Separation and Purification Technology 209 (2019) 892–899

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Separation and Purification Technology

journal homepage: www.elsevier.com/locate/seppur

Identification and role of Opuntia ficus indica constituents in the flocculation T

mechanism of colloidal solutions
⁎
Omar Bouaouinea,b, Isabelle Bourvena, Fouad Khalilb, Philippe Bressollierc, Michel Baudua,
a
Groupement de Recherche Eau, Sol et Environnement (GRESE), University of Limoges, 123 avenue Albert Thomas, 87060 Limoges, France
b
Laboratory of Applied Chemistry (LCA), University Sidi Mohamed Ben Abdellah of Fez, Immouzer Road, BP 2202, Fez, Morocco
c
University of Limoges, Laboratory of Chemistry of Natural Substances (LCSN), IUT of Limousin, Allée André Maurois, 87065 Limoges Cedex, France

A R T I C LE I N FO A B S T R A C T

Keywords: Opuntia ficus indica has been identified for its bioflocculant properties in water treatment; however, its under-
Bioflocculation lying mechanism and active compounds have not been clearly identified. Flocculent molecules of cactus solid
Opuntia ficus indica material (CSM) under alkaline conditions were extracted at pH 10 and then precipitated under neutral condi-
Quercetin tions (pH 7). The precipitate was fractionated by ultrafiltration systems and analyzed using inverted phase
Starch
chromatography and enzymatic treatments. This approach revealed that quercetin and starch constitute the
Model
active agents found in the fractionated parts at ≤3,000 and ≥10,000 Da, respectively. The use of quercetin or
(potato) starch alone at 18 mg/L yielded 72% ± 2% and 54% ± 3% of turbidity removal, respectively. With a
combination of both these components, a higher flocculation activity (84% ± 2%) could be obtained. From
these experimental results, a flocculation model based on identified active constituents is being proposed in
order to improve process knowledge.

1. Introduction ficus indica (galactose, rhamnose and arabinose) only allowed for a
limited removal of turbidity for a synthetic kaolin solution (around
The use of natural flocculants has recently become a promising 50%); moreover, to date, no data from the literature has pointed to
solution in the wastewater treatment of various polluted effluents by active constituents contributing to high turbidity removal rates.
virtue of their coagulation-flocculation mechanisms [1-4]. The in- Bouaouine et al. [15] investigated the action of all cactus-extracted
creasing demand for environmentally friendly technologies has focused materials for turbidity removal in a synthetic solution with both kaolin
attention on various natural polyelectrolytes, which are being used as and humic acid. Results showed an optimal coagulation flocculation at
either coagulants or flocculants in water and wastewater treatment and pH 10 in addition to chemical groups, especially phenol groups, being
moreover are capable of being substituted for metal salts and all syn- involved in the flocculation. Phenol groups were indeed identified as
thetic polyelectrolyte flocculants. Examples include plant organism the most active extracted constituents providing the greatest efficiency
preparation from Moringa oleifera seed or from cactus as well as the in the coagulation-flocculation mechanism.
application of specific biochemical families extracted from plant or- Polyphenols are polycyclic compounds with hydroxyl groups con-
ganisms [5,6]. Bioflocculants are used as mucilage (polysaccharide taining phenol, whether substituted or not, and mainly exist in asso-
polymers): starch from rice and maize [7], polysaccharide from Ocimum ciation with sugars (glucose, galactose, arabinose, etc.). Some poly-
basilicum [8], or sodium alginate from algae [4]. However, other bio- phenols can form complexes with macromolecules, in particular
chemical families are also involved, such as protein from fava beans and proteins, via hydrogen bonds or hydrophobic interactions [17]. Poly-
from Cocos nucifera [10], or polyphenols from grape seed or Acacia phenols have already been used in wastewater treatment applications
mearnsii [9–12]. as flocculants: Jeon et al. [11] demonstrated that catechin (flavonoid)
The high reactivity of Opuntia ficus indica in synthetic surface water and tannic acid (tannin) were both involved in the molecular floccu-
treatment during the coagulation-flocculation process has been con- lation mechanism.
firmed by many authors [6,13–15]. Opuntia ficus indica contains natural In this study, our aim is twofold: (i) identify the active biochemical
polysaccharides (mucilage), proteins and polyphenol [16]. Miller et al. constituents of Opuntia ficus indica involved in flocculation, and (ii)
[6] demonstrated that the mix of monomer sugars contained in Opuntia propose a flocculation model of colloidal suspension under alkaline

⁎
Corresponding author.
E-mail address: Michel.baudu@unilim.fr (M. Baudu).

