CHEMICAL AND ANTIMICROBIAL PROPERTIES OF MORINGA (MORINGA
OLEIFERA) ROOT AND ITS IMPACT ON WATER QUALITY
by
Chandehl R. Morgan
BSc., University of Northern British Columbia, 2014

THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTERS OF SCIENCE
IN
NATURAL RESOURCES AND ENVIRONMENTAL STUDIES

UNIVERSITY OF NORTHERN BRITISH COLUMBIA
January, 2020
© Chandehl R. Morgan 2020

ii
Abstract
Moringa oleifera is a tropical tree with nutritious, anti-inflammatory, and antimicrobial
properties. Moringa seeds have been studied for their ability to purify water, however roots have
not. This study identified the nutrient composition of Moringa roots grown in a greenhouse, and
tested whether the roots improved water quality. Moringa roots were dried, powdered and added
to contaminated water to test their impact on E. coli, pH, turbidity, and electrical conductivity. The
chemical composition of Moringa roots were measured using ICP-MS. The five main elements
observed were potassium, phosphorus, magnesium, sodium and calcium. None of the elements
extracted were of health concern for drinking water quality. Electrical conductivity and pH
remained within drinking water quality guidelines. Moringa root powder resulted in a significant
increase in turbidity. Moringa concentration of 600 mg/L removed up to 87% of E. coli in water.
Moringa root powder shows some potential as a point-of-use water treatment.

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Table of Contents
Abstract ................................................................................................................................. ii
Table of Contents ................................................................................................................. iii
List of Tables ....................................................................................................................... vi
List of Figures ..................................................................................................................... vii
Acknowledgements .............................................................................................................. ix
Chapter 1: Introduction ......................................................................................................... 1
1.1 Introduction to Moringa oleifera .................................................................................... 1
1.2 Rationale for Research .................................................................................................... 2
1.3 Research Objectives ........................................................................................................ 3
1.4 Organization of Thesis .................................................................................................... 4
Chapter 2: Literature Review of Moringa oleifera ............................................................... 5
2.1 Growing Conditions ........................................................................................................ 5
2.2 Rate of Growth ................................................................................................................ 5
2.3 Tree Morphology ............................................................................................................ 5
2.4 Chemical Constituents .................................................................................................... 7
2.5 Research in Nutrition ...................................................................................................... 9
2.6 Antimicrobial Research .................................................................................................. 9
2.7 Moringa in Water Purification ...................................................................................... 11
Chapter 3: Chemical Properties of Moringa Roots............................................................. 15
3.1 Abstract ......................................................................................................................... 15
3.2 Introduction ................................................................................................................... 15
3.3 Methods......................................................................................................................... 18
3.3.1 Planting, Growing, and Processing Moringa ..........................................................18
3.3.2 Chemical Analysis ..................................................................................................19
3.3.3 Solubility Analysis ..................................................................................................20
3.3.4 Statistical Analysis ..................................................................................................21
3.4 Results ........................................................................................................................... 21
3.4.1 Chemical Constituents of Moringa Roots...............................................................21
3.4.2 Water Soluble Elements in Moringa Roots ............................................................25
3.5 Discussion ..................................................................................................................... 28
3.5.1 Chemical Constituents of Moringa Roots...............................................................28
3.5.2 Water Soluble Elements in Moringa Roots ............................................................31

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Chapter 4: Effects of Moringa Root Powder on Contaminated Water ............................... 35
4.1 Abstract ......................................................................................................................... 35
4.2 Introduction ................................................................................................................... 36
4.2.1 Water Crisis ............................................................................................................36
4.2.2 Current Purification Technologies ..........................................................................36
4.2.3 Moringa Seeds in Water Treatment ........................................................................38
4.2.4 Research Questions .................................................................................................41
4.3 Methods......................................................................................................................... 41
4.3.1 Growing and Processing Moringa Root Powder ....................................................41
4.3.2 Development of Methods ........................................................................................41
4.3.3 Water Collection .....................................................................................................41
4.3.4 Isolating Bacteria ....................................................................................................42
4.3.5 Growth Curve Analysis...........................................................................................43
4.3.6 Dilution Analysis ....................................................................................................44
4.3.7 Treatment Application ............................................................................................45
4.3.8 Enumeration of E. coli and total coliforms .............................................................46
4.3.9 Statistical Analysis of Bacteriological Water Quality ............................................47
4.3.10 Sample Preparation for Physical Water Quality Parameters ................................47
4.3.11 pH Measurement ...................................................................................................47
4.3.12 Electrical Conductivity Measurement...................................................................48
4.3.13 Turbidity Measurement .........................................................................................48
4.3.14 Statistical Analysis of Physical Water Quality Parameters ..................................48
4.4 Results ........................................................................................................................... 49
4.4.1 Streak Plates ............................................................................................................49
4.4.2 Growth Curve Results .............................................................................................49
4.4.3 Effectiveness of Moringa root treatment at removing E. coli ................................51
4.4.4 Total Coliforms .......................................................................................................54
4.4.5 pH …………………………………………………………………………………54
4.4.6 Electrical Conductivity ...........................................................................................55
4.4.7 Turbidity..................................................................................................................57
4.5 Discussion ..................................................................................................................... 58
4.5.1 Moringa Root Powder Effect on E. coli and Total Coliforms ................................58
4.5.2 pH............................................................................................................................60
4.5.3 Electrical Conductivity ...........................................................................................61

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4.5.4 Turbidity .................................................................................................................62
Chapter 5: Conclusion............................................................................................................. 64
Literature Cited ....................................................................................................................... 68
Appendix A: Moringa tree growth in a greenhouse ............................................................... 82
Appendix B: Development of Bacteria Analysis Methods ..................................................... 85
B.1 Trial Method 1 .................................................................................................................. 85
B.2 Trial Method 2 .................................................................................................................. 86
B.3 Trial Method 3 .................................................................................................................. 89
B.4 Trial Method 4 ................................................................................................................. 89

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List of Tables
Table 1: The chemical properties and uses of the various parts of Moringa oleifera…..….....8
Table 2: Mean and standard deviation of chemical composition of dried and fresh Moringa
roots. Mean values are expressed in mg/kg.………………………...……….........................23
Table 3: T-test results of difference in amount of elements present in Moringa roots harvested
at 7 months of growth and those harvested after 6 months of growth…………………….…24
Table 4: T-test results of difference in amount of elements present in fresh and dry Moringa
roots harvested at 7 months of growth.………………….………………………...….…...…25
Table 5: Mean (and standard deviation) of chemical elements in water treated with Moringa
root powder as determined by ICP-OES. Mean values are expressed in mg/L..…………….27
Table 6: Bacteria concentration and treatment applied to each sample of contaminated water.
Each sample was prepared in four replicates.……………...……………………………...…46
Table 7: Comparison of physical water quality parameter guidelines to water treated with
Moringa root powder, filtered and left to settle for 1 hour.……………………..…...………63

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List of Figures
Figure 1: Moringa trees grown in greenhouse conditions for 6 months………………………6
Figure 2: Root harvested from a Moringa tree after 6 months of growth in a greenhouse……7
Figure 3: Structure of pterygospermin, adopted from Fahey, 2005…………………………...11
Figure 4: Streak plate of E. coli colonies……..……………………………………..…….......49
Figure 5: Streak plate of total coliform colonies..……………………………………….........49
Figure 6: Mean total coliform growth curve with error bars indicating standard deviation from
three replicates……………………………………………………………...…………….….50
Figure 7: Mean E. coli growth curve with error bars indicating standard deviation from three
replicates……………………………………………………………..………………………51
Figure 8: Colour change observed in quanti pack wells which contain total coliforms…......51
Figure 9: Fluorescence observed in quanti pack wells which contain E. coli…..……….......52
Figure 10: Mean effect of changing Moringa concentration on E.coli with initial bacteria
population of 50 MPN/100 mL from four replicates showing standard deviation……...…...53
Figure 11: Mean effect of changing Moringa concentration on E.coli with initial bacteria
population of 37 MPN/100 mL from four replicates showing standard deviation…………..53
Figure 12: Boxplot representation of the mean effect of three variables, Moringa root
concentration, filtering, and time elapsed on pH of contaminated water showing outliers. pH
significantly decreased with increasing Moringa concentration and it significantly increased
over time…………………..………..………………………………………………………..55
Figure 13: Boxplot representation of the mean effect of three variables, Moringa root
concentration, filtering, and time elapsed on electrical conductivity of contaminated water
showing outliers. Electrical conductivity increased significantly with increasing Moringa
concentration………………………………………………………………………………....56
Figure 14: Boxplot representation of the mean effect of three variables, Moringa root
concentration, filtering, and time elapsed on turbidity of contaminated water showing outliers.
Turbidity significantly decreased over time and significantly increased with increasing
Moringa root concentration. Filtering did not have a significant impact on
turbidity………………………………………………………………………………….…...58
Figure 15: Mean height of 25 Moringa trees in their first six months of growth, showing
standard deviation. In Appendix A…………………………………………………………..83

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Figure 16: Mean crown width of 23 Moringa trees in their first seven months of growth,
showing standard deviation. In Appendix A…………………………………………………84
Figure 17: Mean trunk diameter of 23 Moringa trees in their first seven months of growth,
showing standard deviation. In Appendix A…………………………………………………84

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Acknowledgements
First and foremost, I would like to acknowledge and pay respect to the indigenous peoples
on whose land this research was conducted, and whose knowledge has contributed to this
study. This includes the Lheidli T'enneh people, on whose traditional and unceded lands the
University of Northern British Columbia is built on. Much of this thesis was written on the
traditional, unceded territory of the Coast Salish people, particularly the Musqueam, Squamish,
and Tsleil Waututh. Knowledge of the healing and water clarifying properties of Moringa
oleifera has come from many indigenous groups across the tropics, and I am grateful for the
depth of knowledge from each of these people, without which the research would not have
been possible.
I would like to thank Dr. Chris Opio, as my academic supervisor, for his guidance, support
and advice. I also thank my graduate committee, Dr. Saphida Migabo and Dr. Ché Elkin for
all the ways that they have supported me and contributed to the development of this thesis.
Funding for this research came from NUDF (Northern Uganda Development Foundation)
Special Fund, UNBC Research Grant, Christopher Opio’s Professional Development Fund,
and a research grant from Saphida Migabo.
I am grateful to John Orlowsky and Doug Thompson, for growing the Moringa trees in the
EFL greenhouse. Data was collected in the Northern Analytical Laboratory Services (NALS)
lab at UNBC with the help of Hossein Kazemian, Erwin Rehl, Lon Kerr, and Charles
Bradshaw. In addition to providing this analysis, these individuals also offered invaluable
support and advice throughout this research. Their collaborative nature has helped me to shape
my work and gain skills as a researcher. Early stages of this research took place at the Prince
George Waste Water Treatment Centre, where Joanne Logie lent not only her lab space and
equipment, but also her guidance and support, and training on microbial analytical methods.
Equipment was borrowed from Dr. John Rex and Dr. Mike Rutherford; without which I could
not have collected this data. Amy Ziorio, Lon Kerr and David Morgan were all invaluable as
research assistants. Our source water came from Marlene Whelan, who graciously let us onto
her farm to sample water.
I would like to thank my husband, David Morgan, for his unwavering love and support
through this journey, and for his willingness to participate as research assistant, sounding
board, and editor. I would like to thank my friends and family for the many and various ways
that they have supported, encouraged, and inspired me throughout this process. For my church
communities at First Baptist Church Prince George and Granville Chapel in Vancouver, thank
you for your support and prayers. To my coworkers at the Stanley Park Ecology Society, thank
you for all of the ways that you have supported and challenged me, and helped me grow as a
scientist and an educator. Finally, I would like to thank my Pip, who although she is not yet
born she has already filled me with much joy and inspiration, this thesis is dedicated to you.

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Chapter 1: Introduction
1.1 Introduction to Moringa oleifera
Moringa is a genus that includes 13 species of trees and shrubs (Fahey, 2005). The most
widely distributed of these species is Moringa oleifera, which grows across the tropical zone
(Morton, 1991; Anwar et al., 2007). It originated in the Himalayas and is known by many common
names, including ben oil tree, drumstick tree, and horseradish tree. Moringa has been dubbed “the
miracle tree” because of its many properties that are beneficial to human health and well-being.
Almost all parts of the tree are used, either for nutrition or in traditional medicines (Fahey, 2005).
Moringa is an important component of traditional medicines throughout South Asia and
many parts of Africa. Moringa has been used for pain relief, treatment for headaches, fevers, and
rheumatism and treatment for bug bites (Goyal et al., 2007). More recently, studies on various
parts of Moringa have been undertaken to determine the feasibility of Moringa in the treatment of
cancer (Jung, 2014). In addition to nutritional and medicinal benefits, Moringa is used in fertilizer,
pesticides, contraceptives, perfume, animal food, and as a cleaning agent (Fahey, 2005).
Moringa leaves are particularly nutritious, as they contain gram-for-gram more Vitamin A
than carrots, more calcium than milk, more iron than spinach, more Vitamin C than oranges, and
more potassium than bananas (Fahey, 2005). Moringa leaves are used to treat scurvy and
malnutrition (Gopalakrishnan et al., 2016). Due to the high iron content in the leaves, they have
been prescribed for anemia in the Philippines (Miracle Trees, 2016).
Moringa leaves and seeds have been used to remove contaminants from water. Moringa
seeds are used to create a powder that can eliminate harmful bacteria in water, making it safe for
human consumption (Ndabigengesere et al., 1995). Seeds form a coagulant which reduces the
turbidity of water (Lea, 2010). The seeds also contain a protein which inhibits coliform bacteria

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(Kwaambwa et al., 2010). This protein is the Moringa oleifera cationic protein (MOCP), and it
kills bacteria by fusing the bacteria’s cell membranes (Shebek et al., 2015).
Moringa roots have a high medicinal potency (Igwilo et al., 2014). They are antiinflammatory and, as such, have been used as traditional medicines in Senegal and India to treat
rheumatism, stomatitis and pain caused by arthritis (Igwilo et al., 2014). The roots are pounded
and mixed with salt to form a poultice (Miracle Trees, 2016). The poultice is used to relieve kidney
and lower back pain (Miracle Trees, 2016). Moringa roots are believed to be good for the throat,
bronchitis, and piles (Goyal et al., 2007). Despite its widespread applications, however, little is
known about Moringa roots, particularly its chemical composition and its potential to treat
contaminated water.
1.2 Rationale for Research
Given the importance of Moringa to medicine, nutrition, and culture, further studies on its
composition can yield information that enhances our understanding of the mechanisms through
which the plant is beneficial to humans. A chemical analysis of the roots, one of the lesser studied
aspects of the plant, can increase our knowledge of this ‘miracle tree’.
Studying Moringa roots as a treatment option for contaminated water can be beneficial for
many reasons. Contaminated water is a growing concern in the world; the World Health
Organization (WHO) and the United Nations Children’s Fund (UNICEF) report that 2.1 billion
people lack access to safe drinking water at home (WHO and UNICEF, 2017). Drinking
contaminated water can result in the spread of waterborne diseases such as cholera, typhoid,
diarrhea and gastroenteritis. This results in the death of over 840,000 people each year (PrussUstun et al., 2014). In 2016, 8% or 480,000 children under five died from diarrhea (UNICEF,
2018). Purified water can limit exposure to these preventable diseases, decrease long-term

3
exposure to carcinogenic compounds and heavy metals (Salaudeen et al., 2018), and it can
positively impact food security (Rasul and Sharma, 2015).
Using Moringa roots, instead of its seeds, would be a preferred drinking water treatment
method for many reasons. Moringa can be vegetatively propagated by cuttings. Roots establish
quickly, and the tree can continue to grow after roots have been harvested (Fuglie, 2001). Moringa
roots can be harvested throughout the year, and within the first year of tree growth (this study).
Moringa seeds are harvested when the trees are mature. The roots have the potential to contain a
high concentration of the antimicrobial properties (Tesemma, et al., 2013) and therefore could
require less preparation, potentially leading to more widespread use.
Finally, the use of Moringa roots in water purification would allow the nutrient-rich areas
of the plant, such as the leaves and seeds (Fahey, 2005) to be used as a food source. This is of
particular importance because many countries with the worst water quality are also threatened by
food insecurity. According to the FAO (1996), “Food security exists when all people, at all times,
have physical, social and economic access to sufficient, safe and nutritious food to meet their
dietary needs and food preferences for an active and healthy life”. Food insecurity exists when
those needs are not met. Despite the progress made towards addressing these needs, food insecurity
still affects millions of people. In Sub-Saharan Africa, nearly 218 million people are affected by
food insecurity, roughly one quarter of the population (UNDP, 2012). The availability of the
nutritious Moringa seeds and leaves can provide access to food security.
1.3 Research Objectives
The main objective of this study was to examine the potential use of Moringa root powder
in purifying contaminated drinking water. To fulfill this objective, the following questions were
investigated:

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1. What is the chemical composition of Moringa roots? What is the chemical profile of water
treated with Moringa root powder, and does it meet Canadian and WHO drinking water
quality guidelines?
2. Can the roots of Moringa oleifera be used to treat water for E. coli and total coliforms? Does
Moringa root powder impact the physical properties (pH, electrical conductivity and
turbidity) of contaminated water?
1.4 Organization of Thesis
This thesis is organized as follows. First, research objectives and questions are addressed
in Chapter 1. Chapter 2 is a literature review of Moringa oleifera, detailing its known properties
and uses, as well as previous scientific research conducted on the tree. This chapter highlights the
gaps in current research which this thesis will address. Chapter 3 discusses the process of growing
Moringa oleifera in a greenhouse as well as the methods used to harvest and process the roots to
create the powder used in subsequent treatments. This chapter focuses on the chemical profile of
Moringa root, as determined through Inductively Coupled Plasma Mass Spectrometry (ICP-MS).
In Chapter 4, the effect of Moringa roots on drinking water quality is discussed. The impact on
contaminated water includes a study on how Moringa root powder impacts the indicator organisms
E. coli and total coliforms, and the impact of Moringa root powder on the pH, electrical
conductivity, and water turbidity. Finally, Chapter 5 is a summary and conclusion of the thesis
work, including a discussion of the limitations of this study and suggestions for further research.

