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The present study aimed to develop an effective extraction method for bioflocculants from activated sludge. Microbial polymers obtained from activated sludge by four different extraction methods (steaming, NaOH, SDS and washing extractions) were compared in terms of yield, chemical components, and flocculating activity for kaolin clay suspension. NaOH extraction has the highest yield, while washing extraction resulted to the lowest. The main component of the polymers obtained was protein, involving saccharide and nucleic acid, although the contents were varied considerably for individual polymers. All polymers possessed flocculating activity for kaolin clay containing cations, although the degree of activity varied significantly with extraction method. The activity of the polymer extracted with NaOH was the highest, while the activity of the polymer extracted with washing was the lowest. Among the cations tested, calcium and magnesium ions promoted activity. These results indicate that NaOH extraction was the most effective method for the extraction of bioflocculants from activated sludge
Journal of Water and Environment Technology, Vol.3, No.1, 2005 - 145 - Comparison of Methods for the Extraction of Bioflocculants from Activated Sludge Junichi TSUGE 1 , Masuo NAKANO 2 and Yasunori KUSHI 3 1 Sapporo Otani Junior College N16, E9, Higashi-ku, Sapporo, 065-8567, Japan E-mail: junichi_tsuge@sapporo-otani.ac.jp 2 Faculty of Dairy Science, Rakuno Gakuen University 582 Bunkyodai Midorimachi, Ebetsu, 069-8501, Japan 3 Department of Agricultural and Life Science, Obihiro University of Agriculture and Veterinary Medicine Inada-cho, Obihiro, 080-8555, Japan E-mail: ykushi@obihiro.ac.jp Abstract The present study aimed to develop an effective extraction method for bioflocculants from activated sludge. Microbial polymers obtained from activated sludge by four different extraction methods (steaming, NaOH, SDS and washing extractions) were compared in terms of yield, chemical components, and flocculating activity for kaolin clay suspension. NaOH extraction has the highest yield, while washing extraction resulted to the lowest. The main component of the polymers obtained was protein, involving saccharide and nucleic acid, although the contents were varied considerably for individual polymers. All polymers possessed flocculating activity for kaolin clay containing cations, although the degree of activity varied significantly with extraction method. The activity of the polymer extracted with NaOH was the highest, while the activity of the polymer extracted with washing was the lowest. Among the cations tested, calcium and magnesium ions promoted activity. These results indicate that NaOH extraction was the most effective method for the extraction of bioflocculants from activated sludge. Key words: activated sludge, bioflocculants, flocculating activity Introduction Flocculants are used in a wide range of fields, such as wastewater treatment, dredging, and fermentation. Flocculants are classified into three major groups. Inorganic compounds such as aluminum sulfate, ferric sulfate, and ferric chloride belong to the first category. The second category includes synthetic organic compounds such as polyacrylamide and the third category constitutes natural high-molecular-weight compounds such as microbial polymers (bioflocculants). The first and second flocculant groups are used widely because of their economic advantage and potency. However, these materials can cause environmental pollution. The acrylamide monomer of polyacrylamide, for instance, is a strong carcinogen. In contrast, the third group of flocculants is biodegradable and safe but has flocculating activities too weak for industrial use. Therefore, screening for strong bioflocculants from natural sources is needed. Journal of Water and Environment Technology, Vol.3, No.1, 2005 - 146 - Two types of microbial flocculants exist: first, a material is released into the culture broth, and the flocculant is obtained from the filtrate of the culture broth; and second, a cell surface material associated with the cell wall or adsorbed to the cell surface in an amorphous state is obtained by extraction. Several investigators have isolated bioflocculants from pure cultures of microorganisms (Takagi and Kadowaki, 1985a, b; Kurane et al., 1986; Bar-or and Shilo, 1987; Kurane and Nohata, 1991; Nam et al., 1996; Shimofuruya et al., 1996; Nakata and Kurane, 1999; Kobayashi et al., 2002). However, only a few studies (Brown