Bioresource Technology 171 (2014) 66–70

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Production of a bioflocculant from Aspergillus niger using palm oil mill

effluent as carbon source
Ahmad H. Rajab Aljuboori a,⇑, Yoshimitsu Uemura a, Noridah Binti Osman a, Suzana Yusup b
a
Centre for Biofuel and Biochemical Research (CBBR), Universiti Teknologi PETRONAS, 31750 Tronoh, Perak, Malaysia
b
Biomass Processing Lab, Universiti Teknologi PETRONAS, 31750 Tronoh, Perak, Malaysia

h i g h l i g h t s

 PM-5 is a new bioflocculant produced from Aspergillus niger.

 POME was first time used as carbon source for bioflocculant production.
1
 The maximum PM-5 production was 2.6 g L .
 PM-5 had great potential to treat river water from suspended solids.
2+
 PM-5 combined with Ca acted as stimulating agent.

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

Article history: This study evaluated the potential of bioflocculant production from Aspergillus niger using palm oil mill
Received 10 June 2014 effluent (POME) as carbon source. The bioflocculant named PM-5 produced by A. niger showed a good
Received in revised form 6 August 2014 flocculating capability and flocculating rate of 76.8% to kaolin suspension could be achieved at 60 h of cul-
Accepted 7 August 2014
ture time. Glutamic acid was the most favorable nitrogen source for A. niger in bioflocculant production at
Available online 19 August 2014
pH 6 and temperature 35 °C. The chemical composition of purified PM-5 was mainly carbohydrate and
protein with 66.8% and 31.4%, respectively. Results showed the novel bioflocculant (PM-5) had high
Keywords:
potential to treat river water from colloids and 63% of turbidity removal with the present of Ca2+ ion.
Aspergillus niger
Bioflocculant
Ó 2014 Elsevier Ltd. All rights reserved.
Extracellular biopolymeric substance
Microbial flocculant
Palm oil mill effluent

1. Introduction erties, biodegradability, eco-friendly and free of secondary

Flocculation process is widely used in various fields of indus-
tries, including food production, downstream of fermentation pro-
cess, water purification and wastewater treatment (Wang et al.,
2013a,b). Flocculating agents are generally divided into: inorganic
flocculants (aluminum sulfate and polyaluminium chloride),
organic synthetic flocculants (polyacrylamide), and naturally
occurring flocculants (bioflocculant, chitosan and guar gum)
(Aljuboori et al., 2013). Inorganic and organic synthetic flocculants
are commonly used in water and wastewater industries (Zhao
et al., 2013). However, the excessive use of these chemical floccu-
lants can cause health and environmental problems (Li et al.,
2013). Recently, most of bioflocculants have attracted high bio-
technological attention due to their outstanding flocculation prop-
⇑ Corresponding author. Tel.: +60 5 368 7645; fax: +60 5 368 7649.
E-mail address: ahmaddjla2001@yahoo.com (A.H.R. Aljuboori).
pollution risk (Guo et al., 2013; Zhang et al., 2013). However, high
cost of bioflocculant production related to expensive substrates has
limited the practical application and development of bioflocculant
(Zhao et al., 2012). Therefore, it is necessary to find low cost sub-
strates such as industrial biological waste to replace conventional
media substrates (sucrose, glucose, fructose) and reduce the bio-
flocculant production cost (Wang et al., 2013b; Zhang et al.,
2013; Zhao et al., 2012). For example, Wang et al. (2013b) reported
hydrolysate of corn stover was used as carbon source to produce
the bioflocculant from Ochrobactrum ciceri W2. Moreover, waste
fermenting liquor was successfully used as media substrate to pro-
duce bioflocculant from Bacillus subtilis (You et al., 2008).
Palm oil mill effluent from palm oil extraction process is one of
the most abundant agro-industrial waste containing high organic
matters produced in many countries including Malaysia, Indonesia
and India (Saidu et al., 2013). Typically 1 tonne of Crude Palm Oil
(CPO) production required 5–7.5 m3 of water and more than 50%