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Chapter 2: Literature Review of Moringa oleifera
2.1 Growing Conditions
Moringa is indigenous to Northwest India (Ramachandran et al., 1980), but is now widely
distributed in the tropics throughout Asia-Pacific, Africa, the Caribbean, and Central America
(Fahey, 2005). It thrives in semi-arid conditions at altitudes of 600 m, however, it can grow at a
variety of altitudes, up to 1400 m above sea level (Mohamed et al., 2015). Its temperature range is
also extensive, thriving from 25ºC to 40ºC, but also withstanding both frost and temperatures of
up to 48ºC (HDRA, 2002). It is a resilient tree, requiring an annual rainfall of 250 – 3000 mm
(Anwar et al., 2007). In drought conditions, Moringa will lose leaves, but the tree is able to recover
(HDRA, 2002). Moringa grows well in neutral to slightly acidic soils; it requires well-drained
soils, and cannot withstand water logging. The best soil for Moringa growth is loam to clay-loam,
although it is able to thrive in a variety of soil types (HDRA, 2002).
2.2 Rate of Growth
Moringa can be grown from either seeds or cuttings. Seeds are planted about 2 cm deep
(Verma, 1973). Trees grow quickly, with sprouting taking place only 1-2 weeks after planting.
Seeds do not have a dormant stage, and become less viable with storage (Morton, 1991). When
growing from cuttings, limb cuttings of 4-5 cm in diameter are planted during the rainy season
(Ramachandran et al., 1980). Moringa is a fast-growing tree, able to grow up to seven metres in a
year (Foidl et al., 2001). Full grown Moringa trees can reach heights of up to 12 m.
2.3 Tree Morphology
Moringa has cork-like bark, which peels. It has soft wood, and produces low-quality timber
(Fahey, 2005). The tree produces fragrant, cream-coloured flowers, which first appear after 8
months of growth (Roloff et al., 2009). Moringa leaves are longitudinally cracked, ranging from

6
1.2 to 2 cm long, and are arranged spirally (Roloff et al., 2009). Six month old Moringa trees
grown in greenhouse conditions are pictured below (Figure 1). Moringa fruits are long, triangular
drumstick-shaped pods. These pods vary in size from 20 to 60 cm long and contain 12 to 35 seeds
(Foidl et al., 2001). Moringa seeds are oily. They are brown, round, and surrounded by three white
wings, which are angled (Jamieson, 1939). The seeds weigh approximately 0.3 grams (Makkar
and Becker, 1997), and each tree can produce between 15,000 and 25,000 seeds each year (Foidl
et al., 2001). Moringa has a large tuberous taproot that does not branch (Figure 2), however, it
does have small feeder side roots (HDRA, 2002). The tap root can run deep into the soil,
contributing to the resilience of the tree in drought conditions. The root can be pruned to encourage
shallow lateral growth. The roots of Moringa taste like horseradish, lending the tree one of its
common names, the horseradish tree (Ramachandran et al., 1980).

Figure 1: Moringa trees grown in greenhouse conditions for 6 months.

7

Figure 2: Root harvested from a Moringa tree after 6 months of growth in a greenhouse.
2.4 Chemical Constituents
Interest in Moringa spans across many fields, such as nutrition, medicine, fuel, and water
purification. The chemical and organic constituents of Moringa trees have been studied in order to
better understand and utilize the Moringa plant. Moringa is rich in alkaloids, tannins, flavonoids,
anthocyanins, proanthocyanidins, cinnamates and cardiac glycosides (Goyal et al., 2007;
Alhakmani et al., 2013). These are all phytochemicals which have many medicinal applications.
Table 1 shows the chemical properties of the different parts of the Moringa tree.

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Table 1: The chemical properties and uses of the various parts of Moringa oleifera.
Part of
Medicinal Compound
Uses
Moringa
Seeds
Crude protein, crude fat, carbohydrate, methionine, cysteine, Decreasing liver lipid peroxides,
4-(α-L-rhamnopyranosyloxy)-benzylglucosinolate,
water treatment, treating
benzylglucosinolate, moringyne, mono-palmitic and dioleic
rheumatism.
triglyceride, Vitamin A, beta carotene, terygospermin,
Moringa oleifera cationic protein
Flowers D-mannose, D-glucose, protein, ascorbic acid,
Abortifacient, cholagogue,
polysaccharide.
aphrodisiac, cures inflammations,
muscle diseases, tumors, hysteria,
and spleen enlargement. Lowers
serum cholesterol and decreases
lipid profile of liver and heart.

References
Shebek et al., 2015;
Bennett et al., 2003;
Dahot and Memon,
1985.
Anwar et al., 2007;
Pramanik and Islam,
1998.

Roots

4-(α-L-rhamnopyranosyloxy)-benzylglucosinolate and
benzylglucosinolate

Laxative, circulatory tonic,
antifertility, rubefacient, treating
rheumatism and arthritis, as well
as back and kidney pain.

Anwar et al., 2007,
Goyal et al., 2007;
Bennett et al., 2003.

Leaves

Glycoside niazirin, niazirinin and three mustard oil
glycosides, 4-[4’-O-acetyl- α -L-rhamnosyloxy) benzyl]
isothiocyanate, niaziminin A and B,
quercetin-3-O-glucoside, quercetin-3-O-(6''-malonylglucoside), kaempferol-3-O-glucoside, 3-caffeoylquinic
L-arabinose, D-galactose, D-glucuronic acid, L-rhamnose,
D-mannose, D-xylose and leucoanthocyanin

Nutrition, headache relief, treats
fevers, piles, sore throats, eye and
ear infections, and scurvy.
Controls glucose levels and
reduces swelling.
Dental caries, headache relief,
dysentery, intestinal pain,
abortifacient, treats syphilis and
rheumatism.

Anwar et al., 2007;
Goyal et al., 2007;
Faizi et al., 1995;
Faizi et al., 1994.

4-(α-L-rhamnopyranosyloxy)-benzylglucosinolate

Rubefacient, treats delirium, eye
diseases, relieves earaches,
painkiller.

Anwar et al., 2007;
Bennett et al., 2003.

Gum

Bark

Khare et al., 1997;
Bhattacharya et al.,
1982.

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2.5 Research in Nutrition
Moringa leaves are considered the most nutritious of all tropical legumes (Dhakar et al.,
2011). In a study of eight leafy green vegetables found in Ghana (seven species of sweet potato
leaves and Moringa oleifera), Moringa had the highest content of crude fat, protein, fibre, iron and
calcium (Oduro et al., 2008). Iron and calcium are particularly important for women in
malnourished areas. The high fat content increases the palatability of Moringa leaves compared to
others (Oduro et al., 2008). The leaves also contain all essential amino acids, at concentrations
higher than those recommended by FAO/WHO/UNO for 2-5 year-old children (Makkar and
Becker, 1996). Moringa has been proposed as a simple, effective, and readily available solution to
malnutrition. Moringa is also rich in Vitamin A, Vitamin C, B-complex vitamins, potassium,
magnesium, selenium and zinc (Fuglie, 2001). When compared with other foods, Moringa
contains more Vitamin A than carrots, more calcium than milk, more iron than spinach, more
Vitamin C than oranges, and more potassium than bananas (Fahey, 2005). A nutrient analysis of
Moringa roots has not previously been performed.
2.6 Antimicrobial Research
The application of Moringa in traditional medicines is largely due to its antimicrobial
properties. A compound (referred to as compound-1) produced from chloroform extract of
Moringa demonstrated antimicrobial activity against Shigella boydii, Shigella dysteriae,
Staphylococcus aureus, Bacillus megaterium, Candida albicans and Aspergillus flavus (Nikkon et
al., 2003). Leaf juices and seeds are able to inhibit bacterial growth (Caceres et al., 1991). The
antimicrobial properties of Moringa are so strong that it is recommended as a hand washing
product (Torondel et al., 2014).

10
When various parts of Moringa, such as seed kernels, seed coats, stem bark, unshelled
seeds and dry pod husks, were tested for antimicrobial properties, all components exhibited a zone
of inhibition on several different test organisms (Onsare et al., 2013). When comparing the zones
of inhibition around ethanol extracts from various parts of the Moringa tree, Moringa roots had
significantly higher antibacterial activity against Escherichia coli, Staphylococcus aureus and
Klebsiella pneumoniae than the leaves and seeds (Majali et al., 2015). Moringa roots have
exhibited antimicrobial activity against Shigella boydii, Shigella dysteriae, S. aureus, Bacillus
megaterium, Candida albicans, Aspergillus flavus, Pseudomonas aeruginosa, E. coli,
Pseudomonas aeruginosa and Proteus mirabilis (Raj et al., 2011; Nikkon et al., 2003). The
inhibitory zones are comparable to many standard antibiotics (Onyekaba et al., 2013). Moringa
extracts have been used to cap iron-oxide nanorods, which have antibacterial applications.
Moringa was chosen to enhance these treatments due to their antimicrobial properties, which are
a result of phytochemicals such as alkaloids, tannins, flavonoids, saponins, triterpenoids and
anthraquinones that are present in the tree (Aisida et al., 2019).
One of the main chemical constituents responsible for the antibacterial effects in the roots
is pterygospermin (Tejas et al., 2012). Pterygospermin acts by inhibiting bacteria enzymes in areas
such as transaminase production and interacting with bacteria cell membranes to cause lysis of
bacterial cells (Enwa et al., 2013). Pterygospermin has a low cumulative toxicity, and can be used
for prolonged treatments due to this (Rao and Natarjan, 1949). The structure of pterygospermin is
debated (Horwath and Benin, 2011), however the commonly accepted structures are illustrated
below (Figure 3). Moringa is able to impact both Gram-negative and Gram-positive bacteria. There
are several mechanisms proposed for how the various phytochemical present in Moringa are able
to exhibit their antibacterial activity. Those mechanisms include bacterial enzyme inhibition such

11
as the sortase inhibitory effect, DNA replication, bacterial toxin action, and causing lysis of
bacteria cells (Enwa et al., 2013). It is suggested that peptides interact with the cell membrane in
a way which results in the destabilization of bacteria membranes, and the leakage of cytoplasmic
contents from the bacteria (Omojate et al., 2014). In addition, the permeability of bacteria cell
membranes is changed, there is a loss of membrane potential, and peptides are able to enter the
cells (Enwa et al., 2013).

Figure 3: Structure of pterygospermin, adopted from Fahey, 2005.
2.7 Moringa in Water Purification
One application of Moringa is its use in water treatment. The use of Moringa seeds in water
purification was first reported by Jahn (1977), when he discussed the traditional methods of water
purification in Sudan. In Sudan, the Moringa tree is referred to as a ‘clarifying tree’ (Jahn, 1977).
Based on Jahn’s work, scientists began to study the mechanisms by which Moringa seeds purify
water, as well as how to optimize this water treatment method.
The traditional method of water purification with Moringa seeds begins with crushing the
Moringa seeds, using a mortar and pestle, into a powder. The powder is added to a small amount
of water and stirred thoroughly. The suspension is then added to turbid water, and allowed to sit
until the water becomes clear (Jahn and Dirar, 1979). Using similar methods in laboratory

12
conditions, they found that Moringa is as good a clarifier as alum (Jahn and Dirar, 1979). After
about eight hours, the bacterial count began to rise again (Jahn and Dirar, 1979). The subsequent
increase in bacteria was likely due to the organic components of the Moringa seed treatment acting
as a substrate for the bacteria growth (Jahn and Dirar, 1979).
One of the mechanisms through which Moringa seed powder can clarify water is through
the formation of flocculants (Crump et al., 2004). Flocculation is achieved through adsorption and
charge neutralization processes (Ndabigengesere et al., 1995). The active agents in flocculation
are dimeric cationic proteins which weigh 6.5-13 kDa and have a pH of 10 (Gassenschmidt et al.,
1995). These proteins are small in size, but have a high positive charge, which destabilizes and
attracts colloids and bacteria in turbid water, allowing it to coagulate and settle out. The charges
on the particles are positive, and they act like magnets attracting particles such as clay, silt, and
negatively charged toxic particles (Amagloh and Benang, 2009).
Moringa seed powder is able to flocculate both Gram-positive and Gram-negative bacteria
(Anwar et al., 2007). A wide range of bacteria have been studied and filtered with Moringa seed
powder including Schistosoma mansoni, Salmonella typhimurium, Shigella sonnei, Escherichia
coli, Vibrio cholerae, Streptococcus faecalis and Clostridium perfringens (Olsen, 1987; Madsen
et al., 1987). Moringa seed powder reduces the turbidity of water through flocculation (Muyibi
and Evison, 1995). The coagulation also decreases the pH and conductivity of contaminated water
(Amagloh and Benang, 2009).
In addition to flocculation, the Moringa seed powder also possesses antimicrobial
properties (Olsen, 1987). Seeds are able to remove 90-99% of E. coli and other fecal coliforms,
which are waterborne and can cause diarrheal diseases (Schwarz, 2000). The seeds contain a
protein that inhibits the growth of coliform bacteria (Kwaambwa et al., 2010). This protein is

13
called Moringa oleifera cationic protein (MOCP). MOCP is resistant to heat, and remains active
even after exposure to a temperature of 95°C for 5 hours (Ghebremichael et al., 2005). MOCP has
a net positive charge (Shebek et al., 2015). This protein also has an amphiphilic helix-loop-helix
structure with hydrophobic proline. The molecular weight of MOCP is 6.5 kDa (Shebek, et al.,
2015) and it has isoelectric points above pH 10 (Saini et al., 2016). Amino acid sequencing shows
that it is high in glutamine, arginine and proline (Saini et al., 2016).
The mechanism of MOCP’s antimicrobial activity is primarily membrane fusion. MOCP
fuses the inner and outer membranes of bacteria such as E. coli (Shebek et al., 2015). The positive
charge of MOCP attracts bacteria cells, while the hydrophobic loop incorporates into the bacterial
membrane, causing disruptions which lead to cell death (Shebek et al., 2015; Jerri et al., 2012).
Some of the specific mechanisms associated with this protein are the formation of transmembrane
channels which cause cellular content to leak, depolarization of bacteria membranes, scrambling
of lipid distribution between cell membrane bilayer, and peptide internalization that damages
intracellular targets (Suarez et al., 2005). In addition to MOCP, Moringa contains chitin-binding
lectins which target peptidoglycan in Gram-positive bacteria (Ferriera et al., 2011).
Understanding the mechanisms through which Moringa seed powder is able to purify water
has led to the development of methods to optimize its use in water treatment. One of the issues
with treating water with Moringa seeds was the buildup of organic matter that occurred from
adding the seed powder to water. The organic buildup results in treated water being unable to be
stored for more than 24 hours. Jerri et al. (2012) studied the impact of adding sand to the Moringa
seed powder treatment. The sand treatment removed almost all of the BOD (Biological Oxygen
Demand) in the treated water (2090 ± 250 mg of BOD using just Moringa seed powder and 1.5 ±
0.6 mg of BOD using the sand-Moringa mixture) (Jerri et al., 2012). The reduction of BOD is

14
significant as it allows for safe storage of the treated water. BOD can be reduced by extracting the
oil from the seeds before powdering them (Garcia-Fayos et al., 2016). Using ethanol as a solvent
and the Soxhlet procedure (Method 3540C, 1996), the oil can be extracted from the seeds. The oil
can be used for industrial purposes, and the seed powder is more efficient at treating water (GarciaFayos et al., 2016). The powdered seeds are the most known and researched method of water
purification, however, there is also anecdotal evidence that leaves have been traditionally used in
this role as well.
Many studies have explored antimicrobial effects of Moringa leaf powder on waterborne
bacteria. The leaf powder of Moringa inhibits E. coli and Staphylococcus aureus (Okorondu et al.,
2013). These properties are further shown in its effectiveness in decreasing bacteria when used as
a substitute for hand soap (Torondel et al., 2014). Bacteria inhibition in leaves is through
membrane lysis (Chuang et al., 2007). The cytoplasmic membranes of fungal cells are ruptured by
Moringa extracts – the extracted compounds interact with lipid bilayers, separating the inner and
outer membranes which allows water to enter the cell (Chuang et al., 2007). This causes the
bacteria cell to swell, and leads to cell death (Chuang et al., 2007). Not all parts of the Moringa
tree have been studied for their potential to treat contaminated water. In order to maximize the
Moringa tree’s potential as a point-of-use water treatment, additional parts of the plant should be
studied. This is the central subject of this thesis, and is fully examined in subsequent chapters.

