JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 82, No. 1, 84-87. 1996

Flocculation Properties of Poly(r-Glutamic by Bacillus sub tilis

HARUHIKO YOKOI,* TOMOMI ARIMA, JUN HIROSE, SACHIO HAYASHI,

Acid) Produced
AND

YOSHIYUKI

TAKASAKI

Department of Materials Science, Faculty of Engineering, Miyazaki University, 1-I Nishi, Gakuen-kibanadai, Myazaki 889-21, Japan
Received 23 October 1995IAccepted 10 April 1996

Poly(r_glutamic acid) (T_PGA) prepared from a culture of Bacillussubtilis had a high flocculating activity and its flocculation properties were investigated. Flocculating activity of -/-PGA in a kaolin suspension was stimulated by the addition of Ca*+, Mg*+ or Fe *+ to the suspension. High flocculating activity of I_PGA was induced in a kaolin suspension by addition of Fe3+ or AP+ and pH adjustment to the neutral range. Various inorganic suspensions of active carbon, acid clay, solid soil, and calcium and magnesium compounds were flocculated by r-PGA. Flocculation of organic suspensions such as cellulose and yeast by r_PGA occurred when Fe*+, Fe3+ or A13+ were added and the pHs of the suspensions were adjusted to the neutral range. From these flocculation properties in various suspensions, it may be possible to use r-PGA as a new biodegradable, harmless biopolymer flocculant. [Key words: flocculation properties,
poly(y-glutamic acid), Bacillussubtilis]

Among flocculating agents, widely used synthetic polymer flocculants give rise to environmental problems in that some of them are not readily biodegradable and intermediate products of their degradation are harmful to humans. To resolve these problems, the use of biodegradable biopolymer flocculants has been investigated. Although biodegradable biopolymer flocculants such as protein, glycoprotein and polysaccharide flocculants have been found (l-6), their harmlessness and safety toward humans have not been investigated. Therefore, to date these biopolymer flocculants are not utilized in areas such as drinking-water treatment and downstream processing in food and fermentation industries. In a previous paper, Yokoi et al. (7) reported that Bacillus sp. PY-90 produced a biopolymer flocculant composed of glutamic acid residues and which was possibly poly(yglutamic acid) (r-PGA). Since I-PGA is biodegradable, edible and nontoxic toward humans and the environment, if its flocculation properties and the characteristics of its flocculation are clarified, it will likely be utilized as a new and harmless biopolymer flocculant. In this study, the flocculation properties of authentic T-PGA prepared from a culture of B. subtilis were investigated in detail, and its applicability as a new flocculating agent was evaluated. Preparation of r-PGA B. subtilis IF03335 was cultured in a medium (pH 7.0) consisting of 3.0% glutamic acid, 1.0% NH&l, 1.0% glucose, 0.2% K2HP04, 0.2% yeast extract and 0.2% MgS04.7H20 at 30C for 72 h. After addition of two volumes of cold ethanol to the supernatant of the culture broth, y-PGA was collected by centrifugation and then dissolved in distilled water. After three such ethanol precipitation steps, the I-PGA solution was dialyzed against distilled water and lyophilized. The lyophilized I-PGA contained no protein or polysaccharide as determined by analyses using the Lowry-Folin method (8) and the phenol-sulfuric acid method (9). The I-PGA was confirmed to be composed of glutamic acid, using an amino acid analyzer (Model * Corresponding author.
84

