Biotreatment of naphthalene by PAH-acclimated pure culture with white-rot fungus Phanerochaete chrysosporium

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Pergamon ~I1:

Wal. Sci. Tech. Vol. 34. No. 10. pp. 73-79,1996. CopyrightC> 1996IAWQ. Published by ElsevierScienceLtd PrintedinGreatBritain. All rightsreserved. 0273-1223/96 $15'00 + 0-00

S0273-1223(96)00699-3

BIOTREATMENT OF NAPHTHALENE BY PAH-ACCLIMATED PURE CULTURE WITH WHITE-ROT FUNGUS PHANEROCHAETE CHRYSOSPORIUM Wan-Li Liao and Dyi-HwaTseng Graduate Institute ofEnvironmental Engineering, National Central University, Chungli, Taiwan 32054, Taiwan

ABSTRACT The tolerance of PAH toxicity for a PAH-acclimated pure culture with the fungus P. chrysosporium was evaluated by growth characteristics. The result showed that PAH-acclimated culture could still maintain its microbial activity, whereas the nonacclimated culture showed a declined growth when a high concentration of naphthalene (NTI.) or benzo(a)pyrene (B(a)P) was present. In the treatment of NTI... the PAH-acclimated culture was also superior to the nonacclimated one. More than 90% of NTI.. removal had been attained by the PAH enrichment culture in agitated vessels within 24 h. However. in this experiment. about 20% of NTI.. was adsorbed on the fungal mycelia. After 4 days of reaction in the vessels. some ethyl acetate-extractable metabolites of NTI.. with high polarity in the HPLC elution were detected. One of these polar products was identified to be catechol compound. This study also demonstrated that NTI.. treatment in the aerated batch bioreactor was not comparable to that the agitated vessel owing to low efficiency in oxygen transfer of the system. Copyright @ 1996lAWQ. Published by Elsevier Science Ltd

KEYWORDS Aerated MBR-bioreactor; naphthalene; polycyclic aromatic hydrocarbons; pure culture; white-rot fungus. INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are well-known recalcitrant compounds which recently have been frequently found in the sediments of water systems adjacent to industrial areas. Some of PAHs are mutagenic or carcinogenic to laboratory mammals (Jacob. et al•• 1986; Pahlmann and Pelkonen, 1987) and hence listed as priority pollutants by the U.S. Environmental Protection Agency (Keith and Telliard, 1979). Extensive efforts have been focused on the treatment of waters and wastewaters containing PAH pollutants in the industrialized and developing countries. The use of conventional activated sludge (AS) process to treat PAH compounds is not effective due to their toxicity and resistance to the microbes in AS system. Several investigators had reported that when the AS system was introduced some inducer compounds, the ability of microorganisms could be enhanced to degrade hazardous chemicals (Cardinal and Stenstrom, 1991; Babcook and Stenstrom. 1993). Nevertheless, this alternative for AS system should pay considerable time to determine experimentally available and inexpensive inducer substrates because of the variety of microbia in mixed cultures. In biodegradation or 73

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W.-L. LIAO and D.-R TSENG

detoxification of specific recalcitrants, much efforts have recently been directed toward the use of pure culture systems. Phanerochaete chrysosporium, a white-rot basidiomycete fungus, has been most intensively evaluated in the degradation of persistent organopollutants (Arjrnand and Sandermann, 1985; Eaton, 1985; Bumpus, 1989; Kennedy, et al., 1990; Lin et al., 1991). However, there are still few researchers using the white-rot fungus in a bench-scale bioreactor with atmospheric air supply to investigate the treatment feasibility for PAH-contaminated waters. In this study, the effect of PAH presence on the growth of a PAH-acclimated culture with P. chrysosporium was investigated. The potential of the PAH enrichment culture for N11.. removal was also explored. Experiments were conducted in agitated vessels and an aerated bioreactor with air supply. MATERIALS AND METHODS

