Untitled

ASSESSING THE EFFECTIVENESS OF LEMON GRASS OIL (CYMBOPOGON)
FOR WATER PURIFICATION
BY
MUNDENDE MWENYA
RUSANGU UNIVERSITY
2013

ii
ASSESSING THE EFFECTIVENESS OF LEMON GRASS OIL (CYMBOPOGON)
FOR WATER PURIFICATION
BY
MUNDENDE MWENYA
F008/235
A research report submitted in partial fulfillment of the requirements of a Bachelor of
Science in Environmental Health
RUSANGU UNIVERSITY
SCHOOL OF SCIENCE AND TECHNOLOGY
DEPARTMENT OF ENVIRONMENTAL HEALTH
2013

iii
Declaration
I declare that this work has been composed by myself and has not been accepted in any
previous application for a degree. This work has been done by me and all sources of
information have been acknowledged by means of references
MUNDENDE MWENYA

iv
CERTIFICATION OF THESIS WORK
We the under signed certify that MUNDENDE MWENYA candidate for the degree of
Bachelor of Science in Environmental Health has presented the research project with the
following title.
ASSESSING THE EFFECTIVENESS OF LEMON GRASS OIL (CYMBOPOGON) FOR
WATER PURIFICATION and that the research project is acceptable in form and content,
and that a satisfactory knowledge of the candidate in an oral examination demonstrated a
satisfactory, knowledge of the field covered by the thesis held on 11th
May, 2013.
SUPERVISOR
NAME: MR. D M CHISOWA
SIGNATURE: ……………………………
MAJOR SUPERVISOR
NAME: DR. E KOOMA
SIGNATURE: …………………………….

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DEDICATION
I dedicate this work to my father, Mr. Kasonde Mundende, for being patient, resourceful,
supportive and loving me during my studies. To my late mother, Pamela H. Mundende, I
wish you were here to see me now. To the rest of the family that have been supportive and
caring throughout the entire period I was at Rusangu University.

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ACKNOWLEDGEMENT
I would like to appreciate a lot of people who contributed to the success of this work. I would
like to extend my appreciation to the supervisor, Dr. E. Kooma. Thanking greatly, Mr. D.
Chisowa for his support and assistance with data analysis. A special thanks to the Head of
Department, Environmental Health and members of faculty for their guidance and
constructive critique during this research. This work would not have reached the required
standard without their commitment and co – operation.
I also want to acknowledge the Dean of the School of Engineering, University of Zambia for
giving me the opportunity to use the laboratory for the experiment. Mr. Mutati, the laboratory
technician who helped me carryout the experiments.
I further acknowledge my colleagues; the list is too long to mention you by name for the
input they had one way or another for the completion of this research.
I salute you all.

vii
ABSTRACT
The study was examining the potency of lemon grass oil in destroying bacteria in water at
different dose levels and contact times against the standard chlorine which is the most
common antiseptic. The lemon grass oil doses level used for the research were 2 ml and 4 ml
added to 1 litre of raw water, and the exact was in the case with the standard, chlorine.
The samples were labeled A, B and C. A, representing raw water, B representing water that
was treated with Chlorine and C representing treatment by Lemon grass oil. Sample A, did
was not subjected to any treatment as it acted as a control for the treatments. It is an
interventional study in which the researcher was comparing the means of two groups of
observation.
The treatments (chlorine and lemon grass oil, 2 ml of each) were added to the raw water in
individual containers and given a contact time of 10 minutes for the first experiment.
The same was repeated but saw an increase in the dose and the contact time to 4 ml and a
contact time of 30 minutes for both treatments. This was for the second experiments.
The results for the first experiment indicate that at a significant level (p˂0.05), there was a
significant difference in the reduction of the bacterial load. Chlorine proved more virulent
than lemon grass oil. The second experiment results show that at the same significant level
(p˂0.05), there was no significant difference in the reduction in bacterial load between
chlorine and lemon grass oil.
The study has revealed that lemon grass oil in place of conventional antiseptic can be used to
reduce bacterial load. The study has also indicated that the higher the concentration level of
lemon grass oil added to water, the more bacteria are destroyed.

viii
TABLE OF CONTENTS
PAGE
Title …………………………………………………………………………………. ii
Declaration ………………………………………………………………………….. iii
Certification of thesis work …………………………………………………………. iv
Dedication …………………………………………………………………………… v
Acknowledgements ………………………………………………………………….. vi
Abstract ………………………………………………………………………………. vii
Table of contents ……………………………………………………………………… viii
List of tables ………………………………………………………………………….. x
List of Appendices ……………………………………………………………………. xi
Definition of Terms …………………………………………………………………… 1
CHAPTER 1: INTRODUCTION AND BACKGROUND ………………………… 2
1.1 Background Information …………………………………………………… 2
1.2 Statement of the Problem ………………………………………………….. 4
1.3 Conceptual Framework …………………………………………………….. 4
1.4 Analytical Framework …………………………………………………….... 5
1.5 Significance of the Study …………………………………………………… 6
1.6 Objectives …..………………………………………………………………. 6
1.6.1 General Objective ……………………………………………………. 6
1.6.2 Specific Objectives …………………………………………………… 6
1.7 Research Hypotheses ………………………………………………………. 7
CHAPTER 2: LITERATURE REVIEW ……………………………………………. 8
2.1 Overview …………………………………………………………………… 8
2.2 Water treatment Technologies ……………………………………………… 8
2.2.1 Vegetative Matter ………………………………………………… 8
2.2.2 Chlorine …………………………………………………………… 8
2.2.3 Boiling of Water ………………………………………………… 9
2.2.4 Lemongrass oil …………………………………………………. 10
2.2.5 Extraction of Oil ……………………………………………….. 11
2.2.5.1 Water and Steam Distillation ………………………… 11
2.3 Chemical Composition …………………………………………………. 12