15
Chapter 3: Chemical Properties of Moringa Roots
3.1 Abstract
Many parts of Moringa oleifera are consumed to combat malnutrition, and those
constituents have been studied for the nutritional value. The objective of this study was to
determine the inorganic composition of Moringa roots in order to fill the gap in current research.
The solubility of chemical constituents of Moringa roots was studied, to determine whether water
treated with Moringa roots met Canadian drinking water quality guidelines. The inorganic
constituents of Moringa were determined through inductively coupled plasma mass spectrometry
(ICP-MS). The main chemical constituents were five macrominerals: potassium, phosphorus,
magnesium, sodium and calcium. The concentration of the Moringa root minerals dissolved in
water were determined using inductively coupled plasma optical emission spectrometry (ICPOES). The most abundant elements soluble in water were potassium, phosphorus, sulphur,
magnesium, sodium, and calcium respectively. Each of these elements were either within Canadian
drinking water quality guidelines or did not have a guideline established as they are not considered
a health concern when present in drinking water. Many of these elements were nutrients that could
have potential health benefits.
3.2 Introduction
Many people in the world depend on plants as their primary source of medication. Moringa
oleifera is one such plant that is widely used for its nutritional and medicinal value. Although all
parts of the plant are used medicinally, the roots are of particular interest in phytochemical research
due to their potency. The medicinal uses of Moringa roots include: antilithic, rubefacient, vesiant,
carminative, antifertility, stimulant in paralytic afflictions, and as a cardiac/circulatory tonic
(Anwar et al., 2007). Moringa roots are used to treat rheumatism, inflammation, articular pain,

16
lower back or kidney pain and constipation (Anwar et al., 2007). Roots have anti-spasmodic
activity (Caceres et al., 1992) and hepatoprotective activity (Ruckmani et al., 1998). Moringa roots
have been studied for a potential anticancer role in ovarian cancer (Bose, 2007). Moringa roots
contain isothiocyanates, which are able to induce apoptosis in ovarian cancer, and show antitumor
activity in a variety of other cancers (Bose, 2007). Moringa roots have antibacterial activity and
are rich in antimicrobial agents (Mishra et al., 2011).
Due to the extensive use of Moringa roots in medicine, the phytochemicals present in
Moringa roots have been studied in depth (Furo and Ambali, 2012; Amaglo et al., 2010). In 2012,
the phytochemicals in Moringa roots were studied by preparing various extracts of Moringa roots
(crude, chloro-form, ethyl- acetate, N-butanol and residue); each of these extracts were subjected
to various tests to determine what phytochemicals were present in the roots (Furo and Ambali,
2012). Using this method, it was shown that Moringa roots consist of pharmacologically important
chemical compounds such as carbohydrates, saponins, cardiac glycosides, terpenes, steroids,
flavonoids and alkaloids (Furo and Ambali, 2012).
In a comparison of various phytochemicals found in other parts of the Moringa plant,
roots contain 4-O-(α-l-rhamnopyranosyloxy)-benzylglucosinolate (glucomoringin) and
benzylglucosinolate (glucotropaeolin) (Amaglo et al., 2010). Roots did not contain any
flavonoids that were detected in other parts of the plant; however, they did contain significant
levels of fatty acids. Roots also contain crude protein, ranging from 3.57 - 4.38% (Amaglo et al.,
2010). The roots’ antibacterial and fungicidal activity is due to the presence of 4-α-Lrhamnosyloxybenzyl isothiocyanate and pterygospermin (Ruckmani et al., 1998; Eilert et al.,
1981).

17
Although the organic components of Moringa roots have been studied, the inorganic
elemental components of Moringa roots have not yet been fully explored. Amaglo et al. (2010)
researched the presence of selenium (Se), sodium (Na+), potassium (K+), magnesium (Mg 2+) and
calcium (Ca 2+) in Moringa roots. They found that roots of the vegetative plant contained no
selenium, <0.1% (w/v) sodium, 2.05% (w/v) potassium, no magnesium and 0.3% (w/v) calcium.
In flowering Moringa, there was no selenium in the roots, though the roots had 0.13% sodium,
1.62% potassium, 0.52% magnesium and 0.51% calcium (Amaglo et al., 2010). A full analysis of
all chemicals present in Moringa roots is not currently available.
The chemical composition of the roots is of particular importance when considering their
impact on water quality. The chemical elements present in Moringa roots could determine the
feasibility of Moringa roots as a point-of-use water treatment. The presence of heavy metals or
other harmful elements in roots could indicate that it is not suitable for ingestion, and therefore not
suitable for use in water treatment. Conversely, if the roots contain calcium, iron, or other
beneficial elements, then their use in water purification could have the added benefit of providing
nutrients in clean water.
The objectives for this chapter were to:
1. Determine the chemical elements present in fresh and dried Moringa roots.
2. Determine whether the length of growth time impacts the chemical elements present in
dried Moringa roots by comparing roots grown for six months to roots grown for seven
months.
3. Determine the chemical elements present in water treated with Moringa roots.
4. Compare the chemicals present in Moringa roots with that of a common beverage, 2% cow
milk.

18
3.3 Methods
3.3.1 Planting, Growing, and Processing Moringa
Moringa oleifera was grown in the Enhanced Forestry Lab (EFL) at the University of
Northern British Columbia (UNBC). A soil mixture was prepared by mixing 20 L peat, 20 L coarse
sand, 20 L coir, 60 g of slow-release nutrients (14-14-14), and 3 tablespoons of dolomite. The soil
was divided and placed into 25 one-gallon containers. On October 15, 2015, the seeds were planted
in the center of the one-gallon containers, one-inch-deep in the soil. The containers were
transported to a greenhouse to germinate and grow.
In the greenhouse, the plants received 16 hours of light per day using HPS supplemental
lighting from 0600 to 2200 hours. The greenhouse bay was kept at 24°C with a relative humidity
ranging from 20 to 40%. During the night (2200 to 0600 hours) the temperature was 18°C and the
lights were turned off. The plants were watered three times a week until the pots were saturated.
Two weeks after germination, fertigation was applied once a week using the fertilizer Tune-Up
20-10-20 at 100 ppm. The plants were monitored for diseases and insects. Both thrips and white
flies were detected. These pests were treated with applications of 20 mL/L of Safer’s Soap and
1.28 mL/L of Enstar. After treatment, the infestation was controlled and the trees continued to
grow. The 25 seedlings were grown for six months before harvest.
After six months of growth, 11 of the Moringa trees were randomly selected from the
population of 25 trees to be processed. The trees roots were harvested by first removing each plant
from its one-gallon bucket. The soil was gently brushed off to expose as much of the roots as
possible. The roots were cut off approximately 1 cm up the trunk from the base of the taproot.
Each tree was carefully placed back in the soil and returned to the greenhouse to grow under the
original conditions.

19
After harvesting, the roots were gently washed with water. The root bark was removed
using a stainless steel vegetable peeler. The roots were randomly separated to receive one of two
treatments: denoted as either fresh or dry. The fresh Moringa was ground to smooth consistency
using a blender in order to increase surface area and extract active compounds. Ground Moringa
was labelled, placed in an airtight container and put in a cooler at 4˚C.
Dried Moringa roots were processed as per the methods used by Kasolo et al. (2011). The
peeled roots were oven-dried at 70°C for five days, the point at which a constant weight was
maintained over a 24-hour period. Direct sunlight was avoided to preserve the active compounds
(Kasolo et al., 2011). Dry Moringa was ground using a blender. Since dry roots were much harder
than fresh roots, dry roots were further ground into fine powder using a ball grinder. The powder
was sifted through a sieve with a pore size of 5 mm. The powder was placed in an airtight jar,
labelled and kept at room temperature.
Two weeks after the fresh Moringa was processed and placed in a cooler at 4˚C, they began
to rot. These samples were no longer viable for treatment, so the remaining trees were harvested.
The new samples were harvested after 7 months of growth. They were harvested and prepared in
the same way as the previous fresh samples, but placed in a freezer at -18˚C, rather than a cooler,
to better preserve the sample. New dry samples were processed at the same time to have a
consistent growing time between fresh and dry samples (7 months).
Additional data on the growth of the Moringa trees was collected throughout the six months
of tree growth. This information can be found in Appendix A.
3.3.2 Chemical Analysis
Chemical composition of Moringa roots was analyzed using ICP-MS. Three composite
samples of Moringa were tested. Sample referred to as ‘Dry 6 Months’ was dried roots harvested

20
after growing for six months, ‘Dry 7 Months’ was dried roots which were harvested after seven
months of growth. The final sample, labelled ‘Fresh 7 Months’ was fresh roots that were harvested
after seven months of growth.
A portion of the dried roots from ‘Dry 6 Months’ (~3 g) was digested according to EPA
method 3051A, using 2:1 HNO3: HCl (Method 3051A, 2007). After the acid was added to Moringa
roots, it was placed in a microwave digester. Temperature was increased from ambient temperature
to 180°C within 10 minutes. The temperature was held at 180°C for another 10 minutes and
allowed to cool. The same process was repeated for fresh roots and the batch of dried roots that
were dried for 7 months. The analysis of chemicals present in the composite samples was done in
triplicate. TILL-3, HISS-1 and Tomato Leaves 1573a were used as certified references to ensure
the ICP-MS was calibrated. After digestion, the samples were run through an ICP-MS (Agilent
7500cx). Elements tested for were beryllium, boron, sodium, magnesium, aluminum, phosphorus,
potassium, calcium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium,
germanium, arsenic, selenium, rubidium, strontium, molybdenum, silver, cadmium, tin, antimony,
caesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium,
gadolinium, dysprosium, holmium, erbium, thulium, ytterbium, thallium, lead, thorium and
uranium.
3.3.3 Solubility Analysis
The solubility of Moringa roots was tested using inductively coupled plasma optical
emission spectrometry (ICPOES) (Agilent 5100). Nine different Moringa concentrations were
made: 12.5 mg/L, 27.5 mg/L, 250 mg/L, 1250 mg/L, 2500 mg/L, 4200 mg/L, 8300 mg/L, 12500
mg/L, and 16000 mg/L. A control with no Moringa was prepared. These concentrations were
prepared by dissolving dried Moringa root powder into 400 mL of milliQ water. After Moringa

21
was added to the 400 mL beaker of water, it was stirred with a magnetic stirrer for 30 minutes at
80 rpm and at 30°C. After stirring, 40 mL of the samples were put through a 0.45 µm hydrophilic
nylon filter and dispensed into a 50 mL conical. Samples of each concentration were run through
the ICPOES (Agilent 5100) and matrix matched with 2% HNO3 and 1% HCl. Samples were run
in triplicate, with a 500 ppb standard, a 1 ppm spike, and procedural blanks used for reference.
The chemical composition of 2% cow milk was tested using ICP-MS (Agilent 7500cx).
These data were compared to the chemicals present in the water treated with various concentrations
of Moringa root powder.
3.3.4 Statistical Analysis
The mean concentration of elements present in roots and in water (mg/kg and mg/L) and
standard deviations were calculated. Differences in the concentration of elements analyzed by ICPMS in dry and fresh Moringa roots was compared using a Welch two-sample unpaired t-test. A
Welch two-sample unpaired t-test was used to determine whether there was a significant difference
between chemical elements present in roots harvested and dried after 6 months of growth, and
those harvested and dried after 7 months of grown. Significance was tested with an alpha value of
0.05. Normality was tested with the Shapiro-Wilk normality test. All statistics were analyzed with
R Studio software.
3.4 Results
3.4.1 Chemical Constituents of Moringa Roots
The chemical elements present in Moringa roots were compared between those that were
harvested and dried after six months of growth (Dry 6 Months), Moringa roots that were harvested
and dried after seven months of growth (Dry 7 Months), and fresh Moringa roots harvested at
seven months (Fresh 7 Months). The most abundant element present in the Moringa roots was

22
potassium, which was present in a mean concentration of 17,860 mg/kg in dried roots, and 23,402
mg/kg in fresh roots (Table 2). The roots had high mean concentrations (>1000 mg/kg) of sodium,
magnesium and phosphorus. The roots had a moderate amount of calcium (>600 mg/kg). Boron
and aluminum were also both present in moderate amounts with mean values of over 100 mg/kg
in fresh roots, but below 100 mg/kg for dried roots. Manganese, iron, zinc, strontium and barium
were all present in the roots with mean values between 10 and 100 mg/kg. There were less than 10
mg/kg of copper, gallium, rubidium, tin, chromium, nickel, cerium, cobalt, cadmium and lead.
Cobalt, cadmium and lead were the least abundant of the measured elements with values below 1
mg/kg (Table 2).
Several other elements were tested for but were not detected in the Moringa roots. The
following elements were either not present in the roots, or were present in quantities below the
detectable limit of the ICP-MS: beryllium, selenium, vanadium, germanium, arsenic,
molybdenum, silver, antimony, caesium, lanthanum, praseodymium, neodymium, samarium,
europium, gadolinium, dysporium, holmium, erbium, thulium, thallium, thorium, ytterbium, and
uranium.

