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Keywords:

  • Bacillus licheniformis;
  • calcium alginate;
  • immobilization;
  • tannase

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  The present study was aimed at finding the optimal conditions for immobilization of Bacillus licheniformis KBR6 cells in calcium-alginate (Ca-alginate) beads and determining the operational stability during the production of tannin-acyl-hydrolase (tannase) under semicontinous cultivation.

Methods and Results:  The active cells of B. licheniformis KBR6 were immobilized in Ca-alginate and used for the production of tannase. The influence of alginate concentration (5, 10, 20 and 30 g l−1) and initial cell loading on enzyme production were studied. The production of tannase increased significantly with increasing alginate concentration and reached a maximum enzyme yield of 0·56 ± 0·03 U ml−1 at 20 g l−1. This was about 1·70-fold higher than that obtained by free cells. The immobilized cells produced tannase consistently over 13 repeated cycles and reached a maximum level at the third cycle. Scanning electron microscope study indicated that the cells in Ca-alginate beads remain in normal shape.

Conclusions:  The Ca-alginate entrapment is a promising immobilization method of B. licheniformis KBR6 for repeated tannase production. Tannase production by immobilized cells is superior to that of free cells because it leads to higher volumetric activities within the same period of fermentation.

Significance and Impact of the Study:  This is the first report of tannase production from immobilized bacterial cells. The bacterium under study can produce higher amounts of tannase with respect to other fungal strains within a short cultivation period.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Tannase (tannin-acyl-hydrolase, E.C. 3·1·1·20) is an inducible microbial enzyme. It catalyses the hydrolysis of ester and depside bonds in hydrolysable tannin such as tannic acid, releasing glucose and gallic acid. The enzyme is found to be useful in the manufacture of instant tea, acron wine, coffee-flavoured soft drinks, clarification of beer and fruit juices (Coggon and Sanderson 1972; Chae and Yu 1983; Lekha and Lonsane 1997). Gallic acid is mainly used as an important substrate for the synthesis of propyl gallate, which is widely used as a food antioxidant, and trimethoprim, a pharmaceutical antibacterial agent (Gaathon et al. 1989; Hadi et al. 1994). It is also used as photosensitive resin in semiconductor production (Hadi et al. 1994). Despite the advantages of using such enzymes, its commercial use has been limited owing to its relative instability and high cost. These problems have been solved by immobilizing the tannase (Katwa et al. 1981; Weetal 1985) and the microbial cells (Jouenne et al. 1993; Misro et al. 1997).

The immobilization of growing microbial cells is of particular interest because of their biotransformational and biosynthetic abilities for the production of diverse valuable products like antibiotics (e.g. bacitracin by Bacillus sp.), organic acids (e.g. lactic acid by Lactobacillus sp.), enzymes (e.g. β-amylase by Bacillus megatorium), alcohols (e.g. ethanol by Saccharomyces cerivisiae) and the like (Ramakrishna and Prakasham 1999). Immobilization of whole cells for the production of extracellular enzymes offers many advantages, such as ability to separate cell mass from bulk liquid for possible reuse, facilitating continuous operation over a prolonged period and enhancing reactor productivity (Zhang et al. 1989; Galazzo and Bailey 1990). However, proper selection of immobilization techniques and supporting materials is needed to minimize the disadvantages of immobilization (Ramakrishna and Prakasham 1999). One of the most suitable methods for cell immobilization is entrapment in Ca alginate, because this technique is simple, nontoxic and cheap. Beads of Ca alginate are prepared under mild conditions and used extensively for microencapsulating and entrapping cells (Jamuna and Ramakrishna 1992).

In the present study, cells of Bacillus licheniformis KBR6, a potent tannase producer (Mondal et al. 2000), were immobilized in Ca alginate. The tannase-production and operational stability of the biocatalyst were determined and compared with those of free cells.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Micro-organism

Bacillus licheniformis KBR6 (IMI. No. 379224) has been used in the present study. It was isolated from the lateritic sal forest soil of Midnapore district, West Bengal, India (Mondal and Pati 2000).

Preparation of preinduced inoculum

The selective tannic acid-enriched medium has been used for the preparation of inoculum. The composition of the medium was as follows (in g l−1): tannic acid, 10; NH4Cl, 3; KH2PO4, 0·5; K2HPO4, 0·5; CaCl2, 1·0 and MgSO4, 0·5. The pH of the filter-sterilized media was adjusted to 5·0. The tannic acid medium was inoculated with a loopful of old culture of B. licheniformis and incubated at 35°C in a shaker (160 rev min−1) and grown for 20 h. This preinduced inoculum was used for further study.

