Ecotoxicology and Environmental Safety 121 (2015) 45–50

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Ecotoxicology and Environmental Safety

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

Production and characterization of a thermostable bioflocculant from

Bacillus subtilis F9, isolated from wastewater sludge
Sib Sankar Giri a,d, M. Harshiny a,b, Shib Sankar Sen c, V. Sukumaran a,n, Se Chang Park d,n
a
Deptartment of Biotechnology, Periyar Maniammai University, Thanjavur 613403, Tamil Nadu, India
b
Department of Chemical Engineering, National Institute of Technology, Tiruchirapalli 620015, India
c
School of life Sciences, Jawaharlal Nehru University, New Delhi 110067, India
d
Lab of Aquatic Biomedicine, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 151742, South
Korea

art ic l e i nf o a b s t r a c t

Article history: A bacterium isolated from wastewater sludge, identified as Bacillus subtilis F9, was confirmed to produce
Received 4 December 2014 bioflocculant with excellent flocculation activity. The effects of culture conditions such as initial pH,
Received in revised form temperature, carbon source, nitrogen source, and inoculum size on bioflocculant production were stu-
27 May 2015
died here. The results indicated that 2.32 g/L of purified bioflocculant could be extracted with the fol-
Accepted 5 June 2015
Available online 16 June 2015
lowing optimized conditions: 20 g L
1 sucrose as the carbon source, 3.5 g L
1 peptone as the nitrogen
source, an initial pH of 7.0, and a temperature of 40 °C. The purified bioflocculant consisted of 10.1%
Keywords: protein and 88.3% sugar, including 38.4% neutral sugar, 2.86% uronic acid, and 2.1% amino sugar. The
Bacillus subtilis neutral sugar consisted of sucrose, glucose, lactose, galactose, and mannose at a molar ratio of
Wastewater sludge 2.7:4.7:3.2:9.1:0.8. Elemental analysis of the purified bioflocculant revealed that the weight fractions of
Bioflocclulant
carbon, hydrogen, oxygen, nitrogen, and sulfur were 30.8%, 5.3%, 54.7%, 6.4%, and 2.9%, respectively.
Characterization
Furthermore, the purified bioflocculant was pH tolerant within the range of 2–8 and thermotolerant from
Polysaccharides
10 °C to 100 °C, with optimal activity at pH 7.0 and at a temperature of 40 °C. The purified bioflocculant
showed industrial potential for the treatment of drinking water. Considering these properties, especially
its low molecular weight (5.3  104 Da), this bioflocculant with excellent solubility and favorable floc-
culation activity is particularly suited for flocculating small particles.
& 2015 Elsevier Inc. All rights reserved.

1. Introduction or inorganic flocculants have been widely used in industrial fields

Flocculating agents are widely used in various industrial fields
such as wastewater treatment, textile manufacturing, cosmetol-
ogy, pharmacology, and those involving fermentation processes (Li
et al., 2009; Salehizadeh and Yan, 2014). Flocculants are usually
classified into three groups: (i) organic polymer bioflocculants,
including poly-acrylamide derivatives and poly-acrylic acid; (ii)
inorganic flocculants such as aluminum sulfate and polyaluminum
chloride; and (iii) bioflocculants consisting mainly of microbial
polysaccharide, glycoprotein, protein, lipid, nucleic acid, and nu-
cleoprotein (Aljuboori et al., 2013; Li et al., 2010). Synthetic organic
Abbreviations: BPM, bioflocculant-producing microorganisms; NCBI, national
center for biotechnology information; FA, flocculating activity; HPLC, high-perfor-
mance liquid chromatography; COD, chemical oxygen demand; NTU, nephelo-
metric turbidity unit; COD, chemical oxygen demand
n
Corresponding authors.
E-mail addresses: ssgiri@snu.ac.kr, giribiotek@gmail.com (S.S. Giri),
harshiny37@gmail.com (M. Harshiny), shibsankar.iicb@gmail.com (S.S. Sen),
drvsukumar@gmail.com (V. Sukumaran), parksec@snu.ac.kr (S.C. Park).
due to low production costs and high efficiency; however, their
use may cause environmental and health hazards. For example,
acrylamide monomer is not only a strong carcinogen and neuro-
toxic to humans but is also non-biodegradable (Ruden, 2004). In
addition, there is evidence that aluminum salts may induce Alz-
heimer's disease (Campbell, 2002). Use of synthetic organic poly-
electrolyte bioflocculants has also been restricted in European
countries because they are not readily biodegradable and even
some of their degraded monomers cause health hazards (Li et al.,
2010; Sam et al., 2011). Due to these concerns, bioflocculants
produced by microorganisms have been attracting more attention
in practical application.
