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Development of Protein-Functionalized Magnetic Iron Oxide

Nanoparticles: Potential Application in Water Treatment

Chuka Okoli

Doctoral Thesis

Royal Institute of Technology

School of Biotechnology

Stockholm 2012

© Chuka Christian Okoli

Stockholm 2012


School of Biotechnology

Department of Environmental Microbiology

AlbaNova University Center

SE-106 91 Stockholm

Sweden

and


School of Chemical Science & Engineering

Division of Chemical Technology

SE-100 44 Stockholm

Sweden

Printed by University service US-AB

Stockholm, Sweden

ISBN 978-91-7501-311-4

TRITA-BIO Report 2012:8

ISSN 1654-2312

All rights reserved. No part of this thesis may be reproduced, without the written permission

from the author.

To Queen, Lily and Munachi

Dedication

This work is dedicated to my beloved Mom, Late Mrs. Mabel N. Okoli for her resilience in

insisting to educate and encourage me amidst the absolute critical situation. I pray that you

will continue to rest in the bosom of Almighty God; knowing that I have accomplished that

which you always wanted.

Chuka Okoli

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Abstract

The treatment of water to make it safe for human consumption is a problem of immense

concern, both in developing and developed countries. However, the production of clean water

with chemicals as coagulants has several drawbacks associated with cost, health risks and

complexity in sludge management. The application of nanotechnology in water treatment is a

fast growing discipline proposed as an efficient alternative that will combat these hurdles. The

aim of this thesis is to develop new water treatment strategies in a more eco-friendly manner

based on a bottom-up approach using: (i) a natural coagulant protein from Moringa oleifera

purified with nanoscale magnetic iron oxide nanoparticles for in situ treatment; and (ii) a

protein-functionalized nanoparticle (MOCP-MNPs) system by means of binding the coagulant

protein onto the nanoparticles in order to develop a potential reusable water treatment process.

Magnetic iron oxide nanoparticles with different surface chemistry have been prepared from

co-precipitation in aqueous solution and (water-in-oil and oil-in-water) microemulsion

methods.

The prepared nanoparticles were studied in terms of size, morphology, magnetic

behavior, structure, surface area including surface chemical structure and charges using

different techniques such as TEM, VSM/SQUID, XRD, BET, FT-IR and zeta potential. The

prepared nanoparticles exhibited a size ranging from 2-30 nm with superparamagnetic

properties. The Moringa oleifera coagulant protein (MOCP) with known molecular mass (6.5

kDa) was purified from the crude Moringa oleifera (MO) seed extracts using nanoparticles

prepared from both methods. The obtained MOCP exhibits comparable coagulation activity

with alum in terms of water turbidity removal, implying alternative replacement to chemical

coagulants. This technique can be easily applied where natural materials are available locally.

Studies on the interaction between MOCP and surface modified nanoparticles were

essential to understand the binding mechanism for the development of a protein-

functionalized nanoparticle. Based on in silico investigation, the overall molecular docking

studies reveal the interactions between protein-ligand complexes by electrostatic, van der

Waals and hydrogen-bonding; which imply, that there are at least two binding sites is i.e. one

located at the core binding site (TEOS and APTES ligand) while the other located at the side

chain residues (TSC and Si60-OH).

This work underscores advancement in the development and use of MOCP-MNPs for

potential water treatment. About 70% turbidity removal was achieved gravimetrically using

MOCP-MNPs (60 min) in high and low turbid waters, whereas alum requires 180 min to

reduce the turbidity especially in low turbid waters. The turbidity removal efficiency was

enhanced by the use of MOCP-MNPs under the influence of an external magnetic field. More

than 95% turbidity removal was achieved within 12 min in high and low turbid waters when

MOCP-MNPs were used. The combination of natural coagulant protein and magnetic

nanoparticles as well as the use of applied magnetic field enhanced the performance

coagulating/flocculating properties in the water samples.

These results suggest a successful development of MOCP-MNPs as demonstrated in the

regeneration study. The data shown in this work represent novel potential water treatment

strategies that could be cost-effective, simple, robust and environmentally friendly whilst

utilizing biocompatible materials.

Keywords: magnetic nanoparticles; co-precipitation; microemulsion; Moringa oleifera;

protein purification; water treatment; magnetophoresis; coagulation activity; turbidity

removal.

Development of Protein-Functionalized Magnetic Iron Oxide Nanoparticles: Potential Application in Water Treatment

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Chuka Okoli

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List of Publications

This thesis is based on the following papers:

I. Chuka Okoli, Andrea Fornara, Jian Qin, Muhammet Toprak, Gunnel Dalhammar,

Mamoun Muhammed and Gunaratna Rajarao-Kuttuva. Characterization of

Superparamagnetic Iron Oxide Nanoparticles and Its Application in Protein

Purification. J. Nanosci. Nanotech. 2011, 11, 10201-10206.

II. Chuka Okoli, Magali Boutonnet, Laurence Mariey, Sven Järås and Gunaratna

Rajarao-Kuttuva. Application of Magnetic Iron Oxide Nanoparticles Prepared

From Microemulsions For Protein Purification. J. Chem. Techno. Biotech. 2011,

86, 1386-1393.

III. Chuka Okoli, Margarita Sanchez-Dominguez, Magali Boutonnet, Sven Järås,

Concepción Civera, Conxita Solans and Gunaratna Rajarao-Kuttuva. Comparison

and Functionalization Study of Microemulsion-Prepared Magnetic Iron Oxide

Nanoparticles. Langmuir. 2012, (in press).

IV. Chuka Okoli, Magali Boutonnet, Sven Järås and Gunaratna Rajarao-Kuttuva.

Protein-Functionalized Magnetic Iron Oxide Nanoparticles: Time Efficient

Potential-Water Treatment. 2012, (submitted).

V. Chuka Okoli, Selvaraj Sengottaiyan, Arul N. Murugan, Pavankumar A.

Ramachandran, Hans Ågren and Gunaratna Rajarao-Kuttuva. In Silico Modeling

and Experimental Evidence of Coagulant Protein Interaction with Precursors for

Nanoparticle Functionalization. 2012, (submitted).

VI. Pavankumar R. Ramachandran, Kayathri Rajarathinam, Arul N. Murugan, Zhang

Qiong, Chuka Okoli, Hans Ågren and Gunaratna Rajarao-Kuttuva. Dimerization

of flocculent protein from Moringa oleifera: experimental evidence and in silico

interpretation. 2012, (manuscript).

VII. Ramnath Lakshmanan, Chuka Okoli, Magali Boutonnet, Sven Järås and

Gunaratna Rajarao-Kuttuva. Effect of Magnetic Iron Oxide Nanoparticles for

Surface Water Treatment: Trace Minerals and Microbes. 2012, (manuscript).

Other work not included:

1. Chuka Okoli, Ramnath Lakshmanan, Duke Antwi, Magali Boutonnet, Sven Järås,

and Gunaratna Rajarao-Kuttuva. Investigating the Use of Magnetic Nanoparticles for

Efficient Reduction of Phosphates and Nitrates in Wastewater Treatment Process,

(manuscript).

All papers are reproduced with kind permission from the respective copyright holders.


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Contributions of the author

Paper I. Principal author, involved in planning, performed the experiments, performed part

of sample characterization, evaluation of the results and writing the article.

Paper II. Principal author, involved in planning, performed the experiments, performed part


Paper III. Principal author, involved in planning, performed the experiments, performed part


Paper IV. Principal author, involved in planning, performed the experiments, performed part


Paper V. Principal author, involved in planning, and performed the experimental part,

involved in evaluation of the results and writing part of the article.

Paper VI. Participated in developing ideas and evaluation of results

Paper VII. Participated in planning, synthesis of magnetic nanoparticles, performed part of

experiments, evaluation of results and writing part of the article.

Patent Applications

1. Chuka Okoli, Magali Boutonnet, Laurence Mariey, Sven Järås and Gunaratna

Rajarao-Kuttuva. Application of Magnetic Iron Oxide Nanoparticles Prepared From

Microemulsions For Protein Purification. U.S. Patent and trade office, 61407455,

October 28, 2010

2. Chuka Okoli, Magali Boutonnet, Sven Järås and Gunaratna Rajarao-Kuttuva. Time

Efficient Drinking Water Treatment Using Protein-Functionalized Magnetic Iron

Oxide Nanoparticles. U.S. Patent and trade office, 61599960, February 17, 2012.

Chuka Okoli

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Conference presentations

1. Membrane Technology Conference for Water and Wastewater Treatment; (attended),

September 1-3, 2009. Beijing, P.R.China.

2. Chuka Okoli, Andrea Fornara, Jian Qin, Muhammet S. Toprak, Gunnel Dalhammar,

Mamoun Muhammed and Gunaratna Rajarao-Kuttuva. “Superparamagnetic

nanoscale-based particles for coagulant protein purification” (poster), 2010

International Chemical Congress of Pacific Basin Societies; December 15-20, 2010.

Honolulu, Hawaii, USA.

3. Chuka Okoli, Ramnath Lakshmanan, Magali Boutonnet, Sven Järås and Gunaratna

Rajarao-Kuttuva “Microemulsion magnetic nanoparticles: Synthesis, characterization

and their application” (poster), 94th Canadian Chemistry Conference and Exhibition;

June 5-9, 2011. Montréal, Québec, Canada.

4. Chuka Okoli, Ramnath Lakshmanan, Magali Boutonnet, Sven Järås and Gunaratna

Rajarao-Kuttuva “Magnetic nanoparticles for coagulant protein purification and

water treatment” (oral), Clean water through Bio- and Nano-technology; May 7-9,

2012. Lund, Sweden.


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Abbreviations and Symbols

AAS atomic absorption spectroscopy

AOT Bis(2-ethylhexyl) sulfosuccinate

APTES 3-Aminopropyltriethoxysilane

BET Brunauer-Emmet-Teller

CMC carboxyl methyl cellulose

CTAB cetyltrimethylammonium bromide

Hc coercivity

DBPs disinfectant by-products

Dc critical size thresholds

DOC dissolved organic carbon

Ds superparamagnetism

FDA Food and drug administration

FTIR Fourier transform infrared spectroscopy

GAC granulated active carbon

H applied magnetic field

HLB hydrophilic-lipophilic balance

HRTEM high-resolution transmission electron microscopy

IEX ion exchange

M magnetization

ME-MIONs microemulsion-prepared magnetic iron oxide nanoparticles

MNPs magnetic nanoparticles

MO Moringa oleifera

MO2.1 Moringa oleifera recombinant protein

MOCP Moringa oleifera coagulant protein

MR magnetic remanence

MS saturation magnetization

NF nanofiltration

NMs nanomaterials

NOM natural organic matter

NTU nephelometric turbidity units

o/w oil-in-water

OD optical density

PAC polyaluminium chloride

PEI poly ethylene imine

PMO purified Moringa oleifera

Pzc point of zero charge

SAED selected area electron diffraction

SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis

SENs single-enzyme nanoparticles

SPION superparamagnetic iron oxide nanoparticle

SQUID superconducting quantum interference device magnetometer

TEM transmission electron microscopy


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TEOS tetraethoxysilane

THMs trihalomethanes

TLC thermoelectric temperature controller

TSC trisodium citrate

UV ultraviolet

VSM vibrating sample magnetometer

w/o water-in-oil

WHO world health organization

XRD X-ray diffraction

ζ zeta potential (Zp)

χ magnetic susceptibility

Chuka Okoli

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Table of Contents

1. Introduction ..................................................................................................................................... 1

1.1 Conventional water treatment processes ..................................................................................... 2

1.2 Nanotechnology in water treatment ............................................................................................. 5

1.3 Natural coagulants in water treatment ......................................................................................... 7

1.3.1 Brief History of the Moringa oleifera (MO) treef ................................................................ 7

1.3.2 Water treatment with MO protein ........................................................................................ 8

1.3.2.1 Microbial elimination .................................................................................................... 9

1.3.3 Purification of the MO protein ........................................................................................... 10

1.3.4 Structural prediction ........................................................................................................... 11

1.4 Magnetic nanoparticles .............................................................................................................. 11

1.4.1 Iron oxide nanoparticles ..................................................................................................... 12

1.4.1.1 Magnetic behavior ....................................................................................................... 13

1.4.1.2 Superparamagnetic phenomenon ................................................................................. 14

1.4.2 Synthesis methods .............................................................................................................. 15

1.4.2.1 The co-precipitation method in aqueous solution ........................................................ 16

1.4.2.2 The microemulsion method ......................................................................................... 16

1.4.2.3 The thermal decomposition method ............................................................................ 18

1.4.2.4 The hydrothermal method ........................................................................................... 18

1.4.3 Magnetic separation and mixing ........................................................................................ 19

1.5 Objectives of the present work .................................................................................................. 20

2. Experimental .................................................................................................................................. 23

PART ONE ....................................................................................................................................... 23

Synthesis and characterization .......................................................................................................... 23

2.1 Extraction and analysis of coagulant protein (Papers I & II) .................................................... 23

2.1.1 Preparation of synthetic water ............................................................................................ 24

2.1.2 Coagulation activity test ..................................................................................................... 24

2.2 Synthesis of magnetic iron oxide nanoparticles (Papers I─III) ................................................. 24

2.2.1 Co-precipitation method in aqueous solution ..................................................................... 25

2.2.1.1 Coating with TSC ........................................................................................................ 25

2.2.1.2 Coating with TEOS ..................................................................................................... 26

2.2.1.3 Coating with APTES ................................................................................................... 26

2.2.2 Microemulsion methods ..................................................................................................... 27

2.2.2.1 Synthesis of w/o ME-MION ....................................................................................... 27

2.2.2.1 Synthesis of o/w ME-MION ....................................................................................... 29

2.3 Characterization (Papers I─III) ................................................................................................. 29

2.3.1 Nanoparticles prepared by co-precipitation (Paper I) ......................................................... 30

2.3.2 Nanoparticles prepared by Microemulsion (Papers II-III) ................................................. 31

PART TWO ....................................................................................................................................... 31

2.4 Application of nanotechnology in water treatment (Papers III, IV, V & VII) .......................... 31

2.4.1 Purified MOCP for water treatment (Approach I) .............................................................. 31

2.4.1.1 Purification of the MO coagulant protein with MNPs and activity test ...................... 32

2.4.1.2 Analysis and characterization ...................................................................................... 32

2.4.1.3 Turbidity removal in surface waters ............................................................................ 33

2.4.1.4 Interaction of MOCP and MNPs (Papers III & V) ...................................................... 33


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2.4.1.4.1 Computational and experimental studies .............................................................. 34

2.4.1.4.2 Fluorescence emission study (Paper III) .............................................................. 35

2.4.2 MOCP-MNPs for water treatment (Approach II) ............................................................... 35

2.4.2.1 Development of MOCP-MNPs system ....................................................................... 36

2.4.2.2 Turbidity removal in surface waters ............................................................................ 37

