Applied For Ph.D.

in Chemical Engineering/Chemistry

Candidate Name: Samia Kanwal

Research Proposal Title

Application of Advanced Oxidation Processes (AOPs) for the

Detoxification of Cyanotoxin Contaminated Water

Table of Contents
1. Research Question/Problem Statement
2. Objectives
3. Introduction
4. Literature Survey
4.1. Comparison of AOPs
4.1.1. Degradation efficiency of AOPs
4.1.2. Generation of by-products
5. Methodology
5.1. Bacteria Culture
5.2. Synthesis of TiO2 Anatase Nanomaterials
5.2.1. TiO2 anatase nanoparticles (Ti-A-NP)
5.2.2. TiO2 anatase nanotubes (Ti-A-NT)
5.2.3. TiO2 anatase nanofibers (Ti-A-NF)
5.2.4. PtOx-TiO2 anatase nanomaterials
5.2.5. Photocatalytic experiment
6. Expected Outcomes
7. Timeline
8. References

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 1. Research Question/Problem Statement
The production of drinking water encounters various challenges, with naturally occurring
cyanobacterial toxins posing a significant concern. As pollutants increasingly become more
prevalent and resistant, conventional water treatment methods often fall short in effectively
removing them. Among the emerging technologies, advanced oxidation processes (AOPs) show
promising potential in addressing this specific issue more effectively.
2. Objectives
 Applying various Advanced Oxidation Processes to neutralize cytotoxins.
 Purifying water contaminated with cytotoxins.
 Assessing the effectiveness of tested AOPs and their energy requirements.
 Evaluating the toxicity of resultant end-products.
 Identifying the Transformation Products unveiled during the process.
 Application of Photocatalysis as an Advanced oxidation process.
 Synthesis of Titanium-Platinum based hybrid nanomaterials.
 Characterization of the synthesized nanomaterials
 Application of nanomaterials and UV-C light for the detoxification of cytotoxin strains
such as Nodularia spumigena, Dolichospermum circinale and Aphanizomenon gracile.
3. Introduction
Cyanobacteria, the diverse and widespread phototrophic prokaryotes, have inhabited Earth for
billions of years [1]. They thrive in various terrestrial and aquatic environments, including
unexpected places like Antarctic lakes and hot springs [2]. The rising eutrophication of water
sources and the effects of climate change, impacting nutrient levels and temperatures, contribute
to more frequent and extensive cyanobacterial blooms [1, 3, 4]. While not all blooms are
harmful, around 40 cyanobacterial species produce various secondary metabolites that are toxic
to plants [5], animals, and humans. Consequently, these cyanobacteria and their toxins pose
significant risks to surface waters used for drinking and recreational purposes. Implementing
adequate measures is crucial to prevent or eliminate cyanobacterial blooms and their associated
toxins.
Cyanotoxins can lead to a wide array of clinical symptoms, including acute hepatotoxicosis,
sudden-onset neurotoxicity, gastrointestinal issues, as well as respiratory problems and allergic
reactions [4]. Various cyanobacterial metabolites qualify as cyanotoxins [6], and several
cytotoxins produced by different cyanobacteria are commonly found in drinking water. These
include microcystins, nodularin, Cylindrospermopsin, Anatoxins, Saxitoxins, and β-N-
methylamino-L-alanine [7].

