Low-tech Photocatalysts for Solar Water Disinfection (SODIS)

by

Sarah Marie Larlee

A thesis submitted in conformity with the requirements for the degree of Master of Applied Science Graduate Department of Civil Engineering

University of Toronto

© Copyright by Sarah Marie Larlee (2017)

ii

Low-tech Photocatalysts for Solar Water Disinfection (SODIS) Sarah Marie Larlee

Master of Applied Science

Graduate Department of Civil Engineering

University of Toronto

2017

Abstract Solar water disinfection (SODIS) is a household water treatment technique that involves adding

microbiologically contaminated water to clear bottles and leaving them in full sunlight for at

least 6 hours. While SODIS is a low-cost technique that requires few resources, it is a time-

consuming process. In order to make SODIS more effective, this work investigated the use of

low-tech photocatalysts. Initial experiments examined the ability of fired clay coated with TiO2

and urea to remove colour from a methylene blue solution, but results indicated that bare fired

clay performed similarly to the coated pieces. Therefore, different bare clays were investigated

further as photocatalysts for SODIS. One of the clays, Low Red, was able to inactivate E. coli

within 1 hour of sunlight exposure. Based on the results of these experiments, certain fired clays

may be able to serve as photocatalysts for SODIS.

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Acknowledgments

First and foremost, thank you to my wonderful supervisor, Dr. Susan Andrews. Your kindness and

constant support in this amazing project that we came up with has been one of the most positive

and formative experiences of my life. Thank you to Dr. Ron Hofmann for being the second reader

of my thesis and for helping to make my work better and to Dr. Robert Andrews for his passion

for everything water-related. Funding for this work was generously provided by NSERC CGS-M

and OGS.

The DWRG has an amazing lab support group for which I am very grateful. Thank you to Kerry

Evans-Tokaryk for her support and our meaningful talks about science and everything else. To Liz

Taylor-Edmonds for her knowledge on all things E. coli and to Jim Wang for all his help in the

lab. A big thank you also to Jennifer Lee for making everything in our group run smoothly.

I was fortunate to have the most amazing summer student to help me with my work: Jingyi Han.

Thank you for your help and for being my second (and often more useful) brain during the final

summer of my research.

I am incredibly grateful to my DWRG family for all the amazing experiences we’ve shared. In

particular, thank you to Corinne, Yijing, and Caroline for being there for me. An especially big

thank you to Nathan for making the final year of my MASc very special. Thank you also to the

other Environmental Engineering graduate students who helped make the last two years such a

wonderful experience: to Marisa for being a worthwhile distraction and to Leandra for being my

Alberta friend out east. I would also like to extend a huge thank you to my TiO2 (and life) mentor,

Steph Gora. Your guidance, along with that from Susan, has been so influential on my life. You

are both incredibly amazing women and I admire everything each of you has accomplished. Long

live the S club!

Finally, I cannot begin to say how much I appreciate the constant encouragement I’ve received

from my parents. Thank you to my engineer father who was always eager to help with project

ideas and to my mother who got excited for me when my results went well and listened when they

didn’t. I couldn’t have done this without you two.

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Table of Contents

Abstract ........................................................................................................................................... ii

Acknowledgments.......................................................................................................................... iii

Table of Contents ........................................................................................................................... iv

List of Tables ................................................................................................................................ vii

List of Figures ..................................................................................................................................x

List of Acronyms ......................................................................................................................... xiii

Introduction and research objectives ...........................................................................................1

1.1 Research objectives and rationale ........................................................................................1

1.2 Chapter descriptions.............................................................................................................2

Literature review .........................................................................................................................3

2.1 The need for clean drinking water .......................................................................................3

2.2 SODIS basics and enhancements .........................................................................................4

2.3 Titanium dioxide as a photocatalyst ....................................................................................8

2.3.1 Electronic band theory and the photocatalytic generation of oxidizing species ......9

2.3.2 Solar-active TiO2 ...................................................................................................11

2.3.3 Immobilization of TiO2 ..........................................................................................15

2.3.4 Chemical probes to evaluate photocatalysts ..........................................................16

2.4 Disinfection in solar UV/TiO2 processes ...........................................................................17

2.5 The use of clay in water treatment .....................................................................................18

2.6 Research gaps.....................................................................................................................20

Materials and methods ..............................................................................................................21

3.1 Materials ............................................................................................................................21

3.2 Experimental protocols ......................................................................................................23

3.2.1 Preparation of clays................................................................................................23

3.2.2 Preparation of coated clays ....................................................................................24

3.2.3 Exposure of dyes, E. coli, TPA, and KI to irradiated clay photocatalysts .............25

3.3 Experimental QA/QC.........................................................................................................29

Low-tech C- and N-doped TiO2 for SODIS ..............................................................................30

4.1 Introduction and objective .................................................................................................30

4.2 Experimental ......................................................................................................................32

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4.2.1 Materials ................................................................................................................32

4.2.2 Clay substrate preparation......................................................................................32

4.2.3 Immobilized C-N/TiO2 procedure .........................................................................33

4.2.4 Material characterization .......................................................................................36

4.2.5 Solar simulator system set-up ................................................................................36

4.2.6 Adsorption and photocatalytic activity testing ......................................................37

4.3 Results and discussion .......................................................................................................38

4.3.1 Material characterization .......................................................................................38

4.3.2 Analysis of photocatalytic activity .........................................................................40

4.3.3 Bare Clay Experiments ..........................................................................................43

4.4 Summary ............................................................................................................................45

Fired clay as a photocatalyst for SODIS ...................................................................................46

5.1 Introduction and objective .................................................................................................46

5.2 Experimental ......................................................................................................................48

5.2.1 Materials ................................................................................................................48

5.2.2 Clay substrate preparation......................................................................................49

5.2.3 Material characterization .......................................................................................51

5.2.4 Solar simulator system set-up ................................................................................51

5.2.5 Model dye testing ...................................................................................................52

5.2.6 Bacterial preparation and testing ...........................................................................53

5.2.7 Hydroxyl radical and photogenerated electron hole detection ..............................54

5.3 Results ................................................................................................................................55

5.3.1 Clay characterization .............................................................................................55

5.3.2 Adsorption time requirements................................................................................56

5.3.3 Model dye colour removal .....................................................................................59

5.3.4 Regeneration testing with methylene blue .............................................................63

5.3.5 E. coli inactivation .................................................................................................64

5.3.6 Detection and quantification of hydroxyl radicals and photogenerated electron

holes .......................................................................................................................66

5.4 Summary ............................................................................................................................70

Conclusions ...............................................................................................................................71

6.1 Low-tech C- and N-doped TiO2 for SODIS ......................................................................71

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6.2 Fired clay as a photocatalyst for SODIS ............................................................................71

6.3 Recommendations for future work ....................................................................................72

References .................................................................................................................................74

Appendices ................................................................................................................................84

8.1 Calibration curves ..............................................................................................................84

8.2 Experimental data for Chapter 4 ........................................................................................87

8.3 Experimental data for Chapter 5 ........................................................................................91

8.4 Statistical analysis for Chapter 5......................................................................................101

8.5 Additional information for Chapter 5: Determining the effect of clay weight on MB

colour removal .................................................................................................................106

8.5.1 Methods................................................................................................................106

8.5.2 Results ..................................................................................................................108

8.5.3 Raw Data ..............................................................................................................112

8.6 Clay composition data......................................................................................................114

8.7 Clay safety data sheets from Tucker’s Pottery Supplies ..................................................115

8.8 Experimental procedures .................................................................................................120

8.8.1 Preparation of clays..............................................................................................120

8.8.2 Preparation of coated clays ..................................................................................121

8.8.3 Organic dye concentration measurements ...........................................................122

8.8.4 E. coli culture preparation and enumeration ........................................................124

8.8.5 Hydroxyl radical detection procedure ..................................................................128

8.8.6 Detection and quantification of photogenerated electron holes procedure ..........131

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List of Tables

Table 2-1: Reduction of common bacteria, viruses, and protozoa found in drinking water after

application of SODIS (adapted from Eawag/Sandec, 2016) ........................................................ 7

Table 2-2: Properties of Anatase and Rutile (adapted from Hurum et al., 2003) ........................... 8

Table 3-1: Laboratory equipment ................................................................................................. 21

Table 3-2: Reagents ...................................................................................................................... 22

Table 3-3: Clay firing temperatures .............................................................................................. 24

Table 3-4: Coating solution ratios................................................................................................. 24

Table 4-1: Analysis of clay pieces by XRF .................................................................................. 39

Table 4-2: Coating masses (mg), n=4 ........................................................................................... 40

Table 5-1: Clay compositions (provided by Tucker’s Pottery Supply). Components that have

shown photocatalytic activity are italicized. ................................................................................. 49

Table 5-2: Clay firing temperatures .............................................................................................. 49

Table 5-3: Shrinkage rates of different clays used to determine required wet weight to achieve

similar fired weights ..................................................................................................................... 50

Table 5-4: Analysis of clay pieces by XRF (% Content). Components that have shown

photocatalytic activity are italicized. ............................................................................................ 56

Table 5-5: Correlation coefficients between model dye colour removal and XRF data .............. 62

Table 5-6: Hydroxyl radical rate constant .................................................................................... 68

Table 5-7: Photoelectron hole rate constant .................................................................................. 70

Table 8-1: Clay weights used for coated clay samples, n=24 ....................................................... 87

Table 8-2: Average MB absorbance data for 10 min of dark adsorption, n=2 ............................. 87

Table 8-3: Average MB concentration data for 10 min of dark adsorption, n=2 ......................... 87

Table 8-4: Percent MB colour removal for 10 min of dark adsorption, n=2 ................................ 87

Table 8-5: Average MB absorbance data for 10 min of dark adsorption, followed by 60 min of

sunlight exposure, n=2 .................................................................................................................. 88

Table 8-6: Average MB concentration data for 10 min of dark adsorption, followed by 60 min of

sunlight exposure, n=2 .................................................................................................................. 88

Table 8-7: Percent MB colour removal for 10 min of dark adsorption, followed by 60 min of

sunlight exposure, n=2 .................................................................................................................. 88

Table 8-8: Average MB absorbance data for uncoated clay (raw data), n=4 ............................... 89

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Table 8-9: Average MB concentration data for uncoated clay (raw data), n=4 ........................... 89

Table 8-10: Percent MB colour removal for uncoated clay (raw data), n=4 ................................ 89

Table 8-11: Metal analysis for LR clays (raw data), n=4 ............................................................. 90

Table 8-12: Clay weights used in Chapter 5 ................................................................................. 91

Table 8-13: Average dark absorbance data for MB colour removal from adsorption, n=3.......... 92

Table 8-14: Average concentration data for MB colour removal from dark adsorption, n=3 ...... 92

Table 8-15: Average percent MB colour removal from dark adsorption, n=3 ............................. 93

Table 8-16: Average dark absorbance data for AO7 colour removal from adsorption, n=3 ........ 93

Table 8-17: Average concentration data for AO7 colour removal from dark adsorption, n=3 .... 94

Table 8-18: Average percent AO7 colour removal from dark adsorption, n=3 ............................ 94

Table 8-19: Average MB absorbance data from different clays, n=4........................................... 95

Table 8-20: Average MB concentration data from different clays, n=4 ....................................... 95

Table 8-21: Percent MB colour removal from different clays, n=4 ............................................. 95

Table 8-22: Average AO7 absorbance data from different clays, n=4 ......................................... 96

Table 8-23: Average AO7 concentration data from different clays, n=4 ..................................... 96

Table 8-24: Percent AO7 colour removal from different clays raw data, n=4 ............................. 96

Table 8-25: Average MB absorbance regeneration raw data ADS+SUN, n=4 ............................ 97

Table 8-26: Average MB concentration regeneration raw data ADS+SUN, n=4 ........................ 97

Table 8-27: Percent MB regeneration raw data ADS+SUN, n=4 ................................................. 97

Table 8-28: Average E. coli raw data, n=2, log (CFU/mL) .......................................................... 98

Table 8-29: Hydroxyl radical raw data, n=3 ................................................................................. 99

Table 8-30: Hydroxyl radical rate constant data ........................................................................... 99

Table 8-31: Photogenerated hole raw data, n=3 ........................................................................ 100

Table 8-32: Photogenerated hole rate constant data ................................................................... 100

Table 8-33: ANOVA single factor for MB colour removal ....................................................... 101

Table 8-34: ANOVA single factor for AO7 colour removal ...................................................... 101

Table 8-35: T-test results for MB colour removal. Highlighted results indicate significant

difference between clay types. .................................................................................................... 102

Table 8-36: T-test results for AO7 colour removal. Highlighted results indicate significant

difference between clay types. .................................................................................................... 104

Table 8-37: Wet and fired clay weights starting weight of approximately 7.33 g, n=8 ............. 107

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Table 8-38: Shrinkage rates of different clays used to determine required wet weight to achieve

similar fired weights ................................................................................................................... 107

Table 8-39: Wet and fired clay weights, varying starting weights for final fired weight of 5.31 g,

n=8 .............................................................................................................................................. 108

Table 8-40: Clay weights used in Section 8.4............................................................................. 112

Table 8-41: Percent MB colour removal from different clays raw data (0.85 sun, 7.33 starting

weight), n=4 ................................................................................................................................ 112

Table 8-42: Percent MB colour removal from different clays raw data (0.85 sun, varying starting

weights), n=4 .............................................................................................................................. 113

Table 8-43: Normalization by weight of percent MB colour removal (mg/L of MB removal per

gram of clay) ............................................................................................................................... 113

Table 8-44: Clay content (%) data and firing temperature provided with each clay MSDS (Tucker’s

Pottery Supply) ........................................................................................................................... 114

Table 8-45: Clay content (%) data from XRF analysis............................................................... 114

Table 8-46: Starting clay weights to account for shrinkage ....................................................... 120

Table 8-47: Clay firing temperatures .......................................................................................... 121

Table 8-48: Coating solution ratios............................................................................................. 122

Table 8-49: Model dye calibration standard preparation ............................................................ 123

Table 8-50: Experimental Conditions ......................................................................................... 127

Table 8-51: Preparation of HTPA calibration standards............................................................. 129

Table 8-52: Preparation of I2 calibration standards .................................................................... 131

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List of Figures

Figure 2-1: SODIS process (adapted from Eawag/Sandec, 2016) .................................................. 5

Figure 2-2: Valence band (VB) and conduction band (CB) configuration in conductors,

semiconductors, and insulators ..................................................................................................... 10

Figure 2-3: Band gap of TiO2 and the mechanism of hydroxyl radical creation .......................... 11

Figure 2-4: Mechanism of TiO2 photocatalysis: hν1: pure TiO2; hν2: metal-doped TiO2; hν3: non-

metal doped TiO2 (adapted from Zaleska (2008)) ........................................................................ 12

Figure 2-5: Dye Sensitization (adapted from Zhao et al. (2005)) ................................................. 14

Figure 2-6: UV light and visible light wavelengths ...................................................................... 17

Figure 3-1: Photoemission Tech SS150AA solar simulator ......................................................... 26

Figure 4-1: Coated clay pieces ...................................................................................................... 34

Figure 4-2: Overall C-N/TiO2 preparation process ....................................................................... 35

Figure 4-3: Photoemission Tech SS150AA solar simulator ......................................................... 37

Figure 4-4: Methylene blue samples arranged in solar simulator during testing .......................... 37

Figure 4-5: SEM images (x500) of various coatings: a) 1:1 TiO2:urea, b) 2:1 TiO2:urea, c) 3:1

TiO2:urea, d) 4:1 TiO2:urea, e) 1:0 TiO2:urea, f) uncoated clay ................................................... 38

Figure 4-6: SEM images (x500) from different points on the coated clay (2:1 TiO2:urea ratio

shown) ........................................................................................................................................... 39

Figure 4-7: Percent MB colour removal under 10 min of dark adsorption based on TiO2:urea ratio

and calcination temperature, vertical bars represent maximum and minimum for experimental

replicates where n=2 with the exception of the uncoated clay where n=10 ................................. 41

Figure 4-8: Percent MB colour removal under 10 min of dark adsorption followed by 60 min

sunlight exposure based on TiO2:urea ratio and calcination temperature, vertical bars represent

maximum and minimum for experimental replicates where n=2 with the exception of the uncoated

clay where n=10 ............................................................................................................................ 42

Figure 4-9: Bare clay experiments, vertical bars represent the standard deviation of experimental

replicates where n=4 ..................................................................................................................... 44

Figure 5-1: Chemical structures of methylene blue (left) and acid orange 7 (right) .................... 47

Figure 5-2: Clay piece dimensions ............................................................................................... 51

Figure 5-3: Photoemission Tech SS150AA Solar Simulator ........................................................ 52

Figure 5-4: Methylene blue samples arranged in solar simulator during testing .......................... 52

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Figure 5-5: SEM images (x1,000) of various clays: a) Smooth Raku, b) Thompson Raku, c) Low

Red, d) PHB, e) White Sculpture .................................................................................................. 55

Figure 5-6: Adsorption measurements over time for MB, vertical bars represent the standard

deviation of experimental replicates where n=3 ........................................................................... 57

Figure 5-7: Adsorption measurements over time for AO7, vertical bars represent the standard

deviation of experimental replicates where n=3 ........................................................................... 58

Figure 5-8: MB colour removal after 10 min of dark adsorption (ADS) and 10 min of adsorption

followed by 60 min of sunlight exposure (ADS+SUN) for all clay types, vertical bars represent the

standard deviation of experimental replicates where n=4 ............................................................ 60

Figure 5-9: AO7 colour removal after 10 min of dark adsorption (ADS) and 10 min of adsorption

followed by 60 min of sunlight exposure (ADS+SUN) for all clay types, vertical bars represent the

standard deviation of experimental replicates where n=4 ............................................................ 60

Figure 5-10: MB colour removal after 10 min of dark adsorption followed by 60 min of sunlight

exposure for all clay types over 5 regeneration cycles, vertical bars represent the standard deviation

of experimental replicates where n=4 ........................................................................................... 63

Figure 5-11: E. coli inactivation for WSC and LR clays, vertical bars represent maximum and

minimum for experimental replicates where n=2 ......................................................................... 65

Figure 5-12: Agar plates showing E. coli log-removal after 40 min of sunlight exposure with LR

clay (100 (top) and 10-1 dilutions shown) ...................................................................................... 66

Figure 5-13: Hydroxyl radical generation for each clay type, vertical bars represent the standard

deviation of experimental replicates where n=3 ........................................................................... 67

Figure 5-14: Photoelectron hole generation for each clay type, vertical bars represent the standard

deviation of experimental replicates where n=3 ........................................................................... 69

Figure 8-1: MB Calibration Curve ................................................................................................ 84

Figure 8-2: AO7 Calibration Curve .............................................................................................. 85

Figure 8-3: HTPA Calibration Curve ........................................................................................... 85

Figure 8-4: Iodine Calibration Curve ............................................................................................ 86

Figure 8-5: Metals analysis for LR clays, vertical bars represent the standard deviation of

experimental replicates where n=4 ............................................................................................... 90

Figure 8-6: Clay piece dimensions ............................................................................................. 106

Figure 8-7: Percent MB colour removal for each clay type (Scenario 1), vertical bars represent the

standard deviation of experimental replicates where n=4 .......................................................... 108

xii

Figure 8-8: Percent methylene blue colour removal for each clay type (Scenario 2), vertical bars

represent the standard deviation of experimental replicates where n=4 ..................................... 109

Figure 8-9: Comparison between Scenario 1 and Scenario 2 MB colour removal from 10 min of

dark adsorption and from 10 min of dark adsorption followed by 60 min of sunlight exposure 110

Figure 8-10: Fired clay weights plotted with colour removal..................................................... 110

Figure 8-11: Scenarios 1 and 2 MB colour removal normalized by weight ............................... 111

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List of Acronyms

AO7 Acid orange 7

AOP Advanced oxidation process

C-N/TiO2 Carbon- and nitrogen-doped TiO2

e- electron

E. coli Escherichia coli

h+ Photogenerated hole

hr Hour

HTPA 2-hydroxyterephthalic acid

HWT Household water treatment

OD600 Optical density at 600 nm

PET Polyethylene terephthalate

MB Methylene blue

MDGs Millennium Development Goals

min Minutes

nm Nanometers

ROS Reactive oxygen species

SDGs Sustainable Development Goals

SEM Scanning Electron Microscope

SODIS Solar water disinfection

TiO2 Titanium dioxide

TPA Terephthalic acid

UV Ultraviolet light

UV254 UV light with wavelength of 254 nm

UVA Ultraviolet A (315 nm to 400 nm)

UVB Ultraviolet B (280 nm to 315 nm)

UVC Ultraviolet C (100 nm to 280 nm)

WHO World Health Organization

XRF X-ray Fluorescence

S. M. Larlee 1

Chapter 1 Introduction and research objectives

Introduction and research objectives It is estimated that 663 million people worldwide do not have access to an improved water supply

(WHO/UNICEF, 2015), that is, a water supply that is protected from contamination. Those living

in poor and rural areas are generally more affected than those living in affluent regions. Without

access to clean water, there is a risk of diarrhoeal disease, which kills an estimated 525,000

children under five each year (WHO, 2017). To improve the microbiological quality of water and

reduce diarrhoeal disease, point-of-use water treatment techniques, such as solar water

disinfection, or SODIS, can be used. SODIS is particularly viable in regions that receive long

periods of intense sunlight. In its simplest form, SODIS involves adding potentially contaminated

water to clear plastic or glass bottles and leaving them in the sunlight for a minimum of 6 hr

(Eawag/Sandec, 2016). This method is effective because the optical and heating properties of the

sunlight disinfect the water (McGuigan et al., 1998). However, SODIS is a time-consuming

process and regrowth of bacteria is possible. Therefore, this research investigates the use of clays,

some coated with carbon- and nitrogen-doped TiO2, as low-tech photocatalysts for SODIS. By

using photocatalysts, it was hypothesized that a more complete inactivation of microorganisms

would occur and that less sunlight exposure would be required. Throughout this work, the

complexity of the processes used to make the photocatalysts was considered as another goal of this

work was to create a photocatalyst that can be prepared by SODIS users.

1.1 Research objectives and rationale The overall goal of this research is to find a low-tech photocatalyst that can be prepared and used

where SODIS is currently practiced. More specifically, the objectives are as follows:

1. To prepare a low-tech photocatalyst using readily-available materials

2. To test the photocatalysts using model dyes as a preliminary test to determine

photocatalytic ability

3. To test the most promising photocatalysts with E. coli and determine their disinfection

ability

4. To determine the photocatalytic mechanism of the materials

S. M. Larlee 2

1.2 Chapter descriptions x Chapter 2 provides relevant background information on the need for low-cost, point-of-

use drinking water systems, SODIS practices, TiO2 as a possible photocatalyst for SODIS,

and other possible photocatalysts for SODIS, including metal oxides frequently found in

clay.

x Chapter 3 is an overview of the materials and methods used for the experiments that were

conducted.

x Chapter 4 summarizes the ability of carbon- and nitrogen-doped TiO2 fixed to clay

substrates to remove colour from a methylene blue solution. The characterization of the

materials is presented and the use of the materials as a photocatalyst for SODIS is

discussed.

x Chapter 5 discusses the results of experiments using different types of fired clay as a

photocatalyst for SODIS. The ability of the different clays to remove colour from

methylene blue and acid orange 7 solutions, as well as inactivate E. coli, are discussed. In

addition, using the TPA test, the formation of hydroxyl radicals are investigated.

x Chapter 6 summarizes the major research findings and presents recommendations for

future research.

S. M. Larlee 3

Chapter 2 Literature review

Literature review The following chapter outlines the current state of global access to clean water and how solar water

disinfection, or SODIS, can address this issue on a household scale. Improvements for SODIS will

be discussed, such as the use of solar-active photocatalysts, and how readily available and practical

the improvements are will be considered.

2.1 The need for clean drinking water Infectious agents spread through water contaminated with faeces can cause diarrhoeal disease.

Therefore, access to clean water is crucial. Each year, 1.7 billion cases of diarrhoeal disease occur,

and 525,000 children under five die annually from this preventable and treatable disease (WHO,

2017). Diarrhoea kills more children than AIDS, malaria, and measles each year, combined

(UNICEF/WHO, 2009). It deprives the body of the nutrients and fluids it requires such that

dehydration is the most severe threat posed by diarrhoeal (WHO, 2017). To reduce the number of

cases of diarrhoeal disease, access to safe drinking water, as well as improved sanitation and proper

handwashing techniques, are crucial. Water treatment plants and other common water treatment

techniques provide developed countries with adequate water treatment. However, these options

may not be available to developing nations where resources are scarce and for whom the cost is

prohibitive. In particular, huge disparities exist and those in poor and rural communities are more

seriously affected (United Nations, 2016; WHO/UNICEF, 2015).

The Millennium Development Goals (MDGs) were created in September of 2000 to address goals

in the following areas by 2015: poverty, hunger, universal education, health conditions, lack of

education, gender inequality, and environmental sustainability (WHO, 2015). Included in

environmental sustainability was MDG target 7c, which focused on halving the proportion of

people without sustainable access to safe drinking water and basic sanitation. This target was met

in 2010, and in 2015 91% of the global population used an improved drinking water source

(WHO/UNICEF, 2015). This was an 11% increase from 2000. Although this is a positive result,

663 million people still do not have access to an improved water supply (WHO/UNICEF, 2015).

S. M. Larlee 4

Since the target end-date for the MDGs was 2015, a new set of goals was established called the

Sustainable Development Goals (SDGs). There are 17 SDGs which comprise 169 targets and aim

to address urgent global challenges before 2030. Goal 6 of the SDGs is to “ensure availability and

sustainable management of water and sanitation for all” (United Nations, 2016), which includes

achieving universal and equitable access to safe and affordable drinking water (United Nations,

2015).

