- Email: [email protected]
Screening and flocculating properties of bioflocculant-producing microorganisms
Mineral
Journal of University of Science and Technology Beijing Volume 13, Number 4, August 2006, Page 289
Screening and flocculating properties of bioflocculant-producing microorganisms Yanling Sheng ”, Qiang Zhang ’), Yanru She@, Chengbin Li3),and Huajun Wang’) 1) Civil and Environmental Engineering School, University of Science and Technology Beijing. Beijing 100083, China
2) Nanguan District Teacher Advanced Study School of Changchun, Changchun 130043, China 3) Guangdong Institute of Microbiology, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangzhou 5 10070, China (Received 2005-09-01)
Abstract: Screening of bioflocculant-producing microorganisms was carried out. A strain that secreted excellent bioflocculant was isolated from municipal sewage using the spread plate technique, identified as Klebsiella sp. by the analytical profile index (API)identification system, and named &. Several important factors that had an effect on &’s bioflocculant-producing and flocculating activity were studied. A total of 4 g/L Kaolin suspension was used to measure the flocculating activity of the bioflocculant from 4. It was found that maltose and urea were 4 ’ s best carbon and nitrogen sources, respectively, and the flocculating activity of the flocculating agent from &was markedly increased by the addition of trivalent cations such as Fe3+and A13+;furthermore, the bioflocculant produced by & was most effective when the pH value was 6.0. Key words: bioflocculant; flocculating rate; Klebsiella sp.; screening
1. Introduction Bioflocculant is a type of flocculant that is produced by microorganisms but is readily degradable. Its degradation intermediates are harmless to humans and the environment. In recent years, several kinds of microorganisms, which secreted flocculating biopolymer, have been screened and isolated from activated sludge, soil, and wastewater. The species include bacteria, fungi, actinomyces, and algae. Generally, soil and activated sludge samples are the best sources for isolating bioflocculant-producing microorganisms. Rhodococcus erythropolis was isolated from activated sludge and the bioflocculant from it was highly effective in treating the wastewater generated from industrial processes [ 13. Microorganisms such as Bacillus sp. [2], Enterobacter sp. [3], and Alcaligenes latus B-16 [4] were all isolated from soil samples. Generally, screening of bioflocculant-producing microorganisms is based on the colony morphology (mucoid and ropy) and capsule production. The flocculating efficiency of the bioflocculant is evaluated using measurements such as the ability to remove suspended solids and pigments and the chemical oxygen demand (COD). The bioflocculants from microorganisms were also purified and identified. It was reported that most of them were functional proteins [5-61 or functional polysaccharides [7-111. In this article, the author will elaborate how a bioflocculant-producing strain is screened Corresponding author: Yanling Sheng, E-mail: [email protected]
and isolated from the activated sludge and will also study the bioflocculant produced by it.
2. Materials and methods 2.1. Activated sludge and its maintenance The activated sludge was obtained from Qinghe Municipal Sewage Treatment Plant in Beijing, China. The sludge was maintained in a refrigerator at 4OC.
2.2. Preparation of sample A total of 100 mL of activated sludge was transferred to a 500-mL beaker and was diluted by the addition of 400 mL distilled water. Then the diluted activated sludge was stirred for 30 min at 250 r/min and precipitated for 30 min. The 5-mL supernatant, which was obtained from the beaker, was cultured in a 250mL flask on a conical orbital shaker (150 dmin) at 30°C for 2 days. The culture broth was then used for the isolation of bioflocculant-producing microorganisms.
2.3. Screening and isolation The composition of the isolation medium was as follows: 20 gfL soluble stark, 1 g/L KN03, 0.5 g/L MgS04.7H20,0.5 g/L NaCl, 0.5 g/L K2HP04.3H20, 0.01 g/L FeSO4.7Hz0,and 15-20 g/Lagar powder. Serial concentrations (lo4, lo”, lo“, lo-’) of diluted culture broth were inoculated on agar plates, and the agar plates were cultured in an incubator at 30°C for 2
J. Univ. Sci Technol. Beying, voL13,No.4, Aug 2006
290
or 3 days. After the colonies developed, the colonies whose surfaces were smooth, viscous, and wet were selected and then cultured in a liquid medium for another 3 days.
