ELEMENTAL COMPOSITION AND ANTIBACTERIAL EFFICACY OF MORINGA
OLEIFERA AND ZINGIBER OFFICINALE ROOT POWDERS AGAINST E. COLI

by

Ganeemat Brar
B.Sc., Punjabi University, Patiala, India, 2018

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

UNIVERSITY OF NORTHERN BRITISH COLUMBIA
March 2022

© Ganeemat Brar, 2022

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Abstract
Shortage of clean drinking water is a serious problem faced by the world at present
times, with water borne diseases claiming 5 million deaths globally each year. Many methods
are available to treat contaminated drinking water; however, cultural, economic, and social
factors often impair implementation of these methods, particularly in developing countries.
Moringa and ginger root powders appear to offer a promising alternative to treat contaminated
drinking water. This study examines the nutrient composition of Moringa and ginger roots and
determine their antibacterial efficacy against Escherichia coli (E. coli). After growing Moringa
and ginger plants in a greenhouse for seven months and three years respectively, their roots
were harvested, dried, powdered and analysed for their chemical composition using ICP-MS
and Elemental analysis. The most abundant metal found in both the root powders was
potassium, while calcium, magnesium, sulphur, sodium, and phosphorus were other elements
present in high amounts. However, the concentration of these metals in Moringa and ginger
root powders varied significantly. The abundance of essential elements in the two root powders
justifies their use as a point-of-use water treatment method. For both Moringa and ginger root
powders, 1400 mg/L concentration was determined to be the most effective concentration as it
reduced E. coli in contaminated water by 88.66% and 62.63% respectively. The combination
of the two root powders appeared to have a synergistic effect on E. coli as Moringa and ginger
root powders combined in 1:1 ratio reduced bacterial counts by 94.78% when added to the
contaminated water at 2000 mg/L.

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Table of contents
Abstract…………………………….…………………………………………...……………. ii
Table of contents……………….…………………………………….…………………...…. iii
List of Tables…………………………………….…………………………………………. vii
List of Figures………………………….………………….………….…………….……… viii
Acknowledgements…….………………….……….………….………………….…………. ix
Chapter 1: Introduction …………….…………………………………...………………….…1
Chapter 2: Literature review…………….……………………………….……………………5
2.1 Moringa oleifera.……………….…………………….……………………………….5
2.1.1 Botanical description………………….………….……...……………….…….5
2.1.2 Growing conditions…………………………………………………………….6
2.1.3 Pharmacological potential……………………….…………………….……….6
2.1.4 Food and nutrition….………………………………….……………………….7
2.1.5 Other commercial uses……………………………………...….……….…...…8
2.1.6 Antimicrobial research…………………….………….…………...………...…8
2.1.7 Research in water purification……………………………………...................10
2.2 Zingiber officinale …...…….……………………….…………………………….…11
2.2.1 Botanical description………….…………………...………………………….11
2.2.2 Growing conditions…………………………………………………………...11
2.2.3 Food and nutrition………………………………………………….…………12
2.2.4 Pharmacological effects………….……………….…………………….…….13
2.2.5 Antimicrobial research…………………….…………………………….……14
Chapter 3: Growing Moringa oleifera and Zingiber officinale in a greenhouse and extraction
of their root powders ……………………….………………………………….…………….16
3.1 Abstract……………………………………………………………………………...16
3.2 Moringa oleifera……………………………………………………….……………17
3.2.1 Methods……………………………………………………….……………....17

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3.2.1.1 Planting and growing Moringa in the greenhouse………………………….17
3.2.1.2 Measuring growth of Moringa plants……….………………………………19
3.2.1.3 Harvesting and processing Moringa roots……………………………….…20
3.2.1.4 Extraction of dried Moringa root powder………….……………………….21
3.2.2 Results…………………………………………….…….…………………….22
3.2.2.1 Planting and growing Moringa in the greenhouse……………………….…22
3.2.2.2 Measuring growth of Moringa plants……………………………….………22
3.2.3 Discussion…………………………………………………………………….25
3.3 Zingiber officinale …………….……………………...………………….………….26
3.3.1 Methods……………………………………………………….……………....26
3.3.1.1 Planting and growing ginger in the greenhouse…………………………….26
3.3.1.2 Measuring growth of ginger plants….…….………….…………….………27
3.3.1.3 Harvesting and extraction of ginger powder……….………….……...…….27
3.3.2 Results……………………….………………….…………….………………28
3.3.3 Discussion……………………………………….……………………………29
3.4 Conclusion……….……………………….………………………………………….29
Chapter 4: Chemical analysis of Moringa and ginger root powders……………….………...30
4.1 Abstract……………….………………………………….………………………….30
4.2 Introduction…………….………………….………………….……………….…….31
4.3 Methods……………………………….………...…………….……….………….…34
4.3.1 Determining particle size of Moringa and ginger root powders…………...…34
4.3.2 Determining metal concentrations in ginger and Moringa root powders….....34
4.3.3 Determining total carbon and nitrogen present in Moringa and ginger root
powders…………………………………….………….………………….………….35
4.3.4 Determining concentrations of anions in Moringa and ginger root powders…35
4.3.5 Determining electrical conductivity and pH of the root powders………….…36
4.3.6 Statistical analysis…………………………………………………………….36

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4.4 Results…………….………………………………………………………………....37
4.4.1 Particle size of Moringa and ginger root powders……………………………37
4.4.2 Concentrations of metals present in Moringa and ginger root powders.…......38
4.4.3 Total carbon and nitrogen present in Moringa and ginger root powders….….40
4.4.4 Concentrations of anions present in Moringa and ginger root powders……...40
4.4.5 Electrical conductivity and pH of Moringa and ginger root powders………...42
4.5 Discussion…………………………………………………………………………...42
4.5.1 Particle size of Moringa and ginger root powders….…….……….………….42
4.5.2 Concentrations of metals found in Moringa and ginger root powders….….…42
4.5.3 Total carbon and nitrogen present in Moringa and ginger root powders...…...47
4.5.4 Concentrations of anions present in Moringa and ginger root powders….…..49
4.5.5 Electrical conductivity and pH of Moringa and ginger root powders…….…..53
4.6 Conclusion………………….………….….…………………….…………………...55
Chapter 5: Effect of Moringa and ginger root powders on E. coli population………….……57
5.1 Abstract…………………………………………….…………………….………….57
5.2 Introduction………………………………………………………………………….58
5.3 Methods………………………………….………………….……………………….63
5.3.1 Propagating E. coli strain……………………………….…………………….63
5.3.2 Preparing streak plates of E. coli…………………………………...…………64
5.3.3 Preparing stock potassium dihydrogen phosphate buffer solution……………64
5.3.4 Serial dilution analysis and enumeration of E. coli………………….….…….65
5.3.5 Evaluating antibacterial effect of Moringa and ginger root powders………...67
5.3.6 Statistical analysis…………………………………………………………….69
5.4 Results……………………………………………………………………………….69
5.4.1 Propagating E. coli strain…………………………….……………….………69
5.4.2 Serial dilution analysis……………………….……………………….………70
5.4.3 Antibacterial effect of Moringa and ginger root powders.………………...….71

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5.4.3.1 Moringa root powder treatments……………………………………………71
5.4.3.2 Ginger root powder treatments……………….…………….….……………73
5.4.3.3 Moringa and ginger root powder treatments…………………………….….75
5.5 Discussion………….………………………….………………………….…………78
Chapter 6: Conclusion and Recommendations………….….…………………….………….83
6.1 Conclusion………………………….…………….………….………………………83
6.2 Recommendations………………….…………………………………….………….84
Literature Cited………………………………………….…….……………….….…………86
Appendix A: Extracting E. coli used in bacteriological analysis (Trail method) …...….…..102
Appendix B: Documentation of the research through photos………………………...…….106

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List of Tables
Table 1: Height to diameter ratio (and standard deviation) of Moringa plants planted on two
separate dates— February 27, 2019, and March 20, 2019, over the summer months…….….24
Table 2: Determination of significant differences in height and diameter growth in Moringa
plants over the summer months…………….………...……………………………………….24
Table 3: The average height (in cm), stem diameter (in mm), and height to diameter ratio (H:D
ratio) of the ten ginger plants……………...……….………………………………………….28
Table 4: Mean percentile readings (and standard deviations) of particle size of Moringa and
ginger root powders…………….……………………………………………….……………37
Table 5: Mean distribution (and standard deviation) of Moringa and ginger root powders into
different size classes………………………………………………………………………….37
Table 6: Mean concentrations (and standard deviations) of various metals found in Moringa
and ginger root powders.……….………………………………………………….………….39
Table 7: Means (and standard deviations) of total carbon and nitrogen detected in Moringa and
ginger root powders……………………………….…………………………….……………40
Table 8: Mean concentrations (and standard deviations) of selected anions found in Moringa
and ginger root powders.………………………………………….………………….……….41
Table 9: Means (and standard deviations) of electrical conductivity and pH of the Moringa and
ginger root powders……………………….……………….…………………………………42
Table 10: One-way analysis of variance for different Moringa root powder treatments— 800,
1400 and 2000 mg/L, on mean E. coli counts. A significant difference is considered at p-value
<0.05……………………………………………………………………………………….…72
Table 11: A Tukey’s test showing differences in means, confidence levels (upper and lower
limit) and adjusted p-values for all possible pairs of different concentrations of Moringa root
powder at 95% family-wise confidence level…………….……………….………………….72
Table 12: One-way analysis of variance for different ginger root powder treatments— 800,
1400 and 2000 mg/L, on mean E. coli counts. A significant difference is considered at p-value
<0.05………………………………………………………………………………………….74
Table 13: A Tukey’s test showing differences in means, confidence levels (upper and lower
limit) and adjusted p-values for all possible pairs of different concentrations of ginger root
powder at 95% family-wise confidence level…………………………………………………74
Table 14: One-way analysis of variance for different Moringa and ginger root powder
treatments— 800, 1400 and 2000 mg/L, on mean E. coli counts. A significant difference is
considered at p-value <0.05………………………………….……………………………….76
Table 15: A Tukey’s test showing differences in means, confidence levels (upper and lower
limit) and adjusted p-values for all possible pairs of different concentrations of Moringa and
ginger root powder at 95% family-wise confidence level…………………………………….76

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List of Figures
Figure 1: Indian Moringa oleifera seeds that were grown in UNBC greenhouse (Left). A pot
with three germinating Moringa seedlings (Right)………………….……….……………….18
Figure 2: The Moringa plants on the day of their harvesting (Left). A harvested Moringa root
(Right)……………………………………………………………………….……………….21
Figure 3: Growth in height (m) of Moringa over summer months in 2019, showing standard
deviation as error bars………………………….……………………….……….……………23
Figure 4: Diameter growth (mm) in Moringa over summer months in 2019, showing standard
deviation as error bars……………………………………….……….………………….……23
Figure 5: Grinding of harvested and freeze-dried roots of a ginger plant using Black+Decker
coffee and spice grinder………………….…………………………….………………….….28
Figure 6: A thawed ATCC® mini-cryovial containing 8739TM E. coli……………………….63
Figure 7: Colilert-18 reagent being added to the sample bottles (Left). After mixing Colilert18 reagent in sample bottles, a sample being poured into Quanti-Tray/2000 (Right)….….….66
Figure 8: A Quanti-Tray being sealed using IDEXX Quanti-Tray™ Sealer.…………………67
Figure 9: A streak MUG agar plate with E. coli colonies….…………………………………70
Figure 10: Comparison of sterile LB broth with inoculated LB broth………………………...70
Figure 11: Mean E. coli counts found in different Moringa root powder treatments, with
standard deviation as error bars………………………….……….……………….………….71
Figure 12: Mean E. coli counts found in different ginger root powder treatments, with standard
deviation as error bars…….………………….………………………….…….……………...73
Figure 13: Mean E. coli counts detected in treatments with Moringa and ginger root powder
concentrations, with standard deviation as error bars…….………………….…….…………75
Figure 14: Mean percentage decrease in E. coli numbers observed in different concentrations
of Moringa root powder alone, ginger root powder alone and the combination of the two root
powders, with standard deviation as error bars…………………………………...…………...77
Figure 15: The opening (mouth) of pipe from where water sample was collected, and
environmental parameters of the pond water were measured….……….………………...….103
Figure 16: An E. coli colony (blue in colour) and numerous coliform colonies (red in colour)
observed on a membrane filter with m-ColiBlue media……………………………….….…104
Figure 17: White, cloudy, and circular E. coli colonies observed on MUG agar plates…….104

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Acknowledgements
First and foremost, I am indebted to my supervisor, Dr. Christopher Opio, for his
guidance and supervision throughout the course of this research. I am extremely grateful for
every critical feedback that I received from him, as it has immensely helped me to become a
better researcher. I am also thankful to him for having patience in me and believing in me to
complete this work. Without his moral and academic guidance, this work would not have been
possible. I would like to extend my thanks and appreciation to my committee members, Dr.
Saphida Migabo and Dr. Natalie Linklater, for their encouraging comments and feedback, and
for letting me borrow countless lab equipment from their respective labs all through my
research.
I would like to thank Prof. Amarjeet Bhullar for encouraging me to undertake my
master’s degree at UNBC and for his continual encouragement. I would like to acknowledge
funding support from University of Northern British Columbia through Graduate Project
Award, which made it possible for me to apply myself completely to my project. I would also
like to sincerely thank John Orlowsky and Doug Thompson for their help in the greenhouse;
Lon Kerr for graciously providing an E. coli strain; and Che Elkin for his assistance in statistical
analysis. Data was collected in the Northern Analytical Laboratory Services (NALS) lab at
UNBC with the help of Hossein Kazemian, Erwin Rehl and Charles Bradshaw.
I would also like to thank my family and friends for their moral support which helped
me immensely during this journey. A special thanks to my sister, for always believing in me.

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Chapter 1: Introduction
Quite aptly called the ‘Elixir of Life’ (Mythrey et al., 2012), water is one of the most
important natural resources on this planet (Aulenbach, 1968), without which life would have
never existed on the face of the Earth. Its level of purity determines the quality-of-life people
lead as it has a direct impact on human health (Abiyu et al., 2018). Lack of clean drinking
water, also referred to as potable water, is a huge problem faced by many underdeveloped and
developing countries (Varkey, 2018). According to the World Health Organization (WHO),
water borne diseases claim 5 million deaths globally each year (WHO, 2019). Most of these
deaths are attributed to a wide range of microorganisms including Escherichia coli, a common
microbe found in contaminated water (Cabral, 2010).
Many methods are available to treat contaminated drinking water; however, cultural,
economic, and social factors often impair implementation of these methods, particularly in
developing countries (Morgan et al., 2019). Conventional water treatment procedure used in
developed countries, which includes coagulation, flocculation, sedimentation, filtration, and
disinfection, renders water free of particulate matter such as bacteria, algae, fungi, parasites,
organic matter and metals like iron, arsenic (Abiyu et al., 2018). In addition to the abovementioned procedures, municipal plants in developed countries involve the use of chemical
coagulants such as ferrous chloride (FeCl2) and aluminium sulphate [Al2(SO4)3], for treating
water. These chemical coagulants can adversely affect human health, for instance, aluminium
is a recognized neurotoxin that can cause neurodegeneration (Maya et al., 2016) and it has been
indicated to be a causative agent of neurological diseases like pre-senile dementia (Abiyu et
al., 2018). Therefore, natural coagulants and antimicrobial agents are moving from fringe to
mainstream use with majority of people seeking water treatment remedies free of chemical side
effects.

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Various plants and their products have gained considerable attention for their
potential to purify water without exploiting expensive and detrimental chemicals (Dubey et al.,
2004). These remedial plants appear to offer a promising alternative to treat contaminated water
in developing countries, where most of the rural population depends on contaminated and
turbid water from rivers, streams, and dams (Varkey, 2018).
Moringa oleifera is one of the most useful trees in the world that belongs to
Moringaceae family (Arora et al., 2013). It is a drought-tolerant tree found in tropical and subtropical countries such as India, Mexico, Philippines, Florida and in many parts of Africa
(Daba, 2016). It is often described as a ‘Miracle Tree’ as almost every part of the tree- leaves,
flowers, pods, seeds, seed oil, bark etc., has nutritional, medicinal and industrial applications
(Daba, 2016).
Leaves, flowers, and immature pods of Moringa are consumed by people in countries
such as India, Hawaii, Pakistan and many parts of Africa (Daba, 2016). Various parts of the
tree are used as circulatory and cardiac stimulants; they contain antileptic, antipyretic, antiinflammatory, anti-tumour, cholesterol lowering, antidiabetic, antioxidant, antimicrobial
properties; and are used for treating various diseases in indigenous medicine system (Anwar et
al., 2007).
Various parts of Moringa shoot possess antibacterial properties, especially the seeds,
which have been extensively studied for their antibacterial and water purifying efficacy. Seed
extract of Moringa has shown to kill 90-99.99% of bacteria in turbid water (Lea, 2010) and in
sedimented sludge (Madsen et al., 1987). However, very little research has been done to study
the antimicrobial and water purifying efficacy of Moringa roots. Utilizing Moringa roots in
antimicrobial research would offer additional benefits as roots can be harvested within the first
year of planting the trees (Morgan et al., 2019) and without hindering the growth of the plant

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(Fuglie, 2001). This would also keep in reserve the nutrient-rich parts of the shoot like leaves
and seeds to be used as a food source (Morgan et al., 2019).
The only research done to evaluate the antibacterial efficacy of Moringa root powder
used three concentrations (250, 450 and 600 mg/L) and found that the maximum root powder
concentration (600 mg/L) reduced E. coli colonies by 87% in contaminated pond water
(Morgan et al., 2019). Likewise, antibacterial efficacy of ginger (Zingiber officinale) against
E. coli and numerous other bacteria such as Staphylococcus aureus, Salmonella typhi and
Vibrio cholerae has been reported (Jagetia et al., 2003; Chrubasik et al., 2005; Gupta and
Ravishankar, 2005). Ginger is native to Southeast Asia but is now cultivated in other tropical
and subtropical regions such as the West Indies, Africa, Latin America, and Australia
(Singletary, 2010). It is commonly used in folk medicine and is known to possess a wide range
of pharmacological properties (Afzal et al., 2001).
The objectives of this research were (1) to chemically analyse roots of Moringa and
ginger plants grown in a greenhouse; (2) to determine the individual effects of Moringa and
ginger root powders on E. coli population when they were added to contaminated water at a
concentration higher than 600 mg/L; and (3) to study the combined effects of Moringa and
ginger root powders on E. coli population.
By increasing the concentrations of Moringa and ginger root powders (>600 mg/L)
in contaminated water, the percentage decrease observed in E. coli population was expected to
be more than 87% (more than what was achieved by Morgan et al., 2019). It was also
anticipated that by mixing the two root powders together in 1:1 ratio, they might have a
synergistic effect on E. coli population and depict a higher antibacterial efficacy than attained
by either of the root powders alone, as bioactive components of plant extracts have ability to
modify or block the resistance mechanism of a bacteria when used together (Stefanovic, 2017).

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A solution to the problem of inaccessibility of clean drinking water is sought out by
many organizations and state governments. The findings of this research might provide a
potential solution to the purification of drinking water in underdeveloped countries and rural
areas of developing countries where the problem of contaminated water is quite alarming
(Varkey, 2018). This research will expand the current knowledge on drinking water
purification by identifying the most effective treatment to reduce E. coli population to the
lowest level possible using Moringa and ginger root powders.

