Flocculation Characterization of a Bioflocculant from Bacillus

Mar 2, 2015 - ... are summarized in Table 3, which shows that the present bioflocs are sensitive to HCl, less sensitive to EDTA, and insensitive to ur...
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Flocculation Characterization of a Bioflocculant from Bacillus licheniformis Zhi Wang,† Liang Shen,† Xiaoling Zhuang,† Jiangshui Shi,† Yuanpeng Wang,† Ning He,*,† and You-Im Chang‡ †

Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, People’s Republic of China ‡ Department of Chemical and Material Engineering, Tunghai University, Taichung 40704, Taiwan S Supporting Information *

ABSTRACT: In this work, the effects of different factors on the flocculating activity and flocculation mechanism of bioflocculant XMMBF from Bacillus licheniformis are investigated. The results show that the optimum concentration of bioflocculant for kaolin clay sedimentation is 4 mg/L. Both monovalent and bivalent cations increase the flocculating activity, whereas trivalent cations inhibit it. The optimum pH and concentration of kaolin clay suspension are 5−11 and 2−10 g/L, respectively. The adsorption of XMMBF on kaolin clay is described using the Langmuir adsorption model. Infrared spectrum shows the carboxyl groups, amino groups, and hydrogen bonds in XMMBF prefer the flocculation process. The zeta potential of kaolin clay can be changed from −26.26 to −1.08 mV by both Ca2+ and XMMBF. The formation of ionic bonds in the floc can be related to its sensitivity to HCl. Finally, through the help of SEM pictures, the flocculation mechanism of XMMBF is proposed.

1. INTRODUCTION Flocculation in microbial systems was first reported by Louis Pasteur (1876) for the yeast Levure casseeuse.1 In recent decades, bioflocculants, the macromolecular polymers (e.g., proteins, polysaccharides, and lipids) secreted by microorganisms, have attracted considerable scientific and biotechnological attention due to their biodegradability, harmlessness, and lack of secondary pollution.1,2 Different microorganisms such as algae, bacteria, actinomyces, and fungi have been reported to produce bioflocculants.3 The flocculating activities of bioflocculants are influenced not only by the molecular properties but also by flocculation conditions such as pH, temperature, and ionic strength. Prasertsan et al.4 found that the highest flocculating activity, 91%, of the bioflocculant from Enterobacter cloacae WD7 was obtained with the addition of 2 mg/L bioflocculant and 40 mmol/L CaCl2 at a temperature of 4−50 °C and a pH range of 2−8. Moreover, Okaiyeto et al.5 reported the best flocculating conditions when 0.1 mg/mL of bioflocculant and 1% (w/v) CaCl2 were added at a temperature of 120−100 °C and a pH range of 2−10 for the bioflocculant produced by a bacterial consortium composed of Halomonas sp. Okoh and Micrococcus sp. Leo. The flocculation reaction is a complex physical and chemical process that can be described qualitatively and quantitatively by the DLVO and divalent cation bridging (DCB) theories.6 The DLVO theory, named after its developers, Derjaguin, Landau, Verwey, and Overbeek, was first proposed to describe the stability of colloidal suspensions.7 The basic principle of this classical colloidal theory is that the total interaction energy between two interacting colloidal particles surrounded by a double layer of counterions is the sum of the van der Waals attractive energy and the electrostatic repulsive energy.8 As the ionic strength of the suspension medium increases, the double layer electric field is condensed, which decreases the repulsive © XXXX American Chemical Society

energy among particles, allowing the short-range attractive energy to promote aggregation. The DCB theory was first proposed by McKinney9 and Tezuka.10 According to the DCB theory, divalent cations bridge negatively charged functional groups within the bioflocculant, and this bridging helps to aggregate and stabilize the matrix of biopolymer and microbes, which promote bioflocculation.10 Several researchers have performed experiments that support the DLVO and DCB theory for the cation condensing double layer and bridging. Wang et al.11 investigated the flocculation characterization of bioflocculants from mixed strains of Rhizobium radiobacter F2 and Bacillus sphaeicus F6. The flocculation mechanism was speculated to be that a cation compresses the electrostatic double layer of kaolin clay particles, leading to the adsorption of the bioflocculant onto the kaolin clay particles. Moreover, those cations can incorporate with the carboxyl of bioflocculant for bridging and forming a large floc. He et al.12 and Liu et al.13 reported a bridging mechanism between kaolin particles and biomolecules mediated by Ca2+. Ren et al.14 suggested that Ca2+ could decrease the total interaction energy barrier of photofermentative bacteria, and then greatly promote the bioflocculation of Rhodopseudomonas faecalis RLD-53. In our previous study, a novel bioflocculant, XMMBF, was produced from Bacillus licheniformis CGMCC 2876, with a composition of 89% carbohydrate and 11% protein (w/w).15 This bioflocculant was proven to have potential applications in the sugar refining industry.16 In the present study, the effect of different factors (such as bioflocculant concentration, pH, concentration of kaolin clay particles, temperature, and ionic Received: December 25, 2014 Revised: February 26, 2015 Accepted: March 2, 2015

