Biodegradation of central intermediate compounds produced from ...

Bioprocess Biosyst Eng (2004) 26: 341–345 DOI 10.1007/s00449-004-0364-2

M IN I R E V IE W

O¨zer C¸ınar

Biodegradation of central intermediate compounds produced from biodegradation of aromatic compounds

Received: 6 June 2003 / Accepted: 30 June 2004 / Published online: 6 August 2004 Ó Springer-Verlag 2004

Abstract In this study I consider the incomplete biodegradation of aromatic compounds during the wastewater cycle between aerobic or anaerobic zones in biological nutrient removal processes, including aerobic biodegradation of compounds (such as cyclohex-1-ene1-carboxyl-CoA) produced during the incomplete anaerobic biodegradation of aromatic compounds, and anaerobic biodegradation of compounds (such as catechol, protocatechuate, and gentisic acid) produced during the incomplete aerobic biodegradation of aromatic compounds. Anaerobic degradation of the aerobic central intermediates that result from the incomplete aerobic degradation of aromatic compounds usually leads to benzoyl-CoA. On the other hand, aerobic degradation of the anaerobic central intermediates that result from the incomplete anaerobic degradation of aromatic compounds usually leads to protocatechuate. Keywords Incomplete biodegradation of aromatic compounds Æ Biodegradation of central intermediates Æ Catechol Æ Gentisate Æ Protocatechuate Æ Cyclohex-1-ene-1-carboxyl-CoA

Introduction Developments in science and technology, especially over the last two decades, have increased the amount of aromatic compounds released into the environment. These aromatic compounds are encountered naturally as compounds such as lignin, amino acids, and tannin. Other sources of aromatic compounds include those produced from human activities such as agriculture (insecticides, herbicides, swine waste), domestic sources O¨. C¸ınar Department of Environmental Engineering, _ University, Kahramanmaras¸ Su¨tc¸u¨ Imam Karacasu, Kahramanmaras¸, 46160, Turkey E-mail: [email protected]

(sewage sludge), and industry (solvents, wood preservatives, detergents, oil). The presence of these aromatic compounds in the environment presents some important problems, including: (1) they present a challenge to wastewater treatment plants, and (2) they pollute groundwater and surface water. In order to overcome these adverse effects, some technologies capable of breaking down these compounds have been developed by engineers. One technology, which uses aerobic conditions in which oxygen is the electron acceptor, is simply a metabolism of aromatic compounds by microorganisms. During this aerobic metabolism of aromatic compounds, the microorganisms use molecular oxygen to hydroxylate aromatic compounds and to perform oxidative cleavage of the aromatic ring. During the hydroxylation and cleavage of aromatic compounds, microorganisms produce enzymes called monooxygenases or dioxygenases that transform aromatic compounds into central intermediates such as catechol (1,2-dihydroxybenzene), protocatechuate (3,4-dihydroxybenzoate), and gentisate (2,5-dihydroxybenzoate). These intermediates are then cleaved by dioxygenases [1, 2, 13, 21]. On the other hand, a technology based on anaerobic conditions, in which light or inorganic electron acceptors such as nitrate, sulfate, and carbon dioxide are used, is also used to degrade aromatic compounds. During anaerobic metabolism, microorganisms oxidize aromatic compounds to carbon dioxide mainly via benzoyl-CoA, the most common central intermediate, which is dearomatized by the enzyme called benzoyl-CoA reductase and via other central intermediates such as resorcinol (1,3-dihydroxybenzene) and phloroglucinol (1,3,5-trihydroxybenzene) [6, 14]. Of the electron acceptors used in the anaerobic metabolism of aromatic compounds, nitrate has been paid the most attention because nitrate is naturally produced in wastewater treatment plants using the nitrification process. Another reason that nitrate is commonly used as the electron acceptor in full-scale wastewater treatment plants is that

