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Optimal Conditions for Production of Bacterial Amylase
Zb1. Bakt. Abt. II, Bd. 128, S. 483-490 (1973) [Faculty of Agriculture, Cairo University, Giza, and El-Nasr Pharmaceutical Chemicals Company, Cairo, Egypt]
Optimal Conditions for Production of Bacterial Amylase M. Abd EI-Akher, M. A. EI-Leithy, M. K. EI-Marsafy, and S. A. Kassim With 5 figures Summary The optimal conditions for maximal production of a-amylase by a Bacillus subtilis strain in a selected medium, containing defatted rice bran, corn steep liquor, and superphosphate at p II 7.0, were: inoculum 1%; aeration rate 1: 1 (v/v/min.), and an incubation period of 72 hrs. at 30°C. Sperm oil and rhodersil (0.01 %) were good antifoam agents and had no effect on amylase formation. Ammonium sulphate (50% w/v medium) was the best precipitant for preparation of the enzyme product in a powder form. It recovered 98% of the total activity, produced in culture fluids in laboratory experiments. In pilot plant studies, the processing technique adopted recovered 65-70 % of initial activity. The enzyme preparation contained about 63000 pIg. Introduction In a previous study (EL-LEITHY et a1. 1973) attempts were devoted to obtain a natural medium from Egyptian raw materials and some inexpensive industrial byproducts for commercial production of a-amylase by a strain of Bacillus subtilis. The best medium contained defatted rice bran, corn steep liquor, and superphosphate. However, the enzymes, produced in a medium by different strains, are liable to be influenced by a number of factors other than the composition of the medium, such as temperature and time of incubation, aeration, inoculum size etc. The purpose of this investigation is to define the optimal conditions for industrial production of bacterial amylase in both laboratory and pilot plant scale. Materials and Methods The amylase-producing strain (Bacillus subtilis) , which has been used in the previous study, was employed in this investigation, too. The production medium (DRB-CSL) was composed of defatted rice bran 100/0' corn steep liquor 1.5%, Ca superphosphate 0.1%, pH 7.0 in tap water. Shake culture studies were carried out with 70 m1. portions of the medium in 500 m1. Erlenmeyer flasks which were incubated on a rotary shaker at 200 r.p.m. Amylase production in laboratory fermentors was conducted with 11. portions of the medium in 21. glassfermentors. Pilot plant studies were carried out in a fermentor of 300 1. capacity, provided with a suitable stirrer, and fitted with a system of coils for sterilization by steaming, aeration, cooling, and sampling devices. In all experiments the medium was inoculated with 1% of 24 hours' shake culture, grown in wheat bran extract-peptone medium (PELTIER and BEOKORD 1945). Amylase activity was determined by the technique described in the previous paper. Total sugars, total nitrogen, and pH changes were determined according to A.O.A.C. methods (1960).
484
M. Abd EI-Akher, M. A. EI-Leithy, M. K. El-Marsafy, and S. A. Kassim
100001
30 CO ·35 C 9000
Fig. 1 Effect of Incubation Temperature on Amylase Activity
8000
7000 ..;
.....E :::J
>-
I-
;;;
;:
CJ
«
4001
3000
2000
1000
Fig. 2 Effect of Time on Amylase Production
:4
FERMENTATION TIME {hrs.l
t
• • Activity ..... _._._.-c pH 10000 _._..0-'- ._,-e
9000 8000 7000
E .....
6000
9.0 8.5 8.0 7.5
L._._...,-·_·_·.-J>-
7.0 6.5
5000
6.0
4000
I <;'5
§
3000
5.0
«
2000
:>
~ <..l
1000 000
24
48
72
96
120
144
Fermentation time (hr.s,J
168
192
:r:
0.
485
Optimal Conditions for Production of Bacterial Amylase 10000
9000
8000
7000
6000
!
