Flocculation behavior and mechanism of an exopolysaccharide from the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913

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Bioresource Technology 99 (2008) 6893–6899

Flocculation behavior and mechanism of an exopolysaccharide from the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913 W.W. Li a, W.Z. Zhou a,b,*, Y.Z. Zhang b, J. Wang c, X.B. Zhu a b

a School of Environmental Science and Engineering, Shandong University, Jinan 250100, PR China State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Jinan 250100, PR China c Environment Research Institute, Shandong University, Jinan 250100, PR China

Received 31 August 2007; received in revised form 14 January 2008; accepted 20 January 2008 Available online 18 March 2008

Abstract Flocculation behavior and mechanism of the exopolysaccharide secreted by Pseudoalteromonas sp. SM9913 (EPS SM9913), a psychrophilic bacterium isolated from 1855 m deep-sea sediment, has been studied in this paper. EPS SM9913 showed a peak flocculating activity of 49.3 in 1 g/L kaolin suspension with 4.55 mmol/L CaCl2 and the optimum pH range of 5–8. It appears that the flocculating activity of EPS SM9913 was stimulated by Ca2+ and Fe2+. This study found that EPS SM9913 showed a better flocculation performance than Al2(SO4)3 at salinity of 5–100‰ or temperatures of 5–15 °C. In addition, this EPS was effective to flocculate several other suspended solids. The measured zeta-potentials, the size of flocs formed during the flocculation process and the surface profile of flocs revealed by scan electron micrograph suggest that bridging is the main flocculation mechanism of the studied EPS. Deacetylation of EPS SM9913 resulted in a significant decrease in its flocculating activity indicating that the large number of acetyl groups in EPS SM9913 played an important role in its flocculation performance. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Deep-sea; EPS SM9913; Flocculating activity; Flocculating mechanism; Deacetylation

1. Introduction The flocculants used in water treatment can be classified into three groups: inorganic flocculants such as alum, ferrite flocculants or polyaluminum chloride; synthetic organic flocculants such as polyacrylamide derivatives or polyethylene imine; and naturally occurring flocculants such as sodium alginate or microbial flocculants. Among these flocculants, the use of alum usually leads to the problem of residual aluminum. Recent epidemiological, neuropatho*

Corresponding author. Address: School of Environmental Science and Engineering, Shandong University, Jinan 250100, PR China. Tel./fax: +86 531 88361383. E-mail addresses: [email protected] (W.W. Li), [email protected] (W.Z. Zhou). 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.01.050

logical and biochemical studies suggest a possible link between the neurotoxicity of aluminum and the pathogenesis of Alzheimer’s disease (Banks et al., 2006; Polizzi et al., 2002). Ferrite flocculants can be costly and the resultant excess iron may cause unpleasant metallic taste, odor, color, corrosion, foaming or staining. Although the synthetic organic flocculants are most frequently used because of their cost-effectiveness, they are not readily biodegradable and some of their degraded monomers such as acrylamide are neurotoxic and even show strong human carcinogenic potential (Shih et al., 2001). Because of the limitations of these flocculants, biopolymers produced by microorganisms through the synthesis of extracellular polymers by living cells are investigated as an alternative flocculant. Biopolymers are biodegradable and their degradation intermediates are harmless towards human being and the environment.

