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Anaerobic biodegradation of hydrocarbons
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Anaerobic biodegradation of hydrocarbons Christof Holliger* and Alexander JB Zehndert Anaerobic biodegradation of aliphatic and aromatic hydrocarbons is a promising alternative to aerobic biodegradation treatments in bioremediation processes. It is now proven that, besides toluene, benzene and ethylbenzene can be oxidized under anaerobic redox conditions. Anaerobic bacteria have also been shown capable of utilizing substrates not only in the pure form, but also in complex hydrocarbon mixtures, such as crude oil. In addition, crucial steps in anaerobic treatment processes have been studied in vitro to better understand the enzymes involved in monoaromatic hydrocarbon degradation. Knowledge remains incomplete, however, about the anaerobic degradation of aliphatic and polycyclic aromatic hydrocarbons.
Address *Swiss Federal Institute for EnvironmentalScience and Technology (EAWAG), Limnological Research Center, 6047 Kastanienbaum Switzerland; e-mail: [email protected] tSwiss Federal Institute for EnvironmentalScience and Technology (EAWAG), Ueberlandstrasse, 133 8600 D0bendorf Switzerland; e-mail: [email protected] Current Opinion in Biotechnology 1996, 7:326-330 © Current Biology Ltd ISSN 0958-1669 Abbreviations BTEX benzene,toluene, ethylbenzeneand xylene HPLC high-performanceliquid chromatography PAH polycyclicaromatic hydrocarbon
Introduction Aliphatic and aromatic hydrocarbons are the main constituents of crude oil and fossil fuels. In addition, they are present in creosote, the waste product of coal gasification. Because of leakage of underground storage tanks and pipelines, spills at production wells, refineries, and distribution terminals, and improper disposal and accidents during transport, these compounds have become the most frequently encountered environmental pollutants. O f particular concern are the aromatic hydrocarbons because of their potential carcinogenicity. Two major groups of aromatic hydrocarbons are the monocyclic compounds benzene, toluene, ethylbenzene, and xylene (BTEX) and the polycyclic aromatic hydrocarbons (PAHs) such as naphthalene, anthracene, and phenanthrene.
Although aerobic biodegradation has been successfully applied in bioremediation processes, in certain cases, such a treatment is either not possible or difficult to achieve. Problems commonly encountered with aerobic processes are the production of 'overwhelming' levels of biomass, which may cause clogging in in situ treatment systems, and insufficient supply of oxygen. Therefore, anaerobic treatment processes with nitrate, Fe(III), sulphate, and carbon
dioxide as electron acceptors are interesting alternatives for the bioremediation of hydrocarbon-contaminated sites. First attempts to treat a hydrocarbon-contaminated aquifer by pumping nitrate-containing water into the subsurface were carried out in the early eighties [1]. Additional interest in anaerobic hydrocarbon degradation has arisen as a result of undesired hydrogen sulphide production, a corrosive and toxic agent, by sulphate-reducing bacteria at marine oil production sites. Although 'disappearance' of aliphatic as well as aromatic hydrocarbons has been observed in anoxic environments, little is known about the microbiology involved in the degradation of aliphatic hydrocarbons and PAHs. In the past decade, many advances have been achieved in the understanding of the anaerobic degradation of monocyclic hydrocarbons, especially toluene. Various aspects of these processes, such as the bacteria and the biochemistry involved, have been summarized in several excellent recent reviews [2,3°,4°°,5,6]. Here, we summarize what is described in more detail in those reviews and add the newest findings published in the past 2-3 years. We discuss the anaerobic degradation of the three major groups of hydrocarbons: the aliphatic hydrocarbons, the monocyclic hydrocarbons and PAHs. In every section, knowledge on the bacteria, pathways, and enzymes involved is presented and compared with results obtained in phenomenological studies. Aliphatic hydrocarbons Anaerobic degradation of aliphatic hydrocarbons under sulphate-reducing and denitrifying conditions was first reported in 1944 and 1969, respectively (for references, see [7]). Unfortunately, the enrichments or isolates were not preserved and not much is known about the bacteria responsible for the degradation. Recently, Widdel and colleagues [2,7,8"'] have obtained pure cultures of sulphate-reducing [7,8"'] and denitrifying bacteria [2] that completely oxidize alkanes with six to 20 carbon atoms. A moderately thermophilic sulphate reducer [8"'] and one of the denitrifying strains [2] also grew on crude oil as a substrate, with specific consumption of n-alkanes in accordance with the substrate spectra of the strains. This indicated both that such strains utilize these substrates in the environment, where alkanes are always present as a complex mixture of hydrocarbons, and that the sulphate reducers are indeed able to produce the corrosive and toxic gas hydrogen sulphide with crude oil as a substrate. T h e mechanism of alkane activation in the absence of oxygen is still unknown. After growth of one of the sulphate reducers on alkanes with C-odd chains, C-even fatty acids were dominant [2]. This can best be explained by a removal or an addition of a Ci-unit, possibly carbon monoxide. Even so, results with another sulphate reducer and a denitrifying
Anaerobic biodegradation of hydrocarbons Holliger and Zehnder 327
Figure 1
Schematic pathway of anaerobic degradation of aromatic compounds. Diverse aromatic compounds are first transformed into central intermediates (benzoyI-CoA, resorcinol or phloroglucinol) that are subsequently reduced to alicyclic compounds. The ring is then cleaved by hydrolysis and the non-cyclic products are transformed into the central metabolite acetyl-CoA by 13-oxidation (adapted from [4°°]).
strain showed that other mechanisms may also be involved [2]. With these organisms, C-odd alkanes yielded C-odd fatty acids and C-even alkanes yielded C-even fatty acids. Studies in vitro are still needed and much remains to be discovered concerning the anaerobic degradation of alkanes.
