Biotechnology Letters 21: 249–252, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.
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Production of amylolytic enzymes by Bacillus amyloliquefaciens in pure culture and in co-culture with Zymomonas mobilis Carlos M. Abate1,2,∗ , Guillermo R. Castro1,3 , Faustino Siñeriz1,3 & Danley A.S. Callieri1 1 Planta
Piloto de Procesos Industriales Microbiol´ogicos (PROIMI). Avda. Belgrano y Pasaje Caseros, 4000 Tucum´an, Argentina 2 C´ atedras de Biolog´ıa Celular y Biolog´ıa Molecular 3 C´ atedra de Microbiolog´ıa Superior - Facultad de Qu´ımica, Bioqu´ımica y Farmacia. Universidad Nacional de Tucum´an, Argentina ∗ Author for correspondence (Fax: +54 81 344887; E-mail:
[email protected]) Received 6 January 1999; Accepted 1 February 1999
Key words: mixed cultures, Bacillus amyloliquefaciens, Zymomonas mobilis, α-amylase production, ethanol production, batch cultures
Abstract Mixed cultures of Bacillus amyloliquefaciens MIR-41 and Zymomonas mobilis Flo-B3 showed a 2.5 fold increase in α-amylase production, and a 20 times fold decrease in ethanol production compared with pure cultures. Enhanced α-amylase production by B. amyloliquefaciens in mixed cultures after 24 h could be attributed to the lack of repression in the synthesis of α-amylase by ethanol and protease inhibition by the pH of the culture medium.
Introduction The understanding of the behaviour of microbial populations is useful for the management of microbial mixed cultures during industrial processes, such as the preparation of fermented food and beverages, manufacture of pharmaceutical products, mineral leaching, etc. (Zeikus & Johnson 1991). Zymomonas mobilis is a Gram-negative, aerotolerant bacterium which ferments exclusively glucose, fructose and sucrose mainly to ethanol and CO2 , following the Entner-Doudoroff pathway (Swings & DeLey 1977). The genus Bacillus is the major source of industrial enzymes and Bacillus amyloliquefaciens is one of the most widely used species for the bulk production of α-amylase and proteases (Hardwood 1992). B. amyloliquefaciens MIR-41, a strain which produces extracellular α-amylase in large amounts, and α-glucosidase, was obtained during the course of screening of amylolytic bacteria from soil (Castro et al. 1993b).
Starch is one of the most abundant polysaccharides in nature and several attempts have been reported dealing with the production of useful products by cofermentation of starchy substrates. Mixed cultures of amylolytic yeast and Z. mobilis have been used to convert starch or disaccharides to ethanol and proteins (Abate et al. 1996, Gonzalez et al. 1998). The particular characteristics and capacities of Z. mobilis Flo-B3 and B. amyloliquefaciens MIR-41 induced us to explore the possibility of producing ethanol and αamylase by means of a mixed culture of both strains, using a starchy medium.
