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Characterization of bioflocculant produced by Bacillus sp. DP-152
JOURNAL OFFERMENTATION AND BIOENQINEERING Vol. 84, No. 2, 108-112. 1997
Characterization HYUN-HYO
of Bioflocculant Produced by BaciZZussp. DP-152
SUH,’ GI-SEOK KWON,Z CHANG-HO LEE,’ HEE-SIK KIM,’ HEE-MOCK OH,’ AND BYUNG-DAE YOON’*
Environmental Microbiology Research Unit, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Taejon 305-600’ and Natural Science College, Andong National University, Andong 760-749,= Korea Received 1 July 1996/Accepted 30 May 1997
A strain (designated DP-152) which produces an excellent flocculating substance was isolated from soil samples and identified as Bacillus species. The major flocculating substance (bioflocculant DP-152) produced by Bacillus sp. DP-152 was puriiied by ethanol precipitation and cetylpyridlnium chloride (CPC) precipitation and gel permeation chromatography. In kaolin suspension, the highest flocculating activity was obtained at the bioflocculant concentration of 1 mg/Z. The bioflocculant DP-152, which has an estimated molecular weight of over 2 x 106 daltons, is a novel bioflocculant derived from sugar components consisting of glucose, mannose, galactose, and fucose in an approximate molar ratio of 8 : 4 : 2 : 1. Some of its pbysico-cbemical properties were also determined. [Key words: novel bioflocculant, anionic polysaccharide, Bacillussp.] Flocculating agents are used in a wide range of industrial processes, including wastewater treatment (1). Flocculants are divided into organic synthetic polymer flocculants (polyacrylamide derivatives and polyacrylic acid), inorganic flocculants (aluminium sulfate and polyaluminium chloride), and bioflocculants (chitosan, algin, and microbial flocculants) (2). In particular, polyacrylamides
flocculant-producing bacteria were isolated. The flocculating activities were measured by using a kaolin clay suspension, activated carbon suspension, and industrial wastewater. Identification of strain DP-152 The isolated strain DP-152 was identified from its morphological and physiological properties, according to Bergey’s Manual of Systematic Bacteriology (14). The chemotaxonomical characteristics of the strain were examined by the procedures of Komagata and Suzuki (15), Miller (16), and Tamaoka and Komagata (17). Measurement of flocculating activity Flocculation of kaolin clay Kaolin clay (Junsei Chemical Co.) was used as the suspension material for estimating the flocculating activity. In a lOO-ml beaker, 0.1 ml of 10% (w/v) CaClz .2H20 solution was added to the 100 ml of kaolin clay suspension (S,OOOmg/l) and stirred with a magnetic bar for 1 min (300rpm). After stirring, the culture broth (10 ,&I) was added to the kaolin suspension. The pH of the kaolin suspension was adjusted to pH 7.0 with 1.0 N NaOH. After each addition, the reaction was performed with rapid mixing at 300 rpm for 30 s, followed by slow mixing at 100 rpm for 1 min. The test solution was mixed at room temperature and then kept standing for 5 min. The formation of visible aggregates was observed. The turbidity of the upper phase was measured at 550nm with a spectrophotometer (UV-160A; Shimadzu, Japan). The flocculating activity was calculated by the following equation (4): Flocculating activity = 1/A - 1/B A = optical density of the sample at 550 nm B=optical density of the reference at 550 nm Flocculation of activated carbon Activated carbon flocculation tests were done as above with the kaolin solution (5,OOOmg/f) being replaced by an activated carbon solution (5,000 mg/l). Jar test of industrial wastewaters Wastewaters from the food, textile, and paper industries were used to investigate the flocculating effect of the isolates. In a 250-ml beaker, 0.2 ml of 10% (w/v) CaC12. 2Hz0 solution (co-flocculant) was added to the 200 ml of each type of wastewater, and stirred with a jar tester for 1 min (200rpm). The pHs of each suspension were adjusted to
are frequently used because they are economical and effective flocculating agents (2-5). However, monomers of polyacrylamide have neurotoxic and strong carcinogenic properties (7, 8). Bioproducts produced by microorganisms are expected to be useful flocculating substances because they are safe for the environment. Studies on flocculating substances from microorganisms have examined them from various viewpoints, such as coagulation of kaolin clay and removal of microorganisms in the fermentation industry (2-5). Among these reports, Nakamura et al. (6) reported serial studies on a fungal flocculant from a strain of Aspergillus sojae, Schenck et al. (Balg. patant 822,947) reported that the alga Chlamydomonas mexicana formed flocculating agents which are polysaccharides, and Kurane et al. (3) reported that Rhodococcus erythropolis S-l produced a flocculant which is effective for various colloidal suspensions and coloured pigments. In this paper, the screening and characteristics of a flocculating substance produced by another microorganism are reported. MATERIALS
AND METHODS
FlocScreening of flocculant-producing bacteria culant-producing bacteria were isolated from many kinds of soil samples. The composition of the screening medium was as follows: 40 g glucose, 1.0 g NH803, 0.3 g K2HP0,, 0.3 g KH2P04, 0.1 g MgS04.7Hz0, 0.1 g MnS04. 4Hz0, 0.05 g NaCl, 0.4g CaC03, 0.1 g yeast extract, 0.1 g soytone, and 0.1 g tryptone in 1 1 distilled water. The culture temperature and initial pH were 30°C and 7.0, respectively. By measuring the flocculating activity of the broth cultured in the screening medium for 3 d, * Correspondingauthor. 108
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the floe-forming pH of each type of wastewater. The culture broth was added to each type of wastewater to a concentration of 10ppm. After each addition, the reaction was performed with rapid mixing at 1OOrpm for 1 min, followed by slow mixing at 50 rpm for 2 min, and the test solution then kept standing for 3 min. Chemical oxygen demand (COD) and suspended solids (SS) were selected as the principal tests to determine the extent of flocculation. Physical and chemical properties of bioflocculant The molecular weight of the bioflocculant 152
