Bacterial aerobic degradation of benzene,toluene, ethylbenzene and xylene

Several aerobic metabolic pathways for the degradation of benzene, toluene, ethylbenzene and xylene (BTEX), which are provided by two enzymic systems (dioxygenases and monooxygenases), have been identified. The monooxygenase attacks methyl or ethyl substituents of the aromatic ring, which are subsequently transformed by several oxidations to corresponding substituted pyrocatechols or phenylglyoxal, respectively. Alternatively, one oxygen atom may be first incorporated into aromatic ring while the second atom of the oxygen molecule is used for oxidation of either aromatic ring or a methyl group to corresponding pyrocatechols or protocatechuic acid, respectively. The dioxygenase attacks aromatic ring with the formation of 2-hydroxy-substituted compounds. Intermediates of the “upper” pathway are then mineralized by eitherortho-ormeta-ring cleavage (“lower” pathway). BTEX are relatively water-soluble and there-fore they are often mineralized by indigenous microflora. Therefore, natural attenuation may be considered as a suitable way for the clean-up of BTEX contaminants from gasoline-contaminated soil and groundwater.     

Hydroxylation and carboxylation--two crucial steps of anaerobic benzene degradation by Dechloromonas strain RCB

Benzene is a highly toxic industrial compound that is essential to the production of various chemicals, drugs, and fuel oils. Due to its toxicity and carcinogenicity, much recent attention has been focused on benzene biodegradation, especially in the absence of molecular oxygen. However, the mechanism by which anaerobic benzene biodegradation occurs is still unclear. This is because until the recent isolation of Dechloromonas strains JJ and RCB no organism that anaerobically degraded benzene was available with which to elucidate the pathway. Although many microorganisms use an initial fumarate addition reaction for hydrocarbon biodegradation, the large activation energy required argues against this mechanism for benzene. Other possible mechanisms include hydroxylation, carboxylation, biomethylation, or reduction of the benzene ring, but previous studies performed with undefined benzene-degrading cultures were unable to clearly distinguish which, if any, of these alternatives is used. Here we demonstrate that anaerobic nitrate-dependent benzene degradation by Dechloromonas strain RCB involves an initial hydroxylation, subsequent carboxylation, and loss of the hydroxyl group to form benzoate. These studies provide the first pure-culture evidence of the pathway of anaerobic benzene degradation. The outcome of these studies also suggests that all anaerobic benzene-degrading microorganisms, regardless of their terminal electron acceptor, may use this pathway.     

Anaerobic degradation of benzene, toluene, ethylbenzene, and o-xylene in sediment-free iron-reducing enrichment cultures

Monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylene (BTEX) are widespread contaminants in groundwater. We examined the anaerobic degradation of BTEX compounds with amorphous ferric oxide as electron acceptor. Successful enrichment cultures were obtained for all BTEX substrates both in the presence and absence of AQDS (9,10-anthraquinone-2,6-disulfonic acid). The electron balances showed a complete anaerobic oxidation of the aromatic compounds to CO2. This is the first report on the anaerobic degradation of o-xylene and ethylbenzene in sediment-free iron-reducing enrichment cultures.     

Anaerobic Degradation of Benzene, Toluene, Ethylbenzene, and Xylene Compounds by Dechloromonas Strain RCB

Dechloromonas strain RCB has been shown to be capable of anaerobic degradation of benzene coupled to nitrate reduction. As a continuation of these studies, the metabolic versatility and hydrocarbon biodegradative capability of this organism were investigated. The results of these revealed that in addition to nitrate, strain RCB could alternatively degrade benzene both aerobically and anaerobically with perchlorate or chlorate [(per)chlorate] as a suitable electron acceptor. Furthermore, with nitrate as the electron acceptor, strain RCB could also utilize toluene, ethylbenzene, and all three isomers of xylene (ortho-, meta-, and para-) as electron donors. While toluene and ethylbenzene were completely mineralized to CO₂, strain RCB did not completely mineralize para-xylene but rather transformed it to some as-yet-unidentified metabolite. Interestingly, with nitrate as the electron acceptor, strain RCB degraded benzene and toluene concurrently when the hydrocarbons were added as a mixture and almost 92 μM total hydrocarbons were oxidized within 15 days. The results of these studies emphasize the unique metabolic versatility of this organism, highlighting its potential applicability to bioremediative technologies.     

Anaerobic degradation of toluene and xylene by aquifer microorganisms under sulfate-reducing conditions.

