Effects of creosote compounds on the aerobic bio-degradation of benzene

The inhibitory effect of creosote compounds on the aerobic degradation of benzene was studied in microcosm experiments. A total removal of benzene was observed after twelve days of incubation in microcosms where no inhibition was observed. Thiophene and benzothiophene, two heterocyclic aromatic compounds containing sulfur (S-compounds), had a significant inhibitory effect on the degradation of benzene, but also an inhibitory effect of benzofuran (an O-compound) and 1-methylpyrrole (a N-compound) could be observed, although the effect was weaker. The NSO-compounds also had an inhibitory effect on the degradation of p-xylene, o-xylene, and naphthalene, while they only had a weak influence on the degradation of 1-methylnaphthalene, o-cresol and 2,4-dimethylphenol. The phenolic compounds seemed to have a weak stimulating effect on the degradation of benzene whereas the monoaromatic hydrocarbons and the naphthalenes had no significant influence on the benzene degradation. The inhibitory effect of the NSO-compounds on the aerobic degradation of benzene could be identified as three different phenomena. The lag phase increased, the degradation rate decreased, and a residual concentration of benzene was observed in microcosms when NSO-compounds were present. The results show that NSO-compounds can have a potential inhibitory effect on the degradation of many creosote compounds, and that inhibitory effects in mixtures can be important for the degradation of different compounds.Abbreviations ben benzene - bf benzofuran - bt benzothiophene - dmp 2,4-dimethylphenol - GC gas chromatograph - ind indole - mnap 1-methylnaphthalene - MAHs monoaromatic hydrocarbons - mp 1-methylpyrrole - nap naphthalene - NSO-compounds heterocyclic aromatic compounds containing nitrogen, sulphur or oxygen - o-cre o-cresol - o-xyl o-xylene - PAHs polyaromatic hydrocarbons - phe phenol - p-xyl p-xylene - pyr pyrrole - thi thiophene - qui quinoline     

The influence of creosote compounds on the aerobic degradation of toluene

The inhibiting effect of 14 typical creosote compounds on the aerobic degradation of toluene was studied in batch experiments. Four NSO-compounds (pyrrole, 1-methylpyrrole, thiophene, and benzofuran) strongly inhibited the degradation of toluene. When the NSO-compounds were present together with toluene, little or no degradation of toluene was observed during 16 days of incubation, compared with a total removal of toluene within 4 days when the four compounds were absent. Indole (an N-compound) and three phenolic compounds (phenol, o-cresol, and 2,4-dimethylphenol) also inhibited the degradation of toluene, though the effect was much weaker that of the four NSO-compounds. O-xylene, p-xylene, naphthalene and 1-methylnaphthalene seemed to stimulate the degradation even though the influence was very weak. No effects of benzothiophene (an S-compound) and quinoline (an N-compound) were observed. Benzofuran (an O-compound) was identified as the compound that most inhibited the degradation of toluene. An effect could be detected even at low concentrations (40 μg/l).     

Effects of creosote compounds on the aerobic bio-degradation of benzene

The inhibitory effect of creosote compounds on the aerobic degradation of benzene was studied in microcosm experiments. A total removal of benzene was observed after twelve days of incubation in microcosms where no inhibition was observed. Thiophene and benzothiophene, two heterocyclic aromatic compounds containing sulfur (S-compounds), had a significant inhibitory effect on the degradation of benzene, but also an inhibitory effect of benzofuran (an O-compound) and 1-methylpyrrole (a N-compound) could be observed, although the effect was weaker. The NSO-compounds also had an inhibitory effect on the degradation of p-xylene, o-xylene, and naphthalene, while they only had a weak influence on the degradation of 1-methylnaphthalene, o-cresol and 2,4-dimethylphenol. The phenolic compounds seemed to have a weak stimulating effect on the degradation of benzene whereas the monoaromatic hydrocarbons and the naphthalenes had no significant influence on the benzene degradation. The inhibitory effect of the NSO-compounds on the aerobic degradation of benzene could be identified as three different phenomena. The lag phase increased, the degradation rate decreased, and a residual concentration of benzene was observed in microcosms when NSO-compounds were present. The results show that NSO-compounds can have a potential inhibitory effect on the degradation of many creosote compounds, and that inhibitory effects in mixtures can be important for the degradation of different compounds.     

