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Disinfection of Waterborne Coliform Bacteria by Neem Oil

Article  in  Environmental Engineering Science · September 2009

DOI: 10.1089/ees.2009.0058

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ENVIRONMENTAL ENGINEERING SCIENCE ORIGINAL ARTICLE
Volume 26, Number 9, 2009
ª Mary Ann Liebert, Inc.
DOI: 10.1089=ees.2009.0058

Disinfection of Waterborne Coliform Bacteria by Neem Oil

Robert L. Matthews,1 Michael R. Templeton,2,* Sabitri K. Tripathi,3 and Kiran Bhattarai 4
1
Water and Health Research Centre, Department of Civil Engineering, University of Bristol, Bristol, United Kingdom.
2
Department of Civil and Environmental Engineering, Imperial College London, London, United Kingdom.
3
Department of Science and Humanities; 4Department of Civil Engineering; Nepal Engineering College, Bhaktapur, Nepal.

Received: February 10, 2009 Accepted in revised form: June 5, 2009

Abstract
This study assessed the ability of Neem oil to disinfect an Escherichia coli isolate in pure laboratory-grade water
and total and fecal coliform bacteria in two unfiltered surface waters. Neem oil doses as low as 2.13 g
L1 with
5 min of mixing time achieved high levels of inactivation of the E. coli in pure laboratory-grade water (>99%
inactivation). However, the disinfection of total and fecal coliforms in the unfiltered surface waters was variable
and limited to <70% inactivation typically. Increasing the Neem oil dose and mixing time generally resulted in
an increase in the mean inactivation of total coliform bacteria but not always of fecal coliform bacteria. The
inactivation of total coliforms was consistently greater than the inactivation of fecal coliforms, suggesting a
potential range of sensitivities to Neem oil among coliform bacteria. Reduced effectiveness of the Neem oil in
the unfiltered surface waters compared to the pure laboratory-grade water suggests a possible interference by
natural water constituents (e.g., suspended particles, dissolved organic matter), which may inhibit the anti-
bacterial potential of Neem oil, or that naturally occurring bacteria are more resistant to the antimicrobial effects
of Neem oil. Overall, Neem oil was found to be insufficient on its own for use as a disinfectant of potable water;
however, further investigation is recommended into the performance of Neem oil disinfection when combined
with pretreatment steps (e.g., sand or cloth filtration) and into methods for concentrating the active antimicrobial
ingredients of Neem oil to form more potent disinfectant solutions.

Key words: disinfection; coliform; Neem; essential oils; drinking water; bacteria

Introduction are many other natural essential oils that have been reported
to have antimicrobial properties (e.g., tea tree oil, eucalyptus,

T he Moringa oleifera seed is an example of a naturally

occurring material that is effective as a means for treating
water and has been successfully adopted for use in develop-
ing countries (e.g., Sudan) (Sutherland et al., 1989). The seed
contains substances that act as a coagulant and thereby assist
to settle out particles and pathogens from water. In India,
lentils and seeds from the Tamarind tree and several other
plants have also been proven as effective coagulants for wa-
ters with high turbidity (Schulz and Okun, 1984).
Comparatively little is known about the potential existence
of natural disinfectants (i.e., substances with the ability to
kill=inactivate pathogenic micro-organisms), even though
many herbs and plant extracts are used in traditional medi-
cine and as pesticides in developing countries (Ketkar et al.,
1995; Ross, 1999). One such plant, the Neem tree (Azadirachta
indica), was selected as the focus of this study; however, there
*Corresponding author: Department of Civil and Environmental
Engineering, Imperial College London, London, United Kingdom,
SW7 2AZ. Phone: þ44(0)2075946099; Fax: þ44(0)2075946124; E-mail:
m.templeton@imperial.ac.uk
thyme) (Hammer et al., 1999; Lambert et al., 2001).
The Neem tree grows widely in arid tropical and sub-
tropical areas around the world (Schmutterer, 1995). The
medicinal properties of components of the Neem tree have
been known in India for thousands of years; it is referred to in
Sanskrit texts as ‘‘Arishtha,’’ the reliever of sickness (Ketkar
et al., 1995). Traditional medical uses of Neem products have
covered a vast range of illnesses, from leprosy to intestinal
worms. Neem leaf tea is also often used as a treatment for the
symptoms of malaria and for diarrhoea (Shultz et al., 1992).
Extracts from dried leaves have been reported to be effective
in the treatment of skin infections such as ringworm and
scabies (Biswas et al., 2002).
A substantial number of antibacterial active compounds
have been isolated from various parts of the Neem tree. The
most active compound with regard to pest and insect control is
thought to be azadirachtin (Schmutterer, 1990; Eppler et al.,
1995), while nimbidin extracted from seed oil has been re-
ported to exhibit strong antibacterial properties (Biswas et al.,
2002). The antimicrobial components of essential oils gener-
ally consist of a large group of mainly terpenoid compounds,

