Characterization and flocculating properties of an extracellular biopolymer produced from a Bacillus subtilis DYU1 isolate

Process Biochemistry 42 (2007) 1114–1123 www.elsevier.com/locate/procbio

Characterization and flocculating properties of an extracellular biopolymer produced from a Bacillus subtilis DYU1 isolate Jane-Yii Wu *, Hsiu-Feng Ye Department and Graduate Program of Bioindustry Technology, Dayeh University, Changhua 515, Taiwan Received 1 February 2007; received in revised form 13 April 2007; accepted 7 May 2007

Abstract Biopolymer DYU500 produced from Bacillus subtilis DYU1 was found to have excellent flocculating ability. With the addition of 40 mgDYU500/L and 50 mM CaCO3, the optimum temperature for flocculation performance of DYU500 was 30 8C, giving the highest flocculating activity and rate of 13.5 and 97%, respectively. Analysis with Fourier transform infrared spectrophotometry (FT-IR), nuclear magnetic resonance spectrometry (NMR) and amino acid identification shows that the DYU500 biopolymer mainly possesses the structure of poly-glutamic acid (PGA). The average molecular weight of DYU500 was about (3.16–3.20)  106 Da as determined by gel permeation chromatography. The major components of biopolymer DYU500 were total sugars, uronic acids, proteins and polyamides (homopolymer of glutamic acid), accounting for a weight ratio of approximate 14.9, 2.7, 4.4 and 48.7% (w/w), respectively. The flocculating activity of DYU500 in the kaolin suspension was markedly stimulated by the addition of bivalent cations Ca2+ or Mg2+ in optimum concentration ranges of about 0.15–0.90 and 0.10–0.90 mM, respectively. The synergistic effect of cations was most effective at a weak acidic or neutral pH (6.0–7.0). The flocculating activity of DYU500 linearly decreased with an increase in incubation temperature and the activity was completely lost when heating upon 120 8C, arising from the destruction of the polyamides structure of DYU500. Moreover, mechanisms describing the flocculation process with DYU500 were proposed based on the experimental observations. # 2007 Elsevier Ltd. All rights reserved. Keywords: Biopolymer; Bioflocculant; Bacillus subtilis; Flocculating activity; Polyamides; Kaolin particles

1. Introduction Flocculants have been popularly used in wastewater treatment, food and fermentation industries, drinking-water treatment, and industrial downstream processing [1–3]. In general, flocculants are categorized into synthetic organic flocculants, synthetic inorganic flocculants, and bioflocculants. Among them, the organic synthetic polymers flocculants are widely applied due to their lower cost and higher efficiency [4]. However, the synthetic organic flocculants inherit the drawback of being less biodegradable and producing carcinogenic monomers during degradation. Hence, using those synthetic organic flocculants could raise environmental and health concerns [3,5]. Therefore, the development of safe and biodegradable flocculants is of urgent needs. In wastewater treatment, flocculation is a common and effective method for

* Corresponding author. Fax: +886 4 8511323. E-mail address: [email protected] (J.-Y. Wu). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.05.006

removing suspended solids and metal ions [6]. Bioflocculants act in agreement with metal ions to facilitate the formation and settlement of sludge in both aerobic and anaerobic treatment systems [7]. In addition, bioflocculants can be an alternative to centrifugation and filtration for harvesting microbial cells from broth in food and fermentation industries [8]. Thus, bioflocculants find potential applications in wastewater treatment and other relevant industries. In recent years, the study of bioflocculants has attracted wide attention. Since bioflocculants are biodegradable, harmless, and free of secondary pollution, they could be potential replacement for organic synthetic flocculants in the aforementioned industries when environmental impacts become a major concern [2,6,9]. The bioflocculants produced by microorganisms are mainly composed of high molecular weight biopolymers, such as proteins [10], glycoproteins [11], and polysaccharides [12,13]. Literature shows that a variety of microorganisms are able to produce bioflocculants. For instance, Rhodococcus erythropolis S-1 [14,15], Nacardia amarae YK1 [10], and Bacillus sp. DP-152 [12] produced