https://doi.org/10.1016/j.seppur.2018.09.036
Received 22 May 2018; Received in revised form 26 July 2018; Accepted 10 September 2018
Available online 11 September 2018
1383-5866/ © 2018 Elsevier B.V. All rights reserved.
O. Bouaouine et al. Separation and Purification Technology 209 (2019) 892–899

conditions. Accordingly, the extracted biochemical constituents of reported by the manufacturer is 0.1–7 kDa. The total permeation vo-
cactus under various pH conditions and fractionated by ultrafiltration lume was estimated at 23 mL using NaN3. All measurements were
systems have been investigated with: colorimetric dosage, Inverted conducted using a mobile phase at a flow rate of 0.3 mL·min−1. This
Phase Chromatography (IPC), Size Exclusion Chromatography (SEC), phase was prepared with 25 mM Tris HCl and 100 mM NaCl. The pH
and enzymatic treatment. was adjusted to 10.0 ± 0.1 by a 3 M NaOH solution. The mobile phase
was then filtered through a 0.22-µm cellulose nitrate filter (Sartorius)
2. Materials and methods and degassed before use. In all runs, 100 µL of the extracted cactus
(WEM 10 and PP at 100 mg L−1 for both) were previously filtered with
2.1. Fractionation of active components a 0.22-µm cellulose acetate filter (SPARTAN 13/0.2 RC Whatman). The
apparent molecular weight (aMW) of cactus extract components was
As presented in a previous study [15], isolating the active agents estimated using five proteins as calibration standards: bovine serum
from the cactus solid material (CSM) requires performing a water ex- albumin (BSA) (Sigma) (69.3 kDa), Ribonucleases A (Sigma)
traction under alkaline conditions. This approach entails the water (13.7 kDa), thyrotropin-releasing hormone (TRH) (Sigma) (3.62 kDa),
extraction of soluble constituents from 1 g of dry weight (DW) pow- Tyroglobuline, and tyrosine (Sigma) (0.18 kDa). For the mass calibra-
dered cactus in 100 mL of distilled water at pH 10 (WEM 10). After tion curves, the logarithm of the molecular mass is plotted as a function
stirring for 24 h, the cactus extracts were filtered at 0.45 µm (cellulose of the retention volume (Eq. (1)):
acetate-Whatman). Next, the WEM 10 was acidified to pH 7 to pre-
cipitate active substances, and the precipitate (PP) was recovered by Log (MW ) = −0.132 (Ve ) + 7.692 (r 2 = 0.99)
means of filtration (0.45 µm). The precipitate was lyophilized (Cryotec MW : apparent molecular weight in Daltons;
COSMOS-80), and 1 g was solubilized in 100 mL (pH 10). The soluble Ve : elution volume in mL. (1)
constituents at pH 7 (WEM 7) were then prepared for a comparative
study with other extracts. The PP was further fractionated by ultra- The High Performance Inverted Phase (HP-IP) was used to identify
filtration systems, i.e. Sartorius Stedim ultrafiltration cells-MWCO PES and quantify the flavonoid compounds in cactus extracts. The assay
membranes (GmbH 37070, Germany) at an equivalent 3,000 and system included an automated Agilent (Agilent Technologies, Palo Alto,
10,000 Da. CA) 1100 series, with a diode array detector (DAD). Two mobile phases
were introduced, i.e. an initial phase (A) of 6% acetic acid in 2 mM
sodium acetate (pH 2.57 ± 0.01); and the second phase of acetonitrile.
2.2. Characterization of cactus extracts
The gradient evolution of phase A (in %) can be observed in Fig. 4 (grey
feature). Cactus extract samples were prepared for the polyphenol ex-
2.2.1. Colorimetry
traction in 150 mL of 80% methanol in water at room temperature for
The contents of various biomolecules of the extracted cactus were
60 min. The acid hydrolysis step was prepared using 24 mL of me-
determined using colorimetric assays performed with a spectro-
thanol, 5 mL of concentrated HCl and 1 mL of water, with all solutions
photometer (Cadas 50 S, Dr Lange). The main parameters and refer-
being refluxed for 10 h at 85 °C, as mentioned in literature [24]. Then,
ences of the methods applied herein are summarized in Table 1 [18-23].
before injection, the samples were diluted (50% v/v, with acetonitrile:
Based on the cactus biomolecule contents, proteins, carbohydrates
methanol at 1:3) and an internal standard (hydroxycinnamic acid,
(polysaccharide and uronic acid) and polyphenols (present in flavonoid,
sigma Aldrich) was added (final concentration: 0.25 mg mL−1). The
tannins and lignin in the cactus) were all investigated. More specifi-
injection volume was set at 20 µL. The flow rate was 1 mL·min−1, for a
cally, total tannins, condensed tannins and total flavonoids could also
total run time of 90 min. The detector was set at a specific absorbance
be quantified.
for flavonoid: 368 nm [25]. A calibration for the identification of fla-
The biomolecule contents are expressed with respect to the reagent
vonoid with a relative factor (Rf) was implemented by a series of
used for the standard calibration curve in a “g equivalent (eq) standard
standards: kaempferol (sigma) and quercetin (sigma Aldrich), with an
reagent per g of dry weight (DW) of cactus material, WEM 10, WEM 7
Rf of 1.53 and 1.48, respectively. For the quercetin calibration range,
and precipitate PP”.
the surface area of the quercetin peak (Rf = 1.48) was plotted as a
function of quercetin concentration (g L−1). This range corresponds to
2.2.2. Chromatography the following (Eq. (2)):
High Performance Size Exclusion Chromatography (HP-SEC), cou-
pled to a diode array UV detector (L7455), was carried out with a
Superdex 75 10/300 GL column. The theoretical resolving range

