microbial co-metabolism and degradation of organic compounds … · microbial co-metabolism...

BACTROLOGICAL Ramvws, June 1972, p. 146-155Copyright i 1972 American Society for Microbiology

Vol. 36, No. 2Printed in USA.

Microbial Co-Metabolism and the Degradationof Organic Compounds in Nature

RAYMOND S. HORVATH

Department of Biology, Bowling Green State University, Bowling Green, Ohio 43403

INTRODUCTION ................................................. 146

BIOCHEMICAL MECHANISM OF CO-METABOLISM ............ 147CO-METABOLISM OF ENVIRONMENTAL POLLUTANTS BYPURE CULTURES OF MICROORGANISMS ...... ....................... 149CO-METABOLISM OF ENVIRONMENTAL POLLUTANI'S BYNATURALLY OCCURRING MICROBIALPOPULATIONS.................. 150A NEW LOOK AT MOLECULAR RECALCITRANCE ...... 151CO-METABOLISM AS A BIOCHEMICAL TECHNIQUE ... 153LITERATURE CITED............................................. 154

INTRODUCTION

The phenomenon of co-oxidation was firstreported by Leadbetter and Foster (37) whenthey noted the oxidation of ethane to aceticacid, propane to propionic acid and acetone,and butane to butanoic acid and methyl ethylketone during growth of Pseudomonas meth-anica on methane, the only hydrocarbon ca-pable of supporting growth of the organism.The term "co-oxidation" was used to describethe process in which a microorganism oxidizeda substance without being able to utilize theenergy derived from this oxidation to supportgrowth.

Subsequently, Jensen (34) suggested that themore general term "co-metabolism" be used todescribe this process and to expand the con-cept to include dehalogenation reactions fre-quently carried out by microbial species. How-ever, co-oxidation and co-metabolism havebeen used interchangeably so frequently in theliterature that these terms are now consideredto be synonymous.Although co-metabolism does imply the

concomitant oxidation of a non-growth sub-strate during growth of a microorganism on autilizable carbon and energy source, it alsodescribes oxidation of non-utilizable substratesby resting cell suspensions grown at the ex-pense of substances capable of supporting mi-crobial growth. Therefore, usage of co-metabo-lism refers to any oxidation of substanceswithout utilization of the energy derived fromthe oxidation to support microbial growth anddoes not infer presence or absence of growthsubstrate during the oxidation.

Although this phenomenon appears to occurwidely in microbial metabolism, interpretationof experimental results as indicating co-me-tabolism requires considerable care. For exam-ple, certain hydrocarbons may, in some way,increase the endogenous respiration of the testorganism without being oxidized. If oxygen isconsumed by an organism in the presence of ahydrocarbon, which does not support growth,at a level only slightly higher than endogenous,the question of stimulation of respirationversus co-metabolism must be raised (41). Insuch cases, proof of disappearance of substrateand accumulation of end products is requiredto clearly demonstrate a co-metabolic process.

Caution must also be used not to overlookthe occurrence of co-metabolic phenomena.For example, Broadbent and Norman (8) of-fered no explanation for the fact that soil or-ganic matter became a better source of nu-trient for the microbial soil population whenreadily decomposable organic matter wasadded. It has been demonstrated that microor-ganisms capable of a co-metabolic degradationof organic pollutants can be enriched for byapplication of biodegradable analogues of thepollutant to the microbial ecosystem (26). Thisprocess, called "analogue enrichment," (D. D.Focht, personal communication) appears toaccount for the enhanced rate of decomposi-tion of organic matter noted by Broadbent andNorman (8).Burge (9) reported rapid decomposition of

the herbicide, dalapon, without an increase inbacterial numbers in soil samples. His inter-pretation of these results indicated breakdownof the herbicide by a chemical process or de-

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MICROBIAL CO-METABOLISM

composition by fungi, but the possibility of a

co-metabolic degradation of dalapon appears

to be equally probable. Caseley (11) likewisefailed to consider co-metabolism as the process

responsible for the breakdown of pentachlo-ronitrobenzene (PCNB) by Streptomyces au-

reofaciens, although the fungicide was not uti-lized as a sole source of carbon for growth. Itwas clearly shown that the fungicide was de-graded only during the active growth phase ofthe microorganism, establishing biological par-

ticipation in the decomposition of PCNB, andthe accumulation of the product pentachlo-roanaline in the medium certainly supportsthe occurrence of a co-metabolic process.

