Biosynthesis of thermostable α-amylase by immobilized Bacillus subtilis in batch and repeat batch cultures using fortified date syrup medium

Batch and repeat batch fermentation were performed to evaluate the potential of date syrup and immobilized cells technique for the economical production of α-amylase. The enzyme production by immobilized cells was greater than that of freely suspended cells. Khlas date syrup gave the highest enzyme yield (142.7 Uml) using the immobilized technique after 48 h of fermentation. The highest αamylase production was obtained in a culture grown in a 2% tricalcium phosphate treated medium. The enzyme production was proportional to the amount of date syrup up to 1.0% sugar. Tween 80 was more effective in enhancing cell membrane permeability, and thus increasing enzyme secretion. A 1.4 fold increase in enzyme production was achieved using an optimized medium in a fermenter. The culture activity under repeated batch cultivation remained stable up to the third cycle, and retained about 85% of its initial efficiency during the first five batches when each cycle continued for 48 h. The stimulated activity and the thermo stability were observed in the presence of 10 mM Ca. The results show that date syrup is a promising economic carbohydrate source and cell immobilization is an excellent alternative method for enhanced α-amylase production.


INTRODUCTION
Amylases are one of the most important industrial enzymes and account for nearly 25% of the world's enzyme sales (Rao et al., 2012).These enzymes are of great significance, with extensive biotechnological applications including starch degradation, detergent, food, pharmaceutical, textile and paper manufacturing (Acourene and Ammouche, 2010).Each application of α-amylase (EC 3.2.1.1),requires unique properties with respect to specificity and thermal stability (Konsula and Liakopoulou-Kyriakides, 2004).Improvement in the yield of α-amylase and consequent cost reductions depends on the efficient utilization of agro-industrial residues and by-products as production media (Saad, 2006;Shamala et al., 2012).Kingdom of Saudi Arabia (KSA) is a major date producing country and is ranked the second in the world as per FAO statistics 2010 (El-Habba andAl-Mulhim, 2013).Date syrup is a byproduct of the date industry.It is rich in carbohydrates (75%) with small amounts of protein (1.1%), fat (2.9%), macro-elements in the form of Ca, P, Na, K and Mg and microelements in the form of Fe, Zn, Cu, and Mn (Al-Farsi et al., 2007).The analysis of the sugar content of date syrup shows the presence of fructose (41%), glucose (39%) and traces of sucrose (Khiyami et al., 2008).It can be used as a sweetener and flavouring for human consumption and for microbial fermentation (ethanol, vinegar, antibiotics and single cell protein) because it is readily available and relatively low priced (Radwan et al., 2010).
The enzyme production is greatly affected by the presence of heavy metals which are contained in large amounts in date syrup, so various chemical techniques have been used to precipitate or chelate the ions which cause critical problems during fermentation (Roukas and Kotzekidou, 1997;Saad, 2006).The α-amylase is produced by a wide variety of microorganisms, but on account of its industrial applications, the best commercial producer are Bacillus licheniformis, Bacillus stearothermophilus and Bacillus amyloliquefaciens (Kiran et al., 2012).There is considerable interest in improving the productivity and product economy of α-amylase by using the immobilized cells of Bacillus strains (Singh and Verma, 2008).Cells have been commonly entrapped in a calcium alginate gel through which substrates and products can diffuse easily (Gökhan et al., 2005).The use of immobilized cells offers several advantages over free cells, such as the relative ease of product separation, the re-use of biocatalysts, the prevention of washout, the reduced risk of contamination and operational stability (Shivakumar, 2012).Furthermore, using the entrapment technique, a dense cell culture can be established, leading to improved productivity (Poddar et al., 2011).In the present study, the potential of date syrup and immobilized cells technique for the αamylase production by batch and repeat batch fermentation were investigated.

Source of strain
Bacillus subtilis EM11, identified by phylogenetic analysis, was isolated from soil samples collected from the Jazan region, Saudi Arabia, as described in our previous report (Alamri, 2010).

Pre-treatment of crude date syrup
Date syrup was obtained from two companies in Saudi Arabia, Al-Barak (khlas date syrup, 70% sugars) and Durrah Dates (mixed date syrup, 66% sugars).Date syrup solution was diluted with distilled water in order to obtain 1% (w/v) sugar concentration, and treated with the following methods: 1-Tricalcium phosphate treatment (TCP): date syrup solution was adjusted to pH 7.0 with 1 N NaOH and treated with 1, 2 or 3% (w/v) TCP.The mixture was heated at 100°C for 5 min.The mixture was cooled and then centrifuged at 4,000 g for 20 min.2-Potassium ferrocyanide (PFC): date syrup solution was adjusted to pH 7.0 with 1 N NaOH and sterilized at 121°C for 15 min.The liquid was treated while hot with 25 or 50 μg/ml potassium ferrocyanide to encourage the precipitation of heavy metals.3-Methanol concentration: different methanol concentrations (1, 2, 3 and 4% v/v) were added to the fermentation flasks.

