Carbohydrate Polymers 291 (2022) 119561

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

The structure and flocculation characteristics of a novel exopolysaccharide

from a Paenibacillus isolate
Shijunyin Chen 1, Rui Cheng 1, Xiaodong Xu , Changchang Kong , Lei Wang , Renjie Fu , Jing Li ,
Shiming Wang , Jianfa Zhang *
Center for Molecular Metabolism, Nanjing University of Science & Technology, Nanjing 210094, China

A R T I C L E I N F O A B S T R A C T

Keywords: Bioflocculants like polysaccharides are excellent alternatives to harmful synthetic flocculants and usability under
Bioflocculant stricter levels of ecosystem protection. In this study, an exopolysaccharide producing strain Paenibacillus sp.
Paenibacillus sp. M21629 M21629 was isolated. The exopolysaccharide was abundant in carboxylic groups and named floccuronic acid
Exopolysaccharide
with a high weight average molecular weight of 2.38 × 108 Da. The hexasaccharide repeating units of floccuronic
acid were →3)-α-D-Manp-(1 → 3)-4-Suc-β-D-Manp-(1 → 3)-β-D-Glcp-(1 → 4)-β-D-GlcpA-(1 → 4)[4,6-Pyr-β-D-
Galp-(1 → 3)]-β-D-Galp-(1→. The floccuronic acid at 0.5–1 ppm showed high turbidity removal efficiency in
kaolin suspension (99.8%), coal wastewater (98.8%), and high-turbidity drinking water (89.2%). The floccula­
tion ability of floccuronic acid depended on the instability of solid particles. Owing to these encouraging results,
floccuronic acid is expected to be a useful bioflocculant applied in wastewater treatment and the utilization of
water resources.

1. Introduction mill (Mukherjee et al., 2014) and municipal wastewater in effluent

Flocculation is a popular process to separate colloidal dispersion to
cleaner solution and precipitate. In this process, flocculants are needed
for reducing time consumption and strengthening the formation of flocs.
Therefore, flocculants play an important role in water treatment and can
be traced back to ancient times (Bratby, 2016). Today drinking water
has more strict requirements not only for sustaining human life, also for
color and unpleasant taste (Xia et al., 2018). Pathogen removal is
another requirement as a disinfection treatment (Yang et al., 2014).
Moreover, contaminated wastewater must be treated to keep a friendly
environment and public health (Krishnamurthy et al., 2020). Growing
concerns about the environment problem, flocculants are used with
other processes like membrane filtration to treat wastewater from
different sources, including distilling manufactory (Zhang et al., 2018),
textile dyeing industry (Bisht & Lal, 2019), coal mine (Xu et al., 2017),
oil sands tailings ponds (Gumfekar et al., 2019; Kim et al., 2012), paper
treatment plant (Kanmani & Yuvapriya, 2018). In addition, flocculation
is expected to be low cost, high efficiency and biosafety solution of
microalgae harvest, and these microalgae can be used for potential
functional food with high protein production and valuable biofuels
(Kumar et al., 2019; Li et al., 2019). On the other hand, cyanobacteria
blooms may pollute the freshwater with toxins, which also can be
removed by flocculants (Sun et al., 2015). The application of flocculants
has also been more mentioned with developing high-value products like
monoclonal antibodies (Kang et al., 2014; McNerney et al., 2015).
Flocculants can be obtained from different sources. These include
inorganic salts based on aluminum or ferric ion, artificial syntheses like
polyacrylamide (PAM) derivatives, and natural products like chitosan
and exopolysaccharide. Though inorganic and synthetic flocculants
have been used and developed with a long historical standing, they may
induce safe problems or cause seconded pollution. Aluminum is sus­
pected of relating to Alzheimer's disease, flocculants in drinking water

Abbreviations: PAM, polyacrylamide; FTIR, Fourier transfer infrared; HPLC, high-performance liquid chromatography; PMP, 1-phenyl-3-methyl-5-pyrazolone;
NMR, nuclear magnetic resonance; COSY, correlated spectroscopy; TOCSY, total correlation spectroscopy; HSQC, heteronuclear single quantum coherence; NOESY,
nuclear overhauser effect spectroscopy; HMBC, heteronuclear multiple bond correlation.
* Corresponding author at: Center for Molecular Metabolism, Nanjing University of Science & Technology, 200 Xiaolingwei, Nanjing 210094, China.
E-mail addresses: chensh@njust.edu.cn (S. Chen), chengrui@njust.edu.cn (R. Cheng), xiaodong-xu@njust.edu.cn (X. Xu), kongchang@njust.edu.cn (C. Kong),
315102000263@njust.edu.cn (L. Wang), jingli.seb@njust.edu.cn (J. Li), bioeng@njust.edu.cn (S. Wang), jfzhang@njust.edu.cn (J. Zhang).
1
Shijunyin Chen and Rui Cheng contributed equally to this work.

