HPLC-based analysis of phenol degradation rate and its oxidation products

The analysis of phenol and its oxidation products were carried out with a HPLC (Waters Corporation, Milford, MA, USA). Phenol, hydroquinone, pyrocatechol, resorcinol, benzoquinone and water were all the HPLC-grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The standard phenolic compounds including phenol, hydroquinone, pyrocatechol, resorcinol, benzoquinone solvent were consecutively injected five times to draw calibration curves. The injection volume was 2, 4, 8, 16 and 22 mL, respectively.

The oxidation reaction was carried out as previously described using 50 mL EP tubes [45]. A solution of 500 mg/L of simulation phenol-containing wastewater was made up with dissolving 1.00 g phenol at the constant volume of 2000 mL distilled water. The solution was sterilized at 121 °C for 20 min and then cooled to 30 °C. 5 mL phenol solutions were added into 50 mL EP tubes and add 5, 10 and 15% rGluMnP1 crude enzymes. Reactions are completely at 210 rpm/min and 28 °C for more than 24 h. The determination condition of the samples was set as follows: the column was YMC-pack ODS-A 250 × 4.60 mm S-5.0 μm; the mobile phase adopted in the analysis consists of methanol and water was in the ratio 30:70 (v/v). The separation was conducted in isocratic elution at a flow rate of 0.8 ml/min. The detection wavelength of photo-diode array was set at 280 nm; the column temperature was 30 °C. The injection volume was 20 μL and a data acquisition time of 20 minutes was used [46]. The degradation rate of phenol was calculated according to the following formula: Degradation rate (100%) = (C₀-C)/C₀ × 100%. In the formula, C₀ was the concentration of phenol in the control group; C was the concentration of residual phenol in the treated phenol aqueous solution.

All experiments were performed at least twice using three replicates. The data presented in the text corresponded to the mean values.