Bioresource Technology 127 (2013) 489–493

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Bioresource Technology
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Production and characterization of a bioflocculant produced by Aspergillus flavus

Ahmad H. Rajab Aljuboori a,⇑, Azni Idris a, Norhafizah Abdullah a, Rosfarizan Mohamad b
a
Department of Chemical and Environmental Engineering, University Putra Malaysia, Sri Serdang 43400, Selangor, Malaysia
b
Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, Sri Serdang 43400, Selangor, Malaysia

h i g h l i g h t s

" Cation-independent bioflocculant produced by Aspergillus flavus.

" Sucrose and peptone are the most favorable sources for bioflocculant production.
4
" The bioflocculant mainly consists of polysaccharide and protein with a molecular weight of 2.57  10 Da.
" The bioflocculant is stable at wide ranges of pH and temperature.

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

Article history: The production and characterization of a bioflocculant, IH-7, by Aspergillus flavus was investigated. About
Received 13 December 2011 0.4 g of purified bioflocculant with an average molecular weight of 2.574  104 Da could be obtained
Received in revised form 28 July 2012 from 1 L of fermentation medium. The bioflocculant mainly consisted of protein (28.5%) and sugar
Accepted 7 September 2012
(69.7%), including 40% of neutral sugar, 2.48% of uronic acid and 1.8% amino sugar. The neutral sugar
Available online 14 September 2012
components are sucrose, lactose, glucose, xylose, galactose, mannose and fructose at a molar ratio of
2.4:4.4:4.1:5.8:9.9:0.8:3.1. Fourier-transform infrared spectroscopy analysis revealed that purified IH-7
Keywords:
contained hydroxyl, amide, carboxyl and methoxyl groups. The elemental analysis of purified IH-7
Aspergillus flavus
Bioflocculant
showed that the weight fractions of the elements C, H, O, N and S were 29.9%, 4.8%, 34.7%, 3.3%, and
Microbial flocculant 2.0%, respectively. IH-7 had good flocculating rate in kaolin suspension without cation addition and stable
Bioflocculant stability over wide range of pH and temperature.
Extracellular biopolymeric substance Ó 2012 Published by Elsevier Ltd.

1. Introduction Bioflocculants are mainly composed of macromolecular sub-

Flocculants are useful agents in the aggregation of colloids, cells
and suspended solids and are commonly used for drinking water
production, waste water treatment, fermentation processes, and
food production (Shih et al., 2001). These flocculants are usually
classified into three groups: (1) inorganic flocculants such as
polyaluminum chloride, (2) organic synthetic flocculants such as
polyacrylaminde derivatives and (3) naturally occurring floccu-
lants such as chitosan. Inorganic and organic synthetic flocculants
are widely used in industrial fields because of their cost effective-
ness and efficiency, although their use may incur some environ-
mental and health problems (Liu et al., 2009). In contrast,
bioflocculants, extracellular biopolymeric substances secreted by
bacteria, fungi, algae and yeast (Salehizadeh and Shojaosadati,
2001) are biodegradable and nontoxic flocculants (Dermlim et al.,
1999).
⇑ Corresponding author. Address: Chemical and Environmental Engineering
Department, Faculty of Engineering, University Putra Malaysia. Tel.: +60 12 357
5668; fax: +60 38 656 7120.
E-mail address: ahmaddjla2001@yahoo.com (Ahmad H. Rajab Aljuboori).
stances, such as polysaccharide and protein (Lu et al., 2005; Zheng
et al., 2008). The composition and properties of bioflocculants de-
pend on type of bioflocculant-producing microorganisms (BPMs),
composition of media and environmental conditions. The differ-
ences in the composition and properties of polysaccharides and
proteins lead to differences in the charge of bioflocculant (Bala
Subramanian et al., 2010). The flocculation ability of bioflocculants
produced by Vagococcus sp. (Gao et al., 2006), Halomonas sp. (He
et al., 2010), Bacillus circulans (Li et al., 2009a), Pseudoalteromonas
sp. (Li et al., 2008), Serratia ficaria (Gong et al., 2008), Bacillus
licheniformis (Li et al., 2009b), and Klebsiella mobilis (Wang et al.,
2007) depends on the presence of cations. Ca2+ is known to link
negatively charge groups on flocculants and particles (He et al.,
2010). Addition of cations to the flocculation process can produce
secondary pollution and increase costs. Thus, finding cation-
independent bioflocculants is desirable.
In the present study, the production of a bioflocculant, IH-7,
produced by Aspergillus flavus, was investigated to determine opti-
mal culture medium composition and environmental conditions.
Various factors influencing the production of IH-7, like carbon
source, nitrogen source, C/N ratio, initial pH of culture medium,

