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 anyprevious application for a degree. This work has been done by me and all sources ofinformation 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 ofBachelor of Science in Environmental Health has presented the research project with thefollowing 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, Iwish you were here to see me now. To the rest of the family that have been supportive andcaring 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.

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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 …………………………………………………………………………………. iiDeclaration ………………………………………………………………………….. iiiCertification of thesis work …………………………………………………………. ivDedication …………………………………………………………………………… vAcknowledgements ………………………………………………………………….. viAbstract ………………………………………………………………………………. viiTable of contents ……………………………………………………………………… viiiList of tables ………………………………………………………………………….. xList of Appendices ……………………………………………………………………. xiDefinition of Terms …………………………………………………………………… 1

CHAPTER 1: INTRODUCTION AND BACKGROUND ………………………… 2

1.1 Background Information …………………………………………………… 21.2 Statement of the Problem ………………………………………………….. 41.3 Conceptual Framework …………………………………………………….. 41.4 Analytical Framework …………………………………………………….... 51.5 Significance of the Study …………………………………………………… 61.6 Objectives …..………………………………………………………………. 6

1.6.1 General Objective ……………………………………………………. 61.6.2 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

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CHAPTER 3: RESEARCH METHODOLOGY ………………………………….. 13

3.1 Apparatus Used ………………………………………………………………. 133.2 Variables ……………………………………………………………………… 13

3.2.1 Independent Variables ………………………………………………… 133.2.2 Dependent Variables ………………………………………………….. 13

3.2 Study Design ………………………………………………………………. 133.3 Study Setting ……………………………………………………………… 143.4 Sample Size ………………………………………………………………. 143.5 Plans for Data collection ………………………………………………… 143.6 Plans for Sample Treatment …………………………………………….. 143.7 The Experiment ……………………………………………………….… 143.8 Procedure (Membrane Filtration) ……………………………………….. 153.9 Replication ……………………………………………………………… 15

CHAPTER 4: PRESENTATION AND DISCUSSION OF RESULTS ……… 17

4.1 Introduction ………………………………………………………………. 174.2 Discussion ………………………………………………………………… 194.3 Bacteria Behavior ………………………………………………………… 21

CHAPTER 5: CONCLUSION AND RECOMMENDATIONS …………….. 235.1 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 …………… 267.2 Work Plan/Schedule/Action plan ………………………………………. 267.3 Budget ………………………………………………………………….. 27

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LIST OF TABLES

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

the experiments ……………………………………………………………. 18Table 6: Cost Benefit Analysis …………………………………………………… 20

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LIST OF APPENDICES

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

1

Definition of Terms

1. Total coliform bacteria are commonly found in the environment (e.g., soil orvegetation) and are generally harmless.If only total coliform bacteria are detected in drinking water, the source is probably froma non-pathogenic environmental origin (not likely sewage).

2. Fecal coliform bacteria are a sub-group of total coliform bacteria. They appear in greatquantities in the intestines and faeces of people and animals. The presence of faecalcoliform in a drinking water sample often indicates recent faecal contamination, meaningthat there is a greater risk that pathogens are present than if only total coliform bacteria isdetected.

3. Effectiveness is the capability of producing a desired result. When something is deemedeffective, it means it has an intended or expected outcome or produces a deep, vividimpression.

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 organismcompartment. It is also referred to as Bacteria count.

6. Disinfection is a method applied to reduce the pathogenic amount of disease causingagents by using disinfectants.

7. Drinking water or portable water is water which is safe enough to be consumed byhumans 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 potentialharmful by – products and yet it has been the main source of water purification worldwide. It isbelieved that increased exposure and consumption (that being chlorine mixing with existingchemicals in water) can pose adverse health effects on the human (Dustan et al, 1995). It isagainst this background that the researcher saw the need to pursue alternative, less harmful anduser friendly resource, lemon grass.

Secondly, there is need to provide purifying reagents information about lemon grass which as ofnow may be scanty.

