The alkene monooxygenase from Xanthobacter Py2 is a binuclear non-haem iron protein closely related to toluene 4-monooxygenase

The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter Py2 have been sequenced. The predicted amino acid sequence of the first ORF shows homology with the iron binding subunits of binuclear non-haem iron containing monooxygenases including benzene monooxygenase, toluene 4-monooxygenase (>60% sequence similarity) and methane monooxygenase (>40% sequence similarity) and that the necessary sequence motifs associated with iron co-ordination are also present. Secondary structure prediction based on the amino acid sequence showed that the predominantly α-helical structure that surrounds the binuclear iron binding site was conserved allowing the sequence to be modelled on the co-ordinates of the methane monooxygenase α-subunit. Significant differences in the residues forming the hydrophobic cavity which forms the substrate binding site are discussed with reference to the differences in reaction specificity and stereospecificity of binuclear non-haem iron monooxygenases.     

Initial Studies on the Regulation of Toluene Degradation by Pseudomonas Putida F1

Pseudomonas putida Fl oxidizes toluene through cis-toluene dihydrodiol to 3-methylcatechol. The latter compound is the substrate for “meta” fission of the aromatic nucleus. Kinetic and induction experiments indicate that the genes encoding enzymes for these reactions are part of an operon, designated the tod operon, that is coordinately induced and regulated. Strains unable to utilize toluene as a growth substrate were isolated at high frequencies by using screening procedures that utilize the redox dye, 2,3,5-triphenyl-2H-tetrazolium chloride. Biochemical characterization of strains with mutations in the structural genes of the tod operon showed that toluene induces the first four enzymes in toluene degradation by P. putida Fl. The isolation and characterization of pleiotropicnegative mutants together with mutants altered in terms of their expression of tod genes suggests that the tod operon may be under the control of a positive regulatory element.     

Alternative method for rapidly screening microbial isolates for their potential to degrade volatile contaminants

Summary A method is described for rapidly screening the metabolic potential of bacteria to oxidize semivolatile and volatile compounds as a sole carbon source. The method is based on automated system that utilizes Microplates^TM manufactured by Biolog, Inc. (Hayward, CA, USA). This system detects bacterial respiratory activity from the oxidation of a carbon source introduced in volatile form. This is in contrast to the original design, which is based on inoculating a carbon source directly into each well. The 96-well (MT) microtiter plates contain nutrients and a tetrazolium dye. When a bacterial species is capable of oxidizing a volatile carbon substrate, the dye turns purple, and a spectrophotometric plate reader quantifies the response. As a test of this method 150 isolates, including isolates known to degrade some of the test compounds and negative controls were evaluated for their potential to oxidize carbon tetrachloride, toluene, ando-xylene. Thirty-seven isolates (25%) were qualitatively identified as contaminant oxidizers, and thirteen of these (35%) showed significant degradation capabilities for both toluene ando-xylene.Environmental Sciences Division Publication Number 4277.     

Effects of methylbenzenes on microsomal enzymes in rat liver,kidney and lung

The effects of pretreatment with toluene, o-, m-, p-xylene and mesitylene were investigated on the microsomal enzymes of liver, kidney and lung in rats. The activities of aminopyrine N-demethylase, aryl hydrocarbon hydroxylase, aniline hydroxylase, NADPH-cytochrome c reductase, as well as the concentrations of cytochrome P-450 and cytochrome b₅ were determined. The effects were most marked in the liver, where toluene caused increase in aniline hydroxylase and cytochrome P-450; o-xylene in aminopyrine N-demethylase and cytochrome b₅; m-xylene and mesitylene in all the enzymes investigated. In kidneys, all the compounds increased the activity of aniline hydroxylase; m-xylene induced cytochrome P-450 and b₅ as well as NADPH-cytochrome c reductase; p-xylene induced cytochrome P-450, and mesitylene cytochrome P-450 and b₅. Aminopyrine N-demethylase activity was decreased by toluene. In lungs, only mesitylene caused any significant differences from the controls: increase in aminopyrine N-demethylase and aryl hydrocarbon hydroxylase, decrease in aniline hydroxylase. The methylbenzenes tested induced the microsomal enzymes in a rough correlation to the number of their methyl groups and their hydrophobic properties.     