https://doi.org/10.1016/j.carbpol.2022.119561
Received 20 September 2021; Received in revised form 28 February 2022; Accepted 28 April 2022
Available online 6 May 2022
0144-8617/© 2022 Elsevier Ltd. All rights reserved.
S. Chen et al.
contribute, at least in part, to aluminum intake (Organization, 2010).
Cationic synthetic flocculants are found toxic even less than 1 mg/L, and
the basic anionic synthetic flocculant PAM is also toxic to most fresh­
water species, and is not appropriate under higher levels of ecosystem
protection (Harford et al., 2011). The monomer acrylamide and dime­
thylamine used to synthesize flocculants would remain in PAM products
due to incomplete or reversible reactions. Acrylamide is recognized as a
cumulative neurotoxin and precursor of carcinogen (Organization,
2003), and dimethylamine could react with chlorine and produce
carcinogenic N-nitrosodimethylamine during disinfection (Park et al.,
2007). The absence of biocompatibility makes inorganic and synthetic
flocculants unqualified for bio-related applications, as metal ions are
found to significantly inhibit microalgae growth (Li et al., 2019; Sukias
& Craggs, 2011).
Because of these imperfections in inorganic and synthetic floccu­
lants, bioflocculants like polysaccharides become appealing alterna­
tives. Most bioflocculants are eco-friendly, non-toxic, and
biodegradable, which have been applied grandly in many aspects
mentioned above (Salehizadeh et al., 2018). Compared to poly­
saccharides obtained from plants and animals, exopolysaccharides
secreted by microbes could be produced under controlled conditions
(Ventorino et al., 2019). In this study, we isolated an exopolysaccharide-
producing bacteria from a soil sample. The exopolysaccharide was
characterized with a high molecular weight and an abundance of
carboxyl groups. Thus we hypothesized that the negatively-charged
high-molecular-weight exopolysaccharide has flocculation activity
through the bridging mechanism. The effects of culture conditions on
exopolysaccharide production and flocculation ability were studied, and
the exopolysaccharide was named floccuronic acid. Eventually, the
flocculating capacity of floccuronic acid was analyzed in kaolin sus­
pension, coal wastewater, and high turbid river water.
2. Material and method
2.1. Bacteria, growth conditions, and exopolysaccharide extraction
The strain M21629 was grown on the medium containing sucrose 30
g, KNO3 1.5 g, NaH2PO4 1 g, CaCl2 0.07 g, MgSO4⋅7H2O 0.2 g, FeS­
O4⋅7H2O 0.0375 g, MnSO4⋅H2O 0.003 g, ZnCl2 0.0075 g, agar 15 g, and
H2O 1000 mL, pH 7. Strain cultivation was maintained at 30 ◦ C for 2
days. For the taxonomical identification of the isolate, the genomic DNA
was extracted using a Karroten genomic DNA purification kit (Karroten,
Nanjing, China), then the 16S rRNA gene was amplified by PCR and
sequenced by GENEWIZ Inc. (Suzhou, China). The phylogenetic tree was
constructed using the neighbor-joining method with MEGA X software.
Bootstrap values for 1000 replicates were shown at the node of the tree
(Sun et al., 2020). The effect of different carbon sources on the growth of
bacteria was analyzed with sucrose, maltose, and starch respectively.
The fermentation of the M21629 was incubated in 0.5 L Erlenmeyer
flasks, each containing 100 mL medium, at 28 ◦ C for 20 h under 220
rpm. The turbidity removal rate of fermentation broth was measured by
jar test in kaolin suspension. A kaolin suspension (4 g/L, 100 mL) was
prepared, then CaCl2 (50 g/L, 1 mL) and the fermentation broth (20 μL)
were added. These steps were described in greater detail below. The
crude exopolysaccharide was precipitated from fermentation broth by
adding chilled ethanol (1:3 v/v), drying in a vacuum oven for 24 h at
40 ◦ C to determine the yield. The OD600 of ferment broth was measured
in real-time by a Bacterial Growth Curves Meter (Karroten Scientific,
Nanjing, China).
2.2. Composition analysis
The total sugar and total protein contents of crude exopoly­