835; Hitachi, Tokyo), after hydrolysis with 6N HCl at 110C for 24 h. Its molecular weight was about 1.5 x lo6 as determined by gel chromatography (Model LC-1OA; Shimadzu, Kyoto) using an Asahipak GS-710 column (Shimadzu) at 30C. As a solvent, 4 M guanidine hydrochloride was used at a flow rate of 1.0 ml/min and pullulan (Shodex STANDARD P-82; Showa Denko K.K., Tokyo) and bluedextran (Pharmacia Co., Sweden) were used as molecular weight standards. Assay of flocculating activity Assaying the flocculating activity of T-PGA in a kaolin suspension was performed as reported previously (7). Flocculating activities in active carbon (Wako Pure Chem. Ind. Ltd., Osaka), cellulose (AVICEL SF, Funakoshi Co. Ltd., Tokyo) and acid clay (Wako Pure Chem. Ind. Ltd.) distilled water suspensions, the optical densities at 550nm of which were adjusted to 1.4, were also measured. A solid soil suspension was prepared by addition of soil into distilled water, collecting the upper phase of the muddy water after allowing the suspension to stand for 3 min and adjustment of its optical density to 1.4. A cell suspension of Saccharomyces cerevisiae IF02044 in distilled water having an optical density of 1.4 was used as a yeast suspension. Flocculation properties of r-PGA in inorganic suspenAt first, the effects of various cations on the sions flocculating activity of y-PGA in a kaolin suspension were examined using NaCl, KCl, CaCl* .2Hz0, MgClz. 6Hz0, FeS04. 7H20, FeC& . 6H20 and AlC13. 6H20 as the cation sources. Among these cations, Ca2+, Mg2+ and Fe2+ stimulated the flocculating activity, and optimum combinations of y-PGA and these cations were determined by varying these concentrations from 5 to 40 mg/l and 0 to 20 mM in the reaction mixture, respectively. Figure 1 shows the effects of cation concentration on the flocculating activity of y-PGA present at an optimum concentration. Optimum concentrations of Ca2+ and Mg2+ for the flocculating activity of y-PGA were 10 and 0.2mM, respectively. In the case of Fe2+, the optimum concentration was 0.1 mM and the flocculating activity of y-PGA was inhibited completely at concentra-

VOL. 82, 1996

NOTES

85

0.01

0.1

10

100

Concentration

(mM)
0 10 Time

FIG. I. Effects of cation concentration on flocculating activity of r-PGA in kaolin suspension. Symbols: 0, Ca2+; 0, Mg2+; n, Fe2+. Concentrations of r-PGA in reaction mixtures were 2Omg/l (@)and lOmg/l(@, a).

20
(min)

30

tions higher than 1 mM. It is known that addition of cations to suspensions is necessary to induce the effective flocculation of microbial flocculants (l-6). The microbial flocculants produced by Rhodococcus erythropolis (1) and Alcaligenes cupidus (4) required Ca2+ and A13+ for their high flocculating activity. Flocculating activity of the microbial flocculants produced by Nocardia amarae (2) and Phormidium J-I (6) were stimulated by Na+, Ca2+, A13+ and Fe3+, and Na+ and Mg2+, respectively. Compared with these flocculants, the effects of cations on the flocculating activity of y-PGA were different and value of the flocculating activity of I-PGA in a kaolin suspension was equal or high. Various inorganic suspensions, in addition to kaolin suspensions, could be flocculated by I-PGA. Table 1 shows the flocculating activities of I-PGA regarding active carbon, acid clay and solid soil suspensions in the presence of cations at optimum concentrations. All these suspensions were flocculated to varying degrees and high flocculating activity of y-PGA was observed in the acid clay suspension. Furthermore, r-PGA flocculated suspensions of calcium or magnesium compounds. Suspensions of Ca(OH)2, [Ca3(P04)213.Ca(OH)2, CaC03, Mg(OH)z or MgC03 at a concentration of 5 g/l were flocculated only by addition of I-PGA and the flocculating activity in these suspensions containing 20mg/l I-PGA were 21.3, 5.7, 2.0, 8.1 and 9.3 l/OD, respectively. Therefore, rPGA may be applicable to solid-liquid separation and purification of wastewater containing these inorganic
TABLE 1. Flocculating activity of y-PGA regarding various inorganic suspensions Suspension Active carbon Added cation 8 mM Ca2d 2mM Mg2+ 0.05 mM Fe2 6 mM Ca2 4 mM Mg2+ 0.1 mM FeZ+ 8 mM Ca2+ 8 mM Mg2+ 6 mM FeZi Flocculating activity (1 /OD) 3.3 2.9 2.8 10.9 14.0 34.7 1.1 1.0 1.9

FIG. 2. Changes in relative viscosity, flocculating activity and molecular weight of I-PGA following heat-treatment. After heating of a r-PGA solution (1 g/t) at KKK, the relative viscosity (0), flocculating activity in a kaolin suspension (0) and molecular weight (A) were measured periodically.