“Bioflocculants are natural organic macromolecules that are
derived from microorganisms; they can flocculate particles such as
suspended solids, cells, and colloidal solids out of solution (Sale-
hizadeh and Shojaosadati, 2001).” The degradation products of
bioflocculants are harmless to the ecosystem (Buthelezi et al.,
2010). Furthermore, bioflocculants can be produced economically
on a large scale and be easily recovered from fermentation broth
(Salehizadeh and Shojaosadati, 2001). However, high production

http://dx.doi.org/10.1016/j.ecoenv.2015.06.010
0147-6513/& 2015 Elsevier Inc. All rights reserved.
46 S.S. Giri et al. / Ecotoxicology and Environmental Safety 121 (2015) 45–50
costs and complicated production processes are major restraints of
bioflocculant research (Salehizadeh and Yan, 2014). In addition,
the application of bioflocculants in bioprocessing, wastewater
treatment, and many other industrial operations mainly depends
on further decreasing bioflocculant production costs using cheap
substrates and novel fermentation and recovery strategies (Sale-
hizadeh and Yan, 2014). Hence, research is needed to identify and
select new bioflocculant-producing microorganisms (BPMs) that
are capable of utilizing low-cost substrates, with high activity, high
stability, and non-toxicity (Sharma et al., 2006; Wang et al., 2007).
Thus far, many bacteria, and fungi have been reported to produce
biopolymers such as polysaccharides, glycoproteins, and func-
tional proteins with efficient bioflocculant activity (Zhao et al.,
2012).
Activated sludge, soil and sediments, river and deep-sea water
samples are the best sources for isolating extracellular biopoly-
mer-producing microorganisms (Bala Subramanian et al., 2010;
Salehizadeh and Shojaosadati, 2001). Bioflocculation is a dynamic
process that usually occurs in activated sludge during the aerobic
process (Salehizadeh and Yan, 2014). Organisms such as Bacillus
sp., Halomonas sp., Scenedesmus sp., Chryseobacterium sp., Asper-
gillus sp., and Pseudoalteromonas sp. have been studied for bio-
flocculant production (Aljuboori et al., 2013; Salehizadeh and Yan,
2014; Wang et al., 2013). The composition and properties of bio-
flocculants depend on the type of BPMs, media composition, and
environmental conditions. In most cases, glucose or sucrose has
been used as organic substrates (Zhao et al., 2012); however, the
mechanism of flocculation by bioflocculants is not yet clearly
elucidated. Earlier experiments indicated that organic matter had
substantial effects on flocculation time, floc size, density, and
settling velocity (Elkady et al., 2011). The molecular weights of
bioflocculants are generally over 105 Da. High molecular weight
flocculants do not lead to the complete flocculation of small par-
ticles in suspension and hence, low molecular weight bio-
flocculants would be better in practical use (Liu et al., 2001). Fur-
thermore, the data currently available on the environmental ap-
plication of microbial flocculants (extracellular polymeric sub-
stances) are limited (More et al., 2014).
In the present study, we isolated a BPM from activated sludge.
We investigated the optimal culture medium composition and
various factors influencing flocculation efficiency, such as carbon
source, nitrogen source, C/N ratio, pH, temperature, inoculum size,
and time course. The properties and composition of the bio-
flocculant were determined in order to assess its potential appli-
cation in various industrial processes and in drinking water
purification.
2. Material and methods
2.1. Isolation and identification of bioflocculant bacteria
BPMs were isolated from the wastewater sludge of Periyar
Maniammai University campus, using agar plate techniques on a
basal medium. The composition of the medium was as follows:
glucose, 20 g L
1; yeast extract, 3.5 g L
1; K2HPO4, 5 g L
1;
KH2PO4, 5 g L
1; NaCl, 0.1 g L
1; MgSO4, 0.2 g L
1; and agar–agar,
20 g L
1. The cultures were incubated at 37 °C for 72 h. Colonies
that were established were screened for their ability to produce
bioflocculant based on colony morphology: large, viscous, and
ropy. The selected isolate was cultivated in 250 mL of broth
medium (pH 7.0) in a flask on a rotary shaker at 37 °C for 72 h at
160 rpm.
The selected isolate was identified based on 16S rDNA se-
quencing. The genomic DNA was isolated and subjected to am-
plification of 16S rDNA using universal primers. The amplified
product was gel purified using a QIAGEN gel extraction kit (QIA-
GEN Inc., USA) and the purified products were sequenced as pre-
viously described by Kim et al. (2006). The 16S rDNA sequence
( 1089 bp) was compared with 16S rDNA sequences that are
currently available in the NCBI GenBank database.