1435
1436
such as limonene, carvone, and pinene (Sikkema et al., 1995;
Cotton, 1996). Among the most commonly investigated with
regard to antimicrobial activity are phenolic terpenes such as
eugenol, carvacrol, and thymol ( Juven et al., 1994; Tassou et al.,
2000; Lambert et al., 2001; Arfa et al., 2006; Marwah et al., 2007).
The most important active component of Neem in terms of
bactericidal activity is believed to be the nimbin group of tri-
terpenoids (Kraus and Ermel, 1995; Biswas et al., 2002). The
mechanism by which these compounds affect bacteria is pri-
marily impairment of the cell membrane; the lypophillic
molecules dissolve in the cell membrane, disrupting mem-
brane permeability and resulting in the leakage of ions,
adenosine triphosphate (ATP), nucleic acids, and amino acids
(Sikkema et al., 1995; Griffin et al., 1999; Utlee et al., 1999;
Tassou et al., 2000; Lambert et al., 2001).
There have been only a small number of experimental at-
tempts to assess the antibacterial activity of Neem products.
One study using the disc diffusion method demonstrated the
antibacterial activity of Neem seed oil against various Gram-
negative bacteria including Escherichia coli and some Gram-
positive bacteria such as Staphylococcus pyogenes (Rao et al.,
1986). Reports of the effectiveness of Neem leaf extract are
somewhat conflicting, with some studies reporting antibacte-
rial activity toward certain bacterial species and others re-
porting no effect on any bacterial species (Eppler et al., 1995). A
recent study tested Neem leaf extracts against a range of
foodborne pathogens and found that ethanolic Neem extracts
exhibited antibacterial activity against several Gram-positive
bacteria but no activity against any of the Gram-negative bac-
teria tested, including six strains of E. coli (Hoque et al., 2007). In
contrast, another recent study tested the performance of me-
thanolic Neem leaf extract against various multidrug-resistant
strains of Vibrio cholerae and found significant antibacterial
activity (Thakurta et al., 2007). The latter study also included a
toxicology assay and found that the leaf extract showed no
signs of toxicity in mice following an oral dose of 1,800 mg=kg.
It is difficult to extract conclusions on the antibacterial
properties of Neem components from the published studies to
date, because the studies were performed using vastly dif-
ferent experimental methods, different parts of the Neem tree,
and different types of bacteria. In addition, the majority of the
studies employed agar diffusion techniques that may be un-
reliable for the testing of oils (Mann and Markham, 1997). To
date, there has been no research on Neem extracts specifically
as a potential disinfectant of waterborne pathogens. There-
fore, the specific objectives of this study were:
1. To assess the antibacterial activity of Neem oil against a
sludge-isolated culture of E. coli in pure laboratory-
grade water.
2. To assess the antibacterial activity of Neem oil against
total and fecal coliform bacteria in unfiltered surface
waters.
3. To comment on the practicality of using Neem oil as a
viable water disinfectant based on the required dose
