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Biodegradation of toxic and environmental pollutants
Biodegradation of toxic and environmental pollutants Lilly Y. Young and Max M. Hiiggblom New York University Medical Center, New York, USA Organic chemicals that are toxic to humans and to the environment can be transformed and metabolized by a variety of microorganisms. Such chemicals include trichloroethylene, chloroform, carbon tetrachloride, toluene, phenols, chlorinated phenols, polychlorinated biphenyls and polyaromatic hydrocarbons. This review focuses on some of the most important recent developments in the biodegradation of these toxic chemicals. Depending on the compound and the organism, the extent of our understanding ranges from the molecular level to the conceptual. Current Opinion in Biotechnology 1991, 2:429-435
Introduction The bioremediation used as a part o f the clean-up effort after the EXXON VALDEZaccident in March 1989 has brought the field of biodegradation to the attention of both regulatory and scientific communities, as well as to the general public. These efforts have contributed towards the acceptance of biological means of contaminant destruction as a valid alternative to physical and chemical technologies. Use of microbes in biogradation is as old as composting and has been long accepted as a well established technology, for example in municipal waste treatment. Recent attention, however, has been directed at treating toxic environmental pollutants. The potential for this 'biological technology' is heightened by documentation of the diversity of organisms capable of metabolizing an increasingly wide range of toxic organic compounds. Yet, it should be realized that the natural environment in which it is to be applied has complexities which cannot be readily governed nor quickly understood. Biodegradation is an extremely broad field. Some areas have only recently developed, whereas others have been under investigation for many years. As a consequence, breakthroughs can be at the level of a new paradigm, such as mineral cations serving as electron acceptors for toluene metabolism, as well as at the molecular level, such as a newly constructed metabolic pathway for the breakdown of chlorinated aromatics. By definition, the newer paradigms are less well understood and in some cases may lack appropriate pure cultures with which to work. Rather than detract from the accomplishments, this indicates the richness of possibilities regarding organisms to isolate and new pathways and mechanisms to study. Aerobic metabolism studies continue to involve the skillful construction of new pathways for degradation of chlorinated aromatic compounds [1,2] both in vivo and in vitro. While the construction of new pathways is limited
to the use of genetic material from isolated strains, a vast reservoir of genetic material available in nature has yet to be fully exploited. Thus, new strains are sought after continually and classic techniques for selection on carbon sources have yielded strains with new biodegradative capabilities [3°,4]. Anaerobic conditions, or those in which alternatives to oxygen serve as the electron acceptor, are receiving much attention. With anaerobiosis, there are many conditions under which the selection can take place, effectively increasing the genera of organisms sought after. For the oxidation of toxic chemicals, important environmental electron acceptors such as nitrate, sulfate and carbonate are nosy augmented by the demonstration that reduced iron and manganese also serve as electron acceptors [5"o]. In addition, highly chlorinated molecules such as polychlorinated biphenyls have been proposed to serve as anaerobic electron acceptors while undergoing reductive dechlorination.
Chlorinated aliphatic compounds The versatility of aerobes with noteworthy metabolic properties has been unexpectedly extended by the recent discovery that they are also capable of mediating the cometabolism (or as more recently termed, gramitous metabolism) of chlorinated aliphatic compounds. The widely varying groups of organisms with such capabilities include methanotrophs and nitrifying bacteria. Common to all of these organisms is the induction of mono- or dioxygenases not by the chlorinated aliphatic, but by a different compound altogether. This information is summarized in Table 1. The soluble methane monooxygenase (sMMO) from Metlyylosintts tricioosporittm OB3b appears to have a very
Abbreviations AMO~ammonia monooxygenase;BTX--benzene/toluene/xylenes; CB-~:hlorobenzoate; PCB--polychlorinated biphenyl; PCE~perchloroethylene; sMMO~soluble methane monooxygenase,TCE----trichloroethylene.
(~) Current Biology Ltd ISSN 0958-1669
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Environmental biotechnology Table 1. Oxygenases involved in the degradation of chlorinated aliphatic compounds.
Organisms
Inducer (enzyme)
Substrates
References
Methylosinus trichosporium OB3b
Methane (soluble methane monooxygenase)
Trichloroethylene, monochloroethylene, 1,1-, and cisand trans-l,2-dichloroethylene, dichloromethane, trichloromethane, 1,1- and 1,2-dichloroethane, 1,2-dichloropropane
[6°,7"]
Nitrosomonas europaea
Ammonia (ammonia monooxygenase)
Dichloromethane, trichloromethane, 1,1,2and 1,1,1-trichloroethane, monochloroethylene, dichloroethylenes, trichtoroethylene, 1,2,3-trichloropropane, monohalogenated ethanes
[8%9°1
Nitrosolobus multiformis
Ammonia (ammonia monooxygenase)
1,2-Dichloropropane, 1,2-dibromo-3-chloropropane
[10.1
Pseudomonas cepacia CA
Toluene, phenol (monooxygenase)
Tdchloroethylene
[11,12]
Trichloroethylene
[15"]
Monochloroethylene 1,1-dichloroethylene,cisand trans-l,2-dichloroethylene, trichloroethylene
[16.]
Pseudomonas putida FI [ Escherichia coli Mycobacterium spp.
Alcaligenes eutrophus JMP134 Pseudomonas putida PpG-786
Toluene dioxygenase Propane (propane monooxygenase)
Phenol, 2,4-dichlorophenoxyacetic acid Trichloroethylene Camphor (cytochrome P450 cam)
1,1,2-Trichloroethane
[17] [18]
broad substrate specificity and attacks many but not all of the chlorinated one- and two-carbon compounds. In particular, degradation of trichloroethylene (TCE) is very" rapid [6",7"]. Although this organism also has a second membrane-bound methane monooxygenase, the activity of this enzyme is limited to a few compounds with little observed chloride release. Several lines of evidence support the proposal that sMMO is the enzyme responsible for degradation of chlorinated compounds. First, sMMO is expressed only under conditions of copper limitation, and TCE degradation (as well as that of other chlorinated aliphatics) only occurs in cells grown in the absence of copper [7"]. Second, with rabbit antibodies prepared against purified sMMO, Western blot analysis demonstrated that only cells possessing the sMMO protein were capable of oxidizing TCE [6"]. Although vinyl chloride and chloroform (trichloromethane) were also metabolized by this enzyme, carbon tetrachloride and perchloroethyiene (tetrachloroethene; PCE) were not.
lize most of the chlorinated I and 2 carbon aliphatic compounds. Interestingly, as with sMMO discussed above, vinyl chloride and chloroform could be metabolized but carbon tetrachloride and PCE could not. Results obtained with additional genera of nitrifiers indicated that the soil organisms, N. europaea and Nitrosolobus multiformis are active on all three halocarbon fumigants tested, whereas the marine organism, Nitrosococcus oceanus could metabolize only the methyl bromide and not the chlorinated or brominated propane.s [9"]. The involvement of ammonia monooxygenase (AMO) is suggested by the observations that acetylene and other inh/bitors of AMO also inhibit halocarbon metabolism. In addition, metabolism of the halocarbon reduces the rate of nitrite production from ammonia, implying competition for the active site of AMO [8"]. Using monohalogenated ethanes, it was noted, not unexpectedly, that dehalogenation is inversely proportional to the size of the halogen substituent and to the length of the alkane [10"].
In an extension of work published in 1989, Vannelli et a t [8.] demonstrated that the ammonia-oxidizing nitrifying bacterium, Nitrosomonas europaea, could metabo-
Much interest has focused on the metabolism of TCE mediated by several Pseudomonas species that require induction by toluene or phenol. This was first reported in
Biodegradation of toxic and environmental pollutants Young and Hfiggblom 1987 [11] using Pseudomonas cepacia G4 isolated from the environment. A series of papers on strain G4 have examined the kinetics of TCE-degradation by phenolinduced cells [12], concluding that the toluene degradation proceeds through a new mechanism invoMng two sequential monooxygenations yielding the methylcatechol [13o°], rather than a dioxygenase-mediated reaction. In one of the few reports in which a contaminated site was treated hi situ, G4 was injected into a TCE-contaminated aquifer along with oxygen and nutrients, and this resulted in a reduction of TCE concentrations from 3000ppb to 7 8 p p b in 20days [14]. The effectiveness of using G4 in this pilot study is not convincing, however, because three variables were added without any means of assessing the effect of each one separately; oxygen, nutrients (undefined except as organic and inorganic) and G4 all were added at the same time. As each of the first two variables could themselves stimulate TCE loss, the effectiveness of G4 unfortunately remains obscure.
treated Methanosarchla barkeH cells were able to catalyze the reductive dechlorination ofCC14 to CHCI3 [21o]. Thus, these transformations o f CCI4 may not be enzymatic processes. Reductive dehalogenation can be catalyzed by reduced iron porphyrins, corrinoids and coenzyme F430, all o f which are cellular cofactors that are independently active in vitro and considered to be heat-stable [21o,24°,25]. High levels of these cofactors are found in the strict anaerobes which have the ability to dehalogenate CCI4. Although attributing dehalogenation to the bacterial ceils is not incorrect as they produce the necessary cofactors, the process may not be dependent on living cells (i.e. not enzyme-dependent). Experimental resuits, therefore, should be interpreted with caution, and additional controls should be used to account for the contribution of killed cells.
