Chlorinated hvdrocarbons oxidation in the biosphere Here is a survey of metabolic pathways. prohlents, and limitations involved, and a suggestion that genetic engineering might help to expand microbial capabilities to perform this function
John M. Wood
Gray Fre.rhwatrr Biological Institurr Unicwrsify oJ Minnesota Nararre. Minn. 55392
Modern tcchniqua for the synthai of organic compounds add approximately 200 OOO new chemicals each year to the millions already used by advanced industrial nations ( I ). Microorganisms. which took billions of ycars to evolve their metabolic diversity.arcassud tohave theability to degrade thcce synthctic compounds. many of which b a r no rcscmbl:ince to t h w natural products used ;is nutrients in the microbial world. Sincc m a t of the synthetic chemic;ilr. uscd on a large scale. have been added to the biosphere only in the last 30 years. it scems reasonable to ask the question, “Ilow long will i t take for micro. organisms to acquin the ability to degrade all the new synthetic chemical\ introduced into the environment by modern technology:’” It should be recognized that there arc economic and technical facton that conflict with applying the basic principla of biodepdability in the daign of new chemic;ils for agricultural usc. I:orenample. farmers know that persistent poiicida are much more effective than their biodegradable substituta. Obviously. the longer an insecticide rem:iins in the soil. the more i n w t s it kills: this is why DDT is soefktive. Indeed. economic considerations usually dictate the selection of the more pcrsistcnt chcmicds over safer biodegradable counterparts. I t can be argued that with the in-
creasing world population. there is a grater need for insecticides tocontrol inscct-borne diseases. and for the provision of agricultural chemicals to protect the world food supply. However. this increased use or chemical technolony on a alobal scale incrcascs the risk 6 t h e public hcllth becauscof the toxicity. pcrsistencc. and bioconcentration of many substances currently in use. At this time. we n m l a more systematic approwh to understand t h w basic principles pcrtinent to the removal of synthetic chemicals in the biospherc. Although photochemical reactions play a rolc in the removal of certain compounds (2). biodegradation by microorganisms is of primary importancc. If ii synthetic chemical is bio. dcgradable in a rcasonablc time frame. then that compoundisunlikely t o p a threat to the public health (3. 4 ) . Nevertheless. while microorganisms w n adapt to remove man! toric substances. the great variety of synthetic chemicals used today may disrupt the balanceofthecarboncycle. In thefulure i t will k neccswry to develop microbial systems that a n change this trend. This m i e w will explore the limitations i m m on the biological oxidation of some synthetic compounds. as determined from r m n t studia with enzyma that catalyze theorid:itionof aromatic compounds ( 5 - 9 ) . It will furnish examples of how existing metabolic pathways can adapt to degrade. or partially degrade. a number of widely uscd synthetic chemicals. Special emphasis w i l l be given to the problems confronting microorganisms
lore. pedictlons IQ biodeg8dation cannot Qennally be based on concepts of chemical substituents or
.
chemical reaclivftv.
nlsdanDaaaloauvneltnlme
m t i 0 n - 0 1 a given synttmtic compovnd Wltl fO11Ow ms SBmO metebotic pathway 10 that already dixov-
wed for a shntually similirr natwal P-.
The rniuoblal eoO(0gy of the specitic ecosystem receiving the pollutant is m e uitlcal to its dege dation than are the degrsdative pathways determined wim either w eCUINS
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.i u m i Mmonia w o r n
because nay are likely to lose their p l a m M in complex envkonnmntal situations. soucs Pnwn W o . c k I 14- lbl
in the oxidative degradation of the most persistent chlorinated aromatic compounds such as PCBs. This wellknown problem will be treated from an evolutionary perspective. In addition, the potential application of genetic engineering to the selection of mutants for the purpose of degrading specific pollutants in industrial eftluents will be discussed. For instance, plasmid technology has the potential to produce “super-bugs,” which have better degradative capabilities than do natural populations of microorganisms. This technology involves transfers of genetic material from one organism to another, by isolating extra chromosomal material in the form of circular DNA or RNA. The use of mixed cultures of microorganisms to effect the total degradation of synthetic compounds is an old idea. More than 50 years ago, Gray and Thornton (10) recognized that mixed cultures of bacteria (now called microbial “consortia”) can contain organisms that partially degrade synthetic compounds, without deriving any nutritional benefits, to give partial metabolites that function as growth substrates for other organisms in the consortium. Also, in 1959, Leadbetter and Foster ( I / ) showed that methaneoxidizing bacteria (now called methylotrophs) are capable of the “cooxidation” of a wide variety of synthetic chemicals, all of which fail to function as growth substrates. In the early 1970s, this concept was resurrected and called “cometabolism” (12, 13).
