Chemistry and Ecology

ISSN: 0275-7540 (Print) 1029-0370 (Online) Journal homepage: http://www.tandfonline.com/loi/gche20

Isolation and identification of a bioflocculant-

producing strain and optimisation of cultural
conditions via a response surface model

Mingyan Yang, Yuyan Liang, Yan Dou, Xia Jia & Hongrong Che

To cite this article: Mingyan Yang, Yuyan Liang, Yan Dou, Xia Jia & Hongrong Che (2015)
Isolation and identification of a bioflocculant-producing strain and optimisation of cultural
conditions via a response surface model, Chemistry and Ecology, 31:7, 650-660, DOI:
10.1080/02757540.2015.1075516

To link to this article: http://dx.doi.org/10.1080/02757540.2015.1075516

Published online: 28 Sep 2015.

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 Chemistry and Ecology, 2015
Vol. 31, No. 7, 650–660, http://dx.doi.org/10.1080/02757540.2015.1075516

Isolation and identification of a bioflocculant-producing

strain and optimisation of cultural conditions via a response
surface model
Mingyan Yanga,b∗ , Yuyan Lianga , Yan Doua , Xia Jiaa and Hongrong Chea
a School of Environmental Science and Engineering, Chang’an University, Xi’an, People’s Republic of
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China; b Shaanxi Key Laboratory of Exploration and Comprehensive Utilization of Mineral Resources,
Xi’an, People’s Republic of China

(Received 9 February 2015; final version received 7 July 2015)

A bioflocculant-producing strain named MY6-2 was isolated from a mixed activated sludge by a nitrogen-
free medium. Based on 16S rDNA sequence, biochemical and morphological characteristics, the strain
MY6-2 was identified as Bacillus mucilaginosus. The chemical analysis indicated that the flocculant
MY6-2 was mainly composed of extracellular polysaccharide. The result of the composition of the
medium showed that MY6-2 was able to generate bioflocculant in a nutrient-poor medium that consisted
of 5 g L−1 sucrose and no nitrogen source. Response surface methodology (RSM) was used to optimise
the fermentation condition of MY6-2 for maximum flocculating activity by using central composite design
(CCD). Based on the result, the optimum conditions were as follows: 100 mL of broth in a 250 mL Erlen-
meyer flask, initial pH 8.0 and inoculum concentration 10%, respectively. The highest flocculating rate of
90% was achieved under these conditions by adding 0.5 mL fermentation supernatant to 95 mL of Kaolin
suspension.

Keywords: bioflocculant; Bacillus mucilaginosus; flocculating rate; RSM; fermentation conditions

Introduction

Industrial and municipal wastewater contains large amounts of pollutants that cause water pol-
lution. Therefore, various physical and chemical processes have been used to remove pollutants
from wastewater before discharging it to the environment.[1–5] Among these methods, floc-
culating agents have been widely used in a variety of industrial processes such as wastewater
treatment, municipal water treatment process, downstream processing of food and fermentation
industry due to their economic advantage and efficiency.[6–8] The flocculants can be classified
into three groups: inorganic flocculants such as aluminium salts (aluminium sulphate and poly-
aluminium chloride), chemically synthetic flocculants such as derivatives of polyacrylamide and
polyethylene imines and bioflocculants.[9] While the first two conventional groups are play-
ing dominant roles associated with their effectiveness and low cost, their application has been
restricted due to environmental and health problems. For example, the remaining aluminium is
considered a risk factor for Alzheimer’s disease.[10,11] Furthermore, the acrylamide monomer
is not only a neurotoxin and a strong carcinogen to humans but is also nonbiodegradable .[12]

