Chemosphere 206 (2018) 701e708

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Characterization and coagulation-flocculation performance of a

composite flocculant in high-turbidity drinking water treatment
Xiang Xia a, b, c, Shuhuan Lan a, b, Xudong Li a, b, c, Yifei Xie a, b, c, *, Yajie Liang a, b, c,
Peihan Yan a, b, c, Zhengyang Chen d, Yunxiao Xing e
a
Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, 610041, Chengdu, PR China
b
Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, 610041, Chengdu, PR China
c
University of Chinese Academy of Sciences, 100049, Beijing, PR China
d
Key Laboratory of Synergetic Control and Joint Remediation for Soil & Water Pollution (SEKL-SW), Chengdu University of Technology State Environmental
Protection, Chengdu University of Technology, 610059, Chengdu, PR China
e
College of Chemistry and Materials Science, Sichuan Normal University, 610066, Sichuan, PR China

h i g h l i g h t s

A novel MBF mainly composed of galactan was first proposed in Klebsiella genus.

A new type of composite flocculant (CF) containing MBF and PAFC was prepared.

The CF could reduce the dosage of PAFC by 56e72% in high-turbidity drinking water.

Flocculation mechanism of high turbidity removal capacity was speculated.

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

Article history: Klebsiella variicola B16, a microbial bioflocculant (MBF-B16)-producing bacteria, was isolated and iden-
Received 10 January 2018 tified by its 16S rRNA sequence, biochemical properties, and physiological characteristics. The effects of
Received in revised form culture conditions on MBF-B16 production, including carbon source, nitrogen source, C/N ratio, initial pH,
23 April 2018
and culture temperature, were investigated in this study. Results showed that 6.96 g of MBF-B16 could be
Accepted 26 April 2018
extracted from a 1-L culture broth under optimized conditions. Chemical analysis showed that poly-
Available online 30 April 2018
saccharide and protein were the main components. The neutral sugar consisted of galactose only, which
Handling Editor: W. Mitch was proposed in Klebsiella genus for the first time. In addition, a composite flocculant (CF) that contains
polyaluminum ferric chloride (PAFC) and MBF-B16 for the removal of turbidity and SS in drinking water
Keywords: was optimized by response surface methodology. CF could reduce PAFC dosage by about 56.2e72%.
Microbial bioflocculant Charge neutralization and adsorption bridging effect were the primary flocculation mechanisms.
Klebsiella variicola B16 © 2018 Elsevier Ltd. All rights reserved.
Composite flocculant
Response surface methodology

1. Introduction such treatments would lead to secondary pollution (Okaiyeto et al.,

Water is of vital importance to the sustainable development of
mankind. Sometimes, the turbidity of drinking water is relatively
high, and coagulation/flocculation technology, in which coagulants
such as alum are added, is a common method to purify drinking
water worldwide (Mohtar et al., 2017). However, the sludge from
* Corresponding author. Key Laboratory of Environmental and Applied Microbi-
ology, Chengdu Institute of Biology, Chinese Academy of Sciences, 610041, Chengdu,
PR China.
E-mail address: xieyf@cib.ac.cn (Y. Xie).
2015a). At present, flocculant use is prevalent in various industries,
such as drinking water purification, downstream applications in
food and fermentation processing, textiles, and wastewater treat-
ment (Liu et al., 2013; Li et al., 2015; Yan et al., 2015). Flocculants
can be classified into three groups: chemical flocculants, such as
ferric chloride and aluminum sulfate; chemically synthetic floccu-
lants, such as polyacrylamide (PAM); and bioflocculants (Liu et al.,
2010). Chemical flocculants are widely used because they are
cost-effective and highly efficient; however, their use has become
limited due to health and environmental concerns (Sun et al.,
2015b).