Toluene and the three isomers of xylene were completely mineralized to CO2 and biomass by aquifer-derived microorganisms under strictly anaerobic conditions. The source of the inoculum was gasoline-contaminated sediment from Seal Beach, Calif. Evidence confirming that sulfate was the terminal electron acceptor is presented. Benzene and ethylbenzene were not degraded under the experimental conditions used. Successive transfers of the mixed cultures that were enriched from aquifer sediments retained the ability to degrade toluene and xylenes. Greater than 90% of 14C-labeled toluene or 14C-labeled o-xylene was mineralized to 14CO2. The doubling time for the culture grown on toluene or m-xylene was about 20 days, and the cell yield was about 0.1 to 0.14 g of cells (dry weight) per g of substrate. The accumulation of sulfide in the cultures as a result of sulfate reduction appeared to inhibit degradation of aromatic hydrocarbons.     

Anaerobic degradation of ethylbenzene and toluene in denitrifying strain EbN1 proceeds via independent substrate-induced pathways

Denitrifying strain EbN1 utilizes either ethylbenzene or toluene as the sole source of organic carbon under strictly anoxic conditions. When cells were grown on ethylbenzene, 1-phenylethanol and acetophenone were detected in the culture supernatant. However, these two compounds were not observed when cells were grown on benzoate. Growth on ethylbenzene, 1-phenylethanol, or acetophenone strictly depended on the presence of CO2, whereas growth on benzoate did not. These results suggest that strain EbN1 degrades ethylbenzene via 1-phenylethanol and acetophenone as intermediates, and that acetophenone is subsequently carboxylated. In suspensions of benzoate-grown cells, induction was required for degradation of ethylbenzene, 1-phenylethanol, and acetophenone. Induction was also required for toluene-grown cells to gain activity to degrade ethylbenzene, and, conversely, for ethylbenzene-grown cells to degrade toluene. In accordance with our findings from these studies, two-dimensional gel electrophoretic analysis of extracts of cells grown on benzoate, acetophenone, ethylbenzene, or toluene showed that a number of substrate-specific proteins were induced in strain EbN1. Growth on toluene or ethylbenzene induced a distinct set of proteins. However, some of the induced proteins in ethylbenzene or acetophenone grown cells were identical. This agrees with the finding that acetophenone is an intermediate in the degradation of ethylbenzene.     

Anaerobic degradation of toluene and xylene by aquifer microorganisms under sulfate-reducing conditions.

Toluene and the three isomers of xylene were completely mineralized to CO2 and biomass by aquifer-derived microorganisms under strictly anaerobic conditions. The source of the inoculum was gasoline-contaminated sediment from Seal Beach, Calif. Evidence confirming that sulfate was the terminal electron acceptor is presented. Benzene and ethylbenzene were not degraded under the experimental conditions used. Successive transfers of the mixed cultures that were enriched from aquifer sediments retained the ability to degrade toluene and xylenes. Greater than 90% of 14C-labeled toluene or 14C-labeled o-xylene was mineralized to 14CO2. The doubling time for the culture grown on toluene or m-xylene was about 20 days, and the cell yield was about 0.1 to 0.14 g of cells (dry weight) per g of substrate. The accumulation of sulfide in the cultures as a result of sulfate reduction appeared to inhibit degradation of aromatic hydrocarbons.     

Substrate interactions during the biodegradation of benzene,toluene, ethylbenzene,and xylene (BTEX) hydrocarbons by the fungus Cladophialophora sp. strain T1

The soil fungus Cladophialophora sp. strain T1 (= ATCC MYA-2335) was capable of growth on a model water-soluble fraction of gasoline that contained all six BTEX components (benzene, toluene, ethylbenzene, and the xylene isomers). Benzene was not metabolized, but the alkylated benzenes (toluene, ethylbenzene, and xylenes) were degraded by a combination of assimilation and cometabolism. Toluene and ethylbenzene were used as sources of carbon and energy, whereas the xylenes were cometabolized to different extents. o-Xylene and m-xylene were converted to phthalates as end metabolites; p-xylene was not degraded in complex BTEX mixtures but, in combination with toluene, appeared to be mineralized. The metabolic profiles and the inhibitory nature of the substrate interactions indicated that toluene, ethylbenzene, and xylene were degraded at the side chain by the same monooxygenase enzyme. Our findings suggest that soil fungi could contribute significantly to bioremediation of BTEX pollution.     

Anaerobic benzene degradation by bacteria

Benzene is a widespread and toxic contaminant. The fate of benzene in contaminated aquifers seems to be primarily controlled by the abundance of oxygen: benzene is aerobically degraded at high rates by ubiquitous microorganisms, and the oxygen‐dependent pathways for its breakdown were elucidated more than 50 years ago. In contrast, benzene was thought to be persistent under anoxic conditions until 25 years ago. Nevertheless, within the last 15 years, several benzene‐degrading cultures have been enriched under varying electron acceptor conditions in laboratories around the world, and organisms involved in anaerobic benzene degradation have been identified, indicating that anaerobic benzene degradation is a relevant environmental process. However, only a few benzene degraders have been isolated in pure culture so far, and they all use nitrate as an electron acceptor. In some highly enriched strictly anaerobic cultures, benzene has been described to be mineralized cooperatively by two or more different organisms. Despite great efforts, the biochemical mechanism by which the aromatic ring of benzene is activated in the absence of oxygen is still not fully elucidated; methylation, hydroxylation and carboxylation are discussed as likely reactions. This review summarizes the current knowledge about the ‘key players’ of anaerobic benzene degradation under different electron acceptor conditions and the possible pathway(s) of anaerobic benzene degradation.     