ix
CHAPTER 3: RESEARCH METHODOLOGY ………………………………….. 13
3.1 Apparatus Used ………………………………………………………………. 13
3.2 Variables ……………………………………………………………………… 13
3.2.1 Independent Variables ………………………………………………… 13
3.2.2 Dependent Variables ………………………………………………….. 13
3.2 Study Design ………………………………………………………………. 13
3.3 Study Setting ……………………………………………………………… 14
3.4 Sample Size ………………………………………………………………. 14
3.5 Plans for Data collection ………………………………………………… 14
3.6 Plans for Sample Treatment …………………………………………….. 14
3.7 The Experiment ……………………………………………………….… 14
3.8 Procedure (Membrane Filtration) ……………………………………….. 15
3.9 Replication ……………………………………………………………… 15
CHAPTER 4: PRESENTATION AND DISCUSSION OF RESULTS ……… 17
4.1 Introduction ………………………………………………………………. 17
4.2 Discussion ………………………………………………………………… 19
4.3 Bacteria Behavior ………………………………………………………… 21
CHAPTER 5: CONCLUSION AND RECOMMENDATIONS …………….. 23
5.1 Conclusions ………………………………………………………………. 23
5.2 Recommendations ……………………………………………………….. 23
5.3 Suggestions for Future Research ………………………………………… 23
6.0. REFERENCES ……………………………………………………………. 24
7.0. APPENDICES …………………………………………………………….. 26
7.1 Laboratory results for bacteriological examination of water …………… 26
7.2 Work Plan/Schedule/Action plan ………………………………………. 26
7.3 Budget ………………………………………………………………….. 27

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LIST OF TABLES
Table 1: Summary for the total coliforms ………………………………………… 17
Table 2: Analysis of Variance for Total Coliforms ……………………………...... 17
Table 3: Summary for the fecal coliforms ………………………………………… 17
Table 4: Analysis of Variance for Fecal Coliforms ………………………………. 18
Table 5: Showing the bacteria present/absent in water before and after
the experiments ……………………………………………………………. 18
Table 6: Cost Benefit Analysis …………………………………………………… 20

xi
LIST OF APPENDICES
Appendix 1: Laboratory results for bacteriological examination of water ……….. 26
Appendix 2: Work Plan/Schedule/Action plan …………………………………… 26
Appendix 3: Budget ………………………………………………………………. 27

1
Definition of Terms
1. Total coliform bacteria are commonly found in the environment (e.g., soil or
vegetation) and are generally harmless.
If only total coliform bacteria are detected in drinking water, the source is probably from
a non-pathogenic environmental origin (not likely sewage).
2. Fecal coliform bacteria are a sub-group of total coliform bacteria. They appear in great
quantities in the intestines and faeces of people and animals. The presence of faecal
coliform in a drinking water sample often indicates recent faecal contamination, meaning
that there is a greater risk that pathogens are present than if only total coliform bacteria is
detected.
3. Effectiveness is the capability of producing a desired result. When something is deemed
effective, it means it has an intended or expected outcome or produces a deep, vivid
impression.
4. Purifying is thoroughly cleaning and freeing of or destroying disease causing organisms.
5. Bacterial load is a measurable quantity of bacteria in an object, organism or organism
compartment. It is also referred to as Bacteria count.
6. Disinfection is a method applied to reduce the pathogenic amount of disease causing
agents by using disinfectants.
7. Drinking water or portable water is water which is safe enough to be consumed by
humans or used with low risk of immediate of long term harm (WHO, 2010).
8. Contact time is the specified time allowed for water to mix with a water purifying agent.

2
CHAPTER 1
INTRODUCTION AND BACKGROUND
1.1 Background Information
The goal of water treatment is to reduce or remove all contaminants that are present in the
water. No water, irrespective of the original source, should be assumed to be completely free of
contaminants. Meaning, water used for drinking and cooking should be free of pathogenic
microorganisms that cause such illnesses as typhoid fever, dysentery, cholera, and
gastro – enteritis. Whether a person contracts these diseases from water or not, depends on the
type of pathogen, the number of organisms in the water (density), the strength of the organism
(virulence), the volume of water ingested, and the susceptibility of the individual. Purification of
drinking water containing pathogenic micro – organisms requires specific treatment called
disinfection.
It is assumed that about 15% of people, primarily in rural areas, get their drinking water
from private wells. Having a background that there are no guidelines for testing private water
sources, people that get their water from private wells run a greater risk of illness from all sorts
of natural and man-made water contaminants. Of all the advancements made possible through
science and technology, the treatment and distribution of water for safe use is truly one of the
greatest. Abundant, clean water is essential for good public health. Humans cannot survive
without water; in fact, our bodies are 67% water (www.WaterFiltering.com).
Although several methods eliminate disease-causing microorganisms in water,
chlorination is the most commonly used. Chlorination is effective against many pathogenic
bacteria, but at normal dosage rates it does not kill all viruses, cysts, or worms. When combined
with filtration, chlorination is an excellent way to disinfect drinking water supplies
(www.webdesignpros.net/watertreatment/watertreatment).
Chlorine has now been a major part of Municipal water treatment for nearly 100 years.
About 98% of Municipal water treatment facilities now use chlorine disinfectant as their
disinfectant of choice, and about 200 million U.S. residents receive chlorinated drinking water
through their home faucets (Christman, 2008).