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The primary approach aims to prevent cyanobacterial blooms in surface water by implementing
measures like reducing nutrients, biomanipulation, or using algaecides [3, 8]. Crucially,
especially with toxic cyanobacteria, removing intact cyanobacterial cells becomes vital to
prevent the release of intracellular toxins such as microcystins (MCs), anatoxin-a (ANTX), and
saxitoxin (STX) [4, 9]. The secondary approach involves eliminating cyanobacterial cells and
their metabolites in drinking water treatment facilities, including methods like Advanced
Oxidation Processes (AOPs) [7].
Various oxidants are employed in Advanced Oxidation Processes (AOPs) to degrade cytotoxins.
These include Sulfate radical (SO4 -∙), Hydroxyl radical (∙OH), Ferrate ([FeO 4]2-), Ozone (O3),
Peroxydisulfate (S2O82-), Hydrogen peroxide (H2O2), Peroxymonosulfate (HSO5- or SO52-,
Hydroperoxy radical (HO2∙), Permanganate (MnO4-), Hypochlorous acid (HOCl), Chlorine (Cl2),
Chlorine dioxide (ClO2), Peroxymonosulfate radical (SO5-∙), and Sulfite radical (SO3-∙). Several
AOPs have been utilized in numerous studies, including Hydrogen peroxide, Ozonation and O 3-
based AOPs, Photolysis, Photolysis combined with oxidants, Photocatalysis, Fenton Oxidation,
Non-thermal plasma, Sulfate radical-based AOPs, Electrochemical oxidation, sonolysis, and
Radiolysis [7].
While various reactive chemical species can be produced, the short-lived ∙OH is often regarded
as the most significant species generated in AOPs in water. This perception stems from its
notably high reactivity, as indicated by its redox potential. This highly reactive oxidant
predominantly interacts with organic compounds in two distinct ways: (i) through an
electrophilic attack on electron-rich structures like C=C double bonds, aromatic systems, and
neutral amines, and (ii) by extracting hydrogen from C–H groups [10]. Additionally, at neutral
pH levels, ∙OH can engage in a less favorable kinetic process involving one-electron transfer
[11].
Among the various semiconductor materials studied for the photocatalytic production of
hydrogen, titanium dioxide (TiO2) stands out as one of the most extensively researched. This is
due to its advantageous properties such as photosensitivity, cost-effectiveness, non-toxic nature,
and high chemical as well as thermal stability [12]. TiO2 exists in three distinct crystalline forms:
anatase, rutile, and brookite [13]. Early studies on photocatalysis often indicated that anatase is
the most efficient photocatalyst among the polymorphs of TiO 2 [14]. It's widely acknowledged
that TiO2 polymorphs can be activated primarily through near-UV radiation [15].
For this study, pure TiO2 anatase semiconductor materials can be employed in three different
structural forms: nanoparticles, nanotubes, and nanofibers. These materials can be dispersed with
PtOx (2.0 wt.% nominal loading) nanoparticles. The structural, electronic, and morphological
properties of the calcined materials may be investigated using various techniques, including X-
ray diffraction (XRD), Raman spectroscopy, diffuse reflectance ultraviolet-visible spectroscopy
(DR UV–vis), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy
(TEM), and N2 physisorption methods. The analysis of Tauc plots can reveal the band gap
energy values after the deposition of PtOx over TiO2 nanomaterials.

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 4. Literature Survey
4.1. Comparison of AOPs
4.1.1. Degradation efficiency of AOPs
When assessing different Advanced Oxidation Processes (AOPs), especially in their application
for large-scale water treatment, the efficiency of degradation stands out as a crucial factor. This
efficiency measures the relationship between the energy, oxidant, or catalyst dosage and the
treatment's effectiveness. In terms of energy efficiency, a key metric used is the electrical energy
per order (EEO). EEO is defined as the amount of electrical energy, measured in kWh, required to
reduce a pollutant by 1 order of magnitude (i.e., 90%) in 1 m 3 of water [16]. For oxidants or
catalysts, the "stored electric energy" of a compound can be computed based on electricity costs
(price per kWh) and the cost of the specific compound (price per kg) [17].
Based on a comprehensive review of AOPs for water treatment and their reported energy
efficiency in the literature, Miklos et al. [16] classified emerging AOPs according to their EEO
values (refer to Fig. 1). Most reported E EO values did not consider auxiliary oxidants or catalysts
in their calculations. They classified AOPs into three groups based on their median EEO values:
 The first group comprises AOPs with median E EO values of < 1 kWhm−3, representing a
practical range for full-scale application.
 The second group includes AOPs with median E EO values of 1–100 kWhm−3, requiring
more energy, but these AOPs might offer an attractive solution for specific issues,
including potential large-scale applications.
 The last group encompasses AOPs with median E EO values of > 100 kWhm−3, currently
deemed less energy-efficient. However, ongoing developments might lead to
optimization and reduced energy demands and associated costs.
Regarding the removal of cyanotoxins using AOPs, there is limited reported data on E EO values.
For instance:
 UV/H2O2 treatment of MC-LR and CYN showed EEO values ranging from 4.5×10−3–
6.1×10−3 and 1.6×10−3 kWhm−3, respectively [18, 19].
 UV/PMS and UV/PS treatments were estimated to have E EO values ranging from 10−4 to
10−5 kWhm−3 for CYN, and 0.7 and 0.2 kWhm−3, respectively, for MC-LR [19, 20].
 Electrochemical oxidation of MC-LR had E EO values ranging from 48 to 67 kWhm −3,
depending on the electrode material [21].
These reported EEO values seem to align with the findings of Miklos et al. [16]. However, there
are discrepancies in the reported E EO values for UV/TiO2 treatment. For instance, significantly
lower EEO values (approximately 0.08 to 0.14 kWhm−3 for MC-LR and 0.03 to 0.015 kWhm−3 for
MC-LR, -LA, and -RR) were reported in two independent studies [20, 22]. These values are
notably 2–3 orders of magnitude lower compared to the results reported by Miklos et al. [16].
Interestingly, despite the use of energy-efficient UV-LEDs in the second study [22], the choice

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of light source doesn't seem to have a substantial impact since demanding UV xenon lamps were
utilized in another study by Antoniou et al. [20].