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Chapter 2: Literature review
2.1 Moringa oleifera
2.1.1 Botanical description
Moringa (Moringa oleifera) is native to tropical and sub-tropical regions of South Asia
and Africa (Oliveira et al., 1999) and is the most widely cultivated species of the family
Moringaceae (Arora et al., 2013). It is commonly identified as drumstick tree, horseradish tree,
benzoil tree and cabbage tree in different regions of the world (Koul and Chase, 2015). Moringa
is a deciduous, fast-growing tree which usually grows up to a height of 10-12 meters (Parrotta,
2009) and can be propagated either sexually through seeds or vegetatively through stem
cuttings (Mubvuma et al., 2013). It has an open crown of fragile and drooping branches, giving
the tree canopy an umbrella shape (Parrotta, 2009). The bark is white-grey in colour and has a
thick cork (Parrotta, 2009).
The foliage of Moringa is feathery, consisting of bipinnate or tripinnate leaves, which
can grow up to 45 cm long and are spirally arranged on the twigs (Parrotta, 2009). The flowers
appear after six months of germination and are fragrant, bisexual, having five unequal,
yellowish-white petals (Parrotta, 2009). The fruits of Moringa are dangling, three-sided, 20-45
cm brown pods, which mature after three months of flowering and split to release dark-brown,
globular seeds of one-centimetre diameter (Palanisamy and Kumaresan, 1985). The seeds have
three papery, white wings and are usually dispersed by water and wind. Trees grown from
seeds usually develop deep and thick taproots with wide-spreading system of tuberous and
thick lateral roots; however, trees propagated from cuttings do not develop taproots at all
(Lahjie and Siebert, 1987).

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2.1.2 Growing conditions
Moringa grows in tropical and sub-tropical regions of South Asia and Africa and can
withstand frost and temperatures up to 48 0 C (Oliveira et al., 1999). However, Moringa does
best in the regions where temperature ranges from 26- 40o C and the annual rainfall is at least
500 mm (Goss, 2007). It can endure a wide range of soil types but grows best in well-drained
loam to clay loam soils and neutral to slightly acidic soils as it cannot withstand prolonged
water logging (Padilla et al., 2012).
2.1.3 Pharmacological potential
Moringa is often described as the ‘Miracle Tree’ as almost every part of the tree- leaves,
flowers, pods, seeds, seed oil, bark etc.- have medicinal applications (Daba, 2016). Various
parts of Moringa are used as circulatory and cardiac stimulants, contain antileptic, antipyretic,
anti-inflammatory, anti-tumour, cholesterol lowering, antidiabetic properties, and are used for
treating various diseases in indigenous medicinal systems (Anwar et al., 2007). Moringa is rich
in phytochemicals such as tannins, alkaloids, anthocyanins, proanthocyanins, flavonoids,
cinnamates and cardiac glycosides, which provide the plant its much-researched
pharmacological properties (Goyal et al., 2007; Alhakmani et al., 2013). Anti-inflammatory
activity of Moringa roots is due to the presence of aurantiamide acetate and 1,3- dibenzyl urea
in the roots and root bark of the tree (Mishra et al., 2011).
In-vitro examinations of ethanolic extract of Moringa seeds and various extracts of its
leaves showed anti-tumour activity, which is caused by isothiocyanate and thiocarbonate
related compounds present in those parts of the plant (Nadkarni, 1994; Mishra et al., 2011).
Strong antioxidant activity has been exhibited by aqueous, methanolic and ethanolic extracts
of Moringa leaves, which is induced by Kaemferol (Mishra et al., 2011).

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Isothiocyanate glycosides and thiocarbamate present in the ethanolic extract of
Moringa leaves have been reported to be responsible for antihypertensive activity (Gilani et
al., 1994). Aqueous, methanolic and ethanolic extracts of the leaves have been described to
have anti-ulcer (Mishra et al., 2011), antiepileptic (Amrutia et al., 2011) and anti-diabetic
effects respectively (Suzuki et al., 2007). Alcoholic extracts of Moringa seed kernels have
exhibited anti-asthmatic effect (Mehta and Agrawal, 2008).
2.1.4 Food and nutrition
Moringa is the most nutritious tropical legume (Dhakar et al., 2011). Leaves of Moringa
have high fat content, which increases their palatability (Oduro et al., 2008), and contain all
essential amino acids at concentrations higher than those recommended by the Food and
Agriculture Organisation (FAO) (Makkar and Becker, 1996). Moringa is rich in vitamins such
as A, C, B complex and is also a storehouse of proteins and minerals like iron, calcium,
magnesium, potassium, and zinc (Verma et al., 1976; Fuglie, 2001).
Moringa is reported to have more calcium than milk, more Vitamin A than carrots,
more potassium than bananas and more iron than spinach (Fahey, 2005). Therefore, Moringa
has a potential in combatting nutritional deficiencies and malnutrition in developing countries,
especially in cases of nursing mothers and infants (Srikanth et al., 2014). The leaves of Moringa
provide nutritional security to rural communities of developing countries when it is most
needed, as the leaves of the tree appear towards the end of the dry season, when very few other
sources of leafy green vegetables are available (Fisher, 2011).
Dried or fresh leaves of Moringa are used in various kinds of foods such as salads and
porridges while the flowers are cooked and consumed as a vegetable (Lockett et al., 2000).
Young pods of Moringa are eaten as green beans (Foidl et al., 2001) and the root bark of the

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plant is used as a condiment in some tropical countries (Villafuerte and Villafuerte-Abonal,
2009).
2.1.5 Other commercial uses
Since Moringa is a fast-growing plant, it is planted on a large scale in India to provide
wood to the paper industry. The pulp obtained from Moringa wood is considered suitable for
paper making, wrapping, cellophane and textiles (Ganatra et al., 2012). In Jamaica, exudates
of Moringa are used in the production of blue dye (Verma et al., 1976). A study revealed that
by adding Moringa leaves to livestock feed, daily weight gains of livestock increased by 32%
while milk production increased by 43% (Foidl et al., 2001).
Juice extracted from fresh Moringa leaves can act as a growth hormone and increase
yields of crops by 25-35% (Foidl et al., 2001). Moringa offers a variety of other potential uses,
such as foliar nutrient fertilizer and as green manure (Fuglie, 2001). The ben oil extracted from
Moringa seeds is used in cooking, lubrication, and cosmetic industries (Ramachandran et al.,
1980; Bosch, 2004). Seed oil of Moringa is also a potential contender for biodiesel production,
as it meets almost all the chief specifications of biodiesel standards of Europe, U.S., and
Germany (Mofijur et al., 2014).
2.1.6 Antimicrobial research
Moringa has been widely and extensively used in traditional medicines, mainly
because of its antimicrobial properties. Various studies demonstrate antimicrobial properties
of different parts of Moringa. Using disc diffusion method, fresh leaf juice of Moringa and
aqueous extract of its seeds was found to inhibit growth of Pseudomonas aeruginosa and
Staphylococcus aureus bacteria (Caceres et al., 1991). An experiment conducted in 1995

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reported antifungal activity of seed and leaf extract of Moringa against pathogenic fungi like
Trichophyton rubrum and Trichophyton mentagrophytes (Nwosu and Okafor, 1995).
N-benzyl, S-ethyl thioformate, a compound formed from chloroform extract of
Moringa, showed antimicrobial efficacy against S. aureus, Shigella dysenteriae and Shigella
boydii (Nikkon et al., 2003), while purified dichloromethane extract of Moringa capsules
showed antibacterial efficacy against Escherichia coli, S. aureus, P. aeruginosa and Klebsiella
pneumoniae (Nantachit, 2006). Jamil et al. (2008) studied the antibacterial and antifungal
activity of seed extract of Moringa against microbes like Bacillus subtilis, S. aureus, E. coli,
Rhizopus solani etc., and found that the growth inhibition zone showed greater sensitivity
against bacterial strains as compared to fungal strains.
Antibacterial activities of ethanolic, aqueous and acetone extracts of Moringa leaves
were found to be like antibiotics ciprofloxacin, chloramphenicol and cotrimoxazole (Doughari
et al., 2007). Renitta et al. (2009) studied antibacterial activities of ethanolic extract of flowers,
seeds and leaves of Moringa against E. coli, K. pneumoniae, P. aeruginosa, Enterobacter, S.
aureus and Salmonella typhi with a standard disc diffusion method. Most of the bacteria were
found to be susceptible to the ethanolic extract of Moringa leaves except S. typhi, that was
found to be resistant (Renitta et al., 2009). The ethanolic extract of Moringa seeds was found
to inhibit the growth of S. aureus and K. pneumoniae, but not the other bacteria tested.
Similarly, only K. pneumoniae and E. coli were found to be susceptible to ethanolic extract of
Moringa flowers, while the other tested bacteria were found to be resistant (Renitta et al.,2009).
Moringa oleifera cationic protein (MOCP) which is a low molecular mass protein
extracted from seeds of the Moringa plant, has been found to be an effective coagulating agent
in water treatment (Kwaambwa et al., 2010). It is responsible for inhibiting growth of coliform
bacteria like E. coli by fusing its inner and outer membranes (Shebek et al., 2015). Root powder

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of Moringa reduced E. coli colonies by 87% when added to contaminated water at 600 mg/L
(Morgan et al., 2019). This makes Moringa root powder a promising alternative to purify
contaminated water.
2.1.7 Research in water purification
Traditionally, Moringa seeds were used in various African countries such as
Ethiopia, Uganda, and Sudan to purify contaminated water and removing its turbidity (Jahn,
1977). Seeds of Moringa (also referred to as ‘clarifying tree’ in Sudan) were crushed into a
powder by Sudanese people, and then the powder was added to turbid water and stirred
thoroughly to remove turbidity (Jahn, 1977).
The effectiveness of natural coagulants like Moringa to remove turbidity of water
was found to be similar to chemical coagulants like aluminium sulphate, which are too
expensive and unaffordable by developing countries (Delelegn et al., 2018). The crushed seeds
of Moringa release dimeric cationic proteins to turbid water, which form flocculants with the
water contaminants (Gassenschmidt et al., 1995). These small proteins have a positive charge
and thus, attract negatively charged bacteria, viruses and other colloidal particles found such
as silt and clay, allowing them to coagulate and settle (Amagloh and Benang, 2009).
Various settling times have been proposed for successful water treatment by
Moringa. Doerr (2005) recommended 1-2 hours of settling time for all contaminants to
completely settle at the bottom of container; while Lilliehook (2005) employed a settling time
of 30-120 minutes for water with low, medium, and high turbidity. Mataka et al. (2006)
recommended that mixture of Moringa seed powder and water should be kept undisturbed for
five hours to attain clean drinking water as the lead levels in water decreased exponentially
during the first five hours of reaction.

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The seed powder of Moringa can flocculate various Gram-positive and Gramnegative bacteria such as Salmonella typhimurium, Shigella sonnei, E. coli, Vibrio cholera,
Streptococcus faecalis and Clostridium perfringens (Madsen et al., 1987; Anwar et al., 2007).
In addition to removing turbidity, this flocculation also results in decreasing the conductivity
of contaminated water to log 10 2.34 µS/cm and increasing the pH to 7.2- 7.9, making Moringa
seeds a non-toxic, natural, and effective alternative to purify drinking water (Amagloh and
Benang, 2009). However, Moringa roots showed an inverse effect on conductivity and pH of
contaminated water, as the roots caused significant (p<0.05) increase in conductivity but
decreased the pH from 7.61 to 7.38 (Morgan et al., 2019).
2.2 Zingiber officinale
2.2.1 Botanical description
Ginger (Zingiber officinale) is a member of the plant family Zingiberaceae, which
also includes cardamom and turmeric. It is a two to four feet tall slender monocot, perennial
plant with greenish-yellow flowers (Kaufman, 2016). The leaves of ginger are 15-30 cm long,
arise from sheaths wrapping the stem and are spread alternatively in two vertical rows
(Kaufman, 2016). The inflorescence is solitary, in dense conelike spikes composed of
overlapping green bracts, and each bract encloses a small, purple, and yellow-green flower
(Aleem et al., 2020). The underground stems (known as rhizomes or roots) are thick lobed,
pale yellowish and aromatic (Aleem et al., 2020).
2.2.2 Growing conditions
Ginger is native to Southeast Asia but is now cultivated in other tropical and subtropical
regions such as the West Indies, Africa, Latin America, and Australia (Singletary, 2010). It
requires warm and humid climate with temperature ranging between 19-280 C, high amount of

P a g e | 12
rainfall and relative humidity, and soil with good aeration and water holding capacity for a
promising yield (Teferra et al., 2013). Ginger is sensitive to frost, water logging and salinity,
but is tolerant to drought (Teferra et al., 2013).
2.2.3 Food and nutrition
Rhizomes (or roots) of ginger can be processed into syrup, powder, oleoresin, and
volatile oil, and are widely used in folk medicine, as food flavourings and as dietary
supplements (Bode and Dong, 2011; Gatabazi et al., 2019). Dried and ground ginger is used as
a spice in South-East Asia to flavour breads, curry dishes, confections, sauces, pickles, and
ginger ale, whereas fresh ginger is used in cooking as both a flavouring and a vegetable
(Kaufman, 2016). In Japan, slices of ginger are consumed by people between dishes to clear
the palate (Kaufman, 2016).
The rhizomes of ginger contain carbohydrates (50-70%), lipids (3-8%), terpenes,
phenolic compounds, fibre, proteins, ash, amino acids, vitamins, and volatile oils (Singletary,
2010; Prasad and Tyagi, 2015). The spicy aroma of fresh ginger is mainly due to the presence
of ketones— gingerols, of which [6]-gingerol is most abundant and widely studied in healthrelated scientific research (Bode and Dong, 2011). The pungency of dry ginger results from
shogaols, which are dehydrated gingerols (Ali et al., 2008). The cultivation practices and postharvest treatment of ginger affects the quantity and quality of biologically active constituents
present in the rhizome. Similarly, the chemical components of the rhizome can differ
significantly based on the geographical location of cultivation of ginger, and whether the
product is dried, fresh, or processed (Ali et al., 2008).

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2.2.4 Pharmacological effects
For centuries, ginger has been an essential part of traditional healing strategies in
Middle East, Asia, and Europe. Even today, it is widely used in Ayurvedic, Chinese and TibbUnani herbal medicines for treating a wide range of ailments that include rheumatism, arthritis,
sprains, muscular pains, constipation, sore throat, vomiting, fever, hypertension, and dementia
(Ali et al., 2008).
Evidence primarily from in-vitro and animal studies shows that ginger can be beneficial
for patients suffering from cardiovascular disease as it can counteract hyperlipidaemia,
inflammation, hypertension, and platelet aggregation (Singletary, 2010). The Suekawa group
studied the effect of [6]-gingerol and [6]-shogaol on blood pressure and found that the latter
caused a rapid drop in bradycardia, apnoea, and blood pressure in rats (Chrubasik et al., 2005).
Kadnur and Goyal (2005) reported that methanolic extract of dried ginger rhizomes caused a
significant decrease in fructose-induced elevation of bodyweight, lipid levels, hyperglycaemia,
and hyperinsulinemia. In-vitro studies show that aqueous extract of ginger or its pungent
constituents can effectively inhibit platelet aggregation (Srivas, 1984; Koo et al., 2001).
Previous studies also suggest that intake of ginger powder can significantly reduce platelet
aggregation in patients suffering from coronary artery disease (Bordia et al., 1997) and can
have synergistic effect with nifedipine in hypertensive patients (Young et al., 2006).
Antitumor promoter activity of ginger extracts, [6]-gingerol and [6]-paradol was
demonstrated by Vimala et al. (1999) and Bode et al. (2001). There is suggestive evidence that
ginger powder or an extract can alleviate the symptoms of vomiting and nausea following
pregnancy, cancer therapy, surgery, and motion sickness (Chrubasik et al., 2005; Singletary,
2010). Altman and Marcussen (2001) found ginger to have moderate effect in relieving
osteoarthritic pain, but patients witnessed a significant reduction in knee pain after just 6 weeks

P a g e | 14
of treatment with proprietary ginger extract. Administration of ginger was found to exert
prophylactic and abortive effects in migraine headache without producing any side-effects
(Mustafa and Srivastava, 1990).
2.2.5 Antimicrobial research
Hydroethanolic ginger extract has been reported to suppress the growth of various
Gram-positive and Gram-negative bacteria such as S. aureus, Haemophilus influenzae,
Streptococcus pyogenes, Streptococcus pneumoniae, Salmonella typhimurium, and P.
aeruginosa (Chrubasik et al., 2005). Karuppiah and Rajaram (2012) reported antibacterial
efficacy of ethanolic extracts of ginger rhizomes against E. coli, S. aureus, Bacillus sp., P.
aeruginosa and Proteus sp. with the maximum zone of inhibition displayed against Bacillus
sp. (16.55 mm) followed by E. coli (15.50 mm). Gupta and Ravishankar (2005) found that the
commercially available ginger paste and fresh garlic paste showed better antimicrobial efficacy
against E. coli O157:H7 than turmeric and carrot paste in ground beef and laboratory buffer.
The commercial ginger paste displayed strong antibacterial activity (about 1-2 log CFU/g) in
ground beef at 80 C, while fresh garlic paste showed similar antimicrobial efficacy (1.3 log
CFU/g) against E. coli in buffered peptone water at the same temperature (Gupta and
Ravishankar, 2005).
Gingerols and phenolic metabolites present in ginger rhizome inhibit the growth of
19 strains of Helicobacter pylori and enhance effectiveness of drugs combating gastrointestinal
diseases caused by H. pylori (Mahady et al., 2003). Ginger constituents like shogaol, zingerone
and essential oils have depicted antiviral and antifungal activity against S. typhi, Trichophyton
violaceum, V. cholerae and some filamentous fungi (Jagetia et al., 2003). According to Ficker
et al. (2003), [6], [8] and [10]-gingerols and [6]-gingerdiol are the chief antifungal compounds
present within ginger that are active against 13 human pathogens such as Candida albicans,

P a g e | 15
Trichophyton mentagrophytes and Microsporum gypseum at <1 mg/mL concentrations.
Hydroethanolic and aqueous extract of ginger can effectively inhibit the growth of C. albicans
and Trichomonas vaginalis respectively (Jagetia et al., 2003). However, ginger has shown no
significant action against Aspergillus niger and Aspergillus flavus (Ali et al., 2008).
Not much research has been done on reviewing ginger’s potential to purify
contaminated water other than its potential to inhibit growth of a variety of microorganisms
that might be present in water such as E. coli, P. aeruginosa, Salmonella sp. etc. (Jagetia et al.,
2003; Chrubasik et al., 2005; Gupta and Ravishankar, 2005; Karuppiah and Rajaram, 2012).
Moringa and ginger root powders appear to offer a promising alternative to treat E. coli
colonies present in contaminated water, since Moringa oleifera and Zingiber officinale grow
extensively in underdeveloped and developing countries (Ethiopia, Philippines, India, etc.)
where the problem of contaminated water is quite alarming. In addition to this, various parts of
the plants have been reported to possess antibacterial and water purifying properties.

