ARTICLE IN PRESS

Water Research 39 (2005) 2338–2344

www.elsevier.com/locate/watres

A simple purification and activity assay of the coagulant

protein from Moringa oleifera seed
Kebreab A. Ghebremichaela, K.R. Gunaratnab, Hongbin Henrikssonc,
Harry Brumerc, Gunnel Dalhammarb,
a
Department of Land and Water Resources Engineering, Brinellvägen 32, Royal Institute of Technology (KTH),
100 44 Stockholm, Sweden
b
Division of Applied Environmental Microbiology, Department of Biotechnology, Royal Institute of Technology (KTH),
Albanova University Centre, 106 91 Stockholm, Sweden
c
Mass Spectrometry Laboratory, Department of Biotechnology, Royal Institute of Technology (KTH), Albanova University Centre,
106 91 Stockholm, Sweden
Received 24 February 2004; received in revised form 15 February 2005; accepted 22 March 2005
Available online 25 May 2005

Abstract

Use of extracts from Moringa oleifera (MO) is of great interest for low-cost water treatment. This paper discusses
water and salt extraction of a coagulant protein from the seed, purification using ion exchange, its chemical
characteristics, coagulation and antimicrobial properties. The coagulant from both extracts is a cationic protein with pI
greater than 9.6 and molecular mass less than 6.5 kDa. Mass spectrometric analysis of the purified water extract
indicated that it contained at least four homologous proteins, based on MS/MS peptide sequence data. The protein is
thermoresistant and remained active after 5 h heat treatment at 95 1C. The coagulant protein showed both flocculating
and antibacterial effects of 1.1–4 log reduction. With samples of high turbidity, the MO extract showed similar
coagulation activity as alum. Cecropin A and MO extract were found to have similar flocculation effects for clay and
microorganisms. Simple methods for both the purification and assay of MO coagulating proteins are presented, which
are necessary for large-scale water treatment applications.
r 2005 Elsevier Ltd. All rights reserved.

Keywords: Moringa oleifera; Salt/water extraction; Antimicrobial; Coagulation activity assay; Purification; Characterization

1. Introduction (MO) seed (Jahn, 1986, 1988; Olsen, 1987; Sutherland

Aluminium and iron salts are the most commonly
used coagulants in water treatment. The cost and
environmental side effects of these compounds has
increased interest in the use of organic coagulants
derived from plant material, such as Moringa oleifera
Corresponding author. Tel.: +46 0 8 5537 8300;
fax: +46 0 8 5537 8468.
E-mail address: gunnel@biotech.kth.se (G. Dalhammar).
et al., 1994; Muyibi and Evison, 1995a, b). MO extracts
have been shown to have large effects on turbidity
removal (92–99% reduction) (Jahn, 1986; Muyibi and
Evison, 1995a). Water treated with MO seed extract
produces less sludge volume compared to alum (Nda-
bigengesere and Narasiah, 1998). An additional benefit
of using coagulants derived from MO is that a number
of useful products may be extracted from the seed. In
particular, edible and other useful oils may be extracted
before the coagulant is fractionated. Residual solids may

