Accepted Manuscript Title: Effects of carbohydrate sources on biosorption properties of the novel exopolysaccharides produced by Arthrobacter ps-5 Author: Ye Shuhong Ma Zhiyang Liu Zhaofang Liu Yan Zhang Meiping Wang Jihui PII: DOI: Reference:
S0144-8617(14)00554-2 http://dx.doi.org/doi:10.1016/j.carbpol.2014.05.076 CARP 8943
To appear in: Received date: Revised date: Accepted date:
23-2-2014 14-5-2014 28-5-2014
Please cite this article as: Shuhong, Y., Zhiyang, M., Zhaofang, L., Yan, L., and Jihui, Z. M., ,Wang, Effects of carbohydrate sources on biosorption properties of the novel exopolysaccharides produced by Arthrobacter ps-5, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.05.076 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 3 4 5
Highlights >Carbohydrates from various sources was different on the yield and molecular weight. > EPS produced by addition of glucose presented the highest biosorption amounts. > Ratio of acidic versus neutral EPS was a key factor of the biosorption mechanism.
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Page 1 of 30
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Effects of carbohydrate sources on biosorption properties of the novel
7
exopolysaccharides produced by Arthrobacter ps-5
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Ye Shuhonga, Ma Zhiyanga, Liu Zhaofanga, Liu Yanb, Zhang Meipinga,Wang Jihuia*
11
a
12
b
13 14
*Corresponding author. Tel :+86 13109805039; E-mail address: [email protected](W.Jihui)
Jiangsu Heavy Responsibility Group, Pizhou 221300, China Fax:+86 41186323626
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School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, China
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26 27 28 29
Page 2 of 30
Abstract The crude exopolysaccharides (EPSs) were obtained from Arthrobacter ps-5 fermentation
31
using various carbohydrate sources followed by centrifugation, ethanol precipitation, and the isolated EPSs
32
were further deproteinized and lyophilized. Carbohydrates from various sources resulted in different yield
33
of EPSs from the fermentation and different molecular weight of EPSs. A maximum yield of 0.27 mg/g
34
was achieved by using the culture medium supplemented with sucrose. The EPS produced by
35
glucose-supplemented medium had the maximum content of acidic polysaccharides, subsequently
36
presented the highest biosorption capacity for Cu2+ and Pb2+ at 257.9 mg/g and 331.8 mg/g, respectively.
37
The ratio of acidic to neutral polysaccharides presented in EPSs was a key factor to explicate the
38
biosorption mechanism, the higher the ratio, the stronger the biosorption capacity.
39
Key Words: Carbohydrate-supplemented medium,
40
ps-5
43
44
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Arthrobacter
d
Biosorption,
te
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Exopolysaccharide,
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30
46
47
48
Page 3 of 30
49
1. Introduction
As a result of rapid industrialization heavy metals have been used widely, such as in the industrial
51
processes of electroplating, manufacturing, mining, and automotive. Consequently, heavy metals have
52
been released to the environment. As hazardous pollutants, they are difficult to remove from the
53
environment due to the fact that they are resistant to degradation through chemical, biological and
54
photolytic processes. For instance, copper (Cu) and lead (Pb) are applied in some metabolic processes of
55
microorganisms in the environment, nevertheless, acute exposure to large amounts of Cu2+ and Pb2+ causes
56
health problems such as anemia, damage to liver and kidneys, brain damage and ultimately death (Davis &
57
Vieira, 2000; Davis, Ramirez, Mucci, & Larsen, 2004). As a result, removal of these pollutants from
58
industrial effluents has become a high priority task that is reflected by more stringent environmental
59
regulations enacted in many countries. This creates the demand of efficient and cost-effective treatments in
60
handling large volumes of heavy metal-bearing waste water from mining industries (Davis & Volesky,
61
2000).
cr
us
an
M
d
te
Ac ce p
62
ip t
50
Much of the conventional methods have been employed to remove heavy metals, such as precipitation,
63
oxidation/reduction, ionic exchange, adsorption, filtration, electrochemical processes, membrane
64
separations and evaporation, among which adsorption is highly effective and economical. In addition,
65
biosorption could be an even more economical option, since it utilizes various microorganisms naturally
66
available, including bacteria, fungi, yeast, and algae. These biosorbents possess metal sequestering
67
properties and can effectively decrease the concentration of heavy metal ions in dilute solutions that
68
contain the pollutants at trace levels, leading to aggregation or precipitation of the metals. Therefore
69
biosorption can be an ideal option for treatment of large volumes of waste water which contains heavy
70
metals at relatively low concentrations (Veglio & Beolchini, 1997; Volesky & Holan, 1995).
