Biodegradation of aromatic hydrocarbons under anoxic conditions in a shallow sand and gravel aquifer of the Lower Rhine Valley, Germany

Org. Geochem. Vol. 25, No. 1/2, pp. 41-50, 1996

Pergamon PII: S0146-6380(96)00111-8

Copyright © 1996ElsevierScienceLtd Printed in Great Britain.All rights reserved 0146-6380/96$15.00+ 0.00

Biodegradation of aromatic hydrocarbons under anoxic conditions in a shallow sand and gravel aquifer of the Lower Rhine Valley, Germany R. SCHMITT I, H.-R. L A N G G U T H ~, W. PISITTMANN*, H. P. ROHNS 2, P. ECKERT 2 and J. SCHUBERT 2 'Lehr- und Forschungsgebiet Hydrogeologie RWTH Aachen, Lochnerstr. 4-20, 52064, Aachen, Germany and 2Stadtwerke Diisseldorf AG, Postfach 101136, 40002, Diisseldorf, Germany Abstract--The groundwater in the area of the former gasworks at Diisseldorf in the Lower Rhine Valley, Germany, is contaminated with up to 80 mg l-~ of aromatic hydrocarbons such as benzene, toluene, ethylbenzene, xylenes (BTEX), other alkylated benzenes and low molecular weight PAH. About 100 m downgradient from the contamination source a rapid decrease of these compounds to less than 0.5 mg 1-I is observed due to biodegradation under anoxic conditions. Abundant organic acids are generated as metabolic products of the aromatic hydrocarbons in the contaminant plume. Degradation of the aromatic hydrocarbons is related to nitrate reduction and particularly sulfate reduction. Two possible explanations for the enhanced concentrations of dissolved iron can be given: (1) Biogenic hydrogen sulfide originating from degradation of aromatic hydrocarbons by sulfate reducing bacteria is used for the abiotic ferric iron reduction; (2) Organic acids generated from microbial degradation of aromatic hydrocarbons act as ligands for complexing insoluble Fe(III) oxides in the aquifer. Thereby Fe(III) is made available for iron-reducing bacteria intensifying the degradation process of the aromatic hydrocarbons. Downgradient from the contaminant plume the groundwater is reoxygenated and the dissolved iron is reprecipitated. Copyright © 1996 Elsevier Science Ltd Key words--biodegradation, aromatic hydrocarbons, sand and gravel aquifer, sulfate reduction, iron reduction, organic acids, complexation of iron oxides

INTRODUCTION

al., 1993) and other alkylated benzenes (Edwards et al., 1992; Rueter et al., 1994).

Biodegradation of aromatic hydrocarbons such as benzene, toluene, ethylbenzene, xylenes (BTEX), other alkylated benzenes and PAH of low molecular weight is well known in the presence of molecular oxygen (Gibson et al., 1968). Recently, laboratory and field studies provided evidence that biodegradation of these aromatic hydrocarbons is also possible in anoxic environments under various redox conditions. The degradation of preferentially monoaromatic hydrocarbons under denitrifying conditions has been described by Kuhn et al. (1988), Major et al. (1988), Mihelcic and Luthy (1988), Evans et al. (1991) and Hutchins et al. (1991). The degradation of aromatic hydrocarbons coupled to microbial iron reduction has been reported by Lovley et al. (1989, 1994) and Lovley and Lonergan (1990). Recently, laboratory studies were conducted to demonstrate the capability of sulfate-reducing bacteria to degrade benzene (Lovley et al., 1995), toluene (Haag et al., 1991; Belier et al., 1992; Rabus et