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sulfate-reducing microorganisms need very strict reducing environments, which are relatively expensive to maintain. Biodegradation pathways of aromatic compounds in both aerobic and anaerobic environments are regulated by enzymes. Understanding how microorganisms alter their enzymatic compositions under different environmental conditions is the key to successful biodegradation and bioremediation of aromatic compounds, which is the main concern of this study. An understanding of the regulation of enzymes, achieved by coarse control (regulation of enzyme synthesis) and fine control (regulation of existing enzyme activity), requires very close cooperation between environmental engineers and microbiologists or biochemists who are interested in the biodegradation of aromatic compounds of environmental interest. When biological wastewater treatment plants include both an anoxic tank (nitrate as an electron acceptor) and an aerobic tank (oxygen as an electron acceptor), such as the modified Ludzack–Ettinger (MLE) process that contains one anoxic reactor and one aerobic reactor in series with mixed liquor recirculation (MLR) from aerobic reactor to anoxic reactor, aromatic compounds may not be completely degraded in the anoxic tank and anoxic biodegradation intermediate products may be passed to the aerobic tank or vice versa due to the insufficient hydraulic retention times and/or some inhibitors in these two tanks. Therefore, the aerobic biodegradation of compounds that are produced during the incomplete anoxic biodegradation of aromatic compounds (such as cyclohex-1-ene-1-carboxyl-CoA), and the anaerobic biodegradation of compounds which are produced during the incomplete aerobic biodegradation of aromatic compounds (such as catechol, protocatechuate, and gentisic acid) are considered in this study.

Anaerobic degradation of aerobic central intermediates of aromatic compounds It is very difficult to provide information on the biodegradation of every aromatic compound in aerobic and anaerobic environments. Therefore, benzoic acid was selected for our investigation, because: (1) it is degraded both aerobically and anaerobically; (2) the aerobic and anaerobic degradation pathways are well understood; (3) the coenzyme A thioester form of benzoate is the Fig. 1 Pathway for the anaerobic degradation of catechol by methanogenic consortia involving Methanospirillum hungatei and Methanothrix sohngenii. Compounds: I=catechol, II=phenol, III=cyclohexanol, and IV=cyclohexanone

most common anaerobic central intermediate of many aromatic compounds such as phenol, p-cresol, and aromatic acids [8, 10, 19, 20, 34]; (4) information from the degradation of benzoic acid allows us to extrapolate to the biodegradation of other aromatic compounds, and; (5) the enzymes that metabolize benzoic acid are inducible [22]. If the aerobic biodegradation of benzoic acid is not completed in the aerobic tank (in other words, if the aromatic ring is not aerobically cleaved), the aerobic peripheral degradation intermediates (including 3-hydroxybenzoate, 4-hydroxybenzoate) and the aerobic central intermediates (like gentisic acid, protocatechuic acid, and catechol) will be passed to the anaerobic tank by MLR. Therefore, these compounds will be subjected to anaerobic degradation in the anaerobic tank. In the following section, the anaerobic degradation of these compounds will be reviewed. Anaerobic degradation of catechol Anaerobic biodegradation of phenolic compounds may follow two different routes: (1) if a compound contains more than one hydroxyl group (catechol, say), the microorganisms will dehydroxylate the compound to phenol and then reduce that to hydroxycyclohexane (see Fig. 1) [33], or (2) the phenolic compounds will be carboxylated to a hydroxy aromatic acid (4-hydroxybenzoate for instance) and then reductive elimination of hydroxyl group(s) will occur on these aromatic acids (see Fig. 2) [11]. The pathway for the anaerobic degradation of catechol by methanogenic consortia involving Methanospirillum hungatei and Methanothrix sohngenii is presented in Fig. 1. Phenol is formed as an intermediate during the anaerobic degradation of catechol, phloroglucinol, and hydroquinone. The phenol is then reduced to cyclohexanol and cyclohexanone that are finally metabolized to carbon dioxide and methane [33]. Although the anaerobic biodegradation of catechol has been recognized for a long time [15], there have not been enough studies of this process. In early investigations, it was found that phenol was the first intermediate during the anaerobic degradation of phenol [27], but, recently, no evidence has been found that phenol is the first intermediate [18, 23, 25]. As seen in Fig. 2, Desulfotomaculum sp. strain cat2 requires bicarbonate (indicating participation of carboxylating reaction), uncombined Co-A, and strictly anaerobic conditions (gentle air stream inactivated the enzyme in 3–4 min irreversibly) to convert the catechol to protocatechuyl-CoA. Conversion of protocatechuyl-CoA to

343 Fig. 2 Anaerobic degradation of catechol by Desulfobacterium sp. strain Cat2. Compounds: I=catechol, II=protocatechuate, III=protocatechuyl-CoA, IV=3-hydroxybenzoyl-CoA, and V=benzoyl-CoA

