Microbial biodegradation of aromatic alkanoic naphthenic acids is affected by the degree of alkyl side chain branching

Naphthenic acids (NAs) occur naturally in oil sands and enter the environment through natural and anthropogenic processes. NAs comprise toxic carboxylic acids that are difficult to degrade. Information on NA biodegradation mechanisms is limited, and there are no studies on alkyl branched aromatic alkanoic acid biodegradation, despite their contribution to NA toxicity and recalcitrance. Increased alkyl side chain branching has been proposed to explain NA recalcitrance. Using soil enrichments, we examined the biodegradation of four aromatic alkanoic acid isomers that differed in alkyl side chain branching: (4′-n-butylphenyl)-4-butanoic acid (n-BPBA, least branched); (4′-iso-butylphenyl)-4-butanoic acid (iso-BPBA); (4′-sec-butylphenyl)-4-butanoic acid (sec-BPBA) and (4′-tert-butylphenyl)-4-butanoic acid (tert-BPBA, most branched). n-BPBA was completely metabolized within 49 days. Mass spectral analysis confirmed that the more branched isomers iso-, sec- and tert-BPBA were transformed to their butylphenylethanoic acid (BPEA) counterparts at 14 days. The BPEA metabolites were generally less toxic than BPBAs as determined by Microtox assay. n-BPEA was further transformed to a diacid, showing that carboxylation of the alkyl side chain occurred. In each case, biodegradation of the carboxyl side chain proceeded through beta-oxidation, which depended on the degree of alkyl side chain branching, and a BPBA degradation pathway is proposed. Comparison of 16S rRNA gene sequences at days 0 and 49 showed an increase and high abundance at day 49 of Pseudomonas (sec-BPBA), Burkholderia (n-, iso-, tert-BPBA) and Sphingomonas (n-, sec-BPBA).     

Biodegradation of alkyl branched aromatic alkanoic naphthenic acids by Pseudomonas putida KT2440

The majority of the world’s crude oil reserves consist of highly biodegraded heavy and super heavy crude oils and oil sands that have not yet been fully exploited. These vast resources contain complex mixtures of carboxylic acids known as naphthenic acids (NAs). NAs cause major environmental and economic problems, as they are recalcitrant, corrosive and toxic. Although aromatic acids make up a small proportion of most NA mixtures, they have demonstrable toxicities to some organisms (e.g. some bacteria and algae) and ideally need to be removed or reduced by remediation. The present study analysed the ability of Pseudomonas putida KT2440 to degrade highly recalcitrant aromatic acids, as exemplified by the alkyl phenylalkanoic acid (4′-t-butylphenyl)-4-butanoic acid (t-BPBA) and the more degradable (4′-n-butylphenyl)-4-butanoic acid (n-BPBA). n-BPBA was completely metabolized after 14 days, with the production of a persistent metabolite identified as (4′-n-butylphenyl)ethanoic acid (BPEA) which resulted from removal of two carbon atoms from the carboxyl side chain (beta-oxidation) as observed previously with a mixed consortium. However, when n-BPBA concentration was increased two-fold, degradation decreased by 56% with a concomitant six-fold decrease in cell numbers, suggesting that at greater concentrations, n-BPBA may be toxic to P. putida KT2440. In contrast, P. putida KT2440 was unable to degrade the highly recalcitrant t-BPBA even after 49 days. These findings have implications for NA bioremediation in the environment.     