A

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Industrial & Engineering Chemistry Research strength) that influence the flocculation of clay particle suspensions by XMMBF are investigated, and the mechanism governing this flocculation process is proposed.

and temperature. After equilibrium, the supernatant was collected in a centrifuge tube and then centrifuged at 3000 r/ min for 10 min to remove solid particles. The concentrations of XMMBF were determined by a UV-1800 spectrophotometer (Shimadzu).19 The absorbance of XMMBF aqueous solution was linear for the range 2−250 mg/L at a maximum wavelength of 258 nm. The correlation coefficient was found to be 0.9982. The relationship between the equilibrium concentration of the adsorbate in the liquid phase and in the solid phase was characterized by the Langmuir (eq 2) and Freundlich (eq 3) equations, respectively:

2. MATERIALS AND METHODS 2.1. Strain Cultivation and Materials. B. licheniformis, screened in our laboratory and stored in the China General Microbiological Culture Collection Centre (CGMCC, Beijing, China) with the accession number 2876, was used in the present study. The composition of the cultivation medium and cultivation conditions for B. licheniformis can be found in our previous paper.15 Kaolin clay, purchased from the Fengxian Fengcheng Company (Shanghai, China), was chosen as a test standard material for flocculation. The average diameter of the kaolin clay particle was 1359 nm. 2.2. Production of XMMBF. We produced XMMBF by cultivating B. licheniformis according to the method described by He et al.15 After fermentation, the culture broth was centrifuged at 7000 r/min for 10 min. The supernatant was mixed with two volumes of chilled ethanol and then left to stand still at 4 °C for 12 h. The bioflocculant precipitate was collected by centrifugation at 7000 r/min for 10 min and dissolved in distilled water. We freeze-dried half of the bioflocculant solution to calculate bioflocculant yield. The other part of the bioflocculant solution was stored at 4 °C. Then, the bioflocculant precipitate was dialyzed at 4 °C for 24 h. A 1 g/L bioflocculant solution was prepared according to the concentration of the bioflocculant extract. 2.3. Determination of Flocculating Activity. We measured the flocculating activity as an indicator by using the kaolin clay suspension.17 Briefly, 0.2 g kaolin clay and 40 mL distilled water were added in a 50 mL flask and stirred with a magnetic stirrer. After mixing, the suspension was diluted to 50 mL with 0.2 g/L of CaCl2 and 1 g/L XMMBF bioflocculant. For each run of the flocculation test, the reaction was stirred at 300 rpm for 5 min and then allowed to settle freely for 5 min at room temperature. By measuring the decrease in turbidity in the upper phase of the suspension, the flocculating activity can be expressed as the flocculating rate (FR), which is calculated as FR (%) = ((A − B)/A) × 100

ρe /qe = 1/(bqmax ) + ρe /qmax

(2)

qe = K f ρe1/ n

(3)

where qe is the XMMBF uptake by kaolin clay (mg/g) and ρe is the equilibrium concentration of XMMBF in solution (mg/L). In eq 2, b and qmax are the Langmuir coefficients representing the equilibrium constant for the adsorbate−adsorbent equilibrium and the monolayer capacity. Kf and n in eq 3 are the Freundlich coefficients. The liner Langmuir and Freundlich plots are obtained by plotting (1) ρe/qe vs ρe and (2) lg qe vs lg ρe, respectively, from which the adsorption coefficients could be determined. The essential characteristics of the Langmuir equation also can be expressed in terms of a dimensionless separation factor, RL, which is defined as RL =

1 1 + bρ0

(4)

where b (L/mg) is the Langmuir constant and ρ0 (mg/L) is the highest concentration of XMMBF. The value of RL indicates the shape of the isotherm to be either unfavorable (RL > 1), linear (RL = 1), favorable (0 < RL < 1), or irreversible (RL = 0).20 2.6. Characterization of XMMBF. The zeta-potentials of kaolin clay and XMMBF were analyzed using a Nano-ZS and MPT-2 (Malvern, UK). Scanning electron microscopy (SEM) images were taken using a Zeiss SIGMA SEM (Germany). The purified bioflocculant sample and the bioflocculant combined with kaolin clay were analyzed using a Fourier transform infrared (FT-IR) spectrophotometer (Nicolet IR200, Thermo Electron Corporation, USA) over wavelengths ranging from 4000 to 500 cm−1. To further study the relationship among bioflocculants, kaolin clays and Ca2+, we added the chemicals EDTA (2%, w/v), HCl (1 mol/L) and urea (2%, w/v), respectively, to the flocs.

