Degradation of Phenolic Compounds Using Laccase or Peroxidase 
Enzymatic Treatment 
 
by 
 
Jeremy Dwain Driver 
 
  
 
 
A thesis submitted to the Graduate Faculty of 
Auburn University 
in partial fulfillment of the 
requirements for the Degree of 
Master of Science 
 
Auburn, Alabama 
August 9, 2010 
 
 
 
 
Copyright 2010 by Jeremy Dwain Driver 
 
 
Approved by 
 
Clifford R. Lange, Chair, Associate Professor Civil Engineering 
Mark O. Barnett, Professor Civil Engineering 
Ah Jeong Son, Assistant Professor Civil Engineering 
 
 
 
 
 
ii 
 
 
 
 
 
 
 
Abstract 
 
 
 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 
iii 
 
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-
iv 
 
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. 
 
 
 
 
v 
 
 
 
 
 
Acknowledgments 
First and foremost, I offer my sincerest gratitude to my advisor, Dr. Clifford 
R. Lange, who has supported me throughout my thesis with his patience and 
knowledge whilst allowing me the room to work in my own way. I attribute the level 
of my Masters degree to his encouragement and effort and without him this thesis, 
too, would not have been completed or written. One simply could not wish for a 
better or friendlier advisor.  
Additionally, I would like to express my appreciation to CITGO, Frontier, and 
Imperial Oil for providing refinery wastewater samples for the purposes of research 
and analysis.  
Furthermore, I offer my regards and blessings to my amazing wife Emily for 
all her support and motivation throughout the course of completing this challenging 
educational endeavor. 
Lastly, I would like to thank my parents, other family and friends for 
supporting me throughout all my studies at and prior to Auburn University. 
 
 
 
 
 
 
 
 
 
 
 
vi 
 
 
 
 
 
 
Table of Contents 
 
 
Abstract ......................................................................................................................... ii 
Acknowledgments..........................................................................................................v  
List of Tables .............................................................................................................. vii  
List of Figures ............................................................................................................ viii  
Chapter 1: Introduction  .................................................................................................1 
Chapter 2: Literature Review  ........................................................................................7 
Chapter 3: Materials and Methods  ..............................................................................19 
Chapter 4: Results and Discussion  ..............................................................................31 
Phase One  .........................................................................................................32 
Phase Two  ........................................................................................................59 
Phase Three  ....................................................................................................125 
Chapter 5: Summary and Recommendations .............................................................140 
Bibliography ..............................................................................................................144 
 
 
 
 
 
 
 
 
 
 
 
vii 
 
 
 
 
 
List of Tables 
 
 
Table 3.1: Refinery Wastewater Sources  ....................................................................23 
Table 4.1: Individual Testing First Order Rate Constants ...........................................35 
Table 4.2: First Order Rate Constants for Each Compound in the Defined Mixture ..44 
Table 4.3: Laccase and Peroxidase Rate Constants from Individual Testing and   
       Synthetic Testing for Comparison ........................................................................53 
Table 4.4: First Order Rate Constants for Laccase Treatment of Phenol with  
       Varied H2O2 Concentrations  ................................................................................55 
Table 4.5: First Order Rate Constants for Peroxidase Treatment of Phenol with         
       Varied H2O2 Concentrations .................................................................................58 
Table 4.6: First Order Rate Constants for Laccase and Peroxidase in Frontier  
       Sour Water ............................................................................................................60 
Table 4.7: First Order Rate Constants for Laccase and Peroxidase in CITGO 
       Stripped Sour Water ..............................................................................................72 
Table 4.8: First Order Rate Constants for Laccase and Peroxidase in  
       Frontier Desalter  ..................................................................................................84 
Table 4.9: First Order Rate Constants for Laccase and Peroxidase in  
      CITGO Desalter .....................................................................................................93 
Table 4.10: First Order Rate Constants for Laccase and Peroxidase in the Dartmouth  
       Biox Influent .......................................................................................................105 
Table 4.11: First Order Rate Constants for Each Compound in the Sarnia Biox     
       Effluent ...............................................................................................................117 
 
Table 4.12: Average First Order Rate Constants for Phase Two ...............................142 
 
 
 
 
viii 
 
 
 
 
 
 
 
List of Figures 
 
 
Figure 2.1: Distillation Tower........................................................................................8 
Figure 4.1: Laccase/Peroxidase Treatment of Phenol in De-ionized Water  ...............33 
Figure 4.2: Example Plot .............................................................................................35 
Figure 4.3: Laccase/Peroxidase Treatment of O-cresol in De-ionized Water  ............37 
Figure 4.4: Laccase/Peroxidase Treatment of P-cresol in De-ionized Water ..............38 
Figure 4.5: Laccase/Peroxidase Treatment of 2,4-Dimethyl Phenol in  
       De-ionized Water ..................................................................................................39 
Figure 4.6: Fraction Remaining using Laccase/Peroxide Treatment ...........................40 
Figure 4.7: Fraction Remaining using Peroxidase/Peroxide Treatment ......................41 
Figure 4.8: Laccase Treatment of Phenol in the Defined Mixture ..............................43 
Figure 4.9: Laccase Treatment of O-cresol in the Defined Mixture ............................45 
Figure 4.10: Laccase Treatment of P-cresol in the Defined Mixture ..........................46 
Figure 4.11: Laccase Treatment of 2,4-Dimethyl Phenol in the Defined Mixture  .....47 
Figure 4.12: Peroxidase Treatment of Phenol in the Defined Mixture  .......................49 
Figure 4.13: Peroxidase Treatment of O-cresol in the Defined Mixture .....................50 
Figure 4.14: Peroxidase Treatment of P-cresol in the Defined Mixture  .....................51 
Figure 4.15: Peroxidase Treatment of 2,4-Dimethyl Phenol in the Defined  
       Mixture ..................................................................................................................52 
Figure 4.16: Rate Constants for Laccase/Peroxide with Varied Hydrogen Peroxide  
       Concentrations ......................................................................................................56 
ix 
 
 
Figure 4.17: Rate Constants for Peroxidase/Peroxide with Varied Hydrogen Peroxide  
       Concentrations  .....................................................................................................58 
Figure 4.18: Laccase Treatment of Phenol in Frontier Stripped Sour Water  .............61 
Figure 4.19: Laccase Treatment of O-cresol in Frontier Stripped Sour Water  ...........62 
Figure 4.20: Laccase Treatment of P-cresol in Frontier Stripped Sour Water  ...........63 
Figure 4.21: Laccase Treatment of 2, 4-Dimethyl Phenol in Frontier Stripped  
       Sour Water ............................................................................................................64 
Figure 4.22: Fraction Remaining of Phenolic Compounds using Laccase  
       Treatment on Frontier Stripped Sour Water  ........................................................65 
Figure 423: Peroxidase Treatment of Phenol in Frontier Stripped Sour Water  ..........67 
Figure 4.24: Peroxidase Treatment of O-cresol in Frontier Stripped Sour Water  ......68 
Figure 4.25: Peroxidase Treatment of P-cresol in Frontier Stripped Sour Water  .......69 
Figure 4.26: Peroxidase Treatment of 2,4-Dimethyl Phenol in Frontier Stripped  
       Sour Water ............................................................................................................70 
Figure 4.27: Fraction Remaining of Phenolic Compounds using Peroxidase  
       Treatment on Frontier Stripped Sour Water .........................................................71 
Figure 4.28: Laccase Treatment of Phenol in CITGO Stripped Sour Water  ..............73 
Figure 4.29: Laccase Treatment of O-cresol in CITGO Stripped Sour Water  ...........74 
Figure 4.30: Laccase Treatment of P-cresol in CITGO Stripped Sour Water  ............75 
Figure 4.31: Laccase Treatment of 2,4-Dimethyl Phenol in CITGO Stripped 
       Sour Water ............................................................................................................76 
Figure 4.32: Fraction Remaining of Phenolic Compounds using Laccase  
       Treatment on CITGO Stripped Sour Water   ........................................................77 
Figure 4.33: Peroxidase Treatment of Phenol in CITGO Stripped Sour Water  .........79 
Figure 4.34: Peroxidase Treatment of O-cresol in CITGO Stripped Sour Water  .......80 
Figure 4.35: Peroxidase Treatment P-cresol in CITGO Stripped Sour Water  ............81 
x 
 
 
Figure 4.36: Peroxidase Treatment of 2,4-Dimethyl Phenol in CITGO Stripped  
       Sour Water ............................................................................................................82 
Figure 4.37: Fraction Remaining of Phenolic Compounds using Peroxidase  
       Treatment on CITGO Stripped Sour Water   ........................................................83 
Figure 4.38: Laccase Treatment of Phenol in Frontier Desalter Effluent  ...................85 
Figure 4.39: Laccase Treatment of O-cresol in Frontier Desalter Effluent  ................86 
Figure 4.40: Laccase Treatment of P-cresol in Frontier Desalter Effluent  .................87 
Figure 4.41: Fraction Remaining of Phenolic Compounds using Laccase  
       Treatment on Frontier Desalter Effluent ...............................................................88 
Figure 4.42: Peroxidase Treatment of Phenol in Frontier Desalter Effluent  ..............89 
Figure 4.43: Peroxidase Treatment of O-cresol in Frontier Desalter Effluent  ...........90 
Figure 4.44: Peroxidase Treatment of P-cresol in Frontier Desalter Effluent  ............91 
Figure 4.45: Fraction Remaining of Phenolic Compounds using Peroxidase  
       Treatment on Frontier Desalter Effluent ...............................................................92 
Figure 4.46: Laccase Treatment of Phenol in CITGO Desalter Effluent  ...................94 
Figure 4.47: Laccase Treatment of O-cresol in CITGO Desalter Effluent  .................95 
Figure 4.48: Laccase Treatment of P-cresol in CITGO Desalter Effluent  .................96 
Figure 4.49: Laccase Treatment of 2,4-Dimethyl Phenol in CITGO  
       Desalter Effluent  ..................................................................................................97 
Figure 4.50: Fraction Remaining of Phenolic Compounds using Laccase  
       Treatment on CITGO Desalter Effluent ...............................................................98 
Figure 4.51: Peroxidase Treatment of Phenol in CITGO Desalter Effluent  ...............99 
Figure 4.52: Peroxidase Treatment of O-cresol in CITGO Desalter Effluent  ..........100 
Figure 4.53: Peroxidase Treatment of P-cresol in CITGO Desalter Effluent  ...........102 
Figure 4.54: Peroxidase Treatment of 2,4-Dimethyl Phenol in CITGO Desalter  
       Effluent  ..............................................................................................................103 
xi 
 
Figure 4.55: Fraction Remaining of Phenolic Compounds using Peroxidase  
       Treatment on CITGO Desalter Effluent .............................................................104 
Figure 4.57: Laccase Treatment of Phenol in Dartmouth Biox Influent ...................106 
Figure 4.58: Laccase Treatment of O-cresol in Dartmouth Biox Influent .................107 
Figure 4.58: Laccase Treatment of P-cresol in Dartmouth Biox Influent .................108 
Figure 4.60: Laccase Treatment of 2,4-Dimethyl Phenol in Dartmouth  
       Biox Influent .......................................................................................................109 
Figure 4.61: Fraction Remaining of Phenolic Compounds using Laccase Treatment  
       on Dartmouth Biox Influent ................................................................................110 
Figure 4.62: Peroxidase Treatment of Phenol in Dartmouth Biox Influent ...............112 
Figure 4.63: Peroxidase Treatment of O-cresol in Dartmouth Biox Influent ............113 
Figure 4.64: Peroxidase Treatment of P-cresol in Dartmouth Biox Influent .............114 
Figure 4.65: Peroxidase Treatment of 2,4-Dimethyl Phenol in Dartmouth  
       Biox Influent .......................................................................................................115 
Figure 4.66: Fraction Remaining of Phenolic Compounds using Peroxidase  
       Treatment on Dartmouth Biox Inluent ................................................................116 
Figure 4.67: Laccase Treatment of Phenol in Sarnia Biox Effluent ..........................118 
Figure 4.68: Laccase Treatment of O-cresol in Sarnia Biox Effluent .......................119 
Figure 4.69: Laccase Treatment of P-cresol in Sarnia Biox Effluent ........................120 
Figure 4.70: Fraction Remaining of Phenolic Compounds using Laccase Treatment  
       on Sarnia Biox Influent .......................................................................................121 
Figure 4.71: Peroxidase Treatment of Phenol in Sarnia Biox Effluent .....................122 
Figure 4.72: Peroxidase Treatment of P-cresol in Sarnia Biox Effluent ...................123 
Figure 4.73: Peroxidase Treatment of O-cresol in Sarnia Biox Effluent ...................124 
Figure 4.74: Fraction Remaining of Phenolic Compounds using Peroxidase  
       Treatment on Sarnia Biox Influent .....................................................................125 
Figure 4.75: Ammonia Oxidation using CITGO Stripped Sour Water .....................127 
xii 
 
Figure 4.76: Ammonia Oxidation using Frontier Stripped Sour Water .....................130 
Figure 4.77: Ammonia Oxidation using Dartmouth Biox Influent............................132 
Figure 4.78: Ammonia Oxidation using Nanticoke Desalter .....................................135 
Figure 4.79: Ammonia Oxidation using Sarnia Biox Effluent ..................................137 
 
  
1 
 
 
 
 
Chapter 1 
Introduction  
 
There are approximately 140 operating oil refineries in the United States and 
many more world-wide. An oil refinery must fractionate crude oil into useable 
petroleum products such as diesel and gasoline. To separate these fractions, the crude 
oil must go through one or both of two main processes used at a refinery, separation 
and conversion (Hengstebeck 1959). Unfortunately, these processes form several 
aqueous waste products. Sour water is any wastewater that contains hydrogen sulfide, 
and is the main wastewater from oil refineries (Beychok 1967).  In addition to 
hydrogen sulfide, most sour water also contains high concentrations of phenol, 
substituted phenolic compounds, ammonia, and cyanide (Meyers 1997). Wastewater 
containing high concentrations of nitrite, ammonia, and ammines is a major problem 
at refineries all over the world.  Nitrate is considered a carcinogen and can also have 
harmful effects on human health, such as methylmyoglobia also known as the blue-
baby syndrome (Wang et al. 2007).  Complete removal of ammonia nitrogen from the 
wastewater is required due to its extreme toxicity to most fish species and other 
aquatic life, the high oxygen demand associated with ammonia, and the stimulation of 
aquatic plants and algae (Jorgensen and Weatherley 2003). 
2 
 
Nitrification is the most common way to remove ammonia from wastewater. 
Nitrification is the microbial oxidation of ammonia into nitrites (NO2-), and then into 
nitrate (NO3-). With this method, organic nitrogen and ammonia nitrogen is converted 
into nitrite and nitrate nitrogen in an aerobic environment, subsequently may be 
released into the atmosphere as nitrogen gas after anaerobic denitrification 
(Sintobrator 2002). 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, which happens naturally within the receiving waters. 
This accelerated treatment is usually accomplished by using the activated sludge 
process (Ben-Youssef et al. 2009). Activated sludge was first recognized as a possible 
solution for wastewater treatment in 1914. Edward Arden and William T. Lockett 
noticed that bacterial binding material forms naturally during prolonged aeration of 
urban wastewaters. This allowed the ?activated solids? to be recycled and process 
efficiency to be greatly increased. Thus, the activated sludge process was born. Some 
of the organic matter is changed into biomass (sludge) and the rest is mineralized to 
carbon dioxide and water. The bacterial binding material can then be separated out 
from the treated water using simple settling techniques. In addition to removing 
organics, the activated sludge process has been shown to oxidize ammonia into 
nitrate. This process is referred to as nitrification and occurs concurrently with 
organic carbon oxidation (Ben-Youssef et al. 2009). 
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 
3 
 
disadvantages. One disadvantage is the susceptibility of the nitrifying bacteria to 
several environmental factors such as pH, temperature, alkalinity, and oxygen 
concentration (Liu et al. 2005).  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. Kim et al. (2008) concluded 
that phenol and p-cresol when above 200 and 100 mg/L respectively could noticeably 
inhibit nitrification. Thus, the removal of phenols from refinery wastewater is a 
practical problem of great significance (Singh and Singh 2002). 
There are several methods that can be used to remove phenol from 
wastewater, including; activated carbon, microbial degradation, incineration methods, 
chemical oxidation, solvent extraction, and irradiation (Miland et al. 1996; Tong et al. 
1997). Even though these techniques can be used to remove phenol from the 
wastewater, high cost, long times, and inability to avoid the formation of and possible 
release of hazardous by products top the list of disadvantages (Lante et al. 2000). 
However, bioremediation strategies look to be an environmental-friendly alternative 
to thermal or chemical treatment of phenolic waste (Moeder et al. 2004).   
An enzymatic approach to removing phenols from the wastewater using 
laccase and peroxidase enzymes was proposed as early as 1980 (Klibanov and Alberti 
1981). The process of removing phenols 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 
(Cooper and Nicell 1996).  
4 
 
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, several representative petroleum 
refinery wastewaters were obtained. The petroleum refineries that served as the 
source of test samples included two refineries from the United States (CITGO and 
Frontier), and three Canadian refineries (Imperial Oil). These five refineries were 
chosen because they represent the different types of crude being processed here in 
North America. The US refineries mostly process Texas ?sweet crude? although they 
sometimes process Canadian or Venezuelan tar sand synthetic crude. The three 
Canadian refineries process a mixture of ?sweet crude? and Canadian tar sand 
synthetic crude. ?Sweet crude? is a type of crude that is not very complex and often 
easy to process, where as tar sand synthetic crude comes from adding several water 
soluble compounds such as ethylene glycol or propylene glycol to the tar sand 
mixture to produce the synthetic crude. Samples were taken at various points 
throughout wastewater treatment including: desalter, sour water, coker, DAF effluent, 
biox influent, and biox effluent. These locations were selected because they 
encompass the sources of refinery wastewater.  
5 
 
 The hypothesis of this thesis is that the addition of hydrogen peroxide, and 
either peroxidase or laccase enzymes, can lower the concentration of phenolic 
compounds in actual refinery wastewater matrices.  This hypothesis is based on the 
experimental data and anecdotal information given by previous research. However, 
one the significant limitations of these laboratory studies to refinery wastewater is the 
effect which the complex array of organic and inorganic contaminants in actual 
refinery wastewater have upon the hydrogen peroxide/ enzyme function. In this study 
the five refineries were chosen so that the wastewaters would vary in both phenolic 
compounds and the aqueous matrix, and represent a cross-section of refinery 
wastewater.  
A three phase approach was utilized to determine the efficiency of peroxidase 
and laccase. In phase one of this study, the enzymatic approach to reducing phenols 
was tested in de-ionized water. Hydrogen peroxide and an enzyme were added to a 
synthetic wastewater, and then tested every fifteen minutes on the gas chromatograph. 
Also tested in this phase of the study was the effect of hydrogen peroxide on the 
enzymes. To determine if hydrogen peroxide affected the enzyme?s ability to lower 
phenol concentrations, different concentrations of hydrogen peroxide were added to 
synthetic wastewater along with an enzyme, and then the sample was tested on the 
gas chromatograph over time.  
In the second phase of this research, actual refinery wastewater was used to 
determine peroxidase and laccase enzyme?s ability to reduce the concentration of 
certain phenolic compounds found in the wastewater. An enzyme and hydrogen 
6 
 
peroxide were added to the refinery wastewater samples, and then tested on the gas 
chromatograph over time.  
The third phase of the study considered the effect of the enzymatic treatment 
on nitrification. Treated refinery wastewater was compared against untreated 
wastewater to determine the amount of ammonia oxidized. Since phenol inhibits the 
nitrifying bacteria, the untreated wastewater should have a lower amount of ammonia 
being oxidized.  
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 and the results are presented herein. 
Therefore, by uniting the knowledge obtained from past research, summarized in the 
following chapter (Chapter 2), and that acquired by conducting this thesis (Chapters 3 
and 4), perceptive familiarity with this enzymatic treatment approach should ensue, 
and hopefully will spark interest in connected research topics yet to be explored.  
 