Chambers and Kabler (12) noted in theirstudy of the relationship between chemicalstructure and biodegradability of phenols thatsome cultures had a high oxygen consumptionwith a substance in manometric tests butfailed to utilize the compound as the solesource of carbon and energy for growth in a

mineral-salts medium. If manometric experi-ments repeatedly show oxygen consumption tobe well in excess of the endogenous respiratoryrate, as in this study, the occurrence of co-

metabolism is indicated. Finally, Matsumuraand Boush (40) reported the degradation ofdieldrin by 12 soil isolates growing in man-

nitol-yeast extract medium. The lack ofgrowth of these microorganisms at the expenseof dieldrin, in the absence of the additionalcarbon and energy source, mannitol, indicatesthat co-metabolism was responsible for break-down of this pesticide by the soil isolates.

Evidence for co-metabolism can also be ob-tained by means of enzyme induction experi-ments. It was reported by van Eyk and Bartels(55) that diethoxymethane induced enzymesnecessary for growth of Pseudomonas aerugi-nosa on paraffins and stimulated oxygen con-

sumption to a level far in excess of endogenousrespiration, but failed to support growth. Theability of diethoxymethane to induce enzymesand the high level of oxygen consumption withthis substance indicate that oxygen uptakewith this compound was due to co-metabolismand not merely to stimulation of endogenousrespiration. Demonstration of substrate disap-pearance or product accumulation would haveprovided clear-cut evidence for co-metabolismin this work.

Similarly, Hegeman (22) noted that both p-chloromandelate and p-bromomandelate in-duced enzymes involved in mandelate metabo-lism by P. putida. Both of the halogenatedcompounds were oxidized by enzymes of themandelate group but neither supported growth

of the organism. These results appear to beinterpretable on the basis of co-metabolism.

In spite of the many overlooked examples ofco-metabolism, this phenomenon has beenobserved so frequently that it appears to repre-sent a very important type of microbial metab-olism. Table 1 lists those microorganismswhich have been clearly shown to possess a co-metabolic type of metabolism. The substanceswhich have been studied in regard to this oxi-dative mechanism are listed in Table 2 withthe products resulting from co-metabolism ofeach compound. Those substances for whichthe product of co-metabolism is unknown arenot presented in this table.Undoubtedly, the list of microorganisms

exhibiting this property and the substancesacted upon by this mechanism are far fromcomplete and will be greatly expanded by fu-ture investigation.

BIOCHEMICAL MECHANISM OF CO-METABOLISM

The process of co-metabolism represented tothe microbial physiologist a new oxidativemechanism for which there was no adequateexplanation. Why could a microorganism oxi-dize a substance which it could not use as asource of carbon and energy for growth?

Foster (18) stated that the inability togrow at the expense of a substrate was not

TABLE 1. Microbial species exhibiting thephenomenon of co-metabolism

Microorganism Reference

Achromobacter sp............. 12, 23, 27, 29Arthrobacter sp............... 27Aspergillus niger ............. 53Azotobacter chroococcum ..... 56Azotobacter vinelandii ........ 56Bacillus megaterium .......... 35Bacillus sp................... 27, 40Brevibacterium sp ........... 24, 25, 27Flavobacterium sp ........... 12, 27Hydrogenomonas sp........... 16Microbacterium sp............ 27Micrococcus cerificans ........ 14Micrococcus sp .............. 27Nocardia erythropolis ......... 51, 54Nocardia sp.............. 13, 44Pseudomonas fluorescens ...... 31, 32, 33P. methanica ................ 37P. putida .................... 21, 22Pseudomonas sp.............. 12, 19, 20, 27, 44Streptomyces aureofaciens .... 11, 53Trichoderma viride ........... 40Vibrio sp..................... 6, 10Xanthomonas sp.............. 12, 27

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TABLE 2. Organic substances subject to co-metabolism and accumulated products