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Chapter 3: Growing Moringa oleifera and Zingiber officinale in a greenhouse and
extraction of their root powders
3.1 Abstract
Moringa oleifera (Moringa) and Zingiber officinale (ginger) are two tropical plants
that have been extensively studied and widely used for their nutritional and medicinal
properties all over the world. The objective of this study was to discuss the methods and
techniques used to grow Moringa and ginger plants in the UNBC greenhouse, analyse their
growth, and harvest and process their root powders. Moringa seeds were sown on two separate
dates – February 27, 2019 (three seeds per pot) and March 20, 2019 (one seed per pot). The
seedlings were transplanted to bigger pots in the beginning of May 2019. The height and stem
diameter of the Moringa plants were first measured on May 27, 2019, and then on the same
date of every month until their harvest in October, 2019. By the end of August, the mean height
and diameter of the Moringa plants planted in March slightly surpassed the growth parameters
of the plants planted in February. The Moringa plants had a significant growth over the months
and variability between the Moringa plants was calculated to be small. A massive ginger
rhizome, purchased from a supermarket, was grown in the greenhouse in 2017. In the beginning
of April 2020, ginger plantlets along with their rhizomes were divided and vegetatively
propagated into smaller one-litre pots. The height and stem diameter of the ginger plants was
measured only once, on June 23, 2020. After growing Moringa and ginger plants in the
greenhouse for seven months and three years respectively, their roots were harvested, dried and
ground to a fine powder using Black+Decker coffee and spice grinder.

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3.2 Moringa oleifera
3.2.1 Methods
3.2.1.1 Planting and growing Moringa in the greenhouse
Moringa seeds were obtained from Miracletree Life Science, a privately held organic
farming group based in Madurai, Tamil Nadu, in southern part of India. The sun-dried Moringa
seeds were treated with ‘panchagavya’ before being packed and shipped to Canada.
Panchagavya is a traditional Indian pesticide and a fertilizer made from five cow products (cow
dung, urine, milk, curd, ghee), jaggery, coconut and ripe bananas (Raut and Vaidya, 2018).
Moringa seeds (Figure 1) were planted in the I.K. Barber Enhanced Forestry Lab
(EFL) greenhouse at University of Northern British Columbia, Prince George. A soil mixture
was prepared by mixing 54 L peat, 60 L coarse sand, 69 L coir, 180 g of slow-release nutrients
(21-5-6) and three tablespoons of dolomite. Using calibration buffer pouches and a pH tester,
pH of this soil mixture was measured to be 4.6. One hundred Moringa seeds were sown
manually in one-litre pots on February 27, 2019, each pot containing three seeds. After pushing
the seeds about one cm deep in the soil with a finger, they were covered with soil to level the
soil surface. The pots were randomly placed on the shelves of greenhouse and were given a
light watering.
The greenhouse bay was kept at a temperature of 240 C during the day and 180 C
during the night. The pots were given a light, frequent watering about five times a week to keep
the soil consistently moist, but not wet. After 21 days of sowing, the extra seedlings from pots
that showed two or more germinations (Figure 1) were transplanted to separate one-litre pots
on March 20, 2019. This was done by gently digging them using a narrow trowel while trying
to retain as much of the root ball as possible. Following this, there were a total of 86 pots, each

P a g e | 18
containing one healthy Moringa seedling. On the same day, another batch of 20 Moringa seeds
were sown in 20 one-litre pots.

Figure 1. Indian Moringa oleifera seeds that were grown in UNBC greenhouse (Left). A pot
with three germinating Moringa seedlings (Right). (Photographs by author, taken on February
27 and March 14, 2019, respectively).

Moringa plants received 16 hours of supplemental lighting per day from 0600 to
2200 hours using High Pressure Sodium (HPS) PL lights of 400 watts. The temperature of the
greenhouse was increased to 300 C during the day and 240 C during the night at the beginning
of April, after the leaves of seedlings started to curl and turn yellow. Since the pH of soil was
found to be 4.6, 250 mL of a solution of 10 mL Grotek pro-silicate (0-0-3) well dissolved in
20 L water was added to each pot to increase soil pH to a value adequate for growing Moringa.
This increased the pH of soil to 6. This solution and fertigation with Tune Up 20-10-20 Plus, a
water-soluble fertilizer, was given to all plants once every week from April 5, 2019. As soon
as weeds sprouted, they were removed manually and disposed in the garbage bin outside the
greenhouse to prevent an outbreak.

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On May 2, 2019, 74 healthy Moringa plants were transplanted to bigger four-litre
pots as they outgrew their seed starting containers (one-litre pots). A soil mixture was prepared
by mixing 90 L coarse sand, 107 L peat, 123 L coir, 321 g of slow-release nutrients and seven
tablespoons of dolomite. The newly prepared soil mixture was added to the bottom of four-litre
pots. In order to retain maximum root ball and to give minimum shock to the plants during
transplanting, the one-litre pots were turned upside down and their rims were tapped with one
hand while covering the top of the pots with the other hand to support the plants. Then, the root
balls were gently taken out of the pots. The plants with their root balls were set in the four-litre
pots and the soil mixture was used to cover the root ball and fill the sides of the pots. The plants
were watered immediately after transplanting.
Thrips were first noticed on the undersides of leaves of several Moringa plants on
May 6, 2019. To get rid of thrips, the plants were sprayed with a chemical insecticide, Success
480 SC on May 7, 2019 and then by another chemical insecticide– AVID (Abamectin) on May
17, 2019, when the thrips reappeared. To ensure that the HPS lights did not burn the plants or
interfere with their growth, the lights were removed once the plants reached an approximate
height of one to 1.5 meters.
3.2.1.2 Measuring growth of Moringa plants
The height and stem diameter of the Moringa plants were first measured on May 27,
2019, and then on the same date of every month until their harvest in October. Height of the
plants was measured from the soil surface up to the topmost bud of the plant using a measuring
tape. For measuring stem diameter of a plant, a mark on the stem was made at 3.5 cm above
the soil surface by a sharpie. Using an electronic caliper, three diameter readings were taken
on this mark and averaged to give stem diameter of the plant. The mean height and mean stem
diameter of all Moringa plants were calculated from measurements taken on 27 th of every

P a g e | 20
summer month– May, June, July and August. From the mean height and diameter values of the
Moringa plants, height to diameter ratio (H:D) was calculated since it determines sturdiness,
vigour, and competition in the plants (Opio et al., 2003; Haase, 2008).
One sample T-test was performed to determine if the growth of Moringa was
significant over the months. For this, monthly height measurements of all the Moringa plants
were taken as a sample and were compared to the mean height of the previous month to
calculate t- and p-values of height in each month. Similar calculations were performed to
compute t- and p-values of stem diameter of the Moringa plants during the summer months.
One sample T-test was performed using R Studio software and significance of the test was
determined at a p-value of 0.05.
3.2.1.3 Harvesting and processing of Moringa roots
After about seven months of growing Moringa in the greenhouse, roots of half of the
plants planted on February 27, 2019, were harvested on 4 October (Figure 2) and the remaining
half were harvested on October 8, 2019. The roots of Moringa plants sown later in March were
harvested in mid-October, on October 18, 2019. The roots were harvested by first cutting the
stem of plants at soil surface using a garden lopper, the pots were then inverted, and their rims
were tapped until their root balls dropped (Figure 2). The soil was brushed off to expose as
much of the roots as possible. Pruning shears were used to cut distinctive clumps of roots by
removing thin root ends or remaining trunk base.
The harvested Moringa roots were gently washed with water and their root barks
peeled off using a stainless-steel vegetable peeler. The peeled roots of each Moringa plant were
packed in separate air-tight plastic bags with inscribed plant number, its date of sowing,
transplanting and harvest. These airtight bags were stored in the -18o C freezer of Northern
Analytical Laboratory Services (NALS).

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Figure 2. The Moringa plants on the day of their harvesting (Left). A harvested Moringa root
(Right). (Photographs by author, taken on October 4, 2019).

3.2.1.4 Extraction of dried Moringa root powder
For generating simple random samples, sixteen Moringa root samples were
arbitrarily selected for freeze-drying at the beginning of June 2020. Three of these root samples
were of the Moringa plants sown later in March, while the other 13 root samples were of the
plants planted in February 2019. This was done to ensure proper representation of Moringa
plants from both planting dates in the selected samples.
Excess ice from the airtight containers was dumped before placing the bags in the
drying chamber of FreeZone6 freeze dryer by LabconcoTM. Once the temperature within the
drying chamber dropped to -400 C, the vacuum pump was automatically activated. In about ten
minutes, the vacuum level within the drying chamber stabilized to 0.420 mBar and the
temperature dropped further down to -440 C. The samples were left in the freeze dryer for two
days.

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After two days, Moringa root samples were taken out of the freeze dryer, placed on
a wooden chopping board and were cut into smaller fragments using a plastic knife. These root
fragments were then ground into a fine powder using commercially available, electric
Black+Decker coffee and spice grinder. The stainless-steel bowl and blades of the grinder, the
knife and the chopping board were all cleaned with distilled water and dried with KimTech
Kimwipes before their use. The grinder was run for about a minute until the roots were milled
into a fine powder. The finely ground Moringa root powders were sealed in separate airtight
plastic bags (root powders of each plant in a separate bag) that were then stored in -180 C
freezer at NALS.
3.2.2 Results
3.2.2.1 Planting and growing Moringa in the greenhouse
The Moringa seeds sown on February 27, 2019, showed 86% germination while the
seeds sown on March 20, 2019, showed 90% germination.
3.2.2.2 Measuring growth of Moringa plants
As stated in section 3.1.1.2 of the thesis, the mean height and mean stem diameter of
all Moringa plants were calculated from measurements taken on 27th of every summer month–
May, June, July, and August. Figures 3 and 4 compare the mean height and stem diameter of
Moringa plants planted on February 27, 2019, and March 20, 2019. In the month of August,
the mean height and diameter of Moringa plants planted on March 20 surpassed the mean
height and diameter of the plants grown on February 27, 2019 (Figure 3 and 4).

P a g e | 23

Figure 3. Growth in height (m) of Moringa over summer months in 2019, showing standard
deviation as error bars.

Figure 4. Diameter growth (mm) in Moringa over summer months in 2019, showing standard
deviation as error bars.

P a g e | 24
Height to diameter ratio (H:D) of Moringa plants in the months of May, June, July
and August are shown in Table 1. A significant (p-value <0.05) increase in H:D was detected
in Moringa plants sown on February 27, over the summer months, however, the H:D ratios of
plants sown later on March 20, did not observe a significant increase over the summer months.
Table 1. Height to diameter ratio (and standard deviation) of Moringa plants sown on two
separate dates –February 27, 2019, and March 20, 2019, over the summer months.
Month
May
June
July
August

Plants sown on 27 February 2019
H:D ratio
56.94 (10.55)
73.23 (9.99)
77.42 (10.01)
80.14 (9.46)

Plants sown on 20 March 2019
H:D ratio
67.41 (3.88)
71.99 (9.17)
75.87 (5.47)
77.47 (10.19)

In order to determine if the growth of Moringa was significant over the months, one
sample T-test was performed using R Studio software. The t- and p-values of height and stem
diameter of the Moringa plants during the summer months have been listed in Table 2. T-test
was performed by taking the raw values of the observation month and comparing them to the
mean of the previous month. All the p-values of height and stem diameter of Moringa plants
were found to be less than 0.05 (Table 2).
Table 2. Determination of significant differences in height and diameter growth in Moringa
plants over the summer months.
Variable
Height
(m)
Diameter
(mm)

Month
June
July
August
June
July
August

Plants sown on February 27
t- value
p-value
24.64
<0.001
7.83
<0.001
8.62
<0.001
20.16
<0.001
14.21
<0.001
9.21
<0.001

Plants sown on March 20
t-value
p-value
12.15
<0.001
8.94
<0.001
3.74
0.002
12.64
<0.001
8.97
<0.001
8.66
<0.001

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3.2.3 Discussion
On drawing comparison between the mean height and mean stem diameter of
Moringa plants planted on February 27, 2019 and March 20, 2019, it was noticed that the mean
measurements of the latter slightly surpassed those of the plants planted earlier in February
(Figure 3 and 4). This might be because the seeds sown later in March were sown one in each
pot, had sufficient growing space and thus, the seedlings did not have to compete for light,
water and nutrients with other seedlings in the same pot (Milla et al., 2009) and also did not
have to undergo the shock of first transplanting. This luxurious consumption of water, nutrients
and light by Moringa seeds planted on 20 March could also be the reason behind their greater
germination rate than the seeds planted on 27 February, and their non-significant (p-value
>0.05) increase in H:D ratios. The H:D ratio of Moringa plants increased over the summer
months (Table 1), thus indicating an increasing competition between the plants for light and/or
availability of nutrients and moisture (Opio et al., 2003). Since all the p-values of height and
stem diameter of Moringa plants were found to be less than 0.05 (Table 2), it meant that all the
Moringa plants had a significant growth over the months, regardless of their planting dates.
The size of standard deviation bars was small in all the cases and the data lied within
one standard deviation of the mean (Figure 3 and 4). This meant that all the height and diameter
values were closely spread around their respective mean values, which told us that there was
only minor variability between all the Moringa plants. In addition to this, all the plants received
similar treatment in the greenhouse. A small and insignificant variability between the Moringa
plants meant that their root powders could be mixed before performing various microbial and
chemical tests in the lab. Therefore, all Moringa root powder samples were added to a 1-gallon
Ziploc bag and shaken vigorously for about two minutes until all the samples were thoroughly
mixed to form a homogeneous, composite Moringa root powder sample.

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3.3 Zingiber officinale
3.3.1 Methods
3.3.1.1 Planting and growing ginger in the greenhouse
Ginger was purchased from a supermarket and a single, massive rhizome was grown
in I.K. Barber Enhanced Forestry Lab (EFL) greenhouse at UNBC in 2017. UNBC’s Standard
Potting Mix, which is prepared by mixing three bales of peat, three 20 L buckets of sand, three
20 L buckets of perlite, three 20 L buckets of vermiculture, two cups of Micromax
micronutrients and three litres of 14-14-14 slow-release fertilizer, was used for growing ginger
in the greenhouse. The rhizome was grown about an inch deep in the soil in a single Lisbo
380X300 mm pot.
The pot was kept at a temperature of 200 C during the day and 16 0 C during the night.
In the winter months, they received 10 hours of supplemental lighting per day from 0900 to
1900 hours using High Pressure Sodium (HPS) PL lights of 400 watts. However, in the
summers (2017-2020), no supplemental lighting was given. Ginger received weekly
fertilization with a 20-20-20 ratio fertilizer and was watered when the pot soil appeared dry.
Typically, ginger received watering three times a week in the summers and about two times a
week in the winters. On appearance of mites and whitefly, the ginger plantlets were sprayed
with Avid (abamectin) on December 18 and 28, 2018, and then with Talus (buprofezin) and
Avid 1.9% EC on March 1 and 18, 2019 respectively. On February 14 and 21, 2020, the plant
was sprayed with Orthene 75% (acephate 75%) and Enstar EW (kinoprene 18.42%) to counter
the attack of mealybugs and aphids respectively.
In the beginning of April 2020, ginger plantlets along with their rhizomes were
divided and vegetatively propagated into ten smaller one-litre pots. These pots received

P a g e | 27
treatment similar to the previous crop— 16-200 C temperatures, watering about 3 times each
week and regular fertilization with 20-20-20. To impede the attack of various insects, the ginger
plants were sprayed with Kontos (spirotetramat), Success 480 SC (Spinosad), Pylon
(chlorfenapyr) and Ventigra (afidopyropen) on 16 April, 23 April, 15 May, and June 2, 2020
respectively.
3.3.1.2 Measuring growth of ginger plants
The height and stem diameter of the ginger plants was measured on June 23, 2020,
before harvesting the plants. Heights of the plantlets were measured from the soil surface to
their topmost bud using a measuring tape. Average heights of all the plantlets growing within
a pot was calculated. Similarly, stem diameter of each plantlet was measured 2.5 cm above the
soil surface using an electronic caliper and average stem diameter of all plantlets within a pot
was calculated and regarded as the mean stem diameter of that plant.
3.3.1.3 Harvesting and extraction of ginger powder
Ginger plantlets were harvested on June 23, 2020. To procure the ginger rhizomes,
stems of the plantlets were cut at the soil surface using pruning shears. Then, the soil was
moved towards the edges of each pot to expose the rhizomes. The rhizomes were removed
from the pots and adhering soil was brushed off with hands. Roots and residual stem base were
detached from the rhizomes using pruning shears.
The rhizomes were gently washed with water and were peeled using a stainless-steel
vegetable peeler. The washed, peeled rhizomes were packed in air-tight bags with inscribed
plant number. These air-tight bags were placed in the NALS freezer at -180 C for two days.
The ginger roots were freeze dried and ground into powder using the same methods and
techniques used to process Moringa roots (Figure 5).

P a g e | 28

Figure 5. Grinding of harvested and freeze-dried roots of a ginger plant using Black+Decker
coffee and spice grinder. (Photograph by author, taken on July 6, 2020).

3.3.2 Results
The height (expressed in cm) and stem diameter (in mm) of the ten ginger plants,
measured on June 23, 2020, have been provided in Table 3. Height to diameter ratio (H:D) of
each ginger plant has also been included in Table 3.
Table 3. The average height (in cm), stem diameter (in mm), and height to diameter ratio (H:D
ratio) of the ten ginger plants.
Plant number

Average height (cm)

Average diameter (mm)

Height: Diameter (H:D)

1
2
3
4
5
6
7
8
9
10

36.5
41.6
37.5
48.66
39.5
26.5
36.66
34
47
29

7.37
8.99
8.21
9.77
8.28
5.72
6.82
6.14
8.09
5.85

49.53
46.27
45.68
49.81
47.71
46.33
53.75
55.38
58.10
49.57

P a g e | 29
3.3.3 Discussion
Previous literature on height, stem diameter and H:D ratio of three-year-old ginger
plants could not be found and hence, the growth parameters of the ginger plants (Table 3) could
not be compared with previous studies. As the height, diameter, and H:D ratio of the ginger
plants was measured only once (on the day of their harvesting), a pronouncement regarding
their growth patterns over months, and increasing or decreasing competition between the plants
could not be made. Future studies should document growth of ginger plants over months (like
growth analysis of Moringa plants detailed in section 3.1 of the thesis). This would help to
detect any competition between the plants, to determine if their growth over months is
significant or not, and finally, to calculate variability between the ginger plants.
3.4 Conclusion
Since all the ginger plants were vegetatively propagated from a single ginger
rhizome and all the plants received similar treatment in the greenhouse, the variability between
their roots was assumed as small and insignificant. Similarly, the Moringa plants were found
to have minor variabilities between them (section 3.2.3 of the thesis). Hence, the ground
Moringa and ginger root powders were mixed to form composite samples before performing
chemical analysis and various microbial tests in the lab.

P a g e | 30
Chapter 4: Chemical analysis of Moringa and ginger root powders
4.1 Abstract
Moringa (Moringa oleifera) and ginger (Zingiber officinale) have been extensively
studied and widely used for their nutritional and medicinal properties all over the world. The
objective of this study was to conduct a complete chemical analysis of the root powders
extracted from roots of Moringa and ginger plants grown in the UNBC greenhouse. The most
abundant metal found in both root powders through Inductively coupled plasma- optical
emission spectrometry (ICP-OES) was potassium, with a mean concentration of 6,066.5 mg/kg
in Moringa root powder and 17,484.33 mg/kg in ginger powder. Calcium, magnesium, sulphur,
sodium, and phosphorus were other metals present in the roots in high concentrations. Harmful,
heavy metals such as lead, mercury, arsenic and cadmium were least abundant in the root
powders, with concentrations less than 10 mg/kg. Using Elemental Analysis method, total
carbon and nitrogen found in Moringa root powder was 42.23% (by oven-dry mass) and 0.25%
respectively, and in ginger powder was 41.90% and 3.7% respectively. Results of Ion
Chromatography revealed that concentrations of anions like sulphate, phosphate, chloride, and
nitrate were higher in ginger than in Moringa root powder. This explains the higher Electrical
Conductivity (EC) of ginger (12.45 mS/cm) compared to Moringa root powder, that had 4.74
mS/cm. Both root powders were slightly acidic with pH values ranging from 5 to 6. The
abundance of essential elements and anions, and the scarcity of toxic, heavy metals in the two
root powders justifies their use as a source of food for humans and as a point-of-use water
treatment method.