Mixed-culture technology is now rapidly developing in the private sector, with several companies claiming successes in the improved treatment of both domestic and industrial wastes. They claim that some of their products are remarkably versatile, degrading everything from grease to dioxin her-
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Environ. Sci. Technal., Vol. 16. NO. 5. 1982
FIGURE 1
Nature’s bleach, “chloroperoxidase”: how it works”
I1
bicides. Even PCB-degraders are offered, but very little experimental data is given in support of such claims. To be sure, this technology has a good future, but it is in its infancy, with very little investment in both fundamental research and product quality control. Any qualified microbiologist will say that mixed cultures are difficult to standardize and preserve.
Biological oxidations The evolution of oxygen-based photosynthesis provided the necessary energy for the creation of the earth’s biomass. Photolysis of water by photosynthetic organisms introduced oxygen to the earth’s atmosphere, which in turn led to the evolution of a
great diversity of living creatures that require oxygen. These two critically important evolutionary events set the scene for biological oxidation, because organisms could now derive enormous amounts of energy from the oxidation of organic compounds to that most stable carbon compound, carbon dioxide. For this reason, systems have developed that make use of the direct addition of molecular oxygen to organic substrates; the addition of one atom of the oxygen molecule (hydroxylation); or the addition of one, two, or four electrons to molecular oxygen by electron transfer from a variety of organic compounds. These biological reactions of molecular oxygen are summarized in Table 1. Biological systems are required to “activate” molecular oxygen. Oxygen itself reacts very slowly with organic compounds, because oxygen exists in the triplet state with two unpaired electron spins. By contrast, carbon compounds generally exist in the singlet state; thus, concerted reactions between oxygen and carbon compounds are spin-forbidden (electrons are unable to pair up). Enzymatic “activation” of oxygen from the.triplet state to the singlet state is a crucial prerequisite for biological oxidation to occur. Reaction 3 in Table 1 is interesting in that it leads to the uroduction of the excellent oxidizing ‘agent hydrogen peroxide. Peroxidases use hydrogen
peroxide in a variety of reactions, but perhaps the most unusual one is that of biological halogenation by the enzymes chloroperoxidase and bromop&oxidase. . The reaction nathwav for the halogenation of oiganic Iubstrates by chloroperoxidase is presented in Figure I (17-21). In this reaction pathway, it is believed that halide ion reacts with highly reactive “Compound I” to give coordinated hypochlorite (OCI-). This reaction creates an electrophilic reagent that reacts rapidly with a wide variety of organic substrates. Halogenation is a common reaction in the marine environment, which yields a large number of chlorinated and brominated natural products (22, 23). By comparison, in the terrestrial environment, there are very few examples of biological halogenation (23, 24). In the sea, microorganisms have evolved the ability to degrade these halogenated compounds, sometimes in unusual ways. One example is the volatilization of bromoform in the biodegradation of bromoheptonones by the marine alga Bonnemaisonia hamifera (26). Also, recently, lzac and Sims ( 2 5 ) have isolated a great variety of halogenated aromatic compounds from the genus Laurencia. A survey of the recent literature will show that natural products containing up to three halogen substituents on the aromatic nucleus can be isolated. This number of three substituents might well indicate the limitations for the biodegradation of haloaromatic compounds by marine microorganisms. This indicates the need for a much greater research effort on the secondary metabolism of marine microorganisms, because some of these halophilic organisms are likely to be very effective at the removal of halogenated organic compounds from industrial wastes, an environment in which many terrestrial microorganisms have proved to be dismal failures.