levels observed in this study.
Materials and Methods
Microbiological methods
A culture of E. coli isolated from sewage sludge was
maintained on a plate of Lauria Bertani (LB) agar consisting of
MATTHEWS ET AL.
LB broth (Fisher Scientific, Pittsburgh, PA) and Agar No.1
(Lab M International Diagnostics Group, Lancs, UK). To
prepare the inocula, a loop of culture was transferred to LB
broth and incubated for 18 to 24 h on a shaker plate. After
incubation, the bacteria were washed in phosphate-buffered
saline (PBS) solution (Oxoid) following which the bacte-
ria were refrigerated in PBS and used in experiments within
24 h.
Total and fecal coliform bacteria were used as indicator
organisms to evaluate the disinfection potential of Neem oil
when added to unfiltered surface waters. These groups are
standard indicator organisms for the microbiological quality
of drinking water (WHO, 1993). The membrane filter proce-
dure was chosen as the primary method for all the surface
water testing, according to Standard Methods 9222B and
9222D (APHA, 2005).
It was beyond the aims and scope of this research to ex-
amine the disinfection performance of Neem oil against a
range of other strains of E. coli or other waterborne pathogens
(e.g., viruses, protozoa); however this is recommended for
further investigation.
Water sources
Surface water samples were characterized upon collection
in terms of pH, temperature, ultraviolet absorbance at 254 nm,
and turbidity (Table 1). Samples were transported in an in-
sulated cooler, stored at 48C, and were used in experiments on
the same day as collection. Sample bottles were disinfected
beforehand with Virkonª solution and rinsed with reverse-
osmosis-treated water. All the water required for a particular
test run was taken from a single sample bottle. Each bottle was
agitated to redistribute any settled matter before the sample
volume was dispensed.
Surface waters from England and Nepal (the study coun-
tries) were collected. The surface water from England was a
pond with fecal and organic pollution from a large resident
waterfowl population. The surface water from Nepal was a
river near Kathmandu with high levels of fecal pollution from
the discharge of untreated wastewater and human feces up-
stream. The river water is used for bathing and is known to
contaminate local wells that are used for drinking water.
Neither surface water is used directly as a source of drinking
water, however, they were selected to ensure high enough
bacteria counts to be able to meaningfully quantify the level of
disinfection achieved by the Neem oil. They may also be
considered to represent a worst-case, contaminated drinking
water source in a developing country context.
Table 1. Water Quality Characteristics
of Two Unfiltered Surface Waters Considered
England pond water Nepal river water
Mean Range Mean Range
Parameter
pH 8.6 8.5–8.6 7.1 6.9–7.4
Temperature (oC) 22.4 20–24 22.5 20–24
UV absorbance 0.18 0.17–0.18 N=Aa N=Aa
at 254nm
Turbidity (NTU) 6.4 4.2–14 N=Aa N=Aa
a
It was not possible to measure these parameters in Nepal.
DISINFECTION OF WATERBORNE COLIFORM BACTERIA BY NEEM OIL 1437