Degradation pathways of cyclic alkanes in Rhodococcus sp. NDKK48

The degradation pathways for cyclic alkanes (c-alkanes) in Rhodococcus sp. NDKK48 were investigated. Strain NDKK48 used dodecylcyclohexane as a sole carbon and energy source, and five metabolites in the dodecylcyclohexane degradation pathway were detected by gas-chromatography/mass spectra. The metabolites were identified as cyclohexanecarboxylic acid, cyclohexylacetic acid, 1-cyclohexene-1-acetic acid, 4-dodecylcyclohexanol, and 4-dodecylcyclohexanone. The strain degrades dodecylcyclohexane via a ring oxidation pathway and an alkyl side chain oxidation pathway. Cyclohexanecarboxylic acid was further oxidized to muconic acid via 1-cyclohexene-1-carboxylic acid and benzoic acid, and the muconic acid was finally used by strain NDKK48 for growth. Methylcyclohexane and cyclohexane were co-oxidized with hexadecane by strain NDKK48. Methylcyclohexane was degraded via a ring oxidation pathway, and the degradation pathway contained part of the Baeyer-Villiger oxidation for ring cleavage. Cyclohexane was also degraded by the same pathway as methylcyclohexane. Thus, strain NDKK48 has two pathways for the complete degradation of c-alkanes.     

Degradation of naphthenic acids by sediment micro-organisms

AIMS: Naphthenic acids (NAs) are naturally occurring, linear and cyclic carboxylic surfactants associated with the acidic fraction of petroleum. NAs account for most of the acute aquatic toxicity of oil sands process-affected water (OSPW). The toxicity of OSPW can be reduced by microbial degradation. The aim of this research was to determine the extent of NA degradation by sediment microbial communities exposed to varying amounts of OSPW. METHODS AND RESULTS: Eleven wetlands, both natural and process-affected, and one tailings settling pond in Northern Alberta were studied. The natural wetlands and process-affected sites fell into two distinct groups based on their water chemistry. The extent of degradation of a 14C-labelled monocyclic NA surrogate [14C-cyclohexane carboxylic acid (CCA)] was relatively uniform in all sediments (approximately 30%) after 14 days. In contrast, degradation of a bicyclic NA surrogate [14C-decahydronaphthoic acid (DHNA)] was significantly lower in non process-affected sediments. Enrichment cultures, obtained from an active tailings settling pond, using commercially available NAs as the sole carbon source, resulted in the isolation of a co-culture containing Pseudomonas putida and Pseudomonas fluorescens. Quantitative GC-MS analysis showed that the co-culture removed >95% of the commercial NAs, and partially degraded the process NAs from OSPW with a resulting NA profile similar to that from 'aged wetlands'. CONCLUSIONS: Exposure to NAs induced and/or selected micro-organisms capable of more effectively degrading bicyclic NAs. Native Pseudomonas spp. extensively degraded fresh, commercial NA. The recalcitrant NAs resembled those found in process-affected wetlands. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that it may be possible to manipulate the existing environmental conditions to select for a microbial community exhibiting higher rates of NA degradation. This will have significant impact on the design of artificial wetlands for water treatment.     

Study of biochemical pathways and enzymes involved in pyrene degradation by Mycobacterium sp. strain KMS

Pyrene degradation is known in bacteria. In this study, Mycobacterium sp. strain KMS was used to study the metabolites produced during, and enzymes involved in, pyrene degradation. Several key metabolites, including pyrene-4,5-dione, cis-4,5-pyrene-dihydrodiol, phenanthrene-4,5-dicarboxylic acid, and 4-phenanthroic acid, were identified during pyrene degradation. Pyrene-4,5-dione, which accumulates as an end product in some gram-negative bacterial cultures, was further utilized and degraded by Mycobacterium sp. strain KMS. Enzymes involved in pyrene degradation by Mycobacterium sp. strain KMS were studied, using 2-D gel electrophoresis. The first protein in the catabolic pathway, aromatic-ring-hydroxylating dioxygenase, which oxidizes pyrene to cis-4,5-pyrene-dihydrodiol, was induced with the addition of pyrene and pyrene-4,5-dione to the cultures. The subcomponents of dioxygenase, including the alpha and beta subunits, 4Fe-4S ferredoxin, and the Rieske (2Fe-2S) region, were all induced. Other proteins responsible for further pyrene degradation, such as dihydrodiol dehydrogenase, oxidoreductase, and epoxide hydrolase, were also found to be significantly induced by the presence of pyrene and pyrene-4,5-dione. Several nonpathway-related proteins, including sterol-binding protein and cytochrome P450, were induced. A pyrene degradation pathway for Mycobacterium sp. strain KMS was proposed and confirmed by proteomic study by identifying almost all the enzymes required during the initial steps of pyrene degradation.     