While providing reliable, clean, piped water to each home is ideal, interventions to treat water at

the household level can provide a promising approach to reducing the health risks associated with

consuming unsafe water (Clasen et al., 2015). Point-of-use water treatment, or household water

treatment (HWT), can significantly improve the microbiological quality of the water and empower

communities without access to a piped supply to treat water in their homes. These treatment

techniques involve treating the water at the household level or at the location where the water will

be used (e.g. schools, community locations) (WHO, 2011). Examples of such treatments that have

shown microbiological efficacy and diarrheal disease reduction are (Sobsey et al., 2008):

x Chlorination,

x Combined coagulant and chlorine,

x Ceramic filters,

x Biosand filters, and

x SODIS.

The HWT that is chosen must satisfy the following criteria to be accepted by the community: low

cost, east to use, and sustainable (i.e. not require products that are difficult or costly to obtain)

(McGuigan et al., 2012).

2.2 SODIS basics and enhancements Solar water disinfection (SODIS) is considered a viable HWT. However, since it does not rely on

a commercially manufactured product, nor is there a manufacturing corporation to fund advertising

campaigns to promote the technique, SODIS isn’t considered as often as other HWT techniques

like chlorination or filtration (McGuigan et al., 2012). This method, which is based on the

inactivation of pathogens in water by solar irradiation, was discovered by Professor Aftim Acra in

1980. In the work performed by Prof. Acra, solutions with glucose and salts for rehydration were

S. M. Larlee 5

prepared with contaminated water and exposed to sunlight. After approximately one hour, a zero

coliform count/mL was achieved (Acra et al., 1980).

To standardize SODIS, the Swiss Federal Institute of Aquatic Science and Technology (Eawag)

developed the SODIS method which is shown in Figure 2-1. First, water is added to bottles that

have been thoroughly cleaned. The preferred bottles are clear 2 L PET plastic or glass bottles. If

the water is turbid, the sunlight will not be able to penetrate it to achieve disinfection, and another

disinfection technique should be investigated. The bottles should be left in the open sunlight for 6

hr during intense periods of sun, or for two days under cloudy conditions; SODIS should not be

performed during rainy conditions. Locations where the bottles will not be disturbed, such as

rooftops, are recommended (Eawag/Sandec, 2016). Once the required time has elapsed, the water

should be stored in the SODIS bottles until consumption to reduce the risk of re-contaminating the

water, but it should be consumed within 24 hr to prevent regrowth of bacteria (McGuigan et al.,

2012). Eawag estimates that 5 million people currently use SODIS to disinfect their water

(Eawag/Sandec, 2016).

Figure 2-1: SODIS process (adapted from Eawag/Sandec, 2016)

Sunlight is capable of causing cellular damage to pathogens and disinfecting water because of its

thermal and optical properties. Joyce et al. (1996) investigated the thermal contribution of sunlight

to disinfect water contaminated with E. coli. Under full Kenyan sunshine, water is able to reach a

S. M. Larlee 6

maximum temperature of 55°C, and, after 7 hr, complete disinfection will occur, even in highly

turbid (200 NTU) water. As a follow-up to this work, McGuigan et al. (1998) examined the

inactivation effect of heat and light, both in isolation and in combination. When only the optical

inactivation was examined, inactivation of E. coli occurred even in highly turbid water (200 NTU)

and at low irradiances (10 mW/cm2). Thermal inactivation is only significant when the water

reaches 45°C; below this temperature E. coli inactivation is negligible. At temperatures above

45°C, there is a synergistic effect between the thermal and optical inactivation processes.

Therefore, at or above 55°C, the disinfection is due to thermal inactivation. If the water

temperature is between 45°C and 55°C, complete inactivation cannot occur by thermal activation

alone; high irradiation of at least 70 mW/cm2 is required and the water must not be highly turbid.

It should be noted that PET bottles, the main bottles used for SODIS, block out UVB (280–320

nm) wavelengths, in which case the optical inactivation mechanisms result from UVA and visible

light (McGuigan et al., 1998). While these wavelengths do not modify DNA bases directly, they

aid in the formation of reactive oxygen species (ROS), which will cause damage to DNA

(McGuigan et al., 2012).

The pathogen removal capacity and efficiency of traditional SODIS has been investigated in

numerous studies (Berney et al., 2006; Boyle et al., 2008; Fujioka and Yoneyama, 2002; Kehoe et

al., 2004; McGuigan et al., 1998; Reed, 1997; Smith et al., 2000; Sommer et al., 2014) and has

been shown to be capable of inactivating various bacteria, viruses, and protozoa found in drinking

water (Eawag/Sandec, 2016).

Table 2-1 shows the time required to reduce each of these contaminants by 99.9%. Bacteria are a

major contributor to diarrhoeal disease, so it is important that SODIS be able to achieve a high

level of inactivation. The efficacy of the SODIS method depends on the type and origin of the

pathogenic organism, the intensity of the sunlight, the size and type of bottle used, the turbidity

and organic content of the water, the place and position of the bottle, and the oxygen content and

temperature of the water (Eawag/Sandec, 2016).

S. M. Larlee 7

Table 2-1: Reduction of common bacteria, viruses, and protozoa found in drinking water

after application of SODIS (adapted from Eawag/Sandec, 2016)

Bacteria Health Impact/Indicator Log reduction after 6 hr Escherichia coli Indicator of water quality 2 - 5 Vibrio cholera Cholera 3 - 5 Salmonella species Typhus 2 - 4 Campylobacter jejuni Dysentery > 4 Viruses Bovine rotavirus Diarrhoea, dysentery 0.5 - 1 Polio virus Polio Very low Adenovirus Respiratory illness Very low Protozoa Giardia species Giardiasis 2 - > 3 Cryptosporidium species Cryptospoidiosis 0.3 - > 0.4

Although it is a time-consuming process, SODIS is a simple to use, low-cost technology that is

effective against many pathogenic bacteria. Clasen et al. estimated that SODIS has the lowest cost

amongst all other household-based interventions, with a cost of $0.63/year (2007). However, due

to its reliance on sunlight, SODIS is thought to be a viable technique only in regions between 35

degrees north and south of the equator (Eawag/Sandec, 2016). This is less of a limitation than it

appears because a large portion of the world’s population that lacks access to clean drinking water

lives in areas with constant and intense solar radiation (Mbonimpa et al., 2012).

A number of household techniques have been investigated to improve SODIS including covering

the underside of the bottles with aluminum foil (Kehoe et al., 2001), painting the underside of the

bottles black (Sommer et al., 2014), or shaking the bottle of water before closing it to augment the

dissolved oxygen content (Reed, 1997). In addition, Wilson and Andrews (2011) examined the use

of Moringa oleifera seed emulsion to clarify and decolorize source waters as a pretreatment for

SODIS. However, even with these amendments, SODIS remains a very time-intensive process.

The SODIS manual published by Eawag/Sandec (2016) outlines other advanced designs for

SODIS, which include different bottle and bag designs, SODIS reactors, indicators, and additives.

The use of solar reflectors to concentrate the sunlight can also accelerate the SODIS process

(Eawag/Sandec, 2016). Alternatives to PET bottles have been explored to increase the UV

S. M. Larlee 8

transmittance and treat larger volumes of water such as SODIS bags, which are less bulky and

easier to transport than empty bottles. Electronic and chemical indicators that measure UV

radiation and that produce a signal when the required dose has been reached can provide a more

visual way of determining when the water is sufficiently treated.

Additives have also been suggested as an improvement for SODIS, such as the addition of lemon

or lime juice, hydrogen peroxide, and copper to reduce treatment time (Fisher et al., 2008). Other

additives that have received significant interest are photocatalysts. Of the photocatalysts already

explored, titanium dioxide (TiO2) shows the most promise due to its availability, non-toxicity, high

efficiency, and stability over a wide pH range (Chen et al., 2010). It is also the most investigated

of the photocatalysts used for water treatment applications (McGuigan et al., 2012). The use of

TiO2 as a photocatalyst for water purification will be discussed further in the following sections.

2.3 Titanium dioxide as a photocatalyst Commonly used as a white pigment because of its high refractive index, TiO2 is found in many

products, including paints, cosmetics, sunscreens, toothpaste, and food (Sucher et al., 2012).

Additionally, TiO2 can be used in environmental applications due to its strong oxidation and

reduction ability. This phenomenon was first discovered by Fujishima et al. (1972), who reported

that the photoinduced decomposition of water occured on TiO2 electrodes. Since then, TiO2 has

been used for self-cleaning surfaces, dye-sensitized solar cells, and antipollution coatings (Sucher

et al., 2012). It has also been used for water treatment applications at the small scale (Gelover et

al., 2006; Meichtry et al., 2007) and at an industrial scale (Verma et al., 2014; Zhang et al., 2014).

There are three polymorphs of TiO2: brookite, anatase, and rutile. However, only anatase and rutile

are used in photocatalytic applications. Antase and rutile have different properties, and therefore

perform differently when used as photocatalysts (Hanaor and Sorrell, 2011). The properties of

anatase and rutile are shown in Table 2-2.

Table 2-2: Properties of Anatase and Rutile (adapted from Hurum et al., 2003)

Form of TiO2 Size (nm) Band Gap (eV) Excitation Wavelength (nm)

Anatase 50 3.2 385

Rutile 200 3.0 410

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Anatase is considered the more photochemically active TiO2 species, even though it has a larger

band gap. This is likely because it has a lower recombination rate than rutile (Hurum et al., 2003).

When heated to high enough temperatures, anatase transforms irreversibly to rutile. In general,

this occurs in air at approximately 600°C (Ghosh et al., 2003; Li et al., 2005). However, the

transition temperatures can vary depending on the method used to determine the transition

temperature, raw materials, and processing methods. The most commonly used commercial TiO2

powder is Evonik Aeroxide ® P25, which is a combination of anatase (70%–80%) and rutile

(20%–30%). It has a specific surface area of 50 m2/g and is commonly used as a benchmark against

which new photocatalysts are compared (Castro and Durán, 2015). While the use of a mixture of

rutile and anatase might seem contradictory as anatase is the more photoactive species, Hurum et

al. (2003) determined that the presence of rutile increases the photoactivity of anatase. This is

because when rutile and anatase are used together in mixed-phase TiO2, nanoclusters with small

rutile crystallites interwoven with anatase crystallites are formed. At the transition points between

the rutile and the anatase, rapid electron transfer can occur, producing an effective photocatalyst

(Hurum et al., 2003).

2.3.1 Electronic band theory and the photocatalytic generation of oxidizing species

Most solid materials can be described as having two energetic bands: a valence band (VB), and a

conduction band (CB). Electrons reside in the VB, but once excited, they are promoted to the CB.

The energy difference between these bands is called the band gap. In conductors, the band gap is

either small or nonexistent, so electrons can flow easily between the VB and the CB. In contrast,

insulators have a large band gap, so electrons cannot flow from the VB to the CB. The band gap

in semiconductors is moderately sized (0 to 7 eV), which enables the promotion of electrons from

the VB to the CB when energy, generally from UV or visible light, is applied. The three types of

materials are shown in Figure 2-2.

S. M. Larlee 10

Figure 2-2: Valence band (VB) and conduction band (CB) configuration in conductors,

semiconductors, and insulators

The band gap of TiO2, a common semiconductor, is 3.2 eV (Hanaor and Sorrell, 2011). This band

gap can be exceeded with light with wavelengths between 290 – 380 nm, which is in the UV light

range(Hanaor and Sorrell, 2011). Once the semiconductor is illuminated with photons of energy

equal to or greater than that of the band gap, electrons (e-) move from the VB to the CB, leaving

an electron hole (h+) in the VB. The electron and the electron hole can recombine and dissipate the

energy as heat or react with electron donors and acceptors in the water to form oxidizing agents

such as hydroxyl radicals (OH•) or superoxide (O2•-). At the anodic sites, the light induced holes

and water molecules form hydroxyl radicals and hydrogen ions (H+) (Jaeger and Bard, 1979), as

shown in Equation 2.1

H2O+ h+→ H++OH• 2.1

The electrons at the cathodic sites reduce molecular oxygen to superoxide (Skorb et al., 2008), as

show in Equation 2.2. The photocatalytic generation of oxidizing species is shown in Figure 2-3.

O2+ e-→ O2•- 2.2

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Figure 2-3: Band gap of TiO2 and the mechanism of hydroxyl radical creation

The UV/TiO2 system is considered an advanced oxidation process (AOP), or a process that

produces hydroxyl radicals to remove organics and disinfect water. Other AOPs that are employed

in drinking water treatment include UV/H2O2, UV/O3, and UV/Cl2.

2.3.2 Solar-active TiO2 As TiO2 in its pure form can be activated only under UV light, recent research has focused on how

to make it less energetic active under solar light. The most common methods will be discussed in

the following section.

2.3.2.1 Doped TiO2 Band gap engineering is a strategy to make TiO2 visible light active and involves narrowing the

band gap by elevating the VB maximum or lowering the CB minimum so that activation can occur

with visible light (Wang et al., 2013). An important band gap narrowing strategy is doping, which

involves incorporating another atom into the structure of the TiO2 to create new electronic states

and optical transitions that are not present in pure TiO2. Doping TiO2 is the most intensively

investigated way of realizing the visible light response of TiO2 (Chen et al., 2010). There are two

purposes of doping another element (or elements) into the structure of TiO2. First, doping causes

CB electrons or VB holes to be trapped in defect states, which slows charge recombination (Park

et al., 2013). Additionally, doping enables visible light absorption by creating defect states in the

band gap. These defect states mean that electronic transitions from the VB to defect states, or from

defect states to the CB can occur under lower energy light (i.e. sunlight) (Park et al., 2013).

S. M. Larlee 12

Dopants are generally divided into metal ions and non-metal ions and the dopant selected is

important to the overall photocatalytic activity (Park et al., 2013).

Of the metal dopants, silver (Lee et al., 2005), iron (Vereb et al., 2012), and platinum (Li and Li,

2002), among others, have been examined. Generally, transition metals and noble metals are

examined as dopants, but the former has been investigated more than the latter (Park et al., 2013).

When TiO2 is doped with a metal, it can exhibit visible light photoactivity because a new energy

level is produced in the band gap by the dispersion of metal nanoparticles in the structure of the

TiO2 (Zaleska, 2008). Electrons require less energy to move from the VB to the new energy level,

as shown in Figure 2-4. Therefore, activation can occur in lower energy light, like sunlight.

Many nonmetals have been investigated as dopants. In particular, ntrogen-doped TiO2 has been

extensively investigated (Asahi et al., 2001; Choi et al., 2007; Liu et al., 2009; Mrowetz et al.,

2004; Yang et al., 2010). Other non-metal dopants that have produced visible light photocatalysts

are carbon (Dong et al., 2011), sulfur (Rockafellow et al., 2009), and boron (In et al., 2007). There

are three main opinions regarding the modification of TiO2 when doped with non-metals: 1) band

gap narrowing; 2) impurity energy levels; and 3) oxygen vacancies (Zaleska, 2008). Generally,

when doped with non-metals, a new energy level is created above the VB in the TiO2 and electrons

need only enough energy to move from the new energy level to the CB to produce oxidative

species, as shown in Figure 2-4.

Figure 2-4: Mechanism of TiO2 photocatalysis: hν1: pure TiO2; hν2: metal-doped TiO2; hν3:

non-metal doped TiO2 (adapted from Zaleska (2008))

S. M. Larlee 13

In addition to monodoping, codoping TiO2 has been investigated as a way to prepare a visible light

activated photocatalyst. By codoping, beneficial synergistic effects due to the interaction between

dopants are often observed (Chen et al., 2010). Many combinations of different elements (both

metals and non-metals) have been investigated, including nitrogen- and sulfur-doped TiO2

(Rengifo-Herrera et al., 2009), manganese- and cobalt-doped TiO2 (Venieri et al., 2014), and

nitrogen- and fluoride-doped TiO2 (Wang et al., 2009). However, among the non-metals

investigated as dopants, carbon- and nitrogen-doped TiO2 materials exhibit superior photocatalytic

activity under sunlight (Chen et al., 2007).

Carbon and nitrogen have been investigated as codopants previously to create a solar-active

photocatalyst. Chen et al. (2007) used urea as the nitrogen source and tetrabutylammonium

hydroxide as the carbon source to produce a powder whose ability to bleach methylene blue was

examined. Nawawi and Nawi (2014) used urea as the source of carbon and nitrogen and examined

the ability of the powder to remove colour from methylene blue and reactive red 4 solutions, as

well as to degrade phenol. In the work done by Cong et al. (2006), a microemulsion-hydrothermal

process was used to prepare a powder and the bleaching of rhodamine B and the removal of 2,4-

dichlorophenol was determined. In this case, the nitrogen source was trimethylamine and

tetrabutyltitanate served as the carbon and titanium source. Wang et al. (2011) used a sol-gel

method where titanium butoxide was the titanium and carbon precursor and nitric acid was the

nitrogen source. In this work, the degradation of sulfanilamide was examined under visible light.

Furthermore, in the work by Wang et al. (2012), L-lysine was used as a source of carbon and

nitrogen to prepare nanoparticles whose ability to degrade methyl orange was analysed.

While many examples of carbon- and nitrogen-doped TiO2 photocatalysts exist, there are gaps

associated with the current body of research. First, little research addresses the issue of removing

the photocatalysts from the water. Additionally, the effect of carbon- and nitrogen-doped TiO2 on

bacterial inactivation has not been widely examined. Since a major concern in developing

countries is microbial contamination, for a photocatalyst to be viable in SODIS applications, the

ability of the materials to inactivate bacteria must be examined. Finally, the ease of production of

the photocatalysts must be considered. Since SODIS is a low (and potentially zero) cost water

treatment technique, photocatalysts that involve complex preparations may be prohibitively

expensive and complicated for users.

S. M. Larlee 14

2.3.2.2 Other methods In addition to doping, other strategies have been used to modify TiO2 to produce a photocatalyst

that is solar active such as surface sensitization by organic dyes (Watanabe et al., 1977; Zhao et

al., 2005) and coupling TiO2 with a narrow-band-gap semiconductor (Serpone et al., 1984; Yu et

al., 2003).

The principle of dye sensitization is as follows: a dye is adsorbed onto the surface of the TiO2 and,

when irradiated, the visible light excites the dye (Zhao et al., 2005). Electrons from the excited

dyes then move into the CB of the TiO2 while the VB of the TiO2 is unaffected, as shown in Figure

2-5. In this process, TiO2 does not absorb light directly; instead, the dye acts as an antennae to

absorb light energy (Wang et al., 2013). Once the electrons are injected into the CB of the TiO2,

they can react with the water to yield oxidizing species. To be considered an acceptable

photosensitizer, the dye must have the following properties: an adsorption spectrum that includes

the visible region; photostable; anchoring groups so that the dye molecules are bonded to the

surface of the TiO2; and the excited state of the dye should be higher in energy than the CB edge

of the TiO2 so that the electron transfer between the excited dye and the CB will be

thermodynamically favourable (Hagfeldt et al., 2010). Organic dyes that have been used frequently

for dye sensitization include thiazines and xanthenes (Wang et al., 2013).

Figure 2-5: Dye Sensitization (adapted from Zhao et al. (2005))

S. M. Larlee 15

Another method to modify TiO2 and create a solar active photocatalyst is coupling TiO2 with a

narrow-band-gap semiconductor with a higher CB than that of TiO2 (Robert, 2007). This method

was first proposed by Serpone et al. (1984) where TiO2 was coupled with CdS and demonstrated

an improvement in water splitting and increased the charge separation for the corresponding CBs

and VBs. Similar to dye sensitization, the narrow-band-gap semiconductor acts as a sensitizer and

is excited under visible light (Robert, 2007). Once excited, the electrons generated at the CB of

the sensitizer are injected into the CB of the TiO2 and react with water for form oxidative species.

For a semiconductor to be a candidate for coupling with TiO2, it must have a strong absorption in

the visible region, have a higher CB minimum and VB maximum than the TiO2, and the electron

injection into the narrow-band-gap semiconductor must be fast and efficient (Liu et al., 2010).

Examples of metal oxide semiconductors that have been coupled with TiO2 include CdS, CdSe,

Bi2S3, SnO2, WO3, and Cu2O (Robert, 2007).

2.3.3 Immobilization of TiO2 As TiO2 is a nanomaterial, removing it from the water is challenging due to the small size of the

particles. Filters with small pores can be used, but they are expensive and difficult to acquire,

limiting the application of TiO2 in remote or developing areas (Meichtry et al., 2007). It is

beneficial to use immobilized TiO2 because it is easier to reuse and there is no need to separate it

from the water. Immobilized TiO2 can also achieve better light penetration than elevated

concentrations of suspended material through which light cannot effectively reach the

photocatalyst (Rincón and Pulgarin, 2003). Consequently, many research groups have investigated

the immobilization of TiO2. Coating the inside of plastic PET bottles, which are generally used for

SODIS, with TiO2 has been examined (Fostier et al., 2008; Meichtry et al., 2007). Similarly, Duffy

et al. (2004) investigated coating plastic inserts that can be added to bottles, and Meichtry et al.

(2007) examined coating inexpensive materials such as glass rings, glass rods, and porcelain beads.

It has been observed, however, that attaching the TiO2 to a substrate instead of in suspension

decreases the surface area in contact with the water, which produces fewer active sites for

disinfection. Rincón and Pulgarin (2003) compared suspended TiO2 to immobilized TiO2 and

found that the photocatalytic efficiency of immobilized material varies with the substrate used.

The TiO2 fixed on Nafion® membranes performs similarly to the suspended TiO2, whereas

suspended TiO2 performs better than TiO2 fixed on glass bottles. In addition, Meichtry et al. (2007)

S. M. Larlee 16

noted that an important consideration is the amount of the surface area of the substrate coated in

TiO2 that can be reached by solar light; glass rings were better at harvesting light than glass rods,

beads, or bottles because a larger percentage of the surface area was open to the sunlight.

It is important to consider the long-term stability of the photocatalyst on the support so that no

detachment occurs (Robert et al., 2013). Additionally, the substrate must be able to withstand the

coating process, which may involve high temperatures (Robert et al., 2013).

2.3.4 Chemical probes to evaluate photocatalysts The photocatalytic activity of a photocatalyst is generally tested by measuring its ability to degrade

organic compounds. Model dyes are the most widely used organic compounds due to their rapid

decolorization and ease of detection using spectrophotometric methods, such as UV-vis

spectrophotometers (Bae et al., 2014; Rochkind et al., 2015). However, depending on the

application of the photocatalyst, other model pollutants have been used such as MC-LR

(Likodimos et al., 2013), MS2 (Li et al., 2008), or bacterial pathogens like E. coli and Klebsiella

pneumonia (Venieri et al., 2014)

In a study performed by Bae et al (2014), the ability of a photocatalyst to degrade a model dye was

highly dependent on the dye and the photocatalyst itself. That is, the colour removal observed is

unique to the photocatalyst and dye used. Bae et al. (2014) also found that the dye decolorization

efficiency was not well correlated with the dye mineralization efficiency and did not recommend

generalizing results from specific dye and photocatalysts tests or recommend dye tests for

assessing the activity of visible light photocatalysts. After conducting a review of the literature,

Rochkind et al. (2015) also determined that dyes are not appropriate model compounds for

evaluating photocatalysts and highlighted that some dye degradation may be due to sensitization.

In addition, most commercially available dyes have low purities (70-90%), which can make

comparisons between results measured by different research groups unreliable (Rochkind et al.,

2015).

However, model dyes are still commonly used to assess visible light photocatalysts and provide a

benchmark to which new photocatalysts can be compared. It is recommended that when model

dyes are the only option for assessing activity, dyes that have minimal absorption spectral overlap

S. M. Larlee 17

with the photocatalyst and/or minimal dye sensitization effect should be used (Bae et al., 2014).

The use of multiple dyes is also recommended (Bae et al., 2014). Therefore, in this work, acid

orange 7 (AO7), an anionic dye, and methylene blue (MB), a cationic dye, will be used as they

have different molecular properties and structures. The inactivation of E. coli will also be examined

as the ultimate application of the photocatalysts is to disinfect contaminated water for SODIS

applications.

2.4 Disinfection in solar UV/TiO2 processes The solar spectrum consists of different wavelengths including ultraviolet and visible light, as

shown in Figure 2-6. UV light can be divided into three ranges: UVA, which ranges from 315 nm

to 400 nm; UVB, which ranges from 280 nm to 315 nm; and UVC, which ranges from 200 nm to

280 nm (Bolton, 2001). The biological effect on bacteria differs depending on the type of UV light,

so it is important to treat them as different regions (Jagger, 1985). The majority of UVB radiation

is absorbed by the atmosphere, and, although UVC is considered the most damaging type of UV

radiation, it is entirely filtered out by the atmosphere and does not reach the Earth’s surface.

Included in the UVC range is UV254, considered to be the germicidal wavelength where

microorganisms are killed or mutate through the absorption of radiation by nucleic acids and where

respiration is inhibited (Jagger, 1985). While these effects can be achieved with lower energy UV

light, the doses required are much higher.

Figure 2-6: UV light and visible light wavelengths

S. M. Larlee 18

For SODIS, pathogen inactivation occurs with either UVA or UVB light; UVB light directly

inactivates pathogens by damaging DNA and RNA (Jagger, 1985) while UVA causes the

formation of ROS in water which damage the DNA of the bacteria (Whitlam and Codd, 1986). As

mentioned, UVC does not reach the surface of the Earth and therefore does not contribute to

pathogen inactivation in SODIS. Through the use of staining and flow-cyanometric measurements,

which classify the physiological state of single-celled organisms, Berney et al. (2006) were able

to characterize the loss of cellular functions in E. coli cells under sunlight and UVA light. When

E. coli was irradiated, membrane-function breakdown occured (Berney et al., 2006). In the work

by Bosshard et al. (2009), the effect of SODIS on Shigella flexneri and Salmonella typhimurium

were investigated and the respiratory chain was identified as the likely target of sunlight and UVA

irradiation. With continued irradiation, proteins and enzymes responsible for cellular functions are

damaged and lead to cell inactivation and death (Bosshard et al., 2009). Another finding of this

work was that damage continued to occur during dark storage and the cells could not repair the

damage caused by the light.