2.2. Optimization of culture conditions for bioflocculant production
Various factors such as carbon source, nitrogen source, C/N
ratio, pH, temperature, inoculum size, and culture time could af-
fect bioflocculant production. To determine the effect of carbon
and nitrogen sources on bioflocculant production, glucose was
replaced with sucrose, fructose, lactose, and starch (20 g L
1) as
the carbon source, and yeast extract was replaced with (NH4)2SO4,
NH4NO3, NaNO3, peptone, urea, and glutamic acid (3.5 g L
1 as the
nitrogen source. For the C/N ratio, different concentrations of
glucose were used in order to get different C/N ratios of 0:1–50:1
while yeast extract remained constant. The initial pH of the pro-
duction media was adjusted from 2 to 10. The temperature of the
production media was adjusted from 15 °C to 50 °C. Inoculum size
was adjusted from 0.2 to 10% v/v, while the time course of bio-
flocculant production was adjusted between 0 h and 96 h. All of
the experiments were conducted in triplicates.
2.3. Purification of bioflocculant
The bioflocculant from the broth culture was purified as pre-
viously described by Li et al. (2009), with slight modifications.
Briefly, the culture broth was centrifuged to remove cell pellets
(5000g, 30 min). Thereafter, the supernatant was concentrated
and dialyzed overnight at 4 °C in deionized water. Three volumes
of cold anhydrous ethanol (4 °C) were added to the dialyzed broth.
The precipitate was then dissolved in deionized water followed by
the addition of 10% cetylpyridinium chloride (Sigma-Aldrich, USA)
with continuous stirring. The resulting precipitate was collected by
centrifugation (5000g, 15 min) and dissolved in 0.5 M NaCl. Three
volumes of cold anhydrous ethanol (4 °C) were again added to
obtain the precipitate, which was then washed three times with
75% ethanol and lyophilized to obtain the crude bioflocculant. The
partially purified bioflocculant solution (0.1%) was loaded onto a
Sephacryl S-500 column (16  100 mm2; GE Healthcare Life Sci-
ences, India) and eluted with distilled water at a flow rate of
0.2 mL/min to collect the active fraction, which was lyophilized
until further use.
2.4. Measurement of flocculating activity
A kaolin suspension was used to measure the flocculating rate
of the bioflocculant in culture broth (Aljuboori et al., 2013). In
brief, 2 g of Kaolin clay (Sigma-Aldrich, USA) was suspended in 1 L
of deionized water. One milliliter of culture broth was added to
99 mL of the kaolin suspension in a 400-mL beaker, and the pH
value was adjusted to 7.0 using 1 M NaOH or HCl. The mixture was
stirred at 200 rpm for 1 min, slowly stirred at 100 rpm for 5 min,
and allowed to stand for 5 min. The optical density (OD) of the
supernatant was measured with a spectrophotometer (Perkin El-
mer, USA) at 550 nm. As a control, the above procedures were
repeated using fresh culture medium instead of the culture broth.
The flocculating activity (FA) was calculated according to the fol-
lowing equation:
FA = (B − A/B) × 100
where, ‘A’ is the OD of the sample experiment at 550 nm and ‘B’ is
the OD of the control experiment at 550 nm. Complete removal of
particles yields FA ¼100%. If no flocculation, FA ¼0%.
 S.S. Giri et al. / Ecotoxicology and Environmental Safety 121 (2015) 45–50
2.5. Physical and chemical analysis of purified bioflocculant
The protein content was measured by the Bradford method
with bovine serum albumin as standard (Bradford, 1976). The total
sugar content of the bioflocculant was determined by the phenol–
sulfuric acid using glucose as standard (Chaplin and Kennedy,
1994). The uronic acid was determined using carbazol–sulfuric
acid method with glucouronic acid as standard solution (Chaplin
and Kennedy, 1994). The amino sugars were determined following
the Elson–Morgan method using glucose amine as standard (Gong
et al., 2008). To determine its sugar composition, bioflocculant was
hydrolyzed with trifluoroacetic acid at 121 °C for 1 h. The resulting
sugar was analyzed by HPLC (Perkinelmer, USA) with Rezex RCM
Monosaccharide Ca2 þ column (300  7.8 mm) using deionized
distilled water as eluent at a flow rate of 0.4 mL/min, a column
temperature of 85 °C and back pressure of 331 psi. Elemental
analysis was carried out with a Truspec CHN/CHNS elemental
analyzer (LECO, USA). The average molecular weight of the pur-
ified bioflocculant was determined by HPLC.