Aromatic hydrocarbons As pointed out by Fuchs et al. [4"'], aromatic compounds comprise the second largest group of natural products. Because they are present in large amounts in plant material, they are also the major constituents of crude oil. From all the studies carried out to date with different aromatic compounds, such as phenols, cresols, anilines, benzoates, toluene, benzene, xylenes, nitroaromatic and chlorinated compounds, and many others, it can be concluded that anaerobic bacteria follow a strategy that is similar to that of aerobic bacteria [4°°]. First, the diverse aromatic compounds are transformed into a few central intermediates (Fig. 1). Subsequently, the aromatic ring is activated and cleaved, and the resulting noncyclic compounds are converted into central metabolites. Under anaerobic conditions, the major intermediates are benzoate (or benzoyl-CoA) and, to a lesser extent, resorcinol and phloroglucinol [4"°,6]. Reactions involved in the channelling processes that lead to the central intermediates include carboxylations, decarboxylations, hydroxylations, reductions, reductive dehydroxylations, deaminations, dechtorinations, aryl ether cleavages, and lyase reactions. T h e aromatic central intermediates are reductively attacked, as proposed by Evans in 1977 [9], and cleaved by hydrolysis. The resulting non-cyclic products are transformed by [3-oxidation to central metabolites.
In the following sections, the degradation of the four monocyclic aromatic hydrocarbons of the BTEX group are presented in more detail. In addition, the degradation of benzoate is discussed as this is the central intermediate in BTEX degradation. Finally, a brief summary of the few results published on PAH degradation are given. Benzene, toluene, ethylbenzene environmental samples
a n d x y l e n e in
In all environmental samples tested for their potential to degrade different aromatic hydrocarbons to date, only some of the test compounds have been oxidized and the rest has been persistent [8",10",11,12°,13]. Toluene was degraded in all samples, whereas o-xylene and m-xylene were degraded in some of the samples, but not in others. Benzene, ethylbenzene, and p-xylene were persistent in all of these studies. A mixture of these compounds may have a negative influence on the degradation of a readily degradable aromatic hydrocarbon. For example, toluene degradation in a denitrifying biofilm system is inhibited by o-xylene that has been transformed to the dead-end product 2-methylbenzoate [14]. In an enrichment culture in the presence of sulphate and with crude oil as electron donor, the aromatic hydrocarbons toluene, o-xylene, and m-xylene are specifically consumed and hydrogen sulphide is produced [8°']. This confirms results obtained with alkane-oxidizing sulphate reducers suggesting that these bacteria may be the source of sulphide in oil deposits and oil-production plants. Benzene
Anaerobic degradation of benzene under methanogenic conditions was first reported in 1986 by Vogel and Grbic-Galic [15]. Using H2180, it was shown that the
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initial step in this degradation is the transformation of benzene into phenol. Recently, rapid benzene oxidation has been demonstrated in microcosms under sulphatereducing [16"] and iron-reducing [17,18"'] conditions. Under sulphate-reducing conditions, benzene oxidation is inhibited by molybdate, and electron balances show that sulphate is the main electron acceptor. Complete mineralization has been confirmed through the use of 14C-labelled benzene [16",19]. T h e bacteria involved in benzene degradation under the above different redox conditions have not been identified as yet. Although the degradation pathway is not known, one may postulate that benzene is transformed into the central intermediate benzoate via phenol and p-hydroxybenzoate. Phenol formation from benzene has already been demonstrated in methanogenic cultures [15]. Furthermore, denitrifying bacteria have been shown capable of carboxylation of phenol, and reductive dehydroxylation of p-hydroxybenzoate has already long been known to occur [4"'].