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4.2 Introduction
Moringa (Moringa oleifera) and ginger (Zingiber officinale) have been an integral
part of Asia’s folk medicine for years. Currently, these tropical plants are being extensively
studied and widely used for their nutritional and medicinal properties all over the world.
Various parts of Moringa tree are used as circulatory and cardiac stimulants, and are
known to contain antipyretic, antileptic, anti-inflammatory, anti-tumour, cholesterol lowering
and antidiabetic properties (Anwar et al., 2007). Moringa is rich in phytochemicals such as
alkaloids, tannins, anthocyanins, flavonoids, proanthocyanins, cinnamates and cardiac
glycosides that provide the plant its pharmacological properties (Goyal et al., 2007; Alhakmani
et al., 2013). Moringa roots are used to treat inflammation, rheumatism, articular pain, lower
back pain, constipation (Anwar et al., 2007) and have anti-spasmodic, anti-tumour and
antimicrobial properties (Caceres et al., 1991; Bose, 2007; Mishra et al., 2011). Various
extracts of Moringa roots showed presence of saponins, carbohydrates, cardiac glycosides,
steroids, terpenes, alkaloids, and flavonoids (Furo and Ambali, 2012). Phytochemicals such as
4-O-(α-l-rhamnopyranosyloxy)-benzylglucosinolate (glucomoringin), benzylglucosinolate
(glucotropaeolin) and 3.57- 4.38% crude protein have also been detected in Moringa roots
(Amaglo et al., 2010).
Unlike organic components, inorganic elemental components of Moringa roots have
not been extensively researched (Morgan et al., 2019). Amaglo et al. (2010) detected presence
of <0.1% (w/v) sodium, 2.05% (w/v) potassium and 0.3% (w/v) calcium in Moringa roots. The
first complete analysis of all the inorganic components of Moringa roots was done by Morgan
et al. (2019). Since metal uptake in plants is affected by various factors (soil pH, metal
concentrations in soil, organic matter content, cation exchange capacity etc.), there was a need
to chemically analyse Moringa and ginger root powders before studying their antibacterial

P a g e | 32
efficacy against E. coli. In addition to this, there were significant differences in the handling,
processing, and storage of Moringa roots from the methodology used by Morgan et al. (2019).
Pre- and post-harvest handling of the Moringa plants, such as types of pesticides used,
the procedure of root drying, processing, and storage, in this research varied greatly from
Morgan (2019). Safer’s Soap and Enstar were used by Morgan et al. (2019) to treat thrips and
whiteflies that appeared on Moringa plants, while Success 480 SC and AVID (Abamectin)
were used on the appearance of thrips in this research. Morgan et al. (2019) oven-dried the
Moringa roots at 700 C for five days, whereas in this research, the roots were freeze-dried for
two days (read section 3.1.4 for more details on root processing). The dried Moringa roots were
ground using a ball grinder and then the root powder was stored at room temperature in an
airtight jar by Morgan et al. (2019). But in this research, the roots were ground using
Black+Decker coffee and spice grinder and were stored at -180 C.
Ginger is known to treat a wide range of ailments that include rheumatism, arthritis,
sprains, muscular pains, constipation, sore throat, vomiting, fever, hypertension, and dementia
(Ali et al., 2008). It has been found to contain antioxidant, antimicrobial, and anti-inflammatory
properties. Ginger is rich in bioactive compounds such as gingerols, paradols and shogaols that
provide the plant its much-researched medicinal properties (Mao et al., 2019).
Like Moringa root, not much research has been done to determine the concentrations
of non-essential and essential metals present in ginger, except Wagesho and Chandravanshi
(2015). They found the mean range (µg/g dry weight basis) of various metals found in ginger
to be- Calcium (2000–2540), Magnesium (2700–4090), Iron (41.8–89.0), Zinc (38.5–55.2),
Copper (1.1–4.8), Cobalt (2.0–7.6), Chromium (6.0–10.8), Manganese (184–401), Nickel (5.6–
8.4) and Cadmium (0.38–0.97).

P a g e | 33
The chemical metallic and non-metallic elements present in Moringa and ginger root
powders could affect their viability as a point-of-use water treatment (Morgan et al., 2019).
Presence of beneficial elements such as calcium and iron in the root powders would offer
additional benefits of providing nutrients in water, but detection of heavy metals like lead,
mercury, or toxic anions like fluoride in high amounts would render the root powders
unsuitable for water treatment and consequently unsafe for ingestion.
Concentrations of total carbon and nitrogen (and subsequently C:N ratio) present in
Moringa and ginger root powders were also measured because occurrence of these two
essential nutrients in their roots in adequate amounts would be beneficial for human health as
carbon and nitrogen are the two most essential and vital elements of the human body. The
concentrations of these two elements in Moringa and ginger root powders have not been
previously determined by any study. In addition, physical parameters such as pH and electrical
conductivity of Moringa and ginger root powders were measured as they are important
indicators of a plant’s health that impact mineral nutrition and water absorption in the plants.
An adequate pH of the root powders would ensure that they are safe for human consumption.
A complete chemical analysis of the root powders would also help the future studies to replicate
the antimicrobial tests and obtain consistent results by using Moringa and ginger root powders
with similar chemical composition.
The objectives of this chapter were to determine: (1) concentrations of metals present
in Moringa and ginger root powders by ICP-OES (Inductively coupled plasma-optical
emission spectrometry); (2) total carbon and nitrogen present in the root powders by Elemental
Analysis; (3) concentration of anions by Ion Chromatography; and (4) electrical conductivity
and pH of the Moringa and ginger root powders.

P a g e | 34
4.3 Methods
4.3.1 Determining particle size of Moringa and ginger root powders
Malvern Mastersizer 3000 with Hydro LV module was used to determine particle
size of dried Moringa and ginger root powders. Mastersizer 3000 (by Malvern) uses the laser
diffraction technique to measure particle size of samples, i.e., it measures the intensity of light
that gets scattered when a laser beam passes through a dispersed particulate sample.
Small samples of Moringa and ginger root powders were successively dispersed into
Hydro LV unit to produce stable and completely dispersed suspensions. The samples were run
between 2% and 20% obscuration at 2100 rpm stirrer speed and were each sonicated for 19
seconds prior to their run. The samples were run as per the methods mentioned in Malvern
Mastersizer 3000 instrumentation (Malvern © Instruments Ltd., 2013). The particles of both the
samples were modelled with “Wood Flour” using Refractive Index 1.53. Both Moringa and
ginger root powder samples ran as 5 replicates.
4.3.2 Determining metal concentrations in ginger and Moringa root powders
The ground and homogenised samples of Moringa and ginger roots were dried at 550
C for 48 hours. Four replicates of Moringa root powder sample and three replicates of ginger
powder sample were weighed to 0.2 grams, and block digested in 15 mL digestion tubes for
2.5 hours (45-minute ramp to 950 C). Trace Metal Grade and strong mineral acids—nitric acid
and hydrochloric acid, were used for digestion as these acids can determine lowest contaminant
levels (Caledon Laboratories, 2020). The sample replicates were then diluted to 15 mL with
Type 1 water (18.2MΩ) and run on ICP-OES (Agilent Technologies 5100 ICPOES) to
determine concentrations of metals present in Moringa and ginger root powders.

P a g e | 35
4.3.3 Determining total carbon and nitrogen present in Moringa and ginger root powders
A Costech Elemental Combustion System (ECS) 4010 CHNSO analyser was used
to determine percent by oven-dry mass of total carbon and nitrogen present in the two root
powders. Four replicates of Moringa root powder sample and three replicates of ginger root
powder sample were weighed out to 0.5 grams each in tin capsules.
The samples were combusted in ECS 4010 CHNSO analyser through standard
sequential combustion/ reduction setup recommended by Costech in ECS 4010 brochure
(Costech© Analytical Technologies Inc., 2005). Helium 5.0 with a flowrate of 100 mL per
minute was employed as a carrier gas to bring combustion gases to a gas chromatographic (GC)
separation column and thermal conductivity detector. The combustion oven was set to 1000 0
C, the reduction oven to 650 0 C and the GC oven to 45 0 C. Gases were then separated on a
standard 2 m (Costech 051081) column and quantified with the built-in thermal conductivity
detector (TCD).
4.3.4 Determining concentrations of anions in Moringa and ginger root powders
The procedure from United States Environmental Protection Agency (EPA) Method
300.0 was followed for determining concentration of various anions present in Moringa and
ginger root powders (Pfaff, 1993). Four replicates of dried Moringa root powder and three
replicates of dried ginger powder were weighed to one gram and added to 15 mL tubes. To
each of these tubes, 10 mL (Liquid: Solid ratio= 10:1) of Type 1 water (18.2MΩ) was added.
The tubes were capped and shaken at 80 rpm in end-over-end rotating shaker for 10 minutes,
following which they were centrifuged at 3000 rpm for five minutes using HERMLE Z382K
(by Hermle Labortechnic GmbH). The contents of the tubes were filtered through 0.45 µm
syringe filters and then analysed on Dionex ICS-5000 for determining concentrations of

P a g e | 36
fluoride, chloride, nitrite, sulphate, bromide, nitrate and phosphate in Moringa and ginger root
powders.
4.3.5 Determining electrical conductivity and pH of the root powders
Four replicates of dried Moringa root powder sample and three replicates of dried
ginger root powder sample were weighed to 10 grams and added to 50 mL tubes. To each of
these tubes, 40 mL of MilliQ water (Liquid: Solid ratio= 4:1) was added. The tubes were
capped and shaken at 80 rpm in end-over-end rotating shaker for 60 minutes, after which they
were centrifuged at 3000 rpm for 10 minutes and then decanted. The sample replicates were
centrifuged for double the amount of time (10 minutes) than the time taken for centrifugation
in ion chromatography (5 minutes). This was done to provide sediments enough time to settle
as it was followed by decantation, and not filtration by syringe filters (as in case of ion
chromatography). Electrical conductivity and pH of the root sample replicates was determined
by using OAKTON CON700 (OAKTON® Instruments, 2010) and Orion 420A+ (Orion
Aplus™, 2003) respectively.
4.3.6 Statistical analysis
R Studio software was used to calculate the mean electrical conductivity, pH,
concentrations of metals, non-metallic elements, and anions of the four replicates of Moringa
root powder and three replicates of ginger powder. Standard deviations from the mean values
were also calculated using R Studio software.

P a g e | 37
4.4 Results
4.4.1 Particle size of Moringa and ginger root powders
Table 4 compares the mean standard percentile readings of Moringa and ginger root
powders. Dv 10, Dv 50 and Dv 90 show the maximum particle diameter (in µm) below which
10%, 50% and 90% of sample volume exists (here ‘D’ and ‘v’ stand for diameter and volume
distribution). Particle size of 90% of the Moringa and ginger root powders was under 438 and
364 µm respectively and the most reoccurring particle sizes in the two root powders (mode)
were 22 and 176 µm respectively (Table 4).
Table 4. Mean percentile readings (and standard deviations) of particle size of Moringa and
ginger root powders.
Moringa root powder
Ginger root powder
Volume distribution
Mean percentile
Mean percentile
Dv 10 (µm)
13.0 (0.1)
9.29 (0.03)
Dv 50 (µm)
53.9 (1.8)
73.4 (1.1)
Dv 90 (µm)
438 (16)
364 (2)
Mode (µm)
22 (n/a*)
176 (5)
*Sample mode was identical for all five replicates, resulting in no standard deviation.
Table 5 shows the distribution of particle sizes of Moringa and ginger root powders
into five size classes. About 48% of the Moringa root powder had particle size between 2 to
50 µm, whereas about 50% of the ginger root powder had particle size between 50 to 500 µm.
Table 5. Mean distribution (and standard deviation) of Moringa and ginger root powders into
different size classes.

Particle size class
<2 µm
2- 50 µm
50- 500 µm
500- 1000 µm
>1000 µm

Moringa root powder
Mean percentage
0.91 (0.01)
47.9 (0.5)
43.5 (0.6)
7.3 (0.4)
0.32 (0.33)

Ginger root powder
Mean percentage
1.05 (0.01)
43.3 (0.2)
50.9 (0.4)
4.6 (0.2)
0.13 (0.06)

P a g e | 38
4.4.2 Concentrations of metals present in Moringa and ginger root powders
Inductively coupled plasma- optical emission spectrometry (ICP-OES) was used to
measure concentrations of 27 metals in dried, powdered roots of Moringa and ginger. The
metals to be tested were chosen in a broad sweep, keeping in mind the list of metals used by
Northern Health, British Columbia, to analyse their chemical samples and the metals outlined
in Northern Health Guidelines for the approval of Waterworks (Northern Health, 2020). Table
6 shows the mean concentrations of the 27 metals and the standard deviation found in Moringa
and ginger root powders. The mean concentrations of the metals were expressed in mg/kg.
The most abundant metal found in Moringa roots was potassium, which was present
in a mean concentration of 6,066.5 mg/kg (Table 6). The Moringa roots had high mean
concentrations (>1,000 mg/kg) of calcium, magnesium and sulphur, and moderate
concentrations (>700 mg/kg) of sodium and phosphorus. Barium was present in the Moringa
roots with a mean value of 10.99 mg/kg. There were less than 10 mg/kg of aluminium, arsenic,
boron, iron, mercury, manganese, molybdenum, lead, antimony, selenium, tin, uranium and
zinc. Cadmium, cobalt, chromium, copper, nickel, vanadium and zirconium were the least
abundant of the measured metals with values below 1 mg/kg in the Moringa roots.
In ginger root powder, potassium was the most abundant metal found, with a mean
concentration of 17,484.33 mg/kg (Table 6). The ginger root powder had high mean
concentrations (>1,000 mg/kg) of magnesium, sodium, phosphorus and sulphur, and moderate
mean concentration (>700 mg/kg) of calcium. Barium, iron, manganese and zinc were all
present in the ginger roots with mean values between 10 and 500 mg/kg. There were less than
10 mg/kg of aluminium, arsenic, boron, copper, mercury, nickel, molybdenum, lead, antimony,
selenium, tin and uranium in the ginger roots. Cadmium, cobalt, chromium, vanadium and
zirconium were the least abundant metals present in ginger roots with values below 1 mg/kg.

P a g e | 39
Table 6. Mean concentrations (and standard deviations) of various metals found in Moringa
and ginger root powders.

Aluminium

Moringa root powder
(mg/kg)
<8.25* (0)

Ginger powder
(mg/kg)
<8.25* (0)

Arsenic

<6.75* (0)

<6.75* (0)

Boron

4.31 (0.07)

7.06 (0.09)

Barium

10.99 (0.13)

17.89 (0.23)

Calcium

1,810.25 (19.99)

966.33 (6.66)

Cadmium

<0.23* (0)

0.34 (0.07)

Cobalt

<0.75* (0)

<0.75* (0)

Chromium

<0.23* (0)

<0.26* (0.04)

Copper

<0.77* (0.03)

8.64 (0.23)

Iron

6.88 (0.28)

77.47 (0.25)

Mercury

<3.3* (0)

<3.3* (0)

Potassium

6,066.5 (57.72)

17,484.33 (112.93)

Magnesium

1,183.5 (12.15)

4,414.33 (30.62)

Manganese

9.26 (0.05)

460.81 (5.44)

Molybdenum

<1.05* (0)

<1.07* (0.02)

Sodium

878.75 (5.74)

1,916 (4)

Nickel

<0.83* (0.11)

1.3 (0.09)

Phosphorus

986 (11.80)

6,417.67 (38.79)

Lead

<2.25* (0)

<2.25* (0)

Sulphur

1,232.5 (18.36)

3,977.67 (37.01)

Antimony

<4.1* (0)

<4.1* (0)

Selenium

<9* (0)

<9* (0)

Tin

<6.8* (0)

<6.8* (0)

Uranium

<4* (0)

<4* (0)

Vanadium

<0.75* (0)

<0.75* (0)

Zinc

5.21 (0.10)

46.69 (0.48)

Zirconium

<0.3* (0)

<0.3* (0)

Metals

*The metals with mean values marked with ‘less than’ (<) sign have concentrations below the
method detection limit.

P a g e | 40
Both ginger and Moringa root powders were most abundant in potassium and least
abundant in cadmium, cobalt, chromium, vanadium, and zirconium (Table 6). However, a
direct comparison between the concentration of metals found in the root powders cannot be
made as the Moringa and ginger roots were harvested at different growth periods. Moringa
roots were harvested after about seven months of growing Moringa plants in the greenhouse,
whereas ginger was harvested after about three months of transplanting the rhizomes, that were
previously grown for three years.
4.4.3 Total carbon and nitrogen present in Moringa and ginger root powders
Elemental analysis (EA) was done to determine the amount of two essential
inorganic, non-metallic elements— carbon and nitrogen, present in Moringa and ginger root
powders. The mean percent by oven dry mass of the total carbon and nitrogen present in
Moringa and ginger root powder replicates are given in Table 7. The Carbon: Nitrogen ratio
(C: N) in Moringa root powder was found to be 168.92, whereas in ginger powder, it was found
to be 11.32.
Table 7. Means (and standard deviations) of total carbon and nitrogen detected in Moringa and
ginger root powders.
Total carbon and nitrogen
Total carbon
Total nitrogen

Moringa root powder
(%)
42.23 (0.20)
0.25 (0.01)

Ginger root powder
(%)
41.90 (0.06)
3.7 (0.07)

4.4.4 Concentrations of anions present in Moringa and ginger root powders
Ion chromatography was conducted to determine concentrations of inorganic anions
present in Moringa and ginger root powders. The seven anions mentioned in ‘Part A’ of EPA
Method 300.0 (specified by Environmental Protection Agency of U.S.) were chosen to be

P a g e | 41
analysed for this chemical analysis. These anions are the most commonly investigated anions
while undergoing analysis of drinking water, groundwater, wastewater and solids (Pfaff, 1993).
Table 8 gives the mean concentrations of the seven anions and the standard deviation found in
Moringa and ginger root powder replicates.
Table 8. Mean concentrations (and standard deviations) of selected anions found in Moringa
and ginger root powders.
Moringa root powder
Ginger root powder
(mg/kg)
(mg/kg)
Fluoride
178 (1.16)
182.77 (2.31)
Chloride
454.25 (2.22)
737.33 (7.23)
Nitrite
<10* (0)
<10* (0)
Sulphate
6,035 (334.18)
9,511.33 (46.46)
Bromide
<10* (0)
<10* (0)
Nitrate
53 (0.82)
9,326 (187.74)
Phosphate
1,029.25 (17.02)
12,653.67 (1494.94)
*The anions with mean values marked with ‘less than’ (<) sign have concentrations below the
method detection limit.
Anions

Sulphate was the most abundant anion in Moringa roots with mean concentration of
6,035 mg/kg (Table 8). The roots had high mean concentration (>1,000 mg/kg) of phosphate
and moderate concentrations (>100 mg/kg) of chloride and fluoride. Nitrate was present in
Moringa roots with a mean value of 53 mg/kg. Nitrite and bromide were the least abundant
anions present in the roots with concentrations lower than 10 mg/kg (Table 8).
In ginger root powder, the most abundant inorganic anion was phosphate, with a
mean concentration of 12,653.67 mg/kg. Sulphate and nitrate were also present in the ginger
roots in high amounts, with mean concentrations higher than 9,000 mg/kg (Table 8). The ginger
roots had moderate concentration (>700 mg/kg) of chloride and above 100 mg/kg concentration
of fluoride. Like Moringa root powder, ginger powder was least abundant in bromide and
nitrite, their concentrations being lower than 10 mg/kg (Table 8).