saccharides were estimated by phenol-sulfuric acid and Bradford
methods respectively. For further identification, the crude exopoly­
saccharide was purified by papain and the Sevag method or the heat-
2
Carbohydrate Polymers 291 (2022) 119561
alkaline method (Li et al., 2017; Staub, 1965). In order to identify the
functional groups of the polysaccharide, 10 mg of finely powdered pu­
rified exopolysaccharide was analyzed using a Fourier transfer infrared
(FTIR) spectroscope (Nicolet iS20, Thermo Scientific). The purified
exopolysaccharide was completely hydrolyzed by 2 M trifluoroacetic
acid at 105 ◦ C for 6 h and recovered by vacuum desiccation. Then the
monosaccharide composition was studied by pre-column derivatization
high-performance liquid chromatography (HPLC) method using a Wa­
ters HPLC system (Waters Inc., USA) equipped with a Zorbax SB-Aq
column (4.6 mm × 150 mm, Agilent Technologies, Inc., USA). The
PMP-monosaccharides (detected at 248 nm) were eluted at a flow rate of
0.9 mL/min with acetonitrile/KH2PO4 buffer (30 mM, pH 5.6) at a ratio
of 17.7:82.3 (v/v) (Xu et al., 2018).
2.3. Molar mass determination
The weight average molecular weight (Mw), number average mo­
lecular weight (Mn), and size average molecular weight (Mz) of exo­
polysaccharide purified by papain and the Sevag method were
determined by gel permeation chromatography (GPC) using a Waters
HPLC system (Waters Inc., USA) equipped with a W410 RI detector and a
TSKgel SuperMultiporePW-H column (6.0 mm × 150 mm, Tosoh Corp.,
Japan). The mobile phase was sodium phosphate buffer (PBS, 15 mM,
pH 5.0) with 0.03% sodium azide (NaN3) at a flow rate of 0.6 mL/min
and the eluate was analyzed by the refractive index detector. Freshly
prepared solutions (1 mg/mL) of dextran standards (21–2990 kDa) were
used to draw the calibration curve.
2.4. Methylation analysis
The exopolysaccharide prepared for methylation analysis was puri­
fied by the papain and Savag method, and partly hydrolyzed by sulfuric
acid for improved dissolution in DMSO. To confirm the existence of
uronic acid, the methylation analysis was performed as previously
described with reducing the carboxyl group of uronic acid in the exo­
polysaccharide (Sun et al., 2020). Gas chromatography-mass spec­
trometry (GC–MS) system (Thermo Scientific ISQ LT, USA) equipped
with a TG-200MS capillary column (0.25 mm × 0.25 mm × 300 mm)
was used to analyze partially methylated alditol acetates. The GC pro­
gram was set as 50 ◦ C, 1 min, 40 ◦ C/min to 130 ◦ C, 3 ◦ C/min to 230 ◦ C,
hold 2 min. Helium was used as carrier gas.
2.5. NMR analysis
For NMR experiments, the purified exopolysaccharide was depoly­
merized by 0.1 M TFA at 100 ◦ C for 1 h to lower the viscosity and then
neutralized by 0.1 M NaOH to pH 7.0. The hydrolyzate was dissolved
into D2O. All NMR experiments, including 1H NMR, 13C NMR, COSY,
TOCSY, HSQC, NOESY, and HMBC were performed at 25 ◦ C on a Bruker
Avance 500 MHz spectrometer (Bruker, Karlsruhe, Germany). Chemical
shifts were expressed in parts per million with respect to the 0 ppm point
of sodium-3-trimethylsilyl propionate (TSP), used as internal standard.
2.6. Flocculation experiments
Kaolin clay (Sinopharm Chemical Reagent Co., Ltd., China) was used
as test solids suspension to determine the flocculation activity of floc­
curonic acid. Various parameters such as pH value of suspension, bio­
flocculant concentration, settling time, type of cation were manipulated
to optimize their effects towards flocculation efficiency. The particle
sizes of kaolin clay used in this study were 887.9 nm and the zeta po­
tential was − 31.4 mV, determined by Zetasizer (Nano ZS90, Malvern
Instruments). Briefly, different doses of floccuronic acid solution (1 mg/
mL) and 1 mL of 5% CaCl2 were added in a 100 mL kaolin suspension.
The mixture was vortexed for 2 min and then allowed to stand for 5 min.
Then, the upper portion of the mixture was immediately collected and
S. Chen et al. Carbohydrate Polymers 291 (2022) 119561

