Metabolism of Naphthalene, 1-Naphthol, Indene, and Indole by Rhodococcus sp. Strain NCIMB 12038

The regulation of naphthalene and 1-naphthol metabolism in a Rhodococcus sp. (NCIMB 12038) has been investigated. The microorganism utilizes separate pathways for the degradation of these compounds, and they are regulated independently. Naphthalene metabolism was inducible, but not by salicylate, and 1-naphthol metabolism, although constitutive, was also repressed during growth on salicylate. The biochemistry of naphthalene degradation in this strain was otherwise identical to that found in Pseudomonas putida, with salicylate as a central metabolite and naphthalene initially being oxidized via a naphthalene dioxygenase enzyme to cis-(1R,2S)-1,2-dihydroxy-1,2-dihydronaphthalene (naphthalene cis-diol). A dioxygenase enzyme was not expressed under growth conditions which facilitate 1-naphthol degradation. However, biotransformations with indene as a substrate suggested that a monooxygenase enzyme may be involved in the degradation of this compound. Indole was transformed to indigo by both naphthalene-grown NCIMB 12038 and by cells grown in the absence of an inducer. Therefore, the presence of a naphthalene dioxygenase enzyme activity was not necessary for this reaction. Thus, the biotransformation of indole to indigo may be facilitated by another type of enzyme (possibly a monooxygenase) in this organism.     

Growth and plasmid-encoded naphthalene catabolism of Pseudomonas putida in batch culture

Abstract The growth characteristics of Pseudomonas putida plasmid-harbouring strains which catabolize naphthalene via various pathways in batch culture with naphthalene as the sole source of carbon and energy have been investigated. The strains under study were constructed using the host strain P. putida BS394 which contained various naphthalene degradation plasmids. The highest specific growth rate was ensured by the plasmids that control naphthalene catabolism through the meta-pathway of catechol oxidation. The strains metabolizing catechol via the ortho -pathway grew at a lower rate. The lowest growth rate was observed with strain BS291 harbouring plasmid pBS4 which controls naphthalene catabolism via the gentisic acid pathway. Various pathways of naphthalene catabolism appear to allow these strains to grow at various rates which should be taken into account when constructing efficient degraders of polycyclic aromatic compounds.     

Biosurfactant-Mediated Biodegradation of Polycyclic Aromatic Hydrocarbons—Naphthalene

The present study is aimed at the naphthalene degradation with and without biosurfactant produced from Pseudomonas aeruginosa isolated from oil-contaminated soil. The present study was carried out to isolate the bacterial strains for the naphthalene degradation and also for biosurfactant production. The isolated strains were screened for their ability to degrade the naphthalene by the methods of optimum growth rate test and for the production of biosurfactants by cetyltrimethylammonium bromide, blood agar medium, and thin-layer chromatography. The present study also focused on the effect of biosurfactant for the degradation of naphthalene by isolate-1. Two bacterial strains were isolated and screened, one for biodegradation and another for biosurfactant production. The second organism was identified as Pseudomonas aeruginosa by 16S rRNA analysis. The purified biosurfactant reduces the surface tension of water and also forms stable emulsification with hexadecane and kerosene. The end product of naphthalene degradation was estimated as salicylic acid equivalent by spectrophotometric method. The results demonstrated that Pseudomonas aeruginosa has the potential to produce biosurfactant, which enhances the biodegradation of naphthalene. The study reflects the potential use of biosurfactants for an effective bioremediation in the management of contaminated soils.     

Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10(-5)-10(-4) per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.     

The degradation of biphenyl and chlorobiphenyls by mixed bacterial cultures

Abstract Pseudomonas sp. HV3 grows on naphthalene but not on biphenyl, as the sole source of carbon. When the cells of Pseudomonas sp. HV3 grown on naphthalene were shaken with biphenyl as the carbon source in a mineral salt solution, a yellow metabolite identified as the meta -cleavage product of biphenyl was excreted. The degradation of biphenyl stopped here, but was completed if either 2-methyl-4-chlorophenoxy acetic acid (MCPA)-degrading mixed culture or a Nocardia strain was added to the growth solution. Neither of these uses naphthalene or biphenyl as growth substrate. The mixed culture of Pseudomonas sp. HV3 and Nocardia sp. also degrades the commercial polychlorinated biphenyl (PCB) mixture Aroclor 1221. A yellow metabolite was likewise produced in the degradation, and sometimes two different peaks of the yellow metabolite were observed. The gas chromatography-mass spectrometry (GC-MS) analyses showed that 40–87% of Aroclor 1221 was degraded during an incubation time of 6–21 days. Chlorobenzoic acids were found as metabolites.     