Substrate [ Product Reference

Ethane

Propane

Butane

m-Chlorobenzoate

o-Fluorobenzoate

2-Fluoro-4-nitrobenzoate

4-Chlorocatechol

3,5-Dichlorocatechol

3-Methylcatechol

o-Xylene

p-Xylene

Pyrrolidone

Cinerone

n-Butylbenzene

Ethylbenzene

n-Phopylbenzene

p-Isopropyltoluene

n-Butyl-cyclohexane

2,3, 6-Trichlorobenzoate

2,4,5-Trichlorophenoxy-acetic acid

p,p'-Dichlorodiphenyl methane

1, 1-Diphenyl-2,2,2-trichloroethane

Acetic acid

Propionic acid, acetone

Butanoic acid, methyl ethyl ketone

4-chlorocatechol, 3-chlorocatechol

3-Fluorocatechol, fluoroacteate

2-Fluoroprotocatechuic acid

2-Hydroxy-4-chloro-muconic semialdehyde

2-Hydroxy-3,5-dichloro-muconic semialdehyde

2-Hydroxy-3-methyl-muconic semialdehyde

o-Toluic acid

p-Toluic acid, 2,3-dihydroxy-p-toluic acid

Glutamic acid

Cinerolone

Phenylacetic acid

Phenylacetic acid

Cynnamic acid

p-Isopropylbenzoate

Cyclohexaneacetic acid



p-Chlorophenylacetate

2-Phenyl-3,3,3-trichloropropionic acid

always the result of an organism's inability toattack the substrate but often resulted from itsinability to assimilate the products of oxida-tion. Although this observation was correct, itmerely described the process of co-metabolismand failed to offer an adequate explanation forthe incomplete oxidation of the substrate andaccumulation of end products.Hughes (31) suggested that co-metabolism of

halogenated aromatics could result from aninability of the organism to cleave the halogensubstituent from the benzene ring and carrymetabolism to a point where the carbon couldbe assimilated. This proposal failed to explainthe initial attack on the halogenated-aromaticbut was later supported by the work of Ken-nedy and Fewson (36). These investigatorsshowed that cells possessing the benzoate oii-dase enzyme could oxidize only benzoic acidand the monofluorobenzoates. This was attrib-uted to the similarities of the van der Waalradii of the hydrogen and fluorine atoms, since

chloro- and bromo-substitutions gave com-pounds which were inactive as substrates.This mechanism of co-metabolism does not

appear to be universally applicable in view ofthe many reports of chloro-, bromo-, and io-dobenzoates subject to this action. In addition,co-metabolism of alkyl and aryl-alkyl com-

pounds would tend to eliminate the substit-uent as the sole cause of this phenomenon.

Tranter and Cain (54) had proposed that thefailure of many halogenated aromatic com-

pounds to support growth of bacteria whichcould oxidize them could result from an accu-mulation of toxic products. Horvath (25)showed that co-metabolism of 2,3,6-trichloro-benzoate did result in accumulation of 3,5-dichlorocatechol and development of a toxicenvironment to the cells. However, this expla-nation is again applicable only to halogenatedaromatic compounds. Furthermore, it does notaccount for the accumulation of oxidationproducts which eventually result in production

37

37

37

27, 33, 56

15, 33,54

54

23, 29

23, 29

23, 29

44

44

35

53

13

13

13

13

13

25

24

17

17

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of toxic conditions.An enzymatic explanation of co-metabolism

resulting from work on halogenated catecholSwas offered by Gibson, Koch, Schuld, andKallio (21). They suggested that the halogen-ated dihydroxybenzenes inhibited the enzyme

system catalyzing the incorporation of oxygen

into the aromatic nucleus by chelating the ironat the active center of the enzyme. Althoughthe biochemical mechanism of co-metabolismdid appear to involve enzyme catalysis, inhibi-tion of enzyme action by chelation could notaccount for the co-metabolic attack of suchdiverse organic molecules as ethane, halogen-ated benzoates, halogenated catechols, andalkyl-aryl compounds such as ethylbenzeneand propylbenzene.

Reports that enzymes required for completemetabolism of growth-supporting substratescould be induced by non-growth-supportinganalogues (6, 10, 22, 27, 31, 51, 55) indicatedthat initial co-metabolic attack of the ana-logues involved the same enzyme(s) as thatused for oxidation of the growth substrate. Inaddition, the substrate, in many cases, inducedformation of enzymes which co-metabolicallyoxidized the substituted analogues (27, 33, 36,44, 54). This provided further evidence for an

enzyme common to initial attack of substrateby complete metabolism and co-metabolism ofsubstituted analogues.