Regiospecificity of Two Multicomponent Monooxygenases from Pseudomonas stutzeri OX1: Molecular Basis for Catabolic Adaptation of This Microorganism to Methylated Aromatic Compounds

The pathways for degradation of aromatic hydrocarbons are constantly modified by a variety of genetic mechanisms. Genetic studies carried out with Pseudomonas stutzeri OX1 suggested that the tou operon coding for toluene o-xylene monooxygenase (ToMO) was recently recruited into a preexisting pathway that already possessed the ph operon coding for phenol hydroxylase (PH). This apparently resulted in a redundancy of enzymatic activities, because both enzymes are able to hydroxylate (methyl)benzenes to (methyl)catechols via the intermediate production of (methyl)phenols. We investigated the kinetics and regioselectivity of toluene and o-xylene oxidation using Escherichia coli cells expressing ToMO and PH complexes. Our data indicate that in the recombinant system the enzymes act sequentially and that their catalytic efficiency and regioselectivity optimize the degradation of toluene and o-xylene, both of which are growth substrates. The main product of toluene oxidation by ToMO is p-cresol, the best substrate for PH, which catalyzes its transformation to 4-methylcatechol. The sequential action of the two enzymes on o-xylene leads, via the intermediate 3,4-dimethylphenol, to the exclusive production of 3,4-dimethylcatechol, the only dimethylcatechol isomer that can serve as a carbon and energy source after further metabolic processing. Moreover, our data strongly support a metabolic explanation for the acquisition of the ToMO operon by P. stutzeri OX1. It is possible that using the two enzymes in a concerted fashion confers on the strain a selective advantage based on the ability of the microorganism to optimize the efficiency of the use of nonhydroxylated aromatic hydrocarbons, such as benzene, toluene, and o-xylene.     

Alternative pathways foro-xylene orm-xylene andp-xylene degradation in aPseudomonas stutzeri strain

Pseudomonas stutzeri OX1 is able to grow ono-xylene but is unable to grow onm-xylene andp-xylene, which are partially metabolized through theo-xylene degradative pathway leading to the formation of dimethylphenols toxic to OX1.P. stutzeri spontaneous mutants able to grow onm-xylene andp-xylene have been isolated. These mutants soon lose the ability to grow ono-xylene. Data from HPLC analyses and from induction studies suggest that in these mutantsm-xylene andp-xylene could be metabolized through the oxidation of a methyl substituent.P. stutzeri chromosomal DNA is shown to share homology with pWW0 catabolic genes. In the mutant strains the region homologous to pWW0 upper pathway genes has undergone a genomic rearrangement.Abbreviations BADH benzylalcohol dehydrogenase - cat catechol - C23O catechol 2,3-dioxygenase - 2,3-,3,4-,2,4-,2,6-,3,5-2,5-DMP 2,3-,3,4-,2,4-,2,6-,3,5-,2,5-dimethylphenol - 2-MBOH 2-methylbenzyl alcohol - 3-MBOH 3-methylbenzyl alcohol - 4-MBOH 4-methylbenzyl alcohol - m-,p-tol m-,p-toluate - o-,m-,p-xyl o-,m-,p-xylene     

The roles of intermediates in biodegradation of benzene,toluene, and p-xylene by Pseudomonas putida F1

Several types of biodegradation experiments with benzene, toluene, or p-xylene show accumulation of intermediates by Pseudomonas putida F1. Under aerobic conditions, the major intermediates identified for benzene, toluene, and p-xylene are catechol, 3-methylcatechol, and 3,6-dimethylcatechol, respectively. Oxidations of catechol and 3-methylcatechol are linked to biomass synthesis. When oxygen is limited in the system, phenol (from benzene) and m-cresol and o-cresol (from toluene) accumulate.     