Figure 1. Classification of AOPs according to their E EO values (based on the review by Miklos et
al. [16])
4.1.2. Generation of by-products
A critical consideration in AOP treatment is the generation of toxic disinfection byproducts
(DBPs), particularly halogenated organic and inorganic compounds like trihalomethanes,
haloacetic acids, haloacetonitriles, chlorates, bromates, and others [16]. Due to their harmful
nature, the WHO has recommended guideline values for various DBPs, such as chloroform (300
μg L−1), bromoform (100 μg L−1), perchlorate (70 μg L−1), and bromate (10 μg L−1) [23, 24]. The
formation of DBPs relies on the specific AOP used and the composition of the water, considering
factors like nitrogen presence, organic matter, and halogens [16].
Bromate formation is notable in O3 and O3-based AOPs, where a considerable amount, up to
50%, of bromide (at concentrations > 100 μg L −1) can transform into bromate [16]. Additionally,
∙OH may contribute to bromate formation by around 30–70% [153]. Adjustments such as
lowering pH, reducing O3 or bromide concentration, and introducing H2O2 can help mitigate this
issue [25]. Chlorate generation in O3 and O3-based AOPs is mainly relevant when pre-
chlorination is involved [16].
In most AOPs dominated by ∙OH, bromate formation is typically negligible in the presence of
abundant organic matter or H2O2 due to radical quenching [7, 16]. Chlorate and perchlorate
production occur under specific conditions where reactive chlorine species react with organic
matter, forming halogenated DBPs. Generally, ∙OH-mediated DBP formation is considered
noncritical except in certain instances, such as high-density ∙OH generation at electrode surfaces
in EAOPs [16]. In SR-AOPs, organic matter at low concentrations effectively inhibits bromate
formation [7, 16], although reactions of SO4−∙ with chloride under pH<5 can lead to chlorate
production [7].

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UV irradiation typically does not generate inorganic DBPs but may produce nitrite through
nitrate photolysis, potentially leading to nitrated aromatic compound formation. In UV/chlorine
processes, organic halides can form under alkaline pH and chloride concentrations exceeding 1 g
L−1 [16].
5. Methodology
5.1. Bacteria Culture
Nodularia spumigena, known for producing pentapeptide hepatoxins, along with
Dolichospermum circinale and Aphanizomenon gracile, which are known for producing
neurotoxins, are available for purchase from the microbiology laboratory. These strains can be
cultured and grown within the laboratory for research purposes.
5.2. Synthesis of TiO2 Anatase Nanomaterials
5.2.1. TiO2 anatase nanoparticles (Ti-A-NP)
The Ti-A-NP sample was synthesized using a sol-gel method [15, 26]. In a beaker, 150 mL of
ethanol was added with a specific amount of titanium isopropoxide solution while continuously
stirring. The beaker was kept at room temperature for 60 minutes. The solution's pH was
adjusted to 10 by slowly adding tetrapropyl ammonium hydroxide solution while stirring
consistently. At a pH of 10, a colorless gel was formed, which turned into a white precipitate
upon removal of excess ethanol by heating the gel to 80 °C. The resulting white precipitate was
washed thrice with ethanol and air-dried at 100 °C for 12 hours. Subsequently, it was calcined
under an air stream at 400 °C for 5 hours.
5.2.2. TiO2 anatase nanotubes (Ti-A-NT)
The Ti-A-NT sample was prepared following the alkaline method outlined by Kasuga et al [15,
27]. Initially, 6 grams of the Ti-A-NP sample were dispersed in 120 mL of 10 M NaOH solution.
This suspension was stirred for 30 minutes and then transferred into a Teflon-lined stainless steel
autoclave, sealed, and placed in an electric oven at 130°C for 48 hours.
Afterward, the autoclave was cooled to room temperature, and the resulting material was filtered.
It was then neutralized using a 0.1 M HCl solution and washed five times with deionized water
to eliminate any undesired products. Subsequently, the material was dried at 100°C for 12 hours
and finally subjected to calcination under an airflow at 400°C for 5 hours.
5.2.3. TiO2 anatase nanofibers (Ti-A-NF)
TiO2 anatase material can be purchased from the laboratory. The bulk TiO 2 anatase nanofibers
obtained were subjected to calcination under an airflow at 400°C for 5 hours.
5.2.4. PtOx-TiO2 anatase nanomaterials
In this synthesis procedure, 50 mg of TiO2 anatase nanomaterial and 24.6 mg of polymer
(polyvinyl alcohol) were added to a round bottom flask containing an aqueous solution of
H2PtCl6 (0.2 mg/mL). After achieving a uniform suspension by sonication, 0.5 mL of freshly
prepared sodium borohydride (NaBH4) solution (2 mg/mL) was added to vigorously reduce the

