Influence of the immobilization conditions on the efficiency of α-amylase production by Bacillus licheniformis

Process Biochemistry Vol. 31, No. 3, pp. 229-234, 1996 Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0032-9592/96 S 15.00 + (I.00 ELSEVIER

0032-9592(95)00052-6

Influence of the Immobilization Conditions on the Efficiency of a-Amylase Production by Bacillus

licheniformis E. Dobreva, V. Ivanova,* A. Tonkova & E. Radulova Institute of Microbiology,BulgarianAcademyof Sciences,26 AcademicianG. Bonchev,1113 Sofia,Bulgaria (Received 17 March 1995; accepted 13 June 1995)

Bacillus licheniformis 44MB82-A cells were immobilized in alginate and agar gels. The optimal immobilization parameters (gel concentration, initial cell quantity, biomass age, bead size and solidification prolongation) were determined. The immobilization procedure was most effective at a gel concentration of 4% using cells from a 12 h culture. The optimal initial cell quantity was found to be 0"6-3"0% in agar gel and 0"4% in Ca-alginate gel with bead sizes of 3"Oand 5"0 ram, respectively. A n enzyme yieM of 1100 U ml- 1 culture medium was reached in batch fermentation with agar-entrapped cells under optimal conditions. This activity represented 135% of the corresponding yieM obtained with free cells. Significant increases (2"2-fold) in the enzyme yields in the fourth cycle of repeatedbatch runs with cells entrapped in agar gel pellets with bead size 5"0 mm was observed.

subtilis 5,6,11 and Bacillus amyloliquefaciens. 2, 4,12 However, to our knowledge there are only a few reports on the immobilization of Bacillus licheniformis c e l l s . 3,9, 10 The aim of the present study was to choose optimal conditions for immobilization of growing cells of Bacillus licheniformis 44MB82-A in alginate and agar gels and to evaluate the immobilized biocatalysts in repeated batch fermentations for production of thermostable a-amylase.

INTRODUCTION The possibilities of producing thermostable 1-3 and thermolabile4-6 a-amylases with immobilized Bacillus cells have been investigated. These enzymes are widely used industrially for starch liquefaction. The most frequently used immobilization method is entrapment in gel matrices. 2,5,7,8 Chemical binding on polymer membranes 3,9 and silica carriers 1° have also been studied. The considerable diffusional limitations observed in the biosynthesis of a-amylase 7 can be reduced by application of suitable carriers, microorganisms plus immobilization at optimal conditions. The most extensively studied a-amylase producers in immobilized form are Bacillus

MATERIALS AND METHODS Microbial strain and growth conditions Bacillus licheniformis 44MB82-A, an active producer of thermostable a-amylase, was

*To whomcorrespondence should be addressed. 229

230

E. Dobrevaet al.

described previously.13 Bacteria were cultivated in nutrient broth supplemented with 1.0% (w/v) soluble starch at 35°C on a rotary shaker at 240 rpm and harvested by centrifugation (3000 g, 20 min, 4°C) after 12 or 36 h of growth. This biomass was used both for immobilization and in experiments with free cells after washing with saline solution and recentrifugation. The inoculum concentration in the controls was the same as in the corresponding biocatalysts. The fermentation medium contained (in %, w/v): glucose, 6-0; beef extract, 1.5 (Lab-Lemco powder, Oxoid, Basingstoke, Hampshire, UK); peptone, 1.5 (Oxoid); K2HPO 4, 1.04; comsteep liquor, 0-66; CaC12, 0.11; pH 7-5. Experiments with cells immobilized by ionotropic gelation and the corresponding controls were performed in media without K2HPO 4 to avoid the instability of Ca-alginates in the presence of phosphates. Batch fermentations with immobilized and free cells were carried out over 144 h at 35°C on a rotary shaker at 220 rpm. Repeated-batch fermentations were performed with washed biocatalysts from the previous cycle. The duration of each cycle was 120 h.