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protein flocculants. Alcaligenes latus B-18 [16], Alcaligenes cupidus KT201 [17], and Bacillus sp. DP-152 [12] produced polysaccharide flocculants. In addition, Arathrobacter sp. was found to produce glycoprotein flocculants [18]. Given that most synthetic flocculants are harmful to the organisms and environment, their application could be restricted to a great extent even though they are effective and inexpensive. In contrast, bioflocculants are in general nontoxic, harmless, and biodegradable, and are thus superior to synthetic ones from the environmental aspect. However, high cost and low flocculating activity of bioflocculants have been their major pitfall, limiting the feasibility of their practical applications. Nevertheless, the commercial viability may be enhanced when microbial flocculants are produced economically via large-scale fermentation and the extracellular bioflocculants are harvested from the fermentation broth via simple and cost-effective downstream processing. In this work, biopolymer-producing bacteria processing flocculating activity were isolated, and the biopolymer product (designated as DYU500) was characterized. Furthermore, the flocculation mechanism of DYU500 was also proposed based upon the experimental data. 2. Materials and methods

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collected by centrifugation at 9000  g for 20 min and re-dissolved in distilled water. After the precipitation procedures were repeated three times, the crude biopolymer was obtained, and designated as bioflocculant DYU500.

2.3. Measurement of flocculating activity Kaolin hydrated was purchased from MP Biomedicals Co. (OH, USA). The actual characteristics of kaolin suspension are listed in Table 1. The zeta potential of kaolin particles under different pH was measured using the zeta potential/particle sizer (Nicomp 380/ZLS, CA, USA). The flocculating capability of bioflocculant DYU500 in a kaolin suspension was determined based on the method reported by Yokoi et al. [5] and Deng et al. [6]. In general, 0.1 mL flocculant solution, 0.25 mL cations and 4.65 mL of 5 g/L kaolin suspension were mixed in a test tube. The mixture was stirred with a Vortex mixer for 30 s and then kept still for 5 min. The absorbance of the supernatant and the bioflocculant-free blank control was measured at 550 nm (as OD550 and OD550,blank, respectively). All assays were conducted in triplicates. The flocculating activity and flocculating rate were defined and calculated using the following equations [5,6]: flocculating activity ¼

1 1  OD550 OD550;blank

(1)

flocculating rateð%Þ ¼

OD550;blank  OD550  100 OD550;blank

(2)

2.4. Effect of metal ions, pH and temperature on the flocculating activity of bioflocculant

2.1. Isolation of bioflocculant-producing microorganisms Soil samples collected from a soy sauce manufacturer located in southern Taiwan (Chia-Yi, Taiwan) were suspended in sterile distilled water and boiled for 5 min. An aliquot (0.1–1 mL samples) of each suspension was spread on isolation medium, containing Trypticase Soy Broth (TSB, 30 g/L), potassium nitrate (5 g/L), glucose (10 g/L), L-glutamic acid (20 g/L), and agar (15 g/L). The final pH was adjusted to 7.2. After incubation at 37 8C for 48 h, highly mucoid and fast growth colonies were selected and purified by repetitive dilution. Pure colonies were then inoculated into 100 mL of basal medium supplemented with glucose and L-glutamic acid in a 500 mL conical flask and incubated at 37 8C in a rotary shaker at 150 rpm for 48 h. The basal medium consisted of (g/L): beef extract, 10; yeast extract, 5; glucose, 10; L-glutamic acid, 50. The culture broth bearing bioflocculant appeared to be viscous. The relative viscosity of culture broth from cultivation with different bacterial isolates was measured and compared. Finally, seven promising isolates were selected. Among them, a strain identified as Bacillus subtilis DYU1 (GenBank nucleotide sequence accession number is EF442670) exhibited the highest flocculating activity and was thus chosen for further study. Detailed characterization of B. subtilis DYU1 and strategies for bioflocculant production will be reported elsewhere. The cells of strain DYU1 were collected by centrifugation at 6000  g for 15 min and then suspended in 20 mL of 20% glycerol solution. The cell suspension was stored at 20 8C before use.

