Environmental and Microbiological Problems Arising from Recalcitrant Molecules1

M. ALEXANDER
Laboratory of Soil Microbiologg~, Departrnent of Agronom),, Cornell University, lthaca. New York 14850

A recalcitrant molecule is one that persists for extended periods in all environments tested, regardless of whether the compound is or is not inherently nonbiodegradable [1, 3]. In addition to the intrinsically long-lived chemicals, certain compounds are destroyed remarkably slowly in some localities but are readily metabolized in others. Considerable evidence suggests that the complete conversion of carbonaceous materials to inorganic products results mainly or solely from the activities of microbial communities of natural ecosystems, so that the very existence of molecules that endure for months, years, or decades suggests a microbial inadequacy, a failing that may be evident in all ecosystems owing to the inherent resistance of the substance or one that is observed in only some areas because the potentially active populations are unable to grow or to carry out the needed reactions. The real, potential, or presumed environmental consequences of the presence of refractory substances in nature are numerous. The possible effects probably can be best appreciated after reviewing the types of substances that are kalown to resist microbial destruction in soil, the oceans, or bodies of fresh water. Because of the enormous interest in them, pesticides may be listed first. It is owing to the inability of microorganisms to destroy certain of these pestcontrol agents at reasonable rates that they have been or shortly will be banned in many countries, and the use of chemicals that are effective for insect or weed control and have little mammalian toxicity is thus being terminated. Their unique and common attribute insofar as the environment is concerned is their unsuitability as substrates. For example, DDT, aldrin, lindane, and other chlorinated hydrocarbon insecticides endure for years or decades (Fig. 1), remaining in soils or waters from where they can enter food chains and be subjected

IPresented at a symposium on "Microbial Interactions with Pollutants" at the First International Congress for Bacteriology, Jerusalem, September, 1973. 17 MICROBIAL ECOLOGY, Vol. 2, 17-27 (1975) 9 1975 by Springer-Verlag New York Inc.

18

M. A)ex~ndcr

Sirnazine Monuron(CMU) - - ~ " ? Pentachlorophenol ? Picloram -]~? Aldrin

~- .P D D T

~ 1 ~ ? Lindane . . 4
I

. 8
I

. Years

. 12
I

--~ Parathion I 16

Fig. 1. Illustrative data showing persistence of herbicides ( ), a fungicide ( - - - ) , chlorinated hydrocarbon insecticides ( . . . . . ), and an organophosphate insecticide ( -) in soil. The question mark indicates that the compound was still present at the last sampling time.

to biomagnification or from where they can be disseminated to adjacent or distant sites. Various herbicides also fail to serve as good substrates for heterotrophs in nature, and hence weed-control compounds like simazine, monuron, picloram, and others may endure for periods in excess of a year. Chemicals of these sorts, all recalcitrant to varying degrees, may create problems because their biomagnification occasionally causes bird and fish mortalities. Their persistence in agricultural land may lead to levels in food and feed deemed to be unacceptable by public agencies charged with protecting the food supply. Furthermore, a few herbicides remain in soil until a succeeding plant species in a rotation is seeded into the field, and losses of this crop have resulted thereby. A second category of recalcitrant molecules is represented by the polychlorinated biphenyls, commonly known as PCBs. These biphenyls find many