Chemotaxis of Pseudomonas spp. to the polyaromatic hydrocarbon naphthalene.

Two naphthalene-degrading bacteria, Pseudomonas putida G7 and Pseudomonas sp. strain NCIB 9816-4, were chemotactically attracted to naphthalene in drop assays and modified capillary assays. Growth on naphthalene or salicylate induced the chemotactic response. P. putida G7 was also chemotactic to biphenyl; other polyaromatic hydrocarbons that were tested did not appear to be chemoattractants for either Pseudomonas strain. Strains that were cured of the naphthalene degradation plasmid were not attracted to naphthalene.     

Physiological role of the novel salicylaldehyde dehydrogenase NahV in mineralization of naphthalene by Pseudomonas putida ND6

The classical salicylaldehyde dehydrogenases found in naphthalene-degrading bacteria are denoted as NahF. In addition to NahF, NahV, and its corresponding gene nahV, were found here in multiple naphthalene-degrading bacteria isolated from industrial wastewater polluted with polycyclic aromatic hydrocarbons (PAHs). In this study, we described for the first time the biological function and regulation model of NahV for the mineralization of naphthalene by P. putida ND6 via the construction of nahF-, nahV- and regulatory gene nahR-deficient strains. The two mutants of salicylaldehyde dehydrogenase genes and wild-type Pseudomonas ND6 were compared with respect to growth rate, naphthalene degradation efficiency, protein expression level, and salicylaldehyde dehydrogenase activity. The data showed that the presence of NahV conferred a physiological advantage on P. putida ND6 for the catabolism of naphthalene in the presence of NahF. NahV could facilitate naphthalene degradation by increasing total salicylaldehyde dehydrogenase activity when both dehydrogenases are present and it could replace the function of NahF when nahF gene is deleted or mutated, thus ensuring mutants could survive in naphthalene-containing environments. To investigate regulation model of NahV, we detected the expression levels and salicylaldehyde dehydrogenase activity in the wild-type and the nahR mutant strains following cultivation in the presence of glucose±salicylate. The data demonstrated that just like the classical salicylaldehyde dehydrogenases, NahF, NahV was induced by salicylate in the presence of NahR.     

施氏假单胞菌YC-YH1的萘降解特性及产物分析

【目的】萘是一种重要的环境污染物,它在环境中的积累会对人类健康造成危害,生物降解是解决这一问题的有效方法。本实验室保存的施氏假单胞菌YC-YH1对萘具有较强的降解能力,在此基础上,研究和分析菌株对萘的降解特性、环境因素对萘降解率的影响以及代谢产物。【方法】本文首先采用单因素实验法研究pH、温度、接种量、萘初始浓度对萘降解率的影响;并在单因素实验结果的基础上,利用Design-Expert 8.0.5软件和Box-Behnken设计对pH、温度、接种量3个影响因素进行响应面优化分析,建立环境因素对萘降解率影响的优化模型。利用LC-MS检测萘降解过程中产生的重要代谢产物,从而推测菌株对萘的代谢途径。【结果】响应面分析结果表明,优化模型极显著(P<0.001),拟合度良好,预测结果可信度高。降解实验证明,在培养温度为32.4 °C、pH为7.10、接种量5.74% (体积比)的优化条件下培养3 d即可将浓度为100 mg/L的萘100%降解。LC-MS分析表明,菌株降解萘的过程中,能够被检测到的主要代谢产物有1,2-二羟基萘、水杨酸、邻苯二酚等。【结论】施氏假单胞菌YC-YH1对萘有高的降解效率,pH、温度、接种量3个因素对菌株的降解率有较大影响。利用响应面法优化菌株对萘的降解条件,能够提高YC-YH1菌株对萘的生物降解性能。初步推测菌株YC-YH1对萘的降解是通过水杨酸途径,萘首先被其代谢为1,2-二羟基萘,而后被转化为水杨酸和邻苯二酚,最后进入三羧酸循环被彻底降解。     

