Anaerobic Naphthalene Degradation Research Articles

Naphthalene Carboxylation in the Sulfate-Reducing Enrichment Culture N47 Is Proposed to Proceed via 1,3-Dipolar Cycloaddition to the Cofactor Prenylated Flavin Mononucleotide.

Polycyclic aromatic hydrocarbons are persistent pollutants of anthropogenic or natural origin in the environment and accumulate in anoxic habitats. In this study, we investigated the mechanism of the enzyme naphthalene carboxylase as a model reaction for polycyclic aromatic hydrocarbon activation by carboxylation. An enzyme assay was established with cell extracts of the highly enriched culture N47. In assays without addition of ATP, naphthalene carboxylase catalyzed a stable isotope exchange of the carboxyl group of naphthoate with 13C-labeled bicarbonate buffer, which can only occur via a partial backwards reaction of the naphthalene carboxylase reaction to an intermediate that does not include the carboxyl group. Hence, a new carboxyl group from the labeled bicarbonate is added upon forward reaction to the naphthoate. This indicates that the reaction mechanism consists of two or more steps and that at least the latter steps are reversible and ATP independent. Naphthalene carboxylation assays were carried out in deuterated buffer and revealed the incorporation of 0, 1, 2, or 3 deuterium atoms in the final product naphthoyl-coenzyme A, indicating that the reaction is fully reversible. Putative reaction mechanisms were tested by quantum mechanical calculations. The proposed mechanism of the reaction consists of three steps: the activation of the naphthalene by 1,3-dipolar cycloaddition of the cofactor prFMN to naphthalene, release of a proton and rearomatization producing a stable intermediate, and a carboxylation with a reverse 1,3-dipolar cycloaddition and cleavage of the bond to the cofactor producing 2-naphthoate. IMPORTANCE Pollution with polycyclic aromatic hydrocarbons poses a great hazard to humans and animals, with considerable long-term effects. The anaerobic degradation of polycyclic aromatic hydrocarbons in anoxic zones and anaerobic growth of such organisms is very slow, leading to only poor investigation of the degradation pathways, so far. In this work, we elucidated the mechanism of naphthalene carboxylase, a key enzyme in anaerobic naphthalene degradation. This is the first mechanism proposed for a carboxylase targeting nonsubstituted (polycyclic) aromatic compounds and can serve as a model for the initial activation reaction in the anaerobic degradation of benzene or nonsubstituted polycyclic aromatic hydrocarbons, as well as similar enzymatic reactions from the expanding class of UbiD-like (de)carboxylases.

Anaerobic Degradation of Naphthalene and Pyrene by Sulfate-Reducing Cultures Enriched from Former Manufactured Gas Plant Soil

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) under completely anaerobic sulfate-reducing conditions is an energetically challenging process. To date, anaerobic degradations of only two-ringed naphthalene and three-ringed phenanthrene by sediment-free and enriched sulfate-reducing bacteria have been reported. In this study, sulfate-reducing enrichment cultures capable of degrading naphthalene and four-ringed PAH, pyrene, were enriched from a contaminated former gas plant site soil. Bacterial community composition analysis revealed that a naphthalene-degrading enrichment culture, MMNap, was dominated (84.90%) by a Gram-positive endospore-forming member of the genus Desulfotomaculum with minor contribution (8.60%) from a member of Clostridium. The pyrene-degrading enrichment, MMPyr, was dominated (97.40%) by a species of Desulfotomaculum. The sequences representing the Desulfotomaculum phylotypes shared 98.80% similarity to each other. After 150 days of incubation, MMNap degraded 195 µM naphthalene with simultaneous reduction of sulfate and accumulation of sulfide. Similarly, MMPyr degraded 114 µM pyrene during 180 days of incubation with nearly stochiometric sulfate consumption and sulfide accumulation. In both cases, the addition of sulfate reduction inhibitor, molybdate (20 mM), resulted in complete cessation of the substrate utilization and sulfate reduction that clearly indicated the major role of the sulfate-reducing Desulfotomaculum in biodegradation of the two PAHs. This study is the first report on anaerobic pyrene degradation by a matrix-free, strictly anaerobic, and sulfate-reducing enrichment culture.