Problems Arising From Recalcitrant Molecules

19

uses as coolant-insulant fluids, plasticizers, solvents, and components of insulating materials. Because they withstand microbial action, they endure in natural ecosystems, and the persistence and vast quantities used are reflected by their appearance in many regions of the globe and in fish and birds located in areas remote from the site where the PCBs were initially discharged [4, 5]. The toxicological properties and biological effects of these pervasive and clearly resistant hydrocarbons have attracted considerable attention [17]. Other wastes from manufacturing and industrial activities are discharged directly into waterways. However, until recently, the identities, persistence, behavior in nature, and toxicology of these wastes have not been characterized, and rare is the government that regulates their release into rivers, lakes, or coastal waters. By the application of combined gas chromatography-mass spectrometry, however, a few of the compounds have now been identified, and such characterizations should allow for the establishment of which fail to succumb to rapid enzymatic degradation. Nevertheless, several substances in waters already are known to be refractory. A notable instance is o-chloronitrobenzene, which moves at least 1500 km from the point of its industrial discharge into the Mississippi River, after which the river water enters the sea [16]. It would be inappropriate to ascribe dire consequences to the many chemicals now being found in waterways, yet the number, diversity, occasionally demonstrated persistence, and unknown hazards arising from such an array of substances should prompt a rapid increase in studies of both environmental toxicology and microbiology. In the realms of public health, veterinary medicine, and plant pathology are the refractory constituents contained in the surface layers of human, animal, and plant pathogens. The very fact that the endospores of Clostridium and Bacillus anthracis remain viable in moist soil for years in the absence of spore germination suggests that their surface layers contain components that are not used by neighboring species. The identities of the refractory surface constituents protecting against destruction by parasites have not been established. The cysts of parasitic protozoa also presumably endure, and such durability, if a reality, must likewise be the result of a refractory external layer. Among the soil-borne fungi pathogenic to plants and animals are found resting structures that allow the fungi to remain viable in the presence of numerous heterotrophs that undoubtedly are able to utilize internal constituents of the structure (Table 1); hence the chlamydospore, sclerotium, or other resting structure must be surrounded by an organic coating that, by virtue of its unsuitability as a substrate, protects the fungus. Except for our studies which suggest that melanins [7] and probably individual heteropolysaccharides [6, 18] in fungal surfaces are themselves resistant and shield the organisms from lysis, the biochemical bases for the longevity of such durable structures have yet to be established. A number of refractory compounds are deemed to be objectionable largely on esthetic grounds. Until recently, highly branched alkylbenzene sulfonate sur-

20

M. Alexander Table 1.
Persistence o f Viable Fungal Structures in Soil a

Structure Chlamydospore Teliospore Oospore Sclerotium Microsclerotia

Fungus
Tilletia Ustilago Aphanomyces Phymatotrichum Verticillium

Persistence (yr)
~ 5 8 ~ 10 > 12 14

abased on data summarized in Ref. 131. factants were included in detergent formulations. These surfactants were not quickly decomposed after introduction into streams, rivers, and groundwater, and their presence led to froth and foam in waterways, potable water supplies, and sewage-treatment plants. The public clamor became so intense that useful surfactants were discarded entirely, but at the same time the resulting research led to the establishment of some of the first principles on the effect of chemical structure on biodegradability. Also undesirable from the esthetic viewpoint are the synthetic polymers widely used for the packaging materials which are ever more frequently found on roadsides and in recreational areas. These polymers, in addition, require waste disposal programs of enormous magnitude inasmuch as millions of tons are used per year, not only for packaging purposes but also in the construction industry and in fabrics, bedding, and insulation. The vast bulk of material adds markedly to the problem of solid waste disposal and increases appreciably tile cost of municipal waste disposal. Evidence is also at hand that particles of plastic are accumulating in areas of the open sea [9]. Here, too, the longevity is attributable to a microbial deficiency, an inability to hydrolyze enzymatically the synthetic polymer. Indeed, microorganisms have yet to be found, in either enrichment or axenic culture, which can grow on or extensively metabolize a large number of synthetic polymers (Table 2). Nevertheless, commercial formulations of these substances occasionally are colonized, persumably because the fungi or bacteria use the small molecules in the formulated product for replication. In addition, a number of natural products are quite dramatically resistant. This is shown in Fig. 2. The longevity of these substances is quite variable: a

Problems Arising From RecaZeilrant Molecules Table 2

Polymers Resistant to Microbial Degradation

21

Acetate rayon Polyethylene Polystyrene Polyvinyl chloride Silicones

Dacron Nylon Orlon Saran Teflon

millenium for tanned leather, more than 10,000 years for fungus spores entrapped in lake sediments, tens of millions of years for the terpenoid resin of amber and the succinic acid within the amber, and more than 109 years for porphyrins recovered from sedimentary rocks. Thus, the number of refractory synthetic and natural compounds is large, and their durability in nature is of importance because of their impact on public health, food production, and environmental quality or for aesthetic reasons. Why do such compounds exist? What in the structure of the chemical, in the physiological or morphological characteristics of the microbial cell, or in the

Tanned leather
(in soil)

] [

'Fungus spores (in lake sediments)

Terpenoid resins (of amber) Bound amino acids (in anthracite coal) Chitin (in fossils) Porphyrins (in sedimentary rocks)
I I I 1 I I

j ] ]

Logarithm of years

Fig. 2. Longevity of various products in natural ecosystems [ 3 ].