3-hydroxybenzoyl-CoA by reductive elimination p-hydroxyl group is a very oxygen sensitive step [11]. Acclimation of microorganism plays an important role in the time required in biodegradation of the phenolic compounds. The reason for this might be due to: (1) selection of the microorganisms for the new substrate and increase of these microorganisms in numbers (2) induction or derepression of the necessary enzymes (3) mutation yielding the new genotypes [33]. Acclimation of methanogenic consortia took 32 days to be able to metabolize catechol anaerobically [15]. Anaerobic degradation of protocatechuic acid During the anaerobic degradation of catechol with Desulfotomaculum sp. strain Cat2 grown with catechol and sulfate, protocatechuate was produced as an intermediate from the carboxylation of catechol. During the further anaerobic degradation of protocatechuate, protocatechuate CoA is produced by protocatechuate CoA ligase and then protocatechuate CoA was dehydroxylated to the 3-hydroxybenzoyl-CoA. Finally, 3-hydroxybenzoyl-CoA was again dehydroxylated to the benzoyl-CoA that is the most common central intermediate of anaerobic degradation of aromatic compounds (see Fig. 2). Further anaerobic biodegradation of benzoyl-CoA is followed by very well-established pathways [14]. Anaerobic degradation of gentisic acid Hypothetical fermentative biodegradation pathway of gentisate by strain HQGo1 is presented in Fig. 3 [26]. Gentisate could be converted to salicylate because the elimination of para-hydroxyl group from 3-hydroxybenzoate by fermenting bacteria was demonstrated by Tschech and Schink [29]. Salicylate could then follow either the pathway in which it might be converted to benzoic acid or the pathway in which it might be Fig. 3 Hypothetical fermentative biodegradation pathway of gentisate by strain HQGo1. Compounds: I = gentisate, II = salicylate, III = 2-hydroxycyclohaxane carboxylate

converted to the hydroxycyclohexane carboxyl derivatives [26]. Anaerobic degradation of 3-hydroxybenzoate Although aerobic degradation of 3-hydroxybenzoate has been studied extensively, almost nothing was known about the anaerobic degradation of 3-hydroxybenzoate. Anaerobic degradation of 3-hydroxybenzoate which is the intermediate compound of the anaerobic degradation of catechol by Desulfobacterium sp. strain Cat2 was presented in Fig. 2. In addition to this pathway, the biodegradation of the 3-hydroxybenzoate by nitrate reducing bacteria (strain Asl-3) involves initial step which is activation to 3-hydroxybenzoyl-CoA in an ATP-consuming reaction. The dehydroxylation reaction of 3-hydroxybenzoyl-CoA could not be demonstrated by the nitrate reducing bacteria because of rapid chemical hydrolysis of 3-hydroxybenzoyl-CoA [16]. Moreover, new strains of strictly anaerobic bacteria (fermentative microorganisms) which were enriched with 3-hydroxybenzoate degraded only 3-hydroxybenzoate and benzoate. These new strains of strictly anaerobic bacteria (fermentative microorganisms) convert the 3-hydroxybenzoic acid to benzoate by reductive dehydroxylation (see Fig. 4). Anaerobic degradation of 4-hydroxybenzoate Anaerobic biodegradation pathway of 4-hydroxybenzoic acid is presented in Fig. 5 [7]. The first step in this pathway is the addition of a coenzyme A into a 4-hydroxybenzoate to form 4-hydroxybenzoyl-CoA which is then dehydroxylated into benzoyl-CoA which is the most common anaerobic central intermediate of aromatic compounds.

Aerobic degradation of anaerobic biodegradation intermediate of aromatic compounds Some intermediate compounds during anaerobic degradation of aromatic compounds may pass to an aerobic

344 Fig. 4 Anaerobic degradation pathway of 3-hydroxybenzoate by fermentative microorganisms. Compounds: I = 3-hydroxybenzoate, II = benzoate, III = 2hydroxycyclohexanyl-CoA

Fig. 5 Anaerobic biodegradation pathway of 4-hydroxybenzoic acid. Compounds: I = 4-hydroxybenzoate, II = 4-hydroxybenzoyl-CoA, III = benzoyl-CoA

Fig. 6 Aerobic biodegradation pathway of cyclohexane carboxylate. Compounds: I = cyclohexane carboxylate, II = trans-4-hydroxycyclohexane carboxylate, III = 4-ketocyclohexanecarboxylate, IV = 4-hydroxybenzoate, V = protocatechuate

The similar pathways which are presented in Fig. 6 have been reported by several researchers [5, 17, 24].