2.2. Preparation of bioflocculant The fermentation broth was centrifuged at 9000  g for 20 min. An appropriate amount of supernatant was mixed with ethanol at a volume-tovolume ratio of 1:4 to precipitate the bioflocculant. The resulting precipitate was

To explore the effect of the metal ions on flocculating activity of the tested flocculant, the flocculation experiments were performed using 5 g/L of suspended kaolin supplemented with various metal ions, including Al2(SO4)3, Fe2(SO4)3, CaCO3, CaCl2 and MgSO4. The reaction mixture consisting of 4.65 mL of 5 g/L kaolin suspension, 0.25 mL of various concentrations metal ions (5  103 to 50 mM) and 0.1 mL of 2 g/L DYU500 solution. The final DYU500 concentration in the mixture was 40 mg/L. To estimate the influence of pH values on flocculating activity, the reaction mixture suspension were adjusted to desired pH values using HCl and NaOH. The pH values of the test suspensions ranged from 4.0 to 9.0. The effects of temperature were also examined using kaolin and bioflouccant at various temperatures. The flocculation activity was calculated using Eqs. (1) and (2).

2.5. Analysis of bioflocculant composition and molecular weight The total sugar content of bioflocculant DYU500 was determined by the phenol–sulfuric acid method using glucose as the standard solution [19]. The uronic acid content was measured by cabazoic method [20]. The protein moiety in the flocculant molecule was determined by the Folin-phenol method using bovine serum albumin as a standard. To assay for the amino acids in the bioflocculant, the bioflocculant sample was hydrolyzed with 6N HCl at 100 8C for 5 h in a glass vial under a nitrogen atmosphere with a Pico-Tag apparatus. After removal of residual HCl by evaporation, the hydrolyzed bioflocculant was dissolved in 1 mL of distilled water and the amino acid composition was analyzed by amino acids autoanalyzer (Hitachi L-8800). The average molecular weight of the bioflocculant was measured by gel permeation chromatography (GPC) using a Hitachi L6200 system controller equipped with Shodex KB800 series columns (two KB80M, one KB802.5) and

Table 1 Characteristics of kaolin suspension at pH 7.0 Kaolin concentration (g/L)

Total phosphorus, TP (mg/L)

Total nitrogen, TN (mg/L)

Chemical oxygen demand, COD (mg/L)

Suspended solids, SS (g/L)

1.0 2.5 5.0

0 0 0

0.18 0.42 0.93

0 0 0

0.98 2.46 4.93

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a refractive index (RI) detector (Bischoff, Model 8110). Dextrans standards (Phenomenex, USA; MW: 7.2, 16.2, 35.6, 74.3, 170, 535, 1580, 2754 kDa) were used to construct a calibration curve. The eluant used was de-ionized H2O and the flow rate was set at 1.0 mL/min. The temperature of column oven was set at 50 8C. Test samples used for the FT-IR analysis were first dried and ground into a powder form. The powder was then mixed with KBr (1:100) and pressed into a disk. Analysis was performed on a FT-IR spectrometer (Perkin-Elmer Spectrum RX1 FT-IR System, Buckinghamshire, England). In addition to FT-IR analysis, the composition of bioflocculant DYU500 was also confirmed by the nuclear magnetic resonance (NMR). Analysis of 1H and 13C NMR was conducted with a NMR spectrometer (Varian Unityionva 500 NMR Spectrometer, MO) using DMSO-d6 as an internal reference. The elemental analysis of DYU500 was measured using the Heraeus CHN-OS Rapid elemental analyzer (Heraeus, Germany).