;i
?:
:2
~
5000
4000
3000
2000
Fig. 3 Effect of Inoculum Size on Amylase Production
1000
•
q
n
Fermentation time ChrsJ 10. 1 :1 9000
1.5: 1
8000
0.5: 1
7000
6000
E -. 5000 ::i
>-
:~
~
1.000
3000
2000
Fig. 4 Effect of Aeration on Amylase Production
u
q
Fermentation time ChrsJ
n
486
M. Abd EI-Akher, M. A. El-Leithy, M. K. EI-Marsafy, and S. A. Kassim
Results Amylase production in shake cultures. The optimal conditions for maximum production of a-amylase by B. subtilis in DRB-CSL medium were first assessed in laboratory shakers with respect to incubation temperature, time of fermentation, inoculum size, and effect of additives, i. e., antifoam agents. Incuba tion tempera ture. The maximum amylase activity was produced when the incubation temperature was in the range of 30-35 °C (Fig. 1). Above and below the optimal range the amylase production decreases, However, the rate of increase during the first 48 hrs. of the fermentation period was significantly higher when incubating at 40 or 45°C than at 30-35 °C. Fermentation time. The effect of time of fermentation at the optimum incubation temperature (30°C) is illustrated in Fig. 2. The results show that amylase production increased with time, attaining the peak after 72 hrs. and keeping at that level up to 96 hrs. after which the activity gradually decreased. The pH slightly increased during fermentation. Inoculum size. The results in Fig. 3 show that there was a positive relation between inoculum size and amylase activity during the first 48 hrs. of the fermentation period, but at the end of the 72 hrs. there were no significant differences in the amylase yield. Antifoam agents. Two commercial products, i. e., rhodersil (0.01%) and sperm oil (0.01 %) were tested for their efficiency in foam prevention and their effect on amylase activity. Both materials were quite efficient as antifoam agents and had no inhibitory effect on the amylase production. The sperm oil was chosen for routine use, hecause it is easily obtainable and cheap.
-.E
ACTIVITY TOTAL SLGARS
... '" '"0 ~ .b III
::>
>-
0--._-_ ---0
TOTAL NITROGEN
oil
'c
>
O-----G
pH
i§'"
.l!!
::>
0
~
~I~
10000
9000 9.01 8.5 8000
8.~ 7000[---------o,•••••, 7.5 6000 7.0 5000
'_'_'_.~~.~:.,::_....,.., ---
_--0-----0---
_..0----
·"''::·'''''''-~,O,.C.::::c::::::::.7.CC~'.O,
6.5j4ooo 6.0 3000 5.5 2000 5.0 1000 • 24
48
0,5
4.5
OA5
4D
OA
3.5
0,35
3.0
0.3
•• 2.5
............ ",
72
96
120
144
Fermentation time (hr.sJ
Fig. 5. Biochemical Changes during Fermentation
~
5.0
())5
2D
0.2
15
OJ5
UJ
OJ
05
0.05
487
Optimal Conditions for Production of Bacterial Amylase
Amylase production in laboratory fermentors. The optimal conditions for amylase production and the effect of aeration were verified by the laboratory glass-fermentors. The results in Fig. 4 show clearly the positive relation between aeration and amylase formation. The increase in air volumes (volume medium) minute increased amylase production at different fermentation periods. The optimal aeration rate for maximum activity (9500/-llm!.) was found to be 1: 1 (v/v/min.). The metabolic changes, occurring in the medium, were followed during fermentation in glass-fermentors. Amylase activity, total sugars, total nitrogen, and pH were determined in culture centrifugates every 24 hI's. up to 144 hI's. (Fig. 5). The amylase production curve was similar to that obtained from shake cultures. Three phases could be detectecl, the phase of progressive increase in amylase (24-72 hI's.), the stable or stationary phase (72-96 hI's.), and the decline phase (after 96 hI's.). During fermentation, the pH gradually shifted to the alkaline side, the total sugars decreased as a result of carbohydrate consumption, and so did the total nitrogen. The rate of carbon and nitrogen consumption was at its highest level at the end of the active stage. Separation of the amylase. Ammonium sulphate, sodium chloride, and ethyl alcohol 96 % were investigated as precipitants for amylase separation from culture fluids. After propagating the culture in glass-fermentors, the cells were removed by centrifugation, and the enzyme was precipitated from aliquots of 500 m!. of the supernatant with different compounds. The precipitates were separated by centrifugation, dried overnight in an oven under vacuum at 40°C, weighed, and the amylase activity in each enzyme preparation was determined in /-l/g. From Table 1 it is clearly evident that ammonium sulphate in 50% concentration (wi v medium) was highly efficient for amylase separation. It recovered 98% of the total activity in culture fluids. Ethyl alcohol in a concentration of 2:1 (v/v medium) was less efficient than ammonium sulphate, but definitely better than NaCI which recovered only 5 % of the total activity. However, the use of ethyl alcohol at such concentration is not suitable for large scale production. Table 1. Efficiency of different precipitants in amylase separation from culture fluids Material
-_
..-
----------
Amountl 100 m!. medium
----.