Conclusions tank without being completely degraded. Possible aerobic biodegradation pathway of the intermediate compound is discussed below. Aerobic degradation of cyclohexane carboxylate Although cyclohexane carboxylic acid was earlier reported as an intermediate compound during anaerobic degradation of benzoate [9, 12, 31, 32], recently some other compounds have been reported such as cyclohex-1-ene-1-carboxyl-CoA, (III) 6-hydroxycyclohex-1-ene-1-carboxyl-CoA, and 6-hydroxyl-b-oxocyclohexane-1-carboxyl-CoA; however, no studies have been done on aerobic degradation of the intermediate compounds found lately. Therefore, aerobic biodegradation of cyclohexane carboxylic acid is discussed to be able to give some idea during the aerobic degradation of these compounds. Early studies [3, 4] showed that microorganisms convert cyclohexane carboxylate aerobically to the benzoate, but recent studies showed that during aerobic degradation of cyclohexane carboxylate, the 4-hydroxycyclohexane carboxylate was formed as initial product other than benzoate [5, 28]. Further metabolism of 4-hydroxycyclohexane carboxylate has been reported to occur to form the 4-hydroxybenzoate and then protocatechuate (see Fig. 6) [28].

The findings of several researchers presented in this study point to these conclusions: – Anaerobic degradation of the aerobic central intermediates resulting from incomplete aerobic degradation of aromatic compounds usually leads to the benzoyl-CoA that is the most common central intermediate of anaerobic degradation of aromatic compounds. Further anaerobic biodegradation of benzoyl-CoA is followed by very well-established pathways [14]. – Aerobic degradation of the anaerobic central intermediates resulting from incomplete anaerobic degradation of aromatic compounds usually leads to the protocatechuate that is one of central intermediate of aerobic degradation of aromatic compounds. Further aerobic biodegradation of protocatechuate is followed by very well-established pathways [30]. In the light of these findings from the literature, the incomplete biodegradation of aromatic compounds in either aerobic or anaerobic zones in biological nutrient removal processes (i.e., MLE process) is not likely to be a problem because further biodegradation of these compounds resulting from incomplete biodegradation of aromatic compounds merges the central intermediates compounds (e.g., benzoyl-CoA and protocatechuate) which are easily metabolized by microorganisms.

345 Acknowledgement I would like to thank Emeritus Prof. Dr. C.P. Leslie Grady, Jr. for valuable contributions.

References 1. Altenschmidt U, Oswald B, Steiner E, Herrmann H, Fuchs G (1993) New aerobic benzoate oxidation pathway via benzoylcoenzyme and 3-hydroxybenzoyl-coenzyme A in a denitrifying Pseudomonas-sp. J Bacteriol 175:4851–4858 2. Assinder SJ, Williams PA (1990) The TOL plasmids: determinants of the catabolism of toluene and xylenes. Adv Microb Physiol 31:1–69 3. Babior BM, Bloch K (1966) Aromatization of cyclohexane carboxylic acid. J Biol Chem 241:3643–3651 4. Beer CT, Dickens F, Pearson J (1951) The aromatization of hydrogenated derivatives of benzoic acid in animal tissues. Biochem J 48:222–237 5. Blakley ER (1974) The microbial degradation of cyclohexane carboxylic acid: a pathway involving aromatization to form p-hydroxybenzoic acid. Can J Microbiol 20: 1297–1306 6. Boll M, Fuchs G (1995) Benzoyl-coenzyme A reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism: ATP dependence of the reaction, purification and some properties of the enzyme from Thauera aromatica strain K172. Eur J Biochem 234:921–933 7. Brackmann R, Fuchs G (1993) Enzymes of anaerobic metabolism of phenolic compounds. 4-Hydroxybenzoyl-CoA reductase (dehydroxylating) from a denitrifying Pseudomonas species. Eur J Biochem 213:563–571 8. Dangel W, Brackman R, Lack A, Mohamed M, Koch J, Oswald B, Seyfried B, Tschech A, Fuchs G (1991) Differential expression of enzyme activities initiating anoxic metabolism of various aromatic compounds via benzoyl-CoA. Arch Microbiol 155:256–262 9. Dutton PL, Evans WC (1969) The metabolism of aromatic compounds by Rhodopseudomonas palustris: a new, reductive, method of aromatic ring metabolism. Biochem J 113:525–526 10. Geissler JF, Harwood CS, Gibson J (1988) Purification and properties of benzoate-coenzyme A ligase, Rhodopseudomonas palustris enzyme involved in the anaerobic degradation of benzoate. J Bacteriol 170:1709–1714 11. Gorny N, Schink B (1994) Anaerobic degradation of catechol by desulfobacterium sp strain cat2 proceeds via carboxylation to protocatechuate. Appl Environ Microbiol 60:3396–3400 12. Guyer M, Hegeman G (1969) Evidence for a reductive pathway for the anaerobic metabolism of benzoate. J Bacteriol 99:906– 907 13. Harwood CS, Parales RE (1996) The b-ketoadipate pathway and the biology of self-identity. Ann Rev Microbiol 50:553–590 14. Harwood CS, Burchhardt G, Herrmann H, Fuchs G (1999) Anaerobic metabolism of aromatic compounds via benzoylCoA pathway. FEMS Microbiol Rev 22:439–458 15. Healy JB Jr, Young LY (1978) Catechol and phenol degradation by a methanogenic population of bacteria. Appl Environ Microbiol 35:216–218 16. Heising S, Brune A, Schink B (1991) Anaerobic degradation of 3-hydroxybenzoate by a newly isolated nitrate-reducing bacterium. FEMS Microbiol Lett 68:267–272