The mechanisms leading to cell death or inactivation for the UV/TiO2 process are not fully

understood and the explanations that exist are controversial (Rengifo-Herrera et al., 2013). One

proposed mechanism is that the intracellular coenzyme A is oxidized during the process, inhibiting

cell respiration (Matsunaga et al., 1985). Another proposed mechanism for bacterial inactivation

is that UV/TiO2 causes changes in the cell permeability. This leads to the decomposition of the

cell walls, which allows intracellular constituents to leave the cell, leading to cell death (Saito et

al., 1992). There is also evidence that UVA light can impede the antioxidant defenses of E. coli

and degrade proteins within the cells, which release iron and damage the cell (Kapuscinski and

Mitchell, 1981; Pigeot-Rémy et al., 2011). When discussing how photocatalytic processes

inactivate cells, the synergistic effect of light and ROS is neglected; rather, emphasis is generally

put on how hydroxyl radicals inactivate cells. Instead, it has been proposed that E. coli inactivation

occurs due to the effects of the following three processes: ROS generation, TiO2 nanoparticles

under dark conditions, and the UVA light itself (Rengifo-Herrera et al., 2013).

2.5 The use of clay in water treatment Clay has been used in water treatment for many applications including filtration and as a support

for materials that can disinfect water. The purpose of clay in this research began as a substrate for

S. M. Larlee 19

photocatalysts but transitioned into using clay as a photocatalyst itself. The following section

outlines other applications of clay in water treatment.

The use of ceramic materials to filter water is one of the oldest drinking water treatments, but

recent developments have tried to improve their effectiveness. Ceramic water filters are a low-cost

HWT technique that consist of a pot-shaped ceramic porous media painted with an aqueous silver

nanoparticle solution (van Halem et al., 2009). The ceramic pot filters out pathogens and the silver

nanoparticles inactivate microorganisms (Oyanedel-Craver and Smith, 2008). This technique can

provide significant inactivation of E. coli and protozoan oocysts (van Halem et al., 2009).

However, limitations of this method include relatively high capital cost, a somewhat complex

manufacturing process, and large size and lack of filter durability, which makes transporting the

filters difficult (Ehdaie et al., 2014). As such, Ehdaie et al. (2014) investigated embedding silver

into small ceramic tablets in the form of metallic silver nanopatches as a point of use application.

The tablet was made with clay, water, sawdust, and silver nitrate and when added to water, it

released silver ions that disinfect the water. Silver nitrate was used instead of the silver

nanoparticles used in ceramic water filters as silver nanoparticles are expensive and scarce in areas

that would use ceramic water filters (Ehdaie et al., 2014). Additionally, silver nanoparticles can be

relatively mobile in ceramic porous media, which means that ceramic water filters may not

maintain their effectiveness over time. These tablets were able to achieve a 3-log reduction of E.

coli within 8 hr in 10 L of water and could be used daily for 154 days in the same volume of water.

Natural clays have also been used as a support for TiO2 due to their high adsorption capacity and

low cost (Chong et al., 2010). Hadjltaief et al. (2015) immobilized TiO2 nanoparticles onto two

high surface area materials, natural clay and activated carbon, and examined their ability to degrade

methyl green in wastewaters under UV irradiation. It was noted that the coating of TiO2 onto the

materials resulted in extensive pore blockage and a reduction in adsorption capacity. However, the

TiO2 supported on clay and activated carbon were more active than pure TiO2 powder; the

TiO2/activated carbon and TiO2/natural clay removed 98.6% and 90.2% colour removal in 60 min,

respectively, while the TiO2 powder only achieved 52.8% colour removal during the same time.

Clays have also been introduced into clay-based photocatalysis and have changed the phase of the

semiconductor or to improve the electron and electron hole separation (Liu and Zhang, 2014).

Belessi et al. (2007) studied two TiO2/clay composites (montmorillonite-TiO2 and hectorite-TiO2)

S. M. Larlee 20

and their ability to degrade the herbicide dimethachlor under UV light (λ < 290 nm). In addition

to being removed by sedimentation, the clay materials had better overall removal efficiency per

mass of TiO2 than bare TiO2 produced by a sol-gel method. Zhang et al. (2011) prepared

TiO2/kaolinite composites and examined their removal of acid red G and 4-nitrophenol under UV

light (λ = 253.7 nm). The materials had a higher specific surface area than kaolinite and showed

high photocatalytic activity. Another clay based photocatalyst was prepared by Meshram et al.

(2011), who examined the removal of phenol using a ZnO-bentonite nanocomposite as a

photocatalyst under UV radiation. These clay-based photocatalysts show numerous advantages

over the pure semiconductors: they can be recovered from solution and show enhanced

photocatalytic properties (Liu and Zhang, 2014).

2.6 Research gaps There is a need for a simple, low-cost way to improve SODIS. The focus on doped TiO2 for

harvesting solar light is promising, but more work is required to prepare a doped photocatalyst that

can be created and used where SODIS is practiced. Additionally, there is limited work on the

immobilization of doped TiO2 for SODIS applications. Therefore, the purpose of this work is to

create carbon- and nitrogen-doped TiO2 which will be immobilized on a clay substrates. The

process will be tailored for areas with low-tech facilities so that the doped TiO2 can be made by

SODIS users.

The following sections outline the materials required and the procedure that will be used to create

carbon- and nitrogen-doped TiO2. Urea will be examined as a dopant and the results will be

compared to similar studies in the literature. The ability of the materials to remove colour from a

MB solution will be examined. Based on the results of this study, uncoated clays will also be

investigated as low-tech photocatalysts for SODIS. These uncoated clays will be tested with MB,

AO7, and E. coli. Since a major concern in developing countries is microbial contamination, the

ability of the photocatalysts to inactivate bacteria must be examined to be considered a viable

technique for SODIS applications. Based on the results, the photocatalytic mechanism of the clays

will be investigated.

S. M. Larlee 21

Chapter 3 Materials and methods

Materials and methods This chapter outlines the materials required to prepare carbon- and nitrogen-doped TiO2 and bare

clay photocatalysts. The preparation and testing methods will also be outlined.

3.1 Materials The laboratory equipment and reagents used in the experiments outlined in this document are listed

in Table 3-1: Laboratory equipment Table 3-1 and

Table 3-2, respectively.

Table 3-1: Laboratory equipment

Equipment Manufacturer Product Number

UV-Vis Spectrophotometer Agilent Technologies

(Mississauga, ON) 8453

PET Solar Light Simulator Photo Emission Tech Inc.

(Camarillo, CA) SS150AAA

1100°C Box Furnace

BF51800 Series Muffle

Furnace

Fisher Scientific (Ottawa,

ON) 312060H01

Sybron Thermolyne Furnatol

I Muffle Furnace

Fisher Scientific (Ottawa,

ON) F-A1730

Stir Plates VWR Scientific

(Mississauga, ON)

Standard Multi-Purpose

Stirrer

Analytical Balance OHAUS (Florham Park, NJ) AP2105

Large Capacity Acid

Digestion Vessel 125 mL

PARR Instrument Company

(Moline, IL) 4748

Ultra-sonicator Laval Lab Inc. (Laval, QB) Laborette 17

Biosafety Cabinet Thermo Scientific (Asheville,

NC) SN:10909-B

S. M. Larlee 22

Genie Vortex Mixer Fischer Scientific (Ottawa,

ON) 12-812

Bench Top Shaker Thermo Scientific (Asheville,

NC) 3527

Sterilmatic Autoclave Market Forge Industries

(Everet, MA) 120/208-240

-80°C Freezer Caltec (Mississauga, ON) 8326

CryoELITE™ Cryogenic

Storage Vials (2 mL)

Wheaton Science Products

(Millville, NJ) W985863

Refrigerated Incubator Fischer Scientific (Ottawa,

ON) 815

Biological Safety Cabinet Forma Scientific (Ottawa,

ON) 1126

Cary Eclipse Fluorescence

Spectrophotometer

Agilent Technologies

(Mississauga, ON) G9800A

Heating Stir Plate Thermo Scientific (Asheville,

NC) Cimarec 2

Table 3-2: Reagents

Material or Chemical Manufacturer Product Number

Aeroxide ® TiO2 P25 Sigma-Aldrich (Oakville, ON) 718467

Urea Sigma-Aldrich (Oakville, ON) U5128

Methylene Blue Fisher Scientific (Ottawa, ON) 792949

Acid Orange 7 Fisher Scientific (Ottawa, ON) 195235

Earthenware Low Red Clay Tucker’s Pottery Supply (Richmond Hill,

ON) N/A

Earthenware PHB Clay Tucker’s Pottery Supply (Richmond Hill,

ON) N/A

Smooth Raku Tucker’s Pottery Supply (Richmond Hill,

ON) N/A

S. M. Larlee 23

Thompson Raku Tucker’s Pottery Supply (Richmond Hill,

ON) N/A

White Sculpture Raku Tucker’s Pottery Supply (Richmond Hill,

ON) N/A

E. coli ATCC ® 23631™ Cedarlane Laboratories (Burlington, ON) 23631

Glycerol Simga-Aldrich (Oakville, ON) G5516

Lennox Lysogeny Broth BioShop Canada (Burlington, ON) LBL405.1

Agar BioShop Canada (Burlington, ON) AGR001.500

Dulbecco’s Phosphate

Buffered Saline 10x Sigma-Aldrich (Oakville, ON) D1408

Ringer’s Solution

Calcium chloride

Potassium chloride

Sodium bicarbonate

Sodium chloride

Fisher Scientific (Ottawa, ON)

Caledon Laboratories (Georgetown, ON)

Caledon Laboratories (Georgetown, ON)

Sigma-Aldrich (Oakville, ON)

C77-500

5920-1

7260-1

S5886

Sodium hydroxide pellets Sigma-Aldrich (Oakville, ON) 221465

Terephthalic acid (TPA) Sigma-Aldrich (St. Louis, MO) 185361

2-hydroxyterephthalic acid

(HTPA) Sigma-Aldrich (St. Louis, MO) 752525

Potassium iodide Sigma-Aldrich (Oakville, ON) 746428

Iodine solution (2.5%) Atoma (Llandudno, UK) n/a

3.2 Experimental protocols The experimental procedures used in this thesis are outlined in the following sections, including

how the clays were fired and coated, how they were tested with model dyes and E. coli, and how

hydroxyl radicals and photogenerated holes were measured.

3.2.1 Preparation of clays

Clay was chosen as a substrate for the photocatalytic coating due to its high surface area and

because it is readily available and inexpensive. To prepare the clay pieces, the required amount of

wet clay (6.5 g – 7.5 g) was weighed out based on the clay type and shaped into a 2 cm diameter

S. M. Larlee 24

button-shape. The clay was left to dry for at least 2 days before firing. The day of firing, the muffle

furnace (1100°C Box Furnace BF51800 Series Muffle Furnace, Fisher Scientific) was

programmed to increase by 2°C/min to the manufacturer-specified firing temperature, as shown in

Table 3-3. Clay pieces that required the same firing temperature were fired together.

Table 3-3: Clay firing temperatures

Clay Type Firing Temperature

Smooth Raku (SR) Cone 04 (1060°C)

Thompson Raku (TR) Cone 04 (1060°C)

Low Red (LR) Cone 06 (999°C)

PHB Cone 06 (999°C) or Cone 04 (1060°C)

White Sculpture (WSC) Cone 05 (1046°C)

Once the temperature was reached, the muffle furnace was turned off and the clay pieces cooled

in the furnace for at least 12 hr. The fired clay was weighed after cooling. For more detailed

information on how the clay pieces were prepared, see Section 8.8.1.

3.2.2 Preparation of coated clays

Low red clay was used for the coated clay pieces and was prepared as described in Section 3.2.1.

To make the coating solutions, 10% w/v solutions were prepared with varying weights of TiO2

and urea in 20 mL of Milli-Q® water. Different ratios of TiO2 to urea were prepared as shown in

Table 3-4.

Table 3-4: Coating solution ratios

Ratio (TiO2:urea) Weight of TiO2 (g) Weight of urea (g)

1:1 1.00 1.00

2:1 1.33 0.67

3:1 1.50 0.50

4:1 1.60 0.40

1:0 2.00 -

S. M. Larlee 25

Once the solutions were prepared, they were sonicated for 5 min, then poured into 50 mL beakers.

Two clay pieces were immersed in each slurry for 15 min per side then placed on a drying rack to

allow for excess solution to drip off. Coated clay pieces were left to air dry for one day then dried

at 100°C in the muffle furnace (Sybron Thermolyne Furnatol I Muffle Furnace, Fisher Scientific)

for 1 hour. Once dried, the clay pieces were calcined for 2 hr in the muffle furnace at varying

temperatures (250°C, 300°C, 350°C, 400°C, 450°C). To remove any poorly-adhered coating, the

clay pieces were constantly stirred in 35 mL of Milli-Q® water for 5 min then dried at 100°C for

one hour. After drying, the clay pieces were weighed to determine the weight of the coating. For

more detailed information on coated clay preparation, see Section 8.8.2.

3.2.3 Exposure of dyes, E. coli, TPA, and KI to irradiated clay photocatalysts Model dyes and E. coli were used to test the photocatalytic ability of the clays while TPA and KI

were used to measure hydroxyl radicals and photogenerated holes, respectively. To perform dye,

bacteria, TPA, and KI testing, 35 mL of the required solution was dispensed into a 50 mL beaker,

then placed in the Photoemission Tech SS150AA solar simulator (Camarillo, CA), shown in Figure

3-1. One clay piece was added each beaker, with the exception of the light and dark controls. To

test the dark adsorption of the materials, the beakers were constantly stirred for 10 min in the dark

then samples were analysed. To test the photocatalytic ability of the materials, the beakers were

constantly stirred for 10 min in the dark, and then exposed to sunlight for 60 min for model dyes,

TPA, and KI and 120 min for E. coli. Model dye colour removal and iodine concentration was

determined with the UV-vis spectrophotometer, E. coli inactivation was determined by plating

dilutions on agar plates, and TPA conversion to HTPA was determined with the fluorescence

spectrophotometer. While the solar simulator was in use, dark fabric was used to block ambient

light.

S. M. Larlee 26

Figure 3-1: Photoemission Tech SS150AA solar simulator

3.2.3.1 Organic dye concentration measurements Organic dye measurement procedures were based on work performed by Stephanie Loeb (2013).

According to Beer’s Law, the absorbance of a coloured dye is proportional to its concentration;

higher concentrations will have a higher absorbance. The peak absorption wavelengths using the

UV-vis spectrophotometer are 664 nm and 485 nm for methylene blue (MB) and Acid Orange 7

(AO7), respectively. To determine the concentration of the samples, a calibration curve was

prepared by measuring the absorbance for a series of five solutions with known dye concentrations

(0.625 mg/L, 1.25 mg/L, 2.5 mg/L, 5 mg/L, and 10 mg/L). From the equation of the line, the

concentration of the samples was calculated and, based on the initial concentration (10 mg/L), the

percent colour removal was calculated with Equation 3.1.

% Removal=

initial concentration - concentration at time 't'initial concentration

3.1

For more detailed information on organic dye measurements, see Section 8.8.3.

S. M. Larlee 27

3.2.3.2 E. coli culture preparation and enumeration The procedures used for E. coli preparation and enumeration were adapted from similar works

performed by Stephanie Loeb (2013). An E. coli stock culture (ATCC ® 23631™) was grown

from a seed culture purchased from Cedarlane Laboratories following the recommended procedure

provided by ATCC. Briefly, the stock was revived by adding it to LB broth in a sterile culture tube

and incubated overnight while shaking at 250 rpm. The following day, the starter culture was added

to 50% glycerol to make stocks and frozen at -80°C. These stocks were used for all future tests.

The day before testing, a portion of the frozen stock was reanimated by touching an inoculation

loop to the frozen stock, which was then dipped into 75 mL of LB broth. The broth was then shaken

at 250 rpm overnight in an incubator. The next day, the overnight solution was divided into two

50 mL falcon tubes and centrifuged at 4000 rpm for 15 min. The broth was decanted and the pellet

was rinsed with Ringer’s solution, then centrifuged again. The pellet was rinsed three times, then

added to 25 mL of Ringer’s. The OD600 of the solution was determined, then the amount of solution

to add to 35 mL of Ringer’s was calculated to achieve an initial concentration of ~107 CFU/mL,

which corresponds to an OD600 of ~0.05. This solution was used for the solar experiments.

Aliquots of 0.5 mL were removed at set time intervals and 0.2 mL of this aliquot was added to 1.8

mL of PBS in a culture tube to make a 10-1 dilution. This solution was then vortexed and 0.2 mL

was added to another tube with 1.8 mL of PBS to make a 10-2 dilution. This process was repeated

until a 10-6 dilution was achieved. From these dilutions, 0.1 mL was taken and plated using the

spread plate method. Only the dilutions where a reasonable number of coliform units was expected

were plated. Once plated, the petri dishes were incubated for 24 hr and the CFU/mL was

determined with Equation 3.2.

CFUmL =

colonies counted0.1 mL of sample ×dilution factor

3.2

All solution preparation, dilutions, and plating were performed in an operating biosafety cabinet

in the presence of an ethanol burner following standard aseptic practices. For more detailed

information on E. coli preparation and enumeration, see Section 8.8.4.

S. M. Larlee 28

3.2.3.3 Hydroxyl radical detection The procedures used for hydroxyl radical detection were adapted from similar works performed

by Arlos et al. (2016) and Ishibashi et al. (2000). To determine if hydroxyl radicals were generated

by the clay pieces, terephthalic acid (TPA) at an initial concentration of 0.5 mM was used as a

chemical probe. When hydroxyl radicals are produced, they react with the TPA and form 2-

hydroxyterephthalic acid (HTPA) as shown in Equation 3.3.

C6H4(COOH)2 + ̊OH ==> C6H4(COOH)2OH

TPA HTPA

3.3

HTPA can be detected using fluorescence. Therefore, the concentration of hydroxyl radicals can

be determined from the concentration of HTPA produced during irradiation. To determine the

HTPA generated by the TPA test, a calibration curve was first prepared using an HTPA stock

solution and a working solution was prepared with TPA and NaOH. This working solution was

added to reactors and exposed to sunlight under the PET Solar Simulator. Samples at varying set

time intervals were analysed using the fluorescence spectrophotometer which was set to an

excitation wavelength of 315 nm and an emission wavelength range between 350 nm and 550 nm.

More detailed information on hydroxyl radical detection is found in Section 8.8.4.

3.2.3.4 Detection and quantification of photogenerated electron holes The procedures used for photogenerated hole (h+) detection were adapted from similar works

performed by Turolla et al. (2015). To determine if photogenerated holes were generated by the

clay pieces, potassium iodine was used as a chemical probe at an initial concentration of 50 mM.

When photogenerated holes are produced by the photocatalyst, they react with the iodine ion (I-)

to form iodine (I2) as shown in Equation 3.4. The concentration of photogenerated holes is twice

the concentration of the produced iodine.

2I-+2h+==> I2 3.4

The produced iodine can be detected using the UV-vis spectrophotometer. To determine the iodine

concentration generated by the test, a calibration curve was prepared using an iodine solution. For

the tests with clay, a working solution was prepared with potassium iodide (KI). This working

S. M. Larlee 29

solution was added to reactors and exposed to sunlight under the PET Solar Simulator. Samples at

varying set time intervals were analysed using the UV-vis which was set to a wavelength of 585

nm. More detailed information on photogenerated hole detection is found in Section 8.8.4.

3.3 Experimental QA/QC Experiments with TiO2-coated clay samples discussed in Chapter 4 were prepared and tested in

duplicate. The model dye tests for the uncoated clay samples that are discussed in Chapter 5 were

completed in quadruplicate. The E. coli tests were performed in duplicate and the adsorption time

tests, TPA tests, and iodine tests were performed in triplicate. Average values with vertical bars

showing standard deviation are provided in each results section. When only duplicates were

performed, vertical bars were used to show the maximum and minimum test results.

With respect to the model dye tests, Milli-Q® water was used to blank the UV-vis

spectrophotometer and calibration standards were prepared each testing day to test the validity of

the calibration curve. Samples were analysed immediately whenever possible to avoid additional

degradation from room light. For the E. coli experiments, both the Ringer’s and PBS solutions

were plated each testing day to ensure that samples were free of contamination. If E. coli was

detected in the Ringer’s or the PBS control plates, tests were repeated. TPA and iodine tests were

performed in triplicate. For both tests, the fluorescence spectrophotometer and the UV-vis

spectrophotometer was blanked with Milli-Q® water.

S. M. Larlee 30

Chapter 4 Low-tech C- and N-doped TiO2 for SODIS

Low-tech C- and N-doped TiO2 for SODIS 4.1 Introduction and objective Solar water disinfection, or SODIS, is a point-of-use water treatment technique that is used by an

estimated 5 million people worldwide (Eawag/Sandec, 2016). In its simplest form, SODIS

involves adding potentially contaminated water to transparent bottles and placing them in direct

sunlight for a minimum of 6 hr on days without any cloud cover (Eawag/Sandec, 2016; McGuigan

et al., 2012). After this exposure period, the water has been treated due to the heating and optical

properties of the sunlight (McGuigan et al., 1998). There are many benefits associated with

SODIS: it is a low, or even zero cost treatment technique, as the bottles used are often destined for

refuse. SODIS is also an easy to implement technique; the only material required is the container.

However, SODIS is a time-consuming process and some studies have shown that re-growth of

bacteria is possible (Gelover et al., 2006; Rincón and Pulgarin, 2004).

To enhance the efficiency of SODIS, several household techniques have been investigated

including covering the underside of the bottles with aluminum foil (Kehoe et al., 2001), painting

the underside of the bottle black (Sommer et al., 2014), or shaking the bottle of water before closing

it to augment the dissolved oxygen content (Reed, 1997). In addition, Wilson and Andrews (2011)

examined the use of a Moringa oleifera seed emulsion to clarify and decolorize source waters as

a pretreatment for SODIS. The use of photocatalysts to improve SODIS has also been investigated,

and of the photocatalysts studied, TiO2 shows the most promise due to its chemical stability, high

photocatlytic activity when compared to other semiconductors, and its low cost (2$/kg for TiO2

pigment) (Wang et al., 2013). However, TiO2 is activated by UV light and only approximately 5%

of sunlight lies within the UV range, which makes the use of pure TiO2 with sunlight inefficient.

Thus, it is important to tailor the physicochemical and optical properties of TiO2 so that activation

can occur in sunlight.

Band gap engineering is a strategy to make TiO2 visible light active and involves narrowing the

band gap by elevating the valence maximum or lowering the conduction band minimum so that

activation can occur with visible light (Wang et al., 2013). A common band gap narrowing strategy

S. M. Larlee 31

is doping, which involves incorporating another atom into the structure of the TiO2 to create new

electronic states and optical transitions that are not present in pure TiO2. Doping TiO2 is the most

intensively investigated way of realizing the visible light response of TiO2 (Chen et al., 2010).

Recently, incorporating numerous elements into the structure of the TiO2, or codoping, has

provided beneficial synergistic effects due to the interaction between dopants. Among the non-

metals investigated as dopants, carbon- and nitrogen-doped TiO2 materials exhibit superior

photocatalytic activity under sunlight (Chen et al., 2007).

Carbon and nitrogen have been investigated as codopants previously to create a solar-active

photocatalyst. Chen et al. (2007) used urea as the nitrogen source and tetrabutylammonium

hydroxide as the carbon source to produce a powder whose ability to bleach methylene blue was

examined. Nawawi and Nawi (2014) used urea as the source of carbon and nitrogen and examined

the ability of the powder to remove colour from methylene blue and reactive red 4 solutions, as

well as to degrade phenol. In the work done by Cong et al. (2006), a microemulsion-hydrothermal

process was used to prepare a powder and the bleaching of rhodamine B and 2,4-dichlorophenol

was determined. In this case, the nitrogen source was trimethylamine and tetrabutyltitanate served

as the carbon and titanium source. Wang et al. (2011) used a sol-gel method where titanium

butoxide was the titanium and carbon precursor and nitric acid was the nitrogen source. In this

work, the degradation of sulfanilamide was examined under visible light. Furthermore, in the work

by Wang et al. (2012), L-lysine was used as a source of carbon and nitrogen to prepare

nanoparticles whose ability to degrade methyl orange was analysed.

While many examples of carbon- and nitrogen-doped TiO2 exist, there are gaps associated with

the current body of research. The issue of removing the photocatalysts from the water must be

addressed and the ease of production of the photocatalysts must be considered. Since SODIS is a

low (and potentially zero) cost water treatment technique, photocatalysts that involve complex

preparations may be prohibitively expensive and complicated for users. The purpose of this work

is to prepare carbon- and nitrogen-doped TiO2 (C-N/TiO2) with urea and Aeroxide P25 TiO2, both

accessible materials, coated onto clay, an inexpensive material. Clay was used as a substrate as it

is an accessible material; regions that practice SODIS often have traditional pottery practices. It is

also a material that can be shaped into many different forms to maximize the surface area, and can

withstand the elevated temperatures that are required for calcination of the photocatalytic

S. M. Larlee 32

materials. The use of clay as a support for TiO2 is not new; it has also been used extensively as a

support for photocatalysts because of its high adsorption capacity and low cost (Chong et al.,

2010). Clays have also been used for clay-based photocatalysis and can change the phase of the

semiconductor or improve the electron and electron hole separation (Liu and Zhang, 2014). Belessi

et al. (2007) studied two TiO2/clay composites (montmorillonite-TiO2 and hectorite-TiO2) and

their ability to degrade the herbicide dimethachlor under UV light (λ < 290 nm). Zhang et al. (2011)

prepared TiO2/kaolinite composites and studied their removal of acid red G and 4-nitrophenol

under UV light (λ = 253.7 nm). Another clay based photocatalyst was prepared by Meshram et al.