2.6. pH and thermal stability of the purified bioflocculant
Purified bioflocculant was dissolved in a suitable volume of de-
ionized water to achieve an initial flocculating rate of over 90% and
divided into 9 aliquots. The pH of the aliquots was adjusted to 2, 3,
4, 5, 7, 8, 9, 10 and 11 with 1 M of NaOH or HCl. The aliquots were
kept at 4 °C for 24 h (He et al., 2004) and the flocculating rate was
measured at room temperature using kaolin suspension as de-
scribed above.
Purified bioflocculant was dissolved in a suitable volume of de-
ionized water to achieve an initial flocculating rate of over 90% and
divided into 6 groups with a pH of 3, 4, 5, 6, 7 and 8. Six aliquots of
each pH were treated at temperatures of 10, 20, 40, 50, 60, 70, 80
and 100 °C for 1 h (He et al., 2004) and flocculating rates were
measured at room temperature using kaolin suspension.
2.7. Drinking water flocculation test
Our University tap water was used for this purpose. The tur-
bidity and COD (chemical oxygen demand) of the water sample
were 21 NTU (nephelometric turbidity unit) and 8.3 mg/l, respec-
tively. A sample of 993 ml sample water and 5 ml CaCl2 (10 g/l; pH:
7.) were mixed in a 1000 ml beaker and the pH was adjusted by
adding 0.1 N HCl or NaOH. The flocculant was added to the reac-
tion mixture and stirred vigorously for 5 min. The mixture was
allowed to settle for 10 min and supernatant was collected for
analysis. The turbidity and COD was measured according to ac-
cording to Standard Methods for the Examination of Water and
Wastewater, APHA (APHA et al., 1998).
3. Results and discussion
3.1. Identification of flocculation-producing bacteria
Nineteen BPMs were isolated from the wastewater sludge
sample. Among the isolated bacteria, the most efficient strain, with
higher than 91% flocculation activity, was named F9. F9 was a
gram-positive, rod-shaped, spore-forming bacterium. In compar-
ing the 16S rDNA sequence to the sequences deposited in the NCBI
GenBank database (accession number: KJ661546), the 16S rDNA
sequence is most similar to that of Bacillus subtilis, sharing a 99%
similarity. Based on morphological and biochemical properties and
16S rDNA sequence data, this strain was identified as Bacillus
subtilis F9. Previous research has indicated that many micro-
organisms that secrete extracellular biopolymer flocculants are of
47
the Bacillus genus (Shih et al., 2001; Wu and Ye, 2007; Ryu et al.,
2007; Li et al., 2009; Xiong et al., 2010; Buthelezi et al., 2010;
Elkady et al., 2011).
3.2. Effects of carbon, nitrogen sources, and C/N ratios on bio-
flocculant production
Carbon and nitrogen sources play an important role on bio-
flocculant production because the C/N ratio greatly affects micro-
bial metabolism. The optimal carbon and nitrogen sources and C/N
ratio result in maximum flocculating activity with the shortest
incubation time (Xiong et al., 2010). We found that glucose, su-
crose, and starch were favorable for bioflocculant production,
whereas bioflocculant production was very low when fructose or
lactose was used (Figs. 1A and C). Maximum bioflocculant activity
was obtained with sucrose medium. Glucose, sucrose, or starch
has previously been reported as favorable carbon sources for
bacterial as well as fungal species in the production of bio-
flocculant (Aljuboori et al., 2013, Elkady et al., 2011, Li et al., 2009).
In the present study, organic nitrogen sources (yeast extract and
peptone) were used effectively in producing bioflocculant from B.
subtilis F9, whereas (NH4)2SO4, NaNO3, and glutamic acid resulted
in poor bioflocculant production (Figs. 1B and C). Many strains
were previously reported to effectively use peptone or yeast ex-
tract as nitrogen sources for bioflocculant production, with pep-
tone being one of the most cost-effective nitrogen sources (Alju-
boori et al., 2013; Li et al., 2010; Li et al., 2009). We have de-
termined that maximum bioflocculant production was achieved at
a C/N ratio of 5:1 (Fig. 1D); however, a further increase in C/N ratio
caused a decline in bioflocculant production, which is consistent
with previous studies (Aljuboori et al., 2013). In spite of several
reports on the relationship of the C/N ratio to bioflocculant pro-
duction, there is no fixed favorable C/N ratio in literature. This
might be due to the variety in the type of carbon–nitrogen sources
and microorganisms studied (More et al., 2014).
3.3. Effect of initial pH, temperature, and inoculum size on bio-
flocculant production
The effect of the initial pH of the medium on bioflocculant
production is shown in Fig. 2. The maximum flocculation rate was
achieved between pH 6 and 9, with the production highest at pH 7.
