Degradation of BTEX by anaerobic bacteria: physiology and application

Pollution of the environment with aromatic hydrocarbons, such as benzene, toluene, ethylbenzene and xylene (so-called BTEX) is often observed. The cleanup of these toxic compounds has gained much attention in the last decades. In situ bioremediation of aromatic hydrocarbons contaminated soils and groundwater by naturally occurring microorganisms or microorganisms that are introduced is possible. Anaerobic bioremediation is an attractive technology as these compounds are often present in the anoxic zones of the environment. The bottleneck in the application of anaerobic techniques is the lack of knowledge about the anaerobic biodegradation of benzene and the bacteria involved in anaerobic benzene degradation. Here, we review the existing knowledge on the degradation of benzene and other aromatic hydrocarbons by anaerobic bacteria, in particular the physiology and application, including results on the (per)chlorate stimulated degradation of these compounds, which is an interesting new alternative option for bioremediatio

Isolation and characterization of Alicycliphilus denitrificans strain BC, which grows on benzene with chlorate as the electron acceptor

A bacterium, strain BC, was isolated from a benzene-degrading chlorate-reducing enrichment culture. Strain BC degrades benzene in conjunction with chlorate reduction. Cells of strain BC are short rods that are 0.6 microm wide and 1 to 2 microm long, are motile, and stain gram negative. Strain BC grows on benzene and some other aromatic compounds with oxygen or in the absence of oxygen with chlorate as the electron acceptor. Strain BC is a denitrifying bacterium, but it is not able to grow on benzene with nitrate. The closest cultured relative is Alicycliphilus denitrificans type strain K601, a cyclohexanol-degrading nitrate-reducing betaproteobacterium. Chlorate reductase (0.4 U/mg protein) and chlorite dismutase (5.7 U/mg protein) activities in cell extracts of strain BC were determined. Gene sequences encoding a known chlorite dismutase (cld) were not detected in strain BC by using the PCR primers described in previous studies. As physiological and biochemical data indicated that there was oxygenation of benzene during growth with chlorate, a strategy was developed to detect genes encoding monooxygenase and dioxygenase enzymes potentially involved in benzene degradation in strain BC. Using primer sets designed to amplify members of distinct evolutionary branches in the catabolic families involved in benzene biodegradation, two oxygenase genes putatively encoding the enzymes performing the initial successive monooxygenations (BC-BMOa) and the cleavage of catechol (BC-C23O) were detected. Our findings suggest that oxygen formed by dismutation of chlorite can be used to attack organic molecules by means of oxygenases, as exemplified with benzene. Thus, aerobic pathways can be employed under conditions in which no external oxygen is supplie