1.3 Conceptual Framework

Chlorination

Destruction of Bacteria/reducing bacterial load

Treatment by lemongrass oil

TemperatureVegetative matter

5

1.4 Analytical Framework

Use of lemon grass oilfor water Treatment

Lack of informationon its potency inwater treatment

Possible adversehealth effects ofexisting water

treatmenttechnologies

Inaccessible existingwater treatment

technologies in someparts.

Poor road andcommunication

networks especiallyin rural areas

Due to inaccessiblewater treatment

technologies

Research has beenconducted verifying thatchlorine by products cancause respiratory disease

e.g. Asthma

No research in thisarea has beenundertaken.

Finding alternativewater 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 forceps8. Electronic colony counter9. Two incubators set one at 35 - 37 oC and the other at 44.5oC.

3.2 Variables

3.2.1 Independent Variables

Independent variables considered were:

a. Treatment by Lemon grass oilb. 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 sixsterile 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 0.45microns, made of cellulose compound. The membrane was then incubated on a suitable selectivemedium, allowing the coliform bacteria to reproduce and form colonies.

The number of characteristic colonies produced at 35 oC on a particular medium gives thetotal coliforms (TC) content of the water sample. When incubated at 44.5oC on MFC medium thecolonies 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 filtersupport using a sterile forceps. Then the metal funnel covering the top of the filter support wasplaced and clamped to securely tight. The water samples subjected to treatment of chlorine andlemon 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 thefunnel (which had graduations of 100ml and 200ml inside). The researcher switched on thevacuum pump for suction and eventual filtration.

After the sample of 100ml of the sample had passed through the filter, the pump was switchedoff; the filter was picked and placed on solidified nutrient agar in a petri dish and making sure totrap air bubbles under the filter. The petri dish was the placed in the incubator at the standardtemperature of 35 - 37 ºC.

The same procedure was repeated for testing for feacal coliforms but at the incubationtemperature of 45 ºC.

After an incubation time of 24 hours, the researcher counted the colonies that had developed.

3.9 ReplicationThe experiment was repeated and the researcher increased the dose to be administered to the rawwater 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 RESULTS4.1 Introduction

This chapter presents results and the analysis from the experiments taken. The researcherdecided to combine the presentation and analysis to help potential readers to easily follow thealignment of ideas.

Table 1 below shows the summary for total coliforms. Indicating the coliforms presentafter the raw water was subjected to treatment with chlorine and Lemon grass oil.

Table 1: Summary for the total coliformsTreatment R R Total MeanChlorine 40 0 40 20Lemon grass oil 1200 800 2000 1000= 2040 = 510

Table 2 shows the analysis of variance showing the Fcal and Ftab for the experiment onTotal Coliforms.Table 2: Analysis of Variance for Total ColiformsSource ofVariation

Degrees offreedom

Sum ofSquares

Means ofSquares

Fcal Ftab

Total 3 1 041 200Treatment 1 960 400 960 400 23.77 18.51Error 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 oilin the reduction of total coliforms in water. This is at the dose of 2 ml and with an allowedcontact time of 10 minutes. Therefore, the hypothesis is not supported.

Table 3 below shows the summary for fecal coliforms. Showing the coliforms presentafter the raw water was subjected to treatment with chlorine and Lemon grass oil.Table 3: Summary for the fecal coliformsTreatment R R Total MeanChlorine 0 0 0 0Lemon grass oil 1000 0 1000 500= 1000 = 250

Table 4 shows the analysis of variance showing the Fcal and Ftab for the experiment on TotalColiforms. The critical value is also shown in the table below determining the relationship of thedata.

17

Table 4: Analysis of Variance for Fecal ColiformsSource ofVariation

Degrees offreedom

Sum ofSquares

Means ofSquares

Fcal Ftab

Total 3 750 000Treatment 1 250 000 250 000 1 18.51Error 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 withThe 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 havecontrol on the effectiveness of the antiseptic in bacterial load reduction. Thus indicating thatSodium hypochlorite is more virulent as compared to Citrate.