a 100 Paenibacillus sp. FLOCAN

57 Paenibacillus mucilaginosus strain VKPM B-7519
Paenibacillus solanacearum strain T16R-228
83 Paenibacillus elgii strain SD17
99 Paenibacillus elgii strain NBRC 100335
98 Paenibacillus tyrfis strain MSt1
96
Paenibacillus ehimensis strain IFO 15659
Paenibacillus tianmuensis strain B27
68 Paenibacillus chinjuensis strain WN9
Paenibacillus yunnanensis strain YN2
70 Paenibacillus validus strain JCM 9077
Paenibacillus filicis strain S4
Bacillus funiculus strain NAF001

0.01
b c
1.50 100 10
sucrose Turbidity removal
starch EPS production
1.25 maltose
80 8

EPS production (g/L)

Turbidity removal (%)
Grow th curve, OD600

1.00
60 6
0.75

40 4
0.50

20 2
0.25

0.00 0 0
0 6 12 18 24 30 6 8 10 12 14 16 18
Time (h) Time (h)

Fig. 1. Phylogenetic analysis (a), growth curves (b), and the bioflocculant production (c) by Paenibacillus M21629.

measured the optical density at 550 nm with a UV–vis spectrophotom­
eter. The removal rate of turbidity was calculated according to the
following equation:
The removal rate of turbidity = (A − B)/A × 100%
where A and B are the OD550 of the control and sample, respectively.
To test the flocculation effect on wastewater and drinking water
treatment, turbidity removal of coal wastewater (particle sizes 1932 nm,
zeta potential − 20.9 mV) and high turbidity drinking water (particle
sizes 1208 nm, zeta potential − 15.8 mV) were measured. Coal waste­
water was prepared by coal fly ash powder. High-turbidity drinking
water was prepared by the addition of 100 g of soil to 1 L river water,
vigorously mixing the mixture for 2 h, and then let to rest for 2 h. An
aliquot of 100 mL of the supernatant was added into a beaker, then poly
ferric chloride (10 g/L, 50 μL) and different doses of floccuronic acid
were added (Xia et al., 2018). The detailed procedures and the removal
rate of turbidity were the same as above.
3. Results and discussion
3.1. Identification of the bacteria strain and bioflocculant production
The exopolysaccharide-producing bacterial strain M21629 was iso­
lated from a soil sample, producing slightly translucent colonies with
thick mucoid. The strain M21629 showed sensitivity to various tested
antibiotics, including ampicillin, kanamycin, and chloromycetin. The
16s rDNA sequence was compared with other bacteria through BLAST.
The result suggested that the closest species with 99.9% similarity was
Paenibacillus mucilaginosus, and a phylogenetic tree was constructed by
the neighbor-joining algorithm (Fig. 1a). Hence, the isolate was desig­
nated as Paenibacillus sp. M21629. Growth curves (OD600) were
measured in real-time to investigate the influence of different carbon
sources. The M21629 showed a typical diauxic growth pattern and the
maximum growth rate was achieved with sucrose (Fig. 1b). The first
exponential growth phase started after 3 h of incubation and ended at
14 h, followed by the first stationary phase. The second exponential
growth phase started at 17 h and the second stationary phase initiated at
22 h, which indicates the existence of polysaccharide degrading en­
zymes (Malick et al., 2017). The exopolysaccharide biosynthesis started
at the middle of the first exponential growth phase, and the optimal
exopolysaccharide production of 7.8 g/L was achieved after 16 h at pH
7.0 and 28 ◦ C (Fig. 1c). The fermentation broth was taken for floccula­
tion activity determined by the kaolin test, and the highest removal rate
of turbidity (95.0%) was also achieved after 16 h.
3.2. Biochemical and structural characteristics of exopolysaccharide
The total sugar content of crude exopolysaccharide was estimated to
be 72.1% (w/w), total protein was 23.0% (w/w), and traces of inorganic
salts. The purified polysaccharide showed similar flocculation activity
(99.7%) to crude exopolysaccharide (99.8%) and fermentation broth
(95.0%), implying the polysaccharide a major role in flocculation.
Analyzed by GPC, the number average molecular weight (Mn), weight
average molecular weight (Mw), and size average molecular weight
(Mz) of the polysaccharide were 8.66 × 107 Da, 2.38 × 108 Da, and 3.44
× 108 Da respectively. As far as we know, the average Mw of this
polysaccharide was higher than exopolysaccharides produced by other
Paenibacillus strains (Liang and Wang, 2015). Recently our lab recorded
a novel exopolysaccharide with a high Mw of 1.48 × 107 Da, produced
by Paenibacillus mucilaginosus WL412 (Xu et al., 2017). The Peanibacillus
mucilaginosus TKU032 produced an exopolysaccharide with two mass
peaks with molecular weights of 1.05 × 104 and 1.35 × 104 Da. The
exopolysaccharide produced by Paenibacillus tarimensis REG 0201 M has

3
S. Chen et al. Carbohydrate Polymers 291 (2022) 119561

a Table 1
Methylation analysis of the bioflocculant.
Methylated sugar Glycosidic linkage Mass fragments (m/z)

2,3,4,6-Gal T-Gal 101, 117, 129, 145, 161, 205

2,4,6-Man 1,3-linked-Man 87, 101, 117, 129, 161, 233
2,4,6-Glc 1,3-linked-Glc 87, 101, 117, 129, 161, 233

1727.9

882.8
1245.8
2935.6 2,3,6-Glc 1,4-linked-GlcA 87, 99, 113, 117, 129, 233

1401.5
2,6-Gal 1,3,4-linked-Gal 58, 87, 117, 129, 143, 305
3336.3

1603.5

1155.2
CH3 stretching and bending vibrations (2936.60 cm− 1) (Nep et al.,
Heat-alkaline method 2016). These strong absorption peaks in the range of 1000 cm− 1 and

1070.8
1200 cm− 1 were attributed to the presence of C–O–C and C–O–H

1028.8
Papain and Savag method
stretching vibration, which was ascribed to pyranose ring of poly­
4000 3500 3000 2500 2000 1500 1000 500 saccharide (Tadayoni et al., 2015). Besides, carbonyl moieties showed
Wavenumber (cm-1) peaks at 1727.91 cm− 1, 1603.52 cm− 1, and 1401.51 cm− 1, which were
b 2.5 attributed to carboxylic acid ester form (C–– O), carboxylate anion form
(COO–), and O–C–O symmetric stretching vibration of carboxylate
groups respectively. The peak at 1245.8 cm− 1 was attributed to the
2.0
C–O–C vibration of acyls (Araújo et al., 2020). This peak and peak at
1727.91 cm− 1 were removed in the sample purified by the heat-alkaline
Response (AU)