Immobilization of whole cell

Sodium alginate solutions with a concentration range of 05–30 g l−1 were used for cell immobilization. Alginate was dissolved in boiling water and autoclaved at 121°C for 15 min. The preinduced bacterial cells were resuspended in phosphate buffer saline (PBS) having pH 7·0 and 9 g l−1 NaCl. The concentration of suspended cells was measured by a haemocytometer. Active bacterial cells of appropriate concentration were mixed with the sterile sodium alginate solution. The mixture was dropped into calcium chloride (CaCl2) solution (1 mol) using a peristaltic pump to obtain equal size polymeric beads (3 mm) of Ca alginate, each bead containing about 2 × 108 cells. The entire process was performed aseptically in the laminar airflow chamber. The resultant gel beads were hardened by resuspending into a fresh CaCl2 solution for 24 h at 4°C. Finally, these beads were washed with distilled water to remove excess calcium ions and unentrapped cells. The beads were then transferred to a 50-ml fermentation medium (composition same as described in inoculum preparation) in an Erlenmeyer flask (250 ml) and cultivated for the required time in a rotary shaker (160 rev min−1). All the experiments with immobilized cells have been performed in triplicate and data presented as mean ± SE.

Repeated batch cultivation

Repeated batch fermentation was carried out by decanting the spent medium every 20 h and replacing it by a fresh tannic acid medium (as mentioned earlier) after washing the alginate beads with sterile saline solution. Each fermentation cycle was carried out at standard fermentation condition.

Microscopic examinations

For observation by scanning electron microscopy (SEM), the alginate beads were cut (about 20-μm thickness) and fixed with 2% (v/v) gluteraldehyde. After dehydration in ethanol series (30–100%), the samples were dried, coated with gold (Vassilev et al. 1993) and observed under SEM (Jeol, Model JSM-5200; Japan).

Assay of tannase

Tannase activity in the fermented medium was determined by the colorimetric method of Mondal et al. (2001). For the assay, 0·1 ml of enzyme was incubated with 0·3 ml of substrate tannic acid (1·0% w/v in 0·2 mol l−1 citrate buffer, pH 5·0) at 50°C for 30 min. The reaction was terminated by the addition of 3 ml of bovine serum albumin (BSA) solution (1 mg ml−1), which also precipitates the residual tannic acid. A control reaction was also performed simultaneously with heat-denatured enzyme. The tubes were then centrifuged (5000 g, 10 min) and the precipitate was dissolved in 2 ml of sodium dodecyl sulfate (SDS)-triethanolamine (1% w/v, SDS in 5% v/v, triethanolamine) solution. The absorbency was measured at 530 nm after addition of 1 ml of FeCl3 (0·13 mol l−1).

The specific extinction coefficient of tannic acid at 530 nm was 0·577 (Mondal et al. 2001). Using this coefficient, one unit of tannase activity was defined as the amount of enzyme which is able to hydrolyse 1-mmol l−1 of substrate tannic acid in 1 min at 50°C and pH 5·0.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Effect of alginate concentration

The production of tannase improved with increasing alginate concentration and reached a maximum yield of 0·56 ± 0·03 U ml−1 at 20 g l−1 alginate (Fig. 1). At low alginate concentrations (5·0 g l−1), rapid leakage of the cells from the beads occurred compared with higher alginate concentrations (Fig. 2).

image

Figure 1.  Effect of different alginate concentrations [g l−1: 5 (-□-), 10 (-•-), 20 (-bsl00001-), 30 (-♦-)] in calcium alginate beads on tannase production by Bacillus licheniformis KBR6 cells. Fermentation was carried out in a 250-ml Erlenmeyer flask containing 100 ml medium at 35°C and 160 rev min−1 shaking condition.

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image

Figure 2.  Effect of alginate concentration [g l−1: 5 (-○-), 10 (-•-), 20 (-□-), 30 (-♦-)] in calcium alginate beads on cell leakage of Bacillus licheniformis KBR6 in culture medium. Immobilized cells beads were grown in a 250-ml Erlenmeyer flask containing 100 ml medium at 35°C. The concentration of leaked cell in the fermented medium was optometrically measured and the optical density value was converted into mg ml−1 using a standard curve.

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Effect of initial cell loading

The effect of initial cell loading (ICL) was tested by varying the number of alginate beads from 50 to 500 beads per flask (each bead contained about 2 × 108 viable cells). It was found that optimum enzyme production occurred with ICL of 300 beads per flask. Higher or lower inoculum levels resulted in reduced enzyme yield (Fig. 3).

image

Figure 3.  Effect of initial cell loading on tannase production by immobilized Bacillus licheniformis KBR6. Each bead contains about 2 × 108 bacterial cells. Fermentation was carried out in shake flask for 20 h.

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Effect of substrate concentration

Various tannic acid concentrations (0·5–3·0%, w/v) were used as carbon substrate in the production medium (Fig. 4). The production of tannase increased with increasing tannic acid concentrations and reached a maximum yield of 0·56 ± 0·02 U ml−1 at 2% (w/v), whereas maximum tannase was produced by free cells when culture media contained 1·5% (w/v) tannic acid.

image

Figure 4.  Effect of tannic acid concentration (0·5–3·00%, w/v) on tannase production by Bacillus licheniformis KBR6 cells as free (-○-) as well as immobilized (-•-) in calcium alginate beads. Bioconversion occurred in 250-ml Erlenmeyer flask containing 100 ml of medium and 300 beads at 35°C temperature for 20 h.