Neem oil mixed on a shaker plate at 180 rpm for 1 min, 4 min, 12 min, or
1 h. The samples were then analyzed as described above. The
For the tests in England, Neem oil was obtained from a
positive control consisted of surface water with no Neem oil
commercial source (The Neemteam, Newport, UK). The Neem
addition placed on the shaker plate for 1 h.
oil was produced by cold pressing and was well defined by the
The methodology was kept as similar as possible for the
manufacturer in terms of the concentrations of key active in-
trials in Nepal. The same membrane filter unit and supplies of
gredients (azadirachtin: 1,375 ppm, nimbin: 1,500 ppm, sala-
membrane filters used for the England-based investigation
nin: 1,500 ppm). The same batch was used throughout testing.
were taken to Nepal for the testing.
Neem oil is readily available in Nepal, although it is usually
imported from India. The Neem oil used in this study was
Statistical analyses
manufactured by Shree Baidyanath, Patna, India. It was
darker in color and possessed a stronger bitter smell, sug- All tests were replicated a minimum of three times. The
gesting that it was produced by steam or solvent extraction results are displayed as bar and whisker plots, with the bars
rather than cold pressing (Conrick, 2001). The same batch was indicating the mean values and the whiskers showing the
used for all tests. minimum and maximum values in each data set. The surface
water coliform inactivation results are reported in terms of
percent inactivation, whereas the cultured E. coli inactivation
Disinfection potential of Neem oil against
results are reported as log10 inactivation.
E. coli in lab-grade water
The inocula were prepared at concentrations of 2108 to Results and Discussion
6108 CFU
mL1. The doses of Neem oil trialled were 2.13,
Disinfection performance of Neem oil
4.27, 8.53, and 17.1 g
L1. Inocula (3 mL) was added to 27 mL
of autoclaved PBS in a 125-mL conical flask to create a 101 At a constant mixing time of 5 min, a range of Neem oil
dilution. The Neem oil dose was applied to this dilution, doses from 2.13 to 17.1 g
L1 was found to be effective at
which was then sealed and mixed for 5 min on a shaker plate disinfecting the isolated culture of E. coli in pure laboratory-
at 170 rpm. From the disinfected sample 102, 103, and 104 grade water, with >2-log inactivation (99%) achieved in al-
dilutions were generated. Samples (0.1 mL) of each of these most all cases (Fig. 1). The inactivation of E. coli exhibited no
dilutions were spread-plated on LB agar and incubated for clear dependence on the Neem oil dose, suggesting that a
24 h at 358C to obtain plates with between 20 and 200 count- plateau was reached at the lowest dose tested.
able colonies. Samples for plating were extracted from the When tested against the unfiltered pond water from Eng-
water column below the oil layer; the oil layer was allowed to land, the antibacterial performance of Neem oil was sub-
separate for 1 min before sample collection. It is possible that stantially less successful compared to the observations for the
some fraction of the targeted coliform bacteria were separated E. coli in lab-grade water. The results for varying doses of
into the oil phase, and hence not enumerated in the samples Neem oil at 5 min of mixing time are shown in Fig. 2, and for
collected from the water column below. This hypothesis could 17.1 g L1 Neem oil at varying mixing times are shown in
not be tested because samples taken from the oil layer could Fig. 3. Against total coliform bacteria, all doses of Neem oil
not be analyzed for coliform counts; the oil confounded the resulted in a reduction in average counts; against fecal coli-
membrane filtration technique and would have exerted a forms there was no clear dose-dependence trend.
continued antimicrobial effect during enumeration (i.e., there When tested against the unfiltered river water from Nepal
was no way to ‘‘neutralize’’ the oil). As such, this was accepted the inactivation of total coliform bacteria were similar to those
as a limitation of the experimental method of this study. for the pond water from England, with all Neem oil doses
resulting in reductions in the average total coliform counts but
Disinfection potential of Neem oil against coliform not always in fecal coliform counts (Fig. 4).
bacteria in surface water samples
Surface water samples were mixed with a range of Neem
oil doses over several contact times, to investigate dose and
time dependence of the disinfection performance. As in the
trials with lab-grade water, each sample was left to stand for
1 min to allow the oil and water to separate; a sample of water
containing a minimal amount of oil could then be decanted