Toluene
Toluene is the most readily degraded aromatic hydrocarbon under various anaerobic conditions. Pure cultures of denitrifying [2,20,21,22",23",24,25], iron-reducing [26], and sulphate-reducing [27] bacteria are available that utilize toluene as a carbon and energy source. All denitrifying organisms that have been fully characterized belong to the two genera Thauera and Azoarcus, which cluster in a branch of the I~-subclass of Proteobacteria [20,25]. A study with samples from different environments, such as soils, composts, and aquifers from different geographic regions, has shown that denitrifying toluene degraders are widespread and common in nature [22"]. Geobacter metallireducens strain GS-15 is an iron-reducing bacterium able to oxidize a variety of aromatic compounds [28]; this bacterium was first isolated on acetate as a substrate. A sulphate-reducing bacterium that oxidizes toluene has also been isolated [27], and has been proposed as a new genus/species: Desulfobacula toluolica. Under methanogenic conditions, toluene oxidation has also been observed, but the bacteria responsible for the degradation have not yet been isolated [10",15,19]. Inhibition of this degradation by nitrate or sulphate indicates that organisms other than the above-mentioned bacterial pure cultures may also be involved [10"]. Toluene seems to be degraded via benzoyl-CoA in all toluene-oxidizing bacteria isolated to date; it is apparently transformed to this central intermediate via two main pathways. One involves the oxidation of the methyl group to a carboxy substituent, the other involves an oxidative condensation of the methyl group with the ct-carbon of acetyl-CoA followed by a 13-oxidation to yield benzoyl-CoA [2,4"']. The latter pathway was hypothesized on the basis of the observation that benzylsuccinate and
benzylfumarate are formed as dead-end products by the denitrifying strains T1 [29], T [30"], and K172 [30"], and in a sulphate-reducing enrichment [31]. Evidence for the first pathway, a direct formation of benzoate, was obtained from experiments using non-growth substrates. Fluoro-analogues, chloro-analogues, and methyl-analogues of toluene are transformed to their corresponding benzoates by the denitrifying bacterium Thauera sp. strain K172 [32"']. Similar observations with non-growth substrates have been made using D. toluolica [33] and the denitrifyer strain T [30"]. T h e oxidation of the methyl group is suggested to occur by an initial hydration to form benzyl alcohol followed by two dehydrogenations to benzaldehyde and finally benzoate. Cell suspensions of a denitrifying bacterium that are metabolically blocked by iodoacetamide convert 14C-toluene first to 14C-benzyl alcohol and then to 14C-benzaldehyde [34]. Some of the denitrifying organisms and G. metallireducens are able to grow on benzyl alcohol, whereas other bacteria known to produce benzoate from toluene do not utilize this substrate [2]. The sulphate-reducing bacterium D. toluolica is inhibited by benzyl alcohol at concentrations of 500~tM. If noninhibitory concentrations are applied to cell suspensions, benzyl alcohol is not oxidized, suggesting that benzyl alcohol is not a free intermediate in this bacterium [33]. Isotope-trapping experiments and a high-performance liquid chromatography (HPLC) method for detecting very low concentrations of metabolites have been employed to demonstrate that a mixed methanogenic culture can carry out toluene hydroxylation to benzyl alcohol [35]. Activities of the initial oxidizing enzyme, a benzyl alcohol dehydrogenase, and a benzaldehyde dehydrogenase have been measured in in vitro experiments with strain K172 [32°']. The initial toluene-oxidizing activity is induced by toluene, benzyl alcohol, or benzaldehyde, and this activity is both coupled to nitrate reduction and dependent on the presence of glycerol in the assay. Furthermore, in vitro activity measurements are possible only with 4-fluorotoluene as a substrate, toluene itself is not oxidized in cell extracts. Ethylbenzene
Although ethylbenzene is a substantial component of crude oil and petroleum products, anaerobic degradation of alkylbenzenes with side chains that are longer than a methyl group has not been demonstrated in enrichments. Recently, two previously unknown denitrifying bacteria have been isolated by Rabus and Widdei [23"'] that are able to utilize either ethylbenzene (strain EbN1) or propylbenzene (strain PbN1) as a carbon and energy source. These bacteria completely oxidize alkylbenzenes to carbon dioxide. Growth of strain EbN1 and strain PbN1 on 1-phenylethanol and acetophenone or 1-phenylpropanol and propiophenone, respectively, suggests that the initial attack of ethylbenzene and propyibenzene is a hydroxylation of the alkyl substituent.
Anaerobic biodegradation of hydrocarbons Holliger and Zehnder
Xylenes For all three isomers of xylene, oxidation has been reported either in enrichment cultures or in pure cultures. m-Xylene is a substrate for denitrifying bacteria [22",24] and incubations of cell suspensions at 5°C have shown the accumulation of 3-methylbenzoate, indicating that the initial steps are the same as those for toluene degradation [30"]. T h e ortho-isomer and para-isomer are oxidized by enrichment cultures [10",12"]. In addition, the xylenes can also be co-metabolically transformed to the corresponding methylbenzoates by some denitrifying and sulphate-reducing toluene degraders. Nothing is known about the degradation of the methylbenzoates.
Benzoate Two recent reviews cover the degradation of benzoate in detail [4"',36"]. From the available literature, it can be concluded that not benzoate, but benzoy]-CoA is the substrate of the ring-reducing enzymes. T h e latter compound is formed either directly by benzoate-CoA ligase or indirectly from an aromatic acid that has already been activated by CoA such as phenylpropionyl-CoA. In studies in vitro with 14C-labelled and t3C-labelled compounds, HPLC, thin layer chromatography and onedimensional/two-dimensional NMR analysis, it has been shown that benzoyl-CoA is reduced by two electrons to form cyclohex-l,5-diene-carboxyl-CoA [37,38]. This compound is either reduced to cyclohex-l-ene-carboxyl-CoA or hydrated to 6-hydroxycyclohex-l-ene-carboxyl-CoA. T h e next major intermediate that could be isolated is 3-hydroxypimelyl-CoA, which can be formed from the other two by hydrations, dehydrogenations, and a final hydrolysis. T h e assay in vitro requires strictly anaerobic conditions and a low potential reducing agent such as Ti(III) citrate [38]. The removal of benzoyl-CoA is dependent on the cell extracts used.