Materials and methods Microorganisms Zymomonas mobilis mobilis Flo-B3, a high ethanol producing strain, was isolated from spontaneously fermenting sugar cane juice (Rodríguez & Callieri 1986). Bacillus amyloliquefaciens MIR-41, an α-amylase hy-
250 perproducer strain was isolated from soil (Castro et al. 1993a). Media and growth Z. mobilis was grown in the following medium, in g l−1 : glucose, 50.0; yeast extract, 10.0; (NH4 )2 SO4 , 1.0; MgSO4 · 7H2 O, 1.0; KH2 PO4 , 1.0. B. amyloliquefaciens medium contained in g l−1 : (NH4 )2 SO4 , 1.0; KH2 PO4 , 3.0; K2 HPO4 , 6.0; CaCl2 · 2H2 O, 0.05; FeSO4 · 7H2 O, 0.001; ZnSO4 · 7H2 O, 0.001; MnSO4 · 7H2 O, 0.01; MgSO4 · 7H2 O, 0.01; trisodium citrate, 1.0; soluble starch, 40.0 (Castro et al. 1993b). This medium was also used to perform the co-cultures adding to it 4.0 g l−1 yeast extract to promote a good growth of Z. mobilis. The pH of the media was adjusted to 7.0 with 5.0 M K2 HPO4 solution before sterilization. All reagents were either of analytical or bacteriological grade from Sigma. Triplicate assays were performed in 500 ml shaken flasks (150 rpm) containing 150 ml of medium at 30 ◦ C. A 24 h old culture of the strains was used as inoculum, 5 ml in the case of pure cultures and a mixture of 2.5 ml of each strain in the case of co-cultures. Determinations Bacterial growth was followed spectrophotometrically at 560 nm, diluting the samples with 145 mm NaCl if required. Protein was determined using Coomassie Blue G-250 reagent with BSA (fraction V) as standard (Sedmak & Grossberg 1977). Starch was assayed by the blue index method using soluble starch as standard, and glucose enzymatically as described previously (Castro et al. 1993b). Ethanol was measured in previously distilled samples using a Carl Zeiss Jena immersion refractometer (Rodríguez & Callieri 1986). Alpha amylase (E.C.3.2.1.1) was assayed by determining the dextrinization activity using iodine reagent test (Castro et al. 1993a). One unit is defined as the amount of enzyme required to degrade 10 µg starch in 30 min at 45 ◦ C. Determination of α-glucosidase (E.C.3.2.1.20) was performed measuring the release of glucose using glucose oxidase-peroxidase method (Castro et al. 1993b). One α-glucosidase unit is defined as the amount of enzyme needed to release 1 µmol glucose per min at 45 ◦ C.
Fig. 1. Kinetics of the biomass production: Bacillus amyloliquefaciens MIR-41 in culture medium with ( ) or without ( ) yeast extract, Zymomonas mobilis Flo B3 ( ), B. amyloliquefaciens MIR-41 and Z. mobilis Flo B3 in co-culture ( ). Evolution of the starch concentration in the medium during fermentation by B. amyloliquefaciens in pure culture (×) and in co-culture with Z. mobilis (∗).
N
Results and discussion Specific growth rates of Zymomonas mobilis and Bacillus amyloliquefaciens pure cultures were 0.35 h−1 , and 0.28 h−1 , respectively. Presence of yeast extract increased the growth rate of B. amyloliquefaciens more than 15% (Figure 1). Even so, the overall rate of biomass production was lower than that of the mixed culture, although the medium contained yeast extract. In spite of the difference in growth rate after 48 h of incubation all the cultures reached approximately the same biomass concentration. Kinetics of starch degradation in pure culture of B. amyloliquefaciens, with or without yeast extract in the medium was approximately the same. Starch hydrolysis rates were found in the range of 5.4 to 5.9 g l−1 h−1 in pure and mixed cultures during the first 6 h of incubation. These rates can be attributed to the presence of pullulanase produced by B. amyloliquefaciens MIR41 in the first stages of growth (Castro et al. 1993b), and less than 0.1% of the initial starch remains in the culture after 48 h of cultivation. Ethanol produced by Z. mobilis, detected in mixed cultures, was maximum at 8 h of culture reaching a concentration of 0.8 g l−1 . However, ethanol concentration diminished to 0.27 g l−1 at 24 h of culture. The maximum ethanol concentration is approximately 20 times lower than that corresponding to a pure culture of Z. mobilis in a medium containing the same amount of sugar. The low ethanol concentration measured could be due to its utilisation by B. amyloliquefaciens, since this microorganism
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Fig. 2. Kinetics of production of extracellular alpha amylase: Bacillus amyloliquefaciens MIR-41 in culture medium with ( ) or without yeast extract ( ), B. amyloliquefaciens MIR-41 and Zymomonas mobilis Flo B3 in co-culture ( ).
