- Email: [email protected]
Effect of synthetic and natural culture media on laccase production by white rot fungi
Bioresource Technology 82 (2002) 215–217
Effect of synthetic and natural culture media on laccase production by white rot fungi Sibel S. Kahraman a
a,*
, Ismail H. Gurdal
b
Department of Science, Faculty of Education, Inonu University, 44069 Malatya, Turkey Department of Biology, Institute of Science, Inonu University, 44069 Malatya, Turkey
b
Received 13 August 2001; received in revised form 10 October 2001; accepted 15 October 2001
Abstract Laccase is among the major enzymes of white rot fungi involved in lignocellulose degradation. The present paper reports its production by two white rot fungi (Coriolus versicolor, Funalia trogii) under different nutritional conditions. Various synthetic culture media and natural culture medium (molasses wastewater) were tested. Enzyme production in various synthetic culture media, molasses wastewater (vinasse) culture medium and in the absence or presence of cotton stalk supplements showed that vinasse culture medium was a better laccase-inducer medium than the synthetic culture medium. Addition of cotton stalk to various media enhanced the enzyme production. The highest laccase activity was obtained in vinasse culture medium with cotton stalk. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Cotton stalk; Laccase; Natural culture media; Synthetic culture media; Vinasse; White rot fungi
1. Introduction Laccase (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) is one of the enzymes playing a role in fungal ligninolytic systems. Most fungi, especially white rot fungi, produce high amounts of extracellular laccase. Recent studies indicated that laccase plays an important role in degrading lignin in wood pulp, and could help in decolorizing and detoxifying the vast amount of effluents generated by the pulp and paper industry (Lee et al., 1999). Production of laccase is affected by many typical fermentation factors such as medium composition, carbon and nitrogen ratio, pH, temperature, aeration rate, etc. Moreover, many aromatic compounds have been widely used to stimulate production of laccase (Arora and Gill, 2001; Mansur et al., 1997; Ardon et al., 1996). Laccase activity in fungal cultures can be increased by the addition of different aromatic compounds. Many researchers have been interested in improving the laccase production of white rot fungi (Yesilada et al., 1991; Ardon et al., 1996; Feijoo et al., 1999; Kahraman and Yesilada, 1999; Kaluskar et al., 1999). Platt et al. (1984) reported increased laccase production by Pleurotus ostreatus after adding various phenolic *
Corresponding author. E-mail address: [email protected] (S.S. Kahraman).
substrates to the fungal cultures. Addition of sugarcane bagasse enhanced laccase production in some white rot fungi (Arora and Gill, 2001). Cotton stalk stimulated laccase production of white rot fungi (Ardon et al., 1996; Sık and Unyayar, 1998; Kahraman and Yesilada, 1999). Cotton stalks are available in vast quantities in all cotton-growing countries such as Turkey and generate the largest proportion of local agricultural waste. It has been a topic of concern to find environmentally sound and economically feasible compounds that stimulate laccase production. The use of inexpensive sources (industrial and agricultural wastes) for laccase enzyme production can bring its implementation closer. In this study, the molasses wastewater (vinasse) was used as natural culture medium. Molasses wastewater is the by-product from the distillation of fermented sugar. For every litre of ethanol produced 10–15 l of vinasse are generated (Kumar et al., 1997). The environmental impact of vinasse is very high due to its organic matter content and dark color (Fahy et al., 1997; Yesilada, 1999). The aim of this study was to increase the laccase activity of white rot fungi (Coriolus versicolor and Funlia trogii) in different synthetic culture media and a natural growth medium (with and without cotton stalk). In this study, the production of laccase was studied in these fungi by applying different content of the stock basal medium (SBM) as the synthetic culture media. The stock
0960-8524/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 1 ) 0 0 1 9 3 - 6
216
S.S. Kahraman, I.H. Gurdal / Bioresource Technology 82 (2002) 215–217
Filtered and sterilized (120 °C, 12 min) vinasse was used as natural medium (medium 5) after diluting 20:100. The compositions of synthetic media are shown in Table 1. All of the synthetic culture media and vinasse medium (natural medium or medium 5) were grown in 250 ml flasks containing 30 ml of medium. One gram cotton stalk/30 ml medium was added to these media. Cotton stalk was cut into small chips of about 2–3 cm. All of these media were inoculated with homogenized stock cultures (2 ml) and incubated statically.
basal medium is usually adopted for lignin biodegradation and lignolytic enzyme production studies.