DP-
DP152 was estimated by gel permeation chromatography (GPC) using Sephacryl S-500 (Pharmacia Co.) with dextran as the standard. The infrared spectrum was measured using an IR spectrophotometer (RFX-65; Layser precision analytical, USA) with KBr pellets. The colloidal equivalent weight was measured by colloidal titrimetrics (18). Complete hydrolysis of the bioflocculant DP-152 was carried out with 1.OM trifluoroacetic acid (TFA) at 121°C for 1 h. After hydrolysis, the solution was neutralized with 1.0 N NaOH solution and lyophilized. Sugar costituents in the TFA-hydrolysate were analyzed using high-performance anion-exchange chromatography (HPAE; Dionex Co., USA). The HPAE was fitted with a pulsed amperometric detector (PAD), using a CarboPak PA1 column (4 x 250mm) (Dionex Co., USA). NaOH solution (20mM) was used as the eluent, at a flow rate of 1.0 ml/min. Elemental analysis was conducted with a elemental analyzer (EA1108; Carlo Erba Ins., Italy). The total carbohydrate content of the flocculant was determined by the phenol-sulfuric acid method (19) and expressed as the glucose equivalent. The protein moiety in the llocculant molecule was determined by the Bradford method (20) with bovine serum albumin as a standard. Sugar derivatives were investigated by the carbazole method (21) for uranic acid, the Friedmann method (22) for pyruvic acid, and the hydroxamic acid method (23) for acetic acid. RESULTS AND DISCUSSION Screening of flocculant-producing bacteria For the screening of new flocculant-producing bacteria, more than 200 bacterial strains which excreted mucous material on the agar plate of the screening medium were isolated from soil. The culture broth of each isolate was tested for its ability to flocculate kaolin clay and activated carbon. Flocculating activity was detected in 49 of the isolates (data not shown), 18 of which showed high flocculating activity on the test materials. To evaluate the industrial applicability of these 18 isolates, their flocculating efficiency was examined using wastewaters from the food, textile, and paper industries. In a jar test, coagulation and flocculation were accomplished at room temperature. The reagent dosing, mixing (destabilization), and the subsequent sedimentation (phase separation) took place in the same reaction chamber. The efficiency of the flocculation process in the jar test was evaluated on the basis of the COD and SS before and after treatment. The culture broth of 18 isolates showed high flocculating efficiencies in the industrial wastewaters (data not shown). Among the strains tested, strain DP-152, which showed the highest COD reduction rate (50%) and SS removal rate (90%), was selected for subsequent experiment. These results indicated that the flocculant from strain DP-152 had the ability to flocculate almost all
FROM BACILLUS SP. DP-152
109
solids suspended in an aqueous solution, and that this strain can therefore be considered as a candidate for application in practical wastewater treatment. The morphological Identification of strain DP-152 and physiological characteristics of strain DP-152 were investigated. After incubation for 3 d at 30°C on glucose-nutrient agar medium, colonies were circular, convex, and milky-white. Strain DP-152 was rod shaped (1.2-1.4 pm x 3.5-3.8 ,nm) (Fig. l), gram-positive, and formed spores. Growth occured at 28’C and 45°C but not at 65°C. The isolated strain was able to liquefy gelatin and to form indol. The strain showed a positive reaction in Voges-Proskauer and catalase test, and produced acid from glucose, arabinose, mannitol and xylose. Thus it was considered to belong to Bucilfus sp. as reported by Gorden et al. (24). To elucidate the relationship between strain DP-152 and the genus Bacillus in detail, its chemotaxonomical characteristics were examined. The peptidoglycan of the strain had the meso-type of diaminopimelic acid in the acid hydrolyzate of the cells, indicating that it belongs to Bacillus sp. (14). The main fatty acids were branchedchain fatty acids, such as 13-methyl tetradecanoic acid (iso- : 0) and 12-methyltetradecanoic acid (anteiso15 : 0); this finding is in agreement with that of Suzuki and Komagata (25). The quinone system of strain DP152 was menaquinone-7 (MK-7); the principal menaquinone components of Bacillus sp. are also of the MK-7 type. The mole% G plus C of the DNA of strain DP-152 was found to be 53%; the mole% G plus C of the DNA of Bacillus sp. ranges from 32 to 69 mole% (14). When the keys to the genera listed in Bergey’s Manual (14) were traced on the basis of the these results, strain DP152 was identified as Bacillus species, and designated as Bacillus sp. DP-152. Distribution of flocculating component The distribution of the flocculating activity of the culture broth was examined, and the major constituent with flocculating activity was identified. Figure 2 shows the distribution of the flocculating activity in the culture broth. Most of the flocculating activity was found in the cellfree supernatant, and more than 90% of it was recovered from ethanol precipitation. The major flocculating factor produced by Bacillus sp. DP-152 was identified with the ethanol precipitate.
FIG. 1. Scanning electron micrograph of strain DP-152. The strain was cultured on isolation medium at 30°C for 3 d and observed using a scanning electron microscope (SEM 515; Philips, Netherlands).
110
J. FERMENT. BIOENG.,
SUH ET AL.
l-l
L B
A
C
D
E
FIG. 2. Distribution of flocculating components in culture broth of Bacillus sp. DP-152. The strain was cultured in flocculant-production medium for 50 h. Lanes: (A) culture broth; (El) cell-free supernatant; (C) cell; (D) ethanol precipitate of cell-free supematant; (E) supematant after ethanol precipitation of cell-free supernatant.