P a g e | 42
4.4.5 Electrical conductivity and pH of Moringa and ginger root powders
The mean EC and pH (calculated from values of respective replicates) of ginger and
Moringa root powder are given in Table 9. Ginger powder had 7.71 mS/cm higher electrical
conductivity than Moringa root powder (Table 9). Both the root powders were slightly acidic
with pH values lying between 5 and 6 (Table 9).
Table 9. Means (and standard deviations) of electrical conductivity and pH of the Moringa and
ginger root powders.
Electrical conductivity and pH
Electrical conductivity
pH

Units
mS/cm
-

Moringa root powder
4.74 (0.01)
5.33 (0.01)

Ginger root powder
12.45 (0.33)
5.75 (0.01)

4.5 Discussion
4.5.1 Particle size of Moringa and ginger root powders
Particle size of Moringa and ginger root powders was measured to ensure that the
root powders were ground to such a fine powder that their respective particles would be filtered
out by Whatman 150 mm, grade 2 filter paper before performing antibacterial tests. Whatman
grade 2 filter paper withholds all the particles in the size range of 8 µm and more. Since the
most reoccurring particle sizes in Moringa and ginger root powders were 22 and 176 µm
respectively (Table 4), this meant that Whatman grade 2 filter paper would withhold only
Moringa and ginger root particles but would allow E. coli to pass through, as the average size
of E. coli is known to fall between 1-3 µm (Reshes et al., 2008).
4.5.2 Concentrations of metals found in Moringa and ginger root powders
The most abundant metal found in both the root powders was potassium, with a mean
concentration of 6,066.5 mg/kg in Moringa root powder and 17,484.33 mg/kg in ginger powder

P a g e | 43
(Table 6). Potassium is an essential nutrient required for maintaining normal cell function, acid
and electrolyte balance, and total body fluid volume in humans (WHO, 2012a). According to
WHO, individuals ≥16 years of age should consume at least 3,510 mg of potassium per day
(WHO, 2012a). Uptake of this recommended amount of potassium in humans reduces the risk
of diseases such as coronary heart disease, stroke and cardiovascular disease and will help
regulating blood pressure (WHO, 2012a). The amount of potassium found in ginger and
Moringa root powders was either comparable or higher than the amount found in potassiumrich unrefined foods such as beans and peas, green vegetables, and fruits. According to WHO,
the approximate levels of potassium found in beans and peas, green vegetables and fruits is
13,000 mg/kg, 5,500 mg/kg and 3,000 mg/kg respectively (WHO, 2012a).
Some other metals present in ginger and Moringa root powders in high
concentrations were calcium, magnesium, sulphur, sodium and phosphorus (Table 6). Each of
these metals is deemed important for various biological functions in humans. Magnesium is
required for energy production and is a cofactor in about 300 enzyme systems that regulate
several biochemical reactions in the human body, including blood glucose control, muscle and
nerve function, blood pressure regulation and protein synthesis (ODS, 2020a). The Office of
Dietary Supplements (ODS) of U.S. recommends daily uptake of 400-420 mg magnesium in
adult males and 310-320 mg magnesium in adult females (ODS, 2020a). This requirement can
be easily fulfilled by ginger and Moringa root powders, which both had more than 1,000 mg/kg
of magnesium present in them (Table 6).
Calcium is the most abundant mineral in the human body that is essential for muscle
function, vascular contraction and vasodilation, nerve transmission and hormonal secretion
(ODS, 2020b). Calcium supports the structure and function of bones and teeth in human body
(ODS, 2020b). According to the Office of Dietary Supplements (ODS) of U.S., Recommended

P a g e | 44
Dietary Allowances (RDA) for calcium are about 1,000 mg/day in adults (ODS, 2020b).
Moringa and ginger root powders can help fulfilling this requirement, as they had a mean
concentration of 1,810.25 and 966.33 mg/kg calcium present in them (Table 6).
Phosphorus is a component of teeth, bones, DNA, RNA, cell membrane structure
and of the key energy source of human body- ATP (ODS, 2020c). It plays an important role in
intracellular energy storage, regulating gene transcription and activation of enzymes (ODS,
2020c). Deficiency of phosphorus in human body is quite rare as it is abundant in various foods
(Institute of Medicine [US], 1997). In human adults, 700 mg is the average daily intake of
phosphorus, as recommended by the Office of Dietary Supplements of US (ODS, 2020c).
Ginger and Moringa root powders were abundant in phosphorus, with mean concentrations of
6,417.67 and 986 mg/kg respectively (Table 6).
Sulphur is required for synthesis of essential amino acids— cysteine and methionine,
and some key proteins (Wong, 2020). It is believed that sulphur protects the human body
against osteoarthritis, allergies, muscle soreness, dandruff, rosacea etc. (Wong, 2020).
Although there is no Recommended Dietary Allowance (RDA) for sulphur, a daily intake of
sulphur-containing amino acids has been recommended. The estimated requirement for
methionine (along with cysteine) was determined to be 14 mg/day per kg body weight in adults
(Nimni et al., 2007). Both ginger and Moringa root powders had high concentration (>1,000
mg/kg) of sulphur present in them and can fulfil the dietary requirements of sulphur in human
body (Table 6).
Sodium is an essential element for maintaining pH, fluid balance and cellular
homeostasis in human body (Farquhar et al., 2015). The WHO recommends daily intake of <2
grams of sodium in adults (WHO, 2012b). Intake of higher sodium consumption (>2
grams/day) and insufficient potassium (<3.5 grams/day) can contribute to high blood pressure

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and can increase the risk of stroke and various other heart diseases in adults (WHO, 2012b).
Although one kilogram of ginger and Moringa root powders had only about 1.9 and 0.9 grams
of sodium respectively (Table 6) but adding common table salt to the diet along with these root
powders can fulfil an adult’s daily dietary requirement of sodium. The root powders also had
high concentration of potassium that can counteract the potential negative effects of high
sodium (Morgan et al., 2019).
The most and least abundant metals found in Moringa root powder in this research
were similar to those found by Morgan et al. (2019) in the dried sample of Moringa roots
harvested after 7 months of growth (labelled as ‘Dry 7 Months’). However, the concentrations
of most of these elements were quite low as compared to their levels found by Morgan et al.
(2019). This considerable difference in the concentration of elements might have been caused
due to variation in pre-harvest conditions of the Moringa plants, their post-harvest handling,
processing techniques employed and storage conditions (detailed in section 4.2 of this thesis).
Shewfelt (1990) found that disparities in all the above-mentioned conditions widely affect the
nutrient composition of plants.
The method used to determine concentration of elements present in the Moringa
roots also varied in the two studies. Morgan et al. (2019) used Inductively coupled plasmamass spectrometry (ICP-MS), and in this research, inductively coupled plasma- optical
emission spectrometry (ICP-OES) was used. ICP-OES measures excited atoms and ions at a
wavelength characteristic for specific elements being measured, while ICP-MS measures an
atom’s mass (Thermo Fisher Scientific, 2022). Therefore, the lower detection limit for ICPOES is parts per billion but the lower limit for ICP-MS can extend to parts per trillion (Thermo
Fisher Scientific, 2022). Thus, ICP-MS is used when the elements to be measured are below
or near the lower detection limit of ICP-OES. This might have affected the detected

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concentrations of least abundant metals. Although the Moringa seeds were ordered from India
in both the studies, but the geological region within India from where the seeds were ordered,
their growing conditions or any treatments given to the seeds before shipping might have varied
in the two studies. This might be another reason behind disparity in the concentration of metals
found in the Moringa roots.
The concentrations of iron, zinc, lead, and cadmium found in ginger root powder
were consistent with their concentrations determined by Wagesho and Chandravanshi (2015).
However, magnesium, copper and manganese were present in higher amounts, and calcium,
cobalt, chromium, and nickel were found in concentrations lower than those determined by
Wagesho and Chandravanshi (2015). This discrepancy in the concentration of metals
determined by the two studies can be attributed to differences in mineral concentration of soil
media used to grow ginger, soil pH, organic matter content, cation exchange capacity, varieties
of ginger grown, and age of the rhizomes used for chemical analysis- as all these factors affect
the mineral constitution of a plant (Jung, 2008).
In addition to the essential elements, the concentration of harmful, heavy metals such
as lead, mercury, arsenic, cadmium etc. were also determined in ginger and Moringa root
powders. These metals have been proven to be major health threats as they are harmful for
proper functioning of the human body and can interfere with various metabolic processes
(Jaishankar et al., 2014). All these metals were present in very low amounts in the root powders,
with concentrations less than 10 mg/kg (Table 6). Cadmium was present at <1 mg/kg in the
root powders, whereas lead and mercury were present at <2.25 and <3.3 mg/kg respectively.
The concentration of arsenic in both the root powders was <6.75 mg/kg (Table 6). The
concentration of the heavy metals found in Moringa and ginger root powders was consistent

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with the concentrations observed by Kaba and Goroya (2019) in roots of Moringa stenopetala,
and by Goroya et al. (2019) in ginger roots.
The abundance of essential elements such as potassium, calcium, magnesium,
sulphur, phosphorus, sodium, and the scarcity of toxic heavy metals like lead, mercury and
cadmium in the ginger and Moringa root powders justifies their use as a point-of-use water
treatment. Occurrence of the essential elements in the root powders in high concentrations
would offer additional health benefits and increase the level of nutrients in water.
4.5.3 Total carbon and nitrogen present in Moringa and ginger root powders
Carbon is considered one of the most essential macronutrients as it is required by
plants to form carbohydrates, nucleic acid, proteins etc. and is a vital part of plant biomolecules.
On an average, the dry weight of a plant cell is 50% carbon. Carbon is rarely limiting as a
nutrient, and therefore no specific deficiency symptoms of carbon have been determined in
plants. The carbon content of vegetation has been found to be surprisingly constant across a
vast variety of tissue species and types. Schlesinger and Bernhardt (1991) found that the carbon
content of biomass almost always lies between 45-50% (by oven-dry mass). The results of
elemental analysis of this research support this observation as the total carbon concentration
detected in Moringa and ginger root powders was about 42% (Table 7).
After carbon, nitrogen is one of the important elements present in plants due to its
crucial role in chlorophyll production (Muñoz -Huerta et al., 2013). It is also part of several
enzymatic proteins that regulate and catalyse plant-growth processes (Sinfield et al., 2010).
Furthermore, nitrogen helps in the production of various chemical components that protect
plants against parasites and diseases (Muñoz -Huerta et al., 2013). Crop biomass and yield are
highly affected by total nitrogen present in the plants or nitrogen fertilization given to the plants

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(Tremblay et al., 2011). Total nitrogen is the sum of all different forms of nitrogen, such as
nitrate, nitrite, ammonia, reduced and organic nitrogen etc. present in the plants.
Carbon to nitrogen (C:N) ratio of an organic substrate has a direct impact on its
composting duration, mineralization and release of nitrogen in the soil that gets available for
plant uptake (Brust, 2019). The lower the C:N ratio, more rapidly will the nitrogen be released
in the soil. A C:N ratio greater than 35 results in prolonged composting duration and microbial
immobilization (Brust, 2019). Root C:N ratios are comparatively higher than the C:N ratios of
plant leaves and they do not follow a consistent pattern (Ferlian et al., 2017).
Although differences in climate and conditions such as water availability, soil
fertility affect total carbon and nitrogen content, and hence C:N ratio of plants (Luo et al. 2017),
Zhang et al. (2014) found that the average carbon and nitrogen content of belowground tissues
of most plants growing in Yanqi basin of Northwest China is about 42.8% and 0.7%,
respectively. These amounts are similar to the total carbon and nitrogen detected in ginger and
Moringa root powders through elemental analysis. The average total carbon and nitrogen found
in Moringa root powder was 42.23% (by oven-dry mass) and 0.25%, respectively, and in ginger
powder was 41.90% and 3.7%, respectively (Table 7).
Ginger root powder was found to have about 3.5% more nitrogen (by oven-dry mass)
as compared to Moringa root powder (Table 7). This could have been due to the fact that being
a leguminous plant, ginger is able to fix nitrogen present in soil (Govindan et al., 2009), while
Moringa is unable to do so (Olson, 2014). This can also explain the higher concentration of
total carbon detected in Moringa roots than ginger roots, as leguminous plants (including
ginger) are able to exchange carbon for nitrogen with their nitrogen-fixing symbionts, and thus,
are more responsive to elevated carbon dioxide than non-leguminous plants (Rogers et al.,
2009).

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The presence of adequate amounts of carbon and nitrogen in ginger and Moringa
root powder would be beneficial for human health as carbon and nitrogen are the two most
essential and vital elements of the human body. Carbon is the second most abundant element
that makes up 18.5% of the human body (Chellan and Sadler, 2015). It is a quadrivalent element
that allows building of long and complex chains of molecules. This makes it a fundamental and
vital atom in organic chemistry. Carbon chains are used for building carbohydrates, proteins
and fats, and on breaking, these chains provide energy to the human body (Samir, 2019). On
the other hand, nitrogen is a part of essential biomolecules such as proteins, amino acids,
nucleobases, DNA and RNA (Chellan and Sadler, 2015).
4.5.4 Concentrations of anions present in Moringa and ginger root powders
Various studies have chemically analysed Moringa oleifera and ginger to determine
the concentration of essential nutrients such as calcium, magnesium, iron etc. and
phytochemicals like glucosinolates, carotenoids, flavonoids, gingerols, shogaols etc. present in
their respective plant parts. However, the concentration of various anions found in their plant
parts has not been examined. Cation uptake by plants is known to be affected by nutrient
content of soil, environmental factors such as temperature, light intensity, moisture availability
and genotypic variability of plants (Tang and Rengel, 2003; Anjana and Iqbal, 2007).
Sulphate (SO₄²-) was the most abundant anion present in Moringa root powder with
a mean concentration of 6,035 mg/kg (Table 8). Although it was present in ginger in higher
amounts (9,511 mg/kg), it was the second most abundant anion in ginger root, after phosphate
(Table 8). Sulphate is a vital component of extracellular matrix proteins and assists the human
body in detoxification of drugs, toxic metals and food additives (Schepker, 2017). It also
prevents blood transiting in capillaries from coagulating. Cerebroside sulphate, which is an
integral component of the myelin sheaths that surround neuronal axons in the brain, aids in

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maintaining optimal neurological health (Schepker, 2017). The human body uses inorganic
sulphate in the biosynthesis of essential body compounds (such as dermatan sulphate, keratan
sulphate etc.) that cannot be absorbed intact from foods (Institute of Medicine, 2005).
Deficiency of sulphate is believed to cause cardiovascular diseases (Seneff et al.,
2015), whereas its overconsumption is known to cause diarrhoea, acidosis, and ulcerative
colitis (Institute of Medicine, 2005). Although no-health based guideline value is proposed for
sulphate in foods (WHO, 2004), intake of sulphur rich foods such as coconut oil, olive oil,
legumes, garlic, onion etc. is recommended to prevent sulphate deficiency (Schepker, 2017).
Since ginger and Moringa root powders had high concentrations of sulphate (Table 8), they
can be helpful in preventing sulphate deficiency.
In human body, almost all phosphorus combines with oxygen to form phosphate
(Lewis, 2020). 85% of the body’s phosphate is present in bones, while the rest is located inside
cells, where it aids the process of energy production (Lewis, 2020). Phosphate is essential for
the formation of teeth and bones, and also serves as a building block for cell membranes,
adenosine triphosphate (ATP) and nucleic acids (Lewis, 2020). Phosphate concentration in
blood can become too high and cause hyperphosphatemia or become too low and cause
hypophosphatemia. Although hypophosphatemia is usually asymptomatic, it can cause
anorexia, osteomalacia, fatigue and muscle weakness in cases of severe chronic depletion (Imel
and Econs, 2012). There is no health-based guideline for daily intake of phosphate, and it is
only used as dietary supplement for patients who are unable to get enough phosphorus from
their regular diet (Nelson et al., 2017). Ginger and Moringa root powders were both abundant
in phosphate with 1,029.25 and 12,653.67 mg/kg phosphate present in them respectively (Table
8).

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Chloride is the key extracellular anion in humans that is vital for maintenance of
acid-base balance, serum electroneutrality, osmotic pressure, fluid homeostasis, renal function
and production of hydrochloric acid in gastrointestinal tract (Pfortmueller et al., 2018).
Increased levels of chloride in the human body are known to increase blood pressure and
hypertension (McCallum et al., 2015). It has also been indicated that increased disease severity
in ICU patients is associated with abnormal levels of chloride in their bodies (Pfortmueller et
al., 2018). The tolerable upper limit of chloride, which is the maximum level of daily intake
that is not known to cause any adverse effects, is 3.6 grams per day in adults (Institute of
Medicine, 2005). Both ginger and Moringa root powders had chloride amount lower than its
tolerable upper limit issued by Institute of Medicine. The average concentration of chloride in
ginger and Moringa root powders was found to be 737.33 and 454.25 mg/kg (Table 8).
Fluoride is another anion that is considered beneficial for human body as it maintains
integrity of teeth and bones. When consumed in optimal amounts, fluoride is known to increase
tooth mineralisation, promote dental enamel remineralization, and help reduce dentin
hypersensitivity and enamel demineralization (Palmer and Gilbert, 2012). However, acute
toxicity of fluoride is known to cause dental fluorosis, bloody vomiting, diarrhoea,
hypocalcaemia, hyperkalaemia (Kanduti et al., 2016). In both ginger and Moringa root
powders, fluoride was present in moderate amounts, with its mean concentration being 182.77
and 178 mg/kg in the root powders respectively (Table 8).
Although nitrate exists widely in water, air, soil and plants, but the chief source of
absorbed nitrate in the human body is through food (Ma et al., 2018). Nitric oxide (NO), which
is the metabolic product of dietary nitrate, plays an essential role in protecting the gastric
mucosa and cardiovascular system (Lundberg et al., 2011). When dietary nitrate is unavailable,
the endogenous production of nitrate occurs in mucosa tissues of intestine that performs various

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physiological functions similar to NO, such as blood pressure reduction, vessel protective
effect and platelet aggregation inhibition (Ma et al., 2018). Nitrate has been found to improve
age related hypertension and prevent ischemic heart disease (Ma et al., 2018).
The European Food Safety Authority recommends acceptable daily intake (ADI) of
3.7 mg of nitrate per kg body weight of adults in one day, that is, for a 60 kg human adult, ADI
of nitrate would be 222 mg/day (European Food Safety Authority, 2008). The nitrate content
in ginger and Moringa root powders varied significantly. Where the average nitrate
concentration in Moringa root powder was only 53 mg/kg, it was 9,326 mg/kg in the ginger
powder (Table 8). This means that daily consumption of ginger in very high amounts (>20 g/
day) can be dangerous due to the high concentration of nitrate present in its rhizomes.
The remarkable difference in the amount of nitrate found in the two root powders
can be attributed to genotypic variability between the two plant species or variation in their
watering frequency or variation in light intensity they received (Anjana and Iqbal, 2007).
Moringa plants were watered about five times a week, whereas ginger was watered three times
a week in summers and only two times a week in winters. Anjana and Iqbal (2007) have shown
that plants accumulate less nitrate when they are watered more frequently as frequent irrigation
helps in leaching off excess nitrate. Moringa plants also received six more hours of
supplemental lighting each day than ginger plants. The nitrate content in plants has been found
to be inversely correlated to light intensity received by the plants (Anjana and Iqbal, 2007). On
decreasing light intensity available to plants, synthesis of soluble carbohydrates and organic
acids declines and their role as an osmoticum is taken up by nitrate, which is absorbed and
accumulated by plant roots more readily when light is a limiting factor (Anjana and Iqbal,
2007). Thus, future studies should measure changes caused in nitrate content of ginger roots