This evidence indicated the initial mode ofco-metabolic attack but did not account forthe incomplete oxidation of substrate and,hence, accumulation of end products of co-

metabolism. The actual mechanism of co-me-

tabolism of m-chlorobenzoate by Arthrobactersp. was elucidated by Horvath and Alexander(27) using whole-cell suspensions and cell-freeenzyme preparations. Benzoate-grown cellsoxidized m-chlorobenzoate without a lag, andpreincubation of cells with the halogenatedanalogue induced the cells to metabolize bothbenzoate and m-chlorobenzoate. Also, unin-duced cells exhibited the same lag period inoxygen uptake when incubated with eithercompound. It therefore seemed likely that bothbenzoate and m-chlorobenzoate were metabo-lized by the same enzyme system. These re-

sults were consistent with those describedabove.The product which accumulated in this

study, 4-chlorocatechol, did not inhibit oxygenuptake on benzoate, the substituted analogueor unsubstituted catechol, thus eliminatingtoxicity or enzyme inhibition of the cause ofco-metabolism. The relatively unspecific na-

ture of the benzoate oxidase enzyme and the

specificity of the ring-cleaving enzyme for anunsubstituted catechol appeared to account forco-metabolism by this Arthrobacter (27).This explanation was shown to be applicable

to co-metabolism of 2,3, 6-trichlorobenzoate(2,3,6-TBA) by a Brevibacterium (25). Oxida-tion of this herbicide was catalyzed by induc-ible enzymes through the intermediate prod-ucts 2,3, 6-trichloro-4-hydroxy-benzoate and2,3, 5-trichlorophenol to the end product, 3,5-dichlorocatechol, which accumulated in themedium. This catechol did accumulate to con-centrations which resulted in production of atoxic environment to the cells, but toxicity ofend products was shown to be the result ratherthan the cause of co-metabolism. Again, speci-ficity of the ring-cleaving enzyme for an un-substituted catechol accounted for the accu-mulation of 3, 5-dichlorocatechol and the phe-nomenon of co-metabolism of 2,3,6-TBA bythe Brevibacterium.

This mechanism can be proposed to accountfor the accumulation of alkyl substituted cate-chols as well, but no evidence to support thisview is available at present. It is likely thatspecificity of enzymes which catalyze reactionssubsequent to the initial oxidation is involved,but definitive evidence to verify this is yet tobe obtained.

CO-METABOLISM OFENVIRONMENTAL POLLUTANTS BY

PURE CULTURES OFMICROORGANISMS

Evidence indicating the ecological impor-tance of co-metabolism is rapidly accumu-lating. It has been suggested that co-metabo-lism may account for the degradation of manypesticides which do not sustain microbialgrowth (3), and laboratory data are now avail-able to support this view.Compounds such as diphenyl aliphatics

(DDT and related molecules) and polychloro-aromatics [2,3,6-TBA and 2,4,5-trichloro-phenoxyacetate (2, 4,5-T)] have been de-scribed as recalcitrant molecules because ofrepeated failures to isolate organisms capableof utilizing them as sole sources of carbon andenergy for growth. However, co-metabolism ofthese materials has been shown to occur underlaboratory conditions and may be an impor-tant process in the removal of these pesticidesfrom the environment.

For example, the acaricide chlorobenzilate,a diphenyl aliphatic structurally related toDDT, was subjected to oxidation by the yeastRhodotorula gracilis in a medium containing

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yeast extract and mannitol (42). The initialdegradation step was hydrolysis of chloroben-zilate to 4,4'-dichlorobenzilic acid. This sub-stance was further degraded by decarboxyla-tion and dehydrogenation to the end product,4, 4'-dichlorobenzophenone, which was notsubject to further attack.DDT also appears to undergo microbial deg-

radation by a co-metabolic process. Wede-meyer (57, 58) reported oxidation of DDT top, p'-dichlorodiphenylmethane, a processwhich must certainly be co-metabolic since nocarbon would be available to support growth ofthe microorganism. This substance was shownto be subject to further co-metabolic attack bya Hydrogenomonas (16). The product resultingfrom this oxidation, p-chlorophenylacetic acid,established the susceptibility of at least onering of the DDT molecule to microbialcleavage.

It is obvious from these reports that co-me-tabolism of these pesticides does not result ina complete mineralization of the molecule toinorganic chloride, carbon dioxide, and water,but it does eliminate the toxicity of the pesti-cide in the environment. In addition, co-me-tabolism of the pesticides by natural microbialpopulations present in the environment mayresult in complete degradation of the sub-stances.

This idea derives support from considerationof herbicide degradation by co-metabolism.The herbicide 2,3, 6-TBA was shown to beoxidized by a co-metabolic process to the endproduct 3,5-dichlorocatechol (25). The herbi-cide 2,4, 5-trichlorophenoxyacetate (2,4, 5-T)was also reported to undergo co-metabolic oxi-dation (24). The product resulting from thisoxidation was also identified as 3,5-dichloro-catechol.

This chlorocatechol, resulting from co-me-tabolism of both 2,3, 6-TBA and 2,4, 5-T, hadpreviously been shown to be an intermediatein the degradation of 2, 4-dichlorophenox-yacetic acid (2, 4-D) by an Arthrobacter and tobe completely metabolized by this organism(38). In addition, the co-metabolism of 3,5-dichlorocatechol by Achromobacter sp. hadbeen clearly demonstrated (23, 29). Thus, itappears that both 2,3,6-TBA and 2,4,5-Tmight be completely degraded by the action oftwo or more microbial species by the processesof co-metabolism and metabolism or by a se-ries of co-metabolic reactions.Examples of co-metabolism of environmen-

tally important compounds are by no meanslimited to pesticides. Degradation of the sur-factant alkyl benzene sulfonate (ABS) also

appears to occur via a co-metabolic reactionseries (30). The microbial degradation of ABSrepresents a unique combination of co-metab-olism of a portion of the molecule and com-plete metabolism of the remainder of the mol-ecule. Initial oxidation of the surfactant appar-ently required an expenditure of energy sinceco-metabolism of ABS by the Pseudomonasdid not occur in the absence of an energysource, glucose. Co-metabolism of ABS by thisorganism yielded isopropanol from side-chainoxidation and catechol from aromatic ring oxi-dation. Isopropanol, once formed, was capableof supporting growth of the isolate, indicatingcomplete metabolism of this substance. Desul-fonation of ABS occurred by the process of co-metabolism and involved a coupled reactionbetween phenol, an intermediate product ofABS co-metabolism, and a sulfonated alkylbenzene intermediate. Catechol, the endproduct of aromatic ring co-metabolism, accu-mulated in stoichiometric amounts and ap-peared to result from oxidation of phenol viathe coupled reaction.

Although the laboratory investigations citedindicate that degradation of environmentalpollutants can be accomplished by microorga-nisms isolated from natural habitats, caremust be taken in extrapolating these findingsto natural ecosystems. The co-metabolic phe-nomena discussed have occurred in pure cul-tures in the absence of adverse conditionscaused by competition. Demonstration of asimilar process in soil and aquatic ecosystemsis required to establish susceptibility of thesesubstances to microbial degradation underfield conditions.

CO-METABOLISM OFENVIRONMENTAL POLLUTANTS BYNATURALLY OCCURRING MICROBIAL

POPULATIONSEvidence for the co-metabolic degradation

of environmental pollutants by naturally oc-curring microbial populations is now availableand indicates the importance of this phenom-enon in the ecosystem.

Pure culture studies of Horvath and Koft(30) had established the occurrence of co-me-tabolism in the degradation of ABS, indicatingthe possibility of this degradative mechanismunder mixed culture conditions. Benarde et al.(7) extended these findings by demonstratingthe degradation of ABS under simulated nat-ural conditions, thereby establishing the appli-cability of data obtained from pure culturestudies to the situation obtaining in the bios-phere. Degradation of ABS was shown to occur

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in an aquarium containing lake water andABS, to the extent of 90% in 40 days. The ad-dition of an energy source (glucose) to thissystem resulted in a 100% decrease of the sur-factant concentration in only 20 days. Al-though the participation of co-metabolism wasnot clearly demonstrated in this study, twofactors indicate the occurrence of this phenom-enon. The enhancement of the rate of degrada-tion of ABS by the addition of a utilizable en-ergy source implicates the existence of a co-metabolic process in this system. Secondly,the inability of the investigators to isolatefrom this system a microorganism which couldutilize ABS as its sole source of carbon andenergy for growth indicates that co-metabo-lism is responsible for the decomposition ofABS in this report (7).

Similar results were obtained by Horvath(26) with the herbicide 2,3, 6-TBA. Decompo-sition of this chlorinated aromatic substancewas accomplished by microbial populationspresent in lake water under laboratory condi-tions. Approximately 35% of the 2,3,6-TBAsupplied was degraded in 18 days, and thisdegradation was not accompanied by a signifi-cant increase in microbial numbers. The tech-nique of "analog enrichment" (D. D. Focht,personal communication) was employed andinvolved addition of a biodegradable simulantof the herbicide, benzoic acid, to the system.This technique resulted in an increase in boththe rate of degradation (35% in 4 days) andtotal amount of the herbicide degraded (80% in14 days). The increase in microbial numbersobtained under these conditions was attribut-able only to the concentration of benzoic acidprovided. The lack of microbial growth at theexpense of 2,3, 6-TBA degradation and theincreased rate of decomposition resulting fromthe addition of benzoic acid clearly demon-strate the role of co-metabolism in this investi-gation and establish the importance of the"analog enrichment" (D. D. Focht, personalcommunication) technique in biodegradabilitystudies. Additional evidence of co-metabolismcomes from the fact that the investigator wasunable to isolate, from the natural population,an organism which could grow at the expenseof 2,3,6-TBA as its sole carbon and energysource.The investigations cited indicate that nat-