Degradation of isooctane by Mycobacterium austroafricanum IFP 2173: growth and catabolic pathway

AIMS: Isooctane (2,2,4-trimethylpentane), a major component of gasoline formulations, is recalcitrant to biodegradation probably because of the quaternary carbon group it contains. Information on the biodegradability of this hydrocarbon is essential to evaluate its fate in the environment. For these reasons, the degradation kinetics and the catabolic pathway of isooctane were investigated in Mycobacterium austroafricanum IFP 2173, the only strain characterized to use it as sole carbon and energy source. METHODS AND RESULTS: The selected strain exhibited a rather moderate maximum growth rate (micromax = 0.053 h(-1)) but degraded isooctane up to 99% with a mineralization yield of 45%, indicating attack of the quaternary carbon group. The GC/MS identification of metabolites, 2,4,4-trimethylpentanoic and dimethylpropanoic (pivalic) acids, which transiently accumulated in the cultures indicated that degradation started from the isopropyl extremity of the molecule and subsequently proceeded by catabolism of the tert-butyl moiety. The degradation of putative metabolic intermediates was investigated. The initial isooctane oxidation system was tentatively characterized. CONCLUSIONS: The isooctane-degrading strain harboured two candidate systems for initial alkane oxidation. Although a cytochrome P450 was induced by isooctane degradation, the functional oxidation system was probably a nonheme alkane monooxygenase as indicated by PCR amplification and RT-PCR expression of an alkB gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Isooctane is a recalcitrant branched alkane. A plausible pathway of its degradation by Myco. austroafricanum was put forward.     

The degradation of α-quaternary nonylphenol isomers by Sphingomonas sp. strain TTNP3 involves a type II ipso-substitution mechanism

The degradation of radiolabeled 4(3′,5′-dimethyl-3′-heptyl)-phenol [nonylphenol (NP)] was tested with resting cells of Sphingomonas sp. strain TTNP3. Concomitantly to the degradation of NP, a metabolite identified as hydroquinone transiently accumulated and short-chain organic acids were then produced at the expense of hydroquinone. Two other radiolabeled isomers of NP, 4(2′,6′-dimethyl-2′-heptyl)-phenol and 4(3′,6′-dimethyl-3′-heptyl)-phenol, were synthesized. In parallel experiments, the 4(2′,6′-dimethyl-2′-heptyl)-phenol was degraded more slowly than the other isomers of NP by strain TTNP3, possibly because of effects of the side-chain structure on the kinetics of degradation. Alkylbenzenediol and alkoxyphenol derivatives identified as metabolites during previous studies were synthesized and tested as substrates. The derivatives were not degraded, which indicated that the mineralization of NP does not proceed via alkoxyphenol as the principal intermediate. The results obtained led to the elucidation of the degradation pathway of NP isomers with a quaternary α-carbon. The proposed mechanism is a type II ipso substitution, leading to hydroquinone and nonanol as the main metabolites and to the dead-end metabolites alkylbenzenediol or alkoxyphenol, depending on the substitution at the α-carbon of the carbocationic intermediate formed.     