2. Methods 2.1. Organism and culture conditions C. versicolor ATCC 200801 and F. trogii ATCC 200800 were maintained at 4 °C, after subculturing at 30 °C every 2–3 weeks on Sabouraud dextrose agar (SDA). For inoculum preparation fungi were cultured at 30 °C on slant SDA. After 1 week, conidial suspensions were prepared and used for the cultivation of inoculum. Ten ml of each suspension was transferred into a 250 ml flask with 100 ml Sabouraud dextrose broth (SDB). After a 4day incubation (30 °C) the cultures were homogenized (Kinetic Polytron homogenizer) and used as inocula (2 ml per culture).
2.2. Laccase assay Laccase activity was determined by following the oxidation of syringaldazine at 525 nm. The reaction mixture contained 850 ll of 0.1 M sodium citrate (pH 5), 100 ll syringaldazine (0.1 mM) and 50 ll of the culture filtrate. One unit of enzyme activity (U) was defined as the amount of enzyme that caused an increase of 1.0 in the absorbance per minute under the assay condition (Yesilada and Sam, 1998). Data are means of three replicates.
Table 1 The compositions of synthetic media Medium 1 Medium 2 Medium 3 Medium 4 Glucose (g) NH4 H2 PO4 (g) MgSO4 7H2 O (g) CaCl2 (g) Yeast extract (g) Distilled water (ml)
10 1 0.05 0.01 0.025 1000
10 – – – – 1000
– 1 – – – 1000
– – 0.05 0.01 – 1000
3. Results and discussion C. versicolor and F. trogii cultures in synthetic and natural culture media with and without cotton stalk
Table 2 Laccase production by C. versicolor in different (synthetic, medium 1–4) media, a natural medium (medium 5) with and without cotton stalk Laccase activity (U/ml) Days
C. versicolor Medium 1
4 8 12 16 20
Medium 2
Medium 3
Medium 4
Medium 5
a
b
a
b
a
b
a
b
a
b
0.002 0.007 0.008 0.011 0.012
0.255 0.497 0.333 0.283 0.281
0.049 0.009 0.057 0.056 0.049
0.131 0.145 0.250 0.275 0.239
0.004 0.009 0.016 0.007 0.005
0.305 0.529 0.336 0.294 0.234
0.018 0.033 0.012 0.010 0.010
0.340 0.335 0.150 0.124 0.127
0.005 0.283 0.701 0.946 0.730
0.651 1.002 0.814 4.406 3.806
a – without cotton stalk; b – with cotton stalk; for media see Section 2, medium 5 wastewater medium. Table 3 Laccase production by F. trogii in different (synthetic, medium 1–4) media, a natural medium with and without cotton stalk Laccase activity (U/ml) Days
F. trogii Medium 1
4 8 12 16 20
Medium 2
Medium 3
Medium 4
Medium 5
a
b
a
b
a
b
a
b
a
b
0.006 0.037 0.061 0.054 0.051
0.292 0.347 0.503 0.502 0.504
0.010 0.013 0.025 0.038 0.037
0.059 0.423 0.464 0.496 0.500
0.008 0.005 0.012 0.018 0.051
0.086 0.386 0.401 0.273 0.231
0.014 0.043 0.059 0.077 0.063
0.072 0.620 0.817 0.843 0.917
0.352 0.911 0.868 4.550 4.550
1.159 1.029 3.605 4.880 4.550
a – without cotton stalk; b – with cotton stalk; for media see Section 2, medium 5 wastewater medium.