Chemicals. Naphthalene was purchased from Merck Co., Ltd. (Germany), fluoranthene was from Tokyo Chemical Industry Co., Ltd. (Japan), and benzo(a)pyrene was from Sigma Chemical Co., Ltd. (U.S.A.). High performance liquid chromatography (HPLC)-grade solvents were purchased from Fisher Scientific Co., Ltd. (U.S.A.). Ethyl acetate was purchased from Merck Co., Ltd. Microorganism and culture conditions. The fungus P. chrysosporium ATCC 24725 was obtained from Culture Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan, R.O.C. The fungus was maintained on potato dextrose agar slants at room temperature and routinely reinoculated into fresh P. D. agar every 21 days. The culture was grown in nitrogen-starvation medium that was prepared according to Kirk et al. (1978). The PAH-acclimated culture was grown in the N-limiting medium and supplied with various PAH compounds, including naphthalene, fluoranthene, and benzo(a)pyrene for 2 months. These PAHs were added every 3 days until each PAH attained an accumulative concentration of 10 mg/l. A 2% (w/v) solution of glucose was supplemented every 15 days to the culture during Incubation. Evaluation of Growth activity in the presence of PAll. The fungus spores, when attained 106-107 cell number/ml, were collected from the incubated slants. The cultures were initiated with 25 ml of N-limiting media containing 10% of spore inoculum in 125 ml Erlenmeyer flasks. The flasks were then incubated at 37°C and on a rotary shaker at 175rpm for 30 days. After 4 days of incubation, naphthalene dissolved in N,N-dimethylformamid was added at a concentration of 10 mg/l (17.5 mg/l was fed with B(a)P) to the cultures except the control culture). Samples were taken at designed intervals, and entire contents in the flasks were centifuged for 15 min to separate the aqueous mixture from the mycelia. The collected mycelia were then dried at J05°C for 6 h to determine the dried weight of cells. Reaction in aeration bioreactor. The aeration cultures were carried out in an MBR-bioreactor with 500ml effective volume. For providing oxygen and suspending the mycelia, atmospheric air was injected to the reactor at an initial flow rate of 200mUmin. When the mycelia were grown to a great extent (around 5 days from spore incunation), the air was supplied to 400 mllmin. The aeration reactor was operated at 37°C throughout this experiment. Figure 1 is the schematic of the aeration batch reaction system. Analysis of removal and degradation ofnaphthalene. After incubation for various periods, the cultures with entire contents were extracted with two volume of ethyl acetate. The extracts were condensed by evaporating the solvent, and dissolved in acetonitrile for HPLC analysis. The analysis was performed with Bio-Rad HPLC System equipped with model 1790 programmable UVNIS monitor set at 254 nm and DATA JET Integrator. The HPLC was fitted with a Phenomenex EnviroSep-ppTM 125 x 4.6 mm column. A linear gradient was used with acetonitrile-water (40 : 60) in the first 2 min following injection, then the mobile phase was shifted to 100% acetonitrile in 25 min at a flow rate of 2.0 mUmin. For determining the adsorption of NTL on the fungus, a deactive mycelium was prepared by autoclaving the culture at 121DC for IS min. The deactive mycelia, after being rinsed with deionic water, were resuspended in flasks containing fresh media. The flasks were added 10 mg/l of NTL and shaken at 175 rpm and 37°C.

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Figure I. Schematic of the aeration batch bioreaction system.

RESULTS AND DISCUSSION

The effect ofPAR presence on the microbial activity of suspended cultures. The microbial activity is closely related to the production of certain specific enzymes which control the rate of biochemical reaction in the biological treatment processes. This experiment was to investigate the microbial activities based on the growths of PAH-acclimated culture and nonacclimated culture during the suspended batch incubation with 10 mgll of NTL addition. and the result is shown in Figure 2. When NTL compound was added to the nonacclimated culture after 4-day incubation. the microbial population (on dried weight basis) decreased obviously for 2 days and then the microbial activity restored; however. the biomass in the culture was significantly less than that of the control for the same time interval. The result indicated that NTL dosage might have reached an inhibitory level to the nonacclimated fungus resulting in declining growth of the culture. This experiment had verified the fact that even if P. chrysosporium has an ability to degrade PAH compounds. the nonacclimated fungus was not able to immediately acclimate the PAH added at a high level.