Polyo/clic aromatic hydrocarbons Thus far, naphthalene is the only PAH for which anaerobic degradation has been reported. It is oxidized under denitrifying conditions in microcosms from contaminated aquifers and in slurries of different soils [39,40]. T h e mineralization of this compound has been confirmed by 14CO2 production from 14C-naphthalene. In the first study, -44% of the label was recovered in CO z [40], in the other more than 90% was recovered [39]. Nothing is known about the bacteria and the pathways involved.
Conclusions T h e anaerobic biodegradation of hydrocarbons was still doubted ten years ago and only little was known concerning its microbiology and biochemistry; many of the degradation pathways were largely hypothetical. Recent research demonstrates that hydrocarbons can serve as electron donors and as a carbon source for bacteria functioning under a variety of redox conditions. As biochemical work progresses, new pathways with novel
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and interesting enzymes are unfolding. Because most groups of hydrocarbons intrinsically do not resist anaerobic attack by bacteria, their frequently observed recalcitrance in the environment is the result of unfavourable environmental conditions and their limited availability to the microbes. For successful bioremedial application of hydrocarbon-utilizing microbes under anaerobic conditions, much attention and research needs to be directed towards an understanding of the environmental influences on biodegradation and towards mastering the limitations that prevent the optimal activities of the relevant bacteria in the environment.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • .t
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Fuchs G, Mohamed MES, Altenschmidt U, Koch J, Lack A, Brackmann R, Lochmeyer C, Oswald B: Biochemistry of anaerobic biodegradation of aromatic compounds. In Biochemistry of Microbial Degradation. Edited by Ratledge C. Dordrecht: Kluwer Academic Publishers; 1994:513-553. This excellent review develops an elegant general concept on anaerobic degradation of aromatic compounds before discussing in detail the degradation steps and enzymes involved. 5.
Grbic-Galic D: Anaerobic microbial transformation of nonoxygenated aromatic and alicyclic compounds in soil, subsurface, and freshwater sediments. In Soil Biochemistry. Edited by Bollag J-M, Stotzky G. New York: Marcel Dekker Inc; 1990:117-187.
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Rueter P, Rabus R, Wilkes H, Aeckersberg F, Ralney FA, Jannasch HW, Widdel F: Anaerobic oxidation of hydrocarbons in crude oil by new types of sulphate-reducing bacteria. Nature 1994, 372:455-458. This report shows that the pure cultures that are able to degrade single hydrocarbons also oxidize these compounds when they are present in a mixture of hydrocarbons, such as crude oil. 9.
Evans WC: Biochemistry of the bacterial catabolism of aromatic compounds in anaerobic environments. Nature 1977, 270:1 ?-22.
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Edwards EA, Grbic-Galic D: Anaerobic degradation of toluene and o-xylene by a methanogenic consortium. Appl Environ Microbiol 1994, 60:313-322.
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Although the authors demonstrate degradation only in mixed cultures, the data show that toluene and o-xylene degradation is possible under methanogenic conditions. 11.
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growth on toluene and description of Azoercus tolulyticus sp. nov. Int J Syst Bacteriol 1995, 45:500-506. 26.
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Lovley DR, Giovannoni SJ, White DC, Champine JE, Phillips EJP, Gorby YA, Goodwin S: Geobacter metallireducens 9en. nov. sp. nov., a microorganism capable of coupling the complete oxidation of organic compounds to the reduction of iron and other metals. Arch Microbiol 1993, 159:336-344.
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FrazerAC, Ling W, Young LY: Substrate induction and metebolite accumulation during anaerobic toluene utilization by the denitrlfying strain T1. Appl Environ Microbiol 1993, 59:3157-3160.
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Hutchins SR: Biotransformation and mineralization of alkylbenzenes under denitrifying conditions. Environ Tox Chem 1993, 12:1413-1423.
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Arcangeli J-P, Arvin E: Cometabolic transformation of o-xylene in a biofilm system under nitrate reducing conditions. Biodegradation 1995, 6:19-27.
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Vogel TM, Grbic-Galic D: Incorporation of oxygen from water into toluene and benzene during anaerobic fermentative transformation. Appl Envrion Microbiol 1986, 52:200-202.
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Lovley DR, Coates JD, Woodward JC, Phillips EIP: Benzene oxidation coupled to sulfate reduction. Appl Environ Microbiol 1995, 61:953-958. Using electron balances, inhibitor studies, and 14C-experiments, these authors show that sulphate-dependent benzene oxidation occurs in enrichment cultures from reduced sediments. 1 7,
Lovley DR, Woodward JC, Chapelle FH: Rapid anaerobic benzene oxidation with a variety of chelated Fe(lll) forms. App/ Environ Microbiol 1996, 62:288-291.
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Seyfried B, GIod G, Schocher R, Tschech A, Zeyer J: Initial reactions in the anaerobic oxidation of toluene and m-xylene by denitrifylng bacteria. Appl Environ Microbiol 1994, 60:4047-4052. By carrying out experiments at 5"C and using halogenated substrate analogues, these authors demonstrate that the initial attack in toluene degradation by Pseudomonas sp. strains K172 and T is the oxidation of the methyl group to form benzaldehyde and benzoate. 31.