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Up to now, most of reported biopolymers (bioflocculants) are the exopolysaccharides (EPS) produced by microorganisms that were originated from soil or wastewater sludge. Although the previously studied bioflocculants usually perform well at normal temperatures or neutral pH, their flocculating activity in unfavorable conditions such as low temperatures or high salinity that are commonly encountered in wastewater treatment process often deteriorates or has been rarely studied. Deep sea, an extreme environment with high pressure, low temperature and low nutrient concentration, has numerous diverse microorganisms whose morphological, physiological and metabolic adaptations are significantly different from the microorganisms found on land. It is anticipated that exopolysaccharides from deep sea microorganisms can have a special adaptability to low temperature and high salinity condition and be used as an effective bioflocculant for wastewater treatment. The psychrophilic bacterium Pseudoalteormonas sp. SM9913, a gamma proteobacterium, was extracted from ocean sediments taken at 1855 m depth. Such obtained EPS SM9913 are highly viscous and could help to concentrate proteinaceous particles and metal ions, which provide growth and energy source for the survival of the microorganisms in the nutrient-scarce environment of the deepsea (Qin et al., 2007). There is little published work about the application of deep-sea EPS for environmental protection especially wastewater treatment at low temperature and saline conditions. This paper reports findings of our research on the flocculation behavior of EPS SM9913 as a bioflocculant in kaolin suspension of high salinity and low temperature. The mechanism and potential functional groups were also investigated. 2. Methods 2.1. Cultivating of Pseudoalteromonas sp. SM9913 and preparation of the EPS Pseudoalteromonas sp. SM9913 is a psychrophilic bacterium isolated from deep-sea sediment. Each isolated strain was cultivated in culture medium (75 mL) on a rotary shaker (200 rpm) at 12 °C for 3 days. The medium contains 2% (w/v) corn flour, 1% bran, 2% soybean flour, 0.1% Na2HPO4, 0.03% KH2PO4 and 0.1% CaCl2. The culture was centrifuged at 10,000g for 10 min at 4 °C (Eppendorf Centrifuge 5804R, Germany) to obtain a supernatant, to which cold absolute ethanol (1:1) was added to precipitate the exopolymer. The extracted exopolymer was dissolved in deionized water and decolorized with 5% (w/v) activated charcoal to remove residual culture medium. The Sevag method (Staub, 1965) was used to remove proteins from the exopolymer solution to obtain the crude EPS SM9913. 2.2. Flocculation batch test Batch tests using a suspension of kaolin clay were performed to study the flocculation properties of EPS

SM9913. After the kaolin clay (400 meshes, Sinopharm Chemical Reagent Company of China) was suspended in deionized water at a concentration of 1 g/L, 0.5 mL of 10% CaCl2 solution and 0.5 mL of EPS SM9913 solution were added to the solution. Such prepared mixture was vortexed at 200 rpm for 60 min and rested for 30 min at 25 °C. Two milliliter of supernatant was removed from the upper layer and was measured for its absorbance at 550 nm using a spectrophotometer. The absorbance of a blank sample, replacing EPS SM9913 fraction with deionized water, was also measured. The flocculating activity was calculated as the following (Kurane et al., 1994; Prasertsan et al., 2006): FA ¼ 1=ðOD550Þ  1=ðOD550Þb ; where (OD550) and (OD550)b were the absorbance of the EPS sample and the blank sample. 2.2.1. Factors on the flocculating activity of EPS SM9913 The effects of pH, salt, salinity and temperature on the flocculating activity of EPS SM9913 were examined. To investigate the effect of pH on flocculating activity, the pH of the kaolin suspension was adjusted using HCl and NaOH in the pH range 4–10. To examine the effect of salts, CaCl2 was replaced by various metal salts (KCl, MgCl2, FeSO4, FeCl3 and AlCl3) and the flocculating activity was measured. Different amount of NaCl and CaCl2 (Ca2+ was fixed at 4.55 mmol/L) were added to the kaolin suspension to study the effect of salinity. The studied temperatures ranged from 5 to 40 °C. 2.2.2. Flocculation of various suspended solids The flocculating activities of various inorganic solid suspensions were studied by replacing kaolin solution (1 g/L) with one of activated carbon, diatomite, SiO2, MgO and Al2O3 in each test (1 g/L). The soil suspension was prepared by adding 100 g of local soil to 1 L of deionized water, stirring the solution for 2 min, allowing it to rest for 5 min, and then removing 500 mL of the supernatant that was diluted to 750 mL for the flocculation test. The turbidity of the soil suspension was approximately 850 NTU, comparable to the turbidity of the 1 g/L kaolin solution. 2.3. Flocculation mechanism 2.3.1. Surface structure of flocs Particle interactions and floc surface structure were investigated by scanning electron micrograph with TXA840 scanner. 2.3.2. Zeta-potential measurement The zeta-potential of kaolin suspension (1 g/L) and kaolin clay with EPS SM9913 was analyzed with JS94H electrophoresis system. The zeta-potential of EPS (10 mg/L, the optimum dosage of flocculation) was analyzed with 3000HSA ZetaSizer (Malvern, UK).