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by providing more water and light to the plants. This might help bringing down the nitrate
concentration in ginger roots to a level recommended by European Food Safety Authority.
Bromide and nitrite were the two least abundant anions in ginger and Moringa root
powders, with their average concentrations being less than 10 mg/kg in both the root powders
(Table 8). Since 10 mg/kg was the method detection limit, the exact concentrations of bromide
and nitrite in Moringa and ginger root powders could not be determined. Nitrite was previously
thought to be carcinogenic, and its beneficial functions were ignored. However, most existing
research on tumours and nitrite ignored some complicated compounds in target foods, causing
contradictory conclusions among researchers (Ma et al., 2018). Considering various protective
effects, dietary nitrite seems to play a significant role in physiological functions of the body
through its provision of non-enzymatic NO (Ma et al., 2018). Similarly, bromide was once used
as a sedative and an anticonvulsant as it is known to cause nausea, abdominal pain, vomiting
and even paralysis at large doses, but in smaller amounts, it is considered nutritionally
beneficial (WHO, 2009). The World Health Organisation has recommended the upper limit of
concentration of daily nitrite uptake to 0.06-0.07 mg/kg (Weitzberg and Lundberg, 2013) and
the average daily intake (ADI) of bromide to 0-1 mg/kg body weight (WHO, 2009).
Since the concentration (mg/kg) of anions in ginger and Moringa root powders was
found to be comparable to their desired amounts in food, it can be concluded that the two root
powders can offer additional health benefits and increase the nutrient content of water along
with treating water of E. coli populations.
4.5.5 Electrical conductivity and pH of Moringa and ginger root powders
Early studies on protoplasmic permeability in plants revealed its association to
electrical conductivity (EC). It became evident that the higher the permeability of protoplasm,
the more easily will an electric current pass through it. Hence, EC of protoplasm can determine

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its permeability to ions (Osterhout, 1918). In early 1900s, EC of plant sap was widely measured
to understand problems of plant physiology (Stiles and Jorgensen, 1914) as it was shown that
permeability and EC of plant tissues increases when they are injured from mechanical rupture,
freezing or from use of chemicals (Dexter et al., 1932). Electrical conductivity in plants has
shown to affect water absorption, retention of the absorbed water (Canna©, 2020), water use
efficiency and photosynthetic capacity of plants (Xu et al., 1994).
In contemporary greenhouse studies, EC of plant tissues is measured as it is
proportional to the total amount of ions and fertilizer salts present in that plant part (Nemali,
2018). Kautkar et al. (2014) found that EC in ginger plants increased with increasing
temperature and ionic concentration. Since concentration of all the anions—sulphate, fluoride,
chloride, nitrite, bromide, nitrate and phosphate, was found to be higher in ginger roots than in
Moringa roots (Table 8), EC of ginger was found to be significantly higher than the EC of
Moringa roots (Table 9).
EC is an important indicator of a plant’s health that impacts its nutrient uptake, and
thus consequently, affects the plant’s growth (Ding et al., 2018). High EC of the ginger roots
could be a result of higher concentration of anions (especially nitrate) found in the root powder
and/or overfertilization (Ding et al., 2018). As discussed in section 4.5.4, future studies should
try bringing down the nitrate content, and consequently EC, of the ginger roots by providing
more light and water to the plants to remove excess salt.
Maintaining a stable acid-base status in plant tissues is a fundamental homeostatic
process that is necessary for mineral nutrition (Martiniere et al., 2018) and for preserving the
metabolic function of macromolecules like proteins (Egginton et al., 1999). Plants can regulate
their pH through biochemical or biophysical mechanisms. Biochemical pH regulation involves

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chemical transformations that produce or consume H+, whereas biophysical pH regulation
involves transmembrane fluxes of H +/OH- (Raven, 1985).
The pH of ginger and Moringa root powders was found to be 5.75 and 5.33,
respectively (Table 9). These values are consistent to the pH values of ginger roots determined
by Williams et al. (2003) and Moringa leaves determined by Adetitun et al. (2013). Williams
et al. (2003) found that pH of ginger (Hedychium gardnerianum) lies between 5.5-6.6, whereas
Adetitun et al. (2013) found the pH of aqueous extract of dried Moringa leaves to be 5. Since
pH of human bodies is partially determined by mineral densities of food they consume, food
with pH higher than 4.6 is recommended to reduce chronic diseases and ailments like diabetes,
hypertension, vitamin D deficiency, arthritis, low bone density etc. (Levy, 2020). As both the
root powders had pH higher than 5, it can be concluded that the Moringa and ginger root
powders are safe for human consumption. The pH of Moringa and ginger root powders was
also found to be optimum for their coagulating and water purifying applications as coagulants
with slightly acidic pH values (ranging between 5 and 7) are considered favourable for removal
of organic matter through charge neutralization mechanism (Naceradska et al., 2019).
4.6 Conclusion
While essential elements such as potassium, calcium, magnesium, sodium etc. were
found to be abundant in Moringa and ginger root powders, toxic heavy metals like lead,
mercury and cadmium were present in meagre amounts in the two root powders (Table 6).
Similarly, the root powders were rich in beneficial anions like phosphate, sulphate, chloride
etc. (Table 8) and had pH values optimum for human intake (Table 9). The only anion found
in concentrations higher than the recommended daily intake in ginger roots was nitrate (Table
8), thus suggesting that ginger should be consumed in smaller quantities (<20 g/day). The high
concentration of nitrate in ginger also led to higher EC of ginger roots. Future studies should

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try bringing down the nitrate concentration of ginger rhizomes by providing more water and
light to the plants. Except nitrate, the concentration of all essential elements and anions in
Moringa and ginger root powders were found to be optimal. Therefore, it can be concluded
that Moringa and ginger root powders are safe for human consumption and can offer additional
health benefits and increase the nutrient content of water along with treating water of E. coli
colonies.

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Chapter 5: Effect of Moringa and ginger root powders on E. coli population
5.1 Abstract
Shortage of portable water is a serious problem faced by the world today. Many
water treatment methods are available; however, economic, and cultural factors often impair
implementation of these methods, particularly in underdeveloped and developing countries.
Moringa and ginger root powders appear to offer a promising alternative to treat contaminated
water. The objectives of this study were to determine the sole and the combined effects of
Moringa and ginger root powders on ATCC® 8739™ Escherichia coli population. The
antibacterial efficacy of three replicates of Moringa root powder concentrations—800, 1400
and 2000 mg/L, three ginger root powder concentrations—800, 1400 and 2000 mg/L, and three
concentrations of the two root powders combined in 1:1 ratio to form 800, 1400 and 2000 mg/L
was assessed using IDEXX Colilert-18 Test/Quanti-Tray/2000 Method. The treatments were
added to 100 mL buffered milliQ water containing E. coli diluted to the factor of 1x10-8, stirred,
and made to sit for an hour before filtering into sample bottles and conducting bacteriological
analysis. The mean E. coli numbers decreased significantly (p<0.05) on adding the root powder
concentrations to contaminated water. The Moringa roots were found to be more effective in
reducing E. coli population than ginger roots. The bacterial counts decreased by 84.35%,
88.66% and 86.94% on adding 800, 1400 and 2000 mg/L Moringa root powder concentrations,
respectively, and by 57.82%, 62.63% and 60.44% on adding the same concentrations of ginger
root powder. The combination of the two root powders seemed to have a synergistic effect on
E. coli as the E. coli counts in contaminated water decreased by 90.65%, 93.79% and 94.78%
on adding 800, 1400 and 2000 mg/L concentrations of Moringa and ginger root powders mixed
in 1:1 ratio.

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5.2 Introduction
Among all of Earth’s natural resources, water is deemed the most important resource
as it forms the basic pivotal medium for origin of life (Dwivedi, 2017). Water is critical for
survival of all living organisms and has a direct impact on their health (Abiyu et al., 2018).
Lack of clean drinking water, also referred to as potable water, is a huge problem faced
currently by many sections of population of the world (Varkey, 2018). The contaminants
leading to impure water include a wide spectrum of pathogens and chemicals (Ahmed and
Ismail, 2018). According to WHO, 80% of the total human diseases are water-borne and claim
5 million deaths globally each year (WHO, 2019). Most of these deaths are attributed to a wide
range of microorganisms including Escherichia coli, a common microbe found in contaminated
water (Cabral, 2010).
Contaminated water has become an environmental issue in urban areas of both
developing and developed countries. Although developing countries are marked and
characterized by lower industrialization, their wastewater and solid waste management
strategies are insufficient, thus facilitating the problem of water contamination (BalthazardAccou et al., 2019). Moreover, unexpected population growth in developing countries results
in water quality degradation and causes an increase in microbial loads in water resources
(Balthazard-Accou et al., 2019). Due to these reasons, developing nations in Africa and South
Asia are the ones worst affected by the problem of water contamination.
Many cost-effective methods are available to treat contaminated drinking water.
These methods are chlorination, boiling, sand filtration and solar disinfection. However,
cultural, economic, and social factors often impair implementation of these methods in
developing and underdeveloped countries (Morgan et al., 2019). Boiling is the oldest and the
most common method of Household Water Treatment (HWT) used by an estimated 1.1 billion

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people in 21% of households in middle- and low-income countries (Juran and MacDonald,
2014). It is an effective method known to kill all classes of water-borne pathogens and is even
known to treat highly turbid water (Agrawal and Bhalwar, 2009). However, boiling requires
consumption of energy that can be costly or not easily available in a lot of regions (Laurent,
2005). It affects the taste of water and does not have any impact on chemical contaminants that
might be present in water. In addition to this, there is a high risk for potential microbial
recontamination during storage of boiled water (Laurent, 2005).
Chlorination is a simple and inexpensive method that provides a stable residual.
Thus, the potential risk of microbial recontamination during storage is lowered (Laurent, 2005).
Chlorination is effective against many waterborne pathogens except Cryptopdoridium parvum,
Cyclospora cayetanensis, Giardia lamblia, helminth eggs and Mycobacteria species (Laurent,
2005). However, the effectiveness of this method is highly dependent on the quality of water
as its efficiency is reduced in highly turbid water. There is also some cultural resistance against
this method regarding the taste of chlorine (Laurent, 2005).
Solar disinfection and filtration through sand, fabric or paper are other widely used
methods for treating water in developing and underdeveloped countries. Solar disinfection
involves use of natural UV irradiation to eliminate pathogens present in water. Although this
method is inexpensive and does not involve addition of any chemical to water, only small
quantities of water can be treated by this method, and it requires good amount of sunshine per
day (Laurent, 2005). Sand filtration, on the other hand, is recommended only at community
level, and not for individual households because of its large apparatus that needs regular
maintenance by trained individuals (Laurent, 2005). Most paper and fabric filters have pore
size greater than the diameters of bacteria and viruses, thus reducing its efficiency against these
microbes (Agrawal and Bhalwar, 2009).

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Water treatment plants in developed and developing countries involve use of
chemical coagulants such as ferrous chloride and aluminium sulphate (alum) that can adversely
affect human health. For example, aluminium is a recognized neurotoxin that can cause
neurodegeneration (Maya et al., 2016) and it has been indicated to be a causative agent of
neurological diseases like pre-senile dementia (Abiyu et al., 2018). Therefore, natural
coagulants and antimicrobial agents are moving from fringe to mainstream use with majority
of people seeking inexpensive water treatment remedies that are free of chemical side effects.
These natural antimicrobial agents can also be used in underdeveloped and developing
countries where most of the rural population depends on contaminated and turbid water from
rivers, streams, and dams (Varkey, 2018).
Moringa (Moringa oleifera) and ginger (Zingiber officinale) appear to offer a
promising alternative to treat contaminated water, since Moringa and ginger grow extensively
in areas where the problem of contaminated water is quite alarming and various parts of the
trees have been reported to possess antibacterial and water purifying properties. Moringa seeds
were traditionally used in various African countries such as Ethiopia, Uganda, and Sudan for
purifying contaminated water and removing its turbidity (Jahn, 1977). Hitherto the seeds have
been extensively studied for their water purifying and antibacterial efficacy. Seed extract of
Moringa has been shown to kill 90-99.99% of bacteria in turbid water (Lea, 2010) and in
sedimented sludge (Madsen et al., 1987).
However, utilizing Moringa roots in the antimicrobial research would offer added
benefits as roots, unlike seeds, can be harvested within the first year of planting the trees
(Morgan et al., 2019) and without damaging the tree (Fuglie, 2001). This would reserve the
nutrient-rich parts of the shoot such as leaves and seeds to be used as a food source (Morgan et
al., 2019). The only research done to evaluate antibacterial efficacy of Moringa root powder

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used three concentrations (250, 450 and 600 mg/L) and found that the maximum concentration
(600 mg/L) reduced E. coli colonies by 87% in contaminated pond water (Morgan et al., 2019).
Likewise, ginger extract has been reported to suppress the growth of various Grampositive and Gram-negative bacteria such as Staphylococcus aureus, Haemophilus influenzae,
Streptococcus pyogenes/ pneumoniae, Salmonella typhimurium, Pseudomonas aeruginosa and
E. coli (Chrubasik et al., 2005; Gupta and Ravishankar, 2005). However, not much research
has been done on ginger’s potential to purify contaminated water other than its potential to
inhibit growth of a variety of microorganisms that might be present in water.
This chapter focussed on determining whether a further reduction in E. coli
population can be achieved by increasing the concentrations of Moringa and ginger root
powders, and whether combining ginger with Moringa will have synergistic effect on E. coli.
Thus, the objectives along with null hypothesis (H0) and alternative hypothesis (Ha) of this
chapter were:
Objective 1: To determine the effect of Moringa root powder on E. coli population when it was
added to the contaminated water at 800, 1,400 and 2,000 mg/L.
H0 (1): Increasing concentrations of Moringa root powder will have no effect on
E. coli population.
Ha (1): Increasing concentrations of Moringa root powder will result in either
decreasing or increasing E. coli counts present in contaminated water.
Objective 2: To determine the effect of ginger powder on E. coli population when it was added
to the contaminated water at 800, 1,400 and 2,000 mg/L.

P a g e | 62
H0 (2): Increasing concentrations of ginger root powder will have no effect on E. coli
population.
Ha (2): Increasing concentrations of ginger root powder will result in either
decreasing or increasing E. coli counts present in contaminated water.
Objective 3: To determine the combined effects of ginger and Moringa root powders on E. coli
population when both the root powders were mixed in 1:1 ratio to form 800, 1,400 and 2,000
mg.
H0 (3): Increasing concentrations of amalgamation of Moringa and ginger root
powders will have no effect on E. coli population.
Ha (3): Increasing concentrations of amalgamation of Moringa and ginger root
powders will result in either decreasing or increasing E. coli counts present in
contaminated water.

P a g e | 63
5.3 Methods
5.3.1 Propagating E. coli strain
For preparing 100 mL of LB broth (Luria-Bertani), 2.5 grams of LB broth (Miller)
powder was dissolved in 100 mL of deionized water. It was stirred at 700 rpm for 15 minutes
using VWR Scientific 220 mini-hot plate stirrer, following which it was autoclaved at 121 o C
for 15 minutes (P1 liquid cycle). Five millilitres of the freshly prepared LB broth were pipetted
into a sterile 16 mL glass test-tube.
For propagating E. coli, ATCC® 8739-MINI-PACK™ was used. Extracting E. coli
from a natural source of contaminated water (pond water shared with domestic animals) was
the first preference for this research. However, some practical and logistical problems (detailed
in Appendix A) were encountered while collecting and isolating E. coli due to which cultured
E. coli strains were used instead.
A

frozen

mini-cryovial

(Figure

6)

containing ATCC® 8739™ E. coli was thawed by
placing it in room temperature for 20 minutes to melt
all the ice crystals present within the vial. Immediately
after thawing, the outside surface of the cryovial was
wiped with 70% ethanol and its entire contents were
emptied into the 16 mL test-tube containing freshly
prepared LB broth. The inoculated broth was
incubated at 370 C for 24 hours.

Figure 6. A thawed ATCC® minicryovial containing 8739™ E.
coli. (Photograph by author,
taken on April 12, 2021).

P a g e | 64
5.3.2 Preparing streak plates of E. coli
For preparing MUG agar plates, 13.86 grams of powdered MUG agar medium was
added to 600 mL of deionized water in a 1000 mL beaker. To mix thoroughly, the solution was
stirred on a VWR mini-hot plate stirrer (Model 220) using a magnetic stir bar and was brought
to boil. When the powder completely dissolved in the solution, the beaker was covered with
aluminium foil and autoclaved at 121 0 C for 15 minutes (P1 liquid cycle).
After autoclaving, the beaker was rested on a counter for 20 minutes until it could
be touched by naked hands and before the nutrient media began to solidify. The nutrient agar
was gently poured in sterile petri dishes until it covered about half the volume of each petri
dish. To prevent condensation, the plates were left on a counter overnight to solidify at room
temperature. The next day, MUG agar plates were inverted and stored in a refrigerator at 4 0 C.
A streak plate of E. coli was prepared by dipping a sterilized metal loop into
inoculated LB broth and streaking it across first quadrant using aseptic technique. The metal
loop was sterilized and the bacteria from first quadrant was streaked across the second
quadrant. Similarly, the third and the fourth quadrants were streaked through, with the
inoculating loop sterilized between each quadrant. The streak plates were then incubated at 370
C for 24 hours.
5.3.3 Preparing stock potassium dihydrogen phosphate buffer solution
Recipe of a buffered dilution water mentioned in the book- Standard Methods for
the Examination of Water and Wastewater (Baird and Bridgewater, 2017), was followed to
prepare a stock phosphate buffer solution. Thirty-four grams of potassium dihydrogen
phosphate (KH2PO4) were added to 500 mL of deionized water. The solution was stirred using
a mini-hot plate stirrer (VWR Scientific 220) and a magnetic stir bar for about 10 minutes until

P a g e | 65
the potassium salt dissolved completely in the water. The pH of the solution was checked using
Orion Star TM A211 Benchtop pH meter and was found to be 4.19. It was adjusted to 7.2 by
adding small quantities of 1 mol/L NaOH solution. Distilled water was added to bring the final
volume of the buffer solution to one litre. The buffer solution was then autoclaved at 121 0 C
for 15 minutes. Once the autoclaved buffer solution reached room temperature, it was stored
in a refrigerator at 40 C. To prepare buffered dilution water, 1.25 mL of the stock potassium
dihydrogen phosphate buffer solution was pipetted to one litre of distilled water.
5.3.4 Serial dilution analysis and enumeration of E. coli
To obtain a countable number of E. coli colonies and to ensure that the same
standardized E. coli numbers are used in each treatment, serial dilution analysis was conducted.
Freshly prepared LB broth (5 mL) was placed in a sterile 16 mL test tube and was inoculated
with E. coli. A single E. coli colony was selected from a cultured MUG agar plate and was
transferred to the LB broth using a sterile inoculating loop. The inoculated LB broth was then
incubated at 370 C for 24 hours so that the bacteria entered their stationary growth phase. The
approximate time for E. coli 8739 to hit the stationary phase has been recorded to be about 6
hours (Wang et al., 2018).
The following day, serial dilutions of 10 -2, 10-4, 10-6, 10-7, 10-8 and 10-9 were
prepared from the inoculated broth. The dilution factor of 10-2 was prepared by pipetting 1 mL
of the inoculated broth into 100 mL of buffered milliQ water. Similarly, 10 -4 dilution was
prepared by pipetting 1 mL of the 10-2 dilution into 100 mL of buffered milliQ water. This was
repeated to make dilutions of 10-6 and 10-8. 10-7 and 10-9 dilutions were prepared by pipetting
0.1 mL from the 10-4 dilution and 1 mL from the 10-7 dilution respectively. The dilutions were
prepared in sterile 120 mL sample bottles. All the dilutions were rested for 3 hours and 30
minutes before they were analysed to enumerate E. coli numbers.