2.4. Measurement of flocculating activity and microbial growth The flocculating activities were measured by calculating the flocculating rate using the modified method of Yokoi [12], in which Kaolin clay was chosen as the solid phase in the 4 g/L suspension. After the pH of the suspension was adjusted to 7.0, 2 mL CaCl, (lwta) was added first. Then 2 mL of the centrifuged upper phase of culture broth was added to the 4 g/L Kaolin suspension and stirred for 2 min. After being allowed to settle for 6 min, sampling was done at a depth of 3 cm under the surface of the suspension and the absorbance (OD) of the sample was measured by a spectrophotometer (752 type) at 550 nm. A control experiment, without the addition of any agent, was measured in the same manner. The flocculating.rate was calculated according to the following equation:
the population is high enough to give detectable turbidity. Therefore, measuring the absorbency of the culture broth is one of the common methods to determine the cell growth. The absorbance of the culture broth correlates with the bacterial concentration; therefore, the absorbance reflects the growth of bacteria. In this study, the absorbance of the culture broth was measured with a spectrophotometerat 550 nm.
3. Results and discussion 3.1. Isolated bacteria and flocculating various suspensions of bioflocculant from them Three bacteria with excellent ability to flocculate were isolated, among which the best one was named As and was used in this study. 4 was identified as Klebsiellu sp. by the analytical profile index (API) identification system and its staining photograph is shown in Fig. 1.
loo%, Flocculating rate=[(OD550)c-OD550]/(OD550)c~ where OD550 is the absorbance of the sample at 550 nm and (oD550)~is the absorbance of the control at 550 nm. In the following experiment, the flocculating activities were measured in the Kaolin clay suspensions using the same procedure described above. The amount of scattering in the culture broth is directly proportional to the biomass of cells present and indirectly related to cell number. The extent of light scattering can be measured by a spectrophotometer and is almost linearly related to bacterial concentration at low absorbance level. Thus, the population growth can be easily measured spectrophotometrically as long as
Fig. 1. &'s staining photograph.
The experiment showed that the bioflocculant produced by A9 could flocculate various suspensions, such as bentonite, hematite, bauxite, and coal slurry water. The results are listed in Table 1.
Table 1. Results of the flocculation of various suspensions Suspensions Flocculating rate / %
Bentonite 86.32
The bioflocculant from A9 was more effective in flocculating minerals, and its application to process minerals was worthy of further investigation.
3.2. Effect of carbon sources and nitrogen sources on the flocculating rate It had been proved [131 that changing the carbon and nitrogen sources highly affected the growth of bacteria and the production of bioflocculant. It was reported that the addition of organic acids such as 2-keto gluconic acid could increase the flocculating activity, whereas in the presence of certain inorganic nitrogen
Hematite 88.48
Bauxite 82.36
Coal slurry water 75.69
compounds, the mycelial growth was poor and no flocculating activity could be detected. In this study, the effects of carbon sources and nitrogen sources on the flocculating activity are shown in Tables 2 and 3. Carbon was one of the necessary sources in the bacterial living surroundings. The results from Table 2 showed that maltose was the best carbon source, which was favorable to the enhancement of the flocculating activity and to the growth of 4.Fig. 2 shows the result of growth and flocculating rate variation when maltose is used as the carbon source.
Y.L Sheng et oL, Screening and flocculating properties of bioflocculant-producingmicroorganisms Table 2. Effect of carbon sources on flocculating activity ~
~~
Carbon sources Glucose Fructose Sucrose Maltose
Flocculating rate / % 76.88 80.56
0.8
88.12 82.39
E Oa6
Table 3. Effect of nitrogen sources on flocculating activity Nitrogen sources Urea
24
f 3
2
0.4 0.2
Flocculating rate / %
O’Or2b
94.54 91.84
(NH,),SO, NH,Cl Peptone Y eastrel
or
ferent metal ions. In this experiment, the effects of KC1, NaC1, CaC12,MgSO,, MnSO,, FeCl,, and &(so4),on the flocculating activity were studied. The results are shown in Fig. 4.
