Abstract: Tannin from Cassia siamea was fermented in a bioreacter by Aspergillus aculeatus DBF9 for gallic acid production. Production of gallic acid was observed to be highest at 30°C with an initial pH of 5.5 after 36 h. Aeration at the rate of 4 L min-1 and agitation speed of 100 rpm was found to be suitable for maximum gallic acid production. Medium containing 3% tannin was found suitable for highest gallic acid production.
INTRODUCTION
Gallic acid (3,4,5-tri hydroxy benzoic acid), one of the byproducts of tannin hydrolysis has several applications in chemical and pharmaceutical industries. It has huge demand in India though it is an imported item. It is mainly used for manufacture of propyl gallate (Weetal, 1985; Gathon et al., 1989) and preparation of trimethoprim (Hadi et al., 1994). Gallic acid is also used in the manufacture of pyrogallol, inks, photographic developer, in testing free mineral acids, dihydroxy acetone and alkaloids (Budavari, 1989). The worldwide annual demand of gallic acid is about 8000 tons. Conventionally gallic acid is produced by acid hydrolysis of tannins, but this process releases a large amount of toxic effluent that causes environmental hazards. Production of gallic acid through the fermentation of tannic acid using suitable tannase producing microorganism is preferred today.
A different fungal (Banerjee et al., 2001; Mukherjee and Banerjee, 2004; Misro et al., 1997; Pourrat et al., 1982, 1985; Vermeire and Vandamme, 1990) and bacterial (Deschamps et al., 1980; Deschamps and Lebeault, 1984; Mondal et al., 2001a) strain has been utilized for tannase and gallic acid production. Pourrat et al. (1985) recovered 30% gallic acid through fermentation of tara pod powder by A. niger. Deschamps et al. (1980) studied chestnut tannin degradation by Corynebacterium sp. in a 2 L jar-fermenter. Production of gallic acid by Aspergillus sp. from gallo-tannin was studied by Vermeire and Vandamme (1990). Misro et al. (1997) studied the production of gallic acid using the immobilized cells of Rhizopus oryzae. They found that 78.5% tannin conversion was possible after 4 day of incubation. Mukherjee and Banerjee (2004) reported gallic acid production from tannins of myrabolan and teri pod powder through modified solid state fermentation by two fungal strains Rhizopus oryzae and Aspergillus foetidus. Therefore it has been found that production of gallic acid through fermentation is possible provided that plenty of raw material (tannin) and desired organism for bioconversion are in hand.
In the present investigation for the first time gallic acid production by Aspergillus aculeatus DBF9 has been carried out in Eyela 5 L bioreactor through liquid submerged fermentation of locally available raw plant tannin.
MATERIALS AND METHODS
Microorganism
One of a potent gallic acid producing fungal strain Aspergillus aculeatus
DBF9, has been used in the present study.
Culture Condition
Production of gallic acid was made from raw tannin through fermentation.
Fermentation was carried out in a 5 L jar-fermenter (Eyela, MBF-500PE, Japan)
connected with automatic control devices consisting of a standard cylindrical
glass culture vessel with a working volume of 3 L. The tank diameter was 15
cm and height to vessel diameter ratio was 1.6. The culture medium consisted
of raw tannin (Cassia siamea plant extract), 20.0 g L-1; K2HPO4,
0.5 g L-1; KH2PO4, 0.5 g L-1; MgSO4.7H2O,
1.0 g L-1 and (NH4)2 HPO4, 3.0 g
L-1. For fermentation, the culture medium was inoculated with active
preculture of A. aculeatus spores and was cultivated for a period of
72 h. Fermented broth was removed from the vessels at every 6 h interval by
a hand pump for analysis.
Effect of pH on fermentation was studied by adjusting the initial pH of the medium at various values (5.0, 5.5 and 6.0). Fermentation was conducted at different temperature (30-40°C), aeration (4.0-6.0 L min-1) and agitation (50-150 rpm). Effect of different concentration of tannin (1.5-6.0%) was examined with raw tannin from Cassia siamea plant.
Tannase Activity
Tannase activity was determined following the method of Mondal et al.
(2001b) Enzyme solution (0.05 mL) was incubated with 0.3 mL of 1.0% (w/v) tannic
acid, in 0.2 M acetate buffer (pH 5.0) at 40°C for 10 min and then the reaction
was stopped by addition of 2.0 mL bovine serum albumin (BSA) (1 mg mL-1),
which precipitated the remaining tannic acid. A control reaction was done side
by side with heat denatured enzyme. The tubes were then centrifuged (5,000 x
g, 10 min) and the precipitate was dissolved in 2.0 mL of SDS-triethanolamine
(1% w/v SDS in 5% v/v triethanolamine) solution and the absorbency was measured
at 550 nm after addition of 1.0 mL of FeCl3 (0.13 M) (Systronics
spectrophotometer 105).
One unit of tannase activity is defined as the amount of enzyme required to hydrolyze 1.0 μmol of ester linkage of tannic acid in 1 min under specified condition.
Estimation of Gallic Acid
Gallic acid in the culture broth was estimated by the method of Bajpai and
Patil (1996). Culture supernatant was diluted to 100 fold in 0.2 M acetate buffer,
pH 5.0. The absorbance was recorded at two selective wavelengths of 254.6 and
293.8 nm. The concentration of gallic acid was measured using specific extinction
coefficient, by the following equation; Concentration of gallic acid (μg
mL-1) = 21.77 (A254.6)-17.17 (A293.8).
Biomass Estimation
Growth of the organism was estimated after drying the biomass at 60°C
for 24 h.
All the experiments were performed in triplicate and the results represented here are the mean of the three.