3
It is now recognized that chlorine forms some potentially harmful by – products. The
disinfection by – products of chlorine disinfection are by far the most thoroughly studied.
While the available evidence does not prove that disinfection by – products in drinking
water cause adverse health effects in humans, high levels of these chemicals are certainly
undesirable. Cost – effective methods to reduce disinfection byproducts formation are available
and should be adopted where possible. However, the International Programme on Chemical
Safety (IPCS), a joint venture of the United Nations Environment Programme (UNEP), the
International Labor Organization (ILO), and the World Health Organization (WHO) strongly
cautions:
The health risks from these by – products at the levels at which they occur in drinking
water are extremely small in comparison with the risks associated with inadequate disinfection.
Thus, it is important that disinfection not be compromised in attempting to control such by –
products.
National Toxicology Program (1992) Untreated water can contain a large number of
compounds that react with chlorine, including inorganic reducing agents; ammonia, amines and
amino acids; humic substances (complex polymers of natural origin); and other forms of organic
nitrogen. The principal result of the reaction of chlorine with these compounds is the formation
of halogenated byproducts, particularly trihalomethanes, halogenated acetic acids, halogenated
acetonitriles, chlorinated ketones, halogenated hydrocarbons, and others. Bromide can also be
present in untreated water and react with compounds in the water to form brominated
byproducts.
Recent regulations have further limited disinfection byproducts in drinking water. Most
water systems are meeting these new standards by controlling the amount of natural organic
matter prior to disinfection, while ensuring that microbial protection remains the top priority
(International Programme on Chemical Safety: 2000).
According to the Water Quality and Health Council (WQHC), scientists are now
beginning to examine the possible by – products and side effects of using chlorine in drinking
water. Chlorine is listed as a known poison; it undoubtedly has an adverse effect on our body
systems. Chlorinated water has been linked to the aggravation and cause of respiratory diseases

4
like asthma. Also, because chlorine vaporizes at a much faster rate than water, chlorinated water
presents a significant threat to the respiratory system when used for showering. Recent
discoveries of the health concerns of chlorine have led many researchers to venture in trying to
find safer alternatives for water treatment (www.worldchlorine.com).
1.2 Statement of the Problem
Although chlorine has been commonly used as a water treatment agent, it forms potential
harmful by – products and yet it has been the main source of water purification worldwide. It is
believed that increased exposure and consumption (that being chlorine mixing with existing
chemicals in water) can pose adverse health effects on the human (Dustan et al, 1995). It is
against this background that the researcher saw the need to pursue alternative, less harmful and
user friendly resource, lemon grass.
Secondly, there is need to provide purifying reagents information about lemon grass which as of
now may be scanty.
1.3 Conceptual Framework
Chlorination
Destruction of Bacteria/
reducing bacterial load
Treatment by lemon
grass oil
Temperature
Vegetative matter

5
1.4 Analytical Framework
Use of lemon grass oil
for water Treatment
Lack of information
on its potency in
water treatment
Possible adverse
health effects of
existing water
treatment
technologies
Inaccessible existing
water treatment
technologies in some
parts.
Poor road and
communication
networks especially
in rural areas
Due to inaccessible
water treatment
technologies
Research has been
conducted verifying that
chlorine by products can
cause respiratory disease
e.g. Asthma
No research in this
area has been
undertaken.
Finding alternative
water treatment
technologies

6
1.5 Significance of the Study
The completion of this study may provide information to among the many, Water Utility
Companies, the Ministry of Health (MoH), Non-Governmental Organizations (NGOs), and Rural
Communities.
MoH and Water Providing Companies need to have alternative forms of water treatment to
ensure that people are provided with clean water and are free from waterborne diseases.
NGOs and the private sector to partner with the government, so they need this information to
bring low-tech, low cost, and user-friendly solutions to people who must treat water in their
homes and may not be able to procure chlorine.
The findings of this study will attract further discoveries by scientists which will be used for
the betterment of humanity.
1.6 Objectives
1.6. 1 General Objective
The general objective of this study was to assess the effectiveness of lemon grass as a
drinking water purifying agent.
1.6. 2 Specific Objectives
The specific objectives for this study were:
 To compute the bacterial load in water before and after treatment with lemon grass.
 To identify specific species of bacteria destroyed by lemon grass in water.
 To compare the species of bacteria destroyed by lemon grass and those destroyed
chlorine treatment.

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1.7 Research Hypotheses
: There is no significant difference in the microbial load before and after treatment of
water by lemon grass.
: There is no significant difference in the type of species of bacteria destroyed by lemon
grass in the water and those destroyed by chlorine.
: There is no significant difference in the effectiveness of lemon grass and chlorine in
treating water for drinking.

8
CHAPTER 2
LITERATURE REVIEW
2.1 Overview
Shaw (2007) indicates that there is need for water that is of a high enough quality to
drink, but there is still a vast quantity that needs to be drawn and usually processed in some
way before use. According to the International Centre for Intergrated Sustainable
development (ICIS), as the world becomes more populous, water is becoming more scarce.
There is strong growth potential for all types of water treatment technologies, but some
could do better as countries bid to quench their thirst in a cheap and environmentally friendly
way (www.worldchlorine.com).
2.2 Water treatment Technologies
2.2.1 Vegetative Matter
Baker (2006) observes that other vegetative matter has been used as an adsorbent for
water treatment.
Droste and McJunkin (2002) state that the application of these materials has been in the
form of a granular or leave medium filter. Use of this water treatment medium is still limited,
primarily to those parts of the world where agriculture is widely practiced and where other
filter media are not readily available at low cost. However, these adsorbent materials and
their technologies require further development, evaluation and dissemination before they can
be recommended for household water treatment in other parts of the world.
2.2.2 Chlorine
Shaw (2007) furthermore shows a WHO report indicating that:
Chlorine is most widely and easily used, and the most affordable of the drinking water
disinfectants. It is also highly effective against nearly all waterborne pathogens.
However, water treatment represents only a tiny proportion of global chlorine demand.

