Metabolism of phenols by Ochromonas danica

Transcription

1 ELSEVIER FEMS Microbiology Letters 133 (1995) Metabolism of phenols by Ochromonas danica Kirk T. Semple *, Ronald B. Cain EnrGronmental Microbiology Laboratory, Department of Biological and Nutritional Sciences, The Uniwrsity, Newcastle upon Tvne. NE1 7RU, UK Received 31 July 1995; revised 18 September 1995; accepted 18 September 1995 Abstract This study investigated the catabolic potential of a eukaryotic alga to degrade one of the most common organic pollutants, phenol. The alga, Ochromonas dunica (993/28), was selected for study after screening for its heterotrophic capabilities. The catabolic versatility of the alga was elucidated by incubating with a variety of phenolic compounds. The alga removed phenol, all the cresol isomers and 3,4-xylenol from its incubation media, with phenol being removed more rapidly than any of its methylated homologues. Consequently, the alga was found to have a greater specificity for phenol than for o- or p-cresols. This study shows that 0. dunica could catabolize phenol and its methylated homologues. Kewords: Ochromonas danica; Phenol: Cresol; Xylenol; Catabolism 1. Introduction Before the beginning of man s industrial activities, concentrations of organic compounds on the surface of the planet remained fairly constant with biosynthesis and biodegradation kept in equilibrium by the activities of animals, plants and microorganisms [ 11. With the escalation of industrial processes, there has been a vast increase in the amount of potentially toxic, carcinogenic and/or mutagenic compounds released into the environment [2]. Natural ecosystems are thus faced with the problem of dealing with compounds which either do not fit naturally into the global cycles of carbon, nitrogen, * Corresponding author. Present address: Institute of Environmental and Biological Science, Environmental Science Division, Lancaster University, Lancaster, LA1 4YQ, UK. Tel: +44 (1524) : fax: t44 (1524) sulphur or other elements, or have deleterious effects on the environment. Environmental pollution has been considered an inevitable side effect of industrial societies. Phenolic pollution is characteristic of manufacturing (pesticides, pharmaceuticals, plastics) and raw materials (crude oil, coal) conversion processes. Phenol, itself, is among the compounds most frequently found in rivers, industrial effluent outfalls and landfill runoff waters in the United States, UK and many other European countries [3]. Biodegradative studies of pollutants have concentrated, almost exclusively, on the role of bacteria and fungi in the degradative processes. Eukaryotic algae remain the poor relations of the environmental microbiologist, in spite of their ubiquitous distribution, their central role in the fixation and turnover of carbon and other nutrient elements and recognition of their heterotrophic abilities. This study aims to elucidate the biodegradative capabilities of Federation of European Microbiological Societies SSDI (95)00363-O

2 254 K. T. Semple, R.B. Cain / FEMS Microbiology Letters I33 (1995) Ochromonas danica, a nutritionally versatile chrysophyte [4,5], in the degradation of phenol and its methylated derivatives. 2. Materials and methods 2.1. Eukaryotic alga Ochromonas danica (CCAP 933/28), a golden brown chrysophyte alga, was selected for study after the heterotropic capabilities of the alga were compared against two green algae, Chlamydomonas ulvaensis (CCAP 11/58) and Scenedesmus brasiliensis (276/1B), on a range of substrates including acetate, glucose, succinate, phenol, o- and p-cresols. 0. danica was found to be the most competent heterotroph [9]. All the algal cultures were obtained from the Culture Collection for Algae and Protozoa at the Institute of Freshwater Ecology, Ambleside, Cumbria. The cultures were purified before any studies were carried out [6] Growth conditions 0. danica was grown in Jaworski s medium [7] and stock cultures were maintained in an illuminated incubator at a light intensity of 60 pmol photons -2 m s- at 25 C. Algal cultures were routinely monitored for bacteria using light and phase contrast microscopy and incubation nutrient agar (Oxoid, Unipath Ltd, Basingstoke, UK) and on minimal salts agar containing 2 mm phenol Harvesting of cells Cultures were harvested in the late exponential phase of growth on phenol (1 mm) by centrifugation at 4000 X g for 5 min at 4 C. The cells were washed twice with cold (0-4 C) 0.2 M sodium phosphate buffer, ph 7.2. The washed cell pellet was resuspended in 5-10 ml of washing buffer to a cell density of 1 X lo8 cells ml-r and the cell suspension was placed on ice. The algal suspensions were tested for purity by filtering them, aseptically, through separate 0.2 pm membranes and then placing the membranes on nutrient agar and on minimal salts agar containing phenol and incubating at 25 C and 37 C for 72 h Turnover studies involving substrate utilization Cell suspensions (20 ml containing approximately 5 X lo6 cells ml- ) in 0.2 M sodium phosphate buffer, ph 7.2, were incubated in a 50 m conical flask at 25 C and shaken at 150 rpm in a water bath. One flask was used per substrate or combination of substrates (phenol, cresols or xylenols) to be tested which had starting concentrations of 250 PM or 500 PM per flask [8]. An aliquot of the working cell suspension was boiled for 15 min and used in control incubations in the turnover studies to eliminate the effect of biomass on the substrate(s) concentration or the possible unspecific binding of the phenols to the biomass. Each experiment was carried out twice, with each test incubation done with three replicates. All the chemicals used in this study were obtained from Fisons Scientific Equipment, Loughborough, UK, and were of analytical reagent (AR) grade Substrate determinations High pressure liquid chromatography (hplc) was used both to resolve and determine phenol concentrations. Culture medium (1 ml) was centrifuged in a microfuge at 12,000 X g for 10 min. The pellet was discarded and 0.5 ml of the clear supematant was mixed with 0.5 ml of acetonitrile and 20 ~1 was injected into a LDC/Milton Roy hplc system (LDC/Milton Roy, Stone, Staffs, UK). Resolution of the phenolics was effected with a Merck-Hibar Lichrosorb (5 pm particle size) RP-18 column (250 X 4mm i.d.). The isocratic solvent system used in the hplc was 60:40 acetonitrile:water. 3. Results 3.1. Turnover studies involving substrate utilization A series of incubations with cell suspensions were used to elucidate the versatility of 0. danica in removing individual phenolic compounds (500 PM) from the growth medium. Fig. 1 shows the disappearance of the phenol, m-, o- and p-cresol isomers being completely removed from the incubation medium. The alga removed phenol more rapidly than any of the cresol isomers. Preferential removal of the cresol isomers was related to the position of the