*Corresponding author. Email: yangmingyan67@163.com

© 2015 Taylor & Francis

 Chemistry and Ecology 651

In recent years, bioflocculants that originated from microorganisms have attracted increased
attention as a promising substitute for chemical flocculants due to their biodegradability, lack
of toxicity and free of secondary pollution risks.[13] Many bacteria and fungi (including actino-
myces and yeast) have been found to produce macromolecular substances, such as glycoproteins,
proteins, polysaccharides, lipids and glycolipids .[14,15] However, low flocculating activity, high
cultivation cost and low production yield are still key factors limiting the wide-scale application
of bioflocculants. To overcome these impediments, emphasis is now placed on screening for
microorganisms with high activity, utilisation of inexpensive nutritional sources, cultural condi-
tion and fermentation using microbes in consortia. Liu et al. reported a bioflocculant-producing
strain W6 using a low nutritional medium that contained 1 g L−1 glucose and 2 g L−1 tryptone.
Low nutrition culture can reduce the medium cost of W6 for about 50–80% compared with
other media.[16] Yang et al. used the composite flocculant of MBFGA1 and PAC to decrease
the dosage of PAC required and to increase the effectiveness of the bioflocculant.[17] Zhang
et al. used strain BAFRT4 identified as Staphylococcus sp. and strain CYGS1 identified as Pseu-
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domonas sp. as a consortium for the production of bioflocculant. The mixed culture fermentation
produced bioflocculant with higher yield and flocculation activity than that of the bioflocculant
produced by either of the strains individually under the same conditions.[18] However, most of
these strains required nitrogen sources, which increased the production cost. Lian et al. reported
a flocculant-producing strain GY03 identified as Bacillus mucilaginosus. When cultivated in a
nitrogen-free medium, the flocculation material produced by GY03 was regarded as a bacterium–
mineral complex and mainly distributed in the surface of the bacterium.[19,20] Other reports
focus on the cultural conditions such as the effect of the culture time, initial pH of the production
medium, culture temperature, shaking speed and inoculum size on the flocculating activity of
the bioflocculants. Xia et al. reported a high flocculating activity produced by Proteus mirabilis
TJ-1. Based on cultural condition experiments, the optimal conditions under which a flocculating
activity of 93.13% was reached include inoculum size 2% (v/v), initial pH 7.0, culture temper-
ature 25°C and shaking speed 130 rpm.[21] Gong et al. conducted a series of experiments to
investigate the effect of cultural conditions on the flocculating activity of bioflocculants. Under
the optimised conditions of inoculum size of 1% (v/v), initial pH 6–8 and 72 h cultivation, a
flocculating activity of 95.4% was obtained.[22]
In this study, a bioflocculant-producing strain named MY6-2 selected by a nitrogen-free
medium was identified as B. mucilaginosus based on 16S rDNA sequence, as well as biochemical
and morphological characteristics. MY6-2 was able to generate bioflocculant in a nutrient-poor
medium that consisted of 5 g L−1 sucrose and no nitrogen source. The chemical analysis indi-
cated that the flocculant produced by MY6-2 was an extracellular polysaccharide that was mainly
distributed in the fermentation supernatant. Response surface methodology (RSM) was used to
optimise the fermentation condition of MY6-2 for maximising the flocculating activity. Based
on the central composite design (CCD) results, the optimum conditions were as follows: 100 mL
broth in 250 mL Erlenmeyer flask, initial pH 8.0 and inoculum size 10%. The highest flocculat-
ing rate (FR) of 90% was achieved under this culture condition by adding 0.5 mL fermentation
supernatant to 95 mL of Kaolin suspension.

Methods

Isolation and culture of strain MY6-2

Bioflocculant-producing strains were isolated from the mixed activated sludge collected from
wastewater treatment tank in a paper mill in Xi’an, China. The composition of the selective
medium (nitrogen-free medium) was as follows: 5.0 g sucrose, 0.5 g MgSO4 .7H2 O, 0.1 g
 652 M. Yang et al.

CaCO3 , 2.0 g Na2 HPO4 , 0.005 g FeCl3 , 1.0 g illite powder, 18–20 g agar, 1000 mL distilled
water and pH 7.5–8.0. 5 g samples were added into 250 mL Erlenmeyer flasks containing 95 mL
culture medium and incubated in a shaker at 150 rpm for 3 days at 28°C to enrich the quantity
of the aimed bacteria. Then the sample was spread on the isolation medium after serial dilution.
Strains with a large diameter, fast growth, lack of colour and high viscosity were selected and
inoculated into 250 mL flasks containing 100 mL selecting medium, then incubated in a shaker at
180 rpm for 3 days at 28°C. Then 1 mL culture solution was used to detect flocculating activity.
Strains with high flocculating ability were selected for further studies.

Identification of bioflocculant-producing strain MY6-2

Cell forms and colony characteristics of the strain on selected agar were observed after 2 days
of incubation. The 16S rDNA sequence of the strain was analysed to identify the bacterium. The
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bacterial genomic DNA of MY6-2 was extracted using a commercially available extraction kit
(Genomic DNA Purification Kit, Shanghai Generay Biotech Co., Ltd). PCR amplification of the