Immobilization conditions Sodium alginate (LS 20/60, Protan, Drammen, Norway) and agar (Fluka Chemie AG, Buchs, Switzerland) were used at concentrations in the range 2.0-5.0% w/v. Wet cells (0.2-1-6 g) were mixed with 50 ml of each gel at ambient temperature (alginate) or at 50°C (agar). The slurry (15 ml) was made into spherical beads of 3.0 or 5.0 mm in diameter by dropping into sterile 2.5% (w/v) CaC12 solution ~4and sunflower oil, 15 respectively. The biocatalysts were solidified in these solutions for 30 min (agar), or 3 or 24 h (alginate) and then the gel beads were transferred into 50 ml of fermentation medium after flushing with sterile water. Analytical methods a-Amylase activity in the medium was measured by a starch-iodine method using 1.0% (w/v) soluble potato starch as a substrate (Lintner starch, Serve Feinbiochemica, Heidelberg, Germany), dissolved in 0-066 i phosphate buffer, pH 6"5) 6 One unit of activity was defined as the amount of enzyme producing one microequivalent of anhydrous glucose (0-162 mg) per rain from soluble starch at 30°C.

The concentration of free and released cells was determined by absorbance at 650 nm.

RESULTS

Optimization of immobilization in Ca-alginate The effects of different immobilization parameters (biomass age, initial cell quantity, gel concentration, bead size and solidification time) on the amylase yields are shown in Fig. 1. The course of a-amylase biosynthesis from biocatalysts containing immobilized cells of different ages (12 and 36 h, Fig. I(A)) was similar and maximal amylase activity was established after 144 h of cultivation. The optimal initial biomass concentration was investigated by preparing beads containing 0.4-3-2% cells (wet wt). Initial cell loading (ICL) did not exceed 0.4% w/v. At higher initial biomass concentrations cell growth in the gel beads was probably retarded as a decrease in enzyme secretion was observed (Fig. I(B)). Gel concentrations from 3"0 to 5"0% were tested and highest a-amylase activity was obtained from Bacillus licheniformis entrapped in 4% alginate gels. The pellet durability was improved at gel concentrations > 4"0%, but the diffusional limitations, due to strong gel consistency, decreased the enzyme yield (Fig. 1(C)). The effect of bead size on the fermentation was tested under the optimalized experimental conditions discussed earlier. Bead size had a significant influence on amylase biosynthesis (Fig. I(D)). At a pellet size of 3.0 mm, the amylase yield was low up to 96 h of fermentation. A decrease in amylase yield was also observed after 120 h of cultivation. In order to analyse the influence of the biocatalyst solidification on amylase biosynthesis, experiments were carried out with beads, obtained under optimal conditions and solidified for 3 or 24 h in 2.5% CaC12 solution (Fig. I(E)). The prolongation of the solidification process led to significant increases in amylase yield at all stages of the process. This was probably due to improvement of the gel solidity and was connected with the consequent decrease of cell release from the beads. Optimization of immobilization in agar The age of the immobilized cells affected amylase biosynthesis and its increase led to a lowering of amylase yield (Fig. 2(A)). The effect of the initial

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cell biomass concentration was also studied (Fig. 2(B)) and the biocatalysts produced the same enzyme quantity independently of the initial cell loading (ICL) in the range of 0"6-3"0% (wet wt) cells in the gel. A significant influence on the enzyme yield was observed when different gel concentrations were tested (Fig. 2(C)). While at higher gel concentrations the pellet consistency was also higher and therefore substrate mass-transfer limitations could be expected, amylase biosynthesis was improved. A gel concentration of 4"0% provided an enzyme yield which was approximately 35%

higher than that obtained from cells immobilized in 3"0% agar. Significant differences in the quantifies of cells released from the pellets were not found. Further experiments were carried out with 4"0% agar, since pellet formation was hindered at gel concentrations higher than 4%. In contrast to results obtained with Ca-alginate, immobilization in small pellets (3 mm) was more effective with agar. Generally, the a-amylase activities obtained were higher in cells immobilized in agar gel due to the presence of KEHPO 4 in the fermentation medium. 17

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Optimal immobilization conditions and yield of amylase