D-Man
1.5 method because of the instability of acyls groups as previous research
reported (Sutherland, 1997; van Casteren et al., 2000).
L-Rha D-Gal The monosaccharide composition of the polysaccharide was identi­
1.0
D-GlcA D-Glc fied by TLC and HPLC. Compared with available authentic mono­
saccharides standards in Fig. 2b, the HPLC spectrum of acid hydrolysate
0.5 of floccuronic acid samples in Fig. 2c shows it was composed of
mannose, galactose, glucose, and gluconic acid with the molar ratio of
2:2:1:1. Moreover, methylation analysis was carried out to determine
0.0 the glycosyl linkage pattern. As summarized in Table 1, the GC–MS re­
0 10 20 30 40 sults indicated that there were five partially methylated alditol acetates
Time (min)
c 2.5 (PMAAs) identified as 2,3,4,6-Me4-Galp, 2,4,6-Me3-Manp, 2,4,6-Me3-
D-Man Glcp, 2,3,6-Me3-Glcp, and 2,6-Me2-Galp. As shown in Fig. S1, the
carboxyl-reduced sample showed a higher peak at the position of 2,3,6-
2.0 Me3-Glcp, then this peak was identified as glucuronic acid (Sims et al.,
2018). The methylation analysis showed no trace of pyruvic acid groups,
Response (AU)

1.5
1.0 D-GlcA
0.5
0.0
0 10 20 30
Time (min)
Fig. 2. (a) FTIR spectroscopies of the bioflocculant purified by the papain and
Savag method (black) and the heat-alkaline method (red), the peaks of acyls
groups were removed in the latter. (b) HPLC spectroscopy of monosaccharide
standards. (c) HPLC spectroscopy of the bioflocculant. (For interpretation of the
references to color in this figure legend, the reader is referred to the web
version of this article.)
an Mw of 1.718 × 106 Da (Boukhelata et al., 2019). A strain of Paeni­
bacillus jamilae produced two kinds of exopolysaccharide, and the larger
group was estimated to be more than 2 × 107 Da (Morillo et al., 2007).
High molecular weight polymers were reported to show a good floccu­
lating rate by bridging mechanism (Razali et al., 2011). Longer chains
tend to form more loops and tails when they bind particles, so loops tend
to extend further and more likely attach to a second particle, leading to
bridging between particles.
The FTIR spectrums of polysaccharides purified by different methods
were shown in Fig. 2a. They were typical of carbohydrate polymer, with
the characteristic broad absorption peak of hydroxyl groups
(3000–3700 cm− 1), weak peak of C–H stretching band of CH, CH2, and
because the sample was partly hydrolyzed for improving dissolution in
D-Gal DMSO, which also cleaved the pyruvic acid (Kohno et al., 2009).
The structure of the polysaccharide was established based on 1D (13C
D-Glc in Fig. 3a, DEPT-135 in Fig. 3b, and 1H in Fig. 3c), 2D homonuclear (1H,
1
H COSY in Fig. 4a, TOCSY in Fig. 4b, and NOESY in Fig. 4c) and het­
eronuclear (1H, 13C HSQC in Fig. 4d and HMBC in Fig. 4e) NMR spectra.
As shown in Fig. 3A and B, most carbon and proton signals of the
polysaccharide were obtained in 60–110 ppm and 3.5–5.50 ppm
1 respectively. The 1H NMR spectrum shows anomeric proton signals at
40 5.2–4.4 ppm as well as the 13C NMR spectra showed anomeric carbon
signals at 102–106 ppm. Corresponding with the HSQC spectrum, six
anomeric signals were labeled with a capital letter (A–F) and determined
anomeric configuration by chemical shift. Then these hexapyranose spin
systems were distinguished according to cross-peaks in 2D NMR and
accurately assigned chemical shifts of each glycoside were shown in
Table 2. In addition to the above range, the 13C NMR spectra show peaks
of carboxyl groups in 175–185 ppm, which were greatly considered for
helping flocculation with the cation bridging effect (Bratby, 2016;
Kaarmukhilnilavan et al., 2020; Nontembiso et al., 2011). The peak at
179.30 ppm was assigned to C6 of GlcA, the peak at 178.66 ppm was
assigned to the carboxylic acid of pyruvate residue, and the peaks at
183.98 ppm and 178.34 ppm were assigned to succinyl residue. The
peaks at 1.48–27.89 ppm and 103.63 ppm, and the peaks at 2.51/34.76
ppm and 2.66/33.26 ppm were assigned to other atoms of succinyl
residue. The position of the pyruvate group was determined by the
downfield chemical shift of F4 and F6 (Shashkov et al., 2021). All the
above data indicated the polysaccharide composed of hexasaccharide
repeating units with the following structure: →3)-α-D-Manp-(1 → 3)-4-
Suc-β-D-Manp-(1 → 3)-β-D-Glcp-(1 → 4)-β-D-GlcpA-(1 → 4)[4,6-Pyr-β-D-
Galp-(1 → 3)]-β-D-Galp-(1→. The Haworth structure of the