Belier HR, Reinhard M, Grbic-Galic D: Metabolic by-products of anaerobic toluene degradation by sulfate-reducing enrichment cultures. Appl Environ Microbiol 1992, 58:3192-3195.
Lovley DR, Woodward JC, Chapelle FH: Stimulated anoxic blodegradation of aromatic hydrocarbons using Fe(lll) ligands. Nature 1994, 370:128-131. This report demonstrates that hydrocarbon-contaminated soils and aquifers may be bioremediated by stimulating Fe(lll) reduction in the subsurface using Fe(lll) ligands. This offers the opportunity of utilizing for bioremediation an electron acceptor that is often present in sufficient amounts in the environment.
Biegert T, Fuchs G: Anaerobic oxidation of toluene (analogues) to benzoate (analogues) by whole cells and by cell extracts of a denitrifying Thauera sp. Arch Microbiol 1995, 163:407-417. This is the first report on a study in vitro of the initial oxidative attack of the methyl group of toluene by a denitrifying bacterium. 33.
Rabus R, Widdel F: Conversion studies with substrate analogues of toluene in a sulfate-reducing bacterium, strain To12. Arch Microbiol 1995, 164:448-451.
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Grbic-Galic D, Vogel T: Transformation of toluene and benzene by mixed methanogenic cultures. App/Environ Microbio/198?, 53:254-260.
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Altenschmidt U, Fuchs G: Anaerobic toluene oxidation to benzyt alcohol and benzaldehyde in a denitrlfylng Pseudomonas strain. J Bacterio/1992, 174:4860-4862.
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Anders H-J, Ksetzke A, K~rnpfer P, Ludwig W, Fuchs G: Taxonomic position of aromatic-degrading denitrlfylng pseudomonad strains k 172 and kb 740 and their description as new members of the genera Thauera, as Thauera aromaUca sp. nov., and Azoarcus, as Azoarcus evansii sp. nov., respectively, members of the beta subclass of the Proteobacteria. Int J Syst Bacterio/1995, 45:327-333.
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Edwards EA, Edwards AM, Grbic-Galic D: A method for detection of aromatic metabolites at very low concentrations: application to detection of metabolites of anaerobic toluene degradation. Appl Environ Microbiol 1994, 60:323-327.
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EvansPJ, Mang DT, Shin Kim K, Young LY: Anaerobic degradation of toluene by a denitrifying bacterium. App/ Environ Microbiol 1991, 57:1139-1145.
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Fries MR, Zhou J, Chee-Sanford J, Tiedje JM: Isolation, characterization, and distribution of denitrifylng toluene degraders from a variety of habitats. App/Environ Microbiol 1994, 60:2802-2810. This detailed study shows that denitrifying toluene degraders are widespread and that this degradation capacity is common in nature. Rabus R, Widdel F: Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons by new denitrifying bacteria. Arch Microbio/1995, 163:96-103. Here, it is shown for the first time that anaerobic bacteria in pure culture are able to oxidize alkylbenzenes with side chains longer than a methyl group.
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Elder 13JE,Kelly DJ: The bacterial degradation of benzoic acid and benzenoid compounds under anaerobic conditions: unifying trends and new perspectives. FEMS Microbic)/Rev 1994, 13:441-488. A well referenced review summarizing knowledge of anaerobic benzoate degradation with special emphasis on the degradation by Rhodopseudomonas palustris. 3?.
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Anaerobic biodegradation of hydrocarbons Christof Holliger* and Alexander JB Zehndert Anaerobic biodegradation of aliphatic and aromatic hydrocarbons is a promising alternative to aerobic biodegradation treatments in bioremediation processes. It is now proven that, besides toluene, benzene and ethylbenzene can be oxidized under anaerobic redox conditions. Anaerobic bacteria have also been shown capable of utilizing substrates not only in the pure form, but also in complex hydrocarbon mixtures, such as crude oil. In addition, crucial steps in anaerobic treatment processes have been studied in vitro to better understand the enzymes involved in monoaromatic hydrocarbon degradation. Knowledge remains incomplete, however, about the anaerobic degradation of aliphatic and polycyclic aromatic hydrocarbons.
Address *Swiss Federal Institute for EnvironmentalScience and Technology (EAWAG), Limnological Research Center, 6047 Kastanienbaum Switzerland; e-mail: [email protected] tSwiss Federal Institute for EnvironmentalScience and Technology (EAWAG), Ueberlandstrasse, 133 8600 D0bendorf Switzerland; e-mail: [email protected] Current Opinion in Biotechnology 1996, 7:326-330 © Current Biology Ltd ISSN 0958-1669 Abbreviations BTEX benzene,toluene, ethylbenzeneand xylene HPLC high-performanceliquid chromatography PAH polycyclicaromatic hydrocarbon
Introduction Aliphatic and aromatic hydrocarbons are the main constituents of crude oil and fossil fuels. In addition, they are present in creosote, the waste product of coal gasification. Because of leakage of underground storage tanks and pipelines, spills at production wells, refineries, and distribution terminals, and improper disposal and accidents during transport, these compounds have become the most frequently encountered environmental pollutants. O f particular concern are the aromatic hydrocarbons because of their potential carcinogenicity. Two major groups of aromatic hydrocarbons are the monocyclic compounds benzene, toluene, ethylbenzene, and xylene (BTEX) and the polycyclic aromatic hydrocarbons (PAHs) such as naphthalene, anthracene, and phenanthrene.