Performance evaluation and microbial community analysis of the composite filler micro-embedded with Pseudomonas putida for the biodegradation of toluene

The composite filler micro-embedded with Pseudomonas putida (P. putida) was prepared and the biodegradation performance of the filler was evaluated in a biofilter. Five phases were set up to evaluate the performance of the biofilter under different toluene inlet loadings and transient shock loadings. In particular, the microbial community structure in the biofilms and fillers was measured by sequence analysis of the 16S rRNA gene. The results show that the biofilter packed with the composite fillers was suitable for the biodegradation of toluene. The biofilter could start up quickly with high removal efficiency (RE), and remain above 90 % RE when the empty bed residence time (EBRT) was 18 s and the inlet loading rates were not higher than 41.4 g/(m³·h). Moreover, the biofilter could tolerate substantial transient shock loadings. The high removal efficiency and elimination capacity contributed to rich bacterial communities for the efficient degradation of toluene. The dominant microbial communities at the phylum level were mainly Firmicutes, Actinobacteria and Proteobacteria. It is noteworthy that the abundance of Bacteroidetes at phylum level and Chungangia and Stenotrophomonas at genus level increased significantly during the re-start period.     

Exophiala macquariensis sp. nov., a cold adapted black yeast species recovered from a hydrocarbon contaminated sub-Antarctic soil

A new black yeast species, Exophiala macquariensis is described that is a member of the ascomycete family Herpotrichiellaceae, order Chaetothyriales. The genus Exophiala is comprised of opportunistic pathogens isolated from clinical specimens as well as species recovered from hydrocarbon contaminated environments. Several species have been reported to be able to degrade benzene, toluene, ethylbenzene and xylenes. Here, a novel species of Exophiala (CZ06) previously isolated from a Sub-Antarctic, Macquarie Island soil that was spiked with Special Antarctic Blend diesel fuel (SAB) is described. This isolate has the capacity of toluene biodegradation at cold temperatures. Multilocus sequence typing showed that this fungus was closely related to the pathogenic species Exophiala salmonis and Exophiala equina. With the capacity to utilise hydrocarbons as a sole carbon source at 10 °C, this fungus has great potential for future bioremediation applications.     

Substrate specificities and electron paramagnetic resonance properties of benzylsuccinate synthases in anaerobic toluene and<Emphasis Type="Italic"> m</Emphasis>-xylene metabolism

The anaerobic degradation pathways of toluene and m-xylene are initiated by addition of a fumarate cosubstrate to the methyl group of the hydrocarbon, yielding (R)-benzylsuccinate and (3-methylbenzyl)succinate, respectively, as first intermediates. These reactions are catalyzed by a novel glycyl-radical enzyme, (R)-benzylsuccinate synthase. Substrate specificities of benzylsuccinate synthases were analyzed in Azoarcus sp. strain T and Thauera aromatica strain K172. The enzyme of Azoarcus sp. strain T converts toluene, but also all xylene and cresol isomers, to the corresponding succinate adducts, whereas the enzyme of T. aromatica is active with toluene and all cresols, but not with any xylene isomer. This corresponds to the capabilities of Azoarcus sp. strain T to grow on either toluene or m-xylene, and of T. aromatica to grow on toluene as sole hydrocarbon substrate. Thus, differences in the substrate spectra of the respective benzylsuccinate synthases of the two strains contribute to utilization of different aromatic hydrocarbons, although growth on different substrates also depends on additional determinants. We also provide direct evidence by electron paramagnetic resonance (EPR) spectroscopy that glycyl radical enzymes corresponding to substrate-induced benzylsuccinate synthases are specifically detectable in anoxically prepared extracts of toluene- or m-xylene-grown cells. The presence of the EPR signals and the determined amount of the radical are consistent with the respective benzylsuccinate synthase activities. The properties of the EPR signals are highly similar to those of the prototype glycyl radical enzyme pyruvate formate lyase, but differ slightly from previously reported parameters for partially purified benzylsuccinate synthase.     