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On the other hand. the microbial growth in the PAH-acclimated culture was affected to a limited extent. and no apparent lag phase occurred when 10 mgll NTL was added to the culture. In addition, with further exposure to NTL with 20 mgll, the acclimated culture also performed good acclimation to the naphthalene toxicity (data not shown). When exposed to 17.5 mgll ofbenzo(a)pyrene, which is more toxic than NTL to of P. chrysosporium (Liao et al.• 1995). PAH-acclimated culture could still maintains its microbial activity. whereas a declined growth occurred in the nonacclimated culture (Fig. 3). Similar findings were reported by

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W.-L. L1AO and D.-H . TSENG

Cardinal and Stenstrom (1991), that a culture acclimated to a target chemical co uld be continuously exposed to high concentrations of the target compound. It is worthy noting that P. chrysosporium in the PAH acclimated culture exposed with high concentration of NTL, did not shift its growth phases, especially in the stationary phase, when compared with the growth of the control (Fig . 2). According to previous reports, the ligninolytic enzyme system of the P. chrysosporium plays a key role in the degradation of recalcitrant compounds (Bumpus and Aust, 1986; Hammel and Tardone, 1988; Vall i et al., 1990 ). And the se extracellular enzymes were produced at the end of the logarithmic growth and the onset of the stationary phase (Faison and Kirk, 1985) . Therefore, the use of PAH-acclimated culture could decrease the detrimental effects of PAH on the microbial activity of P. chrysosporium. 0.3 adding 17.5 mgll B(a)p 0.25 OIl

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The enhancement of naphthalene degradation by PAH-acclimated culture. The removal of naphthalene from the PAH-acclimated and nonacclimated cultures in agitaed flasks was investigated, and the results are shown in Figure 4. In PAH-acclimated culture, 91.5 % of NTL was rapidly removed within 24 h and, after 5 days of reaction. approximately 99% of NTL was removed from the culture. On the other hand, only 58% of NTL was removed from the nonacclimated culture within 24 h, and 82 .8% of NTL removal was achieved by this culture after 5 days of NTL addition. The ma intenance of microbial activity as well as the capability of ligninolytic enzymes in the PAH -acclimated culture might account for its good performance in NTL removal. To investigate the metabolites of naphthalene after treatment by the PAH-acclimated culture (unle ss otherwise indicated, hereinafter this culture is used in the following text), the mixtures with entire content in the flasks were sampled at 24 h intervals for 5 days . The samples were extracted by ethyl acetate. The extracts from five replicate ves sels were analyzed by HPLC. When contrasted with the elution profile of the

Biotreatment of naphthalene

77

control without NTL fed into the culture, the extracts from every 24 h sample might be found to contain some suspected metabolites of NTL. According to the retention time of elution from the reverse-phase HPLC column, these metabolites were polar compounds. One of these intermediates was identified to be catechol when referred to the elution pofile of NTL standard with a retention time of 2.09 min (Fig. 5). Catechol is known to be one of the last remaining aromatic hydrocarbons of PAHs before benzene ring fission occurs with microorganisms (Cerniglia, 1984; Gibson and Subramanian, 1984). Thus naphthalene, a dicyclic aromatic compound, might have been biodegraded into a mono-ring catechol in this experiment.

suspected NTL metabolites catechol

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According to Cerniglia et al. (1982) and Gibson (1982), the initial step in naphthalene biodegradation is to incorporate a molecular oxygen into the benzenoid nucleus and to form a dihydrodiol subsequently. This study hence had tried to detect the possible naphthalene dihydrodiols including cis- or trans-naphthalene by HPLC but did not succeed. Since the major fraction of biodegraded PAH are polar metabolites (Shiaris, 1989) moreover, some ethyl acetate-extractable and polar metabolites were found in the elution of HPLC in this experiment, thus it could be suggested that naphthalene including its dihydrodiol compounds might be further degraded to other soluble ring-cleavage products by the PAH enrichment culture of this study. However. further analysis using thin-layer chromatography and gas chromatography-mass spectrometry to isolate and identify these metabolites are necessary.