Acid clay

Solid soil

Concentrations of I-PGA in reaction mixtures containing Ca2+, Mg2+ or Fe2+ were 20, 10 and 10 mg/l, respectively.

compounds. Optimum temperature for flocculation was approximately 70C in a kaolin suspension containing Caz+, Mg*+ or Fe2+, and flocculating activity decreased markedly at temperatures higher than 80C (data not shown). As shown in Fig. 2, when 1.Og/l r-PGA solution was heated at lOOC, its viscosity relative to that of distilled water measured at 35C by an Ostwald viscosimeter and its flocculating activity decreased markedly with an increase in heating time. In spite of the marked decrease in the relative viscosity, the decrease in the molecular weight distribution was very slight. Furthermore, when heat-treated I-PGA was reprecipitated using ethanol and then redissolved in distilled water, the relative viscosity and flocculating activity returned almost to normal levels. It is assumed that the temporary decrease in the relative viscosity and flocculating activity is not caused by an irreversible change in the chemical structure of I-PGA such as degradation to a lower molecular weight structure. Flocculation activity was affected by the pH of reaction mixtures. The reaction mixtures composed of a kaolin suspension, I-PGA and various cations, Ca*+, Mg2+, Fe*+, Fe3+ or A13+, were adjusted to predetermined pH values using HCI and NaOH, and then flocculating activities were measured. Optimum pH for Ca2+ was in the weakly acidic range of 4.0 to 5.0, and that for Mg2+, Fe2+, Fe?+ and A13+ was 6.0 to 7.0 (Fig. 3). It is assumed that Ca2+ and Mg*+ ions stimulate flocculation by neutralization and stabilization of residual negative charges of carboxyl groups in I-PGA forming bridges which bind kaolin particles to each other. When solutions of FeS04. 7&o, FeC13.6H2O or AlCl3.6HZO were added to reaction mixtures as cation sources, pHs of the reaction mixtures dropped to approximately 3.0 and extremely low flocculating activity of I-PGA was observed. However, the flocculating activity of y-PGA was stimulated by raising the pH of reaction mixtures to the neutral range. At or near neutral pH, i.e. 6.0 to 8.0, Fe2+, Fe3+ and A13+ were predominantly present as hydroxides such as Fe(OH)2, Fe(OH)3 and Al(OHh. It is assumed that these hydroxides are effective in stimulating flocculat-

86

YOKOIETAL.
Cellulose

J. FERMENT. BIOENG.,
Yeast

_ 1.5
& z1.c $ 0.5 C

7 PH (-)

11

1.: & : 1.f 8 0.: ifI3

FIG. 3. Effect of pH of kaolin suspensions containing various cations on flocculating activity of I-PGA. Symbols: 0, Ca2+; l , Mg2+; A, F.$+; A, Fe3+; 0, Al>+. Concentrations of I-PGA in reaction mixtures were 20 mg/l (0, A, 0 ) and 10 m&l (0, A). Concentrations of Ca2+, Mg2+, Fe2+, Fesf or Al+ in reaction mixtures were 10, 0.2, 0.1, 0.15 and 0.15 mM, respectively. 3 ing activity of y-PGA 5 7 9 PH (-) 1I

Flocculation properties of I-PGA in organic suspenOrganic suspensions such as yeast or cellulose sions

at or near neutral pH.