Inoculum preparation
Five millilitres of sterile distilled water was added to a 24 h old slant of B. subtilis.The resulting cell suspension was transferred into 250 ml Erlenmeyer flasks containing 50 ml of Luria Bertani medium: 1% tryptone, 0.5% yeast extract and 0.5% NaCl, and cultivated at 45°C /200 rpm on an incubation shaker for 24 h.Cells were harvested by centrifugation (4500 rpm, 20 min), and were used both for immobilization and in experiments with free cells (Konsoula and Liakopoulou-Kyriakides, 2004).

Immobilization
The alginate entrapment of cells was performed according to the method suggested by Konsoula and Liakopoulou-Kyriakides (2006).About 20 mg of wet cells and 12.5 ml of 2% (w/v) sodium alginate solution were mixed and stirred for 10 min to obtain a uniform mixture.The mixture obtained was extruded drop-wise through a 10 ml syringe into a 25 ml of 3.5% (w/v) CaCl 2 solution.Alginate drops were solidified upon contact with CaCl 2 forming capsules, and thus entrapping the bacterial cells.The capsules were allowed to harden for 30 min and then were washed with sterile saline solution (0.9% NaCl) 3 to 4 times to remove excess Ca 2+ and cells.When the capsules were not used, they were preserved in a CaCl 2 solution in the refrigerator.All operations were carried out aseptically within a laminar flow unit

Fermentation
Frementation was done in 250 ml Erlenmeyer flasks, each containing 50 ml of date syrup medium (1% sugar) fortified with peptone 0.5%, K 2 HPO 4 0.6%, MgSO 4 .7H 2 O 0.02% and CaCl 2 .2H 2 O 0.05% (Aqeel and Umar, 2010).Each flask was inoculated with the capsules obtained from 12.5 ml of alginate gel.For the free cell cultures, the production medium was inoculated with bacterial cells equivalent to those used in the immobilized cultures.The fermentation with free and immobilized cells was conducted at 45°C for 84 h under shaking conditions (200 rpm).For evaluation of the effect of surfactants on amylase production, 0.5% of different filter sterilized surfactants (sodium dodecyl sulphate (SDS), Triton-X100, Tween 40 and Tween 80) were added separately to the production medium.A 7.5 L jar fermenter (BioFlo/CelliGen 115 Benchtop Fermentor & Bioreactor) was employed for the fermentor operation with a culture volume of 2.5 L. The agitation speed and incubation temperature were controlled at 200 rpm, 45°C.The culture pH in the fermenter was controlled at 7.5 with an air flow of 4 v/v/min.

Repeated batch fermentation with free and immobilized cells
One of the advantages of using immobilized biocatalysts is that they can be used repeatedly and continuously.Therefore, the reusability of cells immobilized in an alginate matrix was examined.Repeated batch fermentations were conducted by running the fermentation for 48 h.At the end of each cycle, the production medium was recovered, and the immobilized cells were washed with 2% (w/v) CaCl 2 solution for 30 min, fresh production medium was added and the fermentation was continued.Curing the gel capsules with 2% CaCl 2 , after each fermentation batch prevented the disruption of the capsules, maintained their mechanical structure and significantly increased the productive life of the biocatalysts (Konsoula and Liakopoulou-Kyriakides, 2006).

Biomass
Both cell growth in freely suspended cultures and immobilized cells in alginate matrix were determined as dry weight.The alginate capsules were dried and the biomass was determined from the difference between the total weight of the alginate gel capsules containing cells and that of alginate capsules without cells (blank) prepared under the same conditions of immobilization.
α-Amylase assay α-Amylase was assayed by adding 1 ml of enzyme to 1 ml soluble starch (1%) in an acetate buffer with pH 5, and incubated at 50°C for 15 min.The reaction was stopped by the addition of 2 ml of a 3,5-dinitrosalicylic acid reagent (Bernfeld, 1955).The absorbance was measured using a double beam UV/Vis scanning spectrophotometer (Model: Shimadzu, 1601PC) at 550 nm.One enzyme unit (Uml -1 ) is defined as the amount of enzyme which releases 1 μ mole of glucose.