23
Table 2: Mean and standard deviation of chemical composition of dried and fresh Moringa roots.
Mean values are expressed in mg/kg.
Element
Dry 6 Months
Dry 7 Months
Fresh 7 Months
68.91 (16.02)
Aluminum
70.24 (3.38)
133.55 (9.56)
Barium
19.86 (2.02)
10.78 (0.37)
15.52 (0.56)
71.21
(5.43)
71.44
(6.11)
Boron
175.3 (5.44)
<dl
0.03 (0.02)
Cadmium
0.04 (0.01)
975.06 (111.70)
601.35 (20.56)
697.59 (24.73)
Calcium
Cerium
0.54 (0.10)
0.63 (0.06)
1.51 (0.15)
<dl
0.71 (0.13)
1.51 (0.08)
Chromium
0.19
0.10 (0.02)
0.15 (0.05)
Cobalt
8.08
5.27 (0.43)
7.86 (5.40)
Copper
Gallium
4.00 (0.32)
2.39 (0.07)
3.57 (0.36)
Iron
40.52 (24.38)
34.90 (8.81)
46.39
Lead
0.35 (0.01)
0.87 (0.27)
0.57 (0.12)
2688.2 (92.14)
1902.6 (53.38)
2349.4 (74.46)
Magnesium
18.35
13.80 (0.57)
Manganese
11.05 (0.77)
1.41
0.82 (0.05)
1.08 (0.01)
Nickel
Phosphorus
3112.52 (129.73)
3952.11 (91.48)
4521.11 (302.2)
Potassium
24190.74 (1316.68)
17860.63 (1094.32)
23402.85 (133.76)
Rubidium
6.51 (0.53)
4.80 (0.36)
4.72 (0.71)
1321.98 (43.72)
969.64 (26.22)
1577.29 (9.05)
Sodium
Strontium
22.90 (3.06)
11.78 (0.41)
15.15 (1.08)
1.16 (0.10)
2.35 (0.16)
Tin
1.08 (0.10)
Zinc
21.33 (2.17)
16.11 (0.92)
21.56 (1.90)
Values expressed as mean (SD). Samples measuring below the detection limit (dl) are represent
with <dl; Chromium < 0.4 mg/L, Cadmium < 0.03 mg/L. Values with no standard deviation had
only one sample measure above the detection limit.
The comparison between the dried roots that were harvested after six months and those that
were harvested after seven months revealed that the roots that were harvested after seven months
had a significantly lower concentration (p < 0.05) of sodium, magnesium, phosphorus, potassium,
calcium, manganese, lead, nickel, copper, zinc, gallium, rubidium, strontium, and barium (Table
3). There was no significant difference in the concentration of boron, aluminum, iron, cobalt, tin
and cerium between roots harvested at seven months and those harvest after six months of growth
(Table 3).

24
The chemical composition of the fresh roots that had been grown for seven months was
compared to that of the dried roots that had been harvested at seven months. The fresh roots
contained a significantly higher concentration (p < 0.05) of boron, sodium, aluminum, lead,
cerium, tin, magnesium, phosphorus, potassium, calcium, manganese, nickel, gallium, zinc,
strontium and barium (Table 4). There was no significant (p > 0.05) difference in concentration
for chromium, iron, cobalt, copper and rubidium between the dry and fresh roots (Table 4).
Table 3: T-test results of difference in amount of elements present in Moringa roots harvested at
7 months of growth and 6 months of growth.
Element
dF
T- test statistic
p value
Aluminum
4
-0.14
0.90
Barium
4
-7.77
1.48e-3
Boron
4
0
1
Calcium
4
-5.70
4.68e-3
Cerium
4
-1.34
0.25
Cobalt
4
-2.63
0.058
Copper
4
-4
0.016
30
Gallium**
(1, 4)
4.42e
2.2e-16
Iron
4
-0.39
0.72
Lead
4
-3.02
0.039
Magnesium
4
-12.76
2.17e-4
Manganese
4
-5.5
5.33e-3
Nickel
4
-8.5
1.05e-3
Phosphorus
4
-7.42
1.76e-3
Potassium
4
-6.40
3.06e-3
Rubidium
4
-3.54
0.024
Sodium
4
-11.99
2.78e-4
Strontium
4
-6.43
3.02e-3
Tin
4
-0.38
0.72
Zinc
4
-4
0.016
Bold values are statistically significant at an alpha value of 0.05.
Due to no variation among samples an ANOVA was used to calculate the p-value for gallium.

25
Table 4: T-test results of difference in amount of elements present in fresh and dry Moringa roots
harvested at 7 months of growth.
Element

dF

T test statistic

Aluminum
4
-5.95
Barium
4
-9.90
Boron
4
-21.26
Calcium
4
-5.18
Cerium
4
-8.22
Chromium
4
-1.87
Cobalt
4
-1.65
Copper
4
-0.88
Gallium
4
-5
Iron
4
-0.38
Lead
4
-3.41
Magnesium
4
-8.47
Manganese
4
3.5
Nickel
4
-8
Phosphorus
4
-9.15
Potassium
4
-8.71
Rubidium
4
0
Sodium
4
-38.24
Strontium
4
-5
Tin
4
-10.85
Zinc
4
-4
Bold values are statistically significant at an alpha value of 0.05.

p value
4.01e-3
5.84e-4
2.89e-5
6.60e-3
1.19e-3
0.13
0.18
0.43
7.49e-3
0.72
0.027
1.07e-3
0.025
1.32e-3
7.92e-4
9.58e-4
1
2.79e-6
7.49e-3
4.09e-4
0.016

3.4.2 Water Soluble Elements in Moringa Roots
Elements present in Moringa-treated water were recorded by ICP-OES. Ten concentrations
were tested, ranging from 0 mg to 16,000 mg Moringa/L. As the Moringa root concentration
increased through this range, the amount of each element that was present in the water continued
to increase (Table 5).
The most abundant elements dissolved in water were potassium, phosphorus, sulphur,
magnesium, sodium, and calcium. A full list of elements found for each Moringa concentration is
shown in Table 5. Elements tested for but not found in the Moringa-treated water were: aluminum,
arsenic, boron, cadmium, cobalt, chromium, mercury, molybdenum, nickel, lead, antimony,
selenium, tin, uranium and vanadium.

26
The most abundant elements in water treated with Moringa for each concentration
(potassium, phosphorus, sulphur, magnesium, sodium, and calcium) were the same elements found
to be the most abundant in the roots, with the exception of sulphur (Table 2).
In addition to testing the elements present in water treated with Moringa, the elements
present in 2% cow milk were also tested. Milk contained more calcium, potassium, magnesium,
sodium, phosphorus, strontium and zinc than the Moringa-treated water (Table 5). However,
Moringa water had higher levels of barium. The results for manganese and iron were both
inconclusive, as the ICP-MS had different detection limits than the ICP-OES. For these elements,
Moringa water ranged from 0.02 to 0.12 mg/L, and 0.05 to 0.26 mg/L respectively. Milk was
below the detection limit for both of these elements, however, the detection limit for manganese
on the ICP-MS was 0.2 mg/L and for iron it was 3 mg/L. The lower Moringa concentrations (12.5
– 12,500 mg/L) contained less copper than milk, however, the highest Moringa concentration
(16,000 mg/L) contained the same amount of copper as milk (0.05 mg/L). Sulphur and mercury
were both measured by the ICP-OES, but not the ICP-MS.

27
Table 5: Mean (and standard deviation) of chemical elements in water treated with Moringa root powder as determined by ICP-OES.
Mean values are expressed in mg/L.
[Moringa] (mg/L)
Barium
Calcium
Copper
Iron
Potassium
Zinc
0.02 (0.0098)
0.29 (0.0874)
<dl
0.05
<dl
0.00 (0.0030)
0
0.03 (0.0031)
0.28 (0.0096)
<dl
<dl
<dl
0.04 (0.0023)
12.5
0.04 (0.0041)
0.30 (0.0230)
0.01 (0.0013)
<dl
0.65 (0.0850)
0.05 (0.0011)
27.5
0.07 (0.0094)
0.72 (0.4748)
0.01 (0.0011)
0.26 (0.2991)
7.42 (0.1289)
0.16 (0.1432)
250
0.07 (0.0141)
1.34 (0.3183)
0.01 (0.0024)
0.08
35.71 (1.3970)
0.12 (0.0396)
1,250
0.08 (0.0100)
2.20 (0.0871)
0.01 (0.0003)
<dl
72.70 (1.1674)
0.17 (0.0300)
2,500
0.09 (0.0268)
2.18 (0.2938)
0.02 (0.0037)
0.13
75.04 (1.8800)
0.17 (0.0751)
4,200
0.11 (0.0110)
4.37 (0.0374)
0.03 (0.0029)
0.06 (0.0021)
149.94 (1.2733)
0.27 (0.0055)
8,300
0.10 (0.0043)
6.27 (0.8471)
0.04 (0.0007)
0.13 (0.0819)
207.34 (7.2078)
0.39 (0.0164)
12,500
0.13 (0.0198)
8.72 (0.1428)
0.05 (0.0017)
0.13 (0.0024)
279.96 (1.2186)
0.57 (0.0649)
16,000
<0.06
407.21
0.05
<3
1860.30
4.45
Milk (2%)
[Moringa] (mg/L)
Magnesium
Manganese
Sodium
Phosphorus
Strontium
Sulphur
0.05 (0.051)
<dl
0.50 (0.264)
0.10
<dl
<dl
0
0.05 (0.003)
<dl
0.62 (0.011)
0.10
<dl
<dl
12.5
0.08 (0.002)
<dl
0.63 (0.027)
0.10 (0.002)
<dl
<dl
27.5
0.59 (0.075)
0.02
0.90 (0.025)
1.35 (0.061)
<dl
<dl
250
2.68 (0.189)
0.01 (0.0005)
1.98 (0.332)
7.05 (0.374)
0.01 (0.0006)
5.99 (0.440)
1,250
5.55 (0.097)
0.03 (0.0004)
3.29 (0.253)
14.79 (0.205)
0.01 (0.0002)
12.23 (0.182)
2,500
5.34 (0.199)
0.03 (0.0072)
3.17 (0.132)
13.86 (0.443)
0.01 (0.0013)
12.29 (0.164)
4,200
11.52 (0.171)
0.06 (0.0003)
6.21 (0.087)
29.26 (0.458)
0.02 (0.0000)
24.57 (0.253)
8,300
16.06 (1.297)
0.08 (0.0112)
8.50 (0.0101)
39.84 (2.549)
0.02 (0.0026)
32.89 (3.754)
12,500
22.60 (0.242)
0.12 (0.0007)
11.93 (0.409)
54.86 (0.901)
0.03 (0.0010)
45.15 (1.534)
16,000
135.37
<0.2
492.1
1093
0.43
Milk (2%)
Samples measuring below the detection limit (dl) are represent with <dl; Copper < 0.01 mg/L, Manganese < 0.01 mg/L, Iron < 0.05
mg/L, Strontium < 0.002 mg/L, Potassium < 0.4 mg/L, Sulphur < 1.8 mg/L. Values with no sd had one sample measure above the dl.

28
3.5 Discussion
3.5.1 Chemical Constituents of Moringa Roots
The five most abundant elements found in the Moringa roots were potassium, sodium,
magnesium, phosphorus and calcium for all samples (Table 2). Potassium is essential for the
maintenance of cell function and body fluid volume (WHO, 2012a). Lack of potassium is
associated with cardiovascular diseases and high blood pressure. The WHO recommends that
adults (people >16 years of age) consume 3,510 mg of potassium per day (WHO, 2012a).
Potassium was the most abundant element in the Moringa root, with a mean concentration of
23,402.84 mg/kg in the fresh roots. In comparison, one kilogram of white beans (a vegetable high
in potassium) contains only 5,610 mg of potassium. A medium sized banana, which is a fruit
known for its high levels of potassium, contains about 350 mg of potassium (Sampath Kumar et
al., 2012).
Sodium is an important nutrient in the body that maintains plasma volume, balances the
body’s pH, transmits nerve impulses, and promotes normal cell function (WHO, 2012b). Sodium
is abundant in a variety of foods, and it is easy for most adults to acquire their recommended 2 g
of sodium per day (WHO, 2012b). Due to easy access of sodium in diets, most health risks
associated with sodium are through an excess consumption of sodium. Increased sodium can
increase blood pressure and risk of hypertension. Increased risk of stroke and heart disease are
related to an increase in sodium. Although sodium was one of the most abundant elements in
Moringa roots, an entire kilogram of the roots still contains less than the recommended 2 g/day of
sodium. The high levels of potassium in the roots would work to counteract the potential negative
effects of diets high in sodium (WHO, 2012a).

29
Magnesium is heavily involved in the body’s energy metabolism and the synthesis of
proteins, RNA, and DNA (WHO/FAO, 2004). Magnesium also plays an important role in
regulating both potassium and calcium (Classen, 1984). Although magnesium deficiency is rare,
it has many pathological consequences. The neurological and neuromuscular results of magnesium
deficiencies relate to its role in regulating potassium and result in conditions such as nausea,
muscular weakness, lethargy, staggering and weight loss (Shils, 1988). This can progress into
muscular spasms and convulsions and cardiac arrhythmia (Shils, 1988).
Phosphorus is the second most abundant mineral in the body, second only to calcium. Like
calcium, the main role of phosphorus in the body is the formation of bones and teeth. In addition
to bone formation, phosphorus is necessary for the growth, maintenance and repair of cells. It also
assists in kidney function, muscle contractions, nerve signaling and regular heartbeat maintenance
(Moshfegh et al., 2016.). Phosphorus is an essential constituent of all known protoplasm.
Inadequate phosphorus levels can result in anorexia, anemia, muscle weakness, bone pain, rickets
and osteomalacia (Institute of Medicine (US), 1997). Phosphorus however, is abundant in various
foods and a deficiency in it is rare (Institute of Medicine (US), 1997).
Calcium provides rigidity to the skeleton. Calcium ions are an important part of virtually
all metabolic processes. Lack of calcium in the body can lead to conditions such as osteoporosis
(WHO/FAO, 2004).
The roots that were harvested after 6 months had significantly higher levels of sodium,
magnesium, phosphorus, potassium, calcium, manganese, lead, nickel, copper, zinc, gallium,
rubidium, strontium, and barium than those harvested after seven months of growth. This could be
due to the fact that roots of younger plants generally display a greater ability to absorb ions than
older plants do (Tangahu et al., 2011). As trees mature, the concentration of lignin in their cells

30
walls increases (Jha et al., 2017). Lignin is a three-dimensional polymer that provides strength,
stability, and defense to plants (Heldt and Piechulla, 2011). Hardwoods generally have a lignin
content of 25  5% (McDonald and Donaldson, 2001). If the seven month old plants have a higher
concentration of lignin in their cell walls, this would mean reduced space in the cells, and so a
lower concentration of stored elements. Future studies could determine the lignin content of
Moringa roots at various ages, to determine how the concentration of lignin changes in maturing
Moringa roots. The nutritional content of Moringa leaves follow a similar trend, where plant
maturity has a negative influence on the nutritional composition (Quintanilla-Medina et al., 2018).
Leaves harvested at increasing maturity had a downward trend in concentration for most elements,
and this decrease in quality is more rapid in stems due to the increasing cell wall and lignin content
(Quintanilla-Medina et al., 2018).
Fresh roots contained a significantly higher concentration of boron, sodium, aluminum,
lead, cerium, tin, magnesium, phosphorus, potassium, calcium, manganese, nickel, gallium, zinc,
strontium, and barium. This could be because dry roots have a higher concentration of plant
components such as cellulose and lignin, but lower concentrations of some nutrients (Norton and
Ahn, 1997). The fresh roots could also have higher concentrations of nutrients due to a loss during
the drying process. In a comparison of different drying methods of Moringa leaves (oven drying,
sun drying, air drying and freeze drying), oven drying was found to be the least effective method
at preserving the nutraceutical properties of Moringa leaf (Ademiluyi et al., 2018). In Ademiluyi
et al.’s study, oven dried leaves were dried at 40C, which is lower than the temperature used in
this study (70C) for drying Moringa roots. Moringa leaves underwent partial breakdown and
structural changes when dried at 50C (Potisate et al., 2014). It is likely that some nutrients could
be lost in the drying process, perhaps by UV or heat-induced destruction of some labile constituents

31
(Ademiluyi et al., 2018). Further studies comparing different drying methods have determined that
oven drying is not ideal compared to shade drying or freeze drying, due to loss of some elements
such as fibre-bound nitrogen and sulphur (Ramsumair et al., 2014; Grundon and Asher, 2008).
3.5.2 Water Soluble Elements in Moringa Roots
The Moringa root powder was dissolved in water to determine the concentration of these
chemical elements in water. The concentrations of Moringa roots ranged from 12.5 - 16,000 mg/L
of Moringa. The amount of each element present in the Moringa root water continued to increase
with increasing concentration, showing that we had not reached the saturation point for the
Moringa root. Nine of the elements present in Moringa roots were not present in the Moringa root
water, showing that these nine elements in roots did not dissolve in water. Those nine elements
were aluminum, boron, cadmium, cobalt, chromium, iron, nickel, lead, and tin.
The majority of the elements in Moringa roots that were dissolved in water were
macrominerals. Potassium, sodium, magnesium, phosphorus and calcium were the most abundant.
These minerals have many health benefits as discussed previously. Sulphur was present in high
quantities. Sulphur is a component of some amino acids, and as such is involved in protein
synthesis. It also helps with production of collagen. The presence of sulphur in the water, though
none was found in the roots themselves, is due to the difference in measuring equipment. The
solubility analysis was conducted with the more robust ICP-OES, while the solid sample analysis
was performed with ICP-MS, which did not test for sulphur.
The other elements including copper all had less than 1 mg/L soluble in the water. The
WHO states that the range for copper in drinking water from Europe, Canada and the USA is ≤
0.005 to > 30 mg/L (WHO, 2004). Water treated with Moringa root is within that range (0.05
mg/L) for the highest Moringa concentration. Copper is an important component of red blood cells