Perhaps the other assumption could be due to the sources. The first samples werecollected from the Upper side of the lake and the other form the Lower side. The movement ofwater from the Upper to the Lower could see in increase in the bacterial load and contribute tothe reduction in effectiveness of Lemon grass oil treatment.

This, therefore, is the major source of the variation in the data between chlorine andlemon grass oil.

Table 5 below shows the bacteria present in both experiment 1 and experiment before andafter 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 2Chlorine Lemon grass oil Chlorine Lemon grass oil

BacteriaDetected

Beforetreatment

Aftertreatment

Beforetreatment

Aftertreatment

Beforetreatment

Aftertreatment

Beforetreatment

Aftertreatment

Bacillusspecies

Present Absent Present Absent Present Absent Present Absent

EscherichiaColi (E. Coli)

Present Absent Present Present Present Absent Present Absent

Salmonellaspecies

Present Present Present Present Present Absent Present Present

Enterobacterspecies

Present Absent Present Absent Present Absent Present Absent

Enterococcusfaecalis

Present Absent Present Absent

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4.2 DiscussionThe 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 waterwithin a contact time of 10 minutes. The following were the results:

Sample A recorded about 66, 000 coliforms/100ml of total coliforms and 58, 000coliforms/100ml of feacal coliforms. This is representing the raw water that was not subjected toany treatment. Therefore, the coliform percentage for raw water is 100% for both feacal and totalcoliforms.

Sample B (which was treatment by chlorine) recorded 40 coliforms/100ml total coliformsand 0 coliforms/100ml for feacal coliforms. The results mean that the effectiveness of chlorineon feacal coliforms was 100%, whereas when compared to its effectiveness on total coliformswas 99.93% because 40 coliforms of total coliforms were found. Perhaps the reason why all thetotal coliforms were not completely destroyed was the limited contact time. Indicative thatcertain 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 toundergo treatment. Another aspect to consider is that feacal coliforms are made most of onebacterial strain (E. coli) whereas total coliforms contains any other bacteria able to survive inwater.

Sample C (which was treatment by lemon grass oil) recorded 1200 coliforms/100ml for totalcoliforms and 1000 coliforms/100ml for feacal coliforms. The results show that the effectivenessof lemon grass oil on feacal coliforms was 98.18% and compared to its effectiveness on totalcoliforms which was 98.28%. Firstly an assumption can be made that lemon grass is much moreeffective on feacal coliforms as compared to its effectiveness on total coliforms. Similarly toSample B, perhaps the reasons why all the total coliforms and feacal coliforms were notcompletely destroyed were:a. The limited contact time. Meaning 10 minutes was not sufficient for the complete contact

between bacteria and lemon grass oilb. 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 waterwithin a contact time of 30 minutes. The following were the results:

Sample A recorded about 62, 000 coliforms/100ml for total coliforms and 56, 000coliforms/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 totalcoliforms.

Sample B (which was treatment by chlorine) recorded 0 coliforms/100ml total coliformsand 0 coliforms/100ml for feacal coliforms. This clearly indicates that chlorine at 4ml in waterand at a contact of 30 minutes was lethal to all the coliforms which were present in the water. Byrate of percentage chlorine destroyed 100% of both existing feacal and total coliforms in thewater. Therefore, at a dose of 4ml of chlorine and 30minutes contact, no bacteria present in thewater survived.

Sample C (which was treatment by Lemon grass oil) recorded 800 coliforms/100ml fortotal coliforms and 0 coliforms/100ml for feacal coliforms. A reduction in the bacteria load isnoted when the dose and contact time are increased of lemon grass oil. The results show that theeffectiveness of lemon grass oil on feacal coliforms was 100%, whereas when compared to itseffectiveness on total coliforms was 98.71% because 800 coliforms of total coliforms werefound. As earlier established, lemon grass oil may have more effect on feacal coliforms ascompared to total coliforms. Perhaps the reason why feacal coliforms could easily have beendestroyed is that the coliforms consist mainly of a single bacteria strain which is E. coli and thattotal 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 aninsect 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 lemongrass 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 theexperiments. It has proven that at higher doses, it can remove all feacal coliforms. However, thisdoes 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 isreduced greatly.