S.S. Kahraman, I.H. Gurdal / Bioresource Technology 82 (2002) 215–217
were able to produce high amounts of laccase. Laccase activities were detected in 4–8–12–16–20-day old cultures. Addition of cotton stalk to different culture media enhanced the laccase production to variable levels ranging from about 3–100-fold in C. versicolor and F. trogii. Enzyme production in various synthetic culture medium, vinasse culture medium (natural medium) and in the presence of cotton stalk supplements showed that vinasse culture medium was a better laccase inducer medium than the synthetic culture media. The highest laccase activity (4,406 U/ml) of C. versicolor was obtained in medium 5 + cotton stalk. The highest laccase activity (4,880 U/ml) of F. trogii was obtained in medium 5 + cotton stalk. Laccase activity in cultures without cotton stalk remained low (Tables 2 and 3). Obviously, high amounts of laccase were produced by C. versicolor and F. trogii mainly in their secondary metobolism phases. The maximum laccase activities were reached within 12–16 days cultures. It is known that in the presence of inducers many white rot fungi can excrete high amounts of laccase into the medium. Cotton stalk extract stimulated laccase production of white rot fungi in synthetic medium (Ardon et al., 1996). This study shows that the wastewater (vinasse culture medium) and addition of cotton stalk significantly enhances the laccase activity of C. versicolor and F. trogii. So, it would be possible to use wastewater with cotton stalk in order to increase commercial laccase production. These two substrates (cotton stalk and vinasse) are safe and cheap, and could be suggested for prospective application in the production of important lignocellulolytic and other biotechnological enzymes. Also, it will be financially very attractive to use them for the production of enzymes for biotechnological use.
Acknowledgements This research was supported by Turkish Republic Prime Ministry State Planning Organization (Project No. 97K121470).
217
References Ardon, O., Kerem, Z., Hadar, Y., 1996. Enhancement of laccase activity in liquid cultures of the ligninolytic fungus Pleurotus ostreatus by cotton stalk extract. J. Biotechnol. 51, 201–207. Arora, D.S., Gill, P.K., 2001. Effects of various media and supplements on laccase production by some white rot fungi. Biores. Technol. 77, 89–91. Fahy, V., Fitz Gibbon, F.J., McMullan, G., Singh, D., Marchant, R., 1997. Decolorization of molasses spent wash by Phanerochaete chrysosporium. Biotechnol. Lett. 19 (1), 97–99. Feijoo, G., Moreira, M.T., Roca, E., Lema, J.M., 1999. Use of cheese whey as a substrate to produce manganase peroxidase by bjerkandera sp BOS 55. J. Ind. Microbiol. Biotechnol. 23, 86– 90. Kahraman, S., Yesilada, O., 1999. Effect of industrial and agricultural wastes on laccase activity of white rot fungi. J. Biotechnol. 23 (2), 139–144. Kaluskar, M.V., Kapandis, P.B., Jasper, C.H., Penninckx, J.M., 1999. Production of laccase by immobilized cells of Agaricus sp. Appl. Biochem. Biotechnol. 76, 161–170. Kumar, V., Wati, L., Fitz Gibbon, F., Nigam, P., Banat, I.M., Singh, D., Marchant, R., 1997. Bioremediation and decolorization of anaerobically digested distillery spent wash. Biotechnol. Lett. 19, 311–313. Lee, Y.I., Jung, K.H., Lee, C.H., Park, Y.H., 1999. Enhanced production of laccase in Trametes versicolor by the addition of ethanol. Biotechnol. Lett. 21, 965–968. Mansur, M., Suarez, T., Fernandez-Larrea, J.B., Brizuela, M.A., Gonzales, A.E., 1997. Identification of a laccase gene family in the new lignin-degrading basidiomycete CECT 20197. Appl. Environ. Microbiol. 63, 2637–2646. Platt, M.W., Hadar, Y., Chet, I., 1984. Fungal activities involved in lignocellulose degradation by Pleurotus. Appl. Microbiol. Biotechnol. 20, 150–154. Sık, S., Unyayar, A., 1998. Phanerochaete chrysosporium and Funalia trogii for the degradation of cotton stalk and their laccase, peroxidase, ligninase and cellulase enzyme activities under semisolid state conditions. Turk. J. Biol. 22, 287–298. Yesilada, O., Unyayar, A., Fiskin, K., 1991. Determination of the laccase and peroxidase enzyme activities of Coriolus versicolor in the vinasse medium. Tr. J. Biol. 15, 152–157. Yesilada, O., Sam, M., 1998. Toxic effects of biodegraded and detoxified olive oil mill wastewater on the growth of Pseudomonas aeruginosa. Toxicol. Environ. Chem. 65, 87–94. Yesilada, E., 1999. Genotoxic activity of vinasse and its effect on fecundity and longevity of Drosophila melanogaster. Bull. Environ. Contam. Toxicol. 63, 553–559.