The initial pH of the culture medium is important because it may
affect nutrient absorption and enzymatic reaction of BPMs (Xia
et al., 2008). In the case of B. licheniformis, the optimal pH for
bioflocculant production was between 6.5 and 9.0, with produc-
tion highest at pH 7 and above (Li et al., 2009). Moreover, alkaline
pH in the range of 7–12 was favored for bioflocculant production
by Bacillus megaterium, and maximum yield of bioflocculant was
obtained at pH 9.0 (Zheng et al., 2008). Therefore, the effect of pH
on bioflocculant production varies between different micro-
organisms, operational conditions, and medium composition (Shu
and Lung, 2004).
Temperature is one of the most important parameters influ-
encing bioflocculant production (More et al., 2014). The metabo-
lism of culture temperature is directly related to the metabolism of
microorganisms (Salehizadeh and Shojaosadati, 2001). In the
present study, cultures grown at 30 °C or above showed increasing
flocculating rates that were highest at 40 °C (83.6%) (Fig. 3). In a
similar study, the optimal temperature for bioflocculant produc-
tion from B. licheniformis X14 was 37 °C (Li et al., 2009). B. subtilis
PUL-A maximum at temperature 42 °C (Ryu et al., 2007). Thus, the
thermostability of the purified bioflocculant is suitable for water
and wastewater treatment in industry.
In the present study, the use of 2% (v/v) of B. subtilis F9 in-
oculum size in production medium resulted in the highest
48 S.S. Giri et al. / Ecotoxicology and Environmental Safety 121 (2015) 45–50

Fig. 1. The effect of carbon sources, nitrogen sources, C/N ratio on Bacillus subtilis F9 biomass and flocculant production: (A) The effect of carbon sources on bioflocculant
production with yeast extract used in the medium as carbon source; (B) the effect of nitrogen sources on bioflocculant production with glucose used in the medium as carbon
source; (C) the effect of various carbon and nitrogen sources on flocculating rate; and (D) the effect of C/N ratio on the production of bioflocculant. Error bars indicate
standard deviation (n¼ 3).

Fig. 3. Effect of temperature of the medium on the production of bioflocculant.

Error bars indicate standard deviation (n ¼3).
Fig. 2. The effect of initial pH of the medium on the production of bioflocculant.
Error bars indicate standard deviation (n¼ 3).
3.4. Time course of bioflocculant production

Bioflocculant production during the growth of B. subtilis F9 is

flocculating rate (data not shown). In accordance with our results,
Aljuboori et al. (2013) investigated inoculum sizes ranging from
0.2% to 10% (v/v) with A. flavus, and obtained maximum bio-
flocculant production with 2% (v/v) inoculum size. Inoculum size is
an important parameter for bioflocculant production because a
small inoculum size would prolong the lag phase, while a large
inoculum size would lead to excessive overlap between niches of
bacterial strains, restraining bioflocculant production (Salehizadeh
and Shojaosadati, 2001; Salehizadeh and Yan, 2014).
shown in Fig. 4. The strain began to grow in culture after a short
lag phase. A rapid increase in flocculation activity was detected
during growth that was caused by the production of bioflocculant
in the log phase, between 36 h and 72 h, with the highest activity
at 60 h (84.1%). The same phenomenon was observed in the cul-
tivation of Ochrobactrum ciceri W2 (Wang et al., 2013), En-
terobacter aerogenes (Lu et al., 2005), and Bacillus subtilis DYU1
(Wu and Ye, 2007). At the late stationary phase, the flocculating
rate started to decrease, which may be due to the enzymatic
 S.S. Giri et al. / Ecotoxicology and Environmental Safety 121 (2015) 45–50
Fig. 4. Time course of the production of bioflocculant. Error bars indicate standard
deviation (n¼ 3).
activities of the flocculant (Li et al., 2009). At 72 h, B. subtilis F9
reached its early death phase, and consequently the flocculating
rate decreased slowly. The pH profile showed that pH declined
from 7 to 4.8 within 60 h, and increased again to 5.7 at 96 h.
Therefore, a period of 60 h was chosen as the culture time for
subsequent experiments.
Using optimal culture conditions and composition, approxi-
mately 2.32 g of pure bioflocculant could be obtained from 1 L
culture broth of B. subtilis F9. In comparison to the findings of
previous studies, 3.8 g/L of biopolymer was produced by Ochro-
bactrum ciceri W2 (Wang et al., 2013), while 1.3 g/L of the bio-
flocculant WF-1 was produced by Enterobacter aerogenes W23 (Lu
et al., 2005). In the present study, flocculating activity increased
with the increasing concentrations of bioflocculant, which was
similarly reported previously (Aljuboori et al., 2013; He et al.,
2004).