Table 1
Main parameters of the colorimetric methods used for the biochemical quantification of cactus extracts.
Extract content Calibration curve concentration (mg Wavelength (nm) Reagent used Standard Reference
L−1)

*
Proteins 0–200 650 Folin's reagent Copper sulfate 0.5% (w/ Bovine albumin serum (96%, 18
w) sigma)
*
Phenols 0–200 650 Folin's reagent Gallic acids (Aldrich) 18
Polysaccharides 0–100 492 Phenol 5% (w/w) Glucose (recaptur, prolabo) 19
Sulfuric acid 95%
Uronic acids 0–250 520 Sodium tetraborate 12.5 mM Glucuronic acid (99%, Aldrich) 20
Sulfuric acid 95%
Total tannins 0–100 550 Ethanol 96%, Hydrochloric acid 37% Catechin (97%, Sigma Aldrich) 21
**
Total flavonoids 0–200 420 AlCl3 2% (w/w) Absolute ethanol Catechin (Sigma Aldrich) 22
Total condensed tannins 0–100 500 Vanillin 4% (w/w) Hydrochloric acid Catechin (99%, Sigma Aldrich) 23
37%

* Frolund's method was applied in order to correct the colorimetric detection interference between the peptide bond from the protein and phenol group and the
bond from the protein and humic acid in a complex matrix like biological sludge (the polyphenol standard was changed to “humic acid” from “gallic acid”).
** The total hydrolyzed flavonoid method was the same as that cited by Jamila Hadj Salem et al., 2010, in adding 5 mL of HCl (37%) and heating for 10 h at 85 °C.

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O. Bouaouine et al. Separation and Purification Technology 209 (2019) 892–899

Fig. 1. Flocculation results of the size-fractionated parts of the PP (precipitate), compared to CSM (cactus solid material) under optimal conditions (35 mg L−1, pH
10). n = 3.

Surface area of the quercetin peak fraction < 3,000 Da and tested in the flocculation process by Jar Test
Rf 1.48 = 33, 696, 321·[quercetin] + 3, 832, 065 (r 2 = 0.99) under optimal conditions. Next and in order to compare these results,
Surface area of the quercetin peak, Rf 1.48, in A. U . we performed another control test under the same conditions. Control 1
represents the final test, except for the fact that the enzymes were
Quercetin, in g L−1 (2)
prepared alone without adding PP > 10,000 Da. On the other hand,
Control 2 offered a confirmation of the flocculation process by the PP
fraction > 10,000 Da, except that this sample was incubated under the
2.3. Jar test
same conditions without adding any enzymes.
The flocculation process made use of a synthetic saline solution of
kaolinite (1 g L−1) and humic acid (10 mg L−1) (Sigma Aldrich) in 2.5. Size of colloids and flocs
order to achieve a turbidity of between 300 and 350 nephelometric
turbidity units (NTU) [15]. The particle size distribution of the colloidal solution and flocs
A rapid stirring at 250 rotations per minute (rpm) for 10 min, fol- formed after the coagulation-flocculation process was determined by
lowed by a slower speed reduced to 50 rpm for 30 min and lastly the 2- diffracting a LED laser light of 10 mW, 470 nm, 4 mW He-Ne, and
hour sedimentation step allowed for the formation of flocs. pH adjust- 632.8 nm for a blue and red light source respectively, with a size range
ment extending from 10 nm to 3.5 mm (Malvern Master sizer 3000 and
The flocculent efficiency was measured by determining turbidity Malvern Master zetasizer 3000 devices). All samples were set at an
removal in a synthetic solution by means of a turbidimeter (Hanna HI optimal flocculation pH of 10 for 2 h with 50 mg L−1 before conducting
88713). Turbidity (Turb) removal was calculated as follows: the measurements.
InitialTurb−ResidualTurbaftertreatment
Removal (%) = X 100
InitialTurbofthesyntheticsolution (3) 3. Results and discussion

