Naphthalene Degradation Pathway Research Articles

A CoA-Transferase and Acyl-CoA Dehydrogenase Convert 2-(Carboxymethyl)cyclohexane-1-Carboxyl-CoA During Anaerobic Naphthalene Degradation.

The CoA thioester of 2-(carboxymethyl)cyclohexane-1-carboxylic acid has been identified as a metabolite in anaerobic naphthalene degradation by the sulfate-reducing culture N47. This study identified and characterised two acyl-CoA dehydrogenases (ThnO/ThnT) and an intramolecular CoA-transferase (ThnP) encoded within the substrate-induced thn operon, which contains genes for anaerobic degradation of naphthalene. ThnP is a CoA transferase belonging to the family I (Cat 1 subgroup) that catalyses the intramolecular CoA transfer from the carboxyl group of 2-(carboxymethyl)cyclohexane-1-carboxyl-CoA to its carboxymethyl moiety, forming 2-carboxycyclohexylacetyl-CoA. Neither acetyl-CoA nor succinyl-CoA functions as an exogenous CoA donor for this reaction. The flavin-dependent homotetrameric dehydrogenase ThnO is specific for (1R,2R)-2-carboxycyclohexylacetyl-CoA with an apparent Km value of 61.5 μM, whereas ThnT is a promiscuous enzyme catalysing the same reaction at lower rates. Identifying these three enzymes confirmed the involvement of the thn gene cluster in the anaerobic naphthalene degradation pathway. This study establishes a modified metabolic pathway for anaerobic naphthalene degradation upstream of 2-(carboxymethyl)cyclohexane-1-carboxyl-CoA and provides further insight into the subsequent second-ring cleavage reaction.

The evolution of autonomy from two cooperative specialists in fluctuating environments

From microbes to humans, organisms perform numerous tasks for their survival, including food acquisition, migration, and reproduction. A complex biological task can be performed by either an autonomous organism or by cooperation among several specialized organisms. However, it remains unclear how autonomy and cooperation evolutionarily switch. Specifically, it remains unclear whether and how cooperative specialists can repair deleted genes through direct genetic exchange, thereby regaining metabolic autonomy. Here, we address this question by experimentally evolving a mutualistic microbial consortium composed of two specialists that cooperatively degrade naphthalene. We observed that autonomous genotypes capable of performing the entire naphthalene degradation pathway evolved from two cooperative specialists and dominated the community. This evolutionary transition was driven by the horizontal gene transfer (HGT) between the two specialists. However, this evolution was exclusively observed in the fluctuating environment alternately supplied with naphthalene and pyruvate, where mutualism and competition between the two specialists alternated. The naphthalene-supplied environment exerted selective pressure that favors the expansion of autonomous genotypes. The pyruvate-supplied environment promoted the coexistence and cell density of the cooperative specialists, thereby increasing the likelihood of HGT. Using a mathematical model, we quantitatively demonstrate that environmental fluctuations facilitate the evolution of autonomy through HGT when the relative growth rate and carrying capacity of the cooperative specialists allow enhanced coexistence and higher cell density in the competitive environment. Together, our results demonstrate that cooperative specialists can repair deleted genes through a direct genetic exchange under specific conditions, thereby regaining metabolic autonomy.

Discovery of the 1-naphthylamine biodegradation pathway reveals a broad-substrate-spectrum enzyme catalyzing 1-naphthylamine glutamylation.

1-Naphthylamine (1NA), which is harmful to human and aquatic animals, has been used widely in the manufacturing of dyes, pesticides, and rubber antioxidants. Nevertheless, little is known about its environmental behavior and no bacteria have been reported to use it as the growth substrate. Herein, we describe a pathway for 1NA degradation in the isolate Pseudomonas sp. strain JS3066, determine the structure and mechanism of the enzyme NpaA1 that catalyzes the initial reaction, and reveal how the pathway evolved. From genetic and enzymatic analysis, a five gene-cluster encoding a dioxygenase system was determined to be responsible for the initial steps in 1NA degradation through glutamylation of 1NA. The γ-glutamylated 1NA was subsequently oxidized to 1,2-dihydroxynaphthalene which was further degraded by the well-established pathway of naphthalene degradation via catechol. A glutamine synthetase-like (GS-like) enzyme (NpaA1) initiates 1NA glutamylation, and this enzyme exhibits a broad substrate selectivity toward a variety of anilines and naphthylamine derivatives. Structural analysis revealed that the aromatic residues in the 1NA entry tunnel and the V201 site in the large substrate-binding pocket significantly influence NpaA1's substrate preferences. The findings enhance understanding of degrading polycyclic aromatic amines, and will also enable the application of bioremediation at naphthylamine contaminated sites.

