In order to assess the role of yeasts in the natural detoxification process of sediments polluted with biaryl compounds, indigenous yeast species able to degrade biphenyl (BP) were isolated and identified. The degradation ability of 24 strains of the genera Candida spp., Cryptococcus spp., Pichia spp., Rhodotorula spp., Trichosporon spp. and Yarrowia spp. was evaluated by the identification of the BP-metabolites, by HPLC analysis. 4-Hydroxybiphenyl was the main derivative in the Candida krusei, C. tenuis, C. tropicalis, Pichia haplophila, Rhodotorula glutinis, Trichosporon pullulans and Yarrowia lipolytica cultures. 3-Hydroxybiphenyl was detected in minor amounts in the culture supernatant of C. tropicalis, C. krusei strains and R. glutinis. Further hydroxylation led to 3,4-dihydroxy and 2,3-dihydroxybiphenyl; the former in C. tropicalis, C. krusei and R. glutinis cultures, and the latter only in the R. glutinis assays. The cleavage product 4-phenyl-2-pyrone-6-carboxylic acid, was observed in R. glutinis and Y. lipolytica cultures. The degradation ability of the R. glutinis isolates was noteworthy; as four hydrolxylated intermediates and a ring-cleavage product were obtained in both strain cultures. The species studied in this report were dominant in polluted sediments; furthermore, R. glutinis had been mentioned as able to degrade other aromatic hydrocarbons and had high relevance in bioremediation experiments.Instituto de Botánica "Dr. Carlos Spegazzini