 
 
 
 
 
 
 
 
 
 
7 
 
 
 
Chapter 2 
Literature Review 
 
General Info 
Crude oil can be described not as a chemical compound, but a mixture of 
many different chemical compounds. Some as simple as methane [CH4]; others are 
very complex hydrocarbons. Most significantly though is when it is heated to a 
boiling point and held there, unlike water it will not all evaporate (Leffler 1979). 
According to the U.S. Energy Information Association (2009), crude oil can be used 
in many ways, the world uses petroleum products to help move people and 
merchandise by using gasoline and diesel in engines, and to help make plastics, and to 
even make things like crayons, CD?s, ink, and bubble gum (Gary and Handwerk 
1984).  
In order to make a wide range of consumer products, different parts of the 
crude oil are separated into useable petroleum feed stocks at an oil refinery. There are 
around 165 operating oil refineries in North America. Today, most refineries process 
over half of every 42-gallon barrel of crude oil into gasoline (U.S. 2008).  Since the 
properties of crude oils are so diverse and the number of products so abundant the 
name ?petroleum refinery? gives little indication of what operations or equipment is 
used at a refinery. Most petroleum refineries have two main processes; separation 
8 
 
including distillation and conversion including cracking and reforming (Hengstebeck 
1959). 
In separation or distillation, the difference in heavy and light petroleum 
components or fractions allows the various chemical components to separate out. 
Modern separation involves sending the petroleum through a hot furnace (Waddams 
1980). Then the resulting vapors and liquids are discharged into distillation towers. 
Once in these towers the vapors and liquids separate according to their boiling points. 
Figure 2.1 shows the different components of petroleum separating out of a 
distillation tower. The lightest components such as gasoline and liquid petroleum gas 
(LPG) vaporize and rise to the very top of the distillation tower where they then 
condense back to liquids. Kerosene, diesel oil distillates, naphtha and other medium 
weight liquids stay in the middle of the tower. Heavier liquids with the highest 
boiling points such as gas oils settle out at the bottom (Gary and Handwerk 1984).  
 
Figure 2.1: Distillation Tower (Huiyi) 
9 
 
 
The most commonly used conversion method is cracking, because it uses heat 
and pressure to "crack" complex and heavy hydrocarbon molecules into lighter ones. 
Cracking has been a part of the refinery process for a long time. Catalytic cracking, 
since its first successful application in 1936, has become the largest of all catalytic 
operations (Hengstebeck 1959). This cracking and rearranging takes the output from 
an earlier process, the heavy, low-valued feedstock, and changes it to something such 
as gasoline, a higher valued output. This is where output components from the 
distillation towers are transformed into intermediate components that ultimately are 
changed into the end products (Gary and Handwerk 1984).  
Cracking is not the only form of conversion used in refining. Some processes 
rearrange the molecules to add value instead of splitting them. Take alkylation for 
example, which hit the industrial scene in 1955 (Hengstebeck 1959). In the alkylation 
process, which is basically cracking in reverse, several gaseous byproducts are 
combined to form different gasoline components (Meyers 1997).   
Catalytic reforming is another form of conversion. Introduced in the 1950?s, 
reforming was pioneered to help improve the octane number of gasoline. This process 
uses pressure, catalysts, and heat to convert poly aromatic hydrocarbons or naphtha (a 
low-value component of distillation) into high-octane liquid products called 
reformates. These reformates are components of gasoline (Leffler 1979).   
Wastewater Characteristics  
 Sour condensate or sour water is the main waste water source by volume from 
petroleum plants. Sour water is defined as any water that has hydrogen sulfide in it 
10 
 
(CASTion 2008). The sour water from a refinery usually comes from using steam as a 
stripping medium in distillation and specifically the distillation reflux drum. Some 
refineries also inject wash water or steam to absorb corrosive compounds or control 
pH (Beychok 1967). Thus any steam or wash water that comes in contact with 
hydrogen sulfide is considered sour water (Meyers 1997). Furthermore, there is most 
always free NH3 in the water thus causing the water to contain mostly NH4SH rather 
than free H2S or free NH3. There are several points in the refining process that 
produce sour water; crude oil distillation, hydrotreating, catalytic reforming, catalytic 
cracking, thermal cracking, and hydrocracking (Beychok 1967). In addition to 
hydrogen sulfide and ammonia, sour water can also have phenol, substituted phenolic 
compounds, and cyanide (Meyers 1997). Traditionally, the sour water is treated to 
eliminate the hydrogen sulfide before being released. However, due to current 
regulations reducing nitrogen in estuaries, the removal of ammonia is now essential 
(CASTion 2008).  The ammonia in refinery sour water comes from either the nitrogen 
in the crude oil or form when ammonia and/or amines are added into the crude 
fractionator in order to cut down on sulfide catalyzed corrosion (Meyers 1997). 
Ammonia Removal 
 The pollution of ground water with nitrate is a severe problem in many places 
all over the world.  Nitrate is considered a carcinogen and can also have harmful 
effects on human health, such as methemoglobinemia (blue-baby syndrome) (Wang 
et al. 2007).  Complete removal of ammonia is required due to these harmful effects 
as well as extreme aquatic toxicity, and the high oxygen demand. (Jorgensen and 
Weatherley 2003). There are various methods of removing nitrogen, each with 
11 
 
advantages and disadvantages. Although, nitrification, the biological treatment 
method, is the most commonly used at oil refineries, other methods are also 
employed, including: sour water stripping and ion exchange. These processes 
available for ammonia removal, and their advantages and disadvantages, will be 
discussed in the following sections.  
Nitrification 
The most common way to remove ammonia from refinery wastewater is 
through the microbial oxidation of ammonia into nitrite, and then into nitrate, which 
is referred to as nitrification. In nitrification, organic nitrogen and ammonia nitrogen 
is converted into nitrite and nitrate nitrogen in an aerobic environment, and is 
disposed of into the atmosphere as nitrogen gas following anaerobic denitrification 
(Sintobrator 2002). Russian microbiologist Sergei Winogradsky discovered that by 
isolating certain soil bacteria he could clearly show the process of nitrification. Thus, 
he concluded that this process was the result was due to bacterial action.  In the first 
step of nitrification, ammonia-oxidizing bacteria oxidize ammonia to nitrite according 
this equation:  NH3+O2? NO 2-+ 3H++ 2e-  (Watson et al. 1981).  Of all the bacteria 
that can perform this step, Nitrosomonas is the most commonly known genus 
associated with this process, and is also usually used in activated sludge (Fang et al. 
1993). In the second step of nitrification, nitrite oxidizing bacteria oxidize nitrite to 
nitrate according to this equation: NO2-+ H2O ? NO 3-+ 2H++2e-   (Watson et al. 
1981).  Nitrobacter is the main genus isolated from nitrifying activated sludge (Fang 
et al. 1993).  
12 
 
Biological treatment, using suspended biomass, is currently the most common 
treatment solution used to treat wastewaters. The main purpose is to accelerate the 
oxidation process of the organic matter, which happens naturally within the receiving 
waters. This accelerated treatment is usually accomplished by using the activated 
sludge technique (Ben-Youssef et al. 2009). Activated sludge was first recognized as 
a possible solution in 1914. Edward Arden and William T. Lockett noticed that 
bacterial binding material forms naturally during prolonged aeration of urban 
wastewaters. Thus the activated sludge process was born. Some of the organic matter 
is changed into biomass and the rest is mineralized to carbon dioxide and water. The 
bacterial binding material can then be separated out from the treated water using 
simple settling techniques. Originally, both aeration and sedimentation phases were 
done in the same tank or container. This process is currently referred to as sequencing 
batch reactor (SBR) technology. Presently, the sedimentation phase is preformed in a 
second tank to allow for a continuous operating process. Today the activated sludge 
process consist of two different tanks: an aeration tank with suitable biomass for 
settling and a settling tank where the purified water is separated from the activated 
sludge through settling.  
Although this method of treatment may be preferred in some places due to its 
simplicity and economic benefits, this process does have its disadvantages. Nitrifying 
bacteria such as ones listed above are susceptible to several environmental factors 
including pH, substrate concentration, temperature, oxygen concentration, light, 
heavy metals, and heavy organic compounds or solvents (Liu et al. 2005). Kim et al. 
concluded that phenol and p-cresol when above 200 and 100 mg/L respectively could 
13 
 
noticeably inhibit nitrification (2008). Temperature is also a major problem since 
refinery wastewater is often 100? F or higher.  
Sour Water Stripping 
Sour water stripping or ammonia stripping is one way to remove ammonia 
from industrial wastewater. Through this process ammonia is separated from the 
aqueous solution due to their differences in boiling points and vapor pressures (Elston 
and Karmarkar 2007).  However, the sour water must first be prepped by first being 
pumped into a flash drum to remove hydrocarbon vapors and liquids.  The sour water 
is then pumped to a storage tank to allow for further hydrocarbon removal and longer 
mixing time to minimize feed composition fluctuations. The hydrocarbons need to be 
fully removed before the next step otherwise the sour water stripper would become a 
volatile hydrocarbon stripper. From the storage tank, the sour water is pumped on to 
sour water stripper. The sour water is heated in a heat exchanger by hot stripped water 
from the stripper bottoms and then fed into the stripping tower. As the sour water fall 
down the down the tower, ammonia and also hydrogen sulfide are stripped by steam 
entering the tower from below. Thus, the hydrogen sulfide, ammonia, and steam rise 
to the cooling section of the tower, which is cooled by sour water from the middle 
part of the tower being pumped around it. After being cooled, stripped water is then 
sent off site for further water processing (Armstrong et al. 1996).  
Among the several at-source treatment options, steam stripping may offer a 
general and simple method. In addition to its ability to remove several organic 
compounds, steam stripping offers several advantages over other treatment methods. 
A stripper can be built with well developed, but simple equipment (Hwang et al. 
14 
 
1992). However, when designing a stripper it is important to balance the investment 
costs with the operating costs. There are also several operating variables that can 
affect the stripping of the ammonia and hydrogen sulfide in the water. These include 
the steam rate, the tower pressure, and the pH of the sour water (Lee et al. 2002). 
Increasing the stripping rate is one way o raise the stripping rate of ammonia and 
hydrogen sulfide, although the recommended steam rate is about three pounds of 
steam per gallon of sour water (Armstrong et al. 1996). pH is a little complicated. 
Raising the pH will aid in releasing ammonia, but hydrogen sulfide tends to stay in 
solution. Conversely a low pH will tend to hold ammonia in solution, and improve the 
stripping of hydrogen sulfide. Therefore, it is essential to determine what specific 
species are present and at what concentrations levels in the sour water (Melin et al. 
1975).  
Ion Exchange 
Today, biological treatment is the traditional method for ammonia and organic 
removal, however, ion exchange has been around since the early 1850s, when 
agriculturist Harry S. Thompson and chemist John Holmes Way independently 
reported the phenomenon. They discovered that upon treating soil with either 
ammonium sulfate or ammonium carbonate, the majority of the ammonia was 
absorbed and calcium was released into solution.  Ion exchange can be defined as the 
as a reversible exchange of ions involving a solid and a liquid in which the structure 
of the solid is not significant altered. Also the ion exchanger can be regenerated or 
overloaded with desirable ions by washing with an excess of these ions (Dow 
Chemical Company. 1958).  
15 
 
In municipal and industrial wastewater treatment systems designed to remove 
ammonium ions, organic pollutants are likely to be present. Some heterotrophic 
bacteria that can consume these organic species hinder the growth and activity of 
nitrifying bacteria used in biological treatment. In this case, ion exchange can offer an 
alternative solution to the removal of ammonia.  The most common used ion 
exchanger is zeolites, due to their porous structure and high cation-exchange capacity 
(Wang and Peng 2010). There are many different natural zeolites, ones such as 
clinoptilolite, which is the most abundant and has a high affinity for ammonium ions, 
sepiolite, and select other natural zeolites can effectively remove ammonia from 
wastewater (Jorgensen and Weatherley 2003).  In one study, high concentrations of 
ammonia were reduced by around 60% through the use of sepiolite as the ion 
exchanger (Balci and Dincel 2002). In another, clinoptilolite was used as the ion 
exchanger and a removal of 80% was reached (Park et al. 2002).   
Ion exchange has drawn interest from researchers as a method of removing 
ammonia due since it is hardly influenced by temperature change, its ease of 
operation, and it has a relative low cost (Wang et al. 2007). Although ion exchange 
has these advantages, one of the most significant issues in the application of it in 
industrial wastewater is the lack of research on the effects of other contaminants in 
the wastewater on the techniques used (Jorgensen and Weatherley 2003).  Another 
disadvantage is that free product (NAPL oil) can interfere with ion exchange. Also 
the zeolites that are used either need to be discarded or regenerated, and the major 
drawback to this is the high cost (Li et al. 2010).  
 
16 
 
Phenol Removal 
Phenols can occur in a number of industrial wastewaters, wastewater such as 
that from petroleum refining, wood and dye industries, high temperature coal 
conversion, and resin and plastic industries (Tong et al. 1997). It is known to be a 
carcinogen and toxic at high levels (Miland et al. 1996). Thus, the removal of phenols 
from industrial wastewater is a practical problem of great significance (Singh and 
Singh 2002). 
When it comes to removing phenol from wastewater, there are several 
methods that can be used including; using activated carbon, microbial degradation, 
incineration methods, chemical oxidation, solvent extraction, and irradiation are 
available (Miland et al. 1996; Tong et al. 1997). Although these techniques can 
remove phenol from an aqueous matrix, the formation of and possible release of 
hazardous by-products from the polychlorophenols during incineration or even 
composting is often tough to avoid. Other conventional techniques such as solvent 
extraction, adsorption on to activated carbon, and chemical oxidation also have some 
serious disadvantages. High cost, incompleteness of purification, and again the 
formation of hazardous by-products top the list of disadvantages (Lante et al. 2000). 
However, biological treatment strategies look to be an environmental-friendly 
alternative to thermal or chemical treatment of phenolic waste (Moeder et al. 2004).  
There are several enzymatic approaches to removing the phenols from the 
industrial wastewater using laccase and peroxidase enzymes, plus ways using other 
enzymes such as phenolase. This approach was first proposed in the early 1980s, and 
was able to remove over 30 different phenols from water with an efficiency of 99% in 
17 
 
some wastewaters (Klibanov and Alberti 1981). A catalytic process of removing 
phenol by using peroxidase and laccase has been research by several researchers 
(Alemzadeh and Nejati 2009). This research demonstrated 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 
(Cooper and Nicell 1996).   In this process, phenols are oxidized to form the 
equivalent free radicals in the presence of hydrogen peroxide as an oxidant. The free 
radicals are then polymerized into less soluble compounds than the original matter 
and are then removed via settling and/or sorption methods (Singh and Singh 2002). 
This process has many advantages over the current methods listed above, and has 
been shown to be applicable for the treatment of many industrial wastewaters 
(Nakamoto and Machida 1992; Nicell et al. 1993). However, enzyme cost has to be 
significantly reduced in order to make this treatment option economically competitive 
(Cooper and Nicell 1996). The following is several case studies done on this 
enzymatic approach.  
 By using immobilized turnip seed peroxidase in the presence of hydrogen 
peroxide, Singh and Singh (2002) were able to treat and remove 95 % of phenol from 
a synthetic wastewater.  In their experiments 200 ?L of hydrogen peroxide and 90-
100 units of peroxidase enzyme were added to 10 mL of synthetic industrial 
wastewater.  In another study by Almezdeh and Nejati (2009) focused on 
immobilized horseradish peroxidase?s ability to removal phenol in synthetic 
wastewater. Cooper and Nicell (1996) studied the effects of horseradish peroxidase 
on foundry wastewater. They used two different enzyme stocks of different purities, 
18 
 
and results showed that 97 and 99% of phenolic compounds were removed. Klibanov 
and Morris (1981)successfully utilized horseradish peroxidase in polymerizing phenol 
in the effluent of coal-conversion wastewater. They also showed that this enzymatic 
treatment was effective over a wide range of pH, temperatures, and phenol 
concentrations (Alberti and Klibanov 1981; Klibanov 1982). In a study by Moeder et 
al. (2004) laccase and horseradish peroxidase were used to study the degradation of 
several phenolic compounds. They found that most of the phenols in the buffer 
solution were efficiently removed within 48 hrs. In another study, immobilized 
commercial laccase was investigated. This laccase was tested in a model phenol 
solution containing several phenolic substrates. The enzyme laccase could remove 
phenols from this solution although it also can convert them in to intermediates such 
as catechol or guaiacol.  
 
 
 
 
 
 
 
 
 
 
 
19 
 
 
 
Chapter 3 
Materials and Methods 
 
 The goal of the research presented in this thesis was to determine if the 
addition of laccase and peroxidase enzymes along with hydrogen peroxide can reduce 
the concentration of phenolic compounds commonly found in refinery wastewater. To 
accomplish this goal, several representative petroleum refinery wastewaters were 
obtained. The petroleum refineries that served as the source of test samples included 
two refineries from the United States (CITGO and Frontier), and three Canadian 
refineries (Imperial Oil). These five refineries were chosen because they represent the 
different types of crude being processed here in North America. The US refineries 
mostly process Texas ?sweet crude? although they sometimes process Canadian or 
Venezuelan tar sand synthetic crude. The three Canadian refineries process a mixture 
of ?sweet crude? and Canadian tar sand synthetic crude. ?Sweet crude? is a type of 
crude that is not very complex and often easy to process, where as tar sand synthetic 
crude comes from adding several water soluble compounds such as ethylene glycol or 
propylene glycol to the tar sand mixture to produce the synthetic crude. Samples were 
taken at various points throughout wastewater treatment including: desalter, sour 
water, coker, DAF effluent, biox influent, and biox effluent. These locations were 
selected because they encompass the sources of refinery wastewater.  
20 
 
The hypothesis of the research is that the addition of hydrogen peroxide and 
either laccase or peroxidase enzymes can reduce the concentration of phenolic 
compounds in these wastewaters. This hypothesis is based on the experimental data 
and anecdotal information given by previous research. However, one the significant 
limitations of these laboratory studies to refinery wastewater is the effect which 
contaminants in actual refinery wastewater have upon the technique used. In this 
study five refineries were chosen so that the wastewaters would vary in both phenolic 
compounds and the aqueous matrix, and any adverse effects could be seen. Therefore, 
to test the hypothesis, the wastewater samples were exposed to various concentrations 
of the enzymes and hydrogen peroxide. The resulting changes in phenol and phenolic 
compound concentrations were measured using gas chromatography. The 
experimental study approach is outlined in this section.  
Experimental Study Approach  
 The research was conducted in three phases. The first phase involved the 
enzymatic treatment of phenolic compounds in de-ionized water. Enzyme and 
hydrogen peroxide were added to this synthetic wastewater, and then the sample was 
tested on the gas chromatograph over time. Also studied in this phase was the effect 
of hydrogen peroxide on the enzymes. 
The second phase involved the enzymatic treatment of refinery wastewater. 
Enzyme and hydrogen peroxide were added to the refinery wastewater samples. The 
sample was then tested on the gas chromatograph over time.   
21 
 
The third phase of the study investigated the effect of enzymatic treatment on 
nitrification. This was determined by analyzing the amount of ammonia being 
oxidized in each of the samples.  
Phase 1: Synthetic Wastewater  
 In this phase of the research, the laccase and peroxidase were tested fro the 
ability to remove common phenolic compounds individually and in mixtures of 
simulated wastewater. Phenol, 3-methyl phenol (m-cresol), 4-methyl phenol (p-
cresol), and 2, 4-dimethyl phenol were chosen for the study based on their high 
concentrations and common occurrence in refinery wastewater. For each of the 
individual test, 100 ppm of the selected compound was added to de-ionized water. 
This mixture was tested via the gas chromatograph (GC) to determine the starting 
concentrations. Once complete, an enzyme and hydrogen peroxide were added and 
tested in 15 minute intervals over the span of one hour. The make-up of the synthetic 
wastewater was as follows: 100 ppm phenol, 100 ppm m-cresol, 100 ppm p- cresol, 
and 50 ppm 2-4 dimehyl phenol in 100mL de-ionized water. This mixture was tested 
via the gas chromatograph to determine intial concentrations of the compounds 
added. Then 0.1 ml of enzyme (approximately 2,600 units of peroxidase or 200 units 
of laccase) and 1 mL of hydrogen peroxide (300 mg/L) were added to the sample. 
The sample was then test over time every 15 minutes for 60 minutes via the gas 
chromatograph.  
Also tested in this phase of the study was the effect of hydrogen peroxide on 
the enzymes. A synthetic wastewater was created using 100ppm of phenol, 100ppm 
of o-cresol, 100ppm of p-cresol, and 50ppm of 2,4-dimethyl phenol. Once the 
22 
 
synthetic wastewater was made, 2600 units of peroxidase or 200 units of laccase and 
varied concentrations of hydrogen peroxide were added to the sample. This testing 
was done to determine if the concentration of hydrogen peroxide affected the 
reduction of these phenolic compounds by the enzyme. The concentrations used in 
this part of testing were 600 mg/L, 300 mg/L, 150 mg/L, and 75 mg/L of hydrogen 
peroxide. Samples were also tested with peroxide strips to determine the hydrogen 
peroxide concentrations in the sample over time intervals of 0, 15, 30, 45, and 60 
minutes.  The reason for this test was to ensure that hydrogen peroxide was not 
limiting the enzymes? ability to oxidize the phenols.  
Phase 2: Refinery Wastewater 
 In phase 2, the ability of laccase and peroxidase enzymes to remove the four 
target phenolic compounds from refinery wastewater obtained from five working 
refineries was tested. The experiments differed from phase one testing by the fact that 
the phenolic compounds were in an actual refinery wastewater matrix. The 
wastewaters tested were obtained from various sites in the refineries, including: i) 
coker, ii) desalter, iii) Dissolved Air Floatation (DAF) effluent, iv) Biological 
treatment (Biox) influent, and v) Biox effluent. Since the refineries differ in 
production and wastewater treatment operations, not all of these samples were 
obtained from each refinery. The individual samples tested for each of the five 
refineries are summarized in Table 3.1.  
 