The 5,6,7,8-Tetrahydro-2-Naphthoyl-Coenzyme A Reductase Reaction in the Anaerobic Degradation of Naphthalene and Identification of Downstream Metabolites.

Anaerobic degradation of polycyclic aromatic hydrocarbons has been investigated mostly with naphthalene as a model compound. Naphthalene degradation by sulfate-reducing bacteria proceeds via carboxylation to 2-naphthoic acid, formation of a coenzyme A thioester, and subsequent reduction to 5,6,7,8-tetrahydro-2-naphthoyl-coenzyme A (THNCoA), which is further reduced to hexahydro-2-naphthoyl-CoA (HHNCoA) by tetrahydronaphthoyl-CoA reductase (THNCoA reductase), an enzyme similar to class I benzoyl-CoA reductases. When analyzing THNCoA reductase assays with crude cell extracts and NADH as electron donor via liquid chromatography-mass spectrometry (LC-MS), scanning for putative metabolites, we found that small amounts of the product of an HHNCoA hydratase were formed in the assays, but the downstream conversion by an NAD+-dependent β-hydroxyacyl-CoA dehydrogenase was prevented by the excess of NADH in those assays. Experiments with alternative electron donors indicated that 2-oxoglutarate can serve as an indirect electron donor for the THNCoA-reducing system via a 2-oxoglutarate:ferredoxin oxidoreductase. With 2-oxoglutarate as electron donor, THNCoA was completely converted and further metabolites resulting from subsequent β-oxidation-like reactions and hydrolytic ring cleavage were detected. These metabolites indicate a downstream pathway with water addition to HHNCoA and ring fission via a hydrolase acting on a β'-hydroxy-β-oxo-decahydro-2-naphthoyl-CoA intermediate. Formation of the downstream intermediate cis-2-carboxycyclohexylacetyl-CoA, which is the substrate for the previously described lower degradation pathway leading to the central metabolism, completes the anaerobic degradation pathway of naphthalene.IMPORTANCE Anaerobic degradation of polycyclic aromatic hydrocarbons is poorly investigated despite its significance in anoxic sediments. Using alternative electron donors for the 5,6,7,8-tetrahydro-2-naphthoyl-CoA reductase reaction, we observed intermediary metabolites of anaerobic naphthalene degradation via in vitro enzyme assays with cell extracts of anaerobic naphthalene degraders. The identified metabolites provide evidence that ring reduction terminates at the stage of hexahydro-2-naphthoyl-CoA and a sequence of β-oxidation-like degradation reactions starts with a hydratase acting on this intermediate. The final product of this reaction sequence was identified as cis-2-carboxycyclohexylacetyl-CoA, a compound for which a further downstream degradation pathway has recently been published (P. Weyrauch, A. V. Zaytsev, S. Stephan, L. Kocks, et al., Environ Microbiol 19:2819-2830, 2017, https://doi.org/10.1111/1462-2920.13806). Our study reveals the first ring-cleaving reaction in the anaerobic naphthalene degradation pathway. It closes the gap between the reduction of the first ring of 2-naphthoyl-CoA by 2-napthoyl-CoA reductase and the lower degradation pathway starting from cis-2-carboxycyclohexylacetyl-CoA, where the second ring cleavage takes place.

Anaerobic degradation of phenanthrene by a sulfate-reducing enrichment culture.