79.63
Ethanol
291
92.36 89.30
i2
418 !I6 Culture time / h
IiO
I$
0
Fig. 3. As’s growth and flocculating rate variation.
88.45
48
i2
96
120
0 Id4
2
Culture time / h Fig. 2. As’s growth and flocculating rate variation. Fig. 4. Effect of metal ions on the flocculating rate.
As shown in Fig. 2, when cultured on the base of maltose, bioflocculant produced by A9 was most effective after 72 h of cultivation. In addition, it was found that secreted bioflocculant during its cell growth and the activity of bioflocculant reached the maximal efficiency after 72 h of culture. The KNO, in the medium was replaced by different nitrogen compounds; the effects of the nitrogen sources, such as urea, (m4)2s04, NH,Cl, peptone, and yeastrel, on the flocculating activity and cell growth are presented in Table 3. The results from the experiments showed that organic nitrogen sources (except urea) were able to stimulate the cell growth greatly and that the culture broth was far more turbid. However, they had little effect on the increase of the flocculating rate. Although urea and inorganic nitrogen compounds such as (m4)2so4and NH4Cl could stimulate the flocculating activity of the bioflocculant produced by A9, they had no effect on the cell growth. The growth of 4 and the flocculating rate variation when urea was used as the nitrogen source are shown in Fig. 3.
3.3. Effect of metal ions on the flocculating rate Similar to traditional flocculating agent, bioflocculant’s flocculating activity was strongly affected by dif-
It is generally accepted that the flocculation induced by bioflocculant can occur by bridging and charge neutralization [ 141. The flocculating rate was generally increased when different metal ions except Mgz+were added, as shown in Fig. 4. In particular, A13+and Fe3+ increased the flocculating rate. There might be two reasons to account for these results. First, adding cations to the Kaolin suspension may decrease the negative electrical charge of the particles. Second, through cation bridging, bioflocculant can absorb onto the clay particles more effectively and flocculate them easily. The difference between this experiment and other investigations [15] was that Ca2’ had little effect on the flocculating activity when compared with other metal ions.
3.4. Effect of pH values on the flocculating rate The pH value of the suspension had an extreme effect on the function of flocculating agent including bioflocculant because the surface charge of the dispersed phase changed according to various pH values. The variation of the flocculating rate depending on different pH values of the suspension is shown in Fig. 5 . From Fig. 5, it is concluded that the flocculating rate reaches the maximum when the pH value is 6.0, and goes down to the minimum when the pH value is 7.0. The reason
.
292
J. Univ. Sci Technol. Beijing, VoLI3, No.4, Aug 2006
for such a phenomena is worthy further investigation.
PH
Fig. 5. Variation of the flocculating rate vs. the pH value.
4. Conclusions One strain named A, with excellent flocculating ability was screened and isolated using the spread plate technique. A9 was identified as Klebsiella sp. by the AF'I identification system, and the factors that affected the bioflocculant produced from it were investigated. The results show that maltose is A,'s best carbon source and urea is its best nitrogen source. The bioflocculant produced by & becomes more efficient when the pH value is 6.0. Furthermore, trivalent cations Fe3+and A13' have a high synergistic effect on the bioflocculant.