 
 
23 
 
Table 3.1: Refinery Wastewater Sources 
 
 
The concentrations of phenolic compounds were measured in the wastewater 
using gas chromatography. In addition, the concentrations of other organic 
compounds (phenolic and non-phenolic) in the wastewater were measured. Once 
initial concentrations were determined, 2600 units of peroxidase or 200 units of 
laccase (0.1 mL of enzyme) were added along with 300 mg/L (1 mL) of hydrogen 
peroxide to 100 mL of the selected refinery wastewater. This mixture was then tested 
at time intervals of 0, 15, 30, 45, and 60 minutes on the gas chromatograph.  
Phase 3: Nitrifying  
 In phase three of this study, the samples used in phase two were also tested to 
determine if nitrification was being affected by this enzymatic treatment. This can be 
shown by the amount of ammonia being oxidized in the sample. It is hypothesized 
that the amount of ammonia being oxidized will be lower in the control wastewater, 
or the non-treated sample, verses the samples treated with peroxidase or laccase. 
Samples included in this phase of testing were: CITGO stripped sour water, Frontier 
Refinery 
Sour 
Water Coker Desalter DAF 
Biox 
influent 
Biox 
effluent 
Lake Charles 
(CITGO) X X X X   
Cheyenne, WY 
(Frontier) X  X    
Dartmouth 
(Imperial Oil)   X X X  
Nanticoke 
(Imperial Oil)   X  X X 
Sarnia  
(Imperial Oil)  X X  X X 
24 
 
stripped sour water, Dartmouth biox influent, Nanticoke biox influent, and Sarnia 
biox influent.  
 To test this hypothesis, 5 mL of Nitrosomonas was transferred to an empty 
beaker. Then a 15 mL solution of the wastewater to be tested was added to the beaker, 
followed by the addition of a 9.5 mL buffer solution. Finally, a 0.5 mL ammonium 
solution was added to a concentration of 50 mg NH4 ? N L-1 in the beaker. The total 
test volume in the beaker was 30 mL and the pH at the start of testing was 8.3. The 
beakers were incubated on a rotary shaker in darkness for one hour. Aliquots of 1 mL 
were taken and the ammonium concentration was analyzed.  
 The buffer solution (pH ~ 9.0) was made of equal parts of 0.5 M phosphate 
buffer and 0.6 M carbonate buffer according to Sato et al. (1985). The ammonium 
solution added to the beakers contained 5 g N L-1 as (NH4)2SO4. The wastewater 
samples were treated with 2600 units of peroxidase or 200 units of laccase and 300 
mg/L of hydrogen peroxide over a 30 minute reaction time. Dissolved oxygen was 
maintained above 4.0 ppm during the testing by vigorous shaking.  
Analytical Methods 
 An Agilent 6890 Series gas chromatograph (GC) with an FID detector was 
used to determine the concentration of the phenolic compounds tested in this study. 
The column used in the gas chromatograph was a HP5 30m by 320?m by 0.25?m 
with 5 percent phenyl methyl siloxane. For full methodology refer to appendix A1.  
 EM Quant 10081-1 Peroxide Test strips were used to determine the amount of 
peroxide in the samples colorimetrically. The test strips could be used for 
concentrations from 1 ? 100 mg/L of hydrogen peroxide.  
25 
 
Chemicals  
The two enzymes used in this study were obtained from Novozymes, Inc. The 
first enzyme used in the study was peroxidase (Novozyme ? 51004). Once activated 
by hydrogen peroxide, the peroxidase enzyme catalyzes the oxidation of aqueous 
aromatic compounds to produce high molecular weight polymers of low solubility. 
The enzyme was produced by submerged fermentation of a genetically modified 
microorganism, which produces the peroxidase at a high rate. The enzyme protein, 
which in itself is not genetically modified, is separated and purified from the 
production organism. The production organism was a fungi, Aspergillus oryzae. The 
declared activity is 10,000 POXU/g. The color of the enzyme solution is brown. 
Although the color can vary from batch to batch, it is not an indication of enzyme 
activity. The physical form of the enzyme is liquid, with an approximate density of 
1.28 g/mL. For optimum results peroxidase should be stored with in the temperature 
range of 0-25? C (32-77? F). The enzyme has a shelf life in excess of one year.  
One peroxidase unit (POXU) is defined as the amount of enzyme which 
catalyzes the conversion of one micromole of hydrogen peroxide per minute at 30 
degrees Celsius in a 0.1 molar phosphate buffer at pH 7.0, using 0.88 millimoles 
hydrogen peroxide, and 1.67 millimoles of 2,2-azino-bis (3-ethylbenzothiazoline-6-
sulfonate) (ABTS). After 15 seconds of mixing, the reaction is followed for 60 
seconds by the change in absorbance at 418 nanometers, which should be in the range 
of 0.15 to 0.30. For the calculation of activity, an absorption coefficient of oxidized 
ABTS of 36 mM-1cm-1 and a stoichiometry of one micromole of hydrogen peroxide 
converted per two micromoles ABTS oxidized are used (Baaziz, 1989). 
26 
 
 The second enzyme used in this study was laccase (Novozymes ? 51003). 
Laccase was also produced by submerged fermentation of a genetically modified 
microorganism, Aspergillus oryzae (fungi). The enzyme protein, which in itself is not 
genetically modified, is separated and purified from the production organism. Laccase 
activity is expressed in Laccase Myceliophthora Units (LAMU). One LAMU is 
defined as the amount of enzyme that under standard conditions (pH 7.5; 30?C) 
oxidizes 1 micromole syringaldazine per minute. The declared activity the enzyme 
used in this study is 1,000 LAMU/g. The color of the laccase enzyme solution is 
brown. Once more, the color can vary from batch to batch and is not an indication of 
enzyme activity. The physical form of the enzyme is liquid, with an approximate 
density of 1.05 g/mL. For optimum results laccase should be stored with in the 
temperature range of 0-25?C (32-77?F).   
 Phenol, m-cresol, p-cresol, and 2-4 dimethyl phenol were also used in this 
study. The particular phenolic compounds were chosen as these are the most common 
phenols found in refinery wastewater. The phenol was obtained from Acros organics. 
The cresols and the 2-4 dimethyl phenol were obtained from Aldrich Chemical 
Company.  
Refinery Descriptions 
 Five refineries were chosen for this study. Two refineries are located in the 
United States, and three are located in Canada. They were chosen to represent the 
different types of wastewater that can be found at a refinery, as they each have their 
own unique mixture of crudes that are processed at each refinery.  The two main 
types of crude that these refineries use are Texas/Gulf Coast ?sweet crude? and 
27 
 
Canadian or Venezuelan synthetic crude which is derived from tar sand.  The 
following sections are brief descriptions of the five refineries used in this study.  
CITGO  
 CITGO is one of the leading manufacturers of high quality transportation 
fuels, lubricants, and petrochemicals. CITGO?s Lake Charles Refinery in Louisiana is 
a modern, deep-conversion facility that has a crude oil capacity of 425,000 - 440,000 
barrels per day. This refinery processes mostly Texas ?sweet crude? although some 
imported Venezuelan synthetic crude is also processed. The complex is located on the 
banks of the Calcasieu Ship Channel, and encompasses 2,000 acres. Lake Charles 
Refinery is the fourth largest refinery in the nation, and is primarily suited to process 
heavy crudes into high-octane, unleaded gasoline. However, it also produces turbine 
fuel and heating oils. Additionally the CITGO refinery produces petrochemicals such 
as benzene and propylene that are used to manufacture numerous everyday products.  
Frontier 
 The Frontier Refinery, located in Cheyenne, Wyoming, has a permitted 
capacity of 52,000 barrels of crude oil per day. The crude oil used at the Cheyenne 
Refinery is obtained from local producers (Texas and Midwest ?sweet crude?) as well 
as imported from Canada via the Express Pipeline (Canadian synthetic crude oil). The 
Cheyenne Refinery can process extensive amounts of heavy crude oil for use as a 
feedstock due to the coking unit on site. Although capable of utilizing heavy crude, 
this process mostly uses Texas ?sweet crude? and some Canadian synthetic crude oil. 
The ability to process heavy crude oil lowers the cost of feed stock because lighter 
types of crude oil are usually more costly than heavy crude oil. The Cheyenne 
28 
 
refinery has historically used heavy crude oil, as it has accounted for 85 percent or 
more of the total crude oil charge. The Cheyenne Refinery?s product mix usually 
consist of 41% gasoline, 30% diesel fuel, and asphalt and other refined petroleum 
products make up the remaining 29%.   
Imperial Oil 
Imperial Oil (Esso) began in 1880 and is one of Canada's top corporations. It 
has been a leading member of the petroleum industry for more than 125 years. 
Through these years Imperial Oil has been a contributor to the growth of the 
petroleum industry and to Canada's economic and social development. Imperial Oil is 
also an internationally renowned industrial cooperative, devoted to environmental 
protection. Imperial Oil produces several hundred different products. The crude 
processed to make these products is mainly synthetic crude that is derived from the 
tar sands there in Canada; however, these refineries do process some ?sweet crude? 
from the United States or the Middle East as well. Samples from Imperial Oil 
included sample sets from three separate refineries: the Dartmouth Refinery located 
in Halifax, Nova Scotia; the Nanticoke Refinery located on the northern shore of 
Lake Erie just southwest of Hamilton, Ontario; and the Sarnia Refinery located 
southwestern Ontario, in the city of Sarnia.  
The Dartmouth Refinery began production in 1918 so that the demands of 
World War I would be met. Since that time, it has grown continually, and now 
encompasses 650 acres. Dartmouth Refinery now has a processing capacity of 89,000 
barrels of crude oil per day, producing 52 different products.  These products include 
gasoline, jet fuel, diesel, and several heating oils and petrochemicals. The main crude 
29 
 
oil processed at Dartmouth is Canadian tar sand synthetic crude oil. Facilities at the 
Dartmouth Refinery include crude distillation and catalytic cracking.  
The Nanticoke Refinery began shipping product in November of 1978 as part 
Texaco Canada. Imperial Oil purchased the Nanticoke Refinery in 1989 as part of its 
merger with Texaco. Since that time several investments have been made to try and 
improve the gasoline and diesel fuel being produce. Facilities have been upgraded 
and added to improve and reduce the sulfur levels in the fuels. The refinery has a 
processing capacity of 120,000 barrels of crude oil per day. Key products include 
unleaded gasoline, jet fuel, diesel, heavy fuel oil, asphalt and petrochemicals. Crude 
distillation, catalytic reforming, catalytic cracking, and sulfur recovery are the key 
facilities at Nanticoke.  
The Imperial Oil?s Sarnia Refinery was commissioned in 1897, and was the 
largest refinery in Canada with a 900 barrel per day capacity. Today Sarnia is still one 
of the most complex refineries in Canada, and has a capacity of 119,000 barrels per 
day of crude oil. Several improvements since the 1980s has increased its ability to 
process a wide range of crude oil into many quality products such as gasoline, jet 
fuel, diesel, and many other fuels, oils, and petrochemicals. One unique development 
is that since 2003 the Sarnia and Nanticoke refineries have been connected or 
integrated. Through this two refinery integrated system, Sarnia has been able to 
produce ultra low-sulfur gasoline and diesel, less than 30 ppm and 15 ppm 
respectively. Sarnia also manufactures over 250 lubricating oils, 60% is transported to 
customers via tank trucks and rail cars, and the other 40% is packaged into small 
containers on site. The chemical division at Sarnia and Nanticoke is one of Canada?s 
30 
 
largest producers of petrochemicals, and today produces over one million tons of 
production per year. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
31 
 
 
 
Chapter 4 
Results and Discussion  
  
It is hypothesized that the addition of hydrogen peroxide coupled with either 
laccase or peroxidase enzymes can reduce the concentration of selected phenolic 
compounds in refinery wastewaters. This hypothesis is based on the experimental 
data and anecdotal information given by previous research. However, one the major 
limitations of these laboratory studies to refinery wastewater is that this research was 
conducted in either buffer solution or de-ionized water. A second limitation is that 
single compounds or a mixture of several compounds were employed rather than the 
complex mixtures actually present in refinery wastewater. Thus the effect which 
contaminants in actual refinery wastewater have upon this enzymatic approach is not 
reflected in these studies.  
The research of this thesis was conducted in three phases. The first phase 
involved the enzymatic treatment of phenolic compounds in a simple matrix (de-
ionized water). The second phase involved the enzymatic treatment of various 
refinery wastewaters, and the third phase of the study considered the effect of 
enzymatic treatment on nitrification. The results of these three phases of testing are 
present below. 
 
 
32 
 
Phase One: Synthetic Wastewater 
It was theorized that the concentration of phenolic compounds in refinery 
wastewater could be reduced using hydrogen peroxide coupled with either laccase or 
peroxidase. In the first phase of testing this hypothesis, the enzymatic approach was 
tested in synthetic wastewater created by adding the phenolic compounds to de-
ionized water. There were three different studies conducted in phase one using a 
simple matrix. The first study examined the efficiency of laccase and peroxidase to 
reduce the individual phenolic compounds in de-ionized water. The second study 
determined the enzyme?s ability to lower the concentrations of a mixture of phenolic 
compounds in a synthetic wastewater. The final study conducted in phase one was to 
determine the effect of hydrogen peroxide concentration on the efficiency of the 
enzymatic treatment of phenolic compounds. The results of this phase are presented 
in the following sections for each enzyme, and then summarized for both.  
Individual Testing of Each Compound in a simple Matrix  
 The efficacy of laccase and peroxidase enzymes for removing phenol, o-
cresol, p-cresol, 2,4-dimethyl phenol was studied using individual compounds in a 
synthetic de-ionized water solution. The synthetic solution used in this phase of the 
study was created by adding each individual compound to de-ionized water. Laccase 
and peroxidase treatment were tested on the individual samples to examine the 
efficiency of removal. The samples were tested using gas chromatography with a 
flame ionization detector to determine the initial concentrations of each compound. 
Either 200 units of laccase or 2600 units of peroxidase were added along with 300 
mg/L of hydrogen peroxide. Concentrations of each compound were then determined 
33 
 
by gas chromatography at 15 minute intervals. The results of this experiment are 
discussed in this section.  
 The effects of laccase and peroxidase on a pure synthetic solution containing 
phenol are shown in Figure 4.1. Laccase reduced the concentration of phenol from 
130.7 mg/L to 33.6 mg/L over 60 minutes. The reduction in phenol by laccase was 
74.3 percent after 60 minutes. The concentration of phenol decreased from 127.3 
mg/L to 31.8 mg/L using the peroxidase enzyme. Over the 60 minutes of reaction 
time peroxidase lowered the concentration of phenol by 75 percent.   
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
o
n
c
e
n
t
r
a
t
i
o
n
 
(
m
g
/
L
)
0
20
40
60
80
100
120
140
L a c c a s e  T r e a m t m e n t
P e r o x i d a s e  T r e a t m e n t
Figure 4.1: Laccase/Peroxidase Treatment of Phenol in De-ionized Water 
Since the reaction is believed follow first order kinetics, the rate constant for 
each reaction was determined. The equation describing first order kinetics is: ln [A] = 
34 
 
-kt + ln [A0], where A0 equals the initial concentration, A equals the concentration at 
time t, and k is equal to the first order rate constant. The first order rate constant is 
equal to the slope of the line given by ln [A], or natural log of the concentration, 
verses time. An example of the plot used to determine the first order rate constants (k) 
for phenol is shown in Figure 4.2. Table 4.1 summarizes the first order rate constants 
and R2 values for both enzymes for each compound. The first order rate constant for 
laccase reduction of phenol was found to be -0.0223 min-1 with an R2 value of 0.90. 
The first order rate constant for peroxidase was determined to be -0.0248 min-1 with 
an R2 of 0.91, which is very similar to the rate obtained by using laccase. Thus both 
enzymes appeared to be equally effective in a simple matrix. The high values of R2 
(>0.9) indicate that first order fit is acceptable.  
 
 
 
 
 
 
 
 
 
 
 
 
35 
 
T im e ( m inut es)
0 20 40 60
Ln 
[
C
]
3. 2
3. 4
3. 6
3. 8
4. 0
4. 2
4. 4
4. 6
4. 8
5. 0
La cc ase
P eroxi da se
Li ne ar ( P eroxi da se)
Li ne ar ( La cc ase)
 
 
 
         
        Peroxidase 
        y = -0.0248x + 4.895 
        R2 = 0.9803 
  Laccase 
  y = -0.0223x + 4.6833 
  R2 = 0.9084 
 
 
 
 
 
Figure 4.2: Example Plot for Determining Rate Constant, K 
Table 4.1: Individual Testing First Order Rate Constants 
 
The reduction of o-cresol by laccase and peroxidase catalyzed per oxidation is 
shown in Figure 4.3. The concentration of o-cresol was decreased by laccase from 
67.6 mg/L to 32.9 mg/L in 60 minutes. This corresponds to a reduction in o-cresol by 
Compound 
Laccase 
k  (min-1) 
Peroxidase 
k (min-1) 
Phenol -0.0223 (R2 = 0.91) -0.0248 (R2 = 0.98) 
O-cresol -0.0114 (R2 = 0.97) -0.0134 (R2 = 0.99) 
P-cresol -0.0107 (R2 = 0.95) -0.0157 (R2 = 0.98) 
2,4-Dimethyl Phenol -0.0201 (R2 = 0.92) -0.0243 (R2 = 0.90) 
36 
 
laccase of 51.3 percent. Similarly, using the peroxidase enzyme, the concentration of 
o-cresol decreased from 61.2 mg/L to 27.2 mg/L. Peroxidase reduced the 
concentration of o-cresol by 55.6 percent over the 60 minutes of reaction time. The 
first order rate constants for laccase and peroxidase for the removal of o-cresol are 
shown in Table 4.1. Peroxidase/hydrogen peroxide treatment had a slightly higher 
rate of o-cresol removal than the laccase system. The first order rate constant for 
laccase was determined to be -0.0107 min-1 with an R2 value of 0.97. Peroxidase had 
a first order rate constant of -0.0134 min-1 and an R2 value of 0.99. The rate of 
removal for o-cresol was approximately one half than that of phenol removal for both 
enzymes.  
 