Anaerobic degradation processes are very important to attenuate polycyclic aromatic hydrocarbons (PAHs) in saturated, anoxic sediments. However, PAHs are poorly degradable, leading to very slow microbial growth and thus resulting in only a few cultures that have been enriched and studied so far. Here, we report on a new phenanthrene-degrading, sulfate-reducing enrichment culture, TRIP1. Genome-resolved metagenomics and strain specific cell counting with FISH and flow cytometry indicated that the culture is dominated by a microorganism belonging to the Desulfobacteraceae family (60% of the community) and sharing 93% 16S rRNA sequence similarity to the naphthalene-degrading, sulfate-reducing strain NaphS2. The anaerobic degradation pathway was studied by metabolite analyses and revealed phenanthroic acid as the major intermediate consistent with carboxylation as the initial activation reaction. Further reduced metabolites were indicative of a stepwise reduction of the ring system. We were able to measure the presumed second enzyme reaction in the pathway, phenanthroate-CoA ligase, in crude cell extracts. The reaction was specific for 2-phenanthroic acid and did not transform other isomers. The present study provides first insights into the anaerobic degradation pathways of three-ringed PAHs. The biochemical strategy follows principles known from anaerobic naphthalene degradation, including carboxylation and reduction of the aromatic ring system.

Investigation of the Coupling Mechanism Between Naphthalene Degradation and Denitrification of a Naphthalene Degraded Bacterial Consortium Under Denitrification

To investigate the coupling mechanism between naphthalene degradation and denitrification using a liquid media containing naphthalene (sole carbon source) and nitrate, a naphthalene degradation bacterial consortium under denitrification was enriched from a soil with potential risk of PAH (polycyclic aromatic hydrocarbon) contamination. The bacterial community composition of the enriched consortium was analyzed by Illumina MiSeq Sequencing. Subsquently, the enriched consortium was cultured under anaerobic conditions for 9 days, and the concentrations of electron acceptors (nitrate and nitrite) for denitrification, gaseous reduction products (N2O and N2) involved in denitrification, and abundances of denitrification concerned genes (narG:periplasmic nitrate reeducates gene; nirS:cd1-nitrite reductase gene) were detected at days 1, 3, 7, and 9. The result of Illumina MiSeq Sequencing showed that Pseudomonas (Proteobacteria) was the most dominant genus in this enriched consortium. Under anaerobic conditions, the naphthalene removal rate of this enriched consortium was 49.11% within 9 days. Relative higher naphthalene degradation rates were found both at the beginning (day 1-3) and the end (day 7-9) of incubation, and these were significantly higher than at the middle (day 3-7) of the incubation stage (P<0.05). The concentration of nitrate decreased during the whole culture period, while the concentration of nitrite increased during the initial incubation (day 1-3) and rapidly decreased from day 3 to 9. Furthermore, obvious productions of N2O[3.39 μg ·(L ·h)-1] and N2[8.97 μg ·(L ·h)-1] were only measured at the end of incubation (day 7-9). The abundances of both narG and nirS increased during the incubation, indicating the continuous growth of denitrifiers in the enriched consortium during the incubation period. In summary, this study illustrated that both the nitrate reducing stage and gas producing steps of denitrification could be coupled with anaerobic naphthalene degradation, which might be helpful for a deeper investigation regarding the coupling mechanism between denitrification and anaerobic PAH degradation.

Monitoring Microbial Mineralization Using Reverse Stable Isotope Labeling Analysis by Mid-Infrared Laser Spectroscopy

Assessing the biodegradation of organic compounds is a frequent question in environmental science. Here, we present a sensitive, inexpensive, and simple approach to monitor microbial mineralization using reverse stable isotope labeling analysis (RIL) of dissolved inorganic carbon (DIC). The medium for the biodegradation assay contains regular organic compounds and 13C-labeled DIC with 13C atom fractions (x(13C)DIC) higher than natural abundance (typically 2–50%). The produced CO2 (x(13C) ≈ 1.11%) gradually dilutes the initial x(13C)DIC allowing to quantify microbial mineralization using mass-balance calculations. For 13C-enriched CO2 samples, a newly developed isotope ratio mid-infrared spectrometer was introduced with a precision of x(13C) < 0.006%. As an example for extremely difficult and slowly degradable compounds, CO2 production was close to the theoretical stoichiometry for anaerobic naphthalene degradation by a sulfate-reducing enrichment culture. Furthermore, we could measure the aerobic degradation of dissolved organic carbon (DOC) adsorbed to granular activated carbon in a drinking water production plant, which cannot be labeled with 13C. Thus, the RIL approach can be applied to sensitively monitor biodegradation of various organic compounds under anoxic or oxic conditions.