P a g e | 66
IDEXX Colilert-18 Test/Quanti-Tray/2000 Method (IDEXX® Laboratories, 2013)
was used for enumeration of E. coli by determining the Most Probable Number (MPN) of E.
coli present in each of the dilutions. The powdered Colilert-18 reagent was aseptically added
to each sample bottle, following which all the bottles were vigorously shaken for two minutes
(Figure 7). The dilutions were then poured into sterile Quanti-Trays/2000 (Figure 7), which
were then sealed using IDEXX Quanti-Tray™ Sealer (Figure 8). The Quanti-Trays were placed
in an incubator at 370 C for 24 hours.

Figure 7. Colilert-18 reagent being added to the sample bottles (Left). After mixing Colilert18 reagent in sample bottles, a sample being poured into Quanti-Tray/2000 (Right).
(Photographs by author, taken on May 13, 2021).

P a g e | 67

Figure 8. A Quanti-Tray being sealed using IDEXX Quanti-Tray™ Sealer. (Photograph by
author, taken on May 13, 2021).

After 24 hours, the Quanti-Trays were removed from the incubator and viewed under
UV light. The number of wells that fluoresced under the UV light were counted for each
Quanti-Tray. The information was compared with IDEXX MPN table to determine MPN of E.
coli present in each 100 mL of dilution samples. The serial dilution factor of 1x10 -8 was found
to be a suitable dilution factor that resulted in countable MPN of E. coli per 100 mL of sample.
5.3.5 Evaluating antibacterial effect of Moringa and ginger root powders
Antibacterial efficacy of three concentrations of Moringa root powder alone (800,
1400 and 2000 mg/L), three concentrations of ginger root powder alone (800, 1400 and 2000
mg/L), and three concentrations of Moringa and ginger root powders combined in 1:1 ratio to
form 800, 1400 and 2000 mg/L was assessed. Three replicates of each the above-mentioned
concentration were prepared and evaluated for their antibacterial effect on E. coli.
In 12, 250 mL glass beakers, 100 mL milliQ water was poured. To each beaker, 125
µL of potassium dihydrogen phosphate stock buffer solution was added and mixed thoroughly

P a g e | 68
using stirring rods. From each beaker containing 100 mL buffered milliQ water, 10 mL was
pipetted out into an empty 250 mL beaker using a glass pipette. All 24 beakers were autoclaved
at 121 0 C for 15 minutes.To nine beakers each containing 10 mL milliQ water, 0.08 g, 0.14 g
and 0.2 g of Moringa root powder was added (each root powder concentration in three beakers).
The remaining three beakers with 10 mL of buffered milliQ water and no Moringa root powder
were used as a control. All the 12 treatments were simultaneously stirred at 500 rpm for 15
minutes on mini hot-plate stirrers (VWR Scientific 220) using magnetic stir bars, after which
they were added to the 12 beakers containing 90 mL buffered milliQ water to form 12, 100 mL
samples.
Fresh serial dilutions of 10-2, 10-4 and 10-6 were prepared from an inoculated 5 mL
LB broth (that had been incubated for 24 hours after its inoculation). One millilitre from the
dilution factor of 1x10-6 was pipetted into each 100 mL sample to give rise to standardized
bacterial concentration of a dilution factor 1x10-8 per 100 mL sample. All the 12 samples were
stirred at 500 rpm for 10 minutes on VWR mini hot-plate stirrer to thoroughly mix the bacterial
concentrations with the Moringa root powder. After this, the samples were left to sit for one
hour before performing bacterial enumeration procedures.
After one hour, the samples were filtered into sterile 120 mL sample bottles using
Whatman 150 mm, grade 2 filter paper (catalogue number: 1002-147). Total time taken by the
samples to filter through the filter paper varied from about 45 minutes to two hours, depending
on the concentration of root powders added to the samples. While the controls (consisting of
only milliQ water and no root powder concentration) took about 45 minutes to filter through
the Whatman grade 2 filter paper, samples with 2000 mg/L Moringa root powder took about
two hours to complete the filtration process. On completing 3 hours and 30 minutes after
making the standardized bacterial concentrations, the sample bottles were capped,

P a g e | 69
appropriately labelled, and analysed using IDEXX Colilert-18 Test/Quanti-Tray/2000 Method
(IDEXX® Laboratories, 2013). After 24 hours, the Quanti-trays were removed from the
incubator and E. coli present in each sample were enumerated using MPN reference table.
Similarly, the antibacterial effect of ginger root powder concentrations (three
replicates of 0, 800, 1400 and 2000 mg/L each) and the combined effect of the two root powders
mixed (three replicates of 0, 800, 1400 and 2000 mg/L each) on standardized E. coli numbers
was evaluated. To study the synergistic antibacterial effect of the two root powders, Moringa
and ginger root powders were mixed in 1:1 ratio. For instance, 400 mg each of Moringa and
ginger root powder was mixed to evaluate the effect of their combined concentration (800
mg/L) on E. coli population.
5.3.6 Statistical analysis
Statistical analysis of the data was performed by R Studio software. Mean and
standard deviation, which were used to present results graphically, were calculated. A one-way
Analysis of Variance (ANOVA) and a Tukey post hoc comparison test was performed to
ascertain the significance of the effect of Moringa and ginger root powders on E. coli
population. Significance of all tests was determined at a p-value of 0.05.
5.4 Results
5.4.1 Propagating E. coli strain
ATCC® 8739™ E. coli was grown on MUG agar plates using streak plate technique.
The E. coli colonies on MUG agar plate appeared off-white and creamy in colour and had a
shiny texture. The colonies were circular, smooth, opaque and were slightly raised (Figure 9).
The E. coli colonies growing on MUG agar plate fluoresced when viewed under UV light, thus
confirming that the bacterial growth on MUG agar plates consisted of pure E. coli colonies.

P a g e | 70

Figure 9. A streak MUG agar plate with
E. coli colonies. (Photograph by
author, taken on May 6, 2021).

Figure 10. Comparison of sterile LB broth
with inoculated LB broth. (Photograph by
author, taken on May 12, 2021).

When a single E. coli colony was selected from MUG agar plate and transferred to
sterile LB broth, the inoculated LB broth was incubated for 24 hours before using the broth for
subsequent analysis. After 24 hours, the inoculated LB broth appeared cloudy with sediments
settled at the bottom of the test-tube (Figure 10), whereas sterile LB broth was clearer and
transparent.
5.4.2 Serial dilution analysis
The dilution factor of 1x10-8 was found to be suitable and resulted in a countable
1299.7 MPN of E. coli per 100 mL of sample. The Quanti-trays with the samples of dilution
factors— 1x10-2, 1x10-4, 1x10-6 and 1x10-7 were all saturated and showed E. coli counts
(>2419.6 MPN of E. coli/100 mL of dilution sample) higher than the bacterial detection limit
of IDEXX Colilert-18. The dilution factor of 1x10-9 resulted in 11.8 MPN E. coli/100 mL of
sample. Hence, the 1x10-8 dilution factor was found to be adequate and was used in all
antibacterial analyses.

P a g e | 71
5.4.3 Antibacterial effect of Moringa and ginger root powders
As described in part 5.3.5 of this thesis, the antibacterial effect of three Moringa root
powder concentrations— 800, 1400 and 2000 mg/L, was determined on standardized E. coli
numbers. Three replicates of each Moringa root powder concentration along with three
replicates of control (0 mg/L of Moringa in 100 mL of milliQ water) were analysed using
IDEXX Colilert-18/Quanti-Tray/2000 Method.
5.4.3.1 Moringa root powder treatments
The mean E. coli counts decreased from 677.33 MPN to 104.13, 74.6 and 87.53
MPN on adding 800, 1400 and 2000 mg/L Moringa root powder to the contaminated water,
respectively (Figure 11), thus rejecting the null hypothesis [H0 (1)].
900

Mean E.coli counts (MPN/100mL)

800
700
600

500
400
300
200
100
0

0 mg/L

800 mg/L

1400 mg/L

Moringa cocentration (mg/L)

2000 mg/L

Figure 11. Mean E. coli counts found in different Moringa root powder treatments, with
standard deviation as error bars.

P a g e | 72
A one-way ANOVA revealed that a significant decrease (F (3) =136.38, p= 3.30e07) in E. coli population was observed on adding Moringa root powder concentrations to
contaminated water (Table 10). A post hoc Tukey test showed that E. coli counts in each of the
Moringa concentration— 800, 1400 and 2000 mg/L, varied significantly (p<0.05) from the
bacterial counts in control (Table 11). However, a significant difference in E. coli numbers was
not observed within 800 and 1400 mg/L (p= 0.84), 800 and 2000 mg/L (p= 0.97), and 1400
and 2000 mg/L Moringa treatments (p= 0.98).
Table 10. One-way analysis of variance for different Moringa root powder treatments— 800,
1400 and 2000 mg/L, on mean E. coli counts. A significant difference is considered at p-value
<0.05.
Source of variation
Moringa treatments
Residuals

Degrees of
freedom
3
8

Sum of
squares
780768.6
15265.94

Mean
square
260256.2
1908.2425

F-value

p-value

136.38
-

3.30e-07
-

Table 11. A Tukey’s test showing differences in means, confidence levels (upper and lower
limit) and adjusted p-values for all possible pairs of different concentrations of Moringa root
powder at 95% family-wise confidence level.
Treatment groups

Difference in

Lower limit

Upper limit

means

Adjusted pvalue*

800 and 0 mg/L

-573.20000

-687.4195

-458.98051

0.0000011

1400 and 0 mg/L

-602.73333

-716.9528

-488.51384

0.0000007

2000 and 0 mg/L

-589.80000

-704.0195

-475.58051

0.0000009

1400 and 800 mg/L

-29.53333

-143.7528

84.68616

0.8399143

2000 and 800 mg/L

-16.60000

-130.8195

97.61949

0.9645943

2000 and 1400 mg/L

12.93333

-101.2862

127.15282

0.9825329

* Significant at p-value <0.05

P a g e | 73
5.4.3.2 Ginger root powder treatments
The antibacterial effect of three ginger root powder concentrations— 800, 1400 and
2000 mg/L, and a control with 100 mL of milliQ water and no ginger powder (three replicates
of each treatment) was determined using IDEXX Colilert-18. The mean E. coli numbers in
control were found to be 1085.07 MPN, but the bacterial counts decreased significantly (F (3)
=12.75, p= 0.002) in treatments with ginger root powder (Table 12), thus rejecting the null
hypothesis [H0 (2)]. The mean E. coli counts in treatments with 800, 1400 and 2000 mg/L
ginger root powder concentrations were found to be 433.4, 389.1 and 427.87 MPN respectively
(Figure 12).

Mean E.coli counts (MPN/100mL)

1600
1400

1200
1000
800
600
400
200
0

0 mg/L

800 mg/L

1400 mg/L

2000 mg/L

Ginger cocentration (mg/L)

Figure 12. Mean E. coli counts found in different ginger root powder treatments, with standard
deviation as error bars.

P a g e | 74
Table 12. One-way analysis of variance for different ginger root powder treatments— 800,
1400 and 2000 mg/L, on mean E. coli counts. A significant difference is considered at p-value
<0.05.
Source of variation
Ginger treatments
Residuals

Degrees of
freedom
3
8

Sum of
squares
1008335.13
210849.39

Mean
square
336111.71
26356.17

F-value

p-value

12.75
-

0.002
-

A post hoc Tukey test revealed that the E. coli numbers in the control were
significantly higher than in the treatments with 800 mg/L (p= 0.005), 1400 mg/L (p= 0.003)
and 2000 mg/L ginger powder (p= 0.005). However, a statistically significant difference in E.
coli numbers was not observed within treatments with ginger root powder concentrations as
adjusted p-value was found to be higher than 0.05 in all these cases (Table 13).
Table 13. A Tukey’s test showing differences in means, confidence levels (upper and lower
limit) and adjusted p-values for all possible pairs of different concentrations of ginger root
powder at 95% family-wise confidence level.
Treatment groups

Difference in

Lower limit

Upper limit

means

Adjusted pvalue*

800 and 0 mg/L

-651.666667

-1076.1539

-227.1794

0.0051241

1400 and 0 mg/L

-695.966667

-1120.4539

-271.4794

0.0034215

2000 and 0 mg/L

-657.200000

-1081.6872

-232.7128

0.0048683

1400 and 800 mg/L

-44.300000

-468.7872

380.1872

0.9861884

2000 and 800 mg/L

-5.533333

-430.0206

418.9539

0.9999715

2000 and 1400 mg/L

38.766667

-385.7206

463.2539

0.9906214

*Significant at p-value <0.05

P a g e | 75
5.4.3.2 Moringa and ginger root powder treatments
The mean E. coli numbers decreased from 1901.87 MPN (in control) to 169.87,
118.1 and 96.17 MPN on adding 800, 1400 and 2000 mg/L Moringa and ginger root powders
combined in 1:1 ratio to contaminated water respectively (Figure 13), thus rejecting the null
hypothesis [H0 (3)].

3000

Mean E.coli counts (MPN/100mL)

2500

2000

1500

1000

500

0

0 mg/L

800 mg/L

1400 mg/L

2000 mg/L

Moringa + Ginger cocentration (mg/L)

Figure 13. Mean E. coli counts detected in treatments with Moringa and ginger root powder
concentrations, with standard deviation as error bars.

The E. coli counts decreased significantly (F (3) =44.46, p= 2.47e-05) on adding the
combination of both Moringa and ginger root powder to contaminated water (Table 14). Table
15 shows results of the Tukey’s test, according to which E. coli counts in each of the treatments
with the root powders varied significantly (p<0.05) from the bacterial counts in control, but a
significant difference in E. coli numbers was not observed within treatments with 800 and 1400

P a g e | 76
mg/L (p= 0.99), 800 and 2000 mg/L (p= 0.98), and 1400 and 2000 mg/L Moringa and ginger
root powders (p= 0.99).
Table 14. One-way analysis of variance for different Moringa and ginger root powder
treatments— 800, 1400 and 2000 mg/L, on mean E. coli counts. A significant difference is
considered at p-value <0.05.
Source of
variation
Moringa+ ginger
treatments
Residuals

Degrees of
freedom
3

Sum of
squares
7088094.42

Mean
square
2362698.14

F-value

p-value

44.46

2.47e-05

8

425112.47

53139.05

-

-

Table 15. A Tukey’s test showing differences in means, confidence levels (upper and lower
limit) and adjusted p-values for all possible pairs of different concentrations of Moringa and
ginger root powder at 95% family-wise confidence level.
Treatment groups

Difference in

Lower limit

Upper limit

means

Adjusted pvalue*

800 and 0 mg/L

-1732.00000

-2334.7405

-1129.2595

0.0000731

1400 and 0 mg/L

-1783.76667

-2386.5072

-1181.0262

0.0000588

2000 and 0 mg/L

-1805.70000

-2408.4405

-1202.9595

0.0000538

1400 and 800 mg/L

-51.76667

-654.5072

550.9738

0.9921605

2000 and 800 mg/L

-73.70000

-676.4405

529.0405

0.9782437

2000 and 1400 mg/L

-21.93333

-624.6738

580.8072

0.9993845

*Significant at p-value <0.05

The mean percentage decreases observed in E. coli counts on adding different
concentrations of Moringa root powder alone, ginger root powder alone and the combination
of the two root powders were calculated and compared. In Moringa root powder
concentrations, maximum percentage decrease in E. coli numbers was observed in 1400
mg/L— 88.66%, as compared to 800 and 2000 mg/L concentrations that caused 84.35% and
86.94% reduction in E. coli respectively (Figure 14). Although the E. coli numbers present in

P a g e | 77
1400 mg/L treatment were not significantly different from the bacterial counts in 800 and 2000
mg/L treatments (Table 11), 1400 mg/L was considered the most effective concentration of
Moringa root powder as it caused higher percentage decrease in E. coli population than
achieved by 800 and 2000 mg/L concentrations (Figure 14). Like in the case of Moringa root
powder, 1400 mg/L ginger root powder concentration was found to be the most effective at
reducing E. coli numbers. Where 1400 mg/L ginger root powder concentration reduced E. coli
numbers by 62.63%, treatments with 800 and 2000 mg/L concentrations reduced bacterial
counts by just 57.82% and 60.44% respectively (Figure 14).

Moringa

Ginger

Moringa+ Ginger

100

Mean % decrease

90
80
70
60
50
40

800 mg/L

1400 mg/L

2000 mg/L

Concentration of treatments (mg/L)

Figure 14. Mean percentage decrease in E. coli numbers observed in different concentrations
of Moringa root powder alone, ginger root powder alone and the combination of the two root
powders, with standard deviation as error bars.

Moringa and ginger root powders combined in 1:1 ratio, reduced bacterial counts in
contaminated water more efficiently than either of the two root powders alone. Out of the three
concentrations of combined Moringa and ginger root powders, maximum percentage decrease
in E. coli numbers was caused by 2000 mg/L— 94.78%, as compared to 800 and 1400 mg/L
concentrations that reduced E. coli counts by 90.65% and 93.79% respectively (Figure 14).

P a g e | 78
5.5 Discussion
Moringa root powder was found to be more effective in reducing E. coli numbers in
contaminated water than ginger root powder. The Moringa root powder concentration with
highest antibacterial efficacy against E. coli was found to be 1400 mg/L. It reduced the E. coli
counts in contaminated water by 88.66%, whereas 800 and 2000 mg/L Moringa root powder
concentrations reduced the bacterial counts by 84.35% and 86.94% respectively (Figure 14).
These values were similar to the literature values for percentage reduction in bacteria caused
by seed paste or seed extract of Moringa oleifera. Madsen et al. (1987) reported a 90%
reduction of bacterial load in water treated with Moringa seed paste and Oluduro and Aberiye
(2007) reported that Moringa seed extract reduced about 88% of the total bacteria in surface
water.
The percentage decrease observed in E. coli was similar to the values reported by
Morgan et al. (2019), who determined that Moringa root powder had a maximum efficiency of
87% reduction in E. coli when added to contaminated water at 600 mg/L. A similar percentage
decrease (88.66%) in E. coli numbers was achieved by adding more than double the amount of
Moringa root powder used by Morgan et al. (2019). This differential antibacterial efficacy of
the Moringa root powder concentrations in the two studies might have arisen due to variation
in pre- and postharvest handling of the Moringa plants used in the two studies and due to the
differences in concentrations of various chemical components reported in their root powders.
The E. coli strain used in the antibacterial tests might be different in the two studies as well.
While ATCC® 8739™ strain of E. coli was used in this research, Morgan et al. (2019) extracted
an unspecified strain of E. coli from an agricultural pond. This might be another reason behind
the discrepancy in antibacterial effect displayed by Moringa root powder concentrations in the
two studies.

