Bioremediation of Hydrocarbon Contaminants in Subsurface Groundwater

By
Klaudine Monika Oronan Estepa

A Thesis
Presented to
The University of Guelph

In partial fulfillment of requirements

for the degree of
Master of Applied Science
in
Engineering

Guelph, Ontario, Canada

© Klaudine Monika Oronan Estepa, May, 2020

 ABSTRACT

BIOREMEDIATION OF HYDROCARBON CONTAMINANTS IN SUBSURFACE

GROUNDWATER

Klaudine Monika Oronan Estepa Advisor:

University of Guelph, 2020 Dr. Erica Pensini

Hydrocarbons are common contaminants in the subsurface as a result of spills caused by the

petroleum and chemical industry. Current remediation techniques require exhaustive

construction to either excavate contaminated soil to be treated ex-situ or to install impermeable

barriers to contain contaminants preventing plume migration or permeable barriers to treat in-

situ. This thesis will explore two novel approaches that simultaneously contain and bioremediate

naphthalene, a model hydrocarbon, without the requirement of traditional barrier installation.

The first part of this study utilizes a pumpable double emulsion system that envelops

Pseudomonas sp. cells that gels in the subsurface creating an injectable reactive barrier in the

subsurface. The remainder of this thesis looks at the sorption properties of naphthalene on zein

substrates as a method of sequestering PAHs.

 iii

ACKNOWLEDGMENTS

I would like to thank Dr. Erica Pensini for the opportunity to join and work in her group and for
her constant support and advice throughout my research. In addition, I would like to thank the
members of the Pensini group especially Kristine Lamont for being part of my graduate
experience. As a result, have had an unforgettable experience that has fostered both personal and
academic growth I am grateful for.

Further thanks to Dr. Michael K. Denk and Dr. Maria Corradini for reviewing my thesis as
members of my committee and their continued support. Dr. Abdallah Elsayed for fulfilling the
role of committee chariot for my defence. As well as Dr. Kate Stuttaford for allowing me to keep
my chemistry roots by giving me the opportunity to TA in the chemistry department.

Pseudomonas sp. were cultured, isolated, purified, and identified by Dr. Athanasios.
Fluorescence spectroscopy data were obtained with the support of Dr. Maria Corradini and Louis
Colaruotolo with equipment in Dr. Maria Corradini’s laboratory. Confocal microscopy images
were obtained using the Molecular and Cellular Imaging Facility at the Advanced Analysis
Centre of the University of Guelph, with the help of Michaela Strüder-Kypke. Rheology was
completed utilizing equipment in Dr. Alejandro Marangoni’s laboratory.

Thank you to the Estepa family and Srdjan Malicevic for supporting me each step in my post
graduate journey.
 iv

TABLE OF CONTENTS

ABSTRACT.................................................................................................................................... II

ACKNOWLEDGMENTS ............................................................................................................. iii

TABLE OF CONTENTS ............................................................................................................... iv

LIST OF TABLES ........................................................................................................................ vii

LISTS OF FIGURES ................................................................................................................... viii

LISTS OF EQUATIONS ............................................................................................................... ix

LIST OF APPENDICES ................................................................................................................. x

LIST OF SYMBOLS, ABBREVIATIONS, OR NOMENCLATURE ......................................... xi

INTRODUCTION ............................................................................................................... 1

Thesis Structure ................................................................................................................ 1

Context ............................................................................................................................. 1

1.2.1 History of Naphthalene ............................................................................................. 1

1.2.2 Structure and Physicochemical Properties of Naphthalene ...................................... 2

1.2.3 Production and Uses of Naphthalene ........................................................................ 3

1.2.4 Toxicity and Regulation of Naphthalene .................................................................. 5

LITERATURE REVIEW IN CURRENT REMEDIATION TECHNIQUES..................... 7

Current Remediation Processes for PAHs ....................................................................... 7

2.1.1 Bioremediation for PAHs ......................................................................................... 7

2.1.2 Chemical Remediation of PAHs ............................................................................. 11

2.1.3 Surfactant enhanced remediation of PAHs ............................................................. 11

2.1.4 Barriers Utilized in the Remediation of PAHs ....................................................... 13

Problem Statement/Objective ......................................................................................... 14

 v

CHITOSAN-BASED BIOGELS: A POTENTIAL APPROACH TO TRAP AND

BIOREMEDIATE NAPHTHALENE .......................................................................................... 16

Abstract .......................................................................................................................... 16

Introduction .................................................................................................................... 17

Method and Materials..................................................................................................... 18

3.3.1 Materials ................................................................................................................. 18

3.3.2 Emulsion Preparation .............................................................................................. 18

3.3.3 Bacteria Isolation and Characterization .................................................................. 19

3.3.4 Bacterial Growth and Survival................................................................................ 20

3.3.5 Optical and Confocal Microscopy .......................................................................... 20

3.3.6 Shear Rheology Experiments (Strain Sweeps) ....................................................... 21

3.3.7 Steady State Fluorescence Spectroscopy Measurements ........................................ 21

Results and Discussion ................................................................................................... 22

Conclusions .................................................................................................................... 26

ZEIN SORPTION OF NAPHTHALENE ......................................................................... 27

Abstract .......................................................................................................................... 27

Introduction .................................................................................................................... 27

Methods and Materials ................................................................................................... 28

4.3.1 Materials ................................................................................................................. 28

4.3.2 Zein and Naphthalene Preparation and Imaging ..................................................... 29

4.3.3 Kinetic Reaction Experiments ................................................................................ 29

4.3.4 GCMS Analysis ...................................................................................................... 29

4.3.5 Spectroscopic Analysis ........................................................................................... 29

4.3.6 Kinetic Reaction Models......................................................................................... 30

Results and Discussions ................................................................................................. 30

 vi

Conclusions .................................................................................................................... 34

CONCLUSIONS AND FUTURE WORK ........................................................................ 35

Conclusions .................................................................................................................... 35

Future Work ................................................................................................................... 36

REFERENCES ............................................................................................................................. 37

APPENDIX A ............................................................................................................................... 50
APPENDIX B ............................................................................................................................... 54
 vii

LIST OF TABLES

Table 1. Physicochemcial Properties of Naphthalene..................................................................... 2

Table 2. Bioremediation techniques for PAHs ............................................................................... 7
 viii

LISTS OF FIGURES

Figure 1. Chemical structure of naphthalene; C10H8. ..................................................................... 2

Figure 2. Products synthesized utilizing naphthalene as a chemical building block [8]. ............... 4
Figure 3. First step of P450 degradation of naphthalene [7, 59, 60]. ............................................. 9
Figure 4. Metabolic pathways for naphthalene biological degradation via bacteria and algae/fungi
derived enzymes [25, 60]. ............................................................................................................. 10
Figure 5. Graphic illustration of PAH solubility after the CMC [75]. .......................................... 12
Figure 6. Diagram of pumpable surfactant triggered barriers installed to mitigate contamination
migration once the solubility of the pollutant is enhanced with surfactants [72]. ........................ 13
Figure 7. Naphthalene contamination migration via groundwater flow [29]. .............................. 15
Figure 8. Naphthalene degradation over 48 hrs, as probed with fluorescence spectroscopy. ...... 23
Figure 9. Confocal microscopy images of double W/O/W emulsions (top) and CL-W/O
emulsion. The images on the left hand-side (fluorescence images) show the difference between
the oil phase (deep red, dyed with Nile Red) and the water phase (black, without dye).
Brightfield images are shown on the right hand side. The scalebar in the images is 100 m. ..... 24
Figure 10. Schematics of W/O/W double emulsion preparation and gelation. ............................ 25
Figure 11. Optical microscopy images of zein during the coagulation process. .......................... 31
Figure 12. Zein substrate 10 minutes after coagulation with CaCl2. ............................................ 31
Figure 13. Pseudo First Order and Pseudo Second Order Comparison of Naphthalene Zein
Adsorption Kinetic Study. ............................................................................................................ 33
Figure 14. FTIR comparison of zein substrate with and without naphthalene sorption. .............. 34
Figure 15. Naphthalene fluorescence intensity as a function of naphthalene concentration. ....... 51
Figure 16. Pseudomonas sp. isolated from garden soil (ON, Canada). ........................................ 52
Figure 17. W/O/W double emulsion system after 24 h before mixing with CAPB. .................... 53
Figure 18. Naphthalene response ratio (RF) as a function of naphthalene concentration (ppm);
R2 = 0.993; RSS = 4.06x1010 RF; RSME = 82231.2 RF. ............................................................. 54
 ix

LISTS OF EQUATIONS

Equation 1. Non-linear Pseudo First Order Rate Model. .............................................................. 30

Equation 2. Non-linear Pseudo Second Order Rate Model. ......................................................... 30
 x

LIST OF APPENDICES

APPENDIX A ............................................................................................................................... 50
APPENDIX B ............................................................................................................................... 54
 xi