Mechanistic insights into the activation of nano calcium peroxide by pyrite coupled with citric acid for efficient naphthalene degradation: Performance and kinetics

In this research, pyrite was applied in synergy with citric acid for the activation of homemade nano-calcium peroxide to degrade naphthalene in aqueous solution. Results showed that over 90% of naphthalene could be removed within 180 min at a pH range of 3–9 due to the proton-releasing ability of pyrite. Hydroxyl radical, alkyl radical, and singlet oxygen were all detected by electron paramagnetic resonance spectra, with hydroxyl radical playing a dominant role in naphthalene degradation. The nano-calcium peroxide was mainly activated by Fe(II) leached from pyrite in homogeneous ways and the vital role of Fe(III) was also revealed. The superior naphthalene degradation in this system was attributed to the excellent electron-donating capacity of reductive sulfur species in Fe(II)/Fe(III) cycle and the increased concentration of available iron species by the chelating effect of citric acid. Meanwhile, the system showed high tolerance to complex water matrixes and excellent degradation performance for other polycyclic aromatic hydrocarbons and chlorinated olefins. In addition, the naphthalene degradation pathways and the toxicity of detected intermediates were investigated. This research revealed the mechanism of nano-calcium peroxide activation by pyrite coupled with citric acid and demonstrated the great application potential of this system in the treatment of polluted groundwater.

Molecular Basis and Evolutionary Origin of 1-Nitronaphthalene Catabolism in Sphingobium sp. Strain JS3065.

Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) enter the environment from natural sources and anthropogenic activities. To date, microorganisms able to mineralize nitro-PAHs have not been reported. Here, Sphingobium sp. strain JS3065 was isolated by selective enrichment for its ability to grow on 1-nitronaphthalene as the sole carbon, nitrogen, and energy source. Analysis of the complete genome of strain JS3065 indicated that the gene cluster encoding 1-nitronaphthalene catabolism (nin) is located on a plasmid. Based on the genetic and biochemical evidence, the nin genes share an origin with the nag-like genes encoding naphthalene degradation in Ralstonia sp. strain U2. The initial step in degradation of 1-nitronaphthalene is catalyzed by a three-component dioxygenase, NinAaAbAcAd, resulting in formation of 1,2-dihydroxynaphthalene which is also an early intermediate in the naphthalene degradation pathway. Introduction of the ninAaAbAcAd genes into strain U2 enabled its growth on 1-nitronaphthalene. Phylogenic analysis of NinAc suggested that an ancestral 1-nitronaphthalene dioxygenase was an early step in the evolution of nitroarene dioxygenases. Based on bioinformatic analysis and enzyme assays, the subsequent assimilation of 1,2-dihydroxynaphthalene seems to follow the well-established pathway for naphthalene degradation by Ralstonia sp. strain U2. This is the first report of catabolic pathway for 1-nitronaphthalene and is another example of how expanding the substrate range of Rieske type dioxygenase enables bacteria to grow on recalcitrant nitroaromatic compounds. IMPORTANCE Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) have been widely detected in the environment and they are more toxic than their corresponding parent PAHs. Although biodegradation of many PAHs has been extensively described at genetic and biochemical levels, little is known about the microbial degradation of nitro-PAHs. This work reports the isolation of a Sphingobium strain growing on 1-nitronaphthalene and the genetic basis for the catabolic pathway. The pathway evolved from an ancestral naphthalene catabolic pathway by a remarkably small modification in the specificity of the initial dioxygenase. Data presented here not only shed light on the biochemical processes involved in the microbial degradation of globally important nitrated polycyclic aromatic hydrocarbons, but also provide an evolutionary paradigm for how bacteria evolve a novel catabolic pathway with minimal alteration of preexisting pathways for natural organic compounds.

