Folia Microbiol.37 (1), 17-23 (1992)

Cell Growth and a-Amylase Production Characteristics of Bacillus subtilis K. DERCOV,~, J. AUGUSTiN, and D. KRAJt~OVA Department of Biochemical Technology,Facultyof Chemical Technology, Slovak Technical University, 812 37 Bratislava, Czechoslovakia Received May 24, 1991

ABSTRACT. Growth,differential rate of a-amylase synthesisand production characteristics of Bacillus subtilis DP 1 (isolate from starch materials) in comparisonwith 10 Bacillus strains w e r e examined in batch fermentation. The effect of the carbon and nitrogen sourcewas evaluatedwith regard to cell growthand enzymeproduction. The pH optimum of enzymeactivitywas 6.5 and temperature optimum60~ Induction and repression are known to be active in a-amylase (EC 3.2.1.1) synthesis (Priest 1977; Toda 1981). The synthesis of extracellular a-amylase, as well as alkaline proteinase, is initiated by limitation of C or N sources and controlled by catabolite repression (Liebs et al. 1988). Some strains of Bacillus produce a-amylase during the growth phase and some produce it after the growth phase. It is not clear from the literature whether the enzyme production pattern depends exclusively on the strain, environmental conditions, or both. During exponential phase only a little amount of a-amylase is excreted, sufficient for starch utilization as an energy source. At the end of the exponential phase of growth the production of this enzyme substantialy increases (Yoo et al. 1988). The a-amylases produced by different Bacillus strains differ not only in their type but also in the range of pH and temperature for optimal activity. The medium composition, mostly the carbon and nitrogen source, is known to influence the metabolite formation which in turn can have a modulating effect on a-amylase synthesis by changing the pH of the system. Results for two types of experiments are reported here, namely (1) batch experiments under uncontrolled pH conditions in shake flasks (working volume 200 mL), (2) experiments under controlled pH conditions in laboratory fermentor L F 2 (working volume 2 L). MATERIALS AND METHODS Microorganisms. Bacillus subtilis strains CCM 2267, CCM 2268, CCM 2744, CCM 2216, CCM 1718, CCM 2794, CCM 2722, NA 64; B. lichertiformis CCM 2145, B. stearothermophilus CCM 2183, all strains from the Czechoslovak Collectiort of Microorgartisms in Brno and B. subtilis DP 1 (isolate from starch materials) were used. The strains were maintained on nutrient agar slants and transferred at 30-d intervals. Culture conditions. Inoculum was prepared from nutrient agar slants on a liquid complex medium (100 mL) in 500-mL flasks. Cultivation was carried out in shake flasks (500 mL, working volume 200 mL). The pattern of enzyme production and growth was also studied in 5-L laboratory fermentor LF 2 (Development Workshops, Czech. Acad. Sci.) with regulation of pH (working volume 2 L). The aeration was maintained at 1 VVM per min, and the agitation frequency was kept constant at 8.3 Hz. A Slovanik oil T-610 (Czechoslovakia) was used to check foam formation. The effect of the antifoaming agent was negligible as to growth and enzyme production. The size of the inoculum being 2 or 10 % (V/V), respectively. Bacteria were grown at 37 ~ and pH 6.5 under aerobic conditions on a rotary shaker at 3 Hz for 60 h, centrifuged for 10 min at 70 Hz (MPW 340, Poland) and the supernatant served as enzyme source. Nutrient medium for flasks and laboratory fermentor studies contained g/L) (Yoo et al. 1988): carbon source 20 (soluble starch, D-glucose (Lachema, Czechoslovakia), maltose (Sojuzkhimexport, USSR), (NH4)2804 5, K2HPO4 1, MgSO4 0.5, sodium citrate dihydrate 0.5; FeSO4 0.1, MnSO4 0.1, yeast extract 1 (Imuna, Czechoslovakia); pH was adjusted to 6.5 before sterilization. Enzyme assay. Reducing sugars were determined according to Bernfeld (1955). Amylase activities assayed by reducing sugars liberated from 1 % soluble starch following incubation of enzyme solution with substrate for 10 min in thermostat water bath at 40 ~ (MLW M, Germany). The reaction

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K. DERCOV,~ et al.