Effect of temperature on the activity and stability in the presence and absence of 10 mM CaCl 2
The optimum temperature of the enzyme was evaluated by assaying activity between 40 and 100°C for 15 min in the presence and absence of 10 mM CaCl 2 .Thermal stability was investigated by measuring the residual activity after 60 min of pre-incubation at temperatures ranging from 40 to 100°C in the presence and absence of 10 mM CaCl 2 (Asgher et al., 2007).

Statistical analysis
Firstly, parametric testing was performed and, then, the analysis of variance was used to compare the data from the different treatments.
All analyses were performed at p≤0.05 using MINITAB, version 13.1.

Enzyme production by free and immobilized cells using khlas date syrup
As shown in Figure 1, rapid cell growth in the free culture was observed in the first 24 h of cultivation, and a constant cell concentration (2.4 mg ml -1 ) was attained thereafter.On the other hand, the rapid increase in biomass entrapped in the gel capsules continued for 36 h, reaching a cell concentration of 4.7 mg ml -1 .The time required for the stabilization of the cell content in the gel capsules was extended to 60 h of cultivation.The productivity of αamylase in terms of the immobilized cells was significantly greater than that of the freely suspended cells (about 2.35 fold).Although maximal enzyme production was attained after 36 h for the free cells, it was achieved after 48 h of cultivation for the immobilized cells.Similar observations have been reported for various Bacillus species.Most researchers attribute the alteration of the enzyme synthesis mechanism to the stress conditions imposed by immobilization, and to the changes in microenvironmental conditions, and to some metabolic and morphological alterations in the cells (Górecka and Jastrzębska 2011) On the other hand, other researchers have suggested that immobilized biocatalysts produce lower levels of enzyme in comparison with free cells due to diffusion barriers and reduced oxygen availability to immobilized aerobic cells (Konsula and Liakopoulou-Kyriakides, 2004).

Enzyme production by free and immobilized cells using mixed date syrup
The results illustrated in Figure 2 indicate that the growth of cells entrapped in a calcium alginate matrix increased gradually up to 48 h of incubation, whereas with free cells, gradual growth was observed only up to 36 h.Although maximal enzyme production was attained after 48 h of cultivation in both cases, a difference in the kinetics of α-amylase biosynthesis was observed.The enzyme production was higher with immobilized cells (105.5 U ml -1 ) than with free cells (65.3 U ml -1 ).The cell immobilization technique results in a higher production rate within the first 24 h of cultivation (63.7%), while in the case of free cells, only 44.5% was produced during the same period.After 36 h of growth, the immobilized cells produced more than 83.8% of the maximal α-amylase yield, while the production by free cells was restricted to 61.5%.
The increased α-amylase production by the immobilized cells of B. subtilis may be due to the presence of a higher concentration of calcium ions as well as to the formation of strong gel capsules possessing high substrate mass transfer rates and low rates of cell leakage, as reported by Konsoula and Liakopoulou-Kyriakides (2006).Furthermore, according to Acourene et al. (2013), α-amylase production is induced by the presence of starch in the production medium, which unfortunately cannot be used efficiently as a substrate due to the decreased mass transfer rate of this polysaccharide.In our study, the effect on the substrate mass transfer rate was reduced by the replacement of starch with date syrup.

Effect of the pre-treatment of crude date syrup on enzyme production
Khlas date syrup was treated using different chemical methods to precipitate the heavy metals which affect αamylase production.The results presented in Figure 3 indicate that all treatments gave a remarkable increase in α-amylase production and bacterial growth, in comparison with the results involving untreated date syrup.The highest value of α-amylase production was obtained in a culture grown in 2% (w/v) tricalcium phosphate treated medium.The production of α-amylase using a pre-treated date syrup medium was 33.8% higher than that associated with the untreated one.These results are possibly attributed to the presence of two phosphate groups rich in negatively charged oxygen atoms in tricalcium phosphate, that act as attractants for the positively charged elements present in molasses as reported by Mayilvahanan et al. (1996).They reported also that the pre-treatment with tricalcium phosphate dramatically reduced the concentrations of a number of heavy metal ions that may retard the utilization of molasses.3% (v/v) methanol was the second best date syrup treatment.The high stimulition effect of methanol can be attributed to the increase in the microorganisms' tolerance to high levels of heavy metals (Mehyar et al., 2005).Furthermore, Mostafa and Alamri (2012) reported that undesirable substances such as iron, zinc and copper which are contained in great amounts in date syrup can cause critical problems during the fermentation process.It inhibits the growth of microorganisms, influences the ionic strength, the pH of the medium and is involved in the inactivation of the enzymes associated with metabolism.