http://dx.doi.org/10.1016/j.biortech.2014.08.038
0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
 A.H.R. Aljuboori et al. / Bioresource Technology 171 (2014) 66–70
of the water ends up as POME. Malaysia is the first exporter and
second producer of CPO produced around 52.6, 51.4, 49.8, 55.7
and 55.4 million tonnes of POME in 2008, 2009, 2010, 2011 and
2012, respectively (Aljuboori, 2013). Thus, POME could be the most
available and sustainable organic source for biological products
industries, especially for bioflocculant production.
Therefore, the aim of this study are to investigate the bioflocc-
ulant production by A. niger using POME as carbon source, to deter-
mine optimal chemical and environmental conditions for
bioflocculant production and to investigate the composition and
flocculating properties of bioflocculant.
2. Methods
2.1. Substrate preparation
Raw POME collected from Felcra Nasaruddin, Felcra Oil Mill,
Bota, Perak, Malaysia, was used as substrate (carbon source) with
characteristics as follows: Total Carbon (TC) 11,891 mg L1; Total
Organic Carbon (TOC) 11,794 mg L1; Total Nitrogen (TN)
290.4 mg L1 and pH 4.6 (this sample was used for optimization
of PM-5 production).
Additionally, two samples of POME collected from different
palm oil mills were used to study the effect of POME chemical con-
tent on PM-5 production. The first sample was collected from Fel-
cra Berhad Bidor mill (B-POME), Perak, Malaysia, with
characteristics as follows: Total Carbon (TC) 12,720 mg L1; Total
Organic Carbon (TOC) 12,710 mg L1; Total Nitrogen (TN)
570.7 mg L1 and pH 4.5. The second sample was collected from
Felcra Berhad Seberang mill (S-POME), Perak, Malaysia, with char-
acteristics as follows: total Carbon (TC) 9722.5 mg L1; Total
Organic Carbon (TOC) 9715 mg L1; Total Nitrogen (TN)
402.1 mg L1 and pH 4.75. Samples were analyzed in triplicate.
The collected POME samples were stored at temperature of 4 °C.
2.2. Microorganism and growth conditions
A. niger, isolated by the Department of Biotechnology and pre-
served at the Microbial Culture Collection Unit (UNiCC), Laboratory
of Industrial Biotechnology, Institute of Bioscience, University
Putra Malaysia (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 L1): potato extract, 4; glu-
cose, 20 and agar, 15. Meanwhile the initial pH was adjusted to
5.6 ± 0.2.
2.3. Production of bioflocculant
The production medium consisted of (g L1): POME, 10 (TOC);
glutamic acid, 7.92; MgSO47H2O, 0.5; KCl, 0.5; FeSO4, 0.01;
K2HPO4, 1.0; and its initial pH was adjusted to 6.0. The fungus
was cultured in 100-mL Erlenmeyer flasks containing 50 ml of
medium and incubated in a shaker at 150 rpm for 3 days at
32 °C. Samples were taken at different time intervals to determine
flocculation rate and fungal biomass weight. The biomass samples
were filtered and dried at 105 °C in an oven for 2 h. Distilled water
was used to prepare all medium solutions and the media were
sterilized at 121 °C for 20 min.
2.4. Optimization of culture conditions of A. niger for bioflocculant
production
Six factors inclusive nitrogen source, C/N ratio, initial pH, cul-
ture temperature, metal ions and culture time were investigated.
To determine the effect of nitrogen sources on bioflocculant pro-
67
duction, glutamic acid was replaced with (NH4)2SO4, NH4NO3,
NaNO3, urea and yeast extract (1 g L1 nitrogen source).
For the C/N ratio, different concentrations of POME (TOC) were
used in order to get the different C/N ratio of 0/1–40/1. The initial
pH of production media were adjusted at 3–10. Temperature of
production media were adjusted at 25–40 °C. To study the effect
of metal ions on bioflocculant production, KCl was replaced with
NaCl, CaCl2, MgCl2, MnCl2 and FeCl3 at the same concentration.
The effect of time course of bioflocculant production was investi-
gated between 0 and 96 h. All experiments were conducted in
duplicate.
2.5. Purification of the bioflocculant
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 precipitate was collected and dissolved in 100 mL of
0.5 M NaCl. Two volumes of cold ethanol were added and the pre-
cipitate was washed with ethanol, dissolved in 5 mL of deionized
water and vacuum-dried (Aljuboori et al., 2013; Deng et al., 2005).
2.6. Analysis of bioflocculant
The total sugar content of bioflocculant was determined accord-
ing to the phenol sulfuric acid method using glucose as standard
(Dubois et al., 1956; Krishnaveni et al., 1984). The total protein
content was determined by the Bradford method with bovine
serum albumin as standard (Bradford, 1976). The functional groups
of bioflocculant were determined with a Spectrum One FT-IR spec-
trometer (PerkinElmer, USA). Elemental analysis of PM-5 was car-
ried out with a PerkinElmer Series II CHNS/O 2400 elemental
analyzer (USA).
2.7. Determination of flocculating rate of bioflocculant
A kaolin suspension was used to determine the flocculating rate
of the bioflocculant in culture broth. 2 g of Kaolin clay (Merck,
Germany) was suspended in 1 L of deionized water. One ml of cul-
ture broth and 0.5 ml of CaCl2 (10 mmol/L) were added to 198.5 ml
of kaolin suspension in a 500-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 60 rpm for 5 min, and
allowed to stand for 10 min using jar tester (FC6S, VELP SCIENTIF-
ICA, Italy). The optical density (OD) of the supernatant was mea-
sured with a spectrophotometer (DR5000 UN-VIS, HACH, 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 rateð%Þ ¼ ðA550  B550Þ=A550  100 ð1Þ
where A550 and B550 were the OD550 (optical density at 550 nm)
of control and sample supernatant, respectively.
2.8. Purification of river water
Different concentration (1–50 mg L1) of PM-5 bioflocculant and
CaCl2 (5 mmol/L) were added into 200 ml real river water obtained
from Perak river, Perak, Malaysia, mixed at 200 rpm for 1 min, and
then at 60 rpm for another 5 min, then allowed to stand for 10 min
using 6-breaker jar tester (FC6S, VELP SCIENTIFICA, Italy), and the
supernatant was taken for analysis. Turbidity was measured
according to the procedure of turbidity meter (2100Q, HACH,
USA). The pH value of water was measured by a pH meter (OAKTON,
EUTECH INSTRUMENTS, SINGAPORE). The Total suspended solids
68 A.H.R. Aljuboori et al. / Bioresource Technology 171 (2014) 66–70