3 K.T. Semple. R.B. Cain / FEMS Microbiology Letters 133 (1995) methyl groups on the aromatic ring. p-cresol was removed faster than the orrho isomer which, in turn, disappeared faster than m-cresol. Similar specificity for the removal was shown when the alga was incubated with all the xylenol isomers (Fig. 2). The only xylenol isomer to be completely removed from the incubation medium was 3,4-xylenol. The other xylenols were only partially removed by the alga. This ranged from 40% removal of 2,3-xylenol to 20% for the 2,6 isomer. Further investigation into the versatility of the alga s degradative potential was investigated in incubations with mixtures of phenol (250 PM) with o- or p-cresol(250 PM) or 3,4-xylenol(250 PM) (Fig. 3). In every case, the alga preferentially removed the phenol before the secondary substrate (cresol or xylenol). The phenol was removed after 2 h incubation while p-cresol was removed after 2.5 h and both o-cresol and 3,4-xylenol were removed after 3 h. The specificity of the alga for phenol against that of o-cresol and p-cresol was studied (Figs. 4 and 5). The alga was incubated with either o- or p-cresol I 80 -:L _I Fig. 2. Removal of 500 /.LM 2,3- ( - ), 500 /.LM 2,4- (0 ), 500 PM 2,5- (B), 500 FM 2,6- (0). 500 /.LM 3,4- (A) and 500 PM 3,5-xylenols (A ) by washed suspensions of Ochromonas a anica. and the alga was allowed to remove the cresol substrate. After 1.5 h, phenol was introduced into the incubation medium, at which point the phenol was removed rapidly, while the rates of removal of the cresol isomers were reduced until all the phenol had been removed. 4. Discussion Fig. I. Removal of 500 FM phenol ( - 1, 500 PM m-cresol (0). 500 PM o-cresol ( ) and 500 PM p-cresol (0) by washed suspensions of Ochromonas danica. The error bar represents the largest SEM (n = 3) for the data The versatility of 0. &mica to attack methylated analogues of phenol was investigated showing that these molecules became increasingly recalcitrant with increased methylation. The organism was found to possess catabolic apparatus which would only attack the phenolics after it had been induced by phenol or p-cresol [6]. Th is inducible enzymic mechanism has also been found in bacteria [IO], yeasts [ 1 I] and fungi [12]. The initial enzyme in the alga s catabolic pathway was tentatively identified as a phenol hydroxylase (monooxygenase) 161. The position and