37 
 
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
o
n
c
e
n
t
r
a
t
i
o
n
 
(
m
g
/
L
)
0
20
40
60
80
L a c c a s e  T r e a m t m e n t
P e r o x i d a s e  T r e a t m e n t
 
Figure 4.3: Laccase/Peroxidase Treatment of O-cresol in De-ionized Water  
 
Changes in p-cresol concentration, catalyzed by laccase or peroxidase, are 
presented in Figure 4.4. Laccase reduced the initial concentration of p-cresol from 
56.3 mg/L to 27.9 mg/L. Laccase treatment reduced the concentration in p-cresol by 
50.4 percent in 60 minutes. The first order rate constant was determined to be -0.107 
min-1 for laccase with an R2 value of 0.95 (Table 4.1).  The peroxidase enzyme 
lowered the concentration of p-cresol from 53.2 mg/L to 21.7 mg/L. The reduction in 
the concentration of p-cresol by peroxidase was 59.3 percent over the 60 minutes of 
reaction time. The first order rate constant for peroxidase was found to be -0.0157 
38 
 
min-1 with an R2 value of 0.98 (Table 4.1). These rate constants are somewhat similar, 
although peroxidase is a little higher rate. The first order reduction rate constant for p-
cresol was comparable to that of o-cresol, but was nearly half that of phenol and 2,4-
dimethyl phenol.  
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
o
n
c
e
n
t
r
a
t
i
o
n
 
(
m
g
/
L
)
0
10
20
30
40
50
60
L a c c a s e  T r e a m t m e n t
P e r o x i d a s e  T r e a t m e n t
 
Figure 4.4: Laccase/Peroxidase Treatment of P-cresol in De-ionized Water  
 
The effects of laccase and peroxidase on 2,4-dimethl phenol in a synthetic 
solution is shown in Figure 4.5. Laccase decreased the concentration of 2,4-dimethyl 
phenol from 31.3 mg/L to 10.2 mg/L. This reduction in 2,4-dimethyl phenol by 
laccase treatment was 67.4 percent over 60 minutes. The first order rate constant, 
39 
 
shown in Table 4.1, for 2,4-dmp treated with laccase was found to be -0.0201 min-1 
with an R2 value of 0.92. The concentration of 2,4-dimethyl phenol decreased from 
34.2 mg/L to 7.9 mg/L using the peroxidase enzyme with 60 minutes reaction time. 
The reduction in 2,4-dimethyl phenol by peroxidase was 76.9 percent. The first order 
rate constant for peroxidase was found to be -0.0243 min-1 with an R2 of 0.90 (Table 
4.1). These rates appear to be similar. However, the peroxidase/peroxide removed the 
2,4-dmp at a rate that was almost 20 percent faster than laccase/peroxide.  The rates 
are comparable to that of phenol, and almost double the rates of o-cresol and p-cresol.   
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
o
n
c
e
n
t
r
a
t
i
o
n
 
(
m
g
/
L
)
0
10
20
30
40
L a c c a s e  T r e a m t m e n t
P e r o x i d a s e  T r e a t m e n t
 
Figure 4.5: Laccase/Peroxidase Treatment of 2,4-Dimethyl Phenol in  
De-ionized Water  
 
40 
 
To facilitate comparison of the effectiveness of laccase/peroxide and 
peroxidase/peroxide treatment on the removal of the target phenolic compounds, the 
concentrations for each compound were normalized to the initial concentration and 
presented in the same figure to show the fraction remaining. Figure 4.6 shows the 
fraction remaining of each compound removed by the laccase/peroxide treatment over 
the 60 minutes. The figure shows that laccase/peroxide treatment initially removed 
phenol at the fastest rate. 2,4-dmp was removed to almost the same degree as phenol 
after 60 minutes. However, there was an initial lay. O-cresol and p-cresol on the hand 
were removed at a much lower rate and to a lesser degree than phenol and 2,4-dmp.  
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
o
n
c
e
n
t
r
a
t
i
o
n
 
o
v
e
r
 
I
n
i
t
i
a
l
 
C
o
n
c
e
n
t
r
a
t
i
o
n
 
(
C
/
C
o
)
0 . 0
0 . 1
0 . 2
0 . 3
0 . 4
0 . 5
0 . 6
0 . 7
0 . 8
0 . 9
1 . 0
1 . 1
P h e n o l
O - c r e s o l
P - c r e s o l
2 , 4 - d i m e t h y l  p h e n o l
 
Figure 4.6: Fraction Remaining using Laccase/Peroxide Treatment 
41 
 
 
The fraction remaining of each compound reduced by peroxidase/peroxide 
treatment is shown in Figure 4.7. Peroxidase/peroxide treatment initial removed 2,4-
dmp at a faster rate than the other compounds. Although phenol was initially reduced 
at a lower rate than 2,4-dmp, they were removed to almost the same degree after the 
60 minutes. P-cresol and o-cresol were removed to a lesser degree and a slower rate 
than the phenol and 2,4-dmp. The p-cresol was initially removed at a slightly faster 
rate than o-cresol, but over the 60 minutes, both were removed to a similar degree.   
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
o
n
c
e
n
t
r
a
t
i
o
n
 
o
v
e
r
 
I
n
i
t
i
a
l
 
C
o
n
c
e
n
t
r
a
t
i
o
n
 
(
C
/
C
o
)
0 . 0
0 . 1
0 . 2
0 . 3
0 . 4
0 . 5
0 . 6
0 . 7
0 . 8
0 . 9
1 . 0
1 . 1
P h e n o l
O - c r e s o l
P - c r e s o l
2 , 4 - d i m e t h y l  p h e n o l
 
Figure 4.7: Fraction Remaining using Peroxidase/Peroxide Treatment 
 
42 
 
 
Defined Mixture of Phenolic Compounds in a Simple Matrix (Synthetic Wastewater) 
 The second part of phase one was to determine the enzyme?s capacity for 
reducing a mixture of phenolic compounds in a synthetic wastewater. This testing 
was preformed to check for synergism or antagonism between compounds. The 
synthetic wastewater was created by adding 100 ppm phenol, 100 ppm o-cresol, 100 
ppm p-cresol, and 100 ppm 2,4-dimethyl phenol to de-ionized water. The samples 
were then tested using gas chromatography to determine the initial concentrations of 
each compound. An initial concentration of 300 mg/L of hydrogen peroxide and 
either 200 units of laccase or 2600 units of peroxidase was used to initiate the testing. 
Concentrations were determined at 15 minute intervals after enzyme addition. The 
results of this experiment are discussed in this section.  
The efficiency of laccase at reducing the concentration of phenol over time in 
the mixed wastewater is shown in Figure 4.8. The initial phenol concentration in the 
synthetic wastewater was 111.2 mg/L. The laccase treatment lowered the 
concentration of phenol to 43.7 mg/L (60.7 percent) over 60 minutes.  
43 
 
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
o
n
c
e
n
t
r
a
t
i
o
n
 
(
m
g
/
L
)
0
20
40
60
80
100
120
 
Figure 4.8: Laccase Treatment of Phenol in the Defined Mixture 
 
Table 4.2 contains the first order rate constants and R2 values for both 
enzymes for each compound. Again due to the reaction following first order kinetics, 
the rate constant for each reaction could be determined. In first order kinetics ln [A] = 
-kt + ln [A0], where A0 equals the initial concentration, A equals the concentration at 
time t, and k is equal to the first order rate constant. Thus, the first order rate constant 
is equal to the slope of the line given by ln [A], or natural log of the concentration, 
verses time. The first order rate constant was found to be -0.0151 min-1 and had an R2 
44 
 
value of 0.94. The rate constant for phenol was similar to that of 2,4-dimethyl phenol, 
but somewhat higher than the first order rate constant for o-cresol and p-cresol.  
 
Table 4.2: First Order Rate Constants for Each Compound in the Defined Mixture  
Compound Laccase k
   (min-1) 
Peroxidase 
k  (min-1) 
Phenol -0.0151 (R2 = 0.94) -0.0157 (R2 = 0.99) 
O-cresol -0.0103 (R2 = 0.95) -0.0067 (R2 = 0.95) 
P-cresol -0.0087 (R2 = 0.99) -0.0093 (R2 = 0.98) 
2,4-Dimethyl Phenol -0.0162 (R2 = 0.97) -0.0172 (R2 = 0.999) 
 
 
 Figure 4.9 shows the decrease in o-cresol concentration over time. O-cresol 
concentration in the synthetic wastewater was reduced by laccase 43.6 percent. The 
concentration dropped from 46.8 mg/L initially to 26.4 mg/L after 60 minutes. The 
decrease in o-cresol is similar to that of p-cresol, but lower than that of phenol and 
2,4-dimethyl phenol. Laccase reduction of o-cresol has a first order rate constant of -
0.0103 min-1 and a R2 value of 0.95 (Table 4.2). The reduction rate constant is 
slightly high than the rate constant for p-cresol, but significantly lower than that of 
phenol and 2,4-dimethyl phenol.  
45 
 
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
o
n
c
e
n
t
r
a
t
i
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(
m
g
/
L
)
0
10
20
30
40
50
 
Figure 4.9: Laccase Treatment of O-cresol in the Defined Mixture 
 
 The reduction of p-cresol by laccase over time is shown in Figure 4.10. The 
concentration of p-cresol decreased from 31.6 mg/L to 18.9 mg/L in 60 minutes. The 
reduction in p-cresol by laccase was 40.2 percent. Again this drop in concentration 
resembles that of o-cresol, but is much less than the phenol and 2,4-dimethyl phenol. 
Table 4.2 shows how the first order rate constant of laccase for decreasing p-cresol 
concentration compares to the other compounds tested in the synthetic wastewater. 
The first order rate constant was found to be -0.0087 min-1 with an R2 value of 0.99. 
This is the lowest first order rate constant for laccase in this part of phase one testing, 
46 
 
and the first order rate constant for phenol is nearly double that of p-cresol. Another 
note is that the rate constant for peroxidase reducing p-cresol is very similar to the 
laccase rate constant, thus both enzymes appear to be equally effective at reducing p-
cresol in concentration with other compounds.  
 
 
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
o
n
c
e
n
t
r
a
t
i
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n
 
(
m
g
/
L
)
0
5
10
15
20
25
30
35
 
Figure 4.10: Laccase Treatment of P-cresol in the Defined Mixture 
 
The first order rate constants for each compound in the synthetic wastewater 
are shown in Table 4.2. The first order rate constant for 2,4-dimethyl phenol was 
47 
 
found to be -.0162 min-1 with a R2 value equal to 0.97. This is close to the rate 
constant for phenol, but is higher than the rate constant for o-cresol and nearly double 
the rate constant for p-cresol. Figure 4.11 shows the decrease in 2,4-dimethyl phenol 
concentration over time. 2,4-dimethyl phenol was lowered by laccase treatment from 
a concentration of 23.4 mg/L to 8.9 mg/L after 60 minutes. The reduction of 2,4-
dimethyl phenol by laccase was 62 percent. The percent reduction is very close to that 
of phenol.   
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
o
n
c
e
n
t
r
a
t
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(
m
g
/
L
)
0
5
10
15
20
25
 
Figure 4.11: Laccase Treatment of 2,4-Dimethyl Phenol in the Defined Mixture 
 
48 
 
The reduction of phenol by the peroxidase enzyme can be seen in Figure 4.12. 
The concentration of phenol was reduced from an initial concentration of 110 mg/L to 
42.9 mg/L by peroxidase treatment. Over the 60 minutes reaction time phenol was 
reduced by 61 percent. The first order rate constant for the peroxidase enzyme?s 
ability to remove phenol can be seen in Table 4.2. The reduction rate constant was 
found to be -0.0157 min-1 with a R2 value of 0.99. The rate constant for peroxidase is 
almost identical to that of laccase, thus they seem to be equally efficient. However, 
the rate for reducing phenol is similar to that of 2,4-dimethyl phenol, and much higher 
than the first order rate constant for reducing p-cresol and well over two times that of 
o-cresol.  
 
49 
 
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
o
n
c
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t
r
a
t
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(
m
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/
L
)
0
20
40
60
80
100
120
 
Figure 4.12: Peroxidase Treatment of Phenol in the Defined Mixture 
 
 The concentrations of o-cresol over time are shown in Figure 4.13. O-cresol 
was lowered only 34.3 percent by peroxidase treatment. The concentration dropped 
from 41.1 mg/L initially to 27 mg/L after 60 minutes time. O-cresol had the lowest 
percent reduction by the peroxidase enzyme.  The percent reduction was close to half 
of that observed for phenol and 2,4-dimethyl phenol.  The first order rate constant for 
peroxidase reduction of o-cresol was found to be -0.0067 min-1 with R2 equal to 0.95 
(Table 4.2). This rate constant was the lowest first order rate constant for peroxidase 
50 
 
and the lowest for this part of this phase one. The reduction rate constant is one and a 
half times lower that that of phenol and one third that of 2,4-dimethyl phenol.  
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
o
n
c
e
n
t
r
a
t
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(
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/
L
)
0
10
20
30
40
50
 
Figure 4.13: Peroxidase Treatment of O-cresol in the Defined Mixture 
  
P-cresol concentration over time can be seen in Figure 4.14.  P-cresol 
concentration dropped 42.3 percent over the 60 minutes. The synthetic wastewater 
was found to have an initial concentration of 30.7 mg/L and that was reduced by 
peroxidase to 17.7 mg/L. The percent reduced was very similar to that of o-cresol, 
and lower than that of phenol or 2,4-dimethyl phenol. Table 4.2 shows the first order 
rate constant of peroxidase for decreasing the concentration of each compound in the 
51 
 
synthetic wastewater. The first order rate constant for reducing p-cresol was found to 
be -0.0093 min-1 with an R2 value of 0.98. The first order rate constant for p-cresol is 
nearly half that of 2,4-dimethyl phenol.  
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
o
n
c
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t
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a
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(
m
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)
0
5
10
15
20
25
30
35
 
Figure 4.14: Peroxidase Treatment of P-cresol in the Defined Mixture 
 
The reduction in 2,4-dimethyl phenol concentration by peroxidase can be seen 
in Figure 4.15.Peroxidase reduced 2,4-dimethyl phenol from a concentration of 21.8 
mg/L to 7.8 mg/L after 60 minutes. The reduction of 2,4-dimethyl phenol by 
peroxidase was 64.2 percent. The percent reduction is similar to that of phenol, and 
nearly doubles that of o-cresol. The first order rate constant for the peroxidase 
52 
 
enzyme?s ability to remove phenolic compounds can be seen in Table 4.2. The 
reduction rate constant for reducing 2,4-dimethyl phenol was found to be -0.0172 
min-1 with a R2 value of 0.999. The rate constant for peroxidase is similar to that of 
laccase, thus they seem to be equally efficient. However, the rate for reducing 2,4-
dimethyl phenol is similar to that of phenol, and is almost double than the first order 
rate constant for reducing p-cresol and almost three times that of o-cresol.  
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
o
n
c
e
n
t
r
a
t
i
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(
m
g
/
L
)
0
5
10
15
20
25
  
Figure 4.15: Peroxidase Treatment of 2,4-Dimethyl Phenol in the Defined Mixture 
 
Upon initial comparison it appears that in this particular case the two enzymes 
are to some extent equally efficient, albeit peroxidase does have a slightly higher rate 
53 
 
constant for all compounds except o-cresol. The first order reduction rate constant is 
however lower when all compounds are in the synthetic wastewater verses the 
enzymatic treatment of just the single compound in de-ionized water, which is shown 
in Table 4.3. This difference in rate could be due to antagonism between the different 
compounds, meaning the compounds compete for the enzyme, making the rate of 
reduction lower for the mixture.  It is hypothesized that in real refinery wastewater 
the competition for the enzyme will be even greater than in the de-ionized mixture 
due to the other compounds in the wastewater besides the target phenols, thus the 
rates for laccase and peroxidase may be smaller.  
Table 4.3: Laccase and Peroxidase Rate Constants from Individual Testing and 
Synthetic Testing for Comparison 
 
Compound 
Laccase 
Single 
k (min-1) 
Laccase 
Mixture 
k (min-1) 
Peroxidase 
Single 
k (min-1) 
Peroxidase 
Mixture 
k (min-1) 
Phenol -0.0223 -0.0151 -0.0248 -0.0157 
O-cresol -0.0114 -0.0103 -0.0134 -0.0067 
P-cresol -0.0107 -0.0087 -0.0157 -0.0093 
2,4-Dimethyl 
Phenol -0.0201 -0.0162 -0.0243 -0.0172 
 
Hydrogen Peroxide Testing in a Simple Matrix 
 In part three of phase one, the effect of hydrogen peroxide on the enzymes 
was examined. Phenol, 2,4-dimethyl phenol, o-cresol, and p-cresol were added to de-
ionized water to create a synthetic wastewater. To determine if the concentration of 
54 
 
hydrogen peroxide produced any variances in the reaction rate of laccase and 
peroxidase, tests were done using doses of: 600 mg/L, 300 mg/L, 150 mg/L, and 75 
mg/L of hydrogen peroxide. The selected hydrogen peroxide dosage was combined 
with either 2600 units of peroxidase or 200 units of laccase and was added to 
synthetic wastewater. The samples were then tested for individual phenolic 
compound concentrations on the gas chromatograph in fifteen minute intervals for 
thirty minutes.  
The first order rate constants for each compound and each hydrogen 
peroxidase dose were examined using laccase/peroxide treatment (Table 4.4). To 
determine the first order rate constant, the natural log of the concentration at time t, is 
plotted verses time t. The slope of the line created is equal to the first order rate 
constant. The governing equation is ln [A] = -kt + ln [A0], where A0 equals the initial 
concentration, A equals the concentration at time t, and k is equal to the first order 
rate constant.  
As seen in Table 4.4 the hydrogen peroxide concentration can affect the rate at 
which the phenol concentration is reduced. The R2 values show how well the data fits 
the first order model. In this part of the study the fit was moderate (0.80) to very good 
(> 0.95). Laccase/peroxide treatment had the highest reduction rate constant when 
using 600 mg/L hydrogen peroxide to remove the phenolic compounds. The rate for 
600 mg/L rate was on average 44 percent better than the rate for 300 mg/L of 
hydrogen peroxide, over two times higher than the rate when 150 mg/L of hydrogen 
peroxide was used, and over five times higher than the rate when 75 mg/L of 
hydrogen peroxide was used. Although 600 mg/L causes the laccase enzyme to 
55 
 
reduce the total phenolic compound concentrations the most, 300 mg/L is still an 
excess amount of hydrogen peroxide for the reduction to occur at a satisfying rate. 
 The effect of varying hydrogen peroxide concentration on the destruction of 
each of the phenolic compounds by laccase is shown in Figure 4.16. The first order 
rate constants, derived from the data presented in Figure 4.16, are summarized in 
Table 4.4. The first order rate constant increased as the initial hydrogen peroxide dose 
increased. The highest rate constant was determined for hydrogen peroxide 
concentration of 600 mg/L. This demonstrates the dependence of the degradation rate 
on peroxide dose. It should be noted that hydrogen peroxide doses above 100 ppm are 
considered impractical at the temperatures and pressures typically encountered in 
refineries due to safety concerns.  
 
 
Table 4.4: First Order Rate Constants for Laccase Treatment of Phenol with Varied 
H2O2 Concentrations 
 
Hydrogen Peroxide 
Concentration  Phenol O-cresol P-cresol 
2,4-Dimethyl 
Phenol 
600 mg/L -0.0330  (R2 = 0.93) -0.0356 (R2 = 0.95) -0.0421 (R2 = 0.91) -0.0385 (R2 = 0.88) 
300 mg/L -0.0268 (R2 = 0.97) -0.0209 (R2 = 0.99) -0.0307 (R2 = 0.92) -0.0269 (R2 = 0.99) 
150 mg/L -0.0180 (R2 = 0.80) -0.0169 (R2 = 0.92) -0.0114 (R2 = 0.97) -0.0130 (R2 = 0.97) 
75 mg/L -0.0092 (R2 = 0.99) -0.0067 (R2 = 0.95) -0.0054 (R2 = 0.98) -0.0097 (R2 = 0.87) 
 
56 
 
H y dr og en Pe r ox ide C oncent r at ion ( m g / L)
0 100 200 300 400 500 600 700
R
at
e 
C
onstan
t
0. 00
0. 01
0. 02
0. 03
0. 04
0. 05
P he no l
O- cr esol
P - cr esol
2, 4- D i m et hy l  P he no l
 
 
Figure 4.16: Rate Constants for Laccase/Peroxide with  
Varied Hydrogen Peroxide Concentrations 
 
The efficiency of the peroxidase enzyme to reduce the selected phenolic 
compounds over thirty minutes using varied hydrogen peroxide concentrations was 
also examined. The concentrations of hydrogen peroxide tested were again 600 mg/L, 
300 mg/L, 150 mg/L, and 75 mg/L. The selected hydrogen peroxide dosage was 
combined with either 2600 units of peroxidase. The samples were then tested for 
individual phenolic compound concentrations on the gas chromatograph in fifteen 
minute intervals for thirty minutes.  
The first order reduction rate constants for the peroxidase/peroxide treatment 
were examined. The rate constant for each individual phenolic compound for each 
hydrogen peroxide concentration is presented in Table 4.5. As before it is very clear 
that the hydrogen peroxide concentration can affect the rate at which the phenolic 
57 
 
compounds in the solution are oxidized or reduced. The R2 values show how well the 
data fits the first order model. For the peroxidase/peroxide testing the fit to the first 
order model was moderate (0.84) to very good (> 0.95).  
When using 600 mg/L of hydrogen peroxide the highest reduction rate for the 
Peroxidase/peroxide treatment removing the phenolic compounds was achieved. The 
rate for 600 mg/L rate was again on average 40 percent better than the rate for 300 
mg/L of hydrogen peroxide. The rate for 600 mg/L hydrogen peroxide was also 
higher than that when 150 mg/L of hydrogen peroxide was used  and significantly 
higher than the rate when 75 mg/L of hydrogen peroxide was used, approximately 
three times and just under four times respectively. Even though 600 mg/L causes the 
peroxidase enzyme to reduce the total phenolic compound concentration the most, 
300 mg/L is still an excess amount of hydrogen peroxide for the reduction to occur at 
a satisfying rate.  
The effect of varying hydrogen peroxide concentration on the destruction of 
each of the phenolic compounds by peroxidase is shown in Figure 4.17. The first 
order rate constants, derived from the data, are summarized in Table 4.5. The first 
order rate constant again increased as the initial hydrogen peroxide dose increased. 
The highest rate constant was determined for hydrogen peroxide concentration of 600 
mg/L. This demonstrates the dependence of the degradation rate on peroxide dose. 
 