7 9 PH (-1

11

could not be flocculated only by the addition of cations and r-PGA. However, when the pH of the reaction mixtures containing Fez+, Fe3+ or AP+ was varied from 4.0 to 11.0, flocculation of organic suspensions was observed to occur in the neutral pH range. Figure 4 indicates the effects of reaction mixture pH on the change of optical densities of the supernatant of reaction mixtures, containing a cellulose or yeast suspension, 0.15 mM Fe3+ or AP+ and 20 mg/Z I-PGA, 5 min after mixing. A slight decrease in the optical density was caused by the addition of Fe3+ or A13+ only to the reaction mixture of cellulose or yeast at or near neutral pH. However, the optical density was further decreased by the coaddition of y-PGA at pHs from 6.0 to 9.0, indicating a stimulative effect of I-PGA on the flocculation of these organic suspensions. It is assumed that hydroxides of Fe3+ and A13+ present at or near neutral pH cooperate with yPGA to effect the flocculation of organic suspensions as well as inorganic suspensions. Optimum concentrations of Fe2+, Fe3+ and AP+ in a reaction mixture of cellulose (pH7.0) containing 20mg/l y-PGA were 0.5, 5.0 and 5.0 mM, respectively, and the flocculating activities of yPGA were approximately 18, 40 and 125 UOD, respectively (data not shown). Therefore, r-PGA effected the flocculation of not only inorganic suspensions but also organic ones when suitable cations were selected and the pH of the reaction mixture was adjusted to the neutral range. In this study, it was confirmed that y-PGA possesses flocculating activity in various inorganic and organic suspensions, and its flocculation properties were investigated. To date there have been no microbial flocculants like r-PGA which are certified to be harmless toward humans and edible. Among anionic biopolymer flocculants having carboxylic acid as part of their chemical structure, alginic acid is a polysaccharide flocculant extracted from brown algae. Compared with use of alginic acid, use of I-PGA should be economical in that it can be produced easily and extracellularly in high yield by

FIG. 4. Stimulative effects of I-PGA on flocculation of cellulose and yeast suspensions. Optical densities of supernatant at 550nm of reaction mixtures containing 0.15 mM Fe+ (A) or 0.15 mM Al)+ (B) were measured 5min after mixing. Symbols: 0, control; 0, 0.15 mM cation; A, 0.15 mM cation+20mg/l I-PGA.

culturing of bacteria in a fermentor (10). Therefore, it is anticipated that I-PGA will be utilized in the areas of wastewater treatment, drinking-water processing and downstream processing in food and fermentation industries as a new biopolymer flocculant which is harmless toward humans and the environment.
This work was supported in part by a Grant-in-Aid for Scientific Research (No. 07680613) from the Ministry of Education, Science and Culture of Japan. REFERENCES 1. Takeda, M., Kurane, R., Koizumi, J., and Nakamura, 1.: A protein bioflocculant produced by Rhodococcus erythropolis. Agric. Biol. Chem.. 55. 2663-2664 (1991). 2. Takeda, M., Koizhi; J., Matsuoka, h., and Hikuma, M.: Factors affecting the activity of a protein bioflocculant produced by Nocardia amarae. J. Ferment. Bioeng., 74, 408-409 (1992). 3. Lee, S. IL, Lee, S. O., Jang, K. L., and Lee, T. H.: Microbial flocculant from Arcuadendron sp. TS-49. Biotechnol. Lett., 17, 95-100 (1995). 4. Toeda, K. and Kurane, R.: Microbial flocculant from Alcaligenes cupidas KT201. Agric. Biol. Chem., 55, 2793-2799 (1991). 5. Kurane, R. and Nohata, Y.: Microbial flocculation of waste liquids and oil emulsion by a bioflocculant from Alcaligenes laius. Agric. Biol. Chem., 55, 1127-1129 (1991). 6. Fattom. A. and SbBo. M.: Phormidium J-l bioflocculant: production and activity. Arch. Microbial., 139, 421-426 (1984). 7. Yokoi, H., Natsuda, O., Hirose, J., Hayashi, S., and of a biopolymer flocculant Takasaki, Y.: Characteristics produced by Bacillus sp. PY-90. J. Ferment. Bioeng., 79, 378380 (1995). 8. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.: Protein measurement with the Folin phenol reagent. J.

VOL. 82, 1996 Biol. Chem., 196, 265-275 (1951). 9. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F.: Calorimetric method for determination of sugars and related substances. Anal. Chem., 28, 350-356 (1956).

NOTES

87

10. Kubota, H., Matsunobu, T., Uotani, K., Takebe, H., Satoh, A., Tanaka, T., and Taniguchi, M.: Production of poly(rglutamic acid) by Bacillus subtilis F-2-01. Biosci. Biotech. Biochem., 57, 1212-1213 (1993).