3.5. Characterization of purified bioflocculant
Chemical analysis of the bioflocculant revealed that the pro-
portions of total sugar and total protein content were 88.3% and
10.1% (w/w), respectively. Further analysis revealed that the total
sugar composition was 38.4% neutral sugar, 2.86% uronic acid, and
2.1% amino sugar. The neutral sugar components of the bio-
flocculant included sucrose, glucose, lactose, galactose, and man-
nose at a molar ratio of 2.7:4.7:3.2:9.1:0.8. Elemental analysis of
bioflocculant revealed that the weight fractions of carbon, hydro-
gen, oxygen, nitrogen, and sulfur were 30.8%, 5.3%, 54.7%, 6.4%, and
2.9%, respectively. To date, the most recently analyzed bio-
flocculants have been polysaccharide-like substances (Elkady et al.,
2011; Li et al., 2009; Wang et al., 2013, 2007), however, several
bioflocculants containing proteins or lipids have also been re-
ported (Salehizadeh and Shojaosadati, 2001; Li et al., 2009; Sale-
hizadeh and Yan, 2014). The higher nitrogen and oxygen content in
the purified bioflocculant suggests that there are many functional
groups containing nitrogen and oxygen atoms that are preferred
for flocculation (Li et al., 2009).
HPLC analysis indicated that the molecular weight of the pur-
ified bioflocculant was 5.3  104 Da. Low molecular weight bio-
flocculants have been previously purified from many organisms
such as Aspergillus flavus (Aljuboori et al., 2013), Agrobacterium sp.
(Li et al., 2010), and Bacillus mojavensis (Li et al., 2009). B. subtilis
PUL-A produced a biopolymer of molecular weight of 1.3  106
(Ryu et al., 2007). Flocculation with high molecular weight bio-
flocculants involves more adsorption points, stronger bridging,
and higher flocculating activities than with low molecular weight
bioflocculants (Salehizadeh and Shojaosadati, 2001). However,
49
Fig. 5. Bioflocculant stability: (A) The pH stability of bioflocculant and (B) the
thermo stability of bioflocculant at various pH values. Error bars indicate standard
deviation (n ¼3).
elemental analysis of the purified bioflocculant indicated that it
has higher content of nitrogen and oxygen than do other pre-
viously reported bioflocculants (Lu et al., 2005; Yim et al., 2007),
suggesting that there are many functional groups containing ni-
trogen and oxygen atoms that are preferred for flocculation (Li
et al., 2009).
The purified bioflocculant was treated at different pH levels
(Fig. 5A), revealing that the purified bioflocculant was stable with
a wide range of pH, between 2 and 8, and achieved greater than
90% flocculation. Beyond pH 8, flocculation decreased gradually.
This may be that the bioflocculant shows different electric states at
different pH levels, which affects the flocculation ability of bio-
flocculant for kaolin particles (Yong et al., 2009). The stability of
the bioflocculant produced by B. mojavensis 32A was observed at a
pH range of 5–9 and peaked at pH 7.0 (Elkady et al., 2011). In line
with earlier reports, our study suggests that this bioflocculant is
suitable for application in acidic, neutral, and weakly alkaline
conditions.
The purified bioflocculant was thermostable (Fig. 5B) and re-
tained more than 89% flocculation rate at the temperature range of
10–100 °C, when the pH of the bioflocculant was in the range of
3.0–8.0. The thermal stability of the bioflocculant might be due to
that the main backbone of the flocculant consists of poly-
saccharides. With increasing temperature, the polysaccharide
chains of bioflocculant extend, exposing more flocculating sites
and increasing flocculation activity (Li et al., 2010; Lu et al., 2005).
The flocculation activity of the bioflocculant produced by Vago-
coccus sp. W31 remained at 86.5% at 100 °C, and flocculation ac-
tivity was stable over a pH range of 7–10 (Gao et al., 2006). The
superior pH and temperature stability of the purified bioflocculant
indicates that it would be useful in many extreme environments.
50 S.S. Giri et al. / Ecotoxicology and Environmental Safety 121 (2015) 45–50
3.6. Drinking water treatment
The effect of the purified bioflocculant on the treatment of
drinking water was investigated in this study. It was found that
when CaCl2 was added to the water, the flocculation rate in-
creased. However, with the addition of CaCl2 and purified bio-
flocculant, the settling of solid particles was much higher and
faster. After settling, the turbidity and COD of the drinking water
treated with purified flocculant were 1.9 NTU (removal rate
90.96%) and 3.5 mg/L (removal rate 57.58%), respectively. Obtained
values were almost similar to those of native bioflocculants (Gong
et al., 2008). Therefore, this bioflocculant could be used for the
treatment of drinking water and may be an alternative to chemical
flocculants. Furthermore, its low molecular weight and poly-
saccharide composition endowed it with excellent solubility and
favorable flocculation activity, especially for small particulates (Li
et al., 2010). However, further studies should evaluate the ability of
the purified bioflocculant to flocculate other particles.