The study has also shown that the efficacy of lemon grass oil is much more on Grampositive bacteria and on certain strains of Gram negative bacteria, as observed by authors likeSingh et al (2011).

Furthermore, it is shown the resistance to lemon grass oil treatment by some bacteriastrains. Salmonella species were able to survive the treatment of lemon grass oil even at bothlower and higher doses. There is need for further study to identify why Salmonella species areable to resist dose by lemon grass oil.

Table 6: Cost Benefit Analysis

20

Organic (Lemon

Grass oil)

Conventional

(Chlorine)

Price (K) 550 (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 waterbefore and after the experiments. The results in table 5 above show the bacteria present beforeand after the treatment with both chlorine and lemon grass oil. This perhaps would help indetermining the resistance of certain bacteria to lemon grass oil. The above bacteria weredetected in the raw water that was sampled from the Goma Lakes.

At 2ml dose of chlorine almost destroyed all bacteria except for Salmonella specieswhich survived the treatment. The reason perhaps could be that the contact time allowed was notto enough to completely eliminate all bacteria present.

At the same dose lemon grass oil was able to eliminate Enterobacter species and bacillusspecies but could not completely eliminate Salmonella species and E. coli. The reasons perhapscould be:

a. The contact time was not enough to facilitate for complete remove the all bacteriapresent.

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 butsometimes 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 towhich bacteria survived. At 4ml and a contact time of 30 minutes, chlorine destroyed all thebacteria that were detected in the water.

21

At the same dose, lemon grass oil was able to destroy all the bacteria except Salmonellaspecies, thus confirming the Singh et al (2011)’s findings on the resistance exhibited inSalmonella species.

Singh et al (2011) furthermore shows that most Gram negative bacteria not resistant to lemongrass oil as compared to other. This is visible even from the results, that E. coli and Enterobacterspecies are not resistant to the oil though being Gram negative and some species can be resistantas seen by Salmonella species. Gram positive bacteria have no resistance to lemon grass oil, thusboth Bacillus species and Enterococcus faecalis were both destroyed.

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CHAPTER 5

CONCLUSION AND RECOMMENDATIONS

5.1 CONCLUSIONS

The research aimed at testing the potency of lemon grass as an alternative treatment tochlorine in water treatment by testing its efficacy in bacterial load reduction.

The research concludes that, lemon grass oil has the capabilities of a water purifyingagent however; it is not as effective as other conventional purifying agents such chlorine, in thatit requires higher doses and does not completely destroy coliforms.

It is hoped that this study has provided reliable information on which health practitionerscan 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 fordestroying bacteria in water. This could be done a health centres/ or post, throughmedia; newspaper, health documentaries on both television and radio and throughthe social media as well.

2. Health practitioners to facilitate for the availability of lemon grass oil to those inregions where it cannot be accessed. By acquiring the oil through the Nationalbudget 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 contacttime.

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 especiallyin peri – urban and rural areas.

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6.0. REFERENCES

Baker, M. N., (Ed). (2008). The Quest for Pure Water: the History of Water Purificationfrom 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: WaterSupply and Sanitation in Developing Countries. E. J. Schiller and R. L. Droste (Eds.). AnnArbor, Ann Arbor Science: 101-122.

Dustan R.H et al. (1995) “A Preliminary Investigation of Chlorinated Hydrocarbons andChronic 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 inMalawi. 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 AndCarcinogenesis Studies Of Chlorinated Water (Cas Nos. 7782-50-5 And 7681-52-9) AndChlorinated 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 andother 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 andWastewater APHA, 1998”.

2. Work Plan/Schedule/Action plan

Parameter Raw Water Treated WithChlorine

Treated With

Lemon

Grass Oil

Volume Added /LChlorine (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 /LChlorine (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 daysProject proposal March, 2013 Researcher

Data collection April, 2013 Researcher 21

Data analysis June, 2013 Researcher/Supervisor 07

Report writing anddraft report

May, 2013 Researcher 14

Finalize report andsubmission

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