Nowadays, environments in which contaminants are merely transferred from one medium to another are no longer acceptable. It is no surprising that inorganic molecules with oxidizing activity such as ozone or hydrogen peroxide should be so widely used. Both can act individually at high concentrations or in conventional biological treatments including this as pre-treatment to degrade toxic, refractory or bio-inhibitory organics and make them more amenable to biodegradation. Hydrogen peroxide can be also used at low concentration in a polishing step with enzymatic reactions using peroxidases. In this approach, hydrogen peroxide can destroy trace levels of recalcitrant organics pollutants that could easily pass through bio-treatments (Annachatre and Gheewala, 1996; Yee and Wood, 1997; Wagner and Nicell, 2002).

Nevertheless, the use of hydrogen peroxide in biocatalysis as co-substrate for peroxidase activity is limited due to its inhibitory effect at high concentrations above 25 mM and slow deleterious effects at long time with lower concentrations (Nicell and Wright, 1997; Park and Clark, 2006). One solution for this problem is the use of very low and controlled hydrogen peroxide supplementation, necessary just for the maintenance of an effective oxidation level, with no inhibitory effects. This can be achieved using direct pulsated addition (DA) or by in situ electrogeneration (EG). The electroenzymatic approach provides a significantly lower and easily controllable hydrogen peroxide formation rate than any other so far. The electrochemical production of hydrogen peroxide results from the dissolved oxygen reduction present in the reaction mixture. This oxygen reduction produces, as final product, water, with the consumption of 4 electrons. EG was already applied to some biocatalytical oxidations, such as, in the asymmetric oxidation of thioanisole by CPO (Lütz et al. 2004), the oxidation of pentachlorophenol (Kim and Moon, 2005), and the oxidation of dimethylaniline by HRP (Chen and Nobe, 1993).

The purpose of this study was to compare the conventional enzymatic degradation of phenolic compounds yet reported for HRP (Nicell at al. 1993) and CPO (La Rotta and Bon, 2002) with the electroenzymatic oxidation. Results were compared in terms of hydrogen peroxide production, electrochemical conditions, residual phenol concentration (oxidation efficiency), precipitate formation (removal efficiency), and residual enzyme activity after each treatment.

Experimental

The enzyme chloroperoxidase (CPO) was obtained from the submerged culture of Caldariomyces fumago, purified and then modified chemically in a way to enhance its stability and work life-time. This modified biocatalyst was used during the oxidation of several chlorinated phenols (CP) commonly found in industrial waste waters in the presence of hydrogen peroxide. This study compares the direct addition of hydrogen peroxide as single or pulsed addition (DA) with its continuous electrogeneration (EG) during the enzymatic oxidation of CP. Reaction mixtures were studied containing chemically modified CPO, hydrogen peroxide (added or electrochemically generated) and the phenolic substrates: phenol (P), 4-chlorophenol (4-CP), 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP) and pentachlorophenol (PCP), in 100 mM sodium-potassium phosphate buffer pH 6.0, at 25ºC.

For the hydrogen peroxide electrogeneration a bicompartmented reactor used in this study is described in Figure 1. The electrochemical system was composed by a platinum wire as auxiliary electrode (AE) and saturated calomel electrode (SCE) as reference. Graphite felt (GF) and reticulated vitreous carbon foam (RVCF) , both with 2.0 cm² of apparently surface area and an electrolytic copper web of 27 cm² were evaluated as working electrodes (WE). Cathodic potentials from -120 to -820 mV, were evaluated in terms of the hydrogen peroxide production as well as two strategies for air supplementation: Discontinuous aeration by 1 hr with pure O₂ (5.0 mL∙min^-1) and continuous aeration with pure O₂ (5.0 mL∙min^-1) along the 4 hrs of reaction. Continuous magnetic stirring at 200 rpm was maintained in all cases. To evaluate H₂O₂ generation, samples during 4 hrs from the cathodic chamber were taken, and then H₂O₂ was quantified.

In DA experiments, the maximum degradation levels were observed for P and 4-CP, when a multiple hydrogen peroxide addition of 5 pulses was used. Lower degradation levels with this addition of 88, 66 and 59% for 2,4-DCP, 2,4,6-TCP and PCP, were observed respectively. Hydrogen peroxide addition also caused chemical oxidation up to 12% depending on the phenolic compound. This observation corroborates the previous studies where the oxidation of 4-CP was studied (La Rotta and Bon, 2002).