Moreover, several studies provide evidence for a methanogenic transformation of aromatic hydrocarbons (Wilson et al., 1986; Vogel and Grbic-Galic, 1986; Grbic-Galic and Vogel, 1987; Grbic-Galic, 1990). Biodegradation of crude oil hydrocarbons in an otherwise pristine shallow sand and gravel aquifer has been described in detail by Baedecker et al. (1993), Bennett et al. (1993) and Eganhouse et al. (1993). They found methanogenesis and iron/ manganese reduction to be the most important processes in the aquifer connected with the biodegradation of the hydrocarbons. This paper describes a series of hydrochemical investigations carried out during January to June 1994 on a sand and gravel aquifer contaminated with aromatic hydrocarbons by a former gasworks plant. In order to evaluate the bioremediation potential at the contaminated site, examinations were concentrated on: (1) The composition and distribution of the organic constituents; and (2) The inorganic aqueous chemistry in the contaminant plume. Particular attention was paid to a possible link *Present address: J. W. Goethe-Universit/it, FB between the formation of metabolic products from Geowissenschaften-Umweltanalytik-, Georg-Voigt-Str. aromatic hydrocarbons and variations in the ferrous 16, 60054 Frankfurt a.M., Germany. iron and sulfate concentrations in the plume. 41

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Site description The study site is located in the city of Diisseldorf, Germany, near the eastern margin of the Lower Rhine Basin (Fig. 1). The city is located on the lower terrace of the Rhine river at an altitude of about 40 m above sea level. The study site is about 3 km east of the city center in the area of the former urban gasworks (51°13'28"N lat., 6°49'11"E long.). Industrial activities in this area are recorded back to the last century. Gas and further coal-derived oils and tars were produced by hard coal coking until the plant was shut down in 1968. First investigations in 1982 found soil and groundwater to be highly polluted by aromatic hydrocarbons around the former benzene washing facility (Fig. 1) (Eckert et al., 1994). The contamination derived from chronic leaks of tanks and spills during the benzene production. Also, the benzene washing facility had been a target for aerial attacks during World War II. For ground-water monitoring and further investigations, piezometer nests were installed to screen two different depths of the aquifer (Figs 1 and 2).

Geology and hydrogeology The aquifer at the study site consists of late Pleistocene fluvial deposits with medium to coarse grained sand and gravel. Their thickness varies along the investigated area between 12 and 18 m (Fig. 2). The aquifer is underlain by Oligocene silty and glauconitic fine-grained sands. The aquifer is superposed by Holocene mainly silty flood plain deposits reaching a thickness up to 5 m. The uppermost bed is formed of artificial backfill with rubble and cinder material cutting at some places even through the silty flood plain deposits and thus getting in contact with the aquifer. Normal precipitation at Diasseldorf is about 7 5 0 m m a -1 and regional ground-water recharge from infiltration is about 71s -1 km -2. During the investigation, the depth to the water table of the unconfined aquifer ranged from about 5 to 7 m with largely horizontal ground-water flow in a western direction towards the Rhine River (Figs 1 and 2). The aquifer's hydraulic conductivity was determined by pumping test to 1.3 x 10-3 m s-1. The calculated mean apparent flow velocity in the study area was about 2 m per day at an average hydraulic gradient of 2.9 x 10-3. The groundwater in the region is a Ca2+-HCO~ water which exhibits enhanced concentrations of chloride, nitrate and sulfate due to human activities and leaching of construction wastes.

EXPERIMENTAL

Sampling Two series of ground-water sampling were performed in January and in April 1994. More samples

43

had been taken before and after these dates which confirmed the data presented in this paper. As the sampling series in April 1994 was the most complete concerning the number of sampled wells and the performed analyses, the data shown in this paper refer only to this date. Water samples were collected by pumping from observation wells along a flow path upgradient from, around and downgradient from the contamination source (Fig. 1). The drilling of these wells had been performed using cable-tool drilling techniques with bailer and without drilling fluids. Most wells were constructed using PVC screens and casings of 5 cm in diameter. Most of the sampled wells had a screen length of 4 m to 6 m. The screen length of the other sampled wells were 1 m, 2m, and 8m. Sampling wells with screened intervals are shown in Fig. 2. For sampling, the wells were first purged by down-hole pumping with an endpoint being the stabilization of Eh. Temperature, electric conductivity, pH, Eh and Oz were determined in a flow cell connected directly to the pump discharge. The water samples were filled into brown glass bottles without filtration. To 250 ml of each sample collected for analysis of iron 5 ml of HCI (30%) and 3 drops of H202 were added. All samples were stored at 4 °C until analyzed within 48 h after sampling.