0960-8524/$ - see front matter Ó 2012 Published by Elsevier Ltd.

http://dx.doi.org/10.1016/j.biortech.2012.09.016
490 Ahmad H. Rajab Aljuboori et al. / Bioresource Technology 127 (2013) 489–493
culture temperature, metal ions, inoculum size and time course
were investigated. The composition and properties of the IH-7
were investigated as well.
2. Materials and methods
2.1. Microorganism and culture conditions
A. flavus S44-1, isolated by the Department of Biotechnology
and preserved at the Microbial Culture Collection Unit (UNiCC),
Laboratory of Industrial Biotechnology, Institute of Bioscience, Uni-
versity Putra Malaysia (Mohamad and Ariff, 2007) (Selangor,
Malaysia), was maintained on slant media at 4 °C and sub-cultured
every 30–40 days. The medium for slant and subculture consisted
of (g/l): potato extract, 4; glucose, 20; agar, 15; and the initial pH
was adjusted to 5.6 ± 0.2. The production medium consisted of
(g/l): sucrose, 30; peptone, 3.0; MgSO47H2O, 0.5; KCl, 0.5; FeSO4,
0.01; K2HPO4, 1.0; and the initial pH was adjusted to 6.0. The fun-
gus was cultured in 100-mL Erlenmeyer flasks containing 50 ml of
medium and incubated in a shaker at 180 rpm for 3 days at 30 °C.
Samples were taken at different time intervals to determine floccu-
lation rate and fungal biomass weight. The biomass samples were
filtered and dried at 80 °C in an oven for 4 h (Deng et al., 2005). Dis-
tilled water was used to prepare all medium solutions and media
were sterilized at 121 °C for 20 min.
2.2. Optimization of culture conditions of A. flavus for IH-7 production
Nine factors including carbon source, nitrogen source, C/N ratio,
initial pH, culture temperature, inoculum size, metal ions and cul-
ture time were investigated. To determine the effect of carbon and
nitrogen sources on bioflocculant production, sucrose was replaced
with glucose, fructose, lactose, starch, and glycerol (30 g/l for each
type of carbon source) and peptone was replaced with (NH4)2 SO4,
NH4 NO3, NaNO3, yeast extract, urea and glutamic acid (3 g/l for
each type of nitrogen source). To study the effect of metal ions
on bioflocculant production, KCl was replaced with NaCl, CaCl2,
MgCl2, MnCl2 and FeCl3 at same concentration. For the C/N ratio
different concentrations of sucrose were used in order to get the
different C/N ratio of 0/1–50/1 while peptone kept constant. In ini-
tial pH of production media were adjusted at 2–9. Temperature of
production media were adjusted at 15–45 °C. Inoculum size used
0.2–10% v/v. The effect of time course of IH-7 production was
investigated between 0 and 96 h. All the experiments were con-
ducted in triplicate.
2.3. Bioflocculant purification
Two volumes of cold ethanol (at 4 °C) were added to 1 L culture
broth (supernatant).
The precipitate was dissolved in 100 ml deionized water and
50 mL of 2% cetylpyridinium chloride solution (CPC) was added
to the solution and thoroughly mixed. After three hours, the pre-
cipitate was collected and dissolved in 100 mL of 0.5 M NaCl.
Two volumes of cold ethanol were added and the precipitate was
washed with ethanol, Dissolved in 5 mL of deionized water and
vacuum-dried (Deng et al., 2005).
2.4. Physical and chemical analysis of IH-7 bioflocculant
The total sugar content of IH-7 bioflocculant was determined
according to the phenolsulfuric acid method using glucose as stan-
dard (Chaplin and Kennedy, 1994). The total protein content was
determined by the Bradford method with bovine serum albumin
as standard (Bradford, 1976). The uronic acid was determined
using the carbazole–sulfuric acid method (Chaplin and Kennedy,
1994). Amino sugars were determined using the Elson–Morgan
method with glucose amine as standard (Chaplin and Kennedy,
1994). In order to determine of its sugar composition, IH-7 was
hydrolyzed with trifluoroacetic acid at 121 °C for 1 h. The resulting
sugars were analyzed by high-performance liquid chromatography