3. Results and discussion 3.1. Effects of metal ions and pH on the flocculating capability Flocculation is due to decline of charge density by supplied cations, leading to inter-particle bridging between kaolin particles [10]. The promoting effect of the added cations on flocculation is highly dependent on both the concentration and valence of the ions [21]. The pH of reaction mixtures is also known to be a key factor influencing flocculating activity [22]. Therefore, the effect of metal ions and pH was determined by adding various metal ions concentration to kaolin suspension containing DYU500 at different pH values. As shown Fig. 1, the flocculating activity clearly increased with the addition of 50 mM Ca2+ or 20.3 mM Mg2+ at a pH range of 4–9, suggesting highly synergistic effects with the addition of bivalent cations. However, addition of trivalent cations (i.e., Al3+ and Fe3+) resulted in marginal improvement on the flocculating activity. These results are in agreement with a previous study reporting that oversupply of Al2(SO4)3 or Fe2(SO4)3 led to inactivation of bioflocculants, due probably to excessive absorption of FeCl3 [10]. Moreover, addition of monovalent cations, such as K+ and Na+, had slight enhancing effect on flocculating capability. On the other hand, the optimal flocculating activity for both Ca2+ and Mg2+ occurred near a neutral pH, while the flocculating activity decreased when the pH value was higher than 8.0 or lower than 5.0. At pH 7.0, the maximum flocculating activity (13.5) and flocculating rate (97%) were obtained with the addition of 50 mM Ca2+ and 40 mg-DYU500/L. The preferable pH for flocculation obtained with DYU500 is slightly different from that for bioflocculant produced by Rhodovulum sp., as the optimal pH was approximately 8.0 in the presence of Ca2+ or Mg2+ [21]. Salehizadeh et al. [3] indicated that the activity of bioflocculant produced from B. coagulants was stimulated by addition of Al3+, Fe3+ and Ca2+ at a concentration of 0.2, 0.25 and 8 mM, respectively. Moreover, bioflocculant PGA produced from B. licheniformis [23] gave a maximum flocculating activity of 7.1, 6.4 and 7.0 with the addition of Al3+, Fe3+ and Ca2+, respectively. The optimal supplemental concentration of Ca2+ for the best flocculating activity was 13.5 mM [23].

3.2. Effects of bioflocculant concentrations on the flocculating capability The foregoing results clearly show that even supplying in large quantities, the bivalent cations could stimulate the flocculating activity of DYU500, whereas excessive addition of trivalent cations would decrease the activity. Thus, experiments using lower cations concentration were conducted to further examine the cation effect on flocculating activity. Flocculation tests were performed at different DYU500 concentration ranging from 10 to 80 mg/L with an addition of a smaller quantity of cations (Fig. 2). For lower concentration range of trivalent cations, the flocculating activity increased with increasing DYU500 concentration from 10 to 30 mg/L, but it decreased once DYU500 concentration exceeded 40 mg/L. Hence, the trivalent cations could still enhance the flocculating activity if they are supplied at an appropriate amount. In contrast to trivalent cations, the flocculating capability of DYU500 with a low dosage of divalent cations (Fig. 2d) was lower than with a high dosage (Fig. 1d). These results indicate that at the same concentration of bioflocculant, the optimal addition quantity of cations for promoting flocculation tended to decrease with an increase in valence number of the cations. That is to say, the flocculation is due probably to the change in charge density, as the cation effect could result in neutralization of the zeta potential, which is in accordance with Schulze Hardy’s law [24]. The results shown in Figs. 1 and 2 indicate the promoting effect of cations on flocculating activity of DYU500, but also revealed that each cation may have its optimal dosage. 3.3. Effects of metal ions concentrations on the flocculating capability Cations stimulate flocculating activity by neutralizing and stabilizing the residual negative charge of functional groups by forming bridging between particles [4,10,15,25,26]. Fig. 3 shows highly synergistic effects with addition of bivalent or trivalent cations over different cation concentration ranges. Although the synergistic effects were exerted at different pH depending on the cations, the extent of synergistic effects decreased in the order of trivalent > bivalent  monovalent. The optimal cation concentration for enhancing flocculating activity of DYU500 was >10 mM for monovalent ions (Na+ or K+), 0.10–0.90 mM for bivalent ions (Ca2+ or Mg2+), and <0.005 mM for trivalent ions (Al3+ and Fe3+) (Fig. 3). Moreover, the synergistic effect on flocculation of the trivalent cations disappeared when cation concentration was over 0.02 mM (Figs. 1, 2 and 3c). It is obvious that Al3+ and Fe3+ could increase flocculating ability of DYU500 only at low concentrations while high concentrations would inhibit the activity of DYU500. Addition of bivalent cations such as Ca2+ and Mg2+ was able to enhance the flocculating capability. Meanwhile, the preferable dosage of bivalent cations was higher than trivalent cations (Fig. 3b). On the other hand, monovalent cations could also promote the flocculating activity but the effect was less significant. Meanwhile, more than 20 or

