonal lattice with the same center to center distance of 102 +/- 3 A as was observed in the native membranes. Image processing of the micrographs revealed a ring-like structure of the B880-complex at 26 A resolution and suggests that the B880-complex consists of 5 or 6 subunits. For the first time it can be shown that an isolated core-complex is in a stable, ring-like structure even without the reaction center which is supposed to be located in the middle of the B880-ring. The data indicate that the isolated B880-complex exhibits the same structure as in the native membrane.