(2011), who examined the removal of phenol using a ZnO-bentonite nanocomposite as a

photocatalyst under UV radiation. These clay-based photocatalysts show numerous advantages

over the pure semiconductors as they can be recovered from solution and show enhanced

photocatalytic properties (Liu and Zhang, 2014).

Ultimately, the goal of this work is to prepare C-N/TiO2 with accessible materials so that it can be

prepared by users. The doping procedure is based on the work of Rengifo-Herrera et al. (2009) and

by Nawawi and Nawi (2014), while the immobilization is based on the work of Meichtry et al.

(2007). The ability of the materials to remove colour from a methylene blue (MB) solution will

be investigated.

4.2 Experimental 4.2.1 Materials Low Red (LR) clay was purchased from Tucker’s Pottery Supply (Richmond Hill, ON). Aeroxide

P25 TiO2 (21 nm primary particle size, ≥99.5% trace metals basis) from Sigma Aldrich was used

as the starting material for the preparation of C-N/TiO2. ACS reagent urea (99.0% - 100.5%,

chemical formula: NH2CONH2, MW: 60.06 g/mol) purchased from Sigma Aldrich was used as

both the carbon and nitrogen precursor. To determine the photocatalytic activity of the materials,

methylene blue (MB) dye (92%, chemical formula: C16H18ClN8O14S4, MW: 995.23 g/mol) from

Fisher Scientific was used. All solutions were prepared using Milli-Q® water (18.2 MΩ/cm).

4.2.2 Clay substrate preparation Clay pieces were weighed out as moist clay and shaped into a button-shape with a diameter of

approximately 2 cm. Each piece was approximately 7.33 g when wet. This weight was chosen as

S. M. Larlee 33

when shaped into button-shapes, the clay pieces could fit into testing reactors without touching the

stir bars and still remain below the surface of the model dye solution. They were then left for a

minimum of 2 days to air dry, then fired in the muffle furnace. According to the manufacturer, LR

clay can be fired between 999°C and 1120°C. For these experiments, the clay was fired to 999°C

over 8 hr in an 1100°C Box Furnace BF51800 Series Muffle Furnace (Fisher Scientific) and then

allowed to cool for a minimum of 12 hr. The weight of the fired clay was approximately 5.31 g.

4.2.3 Immobilized C-N/TiO2 procedure TiO2 and TiO2-urea mixtures were coated onto clay substrates, described in Section 4.2.2. To

determine the optimal ratio of TiO2 to urea, TiO2 and urea were added to 20 mL of Milli-Q® water

in the following ratios (by weight) ratios to produce a 10% w/v coating solution: 1:1, 2:1, 3:1, 4:1,

and 1:0. The coating solution concentration was used in the work by Meichtry et al. (Meichtry et

al., 2007). TiO2 to urea ratios were chosen as they are commonly tested in the literature and tend

to include higher amounts of TiO2; Nawawi and Nawi (2014) determined that high urea content

leads to lower photocatalytic activity. Urea is soluble in water, so the powders were not initially

mixed together before being added to the water. The TiO2 was weighed in a fume hood in an

aluminum weighing dish to avoid material loss.

To ensure even distribution of particles, the coating solutions were sonicated for 5 min before

coating the clay pieces. Each clay substrate was immersed in one of the coating solution for 15

min per side, then removed and placed on a drying rack so that the excess solution could drain.

The coated substrates air dried for one day and were then calcined in acid digesters for 3 hr in a

muffle furnace. To determine the optimal calcination temperature, the following, commonly tested

temperatures were tested: 250°C, 300°C, 350°C, 400°C, and 450°C (Nawawi and Nawi, 2014;

Rengifo-Herrera et al., 2009). Four clay pieces were coated with each solution and two were tested

for dark adsorption while the other two were tested for dark adsorption followed by sunlight

irradiation. Although care was taken to produce as consistent of a substrate as possible, the clay

substrates were made by hand so there was some difference between the size, and therefore the

surface area, of each substrate. Figure 4-1 shows one batch of coated clay pieces. For each batch,

four clay pieces were left uncoated and did not undergo the calcination process.

S. M. Larlee 34

Figure 4-1: Coated clay pieces

After calcination, the coated clay substrates were added to 35 mL of Milli-Q® water and stirred

constantly for 5 min to remove any excess powder. Next, the substrates were dried in a 105°C oven

for 1 hour. The substrates were then weighed to determine the mass of coating. The overall process

is shown in Figure 4-2.

S. M. Larlee 35

Figure 4-2: Overall C-N/TiO2 preparation process

S. M. Larlee 36

4.2.4 Material characterization The set of clay pieces that were calcined at 450°C were analysed by JOEL JSM6610-LV scanning

electron microscope (SEM) in Secondary Electron Imaging mode to examine the consistency of

the coatings. The bare clay was also analysed by X-ray fluorescence (XRF) to determine its

elemental composition. The XRF was performed using a Philips PW 2404 x-ray spectrometer

using SuperQ to measure and analyse components.

4.2.5 Solar simulator system set-up Methylene blue testing was performed in a Photoemission Tech SS150AA solar simulator

(Camarillo, CA), shown in Figure 4-3. The system produced a field of illumination which was 22

cm by 22 cm and for all experiments, the intensity of the solar simulator was equivalent to 0.85 of

one sun (85 mW/cm2) at the level of the stir plates, as determined using a multi-meter. The spatial

non-uniformity was 0.64% across both the x- and y-axis. The maximum non-uniformity across the

illuminated area was 1.46%. All non-uniformity coefficients were below 2%, which is within the

requirements for Class A systems for international standards (Photo Emission Tech Inc., 2012).

While the solar simulator was in use, dark fabric was used to block ambient light. The temperature

of a 35 mL of Milli-Q® water sample increased from room temperature (19.5°C) to an average of

27°C over the first 60 min, then remained relatively constant for the following 60 min. Four

samples were tested at a time on a stir plate, as shown in Figure 4-4.

S. M. Larlee 37

Figure 4-3: Photoemission Tech SS150AA

solar simulator

Figure 4-4: Methylene blue samples

arranged in solar simulator during testing

4.2.6 Adsorption and photocatalytic activity testing The materials were tested based on their ability to remove colour from a 10 mg/L MB solution as

it is one of the most commonly used model dyes for testing the photocatalytic ability of

photocatalysts (Rochkind et al., 2015). To calculate concentration data form the absorbance values

from the UV-vis spectrophotometer, a calibration curve was generated by determining the

absorption values of solutions of known concentration. By dividing the absorption values by the

slope of the line, the unknown concentration of the test solution can be calculated then, based on

the concentration of the solution, the percent colour removal can be calculated.

To perform testing, one clay piece was added to a 50 mL beaker containing 35 mL of the MB

solution. To test the dark adsorption of the materials, one clay piece was added to a beaker and

was constantly stirred for 10 min in the dark. After 10 min, the colour removal was determined

S. M. Larlee 38

using a UV-vis spectrophotometer. To test the photocatalytic ability of the materials, the MB

solution was constantly stirred for 10 min in the dark, and then exposed to sunlight in the PET

solar light simulator (Photo Emission Tech Inc., Camarillo, CA) for 60 min. The colour removal

was then determined.

4.3 Results and discussion 4.3.1 Material characterization The different clay types were analysed by JOEL JSM6610-LV scanning electron microscope

(SEM) in Secondary Electron Imaging mode to characterize the surface morphology of the clays,

as shown in Figure 4-5. The last image is uncoated clay.

a)

b)

c)

d)

e)

f)

Figure 4-5: SEM images (x500) of various coatings: a) 1:1 TiO2:urea, b) 2:1 TiO2:urea, c) 3:1

TiO2:urea, d) 4:1 TiO2:urea, e) 1:0 TiO2:urea, f) uncoated clay

It is evident that the urea and TiO2 resulted in changing the appearance of the clay surface.

However, it was also noted that the coatings were inconsistent; Figure 4-6 shows the SEM images

S. M. Larlee 39

of the same piece of coated clay, but at different points on the clay piece. The first image shows a

much more even coating, while the second shows flakes of coating.

Figure 4-6: SEM images (x500) from different points on the coated clay (2:1 TiO2:urea ratio

shown)

The composition of the clay pieces was analysed by XRF. Results are shown in Table 4-1. The

italicized rows indicate photocatalytically active components (CaO, MgO, Fe2O3, TiO2, and

Al2O3).

Table 4-1: Analysis of clay pieces by XRF

% Content CaO 3.79 MgO 5.53 K2O 2.79 Na2O 2.50 Fe2O3 5.98 TiO2 0.96 Al2O3 19.3 SiO2 58.4 P2O5 0.15

After the coating procedure was complete, the coated clay samples were weighed and the weight

of the coating was determined by subtracting the fired clay weight from the coated weight. The

coating weights are shown in Table 4-2.

S. M. Larlee 40

Table 4-2: Coating masses (mg), n=4

Ratio (TiO2:urea)

Calcination Temperature (°C) 250 300 350 400 450

1.1 31.8±2.0 28.1±25.2 30.3±2.8 22.4±1.0 26.1±1.7 2.1 28.5±6.9 25.4±237.9 19.4±2.8 20.2±3.3 24.7±25.5 3.1 36.5±3.3 -23.2±24.6 16.9±6.8 17.6±2.0 32.3±9.2 4.1 22.3±3.1 74.5±29.8 10.1±1.3 22.1±2.4 25.1±3.1 1.0 19.9±1.0 9.8±1.7 9.8±1.6 18.2±6.5 18.9±2.0

As was shown in Figure 4-6, there was much variation in the clay coatings. This can also be seen

in the coating weights; no trend was visible for clay weights with respect to coating ratio or

calcination temperature. This may be explained by the clay preparation process; because the clay

pieces were made by hand, they all had variable surface areas and therefore variable surfaces to be

coated with TiO2 and urea.

4.3.2 Analysis of photocatalytic activity Samples were analysed based on their ability to remove colour from a 10 mg/L MB solution. For

the dark adsorption tests, clay pieces were added to 35 mL of the MB solution and stirred for 10

min in the dark. The solution was then analysed with the UV-vis spectrophotometer to determine

colour removal from dark adsorption. Results from 10 min of dark adsorption are shown in Figure

4-7. It should be noted that the uncoated clay pieces were not processed after their initial firing.

Each batch was calcined at a separate calcination temperature, but from each batch, four clay

pieces were kept uncoated. Of the four uncoated pieces, two pieces were tested for adsorption

alone while the other two were tested under sunlight. Therefore the uncoated bar shown in Figure

4-7 represents the average from the uncoated clay samples prepared with each batch.

S. M. Larlee 41

Figure 4-7: Percent MB colour removal under 10 min of dark adsorption based on TiO2:urea

ratio and calcination temperature, vertical bars represent maximum and minimum for

experimental replicates where n=2 with the exception of the uncoated clay where n=10

In general, all coatings and calcination temperatures reduced the concentration of the MB solution

by a similar amount: 2-8%. There was one exception: the clay piece which had a ratio of 4:1 and

was calcined at 350°C had a 12% colour removal. However, this appears to be an outlier. Based

on the data, there does not appear to be an effect of the TiO2:urea ratio or of calcination temperature

on the dark adsorption of the materials. In addition, the uncoated clays remove a similar amount

of colour from the MB solution as the coated clays. Although clay has a high surface area available

for adsorption, it is possible that by coating the clay pieces the surface area was reduced. However,

it is known that TiO2 can be used as an adsorbent (Wiszniowski et al., 2002). In the work performed

by Matthews (1991), the degradation of model dyes by TiO2 supported on sand was investigated.

In 60 min, the coated sand was able to reduce the concentration of an MB solution from 10 µM to

approximately 9 µM (i.e. a 10% colour reduction). Therefore, although coating the clays may

reduce the surface area available to adsorb the dye in the dark, because the clay pieces were coated

with TiO2 which has some adsorption capacity, both the uncoated and the coated clay pieces

behaved similarly.

0.0%

4.0%

8.0%

12.0%

1:1 2:1 3:1 4:1 1:0 uncoated

Perc

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TiO2:urea ratio (by weight)

Calcined at 250°C Calcined at 300°C Calcined at 350°C Calcined at 400°C Calcined at 450°C

S. M. Larlee 42

For the visible light tests, the same procedure was followed but after the 10 min of dark adsorption,

the samples were irradiated under the solar simulator for 60 min and then analysed with the UV-

vis. Results are shown in Figure 4-8. While different clay pieces were used for the adsorption tests

and the adsorption plus sunlight exposure tests, it was assumed that the adsorption results would

be representative of what the clay pieces which were exposed to sunlight would have achieved.

Figure 4-8: Percent MB colour removal under 10 min of dark adsorption followed by 60 min

sunlight exposure based on TiO2:urea ratio and calcination temperature, vertical bars

represent maximum and minimum for experimental replicates where n=2 with the exception

of the uncoated clay where n=10

Based on the data, all coated pieces, regardless of the coating ratio or the calcination temperature,

were able to achieve a percent MB colour removal between 30 and 55%. However, of particular

interest was how well the uncoated clay pieces performed. The colour removal for the uncoated

clay pieces ranged from 43 to 93%. This may be due to the difference in the surface area of the

clay pieces. As previously mentioned, the uncoated clay pieces were not processed after their initial

firing and there was no difference in the process used to make the uncoated clay pieces. While

some uncoated clay pieces did not achieve a colour removal as high as some of the coated clay

pieces, if uncoated clay can act as a photocatalyst for SODIS, they would require less resources

0%

20%

40%

60%

80%

100%

1:1 2:1 3:1 4:1 1:0 uncoated

Perc

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TiO2:urea ratio (by weight)

Calcined at 250°C Calcined at 300°C Calcined at 350°C Calcined at 400°C Calcined at 450°C

S. M. Larlee 43

than if the clay pieces were coated. It has been noted that natural clays like bentonite, sepiolite,

montmorillonite, zeolite, and kaolinite are catalytically inactive (Chong et al., 2010). However, it

is possible that the particular composition of the Low Red clay is photocatalytically active as it

contains known photocatalysts such as CaO, MgO, Fe2O3, TiO2, and Al2O3.

4.3.3 Bare Clay Experiments Based on the data collected for the coated clay materials, the next obvious experimental step was

to examine the uncoated clay pieces to determine whether adsorption or photocatalysis, or both, is

responsible for the colour removal.

A new batch of 20 clay pieces was prepared, all with a starting weight of 7.33 g, and fired to 999°C.

Of the 20 pieces, four clay pieces were tested for 10 min of dark adsorption only, and another four

were tested for 10 min of adsorption followed by 60 min of sunlight exposure. Although these tests

were already performed in the previous section, since there was some batch-to-batch variation of

the clay pieces, the clay pieces were prepared again so that all bare clay experiments would be

from the same batch. The colour removal for a MB solution without clay was also examined for

10 min in the dark, followed by 60 min of sunlight exposure. In addition, four clay pieces were

tested for 70 min in the dark. The last 8 clay pieces of the batch of 20 were calcined in the acid

digesters at 450°C to determine if calcination of the clays affects the colour removal. Four of these

8 pieces were tested for 10 min of adsorption alone, and the other four were tested for 10 min of

adsorption followed by 60 min of sunlight exposure. The results of these tests are shown in Figure

4-9.

S. M. Larlee 44

Figure 4-9: Bare clay experiments, vertical bars represent the standard deviation of

experimental replicates where n=4

Based on the results shown in Figure 4-9, the clay exhibits some photocatalytic activity. When

fired bare clay is added to an MB solution for 70 min in the dark, only 6.0% colour removal is

achieved, which is only 0.8% more than what is achieved within 10 min of dark adsorption (5.2%).

However, when illuminated for 60 min after 10 min of dark adsorption, 59.8% colour removal is

achieved. Additionally, the clay that was calcined after being fired behaved almost identically to

the clay that was only fired and not calcined, both for the 10 min of dark adsorption and for the 10

min of dark adsorption followed by 60 min of sunlight exposure. Therefore, no further processing

of the clay after firing is required. It should also be noted that when no clay is added to the MB

solution, after 10 min of stirring in the dark followed by 60 min of sunlight exposure, 4.7% colour

was removed from the MB solution by direct photolysis.

When attempting to understand how the bare clays were able to remove colour from the model

dyes, a metal analysis was performed using ICP-AES. The presence of metals in solution might

contribute to the photocatalytic activity, such as iron for a Photo-Fenton reaction. To do this, 35

mL of Milli-Q® water was added to a reactor with a piece of uncoated LR clay. After 10 min of

dark adsorption followed by 60 min of sunlight exposure, 14 mL of the sample was added to a

5.2%

59.8%

6.0% 5.5%

57.9%

4.7%

0%

20%

40%

60%

80%

ADS, fired only ADS+SUN, firedonly

Dark control, fired(70 mins)

ADS, calcined at450°C

ADS+SUN,calcined at 450°C

ADS+SUN, no clay

Perc

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S. M. Larlee 45

falcon tube with 0.56 mL of nitric acid then filtered after 3 days for analysis of total metals.

Another 14 mL of the sample was filtered and then added to a falcon tube with the same volume

of acid for analysis of desorbed metals. Results indicated that metals were not released into solution

from the clays; instead, it is likely that reactions are occurring on the surface of the clays pieces.

For all results from the ICP-AES tests, refer to Section 8.2.

4.4 Summary In order to find a low-tech photocatalyst to be prepared and used where SODIS is practiced, fired

clay pieces were coated in varying TiO2 to urea ratios and calcined at different temperatures.

Neither the TiO2 to urea ratio, nor the calcination temperature affected the photocatalytic activity;

all photocatalysts performed similarly. However, the uncoated clay performed similarly, and often

better, than most coated samples. After further investigation, this was due to be a combination of

photocatalysis and dark adsorption. The photocatalytic activity may be due to the composition of

the clay, which contains the following known photocatalytic components: CaO, MgO, Fe2O3,

TiO2, and Al2O3). Bare clays with different compositions will be investigated further as a potential

improvement for SODIS.

S. M. Larlee 46

Chapter 5 Fired clay as a photocatalyst for SODIS

Fired clay as a photocatalyst for SODIS 5.1 Introduction and objective Although the Millennium Development Goal for drinking water was met in 2010 and 91% of the

global population now uses an improved drinking water source, that is, a water supply that is

protected from contamination, 663 million people still do not have access to an improved water

supply (WHO/UNICEF, 2015). In addition, huge disparities exist and those in poor and rural

communities are more seriously affected (WHO/UNICEF, 2015). To address the lack of clean

water, households often rely on household water treatment techniques. First discovered by Acra

et al (1980), solar water disinfection, or SODIS, is recognized as a viable household water

treatment technique (Eawag/Sandec, 2016). It is an effective and low-cost method for improving

water quality in locations where intense sunshine is available (McGuigan et al., 1998). In its

simplest form, SODIS involves adding potentially contaminated water to clean, transparent, 2 L

PET bottles and leaving the bottles in direct sunlight for at least 6 hr on sunny days, or 2 days

when there is 50% or more cloud cover (Eawag/Sandec, 2016). Bacterial reduction results from

optical inactivation, and at temperatures above 45°C, a combination of optical inactivation and

thermal inactivation (McGuigan et al., 1998). SODIS has little or no cost and is easy to apply and

understand, but it requires long treatment times and has limited effectiveness against some viruses

and protozoa (Eawag/Sandec, 2016). While the available literature is inconclusive, there is also

some evidence of regrowth of bacteria (Gelover et al., 2006; Rincón and Pulgarin, 2004).

Therefore, the possibility of regrowth is considered a limitation of SODIS (Eawag/Sandec, 2016).

In an effort to make SODIS more effective and less time-consuming, alterations have been

proposed such as covering the underside of the bottles with aluminum foil (Kehoe et al., 2001),

continuous-flow solar UVB disinfection systems (Mbonimpa et al., 2012), and pre-treatment using

locally-available coagulants (Wilson and Andrews, 2011). When designing an improvement for

SODIS, the complexity of the treatment process must be considered, as well as the supply chain

required to obtain the required materials. Therefore, it is best to rely on locally-available resources.

S. M. Larlee 47

The use of photocatalysts for a faster and more complete oxidation of contaminants has also been

investigated. The most commonly researched photocatalyst is TiO2 (Fujishima et al., 2008). This

is due to its efficient photoactivity and low cost relative to other photocatalysts (Hashimoto et al.,

2007). It is also an attractive material as it has a high chemical stability over a wide pH range

(Chen et al., 2010). Considered a semiconductor, when TiO2 is irradiated with UV light, electrons

move from the valence band to the conduction band and react with the water to form oxidative

species, which remove organic material and disinfect the water. In addition to TiO2, a number of

other photocatalytic species have been identified, including CaO (Kornprobst and Plank, 2012),

MgO (Mageshwari and Sathyamoorthy, 2012), Fe2O3 (Karunakaran and Senthilvelan, 2006;

Mishra and Chun, 2015), and Al2O3 (Leow et al., 2017). Clay is composed of these established

photocatalytic components, and is also an accessible material; regions that practice SODIS often

have traditional pottery practices. It is a material that can be shaped into many different forms then

fired to maximize the surface area. Therefore, fired clay may be able to serve as a photocatalyst

for SODIS.

The current work will examine the ability of different fired clays to remove colour from model

dyes when exposed to sunlight. The dyes selected for testing were methylene blue (MB), a cationic

dye, and acid orange 7 (AO7), an anionic dye. These dyes were selected as they are commonly

used dyes with different molecular properties and structure, which can provide more information

regarding the photocatalytic ability of the photocatalysts. In addition, methylene blue is one of the

most commonly used model dyes for testing the photocatalytic ability of photocatalysts (Rochkind

et al., 2015) and the results can therefore be compared to the results of other photocatalytic

materials. The structures of the model dyes are shown in Figure 5-1.

Figure 5-1: Chemical structures of methylene blue (left) and acid orange 7 (right)

S. M. Larlee 48

In addition, since a major concern in developing countries is microbial contamination, for clay to

be a viable photocatalyst for SODIS applications, the ability of the materials to inactivate bacteria

will be examined. It is hypothesized that certain clay components drive the photocatalytic process.

The clays will be characterized by testing the hydroxyl radical and photogenerated hole production

and the results will be discussed in relation to the model dye and E. coli test results. Based on the

results, the possibility of using clay as a photocatalyst for SODIS is discussed.

5.2 Experimental 5.2.1 Materials All clays were purchased from Tucker’s Pottery Supply (Richmond Hill, ON). The composition

of each clay was provided by the manufacturer and can be found in Table 5-1. Components that

have shown photocatalytic activity in the literature are italicized. To determine the overall

photocatalytic activity of the clays, methylene blue (MB) (92%, chemical formula:

C16H18ClN8O14S4, MW: 995.23 g/mol) and acid orange 7 (AO7) (≥85%, chemical formula:

C16H11N2NaO4C, MW: 350.32 g/mol) from Fisher Scientific were used as model dyes. All model

dye solutions were prepared using Milli-Q® water (18.2 MΩ/cm). Escherichia coli (E. coli) tests

were performed using a lyophilized Escherichia. Coli (E. coli) stock culture (ATCC ® 23631)

purchased from Cedarlane Laboratories. Hydroxyl radical tests were performed using TPA and

HTPA, both from Sigma-Aldrich, the photogenerated holes test was performed using potassium

iodide (Sigma-Aldrich) and an iodine solution (Atoma).

S. M. Larlee 49

Table 5-1: Clay compositions (provided by Tucker’s Pottery Supply). Components that have

shown photocatalytic activity are italicized.

Clay composition (%) by weight

Component Low Red PHB Thompson Raku

Smooth Raku

White Sculpture Raku

BaO 0.40 0.30

CaO 0.25 0.25 2.19 0.33 0.20 MnO2 0.01

MgO 6.56 6.50 2.01 0.16 0.16 K2O 2.64 2.20 0.75 1.45 0.84 Na2O 0.10 0.11 0.59 2.39 1.40 Fe2O3 5.75 4.01 1.15 0.86 0.73 TiO2 1.00 1.21 1.52 0.83 0.79 Al2O3 17.10 20.76 32.21 34.48 32.46 SiO2 66.17 64.62 59.49 59.50 63.37 P2O5 0.02 0.02 0.08 0.01 0.06

5.2.2 Clay substrate preparation Preliminary tests showed that 7.33 g of LR wet clay resulted in a fired weight of approximately

5.31 g, which was appropriately sized for testing in the reactors used in the solar simulator. To

achieve the same final fired weight for each clay, the shrinkage rate for each clay type was

determined by weighing out 7.33 g of each type of wet clay then firing the clay piece at the

manufacturer-specified temperature (shown in Table 5-2). By dividing the average wet weight by

the average fired weight the shrinkage rate was determined and the required wet weight to achieve

a final fired weight of 5.31 g was calculated. Table 5-3 shows the average shrinkage weight for

each clay type and the required wet weight to achieve a final fired weight of 5.31 g.