Page 4 of 30
Biopolymers naturally generated by microgrnisms are biodegradable and are degradation
72
intermediates, which are harmless to human being and the environment. They can be used as biosorbents in
73
heavy metals treatment and performed well at the common conditions. Among biopolymers
74
exopolysaccharides (EPSs) are sugar polymers that are produced by mainly bacteria and cyanobacteria,
75
which have been proved to be effective in aggregation and binding of heavy metals (Freire-Nordi,
76
Henriques Vieira, & Nascimento, 2005). Adsorption of heavy metals by EPSs is considered non-metabolic
77
and energy independent as it is resulted from interaction between metal cations and negative charge of
78
acidic functional groups of EPSs (Kim, Kim, Kim, & Oh, 1996; Francois et al., 2012). During the bacterial
79
growth, EPSs are released and consequently bind the metal ions in the environment. The structures of the
80
EPSs are typically found with long-chain polysaccharides containing branched and sugars or sugar
81
derivatives such as glucose, fructose, mannose and galactose as distinct units.
M
an
us
cr
ip t
71
Carbohydrates are the major sources of cellular carbon and energy in all living organisms.
83
Composition of carbohydrates from various sources exhibits significant effects on the yield,
84
monosaccharide composition and molecular mass of EPSs during microorganism fermentation (Cerning et
85
al., 1994; Tallon, Bressollier, & Urdaci, 2003; Torino, Hébert, Mozzi, & Valdez, 2005), and subsequently
86
impacts on other physical and chemical properties of EPSs (Fukuda et al., 2010; Kaplan, Christian, & Arod,
87
1987). However, the mechanisms of how source carbohydrates may alter monosaccharide composition of
88
EPSs that are generated from strains cultivation remain unclear. In this paper, we aimed to evaluate the
89
effects of different carbohydrate sources used in culture media on the yield, chemical structure and
90
biosorption capacity of the EPSs produced by Arthrobacter ps-5. Possible determinants leading to the
91
biosorption of Cu2+ and Pb 2+ in the EPSs will be discussed.
92
2. Materials and methods
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2.1.
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Cultivation of Arthrobacter ps-5 and reagents Arthrobacter ps-5 was screened from the rhizosphere soil of ginkgo biloba trees and maintained in
our laboratory, which can produce EPSs during the fermentation. The strain was cultivated on basic
96
medium consisting of (per liter) 5.0 g glucose, 3.0 g beef extract, 10.0 g peptone, 5.0 g sodium chloride,
97
20.0 g agar and then maintained at 4 oC. Seed culture was carried out as described in our previous report
98
(Ye et al., 2014).
cr
ip t
95
us
All reagents used were analytical grade. Synthetic stock solution of Cu2+ and Pb2+ (100 mg/L) was
99
prepared by dissolving calculated quantity of copper sulphate and lead nitrate (AR Grade) in double
101
distilled water.
102
2.2.
Isolation and purification of the EPS
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an
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The culture medium prepared as mentioned above was used to ferment Arthrobacter ps-5 and for
104
harvesting EPSs. Arthrobacter ps-5 was propagated in 250 mL conical flasks with 50 mL of liquid medium,
105
using a rotary shaker (HZQ-X100A) when operated at 160 rpm for 48 h. Sucrose, glucose or starch were
106
added in the culture medium as different carbohydrate sources with a final concentration of 2% (w/v). The
107
fermentation temperature, initial pH, inoculation proportion were held at 28 oC, 7.0 and 4.0% (v/v),
108
respectively, based on our previous study results (Ye et al., 2014). According to the culture medium
109
supplemented with different carbohydrates, such as sucrose, glucose and starch, these EPSs obtained were
110
named EPSSuc, EPSGlu and EPSSta respectively.
Ac ce p
te
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103
111
Crude EPS and its purified fractions were prepared as described in our previous report (Ye et al.,
112
2014), using DEAE-52 anion-exchange chromatography column and Sephadex G-100 column. Then acidic
113
or neutral polysaccharides were obtained (Chang, 1987).
Page 6 of 30
114
2.3.