 
 
 
 
 
 
 
58 
 
 
 
Table 4.5: First Order Rate Constants for Peroxidase Treatment of Phenol with 
Varied H2O2 Concentrations 
 
Hydrogen Peroxide 
Concentration  Phenol O-cresol P-cresol 
2,4-Dimethyl 
Phenol 
600 mg/L -0.0399  (R2 = 0.99) -0.0370 (R2 = 0.99) -0.0314 (R2 = 0.91) -0.0224 (R2 = 0.84) 
300 mg/L -0.0319 (R2 = 0.99) -0.0322 (R2 = 0.91) -0.0225 (R2 = 0.99) -0.0123 (R2 = 0.93) 
150 mg/L -0.0214 (R2 = 0.99) -0.0072 (R2 = 0.99) -0.0105 (R2 = 0.95) -0.0079 (R2 = 0.94) 
75 mg/L -0.0150 (R2 = 0.98) -0.0066 (R2 = 0.95) -0.0089 (R2 = 0.98) -0.0056 (R2 = 0.89) 
 
H y dr og en Pe r ox ide C oncent r at ion ( m g / L)
0 100 200 300 400 500 600 700
R
at
e 
C
onstan
t
0. 00
0. 01
0. 02
0. 03
0. 04
0. 05
P he no l
O- cr esol
P - cr esol
2, 4- D i m et hy l  P he no l
  
Figure 4.17: Rate Constants for Peroxidase/Peroxide with  
Varied Hydrogen Peroxide Concentrations 
59 
 
 
Phase Two: Refinery Wastewater 
 In the second phase of testing, the research was conducted to determine how 
well laccase and peroxidase removed phenols from refinery wastewaters, and if there 
were any effects from the other contaminants in the wastewater. The results of this 
phase of the study are presented in this section for each sample tested, as then 
summarized for all samples.  
Frontier Stripped Sour Water 
The efficacy of laccase enzyme for reducing phenolic compounds in stripped 
sour water from the Frontier refinery was examined. The concentration of major 
phenolic constituents was determined by gas chromatography. A dose of 200 units of 
laccase and 300 mg/L of hydrogen peroxide were added to the Frontier sour water 
sample and the concentration of the phenolic compounds were determined at fifteen 
minute intervals over a one hour test period. Six replicates of the frontier sour water 
were tested using the laccase enzyme. These six replicates were averaged and it was 
determined that the sour water initially contained 56.6 mg/L of phenol, 22.3 mg/L of 
o-cresol, 32.7 mg/L of p-cresol, and 1.6 mg/L of 2, 4-dimethy phenol. Thus, the total 
amount of phenols in the sample was 113.2 mg/L.  
Table 4.6 contains the first order rate constants and R2 values for both 
enzymes for each compound. Since the reaction follows first order kinetics, the rate 
constant for each reaction could be determined by plotting the natural log of the 
concentration verses time. In first order kinetics ln [A] = -kt + ln [A0], where A0 
equals the initial concentration, A equals the concentration at time t, and k is equal to 
60 
 
the first order rate constant. The first order rate constant is equal to the slope of the 
line given by ln [A], or natural log of the concentration, verses time.  
Table 4.6: First Order Rate Constants for Laccase and Peroxidase in  
Frontier Sour Water 
 
 
Compound 
 
Laccase 
k (min-1) 
 
Peroxidase 
k (min-1) 
Phenol -0.0122 (R2 = 0.83) -0.0135 (R2 = 0.96) 
O-cresol -0.0085 (R2 = 0.84) -0.0083 (R2 = 0.97) 
P-cresol -0.0060 (R2 = 0.94) -0.0048 (R2 = 0.81) 
2,4-Dimethyl Phenol -0.0183 (R2 = 0.82) -0.0164 (R2 = 0.87) 
 
Figure 4.18 shows the reduction of phenol concentrations over time. The 
concentration of phenol decreased from 56.6 mg/L to 24.0 mg/L in 60 minutes. The 
overall reduction in phenol by laccase was 57.6 percent. It is important to note that 
only a small reduction in phenol was observed over the first 45 minutes of the 
reaction, and the majority of phenol oxidation occurred between 45 and 60 minutes. 
This differs from the testing with phenol in a ?clean matrix? where phenol 
degradation occurred quickly (see Figure 4. __). As shown in Table 4.6, the first order 
reduction rate constant was found to be -0.0122 min-1 with R2 equal to 0.83. The R2 
value indicates a moderate fit using first order kinetics.  
61 
 
T i m e  ( m i n u t e s )
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40
50
60
 
Figure 4.18: Laccase Treatment of Phenol in Frontier Stripped Sour Water 
 
The reduction of o-cresol by laccase is presented in Figure 4.19. O-cresol was 
reduced by laccase from an initial concentration of 19.5 mg/L to 12.1 mg/L after 60 
minutes of reaction time.  The reduction of o-cresol by laccase treatment was 37.9 
percent. Like for phenol, a majority of the reduction occurred after 30 minutes and 
was only a little over half of that observed for phenol. The first order rate constant for 
o-cresol was found to be -0.0085 min-1 with a R2 value of 0.84. The rate is lower 
than that of phenol.  
62 
 
T i m e  ( m i n u t e s )
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5
10
15
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25
 
Figure 4.19: Laccase Treatment of O-cresol in Frontier Stripped Sour Water 
 
 For p-cresol, a 33 percent reduction in concentration was observed (Figure 
4.20). The concentration decreased from an initial concentration of 32.7 mg/L to 21.9 
mg/L after 60 minutes of reaction. The percent reduction was similar to that of o-
cresol, and again close to half of the observed reduction of phenol. P-cresol had the 
lowest first order rate constants shown in Table 4.6. The observed rate constant for 
laccase enzyme?s reduction of p-cresol was -0.0060 min-1 and the R2 value was 0.94. 
The first order rate constant for laccase enzyme?s reduction of p-cresol was the lowest 
observed for any individual phenolic compound.  
63 
 
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
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0
5
10
15
20
25
30
35
 
Figure 4.20: Laccase Treatment of P-cresol in Frontier Stripped Sour Water 
 
Figure 4.21 shows the concentration over time for 2,4-dimethyl phenol. The 
concentration of 2, 4-dimethyl phenol was reduced from 1.6 mg/L to 0.54 mg/L over 
a period of 60 minutes. Laccase decreased the amount of 2, 4-dimthyl phenol by 66.3 
percent. The percent reduction of 2, 4-dimethyl phenol closely matched that of phenol 
and was almost double that of o-cresol and p-cresol. The first order rate constant for 
the laccase enzyme?s ability to remove phenolic compounds can be seen in Table 4.6. 
The reduction rate constant for reducing 2,4-dimethyl phenol was found to be -0.0183 
min-1 with a R2 value of 0.82. The rate for reducing 2,4-dimethyl phenol is the highest 
64 
 
for the monitored phenolic compounds, and more than double than the first order rate 
constant for reducing o-cresol and over three times that of p-cresol. However, this 
may be skewed by the lower initial concentration.  
 
T i m e  ( m i n u t e s )
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C
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0 . 0
0 . 2
0 . 4
0 . 6
0 . 8
1 . 0
1 . 2
1 . 4
1 . 6
1 . 8
 
Figure 4.21: Laccase Treatment of 2,4-Dimethyl Phenol in Frontier  
Stripped Sour Water 
 
The concentrations of each compound tested were normalized to show the 
percent remaining of each compound tested with the laccase enzyme (Figure 4.22). 
Phenol and 2,4-dimethyl phenol have the lowest remaining concentrations with 42.4 
and 31.8 percent respectively. O-cresol and p-cresol were significantly higher 
65 
 
fractions remaining and nearly double that of phenol and 2,4-dimethyl phenol. The 
percent remaining for o-cresol and p-cresol were 62.1 and 67 percent respectively. 
One important thing to note from these tests using laccase is that the majority of the 
phenolic reduction occurs between the reaction time of 30 and 60 minutes. This is 
different than for the clean system, where phenolic reduction occurred during the first 
15 minutes. One likely explanation is that laccase/peroxide was initially expended 
oxidizing other wastewater constituents.  
T i m e  ( m i n u t e s )
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C
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I
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(
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0 . 0
0 . 2
0 . 4
0 . 6
0 . 8
1 . 0
1 . 2
P h e n o l
O - c r e s o l
P - c r e s o l
2 , 4 - d i m e t h y l  p h e n o l
 
Figure 4.22: Fraction Remaining of Phenolic Compounds using Laccase Treatment 
 
66 
 
The Frontier sour water was treated with the peroxide enzyme to determine 
the efficiency for reducing phenolic compounds from the wastewater. An initial dose 
of 2600 units of peroxidase where added along with 300 mg/L of hydrogen peroxide 
to the sour water and the concentrations where measured with the gas chromatograph 
over fifteen minute intervals. As before, six replicate samples were tested using the 
peroxidase enzyme and the average initial concentrations contained in the sour water 
were: 35.8 mg/L of phenol, 18.2 mg/L of o-cresol, 24.1 mg/L of p-cresol, 1.9 mg/L of 
2, 4-dimethyl phenol, and 77.1 mg/L total phenol. The first order rate constants were 
also determined for peroxidase and are shown in Table 4.6 as well.  
The average concentration of phenol in the Frontier sour water was lowered 
from 35.8 mg/L to 15.6 mg/L over one hour (Figure 4.23). Peroxidase reduced the 
amount of phenol in the sour water by 56.4 percent.  The first order rate constant for 
reducing phenol was found to be -0.0135 min-1 with a value of 0.96 for R2 (Table 
4.6). The R2 value indicates a very good fit with the first order model. Unlike, the 
laccase enzyme, peroxidase catalyzed a rapid oxidation of phenol with significant 
reduction in concentration occurring over the first 30 minutes.  
67 
 
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10
20
30
40
 
Figure 4.23: Peroxidase Treatment of Phenol in Frontier Stripped Sour Water 
 
The reduction of o-cresol in Frontier sour water by peroxidase is shown in 
Figure 4.24. O-cresol was found to have an initial concentration of 18.2 mg/L. Over a 
reaction time of 60 minutes, peroxidase reduced the amount of o-cresol from 18.2 
mg/L to 11.2 mg/L. Corresponding to a 38.5 percent decrease in concentration. The 
first order rate constant for peroxidase reduction of o-cresol was found to be -0.0083 
min-1 with R2 equal to 0.97 (Table 4.6). The reduction rate constant is over one and a 
half times lower than that of phenol. Again the first order model yields a good fit for 
the data. 
68 
 
 
T i m e  ( m i n u t e s )
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2
4
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8
10
12
14
16
18
20
 
Figure 4.24: Peroxidase Treatment of O-cresol in Frontier Stripped Sour Water 
 
 The decrease in p-cresol in Frontier sour water by peroxidase over time can be 
seen in Figure 4.25. P-cresol concentration was reduced from an initial concentration 
of 24.1 mg/L to a concentration of 17.1 mg/L (29 percent) over the one hour reaction 
period. Once again, the peroxidase enzyme produced significant reduction in p-cresol 
over the first 30 minutes of reaction. This is different from the results determined 
using laccase, where the majority of the reduction occurred during the last 15 to 30 
minutes of the reaction. The first order rate constant for peroxidase reduction of p-
69 
 
cresol was found in Table 4.6 was -0.0048 min-1 with R2 equal to 0.81. The reduction 
rate constant for p-cresol is almost three times lower than that of phenol. 
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
o
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5
10
15
20
25
30
 
Figure 4.25: Peroxidase Treatment of P-cresol in Frontier Stripped Sour Water 
 
The peroxidase enzyme was able to remove 66.3 percent of the 2,4-dimethyl 
phenol in the Frontier stripped sour water (Figure 4.26). The concentration of 2,4-
dimethyl phenol was reduced from 1.9 mg/L to .64 mg/L over a 60 minute reaction 
time. The decrease in concentration for 2,4-dimethyl phenol was the highest of the 
four compounds tested at 67 percent, and almost double that of p-cresol and o-cresol. 
However, it is important to note that 2,4-dimethyl phenol made up only a small 
70 
 
fraction of the total phenolics in the sample. The first order rate constant found in 
Table 4.6 for peroxidase enzyme?s reduction of 2,4-dimethyl phenol is -0.0164 min-1 
with a R2 value of 0.87. The peroxidase reduction rate for 2,4-dimethyl phenol is the 
highest rate for peroxidase in this particular refinery matrix.  
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
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(
m
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0 . 0
0 . 5
1 . 0
1 . 5
2 . 0
2 . 5
 
Figure 4.26: Peroxidase Treatment of 2, 4-Dimethyl Phenol in Frontier  
Stripped Sour Water 
 
In Figure 4.27 the concentrations of each compound are normalized to show 
the percent remaining of each compound. Phenol and 2,4-dimethyl phenol had the 
lowest remaining concentrations with 43.6 and 33 percent respectively. Since phenol 
is the largest contributor to the total phenolics in the sample, the high fraction 
71 
 
removed is much more important than that observed for 2,4-dimethyl phenol. One 
important observation from the graph is that with the peroxidase enzyme the majority 
of the reduction occurs during the first 30 minutes of the reaction. This is quite 
different than the trend observed for the laccase/peroxide treatment system.  
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
o
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c
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I
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C
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(
C
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C
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)
0 . 0
0 . 2
0 . 4
0 . 6
0 . 8
1 . 0
1 . 2
P h e n o l
O - c r e s o l
P - c r e s o l
2 , 4 - d i m e t h y l  p h e n o l
 
Figure 4.27: Fraction Remaining of Phenolic Compounds using Peroxidase 
Treatment 
 
CITGO Stripped Sour Water 
 Stripped sour water from CITGO?s Lake Charles Refinery in Louisiana was 
used to test the laccase enzyme?s removal efficiency of phenolic compounds. An 
initial dose of 200 units of laccase and 300 mg/L of hydrogen peroxide were added to 
72 
 
the sample and the concentrations of phenol, o-cresol, p-cresol, and 2,4-dimethyl 
phenol were determined at fifteen minute intervals. Six replicate sour water tests were 
run using the laccase enzymatic treatment. The CITGO sour water was found to 
contain an average of 63.7 mg/L of phenol, 14.6 mg/L of o-cresol, 36.8 mg/L of p-
cresol, and 2.1 mg/L of 2,4-dimethyl phenol. The total amount of phenols in the 
CITGO stripped sour water was averaged 117.2 mg/L.  
To better compare the reduction of laccase and peroxidase the first order rate 
constants for each was examined. These first order rate constants are found in Table 
4.7. In first order kinetics ln [A] = -kt + ln [A0], where A0 equals the initial 
concentration, A equals the concentration at time t, and k is equal to the first order 
rate constant. The first order rate constant is equal to the slope of the line given by ln 
[A], or natural log of the concentration, verses time.  
Table 4.7: First Order Rate Constants for Laccase and Peroxidase in CITGO Stripped 
Sour Water 
 
Compound Laccase k  (min-1) Peroxidase k 
 (min-1) 
Phenol -0.0100 (R2 = 0.94) -0.0125 (R2 = 0.95) 
O-cresol -0.0058 (R2 = 0.96) -0.0043 (R2 = 0.96) 
P-cresol -0.0065 (R2 = 0.92) -0.0063 (R2 = 0.95) 
2,4-Dimethyl Phenol -0.0122 (R2 = 0.94) -0.0122 (R2 = 0.95) 
 
 The change over time in phenol concentration cause by laccase/peroxide 
treatment can be seen in Figure 4.28. The initial phenol concentration was 63.7 mg/L 
for the CITGO stripped sour water, and was reduced to 36.6 mg/L over a 60 minute 
reaction time. Thus the phenol concentration was lowered by 42.5 percent. Little 
73 
 
reduction in phenol occurred over the first 15 minutes. The first order rate constant 
for this 42.5 percent reduction in phenol by laccase was found to be -0.0101 with a R2 
equal to 0.94.  
T i m e  ( m i n u t e s )
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50
60
70
 
Figure 4.28: Laccase Treatment of Phenol in CITGO Stripped Sour Water 
 
Shown in Figure 4.29 is the concentration of o-cresol over the 60 minute 
reaction time. The concentration of o-cresol decreased from 14.6 mg/L to 10.6 mg/L 
in 60 minutes. The reduction in o-cresol caused by laccase/peroxide treatment was 
27.4 percent. As for phenol, little reduction of o-cresol occurred during the first 15 
minutes of the reaction. The first order rate constant for laccase reducing o-cresol was 
74 
 
found to be -0.0058 min-1 with an R2 value of 0.96 (Table 4.7). The laccase enzyme?s 
first order reduction rate constant for reducing o-cresol was nearly half that of phenol.  
 
T i m e  ( m i n u t e s )
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C
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8
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12
14
16
 
Figure 4.29: Laccase Treatment of O-cresol in CITGO Stripped Sour Water 
 
The p-cresol concentration over time is shown in Figure 4.30. P-cresol 
concentration decreased 32.9 percent over the 60 minutes when using 
laccase/peroxide treatment. The stripped sour water was found to have an initial 
concentration of 36.8 mg/L and that was reduced by laccase to 24.7 mg/L. The 
percent reduced was very similar to that of o-cresol, and somewhat lower than that of 
75 
 
phenol. The first order rate constant for p-cresol reduced by laccase was found to be -
0.0065 min-1 with an R2 value of 0.92 (Table 4.7). The rate constants for laccase and 
peroxidase for reducing p-cresol were essentially equal; therefore the enzymes are 
equally effective. Although the first order reduction rate constant for p-cresol was 
equivalent to that of o-cresol, the rate was nearly half that of phenol.  
T i m e  ( m i n u t e s )
0 15 30 45 60 75
C
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10
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30
40
 
Figure 4.30: Laccase Treatment of P-cresol in CITGO Stripped Sour Water 
 
The reduction of 2,4-dimethyl phenol by laccase is shown in Figure 4.31. 2,4-
dimethyl phenol was lowered by laccase treatment from a concentration of 2.11 mg/L 
to 1.02 mg/L after 60 minutes of reaction time. The reduction of 2,4-dimethyl phenol 
76 
 
was 51.7 percent. Although 2,4-dimethyl phenol was had the highest percent 
reduction by the laccase enzyme, since phenol is a much more significant component 
of the wastewater, its reduction is much more important. The first order rate constant 
for the oxidation of 2,4-dimethyl phenol by laccase and hydrogen peroxide is -0.0122 
min-1 with R2 equal to .94 and can be found in Table 4.7. The laccase reduction rate 
for 2,4-dimethyl phenol was almost double the rate for o-cresol and p-cresol, and 
about 25 percent higher than the oxidation rate of phenol.   
T i m e  ( m i n u t e s )
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(
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0 . 0
0 . 5
1 . 0
1 . 5
2 . 0
2 . 5
 
Figure 4.31: Laccase Treatment of 2,4-Dimethyl Phenol in 
CITGO Stripped Sour Water 
 
77 
 
In Figure 4.32 the concentrations of each compound are normalized to show 
the percent remaining of each compound. The compounds with the highest percent 
remaining were o-cresol and p-cresol as they were significantly higher than that of 
phenol and 2,4-dimethyl phenol. Phenol and 2,4-dimethyl phenol had the lowest 
remaining concentrations with 57.5 and 47.6 percent respectively. One important 
point seen in the graph is that the major drop in concentrations occurred after 15 
minutes, and this lay in total phenols removed was not observed in the clean system.  
T i m e  ( m i n u t e s )
0 15 30 45 60 75
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0 . 0
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1 . 0
1 . 2
P h e n o l
O - c r e s o l
P - c r e s o l
2 , 4 - d i m e t h y l  p h e n o l
 
Figure 4.32: Fraction Remaining of Phenolic Compounds using Laccase Treatment 
on CITGO Stripped Sour Water 
 
78 
 
The effectiveness of peroxidase enzyme for decreasing concentrations of 
phenolic compounds in stripped sour water from CITGO?s Lake Charles Refinery in 
Louisiana was studied. An enzyme dose of 2600 units of peroxidase and 300 mg/L of 
hydrogen peroxide were added to the sample and the concentrations of individual 
phenolic compounds were determined at fifteen minute intervals. Six replicate sour 
water tests were conducted using the peroxidase for enzymatic treatment. The CITGO 
sour water was found to initially contain an average of 51.9 mg/L phenol, 10.1 mg/L 
o-cresol, 24.5 mg/L p-cresol, and 1.9 mg/L 2,4-dimethyl phenol. The total average 
amount of phenolic compounds in the stripped sour water was 88.3 mg/L.  
The reduction of phenol by peroxidase over time is shown in Figure 4.33. 
Phenol was decreased, by peroxidase/peroxide treatment, from a concentration of 
51.9 mg/L to 24.9 mg/L after 60 minutes. The one hour reduction of phenol was 52 
percent. The first order rate constant for peroxidase treatment of phenol was 
determined to be -0.0125 min-1 with a R2 value of 0.95 (Table 4.7). There was very 
little reduction in phenol over the first 15 minutes of the reaction. Linear reduction in 
phenol was observed after 15 minutes.  
 