Adjustment of pH was done by using 1.0 M NaOH and all floccu- A previous study [15] found that active molecules in coagulation-
lation tests were conducted at pH 10 (for optimal flocculation). For this flocculation by a cactus material are soluble under alkaline conditions
pH value the sedimentation of kaolinite without any flocculant addition and must be isolated by means of solubilization at pH 10 and pre-
corresponds to < 8% of turbidity reduction. cipitation at pH 7. The measurement of zeta potential of both kaolinite
and insoluble cactus material showed a strongly negative potential of
2.4. Enzymatic treatment the two colloidal solutions, respectively −31 mV and −23 mV. So
biochemical constituents of Opuntia ficus indica action occurs through a
To select the active macromolecules in the 10,000+ Da fraction flocculation mechanism with adsorption and bridging between parti-
precipitates (PP), we tested three hydrolytic enzymes for the degrada- cles. For the identification of active molecules, extracted materials were
tion of three compounds found in the cactus (lignin, sugars and pro- compared for the optimal flocculation conditions of a synthetic solu-
teins) due to lignin peroxidase (Sigma Aldrich), α amylase (Sigma tion. It was observed that the alkaline soluble material (WEM 10) and
Aldrich) and the protease alcalase from Bacillus licheniformis (Sigma PP (precipitate) presented a nearly identical highest efficiency com-
Aldrich), respectively. Each enzyme (1% (W/W)) was added to the PP pared to the CSM (cactus solid material): 91% ± 2%, 93% ± 1%, and
fraction > 10,000 Da (1.7 g L−1, final volume: 10 mL). All samples 96% ± 2%, respectively (n = 3). A weak synergistic effect of insoluble
were incubated for 2 h at different pH and temperature values, which CSM material seems to complement the effect of WEM 10. To identify
were established based on the nature of each enzyme. Afterwards, the the most reactive part in the precipitate, we decided to split this extract
10-mL preparation was added to 1 L of a synthetic solution of kaolin into three fractions. Fig. 1 shows the variation in turbidity removal by a
containing 17 mg of the active molecule present in the PP Jar test experiment with fractionated parts of the precipitate compared

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Fig. 2. Size distribution of WEM 10 (dotted line) and PP (continuous line) by SEC (HPSEC) at λ = 220 nm.

to CSM under optimal conditions (35 mg L−1, pH 10). These results reveal that the molecules responsible for the adsorp-
The fraction < 3,000 Da proved to be the most efficient tion and bridging mechanism have been included in the total flavonoids
(74% ± 2%), followed by a nearly similar turbidity removal from both or condensed tannins. Nevertheless, (i) as the SEC fingerprint of WEM
the fraction > 10,000 Da and the one between 3,000 Da and 10,000 Da 10 and PP (Fig. 2) showed, the active compound in the flocculation
(24% ± 1% and 26% ± 1%, respectively). This figure also suggests process has a very low aMW [300–800 Da] and (ii) as cactus opuntia
that the molecular weight of active molecule(s) is < 3000 Da. However, ficus indica contains polyphenols, especially flavonoids [16]; hence, we
the molecules included in the fraction > 3000 Da most likely contribute decided to pursue the analysis with a monomeric molecule such as
through a synergistic effect up to the optimum yield observed with flavonoid.
CSM. Fig. 4 indicates that two polyphenolic forms were detected, namely
Afterwards, we performed a size fractionation by HPLC-SEC, cou- quercetin (Rf = 1.48) and kaempferol (Rf = 1.54), in addition to an-
pled with a UV (220 nm) detection, to identify the apparent molecular other unknown identified type of polyphenol (Rf = 1.55). The % of
weight (aMW) fraction involved in the flocculation mechanism. Fig. 2 each peak detected account for 95%, 2.5% and 2.3% of the total
displays the SEC fingerprints of both WEM 10 and PP. summed area of the detected peak for quercetin, kaempferol and the
Fig. 2 indicates that the soluble part at pH 10 (WEM 10) contains a unknown polyphenol, respectively. The “quercetin” flavonoid is thus
large amount of small aMW compounds, averaging from 250 to 450 Da the major compound in the PP. Fatombi [10] demonstrated the efficacy
in size. In the precipitate (PP), two peaks are distinctly detected with an of tannin groups, especially flavonoids like catechin, in the flocculation
aMW of between 300 and 800 Da. The active compound found in process.
the < 3000 Da PP fraction therefore actually contains a very low aMW The content (g·g−1 of cactus fraction extract DW) of quercetin in all
(300–800 Da). The major components in cactus extracts are determined active CSM extracts, notably WEM 10 and the precipitate, was de-
by colorimetric dosage (in equivalent standard g/g of dry weight cactus termined after the HP IP/273 nm analysis. The quercetin content
extract), with the two ratios (R) WEM 10/WEM 7 and PP/WEM 10 equaled: 0, 61, and 333 mg·g−1 of DW for WEM 7, WEM 10 and PP,
being calculated (Fig. 3). respectively. The tremendous increase in quercetin content from WEM
Let's now observe that the WEM 10/WEM 7 ratios for proteins, 7 to WEM 10 and then to PP confirms that quercetin constitutes an
polysaccharides and phenols are > 1, thus resulting in a preferential active constituent responsible for the flocculation phenomenon.
extraction under alkaline conditions for both these classes of molecules. At pH 10, clays exhibit a high anionic and cationic exchange ca-
This preferential extraction has also been verified for tannins (R = 1.3). pacity [26]. Cationic sites may be present on the surface of clays and
Soluble and active molecules in the flocculation process at pH 10 are allow for anion absorption under divergent pH conditions; they are
included in these compound classes. The PP (extractible materials at pH attributed to oxides of iron, aluminum or calcium [26,27].
10 and insoluble at pH 7) is highly enriched in tannins compared to the To confirm these results, we compared the turbidity removal by
alkaline-soluble part (R = 1.5). It would therefore appear that tannins flocculation with various doses of quercetin or precipitate (Fig. 5).
constitute a particularly active group at pH 10. We observed an increase in turbidity removal for approximately the
In order to specify the tannin biochemical families responsible for same dose (20 mg L−1), with a better efficiency of PP (93%) compared
the flocculation phenomenon, we determined by means of colorimetric to quercetin (70%). For a high concentration of material introduced
assay the condensed tannins and total flavonoids for WEM 10 and PP. (> 60 mg L−1), the efficiency is low and similar for both.
Table 2 lists the concentrations of the main tannin components. If quercetin is the active compound, it presents a lower efficiency
Like in the previous results (Fig. 3), the concentration of total tan- (see also the black bar in Fig. 6) than precipitate, hence another active
nins increased between WEM 10 and PP, from 220 mg·g−1 to molecule must be contributing to the flocculation process. This other
320 mg·g−1 of DW (factor < 1/1.5). For condensed tannins, a rise in molecule, soluble under alkaline conditions, must be a macromolecule
concentration was also found between WEM 10 and PP, from 6 mg·g−1 (i.e. polymer with high molecular weight) involved in the flocculation
to 12 mg·g−1 of DW (factor = 1/2). This same trend was observed for mechanism. To confirm this hypothesis, we tested adding the PP frac-
total flavonoids, with a strong increase between the WEM 10 and PP, tion > 10,000 Da (17 mg) to quercetin (18 mg), and the same turbidity
from 190 mg·g−1 to 310 mg·g−1 of DW (factor > 1/1.6). removal rate (93% ± 1%) as that in the15 study was found (Fig. 6).