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Fig. 1. Effect of metal ions and pH on flocculating activity and flocculating rate using DYU500 (40 mg/L). Al2(SO4)3: 37.5 mM; Fe2(SO4)3: 12.5 mM; FeSO4: 32.9 mM; MgSO4: 20.3 mM; CaCO3: 49.9 mM; NaH2PO4: 32 mM; KH2PO4: 36.7 mM.

10 mM of Na+ and K+, respectively, was required for stimulating flocculation (Figs. 1 and 3a). The charge bridging between the bioflocculants leads to an increase in floc density, floc size and the floc resistance to shear. However, since monovlent cations reduce the strength of the bonds and cause a loose structure of flocs, thus resulting in a decrease in floc density, size and the floc resistance to shear. This explains why the trivalent and bivalent cations have stronger synergistic effect for flocculation. Nevertheless, an excessive addition of bivalent or trivalent cations may eliminate the promoting effect. Table 2 summarizes the bioflocculantproducing microorganisms and their optimum flocculating conditions reported in the literature. The information shows that the characteristics and structure components of bioflocculants are highly dependent on their microbial origin. The same metal ions might have different effects on different bioflocculants, as the promoting effect of the added cations varied according to both the concentration and the valence of cations. 3.4. Effect of temperature on flocculating activity To understand the effect of temperature on flocculating capability of DYU500, the bioflocculant was incubated at different temperatures and the flocculating activity of resulting

samples was determined. The temperature dependence of flocculating capability of DYU500 is depicted in Fig. 4, showing that the flocculating activity and flocculating rate linearly decreased with an increase in incubation temperature. The optimal temperature was 30 8C, giving the best flocculating activity and flocculating rate of 5.2 and 90%, respectively. Yokoi et al. [35] reported an optimum temperature range of 20– 40 8C for flocculating activity of pectin with the highest activity also taking place at 30 8C [35]. The flocculating activity of DYU500 decreased by 50% after incubation at 60 8C for 15 min, while the bioflocculant was completely inactivated when it was heated at 120 8C (Fig. 4). Therefore, the DYU500 could be classified as thermal-sensitive bioflocculant. If the major component of a bioflocculant is a glycoprotein, its stability will depend on the relative contents of protein and polysaccharide [6]. In general, bioflocculants composed of sugars in the structure are thermally stable, but with protein or peptide backbone in the structure are usually susceptible to heat [36]. The structure of protein bioflocculants would be destroyed while heating, hence the protein bioflocculants are considered thermally unstable. For instance, when heating at 100 8C for 30 min, the protein bioflocculant NOC-1 produced from Rhodococcus erythropolis was inactivated with a 50% decrease in flocculating capability [15]. In addition, the flocculating

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Fig. 2. Effects of low metal ions concentration and DYU500 concentration on flocculating activity and flocculating rate at pH 7.0. Al2(SO4)3: 5  103 mM; Fe2(SO4)3: 5  103 mM; CaCO3: 5  103 mM; CaCl2: 5  103 mM; MgSO4: 5  103 mM. DYU500 concentration in mg/L: (a) 10; (b) 20; (c) 30; (d) 40; (e) 50; (f) 60; (g) 70; (h) 80.