and filtered. This careful separation was necessary, as other-
wise the oil accumulated on the surface of the membrane fil-
ter, either causing it to clog or interfering with colony growth.
The total coliform and fecal coliform filter trials for a given
Neem oil dose were performed using the same sample. A
positive control consisted of following an identical method
but without the addition of Neem oil, and a negative control
consisted of Neem oil added to deionized water with no
bacteria.
To test varying mixing times at a fixed concentration, the FIG. 1. Log inactivation of E. coli in phosphate-buffered
disinfected sample was prepared in batches of 250 mL with water, with increasing Neem oil dose and 5 min of mixing
the application of 17.1 g
L1 Neem oil. The samples were then time.
1438
FIG. 2. Percent inactivation of total coliforms and fecal
coliforms in the pond water from England, with increasing
Neem oil dose and 5 min of mixing time.
The tests of 17.1 g L1 Neem oil with increasing contact
time for the unfiltered pond water from England showed that
the average reduction in total coliforms increased with time
up to 12 min, whereas the reduction in fecal coliforms did not
consistently increase with time (Fig. 3).
These results suggest that fecal coliforms may be less sus-
ceptible to the antibacterial action of Neem oil than nonfecal
coliforms. To date, there has been no research to compare the
antibacterial activity of Neem oil against specific fecal coli-
form and nonfecal coliform bacteria. However, previous re-
search into the antibacterial properties of Neem oil and other
Neem components has indicated that the active components
of Neem are selective in the micro-organisms that are targeted
(Eppler et al., 1995; Hoque et al., 2007). One such study using
the disc diffusion method showed that Neem oil was unable
to inhibit Shigella species but exhibited a moderate to good
inhibition of E. coli (Eppler et al., 1995). This also supports the
findings of research into a wide range of essential oils, most of
which have exhibited selective antimicrobial performance
against different bacterial species (Smith-Palmer et al., 1998;
Hammer et al., 1999; Chen, 2004; Romeo et al., 2008). The
ability of the active compounds to disrupt the cell membrane
will depend to some extent on composition and structure
of the membrane of the targeted organism (Canillac and
Mourey, 2004). Also, essential oils are in general reported to be
FIG. 3. Percent inactivation of total coliforms and fecal
coliforms in the pond water from England, with 17.1 g
L1
Neem oil and increasing mixing time.
MATTHEWS ET AL.
FIG. 4. Percent inactivation of total coliforms and fecal
coliforms in the river water from Nepal, with increasing
Neem oil dose and 5 min of mixing time.
more effective against Gram-positive than Gram-negative
bacteria (Tassou et al., 2000).
Comparison of results in surface waters
and pure lab-grade water
Given that E. coli is a major component of the group of fecal
coliform bacteria, a similar performance of Neem oil against
fecal coliforms and E. coli was expected. However, the inac-
tivation of E. coli in the pure laboratory-grade water (>99%
inactivation) was substantially higher than that of fecal coli-
forms in the unfiltered surface waters (typically <50% inac-
tivation). There are several factors that can affect the
antimicrobial activity of an essential oil ( Juven et al., 1994;
Hammer et al., 1999; Singh 1987; Winward et al., 2008), and
it is possible that this difference in disinfection performance
between the trials with pure laboratory-grade water and the
surface water trials is due to one or a combination of these
factors, which are discussed in this section.
As suggested previously, the antimicrobial components of
Neem oil appear to be effective against some bacterial species
more than others. In general, fecal coliforms and E. coli can be
regarded as equivalent, but a small number of nonfecal coli-
forms are thermotolerant and can grow at 448C (Mara and
Horan, 2003). It is possible that some of these are unaffected
by Neem oil; however, this would likely account for only a
small if not insignificant portion of the observed difference.
Growth conditions can alter the resistance of bacterial cells.
Nutrient deficiency, subinhibitory presence of antimicrobial
agents, and other environmental stresses can substantially
reduce susceptibility to disinfectants such as chlorine as well
as antibiotics (Lisle et al., 1998; Pellet et al., 2006). E. coli cul-
tured in the laboratory overnight in nutrient broth with fa-
vorable environmental conditions has been subjected to
minimal stress, and may as a result have much lower anti-
microbial resistance compared to naturally occurring strains
