Date of Award


Publication Type

Doctoral Thesis

Degree Name



Civil and Environmental Engineering

First Advisor

Biswas, Nihar

Second Advisor

Taylor, Keith


Aniline, Azo-Dye, Peroxidase, Soybean, wastewater, water




Azo-dyes are the largest group of colourants produced, and they are applied in many industries. In the environment they are recalcitrant, and under anaerobic conditions can break down to toxic or even carcinogenic aromatic amines. Aerobic treatment of azo-dye-contaminated waters has been shown to be ineffective. Thus, enzyme-catalyzed polymerization and precipitation of azo-dyes and their reduction products was studied and optimized in this dissertation. Additionally, zero-valent iron reduction of azo-dyes under anaerobic conditions followed by soybean peroxidase (SBP) enzymatic treatment was investigated. The use of additives to reduce enzyme requirement and enhance the removal of anilines was also studied. Azo-dyes and authentic anilines were treated at 1 mM, while the anilines recovered from zero-valent iron reduction were treated at 0.5 mM. All experiments were conducted in batch reactors, and the parameters: pH, hydrogen peroxide to substrate ratio, enzyme concentration and additive concentration were optimized. Enzymatic treatment was successful in removal of 95% of both aniline and o-anisidine. The use of additives, sodium dodecyl sulfate (SDS), sodium dodecylbenzenesulfonate (SDBS), Triton X-100, and sodium dodecanoate (SDOD), reduced enzyme dose requirement, while the use of polyetheylene glycol (PEG, average molar mass of 3350 g/mole) had no effect on the required enzyme dose. In addition, the presence of SDS also enhanced treatment by improving precipitation and removing colour. Azo-dyes treated with SBP directly were successfully decolourized, with 85% colour removal of Acid Red 4 (AR4) and 95% for Crocein Orange G (COG). The pretreatment of AR4 with zero-valent iron, was able to achieve an even higher percent of decolourization 95%, while the second stage of treatment with SBP removed >95% of the recovered o-anisidine and further decolourized the water.