(HPLC) with a Rezex RCMMonosaccharide Ca2+ 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 pres-
sure of 331 psi. Elemental analysis was carried out with a Truspec
CHN/CHNS elemental analyzer (LECO, USA). The functional groups
of IH-7 were determined with a Spectrum 100 FTIR spectrometer
(PerkinElmer, USA). The molecular weight of IH-7 bioflocculant
was measured by a Waters-Alliance 2695 GPC system (Waters,
USA), equipped with a Waters 2410 Reactive Index Detector
(RID) and a Waters Ultrahydrogel, linear column (7.8  300 mm).
The mobile phase was 0.1 M sodium nitrate at a flow rate of
0.6 ml/min and the column was operated at room temperature.
The injection volume was 20 lL. A calibration curve was con-
structed using dextran T standards. The average molecular weight
was calculated using Empower soft ware (System Software, Em-
power option GPC, Waters Co.)
2.5. Determination of flocculating rate
A kaolin suspension was used to determine the flocculating rate
of the bioflocculant in culture broth. Two gram of Kaolin clay
(Merck, Germany) was suspended in 1 L of deionized water. One
ml of culture broth was added to 99 ml of 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 80 rpm for 5 min, and allowed to stand for 5 min using
jar tester (JLT6, VELP SCIENTIFICA, Italy). The optical density (OD)
of the supernatant was measured with a spectrophotometer
(GENESYS 10 UV, Thermo Scientific, USA) at 550 nm. In the control
experiment, 1 ml of culture broth was replaced with 1 ml of fresh
culture medium. The flocculating rate was calculated according to
the following equation:
Flocculating rate ð%Þ ¼ ðA550  B550Þ=A550  100
where A550 and B550 were the OD550 (optical density at 550 nm)
of control and sample supernatant, respectively.
2.6. pH stability of purified IH-7
Pure IH-7 was dissolved in a suitable volume of deionized water
to achieve an initial flocculating rate of over 90% and divided into
eight aliquots. The pH of the aliquots was adjusted to 3, 4, 5, 6, 7, 8,
9 and 10 with 1 M of NaOH or HCl. The aliquots were kept at 4 °C
for 24 h (He et al., 2004), and the flocculating rates were measured
at room temperature, kaolin suspension 2 g/l adjusted at pH 7, the
mixture stirred at 200 rpm for 1 min, slowly stirred at 80 rpm for
5 min, and allowed to stand for 5 min.
2.7. Thermo-stability of purified IH-7
Pure IH-7 was dissolved in a suitable volume of deionized water
to achieve an initial flocculating rate of over 90%, and divided into
six 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, 60, 80 and 100 °C for
1 h in a water bath (He et al., 2004), and the flocculating rates were
measured at room temperature, kaolin suspension 2 g/l adjusted at
pH 7, the mixture stirred at 200 rpm for 1 min, slowly stirred at
80 rpm for 5 min, and allowed to stand for 5 min.
 Ahmad H. Rajab Aljuboori et al. / Bioresource Technology 127 (2013) 489–493 491

3. Results and discussion

3.1. IH-7 production

3.1.1. Effects of carbon, nitrogen sources and C/N ratio on IH-

7production
Fig. 1(a and c) shows the flocculating rate of IH-7 after 72 h of
cultivation in media containing glucose, fructose, lactose, starch
or glycerol instead of sucrose. Sucrose, glucose and starch were
favorable for IH-7 production, while the production of IH-7 was
relatively low when fructose and glycerol were used. The highest
production of IH-7 was achieved in sucrose medium. Starch,
sucrose and glucose were also reported as the favorable carbon
sources for Aspergillus parasiticus in the production of bioflocculant
(Deng et al., 2005). Organic nitrogen sources (peptone, yeast ex-
tract and urea) were effectively used in the production of IH-7 by
A. flavus, while (NH4)2SO4 and NaNO3 led to poor IH-7 production
(Fig. 1c). At a fixed concentration of peptone of 7.521 g/L (1 g N/
l), a rapid increase in IH-7 production was noticed when the C/N
ratio was increased up to 5/1 (Fig. 1d), but a further increase in
the C/N ratio caused a decrease in the production of IH-7.

