The availability of portable water is an indispensable feature for preventing
diseases and improving the quality of life (Aderiye et al., 1992). More
than 80% of all human diseases are attributed to use of unsafe water (Effler
et al., 2002; Oslen et al., 2002). Consequently,
about 1.25 billion people in the world especially the tropics suffer from major
water related diseases such as cholera, typhoid, dracunculiasis, schistosomiasis
among others (Odeyemi, 1988). Most rural settings and
villages in developing countries do not have access to portable water and sanitation
facilities and the conventional method of water treatment is expensive (Lewis,
In Nigeria, most rural dwellers depend on water from hand-dug wells/bore holes,
streams/rivers, ponds/lakes and rain when available, for domestic purpose. Most
of the microorganisms isolated in water drawn from wells in Owo; running streams
and even in storage tanks at Ado-Ekiti, southwest Nigeria were found to be members
of the Enterobacteriaceae such as Bacillus sp., Enterobacter sp.,
Escherichia coli, Klebsiella sp., Proteus sp., Pseudomonas
sp., Staphylococcus sp. and Streptococcus sp. (Aderiye
et al., 1992, 2005; Oluyege
and Famurewa, 2005).
The presence of coliforms in water suggests previous human interaction and
faecal pollution which may lead to serious water-borne diseases when consumed
without any form of treatment. Methods usually employed for treating water for
consumption include sedimentation, boiling, filtration, coagulation by addition
of chemical agents such as alum and incorporation of plant parts such as Moringa
oleifera in turbid water (Ndabigensere et al.,
1995). Natural coagulants have been detected and extracted from seeds of
seven different Moringa species viz., M. stenopetala (Kenya)
M. peregina (Egypt), M. drouhardii (Madagascar), M. longituba
(Somalia), M. ovalifolia (Namibia), M. concanesis (India and Pakistan)
and M. oleifera (Sudan) (Jahn, 1988). Of all
the species, the use of M. oleifera seed as a natural coagulant for water
treatment stands out in Sudan and other parts of Africa (Eliert
et al., 1981; Anonymous, 1987).
The plant is known to have a wide application in therapy ranging from its use
as antiscorbutic and anti-irritant in Nigeria, to the treatment of diarrhea,
rheumatism and goiter in Mauritius and the treatment of nervous debility and
leprosy in India (Olayemi and Alabi, 1994). The seed
can be consumed after drying and as condiment and garnish in food. As a multipurpose
tree, it is also of great environmental interest in plantations, in private
premises, around fields and on nursery plots (Madsen et
Provision of safe and clean water to rural villages remains a central objective
of the World Health Organization (Pollard et al.,
1995). A substantial research effort has focused on low cost approaches
to water and waste treatment in less developed countries through the application
of indigenous natural products of soil origin which offers genuine localized
and appropriate solutions to water quality problem (Pollard
et al., 1995). This study therefore examines the efficacy of M.
oleifera seed extract treatment on the microflora of water intended for
MATERIALS AND METHODS
Source of Plant Material
Moringa oleifera seed pods were obtained from the trees planted around
the Doctors residential quarters at the State Specialist Hospital complex
in Ado-Ekiti, Nigeria. The pods were broken manually to release the seeds which
were later spread in the open air to dry. The seed was identified and voucher
sample deposited at the herbarium unit of the Department of Plant Science, University
of Ado-Ekiti, Nigeria.
Source of Microorganisms
Pure cultures of bacterial isolates obtained were identified as described
by Barrow and Feltham (1993) and compared to standard
stock cultures obtained from the Department of Microbiology, Obafemi Awolowo
University (OAU) Ile-Ife, Nigeria. The organisms identified include Bacillus
cereus, Escherichia coli, Pseudomonas aeruginosa, Salmonella
typhi, Shigella dysenteriae, Staphylococcus aureus and
Streptococcus faecalis. The bacteria were maintained on nutrient and selective
agar at 4°C. When needed, the organisms were cultivated on minimal broth
medium and stored at 37°C for 24 h.