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2.4. Deacetylation of EPS SM9913 and flocculating activity of the deacetylated EPS EPS SM9913 contains a large number of acetyl groups. The function of the acetyl groups during flocculation was studied by deacetylating EPS SM9913 as the following: vacuum-dried EPS SM9913 was ground into powder and added into 45% NaOH to react for 15 min at 100 °C. After cooling and addition of absolute ethanol (1:1), the mixture was centrifuged at 10,000g for 10 min at 4 °C. Such obtained sediment was dried and determined by 20SX fourier transform-infrared spectrophotometer. The flocculating activity of deacetylated EPS was measured following the same procedures described in Section 2.2. 3. Results and discussion 3.1. Flocculating properties of EPS SM9913 3.1.1. Effect of the EPS concentration Flocculation reactions were performed at different EPS concentrations in the range of 2–30 mg/L. Flocculating activity increased as the EPS concentrations increased from 2 to 10 mg/L and decreased thereafter. The peak flocculating activity of 49.34 occurred at an EPS concentration of 10 mg/L. Reported flocculating activities of EPS are in a wide range. Except Enterobacter cloacae WD7 (Prasertsan et al., 2006), Enterobacter sp. (Yokoi et al., 1997) and Pestalotiopsis sp. KCTC 8637P (Kwon et al., 1996), few of the reported flocculating activities are higher than 50. Apparently, EPS SM9913 provides a relatively high flocculating activity. To neutral or like-charged bioflocculants, bridging may be used to describe their flocculation mechanism. Bridging is formed when biopolymeric flocculants extend from particles’ surface into the solution for a distance that is greater than the effective distance of inter-particle repulsion. Due to bridging, the biopolymers adsorbed to particles’ surface help to form flocs (Yim et al., 2007). The SEM image of flocs illustrated the bridging effect of EPS SM9913. The EPS SM9913 was absorbed by several particles and therefore caused aggregation of particles. On the other hand, the presence of excessive biopolymers can restabilize particles because there will be no more vacant sites on particle surface to accept biopolymers that can help to form binding among particles. In addition, the settling of flocculated particles can be negatively affected due to the high viscosity from the excessive level of biopolymers in the solution.

3.1.2. Effect of pH The flocculating activities of EPS SM9913 enhanced with the addition of Ca2+, maintained high levels (43.2– 56.5) in pH range of approximately 6–8, reached the maximum of 56.5 at pH 7, but decreased when pH was greater than 8 or lower than 6. In contrast, the flocculating activities of EPS SM9913 without Ca2+ (the control) remained relatively constant at lower than 3. Apparently, cation is needed in the reaction mixture to induce effective flocculation, because polysaccharide and kaolin clay need the mediation of cation to form complexes (Kurane and Matsuyama, 1994). At high pH, hydroxide ions (OH) may interfere with the complex formed between the polysaccharide and kaolin particles mediated by Ca2+ resulting in the suspension of kaolin particles in the mixture (Prasertsan et al., 2006). At low pH, both EPS (zeta-potential of 19.3 mV at neutral pH) and kaolin particles (zetapotential of 45.7 mV at neutral pH) likely adsorb H+, which weakens the complex of polysaccharide and kaolin particles mediated by Ca2+, resulting in lower flocculating activities. The mediating effect of Ca2+ appears to be strongest at neutral pH.

3.1.3. Effect of cation types and concentrations Cations can neutralize negative charges of both polysaccharide and suspended particles and increase the adsorption of polysaccharide onto suspended particles (Wu and Ye, 2007). As shown in Fig. 1, divalent cations Fe2+, Ca2+ and Mg2+ were more effective than monovalent cations K+ and Na+ and trivalent cations Fe3+ and Al3+ in inducing the flocculating activity of EPS SM9913. Because of the large numbers of carboxylate groups of EPS SM9913 can serve as binding sites for divalent cations (Prasertsan et al., 2006), the polysaccharide and kaolin clay could form solid complexes mediated by Fe2+, Ca2+ and Mg2+. It appears that larger atomic weight lead to higher flocculat+

Na +

K 100

2+

Ca 90

Fe

80

Mg

2+ 2+

Flocculating activity

2.3.3. Change of flocs size during flocculation process The average size of flocs that were formed in a stirring cell during the flocculation process was determined with ZetaSizer 2000 (Malvern, UK). The stirring cell is connected to the particle size analyzer by a pipe. The measurement of floc size was initiated when stirring (200 rpm) started and the reading of the floc size was taken every minute.

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3+

Al 70

3+

Fe 60 50 40 30 20 2

4

6

8

10

concentration (mmol/L) Fig. 1. Effect of various cations on flocculating activity.

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ing activities. Monovalent cations such as K+ and Na+ and trivalent cations such as Fe3+ and Al3+ were less effective than divalent cations in promoting flocculation, likely due to their weaker static force of pulling between cations and the EPS.