and Lester, 1980; Sato and Ose, 1980) have been reported regarding the screening for flocculants from natural activated sludge, which is a complex microbial system. A strong flocculant from natural activated sludge might economically provide an effective use of excess sludge. In a previous paper (Tsuge and Nakano, in press), we reported that an extracellular polymer extracted by sonication from activated sludge possessed flocculating activity for several kinds of suspensions, although the polymer yield was low. Here, we compared four different extraction methods, measured polymer yields, determined the components of the polymers, and examined flocculating properties in an effort to develop an effective extraction method for bioflocculants from the microbial cell surface. Materials and Methods Activated sludge samples. Activated sludge samples were collected from an aeration tank of the Obihiro-South local domestic sewage treatment plant, and stored at 4°C for subsequent experiments. All samples were washed 3 times with distilled water before extractions. Extraction of microbial polymers. Steaming extraction. Activated sludge (39.86 g) concentrated by centrifugation (5,000 rpm, 20 min) was suspended in distilled water (300 ml) and autoclaved at 121°C for 10 min followed by centrifugation (7,500 rpm, 30 min). Three volumes of cold ethanol was added to the supernatant with mechanical stirring and allowed to stand at 4°C overnight. The precipitate obtained by centrifugation (10,000 rpm, 15 min) was dissolved in distilled water (100 ml) and stirred mechanically at 4°C overnight. Insoluble materials were removed by centrifugation (10,000 rpm, 30 min). NaOH extraction. Activated sludge (28.08 g) concentrated by centrifugation (5,000 rpm, 20 min) was suspended in cold 0.5 N NaOH (200 ml), stirred mechanically at 4°C for 24 h, and centrifuged (8,000 rpm, 30 min). The supernatant was neutralized with acetic acid and dialyzed against distilled water at 4°C overnight. Sodium acetate was then added to give a concentration of 3% and 5 volumes of cold ethanol was added with mechanical stirring. After allowing it to stand at 4°C overnight, the precipitate was collected by centrifugation (8,000 rpm, 15 min). The precipitate obtained was dissolved in distilled water (80 ml) and stirred mechanically at 4°C overnight. The insoluble Journal of Water and Environment Technology, Vol.3, No.1, 2005 - 147 - materials were removed by centrifugation (10,000 rpm, 30 min). Sodium dodecyl sulfate (SDS) extraction. Activated sludge (55.61 g) concentrated by centrifugation (5,000 rpm, 20 min) was suspended in 2.5% SDS (300 ml), heated at 70°C for 30 min, and then centrifuged (6,000 rpm, 45 min). The supernatant was dialyzed against distilled water at 4°C until SDS was removed completely. Washing extraction. Activated sludge (63.85 g) concentrated by centrifugation (5,000 rpm, 20 min) was suspended in distilled water (300 ml) and stirred mechanically at 4°C for 30 min. The supernatant was obtained by centrifugation (6,000 rpm, 30 min). All the polymers obtained by these procedures were lyophilized and stored in a freezer after dialysis against distilled water at 4°C. Analysis of constituent neutral sugars. GLC analysis of constituent neutral sugar monomers in the polymers was performed after conversion into their corresponding alditol acetates as described previously (Tsuge and Nakano, in press). Aliphatic compounds were extracted with n-hexane from the hydrolysis reaction mixture and subjected to fatty acid analysis. Analysis of fatty acids. The n-hexane phases obtained by hydrolysis of the polymers were analyzed as fatty acid methyl esters by GLC using a glass column (0.3×200 cm) packed with 10% DEGS at 180°C. The detection temperature was 270°C, and injection port temperature was 300°C. Electrophoretic analysis. SDS slab-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the method of Laemmli (1970). A separating gel was formed with 12.5% acrylamide and 0.1% SDS, with a 2.5% acrylamide stacking gel. The sample solution (5 mg/0.525 ml) was mixed with 10% SDS (0.2 ml), 2-mercaptoethanol (0.05 ml) and 70% glycerol (0.1 ml). After the mixture was heated at 100°C for 5 min, 5 µl of 0.1% bromophenol blue was added, and a 10 µl portion was applied to the gel. The electrode buffer was 0.025 M Tris-HCl (pH 6.8) containing 0.192 M glycine and 0.1% SDS. Electrophoresis was performed with 10 mA until the tacking dye entered the separating gel, followed by 20 mA. After SDS-PAGE, components on the gel were detected by Ag-protein stain (Oakley