In order to Purification of bioflocculant DP-152 remove the bacterial cells, the culture broth (5 r) of Bacillus sp. DP-152 was diluted with ten vol. of distilled water. Most of the bacterial cells were removed by centrifugation at 9,000 x g for 30 min. The cell-free culture broth was concentrated to 1.8 1. The concentrated supernatant (1.8 l) was precipitated by the addition of two vol. of ethanol. The precipitated crude flocculant was dried with a vacuum evaporator (2.3 g), and redissolved in distilled water. Then, 10% CPC solution was added until no more precipitate was formed. The insoluble acidic flocculant-CPC complex was collected by centrifugation, and redissolved in a 10% sodium chloride solution. After dialysis against distilled water, the flocculant was precipitated by the addition of two vol. of ethanol and dissolved in distilled water. The acidic flocculant ob.7 .6
0
10
20
30
40
50
Fraction number (5.0 ml/tube) FIG. 3. Gel permeation chromatogram of bioflocculant DP-152 with Sephacryl S-500. Symbols: ( l) polysaccharide, (0) flocculating activity.
Concenbatbn of fhccubnt ( mg I I ) FIG. 4. Effect of flocculant concentration of flocculants on the flocculating activity. Flocculating reactions were performed at different concentrations in kaolin suspension (4,800 mg/l) containing 6.8 mM CaC12.2H20. Symbols: 0, bioflocculant DP-152; 0, polyacrylamide (PAM); n , zooglan (from Zoogloea ramigera).
tained was dialyzed against distilled water and lyophilized. Purified flocculant solution (0.1%) was loaded onto a Sephacryl S-500 column (4.3 x 62 cm) and eluted with distilled water. Fractions of 5 ml each were collected and examined by the phenol-sulfuric acid method (19). Figure 3 shows the Sephacryl S-500 column chromatogram of the flocculant. The peak of flocculating activity agreed in position with that of a polysaccharide, indicating that the main flocculating factor is a polysaccharide. It has been reported that the main flocculating factors of the flocculants produced from R. erythropolis (9) and Pacilomyces sp. (10) are proteins, while Zoogloea sp. (12) and A. cupidus (4) like Bacillus sp. DP-152, produced extracellular polysaccharide as a flocculant. Fractions containing polysaccharide were combined and lyophilized (the final preparation was named bioflocculant DP-152), and then subjected to both chemical and biological analyses. Flocculation Effect of bioflocculant concentration reactions were performed at different concentrations ranging from 0.1 to 5.0 mg/l in kaolin suspensions (4,800 mg/l) containing 6.8 mM CaC12.2Hz0. The flocculating activity of the bioflocculant DP-152 was compared with anionic commercial flocculant (polyacrylamide, PAM) and a bioflocculant (zooglan from Zoogloea ramigera). Some typical flocculation curves of these flocculants in kaolin suspension are shown in Fig. 4. The flocculating activity was highest at 1.0 mg/l and decreased at the higher flocculant concentrations. The flocculating activities of polyacrylamide (PAM) and zooglan were highest at 1.O and 3.0 mg/l, respectively. At a high concentration of flocculant, the floe size increased (data not shown) and the flocculating activity gradually decreased. As shown in Fig. 5, the flocculating activity initially increased with increasing flocculant dosage, but then decreased as the adsorption of excess flocculant restabilized the particles. Because of incomplete dispersion of excess flocculants, only particles around flocculants participated in the flocculating reac-
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VOL. 84, 1997
TABLE 1.
FROM BACILLUS
SP. DP-152
111
Comparison of bioflocculants produced from microorganisms
Flocculant DP-152 (Bacillus sp.) NOC-1 (R. erythropolis) PY-90 (Bacillus sp.)
Optimum concentration (mg/l) 1
Flocculating activity (V0.D.)
20 20
43 33 15
Reference This study 10 12
Flocculating activity was measured with kaolin clay suspension.
Wavenumber (cm-J) FIG. 5. KBr.
Infrared absorption spectrum of bioflocculant DP-152 in
tion in a moment. Therefore, other particles did not participate in the flocculating reaction and the flocculating activity decreased. The bioflocculant DP-152 showed a higher flocculating activity than that of a protein flocculant from R. erythropolis (9) and a polysaccharide flocculant from Bacillus sp. PY-90 (11) (Table 1). The bioflocculant DP-152 was especially effective in the flocculating reaction at a low flocculant concentration. The relationship between the flocculant concentration and flocculating activity of bioflocculant DP-152 was similar to that of polyacrylamide. These results thus suggested that the bioflocculant DP-152 can be substituted for polyacrylamide in respect to flocculation. Physical and chemical properties of bioflocculant DPThe molecular weight of bioflocculant DP-152 152
was estimated to be over 2 x lo6 daltons. Generally, the molecular weight of a flocculant is related to the chain length of the polymer as an important factor in the flocculating reaction. A larger flocculant molecular weight causes a larger floe size in the flocculating reaction (1). As a large-molecular-weight flocculant has more functional groups, it forms a strong floe, coagulating many suspended particles in solution (1). The molecular weights of available flocculants range from a few thousand to several million (13). Michaels (13) reported that an anionic polyacrylamide with a higher molecular weight was more effective in removing suspended particles using clay suspensions. From the infrared spectrum of the bioflocculant DP152 with a KBr pellet, the characteristic chemical groups were analyzed (Fig. 5). The absorption peak at 3,400 -l was characteristic of OH stretching from the itund hydroxyl group, and adsorbed water molecules. The peaks in the range from 2,900 to 2,800 cm-l were an indication of aliphatic C-H stretching. The absorption peaks around 1,620 cm-l and 1,060 cm-l were characteristics of C =0 and C-O groups. The strong absorption peaks observed in the range from 1,000 to 1,200 cm-’ are generally known to be typical characteristics of all sugar derivatives. The infrared spectrum of this flocculant thus shows the presence of carboxyl, hydroxyl, and amino groups. Zajic et al. (5) reported that functional groups known to contribute to the flocculation of clays are the carboxyl, hydroxyl, and amino groups. The bioflocculant DP-152 reacted with the cationic colloid reagent poly (diallyldimethylammonium) chloride, resulting in an anionic flocculant having the charge density of
- 1.75 meq/g. This result is similar to the charge density (- 1.69 meq/g) of Al-201 from Alcaligenes cupidus KT201 (4). The sugar constituents in the TFA-hydrolysate were glucose, mannose, galactose, and fucose in an approximate molar ratio of 8 : 4 : 2 : 1. According to an elemental analysis, the constituent elements were 38.0% carbon, 6.7% hydrogen, 43.7% oxygen, and 0.6% nitrogen. The phenol-sulfuric acid method (19) showed that the polysaccharide contained 82.4% (w/w) of total sugar. In the hydrolysate of DP-152 flocculant, 4.27% acetic acid, 6.20% pyruvic acid, and 32.83% uranic acid were detected. Therefore, bioflocculant DP-152 is an acidic polysaccharide consisting of glucose, mannose, galactose, fucose, uranic acid, acetic acid, and pyruvic acid. It is supposed that the negatively charged groupsuranic acid, acetic acid and pyruvic acid were also found as components of the bioflocculant react at the cationic site of the suspended particles. The results suggest that the bioflocculant DP-152 acts as a polyelectrolyte, which aggregates suspended solids into a floe network by the formation of a chemical bridge. Because the components and composition of the bioflocculant DP-152 differs from those of other polysaccharides produced from Bacillus species or other bacteria, it is believed to be a newly discovered polysaccharide flocculant. The biopolymer flocculant from Bacillus sp. DP-152 is expected to be widely applied in various industrial wastewater treatments as a novel flocculating agent because of its harmlessness toward humans and the environment.
