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In this work, three xenobiotics (orange II, phenol, and bisphenol A) were oxidized by hydrogen peroxide in the presence of a horseradish peroxidase (HRP) using a fed-batch system. During the experiments, the oxidation− reduction potential (ORP) of the reaction mixture was measured continuously. Results demonstrate that ORP values only increased when both substrates of the enzyme (hydrogen peroxide and the target compound) were present in the reaction mixture. For all of the tested pollutants, the continuous addition of hydrogen peroxide caused an increase in ORP values. When the reducing substrate was depleted, the addition of an excess of hydrogen peroxide caused a decrease of ORP values. The time at which ORP reached a maximum represented the end of the oxidation process. This maximum could be easily detected by means of the derivative of ORP as a function of time. To extend the application of the developed technique, the enzymatic oxidation of a binary mixture of BPA and OII was also followed using ORP measurements. Results were similar to those observed with only one reducing substrate. This work demonstrates that ORP measurements can be useful to maximize hydrogen peroxide efficiency through the controlled addition of the oxidant during the oxidation of OII, phenol, and BPA catalyzed by HRP. This approach allows a minimization of time and process costs since the reaction end-point can be easily detected on a real-time basis.

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A simple technique to determine biomass concentration as chemical oxygen demand (COD) was developed as an alternative to the standard volatile suspended solid (VSS) method. The proposed technique for biomass measurement as COD is based on the determination of the biomass COD (COD B) as the difference between total COD (COD T) and the soluble COD (COD S) of the sample. The obtained results show that this technique was quicker and simpler than the traditional VSS method. The validity of the proposed methods was tested with pure cultures of a filamentous microorganisms (Sphaerotilus natans), a floc-forming bacteria and activated sludges. The method was also used for estimating the conversion factor (f CV) from VSS to COD units. A modification of the standard VSS technique was also proposed using two membranes in the filtration device; this technique allowed the biomass determination in 1 µm size bacteria cultures that cannot be detected by the standard VSS method because cells are not retained by the 1.5 µm diameter pore glass-fibre filter. Notation BOD = biochemical oxygen demand (mg•l-¹) COD = chemical oxygen demand (mg•l-¹) VSS = volatile suspended solid (mg•l-¹) f CV = conversion factor = ratio between COD B and VSS COD B = biomass as COD (mg•l-¹) COD T = total COD of the sample containing the biomass (mg•l-¹) COD S = soluble COD (mg•l-¹) CV = coefficient of variation

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