(Brown and Williams, 1985).
It was also observed that Neem oil dispersed more effec-
tively in the laboratory-grade water (i.e., PBS) than in the
surface water samples. In the former, an oil-in-water emulsion
was formed, whereas in the latter a water-in-oil emulsion
‘‘scum layer’’ formed after approximately 1 min of mixing.
This is likely due to the presence of Na2HPO4 in PBS, which
DISINFECTION OF WATERBORNE COLIFORM BACTERIA BY NEEM OIL
acts as an emulsifying agent. It has been suggested that only
hydrocarbon molecules that are dissolved in the aqueous
phase are available for intracellular metabolism (Sikkema
et al., 1995). This improved dispersion may have contributed
to the higher inactivation of E. coli in PBS by increasing the
availability of the active components of the Neem oil.
The antimicrobial action of essential oils is centred on
disruption of the cell membrane by various lypophilic
compounds. The presence of organics has been reported to
be detrimental to this process ( Juven et al., 1994; Hammer
et al., 1999; Singh 1987). PBS contains no organic matter,
whereas the surface waters contained organic compounds
(as evidenced by the ultraviolet absorbance measurements
and observed color). Much of the research into the antibac-
terial properties of essential oils has been directed toward
their use as food preservatives, showing that fats and pro-
teins can detract from their antibacterial performance ( Juven
et al., 1994; Singh 1987). For example, the presence of organic
matter has been shown to interfere with the antibacterial
activity of tea tree oil (Hammer et al., 1999) and to reduce
the effectiveness of essential oil disinfection of greywater
(Winward et al., 2008). Proposed explanations are that the
organic matter bonds to the active sites of the antimicrobial
agents thereby reducing the effective concentration, or that
it protects the micro-organisms from exposure to the anti-
microbial substance by hydrostatically bonding to the cell
membrane ( Juven et al., 1994; Hammer et al., 1999); organic
matter may also reduce the dissolution of Neem oil into
water and thereby limit its disinfection performance.
The presence of suspended particles can also significantly
increase the resistance of bacteria to disinfection (e.g., by
chlorine), due to the shielding of bacteria that are agglomer-
ated together through attachment to particle surfaces (Ridg-
way and Olson, 1982). Particles have also been found to
detract from the performance of essential oils used to disinfect
greywater, especially particles >440 mm in diameter (Win-
ward et al., 2008). PBS contains very few particles compared to
the unfiltered surface waters that were considered in this
study.
The presence of NaCl has been reported to reduce the
bactericidal concentration required for other essential oils, due
to an increased hydrophobicity of the bacterial surface attrib-
uted to the increased salinity causing greater contact between
the micro-organisms and the antimicrobial agent (Kurita and
Koike, 1982; Canillac and Mourey, 2004). NaCl may also
increase the dissolution of Neem oil in water and thereby
increase the disinfection performance. The temperature-
compensated conductivity (TCC) of the pond water from
England was 1.1 mS while the TCC of PBS was 14.1 mS, which
indicates that there was a substantially higher concentration of
dissolved salts in PBS.
Another factor that can influence the performance of es-
sential oils is pH, with some studies suggesting that basic pH
is preferable and others the opposite relationship ( Juven et al.,
1994; Canillac and Mourey, 2004). The optimum pH could
depend on both the essential oil and the specific microor-
ganism being targeted. This is supported by a study of the
antimicrobial activity of caffeic acid, wherein the maximum
activity was obtained at different pH values for a range of
different micro-organisms (Almajano et al., 2007). The effect of
pH on antimicrobial activity may also be attributed to changes
in the surface charges of the microbial cells. The average pH of
1439
the samples from the pond water from England was 8.6, the
pH of the Nepal river water 7.1, and the pH of the PBS was 7.3;
pH was therefore not identified as a definite contributing
factor on coliform inactivation by Neem oil in this study.
Practical aspects of using Neem oil
as a water disinfectant
In this study, Neem oil was shown capable to produce a
reduction in total coliform counts and, less consistently, in
fecal coliform counts in the unfiltered surface waters. How-
ever, the treated waters were not able to meet requirements
for drinking water disinfection, such as WHO guidelines
which state that E. coli or fecal coliforms must not be present in
