Title of Thesis
Optimization of Cultural Conditions on the
Biosynthesis of Xylanase by Locally Isolated Aspergillus Niger
Department of Botany / University of the Punjab,
|Number of Pages|
|Keywords (Extracted from title, table of contents and
abstract of thesis)|
Optimization, Cultural, Conditions,
Biosynthesis, Xylanase, Isolated, Aspergillus Niger, petriplates
The present study is concerned with isolation, selection and optimization of
cultural conditions for the production of enzyme xylanase by Asperhillus niger. One hundred and four strains of A. niger were isolated from different soil samples and tested for the production of enzyme. Of all the strains tested, A. niger GCBT-35 gave maximum production of xylanase. This strain was selected for optimization of cultural conditions in shake flask. Among the four different culture media evaluated, the maximum enzyme production (225 U/ml) was obtained with M-4. Cultural conditions such as rate of enzyme synthesis (48 h) and pH (4.5) were optimized.
After optimization of the cultural conditions, the parental strain GCBT-35 was subjected to UV irradiation for 5-30 minutes. Ninety-four mutants were isolated by observing the zones of xylan hydrolysis due to xylanase activity in the petriplates. Of all the mutants tested for enzyme production, mutant BRCUV-45 gave maximum production (319 U/ml) of xylanase. The selected UV mutated strain was further improved after treatment with MNNG (50-300 µg/ml) for 10-40 min. Seventy-four chemically mutated strains of A. niger were picked up and evaluated for xylanase production in 250 nil shake flask. The mutant A. niger strain GCBTMNNG-30, being a better producer of xylanase (493 U/ml) was selected for further studies.
In shake flask, cultural conditions and nutritional requirements such as rate
of enzyme synthesis (48 h), initial pH (4.5) and level of meat extract ( 1.0 %, w/v)
as a nitrogen supplement were optimized. In another study, different agricultural by-products were tested for the production of enzyme by solid-state fermentation.
Of all the substrates examined, wheat bran moistened with distilled water gave
optimal production of xylanase (1850 U/g). The productivity of enzyme was further improved (2480 U/g) by the addition of starch (2.0 %) and ammonium
sulphate (0.2 %) to the fermentation medium. The production of enzyme in solid state fermentation was found to be maximum 72 h after inoculation.
Xylanase fermentation was carried out by five-repeated batch culture after
immobilization of A. niger conidia in sodium alginate or polyurethane foam. The
production of xylanase was increased in the second batch by A. niger strain
immobilized in the polyurethane foam. The conidia of A. niger mutant
GCBTMNNG-30 were immobilized and cultivated for xylanase production for 24 and 48 h. The production of xylanase was significantly increased as A. niger was pre germinated for 24 h and reached maximum 48 h after inoculation.
Effect of re-use of mycelium of A. niger obtained by aseptic centrifugation
of fermented broth from Ole previous batch, for the production of xylanase was
studied. The rate of substrate utilization was enhanced in the first batch of this
experiment. When mycelia were further re-used in next two batches, a decreased
rate of substrate bioconversion and production of xylanase was noticed.
Scales up studies were carried out in a 7.5 L stirred fermentor. The cultural conditions such as incubation temperature (30oC), initial pH (4.0) and size of 24 h old vegetative inoculum (4.0 %, v/v) were optimized. The optimal production of xylanase (781.4 U/ml) was achieved 48 h after the inoculation when agitation intensity was kept at 200 rpm and air supply at 2 vvm (dissolved oxygen 0.7 %). The production of xylanase was also carried out by fed-batch system in the stirred fermentor. At the end of fermentation, 80-90 % of the fermented broth was replaced by fresh sterilized medium. The batches were repeated four to five times. The product ion of xylanase was decreased sharply after the 4th batch.