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Comparisons of tannase production by free and immobilized Bacillus licheniformis KBR6

In the case of free cells, tannase production reached a maximum level of 0·33 ± 0·02 U ml−1 at 18 h, remained constant up to 20 h, and then declined (Fig. 5). Significant tannase production from immobilized B. licheniformis cells was also observed at the beginning of the cultivation process and it remained at a higher level after 20 h (Fig. 5). At 20 h of cultivation, tannase production by immobilized cells was nearly 1·7 times more than that from the free cells (Fig. 5).

image

Figure 5.  Kinetics of tannase production by free (-○-) and immobilized (-•-) Bacillus licheniformis KBR6 cells. Bioconversion was carried out in 250-ml Erlenmeyer flask containing 100 ml of enriched tannic acid medium with initial same quantity (c. 5 × 1010 cell) of free and immobilized cells.

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Reuse of the immobilized cells

The semicontinuous fermentation was carried out to investigate the ability of the immobilized bacteria for tannase production. The highest activity of 0·55 ±0·02 U ml−1 was obtained at the third repeated cycle. Significant tannase production (0·32–0·55 U ml−1) was noted between the first ten cycles (Fig. 6).

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Figure 6.  Study of repeated batch fermentation of immobilized Bacillus licheniformis for tannase production. Each cycle was carried out for 20 h in a rotary shaker. After each cycle the beads were washed with sterilized saline (0·9% NaCl) and incubated with fresh tannic acid medium.

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SEM studies

The structure of immobilized B. licheniformis KBR6 cells was studied under an SEM. It was observed from the SEM photographic plates that in alginate beads, cells were randomly distributed and remained in normal shape and size (Fig. 7).

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Figure 7.  Scanning electron microscopic observations of entrapped Bacillus licheniformis KBR6 cells in calcium alginate beads [Near the surface of the bead (a), the cross section of bead (b)].

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cell immobilization is a useful technology in the biotechnology industry for the production of continuous extracellular enzymes. There is growing demand and application of tannase in different sectors, especially tea, dye and pharmaceutical industries.

Immobilized B. licheniformis KBR6 cells indicated that: (i) the tannase production was comparatively higher from immobilized cells in Ca-alginate beads than that by free cells; (ii) the operational stability of the immobilized cells for tannase synthesis was retained at a higher level for long time.

In the present study, biocatalyst beads were initially prepared by varying the concentration of alginate solution with the aim of determining the impact of different constraints on the physiological behaviour of immobilized B. licheniformis cells. It has been found that increase of alginate concentration beyond 20 g l−1 was accompanied by a decrease in yield of tannase. Simultaneously, the concentration of leaked cells into the cultured media gradually decreased with the increase of the alginate concentration. This revealed that the strength of the bead was improved at higher alginate concentrations, which limited the growth of cells and enzyme release. In contrast, beads with low alginate concentrations were relatively soft and favoured leakage of entrapped bacteria. These results are in agreement with other investigations (Martinsen et al. 1992).

The effect of ICL in the form of the number of alginate beads per flask on tannase production was studied. A correlated improvement of enzyme production was observed by increasing the cell loading up to 300 beads per flask. This indicates that the amount of tannic acid per cell (nutrient per bead) ratio is an important determining factor for tannase biosynthesis. Beshay (2003) reported similar data on alkaline protease production.

The present study reveals that free bacterial cells produced maximum enzyme in 1·5% (w/v) tannic acid in culture media, whereas immobilized cells, in 2%. Generally, tannic acid is considered a bactericidal compound, as it contains numerous phenolic groups that can easily bind and inactivate proteins and other macromolecules (Scalbert 1991). This result indicates that the Ca-alginate layer protects the bacterial cells from cytotoxic attack of tannic acid. This is a useful characteristic of immobilized cells and it can be exploited for de-tannification or bioconversion of higher tannin containing materials. Similar observations are explicitly reported by Misro et al. (1997) in the case of tannase production from free and immobilized Rhizopus oryzae.

The tannase production increased gradually up to the third repeated batch cycles. Increased tannase productivity of the immobilized cells during the early cycles may be because of the proper adaptation in this microenvironment and appropriate growth of cells in the bead. Microscopic photographs revealed that cells were randomly distributed in Ca-alginate beads without any morphological change. This indicated that Ca-alginate became a suitable and healthy carrier system for immobilization of this bacterium.

To conclude, the results showed that Ca-alginate entrapment is a promising method of immobilization of B. licheniformis for tannase production. It is significant that this is the first report of bacterial cell immobilization for tannase production. Alginate is a nontoxic product and therefore, immobilized bacterial cells can be useful for large-scale production of tannase. Its applications for de-tannification in food industries cannot be denied.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Financial support offered by the University Grant Commission, New Delhi, India is thankfully acknowledged.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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