ural microbial populations are capable of ef-fecting a co-metabolic degradation of environ-mental pollutants. A major objection however,is that these investigations employed condi-tions which deviate from the natural environ-ment in that both studies involved closed sys-

tems. A constant flow system would moreclosely resemble the conditions found in na-ture, and research in this area is necessary be-fore definite conclusions can be drawn.

This objection appears to have been over-come in the report of Sethunathan and Pathak(50). These investigators noted a rapid inacti-vation of diazinon within 3 to 5 days of itsincubation with water from a rice field thathad received several applications of the sub-stance. Degradation of this pesticide in waterfrom an untreated rice field was not significantduring the same time period. The data clearlyestablish that diazinon treatment had induceda microbial population capable of degradingthis compound. Of special significance was thefinding that the Arthrobacter isolated frompaddy water of treated fields was capable ofdegrading diazinon only in the presence ofadditional carbon and energy sources, specifi-cally ethyl alcohol or glucose. This clearlyshows a co-metabolic decomposition of thepesticide by microbial action under naturalenvironmental conditions.

Admittedly, reports concerning the occur-rence of microbial co-metabolism under nat-ural conditions are few in number, but they dodemonstrate and emphasize the importance ofthis phenomenon in the environment. Mi-crobial populations are clearly capable of de-grading pollutants by the process of co-metab-olism. Furthermore, microorganisms capable ofthis co-metabolic degradation can be enrichedfor by repeated applications of the substance(50) or by application of biodegradable analogsof the pollutant (26) to the microbial eco-system.The method of "analog enrichment" (D. D.

Focht, personal communication) may present avaluable technique to be used during the appli-cation of pesticides to natural ecosystems.Application of both the pesticide and a biode-gradable simulant of the pesticide may allowman to have both the benefit of action of thepest control agent and a rapid oxidation of thecompound, thus eliminating the environmentalhazard which might otherwise accompany itsuse (26).

A NEW LOOK AT MOLECULARRECALCITRANCE

In light of the available data indicating therole of co-metabolism in the microbial degra-dation of environmentally important com-pounds, a reexamination of the concept ofmolecular recalcitrance (2) appears to be inorder. This concept assumes that there are

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many compounds which microorganisms areunable to degrade, either under any circum-stances or at sufficiently rapid rates to preventtheir accumulation in the environment (3).The term "recalcitrant" has been used to de-scribe organic pollutants that remain un-

changed because of the fallibility of microorga-nisms (3).

It should be noted that investigations con-

ceming the basis for recalcitrance of com-

pounds are relatively recent, and definitiveconclusions cannot yet be drawn (3). In addi-tion, only a few non-biodegradable substanceshave been identified (5), and there is now some

question as to the validity of this label of re-calcitrance. Are these compounds truely recal-citrant due to microbial fallibility or are themethods employed in biodegradability studiesfallible, thus leading to false conclusions re-garding the microbial decomposition of thesubstances?

It has been stated that microbiologists canfind evidence for microbial fallibility in theirown discipline. For example, bacterial sporesand certain fungi and protozoa can remainviable in a dormant state for years, thus indi-cating an inability of microorganisms to de-grade these structures (4). This evidence formolecular recalcitrance appears to stem fromhuman fallibility rather than microbial falli-bility. The term recalcitrant, if it is to be usedat all, should be applied to individual organiccompounds, not to living systems.However, the failure to demonstrate biodeg-

radation of some pesticides and detergents didappear to be significant and was interpreted as

indicating that microorganisms cannot decom-pose many of the materials provided to them(3). It now appears that this failure may againbe attributed to human rather than microbialfallibility. Application of the term recalcitrantto detergents of the ABS type and pesticidessuch as 2,3,6-TBA, 2,4,5-T, and DDT may bethe result of fallible procedures which do notconsider the co-metabolic phenomenon, ratherthan to inactivity on the part of microorga-nisms.Demonstration of biodegradability has been

based on three approaches (43), none of whichtakes into account the possibility of a co-met-abolic degradation of the substance under in-vestigation. The first approach is a determina-tion of the ability of some commonly stockedprototrophic microorganisms to grow at theexpense of the organic material. Because co-metabolism of a substance yields neithercarbon nor energy to the microorganisms in-volved, this approach clearly eliminates con-