References [l] R. Kurane and H. Matsuyama, Production of a bioflocculant by mixed culture, Biosci. Biotechnol. Biochem., 58 (1994), p. 1590. [2] H. Suh, G Kwon, C. Lee, H. Kim, H. Oh, and B. Yoon, Characterization of bioflocculant produced by Bacillus sp. DP- 152, Ferment Bioeng., 84(1997),p. 109. [3] H. Yokoi, T. Yoshida, S. Mori, J. Hirose, S. Hayashi, and Y. Takasaki, Biopolymer flocculant produced by an Enterobacter sp., Biotechnol. k t t . , 19(1996), p.572. [4] R. Kurane and Y. Nohata, A new water-absorbing polysac-
charide from Alcaligenes latus, Biosci. Biotechnol. Biochem., 58(1994), p.236. [5] J.I. Koizui, M. Takeda, R. Kurane, and J. Nakamura, Synergetic flocculation of the bioflocculant FIX extracellularly produced by Nocardia amarae, Gen. Appl. Microbial., 37(1991), p.447. [6] R. Kurane, K. Habmochi, T. Kakuno, M. Kiyohara, M. Hirano, and Y. Taniguchi, Production of a bioflocculant by Rhodococcus erythropolis S- 1 grown on alcohols, Biosci. Biotechnol. Biochem., 58( 1994), p.428. [7] N. He, Y. Li, J. Chen, and S.Y. Lun, Identification of a novel bioflocculant from a newly isolated Corynebacteriurn glutamicum, Biochem. Eng. J., 11(2002), p.141. [8] H. Salehizadeh, M. Vossoughi, and I. Alemzadeh, Some investigations on bioflocculant producing bacteria, biochem. Eng. J., 5(2000), p.39. [9] I.L. Shih, Y.T. Van, L.C. Yeh, H.G Lin, and Y.N. Chang, Production of a biopolymer flocculant from Bacillus licheniformis and its flocculation properties, Bioresource Technol., 78(2001), p.270. [ 101 K. Toeda and R. Kurane, Microbial flocculant from Alcaligenes cupidus KT201, Agric. Biol. Chem., 55( 199l), p.2793. [ 111 N. Levy, Y. Baror, and S. Magdassi, Flocculation of bentonite particles by a Cyanobacterial bioflocculant, Colloids. Su$, 48(1990), p.337. [12] H. Yokoi, 0. Natsuda, J. Hirose, S. Hayashi, and Y. Takasaki, Characteristics of a biopolymer flocculant produced by Bacillus sp. PY-90, J. Ferment. Bioeng., 82( 1999, p.84. [13] H. Salehizadeh and S.A. Shojaosadati, Extracellular biopolymeric flocculants recent trends and biotechnological importance, Biotechnol. Adv., 19(2001),p375. [141 T. B a n g and Z. Lin, Microbial flocculant and its application in environmentalprotection,J. Environ. Sci., 11(1999), p.4. [15] H. Salehizadeh and S.A. Shojaosadati, Extracellular biopolymeric flocculants recent trends and biotechnological importance, Biotechnol. Adv., 19(2001),p.379.
Journal of University of Science and Technology Beijing Volume 13, Number 4, August 2006, Page 289
Screening and flocculating properties of bioflocculant-producing microorganisms Yanling Sheng ”, Qiang Zhang ’), Yanru She@, Chengbin Li3),and Huajun Wang’) 1) Civil and Environmental Engineering School, University of Science and Technology Beijing. Beijing 100083, China
2) Nanguan District Teacher Advanced Study School of Changchun, Changchun 130043, China 3) Guangdong Institute of Microbiology, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangzhou 5 10070, China (Received 2005-09-01)
Abstract: Screening of bioflocculant-producing microorganisms was carried out. A strain that secreted excellent bioflocculant was isolated from municipal sewage using the spread plate technique, identified as Klebsiella sp. by the analytical profile index (API)identification system, and named &. Several important factors that had an effect on &’s bioflocculant-producing and flocculating activity were studied. A total of 4 g/L Kaolin suspension was used to measure the flocculating activity of the bioflocculant from 4. It was found that maltose and urea were 4 ’ s best carbon and nitrogen sources, respectively, and the flocculating activity of the flocculating agent from &was markedly increased by the addition of trivalent cations such as Fe3+and A13+;furthermore, the bioflocculant produced by & was most effective when the pH value was 6.0. Key words: bioflocculant; flocculating rate; Klebsiella sp.; screening
1. Introduction Bioflocculant is a type of flocculant that is produced by microorganisms but is readily degradable. Its degradation intermediates are harmless to humans and the environment. In recent years, several kinds of microorganisms, which secreted flocculating biopolymer, have been screened and isolated from activated sludge, soil, and wastewater. The species include bacteria, fungi, actinomyces, and algae. Generally, soil and activated sludge samples are the best sources for isolating bioflocculant-producing microorganisms. Rhodococcus erythropolis was isolated from activated sludge and the bioflocculant from it was highly effective in treating the wastewater generated from industrial processes [ 13. Microorganisms such as Bacillus sp. [2], Enterobacter sp. [3], and Alcaligenes latus B-16 [4] were all isolated from soil samples. Generally, screening of bioflocculant-producing microorganisms is based on the colony morphology (mucoid and ropy) and capsule production. The flocculating efficiency of the bioflocculant is evaluated using measurements such as the ability to remove suspended solids and pigments and the chemical oxygen demand (COD). The bioflocculants from microorganisms were also purified and identified. It was reported that most of them were functional proteins [5-61 or functional polysaccharides [7-111. In this article, the author will elaborate how a bioflocculant-producing strain is screened Corresponding author: Yanling Sheng, E-mail: [email protected]
and isolated from the activated sludge and will also study the bioflocculant produced by it.