 
79 
 
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50
60
 
Figure 4.33: Peroxidase Treatment of Phenol in CITGO Stripped Sour Water 
 
The concentrations over time of o-cresol in CITGO stripped sour water by 
peroxidase/peroxide treatment are shown in Figure 4.34. The concentration decreased 
from 10.1 mg/L initially to 7.8 mg/L (22.8 percent) after 60 minutes time. The rate of 
o-cresol change was consistent over the 60 minute test period. The percent reduction 
was less than half of that observed for phenol.  The first order rate constant  for 
peroxidase reduction of o-cresol was -0.0043 min-1 with R2 equal to 0.96 (Table 4.7). 
The reduction rate constant for o-cresol is almost three times lower than that of 
phenol.  
80 
 
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8
10
12
 
Figure 4.34: Peroxidase Treatment of O-cresol in CITGO Stripped Sour Water 
 
The decrease in p-cresol concentration over time by peroxidase oxidation is 
shown in Figure 4.35. The peroxidase enzyme lowered the concentration of p-cresol 
from 24.5 mg/L to 16.8 mg/L. This corresponds to a reduction in p-cresol by 
peroxidase of 31.4 percent. The percent reduction of p-cresol is comparable to o-
cresol, and is almost half that of phenol.. The first order rate constant for peroxidase 
reduction of p-cresol was found to be -0.0063 min-1 with R2 equal to 0.95 (Table 4.7). 
This rate constant was slightly higher than the first order rate constant for peroxidase 
reduction of o-cresol. The reduction rate constant for p-cresol is about two times 
81 
 
lower though that that of phenol. P-cresol degradation began during the first 15 
minutes of the reaction.   
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Figure 4.35: Peroxidase Treatment P-cresol in CITGO Stripped Sour Water 
 
2,4-dimethyl phenol was lowered by peroxide treatment from a concentration 
of 1.87 mg/L to 0.92 mg/L after 60 minutes (Figure 4.36). The reduction of 2,4-
dimethyl phenol was 50.8 percent. Most of the 2,4-dmp degradation occurred during 
the first 30 minutes of reaction. The percent reduction is close to that of phenol. 
However, there was much more phenol in the waste than 2,4-dmp. The first order rate 
constants for each compound in the synthetic wastewater are shown in Table 4.7. The 
82 
 
first order rate constant for 2,4-dimethyl phenol was found to be -.0162 min-1 with a 
R2 value equal to 0.97 using peroxidase enzymatic treatment. This is very close to the 
rate constant for phenol, but is about two times higher than the rate constant for o-
cresol and p-cresol. 
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Figure 4.36: Peroxidase Treatment of 2,4-Dimethyl Phenol in 
CITGO Stripped Sour Water 
 
For each individual phenolic compound the concentrations were normalized to 
the initial concentration so that the fraction remaining is shown (Figure 4.37). Phenol 
and 2,4-dimethyl phenol had the lowest remaining concentrations with 57.5 and 47.6 
83 
 
percent respectively. O-cresol and p-cresol were the phenolic compounds with the 
highest fraction remaining as they were notably higher than that of phenol and 2,4-
dimethyl phenol. Unlike laccase/peroxide treatment, there was no lay with 
peroxidase/peroxide treatment.  
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P h e n o l
O - c r e s o l
P - c r e s o l
2 , 4 - d i m e t h y l  p h e n o l
 
Figure 4.37: Fraction Remaining of Phenolic Compounds using Peroxidase Treatment 
on CITGO Stripped Sour Water 
 
The first order rate constants for the oxidation of each selected compound 
reduced by peroxidase are found in Table 4.7. In this particular refinery wastewater 
matrix the reduction rates were significantly lower than the rates for each compound 
tested in de-ionized water (Table 4.1 and Table 4.2). Again an important thing to note 
84 
 
is that in this particular case the rate constants for laccase and peroxidase are 
essentially the same, thus the enzymes are more or less equally efficient.   
Frontier Desalter Effluent 
 Desalter effluent from Frontier?s Cheyenne Refinery in Wyoming was used to 
test the laccase enzyme?s removal efficacy of phenolic compounds. An initial dose of 
200 units of laccase and 300 mg/L of hydrogen peroxide were added to the sample 
and the concentrations of phenol, o-cresol, p-cresol, and 2,4-dimethyl phenol were 
determined at fifteen minute intervals. There were three replicate tests run using the 
laccase enzymatic treatment. The desalter effluent initially contained none of the 
target compounds. Therefore, 100ppm of phenol, o-cresol, and p-cresol were added to 
the sample. The sample was then tested using the gas chromatograph and was found 
to contain 103.8 mg/L of phenol, 98.6 mg/L of o-cresol, and 76.9 mg/L of p-cresol. 
The total amount of phenols in the Frontier desalter water was initially 279.3 mg/L.  
 
Table 4.8: First Order Rate Constants for Laccase and Peroxidase in Frontier Desalter 
 
Compound Laccase k
 (min-1) 
Peroxidase 
k (min-1) 
Phenol -0.0118 (R2 = 0.95) -0.0122 (R2 = 0.95) 
O-cresol -0.0131 (R2 = 0.94) -0.0135 (R2 = 0.96) 
P-cresol -0.0116 (R2 = 0.95) -0.0095 (R2 = 0.97) 
 
 
85 
 
The change in phenol concentration over time by laccase/peroxide treatment 
in the Frontier desalter effluent is shown in Figure 4.38. The initial phenol 
concentration was 103.8 mg/L and was reduced by the laccase enzymatic treatment to 
50.4 mg/L over a 60 minute reaction time. Thus the phenol concentration was 
lowered by 51.4 percent. One observation is that more phenol reduction occurred in 
the first 30 minutes of the reaction. The first order rate constant for laccase reduction 
of phenol was found to be -0.0118 min-1 with R2 equal to 0.95 (Table 4.8).  
T i m e  ( m i n u t e s )
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Figure 4.38: Laccase Treatment of Phenol in Frontier Desalter Effluent 
 
The decrease in o-cresol concentration by laccase can is shown in Figure 4.39. 
The laccase enzymatic treatment reduced the concentration of o-cresol from 98.6 
86 
 
mg/L to 42.5 mg/L over a 60 minute reaction time. The reduction in o-cresol 
concentration was lowered by 56.9 percent. Like phenol, the reduction of o-cresol 
occurred in the first 30 minutes of the reaction. The first order rate constants for o-
cresol are found in Table 4.8. The first order rate constant for laccase reduction of o-
cresol was found to be -0.031 min-1 with R2 value of 0.95. The reduction rate of o-
cresol was the highest rate for laccase in the Frontier desalter effluent. The rate for 
laccase oxidation of o-cresol was slightly higher than that of phenol.  
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Figure 4.39: Laccase Treatment of O-cresol in Frontier Desalter Effluent 
 
Figure 4.40 shows the concentration over time for p-cresol using the laccase 
enzymatic treatment. P-cresol concentration decreased from an initial concentration 
87 
 
of 76.9 mg/L to 39 mg/L over the 60 minutes, a reduction of 49.3 percent. The first 
order reduction rate constant for laccase/peroxide reduction of p-cresol found in 
Table 4.8 is -0.0116 min-1 with R2 equal to 0.95. The rate constant for p-cresol is 
essentially the same as that of phenol, but slightly lower than the rate constant for o-
cresol.    
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Figure 4.40: Laccase Treatment of P-cresol in Frontier Desalter Effluent 
  
In Figure 4.41 the concentrations for each compound added are normalized to 
show the percent remaining for each phenolic compound in the Frontier desalter 
effluent. O-cresol had the lowest percent remaining with 43.1 percent, while phenol 
and p-cresol had similar percent remaining with 49.6 and 50.7 percent respectively.  
88 
 
The majority of the reduction in these compounds occurred during the first thirty 
minutes of the reaction time, which is closer to what was observed in the ?clean 
matrix? system of phase one.  
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0 . 0
0 . 2
0 . 4
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1 . 2
P h e n o l
O - c r e s o l
P - c r e s o l
 
Figure 4.41: Fraction Remaining of Phenolic Compounds using Laccase Treatment 
on Frontier Desalter Effluent 
 
The efficiency of the peroxidase enzyme for reducing phenolic compounds 
from the wastewater was tested in the same Frontier Desalter Effluent. An initial 
amount of 2600 units of peroxidase where added along with 300 mg/L of hydrogen 
peroxide to the effluent. As before, three replicates were tested using the peroxidase 
enzyme and the average initial concentrations contained in the desalter effluent were: 
89 
 
105.6 mg/L of phenol, 56.0 mg/L of o-cresol, 59.2 mg/L of p-cresol, and 220.8 mg/L 
total phenol. The first order rate constants for the oxidation of each selected 
compound reduced by peroxidase are also shown in Table 4.8.  
Figure 4.42 shows the decrease in concentration of phenol by 
peroxidase/peroxide treatment over the 60 minutes of reaction. The concentration of 
phenol was reduced from 105.6 mg/L to 46.8 mg/L in 60 minutes, a reduction of 55.7 
percent. The first order rate constant for peroxidase reduction of phenol was found to 
be -0.0122 min-1 with R2 value of 0.95 (Table 4.8). One observation is that the 
reduction of phenol in this particular matrix occurred abruptly in the first 15 minutes, 
leveled off, and then decreased again in the last 15 minutes.   
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Figure 4.42: Peroxidase Treatment of Phenol in Frontier Desalter Effluent 
90 
 
 
Figure 4.43 shows the concentration of o-cresol in the Frontier deslater 
effluent over time reduced by peroxidase treatment. O-cresol concentration decreased 
58.8 percent from 56.0 mg/L initially to 23.1 mg/L in 60 minutes. This reduction 
resembles that of phenol. The first order rate constant for o-cresol reduced by 
peroxidase was found to be -0.0135 min-1 with an R2 value of 0.96 (Table 4.8). The 
first order reduction rate constant for o-cresol was higher than that of phenol by 
approximately 10 percent.  
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Figure 4.43: Peroxidase Treatment of O-cresol in Frontier Desalter Effluent 
 
91 
 
The reduction of p-cresol concentration in the Frontier desalter effluent over 
time by peroxidase is shown in Figure 4.44. Peroxidase treatment lowered the 
concentration from 59.2 mg/L to 33.9 mg/L in 60 minutes. This percent reduction 
corresponds to a 42.7 percent decrease and is lower than that of phenol and o-cresol. 
The first order rate constant for p-cresol reduced by peroxidase was found to be -
0.0095 min-1 with an R2 value of 0.97 (Table 4.8). The first order reduction rate 
constant for p-cresol was lower than that of phenol and o-cresol, which were 
approximately 30 percent and 45 percent higher respectively.   
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Figure 4.44: Peroxidase Treatment of P-cresol in Frontier Desalter Effluent 
 
92 
 
In Figure 4.45 the concentrations of phenol, o-cresol, and p-cresol were 
normalized to show the fraction remaining of each compound tested with the 
peroxidase enzyme. Phenol and o-cresol have the lowest remaining concentrations 
with 44.3 and 41.2 percent respectively. P-cresol was slightly higher with 57.3 
percent remaining. One important thing to note from this graph is that a big reduction 
occurs between the reaction time 0 and 15 minutes and then again between 45 and 60 
minutes.  
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0 . 0
0 . 2
0 . 4
0 . 6
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1 . 0
1 . 2
P h e n o l
O - c r e s o l
P - c r e s o l
 
Figure 4.45: Fraction Remaining of Phenolic Compounds using Peroxidase Treatment 
on Frontier Desalter Effluent 
 
 
 
93 
 
CITGO Desalter Effluent 
The efficiency of laccase enzyme to remove selected phenolic compounds was 
examined using desalter effluent from CITGO?s Lake Charles Refinery in Louisiana.  
A dose of 200 units of laccase and 300 mg/L of hydrogen peroxide were added to the 
sample and then tested at fifteen minute intervals on the gas chromatograph to 
determine the concentrations of phenol, o-cresol, p-cresol, and 2,4-dimethyl phenol. 
Three different tests were run using the laccase enzymatic treatment. The desalter 
effluent initially contained 41.6 mg/L of phenol, 20.3 mg/L of o-cresol, 37.1 mg/L of 
p-cresol, and 2.6 mg/L of 2,4-dimethyl phenol. This data was used to determine the 
first order rate constants. These first order rate constants and R2 values for both 
enzymes for each compound are shown in Table 4.6. An R2 value greater than 0.90 
insures the data fits the first order kinetic model well.  
Table 4.9: First Order Rate Constants for Laccase and Peroxidase in  
CITGO Desalter 
 
Compound Laccase k
 (min-1) 
Peroxidase 
k (min-1) 
Phenol -0.0141 (R2 = 0.97) -0.0173 (R2 = 0.89) 
O-cresol -0.0079 (R2 = 0.83) -0.0119 (R2 = 0.99) 
P-cresol -0.0092 (R2 = 0.90) -0.0111 (R2 = 0.79) 
2,4-Dimethyl Phenol -0.0147 (R2 = 0.97) -0.0214 (R2 = 0.97) 
 
The decrease in concentration of phenol over the 60 minute reaction time is 
shown in Figure 4.46. The CITGO desalter effluent contained an initial phenol 
concentration of 41.6 mg/L. Phenol concentration was reduced 58.4 percent by the 
laccase enzymatic treatment to a concentration of 17.3 mg/L. The first order rate 
94 
 
constant for laccase reduction of phenol was found to be -0.0141 min-1 with R2 value 
of 0.97 (Table 4.9).  
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Figure 4.46: Laccase Treatment of Phenol in CITGO Desalter Effluent 
 
The reduction in o-cresol concentration by laccase treatment is shown in 
Figure 4.47. O-cresol was lowered by laccase treatment from a concentration of 20.3 
mg/L to 13.1 mg/L after 60 minutes. This corresponds to a 35.5 percent reduction of 
o-cresol. Most of the o-cresol degradation occurred during 15 and 30 minutes of 
reaction. The percent reduction is significantly less than phenol. The first order rate 
constant for laccase reduction of o-cresol found in Table 4.9 was -0.0079 min-1 with 
95 
 
R2 equal to 0.83. This rate constant is almost two times lower than that of phenol. 
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Figure 4.47: Laccase Treatment of O-cresol in CITGO Desalter Effluent 
 
The concentration of p-cresol over time reduced by laccase is shown in Figure 
4.48. Laccase treatment reduced the P-cresol from a concentration of 37.1 mg/L to 
22.5 mg/L over the span of 60 minutes, a reduction of 39.4 percent. This percent 
resembles that of o-cresol, and is lower than phenol. Similar to o-cresol, the majority 
of p-cresol degradation occurred during the first 30 minutes. The first order rate 
constant for laccase reduction of p-cresol was found to be was -0.007992 min-1 with 
R2 value of 0.90. This rate constant was lower than the first order rate constants for 
96 
 
laccase reduction of phenol, but slightly higher than the removal rate of o-cresol. 
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Figure 4.48: Laccase Treatment of P-cresol in CITGO Desalter Effluent 
 
Figure 4.49 shows the concentration of 2,4-dimethyl phenol in the desalter 
effluent over the 60 minutes. 2,4-dimethyl phenol decreased from a concentration of 
2.6 mg/L to 1.1 mg/L due to laccase/peroxide treatment. This corresponds to a 
reduction of 57.7 percent. The percent reduction is very close to that of phenol, and is 
much higher than p-cresol and o-cresol. However, there was much more phenol in the 
CITGO desalter effluent than 2,4-dimethyl phenol. The first order rate constant for 
laccase reduction of 2,4-dimethyl phenol was determined to be -0.0147 min-1 with a 
97 
 
great R2 value of 0.97. This rate constant for 2,4-dimethyl phenol was the highest first 
order rate constant for the laccase/peroxide treatment. The reduction rate constant for 
2,4-dimethyl phenol is almost two times high that that of o-cresol and p-cresol, and is 
slightly higher than the rate constant for phenol. 
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3 . 0
 
Figure 4.49: Laccase Treatment of 2,4-Dimethyl Phenol in CITGO Desalter Effluent 
 
In Figure 4.50 the individual concentrations for each compound were 
normalized to the initial concentration to show the fraction remaining. Phenol and 
2,4-dimethyl phenol had the smallest percent remaining with 41.6 and 42.3 percent 
respectively. This percent was slightly higher than the percent remaining for o-cresol 
98 
 
and p-cresol, which were 64.5 and 60.6 percent respectively. Like the clean system, 
the most degradation occurred during the first 30 minutes of the reaction, however, 
with o-cresol and p-cresol there was a lay of 15 minutes.  
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P h e n o l
O - c r e s o l
P - c r e s o l
2 , 4 - d i m e t h y l  p h e n o l
 
Figure 4.50: Fraction Remaining of Phenolic Compounds using Laccase Treatment 
on CITGO Desalter Effluent 
 
The efficacy for reducing phenolic compounds from the wastewater was 
tested in the same Frontier Desalter Effluent using the peroxidase enzyme. An initial 
amount of 2600 units of peroxidase where added along with 300 mg/L of hydrogen 
peroxide to the desalter effluent. As before, three duplicate samples were tested using 
the peroxidase enzyme and the average initial concentrations contained in the water 
99 
 
were: 62.4 mg/L of phenol, 29.5 mg/L of o-cresol, 58.2 mg/L of p-cresol, 4.4 mg/L of 
2,4-dimethyl phenol, and 220.8 mg/L total phenol. The first order rate constants for 
peroxidase were also examined and are shown in Table 4.9.  
Figure 4.51 shows the decrease in phenol concentration over time by 
peroxidase/peroxide treatment. The CITGO desalter effluent contained an initial 
phenol concentration of 62.4 mg/L, and this decreased to 22.1 over 60 minutes. This 
corresponds to a reduction in phenol of 64.6 percent. The decrease in phenol over 
time followed an exponential reduction, and a majority of the reduction occurred in 
the first 30 minutes of the reaction. The first order rate constant for peroxidase 
reducing phenol was found to be -0.0173 min-1 with an R2 value of 0.89 (Table 4.9).  
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Figure 4.51: Peroxidase Treatment of Phenol in CITGO Desalter Effluent 
100 
 
The reduction in concentration of o-cresol by the peroxidase enzyme is shown 
in Figure 4.52. The desalter effluent contained an initial o-cresol concentration of 
29.5 mg/L and was lowered to 14.1 mg/L after 60 minutes by peroxidase/peroxide 
treatment. This corresponds to reduction of o-cresol by peroxidase of 52.2 percent. 
The percent reduction is somewhat less than that of phenol. Although like phenol, the 
majority of the reduction occurred in the first 30 minutes of the reaction. The first 
order rate constant for peroxidase/peroxide reducing o-cresol was determined to be -
0.0119 min-1 with an R2  value of 0.99 (Table 4.9). Again, the high R2 value shows 
that the first order model yields a very good fit for the data. The peroxidase enzyme?s 
first order reduction rate constant for o-cresol was nearly half that of phenol.  
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30
35
 
Figure 4.52: Peroxidase Treatment of O-cresol in CITGO Desalter Effluent 
101 
 
 
The concentration over time for peroxidase/peroxide treatment of p-cresol 
over time is shown in Figure 4.53. Peroxidase/peroxide treatment reduced the p-
cresol in the desalter effluent from a concentration of 58.2 mg/L to 26.2 mg/L over 
the span of 60 minutes, a 55 percent reduction. This percent resembles that of o-
cresol., but again is less that the percent removal for phenol. Similar to phenol and o-
cresol, most of the degradation of p-cresol occurred in the first 15 minutes of the 
reaction. The lay after the 15 minutes could be due to low hydrogen peroxide levels in 
the sample, or more realistically, that the peroxidase/peroxide was oxidizing other 
compounds in the matrix other than p-cresol. The first order rate constant for 
peroxidase was found to be -0.0111 min-1 with an R2 value of 0.79 (Table 4.9). The 
first order reduction rate constant for p-cresol was comparable to that of o-cresol, but 
was nearly half that phenol. The lower R2 value indicates a moderate fit with the first 
order kinetic model.  
102 
 
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Figure 4.53: Peroxidase Treatment of P-cresol in CITGO Desalter Effluent 
 
The concentration of 2,4-dimethyl phenol over time reduced by 
peroxidase/peroxide is shown in Figure 4.54. 2,4-dimethyl phenol was reduced by the 
treatment from an initial concentration of 4.4 mg/L to 1.2 mg/L. The peroxidase 
lowered the 2,4-dimethyl phenol concentration by 72.7 percent for the 60 minutes. 
This decrease in 2,4-dimethyl phenol concentration is more than any of the other 
three compounds, however it is important to note that 2,4-dimethyl phenol made up 
only a small fraction of the total phenolics in the sample. The first order rate constant 
for peroxidase reducing 2,4-dimethyl phenol was found to be -0.0214 min-1 with an 
103 
 
R2 value of 0.97, shown in Table 4.9. The rate constant is approximately 25 percent 
higher than that of phenol, and close to two times that of p-cresol and o-cresol.  
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Figure 4.54: Peroxidase Treatment of 2,4-Dimethyl Phenol in  
CITGO Desalter Effluent 
 
The concentrations of each individual compound tested were normalized to 
the initial concentration to show the fraction remaining using the peroxidase enzyme 
(Figure 4.55). The percent remaining for o-cresol and p-cresol was 47.8 and 45 
percent respectively. O-cresol and p-cresol fraction remaining was nearly double that 
of phenol and 2,4-dimethyl phenol. Phenol and 2,4-dimethyl phenol had the lowest 
remaining concentrations with 35.4 and 27.3 percent respectively. Similar to 
104 
 
laccase/peroxide treatment, the majority of the degradation occurred during the first 
30 minutes of the reaction, however with peroxidase/peroxide treatment there was no 
lay in the reduction.  
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i
t
i
a
l
 