Table 5-2: Clay firing temperatures

Clay Type Firing Temperature (°C)

Smooth Raku (SR) 1060

Thompson Raku (TR) 1060

Low Red (LR) 999

PHB 999 or 1060

White Sculpture (WSC) 1046

S. M. Larlee 50

Table 5-3: Shrinkage rates of different clays used to determine required wet weight to

achieve similar fired weights

Clay Type Shrinkage Rate1 Required Wet Weight (g)2

Smooth Raku (SR) 1.30 6.89

Thompson Raku (TR) 1.33 7.06

Low Red (LR) 1.38 7.33

PHB 1.42 7.54

White Sculpture (WSC) 1.31 6.98

To prepare the clay pieces for photocatalytic experiments the required amount of wet clay was

weighed out based on the clay type (Table 5-3) and shaped into a 2 cm diameter button-shape, as

shown in Figure 5-2. The pieces were air dried for at least 2 days, then fired in a muffle furnace

(1100°C Box Furnace BF51800 Series Muffle Furnace, Fisher Scientific) which was programmed

to increase by 2°C/min to the manufacturer-specified firing temperature. Clay pieces that required

the same firing temperature were fired together. Once the temperature was reached, the muffle

furnace was turned off and the clay pieces cooled in the furnace for at least 12 hr. The fired clay

was weighed after cooling to confirm a final fired weight of approximately 5.31 g.

1 Shrinkage rate = Average wet weight ÷ Average fired weight

2 Required wet weight = Shrinkage rate × 5.31 g

S. M. Larlee 51

Figure 5-2: Clay piece dimensions

5.2.3 Material characterization The different clay types were analysed by JOEL JSM6610-LV scanning electron microscope

(SEM) in secondary electron imaging mode to characterize the surface morphology of the clays.

The clays pieces were also analysed by X-ray fluorescence (XRF) to determine their elemental

composition. The XRF was performed using a Philips PW 2404 x-ray spectrometer using SuperQ

to measure and analyse components. Although the composition of the clays was provided by the

manufacturer, XRF was still performed to check accuracy.

5.2.4 Solar simulator system set-up All tests were performed in a Photoemission Tech SS150AA solar simulator (Camarillo, CA),

shown in Figure 5-3. The intensity of the solar simulator was adjusted to a level of one sun at the

level of the stir plates according to the calibration instructions for the instrument. Briefly, the

intensity was adjusted to 71.6 mV, as noted on the reference cell for the solar simulator, using a

multi-meter, which corresponds to an irradiance intensity of 100 mW/cm2. The system produced

a field of illumination which was 22 cm by 22 cm. The spatial non-uniformity was 0.64% across

both the x- and y-axis, as determined using a multi-meter. The maximum non-uniformity across

the illuminated area was 1.46%. All non-uniformity coefficients were below 2%, which is within

the requirements for Class A systems for international standards (Photo Emission Tech Inc., 2012).

S. M. Larlee 52

While the solar simulator was in use, dark fabric was used to block ambient light from reaching

the samples. The temperature of a 35 mL Milli-Q® water sample in a reactor was monitored and

the temperature increased from room temperature (19.5°) to 27°C during the first 60 min, then

remained relatively constant for the following 60 min. Figure 5-4 shows the configuration of the

MB samples in the solar simulator during testing.

Figure 5-3: Photoemission Tech SS150AA

Solar Simulator

Figure 5-4: Methylene blue samples

arranged in solar simulator during testing

5.2.5 Model dye testing The clay pieces were tested based on their ability to remove colour from a 10 mg/L model dye

solution. To perform the testing, one clay piece was added to a 50 mL beaker containing 35 mL of

either a MB or AO7 solution. To test the dark adsorption of the materials, the beakers were

constantly stirred for 10 min in the dark and the colour removal was determined using a UV-vis

spectrophotometer (Agilent Technologies, Mississauga, ON). The peak detection wavelength was

S. M. Larlee 53

664 nm for MB and 485 nm for AO7. To test the photocatalytic ability of the materials, the dye

solution was constantly stirred for 10 min in the dark to allow for adsorption to occur, and then

exposed to sunlight in the solar light simulator for 60 min while being constantly stirred. The

colour removal was then tested with the UV-vis spectrophotometer. The model dyes were also

tested in the absence of clay as a control.

5.2.6 Bacterial preparation and testing An E. coli stock culture (ATCC ® 23631™) was grown from a seed culture purchased from

Cedarlane Laboratories. The seed culture was revived by adding it to 5 mL of LB broth in a sterile

culture tube and incubating it overnight at 37°C while shaking at 250 rpm. The following day, 0.75

mL of the starter culture was added to 0.75 mL of 50% glycerol and frozen at -80°C. These stocks

were used for all tests.

The day before testing, a portion of the frozen stock was reanimated by touching an inoculation

loop to the frozen stock, which was then dipped into 75 mL of LB broth. This solution was shaken

at 250 rpm overnight in a 37°C incubator. The next day, the overnight solution was centrifuged at

4000 rpm for 15 min to remove the growth medium. The pellet was then rinsed with 7.5 mL of

Ringer’s solution and centrifuged again. This rinsing process was repeated three times, after which

the pellet was resuspended in 25 mL of Ringer’s solution. A small volume of this solution was

added to 35 mL of Ringer’s solution to achieve an initial testing solution concentration of ~107

CFU/mL, which corresponds to an OD600 of ~0.05. A 0.1 g/L TiO2 solution was also tested as a

positive control.

Volumes of 0.5 mL were removed from the testing beakers at set time intervals and 0.2 mL of this

aliquot was added to 1.8 mL of PBS in a culture tube. This solution was vortexed and used to make

serial dilutions. 100 µL of the dilutions were plated onto agar plates where it was estimated that a

reasonable number of coliform units would grow. Once prepared, the petri dishes were incubated

for 24 hr and the CFU/mL was determined by counting the colonies and considering the dilution

factor. All solution preparation, dilutions, and plating were performed in an operating biosafety

cabinet in the presence of an ethanol burner following standard aseptic practices.

S. M. Larlee 54

5.2.7 Hydroxyl radical and photogenerated electron hole detection Two common reactive oxidative species generated by photocatalysts are hydroxyl radicals and

photogenerated holes (h+). Hydroxyl radicals can be detected using TPA as a chemical probe and

photogenerated holes can be detected using potassium iodide as a chemical probe.

The procedures used for hydroxyl radical detection were adapted from similar works performed

by Ishibashi et al. (2000) and Arlos et al. (2016). To determine if hydroxyl radicals are generated

by the clay pieces and responsible for the colour removal from the model dyes, terephthalic acid

(TPA) was used as a chemical probe. When hydroxyl radicals are produced, they react with the

TPA and form 2-hydroxyterephthalic acid (HTPA) which can be detected using fluorescence.

Therefore, the concentration of hydroxyl radicals can be determined from the concentration of

HTPA produced during irradiation.

The procedures used for photogenerated hole (h+) detection were adapted from similar works

performed by Turolla et al. (2015). To determine if photogenerated holes were generated by the

clay pieces, potassium iodine was used as a chemical probe. When photogenerated holes are

produced by the photocatalyst, they react with the iodine ion (I-) to form iodine (I2); the

concentration of photogenerated holes is twice the concentration of the produced iodine.

Testing was performed by adding one clay piece to a 50 mL beaker containing 35 mL of the TPA

solution or 35 mL of the potassium iodide solution, depending on the experiment. The samples

were stirred for 10 min in the dark. Then, a 3.5 mL sample was removed, analysed by a

fluorescence spectrophotometer (Agilent Technologies, Mississauga, ON) for the TPA test, or the

UV-vis spectrophotometer for the iodine test, and added back to the reactors as quickly as possible.

The samples were then exposed to sunlight for 60 min during which samples were taken every 20

min, analysed, and added back to the reactors. The fluorescence spectrophotometer was set to an

excitation wavelength of 315 nm and an emission wavelength range between 350 nm and 550 nm.

The UV-vis spectrophotometer was set to a detection wavelength of 585 nm. For the TPA test, a

calibration curve was generated using an HTPA solution to determine the concentration of each

sample. For the iodine test, a calibration curve was generated with an iodine solution to determine

the concentration of each sample.

S. M. Larlee 55

5.3 Results 5.3.1 Clay characterization SEM images of each of the clays are shown in Figure 5-5. Each image is a 1,000 magnification.

The purpose of analysing the clays by SEM was to examine their surface morphology and

determine if one clay had a higher surface area than the others, which might indicate more similar

surface morphologies. Therefore, the different photocatalytic performances may be due to clay

composition rather than surface area.

a)

b)

c)

d)

e)

Figure 5-5: SEM images (x1,000) of various clays: a) Smooth Raku, b) Thompson Raku, c)

Low Red, d) PHB, e) White Sculpture

S. M. Larlee 56

The composition of the clay pieces was analysed by XRF and are shown in Table 5-4. In general,

the XRF data was similar to that specified by the manufacturer. However, the CaO content was

higher than that specified by the manufacturer; XRF results indicated that the composition ranged

from approximately 5 (TR) to 22 (PHB) times the content indicated by the manufacturer. Columns

denoted with TPS refer to the manufacturer data, while columns denoted by XRF refer to the

results for X-ray fluorescence.

Table 5-4: Analysis of clay pieces by XRF (% Content). Components that have shown

photocatalytic activity are italicized.

Low Red PHB Thompson

Raku Smooth Raku White Sculpture Raku

TPS XRF TPS XRF TPS XRF TPS XRF TPS XRF CaO 0.25 3.74 0.25 5.44 2.19 10.43 0.33 2.76 0.20 3.61 MgO 6.56 5.53 6.50 5.16 2.01 3.23 0.16 0.77 0.16 1.45 K2O 2.64 2.79 2.20 1.86 0.75 0.60 1.45 1.40 0.84 0.83 Na2O 0.10 2.50 0.11 -- 0.59 -- 2.39 2.60 1.40 2.41 Fe2O3 5.75 5.98 4.01 4.01 1.15 1.62 0.86 0.88 0.73 0.79 TiO2 1.00 0.96 1.21 1.19 1.52 1.70 0.83 1.02 0.79 0.71 Al2O3 17.10 19.28 20.76 19.87 32.21 22.08 34.48 26.47 32.46 25.66 SiO2 66.17 58.42 64.62 60.11 59.49 59.38 59.50 63.26 63.37 64.04 P2O5 0.02 0.15 0.02 0.00 0.08 0.19 0.01 0.07 0.06 0.29

5.3.2 Adsorption time requirements For the testing of many photocatalysts, a dark adsorption step is generally employed so that the

materials can reach equilibrium. The duration of the dark adsorption step varies (e.g. 10 min

(Zhang et al., 2011), 30 min (Bae et al., 2014), 1 hr (Wang et al, 2011; Yang et al 2008), or 2 hr

(Yang et al., 2010)) and is assumed by the investigator. In some cases, the dark adsorption step is

mentioned, but not quantified. However, experiments are rarely performed or reported to

determine what the optimal adsorption time is for a given material. Instead, commonly used

adsorption times from other experiments are generally used. Therefore, the adsorptive capacity of

the clay materials was investigated with MB and AO7.

For each clay type, three clay pieces were added to three beakers with 35 mL of 10 mg/L MB of

AO7 and were constantly stirred in the dark. In initial tests, 3.5 mL samples were removed from

the beakers at the following time intervals: 2, 4, 6, 8, 10, 20, 30, 40, 60, and 120 min. Because

S. M. Larlee 57

those results indicated that the dark adsorbance continued to increase for the MB samples,

additional tests were performed at 6 and 24 hr for this dye as well. Samples were analysed using

the UV-vis spectrophotometer then returned to the beakers, usually within 1 min, to continue tests.

The results for MB are shown in Figure 5-6 and the results for AO7 are shown in Figure 5-7.

Figure 5-6: Adsorption measurements over time for MB, vertical bars represent the standard

deviation of experimental replicates where n=3

0%

20%

40%

60%

80%

100%

0 200 400 600 800 1000 1200 1400 1600

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Time (min)

LR SR TR PHB WSC

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Figure 5-7: Adsorption measurements over time for AO7, vertical bars represent the

standard deviation of experimental replicates where n=3

Adsorption of the model dyes onto the clays was different depending on the model dye employed.

For MB adsorption, all clays showed a linear trend over the first 2 hr. SR achieved the highest

adsorption over the 2 hr with an average of 35% colour removal followed by TR and WSC which

had a colour removal of 25% and 24%, respectively. LR and PHB had the lowest dark adsorption

colour removal with 12% and 8%, respectively. When longer dark adsorption tests were

performed, the colour removal decreased for the LR and PHB samples, but continued to increase

for the TR, SR, and WSC samples although not in the same linear trend as they did in the first 2

hr. The LR and PHB clays performed similarly over the course of the dark experiments and are

the only two samples with high iron content, and a resulting more orange-brown colouring.

For AO7, all clays reached equilibrium for dark adsorption within the first 30 to 40 min and

remained constant for the remainder of the time, with the exception of the LR clay, which

decreased at the 2 hr mark. However, this was assumed to be an anomaly; it is likely that the

absorbance did not change after reaching equilibrium after the 30 to 40 min. LR was able to

achieve by far the highest dark adsorption during the two hr: 32% colour removal after 40 min.

0%

10%

20%

30%

40%

0 20 40 60 80 100 120 140

Perc

ent A

O7

Col

our

Rem

oval

Time (min)

LR SR TR PHB WSC

S. M. Larlee 59

The next highest dark adsorption was achieved by PHB which was 10% after 40 min. SR, TR, and

WSC all performed similarly and had a maximum adsorption between 3% and 5% at 30 to 40 min.

Based on the MB data, it depends on the clay type when determining how much time is required

to reach equilibrium. For all clays in AO7, a dark adsorption time of 30 to 40 min is required. This

is an important consideration as compounds that behave like MB may require different adsorption

times than compounds that behave like AO7. However, in addition to ensuring that adsorption

occurs and equilibrium is established, one must also make practical considerations; a SODIS user

is unlikely to plan a long adsorption time. Therefore, a 10 min adsorption period was used as this

was thought to be a practical waiting time.

5.3.3 Model dye colour removal Two batches of clay were prepared and tested; one batch was tested with MB and the other with

AO7. After testing in the solar simulator, analysis was performed using a UV-vis

spectrophotometer. Figure 5-8 shows the percent colour removal of the MB solution achieved by

each clay type for adsorption only and for adsorption plus sunlight. Figure 5-9 shows the results

from the AO7 testing.

S. M. Larlee 60

Figure 5-8: MB colour removal after 10 min of dark adsorption (ADS) and 10 min of

adsorption followed by 60 min of sunlight exposure (ADS+SUN) for all clay types, vertical

bars represent the standard deviation of experimental replicates where n=4

Figure 5-9: AO7 colour removal after 10 min of dark adsorption (ADS) and 10 min of

adsorption followed by 60 min of sunlight exposure (ADS+SUN) for all clay types, vertical

bars represent the standard deviation of experimental replicates where n=4

0%

20%

40%

60%

80%

SR TR LR PHB WSC No clay

Perc

ent M

B C

olou

r Rem

oval

Clay Type

Average Reduction (ADS) Average Reduction (ADS+SUN)

-10%

0%

10%

20%

30%

SR TR LR PHB WSC No clayPerc

ent A

O7

Col

our R

emov

al

Clay Type

Average Reduction (ADS) Average Reduction (ADS+SUN)

S. M. Larlee 61

With respect to the MB tests, all clays were able to achieve between 5% and 11% for dark

adsorption, which was consistent with the adsorption time requirements outlined in the previous

section. When exposed to one hour of sunlight after 10 min of adsorption, the highest colour

removal was achieved by the LR and WSC clays with 51.0% and 47.4%, respectively. However,

at the 95% confidence level, no significant difference was observed between the two. The SR clay

performed significantly worse than the LR and WSC clays, but significantly better than the other

clays with a colour removal of 43.1%. The TR and PHB performed similarly with 32.7% and

30.4% colour removal, respectively. All clays performed significantly better than the sun only

samples at the 95% confidence level.

When the colour removal from the dark adsorption experiments are compared to the solar

experiments, the colour removal is consistently higher after one hour of sunlight exposure than it

is for dark adsorption only. The SR clay achieved the highest colour removal after one hour of

dark adsorption (23%), but when exposed to sunlight, achieved 48% colour removal. Similar

results were found for all other clays. From this, it can be concluded that there is photocatalytic

activity.

As was seen in the dark adsorption tests, the LR clay achieved the highest AO7 colour removal in

the first 10 min. Once exposed to sunlight, all clays, with the exception of LR, showed negative

AO7 colour removal. This is likely due to the interaction between the dyes and the photocatalyst;

clay is negatively charged and therefore will attract the MB and repel the AO7. The only clay that

was able to achieve any positive AO7 colour removal was the LR clay. However, the majority of

the colour removal for the LR clay was due to adsorption; the adsorption colour removal was

17.6% and the adsorption followed by sunlight colour removal was 19.3%. This is further

supported when the dark adsorption results are analysed; after one hr of dark adsorption, LR was

able to achieve over 30% colour removal. Therefore, the colour removal may be solely due to

adsorption.

It should also be noted that the dark adsorption achieved by the LR clay was lower than that

achieved in the initial dark adsorption tests discussed in Section 5.3.15.3.1. This is likely because

each test was performed with different clay pieces and, since the clay pieces were prepared by

hand, some difference between batches of clay is expected.

S. M. Larlee 62

After analysing the AO7 sunlight data using t-tests, in all cases, all clays performed significantly

better than the sun only samples at the 95% confidence level. In addition, all clays performed

significantly worse than the sunlight only sample with the exception of the LR clay, which

performed significantly better than all clays and the sunlight only samples.

Without clay, 12.6% colour was removed from the MB solution and 4.0% colour was removed

from the AO7 solution under sunlight.

Based on these results, it can be concluded that each dye interacts differently with the clay pieces.

Model dyes are chemical probes to determine the photocatalytic activity of materials, but it should

be noted that the results are specific to the photcatalyst-dye system and generalizing the results to

other compounds should be done with caution.

When the XRF data is correlated with the MB and AO7 percent removal data, correlation

coefficients can be calculated as shown in Table 5-5. A correlation coefficient of -1 refers to a

negative correlation, 0 refers to no correlation, and 1 refers to a positive correlation. An absolute

correlation value of 0.7 generally refers to a strong linear relationship.

Table 5-5: Correlation coefficients between model dye colour removal and XRF data

Photocatalytic Component MB Correlation Coefficient

AO7 Correlation Coefficient

CaO -0.68 -0.22 MgO -0.18 0.57 Fe2O3 0.15 0.78 TiO2 -0.75 -0.22 Al2O3 0.23 -0.55

With respect to the MB tests, noteworthy correlation coefficents are those for CaO (-0.68) and

TiO2 (-0.75). Interestingly, these correlation coefficients were both negative, which means that as

their % composition increases, the percent MB colour removal decreases. A significant positive

correlation coefficient for AO7 was Fe2O3 (0.78). From the correlation data, however, there does

not appear to be a clear relationship between individual photocatalic components and colour

removal.

S. M. Larlee 63

5.3.4 Regeneration testing with methylene blue The regeneration and reusability of the materials is an important parameter to examine as it will

determine how often the user will need to prepare or purchase the materials; clay pieces that need

to be prepared or purchased often will likely increase the cost to the user and reduce user uptake.

The clay pieces that were used to generate Figure 5-8 were tested based on their ability to

repeatedly remove colour from a 10 mg/L methylene blue solution under the equivalent of one

sun. All five regeneration tests were performed within 5 days; that is, one regeneration was

performed each day. After each round of regeneration testing, each clay took up the colour of the

MB. The clay pieces were kept in open air overnight and the colour faded, but some blue colouring

remained. The percent colour reduction of the MB solution achieved by each clay substrate over

the 6 trials is shown in Figure 5-10.

Figure 5-10: MB colour removal after 10 min of dark adsorption followed by 60 min of

sunlight exposure for all clay types over 5 regeneration cycles, vertical bars represent the

standard deviation of experimental replicates where n=4

The colour removal for the SR decreased by 4.1% after the first regeneration, but remained at

approximately 39% colour removal for all other regeneration cycles. Both the TR and WSC

remained at approximately the same colour removal, 33% and 47%, respectively, not only for the

first regeneration, but for all regeneration cycles that followed. After the first regeneration, the

0%

20%

40%

60%

80%

Smooth Raku Thompson Raku Low Red PHB White Sculpture

Perc

ent M

B c

olou

r re

mov

al

Clay Type

Original First Regeneration Second Regeneration

Third Regeneration Fourth Regeneration Fifth Regeneration

S. M. Larlee 64

colour removal of the LR decreased by an average of 19.3%. For the second regeneration, the

average colour removal decreased by 4.6%, but then remained at approximately 27% for all

remaining regenerations. The PHB colour removal decreased by 6.8% after the first regeneration

cycle, but remained around 24% for all following regeneration cycles. While the colour removal

for some clays decreased after the first regeneration cycle, in general, after the first regeneration,

the ability of the clays to remove colour remained constant over five regeneration cycles.

Regeneration is an important, often overlooked, aspect of photocatalyst. Hadjltaief et al. (2015)

examined the ability of TiO2 supported on natural clays and on activated carbon to repeatedly

degrade methyl green under optimized conditions (initial dye concentration, irradiation time,

photocatalyst concentration). The decrease in degradation efficiency was not significant over 5

trials but the TiO2 powder showed a decrease in activity over the 5 trials. In the work performed

by Miranda-García et al. (2014), different strategies for regeneration were investigated and heat

treatment and H2O2 under UV light were the most effective regeneration methods. However, these

required access to materials and equipment, which might decrease user uptake. As such, it is

beneficial that the clay pieces can be reused without treatment at least 5 times and maintain the

majority of their photocatalytic activity.

5.3.5 E. coli inactivation To perform E. coli testing, WSC and LR clays were prepared as they removed the most colour in

the MB and AO7 tests. A 0.1 g/L TiO2 solution was also tested as a positive control. The results

from E. coli testing are shown in Figure 5-11. A control containing only Ringer’s solution was

performed to ensure that the experimental setup did not contribute contamination.

S. M. Larlee 65

Figure 5-11: E. coli inactivation for WSC and LR clays, vertical bars represent maximum

and minimum for experimental replicates where n=2

The E. coli solution under sun only conditions only had an inactivation of 0.25-log. It should be

noted that while the temperature did increase from 19.5°C to 27°C in the solar simulator, in real

SODIS conditions, the temperature increase would likely be much greater; temperatures typically

reached in SODIS bottles are approximately 40°C (Eawag/Sandec, 2016). Therefore, higher

inactivation may be observed in true SODIS conditions.

The Ringer’s only samples consistently showed zero CFU, meaning that no contamination was

detected in the experimental setup. The positive control TiO2 had over 4-log removal during the 2

hr period. During the same time period, WSC under sunlight had a 1-log removal. Interestingly,

for the LR samples under sunlight, the E. coli was below the detection limit, even without dilutions,

after one hour of sunlight exposure. The 40 min plates are shown in Figure 5-12. To ensure that

the E. coli removal was not just due to adsorption, dark controls for both clays were performed.

WSC showed no removal of E. coli in the dark over the 2 hr, while the LR showed similar removal

0

1

2

3

4

5

6

7

8

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

log

CFU

/mL

Time (min)

SUN only LR SUN LR DARK WSC SUN

WSC DARK TiO2 Control (0.1 g/L) Ringers only

DARK

S. M. Larlee 66

to the sun only condition. Therefore, it appears that clay can act as a photocatalyst for SODIS, and

in the case of the LR clay, can be more effective than TiO2. This is a very positive finding as the

clay can be easily removed from the water, unlike TiO2.

Figure 5-12: Agar plates showing E. coli log-removal after 40 min of sunlight exposure with

LR clay (100 (top) and 10-1 dilutions shown)

5.3.6 Detection and quantification of hydroxyl radicals and photogenerated electron holes

TPA was used as a probe molecule in order to detect the formation of hydroxyl radicals. TPA

interacts with hydroxyl radicals to produce HTPA, a compound that can be detected by

fluorescence (Ishibashi et al., 2000). The results of the TPA test are found in Figure 5-13.

S. M. Larlee 67

Figure 5-13: Hydroxyl radical generation for each clay type, vertical bars represent the

standard deviation of experimental replicates where n=3

For all clays and for the sun only samples, hydroxyl radical concentrations increased linearly over

time. While the SR and WSC clays produced the highest concentration of hydroxyl radicals at

0.112 and 0.111 mM HTPA, respectively, they were slightly less than the concentration produced

by the sun only samples (0.115 mM HTPA). Solar irradiation is known to produce hydroxyl

radicals in water; this is one of the mechanisms by which SODIS disinfects water (McGuigan et

al., 2012). The clays that produced the least amount of hydroxyl radicals, PHB, LR, and TR

produced 0.101, 0.097, and 0.090 mM HTPA, respectively. When the final HTPA concentration

was analysed using t-tests, no significant difference at the 95% confidence level was found

between the sun only samples and SR, PHB, and WSC clays. The rate constants for each of the

clay types are shown in Table 5-6. Based on the data, the concentration of HPTA increases linearly

over time. Therefore, zero-order rate constants were calculated.

0.05

0.07

0.09

0.11

0.13

0 10 20 30 40 50 60 70 80

HTP

A C

once

ntra

tion

(µM

)

Time (min)

TR SR PHB WSC LR Sun only

DARK

S. M. Larlee 68

Table 5-6: Hydroxyl radical rate constant

Rate Constant (µM/min) r2 WSC -0.0007 0.9952 TR -0.0004 0.9632 SR -0.0005 0.9858 PHB -0.0007 0.9959 LR -0.0005 0.9970 Sun only -0.0006 0.9076

When compared to other materials that have been analyzed with the TPA test, the clay samples

produce a low concentration of hydroxyl radicals; during the same amount of time, Arlos et al.