0043-1354/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2005.04.012
 ARTICLE IN PRESS
K.A. Ghebremichael et al. / Water Research 39 (2005) 2338–2344
be used as animal feed and fertilizer, while the shell of
the seed may be activated and used as an adsorbent. The
coagulant is thus obtained at extremely low or zero net
cost.
The main concern in using MO for water treatment is
the significant increase in organic load (Ndabigengesere
and Narasiah, 1998; Okuda et al., 2001b). Jahn (1988)
reported that water treated with crude MO extract
should not be stored for more than 24 h. The crude
extract is therefore not generally suitable for large water
supply systems where the hydraulic residence time is
very high. Two approaches may allow the use of MO
seed in such systems. Adsorption can be used to remove
the organic load from the crude seed extract. Alterna-
tively, the active coagulating component may be
extracted from the seed and used in pure or semi-pure
form, thus reducing the total amount of organic material
added to the treatment process.
The extraction of coagulants from MO has been
described previously, although it is somewhat unclear
what the exact nature of the compounds are. Several
reports have described the main water-extractable
component as proteinaceous. It was described as a
water-soluble protein with a net positive charge (Nkha-
ta, 2001), as dimeric cationic proteins with molecular
mass of 12–14 kDa and isoelectric point (pI) between 10
and 11 (Ndabigengesere et al., 1995). Others reported a
molecular mass of 6.5 kDa and a pI greater than 10
(Gassenschmidt et al., 1995). On the other hand, Okuda
et al. (2001a) reported that the active component from
an aqueous salt extraction was not a protein, poly-
saccharide or lipid, but an organic polyelectrolyte with
molecular weight of about 3.0 kDa. This suggests that
the water and salt extract may be of different nature.
The varying reports on the nature and properties of
the coagulant protein from MO thus necessitate further
study. Additionally, currently used purification methods
involve multiple steps, which complicate the use of MO
seed extracts in large-scale treatment applications. In the
present study, a simple, scalable purification method and
a convenient coagulation activity assay have been
developed which allow the straightforward comparison
of the characteristics and coagulation properties of
water and salt MO extracts relative to alum. In addition,
the antimicrobial properties of MO extract against a few
gram-positive and gram-negative bacteria are discussed.
2. Materials and methods
2.1. Water sample
Kaolin clay (10 g) was added to 1 L tap water, stirred
for 30 min and allowed to settle for 24 h to allow
complete hydration. Desired turbidity was obtained by
dilution.
2339
2.2. Extraction
Dried MO seeds were obtained from Senegal and
stored at room temperature. The seeds were shelled just
before the extraction and the kernel was ground using a
kitchen blender. Oil was extracted with 95% ethanol and
the solids were dried at room temperature. Samples (5%,
w/v) were prepared from the dried solids using distilled
water or 0.5 M NaCl solution, stirred for 30 min and
filtered through Whatman paper No. 3 and 0.45 mm
fiberglass. The filtrate is termed the crude extract.
Aqueous aluminium sulphate (alum) was prepared as a
5% (w/v) solution.
2.3. Purification
MO coagulant protein (MOCP) was purified using a
High-trap CM FF 1 mL cation exchanger column on an
Äkta explorer (Pharmacia Biotech). A flow rate of
2 mL min 1 was applied and 1 mL samples were
collected with an automatic fraction collector. The
column was equilibrated with 50 mM ammonium
acetate solution, pH 7 (solution A). Step elution using
1 M ammonium acetate, pH 7 (solution B) was
performed as follows. Crude water extract (CWE):
0–35% B over 1 min, hold for 10 min, then 35–100% B
over 15 min. Crude salt extract (CSE): 0–35% B over
1 min, hold for 10 min, then 35–65% B over 5 min, hold
for 10 min, 65–85% B over 10 min, hold for 10 min, then
to 100% B over 5 min. Coagulation activity was
measured for each fraction and the active fractions were
collected for further analysis.
2.4. Characterization
Native PAGE was carried out according to Hultmark
et al. (1983) using a Mini-PROTEAN 2 apparatus (Bio-
Rad). After electrophoresis, the 0.5 cm gel pieces were
cut horizontally and the protein was eluted into either
milli-Q water or 50 mM phosphate buffer. The coagula-
tion activity of each fraction was measured. Protein
content was estimated by Bradford method (Bradford,
1976) with bovine serum albumin as a standard.
Molecular mass was determined by SDS-PAGE on a
10% mini gel and tris-tricine SDS-PAGE. The pI was
determined from isoelectric focusing (IEF) (Model 111
mini IEF cell, BioRad) run with ampholytes in the pH
range 8–10. Markers with a pI range from 4.45 to 9.6
were used.
Thermal resistance of the coagulant protein was
studied by incubating crude extracts at temperatures
ranging from 60 to 100 1C for 0.5 to 5 h. Samples were
filtered through 0.45 mm fiber glass and tested for
coagulation activity.
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2340 K.A. Ghebremichael et al. / Water Research 39 (2005) 2338–2344
2.5. MS analysis