https://doi.org/10.1016/j.chemosphere.2018.04.159
0045-6535/© 2018 Elsevier Ltd. All rights reserved.
702 X. Xia et al. / Chemosphere 206 (2018) 701e708
In contrast, bioflocculants have received considerable attention
recently because they are usually biodegradable, nontoxic (Deng
et al., 2003), and not associated with secondary pollution (Zheng
et al., 2008). Bioflocculants are natural organic macromolecules
produced and secreted by microorganisms during active meta-
bolism and cell lysis. These molecules can flocculate suspended
solids (SS), cells, and colloid materials (Lian et al., 2008). The most
common backbones of these macromolecules are polysaccharides,
poly-g-glutamic acid (g-PGA), proteins, and lipids (Guo et al., 2014;
Liu et al., 2015a; Yan et al., 2015). Previous research has reported
that a diversity of microorganisms produce these macromolecular
substances, such as bacteria, fungi, and algae (Gong et al., 2003;
Aljuboori et al., 2015; Tiwari et al., 2015). Low flocculation effi-
ciency and high production costs are the primary impediments for
the practical application of bioflocculants (Sun et al., 2015a). Pre-
vious studies have mostly focused on screening new isolates and
optimizing culture conditions for high yield production (Xia et al.,
2008; Aljuboori et al., 2013; Liu et al., 2013). Thus, it is important
to isolate microorganisms that have a high production yield,
thereby reducing the cost.
Alternatively, the use of a composite flocculant, which contains
both chemical and bioflocculants, can overcome the weaknesses of
the individual flocculants while decreasing the production cost and
increasing the flocculation efficiency (Sun et al., 2015a). Yang et al.
(2009) reported that a composite containing polyaluminum chlo-
ride (PAC) and microbial bioflocculant (MBF) achieved desirable
flocculating activity when treating a kaolin suspension. Sun et al.
(2015b) reported that a composite composed of extracellular
polymeric substance (EPS) and CaCl2 showed a high flocculation
efficiency in a kaolin and Microcystis aeruginosa suspension.
Furthermore, composite flocculants reduce chemical input into the
water, thereby reducing the health and environmental risks. Bio-
flocculant was combined with poly(acrylamide[2-(meth-
acryloyloxy)ethyl]-trimethylammonium chloride (P(AM- DMC)) in
order to decrease the chemical dosage, which ultimately enhanced
the sludge dewatering process (Guo et al., 2015a). Sun et al. (2015a)
observed that a chemical and bioflocculant composite for the pur-
pose of M. aeruginosa removal achieved a high flocculating activity
at a low component dosage. To date, a composite flocculant
including bioflocculant for the treatment of drinking water has
never been reported.
In the present study, we isolated a bioflocculant-producing
strain, Klebsiella variicola B16, from activated sludge. The optimal
culture conditions and medium components were investigated.
Response surface methodology (RSM) was employed to optimize
the composite flocculant (CF) variables for the treatment of high-
turbidity drinking water, and the flocculation mechanism
involved was investigated. The results of our study provided a new
way to reduce the chemical flocculant utilization for the treatment
of drinking water.
2. Materials and methods
2.1. Isolation and identification of a microbial bioflocculant-
producing strain
Microbial bioflocculant (MBF)-producing strains were isolated
from soil and activated sludge samples. Samples (10 g) were diluted
with sterile water (90 mL), thoroughly stirred, and then let to rest
for 1 min. The solutions were aliquoted onto a screening medium
composed of yeast extract (5.0 g L1), peptone (10.0 g L1), NaCl
(5.0 g L1), and agar (20.0 g L1). Selected isolates were inoculated
into an Erlenmeyer flask (250 mL) containing production medium
(50 mL) and incubated at 30  C on a rotating shaker (160 rpm) for
48 h. The composition of the production medium (pH 7.0) was as
follows: glucose (10 g L1), urea (0.5 g L1), yeast extract (0.5 g L1),
K2HPO4 (5 g L1), KH2PO4 (2 g L1), MgSO4 (0.2 g L1), and NaCl
(0.1 g L1) (Xia et al., 2008). The 16S rRNA gene fragment of the
selected strain was amplified by colony PCR. The PCR was per-
formed using forward primer 27F (50 -GAGAGTTTGA
TCCTGGCTCAG-3 ) and reverse primer 1492R (50 -CTACGGC-
0
TACCTTGTT ACGA-30 ) (Liu et al., 2010). The PCR products were
sequenced by Sangon Biotech Co. Ltd (Shanghai, China). Sequencing
results were compared with the 16S rRNA sequences available in
the NCBI Genbank database.
2.2. Production and purification of MBF-B16
The activated strain B16 was inoculated (2.5 mL) into an Erlen-
meyer flask (250 mL) containing the production medium (50 mL)
and cultured at 30  C on a rotating shaker (160 rpm) for 48 h. Cells
were removed from the culture broth by centrifugation (3000g,
20 min); the resulting supernatant contained MBF-B16 produced
by strain B16. To precipitate the bioflocculant, the supernatant was
mixed with 3 vol of cold anhydrous ethanol and then stored at 4  C
overnight. The resulting precipitate was collected by centrifugation
(3000g, 10 min) and lyophilized. The crude MBF-B16 (1 g) was
dissolved in distilled water (100 mL) and then thoroughly mixed
with 2% cetylpyridinium chloride (75 mL) for 2 h. The precipitate
was collected by centrifugation and then dissolved in 0.5 M NaCl
solution (50 mL). Three volumes of ethanol were added to recover
the MBF-B16, which was washed twice using 75% ethanol and then
re-dissolved in distilled water. To obtain the purified form, MBF-
B16 was dialyzed against distilled water overnight and then
lyophilized (Aljuboori et al., 2013).
2.3. Measurement of flocculating activity
The flocculating activity of MBF-B16 was determined using the
kaolin suspension model (Liu et al., 2015a). A kaolin suspension
(5 g L1) was subjected to high-speed revolution for 5 min. The pH
of the solution was then adjusted to 7.0e7.2 using NaOH and HCl.
The kaolin suspension (20 mL) was amended with CaCl2 (10 g L1;
1 mL) and culture broth (0.25 mL), slightly mixed, and then let to
rest for 5 min. The optical density of the supernatant was deter-
mined at 550 nm (OD550) using UVevisible spectrophotometer
(TU-1901, Beijing Purkinje General Instrument Co. LTD, China).
Fresh medium was used as a blank control. The flocculating activity
was calculated according to the following equation:
Flocculating activity ¼ (A  B)/A  100% (1)
where A and B are the OD550 of the control and sample,
respectively.
2.4. Jar test
High-turbidity drinking water was prepared by the addition of
100 g of soil to 1 L tap water, vigorously mixing the mixture over-
night, and then let to rest for 2 h. An aliquot of 50 mL of the test
sample was added into a beaker. First, the water sample was mixed
for 5 min. Then, one portion of the coagulant was added into the
beaker, followed by the addition of the other portion after 30 s.
After that, the sample was stirred for 4.5 min and settled for 20 min.
Finally, the supernatant was collected and measured using a scat-
tering light turbidimeter (WGZ-1A, Shanghai Xin rui Instruments &
meters Co. Ltd, China). Controls were conducted the same way as
both experimental groups, without the use of either coagulant or
composite flocculant.
Suspended solids (SS) is measured using equation (2):
 X. Xia et al. / Chemosphere 206 (2018) 701e708 703