Naphthalene removal by aeartlon batch bioreactor. Figure 6 shows the results of 10 mgll NTL removal from the PAH enrichment culture in an aeration batch bioreactor. The removal ratio obtained from the supernatant into the bioreactor was around 75% of NTL added in the rust 3 days of NTL addition and. after 7 days, more than 95% was removed from the culture. To investigate the removal by adsorption. the deactive mycelia of P. chrysosporium were prepared to adsorb NTL fed with identical concentration in the aerated reactor. In Figure 6, the adsorption of naphthalene onto the cells typically was less than 20% during the experimental period. Because the adsorbed NTL on the fungal mycelia could readily be extracted by

W.-L. UAO and D.-H. TSENG

78

ethyl acetate solvent, it could be suggested that van der Waals' forces or dipole moment is considered to be the dominant mechanism for such mycelium adsorption. On the other hand, when compared with the performance of NTL removal in the agitated vessels (Fig. 2), the treatment of NTL in the aeration batch bioreactor had a relatively low efficiency. To detect dissolved oxygen in these cultures, the average D.O. (at 37¢J) in the supernatant of 4-ilay-old culture from the aerated reactor was 2.83 mgll, whereas the D.O. in the culture from the agitated flask incubated with the same period was 6.05 mgll. Faison and Kirk (1985) had reported that the activity of ligninolytic enzymes of P. chrysosporium affect the degradation of recalcitrant compounds to a great extent, moreover, several researchers indicated that the ligninolytic system is particularly active in the culture grown under high oxygen tension (Kirk et al., 1978; Bar-Lev and Kirk, 1981; Leisola et al., 1983). Therefore, the treatment performance in an aerated bioreactor was not comparable to the shaken flask with shallow culture.

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CONCLUSION The detrimental effect of PAH for given concentrations on the microbial activity of nonacclimated culture with P. chrysosporium has been shown in this study. The use of a PAH-acclimated culture might not only maintain the microbial activity but enhance the treatment efficiency of NTL. The PAH enrichment culture had been shown to degrade NTL to catechol and other suspected metabolites with high polarity by HPLC analysis. However, further experiment on isolation and identification for these NTL metabolites would be necessary. NTL treatment in the aerated batch bioreactor was not comparable to the agitated vessel owing to low oxygen transfer in the system. Therefore. for improving the performance of the MBR-bioreactor. the oxygen transfer efficiency should beincreased including the use of fine bubble diffuser. ACKNOWLEDGEMENT This research was supported by the National Science Council of the Republic of China under contract No. NSC 84 - 2211 • E - 008 - 005. REFERENCES Arjmand, M.. Sandermann, H., Jr. (198S), Mineralization of chloroaniline/lignin conjugates and of free chloroanilines by the white rot fungus Phanerochaete chrysosponum. J. Agric. Food ChenL. 33. I ass-I 060. Babcock, R. W.. Jr .. Stenstrom, M. K. (1993) . Use of inducer compounds in the enricher-reactor process for degradation of 1naphthylamine wastes. Water Environ. Res., 65, 26-33. Bar-Lev. S. S.• Kirk, T. K. (1981) . Effects of molecular oxygen on lignin degradation by Phanerochaete chrysosporium. Biochem: Biophys. Res. Commun ., 99, 373-378.