Effect of synthetic and natural culture media on laccase production by white rot fungi Sibel S. Kahraman a
a,*
, Ismail H. Gurdal
b
Department of Science, Faculty of Education, Inonu University, 44069 Malatya, Turkey Department of Biology, Institute of Science, Inonu University, 44069 Malatya, Turkey
b
Received 13 August 2001; received in revised form 10 October 2001; accepted 15 October 2001
Abstract Laccase is among the major enzymes of white rot fungi involved in lignocellulose degradation. The present paper reports its production by two white rot fungi (Coriolus versicolor, Funalia trogii) under different nutritional conditions. Various synthetic culture media and natural culture medium (molasses wastewater) were tested. Enzyme production in various synthetic culture media, molasses wastewater (vinasse) culture medium and in the absence or presence of cotton stalk supplements showed that vinasse culture medium was a better laccase-inducer medium than the synthetic culture medium. Addition of cotton stalk to various media enhanced the enzyme production. The highest laccase activity was obtained in vinasse culture medium with cotton stalk. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Cotton stalk; Laccase; Natural culture media; Synthetic culture media; Vinasse; White rot fungi
1. Introduction Laccase (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) is one of the enzymes playing a role in fungal ligninolytic systems. Most fungi, especially white rot fungi, produce high amounts of extracellular laccase. Recent studies indicated that laccase plays an important role in degrading lignin in wood pulp, and could help in decolorizing and detoxifying the vast amount of effluents generated by the pulp and paper industry (Lee et al., 1999). Production of laccase is affected by many typical fermentation factors such as medium composition, carbon and nitrogen ratio, pH, temperature, aeration rate, etc. Moreover, many aromatic compounds have been widely used to stimulate production of laccase (Arora and Gill, 2001; Mansur et al., 1997; Ardon et al., 1996). Laccase activity in fungal cultures can be increased by the addition of different aromatic compounds. Many researchers have been interested in improving the laccase production of white rot fungi (Yesilada et al., 1991; Ardon et al., 1996; Feijoo et al., 1999; Kahraman and Yesilada, 1999; Kaluskar et al., 1999). Platt et al. (1984) reported increased laccase production by Pleurotus ostreatus after adding various phenolic *
Corresponding author. E-mail address: [email protected] (S.S. Kahraman).
substrates to the fungal cultures. Addition of sugarcane bagasse enhanced laccase production in some white rot fungi (Arora and Gill, 2001). Cotton stalk stimulated laccase production of white rot fungi (Ardon et al., 1996; Sık and Unyayar, 1998; Kahraman and Yesilada, 1999). Cotton stalks are available in vast quantities in all cotton-growing countries such as Turkey and generate the largest proportion of local agricultural waste. It has been a topic of concern to find environmentally sound and economically feasible compounds that stimulate laccase production. The use of inexpensive sources (industrial and agricultural wastes) for laccase enzyme production can bring its implementation closer. In this study, the molasses wastewater (vinasse) was used as natural culture medium. Molasses wastewater is the by-product from the distillation of fermented sugar. For every litre of ethanol produced 10–15 l of vinasse are generated (Kumar et al., 1997). The environmental impact of vinasse is very high due to its organic matter content and dark color (Fahy et al., 1997; Yesilada, 1999). The aim of this study was to increase the laccase activity of white rot fungi (Coriolus versicolor and Funlia trogii) in different synthetic culture media and a natural growth medium (with and without cotton stalk). In this study, the production of laccase was studied in these fungi by applying different content of the stock basal medium (SBM) as the synthetic culture media. The stock
0960-8524/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 1 ) 0 0 1 9 3 - 6
216
S.S. Kahraman, I.H. Gurdal / Bioresource Technology 82 (2002) 215–217
Filtered and sterilized (120 °C, 12 min) vinasse was used as natural medium (medium 5) after diluting 20:100. The compositions of synthetic media are shown in Table 1. All of the synthetic culture media and vinasse medium (natural medium or medium 5) were grown in 250 ml flasks containing 30 ml of medium. One gram cotton stalk/30 ml medium was added to these media. Cotton stalk was cut into small chips of about 2–3 cm. All of these media were inoculated with homogenized stock cultures (2 ml) and incubated statically.
basal medium is usually adopted for lignin biodegradation and lignolytic enzyme production studies.