4
S. Chen et al.
a Pyr
FE DA BC
Pyr
C6
Suc
110 108 106 104 102
200 150
200 150
A B C DEF
5 4
13
Fig. 3. The C (a), DEPT-135 (b), and 1H (c) NMR spectrum of the bioflocculant.
polysaccharide was shown in Fig. 4f. Therefore, the polysaccharide was
named floccuronic acid.
3.3. Optimization of the flocculation parameters
The effects of various cations, pH, and dosage were tested in kaolin
suspension. As shown in Table S1, the floccuronic acid displayed the
highest flocculation activity with Ca2+ and Fe2+, reduced value with
trivalence cations (Al3+ and Fe3+) and lowest value with Mg2+ and
monovalent cations (Na+, K+, and Li+). The reason for improved effi­
ciency with Ca2+ and Fe2+ can be attributed to neutralizing and stabi­
lizing the residual negative charge of functional groups and thereby
forming bridges between particles described above (Nontembiso et al.,
2011). Besides the cation bridging effect, cations can reduce the size of
the polyelectrolyte coil, thus helping more polyelectrolyte chains to be
accommodated on the particle surface (Bratby, 2016). The flocculation
efficiency of floccuronic acid to kaolin solution at different pH was
measured and results were depicted in Fig. 5 (a–e). Floccuronic acid
showed a fast and high removal rate from pH 4 to 10. A higher dose of
floccuronic acid shows a shorter flocculation time, but the highest
Carbohydrate Polymers 291 (2022) 119561
Pyr
100 50 [ppm]
F6
100 50 [ppm]
Pyr
3 2 [ppm]
turbidity removal rate (99.8%) was achieved with 1 ppm of floccuronic
acid in acidic conditions. The measured zeta potential value of kaolin
suspension with CaCl2 was − 18.7 mV at pH 7 and this value decreased to
− 4.2 mV at pH 4, as shown in Fig. 5f. Negatively surface charge of kaolin
was acknowledged to undergo partial charge neutralization with posi­
tively charged acidic ions, which make kaolin unstable in acidic con­
ditions (Black & Walters, 1964; Thombare et al., 2017). Noticeably zeta
potential with floccuronic acid showed higher negative values in acidic
conditions and no obvious change with floccuronic acid in alkaline
conditions. This can be interpreted by the carboxylic acid content in
floccuronic acid. Therefore, the high flocculation activity of floccuronic
acid has little relation with charge neutralization and electrostatic patch
mechanism, which occurs when charged flocculant is absorbed to the
colloidal particle surface with the opposite sign and cause destabiliza­
tion of particles (Salehizadeh et al., 2018).
3.4. Flocculation performance with coal wastewater
Coal washing wastewater is the tailwater in coal processing and can
lead to serious-environment pollution (Lemly, 2018). So we use it as a
5
S. Chen et al. Carbohydrate Polymers 291 (2022) 119561

a b

F1 [ppm]

F1 [ppm]
F6

F1 [ppm]
csjy 37 1 "D:\Program install free\bruker2.1" exam

F1
28

34 32 30
Pyr

30
A2,3 Suc

32
D2,3

4.0

4.0
B4

34
2.6
2.6 2.4 2.2

2.2
2.0 1.8

1.8
1.6 F2 [ppm]

F2 E4 A3

B3 C4+E3

4.5

4.5
E1,2 D3
F1,2
D1,2 C1
B1
A1 D1
E1

5.0

5.0
F1

5.0 4.5 4.0 F2 [ppm] 5.0 4.5 4.0 F2 [ppm]

c F1 [ppm] d

F1 [ppm]
B2,5

B2,3
4.0

4.0
A1B3
B2,4 B1D3
E1A3
E1,2
4.5

4.5
F1 [ppm]

182 178 174 F1

csjy 39 1 "D:\Program install free\bruker2.1" exam

D1,3
172

D1,5 Suc
174

D1,2 Pyr
176
178
180
5.0

5.0
182

A1,5 A1,3 A1,4

184

3.5 3.0 2.5 2.0 F2 [ppm]

3.5 3.0 2.5 2.0 F2

5.0 4.5 4.0 F2 [ppm] 5.0 4.5 4.0 F2 [ppm]

e f
F1 [ppm]

CH2OH CH2OH CH2OH COOH CH2OH

O O O O O O
4.0

O O O
A OH B OH D O HO C E
O
OH O OH

F1E4 Suc
OH OH OH n
O
4.5

O
D1C3 Pyr
O O
B1D3 B1D2
HO F

A1B3
5.0

A1B2 A1B4
OH

5.0 4.5 4.0 F2 [ppm]

Fig. 4. The 2D NMR spectroscopy and the predicted structure of the bioflocculant. (a) COSY. (b) HSQC. (c) TOCSY. (d) HMBC. (e) NOESY. (f) Haworth structure of
the bioflocculant. A–F labeled in these spectra represent the residues corresponding to Table 2.