activity of a homopolymer bioflocculant produced by Bacillus sp. PY-90 also decreased rapidly at elevated temperatures and almost completely inactivated while heating at 100 8C for 40 min [5]. In contrast, polysaccharide bioflocculants are more thermally stable, as polysaccharide bioflucculants MBFA9 (produced by B. mucilaginosus) [6] and PF-101 (produced by Paecilomyces sp.) [34,37] all display high thermal stability. Since the results indicated that DYU500 was not thermally stable, two possibilities are proposed: (1) DYU500 is not a polysaccharide bioflocculant, but instead belongs to a protein biofocculant or (2) DYU500 is a glycoprotein bioflocculant containing protein as its major component. 3.5. Composition of bioflocculant DYU500 The composition analysis of bioflocculant DYU500 was performed to identify whether it is a protein or a glycoprotein. The results show that DYU500 had a total sugar content of 14.9% (w/w). Moreover, the hydrolyate of DYU500 also contained uronic acid (3.0%, w/w) as well as a variety of amino

acids, including glutamic acid (Glu) and phenylalanine (Phe) with a weight fraction of 91.0 and 1.2%, respectively (Fig. 5). In addition, the ratio of D/L glutamic acid in DYU500 was about 98:2, indicating that D-glutamic acids were the major constituents of the PGA produced by B. subtilis DYU1. Therefore, it is proposed that DYU500 is mainly a homopolyamide, consisting of one type of monomers (glutamic acid) (Table 3). The protein and poly(amino acid) content in DYU500 was 4.4 and 48.7% (w/w), respectively. Although nearly 30 wt% of the components in DYU500 remained unknown, the elemental analysis of DYU500 revealed that the weight fraction of the elements C, H, O, N, and S was 36.2, 11.84, 39.75, 11.14, and 1.07%, respectively. Moreover, analysis with Fourier transform infrared spectrum (FT-IR) displayed absorption peaks at 3410, 1620 and 1544 cm1 (Fig. 6). The peaks located in the range from 3400 to 3500 cm1 indicate the presence of OH and NH2 groups [3]. The peak at 3410 cm1 could be a NH2 group [38]. In addition, the absorption peak at 1620 cm1 is characteristic of a C O group [12], while the COO stretching absorption bond is observed at 1544 cm1. In summary, the

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Fig. 4. Effect of temperature on flocculating activity and flocculating rate using DYU500 (40 mg/L) at pH 7.0.

infrared spectrum of DYU500 shows the presence of amine, carbonyl and carboxyl groups, which are all preferable functional groups for the flocculation process in polyelectrolytes [39]. Moreover, the NMR spectrum of DYU500 shows 1H chemical shifts (in ppm) at 1.7–1.9 (b-CH2), 2.1–2.2 (g-CH2), 3.9–4.0 (CH), and 7.8 (NH) (Fig. 7a) as well as 13C chemical shifts (in ppm) at 27.9–30.8 (b-CH2), 31.5–35.4 (g-CH2), 52.1– 57.8 (CH), 175.2–178.2 (CO), and 178.6–181.5 (COOH) (Fig. 7b). This NMR spectrum is quite similar to that of polyglutamic acid (PGA) reported by Pe´rez-Camero et al. [40]. Hence, there is a good possibility that bioflocculant DYU500 produced from B. subtilis DYU1 contained the structure of PGA. Further analysis shows that the ratio of total sugars,

Fig. 3. Effects of cation concentrations on flocculating activity of bioflocculant DYU500 (40 mg/L) using kaolin suspension at pH 7.0: (a) monovalent cations; (b) bivalent cations; (c) trivalent cations.

Table 2 Optimal dosage of different bioflocculants for various solid suspensions with cation supplements Bioflocculant-producing bacterium

Bioflocculants structure

Molecular weight (Da)

Bacillus sp. DYU1

Major component: polyamides (poly-glutamic acid) Polysaccharide Polysaccharide Polysaccharide Poly-glutamic acid Polysaccharide Polysaccharide Poly-glutamic acid Poly-glutamic acid Polysaccharide Polysaccharide Polysaccharide Polysaccharide Polysaccharide Polysaccharide