REFERENCES 1. Gut&o, S.: Waste treatment with polyelectrolytes and other flocculants. 0.1-37. Noves Data Corn.. New _ Park Ridge, Jersey (1977): 2. Kurane, R. and Matsuyama, H.: Production of a bioflocculant by mixed culture. Biosci. Biotech. Biochem., 58, 1589-1594 (1994). 3. Kurane, R., Takeda, K., and Suzuki, T.: Screening for and characteristics of microbial flocculants. Agr. Biol. Chem., 50, 2301-2307 (1986). 4. Toeda, K. and Kurane, R.: Microbial flocculant from Alcaligenes cupidus KT201. Agr. Biol. Chem., 55, 2793-2799 (1991). 5. Zajic, J. E. and Knettig, E.: Flocculants from paral%nic hydrocarbons. Developments in industrial microbiology, p. 87-98. American Institute of Biological Science, Washington DC (1970). 6. Nakamura, J., Miyashiro, S., and Hirose, Y.: Conditions for production of microbial cell flocculant by Aspergillus sojae AJ7002. Aar. Biol. Chem., 40. 1341-1347 (1976). Vanhoric, M. and Moe&, w.: Carcinogen of acrylamide. Carcinogensis, 4, 1459-1463 (1983). Dearfield, K. L. and Ambermatby, C. 0.: Acrylamide: its metabolism, developmental and reproductive effects, genotoxicity, and carcinogenicity. Mutant Res., 195, 45-77 (1988). Takeda, M., Kurane, R., Koiznmi, J., and Nakamura, I.: A
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protein bioflocculant produced by Rhodococcus erythropolis. Agr. Biol. Chem., 55, 2663-2664 (1991). by 10. Takagi, H. and Kadowaki, K.: Flocculant production Pacilomyces sp. Taxonomic studies and culture conditions for production. Agr. Biol. Chem., 49, 3151-3157 (1985). 11. Yokol, H., Natsuda, O., Hiiose, J., Hayasbi, S., and Takasaki, Y.: Characteristics of a biopolymer flocculant produced by Bacillus sp. PY-90. J. Ferment. Bioeng., 79, 378-380 (1995). 12. Unz, R. F. and Farrah, S. R.: Exopolymer production and flocculation by Zoogloea MP6. Appl. Eviron. Microbial., 31, 623626 (1976). 13. MIchaels, A.S.: Aggregation of suspensions by polyelectrolytes. Ind. Eng. Chem., 46, 1485 (1954). 14. Peter, H. A. S., Nicholas, S.M., Sharpe, M. E., and Holt, J. G.: Bergey’s manual of systematic bacteriology, vol. 2, p. 1104-1139. Williams and Wilkins Co., Baltimore (1986). 15. Komagata, K. and Snzakl, K. I.: Lipid and cell-wall analysis in bacterial systematics, p. 161-207. In Colwell, R. R. and Grigorova, R. (ed.), Methods in microbiology, vol. 19. Academic Press, London (1987). 16. Miller, L.: A single derivatization method for bacterial fatty acid methyl esters including hydroxy acid. J. Clin. Microbial., 16, 584-586 (1982). 17. Tamaoka, K. and Komagata, K.: Determination of DNA base
18. 19. 20.
21. 22.
23.
24.
25.
composition by reversed-phase high-performance liquid chromatoaranhv. FEMS Microbial. Lett., 25. 125-128 (1984). Korea Standards Association: Colloidal titrimefrics,’ Seoul, KSM 0001 (1992). Chaplin, M. F. and Kennedy, J. F.: Carbohydrate analysis, p. 2. IRL Press, Washington, D.C. (1986). Bradford, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-254 (1976). Chaplin, M. F. and Kennedy, J. F.: Carbohydrate analysis, p. 5. IRL Press, Washington, D.C. (1986). Frideman, T. E. and Haugen, G. E.: The determination of keto acids in blood and urine. Pyruvic acid, II. J. Bid. Chem., 147, 415-442 (1943). McComh, E. A. and McCready, R. M.: Determination of acetyl in pectin and in acetylated carbohydrated polymers. Anal. Chem., 29, 819-821 (1957). Gorden, R. E., Heynes, W. C., and Pang, C. H.: The genus Bacillus. Agriculture handbook stock number 202-275-2091. United States Department of Agriculture, Washington, D.C. (1975). Suzuki, K. and Komagata, K.: Taxonomic significance of cellular fatty acid composition in some coryneform bacteria. Int. J. Syst. Bacterial., 33, 188-200 (1983).