Mass spectrometric analysis was carried out on a Q-
TOFTM II mass spectrometer fitted with a nano Z spray
source (Waters Corporation, Micromass MS Technolo-
gies, Manchester, UK). The instrument was operated at
a resolution of 410,000 full width at half maximum
(FWHM). Mass calibration was obtained over the m/z
range 50–2000 using a solution of NaI (1.5 mg/mL) in
1:1 2-propanol/water.
Peptides from in-gel trypsin digestions of SDS-PAGE
samples were desalted and transferred to 1:1 acetonitrile/
water containing 0.1% formic acid using C-18 ZipTipTM
units (Millipore) according to the manufacture’s instruc-
tions. Samples were introduced into the mass spectro-
meter from borosilicate nanospray needles (source
voltage 1000 V). The quadrupole mass filter of the Q-
TOFTM II was operated in a wide band pass (RF only)
mode when collecting time of flight MS data. For
collision-induced dissociation MS/MS analysis, a quad-
rupole resolution of ca. 2 m/z was used when selecting
precursor ions. Maximum EntropyTM 3 (MaxEnt3) was
used to simplify the raw combined MS/MS data by
deconvolution of isotopic and charge state information
to produce centroid spectra containing only monoiso-
topic, single-charged peaks. De novo sequencing was
performed using PepSeqTM.
2.6. Coagulation activity assay
Optimum coagulant dosage is commonly estimated
from jar test analysis using 1 or 2 L volume beakers
equipped with mechanical stirrers. Since jar test analysis
requires large volumes of sample and coagulant dosage,
it may not be convenient for studying and comparing
large number of samples. In order to facilitate the
biochemical studies, it was necessary to develop a small
volume coagulation assay. This was performed by
measuring optical density (OD) of clay suspension at
500 nm (OD500) in a 1 mL semi-micro plastic cuvette
(10  4  45 mm, Sarsted Aktiengesellschaft & Co,
Germany). This method not only reduces the volume
of clay suspension sample and coagulant dosage
requirements, but also makes simultaneous analysis of
large number of samples possible. It is suitable to easily
screen out active and non-active coagulants and to
observe settling characteristics of the flocs by continu-
ously recording OD500.
Coagulant solutions (10 mL) were added to high
turbidity 1 mL clay suspension (250–300 NTU) in the
1 mL cuvette and homogenized instantly. This was
allowed to settle for 1 h and absorbance was measured
at 500 nm using a UV–Visible spectrophotometer (Cary
50 Bio). In order to reduce the background effect, a
sample volume of 200 mL from the top was transferred
to a quartz glass cuvette (type 105.200-QS, 10 mm light
path, HELLMA) for absorbance measurements. The
reduction in absorbance relative to the control defines
coagulation activity.
2.7. Antimicrobial tests
An antimicrobial study was performed using Escher-
ichia coli D31 (obtained from H. G. Boman, Institute of
Microbiology, University of Stockholm, Sweden) and
K12, Pseudomonas aeruginosa (obtained from SMI
Karolinska Institute, Stockholm Sweden) and Bacillus
thuringiensis Bt7 and Bt75 (Siden et al., 1979). Cultures
were grown in 0.2 M phosphate Buffered Lauryl Broth
(LBB) medium at 37 1C overnight with continuous
shaking. Cultures were centrifuged and the pellet was re-
suspended in 50 mM phosphate buffer or a 1:10 dilution
of LBB to OD620 of 0.2. MOCP and cecropin A were
added to the suspension culture, mixed well and incubated
at 37 1C for 2 h. Cell aggregations were analysed under the
microscope (Olympus BX51 with AnalySIS) and images
were captured using a Sony PC 120 camera. For growth
inhibition tests, samples from the incubated culture were
diluted 10- to 106-fold. Aliquots were uniformly spread on
nutrient agar plates and incubated at 37 1C overnight,
followed by colony counting.
3. Results and discussion
3.1. Purification
The high pI value of MOCP has been advantageously
used for simple purification using a cation exchange resin.
The absorbance spectrum at 280 nm of the bound protein
and corresponding coagulation activity are shown in Fig.
1. Salt and water extract samples showed three protein
peaks (a, b and c) where peaks b and c showed good
coagulation activity. The presence of two active peaks
may arise from the heterogeneity of one or more active
proteins in the extract. The two fractions had similar
molecular mass distributions by SDS-PAGE (Fig. 2a), but
showed differences in pI (result not shown), as expected
from their differential elution from the cation exchange
resin. It is interesting to note that the salt extract contains
a larger proportion of peak c, which implies that this
fraction is more tightly bound to the matrix, perhaps due
to stronger ionic interactions, which can only be disrupted
under conditions of high ionic strength.
These results indicate that a single fractionation step
is sufficient to remove the majority of proteins from
crude MO extracts to produce a sample enriched in
coagulating proteins. The use of standard cation
exchange chromatography, which is well established in
batch and continuous flow applications, will allow the
facile scale-up of the method to meet the needs of large
volume applications.
 ARTICLE IN PRESS
K.A. Ghebremichael et al. / Water Research 39 (2005) 2338–2344 2341