(2016) found that their TiO2 immobilized on porous titanium sheets produced approximately 10

times the concentration of hydroxyl radicals under UV-LED irradiation. Turolla et al. (2015) used

the same test and examined Aeroxide P25 TiO2 under UVA irradiation. During a 30 minute

irradiation period, approximately 10 times the total hydroxyl radical concentration of the clays was

achieved. Based on the hydroxyl radical detection data, it is likely that the model dye bleaching

and E. coli inactivation is not based on hydroxyl radical production from the clay because the sun

only samples produced the highest concentration.

Potassium iodine was used to detect the presence of h+. The results of the iodine test are found in

Figure 5-14. As previously noted, the concentration of photogenerated holes is twice the

concentration of the produced iodine.

S. M. Larlee 69

Figure 5-14: Photoelectron hole generation for each clay type, vertical bars represent the

standard deviation of experimental replicates where n=3

Generally all samples showed an increasing linear trend, with the exception of PHB, which showed

a slight decrease at the 70 min mark. All final iodine concentrations were all within a very narrow

range: 0.016 to 0.040 mM. The sun only sample fell in the middle of the range with a final iodine

concentration of 0.021 mM. Because all concentrations were within a small range, and at the

middle of that range was the sun only samples, it cannot be concluded that the photogenerated

holes are produced by the clay, or that the clay produces a higher concentration than the sun only.

When analysed using the ANOVA test, no significant difference was detected between all the clays

and the sun only samples (95% confidence interval). The rate constants for each of the clay types

are shown in Table 5-7. As with the HTPA data, the iodine concentration increased linearly over

time, so zero-order rate constants were calculated.

0

0.02

0.04

0.06

0.08

0 10 20 30 40 50 60 70 80

I 2C

once

ntra

tion

(mM

)

Time (min)

TR SR PHB WSC LR Sun only

DARK

S. M. Larlee 70

Table 5-7: Photoelectron hole rate constant

Rate Constant (mM/min) r2 WSC -0.0003 0.9797 TR -0.0004 0.9937 SR -0.0004 0.9790 PHB -0.0003 0.6494 LR -0.0006 0.9409 Sun only -0.0003 0.9785

Results from the iodine tests are lower than the concentrations achieved in Turolla et al. (2015);

under the same experimental conditions described above for the hydroxyl radical test,

approximately double the iodine concentration was achieved by P25 under UVA light.

Based on both the hydroxyl radical detection data and the photogenerated holes data, it is likely

that the model dye bleaching and E. coli inactivation is not only due to these components.

5.4 Summary When considering SODIS improvements, it is important to be aware that SODIS is already a low-

cost water treatment technique and that any improvements must be inexpensive and easy to apply.

The goal of this work was to use fired clay, an inexpensive and accessible material, as a

photocatalyst for SODIS. Five different clay types were investigated, all with varying

compositions. The clays were shaped by hand, fired and then analysed based on their ability to

remove colour from tow model dye solutions (MB and AO7) and inactivate E. coli. Clays were

characterized using SEM and XRF and the concentration of hydroxyl radicals and photogenerated

holes was determined to understand the photocatalytic mechanism of the clays. The clay that

performed the best was the LR clay; it had the highest colour removal for both MB and AO7 and

was able to reduce E. coli to below the detection limit within one hour of sunlight exposure. Based

on dark adsorption tests and sunlight tests, it was determined that the clay is photocatalytic.

However, the hydroxyl radical and photogenerated hole test results were inconclusive as to how

the clay is able to bleach model dyes and inactivate E. coli. The results of this study indicate that

the particular characteristics of LR clay make it a possible photocatalyst for SODIS.

S. M. Larlee 71

Chapter 6 Conclusions

Conclusions SODIS is currently practiced by over 5 million people worldwide and involves adding potentially

contaminated water to clear bottles and leaving them in full sunlight for a minimum of 6 hr. It is a

low-cost process but is time-consuming, has limited effectiveness against certain microorganisms,

and there is potential for regrowth. Therefore, the goal of this thesis was to find a low-tech,

accessible photocatalyst that can be prepared and used by those that practice SODIS.

6.1 Low-tech C- and N-doped TiO2 for SODIS A commonly investigated material for improving SODIS is TiO2, a photocatalyst that is activated

under UV light. However, only approximately 5% of sunlight is within the UV range. Doping is a

common strategy used to make TiO2 solar active. It involves incorporating other elements into the

structure of the TiO2 and, of the dopants commonly investigated, carbon and nitrogen have been

found to create a promising photocatalyst for use in sunlight applications. In this work, urea was

used as the source of carbon and nitrogen and combined with TiO2 then coated onto fired clay

pieces. Different ratios of TiO2 to urea were investigated, as well as different calcination

temperatures. The pieces were analysed by SEM and the ability of the coated clay pieces to remove

colour from an MB solution was tested. Based on the SEM images, the coating was irregular. The

MB results indicated that neither the calcination temperature, nor the TiO2 to urea ratio improved

the colour removal; all coated clays removed between 30% and 57% colour removal. Surprisingly,

the uncoated clay performed just as well, if not better, than the coated clay pieces. The bare fired

clay was then investigated on its ability to remove colour from an MB solution. The clay that was

not exposed to sunlight only removed 6.0% colour within 70 min, while the clay pieces that were

exposed to sunlight removed 59.8% colour within 70 min. Therefore, it was concluded that the

fired clay was photocatalytic.

6.2 Fired clay as a photocatalyst for SODIS Based on the results of previous experiments, clay may be able to serve as a photocatalyst for

SODIS. Therefore, five clays with varying compositions were fired and investigated as possible

photocatalysts. They were evaluated based on their ability to remove colour from MB and AO7

S. M. Larlee 72

model dye solutions and their ability to inactivate E. coli. All clays were characterized using SEM

and XRF. To determine how the clays were able to bleach model dyes and inactivate E. coli, the

concentration of hydroxyl radicals and photogenerated holes generated by the clays were

quantified.

Both model dyes interacted differently with the clays. All clays removed between 33% and 51%

MB after one hr of sunlight exposure. However, the only clay that achieved a positive AO7

removal was LR (19% during one hr of sunlight); the other clays all had negative removals between

-1% and -3%.

Based on the clays that removed the most colour in the model dye tests, two clays were chosen to

test with E. coli: LR and WSC. The E. coli was undetectable after 60 min of sunlight exposure

with the LR clay. This was better removal than the positive control used, TiO2, which achieved a

4.5-log reduction after 2 hours. During the 2 hr exposure period, the WSC only achieved 1-log

reduction.

After analysing the clays by SEM, little difference was observed between the surface areas of the

clays. The XRF analysis provided the clay compositions and were correlated to model dye removal

results. With respect to the MB tests, significant correlation coefficients were those for CaO (-

0.68) and TiO2 (-0.75). However, negative correlations indicate that as the % composition

increases, the percent MB colour removal decreases. A significant positive correlation coefficient

for AO7 was Fe2O3 (0.78). Based on the correlation data, there does not appear to be a clear relationship

between individual photocatalic components and colour removal.

Although the mechanism by which the bare clays are able to bleach model dye solutions and

inactivate E. coli are not well understood at this point, the results of this work suggest that clay,

and in particular LR clay, may be able to serve as a low-tech and inexpensive photocatalyst for

SODIS.

6.3 Recommendations for future work The use of clays, in particular those with compositions similar to the LR clay used in this work,

may be a promising improvement for SODIS. Understanding the photocatalytic activity of the

S. M. Larlee 73

materials is important as it could provide information to the limitations of the clays. Therefore,

more testing to ascertain the photocatalytic capabilities of the clay components is important.

Determining photon efficiencies and treatment efficiencies with respect to fluence experienced by

the clay and the water are important. In addition, to be more certain of the photocatalytic ability of

the clay and to ensure that the materials will work in the field in SODIS conditions, the following

section outlines recommendations for future work.

Some research has found that regrowth of bacteria is possible with SODIS. Therefore, it is

recommended that regrowth of E. coli after exposure to the clays under sunlight be examined.

Although the reusability of the clays with MB was discussed in this work, the reusability with

respect to E. coli should also be studied. It may also be valuable to examine other strains of

bacteria, such as Salmonella typhimurium, that are not easily removed by SODIS alone.

Other practical considerations include the design of the fired clay and the type of water. SODIS is

generally practiced in clear bottles. Therefore, finding a design for the clay that can fit into bottles

without reducing the volume of the bottles drastically should be investigated. The water used in

these experiments was Milli-Q® water. Future experiments should be performed in natural waters

which contain natural organic matter or ions that could interfere with bacteria inactivation.

S. M. Larlee 74

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Appendices 8.1 Calibration curves

The following calibration curves were used to generate the data in the following Appendices.

Figure 8-1: MB Calibration Curve

y = 0.2131xR = 0.9971

0.00

0.50

1.00

1.50

2.00

2.50

0.0 2.0 4.0 6.0 8.0 10.0 12.0

Abs

orba

nce

at 6

64 n

m

Concentration (mg/L)

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Figure 8-2: AO7 Calibration Curve

Figure 8-3: HTPA Calibration Curve

y = 0.071xR = 0.9998

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0

Abs

orba

nce

at 4

85 n

m

Concentration (mg/L)

y = 130.72xR = 0.9996

0

20

40

60

80

100

120

140

0 0.2 0.4 0.6 0.8 1 1.2

Res

pons

e

Concentration (uM)

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Figure 8-4: Iodine Calibration Curve

y = 2.555xR = 0.8377

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18

Abs

orba

nce

at 5

85 n

m

Concentration (mM)

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8.2 Experimental data for Chapter 4 The following tables show the raw data for Chapter 4.

Table 8-1: Clay weights used for coated clay samples, n=24

Batch and Calcination Temperature Wet Weight (g) Fired Weight (g) Calcined at 250°C (Batch 14) 7.3301±0.0039 5.3087±0.0265 Calcined at 300°C (Batch 11) 7.3303±0.0019 5.3221±0.0254 Calcined at 350°C (Batch 12) 7.3303±0.0024 5.3176±0.0195 Calcined at 400°C (Batch 13) 7.3307±0.0030 5.3192±0.0152 Calcined at 450°C (Batch 15) 7.3302±0.0025 5.2872±0.0228

Table 8-2: Average MB absorbance data for 10 min of dark adsorption, n=2

Ratio (TiO2:urea)

Calcined at 250°C (Batch 14)

Calcined at 300°C (Batch 11)

Calcined at 350°C (Batch 12)

Calcined at 400°C (Batch 13)

Calcined at 450°C (Batch 15)

1:1 1.9549 1.8535 1.9153 1.9695 1.9941 2:1 2.0380 1.8554 1.9482 1.9813 1.9933 3:1 2.0112 1.8522 1.9109 1.9922 1.9992 4:1 1.9597 1.8653 1.8430 1.9825 1.9897 1:0 1.9539 1.9219 1.9238 1.9894 1.9977 uncoated 1.9970 1.9695 1.9510 2.0232 2.0209

Table 8-3: Average MB concentration data for 10 min of dark adsorption, n=2

Ratio (TiO2:urea)

Calcined at 250°C (Batch 14)

Calcined at 300°C (Batch 11)

Calcined at 350°C (Batch 12)

Calcined at 400°C (Batch 13)

Calcined at 450°C (Batch 15)

1:1 9.2978 9.3916 9.1979 9.4882 9.3346 2:1 9.6930 9.5795 9.3562 9.5448 9.3518 3:1 9.5658 9.3850 9.3397 9.4413 9.3796 4:1 9.3205 9.4512 8.8132 9.3954 9.3349 1:0 9.2933 9.2654 9.4030 9.4282 9.3725 uncoated 9.4980 9.4949 9.3301 9.5881 9.4812

Table 8-4: Percent MB colour removal for 10 min of dark adsorption, n=2

Ratio (TiO2:urea)

Calcined at 250°C (Batch 14)

Calcined at 300°C (Batch 11)

Calcined at 350°C (Batch 12)

Calcined at 400°C (Batch 13)

Calcined at 450°C (Batch 15)

1:1 7.0 ± 0.0% 6.1 ± 0.1% 8.0 ± 0.4% 5.1 ± 0.2% 6.7 ± 0.6% 2:1 2.5 ± 2.5% 4.2 ± 0.2% 6.4 ± 0.0% 4.6 ± 0.5% 6.5 ± 0.1% 3:1 4.3 ± 0.8% 6.1 ± 0.5% 6.6 ± 0.1% 5.6 ± 0.1% 6.2 ± 0.6% 4:1 6.8 ± 0.3% 5.5 ± 0.1% 11.9 ± 0.1% 5.7 ± 0.5% 6.3 ± 0.3% 1:0 7.1 ± 0.3% 7.3 ± 0.0% 6.0 ± 0.0% 6.0 ± 0.1% 6.7 ± 0.2% uncoated 5.0 ± 0.3% 5.1 ± 0.4% 6.7 ± 0.1% 4.1 ± 0.4% 5.2 ± 0.7%

S. M. Larlee 88

Table 8-5: Average MB absorbance data for 10 min of dark adsorption, followed by 60 min

of sunlight exposure, n=2

Ratio (TiO2:urea)

Calcined at 250°C (Batch 14)

Calcined at 300°C (Batch 11)

Calcined at 350°C (Batch 12)

Calcined at 400°C (Batch 13)

Calcined at 450°C (Batch 15)

1:1 1.3032 1.2830 1.4214 1.4100 1.2907 2:1 1.1835 1.2891 1.3388 1.4648 1.2880 3:1 1.1940 1.1715 1.2376 1.1614 0.9975 4:1 1.2532 1.3399 1.2868 1.1991 0.9805 1:0 1.2028 1.3076 0.8788 1.2488 0.9478 uncoated 0.5991 1.1878 0.9806 0.7384 0.1582

Table 8-6: Average MB concentration data for 10 min of dark adsorption, followed by 60

min of sunlight exposure, n=2

Ratio (TiO2:urea)

Calcined at 250°C (Batch 14)

Calcined at 300°C (Batch 11)

Calcined at 350°C (Batch 12)

Calcined at 400°C (Batch 13)

Calcined at 450°C (Batch 15)

1:1 6.1982 6.5009 6.8260 6.7925 6.0285 2:1 5.6290 6.4874 6.5434 7.0567 6.0155 3:1 5.6232 5.9358 6.0493 5.7787 4.6590 4:1 5.9020 6.7890 6.1535 5.9661 4.5793 1:0 5.6646 6.3040 4.2952 6.2134 4.4269 uncoated 2.8216 5.7264 4.6896 3.6741 0.7391

Table 8-7: Percent MB colour removal for 10 min of dark adsorption, followed by 60 min of

sunlight exposure, n=2

Ratio (TiO2:urea)

Calcined at 250°C (Batch

14)

Calcined at 300°C (Batch

11)

Calcined at 350°C (Batch

12)

Calcined at 400°C (Batch

13)

Calcined at 450°C (Batch

15) 1:1 38.0 ± 0.2% 35.0 ± 1.0% 31.7 ± 0.5% 32.1 ± 1.2% 39.7 ± 2.0% 2:1 43.7 ± 0.5% 35.1 ± 1.6% 34.6 ± 1.3% 29.4 ± 3.8% 39.8 ± 0.5% 3:1 43.8 ± 1.0% 40.6 ± 0.2% 39.5 ± 1.9% 42.2 ± 0.3% 53.4 ± 3.5% 4:1 41.0 ± 1.6% 32.1 ± 1.1% 38.5 ± 0.4% 40.3 ± 3.7% 54.2 ± 1.1% 1:0 43.4 ± 0.4% 37.0 ± 1.2% 57.0 ± 1.0% 37.9 ± 3.9% 55.7 ± 0.2% uncoated 71.8 ± 3.7% 42.7 ± 7.9% 53.1 ± 0.3% 63.3 ± 3.2 92.6 ± 2.6%

S. M. Larlee 89

Table 8-8: Average MB absorbance data for uncoated clay (raw data), n=4

Regimen Average Absorption ADS, fired only 2.035 ± 0.008 ADS+SUN, fired only 0.864 ± 0.347 MB only 1.870 ± 0.017 Dark control, fired (70 min) 1.955 ± 0.016 ADS, calcined at 450°C 2.027 ± 0.019 ADS+SUN, calcined at 450°C 0.904 ± 0.293

Table 8-9: Average MB concentration data for uncoated clay (raw data), n=4

Regimen Average Concentration ADS, fired only 9.484 ± 0.035 ADS+SUN, fired only 4.024 ± 1.617 MB only 9.534 ± 0.087 Dark control, fired (70 min) 9.403 ± 0.075 ADS, calcined at 450°C 9.445 ± 0.089 ADS+SUN, calcined at 450°C 4.212 ± 0.087

Table 8-10: Percent MB colour removal for uncoated clay (raw data), n=4

Regimen Average Reduction ADS, fired only 5.2 ± 0.4% ADS+SUN, fired only 59.8 ± 16.2% MB only 4.7 ± 0.6% Dark control, fired (70 min) 6.9 ± 2.0% ADS, calcined at 450°C 5.5 ± 0.9% ADS+SUN, calcined at 450°C 57.9 ± 13.7%

S. M. Larlee 90

Table 8-11: Metal analysis for LR clays (raw data), n=4

Total Metals Average (mg/L) Total Desorbed Metals (mg/L) Al 396.153 0.1766±0.0389 0.1640±0.0372 Ba 233.527 0.1353±0.0263 0.1275±0.0340 Be 313.042 0.0000±0.0000 0.00000.0000 Cu 327.393 0.0094±0.0057 0.0004±0.0004 Fe 238.204 0.0040±0.0005 0.0014±0.0018 Mg 285.213 0.2523±0.1393 0.2140±0.1491 Mn 257.610 0.0001±0.0000 0.0000±0.0000 P 213.617 0.0004±0.0003 0.0000±0.0000 Na 589.592 0.5803±0.0803 0.5055±0.0651 Zn 206.200 0.0529±0.0241 0.0053±0.0025 K 766.490 0.1076±0.0068 0.1014±0.0071 Si 251.611 1.4291±0.3099 1.3704±0.3013 Ti 334.940 0.0183±0.0159 0.0000±0.0000

Figure 8-5: Metals analysis for LR clays, vertical bars represent the standard deviation of

experimental replicates where n=4

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Al396.153

Ba233.527

Be313.042

Cu327.393

Fe238.204

Mg285.213

Mn257.610

P213.617

Na589.592

Zn206.200

K766.490

Si251.611

Ti334.940

Con

cent

ratio

n (m

g/L)

Total Metals Average Total Desorbed Metals

S. M. Larlee 91

8.3 Experimental data for Chapter 5 The following tables show the raw data for Chapter 5.

Table 8-12: Clay weights used in Chapter 5

Batch No.

19 20 21 22 23 Wet Weight (g) 7.3287±0.0026 7.3288±0.0025 7.3295±0.0027 7.3297± 0.0034 Fired Weight (g) 5.2892±0.0190 5.2774± 0.0133 5.2702±0.0065 5.3086± 0.0171

n 8 8 8 8 Wet Weight (g) 6.8872±0.0215 6.8894±0.0028 6.8899±0.0023 6.8946± 0.0084 Fired Weight (g) 5.2915±0.0215 5.2907±0.0253 5.2636±0.0053 5.3019±0.0243

n 8 8 8 8 Wet Weight (g) 7.0595±0.0117 7.0596±0.0020 7.0728±0.0359 7.0569±0.0045 Fired Weight (g) 5.3014±0.0117 5.3039±0.0150 5.2936±0.0066 5.3089±0.0078

n 8 8 8 8 Wet Weight (g) 6.9808±0.0085 6.9818±0.0032 6.9811±0.0017 6.9788±0.0040 6.9791±0.0051 Fired Weight (g) 5.3103±0.0085 5.3054±0.0090 5.3010±0.0110 5.3052±0.0056 5.3309±0.0131

n 8 8 8 8 8 Wet Weight (g) 7.5395±0.0178 7.5399±0.0024 7.5403±0.0028 7.5406±0.0054 Fired Weight (g) 5.2970±0.0178 5.2950±0.0099 5.2618±0.0078 5.3466±0.0174

n 8 8 8 8

Batch 19 was used for all MB sunlight experiments (including regeneration), Batch 20 was used for AO7 sunlight experiments, Batch 21

was used for OH radical and E. coli experiments, Batch 22 was used for E. coli experiments, and Batch 23 was used for MB and AO7 dark

adsorption and iodine experiments.

S. M. Larlee 92

Table 8-13: Average dark absorbance data for MB colour removal from adsorption, n=3

Time LR SR TR PHB WSC Initial 1.8763 ±0.0097 1.8799 ±0.0356 1.8642 ±0.0274 1.8355 ±0.0100 1.8882 ±0.0329

2 1.8566 ±0.0130 1.8363 ±0.0048 1.8304 ±0.0073 1.8445 ±0.0211 1.8707 ±0.0347 4 1.8751 ±0.0089 1.8303 ±0.0036 1.8250 ±0.0094 1.8457 ±0.0263 1.8473 ±0.0402 6 1.8685 ±0.0074 1.8131 ±0.0259 1.8164 ±0.0276 1.8464 ±0.0118 1.8206 ±0.0289 8 1.8682 ±0.0032 1.8035 ±0.0160 1.7991 ±0.0079 1.8286 ±0.0234 1.8048 ±0.0234

10 1.8712 ±0.0065 1.7715 ±0.0265 1.7802 ±0.0163 1.8494 ±0.0123 1.7856 ±0.0179 20 1.8494 ±0.0031 1.7121 ±0.0249 1.7391 ±0.0102 1.8266 ±0.0159 1.7145 ±0.0249 30 1.8274 ±0.0016 1.6492 ±0.0297 1.7067 ±0.0212 1.8103 ±0.0175 1.6465 ±0.0158 40 1.8095 ±0.0046 1.5921 ±0.0249 1.6629 ±0.0098 1.7989 ±0.0117 1.5790 ±0.0198 60 1.8005 ±0.0178 1.4919 ±0.0271 1.6034 ±0.0164 1.7799 ±0.0372 1.4627 ±0.0280

120 1.6956 ±0.0249 1.2560 ±0.0327 1.4514 ±0.0185 1.7157 ±0.0314 1.1591 ±0.0626 360 1.6036 ±0.0457 0.8664 ±0.0862 1.2881 ±0.0202 1.7646 ±0.0378 0.6759 ±0.0422

1440 1.7001 ±0.1382 0.1776 ±0.0078 0.8006 ±0.0297 1.8265 ±0.0922 0.0509 ±0.0090

Table 8-14: Average concentration data for MB colour removal from dark adsorption, n=3

Time LR SR TR PHB WSC Initial 9.8178 ±0.0507 9.8366 ±0.1863 9.7548 ±0.1431 9.6047 ±0.0521 9.8801 ±0.1724

2 9.7149 ±0.0679 9.6087 ±0.0251 9.5778 ±0.0381 9.6515 ±0.1105 9.7887 ±0.1813 4 9.8117 ±0.0466 9.5774 ±0.0186 9.5497 ±0.0490 9.6580 ±0.1377 9.6662 ±0.2105 6 9.7770 ±0.0388 9.4873 ±0.1353 9.5044 ±0.1443 9.6615 ±0.0616 9.5267 ±0.1512 8 9.7756 ±0.0167 9.4372 ±0.0835 9.4142 ±0.0415 9.5685 ±0.1224 9.4438 ±0.1224

10 9.7911 ±0.0342 9.2694 ±0.1388 9.3153 ±0.0854 9.6770 ±0.0646 9.3434 ±0.0934 20 9.6774 ±0.0162 8.9586 ±0.1305 9.0999 ±0.0534 9.5579 ±0.0834 8.9712 ±0.1305 30 9.5621 ±0.0082 8.6298 ±0.1556 8.9305 ±0.1111 9.4728 ±0.0916 8.6157 ±0.0828 40 9.4683 ±0.0241 8.3310 ±0.1304 8.7013 ±0.0511 9.4130 ±0.0615 8.2625 ±0.1034 60 9.4215 ±0.0933 7.8067 ±0.1417 8.3900 ±0.0857 9.3135 ±0.1946 7.6536 ±0.1466

120 8.8724 ±0.1304 6.5723 ±0.1710 7.5948 ±0.0970 8.9774 ±0.1643 6.0653 ±0.3276 360 8.3908 ±0.2392 4.5334 ±0.4509 6.7400 ±0.1057 9.2333 ±0.1978 3.5366 ±0.2209

1440 8.8960 ±0.7233 0.9291 ±0.0409 4.1892 ±0.1556 9.5572 ±0.4826 0.2663 ±0.0472

S. M. Larlee 93

Table 8-15: Average percent MB colour removal from dark adsorption, n=3

Time LR SR TR PHB WSC Initial 2.64% ±0.51% 2.46% ±1.86% 3.27% ±1.43% 4.76% ±0.52% 2.03% ±1.72%

2 3.67% ±0.68% 4.72% ±0.25% 5.02% ±0.38% 4.29% ±1.11% 2.93% ±1.81% 4 2.71% ±0.47% 5.03% ±0.19% 5.30% ±0.49% 4.23% ±1.38% 4.15% ±2.11% 6 3.05% ±0.39% 5.92% ±1.35% 5.75% ±1.44% 4.19% ±0.62% 5.53% ±1.51% 8 3.06% ±0.17% 6.42% ±0.83% 6.65% ±0.41% 5.12% ±1.22% 6.35% ±1.22%

10 2.91% ±0.34% 8.08% ±1.39% 7.63% ±0.85% 4.04% ±0.65% 7.35% ±0.93% 20 4.04% ±0.16% 11.16% ±1.31% 9.76% ±0.53% 5.22% ±0.83% 11.04% ±1.31% 30 5.18% ±0.08% 14.42% ±1.56% 11.44% ±1.11% 6.07% ±0.92% 14.56% ±0.83% 40 6.11% ±0.24% 17.39% ±1.30% 13.72% ±0.51% 6.66% ±0.61% 18.07% ±1.03% 60 6.57% ±0.93% 22.59% ±1.42% 16.80% ±0.86% 7.64% ±1.95% 24.11% ±1.47%