LIST OF SYMBOLS, ABBREVIATIONS, OR NOMENCLATURE

BAC-W/O Pseudomonas sp. integrated water in oil primary emulsion

CAPB Cocamidopropyl betaine
Ca-Stearate Calcium Stearate
CL-W/O Sodium citrate integrated water in oil primary emulsion
DI Deionized water
EC10 Ethyl cellulose
EPA Environmental Protection Agency
ERL Sediment Quality Criteria
EtOH Ethanol, anhydrous
G6PD Glucose-6-Phosphate Dehydrogenase
GAA Glacial Acetic Acid
HLB Hydrophilic-Lipophilic Balance
IDLH Immediate Danger to Life and Health
KCl Potassium chloride
LD50 Lethal Dose 50%
NADPH Nicotinamide Adenine Dinucleotide Phosphate
NaOH Sodium hydroxide
NIOSH National Institute for Occupational Safety and Health
OSHA Occupational Safety and Health Administration
PAH Polycyclic Aromatic Hydrocarbons
PFO Pseudo First Order Reaction Rate Models
PSO Pseudo Second Order Reaction Rate Models
REL Occupational Exposure Level
RfC Reference Concentration
TVL-TWA Threshold Limited Value – Time-Weighted Average
W/O Water in oil emulsion
W/O/W Water in oil in water, multiple emulsion
WHO World Health Organization
 1

INTRODUCTION

Thesis Structure

This thesis is organised in five chapters. The first chapter introduces the topic of polycyclic
aromatic hydrocarbons, specifically naphthalene, and related issues. Chapter two is a literature
review of current methods of managing polycyclic aromatic hydrocarbon contamination in the
groundwater in the subsurface and where further research into novel technologies may make
current industry remediation less exhaustive and more efficient. Chapter three is a presently
submitted manuscript to the journal of Colloids & Surfaces A of a novel approach to contain and
bioremediate naphthalene in situ. Chapter four is unpublished work in publication format on a
zein substrate engineered to sorb and remediate naphthalene. The final chapter of this thesis
summarizes the findings and possible future work on this research.

Context

1.2.1 History of Naphthalene

The origin and unintentional initial production of naphthalene originated from the processing of
coal tar at a time when the by products of coal were being explored as chemical building blocks
of synthetic purple dyes to replace the traditional purple dyes that have been in demand and used
as early as 1400 BC [1, 2]. Purple dyes used in this time period were only produced through a
labor-intensive harvesting and processing of Murex genus of mollusc glands; making purple dyes
exclusive to royalty and the like [1]. At a time in Britain where the demand for coal to supply the
steel and electricity industry was at a high, coal tar presented as a new chemical building block,
and naphthalene was being industrially produced as a by-product [2]. W.H. Perkin started an
entire industry of commercially available synthetic dyes in 1856 when, in the process of
synthesizing quinine from coal tar, he synthesized mauvine derived from aniline [1]. Following
W.H. Perkin’s first discovery of synthesized dyes from coal tar by-products his company then
went on to produce alizarin from anthracene in 1869 and a variation of indigo based from toluene
[1]. K. Heumann was able to synthesize indigo from derivatives of benzene and naphthalene [1].

In 1821 the isolation and physical properties of naphthalene were first described in a publication
of the Philosophical Transactions of the Royal Society of London [3]. Michael Faraday then
 2

determined the chemical formula [4], and Emil Erlenmeyer the bifused benzene ring structure in
1866 [5].

In the present-day, commercial naphthalene is still produced from coal tar, however as the
quality and purity of naphthalene is higher when derived from petroleum, commercial
naphthalene is likely sourced from petroleum [6].

1.2.2 Structure and Physicochemical Properties of Naphthalene

Naphthalene (CAS: 91-20-3) is a polycyclic aromatic hydrocarbon (PAH), consisting of two
fused benzene rings, as seen in Figure 1. Physicochemical properties of naphthalene are
summarized in Table 1 [7].

Figure 1. Chemical structure of naphthalene; C10H8.

Table 1. Physicochemcial Properties of Naphthalene

Physicochemical Properties Value Unit

Molecular Weight 128.17 g/mol
Melting Point 80.2 °C
Boiling Point 218 °C
Vapour Density 45.432 (at 293.15 °K/1 atm) g/cm3
Vapour Pressure 10 (at 298.15 °K) Pa
Atmosphere Life Cycle 3-8 Hours
Diffusion Coefficient 7.2x10-2 (at 298 °K) cm2/s
Ethanol, acetate, benzene,
toluene, ether, and
Solubility chloroform. N/A
Note: Very limited
solubility in water
 3

1.2.3 Production and Uses of Naphthalene

Naphthalene is still a large by-product of the crude and oil industry as coal tar and crude oil
contains 10% and 1% naphthalene respectively [8-10]. According to the UN data just under 2950
metric tons of coal tar were produced in 2017 by Australia, Austria, Belgium, Chile, Czechia,
Finland, France, Hungary, Italy, Japan, Korea, Mexico, Netherlands, Poland, Russia, Slovakia,
Spain, Turkey, and the United Kingdoms [11]. It is important to note this data excludes large
contributors such as India, China, United States, and Canada. China produces over 20 million
tons of coal tar every year [12]. In 2017, 1124 tons of naphthalene was released in the United
States and 76 tons in the province of Ontario, Canada [13, 14].

True to naphthalene’s original uses in the 1800s, the compound is still used as a chemical
building block for phthalic anhydride [15]. Other chemicals that utilize naphthalene as a
chemical building block include, but are not limited to, azo dyes, surfactants, pesticides,
fumigants, and plasticizers as seen in Figure 2 (an adapted chemical flow chart from Collin et
al.) [8, 16]. However, naphthalene is easily recognizable in common households through its use
in mothballs due to its insecticide properties [17]. Though it has been largely replaced by
paradichlorobenzene due to the hazards associated with naphthalene [18]. Other indoor sources
that may also expose common populations to naphthalene include building materials (such as
paint, vinyl flooring, and carpeting), vinyl furniture, and foam materials [17]. Outdoor sources
consist of, but are not limited to, cigarettes, gasoline/diesel engines, vehicle emissions, coke
ovens, and wood combustion [19]. Naphthalene is the most abundant PAH in both indoor and
outdoor sources [19, 20]. The association between PAH composition (naphthalene specifically
making a large contribution to this category) and pollution produced from outdoor
(anthropological) human activity is strong enough to create a PAH fingerprint in sources of
pollution [19]. This has been used to create a chemical mass balance model of pollution
migration in the atmosphere and estimate population exposure to pollutants produced as a result
of combustibles and gas emissions [20].
 4

Figure 2. Products synthesized utilizing naphthalene as a chemical building block [8].

 5

1.2.4 Toxicity and Regulation of Naphthalene

Previous studies have noted evidence that naphthalene can be absorbed through oral, dermal, and
inhalation exposure [7]. The LD50 of naphthalene in adult mice is reported to be 380 mg/kg [7].
Naphthalene has been identified to affect the liver, kidneys, lungs, gastrointestinal system, and
neurological system causing symptoms including, (but not limited to vomiting) abdominal pain,
hematuria, renal tubular acidosis, convulsions, hepatomegaly, and ocular nerve atrophy [21].
Acute effects of naphthalene include respiratory distress starting at levels of 2 ppm and
haemolytic anaemic [21]. People with genetic deficiencies of glucose-6-phosphate
dehydrogenase (G6PD) have been observed to have an increased risk of haemolysis [22].
Naphthalene poisoning has commonly resulted in methaemoglobinaemia, where haemoglobin
becomes oxidized converting the Fe+2 into Fe+3 affecting the protein’s ability to bind with
oxygen [21].

Aside from ensuring proper ventilation to eliminate volatilized naphthalene, treatment can
include the use of methylene blue, in the presence of Nicotinamide Adenine Dinucleotide
Phosphate (NADPH), to revert the change of methaemoglobin to haemoglobin [23]. NADPH
acts an electron acceptor forming leucomethylene blue which reduces the methaemoglobin [23].
In patients with a genetic deficiency in G6PD, N-acetylcysteine can be used in place of
methylene blue as the reducing agent to prevent possible haemolysis caused from the methylene
blue in these G6PD deficient patients [24].

Toxic metabolites of naphthalene, produced as a result of degradation from P450

monooxygenase from the CYP27A1 gene, can include naphthalene epoxides, naphthalene
diepoxides, 1,2-naphthaoquinione, and 1,4 – naphthoquinone [7]. The metabolite pathway for
these specific compounds are later discussed in 2.1.1.1. Previous studies have found evidence of
naphthalene oxide metabolites increasing the risk of tissue damage, in organs such as the lung
and liver, by exhausting thiols such as glutathione [25, 26]. These thiols are converted into
glutathione naphthalene conjugates such as 1-napthol glucuronide, as seen in Figure 4, and have
been used to assess population exposure to naphthalene [25, 26].