Similar coagulation activity was observed in samples and IEX had similar molecular weight (Fig. 2b) and
purified by native PAGE. In native PAGE, coagulation other properties.
activity was found in two gel-extracted fractions close to
the end of the gel phase. Fractions from native PAGE 3.2. Characterization of MOCP

Bradford method and absorbance at 280 nm showed

141 0.8 that the coagulants from both water and salt extracts
c Salt extract
were proteins with similar characteristics. Others re-
0.7
Absorbance at 280nm (mAbs)

121 ported that the coagulant from water extract is a cationic

0.6 protein (Nkhata, 2001; Ndabigengesere et al., 1995;
101
Gassenschmidt et al., 1995). Our finding is at variance
0.5
with results of Okuda et al. (2001a), who stated the active

Activity
81
0.4 component from salt extract as an organic compound,
61 which was neither protein, polysaccharide nor lipid. The
a 0.3
b molecular mass of purified MOCP was less than 6.5 kDa,
41
UV-280 0.2 as determined by SDS-PAGE with Coomassie staining
21 Activity 0.1
(Fig. 2a) and tris-tricine SDS-PAGE (data not shown).
Earlier studies have reported molecular weights of 6.5
1 0.0 and 12–14 kDa (Ndabigengesere et al., 1995; Gassensch-
144 154 165 176 186 197 208 218 229 240 250 midt et al., 1995). It was also observed that the two active
(a) Elution volume (mL) peaks from the cation exchange column had different pI
0.8
values, both of which were above 9.6.
180
a The coagulant protein is thermoresistant and remained
160 Water extract 0.7 active after 5 h heat treatment at 95 1C. Coagulation
140 b efficiencies of the heat-treated samples were slightly higher
0.6
than the raw samples. For boiling times of 30 min to 5 h
120
0.5 reduction in absorbance ranged from 79–87% whereas
100 that of the raw sample was 79%. Such a high thermal
0.4
80 stability renders it easy to process and handle and heat
0.3 treatment could be used to remove oil before the
60 UV-280 purification process. One of the options for reducing
c 0.2
40 Activity organic load in water treatment from the use of MO is
0.1 carbon adsorption. In such cases heat treatment could be
20
used to break down large molecules for better adsorption.
0 0.0
(b) 62 67 73 78 84 89 95 100 3.3. MS sequence analysis
Fig. 1. High-Trap CM FF IEX chromatography and coagula-
tion activity for salt extract (a) and water extract (b). From the SDS-PAGE and IEF analysis, it is clear that
Ammonium acetate buffer (50 mM) (pH 7) was used for each protein fraction obtained after cation exchange
equilibration and it was eluted with step gradient up to 1 M chromatography does not consist of a single, homo-
concentration of the same buffer. geneous protein, but is a mixture of proteins with similar