2.4.2.3 Separation by gravity and magnetic field .................................................................... 37

2.4.2.4 Reusability of MOCP-MNPs system ........................................................................... 37

3. Results and discussion ................................................................................................................... 39

PART ONE ....................................................................................................................................... 39

Synthesis and characterization .......................................................................................................... 39

3.1 Investigation of the active protein (Paper I) .............................................................................. 39

3.2 Characterization of MNPs (Papers I─III) .................................................................................. 40

3.2.1 Size and morphology studies ............................................................................................. 40

3.2.2 Structural studies ............................................................................................................... 41

3.2.3 Magnetic studies ................................................................................................................ 42

3.2.4 Surface charge studies ........................................................................................................ 43

PART TWO ....................................................................................................................................... 44

3.3 Application of nanotechnology in water treatment ................................................................... 44

3.3.1 Purification of the MO protein with MNPs and characterization (Papers I─III) ............... 44

3.3.1.1 Turbidity removal in surface waters (Approach I) ...................................................... 45

3.3.2 Interaction of MOCP and MNPs (Papers III & V) ............................................................. 46

3.3.2.1 Computational and experimental studies ..................................................................... 46

3.3.2.2 Fluorescence emission study ....................................................................................... 50

3.3.3 Turbidity removal with the developed MOCP-MNPs system (Approach II) ..................... 51

(Paper IV & V) .............................................................................................................................. 51

3.3.3.1 Separation by gravity and magnetic field .................................................................... 53

3.3.3.2 Reusability of MOCP-MNPs system ........................................................................... 54

4. Conclusions .................................................................................................................................... 57

Further studies ..................................................................................................................................... 58

Acknowledgement ............................................................................................................................... 59

References ............................................................................................................................................ 61

Appendix .............................................................................................................................................. 71

Chuka Okoli

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1. Introduction

Access to clean and safe drinking water is a human right; however, the availability of potable

water is a major concern in both developed and developing countries.1 Water treatment offers

the benefit of potable water in terms of quality (reduced level of contaminants) and quantity

(availability). The world is facing formidable challenges in meeting the rising demands for

safe drinking water supply due to population growth, increasing pollution of water bodies

from several industrial and agricultural activities, drought and competing demands from a

variety of users.1-5

As a result of this water scarcity, children and elderly people are more

vulnerable to water-borne diseases. According to the World Health Organization (WHO),

more than 1 billion people especially in the developing countries still do not have access to an

adequate supply of drinking water.5 As a consequence, the quality of health and welfare

vulnerable groups (children, the elderly and the poor) are dependent on the availability of a

safe and affordable water supply.5, 6

It has been predicted that in the year 2025, about 3.5

billion people, 48% of the world’s population, will have an inadequate water supply.7

The provision of potable water from most raw water sources involves the use of coagulants

introduced during the coagulation/flocculation step to remove turbidity in the form of

dissolved or suspended materials. Chemical coagulants such as aluminum salts (AlCl3),

organic polyaluminum chloride (PAC)8 and ferric chloride (FeCl3) are frequently used to

enhance coagulation and flocculation.9 Nevertheless, given that the choice of chemicals in

water treatment is decisive, treatment processes with synthetic coagulants or disinfectants

may add substances such as disinfectant by-products (DBPs) to the treated water, which can

increase the risk of disease.10

In spite of their high efficiency, the presence of residual DBPs11

or aluminum residues12

in treated water is a huge concern due to their neurotoxicity and

carcinogenic properties such as those found in Parkinson’s, Alzheimer’s and cancer diseases.9,

10, 12-14 Moreover, the existence of large volumes of sludge, the increase in water pH as well as

the high cost of importing these chemicals is still a challenge.15

Considering the importance of potable water globally, and keeping in mind concerns about

the feasibility of recent practices to meet the rising water demands, there is a pressing need to

develop novel technologies and materials that are associated with natural coagulants which

will combat these challenges whilst reducing or replacing the use of chemicals in water


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treatment. While new technologies are being developed today, it is considered paramount to

develop biodegradable, cost effective, user-friendly, robust and more efficient systems than

the existing techniques for the removal of water contaminants either in water treatment

processes or in situ.16

The emergence of nanotechnology has been identified as a promising

technology that could play a major role in providing potable water.5

The use of natural coagulants such as the Moringa oleifera coagulant protein (MOCP)17-20

associated with magnetic nanomaterials16, 21, 22

is one way in which nanotechnology can play a

vital role in providing potable water. Functionalized nanomaterials, such as magnetic iron

oxide nanoparticles, are known to display novel and significant physicochemical as well as

biological properties due to their unique size, structure and large surface-to-volume ratio.21, 23,

24 Protein-functionalized magnetic nanoparticles offer several advantages such as fast

separation, enhanced efficiency, and/or significant reduction in sludge volume and material

recycling. In particular, the incorporation of natural coagulant proteins on magnetic

nanoparticles constitutes an excellent alternative to chemical remediation in water treatment

processes.

1.1 Conventional water treatment processes

Globally, in many water treatment plants, the combination of coagulation/flocculation,

sedimentation, filtration and disinfection is a widely applied water treatment technology

(Figure 1.1) for the production of potable water. This practice has been in use since the early

20th century.16

However, coagulation/flocculation is an important primary step in the water

treatment processes and as such, is critical for other successive steps including the amount of

chemical usage and the overall production cost.

Figure 1.1 Scheme of general water treatment processes.

Coagulation/ flocculation is a process that involves the removal of suspended and dissolved

particles by the addition of salts such as aluminum sulphate, ferric chloride or organic

polyaluminum chloride followed by rapid mixing. These chemicals, which are positively

Incoming water

Coagulation/

Flocculation Sedimentation Filtration Disinfection Outlet

Sludge

Chuka Okoli

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charged, act as coagulants. Particles in water are negatively charged and for this reason repel

each other when they come in close contact. This will force them to remain in suspension

rather than clump together and settle out of the water. The addition of positively charged

coagulants destabilizes the negative charges of the suspended and dissolved particles in the

water.25

When this reaction occurs, the particles bind together (coagulation); this process is

also referred to as flocculation. The large particles (flocs) formed are denser and quickly settle

to the bottom of the vessel.15

This settling process is called sedimentation. The heavy

particles are then removed, and the water moves to the next step.

Nonetheless, coagulation/flocculation and sedimentation are crucial for successful removal of

large amounts of organic compounds, including dissolved organic materials such as Dissolved

Organic Carbon (DOC) or Natural Organic Matter (NOM). Large amounts of these dissolved

organic materials are responsible for undesirable water odor, taste, color and the presence of

microorganisms.26

Previous studies show that coagulation can remove suspended and

dissolved particles by adsorption and charge neutralization, adsorption and inter-particle

bridging, and precipitation.27-29

The most common mechanism in coagulation/flocculation

processes is the inter-particle bridging and charge neutralization.30

Due to the inter-related

properties of coagulation/flocculation processes, the optimization of raw water treatment with

coagulant/coagulant aids can sometimes be very difficult to achieve; thus some factors may

influence the performance of the coagulants in water treatment processes.

These factors include the source water characteristics, raw water pH, choice of

coagulant/coagulant aids and their order of addition, temperature, alkalinity, coagulant dosage

and the degree and mixing condition, including the flocculation period. Keeping track of the

pH and alkalinity is very critical for good performance.31

Alkalinity of water is referred to as

its capacity to neutralize acids. Water alkalinity may be due to the presence of one or more

ions like hydroxides, carbonates and bicarbonates. For instance, raw water with low alkalinity

will utilize most of the available alkalinity by the addition of a coagulant such as ferric salts,

thereby lowering the water pH to a level that hampers effective treatment. Conversely, water

with high alkalinity may require additional coagulants in order to lower the pH to the

acceptable range for coagulation. Therefore, it is more cost-effective to treat water with

acceptable alkalinity. The coagulation/flocculation process can also be affected by

temperature. The coagulant stability, increasing water viscosity as well as hindering the

kinetics of hydrolysis reaction and particle flocculation are all attributes of low water

temperature. Organic coagulants seem to be more effective in cold water since they are pre-


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hydrolyzed.15, 32

Given that the aforementioned coagulants are efficient and widely used in

the first step of water treatment, there are still huge concerns about their adverse health effects

associated with residual chemicals and, increase in water pH, including increased sludge

volume and complexity.

Sludge conditioning and dewatering: inappropriate disposal of sludge from water treatment

plants with little or no treatment is a common practice in many places. However, the direct

disposal of untreated sludge is considered a risk to recipients due to the chemical composition

of the sludge.33

Sludge from water comprises more than 90% of water; thus, it is a big

challenge to dewater.34

Methods such as centrifugation, chemical precipitation and heat

treatment are commonly used sludge conditioning systems. However, these methods are

somewhat complicated and require experienced operators; moreover, the energy needed for

the heat treatment and the high cost of chemicals is a big concern for both developing and

developed countries. Nonetheless, the environmental impact of commonly used chemical

conditioners may be averted if locally available natural materials can be employed.

Filtration is the second step in a conventional water treatment process. This step removes

particulate matter from water by means of forcing the water to percolate and pass via porous

media. The filtration system often consists of sand, gravel and charcoal as filters, as well as

microfiltration or ultrafiltration membranes with varying sizes of pores. The particles,

including microorganisms and ions, that are removed from water during filtration depend on

the pore size of the filter used.35

Membrane filtration or granulated active carbon (GAC)36

is

often used, but the drawback is the high cost of maintenance. For surface water, sand filtration

is usually preferred in the treatment process. Basically, two types of sand filtration exist, slow

sand filtration and rapid sand filtration. Rapid sand filtration is a physical process that

involves the removal of suspended solids. Compared to slow sand filtration, this process is

common; it has fairly high flow rates and requires less space. Slow sand filtration is a

biological treatment and the most preferred method, because it uses bacteria to treat the water.

The surface of the sand filtration is designed in such a way that the bacteria establish a

community on the top layer (biofilm) of sand, whilst cleaning the water by digesting the

contaminants as it passes through the sand filters.37

The process requires considerable

operational space and must be situated in the open air, which makes it vulnerable to other

contaminants. It also requires long hours including periodic cleaning.

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Disinfection is the final step in water treatment processes before sending the water to the end

users. Frequently used disinfectants in water treatment processes include chlorine,

chloramines, and ozone. Chlorine or and its derivatives are relatively cheap and effective; in

spite of their high efficiency, the presence of residual Disinfectant By-Product (DBPs) such as

trihalomethanes (THMs) in the treated water is a major concern for public health due to their

neurotoxicity and carcinogenic properties such as those found in Parkinson’s, Alzheimer’s

and cancer diseases.10, 11

Another effective disinfectant alternative to chlorine compounds is

the use of UV light and ozone treatment; however, this method consumes high energy and

also increases the emission of carbon. For these reasons, it is not considered cost-effective and

eco-friendly.11

Taking into account various limitations and concerns in water treatment

processes, the need for the production of potable water have not yet been met.

The outbreaks of water-borne diseases are increasing exponentially. For instance, recent

outbreaks were reported in places like Östersund, Sweden in 2011 and Manila, the Philippines

in 2012. The presence of different organic contaminants and highly toxic metallic cations

such as arsenic, lead, cadmium, or inorganic anions like nitrates, perchlorate are of major

concern to public health as well as the ecosystem. Moreover, the use of chemicals in water

treatment has resulted in the generation of large amounts of effluents that are not

biodegradable including disinfectant by-products. The potential application of

nanotechnology for novel material development in order to combat these challenges is

deemed necessary for the production of potable water in terms of quality and quantity.

1.2 Nanotechnology in water treatment

Nanotechnology is the engineering and art of manipulating matter at the nanoscale, basically

at the dimensions between 1─100 nm, where unique phenomena enable novel applications in

various disciplines including Biology. Nanotechnology offers the potential of novel nanoscale

materials for the treatment of all kinds of waters including surface water and wastewater.5

Due

to their unique properties towards various water contaminants and flexible applications, many

nanomaterials (NMs) are currently being developed. The key potential impact areas for

nanotechnology in water treatment applications can be divided into three groups, namely

treatment and remediation, sensing and detection, and pollution control.16

The first

mentioned is likely to contribute to long-term water quality, availability (quantity), and

viability of water resources, such as through the use of nanoreactive and functionalized

membranes, and advanced filtration materials that enable greater water reuse, recycling, and


6

desalination. Within the category of sensing and detection, NMs can promote the

development of new and enhanced sensors to detect biological and chemical contaminants

even at very low concentration levels in the assayed water sample. An example of such

material is single-enzyme nanoparticles (SENs).38, 39

Recent research on the synthesis of

different nanomaterials includes nanosorbents, nano-structured/reactive membranes,

nanocatalysts and bioactive nanoparticles.5, 40

The nanosorbents or membrane process

techniques are seen as the main driving force in the advancement made in water purification

and desalination technologies, since conventional water treatment methods are not effective in

removing organic pollutants. Trial experiments with some of these materials are ongoing in

Africa.16

Nanomaterials such as nanoparticles, carbon nanotubes and dendrimers (Figure 1.2)

contribute to the development of more robust, efficient and cost-effective water filtration

processes. Two types of membranes that could serve as effective water treatment techniques

are (i) nanostructured filters, where either carbon nanotubes or nanocapillary arrays provide

the basis for nanofiltration (NF); and (ii) nanoreactive membranes, where functionalized

nanoparticles enhance the filtration process.41-43

The synthesis of dendritic polymers such as

cyclodextrins, poly(propylene imine) and poly(amidoamine) dendrimers offers opportunities

to refine as well as to develop operative filtration processes for purification of water

contaminated by different organic solutes and inorganic pollutants.5

Figure 1.2 Nanotechnology-based materials for water treatment: (a) carbon nanotube (1-3

nm), (b) dendritic polymer as nanoreactive NMs; rectangular shape (blue) represents different

functional groups, and (c) Nanofiltration membranes with pore sizes of 10, 20 and 50 nm.

The use of bioactive nanoparticles for water disinfection is another key area of

nanotechnology in water sanitization.

(a) (c) (b)

Chuka Okoli

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Nanobiocides may present a reasonable alternative that will reduce the amount of sludge

produced during water treatment whilst underscoring new chlorine-free biocides that are

environmentally friendly. Among the most promising antimicrobial NMs are metallic and

metal oxide nanoparticles, especially functionalized iron oxide, polyethyleneimine (PEI),

chitosan, silver nanoparticles (AgNPs) and titanium dioxide catalysts for photocatalytic

disinfections.16, 44

Moreover, current research is being focused on the use of natural coagulant

materials as alternative sources in order to meet the above-mentioned challenges.