Table 1. Optimal conditions for immobilizationof Bacillus

The optimal conditions for immobilization of Bacillus licheniformis 44MB82-A cells for maximal production of thermostable a-amylase are compared in Table 1. It is evident that the optimization of these parameters is an important factor for the effective use of the immobilized biocatalysts. Only the age of the entrapped cells was equivalent and dependent on the strain of Bacillus licheniformis. The gel and biomass concentrations and the bead size have values determined by the properties of the gels employed. It is therefore important to define these parameters before each application of immobilized cells in repeated-batch or continuous fermentations. The a-amylase production by alginate and agar-entrapped cells under optimal conditions is compared in Fig. 3. Amylase activity during 72-144 h of fermentation was 35-45% higher in agar-entrapped cells than in the free cells (control). The activity of alginate-entrapped cells was about 10% higher than the control after 144 h of fermentation.

Conditions

licheniformis 44MB82-Ain calciumalginateand agar gels

Gel concentration(%) Biomassconcentration (g wet wt in 100 ml of 4.0% gel) Bead size (mm) Age of the cells (h) Solidificationtime (h)

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Operational stability of the biocatalysts The possibility of using immobilized ceils of Bacillus licheniformis 44MB82-A (bead size 5"0 nun) repeatedly for a-amylase production was studied over a period of 30 days (five cycles) (Fig. 4). There was a significant increase (2.2-fold) of enzyme yield from cells immobilized in agar gels after three cycles. This increased production was retained up to the last cycle studied (the fifth). A similar but lesser effect was also established for the alginate-immobilized biocatalyst (15-20% increase in yield).

a-Amylase production by Bacillus licheniformis

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233

agar gel. In contrast, in Ca-alginate gel, the optimal bead size was 5 mm. The dependence of enzyme yield on initial cell loading (ICL) is subject to conflicting reports. 7,s In the case of alginate-entrapped cells, enzyme yield decreased at high initial cell density. The amylase yield from agar immobilized cells was 35-45% higher than the yield from the free cell control. These results are similar to observations 5,12 from other reports with free and immobilized cells in batch and continuous fermentations. Moreover, the high operational stability of this biocatalyst indicated the possibility of the application of agar-entrapped cells of Bacillus licheniformis 44MB82-A in batch, repeatedbatch and continuous processes for the production of thermostable a-amylase.

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ACKNOWLEDGEMENT

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This work was supported by grant K24 of the National Scientific Foundation, Bulgarian Ministry of Science and Education.

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REFERENCES

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DISCUSSION Gel concentration has a significant effect on enzyme production. In experiments with alginate, pellet solidity was improved at a gel concentration of > 4.0%, but diffusional limitations decreased enzyme yield. These results are in agreement with other investigations 9,12 and it as been shown that at higher gel concentrations the rate of substrate (starch) mass transfer and the enzyme yield decreasedJ 2 Solidification of the alginate pellet improved the enzyme yield, presumably owing to an improvement in gel consistency and durability and the consequent decrease of the cell release from the beadsJ 8 Similar data were obtained in our experiments with alginate-entrapped cells. Amylase biosynthesis is also dependent on bead size, the immobilization in small pellets (3 mm) being more effective in experiments with

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ionotropic gelation. In Enzyme Technology, ed. R. M. Lafferty. Springer, Berlin, 1983, pp. 219-34. Nilsson, K., Birnbaum, S., Flygare, S., Linse, L., Sehr6der, U., Jeppson, U., Larsson, O.-O., Mosach, K. & Brodelius, P., A general method for the immobilization of cells with preserved viability. Eur. J. Appl. Microbiol. Biotechnol., 17 (1983) 319-26. Pantchev, C., Klenz, G. & Hiifner, B., Vergleiehende Characterisierung von alpha-Amylasepraparaten. Lebensmittelind., 28 ( 1981 ) 71-4. Emanuilova, E., Dobreva, E., Tonkova, A., Kambourova, M. & Kosturkova, P., Phosphate effects on production and thermostability of Bacillus licheniforrnis 44MB82-A a-amylase. Biotechnol. BioEd, B (1992) 38-40. Vorlop, K. D., Entwicklung von Verfahren zur Polymerfixierung von Mikroorganismen und Anvendung der Biokatalysatoren zur Spaltung von Penicillin G und Synthese von L-Tryptophan. PhD Thesis, TU Braunschweig, 1984.