Characterization HYUN-HYO
of Bioflocculant Produced by BaciZZussp. DP-152
SUH,’ GI-SEOK KWON,Z CHANG-HO LEE,’ HEE-SIK KIM,’ HEE-MOCK OH,’ AND BYUNG-DAE YOON’*
Environmental Microbiology Research Unit, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Taejon 305-600’ and Natural Science College, Andong National University, Andong 760-749,= Korea Received 1 July 1996/Accepted 30 May 1997
A strain (designated DP-152) which produces an excellent flocculating substance was isolated from soil samples and identified as Bacillus species. The major flocculating substance (bioflocculant DP-152) produced by Bacillus sp. DP-152 was puriiied by ethanol precipitation and cetylpyridlnium chloride (CPC) precipitation and gel permeation chromatography. In kaolin suspension, the highest flocculating activity was obtained at the bioflocculant concentration of 1 mg/Z. The bioflocculant DP-152, which has an estimated molecular weight of over 2 x 106 daltons, is a novel bioflocculant derived from sugar components consisting of glucose, mannose, galactose, and fucose in an approximate molar ratio of 8 : 4 : 2 : 1. Some of its pbysico-cbemical properties were also determined. [Key words: novel bioflocculant, anionic polysaccharide, Bacillussp.] Flocculating agents are used in a wide range of industrial processes, including wastewater treatment (1). Flocculants are divided into organic synthetic polymer flocculants (polyacrylamide derivatives and polyacrylic acid), inorganic flocculants (aluminium sulfate and polyaluminium chloride), and bioflocculants (chitosan, algin, and microbial flocculants) (2). In particular, polyacrylamides
flocculant-producing bacteria were isolated. The flocculating activities were measured by using a kaolin clay suspension, activated carbon suspension, and industrial wastewater. Identification of strain DP-152 The isolated strain DP-152 was identified from its morphological and physiological properties, according to Bergey’s Manual of Systematic Bacteriology (14). The chemotaxonomical characteristics of the strain were examined by the procedures of Komagata and Suzuki (15), Miller (16), and Tamaoka and Komagata (17). Measurement of flocculating activity Flocculation of kaolin clay Kaolin clay (Junsei Chemical Co.) was used as the suspension material for estimating the flocculating activity. In a lOO-ml beaker, 0.1 ml of 10% (w/v) CaClz .2H20 solution was added to the 100 ml of kaolin clay suspension (S,OOOmg/l) and stirred with a magnetic bar for 1 min (300rpm). After stirring, the culture broth (10 ,&I) was added to the kaolin suspension. The pH of the kaolin suspension was adjusted to pH 7.0 with 1.0 N NaOH. After each addition, the reaction was performed with rapid mixing at 300 rpm for 30 s, followed by slow mixing at 100 rpm for 1 min. The test solution was mixed at room temperature and then kept standing for 5 min. The formation of visible aggregates was observed. The turbidity of the upper phase was measured at 550nm with a spectrophotometer (UV-160A; Shimadzu, Japan). The flocculating activity was calculated by the following equation (4): Flocculating activity = 1/A - 1/B A = optical density of the sample at 550 nm B=optical density of the reference at 550 nm Flocculation of activated carbon Activated carbon flocculation tests were done as above with the kaolin solution (5,OOOmg/f) being replaced by an activated carbon solution (5,000 mg/l). Jar test of industrial wastewaters Wastewaters from the food, textile, and paper industries were used to investigate the flocculating effect of the isolates. In a 250-ml beaker, 0.2 ml of 10% (w/v) CaC12. 2Hz0 solution (co-flocculant) was added to the 200 ml of each type of wastewater, and stirred with a jar tester for 1 min (200rpm). The pHs of each suspension were adjusted to
are frequently used because they are economical and effective flocculating agents (2-5). However, monomers of polyacrylamide have neurotoxic and strong carcinogenic properties (7, 8). Bioproducts produced by microorganisms are expected to be useful flocculating substances because they are safe for the environment. Studies on flocculating substances from microorganisms have examined them from various viewpoints, such as coagulation of kaolin clay and removal of microorganisms in the fermentation industry (2-5). Among these reports, Nakamura et al. (6) reported serial studies on a fungal flocculant from a strain of Aspergillus sojae, Schenck et al. (Balg. patant 822,947) reported that the alga Chlamydomonas mexicana formed flocculating agents which are polysaccharides, and Kurane et al. (3) reported that Rhodococcus erythropolis S-l produced a flocculant which is effective for various colloidal suspensions and coloured pigments. In this paper, the screening and characteristics of a flocculating substance produced by another microorganism are reported. MATERIALS
AND METHODS
FlocScreening of flocculant-producing bacteria culant-producing bacteria were isolated from many kinds of soil samples. The composition of the screening medium was as follows: 40 g glucose, 1.0 g NH803, 0.3 g K2HP0,, 0.3 g KH2P04, 0.1 g MgS04.7Hz0, 0.1 g MnS04. 4Hz0, 0.05 g NaCl, 0.4g CaC03, 0.1 g yeast extract, 0.1 g soytone, and 0.1 g tryptone in 1 1 distilled water. The culture temperature and initial pH were 30°C and 7.0, respectively. By measuring the flocculating activity of the broth cultured in the screening medium for 3 d, * Correspondingauthor. 108
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the floe-forming pH of each type of wastewater. The culture broth was added to each type of wastewater to a concentration of 10ppm. After each addition, the reaction was performed with rapid mixing at 1OOrpm for 1 min, followed by slow mixing at 50 rpm for 2 min, and the test solution then kept standing for 3 min. Chemical oxygen demand (COD) and suspended solids (SS) were selected as the principal tests to determine the extent of flocculation. Physical and chemical properties of bioflocculant The molecular weight of the bioflocculant 152