Composition analysis of the EPSs
The yield of EPSs was determined by an anthrone colorimetric method (Li, Zhou, Zhang, Wang, &
116
Zhu, 2008), through sulphuric acid hydrolysis of EPSs in the presence of anthrone agent at 100 oC. The
117
reaction solution had a blue-green color. The absorbance of the sample solution was measured at 620 nm
118
and calibrated using glucose as a standard reference (Leung, Shuna, Ho, & Wu, 2009). The carbohydrate
119
content was then quantified using the anthrone colorimetric method as described above.
120
2.4.
us
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UV analysis of the EPSs
The UV absorption spectrum (190 nm -500 nm) was recorded using a Lambda 35 spectrophotometer
122
(Perkin Elmer, U.S.) to examine the existence of proteins and nucleic acids. The detailed protocols were
123
reported previously (Wang, Cui, & Xu, 2007).
124
2.5.
125
2.5.1.
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Characterization of the EPSs
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Estimation of molecular mass of the EPSs
The molecular mass distribution of EPSs was estimated using high liquid chromatography (HPLC).
127
The purified EPS was dissolved in water (1mg/mL) and this solution was loaded onto a TSK-Gel
128
G3000SWXL column (7.5 × 300 nm, Waters-2690, USA). The column was eluted with 0.05 M NaCl
129
solution at a flow rate of 0.4 mL/min. The eluent was monitored with a refractive index detector (RID)
130
(Lin, Wang, Chang, Inbaraj, & Chen, 2009). A series of dextrans with known molecular weights were used
131
as the standard (AOAC 980.13).
132
2.5.2.
Monosaccharide components of EPS
Page 7 of 30
Determination of monosaccharide components was followed as per the methods of Xu et al. (Xu, Zhu,
134
Jin, & Yu, 2010) and Fukuda et al (2010) with some modifications. 5 mg of the purified EPS was dissolved
135
in 1 mL of 12 M hydrochloric acid (HCl) and the EPS solution was hydrolyzed in 250 µL by autoclaving at
136
100 oC for 5h. Excess HCl was neutralized by NaOH solution, and the hydrolysate was washed thoroughly
137
with deionized water and then lyophilized. The lyophilized powder was dissolved in 100 µL of deionized
138
water, and 5 µL aliquot was used for TLC. A mixed solvent system (n-butanol, ethanol, water) was used at
139
a ratio of 2:1:1(v/v) for separation of carbohydrates. Carbohydrates were visualized on the TLC plate (20 ×
140
20 cm) by heating (60 oC) after spraying with sulfuric acid (5%, v/v). The commercial sugars, including
141
glucose, galactose and mannose were used as standards for identification of single sugar composition
142
present in the sample. The molar fraction of the hydrolysate was analyzed using an HPLC (Waters-2690,
143
USA) equipped with RID and a Sugar-pak-1 column (Waters, USA). The molar ratio of glucose versus
144
galactose in each sample was calculated from the peak areas. Detailed procedures are reported previously
145
(Ye et al., 2014).
146
2.6.
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Quantification of metal adsorption
In a 250 mL flask, 50 mL of 10 mg/L metal solution was mixed with 2.0 mL crude EPS (the purity of
148
EPS was 82.3% , w/v) solution that has a concentration of 10 mg/L). The mixing was operated at 25 oC and
149
200 rpm in the dark for 4h. After centrifugation at 10,000 rpm for 10min, the concentration of Cu2+ and
150
Pb2+ was determined by atomic absorption spectrophotometer (models 180-80, Hitachi, Japan).
151
All tests were replicated and a blank sample, replacing EPS fraction with deionized water was used to
152
calibrate the AAS. All experimental data were processed using Excel and presented as mean values
153
±standard deviations.
Page 8 of 30
154
The metal uptake, Q (mg/g), was then calculated by the following equation:
Q
155
V (C 0 C e ) W
Where C0 and Ce are the initial and end concentrations of metals in the sample solutions (mg/L),
157
respectively, V is the volume of sample solutions (L), and W is the mass of sorbent (crude EPS) added in
158
the metal solutions (g).
159
2.7.