9
Data indicates that only a 5% maximum of chlorine is used for water treatment, including
drinking water.
WHO (2010) records that of the United Nations’ Millennium Development Goals is to
reduce by half the proportion of people without sustainable access to safe drinking water by
2015. One billion people lack access to safe drinking water, two and a quarter billion to
adequate sanitation. To achieve this target, an additional one and a half billion people will
require access to some form of improved water supply by 2015. That is an additional hundred
million people each year (or 274,000 people/day) until 2015.
2.2.3 Boiling of Water
Boiling water is generally not effective in removing chemical contaminants. In fact, it
generally increases their concentration a bit. Boiled water also runs the risk of
recontamination during the cooling process if not properly protected and stored.
Additionally, boiling water requires a significant amount of fuel, which can exact a toll both
financially and environmentally. Despite these limitations, boiling is still a standard
treatment when any pathogen is at issue.
According to Cairncross et al (2008), over the last several decades, new and innovative
household water treatment systems have been developed by government agencies, NGOs,
and the private sector to bring low-tech, low cost, and user-friendly solutions to people who
must treat water in their homes.
UNICEF (2012) revealed that only 64 % of Zambians have access to safe water sources.
In some poor areas, families rely on shallow, contaminated wells for water. As a result,
incidents of diarrhea are widespread in Zambia, and diarrheal illnesses are the leading cause
of death among children. In the late 1990s, a new household chlorination system was
introduced in Zambia. This product can treat up to 1,000 liters of water and costs about 60
times less than the equivalent amount of charcoal needed to treat this volume of water. This
system has helped to significantly reduce the incidents of diarrhea and is currently used in an
estimated two million households (www.nae.edu/nae/grainger.nsf).

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2.2.4 Lemon grass oil
Mukherjee (2009) indicates that various researches have been conducted to examine the
potency lemon grass oil of as a insect repellant, an anti malarial, an anticancer, an
antibacterial, and an antifungal property. Currently, there is scientific evidence investigating
the use of lemon grass oil in humans.
The anti-septic property of lemon grass oil makes it a good application for external and
internal wounds as well as an ingredient of the anti-septic lotions and creams. The anti-septic
properties of this oil do not let the external and internal cuts and wounds go septic.
Minami et al (2003), indicates that the antiviral of essential oils on herpes simplex virus
type – 1 (HSV – 1) replication was examined in vitro. The replication ability of HSV-1 was
suppressed by incubation of HSV-1 with 1% of lemon grass oil at 4ºC for 24 hours. Lemon
grass oil completely inhibited the viral replication even at a concentration of 0.1% and its
antiviral activity was dependant on the concentrations.
Eifert (2004) adds that when it comes to insect repelling, lemon grass oil with a
concentration of upto 15%, is 30% less effective than synthetic insect repellant. This of
course applies to mosquitoes. The best part of lemon grass oil is that it does not have the
same risks associated with some of the insect killing chemical currently on the market. You
can spray these about yourself and the house and not worry about any physical effect on
people.
Ohno (2003), shows that Lemon grass oil completely inhibits the growth of Helicobacter
pylori at a concentration of 0.1%. Lemon grass was bactericidal against H. pylori, even at
lesser concentrations of 0.01% and the bacterium was unable to develop resistance.
Kamali et al (2008) state that essential oil vapors (what one would create through using a
nebulizing diffuser, essential oils Lemon grass were shown effective even against bacteria.
This may prove useful in hospital settings where such bacteria have become resistant against
conventional antibiotics. Due to its interaction with the immune system, and broad-spectrum

11
anti-microbial action, diffuser use of Lemon grass may be an excellent means of 'disinfecting
the air' in one's home or office.
Onawunmi (1989) illustrates the antifungal activity of lemon grass oil (LGO) using
fungistatic (MIC and agar diffusion tests) and fungicidal (spore germination) studies.
Appreciable activity was observed against various isolates of Candida and Aspergillus. Exposure
of the Aspergillus species to 0.1% of LGO for 5 minutes resulted into 93% of spores not
germinating.
Shadab et al (1992) furthermore shows that a naturally occurring fungus (Candida) in our
bodies which can 'overgrow' due to dietary imbalances (possibly too high a sugar intake),
resulting in vaginal irritation, rashes (particularly on the feet) and the like. Topical application
may be best to utilize these properties, in a 5-10% dilution in any carrier oil.
Extraction of Oil
There are mainly three ways of extracting lemon grass oil from lemon grass. The most
commonly used though is the water and steam distillation. This is so because it produces a high
oil yield after extraction.
Water and Steam Distillation
Lemon grass oil is obtained by steam distillation of Lemon grass (Cymbopogon spp.). It
is the most common and cheapest available in the market.
The extraction is done in a Steam distillation Unit. The walls of this unit are slightly tapering
from top to bottom for uniform mixing between the steam and plant materials.
Lemon grass (chopped or unchopped) is filled in the distillation still and its lid is fitted
tightly by bolts, so that the oil and vapour do not leak out. The steam is injected in the still by the
help of steam spargers provided at the bottom of the vessel. The upcoming steam carries away
the oil from the plant material i.e. lemon grass and both oil as well as steam pass to the
condenser through line vapour line, where these vapours get condensed and oil is seperated from
the water in the seperators.
The oil thus obtained is lemon grass oil with 80 – 85% 0f citral.