DP-
DP152 was estimated by gel permeation chromatography (GPC) using Sephacryl S-500 (Pharmacia Co.) with dextran as the standard. The infrared spectrum was measured using an IR spectrophotometer (RFX-65; Layser precision analytical, USA) with KBr pellets. The colloidal equivalent weight was measured by colloidal titrimetrics (18). Complete hydrolysis of the bioflocculant DP-152 was carried out with 1.OM trifluoroacetic acid (TFA) at 121°C for 1 h. After hydrolysis, the solution was neutralized with 1.0 N NaOH solution and lyophilized. Sugar costituents in the TFA-hydrolysate were analyzed using high-performance anion-exchange chromatography (HPAE; Dionex Co., USA). The HPAE was fitted with a pulsed amperometric detector (PAD), using a CarboPak PA1 column (4 x 250mm) (Dionex Co., USA). NaOH solution (20mM) was used as the eluent, at a flow rate of 1.0 ml/min. Elemental analysis was conducted with a elemental analyzer (EA1108; Carlo Erba Ins., Italy). The total carbohydrate content of the flocculant was determined by the phenol-sulfuric acid method (19) and expressed as the glucose equivalent. The protein moiety in the llocculant molecule was determined by the Bradford method (20) with bovine serum albumin as a standard. Sugar derivatives were investigated by the carbazole method (21) for uranic acid, the Friedmann method (22) for pyruvic acid, and the hydroxamic acid method (23) for acetic acid. RESULTS AND DISCUSSION Screening of flocculant-producing bacteria For the screening of new flocculant-producing bacteria, more than 200 bacterial strains which excreted mucous material on the agar plate of the screening medium were isolated from soil. The culture broth of each isolate was tested for its ability to flocculate kaolin clay and activated carbon. Flocculating activity was detected in 49 of the isolates (data not shown), 18 of which showed high flocculating activity on the test materials. To evaluate the industrial applicability of these 18 isolates, their flocculating efficiency was examined using wastewaters from the food, textile, and paper industries. In a jar test, coagulation and flocculation were accomplished at room temperature. The reagent dosing, mixing (destabilization), and the subsequent sedimentation (phase separation) took place in the same reaction chamber. The efficiency of the flocculation process in the jar test was evaluated on the basis of the COD and SS before and after treatment. The culture broth of 18 isolates showed high flocculating efficiencies in the industrial wastewaters (data not shown). Among the strains tested, strain DP-152, which showed the highest COD reduction rate (50%) and SS removal rate (90%), was selected for subsequent experiment. These results indicated that the flocculant from strain DP-152 had the ability to flocculate almost all
FROM BACILLUS SP. DP-152
109
solids suspended in an aqueous solution, and that this strain can therefore be considered as a candidate for application in practical wastewater treatment. The morphological Identification of strain DP-152 and physiological characteristics of strain DP-152 were investigated. After incubation for 3 d at 30°C on glucose-nutrient agar medium, colonies were circular, convex, and milky-white. Strain DP-152 was rod shaped (1.2-1.4 pm x 3.5-3.8 ,nm) (Fig. l), gram-positive, and formed spores. Growth occured at 28’C and 45°C but not at 65°C. The isolated strain was able to liquefy gelatin and to form indol. The strain showed a positive reaction in Voges-Proskauer and catalase test, and produced acid from glucose, arabinose, mannitol and xylose. Thus it was considered to belong to Bucilfus sp. as reported by Gorden et al. (24). To elucidate the relationship between strain DP-152 and the genus Bacillus in detail, its chemotaxonomical characteristics were examined. The peptidoglycan of the strain had the meso-type of diaminopimelic acid in the acid hydrolyzate of the cells, indicating that it belongs to Bacillus sp. (14). The main fatty acids were branchedchain fatty acids, such as 13-methyl tetradecanoic acid (iso- : 0) and 12-methyltetradecanoic acid (anteiso15 : 0); this finding is in agreement with that of Suzuki and Komagata (25). The quinone system of strain DP152 was menaquinone-7 (MK-7); the principal menaquinone components of Bacillus sp. are also of the MK-7 type. The mole% G plus C of the DNA of strain DP-152 was found to be 53%; the mole% G plus C of the DNA of Bacillus sp. ranges from 32 to 69 mole% (14). When the keys to the genera listed in Bergey’s Manual (14) were traced on the basis of the these results, strain DP152 was identified as Bacillus species, and designated as Bacillus sp. DP-152. Distribution of flocculating component The distribution of the flocculating activity of the culture broth was examined, and the major constituent with flocculating activity was identified. Figure 2 shows the distribution of the flocculating activity in the culture broth. Most of the flocculating activity was found in the cellfree supernatant, and more than 90% of it was recovered from ethanol precipitation. The major flocculating factor produced by Bacillus sp. DP-152 was identified with the ethanol precipitate.
FIG. 1. Scanning electron micrograph of strain DP-152. The strain was cultured on isolation medium at 30°C for 3 d and observed using a scanning electron microscope (SEM 515; Philips, Netherlands).
110
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l-l
L B
A
C
D
E
FIG. 2. Distribution of flocculating components in culture broth of Bacillus sp. DP-152. The strain was cultured in flocculant-production medium for 50 h. Lanes: (A) culture broth; (El) cell-free supernatant; (C) cell; (D) ethanol precipitate of cell-free supematant; (E) supematant after ethanol precipitation of cell-free supernatant.