Conversion of cis-2-carboxycyclohexylacetyl-CoA in the downstream pathway of anaerobic naphthalene degradation.

The cyclohexane derivative cis-2-(carboxymethyl)cyclohexane-1-carboxylic acid [(1R,2R)-/(1S,2S)-2-(carboxymethyl)cyclohexane-1-carboxylic acid] has previously been identified as metabolite in the pathway of anaerobic degradation of naphthalene by sulfate-reducing bacteria. We tested the corresponding CoA esters of isomers and analogues of this compound for conversion in cell free extracts of the anaerobic naphthalene degraders Desulfobacterium strain N47 and Deltaproteobacterium strain NaphS2. Conversion was only observed for the cis-isomer, verifying that this is a true intermediate and not a dead-end product. Mass-spectrometric analyses confirmed that conversion is performed by an acyl-CoA dehydrogenase and a subsequent hydratase yielding an intermediate with a tertiary hydroxyl-group. We propose that a novel kind of ring-opening lyase is involved in the further catabolic pathway proceeding via pimeloyl-CoA. In contrast to degradation pathways of monocyclic aromatic compounds where ring-cleavage is achieved via hydratases, this lyase might represent a new ring-opening strategy for the degradation of polycyclic compounds. Conversion of the potential downstream metabolites pimeloyl-CoA and glutaryl-CoA was proved in cell free extracts, yielding 2,3-dehydropimeloyl-CoA, 3-hydroxypimeloyl-CoA, 3-oxopimeloyl-CoA, glutaconyl-CoA, crotonyl-CoA, 3-hydroxybutyryl-CoA and acetyl-CoA as observable intermediates. This indicates a link to central metabolism via β-oxidation, a non-decarboxylating glutaryl-CoA dehydrogenase and a subsequent glutaconyl-CoA decarboxylase.

Anaerobic naphthalene degradation by sulfate-reducing Desulfobacteraceae from various anoxic aquifers.

Polycyclic aromatic hydrocarbons (PAH) are widespread and persistent environmental contaminants, especially in oxygen-free environments. The occurrence of anaerobic PAH-degrading bacteria and their underlying metabolic pathways are rarely known. In this study, PAH degraders were enriched in laboratory microcosms under sulfate-reducing conditions using groundwater and sediment samples from four PAH-contaminated aquifers. Five enrichment cultures were obtained showing sulfate-dependent naphthalene degradation. Mineralization of naphthalene was demonstrated by the formation of sulfide concomitant with the depletion of naphthalene and the development of (13)C-labeled CO2 from [(13)C6]-naphthalene. 16S rRNA gene and metaproteome analyses revealed that organisms related to Desulfobacterium str. N47 were the main naphthalene degraders in four enrichment cultures. Protein sequences highly similar to enzymes of the naphthalene degradation pathway of N47 were identified, suggesting that naphthalene was activated by a carboxylase, and that the central metabolite 2-naphthoyl-CoA was further reduced by two reductases. The data indicate an importance of members of the family Desulfobacteraceae for naphthalene degradation under sulfate-reducing conditions in freshwater environments.

Two distinct old yellow enzymes are involved in naphthyl ring reduction during anaerobic naphthalene degradation.