12
2.3 Chemical Composition.
Lemon grass contains 80 – 85% citral which is the main constituent of lemon grass oil.
Myrcene, an antibacterial and pain reliever which ranges between 8% - 11. Hydro steam
distillation, condensation and cooling were used to separate the oil from the water. Hydrosol or
Hydrolat, as a by-product of the distillation process, is a pure natural water or plant water
essence used for the production of skin care products such as lotions, creams, and facial
cleansing toner in its pure form. (Masamba et al, 2003)

13
CHAPTER 3
RESEARCH METHODOLOGY
Below is how this study was carried out:
3.1 APPARATUS USED
1. Sterile Sartorius funnel.
2. 2 litre suction flask.
3. Electric vacuum pump or water jet.
4. Sterile membrane filters 50mm diameter.
5. Glass Petri dishes with culture medium.
6. Sterile pipettes.
7. A pair of forceps
8. Electronic colony counter
9. Two incubators set one at 35 - 37 o
C and the other at 44.5o
C.
3.2 Variables
3.2.1 Independent Variables
Independent variables considered were:
a. Treatment by Lemon grass oil
b. Temperature
3.2.2 Dependent Variables
Dependent variables considered were:
a. Number and species of bacteria destroyed per experiment.
b. Background chemical concentration.
3.3 Study Design
The research was an experimental study.
It was an interventional study in which the researcher was comparing the means of two groups of
observation. In this case the researcher had Lemon grass oil as the experiment and Chlorine as
the control. The chosen experimental design was effective in helping to achieve the research
objectives and to produce the desired results.

14
3.4 Study Setting
The research was conducted at the University of Zambia. The researcher used the Goma Lakes
as the source of the samples for the contaminated water.
3.5 Sample Size
Samples were taken from the first (Upper) Goma Lakes near its inlet. The sample had six
sterile one litre containers in which the raw water was placed.
The researcher at each time drew one litre of raw water from Goma Lake until the researcher
had collected three litres in different containers for the first experiment and three litres in
different containers for the second experiment. The raw water was immediately taken to the
Environmental Engineering Laboratory were the actual bacteriological experiment was done.
3.6 Plans for Data collection
Bacterial species were identified through Gram staining; microscopic examination and colony
characteristics observation (color, size and shape) before and after treatment with Lemon grass
oil (Cymbopogon) in order to know the species that survive treatment.
3.7 Plans for Sample Treatment
A measured volume of 100ml of water was filtered through a membrane of pore size 0.45
microns, made of cellulose compound. The membrane was then incubated on a suitable selective
medium, allowing the coliform bacteria to reproduce and form colonies.
The number of characteristic colonies produced at 35 o
C on a particular medium gives the
total coliforms (TC) content of the water sample. When incubated at 44.5o
C on MFC medium the
colonies represent the faecal coliforms (FC) of the sample.
3.8 The Experiment
Experiment 1
The sampled raw water were labeled A, B and C.
For the first experiment;
Sample A contained raw water without any treatment.
Sample B had 2 milliliters (ml) of Chlorine added to it.
Sample C had 2 milliliters (ml) of Lemon grass oil added to it.

15
Nutrient Agar used
Nutrient agar for;
Total coliforms: m – Endo Total Coliform Broth
Feacal coliforms: mFC (m – Faekal Coliform) agar
Procedure (Membrane Filtration)
To test for total coliforms present, the researcher placed a sterile membrane filter on the filter
support using a sterile forceps. Then the metal funnel covering the top of the filter support was
placed and clamped to securely tight. The water samples subjected to treatment of chlorine and
lemon grass oil were vigorously shaken and allowed a contact time of ten minutes.
After ten (10) minutes, 100ml of the water with the water purifier was poured directly into the
funnel (which had graduations of 100ml and 200ml inside). The researcher switched on the
vacuum pump for suction and eventual filtration.
After the sample of 100ml of the sample had passed through the filter, the pump was switched
off; the filter was picked and placed on solidified nutrient agar in a petri dish and making sure to
trap air bubbles under the filter. The petri dish was the placed in the incubator at the standard
temperature of 35 - 37 ºC.
The same procedure was repeated for testing for feacal coliforms but at the incubation
temperature of 45 ºC.
After an incubation time of 24 hours, the researcher counted the colonies that had developed.
3.9 Replication
The experiment was repeated and the researcher increased the dose to be administered to the raw
water and the contact time.
Experiment 2
The sampled raw water were labeled A, B and C.
For the first experiment;
Sample A contained raw water without any treatment.
Sample B had 4 milliliters (ml) of Chlorine added to it.
Sample C had 4 milliliters (ml) of Lemon grass oil added to it.
The same procedure was used in the replication and the contact time was increased from ten (10)
minutes to thirty (30) minutes.

