Eva Annweiler

Anaerobic Naphthalene Degradation by a Sulfate-Reducing Enrichment Culture

ABSTRACT 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 [1-13C]naphthalene or deuterated D8-naphthalene, all metabolites mentioned derived from the introduced labeled naphthalene. When a [13C]bicarbonate-buffered growth medium was used in conjunction with unlabeled naphthalene, 13C 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.

Anaerobic Degradation of 2-Methylnaphthalene by a Sulfate-Reducing Enrichment Culture

ABSTRACT 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 μM 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.

Identical Ring Cleavage Products during Anaerobic Degradation of Naphthalene, 2-Methylnaphthalene, and Tetralin Indicate a New Metabolic Pathway

ABSTRACT 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 C1 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 C11H16O4 (C11H16O4-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 β-oxidation of one of the side chains of the C11H16O4-diacid. Stable isotope-labeling growth experiments with either 13C-labeled naphthalene, per-deuterated naphthalene-d8, or a 13C-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 by a Sulfate-Reducing Enrichment Culture and in a Tar-Oil-Contaminated Aquifer

ABSTRACT 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 [13C]bicarbonate-buffered culture medium, carboxybenzothiophenes which were significantly enriched in the13C content of the carboxyl group were formed, indicating the addition of a C1 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.

The use of a solid adsorber resin for enrichment of bacteria with toxic substrates and to identify metabolites: degradation of naphthalene, O-, and m-xylene by sulfate-reducing bacteria.

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.

Desorption controlled mobility and intrinsic biodegradation of anthracene in unsaturated soil

Abstract Desorption, sorption and biodegradation control the fate of polycyclic aromatic hydrocarbons (PAH) in contaminated soils. The interaction of these processes was studied in a two-layer column experiment with uncontaminated and spiked A-horizon material (13C anthracene) at unsaturated water flow conditions. During constant irrigation (part I of the experiment) the development of a stationary effluent composition was observed. The onset of anthracene breakthrough was delayed by 350 pore volumes (pvs) indicating high affinity sorption towards soil organic matter. The coincidence of anthracene breakthrough with increasing dissolved organic carbon (DOC) concentrations pointed to a carrier-mediated transport of the contaminant. Flow interruptions of varied duration (part II of the experiment) yielded a pronounced response of solution phase parameters. Effluent DOC increased linearly with time of flow interruption, indicating a zero-order rate-limited release. In contrast, anthracene concentrations showed a sharp drop after the stopped-flow events. Thus, the effluent anthracene concentration observed in part I may have been controlled by rate-limited sorption in the uncontaminated layer. Biodegradation of anthracene was evident from 13C-labelled metabolites and the complete decline of effluent anthracene after 1400 pvs. Although PAH seepage in soils is enhanced by rate-limited sorption it may be effectively counteracted by microbial activity.

Organic Pollutants Associated with Macromolecular Soil Organic Matter and the Formation of Bound Residues

All anthropogenic organic chemicals form non-extractable residues to some extent after entering soils. This well known phenomenon has been studied intensively in the field of soil agrochemistry. Similar processes have been observed during the bioremediation of oil-contaminated soils. Thus, the residue formation of toxic and carcinogenic polycyclic aromatic hydrocarbons is of particular concern. Beside mineralisation by microbial activity, the formation of non-extractable residues is a major sink for anthropogenic pollutants in soils. Microbial activity often stimulates the formation of bound residues. On a molecular scale, bound residue formation is suggested to be a covalent binding of xenobiotic substances and their metabolites to macromolecular natural organic matter in soils and sediments. When this binding occurs, the xenobiotic substance loses its chemical identity. A strong reduction of the bioavailability of xenobiotic carbon is one major consequence of bound residue formation in soils. In this overview we will discuss the present state and new trends in the field of bound residue research regarding the application of isotopically labeled tracer substances. Carbon budgets are emphasized to study the fate of isotopically labeled PAH in soils, including bound residue formation.

13C/12C Stable Isotope Fractionation of Toluene by Anaerobic Degradation

In microbial degradation experiments with soil percolation columns and a pure bacterial culture the consumption and the13C/12C stable carbon isotope composition of aromatic hydrocarbons were monitored in order to evaluate if isotopic fractionation is applicable as a parameter to evaluate biodegradation processes in field studies.

The Significance of Bound Residues in the Bioremediation Process of PAH Contaminated Sites

The formation of bound residues has intensively been studied in agrochemistry with the main focus on the fate of pesticides in soil. It is assumed that nearly all xenobiotics entering the soil environment build up these residues, and for many classes this has already been proven (Calderbank 1989).