Health impacts of naphthalene from outdoor sources are difficult to identify as exposure to
population rarely occurs with pure naphthalene where naphthalene would be integrated in a
mixture of other PAHs [7].
 6

The EPA has set an inhalation Reference Concentration (RfC) of 3 μg/m3 [9]. The WHO has set
a guideline value of naphthalene concentrations indoors of 0.01 mg/m3 annually [7]. In the
absence of mothballs, indoor concentrations of naphthalene are about 0.001 mg/m3 [7]. Due to
the pungent aromatic smell of naphthalene the odour threshold has been published to be 0.0075
mg/m3 and has been categorized as class B of the odour safety class factor [27]. The odour safety
class factor of B describes a compound at which 50% to 90% of inattentive population would
sense a compound approaching Threshold Limited Value – Time-Weighted Average (TLV-
TWA) levels [27]. The NIOSH has set an Occupational Exposure Level (REL) of 50 mg/m3; and
has stated an Immediate Danger to Life and Health (IDLH) value of 250 ppm [28].

PAHs, including naphthalene, has been previously observed to alter the diversity of
microorganisms in contaminated soil and groundwater [29]. However, the top down food chain
effects have yet to be more thoroughly explored [30]. Naphthalene has been determined to affect
the sexual maturity and reproductive organs of certain species of fish (Micropogonias undulates)
and to be toxic to water fleas (Daphnia magna), grass shrimp (Pelaemonetes pugio), and
Chlorella vulgaris algae [31-33].

As a result, several governing bodies such as the United Kingdom has banned the use of
naphthalene as a pesticide and fumigant in the early 2000s [34].
 7

LITERATURE REVIEW IN CURRENT REMEDIATION

TECHNIQUES

Current Remediation Processes for PAHs

2.1.1 Bioremediation for PAHs

Significant research efforts have focused on developing effective bioremediation methods to
eliminate PAHs [35]. A well-known disadvantage of bio remedial techniques are slow
degradation rates. This is reflected in Table 2 as most examples of bioremediation techniques
have equilibrium times in the magnitude of tens to hundreds of hours. Table 2 is adapted from
Oluwadara et al. and summarizes bioremediation techniques [35].

Table 2. Bioremediation techniques for PAHs

Bioremediation Naphthalene
Dominant Mechanism(s) Naphthalene Removal
Technique Example
348 nanomoles of
Oxidation of benzene ring forms dihydrodiols that are
naphthalene per minute per
Aerobic then dehydrogenated and metabolized to CO2 and Pseudomonas [38]
mg of protein (identified
water [36, 37]
via Lowry Protein Assay)
100% reduction in
Use nitrate, sulphate, iron as electron acceptors to
Anaerobic Mixed bacteria [41] naphthalene concentration
break down PAH [39], as well as fermentation [40]
from 13 mg/L in 480 hours
100% reduction of residual
Using PAHs as a source of energy, degrade and Burkholderia
Bacterial* naphthalene from 215 mg/L
sequester [42, 43] cepacian [44]
naphthalene in 50 hours

Secrete extracellular enzymes that oxidise PAHs [45] Reduction to 200 mg/kg
P. chrysosporium
Fungal May adsorb PAHs onto a hydrophobic cell wall [46] naphthalene from 600
[47]
(biosorption) mg/kg

Agmenellum 1.4% of added naphthalene

Oxidize PAHs under photoautotrophic conditions to
Algal quadruplicatum was oxidized at 2 M initial
form safer metabolites [48]
[49] over 48 hours
Microbial rhizosphere flora degrade or sequester Poaceae (foxtail Significant removal after
Phytoremediation
PAHs [50] barley) [51] 336 hours
U. prolifera detritus 1.27mg/kg at 10 µg/L
Biosorption Partitioning into algae detritus (algal waste) [52]
[52] initial over ~36 hours
*List of bacteria that are able to degrade naphthalene:
Alcaligenes, Burkholderia, Mycobacterium, Polaromonas, Pseudomonas, Ralstonia,
Rhodococcus, Sphingomonas, Streptomyces [43] Bacillus firmus-APIS272, Pseudomonas
alcaligenes-DAFS311 and Bacillus subtilis-SBS526 [53].
 8

A bio remedial technique that can irreversibly sorb and trap naphthalene would allow bacteria
ample time to degrade it into less harmful forms. This consideration motivates our research,
which used natural materials to trap or sorb naphthalene, as further described in the following
chapters.

Laccase (E.C.1.10.3.2) is a multicopper protein enzyme, naturally produced from fungi like
Trametes versicolor, heavily studied for its ability to oxidize hydrogen donating compounds via
the reduction of oxygen to water [54, 55]. The immobilization of enzymes, like laccase, on
surfaces is highly desired in industry as it increases reusability and can reduce instability in
enzymes compared to freely suspended enzymes [56, 57]. Entrapment of enzymes can also
physical protect them from their surroundings that would otherwise render freely suspended
enzymes inactive [57, 58]. It is important to note that immobilization of enzymes is only useful if
it the scaffold is biocompatible and does not impede the enzymes bioactivity: allowing substrates
to access the active site of the enzyme freely [57].

2.1.1.1 Metabolite Pathways for Naphthalene Degradation

Mammals metabolize naphthalene through an initial oxidation step via P450 monooxygenase,
produced from the CYP27A1 gene [7]. The first metabolic step catalysed by P450 creates an
unstable epoxide [7]. After naphthalene-1,2,-epoxide is formed it can undergo several possible
subsequent transformations facilitated through enzymes or through thermodynamic
rearrangements, as seen in Figure 3 [7, 59, 60]. P450 can further oxidise the epoxide to a
diepoxide, microsomal epoxide hydrolases can open the epoxide to trans-dihydrodiols, and
glutathione transferases can form glutathione conjugates which are then excreted mostly as
mercapturic acid [7]. Non enzyme facilitated transformation of naphthalene-1,2,-epoxide include
1-napthol and 2-napthol [7, 59, 60]. 1-napthol glucuronide is one of the main naphthalene
metabolites detectable in urine and has been used to gauge the population exposed to
naphthalene contamination [7].
 9

Figure 3. First step of P450 degradation of naphthalene [7, 59, 60].

Bacterial and enzymatic degradation of naphthalene has been previously reported to take three
possible metabolic pathways as seen in Figure 4 [25, 37, 43, 49, 60]. Fungi and algae that
produce P450, or other types of monooxygenase, have initial remedial products similar to those
of mammals as mentioned previously [25, 49]. Fungi that produce enzymes like laccase and
lignin oxidizers breakdown naphthalene by creating free radicals which result in a variety of
naphthoquinones which then undergo ring fission [25, 37]. Bacteria process naphthalene utilizing
 10

dioxygenase creating a cis-dihydrodiol, which is then be dehydrogenated to 1,2-

dihydroxynaphthalene [25, 60]. This dihydroxyl product can be nonenzymatically oxidized to
1,2-naphthoquinone, however, its major products ultimately is metabolized to salicylate which
can be converted to gentisate or be decarboxylated to catechol [25, 60]. Catechol can undergo
ring fission resulting in acetaldehyde, pyruvate, acetyl CoA, or succinate [25, 60].

Figure 4. Metabolic pathways for naphthalene biological degradation via bacteria and algae/fungi derived
enzymes [25, 60].
 11

2.1.2 Chemical Remediation of PAHs

About 20 years ago solvents such as propane, butane, acetone, methanol, and dimethyl ether
were used in-situ for the removal of PAHs [61, 62]. These treatment methods created a large
concern for the impact of these extraction agents on the environment in regards to toxicity and
effect on the surrounding ecosystem [61, 62].

With the influx of research into in-situ chemical oxidation of PAHs improvements such as
applicable pump-and-treat technology have been and are now extensively studied with many
efforts focused towards strong oxidizers such as permanganates, persulfates, ozone, and
catalyzed H2O2 propagations [62]. Naphthalene has been oxidized or neutralized by a variety of
methods including the Fenton’s degradation of a naphthalene derivative [63], degradation using
hydrogen peroxide catalytically enhanced by V-Zr [64], in-situ persulfate oxidation via iron-
chelate activated persulfate [65], permanganates [66, 67] and hydrogen peroxide [66]. Other
similar pump-and-treat technologies include the flushing of soil with cyclodextrin [68] or
biosurfactants [69], extraction via sunflower oils [70] or organic solvents/co-solvents such as
ethanol/propanol and water mixtures [71].

Despite the widespread popularity of chemical remediation methods for PAHs there is much
room for improvement in terms of its efficiency and application as solubility of PAHs and the
containment of these pollutant plumes often cannot be fully address with in-situ chemical
treatment alone [61, 62, 72].

2.1.3 Surfactant enhanced remediation of PAHs

Surfactant-enhanced remediation is the application of a surfactant to a surface that is primarily
used to increase the solubility of the PAH [39]. Surfactants increase the solubility of PAHs when
the surfactant concentration is above a certain critical micelle concentration (CMC) that causes
additional surfactant to form micelles that accumulate PAHs and increase solubility [73, 74].
Figure 5 is an modified table from Mulligan et al. that illustrates the effect of increasing
surfactant concentration on the solubility of PAHs [75].
 12

Figure 5. Graphic illustration of PAH solubility after the CMC [75].

Gemini surfactants are combinations of hydrophobic and hydrophilic groups connected by an

alkyl spacer. Gemini surfactants are of note as they have ten to one-hundred times lower CMC
values than monomeric surfactants [76].

