Degradation of Car Engine Base Oil by Rhodococcus sp. NDKK48 and Gordonia sp. NDKY76A

Two microorganisms (NDKK48 and NDKY76A) that degrade long-chain cyclic alkanes (c-alkanes) were isolated from soil samples. Strains NDKK48 and NDKY76A were identified as Rhodococcus sp. and Gordonia sp., respectively. Both strains used not only normal alkane (n-alkane) but also c-alkane as a sole carbon and energy source, and the strains degraded more than 27% of car engine base oil (1% addition).     

Degradation of phenanthrene and anthracene by Nocardia otitidiscaviarum strain TSH1, a moderately thermophilic bacterium

Aims:  The metabolism of phenanthrene and anthracene by a moderate thermophilic Nocardia otitidiscaviarum strain TSH1 was examined.
Methods and Results:  When strain TSH1 was grown in the presence of anthracene, four metabolites were identified as 1,2-dihydroxy-1,2-dihydroanthracene, 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, 2,3-dihydroxynaphthalene and benzoic acid using gas chromatography-mass spectrometry (GC-MS), reverse phase-high performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC). Degradation studies with phenanthrene revealed 2,2'-diphenic acid, phthalic acid, 4-hydroxyphenylacetic acid, o -hydroxyphenylacetic acid, benzoic acid, a phenanthrene dihydrodiol, 4-[1-hydroxy(2-naphthyl)]-2-oxobut-3-enoic acid and 1-hydroxy-2-naphthoic acid (1H2NA), as detectable metabolites.
Conclusions:  Strain TSH1 initiates phenanthrene degradation via dioxygenation at the C-3 and C-4 or at C-9 and C-10 ring positions. Ortho -cleavage of the 9,10-diol leads to formation of 2,2'-diphenic acid. The 3,4-diol ring is cleaved to form 1H2NA which can subsequently be degraded through o -phthalic acid pathway. Benzoate does not fit in the previously published pathways from mesophiles. Anthracene metabolism seems to start with a dioxygenation at the 1 and 2 positions and ortho -cleavage of the resulting diol. The pathway proceeds probably through 2,3-dicarboxynaphthalene and 2,3-dihydroxynaphthalene. Degradation of 2,3-dihydroxynaphthalene to benzoate and transformation of the later to catechol is a possible route for the further degradation of anthracene.
Significance and Impact of the Study:  For the first time, metabolism of phenanthrene and anthracene in a thermophilic Nocardia strain was investigated.     

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.     

Degradation of phenanthrene by the rhizobacterium Ensifer meliloti

The biodegradation of the polycyclic aromatic hydrocarbon phenantherene by the rhizobacterial strain Ensifer meliloti P221, isolated from the root zone of plant grown in PAH-contaminated soil was studied. Bacterial growth and phenanthrene degradation under the influence of root-exuded organic acids were also investigated. Analysis of the metabolites produced by the strain by using thin-layer chromatography, gas chromatography, high-pressure liquid chromatography, and mass-spectrometry revealed that phenanthrene is bioconverted via two parallel pathways. The first, major pathway is through terminal aromatic ring cleavage (presumably at the C3–C4 bond) producing benzocoumarin and 1-hydroxy-2-naphthoic acid, whose further degradation with the formation of salicylic acid is difficult or is very slow. The second pathway is through the oxidation of the central aromatic ring at the C9–C10 bond, producing 9,10-dihydro-9,10-dihydroxyphenanthrene, 9,10-phenanthrenequinone, and 2,2′-diphenic acid. This is the first time that the dioxygenation of phenanthrene at the C9 and C10 atoms, proven by identification of characteristic metabolites, has been reported for a bacterium of the Ensifer genus.     

Degradation of biphenyl by Mycobacterium sp. strain PYR-1

The metabolism of biphenyl by Mycobacterium sp. PYR-1 was investigated. The Mycobacterium sp. degraded >98% of the biphenyl added within 72 h. Analysis of ethyl acetate extracts of the culture medium by HPLC indicated that benzoic acid was the major metabolite. Other products were 4-hydroxybiphenyl, 4-hydroxybenzoic acid, and 5-oxo-5-phenylpentanoic acid. The metabolites were characterized by mass and 1H NMR spectrometry. Identification of benzoic acid and 5-oxo-5-phenylpentanoic acid indicates that biphenyl degradation by Mycobacterium sp. PYR-1 is generally similar to known pathways. A novel alternative metabolic pathway consisted of monooxygenation at C-4 of biphenyl to give 4-hydroxybiphenyl, with subsequent degradation via ring cleavage to 4-hydroxybenzoic acid.     

