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Bioresource Technology 99 (2008) 4668–4674

Bioflocculant production by culture of Serratia ficaria

and its application in wastewater treatment
Wen-Xin Gong, Shu-Guang Wang *, Xue-Fei Sun, Xian-Wei Liu, Qin-Yan Yue, Bao-Yu Gao
School of Environmental Science and Engineering, Shandong University, Jinan, 250100, China

Received 13 July 2007; received in revised form 27 September 2007; accepted 28 September 2007
Available online 19 November 2007

Abstract

A bioflocculant-producing bacterium was isolated from soil and identified as Serratia ficaria. Using optimized culture conditions a
flocculating activity of 95.4% was obtained. It was found to be effective for flocculation of a kaolin suspension over weakly acidic
pH (5–7); divalent cations (Ca2+ and Mg2+) enhanced the flocculating activity, while the co-presence of Al3+ and Fe3+ resulted the neg-
ative effect. Measurements of zeta potential revealed that charge neutralization played an important role in the flocculation. It could
flocculate a variety of real wastewaters, including river water, brewery wastewater, meat processing wastewater and soy sauce brewing
wastewater. The bioflocculant was also used to treat pulp effluent, and the removal rate of color and chemical oxygen demand (COD)
were up to 99.9% and 72.1%, respectively, which were better than traditional chemical flocculants.
 2007 Elsevier Ltd. All rights reserved.

Keywords: Bioflocculant; Serratia ficaria; Flocculation; Real wastewater

1. Introduction wastewater, in downstream processing, and in processing

Bioflocculant is a kind of biodegradable macromolecu-
lar flocculant secreted by microorganisms. Because of their
biodegradability, harmlessness and lack of secondary pol-
lution, bioflocculants have gained much wider attention
and research to date (Li et al., 1999). Most of research
focused on screening for microorganisms, culture condi-
tions, mechanism of flocculation, chemical structure, and
so on (Jang et al., 2001; Salehizadeh and Shojaosadati,
2001; Shih et al., 2001; He et al., 2004; Deng et al., 2005;
Wu and Ye, 2007). Mutational methods to get new efficient
mutant and seeking for low-cost substrates to reduce the
production cost were also investigated (Al-Shahwani and
Al-Rawi, 1989; Wang et al., 2007; Yang et al., 2007). Bio-
flocculants are widely useful in the treatment of water and
*
Corresponding author. Tel.: +86 531 88362802; fax: +86 531 88364513.
E-mail address: wsg@sdu.edu.cn (S.-G. Wang).
of food and chemicals (Salehizadeh and Shojaosadati,
2001).
In wastewater treatment, bioflocculants have been used
to treat dyes solution (Zhang et al., 2002; Deng et al.,
2005), inorganic solid suspensions (bentonite, activated
carbon, solid clay, Ca(OH)2, and aluminum oxide) (Levy
et al., 1992; Shih et al., 2001; Yim et al., 2007), humic acids
(Zouboulis et al., 2004) and other suspensions which are
synthetic (Salehizadeh and Shojaosadati, 2002; Lu et al.,
2005). The knowledge on the treatment of real wastewaters
is lacking or scarce (Deng et al., 2003). And the removal
efficiency was mostly based on flocculating activity. Param-
eters relating with wastewater standard, such as chemical
oxygen demand (COD) and turbidity were not determined.
The application of bioflocculants cannot be availably and
systematically assessed in the wastewater treatment.
In this paper, a bioflocculant-producing bacterium,
named S-14, was isolated from soil and identified as Serra-
tia ficaria. A series of experiments were performed to inves-
tigate the bioflocculant production and flocculation