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Fourier transform infrared spectroscopy analysis
The Infrared (IR) spectra of EPS before and after metal adsorption tests were determined using a
161
Fourier transform infrared (FTIR) spectrophotometer (Spectrum One-B, Perkin Elmer, U.S) equipped with
162
detectors for differentiating various functional groups. The samples were freeze-dried, mixed with dry KBr
163
powder and pressed into pellets. The spectrum was recorded between 400 and 4000 cm−1 (Paulraj, Satish
164
Kumar, Yuvaraj, Paari, Pattukumar, & Venkatesan, 2011).
165
3. Results
166
3.1.
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Effects of different carbon sources on production of EPSs
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an
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Production of EPSs was carried out through fermentation of Arthrobacter ps-5 using a culture
168
medium supplemented with the selected carbohydrates, i.e., sucrose, glucose, starch, fructose or lactose, at
169
a pre-determined concentration level of 2% (w/v). After 48h cultivation, the maximum EPSs production
170
was achieved at 0.27 g/L in the culture medium supplemented with sucrose. EPSs production was 0.24 g/L
171
in the culture medium supplemented with glucose (Fig.1). Next, the EPSs produced from the Carbohydrate
172
-enriched culture media were separated and purified for further studies of their effects on monosaccharide
173
composition and biosorption capacity of EPSs.
Page 9 of 30
174
3.2.
175
3.2.1.
Characteristics of crude and purified EPSs
Isolution and purification of EPSs
The EPSSuc was separated into two independent elution peaks after DEAE anion exchanger, i.e.,
177
sub-fractions Su-1 and Su-2 (Fig.2 Su-A). Sub-fraction Su-1 was a neutral polysaccharide as it was eluted
178
out with water, while sub-fraction Su-2 was more likely acidic as it was eluted with NaCl solution. This is
179
in agreement with other literature indications (Chang, 1987). Su-1 was the major component, shown by its
180
greater peak area in anion-exchange chromatography (Fig.2 Su-A). The ratio of Su-2 and Su-1 was 0.22.
181
Su-1 was collected and further purified through a Sephadex G-100 column and then renamed as Su-1-1 for
182
purified sample (Fig.2 Su-B).
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The EPSGlu was separated into two independent elution fractions, named as sub-fractions Gl-1 and
184
Gl-2 (Fig.2 G-A). Gl-1 was a neutral polysaccharide, and Gl-2 was an acidic polysaccharide, whereas the
185
ratio of Gl-2 and Gl-1 was 1.82. Gl-1 and Gl-2 fractions were collected respectively and further fractioned
186
through a Sephadex G-100 column eluted with 0.05 M NaCl solution, resulting in two purified samples of
187
Gl-1-1 and Gl-2-1 (Fig.2 G-B).
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d
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The EPSSta was also separated into two independent elution fractions St-1and St-2 (Fig.2 St-A).
189
Similarly, St-1was a neutral polysaccharide and St-2 was an acidic polysaccharide. The ratio of St-2 and
190
St-1 was 1.08. St-1 and St-2 fractions were collected and further purified respectively, resulting in two
191
purified samples of St-1-1 and St-2-1 for further tests (Fig.2 St-B).
192
193
The ratio of acidic polysaccharide/neutral polysaccharide in all crude EPSs was shown in Table 1.
3.2.2.
Physical properties of the EPS
Page 10 of 30
The purified EPSs were light yellow in color and in a form of odorless powder. It was soluble in
195
water but insoluble in ethanol and other organic solvents, such as ether and acetone, which conferred with
196
general properties of polysaccharides. The aqueous solutions prepared by all purified EPSs were clear,
197
homogeneous liquids with a color of light yellow and no precipitation occurred after centrifugation. An
198
ultraviolet scan spectrum analysis of the EPSs was shown in Fig.3, indicating the absence of proteins and
199
nucleic acids due to a negative response - no absorption peaks at 260 or 280 nm. So the EPSs were
200
thought to be pure.
201
3.2.3.
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Estimation of EPSs molecular mass
The molecular mass of sub-fractions, Su-1, Gl-1, Gl-2, St-1 and St-2 was measured by HPLC with a
203
size exclusion column. Based on a calibration curve obtained using dextrans as references at various
204
molecular weight cut-off points, the average molecular weights of Su-1, Gl-1, Gl-2, St-1 and St-2 were
205
estimated to be 8.83×105, 3.48×105, 1.21×106, 1.01×105 and 7.36×105 Da, respectively. Molecular mass
206
distribution followed by the order of EPSGlu < EPSsta
S0144-8617(14)00554-2 http://dx.doi.org/doi:10.1016/j.carbpol.2014.05.076 CARP 8943
To appear in: Received date: Revised date: Accepted date:
23-2-2014 14-5-2014 28-5-2014
Please cite this article as: Shuhong, Y., Zhiyang, M., Zhaofang, L., Yan, L., and Jihui, Z. M., ,Wang, Effects of carbohydrate sources on biosorption properties of the novel exopolysaccharides produced by Arthrobacter ps-5, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.05.076 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 3 4 5
Highlights >Carbohydrates from various sources was different on the yield and molecular weight. > EPS produced by addition of glucose presented the highest biosorption amounts. > Ratio of acidic versus neutral EPS was a key factor of the biosorption mechanism.
Ac ce p
te
d
M
an
us
cr
ip t
6
Page 1 of 30
6
Effects of carbohydrate sources on biosorption properties of the novel
7
exopolysaccharides produced by Arthrobacter ps-5
ip t
Ye Shuhonga, Ma Zhiyanga, Liu Zhaofanga, Liu Yanb, Zhang Meipinga,Wang Jihuia*
11
a
12
b
13 14
*Corresponding author. Tel :+86 13109805039; E-mail address: [email protected](W.Jihui)
Jiangsu Heavy Responsibility Group, Pizhou 221300, China Fax:+86 41186323626
an
15 16
M
17
22 23 24 25
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School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, China
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26 27 28 29
Page 2 of 30
Abstract The crude exopolysaccharides (EPSs) were obtained from Arthrobacter ps-5 fermentation
31
using various carbohydrate sources followed by centrifugation, ethanol precipitation, and the isolated EPSs
32
were further deproteinized and lyophilized. Carbohydrates from various sources resulted in different yield
33
of EPSs from the fermentation and different molecular weight of EPSs. A maximum yield of 0.27 mg/g
34
was achieved by using the culture medium supplemented with sucrose. The EPS produced by
35
glucose-supplemented medium had the maximum content of acidic polysaccharides, subsequently
36
presented the highest biosorption capacity for Cu2+ and Pb2+ at 257.9 mg/g and 331.8 mg/g, respectively.
37
The ratio of acidic to neutral polysaccharides presented in EPSs was a key factor to explicate the
38
biosorption mechanism, the higher the ratio, the stronger the biosorption capacity.
39
Key Words: Carbohydrate-supplemented medium,
40
ps-5
43
44
45
cr
us
an
M
Arthrobacter
d
Biosorption,
te
42
Exopolysaccharide,
Ac ce p
41
ip t
30
46
47
48
Page 3 of 30
49
1. Introduction
As a result of rapid industrialization heavy metals have been used widely, such as in the industrial
51
processes of electroplating, manufacturing, mining, and automotive. Consequently, heavy metals have
52
been released to the environment. As hazardous pollutants, they are difficult to remove from the
53
environment due to the fact that they are resistant to degradation through chemical, biological and
54
photolytic processes. For instance, copper (Cu) and lead (Pb) are applied in some metabolic processes of
55
microorganisms in the environment, nevertheless, acute exposure to large amounts of Cu2+ and Pb2+ causes
56
health problems such as anemia, damage to liver and kidneys, brain damage and ultimately death (Davis &
57
Vieira, 2000; Davis, Ramirez, Mucci, & Larsen, 2004). As a result, removal of these pollutants from
58
industrial effluents has become a high priority task that is reflected by more stringent environmental
59
regulations enacted in many countries. This creates the demand of efficient and cost-effective treatments in
60
handling large volumes of heavy metal-bearing waste water from mining industries (Davis & Volesky,
61
2000).
cr
us
an
M
d
te
Ac ce p
62
ip t
50
Much of the conventional methods have been employed to remove heavy metals, such as precipitation,
63
oxidation/reduction, ionic exchange, adsorption, filtration, electrochemical processes, membrane
64
separations and evaporation, among which adsorption is highly effective and economical. In addition,
65
biosorption could be an even more economical option, since it utilizes various microorganisms naturally
66
available, including bacteria, fungi, yeast, and algae. These biosorbents possess metal sequestering
67
properties and can effectively decrease the concentration of heavy metal ions in dilute solutions that
68
contain the pollutants at trace levels, leading to aggregation or precipitation of the metals. Therefore
69
biosorption can be an ideal option for treatment of large volumes of waste water which contains heavy
70
metals at relatively low concentrations (Veglio & Beolchini, 1997; Volesky & Holan, 1995).
