Date of Award


Publication Type

Doctoral Thesis

Degree Name



Civil and Environmental Engineering

First Advisor

Biswas, Nihar

Second Advisor

Taylor, Keith


azo-dyes, decoloration, enzymatic treatment, wastewater treatment, zero-valent iron reduction




The presence of azo-dyes in water bodies represents an environmental problem due to their recalcitrant, toxic and in some cases carcinogenic characteristics. In this dissertation, a more complete analysis is presented which includes color removal, dye conversion, total amines (as aniline) and product degradation. Enzymatic treatment with soybean peroxidase (SBP) was studied to decolorize and degradate two impure azo-dyes, Acid Blue 113 (AB113; a di-azo dye) and Direct Black 38 (DB38; a tri-azo dye). A single-step process (direct enzymatic treatment) and a two-step process (zero-valent iron (Fe°) reduction followed by enzymatic treatment) were compared to obtain the optimal conditions of pH, H2O2 concentration, enzyme concentration and reaction time for maximum decoloration, dye and products degradation as well as total amines removal. More than 95% decoloration and dye degradation was achieved for both dyes after single- and two-step processes. A two-step process was preferred for DB38, because, after Fe° reduction, the products (aniline and benzidine) were SBP substrates which needed low SBP and H2O2 concentrations for 95% removal. The lowest KM value (data obtained from Michaelis-Menten plot) was for a simple and highly pure reference dye, Crocein Orange G (COG), followed by AB113 and then DB38. Evidence for azo-cleavage of COG due to direct enzymatic treatment was obtained, identified and quantified by high-performance liquid chromatography (HPLC) where aniline was produced. Mass spectrometry was used to confirm the presence of aniline after treatment. An alternative for reducing the number of experimental runs during optimization and obtain valuable statistical parameters, response surface methodology (RSM; software program Minitab), was used to optimize and characterize the decoloration of AB113 and DB38. These methods showed that the parameters: pH, H2O2 and enzyme concentrations were statistically significant in the removal of azo-dye; in addition, the presence of curvature in the response surface, indicated a preference for a second-order model. Decoloration of more than 95% was obtained by the RSM model. For AB113 the optimal response obtained with Minitab model was 5.7 % and the experimental value under the same conditions was 8.0% (2.3% difference) while for DB38 the optimal Minitab response was 3.6% and 5.1% for the experimental value (1.5% difference). Equations were obtained to determine percent color remaining within the study area (for AB113 pH 3.6-5.3; enzyme 1.0-2.0 U/mL; H2O2 1.0-3.0 mM and for DB38 pH 3.0-5.0; enzyme 2.5-3.5 U/mL; H2O2 2.5-3.5 mM). A positive RSM result shows the reduction of experimental runs compared to the optimization of one-parameter-at-a-time which represent diminished analysis time.