17. Kaneda T (1974) Enzymatic aromatization of 4-ketocyclohexane carboxylic acid to p-hydroxybenzoic acid. Biochem Biophys Res Commun 58:140–144 18. Kuever J, Kulmer J, Jansen S, Fischer U, Blotevogel KH (1993) Isolation and characterization of a new spore-forming sulfatereducing bacterium growing by complete oxidation of catechol. Arch Microbiol 159:282–288 19. Merkel SM, Eberhand AE, Gibson J, Harwood CS (1989) Coenzyme A thioesters are involved in the anaerobic metabolism of 4-hydoxybenzoate by a Rhodopseudomonas palustris. J Bacteriol 171:1–7 20. Nozawa T, Maruyama Y (1988) Anaerobic metabolism of phthalate and other aromatic compounds by denitrifying bacterium. J Bacteriol 170:5778–5784 21. Powlowski J, Shingler V (1994) Genetics and biochemistry of phenol biodegradation by Pseudomonas sp. CF600. Biodegradation 5:219–236 22. Rudolph JM (1999) Bacterial responses to mixtures of glucose, benzoate and salicylate in continuous culture. PhD Dissertation, Clemson University, Clemson, SC 23. Schnell S, Bak F, Pfenning N (1989) Anaerobic degradation of aniline and dihydroxybenzenes by newly isolated sulfate reducing bacteria and description of Desulfobacterium aniline. Arch Microbiol 152:556–563 24. Smith DI, Callely AG (1975) The microbial degradation of cyclohexane carboxylic acid. J Gen Microbiol 91:210–212 25. Szewzyk R, Pfenning N (1987) Complete oxidation of catechol by the strictly anaerobic sulfate-reducing Desulfobacterium catecholicum sp. nov. Arch Microbiol 147:163–168 26. Szewzyk U, Schink B (1989) Degradation of hydroquinone, gentisate, and benzoate by fermenting bacterium, in pure and defined mixed culture. Arch Microbiol 151:541–545 27. Szewzyk U, Szewzyk R, Schink B (1985) Methanogenic degradation of hydroquinone and catechol via reductive dehydroxylation to phenol. FEMS Microbiol Ecol 31:79–87 28. Taylor DG, Trudgill PW (1978) Metabolism of cyclohexane carboxylic acid by Alcaligenes strain W1. J Bacteriol 134:401– 411 29. Tschech A, Schink B (1986) Fermentative degradation of monohydroxybenzoates by defined syntrophic cocultures. Arch Microbiol 145:396–402 30. Wheelis ML, Palleroni NJ, Stanier RY (1967) The metabolism of aromatic acids by Psedomonas testosteroni and Pseudomonas acidovorans. Arch Microbiol 59:302–314 31. Whittle PJ, Lunt DO, Evans WC (1976) Anaerobic photometabolism of aromatic compounds by Rhodopseudomonas sp. Biochem Soc T 4:490–491 32. Williams RJ, Evans WC (1975) The metabolism of benzoate by Moraxella species through anaerobic nitrate respiration: evidence for reductive pathway. Biochem J 148:1–10 33. Young LY, Rivera MD (1985) Methanogenic degradation of four phenolic compounds. Water Res 19:1325–1332 34. Zeigler K, Braun K, Bockler A, Fuchs G (1987) Studies on the anaerobic degradation of benzoic acid and 2-aminobenzoic acid by a denitrifying Pseudomonas strain. Arch Microbiol 149:62