2. Materials and methods 2.1. Activated sludge and its maintenance The activated sludge was obtained from Qinghe Municipal Sewage Treatment Plant in Beijing, China. The sludge was maintained in a refrigerator at 4OC.
2.2. Preparation of sample A total of 100 mL of activated sludge was transferred to a 500-mL beaker and was diluted by the addition of 400 mL distilled water. Then the diluted activated sludge was stirred for 30 min at 250 r/min and precipitated for 30 min. The 5-mL supernatant, which was obtained from the beaker, was cultured in a 250mL flask on a conical orbital shaker (150 dmin) at 30°C for 2 days. The culture broth was then used for the isolation of bioflocculant-producing microorganisms.
2.3. Screening and isolation The composition of the isolation medium was as follows: 20 gfL soluble stark, 1 g/L KN03, 0.5 g/L MgS04.7H20,0.5 g/L NaCl, 0.5 g/L K2HP04.3H20, 0.01 g/L FeSO4.7Hz0,and 15-20 g/Lagar powder. Serial concentrations (lo4, lo”, lo“, lo-’) of diluted culture broth were inoculated on agar plates, and the agar plates were cultured in an incubator at 30°C for 2
J. Univ. Sci Technol. Beying, voL13,No.4, Aug 2006
290
or 3 days. After the colonies developed, the colonies whose surfaces were smooth, viscous, and wet were selected and then cultured in a liquid medium for another 3 days.
2.4. Measurement of flocculating activity and microbial growth The flocculating activities were measured by calculating the flocculating rate using the modified method of Yokoi [12], in which Kaolin clay was chosen as the solid phase in the 4 g/L suspension. After the pH of the suspension was adjusted to 7.0, 2 mL CaCl, (lwta) was added first. Then 2 mL of the centrifuged upper phase of culture broth was added to the 4 g/L Kaolin suspension and stirred for 2 min. After being allowed to settle for 6 min, sampling was done at a depth of 3 cm under the surface of the suspension and the absorbance (OD) of the sample was measured by a spectrophotometer (752 type) at 550 nm. A control experiment, without the addition of any agent, was measured in the same manner. The flocculating.rate was calculated according to the following equation:
the population is high enough to give detectable turbidity. Therefore, measuring the absorbency of the culture broth is one of the common methods to determine the cell growth. The absorbance of the culture broth correlates with the bacterial concentration; therefore, the absorbance reflects the growth of bacteria. In this study, the absorbance of the culture broth was measured with a spectrophotometerat 550 nm.
3. Results and discussion 3.1. Isolated bacteria and flocculating various suspensions of bioflocculant from them Three bacteria with excellent ability to flocculate were isolated, among which the best one was named As and was used in this study. 4 was identified as Klebsiellu sp. by the analytical profile index (API) identification system and its staining photograph is shown in Fig. 1.
loo%, Flocculating rate=[(OD550)c-OD550]/(OD550)c~ where OD550 is the absorbance of the sample at 550 nm and (oD550)~is the absorbance of the control at 550 nm. In the following experiment, the flocculating activities were measured in the Kaolin clay suspensions using the same procedure described above. The amount of scattering in the culture broth is directly proportional to the biomass of cells present and indirectly related to cell number. The extent of light scattering can be measured by a spectrophotometer and is almost linearly related to bacterial concentration at low absorbance level. Thus, the population growth can be easily measured spectrophotometrically as long as
Fig. 1. &'s staining photograph.