et al., 1980) and Ag-LPS stain (Tsai and Frasch, 1982). Gel filtration of polymers. Gel filtration of polymers was performed in a column (26×450 mm) of Bio-Gel P-100 previously equilibrated with 10 mM Tris-HCl buffer, pH 8.0. Elution was performed with the same buffer at a flow rate of 9 ml/h and 5 ml fractions were collected. The void volume of the column was determined by the elution of Blue Dextran (Pharmacia). Other analyses. Journal of Water and Environment Technology, Vol.3, No.1, 2005 - 148 - Total saccharide was determined using the anthrone-H 2 SO 4 method (Ough, 1964) with a glucose/galactose 1:1 standard. Uronic acid was determined using the method of Blumenkrantz and Asboe-Hansen (1973) with a glucuronic acid/galacturonic acid 1:1 standard. Hexosamine was determined by the method of Blix (1948) with a glucosamine standard. Phosphorus was determined using the molybdenum-H 2 SO 4 method (Schnittger et al., 1959). Protein was determined according to the method of Lowry et al. (1951) using a bovine serum albumin standard. Nucleic acid and protein contents in the gel filtration eluate were determined by measuring absorbances at 260 nm and 280 nm, respectively. All analyses were performed in triplicates. Estimation of flocculating activity. Flocculating activity was estimated using a kaolin clay suspension. The kaolin clay was treated with ethylenediaminetetra-acetate (EDTA) to prevent the influence of contaminating cations. Kaolin clay was suspended in 40 mM EDTA and stirred mechanically for 1 h at room temperature, followed by centrifugation at 3,000 rpm for 10 min. The kaolin clay collected was washed 3 times with distilled water and dried at 80°C in an oven. Two milliliters of kaolin clay suspension (5,000 ppm) prepared in 5 mM CaCl 2 . 2H 2 O was mixed in a test tube with the polymer solution at a final concentration of 5 ppm. After sufficient shaking, absorbance at 660 nm (A 660 ) was measured for 5 min, and the relative A 660 against the control test was calculated every minute using the following formula: testcontrolofA testngflocculatiofA ArelativeThe 660 660 660 = For the control, the polymer solution was replaced by distilled water. To estimate polymer concentration, the polymer concentration in a test tube was increased from 2.5 to 20 ppm. Effect of cations on flocculating activity. To estimate the effect of cations on flocculating activity, 2 ml of kaolin clay suspension (5,000 ppm) prepared in 5 mM solutions of various cations (NaCl, KCl, CaCl 2 . 2H 2 O, MgCl 2 . 6H 2 O, CoCl 2 . 6H 2 O, BaCl 2 . 2H 2 O, FeCl 3 . 6H 2 O and Al 2 (SO 4 ) 3 . 13 . 18H 2 O) was mixed with NaOH-extracted polymer at a final concentration of 5 ppm, and the relative A 660 was calculated as described above. To estimate the concentration of cations, kaolin clay suspension (5,000 ppm) prepared in 0 to 50 mM calcium ion solution was mixed with NaOH-extracted polymer solution. For the control, kaolin clay suspension was prepared with distilled water. Chemicals. Bio-Gel P-100 was the product of Bio Rad Laboratories while blue dextran was the product of Pharmacia Fine Chemicals. ECNSS-M and DEGS were obtained from Gas-Chro Kogyo Co. Tokyo. Other chemicals used in this study were reagent grade. Results and Discussion Yields and chemical components. Journal of Water and Environment Technology, Vol.3, No.1, 2005 - 149 - Table 1. Yields and Chemical Components of Polymers Extracted from Activated Sludge. Steaming 1 NaOH 2 SDS 3 Washing 4 (mg/g, dry sludge) Yields 194.3 239.5 192.3 13.3 (µg/mg, polymer) Protein 294.3 685.0 598.3 336.0 Saccharide 245.7 168.4 49.2 106.0 Hexosamine 37.2 31.0 16.0 24.9 Uronic acid 50.4 44.9 13.5 20.9 Phosphorus 32.1 9.8 9.9 35.7 1 steaming-extracted polymer, 2 NaOH-extracted polymer, 3 SDS-extracted polymer, 4 washing-extracted polymer Table2. Molar Ratios of Constituent Neutral Sugars in Polymers Extracted from Activated Sludge. Sugars Steaming 1 NaOH 2 SDS 3 Washing 4 Rhamnose 0.01 ND ND ND Fucose 0.21 0.21 0.34 0.14 Ribose 0.89 0.23 1.12 0.07 Arabinose 0.11 0.20 0.22 0.14 Xylose 0.14 0.23 0.20 0.15 Mannose 0.24 0.31 0.70 0.17 Galactose 0.31 0.40 0.62 0.24 Glucose 1.00 1.00 1.00 1.00 ND; not detected, 1 steaming-extracted polymer, 2 NaOH-extracted polymer, 3 SDS-extracted polymer, 4 washing-extracted polymer Table 3. Compositions of Fatty Acids in Polymers Extracted from Activated Sludge. Fatty acids Steaming 1 NaOH 2 SDS 3 Washing 4 % Palmitic acid (16:0) 36.8 38.6 30.1 30.7 Palmitoleic acid (16:1) 22.0 25.6 18.9 11.4 Stearic acid (18:0) 12.9 7.6 11.4 15.7 Oleic acid (18:1) 21.8 