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

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.     

Methylation is the initial reaction in anaerobic naphthalene degradation by a sulfate-reducing enrichment culture

The sulfate-reducing culture N47 can utilize naphthalene or 2-methylnaphthalene as the sole carbon source and electron donor. Here we show that the initial reaction in the naphthalene degradation pathway is a methylation to 2-methylnaphthalene which then undergoes the subsequent oxidation to the central metabolite 2-naphthoic acid, ring reduction and cleavage. Specific metabolites occurring exclusively during anaerobic degradation of 2-methylnaphthalene were detected during growth on naphthalene, i.e. naphthyl-2-methyl-succinate and naphthyl-2-methylene-succinate. Additionally, all three enzymes involved in anaerobic degradation of 2-methylnaphthalene to 2-naphthoic acid that could be measured in vitro so far, i.e. naphthyl-2-methyl-succinate synthase, succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase and naphthyl-2-methyl-succinyl-CoA dehydrogenase were also detected in naphthalene-grown cells with similar activities. Induction experiments were performed to study the growth behaviour of the cell when transferred from naphthalene to 2-methylnaphthalene or vice versa. When the cells were transferred from naphthalene to 2-methylnaphthalene they grew immediately, indicating that no new enzymes had to be induced. On the contrary, the transfer of cells from 2-methylnaphthalene to naphthalene caused a lag-phase of almost 100 days demonstrating that an additional catabolic enzyme has to be activated in this case. We propose the methylation as a novel general mechanism of activation reactions in anaerobic degradation of unsubstituted aromatic hydrocarbons.     

Anaerobic naphthalene degradation by Gram-positive, iron-reducing bacteria

An anaerobic naphthalene-degrading culture (N49) was enriched with ferric iron as electron acceptor. A closed electron balance indicated the total oxidation of naphthalene to CO(2). In all growing cultures, the concentration of the presumed central metabolite of naphthalene degradation, 2-naphthoic acid, increased concomitantly with growth. The first metabolite of anaerobic methylnaphthalene degradation, naphthyl-2-methyl-succinic acid, was not identified in culture supernatants, which does not support a methylation to methylnaphthalene as the initial activation reaction of naphthalene, but rather a carboxylation, as proposed for other naphthalene-degrading cultures. Substrate utilization tests revealed that the culture was able to grow on 1-methyl-naphthalene, 2-methyl-naphthalene, 1-naphthoic acid or 2-naphthoic acid, whereas it did not grow on 1-naphthol, 2-naphthol, anthracene, phenanthrene, indane and indene. Terminal restriction fragment length polymorphism and 16S rRNA gene sequence analyses revealed that the microbial community of the culture was dominated by one bacterial microorganism, which was closely related (99% 16S sequence similarity) to the major organism in the iron-reducing, benzene-degrading enrichment culture BF [ISME J (2007) 1: 643; Int J Syst Evol Microbiol (2010) 60: 686]. The phylogenetic classification supports a new candidate species and genus of Gram-positive spore-forming iron-reducers that can degrade non-substituted aromatic hydrocarbons. It furthermore indicates that Gram-positive microorganisms might also play an important role in anaerobic polycyclic aromatic hydrocarbon-degradation.     

Identical ring cleavage products during anaerobic degradation of naphthalene, 2-methylnaphthalene, and tetralin indicate a new metabolic pathway

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 naphthalene degradation by a sulfate-reducing enrichment culture

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.     

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

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 Naphthalene Degradation by a Sulfate-Reducing Enrichment Culture

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.     