The mutagenic effect of benzene,toluene and xylene studied by the SCE technique

In experiments in vitro, neither benzene, toluene nor xylene changed the number of sister-chromatid exchanges (SCEs) or the number of chromosomal aberrations in human lymphocytes. Toluene and xylene caused a significant cell growth inhibition which was not observed with benzene in the same concentrations.     

The mutagenic effect of benzene, toluene and xylene studied by the SCE technique.

In experiments in vitro, neither benzene, toluene nor xylene changed the number of sister-chromatid exchanges (SCEs) or the number of chromosomal aberrations in human lymphocytes. Toluene and xylene caused a significant cell growth inhibition which was not observed with benzene in the same concentrations.     

Anaerobic degradation of toluene by a denitrifying bacterium

A denitrifying bacterium, designated strain T1, that grew with toluene as the sole source of carbon under anaerobic conditions was isolated. The type of agar used in solid media and the toxicity of toluene were determinative factors in the successful isolation of strain T1. Greater than 50% of the toluene carbon was oxidized to CO2, and 29% was assimilated into biomass. The oxidation of toluene to CO2 was stoichiometrically coupled to nitrate reduction and denitrification. Strain T1 was tolerant of and grew on 3 mM toluene after a lag phase. The rate of toluene degradation was 1.8 mumol min-1 liter-1 (56 nmol min-1 mg of protein-1) in a cell suspension. Strain T1 was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar biochemical pathway of oxidation. In addition, o-xylene was transformed to a metabolite in the presence of toluene but did not serve as the sole source of carbon for growth of strain T1. This transformation was dependent on the degradation of toluene.     

Anaerobic benzene degradation by Gram-positive sulfate-reducing bacteria

Despite its high chemical stability, benzene is known to be biodegradable with various electron acceptors under anaerobic conditions. However, our understanding of the initial activation reaction and the responsible prokaryotes is limited. In the present study, we enriched a bacterial culture that oxidizes benzene to carbon dioxide under sulfate-reducing conditions. Community analysis using terminal restriction fragment length polymorphism, 16S rRNA gene sequencing and FISH revealed 95% dominance of one phylotype that is affiliated to the Gram-positive bacterial genus Pelotomaculum showing that sulfate-reducing Gram-positive bacteria are involved in anaerobic benzene degradation. In order to get indications of the initial activation mechanism, we tested the substrate utilization, performed cometabolism tests and screened for putative metabolites. Phenol, toluene, and benzoate could not be utilized as alternative carbon sources by the benzene-degrading culture. Cometabolic degradation experiments resulted in retarded rates of benzene degradation in the presence of phenol whereas toluene had no effect on benzene metabolism. Phenol, 2-hydroxybenzoate, 4-hydroxybenzoate, and benzoate were identified as putative metabolites in the enrichment culture. However, hydroxylated aromatics were shown to be formed abiotically. Thus, the finding of benzoate as an intermediate compound supports a direct carboxylation of benzene as the initial activation mechanism but additional reactions leading to its formation cannot be excluded definitely.     

Anaerobic degradation of toluene by a denitrifying bacterium.

A denitrifying bacterium, designated strain T1, that grew with toluene as the sole source of carbon under anaerobic conditions was isolated. The type of agar used in solid media and the toxicity of toluene were determinative factors in the successful isolation of strain T1. Greater than 50% of the toluene carbon was oxidized to CO2, and 29% was assimilated into biomass. The oxidation of toluene to CO2 was stoichiometrically coupled to nitrate reduction and denitrification. Strain T1 was tolerant of and grew on 3 mM toluene after a lag phase. The rate of toluene degradation was 1.8 mumol min-1 liter-1 (56 nmol min-1 mg of protein-1) in a cell suspension. Strain T1 was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar biochemical pathway of oxidation. In addition, o-xylene was transformed to a metabolite in the presence of toluene but did not serve as the sole source of carbon for growth of strain T1. This transformation was dependent on the degradation of toluene.     