20.1 25.7 21.7 Linoleic acid (18:2) 6.5 5.5 8.6 9.9 Arachic acid (20:0) Trace Trace 1.2 3.6 Behenic acid (22:0) Trace Trace 0.6 2.9 Lignoceric acid (24:0) Trace 2.6 3.4 3.9 1 steaming-extracted polymer, 2 NaOH-extracted polymer, 3 SDS-extracted polymer, 4 washing-extracted polymer Journal of Water and Environment Technology, Vol.3, No.1, 2005 - 150 - Yields and chemical components of polymers obtained from activated sludge by the four extraction methods are summarized in Table 1. Yield varied significantly with extraction method, with NaOH extraction having the highest yield (239.5 mg/g, dry sludge), and washing extraction the lowest (13.3 mg/g, dry sludge). Protein was the main component of all the polymers, although the content varied significantly with the polymer. The NaOH- and SDS-extracted polymers included large amounts of protein (68.5% and 59.8%, respectively); in contrast, the steaming-extracted polymer included a larger amount of saccharides (24.6%) compared to the other methods. The SDS-extracted polymer included a small amount of material other than protein. Furthermore, the steaming- and washing-extracted polymers contained large amounts of phosphorus (of 3.2% and 3.6%, respectively). Table 2 shows molar ratios of the constituent neutral sugars of the polymers. Although the molar ratio varied by polymer, the main constituent sugar was glucose for all the polymers. The steaming-extracted polymer contained large amounts of ribose and phosphorus (as shown in Table 1), suggesting that nucleic acids were a major component of this polymer. The main constituent sugars of the polymers agreed with data reported previously (Steiner et al., 1976; Sato and Ose, 1980; Horan and Eccles, 1986; Nakata and Kurane, 1999). Table 3 shows the composition of fatty acids released by acid hydrolysis of the polymers. The composition and ratios were similar, i.e., the main fatty acid was palmitic acid, followed by oleic acid, palmitoleic acid, stearic acid, and linoleic acid. Other fatty acids were present only in small or trace amounts. Electrophoresis. Figure 1 shows the SDS-PAGE profiles of polymers. Although bands were not detected clearly Fig. 1. SDS-PAGE of Polymers Extracted from Activated Sludge. St; molecular weight marker including lysozyme (14,400), soybean trypsin inhibitor (21,500), carbonic anhydrase (31,000), ovalbumin (42,699), bovine serum albumin (66,200) and phosphorylase b (97,400), 1; steaming-extracted polymer, 2; NaOH-extracted polymer, 3; SDS-extracted polymer, 4; washing-extracted polymer. Journal of Water and Environment Technology, Vol.3, No.1, 2005 - 151 - using Ag-protein stain, the components varied greatly by polymer. Several bands demonstrated the same mobility in both stains, suggesting that these bands were complexes of protein and saccharide. These included the band corresponding to a molecular weight of 103,000 in the steaming-extracted polymer (1), the band corresponding to 38,000 in the NaOH-extracted polymer (2), the band corresponding to 24,700 in the SDS-extracted polymer (3), and the bands corresponding to 10,700, 29,200, and 39,000 in the washing-extracted polymer (4). Flocculating activity. Flocculating activities of polymers for kaolin clay suspensions are compared in Fig. 2. All polymers possessed activity at a concentration of 5 ppm for kaolin clay suspension in the presence of 5 mM calcium, although the level of activity was different for each polymer. The NaOH- and steaming-extracted polymers possessed strong activities (relative A 660 values at 3 min were 0.337 and 0.421, respectively). In contrast, activities of the washing- and SDS-extracted polymers were weak (relative A 660 values at 3 min were 0.754 and 0.697, respectively). Brown and Lester (1980) reported that NaOH treatment caused extensive disruption in microbial cultures, and steaming was the most effective extraction method for activated sludge. However, cell disruption is not effective for the isolation of bioflocculants. Thus, NaOH extraction was the most effective method for the extraction of bioflocculants from activated sludge, followed by steaming extraction. SDS and washing extraction methods were ineffective because of low flocculating activity, and low yield and low activity, respectively. Although the finding that NaOH-extracted polymer composed mainly of protein possessed satisfactory flocculating activity agreed with a previous report by Willén et al. (2003), the low activity of the SDS-extracted polymer, which was also composed mainly of protein, was unexpected. Figure 3 shows the effect