That the metabolism of TCE is mediated specifically by toluene dioxygenase in Pseudomonasptttida F1, and by no other enzymes of the pathway, was confirmed by cloning the four genes necessary for the dioxygenase into Eschericbia coli [15"]. TCE metabolism was mediated by the recombinant E. coli and not by the control strain which possessed the vector but not the genes. In a survey of widely differing strains that express a variety of monoand dioxygenases, all ~'e Mycobacterium species which grow on propane as a sole source of carbon were found to mediate TCE metabolism [16.]. TCE activity was associated with propane monooxygenase as uninduced cells did not metabolize TCE. Although the dichloroethylene derivatives and vinyl chloride were metabolized, PCE was not. It was noted in this paper [16 °] that P. putida F1 did not attack vinyl chloride. Degradation of chlorinated aliphatic compounds is also mediated by oxgenases induced by other aromatic compounds such as phenol, 2,4dichlorophenoxyacetic acid and camphor [17,18].
Chlorinated aromatic compounds Polychlorinatedbiphenyls
Several examples of strict anaerobic bacteria that can mediate the dechlorination of carbon tetrachloride (CCI 4) and chloroform (trichloromethane, CHCI3) [19oo,20,21 °] suggest that this ability may be rather widely dispersed. CCI4 transformation to CO 2 was also reported with a denitrifying pseudomonad growing on acetate [22]. Sulfate-reducing enrichments grown on lactate exhibited PCE dechlorination to tfichloroethene and dichloroethene. Although the evidence suggests that methanogens were not active, the group of organisms responsible was not identified [23]. A cautioriary note regarding these anaerobic dehalogenations was struck by the observation that sterilized controis can also display dechlorinating activity. This was noted with autoclaved cells of Acetobacterium woodii and Methanobacterium thermoautotrophicum. In experiments using 14CC14, both species formed the same amount o f 14COa in autoclaved and in active cultures and the M. tbennoautotrophicum also formed 14CHCI3. The rates of this transformation in both whole cells and cell extracts were also the same for autoclaved and unautoclaved preparations [19"']. Furthermore, heat-
Recent work on polychlorinated biphenyls (PCBs) has been directed at the anaerobic or reductive dechlorination of these highly chlorinated compounds. This has drawn attention away from the still very interesting aerobic metabolism of this class o f molecules. Aerobic degradation of PCBs is initiated by dioxygenase attack, usually at carbons 2 and 3, but sometimes at positions 3 and 4. The dihydroxychlorobiphenyls undergo ring fission to yield chlorinated benzoic acids or acetophenones. Monochlorobiphenyls are the only PCBs shown to be used as growth substrates. In a study using eight PCBdegrading bacterial strains, considerable differences between the strains were noted, with respect to both the choice of ring attacked and the relative rates of primary degradation [26,P1*]. Six o f the strains apparently only degrade PCBs by the 2,3-dioxygenase pathway, whereas two strains have both 2,3- and 3,4-dioxygenase activity. All of the strains preferentially metabolize an unchlorinated ring. Under reductive dechlorinating conditions, the rate, extent and pattern of dechlorination of commercial PCB mixtures (Arochlor 1242, 1248, 1254 and 1260) by sediment anaerobes has received much attention. All four Arochlors were dechlorinated with loss of chlorines in the meta- and para- positions, resulting in accumulation of the ortho-chlorinated congeners [27°°]. The extent of dechlorination was 15-85 % of total chlorines in 16 weeks, and the rate was inversely related to the degree of chlorination. Addition of organic substrates such as methanol, glucose or acetone stimulated dechlorination, whereas little stimulation was observed in parallel sediment samples that received no organic amendments [28°]. Again, meta- and parachlorines were preferentially removed. Implicit in these studies is the concept that a sequential anaerobic-aerobic treatment could be developed for bioremediation of Contaminated sites. It should be noted, however, that publications on reductive dechlorination
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Environmentalbiotechnology to date describe laboratory studies in which high concentrations (300-700 ppm) of Arochlors were added for the purposes of the experiments. Two points should be taken into account. First, laboratory results suggest that reductive dechlorination of PCBs is less effective at concentrations below 200 ppm and that, in situ, the PCBs are designated as hazardous at much lower concentrations than those used in the laboratory studies. Second, the availability from soils or sediment of newly added PCBs will be different from those that have aged in their natural environment and may be tightly bound. Thus, the real effectiveness of biotreatment on in situ and aged PCBs remains to be seen.
Chlorophenols and chlorobenzoic acids The aerobic degradation of mono- and dichlorinated phenols and benzoates generally proceeds by dioxygenation to chlorocatechols, with dehalogenation occurring after ring fission [29]. Evidence obtained with a Pseudomonas cepacia 2CBS isolated from the environment indicates the existence of a different mechanism in which dechlorination of 2-chlorobenzoate (CB) takes place during the initial dioxygenation step yielding catechol [30°]. A partially purified enzyme which catalyzed.the reaction was obtained, and required molecular oxygen, NADH and Fe 2+ for activity. Stoichiometric amounts of chloride were released and experiments using 180 2 confirmed that this was a dioxygenase mechanism. Aerobic dechlorination of the aromatic ring was also observed with Pseudomonasaerug#msaJB2 isolated from soil [4]. Along with other mono-, di- and trichlorobenzoates, 2,3and 2,5-dichlorobenzoate can serve as growth substrates. During the metabolism of these last two compounds, 4-chlorocatechol was detected as a metabolite, supporting the assumption that an initial dechlorination had occurred. Most of the recent studies on the anaerobic degradat_ion o f chloroaromatic compounds have been carried out under methanogenic conditions which seem to readily generate dechlorinating activity. Although the oxidationreduction pote.ntial for sulfate-reducing conditions is similar to that for methanogenesis ( - 250-- - 3 5 0 mY), the presence or addition of sulfate has been shown to inhibit dechlorinating activity [31]. However, chlorophenol degradation coupled to sulfate reduction in enrichments was reported for all three monochlorophenol isomers [32]. Evidence that supports a role for sulfate reduction includes stoichiometric utilization of sulfate for chlorophenol oxidation to CO 2, inhibition of chlorophenol degradation in the presence of molybdate, a specific inhibitor of sulfate reduction, and formation of sulfide during chlorophenol degradation. Whether it is the sulfate-reducers utilizing the chlorophenols or whether it is another organism tighdy coupled to the sulfate-reducer in a consortium arrangement is not known at this time. Several independent isolates o f the photosynthetic bacterium, Rhodopsettdomonaspalustriswere able to utilize 3-CB for growth in the presence of light and in the absence of oxygen [33], as illustrated by the conversion
of radiolabeled CB to radiolabeled CO z. This extends the aromatic substrate range for this species, which already includes phenols and aromatic acids and aldehydes. Chlorinated aromatic compounds thus appear to be susceptible to biodegradation by a number of physiologically different species, and this can take place under a variety of anaerobic conditions.
Energetics of dechlorination Thermodynamic data indicate that the reductive dechlorination of chlorinated aromatic compounds is an exergonic reaction which may yield biologically useful energy. Support for this hypothesis comes from studies with the anaerobic dechlorinating bacterium, Desulfomonile tiedje strain DCB1 [34"', 35]. DCB1 can only reductively remove the chlorine from 3-CB. In the presence of formate and acetate as electron donors and 3-CB as the electron acceptor, the cell yield increased stoichiometrically with the amount of 3-CB added. No growth was obtained if benzoate was substituted for 3-CB, or in the absence of 3-CB. Increased ATP levels were also obtained in response to the addition of 3-CB. Because strain DCB1 is the only anaerobic dechlorinating strain in pure culture, it will be interesting to learn whether this energy-generating mechanism also holds for other organisms and for other chlorinated compounds.
Cell-free dechlorinating enzyme Strain DCB1 was also the source for cell extracts and enzyme studies, in which Deweerd and Suflita [36"] reported the first cell-free dehalogenation system in an obligate anaerobe. Dehalogenation activity is heat-labile, dependent on reduced methyl viologen, an electron carrier, and stimulated by formate, carbon monoxide and hydrogen. It is membrane-associated and inhibited by oxygen. Extracts from cells grown in the presence of 3-CB had 10-fold greater activity than those from cells grown in its absence, suggesting that it is an induced enzyme. The cell extract also dehalogenated 3-bromo- and 3-iodobenzoate, but not 3-fluorobenzoate. Although strain DCB1 is a sulfidogen and can grow via reduction of sulfur oxTailions, sulfite or thiosulfate but not sulfate inhibit dehalogenation. This may seem contradictory, but it is consistent with the authors' suggestion that the sulfur oxyanions may be competing with dehalogenation for reducing equivalents. It should be noted that reductive dehalogenation is not limited to anaerobes; it has also been reported in cell extracts o f aerobic bacteria where it is not inhibited by oxygen [29].