Degradation of benzene and other aromatic hydrocarbons by anaerobic bacteria

Accidental spills, industrial discharges and gasoline leakage from underground storage tanks have resulted in serious pollution of the environment with monoaromatic hydrocarbons, such as benzene, toluene, ethylbenzene and xylene (so-called BTEX). High concentrations of BTEX have been detected in soils, sediments and groundwater. The mobility and toxicity of the BTEX compounds are of major concern. In situ bioremediation of BTEX by using naturally occurring microorganisms or introduced microorganisms is a very attractive option. BTEX compounds are known to be transformed (or degraded) by microorganisms under aerobic and anaerobic conditions. As BTEX compounds are often present in the anaerobic zones of the environment, anaerobic bioremediation is an attractive remediation technique. The bottleneck in the application of anaerobic techniques is the lack of knowledge about the anaerobic biodegradation of benzene. In particular, little is known about the bacteria involved in anaerobic benzene degradation and the anaerobic benzene degradation pathway has still not been elucidated. The aim of the research presented in this thesis was to gain more insight in the degradation of benzene and other aromatic hydrocarbons by anaerobic bacteria. In particular, the physiology and phylogeny of the bacteria responsible for the degradation were studied and the results have been presented in this thesis. Anaerobic benzene and toluene degradation was studied with different electron acceptors in batch experiments inoculated with material from an aquifer polluted by BTEX-containing landfill leachate (Banisveld landfill near Boxtel, The Netherlands). Benzene was not degraded during one year of incubation. Toluene degradation, on the other hand, was observed with nitrate, MnO2 and Fe(III)NTA as electron acceptors. After further enrichment and several isolation attempts, a novel betaproteobacterial bacterium, strain G5G6, was obtained in pure culture. Strain G5G6 is able to grow with toluene as the sole electron donor and carbon source, and amorphous and soluble Fe(III)-species, nitrate and MnO2 as electron acceptors. Strain G5G6 has several other interesting physiological and phylogenetic characteristics, which will be subject of future research. Strain G5G6 represents a novel species in a novel genus for which we propose the name Georgfuchsia toluolica. In general, aerobic degradation of BTEX is a faster process than anaerobic BTEX degradation. However, for a number of reasons application of oxygen-dependent processes in the subsurface are technically and financially often not appealing. Therefore, an alternative bioremediation strategy would be to introduce oxygen in an alternative way, e.g. by in situ production. Chlorate reduction is a way to produce molecular oxygen in situ under anaerobic conditions. The formation of oxygen during chlorate reduction may result in rapid oxidation of compounds which are slowly degraded under anaerobic conditions; an example of such a compound is benzene. Therefore, benzene degradation coupled to the reduction of chlorate (ClO3-) was studied in this thesis. With mixed material from a wastewater treatment plant and soil samples obtained from a location contaminated with benzene, a benzene-degrading chlorate-reducing stable enrichment culture was obtained. This stable enrichment consisted of about five different bacterial species. Cross feeding involving interspecies oxygen transfer is a likely mechanism in this enrichment. One of these species, strain BC, was obtained in pure culture. Phylogenetic analysis showed that strain BC is a Alicycliphilus denitrificans strain. Strain BC is able to degrade benzene in conjunction with chlorate reduction. Oxygenase genes putatively encoding the enzymes performing the initial steps in aerobic degradation of benzene, were detected in strain BC. This demonstrates the existence of aerobic benzene bacterial biodegradation pathways under essentially anaerobic conditions. Thus, aerobic pathways can be employed under conditions where no external oxygen is supplied. The new insights into toluene degradation under anaerobic conditions and benzene degradation coupled to chlorate reduction, as described in this thesis, can be applied for the improvement or development of in situ bioremediation strategies for BTEX contamination. <br/

Microbial degradation of aliphatic and aromatic hydrocarbons with (per)chlorate as electron acceptor

"Water is life!" All active cellular systems require water as the medium and solvent of their metabolic activities. Hydrophobic compounds and structures, which tend to exclude water, though providing inter alia excellent sources of energy and a means of biological compartmentalization, present problems of cellular handling, poor bioavailability and, in some cases, toxicity. Microbes both synthesize and exploit a vast range of hydrophobic organics, especially petroleum oil hydrocarbons and industrial pollutants, and the underlying interactions not only have major consequences for the lifestyles of the microbes involved, but also for biogeochemistry, climate change, environmental pollution, human health and a range of biotechnological applications. The aim of this handbook is to be the definitive resource of current knowledge on the diverse and multifaceted aspects of these interactions, the microbial players, and the physiological mechanisms and adaptive strategies characteristic of the microbial lifestyle that plays out at hydrophobic material: aqueous liquid interface

Denitrification in aqueos FeEDTA solutions

The biological reduction of nitric oxide (NO) in aqueous solutions of FeEDTA is an important key reaction within the BioDeNOx process, a combined physico-chemical and biological technique for the removal of NO, from industrial flue gasses. To explore the reduction of nitrogen oxide analogues, this study investigated the full denitrification pathway in aqueous FeEDTA solutions, ie the reduction of NO3-, NO2-, NO via N2O to N-2 in this unusual medium. This was done in batch experiments at 30degreesC with 25 mmol dm(-3) FeEDTA solutions (pH 7.2 +/- 0.2). Also Ca2+ (2 and 10 mmol dm(-3)) and Mg2+ (2 mmol dm(-3)) were added in excess to prevent free, uncomplexed EDTA. Nitrate reduction in aqueous solutions of Fe(III)EDTA is accompanied by the biological reduction of Fe(III) to Fe(II), for which ethanol, methanol and also acetate are suitable electron donors. Fe(II)EDTA can serve as electron donor for the biological reduction of nitrate to nitrite, with the concomitant oxidation of Fe(II)EDTA to Fe(III)EDTA. Moreover, Fe(II)EDTA can also serve as electron donor for the chemical reduction of nitrite to NO, with the concomitant formation of the nitrosyl-complex Fe(II)EDTA-NO. The reduction of NO in Fe(II)EDTA was found to be catalysed biologically and occurred about three times faster at 55 degreesC than NO reduction at 30 degreesC. This study showed that the nitrogen and iron cycles are strongly coupled and that FeEDTA has an electron-mediating role during the subsequent reduction of nitrate, nitrite, nitric oxide and nitrous oxide to dinitrogen gas. (C) 2004 Society of Chemical Industry