Biodegradation of methyl tert-butyl ether and other fuel oxygenates by a new strain,Mycobacterium austroafricanum IFP 2012

A strain that efficiently degraded methyl tert-butyl ether (MTBE) was obtained by initial selection on the recalcitrant compound tert-butyl alcohol (TBA). This strain, a gram-positive methylotrophic bacterium identified as Mycobacterium austroafricanum IFP 2012, was also able to degrade tert-amyl methyl ether and tert-amyl alcohol. Ethyl tert-butyl ether was weakly degraded. tert-Butyl formate and 2-hydroxy isobutyrate (HIBA), two intermediates in the MTBE catabolism pathway, were detected during growth on MTBE. A positive effect of Co2+ during growth of M. austroafricanum IFP 2012 on HIBA was demonstrated. The specific rate of MTBE degradation was 0.6 mmol/h/g (dry weight) of cells, and the biomass yield on MTBE was 0.44 g (dry weight) per g of MTBE. MTBE, TBA, and HIBA degradation activities were induced by MTBE and TBA, and TBA was a good inducer. Involvement of at least one monooxygenase during degradation of MTBE and TBA was shown by (i) the requirement for oxygen, (ii) the production of propylene epoxide from propylene by MTBE- or TBA- grown cells, and (iii) the inhibition of MTBE or TBA degradation and of propylene epoxide production by acetylene. No cytochrome P-450 was detected in MTBE- or TBA-grown cells. Similar protein profiles were obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extracts from MTBE- and TBA-grown cells. Among the polypeptides induced by these substrates, two polypeptides (66 and 27 kDa) exhibited strong similarities with known oxidoreductases.     

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.     

Metabolism of viprostol--a synthetic vasodilator PGE2 analog

Metabolic studies were done with 14C-Viprostol (I) administered by various routes (I.V., oral and topical) to six animal species and to man. Total radioactivity and metabolic profiles were analyzed in plasma, tissues and excreta. The main metabolites were isolated and identified by capillary GC/MS. Plasma and urinary metabolic profiles were qualitatively similar across species, with two major metabolic reactions being predominant: rapid hydrolysis to the pharmacologically active free acid (II) and oxidation of the alpha-chain to dinor and tetranor acids (III, IV). In the monkey and man, reduction of the 9-keto group lead to PGF2 type metabolites (VI-VIII). In the rat, omega oxidation of the beta-chain occurred as well, resulting in the formation of dicarboxylic acids (V).     

Changes in fatty acid composition of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> KB2 during co-metabolic degradation of monochlorophenols

The changes in the cellular fatty acid composition of Stenotrophomonas maltophilia KB2 during co-metabolic degradation of monochlorophenols in the presence of phenol as well as its adaptive mechanisms to these compounds were studied. It was found that bacteria were capable of degrading 4-chlorophenol (4-CP) completely in the presence of phenol, while 2-chlorophenol (2-CP) and 3-chlorophenol (3-CP) they degraded partially. The analysis of the fatty acid profiles indicated that adaptive mechanisms of bacteria depended on earlier exposure to phenol, which isomer they degraded, and on incubation time. In bacteria unexposed to phenol the permeability and structure of their membranes could be modified through the increase of hydroxylated and cyclopropane fatty acids, and straight-chain and hydroxylated fatty acids under 2-CP, 3-CP and 4-CP exposure, respectively. In the exposed cells, regardless of the isomer they degraded, the most important changes were connected with the increase of the contribution of branched fatty acid on day 4 and the content of hydroxylated fatty acids on day 7. The changes, particularly in the proportion of branched fatty acids, could be a good indicator for assessing the progress of the degradation of monochlorophenols by S. maltophilia KB2. In comparison, in phenol-degrading cells the increase of cyclopropane and straight-chain fatty acid content was established. These findings indicated the degradative potential of the tested strain towards the co-metabolic degradation of persistent chlorophenols, and extended the current knowledge about the adaptive mechanisms of these bacteria to such chemicals.     

Degradation of pyrene, benz[a]anthracene, and benzo[a]pyrene by Mycobacterium sp. strain RJGII-135, isolated from a former coal gasification site.