Clearly, surfactants are a powerful tool to increase the solubility of PAHs and result in higher
rates of degradation or adsorption. Increasing the solubility of a PAH in a groundwater system,
however, may cause more PAHs to enter the groundwater system and should only be used once
PAH barriers that prevent the movement of the contaminant have been established as seen in
Figure 6 [72].
 13

Figure 6. Diagram of pumpable surfactant triggered barriers installed to mitigate contamination migration
once the solubility of the pollutant is enhanced with surfactants [72].

2.1.4 Barriers Utilized in the Remediation of PAHs

Impermeable barriers have been used in ground water as slurry walls, grout curtains, and steel
sheet piles [77]. These barriers require a well established connection to an impermeable bottom
to warrant total impermeability and ensure no contaminated groundwater leak [77]. It is
important to note that these barriers do not directly treat the contaminant and a secondary step of
remediation is usually employed [77]. A second class of barriers used in the remediation of
pollutants in ground water are permeable reactive barriers [62, 78, 79]. These barriers are often
used in saturated zones in ground water to prevent the migration of untreated contaminants in the
subsurface [62, 78, 79]. The reactive barrier are driven by the natural groundwater flow and are
placed either downgradient or adjacent to contaminated sites utilizing either adsorbents or
chemical treatments to allow the remediation of contaminants whilst the contaminates migrate in
the subsurface [62, 78, 79]. These barriers have been found to be most effective when installed
directly adjacent to the contamination site, opposed to down gradient [77]. Adsorbents used in
these barriers can consist of graphene, sand, fly ash, peat, and zeolite [80]. Chemicals integrated
into these permeable reactive barriers consists of iron or other oxidizers that would commonly be
 14

used to treat the contaminated soil in-situ or ex-situ as discussed in 2.1.2 [79]. There is the risk
that a permeable barrier may become unreactive after a period of time allowing untreated
pollutants to migrate to the surrounding area [78]. Both type of barriers require intensive
construction and installation and have a large initial cost that increases considerably the deeper
the barriers are implemented [78, 81]. Injectable reactive barriers can be implemented deeper and
with less construction and upfront costs [80, 81]. However, these barriers do not contain the
pollutant and thus risk the migration of untreated pollutants [80, 81]. This motivates this thesis to
address the lack of a groundwater remediation technique that can both contain and treat
pollutants in the subsurface without rigorous installation and construction.

Problem Statement/Objective

Several studies have found a relationship between the increase of PAH loading in the atmosphere
and anthropogenic activities [82]. Combustion particulates containing PAHs, sourced from coal,
biomass, and oil emissions from human activity, have been observed to travel via air currents
affecting air quality in other regions [83]. As human activity increases, so has the frequency that
naphthalene has been accidentally released to the environment [82].

Abundances of PAHs, naphthalene consisting a majority of the mixture, have been detected in
the environment such as the Shadegan wetland in the Persian Gulf surpassing the sediment
quality criteria (ERL), posing a risk to ecosystem and the surrounding population, as a result of
oil spills from the transportation of oil, fuel smuggling, leakage from petroleum
factories/pipelines [84]. Naphthalene contaminations will travel faster though soil without the
presence of other hydrocarbons, but soil and sediment sorb naphthalene when there is an
increased organic carbon contents present by other PAHs [25]. Studies have stated the
importance of increasing the solubility of PAHs in order to increase the rate of biodegradation
[25]. As studies have identified sites of PAH contamination, an influx of research has been
conducted to efficiently remediate and targeted specific PAH pollutants in groundwater [82, 84].
As a result, several chemical oxidants, surfactants, and biodegradation tools have been explored
[35, 39]. Figure 7 is an adapted illustration from Madsen et al. that describes pollutant migration
in groundwater. As seen in this figure PAH contamination in the subsurface does not only need
to be treated it also needs to be contained as groundwater flow may stimulate pollutant plumes to
further contaminate the surrounding area [29].
 15

Figure 7. Naphthalene contamination migration via groundwater flow [29].

Barriers are commonly used to mitigate pollutant migration in groundwater however these
barriers do not remediate PAHs and involve extensive construction for implementation [77].
Reactive barriers that remediate PAHs have been investigated, however they do not trap the
pollutant [78]. There exists a gap where an impermeable barrier that contains and simultaneously
remediates the contaminant whilst requiring limited installation and construction has yet to be
explored and inspires this thesis.
 16

CHITOSAN-BASED BIOGELS: A POTENTIAL APPROACH TO TRAP

AND BIOREMEDIATE NAPHTHALENE

This chapter has been submitted to Colloids and Surfaces A.

Abstract

Naphthalene-degrading bacteria (Pseudomonas sp.) were isolated from a naphthalene

contaminated site. Bacterial degradation of naphthalene in water was confirmed using steady
state fluorescence spectroscopy. Pseudomonas sp. bacteria were incorporated into a double
emulsion system (water in oil in water, W/O/W), which can be injected in the subsurface to trap
and biotreat naphthalene. The outermost phase of the W/O/W emulsion was a 1.65 wt.% aqueous
solution of chitosan, with Tween 20 (used as emulsifier) and KCl (used to balance the osmotic
pressure). Two separate primary water in oil (W/O) emulsions were emulsified in chitosan. W/O
emulsions were prepared with canola oil (containing ethyl cellulose and calcium stearate as
emulsifiers), and aqueous solutions of either sodium citrate (crosslinker) or Pseudomonas sp. in
minimal broth. The formation of W/O/W double emulsions was verified with optical and
confocal microscopy. W/O/W emulsions were stable and mainly viscous under shear. The shear
elastic modulus (G’ = 1.90 ± 0.39 Pa) and the shear viscous modulus (G” = 5.76 ± 0.06 Pa) were
low, to facilitate pumping in the subsurface. Naphthalene could be dispersed in the chitosan
phase of W/O/W emulsions. Emulsions were destabilized on demand using the surfactant
cocamidopropyl betaine, to release citrate (crosslinker) and Pseudomonas sp. in the chitosan
phase, where naphthalene was dispersed. Mixing chitosan with citrate led to its gelation, trapping
naphthalene. Mixing naphthalene with Pseudomonas sp. led to its bioremediation. Gelation was
demonstrated by the increase in the viscoelastic moduli, with G’ = 111.02 ± 1.67 Pa > G” = 8.66
± 0.10 Pa. With naphthalene, Pseudomonas sp. survival in gelled chitosan was confirmed by
plating bacteria sampled from the gel. Without naphthalene, Pseudomonas sp. did not survive in
chitosan.

Keywords: Chitosan, Naphthalene, Emulsion, Bioremediation, Biogel

 17

Introduction

Naphthalene is a double ringed aromatic compound produced as a major by-product in the

chemical industry and found in consumer products such as mothballs [85, 86]. Naphthalene is
also contained in crude oil [86]. Naphthalene is acutely toxic and carcinogenic to humans, and it
also is heavily toxic to aquatic environments [60].

In situ remediation of hydrocarbons has been successfully accomplished with different

approaches, including chemical and biological methods. Examples of hydrocarbon chemical
treatment methods include the use of Fenton’s reagents [87], ozone [88], slow release oxidizers
[89], oxidizers delivered by electrokinetics [90], oxidizing nanoparticles [91] and ferrate [92],
and surfactant enhanced oxidizers [93]. Examples of hydrocarbon biological treatment methods
include bioremediation using bacteria [94, 95] and phytoremediation [51]. Biological treatment
methods are attractive because they are safe and require low energy [96]. In the subsurface, the
use of bacteria is better suited than phytoremediation for contaminants that are too deep to be
reached by plant roots [97]. Bacterial degradation of naphthalene was previously reported [98]
and the injection of bacteria to enhance hydrocarbon remediation is a well-established strategy
[99, 100].

In the case of an oil spill, plume migration can result in spreading of groundwater contamination,
with risks to human and ecological downstream receptors [86]. Contaminant containment during
its remediation is essential to mitigate this risk [101, 102]. Cutoff walls [103], permeable reactive
barriers [104], sorbent barriers [80, 105, 106] and oxidizing barriers [107-111] can effectively
impede contaminant migration downstream. Permeable biobarriers developed using wheat straw
and coconut shell biochar could also degrade hydrocarbons [112]. However, soil excavation is
required for the installation of all these barriers, rendering their use challenging when
contaminants are deep [101, 102]. Pumpable barriers were proposed to trap and treat Cr(VI) by
reducing it to Cr(III) [113, 114] as well as to impede the migration of hydrocarbons, but not
degrade them [101, 102]. These barriers were polymer-based fluids or alginate-based double
emulsions that gelled only in contaminant proximity or upon mixing with selected surfactants
[101, 102, 113-115]. Gelation was obtained through the targeted release of crosslinking agents
contained in the double emulsions [101, 102]. While reversible double emulsions are known
tools for the targeted delivery of drugs [116], their use in the environmental sector has only been
 18

recently explored [101, 102]. Emulsified polycolloids injected in the soil were used to promote
both toluene sorption onto soil grains and its bacterial degradation, forming a bio-barrier [117].
A pumpable emulsified oil formulation was also used to obtain pumpable biobarriers that
promoted bacterial growth, to degrade nitramine explosives and perchlorate [118]. However,
pumpable bio-barriers were not previously developed to degrade naphthalene in contaminated
sites. Also, previous bio-barriers did not fully impede flow, with potential unresolved risks for
downstream receptors.