P a g e | 79
Comparing the antibacterial efficacy of Moringa root powder found in this study
with the results attained by Morgan et al. (2019), it appears that very little incremental death in
E. coli population is attained by increasing the Moringa root powder concentrations past 600
mg/L. Morgan et al. (2019) found significant (F (1,6) =74.27, p= 0.0001) reduction of 87% in
E. coli by increasing Moringa root concentration from 0 to 600 mg/L. However, past 600 mg/L,
not much further reduction in E. coli was achieved, as 800, 1400 and 2000 mg/L Moringa root
powder concentrations were able to achieve only 84.35%, 88.66% and 86.94% percentage
decrease in E. coli numbers (Figure 14). A significant difference in E. coli numbers was not
observed between 800 and 1400 mg/L (p= 0.84), 800 and 2000 mg/L (p= 0.97), and 1400 and
2000 mg/L (p= 0.98) Moringa treatments (Table 11). With little increment observed in death
of E. coli after adding Moringa root powder concentrations past 600 mg/L, and as the bacterial
population increased after increasing the root powder concentration from 1400 mg/L to 2000
mg/L, it seems like 1400 mg/L is the most effective and the highest Moringa root powder
concentration that can reduce E. coli colonies in contaminated water.
The bioactive component of Moringa roots responsible for killing E. coli in
contaminated water was not ascertained in this research. Eilert et al. (1981) identified 4-(α-Lrhamnopyranosyloxy) benzyl isothiocyanate as the active antibacterial component present in
seeds of M. oleifera and Moringa stenopetala. Benzyl isothiocyanate is known to target cellular
proteins of bacteria, resulting in disruption of major metabolic processes and eventually leading
to cell death (Dufour et al., 2013). Ganatra et al. (2012) found 4-(α-L-rhamnopyranosyloxy)
benzyl isothiocyanate, 4-(α-L-rhamnopyranosyloxy) benzyl glucosinolate and pterygospermin
to be the chief chemical constituents accountable for antibacterial activity of M. oleifera Lam.
Out of these three constituents, roots of M. oleifera are known to contain 4-(α-Lrhamnopyranosyloxy) benzyl glucosinolate (Ganatra et al., 2012). Thus, the bioactive
component of Moringa roots responsible for killing E. coli might be 4-(α-L-

P a g e | 80
rhamnopyranosyloxy) benzyl glucosinolate. However, further research needs to be done to
confirm this proposition and to determine if its mode of action is similar to benzyl
isothiocyanate or Moringa oleifera cationic protein (described in section 2.1.6 of the thesis).
Ginger root powder was found to be less effective at reducing E. coli counts in the
contaminated water than Moringa root powder (Figure 14). However, like Moringa root
powder, 1400 mg/L concentration of ginger root powder caused higher percentage decrease in
E. coli numbers than 800 and 2000 mg/L ginger concentrations, thus making 1400 mg/L the
highest and the most effective ginger root powder concentration capable of reducing E. coli
colonies in contaminated water. Ginger root powder reduced E. coli numbers by 57.82%,
62.63% and 60.44% when added to contaminated water at 800, 1400 and 2000 mg/L
concentration respectively (Figure 14). This is in accordance with various previous studies that
determined antibacterial effect of ginger extracts on numerous bacteria (Malu et al., 2009;
Karuppiah and Rajaram, 2012; Islam et al., 2014; Yadufashije et al., 2020; Wang et al., 2020).
However, a direct comparison of percentage decrease in E. coli caused by ginger root powder
cannot be drawn with the previous studies as all the above-mentioned studies investigated
inhibition zone caused in bacterial growth by ginger extracts or powder. None of the previous
studies enumerated E. coli numbers before and after applying ginger powder concentrations to
the contaminated water.
This research did not identify the bioactive compound of ginger rhizomes
responsible for killing the E. coli population. Mao et al. (2019) found phenolic compounds
such as gingerols, shogaols and paradols to be the chief bioactive components present in ginger.
Zingerone and 6-shogaol (both formed from gingerol), zingiberene, 6-gingerol, α-curcumene
(present in ginger’s essential oil), 10-gingerol and 12-gingerol are some of the bioactive
components responsible for antibacterial efficacy of ginger (Mao et al., 2019; Wang et al.,

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2020). While ginger essential oil and its components are known to inhibit gene expression of
bacterial energy metabolism, cell membrane related proteins, and DNA metabolism (Wang et
al., 2020), gingerol and its components are believed to affect membrane integrity and inhibit
biofilm formation in bacterial cells (Mao et al., 2019). Further research needs to be done to
identify the specific bioactive compound (out of the above-mentioned compounds) present in
ginger rhizomes that kills E. coli colonies in contaminated water and determine its mode of
action.
In both the cases of Moringa and ginger, the antibacterial effect of different
concentrations of the root powders on E. coli showed an inverted bell-shaped trend (Figure 11,
12). That is, the E. coli numbers decreased on adding 800 mg/L of the root powders to
contaminated water. A further decrease in E. coli population was noticed when the
concentration of the root powders was increased from 800 to 1400 mg/L. However, following
1400 mg/L, no further increment in the percentage reduction of E. coli was observed and thus,
the E. coli counts enumerated in treatments with 2000 mg/L concentration of the two root
powders was higher than the bacterial numbers found in treatments with 1400 mg/L (Figure
11,12). Thus, the antibacterial properties of Moringa and ginger root powders appear to be dose
dependent. This can either be because at higher concentrations, the nutritional potency of root
powders surpasses their antibacterial efficacy, or at higher concentrations, the phytochemical
constituents of the root powders alter the water conditions in a way that favours E. coli growth.
Further research needs to be done to understand why after a certain concentration, the
antibacterial effect of Moringa and ginger root powders on E. coli population begins to drop.
Moringa and ginger root powders appeared to have a synergistic effect on E. coli.
The combination of the two root powders mixed in 1:1 ratio showed a higher percentage
reduction in E. coli numbers than shown by either of the root powders alone. On adding 800,

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1400 and 2000 mg/L concentration of the two root powders mixed in 1:1 ratio, the E. coli
counts in contaminated water decreased by 90.65%, 93.79% and 94.78% respectively (Figure
14). According to Stefanovic (2017), bioactive components of plant extracts have ability to
modify or block the resistance mechanism of a bacteria, thus making the bacteria sensitive to
another plant extract used along with it or an antibiotic. Thus, the antibiotic (or another plant
extract) can produce similar antibacterial results at lower concentration when added along with
a plant extract (Stefanovic, 2017). Future studies should try to examine the mode of actions of
Moringa and ginger root powders on E. coli, and the combined mode of action of their
respective bioactive compounds on E. coli. This would explain the rationale behind synergistic
effect of amalgamation of the two root powders on E. coli.
Standard deviation, displayed as error bars in the Figures 11, 12 and 13, varies
greatly among different concentrations of Moringa and ginger root powder. Although
standardized E. coli numbers were used in each replicate of a treatment (obtained by adding
the same dilution factor—1x10-8), the initial population of E. coli pipetted into each sample
could have varied to some extent, thus resulting in greater standard deviation among some
treatments than others. Another reason behind disparity in E. coli numbers reported in treatment
replicates might be the discrepancy in weights of root powders used. The sartorius weighing
balance used to weigh root powders displayed values up to two decimal places. For instance,
0.08 gram of root powder was added to 100 mL of sample for preparing 800 mg/L treatment
concentration. So, the root powder used in different replicates of 800 mg/L concentration could
have varied from 0.081 gram to 0.089 grams, or from 81 mg to 89 mg. Future studies should
use a weighing balance capable of weighing root powders up to three decimal places to ensure
less discrepancy among replicates of a single root powder concentration.

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Chapter 6: Conclusion and Recommendations
6.1 Conclusion
This study aimed to document the methods and techniques used to grow Moringa
oleifera and Zingiber officinale (ginger) plants in a greenhouse, analyse their growth, harvest,
and process their roots, study inorganic elemental composition of their root powders and
determine their antibacterial efficacy against E. coli. The Moringa seeds were obtained from
India and were planted in I.K. Barber Enhanced Forestry Lab (EFL) greenhouse at UNBC for
seven months, whereas the ginger rhizomes were purchased from a supermarket and were
grown in the greenhouse for about three years. The Moringa and ginger roots were harvested,
washed, peeled, freeze-dried, and ground to a fine powder using an electronic grinder.
Inductively coupled plasma- optical emission spectrometry (ICP-OES) was used to
measure concentrations of 27 metals in the root powders of Moringa and ginger. The most
abundant metal found in both the root powders was potassium, while calcium, magnesium,
sulphur, sodium, and phosphorus were present in high concentrations (each >1,000 mg/kg).
Harmful and heavy metals such as lead, mercury, arsenic and cadmium were present in the root
powders in very low concentrations— each less than 10 mg/kg. Concentrations of anions such
as sulphate, phosphate, chloride, and nitrate were found to be significantly higher in ginger
than in Moringa root powder. Both the root powders were found to be slightly acidic with pH
values lying between 5 and 6.
The antibacterial efficacy of the root powders was determined using IDEXX
Colilert-18 Test/Quanti-Tray/2000 Method. The mean E. coli numbers decreased significantly
(p<0.05) on adding different concentrations of Moringa and ginger root powder to
contaminated water. The Moringa roots were more effectual in reducing E. coli population than
ginger roots. For both Moringa and ginger root powder, 1400 mg/L concentration was

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determined to be the most effective concentration as it reduced E. coli in contaminated water
by 88.66% and 62.63% respectively. The combination of the two root powders was found to
have a synergistic effect on E. coli as Moringa and ginger root powder combined in 1:1 ratio
reduced bacterial counts more proficiently than either of the two root powders alone. The E.
coli counts in contaminated water decreased by 90.65%, 93.79% and 94.78% on adding 800,
1400 and 2000 mg/L concentrations of mixed Moringa and ginger root powder.
This research sought to explore the potential use of Moringa and ginger roots in
treating contaminated water, which is a very common problem in underdeveloped and
developing countries. The abundance of essential elements and the scarcity of toxic, heavy
metals in the two root powders validates their use as a point-of-use water treatment. The
presence of essential elements in the root powders in high concentrations will offer additional
health benefits along with treating water of E. coli colonies.
6.2 Recommendations
This research determined 1400 mg/L as the most effective concentration for both
Moringa and ginger root powders in reducing E. coli counts. However, the most effective
concentration of the combination of Moringa and ginger root powders (mixed in 1:1 ratio)
could not be determined as the E. coli numbers still seemed to be decreasing by increasing the
concentration of amalgamation of the root powders following 2000 mg/L (the highest
concentration of the root powders tested). Thus, further studies need to be conducted to
determine a specific concentration of amalgamation of Moringa and ginger root powders
which, when added to contaminated water, reduces the E. coli counts to the lowest number
possible.
Future studies should conduct a direct comparison between antibacterial and
chemical properties of Moringa and ginger root powders by growing Moringa and ginger plants

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in a greenhouse under similar conditions and for equal length of time. The bioactive compounds
within Moringa and ginger roots responsible for killing E. coli colonies in contaminated water
and their respective mode of actions should also be investigated in the future.
Since E. coli population in contaminated water did not reach WHO’s required
standard of <1 CFU/100 mL (WHO and UNICEF 2017), further research needs to be done to
develop Moringa and ginger roots as a water treatment method that can produce more viable
results. The two root powders could be combined with crushed Moringa seeds, leaves or with
other plant extracts known to possess antibacterial properties. A comparison could also be
drawn between antibacterial efficacy of the Moringa and ginger roots harvested from plants
belonging to different age groups. Lastly, water treated with Moringa and ginger root powders
should be assessed for physical water quality parameters such as turbidity, pH etc., palatability
and social acceptance in order to successfully develop the two root powders as a point-of-use
water treatment method in underdeveloped and developing countries.

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Appendix A. Extracting E. coli used in bacteriological analysis (Trial method)
Since the primary aim of this research was to determine the effect of Moringa and
ginger root powder on E. coli population, extracting E. coli from a natural source of water was
the first preference. The E. coli strains extracted from a natural water source would be
genetically closer and behave similar to the E. coli strains found in open water sources in
underdeveloped and developing countries, than the K-12 E. coli or other cultured strains of E.
coli belonging to Risk Group 1. Generally, open water sources are used to draw drinking water
in countries where Moringa and ginger grow naturally and where the problem of contaminated
water is quite alarming.
Water was collected from a pond located in an agricultural farm close to UNBC and
in the north of Prince George, on November 18, 2020. This pond was selected due to the natural
presence of E. coli in its water, as determined by Morgan et al. (2019). In addition to this, the
bacteriological contamination and the physical properties of the pond water represented the
real-world water quality conditions in open water sources (Morgan et al. 2019). Five litres of
pond water were collected from mouth of a pipe feeding the frozen pond (Figure 15) in a 20L
sterilized plastic container (Reliance Rectangular Aqua Pak water container). The container
was rinsed thrice with the pond water before collecting the water sample according to CCME
standards (CCME 2011).
The environmental parameters—temperature, pH, turbidity, and electrical
conductivity, of the pond water were measured on the same day to understand the baseline
characteristics of the water used for testing. Three readings of each environmental parameter
were taken and a mean value for each of them was calculated. The mean temperature, pH,
electrical conductivity and IR ratio (turbidity) of the pond water was calculated to be 0.23 0 C,
6.85, 71.28 µS/cm and 9.89 respectively.

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Figure 15. The opening (mouth) of pipe from where water sample was collected, and
environmental parameters of the pond water were measured. (Photograph by author, taken on
November 19, 2020).

The pond water sample was brought back to UNBC. The plastic container was
shaken and swirled for a few minutes to thoroughly mix the water sample before removing 400
mL of the sample. This 400 mL water sample was used to extract and isolate E. coli using serial
dilutions and membrane filtration. The water sample was diluted by a factor of 2, 10 and 100
using milliQ water. These dilutions along with an undiluted pond water sample were run
through 0.45 µm membrane filters. Using sterile forceps, the membrane filters were transferred
to petri-dishes with poured m-ColiBlue® broth ampules. m-coli blue media was used for
selecting E. coli and coliform colonies growing on the filter. The petri-dishes were incubated
for 24 hours at 350 C.
After 24 hours, a single E. coli colony (blue in colour) and a few coliform colonies
(red in colour) were observed on filter with water sample diluted by a factor of 2 (Figure 16).

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Water samples diluted by a factor of 10 and 100 showed no coliform or E. coli growth, whereas
the undiluted sample depicted growth of a few coliform colonies, but not a single E. coli colony.
Extraction of just one E. coli colony from four different dilutions of pond water could be a
result of low temperature of pond water used for isolating E. coli. Since the pond water sample
was collected in the middle of November 2020, most parts of the pond were frozen. Due to
this, the water had to be collected from mouth of a pipe feeding the pond as it was the only spot
with flowing water.

Figure 16. An E. coli colony (blue in
colour) and numerous coliform colonies
(red in colour) observed on a membrane
filter
with
m-ColiBlue
media.
(Photograph by author, taken on
November 20, 2020).

Figure 17. White, cloudy, and circular E.
coli colonies observed on MUG agar
plates. (Photograph by author, taken on
November 21, 2020).

A streak plate was made by transferring the E. coli colony to MUG agar plate and
streaking it across the quadrants using sterile techniques. The streak plate was then incubated
at 370 C for 24 hours. The next day, large, white, cloudy, and circular E. coli colonies were
observed on the first and second quadrant of the MUG agar plate (Figure 17). A single E. coli
colony was selected from the MUG agar plate with an inoculating loop and was transferred to

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a sterilized flask with 60 mL of freshly prepared LB broth. The inoculated LB broth was then
incubated at 370 C for 24 hours, after which dilution analysis was done to determine which
serial dilution factor of the inoculated broth will result in countable E. coli numbers that can be
used in bacteriological analysis.
Serial dilution analysis was conducted following the methods described in the
section 5.3.4 of this thesis. Enumeration of E. coli numbers through IDEXX Colilert-18
Test/Quanti-Tray/2000 Method revealed that the serial dilutions of 10 -2 and 10-4 each had
2419.6 MPN of E. coli per 100 mL and the serial dilutions of 10-5, 10-6, 10-7, 10-8 and 10-9
showed <1 MPN E. coli/ 100 mL of sample. 2419.6 MPN is the highest number of E coli that
can be detected by IDEXX Colilert-18. The serial dilution analysis was conducted the second
time using potassium dihydrogen phosphate buffer solution to ensure the bacteria cells were
not being lysed by milliQ water. But all the serial dilutions greater than 10 -4 again showed <1
MPN E. coli/ 100 mL of sample.
However, the IDEXX trays displayed fluorescent-coloured wells after three days of
incubating them. Since colilert-18 media is selective, only wells that are positive (with
fluorescent wells) after 24-28 hours of incubating the IDEXX trays can be confirmed as wells
with E. coli growth (IDEXX® Laboratories 2013). Fluorescent IDEXX wells observed after
three days of conducting serial dilution analysis indicated that the bacterial growth on MUG
agar plates did not consist of pure E. coli colonies. Inability to collect pond water the second
time due to ongoing winters led to choosing cultured E. coli strains for the bacteriological
research.

(Left to right) After sowing the Moringa seeds on February 27, 2019, the pots received light watering; The Moringa seeds
showed 86% germination success; The seedlings were transplanted into larger four-litre pots on May 2, 2019. (Photographs
by author).

(Left to right) Moringa oleifera seeds that were grown in the EFL greenhouse at UNBC; Preparation of soil mixture by adding
adequate quantities of peat, coarse sand, coir, slow-release nutrients, and dolomite; Adding freshly prepared soil mixture in
one-litre pots. (Photographs by author).

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Appendix B. Documentation of the research through photos

(Left to right) Moringa seedlings during the last week of April 2019; the Moringa plants during the first week of July 2019; Moringa plants
during the last week of August 2019; A harvested Moringa root on September 27, 2019. (Photographs by author).

(Left to right) The harvested, washed and peeled Moringa and ginger roots were freeze-dried using Labconco FreeZone6 freeze dryer in the
beginning of June 2020; The freeze-dried roots were cut into smaller pieces using a knife and a wooden board; The freeze-dried roots were
ground into a fine powder using Black+Decker coffee and spice grinder; Composite samples of Moringa and ginger roots were created by
mixing the root powders of different Moringa and ginger plants respectively. (Photographs by author).

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(Left to right) Appropriate quantities of Moringa and ginger root powders were added to 10mL buffered milliQ water and stirred for 15 minutes
on VWR mini hot-plate stirrers using magnetic stir bars; After adding Moringa and ginger root powders to 100ml milliQ water with
standardized E. coli numbers, the treatments were filtered into sample bottles through Whatman 150 mm, grade 2 filter paper; After filtering
the treatments, Colilert-18 reagent was added to each sample bottle; The treatments were then vigorously shaken and poured into sterile QuantiTrays/2000. (Photographs by author).

(Left to right) A thawed ATCC® mini-cryovial containing 8739™ E. coli; VWR’s powdered LB Broth (Luria-Bertani) used for preparing LB
broth; Criterion’s dehydrated culture media of MUG nutrient agar used for preparing MUG agar plates; Comparison of sterile LB broth with
inoculated LB broth; A streak MUG agar plate with E. coli colonies. (Photographs by author).

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(Left to right) Quanti-Trays were sealed using IDEXX Quanti-Tray™ Sealer; Sealed QuantiTrays were stacked and placed in the incubator at 37 0 C for 24 hours; After 24 hours, the QuantiTrays were viewed under UV light and the number of fluoresced wells were counted and the
information was compared with IDEXX MPN table to determine MPN of E. coli present in
each Quanti-Tray. (Photographs by author).

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