Arambarri, Angélica Margarita

Cazau, María Cecilia

Hammer, Elke

Romero, María Cristina

World Journal of Microbiology and Biotechnology

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mm World Journal of Microbiology & Biotechnology 17: 591-594, 2001. 591© 2001 Kluwer Academic Publishers. Printed in the Netherlands.Selection of autochthonous yeast strains able to degrade biphenylM. Cristina Romero1’*, Elke Hammer2, M. Cecilia Cazau1 and Angélica M. Arambarri11 Instituto Spegazzini, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, La Plata, Argentina^Instituí für Mikrobiologie and Molekularbiologie, Ernst-Moritz-Arndt, Universitãt Greifswald, Greifswald, Germany * Author for correspondence: Fax: + 54-221-422-2904, E-mail: anmabarr@museo.fcnym.unlp.edu.arReceived 15 December 2000; accepted 18 July 2001Keywords: Autochthonous yeasts, biphenyl degraders, cleavage product, metabolitesSummaryIn order to assess the role of yeasts in the natural detoxification process of sediments polluted with biaryl compounds, indigenous yeast species able to degrade biphenyl (BP) were isolated and identified. The degradation ability of 24 strains of the genera Candida spp., Cryptococcus spp., Pichia spp., Rhodotorula spp., Trichosporon spp. and Yarrowia spp. was evaluated by the identification of the BP-metabolites, by HPLC analysis. 4-Hydroxybiphenyl was the main derivative in the Candida krusei, C. tenuis, C. tropicalis, Pichia haplophila, Rhodotorula glutinis, Trichosporon pullulans and Yarrowia lipolytica cultures. 3-Hydroxybiphenyl was detected in minor amounts in the culture supernatant of C. tropicalis, C. krusei strains and R. glutinis. Further hydroxylation led to 3,4-dihydroxy and 2,3-dihydroxybiphenyl; the former in C. tropicalis, C. krusei and R. glutinis cultures, and the latter only in the R. glutinis assays. The cleavage product 4-phenyl-2-pyrone-6-carboxylic acid, was observed in R. glutinis and Y. lipolytica cultures. The degradation ability of the R. glutinis isolates was noteworthy; as four hydrolxylated intermediates and a ring-cleavage product were obtained in both strain cultures. The species studied in this report were dominant in polluted sediments; furthermore, R. glutinis had been mentioned as able to degrade other aromatic hydrocarbons and had high relevance in bioremediation experiments.IntroductionBiphenyl (BP) and polychlorinated BPs are widespread environmental pollutants because of their extensive industrial and commercial use. BP has been used as a model compound to study the metabolism of higher chlorinated xenobiotics; but most researches have been focused on bacterial metabolism and its mineralization potential (Mohn et al. 1997). On the other hand, catabolism of aromatics by yeasts was reviewed by Middelhoven (1993), but detailed biochemical studies have been carried out only recently and they pointed to the synthesis of hydroxylated intermediates (Salvo et al. 1990; Oudin et al. 1999).Other studies showed that BP enrichment of cultures enhanced the microbial ability to degrade more toxic contaminants, such as the polychlorobiphenyls, so these microorganisms can be relevant for a practical approach to bioremediation technologies (Chaudhry & Chapala- madugu 1991; Sondossi et al. 1992). Although yeasts are the dominant fungi in soils and sediments polluted with aromatic compounds, the biodegradation capacity of the biarylic structure is still uncertain; it is partially due to the scarcity of data on this subject and to the few tested yeast species (Hammer et al. 1998).The biostatic and biocidal properties of BP and its derivatives adversely affected the biodegradation re­searches. In order to assess the role of yeasts in the natural detoxification process, the objectives of this study were to isolate indigenous yeast species able to degrade BP, and to evaluate this ability by the identi­fication of the intermediate metabolites produced during BP incubation experiments.Materials and methodsIsolation and identification of yeastsYeasts were isolated from heavy polluted sediments collected from an industrial area, near the oil YPF- Refinery, La Plata, Argentina. Decimal dilutions of the samples were spread on a basal medium (BM) contain­ing per litre of distilled water: (NH4)2SO4 1000 mg, K2HPO4 800 mg, KH2PO4 200 mg, MgSO4 • 7H2O 200 mg, CaCl-2H2O 100 mg, FeSO4-7H2O 5 mg, 2% agar, 0.03 pg streptomycin, pH 6.8. The plates were incubated in a closed vessel, and 5 g phenol was put in a small open flask inside the bigger one, to provide it as 592 M.C. Romero et al.carbon source in vapour phase, at 27 ± 1 °C during 20 days.The phenol-tolerant fungi were selected on the basis of their prevailing growth on subsequent platings. Sterile controls with phenol were simultaneously incubated, in triplicate. Yeasts are enumerated by standard spread plate methods on the same medium. Stock cultures of the isolated yeasts were maintained on selective medium at 4 °C and stored in the culture collection at the Instituto Spegazzini (LPSC), Universidad Nacional de La Plata, Argentina.The yeasts were identified by colony appearance, cell morphologies, pseudo-true hyphae, assimilation and physiological differences (Kurtzman & Fell 1998). D- Glucuronate assimilation in liquid medium, growth on n-hexadecane and coenzyme Q-system, were also assesed (Kreisel & Schauer 1987). The results were confirmed by the Yeast identification PC program (Barnett et al. 1996).Growth and incubation conditionsYeasts were precultivated on 40 ml of liquid Sabouraud media, for 48 h at 180 rev/min and 30 °C, for 2 days. For degradation experiments 1 ml of this culture was incubated in 500 ml-shake flasks