The 2-naphthoyl-coenzyme A (NCoA) reductase (NCR) is so far the only characterized enzyme involved in the anaerobic degradation of the environmentally relevant polycyclic aromatic hydrocarbons. The old yellow enzyme (OYE) family member apparently reduced the nonactivated naphthyl ring to 5,6,7,8-tetrahydro-2-napthoyl-CoA (THNCoA). In this work, the candidate genes of three NCRs from the sulphate-reducing, naphthalene-degrading N47 and NaphS2 cultures were expressed in Escherichia coli. The isolated products contained flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) a [4Fe-4S] cluster and catalyzed only the two-electron reduction of NCoA to 5,6-dihydro-2-naphthoyl-CoA (5,6-DHNCoA) at a very negative E°' = -493 mV. All NCRs exhibited high NCoA-forming DHNCoA oxidase activities that are proposed to be involved in oxygen-detoxification during naphthalene degradation. Extracts of N47 and NaphS2 catalyzed the reduction of 5,6-DHNCoA to THNCoA. Genes putatively coding for 5,6-DHNCR from N47 and NaphS2 were heterologously expressed in E. coli. The enriched enzyme products specifically catalyzed the reduction of 5,6-DHNCoA to THNCoA at E°' = -375 mV. With the three NCRs and two 5,6-DHNCRs, five OYEs have been characterized that are involved in the reduction of the nonsubstituted naphthyl-ring system; these unprecedented enzymatic reactions expand our knowledge of the functional diversity of OYE.

A PCR-based assay for the detection of anaerobic naphthalene degradation

Polycyclic aromatic hydrocarbons (PAH) are widespread environmental pollutants of considerable risk to human health. The aerobic degradation of PAH via oxygenase reactions has been studied for several decades. In contrast, it was not until very recent that the first key enzyme involved in anaerobic PAH degradation, the dearomatizing 2-naphthoyl-CoA reductase, was isolated and characterized. In this work, a PCR-based functional assay was developed to detect microorganisms that have the ability to anaerobically degrade naphthalene, as a model for larger PAH. The degenerative oligonucleotide probes introduced here amplified a highly conserved region of the gene encoding 2-naphthoyl-CoA reductase (Ncr) in numerous sulfate-reducing pure cultures and environmental enrichments. The assay provides the first molecular tool for monitoring the anaerobic degradation of a model PAH.

ATP‐dependent/‐independent enzymatic ring reductions involved in the anaerobic catabolism of naphthalene

Polycyclic aromatic hydrocarbons are among the most hazardous environmental pollutants. However, in contrast to aerobic degradation, the respective degradation pathways in anaerobes are greatly unknown which has so far prohibited many environmental investigations. In this work, we studied the enzymatic dearomatization reactions involved in the degradation of the PAH model compounds naphthalene and 2-methylnaphthalene in the sulfate-reducing enrichment culture N47. Cell extracts of N47 grown on naphthalene catalysed the sodium dithionite-dependent four-electron reduction of the key intermediate 2-naphthoyl-coenzyme A (NCoA) to 5,6,7,8-tetrahydro-2-naphthoyl-CoA (THNCoA). The NCoA reductase activity was independent of ATP and was, surprisingly, not sensitive to oxygen. In cell extracts in the presence of various electron donors the product THNCoA was further reduced by a two-electron reaction to most likely a conjugated hexahydro-2-naphthoyl-CoA isomer (HHNCoA). The reaction assigned to THNCoA reductase strictly depended on ATP and was oxygen-sensitive with a half-life time between 30 s and 1 min when exposed to air. The rate was highest with NADH as electron donor. The results indicate that two novel and completely different dearomatizing ring reductases are involved in anaerobic naphthalene degradation. While the THNCoA reducing activity shows some properties of ATP-dependent class I benzoyl-CoA reductases, NCoA reduction appears to be catalysed by a previously unknown class of dearomatizing aryl-carboxyl-CoA reductases.