Flocculating activity

85

3.1.4. Effect of salinity Typical seawater has an average salinity of 35‰ (by weight), 61.2% of which is NaCl (Swenson, 1983). Pseudoalteromonas sp. SM9913 was screened from deep-sea sediment. The flocculation property of EPS secreted by this strain may endure well to high salinity. As shown in Fig. 2, the flocculating activities of EPS SM9913 (at optimum dosage of 10 mg/L) are higher than that of Al2(SO4)3 (at optimum dosage of 6 mg/L) when the salinity was higher than 5‰. The peak flocculating activity (37.4) of EPS SM9913 occurred at salinity of 35‰. However, addition of NaCl (which is monovalent) still reduced the flocculating activity relative to addition of CaCl2 alone. 3.1.5. Effect of temperature The temperature’s effect on flocculation properties is shown in Fig. 3. Flocculating activities of EPS SM9913 are 50.24–59.25 when temperatures were in 5–15 °C, dropped to 28.2 at 20 °C, and then recovered to 49.34– 57.45 when temperatures rose to 25–35 °C. The flocculating activities of EPS SM9913 were higher than that of Al2(SO4)3, when temperatures were 15 °C or lower, but lower than the flocculating activities of Al2(SO4)3 when temperatures were above 15 °C. The findings suggest that EPS SM9913 can be a more effective flocculant than alum for wastewater treatment application at low temperatures. 3.1.6. Flocculation of various suspended solids The measured flocculating activities for various suspended solids in aqueous solution are shown in Table 1. The suspensions of kaolin, activated carbon and soil solid

40 35

Flocculating activity

EPS Al2(SO4)3

30 25 20 15 10 5 0

20

40

60

80

salinity ‰ Fig. 2. Effect of salinity on flocculating activity.

100

80

EPS

75

Al2(SO4)3

70 65 60 55 50 45 40 35 30 25 0

5

10

15

20

25

30

35

40

45

Temperature ˚C Fig. 3. Effect of temperature on flocculating activity.

Table 1 The flocculating activity of EPS from Pseudoalteromonas sp. SM9913 on different suspended solids at neutral pH Solid suspensions

Flocculating activitya (1/OD)

Optimum dosage (mg/L)

Maximum flocculating activity

Kaolin clay Activated carbon Soil solid Diatomite SiO2 MgO b Al2O3 b

49.34 20.45 7.95 2.12 4.29 87.30 280.70

10 20 25 18 10 10 4–20

49.34 75.00 41.67 6.42 4.29 87.30 280.70

a

Activity was measured with 10 mg/L bioflocculant and 4.55 mmol/L CaCl2. b Activity was measured without CaCl2.

could be flocculated effectively with addition of Ca2+. At 10 mg/L of bioflocculant, the flocculating activities of activated carbon and soil solid are lower than that of kaolin clay. However, at optimum dosage of bioflocculant, the peak flocculating activities of activated carbon and soil solid are close to or higher than that of kaolin clay. Because of its strong adsorption ability, activated carbon can either easily adsorb Ca2+ to form complex with polysaccharide or can adsorb polysaccharide directly without Ca2+ to form complex. For soil solid, the inorganic ions or organic compound in soil may interfere with the complexation of polysaccharide with sand or clay solid in soil, resulting in flocculating activities in soil lower than that in kaolin clay. The MgO and Al2O3 suspensions were flocculated by the addition of only polysaccharide without Ca2+, which was likely due to the mediating function of Mg(II) and Al(III). Both SiO2 and diatomite suspensions possess strong negative charges and they are chemically stable. As a result, it is difficult for them to adsorb Ca2+ and polysaccharide resulting in lower level of flocculation.

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3.2. Flocculation mechanism of EPS SM9913 3.2.1. Zeta-potentials and size changes of flocs during the flocculation process The measured zeta-potentials of kaolin clay and EPS SM9913 were 45.7 mV and 19.3 mV, respectively. The zeta-potential of kaolin particles flocculated with EPS SM9913 was 16.0 mV, when the highest flocculating activity was found. These findings suggest the static repulsive forces among the flocculated particles and indicate that

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charge neutralization would not be the main mechanism to describe the flocculation process. The particle sizes determined with ZetaSizer 2000 throughout the flocculation process revealed continued growth of the mean sizes of flocs during the first 45 min, which was followed by a slight decrease in floc sizes during the subsequent 15 min. The growth of floc sizes in the first 45 min was likely due to the aggregations of particles induced by the added EPS SM9913. The extended stirring may lead to the break-up of flocs, resulting in the slight