6
S. Chen et al. Carbohydrate Polymers 291 (2022) 119561

Table 2 model to test the performance of floccuronic acid with industry waste­
The 1H NMR and 13C NMR shifts (ppm) of the bioflocculant. Bold values water. As shown in Fig. 6a, the highest turbidity removal rate (98.8%)
represent glycosylation sites. was reached with 1 ppm floccuronic acid in coal-washing wastewater.
Residues Chemical shifts (ppm) The flocculation accelerates with a higher dose of floccuronic acid
H1/C1 H2/C2 H3/C3 H4/C4 H5/ H6/C6
without an obvious change in turbidity removal rate. The negative zeta
C5 potential value of treated wastewater illustrated in Fig. 6c tended to
increase initially, indicating the same conclusion as mentioned above.
[A] → 3)α-D- 5.16 4.20 3.76 3.65 4.11 4.20
Manp(1→ 104.23 73.59 79.59 68.87 75.69 73.59 However, zeta potential showed a lower negative value with 2 ppm
[B] → 3)4-Suc- 4.94 4.28 3.88 3.65 3.50 3.75 floccuronic acid, implying the excess floccuronic acid was also attached
β-D-Manp(1→ 103.05 73.69 83.89 68.80 79.00 63.90 in flocs and caused the faster settling speed. The size and structure of
[C] → 4)β-D- 4.63 3.49 4.06 3.71 3.80 – flocs are considered fundamental in industrial wastewater treatment, as
GlcpA(1→ 102.46 75.26 72.95 83.03 79.17 179.30
[D] → 3)β-D-Glcp 4.54 3.43 3.75 3.44 3.52 3.98
large compact flocs can settle fast in low turbidity wastewater and
(1→ 104.86 75.95 86.90 71.37 78.12 63.13 porous flocs can aid filtration (Fan et al., 2020; Saxena & Brighu, 2020).
[E] → 3,4)β-D- 4.47 3.66 3.71 3.78 3.80 3.78 The surface morphology of flocs was observed and shown in Fig. 6b.
Galp(1→ 105.71 73.38 83.03 78.99 79.17 63.85 Compared to different doses of floccuronic acid, flocs became larger
[F] T-4,6-Pyr-β-D- 4.44 3.56 3.92 3.96 3.65 4.03,
with a higher dose, and reached a peak with 2 ppm floccuronic acid
Galp(1→ 3.97
105.85 78.01 74.67 71.45 68.87 67.76 (0.357 mm). Fractal dimension showed high values of 1.80 and 1.81 at
Succinyl 2.66 2.51 – – 0 and 0.5 ppm respectively. However, these compact flocs are caused by
33.26 34.76 178.34 183.98 small size and imply insufficient floccuronic acid cannot bind all parti­
Pyruvate 1.48 – – cles, as small flocs like non-treated wastewater can be found in waste­
27.89 103.63 178.66
water treated with 0.5 ppm floccuronic acid. The lowest fractal

a 100 b 100
Turbidity removal (%)
Turbidity removal (%)

80 80

60 60

40 40

20 20

0 0
0.5 1 2 3 4 5 0.5 1 2 3 4 5
Time (min) Time (min)

c 100 d 100
Turbidity removal (%)
Turbidity removal (%)

80 80

60 60

40 40

20 20

0 0
0.5 1 2 3 4 5 0.5 1 2 3 4 5
Time (min) Time (min)

e 100 f -2 0 ppm
1 ppm
Turbidity removal (%)

80 -6
Zeta potential (mV)

5 ppm
60 -10

40 -14

20 -18

0 -22
0.5 1 2 3 4 5 4.0 5.5 7.0 8.5 10.0
Time (min) pH

Fig. 5. Flocculation abilities of floccuronic acid with kaolin suspension at pH 4 (a); pH 5.5 (b); pH 7 (c); pH 8.5 (d); pH 10 (e). Comparative zeta potential at different
doses and pH (f).

7
S. Chen et al. Carbohydrate Polymers 291 (2022) 119561

a b
0 0.1 0.5 2 (min)

0 0

200μm

0.5 0.5

1 1

2 (ppm) 2 (ppm)

c d
0.8 Fractal dimension Median Line 1.9
100 0.5 min -18
2 min Range within 1.5IQR Outliers
25%~75% Mean
-20 1.8
Turbidity removal (%)

80
Fractal dimension

0.6
Zeta potential (mV)

Floc size (mm)

-22
60 1.7
-24 0.4
40 1.6
-26
0.2
20 1.5
-28

0 -30 0.0 1.4

0 0.5 1 2 0 0.5 1 2
Dose (ppm) Dose (ppm)

Fig. 6. Flocculation abilities of different doses of floccuronic acid with coal wastewater: (a) photographs of coal wastewater with varying settling times; (b) optical
images of flocs; (c) turbidity removal of the wastewater; (d) particle size and fractal dimension of the wastewater.

