Question: What is the residual formaldehyde content (wt% or ppm) in Mannich-reaction tannin-based coagulant products?

Industrial Crops & Products 176 (2022) 114336

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Sustainable tannin-based coagulants synthesized through Mannich reaction
using melamine as an amine source for water treatment applications

*
Adedayo Bello”, Tiina Leiviskä

University of Oulu, Chemical Process Engineering, P.O. Box 4300, FI-90014 Oulu, Finland

ARTICLE INFO

ABSTRACT

Keywords:
Melamine

This study explored the potential of melamine, as the nitrogenating source for the cationization of pulverized
quebracho and spruce tannins. The influence of modification conditions on the properties of the coagulants was
studied by varying the formaldehyde and melamine ratios with different activation times and temperatures.
Based on considerations of charge density and shelf life, the most viable modifications were established as co-
agulants synthesized with a 1:0.52 formaldehyde to melamine molar ratio at 70 °C. At optimal conditions, the
charge density of the quebracho and spruce coagulants was 2.22 meq/g and 1.04 meq/g, respectively, and the
residual formaldehyde content in the coagulants was low. The developed synthesis of this study demonstrated a
clear advantage over previous methods due to the rapid modification step. X-ray photoelectron spectroscopy
(XPS) confirmed the emergence of an amine which signifies a successful Mannich reaction in the coagulant.
Although optimal modification conditions for the coagulants were established at 70 °C (5-min activation time),
the average molecular weight could not be determined for these conditions. Nevertheless, electrospray ionization
mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass
spectrometry measurements revealed that the tannin-based coagulants obtained at lower temperatures (23 °C
and 45 °C) possessed low average molecular weight (approx. 800-900 Da). Furthermore, ESI-MS and MALDI-
TOF spectra showed that the Mannich modification resulted in the depolymerisation of the quebracho tannin,
leading to a reduction in units of higher mass fractions in the synthesized coagulant, which contrasted with the
spruce tannin. Jar test experiments with surface and industrial process waters demonstrated that the tannin
coagulants enhanced particle settling effectively.

Residual formaldehyde

Mannich reaction

Biocoagulants
Process waters

mechanisms used for this process are mainly charge neutralization and
sweep coagulation (Hussain et al., 2019; Zhu et al., 2011) while
adsorption, patch coagulation and bridging flocculation are also
employed depending on the coagulant (Roussy et al., 2005; Leiviskä and
Rämö, 2008). For a chemical to be considered adequate for coagulation,
it should be able to achieve a significant turbidity reduction within a
short period of time (Sincero and Gregoria, 2003). However, the effi-
ciency of the coagulation-flocculation is dictated by several factors,
which include coagulant type and dosage, pH, concentration of organic
matter and dissolved solids, ionic strength and temperature of the
effluent (Zhu et al., 2011; Sher et al., 2013).
Aluminium and iron salts are the most frequently used coagulants in
wastewater treatment, and their efficiency in improving the aesthetic
condition of water and wastewaters are well reported in the literature
(Alexander et al., 2012; Zhao et al., 2011). However, the major draw-
backs in the use of these salts include the generation of a large amount of

1. Introduction

Water is a key commodity for many industrial processes, and one
main concern is the remediation of industrial effluents, which are usu-
ally characterised by high turbidity. Waters with elevated turbidity
induced mostly by the presence of colloids and suspended solids cannot
be released directly into the environment without pre-treatment. This
concern has been further exacerbated by the changing and more strin-
gent water quality standards that individual point source dischargers
such as industrial facilities need to comply with. Coagulation-
flocculation is considered the most efficient pre-treatment technique
for turbidity removal (Daifa et al., 2019; Teh et al., 2016).
The coagulation-flocculation process is initiated by the addition of
chemicals to alter the physical state of pollutants, facilitating the
agglomeration of the colloids and subsequent removal through a sedi-
mentation or flotation process (Alexander et al., 2012). The removal

Corresponding author.

E-mail address: adedayo.bello@oulu.fi (A. Bello).

https://doi.org/10.1016/j.indcrop.2021.114336

Received 23 August 2021; Received in revised form 22 November 2021; Accepted 25 November 2021

Available online 9 December 2021

0926-6690/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

A. Bello and T. Leiviskä

Industrial Crops & Products 176 (2022) 114336

toxic sludge, residual metal in treated waters, ineffectiveness over a
wide range of pH values and poorer performance during the treatment of
cold waters, detrimental effects to human health and the environment
(Bratby, 2016; Haarhoff and Cleasby, 1988). Another widely used type
of coagulant is synthetic polymers. In comparison to aluminium and iron
salts, synthetic polymers counteract some of the disadvantages associ-
ated with the use of inorganic salts, such as the generation of smaller
volumes of sludge and the absence of residual metals in sludge and
treated waters. Nevertheless, the applicability of these synthetic poly-
mers has been marred by high cost, a low degree of biodegradability,
and the fact that little is known of the toxicity of their monomers (Per-
eira et al., 2018; Harford et al., 2011). This has necessitated the need for
an efficient and environmentally friendly coagulant to replace these
conventional coagulants.
Organic coagulant derived from both animal- and plant-based re-
sources provides a viable solution to the problems associated with fully
synthetic coagulants. Chitosan, which is obtained by the deacetylation
of chitin extracted from crustacean shells, is an example of animal-based
coagulants whose impressive coagulative performance has been exten-
sively reviewed (Lichtfouse et al., 2019; Yang et al., 2016). However, the
limited availability of chitin resources seems to have reduced their po-
tential for replacing chemical coagulants. On the other hand,
plant-based coagulants are largely non-hazardous, biodegradable and
renewable coagulants, which are naturally derived from various plant
components. In recent years, a group of plant-based coagulants that has
attracted increasing interest is tannin-based coagulants (Arismendi
et al., 2018; Beltrán-Heredia et al., 2010; Sánchez-Martín et al., 2014;
Graham et al., 2008; Grenda et al., 2020; Bello et al., 2020). This is
because tannins are widely available in almost every part of a plant and
are water-soluble, non-toxic polyphenols (Chung et al., 1998),
Tannins can be easily extracted with water and then cationized
through the Mannich reaction to produce an effective coagulant. The
Mannich reaction is a process that involves the condensation of an
aldehyde with a primary or secondary amine to produce an iminium ion,
which then introduces a positive charge into the polyphenolic matrix of
a tannin (Teh et al., 2016; Grenda et al., 2020). However, formaldehyde
and the amines used during the cationization process are toxic. For
example, the International Agency for Research on Cancer classified
formaldehyde as a human leukemogenic (IARC, 2012), and several
studies have found positive statistical significance between formalde-
hyde exposure and different types of cancer (IARC, 2012; Hauptmann
et al., 2009; Zhang et al., 2010). Despite this, no study was found to have
conducted a residual analysis of formaldehyde in tannin-based co-
agulants. Furthermore, previous studies on the synthesis of tannin-based
coagulants have performed the Mannich reaction with the aid of amines
such as diethanolamine (Arismendi et al., 2018; Beltrán-Heredia et al.,
2010; Bello et al., 2020), ethanolamine (Arismendi et al., 2018;
Beltrán-Heredia et al., 2010; Bello et al., 2020), dimethylamine hydro-
chloric acid (Grenda et al., 2020) and ammonium chloride (Arismendi
et al., 2018; Beltrán-Heredia et al., 2010). The amines mentioned above
have different toxicity levels with detrimental effects on human health
and the environment and could only be employed in real applications
under stringent guidelines (Brooks, 2008; Poste et al., 2014). Melamine,
on the other hand, is a highly nitrogenous compound considered not to
be acutely toxic and to have no notable environmental effect (Rajpoot
et al., 2020; ECCC, 2016). However, due to the formation of urinary
stones, which could lead to kidney failure, melamine is prohibited as a
direct additive in food products (FAO, 2019). Melamine reacts with
formaldehyde to produce resins with impressive physical and chemical
properties and is used in many industrial and biotechnological appli-
cations (Brettebauer and Schwarzinger, 2012). The reaction of mel-
amine and formaldehyde occurs mostly at high temperatures (>90 °C)
and is achieved via a two-phase reaction mechanism of methylolation
and condensation. During the reaction, up to six molecules of formal-
dehyde can be attached to a melamine molecule to form two methylol
compounds in the initial methylolation phase, which is succeeded by a