Table 2 Main functional groups and their relative quantities Vibration type

EPS SM9913

Deacetylated EPS 1

OAH stretching CAH stretching C@O stretching CAH bending CAO stretching

Wavenumber (cm )

Peak area

Relative quantities (%)

Wavenumber (cm1)

Peak area

Relative quantities (%)

3445.47 2920.49 1653.17 1398.28–1457.68 1047.22–1159.19

20565 3015.7 8605.2 9651.8 13945

36.87 5.42 15.43 17.30 25.00

3422.50 2915.01 1637.83 1391.87–1474.91 1030.26–1084.33

8535.8 2952.8 1226.1 7603.6 2748.3

37.01 12.80 5.32 32.96 11.92

a

EPS 50

Flocculating activity

Al2(SO4)3 40

deacetylated EPS 30

20

10

0 0

5

10

15

20

25

30

EPS mg/L

c

b

80

Flocculating activity

40

60

30

40

20 20

10 0 0

20

40

60

salinity ‰

80

100

0

5

10

15

20

25

30

35

temperature ˚C

Fig. 4. The effect of dosage (a), salinity (b), and temperature (c) on the flocculating activity of deacetylated EPS.

40

45

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decrease in floc sizes during the later 15 min in flocculation. The measured mean sizes of flocs decreased quickly and significantly when stirring stopped, and this may be because large flocs settled out prior to getting into the pipe circuit to be measured. 3.2.2. Deacetylation of EPS SM9913 and flocculating activity of the deacetylated EPS The structure of EPS from Pseudoalteromonas sp. SM9913 is characterized as a linear arrangement of a-(1,6)?Glc and high degree of acetylation (Qin et al., 2007). The large number of acetyl groups in EPS SM9913 makes it easy to absorb cations and other positively charged materials. The function of acetyl groups on flocculating activity was investigated by deacetylating EPS SM9913. The main chemical reaction is as the following: OHCH3 C OR CH3 C OH + ROH 100 oC O O

SM9913 are essential to promote effective flocculation in model saline water or low temperature wastewater. 4. Conclusions This study found that the EPS from Pseudoalteromonas sp. SM9913 can flocculate various suspended solids such as kaolin clay, activated carbon, soil, MgO and Al2O3. The flocculating activities of EPS SM9913 can be enhanced by the addition of bivalent cations such as Ca2+ and Fe2+ because of these cations’ mediating function. Results indicated that EPS SM9913 delivers better flocculation performance than alum at low temperature (5–15 °C) or in high-salinity (5–100‰) water. Such findings suggest that EPS SM9913 can be used as an effective flocculant for wastewater treatment at low temperature and/or salinity. Absorbing and bridging are likely the main flocculation mechanism of EPS SM9913. It was found that the high degree of acetylation in EPS SM9913 plays an important role in affecting its flocculation performance, especially in model saline water or low temperature wastewater. Acknowledgements

Because the FIIR spectra of EPSs were not quantitatively determined, the transmittance intensity can not be used to quantify the measured functional groups and the peaks of the two curves can not be compared directly. Instead, the area ratios of each peak to all peaks of the same curve were calculated following a Gaussian curve fit, because such ratios represent the relative quantities of the peaks. The main functional groups and their relative quantities are listed in Table 2. It was found that acetyl groups in EPS SM9913 were partly removed by deacetylation because the relative quantity of acetyl (C@O) decreased from 15.43% to 5.32% and the relative quantity of CAO decreased from 25.00% to 11.92%. The flocculating activities of deacetylated EPS and untreated EPS were compared in Fig. 4. At the optimum dosage of 20 mg/L, the highest flocculating activity of the deacetylated EPS was 27.8, which was 43.6% lower than the highest flocculating activity of 49.3 from the untreated EPS at its the optimum dosage of 10 mg/L. The flocculating activities of deacetylated EPS were all lower than that of untreated EPS in the tested range of salinity (5– 100‰). However, when the salinity was higher than 35‰, the flocculating activity of deacetylated EPS was still higher than that of Al2(SO4)3. Experimental results indicated that higher temperatures resulted in increased flocculating activities of acetylated EPS (except 20 °C), which however, were lower than that of untreated EPS or Al2(SO4)3 in the tested range of the temperature (5–40 °C). Such findings suggest that the destruction of acetyl groups in EPS SM9913 weakened the advantage of EPS SM9913 as a high performance flocculant especially at low temperature and high salinity conditions, because large number of acetyl groups in EPS

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