16
CHAPTER 4
PRESENTATION AND DISCUSSION OF RESULTS
4.1 Introduction
This chapter presents results and the analysis from the experiments taken. The researcher
decided to combine the presentation and analysis to help potential readers to easily follow the
alignment of ideas.
Table 1 below shows the summary for total coliforms. Indicating the coliforms present
after the raw water was subjected to treatment with chlorine and Lemon grass oil.
Table 1: Summary for the total coliforms
Treatment R R Total Mean
Chlorine 40 0 40 20
Lemon grass oil 1200 800 2000 1000
= 2040 = 510
Table 2 shows the analysis of variance showing the Fcal and Ftab for the experiment on
Total Coliforms.
Table 2: Analysis of Variance for Total Coliforms
Source of
Variation
Degrees of
freedom
Sum of
Squares
Means of
Squares
Fcal Ftab
Total 3 1 041 200
Treatment 1 960 400 960 400 23.77 18.51
Error 2 80 800 40 400
Fcal ˃ Ftab, p˂0.05 CV = 115%
This shows that there is a significant difference in the efficiency of chlorine and lemon grass oil
in the reduction of total coliforms in water. This is at the dose of 2 ml and with an allowed
contact time of 10 minutes. Therefore, the hypothesis is not supported.
Table 3 below shows the summary for fecal coliforms. Showing the coliforms present
after the raw water was subjected to treatment with chlorine and Lemon grass oil.
Table 3: Summary for the fecal coliforms
Treatment R R Total Mean
Chlorine 0 0 0 0
Lemon grass oil 1000 0 1000 500
= 1000 = 250
Table 4 shows the analysis of variance showing the Fcal and Ftab for the experiment on Total
Coliforms. The critical value is also shown in the table below determining the relationship of the
data.

17
Table 4: Analysis of Variance for Fecal Coliforms
Source of
Variation
Degrees of
freedom
Sum of
Squares
Means of
Squares
Fcal Ftab
Total 3 750 000
Treatment 1 250 000 250 000 1 18.51
Error 2 500 000 250 000
Fcal ˃ Ftab, p˂0.05 CV = 200%
The results show that there is no significant difference in the efficiency of chlorine and
lemon grass oil in the reduction of fecal coliforms in water. This therefore, is in agreement with
The CV in both experiments is higher than the normal because the researcher had no
influence on the effectiveness of the active ingredients in both chlorine (Sodium hypochlorite)
and lemon grass oil (citrate). Though at the same dose, it is impossible for the researcher to have
control on the effectiveness of the antiseptic in bacterial load reduction. Thus indicating that
Sodium hypochlorite is more virulent as compared to Citrate.
Perhaps the other assumption could be due to the sources. The first samples were
collected from the Upper side of the lake and the other form the Lower side. The movement of
water from the Upper to the Lower could see in increase in the bacterial load and contribute to
the reduction in effectiveness of Lemon grass oil treatment.
This, therefore, is the major source of the variation in the data between chlorine and
lemon grass oil.
Table 5 below shows the bacteria present in both experiment 1 and experiment before and
after being subjected to treatment of Chlorine and Lemon Grass Oil.
Table 5: Showing the bacteria present/absent in water before and after the experiments
Experiment 1 Experiment 2
Chlorine Lemon grass oil Chlorine Lemon grass oil
Bacteria
Detected
Before
treatme
nt
After
treatme
nt
Before
treatme
nt
After
treatme
nt
Before
treatme
nt
After
treatme
nt
Before
treatme
nt
After
treatment
Bacillus
species
Present Absent Present Absent Present Absent Present Absent
Escherichia
Coli (E. Coli)
Present Absent Present Present Present Absent Present Absent
Salmonella
species
Present Present Present Present Present Absent Present Present
Enterobacter
species
Present Absent Present Absent Present Absent Present Absent
Enterococcus
faecalis
Present Absent Present Absent

18
4.2 Discussion
The efficacy of lemon grass oil in comparison with conventional antibacterial (chlorine) when
applied to raw water with the same dose and same contact time proves slightly less effective. The
research was done in two experiments as earlier mentioned. The results were as follows.
Experiment one had 2ml of both chlorine and lemon grass oil added to 100ml of water
within a contact time of 10 minutes. The following were the results:
Sample A recorded about 66, 000 coliforms/100ml of total coliforms and 58, 000
coliforms/100ml of feacal coliforms. This is representing the raw water that was not subjected to
any treatment. Therefore, the coliform percentage for raw water is 100% for both feacal and total
coliforms.
Sample B (which was treatment by chlorine) recorded 40 coliforms/100ml total coliforms
and 0 coliforms/100ml for feacal coliforms. The results mean that the effectiveness of chlorine
on feacal coliforms was 100%, whereas when compared to its effectiveness on total coliforms
was 99.93% because 40 coliforms of total coliforms were found. Perhaps the reason why all the
total coliforms were not completely destroyed was the limited contact time. Indicative that
certain bacteria strains may need a longer contact time with the reagent for complete destruction.
Perhaps another aspect would have to do with increasing the dosage administered to the water to
undergo treatment. Another aspect to consider is that feacal coliforms are made most of one
bacterial strain (E. coli) whereas total coliforms contains any other bacteria able to survive in
water.
Sample C (which was treatment by lemon grass oil) recorded 1200 coliforms/100ml for total
coliforms and 1000 coliforms/100ml for feacal coliforms. The results show that the effectiveness
of lemon grass oil on feacal coliforms was 98.18% and compared to its effectiveness on total
coliforms which was 98.28%. Firstly an assumption can be made that lemon grass is much more
effective on feacal coliforms as compared to its effectiveness on total coliforms. Similarly to
Sample B, perhaps the reasons why all the total coliforms and feacal coliforms were not
completely destroyed were:
a. The limited contact time. Meaning 10 minutes was not sufficient for the complete contact
between bacteria and lemon grass oil
b. The dose enough to deal with bacteria especially at 2ml. Thereby showing that an increase in
the dosage could produce a different a different result.
Experiment two had 4ml of both chlorine and lemon grass oil added to 100ml of water
within a contact time of 30 minutes. The following were the results:
Sample A recorded about 62, 000 coliforms/100ml for total coliforms and 56, 000
coliforms/100ml for feacal coliforms. This once again is representing the raw water that did not