0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.09.077
 W.-X. Gong et al. / Bioresource Technology 99 (2008) 4668–4674
characteristics. The application in treatment of various real
wastewaters and comparison with chemically synthesized
flocculants were investigated.
2. Methods
2.1. Screening and culturing of microorganism
Bioflocculant-producing strains were screened from soils
and activated sludges. The culture medium consisted of (g/
l): glucose, 10; yeast extract, 0.5; urea, 0.5; NaCl, 0.1;
KH2PO4, 5. The medium for slant contained (g/l): glucose,
20; yeast extract, 0.5; urea, 0.5; NaCl, 0.1; K2HPO4, 5;
KH2PO4, 2; agar, 20. The initial pH of media was adjusted
to 7.0–7.2.
Cultures from a slant were inoculated into 250 ml flasks
containing 50 ml culture medium and incubated on a reci-
procal shaker at 120 rpm. For flask tests, the carbon and
nitrogen sources, concentration of salts, addition of other
bioflocculant-producing bacterium, pH of culture medium
and inoculum size (%, v/v) were varied to determine the
optimum culture conditions. Some agricultural product
processing wastewaters, brewery wastewater (BW), dairy
wastewater (DW), meat processing wastewater (MW),
and soy sauce brewing wastewater (SW) from local plants
(Jinan, China) were used to investigate the utilization of
low-cost medium. In the culture 5% (v/v) ethanol
(P99.7%, w/w) was added in the wastewaters medium.
2.2. Bioflocculant purification
The viscous culture broth was diluted with the two vol-
umes of distilled water and then centrifuged at 5000g for
10 min to remove cell pellets. The supernatant was poured
into four volumes of cold ethanol to precipitate the bio-
flocculant. The resulting precipitate was collected by centri-
fugation at 10,000g for 15 min and re-dissolved in water.
After two such steps, the crude bioflocculant obtained
was purified by dialysis overnight at 4 C in distilled water
and then lyophilized.
2.3. Determination of flocculating activity
The mixture containing 100 ml kaolin clay suspension
(5 g/l, pH 7.0), 0.2 ml sample (supernatant centrifuged at
5000g for 30 min) and 2 ml 1% (w/v) CaCl2 solution, was
stirred with rapid mixing at 200 rpm for 30 s, followed by
slow mixing at 100 rpm for 5 min and left standing for
10 min. The supernatant was measured for absorbance at
550 nm. A control was prepared using the same method
but the sample was replaced by distilled water. The floccu-
lating activity was calculated according to the following
equation:
Flocculating activity ð%Þ ¼ ðA  BÞ=A  100 ð1Þ
where A and B are the optical densities of the control and
the sample, respectively.
4669
2.4. Analytical methods
Cell growth in flask cultures was monitored turbidomet-
rically at 660 nm (OD660). The measurements of COD,
color, turbidity, and total nitrogen (TN) were performed
according to the standard methods issued by the China
National Environmental Protection Agency. Zeta potential
of bioflocculant was determined using Malvern-Zetasizer
3000HS (Malvern, UK).
2.5. Characteristics of bioflocculant
Total sugar and protein content were measured using
the phenol–sulfuric acid and Lowry–Folin methods,
respectively (Salehizadeh et al., 2000). The infrared spec-
trum was obtained using an IR spectrophotometer (Nicolet
20SXFT-IR) with a NaCl pellet and methyl butyl ketone as
standard. The average molecular weight of the biofloccu-
lant was measured by the Research Center of Analysis
and Measurement of Beijing University of Chemical Tech-
nology using gel permeation chromatography (GPC).
Thermal stability of the bioflocculant was also measured
(Salehizadeh and Shojaosadati, 2002; He et al., 2004).
2.6. Flocculation of various wastewaters
To get the knowledge of flocculation characteristics of
bioflocculant, the effects of dosage of bioflocculant, various
salts and pH of solution were examined. Flocculating tests
were conducted using the method described above. The
dosage of bioflocculant was varied from 0 to 1.4 ml per
100 ml solution. Solutions of NaCl, MgCl2, CaCl2, FeCl3
and AlCl3 were used as cations sources and the concentra-
tion and dosage were the same as that of CaCl2. The pH of
the kaolin suspension was adjusted using HCl and NaOH
in the pH range of 3–13.
The river water was collected from Xiaoqing River of
Jinan, Shandong Province and other wastewaters were col-
lected from local industrial plants (Jinan, China). All
wastewaters were stored at 4 C before flocculation. A dose
of 0.4 mg/l bioflocculant and 1 ml 1% CaCl2 solution was
added to the 100 ml wastewaters at pH 7.0. After the addi-
tion of bioflocculant, the compound in the beaker was
mixed using a blender at 200 rpm for 1 min, and then at
40 rpm for another 3 min. The wastewater was left to settle
for 10 min, and then the supernatant was taken for analy-
sis. To be compared with chemically synthesized floccu-
lants, the bioflocculant was replaced by polyacrylamide
(PAM), polyaluminum chloride (PAC) and AlCl3 which
were purchased from Bin Zhou Chemical Co. (Shandong,
China).
The residual COD, turbidity and color were determined,
and the removal efficiency was calculated as follows:
Removal efficiency ð%Þ ¼ ðC 0  CÞ=C 0  100 ð2Þ