Analytical procedures Nitrate and sulfate concentrations were determined by ion chromatography using a Dionex DX 100 ion chromatograph with a Dionex Ion pack AS 4A P/N 37041 column, a Dionex Ion Pack A G 4A 4 m m P/N 37042 (10-32) pre-column to preserve the column from organic compounds, and a conductivity detector. Analysis of iron was performed with Atomic Adsorption Spectroscopy (AAS) using a Varian 400A instrument. To determine the non-volatile DOC (NVDOC) the water samples were stripped with oxygen to remove inorganic C. The non-volatile DOC remaining in the water was converted to CO2 by thermal catalyzed oxidation at 800-900 °C with a Pt-catalyst. The non-volatile DOC was determined in a Dimatoc 100 carbon analyzer using an infra red detector (NDIR). For analysis of organic constituents 100 #g 1,2,3,5-tetramethyl-benzene (isodurene) were added as internal standard to 250ml water from each sample. Then the samples were solvent extracted by shaking first at pH 10 with 2 x 10ml dichloromethane and subsequently at pH 2 with 3 × 30 ml diethyl ether. The extracts obtained at pH 10 were directly analysed for aromatic hydrocarbons using a Carlo Erba 4100 gas chromatograph (GC) equipped with a OV1-CB5 column (25 m x 0.32 mm i.d., universal phase, from CS-Chromatographie Service GmbH)

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Biodegradation of aromatic hydrocarbons and hydrogen as carrier gas. The oven temperature was kept isothermal at 40 °C for 5 min, and then programmed at 4 °C min -1 to 280 °C and held for 20 min. Identification was performed with a Finnigan M A T 8200 mass spectrometer (MS) interfaced to a Varian 3700 GC with helium as carrier gas using the same OV-1-CB5 column and oven temperature program as for GC-analysis. The extracts obtained at pH 2 were dried over anhydrous sodium sulfate, and the ether was removed. Then squalane was added, and the extracts were treated with BSTFA for silylation of polar components and heated at 40 °C for 60 min. For GC analysis a Carlo Erba 5160 Mega Series GC was used equipped with a SE-54 column (25 m x 0.25 mm i.d.) and hydrogen as carrier gas. The oven temperature was isothermal at 40 °C for 5 rain, and then programmed at 4 ° C m i n -~ to 300°C and held for 20 min. Peak identification was performed by the same G C - M S as described previously using the same type of SE-54 column and oven temperature program as described for GC analysis.

RESULTS

Inorganic chemistry At well MP32 which is located upgradient from the contamination source of aromatic hydrocarbons and screens the shallow part of the aquifer from 1.30 m to 5.30 m below the water table, the sampled groundwater is slightly oxidizing. The concentration of molecular oxygen is only 0.5mg1-1, and the measured Eh value is + 150mV (Fig. 3a). In the area of the contamination source and farther downgradient, molecular oxygen is completely depleted at the wells screening the shallow part of the groundwater to about 4-6 m below the water table (wells 19053, MP23). Eh decreases within this area to -100 mV. Farther downgradient (wells MP14, 19051) the shallow part of the groundwater is reoxygenated and contains nearly 3 mg 1-1 of molecular oxygen. Eh is + 200 mV. The concentration of nitrate is nearly 7 mg 1-1 and the concentration of sulfate about 240 mg 1-1 upgradient from the contamination source (well MP32). In the area of the contamination source nitrate is completely depleted and sulfate decreases from approximately 240 mg 1-1 to about 90 mg 1- l (Fig. 3b, c). Farther downgradient in the reoxygenated zone nitrate increases to about 14 mg 1-1 and sulfate to about 120 mg 1-1. Dissolved iron exhibits a concentration of about 6 mg 1-1 upgradient from the contamination source (Fig. 3d). It decreases in the water when sulfate concentrations reach minimum values. Farther downgradient iron concentrations increase to about 12 mg 1-1 (well MP5). In the reoxygenated zone iron is depleted again.