In order to Purification of bioflocculant DP-152 remove the bacterial cells, the culture broth (5 r) of Bacillus sp. DP-152 was diluted with ten vol. of distilled water. Most of the bacterial cells were removed by centrifugation at 9,000 x g for 30 min. The cell-free culture broth was concentrated to 1.8 1. The concentrated supernatant (1.8 l) was precipitated by the addition of two vol. of ethanol. The precipitated crude flocculant was dried with a vacuum evaporator (2.3 g), and redissolved in distilled water. Then, 10% CPC solution was added until no more precipitate was formed. The insoluble acidic flocculant-CPC complex was collected by centrifugation, and redissolved in a 10% sodium chloride solution. After dialysis against distilled water, the flocculant was precipitated by the addition of two vol. of ethanol and dissolved in distilled water. The acidic flocculant ob.7 .6
0
10
20
30
40
50
Fraction number (5.0 ml/tube) FIG. 3. Gel permeation chromatogram of bioflocculant DP-152 with Sephacryl S-500. Symbols: ( l) polysaccharide, (0) flocculating activity.
Concenbatbn of fhccubnt ( mg I I ) FIG. 4. Effect of flocculant concentration of flocculants on the flocculating activity. Flocculating reactions were performed at different concentrations in kaolin suspension (4,800 mg/l) containing 6.8 mM CaC12.2H20. Symbols: 0, bioflocculant DP-152; 0, polyacrylamide (PAM); n , zooglan (from Zoogloea ramigera).
tained was dialyzed against distilled water and lyophilized. Purified flocculant solution (0.1%) was loaded onto a Sephacryl S-500 column (4.3 x 62 cm) and eluted with distilled water. Fractions of 5 ml each were collected and examined by the phenol-sulfuric acid method (19). Figure 3 shows the Sephacryl S-500 column chromatogram of the flocculant. The peak of flocculating activity agreed in position with that of a polysaccharide, indicating that the main flocculating factor is a polysaccharide. It has been reported that the main flocculating factors of the flocculants produced from R. erythropolis (9) and Pacilomyces sp. (10) are proteins, while Zoogloea sp. (12) and A. cupidus (4) like Bacillus sp. DP-152, produced extracellular polysaccharide as a flocculant. Fractions containing polysaccharide were combined and lyophilized (the final preparation was named bioflocculant DP-152), and then subjected to both chemical and biological analyses. Flocculation Effect of bioflocculant concentration reactions were performed at different concentrations ranging from 0.1 to 5.0 mg/l in kaolin suspensions (4,800 mg/l) containing 6.8 mM CaC12.2Hz0. The flocculating activity of the bioflocculant DP-152 was compared with anionic commercial flocculant (polyacrylamide, PAM) and a bioflocculant (zooglan from Zoogloea ramigera). Some typical flocculation curves of these flocculants in kaolin suspension are shown in Fig. 4. The flocculating activity was highest at 1.0 mg/l and decreased at the higher flocculant concentrations. The flocculating activities of polyacrylamide (PAM) and zooglan were highest at 1.O and 3.0 mg/l, respectively. At a high concentration of flocculant, the floe size increased (data not shown) and the flocculating activity gradually decreased. As shown in Fig. 5, the flocculating activity initially increased with increasing flocculant dosage, but then decreased as the adsorption of excess flocculant restabilized the particles. Because of incomplete dispersion of excess flocculants, only particles around flocculants participated in the flocculating reac-
BIOFLOCCULANT
VOL. 84, 1997
TABLE 1.
FROM BACILLUS
SP. DP-152
111
Comparison of bioflocculants produced from microorganisms
Flocculant DP-152 (Bacillus sp.) NOC-1 (R. erythropolis) PY-90 (Bacillus sp.)
Optimum concentration (mg/l) 1
Flocculating activity (V0.D.)
20 20
43 33 15
Reference This study 10 12
Flocculating activity was measured with kaolin clay suspension.
Wavenumber (cm-J) FIG. 5. KBr.
Infrared absorption spectrum of bioflocculant DP-152 in
tion in a moment. Therefore, other particles did not participate in the flocculating reaction and the flocculating activity decreased. The bioflocculant DP-152 showed a higher flocculating activity than that of a protein flocculant from R. erythropolis (9) and a polysaccharide flocculant from Bacillus sp. PY-90 (11) (Table 1). The bioflocculant DP-152 was especially effective in the flocculating reaction at a low flocculant concentration. The relationship between the flocculant concentration and flocculating activity of bioflocculant DP-152 was similar to that of polyacrylamide. These results thus suggested that the bioflocculant DP-152 can be substituted for polyacrylamide in respect to flocculation. Physical and chemical properties of bioflocculant DPThe molecular weight of bioflocculant DP-152 152
was estimated to be over 2 x lo6 daltons. Generally, the molecular weight of a flocculant is related to the chain length of the polymer as an important factor in the flocculating reaction. A larger flocculant molecular weight causes a larger floe size in the flocculating reaction (1). As a large-molecular-weight flocculant has more functional groups, it forms a strong floe, coagulating many suspended particles in solution (1). The molecular weights of available flocculants range from a few thousand to several million (13). Michaels (13) reported that an anionic polyacrylamide with a higher molecular weight was more effective in removing suspended particles using clay suspensions. From the infrared spectrum of the bioflocculant DP152 with a KBr pellet, the characteristic chemical groups were analyzed (Fig. 5). The absorption peak at 3,400 -l was characteristic of OH stretching from the itund hydroxyl group, and adsorbed water molecules. The peaks in the range from 2,900 to 2,800 cm-l were an indication of aliphatic C-H stretching. The absorption peaks around 1,620 cm-l and 1,060 cm-l were characteristics of C =0 and C-O groups. The strong absorption peaks observed in the range from 1,000 to 1,200 cm-’ are generally known to be typical characteristics of all sugar derivatives. The infrared spectrum of this flocculant thus shows the presence of carboxyl, hydroxyl, and amino groups. Zajic et al. (5) reported that functional groups known to contribute to the flocculation of clays are the carboxyl, hydroxyl, and amino groups. The bioflocculant DP-152 reacted with the cationic colloid reagent poly (diallyldimethylammonium) chloride, resulting in an anionic flocculant having the charge density of
- 1.75 meq/g. This result is similar to the charge density (- 1.69 meq/g) of Al-201 from Alcaligenes cupidus KT201 (4). The sugar constituents in the TFA-hydrolysate were glucose, mannose, galactose, and fucose in an approximate molar ratio of 8 : 4 : 2 : 1. According to an elemental analysis, the constituent elements were 38.0% carbon, 6.7% hydrogen, 43.7% oxygen, and 0.6% nitrogen. The phenol-sulfuric acid method (19) showed that the polysaccharide contained 82.4% (w/w) of total sugar. In the hydrolysate of DP-152 flocculant, 4.27% acetic acid, 6.20% pyruvic acid, and 32.83% uranic acid were detected. Therefore, bioflocculant DP-152 is an acidic polysaccharide consisting of glucose, mannose, galactose, fucose, uranic acid, acetic acid, and pyruvic acid. It is supposed that the negatively charged groupsuranic acid, acetic acid and pyruvic acid were also found as components of the bioflocculant react at the cationic site of the suspended particles. The results suggest that the bioflocculant DP-152 acts as a polyelectrolyte, which aggregates suspended solids into a floe network by the formation of a chemical bridge. Because the components and composition of the bioflocculant DP-152 differs from those of other polysaccharides produced from Bacillus species or other bacteria, it is believed to be a newly discovered polysaccharide flocculant. The biopolymer flocculant from Bacillus sp. DP-152 is expected to be widely applied in various industrial wastewater treatments as a novel flocculating agent because of its harmlessness toward humans and the environment.