SS ¼ m/V*1000 (2) indicated that several microorganisms that produced extracellular

where m is the weight of filter cake of supernatant after drying at
105  C for 8 h and V is the volume of the supernatant.
2.5. Characteristics of the bioflocculant
biopolymer flocculants belonged to the Klebsiella genus (Wang
et al., 2007; Yin et al., 2014).
3.2. Optimal medium and culture conditions for MBF-B16
production

The total sugar content of the microbial bioflocculant was 3.2.1. Effects of carbon sources and nitrogen sources on MBF-B16
determined by the phenol-sulfuric acid method (Chaplin and production
Kennedy, 1994). The protein content of the flocculant was The sources of carbon and nitrogen play a vital role in bio-
measured by the Coomassie brilliant blue G-250 dye method with flocculant production because microbial growth and metabolism
bovine serum album as the standard (Bradford, 1976). The uronic are significantly influenced by medium nutrients. The effects of
sugar, neutral sugar, and amino sugar were measured using the various carbon sources on MBF-B16 production were monitored
standard method (Giri et al., 2015). To determine the sugar (Fig. 1A). Soluble starch, sucrose, glucose, mannitol, maltose,
composition of the flocculant, it was hydrolyzed with 2 M tri- ethanol, and lactose were all favorable carbon sources for MBF-B16
fluoroacetic acid at 121  C for 2 h. The resulting sugar was analyzed production; however, sodium citrate did not allow for favorable
by high-performance liquid chromatography with a C18 column biopolymer production. These results suggested that the B16 strain
(4.6  150 mm, GL sciences lnc, Japan) (Xia et al., 2018). A Fourier could make use of extensive carbon sources to produce bio-
transform infrared spectrometer was used in the frequency wave flocculant. A similar result was reported by Liu et al. (2010), in
range of 4000e400 cm1 (Spectrum, PerkinElmer, America). which the B16 strain could utilize mannose, glucose, maltose,
ethanol, and arabinose for bioflocculant production. In this study,
2.6. Optimization of culture conditions for MBF-B16 production the highest MBF-B16 production was achieved in glucose medium;
hence, glucose was selected as the carbon source for all subsequent
Many factors affect MBF-B16 production; however, cultivation
conditions and medium components are the most important (Liu
et al., 2013). The effect of various carbon sources on bioflocculant
production was investigated by replacing glucose with soluble
starch, sucrose, mannitol, sodium citrate, maltose, ethanol, and
lactose (at 10 g L1 each). Similarly, the effect of various nitrogen
sources was tested by replacing yeast extract þ urea with NaNO3,
urea, (NH4)2SO4, NH4NO3, ammonium acetate, peptone, and yeast
extract (at 1 g L1 each). The initial pH of the medium was adjusted
from 3.0 to 12.0, and the temperature was adjusted from 10 to
45  C. To determine the optimal incubation time, MBF-B16 pro-
duction was monitored between 0 and 144 h.