Metabolic pathways responsible for consumption of aromatic hydrocarbons by microbial associations: Molecular-genetic characterization

Genes for catechol 1,2- and 2,3-dioxygenases were cloned. These enzymes hold important positions in the ortho and meta pathways of the metabolism of aromatic carbons by microbial associations that consume the following volatile organic compounds in pilot minireactors: toluene, styrene, ethyl benzene, o-xylene, m-xylene, and naphthalene. Genes of both pathways were found in an association consuming m-xylene; only genes of the ortho pathway were found in associations consuming o-xylene, styrene, and ethyl benzene, and only genes of the meta pathway were found in associations consuming naphthalene and toluene. Genes of the ortho pathway (C12O) cloned from associations consuming o-xylene and ethyl benzene were similar to corresponding genes located on the pND6 plasmid of Pseudomonas putida. Genes of the ortho pathway from associations consuming o-xylene and m-xylene were similar to chromosomal genes of P. putida. Genes of the meta pathway (C23O) from associations consuming toluene and naphthalene were similar to corresponding genes formerly found in plasmids pWWO and pTOL.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 3, 2005, pp. 298–302.Original Russian Text Copyright © 2005 by Khomenkov, Shevelev, Zhukov, Kurlovich, Zagustina, Popov.     

Cometabolic transformation ofo-xylene in a biofilm system under nitrate reducing conditions

The purpose of this work was to investigate the anaerobic transformation ofo-xylene in a laboratory biofilm system with nitrate as an electron acceptor.o-Xylene was degraded cometabolically with toluene as primary carbon source. A mass balance showed thato-xylene was not mineralized but transformed.o-Methyl-benzalcohol ando-methyl-benzaldehyde were identified as intermediates ofo-xylene transformation which resulted in the formation ofo-methyl-benzoic acid as an end product. A cross inhibition phenomenon was observed between toluene ando-xylene. The presence of toluene was necessary for stimulation ofo-xylene transformation, but above a toluene concentration of 1–3 mg/L theo-xylene removal rate dramatically decreased. In returno-xylene inhibited the toluene degradation at concentrations above 2–3 mg/L.     

Mesophilic and thermophilic BTEX substrate interactions for a toluene-acclimatized biofilter

Benzene, toluene, ethylbenzene and xylene (BTEX) substrate interactions for a mesophilic (25°C) and thermophilic (50°C) toluene-acclimatized composted pine bark biofilter were investigated. Toluene, benzene, ethylbenzene, o-xylene, m-xylene and p-xylene removal efficiencies, both individually and in paired mixtures with toluene (1:1 ratio), were determined at a total loading rate of 18.1 g m^–3 h^–1 and retention time ranges of 0.5–3.0 min and 0.6–3.8 min for mesophilic and thermophilic biofilters, respectively. Overall, toluene degradation rates under mesophilic conditions were superior to degradation rates of individual BEX compounds. With the exception of p-xylene, higher removal efficiencies were achieved for individual BEX compounds compared to toluene under thermophilic conditions. Overall BEX compound degradation under mesophilic conditions was ranked as ethylbenzene >benzene >o-xylene >m-xylene >p-xylene. Under thermophilic conditions overall BEX compound degradation was ranked as benzene >o-xylene >ethylbenzene >m-xylene >p-xylene. With the exception of o-xylene, the presence of toluene in paired mixtures with BEX compounds resulted in enhanced removal efficiencies of BEX compounds, under both mesophilic and thermophilic conditions. A substrate interaction index was calculated to compare removal efficiencies at a retention time of 0.8 min (50 s). A reduction in toluene removal efficiencies (negative interaction) in the presence of individual BEX compounds was observed under mesophilic conditions, while enhanced toluene removal efficiency was achieved in the presence of other BEX compounds, with the exception of p-xylene under thermophilic conditions.     

Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for enhanced chlorinated ethene degradation and o-xylene oxidation

Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 has been shown to degrade all chlorinated ethenes individually and as mixtures. Here, DNA shuffling of the alpha hydroxylase fragment of ToMO (TouA) and saturation mutagenesis of the TouA active site residues I100, Q141, T201, F205, and E214 were used to enhance the degradation of chlorinated aliphatics. The ToMO mutants were identified using a chloride ion screen and then were further examined by gas chromatography. Escherichia coli TG1/pBS(Kan)ToMO expressing TouA saturation mutagenesis variant I100Q was identified that has 2.8-fold better trichloroethylene (TCE) degradation activity (apparent V _max of 1.77 nmol min⁻¹ mg⁻¹ protein⁻¹ vs 0.63 nmol min⁻¹ mg⁻¹ protein⁻¹). Another variant, E214G/D312N/M399V, has 2.5-fold better cis-1,2-dichloroethylene (cis-DCE) degradation activity (apparent V _max of 8.4 nmol min⁻¹ mg⁻¹ protein⁻¹ vs 3.3 nmol min⁻¹ mg⁻¹ protein⁻¹). Additionally, the hydroxylation regiospecificity of o-xylene and naphthalene were altered significantly for ToMO variants A107T/E214A, T201G, and T201S. Variant T201S produced 2.0-fold more 2,3-dimethylphenol (2,3-DMP) from o-xylene than the wild-type ToMO, whereas variant A107T/E214A had 6.0-fold altered regiospecificity for 2,3-DMP formation. Variant A107T/E214A also produced 3.0-fold more 2-naphthol from naphthalene than the wild-type ToMO, whereas the regiospecificity of variant T201S was altered to synthesize 3.0-fold less 2-naphthol, so that it made almost exclusively 1-naphthol (96%). Variant T201G was more regiospecific than variants A107T/E214A and T201S and produced 100% 3,4-DMP from o-xylene and >99% 1-naphthol from naphthalene. Hence, ToMO activity was enhanced for the degradation of TCE and cis-DCE and for the regiospecific hydroxylation of o-xylene and naphthalene through DNA shuffling and saturation mutagenesis.     

Transformation ofo-xylene too-methyl benzoic acid by a denitrifying enrichment culture using toluene as the primary substrate

A highly enriched denitrifying mixed culture transformedo-xylene cometabolically along with toluene by methyl group oxidation.o-Methyl benzaldehyde ando-methyl benzoic acid accumulated transiently as metabolic products ofo-xylene transformation. Transformation ofo-methyl benzyl alcohol ando-methyl benzaldehyde occurred independently of toluene degradation and resulted in the formation of a compound coeluting witho-methyl benzoic acid on a gas chromatograph. The cometabolic relationship between toluene ando-xylene could be attributed to a mechanism linked to the initial oxidation of the methyl group.     

Degradation of <Emphasis Type="Italic">o</Emphasis>-xylene and <Emphasis Type="Italic">m</Emphasis>-xylene by a novel sulfate-reducer belonging to the genus <Emphasis Type="Italic">Desulfotomaculum</Emphasis>

A strictly anaerobic bacterium, strain OX39, was isolated with o-xylene as organic substrate and sulfate as electron acceptor from an aquifer at a former gasworks plant contaminated with aromatic hydrocarbons. Apart from o-xylene, strain OX39 grew on m-xylene and toluene and all three substrates were oxidized completely to CO₂. Induction experiments indicated that o-xylene, m-xylene, and toluene degradation were initiated by different specific enzymes. Methylbenzylsuccinate was identified in supernatants of cultures grown on o-xylene and m-xylene, and benzylsuccinate was detected in supernatants of toluene-grown cells, thus indicating that degradation was initiated in all three cases by fumarate addition to the methyl group. Strain OX39 was sensitive towards sulfide and depended on Fe(II) in the medium as a scavenger of the produced sulfide. Analysis of the PCR-amplified 16S rRNA gene revealed that strain OX39 affiliates with the gram-positive endospore-forming sulfate reducers of the genus Desulfotomaculum and is the first hydrocarbon-oxidizing bacterium in this genus.     