Enhanced tolerance to naphthalene and enhanced rhizoremediation performance for Pseudomonas putida KT2440 via the NAH7 catabolic plasmid

In this work, we explore the potential use of the Pseudomonas putida KT2440 strain for bioremediation of naphthalene-polluted soils. Pseudomonas putida strain KT2440 thrives in naphthalene-saturated medium, establishing a complex response that activates genes coding for extrusion pumps and cellular damage repair enzymes, as well as genes involved in the oxidative stress response. The transfer of the NAH7 plasmid enables naphthalene degradation by P. putida KT2440 while alleviating the cellular stress brought about by this toxic compound, without affecting key functions necessary for survival and colonization of the rhizosphere. Pseudomonas putida KT2440(NAH7) efficiently expresses the Nah catabolic pathway in vitro and in situ, leading to the complete mineralization of [(14)C]naphthalene, measured as the evolution of (14)CO(2), while the rate of mineralization was at least 2-fold higher in the rhizosphere than in bulk soil.     

Physiological role of NahW, the additional salicylate hydroxylase found in Pseudomonas stutzeri AN10

The physiological role of NahW, the second salicylate hydroxylase of Pseudomonas stutzeri AN10, has been analysed by gene mutation and further complementation. When grown on naphthalene as a unique carbon and energy source, the nahW mutant showed a strong decrease in salicylate hydroxylase activity when compared with the wild-type strain, exhibited lower specific growth rates and accumulated salicylate in culture supernatants. Similarly, lower specific growth rates and salicylate accumulation were observed for the nahW mutant when growth on naphthalene supplemented with succinate or pyruvate. When P. stutzeri AN10 was grown in Luria–Bertani medium in the presence of salicylate, or was cultivated on minimal medium supplemented with salicylate as a unique carbon and energy source, an increase in the lag phase and a decrease in the specific growth rate were observed on increasing the salicylate concentrations, suggesting a plausible toxic effect. This toxic effect of salicylate was much more evident for the nahW mutant than for the wild-type strain. Complementation of the nahW mutant restored all growth parameters. These results indicate that NahW may have two functions in P. stutzeri AN10: (1) to improve its capacity to degrade naphthalene and (2) effectively convert the salicylate produced during naphthalene degradation to tricarboxylic acid cycle intermediates, preventing its toxic effect.     

Efficiency of naphthalene and salicylate degradation by a recombinant Pseudomonas putida mutant strain defective in glucose metabolism

Metabolically engineered microorganisms may have tremendous potential in removing toxic compounds from nature. In general, microorganisms prefer to utilize simpler carbon sources over toxic compounds when both are present in an environment and, therefore, the presence of simpler carbon sources may greatly reduce the efficiency of a microorganism towards toxic compounds. If a microorganism is prevented from utilizing simpler carbon sources, thereby making it totally dependent upon the toxic compounds, it should increase the specificity for and efficiency of degradation of the toxic compounds in the presence of other, simpler carbon sources. To test this hypothesis, the efficiency of naphthalene and salicylate degradation in the presence of glucose by a recombinant Pseudomonas putida strain mutated in glucose metabolism was determined and compared to the non-mutated strain. Results obtained indicate that the impairment of glucose metabolism leads to better degradation of naphthalene and salicylate in the presence of glucose.     

Maintenance and induction of naphthalene degradation activity in Pseudomonas putida and an Alcaligenes sp. under different culture conditions.

