Date of Award


Publication Type

Doctoral Thesis

Degree Name



Civil and Environmental Engineering

First Advisor

Nihar Biswas

Second Advisor

Keith Taylor


Arylamine, Contaminants, HPLC, Mass spectrometry, Soybean peroxidase enzyme, Water treatment




Oxidative polymerization of hazardous aqueous pollutants that are suspected to be human carcinogens by U.S. EPA 2018, including poly-substituted monocyclic anilines (p-cresidine and 4-chloro-o-toluidine) and atom-bridged bis-anilines (4,4'-oxydianiline, 4,4'-methylenedianiline, 4,4’-thiodianiline and 4,4'-methylenebis(2-chlororaniline)), were catalyzed by a crude form of soybean peroxidase (SBP), isolated from soybean seed hulls, in the presence of hydrogen peroxide. In this work, harmful arylamines present in water were treated and removed for the first time using SBP. Firstly, experiments were conducted to achieve the optimal operating conditions with respect to pH, hydrogen peroxide-to-substrate concentration ratio and enzyme activity to achieve ≥ 95% removal of these compounds from water in a 3-hour period monitored by a high-performance liquid chromatography system with ultraviolet detection capability (HPLC-UV). 4-Chloro-o-toluidine required the lowest enzyme activity (0.009 U/mL), whereas 4,4'-methylenedianiline required the highest enzyme activity (0.700 U/mL). The reaction time profiles, directly related to the economic consideration of the treatment process, were monitored from which the half-lives and rate constants for removal of the compounds were estimated. Investigation of these kinetic parameters is also expected to play an important role in the design of wastewater treatment plants. Secondly, a kinetic study involving pollutant removal was performed using the Michaelis-Menten model to determine the affinity of the enzyme for the substrates by investigating the Michaelis constant (KM) and the maximum velocity of the reaction (Vmax). Thirdly, an investigation was performed on the fate of the analytes by determining the products generated after the enzymatic treatment using mass spectrometry techniques. The results indicated formation of product oligomers that could be removed in subsequent treatment processes. UV-VIS characteristics were used to confirm the formation of the azo products that were detected using mass spectrometry. And lastly, a pro-forma cost analysis for the feasibility of commercializing enzymatic treatment in comparison to other conventional treatment methods was performed. It was estimated that the total cost of enzymatic treatment of these pollutants was nearly 2 to 8 times less than that of the conventional treatment processes. It is recommended that enzymatic treatment could be employed to pretreat the effluent consisting of these toxic chemicals before they are released and/or mixed with wastewater from other industrial sources.