Journal of Ecological Engineering
Journal of Ecological Engineering 2024, 25(11), 134–142 Received: 2024.08.20
https://doi.org/10.12911/22998993/192713 Accepted: 2024.09.23
ISSN 2299–8993, License CC-BY 4.0 Published: 2024.10.01
Antibacterial Efficacy of Moringa oleifera Seeds for Water Purification
Nadia Sandra Kacem1*, Rahma Derdour1, Abdelhamid Djekoun1
1
Genetic, Biochemistry and Plant Biotechnology Laboratory, Faculty of Natural Sciences, Constantine 1 Frères
Mentouri University, Route Ain El Bey, Constantine 25000, Algeria
* Corresponding author’s e-mail:
[email protected]ABSTRACT
This study investigated the antibacterial efficacy of Moringa oleifera (M.oleifera) seeds as a natural coagulant for
water purification, addressing the critical need for clean water due to its significant impact on human health and dis-
ease prevalence linked to contaminated water. The research was carried out to observe the effect of M. oleifera on the
microbial load of Raw Water (RW) and treated water (TW) sourced from the Ibn Ziad wastewater treatment facility
in Constantine. Results of bacteriological analyses show that the raw water is heavily polluted with high levels of the
targeted bacteria: 4.4 × 10⁹ CFU/ml in total germs, 2.4 × 10⁴ CFU/100 ml in total coliforms and total streptococci,
and 1.1 × 10⁴ CFU/100ml in fecal coliforms, Escherichia coli, and fecal streptococci, with no decrease observed for
sulfite-reducing clostridia. However, the water treated with M. oleifera, at a dose of 20 g·L⁻¹, demonstrates a signi-
ficant reduction of 99% in total germs, 96% in total coliforms, and 98% in fecal coliforms, with a complete absence
of Escherichia coli, streptococci, as well as a complete elimination of total sulfite-reducing clostridia, which falls
with the standard guidelines for using this water in irrigation. The obtained results confirmed that M. oleifera seeds
positively impact the reduction of pathogenic microorganisms found in wastewater.
Keywords: antibacterial activity, microbes, Moringa oleifera, seeds, water purification.
INTRODUCTION pathogenic microorganisms, including bacteria,
viruses, and parasites. This critical process is the
Water plays a crucial role in all aspects of hu- first step in conventional water treatment and
man life, industrial processes, and agriculture, all typically uses aluminum sulfate as the coagulant
of which are increasingly strained by socio-eco- (Degremont, 2005). In addition to aluminum sul-
nomic development and a growing global popu- fate, inorganic coagulants such as ferric salts and
lation. The World Health Organization estimates polyaluminum chloride are frequently employed
that up to 80% of diseases and illnesses worldwide in wastewater treatment plants for coagulation.
are linked to inadequate sanitation, unsafe water, However, these synthetic coagulants have notable
or water scarcity (Delelegn et al., 2018; Kenea drawbacks, including the production of inorganic
et al., 2023). Contaminated water contains vari- sludge that necessitates complex and costly man-
ous water-borne diseases like diarrhoea, typhoid, agement, as well as significant variations in pH
cholera, amoebic dysentery, giardiasis and certain following treatment (Andrade et al., 2021). The
neurological disorders. It has been estimated that use of natural coagulants, such as Moringa ole-
about 6 million children die from diarrhoea ev- ifera seeds, emerges as a promising solution for
ery year in developing countries. This alarming treating wastewater before its reuse for irrigation.
situation is essentially due to the lack of effective This plant, originally from Asia, has now spread
processes (WHO, 2015). widely across Africa (Yamaguchi et al., 2021), at-
Coagulation-flocculation is an essential tech- tributed to its drought resistance, rapid growth,
nique for removing turbidity from water, which and nutritional value, making Moringa increas-
consists of suspended particles (both inorganic ingly cultivated. M. oleifera roots, leaves, seed,
and organic), dissolved organic materials, and fruit, fowers, bark and immature pods are used as
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Journal of Ecological Engineering 2024, 25(11), 134–142
cardiac and circulatory stimulants, have antipyret- the inlet and outlet of the plant using sterile 1-liter
ic, antiepileptic, antitumor, antiinfammatory, anti- glass bottles with stoppers. The most commonly
ulcer, diuretic, antihypertensive, cholesterol lower- employed sampling method involves completely
ing, antispasmodic, antidiabetic, hepatoprotective, filling the bottles without shaking them, while
antioxidant, antibacterial and antifungal proper- minimizing contact with air as much as possible.
ties, and are being used for the treatment of vari- This process adheres to the guidelines outlined in
ous ailments in the indigenous system of medicine ISO 5667-1:2006, which provides international
(Anwar et al., 2007). Several studies have investi- recommendations for the microbiological analy-
gated the efficacy of M. oleifera seeds in wastewa- sis of wastewater.
ter treatment. Research has demonstrated that the
seed extracts can significantly reduce bacterial Plant material
load in wastewater, thereby improving the overall
water quality (Ghebremichael et al., 2005; Anwar The mature seeds of M.oleifera obtained from
et al., 2007; Shebek et al., 2015, Al-Kindi and Al- southern Algeria (Ghardaia nursery) were de-
Haidri 2021; Freire et al., 2015; Yamaguchi et al., shelled and dried at ambient temperatures (25 °C)
2021; Shah et al., 2023). Therefore, the aim of for 5 days before milling. The white kernels
this study was to evaluate the antibacterial effi- were ground into using a mortar and pestle and
cacy of M.oleifera seeds in wastewater treatment, then passed through a 2 mm sieve to obtain the
focusing on various bacteriological indicators. fine powder. Two grams of this fine seed pow-
der was added to 100 mL of distilled water. The
obtained mixture was agitated for 1 hour to ex-
MATERIALS AND METHODS tract the coagulant used to treat the waters and
filtered using Whatman N°1 before using. The
Sample preparation filtrate was shaken again (Ndabigengesere et al.,
1995; Shahzad et al., 2014).
The Ibn Ziad wastewater treatment plant is
located 12 km from the city of Constantine, in Decimal dilutions
the Hamma Bouziane district, along the road to
Mila; it covers an area of 12 hectares (Figure 1). The selection of the number of dilutions lar-
Tests were conducted on two types of water: raw gely depends on the microbial load of the water
wastewater and treated wastewater. These sam- being analyzed. According to Dellaras (2010),
ples were sourced from the Iben Ziad wastewater the procedure for preparing decimal dilutions is
treatment plant in the Constantine province (Al- as follows: 10 ml of the water sample is asepti-
geria). Water samples were collected from both cally introduced into a glass bottle containing 90
Figure 1. Study site and location of sampling station
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Journal of Ecological Engineering 2024, 25(11), 134–142
ml of sterile distilled water, using a graduated • 10 ml into three tubes containing 10 ml of
pipette. Once the bottle is closed, it is shaken to BCPL D/C medium.
ensure thorough mixing, resulting in a 1/10 dilu- • 1 ml into three tubes containing 10 ml of
tion. Then, 10 ml from this 10¹ dilution is taken BCPL S/C medium.
and transferred to a second bottle to produce a 10² • 0.1 ml into three tubes containing 10 ml of
dilution. This process should be repeated until the BCPL S/C medium.
desired dilution is achieved.
After transferring the samples, the tubes were
gently shaken to homogenize the contents wi-
Bacteriological analyses thout introducing air into the Durham tube. All
prepared tubes were then incubated at 37 °C for
Bacteriological quality is one of the impor-
24 to 48 hours. Following incubation, the tubes
tant parameters of water potability. It is mea-
that are considered positive showed turbidity,
sured by the presence of a pollution indicator of
with a color change from violet to yellow, and gas
organisms, in particular, total germs and fecal
production in the Durham tube (ISO 2012).
coliforms (Escherichia coli). The bacteriolo-
gical analyses of raw wastewater, treated was-
Fecal coliforms and Escherichia coli
tewater, and wastewater treated with M.oleifera
were conducted using the most probable number Fecal coliforms and E. coli were quantified
method, which aims to detect and quantify the following the ISO 9308 (2012) standard. Positive
following germs: total germs (TG), total coli- BCPL tubes identified during coliform enumera-
forms (TC), fecal coliforms (FC) and Escheri- tion were subcultured by transferring a loopful
chia Coli, total streptococci (TS), fecal strepto- into a tube containing Schubert medium with a
cocci (FS) and sulfite-reducing clostridia (SRC). Durham tube. The gas in the Durham tube was
The method used to isolate and determine these expelled, and the medium was thoroughly mixed.
bacteria is the NPP liquid count technique in line The tubes were then incubated at 44 °C for 24
with the AFNOR NF T90-413 standard (1985). to 48 hours. Tubes showing both bacterial growth
and gas production were deemed positive. The
Total germs presence of Escherichia coli was confirmed by
the formation of a red ring at the surface after ad-
This measure provides an overall assessment of
ding 2 to 3 drops of Kovacs reagent (ISO 2012).
the harmful nature of the water, without identifying
the specific sources of contamination. E. coli is a
Total streptococci
key bacterium used to indicate fecal contamination
(Edberg et al., 2000). Both total bacteria and E. coli To assess total streptococci counts from the
serve as indicators to assess pollution levels and water sample, 10 ml was aseptically transferred
water quality (Adamou et al., 2020). into three tubes containing 10 ml of Rothe D/C
To assess total bacterial counts from the water medium. Additionally, 1 ml was transferred into
sample, 1 ml was transferred into two prepared three tubes containing 10 ml of Rothe S/C me-
and numbered Petri dishes. Each dish was then dium, and 0.1 ml was also transferred into three
filled with approximately 15 ml of nutrient agar tubes containing 10 ml of Rothe S/C medium.
and gently mixed by rotating before allowing it The tubes were then shaken to ensure homogeni-
to solidify. The prepared dishes were incubated zation while avoiding the introduction of air into
under two different conditions: one at 37 °C for the Durham tube. Following this, the prepared
24 to 48 hours and the other at 22 °C for 72 hours. tubes were incubated at 37°C for 24 to 48 hours.
The results are expressed as the number of germs After incubation, the tubes that exhibited micro-
per milliliter (ISO 6222:1999) (ISO 1999). bial turbidity were considered positive 4 (ISO
7899-2 (2012)).
Total coliforms
Fecal streptococci
Total coliforms are quantified according
to ISO 9308 (2012). From the water sample to Fecal streptococci were counted following
be analyzed (mother solution or the three suc- the ISO 7899 (2012) standard: The positive Ro-
cessive dilutions), the following are aseptically the tubes identified during the streptococci en-
transferred: umeration were subcultured using a loop into a
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Journal of Ecological Engineering 2024, 25(11), 134–142
tube containing LITSKY EVA medium. Any gas followed by a Fisher comparison of means test at
present in the Durham tube should be expelled, 5% levels. The homogeneous groups were separ-
and the medium should be mixed thoroughly. The ated using the Newman-Keuls test at 5% levels.
tubes were then incubated at 37 °C for 24 hours.
Tubes were considered positive if they displayed
both microbial turbidity and a violet or whitish RESULTS
disc at the bottom (ISO 2012)
Total germs
Sulfite reducing clostridia
The results shown in Figure 2 indicate a high
The enumeration of sulfate-reducing anaerobic concentration of total germs in the untreated
bacteria follows a specific procedure as outlined in wastewater, with an average of 4.4 × 10⁹ CFU/ml
the NFT 90-415 standard (1985). First, approxima- at 22 °C and 2.4 × 10⁸ CFU/ml at 37 °C. Follow-
tely 250 ml of the water sample (whether the ori- ing treatment, this concentration significantly
ginal solution or three successive dilutions) were decreased, achieving a removal rate of 99.96%
placed into a sterile tube and heated at 75 °C for at 22 °C and 99.95% at 37 °C. Treatment with
15 minutes to destroy any vegetative forms of sul- Moringa seeds led to an even greater reduction
fate-reducing anaerobic bacteria. After heating, the (p < 0.001), with removal rates of 99.99% at 22 °C
tube was rapidly cooled under running water. The and 99.97% at 37 °C.
sample was then divided into four sterile tubes,
with 5 ml per tube, and 18 to 24 ml of meat-pep- Total coliforms
tone agar is added to each. The tubes were gently The results shown in Figure 3 indicate that the
mixed to prevent the introduction of air bubbles total coliform concentrations in the raw water are
and oxygen, then allowed to solidify for 30 mi- 2.4 × 10⁴ CFU/100 ml. Following treatment, the ef-
nutes before being incubated at 37 °C for 48 hours. fluent showed a highly significant reduction, with
Observations are made at 24 and 48 hours. Finally, coliform levels decreasing to 1.1 × 10⁴ CFU/100
all black colonies were counted, and the total nu- ml, reflecting a 54.16% removal rate. On the other
mber of colonies from the four tubes was reported hand, treatment with M. oleifera seeds significant-
per 20 ml of the analyzed water. ly improved removal rates, achieving 80.83% for
raw wastewater and 96.35% for treated water.
Statistical analysis
Fecal coliforms and Escherichia coli
The data were analyzed using MINITAB 18
software. The analysis was carried out using an- The results presented in Figure 3 indicate an
alysis of variance (ANOVA). For each parameter average concentration of fecal coliforms of 1.1 × 10⁴
three experimental replicates were performed, CFU/100 ml in the untreated wastewater. Following
Figure 2. Estimation of total germ counts at 22 °C and 37 °C in raw wastewater (RW) as
well as treated wastewater (TW) before and after the addition of M. oleifera seeds.
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Journal of Ecological Engineering 2024, 25(11), 134–142
Figure 3. Estimation of total coliforms (TC), fecal coliforms (FC), and Escherichia coli in raw
wastewater (RW) and treated wastewater (TW) before and after the addition of M.oleifera seeds
treatment, there was a significant reduction, achiev- 10² CFU/100 ml) at the inlet and a complete elimin-
ing a removal rate of 58.18%. In contrast, the water ation (100%) at the outlet.
treated with M. oleifera seeds demonstrated sig-
nificantly higher fecal coliform removal rates (p < Streptococci
0.001); the removal rate in raw water was 92.09%,
while in treated water, it increased to 97.10%. The analyses indicate relatively high concen-
Bacteriological analyses of Escherichia coli trations of total streptococci at the entrance of the
indicated elevated levels at the entrance of the waste- wastewater treatment plant, measuring 2.4 × 10⁴
water treatment plant, with an average concentration CFU/100 ml. In contrast, fecal streptococci con-
of 1.1 × 10⁴ CFU/100 ml. The reduction in fecal coli- centrations are lower, approximately 1.1 × 10⁴
forms was statistically significant at the 5% level, CFU/100 ml in the raw water. Following treat-
showing a decrease of 58.18%, with concentrations ment, these concentrations decrease, with a re-
around 4.6 × 10³ CFU/100 ml, which still excee- duction rate of 54.16% for total streptococci and
ded acceptable limits for safe reuse (Blumenthal et 58.18% for fecal streptococci (Figure 4). Nota-
al. 2000). In contrast, the treatment with M.oleifera bly, the treatment with M.oleifera seeds results
seeds exhibited exceptionally high reduction rates, in the complete elimination of these microorgan-
achieving a remarkable 98.03% reduction (2.16 × isms, achieving a 100% reduction rate.
Figure 4. Estimation of total streptococci (TS) and fecal streptococci (FS) in raw wastewater
(RW) and treated wastewater (TW) before and after the addition of M.oleifera seeds
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Journal of Ecological Engineering 2024, 25(11), 134–142
Sulfite-reducing clostridia levels of various indicator bacteria, such: total
germs, total and fecal coliforms, Escherichia coli,
The enumeration results (Table 1) indicate total streptococci, and sulfite-reducing clostridia
that sulfite-reducing clostridia concentrations are (Figure 5) signaling significant contamination. The
significantly elevated at both the inlet and outlet very high concentrations of these indicators are due
of the wastewater treatment facility. Interesting- to the abundance of nutrients, sufficient dissolved
ly, the results from both raw and treated wastew- oxygen, alkaline pH, and moderate temperatures,
ater samples treated with M.oleifera seeds dem- all of which create ideal conditions for bacterial
onstrated complete elimination of these spores, growth and proliferation (Smith et al., 2022).
achieving a 100% reduction rate. After conventional treatment, all measured
parameters showed significant reductions; howe-
ver, they still remained above the acceptable li-
DISCUSSION mits for safe reuse in irrigation (Blumenthal et al.,
2000). In contrast, the water treated with M.olei-
Results of bacteriological analyses indicate fera seeds exhibited even greater reductions and
that raw wastewater is heavily polluted with high in accordance with standard norms (Blumenthal
Table 1. Enumeration of sulfite-reducing clostridia
Types of water
Parameter
RW TW RWM TWM
SRC (spores/20 ml) Innumerable Innumerable 0 0
Note: RW – raw water, TW – treated water, RWM – raw water treated with MO seeds, TWM – treated water
treated with MO seeds, SRC – sulfite-reducing clostridia.
Figure 5. Demonstrations of some enumerations of total germs (A), sulfite-reducing Clostridium
(B), total coliforms (C), fecal coliforms and E. coli (D), and total and fecal streptococci
(E) in raw water. MO (+) presence of M. oleifera, MO (-) absence of M. oleifera
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Journal of Ecological Engineering 2024, 25(11), 134–142
et al., 2000, WHO, 2006). The findings clearly in- 4-(L-rhamnopyranosyloxy) benzylglucosinolate
dicate that Moringa seed powder can effectively (Xiao et al., 2020). Adji et al. (2020) reported that
influence a variety of bacterial species, including the seed and leaf extract of M. oleifera played a
total germ, total coliforms, fecal coliforms, E. role in preventing some of the effects of the pa-
coli, total streptococci, fecal streptococci, and thogenesis of diarrhea due to bacterial infection.
sulfite-reducing clostridia. Methanol, N-hexane, ethyl acetate, flavonoids,
While total coliforms are a broad group that phenols, saponins, alkaloids, tannins, and steroids
includes bacteria from various sources, fecal coli- from the seed and leaf extract of M. oleifera have
forms are a more specific subset that is better suit- antibacterial effects.
ed as an indicator of recent fecal pollution from The antibacterial properties of M. oleifera can
warm-blooded animals (Li et al., 2021). These also be ascribed to the presence of a natural ca-
results align with the findings of Asrafuzzaman et tionic protein (MOCP) that acts as a flocculant,
al. (2011), who reported a fecal coliform removal decreasing the turbidity and removing negatively
rate of up to 96% using Moringa seeds. The ob- charged particles, including bacteria (Ndabigen-
tained result aligns also with the findings of Suth- gesere et al., 1995). The cationic proteins found
erland et al. (2022) who found that the addition in M. oleifera seeds are initially attracted to the
of M. oleifera seeds to raw water resulted in sig- bacteria through electrostatic forces. Once ad-
nificant microbial reductions, achieving removal hered, the protruding hydrophobic loop on the
rates of 85–95% for fecal coliforms and up to protein may penetrate the bacterial cell wall and
100% for Escherichia coli. destroy the bacteria (Suarez et al. 2005; Jerri et al.
The findings of this study also indicate the 2012; Shebek et al., 2015).
complete elimination of streptococci following
the addition of M. oleifera seeds. Similar results
were documented by Aitmelloul et al. (2020) CONCLUSIONS
and Vunain et al. (2019), who reported a 100%
reduction in fecal streptococci. Contamination The investigation into the antibacterial efficacy
of water supplies by streptococci is indicative of the M. oleifera seeds for water purification has
of fecal pollution, typically arising from animal demonstrated promising results, highlighting the
waste, and can also occur in human as well as potential of these seeds as an effective natural co-
plant sources (Sinton et al., 2007) agulant for wastewater treatment. The study dem-
Sulfite-reducing clostridia are frequently used onstrates the superior antibacterial properties of M.
as indicators of historical or intermittent fecal oleifera seeds compared to conventional wastew-
contamination in water sources. Their persistence ater treatment methods. However, further research
suggests a failure at a specific point in the natural is necessary to evaluate the effectiveness of this
filtration process, potentially allowing contamin- plant in the wastewater purification process, with
ants to pass through (Robles et al., 2000). Studies by additional analyses focused on improving the treat-
Wéry et al. (2009) and Wen et al. (2010) highlight ment technique utilizing M. oleifera seed powder.
the significant environmental dispersal of Clostri-
diaceae via wastewater treatment plant effluents. Acknowledgement
The persistence of these bacteria in the environ-
This study was supported by the SEACO
ment can be attributed to their extraordinary resili-
Laboratory in Ain Smara, Constantine, Algeria,
ence to produce spores that can endure challenging
and the laboratory of Genetics, Biochemistry,
environmental conditions, allowing them to persist
and Plant Biotechnology at Constantine 1 Frères
in water sources. While there may not be a direct
Mentouri University. We thank the both institu-
citation confirming a 100% reduction in sulfite-re-
tions for their assistance.
ducing clostridia specifically, the existing research
highlights the significant antibacterial properties of
M. oleifera seeds and their effectiveness in reducing
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