22

M. Alexander

conditions of a particular environment dictates that a compound or natural product fails to succumb to biodegradation? Although knowledge of catabolic pathways and microbial physiology is extensive, information on the reasons for the absence of a biodegradative sequence is meager. Indeed, research on the biochemistry of improbable or impossible biological reactions is not likely to attract investigators who seek positive answers to their questions. Nevertheless, several hypotheses-tentative though they must be-can be advanced to account for the persistence of molecules in nature. Since this meeting is being held in the land of the people from whom mankind first learned the ten commandments, the mechanisms might be formulated, facetiously to be sure, as commandments. The commandments are not designed as guides for the actions of man but rather for the inactions of the communities of microscopic creatures often essential for the maintenance of environmental quality. The code is now in the form of a decalogue, an extension of the earlier but incomplete six commandments dictating what microbial communities could not do or could not do readily [2]. These ten generalizations are an abbreviation from a longer list of postulated mechanisms of recalcitrance [3].

1. Thou shalt not degrade a compound for which there exists no enzyme (or enzymes) catalyzing an initial reaction (or reaction sequence) in the catabolic pathway. Experimental confirmation of this hypothesis is unlikely because proof that an enzyme does not exist is difficult, if not impossible, to provide. However, studies of enzyme specificity show how a slight change in a substrate renders it unavailable to an enzyme, and many synthetic compounds of environmental importance differ only slightly from either intermediates in biochemical pathways or compounds that are excellent substrates for microbial growth. Thus, PCBs and DDT, both of which are persistent, are closely related to diphenylmethane, which is an excellent substrate for bacterial growth [11].
If biodegradation of a class of compounds proceeds in nature principally or solely by a particular metabolic sequence, a structural change in a member of that class that renders the molecule unavailable as a substrate to the enzyme system is an alteration that renders resistant what was previously a suitable substrate. The molecule would not be destroyed unless an enzyme were present to reverse the change or an organism would proliferate that had such an enzyme or possessed a different catabolic pathway to effect the decomposition. This is exemplified by a methyl or chloro substituent introduced onto fatty acids, which are generally destroyed by /3-oxidation. Such a substituent, if on the a- or/3carbon, prevents /3-oxidation and thus retards or precludes destruction of the substituted analog of the biodegradable aliphatic acid (Table 3).

2. Thou shalt not readily degrade a substrate which provides insufficient energy or no carbon for growth. Some pollutants which are metabolized are not known to support the growth of any bacterium, actinomycete, or fungus. Should the active species not multiply at the expense of the substrate and should its

Problems Arising From Recalcitrant Molecules Table 3 Relative Rates of Biodegradation by Sewage or Soil Organisms a Chemical class Butyric acid Relative biodegradability Moderate

23

Susceptible Unsubstituted 4-Chlorobutyric

Resistant 2,2-Dimethylbutyric

3-Chlorobutyric 2-Methylvaleric 5-Chlorovaleric

2-Chlorobutyric 2,2-Dimethylvaleric 2-Chlorovaleric 2-Bromocaproic

Valeric acid

Unsubstituted

Caproic acid

6-Bromocaproic

a From Dias and Alexander [101 and Hammond and Alexander [12]. population density be low, the rate of substrate turnover will be slow. This may explain why chlorinated hydrocarbon insecticides are long-lived in nature, despite the fact that species capable of metabolizing them have been isolated. The ease of finding microorganisms able to metabolize a molecule that does not give the cell energy or an element essential for growth is important in any consideration of the transformations of persistent chemicals. Such organisms may be responsible for inducing modifications in pollutants that endure in soils, waters, or sediments, as in the case of the structural changes brought about in DDT [19]. The products that seem to be generated by these populations frequently appear to be persistent so that the incomplete transformations may give rise to new and significant refractory pollutants.
3. Thou shalt not degrade a compound in environments where at least one essential nutrient is missing. The lack of free water or the absence of molecular oxygen needed either as an electron acceptor for the potentially active species or as a reactant in initial phases of hydrocarbon metabolism undoubtedly accounts for the persistence in some environments of compounds that are readily decomposed in other localities. Thus, carbonaceous materials are preserved in arid regions, and hydrocarbon deposits remain undisturbed by biotic communities for enormously long time periods. 4. Thou shalt not destroy a substrate in environments containing factors inimical to microbial proliferation. This commandment, too, applies to compounds that are intrinsically biodegradable but which have been deposited in a milieu where species having the requisite enzymes are inhibited or where extracellular enzymes responsible for key steps in the degradation are unable to