Denitrification in aqueos FeEDTA solutions

The biological reduction of nitric oxide (NO) in aqueous solutions of FeEDTA is an important key reaction within the BioDeNOx process, a combined physico-chemical and biological technique for the removal of NO, from industrial flue gasses. To explore the reduction of nitrogen oxide analogues, this study investigated the full denitrification pathway in aqueous FeEDTA solutions, ie the reduction of NO3-, NO2-, NO via N2O to N-2 in this unusual medium. This was done in batch experiments at 30degreesC with 25 mmol dm(-3) FeEDTA solutions (pH 7.2 +/- 0.2). Also Ca2+ (2 and 10 mmol dm(-3)) and Mg2+ (2 mmol dm(-3)) were added in excess to prevent free, uncomplexed EDTA. Nitrate reduction in aqueous solutions of Fe(III)EDTA is accompanied by the biological reduction of Fe(III) to Fe(II), for which ethanol, methanol and also acetate are suitable electron donors. Fe(II)EDTA can serve as electron donor for the biological reduction of nitrate to nitrite, with the concomitant oxidation of Fe(II)EDTA to Fe(III)EDTA. Moreover, Fe(II)EDTA can also serve as electron donor for the chemical reduction of nitrite to NO, with the concomitant formation of the nitrosyl-complex Fe(II)EDTA-NO. The reduction of NO in Fe(II)EDTA was found to be catalysed biologically and occurred about three times faster at 55 degreesC than NO reduction at 30 degreesC. This study showed that the nitrogen and iron cycles are strongly coupled and that FeEDTA has an electron-mediating role during the subsequent reduction of nitrate, nitrite, nitric oxide and nitrous oxide to dinitrogen gas. (C) 2004 Society of Chemical Industry

Anaerobic reduction and oxidation of quinine moieties and the reduction of oxidized metals by halorespiring and related organisms

Halorespiring microorganisms have been detected in soils that were not polluted with chlorinated compounds. In this study, we describe alternative electron acceptor utilization by some halorespiring bacteria and phylogenetically related bacteria. It appears that oxidized metals like selenate, arsenate and manganese are rather common electron acceptors for halorespiring species of Desulfitobacterium and Sulfurospirillum and related bacteria. All tested microorganisms are able to reduce anthraquinone-2,6-disulfonate (AQDS) and four tested organisms (Desulfobacterium hafniense DP7, Sulfurospirillum barnesii, Sulfurospirillum deleyianum and Sulfurospirillum arsenophilum) are able to oxidize reduced anthrahydroquinone-2,6,-disulfonate (AH(2)QDS) as well. The characteristic to reduce oxidized metals, and to reduce and oxidize quinone moieties coupled to energy conservation is a likely explanation for the presence of halorespiring microorganisms in unpolluted soils. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved

Combined carbon and hydrogen isotope fractionation investigations for elucidating benzene biodegradation pathways

Recently, combined carbon and hydrogen isotope fractionation investigations have emerged as a powerful tool for the characterization of reaction mechanisms relevant for the removal of organic pollutants. Here, we applied this approach in order to differentiate benzene biodegradation pathways under oxic and anoxic conditions in laboratory experiments. Carbon and hydrogen isotope fractionation of benzene was studied with four different aerobic strains using a monooxygenase or a dioxygenase for the initial benzene attack, a facultative anaerobic chlorate-reducing strain as well as a sulfate-reducing mixed culture. Carbon and hydrogen enrichment factors (epsilon(C), epsilon(H)) varied for the specific pathways and degradation conditions, respectively, so that from the individual enrichment factors only limited information could be obtained for the identification of benzene biodegradation pathways. However, using the slope derived from hydrogen vs carbon isotope discriminations or the ratio of hydrogen to carbon enrichment factors (lambda = deltaH/ deltaC approximately epsilon(H)/epsilon(C)), benzene degradation mechanisms could be distinguished. Although experimentally determined lambda values partially overlapped, ranges could be determined for different benzene biodegradation pathways. Specific lambda values were 17 for anaerobic degradation. Moreover, variations in lambda values suggest that more than one reaction mechanism exists for monohydroxylation as well as for anaerobic benzene degradation under nitrate-reducing, sulfate-reducing, or methanogenic conditions. Our results show that the combined carbon and hydrogen isotope fractionation approach has potential to elucidate biodegradation pathways of pollutants in field and laboratory microcosm studie