3.1.2. Effect of the initial pH, temperature, inoculum size and metal
ions on IH-7 production
Fig. 2 shows the effect of the initial pH of the culture medium on
IH-7 production. The initial pH of the culture medium can affect
the nutrient absorption and enzymatic reaction of bioflocculant-
producing microorganisms (Xia et al., 2008). The highest flocculat-
ing rate was recorded between 5 and 9, and the production was
highest at pH 7. Fig. 3a shows the effect of culture temperatures
on the production of IH-7. Cultures grown at 25 °C and above of
culture temperature, showed increasing flocculating rates that
reached 86.2% at 40 °C. The flocculating rate of IH-7 obtained from
cultures inoculated with 0.2–10% (v/v) are shown in Fig. 3b. The
use of 2% of A. flavus inoculum size in production medium recorded
the highest flocculating rate of 86.6%. IH-7 production by A. flavus
was stimulated in the presence of Na+, K+, Ca2+, Mg2+ and Mn2+ in
culture medium, while IH-7 production was inhibited by Fe3+
(Fig. 4) even though Fe3+ was the most favorable metal ion for
the biomass growth.

3.1.3. Time course of the production of IH-7

Fig. 5 shows IH-7 production during growth of A. flavus. Overall,
the production of IH-7 paralleled the growth of the biomass. The
flocculating rate of culture supernatants from early stationary cul-
tures was the highest (87.2% at 60 h). The same phenomenon was
reported in the cultivation of A. parasiticus (Deng et al., 2005),
Bacillus mucilaginosus (Deng et al., 2003), Bacillus subtilis DYU1(Wu
and Ye, 2007) and Citrobacter sp. TKF04 (Fujita et al., 2000). At late
stationary phase, the flocculating rate started to decrease, it may
be due to the deflocculation enzymes activities (Li et al., 2009b).
After 72 h, the A. flavus reached its early dead phase, consequently,
the flocculating rate of the IH-7 decreased slowly. Corresponding
to the profile of flocculating rate, the pH profile showed that the
pH decreased from 7.0 to 5.3 within 48 h, then the pH slightly
dropped and increased again to 5.6 at 96 h. Consequently, a period
of 60 h was chosen as the culture time for the subsequent
experiments. Additionally, using the optimal culture composition
and conditions, about 0.4 g of pure IH-7 bioflocculant could be
obtained from 1 L culture broth. Fig. 1. The effect of carbon sources, nitrogen sources and C/N ratio on A. flavus
This study found the flocculating rate was increased as the bio- biomass and production of IH-7 bioflocculant: (a) The effect of carbon sources on
flocculant concentration increased. The same phenomenon has IH-7 production with peptone used in the medium as nitrogen source; (b) The effect
of nitrogen sources on IH-7 production with sucrose used in the medium as carbon
been reported by Li et al. (2008) where the flocculating activities source; (c) The effect of different carbon sources and nitrogen sources on
increased as the concentration of bioflocculant produced by bioflocculating rate; (d) The effect of C/N ratio of the production of IH-7. Error
Pseudoalteromonas sp. SM9913 increased. However, at very low bars indicate standard deviation of triplicate experiments.
492 Ahmad H. Rajab Aljuboori et al. / Bioresource Technology 127 (2013) 489–493

Flocculating rate Biomass 100 15

100 25 Flocculating rate 14
90
90 Biomass 13
80 pH 12
80 20
11
Flocculating rate (%)

70 70

Biomass )g/L)

Flocculating rate (%)