Combined Genomic and Proteomic Approaches Identify Gene Clusters Involved in Anaerobic 2-Methylnaphthalene Degradation in the Sulfate-Reducing Enrichment Culture N47

The highly enriched deltaproteobacterial culture N47 anaerobically oxidizes the polycyclic aromatic hydrocarbons naphthalene and 2-methylnaphthalene, with sulfate as the electron acceptor. Combined genome sequencing and liquid chromatography-tandem mass spectrometry-based shotgun proteome analyses were performed to identify genes and proteins involved in anaerobic aromatic catabolism. Proteome analysis of 2-methylnaphthalene-grown N47 cells resulted in the identification of putative enzymes catalyzing the anaerobic conversion of 2-methylnaphthalene to 2-naphthoyl coenzyme A (2-naphthoyl-CoA), as well as the reductive ring cleavage of 2-naphthoyl-CoA, leading to the formation of acetyl-CoA and CO₂. The glycyl radical-catalyzed fumarate addition to the methyl group of 2-methylnaphthalene is catalyzed by naphthyl-2-methyl-succinate synthase (Nms), composed of α-, β-, and γ-subunits that are encoded by the genes nmsABC. Located upstream of nmsABC is nmsD, encoding the Nms-activating enzyme, which harbors the characteristic [Fe₄S₄] cluster sequence motifs of S-adenosylmethionine radical enzymes. The bns gene cluster, coding for enzymes involved in beta-oxidation reactions converting naphthyl-2-methyl-succinate to 2-naphthoyl-CoA, was found four intervening open reading frames further downstream. This cluster consists of eight genes (bnsABCDEFGH) corresponding to 8.1 kb, which are closely related to genes for enzymes involved in anaerobic toluene degradation within the denitrifiers “Aromatoleum aromaticum” EbN1, Azoarcus sp. strain T, and Thauera aromatica. Another contiguous DNA sequence harbors the gene for 2-naphthoyl-CoA reductase (ncr) and 16 additional genes that were found to be expressed in 2-methylnaphthalene-grown cells. These genes code for enzymes that were supposed to catalyze the dearomatization and ring cleavage reactions converting 2-naphthoyl-CoA to acetyl-CoA and CO₂. Comparative sequence analysis of the four encoding subunits (ncrABCD) showed the gene product to have the closest similarity to the Azoarcus type of benzoyl-CoA reductase. The present work provides the first insight into the genetic basis of anaerobic 2-methylnaphthalene metabolism and delivers implications for understanding contaminant degradation.Polycyclic aromatic hydrocarbons (PAHs) are constantly released into the environment by anthropogenic activities such as industrial use or by accidental contamination. Due to the low chemical reactivity caused by the resonance energy of the aromatic ring structure and the low bioavailability of PAHs, they are persistent in the environment (15). The understanding of microbial metabolic capabilities in terms of anaerobic PAH degradation is in its infancy. However, natural amelioration of contaminated sites relies on the degradation capacities of microorganisms, and therefore, it is an essential prerequisite to broaden knowledge about the microorganisms involved and their potentials concerning PAH breakdown.Numerous microorganisms that can degrade PAHs under aerobic conditions have already been identified, but only a small number of anaerobic cultures that degrade PAHs like naphthalene, 2-methylnaphthalene, and phenanthrene have been isolated so far (17, 20, 24, 31, 46-48, 50, 52, 66). It has been shown that these anaerobic degraders activate aromatic hydrocarbons by very unusual biochemical reactions which differ completely from those of aerobic degradation. The peripheral pathway of 2-methylnaphthalene degradation occurs in analogy to anaerobic toluene degradation by the addition of fumarate to the methyl group, catalyzed by the glycyl radical enzyme naphthyl-2-methyl-succinate synthase (Nms) (Fig. ​(Fig.1)1) (3). In subsequent reactions, naphthyl-2-methyl-succinate is activated to yield the coenzyme A (CoA) ester and oxidized to form naphthyl-2-methylene-succinyl-CoA. The following beta-oxidation of the side chain results in the formation of 2-naphthoyl-CoA and succinate (3, 53). The first three enzyme reactions of this pathway have been