16S rDNA was carried out using universal primers, 27F 5 -AGAGTTTGATCCTGGTCAGAAC-
  
3 and 1492R 5 -TACGGCTACCTTGTTACGACTT-3 . The conditions for PCR were as follows:
5 min of denaturation at 94°C, followed by 30 cycles of 94°C for 1 min, 55°C for 1 min, 72°C
for 1 min and a final extension at 72°C for 10 min. PCR products were purified and sequenced
commercially. The resultant sequence was submitted to the GenBank database and analysed
using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Alignment of related sequences was car-
ried out via Clustal X 1.81. Phylogenetic trees were constructed by the neighbour-joining and
maximum-parsimony algorithms using MEGA (version 4.1).[23]

Determination of flocculating activity

Flocculating activity was calculated by FR using the modified method of Kurane et al. [24]
in which Kaolin clay was chosen as the solid phase in the 3 g L−1 suspension. After the pH
of the suspension was adjusted to 7.2–7.5, 2.0 mL of 1% CaCl2 and 1.0 mL culture solution
were added to 100 mL Kaolin suspension in turn. The mixture was stirred at 50 rpm for 3 min
and held still for 1 min. The supernatant portion was collected to measure its OD550 by a 751
spectrophotometer. The control was prepared by a similar procedure except that 1 mL of distilled
water was replaced by the culture solution. The FR was calculated according to the following
equation:
(A − B)
FR = ,
A
where A is the optical density of the blank control and B is the optical density of sample.

Crude extraction of bioflocculant MY6-2 and chemical analysis

Strain MY6-2 was introduced into a 250 mL Erlenmeyer flask containing 100 mL of broth
medium and incubated in a shaker with 180 rpm at 28°C. After incubation for 60 h, the cul-
ture broth was centrifuged at 6000 rpm for 20 min to remove bacterial cells. Three volumes of
cold ethanol were added to the cell-free supernatant to precipitate the bioflocculant. The mix-
ture was allowed to settle overnight at 4°C to enhance the precipitation. The deposit was then
collected by centrifugation at 6000 rpm for 20 min and washed twice by ethanol and dehydrated
at 40°C to yield the raw bioflocculants. The protein content was measured by the Coomassie
brilliant blue reaction using bovine serum albumin as the standard.[25] The total sugar content
was determined by the phenol–sulphuric acid method with glucose as the standard solution.[26]
 Chemistry and Ecology 653

The presence of nucleic acid and protein were measured qualitatively by UV spectrophotometer
within the range of 200–400 nm.

Optimisation of culture conditions of MY6-2

Fermentation time and nutrition sources were the most important factors influencing the cost and
yield of bioflocculant production. The growth curve and the effects of various carbons on the
production and flocculating activity of MY6-2 were studied. Although the strain was selected by
a nitrogen-free medium, different types of inorganic and organic nitrogen sources were used to
assess the impact of nitrogen sources on the production and flocculating activity.
RSM is an empirical statistical modelling technique used in multiple regression analyses of
quantitative data to solve multivariable equations simultaneously.[27] RSM was used to optimise
the culture conditions to maximise the flocculating activity of MY6-2. In the present work, single
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factor experiments for cultural conditions were used to evaluate the effects on the flocculation
activity of MY6-2. Based on the result of these experiments, three factors (broth content, inocu-
lum size and initial pH) that significantly affected flocculation activity of MY6-2 were optimised
via a CCD. The experiments were conducted three times, and the average flocculating activity at
each run was used as the response variable.

Results and discussion

Isolation and identification of bioflocculant-producing strain

Ninety different strains were isolated from the mixed activated sludge by a selective nitrogen-
free medium. Their flocculating activities were tested by the Kaolin suspension method. Among
these strains, a bioflocculant-producing bacterium (named MY6-2) with a FR exceeding 90%
was screened out. Therefore, the strain MY6-2 was selected as the aimed strain for further study.
The colony of strain MY6-2 was found to be round, transparent, smooth and colourless after 48
h of aerobic incubation (Figure 1(a)). Some of its biochemical and physiological characteristics
are as follows: Gram stain ( − ), catalase ( − ), peroxidise ( − ), the Voges–prokauer test ( − ),
Methyl red reaction ( − ), indole test ( − ), glucose fermentation (gas and acid), citrate test ( − ),

(a) (b)

Figure 1. Morphological and characteristic features of MY6-2: 1 Colony characteristic; 2 Microscopic characteristic
( × 1600).
 654 M. Yang et al.

phenylalanine test ( − ), Gelatin hydrolysate ( − ), amylolysis ( + ), litmus milk ( + ) and licithase