sideration of this metabolic process in the de-composition of the compound.The second method for evaluating degrada-

bility is the "die-away" test (43). This proce-dure has generally involved relatively rapidspectrophotometric methods (39) to follow thedisappearance of ultraviolet absorbance causedby cleavage of the aromatic nucleus of deter-gents and chloro-aromatic pesticides. Theprocess of co-metabolism has been shown toaccount for a significant alteration of the ABSmolecule, the 2,3, 6-TBA molecule, and the2,4, 5-T molecule with a catechol-like com-pound resulting as the end product in eachcase. Obviously, spectrophotometric methodswould be inadequate for the measurement ofdegradation of each compound since the aro-matic nucleus was not cleaved, and thus nosignificant change in ultraviolet absorbancecould be recorded. Thus, this method wouldnot have detected the oxidation of each com-pound discussed previously. In addition, use ofthe die-away test under pure culture condi-tions eliminates the possibility of completemineralization by co-metabolism, which ap-pears to be accomplished by mixed cultures(26, 50).

Nevertheless, a die-away evaluation proce-dure can be employed to detect co-metabolicdegradation of a substance if gas chromato-graphic analysis is used. This method allowsdetection of any alteration of the moleculeunder consideration as well as a quantitativemeasurement of degradation of the substance.The use of natural microbial populations suchas found in soil, lake water, or sewage oxida-tion lagoons is to be encouraged in thesestudies for obvious reasons.The third approach in demonstrating biode-

gradability involves the elective culturemethod (43). Those organisms capable of uti-lizing the compound of interest should in-crease in number during the enrichment pe-riod due to the competitive advantage theyenjoy in this system. Isolation, by this proce-dure, of microorganisms capable of utilizingthe substance for growth has generally beenconsidered to be a necessary step in estab-lishing that the microbial population is in-volved in the decomposition process (1). Forexample, 2,4, 5-T has been regarded as a recal-citrant molecule because an isolate capable ofgrowing at the expense of this substance hasnot been isolated by the elective culturemethod (1). This procedure again completelyignores co-metabolism as a factor in the de-composition of organic materials, and in factrules out co-metabolism as a mechanism by

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virtue of the requirement for growth at theexpense of the substance. The existence of co-metabolism represents a severe drawback tothe use of growth as a criterion of biodegrada-bility of substrates. Compounds which hadbeen previously designated as recalcitrant havenow been clearly shown to be subject to co-metabolic degradation.Thus, the inability to demonstrate microbial

transformations of certain compounds mayresult from an emphasis on techniques de-signed to select for microorganisms that canutilize such compounds as the sole source ofcarbon and energy rather than from an ina-bility of the microorganisms to actively oxidizethese compounds. Results obtained with co-metabolism indicate that the concept of mo-lecular recalcitrance (2) may no longer bevalid.

CO-METABOLISM AS A BIOCHEMICALTECHNIQUE

The process of co-metabolism presents tothe microbial physiologist more than merely anew metabolic phenomenon to be investigated.It has been employed as a technique for thebiochemical study of microbial aromatic me-tabolism, and in this regard, may prove to beas valuable a tool as simultaneous adaptationexperiments have been (52).

Gibson et al. (21) used co-metabolism as atechnique to isolate and identify products re-sulting from oxidation of halogenated benzenesand p-chlorotoluene when these substanceswere incubated with Pseudomonas putida.This technique was necessary because bothbenzene and toluene were oxidized so rapidlyby this organism that reaction products failedto accumulate (19, 20). Use of co-metabolismaided study of the mechanism of oxygen fixa-tion into the aromatic nucleus by allowing forisolation of early intermediates in the degrada-tion of these compounds.

Focht and Alexander (16) also employed co-metabolism as a technique in their investiga-tion of the microbial degradation of DDT me-tabolites. Their findings indicated that p,p'-dichlorodiphenylmethane, a known metabolitein DDT metabolism (57, 58), was oxidized top-chlorophenylacetic acid by the Hydrogeno-monas organism used, thus establishingcleavage of one of the two rings of this sub-strate.The microbial oxidation of 2,3,6-TBA was

also elucidated by means of co-metabolic tech-niques (25). The accumulation of 3,5-dichloro-catechol, in stoichiometric amounts, assuredcomplete recovery of the end product and al-

lowed for demonstration of the pathway em-ployed by the Achromobacter. Because co-metabolism involves very few oxidative steps,it was possible to determine the sequence ofoxygenation, decarboxylation, and dehalogena-tion reactions involved in 2,3,6-TBA degrada-tion.