In response to these gaps, the current study uses stable, injectable chitosan-based W/O/W
emulsions with low viscoelasticity to trap and treat (biodegrade) subsurface naphthalene
contamination. The low viscoelasticity of these emulsions should favor their injection, avoiding
soil excavation. W/O/W emulsions were destabilized and gelled on demand only upon mixing
with a trigger (cocamidopropyl betaine), to trap naphthalene by impeding flow. These W/O/W
emulsions able to gel on demand also contained naphthalene-degrading Pseudomonas sp.
bacteria, to allow naphthalene bioremediation.

Method and Materials

3.3.1 Materials
Calcium stearate (Ca-stearate) (5.9 to 7.1% calcium, powder, ACROS Organics), Tween 20
(Ultrapure grade, Thermo Scientific), ethyl cellulose (EC10) (purity: 48% ethoxyl content, 10 cP,
ACROS Organics), potassium chloride (KCl) salt (99.5% pure), chitosan (75% deacetylated,
ACROS Organics), glacial acetic acid (GAA) (99.7% pure), sodium citrate dihydrate (99.0%
pure), and naphthalene (98.5%, ACROS Organics) were purchased from Fisher Scientific.
Cocamidopropyl betaine (CAPB) (Candora Soap brand) and canola oil (Selection brand) were
purchased from local markets. Fluorescent Nile Red (technical grade, 95% pure) and M9
minimal salts (x5) were purchased from Sigma Aldrich. Methylene blue dye (used to dye
Pseudomonas sp. bacteria) was purchased from Fisher Scientific. Deionised (DI) water was used
in all experiments.

3.3.2 Emulsion Preparation

A hotplate was used to heat and dissolve the emulsifiers EC10 (10 g/L) and Ca-stearate (2.5 g/L)
in canola oil (at 150 °C) for approximately 20 min. An aqueous solution was prepared at room
 19

temperature (23 °C) using sodium citrate dihydrate (0.204 M, used as crosslinker) and minimal
M9 salt (33.84 g/L, containing essential elements to support bacterial growth). The pH of the
sodium citrate/M9 solution was pH 7. The EC10/Ca-stearate solution in canola oil (15 mL, kept
at 150 °C) was emulsified with the citrate/M9 solution in DI water (10 mL, kept at 23 °C) in
glass vials having 1 cm diameter, using a homogenizer (Cole Parmer Hand-Held Homogenizer,
110 V) at high speed (speed setting 2 for 2 min). Emulsions of citrate/M9 solutions in canola oil
(CL-W/O) were then cooled to 23 °C. A second W/O emulsion containing Pseudomonas sp.
bacteria was also prepared (BAC-W/O). EC10 (6.25 g/L) was dissolved in canola oil at 150 °C,
cooled to 23 °C, and then emulsified with aqueous solutions of Pseudomonas sp. in M9 broth
(11.28 g/L) (also at 23 °C, pH 7). Emulsification was achieved by vigorous manual agitation for
10 min in glass vials having 1 cm diameter, using 10 mL of aqueous Pseudomonas sp. in M9
solution and 15 mL of EC10 solution in canola oil. Hand mixing and room temperatures were
used for the preparation of emulsions containing Pseudomonas sp. to avoid harming the bacteria.
Chitosan was dispersed in water at 1.65 wt.% concentrations at pH 4 (adjusted with GAA).
Tween 20 (2.5 mL/L, used as emulsifier) and KCl (34 g/L, used to balance the osmotic pressure)
were then added to the chitosan solution. CL-W/O (500 mL) and BAC-W/O (100 mL) were hand
mixed with a chitosan solution (1 L, pH 4) for 1 min using a spatula, to obtain double W/O/W
emulsions.

3.3.3 Bacteria Isolation and Characterization

Bacteria were isolated from garden soil (Hamilton, Ontario, Canada). The soil was dispersed in
water and agitated in glass jars. The soil was then allowed to settle, and the supernatant was used
to streak agar plates onto which naphthalene was added through sublimation as sole carbon
source. Agar plates without naphthalene were also streaked for comparison. Minimal broth M9
(11.28 g/L) was added to 1.5 wt.% agar. Bacterial colonies able to survive and grow in the
presence of naphthalene were then dispersed in M9 broth solutions in water (11.28 g/L) with
naphthalene (0.01g/L). Without naphthalene, growth was negligible. The microbial species ID
determination was based on 16S rRNA gene sequencing (V3-V4 region) using the Sanger
method. Briefly, the V3-V4 region was amplified by traditional PCR and the product qualitative
detected through agarose gel electrophoresis. The DNA cycle sequencing was performed at the
MOBIX Lab (McMaster University, Faculty of Health Sciences) using ABI BigDye terminator
chemistry on a 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences
 20

obtained, were blasted with the GenBank database (http://www.ncbi.nlm.nih.gov/) for species or
genus assignment. The highest genus identity was identified as Pseudomonas sp.

3.3.4 Bacterial Growth and Survival

Bottle tests were conducted to assess the survival and growth of Pseudomonas sp. with
individual components of the W/O/W emulsion (Ca-stearate, Tween 20, EC10, KCl, chitosan,
GAA, sodium citrate dihydrate, CAPB, and canola oil) and in the complete W/O/W system,
before and after gelation. Tests conducted with the individual components involved inoculating
autoclaved aqueous M9 broth (11.28 g/L) with Pseudomonas sp. and mixing it with the
component of interest, with and without naphthalene. The following concentrations were used:
Ca-stearate = 2.5 g/L, Tween 20 = 2.5 mL/L, EC10 = 10 g/L, KCl = 34 g/L, chitosan = 16.5 g/L,
GAA = 4 mL/L, sodium citrate dihydrate = 60 g/L, CAPB = 9.375 mL/L, and 100% canola oil.
Samples were incubated at 29 ⁰C in sterile glass vials after inoculation. Bacterial growth and
survival in the vials were assessed by qualitatively observing changes in the optical properties of
the sample (e.g. turbidity) and by plating at 3-day intervals in agar with M9 broth at 29 ⁰C, for up
to a one-month period. Bacterial growth and survival tests in W/O/W emulsions and gels were
studied over a one-month period, at 29 ⁰C. At the end of each test, 1.5 g of sample (W/O/W
emulsion or gel) was centrifuged it for a minimum of 3 min. The supernatant was then streaked
on 1.5 wt.% agar Petri dishes, in the presence of naphthalene, and bacterial growth was visually
assessed.

3.3.5 Optical and Confocal Microscopy

An optical VHX-5000 digital microscope (Keyence, Canada) was used to image Pseudomonas
sp. bacteria, dyed with methylene blue for visual contrast. W/O and W/O/W emulsions were also
imaged with this optical microscope and with an upright Leica TCS SP5 Laser Scanning
Confocal Microscope (Leica, Mannheim, Germany), which was equipped with a 20x glycerol
objective and an Argon laser with excitation at 476 nm and emission range of 555 – 620 nm. The
oil phase was stained with Nile red, a lipophilic dye for confocal microscopy imaging, to
differentiate between the oil phase (which fluoresced red) from the water phase (which appeared
black).
 21

3.3.6 Shear Rheology Experiments (Strain Sweeps)

The shear rheological behaviour of chitosan-based W/O/W emulsions was studied at 23 °C
before and after hand-mixing for 60 s with CAPB (9.375 mL/L), using a rotational torque-
controlled (i.e. combined-motor-transducer type) rheometer (MCR302 Anton Paar, Graz,
Austria). Strain sweeps were conducted at a constant frequency of 6 rad/s, increasing the strain
from 0.01% to 1000%. Measurements with W/O/W emulsions before CAPB addition were using
single-gap concentric cylinder geometry (inner radius = 13. 33 mm and height = 40 mm).
Measurements with W/O/W emulsions after CAPB addition were conducted using parallel plate
geometry (diameter = 50 mm), onto which sandpaper (600 grit) was affixed to minimize slip. In
shear rheology measurements, the linear viscoelastic region was identified as the region in which
the viscous (G”) and elastic (G’) moduli were independent of strain.