N
can grow in a medium containing ethanol as the sole carbon source (data not shown). Then, ethanol concentration detected was low during fermentation, either because it was scarcely produced or because it was simultaneously consumed. Oligosaccharides are the major products of αamylase activity, and are substrates for α-glucosidase to produce glucose. In pure cultures of B. amyloliquefaciens, 20 U α-glucosidase/l were detected at the end of exponential growth phase (Castro et al. 1993b). Similar profiles of α-glucosidase production were found in mixed cultures with Z. mobilis but, in stationary phase of growth the activity of αglucosidase decreased to 1.8 U l−1 . Addition of yeast extract to synthetic medium in pure cultures of B. amyloliquefaciens decreased the maximum α-glucosidase production by 3.5 times (data not shown). It seems possible that the release of glucose and oligosaccharides from starch could be the rate-limiting step in the microorganisms growth because glucose release during fermentation was rapidly consumed. Glucose concentration reached approximately 100 mg l−1 in pure cultures of B. amyloliquefaciens at 24 h. On the contrary, low α-glucosidase activity expressed in pure cultures of B. amyloliquefaciens supplemented with yeast extract can be correlated with 10 mg l−1 glucose concentration at the same growth time. Production of α-amylase by B. amyloliquefaciens in minimal medium was detected at the beginning of the exponential growth phase (Figure 2), and increased slowly, reaching about 10 kU l−1 after 48 h. Addition of yeast extract to the medium strongly improved the enzyme rate and specific production (U enzyme/g biomass) to about 5.5 times the original activity. Af-
ter 24 h of incubation the α-amylase released in the medium was 4.4 times higher than that found in the medium without yeast extract. However, presence of high levels of protease activities concomitant with the sporulation process in cultures of B. amyloliquefaciens supplemented with yeast extract could be the cause for α-amylase inactivation after 24 h culture at end of the exponential growth phase. On the contrary, pure cultures of B. amyloliquefaciens without yeast extract showed a 25 fold lower protease activity at the same culture times (data not shown), therefore no α-amylase inactivation was detected. In mixed cultures, the maximal production of α-amylase occurred with different kinetic pattern (Figure 2). During the first 24 h incubation in mixed cultures, evolution of α-amylase concentration was similar to that found in pure culture of B. amyloliquefaciens in medium deprived of yeast extract. After 24 h, there was an abrupt increase of α-amylase concentration from 5 to 25 kU l−1 . In mixed culture, the protease activity was approximately the same that was found in pure culture supplemented with yeast extract (data not shown). However, the pH in the fermentation medium was 5.6. In this condition, the protease activity is only less than 10% of the values of activity at pH 7. This fact could explain why α-amylase is not degraded in mixed culture (Figure 2). The influence of ethanol on the α-amylase activity and synthesis was also tested. It was found that at 30 ◦ C, 350 g l−1 of ethanol were needed to reduce by 50% the activity of the enzyme. Therefore, it can be concluded that the concentration of ethanol during fermentation, less than 1 g l−1 , was too low to impair the activity of the enzyme. Using 5.0 g l−1 ethanol as carbon source in a synthetic and supplemented medium with yeast extract, B. amyloliquefaciens showed no α-amylase activity in 24 h old cultures. These results suggest that α-amylase synthesis is strongly repressed by ethanol (catabolite repression). So, the synthesis of α-amylase in mixed culture could be partially repressed during the exponential growth phase by the presence of ethanol produced by Z. mobilis at the expense of glucose. After 24 h of growth, and concomitant by the glucose and ethanol concentrations decrease in the media, α-amylase synthesis was re-started. To find out if the positive influence of Z. mobilis on α-amylase production in mixed culture was due to some metabolite produced by this microorganism, a set of experiments was performed using B. amyloliquefaciens in minimal medium supplemented with increasing amounts of cell free supernatant of a pure
252 culture of Z. mobilis to 50%. No modification in the amount and kinetics of the enzyme production was observed. It was previously found that the synthesis of extracellular enzymes is repressed by the presence of readily metabolizable carbon sources in the culture medium (Fisher & Sonensheim 1991). In particular, synthesis and activity of α-amylase in B. subtilis was described subject to carbon catabolite repression and enzyme inhibition by glucose and glycerol (Saito & Yamamoto 1975). Similar results were found with Bacillus licheniformis grown in batch as well as in continuous culture with glucose as carbon source (Priest & Thirunavukkarasu 1985). Both species are highly related to B. amyloliquefaciens, and belongs to the same cluster (Priest et al. 1987).
Acknowledgements The financial support from International Foundation for Science (Sweden), CONICET and Fundación Antorchas (Argentina) is gratefully acknowledged.
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