19
undergo any treatment. The coliform percentage for raw water is 100% for both feacal and total
coliforms.
Sample B (which was treatment by chlorine) recorded 0 coliforms/100ml total coliforms
and 0 coliforms/100ml for feacal coliforms. This clearly indicates that chlorine at 4ml in water
and at a contact of 30 minutes was lethal to all the coliforms which were present in the water. By
rate of percentage chlorine destroyed 100% of both existing feacal and total coliforms in the
water. Therefore, at a dose of 4ml of chlorine and 30minutes contact, no bacteria present in the
water survived.
Sample C (which was treatment by Lemon grass oil) recorded 800 coliforms/100ml for
total coliforms and 0 coliforms/100ml for feacal coliforms. A reduction in the bacteria load is
noted when the dose and contact time are increased of lemon grass oil. The results show that the
effectiveness of lemon grass oil on feacal coliforms was 100%, whereas when compared to its
effectiveness on total coliforms was 98.71% because 800 coliforms of total coliforms were
found. As earlier established, lemon grass oil may have more effect on feacal coliforms as
compared to total coliforms. Perhaps the reason why feacal coliforms could easily have been
destroyed is that the coliforms consist mainly of a single bacteria strain which is E. coli and that
total coliforms is a representative a lot of bacteria able to be detected in water.
Another view could be that certain bacteria are resistant to lemon grass oil.
This is in agreement with Eifert (2004) who focused on comparing lemon grass oil as an
insect repellant. It was proved that the oil is 30% less effective than synthetic insect repellant.
Eifert continues to say that to achieve a better result in terms of effectiveness, the dose of lemon
grass oil would have to be at higher dose than the synthetic or conventional treating.
Lemon grass oil has the capability of destroying bacteria in water as seen in the
experiments. It has proven that at higher doses, it can remove all feacal coliforms. However, this
does not leave out the fact that lemon grass was unable to completely eradicate total coliforms.
Implying, at higher doses of lemon grass oil, and a long contact time, the bacterial load is
reduced greatly.
The study has also shown that the efficacy of lemon grass oil is much more on Gram
positive bacteria and on certain strains of Gram negative bacteria, as observed by authors like
Singh et al (2011).
Furthermore, it is shown the resistance to lemon grass oil treatment by some bacteria
strains. Salmonella species were able to survive the treatment of lemon grass oil even at both
lower and higher doses. There is need for further study to identify why Salmonella species are
able to resist dose by lemon grass oil.
Table 6: Cost Benefit Analysis

20
Organic (Lemon
Grass oil)
Conventional
(Chlorine)
Price (K) 5
50 (Ng)
Dose for treatment (ml) 4 or higher 2
Availability Needs to processed Available
Chlorine proves to be cheaper because it has been included in the national budget and is
dispensed or sold at a subsidize price. Equally lemon grass oil could be the same once the
information in this research is communicated to policy makers and implementers. To have a
deliberate agricultural policy to grow lemon grass and process it for the purpose on treating
water.
4.3 Bacteria Behavior
The researcher also wanted to find out the types of bacteria that were present in the water
before and after the experiments. The results in table 5 above show the bacteria present before
and after the treatment with both chlorine and lemon grass oil. This perhaps would help in
determining the resistance of certain bacteria to lemon grass oil. The above bacteria were
detected in the raw water that was sampled from the Goma Lakes.
At 2ml dose of chlorine almost destroyed all bacteria except for Salmonella species
which survived the treatment. The reason perhaps could be that the contact time allowed was not
to enough to completely eliminate all bacteria present.
At the same dose lemon grass oil was able to eliminate Enterobacter species and bacillus
species but could not completely eliminate Salmonella species and E. coli. The reasons perhaps
could be:
a. The contact time was not enough to facilitate for complete remove the all bacteria
present.
b. Salmonella species have some resistance to lemon grass oil. According to Singh et al
(2011), Salmonella, a Gram negative strain of bacteria, is sensitive to lemon grass oil but
sometimes can exhibit resistance of upto 60% towards the same.
However, an increase in the dose and the contact time for both indicate different results as to
which bacteria survived. At 4ml and a contact time of 30 minutes, chlorine destroyed all the
bacteria that were detected in the water.

21
At the same dose, lemon grass oil was able to destroy all the bacteria except Salmonella
species, thus confirming the Singh et al (2011)’s findings on the resistance exhibited in
Salmonella species.
Singh et al (2011) furthermore shows that most Gram negative bacteria not resistant to lemon
grass oil as compared to other. This is visible even from the results, that E. coli and Enterobacter
species are not resistant to the oil though being Gram negative and some species can be resistant
as seen by Salmonella species. Gram positive bacteria have no resistance to lemon grass oil, thus
both Bacillus species and Enterococcus faecalis were both destroyed.

22
CHAPTER 5
CONCLUSION AND RECOMMENDATIONS
5.1 CONCLUSIONS
The research aimed at testing the potency of lemon grass as an alternative treatment to
chlorine in water treatment by testing its efficacy in bacterial load reduction.
The research concludes that, lemon grass oil has the capabilities of a water purifying
agent however; it is not as effective as other conventional purifying agents such chlorine, in that
it requires higher doses and does not completely destroy coliforms.
It is hoped that this study has provided reliable information on which health practitioners
can base their ideas to adopt this for possible future implementation.
5.2 RECOMMENDATIONS
1. Awareness in both urban and rural about the use of lemon grass oil, not just for
destroying bacteria in water. This could be done a health centres/ or post, through
media; newspaper, health documentaries on both television and radio and through
the social media as well.
2. Health practitioners to facilitate for the availability of lemon grass oil to those in
regions where it cannot be accessed. By acquiring the oil through the National
budget and distributing the oil to the people as done with chlorine.
5.3 Suggestions for Future Research
Further studies should be conducted to find solutions to issues which may include:
1. The efficiency of lemon grass oil on bacteria at higher doses and with a longer contact
time.
2. The long term effect on human health of consuming chlorine and/or lemon grass oil.
3. Why lemon grass oil has more virulence on Gram positive bacteria as compared to Gram
negative.
4. The effect of lemon grass oil on other organisms i.e. protozoa, viruses, fungi and insects
in Zambia.
5. More natural herbs and artificial antiseptic that can help improve water quality especially
in peri – urban and rural areas.