2. Methods 2.1. Organism and culture conditions C. versicolor ATCC 200801 and F. trogii ATCC 200800 were maintained at 4 °C, after subculturing at 30 °C every 2–3 weeks on Sabouraud dextrose agar (SDA). For inoculum preparation fungi were cultured at 30 °C on slant SDA. After 1 week, conidial suspensions were prepared and used for the cultivation of inoculum. Ten ml of each suspension was transferred into a 250 ml flask with 100 ml Sabouraud dextrose broth (SDB). After a 4day incubation (30 °C) the cultures were homogenized (Kinetic Polytron homogenizer) and used as inocula (2 ml per culture).
2.2. Laccase assay Laccase activity was determined by following the oxidation of syringaldazine at 525 nm. The reaction mixture contained 850 ll of 0.1 M sodium citrate (pH 5), 100 ll syringaldazine (0.1 mM) and 50 ll of the culture filtrate. One unit of enzyme activity (U) was defined as the amount of enzyme that caused an increase of 1.0 in the absorbance per minute under the assay condition (Yesilada and Sam, 1998). Data are means of three replicates.
Table 1 The compositions of synthetic media Medium 1 Medium 2 Medium 3 Medium 4 Glucose (g) NH4 H2 PO4 (g) MgSO4 7H2 O (g) CaCl2 (g) Yeast extract (g) Distilled water (ml)
10 1 0.05 0.01 0.025 1000
10 – – – – 1000
– 1 – – – 1000
– – 0.05 0.01 – 1000
3. Results and discussion C. versicolor and F. trogii cultures in synthetic and natural culture media with and without cotton stalk
Table 2 Laccase production by C. versicolor in different (synthetic, medium 1–4) media, a natural medium (medium 5) with and without cotton stalk Laccase activity (U/ml) Days
C. versicolor Medium 1
4 8 12 16 20
Medium 2
Medium 3
Medium 4
Medium 5
a
b
a
b
a
b
a
b
a
b
0.002 0.007 0.008 0.011 0.012
0.255 0.497 0.333 0.283 0.281
0.049 0.009 0.057 0.056 0.049
0.131 0.145 0.250 0.275 0.239
0.004 0.009 0.016 0.007 0.005
0.305 0.529 0.336 0.294 0.234
0.018 0.033 0.012 0.010 0.010
0.340 0.335 0.150 0.124 0.127
0.005 0.283 0.701 0.946 0.730
0.651 1.002 0.814 4.406 3.806
a – without cotton stalk; b – with cotton stalk; for media see Section 2, medium 5 wastewater medium. Table 3 Laccase production by F. trogii in different (synthetic, medium 1–4) media, a natural medium with and without cotton stalk Laccase activity (U/ml) Days
F. trogii Medium 1
4 8 12 16 20
Medium 2
Medium 3
Medium 4
Medium 5
a
b
a
b
a
b
a
b
a
b
0.006 0.037 0.061 0.054 0.051
0.292 0.347 0.503 0.502 0.504
0.010 0.013 0.025 0.038 0.037
0.059 0.423 0.464 0.496 0.500
0.008 0.005 0.012 0.018 0.051
0.086 0.386 0.401 0.273 0.231
0.014 0.043 0.059 0.077 0.063
0.072 0.620 0.817 0.843 0.917
0.352 0.911 0.868 4.550 4.550
1.159 1.029 3.605 4.880 4.550
a – without cotton stalk; b – with cotton stalk; for media see Section 2, medium 5 wastewater medium.
