ISSN 1063455X, Journal of Water Chemistry and Technology, 2009, Vol. 31, No. 2, pp. 135–138. © Allerton Press, Inc., 2009. Original Russian Text © I.I. Romanovskaya, Yu.A. Shesterenko, O.V. Sevast’yanov, 2009, published in Khimiya i Tekhnologiya Vody, 2009, Vol. 31, No. 2, pp. 235–241.

BIOLOGICAL METHODS OF WATER TREATMENT

Elimination of Phenol with the Use of Tyrosinase of Fungi I. I. Romanovskaya, Yu. A. Shesterenko, and O. V. Sevast’yanov Bogatskii PhysicoChemical Institute, National Academy of Sciences of Ukraine, Odessa Received October 24, 2007

Abstract—It was shown that partially purified preparation of tyrosinase of fungi Agaricus bisporus trans form phenol with the formation of watersoluble polymer products. The article defined conditions of oxi dizing phenol leading it to 98% of bioconversion. The paper proposed inorganic coagulants (potash, ammonium, or iron alums) whose use will make it possible to completely remove the reaction products within a broad range of the initial concentrations of phenol (0.5–10 mmol/dm3); the elimination degree in all the cases was in excess of 97%. DOI: 10.3103/S1063455X09020088

INTRODUCTION Phenols contained by wastewaters of a number of industrial sectors (oilprocessing, chemical recovery coke, woodworking, metallurgy, textile, pharmaceutical) possess high toxicity and are dangerous pollutants of the environment. Their maximum admissible concentration (MAC) varies 0.001 to 0.1 mg/dm3. The existing methods of purifying wastewaters of phenols (chemical oxidation, microbial degradation, adsorption on activated carbon, extraction by solvents) despite their efficiency possess a number of substantial drawbacks: high costs, incomplete treatment, formation of toxic products. In this connection a substantial interest is generated by alternative technologies, in the first place, fermentative methods thanks to their selec tivity, high degree of treatment and the possibility of using within a broad interval of the pH, temperature, and concentration of phenol. Therefore an important task is the use for the removal of phenols of tyrosinase—an enzyme of the class of oxidoreductases (K.F. 1.14.18.1), catalyzing oxidation of phenol substrates in the presence of molecular oxy gen [2]. Tyrosinase possesses two types of activity: monophenolase one—orthohydroxilation of monophe nols and diphenolse one—oxidation of diphenols to oquinones. Commercial preparations of tyrosinase are extremely expensive, which limits the possibility of their prac tical use, therefore it is expedient to use partially tinted fermentative preparations. As a result of oxidation of phenol catalyzed by tyrosinase soluble darktinted compounds are formed. These compounds represent oligomer products of oquinone condensation, which entails the necessity of using coagulants for their removal. Aluminum sulfate and cationic polymer coagulants: copolymer of hexamethyl diamine with epichlorohydrine, polyethylenimine, chitosan, chitin, etc. were used as coagulants [3–5]. How ever, their use in a number of cases did not allow the removal of the reaction products completely. The objective of the given paper is a research of elimination of phenol by means of the partially purified preparation of tyrosinase from fungi Agaricus bisporus and inorganic coagulants. EXPERIMENTAL The partially purified preparation of tyrosinase from fungi Agaricus bisporus isolated according to [6]: 300 g of fungi were homogenized with 600 cm3 of a cooled extractant (an aqueous solution containing 1% of ascor bic acid and 0.2% of benzoic acid) was stirred for 1 h and after that the resultant extract was centrifuged at 11000 rpm (30 min). The enzyme was precipitated by saturation of the supernatant liquor of ammonium sul fate to 80% and centrifuged under analogous conditions. The precipitate was dissolved in 15 cm3 of distilled water and then dialysis at 0°C for three days was carried out with the aim of isolating the tyrosinase preparation in which the content of protein was determined by the Lori method in the Hartree modification [7], activity by tyrosinase [8] and pyrogallol [9]. 135