Chemotaxis of Pseudomonas stutzeri OX1 and Burkholderia cepacia G4 toward chlorinated ethenes

The chemotactic responses of Pseudomonas putida F1, Burkholderia cepacia G4, and Pseudomonas stutzeri OX1 were investigated toward toluene, trichloroethylene (TCE), tetrachloroethylene (PCE), cis-1,2-dichloroethylene (cis-DCE), trans-1,2-dichloroethylene (trans-DCE), 1,1-dichloroethylene (1,1-DCE), and vinyl chloride (VC). P. stutzeri OX1 and P. putida F1 were chemotactic toward toluene, PCE, TCE, all DCEs, and VC. B. cepacia G4 was chemotactic toward toluene, PCE, TCE, cis-DCE, 1,1-DCE, and VC. Chemotaxis of P. stutzeri OX1 grown on o-xylene vapors was much stronger than when grown on o-cresol vapors toward some chlorinated ethenes. Expression of toluene-o-xylene monooxygenase (ToMO) from touABCDEF appears to be required for positive chemotaxis attraction, and the attraction is stronger with the touR (ToMO regulatory) gene.     

Organization and Regulation of meta Cleavage Pathway Genes for Toluene and o-Xylene Derivative Degradation in Pseudomonas stutzeri OX1

Pseudomonas stutzeri OX1 meta pathway genes for toluene and o-xylene catabolism were analyzed, and loci encoding phenol hydroxylase, catechol 2,3-dioxygenase, 2-hydroxymuconate semialdehyde dehydrogenase, and 2-hydroxymuconate semialdehyde hydrolase were mapped. Phenol hydroxylase converted a broad range of substrates, as it was also able to transform the nongrowth substrates 2,4-dimethylphenol and 2,5-dimethylphenol into 3,5-dimethylcatechol and 3,6-dimethylcatechol, respectively, which, however, were not cleaved by catechol 2,3-dioxygenase. The identified gene cluster displayed a gene order similar to that of the Pseudomonas sp. strain CF600 dmp operon for phenol catabolism and was found to be coregulated by the tou operon activator TouR. A hypothesis about the evolution of the toluene and o-xylene catabolic pathway in P. stutzeri OX1 is discussed.     

Anaerobic Degradation of p-Xylene by a Sulfate-Reducing Enrichment Culture

A strictly anaerobic enrichment culture was obtained with p-xylene as organic substrate and sulfate as electron acceptor from an aquifer at a former gasworks plant contaminated with aromatic hydrocarbons. p-Xylene was completely oxidized to CO₂. The enrichment culture depended on Fe(II) in the medium as a scavenger of the produced sulfide. 4-Methylbenzylsuccinic acid and 4-methylphenylitaconic acid were identified in supernatants of cultures indicating that degradation of p-xylene was initiated by fumarate addition to one of the methyl groups. Therefore, p-xylene degradation probably proceeds analogously to toluene degradation by Thauera aromatica or anaerobic degradation pathways for o- and m-xylene.     

Modelling of cometabolic transformation ofortho-xylene in a denitrifying biofilm system

A model describing the cometabolic biotransformation ofo-xylene with toluene as primary carbon source in a continuously fed fixed biofilm reactor is presented. The model is based on the concept of competitive inhibition betweeno-xylene and toluene. The proposed model simulated successfully the transformation ofo-xylene and the associated by-products formation, as well as the toluene degradation. However, it appears that an accurate measurement of active biomass density and distribution in the biofilm is needed, since these factors dramatically affects the modelling. The modelling of various kinetic experiments indicates that the active biomass (or toluene degraders) is accumulated on the top of the biofilm, leading to the conclusion that only a minor part of the biofilm thickness was active. The calibrated model is able to predict the removal of toluene ando-xylene for concentrations ranging from 0 to 30 mg/L. For higher concentrations toxicity phenomena may decrease the accuracy of the model.     