measured in vitro (3, 53). Recently, Musat et al. (48) identified the gene coding for the α-subunit of a putative naphthyl-2-methyl-succinate synthase (nmsA) in 2-methylnaphthalene-grown bacterial cultures. The molecular composition of the nmsA gene is analogous to that of the benzylsuccinate synthase α-subunit gene (bssA). The Bss enzyme is a well-investigated close homolog of Nms, catalyzing fumarate addition in the initial reaction of anaerobic toluene degradation (34, 40). Based on findings from comparative sequence studies, glycine radical-catalyzed fumarate addition has been shown to be a widely distributed initial reaction mechanism for anaerobic hydrocarbon degradation involving toluene and 2-methylnaphthalene, n-alkanes (12, 13, 25, 51), m-xylene (33), m- and p-cresols (9), and ethylbenzene (32).Open in a separate windowFIG. 1.Proposed pathway for anaerobic 2-methylnaphthalene degradation and reductive dearomatization of 2-naphthoyl-CoA (3, 4, 53). Genes found in the N47 genome encode the following enzymes (shown in gray boxes): NmsABC, naphthyl-2-methyl-succinate synthase; BnsEF, naphthyl-2-methyl-succinate CoA transferase; BnsG, naphthyl-2-methyl-succinyl-CoA dehydrogenase; BnsH, naphthyl-2-methylene-succinyl-CoA hydratase; BnsCD, naphthyl-2-hydroxymethyl-succinyl-CoA dehydrogenase; BnsAB, naphthyl-2-oxomethyl-succinyl-CoA thiolase; and NcrABCD, 2-naphthoyl-CoA reductase. The position of the double bond is not known for octahydro-2-naphthoyl-CoA. COSCoA, thioester of CoA and the respective carboxyl group.In a process analogous to the anaerobic benzoyl-CoA degradation pathway (7), 2-naphthoyl-CoA is subjected to aromatic ring reduction by a putative naphthoyl-CoA reductase, probably generating 5,6,7,8-tetrahydro-naphthoyl-CoA and further octahydro-2-naphthoic acid (4, 46). In the subsequent reactions, the ring system should be thiolytically cleaved and subjected to beta-oxidation, leading to the formation of acetyl-CoA and CO₂.In contrast to the first enzymatic reaction in the degradation of methylated aromatics, the first enzymatic reaction in anaerobic degradation of unsubstituted aromatic compounds such as naphthalene is still unresolved. In order to determine the initial activation reaction of anaerobic naphthalene degradation, studies based on the analysis of metabolites have been performed. Zhang and Young (66) observed the incorporation of ¹³C-labeled bicarbonate from the buffer into the carboxyl group of 2-naphthoic acid, hypothesizing that carboxylation is the initial activation reaction of anaerobic naphthalene degradation in the culture studied. Recently, Safinowski and Meckenstock (54) identified the deuterated metabolites naphthyl-2-methyl-succinate and naphthyl-2-methylene-succinate, which are exclusive intermediates of anaerobic 2-methylnaphthalene degradation, in the enrichment culture N47 when the culture was cultivated on fully deuterated naphthalene. Moreover, specific enzyme activities of the anaerobic 2-methylnaphtahlene degradation pathway have been detected in naphthalene-grown cells (54). Therefore, methylation of naphthalene to yield 2-methylnaphthalene as the initial activation reaction and subsequent degradation via the 2-methylnaphthalene pathway were proposed for this bacterial culture. The elucidation of 2-methylnaphthalene degradation may therefore reveal an important part of the naphthalene degradation pathway. However, Musat et al. (48) questioned methylation as the first reaction in naphthalene degradation for their marine naphthalene-degrading deltaproteobacterial NaphS strains.Whereas molecular components involved in anaerobic degradation of monoaromatic hydrocarbons are well known, knowledge about genes and enzymes involved in anaerobic PAH degradation is still missing (14). Here, we provide the first results of a whole-proteome- and whole-genome-based investigation of the sulfate-reducing enrichment culture N47 degrading naphthalene and 2-methylnaphtalene. We have identified some gene clusters encoding enzymes involved in 2-methylnaphthalene degradation, 2-naphthoyl-CoA dearomatization, and subsequent ring cleavage reactions in 2-methylnaphthalene-grown N47 cells.     