24

M. Alexander

function. Extremes of salinity, osmotic pressure, and temperature and environments containing organic or inorganic toxins are common in nature. Preservation of natural products is frequent in such surroundings. Clays, other colloidal matter, and polyaromatics are also found in many terrestrial habitats, and these substances are known to protect natural products and presumably also synthetic chemicals.
5. Thou shalt not metabolize a large molecule that fails to penetrate thy cells. Many high-molecular-weight compounds do not pass through the cell membrane, but they are still destroyed because they are first depolymerized by extracellular hydrolases. But consider the case of a homologous series of chemicals in which the low-molecular-weight representatives penetrate the cell, where they are decomposed, but the higher homologs do not pass through the membrane and are also not modified by extracellular enzymes. They would thus be refractory. Although impermeability has yet to be shown directly to be of ecological significance, the capacity of aerobic microorganisms to destroy simple alkanes in soil and water and their inability to degrade polyethylenes, which are long-chain alkanes, suggest that permeability may be responsible; i.e., the responsible enzymes are solely intracellular and no extracellular depolymerizing mechanism exists. 6. Thou mayest have difficulty in degrading a compound present in aqueous solution in exceedingly low concentrations. Many substances that are poorly soluble in Water and others that are soluble but exist at low levels are slowly destroyed. Exceptions to this generalization are not uncommon because some highly insoluble polysaccharides, for example, are depolymerized rapidly, but several studies show that insoluble aromatic hydrocarbons [22] and readily utilizable, water-soluble substrates [13] are acted upon very slowly or not at all when the concentration in aqueous solution is quite low. 7. Thou shalt not degrade a chemical that fails to induce the formation of. enzymes needed for its breakdown. Many synthetic compounds are acted upon by inducible enzymes, and the failure of the enzymes to be formed in the cell, possibly owing to a tow concentration or poor solubility of the inducer, might be the reason for the lack of biodegradation. 8. Thou mayest not be able to decompose rapidly a molecule whose degradation requires several extracellular enzymes produced by different populations. Consider a polymer with dissimilar linkages between the monomers and possible even different monomers; its depolymerization would probably usually require an array of enzymes excreted by different organisms. Such a reaction sequence would likely be slow either because of the need for each organism to alight near the substrate or because of the slow release of products that permeate the cell and provide it with energy so that the population density will rise. Such a need for various populations synthesizing a group of enzymes acting at differ-

Problems Arising From Recalcitrant Molecules

25

ent sites in a polymer has been proposed as the basis for the slow microbial utilization of components of soil organic matter [20] and of the cell walls of fungal hyphae that apparently remain intact for some time in soil [6].
9. Thou shalt have trouble dealing with inaccessible substrates. A substrate normally metabolized with ease may be protected from transformation if: (a) it is complexed with tannin, lignin, or certain other organic molecules; (b) it is entrapped within the lattice structure of a clay mineral or coated with a refractory substance; or (c) it is deposited at a microsite sufficiently remote or so small that microorganisms cannot penetrate. Some evidence exists to show that 9persistence occasionally is attributable to a shielding of these sorts [3].

10. Thou shalt similarly find difficulty in cleaving a molecule when the sites to be cleaved are not readily accessible. Indications that this is a valid commandment are easy to find. Highly cross-linked polymers, be they proteins or synthetic polymers, are commonly refractory, and it is easy to visualize that a molecule as large as an enzyme encounters difficulty in combining with potentially cleavable bonds buried within an extensively cross-linked structure. For example, the highly cross-linked protein, keratin, is only slowly destroyed by proteolytic enzymes, but its hydrolysis is markedly enhanced when the disulfide bonds important to keratin's structural integrity are ruptured [ 15]. A number of investigations have demonstrated that the recalcitrance inadvertently introduced into chemicals of great practical importance can be eliminated, while still maintaining the usefulness of that class of compounds. For example, slight structural modifications in refractory surfactants included in detergent preparations often make them readily biodegradable [21], and modest alterations in the structures of persistent but useful pesticides convert them into short-lived relatives [8, 14]. The metabolic reason for the change from resistance to susceptibility occasionally is readily evident; more often the data cannot yet be adequately explained. Because recalcitrant molecules are important in public health, food production, industry, and environmental quality, investigations of the reasons for their persistence have great practical importance. Furthermore, because the very resistance of these molecules runs counter to the widely held belief of microbial omnipotence and because information establishing the physiological or environmental reasons underlying their persistence should contribute to knowledge of microbial behavior, such studies should have considerable significance for basic science. It is appropriate in the context of the pressing need for and relevance of these investigations to refer to one of the ancient Hebrew sages, Hillel, who resided in this very locality. He concluded one of his sayings with the question, "If not now, then when?"