Hydrocarbons
AIkylbenzenes Benzene, toluene and xylenes (BTX) have been identiffed by the US Environmental Protection Agency as among the 17 chemicals posing the greatest threats to human health. Earlier this year, the largest industrial users
Biodegradation of toxic and environmental pollutants Young and Hfiggblom were asked to voluntarily reduce their release of these chemicals over the next 3 years. Metabolism by aerobic bacteria has been well characterized for the BTX and in fact the organisms and the enzymes involved have additional value because of their ability to cometabolize the chloroalkanes and alkenes (see above). Because o f the lack of pure cultures for study, metabolism by anaerobic organisms is much less well understood. This may be remedied by three different reports of pure cultures which metabolize toluene [5",37",38"]. The organism GS-15 catalyzes a conceptually novel set of reactions, namely, the oxidation of toluene coupled to the reduction of Fe3+ [5"']. With radiolabeled toluene and stoichiometric calculations, it was shown that toluene mineralization is dependent on the reduction o f Fe3 + to Fe 2+. As GS-15 was isolated originally for its ability to reduce iron while using acetate, the ability to use toluene without oxygen may not be unique. In fact, a variety of environmental sources were found to have denitrifying toluene utilizers [39]. A second organism, tentatively identified as a Pseudomonas sp., utilized toluene under denitrifying conditions. With use of radio- labeled toluene, it was shown that more than half the radiolabeled ring carbons were recovered as CO 2 [37"]. The organism was isolated from a laboratory soil column which was enriched for alkytbenzene degradation under denitrifying conditions. The third isolated organism, referred to as strain T1, is not a pseudomonad and, under denitrifying conditions, stoichiometricalty converts toluene to cell mass and COg [38"]. All three independently isolated toluene-utilizers also metabolized other substituted aromatic compounds, including pcresol, />hydroxybenzaldehyde, phydroxybenzoate and benzoate. Although growth on these compounds may be consistent with proposed pathways in which pcresol or benzoate are intermediates [5"'], the evidence is insufficient to prove their existence.
orescens isolated from a contaminated site [42"]. The site of insertion was such that the resulting plasmid conferred the ability to produce luciferase-mediated light production in the organism on exposure to naphthalene, or to salicylate, a regulatory inducer metabolite. In strains harboring the plasmid, induction of bioluminescence coincided with naphthalene degradation, and light emission reflected naphthalene exposure [42",43]. This technology was demonstrated in a laboratory experiment using contaminated soil into which the strain Carrying the plasmid was mixed. Bioluminescence was measured within several hours of exposure, whereas none could be detected in uncontaminated soils. Certainly, naphthalene also can be measured directly by chemically analyzing a soil sample. Thus, the benefits of this bioluminescence approach have yet to be proven. It can be argued, however, that reporter strains allow the detection of bioavailable naphthalene, which direct chemical analysis does not. The authors suggested that potential application of bioluminescent reporter plasmicls could involve sensor technology using immobilized cells and fiber optics.
Conclusion Progress this past year in extending the concepts of bitdegradation has been significant. Findings include the following: a number o f different oxygenases also mediate dechlorination of halogenated aliphatic compounds; reductive dechlorination serves as an energy-generating mechanism; and aldylbenzene is completely degraded by newly isolated strains of denitrifying bacteria. Anaerobic environments also provide a wide variety of electron acceptors including sulfate and Fe3+, two newly identitied electron acceptors coupled to the biodegradation of toxic chemicals. This cumulative information is providing a broad base of knowledge for the future development of biological treatment technologies.
Polycyclic aromatic hydrocarbons Biodegradation of lower molecular weight polyc3,clic aromatic hydrocarbons by aerobic bacteria and fungi has been well documented [40]. Compounds with three or more fused rings, however, tend to be more persistent. This is partly because of their very low solubility which limits their availability to the microorganisms. However, a strain of Pseudomonas paucimobilis isolated from a creosote-contaminated soil utilized fluoranthene as a sole source o f carbon and energy. Growth was also supported by naphthalene, 2,3-dimethylnaphthalene, phenanthrene, anthracene and benzo[b]fluorene [3"]. Independently, a P. paucimobilis, P. vesicularis and Alcaligenes denitrificans were isolated on phenanthrene, tluorene and fluoranthene, respectively, with evidence supporting the com. plete degradation of these compounds [41"]. The expression of genes has been measured using a variety of gene fusions and biochemical assays for the gene products. Using bioluminescence as the genetic marker, the htxgenes have been inserted into a naphthalene catabolic plasmid (pKA1) from a Pseudomonasflu-
Acknowledgements We ~ish to acknowledge the support, in part, by the US Emironmental Protection Agency grants CR-814611, R-816483, and by the US National Institute of Emironmental Health Sciences grant ES0 4895. We thank Shay Cravd'ord for her assistance in typing.
References and recommended reading Papers of special interest, published within the annual period of re-dew, have been highlighted as:
• •,
of interest of outstanding interest ABRIL M-A, MICHAN C, TD.IMLS KN, I{A.MOSJL: Regulator and Enzyme Specificities of the TOL Plasmid-Encoded Upper Pathway for Degradation of Aromatic ttydrocarbons and Expansion of the Substrate Range of the Pathway. J Bacte riol 1989, 171:6782-6790.
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Environmental blotechnology 2.
HAUGLANDRA, SCH~ESL~I DJ, LYOUNS RP IiI, SFERRA PR, CHAKRABAmYAM: Degradation of the Chlorinated Phenoxyacetate Herbicides 2,4-Dichlorophenoxy-acetie Acid and 2,4,5-Trichlorophenoxyacetic Acid by Pure and Mixed Bacterial Cultures. Zppl Environ Microbiol 1990, 56:1357-1362.
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MUELLERJG, CHAPMAN PJ, BLA'rI'~t~NN BO, PRrrCHARD PIt: Isolation and Characterization of a Huoranthene-UtiUzing Strain of Pseudomonas paucimobilis~ Appl Environ Micrc~ biol 1990, 56:1079-1086. By using a non-ionic detergent which increased substrate availability, this strain of P. paucimobilis xx~s isolated on fluoranthese as the sole carbon source. It also degraded several other pol)~dic aromatic hydrocarbons. 4.
HICKEYWJ, FOCHT DD: Degradation of Mono-, Di-, and Trihalogenated Benzoic Acids by Pseudomonas aeruginosa JB2. Appl Environ MicrobioL 1990, 56:3842-3852.
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I.O'd.EYDR, LO.','EGRANDJ: Anaerobic Oxidation of Toluene, Phenol and p-Cresol by the Dissimilatory Iron-Reducing Organism, GS-15. Appl Environ Microbiol 1990, 56:1858-1864. GSd5 mediated the degradation of toluene, coupling it to Fe3+ reduction to Fez+. This extends the range of em~ronmental electron acceptors for anaerobic degradation of aromatic compounds to mineral cations. 6. •
TSIEN HC, BRUSSEAUGA, tlANSONRS, WACKETrLP: Biodegradation of Trichloroethylene by Methylosinus trichosporium OB3b. Appl Environ Microbiol 1989, 55:3155-3161. The methanotroph, 21/. trictxx~)orium has a sMMO that is capable of oxidizing TCE and other chlorinated one and t~x3-carbon compounds.
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OLDENHUISR, VL\X RLJM, JANSSEN DB, WITHOLT B: Degradation of Chlorinated Aliphatic Hyrdroearbons by Meth)~ losinus trichosporium OB3b Expressing Soluble Methane Monooxygenase. Appl Environ Microbiol 1989, 55:2819-2826. 21I. t r i ~ r i u m also degrades TCE with a sMMO under co-metabolic conditions.
14.
NELSONMJK, K~SELLAJV, MO.'ffOYA,T: In Situ Biodegradation of TCE Contaminated Groundwater. Environ Prog 1990, 9:191-196.
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ZYtSTRAGJ, WACKETTP, GIBSONDT: Trichloroethylene Degradation by Escherichia coil Containing the Cloned Pseudotnonas p u t i d a F1 Toluene Diox}genas¢ Genes. Appl Environ Microbiol 1989, 55:3162-3166. The toluene diox3~genasefrom the pseudomonad is the enzyme specifically responsible for TCE degradation. This was confirmed by expression of the cloned genes for this enzTme in K colt 16. .
WACKETrLP, BRUSSEAUGA, HOUSEItOLDERSR, I~N'SON RS: Survey of Microbial Oxygenases: Trichloroethylene Degradation by Propane-Oxidizing Bacteria- Appl Environ Microbiol 1989, 55:2960-2964. From a range of species ~ahich express oxTgenases, ~'e propanedegrading MycobacteHum strains could degrade TCE. 17.
HXa~R AR, Ktxt YK: Trichloroethylene Degradation by Two Independent Aromatic-Degrading Pathways in Alcalb genes eutrophus JMPI34. Appl Environ Microbiol 1990, 56:1179--1181.
18.
CASTROCE, BEISER NO: Biodehalogenation: Oxidative and Reductive Metabolism of l,l,2-Trichloroethane by Pseu. domonas p u t i d a m Biogeneration of Vinyl Chloride. Ent~ iron Toxicol Chem 1990, 9:707-714.
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EGU C, STRO3~YERS, COOKAM, LEISINGERT: Transformation of Tetra-and Trichloromethane to CO 2 by Anaerobic Bacteria is a Non-Enz)anic Process. FE~IS Microbiol Lett 1990, 68:207-212. Autoclaved cells of two strict anaerobes catabxed the dechlorination of CCI4 and chloroform to CO2. Non-enz)vnatic processes, therefore, are thought to be im-olved in dehalogenation reactions. 20.
MIKESELLMD, BOYD S& Dechlorination of Chloroform by Metbanosarcina Strains.Appl Environ Microbio11990, 56:1198 -1201.
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VANNELLI T, LOG&'qM, ARCIERODM, HOOPERAB: Degradation of Halogenated Aliphatic Compounds by the Ammonia-Oxidizing Bacterium Nitrosomonas europaea. Appl Environ Microbiol 1990, 56:1169-1171. Metabolism of chlorinated aliphatic compounds is catabxed by the AMO present in the nitrif)ing bacterium, N. europae,z
KRONEUE, LAUFERK, THAUER RK, HOGENKAMPHPC: Coenz)ane I:430 as a Possible Catal)~t for the Reductive Dehalogenation of Chlorinated C1 Hydrocarbons in Methanogenic Bacteria. Biochemistry 1989, 28:10061-10065. Coenz3rne F430 in either live or heat-treated Metbanosarcina barkeri catab'zed reductive dechlorination of CCI4.