Source of Water
Samples of raw water were obtained aseptically from a concrete ring-walled
well and a slow running stream. The well about 7 m deep is located 120 m from
a seasonal water logged area along the Housing Corporation estate, Ado-Ekiti.
The Elemi stream is also seasonal and situated north-east of the University
of Ado-Ekiti campus, along Iworoko road, Ado-Ekiti. Water sample obtained from
both the well and stream was designated as underground and surface water, respectively.
Sampling was carried out daily between 7:30 and 8:00 am in July through September
for three consecutive years, 2004-2006. Treatment and analyses of water samples
were carried out at the Laboratory of Microbiology Department, University of
Ado-Ekiti within 2 h of collection.
Preparation of Moringa Seed Suspension
The seed suspension was prepared as described by Olayemi
and Alabi (1994) and Jahn (1988). Fifty grams of
the dried seed were ground into powder and kept aseptically at room temperature
until when needed. Two grams of the seed powder were soaked in 100 mL distilled
water for about 1 h. Later, the suspension was thoroughly shaken and filtered
through a Whatman filter paper No. 1. The filtrate served as the crude extract.
Treatment of Water Samples and Bacteriological Analysis
A known quantity (ca 100 mL) of varied concentrations (20, 40 and 60 μg
L-1) of Moringa seed extract was aseptically added to 1 L
of each water sample. The untreated water samples served as control.
Water samples were cultured on the following media; nutrient agar, Salmonella
and Shigella agar, Slanetz and Barthley medium and mannitol salt
agar using the pour plate method (Barrow and Felthan, 1993).
Bacteria counts were determined hourly for 24 h and also at 12 h interval for
The determination of the Minimum Inhibitory Concentration (MIC) of the seed
extract on the bacterial pathogens was carried out using the agar diffusion
technique (Dahot, 1998). The estimation of the total
bacteria and coliforms was carried out by serial dilution using the pour plate
method. The inhibitory rate (cfu h-1) was the difference between
the microbial concentration of water sample after 24 h seed treatment and that
of fresh water sample over 24 h.
Data obtained were statistically analysed using the t-test and analysis
of variance. The level of significant difference was determined at p≤0.05.
Twenty six percent of the total bacteria cells present in the untreated surface water sample were precipitated within 24 h (Table 1). When water was treated with 20 μg mL-1 M. oleifera seed extract, there was 93.75% reduction in total bacteria count at an inhibitory rate of 7.5x102 cfu h-1. About 26.67% coliforms were precipitated in the untreated surface water sample after 24 h storage while 97.4% coliforms in Moringa seed treated water sample were precipitated at the rate of 3.04x102 cfu h-1.
A 32.9% reduction in total bacteria load was observed in the fresh underground water after 24 h storage as against 89.4% in Moringa seed treated water precipitated at the rate of 3.17x102 cfu h-1. Meanwhile, the coliforms were reduced by 94.29% at the rate of 1.38x102 cfu h-1 after 24 h treatment with the seed extract.
With 30 μg mL-1 seed extract treatment and 72 h storage period in surface and underground water samples, the total bacteria count reduced drastically to 1000 cells mL-1 (Table 2). No growth was recorded on any specialized media for the cultivation of coliforms even at concentrations as low as 10 μg mL-1 of the seed extract after 48 h storage.
|| Effect of Moringa oleifera1 seed extract
on bacterial load* of surface and underground water samples
|120 μg mL-1 M. oleifera
seed extract was used; * (104 cfu mL-1); Frh: Fresh
(untreated) water sample; TB: Total Bacterial count; CO: Coliform count
Within 6 h of seed treatment, the inoculum sizes reduced by 98.6% in E.
coli, 97.6% in P. aeruginosa, 97.2% in S. faecalis, 94% in
S. typhi and 36.4% in S. dysenteriae. The bacterial population
of P. aeruginosa and S. faecalis remained constant after 7 h storage
(Table 3). However there was a tremendous increase in the
cell population by 98.4% in E. coli, 98.5% in S. typhi and 99.4%
in S. dysenteriae after 24 h. There was a significant difference in the
increase of the bacterial population in the different microbes at p<0.05.