vate oxygen as a prerequisite for oxygen-oxygen bond cleavage. But there are certain constraints for this reaction: The reaction must proceed at ambient temperatures. The activation energy for the reaction must be low, so that it can be supplied by oxygen binding to the enzyme active site. The enzyme-02 complex must be very reactive, and have the ability to form a singlet-bond; therefore, this enzyme-02 complex should be active as sinklet oxygen, the superoxide ion, or the peroxide ion. In biological systems, the peroxide ion pathway appears to be the route most often chosen (28, 29). There is overwhelming support for the intermediate formation of peroxide ions in both mixed function oxygenase and dioxygenase catalyzed reactions. For example, organic hydroperoxides, peracids, and even hydrogen peroxide itself will drive the cytochrome P450 (a heme-containing enzyme that can hydroxylate certain natural and synthetic organic chemicals) containing mixed function oxygenases (29, 30). Also, recent evidence suggests the formation of an organic peroxide as a
prerequisite for cleavage of the aromatic nucleus. The interaction of aromatic compounds with phenolic dioxygenases depends on the nature and the position of substituents in the benzene ring. The presence of substituents that withdraw electrons from the aromatic nucleus places the greatest constraint on the ability of the benzene ring to be cleaved by oxygen to give easily degraded aliphatic products. Dioxygenases Dioxygenases are responsible for the fixation of oxygen directly into organic compounds. These enzymes are commonly found in aerobic bacteria and fungi, and play an important role in the degradation of natural products such as lignins, alkaloids, flavanoids, xanthanes, and terpenes. In addition, dioxygenases, which function in the oxidation of thiols and in the hydroperoxidation of polyunsaturated fatty acids, have been isolated. Mechanistic studies on the dioxygenase that cleaves 3,4-dihydroxy benzoic acid (protocatechuic acid 3,4-oxygenase) provide insight into the constraints placed (on the fixation of molecular oxygen) by substituents on
FIGURE 2
Catalytic cycle for protocatechuate 3,hxygenase.s (proposed)
Biochemical constraints The biological oxidation of organic compounds requires the cleavage of oxygen (that is, the separation of the oxygen atoms). The enzymes that catalyze this reaction are in three major groups: oxidases (4e-), monooxygenases (2e-), and dioxygenases (Oe-). These enzymes contain a variety of cofactors such as hemes (camplexed Fe in hemoglobin-type molecules) a, b, c, and d; flavins; pterins; conoer: .. . manganese: and nonheme iron centers (27); The common feature of all of these active enzymes is their ability to actiEnviron. Scl. Technol.. VoI. 16. NO. 5. 1982
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the benzene ring (31-35). Although there is some discussion on the final cleavage pathway, the following mechanism has gained general acceptance, and is now offered in contemporary biochemistry textbooks (36) (Figure 2). Coordination of the parahydroxyl group of the substrate to the high-spin ferric active site stabilizes the ketoform of protocatechuic acid, and increases the electron density at carbon 4 of the aromatic ring. Electrophilic attack by oxygen produces an oxygen anion that couples with the ferric cation to give an organo-peroxy intermediate. If the benzene ring contains a number of strong electron with-
(which reflect the electronegativity of groups) for different substituents (31-33). This basic study of the mechanism of action of a dioxygenase provides a rationale for the resistance of multihalogenated aromatic compounds to biological oxidation. (There is no p i tion on the benzene ring where the electron density is sufficient to allow electrophilic attack by oxygen.) Therefore, dehalogenation reactions are important prerequisites for the ring-cleavage of multihalogenated aromatic compounds. However, the important question remains: “How many chlorine substituents can be tolerated by microorganisms on the aromatic ring?” One substituent ..-.-..._I should easily be handled through bio7 logical oxidation-but what about live TABLE 2 or six? Haloaromatlcs metabolized by terrestrial microorganisms Biological dehalogenation C-lWOUnh RelermS.. Although many naturally occurring halogenated organic compounds are 2.4.5-Trichlorophenoxy 51. 38. 39 synthesized in the marine environacetic acid ment, very few halogenated secondary (2.4S-T) metabolites are produced in the ter2.4Dichiorophenoxy 40.41 restrial system. This is probably the acetic acid reason why terrestrial microorganisms (2.4-0) have relatively poor rates for the 4Chlorc-2-meihyl42 transformation of xenobiotic halophenoxyacetic acid genated organic compounds. Table 2 4-Chlorophenoxy 47 provides a summary of those haloaroacetic acid matic compounds found to be metab 3.5-Dichlorobenzoic 43 or partially metabolized, by olized, acid terrestrial microorganisms. 