4. Conclusion
In summary, bioflocculant produced by the newly isolated
bacteria B. subtilis F9 has high flocculating activity with unique
characteristics such as pH tolerance and thermal stability. Its
constituent was mainly polysaccharides (88.3%; w/w) that explain
its stability in organic solvents. Further, its higher content of ni-
trogen and oxygen indicated the presence of many functional
groups containing nitrogen and oxygen atoms, which are preferred
for flocculation. In addition, its low molecular weight added ad-
vantage for solubility and flocculation of small molecules. The
bioflocculant showed good performance in treating the drinking
water. However, further studies (e.g., toxicity test, ability to floc-
culate other particles) are required before it is implemented in
field processes. Moreover, the flocculation mechanism of bio-
flocculants should be investigated further.
Acknowledgments
Currently, 1st author is a BK21 PLUS Post-doctoral fellow at
SNU. SSS is a receipt of DSKPDF from UGC, GoI. Authors declare no
conflict of interest.
References
Aljuboori, A.H.R., Idris, A., Abdullah, N., Mohamad, R., 2013. Production and char-
acterization of bioflocculant produced by Aspergillus flavus. Bioresour. Technol.
127, 489–493.
APHA, AWWA, WEF, 1998. 2nd edStandard Methods for the Examination of Water
and Waste Water, American Public Health Association, American Water Works
Association, Water Environment Association, Washington, DC, pp. 413-428.
Bala Subramanian, S., Yan, S., Tyagi, R.D., Surampalli, R.Y., 2010. Extracellular
polymeric substances (EPS) producing bacterial strains of municipal waste-
water sludge: isolation, molecular identification, EPS characterization and
performance for sludge settling and dewatering. Water Res. 44, 2253–2263.
Bradford, M.M., 1976. A rapid and sensitive method for the quantification of mi-
crogram quantities of protein utilizing the principle of protein dye binding.
Anal. Biochem. 72, 248–254.
Buthelezi, S.P., Olaniran, A.O., Pillay, B., 2010. Production and characterization of
bioflocculants from bacteria isolated from wastewater treatment plant in South
Africa. Biotechnol. Bioprocess. Eng. 15, 874–881.
Campbell, A., 2002. The potential role of aluminium in Alzhemer’s disease. Nephrol.
Dial. Transplant. 17, 17–20.
Chaplin, M.F., Kennedy, J.F., 1994. Carbohydrate Analysis. Oxford University Press,
New York.
Elkady, M.F., Soha, Farag, Sahar, Zaki, Gadallah, A.-E., Desouky, A.-E.-H., 2011. Ba-
cillus mojavensis strain 32A, a bioflocculant-producing bacterium isolated from
an Egyptian salt production pond. Bioresour. Technol. 102, 8143–8151.
Gao, J., Bao, Y., Xin, M.-X., Liu, Y.-X., Li, Q., Zhang, Y.-F., 2006. Characterization of a
bioflocculant from a newly isolated Vagococcus sp. W31. Zhejiang Univ. Sci. B7,
186–192.
Gong, W.X., Wang, S.G., Sun, X.F., Liu, X.W., Yue, Q.Y., Gao, B.Y., 2008. Bioflocculant
production by culture of Serratia ficaria and its application in wastewater
treatment. Bioresour. Technol. 99, 4668–4674.
He, N., Li, Y., Chen, J., 2004. Production of a novel polygalacturonic acid bio-
flocculant REA-11 by Corynebacterium glutamicum. Bioresour. Technol. 94,
99–105.
Kim, L.S., Hong, S.J., Son, M.K., Lee, Y.H., 2006. Polymeric and compositional prop-
erties of novel extracellular microbial polyglucosamine biopolymer from new
strain of Citrobacter sp. BL-4. Biotechnol. Lett. 28, 241–245.
Li, Q., Liu, H.-l, Qi, Q.-s, Wang, F.-s, Zhang, Y.-z, 2010. Isolation and Characterization
of temperature and alkaline stable bioflocculant from Agrobacterium sp.
M-503. New Biotechnol. 27 (3), 789–794.
Li, Z., Zhong, S., Lei, H.-y, Chen, R.-w, Yu, Q., Li, H.-L., 2009. Production of a novel
bioflocculant by Bacillus licheniformis X14 and its application to low tem-
perature drinking water treatment. Bioresour. Technol. 100, 3650–3656.