( + ).
Microscopic examination showed that MY6-2 was a bacilliform with thick capsules in the cell
surroundings. The capsule size was generally 10–20 times larger than the size of the bacterial
body (Figure 1(b)). Individual cells were seen to be linked by the capsule to form zoogloea.
The 16s rDNA were sequenced following PCR amplification. The result of the PCR product
in agarose gel electrophoresis is shown in Figure 2. The 16s rDNA sequences of strain MY6-2
was determined to be 1501 bp long. The BLAST search program was used to look for nucleotide
sequence homology. The highest homology and total score were noted for further analysis. The
full length of 16s rDNA sequences were aligned by Clustal X1.81 using MEGA version 4.1
software, and a phylogenetic tree was constructed according to the neighbour-joining algorithm
(Figure 3). According to the morphological characteristics and 16s rDNA sequence analysis,
MY6-2 could be identified as B. mucilaginosus.
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Figure 2. Result of the PCR product in agarose gel electrophoresis Lane M: DNA marker; Lane1: MY6-2 PCR product.

Figure 3. Neighbour-joining phylogeny of 16s rDNA sequences of MY6-2.

 Chemistry and Ecology 655

Extraction and analysis of the composition of MY6-2

The extracted and dried raw flocculation material appeared as a white powder. The extraction
rate of the exocellular polymer was about 11 g L−1 culture liquid due to difference culture con-
ditions. This rate was far higher than the reported one of 1.58–2.19 g L−1 culture liquid by Lian
et al.[19] The UV spectrum scanning result showed that there were no characteristic absorption
peaks at 260 or 280 nm, indicating that the bioflocculant may be free of proteins and nucleic
acids. The total sugar content was about 65.08%, confirming that the effective component of this
flocculation material was primarily extracellular polysaccharides.

Factors affecting the bioflocculant production

The dry cell weight, production of bioflocculant, and pH of MY6-2 are illustrated in Figure 4.
After the first lag period, both the dry cell weight and production of bioflocculant increased
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sharply and reached their maxima at 60 h, then decreased slowly during the stationary phase. The
profile of the bioflocculant yield showed a good correlation to the growth curve of strain MY6-2,
indicating that the bioflocculant was formed during its growth, not during cell autolysis. The
pH value decreased gradually during the fermentation period, indicating that some acid material
was secreted during MY6-2 fermentation. The growth curve of the strain, pH variation of the
culture broth and the flocculant production were similar to those of the bioflucculants MBFA9
and MBFF19, as reported by Deng et al. [28] and Zheng et al.,[8] suggesting that the mechanism
of bioflucculant production by different microorganisms is similar. However, the harvest time,
production level and flocculating activity are different. For example, the harvest time is shorter
and the yield is higher of MY6-2 than that of MBFA9 and MBFF19. Thus, the 60 h culture time
was chosen in the following studies for bioflocculant harvest with high biomass and flocculant
production.
The effects of various carbons on flocculating activity and flocculant production of MY6-
2 were investigated. As shown in Figure 5, the flocculant production of about 10 g L−1 was

Figure 4. Growth curve, pH variation and flocculant production of strain MY6-2 on a shaker at 180 rpm 28°C for
72 h.
 656 M. Yang et al.
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Figure 5. Effect of different carbon sources on flocculating rate and flocculant production of MY6-2. The carbon
concentration was 5 g L−1 .

achieved by using six different sugars, indicating that strain MY6-2 can make full use of a variety
of different sugars to produce bioflocculants. However, the resultant flocculating activities were
different. Among these sugars, sucrose can achieve both higher flocculant production and higher
FR. Thus, sucrose was used as the carbon source during additional experiments.

Figure 6. Effect of different nitrogen sources on flocculating rate and flocculant production of MY6-2. The nitrogen
concentration was 2 g L−1
 Chemistry and Ecology 657

The effects of nitrogen sources on flocculating activity and flocculant production of MY6-2
are shown in Figure 6. Organic nitrogen sources such as peptone, carbamide and beef extract
resulted in lower flocculating activity than that of inorganic nitrogen resources such as NaNO3
and (NH4 )2 SO4 . However, flocculating activity and flocculant production in a nitrogen-free
medium were higher than that of the two nitrogen groups, indicating that strain MY6-2 can fix
nitrogen biologically and produce bioflucculant efficiently in a nitrogen-free environment. This
characteristic greatly reduces the cost of fermentation and may have a potential application for
actual waste water treatment.

Optimisation of Bioflocculant MY6-2 with RSM

A CCD model of 3-factor-3 level with 17 experiments was carried out. Experimental trials were
all carried out in triplicate and the average of both flocculating activity at each run was used as
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the response variable (Table 1).