where C0 is the initial value and C is the value after the floc-
culation treatment.
4670 W.-X. Gong et al. / Bioresource Technology 99 (2008) 4668–4674
3. Results and discussion
3.1. Screening and identification of bioflocculant-producing
bacteria
About 17 types of bioflocculant-producing bacteria were
screened from soil and activated sludge samples on the
basis of a flocculation activity over 80%. Among these bac-
teria, strain S-14, with a flocculating activity exceeding 90%
was the most effective bacterium. The colony of S-14 was
circular, milk white, and smooth. The bacterium was rod-
shaped, Gram-negative, with flagellum and motile. The
strain was identified as S. ficaria using BIOLOG by the
State Key Lab Microbial Technology (Jinan, China). The
bioflocculant produced by strain S-14 was named as SF-1.
3.2. Bioflocculant production
3.2.1. Effect of carbon source, nitrogen source and various
salts
The composition of medium was optimized, including
carbon source, nitrogen source and salts. As shown in
Table 1, it is evident that lactose, glucose and ethanol were
suitable for the production of bioflocculant by S-14, with
the flocculating efficiency exceeding 90% after 72 h cultiva-
tion. And lactose was the best carbon source for the pro-
duction of the bioflocculant, and the flocculating activity
and bioflocculant production achieved were 97.15% and
2.41 g/l, respectively.
Table 1 also showed the flocculation activity after 72 h
cultivation, with 1% (w/v) nitrogen sources (beef extract,
urea, peptone, (NH4)2SO4 and yeast extract) replacing the
combination of beef extract (0.05%) and urea (0.05%).
Urea, peptone, (NH4)2SO4 were not favorable for bioflocc-
ulant production. In contrast, beef extract and yeast extract
were more suitable for bioflocculant production. But,
higher flocculating activity (94.1%) was obtained when com-
plex nitrogen source consisting of beef extract and urea was
used. So, complex nitrogen source was better than the sole
inorganic or organic nitrogen sources for S-14.
The effect of various salts on bioflocculant production
was examined (Table 1). KH2PO4 was replaced by various
salts at the same concentration. The flocculating activity
was stimulated by KCl, FeCl3, Ba(NO3)2, CaCl2 but was
negatively affected by CuSO4 and MgSO4. Combination
Table 1
Effects of composition of medium on the bioflocculant production
Carbon source Starch Glucose
Flocculating activity (%) 56.76 96.69
Nitrogen source Beef extract Urea
Flocculating activity (%) 87.6 45.9
Salts KCl FeCl3
Flocculating activity (%) 76.83 74.6
Salts MgSO4 K2HPO4
Flocculating activity (%) 41.59 53.33
of KH2PO4 and K2HPO4 provided the best inorganic salts
for S-14 in the production of the bioflocculant. Further
investigation revealed that the pH of the culture broth
reduced after cultivation. Combination of KH2PO4 and
K2HPO4 is a good buffer; it could alleviate the effect of
pH change during cultivation.
3.2.2. Utilization of some cost-effective medium
Bioflocculants may be produced relatively inexpensively
by means of some cost-effective fermentation medium (He
et al., 2004). Four wastewaters were used as a substrate for
the production of bioflocculant and results were shown in
Table 2. Dairy wastewater, soy sauce brewing wastewater
and meat processing wastewater were each deemed as suit-
able substrates for bioflocculant production in presence of
5% ethanol. Unknown fermentation products in brewery
wastewater are not biodegradable and may suppress the
microorganism. The highest flocculating activity of 95.3%
was observed for the dairy wastewater medium. Table 2
also showed that no additional nitrogen was needed. So,
dairy wastewater with 5% added ethanol is a candidate of
low cost substrate for bioflocculant production.
3.2.3. Effects of pH and inoculum size
The effect of the initial pH of the medium and inoculum
size on the flocculating ability of the culture broth was
examined (details not shown). Over the pH range of 4.5–
10, the flocculating efficiency was 63–95%. The optimal
pH for the bioflocculant production was in the range of
6–8. Lower pH was seemly more favorable for the bioflocc-
ulant production. The flocculating activity initially
increased with inoculum size. At the inoculum size of 1%,
the maximum flocculating activity was obtained. However,
any further increase in inoculum size did not result in any
higher flocculating activity. These results are in agreement
with previous findings (Zhong and Yoshida, 1995; Kallos
and Behie, 1999; Jang et al., 2001). As a result, an inoculum
size of 1% was used for all subsequent cultures.
3.2.4. Time course of the production of SF-1
Fig. 1 showed how bioflocculant production varied dur-
ing a growth curve of S. ficaria. The flocculating activity
reached its maximum flocculating activity in early station-
ary phase (at 72 h), which indicated that the bioflocculant
was produced by biosynthesis during its growth. Due to
Sucrose Lactose Ethanol
62.16 97.15 91.11
Peptone (NH4)2SO4 Yeast extract
56.3 75.2 89.3
CaCl2 Al2(SO4)3 CuSO4
69.84 48.15 42.22
Na2HPO4 Ba(NO3)2 0.2% K2HPO4 + 0.5% KH2PO4
57.14 73.33 96.34
 W.-X. Gong et al. / Bioresource Technology 99 (2008) 4668–4674 4671