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M. Alexander Acknowledgment

The author's research on recalcitrant molecules was supported in part by the Office of Naval Research.

References 1. 2. 3. 4. Alexander, M. 1965. Biodegradation: Problems of molecular recalcitrance and microbial fallibility. Advan. AppL Microbiol. 7: 35-80. Alexander, M. 1966. Biodegradation of pesticides. In: Pesticides and Their Lffects on Soils and Water. Amer. Soc. Agron., Madison, Wisconsin. pp. 78-84. Alexander, M. 1973. Nonbiodegradable and other recalcitrant molecules. Biotechnol. Bioeng. 15:611-647. Bache, C. A., J. W. Serum, W. D. Youngs, and D. J. Lisk. 1972. Polychlorinated biphenyl residues: Accumulation in Cayuga Lake trout with age. Science 177: 1191-1192. Bailey, S., and P. J. Bunyan. 1972. Interpretation of persistence and effects of polychlorinated biphenyls in birds. Nature (London} 236: 34-36. Ballesta, J.-P. G., and M. Alexander. 1971. Resistance of Zygorhynchus species to lysis. J. Bacteriol. 106: 938-945. Bloomfield, B. J., and M. Alexander. 1967. Melanins and resistance of fungi to lysis. J. Bacteriol. 93: 1276-1280. Burger, K., I. C. MacRae, and M. Alexander. 1962. Decomposition of phenoxyalkyl carboxylic acids. Soil ScL Soe. Amer. Proc. 26: 243-246. Carpenter, E. J., and K. L. Smith, Jr. 1972. Plastics on the Sargasso Sea surface. Science 175: 1240-1241. Dias, F. F., and M. Alexander. 1971. Effect of chemical structure on the biodegradability of aliphatic acids and alcohols. AppL Microbiol. 22:1114-1118. Focht, D. D., and M. Alexander. 1970. Bacterial degradation of diphenyhnethanc, a DDT model substrate. Appl. Microbiol. 20:608-611. Hammond, M. W., and M. Alexander. 1972. Effect of chemical structure on microbial degradation of methyl-substituted aliphatic acids. Environ. Sei. Technol. 6: 732-735. Jannasch, H. W. 1967. Growth of marine bacteria at limiting concentrations of organic carbon in seawater. Lirnnol. Oceanogr. 12:264-271. Kaufman, D. D. 1966. Structure o1' pesticides and decomposition by soil microorganisms. In: Pesticides and Their Ef]ect on Soils and Water. Amer. Soc. Agron., Madison, Wisconsin. pp. 85-94. Mercer, E. H. 1961. Keratin and Keratinization. Pergamon Press, New York. Middleton, F. M. 1963. The problem of refractory substances in water and wastes. In: Pollution Control Engineering, A. B. Mindler and T. E. Brenner, editors. Vol. 59, No. 45. Amcr. Inst. Chem. Engineers, New York. pp. 26-33. Panel on Hazardous Trace Substances. 1972. Polychlorinated biphenyls: Environmental impact. Environ. Research 5: 249-362.

5. 6. 7. 8. 9. 10. 11. 12.

13. 14.

15. 16.

17.

Problems Arising From Recalcitran! Molecules 18. 19.

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Pengra, R. M., M. A. Cole, and M. Alexander. 1969. Cell walls and lysis of Mortierella parvispora hyphae. J. Bacteriol. 97:1056-1061. Pfaender, F. K., and M. Alexander. 1972. Extensive microbial degradation of DDT in vitro and DDT metabolism by natural communities. J. Agr. Food Chem. 20: 842-846. Swaby, R. J., and J. N. Ladd. 1966. Stability and origin of soil humus. In: The Use oJ" Isotopes in Soil Organic Matter Studies. R. A. Silow, editor. Pergamon Press, Oxford. pp. 153-157. Swisher, R. D. 1970. Surfaetant Biodegradation. Dekker, New York. Wodzinski, R. S., and M. J. Johnson. 1968. Yields of bacterial cells from hydrocarbons. Appl. Microbiol. 16: 1886-1891.

20.

21. 22.