3Chlwobenzoic acid 44,45 The majority of the metabolic 4-Chlwobenzoic acid 49 transformations for the haloaromatic 2-Fluorobenzoic acid 46, 47 compounds presented in Table 2 leave 4-Fluorobenzoic acid 48 the carbon-halogen bond intact until SChluo~alicylicacid 50 halocatechols are produced and cleaved by dioxygenases (Figure 3). 2.4.5-Trichlorophenoi 51 Therefore, dehalogenation usually 2.4-Dichlcfophenol 52 occurs by elimination of the halogen as 4-Chlnophenol 53 the hydrogen halide, with subsequent Halogenated benzenes 54 double-bond formation on the aliChlorcanalines 55 phatic intermediate. Examples of this mechanism are presented in Figure 4, with the metabolism of 3.5-dichlorodrawing substituents, such as chlorine, catechol and 5-chlorosalicylic acid. A then electrophilic attack by oxygen is similar mechanism is employed in the inhibited. On the other hand, the or- dehalogenation of ring fission products gano-peroxy complex is readily in 3-chloro- and 4-chlorocatechol formed, but over-stabilized, if strong degradation. A second mechanism electron donating substituents are involves halide substitution on the intact aromatic ring. Such substitution present. Ring cleavage is believed to be reactions, by which the halide is recaused by the same mechanism de- placed by a hydroxyl group, occur termined for the rearrangement of quite frequently with the involvement transdecalin peresters (37). This re- of either mixed-function oxygenases or arrangement reaction has been shown dioxygenases (Figure 5). to be very sensitive to ring substituents Reductive mechanisms that involve (37). The rate of ring cleavage of the nucleophilic displacement of a variety of aromatic catechols by di- halogen substituents on aromatic oxygenase has been shown to correlate compounds are energetically unfawell with Hammet up constants vorable reactions. However, there is 2S4A
Envlron. Sci. Teclnwl.. Vol. 16. No. 5.1982
some evidence for reductive mechanisms in the dehalogenation of aliphatic alkyl halides, including DDT and DDE (55.56). Also, there is some preliminary evidence for extremely slow reductive dehalogenation of haloaromatic compounds in anaerobes (57) (Figure 6). The majority of metabolic pathways presented in Figures 3 through 6 a p pear to be adaptations of those pathways determined for structurally similar natural products such as salicylic acid, benzoic acid, protocatechuic acid, gentisic acid, and catechol. Multihalogenated aromatics The biological oxidation of multihalogenated aromatic compounds is hampered by the lack of metabolic versatility, and by the low enzyme levels present in microbial populations. I n many cases in which partial metabolism does occur, the biochemical pathways remain to be rigorously elucidated (57). The lack of information on metabolic pathways has, in turn, slowed progress on the application of gene technology in thecreation of mutants with greater diversity of metabolic capabilities. Both 2.4-D and 2 , 4 3 7 are degraded by mixed populations of soil microorganisms. In fact, thedegradation of 2 , 4 - ~is normal for soil bacteria, and yields 2.4-dichlorophenol as an intermediate (40.41). The latter compound has been shown to be a natural secondary metabolite of some fungi - (14). (Figure 7). However. the deeradation of 2 . 4 5 ~ appears to be quite limited. This persistent herbicide has been reported to be responsible for several public health problems including infertility in males, spontaneous abortions, and birth defects (59). Most of these health effects have been associated with the formation of 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD), which is a contaminant of 2 , 4 5 7 synthesis (60). 2,4,5-~is degraded very slowly by mixed cultures of soil bacteria, but the metabolic pathway that has been proposed for its degradation is highly speculative; dehalogenation mechanisms are little understood. Chapman has pointed out the difficulties encountered with the dehalogenation of trichloroaromatic compounds (40) (Figure 8). Effective dehalogenations would require all three mechanisms (hydroxylation, elimination, and reductive cleavage). Perhaps the most exciting new research observations on 2.4.5-T degradation come from Chakrabarty’s laboratory. This research group has iso-
FIGURE 5
Dehalogenation by hydroxylation
* -
FIGURE 3
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Production of chlorocatechols by two routes v"sur-
.