Page 4 of 30
Biopolymers naturally generated by microgrnisms are biodegradable and are degradation
72
intermediates, which are harmless to human being and the environment. They can be used as biosorbents in
73
heavy metals treatment and performed well at the common conditions. Among biopolymers
74
exopolysaccharides (EPSs) are sugar polymers that are produced by mainly bacteria and cyanobacteria,
75
which have been proved to be effective in aggregation and binding of heavy metals (Freire-Nordi,
76
Henriques Vieira, & Nascimento, 2005). Adsorption of heavy metals by EPSs is considered non-metabolic
77
and energy independent as it is resulted from interaction between metal cations and negative charge of
78
acidic functional groups of EPSs (Kim, Kim, Kim, & Oh, 1996; Francois et al., 2012). During the bacterial
79
growth, EPSs are released and consequently bind the metal ions in the environment. The structures of the
80
EPSs are typically found with long-chain polysaccharides containing branched and sugars or sugar
81
derivatives such as glucose, fructose, mannose and galactose as distinct units.
M
an
us
cr
ip t
71
Carbohydrates are the major sources of cellular carbon and energy in all living organisms.
83
Composition of carbohydrates from various sources exhibits significant effects on the yield,
84
monosaccharide composition and molecular mass of EPSs during microorganism fermentation (Cerning et
85
al., 1994; Tallon, Bressollier, & Urdaci, 2003; Torino, Hébert, Mozzi, & Valdez, 2005), and subsequently
86
impacts on other physical and chemical properties of EPSs (Fukuda et al., 2010; Kaplan, Christian, & Arod,
87
1987). However, the mechanisms of how source carbohydrates may alter monosaccharide composition of
88
EPSs that are generated from strains cultivation remain unclear. In this paper, we aimed to evaluate the
89
effects of different carbohydrate sources used in culture media on the yield, chemical structure and
90
biosorption capacity of the EPSs produced by Arthrobacter ps-5. Possible determinants leading to the
91
biosorption of Cu2+ and Pb 2+ in the EPSs will be discussed.
92
2. Materials and methods
Ac ce p
te
d
82
Page 5 of 30
93
2.1.
94
Cultivation of Arthrobacter ps-5 and reagents Arthrobacter ps-5 was screened from the rhizosphere soil of ginkgo biloba trees and maintained in
our laboratory, which can produce EPSs during the fermentation. The strain was cultivated on basic
96
medium consisting of (per liter) 5.0 g glucose, 3.0 g beef extract, 10.0 g peptone, 5.0 g sodium chloride,
97
20.0 g agar and then maintained at 4 oC. Seed culture was carried out as described in our previous report
98
(Ye et al., 2014).
cr
ip t
95
us
All reagents used were analytical grade. Synthetic stock solution of Cu2+ and Pb2+ (100 mg/L) was
99
prepared by dissolving calculated quantity of copper sulphate and lead nitrate (AR Grade) in double
101
distilled water.
102
2.2.
Isolation and purification of the EPS
M
an
100
The culture medium prepared as mentioned above was used to ferment Arthrobacter ps-5 and for
104
harvesting EPSs. Arthrobacter ps-5 was propagated in 250 mL conical flasks with 50 mL of liquid medium,
105
using a rotary shaker (HZQ-X100A) when operated at 160 rpm for 48 h. Sucrose, glucose or starch were
106
added in the culture medium as different carbohydrate sources with a final concentration of 2% (w/v). The
107
fermentation temperature, initial pH, inoculation proportion were held at 28 oC, 7.0 and 4.0% (v/v),
108
respectively, based on our previous study results (Ye et al., 2014). According to the culture medium
109
supplemented with different carbohydrates, such as sucrose, glucose and starch, these EPSs obtained were
110
named EPSSuc, EPSGlu and EPSSta respectively.
Ac ce p
te
d
103
111
Crude EPS and its purified fractions were prepared as described in our previous report (Ye et al.,
112
2014), using DEAE-52 anion-exchange chromatography column and Sephadex G-100 column. Then acidic
113
or neutral polysaccharides were obtained (Chang, 1987).
Page 6 of 30
114
2.3.