The degradation of three polycyclic aromatic hydrocarbons (PAH), pyrene (PYR), benz[a]anthracene (BAA), and benzo[a]pyrene (BaP), by Mycobacterium sp. strain RJGII-135 was studied. The bacterium was isolated from an abandoned coal gasification site soil by analog enrichment techniques and found to mineralize [14C]PYR. Further degradation studies with PYR showed three metabolites formed by Mycobacterium sp. strain RJGII-135, including 4,5-phenanthrene-dicarboxylic acid not previously isolated, 4-phenanthrene-carboxylic acid, and 4,5-pyrene-dihydrodiol. At least two dihydrodiols, 5,6-BAA-dihydrodiol and 10,11-BAA-dihydrodiol, were confirmed by high-resolution mass spectral and fluorescence analyses as products of the biodegradation of BAA by Mycobacterium sp. strain RJGII-135. Additionally, a cleavage product of BAA was also isolated. Mass spectra and fluorescence data support two different routes for the degradation of BaP by Mycobacterium sp. strain RJGII-135. The 7,8-BaP-dihydrodiol and three cleavage products of BaP, including 4,5-chrysene-dicarboxylic acid and a dihydro-pyrene-carboxylic acid metabolite, have been isolated and identified as degradation products formed by Mycobacterium sp. strain RJGII-135. These latter results represent the first example of the isolation of BaP ring fission products formed by a bacterial isolate. We propose that while this bacterium appears to attack only one site of the PYR molecule, it is capable of degrading different sites of the BAA and BaP molecules, and although the sites of attack may be different, the ability of this bacterium to degrade these PAH is well supported. The proposed pathways for biodegradation of these compounds by this Mycobacterium sp. strain RJGII-135 support the dioxygenase enzymatic processes reported previously for other bacteria. Microorganisms like Mycobacterium sp. strain RJGII-135 will be invaluable in attaining the goal of remediation of sites containing mixtures of these PAH.     

Production of metabolites in the biodegradation of phenanthrene, fluoranthene and pyrene by the mixed culture of Mycobacterium sp. and Sphingomonas sp

The effects of the mixed culture of Mycobacterium sp. strain A1-PYR and Sphingomonas sp. strain PheB4 on the degradation characteristics of single polycyclic aromatic hydrocarbon were investigated. In the mixed bacterial culture, phenanthrene, fluoranthene and pyrene were degraded by 100% at Day 3, 71.2% and 50% at Day 7, respectively. Compared to their respective pure cultures, the degradation of phenanthrene and fluoranthene decreased, but that of pyrene increased significantly. Based on GC-MS analysis, eight and six new metabolites were produced from the biodegradation of phenanthrene and fluoranthene, respectively, while only two new metabolites were formed from pyrene. To our knowledge, this is the first report that the mixed bacterial culture could increase the diversity of metabolites from PAH, but the diverse metabolite pattern was not necessarily beneficial to the degradation of the recalcitrant PAH. The enhancement on pyrene degradation was possibly attributed to the rapid growth of strain PheB4.     

Species differences in the metabolism of norephedrine in man, rabbit and rat

1. (+/-)-2-Amino-1-phenyl[1-(14)C]propan-1-ol ([(14)C]norephedrine) was administered orally to man, rat and rabbit and the metabolites excreted in the urine were identified and measured. Pronounced species differences in the metabolism of the drug were found. 2. Three male human subjects, receiving 25mg each of [(14)C]norephedrine hydrochloride, excreted over 90% of the (14)C in the first day. The main metabolite was the unchanged drug (86% of the dose) and minor metabolites were hippuric acid and 4-hydroxynorephedrine. 3. In rats given 12mg of the drug/kg almost 80% of the (14)C administered was excreted in the first day. The major metabolites in the urine were the unchanged drug (48% of the dose), 4-hydroxynorephedrine (28%) and trace amounts of side-chain degradation products. 4. Rabbits given 12mg of the drug/kg excreted 85-95% of the dose of (14)C in the urine in the first 24h after dosing. The major metabolites in the urine were conjugates of 1,2-dihydroxy-1-phenylpropane (31% of the dose) and of 1-hydroxy-1-phenylpropan-2-one (27%) and hippuric acid (20%). The unchanged drug was excreted in relatively small amounts (8%).     