A mutagenic analysis of NahE, a hydratase-aldolase in the naphthalene degradative pathway

NahE is a hydratase-aldolase that converts o-substituted trans-benzylidenepyruvates (H, OH, or CO2−) to benzaldehyde, salicylaldehyde, or 2-carboxybenzaldehyde, respectively, and pyruvate. The enzyme is in a bacterial degradative pathway for naphthalene, which is a toxic and persistent environmental contaminant. Sequence, crystallographic, and mutagenic analysis identified the enzyme as a member of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. As such, it has a conserved lysine (Lys183) and tyrosine (Tyr155), for Schiff base formation, as well as a GXXGE motif for binding of the pyruvoyl carboxylate group. A crystal structure of the selenomethionine derivative of NahE shows these active site elements along with nearby residues that might be involved in the mechanism and/or specificity. Mutations of five active site amino acids (Thr65, Trp128, Tyr155, Asn157, and Asn281) were constructed and kinetic parameters measured in order to assess the effect(s) on catalysis. The results show that the two Trp128 mutants (Phe and Tyr) have the least effect on catalysis, whereas amino acids with bulky side chains at Thr65 (Val) and Asn281 (Leu) have the greatest effect. Changing Tyr155 to Phe and Asn157 to Ala also hinders catalysis, and the effects fall in between these extremes. These observations are put into a structural context using a crystal structure of the Schiff base of the reaction intermediate. Trapping experiments with substrate, Na(CN)BH3, and wild type enzyme and selected mutants mostly paralleled the kinetic analysis, and identified two salicylaldehyde-modified lysines: the active site lysine (Lys183) and one outside the active site (Lys279). The latter could be responsible for the observed inhibition of NahE by salicylaldehyde. Together, the results provide new insights into the NahE-catalyzed reaction.

Co-substrate promoting the biodegradation of refractory DOM in semi-coking wastewater: DOM evolution and microbial community

Semi-coking wastewater treatment has become a difficult issue because of its complexity, heterogeneity and toxicity. In this study, co-metabolism with ethanol or sodium acetate as co-substrates was applied to improve the biological degradation of refractory dissolved organic matter (DOM) in semi-coking wastewater. Kinetic model simulation results indicated that both co-substrates addition improved COD removal efficiency and biodegradation rate. Excitation-emission matrix (EEM) spectroscopy and size exclusion chromatography with organic carbon detector (SEC-OCD) analysis were applied to evaluate the DOM biodegradation in terms of fluorescence and molecular weight (MW). Although co-substrates resulted in a comparable enhanced removal efficiency of protein-like and humic-like fluorophores, there are distinct differences in their specific protein-like fluorescent peaks (Peak C1&C2). Ethanol addition was more conducive to the removal of humic substance with apparent molecular weight (AMW) ranging from 1.1 kDa to 20 kDa. Acetate addition resulted in higher removal of building blocks of humic substances (BB, AMW 0.45–1.1 kDa) and proteinaceous biopolymers (BP, AMW >20 kDa). Co-metabolism triggered the shift of microbial community structure and metabolic functions. Using co-occurrence network analysis, nine keystone genera were identified, which maintained the functional stability of bacterial community responding to organic toxicants stress. LefSe analysis elucidated that the potential major genera involved in refractory organics biodegradation with acetate addition were Stenotrophomonas (40.3%) and Pseudomonas (21.2%), while Pseudoxanthomonas (10.4%) with ethanol addition. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt2) analysis showed that benzoate, styrene, xylene and naphthalene degradation pathway were enhanced by acetate addition as compared to ethanol.

Reutilization of waste self-heating pad by loading cobalt: A magnetic and green peroxymonosulfate activator for naphthalene degradation