8
S. Chen et al. Carbohydrate Polymers 291 (2022) 119561

a
0.5 1 2 3 4 5 (min)

0.05

0.5

1 (ppm)

b c
0.5 1 2 3 4 5 min Fractal dimension Median Line
100 -8 0.8 Range within 1.5IQR Outliers 2.0
ZP
25%~75% Mean

80 -10
Turbidity removal (%)

Zeta potential (mV)

0.6 1.8

Fractal dimension
Floc size (mm)

60 -12
0.4 1.6
40 -14

0.2 1.4
20 -16

0 -18 0.0 1.2

0 0.05 0.5 1 0 0.05 0.5 1
Dose (ppm) Dose (ppm)
d
0 0.05

200μm

0.5 1 (ppm)

Fig. 7. Flocculation abilities of different doses of floccuronic acid with high-turbidity drinking water: (a) photographs with varying settling times; (b) turbidity
removal of the water sample; (c) particle size and fractal dimension of the water sample; (d) optical images of flocs.

9
S. Chen et al.
Table 3
The flocculation ability of floccuronic acid in various suspensions.
Type of suspension Zeta potential Mean of particle 10th percentile of particle size
(mV) size (nm) distribution (nm)
Coal wastewater − 20.9 1932 615
Kaolin clay − 31.4 1291 396
High-turbidity − 15.8 1208 255
drinking water
dimension value of 1.65 was achieved at 1 ppm, which means most small
flocs were bound and the porous flocs were formed. A further higher
dose of floccuronic acid seemed to form larger and denser flocs with a
fractal dimension of 1.75, thus larger fractal dimension and faster
settling time were reached. As mentioned above, turbidity removal rates
show no obvious difference between 1 and 2 ppm floccuronic acid,
implying the size and compaction of flocs can influence the settling
speed but have little effect on turbidity removal rate.
3.5. Flocculation performance with high-turbidity drinking water
The pH, residual chlorine, and turbidity are the most commonly used
indicators for water quality (Engelhardt, 2010). To remove the turbidity
of potable water, flocculants are widely used in conjunction with a
primary coagulant, as primary coagulant specializes in destabilizing
particles by charge neutralization and flocculant enhance clarified water
quality by better floc characteristics (Zhang et al., 2020). This study uses
a low-dose poly ferric chloride (PFC) of 5 ppm as the primary coagulant.
The coagulation-flocculation performance of PFC and floccuronic acid in
high-turbidity drinking water are shown in Fig. 7. Similar to the result of
kaolin suspension and coal washing wastewater, the flocculation speeds
up with a higher dose of floccuronic acid, and the highest turbidity
removal rate (89.2%) was reached with 0.5 ppm floccuronic acid. The
zeta potential value decreased from − 15.8 mV to − 13.6 mV with PFC,
indicating that the low dose of primary coagulant may only partially
absorb and neutralize particles. Similar to coal wastewater, the slight
amount of floccuronic acid showed a higher negative zeta potential
value, then lower values were achieved with a higher dose of floccuronic
acid. However, the flocs morphology shows a different trend to coal
wastewater. Flocs became larger from 0.05 ppm to 0.5 ppm floccuronic
acid and the largest value was 0.41 mm, then reduced to 0.33 mm at the
dose of 1 ppm, indicating the flocs were breaking down due to large size.
The fractal dimension of the sample with floccuronic acid show values
higher than 1.79, indicating compact flocs were formed with floccuronic
acid, which may cause a faster settling speed than using only primary
coagulant.
4. Conclusion
In this study, a novel bioflocculant floccuronic acid from Paeniba­
cillus sp. M21629 was investigated for its prospective applications in
wastewater and water treatment. The floccuronic acid was characterized
as a high molecular weight polysaccharide with an abundance of
carboxyl groups, which strongly associated with the bridging mecha­
nism of flocculation. The floccuronic acid shows a great flocculation
performance with a large range of pH values. The flocculation ability of
floccuronic acid depended on the instability of solid particles and
required cations or coagulant for relatively stable suspension (the details
were shown in Table 3). This finding indicates floccuronic acid is an
alternative flocculant with a low dose of 0.5 to 1 ppm.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.carbpol.2022.119561.
10
Carbohydrate Polymers 291 (2022) 119561
90th percentile of particle size Requirement
distribution (nm)
3091 No
2305 Require Ca2+ for cation
bridging effect
1990 Require PFC for destability
CRediT authorship contribution statement
Shijunyin Chen: Formal analysis, Investigation, Data curation,
Writing – original draft, Writing – review & editing. Rui Cheng: Vali­
dation, Visualization. Xiaodong Xu: Methodology, Investigation.
Changchang Kong: Methodology, Investigation. Lei Wang: Method­
ology, Investigation. Renjie Fu: Investigation, Data curation. Jing Li:
Methodology, Resources. Shiming Wang: Validation, Resources. Jianfa
Zhang: Conceptualization, Resources, Writing – review & editing, Su­
pervision, Project administration, Funding acquisition.
Declaration of competing interest
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.
Acknowledgments
This work was supported by the National Natural Science Foundation
of China (31871178) and the Fundamental Research Funds for the
Central Universities (30920031102).
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