Roles of tert-butyl formate,tert-butyl alcohol and acetone in the regulation of methyl tert-butyl ether degradation by Mycobacterium austroafricanum IFP 2012

Mycobacterium austroafricanum IFP 2012 is a Gram-positive strain able to grow on methyl tert-butyl ether (MTBE) as a sole carbon and energy source. The effect of two downstream metabolites of MTBE, tert-butyl formate (TBF) and tert-butyl alcohol (TBA) on MTBE degradation was investigated using resting cells. The addition of low concentrations of TBF decreased the MTBE degradation rate by about 30%. In contrast, the addition of TBA did not have a significant effect on MTBE degradation rate, even at high concentrations; and it was also shown that TBA degradation occurred only once MTBE was exhausted. At neutral pH, TBF hydrolysis involved mainly an esterase-type activity regulated by the presence of TBA. The TBF degradation rate was about four times lower than the MTBE degradation rate. Furthermore, acetone was identified as an intermediate during TBA degradation. An acetone mono-oxygenase activity, inhibited by methimazole but not by acetylene, was suggested. It was different from the MTBE/TBA mono-oxygenase and, thus, acetone did not appear to compete with MTBE and TBA for the same enzyme. These new results show that the metabolic regulation of the early steps of MTBE degradation by M. austroafricanum IFP 2012 is complex, involving inhibition and competition phenomena.     

Degradation of anthracene by Mycobacterium sp. strain LB501T proceeds via a novel pathway,through o-phthalic acid

Mycobacterium sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of o-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through o-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in Mycobacterium sp. LB501T.     

Aerobic biodegradation of methyl tert-butyl ether by aquifer bacteria from leaking underground storage tank sites.

The potential for aerobic methyl tert-butyl ether (MTBE) degradation was investigated with microcosms containing aquifer sediment and groundwater from four MTBE-contaminated sites characterized by oxygen-limited in situ conditions. MTBE depletion was observed for sediments from two sites (e.g., 4.5 mg/liter degraded in 15 days after a 4-day lag period), whereas no consumption of MTBE was observed for sediments from the other sites after 75 days. For sediments in which MTBE was consumed, 43 to 54% of added [U-(14)C]MTBE was mineralized to (14)CO(2). Molecular phylogenetic analyses of these sediments indicated the enrichment of species closely related to a known MTBE-degrading bacterium, strain PM1. At only one site, the presence of water-soluble gasoline components significantly inhibited MTBE degradation and led to a more pronounced accumulation of the metabolite tert-butyl alcohol. Overall, these results suggest that the effects of oxygen and water-soluble gasoline components on in situ MTBE degradation will vary from site to site and that phylogenetic analysis may be a promising predictor of MTBE biodegradation potential.     

Biodegradation of ether pollutants by Pseudonocardia sp. strain ENV478

A bacterium designated Pseudonocardia sp. strain ENV478 was isolated by enrichment culturing on tetrahydrofuran (THF) and was screened to determine its ability to degrade a range of ether pollutants. After growth on THF, strain ENV478 degraded THF (63 mg/h/g total suspended solids [TSS]), 1,4-dioxane (21 mg/h/g TSS), 1,3-dioxolane (19 mg/h/g TSS), bis-2-chloroethylether (BCEE) (12 mg/h/g TSS), and methyl tert-butyl ether (MTBE) (9.1 mg/h/g TSS). Although the highest rates of 1,4-dioxane degradation occurred after growth on THF, strain ENV478 also degraded 1,4-dioxane after growth on sucrose, lactate, yeast extract, 2-propanol, and propane, indicating that there was some level of constitutive degradative activity. The BCEE degradation rates were about threefold higher after growth on propane (32 mg/h/g TSS) than after growth on THF, and MTBE degradation resulted in accumulation of tert-butyl alcohol. Degradation of 1,4-dioxane resulted in accumulation of 2-hydroxyethoxyacetic acid (2HEAA). Despite its inability to grow on 1,4-dioxane, strain ENV478 degraded this compound for > 80 days in aquifer microcosms. Our results suggest that the inability of strain ENV478 and possibly other THF-degrading bacteria to grow on 1,4-dioxane is related to their inability to efficiently metabolize the 1,4-dioxane degradation product 2HEAA but that strain ENV478 may nonetheless be useful as a biocatalyst for remediating 1,4-dioxane-contaminated aquifers.     