100 mL (WHO, 1993); in these experiments, total and fecal
coliforms in the surface water samples were typically reduced
from between 100 and 300 colony-forming units (CFU) per
100 mL in the untreated surface water samples to between 20
and 100 CFU per 100 mL in the final treated samples. In ad-
dition, the taste and appearance at the dose levels required to
achieve measurable inactivation in the unfiltered surface
waters in this study would be unappealing to consumers of
the treated water. It should therefore be concluded that Neem
oil is not directly suitable for use as a drinking water disin-
fectant of unfiltered surface waters.
That said, the fact that Neem oil exhibited a substantial
reduction of isolated E. coli and, in some cases, a reduction of
average total coliform counts and fecal coliform counts, albeit
lower than would be required for drinking water disinfection,
indicates that there is potential for a natural drinking water
disinfectant based on or including Neem extracts. Due to the
inconsistent performance exhibited by most essential oils
against different bacterial species, an effective natural disin-
fectant may need to consist of a mixture of several plant spe-
cies (Bhattarai, 1989). Indeed, synergistic effects of combined
essential oils have been reported (Lachowicz et al., 1998).
A natural disinfectant has the potential to provide advan-
tages over the conventional small-scale (i.e., household level)
disinfectant options currently available in developing coun-
tries. A natural disinfectant could be produced using locally
grown plants, with no need to import materials. Further,
by making use of plants that are traditionally used for me-
dicinal or other purposes, the natural disinfectant may be
culturally more acceptable to the local community than im-
ported chemicals. Making use of locally available plants
would increase the availability of the disinfectant product in
geographically remote areas. It may also be possible to de-
velop a natural disinfectant product that does not suffer from
the taste and odour issues sometimes associated with chlo-
rine; the choice of plants used for flavoring could be tailored to
meet the esthetic preferences of a specific country or region.
There is also the potential to combine the application of a
natural disinfectant with other domestic or small-scale water
treatment processes, such as filtration or coagulation=settling.
By partially treating the water before the disinfection step,
some of the influence of natural water constituents that ap-
pear to inhibit the antibacterial properties of Neem oil could
be reduced, potentially allowing lower Neem oil doses to be
used. The principal role of the natural disinfectant would then
be to inactivate any remaining bacteria after filtration and
to prevent regrowth. Further investigation into potential
methods for isolating and concentrating the active chemical
1440
substances in Neem oil (e.g., nimbidin) to create a more potent
disinfectant solution is also recommended.
Conclusions
The main conclusions of this study were:
1. Neem oil doses as low as 2.13 g
L1 with 5 min of
mixing time achieved high levels of inactivation of the
isolated culture of E. coli in pure laboratory-grade water
(>99% inactivation). This is evidence in support of
the previously reported antimicrobial properties Neem
oil and demonstrates its potential as a natural disin-
fectant.
2. The disinfection of total and fecal coliforms in unfiltered
surface waters was variable and limited to <70% inac-
tivation typically. The inactivation of total coliforms
was consistently greater than the inactivation of fecal
coliforms, suggesting a potential range of sensitivities to
Neem oil among coliform bacteria.
3. The reduced effectiveness of the Neem oil in the unfil-
tered surface waters compared to the pure laboratory-
grade water suggests a possible interference by natural
water constituents (e.g., suspended particles, dissolved
organic matter) which may inhibit the antibacterial
potential of Neem oil, or that naturally occurring bac-
teria are more resistant to the antimicrobial effects of
Neem oil. The performance of Neem oil disinfection
combined with a pretreatment step (e.g., sand or cloth
filtration) and=or application of Neem oil in a mixture
with other essential oils are recommended for further
investigation.
Acknowledgments
The authors acknowledge the British Council=Department
for International Development (DfID) ‘‘Development of
Partnerships in Higher Education’’ (DelPHE) program for
facilitating the link between the UK and Nepal partners on
this research.
Author Disclosure Statement
The authors declare that no conflicting financial interests
exist.
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