5 CHAPTER I INTRODUCTION GENERAL PATHWAYS OF MICROBIAL DEGRADATION OF AROMATIC COMPOUNDS A number of microorganisms can perform various types of reactions (Fonken and Johnson, 1972; Beukers et al., 1972) such as oxidation, reduction, hydrolysis, esterification, phosphorylation etc. These enzymatic reactions enable the microorganism to either synthesize or degrade complex organic compounds with ease. Such studies have become important from both fundamental and applied considerations. The microorganisms which are frequently used are fungi or bacteria. A great deal of work has been done on the microbiological transformation of steroids (Charney and Herzog, 1967) and a number of transformations have actually been exploited for commercial applications (Briggs and Brotherton, 1970). To cite an example, cortisone (l) an adrenocorticoid hormone which was effective against rheumatoid arthritis, a grave crippling disease was prepared by a sequence of chemical reaction from bile acids involving 32 steps (Peterson, 1963; Briggs and Brotherton, 1970). The formidable step was the introduction of an oxygen function (Carbonyl group) in the position 11 of the steroidal nucleus. With the combined chemical and microbial methods, the sequence of steps was reduced to only 12. Amongst steroids, the compounds studied are hormones of androstane Biit ocidt and pregnane series, estrogens, bile acids, cardenolides and bufadienolides. Studies have also been carried out on the microbial transformation of terpenes (Ciegler, 1969; Abbott and Cledhill, 1971), carbohydrates (Vezinae et al., 1968) and alkaloids (lizuka and Naito, 1967; Vining, 1969). Besides these compounds the microbial transforma­ tion studies were also reported on antibiotics (Sebek and Perlman, 1971; Sebek, 1974), herbicides (Kaufman and Kearney, 1976) and pesticides (Bollag, 1974) etc. In recent years, there are also some reports on the microbial utilization of naturally occurring lignins (Crawford and Crawford, 1980), tannins and flavonoids (Barz and Hosel, 1975). In the present chapter attempt will be made to describe briefly the bio-degradation of aromatic compounds by microorganisms. However, a more detailed summary on the microbiological transformations of flavonoids which is the main subject of this dissertation is also included in this chapter. DEGRADATION OF AROMATIC CONPOUNDS Studies related to the microbiological transformation of aromatic compounds have grown considerably during the last thirty years. Degradation of aromatic compounds is a fascinating subject of research for several reasons. It provides an excellent example of the part played by the microbes in maintaining the balance of chemical compounds available to living systems. Microbes also degrade the aromatic compounds that are used as hormones, herbicides, pesticides and detergents, thus avoiding serious ecological changes. Interest in the microbial degradation of aromatic compounds centers around: (1) Studies on the metabolic intermediates (Abbott and Cledhill, 1971; Evans, 1969; Dagley, 1971; Chapman, 1972; Sugumaran and Vaidyanathan, 1978) in the degradation of different aromatic substrates; (ii) Enzymatic mechanisms of hydroxylation and ring fission (Hayaishi, 1964; 1968; Gibson, 1968); (ill) Investigations into the control of enzymes talcing part in the metabolism of aromatic compounds (Stanier and Ornston, 1973). Bacteria (Evans, 1969) are quite versatile in the breakdown of aromatic compounds, but several yeasts and fungi can also degrade a limited range of benzenoid structures. Among the eubacteria, representatives of the families Coccaceae, Mycpbacteriaceae, Pseudo- monadaceae, Splrillaceae, Bacteriaceae and Bacillaceae are able to utilize a large number of benzenoid compounds. Some of the yeasts like Oospora, Candida, Debaromyces, Pichia and Saccharomyces can utilize simple phenols as sole carbon source. While higher fungi like Aspergillus, PeniciIlium and Neurospora can attack benzenoid compounds. A variety of soil and wood-rotting fungi degrade the aromatic polymer of plant origin like flavonoids, tannins and lignins, In all these cases of aromatic ring metabolism, molecular oxygen is an obligatory oxidant. DEGRADATION UNDER AEROBIC CONDITIONS Degradation of substituted or polynuclear aromatic confounds to dihydroxyphenolst Microbes must manipulate a large variety of aromatic compounds into either ortho- or para-di hydroxy derivatives (Gibson, 1968), before cleavage can occur to give aliphatic compounds which are energy yielding reactions. The most common intermediates in the microbial degradation of many aromatic compounds are catechol (2), and protocatechuic acid (3) which are ortho-dihydroxyphenols or gentisic acid (4), which is a para-dihydroxyphenol. Microbes must degrade/ transform various kinds of aromatic compounds to the above dihydroxy compounds or their derivatives before ring cleavage occurs. Some typical examples of manipulation of aromatic compounds to dihydroxy- phenol derivatives by microbes are given below. (i) Aromatic acidic compounds; Aromatic acids can either be metabolised directly to catechol (2) or undergo hydroxylations, followed by decarboxylation COOH aOH I COOH OH 2 3 4 to form protocatechuic acid (3) (Fig. 1.1). Thus salicylic acid (5) is metabolised (Katagiri et al., 1965) by a Pseudomonas sp. to PatudomoncLG •OH GP OH COOH \j - COOH /r/eb/'-sej/a. a.eromenta OH OH ! 