23
6.0. REFERENCES
Baker, M. N., (Ed). (2008). The Quest for Pure Water: the History of Water Purification
from the Earliest Records to the Twentieth Century. American Water Works Association,
Denver.
Christman, K. (2008). The history of chlorine. Waterworld. 14 (8), 66-67.
Cairncross, S., I. Carruthers, et al. (2008). Evaluation for Village Water Supply Planning.
New York, John Wiley & Sons.
Droste, R. L. and F. E. McJunkin, (Ed) (2002). Simple Water Treatment Methods. In: Water
Supply and Sanitation in Developing Countries. E. J. Schiller and R. L. Droste (Eds.). Ann
Arbor, Ann Arbor Science: 101-122.
Dustan R.H et al. (1995) “A Preliminary Investigation of Chlorinated Hydrocarbons and
Chronic Fatigue Syndrome.” The Medical Journal of Australia, September 18; 163:294-297.
Eifert J. R (2004), The Effectiveness of Lemon Grass as a Natural Mosquito Repellent,
California, USA.
El Kamali HH, Hamza MA, El Amir MY (2005). Antibacterial activity of the essential oil
from Cymbopogon nervatus inflorescence. Fitoterapia; 76(5):446-449
International Programme on Chemical Safety (2000). Disinfectants and disinfectant
byproducts, Environmental Health Criteria 216.
Masamba W, et al (2003). Extraction and analysis of Lemongrass (Cymbopogon Citratus) oil:
An essential oil with potential to control the Larger Grain Borer in stored products in
Malawi. University of Malawi, Lilongwe, Malawi.
Minami M, et al (2003). The Inhibitory effect of essential oils on herpes simplex virus type-1
replication in vitro. Prefectural University of Medicine, Kyoto, Japan.
Mukherjee, A (2009). Health benefits of lemon grass essential oil. Organic Facts
National Toxicology Program (1992) Technical Report On The Toxicology And
Carcinogenesis Studies Of Chlorinated Water (Cas Nos. 7782-50-5 And 7681-52-9) And
Chlorinated Water (Drinking Water Studies). USA

24
Onawunmi G, O. (1989). Evaluation of the Antifungal Activity of Lemon Grass Oil.
Pharmaceutical Biology
Ohno T, et al. (2003). Antimicrobial activity of essential oils against Helicobacter pylori.
Prefectural University of Medicine, Kyoto, Japan.
Shadab, Q., Hanif, M. & Chaudhary, F.M. (1992). Antifungal activity by lemongrass essential
oils. Pak. J. Sci. India. Res. 35, 246-249.
Singh B, Singh V, Singh K and Ebibeni N (2011). Antimicrobial activity of lemongrass
(Cymbopogon citratus) oil against microbes of environmental, clinical and food origin. India
Shaw C (2007). Global Demand For Clean Water Set To Rocket. Online; 23rd
June, 2010.
UNICEF (2012). Water, Sanitation and Hygiene in Zambia. UNICEF Zambia Fact Sheets.
Zambia
World Health Organization (1996) Guidelines for drinking-water quality, Health criteria and
other supporting information. Geneva. 2nd ed. Vol.2.
World Health Organization Report (2010)”. Millennium Development Goals. Washington
D.C.USA.
www.nae.edu/nae/grainger.nsf
www.WaterFiltering.com. Visited on 30 April, 2012.
www.webdesignpros.net/watertreatment/watertreatment. Visited on 6 January, 2012.
www.worldchlorine.com. Visited on 7 October, 2011.

25
7.0. APPENDICES
1. Laboratory results for bacteriological examination of water
Tests carried out in conformity with “Standard Methods for the Examination of water and
Wastewater APHA, 1998”.
2. Work Plan/Schedule/Action plan
Parameter Raw Water Treated With
Chlorine
Treated With
Lemon
Grass Oil
Volume Added /L
Chlorine (ml)
0 2 2
Volume Added /L Oil
(ml)
0 2 2
Total coliforms (#/100ml) 66000 40 1200
Feacal coliforms (#/100ml) 58000 0 1000
Volume Added /L
Chlorine (ml)
0 4 4
Volume Added /L Oil
(ml)
0 4 4
Total coliforms (#/100ml) 62000 0 800
Feacal coliforms (#/100ml) 56000 0 0
Task Month/Dates Person Responsible Number of days
Project proposal March, 2013 Researcher
Data collection April, 2013 Researcher 21
Data analysis June, 2013 Researcher/Supervisor 07
Report writing and
draft report
May, 2013 Researcher 14
Finalize report and
submission
July, 2013 Researcher 14

26
3. Budget
In order to achieve the objectives of this research the following budget was followed
Description Quantity Unit Cost
(ZMK)
Total
Transport
Within Lusaka
300.00
Stationary
Printing and binding
services
Copies 30.00 300.00
Laboratory Fees 6 water bottles 100.00 600.00
Miscellaneous 300.00 300.00
Total K 1 500.00

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