The disposal and recovery of solid wastes and the remediation of polycyclic aromatic hydrocarbons (PAHs) are the key issues of environmental pollution control. In this study, micro cobalt loaded on iron-carbon-vermiculite composite (Co-ICV) was prepared for the first time by the reutilization of waste self-heating pad as a carrier of cobalt catalyst, which exhibited better performance than bulk cobalt catalyst in peroxymonosulfate (PMS) activation for the degradation of naphthalene (NAP) in water. Above 98% of NAP (2.0 mg/L) was effectively eliminated within 15 min by the Co-ICV (0.2 g/L) activated PMS (0.5 mmol/L) in a pH range of 5.0–9.0. High magnetism and very limited cobalt leaching realized the convenient separation and stable reusability of Co-ICV. Mechanism investigation indicated that Co(II) species were the main active sites to activate PMS decomposition for the generation of SO4•− and •OH, contributing to the rapid degradation of NAP. Meanwhile, the NAP degradation pathways were deduced via combining the identification of intermediates and the calculation of frontier electron densities (FEDs). Furthermore, the ability of the Co-ICV/PMS system for the NAP degradation in actual lake water and the removal of other refractory pollutants demonstrated that the combination of Co-ICV and PMS was a prospective method for the removal of PAHs. Overall, Co-ICV is a green and promising activator of PMS, and the future development will provide more insights into the comprehensive utilization of solid wastes for the remediation of wastewater containing PAHs.

Expression, purification and crystallization of a novel metagenome-derived salicylaldehyde dehydrogenase from Alpine soil.

Salicylaldehyde dehydrogenase (SALD) catalyses the last reaction in the upper pathway of naphthalene degradation: the oxidation of salicylaldehyde to salicylate. This enzyme has been isolated and studied from a few organisms that belong to the betaproteobacteria and gammaproteobacteria, predominantly Pseudomonas putida. Furthermore, there is only one crystal structure of this enzyme, which was obtained from P. putida G7. Here, crystallographic studies and analysis of the crystal structure of an Alpine soil metagenome-derived SALD (SALDAP) from an alphaproteobacterium are presented. The SALDAP gene was discovered using gene-targeted sequence assembly and it was cloned into a pLATE51 vector. The recombinant protein was overexpressed in Escherichia coli BL21 (DE3) cells and the soluble protein was purified to homogeneity. The protein crystallized at 20°C and diffraction data from the crystals were collected at a resolution of 1.9 Å. The crystal belonged to the orthorhombic space group C2221, with unit-cell parameters a = 116.8, b = 121.7, c= 318.0 Å. Analysis of the crystal structure revealed its conformation to be similar to the organization of the aldehyde dehydrogenase superfamily with three domains: the catalytic, NAD+-binding and bridging domains. The crystal structure of NahF from P.putida G7 was found to be the best structural homologue of SALDAP, even though the enzymes share only 48% amino-acid identity. Interestingly, a carboxylic acid (protocatechuic acid) was found to be a putative ligand of the enzyme and differential scanning fluorimetry was employed to confirm ligand binding. These findings open up the possibility of studying the mechanism(s) of product inhibition and biocatalysis of carboxylic acids using this enzyme and other related aldehyde dehydrogenases.

Transcriptomic Analysis of Degradative Pathways for Azo Dye Acid Blue 113 in Sphingomonas melonis B-2 from the Dye Wastewater Treatment Process.

Background: Acid Blue 113 (AB113) is a typical azo dye, and the resulting wastewater is toxic and difficult to remove. Methods: The experimental culture was set up for the biodegradation of the azo dye AB113, and the cell growth and dye decolorization were monitored. Transcriptome sequencing was performed in the presence and absence of AB113 treatment. The key pathways and enzymes involved in AB113 degradation were found through pathway analysis and enrichment software (GO, EggNog and KEGG). Results: S. melonis B-2 achieved more than 80% decolorization within 24 h (50 and 100 mg/L dye). There was a positive relationship between cell growth and the azo dye degradation rate. The expression level of enzymes involved in benzoate and naphthalene degradation pathways (NADH quinone oxidoreductase, N-acetyltransferase and aromatic ring-hydroxylating dioxygenase) increased significantly after the treatment of AB113. Conclusions: Benzoate and naphthalene degradation pathways were the key pathways for AB113 degradation. NADH quinone oxidoreductase, N-acetyltransferase, aromatic ring-hydroxylating dioxygenase and CYP450 were the key enzymes for AB113 degradation. This study provides evidence for the process of AB113 biodegradation at the molecular and biochemical level that will be useful in monitoring the dye wastewater treatment process at the full-scale treatment.