3.3.7 Steady State Fluorescence Spectroscopy Measurements

Steady state fluorescence spectroscopy was used as a non destructive way to monitor naphthalene
consumption by the bacteria using a FluoroMax-4 spectrofluorometer (Horiba Scientific Inc., Edison, NJ,
USA). The samples were loaded in 1 cm light path quartz cuvettes (Firefly Sci, Staten Island, NY, USA).
Samples were excited at 270 nm, and the spectra were collected over an emission range from 290 nm –
500 nm. The excitation and emission slits were set to 1 and 2, respectively. The naphthalene fluorescence
intensity was correlated to the naphthalene concentration [119], as further described in the supporting
information (Appendix A). Naphthalene stock solutions were prepared by saturating M9 broth (11.28 g/L)
with naphthalene, by adding a large excess of naphthalene (5 g/L) to the M9 broth at 23 ⁰C for 72 hrs
while stirring on a magnetic plate in air-tight containers (to prevent naphthalene volatilization).
Naphthalene crystals were filtered from the solution before all measurements, using a Whatman™ 1005-
070 Grade 5 qualitative filter paper and a vacuum filtration apparatus. The naphthalene stock solution was
then diluted to different concentrations to obtain a calibration curve (R2 = 0.996, Figure 15). Sequential
dilutions were performed to avoid inner filter effects. The naphthalene stock solution was also inoculated
with Pseudomonas sp. to assess naphthalene degradation over time. After bacterial inoculation, the
solution was stirred for 24 hrs and 48 hrs at 23⁰C in an air-tight container (to prevent naphthalene
volatilization), after which naphthalene concentrations were measured. Samples without bacteria were
also tested, to verify the lack of naphthalene degradation in the absence of the biological agent. Control
samples were thoroughly scanned to assess any background contributions.
 22

Results and Discussion

In aqueous M9 solutions, Pseudomonas sp. could only growth in the presence of naphthalene.
With naphthalene, M9 solutions inoculated with bacteria became turbid due to bacterial growth.
Without naphthalene, they remained clear. Plates streaked with Pseudomonas sp. solutions and
incubated for a week at 29 ⁰C confirmed that bacteria survived with naphthalene only.
Fluorescence spectroscopy showed that Pseudomonas sp. (Figure 16) could effectively degrade
naphthalene, as reflected in the progressive decrease in the fluorescence intensity (Figure 8). The
concentration of naphthalene decreased by 76.1% over 24 hrs and by 92.5% over 48 hrs, as
calculated based on naphthalene’s extinction coefficient (Appendix A, SI.A.1). Different
Pseudomonas strains are reported to biodegrade naphthalene, including Pseudomonas putida G7
and Pseudomonas sp. strain NCIB 9816-4 [120], Pseudomonas sp. strain ND6 [121],
Pseudomonas mendocina [122], Pseudomonas stutzeri AN10 [123], and Pseudomonas gessardii
LZ-E [124]. It has been observed that microbial naphthalene degradation is mainly aerobic [125].
Previous studies showed that bacteria use dioxygenases to incorporate molecular oxygen into
naphthalene, to form cis-dihydrodiol [125]. Dihydroxylated intermediates undergo ring cleavage,
forming salicylate [125]. Salicylate then undergoes oxidative decarboxylation, forming first
catechol and then tricarboxylic acid cycle intermediates [125]. Diverse methods have been
proposed to introduce oxygen in the subsurface, including oxygen-releasing beads [126],
nanocomposites [91] and other slow oxygen-releasing compounds [127]. In this study, the
oxygen initially present in aqueous naphthalene solutions was sufficient to sustain bacterial
metabolism. Future research should focus on methods to supply the Pseudomonas sp. bacteria
isolated in this study with oxygen, to sustain their metabolism in the subsurface.
 23

350000

300000
Fluorescence Intensity (CPS)

t=0
250000

200000

150000

100000 t=24 hrs

t=48 hrs
50000

0
280 330 380 430 480 530
Wavelength (nm)

Figure 8. Naphthalene degradation over 48 hrs, as probed with fluorescence spectroscopy.

Naphthalene can migrate in aquifers, posing risks to downstream receptors [128]. Therefore,
Pseudomonas sp. bacteria were incorporated in double W/O/W chitosan-based emulsions, which
also contained a crosslinker (citrate). Double W/O/W formation was confirmed with confocal
(Figure 9) and optical microscopy (Figure 17). Before mixing with CAPB (which destabilized
W/O/W emulsions), the double W/O/W emulsions were stable under quiescent conditions over 3
days (longer observations were not conducted) and upon manual agitation. Also, they could be
poured from a beaker, and they were mainly viscous with low shear viscoelastic moduli (elastic
modulus G’ = 1.90 ± 0.39 Pa, viscous modulus G” = 5.76 ± 0.06 Pa in the linear region, which
extended to a strain of approximately 1%). Polymeric fluids with low viscoelastic moduli and G”
> G’ flow easily, as opposed to gels with high viscoelastic moduli and G’ > G” [129]. The
rheological behaviour of double W/O/W before mixing with CAPB should therefore facilitate
pumping in the polluted subsurface regions.
 24

Figure 9. Confocal microscopy images of double W/O/W emulsions (top) and CL-W/O emulsion. The
images on the left hand-side (fluorescence images) show the difference between the oil phase (deep red,
dyed with Nile Red) and the water phase (black, without dye). Brightfield images are shown on the right
hand side. The scalebar in the images is 100 m.

Mixing with CAPB destabilized the chitosan-based double W/O/W emulsions, causing gelation
(Figure 10). Upon gelation, the shear viscoelastic moduli increased (G’ = 111.02 ± 1.67 Pa > G”
= 8.66 ± 0.10 Pa) and the chitosan-based material could not flow. It is envisioned that W/O/W
emulsion injection in naphthalene-polluted areas should be followed by CAPB injection, to
impede naphthalene migration in aquifers (naphthalene trapping). In this study, contact of CAPB
with W/O/W emulsions was obtained through hand-mixing. In the field, CAPB pulsed injection
could be used as a strategy to promote mixing with W/O/W emulsions [130, 131]. Gelation was
attributed to the crosslinking of the deacylated amine groups in the chitosan structure by citrate
 25

ions [132]. Upon mixing with CAPB, emulsion destabilization is attributed to the change in the
hydrophilic lipophilic balance (HLB) number of the chitosan-based W/O/W emulsion system.
Gelation due to HLB changes with surfactant addition is reported for other double emulsion
systems [72]. Gelation could not be achieved when incorporating BAC-W/O emulsions
(containing Pseudomonas sp., M9 broth and canola oil) into previously developed alginate-based
double W/O/W emulsions [101, 102]. This is because alginate is crosslinked by calcium ions,
which formed complexes with the phosphates contained in M9 broth (which are required for
bacterial survival). Calcium complexation by the phosphates in M9 broth was confirmed by the
formation of precipitates upon mixing M9 with CaCl2 in water. Binding between calcium and
phosphates was previously reported [102]. Instead, M9 broth did not interfere with chitosan-
citrate crosslinking.

Figure 10. Schematics of W/O/W double emulsion preparation and gelation.

Pseudomonas sp. could not survive at pH 4. This was verified by streaking naphthalene-
containing agar Petri dishes with bacteria inoculated in M9 aqueous solutions at pH 4 (adjusted
with GAA). Instead, Pseudomonas sp. survived at pH 5.5 - 7. For this reason, Pseudomonas sp.
was not dispersed directly in the chitosan phase of the W/O/W emulsion (which was at pH 4).
Instead, before CAPB addition, Pseudomonas sp. was protected in the primary BAC-W/O
emulsion droplets, in aqueous M9 broth at pH 7, surrounded by canola oil and EC10. After
CAPB addition, mixing between the chitosan (pH 4) and the water phase of the primary
emulsions (pH 7) yielded pH 6.5, allowing Pseudomonas sp. survival. Pseudomonas sp. survival
 26

in chitosan-based W/O/W emulsions was verified before and after mixing with CAPB, with
naphthalene added. Bacterial growth was observed on agar plates streaked with one-month old
W/O/W sample. Without naphthalene, bacterial growth on agar plates was negligible, in either
gelled and non-gelled samples. These results confirmed that Pseudomonas sp. used naphthalene
as sole carbon source in the system studied, allowing its bioremediation. While chitosan did not
hinder the survival of naphthalene-degrading Pseudomonas sp. used here, it has bactericide
properties [133]. The bactericide properties of chitosan should promote chitosan-barrier integrity
and resistance to bacterial degradation by autochthonous bacteria in groundwater. This important
aspect should be verified on the field, along with the effect of chemical species present in
groundwater. Pseudomonas sp. survival and metabolisms in the field should also be studied.

Conclusions

Pseudomonas sp. was isolated from a naphthalene contaminated site and dispersed in a chitosan-
based W/O/W double emulsion to trap and biotreat naphthalene in water. Naphthalene
degradation in water was verified using steady state fluorescence spectroscopy. Pseudomonas sp.
survived exclusively in the presence of naphthalene, both in aqueous M9 broth solutions or
W/O/W emulsions (either with or without CAPB). This observation was indirect evidence of
naphthalene biodegradation. Before mixing with the surfactant CAPB, W/O/W double emulsions
were stable and mainly viscous, with low shear viscoelastic moduli (G’ = 1.90 ± 0.39 Pa < G” =
5.76 ± 0.06 Pa). CAPB was used to reverse W/O/W on demand, releasing the crosslinker citrate
into the outer chitosan phase, hence inducing gelation (G’ = 111.02 ± 1.67 Pa > G” = 8.66 ± 0.10
Pa) and trapping naphthalene. All experiments were conducted in aerobic conditions and in a
controlled lab environment. Future research should investigate Pseudomonas sp. survival in the
field, using either new or existing oxygen delivery methods. Field studies should also elucidate
chitosan-based barrier integrity over extended time periods, in the presence of autochthonous
bacteria and chemical species in groundwater.
 27

ZEIN SORPTION OF NAPHTHALENE

This manuscript will be submitted shortly.