Although aerobic biodegradation has been successfully applied in bioremediation processes, in certain cases, such a treatment is either not possible or difficult to achieve. Problems commonly encountered with aerobic processes are the production of 'overwhelming' levels of biomass, which may cause clogging in in situ treatment systems, and insufficient supply of oxygen. Therefore, anaerobic treatment processes with nitrate, Fe(III), sulphate, and carbon
dioxide as electron acceptors are interesting alternatives for the bioremediation of hydrocarbon-contaminated sites. First attempts to treat a hydrocarbon-contaminated aquifer by pumping nitrate-containing water into the subsurface were carried out in the early eighties [1]. Additional interest in anaerobic hydrocarbon degradation has arisen as a result of undesired hydrogen sulphide production, a corrosive and toxic agent, by sulphate-reducing bacteria at marine oil production sites. Although 'disappearance' of aliphatic as well as aromatic hydrocarbons has been observed in anoxic environments, little is known about the microbiology involved in the degradation of aliphatic hydrocarbons and PAHs. In the past decade, many advances have been achieved in the understanding of the anaerobic degradation of monocyclic hydrocarbons, especially toluene. Various aspects of these processes, such as the bacteria and the biochemistry involved, have been summarized in several excellent recent reviews [2,3°,4°°,5,6]. Here, we summarize what is described in more detail in those reviews and add the newest findings published in the past 2-3 years. We discuss the anaerobic degradation of the three major groups of hydrocarbons: the aliphatic hydrocarbons, the monocyclic hydrocarbons and PAHs. In every section, knowledge on the bacteria, pathways, and enzymes involved is presented and compared with results obtained in phenomenological studies. Aliphatic hydrocarbons Anaerobic degradation of aliphatic hydrocarbons under sulphate-reducing and denitrifying conditions was first reported in 1944 and 1969, respectively (for references, see [7]). Unfortunately, the enrichments or isolates were not preserved and not much is known about the bacteria responsible for the degradation. Recently, Widdel and colleagues [2,7,8"'] have obtained pure cultures of sulphate-reducing [7,8"'] and denitrifying bacteria [2] that completely oxidize alkanes with six to 20 carbon atoms. A moderately thermophilic sulphate reducer [8"'] and one of the denitrifying strains [2] also grew on crude oil as a substrate, with specific consumption of n-alkanes in accordance with the substrate spectra of the strains. This indicated both that such strains utilize these substrates in the environment, where alkanes are always present as a complex mixture of hydrocarbons, and that the sulphate reducers are indeed able to produce the corrosive and toxic gas hydrogen sulphide with crude oil as a substrate. T h e mechanism of alkane activation in the absence of oxygen is still unknown. After growth of one of the sulphate reducers on alkanes with C-odd chains, C-even fatty acids were dominant [2]. This can best be explained by a removal or an addition of a Ci-unit, possibly carbon monoxide. Even so, results with another sulphate reducer and a denitrifying
Anaerobic biodegradation of hydrocarbons Holliger and Zehnder 327
Figure 1
Schematic pathway of anaerobic degradation of aromatic compounds. Diverse aromatic compounds are first transformed into central intermediates (benzoyI-CoA, resorcinol or phloroglucinol) that are subsequently reduced to alicyclic compounds. The ring is then cleaved by hydrolysis and the non-cyclic products are transformed into the central metabolite acetyl-CoA by 13-oxidation (adapted from [4°°]).
strain showed that other mechanisms may also be involved [2]. With these organisms, C-odd alkanes yielded C-odd fatty acids and C-even alkanes yielded C-even fatty acids. Studies in vitro are still needed and much remains to be discovered concerning the anaerobic degradation of alkanes.
Aromatic hydrocarbons As pointed out by Fuchs et al. [4"'], aromatic compounds comprise the second largest group of natural products. Because they are present in large amounts in plant material, they are also the major constituents of crude oil. From all the studies carried out to date with different aromatic compounds, such as phenols, cresols, anilines, benzoates, toluene, benzene, xylenes, nitroaromatic and chlorinated compounds, and many others, it can be concluded that anaerobic bacteria follow a strategy that is similar to that of aerobic bacteria [4°°]. First, the diverse aromatic compounds are transformed into a few central intermediates (Fig. 1). Subsequently, the aromatic ring is activated and cleaved, and the resulting noncyclic compounds are converted into central metabolites. Under anaerobic conditions, the major intermediates are benzoate (or benzoyl-CoA) and, to a lesser extent, resorcinol and phloroglucinol [4"°,6]. Reactions involved in the channelling processes that lead to the central intermediates include carboxylations, decarboxylations, hydroxylations, reductions, reductive dehydroxylations, deaminations, dechtorinations, aryl ether cleavages, and lyase reactions. T h e aromatic central intermediates are reductively attacked, as proposed by Evans in 1977 [9], and cleaved by hydrolysis. The resulting non-cyclic products are transformed by [3-oxidation to central metabolites.