Transformation of 1- and 2-methylnaphthalene by Cunninghamella elegans.

Cunninghamella elegans metabolized 1- and 2-methylnaphthalene primarily at the methyl group to form 1- and 2-hydroxymethylnaphthalene, respectively. Other compounds isolated and identified were 1- and 2-naphthoic acids, 5-hydroxy-1-naphthoic acid, 5-hydroxy-2-naphthoic acid, 6-hydroxy-2-naphthoic acid, and phenolic derivatives of 1- and 2-methylnaphthalene. The metabolites were isolated by thin-layer and reverse-phase high-pressure liquid chromatography and characterized by the application of UV-visible absorption, 1H nuclear magnetic resonance, and mass spectral techniques. Experiments with [8-14C]2-methylnaphthalene indicated that over a 72-h period, 9.8% of 2-methylnaphthalene was oxidized to metabolic products. The ratio of organic-soluble in water-soluble metabolites at 2 h was 92:8, and at 72 h it was 41:59. Enzymatic treatment of the 48-h aqueous phase with either beta-glucuronidase or arylsulfatase released 60% of the metabolites of 2-methylnaphthalene that were extractable with ethyl acetate. In both cases, the major conjugates released were 5-hydroxy-2-naphthoic acid and 6-hydroxy-2-naphthoic acid. The ratio of the water-soluble glucuronide conjugates to sulfate conjugates was 1:1. Incubation of C. elegans with 2-methylnaphthalene under an 18O2 atmosphere and subsequent mass spectral analysis of 2-hydroxymethylnaphthalene indicated that hydroxylation of the methyl group is catalyzed by a monooxygenase.     

Identification of new enzymes potentially involved in anaerobic naphthalene degradation by the sulfate-reducing enrichment culture N47

The sulfate-reducing highly enriched culture N47 is capable to anaerobically degrade naphthalene, 2-methylnaphthalene, and 2-naphthoic acid. A proteogenomic investigation was performed to elucidate the initial activation reaction of anaerobic naphthalene degradation. This lead to the identification of an alpha-subunit of a carboxylase protein that was two-fold up-regulated in naphthalene-grown cells compared to 2-methylnaphthalene-grown cells. The putative naphthalene carboxylase subunit showed 48% similarity to the anaerobic benzene carboxylase from an iron-reducing, benzene-degrading culture and 45% to alpha-subunit of phenylphosphate carboxylase of Aromatoleum aromaticum EbN1. A gene for the beta-subunit of putative naphthalene carboxylase was located nearby on the genome and was expressed with naphthalene. Similar to anaerobic benzene carboxylase, there were no genes for gamma- and delta-subunits of a putative carboxylase protein located on the genome which excludes participation in degradation of phenolic compounds. The genes identified for putative naphthalene carboxylase subunits showed only weak similarity to 4-hydroxybenzoate decarboxylase excluding ATP-independent carboxylation. Several ORFs were identified that possibly encode a 2-naphthoate-CoA ligase, which is obligate for activation before the subsequent ring reduction by naphthoyl-CoA reductase. One of these ligases was exclusively expressed on naphthalene and 2-naphthoic acid and might be the responsible naphthoate-CoA-ligase.     

Anaerobic Degradation of p-Xylene by a Sulfate-Reducing Enrichment Culture

A strictly anaerobic enrichment culture was obtained with p-xylene as organic substrate and sulfate as electron acceptor from an aquifer at a former gasworks plant contaminated with aromatic hydrocarbons. p-Xylene was completely oxidized to CO₂. The enrichment culture depended on Fe(II) in the medium as a scavenger of the produced sulfide. 4-Methylbenzylsuccinic acid and 4-methylphenylitaconic acid were identified in supernatants of cultures indicating that degradation of p-xylene was initiated by fumarate addition to one of the methyl groups. Therefore, p-xylene degradation probably proceeds analogously to toluene degradation by Thauera aromatica or anaerobic degradation pathways for o- and m-xylene.     

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.     