Vol. 37

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1992

CELL GROWTH AND a-AMYLASE PRODUCTION

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Fig. 1. Time course of starch utilization S (g/L), dry biomass B (g/L, c l o s e d circles), reducing sugars R (mmol/L, s e m i c l o s e d circles), amylase activity A (U/mL, o p e n triangles), and pH changes (pH, c l o s e d triangles) during 60 h of batch fermentation of B a c i l l u s strains under uncontrolled pH conditions in shake flasks; A - B. subtilis B - B . subtilis C - B. subtilis D - B . subtilis

CCM 2267 CCM 2268 CCM 2744 DP 1

E - B. s t e a r o t h e r m o p h i l u s F - B. subtilis

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2722 1718 2794 2216 CCM 2145

NA 64

was stopped by addition of 3,5-dinitrosalicylic reagent. After 7 min of boiling at 100 ~ and cooling, reducing sugars were determined spectrophotometrically at 525 nm (Specol 11, Germany). One unit of a-amylase activity was defined as the amount of enzyme releasing reducing power equivalent to 1/zmol glucose per rain per mL under conditions described above. Analytical methods. The absorbance of cell mass was measured at 620 nm and expressed as dry mass. Residual starch in the medium was determined by the iodine method at 620 nm (De Mot et al. 1984). Glucose concentration was measured enzymically with by the Oxo-chrom test at 498 nm (BIOLA test, Czechoslovakia). pH and temperature optimum of enzyme activity were determined on incubating the cell-free supernatant for 10 min at 40 ~ with buffered starch ( 1 % W/V soluble starch, 0.2 mol/L phosphate buffer). The pH range was 4.0-8.0, the temperature range 40-90 ~ at pH 6.4. Amylase activity was assayed as described above.

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RESULTS AND DISCUSSION

Identificatian of microorganism The isolate was identified as Bacillus subtilis based on the morphological and physiological properties.

Production of enzyme in shake flasks The growth pattern of B. subtilis DP 1 in comparison with 10 other Bacillus strains was observed after 60 h in basal medium with 2 % soluble starch as carbon source in 500 m L flasks on a rotary shaker. The concentration profiles for cell mass, starch, reducing sugars and amylase activity are given in Fig. 1. According to Takehito (1988) secretion of the a-amylase occurs most actively between the end of the exponential phase to an early stationary phase. In agreement with Yoo et al. (1988) and Bajpai and Bajpai (1989), maximum amylase production by Bacillus strains occurs after the cell population has reached its peak. Decrease of the amylolytic activity of the cultivation media observed in the stationary phase of growth can be explained by an inactivation of the enzyme by proteolysis a n d / o r by an acidification of the media. Bacillus strains produce both amylolytic and proteolytic enzymes simultaneously. Acidification of cultivation media were observed mainly at the end of the stationary phase when the buffering capacity was overloaded (Fig. 1). Results in Fig. 4 indicate that inactivation of amylolysis is obvious at pH < 5 and reaction time 10 min. Table I shows the differential rate of a-amylase synthesis from the exponential phase of growth. The relationship between the rate of enzyme synthesis and the biomass concentration can be explained by a simple mathematical formula. A more detailed study based on continuous cultivation would be necessary for solving such correlations. I. Specificamylaseformation (U/mg) and differential rates (U mg-1 h-1) of a-amylase synthesisa Table

Strain

B. subtilis

CCM 2267 CCM 2268 CCM 2744 CCM 2794 NA 64 CCM 2722 CCM 2216 CCM 1718 DP 1 B. licheniformis CCM 2145 B. stearothemwphillusCCM 2183

AE U/mL 5.14 5.10 6.42 6.42 7.90 34.17 8.40 7.52 14.68 6.72 4.72

AX

g/L 0.898 0.490 1.045 1.013 0.655 0.477 0.971 0.980 0.648 1.120 0.142

U/mg

U mg-1 h -1

5.72 10.41 6.14 6.34 12.06 71.63 8.65 7.67 22.65 6.00 33.24

0.286 0.520 0.307 0.317 0.600 3.581 0.432 0.383 1.132 0.300 1.662

aAE - increase of enzymeactivityin 1 mL of supernatant. AX - increase of biomass in 1 mL of cultivation medium in the range 9- 29 h.

Production of enzyme in the L F 2 fermentor Figs 2 and 3 show the production of a-amylase in the LF 2 laboratory fcmentor with regulation of pH in optimizcd conditions. The growth and enzyme production rates in the fcrmentor were higher compared to those in shake flasks. Also, the cnzyme activity was about 10 times higher. This fact may bc attributed to better acration and p H control in the fcrmentor. Maximum cnzyme production was achicvcd by B. subtilis DP 1 aflcr 34 h whcre thc activity was 22 U / m E Soluble starch as a carbon source in concentration 20 g / L was completely hydrolyzed within 15 h.

1992

CELL GROWTH AND a-AMYLASE PRODUCTION

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Fig. 2. Time course of a-amylase batch fermentation by B. subtilis DP 1 under controlled pH conditions (pH 6.5) in LF 2 fermentor; for symbols see Fig. 1.

0

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Fig. 3. Time course of a-amylase batch fermentation by B. licheniformis CCM 2145 under controlled pH conditions (pH 6.5) in LF 2 fermentor; for symbols s e e Fig. 1.