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--x T -
=-w
FIGURE 6
Reductive dehalogenation of aliphatic substituents by two electron reductants
FIGURE 4
Eliminating chlorine from organic compounds"
FIGURE 7
Dehalogenation of 2,4-D'
Ironically,, traditional organic chemists are discovering that fermentation technology can be exceedingly useful in the manufacture of valuable industrial chemicals from less expensive substrates, as well as in the removal of troublesome constituents from raw materials such as oil. Ten years ago, the prospect of screening 200 000 new chemicals each year would appear to be a hopeless exercise, but now at least there are some foundations for understanding the constraints placed by biological systemson biodegradability.
FIGURE 8
Dehalogenation of 2,4,5-T' OCHSOOH
c1z3cl NADH
0 2
NAD'
H20
YHO COOH
Q OH CI@ CI
CI
H
dehalogenallon b
c d e
~
Reduction Hydration Ellmlnatmn
MiXed.IUnCtIon oxygenation
2.4-D CI
lated a strain of Pseudomonas cepacia (Ac 1100). which grows on 2.4.5-Tas a sole carbon and energy source (38, 6 1 ) . Thisorganism wasseparated from a mixed culture grown in a chemostat that received increasing concentrations of 2.4.5-T for several months. Pseudomonas cepacia grows well on 2,4,5-~, and releases 90%of the chlorine as chloride ion in six days. It is clear that this Pseudomonad has evolved the necessary metabolic capability by acquiring the necessary plasmids to extend its metabolism. This organism has been shown to remove 2.4.5-T from contaminated soil ( 3 8 , 6 / ) .Clearly there is great potential for the use of such organisms to degrade persistent haloaromatic compounds in critical areas such as Love Canal. Success stories of this kind should stimulate a wider search for laboratory strains with the meta.bolic armory to remove some of the more dangerous persistent pollutants.
The future It is difficult to predict the trends of the chemical industry in its frantic search for new synthetic chemicals. In BOA
metabolic pathway
Envirm. Scl. TscmOl.. VoI. 16, No. 5,1982
the past, scientific laboratories in academic institutions have not been able to develop a systematic approach to decide the determinants for biodegradability. Indeed, it took the Gray Freshwater Biological Institute research group almost 15 years to develop a rudimentary understanding of the principles involved in the oxidative cleavage of the benzene nucleus by microorganisms, and this work must be continually reexamined. Even today secondary metabolism is viewed as a subject for the backwaters of biochemistry, unless of course those secondary metabolites happen to be antibiotics. However, in the last three years, fermentation technology and genetic engineering have come of age. It should become possible to advise regulatory agencies on how to regulate the use of persistent chemicals, and on how to treat the chronic pollution situations that currently exist. In short, biologists are beginning to ask serious scientific questions of the traditional industrial organic chemists, questions that never would have been asked IO years ago, because of the lack of biochemical expertise.