The expression of xenobiotic-degradative genes in indigenous bacteria or in bacteria introduced into an ecosystem is essential for the successful bioremediation of contaminated environments. The maintenance of naphthalene utilization activity is studied in Pseudomonas putida (ATCC 17484) and an Alcaligenes sp. (strain NP-Alk) under different batch culture conditions. Levels of activity decreased exponentially in stationary phase with half-lives of 43 and 13 h for strains ATCC 17484 and NP-Alk, respectively. Activity half-lives were 2.7 and 5.3 times longer, respectively, in starved cultures than in stationary-phase cultures following growth on naphthalene. The treatment of starved cultures with chloramphenicol caused a loss of activity more rapid than that measured in untreated starved cultures, suggesting a continued enzyme synthesis in starved cultures in the absence of a substrate. Following growth in nutrient medium, activity decreased to undetectable levels in the Alcaligenes sp. but remained at measurable levels in the pseudomonad even after 9 months. The induction of naphthalene degradation activities in these cultures, when followed by radiorespirometry with 14C-labeled naphthalene as the substrate, was consistent with activity maintenance data. In the pseudomonad, naphthalene degradation activity was present constitutively at low levels under all growth conditions and was rapidly (in approximately 15 min) induced to high levels upon exposure to naphthalene. Adaptation in the uninduced Alcaligenes sp. occurred after many hours of exposure to naphthalene. In vivo labeling with 35S, to monitor the extent of de novo enzyme synthesis by naphthalene-challenged cells, provided an independent confirmation of the results.     

Expression of dibenzothiophene-degradative genes in two Pseudomonas species

The genes encoding dibenzothiophene (DBT) degradation in Pseudomonas alcaligenes strain DBT2 were cloned into plasmid pC1 by other workers. This plasmid was conjugally transferred into a spontaneous variant of Pseudomonas sp. HL7b (designated HL7bR) incapable of oxidizing DBT (Dbt- phenotype). Acquisition of plasmid pC1 simultaneously restored oxidation of DBT and naphthalene to the transconjugant, although the primary DBT metabolite produced by transconjugant HL7bR(pC1) corresponded to that produced by wild-type strain DBT2 rather than that from wild-type strain HL7b. Inducers of the naphthalene pathway (naphthalene, salicylic acid, and 2-aminobenzoate) stimulated DBT oxidation in transconjugant HL7bR(pC1) when present at 0.1 mM concentrations but had no effect on wild-type strain HL7b. Higher concentrations (5 mM) of salicylic acid and naphthalene were inhibitory to DBT oxidation in all strains. DNA-DNA hybridization was not observed between plasmid pC1 and genomic DNA from strains HL7b or HL7bR, nor between authentic naphthalene-degradative genes (plasmid NAH2) and either plasmid pC1 or strain HL7b, despite the observation that the degradative genes encoded on plasmid pC1 functionally resembled broad-specificity naphthalene-degradative genes. Transconjugant HL7bR(pC1) is a mosaic of the parental types regarding DBT metabolite production, regulation, and use of carbon sources.     

Microbial cooxidation of naphthalene for the production of 1,2-dihydroxy-1,2-dihydronaphthalene

A microbial cooxidation process for 1,2-dihydroxy-1,2-dihydronaphthalene from naphthalene has been demonstrated. A Pseudomonas putida it119 mutant strain grown with glucose as the sole carbon and energy source was used to oxidize naphthalene. Growth characteristics of the P. putida mutant strain were studied in both batch and continuous fermentation experiments. The rate of product formation was found to depend on naphthalene particle sizes, initial naphthalene and glucose concentrations. Kinetic models were developed to quantify the microbial cooxidation process and a two-stage fermentation process is proposed for further studies.     

Isolation and characterization of a fluoranthene-utilizing strain of Pseudomonas paucimobilis.

A soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy for growth was purified from a seven-member bacterial community previously isolated from a creosote waste site for its ability to degrade polycyclic aromatic hydrocarbons. By standard bacteriological methods, this bacterium was characterized taxonomically as a strain of Pseudomonas paucimobilis and was designated strain EPA505. Utilization of fluoranthene by strain EPA 505 was demonstrated by increase in bacterial biomass, decrease in aqueous fluoranthene concentration, and transient formation of transformation products in liquid cultures where fluoranthene was supplied as the sole carbon source. Resting cells grown in complex medium showed activity toward anthraquinone, benzo[b]fluorene, biphenyl, chrysene, and pyrene as demonstrated by the disappearance of parent compounds or changes in their UV absorption spectra. Fluoranthene-grown resting cells were active against these compound as well as 2,3-dimethylnaphthalene, anthracene, fluoranthene, fluorene, naphthalene, and phenanthrene. These studies demonstrate that organic compounds not previously reported to serve as growth substrates can be utilized by axenic cultures of microorganisms. Such organisms may possess novel degradative systems that are active toward other compounds whose biological degradation has been limited because of inherent structural considerations or because of low aqueous solubility.     