Isolation and characterization of a thermotolerant bacterium Ralstonia sp. strain PHS1 that degrades benzene, toluene, ethylbenzene, and o-xylene

A thermotolerant bacterium, designated as PHS1, was isolated from a hot spring in Pohang, Korea, on the basis of its ability to grow on benzene, toluene, ethylbenzene, and xylenes (BTEX) as a sole carbon source. Strain PHS1 is a gram-negative, rod-shaped aerobe and grows optimally at 42 degrees C and pH 7.2. According to 16 S rDNA analysis, strain PHS1 showed highest similarity to Ralstonia eutropha (previously named Alcaligenes eutrophus). Unlike its closest known Ralstonia species, however, strain PHS1 was able to utilize toluene, ethylbenzene, o-xylene, and both m- and o-cresol. The degradation of o-xylene by strain PHS1 is particularly important, since o-xylene is a compound of considerable environmental interest, owing to its recalcitrance; and very few microorganisms have been reported to utilize o-xylene as a sole carbon source. It was found that strain PHS1 transformed o-xylene to 2,3-dimethylphenol through direct oxygenation of the aromatic ring. The unique properties of strain PHS1, such as thermotolerance and the ability to degrade o-xylene, may have important implications for the treatment of BTEX-contaminated industrial effluents.     

Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons by new denitrifying bacteria

Anaerobic degradation of alkylbenzenes with side chains longer than that of toluene was studied in freshwater mud samples in the presence of nitrate. Two new denitrifying strains, EbN1 and PbN1, were isolated on ethylbenzene and n-propylbenzene, respectively. For comparison, two further denitrifying strains, ToN1 and mXyN1, were isolated from the same mud with toluene and m-xylene, respectively. Sequencing of 16SrDNA revealed a close relationship of the new isolates to Thauera selenatis. The strains exhibited different specific capacities for degradation of alkylbenzenes. In addition to ethylbenzene, strain EbN1 utilized toluence, but not propylbenzene. In contrast, propylbenzene-degrading strain PbN1 did not grow on toluene, but was able to utilize ethylbenzene. Strain ToN1 used toluene as the only hydrocarbon substrate, whereas strain mXyN1 utilized both toluene and m-xylene. Measurement of the degradation balance demonstrated complete oxidation of ethylbenzene to CO₂ by strain EbN1. Further characteristic substrates of strains EbN1 and PbN1 were 1-phenylethanol and acetophenone. In contrast to the other isolates, strain mXyN1 did not grow on benzyl alcohol. Benzyl alcohol (also m-methylbenzyl alcohol) was even a specific inhibitor of toluene and m-xylene utilization by strain mXyN1. None of the strains was able to grow on any of the alkylbenzenes with oxygen as electron acceptor. However, polar aromatic compounds such as benzoate were utilized under both oxic and anoxic conditions. All four isolates grew anaerobically on crude oil. Gas chromatographic analysis of crude oil after growth of strain ToN1 revealed specific depletion of toluene.     

Simultaneous determination of benzene, toluene, ethylbenzene, and xylenes in urine by thermal desorption–gas chromatography

The determination of metabolites of benzene, toluene, ethylbenzene, and xylenes in urine has been used to assess human exposure to these compounds. The analyses of urine samples for these metabolites are tedious and time consuming. The determination of unmetabolized individual compounds in urine has been studied previously with some success. A simultaneous determination of several unmetabolized VOC compounds in urine by thermal desorption–gas chromatography was conducted to assess the exposure of smokers and nonsmokers to these compounds. The method of thermal desorption–GC was sensitive enough to detect a significant difference in exposure levels due to the contribution of light smoking in the environmentally-exposed group.     

Bioremediation of benzene,toluene, ethylbenzene,xylenes-contaminated soil: a biopile pilot experiment

Aims: In this study, we evaluated the removal efficiency of fuel hydrocarbons from a jet fuel contaminated area using bioaugmentation treatment in biopile. Methods and Results: The hydrocarbon analysis of the sample revealed total hydrocarbons mainly constituted by benzene, toluene, ethylbenzene, xylenes (BTEX) and heavy aliphatic hydrocarbons. Enrichments of soil sample were performed with BTEX, pristane and fuel JP-5, respectively, selected hydrocarbon-degrading strains, namely Acinetobacter sp., Pseudomonas sp. and Rhodococcus sp. Three hundred litres of culture containing 10⁸ cell ml⁻¹ of each strain and nutrients sprayed on the biopile allowed a removal of 90% of total hydrocarbons in 15 days. Bioremediation process was monitored by observation of the respiration rate and the bacterial abundance and GC-MS analysis. Conclusions: The efficiency of the treatment in the biopile was considerable. The assessment of microbial activity during the experiment is necessary for interventions targeted to improve environmental parameters such as humidity, temperature, pH and nutrients for optimization of the bioremediation process. Significance and Impact of the Study: A better knowledge of microbial successions at oil-polluted sites is essential for environmental bioremediation. Data obtained in biopile study improve our understanding of processes occurring during oil pollution.