(1)

where A and B are the optical density values at 550 nm of the control and the sample, respectively.18 2.4. Flocculation Conditions. The effects of the concentration of XMMBF, cationic electrolytes, pH, concentration of kaolin clay particles, and temperature on FR were examined under various conditions as follows: (1) Different XMMBF concentrations of 0, 1, 2, 4, 8, 16, 32, 64, 128, and 256 mg/L were added into the kaolin clay solution. (2) Different amounts of electrolytes of NaCl, KCl, CaCl2, MgCl2, FeCl3, and AlCl3 were used as the sources of cationic electrolytes. (3) The pH values of the kaolin clay suspension were adjusted by using HCl and NaOH of 1 mol/L to pH values of 3, 4, 5, 6, 7, 8, 9, 10, and 11. (4) Various concentrations of kaolin clay particles ranging from 0.5 to 10 g/L were flocculated by the different concentrations of XMMBF adopted above. (5) The study temperatures ranged from 10 to 85 °C. 2.5. XMMBF Adsorption Experiments. The optimized volume of CaCl2, 0.2 g kaolin clay and various volumes of XMMBF solutions (250 mg/L) were added to a 50 mL flask. Then, the aqueous solutions were diluted to 50 mL with distilled water. The aqueous solutions of XMMBF of various concentrations (5−160 mg/L) were adjusted to the desired pH

3. RESULTS AND DISCUSSION 3.1. Effect of the Concentration of XMMBF on Flocculating Activity. Figure 1 shows that flocculating rate increases with increase in XMMBF concentration. The flocculating rate remained at 98% with 4−32 mg/L XMMBF. When the concentration of XMMBF was lower than 4 mg/L or higher than 32 mg/L, the flocculating rate was slightly reduced. Flocculating activity was poor at either low or high concentrations of the bioflocculant because of the shortage of colloid adsorption coverage and adsorption degree.21,22 This result can be evidenced by some previous reports. (1) The linkage of the bioflocculant TJ-F1 chain drawn from Proteus mirabilis23 was difficult to stretch across the liquid at high concentrations; therefore, the surface adsorption sites were reduced, and bridging became difficult. (2) The highest B

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The highest flocculating rate of the bioflocculant drawn from Bacillus sp. PY-9025 was at a concentration of 20 mg/L but decreased at lower or higher concentrations. (4) When the concentration of the bioflocculant drawn from Citrobacter sp. TKF0426 was within 1−10 mg/L, the flocculating rate remained as high as 90%. 3.2. Effect of Cationic Electrolytes on the Flocculating Activity Of XMMBF. The cationic electrolytes can stimulate flocculation by neutralization and stabilization of the negative charges of the carboxyl groups of uronic acid, pyruvic acid, and acetic acid that exist in an acidic polysaccharide, which, in turn, can enhance the bridge formed among kaolin particles in aqueous solution.27 Figure 2 shows that almost all the cationic electrolytes can enhance flocculating activity at a rate higher than 50%, with the exception of Fe3+ and Al3+. Divalent cations (Ca2+, Mg2+) exhibit better flocculating activity than monovalent cations (Na+, K+). This suggests that the electrostatic neutralization ability of divalent cations is higher than that of monovalent cations. As shown in Figure 2, the highest flocculating rate of XMMBF reaches 90% at a concentration of 2.0 mmol/L of MgCl2 and CaCl2. The flocculating rate is

Figure 1. Effect of the concentration of XMMBF on flocculating rate.

flocculating rate of bioflocculant F-1 drawn from Baker’s yeast cells24 could be achieved at a concentration of 10−200 mg/L. However, the rate decreased when the bioflocculant concentration was higher than 1000 mg/L or lower than 2 mg/L. (3)