1.3 Natural coagulants in water treatment

Recent research has focused on the development and use of natural coagulants which can be

extracted or produced from plants, animals or microorganisms due to their presumed safety to

humans and the environment.9 The application of natural materials of plant origin for

clarifying turbid raw water from rivers is an ancient and domestic household practice in

tropical developing countries where these natural materials act as primary coagulants due to

their availability throughout the year.45, 46

Different kinds of natural coagulants obtained from

apricots (beach kernels), groundnut seed, nirmali seed, pumice seed, maize and the Moringa

oleifera (MO) coagulant protein have been described in various reports.9, 25, 26, 47

However,

among the investigated natural materials, water soluble extracts from the seeds of MO have

attracted particular attention since they possess dual functionality in water treatment by acting

as coagulants as well as antimicrobial agents.9, 48

Jahn, S.A.A49, 50

and Madsen et al.,51

were

among the first researchers to study the use of MO as natural coagulants/disinfectants in water

treatment. Since then, numerous studies have been conducted to optimize its use in water

treatment.20, 29, 52

The protein extracted from MO seeds competes quite favorably with

chemical coagulants.20, 53 It is widely accepted from various studies that extracts from MO

seeds possess effective coagulation/flocculation as well as antimicrobial properties, even

though the properties and nature of the active component seem to differ slightly, depending on

the extraction method used48

or the geographical region where the plants were grown.

1.3.1 Brief History of the Moringa oleifera (MO) treef

Moringa oleifera is a multipurpose tree belonging to the family of Moringaceae, a single

family of shrubs with 13 known species.54

It is a tropical plant found throughout Asia, sub-

Saharan Africa and Latin America. MO is widely recognized as a tree with almost every part

of the plant utilized for beneficial purposes. Due to the diverse applications of MO, it is

sometimes referred to as the miracle tree.32

The MO tree (Figure 1.3) is drought-tolerant and


8

is generally used in the developing world as a vegetable, medicinal plant, nutrition

supplement, cattle fodder, fertilizer and a source of oil.54, 55

Moringa oleifera seeds are also

very rich in iron and calcium and they contains 40% by weight of oil which can be used for

cooking, lamp fuel and production of soap.54

It has been reported that the press cake

remaining after oil extraction still contains active coagulants.18

The medicinal and therapeutic

potential of MO is being utilized in the cure of different diseases and ailments.56

Among

many other properties, seeds from MO contain a coagulant protein that can be used for water

clarification.17, 20, 49, 57, 58

The coagulant is obtained at extremely low or zero net cost.59

MO

seed protein is known to be one of the most effective natural coagulants and the study on

treatment of different kinds of waters has been growing recently.32, 58, 60

Figure 1.3 Moringa oleifera tree with pods and seed kernels. The active coagulant is

extracted from the seed kernel.

The active components of MO are water soluble cationic proteins with a molecular weight of

6.5 to 13 kDa and pI values around 10. Ndabigengesere et al.,61

in their study, described the

active coagulating agent of MO as a dimeric cationic protein having a molecular weight of 13

kDa and an isoelectric point between 10 and 11. The MO coagulant protein was identified as a

heterogeneous mixture consisting of sixty amino acid residues.57, 61, 62

Moreover, extracts

from MO seeds possess significant properties in the reduction of sludge volume and bacteria

in contaminated waters63, 64

without affecting the water pH, conductivity and alkalinity,

thereby making the MO protein more attractive than aluminum salts in water treatment.53

Apart from being non-toxic, the MO protein is entirely biodegradable.9, 48

1.3.2 Water treatment with MO protein

The active component can be extracted from seeds by the use of water or salt solutions,

usually NaCl.57, 65

The use of MO seed protein in water treatment can be applied both at

industrial scale where it can be used as a coagulant aid15, 66

or at the household level.67

Several

reports suggest that the MO seed is more efficient when applied in high turbid waters; hence,

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its use in large scale water treatment during the spring season when water turbidity is at its

highest level will benefit the water treatment plants that are forced to shut down due to lack of

funding.67

The coagulation/flocculation mechanism of MO coagulant protein (MOCP) was

described by Ndabigengesere et al.,57

as a mechanism involving adsorption and neutralization

of charges, implying that the positively charged amino acids of this protein bind to the

suspended or dissolved particles that are mainly negatively charged, and this leads to the

formation of negatively and positively charged areas on the particle surface. Inter-particle

neutralization of differently charged sectors and formation of flocs take place due to particle

collision (Figure 1.4).30

Figure 1.4 Mechanism of coagulation/flocculation with Moringa oleifera coagulation

protein (MOCP) showing adsorption and neutralization of the colloidal charges with net-like

structure.

1.3.2.1 Microbial elimination

Traditional water disinfection processes usually make use of chlorinated (chlorine and

chloramine) chemical additives in eliminating the microbial contaminants. While their

benefits are well established, concerns have also been raised about their safety issues.11

Conversely, MO seed extracts are capable of bacterial aggregation and removal. Their

antimicrobial activity may lead to growth inhibition and killing of bacteria, including

antibiotic-resistant human microorganisms.53

On the other hand, Broin et al.,68

showed that a

recombinant protein (MO2.1) of MOCP is capable of flocculating both Gram-Positive and

Gram-negative bacterial cells. Microorganisms can be removed in this case by settling in the


10

same way as the removal of colloids in well coagulated and flocculated water; alternatively,

the protein may also act directly upon the microorganism, resulting in growth inhibition.68

The microbial growth inhibition might be mediated by the interaction of positively charged

amino acids with the negatively charged surface of the microorganism’s membrane.76

1.3.3 Purification of the MO protein

Apart from several advantages of the MO protein over chemical coagulants, the main

drawback in using the crude extracts of MO seeds in water treatment is the release of organic

matter and nutrients to the water. Previous studies show that crude seed extracts increase the

organic, nitrate and phosphate contents in treated water, while the purified form (MOCP) does

not.18, 69

The presence of these organic loads is a source of odor, color and taste in water;

moreover they also facilitate the growth of microorganisms upon storage, thus limiting the use

of crude MO seed extract as a coagulant for water treatment in domestic and industrial

levels.70

In order to overcome these limitations, the MO coagulant protein needs to be

purified.

Consequently, the quest for low cost and simple purification procedures is critical for efficient

maximization of natural coagulant in use. Previous reports imply that the MO purification

process involves extensive methods that require several steps, which makes the purification

system time consuming, complicated and expensive. Furthermore, it has become difficult to

purify the protein on a large scale for water treatment applications.48, 69

Recently, Habauka et

al.,48

employed a purification method that involves more than six steps, including dialysis. A

single-step elution procedure was adopted by Ghebremichael et al.18, 59

They developed a

simple method for the purification of coagulant protein using ion-exchange matrix (IEX).

While these methods are efficient, there are still some unresolved issues, such as the low

binding capacity of the IEX matrix, the high cost of material as in the case of commercial

beads, as well as long process times. These challenges can be a limiting factor which might

potentially detract from the advantages of the aforementioned methods, bearing in mind the

accessibility problem for people in the developing countries.

The application of magnetic nanoparticles could add more benefits over the existing

technologies.71

This fast and easy technique can achieve separations (via magnetophoresis)

that are difficult to achieve practically with conventional methods.72-74

Magnetic separation

eliminates some steps during the purification, thereby promoting gentle, quick, and scalable

alternatives to the conventional methods. The desired targets are captured on magnetic

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nanoparticles coated with a specific surface and then separated from the samples with an

external magnet.75

1.3.4 Structural prediction

Many studies have been carried out with MOCP, but to date, the structure of this protein has

not been solved by X-ray crystallography or NMR spectroscopy. However, Suarez et al76

performed computer simulation modeling to reveal the structure of the MO2.1 peptide. In their

study, they predicted the structure of this protein from the known α-helices modeled structure

of the napin peptide in a consensus model of MO2.1 peptide secondary structure, which depicts

high probability of α-helices in the three regions that form helical structures in the

corresponding parts of napin.76

Studies show that MOCP consists of three α-helices.67

The

structure-function study of the peptides used, reveals the relationship between the water

clarification and the disinfectant activities of the MOCP. Studies show that sedimentation

requires positively charged glutamine and arginine residues of the protein to form aggregate

of particles. The bactericidal activities follow a sequence prone to form a helix-loop-helix

structure, which may consist of a hydrophobic patch containing two prolines surrounded by

positively charged arginines pointing outward from the folded polypeptide chain.

1.4 Magnetic nanoparticles

Magnetic nanoparticles are of great interest for a broad range of applications, including

catalysis,74, 77

biotechnology,78-81

biomedicine,82, 83

data storage,84, 85

magnetic resonance

imaging86-88

and environmental remediation.16, 89, 90

The use of nanoparticles offers several

advantages due to their unique size and physical properties. However, in order to design

magnetic nanoparticles for a specific application, it is important to understand their atomic

structure, surface and magnetic structure or spin dynamics.74

Several magnetic nanoparticles

have been designed for a specific application, ranging from pure metals (Fe, Co, Ni and Mn)

to metal oxides (Fe3O4, γ-Fe2O3), metal alloys (FePt, CoPt) and ferrites (MFe2O4, where M =

Co, Cu, Ni, Mn and Mg).91

Among the investigated magnetic nanoparticles, iron oxide

nanoparticles have been employed in virtually all the discussed fields due to their intrinsic

properties. In fact, magnetic iron oxide nanoparticles are the only magnetic materials

approved for clinical use by the US Food and Drug Administration (FDA), owing to the fact

that they are non-toxic, biocompatible and relatively simple to prepare.92, 93


12

1.4.1 Iron oxide nanoparticles

In nature, iron oxides exist in various forms, the most common being magnetite (Fe3O4),

maghemite (γ-Fe2O3) and hematite (α-Fe2O3).94

Fe3O4 exhibits the strongest magnetism of all

three mentioned iron oxides because it is in a more stable form. It is also known as black

oxide and contains both divalent and trivalent Fe ions. On the other hand, γ-Fe2O3 consists of

magnetite and hematite which reflect the similarity between the magnetite and maghemite

structures. Maghemite (γ-Fe2O3) is reddish brown in color and it occurs in soil as a

weathering product of magnetite. Hematite (α-Fe2O3) is the oldest known of the iron oxides

and is sometimes referred to as ferric oxide. At ambient condition, these iron oxides are

extremely stable and often may occur as the end products of transformation of other iron

oxides.94

Some of their physical and magnetic properties are presented in Table 1.1.

The crystal structure of the aforementioned iron oxides can be described in terms of a close-

packed plane of oxygen anions having iron in octahedral or tetrahedral sites. In magnetite and

maghemite (Figure 1.5b), the oxygen ions occupy cubic closed-pack interstitial positions.

Fe3O4 has inverse spinel structure with Fe ions distributed randomly between tetrahedral and

octahedral positions. The electrons can jump between Fe2+

and Fe3+

ions in the octahedral

positions at room temperature, rendering magnetite an important class of half-metallic

materials. When Fe3O4 crystals are oxidized, their crystal lattice transforms from the inverse

spinel magnetite to the cubic Fe3+

oxide lattice of maghemite.95

Conversely, γ-Fe2O3 has a

spinel structure similar to that of Fe3O4 but with vacancies in the cation sublattice. All the iron

atoms are in the trivalent state and the cation vacancies compensate for the oxidation of Fe2+

.

The eight cations occupy tetrahedral sites while the remaining cations are randomly

distributed over the octahedral sites and the vacancies are confirmed to the octahedral

positions. Their unit cell edge length is a = 0.8347 nm as compared to a = 0.8396 nm in the

magnetite, but both have eight formula units in their individual unit cells. Maghemite has a

composition close to ferric oxide and exhibits strong magnetism and remanence. It can be

considered as an Fe(II)-deficient magnetite. In the crystal structure of hematite, the Fe3+

occupies octahedral positions while oxygen ions are in a close-packed hexagonal arrangement

(Figure 1.5a).

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Figure 1.5 Crystal structures of (a) hematite and (b) magnetite and maghemite. In hematite,

oxygen ions are in hexagonal close-packed arrangements with Fe3+

occupying octahedral

positions. The oxygen ions in magnetite and maghemite are in a cubic close-packed

arrangement.94

Table 1.1: Physical and magnetic properties of iron oxide nanoparticles96

Property

Magnetite

Maghemite

Hematite

Molecular formular Fe3O4 γ-Fe2O3 α-Fe2O3

Lattice parameter (nm) a = 0.8396 a = 0.8347 a = 0.5034

Crystallographic system Cubic Cubic or tetrahedral Hexagonal

Structural type Inverse spinel Defect spinel Corundum

Type of magnetism Ferromagnetic or Ferromagnetic or Weakly ferromagnetic

superparamagnetic superparamagnetic

1.4.1.1 Magnetic behavior

Magnetic materials are usually classified based on their magnetic susceptibility (χ), which is

defined as the ratio between the induced magnetization (M) and the applied magnetic field

(H). The iron atom has a very strong magnetic moment as a result of four unpaired electrons

in its 3d orbital; conversely, when crystals are formed from iron atoms, several magnetic

states can arise.94

Magnetic iron oxide nanoparticles may be broadly divided into three main

classes: paramagnetic, ferromagnetic and superparamagnetic (Figure 1.6).97

A material is said

to be in paramagnetic state when the magnetic dipoles are oriented in random directions at

normal temperature due to unpaired electrons. Some of these single-electron dipoles align to

the direction of applied magnetic field, which causes them to display a low positive

susceptibility (weak attraction) in a magnetic field; however, when the magnetic field is

removed, they do not remain magnetized, because the ambient thermal energy is sufficient to

spin the dipoles in random directions. Ferromagnetic materials, on the other hand, contain


14

unpaired electrons, but unlike the unpaired electrons in paramagnetic materials, these are

arranged into domains consisting of many ions and atoms, whilst each domain is a single

magnetic dipole with dimensions less than 100 nm. The magnetic dipoles of ferromagnets are

equally organized in random directions; however, when a magnetic field is applied, they spin

in the direction of the applied field, and remain aligned even in the absence of field because

the ambient thermal energy is insufficient to flip them back. Since the ferromagnetic materials

depend on their domain structure to remain magnetized even in the absence of an applied

field, their size decreases to less than the domain size (of the order of tens of nanometers)

when their properties undergo a significant change. Magnetic nanoparticles of this size are

said to be superparamagnetic because, even though their dipoles align parallel to an applied

magnetic field, the ambient thermal energy is sufficient to spontaneously disorganize the

direction of their magnetization in the absence of applied magnetic field.74, 97

This unique

property makes iron oxide nanoparticles better candidates for a wide range of applications.

Figure 1.6 Magnetization hysteresis loops of different magnetic materials with induced

magnetization (M) and applied magnetic field (H). Superparamagnetic materials have a high

saturation magnetization (MS) and zero remanence (MR) and coercivity (Hc). In ferromagnets,

a residual magnetic moment remains at zero field. The magnetic susceptibility of

superparamagnetic nanoparticles is much larger than that of paramagnets.

1.4.1.2 Superparamagnetic phenomenon

Superparamagnetism is a form of magnetism, which occurs in small ferromagnetic

nanoparticles (Figure 1.7). It exhibits a behavior similar to paramagnets but differs slightly in

magnetic susceptibility. The ambient thermal energy in the superparamagnetic materials is

high enough to reorient the nanoparticles even at temperatures below the Curie or the Néel

temperature, i.e. the temperature at which ferromagnetic nanoparticles exhibit paramagnetic

http://www.wikipedia.org/wiki/Magnetic_susceptibility

http://www.wikipedia.org/wiki/Magnetism

http://www.wikipedia.org/wiki/Ferromagnetic

http://www.wikipedia.org/wiki/Nanoparticles


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properties upon heating. In small nanoparticles (1-15 nm), magnetization can randomly spin

direction under the influence of temperature.94

Néel relaxation time is the typical time

between these two spins. If the time used to measure the magnetization of nanoparticles is

much longer than the Néel relaxation time without an external magnetic field, their

magnetization appears to be on average zero; thus, they are said to be in the

superparamagnetic state.74, 98

In this state, similar to paramagnets, an external magnetic field

is able to magnetize the nanoparticles. However, the magnetic susceptibility of

superparamagnetic nanoparticles is much larger than that of paramagnets since the magnetic

moment of the entire nanoparticle tends to align in the direction of the magnetic field,99

unlike

in paramagnets where each individual atom is independently influenced by an external

magnetic field.

Figure 1.7 Scheme showing magnetic behavior of ferromagnetic and superparamagnetic

nanoparticles (a) with and without external magnetic field. Under applied magnetic field, both

ferromagnetic and superparamagnetic nanoparticles align with the applied field but in the

absence of external magnetic field, only ferromagnetic nanoparticles retain a net

magnetization. (b) Size effect and magnetic domain in nanoparticles. Ds and Dc are

superparamagnetism and critical size thresholds respectively.99

1.4.2 Synthesis methods

In recent decades, great efforts have been devoted to the synthesis of superparamagnetic iron

oxide nanoparticles (SPION) due to their potential applications in several diverse fields. Since

the SPION behavior strongly depends on size, surface chemistry and state of aggregation of

the particles, preparation methods to produce nanoparticles with unique properties are

required. Particularly during the past few years, many publications have described efficient



16

synthesis routes that offer the opportunity to easily modify process parameters that will lead

to nanoparticles with tailored structure and highly stable monodispersed suspensions with

good magnetic properties. Several common methods including chemical co-precipitation,

microemulsion synthesis, thermal decomposition, and hydrothermal synthesis have been

studied.

1.4.2.1 The co-precipitation method in aqueous solution

This method is a facile and convenient way to prepare magnetic nanoparticles. SPIONs can be

synthesized by co-precipitation of Fe3+

and Fe2+

aqueous salt solutions by addition of bases

under inert atmosphere at room temperature or at elevated temperature. Control over the size,

shape and composition of nanoparticles depends on the type of salt used i.e. chlorides,

nitrates, sulphates, etc., Fe3+

and Fe2+

ratio, ionic strength, and the pH of the medium.100-102

However, if the synthesis conditions are fixed, the quality of the nanoparticles can be

reproducible. SPIONs (either Fe3O4 or γ-Fe2O3) and ferrites are usually prepared in aqueous

medium with this approach. Magnetite nanoparticles are not very stable under ambient

conditions, and are easily oxidized to maghemite or dissolved in an acidic medium.74

The

synthesis of magnetite nanoparticles can be considered to proceed according to the overall

reaction given in Eq. 1.1.

2FeCl3 + FeCl2 + 4H2O + 8NH3 → Fe3O4 + 8NH4Cl (1.1)

According to the above reaction, a complete precipitation of Fe3O4 is expected in the pH

range 7.5-14 while maintaining a molar ratio of Fe3+

/ Fe2+

= 2:1 under a non-oxidizing

environment. The co-precipitation method allows the preparation of large quantities of

nanoparticles in a single batch; however, the size distribution is usually not very narrow.74, 91

1.4.2.2 The microemulsion method

Microemulsions are isotropic, thermodynamically stable single-phase colloidal dispersions

which consist of spherical aqueous nanodroplets called micelles surrounded by surfactant

molecules. Microemulsion systems comprise three components: water, oil and an amphiphilic

molecule, called a surfactant. The surfactant molecule, which could be cationic

(cetyltrimethylammonium bromide, CTAB), anionic, (bis(2-ethylhexyl) sulfosuccinate,

AOT), and nonionic, Synperonic® 6/10), lowers the interfacial tension between the water and

oil, resulting in the formation of a transparent solution. Depending on the ratio of oil and

water and on the hydrophilic-lipophilic balance (HLB) of the surfactant (Figure 1.8),

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microemulsions can exist as oil-swollen micelles dispersed in water (o/w microemulsion),103

or water-swollen inverse micelles dispersed in oil (w/o microemulsion);71, 104

at intermediate

compositions and HLBs, bicontinuous structures can exist.

Figure 1.8 Microstructure of a microemulsion ternary mixture at a given concentration of

water, oil (organic liquid), and surfactants as a function of temperature. A region of isotropic

single-phase solution is observed, which extends from the water-rich to the oil-rich side at

constant surfactant concentration. At increased oil concentrations, a bicontinuous phase with

undefined shape is formed which further transforms into a structure of small water droplets in

a continuous oil phase (reverse micelles) at a higher oil concentration.105

This method is similar to the co-precipitation method but instead of using an aqueous

solution, a microemulsion solution is used. The water or oil nanodroplets which contain

reagents as a nanoreactor undergo rapid coalescence, a precipitation reaction followed by an

aggregation process. These nanoreactors act as a confined environment for particle growth,

which reduce the average size of the particles during the collision and aggregation process.

This phenomenon makes microemulsion methods powerful tools for the preparation of small

magnetic nanoparticles with uniform size distribution, for example magnetic iron oxide

nanoparticles.106-108

However, the size of the spherical nanoparticles can be tailored by

changing the size of the water pool (water-to-surfactant molar ratio).91

Microemulsions can

be used to prepare monodispersed nanoparticles with various morphologies and surface

25

35

40

Te

mp

era

ture

(ºC

)

W/O O/W Bicontinuous

0 0.2 0.4 0.6 0.8 1

Weight fraction = [oil/(water + oil)]

µem + water

µem + oil

µem lam lam

µem + oil

µem + water


18

specificity in order to enhance their efficiency. However, in some cases (w/o), this method

may require a large amount of solvent to prepare an appreciable amount of particles.74

1.4.2.3 The thermal decomposition method

Another method of preparing quality semiconductor nanocrystals and magnetic nanoparticles

with a high level of monodispersity and tunable size is by the use of thermal decomposition.

This is achieved when organometallic precursors such as metal acetylacetonates, metal

cupferronates [FexCupx] where (Cup = N-nitosophenylhydoxyl amine) or carbonyls (iron

pentacarbonyl)109

are heated in high-boiling organic solvents and a surfactant such as oleic

acid or hexadecylamine.110, 111

Metal oxide magnetic nanoparticles including iron oxide

nanoparticles can also be prepared by the thermal decomposition method. To date, two

different approaches have been used for this purpose in order to avoid the use of highly toxic

compounds such as metal carbonyls. Hyeon et al.,112

in their procedure, used cheap and

environmentally friendly FeCl3 and sodium oleate. The first step consists of iron oleate, or

stereate precursors that decompose in a second step by slowly heating the mixture to 320 oC

to form the desired nanoparticles.113

The size and morphology of the magnetic nanoparticles

can be affected by factors like the ratio of the starting reagents, for instance organometallic

compounds, solvents and surfactants. The reaction time, reaction temperature and aging time

are also critical. Even though thermal decomposition seems well suited for the preparation of

controlled size and high crystalline nanoparticles,113

one of the drawbacks of this method is

the uncertainty of whether the prepared nanoparticles can be suspended in aqueous media,

which limits the extent of their applications in biological and environmental fields.

Furthermore, this method usually leads to complicated processes or requires relatively high

temperature and inert atmosphere, thereby making synthesis time-consuming and expensive.91

1.4.2.4 The hydrothermal method

Under hydrothermal synthesis, a wide range of nanoparticles can be formed. These reactions

are performed in an aqueous medium in autoclaves or reactors under high pressure and

temperature. The hydrothermal method has been reported as being environmentally friendly

and versatile since it does not involve the use of any organic solvent;114

thus, it has been

widely studied for the preparation of metal oxide nanoparticles.115

Reduction of metal ions

under hydrothermal conditions is being utilized in the preparation of iron oxide nanoparticles,

using metal salts, polyethylene glycol and sodium acetate.116

The slow reaction kinetics at any

given temperature is one of the limitations of this method; in addition, the engineering of

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nanoparticle surfaces cannot be accomplished in situ and therefore additional post-processing

steps are required.91

Among the above-described methods, co-precipitation and microemulsion methods have

shown success, and are widely utilized in the synthesis of superparamagnetic iron oxide

nanoparticles for biomedical and environmental applications. Normally, they are preferred

due to their facile and convenient preparation approach, low production cost, as well as

reproducibility. Moreover, the working window for these two methods, especially co-

precipitation, is quite large; thus, it promotes the surface chemistry modification of the

nanoparticle during the synthesis (in situ) or after synthesis. Another advantage of co-

precipitation and microemulsion methods is that the reaction temperature and time preparation

are much lower than in the other methods (Table 1.2), hydrothermal and thermal

decomposition.

Table 1.2: Comparative summary of magnetic iron oxide synthesis methods

Synthesis

method Synthesis Solvent Reaction

temp. (oC)

Reaction

period

Size and

shape control Yield

Co-precipitation very simple, ambient

conditions

water 20-90 minutes difficult high/scalable

Microemulsion very simple, ambient

conditions

water-

organic

20-40 minutes-

hours

easy medium

Thermal

decomposition

complicated, inert

atmosphere

organic 100-320 hours-days easy high/scalable

Hydrothermal simple, high pressure water-

organic

220 hours-days easy medium

1.4.3 Magnetic separation and mixing

Magnetic nanoparticles, especially SPIONs, are easily separated from ambient matrices under

mild conditions with the use of simple and cost-effective devices. For this reason, magnetic

iron oxide nanoparticles are used in biological or environmental applications. However,

magnetic separation may be as simple as applying external magnetic fields to the outside

container; such a technique is used in both small- and large-scale separators which allow

magnetic nanoparticles to be retained, whilst supernatant volumes ranging from 10 µl to a few

milliliters are removed manually. However, the automated versions that allow multiple


20

samples to be processed in large volumes, up to several liters, are available from companies

such as Dexter Magnetic Technologies, IL, USA.97

When magnetic iron oxide nanoparticles

are introduced in a sample, they tend to sediment under the influence of gravity; therefore,

mixing is required to keep them in suspension in order to improve performance. Since the

solution volumes used in most of the assays are usually very small, slow mixing or slow-tilt

rotation may be sufficient to keep the nanoparticles well-dispersed in solution. Depending on

the application and magnetic strength of the nanoparticles, permanent or electro magnets may

be applied. For biological or environmental assays with magnetic nanoparticles, a permanent

magnet system is most preferred for magnetophoresis (i.e., particle migration that occurs

when a magnetic field is exerted on a nanoparticle), since most of the laboratory-based

research studies require a simple and portable device, instead of the complicated, on-chip

integrated magnetic device.97, 117, 118

1.5 Objectives of the present work

The aim of this thesis is to develop a simple and efficient water treatment strategy that will

benefit society by making use of available and environmentally-friendly materials. In this

perspective, the proposed technology is based on a bottom-up approach i.e. the synthesis,

characterization and application of magnetic nanoparticles. As a novel water treatment

technique, magnetic nanoparticles (MNPs) for binding and separation of a coagulant protein

were developed. The first part (Part One) of this study aimed at extracting the active

coagulant protein from MO seeds; also, the synthesis of MNPs with suitable surfaces for

protein binding was carried out using two different methods. The prepared MNPs were

characterized with different techniques in order to evaluate their physico-chemical properties

as well as the biological properties of the protein, and the efficiency of the prepared magnetic

nanoparticles in the purification of the MO protein was investigated.

The second part (Part Two) highlights the development of feasible water treatment strategy

(Figure 1.9) for both developing and developed countries using two different approaches.

Approach I is based on the use of MOCP in an in situ water treatment. The crude MO protein

is adsorbed onto MNPs, followed by desorption to obtain the purified protein (MOCP). The

purified MOCP was then used for the treatment of turbid waters. These experiments were

carried out in a batch system. Approach II is based on the use of protein-functionalized

nanoparticles (MOCP-MNPs) for water treatment processes. In this approach, the MOCP

adsorbed onto the MNPs was used. The MOCP-MNPs were separated from the water by

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applying an external magnetic field. This method is particularly appealing because it promotes

material recycling; i.e. the same material can be used several times. In addition, this approach

has the potential of reducing the overall cost of treatment process including time.

Figure 1.9 Scheme showing the development of potential water treatment strategies.

Approach I is based on the use of purified MOCP for water treatment. The MOCP is desorbed

from the MNPs, followed by separation of MNPs with applied magnetic fields. Approach II is

based on the use of MOCP-MNPs for water treatment. The MOCP-MNPs can be reused after

cleaning.

Approach I Approach II

Crude MO proteins MNPs MOCP-MNPs


22

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2. Experimental

This section highlights the experimental procedures that are not described in detail, in the

appended publications, whilst reflecting the data already reported in papers I-VII. Their

results are discussed in section 3.

PART ONE

Synthesis and characterization

2.1 Extraction and analysis of coagulant protein (Papers I & II)

The MO seeds were purchased from Kenya and stored at room temperature. The husks were

removed manually and the seed kernels were ground to a fine powder using mortar and pestle.

The seed powder was defatted by mixing with ethanol (95%) using a magnetic stirrer for 30

min. The supernatant was separated by centrifugation (3000 rpm, 10 min) and the residual

powder dried at room temperature overnight. From the dried powder, the crude coagulant

protein was extracted (5% w/w) using Milli-Q water.59

The mixture was stirred for 30 min

using a magnetic stirrer and then by centrifugation at 3000 rpm for 45 min. The resultant

supernatant was termed as the crude extract. The extraction process of the active component

from crude MO seed is shown in Figure 2.1. The protein content of the crude extract was

estimated using the Bradford method.119

Figure 2.1 Extraction of the crude coagulant protein from MO seeds. The by-products

obtained during the processing are useful at domestic level.


24

2.1.1 Preparation of synthetic water

Synthetic turbid water (150-170 NTU) was prepared from kaolin (clay suspension) in order to

examine the performance of the crude extract. The clay suspension was prepared by mixing

1L of tap water and 10g of Kaolin clay with a magnetic stirrer for 30 min to obtain an even

dispersion of the kaolin particles.59

The solution was allowed to settle for 24 h to achieve a

complete hydration of the kaolin. The clear solution containing the clay suspension was

decanted into a fresh bottle and kept at room temperature prior to use.

2.1.2 Coagulation activity test

A small-volume (1 mL) coagulation activity test was employed using kaolin clay suspension59

in order to show the coagulation effect of the crude MO seed extract. This method can also be

used to study the performance of other coagulants such as alum or purified coagulant protein.

Three sample volumes (2, 5 and 10 µL) were added separately to a clay suspension to a final

volume of 1 mL in a semi-micro plastic cuvette (10x4x45mm). The samples were mixed

instantly and the initial absorbance (time 0) was measured with a Thermo Spectronic UV-

visible spectrophotometer at 500 nm (OD500). The samples were allowed to settle for 1 h and a

final absorbance was measured. A decrease in absorbance relative to control defines

coagulation activity. Eq. 2.1 was used to estimate the percentage coagulation activity of the

samples.

(2.1)

where IAbs is the initial absorbance and FAbs is the finial absorbance.

2.2 Synthesis of magnetic iron oxide nanoparticles (Papers I─III)

In this study, two different methods have been employed in the preparation of magnetic iron

oxide nanoparticles. The synthesis consists of co-precipitation in aqueous solution and

microemulsion methods. For clarity, nanoparticles prepared by the co-precipitation method

will be referred to as SPIONs while ME-MIONs will be used for microemulsion-prepared

magnetic iron oxide nanoparticles.

Chuka Okoli

25

2.2.1 Co-precipitation method in aqueous solution

A facile way to prepare SPIONs is the use of the chemical co-precipitation method. A typical

synthetic procedure according to Massart et al.,120

with some modifications is briefly

described.

The iron source was prepared by dissolving 1 M FeCl3 and 0.5 M FeCl2 in Milli-Q water. A

volume of 35 mL of the iron source was added to 250 mL of 0.7 M ammonia solution at 70 oC

under vigorous stirring (1200 rpm). The reaction mixture was allowed to proceed for 45 min

under N2 atmosphere in a closed system. The black precipitated nanoparticles were collected

with an external magnet and then washed several times with Milli-Q water. The obtained

magnetic nanoparticles were re-suspended in water and stored at 4 oC for further use and

characterization. Part of the synthesized nanoparticles was coated with inorganic precursors

for protein binding and separation.

2.2.1.1 Coating with TSC

Citrate moieties have been extensively used as stabilizing agents for magnetic iron oxide

nanoparticles.121, 122

Their molecular functions by chemical or biological means have been

explored for different applications due to the presence of carboxyl functional groups on the

nanoparticles surface (Figure 2.2). By virtue of the complexation (sorption of oppositely

charged surfaces) occurring between the iron ions on the nanoparticles and the citrate groups,

highly stable magnetic fluid with well-dispersed nanoparticles can be obtained.123

Coating of the SPION with trisodium citrate (TSC) salt was carried out by re-dispersing 20

mL (18 mg/ml) of the magnetic nanoparticles suspension in Milli-Q water. The resultant

solution was subjected to vigorous stirring (620 rpm, 30 min) at 90 oC upon the addition of

trisodium citrate solution (0.2 g). The reaction mixture was cooled to room temperature and

the obtained TSC-coated SPION was re-suspended in water after several washing steps with

water. The TSC-coated SPION core-shell was evaluated by Fourier transform infrared

spectroscopy (FT-IR).112


26

Figure 2.2 Formation of TSC-coated SPION. By using the right concentration of TSC, the

nanoparticles can be charged with more citrate groups, leading to stronger electrostatic

repulsion which prevents the nanoparticles from aggregating.

2.2.1.2 Coating with TEOS

Surface modification of the nanoparticles was achieved following the method described by

Stöber et al.124

Tetraethoxysilane (TEOS) was used as precursor. Briefly, the magnetic

nanoparticles suspension (40 mg) was diluted with Milli-Q water, alcohol and aqueous

ammonia (paper I). The dispersion was homogenized by ultrasonic vibration in water bath.

Upon continuous mechanical stirring, TEOS was slowly added to the reaction mixture (Figure

2.3). After stirring for 12 h, silica was formed on the surface of the SPION through hydrolysis

and condensation reaction.

Figure 2.3 Formation of TEOS-coated SPION. Silica was covalently attached to the surface

of the SPION via hydrolysis and condensation reaction.

2.2.1.3 Coating with APTES

3-Aminopropyltriethoxysilane (APTES) was used as an amino-silane coupling agent for the

surface coating of SPION. APTES is well suited to obtain a high density of –NH2 surface

functional groups.125

The magnetic nanoparticles suspension (72 mg) was dispersed in 35 mL

of Milli-Q water. APTES was added dropwise into the reaction mixture under N2 atmosphere

at 40 °C for 1 h (Figure 2.4). The solution was cooled to room temperature. The prepared

APTES-coated SPION was collected with an external magnet and washed with ethanol,

Fe3O

4

OH OH OH TEOS, RT, 12 h

Si

C2H

5O

C2H

5O

C2H

5O

OC2H

5

Fe3O

4

O O O

Si OH

Fe3O

4 +

Fe3O

4

COO─

C

H2C

HO

Na+

H2C COO

─

COO─

Na+

Na+

30 o

C, 30 min

Chuka Okoli

27

followed by Milli-Q water. Finally, APTES-coated SPION was re-suspended in 20% ethanol

and kept at 4 oC prior to use.

Figure 2.4 Formation of APTES-coated SPION. 3-Aminopropyltriethoxysilane (APTES)

was used as the amino-silane coupling agent.

2.2.2 Microemulsion methods

The preparation of microemulsion-based magnetic iron oxide nanoparticles (ME-MIONs) was

carried out from two different microemulsion systems: water-in-oil (w/o) and oil-in-water

(o/w).

2.2.2.1 Synthesis of w/o ME-MION

For the synthesis of w/o ME-MIONs, two approaches were used (Figure 2.5). Microemulsion

system 1 consists of a surfactant, cetyltrimethyl ammonium bromide (30 wt%, CTAB), a co-

surfactant (11 wt%, 1-butanol), an oil phase (27 wt%, hexanol) and an aqueous phase

containing either of the iron precursors: 31.6 wt%, Milli-Q water plus FeCl3 and FeCl2, 2:1

molar ratio or NH3 solution. The system was termed ME 1-MION.

Microemulsion 2 consists of a surfactant, cetyltrimethyl ammonium bromide (13.2 wt%,

CTAB), a co-surfactant (10.1 wt%, 1-butanol), an oil phase (49.8 wt%, n-octane) and an

aqueous phase containing the iron precursors (26.9 wt%, Milli-Q water plus FeCl3 and FeCl2,

2:1 molar ratio) and a precipitating agent (15 wt% NH3).71

The preparation of ME 1-MION was achieved by mixing two microemulsions (Figure 2.5a).

The two microemulsions respectively consist of an aqueous solution of iron precursor and a

precipitating agent (aq.NH3). Precipitation of magnetic nanoparticles was carried out by

adding the former microemulsion into the latter at room temperature upon vigorous stirring

for 1 h. The pH of the mixture was adjusted to 11 with ammonia. Formation of magnetic

nanoparticles was observed by the resulting black coloration. The obtained magnetic

nanoparticles were separated with an external magnet and then washed by several cycles of

precipitation and re-suspension in a mixture of chloroform and methanol (1:1), and finally

Fe3O

4

OH OH OH APTES, 40

o

C, 1 h

Si

C2H

5O

C2H

5O

C2H

5O

(CH2)3 NH

2

Fe3O

4

O O O

Si (CH2)3 NH

2


28

water. The nanoparticles were either dried at 70 ◦C or suspended in Milli-Q water and kept at

4 ◦C until further use.

Figure 2.5 Schemes for the preparation of inorganic magnetic nanoparticles by the w/o

microemulsion reaction approach by (a) mixing two microemulsions of identical composition

except for reactant I (metal precursors) and reactant II (precipitating agent), (b) using a

microemulsion containing Fe salts (FeCl3:FeCl2), whilst the precipitating agent (aq.NH3) is

added directly.

For the synthesis of ME 2-MION, a single-step mode of preparation was used. In this

approach, only one type of microemulsion solution is needed in the formation of magnetic

nanoparticles (Figure 2.5b). Addition of the precursor solution to the mixture of CTAB/1-

butanol/n-octane leads to the formation of a microemulsion. Formation of magnetic

nanoparticles was achieved by adding the appropriate amount of precipitating agent (aq. NH3)

dropwise to the aforementioned microemulsion containing the precursor upon vigorous

stirring until pH 11 was achieved. The reaction mixture was allowed to stir for 1 h at 25 oC. A

dark brown coloration indicates the formation of magnetic nanoparticles in the reaction

mixture. The obtained magnetic nanoparticles were separated with an external magnet and

then washed by several cycles of precipitation and re-suspension in a mixture of chloroform

Chuka Okoli

29

and methanol (1:1), and finally water. The nanoparticles were either dried at 70 ◦C or

suspended in Milli-Q water and kept at 4 ◦C until further use.

2.2.2.1 Synthesis of o/w ME-MION

The procedure of o/w microemulsion synthesis was developed by Sanchez-Dominquez et

al.103

The microemulsion system consists of a nonionic surfactant (21.5 wt%, Synperonic®

10/6), an oil phase containing the iron precursor (14 wt%, hexane plus Iron (III) 2-

ethylhexanoate), and an aqueous phase (64.5 wt%, Milli-Q water). The microemulsion

containing the corresponding organometallic precursor was prepared by mixing the three

components in the aforementioned proportions and stirring at 30oC until a homogenous,

transparent brownish isotropic phase was obtained. The precipitating agent (30 wt% NH3) was

added directly while stirring the mixture in order to increase the pH and precipitate the

magnetic iron oxide nanoparticles. The reaction mixture was adjusted to pH 11 and kept under

continuous stirring at ~30oC. The obtained magnetic nanoparticles (Figure 2.6) were separated

and washed by several cycles of precipitation and re-suspension in ethanol-water (1:1) and

finally ethanol. The magnetic nanoparticles were either dried at 70 o

C or suspended in Milli-Q

water and kept at 4oC until further use.

Figure 2.6 Scheme for a single-step preparation of inorganic magnetic nanoparticles by the

o/w microemulsion reaction approach. The magnetic nanoparticles were precipitated upon the

addition of an aqueous NH3 solution into the microemulsion containing Fe salts. Figure

adapted from103

.

2.3 Characterization (Papers I─III)

Several techniques have been employed to investigate the size, morphology, magnetic

property and surface chemistry of the nanoparticle. The two types of ME-MION have been

compared with different surface-coated SPIONs prepared by the water-based co-precipitation


30

method. The following subsections discuss the characterization techniques used for each

preparation method.

2.3.1 Nanoparticles prepared by co-precipitation (Paper I)

The average diameters of nanoparticles were estimated from TEM micrographs. Transmission

electron microscopy (TEM) was carried out using a JEM-2100F microscope under an

accelerating voltage of 200 kV. The specimen for TEM imaging was prepared by the

dispersion of magnetic nanoparticles in alcohol. A drop of well dispersed sample was placed

on a carbon-coated 200 mesh copper grid and the sample dried under ambient condition

before loading the grid in the sample holder of the microscope for TEM analysis.

Crystallographic analysis of the sample was performed on dried powder by powder X-ray

diffraction (Xpert PRO, model PW3040/60) to determine the crystal structure. The 2θ data

were collected using a continuous scan mode in the range of 20o-70

o. The average crystalline

sizes of the nanoparticle were calculated using the Scherrer equation:

d = 0.9 λ / β cos θ (2.2)

where d is the particle size (nm), λ: X-ray wavelength (Å), θ: diffraction angle (o) and β: the

full width at half maximum of the diffraction peak at θ (rad).

Atomic absorption spectroscopy (AAS) analysis was used in the quantitative determination of

the nanoparticle concentration using a Spectra AA-200 spectrometer.

Fourier transform infrared spectroscopy (FT-IR) was carried out on a Nicolet Avatar 360 FT-

IR ESP to confirm the presence of carboxyl group in the TSC-coated SPION. The absorption

bands are characteristic of the functional groups present in a sample. Different peaks

(functional groups) were evaluated and compared with uncoated and coated SPION.

In order to determine the magnetic behavior of the nanoparticles, room temperature magnetic

hysteresis loops measurement was carried out using a vibrating sample magnetometer (VSM,

Model 155 EG&G Princeton Applied Research). The magnetic moments of the particles were

determined in field over a range of ± 8kOe.

Zeta potential (Zp) measurement using Zetasizer Nano series equipment was employed to

study the surface charge of the nanoparticles. The pH of surface-coated SPIONs were

adjusted to different pH values ranging from 3 to 12 with 1 M NaOH and 1 M HCl solutions.

The charges were determined and the point of zero charge was estimated from the zeta

potential versus the pH curve.

Chuka Okoli

31

2.3.2 Nanoparticles prepared by Microemulsion (Papers II-III)

X-ray diffraction data of the nanoparticles prepared by microemulsion methods were acquired

between 20o and 70

o on a Siemens D5000 diffractometer, using Cu Kα radiation (Paper I).

The average crystalline sizes of the nanoparticle were calculated using the Scherrer equation

(Eq. 2.2).

The size and morphology of the nanoparticles were examined by High-Resolution

Transmission Electron Microscopy (HRTEM), which was carried out using a Field Emission

Transmission Electron Microscope (JEM-2200FS) at 200 kV, with 0.19 nm resolution in

TEM mode. For this purpose, dry powder samples were dispersed in isopropanol and

deposited on formvar carbon copper grids. Selected area electron diffraction patterns (SAED)

of the nanoparticles were also recorded from the samples.

Room temperature magnetic hysteresis loops of the nanoparticles were measured using a

Superconducting Quantum Interference Device (SQUID) Magnetometer, Quantum Design

MPMS XL. The magnetic moments of the particles were determined in field over a range of ±

60kOe.

Surface area and porosimetry of the nanoparticles were calculated by the Brunauer-Emmet-

Teller (BET) method using Micromeritics ASAP 2010. Specific surface area as well as the

specific pore volume was detected after degassing the sample overnight at 110 oC prior to the

measurement.

PART TWO

2.4 Application of nanotechnology in water treatment (Papers III, IV, V & VII)

2.4.1 Purified MOCP for water treatment (Approach I)

The purpose of this study is to demonstrate the potential of MOCP in turbid water

clarification. For this reason, a novel purification approach based on the use of magnetic

nanoparticles (MNPs) prepared from two different systems (co-precipitation and

microemulsion methods) was developed and investigated. The purification of MOCP for

water treatment as well as its analysis and characterization is discussed in the following

subsections.


32

2.4.1.1 Purification of the MO coagulant protein with MNPs and activity test

Purification of the MO protein was carried out with the aforementioned magnetic

nanoparticles (2-30 nm size) in a batch system (Figure 2.7). The MNPs were equilibrated

using 10 mM ammonium acetate buffer, pH 6.7. Crude extract (2.6 mg/mL) was added to the

equilibrated MNPs and the sample volume was adjusted with 10 mM ammonium acetate

buffer. The sample was mixed and incubated at room temperature for 1 h. Finally, the

adsorbed protein was separated from the mixture using an external magnetic field (6000 G).

The coagulant protein was eluted with different concentrations of NaCl (0.6, 0.8 or 1.0 M) in

10 mM ammonium acetate solution.71, 126

The sample was either stored at -20 oC or freeze-

dried. The IEX and CMC beads were used as controls.126

Coagulation activity of the MOCP

was measured following the procedure already described in section 2.1.2.

Figure 2.7 Batch purification procedure of the MO protein using MNPs. The MO protein

binds to MNPs; unbound proteins (pink and blue) are removed by washing. The MOCP is

desorbed (eluted) from the MNPs. Rapid separation is achieved with the help of an external

magnetic field to separate the MOCP and MNPs. The desorbed MOCP (green) is either used

in suspension or as a freeze-dried powder for in situ water treatment.

2.4.1.2 Analysis and characterization

Protein contents of the MO seed extract and purified protein were determined by the Bradford

assay.119

The protein profile, molecular weight of the purified protein and that of the crude

extract were determined and compared in 10% sodium dodecyl sulphate (SDS)-

polyacrylamide gel electrophoresis (PAGE)126

mini gels stained with Coomassie brilliant blue

to visualize the protein.

Chuka Okoli

33

Mass spectrometric analysis of the purified protein was carried out based on MS/MS peptide

sequence analysis of in-gel trypsin digested protein using a Q-TOFTM

II mass spectrometer.

2.4.1.3 Turbidity removal in surface waters

A simple and quick method to verify the coagulation activity of MOCP in terms of turbidity

removal was carried out. The water samples for this study were collected from three different

lakes in Sweden. Raw surface waters with initial turbidity ranging from 7–17 NTU were

collected from Lake Mälaren in autumn 2009; Arboga (Västmanland) and Vättern-Lake

(Götaland) waters were collected during spring 2010. The choice of seasonal variation in the

collection of raw water samples is essential to evaluate the performance of the coagulant

agent with different turbidity/contaminants. Once collected, the samples were stored at 4 oC to

prevent any bacterial growth. The kaolin clay with an initial turbidity of 150–170 NTU was

used as one of the control sample for comparison purposes. Aluminum sulphate (5%, alum)

was prepared and used as a control. All experiments were carried out in a batch system.

In order to estimate the performance (turbidity removal) of the MOCP or alum, coagulation

activity assays were carried out as described in section 2.1.2. In a typical experimental

procedure, MOCP (0.1-0.5 mg/L) was added to raw surface water or clay suspension. The

sample was mixed rapidly at 50 rpm for 5 min to allow an optimal contact between the sample

waters and MOCP. A small volume of the sample was collected and the initial absorbance

measured at 500 nm (OD500) with a Thermo Spectronic UV-visible spectrophotometer. The

sample was allowed to settle (gravity) for 1 h and the absorbance was measured again (final

absorbance). However, the measurements may be carried out periodically (0-240 min) during

their settling time in order to monitor the optimal time needed to achieve good activity. A

decrease in absorbance relative to the controls defines turbidity removal from the source

water. Alum was used for comparison (positive control) while surface water and a clay

suspension without the protein were used as negative controls. The interaction studies of

MOCP and MNPs with different surface chemistry have been carried out to understand the

binding behavior of MOCP on the surface-coated MNPs. The following section illustrates the

in silico modeling investigation.

2.4.1.4 Interaction of MOCP and MNPs (Papers III & V)

Several studies have been performed in order to elucidate the binding of MOCP onto MNPs

with different modified surfaces. For this purpose, computational modeling study of the

interaction between the protein molecule and the surface-coated SPIONs was performed and


34

the data were compared with experimental results. Furthermore, circular dichroism (CD)

spectroscopy of MO2.1 fluorescence emission of the protein and ME-MIONs were

investigated.

2.4.1.4.1 Computational and experimental studies

The MOCP (MO2.1) secondary structure was examined using circular dichroism (CD)

spectroscopy. The far-UV CD was measured on a Jasco-710 dichrograph previously calibrated

with d-10-camphorsulphonic acid. Measurements were performed in 0.5 cm path-length

cuvette at 22 ºC. The ellipticity was recorded with a 0.5 nm bandwidth and a 2s response.

Sample concentrations were ~1mg/mL, in ammonium acetate buffer pH 6.7, 0.6 M NaCl.

Five accumulations were acquired for the spectrum. The mean residue ellipticity (MRE) was

calculated using:

cl10

MRW obsmr

deg. cm

2.dmol

-1 (2.3)

where MRW is the molecular weight per residue (MRW=MW/(n-1)), θobs: observed ellipticity

in degrees and c: concentration in g/ml.

Theoretical modeling tools have been used to understand the structure of a

coagulating/flocculating protein from MO and its ability to bind to various ligand precursors

(Paper V). Gaussian 03 Software127

was used to generate 3D structures for these ligand

molecules, namely trisodium citrate (TSC), 3-aminopropyl trimethoxysilane (APTES), and

tetraethoxysilane (TEOS). The electrostatic potential of this protein was also calculated.

Different theoretical tools were employed in the following sequence. (i) Homology modeling

was used to obtain the protein structure from the known peptide sequence of MO2.1 (P24303)

through superimposition with the mabinlin sequence (Paper VI);

QGPGRQPDFQRCGQQLRNISPPQRCPSLRQAVQLTHQQQGQVGPQQVRQMYR

VASNIPST.61

(ii) Molecular docking studies were performed to investigate the binding

ability and mode of binding of different organic molecules and hydroxyl groups

functionalized with silicon clusters with the aforementioned protein. (iii) Since the docking

studies mimic the zero temperature situations, molecular dynamics simulations for the docked

protein-ligand structures were carried out in aqueous solution to study finite temperature

effects. Protein-ligand distance profile data was compared with the experimental results.

Basically a computation of the instantaneous distance (defined as RN-ligand where N refers to

Chuka Okoli

35

the residue number and ligand refers to the residues, namely TSC, APTES and TEOS)

between each of the 60 residues of protein and the ligand center of mass was calculated. In

addition, computation of the average RN-ligand was also conducted. This calculation has been

done for 1000 configurations obtained from the molecular dynamics128, 129

2.4.1.4.2 Fluorescence emission study (Paper III)

The binding of MOCP with w/o ME-MION and o/w ME-MION was studied. Emission

fluorescence on a modular PTI Spectrometer with an excitation wavelength of 255 nm was

employed. Emission was recorded at λ= 285 to 475 nm. The excitation and emission slits

were adjusted to 2 nm. Temperature was controlled (22 oC) with a thermoelectric temperature

controller, TLC 50. Cells with a path length of 1 cm were used and all the experiments were

performed at least twice. The average spectra were subtracted from appropriate blanks before

analysis.

2.4.2 MOCP-MNPs for water treatment (Approach II)

Here, a novel approach for water treatment with MOCP-MNPs was developed (Figure 2.8).

The use of MOCP-MNPs enables the separation and recovery of this system from water using

an external magnetic field, and consequently the opportunity to regenerate the system for

further water treatment.


36

Figure 2.8 Scheme of the proposed MOCP-MNPs for water treatment. Treatment:- MOCP-

MNPs is added to water followed by a slow mixing. The impurities in the water bind to the

MOCP-MNPs. Separation can be achieved either by gravity or external magnet. A rapid

separation is achieved with the help of an external magnetic field within 1-2 min. Clean and

potable water is obtained following the separation step. Regeneration:- MOCP-MNPs is

cleaned, separated and reused for another batch of raw water.

2.4.2.1 Development of MOCP-MNPs system

The development of MOCP-MNPs followed a batch system preparatory approach. The MNPs

(2-30 nm size) prepared either by co-precipitation or microemulsions, were equilibrated using

10 mM ammonium acetate buffer, pH 6.7. Crude MO seed extract (2.6 mg/mL) or MOCP

(0.6 mg/mL) was added to the equilibrated MNPs. The samples were kept for 1-2 h in a rotary

mixer (20 rpm) at room temperature. The supernatant was removed by magnetic separation

and the resultant MOCP-MNPs was washed with ammonium acetate buffer to remove the

non-adsorbed protein and other impurities. The MOCP-MNPs was suspended in ammonium

acetate buffer and kept at 4 oC or freeze-dried prior to use. The developed MOCP-MNPs were

termed MOCP+SPION or MOCP+ME-MION systems.

The functionality of the developed MOCP-MNPs was tested in clay suspension according to

the procedure previously described in section 2.1.2.

Chuka Okoli

37

2.4.2.2 Turbidity removal in surface waters

The efficiency of the developed MOCP-MNPs in terms of turbidity removal against surface

water was analyzed following a similar experimental procedure described in section 2.4.1.1.

The clay suspension and alum (5%) were used as controls.

Briefly, MOCP+SPION or MOCP+ME-MION (0.01-0.4 mg/L) were added to the water

samples and the initial absorbance measured. The samples were allowed to settle for 1 h and

the final absorbance was measured. However, the measurements may be carried out

periodically (0-240 min) during their settling time in order to monitor the optimal time needed

to achieve good activity. A decrease in absorbance relative to controls defines turbidity

removal from the source water. The percentage coagulation activity (turbidity removal) was

calculated according to Eq. 2.1.

2.4.2.3 Separation by gravity and magnetic field

A quick method to achieve efficient turbidity removal as well as rapid separation with

MOCP+ME-MION was tested. The coagulation and magnetization properties of MOCP+ME-

MION under the influence of an external magnetic field were compared with the settling of

impurities by gravity. For settling of impurities by gravity, alum was used as a control.

Synthetic clay suspension and natural surface water samples (SW1; 7 NTU and SW2; 17

NTU) from lake Mälaren were used. The MOCP+ME-MION and alum were added separately

in two different flasks containing the turbid water samples and mixed instantly; the initial

absorbance of the mixture was measured at 500 nm. Then the resultant mixture was allowed

to interact for 10 min at room temperature. After the interaction time, MOCP+ME-MIONs

were separated from water with a magnet (1-2 min) to obtain clean water; the water sample

containing alum was decanted (Figure 2.8). Final absorbance of the clean water was measured

and the turbidity removal efficiency calculated.

2.4.2.4 Reusability of MOCP-MNPs system

In this study, the possibility to reuse MOCP-MNPs was investigated. The number of times the

developed system can be reused in water treatment was verified.

In this investigation, the impurities adsorbed on MOCP-MNPs were cleaned with a mild

detergent or a cleaning solution (0.1 wt% Tween 20 in water and 20% ethanol) after each

turbidity removal assay with water samples. A solution of 0.1 wt% Tween 20 or 20% ethanol

was added to the used MOCP-MNPs and the mixture was left for 10 min at room temperature.


38

The MOCP-MNPs were removed by magnetic separation followed by two washes with 10

mM ammonium acetate buffer. The control sample was the initial coagulation activity of

MOCP-MNPs in turbid water sample before cleaning. The coagulation activity test was

performed as described earlier (section 2.1.2).

Chuka Okoli

39

3. Results and discussion

Some of the data presented in this section have not been published; however, they reflect the

results already appended in papers I-VII.

PART ONE

Synthesis and characterization

3.1 Investigation of the active protein (Paper I)

In order to estimate the activity of the crude MO seed extract, a control sample (clay

suspension) was used. The difference between the control sample and the MO protein is

considered as the real coagulation activity. The crude MO seed protein showed good

coagulation activity on clay suspensions. The baseline in Figure 3.1 indicates settling of the

clay suspension by gravity; this can be subtracted from the percentage activity of the active

components of the protein to obtain the real activity. The coagulant protein exhibits good

activity (~ 80%) in all the studied concentrations.32, 130

Given that the MO crude extract

contained the coagulant protein, it was appealing to bind or separate the active protein onto

the right surface of the magnetic nanoparticles in order to develop a simple and feasible water

treatment procedure. The subsequent sections discuss the results obtained from the design and

application of MNPs for water treatment.

Figure 3.1 Coagulation activity of crude MO seed extract (2.5 mg/ml, stock) on a synthetic

clay suspension with 1 h as a setting time. The baseline indicates settling of the clay

suspension by gravity; this can be subtracted from the percentage activity (turbidity reduction)

of the active components of the protein to obtain the real activity.


40

3.2 Characterization of MNPs (Papers I─III)

Magnetic iron oxide nanoparticles (MNPs) were prepared for binding and separation

(purification) of protein for water treatment. The prepared nanoparticles from co-precipitation

(surface-coated SPIONs) and microemulsion (w/o ME-MION and o/w ME-MION) systems

were characterized using different techniques. This section compares their physico-chemical

properties.

3.2.1 Size and morphology studies

TEM images of bare SPION, APTES-coated SPION, w/o ME-MION and o/w ME-MION are

presented in Figure 3.2. As observed from the TEM micrographs, the nanoparticles depict

large aggregates. This may be due to the high surface energy of the nanoparticles; on the other

hand, it can be envisaged that high concentrations of the nanoparticles were deposited on the

TEM grids which affected the micrographs. The average particle size of each nanoparticle

was calculated by measuring the diameter of more than 200 particles using image J software.

The diameter of the bare SPION is in the size range of 6-10 nm (Figure 3.2a). After coating

the SPION with APTES, dense aggregates were observed in some areas with quasi-spherical

morphologies. The average particle diameter of APTES-coated SPION was in the order of 20

nm (Figure 3.2b).

Figure 3.2 TEM images of (a) bare SPION, (b) APTES-coated SPION, (c) w/o ME-MION

and (d) o/w ME-MION.

(a) (b)

50 nm 20 nm

20 nm

(c) (d)

20 nm

Chuka Okoli

41

However, from the TEM micrographs of ME-MION, the w/o ME-MION nanoparticles were

irregular in shape, consisting of elongated, rod-like morphologies with widths ranging from 5-

10 nm (Figure 3.2c). The o/w ME-MION nanoparticles were globular and in the order of 3 nm

with dense aggregates (Figure 3.2d). The results obtained from the TEM size estimation

(Figures 3.2, c and d) were in agreement with the crystalline size calculation of w/o and o/w

ME-MIONs (Paper III) using the Scherrer equation (Eq. 2.2, section 2.3.1) with the XRD data

(Figure 3.3). The present study underscores the fact that nanoparticles with similar

morphology and size can be obtained irrespective of the preparation method. Nevertheless,

there is a considerable overlap and large aggregation in ME-MIONs due to the large surface

energy of the nanoparticles. Aggregation of these nanoparticles may depend on experimental

parameters like temperature, pH, reaction environment and surface condition. If not properly

controlled, these parameters can influence the structure of the obtained iron oxide

nanoparticles.71, 103

3.2.2 Structural studies

The diffraction patterns of nanoparticles prepared from co-precipitation and microemulsion

systems depict the presence of a magnetic phase of either Fe3O4 or γ-Fe2O3 (Figure 3.3);

which is quite typical at 35o for (311) and 63

o for (440).

Figure 3.3 XRD patterns of (a) bare SPION and (b) w/o- and o/w- ME-MION.

The XRD patterns of bare SPION and w/o ME-MION nanoparticles indicate that the particles

are highly crystalline, which confirms the inverse spinel structure of magnetite and

(b)

o/w

20 25 30 35 40 45 50 55 60 65 70

Inte

ns

ity (

arb

. u

nit

)

2θ (o)

(311)

(220) (400)

(511)

(440)

(a)

w/o

(311)

(440)

(511)

(220)

(b)

o/w


42

maghemite (even though it is difficult to distinguish between them from XRD).122, 126

The o/w

ME-MION XRD pattern showed wide peaks characteristic of small nanoparticles suggesting

that the original size of the nanoparticles is very small and fits well to a typical spinel phase of

either magnetite (Fe3O4) or maghemite (γ-Fe2O3) or a combination of both.107, 131

3.2.3 Magnetic studies

The results from magnetization (M) versus applied field (H) measurements at room

temperature (300 K) for bare SPION, w/o- and o/w- ME-MIONs are presented in Figure 3.4.

It can be seen that the magnetization curves of bare SPION and w/o ME-MION () clearly

depict zero remanence and coercivity, confirming a superparamagnetic behavior and larger

particles size as compared to o/w ME-MION (). The recorded saturation magnetization (Ms)

value for bare SPION is 70 emu/g while w/o ME-MION presented MS of 30 emu/g.

Conversely, the magnetic moment of o/w ME-MION exhibited a weak/or incomplete

saturation magnetization (MS) of 10 emu/g, indicating a decrease in mean particle size. The

obtained data imply that the nanoparticle preparation method determines their magnetic

behavior; moreover, the specific magnetization of iron oxide nanoparticles is known to

depend on their size.132

Figure 3.4 Magnetization as a function of magnetic field at 300 K for (a) VSM of SPION.

Magnetic moments of the samples were determined in field over a range ± 8kOe. The

recorded MS value of bare SPION is 70 emu/g; (b) SQUID magnetometer of w/o ME-MION

() and o/w ME-MION (). The recorded MS value for w/o ME-MION is 30 emu/g and 10

emu/g for o/w ME-MION. Magnetic moments of the samples were determined in field over a

range of ± 60kOe.

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Chuka Okoli

43

3.2.4 Surface charge studies

The ζ-potential results of SPION, TSC-coated SPION, TEOS-coated SPION and APTES-

coated SPION are shown in Figure 3.5. The magnetic nanoparticle dispersion was titrated

from pH values of 2 to 12, using HCl (0.1 M) and NaOH (0.1 M) solutions. Bare SPION

(magnetite or maghemite) showed a point of zero-charge (pzc) at a pH of ~7, above which the

surface of SPION is negatively charged and below positively charged. It is envisaged that the

ME-MIONs present similar behavior, implying that magnetite or maghemite exhibits a point

of pzc at pH around 7.5.133

Figure 3.5 Zeta potential (ζ) as a function of pH for bare SPION, TSC-coated SPION,

TEOS-coated SPION and APTES-coated SPION.

The pzc for TSC-coated SPION is observed at pH 5.3, which clearly illustrates that the

surface of SPION was successfully modified with TSC. In the case of APTES-coated SPION,

the amino groups from the surface of SPION displayed a shape almost similar to a sinusoidal

curve with a positive maximum value at ζ=48.1 mV (pH=2) and a negative maximum value at

ζ= ─ 42 mV (pH=6.8). The APTES-coated displayed pzc at pH around 5.85; therefore, it can

be concluded that the dispersion of APTES-coated SPION suspension is stable from pH 2 to

9.134

Conversely, the TEOS-coated SPION exhibited a net negative charge for a wide range

of pH values (pH 2 to 9).


44

PART TWO

3.3 Application of nanotechnology in water treatment

3.3.1 Purification of the MO protein with MNPs and characterization (Papers I─III)

MOCP was separated from the crude seed extract using magnetic nanoparticles (MNPs)

prepared from the two systems (surface-coated SPIONs and w/o and o/w ME-MIONs). The

purification efficiency of MNPs was compared with that of commercial beads (carboxyl

methyl cellulose, CMC) with micrometer size (Paper I). The coagulant protein was eluted

with 0.8 M Nacl. The molecular mass of the purified protein was determined by SDS-PAGE,

using low range molecular weight markers (Papers I-III). Crude seed extracts showed multiple

bands, indicating the presence of other proteins.59

The protein purified with TSC-coated

SPION and TEOS-coated SPION (Paper I) depicts a single band, indicating that the coagulant

protein was eluted from the SPIONs. Similarly, purification with w/o- and o/w- ME-MION

also showed a single band which is in agreement with the data already reported (Paper III).

The MNPs used in this work especially the ME-MIONs, suggest the presence of high surface

area which enhanced their performance in protein purification. The molecular mass of the

MOCP is ~ 6.5 kDa as determined by SDS-PAGE.18, 126

The eluted proteins from the

nanoparticles showed good coagulation activity compared to unbound protein.71, 126

It can be

concluded that the purification efficacy of both systems (surface modified SPIONs and ME-

MIONs) is comparable and similar to commercial beads in terms of their performance.

However, due to the low cost of preparation including the high surface-to-volume ratio of the

investigated MNPs, they can serve as a good alternative to the commercial bead.

To further confirm the purified fractions after SDS-PAGE, mass spectrometric analysis of

TSC-coated SPION and w/o ME-MION purified protein was carried out based on MS/MS

peptide sequence analysis (Table 3.1). The obtained peptide sequences confirmed that the

purified protein is identical or similar to the already published sequence of MOCP.59, 61

However, the specific regions where the amino acid residues appear to be slightly different

from the obtained peptide sequences after purification with MNPs are highlighted in yellow

color (Table 1). Although, it was reported that coagulant protein fraction obtained after

purification may not consist of a single, homogeneous protein, but a heterogeneous protein

with similar physical characteristics;59

Chuka Okoli

45

Table 3.1: Mass spectrometric analysis─ MS/MS determination of the sequence of peptides

from the trypsin digested protein

Purification method

Peptide Sequence

TSC-coated SPION QAVQLTHQQQGQVGPQQVR

w/o ME-MION QAVQLTHQQQGQVGPQQVR

IEX Chromatographya QAVQLETQQQGQVGPQQVR

IEX Chromatographyb QAVQLTHQQQGQVGPQQVR

*a,b

Similar or identical to sequence reported by Ghebremichael

et al. (2005) and Gassenschmidst et al. (1995) respectively.

3.3.1.1 Turbidity removal in surface waters (Approach I)

The performance of the MOCP in terms of turbidity removal efficiency in natural and

synthetic waters is shown in Figure 3.6. The coagulation efficiency of the active protein

eluted from TSC-coated SPION, w/o ME-MION and o/w ME-MION was compared with

alum.

Figure 3.6 Coagulation performance of purified MOCP and alum after a 1 h settling period

in clay suspension (170 NTU) and surface water (17 NTU) from Lake Mälaren. The MOCP 1,

MOCP 2 and MOCP 3 represent protein purified with TSC-coated SPION, w/o ME-MION

and o/w ME-MION respectively. Control is the water sample.

As Figure 3.6 clearly depicts, the coagulation activity of MOCP is significantly higher and

comparable with alum in turbidity removal assay. Their performance follows a similar pattern

in both synthetic clay suspension and natural water, implying that, in a 1 h settling period, ~

50 % turbidity removal can be achieved in low turbid waters (17 NTU) with MOCP, whereas

more than 75 % turbidity removal can be obtained in high turbid water (170 NTU). It can be


46

observed that parameters such as initial water turbidity, coagulant agent dosage and

interaction time have significant influence on the removal of suspended particles from the

water samples.32, 59

This observation may be explained in terms of the increase in suspended

particles available for adsorption and inter-particle bridge formation in clay suspension. The

net effect is increase in particle collision and agglomeration (floc formation).31

This behavior

reveals the variation observed in coagulation performance of clay suspension and surface

water. Muyibi et al.,31

reported that the use of high concentrations (large doses) of the

coagulant agents can lead to overdosing, which may result in the restabilization of the already

destabilized particles due to insufficient number of particles to form more inter-particle

bridges.

3.3.2 Interaction of MOCP and MNPs (Papers III & V)

This section gives an in-depth knowledge of MOCP interactions with MNPs of different

surface chemistry; the present study was critical for the development of robust water

treatment process. Results from the MOCP-ligand interactions are shown and discussed in the

following sections. In silico and in vitro data are reported in section 3.3.2.1 for surface-coated

SPIONs (Paper V) while in section 3.3.2.2, a fluorescence emission study of ME-MIONs and

with MOCP is reported (Paper III).

3.3.2.1 Computational and experimental studies

Based on the homology modeling, the tertiary structure for the MOCP (MO2.1) monomer is

proposed to consist of three α-helices and two loops (Figure 3.7). The hypothetical structure

of the monomer and the stability of dimers were established via homology modeling, protein-

protein docking studies and binding free energy calculations. Paper VI provides in-depth

knowledge on this aspect. Furthermore, the secondary structure of MO2.1 has been obtained

from the circular dichroism (CD) spectroscopy (Figure 3.7c). As seen in Figure 3.7c, MO2.1

depicts typical secondary structure of α-helix protein with two minima (202 and 248 nm

respectively).

However, given that the binding sites of the MOCP protein are poorly understood, it was

essential to understand the molecular interactions of MOCP-ligand chemistry for a wide range

of applications (Paper V).

Chuka Okoli

47

Figure 3.7 Homology modeling of (a) mabinlin2, which was predicted to be highly

homologous to MO2.1. Among the suggested 72 amino acid residues of mabinlin2 involved in

the homologous structure of MO2.1, only 3 helices and 2 loops consist of 55 amino acid

residues (indicated in light blue), (b) predicted homologue after normalizing the sequences

with the 5 amino acids at N-terminal region (indicated in red), to show all the 60 amino acid

residues of MO2.1, and (c) CD spectrum of MO2.1 showing typical secondary structure of α-

helix protein with two minima.

SPIONs were decorated with precursor ligands such as TSC, TEOS and APTES for the

protein-ligand interaction studies (Figure 3.8). The interaction with bare SPION was studied

with Si60 nanoclusters functionalized with hydroxyl groups. The overall molecular docking

studies depict at least two binding sites, whilst the size and charge of the ligand are critical in

localizing the binding sites. Among the two binding sites, one is located at the core of the

protein (i.e. TEOS and APTES ligands at 1st and 2nd helical regions) surrounded by Cys12,

Ala28 and Ala32 residues while the other one (TSC and Si60-OH─) is located at the side chain

of the 3rd helix around Arg48 and Arg52 residues. However, TSC or hydroxyl group

functionalized Si60 ligands bind to the positively charged Arg48 and Arg52 residues via

electrostatic interaction whereas the TEOS and APTES ligands bind to core binding site

where the Cys12 residue of the protein was involved in hydrogen-bonding; this suggests that

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60

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190 210 230 250

Ell

ipti

cit

y (

de

g.c

m2.d

mo

l-1)

Wavelength (nm)

(c)


48

the Cys12 residue was protonated in the aqueous solution at pH 7. The Alanine (Ala) residues,

on the other hand, initiated van der Waals interaction.

Figure 3.8 Docking study of MOCP (MO2.1) interaction with ligand molecules (a) TSC

ligand, (b) TEOS ligand, (c) APTES ligand and (d) Si60 nanoclusters. Important protein

residues involved in binding are highlighted; arginine (Arg, green), cysteine (Cys, yellow),

alanine (Ala, white) and Si60 nanoclusters with OH group (red and white).

The protein residues-ligands distance profile analysis as well as the coagulation activity of

protein-ligand (MOCP-MNPs) are shown in Figure 3.9. The average distance value for each

residue and its distribution with respect to the average value of the studied protein-ligand

interaction (protein-TSC, protein-TEOS and protein-APTES) are shown in Figures 3.9 (a)-(c).

The width is directly related to the amplitude of the residue motion (with respect to the ligand).

A larger r-value for the width means a large amplitude motion for the residue while a smaller r-

value for the width means a localized motion for the particular residue. As can be seen in

Figure 3.9a, the residues Arg48 and Arg52 are in closer contact with the TSC ligand.

Interestingly, a smaller width computed for these two residues (among all 60 residues) was

observed. This clearly shows that the binding of the ligand reduces the dynamics of these two

residues. When compared to TOES and APTES ligands, a relatively smaller value for widths of

Chuka Okoli

49

other residues in the 3rd helix is observed; thus, the effect of ligand binding is in some sense

seen over the whole helix, whilst larger width for the residues that form 1st and 2nd helices

implies increased conformational flexibility for these two helices when compared to 3rd helix.

The phenomenon was critical in the high performance of TSC-modified SPION (Figure 3.9d)

in terms of coagulation activity.

Figure 3.9 Motion distance calculations of the protein residues with different ligands (a)

TSC, (b) TEOS, (c) APTES and (d) experimental results showing coagulation activity of

MOCP attached to different surface-modified SPIONs. Blue arrow depicts the position where

protein residues bind to the precursor ligand.

Conversely, for the other two cases (TEOS and APTES ligands), the 1st and 2nd helices

have restricted dynamics over the third helix since these two helices are involved in binding

with the ligands. Particularly, Cys12 of helix 1 and Ala28 and Ala32 of the protein (helix 2)

appear at smaller r-value in the figure, implying that they are involved in the binding. As

mentioned earlier, in these two cases, a smaller width in the case of Cys12, Ala28 and Ala32

residues was also observed. The features seen in the cases of protein-TEOS and protein-APTES

are almost similar except in the region corresponding to the residues 39-50. A relatively larger

width for the residues was seen in the case of protein-APTES which suggests an increased

conformational flexibility for the 3rd helix; this behavior also explains why the APTES-


50

modified SPIONs presented better performance over TEOS-modified SPION (Figure 3.9d).

However, the protein-ligand distance profile analysis confirms the difference in functional

activity of the surface-modified SPIONs, evidencing a correlation between the computational

model and experimental data.

3.3.2.2 Fluorescence emission study

Emission fluorescence spectra of MOCP, MOCP+w/o ME-MION, and MOCP+o/w ME-

MION are presented in Figure 3.10. A significant interaction between the magnetic

nanoparticles and the protein can be described by changes in fluorescence emission spectra.

The MOCP has a broad peak with apparently two maxima at 310 and 326 nm. A shift to λmax

of 295 nm is observed with MOCP+w/o ME-MION, implying a modification of protein

fluorophore environment. Similar behavior is observed in the absorption spectra of MOCP-

o/w ME-MION. However, the binding of nanoparticles in the ligand binding site of the

protein makes the environment of the aromatic side chains in the binding site less polar.135

The decrease in the fluorescence intensity can be attributed to the adsorption of MOCP onto

the nanoparticles surface. In fact, the small particle size and large surface-to-volume ratio

(Paper III) could be critical for this behavior; hence the density of MOCP+ME-MIONs

increased following the absorption of protein on the particles which initiated large protein

precipitation with the nanoparticles. Adsorbed MOCP still retains its functionality after

binding to ME-MIONs, thus implying the suitability of this phenomenon in the development

of protein-functional nanoparticles for water treatment.

Figure 3.10 Fluorescence emission spectra of MOCP (red), MOCP+w/o- (blue) and o/w-

(green) ME-MION at 255 nm excitation wavelength.

0

0,2

0,4

0,6

0,8

1

1,2

280 300 320 340 360 380 400

No

rmalized

flu

ore

scen

ce

Wavelength (nm)

MOCP

w/o ME-MION

o/w ME-MION

Chuka Okoli

51

3.3.3 Turbidity removal with the developed MOCP-MNPs system (Approach II)

(Paper IV & V)

Limited research has been carried out on developing a method that will promote the reuse of

MOCP in water or wastewater treatment processes. According to our knowledge and to date,

all the investigated applications with MOCP in turbid water clarification have been based only

on one time use,17, 67, 136

as it is difficult practically to recover and reuse the protein. The

coagulation activity of the developed MOCP-MNPs was investigated in the clarification of

surface waters (Figure 3.11). The optimal time required to achieve an efficient coagulation

activity (turbidity removal) with different coagulant agents (MOCP+w/o ME-MION,

MOCP+o/w ME-MION, MOCP+TSC-coated SPION, alum and MOCP) was around 60 min.

The coagulation activity of alum or MOCP depends on the composition or initial turbidity of

the water source; thus, an optimal time for effective turbidity removal may vary between 90

and 120 min.


52

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100

0 20 40 60 80 100 120 140 160 180 200 220 240

Co

ag

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tio

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vit

y (

%)

Time (min)

(b)

(a)

Figure 3.11 Coagulation kinetic studies comparing the performance of MOCP-MNPs,

MOCP and alum in surface water from Lake Mälaren with (a) an initial turbidity of 17 NTU

and (b) an initial turbidity of 7 NTU. The control represents a surface water sample.

However, as shown in Figure 3.12, neither of the coagulant agents demonstrated a significant

increase in coagulation performance at concentrations above 0.2 mg/L, which could represent

the optimal concentration of MOCP-MNPs for surface water turbidity removal. The MOCP-

MNPs (MOCP+w/o ME-MION and MOCP+TSC-coated SPION) exhibited higher

coagulation performance of about 20% compared to alum and MOCP.

The MOCP+w/o ME-MION, MOCP+o/w ME-MION and MOCP+TSC-coated SPION depict

similar patterns in performance, implying a significant decrease in turbidity when tested in

natural water. It has been reported that the performance of most chemical coagulant agents

typically depends on the initial turbidity of the water sample.32

The experimental results show

0

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MOCP+w/o ME-MIONMOCP+o/w ME-MIONMOCP+TSC-coated SPIONAlumMOCPControl

(a)

Chuka Okoli

53

that with MOCP+w/o ME-MION, MOCP+o/w ME-MION and MOCP+TSC-coated SPION,

coagulation activity of ~ 80 % can be achieved.

Figure 3.12 Coagulation performance vs. applied dosage of MOCP-MNPs, alum and MOCP

(mg/L) in terms of turbidity removal in surface water at 1 h interaction time.

However, the difference observed between MOCP-MNPs and other coagulant agents could be

ascribed to the high density mass of the magnetized flocs induced by electrostatic interaction,

which resulted in rapid flocculation in the water sample. In contrast, alum and MOCP were

found to be less effective, especially in water samples with initial turbidity of 7 NTU (Figure

3.11b). Nonetheless, alum and MOCP were effective in surface water with 17 NTU. This

study suggests that the MOCP-MNPs may represent a feasible complementary agent in water

treatment process.

3.3.3.1 Separation by gravity and magnetic field

As shown in Figure 3.13, the magnetization effect of MOCP+ME-MION in the clarification

of turbid waters (coagulation activity) was enhanced under the influence of an external

magnetic field when compared with the turbidity removal by means of gravitational settling

i.e., as in the case of alum. It was reported that (Paper IV), with MOCP+ME-MION, almost

95% turbidity removal can be achieved within 12 min due to the existence of

magnetophoresis of the nanoparticles.137, 138

This implies that when the MOCP+ME-MION

comes in contact with polluted water, it tends to react with the colloidal or suspended particles

thereby transiting from dispersed to aggregated state. According to magnetization curve

theories, when nanoparticles are placed within a steady external magnetic field, the internal

magnetic moment spins in the same direction as the external magnetic field. As a result, the

0

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0 0,1 0,2 0,3 0,4 0,5

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Alum

MOCP

MOCP+TSC-coated SPION

MOCP+w/o ME-MION


54

shifting of magnetic domain wall occurs, which boosts the magnetic properties of the

nanoparticles;74, 91

this phenomenon leads to the aggregation of the water impurities.

Figure 3.13 Separation performance by magnetic field and/or gravity in natural and

synthetic turbid water samples. MOCP+ME-MION and alum were added to water samples

followed by 10 min interaction time and 2 min separation. SW1 and SW2 represent surface

water (7 and 17 NTU respectively) from Lake Mälaren. ME-MION is bare nanoparticle

(control).

However, these dynamics will induce the rapid aggregation of the water contaminants by

MOCP+ME-MION. In contrast to the coagulation/flocculation of impurities by gravity, i.e. in

the case of water containing alum, the combination of MOCP+ME-MION and applied

magnetic field enhanced the effectiveness of the system in forming flocs. Moreover, it was

apparent that even though the bare ME-MION may possess some coagulation potential, a

lower performance was also observed when compared with MOCP+ME-MIONs. This clearly

reveals that the binding of the protein onto the nanoparticle was critical in enhancing their

performance.

3.3.3.2 Reusability of MOCP-MNPs system

From the coagulation performance of the MOCP-MNPs, it is clearly evident that the system

exhibits reusability potential after removing the adsorbed impurities with mild detergent or

cleaning solutions (0.1% Tween 20 and 20% EtOH). As shown in Figure 3.14, the

0

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90

100

Clay SW1 SW2 Clay SW1 SW2 Clay SW1 SW2

Co

ag

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vit

y (

%)

Water samples

MOCP+ME-MION

Alum

ME-MION

Chuka Okoli

55

coagulation activity test performed with MOCP+TSC-coated SPION and MOCP+w/o ME-

MION after cleaning with 0.1% Tween 20 or 20% EtOH suggests that it is possible to reuse

the MOCP-MNPs three times. The control sample is the initial activity of MOCP-MNPs

before cleaning with detergent or cleaning solutions. Cleaning with 20% EtOH or 0.1%

Tween 20 showed almost similar coagulation activity to that of control, especially in the first

reuse. MOCP+TSC-coated SPION and MOCP+w/o ME-MION showed a reduction in activity

in the two successive cleanings with EtOH. The cleaning of MOCP+w/o ME-MION with

Tween 20 demonstrated a high level of consistency in the successive reuses, which is similar

to the control sample; on average, an insignificant reduction in activity was observed.

Figure 3.14 Regeneration of MOCP-MNPs after coagulation test with a clay suspension.

The cleaning capacity of 20% EtOH and 0.1% Tween 20 was compared after a 1h coagulation

activity test. Control sample represents the initial coagulation activity of MOCP-MNPs before

cleaning.

In the case of MOCP+TSC-coated SPION, second and third reuses did not show a consistent

activity after cleaning with Tween 20, even though the variation in reduction (10%) is

somewhat insignificant when compared to MOCP+w/o ME-MION. Nevertheless, the most

probable reason for the reduction in activity with MOCP-MNPs after successive cleaning

could be that the adsorbed impurities were not completely removed from the MOCP-MNPs.

Therefore, it is unlikely that the detergent or cleaning solution will decrease the electrostatic

interaction between the negatively charged nanoparticles and the positively charged protein.

Results from this test indicate that 20% EtOH and 0.1 % Tween 20 may present the highest

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acti

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Cleaning with 20% EtOH Cleaning with 0.1% Tween 20

MOCP+TSC-coated SPION

MOCP+w/o ME-MION


56

capacity to regenerate the MOCP+ME-MION after several reuses. More investigation is being

conducted to fully understand the mechanism of cleaning in order to improve the efficiency of

the system while reducing the overall cost of the treatment process.

Chuka Okoli

57

4. Conclusions

The work presented in this thesis demonstrates advancement in the development of eco-

friendly water treatment strategy using magnetic iron oxide nanoparticles for MOCP

separation (i.e. in situ treatment) as well as the preparation of MOCP-MNPs systems for water

treatment. Magnetic nanoparticles with different surface chemistry (sizes, 2-30 nm) were

successfully prepared from co-precipitation and microemulsion (w/o and o/w) methods. The

characterization of the prepared nanoparticles was critical for protein-nanoparticles interaction

study. The protein-nanoparticle binding mechanism was verified by in silico study, which

validates the experimental (in vitro) data.

A simple, fast and efficient method for the purification of MOCP was developed and used in

an in situ water treatment strategy. This approach is based on the separation of the coagulant

protein with magnetic nanoparticles; the performance of the obtained MOCP (turbidity

removal) was compared with alum for turbid water clarification. Moreover, the purification

method used here is especially appealing since the used materials are obtained at a low cost.

Similarly, the second treatment approach highlights the development and application of

MOCP-MNPs for efficient and rapid water treatment. The strategy used is based on the

magnetophoretic effects of the nanoparticles under the influence of external magnetic field.

The strategy presented in this work (approach II) shows superior performance over alum or

MOCP in the removal of water turbidity, irrespective of the water composition and turbidity

level.

The MOCP-MNPs could effectively remove more than 95% turbidity in surface waters under

the influence of an external magnetic field within 12 min, whereas conventional water

treatment processes require several hours. The combination of natural coagulant protein plus

magnetic nanoparticles and magnetic field enhanced the effectiveness of the system in

coagulating/flocculating impurities in water samples. The present investigation also suggests

the possibility of regenerating the MOCP-MNPs; moreover, the developed strategy is unlikely

to introduce residual ions during water treatment. Similarly, this strategy does not influence

the pH or conductivity of the treated waters (Paper VII). These characteristics make the

MOCP-MNPs appealing in water treatment. To the best of our knowledge and according to

the published data, this is the first report on the separation or binding of MOCP using

magnetic nanoparticles. The data shown in this work represent water treatment approaches

(with safe materials) that are simple to use, robust and environmentally friendly.


58

Further studies

This work endorses the possibilities of using alternative natural coagulant materials (MOCP)

or their association with magnetic nanoparticles (MOCP-MNPs) in the treatment of different

surface waters. In order to appreciate the real integrated effect and to apply these findings to

water treatment plants, pilot and full-scale studies are required. Furthermore, confirmatory

studies are needed to validate that the treatment approaches do not alter the pH or

conductivity of the treated waters, although preliminary results with bare nanoparticles or

MOCP-MNPs (Paper VII) suggest that neither the pH nor the conductivity of the treated

water was changed. It is necessary to investigate the cytotoxicity of the MOCP+MNPs even

though it has been established by the U.S. FDA that iron oxide nanoparticles are the only safe

nanoparticles approved for clinical use.

The development of recombinant MOCP for large-scale protein production is a work in

progress. This procedure will promote large-scale production of the protein whilst, reducing

cost. Current investigation with NMR is ongoing in order to determine the MOCP protein

structure. To date, the highest number of amino acid residues in one of the identified peptide

sequences obtained from MS/MS analysis was 19 (section 3.3.1) which differs from 60

residues reported by Gassenschmidt et al., (1995). However, in-depth study is deemed

necessary to identify the full sequence of this protein, and in particular to identify which

region of the protein is responsible for coagulation and antimicrobial action. Moreover, the

existence of the heterogeneous nature of the protein suggests the need to disclose the full

sequence of the MOCP. Presently, studies are being carried out in our group with in silico

modeling tools to understand and possibly resolve the protein structure.

The present investigation on this work suggests that the MOCP and/or its combination with

MNPs possess other potential applications beside water treatment such as dye removal. A

study on the removal of other water pollutants like industrial effluents is essential in order to

assess the wider application of the developed approaches.

Chuka Okoli

59

Acknowledgement

I wish to express my sincere gratitude to my supervisor, Assoc. Prof. Gunaratna Rajarao-

Kuttuva; your constant support, encouragement and positive criticisms have always been a

source inspiration. Thank you very much for introducing me to the fascinating field of

biology. In fact, I cannot thank you enough.

I am very obliged to my co-supervisors, Assoc. Prof. Magali Boutonnet and Prof. Sven Järås

at the School of Chemical Science & Engineering. You both have encouraged and motivated

me during the years of my study especially in supporting most of my conference and research

trips. I thank you Magali, for introducing me to the synthesis of nanoparticles by

microemulsion methods.

Many thanks to Prof. Per-Åke Nygren, Prof. Vincent Bulone, Prof. Gunnel Dalhammar, Prof.

Lars J Pettersson, and Dr. Arul Murugan for their brilliant suggestions and assistance. I

would like to thank Dr. Margarita Sanchez-Dominguez (CIMAV-Unidad Monterrey, Mexico)

for her fruitful collaboration including her excellent contributions and assistance in material

characterization. I enjoy the company, including the brilliant ideas of my colleagues; Dr. A.R.

Pavankumara, Ida, Ramnath, Delaram and Tobias. To all my colleagues, in the school of

Biotechnology (Plan 1) and at the Division of Chemical Technology; thanks for the quality

time spent with you guys. To Marita Johnsson; thanks for me helping out with administrative

issues. Many thanks to kaj for the I.T support.

My deep gratitude goes to my family; my Dad, Chikodi, Kenneth, Uju, Chinedu, Ifeanyi,

Chekwus, Onyeka, and Ifeoma. My profound appreciation goes to Mr. & Mrs. Ononiwu,

Chike Ilechukwu and Barrister Osonwa; I thank you all for your support and understanding. I

thank my in-laws for their support and best wishes.

Many thanks to Efraim and Ulrika Moström for their prayers and support; it’s a huge blessing

to be around you guys. To Philip and KC Onu, Mr. & Mrs. Rogo, Inno Okeke and his family,

Fred and Chinenye Ijeboi, including all my pals that graduated from KTH; thank you folks for

all sorts of activities, which made it possible to relax outside research.

My sincere appreciation goes to my aunt, Lolo Enuma Ginigeme for her unfailing love and

motivation especially, her constant prayers and support towards my wife and kids while I was

abroad; Aunt, we are proud of you. To Dera, Uche, Kosi and Nnamdi; it’s fun to be around

you guys and I am certain that you all will make Mom proud. My profound appreciation goes

to Mr. & Mrs. Okey Nweke for their kindness and support. My deepest gratitude goes to Rev.

& Mrs. Julius Edah, and all the members of UAPC Dallas, TX for their prayers and support.

Many thanks to my very good friend, Dr. Mfon Inyang and his family.

I am grateful to the Swedish Research Institute; FORMAS for financing this project.

Finally, I wish to express my profound appreciation to the love of my life; my wife, Queen,

who has constantly supported and encouraged me during these years. Without her

understanding and prayers, it would not have been possible for me to complete my research at

this time. I sincerely appreciate your hard work in taking good care of our wonderful kids,

Lily and Munachi.


60

Chuka Okoli

61

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Appendix

Papers I to VII

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