Precipitate*) Weight (g.)
• _ _ _ _ _ _ _ _ _ _ _ _.·H' _ _ _•_
Recovery**) Activity (PIg.)
%
_ _ .. _ _ _
(NH 4h S 04
30 g 40 g 50 g
60 73 75
28000 43200 46000
48 91 98
NaCI
30 g
55
3180
5
30 g
50
3500
5
100 ml 200 ml
57 61
30000 39000
50 68
(NH4hS04
+ NaCl (1: 1)
Ethyl alcohol
*) from 500 ml. fermented liquor containing 7000 /Llml. **) % units recovered in precipitate from total units, present in fermented liquor.
488
M. Abd EI-Akher, M. A. EI-Leithy, M. K. EI-Marsafy, and S. A. Kassim
Pilot plant amylase production. Four experim~IJts were carried out in a pilot plant fermentor of 3001. capacity. The medium (2001.) was prepared in the production fermentor, steemed under normal atmospheric pressure for 10 min. to dissolve the ingredients, pH was adjusted to 7.2 with NaOH 40%, and then sterilized at 121°C for 30 min. After cooling to 30°C, the medium was inoculated with 1 % of 24 hours' seeding culture. Aeration was adjusted at a rate of 1: 1 (v/v/min.). When foam appeared, 0.01 % of sperm oil was added. The amylase activity and pH changes were followed up during fermentation at 30°C, and the results (Table 2) were similar to that obtained from laboratory experiments, indicating the reliability of these data in actual production. Table 2 Amylase production in pilot plant Batch No.
Fermentation period (hI's.)
24 36 48 60 72
1
2
3
4
p,fm1.
pH
film!.
pH
film!.
pH
film!.
pH
2850 3900 5200 7100 9300
7.0 7.2 7.4 7.5 7.7
3100 5000 6900 8350 10000
7.0 7.1 7.3 7.5 7.6
3300 6000 7300 8600 10500
7.1 7.3 7.4 7.5 7.5
4000 5200 6300 7800 10200
7.3 7.4 7.5 7.7 7.8
Final product*) Weight (kg.) 20 Moisture (%) 12 pH (in 1 % solution) 6.5 Activity (fI!g.) 65000 Activity yield %**)
70
21 13 7.1 63000 68
22 11
6.9 62000 66
21 14 6.6 61500 65
*) from 200 1. fermented liquor (72 hI'S.). **) % units recovered in final product from total units, present in 72 hI'S. fermented liquor. The enzyme product was prepared as follows: after cooling the culture fluid to 15°C, the cells and debris were removed by a Delaval centrifuge at 4500 r.p.m., and ammonium sulphate was added gradually while stirring for 1 hr. to give a concentration of 50 %. The precipitate, separated by centrifugation, was placed on trays in thin layers and dried in an aerated room at about 40°C. The weight of the final product, obtained from 2001. fermented liquor, and its amylase activity (p,/g.) arc recorded in Table 2. The processing technique recovered about 63 % of the total activity in culture fluids. The final product contained about 63000/1/g. with a moisture content of about 10-15 %, and the pH of 1 % solution ranged from 6.5 -7.0.