Anaerobic Degradation of m-Cresol by a Sulfate-Reducing Bacterium

m-Cresol metabolism under sulfate-reducing conditions was studied with a pure culture of Desulfotomaculum sp. strain Groll. Previous studies with a sulfate-reducing consortium indicated that m-cresol was degraded via an initial para-carboxylation reaction. However, 4-hydroxy-2-methylbenzoic acid was not degraded by strain Groll, and no evidence for ring carboxylation of m-cresol was found. Strain Groll readily metabolized the putative metabolites of a methyl group oxidation pathway, including 3-hydroxybenzyl alcohol, 3-hydroxybenzaldehyde, 3-hydroxybenzoic acid, and benzoic acid. Degradation of these compounds preceded and inhibited m-cresol decay. 3-Hydroxybenzoic acid was detected in cultures that received either m-cresol or 3-hydroxybenzyl alcohol, and trace amounts of benzoic acid were detected in m-cresol-degrading cultures. Therefore, we propose that strain Groll metabolizes m-cresol by a methyl group oxidation pathway which is an alternate route for the catabolism of this compound under sulfate-reducing conditions.     

Microbial Hydrocarbon Co-oxidation. III. Isolation and Characterization of an alpha, alpha'-Dimethyl-cis, cis-Muconic Acid-producing Strain of Nocardia corallina

A soil isolate identified as a strain of Nocardia corallina accumulated α, α′-dimethyl-cis, cis-muconic acid under co-oxidation conditions employing n-hexadecane for growth and p-xylene as the co-oxidizable substrate. N. corallina V-49 was postulated to have two pathways for the oxidation of p-xylene. One pathway proceeds throughp-benzyl alcohol, p-tolualdehyde, and p-toluic acid to 2, 3-dihydroxy-p-toluic acid, and the other pathway results in ortho ring cleavage of 3, 6-dimethylpyrocatechol and hence accumulation of α,α′-dimethyl-cis, cis-muconic acid.     

Microbial Hydrocarbon Co-oxidation. III. Isolation and Characterization of an α, α′-Dimethyl-cis,cis-Muconic Acid-producing Strain of Nocardia corallina

A soil isolate identified as a strain of Nocardia corallina accumulated α, α′-dimethyl-cis, cis-muconic acid under co-oxidation conditions employing n-hexadecane for growth and p-xylene as the co-oxidizable substrate. N. corallina V-49 was postulated to have two pathways for the oxidation of p-xylene. One pathway proceeds throughp-benzyl alcohol, p-tolualdehyde, and p-toluic acid to 2, 3-dihydroxy-p-toluic acid, and the other pathway results in ortho ring cleavage of 3, 6-dimethylpyrocatechol and hence accumulation of α,α′-dimethyl-cis, cis-muconic acid.     

Aerobic biotransformation of alkyl branched aromatic alkanoic naphthenic acids via two different pathways by a new isolate of Mycobacterium

Naphthenic acids (NAs) are complex mixtures of carboxylic acids found in weathered crude oils and oil sands, and are toxic, corrosive and persistent. However, little is known about the microorganisms and mechanisms involved in NA degradation. We isolated a sediment bacterium (designated strain IS2.3), with 100% 16S rRNA gene sequence identity to Mycobacterium aurum, which degraded synthetic NAs (4'-n-butylphenyl)-4-butanoic acid (n-BPBA) and (4'-t-butylphenyl)-4-butanoic acid (t-BPBA). n-BPBA was readily oxidized with almost complete degradation (96.8% ± 0.3) compared with t-BPBA (77.8% ± 3.7 degraded) by day 49. Cell counts increased fourfold by day 14 but decreased after day 14 for both n- and t-BPBA. At day 14, (4'-butylphenyl)ethanoic acid (BPEA) metabolites were detected. Additional metabolites produced during t-BPBA degradation were identified by mass spectrometry of derivatives as (4'-carboxy-t-butylphenyl)-4-butanoic acid and (4'-carboxy-t-butylphenyl)ethanoic acid; suggesting that strain IS2.3 used omega oxidation of t-BPEA to oxidize the tert-butyl side-chain to produce (4'-carboxy-t-butylphenyl)ethanoic acid, as the primary route for biodegradation. However, strain IS2.3 also produced this metabolite through initial omega oxidation of the tert-butyl side-chain of t-BPBA, followed by beta-oxidation of the alkanoic acid side-chain. In conclusion, an isolate belonging to the genus Mycobacterium degraded highly branched aromatic NAs via two different pathways.     