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H2PtCl6 while stirring. Another 0.5 mL of NaBH 4 solution was added after 2 hours. The solution
was continuously stirred for 12 hours to form a precipitate. The resulting precipitate was
separated via centrifugation and washed three times with deionized water and ethanol. The PtO x-
TiO2 anatase nanomaterial was obtained by drying the powder in air at 100°C for 3 hours [15].
5.2.5. Photocatalytic experiment
This experiment was based on the method proposed by Jin et al. [28]. For cyanobacterial
degradation, a 100 mL suspension of either N. spumigena, D. circinale, or A. gracile cells,
containing 200 mg/L of PtOx-TiO2 anatase nanomaterial, was introduced into separate 200 mL
quartz glass beakers. The protocol remained consistent for each type of anatase nanomaterial
used. Cyanobacterial concentrations were adjusted using distilled water. The beakers containing
cyanobacterial suspensions were placed in an incubator devoid of aeration to maintain a stable
temperature (25℃) and prevent any influence from natural sunlight. UV-C light was emitted by
a Mercury lamp positioned above the surface of the cyanobacterial suspensions within the
incubator, maintaining a distance of 2 cm between the suspension surface and the lamp.
Throughout the experiment, the suspensions were continuously stirred using magnetic stirring at
14 g. All experiments were conducted in triplicate, and the resulting data were expressed as the
mean value ± standard deviation (SD).

Figure 2. Schematic diagram of methodology and its expected outcomes

6. Expected Outcomes
 It is anticipated that the cells of N. spumigena, D. circinale, and A. gracile are sensitive to
UV-C light and to all types of anatase nanomaterials, potentially leading to the
destruction of algal cells and the degradation of released toxins and other metabolites.

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  The effectiveness of PtOx-TiO2 anatase nanomaterial in degrading both algal cells and
the released toxins is expected to be higher due to the efficient incorporation of platinum
into the TiO2 anatase material, compared to other types of anatase nanomaterials.
 The anticipated hierarchy of anatase nanomaterial effectiveness is as follows:
PtOx-TiO2 > Ti-A-NF > Ti-A-NT > Ti-A-NP
 It is expected that each strain's removal rate could exceed 95% due to the application of
nanomaterials and UV-C light, which are effective in degrading the algal strains.
7. Timeline
Table 1. Schematic representation of the timeline in Ph.D.
TASK Year 1 Year 2 Year 3

Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4

Course Work

Research Proposal

Literature Review

Module-1

Paper draft and

submission

Module-2

Paper draft and

submission

Module-3

Paper draft and

submission

Preparation of thesis

Final defense

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  The project timeline spans three years. During the initial year, the focus will be on
completing coursework, formulating a research proposal, and conducting a
comprehensive literature survey. Additionally, efforts will be made to acquire necessary
lab equipment and arrange for required chemicals.
 Moving into the second year, the experimental procedures will commence and progress.
The aim is to complete the experimental phase and further analyze the obtained samples.
 In the final year, the emphasis will be on finalizing the thesis and preparing for the thesis
defense.
8. References
1. Newcombe, G., International guidance manual for the management of toxic
cyanobacteria. 2012: Iwa Publishing.
2. Durai, P., M. Batool, and S.J.M.D. Choi, Structure and effects of cyanobacterial
lipopolysaccharides. 2015. 13(7): p. 4217-4230.
3. Mantzouki, E., et al., Understanding the key ecological traits of cyanobacteria as a basis
for their management and control in changing lakes. 2016. 50: p. 333-350.
4. Buratti, F.M., et al., C yanotoxins: producing organisms, occurrence, toxicity, mechanism
of action and human health toxicological risk evaluation. 2017. 91: p. 1049-1130.
5. Machado, J., et al., Effects of microcystin-LR and cylindrospermopsin on plant-soil
systems: A review of their relevance for agricultural plant quality and public health.
2017. 153: p. 191-204.
6. Meriluoto, J., L. Spoof, and G.A. Codd, Handbook of cyanobacterial monitoring and
cyanotoxin analysis. 2017: John Wiley & Sons.
7. Schneider, M. and L.J.E.S.E. Bláha, Advanced oxidation processes for the removal of
cyanobacterial toxins from drinking water. 2020. 32(1): p. 94.
8. Ibelings, B.W., et al., CYANOCOST special issue on cyanobacterial blooms: synopsis—a
critical review of the management options for their prevention, control and mitigation.
2016. 50: p. 595-605.
9. Westrick, J.A., et al., A review of cyanobacteria and cyanotoxins removal/inactivation in
drinking water treatment. 2010. 397: p. 1705-1714.
10. Schwarzenbach, R.P., P.M. Gschwend, and D.M.J.E.o.c. Imboden, Indirect photolysis:
reactions with photooxidants in natural waters and in the atmosphere. 2002: p. 655-686.
11. Von Sonntag, C. and U.J.I.P. Von Gunten, London, Reactions of hydroxyl and peroxyl
radicals. Chemistry of ozone in water and wastewater treatment: from basic principles to
applications. 2012: p. 225-248.
12. Schneider, J., et al., Understanding TiO2 photocatalysis: mechanisms and materials.
2014. 114(19): p. 9919-9986.
13. Chen, X. and S.S.J.C.r. Mao, Titanium dioxide nanomaterials: synthesis, properties,
modifications, and applications. 2007. 107(7): p. 2891-2959.
14. Li, M., et al., Ultrathin Anatase TiO2 Nanosheets for High‐Performance Photocatalytic
Hydrogen Production. 2017. 13(16): p. 1604115.

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15. Alshehri, A. and K. Narasimharao, PtOx-TiO2 anatase nanomaterials for photocatalytic
reformation of methanol to hydrogen: effect of TiO2 morphology. Journal of Materials
Research and Technology, 2020. 9(6): p. 14907-14921.
16. Miklos, D.B., et al., Evaluation of advanced oxidation processes for water and
wastewater treatment–A critical review. 2018. 139: p. 118-131.
17. Rosenfeldt, E.J., et al., Comparison of the efficiency of OH radical formation during
ozonation and the advanced oxidation processes O3/H2O2 and UV/H2O2. 2006. 40(20):
p. 3695-3704.
18. Liu, J., et al., Effectiveness and intermediates of microcystin-LR degradation by UV/H 2
O 2 via 265 nm ultraviolet light-emitting diodes. 2017. 24: p. 4676-4684.
19. He, X., et al., Destruction of cyanobacterial toxin cylindrospermopsin by hydroxyl
radicals and sulfate radicals using UV-254 nm activation of hydrogen peroxide,
persulfate and peroxymonosulfate. 2013. 251: p. 160-166.
20. Antoniou, M.G., et al., Enhancing photocatalytic degradation of the cyanotoxin
microcystin-LR with the addition of sulfate-radical generating oxidants. 2018. 360: p.
461-470.
21. Tran, N. and P.J.J.o.e.m. Drogui, Electrochemical removal of microcystin-LR from
aqueous solution in the presence of natural organic pollutants. 2013. 114: p. 253-260.
22. Schneider, O.M., et al., Photocatalytic degradation of microcystins by TiO2 using UV-
LED controlled periodic illumination. 2019. 9(2): p. 181.
23. Organization, W.H., Manganese in drinking water: background document for
development of WHO guidelines for drinking-water quality. 2021, World Health
Organization.
24. Organization, W.H., Guidelines for drinking-water quality. Vol. 1. 2004: World Health
Organization.
25. Von Gunten, U., J.J.E.s. Hoigne, and technology, Bromate formation during ozonization
of bromide-containing waters: interaction of ozone and hydroxyl radical reactions. 1994.
28(7): p. 1234-1242.
26. Alqurashi, G.K., A. Al-Shehri, and K.J.R.A. Narasimharao, Effect of TiO 2 morphology
on the benzyl alcohol oxidation activity of Fe 2 O 3–TiO 2 nanomaterials. 2016. 6(75): p.
71076-71091.
27. Kasuga, T., et al., Titania nanotubes prepared by chemical processing. 1999. 11(15): p.
1307-1311.
28. Jin, Y., et al., Application of N-TiO2 for visible-light photocatalytic degradation of
Cylindrospermopsis raciborskii—More difficult than that for photodegradation of
Microcystis aeruginosa? 2019. 245: p. 642-650.

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