2.7. Experimental design using response surface methodology

In this study, statistical analyses were performed using Design

Expert (v. 8.0.6), and the Box-Behnken design (BBD) was employed
to optimize the independent variables. We investigated the in-
teractions between the independent variables at three different
levels (low (1), medium (0), high (þ1)) (Wang et al., 2015a). The
response variables were fitted using a second-order model con-
taining the independent variables studied in the form of a quadratic
polynomial equation:
P P 2
Y ¼ b0 þ biXi þ bijXiXj þ Xi (3)

where Y is the predicted response, b0 is the intercept coefficient, bi

is the linear coefficient, bii is the quadratic coefficient, bij is the term
that reflects the interaction between Xi and Xj, and Xi and Xj are the
input variables that influence the response variables.

3. Results and discussion

3.1. Identification and flocculating activity of strain B16

A total of 200 strains were isolated from soil and activated

sludge samples. Nine strains demonstrated flocculating activity
above 80%, while the flocculating activity of B16 exceeded 90%. On
the basis of the morphology, biochemical properties, and 16S rRNA
Fig. 1. Effects of carbon and nitrogen sources on flocculating activity. (A) Effects of
sequences, the strain was identified as K. variicola. Therefore, this carbon sources on cell growth and flocculating activity; (B) Effects of nitrogen sources
strain and its compound biopolymer flocculant were named as on cell growth and flocculating activity. Flocculating activity (blank columns), cell
K. variicola B16 and MBF-B16, respectively. Previous research growth (dark columns).
704 X. Xia et al. / Chemosphere 206 (2018) 701e708
tests. In addition, the effect of nitrogen sources on MBF-B16 pro-
duction was examined when glucose (10 g L1) was supplied as the
carbon source (Fig. 1B). Among the nitrogen sources studied, all
inorganic nitrogen led to high flocculating activities and good cell
growth. The highest flocculating activity of MBF-B16 was obtained
with (NH4)2SO4 as the nitrogen source. Conversely, organic nitro-
gen sources resulted in poorer biopolymer flocculant production
and higher cell growth. Therefore, we concluded that inorganic
nitrogen sources were better for MBF-B16 production than organic
nitrogen sources. In previous studies, similar phenomena have
been reported. For example, a study by Okaiyeto et al. reported that
(NH4)2SO4, NH4NO3, and NaNO3 were favorable for bioflocculant
production by B. toyonensis AEMREG6, when compared to organic
nitrogen sources (Okaiyeto et al., 2015a). In contrast, Liu et al.
(2015a) observed that organic nitrogen sources were more suit-
able than the inorganic sources for bioflocculant production.
3.2.2. Effects of temperature and initial pH on MBF-B16 production
Temperature is one of the most important factors that influence
both cell growth and bioflocculant yield (Giri et al., 2015). Culture
temperatures of 20  C and above promoted flocculating activity
with the highest activity observed at 30  C; hence, 30  C was used
in the following experiments (Supplementary Fig. S1). At temper-
atures above 40  C, the flocculating activity began to decrease.
These higher temperatures would be harmful to the nucleic acid
and enzymatic systems of the bacteria, thereby affecting MBF-B16
production. Alternately, at lower temperatures, the enzymatic
systems associated with bioflocculant production might not be
fully activated (Xia et al., 2008).
The effects of initial pH of the medium within the range of
3.0e12.0 on MBF-B16 production were investigated. MBF-B16
production occurred within the initial pH range of 7.0e9.0 and
the final pH range of 7.09e8.22, which suggested that K. variicola
B16 was an alkaline-tolerant strain (Supplementary Fig. S2). The
maximum flocculating activity was achieved at an initial pH of 7.0;
this agrees with the finding of a previous study by Liu et al. (2015b).
On the basis of a research reported by Patil et al. (2011), the initial