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22.
CRIDDLECS, DEWrrr JT, GRBIC-G.~d.lCD, McCAR'rgPIz Transformation of Carbon Tetrachloride by Pseudomonas sp Strain KC Under Denitrifieation Conditions. Appl Environ Micrc~ biol 1990, 56:3240-3246.
23.
BAGLEYDM, GOSSETrJM: Tetrachloroethene Transformation to Trichlorocthene and Cis-l,2-Dichloroethene by SulfateReducing Enrichment Cultures. Appl Environ Microbio11990, 56:2511-2516.
RA.~HEME, HY~t~.'q MR, Am' D: Biodegradation of Halogenated tlydrocarbon Fumigants by Nitrify-ing Bacteria. Appl Environ Microbiol 1990, 56:2568-2571. Three genera of nitrif)~ng bacteria can mediate the dehalogenation of halogenated hydrocarbons, at different degrees. RASCHEME, HICKS R, HYMAN IZM Am' DJ: Oxidation of Monohatogenated Ethanes and n-Chlorinated Alkanes by Whole Cells of Nitrosomonas europaea. J Bacteriol 1990, 172:5368-5373. The rate of oxidation of halogenated alkanes by N. europaea is shov~aa to increase ~ t h decreasing size of the halogen substituent and ~ t h decreasing length of the alkane.
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11.
NELSONMJK, MONTGOMERYSO, M.MtAFFEYWR, PRrrCtIARD PH: Biodegradation of Trichlorocthylene and Involvement of an Aromatic Biodegradation Pathway; Appl Environ Microbiol 1987, 53:949-954.
12.
FOLSOM BR, CHAPMAN PJ, PRITCIIARD Ptt: Phenol and Trich/oroethylene Degradation by- Pseudomonas cepacta G4: Kinetics and Interactions Between Substrates. Appl Environ Microbiol 1990, 56:1279-1285.
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SHIELDSMS, MONTGOMERY SO, CHAPMAN PJ, CUSKEY SM, PRITCH.~d~DPtt: Novel Pathway of Toluene Catabolism in the Trichloroethylene-Degrading Bacterium G4. Appl Environ Microbiol 1989, 55:1624-1629. A study on the TCE-degrading bacterium, G4, reveals that two sequential monooxTgenation steps rather than one diox)genation step, )Seld a meth)tcatechol from toluene.
24. .
KRONEUE, TIIAUERRK, HOGEr,q~MP HPC: Reductive Dehalogenation of Chlorinated Cl-tlydrocarbons Mediated by Corrinoids. Bioc~mistry 1989, 28:4908-4914. Different corrinoids in vitro catalyze reductive dechlorination. Their high concentration in many species of strict anaerobes supports the h)l~othesis that reductix~edechlorination is catabxed by the corrinoids present in these organisms. 25.
KIECKAGM, GO,~StORSJ: Reductive Dechlorination of Chlorinated Methanes and Ethanes by Reduced Iron (II) Porphyrins. CIJemosphere 1984, 13:391-402.
26.
BEDARDDI, ttABERLMI- Influence of Chlorine Substitution Pattern on the Degradation of Polychlorinated Biphenyis by Eight Bacterial Strains. AIicrobiol Ecol 1990, 20:87-102.
27. ,•
QUENSENFJ IIl, BOYD SA, TIEDJEJM: Dechlorination of Four Commercial Polychlorinated Biphenyl Mixtures (Arochlors) by Anaerobic Microorganisms from Sediments. Appl Emqron Microbiol 1990, 56:2360-2369. Sediment anaerobes catabxed the reductive dechlorination of commercial PCB mixtures. The meta- and para-chlorines were removed pref-
Biodegradation of toxic and environmental pollutants Young and H~ggblom 435 erentially and the rate and extent of deddorination decreased as the degree of chlorination increased.
The aromatic hydrocarbon, toluene, is completely mineralized by this denitrifying organism in the absence of molecular oxygen.
NIES L, VOGELTM: Effects of Organic Substrates on Dechlorination of Aroclor 1242 in Anaerobic Sediments. Appl Environ Microbiol 1990, 56:2612-2617. Addition of organic substrates to Arochlor 1242 stimulated PCB dechlotination.
EVANSPJ, MANGDT, KIM KS, YOUNG LY: Anaerobic Degradation of Toluene by a Denitrifying Bacterium. Appl Environ Microbiol 1991, 57:1139-1145. This pure culture of an anaerobic denitrifying bacterium grew on 3 mM toluene. The oxidation of toluene to cell mass and CO2 vas stoichiometrically coupled to denitrification.
29.
39.
28. •
tIAGGBLOMH: Mechanisms of Bacterial Degradation and Transformation of Chotrinated Monoaromatic Compounds. J Basic Microbiol 1990, 30:115-141.
FE'IZNERS, MOLLERR, LINGENSF: Degradation of 2-Chlorobenzoate by Pseudomonas cepacia 2CBS. Biol Cbem Hopt~ Seyler 1989, 370:1173--1182. A diox3genase enzyme catalyzed the conversion of 2-CB to catechol. Thus, a dechlorination appears to takes place during the initial dioxygenation step. 30.
•
31.
Kt.rtgqEP, TOWNSEND GT, SUFLITAJM: Effect of Sulfate and Organic Carbon Supplements On Reductive Dehalogenation of Chloroanilines in Anaerobic Aquifer Slurries. Appl Environ Microbiol 1990, 56:2630-2637.
32.
HAGGBLOMM, YOUNG LY: Chlorophenol Degradation Coupled to Sulfate Reduction. Appl Environ Microbiol 1990, 56:3255-3260.
33.
KAMALV, WYNDHAMCR: Anaerobic Phototrophic Metabolism of 3-Chlorobenzoate by Rhodopseudomonas palustris WS17. Appl Environ Microbiol 1990, 56:3871-3873.
34. o,
DOH"~GJ: Reductive Dechlorination of 3-Chlorobenzoate is Coupled to ATP Production and Growth in an Anaerobic Bacterium, Strain DCB-I. Arch Microbiol 1990, 153:264-266. A study of the anaerobic dechlorinating bacterium strain DCB1. Cell yields and ATP levels both increased as a function of the 3-CB concentration added to the ceil extract. As this strain can only dechlorinate t h e ring, it lends credence to the h)pothesis that reductive dechlorination can ~Seld biologically useful energy. 35.
MoL-'qWW, TtEDJE J: Strain DCB-1 Conserves Energy for Growth from Reductive Dechlorination Coupled to Formate Oxidation. Arch Microbiol 1990, 153:267-271.
36. **
DEWEERDKA, SLrFI.rrAJM: Anaerobic AaD'I Reductive Dehalogenation of Halobenzoates by Cell Extracts of Desulfomontle ttedjet. Appl Era,iron Microbio11990, 56:.29")9-3005. Cell extracts were obtained from the strict anaerobe, strain DCB1, and it v~ts confirmed that dechlorination is an enzymaticaUycatabxed process. 37. •
DOLF~GJ, ZEYERJ, EICHER-BINDERP, SCHWARZENBACHRP: Isolation and Characterization of a Bacterium that Mineralizes Toluene in the Absence of Molecular Oxygen. Arch Micr~ biol 1990, 154:336--341.
38. •
EVANSPJ, MANG DT, YOUNG LY: Degradation of Toluene and m-Xylene and Transformation of o-Xylene by Denitrifying Enrichment Cultures. Appl Environ Microbiol 1991, 57:450--454. 40. CERN'IGLIACE: Microbiol Transportation of Aromatic llydrocarbons. In Petroleum Microbiology edited by Arias RM [book]. New York: Macmillan Publishing Company 1984, 3:99-128. 41. WEtSSEhTELSWD, BEYERM, KLEL'4J: Degradation of Phenan• threne, Huorene and Fluoranthene by Pure Bacterial Cultures. Appl Microbiol Biotedmol 1990, 32:479-484. Several pure cultures were isolated v~dch grew on and completely metabolized the pob-c3"clicaromatic hydrocarbons, phenanthrene, fluorene and fluoranthene. 42. KLXGJMtt, DIGRAZlAPM, APPLEGATEB, BURLAGER, SANSEVERINO • J, DUt,q3AR P, IAJ~MER F, SAYLERGS: Rapid Sensitive Bioluminescent Reporter Technology for Naphthalene Exposure and Biodegradation. Science 1990, 249:778--781. A bioluminescent reporter pLasmid for napththaIene degradation v-as developed by insertion of the lux genes into a naphthalene-catabolic plasmid. Light is emitted when naphthalene is degraded by the organism harboring the plasmid. 43. BURLAGERS, SAYLERGS, LARLXIERF: Monitoring of Naphthalene Catabolism by Bioluminescence with Nah-Lux Transcriptional Fusions. J Bacteriol 1990, 172:4749--4757.
Annotated patents • •,
of interest of outstanding interest
P1. BED.M~.DDL, BREr,.'NANMJ JR: Methods for Biodegradation of • PCBs. US Patent 4876201. 24/10/89. This patent describes a method for aerobic degradation of PCBs using a pure culture of an Alcaligenes eutropbusstr'An.