(3.16–3.20)  10 6

Aspergillus parasiticus Bacillus sp. I-450 Bacillus coagulants As-101 B. licheniformis CCRC12826 B. mucilaginosus Bacillus sp. DP-152 Bacillus sp. PY-90 B. subtilis Citrobacter sp. TKF04 Corynebacterium glutamicum Enterobacter aerogenes Gyrodinium impudicum KG03 Klebsiella sp. Paecilomyces sp. I-1

3.2  10 5 2.2  10 6 – 2.0  10 6 2.6  10 6 2  10 6 – 1.5  10 6 3.2  10 5 105 2.4  10 6 1.87  10 6 >2  10 6 >3  10 5

Optimum bioflucculant concentration (mg/L) 40

– 22.2a 40a 3.7 0.1a 1 20 20 100a 25a 90 1 10a 200a

Ions added

Various solid suspensions

Flocculating activity or flocculating rate (%)

References

Ca2+

Kaolin

13.5 (97%)

This study

– Ca2+ Ca2+, Fe3+, Al3+ Ca2+ No added ions Ca2+ Ca2+ Ca2+ No ions Ca2+ Zn2+ Ca2+ Ca2+ Ca2+, Mn2+

Kaolin Kaolin Kaolin Kaolin Kaolin Kaolin Kaolin Kaolin Kaolin Kaolin Trona Kaolin Kaolin Anionic particles

98.1% – (High) 90% 8.5 99% 43 33 21.3 98% 80% 2.92  104 m/sb 90% 1.4 8

[27] [28] [3] [23] [6] [12] [5] [22] [29] [30] [31] [32] [33] [34]

– Not available. a The dosage of bioflocculant for flocculation test suspension was in a unit of mL/L. b The flocculating activity was indicated by the sedimentation rate (setting height per unit time).

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Fig. 6. FT-IR spectra of DYU500 obtained by B. subtilis DYU1.

Fig. 5. Amino acid composition of hydrolyzed bioflocculant DYU500.

uronic acids, proteins and PGA in DYU500 was approximately 14.9, 2.7, 4.4 and 48.7%, respectively. The average molecular weight of bioflocculant DYU500 determined by gel permeation chromatography was ca. (3.16–3.20)  106 Da (Table 3). The

Fig. 7. (a) 1H NMR spectra and (b)

13

molecular weight of DYU500 is much higher than other bioflocculants indicated in previous work (Table 2). On the other hand, the polydispersity (PD) of DYU500 was about 1.03–1.09 (Table 3). The feature of high molecular weight seems to be positive in flocculation performance, as the flocculating activity clearly lower for a PGA compound (purchased from Sigma Chemical) with a lower molecular weight (ca. 1  105 Da).

C NMR spectra of the purified DYU500 from B. subtilis DYU1.

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Table 3 Characteristics of biopolymer DYU500 produced from B. subtilis DYU1 isolate Biopolymer DYU500 concentration (g/L)

PGA content in DYU 500 (g/L)

D/L form glutamic acid in PGA

Viscosity (cPs)

Weight average molecular weight, Mw (106 Da)

Polydispersity, PD

2.5 20.0 60.0

1.2 9.8 29.5

97/3 98/2 96/4

2.1 6.5 46.2

3.16 3.20 3.18

1.03 1.03 1.09

Average content of PGA in DYU500 was about 48.7% (w/w).

However, the optimal concentration of both bioflocculants was similar (data not shown). Comparison between DYU500 ((3.16–3.20)  106 Da) and the commercial PGA (1 105 Da) indicates that the molecular weight of a bioflocculant closely related to the chain length of the polymer is an important factor in influencing the flocculating activity. Bioflocculants with a high molecular weight could effectively make bridging between bioflocculant and kaolin particles because it could form a larger floc size in the flocculating reaction [4,6,12]. All these properties seem to favor the performance of flocculation. Thus, DYU500 obtained from this study is an effective bioflocculant because it possesses a high molecular weight and a high PGA content. The carboxyl groups present on the molecular chain of PGA make the chain stretched out because of electrostatic repulsion. The stretched molecular chains could provide more effective sites for kaolin particle attachment [6]. Furthermore, for the bioflocculant MBFA9 from B. mucilaginosus, the high molecular weight (2.6  106 Da) and appropriate content of uronic acid (19.1%) are also beneficial for flocculation [6].