Identification of naphthalene carboxylase as a prototype for the anaerobic activation of non‐substituted aromatic hydrocarbons

Polycyclic aromatic hydrocarbons such as naphthalene are recalcitrant environmental pollutants that are only slowly metabolized by bacteria under anoxic conditions. Based on metabolite analyses of culture supernatants, carboxylation or methylation of naphthalene have been proposed as initial enzymatic activation reactions in the pathway. However, the extremely slow growth of anaerobic naphthalene degraders with doubling times of weeks and the little biomass obtained from such cultures hindered the biochemical elucidation of the initial activation reaction, so far. Here, we provide biochemical evidence that anaerobic naphthalene degradation is initiated via carboxylation. Crude cell extracts of the sulfate-reducing enrichment culture N47 converted naphthalene and (13)C-labelled bicarbonate to 2-[carboxyl-(13)C]naphthoic acid at a rate of 0.12 nmol min(-1) mg protein(-1) . The enzyme, namely naphthalene carboxylase, catalysed a much faster exchange of (13) C-labelled bicarbonate with the carboxyl group of 2-[carboxyl-(12)C]naphthoic acid at a rate of 3.2 nmol min(-1) mg protein(-1), indicating that the rate limiting step of the carboxylation reaction is the activation of the naphthalene molecule rather than the carboxylation itself. Neither the carboxylation nor the exchange reaction activities necessitate the presence of ATP or divalent metal ions and they were not inhibited by avidin or EDTA. The new carboxylation reaction is unprecedented in biochemistry and opens the door to understand the anaerobic degradation of polycyclic aromatic hydrocarbons which are among the most hazardous environmental contaminants.

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.

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.

Genome Sequence of the Deltaproteobacterial Strain NaphS2 and Analysis of Differential Gene Expression during Anaerobic Growth on Naphthalene

BackgroundAnaerobic polycyclic hydrocarbon (PAH) degradation coupled to sulfate reduction may be an important mechanism for in situ remediation of contaminated sediments. Steps involved in the anaerobic degradation of 2-methylnaphthalene have been described in the sulfate reducing strains NaphS3, NaphS6 and N47. Evidence from N47 suggests that naphthalene degradation involves 2-methylnaphthalene as an intermediate, whereas evidence in NaphS2, NaphS3 and NaphS6 suggests a mechanism for naphthalene degradation that does not involve 2-methylnaphthalene. To further characterize pathways involved in naphthalene degradation in NaphS2, the draft genome was sequenced, and gene and protein expression examined.ResultsDraft genome sequencing, gene expression analysis, and proteomic analysis revealed that NaphS2 degrades naphthoyl-CoA in a manner analogous to benzoyl-CoA degradation. Genes including the previously characterized NmsA, thought to encode an enzyme necessary for 2-methylnaphthalene metabolism, were not upregulated during growth of NaphS2 on naphthalene, nor were the corresponding protein products. NaphS2 may possess a non-classical dearomatizing enzyme for benzoate degradation, similar to one previously characterized in Geobacter metallireducens. Identification of genes involved in toluene degradation in NaphS2 led us to determine that NaphS2 degrades toluene, a previously unreported capacity. The genome sequence also suggests that NaphS2 may degrade other monoaromatic compounds.ConclusionThis study demonstrates that steps leading to the degradation of 2-naphthoyl-CoA are conserved between NaphS2 and N47, however while NaphS2 possesses the capacity to degrade 2-methylnaphthalene, naphthalene degradation likely does not proceed via 2-methylnaphthalene. Instead, carboxylation or another form of activation may serve as the first step in naphthalene degradation. Degradation of toluene and 2-methylnaphthalene, and the presence of at least one bss-like and bbs-like gene cluster in this organism, suggests that NaphS2 degrades both compounds via parallel mechanisms. Elucidation of the key genes necessary for anaerobic naphthalene degradation may provide the ability to track naphthalene degradation through in situ transcript monitoring.