32
and collagen; it is important for nerve and immune system health, and it is an antioxidant (Penn
State, 2015). Iron is another important component of blood, and was present in the Moringa root
solutions.
Many of the elements found in water which had Moringa root powder dissolved in it do
not have established drinking water guideline values, as they are not of health concern in drinking
water (WHO, 2017). Elements found in Moringa root water that are not of health concern were
iron, manganese, potassium, sodium and sulphate (WHO, 2017). The concentration of minerals
and other elements in the Moringa root treated water were compared to the Canadian Drinking
Water Quality Guidelines (CDWQG) (Health Canada, 2017). Three elements present in the
Moringa root treated water are not listed in the guidelines. Those elements are potassium,
phosphorus and strontium. Calcium and magnesium are both listed in the guideline and have no
limit. There is no guideline for sulphur listed either, however, the guideline for sulphate is ≤ 500
mg/L, and the Moringa treated water had sulphur levels below this guideline (45.15 mg/L) (Health
Canada, 2017). The guideline for barium is ≤ 1 mg/L. This guideline is necessary due to the
potential for increased blood pressure caused by barium. Water treated with Moringa root powder
was within the CDWQG for all concentrations. Copper, iron, zinc, manganese and sodium have
guidelines based on aesthetic considerations, such as taste or staining laundry. These guidelines
are not based on health effects. Of these aesthetic considerations, the water treated with Moringa
root powder was below the guidelines for all concentrations for copper, iron, sodium and zinc.
The guideline for manganese is ≤ 0.05 mg/L. The water treated with 16,000 mg/L of
Moringa root powder had a concentration of 0.12 mg/L of manganese. This is above the guidelines.
When water was treated with concentration of Moringa root powder at or below 4,200 mg/L, the
amount of manganese present in the water was within the Canadian Guidelines. If treating water

33
with Moringa root powder, the concentration should be kept below 4,200 mg/L of Moringa root
to ensure that water remains within drinking water quality guidelines.
The milliQ water contained low levels of barium, calcium, iron, zinc, magnesium, sodium
and phosphorus. This procedural control was prepared in the same way that the samples were
prepared – they were placed in a glass beaker, stirred with a magnetic stirrer for 30 minutes at 80
rpm at 30°C. They were then put through a 0.45 µm hydrophilic nylon filter and dispensed into a
50 mL conical before being run through the ICP-OES. This procedure is likely adding low levels
of the aforementioned elements; they are possibly introduced when the samples are heated in the
glassware. As the levels of these elements continue to increase with increasing Moringa
concentration, the amount added by the procedure does not account for all of the ions present in
the Moringa treated water, and these ions are being added to the water from the Moringa roots.
In order to compare these levels to a common beverage that is considered healthy, the
elemental composition of cow milk (2%) was examined. Of the elements tested for, milk had a
higher concentration of each element except for barium and iron. The amount of barium and iron
in milk were each below the detection limit. Moringa treated water contained these elements,
although they were present in small amounts, within the drinking water quality guidelines. This
comparison gives confidence that Moringa root water could be safe for human consumption.
Chemical elements and minerals present in other parts of the Moringa tree (leaves, seeds,
pods) have been studied. Our findings are consistent with the mineral composition of the other
constituents of the Moringa tree. Moringa leaf powder added to different types of flour increased
the amount of potassium, magnesium, and calcium present in the flour (Oyeyinka and Oyeyinka,
2018). A meta-analysis performed by Brilhante et al. (2017) compiled data from many papers to
determine the range of reported values of micronutrients present in leaves, pods and seeds of

34
Moringa. Their analysis showed that for seed pods, the most abundant nutrient was potassium,
followed by sodium, phosphorus, calcium and sulphur. The pods also contained magnesium, iron
and copper, although these were present in smaller amounts.
Moringa leaves were highest in potassium, followed by calcium, magnesium, sulphur,
phosphorus and iron. Moringa leaves also contained small amounts of zinc and copper. The range
of mg/kg of these macronutrients in leaves reported by Brilhante et al. (2017) are consistent with
the findings in this thesis. The mg/kg of potassium, sodium, magnesium and phosphorus measured
in the Moringa roots fall within the range given for leaves. The amount of calcium in seeds is
higher than the range given for calcium present in Moringa leaves. The only four nutrients
analyzed for Moringa seeds were calcium, magnesium, iron and copper (Brilhante et al., 2017).
An additional analysis of micronutrients found in Moringa seeds showed that the most abundant
macrominerals present were magnesium, followed by phosphorus, calcium, copper, then sulphur,
however they did not measure the abundance of potassium present in the seeds (Gopalakishnan et
al., 2016).
The presence of a large quantity of macrominerals in Moringa roots could provide an
additional benefit to using Moringa root powder as a point-of-use water treatment method. Many
areas of the world lacking access to clean water are also areas that experience high levels of
malnutrition. Combining water treatment with increased nutrition has the potential to have
beneficial impacts on human health. The level of all elements present in the Moringa root water
compared to established guidelines were safe for human consumption when Moringa
concentration was at or below 4,200 mg/L.

35
Chapter 4: Effects of Moringa Root Powder on Contaminated Water
4.1 Abstract
Water borne bacteria can have serious human health effects. Effective methods of treating
contaminated drinking water can greatly reduce these risks. Many current methods of water
treatment are underutilized in certain areas, however, Moringa seems to be a promising solution
to this problem. The objective of this study was to test whether Moringa root powder had an impact
on the bacteriological quality of water, pH, electrical conductivity (EC) and turbidity of drinking
water, and to assess whether water treated with Moringa root powder met WHO drinking water
quality guidelines for these parameters. Moringa roots were dried, powdered, and added to
contaminated water. Four replicates were made among Moringa concentrations of 250, 450, 600
and 0 mg/L as a control. For bacteriological analysis, Moringa treatments sat in contaminated
water for one hour, then were filtered. The amount of E. coli present was measured using the
colilert method. To test the impact of Moringa root concentration on the physical parameters of
water quality, each concentration of Moringa was subject to two treatments, filtered and unfiltered.
Electrical conductivity and pH were measured at 1, 2 and 3 hours. Turbidity was measured at 0, 1,
2, 3, 24, and 48 hours. Moringa root powder resulted in significant (p < 0.05) decrease in pH, from
7.61 to 7.45 for filtered samples, and 7.61 to 7.38 for unfiltered samples. The pH remained within
drinking water quality guidelines (6.5 – 8.5). Electrical conductivity increased significantly (p <
0.05) with increasing Moringa root concentration. Electrical conductivity ranged from 209 µS/cm
(250 mg/L, unfiltered after 1 hour) to 244 µS/cm (450 mg/L, filtered after 1 hour). Increasing
Moringa root powder concentration resulted in a significant (p < 0.05) increase in the turbidity of
water. Analysis of variance showed a statistically significant (p < 0.05) 87% reduction of E. coli
in the contaminated water. Moringa root powder is able to reduce the amount of E. coli in water,

36
and could help reduce the negative impact that contaminated drinking water has on human health.
To increase the effectiveness of Moringa root powder as a water treatment, it should be further
tested in conjunction with other substances that may increase its ability to act as a flocculent and
treat turbidity in water.
4.2 Introduction
4.2.1 Water Crisis
Limited access to clean and safe drinking water is one of the biggest human health issues
in the world today. There are 2.1 billion people who do not have access to safe drinking water at
home (WHO and UNICEF, 2017). Lack of access to clean water and sanitation results in the spread
of bacteria and diseases such as cholera, typhoid, diarrhea, and gastroenteritis. This results in the
death of over 840,000 people each year (Pruss-Ustun et al., 2014). In 2016, 8% or 480,000
children under five died from diarrhea (UNICEF, 2018). Purified water can limit exposure to these
preventable diseases, decrease long-term exposure to carcinogenic compounds and heavy metals,
and it can positively impact food security (Hunter et al., 2010). More than 40% of people without
access to safe drinking water live in Sub-Saharan Africa (Freitas, 2013). This region is prone to
water stress due to insufficient infrastructure, impacts of climate change, demanding agricultural
activities, and deforestation (Freitas, 2013). Due to this poor quality of water, almost half of
Africans suffer from water-borne diseases (Freitas, 2013).
4.2.2 Current Purification Technologies
Many methods of water purification exist, such as chlorination, sand filtration, boiling, and
UV filtering. Although many of these methods are considered to be cost effective (Clasen et al.,
2007), they are still not universally utilized. Chlorination is a simple and inexpensive method,
however, it is difficult to access for those in poor and rural areas. It requires a continual purchase

37
of chlorine, which is not always available (CDC/US AID, 2009). Many users are highly opposed
to the taste of chlorine (CAWST, 2009), and even if they do drink it, it does not protect against
parasites, and can lead to toxic by-products in water with high organic content (WHO, 2006).
Boiling is another simple method of water treatment, however, it too has not been found to
be ideal for those in developing countries. Boiling requires consumption of fuel, which can be
costly or time consuming to acquire (Clasen et al., 2008). After boiling, the water often needs to
cool down. As such, boiled water is often stored before being consumed. Improper storage of water
can lead to further contamination of water, even after being boiled (Mintz et al., 2001). Improper
storage or transportation of water has been found to be a significant route of transmission of cholera
and dysentery (Mintz et al., 2001). Although it effectively kills most pathogens, boiling cannot
remove turbidity, chemicals, or change the water’s aesthetics (Clasen et al., 2007).
Solar disinfection (SODIS) has been used with success to improve microbial quality of
water. This method is easy and inexpensive, though it can only treat small quantities of water.
Although inexpensive, it also requires access to plastic polyethylene terephthalate (PET) bottles
(Oates et al., 2003). Another common treatment mechanism is the use of biosand filters. These are
often made from local materials, however, ease of access and cost of these materials can vary from
place to place. The biological layer can take up to a month to develop, and the system must be
used continually (CAWST, 2009). These drawbacks (monetary and time expenses, lack of access
to materials and demand for maintenance) contribute to the underuse of water purification
technologies leaving poor regions in the global south (countries with low or middle income)
disproportionately unable to gain access to clean drinking water.

38
4.2.3 Moringa Seeds in Water Treatment
An accessible and affordable method of water treatment is needed in these regions.
Moringa seems to be a promising method of water treatment. Moringa grows in many areas of the
global south that are in water distress. Moringa oleifera seeds have been shown to kill bacteria in
water. Hendrawati et al. (2016) looked at the effectiveness of Moringa seeds at treating E. coli in
both wastewater and ground water. They found that Moringa seeds reduced E. coli by 80% in
wastewater treatment, and 45% in ground water treatment (Hendrawati et al., 2016). This is
consistent with the impact Moringa seeds have on total coliforms, which were reduced by about
88% (Amagloh and Benang, 2009).
When testing the impact of Moringa on bacteria, often Escherichia coli (E. coli) and other
coliforms are used as these are standard indicator bacteria for fecal contamination. E. coli is a
Gram-negative rod shaped bacteria. It is found in the intestines of endotherms. Some strands of E.
coli can cause diseases such as gastroenteritis (Besser et al., 1999). The WHO standard for E. coli
in drinking water is <1 CFU (Colony Forming Units)/100 mL (WHO, 2017).
In addition to reducing bacteria in water, Moringa seeds can have a positive impact on the
physical water quality parameters: turbidity, pH and electrical conductivity, which are important
parameters to explore for drinking water quality.
Turbidity is an important parameter of water quality as it indicates the amount of
particulates in the water. Turbidity is caused when light is blocked by microorganisms, silt
particles, chemicals, or other debris in water (WHO, 2017). Most particles contributing to turbidity
have no health significance (WHO, 2017). Although particulates may not be harmful, turbidity can
indicate chemical or microbial content in drinking water (WHO, 2017). Due to the appearance of
turbid water, many people choose not to drink turbid water if they have the option. The WHO
guidelines of turbidity for safe drinking water is 5 nephelometric turbidity units (NTU) (WHO,

39
2017). Seeds of Moringa are used in water purification not only due their ability to kill bacteria,
but also because of their ability to act as a flocculent (Shebek, et al., 2015). When flocculants are
formed, they allow particulates in the water to settle out, thereby decreasing turbidity of
contaminated water (Shebek, et al., 2015). Moringa seeds reduced turbidity of wastewater by
98.6%, and reduced turbidity of ground water by 97.5% (Hendrawati et al., 2016). When treating
turbidity with Moringa seed powder, Nkurunziza et al. (2009) found that solutions with the
concentration of Moringa seed powder ranging from 125-150 mg/L were the most effective
concentrations. The lowest turbidity removal among the tested ranges was 83.2%, and the highest
was 99.8% (Nkurunziza et al., 2009).
The pH of water is another important water quality parameter. The WHO (2017) guidelines
for safe drinking water states that water should have a pH between 6.5 and 8.5 (WHO, 2017).
Acidic pH can corrode water pipes. When pH is less than 6.5, water is more likely to leach metal
ions; low pH can also be corrosive to the human body (Health Canada, 2015). A higher pH in
water can be an irritant to skin and mucous membranes (Health Canada, 2015). Alkalinity can also
corrode pipes. This damage can lead to contaminated drinking water, as well as have a negative
impact on the taste and odour of water (WHO, 2017). Moringa seeds have been found to increase
the pH of water when applied as a treatment (Hendrawati et al., 2016; Amagloh and Benang, 2009;
Nkurunziza et al., 2009). This increase is due to the presence of the cationic proteins in the seeds,
which accept protons from the water, resulting in a more basic solution (Amagloh and Benang,
2009). Although the pH was increased in each of these experiments, it remained in the acceptable
range of 6.5-8.5 (WHO, 2017). In some cases, the increase was preferred to other treatments, such
as powdered activated carbon (PAC), which reduced the pH outside of the acceptable range
(Hendrawati et al., 2016).

40
Conductivity is another important parameter of water quality. Conductivity of water is
directly related to the ions present in water. As a measure of the total dissolved solids in water, it
can vary significantly. There is not a standard value of conductivity for drinking water, however,
high levels of conductivity may make water undesirable to consumers, as it can indicate a change
in taste or appearance of water (Health Canada, 2017). Moringa seeds have a positive impact on
the EC of water. Moringa seeds reduce conductivity of wastewater by 10.8%, and the conductivity
of ground water by 53.4% (Hendrawati et al., 2016). This reduction in conductivity indicates that
ions were removed from the water by the Moringa seed treatment, making the water more palatable
for consumers.
While seeds of Moringa have been studied in-depth for their water-treatment capabilities,
other parts of the Moringa tree have not been studied for their potential in the same application
(Hendrawati et al., 2016; Amagloh and Benang, 2009; Nkurunziza et al., 2009). Roots of Moringa
could be explored to determine if they can treat contaminated water. Since Moringa roots
propagate quickly, they may be a more economic water treatment method than the seeds if they
possess the same antimicrobial properties. A large taproot develops from seeds within six months
of growth (this study) and their roots can be harvested at any time of the year. When roots are
harvested, a tree can be re-propagated from a cutting (Palada and Chang, 2003). Moringa trees can
take two to three years to begin producing seed pods. At three years, the trees have not reached
full maturity, and are not yet at optimal seed pod production. Trees produce seeds once a year
(Palada and Chang, 2003). Therefore, the use of roots could be more advantageous than the use of
the seeds in water treatment.