Biodegradation of the gasoline oxygenates methyl tert-butyl ether, ethyl tert-butyl ether, and tert-amyl methyl ether by propane-oxidizing bacteria.

Several propane-oxidizing bacteria were tested for their ability to degrade gasoline oxygenates, including methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME). Both a laboratory strain and natural isolates were able to degrade each compound after growth on propane. When propane-grown strain ENV425 was incubated with 20 mg of uniformly labeled [14C]MTBE per liter, the strain converted > 60% of the added MTBE to 14CO2 in < 30 h. The initial oxidation of MTBE and ETBE resulted in the production of nearly stoichiometric amounts of tert-butyl alcohol (TBA), while the initial oxidation of TAME resulted in the production of tert-amyl alcohol. The methoxy methyl group of MTBE was oxidized to formaldehyde and ultimately to CO2. TBA was further oxidized to 2-methyl-2-hydroxy-1-propanol and then 2-hydroxy isobutyric acid; however, neither of these degradation products was an effective growth substrate for the propane oxidizers. Analysis of cell extracts of ENV425 and experiments with enzyme inhibitors implicated a soluble P-450 enzyme in the oxidation of both MTBE and TBA. MTBE was oxidized to TBA by camphor-grown Pseudomonas putida CAM, which produces the well-characterized P-450cam, but not by Rhodococcus rhodochrous 116, which produces two P-450 enzymes. Rates of MTBE degradation by propane-oxidizing strains ranged from 3.9 to 9.2 nmol/min/mg of cell protein at 28 degrees C, whereas TBA was oxidized at a rate of only 1.8 to 2.4 nmol/min/mg of cell protein at the same temperature.     

Fluoranthene metabolism and associated proteins in Mycobacterium sp. JS14

Fluoranthene is a polycyclic aromatic hydrocarbon (PAH) commonly present in PAH-contaminated soils. We studied fluoranthene catabolism and associated proteins in Mycobacterium sp. JS14, a bacterium isolated from a PAH-contaminated soil in Hilo (HI, USA). Fluoranthene degrades in at least three separated pathways via 1-indanone, 2',3'-dihydroxybiphenyl-2,3,-dicarboxylic acid, and naphthalene-1,8-dicarboxylic acid. Part of the diverse catabolism is converged into phthalate catabolism. An increased expression of 25 proteins related to fluoranthene catabolism is found with 1-D PAGE or 2-DE and nano-LC-MS/MS. Detection of fluoranthene catabolism associated proteins coincides well with its multiple degradation pathways that are mapped via metabolites identified. Among the up-regulated proteins, PAH ring-hydroxylating dioxygenase alpha-subunit and beta-subunit and 2,3-dihydroxybiphenyl 1,2-dioxygenase are notably induced. The up-regulation of trans-2-carboxybenzalpyruvate hydratase suggests that some of fluoranthene metabolites may be further degraded through aromatic dicarboxylic acid pathways. Catalase and superoxide dismutase were up-regulated to control unexpected oxidative stress during the fluoranthene catabolism. The up-regulation of chorismate synthase and nicotine-nucleotide phosphorylase may be necessary for sustaining shikimate pathway and pyrimidine biosynthesis, respectively. A fluoranthene degradation pathway for Mycobacterium sp. JS14 was proposed and confirmed by proteomic study by identifying almost all the enzymes required during the initial steps of fluoranthene degradation.