medium pH could influence the electric charge of cells and the
redox reactions, which impacted the nutrient adsorption and
enzymatic reactions of bioflocculant production.
3.2.3. MBF-B16 production over time
MBF-B16 production varied with the growth curve of strain B16
over a cultivation time of 192 h. As shown in Fig. 2, MBF-B16 pro-
duction reflected the biomass growth, reaching its maximum
Fig. 2. Time course of MBF-B16 production.
production yield in the late logarithm phase (6.96 g L1 at 24 h).
This implied that MBF-B16 was formed during cellular growth; in
other words, MBF-B16 production was not due to cell autolysis
(Ugbenyen et al., 2012). MBF-B16 production was very high in the
initial 24 h, when the cell growth was also high. The cell growth
reached the stationary phase between 24 and 48 h of cultivation
time, after which the growth and flocculating activity declined. The
decrease in flocculating activity after 48 h might also be caused by
the production of deflocculating enzymes or because the bio-
flocculant was consumed by the strain due to lack of food (Li et al.,
2009). Many papers have reported that bioflocculants were
collected during the latter log phase or the early stationary phase.
For example, the maximum flocculating activity of bioflocculant
secreted by B. subtilis F9 (Giri et al., 2015) and P. mirabilis TJ-1 (Liu
et al., 2013) was achieved during the late logarithmic and early
stationary phases, respectively. In this study, the highest produc-
tion yield of MBF-B16 was achieved at 24 h, which was selected for
the following experiments. This was a desirable feature, as less
fermentation time was preferred in industrial applications
(Okaiyeto et al., 2015a). Additionally, the pH profile of the medium
initially decreased from 7.89 to 6.51 after 12 h, suggesting that the
secreted MBF-B16 decreased the pH of the medium; however, after
36 h, it increased to 6.91 and then stabilized for the remainder of
the fermentation period.
A relatively high production yield of 6.96 g could be obtained
from a 1-L fermentation broth under the optimized conditions,
when compared to other published bioflocculant yields of
0.4e4.65 g L1 (Lu et al., 2005; Zheng et al., 2008; Aljuboori et al.,
2013; Giri et al., 2015; Liu et al., 2015a; Okaiyeto et al., 2016);
however, higher bioflocculant yields of 16.55 g L1 and 10 g L1
from B. licheniformis and B. firmus, respectively, were achieved
when the two strains were cultured in 50 g L1 glucose medium
(Devi and Natarajan, 2015). These higher bioflocculant production
yields required higher nutrient concentrations and lower yield ef-
ficiency, when compared to the present study, which could nega-
tively affect its practical application.
3.3. Characteristic analysis of MBF-B16
3.3.1. Characterization of MBF-B16
Chemical analysis showed that the bioflocculant was composed
of 68.1% polysaccharide and 8.4% protein. Further analysis of the
flocculant showed that the polysaccharide was neutral sugar
(62.1%) and uronic acid (4.8%). The component of the neutral sugar
was galactose only which has never been reported in Klebsiella
genus (Zhao et al., 2013).
3.3.2. Fourier transform infrared analysis
The functional groups of partially purified flocculants were
analyzed by FTIR spectroscopy. The intense adsorption peak at
3357 cm1 could be attributed to the vibration of hydroxyl and
amino groups (Cao et al., 2015). A weak adsorption peak at
2930 cm1 indicated the presence of CeH. Bands at 1652 and
1534 cm1 represented the functional group of carbonyl. The weak
symmetrical peak at 1401 cm1 indicated the presence of carboxyl
group. The adsorption peak at 1069 cm1 was the characteristic of
methoxyl groups (Sun et al., 2015a). The peaks between 1000 and
1100 cm1 were the results of all sugar derivatives (Sun et al.,
2015b). In the previous paper, the FTIR spectrum showed that the
amino, hydroxyl, carbonyl, and methoxyl groups were the major
functional groups associated with flocculation (Supplementary
Fig. S3) (Okaiyeto et al., 2015b).
 X. Xia et al. / Chemosphere 206 (2018) 701e708 705