Figure 2. Effect of the cations on the flocculating rate of XMMBF. C

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Industrial & Engineering Chemistry Research lowered when trivalent cations are added. These trivalent cations might change the surface potential of kaolin clay particles from negative to positive and cover most of the adsorption sites of XMMBF because of their high positive charge density. Similar phenomena have been reported for other bioflocculants. He et al.12 found that the flocculating rate of HBF-3 produced by Halomonas sp. V3a′ could be increased from 73.7 to 96.9% when Ca2+ concentration rose from 0 to 11.25 mM, and this result could be attributed to the double layer compression effect. Kumar et al.28 realized that the flocculating activity of the bioflocculant drawn from a haloalkalophilic Bacillus can be significantly enhanced by the addition of divalent cations such as Ca2+, Cu2+, Zn2+, Mn2+, Co2+, and Fe2+ because of the neutralization of the zeta potential and can be decreased efficiently by the addition of Al3+, Fe3+, Ni2+, and Na+. 3.3. Effect of the pH on the Flocculating Activity of XMMBF. With dissociative groups, such as −COOH, −SO3H, and −NH3+, different bioflocculants have corresponding pH requirements. The effect of the pH value of kaolin clay suspension on the flocculating rate of XMMBF is shown in Figure 3. In the presence of Ca2+, the flocculating activity

Figure 4. Effect of the concentration of kaolin clay suspension on flocculating rate.

that the ability of Ca2+ on compressing the electric double layer becomes limited. 3.5. Effect of Temperature on the Flocculating Activity of XMMBF. As shown in Figure 5, the flocculating

Figure 5. Effect of the temperature of kaolin clay solution on flocculating rate.

Figure 3. Effect of the pH value of kaolin clay solution on the flocculating rate of XMMBF.

activities of XMMBF are all above 80% within the examined temperature ranges of 10−85 °C. The flocculating activity of a bioflocculant drawn from Arcuadendron sp. TS-49 was significantly increased at a temperature above 50 °C due to the increase in entropy,32 or this temperature may be the optimum temperature for enhancing the polysaccharide chain to have highly effective flocculation.4 Moreover, the optimum reaction temperature for the acidic polysaccharide produced by Enterobacter sp. BY-29 was found to be 25 °C.33 The wider range of acceleration temperature (10−85 °C) for XMMBF from B. licheniformis CGMCC 2876 may be attributed to its composition of 89% carbohydrate and 11% protein (w/w).15 Polysaccharide consist of crystalline and amorphous regions; generally, the crystalline regions are stronger and more resistant to high temperatures than noncrystalline counters.34 3.6. Characterization and Flocculation Mechanism of XMMBF. 3.6.1. Adsorption Isotherm of XMMBF on Kaolin Clay. Figure 6 shows XMMBF uptake isotherm plotted against final XMMBF concentration, ρe, in aqueous solutions. The experimental data are plotted as ρe/qe vs ρe and lg qe vs lg ρe. The values of Langmuir and Freundlich constants were obtained using linear regression, and the results are shown in Table 1. The Langmuir model fits better than the Freundlich

always maintains above 92% at pH ranging from 5 to 12, which reaches the maximum of 97% at a pH of 8. The increased zeta potentials (i.e., the electrostatic repulsion forces) with the rise of pH values limit XMMBF to be adsorbed on the surfaces of kaolin clay particles and therefore reduce the flocculating rate (see Figure 7 below). The pH is known to be a key factor that influences the flocculating activity in different reaction systems.24,29,30 The optimum environmental pH value for the bioflocculant pKG0322 was reported to be 3−6, whereas that for the bioflocculant NU-231 was pH 12−14. 3.4. Effect of the Concentration of Kaolin Clay Solution on Flocculating Activity. Without adding Ca2+, as shown in Figure 4, the flocculating rate of XMMBF can reach 75% and then decrease to 46% as the concentration of kaolin clay increases, while in the presence of Ca2+, the flocculation rate of XMMBF can reach 95% for all of those kaolin clay concentrations present in Figure 4. This is due to the strong compression ability of Ca2+ on the thickness of the electric double layer. At a 10 g/L concentration of kaolin clay suspension, the flocculation rate decreases slightly, indicating D