Optimal Conditions for Production of I3acterial Amylase
489
Discussion The present investigation describes, in part, the environmental conditions which favour the production of a-amylase by a Bacillus subtilis strain in submerged cultures. Maximum amylase production was achieved at the optimum growth temperature of Bacillus subtilis strains (30-35°C), but the incubation period, needed for maximum activity (72 hI's.), may differ from that reported with other strains (WALLERSTEIN 1939; WALDMAN 1942; BECKaRD et al. 1945, 1946; KNEEN and BECKaRD 1946; MAHMOUD et al. 1967). Differences may be due to the method of cultivation and/or strain variability. The concentration of inoculum, in agreement with BECKaRD et al. (1945), did not appear to be a critical factor for amylase production. The increase in inoculum size did not reveal significant differences in amylase yield at the end of the fermentation. MAHMOUD et al. (1967), however, observed a positive relation between the size of inoculum and the amylolytic activity in 48 hI's.' static cultures, but this conclusion, in fact, does not contrast with the present work, since during the first 48 hI's. of fermentation in submerged cultures a positive correlation was likewise recorded. Aeration, on the other hand, had an appreciable influence on amylase formation. The most favourable aeration rate in glass-fermentors was 1: 1 (v/v/min.). The importance of aeration has been reported by earlier investigators. At optimal conditions, the amylase production curve during fermentation tended to be in parallel with the growth phases of the organism, and confirmed the findings of COLEMAN and ELLIOT (1962). Maximum production was attained at the end of the logarithmic phase where the rate of carbon and nitrogen consumption was at its highest level. The amylase yield remained stable at a maximum during the stationary phase of growth, and with prolonged incubation, a slow decline occurred, perhaps due to slight inactivation. The maintenance of a stable activity for over 24 hI's. (72-96 hI's.) at maximum yield is of particular importance in the industrial technology of amylase production. It restricts considerably the chances of obtaining disappointments, due to a late decision to terminate the fermentation process, and allows a comfortable period for easy preparation of the succeeding handling operations, e. g., filtration, precipitation etc. This contrasts sharply with certain antibiotic fermentation, where the termination is very initial and highly impredictable. The results obtained from the pilot plant define the basis for commercial production of a-amylase by the strain investigated. The methods employed for processing the fermented liquor into a powdered enzyme product recovered about 63% of the total activity in culture fluids, though in laboratory experiments a recovery of 95 % was reached. However, losses in recovery in the pilot plant could be reduced by better handling and shortening of the pipe lines. Anyhow, the process in the whole is successful and economic, and the cost picture of the final product is cheap. Zusammenfassung Die optimalen Bedingungen fiir eine maximale a-Amylaseproduktion durch einen Stamm von Bacillus subtilis in einem Medium, das entfettete Reiskleie, Getreide-Extrakt und Superphosphat bei einem pH von 7,0 entllieh, waren folgende: Inoculum 1 % , Beliiftung 1: 1 (v/v/Min.), Inkubationszeit 72 Std. bei 30°C. Walratiil und Rhodersil waren gute Antischaumungsmittel, die keine vYirkung auf die Amylasebildung hatten. Ammoniumsulfat war das beste Fallungsmittel ZUI' Aufbercitung des Enzymprodukts in Pulverform. Bei Laborexperi-
490
M. Ab d EI-Akher ... , Optimal Conditions for Production of Bacterial Amylase
menten beuug die aus den Kultur£lussigkeiten erzielte Ruckgewinnung 98 % del' gesamten Aktivitat. Bei Studien in Versuchsanlagen wurden mit del' angewendeten Herstellungstechnik 65-70% del' Anfangsaktivitat zuruckgewonnen. Die Enzymbereitung enthielt etwa 63000 Einheiten/g.
Literature A.O.A.C.: Official methods of analysis. 9th ed. Benjamin Franklin Station, Washington 4, D.C. 1960. - BEOKORD, L. D., KNEEN, E., and LEWIS, K. H.: Bacterial amylases j production on wheat bran. Ind. Eng. Chern. 37 (1945), 692-696. - BEOKORD, 1. D., PELTIER, G. L., and KNEEN, E.: Bacterial amylases j production in thin stillage. Ind. Eng. Chern. 38 (1946), 232238. - COLEMAN, G., and ELLIOT, W. H.: a-Amylase formation of Bacillus subtilis. Biochem. J. 83 (1962), 256-263. - EL-LEITHY, M. A., IBRAIDM, A. Y., EL-MARSAFY, M. K., and KASSIM, S. A.: Production of bacterial amylase on some commercial materials. Zbl. Bakt. II 128 (1973), 473-482. - KNEEN, E., and BECKORD, 1. D.: Quantity and quality of amylase, produced by val'ious bacterial isolates. Arch. Biochem.10 (1946), 41-54. - MAHMOUD, S. A. Z., TAHA, S. M., and ATTIA, R. M.: Studies of some factors affecting bacterial amylase production. J. Bot. (U.A.R.) 10 (1967), 33-41. - PELTIER, G. L., and BEoKoRD, L. D.: Sources of amylaseproducing bacteria. J. Bact. 50 (1945),711-714. - WALDMAN, R.: Production of amylolytic enzymes by cultivating bacteria. U.S.-Patent 2,302,379 (1942). - WALLERSTEIN, 1.: Enzyme preparation derived from micro-organisms. Their commercial production and industrial application. Ind. Eng. Chern. 31 (1939), 1218.
Authors' addresses: M. Abd EI-Akherand Prof. Dr. M. A. EI-Leithy, Faculty of Agriculture, Cairo University, Giza, and M. K. EI-Marsafy and S. A. Kassim, El-Nasr Pharmaceutical Chemicals Company, Cairo (Egypt).
Optimal Conditions for Production of Bacterial Amylase M. Abd EI-Akher, M. A. EI-Leithy, M. K. EI-Marsafy, and S. A. Kassim With 5 figures Summary The optimal conditions for maximal production of a-amylase by a Bacillus subtilis strain in a selected medium, containing defatted rice bran, corn steep liquor, and superphosphate at p II 7.0, were: inoculum 1%; aeration rate 1: 1 (v/v/min.), and an incubation period of 72 hrs. at 30°C. Sperm oil and rhodersil (0.01 %) were good antifoam agents and had no effect on amylase formation. Ammonium sulphate (50% w/v medium) was the best precipitant for preparation of the enzyme product in a powder form. It recovered 98% of the total activity, produced in culture fluids in laboratory experiments. In pilot plant studies, the processing technique adopted recovered 65-70 % of initial activity. The enzyme preparation contained about 63000 pIg. Introduction In a previous study (EL-LEITHY et a1. 1973) attempts were devoted to obtain a natural medium from Egyptian raw materials and some inexpensive industrial byproducts for commercial production of a-amylase by a strain of Bacillus subtilis. The best medium contained defatted rice bran, corn steep liquor, and superphosphate. However, the enzymes, produced in a medium by different strains, are liable to be influenced by a number of factors other than the composition of the medium, such as temperature and time of incubation, aeration, inoculum size etc. The purpose of this investigation is to define the optimal conditions for industrial production of bacterial amylase in both laboratory and pilot plant scale. Materials and Methods The amylase-producing strain (Bacillus subtilis) , which has been used in the previous study, was employed in this investigation, too. The production medium (DRB-CSL) was composed of defatted rice bran 100/0' corn steep liquor 1.5%, Ca superphosphate 0.1%, pH 7.0 in tap water. Shake culture studies were carried out with 70 m1. portions of the medium in 500 m1. Erlenmeyer flasks which were incubated on a rotary shaker at 200 r.p.m. Amylase production in laboratory fermentors was conducted with 11. portions of the medium in 21. glassfermentors. Pilot plant studies were carried out in a fermentor of 300 1. capacity, provided with a suitable stirrer, and fitted with a system of coils for sterilization by steaming, aeration, cooling, and sampling devices. In all experiments the medium was inoculated with 1% of 24 hours' shake culture, grown in wheat bran extract-peptone medium (PELTIER and BEOKORD 1945). Amylase activity was determined by the technique described in the previous paper. Total sugars, total nitrogen, and pH changes were determined according to A.O.A.C. methods (1960).
484
M. Abd EI-Akher, M. A. EI-Leithy, M. K. El-Marsafy, and S. A. Kassim
100001
30 CO ·35 C 9000
Fig. 1 Effect of Incubation Temperature on Amylase Activity
8000
7000 ..;
.....E :::J
>-
I-
;;;
;:
CJ
«
4001
3000
2000
1000
Fig. 2 Effect of Time on Amylase Production
:4
FERMENTATION TIME {hrs.l
t
• • Activity ..... _._._.-c pH 10000 _._..0-'- ._,-e
9000 8000 7000
E .....
6000
9.0 8.5 8.0 7.5
L._._...,-·_·_·.-J>-
7.0 6.5
5000
6.0
4000
I <;'5
§
3000
5.0
«
2000
:>
~ <..l
1000 000
24
48
72
96
120
144
Fermentation time (hr.s,J
168
192
:r:
0.
485
Optimal Conditions for Production of Bacterial Amylase 10000
9000
8000
7000
6000
!
;i
?:
:2
~
5000
4000
3000
2000
Fig. 3 Effect of Inoculum Size on Amylase Production
1000
•
q
n
Fermentation time ChrsJ 10. 1 :1 9000
1.5: 1
8000
0.5: 1
7000
6000
E -. 5000 ::i
>-
:~
~
1.000
3000
2000
Fig. 4 Effect of Aeration on Amylase Production
u
q
Fermentation time ChrsJ
n
486
M. Abd EI-Akher, M. A. El-Leithy, M. K. EI-Marsafy, and S. A. Kassim
Results Amylase production in shake cultures. The optimal conditions for maximum production of a-amylase by B. subtilis in DRB-CSL medium were first assessed in laboratory shakers with respect to incubation temperature, time of fermentation, inoculum size, and effect of additives, i. e., antifoam agents. Incuba tion tempera ture. The maximum amylase activity was produced when the incubation temperature was in the range of 30-35 °C (Fig. 1). Above and below the optimal range the amylase production decreases, However, the rate of increase during the first 48 hrs. of the fermentation period was significantly higher when incubating at 40 or 45°C than at 30-35 °C. Fermentation time. The effect of time of fermentation at the optimum incubation temperature (30°C) is illustrated in Fig. 2. The results show that amylase production increased with time, attaining the peak after 72 hrs. and keeping at that level up to 96 hrs. after which the activity gradually decreased. The pH slightly increased during fermentation. Inoculum size. The results in Fig. 3 show that there was a positive relation between inoculum size and amylase activity during the first 48 hrs. of the fermentation period, but at the end of the 72 hrs. there were no significant differences in the amylase yield. Antifoam agents. Two commercial products, i. e., rhodersil (0.01%) and sperm oil (0.01 %) were tested for their efficiency in foam prevention and their effect on amylase activity. Both materials were quite efficient as antifoam agents and had no inhibitory effect on the amylase production. The sperm oil was chosen for routine use, hecause it is easily obtainable and cheap.
-.E
ACTIVITY TOTAL SLGARS
... '" '"0 ~ .b III
::>
>-
0--._-_ ---0
TOTAL NITROGEN
oil
'c
>
O-----G
pH
i§'"
.l!!
::>
0
~
~I~
10000
9000 9.01 8.5 8000
8.~ 7000[---------o,•••••, 7.5 6000 7.0 5000
'_'_'_.~~.~:.,::_....,.., ---
_--0-----0---
_..0----
·"''::·'''''''-~,O,.C.::::c::::::::.7.CC~'.O,
6.5j4ooo 6.0 3000 5.5 2000 5.0 1000 • 24
48
0,5
4.5
OA5
4D
OA
3.5
0,35
3.0
0.3
•• 2.5
............ ",
72
96
120
144
Fermentation time (hr.sJ
Fig. 5. Biochemical Changes during Fermentation
~
5.0
())5
2D
0.2
15
OJ5
UJ
OJ
05
0.05
487
Optimal Conditions for Production of Bacterial Amylase
Amylase production in laboratory fermentors. The optimal conditions for amylase production and the effect of aeration were verified by the laboratory glass-fermentors. The results in Fig. 4 show clearly the positive relation between aeration and amylase formation. The increase in air volumes (volume medium) minute increased amylase production at different fermentation periods. The optimal aeration rate for maximum activity (9500/-llm!.) was found to be 1: 1 (v/v/min.). The metabolic changes, occurring in the medium, were followed during fermentation in glass-fermentors. Amylase activity, total sugars, total nitrogen, and pH were determined in culture centrifugates every 24 hI's. up to 144 hI's. (Fig. 5). The amylase production curve was similar to that obtained from shake cultures. Three phases could be detectecl, the phase of progressive increase in amylase (24-72 hI's.), the stable or stationary phase (72-96 hI's.), and the decline phase (after 96 hI's.). During fermentation, the pH gradually shifted to the alkaline side, the total sugars decreased as a result of carbohydrate consumption, and so did the total nitrogen. The rate of carbon and nitrogen consumption was at its highest level at the end of the active stage. Separation of the amylase. Ammonium sulphate, sodium chloride, and ethyl alcohol 96 % were investigated as precipitants for amylase separation from culture fluids. After propagating the culture in glass-fermentors, the cells were removed by centrifugation, and the enzyme was precipitated from aliquots of 500 m!. of the supernatant with different compounds. The precipitates were separated by centrifugation, dried overnight in an oven under vacuum at 40°C, weighed, and the amylase activity in each enzyme preparation was determined in /-l/g. From Table 1 it is clearly evident that ammonium sulphate in 50% concentration (wi v medium) was highly efficient for amylase separation. It recovered 98% of the total activity in culture fluids. Ethyl alcohol in a concentration of 2:1 (v/v medium) was less efficient than ammonium sulphate, but definitely better than NaCI which recovered only 5 % of the total activity. However, the use of ethyl alcohol at such concentration is not suitable for large scale production. Table 1. Efficiency of different precipitants in amylase separation from culture fluids Material
-_
..-
----------
Amountl 100 m!. medium
----.
Precipitate*) Weight (g.)
• _ _ _ _ _ _ _ _ _ _ _ _.·H' _ _ _•_
Recovery**) Activity (PIg.)
%
_ _ .. _ _ _
(NH 4h S 04
30 g 40 g 50 g
60 73 75
28000 43200 46000
48 91 98
NaCI
30 g
55
3180
5
30 g
50
3500
5
100 ml 200 ml
57 61
30000 39000
50 68
(NH4hS04
+ NaCl (1: 1)
Ethyl alcohol
*) from 500 ml. fermented liquor containing 7000 /Llml. **) % units recovered in precipitate from total units, present in fermented liquor.
488
M. Abd EI-Akher, M. A. EI-Leithy, M. K. EI-Marsafy, and S. A. Kassim
Pilot plant amylase production. Four experim~IJts were carried out in a pilot plant fermentor of 3001. capacity. The medium (2001.) was prepared in the production fermentor, steemed under normal atmospheric pressure for 10 min. to dissolve the ingredients, pH was adjusted to 7.2 with NaOH 40%, and then sterilized at 121°C for 30 min. After cooling to 30°C, the medium was inoculated with 1 % of 24 hours' seeding culture. Aeration was adjusted at a rate of 1: 1 (v/v/min.). When foam appeared, 0.01 % of sperm oil was added. The amylase activity and pH changes were followed up during fermentation at 30°C, and the results (Table 2) were similar to that obtained from laboratory experiments, indicating the reliability of these data in actual production. Table 2 Amylase production in pilot plant Batch No.
Fermentation period (hI's.)
24 36 48 60 72
1
2
3
4
p,fm1.
pH
film!.
pH
film!.
pH
film!.
pH
2850 3900 5200 7100 9300
7.0 7.2 7.4 7.5 7.7
3100 5000 6900 8350 10000
7.0 7.1 7.3 7.5 7.6
3300 6000 7300 8600 10500
7.1 7.3 7.4 7.5 7.5
4000 5200 6300 7800 10200
7.3 7.4 7.5 7.7 7.8
Final product*) Weight (kg.) 20 Moisture (%) 12 pH (in 1 % solution) 6.5 Activity (fI!g.) 65000 Activity yield %**)
70
21 13 7.1 63000 68
22 11
6.9 62000 66
21 14 6.6 61500 65
*) from 200 1. fermented liquor (72 hI'S.). **) % units recovered in final product from total units, present in 72 hI'S. fermented liquor. The enzyme product was prepared as follows: after cooling the culture fluid to 15°C, the cells and debris were removed by a Delaval centrifuge at 4500 r.p.m., and ammonium sulphate was added gradually while stirring for 1 hr. to give a concentration of 50 %. The precipitate, separated by centrifugation, was placed on trays in thin layers and dried in an aerated room at about 40°C. The weight of the final product, obtained from 2001. fermented liquor, and its amylase activity (p,/g.) arc recorded in Table 2. The processing technique recovered about 63 % of the total activity in culture fluids. The final product contained about 63000/1/g. with a moisture content of about 10-15 %, and the pH of 1 % solution ranged from 6.5 -7.0.
Optimal Conditions for Production of I3acterial Amylase
489
Discussion The present investigation describes, in part, the environmental conditions which favour the production of a-amylase by a Bacillus subtilis strain in submerged cultures. Maximum amylase production was achieved at the optimum growth temperature of Bacillus subtilis strains (30-35°C), but the incubation period, needed for maximum activity (72 hI's.), may differ from that reported with other strains (WALLERSTEIN 1939; WALDMAN 1942; BECKaRD et al. 1945, 1946; KNEEN and BECKaRD 1946; MAHMOUD et al. 1967). Differences may be due to the method of cultivation and/or strain variability. The concentration of inoculum, in agreement with BECKaRD et al. (1945), did not appear to be a critical factor for amylase production. The increase in inoculum size did not reveal significant differences in amylase yield at the end of the fermentation. MAHMOUD et al. (1967), however, observed a positive relation between the size of inoculum and the amylolytic activity in 48 hI's.' static cultures, but this conclusion, in fact, does not contrast with the present work, since during the first 48 hI's. of fermentation in submerged cultures a positive correlation was likewise recorded. Aeration, on the other hand, had an appreciable influence on amylase formation. The most favourable aeration rate in glass-fermentors was 1: 1 (v/v/min.). The importance of aeration has been reported by earlier