Liu, Z., Xu, G., Liu, Z., Yang, H., 2001. Composition and structure of bioflocculant
BP25. Acta Microbiol. Sci. 41, 348–352.
Lu, W.Y., Zhang, T., Zhang, D.Y., Li, C.H., Wen, J.P., Du, L.X., 2005. A novel bio-
flocculant produced by Enterobacter aerogenes and its use in defecating the
trona suspension. Biochem. Eng. J. 27, 1–7.
More, T.T., Yadav, J.S.S., Yan, S., Tyagi, R.D., Surampalli, R.Y., 2014. Extracellular
polymeric substances of bacteria and their potential environmental applica-
tions. J. Environ. Manag. 144, 1–25.
Ruden, C., 2004. Acrylamide and cancer risk-expert assessments and the public
debate. Food Chem. Toxicol. 42, 335–349.
Ryu, M.-J., Jang, E.-K., Lee, S.-P., 2007. Physiological properties of a biopolymer
flocculant produced from Bacillus subtilis PUL-A. Korean J. Microbiol. Bio-
technol. 35, 203–209.
Salehizadeh, H., Shojaosadati, S.A., 2001. Extracellular biopolymer flocculants: re-
cent trends and biotechnological importance. Biotechnol. Adv. 19, 371–385.
Salehizadeh, H., Yan, N., 2014. Recent advances in extracellular biopolymer floc-
culants. Biotechnol. Adv. 32, 1506–1522.
Sam, S., Kucukasik, F., Yenigun, O., Nicolaus, B., Oner, E.T., Yukselen, M.A., 2011.
Flocculating performances of exopolysaccharides produced by a halophilic
bacterial strain cultivated on agro-industrial waste. Bioresour. Technol. 102,
1788–1794.
Sharma, B.R., Dhuldhoya, N.C., Merchant, U.C., 2006. Flocculants—an ecofriendly
approach. J. Polym. Environ. 14, 195–202.
Shih, I.L., Van, Y.T., Yeh, L.C., Lin, H.G., Chang, Y.N., 2001. Production of a biopolymer
flocculant from Bacillus licheniformis and its flocculation properties. Bioresour.
Technol. 78, 267–272.
Shu, C.H., Lung, M.-Y., 2004. Effect of pH on the production and molecular weight
distribution of exopolysaccharide by Antrodia camphorate in batch cultures.
Process Biochem. 39, 931–937.
Wang, L., Fang, Ma, Duu-Jong, Lee, Aijie, Wang, Nanqi, Ren, 2013. Bioflocculants
from hydrolysates of corn stover using isolated strain Ochrobactrum ciceri W2.
Bioresour. Technol. 145, 259–263.
Wang, S.G., Gong, W.X., Liu, X.W., Lin, T., Yue, Q.Y., Gao, B.Y., 2007. Production of a
novel bioflocculant by culture of Klebsiella mobilis using dairy wastewater.
Biochem. Eng. J. 36, 81–86.
Wu, J.Y., Ye, H.F., 2007. Characterization and flocculating properties of an extra-
cellular biopolymer produced from a Bacillus subtilis DYU1 isolate. Process
Biochem. 42, 1114–1123.
Xia, S., Zhang, Z., Wang, X., Yang, A., Chen, L., Zhao, J., Leonard, D., Jaffrezic-Renault,
N., 2008. Production and characterization of a bioflocculant by Proteus mirabilis
TJ-1. Bioresour. Technol. 99, 6520–6527.
Xiong, Y., Wang, Y., Yu, Y., Li, Q., Wang, H., Chen, R., et al., 2010. Production and
characterization of a novel bioflocculant from Bacillus lichenoformis. Appl.
Environ. Microbiol. 76, 2778–2782.
Yim, J.H., Kim, S.J., Ahn, S.H., Lee, H.K., 2007. Characterization of a novel bio-
flocculant, p-KG03, from marine dinoflagellate, Gyrodinium impudicum KG03.
Bioresour. Technol. 98, 361–367.
Yong, P., Bo, S., Yu, Z., 2009. Research on flocculation property of bioflocculant PG.
a21 Ca. Mod. Appl. Sci. 3 (6), 106–112.
Zhao, G., Ma, F., Wei, L., Chua, H., 2012. Using rice straw fermentation liquor to
produce bioflocculants during an anaerobic dry fermentation process. Bior-
esour. Technol. 113, 83–88.
Zheng, Y., Ye, Z.L., Fang, X.L., Li, Y.H., Cai, W.M., 2008. Production and characteristics
of a bioflocculant produced by Bacillus sp. F19. Bioresour. Technol. 99,
7686–7691.