好氧条件下Sphingomonas sp．XJ1降解DBP途径的研究

在三角瓶中采用Sphingomonas sp．XJ1对邻苯二甲酸丁酯（DBP）进行好氧降解,以考察DBP的降解途径。分别对降解16h、32h和40h的DBP样品进行代谢产物分析,可判定保留时间为4．79min和5．11min所对应的代谢产物分别为原儿茶酸和邻苯二甲酸。由此可知,菌株Sphingomonassp．XJ1对DBP的降解遵循DBP好氧生物降解途径的一般途径。即在菌株XJI的作用下,DBP首先水解为MBP,继而水解为PA,经由PCA最终完全降解为CO2和H2O。     

Oxidative degradation of purines by the faculative phototrophic bacterium Rhodopseudomonas capsulata

The mechanism of purine degradation was studied in the facultative phototrophic bacterium Rhodopseudomonas capsulata. Using tungstate as an inhibitor of synthesis of an active xanthine dehydrogenase it could be shown in growth experiments that purine compounds are transformed to uric acid as central purine intermediate prior to ring cleavage. Because of its rapid degradation, the mechanism of uric acid conversion was investigated using 1-methyluric acid as substrate. The analogue was partially degraded by whole cells yielding 3-methylallantoin and methylurea. This implicated an oxidative degradation of 1-methyluric acid analogous to oxidation of uric acid to allantoin suggesting uric acid degradation via allantoin. In cell-free extracts, allantoinase, allantoicase, ureidoglycolase and urease activities degrading allantoin to NH₃, CO₂ and glyoxylic acid were detected. Apparently, purine degradation in R. capsulata proceeds in a manner similar to many aerobic microorganisms. It is peculiar to this bacterium, however, that the pathway evidently operates also under anaerobic conditions. In cell extracts, oxidation of uric acid was observed which could be increased by addition of cytochrome c. The basis of this stimulation is still unknown.     

Bioconversion of 2-hydroxy-6-oxo-6-(4'-chlorophenyl)hexa-2,4-dienoic acid, the meta-cleavage product of 4-chlorobiphenyl

Bacterial conversion of 4-chlorobiphenyl (4-CB) usually proceeds through a pathway involving an initial oxidation of the unsubstituted ring in the 2,3 position followed by a 1,2 meta-cleavage. The meta-cleavage product (MCP) is converted through a single hydrolysis step into chlorobenzoic acid. However, several other acidic metabolites that were not expected as part of this pathway have already been described. In this paper, we used strains of Pseudomonas putida carrying cloned genes from Pseudomonas testosteroni B-356 that are involved in polychlorinated biphenyl (PCB) degradation to demonstrate that several acidic metabolites found in the culture media of various bacteria grown in the presence of 4-CB result from alternative novel bioconversion pathways of MCP. The degradation products of MCP through these pathways were identified as analogues with saturated or shorter side chains or as 4'-chlorophenyl-2-picolinic acid; pathways leading to their formation are proposed.     

Biodegradation of synthetic pyrethroids by <Emphasis Type="Italic">Ochrobactrum tritici</Emphasis> strain pyd-1

A synthetic pyrethroid (SP)-degrading bacterium, designated pyd-1, was isolated from SPcontaminated soil. Based on its phenotypic and genotypic properties, the strain was identified as Ochrobactrum tritici. Strain pyd-1 was able to degrade a wide range of SPs, and its degradation efficiencies were dependent on the molecular structure of the SP. Interestingly, the strain degraded cis- and trans-permethrin (cypermethrin) at nearly the same rate and possessed approximately equal hydrolysis activities toward the two enantiomers of fenpropathrin. These results suggest that different isomers of SPs are degraded with equal efficiency by strain pyd-1. We studied the metabolic pathway of fenpropathrin degradation in strain pyd-1 by metabolite identification and enzymatic analysis. Fenpropathrin is degraded by hydrolysis of the carboxylester linkage to yield 2,2,3,3-tetramethylcyclopropanecarboxylic acid and 3-phenoxybenzaldehyde, which is converted to 3-phenoxybenzoic acid (PBA). PBA is further metabolized to 4-hydroxy-3-phenoxybenzoic acid (4-hydroxy-PBA). 4-Hydroxy-PBA is oxidized to protocatechuate and p-hydroquinone. Protocatechuate is further oxidized through an ortho-cleavage pathway, and p-hydroquinone is degraded via 1,2,4-benzenetriol.     