3.4. Application of CF
3.4.1. Preparation of CF by RSM for drinking water
Chemical analysis showed that purified MBF-B16 was composed
of polysaccharides and proteins. To understand the effects of pol-
yaluminum ferric chloride (PAFC) dosage, MBF-B16 dosage, and pH
values on the response variables (Turbidity and SS, Y1 and Y2), a
standard 3-factor-3-level BBD analysis was adopted to optimize the
independent variables in an effort to lower the turbidity and SS of
high-turbidity drinking water. Statistical testing of the two models
was carried out using the Fisher's test for the ANOVA (Guo et al.,
2015b). Results of the ANOVA in terms of the independent
turbidity and SS variables indicated that the two quadratic poly-
nomial models were significant at a 95% confidence level, with
values of “P-value > F” less than 0.05.
The following equation represents the second-order polynomial
relationship between Y1 and the three variables:
Y1 ¼ 14.627 þ 0.426X1  0.020X2  7.317X3  0.004X1X2
 0.214X1X3 þ 0.011X2X3 þ 0.009X21 þ 0.003X22 þ 1.537X23
where Y1 is the predicted turbidity, and X1, X2, and X3 are the PAFC
dosage (mg L1), MBF-B16 dosage (mg L1), and pH value,
respectively.
The R2 (0.9789) and adjusted R2 (0.9518) values suggested good
agreement between the experimental and predicted values, which
reflected the accuracy and ability of RSM to optimize the floccula-
tion process for turbidity removal (Sun et al., 2015b). The adjusted
R2 value of 0.9518 suggested that 95.18% of the total variation in the
flocculation process was ascribed to the independent variables and
that only 4.82% of the total variation could not be explained by this
model. The AP value of 20.65 was desirable. A “Pro > F” value less
than 0.05 is significant. In this case, X1, X3, X1X2, X1X3, and X21 were
found to be significant in the model (Table 1).
The following quadratic equation represents the empirical
relationship between the response Y2 (SS) and the three factors:
Y2 ¼ 24.597 þ 0.378X1  0.366X2 þ 6.953X3  0.004X1X2
þ 0.196X1X3 þ 0.030X2X3 þ 0.008X21 þ 0.006X22 þ X23
adjusted R2 indicates that 97.38% of the total variation in the floc-
culation process was ascribed to the independent variables, with
only 2.62% of the total variation not explained by this model. In
conclusion, the mathematical model was suitable for the experi-
mental SS removal values of the new composite because the SS, X1,
X2, X3, X1X2, X1X3, X21, and X22 were significant in the model
(Supplementary Table S1).
3.4.2. Coagulation performance in drinking water
On the basis of our results, the relationship between turbidity
and SS was linear, with a correlation coefficient of 0.9357; there-
fore, only turbidity removal was investigated in the following ex-
periments. According to the results of RSM, the optimal condition
was 89.40 mg L1 of PAFC, 37.38 mg L1 of MBF-B16, and a pH of
8.44 at a turbidity of 1100 NTU. Fig. 3 shows the effect of coagulant
dosage on the turbidity removal in the treatment of drinking water.
Regardless of the turbidity level, the residual turbidity of CF (the
ratio of PAFC to MBF-B16 was 2.39:1) showed the same trend as
(4) that of PAFC alone, which showed that PAFC was dominated in the
coagulation-flocculation (Huang et al., 2015b). In the treatment of
drinking water with a turbidity of 3000 NTU, the residual turbidity
decreased as the CF dosage increased from 0 to 90 mg L1 and
decreased slightly in the range of 90e150 mg L1. The residual
turbidity of PAFC reduced from 0 to 200 mg L1 with the minimum
value at 200 mg L1. The residual turbidity increased slightly when
the dosage of PAFC was greater than 200 mg L1 for the reason that
excessive coagulants might have intertwined when the dosage
level was too high, thus resulting in a lower removal capacity. For
CF, the residual turbidity could reach 4.9 NTU when the concen-
tration was set as 80 mg L1 (56 mg L1 of PAFC and 24 mg L1 of
MBF-B16). For PAFC, however, the residual turbidity decreased to
6.0 NTU at a dosage of 200 mg L1. Therefore, the PAFC dosage in CF
could be reduced by 72% compared with that in the addition of
PAFC alone to achieve a similar residual turbidity.
For a turbidity of 1100 NTU, the residual turbidity could reach
5.0 NTU with the addition of 50 mg L1 CF (35 mg L1 of PAFC and
15 mg L1 of MBF-B16), while a similar residual turbidity (6.1 NTU)
(5)