Table 2
100
Characteristics and utilization of four low-cost culture mediums

Flocculating Activity (%)

BW DW SW MW
80
COD (mg/l) 953 1092 892.3 1265
TN (mg/l) 352 593 478.1 683
Flocculating activity (%) 75.2 95.5 94.3 92.3 60

40 SF-1 after 1st passage

SF-1 after 2nd passage
100 8 SF-1 after 3rd passage
20
SF-1
Flocculating Activity (%)

80
6 0
0 2 4 6 8 10 12 14 16
60 Time (d)

40
Flocculating Activity
20
pH
0 0
0 1 2 3 4 5
Time (d)
Fig. 1. The time course of SF-1 production.
cell autolysis and enzymatic activity decreasing the floccu-
lating activity started to decrease slowly after 84 h. The
maximum flocculating activity was 94.3% which is a little
lower than 97% of Bacillus sp. DYU1 (Wu and Ye,
2007), 98% of Citrobacter sp. TKF04 (Fujita et al., 2000),
98.1% of Aspergillus parasiticus (Deng et al., 2005) and
99% of Bacillus mucilaginosus (Deng et al., 2003). However,
using the dairy wastewater medium, flocculating activity of
95.3% was observed. Considering the difference of experi-
mental methods and the utilization of low-cost medium,
the potential of SF-1 was in consideration.
Corresponding to the profile of flocculating activity, the
pH profile showed that the pH decreased from 7.0 to 5.3
within early 56 h, followed by a slight drop till the end.
The reciprocal change of pH and flocculating activity con-
firms the production of organic acids in the bioflocculant
production (Dermlim et al., 1999; Deng et al., 2003; Lu
et al., 2005).
pH
4
Fig. 2. Effect of passage on the flocculating activity.
2 ficaria was cultured at the same conditions. As shown in
Fig. 3, at the beginning of the experiment, the flocculating
activity was much lower than that of both strains. The floc-
6 culating activity increased to 89.6% at day 3 and kept at the
average of both strains. After the initial competition for
nutrient and acclimation no significant interaction between
two strains was observed. So, Mixed bioflocculant-produc-
ing strains seems to be available in bioflocculant
production.
3.3. Characteristics of the bioflocculant
Chemical analysis showed that the major component of
the bioflocculant was neutral sugar and did not contain any
protein. From the infrared spectrum, the characteristic
chemical groups were analyzed. The absorption peak at
3425 cml was characteristic of OH stretching from the
hydroxyl group, and adsorbed water molecules. The peak
from 2926 cml was an indication of aliphatic C–H stretch-
ing. The absorption peaks around 1600 cml and
1075 cml were characteristics of C@O and C–O groups,
respectively. The strong absorption peaks observed in the
100
Flocculating Activity (%)