Acknowledgments I wish to dedicate this review to my mentor. ProfessorStanley Dagley. who initiated my interest in aromatic metabolism exactly 20 years ago. He still provides me and his other students, P. J. Chapman and D. T. Gibson, with great ideas, insights. and wit. Also, 1 wish to acknowledge the contributions made by my wlleagues R. L. Crawford, J. D. Lipswmb. and L. Que. Jr. Paul Tomasek is sincerely thanked for his help in reviewing the literature. Some of the rsearch reported in this proposal was supported by a grant from the National Science Foundation (PCM 7820461). Before publication. this article was read and commented on for appropriateness and suitability as an ES&T critical review by Dr. Fumio Matsumura, Pesticide Research Institute, Michigan State University. East Lansing, Mich. 48824; and Dr. Michae1.R. Hoffmann. Environmental Engineering Science, W.M.Keck Laboratories. California Institute of Technology, Pasadena, Calif. 91125.
John M.Wood is rofessor ofbiorhemisfry
R
and ecology a f t e Gray Freshwafer Biological Institute, UniuersifyOJMinnesofa. He was the recipienf of thesynthetic Organic Chemical Manufacturers Associalion award for his work on the biomefhylafionofmercury in 1972. Thafsame year, he received a Cuggenheim Fellowship, and spenf a year in Inorganic Chemisfry at Oxford. I n 1981, he received the E. J. Zimmermann Award from the ACS Jar research in environmental science. W w d is a member of fhe ediforial boards OJ Science and BICAL. He receiued his B.Sc. and Ph.D. in biochemisfryat fhe University of k e d s , England. He is author of over 100 papers in the chemical and biochemical liferature.
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Short-Lived Radionuclides in Chemistry and Biology
Advances in Chemistry Series No. 197 John W. Root, €ditor University of California, Davis Kenneth A. Krohn, €ditor University of California, Davis Based on a symposium cosponsored by the Divisions of Nuclear Chemistry and Technology and Physical Chemistry and iointly sponsored b y the Division of Biological Chemistry of the American Chemical Society. A compendium of recent radiochemistry applications covering a broad spectrum of disciplines in chemistry and the biological sciences In this book, equal consideration is given to biological and environmental research and to chemistry and chemical kinetics. The emphasis is on experiments in which the investigator must produce the tracer on site when it is to be used. Techniques utilizing short-lived nuclides of carbon, nitrogen, oxygen, and the halogen elements are also presented. These 28 chapters are organized into five sections - two dealing with chemical reaction studies, two focusing on radiobiochemistry, and one addressing microbiological and environmental techniques. The book will be of interest to those involved in chemistry, physics, biochemistry, medicine, physiology, microbiology, geochemistry, and agricultural and environmental science. CONTENTS Recoil Chemistry and Mechanistic Studies Radioactive Decay of Muititritiated Molecules Synthesis via Nuclear Recoil Methods Charge Distribution in ’3N Ions Reactions of lQFAtoms with Halocarbons Radiotracers and Molecular Dynamics Studies Rotational Excitation * Unimolecular Energy Transfer # Crossed Beam Studies of Chemical Kinetics Thermal Hydrogen Abstraction Reactions Isotopic Nitrogen as aBiochemicalTracer m”C,’3N.150. l8FwithHeavy-lon Beams Chemical Form of I3N Nitrogen Metabolism in Cyanobacteria ’3N in Denitrification Studies N2 Fixing Plants N and C Asslmilation Nitrate in KiebSie//a pneumoniae ‘3N-LabeIed Substituted Nitrosourea * Ammonia Uptake and Metabolism in Brain * j3N-Labeled L-Amino Acids ”C and 18F Labeling Positron Emission Tomography Myocardial Metabolism In VIVOTracers Tracer Studies wlth 13NH4-,42Ki, and2aMg2’, Vitamin 8 , ~Folate. . & Niacin in Blood Applirations of Mossbauer Spectroscopy Ion Beam Anaiysis of Environmental Samples Stable Aciivabie Tracers in Environmental Science
548 pages (1981) Clothbound US & Canada $72.95 Export $87.95 LC 81-19148 iSBN 0-8412-0603-1 Drder from: Distribution Dept. 25 American Chemical Society 1155 Sixteenth St., N.W. Washington, D.C. 20036 or CALL TOLL FREE 800-424-6747 snd use your credit card.
Environ. Sci. Technoi., Voi. 16, No. 5 , 1982
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