47

Non-volatile DOC The non-volatile DOC is almost 3 mg 1- l upgradient from the contamination source (MP32) and increases to more than 8 mg 1- l in the area of the contamination source (Fig. 3e). Farther downgradient in the reoxygenated zone the non-volatile DOC decreases to less than 3 mg 1-j.

Aromatic hydrocarbons Aromatic hydrocarbons were not detected in the well upgradient from the contamination source (MP32). Farther downgradient up to 80 m g l - l of aromatic hydrocarbons were detected in the shallow part of the groundwater in the zone found to be the most reducing (Fig. 3f, g). Figure 4a shows the gas chromatogram of the extract at pH 10 from the sample of well MP23 which is located 30 m downgradient from the contamination source. Among the detected components, BTEX predominates (up to 70 mg 1-1) associated with further alkylated benzenes, styrene, PAH such as naphthalene, 2-methylnaphthalene, indene and acenaphthene. Additionally, low concentrations of heterocyclic aromatic compounds such as benzothiophene, benzofuran and dibenzofuran were detected in the extracts obtained at pH 2 (not presented in the chromatograms shown). The contaminant plume of BTEX and the other aromatic hydrocarbons is limited mainly to the shallow part of the groundwater, i.e. high concentrations of these compounds are only found at the wells screened until about 4-6 m below the water table. At the wells screened in the groundwater deeper than 78 m below the water table the concentrations of BTEX are only about 0.05 mg 1-1 and other aromatics are below 0.6 mg 1-I. Among the polycyclic aromatic hydrocarbons, acenaphthene is a major component contributing more than 70% of PAH. Horizontally, the contaminant plume extends about 100 m downgradient from the contamination source, and the concentration of aromatic hydrocarbons decreases rapidly (Fig. 3f, g). At a well 120 m downgradient from the contamination source (MP5) only about 0.5 mg 1-1 of aromatics were detected.

Polar compounds Among the polar components, several aromatic acids were identified including benzoic acid, 2hydroxybenzoic acid, phenylacetic acid, 2-hydroxyphenylacetic acid and tolyl acetic acid as well as phenol and o-cresol. Furthermore low molecular weight aliphatic acids such as hydroxyacetic acid, 2hydroxypropanoic acid and 3-hydroxypropanoic acid as well as hexadecanoic acid and octadecanoic acid were detected in the groundwater (Fig. 4b). The highest concentrations of polar compounds were found in the contaminant plume at the wells within 50 m downgradient from the contamination source. Here, about 5 mg 1-1 of polar compounds

48

R. Schmitt et al.

were extracted from the samples (Fig. 3h). At these wells, also the aromatic and low molecular weight aliphatic acids reach their highest individual concentrations of up to 0.05 mg 1-~. Benzoic acid even shows a concentration of about 0.7 mg 1-1 at well MP23. Farther downgradient these acids decrease continuously. Upgradient and downgradient from the contaminant plume as well as in the deeper zone of the groundwater only few of the polar compounds mentioned above were detected such as hexadecanoic acid and octadecanoic acid.

DISCUSSION The aromatic and aliphatic low molecular weight acids detected within the contaminant plume are known as metabolic products generated by the anoxic degradation of aromatic hydrocarbons. Most of them have previously been detected in laboratory experiments as well as in field studies (Grbic-Galic and Vogel, 1987; Grbic-Galic, 1990; Grbic-Galic et al., 1990; Cozzarelli et al., 1990, 1994). Aromatic acids occur during the first steps of transformation followed by aliphatic low molecular weight acids as products of further degradation prior to mineralization. The aromatic and aliphatic low molecular weight acids detected in this study provide evidence for the different stages of biodegradation of the aromatic hydrocarbons under anoxic conditions. In the center of the contaminant plume, showing the highest concentrations of aromatic hydrocarbons, nitrate and sulfate reduction are indicated by significant decrease in concentrations of these species. Nitrate reduction can only play a minor role in the biodegradation process, because the nitrate concentration is only about 7 mg 1-1 upgradient from the contamination source. As main elec-