Pathways in the degradation of hydrolyzed alcohols of butyl benzyl phthalate in metabolically diverse Gordonia sp. strain MTCC 4818

In the present study, the metabolic pathways involved in the degradation of benzyl alcohol and 1-butanol, the hydrolyzed products of butyl benzyl phthalate, were investigated by the Gordonia sp. strain MTCC 4818. The strain can utilize both benzyl alcohol and 1-butanol individually as sole carbon sources, where benzyl alcohol was found to be metabolized via benzaldehyde, benzoic acid and catechol, which was further degraded by ortho-cleavage dioxygenase to cis,cis-muconic acid and subsequently to muconolactone leading to tricarboxylic acid cycle. On the other hand, 1-butanol was metabolized via butyraldehyde and butyric acid, which was channeled into the tricarboxylic acid cycle via the beta-oxidation pathway. Numbers of dehydrogenases, both NAD+-dependent and NAD+-independent, were found to be involved in the degradation of benzyl alcohol and 1-butanol, where several dehydrogenases exhibited relaxed substrate specificity. Both 2,3- and 3,4-dihydroxybenzoic acids were utilized by the test organism for growth and metabolized by the ortho-cleavage pathway by the cell-free extract of benzoate-grown cells, similar to catechol, suggesting possible broad substrate specificity of the ring cleavage dioxygenase. Moreover, the test organism can utilize various primary and secondary alcohols, aliphatic aldehydes and acids in the C2-C5 range besides n-hexadecane, 1,4-butanediol and cyclohexanol individually as the sole carbon sources indicating metabolic diversity in the Gordonia sp. strain MTCC 4818.     

Degradation of chlorobenzene by strain <Emphasis Type="Italic">Ralstonia pickettii</Emphasis> L2 isolated from a biotrickling filter treating a chlorobenzene-contaminated gas stream

A Ralstonia pickettii species able to degrade chlorobenzene (CB) as the sole source of carbon and energy was isolated from a biotrickling filter used for the removal of CB from waste gases. This organism, strain L2, could degrade CB as high as 220 mg/L completely. Following CB consumption, stoichiometric amounts of chloride were released, and CO₂ production rate up to 80.2% proved that the loss of CB was mainly via mineralization and incorporation into cell material. The Haldane modification of the Monod equation adequately described the relationship between the specific growth rate and substrate concentration. The maximum specific growth rate and yield coefficient were 0.26 h⁻¹ and 0.26 mg of biomass produced/mg of CB consumed, respectively. The pathways for CB degradation were proposed by the identification of metabolites and assay of ring cleavage enzymes in cell extracts. CB was degraded predominantly via 2-chlorophenol to 3-chlorocatechol and also partially via phenol to catechol with subsequent ortho ring cleavage, suggesting partially new pathways for CB-utilizing bacteria.     

Degradation of Mono-chlorinated Dibenzo-p-Dioxins by Janibacter sp. Strain YA Isolated from River Sediment

Strain YA was newly isolated from an enrichment culture of river sediment and was identified as Janibacter sp. It was able to utilize dibenzofuran as the sole source of carbon and energy. Strain YA degraded > 90% of 1-chloro-dibenzo-p-dioxin (1-CDD) and > 80% of 2-chloro-dibenzo-p-dioxin in 18 hours with each initial concentration at 40 mg/L. A novel metabolite, 2-chloro-2′,6-dihydroxydiphenylether, was observed in 1-CDD degradation. From the metabolites detected by gas chromatography–mass spectrometry, strain YA was supposed to have at least two types of oxidation pathways in 1-CDD degradation.     