120 12.02% ±1.30% 34.83% ±1.71% 24.69% ±0.97% 10.98% ±1.64% 39.86% ±3.28% 360 16.79% ±2.39% 55.05% ±4.51% 33.16% ±1.06% 8.44% ±1.98% 64.93% ±2.21% 1440 11.79% ±7.23% 90.79% ±0.41% 58.46% ±1.56% 5.23% ±4.83% 97.36% ±0.47%

Table 8-16: Average dark absorbance data for AO7 colour removal from adsorption, n=3

Time LR SR TR PHB WSC Initial 0.6788 ±0.0158 0.6613 ±0.0001 0.6935 ±0.0129 0.6921 ±0.0018 0.6839 ±0.0097

2 0.6384 ±0.0098 0.6624 ±0.0005 0.6858 ±0.0024 0.6921 ±0.0024 0.6839 ±0.0019 4 0.6041 ±0.0136 0.6628 ±0.0015 0.6846 ±0.0030 0.6842 ±0.0067 0.6749 ±0.0020 6 0.5775 ±0.0115 0.6628 ±0.0014 0.6837 ±0.0021 0.6713 ±0.0094 0.6749 ±0.0027 8 0.5591 ±0.0128 0.6630 ±0.0029 0.6840 ±0.0033 0.6603 ±0.0130 0.6743 ±0.0025

10 0.5414 ±0.0121 0.6645 ±0.0016 0.6863 ±0.0033 0.6532 ±0.0157 0.6746 ±0.0023 20 0.4975 ±0.0136 0.6660 ±0.0027 0.6830 ±0.0011 0.6477 ±0.0216 0.6743 ±0.0040 30 0.4813 ±0.0140 0.6684 ±0.0021 0.6817 ±0.0009 0.6319 ±0.0302 0.6751 ±0.0070 40 0.4732 ±0.0147 0.6704 ±0.0026 0.6821 ±0.0015 0.6230 ±0.0330 0.6746 ±0.0082 60 0.4717 ±0.0135 0.6748 ±0.0017 0.6833 ±0.0035 0.6242 ±0.0331 0.6768 ±0.0074 120 0.5343 ±0.0369 0.6899 ±0.0036 0.6933 ±0.0053 0.6285 ±0.0077 0.6813 ±0.0070

S. M. Larlee 94

Table 8-17: Average concentration data for AO7 colour removal from dark adsorption, n=3

Time LR SR TR PHB WSC Initial 9.7689 ±0.2270 9.5172 ±0.0012 9.9802 ±0.1856 9.9603 ±0.0262 9.8430 ±0.1403

2 9.1876 ±0.1412 9.5333 ±0.0070 9.8707 ±0.0348 9.9603 ±0.0344 9.8430 ±0.0268 4 8.6937 ±0.1956 9.5386 ±0.0210 9.8524 ±0.0425 9.8466 ±0.0966 9.7137 ±0.0285 6 8.3114 ±0.1648 9.5389 ±0.0200 9.8402 ±0.0301 9.6612 ±0.1357 9.7138 ±0.0386 8 8.0469 ±0.1840 9.5419 ±0.0414 9.8446 ±0.0479 9.5027 ±0.1872 9.7040 ±0.0360

10 7.7919 ±0.1737 9.5629 ±0.0231 9.8767 ±0.0473 9.4008 ±0.2266 9.7087 ±0.0329 20 7.1600 ±0.1953 9.5851 ±0.0382 9.8295 ±0.0163 9.3211 ±0.3114 9.7043 ±0.0579 30 6.9264 ±0.2014 9.6201 ±0.0309 9.8104 ±0.0135 9.0939 ±0.4346 9.7157 ±0.1003 40 6.8100 ±0.2120 9.6479 ±0.0380 9.8175 ±0.0218 8.9656 ±0.4753 9.7092 ±0.1176 60 6.7890 ±0.1938 9.7116 ±0.0246 9.8340 ±0.0506 8.9833 ±0.4759 9.7401 ±0.1064 120 7.6893 ±0.5304 9.9286 ±0.0525 9.9784 ±0.0758 9.0457 ±0.1108 9.8056 ±0.1008

Table 8-18: Average percent AO7 colour removal from dark adsorption, n=3

Time LR SR TR PHB WSC Initial 2.21% ±2.27% 4.73% ±0.01% 0.09% ±1.86% 0.29% ±0.26% 1.47% ±1.40%

2 8.03% ±1.41% 4.57% ±0.07% 1.19% ±0.35% 0.29% ±0.34% 1.47% ±0.27% 4 12.97% ±1.96% 4.51% ±0.21% 1.37% ±0.42% 1.43% ±0.97% 2.76% ±0.28% 6 16.80% ±1.65% 4.51% ±0.20% 1.50% ±0.30% 3.29% ±1.36% 2.76% ±0.39% 8 19.45% ±1.84% 4.48% ±0.41% 1.45% ±0.48% 4.87% ±1.87% 2.86% ±0.36%

10 22.00% ±1.74% 4.27% ±0.23% 1.13% ±0.47% 5.89% ±2.27% 2.81% ±0.33% 20 28.33% ±1.95% 4.05% ±0.38% 1.60% ±0.16% 6.69% ±3.11% 2.86% ±0.58% 30 30.66% ±2.01% 3.70% ±0.31% 1.79% ±0.14% 8.97% ±4.35% 2.74% ±1.00% 40 31.83% ±2.12% 3.42% ±0.38% 1.72% ±0.22% 10.25% ±4.75% 2.81% ±1.18% 60 32.04% ±1.94% 2.78% ±0.25% 1.56% ±0.51% 10.07% ±4.76% 2.50% ±1.06%

120 23.03% ±5.30% 0.61% ±0.52% 0.11% ±0.76% 9.45% ±1.11% 1.84% ±1.01%

S. M. Larlee 95

Table 8-19: Average MB absorbance data from different clays, n=4

Clay Type Average Reduction (ADS)

Average Reduction (ADS+SUN)

Smooth Raku 1.908±0.008 1.203±0.029 Thompson Raku 1.947±0.009 1.424±0.034 Low Red 1.960±0.016 1.034±0.214 PHB 1.974±0.011 1.471±0.047 White Sculpture 1.886±0.011 1.114±0.050 No clay 1.939±0.009 1.767±0.043

Table 8-20: Average MB concentration data from different clays, n=4

Clay Type Average Reduction (ADS)

Average Reduction (ADS+SUN)

Smooth Raku 9.153±0.040 5.685±0.135 Thompson Raku 9.341±0.045 6.730±0.161 Low Red 9.403±0.076 4.890±1.012 PHB 9.469±0.051 6.953±0.224 White Sculpture 8.914±0.054 5.262±0.235 No clay 9.587±0.043 8.734±0.213

Table 8-21: Percent MB colour removal from different clays, n=4

Clay Type Average Reduction (ADS)

Average Reduction (ADS+SUN)

Smooth Raku 8.5 ± 0.4% 43.1 ± 1.3% Thompson Raku 6.6 ± 0.5% 32.7 ± 1.6% Low Red 6.0 ± 0.8% 51.1 ± 10.1% PHB 5.3 ± 0.5% 30.5 ± 2.2% White Sculpture 10.9 ± 0.5% 47.4 ± 2.4% No clay 4.1 ± 0.4% 12.7 ± 2.1%

S. M. Larlee 96

Table 8-22: Average AO7 absorbance data from different clays, n=4

Clay Type Average Reduction (ADS)

Average Reduction (ADS+SUN)

Smooth Raku 0.7030±0.002 0.7224±0.007 Thompson Raku 0.7045±0.002 0.7152±0.011 Low Red 0.5848±0.016 0.5726±0.074 PHB 0.6972±0.005 0.729±0.008 White Sculpture 0.7054±0.001 0.7184±0.010 No clay 0.7026±0.000 0.6810±0.013

Table 8-23: Average AO7 concentration data from different clays, n=4

Clay Type Average Reduction (ADS)

Average Reduction (ADS+SUN)

Smooth Raku 9.906±0.025 10.18±0.092 Thompson Raku 9.927±0.032 10.08±0.158 Low Red 8.240±0.227 8.068±1.040 PHB 9.825±0.072 10.27±0.111 White Sculpture 9.939±0.011 10.12±0.145 No clay 9.900±0.006 9.595±0.179

Table 8-24: Percent AO7 colour removal from different clays raw data, n=4

Clay Type Average Reduction (ADS)

Average Reduction (ADS+SUN)

Smooth Raku 0.9 ± 0.2% -1.8 ± 0.9% Thompson Raku 0.7 ± 0.3% -0.8 ± 1.6% Low Red 17.6 ± 2.3% 19.3 ± 10.4% PHB 1.7 ± 0.7% -2.7 ± 1.1% White Sculpture 0.6 ± 0.1% -1.2 ± 1.4% No clay 1.0 ± 0.1% 4.0 ± 1.8%

S. M. Larlee 97

Table 8-25: Average MB absorbance regeneration raw data ADS+SUN, n=4

Clay Type Original First Regeneration

Second Regeneration

Third Regeneration

Fourth Regeneration

Fifth Regeneration

Smooth Raku 1.203±0.029 1.237±0.022 1.237±0.040 1.298±0.025 1.348±0.038 1.298±0.033 Thompson Raku 1.424±0.034 1.363±0.030 1.368±0.028 1.448±0.042 1.470±0.037 1.366±0.043 Low Red 1.034±0.214 1.383±0.082 1.467±0.018 1.524±0.021 1.551±0.011 1.553±0.032 PHB 1.471±0.047 1.546±0.031 1.474±0.017 1.606±0.021 1.614±0.016 1.562±0.033 White Sculpture 1.114±0.050 1.035±0.030 1.035±0.049 1.167±0.026 1.167±0.016 1.136±0.029

Table 8-26: Average MB concentration regeneration raw data ADS+SUN, n=4

Clay Type Original First Regeneration

Second Regeneration

Third Regeneration

Fourth Regeneration

Fifth Regeneration

Smooth Raku 5.685±0.135 6.102±0.110 6.134±0.200 6.158±0.118 6.462±0.183 6.294±0.159 Thompson Raku 6.730±0.161 6.722±0.149 6.785±0.139 6.874±0.201 7.046±0.176 6.624±0.208 Low Red 4.890±1.012 6.822±0.402 7.275±0.090 7.232±0.101 7.435±0.052 7.530±0.157 PHB 6.953±0.224 7.627±0.154 7.308±0.083 7.623±0.097 7.734±0.075 7.571±0.162 White Sculpture 5.262±0.235 5.103±0.147 5.131±0.241 5.538±0.121 5.594±0.077 5.509±0.142

Table 8-27: Percent MB regeneration raw data ADS+SUN, n=4

Clay Type Original First Regeneration

Second Regeneration

Third Regeneration

Fourth Regeneration

Fifth Regeneration

Smooth Raku 43.1 ± 1.3% 39.0 ± 1.1% 38.7 ± 2.0% 38.4 ± 1.2% 35.4 ± 1.8% 37.1 ± 1.6% Thompson Raku 32.7 ± 1.6% 32.8 ± 1.5% 32.1 ± 1.4% 31.3 ± 2.0% 29.5 ± 1.8% 33.8 ± 2.1% Low Red 51.1 ± 10.1% 31.8 ± 4.0% 27.2 ± 0.9% 27.7 ± 1.0% 25.6 ± 0.5% 24.7 ± 1.6% PHB 30.5 ± 2.2% 23.7 ± 1.5% 26.9 ± 0.8% 23.8 ± 1.0% 22.6 ± 0.7% 24.3 ± 1.6% White Sculpture 47.4 ± 2.4% 49.0 ±1.5% 48.7 ± 2.4% 44.6 ± 2.0% 44.1 ± 0.8% 44.9 ± 1.4%

S. M. Larlee 98

Table 8-28: Average E. coli raw data, n=2, log (CFU/mL)

Time LR (sun) LR (dark) WSC (sun) WSC (dark) 0 7.06E+00 ±1.95E-01 7.113943 ±7.26E-02 6.39794 ±1.94E-01 7.190332 ±3.45E-01

10 7.40E+00 ±6.45E-02 7.30103 ±2.22E-01 6.267172 ±0.00E+00 6.977724 ±1.33E-01 40 3.50E+00 ±3.01E-01 7.060698 ±5.84E-01 6.113943 ±2.11E-01 6.778151 ±4.77E-01 70 0.00E+00 ±0.00E+00 6.90309 ±3.01E-01 5.667453 ±4.12E-01 7.029384 ±3.86E-01

100 0.00E+00 ±0.00E+00 6.954243 ±5.12E-02 5.469822 ±1.08E-01 7.130334 ±1.64E-02 130 0.00E+00 ±0.00E+00 6.916454 ±6.15E-01 5.469822 ±8.96E-02 7.361728 ±0.00E+00

Time Sun only TiO2 control (0.1 g/L) Ringer's only

0 7.28E+00 ±1.19E-01 6.69897 ±2.22E-01 0 ±0 10 7.32E+00 ±6.69E-02 7.20412 ±7.57E-02 0 ±0 40 7.27E+00 ±1.16E-02 4.021189 ±1.02E+00 0 ±0 70 7.00E+00 ±1.46E-01 3.770852 ±0.00E+00 0 ±0

100 7.27E+00 ±9.46E-02 3.897627 ±7.51E-01 0 ±0 130 7.02E+00 ±1.25E-01 2.190332 ±2.36E-01 0 ±0

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Table 8-29: Hydroxyl radical raw data, n=3

Clay Type Time Average Fluorescence

Intensity Average Concentration (mM/L HPTA)

TR 10 min dark 10.496 ±0.603 0.070 ±0.007 30 min 11.452 ±0.465 0.075 ±0.007 50 min 12.876 ±0.676 0.086 ±0.002 70 min 14.556 ±0.640 0.090 ±0.002 SR 10 min dark 10.496 ±0.603 0.081 ±0.005 30 min 11.452 ±0.465 0.088 ±0.004 50 min 12.876 ±0.676 0.099 ±0.005 70 min 14.556 ±0.640 0.112 ±0.005 PHB 10 min dark 7.993 ±0.154 0.062 ±0.001 30 min 9.666 ±0.181 0.075 ±0.001 50 min 11.114 ±0.555 0.086 ±0.004 70 min 13.120 ±1.346 0.101 ±0.010 WSC 10 min dark 8.601 ±0.281 0.066 ±0.002 30 min 10.894 ±0.092 0.084 ±0.001 50 min 12.630 ±0.402 0.098 ±0.003 70 min 14.389 ±0.666 0.111 ±0.005 LR 10 min dark 8.478 ±0.505 0.065 ±0.004 30 min 9.668 ±0.523 0.075 ±0.004 50 min 11.083 ±0.337 0.086 ±0.003 70 min 12.604 ±0.855 0.097 ±0.007 Sun only 10 min dark 10.309 ±1.428 0.080 ±0.011 30 min 13.136 ±1.178 0.101 ±0.009 50 min 13.730 ±1.328 0.106 ±0.010 70 min 14.932 ±1.004 0.115 ±0.008

Table 8-30: Hydroxyl radical rate constant data

Slope Intercept r2 WSC 0.0007 0.0603 0.9952 TR 0.0004 0.066 0.9632 SR 0.0005 0.0744 0.9858 PHB 0.0007 0.0549 0.9959 LR 0.0005 0.0595 0.997 Sun only 0.0006 0.0783 0.9076

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Table 8-31: Photogenerated hole raw data, n=3

Clay Type Time Average Absorbance Average Concentration (mM I2) TR 10 min dark 0.0174 ±0.0025 0.0068 ±0.0010 30 min 0.0389 ±0.0048 0.0152 ±0.0019 50 min 0.0596 ±0.0069 0.0233 ±0.0027 70 min 0.0747 ±0.0066 0.0292 ±0.0026 SR 10 min dark 0.0204 ±0.0106 0.0080 ±0.0042 30 min 0.0486 ±0.0142 0.0190 ±0.0056 50 min 0.0640 ±0.0087 0.0250 ±0.0034 70 min 0.0810 ±0.0090 0.0317 ±0.0035 PHB 10 min dark 0.0054 ±0.0029 0.0021 ±0.0011 30 min 0.0174 ±0.0120 0.0068 ±0.0047 50 min 0.0574 ±0.0465 0.0225 ±0.0182 70 min 0.0405 ±0.0320 0.0159 ±0.0125 WSC 10 min dark 0.0133 ±0.0063 0.0052 ±0.0025 30 min 0.0318 ±0.0048 0.0125 ±0.0019 50 min 0.0420 ±0.0016 0.0164 ±0.0006 70 min 0.0533 ±0.0021 0.0209 ±0.0008 LR 10 min dark 0.0110 ±0.0081 0.0043 ±0.0032 30 min 0.0186 ±0.0073 0.0073 ±0.0029 50 min 0.0628 ±0.0555 0.0246 ±0.0217 70 min 0.0981 ±0.0569 0.0384 ±0.0223 Sun only 10 min dark 0.0101 ±0.0055 0.0039 ±0.0022 30 min 0.0295 ±0.0090 0.0115 ±0.0035 50 min 0.0446 ±0.0124 0.0175 ±0.0048 70 min 0.0542 ±0.0088 0.0212 ±0.0034

Table 8-32: Photogenerated hole rate constant data

Slope Intercept r2 WSC 0.0003 0.0036 0.9797 TR 0.0004 0.0036 0.9937 SR 0.0004 0.0055 0.9790 PHB 0.0003 0.0004 0.6494 LR 0.0006 -0.0053 0.9409 Sun only 0.0003 0.0020 0.9785

S. M. Larlee 101

8.4 Statistical analysis for Chapter 5 Table 8-33: ANOVA single factor for MB colour removal

SUMMARY Groups Count Sum Average Variance

SR 4 1.725844 0.431461 0.000182 TR 4 1.307838 0.326959 0.00026 LR 4 2.043952 0.510988 0.010239 PHB 4 1.218926 0.304731 0.000504 WSC 4 1.895086 0.473772 0.000552 No clay 4 0.506477 0.126619 0.000454 ANOVA Source of Variation SS df MS F P-value F crit

Between Groups 0.397703 5 0.079541 39.14479 4.67E-

09 2.772853 Within Groups 0.036575 18 0.002032 Total 0.434278 23

Table 8-34: ANOVA single factor for AO7 colour removal Anova: Single Factor SUMMARY

Groups Count Sum Average Variance SR 4 -0.07188 -0.01797 8.43E-05 TR 4 -0.03103 -0.00776 0.00025 LR 4 0.772753 0.193188 0.010819 PHB 4 -0.10929 -0.02732 0.000124 WSC 4 -0.04896 -0.01224 0.000209 No clay 4 0.161886 0.040471 0.000319 ANOVA Source of Variation SS df MS F P-value F crit

Between Groups 0.142058 5 0.028412 14.43872 9.21E-

06 2.772853 Within Groups 0.035419 18 0.001968 Total 0.177477 23

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Table 8-35: T-test results for MB colour removal. Highlighted results indicate significant difference between clay types.

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Table 8-36: T-test results for AO7 colour removal. Highlighted results indicate significant difference between clay types.

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8.5 Additional information for Chapter 5: Determining the effect of clay weight on MB colour removal

Initial experiments were performed to determine the effect of the fired clay weight on its ability to

remove colour from an MB solution. The following sections describe how the different weights of

clay pieces were determined, the colour removal each was able to achieve, as well as the raw data.

8.5.1 Methods Clay substrates were shaped by hand and weighed out as wet clay. Initially, approximately 7.33 g

of each clay was weighed and shaped into a circular “button” shape, which was approximately 2

cm in diameter as shown in Figure 8-6. Eight pieces were made from each clay type.

Figure 8-6: Clay piece dimensions

The clay pieces air dried for a minimum of 2 days, then were fired at 2°C/min in a 1100°C Box

Furnace BF51800 Series Muffle Furnace (Fisher Scientific) to the temperature recommended by

the manufacturer. Low Red (LR) clay was fired to Cone 06 (999°C), Thompson Raku (TR) and

Smooth Raku (SR) were fired to Cone 04 (1060°C), and PHB and White Sculpture Clay (WSC)

was fired to Cone 05 (1046°C). The clay pieces cooled for a minimum of 12 hr. The wet and fired

clay weights are shown in Table 8-37.

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Table 8-37: Wet and fired clay weights starting weight of approximately 7.33 g, n=8

Clay Type Average Wet Weight (g) Average Fired Weight (g)

Smooth Raku (SR) 7.3277 ± 0.0029 5.6468 ± 0.0210

Thompson Raku (TR) 7.3309 ± 0.0028 5.5068 ± 0.0185

Low Red (LR) 7.3292 ± 0.0025 5.3131 ± 0.0161

PHB 7.3281 ± 0.0019 5.1645 ± 0.0123

White Sculpture (WSC) 7.3274 ± 0.0018 5.5796 ± 0.0112

Based on the clay weight data in Table 8-37, the shrinkage rate of each clay was calculated by

dividing the average wet weight by the average fired weight. To determine the effect of clay weight

on the photocatalytic ability of the clay materials, new clay pieces were prepared by calculating

the new starting weight based on the shrinkage rate so that each clay piece would have a similar

fired clay weight. 5.31 g was chosen as the desired fired weight as all previous clay experiments

were performed with LR clay, which was weighed out as 7.33 g when wet and weighed

approximately 5.31 g when fired. Table 8-38 shows the data used to calculate the required wet

weights.

Table 8-38: Shrinkage rates of different clays used to determine required wet weight to

achieve similar fired weights

Clay Type Shrinkage Rate Required Wet Weight (g)

Smooth Raku (SR) 1.30 6.89

Thompson Raku (TR) 1.33 7.06

Low Red (LR) 1.38 7.33

PHB 1.42 7.54

White Sculpture (WSC) 1.31 6.98

Eight clay pieces were made from each clay type to account for shrinkage and were fired in the

same way as all previous clay pieces. The wet and fired clay weights are shown in Table 8-39. In

the following sections, Scenario 1 will refer to the clay pieces which all had a starting weight of

7.33 g, while Scenario 2 will refer to the clay pieces which had varying starting weights and a final

fired weight of approximately 5.31 g.

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Table 8-39: Wet and fired clay weights, varying starting weights for final fired weight of 5.31

g, n=8

Clay Type Average Wet Weight (g) Average Fired Weight (g)

Smooth Raku (SR) 6.8904 ± 0.0025 5.3134 ± 0.0170

Thompson Raku (TR) 7.0616 ± 0.0021 5.3210 ± 0.0098

Low Red (LR) 7.3283 ± 0.0029 5.3040 ± 0.0308

PHB 7.5373 ± 0.0018 5.3188 ± 0.0188

White Sculpture (WSC) 6.9796 ± 0.0027 5.3252 ± 0.0092

8.5.2 Results Results from 10 min of dark adsorption, as well as 10 min of dark adsorption plus 60 min of

sunlight exposure for each clay type under Scenario 1 are shown in Figure 8-7. Figure 8-8 shows

the same test results for Scenario 2.

Figure 8-7: Percent MB colour removal for each clay type (Scenario 1), vertical bars

represent the standard deviation of experimental replicates where n=4

6.9% 4.4% 4.1% 4.9%

12.7%

36.5%43.0% 42.2% 40.2%

49.2%

0%

20%

40%

60%

80%

100%

Smooth Raku Thompson Raku Low Red PHB White Sculpture Clay

Perc

ent M

B c

olou

r rem

oval

Clay Type

Average Reduction (ADS) Average Reduction (ADS+SUN)

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Figure 8-8: Percent methylene blue colour removal for each clay type (Scenario 2), vertical

bars represent the standard deviation of experimental replicates where n=4

Figure 8-9 compares the percent colour removal of the MB solution achieved by each clay weight in

Scenario 1 to each clay weight in Scenario 2.

8.8% 7.0% 4.6% 6.4%11.4%

35.2%28.7% 27.0% 27.7%

43.7%

0%

20%

40%

60%

80%

100%

Smooth Raku Thompson Raku Low Red PHB White Sculpture

Perc

ent M

B C

olou

r Rem

oval

Clay Type

Average Reduction (ADS) Average Reduction (ADS+SUN)

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Figure 8-9: Comparison between Scenario 1 and Scenario 2 MB colour removal from 10 min of

dark adsorption and from 10 min of dark adsorption followed by 60 min of sunlight exposure

To better visualize the effect of the clay weight on the colour removal, Figure 8-10 was generated.

It compares the adsorption plus sunlight data to the fired clay weight data.

Figure 8-10: Fired clay weights plotted with colour removal

0%

20%

40%

60%

Smooth Raku Thompson Raku Low Red PHB White Sculpture

Perc

ent M

B c

olou

r re

mov

al

Clay Type

Scenario 1, ADS only Scenario 2, ADS only Scenario 1, ADS+SUN Scenario 2, ADS+SUN

5.65 5.51 5.31 5.165.58

37% 43% 42% 40% 49%

5.31 5.32 5.30 5.32 5.33

35% 29% 27% 28% 44%

Smooth Raku Thompson Raku Low Red PHB White Sculpture

Scenario 1 Fired Clay Weight (g) Scenario 1 Colour Removal (ADS+SUN)

Scenario 2 Fired Clay Weight (g) Scenario 2 Colour Removal (ADS+SUN)

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The percent colour removal decreased for all clay types from Scenario 1 to Scenario 2. This result

was unexpected for the PHB as the wet weight increased, but the colour removal decreased from

40.2% to 27.7%. This was also unexpected for the LR clay because the wet and fired weights did

not change between Scenario 1 and Scenario 2, but the colour removal decreased by 15.2%.