10
60 15
60 9

Biomass (g/L)
50 8
50

pH
40 10 7
40 6
30
5
20 5 30
4
10 20 3

0 0 2
10
1 3 5 7 9 1
pH
0 0
12 24 36 48 60 72 84 96
Fig. 2. The effect of initial pH of the medium on production of IH-7. Error bars Incubation period (h)
indicate standard deviation of triplicate experiments.
Fig. 5. Time course of the production of IH-7. Error bars indicate standard deviation
of triplicate experiments.
100 20

90 Flocculating rate 18
Biomass
3.2. Characterization of the purified bioflocculant IH-7
80 16
Flocculating rate (%)

70 14 3.2.1. Composition analysis of IH-7

Biomass (g/L)

Purified IH-7 consisted of 28.5% protein and 69.7% sugar, includ-

60 12
ing 40% neutral sugar, 2.48% uronic acid and 1.8% amino sugar. The
50 10 neutral sugar components of hydrolyzed IH-7 included sucrose,
40 8 lactose, glucose, xylose, galactose, mannose and fructose at a molar
ratio of 2.4:4.4:4.1:5.8:9.9:0.8:3.1. The elemental analysis of puri-
30 6
fied IH-7 showed that the mass proportion of C, H, O, N, and S were
20 4 29.9%, 4.8%, 34.7%, 3.3%, and 2.0% (w/w), respectively. Gel perme-
10 2 ation chromatography indicated that the average molecular weight
of IH-7 was 2.574  104 Da.
0 0
15 20 25 30 35 40 45

Temperature ( ° C) 3.2.2. Functional group analysis of IH-7

The IR spectrum of purified IH-7 bioflocculant displayed a broad
Fig. 3. (a) The effect of temperature of the medium on production of IH-7; (b) The
effect of inoculum size on production of IH-7. Error bars indicate standard deviation
stretching peak at around 3285 cm1, which indicates characteris-
of triplicate experiments. tic of hydroxyl groups, and a weak C–H stretching band at
2927 cm1. Other peaks at 1333 and 1633 cm1 were confirmed
to be the consequence of COO– bond asymmetric oscillation (Lian
Flocculating rate Biomass et al., 2008). The peak at 1538 cm1 could be attributed to NH
100 10
bending vibrations and the strong absorption peak presented at
90 9
1007 cm1 indicates C–O stretching vibration and the presence of
80 8 methoxyl groups, which are known to be typical characteristics
Flocculating rate (%)

70 7 of sugar derivatives (Zheng et al., 2008).

Bimass (g/l)

60 6
50 5
40 4
3.2.3. pH stability of IH-7
Fig. 6a shows that IH-7 was quite stable at wide range of pH be-
30 3
tween 3 and 7 and more than 90% flocculation was achieved at this
20 2
range. Thus, this bioflocculant is suitable to be applied in acidic and
10 1
neutral circumstances. While a pH higher than 7.0 decreased the
0 0
NaCl KCl CaCl MgCl MnCl FeCl Control
flocculation rate dramatically. This may be due to the bioflocculant
Metal ions
shows different electric states at different pH and that will affect
the flocculation ability of the bioflocculant for the kaolin particles
Fig. 4. The effect of metal ions on production of IH-7. Error bars indicate standard (Yong et al., 2009).
deviation of triplicate experiments.

3.2.4. Thermal stability of IH-7

concentration of IH-7, the flocculating rate was low. Because of Over 90% of flocculating rate was maintained at temperatures
insufficient concentration of bioflocculant was used to neutralize range of 10–100 °C, when the pH of the IH-7 was in the range of
the negative charges on kaolin particles. While, excessive concen- 3.0–7.0. The thermal stability of this bioflocculant might be be-
tration of bioflocculant led to restabilize the kaolin particles and cause the main backbone of IH-7 bioflocculant is a polysaccharide
decreased the flocculating rate (data not shown). (Lu et al., 2005).
 Ahmad H. Rajab Aljuboori et al. / Bioresource Technology 127 (2013) 489–493 493

research university grant (RUGS. 9199673). We would like to thank

a 100
Ibn Abubakar, B.S.U. for his valuable comments to this work.
90

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This study shows that A. flavus is a bioflocculant-producing
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