Isolation and characterization of a fluoranthene-utilizing strain of Pseudomonas paucimobilis

A soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy for growth was purified from a seven-member bacterial community previously isolated from a creosote waste site for its ability to degrade polycyclic aromatic hydrocarbons. By standard bacteriological methods, this bacterium was characterized taxonomically as a strain of Pseudomonas paucimobilis and was designated strain EPA505. Utilization of fluoranthene by strain EPA 505 was demonstrated by increase in bacterial biomass, decrease in aqueous fluoranthene concentration, and transient formation of transformation products in liquid cultures where fluoranthene was supplied as the sole carbon source. Resting cells grown in complex medium showed activity toward anthraquinone, benzo[b]fluorene, biphenyl, chrysene, and pyrene as demonstrated by the disappearance of parent compounds or changes in their UV absorption spectra. Fluoranthene-grown resting cells were active against these compound as well as 2,3-dimethylnaphthalene, anthracene, fluoranthene, fluorene, naphthalene, and phenanthrene. These studies demonstrate that organic compounds not previously reported to serve as growth substrates can be utilized by axenic cultures of microorganisms. Such organisms may possess novel degradative systems that are active toward other compounds whose biological degradation has been limited because of inherent structural considerations or because of low aqueous solubility.     

Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10^?5–10^?4 per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.     

Naphthalene oxidation by a Pseudomonas putida strain carrying a mutant plasmid

Naphthalene oxidation by a parent and a mutant strain of Pseudomonas putida was studied. The parent strain contained a plasmid NPL-1 which controlled oxidation of naphthalene to salicylic acid and was capable of oxidizing salicylate. The mutant strain did not oxidize salicylate because of a mutation in salicylate hydroxylase; it contained also a mutant plasmid NPL-41 which determined constitutive synthesis of naphthalene oxygenase. Salicylic acid which accumulated as a product of naphthalene catabolism in the cultural broth of the wild strain was found to undergo further oxidation by the population of growing cells. The content of salicylic acid in the cultural broth of the mutant strain reached maximum and then remained constant. An anion-exchange resin was tested in order to prevent the inhibition of naphthalene oxygenase by salicylate and to increase the yield of salicylic acid. The transmissible character of the mutant plasmid NPL-41 makes it possible, with the aid of conjugation, to construct Pseudomonas strains which would oxidize naphthalene to salicylic acid without further degradation of this compound.     

Growth kinetics of Pseudomonas putida G7 on naphthalene and occurrence of naphthalene toxicity during nutrient deprivation

The objectives of this work were (1) to demonstrate how the chemostat approach could be modified to allow determination of kinetic parameters for a sparingly soluble, volatile substrate such as naphthalene and (2) to examine the influence of the interactions of various nutrients on possible growth-inhibitory effects of naphthalene. Pseudomonas putida G7 was used as a model naphthalene-degrading microorganism. Naphthalene was found to be toxic to P. putida G7 in the absence of a nitrogen source or oxygen. The death rate of cells grown on minimal medium plus naphthalene and then exposed to naphthalene under anoxic conditions was higher than that observed under oxic conditions in the absence of a nitrogen source. The presence of necessary nutrients for the biodegradation of PAH compounds is indicated to be important for the survival of microorganisms that are capable of PAH degradation. The amounts of ammonia and oxygen necessary for naphthalene biodegradation and for suppression of naphthalene toxicity were calculated from growth yield coefficients. A chemostat culture of P. putida G7 using naphthalene as a carbon and energy source was accomplished by using a feed augmented with a methanol solution of naphthalene so as to provide sufficient growth to allow accurate evaluation of kinetic parameters. When naphthalene was the growth-limiting substrate, the degradation of naphthalene followed Monod kinetics. Maximum specific growth rate (micrometer) and Monod constant (Ks) were 0.627 +/- 0.007 h-1 and 0.234 +/- 0.0185 mg/L, respectively. The evaluation of biodegradation parameters will allow a mathematical model to be applied to predict the long-term behavior of PAH compounds in soil when combined with PAH transport parameters.