LYYoung, Department of Microbiology and Institute of Em'iromnental Medicine, NYU Medical Center, 550 First Avenue, New York, New York 10016, USA. MM tt~iggblom, Department of Microbiology, NYU Medical Center, 550 First Avenue, New York, New York 10016, USA.
Introduction The bioremediation used as a part o f the clean-up effort after the EXXON VALDEZaccident in March 1989 has brought the field of biodegradation to the attention of both regulatory and scientific communities, as well as to the general public. These efforts have contributed towards the acceptance of biological means of contaminant destruction as a valid alternative to physical and chemical technologies. Use of microbes in biogradation is as old as composting and has been long accepted as a well established technology, for example in municipal waste treatment. Recent attention, however, has been directed at treating toxic environmental pollutants. The potential for this 'biological technology' is heightened by documentation of the diversity of organisms capable of metabolizing an increasingly wide range of toxic organic compounds. Yet, it should be realized that the natural environment in which it is to be applied has complexities which cannot be readily governed nor quickly understood. Biodegradation is an extremely broad field. Some areas have only recently developed, whereas others have been under investigation for many years. As a consequence, breakthroughs can be at the level of a new paradigm, such as mineral cations serving as electron acceptors for toluene metabolism, as well as at the molecular level, such as a newly constructed metabolic pathway for the breakdown of chlorinated aromatics. By definition, the newer paradigms are less well understood and in some cases may lack appropriate pure cultures with which to work. Rather than detract from the accomplishments, this indicates the richness of possibilities regarding organisms to isolate and new pathways and mechanisms to study. Aerobic metabolism studies continue to involve the skillful construction of new pathways for degradation of chlorinated aromatic compounds [1,2] both in vivo and in vitro. While the construction of new pathways is limited
to the use of genetic material from isolated strains, a vast reservoir of genetic material available in nature has yet to be fully exploited. Thus, new strains are sought after continually and classic techniques for selection on carbon sources have yielded strains with new biodegradative capabilities [3°,4]. Anaerobic conditions, or those in which alternatives to oxygen serve as the electron acceptor, are receiving much attention. With anaerobiosis, there are many conditions under which the selection can take place, effectively increasing the genera of organisms sought after. For the oxidation of toxic chemicals, important environmental electron acceptors such as nitrate, sulfate and carbonate are nosy augmented by the demonstration that reduced iron and manganese also serve as electron acceptors [5"o]. In addition, highly chlorinated molecules such as polychlorinated biphenyls have been proposed to serve as anaerobic electron acceptors while undergoing reductive dechlorination.
Chlorinated aliphatic compounds The versatility of aerobes with noteworthy metabolic properties has been unexpectedly extended by the recent discovery that they are also capable of mediating the cometabolism (or as more recently termed, gramitous metabolism) of chlorinated aliphatic compounds. The widely varying groups of organisms with such capabilities include methanotrophs and nitrifying bacteria. Common to all of these organisms is the induction of mono- or dioxygenases not by the chlorinated aliphatic, but by a different compound altogether. This information is summarized in Table 1. The soluble methane monooxygenase (sMMO) from Metlyylosintts tricioosporittm OB3b appears to have a very
Abbreviations AMO~ammonia monooxygenase;BTX--benzene/toluene/xylenes; CB-~:hlorobenzoate; PCB--polychlorinated biphenyl; PCE~perchloroethylene; sMMO~soluble methane monooxygenase,TCE----trichloroethylene.
(~) Current Biology Ltd ISSN 0958-1669
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Environmental biotechnology Table 1. Oxygenases involved in the degradation of chlorinated aliphatic compounds.
Organisms
Inducer (enzyme)
Substrates
References
Methylosinus trichosporium OB3b
Methane (soluble methane monooxygenase)
Trichloroethylene, monochloroethylene, 1,1-, and cisand trans-l,2-dichloroethylene, dichloromethane, trichloromethane, 1,1- and 1,2-dichloroethane, 1,2-dichloropropane
[6°,7"]
Nitrosomonas europaea
Ammonia (ammonia monooxygenase)
Dichloromethane, trichloromethane, 1,1,2and 1,1,1-trichloroethane, monochloroethylene, dichloroethylenes, trichtoroethylene, 1,2,3-trichloropropane, monohalogenated ethanes
[8%9°1
Nitrosolobus multiformis
Ammonia (ammonia monooxygenase)
1,2-Dichloropropane, 1,2-dibromo-3-chloropropane
[10.1
Pseudomonas cepacia CA
Toluene, phenol (monooxygenase)
Tdchloroethylene
[11,12]
Trichloroethylene
[15"]
Monochloroethylene 1,1-dichloroethylene,cisand trans-l,2-dichloroethylene, trichloroethylene
[16.]
Pseudomonas putida FI [ Escherichia coli Mycobacterium spp.
Alcaligenes eutrophus JMP134 Pseudomonas putida PpG-786
Toluene dioxygenase Propane (propane monooxygenase)
Phenol, 2,4-dichlorophenoxyacetic acid Trichloroethylene Camphor (cytochrome P450 cam)
1,1,2-Trichloroethane
[17] [18]
broad substrate specificity and attacks many but not all of the chlorinated one- and two-carbon compounds. In particular, degradation of trichloroethylene (TCE) is very" rapid [6",7"]. Although this organism also has a second membrane-bound methane monooxygenase, the activity of this enzyme is limited to a few compounds with little observed chloride release. Several lines of evidence support the proposal that sMMO is the enzyme responsible for degradation of chlorinated compounds. First, sMMO is expressed only under conditions of copper limitation, and TCE degradation (as well as that of other chlorinated aliphatics) only occurs in cells grown in the absence of copper [7"]. Second, with rabbit antibodies prepared against purified sMMO, Western blot analysis demonstrated that only cells possessing the sMMO protein were capable of oxidizing TCE [6"]. Although vinyl chloride and chloroform (trichloromethane) were also metabolized by this enzyme, carbon tetrachloride and perchloroethyiene (tetrachloroethene; PCE) were not.
lize most of the chlorinated I and 2 carbon aliphatic compounds. Interestingly, as with sMMO discussed above, vinyl chloride and chloroform could be metabolized but carbon tetrachloride and PCE could not. Results obtained with additional genera of nitrifiers indicated that the soil organisms, N. europaea and Nitrosolobus multiformis are active on all three halocarbon fumigants tested, whereas the marine organism, Nitrosococcus oceanus could metabolize only the methyl bromide and not the chlorinated or brominated propane.s [9"]. The involvement of ammonia monooxygenase (AMO) is suggested by the observations that acetylene and other inh/bitors of AMO also inhibit halocarbon metabolism. In addition, metabolism of the halocarbon reduces the rate of nitrite production from ammonia, implying competition for the active site of AMO [8"]. Using monohalogenated ethanes, it was noted, not unexpectedly, that dehalogenation is inversely proportional to the size of the halogen substituent and to the length of the alkane [10"].
In an extension of work published in 1989, Vannelli et a t [8.] demonstrated that the ammonia-oxidizing nitrifying bacterium, Nitrosomonas europaea, could metabo-
Much interest has focused on the metabolism of TCE mediated by several Pseudomonas species that require induction by toluene or phenol. This was first reported in
Biodegradation of toxic and environmental pollutants Young and Hfiggblom 1987 [11] using Pseudomonas cepacia G4 isolated from the environment. A series of papers on strain G4 have examined the kinetics of TCE-degradation by phenolinduced cells [12], concluding that the toluene degradation proceeds through a new mechanism invoMng two sequential monooxygenations yielding the methylcatechol [13o°], rather than a dioxygenase-mediated reaction. In one of the few reports in which a contaminated site was treated hi situ, G4 was injected into a TCE-contaminated aquifer along with oxygen and nutrients, and this resulted in a reduction of TCE concentrations from 3000ppb to 7 8 p p b in 20days [14]. The effectiveness of using G4 in this pilot study is not convincing, however, because three variables were added without any means of assessing the effect of each one separately; oxygen, nutrients (undefined except as organic and inorganic) and G4 all were added at the same time. As each of the first two variables could themselves stimulate TCE loss, the effectiveness of G4 unfortunately remains obscure.
treated Methanosarchla barkeH cells were able to catalyze the reductive dechlorination ofCC14 to CHCI3 [21o]. Thus, these transformations o f CCI4 may not be enzymatic processes. Reductive dehalogenation can be catalyzed by reduced iron porphyrins, corrinoids and coenzyme F430, all o f which are cellular cofactors that are independently active in vitro and considered to be heat-stable [21o,24°,25]. High levels of these cofactors are found in the strict anaerobes which have the ability to dehalogenate CCI4. Although attributing dehalogenation to the bacterial ceils is not incorrect as they produce the necessary cofactors, the process may not be dependent on living cells (i.e. not enzyme-dependent). Experimental resuits, therefore, should be interpreted with caution, and additional controls should be used to account for the contribution of killed cells.
That the metabolism of TCE is mediated specifically by toluene dioxygenase in Pseudomonasptttida F1, and by no other enzymes of the pathway, was confirmed by cloning the four genes necessary for the dioxygenase into Eschericbia coli [15"]. TCE metabolism was mediated by the recombinant E. coli and not by the control strain which possessed the vector but not the genes. In a survey of widely differing strains that express a variety of monoand dioxygenases, all ~'e Mycobacterium species which grow on propane as a sole source of carbon were found to mediate TCE metabolism [16.]. TCE activity was associated with propane monooxygenase as uninduced cells did not metabolize TCE. Although the dichloroethylene derivatives and vinyl chloride were metabolized, PCE was not. It was noted in this paper [16 °] that P. putida F1 did not attack vinyl chloride. Degradation of chlorinated aliphatic compounds is also mediated by oxgenases induced by other aromatic compounds such as phenol, 2,4dichlorophenoxyacetic acid and camphor [17,18].