Co-metabolism appears to be common in themicrobial oxidation of halogenated aromaticsubstances; thus, it should be possible to ob-tain accumulation of a variety of halogen-sub-stituted metabolites (28). It has also been ob-served that bacteria capable of growing on hal-ogenated compounds co-metabolize the corre-sponding unsubstituted products (38). Thus,accumulation of non-halogenated metabolitesshould also be possible by exploitation of co-metabolism.

For example, Ribbons and Senior (47) em-ployed this technique for the quantitative esti-mation of 2,3-dihydroxy-p-toluate by spectro-photometric measurement. This compoundoccurs as a metabolite in certain aromatic hy-drocarbon fermentations, and use of co-me-tabolism allowed a rapid, sensitive, and spe-cific method of assay for the substance. Thistechnique may thus offer a simple and rapidmethod for the preparation of biochemicalsuseful for tracing metabolic pathways (28).Refinements in co-metabolic procedures

may also offer an attractive means of accumu-lating products important in the fermentationindustry. Raymond, Jamison, and Hudson(45) reported the use of anion-exchange resinsto significantly increase the yields of acidicproducts in co-metabolic systems. When theresin was incorporated in shake flasks or agarplates, p-toluic acid, 2, 3-dihydroxy-p-toluicacid, and a, a -cis, cis-dimethylmuconic acidresulting from the oxidation of p-xylene byNocardia sp. accumulated on the resin. Finalproduct concentration was shown to increasewith increasing resin concentration, and min-eral balances were not affected if the resin wasproperly conditioned before use.

Co-metabolic accumulation of at least oneproduct important in the pharmaceutical in-dustry has been shown to be economicallyfeasible. Co-metabolism was used in the aro-matization of steroids into equilin by Nocardiarubra (49). Resting cell suspensions of this or-ganism converted 19-hydroxy-androsta-4, 7-diene-3, 17-dione into equilin with yields of40% equilin for a substrate concentration of1 g/liter.Co-metabolism appears to be an ideal tech-

nique for aromatization, and specific hydroxy-lation of many compounds produced by the

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fermentation industry and future researchshould provide many examples of presentlyused processes which might be carried outmore efficiently or more easily by the tech-nique of co-metabolism.The importance of co-metabolism is not

limited to the accumulation of biochemicalproducts. Co-metabolism has also been usedfor the detection and demonstration of spe-cific enzyme action. Demonstration of theaction of catechol-1, 6-dioxygenase, a newmetacleaving enzyme possessed by an Achro-mobacter, was accomplished by use of thetechnique of co-metabolism (23, 29). The ac-cumulation of end products allowed for theisolation and identification of metabolites andthe determination of specific bonds of substi-tuted catechols which were cleaved by theco-metabolic process. The products identifiedas 3, 5-dichloro-2-hydroxymuconic semialde-hyde, 3-methyl-2-hydroxymuconic semialde-hyde, and 4-chloro-2-hydroxymuconic semial-dehyde which resulted from co-metabolism of3, 5-dichlorocatechol, 3-methylcatechol, and4-chlorocatechol, respectively, could beformed only by cleavage of the bond betweencarbon atoms 1 and 6 of the aromatic ring. Theimportance of co-metabolism as a techniquefor the study of microbial enzyme specificityand action is clearly indicated by this report(23, 29).

Ribbons and Senior (46, 48) also utilized co-

metabolism to determine the site of cleavageof 2, 3-dihydroxy-benzoate. These investigatorsused an analogue, 2, 3-dihydroxy-p-toluate,which had been isolated as a co-metabolicproduct from p-xylene oxidation (44) as a sub-strate for 2,3-dihydroxybenzoate oxygenase.Whole cells co-metabolized this analogue andaccumulated 2, 6-dioxoheptenoic acid. Theseresults were consistent only with ring cleavageof 2,3-dihydroxy-p-toluate in the 3,4-positionand indicated that the site of cleavage of 2,3-dihydroxybenzoate was also in the 3, 4-posi-tion.

Applications of co-metabolism as a tech-nique for biochemical and metabolic studiesappear to be limited only by the imaginationof investigators. Future studies should estab-lish many more uses for this new and poten-tially valuable technique.

ACKNOWLEDGMENTSThe author was a Faculty Research Awardee of the

Bowling Green State University Faculty Research Com-mittee.

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