Fig. 2. SDS-PAGE images of the various fractions from IEX and native PAGE. (a) molecular weight determination using BioRad
protein marker and cecropin A. The two active protein peaks (peaks b and c) show the same size (b) Compares sizes of protein samples
from IEX (peak c) and native PAGE (lower fraction).
 ARTICLE IN PRESS
2342 K.A. Ghebremichael et al. / Water Research 39 (2005) 2338–2344

physical characteristics. To further analyse the composi- 2.0

tion of these mixtures, MS/MS experiments were

Absorbance at 500 nm, Abs

1.8 High turbidity
performed to obtain peptide sequence information from 1.6
the samples after SDS-PAGE. As is shown in Table 1, a 1.4
number of peptide sequences were obtained which are 1.2
similar or identical to the known sequence of an MOCP 1.0
(Gassenschmidt et al., 1995). It has been reported that 0.8
more than one protein family with flocculation activity 0.6 Control
is present in MO seed (Gassenschmidt et al., 1995). 0.4 Alum
Indeed, ongoing genomics projects around the world 0.2 CWE
Cecropin A
continue to demonstrate that plants express many 0.0
closely related proteins during different developmental 0 5 10 20 25 35 45 60 100 135 145
stages (Dong et al., 2004) so it is not unexpected that (a) Time, min
MO produces a range of sequence variants of seed 0.7
storage proteins with coagulant activity. Further work