Chlorinated aromatic compounds Polychlorinatedbiphenyls
Several examples of strict anaerobic bacteria that can mediate the dechlorination of carbon tetrachloride (CCI 4) and chloroform (trichloromethane, CHCI3) [19oo,20,21 °] suggest that this ability may be rather widely dispersed. CCI4 transformation to CO 2 was also reported with a denitrifying pseudomonad growing on acetate [22]. Sulfate-reducing enrichments grown on lactate exhibited PCE dechlorination to tfichloroethene and dichloroethene. Although the evidence suggests that methanogens were not active, the group of organisms responsible was not identified [23]. A cautioriary note regarding these anaerobic dehalogenations was struck by the observation that sterilized controis can also display dechlorinating activity. This was noted with autoclaved cells of Acetobacterium woodii and Methanobacterium thermoautotrophicum. In experiments using 14CC14, both species formed the same amount o f 14COa in autoclaved and in active cultures and the M. tbennoautotrophicum also formed 14CHCI3. The rates of this transformation in both whole cells and cell extracts were also the same for autoclaved and unautoclaved preparations [19"']. Furthermore, heat-
Recent work on polychlorinated biphenyls (PCBs) has been directed at the anaerobic or reductive dechlorination of these highly chlorinated compounds. This has drawn attention away from the still very interesting aerobic metabolism of this class o f molecules. Aerobic degradation of PCBs is initiated by dioxygenase attack, usually at carbons 2 and 3, but sometimes at positions 3 and 4. The dihydroxychlorobiphenyls undergo ring fission to yield chlorinated benzoic acids or acetophenones. Monochlorobiphenyls are the only PCBs shown to be used as growth substrates. In a study using eight PCBdegrading bacterial strains, considerable differences between the strains were noted, with respect to both the choice of ring attacked and the relative rates of primary degradation [26,P1*]. Six o f the strains apparently only degrade PCBs by the 2,3-dioxygenase pathway, whereas two strains have both 2,3- and 3,4-dioxygenase activity. All of the strains preferentially metabolize an unchlorinated ring. Under reductive dechlorinating conditions, the rate, extent and pattern of dechlorination of commercial PCB mixtures (Arochlor 1242, 1248, 1254 and 1260) by sediment anaerobes has received much attention. All four Arochlors were dechlorinated with loss of chlorines in the meta- and para- positions, resulting in accumulation of the ortho-chlorinated congeners [27°°]. The extent of dechlorination was 15-85 % of total chlorines in 16 weeks, and the rate was inversely related to the degree of chlorination. Addition of organic substrates such as methanol, glucose or acetone stimulated dechlorination, whereas little stimulation was observed in parallel sediment samples that received no organic amendments [28°]. Again, meta- and parachlorines were preferentially removed. Implicit in these studies is the concept that a sequential anaerobic-aerobic treatment could be developed for bioremediation of Contaminated sites. It should be noted, however, that publications on reductive dechlorination
431
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Environmentalbiotechnology to date describe laboratory studies in which high concentrations (300-700 ppm) of Arochlors were added for the purposes of the experiments. Two points should be taken into account. First, laboratory results suggest that reductive dechlorination of PCBs is less effective at concentrations below 200 ppm and that, in situ, the PCBs are designated as hazardous at much lower concentrations than those used in the laboratory studies. Second, the availability from soils or sediment of newly added PCBs will be different from those that have aged in their natural environment and may be tightly bound. Thus, the real effectiveness of biotreatment on in situ and aged PCBs remains to be seen.
Chlorophenols and chlorobenzoic acids The aerobic degradation of mono- and dichlorinated phenols and benzoates generally proceeds by dioxygenation to chlorocatechols, with dehalogenation occurring after ring fission [29]. Evidence obtained with a Pseudomonas cepacia 2CBS isolated from the environment indicates the existence of a different mechanism in which dechlorination of 2-chlorobenzoate (CB) takes place during the initial dioxygenation step yielding catechol [30°]. A partially purified enzyme which catalyzed.the reaction was obtained, and required molecular oxygen, NADH and Fe 2+ for activity. Stoichiometric amounts of chloride were released and experiments using 180 2 confirmed that this was a dioxygenase mechanism. Aerobic dechlorination of the aromatic ring was also observed with Pseudomonasaerug#msaJB2 isolated from soil [4]. Along with other mono-, di- and trichlorobenzoates, 2,3and 2,5-dichlorobenzoate can serve as growth substrates. During the metabolism of these last two compounds, 4-chlorocatechol was detected as a metabolite, supporting the assumption that an initial dechlorination had occurred. Most of the recent studies on the anaerobic degradat_ion o f chloroaromatic compounds have been carried out under methanogenic conditions which seem to readily generate dechlorinating activity. Although the oxidationreduction pote.ntial for sulfate-reducing conditions is similar to that for methanogenesis ( - 250-- - 3 5 0 mY), the presence or addition of sulfate has been shown to inhibit dechlorinating activity [31]. However, chlorophenol degradation coupled to sulfate reduction in enrichments was reported for all three monochlorophenol isomers [32]. Evidence that supports a role for sulfate reduction includes stoichiometric utilization of sulfate for chlorophenol oxidation to CO 2, inhibition of chlorophenol degradation in the presence of molybdate, a specific inhibitor of sulfate reduction, and formation of sulfide during chlorophenol degradation. Whether it is the sulfate-reducers utilizing the chlorophenols or whether it is another organism tighdy coupled to the sulfate-reducer in a consortium arrangement is not known at this time. Several independent isolates o f the photosynthetic bacterium, Rhodopsettdomonaspalustriswere able to utilize 3-CB for growth in the presence of light and in the absence of oxygen [33], as illustrated by the conversion
of radiolabeled CB to radiolabeled CO z. This extends the aromatic substrate range for this species, which already includes phenols and aromatic acids and aldehydes. Chlorinated aromatic compounds thus appear to be susceptible to biodegradation by a number of physiologically different species, and this can take place under a variety of anaerobic conditions.
Energetics of dechlorination Thermodynamic data indicate that the reductive dechlorination of chlorinated aromatic compounds is an exergonic reaction which may yield biologically useful energy. Support for this hypothesis comes from studies with the anaerobic dechlorinating bacterium, Desulfomonile tiedje strain DCB1 [34"', 35]. DCB1 can only reductively remove the chlorine from 3-CB. In the presence of formate and acetate as electron donors and 3-CB as the electron acceptor, the cell yield increased stoichiometrically with the amount of 3-CB added. No growth was obtained if benzoate was substituted for 3-CB, or in the absence of 3-CB. Increased ATP levels were also obtained in response to the addition of 3-CB. Because strain DCB1 is the only anaerobic dechlorinating strain in pure culture, it will be interesting to learn whether this energy-generating mechanism also holds for other organisms and for other chlorinated compounds.
Cell-free dechlorinating enzyme Strain DCB1 was also the source for cell extracts and enzyme studies, in which Deweerd and Suflita [36"] reported the first cell-free dehalogenation system in an obligate anaerobe. Dehalogenation activity is heat-labile, dependent on reduced methyl viologen, an electron carrier, and stimulated by formate, carbon monoxide and hydrogen. It is membrane-associated and inhibited by oxygen. Extracts from cells grown in the presence of 3-CB had 10-fold greater activity than those from cells grown in its absence, suggesting that it is an induced enzyme. The cell extract also dehalogenated 3-bromo- and 3-iodobenzoate, but not 3-fluorobenzoate. Although strain DCB1 is a sulfidogen and can grow via reduction of sulfur oxTailions, sulfite or thiosulfate but not sulfate inhibit dehalogenation. This may seem contradictory, but it is consistent with the authors' suggestion that the sulfur oxyanions may be competing with dehalogenation for reducing equivalents. It should be noted that reductive dehalogenation is not limited to anaerobes; it has also been reported in cell extracts o f aerobic bacteria where it is not inhibited by oxygen [29].
