Biological removal of 4-chlorophenol by phytodegradation using different peroxidases

D. S. BARBOSA; E. GÓMEZ; P. S. GONZÁLEZ; E. AGOSTINI

Sevilla

Congreso; Congreso Internacional de Ingeniería Química de ANQUE (ANQUE-ICCE 2012); 2012

Biological removal of 4-chlorophenol by phytodegradation using different peroxidases D. S. Barbosa1, E. Gómez1(*), M.D. Murcia1, P. S. González2, E. Agostini2 1 Departamento de Ingeniería Química, Universidad de Murcia, 30071 Murcia, Spain, Tel:+34 868887352. Fax: + 34868884148 (*)e-mail: egomez@um.es 2 Departamento de Biología Molecular, FCEFQN, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, CP 5800 Río Cuarto, Córdoba, Argentina Tel: +54 358 4676537; fax: +54 358 4676232 T9. Bioprocesses and Biocatalysis: Characterization and Improvement of Biocatalysts. Introduction Phytoremediation is an interesting strategy because of the ability of some plants to adsorb, accumulate and/or tolerate high concentrations of pollutants such as heavy metals, organic compounds and radioactive elements. In the particular case of phenolic compounds, two different methods are usually employed; (a) the phytoextraction, in which roots capture these organic compounds which are accumulated in roots, stems and leaves and can be afterwards precipitated and concentrated; (b) the phytodegradation, whereby aquatic and terrestrial plants capture, store and degrade organic compounds to less toxic or harmless byproducts [1].In the present work, the phytodegradation of 4-chlorophenol using different peroxidases i.e: two Total Peroxidase Extracts (TPE), obtained from turnip (Brassica napus) and tobacco (Nicotiana tabacum cv. Wisconsin) hairy roots (HR) respectively, and a commercial soybean peroxidase (SBP), has been investigated. In addition, a complete characterization and a comparative study of these peroxidases have been carried out. Materials and methods The turnip and tobacco peroxidases were obtained from total enzyme extraction of the corresponding HR [1] and the commercial SBP was hydrated before use. Peroxidase activity was determined with o-dianisidine as substrate. Activity was measured following the increase in A460 at 37 ºC due to the appearance of o-dianisidine oxidation product (460 = 11.4 mM-1 cm-1), allowing at the same time to determine the stability of the peroxidases (activity after 15 and 30 days of storage). On the other hand, the isoelectric point (pI) of the peroxidase isoenzymes was determined by isoelectric focusing (IEF), the Reinheitszahl index (Rz) by determining the absorbance ratio A403/A280, and the molecular weight (MW) by SDS-PAGE electrophoresis. The comparative study of 4-clorophenol elimination has been carried out in a discontinuous stirred (100 rpm) tank reactor (50 ml), working at room temperature (≈ 25 ºC). A 5 ml solution of 2.0 mM of 4-clorophenol, 2.0 mM of hydrogen peroxide and 100*103 U/ml of the different tested peroxidases was used. To follow the time course of the reaction the concentration of residual 4-chlorophenol was measured using the aminoantipirine (AAP) colorimetric method [3]. Results and discussion Activity measurements showed that all the studied peroxidases have a high enzymatic activity (i.e. higher than 200*103 U/ml) and that TPE from tobacco has the higher activity, followed by TPE from turnip and finally the commercial SBP. Enzymes stability evaluation has demonstrated that after 30 days of storage, both TPE still have a remaining activity of 80% compared to the fresh peroxidases, while the activity of the SBP decrease to 30%. Concerning the Reinheitszahl index, as it was to expected, the commercial SBP has the higher purity grade with a Rz value of about 1.80, while the non-purified TPE showed low Rz values of 0.16 and 0.59 for turnip and tobacco peroxidasas, respectively. According to the isoelectric point determination, the commercial SBP presents a main isoenzyme which has an almost neutral pI of 6.7, while both TPE present two main basic peroxidase isoenzymes (i.e. pIs of 10.1 and 8.8 for turnip TPE and 10.0 and 8.0 for tobacco TPE). The molecular weight (MW) determination showed that commercial SBP has a main band corresponding to MW of 44,90 kDa, which is typical for this kind of peroxidase. On the other hand, turnip TPE presents two important bands, the majority one having a MW of 56.80 kDa, while tobacco TPE presents at least five important bands which have MWs included between 12.34 and 39.93 kDa. Figure 1. 4-chlorophenol conversion versus operational time for three different peroxidases. ♦ SBP, ■ TPE from tobacco HR and ▲ TPE from turnip HR. Finally, as it can be seen in Figure 1, according to the results obtained in a discontinuous stirred tank reactor, both TPE are more efficient than commercial SBP to remove 4-chlorophenol from wastewater. This result could be explain by the fact that TPE have several peroxidase isoenzymes which would have more affinity for hydrogen peroxide as substrate than SBP, producing the initiator agent of the polymerization reaction, and consequently increasing the efficiency of the removal of the 4 chlorophenol in wastewater. References [1] Talano M. A., Frontera S., González P., Medina M. I., Agostini E. (2010). Removal of 2, 4-diclorophenol from aqueous solutions using tobacco hairy root cultures. Journal of Hazardous Materials. 176, 784?791. [2] Standard Methods for the examination of water and wastewater, 19th Edition. (1995). Method 5530, Section 5, 36-39. Acknowledgements This work was supported by 08683/PI/08 from Fundación Séneca (Spain); CONICET (Argentine); SECyT-UNRC (Argentine). Besides D. S. Barbosa is beneficiary of scholarships from MICINN.