with 100 ml BM, supplemented with 2% glucose, 1 ml vitamin solution and pH 6.8. After incubation for 3 days at 30 °C and 180 rev/min on a rotary shaker, cells were harvested by centrifugation (5000 x g, 5 min), washed twice with sterile BM and the pellets were resuspended to an optical density of 6 (600 nm). BP (10 mg) was added to 100 ml BM with the cell suspensions and incubated as described above.Additional cultures with 100 ml BM and 1 ml cell suspensions were incubated with 10 mg of 2-hydroxy- biphenyl, 3-hydroxybiphenyl and 4-hydroxybiphenyl, to enrich the yield of the other intermediates. The toxic effects of BP and the derivatives were estimated by the decrease on cells densities of cultures with 2% glucose as growth substrate, by comparing the OD of these cultures grown with and without the pollutants.This methodology was also employed in inhibition experiments, but adding 10 mg BP or 10 mg 4-hydroxy- biphenyl as substrates, and 10 mg 4-aminobenzotriazole as enzymatic inhibitor. Cultures with the inoculum of yeast cells in BM, but without substrate, were used as controls; and all the assays were performed in duplicate.Metabolism of BPFor kinetic studies of BP degradation and isolation of metabolites, at different sampling periods, 2 ml of each flask were centrifuged (5000 x g, 5 min) and 100 /d of the supernatant were analysed. The BP and metabolite contents were estimated by HPLC analysis (Hewlett- Packard, Bad Homburg, Germany), apparatus 1050 M equipped with a quaternary pump system, a diode array detector 1040 M series I, and an HP Chemstation. The separation was achieved with a LiChroCart 125-4 RP-18 end-capped (5 /an) column (Merck, Darmstadt, Germa­ny). The initial solvent composition was 30% CH3OH 70% H3PO4 (0.1%), reaching 100% methanol after 14 min at a flow rate of 1 ml/min.The u.v.-visible absoption spectra of the products were determined in a diode array detector. The mono­hydroxylated metabolites were identified by comparison of analytical data with commercial standards, the di­hydroxylated ones and ring-fission product by other authors data (Lange et al. 1998); by their u.v.-visible spectra during detection with a diode array detector. The BP and 2-hydroxybiphenyl were purchased from Merck; 3-hydroxybiphenyl, 4-hydroxybiphenyl, 4-aminobenzo- triazole and phenol were obtained from Aldrich-Chemie; all the chemicals and solvents were of the highest purity available.ResultsTo verify that culturable indigenous phenol-tolerant yeasts were able to transform BP, isolation on selective agar plates were performed with sediment samples from chronic polluted sites, with 156 mg BP/1. Although 24 phenol-tolerant yeast strains were isolated, only 10 of them were able to degrade BP, and represented the 41.6% of the isolates.Three Candida krusei strains, C. tenuis, C. tropicalis, two Cryptococcus laurentii strains, P. Lichia membran- aefaciens, two P. cactophila strains, two P. anómala strains, P. haplophila, two Rhodotorula mucilaginosa strains, R. aurantiaca, four R. glutinis, R. minuta, two Trichosporon pullulans strains and Yarrowia lipolytica were assayed to confirm their ability to grow on BP as carbon source.BP was not a growth substrate as a decrease in the flask with this pollutant as sole C-substrate was ob­served in some yeast experiments; though BP was metabolized as two C. krusei strains, C. tenuis, C. tropicalis, P. haplophila, two R. glutinis strains, two T. pullulans strains and Y. lipolytica produced BP-meta- bolites (Table 1).The degradation of BP resulted in the formation of 4- hydroxybiphenyl and it was the only product in the C. tenuis, P. haplophila and T. pullulans cultures. In C. tropicalis, C. krusei, C. tenuis and P. haplophila cultures the 4-hydroxybiphenyl was observed at day 4, though in the R. glutinis, T. pullulans and Y. lipolytica experiments it was detected on day 1 of the incubation time.In addition to 4-hydroxybiphenyl, four different degradation products were detected in C. tropicalis, C. krusei strains, Y. lipolytica and R. glutinis assays. A second monohydroxylated compound was found in minor amounts in the culture supernatant of C. tropi­calis, C. krusei strains and R. glutinis, and it was identified as 3-hydroxybiphenyl; 2-hydroxybiphenyl was not detected at all. Further hydroxylation led to 3,4- dihydroxybiphenyl and 2,3-dihydroxybiphenyl, the for-Selection of degrading biphenyl yeasts 593Table 1. Yeast isolates and maximum amounts of each metabolite formed with BP.Yeast species BP metabolites (/ig/1)3-OH-BP 4-OH-BP 3,4-diOH-BP 2,3-diOH-BPC. krusei LPS 589 22.8 155.0 120.0 -C. krusei LPS 590 18.4 98.0 105.0 -C. tenuis LPS 604 - 39.5 - -C. tropicalis LPS 610 18.3 19.5 76.5 -P. haplophila LPS 606 - 45.5 - -R. glutinis LPS 603 29.0 215.3 200.5 98.5R. glutinis LPS 597 21.0 175.5 105.0 45.3T. pullulans LPS 602 - 130.2 - -T. pullulans LPS 608 - 98.5 - -Y. lipolytica LPS 605 - 250.5 125.5 -mer in C. tropicalis, C. krusei strains and both R. glutinis cultures and the second metabolite only in the R. glutinis assays (Figure 1). The 3,4-dihydroxybiphenyl can be derived from 4-hydroxybiphenyl, and 2,3-dihydro- xybiphenyl from 3-hydroxybiphenyl; 3,4-dihydro- xybiphenyl was found in Y. lipolytica. C. tropicalis and C. krusei cultures and both metabolites in R. glutinis strains.The ring cleavage product 4-phenyl-2-pyrone-6-carb- oxylic acid was observed in R. glutinis and Y. lipolytica cultures (Figure 2). It was detected earlier in the R. glutinis assays in respect to the Y. lipolytica ones, after 2 and 4 days incubation time, respectively.To enhance the ability of R. glutinis and Y. lipolytica strains to transform BP derivatives, in a subsequent