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kaolin clay particles could intimately entwine with each other in the suspension. The three-dimensional structure of the floc could also raise the flocculating activity by net catching particles. Meanwhile, the sweep flocculation mechanism can be speculated from the phenomenon that the flocculating rate is always higher than 90% when the concentration of XMMBF is increased.36 Compared with the bioflocculant TJ-F1 (∼4 μm) from Proteus mirabilis TJ-1,37 as shown in Figure 8a, the linear bioflocculant XMMBF (>10 μm) is longer. In Figure 8c, the formation of large flocs is similar to the flocs formed by a bioflocculant derived from the consortium of Cobetia sp. OAUIFE and Bacillus sp. MAYA.38 This large floc settles easily due to its greater gravity, which reveals its excellent flocculating performance. 3.6.4. Infrared Spectrum of the Flocculation Behavior. The infrared spectra of kaolin suspensions for the present bioflocculation experiments are also measured. As shown in Figure 9, there is a shift on the characteristic peak of −NH2, changing from 3423 to 3448 cm−1. Part of the −NH2 involved in the adsorption and the original hydrogen bonds is damaged, which will lead to the increased charge density around the -NH2 groups. In addition, because of the decreased charge density after the coordination bond formatted between −COO− and Ca2+, we note that there is a shift on the vibration absorption peaks of −COO−, changing from 1641 to 1630 cm−1. Table 2 shows that the flocculating rate reaches 40% only when Ca2+ is present in the kaolin clay suspension. A possible reason could be drawn from the fact that Ca2+ compresses the electric double layer of clay particles and therefore reduces the electrostatic repulsion force interacting between the particles, which consequently causes clay particle suspension to become unstable. The coordination ability of Ca2+, which bridges kaolin clay particles directly, is another possible reason. Because of the finite number of adsorption sites on kaolin clay, the Ca2+ could be absorbed on the clay particles, which then caused slight flocculation of kaolin clay.39 As shown in Table 2, the flocculating rate reaches 62% only when XMMBF is present in the kaolin clay suspension. This is because the amino and carboxyl groups of XMMBF facilitate a chemical type of adsorption on the kaolin clay surfaces and then form a larger floc to produce easier flocculation. Additionally, because the zeta potential of the floc decreases from −26.26 to −1.08 mV in the presence of XMMBF and Ca2+, the flocculating rate reaches 98%. The effects of HCl, EDTA, and urea on the present bioflocculation experiments are summarized in Table 3, which shows that the present bioflocs are sensitive to HCl, less sensitive to EDTA, and insensitive to urea. Generally, EDTA and HCl can destroy the formation of ionic bonds, and urea can destroy hydrogen bonds. It is easier for EDTA to coordinate with Ca2+ by destroying the ionic bonds in the flocs. When adding the flocs into the solution of HCl, the carboxyl ion (−COO−) of XMMBF prefers to accept the H+ of the solution and the −COOH group can be formed, which destroys the ionic bonds formed between Ca2+ and the bioflocculant and consequently leads to the collapse of the flocs. However, there is no collapse observed for those flocs present in the urea solution, which indicates that no hydrogen bond will be formed in this bioflocculation experiment. All these results prove that Ca2+ not only plays a role in compressing the electric double layer but also plays a significant part in coordinating the bridge

Figure 6. Adsorption of XMMBF on kaolin clay at temperature 25 °C and initial pH 7.0.

Table 1. Parameters for Langmuir and Freundlich Isotherms for XMMBF Adsorption Freundlich coefficients

Langmuir coefficients (1−1

R2

n

Kf (mg /n) 1/n L /g)

R2

b (L/mg)

qmax (mg/g)

RL

0.8050

3.306

78.168

0.9943

0.3748

263.158

0.0106

model with the experimental data due to the higher correlation coefficient (R2). The obtained RL value for the Langmuir model is between 0 and 1, which indicates that the adsorption of XMMBF on the clay surfaces with finite number of homogeneously distributed adsorption sites is favorable for the type of monolayer adsorption.35 3.6.2. Zeta-Potentials of XMMBF and Kaolin Clay Solution. The surface electrical properties of the kaolin clay and XMMBF for different pH values are shown in Figure 7; both kaolin clay and bioflocculant surfaces are negatively charged in the neutral environment.

Figure 7. Zeta-potential of XMMBF and kaolin clay solutions.