Absorbance at 500 nm, Abs

0.6 Low turbidity
on the genome and proteome of MO is clearly necessary
to identify the exact nature. 0.5
0.4
3.4. Coagulation study 0.3
0.2 Control
3.4.1. Small volume assay
Alum
In this study we developed a simple and quick method 0.1
CWE
to test coagulation activity with very small sample
0.0
volumes (1 mL) and coagulant dosage. This method can 0 5 10 20 25 35 45 60 100 135 145
be generally applied for easy screening of active (b) Time, min
coagulants from large number of samples. A coagula-
tion kinetics study could also be easily performed to Fig. 3. Coagulation kinetics of MO, cecropin A and alum for
monitor settling rate behaviour of different coagulants. high- and low-initial turbidity waters.
Such data can be used to distinguish active and non-
active coagulants in a short time period. A significant
difference in attenuance (OD500) was observed between the clear solution from the top is transferred to a quartz
the coagulants and the control already in 10–20 min glass cuvette, the background effect is reduced thereby a
(Fig. 3a). higher reduction in attenuance could be observed.
Attenuance measurements were made either directly
on the 1 mL cuvette or by taking a 200 mL sample to a 3.4.2. Comparative studies
quartz glass cuvette. For comparison and screening A jar test study of the CSE and the CWE revealed that
purposes the direct measurement could be used with coagulation activity is higher in the CSE, which has been
satisfactory results, such as for identifying the active reported previously (Ghebremichael and Hultman,
coagulant fractions in a large number of samples. When 2004; Okuda et al., 1999). Possible explanations for this
observation are: (a) when the CSE was mixed with water
or low molar buffer solution, precipitates were observed.
Table 1 The precipitates could act as nuclei for floc formation.
Electrospray ionization—MS/MS determination of the se- (b) The amount of protein was higher (two-fold) in the
quences of peptides from the trypsin digested protein CSE than in the CWE.
Although the protein concentration in CSE was
Observed Observed Peptide sequence
peptide charge higher than in CWE, the amount of protein bound to
mass state the ion-exchange matrix during purification was similar
in both extracts. In the purified form, both extraction
1268.62 +2 ANPPVQPDFQR methods showed similar coagulation activity and
2087.09 +3 QAVQLTHQQQGQAGPLQVR physico-chemical properties. This was also observed in
2100.18 +2, +3 QAVQLAHQQQGQVGPQQVR samples from batch cation exchange purification experi-
2122.12 +2, +3 QAVQLETQQQGQVGPQQVR ments, where the eluted proteins from the water and salt
2130.28 +2, +3 QAVQLTHQQQGQVGPQQVRa
extracts had similar protein content and coagulation
a
This is identical to the sequence published by Gassenschmidt activity. Further characterization and comparative tests
et al. (1995). were then carried out using only samples from the CWE.
 ARTICLE IN PRESS
K.A. Ghebremichael et al. / Water Research 39 (2005) 2338–2344
We compared flocculation caused by the CWE and
alum both by the standard jar analysis and the small
volume method developed. At high initial turbidity
(250–300 NTU), both coagulants showed similar activity
whereas at low initial turbidity (76–110 NTU), alum
showed a higher settling rate and lower final turbidity
than the CWE (Fig. 3b). Result of kinetic experiments
using CWE (15 mg/L), alum (15 mg/L) and cecropin A
(5 mg/L) showed similar coagulation activity for high
initial turbidity (Fig. 3a). We observed that there was no
difference in coagulation activity before and after oil
extraction. Thus, it is possible to obtain the coagulant
protein from oil processing residue.
As cecropin A is a small antimicrobial peptide (4 kDa)
with high pI, it is of interest to compare it with MOCP.
Cecropin A showed similar coagulation activity to both
MOCP and alum. This is the first such report to our
knowledge of this effect, which suggests that a number
of other small, basic peptides from plants and animals
could be examined for such purposes. There are several
areas where this result could be applied, the most
appealing of which is to develop efficient coagulation in
water and wastewater treatment systems using locally
available materials.
3.5. Antimicrobial effect
A preliminary study of the antimicrobial effects of
MOCP compared to cecropin-A was conducted in terms
of cell aggregation and growth inhibition. A few gram-
Fig. 4. Flocculation effect of MOCP and cecropin A for D31 and Bt7. Images a, b and c represent control, MOCP treated and
cecropin A treated samples of D31, respectively. The corresponding images for Bt7 are shown in d, e and f, respectively. Note that
cecropin A did not aggregate Bt7 (f).
2343
negative and gram-positive bacteria strains were se-
lected. The effect of MOCP and cecropin A on cell
aggregation are shown in Fig. 4. MOCP was able to
aggregate both E. coli (D31) and B. thuringiensis (Bt7
and Bt75). On the other hand cecropin A was able to
aggregate E. coli (D31) but not B. thuringiensis (Bt7)
(Fig. 4). From viable counts using MOCP, we observed
1.1 log reduction in E. coli (D31) and 4 log reduction in
E. coli (K12), B. thuringiensis (Bt7, Bt75) and P.
aeruginosa. In the case of E. coli (K12) and P. aeruginosa
reduction in viable counts was observed without notice-
able cell aggregation. Cecropin A did not show
aggregation or reduction in viable count for B.
thuringiensis (Bt7). Studies have reported that B.
thuringienesis inhibits the activity of cecropin A by a
protease that degrades the peptide (Dalhammar, 1987).
We could find similarities between MOCP and cecropin
A although there was no resemblance in the sequence
data. Further studies are required to establish the true
nature of MOCP and the mechanism of antimicrobial
action.
4. Conclusion
MOCP has been purified from the native source using
a simple and straightforward technique, which can be
readily applied for water treatment in areas where MO is
available. A key advantage of the purification is that it
reduces the organic load in treatment systems without
 ARTICLE IN PRESS
2344 K.A. Ghebremichael et al. / Water Research 39 (2005) 2338–2344
requiring more complex protein production methods,
such as recombinant (heterologous) expression. More-
over, the high thermostability of MOCP and its ability
to reduce microbial populations contributes to its
attractiveness as a flocculant. Similar results using
cecropin A indicate that plants and animals are fruitful
sources of proteins and peptides for water and waste-
water treatment applications.
Acknowledgments
The authors are grateful to Per Dalhammar and Jaen
Ping for their assistance in the laboratory work and
useful discussions. We would also like to extend our
gratitude to Kaj Kauko for microscopic analysis.
Funding for the purchase of mass spectrometry equip-
ment was from the Wallenberg Consortium North.
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