Effect of sugar concentration on enzyme production
The data presented in Figure 4 indicates that the date syrup was thought to be a good carbon source and is sufficient to support microbial growth and α-amylase production.The maximum production of α-amylase was achieved at 1.0% sugar, and the productivity was propor-tional to the amount of date syrup.A further increase in the sugar concentration did not improve the enzyme production.Swain et al. (2006) and Aqeel and Umar (2010) reported that the α-amylase production by B. subtilis was constitutive, since the biosynthesis of the enzyme took place, not only in the presence of starch, but also with other carbon sources such as maltose, glucose, fructose and date syrup.Pan and Xu (2003) and Radwan et al. (2010) found that the date syrup contains sufficient amounts of sugar that cover the growth of the strain.They reported also that the date syrup contains 39% glucose; therefore, glucose has no effect on the production of amylase in the presence of date syrup.Moreover, enzyme production reached the maximum yield after 48 h, which suggests that the catabolic repression of glucose did not occur.The results also indicate that the strain produced α-amylase constitutively and without sensitivity to catabolite repression or transient repression.On the other hand, Sudharhsan et al. (2007) reported that the synthesis of carbohydrate degrading enzymes in some species of genus Bacillus, leads to catabolic repression by readily metabolizable substrates such as glucose and fructose.

Effect of surfactants on enzyme production
The results illustrated in Figure 5 indicate that the addition of surfactants sometimes either increases or decreases enzyme production.The results shows clearly that the addition of a non-ionic detergent, Tween 80, causes the maximum production of α-amylase (216.3Uml -1 ) followed by Tween 40 (197.5 Uml -1 ) in comparison with the control.These results were in agreement with that of Sankaralingam et al. (2012).They reported that Tween 80 was an excellent surfactant for the production of αamylase.Surfactants reduce the surface tension of the liquid medium, and also provide essential nutrients for the growth of the organism, and increase the secretion of αamylase from the bacterial cells by increasing the cell membrane's permeability (Uelger and Cirakoglu, 2001).The enzyme production was greatly inhibited by the addition of other surfactants, Triton X100 and SDS.It might have been due to the fact that sodium ions, along with sulphate ions, were toxic for bacterial growth and enzyme production as reported by Milner et al. (1996).Also, Serin et al. (2012) reported that Triton X-100 suppressed α-amylase production in Bacillus circulans.On the other hand, Sudharhsan et al. (2007) found that the addition of SDS causes a higher production of amylase, but the other additives such as Triton X100 and Tween 20 decreased the production of α-amylase.

Batch fermentation under optimized conditions in shake flasks and fermenter
To confirm the aforementioned performance of 2% tricalcium phosphate, 1% sugar, 0.5% Tween 80 with the basal medium components, fermentor operations were carried out using a 2.5 L working volume.Under optimized conditions, batch fermentation using shake flasks and a fermenter was carried out for up to 72 h (Figure 6).The production of α-amylase in the fermenter after 36 h of cultivation was higher than that in the shake flask after 48 h, probably mainly due to the improvement in aeration conditions.In other words, there is great potential for an improvement in terms of α-amylase yield by further optimizing the operating conditions in future experiments.An overall 1.34-fold increase in enzyme production was achieved using an optimized medium in a fermentor when compared with a shake flask.This might be due to the fact that this medium provided an adequate amount of essential nutrients for microbial growth, and subsequently for enzyme production.Crueger and Crueger (2000) reported that molasses is one of the cheapest sources of carbohydrates.Besides a large amount of sugar, molasses contain nitrogenous substances, vitamins and trace elements.Our results indicated that the evaluation of a suitable medium is critical for a successful fermentation process by microbes.Similar behaviour was reported by Uma et al. (2007).They found that a 1.6 and a 2.1-fold increase in enzyme production were achieved in an optimized medium in shake flasks and fermenter, respectively.Moreover, Bozic et al. (2011) reported that during the batch fermentation of B. subtilis IP 5832, a 60% higher α-amylase activity was obtained.