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Within the interval of the pH values 3–10 the pH optimum of the phenol oxidation reaction was found using the following buffer solutions: 0.01 mol/dm3 of sodium pyrophosphate—succinic acid (pH 3–5; 8–10), 0.05 mol/dm3 phosphate NaH2PO4–NaOH (pH 5.5–7). The impact of temperature on the degree of phenol bioconversion by tyrosinase was investigated within the interval 2–80°C at pH 6.7 and the substrate concentration 0.5 mmol/dm3. The degree of phenol bioconversion was found by its loss spectrophotometrically by the reaction with 4 aminoantipyrine [10]. Phenol oxidation catalyzed by tyrosinase was investigated within the range of the concentration 0.5– 10 mmol/dm3 at pH 6.5, temperature 25°C and enzyme activity 30–600 Unit/cm3 for three hours. Coagulation of the products of fermentative oxidation of phenol was carried out by means of potash, ammonium, and iron alums with the phenol initial concentration 0.5–10 mmmol/dm3 and concentration of coagulants 1–20 g/dm3. When determining the concentration of the coagulants necessary for water treatment the technique of test coagulation was used [11], this technique imitates coagulation purification followed by settlement. To 10 cm3 of the solution containing products of phenol bioconversion 10–200 mg of coagulants (depending on the concentration) were added. Coagulation was carried out by intensive stirring of the solu tions (1 min) and further weak stirring (3 h) at 25°C followed by filtration of the solutions. The degree of removing the products of phenol oxidation was determined spectrophotometrically by a decrease of optical density at 510 nm [8]. RESULTS AND DISCUSSION Tyrosinase was isolated from the fungi Agaricus bisporus with the yield of protein 2.77 mg/g, specific activ ity 6.5 μmol of purpurogallin/(mg of protein min) and 255 Unit/(mg protein min) respectively by pyrogallol and tyrosinase. When investigating the properties of the resultant enzyme its pH and temperature optimum were studied. From the data given in the table it follows that the high degree of maintaining the activity of tyro sinase is observed at pH 5.5–7 and the temperature 20–50°C. Impact of the pH and temperature of the incubation medium on the degree of phenol bioconversion catalyzed by tyrosinase pH 3 4 5 5.5 6 6.5 7 8 9 10

Degree of phenol bioconversion, % of maximum 1.9 28.3 53.3 90.2 98.1 100.0 97.5 50.5 28.1 2.2

Temperature,°C 2 10 20 25 30 40 50 60 70 80

Degree of phenol bioconversion, % of maximum 23.7 62.3 79.2 88.3 93.4 100.0 90.4 66.0 47.9 31.3

The degree of phenol bioconversion increases with an increase of enzyme activity reaching the maximum level after 1 h of incubation with tyrosinase with the activity 50 Unit/cm3; when using the enzyme of lower activity (30 and 10 unit/cm3) the time necessary for the maximum degree of bioconversion increases respec tively to 3 and 24 h (Fig. 1). By means of the isolated tyrosinase phenol oxidation within a broad range of concentrations (0.5– 10 mmol/dm3), in this case the degree of bioconversion in all cases constituted 98%. During phenol bioconversion by tyrosinase one can see a linear relationship between the initial phenol concentration (0.5–10 mmol/dm3) and activity of tyrosinase (Fig. 2). JOURNAL OF WATER CHEMISTRY AND TECHNOLOGY

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1 2

80 60

3

40 20 0 0

4

8

12

16

20

τ, h

Fig. 1. Relationship between the phenol bioconversion (Bc) degree and the time (phenol concentration: 0.5 mmol/dm3, tem perature: 30°C, pH 6.5); tyrosinase (Unit/cm3): 1—50, 2—30, 3—10. 3

Atyr, Unit/cm 600

400

200

0 0

2

4

6

8

10

3

Cph, mmol/dm

Fig. 2. Relationship between the tyrosinase (Atyr) and the initial phenol concentration (Cph) (time of bioconversion: 3 h, time of bioconversion: 3 h, temperature: 25°C, pH 6.5).

The minimum activity of the enzyme necessary for a 98% phenol bioconversion may be computed by the method of linear regression: Atyr = 57.127 · [Phenol]0 – 14.019; r = 0.9960, where [Phenol]0 is the initial concentration of phenol, mmol/dm3; r is the coefficient of linear correlation. For the removal of soluble tinted phenol oxidation products potash, ammonium, and iron alums were cho sen as coagulants. Alums are widely used for wastewater treatment, but earlier were not used for this process. Coagulants were used for purifying phenol solutions (0.5–10 mmol/dm3) oxidized in the presence of tyrosi nase by more than 98%. Based on experimental data the graphs of the relationship between the degree of removing phenol oxidation products and a decrease of optical density and the coagulant concentration were built (Fig. 3) [8]. For precipitation of the products of fermentative phenol oxidation (0.5 mmol/dm3) coagulants were used in the range of concentrations from 0.3 to 2.66 g/dm3. In the case the concentration of the coagulants to 1.00; 1.33, and 1.66 respectively for potash, ammonium, and iron alums the degree of removing the oxidation prod ucts increased, further increase of the concentration did not lead to its substantial change. By means of the chosen coagulants the removal of oxidation products within a broad range of initial con centration of phenol was carried out and in this case the removal degree in all cases constituted more than 97%. The most effective results were obtained when using ammonium and potash alums. For the removal of phe nol oxidation products with the initial concentration 0.5–10 mmol/dm3, 1.0–17.5 g/dm3 of coagulants were needed, whereas the concentration of iron alum constituted 1.66–20.66 g/dm3 (Fig. 4).

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1 2

80

3

60 40 20 0 0

1

3

Cc, g/dm

2

Fig. 3. Relationship between the degree of removing the phenol oxidation products (Rpr) and the concentration of coagulant (Cc) (Cph—0.5 mmol/dm3, temperature—25°C, pH 6.5, incubation time—3 h): 1—ammonium alum; 2—potash alum; 3— iron alum. 3

Cc, g/dm 25 20

1 15

2 3

10 5 0 0

1

2

3

4

5

6

7

8

9

3

10 Cph, mmol/dm

Fig. 4. Relationship between the concentration of coagulants (Cc) and the initial phenol concentration (Cph) (temperature— 25°C, pH 6.5, incubation time—3 h): 1—iron alum; 2—potash alum; 3—ammonium alum.

CONCLUSIONS Thus, the results of the research conducted demonstrated the possibility of quantitative elimination of phe nol from aqueous solutions (0.5–10 mmol/dm3) by means of oxidation catalyzed by the partially purified preparation of tyrosinase from the fungi Agaricus bisporus followed by precipitation of bioconversion products with potash, ammonium (1.0–17.5 g/dm3), or iron alums (1.66–20.66 g/dm3). REFERENCES 1. Dorofeeva, N.L., Ochistka stochnykh vod i utilizatsiya fenol’nykh soedinenii (Purification of Wastewaters and Utiliza tion of Phenol Compounds), Kiev: UkrNIINTI Gosplana USSR, 1990. 2. Solomon, E., Sundaram, U., and Machokin, T., Chem. Rev., 1996, vol. 96, no. 7, pp. 2563–2603. 3. Wada, S., Ichikawa, H., and Tastsumi, K., Biotecnol. Bioeng., 1995, vol. 45, no. 4, pp. 304–309. 4. Payne, G., Sun, W., and Sohrabi, A., ibid., 1992, vol. 40, no. 9, pp. 1011–1018. 5. Bevilaqua, J., Cammarota, M., Freire, D., et al., Brazil. J. Chem. Eng., 2002, vol. 19, no. 2, pp. 151–158. 6. Cohen, E.M. and Lerner, L. L., US Patent 2 956929, IPC 19668, publ. Oct. 10, 1960. 7. Hartree, E.E., Anal. Biochem., 1972, vol. 48, no. 1, pp. 422–427. 8. Ikehata, K. and Nicell, J., Biotechnol. Prog., 2000, vol. 16, no. 4, pp. 533–540. 9. Patra, H. and Mishra, D., Plant Physiol., 1979, vol. 63, no. 1, pp. 318–323. 10. Korenman, I.M., Fotometricheskii analiz. Medoty opredeleniya organicheskikh soedinenii (Photometric Analysis. Methods of Determining Organic Compounds), Moscow: Nauka, 1983. 11. Yaroshevskaya, N.V., Sergienko, A.N., Murav’ev, V.R., et al., Khimiya i Tekhnologiya Vody, 2005, vol. 27, no. 2, pp. 173–183. JOURNAL OF WATER CHEMISTRY AND TECHNOLOGY

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