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Bumpus. J. A.• Aust, S. D. (1986). Biodegradation of environmental pollutants by the white rot fungus Phanerochaete chrysosporium: involvement of the lignin degrading system. BioEssays. 6. 166-170. Bumpus. J. A. (1989). Biodegradation of Polycyclic Aromatic Hydrocarbons by Phanerochaete chrysosporium. Appl. Environ. Microbiol.• 55, 154-158. Cardinal, L. J.• Stenstrom. M. K. (1991). Enhenced biodegradation of polyaromatic hydrocarbons in the activated sludge process. Res. J. Water Pollut. Control Fed., 63. 950. Cerniglia. C. E., Freeman. J. P.• Mitchum, R. K. (1982). Glucuronide and sulfate conjugation in the fungal metabolism of aromatic hydrocarbons. Appl. Environ. Microbiol.• 43.1070-1075. Cerniglia, C. E. (1984). Microbial metabolism of polycyclic aromatic hydrocarbons. Adv. Appl. Microbial .• 30. 31-71. Eaton. D. C. (1985). Mineralization of polychlorinated biphenyls by Phanerochaete chrysosporium: a ligninolytic fungus. Enzyme. Microb. Technol .• 7.194-196. Faison. B. D., Kirk. T. K. (1985). Factors involved in the regulation of a Iigninase activity in Phanerochaete chrysosporium. Appl. Environ. Microbiol., 49. 299-304. Gibson, D. T. (1982). Microbial degradation of hydrocarbons. Environ. Toxicol. Chern.• 5. 237-250. Gibson. D. T .. Subramanian. V. (1984). Microbial degradation of aromatic hydrocarbons. In: Microbial Degradation of Organic Compounds. D. T. Gibson (Ed.), Marcel Dekker. New York. N. Y.• 181. Hammel, K. E.• Tardone, P. J. (1988). The oxidative 4-dechlorination of polychlorinated phenols is catalyzed by extracellular fungal lignin peroxidases, Biochemistry. 27. 6563-6568. Jacob. J., Karcher. W.• Belliando, J. J., Wagstaffe, P. J. (1986). Polycyclic Aromatic Hydrocarbons of Environmental and Occupational Importance. Fresenius Z. Anal. Chern.• 323. 1-10. Keith. L. H., Telhard, W. A. (1979). Priority Pollutants. I. A Perspective View. Environ. Sci. Technol .•n. 416-423. Kennedy. D. W.• Aust, S. D.• Bumpus. J. A. (1990). Comparative biodegradation of alkyl halide insecticides by the white rot fungus. Phanerochaete chrysosporium (BKM-F-1767). Appl. Environ. Microbiol., 56. 2347-2353. Kirk, T. K., Schultz. E.• Connors. W. J.. Lorenz. L. F.. Zeikus, J. G. (1978). Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporium. Arch. Microbiol.. 117.277-285. Leisola, M., Ulmer. D.• Fiechter, A. (1983). Problem of oxygen transfer during degradation of lignin by Phanerochaete chrysosporium. Eur. J. Appl. Microbiol. Biotechnol.• 17, 113-116. Liao, W. L.. Tseng. D. H.• Tsai, Y. C; Chang. S. C. (1995). Microbial removal of polycyclic aromatic hydrocarbons by Phanerochaete chrysosporium. Proceedings of I.A.W.Q. 5th Asian Regional Conference on Water Quality and Pollution Control. 143-152. Lin, J. E., Hickey, R. F., Wang, H. Y. (1991). Use of coimmobilized biological system to degrade toxic organic compounds. Biotechnol. Bioeng .• 38, 273-280. Pahlmann, R.. Pelkonen, O. (1987). Mutagemcity Studies of Different Polycyclic Aromatic Hydrocarbons: The Significance of Enzymatic Factors and Molecular Structures. Carcinogenesis, 8, 773-778. Shiaris, M. P. (1989). Seasonal biotransformation of naphthalene, phenanthrene. and benzo(a)pyrene in surficial estuarine sediments. Appl. Environ. Microbiol.• 55,1391-1399. Valli. K., Wariishi, H., Gold. M. H. (1990). Oxidation of monomethoxylated aromatic compounds by lignin peroxidase: Role of veratryl alcohol in lignin biodegradation. Biochemistry, 29. 8535-8539.