In the following sections, the degradation of the four monocyclic aromatic hydrocarbons of the BTEX group are presented in more detail. In addition, the degradation of benzoate is discussed as this is the central intermediate in BTEX degradation. Finally, a brief summary of the few results published on PAH degradation are given. Benzene, toluene, ethylbenzene environmental samples
a n d x y l e n e in
In all environmental samples tested for their potential to degrade different aromatic hydrocarbons to date, only some of the test compounds have been oxidized and the rest has been persistent [8",10",11,12°,13]. Toluene was degraded in all samples, whereas o-xylene and m-xylene were degraded in some of the samples, but not in others. Benzene, ethylbenzene, and p-xylene were persistent in all of these studies. A mixture of these compounds may have a negative influence on the degradation of a readily degradable aromatic hydrocarbon. For example, toluene degradation in a denitrifying biofilm system is inhibited by o-xylene that has been transformed to the dead-end product 2-methylbenzoate [14]. In an enrichment culture in the presence of sulphate and with crude oil as electron donor, the aromatic hydrocarbons toluene, o-xylene, and m-xylene are specifically consumed and hydrogen sulphide is produced [8°']. This confirms results obtained with alkane-oxidizing sulphate reducers suggesting that these bacteria may be the source of sulphide in oil deposits and oil-production plants. Benzene
Anaerobic degradation of benzene under methanogenic conditions was first reported in 1986 by Vogel and Grbic-Galic [15]. Using H2180, it was shown that the
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initial step in this degradation is the transformation of benzene into phenol. Recently, rapid benzene oxidation has been demonstrated in microcosms under sulphatereducing [16"] and iron-reducing [17,18"'] conditions. Under sulphate-reducing conditions, benzene oxidation is inhibited by molybdate, and electron balances show that sulphate is the main electron acceptor. Complete mineralization has been confirmed through the use of 14C-labelled benzene [16",19]. T h e bacteria involved in benzene degradation under the above different redox conditions have not been identified as yet. Although the degradation pathway is not known, one may postulate that benzene is transformed into the central intermediate benzoate via phenol and p-hydroxybenzoate. Phenol formation from benzene has already been demonstrated in methanogenic cultures [15]. Furthermore, denitrifying bacteria have been shown capable of carboxylation of phenol, and reductive dehydroxylation of p-hydroxybenzoate has already long been known to occur [4"'].
Toluene
Toluene is the most readily degraded aromatic hydrocarbon under various anaerobic conditions. Pure cultures of denitrifying [2,20,21,22",23",24,25], iron-reducing [26], and sulphate-reducing [27] bacteria are available that utilize toluene as a carbon and energy source. All denitrifying organisms that have been fully characterized belong to the two genera Thauera and Azoarcus, which cluster in a branch of the I~-subclass of Proteobacteria [20,25]. A study with samples from different environments, such as soils, composts, and aquifers from different geographic regions, has shown that denitrifying toluene degraders are widespread and common in nature [22"]. Geobacter metallireducens strain GS-15 is an iron-reducing bacterium able to oxidize a variety of aromatic compounds [28]; this bacterium was first isolated on acetate as a substrate. A sulphate-reducing bacterium that oxidizes toluene has also been isolated [27], and has been proposed as a new genus/species: Desulfobacula toluolica. Under methanogenic conditions, toluene oxidation has also been observed, but the bacteria responsible for the degradation have not yet been isolated [10",15,19]. Inhibition of this degradation by nitrate or sulphate indicates that organisms other than the above-mentioned bacterial pure cultures may also be involved [10"]. Toluene seems to be degraded via benzoyl-CoA in all toluene-oxidizing bacteria isolated to date; it is apparently transformed to this central intermediate via two main pathways. One involves the oxidation of the methyl group to a carboxy substituent, the other involves an oxidative condensation of the methyl group with the ct-carbon of acetyl-CoA followed by a 13-oxidation to yield benzoyl-CoA [2,4"']. The latter pathway was hypothesized on the basis of the observation that benzylsuccinate and
benzylfumarate are formed as dead-end products by the denitrifying strains T1 [29], T [30"], and K172 [30"], and in a sulphate-reducing enrichment [31]. Evidence for the first pathway, a direct formation of benzoate, was obtained from experiments using non-growth substrates. Fluoro-analogues, chloro-analogues, and methyl-analogues of toluene are transformed to their corresponding benzoates by the denitrifying bacterium Thauera sp. strain K172 [32"']. Similar observations with non-growth substrates have been made using D. toluolica [33] and the denitrifyer strain T [30"]. T h e oxidation of the methyl group is suggested to occur by an initial hydration to form benzyl alcohol followed by two dehydrogenations to benzaldehyde and finally benzoate. Cell suspensions of a denitrifying bacterium that are metabolically blocked by iodoacetamide convert 14C-toluene first to 14C-benzyl alcohol and then to 14C-benzaldehyde [34]. Some of the denitrifying organisms and G. metallireducens are able to grow on benzyl alcohol, whereas other bacteria known to produce benzoate from toluene do not utilize this substrate [2]. The