Anaerobic degradation of naphthalene and 2‐methylnaphthalene by strains of marine sulfate‐reducing bacteria

The anaerobic biodegradation of naphthalene, an aromatic hydrocarbon in tar and petroleum, has been repeatedly observed in environments but scarcely in pure cultures. To further explore the relationships and physiology of anaerobic naphthalene-degrading microorganisms, sulfate-reducing bacteria (SRB) were enriched from a Mediterranean sediment with added naphthalene. Two strains (NaphS3, NaphS6) with oval cells were isolated which showed naphthalene-dependent sulfate reduction. According to 16S rRNA gene sequences, both strains were Deltaproteobacteria and closely related to each other and to a previously described naphthalene-degrading sulfate-reducing strain (NaphS2) from a North Sea habitat. Other close relatives were SRB able to degrade alkylbenzenes, and phylotypes enriched anaerobically with benzene. If in adaptation experiments the three naphthalene-grown strains were exposed to 2-methylnaphthalene, this compound was utilized after a pronounced lag phase, indicating that naphthalene did not induce the capacity for 2-methylnaphthalene degradation. Comparative denaturing gel electrophoresis of cells grown with naphthalene or 2-methylnaphthalene revealed a striking protein band which was only present upon growth with the latter substrate. Peptide sequences from this band perfectly matched those of a protein predicted from genomic libraries of the strains. Sequence similarity (50% identity) of the predicted protein to the large subunit of the toluene-activating enzyme (benzylsuccinate synthase) from other anaerobic bacteria indicated that the detected protein is part of an analogous 2-methylnaphthalene-activating enzyme. The absence of this protein in naphthalene-grown cells together with the adaptation experiments as well as isotopic metabolite differentiation upon growth with a mixture of d(8)-naphthalene and unlabelled 2-methylnaphthalene suggest that the marine strains do not metabolize naphthalene by initial methylation via 2-methylnaphthalene, a previously suggested mechanism. The inability to utilize 1-naphthol or 2-naphthol also excludes these compounds as free intermediates. Results leave open the possibility of naphthalene carboxylation, another previously suggested activation mechanism.

Anaerobic degradation of naphthalene by the mixed bacteria under nitrate reducing conditions

Mixed bacteria were enriched from soil samples contaminated with polycyclic aromatic hydrocarbons (PAHs). The anaerobic degradation characteristics by the enriched bacteria with different initial naphthalene concentrations were investigated under nitrate reducing conditions. The results showed that the mixed bacteria could degrade nearly all the naphthalene over the incubations of 25 days when the initial naphthalene concentration was below 30 mg/L. The degradation rates of naphthalene increased with increasing initial concentrations. A high naphthalene concentration of 30 mg/L did not inhibit neither on the bacterial growth nor on the naphthalene degradation ability. The accumulation of nitrite was occurred during the reduction of nitrate, and a nitrite concentration of 50 mg/L had no inhibition effect on the degradation of naphthalene. The calculation of electron balances revealed that most of the naphthalene was oxidized whereas a small proportion was used for cell synthesis.

Anaerobic degradation of naphthalene in a fluvial aquifer: A radiotracer study

A radiotracer study was conducted in a creosote-contaminated aquifer beneath the Fraser River, British Columbia Canada to investigate the in situ degradation of naphthalene. The groundwater is anaerobic, with abundant methane, ferrous iron and carbon dioxide. This study followed earlier work at the site where the contaminant distribution could only be explained by invoking a mass loss through degradation, even though extensive field and laboratory microcosm studies closer to the source zone onshore could not confirm degradation. Accordingly, 14C-naphthalene was injected into the aquifer offshore, further from the source zone where modeling suggested degradation was occurring. During the 230-day monitoring period, 14CO 2 was detected, confirming the degradation of the radio-labeled naphthalene tracer. A zero-order degradation rate of naphthalene of 5 μg/L-day was estimated based on the decrease in 14C-naphthalene concentration with time. While the degradation pathway could not be determined from the radiotracer study alone, the geochemistry of the site suggests that either iron reduction or methanogenesis is the terminal electron accepting processes responsible for naphthalene oxidation.

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.

Combined application of stable carbon isotope analysis and specific metabolites determination for assessing in situ degradation of aromatic hydrocarbons in a tar oil-contaminated aquifer.

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.