The experiment showed that the bioflocculant produced by A9 could flocculate various suspensions, such as bentonite, hematite, bauxite, and coal slurry water. The results are listed in Table 1.
Table 1. Results of the flocculation of various suspensions Suspensions Flocculating rate / %
Bentonite 86.32
The bioflocculant from A9 was more effective in flocculating minerals, and its application to process minerals was worthy of further investigation.
3.2. Effect of carbon sources and nitrogen sources on the flocculating rate It had been proved [131 that changing the carbon and nitrogen sources highly affected the growth of bacteria and the production of bioflocculant. It was reported that the addition of organic acids such as 2-keto gluconic acid could increase the flocculating activity, whereas in the presence of certain inorganic nitrogen
Hematite 88.48
Bauxite 82.36
Coal slurry water 75.69
compounds, the mycelial growth was poor and no flocculating activity could be detected. In this study, the effects of carbon sources and nitrogen sources on the flocculating activity are shown in Tables 2 and 3. Carbon was one of the necessary sources in the bacterial living surroundings. The results from Table 2 showed that maltose was the best carbon source, which was favorable to the enhancement of the flocculating activity and to the growth of 4.Fig. 2 shows the result of growth and flocculating rate variation when maltose is used as the carbon source.
Y.L Sheng et oL, Screening and flocculating properties of bioflocculant-producingmicroorganisms Table 2. Effect of carbon sources on flocculating activity ~
~~
Carbon sources Glucose Fructose Sucrose Maltose
Flocculating rate / % 76.88 80.56
0.8
88.12 82.39
E Oa6
Table 3. Effect of nitrogen sources on flocculating activity Nitrogen sources Urea
24
f 3
2
0.4 0.2
Flocculating rate / %
O’Or2b
94.54 91.84
(NH,),SO, NH,Cl Peptone Y eastrel
or
ferent metal ions. In this experiment, the effects of KC1, NaC1, CaC12,MgSO,, MnSO,, FeCl,, and &(so4),on the flocculating activity were studied. The results are shown in Fig. 4.
79.63
Ethanol
291
92.36 89.30
i2
418 !I6 Culture time / h
IiO
I$
0
Fig. 3. As’s growth and flocculating rate variation.
88.45
48
i2
96
120
0 Id4
2
Culture time / h Fig. 2. As’s growth and flocculating rate variation. Fig. 4. Effect of metal ions on the flocculating rate.
As shown in Fig. 2, when cultured on the base of maltose, bioflocculant produced by A9 was most effective after 72 h of cultivation. In addition, it was found that secreted bioflocculant during its cell growth and the activity of bioflocculant reached the maximal efficiency after 72 h of culture. The KNO, in the medium was replaced by different nitrogen compounds; the effects of the nitrogen sources, such as urea, (m4)2s04, NH,Cl, peptone, and yeastrel, on the flocculating activity and cell growth are presented in Table 3. The results from the experiments showed that organic nitrogen sources (except urea) were able to stimulate the cell growth greatly and that the culture broth was far more turbid. However, they had little effect on the increase of the flocculating rate. Although urea and inorganic nitrogen compounds such as (m4)2so4and NH4Cl could stimulate the flocculating activity of the bioflocculant produced by A9, they had no effect on the cell growth. The growth of 4 and the flocculating rate variation when urea was used as the nitrogen source are shown in Fig. 3.
3.3. Effect of metal ions on the flocculating rate Similar to traditional flocculating agent, bioflocculant’s flocculating activity was strongly affected by dif-
It is generally accepted that the flocculation induced by bioflocculant can occur by bridging and charge neutralization [ 141. The flocculating rate was generally increased when different metal ions except Mgz+were added, as shown in Fig. 4. In particular, A13+and Fe3+ increased the flocculating rate. There might be two reasons to account for these results. First, adding cations to the Kaolin suspension may decrease the negative electrical charge of the particles. Second, through cation bridging, bioflocculant can absorb onto the clay particles more effectively and flocculate them easily. The difference between this experiment and other investigations [15] was that Ca2’ had little effect on the flocculating activity when compared with other metal ions.