tron acceptor in the center of the contaminant plume, sulfate is indicated which is depleted by 150mg1-1 in the groundwater (Fig. 3c). The decreased iron concentration at well MP23 indicates that reduced sulfur species probably react with the reduced iron precipitating as iron sulfides (Fig. 3d). For explanation of the enhanced iron concentrations, two different hypotheses are proposed. The first hypothesis is based on the laboratory study of Beller et al. (1992). They found ferric iron reduction proceeding concurrently with toluene degradation and sulfate reduction. Based on stoichiometric data and other observations, iron reduction was indicated not to be directly coupled to toluene oxidation but to be a secondary, presumably abiotic, reaction between ferric iron and biogenic hydrogen sulfide. Transferred to the study presented here, the enhanced iron concentrations would be a secondary, presumably abiotic, product of the biodegradation of the aromatic hydrocarbons coupled to sulfate reduction. The second hypothesis is based on the laboratory study of Lovley et al. (1994). They demonstrated that Fe(III) oxides which are abundantly present but usually insoluble in shallow aquifers can be mobilized via complexation. Lovley et al. (1994) used NTA (nitrilotriacetic acid, C6H9NO6) as organic ligand complexing Fe(III) oxides. Thus, the bioavailibility of Fe(III) increased drastically and aromatic hydrocarbons such as toluene and even benzene were rapidly degraded. In the study presented here, no organic ligands were added to the groundwater. But the identified organic acids produced as metabolic intermediates from the initial degradation of the aromatic hydrocarbons under nitrate and sulfate reducing conditions might be suitable complexation agents according to their structural similarity with the agents used by Lovley et al. (1994) (Fig. 5).

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49

Biodegradation of aromatic hydrocarbons They might mobilize insoluble Fe(III) in the aquifer which is then available for microbial iron reduction intensifying the degradation process of the aromatic hydrocarbons. Thereby, Fe(II) concentrations increase in the groundwater (well MP5 Fig. 3d, f, g). Consequently, we hypothesize that the biodegradation process of the aromatic hydrocarbons occurs in three major stages: (1) Initially high concentrations of aromatic hydrocarbons are partly degraded under nitrate and sulfate reducing conditions forming aromatic and aliphatic low molecular weight organic acids as metabolic intermediates. (2) These organic acids act as ligands complexing insoluble Fe(III) oxides in the aquifer and mobilizing Fe(III). (3) The mobilized Fe(III) is now available for iron reducing bacteria and intensifies the degradation of the aromatic hydrocarbons.

CONCLUSIONS Introduction of aromatic hydrocarbons from former gasworks activities into a mineralized and slightly oxidizing aquifer environment causes the complete depletion of molecular oxygen and nitrate in the groundwater. The correlation of organic and inorganic parameters indicates biodegradation of the aromatic hydrocarbons in the groundwater under anoxic conditions. Several aromatic and low molecular weight aliphatic organic acids are generated as metabolic intermediates of the biodegradation of the aromatics. Sulfate appears to be the dominant electron acceptor as indicated by its significant depletion relative to the concentration upgradient from the contamination. Two hypotheses are proposed to explain enhanced iron concentrations. The enhanced iron concentrations can either be a secondary, presumably abiotic, product of the biodegradation of aromatic hydrocarbons coupled to sulfate reduction or might be due to microbial iron reduction via organic acids acting as ligands complexing insoluble Fe(III) oxides and mobilizing them. The contaminant plume does not extend more than about 100 m downgradient from the contamination source due to biodegradation. Farther downgradient aromatic hydrocarbons and metabolic products are almost completely depleted and the water is reoxygenated. Acknowledgements--Use of analytical instrumentation was possible at Lehrstuhl fiir Geologic, Geoehemie and Lagerst~itten des Erd61s und der Kohle, RWTH Aachen, Germany. We would like to thank Dr Kissel, Stadtwerke

Diisseldorf AG, Germany, for providing data of inorganic parameters.

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