3.6.3. SEM Observation. Figure 8 presents SEM pictures of XMMBF, kaolin clay suspension, and flocculation flocs. As shown in Figure 8a, the morphology of the XMMBF is an interleaving linear chain-like structure. The formation of large flocs is shown in Figure 8c, which is like a tightly intertwined net and provides evidence that the present kaolin clay suspension can be flocculated by XMMBF efficiently. This result suggests that the chain-like XMMBF connected with E

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Figure 8. SEM analysis of (a) purified bioflocculant, (b) kaolin clay and, (c) kaolin clay suspension flocculated by XMMBF.

obtained either between kaolin clay particles and biofloccuant XMMBF or between kaolin clay particles themselves. The previous measurements reported that zeta potentials of kaolin clay and kaolin clay flocculated by SM9913 are −45.7 and −19.3 mV, respectively, which indicates that the electrostatic repulsion forces are essential in flocculating clay particles and that the charge neutralization mechanism is not the main mechanism of flocculation.40 Flocs of Pseudomonas strain C-120 can be either deflocculated in HCl or reversibly deflocculated by urea.41 The phenomena that Pb (II)-loaded MBFGA1 dissolved gradually in EDTA and dissolved rapidly in HCl, indicating that ionic bonding formation is the main meachanism of combining MBFGA1 with Pb (II).42 3.6.5. Hypothesis of the Bioflocculation Mechanism. The flocculation model of bioflocculant XMMBF in kaolin clay solution is hypothesized in Figure 10. First, Ca2+ incorporates with the carboxyl of XMMBF. Then, the kaolin clay particle can effectively connect with the XMMBF through ionic bonding. Finally, the chain-like XMMBF twists together and sinks. During the settlement process, free kaolin clays are net-caught. In addition, Ca2+ compresses the electric double layer of kaolin clay particles, which benefits the adsorption of XMMBF and therefore will help increase flocculating activity. Hence, Ca2+ plays an indispensable role in the present flocculating process. Generally, DLVO and DCB theory were used to explain the flocculation process, mostly with both biological and chemical flocculants.6 The DLVO theory indicates that the addition of any cations such as Ca2+ and Fe3+ will increase the solution ionic strength and compress the double layer thereby decreasing the electrostatic repulsive energy among particles. Whereas, our results for the case of adding the trivalent cations Fe3+ and Al3+ are not in conformity to the DLVO theory (Figure 2) and Xu et al.43 found a similar result. However, Ca2+ can decrease the absolute value of kaolin clay potential from 26.26 to 13.58 mV (Table 2) due to reducing the size of the electric double layer. It is indicated by the divalent cation

Figure 9. Infrared spectrum of XMMBF bioflocculant and flocs.

Table 2. Relationship of Kaolin Clay, XMMBF Bioflocculant and Ca2+ in the Flocculation Process kaolin clay Abs flocculating rate (%) zeta potential (mV)

2.398 0 −26.26

kaolin clay + Ca2+ 1.435 40 −13.58

kaolin + bioflocculant 0.914 62 −19.42

kaolin + Ca2+ + bioflocculant 0.047 98 −1.08

Table 3. Results of Treatment of Flocs by Different Chemical Reagents reagent phenomenon

HCl flocculent body collapsed clearly

urea no phenomenon of collapse of the flocculant body

EDTA flocculent body collapsed, but not clearly

Figure 10. Flocculation model of kaolin clay−Ca2+−XMMBF bioflocculant (not to scale). F

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bridging (DCB) theory that the divalent cations can bridge those negatively charged functional groups that existed on the surfaces of flocculants, and this bridging effect efficiently aids the flocculation process. In the present XMMBF flocculation experiment, Ca2+ and Mg2+ produced the same improvement effect, which suggests that the interaction between XMMBF and those divalent cations is a specific bridging phenomenon based on all the cations. In addition, there are several other reports. The IH-7, pKG03 and MBF-6 were not enhanced by the addition of any cation including Ca2+.22,44,45 The charge neutralization mechanism played an important role during the flocculation process of the sludge bioflocculant PSB-2.46 The selfflocculation of the bioflocculant from Arthrobacter sp. B4 was mediated by ionization and charge neutralization mechanism.47

4. CONCLUSIONS Our study shows a much lower optimum dosage of XMMBF, 4 mg/L, in kaolin clay solution with high thermal and pH stability, which might indicate an economical industrial application potential. Both Ca2+ mediated adsorption bridging through ionic bond and compressing the electric double layer play the leading role in the flocculation process by the bioflocculant XMMBF. Meanwhile, the net-catch can promote the flocculation of kaolin clay.

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ASSOCIATED CONTENT

S Supporting Information *

Langmuir and Freundlich isotherms for XMMBF adsorption on kaolin clay. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-592-2185495. Fax: +86-592-2184822. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (51378444) and the program for New Century Excellent Talents of Education Ministry of China (ncet-13-0501). We also gratefully acknowledge the American Journal Experts (AJE) for revising our English.

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DOI: 10.1021/ie5050204 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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DOI: 10.1021/ie5050204 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX