Production of tannase by aspergillus niger using mangrove (rhizophora apiculata) bark under solid substrate fermentation.
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Date
2010-08
Authors
Wee Yee, Tan
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Abstract
Rhizophora apiculata bark is tannin rich waste material from charcoal
industry, and was utilized as the solid substrate for the production of tannase in solid
substrate fermentation system. Upon improvement of physical and chemical
parameters via a simple shake flask system, combination of0.75 mm and 2.00 mm of
substrate particles, Czapek-Dox medium (pH7) as moisturizing agent, initial
moisture content of 1:1.5 (w/v), incubation temperature at 30° C, mixing at every 24
hours and inoculum size of 1 x 106 spores/ml showedtannaseachieved4.97 U/g of
fermented substrate on the 61
h day of cultivation time. After supplementation of 5%
(w/w) tannic acid and 1% (w/w) of urea, tannase achieved 39.34 U/g of fermented
substrate after 6 days of cultivation. Enzyme activity increased 1280.35% compared
to initial profile (2.85 U/g of fermented substrate). A tray system (15.0 em x 15.0 em
x 7.5 em) was carried out to scale-up the production of enzyme. After improvement
of several key process parameters, substrate bed height of 0.6 em (50 g of solid
substrates), moisture content at 1:1.5 (w/v), mixing at every 48 hours during the
cultivation period, inoculum size of 1 x l 06 spores/ml and 6 days of cultivation time
showed that tannase was 63. 24 U/g of fermented substrate. A further scale-up with 1
XXV
kg of solid substrates via a bigger tray system (32 em x 22 em x II em) showed that
tannase production achieved 127. 13 U/g of fermented substrate on the day 14 of
cultivation. Enzyme activity increased 4360.70 % compared to initial profile
(2.85U/g) from shake flask system. Crude enzyme was concentrated and
purifiedabout 55 fold with specific activity of 1.1 0, recovery at 0.02 % and molecular
weight of 55.92 kDa. Optimum reaction temperature for both crude and purified
enzymes was 70°C and stable at the range of 30-70°C around 1 hour. Optimum pH
for crude and purified enzyme was pH 6 and pH 5.5, respectively. They were stable
at pH 5.0 and 5.5, respectively, around 1 hour. Tannic acid at 0.4% (w/v) was
specific substrate for both crude and purified tannase. Metal ions of At3+, Ba2+, Ca2+,
Co2
+, Cu2+, Fe2+, K\ Li2+, Mg2+, Mn2+, Na+ an~ Zn2+ inhibited the crude and purified
tannase activities. Substrate degradation evaluation with the aid of light microscope,
SEM and TEM showed the effect ofbiodegradation by Aspergillus niger and activity
of tannase on fermented substrate.
Description
Keywords
Aspergillus niger , (Rhizophora apiculata) , Solid substrate fermentation.