Methanogenic toluene metabolism: community structure and intermediates

Toluene is a model compound used to study the anaerobic biotransformation of aromatic hydrocarbons. Reports indicate that toluene is transformed via fumarate addition to form benzylsuccinate or by unknown mechanisms to form hydroxylated intermediates under methanogenic conditions. We investigated the mechanism(s) of syntrophic toluene metabolism by a newly described methanogenic enrichment from a gas condensate-contaminated aquifer. Pyrosequencing of 16S rDNA revealed that the culture was comprised mainly of Clostridiales. The predominant methanogens affiliated with the Methanomicrobiales. Methane production from toluene ranged from 72% to 79% of that stoichiometrically predicted. Isotope studies using (13)C(7) toluene showed that benzylsuccinate and benzoate transiently accumulated revealing that members of this consortium can catalyse fumarate addition and subsequent reactions. Detection of a BssA gene fragment in this culture further supported fumarate addition as a mechanism of toluene activation. Transient formation of cresols, benzylalcohol, hydroquinone and methylhydroquinone suggested alternative mechanism(s) for toluene metabolism. However, incubations of the consortium with (18)O-H(2)O showed that the hydroxyl group in these metabolites did not originate from water and abiotic control experiments revealed abiotic formation of hydroxylated species due to reactions of toluene with sulfide and oxygen. Our results suggest that toluene is activated by fumarate addition, presumably by the dominant Clostridiales.     

Methanogenic biodegradation of two-ringed polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAH) are widespread in methane-rich subsurface environments, such as oil reservoirs and fuel-contaminated aquifers; however, little is known about the biodegradation of these compounds under methanogenic conditions. To assess the metabolism of PAH in the absence of electron acceptors, a crude oil-degrading methanogenic enrichment culture was tested for the ability to biodegrade naphthalene, 1-methylnaphthalene (1-MN), 2-methylnaphthalene (2-MN), and 2, 6-dimethylnaphthalene (2, 6-diMN). When methane was measured as an indicator of metabolism, nearly 400 μmol of methane was produced in the 2-MN- and 2, 6-diMN-amended cultures relative to substrate-unamended controls, which is close to the amount of methane stoichiometrically predicted based on the amount of substrate added (51-56 μmol). In contrast, no substantial methane was produced in the naphthalene- and 1-MN-amended enrichments. In time course experiments, metabolite analysis of enrichments containing 2-MN and 2, 6-diMN revealed the formation of 2-naphthoic acid and 6-methyl-2-naphthoic acid, respectively. Microbial community analysis by 454 pyrosequencing revealed that these PAH-utilizing enrichments were dominated by archaeal members most closely affiliated with Methanosaeta and Methanoculleus species and bacterial members most closely related to the Clostridiaceae, suggesting that these organisms play an important role in the methanogenic metabolism of the substituted naphthalenes in these cultures.     

Anaerobic degradation of benzene, toluene, ethylbenzene, and o-xylene in sediment-free iron-reducing enrichment cultures

Monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylene (BTEX) are widespread contaminants in groundwater. We examined the anaerobic degradation of BTEX compounds with amorphous ferric oxide as electron acceptor. Successful enrichment cultures were obtained for all BTEX substrates both in the presence and absence of AQDS (9,10-anthraquinone-2,6-disulfonic acid). The electron balances showed a complete anaerobic oxidation of the aromatic compounds to CO2. This is the first report on the anaerobic degradation of o-xylene and ethylbenzene in sediment-free iron-reducing enrichment cultures.     

