This Is AuburnElectronic Theses and Dissertations

Degradation of Phenolic Compounds Using Laccase or Peroxidase Enzymatic Treatment




Driver, Jeremy D.

Type of Degree



Civil Engineering


To remove ammonia from wastewater most refineries use biological treatment employing suspended biomass because there is no secondary pollutant. The main purpose of biological treatment is to accelerate the oxidation process of the organic matter (nitrification), which happens naturally within the receiving waters. This accelerated treatment is usually accomplished by using the activated sludge process. Although this method of treatment may be the preferred way to remove ammonia due to its simplicity and economic benefits, this process does have some disadvantages. One of the major problems associated with treating refinery wastewater is the potential for inhibition of the nitrifying bacteria due to several phenolic compounds found in the wastewater. The process of removing these phenolic compounds by using peroxidase and laccase has been researched by several researchers. This research has shown that these enzymes can react with aqueous phenolic compounds to create non-soluble compounds that can simply be removed from the aqueous phase using any conventional removal method. It has been hypothesized that the addition of hydrogen peroxide and either laccase or peroxidase enzymes can reduce the concentration of selected phenolic compounds in refinery wastewater. This enzymatic approach had been researched before; however, the majority of the previous research was done using a synthetic wastewater matrix, either de-ionized water or a buffer solution. Therefore, the main objective of this thesis was to determine the efficiency of the laccase and peroxidase enzymes at reducing the amount of phenols in an actual refinery wastewater matrix. To accomplish the objective of this thesis, this study was conducted in three phases. The first phase involved the enzymatic treatment of phenolic compounds in a simple matrix (de-ionized water) to determine both the efficiency of the enzymes and also the effect of hydrogen peroxide on the enzymes. The second phase involved the enzymatic treatment of various refinery wastewaters (CITGO, Frontier, and Imperial Oil). Lastly, the third phase investigated the effect of enzymatic treatment on nitrification. Phase one considered the efficiency of laccase and peroxidase treatment in both a simple matrix with only one individual compound present and a simple matrix with a defined mixture of phenolic compounds present. The testing of the individual compounds showed that the peroxidase/peroxide treatment had a slightly higher rate of removing the compounds from the simple matrix than the laccase/peroxide treatment (refer to Table 4.1). However, the overall percent remaining for all compounds were similar for both treatment options. The phenol and 2,4-dmp were reduced to about 25 to 30 percent remaining after one hour, whereas o-cresol and p-cresol were reduced to a remaining percent of about 45 to 50 percent (see Figures 4.5 and 4.6) In the simple matrix with defined concentrations of phenolic compounds it was determined that the peroxidase/peroxide treatment had a faster rate of removal and was more efficient. Phenol and 2,4-dmp had higher reduction rates than the o-cresol and p-cresol. Also noticed in this part of phase one is that the rates for removing the phenolic compounds in the defined mixture were lower than the rate for removing the compounds individually. In Phase Two the results for each individual wastewater differed slightly from each other. One important observation is that the rates for reducing the target phenols were generally lower than the rates determined in Phase One. Another difference is with most of the wastewater samples, the majority of the reduction did not occur until the last 15 to 30 minutes of the reaction; whereas, the reaction occurred in the first 15 to 30 minutes in Phase One with the clean system. Table 4.12 shows the average first order reduction rate constants for all compounds in the defined mixture. To confirm the finding in Phases One and Two, Phase Three testing was conducted using a commercially available culture of Nitrosomonas and Nitrobacter known as Nitrotox. Dilutions of treated wastewater were made over the range expected in the wastewater treatment process. The rate of nitrification in these dilutions was measured as the reduction in ammonia over time. Compared to the control (no enzymatic treatment), the ammonia reduction in the peroxidase/peroxide and laccase/peroxide treated samples was significantly higher. The results of this research increase the overall understanding of this enzymatic approach to removing phenol from refinery wastewater. The reduction of phenolic compounds by laccase and peroxidase in synthetic and refinery wastewater are explored throughout this investigation.