80
3.2.5. Effect of passage and competition of other
bioflocculant-producing bacterium 60
SF-1 production from S. ficaria after 3 passages was S-1
shown in Fig. 2. The flocculating activity of SF-1 from sub-
40 KL
cultured micrograms fluctuated between 78% and 95%,
close to that of primordial bacterium. And the time courses S-1+KL
20
of S. ficaria after 3 passages are similar to that of seed
bacterium. The passaged cells keep their fine biological
behavior, which could be further served as seeds. So the 0
1 2 3 4 5 6 7
pilot-scale and large-scale production may be available.
Time (d)
In previous study Klebsiella mobilis was screened as a
bioflocculant-producing bacterium (Dermlim et al., 1999; Fig. 3. Effect of other bioflocculant producing bacterium Klebsiella
Wang et al., 2007). The mixed strain of K. mobilis and S. mobilis (KL) on the flocculating activity.
4672 W.-X. Gong et al. / Bioresource Technology 99 (2008) 4668–4674

range from 1000 to 1200 cml were generally known to be
typical characteristics of all sugar derivatives. The infrared
spectrum of SF-1 thus indicates the presence of carboxyl
and hydroxyl groups (Suh et al., 1997; Deng et al., 2003;
Lu et al., 2005; Yim et al., 2007). This is in agreement with
the chemical analysis results that SF-1 is polysaccharide.
The weight average molecular weight (Mw) of SF-1 was
determined as 3.13 · 105 Da by GPC. Compared to that
of other bacteria, Mw is a little low in the Mw range of poly-
saccharide (Deng et al., 2005; Wu and Ye, 2007).
3.4. Application of bioflocculant
3.4.1. Distribution of the flocculating activity in the culture
To investigate the distribution of the flocculating activ-
ity the flocculating activity of culture broth, cell free super-
natant supernatant (centrifuged at 5000g for 30 min) and
supernatant after ethanol precipitation of cell-free superna-
tant was determined. The flocculating activity of culture
broth and cell-free supernatant was 12% and 96%, respec-
tively, which indicts that most of the flocculating activity
was in the supernatant. Similar to previous findings (Suh
et al., 1997) after ethanol precipitation the flocculating
activity was only 8%. The supernatant after centrifugation
showed the best performance and could be directly used in
practice.
3.4.2. Thermal stability of the bioflocculant
Effect of heat on the SF-1 was shown in Fig. 4. The floc-
culating activity of the bioflocculant decreased by 15%
after being heated to 100 C for 15 min and by 20% after
being heated to 50 C for 30 min. After being heated to
80 C for 30 min the flocculating activity fluctuated. The
thermal stability was presumably because the main back-
bone of this bioflocculant was a polysaccharide (Lu et al.,
2005). Higher temperature induced higher flocculating
activity. This may be explained by the release of extra-
3.4.3. Flocculating characteristics
The effect of bioflocculant dosage (Fig. 5) showed that
flocculating activity was over 90% in the range of 0.2–
0.4 ml and the maximum flocculating activity of 96.1%
was observed at an optimum bioflocculant dosage of
0.4 ml. The dosage of bioflocculant in the form of culture
broths was 4 ml/l which is low in the range of 1.0–
150 ml/l (Deng et al., 2003). Fig. 5 also showed that higher
or lower dosage induced the lower efficiency. When bio-
flocculant is insufficiency, the bridging phenomena can
not effectively form. And the restabilization of kaolin par-
ticles can be explained by the change of charge of kaolin
particle after over addition of bioflocculant. Over addition
of negatively charged bioflocculant cause the competition
and repulsion of negatively charged kaolin particles and
reduced effective volume also lead to poor settability.
To achieve high flocculating activity, metal cations are
often added. Table 3 showed the effect of cations on floccu-
lation. Apparently, divalent cations (Ca2+ and Mg2+) were
more effective than monovalent (Na+) and trivalent cations
(Al3+ and Fe3+). Ca2+ and Mg2+ could destabilize the neg-
atively charged kaolin particle by neutralizing and bridg-
ing. However, trivalent cations could change the surface
charge of kaolin particle and cover the adsorb sites. The
competition of the positively charged particles and less
adsorb sites induce the low flocculating activity. And corre-
sponding to the high flocculating activity (Fig. 5 and Table
3) the zeta potential is in some range. So, the charge neu-
tralization plays an important role in the flocculation.
The bioflocculant SF-1 significantly improved the sepa-
ration of kaolin particle in the presence of Ca2+. The opti-
mization of dosage of Ca2+ showed that the optimum Ca2+
100 0
Flocculating Activity (%)