Anaerobic degradation of 1-methylnaphthalene by a member of the <Emphasis Type="Italic">Thermoanaerobacteraceae</Emphasis> contained in an iron-reducing enrichment culture

An anaerobic culture (1MN) was enriched with 1-methylnaphthalene as sole source of carbon and electrons and Fe(OH)₃ as electron acceptor. 1-Naphthoic acid was produced as a metabolite during growth with 1-methylnaphthalene while 2-naphthoic acid was detected with naphthalene and 2-methylnaphthalene. This indicates that the degradation pathway of 1-methylnaphthalene might differ from naphthalene and 2-methylnaphthalene degradation in sulfate reducers. Terminal restriction fragment length polymorphism and pyrosequencing revealed that the culture is mainly composed of two bacteria related to uncultured Gram-positive Thermoanaerobacteraceae and uncultured gram-negative Desulfobulbaceae. Stable isotope probing showed that a ¹³C-carbon label from ¹³C₁₀-naphthalene as growth substrate was mostly incorporated by the Thermoanaerobacteraceae. The presence of putative genes involved in naphthalene degradation in the genome of this organism was confirmed via assembly-based metagenomics and supports that it is the naphthalene-degrading bacterium in the culture. Thermoanaerobacteraceae have previously been detected in oil sludge under thermophilic conditions, but have not been shown to degrade hydrocarbons so far. The second member of the community belongs to the Desulfobulbaceae and has high sequence similarity to uncultured bacteria from contaminated sites including recently proposed groundwater cable bacteria. We suggest that the gram-positive Thermoanaerobacteraceae degrade polycyclic aromatic hydrocarbons while the Desulfobacterales are mainly responsible for Fe(III) reduction.     

Anaerobic Degradation of Benzene, Toluene, Ethylbenzene, and o-Xylene in Sediment-Free Iron-Reducing Enrichment Cultures

Monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylene (BTEX) are widespread contaminants in groundwater. We examined the anaerobic degradation of BTEX compounds with amorphous ferric oxide as electron acceptor. Successful enrichment cultures were obtained for all BTEX substrates both in the presence and absence of AQDS (9,10-anthraquinone-2,6-disulfonic acid). The electron balances showed a complete anaerobic oxidation of the aromatic compounds to CO₂. This is the first report on the anaerobic degradation of o-xylene and ethylbenzene in sediment-free iron-reducing enrichment cultures.     

Evidence for aromatic ring reduction in the biodegradation pathwayof carboxylated naphthalene by a sulfate reducing consortium

Naphthalene was used as a model compound in order to study the anaerobic pathway of polycyclic aromatic hydrocarbon degradation. Previously we had determined that carboxylation is an initial step for anaerobic metabolism of naphthalene, but no other intermediate metabolites were identified (Zhang & Young 1997). In the present study we further elucidate the pathway with the identification of six novel naphthalene metabolites detected when cultures were fed naphthalene in the presence of its analog 1-fluoronaphthalene. Results from cultures supplemented with either deuterated naphthalene or non-deuterated naphthalene plus [¹³C]bicarbonate confirm that the metabolites originated from naphthalene. Three of these metabolites were identified by comparison with the following standards: 2-naphthoic acid (2-NA), 5,6,7,8-tetrahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. The presence of 5,6,7,8-tetrahydro-2-NA as a metabolite of naphthalene degradation indicates that the first reduction reaction occurs at the unsubstituted ring, rather than the carboxylated ring. The overall results suggest that after the initial carboxylation of naphthalene, 2-NA is sequentially reduced to decahydro-2-naphthoic acid through 5 hydrogenation reactions, each of which eliminated one double bond. Incorporation of deuterium atoms from D₂O into 5,6,7,8-tetrahydro-2-naphthoic acid suggests that water is the proton source for hydrogenation.     

Benzylsuccinate Formation as a Means of Anaerobic Toluene Activation by Sulfate-Reducing Strain PRTOL1

Permeabilized cells of toluene-mineralizing, sulfate-reducing strain PRTOL1 catalyzed the addition of toluene to fumarate to form benzylsuccinate under anaerobic conditions. Recent in vitro studies with two toluene-mineralizing, denitrifying bacteria demonstrated the same fumarate addition reaction and indicated that it may be the first step of anaerobic toluene degradation. This study with strain PRTOL1 shows that anaerobic toluene activation by fumarate addition occurs in bacteria as disparate as sulfate-reducing and denitrifying species (members of the delta and beta subclasses of the Proteobacteria, respectively).