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Effect of carbon source The effect of different carbon sources such as soluble starch, glucose and maltose as an alternate carbon source on a-amylase production was studied. Table II. Effect of carbon source (20 g/L) on maximum a-amylase activity in cultivation media (Emax, U/mL) by B. subtilis DP 1 and B. subtilis CCM 2722 Strain CCM 2722 DP 1

Starch

Maltose

Glucose

39.0 63.7

29.3 28.6

10.8 10.5

The effect of carbon sources was evaluated with regard to the cell growth and the enzyme production. The data (Table II) show the effect of different carbon source on a-amylase activity by two strains with the highest production of enzyme. Starch and maltose were found to be inducer of a-amylase synthesis and the synthesis of a-amylase was suppressed, when B. subtilis was grown on glucose.

Effect of nitrogen source In a recent study (Yoon et al. 1989) the concentration of yeast extract was found to be an important factor in a-amylase synthesis by B. amyloliquefaciens. Alam et al. (1989) indicate, that there is an optimum yeast extract concentration for a-amylase production. The aim of our work was to investigate the effect of the concentration of yeast extract on a-amylase synthesis by B. subtilis DP 1 in comparison with some other bacterial strains under controlled and uncontrolled pH conditions. Batch experiments were carried out in shake flasks under uncontrolled pH conditions with 1 and 2 g/L of yeast extract and in a fermentor with 2 and 5 g/L. Under uncontrolled pH fermentation with 2 g/L of yeast extract it an increase of extracellular amylase activity can be observed, but pH fell down significantly in comparison with fermentation at 1 g/L of yeast extract. This results in a complete

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K. DERCOVA et al.

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repression of enzyme synthesis. Under controlled pH conditions in the LF 2 bioreactor the a-amyl-ase activity in the medium containing yeast extract 5 g/L was comparable with the experiment at 2 g/L (not shown). Temperature and pH profiles of the studied Bacillus strains are given in Fig. 4. The pH optimum of a-amylase activity B. subtilis DP 1 was observed at 6.5 and temperature optimum at 60 ~

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Fig. 4. Temperature ( t o p ) and pH ( b o t t o m ) profiles of a-amylase activityA (U/mL); CCM 2267 CCM 2722 subtilis CCM 2794 l i c h e n i f o r m i s CCM 2145 subtilis CCM 2268 subtilis CCM 2744

1 - B. subtilis

2 - B. subtilis 3 - B. 4 - B. 5 - B. 6 - B.

DP 1 NA 64 9 - B . subtilis CCM 1718 1 0 - B . subtilis CCM 2216 7 - B. subtilis

8 - B. subtilis

11 - B. stearothermoplu'lus

CCM 2183

It can be assumed that regulation of physiological conditions influences extracellular a-amylase production by B. subtilis DP 1. A strict pH control is required in complex media containing high level of yeast extract for studying a-amylase synthesis. Strain B. subtilis DP 1 can be considered as a good model for kinetic study of a-amylase synthesis. We suppose that using further optimization of fermentation conditions or genetic manipulation a higher amylolytic activity will be achieved than in the original strain. The authors wish to acknowledge the valuable assistance of the C z e c h o s l o v a k C o l l e c t i o n o f M i c r o o r g a n i s m s in Brno in identifying the bacteria.

1992

CELL GROWTH AND a-AMYLASE PRODUCTION

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REFERENCES ALAM S., HONG J., WEIGANDW.A.: Effect of yeast extract on a-amylase synthesis by Bacillus amyloliquefaciens. Biotechnol.Bioeng. 33, 780-785 (1989). BAJPAIP., BAJPAI P.K.: High temperature alkaline a-amylase from Bacillus licheniformis TCRDC-B13. BiotechnoLBioeng. 33, 72-78 (1989). BERr~IrELDP.: Amylases a and fl, pp. 149-158 in Methods in Enzymology, VoL 1 (N. Kaplan, Ed.). Academic Press, New York 1955. DE MOT IL, ANDRIESK., VERACHTERTH.: Comparative study of starch degradation and amylase production by ascomyceteous yeast species. System.Appl.Microb. 5, 106-118 (1984). LIEBS P., RIEDELK., GRABAJ.P., SCHRAPELD., TISCHLERU.: Formation of some extracellular enzymes during the exponential growth of Bacillus subtilis. Folia Microbiol. 33, 88-95 (1988). PRIES'r F.G.: Extracellular enzyme synthesis in the genus Bacillus. BacterioLRev. 41, 711 -753 (1977). T^KF_~rro Y.: Data on individual amylases, pp. 18-125 in Handbook of Amylases and Related Enzymes (The Amylase Research Society of Japan, Ed.). Pergamon Press, Oxford 1988. TODA K.: Induction and repression of enzymes in microbial culture.J.Chem.Technol.Bioteclmol. 31, 775-790 (1981). Yoo YJ., CADMANT.W., HONG J., HATCH R.T.: Kinetics of a-amylase synthesis from Bacillus amyloliquefaciens. BiotechnoLBioeng. 31, 357-365 (1988). YOON M.Y., Yoo Y.J., C^DM~ T.W.: Phosphate effects in the fermentation of a-amylase by Bacillus amyloliquefaciens. Biotech.Lett. 11, 57-60 (1989).