of concentration of the NaOH-extracted polymer (2.5 to 20 ppm) on activity in the presence of 5 mM calcium ions. The activity correlated with polymer Journal of Water and Environment Technology, Vol.3, No.1, 2005 - 152 - concentration, although this effect was not observed at concentrations greater than 5 ppm. Effects of cations on flocculating activity. Figure 4 shows the effect of cations at a concentration of 5 mM. Although all cations affected flocculating activity, the effect varied significantly by cation. Little flocculation of kaolin clay occurred in the presence of monovalent cations such as sodium and potassium (relative A 660 values at 3 min were 0.970 and 0.909, respectively). The effect of ferric ions was small (relative A 660 at 3 min was 0.796). In contrast, calcium and magnesium ions promoted activity (relative A 660 values at 3 min were 0.442 and 0.490, respectively) to a greater degree than the trivalent cation of aluminum (relative A 660 at 3 min was 0.583). These data suggest that calcium and magnesium ions play a role in the floc formation of activated sludge and the adsorption of pollutants on the floc surface. Tezuka (1969) reported that the Flavobacterium sp. dominant in activated sludge flocculated when calcium and magnesium were added to the culture medium and proposed that bacterial cells aggregated through a mechanism involving linkage among these cations. Sato and Ose (1980) reported that the calcium content in ash of floc-forming substances extracted from activated sludge was much greater than the magnesium content; thus, calcium may be more important than magnesium. Dermlin et al. (1999) reported that the polysaccharide produced by Klebsiella sp. S11 possessed the ability to flocculate a kaolin suspension in the presence of 1% CaCl 2 . Kurane et al. (1986) reported that the flocculating activity for kaolin clay of the microbial flocculant obtained from the culture broth of Rhodococcus erythropolis increased significantly by the addition of calcium and aluminum ions, and that this activity was not affected by magnesium ions and was slightly stimulated by ferric ions. However, the present study found that magnesium ions significantly affected flocculating activity. Figure 5 shows the effect of calcium ion concentration (0.01 to 50 mM) at a NaOH-extracted polymer concentration of 5 ppm. Journal of Water and Environment Technology, Vol.3, No.1, 2005 - 153 - Kaolin clay did not flocculate when calcium ion concentration was less than 0.1 mM. The flocculating activity correlated with calcium ion concentration up to 10 mM, confirming that polyvalent cations are required for the polymer to exhibit flocculating activity. However, the activity did not increase at calcium concentrations greater than 10 mM. These results suggest that a balance between polymer and cation concentrations is important for effective flocculation. Gel filtration. The steaming- and NaOH-extracted polymers, which possessed satisfactory flocculating activities, were subjected to Bio-Gel P-100 column chromatography. Figure 6 shows the elution profiles. The profile of steaming-extracted polymer (A) contained two saccharide peaks (at elution volumes of 70 ml and 90 ml) and one UV-absorbing peak (at elution volume of 95 ml). The A 280 to A 260 ratio of the UV-absorbing peak indicated it was composed mainly of nucleic acid. In the profile of the NaOH-extracted polymer (B), the elution volume (80 ml) of saccharide corresponded to that of the UV-absorbing peak. In this case, the UV-absorbing peak was mainly protein. These results agreed well with analytical data of the polymer components shown in Table 1. Conclusions Four different microbial polymer extraction methods (steaming extraction, NaOH extraction, SDS extraction, and washing extraction) were compared to develop an effective extraction method for bioflocculants from activated sludge. The NaOH extraction produced the highest yield, while the washing extraction produced the lowest. Although all polymers possessed flocculating activity for kaolin clay containing cations, the NaOH-extracted polymer exhibited the greatest activity, and the washing-extracted polymer possessed the lowest. Among the cations tested, calcium and magnesium ions showed the greatest benefit on activity. These results indicated that NaOH extraction was the most effective method in obtaining bioflocculants from activated sludge. References Journal of Water and Environment Technology, Vol.3, No.1, 2005 - 154 - Bar-or, Y. and Shilo, M. 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