Composition analysis of the EPSs
The yield of EPSs was determined by an anthrone colorimetric method (Li, Zhou, Zhang, Wang, &
116
Zhu, 2008), through sulphuric acid hydrolysis of EPSs in the presence of anthrone agent at 100 oC. The
117
reaction solution had a blue-green color. The absorbance of the sample solution was measured at 620 nm
118
and calibrated using glucose as a standard reference (Leung, Shuna, Ho, & Wu, 2009). The carbohydrate
119
content was then quantified using the anthrone colorimetric method as described above.
120
2.4.
us
cr
ip t
115
an
UV analysis of the EPSs
The UV absorption spectrum (190 nm -500 nm) was recorded using a Lambda 35 spectrophotometer
122
(Perkin Elmer, U.S.) to examine the existence of proteins and nucleic acids. The detailed protocols were
123
reported previously (Wang, Cui, & Xu, 2007).
124
2.5.
125
2.5.1.
d
te
Ac ce p
126
Characterization of the EPSs
M
121
Estimation of molecular mass of the EPSs
The molecular mass distribution of EPSs was estimated using high liquid chromatography (HPLC).
127
The purified EPS was dissolved in water (1mg/mL) and this solution was loaded onto a TSK-Gel
128
G3000SWXL column (7.5 × 300 nm, Waters-2690, USA). The column was eluted with 0.05 M NaCl
129
solution at a flow rate of 0.4 mL/min. The eluent was monitored with a refractive index detector (RID)
130
(Lin, Wang, Chang, Inbaraj, & Chen, 2009). A series of dextrans with known molecular weights were used
131
as the standard (AOAC 980.13).
132
2.5.2.
Monosaccharide components of EPS
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Determination of monosaccharide components was followed as per the methods of Xu et al. (Xu, Zhu,
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Jin, & Yu, 2010) and Fukuda et al (2010) with some modifications. 5 mg of the purified EPS was dissolved
135
in 1 mL of 12 M hydrochloric acid (HCl) and the EPS solution was hydrolyzed in 250 µL by autoclaving at
136
100 oC for 5h. Excess HCl was neutralized by NaOH solution, and the hydrolysate was washed thoroughly
137
with deionized water and then lyophilized. The lyophilized powder was dissolved in 100 µL of deionized
138
water, and 5 µL aliquot was used for TLC. A mixed solvent system (n-butanol, ethanol, water) was used at
139
a ratio of 2:1:1(v/v) for separation of carbohydrates. Carbohydrates were visualized on the TLC plate (20 ×
140
20 cm) by heating (60 oC) after spraying with sulfuric acid (5%, v/v). The commercial sugars, including
141
glucose, galactose and mannose were used as standards for identification of single sugar composition
142
present in the sample. The molar fraction of the hydrolysate was analyzed using an HPLC (Waters-2690,
143
USA) equipped with RID and a Sugar-pak-1 column (Waters, USA). The molar ratio of glucose versus
144
galactose in each sample was calculated from the peak areas. Detailed procedures are reported previously
145
(Ye et al., 2014).
146
2.6.
cr
us
an
M
d
te
Ac ce p
147
ip t
133
Quantification of metal adsorption
In a 250 mL flask, 50 mL of 10 mg/L metal solution was mixed with 2.0 mL crude EPS (the purity of
148
EPS was 82.3% , w/v) solution that has a concentration of 10 mg/L). The mixing was operated at 25 oC and
149
200 rpm in the dark for 4h. After centrifugation at 10,000 rpm for 10min, the concentration of Cu2+ and
150
Pb2+ was determined by atomic absorption spectrophotometer (models 180-80, Hitachi, Japan).
151
All tests were replicated and a blank sample, replacing EPS fraction with deionized water was used to
152
calibrate the AAS. All experimental data were processed using Excel and presented as mean values
153
±standard deviations.
Page 8 of 30
154
The metal uptake, Q (mg/g), was then calculated by the following equation:
Q
155
V (C 0 C e ) W
Where C0 and Ce are the initial and end concentrations of metals in the sample solutions (mg/L),
157
respectively, V is the volume of sample solutions (L), and W is the mass of sorbent (crude EPS) added in
158
the metal solutions (g).
159
2.7.
cr
ip t
156
us
Fourier transform infrared spectroscopy analysis
The Infrared (IR) spectra of EPS before and after metal adsorption tests were determined using a
161
Fourier transform infrared (FTIR) spectrophotometer (Spectrum One-B, Perkin Elmer, U.S) equipped with
162
detectors for differentiating various functional groups. The samples were freeze-dried, mixed with dry KBr
163
powder and pressed into pellets. The spectrum was recorded between 400 and 4000 cm−1 (Paulraj, Satish
164
Kumar, Yuvaraj, Paari, Pattukumar, & Venkatesan, 2011).