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Fig. 3. Ratio (R) of the content of specific biochemical molecules between two cactus extract fractions: the WEM 10/WEM 7 and precipitate/WEM 10 ratios. n = 3.

Table 2 To identify the active biochemical class of compounds present in the

Contents of specific polyphenols: total tannin condensed tannin and flavonoids PP fraction > 10,000 Da, we treated this active fraction with three
from cactus extracts, as determined by colorimetric methods. hydrolytic enzymes (i.e. α amylase, lignin peroxidase and protease al-
Sample Tannins mg·g−1 of Condensed tannins, in Flavonoids (in calase). If the active polymer is hydrolyzed, then the mix of modified PP
DW (Catechin Eq.) mg·g−1, of DW mg·g−1) of DW fraction > 10,000 Da and quercetin must yield a lower efficiency for
(Catechin Eq.) (Catechin Eq.) turbidity removal after the flocculation process. The test preparation
with enzymes and PP > 10,000 Da, incubated under optimal enzyme
WEM 10 220 ± 1 6±1 190 ± 1
Precipitate 320 ± 2 12 ± 2 310 ± 2 activity conditions, has been added to quercetin and the synthetic so-
lution for this flocculation process. Two controls were performed to
*n = 3 validate this result: Control 1 (preparation with PP
DW: dry weight fraction > 10,000 Da alone), and Control 2 (preparation with the en-
zyme alone). The three series of Control 1 and Control 2 show a tur-
bidity removal efficiency at 93% ± 1% and 68% ± 1%, respectively.
Flocculation results after degradation by the three enzymes reveal

Fig. 4. HP-IP fingerprint of PP, coupled with a 273-nm absorbance detection: Isolation and identification of the main polyphenolic substances in the precipitate.

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Fig. 5. Comparison of turbidity removal rates between quercetin and the precipitate (PP) by means of flocculation and settling at pH 10 for various material doses
(mg). n = 3.

Fig. 6. Comparison of turbidity removal rates after the flocculation process with a combination of quercetin and the precipitate > 10,000 Da or glucosidic mac-
romolecules. n = 2.

Table 3 amylase act upon α-glycosidic bonds and hydrolyze these bonds to
The size distribution of colloids and flocs formed after various cactus treatment, produce α-anomeric mono- or oligosaccharides [28].
and removal turbidity. These results demonstrate that macromolecules containing glucose
Samples Colloids (from quercetin precipitate CSM and are involved in the flocculation process under alkaline conditions.
synthetic saline and colloid (PP) and colloid Among such polymers, we tested starch (as previously indicated by
solution of colloid Lamghari [29] in cactus). Starch is a mixture of amylose and amylo-
kaolinite) pectin (> 10,000 Da). The glucose units are linked by α (1–4) bonds
Size of floc, in 0.05–0.15 1–10 100–200 300–400
and α (1–6) bonds, which are responsible for ramifications in the mo-
µm lecular structure. Amylose is slightly branched, whereas amylopectin is
Turbidity 0 72 ± 1 93 ± 1 96 ± 2 a branched molecule with long branches every 24–30 glucose units via
removal α (1–6) bonds.
(%) at pH
The ratio of amylose to amylopectin depends on the botanical
10
source of the starch. Starch isolation has proven to be best at pH 10
[30]. We also tested another synthetic glucosidic polymer: a dextran
no effect for lignin peroxidase and protease alcalase, with a 91% ± 1% formed by glucose units linked by α (1–6) bonds but with no branching.
and 90% ± 2% turbidity removal rate, respectively. For α amylase Next, we tested a combination of quercetin, starch extracted from po-
however, the removal of turbidity was reduced to 64% ± 2%. The α tatoes and dextran (Fig. 6).

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Fig. 7. Potential chemical interactions between quercetin, amidon and kaolinite involved in adsorption/bridging flocculation mode.

Results showed that the mix of quercetin and starch is better than evolution of the turbidity reduction shows that, starting from a dose of
the combination of quercetin and dextran, with a turbidity removal rate 10 mg L−1, quercetin begins to destabilize the colloidal solution of
of 84% ± 2% and 46% ± 3%, respectively, compared to quercetin kaolinites. The reduced efficiency observed from 40 mg L− 1, (Fig. 5)
with the precipitate fraction > 10,000 Da (93% ± 2%). This same can be due to a large coating of colloidal surface by quercetin accent-
trend was observed when using both of them alone without quercetin, uating the negative charge and leading a partial restabilization of the
yielding 54% ± 2% and 42% ± 1% for starch and dextran, respec- already structured flocs. Starch or cellulose can also be adsorbed on the
tively. The active glucosidic polymer must therefore be a branching kaolinite by hydrogen bonds (Fig. 7) and contribute to the quality of the
polymer. The efficiency of starch or PP > 10,000 Da, in combination flocs by bridging and entrainment. Mateus et al. [31] suggest the pre-
with quercetin, is very similar, and the lower efficiency with starch sence of hydrogen bonds between carbohydrate oxygen atoms and the
might be due to the nature of the molecules used in our experiments hydroxyl group of polyphenols, as well as hydrophobic interactions.
(extracted from potatoes). Starch is indeed found in cactus Opuntia ficus
indica [29], but the percentage of amylase and amylopectine and hence 4. Conclusion
the percentage of branching, may differ from that of potatoes. The
presence of hydrogen bonds between the carbohydrate oxygen atoms Opuntia ficus indica, is a bioresource known to act as a bioflocculant.
and the hydroxyl group of polyphenols, along with hydrophobic type We identified the molecules involved in the bridging absorption floc-
interactions, could explain the interaction between quercetin and the culation mechanism using a synthetic kaolin effluent at optimal con-
glucosidic polymer [31]. dition of pH 10. Two molecules present in the same mass contents,
The table 3 presents size distribution of colloids and flocs formed by quercetin - a flavonoid-with polyphenol groups and starch-a branched
CSM and all fractionated parts of cactus Opuntia ficus indica and their polymer of glucose with hydroxyl functions - have a synergistic floc-
removal turbidity. culating power and act in a bridging adsorption mode (70% turbidity
It shows that under optimal conditions (pH 10, 18 mg L−1 of quer- removal for quercetin alone for 93% total removal when adding starch).
cetin or PP < 3,000 Da), an increasing particle size (from 0.15 µm to For adsorption, a chemical interaction model was proposed between
10 µm) and 72% ± 1% turbidity removal after adding quercetin to the kaolinite and quercetin (ionic interaction), between kaolinite and
synthetic colloidal solution. Quercetin is thus acting as a flocculant starch (hydrogen bond) and between quercetin and starch (hydrogen
after the adsorption phenomenon. A flocculation by bridging was ob- bond).
served through the formation of large flocs between 100 and 200 µm
after adding 17 mg L−1 of branching starch or PP > 10,000 Da, which References
led to obtaining a high turbidity removal rate (84% ± 2% for potato
starch and 93% ± 1% for PP). A solid effect increased the efficiency to [1] M. Šćiban, M. Klašnja, M. Antov, B. Škrbić, Removal of water turbidity by natural
96% ± 2% by forming more large flocs between 300 and 400 µm. coagulants obtained from chestnut and acorn, Bioresour. Technol. 100 (2009)
6639–6643, https://doi.org/10.1016/j.biortech.2009.06.047.
As the two molecules, quercetin and starch act in a bridging ad- [2] N. Sanyano, P. Chetpattananondh, S. Chongkhong, Coagulation–flocculation of
sorption mode, we propose a model of chemical interactions involved in marine chlorella sp. for biodiesel production, Bioresour. Technol. 147 (2013)
adsorption step. Quercetin (and tannins) which is ionized in a very 471–476, https://doi.org/10.1016/j.biortech.2013.08.080.
[3] M. Chethana, L.G. Sorokhaibam, V.M. Bhandari, S. Raja, V.V. Ranade, Green ap-
basic medium (pKa ≈ 9). Kuntic et al., [32] can establish ionic bonds proach to dye wastewater treatment using biocoagulants, ACS Sustain. Chem. Eng.
with the element Al of kaolinite. This interaction may be a direct ionic 4 (2016) 2495–2507, https://doi.org/10.1021/acssuschemeng.5b01553.
interaction or be related to anion exchange (Fig. 7). According to many [4] T.K.F.S. Freitas, C.A. Almeida, D.D. Manholer, H.C.L. Geraldino, M.T.F. de Souza,
J.C. Garcia, Review of utilization plant-based coagulants as alternatives to textile
authors [33] the adsorption of the anions would be done essentially by wastewater treatment, Springer, Singapore, 2018, pp. 27–79, https://doi.org/10.
replacing the hydroxyls of clays. Wey et al. [34] showed an agreement 1007/978-981-10-4780-0_2.
between the anion exchange capacity and the Al3+ ions. Moreover, the [5] L. Gopalakrishnan, K. Doriya, D.S. Kumar, Moringa oleifera: a review on nutritive

898
O. Bouaouine et al. Separation and Purification Technology 209 (2019) 892–899

importance and its medicinal application, Food Sci. Human Wellness 5 (2016) [19] M. DuBois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method
49–56, https://doi.org/10.1016/j.fshw.2016.04.001. for determination of sugars and related substances, Analyt. Chem. 28 (1956)
[6] S.M. Miller, E.J. Fugate, V.O. Craver, J.A. Smith, J.B. Zimmerman, Toward, 350–356, https://doi.org/10.1021/ac60111a017.
Understanding the efficacy and mechanism of opuntia spp. as a natural coagulant for [20] N. Blumenkrantz, G. Asboe-Hansen, New method for quantitative determination of
potential application in water treatment, Environ. Sci. Technol. 42 (2008) uronic acids, Analyt. Biochem. 54 (1973) 484–489, https://doi.org/10.1016/0003-
4274–4279, https://doi.org/10.1021/es7025054. 2697(73)90377-1.
[7] S. Abdelaal, Y. Gad, A. Dessouki, Use of rice straw and radiation-modified maize [21] E.C. Bate-Smith, Recent progress in the chemical taxonomy of some phenolic con-
starch/acrylonitrile in the treatment of wastewater, J. Hazard. Mater. 129 (2006) stituents of plants, Bulletin de la Société Botanique de France 112 (1965) 16–28,
204–215, https://doi.org/10.1016/j.jhazmat.2005.08.041. https://doi.org/10.1080/00378941.1965.10838284.
[8] S. Shamsnejati, N. Chaibakhsh, A.R. Pendashteh, S. Hayeripour, Mucilaginous seed [22] J.H. Salem, C. Humeau, I. Chevalot, C. Harscoat-Schiavo, R. Vanderesse,
of ocimum basilicum as a natural coagulant for textile wastewater treatment, Ind. F. Blanchard, M. Fick, Effect of acyl donor chain length on isoquercitrin acylation
Crops Products 69 (2015) 40–47, https://doi.org/10.1016/j.indcrop.2015.01.045. and biological activities of corresponding esters, Process Biochem. 45 (2010)
[9] D.V. Kukic, M.B. Šciban, J.M. Prodanovic, A.N. Tepic, M.A. Vasic, Extracts of fava 382–389, https://doi.org/10.1016/j.procbio.2009.10.012.
bean (Vicia Faba L.) seeds as natural coagulants, Ecolog. Eng. 84 (2015) 229–232, [23] B. Sun, J.M. Ricardo-da-Silva, I. Spranger, Critical factors of vanillin assay for ca-
https://doi.org/10.1016/j.ecoleng.2015.09.008. techins and proanthocyanidins, J. Agri. Food Chem. 46 (1998) 4267–4274, https://
[10] J.K. Fatombi, B. Lartiges, T. Aminou, O. Barres, C. Caillet, A natural coagulant doi.org/10.1021/jf980366j.
protein from copra (cocos nucifera): isolation, characterization, and potential for [24] R. Bobinaitė, P. Viškelis, A. Šarkinas, P.R. Venskutonis, Phytochemical composition,
water purification, Sep. Purific. Technol. 116 (2013) 35–40, https://doi.org/10. antioxidant and antimicrobial properties of raspberry fruit, pulp, and marc extracts,
1016/j.seppur.2013.05.015. CyTA – J. Food. 11 (2013) 334–342, https://doi.org/10.1080/19476337.2013.
[11] J.-R. Jeon, E.-J. Kim, Y.-M. Kim, K. Murugesan, J.-H. Kim, Y.-S. Chang, Use of grape 766265.
seed and its natural polyphenol extracts as a natural organic coagulant for removal [25] R. Tsao, R. Yang, J.C. Young, H. Zhu, Polyphenolic profiles in eight apple cultivars
of cationic dyes, Chemosphere 77 (2009) 1090–1098, https://doi.org/10.1016/j. using high-performance liquid chromatography (HPLC), J. Agri. Food Chem. 51
chemosphere.2009.08.036. (2003) 6347–6353, https://doi.org/10.1021/jf0346298.