Modeling of growth kinetics for <Emphasis Type="Italic">Pseudomonas putida</Emphasis> during toluene degradation

Glucose has been often used as a secondary substrate to enhance the degradation of primary substrate as well as the increase of biomass, especially for the inhibitory range of substrate concentration. In this study, we investigated the effect of glucose concentration on growth kinetics of Pseudomonas putida during toluene degradation for a wide concentration range (60–250 mg/l). Batch microcosm studies were conducted in order to monitor bacterial growth for three different initial concentrations (2, 5, 10 mg/ml) of glucose for a given toluene concentration. Modeling of growth kinetics was also performed for each growth curve of glucose dose using both Monod and Haldane kinetics. Batch studies revealed that bacterial growth showed a distinct inhibitory phase above some limit (∼170 mg/l) for the lowest (2 mg/ml) glucose dose, but the degree of inhibition decreased as the glucose dose increased, leading to three different growth patterns. The bacterial growth followed each of the modified Wayman and Tseng, Wayman and Tseng, and Luong model as the glucose dose increased from 2 to 10 mg/ml. This indicates that glucose has a prominent influence on bacterial growth during toluene degradation and that different kinetics should be adopted for each broth condition.     

Toluene biodegradation by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> F1: targeting culture stability in long-term operation

The stability of Pseudomonas putida F1, a strain harbouring the genes responsible for toluene degradation in the chromosome was evaluated in a bioscrubber under high toluene loadings and nitrogen limiting conditions at two dilution rates (0.11 and 0.27 h⁻¹). Each experiment was run for 30 days, period long enough for microbial instability to occur considering previously reported studies carried out with bacterial strains encoding the catabolic genes in the TOL plasmid. At all tested conditions, P. putida F1 exhibited stable performance as shown by the constant values of the specific toluene degradation yield, CO₂ produced versus toluene degraded yield, and biomass concentration within each steady state. Benzyl alcohol, a curing agent causing TOL plasmid deletion in Pseudomonas strains, was present in the cultivation medium as a result of the monooxygenation of toluene by the diooxygenase system of P. putida F1. However, no mutant population growing at the expense of the extracellular excreted carbon or lysis products was established in the chemostat as confirmed by the constant dissolved total organic carbon (TOC) concentration and fraction of toluene degrading cells (approx. 100%). In addition, batch experiments conducted with both lysis substrate and toluene simultaneously confirmed that P. putida F1 preferentially consumed toluene rather than extracellular excreted carbon.     

Determination of monobromobimane derivatives of phenylmercapturic and benzylmercapturic acids in urine by high-performance liquid chromatography and fluorimetry

A method was developed for the determination in human urine of S-phenylmercapturic (PMA) and S-benzylmercapturic (BMA) acids, metabolites respectively of benzene and toluene. PMA and BMA were determined, after alkaline hydrolysis, to give respectively thiophenol and benzylmercaptan, and coupling of the thiol-containing compounds with monobromobimane (MB), by reversed-phase HPLC on a diphenyl-silica bonded cartridge (100×4.6 mm I.D., 5 μm particle size) with fluorimetric detection. Wavelengths for excitation and emission were 375 and 480 nm, respectively. The recovery of PMA and BMA from spiked urines was >90% in the 10–500 μg/l range; the quantification limits were respectively 1 and 0.5 μg/l; day-to-day precision at 42 μg/l was C.V. <7%. The suitability of the proposed procedure for the biological monitoring of exposure to low-level airborne concentrations of benzene and toluene, was evaluated by analyzing the urinary excretion of PMA and BMA in subjects exposed to different sources of aromatic hydrocarbons, namely occupationally-unexposed referents (non-smokers, n=15; moderate smokers, n=8; mean number of cigarettes smoked PER-DAY=17 cig/day) and non-smoker workers occupationally exposed to toluene in maintenance operations of rotogravure machines (non-smokers, n=17). Among referents, non-smokers showed values of PMA ranging from <1 to 4.6 μg/l and BMA from 1.0 to 10.4 μg/l; in smokers, PMA values ranging from 1.2 to 6.7 μg/l and BMA from 9.3 to 39.9 μg/l, were observed. In occupationally exposed non-smoker subjects, BMA median excretion value (23.6 μg/l) was higher than in non-smoker referents (3.5 μg/l) (P<0.001) and individual BMA values (y, μg/l) were associated and increased with airborne toluene concentration (x, mg/m³) according to the equation y=6.5+0.65x (r=0.69, P<0.01, n=17). The proposed analytical method appears to be a sensitive and specific tool for biological monitoring of low-level exposure to benzene and toluene mixtures in occupational and environmental toxicology laboratory.