41
4.2.4 Research Questions
To explore the feasibility of Moringa roots as a point-of-use water treatment method, the
impact of Moringa roots on E. coli and total coliform colonies in contaminated water was tested.
The impact of powdered Moringa roots on turbidity, pH, and electrical conductivity of
contaminated water were also tested. The research questions explored were:
1. Does Moringa root powder reduce the amount of E. coli and total coliforms in
contaminated water?
2. Does Moringa root powder affect the physical water quality parameters (turbidity, pH,
and electrical conductivity)?
4.3 Methods
4.3.1 Growing and Processing Moringa Root Powder
Moringa root was grown and processed according to the methods found in Chapter 3.3.1
of this thesis. The roots used to test the impact of Moringa root powder on water quality were
grown for seven months and dried.
4.3.2 Development of Methods
To develop the methods used in this study, pre-trials were performed. The method was
adjusted several times while determining an effective and robust way to apply the Moringa root
powder to contaminated water, and to test the impact Moringa root concentrations had on bacteria
levels in water. The details of these pretrial methods are discussed in Appendix B.
4.3.3 Water Collection
Five litres of pond water was collected from a pond located at 54° 4' 23'' N, 122° 45' 19''
W, on a mixed livestock farm north of Prince George, BC in January of 2018. A creek on the west
side of the pond fed the pond through a culvert. The pond was frequented by many animals, either

42
directly at, or upstream of the sample site. Animals using the pond included horses, cows, beavers
and chickens. The pond was also used by local wildlife species. This pond was chosen as
preliminary tests (Appendix B) showed the natural presence of E. coli and other fecal bacteria in
the water.
The environmental parameters of the pond water (temperature, pH and electrical
conductivity) were measured in order to understand the baseline characteristics of the water used
for testing. Environmental data was collected on the same day that water samples were obtained.
These parameters were measured at four points along the shore of the pond. Measurements were
taken approximately 7 m apart. Temperature and pH were both measured with a pH/ temperature
probe (VWR Scientific Products Model 2180 pH/Temperature/mV Meter). The mean temperature
of the pond was 0.45°C and the mean pH was 5.75. The electrical conductivity was measured using
a calibrated Thermo Scientific Field Conductivity Meter. The mean electrical conductivity of the
pond was 130µS/cm.
Water was collected in a sterile plastic container, which was rinsed three times with pond
water (Penn State, 2017) before collecting the sample according to CCME standards (CCME,
2011). The pond water was brought to the lab located at UNBC. In the lab, 200 mL of pond water
was removed from the container. This 200 mL sample was used to isolate bacteria using methods
described in section 4.3.4, while the remaining water was used for the analysis in section 4.3.10.
4.3.4 Isolating Bacteria
Coliforms and E. coli were isolated from the pond water using serial dilutions and
membrane filtration. The dilutions were made from 200 mL of pond water removed from the
original container. The pond water was diluted with milliQ water by a factor of 2, 10, and 100, as
well as one undiluted sample. These dilutions were run through a 0.45 µm membrane filter. The

43
membrane was then placed on a petri dish with m-coli blue media, which selects for E. coli and
total coliforms. The samples were incubated for 24 hours at 35˚C.
After 24 hours, the development of E. coli and total coliform colonies were observed. A
streak plate of E. coli was made by selecting an E. coli colony from the samples, transferring that
colony to MUG agar plates using sterile technique, and streaking it across a quadrant. The
inoculating loop was sterilized and bacteria from the first quadrant was streaked across to the next
quadrant. Each quadrant was streaked through, with the loop sterilized between each quadrant.
The same method was repeated for a coliform colony. The MUG plates were incubated for 24
hours at 37˚C and the individual colonies were observed the next day.
4.3.5 Growth Curve Analysis
The growth curve was determined for both total coliforms and E. coli in LB broth. Six 125
mL flasks were prepared with 30 mL of LB broth each. Three flasks were used for E. coli and the
remainder for coliforms. An individual colony of E. coli was chosen from the MUG plate. A sterile
inoculating loop was used to transfer an E. coli colony into the flask containing LB broth. The
inoculating loop was swirled in the broth for 30 seconds. The flask was covered and placed in an
incubator at 37˚C. While incubating, the flask was shaken at 150 rpm. The inoculating loop was
sterilized and the procedure repeated for two more flasks. The same procedures were repeated for
the remaining three flasks, but were inoculated with total coliforms rather than E. coli.
The bacterial growth was analyzed by measuring the absorbance (OD) of bacteria in LB
broth with an Ultraspec 2100 pro UV/Visible Spectrophotometer. A 1 mL cuvette filled with sterile
LB broth was used as a reference, at a wavelength of 600 nm. Every 20 minutes, 1 mL of broth
was removed from the incubating broth and placed in a cuvette. The OD of each sample was
measured by the spectrophotometer. Measurements were taken until the bacteria growth was no

44
longer exponential, which occurred after approximately six hours. The results were plotted to
determine the point where E. coli and total coliforms moved from their exponential phase of
growth to the stationary phase.
4.3.6 Dilution Analysis
To determine how diluted the E. coli culture needed to be in order to analyze the impact
that Moringa root powder had on E. coli, a dilution analysis was performed. Sterile LB broth (30
mL) was placed in a 125 mL flask. The flask was inoculated with E. coli cultured on a MUG plate.
The inoculated broth was incubated at 37˚C and shaken at 150 rpm. The broth was incubated until
it reached its stationary phase, as determined by the growth curve analysis. The same process was
repeated for total coliforms. There were two replicates for each bacteria.
Serial dilutions of each broth were prepared. Dilutions were prepared by pipetting 1 mL of
inoculated broth into 99 mL of milliQ water to make a 10-2 sample. The next dilution was prepared
by pipetting 1 mL of the 10-2 sample into another 99 mL of milliQ water. This was repeated to
give dilutions of 10-4, 10-6, 10-8, and 10-10 for each sample. Dilutions were made in high density
polyethylene (HDPE) bottles. The dilutions sat for three hours before processing. After three
hours, each 100 mL sample was analyzed to determine the Most Probable Number (MPN) of E.
coli and total coliform colonies in the sample. The presence of E. coli was determined using the
IDEXX Colilert Test/Quanti-Tray/2000 Method (IDEXX® Laboratories, 2013). The powdered
Colilert reagent was aseptically opened, added to each 100mL sample and shaken vigorously for
two minutes to dissolve the powdered reagent. The mixture was poured into a sterile plastic quantitray. The quanti-tray was sealed and placed in an incubator at 37˚C for 24 hours. The remaining
inoculated broth was placed in a refrigerator at 4˚C.

45
After 24 hours the trays were removed from the incubator. The number of wells that
exhibited a colour change (clear to yellow) were recorded. This information was compared with
the quanti-pack conversion chart to determine the MPN/100mL of total coliforms. The quantitrays were then placed under a UV light and the number of wells that fluoresced were counted.
The information was compared with the chart to determine the MPN/100 mL of E. coli. A serial
dilution factor of 1x10-10 was found to be an adequate dilution factor which resulted in a countable
MPN/100 mL of both E. coli and total coliforms using the colilert method. This dilution factor was
used to calculate the specific dilutions needed to result in approximately 30 MPN/100 mL and 50
MPN/100 mL of E. coli and 60 MPN/100 mL and 100 MPN/100 mL of total coliforms.
4.3.7 Treatment Application
To test the effect of Moringa root powder on E. coli, fresh bacteria dilutions were prepared
to produce two bacteria concentrations, the final concentrations of which were 50 MPN/100 mL
and 37 MPN/100 mL. Four replicates (500 mL) for each bacteria concentration were made. Each
500 mL solution was further subdivided into four 90 mL samples. Therefore, a total of 16 (4
treatments x 4 replicates), 90 mL samples per bacteria concentration were created.
Three different Moringa treatment concentrations were prepared using 0.025 g, 0.045 g and
0.060 g of dried Moringa root powder. Each sample of Moringa root powder was added to 10 mL
of milliQ water. Eight replicates of each Moringa concentration were prepared. A control
containing 10 mL of milliQ water with no Moringa root powder was also prepared. These
treatments were set on a heat source with a magnetic stir rod and heated to 30°C while stirring at
60 rpm for 15 minutes. Table 6 displays the Moringa treatments applied to each contaminated
water sample.

46
The Moringa preparations were added to the prepared bacterial samples. For each bacterial
concentration (50 MPN/100 mL and 37 MPN/100 mL), there were four Moringa treatments
(0.025, 0.045, 0.060 g and control), each replicated four times. The final concentration of each
treatment was 0, 250, 450 and 600 mg/L for the control. Water was poured back and forth between
the two beakers to ensure that all of the Moringa root powder was well incorporated into each
bacteria sample. Each sample was stirred for 10 minutes and left to sit for one hour before
performing bacteria enumeration procedures.
Table 6: Bacteria concentration and treatment applied to each sample of contaminated water.
Each sample was prepared in four replicates.
Sample
Bacteria
Moringa Root
Moringa Concentration
Concentration
Powder Weight (g)
(mg/L)
(MPN/100 mL)
A1
37
0
0
A2

50

0

0

B1

37

0.025

250

B2

50

0.025

250

C1

37

0.045

450

C2

50

0.045

450

D1

37

0.06

600

D2

50

0.06

600

4.3.8 Enumeration of E. coli and total coliforms
After one hour, each sample was filtered into sterilized HDPE bottles. Filtering was done
using Whatman 150 mm, quality 1 filter paper. The presence of E. coli and total coliforms for
each bacteria concentration and Moringa treatment was determined using the IDEXX Colilert
Test/Quanti-Tray/2000 method.
After 24 hours, the trays were removed from the incubator. The MPN/ 100 mL of total
coliforms and E. coli were counted.

47
4.3.9 Statistical Analysis of Bacteriological Water Quality
Statistical analysis was done using R Studio software. Normality was tested using the
Shapiro-Wilk test. The mean and standard deviations were calculated. A one-way ANOVA was
performed to determine the significance of the effect of Moringa root concentration on
bacteriological water quality. Significance of all tests were assessed at a p-value of 0.05.
4.3.10 Sample Preparation for Physical Water Quality Parameters
Four different concentrations of Moringa root powder were tested: 0, 250, 450 and 600
mg/L. The three treatments receiving Moringa root powder were prepared by weighing 25 mg, 45
mg, and 60 mg of Moringa root powder separately. The weighed Moringa was placed in 10 mL of
milliQ water, which was heated to 30°C and stirred for 30 minutes at 60 rpm. For the control (0
mg/L), 10 mL of milliQ water was heated and stirred in the same way. Each concentration was
prepared eight times, and divided into four replicates of filtered and unfiltered treatments. The
treatments were added to 90 mL of pond water which had been stored at room temperature, and
stirred for two minutes at 60 rpm. The suspensions were left to sit for one hour. After one hour,
half of the samples were filtered, using Whatman quality 1 filter paper, into sterile 100 mL beakers.
The other half of the samples remained unfiltered, giving four replicates for each treatment. All
physical water quality parameters were measured using Standard Methods (APHA et al., 1992).
4.3.11 pH Measurement
The pH of each of the 32 samples (4 replicates each of unfiltered 0 mg/L, 250 mg/L, 450
mg/L and 600 mg/L treatments, and 4 replicates each of filtered 0 mg/L, 250 mg/L, 450 mg/L and
600 mg/L treatments) was measured by a calibrated Thermo Orion 420 pH meter. In each sample,
the pH probe was inserted without touching the side or bottom of the beaker and pH reading was
taken once the reading stabilized. Readings were taken after one, two and three hours.

48
4.3.12 Electrical Conductivity Measurement
The same 32 samples used in the pH measurements were used to measure electrical
conductivity. After taking the pH reading, a calibrated YSI 3100 conductivity meter was inserted
into the sample without touching the sides or bottom of the beaker. The conductivity reading was
recorded after the display stabilized. Readings were taken after one, two and three hours.
4.3.13 Turbidity Measurement
Due to time limitations, separate samples of treated pond water were prepared on a different
day for turbidity analysis. Samples were prepared in the same way as those used in the pH and
electrical conductivity experiments, using 4 replicates. A calibrated Analite NEP 160 turbidity
meter by McVan instruments was inserted in the sample, and turbidity recorded after the display
stabilized. The probe was set up on a clamp, and inserted at an angle to prevent any bubbles
forming on the probe surface. The probe was set to take the average readings over 8 seconds.
Readings were taken immediately after samples were prepared and after one, two, three, four, 24,
and 48 hours.
4.3.14 Statistical Analysis of Physical Water Quality Parameters
Data was analyzed using R software. A three-way repeated measures ANOVA was
performed for each dependent variable (pH, electrical conductivity, and turbidity). Significance
was determined using α = 0.05. The independent variables tested were time, Moringa
concentration (0, 250, 450 and 600 mg/L), and treatment type (filtered or unfiltered). The
interactions between these variables were analyzed as well. To ascertain where the differences
existed, a post-hoc Tukey test was computed. Boxplots portraying the dependent variables as
factors of three independent variables were created in R. Mean values of the four replicates were
calculated for each Moringa root powder concentration for the trials that were filtered and left to

49
sit for one hour, in order to compare these values to the WHO (2017) drinking water quality
guidelines. The filtered trials after one hour were chosen as this was the same method and treatment
time used to study the impact of Moringa root concentration on bacteria in contaminated water.
4.4 Results
4.4.1 Streak Plates
Bacteria colonies were confirmed to be E. coli and total coliforms based on their initial
colour on the M-coli blue agar (red for coliforms and blue for E. coli) as well as their impact on
the MUG plate (the E. coli fluoresced under a UV light on the MUG agar, but the total coliforms
did not).
The E. coli colonies were white, circular, raised, smooth, and glistening (Figure 4). They
had an opaque centre and translucent margins. The total coliform colonies were smaller, white,
round, opaque, and smooth (Figure 5).

Figure 4: Streak plate of E. coli colonies.

Figure 5: Streak plate of total coliform colonies.

4.4.2 Growth Curve Results
Figure 6 shows the growth curve of total coliforms. Total coliforms growing in LB broth
at 37°C moved from the exponential phase of growth to the stationary phase between 260 and 280

50
minutes. All trials resulted in low standard deviations. The growth curve of E. coli is shown in
Figure 7. E. coli moved into its stationary phase between 280 and 300 minutes. The standard
deviations for E. coli were higher than the standard deviations for total coliforms. The growth
curves were measured and plotted to determine the point at which the bacteria move into their
stationary phase.

2.5

OD600 ( )

2
1.5
1
0.5
0
-0.5

0

50

100

150

200

250

300

350

400

Time (minutes)

Figure 6: Mean total coliform growth curve with error bars indicating standard deviation
from three replicates.

51
2.5

OD600 ()

2
1.5
1
0.5
0
-0.5

0

50

100

150

200

250

300

350

400

Time (minutes)

Figure 7: Mean E. coli growth curve with error bars indicating standard deviation from three
replicates.
4.4.3 Effectiveness of Moringa root treatment at removing E. coli
The colour change and fluorescence of the quanti-trays was counted to determine the MPN/ 100
mL of E. coli and total coliforms. Examples of these changes can be observed in Figures 8 and 9.

Figure 8: Colour change observed in quanti-tray wells which contain total coliforms.

52

Figure 9: Fluorescence observed in quanti-tray wells which contain E. coli.
The effect of Moringa root powder concentration on E. coli in contaminated water was
tested using two different initial E. coli concentrations (Figure 10 and Figure 11). The higher initial
E. coli concentration (50 MPN/100 mL) had a significant (F(1, 6) = 74.27, p = 0.0001) overall
reduction in E. coli from 0 mg/L to 600 mg/L of Moringa concentration of 87%. Significant (F(1,
6) = 49.73, p = 0.0004 and F(1, 6) = 66.3, p = 0.0002) reductions, respectively, were observed
with Moringa treatment from 0 mg/L to 250mg/L and from 0 mg/L to 450 mg/L for 50 MPN/100
mL. A bacteria reduction of 77% occurred from 0 mg/L to 250 mg/L. In the lower bacteria
concentration (37 MPN/100 mL), maximum and significant (F(1, 6) = 11.14, p = 0.0157) reduction
of 74% in E. coli was observed at 450 mg/L concentration.
In both bacteria concentrations, increased concentration of Moringa root powder in the
water reduced E. coli, although at a lower rate at higher concentrations of 450 mg/L and 600 mg/L
(Figure 10). For the initial E. coli population of 37, the decrease that occurred between 250 mg/ L
and 450 mg/L of Moringa was significant (F(1, 6) = 5.99, p = 0.05). For the initial E. coli
population of 50, the decrease that occurred between these Moringa populations was not
significant (F(1, 6) = 2.70, p = 0.15). There were no significant differences between the 450 mg/L
and 600 mg/L in reduction of E. coli colonies in both 37 MPN/100 mL (F(1, 6) = 0.16, p = 0.70)

53
and 50 MPN/100 mL (F(1, 6) = 0.036, p = 0.86) bacteria concentrations. Although there was a
slight increase in E. coli population in 37 MPN/100 mL when the concentration of Moringa
increased from 450 mg/L to 600 mg/L, this increase was not significant.
70
Number of E.coli colonies
(MPN/100mL)

60
50
40
30
20
10

0

250
450
600
Moringa concentration (mg/L)
Figure 10: Mean effect of changing Moringa concentration on E. coli with initial bacteria
population of 50 MPN/100 mL from four replicates showing standard deviation.
Number of E. coli colonies (MPN/100
mL)

0

60
50
40
30
20
10
0

0

250
450
Moringa concentration (mg/L)

600

Figure 11: Mean effect of changing Moringa concentration on E. coli with initial bacteria
population of 37 MPN/100 mL from four replicates showing standard deviation.

