Ecological Engineering 49 (2012) 48–52

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Ecological Engineering
journal homepage: www.elsevier.com/locate/ecoleng

Short communication

Evaluation of the efficiency of natural coagulant obtained by ultrafiltration of

common bean seed extract in water turbidity removal
Mirjana G. Antov ∗ , Marina B. Šćiban, Jelena M. Prodanović
University of Novi Sad, Faculty of Technology, Blvd. Cara Lazara 1, 21000 Novi Sad, Serbia

a r t i c l e i n f o a b s t r a c t

Article history: Efficiency of natural coagulant obtained by ultrafiltration of common bean (Phaseolus vulgaris) seed extract
Received 9 March 2012 in water turbidity removal was experimentally evaluated. Prepared ultrafiltration fractions have shown
Received in revised form 6 July 2012 differences in behavior regarding pH values and dosages at which they expressed their highest efficiency
Accepted 10 August 2012
in turbidity removal. The highest obtained coagulation activities were at pH 9.5 with crude extract and
Available online 28 September 2012
fraction containing molecules with molecular weight less than 10 kDa, 50.6% and 49.1%, respectively. The
organic load that remained in water after treatment was reduced when natural coagulant processed by
Keywords:
ultrafiltration instead of crude extract was applied. Moreover, fraction with molecules between 10 kDa and
Natural coagulant
Ultrafiltration
30 kDa, decreased chemical oxygen demand by 9% relative to blank at conditions this fraction expressed
Coagulation activity its highest coagulation activity.
Organic load © 2012 Elsevier B.V. All rights reserved.
Common bean seed

1. Introduction Thus, in water treatment, the use of natural coagulants could

Water question is surely one of the main factors that are
involved in the human development considering its influence on
human lives. Among the new techniques for water and wastewa-
ter treatment there is the use of natural coagulants, aiming at a
better quality of treated water by reducing the use of chemicals.
Namely, coagulation/flocculation step which is essential process
in the treatment of both surface water and industrial wastewa-
ter, includes removal of dissolved organic species and turbidity
from water most commonly via addition of conventional chemical-
based coagulants – alum, ferric chloride and synthetic organic
polymers. While the effectiveness of these chemicals as coagu-
lants is well-recognized (Edzwald, 1993; Kang et al., 2003) there
are, nonetheless, disadvantages associated with their usage such
as ineffectiveness in low-temperature water (Haaroff and Cleasby,
1988), relatively high procurement costs, detrimental effects on
human health, production of large sludge volumes and the fact that
they significantly affect pH of treated water. There is also strong evi-
dence linking aluminum-based coagulants to the development of
Alzheimer’s disease in human beings (Flaten, 2001). It is therefore
desirable to replace these chemical coagulants with plant-based
coagulants to counteract the aforementioned drawbacks.
∗ Corresponding author. Tel.: +381 21 485 3647; fax: +381 21 450 413.
E-mail address: mantov@uns.ac.rs (M.G. Antov).
be an option with many advantages over chemical agents, partic-
ularly the biodegradability, low toxicity and low residual sludge
production (Narasiah et al., 2002). Biopolymers may be of great
interest since they are natural low-cost products, characterized
by their environmental friendly behavior and usually have large
number of surface charges that increase the efficiency of the coag-
ulation process. These advantages are especially augmented if the
plant from which the coagulant is extracted is indigenous to local
community. In recent numerous studies variety of plant materials
has been reported as a source of natural coagulants (Raghuwanshi
et al., 2002; Diaz et al., 1999; Miller et al., 2008; Šćiban et al., 2009)
but the most studied is Moringa oleifera whose efficiency has been
approved for turbidity removal (Ndabigengensere and Narasiah,
1998; Okuda et al., 2001; Ghebremichael et al., 2006) as well as
antimicrobial properties (Ghebremichael et al., 2005).
During the course of plants’ screening program in our labora-
tory, crude extract from common bean (Phaseolus vulgaris) seed
was applied for water turbidity removal while partially purified
coagulant has shown high coagulation efficiency followed by low
organic load (Antov et al., 2010). Seed of common bean has food
grade nature and contains no oil, so, delipidation step is not nec-
essary which is beneficial for both economic and environmental
reasons. Considering this common bean is a promising source of
natural coagulant for water treatment.
The objective of the study was to experimentally evaluate the
efficiency of different fractions prepared by ultrafiltration of com-
mon bean seed extract in water turbidity removal. Membrane

0925-8574/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ecoleng.2012.08.015
 M.G. Antov et al. / Ecological Engineering 49 (2012) 48–52
separation technique was applied instead of isolation and purifi-
cation multiple steps procedure to examine its suitability for the
processing of natural coagulant. Coagulation efficiencies of pre-
pared fractions were measured at different pH values and dosages,
and have been compared to the one of crude extract. In addition,
suitability of prepared natural coagulants was tested in respect of
organic load that remained in water after treatment.
2. Materials and methods
2.1. Extraction of active components from common bean seed
The locally obtained common bean (P. vulgaris) dry seed was
ground to a fine powder by using a laboratory mill and sieved
through 0.4 mm sieve. The fraction with particle size less than
0.4 mm was used in experiments. Ten grams of seed powder
was suspended in 1 L of NaCl water solution (0.5 mol/L). The
suspension was stirred using a magnetic stirrer for 10 min to
accomplish extraction and then filtered through a rugged filter
paper (Macherey-Nagel, MN 651/120) to obtain filtrate – crude
extract of active components.
2.2. Preparation of fractions of natural coagulant by
ultrafiltration
The coagulation active components from common bean extract
were further processed by ultrafiltration in Amicon stirred cell
(model 8200, MilliporeTM ) with polyethersulfone membranes
(Biomax, MilliporeTM ) having cut-off 10,000 and 30,000 Da. Ultra-
filtration was performed under constant pressure of inert gas
(2.5 bar) and gentle magnetic stirring (30 rpm) at constant room
temperature (21 ◦ C). The flow rate through membranes with 10,000
and 30,000 Da cut-off was 13.5 and 35 mL/h, respectively.
Fractionation of components was conducted using membranes
in consecutive manner – ultrafiltration through lower cut-off mem-
brane was followed by the one through membrane having higher
used cut-off. In both steps ultrafiltration was running until volume
of retentates reached 10% of initial volume. After each ultrafiltra-
tion step, volumes of retentates were adjusted to value of initial
volume with 0.5 mol NaCl/L. Permeate obtained by ultrafiltration
with membrane cut-off 10,000 was assigned as the 1st fraction,
fraction containing components having approximate molecular
weight between 10,000 and 30,000 Da was assigned as the 2nd
fraction, and retentate from ultrafiltration with 30,000 cut-off as
the 3rd fraction.
2.3. Preparation of turbid water
Turbid water for coagulation tests was prepared by adding 1 g
kaolin to 1 L tap water. The suspension was stirred for 1 h to achieve
uniform dispersion of kaolin particles, and then it was allowed to
remain for 24 h for completing hydration of the particles. This sus-
pension was used as the stock suspension. Turbid water having
35 nephelometric turbidity units (NTU) was prepared by diluting of
stock suspension to 1000 mL tap water just before the coagulation
test. The initial pH of the synthetic water was adjusted to 9.0, 9.5 or
10.0 with 1 mol/L NaOH, in accordance to previous investigations
(Šćiban et al., 2005) just before experiments.
2.4. Coagulation test
Coagulation activity of fractions of natural coagulant prepared
by ultrafiltration as well as crude extract was evaluated in jar tester
VELP model FC6S. Samples were added to the beakers at different
dosages (from 0.125 to 2.5 mL/L turbid water) and the content was
49
stirred at 200 rpm for 1 min. The mixing speed was then reduced
to 60 rpm and was kept for 30 min. Then, the suspensions were
left to allow sedimentation. After 1 h of sedimentation, upper clar-
ified liquid was collected and residual turbidity was measured. The
residual turbidity of sample was RTS . The same coagulation test was
performed with no coagulant as the blank. The residual turbidity
in the blank was RTB . Coagulation activity was calculated as:
RTB − RTS
Coagulation activity (%) = · 100 (1)
RTB
2.5. Analytical methods
Protein concentration was measured according to Bradford
(1976) with bovine serum albumin as standard. Turbidity was mea-
sured using a turbidimeter (WTW TURB 550/550 IR) and it was
expressed in nephelometric turbidity units (NTU). Chemical oxygen
demand was determined according to Standard Methods (APHA-
AWWA-WEF, 1998).
All experiments were run in duplicate (the accuracy is consid-
ered to be ±5%) and the mean value is presented herein.
3. Results and discussion
3.1. Ultrafiltration of common bean seed extract
Our previous investigations have shown that proteins from
common bean seed expressed coagulation activity (Antov et al.,
2010). This was the reason for crude extract and fraction prepared
by ultrafiltration to be characterized by measuring protein con-
centration although it is clear that they contained numerous water
soluble components from the seed as well (Morales-de Leon et al.,
2007).
Results of ultrafiltration regarding protein concentration
revealed that majority of extracted proteins was in the third
fraction, 0.47 mg/mL, which mostly contained molecules having
molecular weight above 30 kDa, while the concentration of pro-
teins in the second fraction with molecules between approximately
10 kDa and 30 kDa was the lowest one, amounting 0.04 mg/mL.
Protein concentration in crude extract and the first fraction were
0.86 mg/mL and 0.36 mg/mL, respectively. On the base of these
results it can be calculated that content of proteins in the 1st and
the 3rd ultrafiltration fractions amounted about 40% and 55% of
total extracted protein, respectively. It is known that common bean
(P. vulgaris) seed is a valuable source of protein which content
is approximately 20–30%. The storage globulin phaseolin repre-
sents more than half of the total protein content in the seed and
it is a trimer with subunit Mw ∼ 50 kDa, while albumin, prolamin
and glutelin proteins represent the other seed protein fractions.
According to obtained results distribution of proteins in fractions
prepared by ultrafiltration corresponded in great extent to com-
mon bean proteins’ characteristics regarding both their molecular
weights and content in seed (Morales-de Leon et al., 2007; Osborn
et al., 1988; Montoya et al., 2010).
3.2. Turbidity removal in model water
Efficiency of prepared ultrafiltration fractions in turbidity
removal was investigated in model water at three pH values start-
ing from the turbidity 35 NTU. Coagulation tests were conducted
with different dosages of tested fractions and crude extract, and
obtained results are shown in Fig. 1. Values of pH were chosen in
accordance to our previous results which revealed the highest effi-
ciency of natural coagulant from common bean seed in removal
of turbidity from synthetic water was attained at pH 9–11 (Šćiban
50 M.G. Antov et al. / Ecological Engineering 49 (2012) 48–52

60 60
Crude extract st
1 fraction
50 50
pH 9.0
pH 9.5
Coagulation activity (%)

Coagulation activity (%)

40 pH 10.0 40

30 30

20 20

10 10

0 0
0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5

Dosage (mL/L) Dosage (mL/L)

60 60
nd rd
2 fraction 3 fraction
50 50
Coagulation activity (%)
Coagulation activity (%)

40 40

30 30

20 20

10 10

0 0
0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5

Dosage (mL/L) Dosage (mL/L)

Fig. 1. Coagulation activities of crude extract from common bean seed and fractions prepared by ultrafiltration in relation to coagulant dosages and at different pH values.

et al., 2005). Residual turbidity of the blank varied slightly within
investigated pH range and its mean value was 23.4 ± 0.6 NTU.
Crude extract from common bean seed as well all ultrafiltra-
tion fractions have shown differences in behavior when they were
evaluated for turbidity removal regarding pH and dosages at which
they expressed the highest obtained coagulation activities as well
as the values of this highest activities. These differences might be
explained by differences in kind and characteristics of biomolecules
as well as by their content in fraction and crude extract. Proteins
as well as other extracted molecules separated by ultrafiltration
differ not only in molecular weight but e.g. in pI which may addi-
tionally affect coagulation as consequence of their charge at certain
pH. However, it can be noticed that the higher efficiencies in tur-
bidity removal generally were obtained at higher investigated pH
values and that pH 9 was the least appropriate for common bean
seed molecules to express their coagulation action.
Crude extract has attained its highest coagulation activity,
slightly above 50%, at dosage 1 mL/L and pH 9.5. In comparison
to this result, highest obtained coagulation activities crude extract
has reached at pH 9 and 10 were about 2.4 and 1.5 times lower,
respectively. The highest obtained efficiency in turbidity removal
resulting in coagulation activity just below 50%, the 1st fraction
has shown also at pH 9.5 but at double dosage in comparison to
crude extract. In addition, at pH 10 this fraction containing com-
pounds having molecular weight below 10,000 Da has also showed
high coagulation activity in comparison to other results, 47.6% with
relatively low dosage 0.5 mL/L.
In comparison to crude extract and other two ultrafiltration
fraction, the highest coagulation activity of the 2nd fraction was
the lowest one, 45.3%. It was achieved at the highest investigated
pH value, pH 10, which appeared to be the most appropri-
ate for molecules contained in this fraction to act as turbidity
removing agents. However, at the same conditions, this frac-
tion exhibited the best performance regarding COD removal (see
Table 1). As for the 3rd fraction containing biomolecules above
30 kDa and having the highest protein concentration, maximum
of coagulation activity was recorded at pH 9.5 with relatively
small volume of fraction added to turbid water, 0.25 mL/L. In
comparison to this result, the highest coagulation activities this
fraction has shown at pH 9 and pH 10 were about 2.4 and 1.4
times lower, respectively, which were achieved at higher dosage,
1 mL/L.
 M.G. Antov et al. / Ecological Engineering 49 (2012) 48–52
Table 1
COD values in water treated by crude extract from common bean seed or frac-
tions prepared by ultrafiltration at conditions expressing their highest coagulation
activities (COD for blank 2.02 mg O2 /L).
Natural coagulant Crude extract 1st fraction 2nd fraction 3rd fraction
Dosage (mL/L) 1.0 2.0 1.0
pH 9.5 9.5 10.0
Coagulation activity (%) 50.6 49.1 45.3
COD (mg O2 /L) 3.74 3.33 1.84
Evaluation of crude extract of common bean and ultrafiltration
fraction for turbidity removal revealed that the most efficient was
the crude extract. Although lower than that of crude extract, the
highest coagulation activities of all fractions were close to this high-
est obtained result. Efficiency of crude extract and fraction prepared
by ultrafiltration in turbidity removal were lower than those for
natural coagulants from the most efficient M. oleifera and other
plant materials (Ndabigengensere and Narasiah, 1998; Diaz et al.,
1999; Okuda et al., 2001; Sanchez-Martin et al., 2010). However,
this lower efficiency can be also perceived from the aspect that
it was evaluated at low water turbidity and considering that M.
oleifera is also not efficient coagulant for low-turbidity water (Lea,
2010).
3.3. Organic load in treated water
The crude extracts which are used as natural coagulants con-
tain biomolecules and inorganic substances which may be released
in water leading to increased COD (Ghebremichael et al., 2006).
Besides, the use of natural coagulants may increase the organic
load in water which may result in increased microbial activ-
ity (Ndabigengensere and Narasiah, 1998; Okuda et al., 2001).
In addition, the organic matter might consume additional chlo-
rine or other disinfectants in the water treatment plant and
can acts as a precursor of byproducts during the disinfection
process. In order to solve these problems and minimize the
value of unnecessary organic material which might adversely
affect quality of the water, purification of the active compo-
nents is usually recommended (Okuda et al., 2001; Ghebremichael
et al., 2006; Antov et al., 2010).
Organic matter in water before and after coagulation tests was
measured to establish the increase in organic load when crude
extract and ultrafiltration fractions were added to turbid water at
dosages exhibiting their highest coagulation activity. Results pre-
sented as relative increase in COD in comparison to blank (Table 1)
have shown that the increase in organic matter that remained
in water after coagulation was the highest when crude extract
was used. Substantially lower relative increase in COD in com-
parison to that for crude extract was caused by the use of the
3rd fraction. Contrary to that, when the 2nd fraction was used for
turbidity removal, COD was even decreased, relatively to blank,
by 9%. Obtained results might be explained by lower proteins
concentration in tested fraction because of their fractionation by
ultrafiltration but also by fractionation of other compounds from
crude sample which caused reduced organic load in comparison to
crude extract. As for the 2nd fraction, it might be suggested that
its composition as well as conditions for the removal of turbidity
regarding pH and dosage were beneficial resulting in even lower
chemical oxygen demand in comparison to blank.
Obtained results might be promising in respect of obtaining nat-
ural coagulant that is suitable for application in turbidity removal
regarding the problem of organic load. Instead of complicated
and demanding multistep procedures of isolation and purifica-
tion of coagulation active components which can be expensive and
have low yield, a membrane separation technique can be applied
51
yielding in fractions that are efficient in turbidity removal giving,
at the same time, diminished organic load.
4. Conclusion
0.25
9.5 Evaluation of fractions obtained by ultrafiltration of crude
47.7 extract of common bean seed for their suitability for turbid-
2.32
ity removal revealed that they were efficient in it although
their coagulation activities were slightly lower that of crude
extract. Obtained fractions brought reduced organic load into
the water after the treatment in comparison to crude extract or
even diminished it in comparison to blank. According to these
results ultrafiltration appeared to be promising technique for
processing crude extract of common bean seed in respect of its
simplicity in comparison to multistep isolation and purification
procedures.
Acknowledgment
The financial support from Ministry of Education and Science,
Republic of Serbia (Project No. 31002) is greatly acknowledged.
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