C
o
n
c
e
n
t
r
a
t
i
o
n
 
(
C
/
C
o
)
0 . 0
0 . 2
0 . 4
0 . 6
0 . 8
1 . 0
1 . 2
P h e n o l
O - c r e s o l
P - c r e s o l
2 , 4 - d i m e t h y l  p h e n o l
 
Figure 4.55: Fraction Remaining of Phenolic Compounds using Peroxidase Treatment 
on CITGO Desalter Effluent 
 
Dartmouth Biox Influent 
The laccase enzyme?s ability to reduce the concentration of major phenolic 
constituents in biox influent from Imperial Oil?s Dartmouth Refinery in Nova Scotia 
was examined. An initial dose of 200 units of laccase and 300 mg/L of hydrogen 
peroxide were added to the biox influent. Gas chromatography was used to determine 
105 
 
the concentrations of the phenol, o-cresol, p-cresol, and 2,4-dimethyl phenol at fifteen 
minute intervals. Four replicate tests were run using the laccase/peroxide enzymatic 
treatment.  The biox influent was found to contain a non-detectable amount of the 
target phenolic compounds; therefore, 100ppm of phenol, 100ppm of o-cresol, 
100ppm of p-cresol, and 50ppm of 2,4-dimethyl phenol were added to the biox 
influent. The biox influent was then test and initially contained 69.9 mg/L of phenol, 
63.8 mg/L of o-cresol, 34.6 mg/L of p-cresol, and 2.1 mg/L of 2,4-dimethyl phenol, a 
total amount of 172.8 mg/L phenolics. This data was used to determine the first order 
rate constants and R2 values for laccase/peroxide reduction of each individual 
compound; these are shown in Table 4.10.  
Table 4.10: First Order Rate Constants for Laccase and Peroxidase  
in the Dartmouth Biox Influent  
 
Compound Laccase k
 (min-1) 
Peroxidase 
k (min-1) 
Phenol -0.0113 (R2 = 0.83) -0.0141 (R2 = 0.95) 
O-cresol -0.0117 (R2 = 0.80) -0.0154 (R2 = 0.98) 
P-cresol -0.0104 (R2 = 0.92) -0.0149 (R2 = 0.91) 
2,4-Dimethyl Phenol -0.0119 (R2 = 0.97) -0.0148 (R2 = 0.91) 
 
The decrease in phenol concentration by the laccase enzyme over time is 
shown in Figure 4.56. The initial 69.9 mg/L of phenol in the water was lowered to 
36.0 mg/L over the 60 minutes. The concentration decrease of phenol in the biox 
influent corresponds to a 48.5 percent reduction by laccase/peroxide treatment. It is 
important to note that the majority of the degradation of phenol occurred during the 
first 30 minutes of the reaction, very little occurred after that time. For reducing 
106 
 
phenol laccase/peroxide had a first order rate constant of -0.0113 min-1 and an R2 
value of 0.83 (Table 4.10). The low R2 value indicates that the data had a moderate fit 
with the first order model.  
Ti m e  (m in u te s)
0 15 30 45 60 75
C
o
n
ce
n
tra
tio
n
 (m
g
/L
)
0
20
40
60
80
 
Figure 4.57: Laccase Treatment of Phenol in Dartmouth Biox Influent 
 
Figure 4.58 shows the reduction of o-cresol over time caused by the 
laccase/peroxide addition. The Dartmouth biox influent contained an average initial 
o-cresol concentration of 63.8 mg/L, which was reduced to 32.5 mg/L over 60 
minutes reaction time. This corresponds to reduction of 49.1 percent. Like the 
decrease in phenol, a majority of the reduction of o-cresol also occurred during the 
first 30 minutes of the reaction. Laccase had a first order rate constant of -0.0117 min-
107 
 
1 and an R2 value of 0.80 (Table 4.10). Also like phenol, the first order rate constant 
was very similar (less than 5 percent better) to phenol and the data also yield a 
moderate fit with the first order model.  
Ti m e  (m in u te s)
0 15 30 45 60 75
C
o
n
ce
n
tra
tio
n
 (m
g
/L
)
0
10
20
30
40
50
60
70
 
Figure 4.58: Laccase Treatment of O-cresol in Dartmouth Biox Influent 
 
Figure 4.59 shows the decrease in p-cresol concentration over time due to 
laccase/peroxide treatment. The p-cresol concentration in the biox influent decreased 
from 34.6 mg/L to 17.7 mg/L after 60 minutes, a 48.8 percent reduction. This 
reduction is very similar to that of phenol and o-cresol. The first order rate constant 
for p-cresol was determined to be -.0104 min-1 with a R2 value of 0.92 (Table 4.10). 
108 
 
The rate constant for p-cresol was lower than the rate constant of o-cresol and phenol 
by approximately 10 percent.  
Ti m e  (m in u te s)
0 15 30 45 60 75
C
o
n
ce
n
tra
tio
n
 (m
g
/L
)
0
10
20
30
40
 
Figure 4.59: Laccase Treatment of P-cresol in Dartmouth Biox Influent 
 
The change in concentration of 2,4-dimethyl phenol over time caused by 
laccase/peroxide is shown in Figure 4.60. The concentration of 2,4-dimethyl phenol 
decreased from an initial concentration of 2.1 mg/L to 0.99 mg/L after 60 minutes 
(52.4 percent). Although 2,4-dimthyl had the highest percent reduction using the 
laccase/peroxide treatment, however, the 48.5 percent removal for phenol is much 
more important because phenol is the largest contributor to the total phenolics. The 
first order rate constant for laccase reducing 2,4-dimethyl phenol was found to be -
109 
 
.0119 min-1 with a R2 equal to 0.97 (Table 4.10). Although this is the highest rate for 
laccase/peroxide treatment, it is less than 5 percent better than the rate constants of o-
cresol and phenol.  
Ti m e  (m in u te s)
0 15 30 45 60 75
C
o
n
ce
n
tra
tio
n
 (m
g
/L
)
0 . 0
0 . 5
1 . 0
1 . 5
2 . 0
2 . 5
 
Figure 4.60: Laccase Treatment of 2,4-Dimethyl Phenol in Dartmouth Biox Influent 
 
To compare the decrease in the individual phenolic compounds, the 
concentrations of each enzyme were normalized to the initial concentration and are 
shown in Figure 4.61. This then shows the percent remaining for each compound over 
the 60 minutes. In this case, phenol had the highest percent remaining with 51.5, 
while 2,4-dimethyl phenol had the lowest with 47.6 percent. O-cresol and p-cresol 
had 50.9 and 51.2 percent remaining, respectively. An important observation from the 
110 
 
graph is that with the laccase/peroxide treatment the majority of the reduction 
occurred during the first 30 minutes of the reaction, similar to that observed in a clen 
system. 
T im e ( m inut es)
0 15 30 45 60 75
C
oncent
r
at
ion 
ov
er
 
I
nit
ial 
C
oncent
r
at
ion 
(
C
/
C
o)
0. 0
0. 2
0. 4
0. 6
0. 8
1. 0
1. 2
P he no l
O- cr esol
P - cr esol
2, 4- D i m et hy l  P he no l
 
Figure 4.61: Fraction Remaining of Phenolic Compounds using Laccase Treatment 
on Dartmouth Biox Influent 
 
The peroxidase enzyme was tested on the same biox influent from Dartmouth 
for its efficiency to remove the same phenolic compounds.  An initial dose of 2600 
units of peroxidase and 300 mg/L of hydrogen peroxide were added to the biox 
influent sample.  Like the laccase testing 100ppm of phenol, 100ppm of o-cresol, 
100ppm of p-cresol, and 50ppm of 2,4-dimethyl phenol were added to the sample 
since the original concentrations were non-detectable. Once added the concentrations 
111 
 
of phenol, o-cresol, p-cresol, and 2,4dimethyl phenol where measured with the gas 
chromatograph over fifteen minute intervals. There were three replicate test run using 
the peroxidase enzyme; and the average initial concentrations contained in the biox 
influent after the addition of phenols were: 101.3 mg/L of phenol, 38.4 mg/L of o-
cresol, 39.8 mg/L of p-cresol, 2.2 mg/L of 2,4-dimethyl phenol, and 177.9 mg/L total 
phenol. The first order rate constants were also determined for peroxidase and are 
shown in Table 4.10.  
Figure 4.62 shows the decrease in phenol concentration over time using the 
peroxidase enzyme coupled with hydrogen peroxide. The phenol concentration in the 
Dartmouth biox influent was reduced by peroxidase 61 percent from an initial phenol 
concentration of 101.3 mg/L to 22.1 mg/L after 60 minutes. The majority of the 
reduction occurred during the first and last 15 minutes of the reaction, with a lay in 
the middle. The first order rate constant for peroxidase for decreasing phenol was 
found to be -.0141 min-1 with a R2 value of 0.95 (Table 4.10).  
112 
 
 
Ti m e  (m i n u te s)
0 15 30 45 60 75
C
o
n
ce
n
tra
tio
n
 (m
g
/L
)
0
20
40
60
80
100
120
 
Figure 4.62: Peroxidase Treatment of Phenol in Dartmouth Biox Influent 
 
The initial concentration of o-cresol contained in the biox influent was 38.4 
mg/L (Figure 4.63). Peroxidase/peroxide decreased the concentration to 15.6 mg/L 
after 60 minutes. This corresponds to a reduction of o-cresol of 59.4 percent. The 
percent reduction is very close to that of phenol. The first order rate constant for 
peroxidase reducing o-cresol was found to be -.0154 min-1 with a R2 equal to 0.98 
(Table 4.10). The first order rate constant for o-cresol is close to 10 percent better 
than the rate for phenol. 
113 
 
T im e (m inut es)
0 15 30 45 60 75
Concent
ratio
n 
(m
g
/
L)
0
10
20
30
40
50
 
Figure 4.63: Peroxidase Treatment of O-cresol in Dartmouth Biox Influent 
 
The change in concentration over time for p-cresol is shown in Figure 4.64. 
Peroxidase treatment reduced the P-cresol in the biox influent from a concentration of 
39.8 mg/L to 15.9 mg/L over the 60 minutes, a 60.1 percent reduction. This percent 
resembles that of o-cresol and phenol. However, unlike phenol and o-cresol the major 
degradation of p-cresol occurred after 30 minutes reaction. The first order rate 
constant for p-cresol was found to be -.0149 min-1 with a R2 equal to 0.91 (Table 
4.10). The rate constant of p-cresol was about 5 percent higher than that of phenol 
and approximately 5 percent lower than that of o-cresol.  
114 
 
T im e (m inut es)
0 15 30 45 60 75
Concent
ratio
n 
(m
g
/
L)
0
10
20
30
40
50
 
Figure 4.64: Peroxidase Treatment of P-cresol in Dartmouth Biox Influent 
 
The change in concentration of 2,4-dimethyl phenol over time caused by 
peroxidase/peroxide is shown in Figure 4.65. The 2,4-dimethyl phenol was reduced 
from an initial concentration of 2.2 mg/L to .94 mg/L. The peroxidase lowered the 
2,4-dimethyl phenol concentration by 59.1 percent for the 60 minutes. Similar to o-
cresol the majority of the reduction occurred during the first 30 minutes of the 
reaction. Although the percent reduced is similar to that of the other phenolic 
compounds, since the other phenolic compounds make up 99 percent of the total 
phenolics their reduction is much more important. The first order rate constant for 
phenol reducing 2,4-dimethyl phenol was found to be -.0148 min-1 with a R2 equal to 
115 
 
0.91 (Table 4.10). This rate constant is very similar to the rate for reducing p cresol, 
slightly lower than that of o-cresol, and slightly higher than the phenol first order rate 
constant.  
T im e (m inut es)
0 15 30 45 60 75
C
oncent
ratio
n 
(m
g
/
L)
0. 0
0. 5
1. 0
1. 5
2. 0
2. 5
 
Figure 4.65: Peroxidase Treatment of 2,4-Dimethyl Phenol in Dartmouth Biox 
Influent 
 
The concentrations for each individual compound tested were normalized to 
the initial concentration in order to compare the fraction remaining for each 
compound reduced by the peroxidase enzyme (Figure 4. 66). Phenol and p-cresol had 
the lowest remaining concentrations with 39 and 39.9 percent respectively. The 
percent remaining for o-cresol and 2,4-dimethyl phenol was 40.6 and 40.9 percent 
116 
 
respectively.  The peroxidase enzyme essentially reduced all of the compounds the 
same amount.  
T im e ( m inut es)
0 15 30 45 60 75
C
oncent
r
at
ion 
ov
er
 
I
nit
ial 
C
oncent
r
at
ion 
(
C
/
C
o)
0. 0
0. 2
0. 4
0. 6
0. 8
1. 0
1. 2
P he no l
O- cr esol
P - cr esol
2, 4- di m et hy l  ph en ol
 
Figure 4.66: Fraction Remaining of Phenolic Compounds using Peroxidase Treatment 
on Dartmouth Biox Influent 
 
Sarnia Biox Effluent 
The efficacy of the laccase enzyme to remove selected phenolic compounds 
was studied using biox effluent from Imperial Oil?s Sarnia Refinery in Southwestern 
Ontario. The initial testing of the biox effluent revealed a non-detectable amount of 
the target phenolic compounds were present in the biox effluent, therefore 100ppm of 
phenol, 100ppm of o-cresol, and 100ppm of p-cresol were added for testing. An 
initial dose of 200 units of laccase and 300 mg/L of hydrogen peroxide were added to 
117 
 
the sample and then tested at fifteen minute intervals to determine the concentrations. 
Three replicate tests were run using the laccase/peroxide enzymatic treatment and the 
biox effluent was then found to contain an average of 56.2 mg/L phenol, 62.3 mg/L o-
cresol, 59.8 mg/L p-cresol, and a total amount of phenols of 178.3 mg/L. The data 
above was used to determine the first order rate constants for each phenolic 
compound for each enzyme; these are shown in Table 4.11.  
Table 4.11: First Order Rate Constants for Each Compound in the Sarnia Biox 
Effluent  
 
Compound Laccase k
 (min-1) 
Peroxidase 
k (min-1) 
Phenol -0.0153 (R2 = 0.96) -0.0152 (R2 = 0.98) 
O-cresol -0.0119 (R2 = 0.99) -0.0128 (R2 = 0.97) 
P-cresol -0.0060 (R2 = 0.99) -0.0108 (R2 = 0.90) 
 
Phenol concentration in the biox effluent was reduced by laccase/peroxide 
treatment from 56.2 mg/L to 21.1 mg/L over 60 minutes (Figure 4.67). This reduction 
in phenol by the laccase enzymatic treatment corresponds to 62.5 percent. Similar to 
that observed in the clean system, laccase catalyzed a rapid oxidation of phenol with 
significant reduction in concentration occurring over the first 15 minutes. The first 
order rate constant for phenol reduced by laccase/peroxide was found to be -0.0153 
min-1 with a R2 equal to 0.96 (Table 4.11). The high R2 value indicates a very good fit 
with the first order model.  
118 
 
T im e (m inut es)
0 15 30 45 60 75
Concent
ratio
n 
(m
g
/
L)
0
10
20
30
40
50
60
 
Figure 4.67: Laccase Treatment of Phenol in Sarnia Biox Effluent  
 
The decrease in o-cresol concentration over the one hour reaction time is 
shown in Figure 4.68. The Sarnia biox effluent was found to contain an initial o-
cresol concentration of 62.3 mg/L; laccase/peroxide reduced the concentration to 30.5 
over the 60 minute reaction time (51 percent). This reduction was slightly lower than 
phenol, however similar to phenol, the majority of the reduction occurred in the first 
30 minutes. The first order rate constant, found in Table 4.11, for o-cresol reduced by 
laccase/peroxide was -0.0119 min-1 with a R2 equal to 0.99. The rate constant of o-
cresol was lower than the rate for the reduction of phenol by over 25 percent.   
119 
 
T im e (m inut es)
0 15 30 45 60 75
Concent
ratio
n 
(m
g
/
L)
0
10
20
30
40
50
60
70
 
Figure 4.68: Laccase Treatment of O-cresol in Sarnia Biox Effluent 
 
Figure 4.69 shows the change in concentration of p-cresol over time caused by 
laccase/peroxide treatment. The p-cresol decreased from 59.8 mg/L initially to 41.4 
mg/L after 60 minutes. This reduction by laccase/peroxide of p-cresol corresponds to 
31.3 percent; nearly half of the reduction seen for phenol, and significantly smaller 
than that of o-cresol. The first order rate constant for p-cresol reduced by laccase was 
found to be -0.0060 min-1 with a R2 value of 0.99 (Table 4.11). The rate constant of p-
cresol was significantly lower than the rate for phenol and approximately half the rate 
determined for o-cresol. 
120 
 
T im e (m inut es)
0 15 30 45 60 75
Concent
ratio
n 
(m
g
/
L)
0
10
20
30
40
50
60
70
 
Figure 4.69: Laccase Treatment of P-cresol in Sarnia Biox Effluent 
 
The concentrations for each individual phenolic compound were normalized 
to the initial concentration to show the fractions remaining over the 60 minute 
reaction; these are shown in Figure 4.70. Phenol had the smallest fraction remaining 
with 37.5 percent. This percent was about 25 percent lower than the fraction 
remaining for o-cresol and nearly half that of p-cresol, which were 49 and 68.7 
percent respectively. The decrease in phenol occurred in the first 15 minutes of the 
reaction; one explanation for this similarity to the clean system could be that the biox 
effluent has little to no other contaminants.   
121 
 
T im e ( m inut es)
0 15 30 45 60 75
C
oncent
r
at
ion 
ov
er
 
I
nit
ial 
C
oncent
r
at
ion 
(
C
/
C
o)
0. 0
0. 2
0. 4
0. 6
0. 8
1. 0
1. 2
P he no l
O- cr esol
P - cr esol
 
Figure 4.70: Fraction Remaining of Phenolic Compounds using Laccase Treatment 
on Sarnia Biox Influent 
 
The biox effluent from the Sarnia Refinery was also tested using the 
peroxidase enzyme. Once again 100ppm phenol, 100ppm o-cresol, and 100ppm p-
cresol were mixed into the sample since the original concentrations were non-
detectable. An initial dose of 2600 units of peroxidase and 300 mg/L hydrogen 
peroxide were added to the biox effluent sample.  There were three replicate test run 
using the peroxidase enzyme and the average initial concentrations contained in the 
biox effluent after the addition of phenols were: 47.8 mg/L phenol, 66.2 mg/L o-
cresol, 61.4 mg/L p-cresol and 175.4 mg/L total phenol. The first order rate constants 
for peroxidase/peroxide treatment were also examined (Table 4.11). 
122 
 
Figure 4.71 shows the decrease in phenol concentration over time in Sarnia 
biox effluent due to peroxidase/peroxide treatment. The phenol concentration in the 
biox effluent was reduced from 62.4 mg/L to 22.1 mg/L after 60 minutes, a 58.8 
percent reduction. The first order rate constant for phenol reduced by peroxidase was 
found to be -0.0152 min-1 with a R2 value of 0.98 (Table 4.11). The high R2 value 
indicates a very good fit with the first order model. 
T im e (m inut es)
0 15 30 45 60 75
Concent
ratio
n 
(m
g
/
L)
0
10
20
30
40
50
60
 
Figure 4.71: Peroxidase Treatment of Phenol in Sarnia Biox Effluent 
 
The o-cresol concentration in the biox effluent decreased from 66.2 mg/L 
initially to a concentration of 31.7 mg/L after 60 minutes time (Figure 4.72). This 
corresponds to a reduction in o-cresol concentration of 52.1 percent. The percent 
123 
 
reduction is slightly less than phenol. The majority of the degradation occurred during 
the first 45 minutes of the reaction. The first order rate constant for o-cresol was 
examined and was determined to be -0.0128 min-1 with a R2 value equal to 0.97 
(Table 4.11). The rate constant of o-cresol was approximately 20 percent lower than 
the rate constant for phenol. 
T im e (m inut es)
0 15 30 45 60 75
Concent
ratio
n 
(m
g
/
L)
0
10
20
30
40
50
60
70
 
Figure 4.72: Peroxidase Treatment of O-cresol in Sarnia Biox Effluent 
 
The change in p-cresol concentration over time caused by peroxidase/peroxide 
treatment is shown in Figure 4.73. Peroxidase reduced the P-cresol in the Sarnia biox 
effluent from a concentration of 61.4 mg/L to 30.8 mg/L over the 60 minutes, a 
reduction of 49.8 percent. This percent resembles that of o-cresol., but again is less 
124 
 
that the percent removal for phenol. The first order rate constant for peroxidase 
reducing p-cresol was found to be -0.0108 min-1 with a R2 value of 0.90 (Table 4.11). 
The rate constant of p-cresol was the lowest rate for peroxidase/peroxide, 40 percent 
lower than the rate for phenol.  
T im e (m inut es)
0 15 30 45 60 75
Concent
ratio
n 
(m
g
/
L)
0
10
20
30
40
50
60
70
 