80 -3

Zeta Potential (mv)

and inner-cell polysubstances after heat at higher
temperature.
60 -6
Flocculating
40 Activity -9
Zeta
100 Potential
20 -12
Flocculating Activity (%)

80
0 -15
0 0.2 0.4 0.6 0.8 1 1.2
60
Dosage of bioflocculant (ml)
40 Fig. 5. Effect of dosage of bioflocculant on the flocculating activity.
50ºC 80ºC 100ºC
20
Table 3
0 Effect of cations on the flocculation
0 5 10 15 20 25 30 35 Cations Al3+ Fe3+ Ca2+ Mg2+ Na+
Heat Time (min) Flocculating activity (%) 86.81 82.58 94.75 90.53 83.23
Zeta potential (mV) 24.1 26.3 15.8 12.2 18.6
Fig. 4. Thermal stability of the bioflocculant SF-1.
 W.-X. Gong et al. / Bioresource Technology 99 (2008) 4668–4674 4673

Table 4 Table 6
Flocculation of river water using bioflocculant Flocculation of pulp and paper mill effluent using bioflocculant
COD (mg/l) Turbidity Color COD (mg/l) OD660 Color
Blank 205 19.0 135.2 Blank 6535 0.652 30,000
PAC 30.5 (85.1%) 2 (89.5%) 18 (86.7%) AlCl3 2937 (55.1%) 0.012 (98.2%) 34 (99.9%)
PAM 36.1 (82.4%) 2 (89.5%) 15 (88.9%) PAM 2461 (62.3%) 0.011 (98.3%) 24 (99.9%)
SF-1 26.2 (87.1%) 3 (84.2%) 13 (90.4%) SF-1 1823 (72.1%) 0.012 (98.2%) 30 (99.9%)