Hydrocarbons
AIkylbenzenes Benzene, toluene and xylenes (BTX) have been identiffed by the US Environmental Protection Agency as among the 17 chemicals posing the greatest threats to human health. Earlier this year, the largest industrial users
Biodegradation of toxic and environmental pollutants Young and Hfiggblom were asked to voluntarily reduce their release of these chemicals over the next 3 years. Metabolism by aerobic bacteria has been well characterized for the BTX and in fact the organisms and the enzymes involved have additional value because of their ability to cometabolize the chloroalkanes and alkenes (see above). Because o f the lack of pure cultures for study, metabolism by anaerobic organisms is much less well understood. This may be remedied by three different reports of pure cultures which metabolize toluene [5",37",38"]. The organism GS-15 catalyzes a conceptually novel set of reactions, namely, the oxidation of toluene coupled to the reduction of Fe3+ [5"']. With radiolabeled toluene and stoichiometric calculations, it was shown that toluene mineralization is dependent on the reduction o f Fe3 + to Fe 2+. As GS-15 was isolated originally for its ability to reduce iron while using acetate, the ability to use toluene without oxygen may not be unique. In fact, a variety of environmental sources were found to have denitrifying toluene utilizers [39]. A second organism, tentatively identified as a Pseudomonas sp., utilized toluene under denitrifying conditions. With use of radio- labeled toluene, it was shown that more than half the radiolabeled ring carbons were recovered as CO 2 [37"]. The organism was isolated from a laboratory soil column which was enriched for alkytbenzene degradation under denitrifying conditions. The third isolated organism, referred to as strain T1, is not a pseudomonad and, under denitrifying conditions, stoichiometricalty converts toluene to cell mass and COg [38"]. All three independently isolated toluene-utilizers also metabolized other substituted aromatic compounds, including pcresol, />hydroxybenzaldehyde, phydroxybenzoate and benzoate. Although growth on these compounds may be consistent with proposed pathways in which pcresol or benzoate are intermediates [5"'], the evidence is insufficient to prove their existence.
orescens isolated from a contaminated site [42"]. The site of insertion was such that the resulting plasmid conferred the ability to produce luciferase-mediated light production in the organism on exposure to naphthalene, or to salicylate, a regulatory inducer metabolite. In strains harboring the plasmid, induction of bioluminescence coincided with naphthalene degradation, and light emission reflected naphthalene exposure [42",43]. This technology was demonstrated in a laboratory experiment using contaminated soil into which the strain Carrying the plasmid was mixed. Bioluminescence was measured within several hours of exposure, whereas none could be detected in uncontaminated soils. Certainly, naphthalene also can be measured directly by chemically analyzing a soil sample. Thus, the benefits of this bioluminescence approach have yet to be proven. It can be argued, however, that reporter strains allow the detection of bioavailable naphthalene, which direct chemical analysis does not. The authors suggested that potential application of bioluminescent reporter plasmicls could involve sensor technology using immobilized cells and fiber optics.
Conclusion Progress this past year in extending the concepts of bitdegradation has been significant. Findings include the following: a number o f different oxygenases also mediate dechlorination of halogenated aliphatic compounds; reductive dechlorination serves as an energy-generating mechanism; and aldylbenzene is completely degraded by newly isolated strains of denitrifying bacteria. Anaerobic environments also provide a wide variety of electron acceptors including sulfate and Fe3+, two newly identitied electron acceptors coupled to the biodegradation of toxic chemicals. This cumulative information is providing a broad base of knowledge for the future development of biological treatment technologies.
Polycyclic aromatic hydrocarbons Biodegradation of lower molecular weight polyc3,clic aromatic hydrocarbons by aerobic bacteria and fungi has been well documented [40]. Compounds with three or more fused rings, however, tend to be more persistent. This is partly because of their very low solubility which limits their availability to the microorganisms. However, a strain of Pseudomonas paucimobilis isolated from a creosote-contaminated soil utilized fluoranthene as a sole source o f carbon and energy. Growth was also supported by naphthalene, 2,3-dimethylnaphthalene, phenanthrene, anthracene and benzo[b]fluorene [3"]. Independently, a P. paucimobilis, P. vesicularis and Alcaligenes denitrificans were isolated on phenanthrene, tluorene and fluoranthene, respectively, with evidence supporting the com. plete degradation of these compounds [41"]. The expression of genes has been measured using a variety of gene fusions and biochemical assays for the gene products. Using bioluminescence as the genetic marker, the htxgenes have been inserted into a naphthalene catabolic plasmid (pKA1) from a Pseudomonasflu-
Acknowledgements We ~ish to acknowledge the support, in part, by the US Emironmental Protection Agency grants CR-814611, R-816483, and by the US National Institute of Emironmental Health Sciences grant ES0 4895. We thank Shay Cravd'ord for her assistance in typing.
References and recommended reading Papers of special interest, published within the annual period of re-dew, have been highlighted as:
• •,
of interest of outstanding interest ABRIL M-A, MICHAN C, TD.IMLS KN, I{A.MOSJL: Regulator and Enzyme Specificities of the TOL Plasmid-Encoded Upper Pathway for Degradation of Aromatic ttydrocarbons and Expansion of the Substrate Range of the Pathway. J Bacte riol 1989, 171:6782-6790.
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434
Environmental blotechnology 2.
HAUGLANDRA, SCH~ESL~I DJ, LYOUNS RP IiI, SFERRA PR, CHAKRABAmYAM: Degradation of the Chlorinated Phenoxyacetate Herbicides 2,4-Dichlorophenoxy-acetie Acid and 2,4,5-Trichlorophenoxyacetic Acid by Pure and Mixed Bacterial Cultures. Zppl Environ Microbiol 1990, 56:1357-1362.
3. •
MUELLERJG, CHAPMAN PJ, BLA'rI'~t~NN BO, PRrrCHARD PIt: Isolation and Characterization of a Huoranthene-UtiUzing Strain of Pseudomonas paucimobilis~ Appl Environ Micrc~ biol 1990, 56:1079-1086. By using a non-ionic detergent which increased substrate availability, this strain of P. paucimobilis xx~s isolated on fluoranthese as the sole carbon source. It also degraded several other pol)~dic aromatic hydrocarbons. 4.
HICKEYWJ, FOCHT DD: Degradation of Mono-, Di-, and Trihalogenated Benzoic Acids by Pseudomonas aeruginosa JB2. Appl Environ MicrobioL 1990, 56:3842-3852.
5. ••
I.O'd.EYDR, LO.','EGRANDJ: Anaerobic Oxidation of Toluene, Phenol and p-Cresol by the Dissimilatory Iron-Reducing Organism, GS-15. Appl Environ Microbiol 1990, 56:1858-1864. GSd5 mediated the degradation of toluene, coupling it to Fe3+ reduction to Fez+. This extends the range of em~ronmental electron acceptors for anaerobic degradation of aromatic compounds to mineral cations. 6. •
TSIEN HC, BRUSSEAUGA, tlANSONRS, WACKETrLP: Biodegradation of Trichloroethylene by Methylosinus trichosporium OB3b. Appl Environ Microbiol 1989, 55:3155-3161. The methanotroph, 21/. trictxx~)orium has a sMMO that is capable of oxidizing TCE and other chlorinated one and t~x3-carbon compounds.
7. •
OLDENHUISR, VL\X RLJM, JANSSEN DB, WITHOLT B: Degradation of Chlorinated Aliphatic Hyrdroearbons by Meth)~ losinus trichosporium OB3b Expressing Soluble Methane Monooxygenase. Appl Environ Microbiol 1989, 55:2819-2826. 21I. t r i ~ r i u m also degrades TCE with a sMMO under co-metabolic conditions.
14.
NELSONMJK, K~SELLAJV, MO.'ffOYA,T: In Situ Biodegradation of TCE Contaminated Groundwater. Environ Prog 1990, 9:191-196.
15. •
ZYtSTRAGJ, WACKETTP, GIBSONDT: Trichloroethylene Degradation by Escherichia coil Containing the Cloned Pseudotnonas p u t i d a F1 Toluene Diox}genas¢ Genes. Appl Environ Microbiol 1989, 55:3162-3166. The toluene diox3~genasefrom the pseudomonad is the enzyme specifically responsible for TCE degradation. This was confirmed by expression of the cloned genes for this enzTme in K colt 16. .
WACKETrLP, BRUSSEAUGA, HOUSEItOLDERSR, I~N'SON RS: Survey of Microbial Oxygenases: Trichloroethylene Degradation by Propane-Oxidizing Bacteria- Appl Environ Microbiol 1989, 55:2960-2964. From a range of species ~ahich express oxTgenases, ~'e propanedegrading MycobacteHum strains could degrade TCE. 17.
HXa~R AR, Ktxt YK: Trichloroethylene Degradation by Two Independent Aromatic-Degrading Pathways in Alcalb genes eutrophus JMPI34. Appl Environ Microbiol 1990, 56:1179--1181.
18.
CASTROCE, BEISER NO: Biodehalogenation: Oxidative and Reductive Metabolism of l,l,2-Trichloroethane by Pseu. domonas p u t i d a m Biogeneration of Vinyl Chloride. Ent~ iron Toxicol Chem 1990, 9:707-714.
19. **
EGU C, STRO3~YERS, COOKAM, LEISINGERT: Transformation of Tetra-and Trichloromethane to CO 2 by Anaerobic Bacteria is a Non-Enz)anic Process. FE~IS Microbiol Lett 1990, 68:207-212. Autoclaved cells of two strict anaerobes catabxed the dechlorination of CCI4 and chloroform to CO2. Non-enz)vnatic processes, therefore, are thought to be im-olved in dehalogenation reactions. 20.
MIKESELLMD, BOYD S& Dechlorination of Chloroform by Metbanosarcina Strains.Appl Environ Microbio11990, 56:1198 -1201.
8. •
VANNELLI T, LOG&'qM, ARCIERODM, HOOPERAB: Degradation of Halogenated Aliphatic Compounds by the Ammonia-Oxidizing Bacterium Nitrosomonas europaea. Appl Environ Microbiol 1990, 56:1169-1171. Metabolism of chlorinated aliphatic compounds is catabxed by the AMO present in the nitrif)ing bacterium, N. europae,z
KRONEUE, LAUFERK, THAUER RK, HOGENKAMPHPC: Coenz)ane I:430 as a Possible Catal)~t for the Reductive Dehalogenation of Chlorinated C1 Hydrocarbons in Methanogenic Bacteria. Biochemistry 1989, 28:10061-10065. Coenz3rne F430 in either live or heat-treated Metbanosarcina barkeri catab'zed reductive dechlorination of CCI4.