Abstract

Naphthalene is a major polycyclic aromatic hydrocarbon often found where pollutants of fuel
combustion or crude oil may have entered the environment. Adsorbents have been used in
studies and have been identified as a cost effective, energy efficient tool to sequester out targeted
pollutants in the environment. Zein is a hydrophobic protein created as a by-product of the mass
production and processing of corn, for example to manufacture high fructose corn syrup or
ethanol. The hydrophobic properties of zein suggest it may act as an efficient adsorbent for
hydrophobic compounds. This study explores zein as a possible adsorbent for polycyclic
aromatic hydrocarbon, using naphthalene as a model compound. Naphthalene sorption to zein
crosslinked with CaCl2 was found to follow pseudo first order reaction kinetics (R2 = 0.983). The
cross-linked zein was observed to have a loading capacity of 13.91 mg/g of naphthalene per zein
substrate, with a sorption rate constant of 0.0066 min-1 at 21 °C.

Keywords: Zein, Naphthalene, Adsorption

Introduction

Naphthalene is a Polycyclic Aromatic Hydrocarbon (PAH) commonly created as a by-product of

the coal, oil, and chemistry industry [19]. PAHs have gained attention as a pollutant due to its
toxicity, persistence, and volatility posing a risk to the environment and surrounding population
in the case of an accidental release [20]. Studies have thoroughly looked at methods of
bioremediation as a method to handle PAH contamination in the environment as its is low energy
remediation technique [35]. However, the use of adsorbents to sequester contaminants out of
water is another technique explored in literature [102]. Examples of adsorbents used to seize
naphthalene in groundwater flow can include, but are not limited to, porous carbon derived from
sewage sludge, zeolites, and graphene composites [105, 134, 135]. Similar to traditional methods
of remediation, the implementation and recovery of these remedial devices can be cumbersome
and in some case require heavy construction [110].
 28

The kinetics and thermodynamic of an adsorbate and adsorbent can give insight on the sorption
mechanism involved allowing the opportunity to optimize sorption and better understand in what
conditions an adsorbent may be most effective [136]. Chemisorption is the adsorption
mechanism that is dependant on the chemical properties and valence forces of an adsorbent and
adsorbate sharing electrons [136]. This adsorption mechanism often has a large energy threshold
and thus may take longer than physisorption [137]. Physisorption is the mechanism at which an
adsorbent and adsorbate adsorb to one another through interactions such as van der Waals forces
[137]. These interaction in nature have a very low energy threshold and thus form spontaneously
and in a short amount of time [137].

Zein is a protein commonly derived from corn and can exist in four forms: α, β, ƴ, and δ [138].
α-zein is the most common form of zein being produced as the major product and will be the
focused form of zein for this study [138]. This form of zein is majorly comprised of hydrophobic
amino acids, the lack of charged amino acids results in the highly hydrophobic properties of zein
[138]. Natural polymers, like zein, are exceptional candidates for use in environmental
remediation as they are often non-toxic and biocompatible with the environment [56]. In
addition, for similar reasons, they are good options as substrates for the immobilization of
enzymes [56]. As zein is largely hydrophobic it poses as a good candidate for adsorption with
PAHs where the contaminants hydrophobicity has previously posed as a challenge to
remediation techniques [73, 138].

This study will look at the adsorption of naphthalene on zein and its potential to increase the
contact between naphthalene and laccase. Unlike other adsorbents, this zein procedure can be
pumped into the subsurface and coagulated with a secondary step utilizing CaCl2. This process is
non-toxic and biologically inert to the environment and ecosystem due to the fact that zein is a
plant derivative and the naturally occurring concentration of CaCl2 in soil [138].

Methods and Materials

4.3.1 Materials
Purified Zein (98%, powder, ACROS Organics), sodium hydroxide (NaOH) (97%, pellets),
calcium chloride anhydrous (CaCl2) (96%, Alfa Aesar), naphthalene (98.5%, ACROS Organics),
and ethanol anhydrous (EtOH) (95%) were purchased from Fisher Scientific. Laccase from
 29

Trametes versicolor (>0.5 U/mg, powder) was purchased from Sigma Aldrich. Deionized (DI)
water was used in all experiments.

4.3.2 Zein and Naphthalene Preparation and Imaging

Zein substrates were produced by dissolving purified zein (0.03 g/L) in diluted NaOH (0.065 M)
and coagulated drop wise with CaCl2 (2 M) and left to form for 10 minutes.

Naphthalene stock solutions were prepared by saturating DI with naphthalene, by adding a large
excess of naphthalene (3.33 g/L) to the DI at 23 ⁰C for 72 hrs while stirring on a magnetic plate
in gas-tight containers. Naphthalene crystals were filtered from the solution before all
measurements, using a Whatman™ 1005-070 Grade 5 qualitative filter paper and a vacuum
filtration apparatus.

Images were taken with a VHX-5000 digital optical microscope (Keyence).

4.3.3 Kinetic Reaction Experiments

Kinetic studies were conducted to evaluate the sorption of stock naphthalene on the zein
substrate (13.3 g/L). This was completed in duplicates in airtight containers on stir plates at
23°C. Controls to account for the sublimation of the stock naphthalene in the airtight agitated
containers and zein in DI were also observed. 500 μL aliquots were sampled 11 times, at t (mins)
= 0,5,10,20,40,60,120,1130,1515,1890, and 2640, and diluted to 1500 μL with EtOH and
analysed via GCMS.

4.3.4 GCMS Analysis

GCMS data were obtained with an Agilent 6890N GC with a 5975 Inert XL MSD equipped with
a 7683B series autoinjector and G2614A tray. The column utilized for separation was a HP-5MS
UI (5% phenyl polycarborane–siloxane; crosslinked) column purchased from Agilent with
dimensions of 30 m x 250 μm x 0.25 μm film. Oven initial temperature was set to 30 ⁰C for 4
minutes with a ramp of 40 ⁰C/min to 100 ⁰C and held for 1 minute, then a second ramp of 120
⁰C/min to a final temperature for 220 ⁰C and held for 2 minutes. Injection volume was set to a
splitless injection of 0.20 μL and flow rate to 1.0 mL/min.

4.3.5 Spectroscopic Analysis

FTIR spectra was attained utilizing a Shimadzu IRAffinity-1S FTIR equipped with a MIRacle A
ATR attachment.
 30

Fluorescence spectroscopic data was collected using a FluoroMax-4 spectrofluorometer (Horiba

Scientific Inc., Edison, NJ, USA and quartz cuvettes with a pathlength of 1 cm (FireflySci,
Staten Island, NY, USA). Samples were excited at 270 nm, 280 nm, and 295 nm and the spectra
collected within a range of 310 nm to 500 nm. The excitation and emission slit were set at 3nm
and 5nm, respectively.

4.3.6 Kinetic Reaction Models

Pseudo First Order Rate Models (PFO) and Pseudo Second Order Rate Model (PSO) were tested
to provide an appropriate characterization of the kinetic data, under assumptions that the
naphthalene absorption follows first/second order rate law [139].

Equation 1. Non-linear Pseudo First Order Rate Model.

𝑞𝑡 = 𝑞𝑒 (1 − 𝑒 −𝑘1𝑡 ) [140]

Where qe is the adsorbate loading at equilibrium, qt is the absorbate loading at any time t, and k1
being the rate constant [140].

Equation 2. Non-linear Pseudo Second Order Rate Model.

𝑞 2 𝑘2 𝑡
𝑞𝑡 = 1+𝑘𝑒 [140]
2 𝑞𝑒 𝑡

Where qe is the adsorbate loading at equilibrium, qt is the absorbate loading at any time t, and k2
being the rate constant [140].

Results and Discussions

As seen in Figure 11 and Figure 12, the zein forms a very porous shell supported by a filament-
like layer. This allows the zein structure to maximize surface area for sorption of naphthalene. It
was observed that the longer the substrate is allowed to sit the more robust it becomes and upon
contact with water the substrate turned pale white and became very rigid.
 31

Figure 11. Optical microscopy images of zein during the coagulation process.

Figure 12. Zein substrate 10 minutes after coagulation with CaCl2.

Most traditional remediation techniques for naphthalene clean up require heavy construction into
excavating contaminated land and treating it off site [35]. Newer technologies with lower
environmental impact can widely vary in approach such as in situ oxidation, bioremediation, and
surfactant enhanced treatments [39]. However, no technique in the literature has been reported to
efficiently both contain and degrade naphthalene in a way that did not require heavy installation
or adversely affect the surrounding ecosystem. Before coagulation, zein dissolved at basic pH is
fluid and easily injectable into the subsurface. Due to the majority of the amino acids in zein
being non charged, zein results in being a very hydrophobic polymer that can be solubilized with
some pH adjustment [138]. This hydrophobicity will allow PAHs to readily sorb to the zein
substrate as seen in the kinetic study below, Figure 13.
 32

Physisorption generally occurs instantaneously as no energy threshold is required for the

adsorbent to adhere to the absorbates surface [137]. This energy of adherence is comparable to
the energy of condensation to the adsorbate [137]. Chemisorption rates can vary largely on the
energy required for the adsorbent to stick onto the adsorbate [136]. Conventionally studies have
concluded that fitting an adsorption kinetic study to PSO reaction rate models suggests
chemisorption as the primary mechanism of adsorption, however this conclusion is not always
correct as described by Tran et al. [136]. The determination of adsorption mechanisms based on
fitting kinetic studies to PFO/PSO models can be lacking without the support of further studies
such as FTIR and thermodynamic studies [136, 137].