where Y2 is the predicted SS, and X1, X2, and X3 are the PAFC dosage
(mg L1), MBF-B16 dosage (mg L1), and pH value, respectively.
The R2 (0.9885) and adjusted R2 (0.9738) indicated good
agreement between the predicted and experimental values. The

Table 1
ANOVA response surface quadratic model for the optimization of turbidity removal.

Source SS DF MS F-value P-value > F

Model 3008.93 9 334.33 36.12 <0.0001 Significant

X1 1010.25 1 1010.25 109.13 <0.0001
X2 35.7 1 35.7 3.86 0.0903
X3 198.01 1 198.01 21.39 0.0024
X1 X2 113.42 1 113.42 12.25 0.01
X1 X3 660.49 1 660.49 71.35 <0.0001
X2 X3 1.21 1 1.21 0.13 0.7284
X21 835.02 1 835.02 90.2 <0.0001
X22 48.17 1 48.17 5.2 0.0565
X23 50.33 1 50.33 5.44 0.0525
Residual 64.8 7 9.26
Lack of fit 63.83 3 21.28 87.55 0.0004
Pure error 0.97 4 0.24
Cor total 3073.73 16
R2 0.9789 Adj R2 0.9518

SS, sum of squares; MS, mean square; DF, degree of freedom; Adj R2, adjusted R2; F,
Fisher's test; P, probability value; X1, PAFC dosage; X2, MBF-B16 dosage; X3, pH Fig. 3. Comparison of flocculation between PAFC dosage (A) and CF dosage (B) at 3000
value. NTU turbidity.
706 X. Xia et al. / Chemosphere 206 (2018) 701e708

was achieved by the addition of 80 mg L1 PAFC. Thus, the PAFC

dosage could be reduced by 56.2% when using CF compared to that
when using PAFC alone (Fig. 4). For a turbidity of 100 NTU, the
residual turbidity could reach 4.9 NTU with the addition of
15 mg L1 CF (10.5 mg L1 of PAFC and 4.5 mg L1 of MBF-B16),
while to achieve a similar residual turbidity (5.0 NTU), 25 mg L1
of PAFC was required. Therefore, the concentration of PAFC was
reduced by 58% with CF compared with that with PAFC alone
(Fig. 5). The results of experiments demonstrated that a much
lesser amount of PAFC was required for CF compared with that for
PAFC alone to achieve the same flocculation result. A similar finding
was documented by Yang et al., who reported that the composite
flocculant could enhance the flocculating rate and reduce the
flocculent cost (Yang et al., 2009). Although at this stage, the cost of
the bioflocculant was greater than that of chemical flocculants,
with the industrialization and production rate increase, the cost
could further decrease, and the competitiveness of CF would be
stronger in drinking water treatment.

3.5. Flocculation mechanism

3.5.1. SEM results

SEM images showed that MBF-B16 has an amorphous compact
structure. Before the addition of PAFC or CF, soil suspension parti-
cles had a fine and scattered state (Fig. 6). When PAFC was used as Fig. 5. Comparison of flocculation between PAFC dosage (A) and CF dosage (B) at 100
NTU turbidity.
the coagulant, it was first hydrolyzed to be a positively charged
polymer, which could attract the negatively charged soil suspen-
sion particles. As the result, the negatively charged particles Okaiyeto et al. (2015b).
aggregated around the positively charged polymer, thus leading to
the formation of small flocs (Duan and Gregory, 2003). Followed by
3.5.2. Zeta potential
the addition of MBF-B16, the interaction between MBF and soil
The surface electrical properties of flocs were investigated. Soil
particles allowed the small flocs to grow and gradually precipitate
particle surface was negatively charged (15.51 mV) in the natural
due to gravity. SEM image showed that MBF-B16 and soil particle
pH value. The zeta potential of soil particles in the presence of PAFC
could intimately intertwine with each other, thus leading to the
was 10.81 mV, which was higher than that of soil particles in the
formation of three-dimensional structures of larger flocs. The
absence of PAFC. This meant that charge neutralization proceeded
structure indicated that soil particles could be efficiently removed
during the flocculation process. The zeta potential (20.43 mV) of
from the suspension using PAFC and MBF. These results were
suspended particles with the addition of PAFC and MBF-B16 was
similar to the previous results reported by Wang et al. (2015b) and
lower than that of particles in the presence of PAFC, showing that
MBF was negatively charged.