9. .
22.
CRIDDLECS, DEWrrr JT, GRBIC-G.~d.lCD, McCAR'rgPIz Transformation of Carbon Tetrachloride by Pseudomonas sp Strain KC Under Denitrifieation Conditions. Appl Environ Micrc~ biol 1990, 56:3240-3246.
23.
BAGLEYDM, GOSSETrJM: Tetrachloroethene Transformation to Trichlorocthene and Cis-l,2-Dichloroethene by SulfateReducing Enrichment Cultures. Appl Environ Microbio11990, 56:2511-2516.
RA.~HEME, HY~t~.'q MR, Am' D: Biodegradation of Halogenated tlydrocarbon Fumigants by Nitrify-ing Bacteria. Appl Environ Microbiol 1990, 56:2568-2571. Three genera of nitrif)~ng bacteria can mediate the dehalogenation of halogenated hydrocarbons, at different degrees. RASCHEME, HICKS R, HYMAN IZM Am' DJ: Oxidation of Monohatogenated Ethanes and n-Chlorinated Alkanes by Whole Cells of Nitrosomonas europaea. J Bacteriol 1990, 172:5368-5373. The rate of oxidation of halogenated alkanes by N. europaea is shov~aa to increase ~ t h decreasing size of the halogen substituent and ~ t h decreasing length of the alkane.
21. •
10. •
11.
NELSONMJK, MONTGOMERYSO, M.MtAFFEYWR, PRrrCtIARD PH: Biodegradation of Trichlorocthylene and Involvement of an Aromatic Biodegradation Pathway; Appl Environ Microbiol 1987, 53:949-954.
12.
FOLSOM BR, CHAPMAN PJ, PRITCIIARD Ptt: Phenol and Trich/oroethylene Degradation by- Pseudomonas cepacta G4: Kinetics and Interactions Between Substrates. Appl Environ Microbiol 1990, 56:1279-1285.
13. •,
SHIELDSMS, MONTGOMERY SO, CHAPMAN PJ, CUSKEY SM, PRITCH.~d~DPtt: Novel Pathway of Toluene Catabolism in the Trichloroethylene-Degrading Bacterium G4. Appl Environ Microbiol 1989, 55:1624-1629. A study on the TCE-degrading bacterium, G4, reveals that two sequential monooxTgenation steps rather than one diox)genation step, )Seld a meth)tcatechol from toluene.
24. .
KRONEUE, TIIAUERRK, HOGEr,q~MP HPC: Reductive Dehalogenation of Chlorinated Cl-tlydrocarbons Mediated by Corrinoids. Bioc~mistry 1989, 28:4908-4914. Different corrinoids in vitro catalyze reductive dechlorination. Their high concentration in many species of strict anaerobes supports the h)l~othesis that reductix~edechlorination is catabxed by the corrinoids present in these organisms. 25.
KIECKAGM, GO,~StORSJ: Reductive Dechlorination of Chlorinated Methanes and Ethanes by Reduced Iron (II) Porphyrins. CIJemosphere 1984, 13:391-402.
26.
BEDARDDI, ttABERLMI- Influence of Chlorine Substitution Pattern on the Degradation of Polychlorinated Biphenyis by Eight Bacterial Strains. AIicrobiol Ecol 1990, 20:87-102.
27. ,•
QUENSENFJ IIl, BOYD SA, TIEDJEJM: Dechlorination of Four Commercial Polychlorinated Biphenyl Mixtures (Arochlors) by Anaerobic Microorganisms from Sediments. Appl Emqron Microbiol 1990, 56:2360-2369. Sediment anaerobes catabxed the reductive dechlorination of commercial PCB mixtures. The meta- and para-chlorines were removed pref-
Biodegradation of toxic and environmental pollutants Young and H~ggblom 435 erentially and the rate and extent of deddorination decreased as the degree of chlorination increased.
The aromatic hydrocarbon, toluene, is completely mineralized by this denitrifying organism in the absence of molecular oxygen.
NIES L, VOGELTM: Effects of Organic Substrates on Dechlorination of Aroclor 1242 in Anaerobic Sediments. Appl Environ Microbiol 1990, 56:2612-2617. Addition of organic substrates to Arochlor 1242 stimulated PCB dechlotination.
EVANSPJ, MANGDT, KIM KS, YOUNG LY: Anaerobic Degradation of Toluene by a Denitrifying Bacterium. Appl Environ Microbiol 1991, 57:1139-1145. This pure culture of an anaerobic denitrifying bacterium grew on 3 mM toluene. The oxidation of toluene to cell mass and CO2 vas stoichiometrically coupled to denitrification.
29.
39.
28. •
tIAGGBLOMH: Mechanisms of Bacterial Degradation and Transformation of Chotrinated Monoaromatic Compounds. J Basic Microbiol 1990, 30:115-141.
FE'IZNERS, MOLLERR, LINGENSF: Degradation of 2-Chlorobenzoate by Pseudomonas cepacia 2CBS. Biol Cbem Hopt~ Seyler 1989, 370:1173--1182. A diox3genase enzyme catalyzed the conversion of 2-CB to catechol. Thus, a dechlorination appears to takes place during the initial dioxygenation step. 30.
•
31.
Kt.rtgqEP, TOWNSEND GT, SUFLITAJM: Effect of Sulfate and Organic Carbon Supplements On Reductive Dehalogenation of Chloroanilines in Anaerobic Aquifer Slurries. Appl Environ Microbiol 1990, 56:2630-2637.
32.
HAGGBLOMM, YOUNG LY: Chlorophenol Degradation Coupled to Sulfate Reduction. Appl Environ Microbiol 1990, 56:3255-3260.
33.
KAMALV, WYNDHAMCR: Anaerobic Phototrophic Metabolism of 3-Chlorobenzoate by Rhodopseudomonas palustris WS17. Appl Environ Microbiol 1990, 56:3871-3873.
34. o,
DOH"~GJ: Reductive Dechlorination of 3-Chlorobenzoate is Coupled to ATP Production and Growth in an Anaerobic Bacterium, Strain DCB-I. Arch Microbiol 1990, 153:264-266. A study of the anaerobic dechlorinating bacterium strain DCB1. Cell yields and ATP levels both increased as a function of the 3-CB concentration added to the ceil extract. As this strain can only dechlorinate t h e ring, it lends credence to the h)pothesis that reductive dechlorination can ~Seld biologically useful energy. 35.
MoL-'qWW, TtEDJE J: Strain DCB-1 Conserves Energy for Growth from Reductive Dechlorination Coupled to Formate Oxidation. Arch Microbiol 1990, 153:267-271.
36. **
DEWEERDKA, SLrFI.rrAJM: Anaerobic AaD'I Reductive Dehalogenation of Halobenzoates by Cell Extracts of Desulfomontle ttedjet. Appl Era,iron Microbio11990, 56:.29")9-3005. Cell extracts were obtained from the strict anaerobe, strain DCB1, and it v~ts confirmed that dechlorination is an enzymaticaUycatabxed process. 37. •
DOLF~GJ, ZEYERJ, EICHER-BINDERP, SCHWARZENBACHRP: Isolation and Characterization of a Bacterium that Mineralizes Toluene in the Absence of Molecular Oxygen. Arch Micr~ biol 1990, 154:336--341.
38. •
EVANSPJ, MANG DT, YOUNG LY: Degradation of Toluene and m-Xylene and Transformation of o-Xylene by Denitrifying Enrichment Cultures. Appl Environ Microbiol 1991, 57:450--454. 40. CERN'IGLIACE: Microbiol Transportation of Aromatic llydrocarbons. In Petroleum Microbiology edited by Arias RM [book]. New York: Macmillan Publishing Company 1984, 3:99-128. 41. WEtSSEhTELSWD, BEYERM, KLEL'4J: Degradation of Phenan• threne, Huorene and Fluoranthene by Pure Bacterial Cultures. Appl Microbiol Biotedmol 1990, 32:479-484. Several pure cultures were isolated v~dch grew on and completely metabolized the pob-c3"clicaromatic hydrocarbons, phenanthrene, fluorene and fluoranthene. 42. KLXGJMtt, DIGRAZlAPM, APPLEGATEB, BURLAGER, SANSEVERINO • J, DUt,q3AR P, IAJ~MER F, SAYLERGS: Rapid Sensitive Bioluminescent Reporter Technology for Naphthalene Exposure and Biodegradation. Science 1990, 249:778--781. A bioluminescent reporter pLasmid for napththaIene degradation v-as developed by insertion of the lux genes into a naphthalene-catabolic plasmid. Light is emitted when naphthalene is degraded by the organism harboring the plasmid. 43. BURLAGERS, SAYLERGS, LARLXIERF: Monitoring of Naphthalene Catabolism by Bioluminescence with Nah-Lux Transcriptional Fusions. J Bacteriol 1990, 172:4749--4757.
Annotated patents • •,
of interest of outstanding interest
P1. BED.M~.DDL, BREr,.'NANMJ JR: Methods for Biodegradation of • PCBs. US Patent 4876201. 24/10/89. This patent describes a method for aerobic degradation of PCBs using a pure culture of an Alcaligenes eutropbusstr'An.
LYYoung, Department of Microbiology and Institute of Em'iromnental Medicine, NYU Medical Center, 550 First Avenue, New York, New York 10016, USA. MM tt~iggblom, Department of Microbiology, NYU Medical Center, 550 First Avenue, New York, New York 10016, USA.