concentration was 2 ml/100 ml suspension. The effect of
pH was also investigated (data not shown). With the
increase of pH flocculating activity increased to the maxi-
mum value at pH 6.0 and slowly decreased. Optimum pH
was in the weakly acidic or near neutral range of 5.0–7.0.
The optimal acidic pH is similar to results of other bio-
flocculants (Fujita et al., 2000; Yim et al., 2007). This is
due to the functional groups of SF-1. The acidic carboxyl
and hydroxyl groups could keep flocculating ability of
SF-1 in acidic pH.
3.4.4. Flocculation of real wastewaters
In this study, it was confirmed that SF-1 possesses high
flocculating activity in kaolin suspensions, and its floccula-
tion properties in real wastewater were investigated.
According to the content of soluble and particulate sub-
stances, river water, agricultural products processing
wastewater and pulp and paper mill effluent were selected
to assess the flocculating activity of SF-1.
River water is one of typical surface water with low
COD and turbidity. The results of treatment using SF-1
were shown in Table 4. The removal efficiency of COD,
turbidity and color were 87.1%, 84.2%, and 90.4%, respec-
tively. The COD could be reduced below 30 mg/l. The
results were compared with several commonly used chemi-
cally synthetic flocculants and SF-1 achieved the slightly
better flocculating efficiency in the clarification of river
water.
Three agricultural products processing wastewater,
brewery wastewater, meat processing wastewater and soy
sauce brewing wastewater were tested to investigate the
industrial application potential of SF-1. The results of
COD and turbidity removal were shown in Table 5. The
removal efficiency of COD was 60–80% and is similar to
the PAC and PAM. The removal of COD is similar to
the result of starch wastewater treatment using biofloccu-
lant MBFA9 (Deng et al., 2003). The turbidity removal
of SF-1 was 91.8–93.7% which is better than that of PAC
and PAM. And the color removal of SW using PAC,
PAM and SF-1 were 98.2%, 97.5% and 97.1%, respectively
(data not shown). These results show the feasibility of the
use of the bioflocculant in wastewater treatment for some
agricultural products processing industries.
Pulp and paper mill effluent was hard treating wastewa-
ter because of the nonbiodegradable substances and com-
plex composition. Bioflocculant was used to treat the
effluent for the first time in this research and comparison
with flocculants currently used in the wastewater treatment
of the plant was also carried (Table 6). The COD removal of
AlCl3, PAM, and SF-1 was 55.1%, 62.3% and 72.1%,
respectively. The comparison of COD removal reveals that
SF-1 had stronger flocculating activity. The color removal
efficiencies of three flocculants were 99.9%. Though high
removal efficiency was due to the high C0, low residual color
indicates that color removal by bioflocculant is consider-
able. The bioflocculant can effectively decolor some syn-
thetic dye solution (Deng et al., 2005). When real
wastewater was treated the decolorization efficiency of bio-
flocculant from Alcaligenes latus was also high (Kurane and
Nohata, 1991). This could mean that the soluble and partic-
ulate dyes can be simultaneously removed by bioflocculant.
4. Conclusions
A bioflocculant-producing bacterium, named S-14, was
isolated from soil and identified as S. ficaria. Chemical
analysis showed that the novel bioflocculant was a polysac-
charide where no amino acids were detected. Analysis of
GPC revealed that Mw was 3.13 · 105 Da. Bioflocculant
production experiments showed the optimal culture condi-
tions: the carbon of lactose, the complex nitrogen source
consisting of beef extract and urea, combination of
KH2PO4 and K2HPO4, an inoculum size of 1% (v/v), initial
pH 6–8 and 72 h cultivation. Using optimized conditions
for the production of the bioflocculant, a flocculating activ-
ity of 95.4% was obtained. It could utilize some wastewater
in agricultural products processing for its growth as the
carbon sources when 5% ethanol was supplemented. The

Table 5
COD and turbidity removal of agricultural products processing wastewater using bioflocculant
BW MW SW
COD (mg/l) Turbidity COD (mg/l) Turbidity COD (mg/l) Turbidity
Blank 784 88.8 556 121.6 1207 79.0
PAC 65.1% 90.8% 76.4% 88.1% 71.3% 89.9%
PAM 73.1% 93.0% 75.3% 88.9% 72.5% 87.3%
SF-1 80.7% 91.8% 76.3% 93.7% 64.1% 93.7%
4674 W.-X. Gong et al. / Bioresource Technology 99 (2008) 4668–4674
bioflocculant SF-1 was effective for flocculation of a kaolin
suspension over a acidic range of pHs (5–7). The flocculat-
ing activity was enhanced by Ca2+ and Mg2+, but was
reduced by Na+, Al3+ and Fe3+. SF-1 was able to floccu-
late several types of real wastewaters, such as river water,
brewery wastewater, soy sauce brewing wastewater and
meat processing wastewater. When treating pulp effluent
the removal rate of color and COD were up to 99.9%
and 72.1%, respectively. The removal efficiencies of real
wastewaters using SF-1 was similar and even better than
that using chemically synthetic flocculants.
Acknowledgement
This work was sponsored by Jinan Star Program, PR
China.
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