The statistical testing of this model was carried out with the Fisher’s statistical method for
analysis of variance (ANOVA). The result of ANOVA, shown in Table 2, was statistically valid
with a low probability value (pmodel = .0008), indicating that the equation was adequate for pre-
dicting MY6-2 production. The lack of fit value was not significant (p = .0562), indicating that
the equation was adequate for predicting flocculating activity under all conditions. The adequacy
of the model was indicated by the determination coefficient (R2 = 0.9510), which accounted for
95.10% of the response variability.
The graphical representation of the regression equation yields a three-dimensional surface
response plot that is generally used to demonstrate relationships between the response and
experimental levels of each variable. These surface plots, therefore, allow for visualisation of the

Table 1. CCD matrix for cultural condition of MY6-2.

Run A: broth content (mL) B: initial pH C: inoculum size (%) FR (%)

1 100 8.00 10 90.6

2 120 8.00 15 81.0
3 100 8.50 15 86.6
4 80 8.00 5 82.4
5 100 7.50 5 86.0
6 80 8.00 15 82.9
7 100 8.50 5 82.2
8 120 7.50 10 83.4
9 100 8.00 10 89.7
10 120 8.00 5 84.0
11 100 7.50 15 85.9
12 100 8.00 10 89.7
13 100 8.00 10 89.1
14 80 8.50 10 84.0
15 80 7.50 10 82.4
16 100 8.00 10 90.5
17 120 8.50 10 82.0

Table 2. ANOVA for optimisation of MY6-2 flocculating activity.

Source SS DF MS F Pr > F

Lack of fit 7.20 3 2.40 6.12 0.0562

Pure error 1.57 4 0.39
Model 170.31 9 18.92 15.10 0.0008
Residual (error) 8.77 7 1.25
Total 179.08 16
 658 M. Yang et al.

(a)
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(b)

(c)

Figure 7. Response surface plot of fermentation conditions on flocculating activity of MY6-2; (a) Effects of initial
pH, broth content and their mutual interaction; (b) Effects of inoculum concentration, broth content and their mutual
interaction; (c) Effects of inoculum concentration, pH and their mutual interaction
 Chemistry and Ecology 659

optimum levels of each variable to maximise the production of microbial metabolites.[29] The
interaction between broth content, initial pH and inoculums size is visualised in Figure 7(a)–7(c).
From Figure 7(a), it can be seen that an increase in flocculating activity was achieved with an
increase in the initial pH, with the maximum response occurring at pH 8.0. The initial pH of the
culture medium determines the electric charge of the cells and the oxidation–reduction potential,
which can affect nutrient absorption and enzymatic reactions.[21] According to Figure 7(b), the
flocculating activity increased sharply with the inoculum concentration increasing from 5% to
10%, and then decrease slowly. Maximum response occurred when broth content was 100 mL
and inoculum concentration was 10%, respectively. Figure 7(c) depicts the combined effects of
inoculum concentration and pH on the flocculating activity of MY6-2. From the elliptical nature
of the contour plot, a prominent interaction between these variables is apparent.
To sum up, the optimal culture conditions for MY6-2 were 100 mL of broth in a 250 mL
Erlenmeyer flask, initial pH 8.0 and inoculum concentration 10%, respectively. Consequently,
the maximum FR of 90% was achieved under these culture conditions by adding 0.5 mL
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fermentation supernatant to 95 mL of the Kaolin suspension.

Conclusion

The bacterium MY6-2, isolated from paper mill wastewater, was identified as B. mucilaginosus
based on 16S rDNA sequence, and biochemical and morphological characteristics. MY6-2 can
make full use of various kinds of carbon resources and produce bioflocculant in a nitrogen-free
medium. The chemical analysis indicated that the main composition of the MY6-2 flocculant was
extracellular polysaccharide. The result of the CCD showed that the optimal culture conditions
for MY6-2 were 100 mL of broth in a 250 mL Erlenmeyer flask, initial pH 8.0 and inoculum
concentration 10%, respectively. Consequently, a maximum flocculating activity of 90% was
achieved under these conditions by adding 0.5 mL fermentation supernatant to 95 mL of Kaolin
suspension. Although further studies are required, the low nutrient requirements and high floc-
culating activity of strain MY6-2 suggest that it may have promising application in industrial
flocculation.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by The Opening Foundation of Shaanxi Key Laboratory of Explo-
ration, Comprehensive Utilization of Mineral resources [grant number 2014HB003], Shaanxi
Science and Technology Development Project [grant number 2015SF263] and The Fundamen-
tal Research Funds for the Central Universities [grant number 2014G1502030] and the Open
Research Fund of Key Laboratory of Subsurface Hydrology and Ecological Effect in Arid Region
of Ministry of Education [310850142030].

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