(TSS) of river water was determined by 2540 D (APHA, 2005). The was the most favorable nitrogen source for PM-5 production and
turbidity removal was calculated as follows: used in the following experiments.
The effect of C/N ratio on bioflocculant production was investi-
Turbidity removal efficiency ð%Þ ¼ ðC a  C b Þ=C a  100 ð2Þ
gated in this study. Results showed the production of bioflocculant
where Ca is the initial turbidity value and Cb is the turbidity value gradually increased as the C/N ratio increased up to 20/1 with floc-
after treatment. culating rate of 79% (Fig. 1b), but further increased in C/N ratio
slightly decreased the PM-5 production. Thus, 20/1 ratio was cho-
sen for the next experiments.
3. Results and discussion

3.1. Bioflocculant production
3.1.1. Effect of POME, nitrogen sources and C/N ratio on bioflocculant
production
As shown in Fig. 1(a and b), POME was a good carbon source for
bioflocculant production by A. niger with the flocculating rates up to
80%. In order to maximize the bioflocculant production, organic and
inorganic nitrogen sources were used to find the most favorable
nitrogen source for A. niger. Glutamic acid (organic nitrogen) was
efficiently used in the production of PM-5 with the flocculating rate
up to 81%. Although yeast extract and urea were favorable nitrogen
sources for biomass growth, the flocculating rate showed not higher
than 65%. Similarly, Ugbenyen et al. (2012), reported yeast extract
and urea used for bioflocculant production were poorly utilized
by Cobetia sp. Moreover, the low productions of PM-5 and biomass
growth were observed when (NH4)2SO4, NH4NO3 and NaNO3 were
used as inorganic nitrogen sources. This result is in agreement with
Liu et al. (2010) finding, where inorganic nitrogen sources ((NH4)2-
SO4, NH4NO3 and NaNO3) led to poor production of bioflocculant
and cell growth of Chryseobacterium daeguense W6. Glutamic acid
Flocculating Rate
100
90
80
Flocculating rate (%)
3.1.2. Effect of the initial pH, temperature and metal ions on PM-5
production
For most microorganisms the microbial product such as bio-
flocculant is regularly increase between minimum and the opti-
mum pH, and a corresponding regularly decrease in microbial
product between the optimum and the maximum pH. This reflects
the effect of [H+] change on enzymatic reaction rates and nutrient
absorption. Fig. 2 shows the effect of initial pH of culture medium
on PM-5 production. The production of PM-5 dramatically
increased as the pH increased from 3 to 6, and about 77% of highest
flocculating rate was recorded at pH 6. While, culture medium
with initial pH between 7 and 10 showed low bioflocculant pro-
duction. Similarly, Mabinya et al. (2012), reported that biofloccu-
lant produced by Arthrobacter sp. at acidic pH range was higher
than basic pH range. Thus, pH 6 was selected as the initial pH in
the following experiments.
Physical conditions affect the microbial growth and productivi-
ties, as enzymatic activity of microorganism depends on tempera-
ture of culture medium. The production of bioflocculant (PM-5)
rapidly increased as the temperature of culture medium increased
from 25 to 30 °C, and then slightly increased as the temperature
reached 35 °C (Fig. 3). The highest flocculating rate was 76.3% at
Biomass 35 °C, further increased in culture medium temperature (40 °C) sig-
nificantly affected the PM-5 production. This may be due to the
(a) 14
enzymes of bioflocculant production are deactivated at 40 °C (Xia
12
et al., 2008). The optimal temperature range for bioflocculant-pro-

70
ducing microorganisms was reported to be between 25 and 37 °C
Biomass (g/L)

10
60
8 (Nam et al., 1996; Salehizadeh and Shojaosadati, 2001; Wu and
50
Ye, 2007). However, the A. niger growth gradually decreased as the
40 6
temperature increased from 25 to 40 °C. As shown, the optimum
30 4 temperature for PM-5 production was 35 °C, this temperature was
20
2 chosen for the following experiments.
10
In addition, PM-5 production by A. niger was stimulated in the
0 0
Yeast Glutamic Urea (NH ) NH NO NaNO Control presence of different metal ions Mn2+, Ca2+, K+, Mg2+ and Na+ in cul-
Extract acid SO (no ture medium with the flocculating rate of 74.7, 70.6, 69, 66.4 and
nitrogen
Nitrogen Source added) 56.2%, respectively. In contrast, Fe3+ poorly stimulates the PM-5
production with the flocculating rate of 26.1% (Fig. 4). Ugbenyen
Flocculating rate Biomass and Okoh (2013) also reported, metal ions such as Ca2+, Mn2+, K+,
100 Mg2+ and Na+ were significantly stimulated the bioflocculant
90
(b) 14

80 12 Flocculating rate Biomass

Flocculating rate (%)

70 100 20
10
Biomass (g/L)

60 90 18
8
50 80 16
Flocculating rate (%)

40 6 70 14
Biomass (g/L)

30 60 12
4
20 50 10
2 40 8
10
30 6
0 0
0/1 1/1 2/1 4/1 6/1 8/1 10/1 20/1 30/1 40/1 20 4
C/N ratio 10 2
0 0
Fig. 1. The effect of nitrogen sources and C/N ratio on production of PM-5 3 4 5 6 7 8 9 10
bioflocculant and A. niger biomass: (a) the effect of nitrogen sources on PM-5 pH
production with POME used in the medium as carbon source; (b) the effect of C/N
ratio on production of PM-5. Fig. 2. The effect of initial pH of the medium on production of PM-5.
 A.H.R. Aljuboori et al. / Bioresource Technology 171 (2014) 66–70 69

Flocculating rate Biomass gradually thereafter. This may be due to enzymatic activity and cell
100 15 lysis (Aljuboori et al., 2013; Xiong et al., 2010). This phenomenon
90 13 showing the PM-5 produced by biosynthesis during A. niger growth
80 (Okaiyeto et al., 2013). Studies similarly reported that biofloccu-
11
Flocculating rate (%)

70 lants from Rhodococcus R3 (Guo et al., 2013), Serratia ficaria

Biomass (g/L)
60 9 (Gong et al., 2008) and O. ciceri W2 (Wang et al., 2013b) were pro-
50 7 duced by biosynthesis during cell growth. The biomass growth was
40 rapidly increased from 12 to 24 h of culture time then slowly
5
30 increased and reached stationary phase with maximum biomass
20
3 growth of 3.82 g L1. Referring to pH, the pH profile was relatively
10 1 changed during PM-5 production. Results showed as the flocculat-
ing rate increased the pH level decreased from 5.89 to 4.4 within
0 -1
25 30 35 40 72 h, which might be due to the presence of organic acid compo-
Temperature (˚C) nents of the bioflocculant (Lu et al., 2005; Xiong et al., 2010). About
2.6 g L1 of purified PM-5 was obtained from culture broth when
Fig. 3. The effect of temperature of the medium on production of PM-5. the flocculating rate reached the maximum level at 60 h.
Moreover, this study investigated the effect of POME chemical
content on PM-5 production. B-POME and S-POME are two differ-
Flocculating rate Biomass
ent samples with different chemical content were used to produce
100
14 PM-5 by A. niger at optimum conditions. This study found that
90
B-POME produced 2.73 g L1 of purified PM-5 with average floccu-
80 12
lating rate of 77.2%. While S-POME produced 2.21 g L1 of purified
Flocculating rate (%)

70
10 PM-5 with average flocculating rate of 62.6%. These results show
Biomass (g/L)

60
8 that POME with high TOC concentration produced high concentra-
50
tion of PM-5 by A. niger.
40 6
30 3.2. Chemical analysis of PM-5
4
20
2
10 The phenol-sulfuric acid method showed that purified PM-5
0 0 bioflocculant contain 66.8% of carbohydrate and Bradford method
FeCl MnCl CaCl NaCl MgCl KCl Control
(no metal determined about 31.4% of total protein in PM-5. The elemental
Metal ions ion) analysis of purified PM-5 showed that the mass proportion of C,
H, O and N, were 25.2%, 4.6%, 69.1% and 1.1% (w/w), respectively.
Fig. 4. The effect of metal ions on production of PM-5. The IR spectrum of purified PM-5 showed a broad stretching peak
at 3430 cm1 which is an indication of –OH stretching from hydro-
xyl group. A weak peak at 2927 cm1 indicated C–H asymmetrical
production by Bacillus sp. Although Mn2+ was the most favorable stretching vibration and known to be typical of carbohydrate
metal ion for PM-5 production, A. niger growth was the lowest in derivatives and the peak at 1631 cm1 displayed a carboxylate
comparison with other metal ions and control (no metal ion added) anion group. Another peak at 1413 cm1 could be attributed to
culture medium. Hence, as Mn2+ was the most favorable metal ion the symmetric stretching of the –COO group. A weak peak at
and used in culture medium for the following experiments. 1324 cm1 showed carboxyl group. The behavior of the wide peaks
in 1078–1047 cm1 range, represents the stretch vibration of C–O
in the carbohydrate region. The presence of hydroxyl and carboxyl
3.1.3. Time course of PM-5 production
groups in purified PM-5 bioflocculant is preferable functional
Fig. 5 shows the time courses of flocculating rate, growth and
groups for the bioflocculation process and induce high binding
pH of A. niger culture broth. In general, the flocculating rate of
capacity (Guo et al., 2014). Metal ions are stimulating agents for
the culture broth increased in parallel with cell growth. The floccu-
the flocculating activity of cation-dependent bioflocculant. There-
lating rate increased dramatically after 24 h and reached maxi-
fore, Ca2+ ion induced the flocculating activity of PM-5 on kaolin
mum value of 77% at early stationary phase (60 h) and decreased
particles by neutralizing and stabilizing the negative charge of
those functional groups and thereby bridging mechanism occurs
Flocculating rate Biomass pH when the kaolin particles adsorbed onto the bioflocculant chain
90 14 (Gao et al., 2006).
13
80 12
11 3.3. Purification of river water
70
Flocculating rate (%)

10
60
Biomass (g/L)

9 Real river water with initial turbidity of 73.2 NTU, Total

50 8
Suspended Solids of 117 mg L1 and pH 6.9 was used to investigate
pH

7
40 6 the effect of PM-5 on turbidity removal. Results showed PM-5 bio-
30 5 flocculant produced by A. niger could effectively use to reduce the
4
20 3 turbidity of river water. The turbidity removal was increased dra-
10 2 matically as the PM-5 concentration increased from 20 to
1 35 mg L1 and recorded the highest removal of turbidity of 63%.
0 0
0 12 24 36 48 60 72 84 96
This is due to high concentration of PM-5 in the presence of Ca2+
Time (h) ion that provided more bridging sites to colloids and particles
(Doherty et al., 2003). Thereafter the turbidity removal was sharply
Fig. 5. Time course of the PM-5 production. decreased as the PM-5 concentration increased beyond 35 mg L1
70 A.H.R. Aljuboori et al. / Bioresource Technology 171 (2014) 66–70
(overdose), due to restabilization of colloid complex (Renault et al.,
2009).
Li et al. (2009) reported that bioflocculant produced from Bacil-
lus licheniformis X14 was able to remove 95% of turbidity from river
water. Another study showed bioflocculant produced from mixed
culture bacteria produced turbidity removal of up to 96% from river
water (Buthelezi et al., 2009). However, these bioflocculants are
mainly produced from an expensive carbon source such as sucrose,
ethanol and yeast extract. Therefore, in this study the significant
flocculating efficiency of PM-5 in river water treatment and the
conversion of organic pollutant in industrial wastewater (POME)
into value-added chemical (bioflocculant) by A. niger indicated
the feasibility of its commercial use in real wastewater treatment
industries.
4. Conclusions
This study shows that A. niger is a bioflocculant-producing
microorganism, which produced bioflocculant named PM-5. The
new bioflocculant (PM-5) was optimally produced from palm oil
mill effluent as carbon source and glutamic acid as nitrogen source.
PM-5 showed a significant flocculating rate of kaolin suspension in
the presence of Ca2+ ion. Results showed that PM-5 was success-
fully able to treat real river water with turbidity removal of 63%.
Therefore, this study discovered significant potential in the utiliza-
tion of agro-industrial waste as medium culture for bioflocculant
production and improved the feasibility of commercial production.
Acknowledgement
This work was supported by Mitsubishi Corporation Education
Trust Fund (MCETF).
References
Aljuboori, A.H.R., 2013. Oil palm biomass residue in Malaysia: availability and
sustainability. Int. J. Biomass Renew. 2, 13–18.
Aljuboori, A.H.R., Idris, A., Abdullah, N., Mohamad, R., 2013. Production and
characterization of a bioflocculant produced by Aspergillus flavus. Bioresour.
Technol. 127, 489–493.
APHA, 2005. Standard Methods for the Examination of Water and Wastewater, 21st
ed., American Public Health Association, Maryland.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein dye binding.
Anal. Biochem. 72, 248–254.
Buthelezi, S.P., Olaniran, A.O., Pillay, B., 2009. Turbidity and microbial load removal
from river water using bioflocculants from indigenous bacteria isolated from
wastewater in South Africa. Afr. J. Biotechnol. 8, 3261–3266.
Deng, S., Yu, G., Ting, Y.P., 2005. Production of a bioflocculant by Aspergillus
parasiticus and its application in dye removal. Colloids Surf. B Biointerfaces 44,
179–186.
Doherty, W.O.S., Fellows, C.M., Gorjian, S., Senogles, E., Cheung, W.H., 2003.
Flocculation and sedimentation of cane sugar juice particles with cationic
homo- and copolymers. J. Appl. Polym. Sci. 90, 316–325.
Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric
method for determination of sugars and related substances. Anal. Chem. 28,
350–356.
Gao, J., Bao, H.Y., Xin, M.X., Liu, Y.X., Li, Q., Zhang, Y.F., 2006. Characterization of a
bioflocculant from a newly isolated Vagococcus sp. W31. J. Zhejiang Univ. Sci. B
7, 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.
Guo, J., Yang, C., Peng, L., 2014. Preparation and characteristics of bacterial polymer
using pre-treated sludge from swine wastewater treatment plant. Bioresour.