0H OH COOH COOH OH i> Wo HO COOH COOH COOH_ COOH S S^p—CCCH HO HO COOH OH ! y —— y OH 10 OH <«• 1.1. The degradative pathways of aromatic acids by bacteria (Buswell and Clark, 1976; Channa Heddy et al., 1976; Katagiri et ai., 1965; Reiner, 1971; Reiner and Hegeman, 1971; Ribbons and Evans, 1960; WhaUs et al., 1967). catechol (2) by oxidative decarboxylation* Similarly* benzoic acid (6) is transformed (Heiner, 1971; Seiner and Hegeman, 1971) by •tt-lcaligenes eutrophus to catechol (2) via 3,5-cyclohexadiene-1,2- diol-1-carboxylic acid (7). *hile a Pseudomonaa Sp. (Wheelis, et al 1967) metabolized benzoic acid (6) to protocatechuic acid (3) by sequential hydroxylation. Similarly phthalic acid (8) undergoes hydroxylation (Ribbons and Evans. 1960) by a feeudomonas Sp. to 4,5-dihydroxyphtbalate (9) which is then decarboxylated to protocatechuic acid (3). However, a strain of Bacillus transformed p-hydroxybenzoic acid (10) to gentisic acid (4) by an unusual pathway in which the migration of carboxylic acid group to ortho position due to the hydr0xy8*tioa (NIH shift) seems to have occurred (Buswell and Clark, 1976) (Fig. 1.1). Shile protocatechuic acid (3) is decarboxylated to catechol (2) by Klebisella aerogenee (Channa Reddy et al., 1976). (ii) Polynuclear aromatic compoundst Polynuclear aromatic compounds are first transformed to o-dihydroxy compounds which are further degraded finally to aliphatic compounds via catechol (Pig. 1.2). Thus benzene (11) was metabolised (Gibson, 1968) by a Pseudomonas Sp. to 3»5-cyclohexadien-1,2-cis-diol (12) through a cyclic peroxide intermediate. The intermediate (12) is then transformed to catechol (2) by enzymatic dehydrogenation. In a similar patheay, naphthalene (13) is first transformed (Jerina et al., 1971) by a soil Pseudomonas Sp. to cis-1,2-dihydroxy-1»2-dihydro- naphthalene (14) which on dehydrogenation gave 1,2-dihydrOxynaphthalene (15). The latter compound then undergoes ring cleavage (Davies and Evans, 1964) by the disruption of bond between the angular carbon and JH 0 I c$ M-OH 0=~ U II 13 18 */• + /r ft CH5COC0OH IS Vh %+ 25 Fig. 1.2. The pathways of degradation of benzene and other polycyclic aromatic compounds by soil Paeudomonas sp. (Daries and Evans, 1964; Evans et al., 1965; Gibson et al., 1968; Jerina et al., 1971). carbon-1 of the naphthalene nucleus to yield cis-o-hydroxybenzal- pyruvic acid (16) (anion form). The compound (16) is then cleaved to salicylaldehyde (18) and pyruvic acid (19) via i -hydroxy- r-o- hydroxyphenyl-oC-oxobutyric acid (17). Salicylaldehyde is then oxidatively decarboxylated to catechol (2) which is degraded further (Fig. 1.2). In the same way anthracene (20) and phenanthrene (23J are transformed (Evans et al., 1965) by a Pseudomonas Sp. to 1,2- dihydroxy compounds (22) and (25) via 1,2-di hydro-1,2-di hydroxy compounds (21) and (24) respectively. The 1,2-dihydroxy confounds (22) and (25) are degraded to catechol (2) by a similar pathway as described for the degradation of naphthalene (Fig. 1.2). (iii) Alkoxy aromatic compounds* Alkoxy aromatic compounds are dealkylated to give the parent phenol with the concomitant liberation of alkyl moiety as an aldehyde. Thus 4-methoxybenzoic acid (26) (Henderson, 1957), vanillic acid (28) (Cartwright and Smith, 1967; Henderson, 1961), the herbicide, 2,4-dichlorophenoxyacetic acid (29) (Tiedje and Alexander, 1969; Evans et al., 1971a) and 4-chloro-2-methylphenoxy- acetic acid (31) (Gaunt and Evans, 1971) are transformed to 4-hydroxybenzoic acid (27), protocatechuic acid (3), 2,4-dichloro- phenol (30) and 5-chloro-o-cresol (32) respectively by soil bacteria (Fig. 1.3). The phenols are further transformed to o-dihydroxy compounds which are degraded further to aliphatic compounds. OCH OH DM HCMO OH O-CHCOOM GCHjCOOH Fig. 1.3. The degradative pathways of metabolism of alkoxyaromatic compounds by soil bacteria (Cartwright and Smith, 1967; Evans, 1971; Henderson, 1957; 1961; Tiedje and Alexander, 1969). (iv) Alkyl aromatic compounds: Alkyi aromatic compounds are either hydroxylated to catechol systems or undergo successive oxidation of alkyl group to give substituted benzoic acid which is degraded