investigators. At optimal conditions, the amylase production curve during fermentation tended to be in parallel with the growth phases of the organism, and confirmed the findings of COLEMAN and ELLIOT (1962). Maximum production was attained at the end of the logarithmic phase where the rate of carbon and nitrogen consumption was at its highest level. The amylase yield remained stable at a maximum during the stationary phase of growth, and with prolonged incubation, a slow decline occurred, perhaps due to slight inactivation. The maintenance of a stable activity for over 24 hI's. (72-96 hI's.) at maximum yield is of particular importance in the industrial technology of amylase production. It restricts considerably the chances of obtaining disappointments, due to a late decision to terminate the fermentation process, and allows a comfortable period for easy preparation of the succeeding handling operations, e. g., filtration, precipitation etc. This contrasts sharply with certain antibiotic fermentation, where the termination is very initial and highly impredictable. The results obtained from the pilot plant define the basis for commercial production of a-amylase by the strain investigated. The methods employed for processing the fermented liquor into a powdered enzyme product recovered about 63% of the total activity in culture fluids, though in laboratory experiments a recovery of 95 % was reached. However, losses in recovery in the pilot plant could be reduced by better handling and shortening of the pipe lines. Anyhow, the process in the whole is successful and economic, and the cost picture of the final product is cheap. Zusammenfassung Die optimalen Bedingungen fiir eine maximale a-Amylaseproduktion durch einen Stamm von Bacillus subtilis in einem Medium, das entfettete Reiskleie, Getreide-Extrakt und Superphosphat bei einem pH von 7,0 entllieh, waren folgende: Inoculum 1 % , Beliiftung 1: 1 (v/v/Min.), Inkubationszeit 72 Std. bei 30°C. Walratiil und Rhodersil waren gute Antischaumungsmittel, die keine vYirkung auf die Amylasebildung hatten. Ammoniumsulfat war das beste Fallungsmittel ZUI' Aufbercitung des Enzymprodukts in Pulverform. Bei Laborexperi-
490
M. Ab d EI-Akher ... , Optimal Conditions for Production of Bacterial Amylase
menten beuug die aus den Kultur£lussigkeiten erzielte Ruckgewinnung 98 % del' gesamten Aktivitat. Bei Studien in Versuchsanlagen wurden mit del' angewendeten Herstellungstechnik 65-70% del' Anfangsaktivitat zuruckgewonnen. Die Enzymbereitung enthielt etwa 63000 Einheiten/g.
Literature A.O.A.C.: Official methods of analysis. 9th ed. Benjamin Franklin Station, Washington 4, D.C. 1960. - BEOKORD, L. D., KNEEN, E., and LEWIS, K. H.: Bacterial amylases j production on wheat bran. Ind. Eng. Chern. 37 (1945), 692-696. - BEOKORD, 1. D., PELTIER, G. L., and KNEEN, E.: Bacterial amylases j production in thin stillage. Ind. Eng. Chern. 38 (1946), 232238. - COLEMAN, G., and ELLIOT, W. H.: a-Amylase formation of Bacillus subtilis. Biochem. J. 83 (1962), 256-263. - EL-LEITHY, M. A., IBRAIDM, A. Y., EL-MARSAFY, M. K., and KASSIM, S. A.: Production of bacterial amylase on some commercial materials. Zbl. Bakt. II 128 (1973), 473-482. - KNEEN, E., and BECKORD, 1. D.: Quantity and quality of amylase, produced by val'ious bacterial isolates. Arch. Biochem.10 (1946), 41-54. - MAHMOUD, S. A. Z., TAHA, S. M., and ATTIA, R. M.: Studies of some factors affecting bacterial amylase production. J. Bot. (U.A.R.) 10 (1967), 33-41. - PELTIER, G. L., and BEoKoRD, L. D.: Sources of amylaseproducing bacteria. J. Bact. 50 (1945),711-714. - WALDMAN, R.: Production of amylolytic enzymes by cultivating bacteria. U.S.-Patent 2,302,379 (1942). - WALLERSTEIN, 1.: Enzyme preparation derived from micro-organisms. Their commercial production and industrial application. Ind. Eng. Chern. 31 (1939), 1218.
Authors' addresses: M. Abd EI-Akherand Prof. Dr. M. A. EI-Leithy, Faculty of Agriculture, Cairo University, Giza, and M. K. EI-Marsafy and S. A. Kassim, El-Nasr Pharmaceutical Chemicals Company, Cairo (Egypt).