Technol. 152, 490–498.
Guo, J., Yang, C., Zeng, G., 2013. Treatment of swine wastewater using chemically
modified zeolite and bioflocculant from activated sludge. Bioresour. Technol.
143, 289–297.
Krishnaveni, S., Balasubramanian, T., Sadasivam, S., 1984. Sugar distribution in
sweet stalk sorghum. Food Chem. 15, 229–232.
Li, O., Lu, C., Liu, A., Zhu, L., Wang, P.-M., Qian, C.-D., Jiang, X.-H., Wu, X.-C., 2013.
Optimization and characterization of polysaccharide-based bioflocculant
produced by Paenibacillus elgii B69 and its application in wastewater
treatment. Bioresour. Technol. 134, 87–93.
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
temperature drinking water treatment. Bioresour. Technol. 100, 3650–3656.
Liu, W., Wang, K., Li, B., Yuan, H., Yang, J., 2010. Production and characterization of
an intracellular bioflocculant by Chryseobacterium daeguense W6 cultured in
low nutrition medium. Bioresour. Technol. 101, 1044–1048.
Lu, W.Y., Zhang, T., Zhang, D.Y., Li, C.H., Wen, J.P., Du, L.X., 2005. A novel
bioflocculant produced by Enterobacter aerogenes and its use in defecating the
trona suspension. Biochem. Eng. J. 27, 1–7.
Mabinya, L.V., Cosa, S., Nwodo, U., Okoh, A.I., 2012. Studies on bioflocculant
production by Arthrobacter sp. Raats, a freshwater bacteria isolated from Tyume
River, South Africa. Int. J. Mol. Sci. 13, 1054–1065.
Nam, J.S., Kwon, G.S., Lee, S.O., Hwang, J.S., Lee, J.D., Yoon, B.D., Lee, T.H., 1996.
Bioflocculant Produced by Aspergillus sp. JS-42. Biosci. Biotechnol. Biochem. 60,
325–327.
Okaiyeto, K., Nwodo, U.U., Mabinya, L.V., Okoh, A.I., 2013. Characterization of a
bioflocculant produced by a consortium of Halomonas sp. Okoh and Micrococcus
sp. Leo. Int. J. Env. Res. Public Health 10, 5097–5110.
Renault, F., Sancey, B., Badot, P.M., Crini, G., 2009. Chitosan for coagulation/
flocculation processes – an eco-friendly approach. Eur. Polym. J. 45, 1337–1348.
Saidu, M., Yuzir, A., Salim, M.R., Salmiati, Azman, S., Abdullah, N., 2013. Influence of
palm oil mill effluent as inoculum on anaerobic digestion of cattle manure for
biogas production. Bioresour. Technol. 141, 174–176.
Salehizadeh, H., Shojaosadati, S.A., 2001. Extracellular biopolymeric flocculants:
recent trends and biotechnological importance. Biotechnol. Adv. 19, 371–385.
Ugbenyen, A., Cosa, S., Mabinya, L., Babalola, O.O., Aghdasi, F., Okoh, A., 2012.
Thermostable bacterial bioflocculant produced by Cobetia spp. Isolated from
Algoa Bay (South Africa). Int. J. Env. Res. Public Health 9, 2108–2120.
Ugbenyen, A.M., Okoh, A.I., 2013. Flocculating properties of a bioflocculant
produced by Bacillus sp. isolated from a marine environment in South Africa.
Chem. Biochem. Eng. Q 27, 511–518.
Wang, L., Li, Y.C., Zhao, H., Zhang, Z.H., Zhao, B., Zhang, H.W., Cui, L.X., 2013a.
Pretreatment process of nanofiltration for silting density index reduction in
drinking water treatment system. Adv. Mat. Res. 777, 467–471.
Wang, L., Ma, F., Lee, D.-J., Wang, A., Ren, N., 2013b. Bioflocculants from
hydrolysates of corn stover using isolated strain Ochrobactium ciceri W2.
Bioresour. Technol. 145, 259–263.
Wu, J.Y., Ye, H.F., 2007. Characterization and flocculating properties of an
extracellular 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., He, N., 2010. Production and
characterization of a novel bioflocculant from Bacillus Licheniformis. Appl.
Environ. Microbiol. 76, 2778–2782.
You, Y., Ren, N., Wang, A., Ma, F., Gao, L., Peng, Y., Lee, D., 2008. Use of waste
fermenting liquor to produce bioflocculants with isolated strains. Int. J.
Hydrogen Energy 33, 3295–3301.
Zhang, X., Sun, J., Liu, X., Zhou, J., 2013. Production and flocculating performance of
sludge bioflocculant from biological sludge. Bioresour. Technol. 146, 51–56.
Zhao, G., Ma, F., Wei, L., Chua, H., 2012. Using rice straw fermentation liquor to
produce bioflocculants during an anaerobic dry fermentation process.
Bioresour. Technol. 113, 83–88.
Zhao, H., Liu, H., Zhou, J., 2013. Characterization of a bioflocculant MBF-5 by
Klebsiella pneumoniae and its application in Acanthamoeba cysts removal.
Bioresour. Technol. 137, 226–232.