S.S. Kahraman, I.H. Gurdal / Bioresource Technology 82 (2002) 215–217
were able to produce high amounts of laccase. Laccase activities were detected in 4–8–12–16–20-day old cultures. Addition of cotton stalk to different culture media enhanced the laccase production to variable levels ranging from about 3–100-fold in C. versicolor and F. trogii. Enzyme production in various synthetic culture medium, vinasse culture medium (natural medium) and in the presence of cotton stalk supplements showed that vinasse culture medium was a better laccase inducer medium than the synthetic culture media. The highest laccase activity (4,406 U/ml) of C. versicolor was obtained in medium 5 + cotton stalk. The highest laccase activity (4,880 U/ml) of F. trogii was obtained in medium 5 + cotton stalk. Laccase activity in cultures without cotton stalk remained low (Tables 2 and 3). Obviously, high amounts of laccase were produced by C. versicolor and F. trogii mainly in their secondary metobolism phases. The maximum laccase activities were reached within 12–16 days cultures. It is known that in the presence of inducers many white rot fungi can excrete high amounts of laccase into the medium. Cotton stalk extract stimulated laccase production of white rot fungi in synthetic medium (Ardon et al., 1996). This study shows that the wastewater (vinasse culture medium) and addition of cotton stalk significantly enhances the laccase activity of C. versicolor and F. trogii. So, it would be possible to use wastewater with cotton stalk in order to increase commercial laccase production. These two substrates (cotton stalk and vinasse) are safe and cheap, and could be suggested for prospective application in the production of important lignocellulolytic and other biotechnological enzymes. Also, it will be financially very attractive to use them for the production of enzymes for biotechnological use.
Acknowledgements This research was supported by Turkish Republic Prime Ministry State Planning Organization (Project No. 97K121470).
217
References Ardon, O., Kerem, Z., Hadar, Y., 1996. Enhancement of laccase activity in liquid cultures of the ligninolytic fungus Pleurotus ostreatus by cotton stalk extract. J. Biotechnol. 51, 201–207. Arora, D.S., Gill, P.K., 2001. Effects of various media and supplements on laccase production by some white rot fungi. Biores. Technol. 77, 89–91. Fahy, V., Fitz Gibbon, F.J., McMullan, G., Singh, D., Marchant, R., 1997. Decolorization of molasses spent wash by Phanerochaete chrysosporium. Biotechnol. Lett. 19 (1), 97–99. Feijoo, G., Moreira, M.T., Roca, E., Lema, J.M., 1999. Use of cheese whey as a substrate to produce manganase peroxidase by bjerkandera sp BOS 55. J. Ind. Microbiol. Biotechnol. 23, 86– 90. Kahraman, S., Yesilada, O., 1999. Effect of industrial and agricultural wastes on laccase activity of white rot fungi. J. Biotechnol. 23 (2), 139–144. Kaluskar, M.V., Kapandis, P.B., Jasper, C.H., Penninckx, J.M., 1999. Production of laccase by immobilized cells of Agaricus sp. Appl. Biochem. Biotechnol. 76, 161–170. Kumar, V., Wati, L., Fitz Gibbon, F., Nigam, P., Banat, I.M., Singh, D., Marchant, R., 1997. Bioremediation and decolorization of anaerobically digested distillery spent wash. Biotechnol. Lett. 19, 311–313. Lee, Y.I., Jung, K.H., Lee, C.H., Park, Y.H., 1999. Enhanced production of laccase in Trametes versicolor by the addition of ethanol. Biotechnol. Lett. 21, 965–968. Mansur, M., Suarez, T., Fernandez-Larrea, J.B., Brizuela, M.A., Gonzales, A.E., 1997. Identification of a laccase gene family in the new lignin-degrading basidiomycete CECT 20197. Appl. Environ. Microbiol. 63, 2637–2646. Platt, M.W., Hadar, Y., Chet, I., 1984. Fungal activities involved in lignocellulose degradation by Pleurotus. Appl. Microbiol. Biotechnol. 20, 150–154. Sık, S., Unyayar, A., 1998. Phanerochaete chrysosporium and Funalia trogii for the degradation of cotton stalk and their laccase, peroxidase, ligninase and cellulase enzyme activities under semisolid state conditions. Turk. J. Biol. 22, 287–298. Yesilada, O., Unyayar, A., Fiskin, K., 1991. Determination of the laccase and peroxidase enzyme activities of Coriolus versicolor in the vinasse medium. Tr. J. Biol. 15, 152–157. Yesilada, O., Sam, M., 1998. Toxic effects of biodegraded and detoxified olive oil mill wastewater on the growth of Pseudomonas aeruginosa. Toxicol. Environ. Chem. 65, 87–94. Yesilada, E., 1999. Genotoxic activity of vinasse and its effect on fecundity and longevity of Drosophila melanogaster. Bull. Environ. Contam. Toxicol. 63, 553–559.