Insights into naphthalene degradation in aqueous solution and soil slurry medium: Performance and mechanisms

The performance of naphthalene (NAP) degradation in peroxodisulfate (PDS) and peroxymonosulfate (PMS) oxidation systems by nano zero valent iron (nZVI) combined with citric acid (CA) activation was reported in aqueous solution and soil slurry medium. The results in aqueous solution tests indicated that 98.1% and 98.9% of NAP were individually degraded in PDS/nZVI/CA and PMS/nZVI/CA systems within 2 h when the dosages of PDS, PMS, nZVI and CA were 1.0 mM, 0.1 mM, 0.2 mM and 0.1 mM, respectively. The consequences of scavenging tests and electron paramagnetic resonance detection demonstrated that HO• and SO4−• were the key factors on NAP removal. The presence of surfactants could consume ROSs and inhibit NAP removal. In addition, GC-MS was applied for the determination of NAP degradation intermediates, and three possible NAP degradation pathways were proposed in PDS oxidation process and two pathways in PMS oxidation process, respectively. The results in soil slurry medium showed that the presence of CA could promote the dissolution of soil minerals and the desorption of NAP from soil medium. 93.5% and 96.8% degradation of NAP were obtained in PDS/nZVI/CA and PMS/nZVI/CA systems within 24 h. Besides, the existence of DOM in soil could promote Fe(II)/Fe(III) cycle and NAP degradation through electron transfer. Based on the NAP degradation performance in the actual groundwater and soil medium, the above findings could provide basis and strong support for the potential application of PDS/nZVI/CA and PMS/nZVI/CA systems in the remediation of NAP contaminated sites.

Salicylate or Phthalate: The Main Intermediates in the Bacterial Degradation of Naphthalene

Polycyclic aromatic hydrocarbons (PAHs) are widely presented in the environment and pose a serious environmental threat due to their toxicity. Among PAHs, naphthalene is the simplest compound. Nevertheless, due to its high toxicity and presence in the waste of chemical and oil processing industries, naphthalene is one of the most critical pollutants. Similar to other PAHs, naphthalene is released into the environment via the incomplete combustion of organic compounds, pyrolysis, oil spills, oil processing, household waste disposal, and use of fumigants and deodorants. One of the main ways to detoxify such compounds in the natural environment is through their microbial degradation. For the first time, the pathway of naphthalene degradation was investigated in pseudomonades. The salicylate was found to be a key intermediate. For some time, this pathway was considered the main, if not the only one, in the bacterial destruction of naphthalene. However, later, data emerged which indicated that gram-positive bacteria in the overwhelming majority of cases are not capable of the formation/destruction of salicylate. The obtained data made it possible to reveal that protocatechoate, phthalate, and cinnamic acids are predominant intermediates in the destruction of naphthalene by rhodococci. Pathways of naphthalene degradation, the key enzymes, and genetic regulation are the main subjects of the present review, representing an attempt to summarize the current knowledge about the mechanism of the microbial degradation of PAHs. Modern molecular methods are also discussed in the context of the development of “omics” approaches, namely genomic, metabolomic, and proteomic, used as tools for studying the mechanisms of microbial biodegradation. Lastly, a comprehensive understanding of the mechanisms of the formation of specific ecosystems is also provided.

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.

Isolation and Characterization of Genes Responsible for Naphthalene Degradation from Thermophilic Naphthalene Degrader, Geobacillus sp. JF8.

Geobacillus sp. JF8 is a thermophilic biphenyl and naphthalene degrader. To identify the naphthalene degradation genes, cis-naphthalene dihydrodiol dehydrogenase was purified from naphthalene-grown cells, and its N-terminal amino acid sequence was determined. Using a DNA probe encoding the N-terminal region of the dehydrogenase, a 10-kb DNA fragment was isolated. Upstream of nahB, a gene for dehydrogenase, there were two open reading frames which were designated as nahAc and nahAd, respectively. The products of nahAc and nahAd were predicted to be alpha and beta subunit of ring-hydroxylating dioxygenases, respectively. Phylogenetic analysis of amino acid sequences of NahB indicated that it did not belong to the cis-dihydrodiol dehydrogenase group that includes those of classical naphthalene degradation pathways. Downstream of nahB, four open reading frames were found, and their products were predicted as meta-cleavage product hydrolase, monooxygenase, dehydrogenase, and gentisate 1,2-dioxygenase, respectively. A reverse transcriptase-PCR analysis showed that transcription of nahAcAd was induced by naphthalene. These findings indicate that we successfully identified genes involved in the upper pathway of naphthalene degradation from a thermophilic bacterium.

