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Efficient separation of solids from fermentation broth in bacterial α-amylase production
JOURNALOF FERMENTATIONAND BIOENGINEERING Vol. 71, NO. 4, 284-285. 1991
Efficient Separation of Solids from Fermentation Broth in Bacterial o -Amylase Production P R A T I M A B A J P A I , R A J E S H K. G E R A , AND P R A M O D K. BAJPAI*
Chemical and Biochemical Engineering Division, Thapar Corporate Research and Development Center, Patiala 147001, India Received 21 November 1990/Accepted 27 January 1991 Various coagulating-flocculating agents were tried in different combinations for the clarification of bacterial broth having high a-amylase enzyme activity. Best separation was achieved using a combination of calcium chloride, sodium hydroxide and sodium alginate.
Thermostable ~-amylases from Bacillus sp. are of considerable commercial interest. These enzymes are normally produced from complex nitrogen and carbon sources using a submerged fermentation process. The fermented broth contains large a m o u n t s of partially c o n v e r t e d / u n converted nutrients, bacterial biomass, etc., which need to be separated out from the liquid phase containing enzyme. Normally, broths of bacterial fermentations are difficult to process. Moreover, the solids in the broth are colloidal in nature, which present technical difficulties in solid-liquid separation. Addition of flocculating or coagulating agents is needed to aid precipitation. Inorganic salts and synthetic polyelectrolytes have been reported to be good flocculating agents (1, 2). Recently, we have reported the clarification o f bacterial broth containing high ~-amylase activity (3). In this paper, we report a more efficient method for the separation of solids from the bacterial broth in (~-amylase enzyme production. Fermentation broths were obtained by fermentation o f complex medium with Bacillus sp. TCRDC-M1 in Marubishi and Chemap fermentors (2.61 to 71) as described earlier (3). Broth samples were treated with coagulating agents with the help of a variable-speed stirrer fitted with turbine blades. Treated broths were centrifuged for 2 0 m i n at 2,150× g and the supernatant was subjected to different tests. The treated broths were also filtered in a Buchner funnel using W h a t m a n filter paper no. 1 (12.5 cm dia.) under 550 mm Hg vacuum. Turbidity of the clarified liquid was measured by nephelometer using hexamine hydrazine sulphate as standard in terms of nephelometric turbidity units, NTU (4). Viable organism count was determined by standard plating technique. Enzyme activity was estimated in terms of dextrinizing units ( D U N ) / m l according to the procedure described earlier (5, 6). It was found earlier (3) that treatment of bacterial (~amylase enzyme broth with 10g CaC12//, 1 g N a O H / / , 0.3 g F e S O 4 / / a n d 0.3 g p o l y a c r y l a m i d e / / g a v e reasonably good clarity without any loss in enzyme activity. It was possible to reduce the turbidity of broth from 11,800 to 17 NTU. In order to get a clear enzyme solution of acceptable quality ( < 1 0 NTU) for easy downstream processing, it was decided to use other flocculants along with the above
mixture. Treatment with gelatin, guar gum, sodium carboxy methyl cellulose and bentonite, individually, along with the above mixture, did not result in any decrease in turbidity (Table 1). However, treatment with sodium alginate resulted in significant reduction in turbidity without any loss in enzyme activity. The clarity of the enzyme solution depended on the concentration of sodium alginate. The turbidity dropped to 6 and 4 NTU on treatment with 1 and 2 g/I of sodium alginate, respectively. At both the concentrations, very big flocs were produced which settled rapidly. However, no flocculation was noticed earlier when the broth was treated with sodium alginate alone (3). Flocculation with sodium alginate in the presence of other agents indicates the combined effects of different coagulating-flocculating agents. Similar results were also obtained when F e S Q and polyacrylamide were eliminated; only CaCI2 NaOH and sodium alginate were used. Sodium alginate was tried in various concentrations ranging from 0.5 to 5 g/I with CaClz/NaOH mixture. Concentrations higher than 5 g/l were not tried because the broth became viscous. It was found that the floc size increased and the turbidity of the broth decreased with increase in sodium alginate concentration from 0.5 to 5 g/l (Table 2). With 5 g sodium alginate//, it was possible to reduce the turbidity to a great extent, up to 1.4 NTU, without any loss in enzyme activity. More than 959/oo of the TABLE 1. Effectof addition of natural flocculatingagents on clarity of broth treateda with CaCIz+ NaOH + FeSO4+ polyacrylamide Flocculating agent No treatment b Treated ~' Gelatin Sodium carboxy methyl cellulose Guar gum Sodium alginate Bentonite
Concentration (g//) -1.0 2.0 1.0 2.0 1.0 2.0 1.0 2.0 1.0 2.0
Turbidity Enzyme activity (NTU) (DUN/ml) 11800 17 17 17 17 17 17 17 6 4 17 17
4128 4129 4127 4130 4128 4128 4125 4130 4130 4130 4130 4130
~' The broth was treated with l0 g CaCljl, 1g NaOH/I, 0.3 g FeSO4/ / and 0.3 g cationic polyacrylamide//. b The original broth, without any type of treatment.
Corresponding author. 284
VOL, 71, 1991 TABLE 2.
NOTES Effect of addition of sodium alginate on clarity of broth a treated with CaC12+NaOH
TABLE 3.
Effect of addition of sodium alginate on filtration performance
Concentration of sodium alginate (g//)
Turbidity (NTU)
Enzyme activity (DUN/ml)
Concentration of sodium alginate (g//)
Filtration efficiency" (~)
Turbidity
0.0 0.5 1.0 2.0 3.0 4.0 5.0
34.0 10.0 7.0 4.5 3.0 2.0 1.4
4128 4130 4129 4131 4130 4129 4133
0.0 0.5 1.0 2.0 3.0 4.0 5.0
92.0 94.5 93.2 94.1 93.8 94.2 95.1
a The broth was treated with 10 g CaCIJl and 1 g NaOH//before addition of sodium alginate.
b a c t e r i a l l o a d was also e l i m i n a t e d . T h e effects o f s o d i u m a l g i n a t e o n f i l t r a t i o n rate a n d efficiency were also s t u d i e d . T h e r a t e o f f i l t r a t i o n i n c r e a s e d with i n c r e a s e o f s o d i u m a l g i n a t e c o n c e n t r a t i o n in the b r o t h ( T a b l e 3). A t a c o n c e n t r a t i o n o f 5 g/l, the filtration r a t e was 163 I / m 2 . h , a n d t h e filtration efficiency a n d the t u r b i d i t y o f b r o t h were 9 5 % a n d 1.4 N T U , respectively. Alt h o u g h a c c e p t a b l e clarity was o b t a i n e d w i t h 0.5 g s o d i u m a l g i n a t e / / , b e t t e r filtration rates w e r e o b t a i n e d at h i g h e r c o n c e n t r a t i o n s o f s o d i u m alginate. T h e f i l t r a t i o n rates are e x p e c t e d t o be i m p r o v e d f u r t h e r in a large-scale o p e r a t i o n w h e r e c r o s s - f l o w m i c r o f i l t r a t i o n s are u s e d . T h e results s h o w t h a t a l m o s t all the c o l l o i d a l m a t t e r can be r e m o v e d easily w i t h o u t a n y loss in e n z y m e activity b y the t r e a t m e n t o f b r o t h w i t h 10 g CaC12//, 1 g N a O H / / a n d 0.5 g s o d i u m a l g i n a t e / / . The authors are grateful to the Director, Thapar Corporate R & D Centre, Patiala, India for permission to publish these results. Tile valuable assistance provided by the Microbial Biotechnology Centre, Department of Biology, University of Waterloo, Canada during the preparation of the manuscript is gratefully acknowledged.
285
(NTU)
Initial flux (l/m 2. h)
Average flux~ (l/m-' •h)
34.0 10.0 7.0 4.5 3.0 2.0 1.4
-97.8 113.4 ---254.7
-54.4 65.2 81.5 97.8 139.8 163.1
~' Filtration efficiency--percentage of enzyme recovery on filtration. Volume of filtrate collected b Average f l u x Time of filtration, filter area
REFERENCES 1. Gasner, L.L. and Wang, D. I. C.: Microbial cell recovery enhancement through flocculation. Biotechnol. Bioeug,, 12, 873887 (1970). 2. Gregory, J,: Fundamentals of flocculation. CRC Critical Reviews Env. Control., 19, 185-230 (1989). 3. Bajpai, P., Neer, J., and Bajpai, P. K.: Clarification of bacterial broth containing high ~-amylase activity. Biotechnol. Technique, 4, 227-232 (1990). 4. Greenberg, A. E., Trussell, R. H., and Clesceri, L. S. (ed.): Standard methods for the examination of water and wastewater, 16th ed. p. 134-139. Amer. Public Health Assoc., New York (1985). 5. Bajpai, P. and Bajpai, P.K.: High temperature alkaline ~ramylase from Bacillus licheniformis TCRDC-B13. Biotechnol. Bioeng., 33, 72-78 (1989). 6. Bajpai, P., Sharma, U., and Bajpai, P. K.: Effect of corn gluten on cr-amylase production by Bacillus licheniformis TCRDC-B 13. Biotech. Appl. Biochem., it, 610-615 (1989).
Efficient Separation of Solids from Fermentation Broth in Bacterial o -Amylase Production P R A T I M A B A J P A I , R A J E S H K. G E R A , AND P R A M O D K. BAJPAI*
Chemical and Biochemical Engineering Division, Thapar Corporate Research and Development Center, Patiala 147001, India Received 21 November 1990/Accepted 27 January 1991 Various coagulating-flocculating agents were tried in different combinations for the clarification of bacterial broth having high a-amylase enzyme activity. Best separation was achieved using a combination of calcium chloride, sodium hydroxide and sodium alginate.
Thermostable ~-amylases from Bacillus sp. are of considerable commercial interest. These enzymes are normally produced from complex nitrogen and carbon sources using a submerged fermentation process. The fermented broth contains large a m o u n t s of partially c o n v e r t e d / u n converted nutrients, bacterial biomass, etc., which need to be separated out from the liquid phase containing enzyme. Normally, broths of bacterial fermentations are difficult to process. Moreover, the solids in the broth are colloidal in nature, which present technical difficulties in solid-liquid separation. Addition of flocculating or coagulating agents is needed to aid precipitation. Inorganic salts and synthetic polyelectrolytes have been reported to be good flocculating agents (1, 2). Recently, we have reported the clarification o f bacterial broth containing high ~-amylase activity (3). In this paper, we report a more efficient method for the separation of solids from the bacterial broth in (~-amylase enzyme production. Fermentation broths were obtained by fermentation o f complex medium with Bacillus sp. TCRDC-M1 in Marubishi and Chemap fermentors (2.61 to 71) as described earlier (3). Broth samples were treated with coagulating agents with the help of a variable-speed stirrer fitted with turbine blades. Treated broths were centrifuged for 2 0 m i n at 2,150× g and the supernatant was subjected to different tests. The treated broths were also filtered in a Buchner funnel using W h a t m a n filter paper no. 1 (12.5 cm dia.) under 550 mm Hg vacuum. Turbidity of the clarified liquid was measured by nephelometer using hexamine hydrazine sulphate as standard in terms of nephelometric turbidity units, NTU (4). Viable organism count was determined by standard plating technique. Enzyme activity was estimated in terms of dextrinizing units ( D U N ) / m l according to the procedure described earlier (5, 6). It was found earlier (3) that treatment of bacterial (~amylase enzyme broth with 10g CaC12//, 1 g N a O H / / , 0.3 g F e S O 4 / / a n d 0.3 g p o l y a c r y l a m i d e / / g a v e reasonably good clarity without any loss in enzyme activity. It was possible to reduce the turbidity of broth from 11,800 to 17 NTU. In order to get a clear enzyme solution of acceptable quality ( < 1 0 NTU) for easy downstream processing, it was decided to use other flocculants along with the above
mixture. Treatment with gelatin, guar gum, sodium carboxy methyl cellulose and bentonite, individually, along with the above mixture, did not result in any decrease in turbidity (Table 1). However, treatment with sodium alginate resulted in significant reduction in turbidity without any loss in enzyme activity. The clarity of the enzyme solution depended on the concentration of sodium alginate. The turbidity dropped to 6 and 4 NTU on treatment with 1 and 2 g/I of sodium alginate, respectively. At both the concentrations, very big flocs were produced which settled rapidly. However, no flocculation was noticed earlier when the broth was treated with sodium alginate alone (3). Flocculation with sodium alginate in the presence of other agents indicates the combined effects of different coagulating-flocculating agents. Similar results were also obtained when F e S Q and polyacrylamide were eliminated; only CaCI2 NaOH and sodium alginate were used. Sodium alginate was tried in various concentrations ranging from 0.5 to 5 g/I with CaClz/NaOH mixture. Concentrations higher than 5 g/l were not tried because the broth became viscous. It was found that the floc size increased and the turbidity of the broth decreased with increase in sodium alginate concentration from 0.5 to 5 g/l (Table 2). With 5 g sodium alginate//, it was possible to reduce the turbidity to a great extent, up to 1.4 NTU, without any loss in enzyme activity. More than 959/oo of the TABLE 1. Effectof addition of natural flocculatingagents on clarity of broth treateda with CaCIz+ NaOH + FeSO4+ polyacrylamide Flocculating agent No treatment b Treated ~' Gelatin Sodium carboxy methyl cellulose Guar gum Sodium alginate Bentonite
Concentration (g//) -1.0 2.0 1.0 2.0 1.0 2.0 1.0 2.0 1.0 2.0
Turbidity Enzyme activity (NTU) (DUN/ml) 11800 17 17 17 17 17 17 17 6 4 17 17
4128 4129 4127 4130 4128 4128 4125 4130 4130 4130 4130 4130
~' The broth was treated with l0 g CaCljl, 1g NaOH/I, 0.3 g FeSO4/ / and 0.3 g cationic polyacrylamide//. b The original broth, without any type of treatment.
Corresponding author. 284
VOL, 71, 1991 TABLE 2.
NOTES Effect of addition of sodium alginate on clarity of broth a treated with CaC12+NaOH
TABLE 3.
Effect of addition of sodium alginate on filtration performance
Concentration of sodium alginate (g//)
Turbidity (NTU)
Enzyme activity (DUN/ml)
Concentration of sodium alginate (g//)
Filtration efficiency" (~)
Turbidity
0.0 0.5 1.0 2.0 3.0 4.0 5.0
34.0 10.0 7.0 4.5 3.0 2.0 1.4
4128 4130 4129 4131 4130 4129 4133
0.0 0.5 1.0 2.0 3.0 4.0 5.0
92.0 94.5 93.2 94.1 93.8 94.2 95.1
a The broth was treated with 10 g CaCIJl and 1 g NaOH//before addition of sodium alginate.
b a c t e r i a l l o a d was also e l i m i n a t e d . T h e effects o f s o d i u m a l g i n a t e o n f i l t r a t i o n rate a n d efficiency were also s t u d i e d . T h e r a t e o f f i l t r a t i o n i n c r e a s e d with i n c r e a s e o f s o d i u m a l g i n a t e c o n c e n t r a t i o n in the b r o t h ( T a b l e 3). A t a c o n c e n t r a t i o n o f 5 g/l, the filtration r a t e was 163 I / m 2 . h , a n d t h e filtration efficiency a n d the t u r b i d i t y o f b r o t h were 9 5 % a n d 1.4 N T U , respectively. Alt h o u g h a c c e p t a b l e clarity was o b t a i n e d w i t h 0.5 g s o d i u m a l g i n a t e / / , b e t t e r filtration rates w e r e o b t a i n e d at h i g h e r c o n c e n t r a t i o n s o f s o d i u m alginate. T h e f i l t r a t i o n rates are e x p e c t e d t o be i m p r o v e d f u r t h e r in a large-scale o p e r a t i o n w h e r e c r o s s - f l o w m i c r o f i l t r a t i o n s are u s e d . T h e results s h o w t h a t a l m o s t all the c o l l o i d a l m a t t e r can be r e m o v e d easily w i t h o u t a n y loss in e n z y m e activity b y the t r e a t m e n t o f b r o t h w i t h 10 g CaC12//, 1 g N a O H / / a n d 0.5 g s o d i u m a l g i n a t e / / . The authors are grateful to the Director, Thapar Corporate R & D Centre, Patiala, India for permission to publish these results. Tile valuable assistance provided by the Microbial Biotechnology Centre, Department of Biology, University of Waterloo, Canada during the preparation of the manuscript is gratefully acknowledged.
285
(NTU)
Initial flux (l/m 2. h)
Average flux~ (l/m-' •h)
34.0 10.0 7.0 4.5 3.0 2.0 1.4
-97.8 113.4 ---254.7
-54.4 65.2 81.5 97.8 139.8 163.1
~' Filtration efficiency--percentage of enzyme recovery on filtration. Volume of filtrate collected b Average f l u x Time of filtration, filter area
REFERENCES 1. Gasner, L.L. and Wang, D. I. C.: Microbial cell recovery enhancement through flocculation. Biotechnol. Bioeug,, 12, 873887 (1970). 2. Gregory, J,: Fundamentals of flocculation. CRC Critical Reviews Env. Control., 19, 185-230 (1989). 3. Bajpai, P., Neer, J., and Bajpai, P. K.: Clarification of bacterial broth containing high ~-amylase activity. Biotechnol. Technique, 4, 227-232 (1990). 4. Greenberg, A. E., Trussell, R. H., and Clesceri, L. S. (ed.): Standard methods for the examination of water and wastewater, 16th ed. p. 134-139. Amer. Public Health Assoc., New York (1985). 5. Bajpai, P. and Bajpai, P.K.: High temperature alkaline ~ramylase from Bacillus licheniformis TCRDC-B13. Biotechnol. Bioeng., 33, 72-78 (1989). 6. Bajpai, P., Sharma, U., and Bajpai, P. K.: Effect of corn gluten on cr-amylase production by Bacillus licheniformis TCRDC-B 13. Biotech. Appl. Biochem., it, 610-615 (1989).