4 256 K.T. Semple, R.B. Cain / FEMS Microbiology Letters 133 (1995) Fig. 3. Removal of 250 PM phenol ( - ) when incubated with 250 PM o-cresol (H), 250 FM p-cresol (0) or 250 PM 3,4-xylenol (A > by washed suspensions of Ochromonas danica. The error bar represents the largest SEM (n = 3) for the data Fig. 5. Removal of 250 PM p-cresol (0) with the addition of 250 PM phenol ( *) by washed suspensions of Ochromonas danica Fig. 4. Removal of 250 PM o-cresol ( 1 with the addition of 250 pm phenol ( - ) by washed suspensions of Ochromonas danica. number of the methyl groups on the aromatic ring resulted in a decrease in the alga s ability to oxidize the phenolic substrate (phenol > p-cresol > o-cresol). This was highlighted when one of the cresol isomers was incubated with the alga and then phenol was added showing a decrease in the rate of removal of the cresol isomer until the phenol had be removed. Another factor which influenced the metabolism of phenol and its methylated homologues was the ring cleavage of the aromatic ring. In a previous study, 0. dunica was found to use the meta cleavage pathway to cleave the double bond 161. Once again, the position of the methyl substituents was applicable to the order of activity between the ring cleaving enzyme and the catecholic substrate (catechol > 4- methylcatechol > 3-methylcatechol). To further reinforce this, the alga was found to metabolize the ring cleavage product (2-hydroxymuconic semialdehyde) by the NAD+-dependent dehydrogenase route as opposed to the hydrolase branch of the pathway. In bacteria it has been reported that 3-methylcatechol, formed from m- and o-cresols, is degraded after ring

5 K.T. Semple, R.B. Cain / FEMS Microbiology Letters 133 f 1995) cleavage via the hydrolase route [13]. Whereas catechol and 4-methylcatechol, formed from phenol and p-cresol respectively, are metabolized via the NAD+-dependent dehydrogenase route [ 141. In this algal system, this would explain the reluctance of the alga to oxidize 3-methylcatechol and o-cresol and the slower removal of m- and o-cresols in the turnover studies. This study shows that not only do algae possess the ability to degrade a variety of potential environmental organic pollutants, but that they may have a larger role to play in the environment than has been previously thought. References [ 1] Leisinger, T. (1983) Microbial degradation of environmental pollutants. Experientia 39, [2] Ghisalba, 0. (1983) Microbial degradation of chemical waste, an alternative to physical waste disposal. Experentia 39, [3] Water Research Centre. (1980) An inventory of organic pollutants which have been identified in various surface waters, effluent discharges, aquatic animals and plants, and bottom sediments. In Cost Project 64/B: Analysis of Micropollutants in Water, pp Water Research Centre, Stevenage, Her&. [4] Aaronson, S. (1973) Digestion of phytoflagellates. In Lyso- somes in Biology and Medicine (Dingle, J.T., Ed.), Vol 3, pp North Holland Publishing, Amsterdam. [5] Andersson, A., FaJk, S., Samuelsson, G. and Hagstrom, A. (1989) Nutritional characteristics of a mixotrophic nanoflagellate, Ochromonas sp. Micro. Ecol. 17, 25 I-262. [6] Semple, K.T. and Cain, R.B. (1995) Biodegradation of phenolics by a eukaryotic alga. Submitted to Can. J. Microbial. [7] Beakes, G.W., Canter, H.M. and Jaworski, G.H.M. (1988) Zoospore ultrastructure: Zygorhbidium assulens and Z. planktonicum, two chytrids parasitising the diatom Asteri- onella formosa. Can. J. Bot. 66, [8] Newbould, E.C. (1987) Catabolism of naphthalene sulphonic acids by three strains of bacteria. PhD. Thesis. The University of Newcastle upon Tyne, UK. [9] Semple, K.T. (1994) The biodegradation of phenols by a eukaryotic alga. PhD thesis. The University of Newcastle upon Tyne, UK. [IO] Shingler, V., Franklin, F.C.H., Tsuda, M. Holroyd. D. and Bagdasarian, M. (1989) Molecular analysis of a plasmid-en- coded phenol hydroxylase from Pseudomonas CF600. J. Gen. Microbial. 135, [I I] Kahn, M., Neujahr, H.A., Weissmahr, R.N., Sejlitz, T., Johl, R., Fiechter, A. and Reiser, J. (1992) Phenol hydroxylase from Trichosporon cufaneum: gene cloning, sequence analysis, and functional expression in Escherichia coli. J. Bacteriol. 174, [12] Jones, K.H., Trudgill, P.W. and Hopper. D.J. (1993) Metabolism of p-cresol by the fungus Aspergillus fumigatus. Appl. Environ. Microbial. 59, 1 l25- I 130. [I31 Dagley, S. and Gibson, D.T. (1965) The bacterial degradation of catechol. Biochem. J. 95, [14] Sala-Trepat, J.M. and Evans, W.C. (1971) The metabolism of 2-hydroxymuconic semialdehyde by A,-ofobacter species. Biochem. Biophys. Res. Comm