Catalytic mechanism for the conversion of salicylate into catechol by the flavin-dependent monooxygenase salicylate hydroxylase.

Salicylate hydroxylase (NahG) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of salicylate into catechol in the naphthalene degradation pathway in Pseudomonas putida G7. We explored the mechanism of action of this enzyme in detail using a combination of structural and biophysical methods. NahG shares many structural and mechanistic features with other versatile flavin-dependent monooxygenases, with potential biocatalytic applications. The crystal structure at 2.0 Å resolution for the apo form of NahG adds a new snapshot preceding the FAD binding in flavin-dependent monooxygenases. The kcat/Km for the salicylate reaction catalyzed by the holo form is >105 M-1 s-1 at pH 8.5 and 25 °C. Hammett plots for Km and kcat using substituted salicylates indicate change in rate-limiting step. Electron-donating groups favor the hydroxylation of salicylate by a peroxyflavin to yield a Wheland-like intermediate, whereas the decarboxylation of this intermediate is faster for electron-withdrawing groups. The mechanism is supported by structural data and kinetic studies at different pHs. The salicylate carboxyl group lies near a hydrophobic region that aids decarboxylation. A conserved histidine residue is proposed to assist the reaction by general base/general acid catalysis.

Modulation of laccase transcriptome during biodegradation of naphthalene by white rot fungus Pleurotus ostreatus.

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) using Pleurotus ostreatus was investigated in the current study along with the expression levels of laccase genes involved in biodegradation under variable conditions. Biodegradation of PAHs (naphthalene, anthracene, and 1,10-phenanthroline) was detected spectrophotometrically. Recorded data revealed that biodegradation of the tested PAHs was time dependent. Elevated level of naphthalene biodegradation (86.47%) was observed compared to anthracene (27.87%) and 1,10-phenanthroline (24.51%) within 3days post incubation. Naphthalene was completely degraded within 5days. Further incubation enhanced the biodegradation of both anthracene and 1,10-phenanthroline until reaches 93.69% and 92.00% biodegradation of the initial concentration within an incubation period of 11 and 14days, respectively. Naphthalene was selected as a PAH model. HPLC and thin layer chromatography of naphthalene biodegradation products at time intervals proposed that naphthalene was first degraded to α- and β-naphthol which was further metabolized to salicylic and benzoic acid. The metabolic pathway of naphthalene degradation by this fungus was elucidated based on the detected metabolites. The expression profile of six laccase isomers was evaluated using real-time PCR. The transcriptome of the fungal laccase isomers recorded higher levels of transcription under optimized fermentation conditions especially in presence of both naphthalene and Tween 80. The accumulation of such useful metabolites from the biodegradation of PAH pollutants recommended white rot fungus as a potential candidate for production of platform chemicals from PAH wastes.

Genome Analysis of Naphthalene-Degrading Pseudomonas sp. AS1 Harboring the Megaplasmid pAS1.

Polycyclic aromatic hydrocarbons (PAHs), including naphthalene, are widely distributed in nature. Naphthalene has been regarded as a model PAH compound for investigating the mechanisms of bacterial PAH biodegradation. Pseudomonas sp. AS1 isolated from an arsenic-contaminated site is capable of growing on various aromatic compounds such as naphthalene, salicylate, and catechol, but not on gentisate. The genome of strain AS1 consists of a 6,126,864 bp circular chromosome and the 81,841 bp circular plasmid pAS1. Pseudomonas sp. AS1 has multiple dioxygenases and related enzymes involved in the degradation of aromatic compounds, which might contribute to the metabolic versatility of this isolate. The pAS1 plasmid exhibits extremely high similarity in size and sequences to the well-known naphthalene-degrading plasmid pDTG1 in Pseudomonas putida strain NCIB 9816-4. Two gene clusters involved in the naphthalene degradation pathway were identified on pAS1. The expression of several nah genes on the plasmid was upregulated by more than 2-fold when naphthalene was used as a sole carbon source. Strains have been isolated at different times and places with different characteristics, but similar genes involved in the degradation of aromatic compounds have been identified on their plasmids, which suggests that the transmissibility of the plasmids might play an important role in the adaptation of the microorganisms to mineralize the compounds.