condensation reaction to produce melamine-formaldehyde resins (Pizzi
and Mittal, 2017). Some studies (Yubo et al., 2016; Kuo et al., 2019)
have reported the synthesis of resins with superior adhesive character-
istics from a composite of tannin-melamine-formaldehyde compared to
conventional melamine-formaldehyde resins. Also, Bucuroiu and
co-workers (Bucuroiu et al., 2016) investigated the possibility of
applying melamine-formaldehyde polymer directly as a coagulant. This
coagulant was claimed to be moderately effective in treating oily
wastewaters, but limited information is available regarding its synthesis
and performance. To the best of our knowledge, melamine has not been
used in the Mannich modification of tannins into coagulants. The reac-
tion of melamine with formaldehyde may provide additional benefits
such as improved shelf-life of tannin coagulants and tight binding of
formaldehyde.
In the present study, an investigation on the synthesis of sustainable
Mannich modification of tannin extracted from the bark of two tree
species (quebracho and spruce) was conducted by replacing conven-
tional toxic amines with melamine. The effects of modification param-
eters such as formaldehyde and amine concentrations, Mannich
activation time and temperature were evaluated with consideration to
shelf life and charge density to optimize the modification. The residual
formaldehyde content of the optimized products was analysed, and their
average molecular weights were estimated. A series of jar tests were
conducted with effluents of different characteristics to determine the
performance of the biocoagulants in real applications. The jar test ex-
periments were evaluated using major water quality parameters, i.e.,
turbidity, total surface charge (TSC), dissolved organic carbon (DOC)
and specific ultra-violet absorbance (SUVA 254). Electrospray ionization
(ESI), matrix-assisted laser desorption ionization time-of-flight (MALDI-
TOF) and X-ray photoelectron spectroscopy (XPS) characterization were
used to elucidate the changes induced by Mannich modification on the
pristine tannin samples.

2. Materials and methods

2.1. Tannins

Two tannin raw materials were aminomethylated through the
Mannich reaction. Spruce tannin (designated hereinafter as ST) was
supplied by VTT, the Technical Research Centre of Finland. The spruce
tannin was obtained through hot water extraction from the bark of
summer harvested spruce trees (Picea abies) and was pulverized into a
powdered tannin product through spray-drying. A detailed description
of the spruce tannin extraction conditions and procedures has been re-
ported in previous studies (Bello et al., 2020; Kemppainen, 2015).
Quebracho (Schinopsis balansae) tannin (QT), a commercial tannin
product, was provided by Silvachimica Srl (Italy).

2.2. Chemicals and reagents

All chemicals and reagents used during this study were of analytical
grade. Milli-Q ultrapure water was used throughout the experiments
except when indicated. The melamine used as an amine source in cat-
ionization was manufactured by Sigma-Aldrich Chemical Corporation
(USA), and formaldehyde (37% v/w) was supplied by VWR interna-
tional (France). HCl (Merck KGaA) and NaOH (VWR Chemicals) were
employed for pH adjustment. The ferric sulphate (PIX-322) that was
used as reference material in the coagulation experiments was provided
by Kemira Oyj, Helsinki (Finland). BTG Instrument AB, (Sweden), sup-
plied the sodium polyethylene sulphonate (PesNa) and poly-
diallyldimethylammonium chloride (PolyDadmac) solutions employed
to measure the charge density of coagulants and surface charge of water
samples.

2

A. Bello and T. Leiviskä

Industrial Crops & Products 176 (2022) 114336

2.3. Water samples

densities of modified products were estimated in the equivalent of
charge per gram of solid tannin in the standardized volume of
coagulants.
All experiments were conducted in two repeats, and the most viable
conditions for synthesis were evaluated based on the charge density and
shelf life of the coagulants. To evaluate the shelf life, the tannin-based
coagulants were stored at room temperature and evaluated after three
and eight months by visually checking their solubility (no formation of
precipitate) and measuring the charge density.
For comparison, melamine-formaldehyde coagulants (M-FA) were
synthesized with the same method used for tannin-based coagulant,
except that tannin solution was replaced with MQ water. The M-FA
coagulant was used as a reference during the jar test experiment for
kaolin/river water to study the advantage of tannin in the tannin-based
coagulant.

One natural water and two industrial process waters with different
characteristics were selected to evaluate the applicability of the tannin-
based coagulant to remove turbidity. The natural water sample was
collected from the Oulu river (Oulu, Finland) and it had high humic
content and low turbidity. The river water was then spiked with kaolin
to obtain a kaolin/river water with relatively high turbidity (~40 NTU).
Three grams of fine kaolin clay (0.063-0.5 mm) was dispersed in a batch
of 121 of river water to produce a kaolin suspension of 250 mg/l. Other
procedures undertaken to obtain a stable kaolin dispersion in the river
water have been explained in detail in a previous publication (Bello
et al., 2019). The two process wastewaters (PW1 and PW2) were
sampled from a metallurgical factory located in Finland. PW1, con-
taining oil and grease, was taken from the neutralization plant of a cold
rolling mill before the flotation stage. PW2 was water from a gas
scrubber obtained from a ferrochrome smelter. In contrast to the river
water, the industrial wastewaters were characterized by high turbidity.
The water quality of the water samples is presented in Table 1.

2.5. Characterization

Before XPS analysis, all the samples were dried overnight in an oven
at 40 °C to produce a solid product. The products were then ground to
obtain particles of uniform size. XPS characterization was performed
with a Thermo Fisher Scientific ESCALAB 250xi equipped with a
rotating monochromatic Al anode, generating an X-ray beam at 1486.6
eV. The XPS data analysis was performed with Avantage software, and
all spectra were fitted using the Shirley-type background. Charge
correction was performed on the binding energy (BE) with the
assumption that the binding energy of adventitious carbon is 284.8 eV.
X-ray diffraction (XRD) characterization was performed with a
Rigaku SmartLab diffractometer equipped with a Co rotating anode
lamp (40 kV, 135 mA). The speed of acquisition was four degrees per
minute at 0.02 degrees per step. The charge density of the tannin co-
agulants was determined via titration with PesNa using a Mütek particle
charge detector PCD 03 pH (Hersching, Germany). The average of three
titrations was calculated. The pH measurement of the modified tannin
was performed using a VWR pHenomenal® 1100 L instrument.
The weight-average molecular weight of the tannin samples and
tannin-based coagulants were estimated with the following mass spec-
trometry methods: ESI and MALDI-TOF. Prior to molecule weight
measurement by MALDI-TOF and ESI, the samples were dissolved in
50% acetonitrile (ACN)/0.1% formic acid at 1000-fold dilution. For the
ESI method, the analytes were infused at 10 μl/min into a Q-extractive
plus orbitrap mass spectrometer equipped with a standard ESI source.
The orbitrap mass spectrometer was calibrated with a Thermo mass
standard dedicated for Orbi-trap instruments. Mass spectra were recor-
ded in positive mode using the standard setting for source parameters
and data acquisition over a mass range of 160-4000 m/z. The molecular
average weight of the tannin fractions from the ESI spectra was calcu-
lated with the classical polymer parameters presented in Eq. (1).

2.4. Mannich modification

Tannin-based coagulants were synthesized through the amino-
methylation of tannin using formaldehyde (FA) and melamine as the
aldehyde and amine sources. The coagulant modification was achieved
through a dual-step system. The first step involved the preparation of
tannin solution and Mannich solution. For the tannin solution, 2.5 g of
pulverized tannin was dissolved in 10 ml of MQ water at ambient tem-
perature. The tannin was gradually added in batches, and the complete
dissolution of tannin in MQ water was ensured by the continuous mixing
of the solution with a laboratory spoon. In the Mannich solution (MS),
the melamine was allowed to react with formaldehyde at different
dosages to generate the imine ion. To ensure an optimal reaction in MS,
the mixture was kept at 120 °C for 2 h. After that, the MS was acidified
by injecting 1 ml of HCl (37%) into the solution, which reduced the pH
to ~1. Next, the tannin solution and the MS were mixed and reacted over
specific periods and temperatures under continuous agitation. The ob-
tained product was standardized by transferring it to a 50 ml volumetric
flask and filled up to the mark with MQ water. The solid contents of the
coagulant, before and after standardization were ~10% (w/v) and 5%
(w/v), respectively. The modified products were within a pH range of
1.7±0.2 after standardization. The dosage of tannin coagulants was
determined as the active coagulant content of the standardized product.
Chemical dosages and experimental conditions were selected ac-
cording to preliminary trials, which provided information about the
conditions under which undesirable instantaneous gelation of QT co-
agulants (T > 45 °C and high FA to melamine ratio) or the formation of
white crystals (low FA to melamine ratio) occurred. The effects of
chemical dosages and experimental conditions on the properties of the
tannin-based coagulants were then studied in two parts. First, the effect
of activation times (1, 5 and 20 min) was studied with varying melamine
dosages (9.0, 10.4 and 11.4 mmol) while the FA dosage (26.9 mmol) and
temperature (45 °C) were kept constant. This gave FA to melamine
molar ratios of 1:0.33, 1:0.39 and 1:0.42, respectively. Next, the effect of
activation temperature (23, 45 and 70 °C) with different FA dosages
(20.1, 26.9 and 40.4 mmol) was studied with a fixed melamine dosage
(10.4 mmol) and using an activation time of five minutes. The FA to
melamine molar ratios were 1:0.52, 1:0.39, and 1:0.26, and charge

Σ(m/z) 11
Σ(m/z);11

Mw =

(1)

m/z is the mass to charge of the oligomer.
It is the intensity of the oligomer.

The MALDI-TOF spectra were measured with a Bruker Ultra-
flextreme instrument in reflectron mode with protonation in the mass
range of 500-6000 m/z. MALDI was externally calibrated before the
measurement with Bruker's peptide calibration standard II. The MALDI-

Tablee 1

Characteristics of the kaolin/river water and process waters.

DOC (mg/l)

Water sample

TSC (μeq/1)

SUVA (l/mg..m)
3.284

Turbidity (NTU)

UV 254 (1/cm)

pH

Conductivity (µS/cm)

Kaolin/river water

36

7.5

43

0.312

9.5

-45
-18.6

0.181

PW1

50
152

6.3

448

11

1.645
0.904

0.235

PW2

8.4

1735

26

-11.5

3

A. Bello and T. Leiviskä

Industrial Crops & Products 176 (2022) 114336

TOF spectra of tannin samples and tannin coagulants were run with the
same volume of matrix solution (50% v/v) while the matrix solution was
prepared by dissolving 2,5-dihydroxy benzoic acid (DHB) in 50% ACN/
water to a concentration of 10 mg/ml. Subsequently, the matrix and
analyte were subjected to brief ultrasonic mixing, and 1 µl aliquots were
placed on a steel sample stage to air dry. After drying, the steel sample
stage was introduced into the spectrometer. The weight-average mo-
lecular weight of the tannin fraction in the spectra produced from
MALDI-TOF was computed using Eq. (2).

3. Results and discussion

3.1. Effect of activation time and melamine dosage

The effect of activation time (1-20 min) and melamine dosage
(9.0-11.4 mmol) on charge density when the FA dosage (26.9 mmol) and
activation temperature (45 °C) were kept constant is shown in Fig. 1. A
visible charge density progression could be observed as the activation
time and melamine dosage increased in most QT modifications (Fig. 1a).
QT coagulants having activation times of 1 min and 20 min resulted in a
significant increase in charge density when the melamine dosage was
increased. On the other hand, the charge densities of the QT coagulants
having an activation time of 5 min deviated from the trend, and a logical
reason for this deviation could not be established. When comparing the
effect of activation time on recorded charge densities, there was only a
minimal gain in charge density with 10.4 mmol melamine dosage within
the studied timeframe. Coagulants modified with 9.0 and 11.4 mmol
melamine dosages recorded a perceptible 25% (0.24 meq/g) and 8%
(0.14 meq/g) increase in charge densities, respectively, when the acti-
vation time was increased from 1 to 20 min. Nonetheless, the activation
time increase was confirmed not to be statistically significant with all
melamine dosages (t-test, p < 0.05, two-tailed).
The ST coagulant graph displayed in Fig. 1b indicates that there was
no notable correlation between the charge density and melamine dosage,
as coagulant modification with a higher melamine dosage did not result in
a significantly higher charge density. The ST coagulants modified with
shorter activation times (1 and 5 min) clearly showed higher charge
density values at higher melamine dosages as compared to the lowest
melamine dosage, while this was not the case with the 20 min activation
time. It is noteworthy that the most significant increase in charge density
due to melamine dosage variation was recorded when the dosage was
raised from 9.0 to 10.4 mmol (0.29 ± 0.17-0.48 ± 0.18 meq/g) when
compared with 11.4 mmol (0.41 ± 0.14 meq/g). Interestingly, the charge
densities of the modified coagulants increased progressively with the
activation time for coagulants modified with 9.0-10.4 mmol melamine
dosages. Charge densities increased from an average of 0.21 ± 0.11 meq/
g to 0.41 ± 0.17 meq/g when activation time was increased from 1 min
to 5 min, and further increased to 0.55 ± 0.11 meq/g when the activation
time was raised to 20 min.

ΣN;M²
ΣΝ Μ

Mw

(2)

=

Mi is the mass of the oligomer.

NĘ is the number of molecules of a specific molecular weight.
The residual formaldehyde of the tannin coagulants was analysed
using a high-performance liquid chromatograph with diode-array
detection (HPLC-DAD) from Waters (Autosampler: 717 Plus; Quater-
nary pump: 600 Controller; 2998 Detector). The samples were diluted in
water and mixed with 2,4-dinitrophenylhydrazine, whereby aldehydes
and ketones react to give the respective hydrazones. These were sepa-
rated through a reversed-phase HPLC method, detected at 355 nm and
quantified.

2.6. Water analysis

The turbidity of water samples was measured with a Hach 2100Q
portable turbidity metre. The total surface charge of water samples was
measured with a Mütek particle charge detector (PCD 03) by titrating
10 ml of water samples with PesNa or Poly-Dadmac titrant (0.001 N). A
Metrohm 744-pH metre equipped with an epoxy electrode and a Mettler
Toledo conductivity metre were employed to measure the pH and con-
ductivity of the water samples. The ultraviolent absorbance (UVA) of the
water samples was measured at 254 nm with a Shimadzu ultraviolet
spectrometer (model UV-1800), while dissolved organic carbon (DOC)
measurement was performed with a SIEVERS 900 portable TOC analy-
ser. The DOC and UVA measurement was performed after filtration with
0.2-μm filtered (VWR, polyethersulphone membrane) water samples,
and specific ultraviolet absorbance (SUVA) values were calculated from
the UVA and DOC values in accordance with EPA methods (EPA, 2009).
A detailed analysis of water samples was performed in accordance with
standardized methods: NH4-N, NO2-N, NO3-N and PO4-P were measured
with a continuous flow analyser using the SFS-EN ISO 11732:2005, SFS-
EN ISO 13395:1997 and SFS-EN ISO 15681-2:2005 methods, respec-
tively. Cl, F and SO4² were analysed using ion chromatography (SFS-EN
ISO 10304-1:2009), and the concentration of the elements in the water
samples was analysed using an inductively coupled plasma optical
emission spectrometer (ICP-OES) (SFS-EN ISO 17294-2:2016).

On a general note, coagulants with relatively stable high charge
densities were obtained in both tannin modifications when the mel-
amine dosage was kept at 10.4 mmol. When considering the charge
densities obtained from the shortest activation time (1 min), this study
showed a rapid crosslinking between the flavonoid (tannin) units and
iminium ions. A short activation timeframe was chosen for this study
because a prolonged activation time resulted in the gelation of QT co-
agulants modified with 9.0 mmol of melamine at 45 °C. However,
earlier researchers of this topic (Arismendi et al., 2018; Beltrán-Heredia
et al., 2010; Grenda et al., 2020) have ascertained that crosslinking
progresses over time and that a longer time is required to obtain a
complete crosslinking of the two constituents (tannin and MS). A direct
comparison is difficult because different amines were used during
Mannich modification in previous studies. Grenda et al. (2020) pro-
duced tannin coagulants from Acacia mearnsii and quebracho with
dimethylamine as the amine source and reported a 100 and 180-minutes
activation times for an optimal coagulant modification. In a separate
study, Grenda et al. (2018) also recommended a reaction time of 90 min
for Acacia mearnsii based on the products shelf life. In their studies,
Beltrán-Heredia et al. (2010), synthesized a tannin-based coagulant
from quebracho and Acacia mearnsii with diethanolamine, ethanolamine
and ammonium chloride and suggested a 24-hour timeframe for optimal
modification. The synthesis developed in this study has a clear advan-
tage over previous methods due to the rapid modification step. How-
ever, it is essential to state that all QT coagulants synthesized during this
phase of the study gelated after one month of storage, whereas the ST
coagulants retained their liquid state.

2.7. Coagulation experiments

Coagulation experiments were performed in jar tests using a Kemira
flocculator 2000 jar test apparatus equipped with one-litre glass bea-
kers. For the jar tests, the beakers were filled to the 800 ml mark with
water samples and dosed with different dosages of coagulants. The so-
lution was then subjected to rapid mixing (150 rpm) for one minute,
slow mixing (40 rpm) for 20 min and sedimentation for 30 min. The
sedimentation period was followed by the extraction of 200 ml of the
supernatant from 3 cm below the surface of the test water sample for
various analyses. Jar test residues were recovered by first decanting the
excess supernatant, centrifuging the flocs at 2500 rpm for 10 min and
decanting the supernatant again. The flocs were then oven-dried over-
night at 40 °C to produce jar test residues.

4

A. Bello and T. Leiviskä

Industrial Crops & Products 176 (2022) 114336

ST Coagulant

a)

QT Coagulant

b)

2.0

2.0

Melamine 9.0 mmol
Melamine 10.4 mmol

Charge density (meq/g)

Melamine 11.4 mmol

1.5

1.5

1.0

1.0

0.5

0.5

0.0

-0.0

5
Time (min)

20

20

1

5
Time (min)

Fig. 1. Effects of activation time and melamine dosage on charge densities of a) QT and b) ST coagulants. Range of charge densities represents the deviation in two
repeats: temperature 45 °C and FA dosage 26.9 mmol. Charge densities were measured immediately after Mannich modification.

3.2. Effect of activation temperature and formaldehyde dosage

temperatures (23, 45 and 70 °C) and formaldehyde dosages (20.1, 26.9
and 40.4 mmol) were investigated with a constant melamine dosage of
10.4 mmol and a five-minute activation time. The result for the QT co-
agulants, displayed in Fig. 2a, indicates that the activation temperature
had a significant effect only when the lowest FA dosage was used, as the
charge density increased from 0.88 to 1.78 meq/g with increasing
temperature. With higher FA dosages, the activation temperature did

As described in section 3.1, coagulants with considerably high
charge densities can be obtained with short activation times. Besides,
some preliminary experiments in this study had shown that QT Co-
agulants were quite unstable when modified at high temperature and
when using a longer activation time. Next, the effects of the activation

ST Coagulant

QT Coagulant

a)

b)

2.0

2.0

FA 20.1 mmol

FA 26.9 mmol

Charge density (meq/g)

FA 40.4 mmol

1.5

1.5

10

1.0

0.5

0.5

0.0

-0.0

23

70

23

70

45
Temperature (°C)

45
Temperature (°C)

Fig. 2. Effects of temperature and FA-melamine dosages on charge densities of a) QT and b) ST coagulants. Range of charge densities represents the deviation in two
repeats: activation time (5 min) and melamine dosage (10.4 mmol). Charge densities were measured immediately after Mannich modification.

5

A. Bello and T. Leiviskä

Industrial Crops & Products 176 (2022) 114336

not affect the charge density values. The QT coagulant resulted in a high
charge density even when modified at room temperature and using a
medium FA dosage of 26.9 mmol. The reduction in charge density with
the highest FA dosage can be attributed to a reduced iminium ion for-
mation due to an excess of formaldehyde in the reaction. Excess form-
aldehyde to amine has been reported to inhibit the formation of
hemiaminals: an intermediate compound produced before iminium ions
are formed during Mannich reactions (Iwasawa et al., 2007; Short et al.,
1992). Earlier studies on the synthesis of tannin-based coagulants have
noted that a higher formaldehyde dosage is detrimental to the
physico-chemical properties of the biocoagulants, as it results in the
instant gelation of products or coagulants with a relatively low shelf life
(Ibrahim et al., 2021). Also, a physical observation of the QT coagulants
modified with the highest formaldehyde dosage (40.4 mmol) revealed
that they underwent a colour change from a homogeneous dark brown
solution to light brown products characterized by precipitates, during
modification at 70 °C.
In contrast to the QT coagulants, the charge densities of the ST co-
agulants increased with an increase in temperature in most cases
(Fig. 2b), except for the coagulant modified with 20.1 mmol of FA at
45 °C. Also, increasing the formaldehyde dosage did not enhance the ST
coagulant charge densities. High temperature might have distinctively
favoured the crosslinking process in the spruce tannin compared to the
QT tannin. Nevertheless, the range of charge densities obtained from all
the individual modifications for the ST coagulants (0.32–0.90 meq/g)
was still lower than that of the QT coagulants (0.82–1.95 meq/g). This
was probably related to the presence of fewer phenolic functional groups
in the spruce tannin to accept the iminium ion generated in the Mannich
solution (Kemppainen, 2015). Previous studies (Bello et al., 2020) have
corroborated this claim and further elaborated that the content of
proanthocyanidins (condensed tannin) is over 300% higher in
quebracho tannin than in its spruce counterpart. Another factor
considered to have influenced the performance of spruce tannin is the
presence of impurities such as ash and glucosides. These impurities
usually lead to the formation of undesired and competitive by-products
during aminomethylation (Arismendi et al., 2018).

3.3. Evaluation of shelf life of tannin-based coagulants

A viable coagulant in water treatment applications should have a
reasonable shelf life. To evaluate the stability of the Mannich-modified
coagulants, they were stored at room temperature for three months.
After the three-month storage period, it was discovered that all the QT
coagulants modified at the higher formaldehyde dosage (≥26.9 mmol)
had gelated. Surprisingly, gelation was not recorded with any ST bio-
coagulants during the reviewed timeframe. The reason for the selective
coagulant gelation could not be validated during this study. However, it
is widely known that one of the reaction mechanisms behind the ami-
nomethylation of tannin-based polymers is condensation
(Sánchez-Martín et al., 2014; Grenda et al., 2020). This infers that
crosslinking progresses over time, which leads to continuous thickening
and eventual gelation of the modified products (Grenda et al., 2020).
Further studies were performed only on both tannin coagulants
modified with 10.4 mmol melamine dosage and 20.1 mmol FA dosage
(FA to melamine molar ratio 1:0.52). The variations in charge densities
obtained for the coagulants after the three- and eight-month storage
periods are shown in Fig. 3 and Table 2. Both tannin coagulants wit-
nessed an average increase of about 45% in charge densities after the
three-month storage period. However, it is important to note that the
charge density variations were more pronounced in the QT samples. A
comparison between charge densities obtained from the samples after
the three- and eight-month storage periods reveals that the crosslinking
reaction had been completed within three months, resulting in products
with more stable charge densities. Interestingly, coagulants with rela-
tively stable charge densities could be obtained by employing higher
temperature during the activation phase. Thus, higher temperature was
concluded to have accelerated the crosslinking process. It is essential to
note that the liquid-equivalent charge densities (meq/1) of QT co-
agulants were close to those of M-FA coagulants modified with exactly
the same melamine and formaldehyde dosages and subjected to similar
modification conditions (Table S1).
Formaldehyde is categorized as a carcinogen by the International
Agency for Research on Cancer (IARC, 2012), and thus a lower amount

QT Coagulant

a)

ST Coagulant

b)

3.0

3.0

23°C

45°C

Charge density (meq/g)

2.5

70°C

2.0

2.0

1.5

1.5

1.0

1.0

0.5

0.5

0.0

-0.0

3
Time (month)

0

8

3

Time (month)

Fig. 3. Changes in charge densities of a) QT coagulants and b) ST coagulants monitored over time (0, 3 and 8 months) as a function of reaction temperature (23 °C,
45 °C and 70 °C). Range of charge densities represents the deviation in two repeats (two modifications) (FA 20.1 mmol, melamine 10.4 mmol, activation time 5 min).

6

A. Bello and T. Leiviskä

Industrial Crops & Products 176 (2022) 114336

in some fishes (European Food and safety Authority, 2014) to
406 mg/kg in dried mushrooms (Yeh et al., 2013). An estimation of the
concentration of FA content in a treated water sample, if 100 mg/1
dosages of coagulants are applied and assuming all residual FA stays in
the treated water, shows a significantly low residual FA of 1.8 mg/1
(0.00018 wt%) and 4.2 mg/1 (0.00042 wt%) for the QT and ST co-
agulants modified at 70 °C, respectively.

Table 2

Residual formaldehyde, charge densities, and molecular weights of coagulants
after 8 months' storage. Activation time 5 min, FA 20.1 mmol, melamine
10.4 mmol.

Coagulants

Formaldehyde
content (wt% FA/v
(coagulant solution))

Molecular
weight (Da)

Charge density (meq/g
of solid tannin in

coagulant solution)

MALDI ESI

QT 23 °C

2.133 0.005 (pH
4.4)
2.174 0.008 (pH

916.4

928.7

0.08

3.4. XPS analysis of tannin samples and tannin-based coagulants

QT 45 °C

880.1

920.5

0.09

4.4)

XPS analysis was employed to elucidate the elemental change
induced by the Mannich reaction on the tannin samples. XPS revealed
that the tannin samples (QT and ST) consisted mainly of carbon and
oxygen peaks located at binding energy ~285 eV (C1s) and ~533 eV
(01s) in the wide spectra (Fig. S1). Additionally, the C1s peak could be
fitted into two and three carbon components for the quebracho and
spruce tannin, respectively. These were in agreement with a previous
study (Bello et al., 2020). The first two carbon components at 284.8 eV
and 286.4 present in both tannin samples were assigned to
C-C/C=C/C-H (C1 component) and C-OH/C-O (C2 component) bonds
(Bello et al., 2020; Klébert et al., 2017). The third peak at 288.1 eV
detected only in the spruce tannin was assigned to the C=O bond (C3
component) (Awada et al., 2012). The C=O bond might be attributed to
polymeric sugars in the spruce tannin sample (Bello et al., 2020;
Kemppainen, 2015).
Fig. 4a and b show the XPS high-resolution Cls and N1s spectra for
the pristine melamine sample. The two peaks in the Cls spectrum
located at 284.8 eV and 287.8 eV were assigned to C-C/C-H (C1
component) and C-N/ C=N (C2 component) bonds (Yao et al., 2005).
The C1 component is formed due to surface contamination by adventi-
tious carbon (Dementjev et al., 2000). The C-N bonds in the melamine
are located on the low BE side of the C2 component and the C=N bonds

QT 70 °C

2.2170.004 (pH

0.09

N/A

N/A

4.5)

1.227 0.002 (pH

ST 23 °C

0.14

736.4

918.5

4.5)

1.253 0.001 (pH
4.5)
1.040 0.002 (pH
4.5)

ST 45 °C

0.13

830.2

858.6

ST 70 °C

0.21

N/A

N/A

N/A: Not available (The molecular weight of coagulants modified at 70 °C was
disregarded because ESI and MALDI-TOF consistently produced spectra with
low intensities).

of residual formaldehyde is preferred in the modified products. The
amount of residual formaldehyde in the coagulants after eight months is
presented in Table 2. The result indicates a low percentage of residual
formaldehyde in both tannin coagulants. Nonetheless, the formaldehyde
amount was slightly lower in the QT coagulants than that in the ST
coagulants. The residual FA amounts were low when considering the
background level of formaldehyde in many food items. Formaldehyde
exists in the environment due to natural processes and anthropogenic
activities (IPCS, 1989), and a reasonable amount of formaldehyde is
found as a natural constituent in food products ranging from 200 mg/kg

a)

b)

C2

N1
5.

N2

C1

396

402

400

300

295

290

285

280

404

398

BE (eV)

BE (eV)

Fig. 4. High resolution a) Cls and b) N1s XPS spectra of melamine.

7

A. Bello and T. Leiviskä

Industrial Crops & Products 176 (2022) 114336

on the high BE side. In the N1s spectrum, the lower energy component
(N1), located at 398.0 eV, was ascribed to C=N-C (imine), while the N2
component positioned at 399.0 eV was attributed to C-NH2, a type of
nitrogen bond (amine) (Lee et al., 2016).
The XPS survey for the commercial ferric sulphate used as a reference
coagulant confirmed the presence of carbon, oxygen, iron and sulphur
(Fig. S2). In the case of the Mannich-modified tannin coagulants, addi-
tional peaks for Cl and N (besides the peaks observed in tannin spectra)
were observed in the survey spectra at binding energy 198 eV (Cl2p) and
~400 eV (N1s) (Fig. S2). The Cl2p was believed to have originated from
the HCl used for acidification, and the N1s peak was related to mel-
amine. The Cls spectra of the QT and ST coagulant samples (Fig. 5a and
b) were fitted to three and four carbon peaks, respectively. Both tannin-
based coagulants have peaks at 284.8 eV and 286.4 eV, which is
consistent with the peaks of C1 and C2 components identified in the
tannin samples. The third peak for the QT and ST coagulants was located
at 288.3 eV and 287.5 eV, respectively. The ST coagulant was observed
to possess an additional peak at 288.8 eV. The emergence of the peak at
287.5 eV (C3 component) in the ST coagulant is related to the C-N and
C=N bonds of melamine (Son et al., 2020) associated with a successful
Mannich modification (Sahoo et al., 2006). The fourth peak in the ST
coagulant located at 288.8 eV (C4 component) can most likely be
attributed to the emergence of a new carbon bond associated with the
carboxyl groups (O=C-OH, O=C-O) (Awada et al., 2012; Feng et al.,
2017), which might indicate the formation of D-glucosamine derivatives
(Sharma and Peddinti, 2018; Swilem et al., 2017). This peak further
corroborates the formation of competitive by-products during spruce
tannin aminomethylation, hence reducing the regioselectivity of Man-
nich modification, as suggested in previous research studies (Arismendi

et al., 2018; Bello et al., 2020). In the QT coagulant, the third broader
peak (C3/C4 component) on the low BE side included the C-N and C=N
bonds of melamine and possibly carboxyl groups (0=C-0, O=C-OH)
on the high BE side. These O=C-O/O=C-OH bonds could not be
justified in the QT coagulant based on the Cls spectrum of the pristine
quebracho tannin (Fig. S3). However, Kemppainen et al. (2014) char-
acterized the same commercial quebracho tannin with ³¹p nuclear
magnetic resonance and reported a 0.22 mmol/g carboxylic content,
indicating the presence of some impurities.
Fig. 5c and d illustrate the chemical state of nitrogen in the modified
coagulants. The weight percentage of nitrogen in the samples after
Mannich modification was 19.0% and 16.4% for the QT and ST co-
agulants, respectively (Table S3). The N1s high-resolution spectrum of
both tannin modifications were fitted to three peaks, representing imine
(C=N-C in melamine structure, N1 component), deprotonated amine
(N2) and protonated amine (N3) components. For the QT coagulant, the
imine, deprotonated, and protonated amine peaks were located at
398.5 eV (N1), 399.5 eV (N2) and 400.3 eV (N3) binding energies,
respectively, while those of the ST coagulant were located on almost the
same binding energies (398.4 eV (N1), 399.4 eV (N2) and 400.4 eV
(N3)). The presence of the nitrogen peak signifies the success of the
Mannich reaction.

3.5. Molecular weight of tannin samples and modified coagulants

The positive mode ES1 spectra of the pristine tannin samples and the
Mannich-modified coagulants are shown in Fig. 6. The quebracho tannin
(Fig. 6a) possessed polyphenols of higher mass fractions than the spruce
tannin (Fig. 6b). Furthermore, the data indicates that fragmented or

b)

a)

C1

C1

C2
8→

-3

C3/C4

295

300

290

285

280

300

295

290

285

280

BE (eV)

BE (eV)

c)

d)

A

N1

N2

N2

N1

N3

N3

T
396

404

402

400

398

404

402

400
BE (eV)

398

396

BE (eV)

Fig. 5. High resolution C1s XPS spectrum for a) QT and b) ST coagulants and N1s XPS spectrum for c) QT and d) ST coagulants (FA 20.1 mmol, melamine 10.4 mmol,
activation time 5 min, temperature 70 °C).

8

A. Bello and T. Leiviskä

Industrial Crops & Products 176 (2022) 114336

simple phenolic compounds dominate the polyphenol composition of
the spruce tannin. The quebracho tannin produced a spectrum with a
series of procyanidin peaks with a mass difference of 288 Da apart
(1108-1396 m/z) and an oligomer series with a mass of repeated units of
272 Da (563-835 and 683-956 m/z). The pattern of these oligomeric
units was in agreement with the literature for hot water extracted
quebracho tannin (Venter et al., 2012; Pash et al., 2001). The quebracho
tannin fractions were more widely distributed within a 350-2000 m/z
mass range (Fig. 6a). The average molecular weight of the quebracho
tannin was estimated as 1389 Da (by ESI). The fractions of the spruce
tannin were narrowly distributed within the mass range of 150-900 m/z
and possessed an average molecular weight of 612 Da (Fig. 6b).
The mass fractions displayed in Fig. 6c and d for the QT and ST co-
agulants were quite different from their pristine tannin samples. The
obtained mass fractions after Mannich modification were characterized
by lower mass polymers (100-1400 m/z), compared with the tannin
sample for quebracho. The increase in the proportion of lower mass
fractions suggests that the aminomethylation process resulted in the
degradation of interflavonoid bonds in the quebracho tannin (Pash et al.,
2001). This confirms previous findings that, when subjected to a low pH
such as that required for the Mannich reaction, the proanthocyanidins in
quebracho tannin depolymerize, releasing subunits as electrophilic
flavan-3-ol intermediates (Kennedy and Jones, 2001). Also, the
observed reduction in the average molecular weight of the QT coagulant
after Mannich modification (920 Da) was of interest. In contrast, the
depolymerization effect due to acidic treatment witnessed with the QT
coagulant was not recorded in the ST coagulant spectrum. Although it
had a lower relative intensity, the ESI spectrum of the ST coagulant
clearly showed an increase in the mass fractions compared to its pristine
spruce tannin sample, and it produced a higher average molecular
weight of 859 Da. This implies that an increase in polymer chain lengths
might have occurred in the ST coagulant due to the condensation and
crosslinking mechanism associated with the Mannich reaction (Grenda
et al., 2020). It is worth mentioning that both tannin coagulants
possessed higher mass fractions (Fig. S4) and higher average molecular

weights (Table S2) than M-FA coagulants modified under the same
conditions.

The MALDI-TOF analysis displayed in Fig. 7 was in close agreement
with the molecular weight distribution pattern of the tannins and
modified coagulants produced by the ESI. The spectra displayed a series
of peaks ranging from 500 to 2400 m/z. Quebracho and spruce tannin
produced an average molecular weight of 1089 and 769 Da in sequential
order, whereas 880 and 830 Da were obtained for the modified QT and
ST coagulants, respectively. The average molecular weight estimated
from the MALDI-TOF measurements was slightly lower than that ob-
tained from the ESI measurement, except for the pristine spruce tannin.
The disparity in the estimated average molecular weight between the
MALDI-TOF and ESI methods was attributed to the moderate mass ac-
curacy of MALDI-TOF, as signal intensity could differ significantly
within one sample preparation (hot spot phenomenon) (Ole, 1994;
Strupat, 2005).

3.6. Coagulation performance

3.6.1. Kaolin/river water

Fig. 8. displays the dosage curves for turbidity, total surface charge,
ultraviolet absorbance and specific ultraviolet absorbance of treated
kaolin/river water samples. The tannin coagulants and the reference
coagulants (PIX-322 and M-FA) were all able to reduce the concentra-
tion of suspended solids and colloids in the kaolin/river water sample
impressively (>90% turbidity reduction). A lower dosage was required
with the tannin coagulants (QT 25 mg/1 and ST 50 mg/l) and M-FA
coagulant (50 mg/l) for minimum residual turbidity when compared
with the PIX-322 (125 mg/l). It is noteworthy that the QT coagulant
achieved clearly lower residual turbidity and slightly wider optimal
dosage range than the ST and M-FA coagulants.
The pH of the kaolin/river water was close to neutral (7.5 ± 0.2) and
it is well known that ferric sulphate produces the positively charged
species needed for charge neutralization at slightly acidic pH. However,
if enough iron coagulant is added, then the solution pH becomes suitable

a) 60000000-

b) 50

50000000

683 835

295

50000000-

Intensity (au)

Intensity (au)

40000000-

40000000-

30000000-

1108

30000000

956

20000000-

20000000-

1398

595

165

393 563

1687

10000000-

10000000-

835

1944

2331 2507

1109

0

0

500

2500

2500

2000

3000

1000

1500
m/z

500

1500

2000

3000

1000

m/z

0

d) 16000000-

12000000

277

287

10000000-

Intensity (au)

14000000-

Intensity (au)

12000000

415

8000000

10000000-

6000000

8000000

835

169

664

553

6000000-

4000000

663

169 415

4000000-

1108

2000000-

853

962

1057 1321 1453

2000000

1397 1670

0

0

1500
m/z

500

1500
m/z

2000

2500

3000

500

1000

2000

2500

3000

1000

Fig. 6. ESI mass spectrum for a) QT b) ST c) QT coagulant and d) ST coagulant. (FA 20.1 mmol, melamine 10.4 mmol, activation time 5 min, temperature 45 °C).

9

Industrial Crops & Products 176 (2022) 114336

A. Bello and T. Leiviskä

b) 35000-

a)

588

683

120000

30000

Intensity (au)

Intensity (au)

100000

25000

80000

683

20000-

829 955

60000

15000

759

1145

863

40000

1129

10000

557

1401

1963 2235

1691

20000

5000

0

0

600 800 1000 1200 1400 1600 1800 2000 2200 2400

600 800 1000 1200 1400 1600 1800 2000 2200 2400
m/z

m/z

d)

c)

553

554

400000

200000-

Intensity (au)

Intensity (au)

566

150000-

300000

565

100000-

583

200000

985

692

584

842

50000-

100000

704

1123

992 1042

(1273

1273

1493

1423

1280

0

0

600 800 1000 1200 1400 1600 1800 2000 2200 2400
m/z

600 800 1000 1200 1400 1600 1800 2000 2200 2400
m/z

Fig. 7. MALDI-TOF mass spectrum for a) quebracho tannin b) spruce tannin c) QT coagulant and d) ST coagulant. (FA 20.1 mmol, melamine 10.4 mmol, activation
time 5 min, temperature 45 °C).

b)
200-

a) 70

M-FA Coagulant
PIX-322
QT Coagulant
ST Coagulant

M-FA Coagulant

PIX-322

60

QT Coagulant
ST Coagulant

Total surface charge (μeq/l)

150-

50

Turbidity (NTU)

100

40

30

50

20

10

-50

0

150

0

50

100

200

250

300

0

50

100

150

200

250

300

Dosage (mg/l)

Dosage (mg/l)

c) 0.7

d)

0.6

6-

0.5

5.

SUVA (l/mg-m)

UV 254 (1/cm)

0.4

M.FA Coagulant
PIX-322

0.3.

M-FA Coagulant

PIX-322

QT Coagulant
ST Coagulant

QT Coagulant
ST Coagulant

0.2-

0.1

1

0.0

0-

50
50

250

300

150
Dosage (mg/l)

250

0

100

150
200
Dosage (mg/l)

50

100

200

300

Fig. 8. Effects of coagulant dosage on a) turbidity b) total surface charge c) UV254 absorbance and d) SUVA on the kaolin/river water using QT, ST, M-FA and PIX-

322 coagulants.

10

A. Bello and T. Leiviskä

Industrial Crops & Products 176 (2022) 114336

significantly at extreme coagulant dosage due to the evident increase in
UV 254 absorbance, while the increment recorded with PIX-322 was
attributed to low DOC values (data not shown).

for the generation of the required iron species (Bratby, 2016). This
phenomenon was evident during the experiment as the pH of the treated
water was reduced from 7.5 to ~4.8, which was obtained after applying
an iron coagulant dosage of 125 mg/1 before any significant turbidity
reduction was recorded. On the other hand, the tannin-based and co-
agulants were observed to work well at the initial pH of the kaolin/river
water sample and only moderately decreased the pH values to 6.9 and
6.7 at the optimal dosage for the QT and ST coagulants, respectively. The
effectiveness of tannin-based coagulants over a wide pH range has been
previously reported (Grenda et al., 2018). Compared to the tannin co-
agulants, a much lower pH (5.9) was recorded for the M-FA coagulant at
optimal dosage.
The TSC displayed in Fig. 8b indicates that increasing the coagulant
dosage resulted in a decrease in surface charge (TSC becomes less
negative) with all of the tested coagulants. At the optimal dosages of
tannin and M-FA coagulants based on the residual turbidites, the TSC
values were close to zero, while overdosing increased turbidity and
surface charge. The lack of increased turbidity with the PIX-322 coag-
ulant even at excess coagulant concentrations supports the formation of
electroneutral iron species at above optimal coagulant dosage (Bratby,
2016). In such an instance, the increase of coagulant dosage has little or
no effect on the turbidity of the treated water. Fig. 8b shows that the TSC
dosage curve of water samples treated with PIX-322 was more stable
than the other coagulants even at an excess dosage. TSC describes the
charge of the treated water. For PIX-322 coagulant, once the positive
charge required to neutralize the colloid is generated at optimal pH,
overdosing of the iron salt results in the formation of Fe-hydroxide
precipitates, which settles with the flocs. On the other hand, tannin
and M-FA coagulants stay dissolved in the solution and, when over-
dosed, result in a higher TSC for the treated water, as demonstrated in
Fig. 8b. It is noteworthy that the increase in the supernatant's surface
charge was more pronounced with the QT and M-FA coagulants due to
their higher cationic charge.
The fact that TSC was close to zero when optimal turbidity was ob-
tained with both tannin coagulants indicates that the primary mecha-
nism employed by the tannin coagulants was charge neutralization.
Also, this was supported by the lower dosage requirement observed for
the QT coagulant compared to the ST coagulant due to its higher charge
density. This finding was consistent with earlier studies (Fang, 2007)
which presented charge neutralization as the dominant mechanism used
by tannin-based coagulants to destabilize colloids in kaolin/river mix-
tures at neutral pH.
UV 254 and SUVA 254 are vital indicators used to obtain information
about dissolved organic compounds in water samples. As shown in
Fig. 8c, the UV254 in the treated water sample decreases as the coagulant
dosage increases. The decrease in UV254 absorbance translates into a
humic reduction of 90%, 80%, 69% and 68% in treated natural water at
optimal dosage for the PIX-322, QT, M-FA, and ST coagulants, respec-
tively. This is consistent with earlier researchers who reported that ferric
salts are more effective in removal of humic substances than tannin
coagulants (Heiderscheidt et al., 2016). Surprisingly there was a steep
increase in UV 254 absorbance with the M-FA coagulant, which tran-
scended the initial absorbance value of the kaolin/river water when
coagulant concentration exceeded the optimal dosage. This indicates
that when overdosed, the residual M-FA coagulant in the treated water
affect strongly UV254 absorbance. The other coagulants showed a minor
increase in the UV 254 absorbance when optimal dosage range was
exceeded, which suggests good removal of organic compounds despite
overdose. The initial SUVA value (>3 1/mg-m) displayed in Fig. 8d for
the kaolin/river mixture is a typical value of natural waters composed of
hydrophobic, humic and aromatic organic compounds of higher mo-
lecular weight (Edzwald and Van Benschoten, 1990). The SUVA value
reduction below 2 1/mg--m at optimal dosages for all coagulants implies
that the optimally treated river/kaolin waters will later be characterized
by non-humic, hydrophilic and lower molecular weight organic frac-
tions. It is noteworthy that the SUVA value of M-FA increased

3.6.2. Industrial process waters

To evaluate the performance of the coagulants in industrial appli-
cations, the tannin coagulants were used to enhance particle settlement
in two process waters (referred to hereinafter as PW1 and PW2). It is
important to restate that the verifiable difference between the PW1 and
PW2 water samples was that PW2 possessed a higher concentration of
suspended solids, a higher pH value, higher COD, higher UV254 absor-
bance and higher conductivity, as shown in Table 1.
Fig. 9 shows the dosage curves of turbidity reduction, total surface
charge, UV254 and COD for the treated PW1 samples. The QT coagulant
achieved a very high turbidity reduction (>85%) with a relatively low
dosage (10 mg/l). However, with a higher dosage, the ST coagulant was
also able to achieve an impressive turbidity reduction of 76% with a
coagulant dosage of 50 mg/l, demonstrating the effectiveness of the
coagulants in the effluent of concern. In contrast to the kaolin/river
water, the dosage curve obtained from both coagulants showed that the
turbidity of the treated water was stable over a wide range of coagulant
dosage, and charge reversal did not occur even at an extreme coagulant
dosage (Fig. 9b). However, the total surface charge approached zero as
the coagulant dosage increased. The stabilization of the surface charge
over a wide dosage range tends to suggest that electrostatic patch
adsorption was the dominant mechanism for colloid destabilization and
that charge neutralization may have occurred to some extent. For the
electrostatic patch mechanism to occur, chain polymers carrying
opposite charges are believed to be adsorbed onto the colloids' surface,
forming a charge-like mosaic, which is then attracted by other colloids.
Earlier studies have noted that the destabilization of colloids by tannin-
based coagulants is complex and might have been ensured through
various mechanisms (Fang, 2007; Hameed et al., 2016). Hameed et al.
(2016) referred to both charge neutralization and bridging as the
dominant mechanisms for colloid destabilization in their treated
effluent.

The UV254 absorbance reduction with dosage for the PW1 sample is
shown in Fig. 9c. The UV absorbance was partially reduced by the QT
coagulant but not by the ST coagulant. The dosage curve of chemical
oxygen demand (COD) for the treated PW1 sample revealed that, at
optimal dosage for turbidity reduction, the QT and ST coagulants
simultaneously achieved 50% COD reductions with 10 mg/1 and 50 mg/
1 dosages, respectively (Fig. 9d). Both tannin coagulants exhibited an
identical COD reduction pattern at lower coagulant dosages, but after
the dosage exceeded 60 mg/1, the COD was observed to increase with
the ST coagulant while the COD continued to decrease with the QT
coagulant.
To further evaluate the performance of the tannin-based coagulants
in industrial applications, QT and ST coagulants were tested in another
process water (PW2). A very small amount of coagulant was needed to
bring the turbidity to a low level (Fig. 10a). This can probably be
attributed to the availability of a nucleation site for floc formation
provided by the high concentration of suspended solids (Amy and
Chadik, 1983), thereby reducing the coagulation burden and favouring
the precipitation of suspended particles (Zhou et al., 2020). Addition-
ally, the TSC of PW2 was small (−11.5 µeq/1), which indicated a small
cationic demand. The lack of a visible reduction in the total surface
charge of the treated water sample in Fig. 10b (TSC becoming less
negative) further affirms that the cationic charge of modified tannin
coagulants did not make any significant contribution to the destabili-
zation of the colloids. Considering the lower molecular weight charac-
teristic of the coagulants and the water quality of the PW2 sample, it is
reasonable to conclude that the main destabilization mechanism
employed was patching.
Neither of the tannin coagulants was able to reduce the UV absor-
bance and COD of the PW2 sample (Figs. 10c and 10d). This was

11

A. Bello and T. Leiviskä

Industrial Crops & Products 176 (2022) 114336

a) 60

b)

0

60
50

Total surface charge (peq/l)

40

Turbidity (NTU)

30-

20
20

-15

QT Coagulant
ST Coagulant

QT Coagulant
ST Coagulant

10-

-20

550
50

50

100

150

200

100
Dosage (mg/l)

150

200

0

0

Dosage (mg/l)

c) 0.20

d) 50

0.18

10
40

Chemical Oxygen demand (mg/l)

0.16-

0.14-

UV 254(1/cm)

0.12-

30

0.10-

20

0.08

0.06-

10
10

0.04-

QT Coagulant
ST Coagulant

QT Coagulant
ST Coagulant

0.02-
0.00

0

0

100
Dosage (mg/l)

50

50

150

200

0

100

150

200

Dosage (mg/l)

Fig. 9. Effect of coagulant dosage on a) turbidity b) total surface charge c) UV 254 absorbance and d) COD of the PW1 water using QT and ST coagulants.

a) 160

b)
-7

140

Total surface charge (μeq/l)

-8

120

Turbidity (NTU)

100

80
08

1

60-

QT Coagulant
ST Coagulant

40-

QT Coagulant
ST Coagulant

-12

20-

-13

0

120 140 160

0

20

40

60

80
Dosage (mg/l)

100

0

20

40

60

80
Dosage (mg/l)

100

120

140 160

d)80

c) 0.25

70

Chemical oxygen demand (mg/l)

60

0.20-

50

UV 254(1/cm)

0.15-

40

30

0.10-

20

0.05-

QT Coagulant
ST Coagulant

QT Coagulant
ST Coagulant

10

0.00

0

120

140 160

80 100
Dosage (mg/l)

160

0

20

40

60

100
Dosage (mg/l)

80

0

20

40

60

120

140

Fig. 10. Effect of coagulant dosage on a) turbidity b) total surface charge and c) UV254 absorbance d) COD of the PW2 using QT and ST coagulants.

12

A. Bello and T. Leiviskä

Industrial Crops & Products 176 (2022) 114336

attributed to the inability of the tannin coagulants to retain their
cationic charge at higher pH. The pH value of PW2 was 8.6 and
decreased only to 8.2 with the highest used dosage, thus the pH was at a
higher level than with PW1 and kaolin/river water. Earlier studies have
highlighted that the charge possession of tannin-based coagulant is pH-
dependent. Fang (2007) found that the charge density of a tannin-based
coagulant decreased from 3.07 meq/g to 0.2 meq/g with increasing pH
values from 4 to 9. Another possible reason for the coagulants' inef-
fectiveness in reducing the UV absorbance and COD was the quality of
organic matter in the process water. Marhaba and Pipada (2000) have
demonstrated that coagulants are less effective in removing simple and
well-dissolved organic matter, and others have reported that hydrophilic
dissolved organics are not amenable to coagulation (Amy et al., 1987).

QT JR-PW2

ST JR-PW2

SSPW2

3.7. Characterization of jar test residues and suspended solids of process
water samples

T
1040

1030

1050

1020

BE(eV)

Fig. 11. Overlaying comparison of Zn2p spectra for PW2 suspended solids
(SSPW2) and jar test residues (QT JR-PW2 and ST JR-PW2).

XPS analysis of the surface elemental content was performed on the
jar test residues of all of the treated water samples and suspended solids
of the original process water samples. The XPS survey for the jar test
residues obtained from the kaolin/river water showed that they
possessed new peaks located at 103.2 eV and 75 eV, assigned to Si2p and
Al2p, respectively. Al and Si were believed to have originated from the
kaolin introduced to the river water. Further analysis was not performed
on the kaolin/river jar test residue because no additional elements were
detected. Besides, analysis of the river water's nutrient and elemental
content showed a very low concentration of elements (Table S4).
The XPS analysis of surface elemental content of suspended solids in
the PW1 (SSPW1) sample revealed that it contained only C, O and Si.
The Si2p peak was located at 101.5 eV in the Si2p high-resolution XPS
spectrum. The XPS wide spectra revealed that the PW1 jar test residues
(JR-PW1) from both tannin coagulants contained mainly C, N, O and Si.
The high-resolution spectra showed that the Si2p peaks of the jar test
residues were located at 101.7 eV while the N1s peaks were located at
the same BEs as pure tannin coagulants.
The XPS characterization of suspended solids in the PW2 (SSPW2)
sample showed that the particles were composed of several elements
(Ca, C, Mg, N, O, S, Si and Zn). Zn was the element with the most
interesting characteristics since the high-resolution Zn2p spectrum
showed two-component doublets. The fitted Zn2p3/2 peaks were located
at 1022.1 eV and 1024.7 eV and the Zn2p1/2 peaks were positioned at
1045.2 eV and 1047.8 eV, respectively. The fact that PW2 was water
from a gas scrubber obtained from a ferrochrome smelter and a high
resolution of S2p peak at 162.4 eV was attributed to metallic sulfide
(Fantauzzi et al., 2015) suggests the presence of ZnS. Sulphur reacts with
zinc at above hydrothermal temperature to form ZnS (Gao et al., 2015).
Thus, it is reasonable to conclude the sulphur exists as ZnS in the sus-
pended solids. X-ray diffraction (XRD) confirmed the presence of ZnS in
the suspended solids and further suggested that the crystalline zinc
phases of the SSPW2 were wurtzite (~23%), elemental Zn (~15%) and
sphalerite (~12%), supporting the multi-component structure observed
with XPS. The dual-peak structures observed with XPS have been re-
ported as a characteristic of multi-component zinc (Raveendra et al.,
2018; Wang et al., 2015). Raveendra et al. (2018) ascribed the double
component structure to Zn2+ ions in Zn-O species associated with
wurtzite. To understand the changes induced by the coagulants, XPS
analysis was carried out on the PW2 jar test residues (JR-PW2), which
confirmed the presence of all elements earlier detected in the SSPW2
(Fig. S5). Overlaying the Zn2p spectra for the pristine sample and jar test
residues (Fig. 11) showed a broadening of the JR sample peaks by
~1.0 eV towards higher binding energies. The broadening increased the
energy gap between the component peaks of the JR samples by 1.7 eV,
from 2.1 eV (SSPW2) to ~3.8 eV (JR-PW2). Peak fitting of the Zn2p3/2
of the JR-PW2 samples revealed that an additional peak at 1025.9 eV
might be related to Zn-N interactions (Fig. S6, only spectrum of QT
JR-PW2 shown). Yahya et al. (2002) have reported the emergence of

similar peaks as a result of complexation between zinc cation and the
nitrogen in the amine of a chitosan coagulant.
Analysis of the surface elemental content of the PW2 water sample
presented in supplementary Table S5 confirmed that sulphate and zinc
were present in high concentrations. An additional elemental analysis
was performed on the treated water samples (Table S5) to evaluate the
residual elemental concentration after coagulation. The probe into the
residual elemental concentration of the treated PW2 water sample
showed that the two tannin coagulants reduced the sulphate concen-
tration by 20% from 400 mg/1 to ~320 mg/l. The elemental analysis of
the treated water sample also revealed that the QT coagulant reduced
zinc from an initial concentration of 3.8 mg/1 to 1.1 mg/1 (74%). At the
same time, the ST coagulant achieved a zinc reduction of 93% (0.26 μg/
1). Metal sequestration was not of the utmost importance during this
study, and thus, the reason for the impressive performance of the ST
coagulant was not investigated. However, some studies have reported
the ability of N-glycosidic compounds (glucosamine derivatives) pro-
duced as a side product in the ST coagulant to provide chelating sites for
the complexation of various cationic metals (Allscher et al., 2008; Yano
et al., 1999; Yano and Mikata, 2002). More importantly, Yano et al.
(1999) have demonstrated that N-glucoside ligands from D-glucosamine
could effectively ligate zinc atoms.

4. Conclusions

This study has shown that melamine can be used as a nitrogenating
agent during the aminomethylation of tannins. With this synthesis, low-
to-medium charge density and low molecular weight products with low
FA content can be obtained, depending on the tannin raw material. A
higher dosage FA and melamine had a detrimental effect on the stability
of the QT coagulant, while a higher modification temperature acceler-
ated the crosslinking process in both tannin modifications. The results of
the XPS analysis confirmed that iminium ions were successfully
embedded into the tannin matrix after aminomethylation. ESI and
MALDI-TOF analyses revealed that the Mannich modification led to the
depolymerization of the quebracho tannin. In general, the results ob-
tained from the jar tests showed that both types of modified coagulants
were effective in turbidity reduction, with QT slightly outperforming the
ST coagulant due to its relatively higher charge density. Nevertheless,
the ST coagulant could still be considered a potential coagulant, espe-
cially in industrial applications, because of its stability. Coagulation
studies demonstrated that charge neutralization, bridging and patching
mechanisms could be employed by the tannin-based coagulants when
applied as a coagulant, depending on the effluent surface characteristics.
The results of the XPS analysis of jar test residue suggested that metals
sequestered from the treated effluent were bonded within stable

13

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Industrial Crops & Products 176 (2022) 114336

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Declaration of Competing Interest

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The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

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Acknowledgements

This work was funded by University of Oulu graduate school and
Maa- ja vesitekniikan tuki ry. The authors would also like to acknowl-
edge the support of VTT (Technical Research Centre of Finland) and the
Silvachimica team for providing the tannin samples.

ius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252F
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15
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An estimation of the
concentration of FA content in a treated water sample, if 100 mg/1
dosages of coagulants are applied and assuming all residual FA stays in
the treated water, shows a significantly low residual FA of 1.8 mg/1
(0.00018 wt%) and 4.2 mg/1 (0.00042 wt%) for the QT and ST co-
agulants modified at 70 °C, respectively.