Repeated batch fermentation with free and immobilized cells
One of the most important benefits of immobilized cells is their ability in a stable fashion to produce α-amylase under repeated batch cultivation.The α-amylase production by B. subtilis cells for 10 batch cultivations with parallel experiments using free cells as a control are recorded in Figure 7.The results indicate that the free cells lost 35.4% of their productive ability after the first batch.In contrast, the immobilized cells showed high α-amylase productivity upon re-use.The culture activity remains stable up to the third cycle, and retains about 85% of their initial efficiency during the first five batches when each cycle was continued for 48 h.The enzyme production with immobilized cells gradually decreases from the fifth batch onwards.This may occur as a result of beads disin-tegrating during the batch operation.Thus, the repeated batch fermentation with calcium alginate beads was successfully run for 5 batches (10 days).The repeat batch culture technique has many advantages over batch cultivation, such as decreases in the cost of sterilization and the preparation of a fermentor and inoculums preparation.This increases the economic efficiency of enzyme production (Ates et al., 2002).The enzyme production after 5 fermentation cycles showed that the α-amylase yield could be raised from 237.8 units in a free cell state to 1,025.8 units using an immobilized state.These findings were similar to those reported by Kiran et al. (2012) and Konsoula and Liakopoulou-Kyriakides (2006).The lengthy viability of the immobilized cells may be due to the different composition of proteins, nucleic acids and inorganic substances, in comparison with the free cells (Shivakumar, 2012).
Effect of temperature on the activity and stability of α-amylase in the presence and absence of 10 mM CaCl 2 The α-amylase activity was determined at different temperatures ranging from 40 to 100°C in the absence and presence of 10 mM CaCl 2 (Figure 8).The optimum enzyme activity was recorded at 50°C, with the enzyme activity gradually declining at temperatures beyond this value.The enzyme activity sharply decreased to about 50.2% at 70°C.The activity of the enzyme was increased to 128% in the presence of 10 mM Ca 2+ , suggesting that calcium is needed for the optimum activity of the enzyme.Most amylases are known to be metal ion-dependent  enzymes.The calcium ion was reported to increase the amylase activity of Bacillus strains (Asgher et al., 2007).Furthermore, Gupta et al. (2003) and Rao et al. (2012) reported that the α-amylase enzyme contains at least one Ca 2+ ion and the affinity of Ca 2+ is much stronger than that of other ions.The thermal stability of the α-amylase was tested at different temperatures in a range of 40 to 100°C for a period of 1 h in the presence and absence of 10 mM CaCl 2 (Figure 8).The enzyme was thermo-stable over a temperature range of 40 to 70°C, retaining 88.1% of its original activity at 80°C after 1 h of incubation in the absence of CaCl 2 .The stimulated thermo-stability was observed in the presence of 10 mM Ca 2+ .The enzyme was completely active up to 90°C, while 90.8% remained after pre-incubation at 100°C.The stabilizing effect of Ca 2+ on the thermo-stability of the enzyme can be explained due to the salting out of hydrophobic residues by Ca 2+ in the protein, thus causing the adoption of a compact structure (Swetha et al., 2006).Similarly, complete activity at 90°C for 1 h for the amylase from Bacillus sp. has been reported by Teodoro and Martins (2000).Violet and Meunier (1989) reported that α-amylase contains at least one Ca 2+ molecule which is involved in the stabilization of the molecular structure.Moreover, Konsula and Liakopoulou-Kyriakides (2004), Hwang et al. (2013) and Sindhu et al. (2011) found that the thermo-stability of α-amylase from the B. subtilis and Streptomyces avermitilis strain was enhanced in the presence of calcium ion.The stability of the enzyme could be due to its genetic adaptability when it comes to carrying out their biological activities at higher temperatures.Our results confirm the key role of Ca 2+ in terms of enzyme thermo-stability-an important feature for the use of amylolytic enzymes in starch-processing industries.

Conclusion
The productivity of α-amylase by the immobilized cells was greater than that of the freely suspended cells.The activity of the culture remained stable after a repeated batch culture for about five cycles.The potential application of α-amylase in various industries and the need for the development of economic methods for improved production make date syrup an economic carbohydrate source, and whole bacterial cell immobilization, excellent alternative methods for enhanced amylase production.

Figure 1 .
Figure 1.Enzyme production by free and immobilized cells using khlas date syrup.

Figure 2 .
Figure 2. Enzyme production by free and immobilized cells using mix date syrup.

Figure 4 .
Figure 4. Effect of sugar concentration of date syrup on enzyme production.

Figure 5 .
Figure 5.Effect of surfactants on enzyme production.

Figure 6 .
Figure 6.Batch fermentation under optimized conditions in shake flasks and fermenter.

Figure 7 .
Figure 7. Repeated batch fermentation with free and immobilized cells.

Figure 8 .
Figure 8.Effect of temperature on the activity and stability of α-amylase in the presence and absence of 10 mM CaCl 2 .