Concentrations of naphthalene were analysed via GCMS and were quantified with a calibration
curve of R2 = 0.993 (Appendix B). The kinetic study (Figure 13) of naphthalene sorption onto
zein had an R2 = 0.983 match to the PFO model and a R2 = 0.981 fit to the PSO model.
Calculated with Equation 1, the PFO model [140], the kinetic study observed an average loading
capacity of 13.91 mg/g of naphthalene onto zein at equilibrium (500 minutes) at 21 °C and a
sorption rate constant of 0.0066 min-1.
 33

18

PFO Curve
16
PSO Curve

14
Naphthalene Adsorbed per Zein (mg/g)

12

10

0
0 500 1000 1500 2000 2500 3000
Time (min)

Figure 13. Pseudo First Order and Pseudo Second Order Comparison of Naphthalene Zein Adsorption
Kinetic Study.

Zein samples before and after 12 hours of naphthalene adsorption were examined with FTIR, as
seen in Figure 14, noting a unique peak at 1025 cm-1 and 1100 cm-1 circled in the figure below.
These peaks could indicate a C-O stretch and C-N stretch respectively, that would be altered on
the protein structures of zein after the sorption of naphthalene [141]. As the equilibrium time for
naphthalene on the zein substrate was observed to be 500 minutes (at 21 °C) and changes in
FTIR spectra at 1025 cm-1 and 1100 cm-1 are observed before and after sorption (see Figure 14),
it can be speculated the mechanism for adsorption may be chemisorption dominant. However, as
the PFO model has a slightly better fitting R2 = 0.981 to the kinetic data as well as the
established hydrophobic properties of zein and naphthalene, physisorption may closely compete
with chemisorption for the dominant mechanism. The equilibrium time is most likely also
affected by the pores formed on the zein substrate (Figure 11) [136]. Despite physisorption
 34

normally occurring instantaneously if the adsorbent is porous, the time to equilibrium may be
prolonged [139].

0.4

0.35

0.3

0.25

Absorbance
0.2

0.15

0.1

0.05

0
3600 3100 2600 2100 1600 1100 600
-0.05
Wavenumbers (cm-1)

NAP-ZEIN ZEIN

Figure 14. FTIR comparison of zein substrate with and without naphthalene sorption.

Conclusions

This study was able to identify zein as an injectable pumpable adsorbent into the subsurface for
naphthalene pollutants triggered with a second injection of CaCl2 to cause gelation of zein
creating a porous substrate for naphthalene to sorb unto. The zein substrate was found to have a
loading capacity of 13.91 mg/g at 21 °C with a sorption rate constant of 0.0066 min-1. The
kinetics of the study could be described with a PFO model (R2 = 0.983). As the equilibrium time
of 500 minutes it is speculated that the dominant mechanism of sorption is chemisorption. Due to
the hydrophobicity of zein and the hydrophobic nature of naphthalene and all PAHs, zein was
identified as a successful adsorbent and possible scaffold for laccase.
 35

CONCLUSIONS AND FUTURE WORK

Conclusions

Naphthalene has been around since the early 1800 with its inevitable isolation and identification
starting as far back as the colour purple was made and in demand [1]. Since then it has continued
to be produced as a side product of oil and coal processing and remains to be used as a chemical
building block, now creating more than just dyes, including phthalic anhydrides, surfactants,
plasticizers, and fumigants for example [19]. As a result, the current day population is still
unintendedly exposed to naphthalene through contact with mothballs or crude oil/coal tar
pollution [17, 85]. As naphthalene use in mothballs has been largely reduced [17], this study has
focused on the pollutant source of exposure. Current remediation techniques for naphthalene
contamination most often include traditional excavation and off-site treatment of contaminated
soil [39]. Newer remediation techniques involving surfactants, enzymes, bacteria, and chemical
oxidizers have been researched [35]. However, no technique addressed a way to contain
pollution migration in groundwater that would simultaneously trap and treat the contaminant in a
low energy, low construction implementation, environmentally inert way.

The first submitted paper of this thesis successfully engineered a surfactant triggered chitosan
biogel that can contain the contaminant and concurrently treat the naphthalene pollution with the
incorporated bacteria and when untriggered remains as a pumpable double emulsion eliminating
the need for heavy construction of traditional barriers. Before triggering gelation in the double
emulsion (W/O/W) with the surfactant (CAPB) the multiple emulsion remained stable and was
observed to have a low shear viscoelastic moduli (G’ = 1.90 ± 0.39 Pa < G” = 5.76 ± 0.06 Pa).
After emulsion reversal with CAPB, causing gelation due to the release of citrate crosslinker into
the outer chitosan phase, the resulting gel was noted to have a viscoelastic moduli of G’ = 111.02
± 1.67 Pa > G” = 8.66 ± 0.10 Pa trapping naphthalene within the biogel for biodegradation with
the incorporated Pseudomonas sp.

The second unsubmitted work in published format explored zein as a pumpable adsorbent for
sequestering out naphthalene utilizing FTIR and GCMS and was experimentally determined to
have a loading capacity of 13.91 mg/g at 21 °C with a sorption rate constant of 0.0066 min-1 with
a R2 = 0.983 fit to PFO reaction rate models. As the equilibrium time for sorption of naphthalene
 36

on zein was about 500 minutes and FTIR spectra before and after sorption highlighted two
differing peaks it was speculated that chemisorption may be the dominant mechanism of
sorption.

Future Work

Future work for this study includes field trials of the chitosan-based bio gel and the zein
adsorbent. Possible interferences that could occur with the chitosan-based gel that would impede
its ability to work as designed could include salt concentrations found in the contaminated
subsurface. These salt concentrations could create an osmotic pressure in the double emulsion
system causing it to prematurely gel. In addition to adsorbing naphthalene pollution, zein can be
functionalized into a scaffold to immobilize laccase to increase its enzymatic efficiency at
degrading naphthalene. Complications with the zein adsorbent may include interactions with
salts and maintaining the activity and stability of enzymes adhered to the zein scaffold.
 37

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 50

APPENDIX A

Supporting Information for 3.0 CHITOSAN-BASED BIOGELS: A POTENTIAL APPROACH TO TRAP AND BIOREMEDIATE
NAPHTHALENE

This supporting information file contains details regarding steady-state fluorescence spectroscopic experiments used to determine the
changing naphthalene concentration in water as a function of time (SI.A.1), and optical microscopy images of naphthalene-degrading
Pseudomonas sp. (Figure 16) and of W/O/W emulsions (Figure 17) in section SI.A.2.

SI.A.1 Fluorescence spectroscopy

The naphthalene concentration in water was determined using the molar extinction coefficient (3.9x 10-5 L ug-1 cm-1) as reported by
Swartz and Wasik (1976). A concentration study was subsequently conducted to a) verify that no inner filter effect (i.e., re-absorbance
due to excessive concentration of naphthalene) was observed and b) calculate the naphthalene concentration in the treated solutions.
The results of the concentration study are shown in Figure 15.
 51

350000

y = 3E+10x - 1184.1
300000 R² = 0.9959

250000
Fluorescence Intensity (CPS)

200000

150000

100000

50000

0
0.00E+00 2.00E-06 4.00E-06 6.00E-06 8.00E-06 1.00E-05 1.20E-05
Naphtalene Concentration (M)

Figure 15. Naphthalene fluorescence intensity as a function of naphthalene concentration.

The range of concentrations used in this study were within the linear region of the fluorescence intensity vs. concentration relationship
as shown in Figure 15. This allows concluding that the fluorescence intensity reduction in the samples was the result of degradation of
naphthalene due to the application of the treatment.
 52

SI.A.2 Optical Microscopy Imaging of Pseudomonas sp. and W/O/W Emulsions

Figure 16 shows Pseudomonas sp. bacteria, dyed with methylene blue for visual contrast. Figure 17 shows chitosan-based W/O/W
emulsions.

Figure 16. Pseudomonas sp. isolated from garden soil (ON, Canada).
 53

Figure 17. W/O/W double emulsion system after 24 h before mixing with CAPB.
 54

APPENDIX B

Supporting Information for 4.0 ZEIN SORPTION OF NAPHTHALENE

This supporting information file contains the data obtain using GCMS to determine naphthalene concentrations in water (SI.B.1).
SI.B.1 GCMS

Figure 18. Naphthalene response ratio (RF) as a function of naphthalene concentration (ppm);
R2 = 0.993; RSS = 4.06x1010 RF; RSME = 82231.2 RF.