A pathway for biodegradation of 1-naphthoic acid by Pseudomonas maltophilia CSV89

Pseudomonas maltophilia CSV89, a bacterium isolated from soil in our laboratory, grows on 1-naphthoic acid as the sole source of carbon and energy. To elucidate the pathway for degradation of 1-naphthoic acid, the metabolites were isolated from spent medium, purified by TLC, and characterized by gas chromatography-mass spectrometry. The involvement of various metabolites as intermediates in the pathway was established by demonstrating relevant enzyme activities in cell-free extracts, oxygen uptake and transformation of metabolites by the whole cells. The results obtained from such studies suggest that the degradation of 1-naphthoic acid is initiated by double hydroxylation of the aromatic ring adjacent to the one bearing the carboxyl group, resulting in the formation of 1,2-dihydroxy-8-carboxynaphthalene. The resultant diol was oxidized via 3-formyl salicylate, 2-hydroxyisophthalate, salicylate and catechol to TCA cycle intermediates.     

好氧条件下Sphingomonassp.XJ1降解DBP途径的研究

在三角瓶中采用Sphingomonas sp.XJ1对邻苯二甲酸丁酯(DBP)进行好氧降解,以考察DBP的降解途径。分别对降解16h、32h和40h的DBP样品进行代谢产物分析,可判定保留时间为4.79min和5.11min所对应的代谢产物分别为原儿茶酸和邻苯二甲酸。由此可知,菌株Sphingomonas sp.XJ1对DBP的降解遵循DBP好氧生物降解途径的一般途径。即在菌株XJ1的作用下,DBP首先水解为MBP,继而水解为PA,经由PCA最终完全降解为CO2和H2O。     

Biodegradation of the mixtures of 4-chlorophenol and phenol by Comamonas testosteroni CPW301

A 4-chlorophenol (4-CP)-degrading bacterium, strain CPW301, was isolated from soil and identified as Comamonas testosteroni. This strain dechlorinated and degraded 4-CP via a meta-cleavage pathway. CPW301 could also utilize phenol as a carbon and energy source without the accumulation of any metabolites via the same meta-cleavage pathway. When phenol was added as a additional substrate, CPW301 could degrade 4-CP and phenol simultaneously. The addition of phenol greatly accelerated the degradation of 4-CP due to the increased cell mass. The simultaneous degradation of the 4-CP and phenol is useful not only for enhanced cell growth but also for the bioremediation of both compounds, which are normally present in hazardous waste sites as a mixture.     

Pathways for 3-chloro- and 4-chlorobenzoate degradationin Pseudomonas aeruginosa 3mT

A bacterial isolate, Pseudomonas aeruginosa 3mT, exhibited the ability to degrade high concentrations of 3-chlorobenzoate (3-CBA, 8 g l^-1) and 4-chlorobenzoate (4-CBA 12 g l^-1) (Ajithkumar 1998). In this study, by delineating the initial biochemical steps involved in the degradation of these compounds, we investigated how this strain can do so well. Resting cells, permeabilised cells as well as cell-free extracts failed to dechlorinate both 3-CBA and 4-CBA under anaerobic conditions, whereas the former two readily degraded both compounds under aerobic conditions. Accumulation of any intermediary metabolite was not observed during growth as well as reaction with resting cells under highly aerated conditions. However, on modification of reaction conditions, 3-chlorocatechol (3-CC) and 4-chlorocatechol (4-CC) accumulated in 3-CBA and 4-CBA flasks, respectively. Fairly high titres of pyrocatechase II (chlorocatechol 1,2-dioxygenase) activity were obtained in extracts of cells grown on 3-CBA and 4-CBA. Meta-pyrocatechase (catechol 2,3-dioxygenase) activity against4-CC and catechol, but not against 3-CC, was also detected in low titres. Accumulation of small amounts of 2-chloro-5-hydroxy muconic semialdehyde, the meta-cleavage product of 4-CC, was detected in the medium, when 4-CBA concentration was 4 mM or greater, indicating the presence of a minor meta-pathway in strain 3mT. However, 3-CBA exclusively, and more than 99% of 4-CBA were degraded through the formation of the respective chlorocatechol, via a modified ortho-pathway. This defies the traditional view that the microbes that follow chlorocatechol pathways are not very good degraders of chlorobenzoates. 4-Hydroxybenzoatewas readily (and 3-hydroxybenzoate to a lesser extent) degraded by the strain, through the formation of protocatechuate and gentisate, respectively, as intermediary dihydroxy metabolites.