The formation of precipitates was observed during the first two hours. In consequence, 80% of both P and 4-CP, were removed. In the case of 2,4,6-TCP and PCP only an increase in turbidity was observed with no precipitate formation. Since the simultaneous addition of several coagulants such as chitosan gels was already successfully used with other peroxidases during the enzymatic oxidation of phenols (Zhang et al. 1997; Lai and Lins, 2005), we decided to evaluate its addition in a final concentration of 2% (w/v). Consequently, the complete removal of phenol and 4-CP was achieved and increments in removal of 12 and 30% were observed for 2,4-DCP and 4-CP, respectively. Removal quantification was not possible for 2,4,6-TCP and PCP due in part to their solubility features. However, residual CP values (data not shown) indicated the incidence of phenol removal from the non polymerized products generated during the enzymatic oxidation and the addition of chitosan.

Bioelectrodegradation of CP

Figure 2 shows the effect of the cathodic potential over the 4-CP bioelectrochemical degradation. As it can be seen, 4-CP bioelectrooxidation at the cathodic chamber (EG CC) increased with the cathodic overpotential. As a consequence of this, no 4-CP was detected after 4 hrs at -620 mV_SCE. And using -420 and -220 mV_SCE, 7 and 25% of residual 4-CP was detected, respectively. These results demonstrate the dependence of the applied potential over the electroezymatic degradation. It was also observed anodic direct oxidation (EG AC) up to 30% during the bioelectrochemical processes. Cathodic chamber controls (CC C) with no CPO addition showed 18% of chemical oxidation due to the electrogenerated hydrogen peroxide.

Electrodegradation also drives the precipitation only at the cathodic chamber. As a consequence, when a higher H₂O₂ level was produced, the highest precipitate formation occurred. Maximum precipitation was observed for phenol and 4-CP and increases only in turbidity were observed for 2,4-DCP, 2,4,6-TCP and PCP, with no precipitation. On the other hand, when chitosan was added, removal levels achieved 91,5 and 91,3%, for 4-CP and 2,4-DCP, respectively. Although the chitosan addition improves removal levels during EG, could also promotes the electrode passivation, since the flocculated product could remains adhered on the electrode, promoting mass transfer problems and decreasing  the electrode life-time. This result shows that the application of chitosan or other coagulants during EG should be analyzed, probably in a discontinuous process or with a different reactor arrangement.

Evaluation of residual peroxidase activity

Although, both methodologies were similarly efficient in terms of phenol oxidation and precipitate formation, it was observed that CPO loses activity rapidly when the direct addition was employed. This fact is not just related to the presence of hydrogen  peroxide, but is even more remarkable when both oxidized products and hydrogen peroxide were simultaneously present during the reaction with DA or EG, indicating a possible suicide inactivation already observed for oxidases such as tyrosinase (Haghbeen et al. 2004). Thus, up to 60% of initial activity was lost in DA experiments, followed by 40% using EG after 4 hrs. Free and pure CPO in 100 mmoles∙L^-1 potassium phosphate buffer pH 6.0 remained stable up-to 4 hrs at 25ºC. In terms of inactivation rates, CPO was more rapidly inactivated with the direct addition of hydrogen peroxide rather than with its electrogeneration. On the other hand, CPO presented half-life and work-life times two fold-times higher when EG were used instead DA.

The continuous electrogeneration (EG) over the pulsated directaddition (DA)of the hydrogen peroxide offered the possibility to conciliate an effective hydrogen peroxide availability to undisruptive concentrations in reaction mixtures containing phenol and chlorinated phenols. With EG system it was possible to obtain variable hydrogen peroxide concentrations depending on the working electrode material, the applied cathodic potential and the aeration mode. Even though, DA was relatively more efficient in terms of enzymatic oxidation than the electrochemical approach, this last one showed a lower inactivation rates, and longer half-life and work-life times. Deeper studies are needed to identify the optimal conditions for the total detoxification of chlorophenolic solutions and  also the development of novel reactor configurations that prevent the accumulation of polymeric products. Finally, an additional question that needs to be addressed in future studies is how to increase the enzyme stability and re-use. At this point the possibility of enzyme immobilization is stand.

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