REFERENCES 1. Gut&o, S.: Waste treatment with polyelectrolytes and other flocculants. 0.1-37. Noves Data Corn.. New _ Park Ridge, Jersey (1977): 2. Kurane, R. and Matsuyama, H.: Production of a bioflocculant by mixed culture. Biosci. Biotech. Biochem., 58, 1589-1594 (1994). 3. Kurane, R., Takeda, K., and Suzuki, T.: Screening for and characteristics of microbial flocculants. Agr. Biol. Chem., 50, 2301-2307 (1986). 4. Toeda, K. and Kurane, R.: Microbial flocculant from Alcaligenes cupidus KT201. Agr. Biol. Chem., 55, 2793-2799 (1991). 5. Zajic, J. E. and Knettig, E.: Flocculants from paral%nic hydrocarbons. Developments in industrial microbiology, p. 87-98. American Institute of Biological Science, Washington DC (1970). 6. Nakamura, J., Miyashiro, S., and Hirose, Y.: Conditions for production of microbial cell flocculant by Aspergillus sojae AJ7002. Aar. Biol. Chem., 40. 1341-1347 (1976). Vanhoric, M. and Moe&, w.: Carcinogen of acrylamide. Carcinogensis, 4, 1459-1463 (1983). Dearfield, K. L. and Ambermatby, C. 0.: Acrylamide: its metabolism, developmental and reproductive effects, genotoxicity, and carcinogenicity. Mutant Res., 195, 45-77 (1988). Takeda, M., Kurane, R., Koiznmi, J., and Nakamura, I.: A
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protein bioflocculant produced by Rhodococcus erythropolis. Agr. Biol. Chem., 55, 2663-2664 (1991). by 10. Takagi, H. and Kadowaki, K.: Flocculant production Pacilomyces sp. Taxonomic studies and culture conditions for production. Agr. Biol. Chem., 49, 3151-3157 (1985). 11. Yokol, H., Natsuda, O., Hiiose, J., Hayasbi, S., and Takasaki, Y.: Characteristics of a biopolymer flocculant produced by Bacillus sp. PY-90. J. Ferment. Bioeng., 79, 378-380 (1995). 12. Unz, R. F. and Farrah, S. R.: Exopolymer production and flocculation by Zoogloea MP6. Appl. Eviron. Microbial., 31, 623626 (1976). 13. MIchaels, A.S.: Aggregation of suspensions by polyelectrolytes. Ind. Eng. Chem., 46, 1485 (1954). 14. Peter, H. A. S., Nicholas, S.M., Sharpe, M. E., and Holt, J. G.: Bergey’s manual of systematic bacteriology, vol. 2, p. 1104-1139. Williams and Wilkins Co., Baltimore (1986). 15. Komagata, K. and Snzakl, K. I.: Lipid and cell-wall analysis in bacterial systematics, p. 161-207. In Colwell, R. R. and Grigorova, R. (ed.), Methods in microbiology, vol. 19. Academic Press, London (1987). 16. Miller, L.: A single derivatization method for bacterial fatty acid methyl esters including hydroxy acid. J. Clin. Microbial., 16, 584-586 (1982). 17. Tamaoka, K. and Komagata, K.: Determination of DNA base
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composition by reversed-phase high-performance liquid chromatoaranhv. FEMS Microbial. Lett., 25. 125-128 (1984). Korea Standards Association: Colloidal titrimefrics,’ Seoul, KSM 0001 (1992). Chaplin, M. F. and Kennedy, J. F.: Carbohydrate analysis, p. 2. IRL Press, Washington, D.C. (1986). Bradford, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-254 (1976). Chaplin, M. F. and Kennedy, J. F.: Carbohydrate analysis, p. 5. IRL Press, Washington, D.C. (1986). Frideman, T. E. and Haugen, G. E.: The determination of keto acids in blood and urine. Pyruvic acid, II. J. Bid. Chem., 147, 415-442 (1943). McComh, E. A. and McCready, R. M.: Determination of acetyl in pectin and in acetylated carbohydrated polymers. Anal. Chem., 29, 819-821 (1957). Gorden, R. E., Heynes, W. C., and Pang, C. H.: The genus Bacillus. Agriculture handbook stock number 202-275-2091. United States Department of Agriculture, Washington, D.C. (1975). Suzuki, K. and Komagata, K.: Taxonomic significance of cellular fatty acid composition in some coryneform bacteria. Int. J. Syst. Bacterial., 33, 188-200 (1983).