3.5.3. Flocculation mechanism

Fig. 4. Comparison of flocculation between PAFC dosage (A) and CF dosage (B) at 1100
NTU turbidity.
In the case of PAFC, positive multivalent polymeric Fe and Al
species were generated under natural pH condition. In this system,
charge neutralization, sweep, and co-precipitation all worked
together as part of the flocculation mechanisms (Huang et al.,
2015a). Polymeric positively charged Al and Fe could neutralize
the negatively charged particle surface, thus reducing the distance
between either soil particles themselves or between soil particles
and MBF-B16 chain (Wang et al., 2015b). A short distance could
result in the effect of attraction forces, which in turn aggregated
these particles together. If the extension of the MBF molecular
chain from the soil particle surface was greater than the distance
between inter-particle surfaces, bridging effects would occur. Once
a particle was attached to a molecular chain, it could serve the
function as a bridge and attract a few other chains, binding them
together and forming three-dimensional flocs, thus promoting the
turbidity removal rate. Furthermore, the net catching and sweep
flocculation could be attributed to the removal of free suspended
particles in aqueous solution, along with the settlement of large
three-dimensional precipitates (Huang et al., 2015a). Turbidity
removal was obviously improved by MBF in the presence of PAFC,
which could be due to the adsorption and bridging effects of MBF-
B16 because it contained an abundant amount of eCOO and eOH
(Wan et al., 2013).
 X. Xia et al. / Chemosphere 206 (2018) 701e708
Fig. 6. SEM images associated with purified MBF-B16 (A), soil suspension (B), soil suspension flocculated by PAFC (C), soil suspension flocculated by PAFC, and purified MBF-B16 (D).
In the suspended soil coagulation system, Fe-soil and Al-soil
small flocs were formed first, and then, these small flocs grew
into bigger ones by adsorption and bridging during MBF-B16
addition. In addition, the high turbidity removal could also be
due to electrostatic effect. PAFC could be hydrolyzed into positively
charged polymers that adsorbed onto the surface of soil and created
positive charges locally, thus forming small Fe-soil and Al-soil flocs.
The small flocs could adsorb onto the negative group (eCOO) in
the MBF-B16 chain forming particle-PAFC-MBF or particle-PAFC
-MBF-PAFC-particle (Lin et al., 2008). Therefore, charge neutrali-
zation and adsorption-bridging effect were the main flocculation
mechanisms. Furthermore, sweep and net catching were also
involved in the flocculation process.
4. Conclusions
There are some safety problems in the treatment of drinking
water by chemical flocculants. In the present study, a novel com-
posite flocculant for drinking water treatment was provided by
response surface method, which greatly reduced the use of
chemical and improved the safety of drinking water. The optimized
composite was composed of PAFC and MBF-B16, which were pro-
duced by K. variicola B16. The ratio of PAFC to MBF-B16 was 2.39:1.
The composite could decrease the use of chemical flocculants by
56.2e72%. Charge neutralization and bridging effect were the main
flocculation mechanisms. Higher turbidity removal capacity of the
composite flocculant suggested that this novel composite could
provide a safe solution for the treatment of drinking water.
Acknowledgments
This study was supported by the Chinese Academy of Sciences
Strategic Biological Resource Service Network Plan Biological
Resource Derivation Library (ZSYS-005) and Sichuan Science and
Technology Plan Project (2016JY0054). The paper was embellished
by Zhizhi Xu (School of Environmental Science and Engineering,
Shandong University, 250100, PR China).
707
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.chemosphere.2018.04.159.
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