Figure 4.73: Peroxidase Treatment of P-cresol in Sarnia Biox Effluent  
 
The concentrations of each compound added to the biox effluent were 
normalized to the initial concentration to show the fraction remaining after 60 
minutes using the peroxidase enzyme (Figure 4.74). The percent remaining for o-
cresol and p-cresol was 47.9 and 50.2 percent respectively. O-cresol and p-cresol 
percent remaining was a little higher that of phenol. Phenol had the lowest remaining 
125 
 
concentration with 41.2 percent. The decrease in the phenol due to 
peroxidase/peroxide followed a more linear pattern, whereas o-cresol reduction 
occurred during the first 45 minutes, and p-cresol degradation occurred after 30 
minutes of reaction.  
T im e ( m inut es)
0 15 30 45 60 75
C
oncent
r
at
ion 
ov
er
 
I
nit
ial 
C
oncent
r
at
ion 
(
C
/
C
o)
0. 0
0. 2
0. 4
0. 6
0. 8
1. 0
1. 2
P he no l
O- cr esol
P - cr esol
 
Figure 4.74: Fraction Remaining of Phenolic Compounds using Peroxidase Treatment 
on Sarnia Biox Influent 
 
Phase Three: Effects of Enzyme/Peroxide Treatment on Nitrification.  
The use of laccase and peroxidase was shown to reduce the concentration of 
phenols in Phase I and II. To confirm that the enzymatic treatment reduced the 
toxicity of various wastewaters to the nitrification process, Phase III testing was 
conducted using a commercially available culture of Nitrosomonas and Nitrobacter 
126 
 
known as Nitrotox. Wastewater samples were treated with 500 units of laccase or 
peroxidase enzyme, 100 mg/L peroxide, and allowed to react for 30 minutes. Samples 
included in this phase of testing were: CITGO stripped sour water, Frontier stripped 
sour water, Dartmouth biox influent, Nanticoke biox influent, and Sarnia biox 
influent. Dilutions of the 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. 
The rate of nitrification as a function of the percent of CITGO Sour Stripper 
wastewater added to the culture is presented in Figure 4.75.  The control was 
wastewater that was untreated by enzyme and peroxide. With no process wastewater 
added, the rate of nitrification was 29.4 mg NH4-N/L/hr, which is a relatively high 
rate compared to those observed in the field. The rate of nitrification in the control 
sample decreased as the percentage of wastewater increased, indicating that the 
CITGO Sour Stripper water was inhibitory to nitrification. A fifty percent reduction 
in nitrification occurred at a wastewater concentration of just slightly more than 5 
percent.  The nitrification rate continued to decrease with increasing Sour Stripper 
Water percentage. When the wastewater concentration approached 40 percent of the 
total reactor volume, nitrification was almost completely inhibited. This shows that 
the untreated wastewater was very inhibitory to nitrification, even when present as a 
relatively small percentage of the total influent volume. 
127 
 
W as t ew at er, perc ent
0 10 20 30 40 50 60
A
m
m
onia 
Ox
idat
ion 
(
m
g
/
L/
hr)
0
5
10
15
20
25
30
35
C on t r ol
P eroxi da se
La cc ase
  
Figure 4.75: Ammonia Oxidation Using CITGO Stripped Sour Water 
 
The wastewater treated with laccase was also inhibitory to nitrification as 
indicated by the decrease in ammonia oxidation when the percent of wastewater was 
increased. However, the degree of inhibition was significantly less than observed for 
the control. Fifty percent reduction in nitrification occurred when the percent 
wastewater was higher than 15 percent. This was three times higher than observed for 
the control with no enzyme/peroxide treatment.  Almost 20 percent of the non-
inhibited nitrification activity remained in the reactor with the highest (40 %) 
wastewater concentration.  Although significantly inhibited, the activity of 6.7 mg 
NH4-N/L/hr is sufficiently high enough to attain a high degree of ammonia removal 
128 
 
from a process with 12 to 24 hours of hydraulic residence time.   This demonstrates 
that the use of laccase enzyme can decrease the inhibition of nitrification in the 
CITGO sour stripper water.  
The CITGO sour stripper wastewater was also treated with peroxidase and 
peroxide. As shown in Figure 4.75, the peroxidase wastewater was still inhibitory to 
nitrification as indicated by the decrease in ammonia oxidation when the percent of 
wastewater was increased. The degree of inhibition was significantly less than 
observed for the control and lower than that for the laccase treated samples. Fifty 
percent reduction in nitrification occurred when the percent wastewater was higher 
than 25 percent. This concentration was five times higher than observed for the 
control with no enzyme/peroxide treatment and almost twice as high as the laccase 
treated wastewater.   Almost 30 percent of the non-inhibited nitrification activity 
remained in the reactor with the highest expected (40 %) wastewater concentration.  
Although significantly inhibited, the activity of 8.7 mg NH4-N/L/hr is sufficiently 
high enough to attain a high degree of ammonia removal from a process with 12 to 24 
hours of hydraulic residence time.   This demonstrates that the use of peroxidase 
enzyme can decrease the inhibition of nitrification in the CITGO sour stripper water.  
Furthermore, the peroxidase enzyme was more effective in reducing the inhibition of 
process toxicity than the laccase enzyme. This mirrors the findings of Phase II, where 
the peroxidase enzyme was slightly more effective in removing the targeted phenolic 
compounds. 
Figure 4.76 shows the rate of nitrification as a function of the percent of 
Frontier sour stripper wastewater added to the culture.  The control wastewater was 
129 
 
untreated by enzyme and peroxide. With the control, the rate of nitrification was 25.4 
mg NH4-N/L/hr, which is a relatively high rate compared to those observed in the 
field. The rate of nitrification in the control sample decreased as the percentage of 
wastewater increased, indicating that the Frontier stripped sour water was inhibitory 
to nitrification. A fifty percent reduction in nitrification occurred at a wastewater 
concentration of just slightly less than 10 percent.  The nitrification rate continued to 
decrease with more Frontier sour water percentage. When the wastewater 
concentration approached 50 percent of the total reactor volume, nitrification was 
almost completely inhibited (less than 2 mg NH4-N/L/hr). This shows that the 
untreated wastewater was very inhibitory to nitrification, even when present as a 
relatively small percentage of the total influent volume.  
 
130 
 
W as t ew at er, perc ent
0 10 20 30 40 50
A
m
m
onia 
Ox
idat
ion 
(
m
g
/
L/
hr)
0
5
10
15
20
25
30
C on t r ol
P eroxi da se
La cc ase
 
Figure 4.76: Ammonia Oxidation Using Frontier Stripped Sour Water 
 
The wastewater treated with laccase was also inhibitory to nitrification as 
indicated by the decrease in ammonia oxidation when the percent of wastewater was 
increased. However, the degree of inhibition was significantly less than observed for 
the control. Fifty percent reduction in nitrification occurred when the percent 
wastewater was higher than 20 percent. This was about two times higher than that 
observed for the control with no enzyme/peroxide treatment.  Close to 20 percent of 
the non-inhibited nitrification activity remained in the reactor with the 40 percent 
wastewater concentration.  Although significantly inhibited, the activity of 5.9 mg 
NH4-N/L/hr is sufficiently high enough to attain a high degree of ammonia removal 
from a process with 12 to 24 hours of hydraulic residence time.   This demonstrates 
131 
 
that the use of laccase enzyme can lower the amount of inhibition to nitrification in 
the Frontier sour water.  
The Frontier stripped sour wastewater was also treated with peroxidase and 
peroxide. This enzymatic treatment using peroxidase and peroxide was still inhibitory 
to nitrification as indicated in Figure 4.76 by the decrease in ammonia oxidation when 
the percent of wastewater was increased. Nonetheless, the degree of inhibition was 
still significantly less than observed for the control and slightly lower than that for the 
laccase treated samples. Fifty percent reduction in nitrification occurred when the 
percent wastewater was higher close to 30 percent. This concentration was three 
times higher than that observed for the control and almost one and half times higher 
than the laccase treated wastewater.   Almost 30 percent of the non-inhibited 
nitrification activity remained in the reactor with the highest expected wastewater 
concentration of 40 percent.  Although significantly inhibited, the activity of 8.7 mg 
NH4-N/L/hr is sufficiently high enough to attain a high degree of ammonia removal 
from a process with a hydraulic residence time of 12 to 24 hours. This demonstrates 
that the use of peroxidase enzyme can decrease the inhibition of nitrification in the 
Frontier sour water.  As before, the peroxidase enzyme was slightly more effective in 
reducing the inhibition of process toxicity than the laccase enzyme; but this was 
expected as the peroxidase enzyme was slightly more effective in removing the 
targeted phenolic compounds in Phase II. 
In Figure 4.77 the rate of nitrification as a function of the percent of 
Dartmouth biox influent wastewater added to the culture is shown. With the control, 
wastewater untreated by enzyme and peroxide, the rate of nitrification was 23.5 mg 
132 
 
NH4-N/L/hr. The rate of nitrification in the control sample decreased as the 
percentage of wastewater increased, indicating that the Dartmouth biox influent water 
was inhibitory to nitrification.   A wastewater concentration of around 15 percent 
caused a decrease in nitrification of fifty percent. The higher the Dartmouth biox 
influent percentage the more the nitrification rate continued to decrease. When the 
wastewater concentration approached 40 percent of the total reactor volume, 
nitrification was completely inhibited (non-detectable amount). This shows that the 
untreated biox influent was very inhibitory to nitrification, even when present as a 
relatively small percentage of the total influent volume.  
W as t ew at er, perc ent
0 10 20 30 40 50
A
m
m
onia 
Ox
idat
ion 
(
m
g
/
L/
hr)
0
5
10
15
20
25
30
C on t r ol
P eroxi da se
La cc ase
 
Figure 4.77: Ammonia Oxidation using Dartmouth Biox Influent 
 
133 
 
The biox influent was treated using the enzymatic approach above, when 
using the laccase/peroxide treatment the wastewater was also inhibitory to 
nitrification as indicated by the decrease in ammonia oxidation when the percent of 
wastewater was increased (Figure 4.77). Yet, the degree of inhibition was 
significantly less than observed for the control. Fifty percent reduction in nitrification 
occurred when the percent wastewater was almost 30 percent. This was about two 
times higher than that observed for the control.  Also, when the reactor contained a 
wastewater concentration of 40 percent of the total reactor volume (complete 
inhibition in the control) the non-inhibited nitrification activity remained over 35 
percent.  Although significantly inhibited, the activity of 9.6 mg NH4-N/L/hr is 
sufficiently high enough to attain a high degree of ammonia removal from a process 
with 12 to 24 hours of hydraulic residence time.   This demonstrates that the use of 
laccase enzymatic treatment can lower the amount of inhibition to nitrification in the 
Dartmouth biox influent.  
The Dartmouth biox influent was also treated using the peroxidase/peroxide 
treatment method. This enzymatic treatment using peroxidase and peroxide was still 
inhibitory to nitrification, as indicated in Figure 4.77 by the decrease in ammonia 
oxidation when the percent of wastewater was increased. Nevertheless, the degree of 
inhibition was still significantly lower than that observed for the control and that for 
the laccase/peroxide treated samples. Fifty percent reduction in nitrification occurred 
when the percent wastewater was higher close to 30 percent. This concentration was 
over two times that observed for the control and about the same as the laccase treated 
wastewater.   Almost 43 percent of the non-inhibited nitrification activity remained 
134 
 
when the wastewater concentration of 35 percent of the total reactor volume was 
present.  Even though the activity is significantly inhibited at 8.4 mg NH4-N/L/hr, this 
is sufficiently high enough to attain a high degree of ammonia removal from a 
process with a 12 to 24 hour hydraulic residence time. This demonstrates that the use 
of peroxidase/peroxide enzymatic treatment can decrease the inhibition of 
nitrification in the Dartmouth biox influent.  Again, as expected from the results in 
Phase II, the peroxidase enzyme was slightly more effective in reducing the inhibition 
of process toxicity than the laccase enzyme. 
The rate of nitrification as a function of the percent of Nanticoke desalter 
wastewater added to the culture is shown in Figure 4.78.  The control wastewater was 
wastewater untreated by enzyme and peroxide. With no process wastewater added, 
the rate of nitrification was 27.8 mg NH4-N/L/hr. The rate of nitrification in the 
control sample decreased as the percentage of wastewater increased, indicating that 
the Nanticoke desalter water did inhibit nitrification. A wastewater concentration of 
around 5 percent caused a decrease in nitrification of fifty percent. The higher the 
Nanticoke desalter percentage the more the nitrification rate continued to decrease, by 
25 percent the rate of nitrification had decreased to a non-detectable amount. This 
shows that the untreated wastewater was very inhibitory to nitrification, even when 
present as a small percentage of the total influent volume.  
 
135 
 
W as t ew at er, perc ent
0 10 20 30 40 50
A
m
m
onia 
Ox
idat
ion 
(
m
g
/
L/
hr)
0
5
10
15
20
25
30
C on t r ol
P eroxi da se
La cc ase
 
Figure 4.78: Ammonia Oxidation using Nanticoke Desalter 
 
The wastewater was treated using the laccase/peroxide treatment, this was 
also inhibitory to nitrification as indicated by the decrease in ammonia oxidation 
when the percent of wastewater was increased (Figure 4.78). However, the inhibition 
was to a significantly lesser degree than that observed for the control. Fifty percent 
reduction in nitrification occurred when the percent wastewater was almost 10 
percent. This was about two times higher than that observed for the control.  Also, 
when the reactor contained a wastewater concentration of 25 percent of the total 
reactor volume (complete inhibition in the control) the non-inhibited nitrification 
activity remained close to 15 percent.  Although significantly inhibited, the activity of 
136 
 
4.2 mg NH4-N/L/hr is enough to achieve a sufficient degree of ammonia removal 
from a process with 12 to 24 hours of hydraulic residence time.   Thus, by using 
laccase enzymatic treatment, the amount of inhibition to nitrification in the Nanticoke 
desalter can be lowered.  
The Nanticoke desalter was also treated using the peroxidase/peroxide 
treatment method. This enzymatic treatment using peroxidase and peroxide was still 
inhibitory to nitrification, as indicated by the decrease in ammonia oxidation when 
the percent of wastewater was increased as shown in Figure 4.78. The degree of 
inhibition, however, was still significantly lower than that observed for the control 
and that for the laccase/peroxide treated samples. Fifty percent reduction in 
nitrification occurred when the percent wastewater was higher close to 15 percent. 
This concentration was over three times that observed for the control. Almost 20 
percent of the non-inhibited nitrification activity remained when the wastewater 
concentration of 25 percent of the total reactor volume.  Even though the activity is 
significantly inhibited at 5.1 mg NH4-N/L/hr, this is sufficiently high enough to attain 
a high degree of ammonia removal from a process with a 12 to 24 hour hydraulic 
residence time. This demonstrates that the use of peroxidase/peroxide enzymatic 
treatment can decrease the inhibition of nitrification in the Nanticoke desalter.  
Although this particular wastewater was not tested in Phase II, the results continue to 
follow the trend shown in that phase of the study.  
The rate of nitrification as a function of the percent of Sarnia biox effluent 
wastewater added to the culture is shown in Figure 4.79.  The rate of nitrification in 
the control, wastewater untreated by enzyme and peroxide, was 26.8 mg NH4-N/L/hr. 
137 
 
This decreased as the percentage of wastewater increased, indicating that the Sarnia 
biox effluent water did inhibit nitrification. A fifty percent reduction in nitrification 
was seen at a wastewater concentration around 5 percent. The higher the Sarnia biox 
effluent percentage the more the nitrification rate continued to decrease. When the 
wastewater concentration approached 40 percent of the total reactor volume, 
nitrification had decreased to a non-detectable amount. This shows that the untreated 
wastewater was very inhibitory to nitrification, even when present as a relatively 
small percentage of the total influent volume.  
W as t ew at er, perc ent
0 10 20 30 40 50
A
m
m
onia 
Ox
idat
ion 
(
m
g
/
L/
hr)
0
5
10
15
20
25
30
C on t r ol
P eroxi da se
La cc ase
 
Figure 4.79: Ammonia Oxidation using Sarnia Biox Effluent  
 
138 
 
The wastewater treated with the laccase/peroxide treatment was also 
inhibitory to nitrification as indicated in Figure 4.79 by the decrease in ammonia 
oxidation when the percent of wastewater was increased. However, the degree of 
inhibition was significantly less than that observed for the control. When the percent 
wastewater was almost 20 percent, a fifty percent reduction in nitrification occurred. 
This was about four times higher than that observed for the control.  Also, when the 
reactor contained a wastewater concentration of 40 percent of the total reactor volume 
the non-inhibited nitrification activity remained close to 20 percent.  Although 
significantly inhibited, the activity of 5.8 mg NH4-N/L/hr is high enough to achieve a 
sufficient degree of ammonia removal from a process with an hydraulic residence 
time of 12 plus hours.   As a result, the amount of inhibition to nitrification in the 
Sarnia biox effluent can be lowered by using laccase enzymatic treatment.  
The Sarnia biox effluent was also treated using the peroxidase/peroxide 
treatment method. This enzymatic treatment was still inhibitory to nitrification, as 
indicated by the decrease in ammonia oxidation when the percent of wastewater was 
increased (Figure 4.79). The degree of inhibition, however, was still significantly 
lower than that observed for the control and that for the laccase/peroxide treated 
samples. Fifty percent reduction in nitrification occurred when the percent wastewater 
was higher close to 25 percent. This concentration was over five times that observed 
for the control. When the wastewater concentration reached 25 percent of the total 
reactor volume, almost 35 percent of the non-inhibited nitrification activity remained.  
Even though the activity is inhibited, 9.6 mg NH4-N/L/hr is sufficiently high enough 
to attain a high degree of ammonia removal from a process with a 12 to 24 hour 
139 
 
hydraulic residence time. This demonstrates that the use of peroxidase/peroxide 
enzymatic treatment can also decrease the inhibition of nitrification in the Sarnia biox 
effluent.  Furthermore, the peroxidase enzyme was more effective in reducing the 
inhibition of process toxicity than the laccase enzyme. This mirrors the results of 
Phase II, where the peroxidase/peroxide treatment was slightly more effective in 
removing the target phenolic compounds from the wastewater.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
140 
 
 
 
Chapter 5 
Summary and Recommendations  
  
The research presented in this thesis supports the hypothesis in that laccase 
and peroxidase, coupled with hydrogen peroxide, do in fact reduce the concentrations 
of the chosen phenolic compounds (phenol, o-cresol, p-cresol, and 2,4-dimethyl 
phenol) in refinery wastewater. Although this enzymatic approach to phenol 
remediation may appear to be a common subject for research, the matrix in which 
previous research has been done was buffer solutions or de-ionized water. Whereas 
the matrix researched in this thesis was not only DI water, but actual refinery 
wastewater from five different refineries across the US and Canada, each with 
different wastewater matrices. Therefore the results of this thesis will contribute to 
the understanding of how this enzymatic approach can be applied to refinery 
wastewaters. In this chapter, a summary of the findings along with recommendations 
for further work are presented.  
Summary 
 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 
141 
 
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 
142 
 
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.  
Table 4.12: Average First Order Rate Constants for Phase Two 
Compound Laccase  k (min-1) Peroxidase  k (min-1) 
Phenol -0.0125 (R2 = 0.91) -0.0141 (R2 = 0.95) 
O-cresol -0.0098 (R2 = 0.89)  -0.0110 (R2 = 0.97) 
P-cresol -0.0083 (R2 = 0.94) -0.0096 (R2 = 0.89) 
2,4-Dimethyl Phenol -0.0114 (R2 = 0.93) -0.0130 (R2 = 0.93) 
 
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.  
Recommendations 
 In order to reduce the toxicity of phenolic compounds to the nitrification 
process, the amount of phenols in the wastewater being treated needs to be reduced. 
Enzymatic treatment using peroxidase/peroxide and laccase/peroxide is a viable 
option for the degradation of phenols. Throughout the course of this thesis, better 
optimization of parameters was recognized as a potential area for improvement and 
therefore, should be obtained in further studies.  
143 
 
 For instance, it should be determined how pH affects the enzymatic treatment 
process. Moreover, further studies on pH could determine if a constant pH would 
yield a better reduction rate.  
 More studies using other enzymes should be included in this research topic in 
order to determine the most effective enzyme. Additionally, relationships involving 
other contaminants should also be explored. Furthermore, a cost analysis of enzyme 
production for all viable treatment options could provide more insight into real-world 
practical application.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
144 
 
 
 
 
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