experiment they were cultivated under similar condi­tions with 4-hydroxybiphenyl. This was transformed to 3,4-dihydroxybiphenyl and 4,4'-dihydroxybiphenyl, and a 10.5-12.0% increase in the formation of the former metabolite was detected in each yeast culture. So, under these conditions an increase in the 4-hydroxybiphenyl degradation rate and a different intermediate were obtained.To assess the toxic effects of BP and the 2-, 3- and 4- hydroxybiphenyl, the OD of the yeast cultures at the stationary phase were controlled in the assays, in 1 week incubation time. BP showed minor effects on growing cells, as a slight decrease of 5-6% (mean values) in the OD of the R. glutinis and Y. lipolytica cultures were observed. However, the hydroxylated products signifi­cantly inhibited the growth of yeasts; the 4-hydroxy- biphenyl was more toxic than the other compounds, as aFigure 1. HPLC elution profile of the metabolites formed from BP by R. glutinis, at the seventh day incubation period (mAU: absorbance units; RCP: ring cleavage product; BP: biphenyl).Figure 2. HPLC spectrum of the RCP formed from BP by R. glutinis (a) library spectrum, (b) 4-phenyl-2-pyrone-6-carboxylic acid spectrum (mAU: absorbance units).42-48% decrease in the cell densities were obtained with this substrate, in respect to 2- and 3-hydroxybiphenyl (mean values: 12-15 and 22-25%, for R. glutinis and Y. lipolytica cultures, respectively).The formation of 4-hydroxybiphenyl as the major metabolite resembled other fungal and mammalian enzyme systems; so, in order to estimate the similarities of these results, enzyme inhibitor assays were per­formed. The 4-aminobenzotriazole was added as enzy­matic inhibitor of the cytochrome P-450 systems, to R. glutinis cultures enriched with BP or 4-hydroxybiphenyl. As a consequence, the formation of 3,4-dihydroxy­biphenyl decreased by 97 and 92% when the enzyme inhibitor was added to each yeast culture.DiscussionScreening of polluted sediments for BP degrading yeasts revealed that a significant diversity of yeast strains were able to hydroxylate BP; in fact, C. krusei, C. tenuis, C. tropicalis, P. haplophila, R. glutinis, T. pullulans and 594 M.C. Romero et al.Y. lipolytica were confirmed as BP degraders. Indeed, for C. krusei, C. tenuis, P. haplophila, T. pullulans and R. glutinis it is the first mention. The data available concerning this ability are controversial; Smith et al. (1980) suggested that there was no BP transformation in C. tropicalis cultures, however, this species was charac­terized as degraders by Wiseman et al. (1975). Further­more, oxidation of BP by C. lipolytica was also shown by Cerniglia & Crow (1981).The 4-hydroxybiphenyl was the predominant meta­bolite, as also observed by other researchers (Abramowicz 1990; Mobley 1994). While three different hydroxylated metabolites and a ring-cleavage product were detected in our R. glutinis assays, only two of them were found in Debaryomyces vanrijiae (Lange et al. 1998). Moreover, the same fission product mentioned for D. vanrijiae was observed in the R. glutinis and Y. lipolytica assays.Fungal aromatic hydroxylation is of interest because it mimics the cytochrome P-450-mediated metabolism of xenobiotics in mammals (Parkinson & Safe 1982). A BP- transforming cytochrome P-450 had been suggested for C. tropicalis (Wiseman et al. 1975) and D. vanrijiae (Lange et al. 1998); therefore, our results on the enzymatic inhibition of cytochrome-P-450 bring out the fungal BP catabolism on the subject.The ability of the R. glutinis isolates to degrade BP was noteworthy; in as much as four hydrolxylated interme­diates and a ring-cleavage product were obtained, at shorter incubation period in respect to the other yeasts. It must be outstanding, that the species studied in this report were dominant in the fungal population from sediments polluted with aromatic compounds. Further­more, R. glutinis may be of high relevance for a practical approach in environmental biotechnology, due to this species being widely distributed among soil fungi.AcknowledgementsThis research was supported by grants from the Na­tional Council of Scientific and Technological Research (CONICET), and from the National University of La Plata (UNLP), Argentina. We thank Lie. Ana. M. Bucsinszky for providing the cultures maintained under selective conditions. This research was partially realized in the Institut für Mikrobiologie and Molekularbiologie, Ernst-Moritz-Arndt-Universitât Greifswald, Greifs­wald, Germany.ReferencesAbramowicz, D.A. 1990 Aerobic and anaerobic biodegradation of PCBs: a review. CRC Critical Review of Biotechnology 10, 241- 251.Barnett, J.A., Payne, R.W. & Yarrow, D. 1996 Yeast Identification PC program, version 4 - United Kingdon. ISBN 0 9513148 82.Cerniglia, C.E., Crow, S.A. 1981. Metabolism of aromatic hydrocar­bons by yeasts. Archives of Microbiology 129, 9-13.Chaudhry, G.R. & Chapalamadugu, S. 1991 Biodegradation of halogenated organic compounds. Microbiological Review 55, 59- 79.Hammer, E., Krowas, D., Schâfer, A., Specht, M., Francke, W. & Schauer, F. 1998 Isolation and characterization of a dibenzofuran degrading yeast: Identification of oxidation and ring cleavage products. 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Applied and Environmental Microbiology 58, 485-495.Wiseman, A., Gondal, J.A. & Sims, P. 1975 4'-Hydroxylation of biphenyl by yeasts containing cytochrome P-450; radiation and thermal stability, comparison with liver enzyme (oxidized and reduced forms). Biochemistry Society Transactions 3, 278- 281.

Selection of autochthonous yeast strains able to degrade biphenyl

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