Enhancing the production of hydroxyl radicals by Pleurotus eryngii via quinone redox cycling for pollutant removal

Abstract

The induction of hydroxyl radical (OH) production via quinone redox cycling in white-rot fungi was investigated to improve pollutant degradation. In particular, we examined the influence of 4-methoxybenzaldehyde (anisaldehyde), Mn(2+), and oxalate on Pleurotus eryngii OH generation. Our standard quinone redox cycling conditions combined mycelium from laccase-producing cultures with 2,6-dimethoxy-1,4-benzoquinone (DBQ) and Fe(3+)-EDTA. The main reactions involved in OH production under these conditions have been shown to be (i) DBQ reduction to hydroquinone (DBQH(2)) by cell-bound dehydrogenase activities; (ii) DBQH(2) oxidation to semiquinone (DBQ(-)) by laccase; (iii) DBQ(-) autoxidation, catalyzed by Fe(3+)-EDTA, producing superoxide (O(2)(-)) and Fe(2+)-EDTA; (iv) O(2)(-) dismutation, generating H(2)O(2); and (v) the Fenton reaction. Compared to standard quinone redox cycling conditions, OH production was increased 1.2- and 3.0-fold by the presence of anisaldehyde and Mn(2+), respectively, and 3.1-fold by substituting Fe(3+)-EDTA with Fe(3+)-oxalate. A 6.3-fold increase was obtained by combining Mn(2+) and Fe(3+)-oxalate. These increases were due to enhanced production of H(2)O(2) via anisaldehyde redox cycling and O(2)(-) reduction by Mn(2+). They were also caused by the acceleration of the DBQ redox cycle as a consequence of DBQH(2) oxidation by both Fe(3+)-oxalate and the Mn(3+) generated during O(2)(-) reduction. Finally, induction of OH production through quinone redox cycling enabled P. eryngii to oxidize phenol and the dye reactive black 5, obtaining a high correlation between the rates of OH production and pollutant oxidation.

FIG. 1.

Production of H₂O₂ by P. eryngii via anisaldehyde redox cycling. Ten-day-old washed mycelium was incubated with 1 mM anisaldehyde in 50 ml 20 mM phosphate buffer, pH 5. Anisaldehyde was omitted in blank incubations. The error bars indicate standard deviations.

FIG. 2.

Effect of anisaldehyde on OH production by P. eryngii through the redox cycling of several benzoquinones. TBARS levels produced after 120-min incubation of the fungus with 500 μM BQ, MBQ, DBQ, or no quinone (No Q); 1 mM anisaldehyde; 100 μM Fe³⁺-110 μM EDTA; and 2.8 mM 2-deoxyribose are shown. Anisaldehyde was absent in incubation blanks. The error bars indicate standard deviations.

FIG. 3.

Effects of different concentrations of Mn²⁺ on H₂O₂ production and DBQ(H₂) levels during the incubation of P. eryngii with DBQ. Washed mycelium was incubated with 500 μM DBQ in the absence and presence of 20, 100, 250, and 500 μM Mn²⁺. The error bars indicate standard deviations.

FIG. 4.

Effects of different concentrations of Mn²⁺ on OH production by P. eryngii via DBQ redox cycling. The incubation mixtures were as described in the legend to Fig. 3 plus 100 μM Fe³⁺-110 μM EDTA and 2.8 mM 2-deoxyribose. The error bars indicate standard deviations.

FIG. 5.

Time course of oxalate production (•) and glucose consumption (▪) by P. eryngii in glucose-peptone medium. Cultures were carried out in the absence (filled symbols) and presence (open symbols) of 50 μM MnSO₄. Glucose levels without Mn were not significantly different from those with Mn (data not shown). The error bars indicate standard deviations.

FIG. 6.

Effects of oxalate, used as an iron-chelating agent, on DBQ(H₂) levels and OH production during the incubation of P. eryngii with DBQ. The incubation mixtures contained 500 μM DBQ, 2.8 mM 2-deoxyribose, and either 100 μM Fe³⁺-110 μM EDTA or 100 μM Fe³⁺-500 μM oxalate. (A) DBQ (solid lines) and DBQH₂ (dashed lines) levels. (B) TBARS levels in whole incubations (solid lines) and blanks without DBQ (dashed lines). The error bars indicate standard deviations.

FIG. 7.

Effect of the Fe³⁺/oxalate concentration ratio on OH production by P. eryngii via DBQ redox cycling. Washed mycelium was incubated with 500 μM DBQ, 2.8 mM 2-deoxyribose, and 100 μM Fe³⁺ chelated with different oxalate concentrations. The error bars indicate standard deviations.

FIG. 8.

Phenol removal by P. eryngii under conditions producing different levels of OH radicals. Incubations of washed mycelium with 500 μM concentrations of phenol and DBQ were performed in the absence (no Fe) and presence of 100 μM Fe³⁺-110 μM EDTA or 100 μM Fe³⁺-300 μM oxalate. The time courses of phenol disappearance and catechol production are shown (solid and dashed lines, respectively). The error bars indicate standard deviations.

FIG. 9.

Effect of Mn²⁺ on OH production by P. eryngii in incubations with DBQ and Fe³⁺-oxalate. The incubation mixtures contained 500 μM DBQ, 100 μM Fe³⁺-300 μM oxalate, 2.8 mM 2-deoxyribose, and 100 μM Mn²⁺ (Q-Fe-Mn). Incubation blanks without Mn (Q-Fe) or Fe³⁺-oxalate (Q-Mn) were performed. The error bars indicate standard deviations.

FIG. 10.

RB5 removal by P. eryngii under conditions producing different levels of OH radicals. The composition of the reaction mixtures was as described in the legend to Fig. 9, except 2-deoxyribose was replaced by 50 μM RB5. The error bars indicate standard deviations.

FIG. 11.

Scheme showing the main reactions involved in the production of OH radicals by P. eryngii via DBQ redox cycling in the presence of Mn²⁺, the complex Fe³⁺-oxalate, and anisaldehyde. R = OCH₃, and X = EDTA or oxalate (see Discussion for an explanation).