[12] A.L. Missio, B. Tischer, P.S.B. dos Santos, C. Codevilla, C.R. de Menezes, J.S. Barin, [26] P.M. Huang, Retention of arsenic by hydroxy-aluminum on surfaces of micaceous
C.R. Haselein, J. Labidi, D.A. Gatto, A. Petutschnigg, Analytical characterization of mineral colloids1, Soil Sci. Soc. America J. 39 (1975) 271, https://doi.org/10.
purified mimosa (acacia mearnsii) industrial tannin extract: single and sequential 2136/sssaj1975.03615995003900020016x.
fractionation, Sep. Purific. Technol. 186 (2017) 218–225, https://doi.org/10.1016/ [27] H. Xu, B. Allard, A. Grimvall, Influence of pH and organic substance on the ad-
j.seppur.2017.06.010. sorption of As (V) on geologic materials, Water Air Soil Pollut. 40 (1988) 293–305.
[13] H. Betatache, A. Aouabed, N. Drouiche, H. Lounici, Conditioning of sewage sludge [28] M.J.E. van der Maarel, B. van der Veen, J.C. Uitdehaag, H. Leemhuis, L. Dijkhuizen,
by prickly pear cactus (Opuntia Ficus Indica) juice, Ecolog. Eng. 70 (2014) Properties and applications of starch-converting enzymes of the α-amylase family,
465–469, https://doi.org/10.1016/j.ecoleng.2014.06.031. J. Biotechnol. 94 (2002) 137–155, https://doi.org/10.1016/S0168-1656(01)
[14] L. Gomes, E.P. Troiani, G.R.P. Malpass, J. Nozaki, Opuntia Ficus Indica as a poly- 00407-2.
electrolyte source for the treatment of tannery wastewater, Desalin. Water Treat. 57 [29] R. Lamghari El Kossori, C. Villaume, E. El Boustani, Y. Sauvaire, L. Méjean,
(2016) 10181–10187, https://doi.org/10.1080/19443994.2015.1035677. Composition of pulp, skin and seeds of prickly pears fruit (Opuntia ficus indica sp.),
[15] O. Bouaouine, I. Bourven, F. Khalil, M. Baudu, Identification of Functional groups of Plant Foods Human Nutrition 52 (1998) 263–270.
opuntia ficus-indica involved in coagulation process after its active part extraction, [30] N. Lumdubwong, P.A. Seib, Rice starch isolation by alkaline protease digestion of
Environ. Sci. Pollution Res. (2018) 11111–11119, https://doi.org/10.1007/ wet-milled rice flour, J. Cereal Sci. 31 (2000) 63–74, https://doi.org/10.1006/jcrs.
s11356-018-1394-7. 1999.0279.
[16] F.C. Stintzing, R. Carle, Cactus stems (opuntia spp.): a review on their chemistry, [31] N. Mateus, E. Carvalho, C. Luís, V. de Freitas, Influence of the tannin structure on
technology, and uses, Mol. Nutrition Food Res. 49 (2005) 175–194, https://doi.org/ the disruption effect of carbohydrates on protein–tannin aggregates, Analyt. Chim.
10.1002/mnfr.200400071. Acta 513 (2004) 135–140, https://doi.org/10.1016/j.aca.2003.08.072.
[17] Bruneton J. Pharmacognosie, phytochimie, plantes médicinales, Ed. Lavoisier, [32] V. Kuntić, N. Pejić, S. Mićić, D. Malesˇev, Z. Vujic, Determination of dissociation
Paris, 4 (2009). constants of quercetin, Pharmazie (2003) 439–440.
[18] B. Frølund, R. Palmgren, K. Keiding, P.H. Nielsen, Extraction of extracellular [33] Grim E. Ralph, Clay Mineralogy, Mc Graw Hill Book, 1953, p. 384.
polymers from activated sludge using a cation exchange resin, Water Res. 30 (1996) [34] R. Wey, Étude de la rétention des anions phosphoriques par les argiles montmor-
1749–1758, https://doi.org/10.1016/0043-1354(95)00323-1. illonite et kaolinite, Ann. Agron. 7 (1) (1956) 1–62.

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