RESULTS AND DISCUSSION
Selection of a substrate for enzyme and subsequent product formation by fermentation depends on several factors like cost, availability and suitability of the substrate for obtaining the desired product of fermentation and thus requires screening of several agro-industrial residues (Pandey et al., 1999). In the present experiment, cost-effective substrates for biotransformation were selected on the basis of their tannin content and it was found that Cassia siamea plant tannin gives better result (results are not shown). Tannin obtained from Cassia siamea was selected as a substrate for optimization of gallic acid production through fermentation of A. aculeatus DBF9. Mukherjee and Banerjee (2004) used tannin rich plant material myrabolan and teri pod powder as a source of raw material for tannase and gallic acid production by Rhizopus oryzae and Aspergillus foetidus.
Effect of Temperature
The effect of cultivation temperature (25-40°C) on the growth, tannase
and gallic acid production by A. aculeatus in batch fermenter was studied
and is presented in Fig. 1. It was observed that during fermentation
the growth of the organism was highest at temperature 30°C (Fig.
1A). The tannase activity in culture media increased rapidly during the
initial period of cultivation (Fig. 1B). The highest tannase
activity of 3.93 U mL-1 was noted in the medium after 36 h of cultivation
at 30°C. Negligible amount of tannase was produced at 25 and 40°C. It
was also found that production of gallic acid varied with incubation temperature.
| Fig. 1: | Effect of temperature on growth (A), tannase (B) and gallic acid © production (●, 25°C; ■, 30°C; ▲, 35°C; ♦, 40°C) |
Maximum gallic acid (Fig. 1C) of 6.0 mg mL-1 was produced at 30°C after 36 h of incubation. Temperature of the fermentation medium is one of the crucial factors that have direct effect on the production of tannase and gallic acid. Earlier 35°C was found suitable for tannase production by A. niger (Pourrat et al., 1985).
Effect of pH
Effect of pH on gallic acid production was studied in relation to tannase
production for 72 h and is presented in Fig. 2. Maximum amount
of gallic acid (6.21 mg mL-1) was obtained after 36 h when the initial
pH of fermentation medium was kept at 5.5 (Fig. 2B). This
may be due to maximum production of tannase in the same pH. Further increase
of medium pH decreases gallic acid production. The pH is very important factor
for tannase and gallic acid production (Barthomeuf et al., 1994). The
maintenance of a favourable pH is very essential for the successful fermentation
of gallic acid (Vermeire and Vandamme, 1988).
Effect of Aeration
Tannase and gallic acid production by A. aculeatus was tested at
different aeration rate (3.0, 4.0 and 5.0 L min-1); the results are
shown in Fig. 3. The highest tannase production was obtained
at an aeration level of 4 L min-1 (Fig. 3B). But
above or below this aeration level productions of tannase become reduced. Gallic
acid (6.4 mg mL-1) production was also found maximum after 36 h with
the same aeration rate of 4 L min-1. Sufficient aeration of the medium
as well as supply of oxygen is essential for adequate growth of the microorganism
and production of tannase. Pourrat et al. (1987) mentioned that a critical
level must be maintained for aeration as in high oxygenation causes oxidation
of tannins and catabolism of the gallic acid.
| Fig. 2: | Effect of initial medium pH on tannase (A) and gallic acid (B) production (●, 5.0; ■, 5.5; ▲, 6.0) |
| Fig. 3: | Effect of aeration on tannase (A) and gallic acid (B) production (●, 3 L min-1; ■, 4 L min-1; ▲, 5 L min-1) |
| Fig. 4: | Effect of agitation speed on tannase (A) and gallic acid (B) production (●, 50 rpm; ■, 100 rpm; ▲, 150 rpm) |
Highest gallic acid production was recorded after 36 h at aeration of 4 L min-1. Higher aeration (5 L min-1) rate resulting lower gallic acid production. This may be due to the oxidation of tannins. Under favourable conditions the fungal mycelia simultaneously synthesized an antioxidant, which prevented tannin oxidation (Barthomeuf et al., 1994).
Effect of Agitation
Tannase production was enhanced in culture media with the increase in the
agitation speed upto 100 rpm (Fig. 4A). At the same agitation
speed tannase production after 36 h of fermentation was found 4.18 U mL-1
but after 48 h its production was reduced to 3.84 U mL-1. Gallic
acid (6.64 mg mL-1) production reached maximum after 36 h with 100
rpm (Fig. 4B). Lower agitation speed may limit the nutrient
availability to the mycelium and at higher agitation speed, mycelial growth
rate decreased due to the breakage of mycelial wall. Though Barthomeuf et
al. (1994) found that 300-450 rpm agitation speed was suitable for tannase
production in A. niger.
Effect of Tannin Concentration
The effect of tannin concentration (1.5-6.0%, w/v) on tannase and gallic
acid production by Aspergillus aculeatus DBF9 was carried out in 5 L
jar fermenter with different concentrations of Cassia siamea tannin (Fig.
5). The maximum amount of gallic acid (6.8 mg mL-1) was obtained
after 36 h in the medium containing 3% tannin. Production of tannase was found
maximum (4.12 U mL-1) with the same concentration of tannin. Reduction
of tannase production was noticed at higher concentration of tannin. This indicates
that low tannin concentration is a good inducer for tannase biosynthesis. It
has a significant impact on large-scale production of gallic acid in industrial
basis.
| Fig. 5: | Effect of tannin concentration (w/v) on tannase (A) and gallic acid (B) production |
CONCLUSIONS
This is for the first time; gallic acid production has been made from the raw tannin of Cassia siamea through fermentation by A. aculeatus DBF9. Plenty of such cost effective raw material ensured the possibility for exploitation of this organism in large scale production of gallic acid.
ACKNOWLEDGMENT
The author (DB) is thankful to CSIR, New Delhi for fellowship.