3.4. Effect of pH values on the flocculating rate The pH value of the suspension had an extreme effect on the function of flocculating agent including bioflocculant because the surface charge of the dispersed phase changed according to various pH values. The variation of the flocculating rate depending on different pH values of the suspension is shown in Fig. 5 . From Fig. 5, it is concluded that the flocculating rate reaches the maximum when the pH value is 6.0, and goes down to the minimum when the pH value is 7.0. The reason
.
292
J. Univ. Sci Technol. Beijing, VoLI3, No.4, Aug 2006
for such a phenomena is worthy further investigation.
PH
Fig. 5. Variation of the flocculating rate vs. the pH value.
4. Conclusions One strain named A, with excellent flocculating ability was screened and isolated using the spread plate technique. A9 was identified as Klebsiella sp. by the AF'I identification system, and the factors that affected the bioflocculant produced from it were investigated. The results show that maltose is A,'s best carbon source and urea is its best nitrogen source. The bioflocculant produced by & becomes more efficient when the pH value is 6.0. Furthermore, trivalent cations Fe3+and A13' have a high synergistic effect on the bioflocculant.
References [l] R. Kurane and H. Matsuyama, Production of a bioflocculant by mixed culture, Biosci. Biotechnol. Biochem., 58 (1994), p. 1590. [2] H. Suh, G Kwon, C. Lee, H. Kim, H. Oh, and B. Yoon, Characterization of bioflocculant produced by Bacillus sp. DP- 152, Ferment Bioeng., 84(1997),p. 109. [3] H. Yokoi, T. Yoshida, S. Mori, J. Hirose, S. Hayashi, and Y. Takasaki, Biopolymer flocculant produced by an Enterobacter sp., Biotechnol. k t t . , 19(1996), p.572. [4] R. Kurane and Y. Nohata, A new water-absorbing polysac-
charide from Alcaligenes latus, Biosci. Biotechnol. Biochem., 58(1994), p.236. [5] J.I. Koizui, M. Takeda, R. Kurane, and J. Nakamura, Synergetic flocculation of the bioflocculant FIX extracellularly produced by Nocardia amarae, Gen. Appl. Microbial., 37(1991), p.447. [6] R. Kurane, K. Habmochi, T. Kakuno, M. Kiyohara, M. Hirano, and Y. Taniguchi, Production of a bioflocculant by Rhodococcus erythropolis S- 1 grown on alcohols, Biosci. Biotechnol. Biochem., 58( 1994), p.428. [7] N. He, Y. Li, J. Chen, and S.Y. Lun, Identification of a novel bioflocculant from a newly isolated Corynebacteriurn glutamicum, Biochem. Eng. J., 11(2002), p.141. [8] H. Salehizadeh, M. Vossoughi, and I. Alemzadeh, Some investigations on bioflocculant producing bacteria, biochem. Eng. J., 5(2000), p.39. [9] I.L. Shih, Y.T. Van, L.C. Yeh, H.G Lin, and Y.N. Chang, Production of a biopolymer flocculant from Bacillus licheniformis and its flocculation properties, Bioresource Technol., 78(2001), p.270. [ 101 K. Toeda and R. Kurane, Microbial flocculant from Alcaligenes cupidus KT201, Agric. Biol. Chem., 55( 199l), p.2793. [ 111 N. Levy, Y. Baror, and S. Magdassi, Flocculation of bentonite particles by a Cyanobacterial bioflocculant, Colloids. Su$, 48(1990), p.337. [12] H. Yokoi, 0. Natsuda, J. Hirose, S. Hayashi, and Y. Takasaki, Characteristics of a biopolymer flocculant produced by Bacillus sp. PY-90, J. Ferment. Bioeng., 82( 1999, p.84. [13] H. Salehizadeh and S.A. Shojaosadati, Extracellular biopolymeric flocculants recent trends and biotechnological importance, Biotechnol. Adv., 19(2001),p375. [141 T. B a n g and Z. Lin, Microbial flocculant and its application in environmentalprotection,J. Environ. Sci., 11(1999), p.4. [15] H. Salehizadeh and S.A. Shojaosadati, Extracellular biopolymeric flocculants recent trends and biotechnological importance, Biotechnol. Adv., 19(2001),p.379.