165
3. Results
166
3.1.
M
d te
Effects of different carbon sources on production of EPSs
Ac ce p
167
an
160
Production of EPSs was carried out through fermentation of Arthrobacter ps-5 using a culture
168
medium supplemented with the selected carbohydrates, i.e., sucrose, glucose, starch, fructose or lactose, at
169
a pre-determined concentration level of 2% (w/v). After 48h cultivation, the maximum EPSs production
170
was achieved at 0.27 g/L in the culture medium supplemented with sucrose. EPSs production was 0.24 g/L
171
in the culture medium supplemented with glucose (Fig.1). Next, the EPSs produced from the Carbohydrate
172
-enriched culture media were separated and purified for further studies of their effects on monosaccharide
173
composition and biosorption capacity of EPSs.
Page 9 of 30
174
3.2.
175
3.2.1.
Characteristics of crude and purified EPSs
Isolution and purification of EPSs
The EPSSuc was separated into two independent elution peaks after DEAE anion exchanger, i.e.,
177
sub-fractions Su-1 and Su-2 (Fig.2 Su-A). Sub-fraction Su-1 was a neutral polysaccharide as it was eluted
178
out with water, while sub-fraction Su-2 was more likely acidic as it was eluted with NaCl solution. This is
179
in agreement with other literature indications (Chang, 1987). Su-1 was the major component, shown by its
180
greater peak area in anion-exchange chromatography (Fig.2 Su-A). The ratio of Su-2 and Su-1 was 0.22.
181
Su-1 was collected and further purified through a Sephadex G-100 column and then renamed as Su-1-1 for
182
purified sample (Fig.2 Su-B).
M
an
us
cr
ip t
176
The EPSGlu was separated into two independent elution fractions, named as sub-fractions Gl-1 and
184
Gl-2 (Fig.2 G-A). Gl-1 was a neutral polysaccharide, and Gl-2 was an acidic polysaccharide, whereas the
185
ratio of Gl-2 and Gl-1 was 1.82. Gl-1 and Gl-2 fractions were collected respectively and further fractioned
186
through a Sephadex G-100 column eluted with 0.05 M NaCl solution, resulting in two purified samples of
187
Gl-1-1 and Gl-2-1 (Fig.2 G-B).
te
Ac ce p
188
d
183
The EPSSta was also separated into two independent elution fractions St-1and St-2 (Fig.2 St-A).
189
Similarly, St-1was a neutral polysaccharide and St-2 was an acidic polysaccharide. The ratio of St-2 and
190
St-1 was 1.08. St-1 and St-2 fractions were collected and further purified respectively, resulting in two
191
purified samples of St-1-1 and St-2-1 for further tests (Fig.2 St-B).
192
193
The ratio of acidic polysaccharide/neutral polysaccharide in all crude EPSs was shown in Table 1.
3.2.2.
Physical properties of the EPS
Page 10 of 30
The purified EPSs were light yellow in color and in a form of odorless powder. It was soluble in
195
water but insoluble in ethanol and other organic solvents, such as ether and acetone, which conferred with
196
general properties of polysaccharides. The aqueous solutions prepared by all purified EPSs were clear,
197
homogeneous liquids with a color of light yellow and no precipitation occurred after centrifugation. An
198
ultraviolet scan spectrum analysis of the EPSs was shown in Fig.3, indicating the absence of proteins and
199
nucleic acids due to a negative response - no absorption peaks at 260 or 280 nm. So the EPSs were
200
thought to be pure.
201
3.2.3.
us
cr
ip t
194
an
Estimation of EPSs molecular mass
The molecular mass of sub-fractions, Su-1, Gl-1, Gl-2, St-1 and St-2 was measured by HPLC with a
203
size exclusion column. Based on a calibration curve obtained using dextrans as references at various
204
molecular weight cut-off points, the average molecular weights of Su-1, Gl-1, Gl-2, St-1 and St-2 were
205
estimated to be 8.83×105, 3.48×105, 1.21×106, 1.01×105 and 7.36×105 Da, respectively. Molecular mass
206
distribution followed by the order of EPSGlu < EPSsta