Naphthalene biodegradation under oxygen‐limiting conditions: community dynamics and the relevance of biofilm‐forming capacity

SummaryToxic polycyclic aromatic hydrocarbons (PAHs) are frequently released into the environment from anthropogenic sources. PAH remediation strategies focus on biological processes mediated by bacteria. The availability of oxygen in polluted environments is often limited or absent, and only bacteria able to thrive in these conditions can be considered for bioremediation strategies. To identify bacterial strains able to degrade PAHs under oxygen‐limiting conditions, we set up enrichment cultures from samples of an oil‐polluted aquifer, using either anoxic or microaerophilic condition and with PAHs as the sole carbon source. Despite the presence of a significant community of nitrate‐reducing bacteria, the initial community, which was dominated by Betaproteobacteria, was incapable of PAH degradation under strict anoxic conditions, although a clear shift in the structure of the community towards an increase in the Alphaproteobacteria (Sphingomonadaceae), Actinobacteria and an uncultured group of Acidobacteria was observed in the enrichments. In contrast, growth under microaerophilic conditions with naphthalene as the carbon source evidenced the development of a biofilm structure around the naphthalene crystal. The enrichment process selected two co‐dominant groups which finally reached 97% of the bacterial communities: Variovorax spp. (54%, Betaproteobacteria) and Starkeya spp. (43%, Xanthobacteraceae). The two dominant populations were able to grow with naphthalene, although only Starkeya was able to reproduce the biofilm structure around the naphthalene crystal. The pathway for naphthalene degradation was identified, which included as essential steps dioxygenases with high affinity for oxygen, showing 99% identity with Xanthobacter polyaromaticivorans dbd cluster for PAH degradation. Our results suggest that the biofilm formation capacity of Starkeya provided a structure to allocate its cells at an appropriate distance from the toxic carbon source.

Liquid-phase oxidation of naphthalene with $$\hbox {H}_{2}\hbox {O}_{2}$$ H 2 O 2 in the presence of ordered mesoporous $$\hbox {V-}m\hbox {-Al}_{2}\hbox {O}_{3}$$ V- m -Al 2 O 3 catalysts

The ordered mesoporous $$\hbox {V-}m\hbox {-Al}_{2}\hbox {O}_{3}$$ catalysts were successfully synthesized via a facile one-pot evaporation-induced self-assembly (EISA) strategy and applied in the liquid-phase oxidation of naphthalene with hydrogen peroxide in the presence of ascorbic acid as a reductant. The physicochemical properties of the catalysts were investigated using various techniques, like XRD, $$\hbox {N}_{2}$$ sorption, UV-Vis spectra, Raman spectroscopy, XPS, XRF and TEM. Small-angle XRD, $$\hbox {N}_{2}$$ sorption and TEM results show that mesoporous $$\hbox {V-}m\hbox {-Al}_{2}\hbox {O}_{3}$$ catalysts possess a highly ordered mesostructure with large surface areas and narrow pore-size distributions. High-angle XRD, UV-Vis spectra and Raman spectroscopy results indicate that $$\hbox {VO}x$$ species were homogeneously incorporated in the matrix of mesoporous $$\hbox {Al}_{2}\hbox {O}_{3}$$ .The catalytic performance in the liquid oxidation of naphthalene with $$\hbox {H}_{2}\hbox {O}_{2}$$ over $$8\hbox {V-}m\hbox {-Al}_{2}\hbox {O}_{3}$$ catalyst (naphthalene conversion 45.4% and phthalic anhydride selectivity 61.0%) was higher than other catalysts. The vanadium species incorporated in the $$8\hbox {V-}m\hbox {-Al}_{2}\hbox {O}_{3}$$ sample were stable, and its catalytic stability was kept well even after repeated use for 5 times, which indicates a green and economical pathway for naphthalene degradation. SYNOPSIS In the presence of hydrogen peroxide as oxidant and ascorbic acid as reductant, ordered mesoporous $$\hbox {V-}m\hbox {-Al}_{2}\hbox {O}_{3}$$ catalysts were synthesized via a facile one-pot evaporation-induced self-assembly method and successfully applied to the liquid-phase oxidation of naphthalene.

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.