it was found that the zeta potential value was always negative (from 18 to 40 mV) at a pH of 3.0–11.0, and the zeta potential decreased with an increase in pH. The flocculation of negatively charged kaolin particles by anionic bioflocculant DYU500 may also be enabled by cationic bridge formation between particles and flocculant chains (Fig. 8). According to the foregoing results, two flocculation mechanisms are proposed as follows: (1) As indicated in Fig. 8a, the flocculation may be attributed to a decrease in the negative charge on the particle surfaces when cations are present, and eventually the charge may be reversed from negative to positive. Thus, the negatively charged carboxyl group (COO) of the bioflocculant DYU500 could react with the positively charged site of the suspended kaolin particles. (2) It can be assumed that cations stimulate flocculation by neutralization and stabilization of residual negative charges of the carboxyl group of bioflocculant DYU500 forming bridge that binds kaolin particles to each other (Fig. 8b).

3.6. Mechanism of flocculation In general, bioflocculants cause aggregation of cells and particles by bridging and charge neutralization [4]. In this study,

However, no matter which mechanism of flocculation would be, bridging finally occurs after the particles have adsorbed onto the chains of bioflocculant DYU 500. Many kaolin

Fig. 8. Proposed flocculation mechanism for bioflocculant DYU500.

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particles could adsorb to a long molecular chain, and they could adsorb simultaneously by the other chains in bioflocculant DYU500, leading to the formation of three-dimensional flocs capable of rapid settling (Fig. 8). Base on this assumption, the promoting effect of cations on flocculation of DYU500 is highly dependent on both the concentration and valence of the ions, which has been confirmed with the experimental results described in Section 3.3. The role of cations is to increase the adsorption of bioflocculants on the surface of suspended particles by decreasing the negative charge on both the bioflocculant and the particle [25,26]. Flocculation is due to decline of charge density by the added cations, leading to inter-particle bridging between kaolin particles [8]. In accordance with Schulze Hardy’s law, it is suggested that flocculation is a result of changes in the charge density, indicating that cation effects resulted in neutralization of the zeta potential [24]. 4. Conclusions This study attained a novel biopolymer DYU500 from an indigenous flocculant-producing bacterial isolate identified as B. subtilis DYU1. The bioflocculant DYU500 possesses a good flocculating activity, which can be promoted by the addition of trivalent and bivalent cations in kaolin suspension. The synergistic effects of metal cations were most significant at pH 6.0–7.0, and the optimum temperature for flocculation was 30 8C. Composition analysis and NMR spectrum show that the biopolymer mainly contains the structure of PGA. In addition, two mechanisms describing the flocculating process by bioflocculant DYU500 was proposed based on the experimental observations. The carboxyl groups in a PGA structure of DYU500 offer an additional benefit of working as functional moieties to generate new or modified biopolymer variants via polymer engineering or novel formulation design. Moreover, with a good flocculating activity and the feature of being less harmful to human and the environment, DYU500 is expected to be a potential replacement of conventional synthetic flocculants and widely applied in wastewater treatment and downstream processing of food and fermentation industries. Acknowledgement This work was financially supported in part by a grant from the National Science Council of Republic of China under a contract number of NSC 94-2211-E-212-008. References [1] Tong Z, Zhe L, Huai-Lan Z. Microbial flocculant and its application in environmental protection. J Environ Sci 1999;11:1–12. [2] Kurane R, Takeda K, Suzuki T. Screening for and characteristics of microbial flocculants. Agric Biol Chem 1986;50:2301–7. [3] Salehizadeh H, Vossoughi M, Alemzadeh I. Some investigations on bioflocculant producing bacteria. Biochem Eng J 2000;5:39–44. [4] Salehizadeh H, Shokaosadati SA. Extracellular biopolymeric flocculants Recent trends and biotechnological importance. Biotechnol Adv 2001;19: 371–85.

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