54
4.4.4 Total Coliforms
All wells exhibited colour change, indicating the presence of total coliforms. The total
coliforms were too numerous to count and a percent reduction could not be calculated.
4.4.5 pH
The concentration of Moringa root powder had a significant impact on the pH of treated
water (F(1, 84) = 10.84, p = 1.46 x 10-3) (Figure 12). An increase in Moringa concentration
correlated with a decrease in pH (7.45 – 7.61 for filtered samples, and 7.38 – 7.61 for unfiltered
samples). Post hoc comparisons using the Tukey HSD test indicated that the pH of the 250 mg/L
Moringa concentration was significantly different than the 600 mg/L concentration. Time had a
significant impact on the pH of the water (F(1, 84) = 12.92, p = 5.48 x 10-4). The pH of the water
increased over time, and this influence was seen in the control as well. A post hoc Tukey test
showed that each hour resulted in a significant change in pH. The treatment type, whether the
Moringa powder was filtered out or not, did not have a significant impact on the pH of the water
(F(1, 84) = 0.59, p = 0.45). The pH was higher in the samples that were filtered than in unfiltered
samples. The interactions between these variables were analyzed as well. The interaction between
concentration and treatment type was significant (F(1, 84) = 14.75 p = 2.38 x 10-4). No other
interactions had a significant impact on pH. Figure 12 shows the influence of Moringa
concentration, treatment type, and time on the pH of the water.

55

Filtered

Unfiltered

7.6
Moringa
Concentration (mg/L)
0
7.5
pH

250
450
600

7.4

7.3
1

2

3

1

2

3

Time (hours)
Figure 12: Boxplot representation of the mean effect of three variables, Moringa root
concentration, filtering, and time elapsed on pH of contaminated water showing outliers. pH
significantly decreased with increasing Moringa concentration and significantly increased over
time.
4.4.6 Electrical Conductivity
The electrical conductivity (EC) ranged from 209 µS/cm (250 mg/L, unfiltered after 1
hour) to 244 µS/cm (450 mg/L, filtered after 1 hour). The concentration of Moringa root powder
did have a significant impact on the EC of the water (F(1, 84) = 18.89, p = 3.86 x 10-5). As Moringa
root powder concentration increased, EC increased (Figure 13). Significant increases in EC
occurred from 0 mg/L to 450mg/L, 0 mg/L to 600 mg/L and 250 mg/L to 600 mg/L of Moringa.
The treatment type (filtered or unfiltered) did not have a significant impact on EC (F(1, 84) = 1.71,

56
p = 0.19), however the effect of the interaction between concentration and treatment type was
significant (F(1, 84) = 5.05, p = 0.027), with filtered treatments having a higher conductivity than
unfiltered treatments for each concentration. Electrical conductivity was also significantly
impacted by an interaction between the concentration of Moringa root and time (F(1, 84) = 9.41,
p = 2.91 x 10-3). Time had a significant impact (F(1, 84) = 4.50, p = 0.037) on the EC of the water
as well, with EC decreasing over time (Figure 13).
Filtered

Unfiltered

Electrical Conductivity (µS/cm)

240
Moringa
Concentration (mg/L)

230

0
250

220

450
600

210

200

1

2

3

1

2

3

Time (hours)
Figure 13: Boxplot representation of the mean effect of three variables, Moringa root
concentration, filtering, and time elapsed on electrical conductivity of contaminated water showing
outliers. Electrical conductivity increased significantly with increasing Moringa concentration.

57
4.4.7 Turbidity
Changing Moringa concentration had a significant impact on water turbidity (F(1, 160) =
4.69, p = 0.032), with turbidity increasing as Moringa concentration increased.. Treatment type
did not have a significant impact (F(1, 160) = 2.15, p = 0.14). Time had a significant impact on
the turbidity (F(1, 160) = 5.21, p = 0.024). The longer the treated water sat, the less turbid it
became. This trend is most prominent in the unfiltered trials. The change in turbidity between the
initial start time and all subsequent times was significant, as shown in a post-hoc Tukey HSD test.
There was also a significant difference in turbidty from 2 hours to 24 hours, and 2 hours to 48
hours. There was not a significant difference between the other hours. The interactions between
all variables were analyzed as well, although none of these interactions had a significant impact
on turbidity. These trends are displayed in Figure 14.

58

Unfiltered

Filtered

Turbidity (NTU)

50

40

Moringa
Concentration (mg/L)
0

30

250
450
600

20

10

0

1

2

3

4

24

48

0

1

2

3

4

24

48

Time (hours)
Figure 14: Boxplot representation of the mean effect of three variables, Moringa root
concentration, filtering, and time elapsed on turbidity of contaminated water showing outliers.
Turbidity significantly decreased over time and significantly increased with increasing Moringa
root concentration. Filtering did not have a significant impact on turbidity.
4.5 Discussion
4.5.1 Moringa Root Powder Effect on E. coli and Total Coliforms
The growth curves developed for E. coli and total coliforms followed the trends expected
from literature (Sondi and Salopek-Sondi, 2004). Sondi and Salopek-Sondi (2004) grew E. coli in
LB broth, to determine the impact of silver nanoparticles on E. coli growth. The curve they created
from their data for the control in their experiment (no silver nanoparticles added), is similar to the

59
E. coli growth curve in this paper. This shows that E. coli obtained from the pond water is behaving
as expected, with a duplication time of ~20 minutes, and exiting the exponential phase at around
300 minutes (Sondi and Salopek-Sondi, 2004). The larger standard deviation in the growth curve
for E. coli could be due to the low number of replicates. Although measures were taken to
standardize sampling, if one replicate received a larger initial population of E. coli, it could result
in a quicker increase in optical density. This change would be most prominent during the
exponential phase, which is seen in Figure 7. This information aided subsequent analysis as
bacteria were used when they entered the stationary phase.
Moringa root powder was shown to have a significant impact on E. coli in contaminated
water. A higher concentration of Moringa root powder resulted in lower E. coli populations.
Moringa root powder had a maximum efficiency of 87% reduction in E. coli. This reduction is
comparable to the literature values for the percent reduction in bacteria caused by Moringa seeds
(Hendrawati et al., 2016; Amagloh and Benang, 2009). The Moringa root powder was more
efficient at killing E. coli with a higher initial E. coli population, although both follow similar
trends. Both populations dropped to similar numbers (6.95 MPN/100 mL and 9.83 MPN/100 mL)
when treated with 450 mg/L of Moringa. Subsequent changes in E. coli population were not
significant, which could indicate that Moringa root powder is less effective at killing E. coli when
only a small amount of the bacteria is present (< 10 MPN/100 mL). The Moringa root powder may
have been more efficient with the higher initial bacteria population as there was more bacteria
present for the Moringa root proteins to come into contact with, destabilizing the E. coli. When
the populations fell below 10 MPN/100 mL the effectiveness decreased, possibly because Moringa
root powder was less likely to come into contact with the E. coli at these lower populations. More
research is needed to determine exactly why Moringa root powder was more efficient with the

60
higher initial bacteria population. Studies could also be done to determine whether there is a
significant difference in the percent reduction for the different initial bacteria populations. As
shown in Figure 10 and Figure 11, the largest reduction in E. coli for both initial populations
occurred when the concentration of Moringa root powder was increased from 0 mg/L to 250 mg/L,
the rate of decrease in E. coli population then slowed down as Moringa root powder concentration
was increased to 450 mg/L and finally 600 mg/L. This could indicate that the concentration of
Moringa root powder that is most efficient at removing E. coli from contaminated water is between
250 mg/L and 450 mg/L. Moringa seeds are reported to reduce E. coli in water by 80%
(Hendrawati et al., 2016).
The mechanism through which Moringa roots are able to kill E. coli in water was not
studied; it is possible that it possesses MOCP found in the seeds, which kill bacteria through
membrane fusion (Shebek et al., 2015). Moringa root powder can kill E. coli in water. As this
method did not result in the complete elimination of E. coli in water, further studies could be
conducted to try and reach the <1 CFU/100 mL required by WHO drinking water guidelines
(WHO, 2017).
Total coliforms followed a similar growth curve trend as E. coli. The growth curve for total
coliforms developed in this study demonstrated the expected lag phase, log phase, and stationary
phase. A specific growth curve for total coliforms in LB broth was not found in literature to
compare the specific times of transition.
4.5.2 pH
Increased concentration of Moringa root powder resulted in a statistically significant
decrease in the pH of the water. Although this decrease was significant, the pH of the contaminated
water had minimal change, and remained inside the WHO (2017) drinking water quality

61
guidelines. The pH did change over time, however all recorded values were between 7.3 and 7.7,
which is within acceptable drinking water guidelines (6.5-8.5) (WHO, 2017). This has positive
implications for considering Moringa root powder as a potential point-of-use water treatment
method. The protein in the root powder may share a similar structure to that in the seeds, allowing
the Moringa roots to release hydroxyl groups into the water by accepting protons from the water
(Amagloh and Benang, 2009). A neutral pH in drinking water is essential to prevent damage to the
digestive tract, and to prevent leaching of heavy metals into water (Health Canada, 2015). All trials
stayed within a neutral pH range. Table 7 compares the WHO (2017) drinking water quality
guidelines for pH to the pH of water treated with the various concentrations of Moringa root
powder, filtered and left to sit for one hour. The filtered trials after one hour were chosen as this is
the same method and treatment time that was used to study the impact of Moringa root
concentration on bacteria in contaminated water. The other factors (time and filtering) also affected
the pH. These parameters impacted the control in the same way, and did not exhibit any covariance
with changing Moringa root concentration.
4.5.3 Electrical Conductivity
The increase in EC with an increase in Moringa root powder concentration indicates that
the increased Moringa concentration is adding more ions to the water than it is removing. The
higher conductivity is not necessarily a concern when it comes to drinking water quality. Other
beverages have a much higher electrical conductivity. The EC of milk from Dairy Gyr cows is
4900 µS/cm (Boas et al., 2016). The mean literature EC values for normal milk range from 4000
to 5860 µS/cm (Boas et al., 2016). Another study gives a mean EC for raw milk of 4680 µS/cm
(Muccheiti et al., 1994), further confirming this average. In addition, orange juice is known to
have electrical conductivity ranging from 7000 µS/cm to 16,000 µS/cm (Palaniappan and Sastry,

62
1991). However, the increased conductivity of water does mean an increase of ions present, and
this could affect the odour and taste of the water. Odour and taste can affect palatability of water,
making it less likely to be consumed, even if it is otherwise safe. The potential danger of high
water conductivity could come from not knowing what ions are in the water. The samples which
received the filtered treatment had higher EC than the unfiltered samples. Based on the solubility
of Moringa roots found in section 3.4.2 of this paper, the ions in water treated with Moringa root
are likely potassium, calcium, magnesium, and sodium, with trace amounts of other elements.
Phosphorus and sulphur were both found soluble in Moringa root water, however they are not
conductors and would not be contributing to the EC of the solution (Barbalace, 2020).
The significant impact of the interaction between treatment type and concentration
indicates that the filter is reducing the ions in the water, and that this reduction is related to the
Moringa concentration. Filtering is part of the process that has been proposed in this study for
treating water with Moringa roots, which is the method that resulted in lower EC.
4.5.4 Turbidity
Changing Moringa root powder concentration had a significant impact on the turbidity of
the contaminated water. Moringa roots increased turbidity of the water. These results are contrary
to what was expected based on the ability of Moringa seed powder to reduce turbidity in water
(Amagloh and Benang, 2009). Seeds of Moringa oleifera contain water soluble Moringa oleifera
lectin, which is a chitin binding protein. This protein was isolated and shown to decrease the
turbidity in water by 47.56% without filtration, and by 64.16% with filtration (Freitas et al., 2016).
As Moringa roots did not reduce turbidity, it is likely that they do not possess the same ability to
form flocculents that are present in the seeds. This could be further studied to determine if roots
do contain any Moringa oleifera lectin.

63
Part of the water treatment process with Moringa roots that is proposed in this paper is to
filter the Moringa roots out of the water. There was a decrease in turbidity for trials that were
filtered compared to the unfiltered trials, however this decrease was not significant. The decrease
observed was due to the filter removing particulate from the water. Turbidity did decrease
significantly with time, which is likely due to particles settling out of the water the longer the water
sat still.
Table 7 compares the turbidity of the Moringa root treated water with the WHO (2017)
drinking water quality guideline. As the turbidity of Moringa root treated water is above the
drinking water quality guidelines, future studies could look into augmenting Moringa root powder
water treatment with a flocculent. If the Moringa root powder were able to decrease turbidity, it
would not only make water more palatable, but it could also make the root powder even more
effective at treating bacteria in contaminated water.
Some trials had a large amount of error (filtered control samples for the initial turbidity and
the turbidity after 2 hours, and filtered samples with 450 mg/L of Moringa at 2 hours). This error
may have been introduced during sampling, as the turbidity probe is sensitive to any bubbles
present.
Table 7: Comparison of physical water quality parameter guidelines to water treated with
Moringa root powder, filtered and left to settle for 1 hour.
Parameter

WHO
Guideline
(2017)

Control

250 mg/L
Moringa

450 mg/L
Moringa

600 mg/L
Moringa

pH

6.5 - 8.5

7.51

7.49

7.48

7.45

Electrical
Conductivity

N/A

228.38

237.25

242.78

241.73

Turbidity (NTU)

5

9.91

10.99

11.39

10.28

64
Chapter 5: Conclusion
This thesis aimed to document the inorganic elemental composition of Moringa oleifera
roots. The concentration of soluble elements in Moringa root water were compared to 2% milk.
This thesis also sought to explore the potential of using Moringa roots as a point-of-use water
treatment by testing its impact on several parameters of water quality. Experiments were designed
to test if Moringa root powder impacted bacteria in water, pH of water, electrical conductivity,
and turbidity of contaminated water.
The most concentrated element found in the roots through ICP-MS was potassium. This
was followed by sodium, magnesium, phosphorus and calcium. When Moringa roots were
dissolved in water, the most abundant elements in the water were potassium, phosphorus, sulphur,
magnesium, sodium, and calcium. Sulphur was found in the water but not in the roots due to a
difference in measuring equipment – the ICP-MS did not measure sulphur.
The impact of Moringa root powder on water quality was measured in two ways – its
impact on the physical parameters of water quality, and its impact on the bacteriological quality of
water. The Moringa root treatment was prepared by drying and powdering Moringa roots. Moringa
root powder was added to milliQ water, and stirred for ten minutes. The Moringa root solution
was applied to contaminated water. After one hour, the water was filtered and tested for E. coli
and total coliforms.
The Moringa root had a maximum efficiency of reducing E. coli by 87% in the
contaminated water. This efficiency was reached with the higher initial E. coli population (50
MPN/100 mL), and the highest Moringa concentration of 600 mg/L. For the lower bacteria
concentration (37 MPN/100 mL), the E. coli was reduced by 74%. Total coliforms were too
numerous to count.

65
Moringa root powder significantly increased the turbidity of contaminated water. Moringa
root powder increased the pH of water. The pH remained in the acceptable parameters for drinking
water quality. Moringa root powder increased the electrical conductivity of contaminated water.
In addition to these results, this study produced robust methods for treating contaminated
water with Moringa root powder, and testing the treated water for coliforms. The development of
methods of treatment application was amended from Amagloh and Benang (2009). Moringa roots
were more fibrous than Moringa seeds used in Amagloh and Benang’s study, and as a result the
step of filtering out the roots was added to the treatment process. Adding Moringa root powder to
contaminated wastewater was found to have highly variable results. Isolating and culturing
bacteria was the most effective way found to give consistent results without introducing
confounding variables. Due to the high dilution factor, bacteria were lysing in the milliQ water
during the treatment time. When preparing concentrations to determine the dilution factor to use
in treatment, the samples did not sit in the diluted water as long as they did during the actual
treatment. This meant that more of the E. coli were lysing during the treatment. This study
addressed this issue by accounting for the time in spent in treatment, and leaving the E. coli to sit
in the diluted water for the same amount of time when calculating the dilution factor. This could
also be addressed by adding salt to the water to prevent the lysing.
In these trials E. coli population did not reach the WHO required standard of <1 CFU/100
mL (WHO, 2017). Moringa root powder could be further developed as a water treatment method
in order to increase its efficiency. Further research of Moringa root powder water treatment could
involve determining the optimal concentration of Moringa roots. Moringa root powder could be
combined with a flocculating agent such as alum to improve its efficiency as well. This study
tested the impact that Moringa root had on E. coli sourced from an open pond. Other bacteria

66
species could be tested, specifically those known to cause diseases carried through water, such as
Vibrio cholera, or Salmonella typhi. Moringa trees used in this study were grown in a lab for seven
months. To better reflect real-world conditions, it would be beneficial to repeat this study with
Moringa that is grown outdoors, and taking into account various ages of Moringa trees. Further
research into the development of Moringa root as a point-of-use water treatment method should
also include a study on the palatability of water treated with Moringa roots, and the likelihood of
such a treatment being practically implemented.
The mechanism through which the Moringa roots are able to kill E. coli in water should
also be studied, to determine whether it is the same process of membrane fusion that is observed
in Moringa seeds.
The chemical elements of Moringa root could also be further studied. One limitation of
this study was that it only looked at six and seven month old trees that were grown in a greenhouse.
Mature tree roots, or roots from trees grown in the wild may have a different chemical composition.
Testing the elements present and water soluble in roots of different ages and growing conditions
would give better understanding of the potential for any harmful chemicals to enter the roots. The
Moringa trees used in this study were grown in a controlled environment, using augmented soil.
When grown in field conditions the mineral content of the soil could vary widely due to
environmental factors (Melesse et al., 2012). Moringa grown in various types of fertilizer contain
different concentrations of nutrients in the leaves, and the roots may be similarly effected (Dania
et al., 2014; Makinde, 2013). All trees in this study were treated with applications of Safer’s Soap
and Enstar. This study did not examine the impact of these treatments on the nutrient content of
the tree roots. This study also only did an elemental analysis. Further analysis could be done to
determine other compounds present in the roots, as well as phytochemicals present in the roots.

67
This study provides an exploratory look at the potential for the use of Moringa oleifera
roots as a point-of-use water treatment method. The roots showed promising results in their ability
to kill E. coli, and so further research should be conducted to optimize this method and determine
its feasibility as a low cost and low-tech point-of-use water treatment for countries lacking access
to safe drinking water.

68
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Appendix A: Moringa tree growth in a greenhouse
The 25 Moringa seedlings were grown for six months before initial harvest. Of the 25
seeds, 23 plants germinated successfully. The seeds were planted in October, and harvested in
April. During these six months the growth of the Moringa plants was monitored and measured.
Measurements were periodically taken of the diameter of the trunk, and the height and width of
the tree. Diameter and crown width were measured for an additional month after the initial harvest.
The diameter was measured at the same point on each tree, about 2 cm up from the base of the
soil, using calipers. To ensure that the same point was measured each time, a mark was made on
the bark of the tree. The height of the trees was measured using a tape measure from the base of
the tree in the soil to the highest branch.
After six months of growth, the height of the trees ranged from 52 cm to 123 cm. The most
rapid growth occurred between December and January, at which point growth slowed down
between January and March. The growth conditions remained the same as this was a greenhouse
environment, so it is unlikely that this impact was due to the time of year. The slowdown in growth
starting in January is likely due to the white fly infestation. White fly was first seen on the plants
at the end of December, and many leaves on the trees were damaged due to these pests. This growth
trajectory is displayed in Figure 15.
The average mass of the peeled root was 37.13 g. The root mass ranged from 21.45 g to
53.87 g. No significant correlation was observed between root mass and tree height, as confirmed
by an ANOVA (p = 0.74). Drying the roots resulted in a mean dry root mass of 6.98 g. The mean
percent moisture of the roots was 80.8%  2%.
The more mature the trees were, the larger the range of tree height, crown width, and tree
diameters were observed. This can be seen with the increasing standard deviation with time in

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Figure 15, Figure 16, and Figure 17. Higher variability was also introduced in the crown width
after the white fly infestation as some trees lost their leaves entirely by March (Figure 16).
120
100

Height (cm)

80
60
40
20
0
0
Oct

1
Nov

2
Dec

3
Jan

4Feb

5
March

6
April

7

Month
Figure 15: Mean height of 23 Moringa trees in their first six months of growth with line of best fit,
showing standard deviation.

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Mean Moringa tree crown width (cm)

45
40
35
30

25
20
15
10
5
0

0
Oct

1
Nov

2
Dec

3
Jan

4
5
6
7
8
Feb
March
April
May
Month
Figure 16: Mean crown width of 23 Moringa trees in their first seven months of growth, showing
standard deviation.

0
Oct

1
Nov

2
Dec

3
Jan

18
Mean tree diameter (mm)

16
14
12

10
8
6
4
2
0

4
Feb
Month

5
March

6
April

7
May

Figure 17: Mean trunk diameter of 23 Moringa trees in their first seven months of growth,
showing standard deviation.

8

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Appendix B: Development of Bacteria Analysis Methods
B.1 Trial Method 1
As the Moringa root water treatment was exploratory research, the methods of determining
the effect on bacteria had to be developed. A uniform and predictable initial bacteria concentration
was required. Many trials and processes were performed in order to perfect this method.
Initially, the contaminated water used was obtained from Prince George's City Landsdowne
Wastewater Treatment Centre effluent. Serial dilutions were conducted to determine concentration
of bacteria (E. coli, total coliforms and fecal coliforms), in the water. Dilutions tested were 1:1000,
1:10,000 and 1:100,000. Based on those results, a dilution factor was determined, and the effluent
was diluted accordingly (1:2000 for fecal coliforms and 1:50,000 for E. coli and total coliforms).
The diluted water was treated with both fresh and dry Moringa root. Both treatments of Moringa
were tested in six concentrations (0, 25, 50, 100, 150 and 200 mg/L). Each concentration had 20
replicates. These concentrations were prepared by dissolving a 12.5, 25, 50, 75 and 100 mg of
Moringa roots in 30 mL of water and pipetting 3 mL of each concentration into the contaminated
water. The treatment sat for one hour.
After one hour, the samples were processed using the membrane filtration method. In this
method, 2 mL of M-Coli Blue was added to a petri dish to test for E. coli and total coliforms, and
2 mL of m-FC with Rosolic Acid was added to separate petri dishes to test for fecal coliforms. A
45 µm membrane filter was placed on a manifold with a funnel and 100 mL of the sample was run
through the membrane filter. The membrane was placed in the petri dish and placed in an incubator.
E. coli samples were incubated at 37˚C and fecal coliforms were incubated at 44.5˚C. After 24
hours, the number of colonies on the samples were counted.

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The results of this test varied widely, without any discernable trends. We observed that
pieces of Moringa in the samples seemed to interfere with the media, construing the results. In
addition, the effluent from the Wastewater Treatment Centre was heavily contaminated with nonbacterial contamination, including high levels of nutrients, which may have had confounding
results. This test was inconclusive.
B.2 Trial Method 2
The method was amended in two ways. The first was to use water from a pond. This was
done to reduce the high level of other contaminates in the water, and to closer represent the levels
of contamination that would be in an open water source. Open water sources are commonly used
to draw drinking water in many of the countries where Moringa grows naturally. The second
amendment to the method was to change the way the treatment itself was applied.
Water was sampled from five sites on a farm north of Prince George, BC in July 2016 to
determine bacteriological quality. Samples from each of the five sites were brought to the Northern
Analytical Laboratory Service (NALS), located at UNBC, for bacteria analysis using membrane
filtration. Based on these samples, we determined that the site with the highest number of E. coli
and fecal coliforms was the farm pond located at 54° 4' 23'' N, 122° 45' 19'' W. A creek on the
west side of the pond fed the pond through a culvert. The pond is frequented by many animals,
either directly at, or upstream of, the sample site. Animals using the pond include horses, cows,
beavers and chickens. This pond was chosen due to the natural presence of E. coli and other fecal
bacteria in the water. This contamination was necessary to determine the impact that Moringa
roots had on bacteriological water quality. This pond contamination represented the real world
water quality conditions in open water sources.

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The environmental parameters of the pond water (temperature, pH and electrical
conductivity) were measured in order to understand the baseline characteristics of the water used
for testing. Environmental data was collected on the same day that water samples were obtained.
These parameters were measured at four points along the shore of the pond. Measurements were
taken approximately 7 m apart. Temperature and pH were both measured with a pH/ temperature
probe (VWR Scientific Products Model 2180 pH/Temperature/mV meter). The mean temperature
of the pond was 0.45°C and the mean pH was 5.75. The electrical conductivity was measured using
a calibrated Thermo Scientific field conductivity meter. The mean electrical conductivity of the
pond was 130µS/cm.
To ensure Moringa root powder in treatment itself was uniform, the method of preparing
and applying the treatment was adjusted. First, the Moringa powder was put through a 2mm sieve.
15, 25, 35, and 45 mg of Moringa were weighed. This time they were not pipetted into the
contaminated water. Instead, the Moringa treatment was weighed and added to a 10 mL of milliQ
water. The entirety of this Moringa concentration was added to contaminated water. This
eliminated any inconsistency in the treatment, and ensured that a known weight of Moringa was
in the treated water. The concentrations of Moringa were also adjusted to create fewer samples
with a larger spread (0, 150, 250, 350 and 450 mg/L). Only one concentration of fresh Moringa
was prepared (150 mg/L) to determine whether there was a significant difference between the fresh
and dry Moringa treatments.
To eliminate any effect that the Moringa particles were having on the of M-Coli Blue and
m-FC with Rosolic Acid media, the treated water was filtered through Whatman 150 mm, quality
1 filter paper. The filtering occurred after the treatment had sat for one hour. Samples were then
processed in the same way, using the membrane filtration method.

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The preliminary data (three replicates) for this new method showed that there was a trend
between Moringa concentration and bacteria population. Increased Moringa concentration
resulted in a decreased bacteria population. The data for total coliforms showed a significant (p =
0.00014) decrease of 48.5% of total coliforms between the control which received no treatment,
and the samples which received 450mg/L of Moringa root powder. E. coli had a significant (p =
0.00011) decrease of 53.6% between the control and water treated with 450 mg/L of Moringa root
powder. The fecal coliforms decreased by 39.2%.
For the trials with twenty replicates using this new method, four Moringa concentrations
were chosen to be tested. These concentrations were 250 mg/L, 450 mg/L, 600mg/L, and 0mg/L
as a control. These data were inconclusive. A downward trend in the amount of bacteria was
observed, however it was less prominent than in the preliminary trial. The initial bacteria
concentrations for E. coli and fecal coliforms were lower than they had been during the pre-trials.
During the pretrials, there were 28 CFUs for fecal coliforms, and 31 CFUs of E. coli in the initial
populations. For statistical analysis of bacterial enumeration, a population of at least 25 CFU/100
mL is required, and the population should not be above 250 CFU/mL (Tomasiewicz et al., 1980).
The initial populations in these cases were 14, and no statistically significant trend could be
determined. Conversely, the initial total coliform population was 350, which required counting in
quadrants. These trials were also inconclusive regarding the effect of Moringa concentration on
bacteria. However, the data showed that there was no significant difference in treating the water
with dry roots or fresh roots. Due to this fact, the use of fresh treatments ceased in subsequent
trials. Variability may have been introduced due to the time it took to process 20 samples for each
treatment. The processing time may have been introducing another confounding factor. A
reduction in the number of replicates aimed at reducing this variability.

89
B.3 Trial Method 3
The methods of bacteria testing were further amended to reduce the variability in data. In
order to combat the high variability of bacteria in the pond water, five litres of water was collected.
The same day it was collected, the water was tested for bacteria populations. The pond water was
kept at 4°C overnight while the population samples were incubating. The next day, the samples
were analyzed to determine the appropriate dilution factor. The refrigerated pond water was then
used for the trials. The purpose of using water for the treatments that was collected the same day
as the population analysis was to eliminate the effect of the natural daily variability of bacteria
concentration in the pond. Two different bacteria concentrations were made to see how the
Moringa responded with different initial bacteria concentrations. With both a high (~100) and low
(~30) initial bacteria population, the impact of Moringa root on bacteria could be studied more
robustly. The Moringa treatment application remained the same. Once again, the data was
inconclusive. All total coliform samples were too numerous to count. The downward trend that
had been observed in previous trials of E. coli was no longer prominent, and variation in results
was high.
B.4 Trial Method 4
The natural variability of bacteria in the pond water was a major reason for these
confounding results. At this point, the method was adjusted to culture E. coli and total coliforms.
These bacteria were collected from the same pond, and cultured in LB broth. Through serial
dilutions, a known bacteria concentration was prepared. The method of bacteria enumeration was
also changed. The membrane filtration was limiting because bacteria population had to fall
between 30-200 CFU/100 mL. The new method chosen was the colilert quanti-pack method, which
allowed a larger range of bacteria populations to be tested. The colilert method has no statistical

90
difference from standard methods of E. coli enumerations (Macy et al., 2005), but it does reduce
human errors present in counting colonies. This method was done by adding the colilert powdered
reagent to 100 mL of water sample. The sample was mixed thoroughly, then poured into a plastic
package containing 97 wells of two different sizes. After 24 hours of incubation at 37ºC, the
coliforms were counted. Wells containing coliforms exhibited a change in colour from clear to
yellow. Wells containing E. coli fluoresced under UV light. The number of wells containing each
bacteria were counted and compared to a statistically tested chart to determine the most probable
number (MPN) of colony forming units/ 100 mL of water. The colilert system only tests for E. coli
and total coliforms, so fecal coliforms were no longer a testable parameter.
Pretrials for this new method tested 450 mg/L of Moringa and 0 mg/L of Moringa at two
different initial bacteria populations (~100 and ~30). Both bacteria populations had a significant
decrease in both E. coli and total coliforms when Moringa was added. The trials with an initial
concentration of 120.9 MPN/100mL of E. coli decreased to 9.1 MPN/ 100mL after being treated
with 450mg/L of Moringa root powder. This was a significant decrease (p = 0.0026). With an
initial E. coli population of 21.8 MPN/100 mL, there was also a significant (p = 0.00096) decrease
in E. coli down to 3.5 MPN/100 mL. The total coliforms with an initial concentration of 120.9
MPN/100mL also had a significant (p = 0.0027) decrease when treated with Moringa root powder
down to 9.5 MPN/100 mL. The initial total coliforms of 28.2 MPN/100 mL decreased to 8.0
MPN/100 mL, however this decrease was not significant (p = 0.096). This variability was
attributed to one low initial value of 9 MPN/100 mL, which had a large impact on the standard
deviation as there were only three replicates. The cultured bacteria were used for to test a full set
of replicates for all of the treatments. In the subsequent trials initial bacteria populations were

91
extremely low (~4CFU/100mL), and could not be analyzed for statistical significance as they were
outside of the testable range of 25-250 CFU/100 mL (Tomasiewicz et al., 1980).
The cause of this significant dieback in bacteria population was attributed to one of two
possibilities. First, between the pretrials and the last trial, the inoculated LB broth had been in the
fridge for an additional 24 hours. During this time the bacteria may have either died or had their
metabolic rate slow down. Another potential cause of dieback was the length of time that the
bacteria sat in the diluted water during the full test compared to the pretrials. With less replicates
and treatments in the pretrials, bacteria were diluted with milliQ water about one hour before they
were placed in the quanti pack. During the trials with full set of treatment types and replicates, the
total time from when the bacteria was diluted to when it was placed in the quanti packs was closer
to three hours. As the bacteria was diluted with milliQ water, they could be lysing during the three
hours. Two possible solutions to this problem were to either dilute the bacteria with salt water, or
calculate the dilution factor based on a three-hour time period before analysis. In this study, the
dilution factor was calculated by taking into account the three hour wait for main trials. In addition,
the final main trials were conducted without refrigerating the inoculated broth for the additional
24 hours.