Identification and characterization of <Emphasis Type="Italic">o</Emphasis>-xylene-degrading <Emphasis Type="Italic">Rhodococcus</Emphasis> spp. which were dominant species in the remediation of <Emphasis Type="Italic">o</Emphasis>-xylene-contaminated soils

Soils contaminated with o-xylene were more difficult to bioremediate than those contaminated with other BTEX hydrocarbons (benzene, toluene, ethylbenzene, m-xylene and p-xylene). In order to identify microorganisms responsible for o-xylene degradation in soil, microbial community structure analyses were carried out with two soil samples in the presence of o-xylene and mineral nutrients. In two different soil samples, Rhodococcus opacus became abundant. We were also able to isolate o-xylene degrading Rhodococcus species from these soil samples. A primer set was developed to specifically detect a cluster of this Rhodococcus group including isolated Rhodococcus strains, Rhodococcus opacus and Rhodococcus koreensis. The growth of this bacterial group in an o-xylene-contaminated soil was followed by competitive PCR (cPCR). The decrease in o-xylene clearly paralleled the growth of the Rhodococcus group.     

Benzylic and aryl hydroxylations of m-xylene by o-xylene dioxygenase from Rhodococcus sp. strain DK17

Escherichia coli cells expressing Rhodococcus DK17 o-xylene dioxygenase genes were used for bioconversion of m-xylene. Gas chromatography–mass spectrometry analysis of the oxidation products detected 3-methylbenzylalcohol and 2,4-dimethylphenol in the ratio 9:1. Molecular modeling suggests that o-xylene dioxygenase can hold xylene isomers at a kink region between α6 and α7 helices of the active site and α9 helix covers the substrates. m-Xylene is unlikely to locate at the active site with a methyl group facing the kink region because this configuration would not fit within the substrate-binding pocket. The m-xylene molecule can flip horizontally to expose the meta-position methyl group to the catalytic motif. In this configuration, 3-methylbenzylalcohol could be formed, presumably due to the meta effect. Alternatively, the m-xylene molecule can rotate counterclockwise, allowing the catalytic motif to hydroxylate at C-4 yielding 2,4-dimethylphenol. Site-directed mutagenesis combined with structural and functional analyses suggests that the alanine-218 and the aspartic acid-262 in the α7 and the α9 helices play an important role in positioning m-xylene, respectively.     

Induction of aromatic catabolic activity in Sphingomonas aromaticivorans strain F199

Enzyme induction studies with Sphingomonas aromaticivorans F199 demonstrated that both toluene and naphthalene induced expression of both naphthalene and toluene catabolic enzymes. However, neither aromatic compound induced expression of all the enzymes required for complete mineralization of either naphthalene or toluene. Activity measurements in combination with gene sequence analyses indicate that growth on either aromatic substrate in the absence of the other is, therefore, sub-optimal and is predicted to lead to the build-up of metabolites due to imbalance in toluene or naphthalene catabolic enzyme activities. Growth on toluene may be further inhibited by the co-expression of two toluene catabolic pathways, as predicted from gene sequence analyses. One of these pathways may potentially result in the formation of a dead-end intermediate, possibly benzaldehyde. In contrast, either p-cresol or benzoate can support high levels of growth. Analyses of promoter region sequences on the F199 aromatic catabolic plasmid, pNL1, suggest that additional regulatory events are modulated through the interaction of BphR with Sigma54 type promoters and through the binding of a regulator upstream of p-cresol catabolic genes and xylM. We hypothesize that the unusual gene clustering in strain F199 is optimized for simultaneous degradation of multiple aromatic compound classes, possibly in response to the heterogeneous composition of aromatic structures in the fossil organic matter present in the deep Atlantic Coastal Plain sediments from which this bacterium was isolated. Received 26 April 1999/ Accepted in revised form 16 August 1999