Furthermore, in Scenario 1, the average fired weight of the SR pieces was 5.6468 g, and in Scenario

2 the average fired weight was 5.3134 g. This represents a 0.3334 g decrease, but the colour

removal only decreased by 1.3%. WSC clay had the highest percent MB colour removal for both

Scenario 1 and Scenario 2 with 49.2% and 43.7% colour removal, respectively. SR showed the

lowest colour removal for Scenario 1, but had the second highest colour removal for Scenario 2.

In Scenario 1, the TR, the LR, and the PHB performed within the range of 40.2% to 43.0%. While

the colour removal in Scenario 2 was an average of 14.0% lower, all three clays still performed

similarly, all falling within the range of 27.0% to 28.7%.

The data from Scenario 1 and Scenario 2 were normalized based on clay weight shown in Figure

8-11. Because the mg/L of MB removed per gram of clay changes between Scenarios, there is

likely another factor to consider in addition to weight, such as surface area, when determining the

photocatalytic ability of the clays.

Figure 8-11: Scenarios 1 and 2 MB colour removal normalized by weight

-

0.2

0.4

0.6

0.8

1.0

SR LR PHB WSC TR

mg/

L of

MB

rem

oved

per

g o

f cla

y

Clay Type

ADS Average (Scenario 1) ADS+SUN Average (Scenario 1)

ADS Average (Scenario 2) ADS+SUN Average (Scenario 2)

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8.5.3 Raw Data

Table 8-40: Clay weights used in Section 8.4

Batch 16 clay pieces all had starting weights of 7.33 g, while Batch 17 clay pieces had varying

start weights and accounted for shrinkage.

Batch No.

16 17 Wet Weight (g) 7.3309±0.0029 7.3283±0.0029 Fired Weight (g) 5.3130±0.0029 5.3040±0.0308

n 16 8 Wet Weight (g) 7.3277±0.0029 6.8904±0.0025 Fired Weight (g) 5.6468±0.0210 5.3134± 0.0170

n 8 8 Wet Weight (g) 7.3282±0.0029 7.0616±0.0021 Fired Weight (g) 5.5185±0.0210 5.3210±0.0099

n 8 8 Wet Weight (g) 7.3279±0.0023 6.9796±0.0027 Fired Weight (g) 5.5791±0.0112 5.3252±0.0092

n 8 8 Wet Weight (g) 7.3281±0.0019 7.5373±0.0018 Fired Weight (g) 5.1645±0.0123 5.3188±0.0188

n 8 8

Table 8-41: Percent MB colour removal from different clays raw data (0.85 sun, 7.33 starting

weight), n=4

Clay Type Average Reduction (ADS)

Average Reduction (ADS+SUN)

Smooth Raku 6.9 ± 0.8% 36.6 ± 2.4% Thompson Raku 4.5 ± 0.5% 42.9 ± 1.1% Low Red 4.0 ± 1.0% 42.1 ± 1.8% PHB 4.8 ± 0.6% 40.1 ± 3.2% White Sculpture 12.8 ± 1.1% 49.4 ± 1.5%

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Table 8-42: Percent MB colour removal from different clays raw data (0.85 sun, varying

starting weights), n=4

Clay Type Average Reduction (ADS)

Average Reduction (ADS+SUN)

Smooth Raku 8.7 ± 0.8% 35.1 ± 1.6% Thompson Raku 6.9 ± 0.7% 28.6 ± 1.9% Low Red 4.6 ± 0.2% 27.1 ± 5.4% PHB 6.5 ± 0.4% 27.6 ± 1.3% White Sculpture 11.3 ± 1.3% 43.5 ± 1.4%

Table 8-43: Normalization by weight of percent MB colour removal (mg/L of MB removal

per gram of clay)

Scenario 1 Scenario 2

ADS Average (Batch 16)

ADS+SUN Average

(Batch 16)

ADS Average (Batch 17)

ADS+SUN Average

(Batch 17) Smooth Raku 0.12 ± 0.01 0.65 ± 0.04 0.17 ± 0.01 0.66 ± 0.03 Thompson Raku 0.08 ± 0.01 0.80 ± 0.04 0.09 ± 0.01 0.51 ±0.10 Low Red 0.10 ± 0.01 0.78 ± 0.06 0.12 ±0.01 0.52 ± 0.02 PHB 0.23 ± 0.01 0.88 ± 0.03 0.21 ± 0.03 0.82 ± 0.03 White Sculpture 0.08 ± 0.01 0.78 ± 0.02 0.13 ± 0.01 0.54 ± 0.04

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8.6 Clay composition data

Table 8-44: Clay content (%) data and firing temperature provided with each clay MSDS

(Tucker’s Pottery Supply)

Component Clay Type

Low Red PHB Thompson Raku Smooth Raku White

Sculpture BaO 0.4 0.3 CaO 0.25 0.25 2.19 0.33 0.2 MnO2 0.01 MgO 6.56 6.5 2.01 0.16 0.16 K2O 2.64 2.2 0.75 1.45 0.84 Na2O 0.1 0.11 0.59 2.39 1.4 Fe2O3 5.75 4.01 1.15 0.86 0.73 TiO2 1 1.21 1.52 0.83 0.79 Al2O3 17.1 20.76 32.21 34.48 32.46 SiO2 66.17 64.62 59.49 59.5 63.37 P2O5 0.02 0.02 0.08 0.01 0.06

Firing Temperature

Cone 06 (999°C)

Cone 06 (999°C) or Cone 04 (1060°C)

Cone 04 (1060°C)

Cone 04 (1060°C)

Cone 05 (1046°C)

Table 8-45: Clay content (%) data from XRF analysis

Component Clay Type

Low Red PHB Thompson Raku Smooth Raku White

Sculpture CaO 3.794 5.444 10.483 2.756 3.605 MgO 5.53 5.161 3.226 0.77 1.449 K2O 2.792 1.855 0.604 1.401 0.825 Na2O 2.498 2.602 2.409 Fe2O3 5.977 4.014 1.621 0.88 0.794 TiO2 0.957 1.194 1.704 1.017 0.709 Al2O3 19.284 19.865 22.075 26.469 25.656 SiO2 58.419 60.111 59.383 63.256 64.036 P2O5 0.147 0.001 0.186 0.065 0.288

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8.7 Clay safety data sheets from Tucker’s Pottery Supplies

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8.8 Experimental procedures 8.8.1 Preparation of clays Clay was used in the experiments in Chapter 4 as a support for the C-N/TiO2 and in Chapter 5 as

the photocatalyst itself. To prepare the clay, a mass of wet clay was weighed out and shaped into

a button-shape, air-dried, then fired in a muffle furnace that was programmed to act as a kiln. The

following sections outline how weight out clay and how to fire it in the muffle furnace.

Methods

Preparing clay pieces

1. Weigh out required amount of clay onto a weigh dish based on the clay type. If accounting

for shrinkage, the starting weights for each clay to achieve roughly the same fired weight

are shown in Table 8-46.

Table 8-46: Starting clay weights to account for shrinkage

Clay Type Wet weight (g)

Smooth Raku (SR) 6.89

Thompson Raku (TR) 7.06

Low Red (LR) 7.33

PHB 7.54

White Sculpture (WSC) 6.98

2. Shape the clay into a 2 cm diameter button-shape, working as quickly as possible so the

clay does not dry out.

3. Note the exact wet weight of the clay.

4. Allow the clay pieces to air dry for at least 2 days before firing.

Firing clay pieces

1. Program the muffle furnace to increase by 2°C/min.

2. Put the clay pieces that require the same firing temperature in the muffle furnace. Note that

only 16 pieces can fit in the muffle furnace at once. The manufacturer-specified

temperature are identified in Table 8-47.

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Table 8-47: Clay firing temperatures

Clay Type Firing Temperature

Smooth Raku (SR) Cone 04 (1060°C)

Thompson Raku (TR) Cone 04 (1060°C)

Low Red (LR) Cone 06 (999°C)

PHB Cone 06 (999°C) or Cone 04 (1060°C)

White Sculpture (WSC) Cone 05 (1046°C)

3. Once the temperature is reached, turn the muffle furnace off and allow the clay to cool for

at least 12 hr.

4. Remove the clay from the muffle furnace and weigh.

8.8.2 Preparation of coated clays

TiO2 is a photocatalytic material that produces ROS when radiated with UV light. To create a

photocatalyst that is solar-active, the TiO2 was doped with urea to alter the band gap and create

carbon- and nitrogen-doped TiO2. Initially, varying masses of urea and TiO2 were mixed with

Milli-Q® water to make a slurry in which the clay pieces were soaked. The coated clay pieces

were dried and calcined at varying temperatures then tested under the PET solar simulator for their

ability to remove colour from an MB solution. The following sections outline how to prepare

coating solutions, coat the clay pieces, and calcine the coated clay pieces.

Methods

1. Prepare Low Red clay pieces as outlined in Section 8.8.2.

2. Make 10% w/v solutions with the following weights of TiO2 and urea in 20 mL of Milli-

Q® water in glass vials.

S. M. Larlee 122

Table 8-48: Coating solution ratios

Ratio (TiO2:urea) Weight of TiO2 (g) Weight of urea (g)

1:1 1.00 1.00

2:1 1.33 0.67

3:1 1.50 0.50

4:1 1.60 0.40

1:0 2.00 -

3. Sonicate each solution for 5 min then pour into 50 mL beakers.

4. Immerse a clay piece into each slurry and leave for 15 min per side.

5. Remove the substrates from the slurry and place on drying rack so that the excess solution

can drip off.

6. Leave to air dry for one day

7. Dry the substrates at 100°C in the muffle furnace for 1 hour

8. Calcinate the substrates for 2 hr in the muffle furnace at varying temperatures (300°C,

350°C, 400°C, 450°C).

9. Rinse the substrates with 35 mL of Milli-Q® water under constant stirring to remove any

excess coating solution.

10. Allow the substrates to air dry overnight

11. Dry clay substrates in a muffle furnace at 100°C for one hour

12. Weigh each coated substrate once dried to determine the weight of coating

8.8.3 Organic dye concentration measurements

Organic dyes absorb light in proportion to their concentration and the absorption can be measured

with a UV-vis spectrophotometer. The peak absorption wavelength is specific for each dye. For

the two organic dyes selected, methylene blue (MB) and Acid Orange 7 (AO7), the peak absorption

wavelengths are 664 nm and 485 nm, respectively. To determine the percent colour removal from

the organic dye solutions, a high concentration stock solution was prepared. From the stock

solution, a lower concentration, working solution was prepared which was used for testing and for

preparing the calibration standards of known concentration. From the absorption results of the

calibration standards, a calibration curve was prepared from which the concentration of unknown

S. M. Larlee 123

samples could be inferred. The following sections outline how to prepare stock solutions, working

solutions, calibration standards, and how to analyse samples.

Methods

Stock solution (1000 mg/L)

1. Weigh 500 mg of dye and add it to a 500 mL volumetric flask

2. Bring flask to the 500 mL mark with Milli-Q® water. Insert stopper into flask and invert

until the dye is dissolved.

3. Transfer the solution to an amber vial and keep in the 4°C fridge until use.

Working Solution (10 mg/L)

1. Using a pipette, transfer 10 mL of the stock solution to a 1 L volumetric flask.

2. Bring flask to the 1 L mark with Milli-Q® water. Insert stopper into flask and invert to

mix.

Calibration Standards (0.625 mg/L to 5 mg/L)

1. Using a pipette, transfer the required volume of the working solution (see table below) to

a 25 mL volumetric flask.

2.

Table 8-49: Model dye calibration standard preparation

Concentration of calibration standard (mg/L) Volume of 10 mg/L stock (mL)

in 25 mL Milli-Q® water

0.625 1.563

1.25 3.125

2.5 6.25

5 12.5

10 n/a

3. Bring flask to 25 mL mark with Milli-Q® water. Insert stopper into flask and invert to mix.

4. Transfer each solution to amber vials, or clear vials covered in tin foil to protect the

standards from light exposure.

5. Prepare a new calibration standard for each testing day and for each new working solution.

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Analysis of samples and standards with UV-vis spectrophotometer

1. Set the spectrophotometer to detect 485 nm for AO7 analysis and 664 nm for MB analysis.

2. Rinse the cuvette twice with Milli-Q® water, wipe the sides of the cuvette with a Kimwipe,

and run the Milli-Q® blank.

3. Discard the Milli-Q® water and rinse the cuvette with the sample/standard to be analysed.

Fill the cuvette with the sample, wipe the sides of the cuvette with a Kimwipe, and run the

sample. Ensure that the cuvette is placed in the same orientation each time.

4. Record the absorbance from the screen.

5. Rinse the cuvette twice with Milli-Q® water, once with the next sample to be analysed,

wipe the sides of the cuvette with a Kimwipe, and run the sample. Record the measurement.

6. After each 10 samples, re-run a calibration standard to ensure that the calibration curve is

still valid.

8.8.4 E. coli culture preparation and enumeration

A lyophilized E. coli stock culture (ATCC ® 23631™) was purchased from Cedarlane

Laboratories and revived to prepare glycerol stocks, which were frozen at -80°C. These stocks

were used for inoculation the day before experimentation, grown overnight, and used to prepare a

testing solution of ~107 CFU/mL to expose to the clay samples under the solar simulator. Samples

were taken at various time intervals, serial diluted, and plated onto agar plates using the spread

plate method. The plates were incubated overnight and the coliform units were counted the

following day. The counts were used to determine the CFU/mL of the samples. All sample prep

was performed in a biosafety cabinet and all pipettes, tubes, solutions, and equipment were

sterilized by autoclaving at 121°C for 20 min. All solution preparation, dilutions, and plating were

performed in an operating biosafety cabinet in the presence of an ethanol burner following standard

aseptic practices. The following section outlines how to make all solutions, prepare agar plates,

formulate E. coli stocks, make testing solutions, and count colonies.

Methods

LB broth preparation

1. Fill 1 L bottle to approximately half with distilled water.

2. Weigh 20 g of powdered LB and add to distilled water.

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3. Close the bottle and shake vigorously.

4. Fill the bottle to the 1 L mark with distilled water.

5. Autoclave at 121°C for 20 min, ensuring that the cap is loose.

6. Allow solution to cool in the biosafety cabinet and store in the 4°C fridge with the cap

sealed tightly to prevent contamination.

PBS 1x stock preparation

1. Fill 1 L bottle to approximately half with distilled water.

2. Measure 100 mL of PBS 10x solution.

3. Fill the bottle to the 1 L mark with distilled water.

4. Autoclave at 121°C for 20 min, ensuring that the cap is loose.

5. Allow solution to cool in the biosafety cabinet and store in the 4°C fridge with the cap

sealed tightly to prevent contamination.

Agar preparation and plate pouring

1. Fill 1 L bottle to approximately half with distilled water.

2. Weigh 20 g of powdered LB and 15 g of agar and add to bottle.

3. Fill bottle to 1 L mark, close, and shake vigorously.

4. Autoclave at 121°C for 20 min. Ensure that the cap is loose.

5. Allow solution to cool slightly in the biosafety cabinet, but not enough so that it solidifies.

6. Pour agar into 100 mm petri dishes so that the dishes are ¾ full. Let the agar solidify

overnight in the biosafety cabinet.

7. The plates can be put in a sealed bag and left in the 4°C fridge for up to 2 weeks. Ensure

that petri dishes are stored upside down to prevent condensation on agar.

Ringer’s solution ¼ strength preparation

1. Fill 1 L bottle to approximately half with distilled water.

2. Weigh out the following ingredients into the bottle:

a. Calcium chloride: 0.25 g

b. Potassium chloride: 0.42 g

c. Sodium bicarbonate: 0.2 g

d. Sodium chloride: 6.5 g

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3. Fill the bottle to the 1 L mark with distilled water and stir until all reagents are dissolved.

4. Dilute to ¼ strength by removing 250 mL of the solution and adding to another 1 L bottle

and filling to the 1 L mark.

5. Autoclave at 121°C for 20 min, ensuring that the cap is loose.

6. Allow solution to cool in the biosafety cabinet and store in the 4°C fridge with the cap

sealed tightly to prevent contamination.

E. coli stock solution

1. Use the propagation procedure from the ATCC product sheet (see Appendix) to make the

starter culture:

a. Revive the stock by adding 0.5 mL of LB directly to the stock culture and mix well.

b. Transfer the 0.5 mL to a sterile test tube containing 5 mL of LB broth.

2. Incubate overnight at 37°C while shaking at 250 rpm.

3. The next day, prepare glycerol stocks by adding 0.75 mL of 50% glycerol, which has been

filter sterilized or autoclaved, to a CryoELITE™ cryogenic storage vial.

4. Add 0.75 mL of the overnight culture to the glycerol in the CryoELITE™ cryogenic storage

vial.

5. Vortex to ensure even mixing and place on ice immediately.

6. Store in -80°C freezer.

Reanimating E. coli culture from frozen stock

1. The evening prior to testing, reanimate a portion of the E. coli stock by touching a sterile

inoculation loop to the frozen culture then immerse it in 70 mL of LB broth in a 250 mL

Erlenmeyer flask. Incubate the solution overnight at 37°C shaking at 250 rpm.

2. The day of experimentation, transfer half of the stationary culture to two sterile 50 mL

centrifuge tube and spin for 15 min at 4000 rpm. Pour off the supernatant growth media

and add 7.5 mL of ¼ strength Ringer’s solution. Gently rinse the pellet with the Ringer’s

solution.

3. Spin down the solution for 15 min at 4000 rpm and pour off the supernatant. Add 7.5 mL

of ¼ strength Ringer’s solution and rinse the pellet.

4. Repeat step 3 two additional times so that the pellet is rinsed three times

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5. Once the pellet has been rinsed for the third time, pour off the supernatant and add 25 mL

of Ringer’s solution. Rinse the pellet and vortex to ensure even distribution of the bacteria.

6. Take the OD of the solution and use as stock to add to reactors.

Sample testing

The sample conditions are shown in Table 8-50. Two clay pieces were tested under each

condition.

Table 8-50: Experimental Conditions

Condition Clay Type Ringer’s solution only (i.e. no E. coli)

LR WSC No clay No clay

No sunlight

Sunlight

1. Remove agar plates from fridge at the beginning of the day to allow them to reach room

temperature.

2. Sterilize solar simulator, surrounding curtains, and stir plates with 70% ethyl alcohol

solution.

3. Prepare glass tubes with 1.8 mL of PBS for each dilution.

4. Immediately prior to experimentation, add enough volume of the E. coli stock to 35 mL of

¼ Ringer’s solution to achieve a starting concentration of ~107 CFU/mL in each reactor,

which corresponds to an OD600 of approximately 0.05.

5. Add a clay piece and stir bar to the reactors and stir in the dark under the solar simulator

(shutter closed) for 10 min.

6. Remove 0.2 mL from each reactor and add directly to the first prepared glass tube.

7. Open shutter after the 10 min have elapsed to expose samples to sunlight.

8. Remove 0.2 mL from each reactor every twenty min and add directly to the first prepared

glass tube.

9. Make eight serial dilutions in the prepared glass tubes. Vortex each dilution to ensure

proper mixing

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10. Pipette 100 µL of each dilution in the middle of a pre-poured agar plate and manually

spread using a sterile spreader until the entire sample is absorbed on to the agar.

a. Note: not all dilutions need to be plated. Choose the dilutions that are more likely

to bracket the expected CFU/mL.

11. Incubate the plates upside down overnight in a 37°C incubator

12. Count the CFU formed on the plates and calculate the concentration in the samples using

the dilution factors and Equation 8.1:

CFUmL =

colonies counted0.1 mL of sample ×dilution factor

8.1

13. Bag all contaminated materials and discard in regular waste.

8.8.5 Hydroxyl radical detection procedure The commonly accepted mechanism of disinfection and removal of organic matter with

photocatalysts like TiO2 is the generation of hydroxyl radicals. To determine if hydroxyl radicals

are generated by the clay pieces and responsible for the colour removal from the model dyes,

terephthalic acid (TPA) was used as a chemical probe. When hydroxyl radicals are produced, they

react with the TPA and form 2-hydroxyterephthalic acid (HTPA) which can be detected using

fluorescence. Therefore, the concentration of hydroxyl radicals can be determined from the

concentration of HTPA produced during irradiation.

To determine the HTPA generated by the TPA test, a calibration curve was prepared using an

HTPA stock solution. Next, a working solution was prepared with TPA and NaOH which was

added to reactors and exposed to sunlight under the solar simulator. Samples at varying set time

intervals were analysed using the fluorescence spectrophotometer. The following sections outline

how to prepare stock solutions, working solutions, calibration standards, and how to analyse

samples.

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Methods

NaOH Stock Solution

1. Dissolve 40 g of NaOH pellets in 1L of Milli-Q® water.

HTPA Solution Prparation for Calibration Standards

1. Add 1.5 mL of 1 M NaOH to a 250 mL volumetric flask

2. Add 2.7319 mg of powdered HTPA to the same flask.

3. Fill to 250 mL mark then add to an amber bottle. The solution will be 60 PM HTPA.

HTPA Calibration Standards

1. Using a pipette, transfer the required volume of the HTPA solution (see Table 8-51) to a

10 mL volumetric flask.

Table 8-51: Preparation of HTPA calibration standards

Concentration of calibration standard (µM HTPA) Volume of 120 µM stock (µL)

in 5 mL Milli-Q® water

0.0625 5

0.125 10

0.25 21

0.5 42

1 83

2. Bring flask to 10 mL mark with Milli-Q® water. Insert stopper into flask and invert to mix.

3. Transfer each solution to amber vials, or clear vials covered in tin foil to protect the

standards from light exposure.

4. Make duplicate calibration standards.

5. Analyse with fluorescence spectrophotometer.

Sample Analysis

1. Open the “Scan” program on the computer connected to the fluorescence

spectrophotometer.

2. Change the settings in the “set-up” folder:

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a. Set excitation wavelength to 315 nm.

b. Set emission wavelength to range between 350 nm and 550 nm.

3. Zero the instrument using Milli-Q® water.

4. Analyse samples.

Preparation of TPA Working Solution

1. Fill a 1 L volumetric flask to approximately ¼ volume.

2. Add 6 mL of 1 M NaOH to the flask.

3. Dissolve 0.0831 g of TPA.

4. Top up with water up to the line.

5. Transfer into a 1 L amber bottle.

The concentration of NaOH in the solution is 6 mM and the concentration of TPA is 0.5 mM.

Sample Preparation

1. Dispense 35 mL of working solution into each batch reactor.

2. Add stir bar and one piece of clay to each.

3. Place in solar simulator

4. Mix for 10 min in the dark, then remove 3.0 mL of the sample with a pipette and transfer

to an amber vial. Analyse the sample with the fluorescence spectrophotometer as quickly

as possible, then return the analysed working solution to the reactor.

5. Turn on the solar lamp and irradiate samples 20 min. At the 20 min mark, remove 3.0 mL

of the sample with a pipette and transfer to an amber vial and analyse as quickly as possible.

6. Perform the same analysis every 20 min for one hour for a total of 70 min sample time with

4 samples.

Ensure that sun only controls are prepared in addition to the clay samples.

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8.8.6 Detection and quantification of photogenerated electron holes procedure

To determine if photogenerated holes were generated by the clay pieces, potassium iodine was

used as a chemical probe. When photogenerated holes are produced by the photocatalyst, they

react with the iodine ion (I-) to form iodine (I2); the concentration of photogenerated holes is twice

the concentration of the produced iodine. The concentration of iodine can be determined using the

UV-vis spectrophotometer set to a detection wavelength of 585 nm.

To determine the iodine concentration, a calibration curve was prepared using an iodine solution.

For the working solution, a potassium iodide solution was prepared, which was added to reactors

and exposed to sunlight under the solar simulator. Samples at varying set time intervals were

analysed using the UV-vis spectrophotometer. The following sections outline how to prepare stock

solutions, working solutions, calibration standards, and how to analyse samples.

Methods

Iodine Calibration Standards

1. Using a pipette, transfer the required volume of iodine solution (see

2. Table 8-52) to a 50 mL volumetric flask.

Table 8-52: Preparation of I2 calibration standards

Desired Concentration (mM) Volume I2 Stock to Add (𝝁L)

0.015625 7.93125

0.04375 22.2075

0.071875 36.48375

0.1 50.76

0.128125 65.03625

0.15625 79.3125

3. Bring flask to 50 mL mark with Milli-Q® water. Insert stopper into flask and invert to mix.

4. Transfer each solution to amber vials, or clear vials covered in tin foil to protect the

standards from light exposure.

5. Make duplicate calibration standards.

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Sample Analysis

1. Open the program on the computer connected to the UV-vis spectrophotometer. Ensure

that both lamps are turned on.

2. Change the detection wavelength to 585 nm.

3. Zero the instrument using Milli-Q® water.

4. Analyse samples.

Preparation of KI Working Solution (50 mM)

1. Add 8.3 g of KI to 1 L of Milli-Q® water.

2. Mix until dissolved.

3. Store in a 1 L amber bottle.

Sample Preparation

1. Dispense 35 mL of KI working solution into each batch reactor.

2. Add stir bar and one piece of clay to each.

3. Place in solar simulator.

4. Mix for 10 min in the dark, then remove 3.5 mL of the sample with a pipette and transfer

to an amber vial. Analyse the sample with the UV-vis spectrophotometer as quickly as

possible, then return the analysed working solution to the reactor.

5. Turn on the solar lamp and irradiate samples for 20 min. At the 20 min mark, remove 3.5

mL of the sample with a pipette and transfer to an amber vial and analyse as quickly as

possible.

6. Perform the same analysis every 20 min for one hour for a total of 70 min sample time with

3 samples.

Ensure that sun only controls are prepared in addition to the clay samples.