The zones of inhibition increased correspondingly with an increase in the concentration of the seed extract (Table 4). All the pathogens except P. aeruginosa were less affected by the seed extract at concentrations below 40 μg mL-1. It required 50 μg mL-1 of the seed extract as the MIC against S. typhi.
|| Effect of Moringa oleifera seed extract on microbial
load in surface and underground water1
|1Count: x104 cfu mL-1; NG:
|| Antibacterial activity of Moringa oleifera1
|*20 μg mL-1 seed extract used; 1Organisms
cultivated in minimum broth medium; aInoculum size of bacteria
|| Inhibitory potential and Minimum Inhibitory Concentration
(MIC) of Moringa oleifera seed extract*
|*Seed extract incorporated into agar media; +: Poor growth,
++: Slight growth, +++: Dense growth; aInoculum size seeded
as in Table 3; bReadings taken after 24th
incubation at 30°C
In this study, M. oleifera seed extract efficiently precipitated about
90% of the total bacteria and 99% coliforms, an indication of the seed potential
to coagulate and clarify the body of water. Madsen et
al. (1987) also reported a 90% reduction in the bacterial load of water
treated with Moringa seed paste. Meanwhile, Sattaur
(1983) and Grabow et al. (1985) posited that
a considerable hygiene improvement amounting to a primary bacterial reduction
of 90-99% or more was achieved within 1 h of Moringa seed treatment.
The rate at which the total bacteria and coliforms were coagulated was faster
in Moringa seed treated surface water than in underground water; the
surface water being more turbid than the underground water. This might explain
in part the reason for the high efficacy of the seed in highly turbid water
than in less turbid water. The bacterial regrowth observed in S. typhi
(98.4%), S. dysenteriae (99.4%) and E. coli (98.4%) is
an indication of the bacteriostatic ability of M. oleifera seed
on these organisms. However, the seed also possessed bactericidal activity especially
in water samples inoculated with S. faecalis and P. aeruginosa
where there was no regrowth. It is worth noting that treated water when left
to stand may deteriorate in quality than even untreated water due to bacterial
regrowth. Madsen and Schlundt (1989) reported secondary
increases in total bacteria and faecal coliform counts after using Moringa
seed and filtration through Sudanese bentomite clays.
The secondary bacterial growth might be due to the presence of some bacterial
cells which are partially inactivated. Experimental parameters such as time,
temperature and water constituent exert a profound influence on bacterial regrowth
(Madsen et al., 1987). Coliform regrowth conditions
can be expected to occur regularly in polluted tropical waters where temperature
exceeds 20°C, a feature which is reflected in real life situation by multiplication
of some species of faecal bacteria in the environment under favourable conditions
(Madsen et al., 1987). The results obtained in
this study corroborate the report of Aderiye et al.
(2005) and Oluyege and Famurewa (2005) confirming
the endemic presence of these human pathogens in and around Ado-Ekiti. This
may be attributed to poor environmental sanitation where wastes are disposed
off indiscrimately into drainage and environ especially during the rains.
Moringa oleifera seed elicited an appreciable antimicrobial activity
in vitro against both Gram-positive and Gram-negative bacteria. The zones
of inhibition increased correspondingly with an increase in the concentration
of the seed extract. The chemical structure of the bioactive components of M.
oleifera seed may explain the exact chemical reaction on the pathogens vis-à-vis
the bacteriostatic effect and eventual regrowth of these organisms. Studies
on the elucidation of the structure are ongoing in our Laboratory.