sulphate-reducing bacterium D. toluolica is inhibited by benzyl alcohol at concentrations of 500~tM. If noninhibitory concentrations are applied to cell suspensions, benzyl alcohol is not oxidized, suggesting that benzyl alcohol is not a free intermediate in this bacterium [33]. Isotope-trapping experiments and a high-performance liquid chromatography (HPLC) method for detecting very low concentrations of metabolites have been employed to demonstrate that a mixed methanogenic culture can carry out toluene hydroxylation to benzyl alcohol [35]. Activities of the initial oxidizing enzyme, a benzyl alcohol dehydrogenase, and a benzaldehyde dehydrogenase have been measured in in vitro experiments with strain K172 [32°']. The initial toluene-oxidizing activity is induced by toluene, benzyl alcohol, or benzaldehyde, and this activity is both coupled to nitrate reduction and dependent on the presence of glycerol in the assay. Furthermore, in vitro activity measurements are possible only with 4-fluorotoluene as a substrate, toluene itself is not oxidized in cell extracts. Ethylbenzene
Although ethylbenzene is a substantial component of crude oil and petroleum products, anaerobic degradation of alkylbenzenes with side chains that are longer than a methyl group has not been demonstrated in enrichments. Recently, two previously unknown denitrifying bacteria have been isolated by Rabus and Widdei [23"'] that are able to utilize either ethylbenzene (strain EbN1) or propylbenzene (strain PbN1) as a carbon and energy source. These bacteria completely oxidize alkylbenzenes to carbon dioxide. Growth of strain EbN1 and strain PbN1 on 1-phenylethanol and acetophenone or 1-phenylpropanol and propiophenone, respectively, suggests that the initial attack of ethylbenzene and propyibenzene is a hydroxylation of the alkyl substituent.
Anaerobic biodegradation of hydrocarbons Holliger and Zehnder
Xylenes For all three isomers of xylene, oxidation has been reported either in enrichment cultures or in pure cultures. m-Xylene is a substrate for denitrifying bacteria [22",24] and incubations of cell suspensions at 5°C have shown the accumulation of 3-methylbenzoate, indicating that the initial steps are the same as those for toluene degradation [30"]. T h e ortho-isomer and para-isomer are oxidized by enrichment cultures [10",12"]. In addition, the xylenes can also be co-metabolically transformed to the corresponding methylbenzoates by some denitrifying and sulphate-reducing toluene degraders. Nothing is known about the degradation of the methylbenzoates.
Benzoate Two recent reviews cover the degradation of benzoate in detail [4"',36"]. From the available literature, it can be concluded that not benzoate, but benzoy]-CoA is the substrate of the ring-reducing enzymes. T h e latter compound is formed either directly by benzoate-CoA ligase or indirectly from an aromatic acid that has already been activated by CoA such as phenylpropionyl-CoA. In studies in vitro with 14C-labelled and t3C-labelled compounds, HPLC, thin layer chromatography and onedimensional/two-dimensional NMR analysis, it has been shown that benzoyl-CoA is reduced by two electrons to form cyclohex-l,5-diene-carboxyl-CoA [37,38]. This compound is either reduced to cyclohex-l-ene-carboxyl-CoA or hydrated to 6-hydroxycyclohex-l-ene-carboxyl-CoA. T h e next major intermediate that could be isolated is 3-hydroxypimelyl-CoA, which can be formed from the other two by hydrations, dehydrogenations, and a final hydrolysis. T h e assay in vitro requires strictly anaerobic conditions and a low potential reducing agent such as Ti(III) citrate [38]. The removal of benzoyl-CoA is dependent on the cell extracts used.
Polyo/clic aromatic hydrocarbons Thus far, naphthalene is the only PAH for which anaerobic degradation has been reported. It is oxidized under denitrifying conditions in microcosms from contaminated aquifers and in slurries of different soils [39,40]. T h e mineralization of this compound has been confirmed by 14CO2 production from 14C-naphthalene. In the first study, -44% of the label was recovered in CO z [40], in the other more than 90% was recovered [39]. Nothing is known about the bacteria and the pathways involved.
Conclusions T h e anaerobic biodegradation of hydrocarbons was still doubted ten years ago and only little was known concerning its microbiology and biochemistry; many of the degradation pathways were largely hypothetical. Recent research demonstrates that hydrocarbons can serve as electron donors and as a carbon source for bacteria functioning under a variety of redox conditions. As biochemical work progresses, new pathways with novel
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and interesting enzymes are unfolding. Because most groups of hydrocarbons intrinsically do not resist anaerobic attack by bacteria, their frequently observed recalcitrance in the environment is the result of unfavourable environmental conditions and their limited availability to the microbes. For successful bioremedial application of hydrocarbon-utilizing microbes under anaerobic conditions, much attention and research needs to be directed towards an understanding of the environmental influences on biodegradation and towards mastering the limitations that prevent the optimal activities of the relevant bacteria in the environment.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • .t
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Although the authors demonstrate degradation only in mixed cultures, the data show that toluene and o-xylene degradation is possible under methanogenic conditions. 11.
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