THE EFFECTS OF MUTAGENIC TREATMENT ON THE RATE OF CELLULASE PRODUCTION BY ASPERGILLUS NIGER ISOLATED FROM THE SOIL
THE EFFECTS OF MUTAGENIC TREATMENT ON THE RATE OF CELLULASE PRODUCTION BY ASPERGILLUS NIGER ISOLATED FROM THE SOIL
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Date
2007-07
Authors
DAMISA, DURO
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Abstract
Cellulases present great and exciting possibilities for use in several biotechnological
processes especially lignocellulosic bioconversion. Since microbial sources of cellulases
appear to be the cheapest option, the search for strains of microorganisms with robust
characteristics is open ended. Conidia of parental strains of Aspergillus niger isolated from
soil with cellulolytic capabilities on Mandel’s medium were mutagenised with U.V
irradiation, hydroxylamine and acridine orange independently. Visual agar plate detection
system using Mandel-Weber screening medium was used to identify mutants with
increased cellulase activity. The selected mutant coded Aspergillus niger AH3, obtained
after treatment with hydroxylamine was tested for the production of cellulase on
carboxymethylcellulose, bagasse, corn cob and corn straw in shake flask incubated at
320C. The crystalline lignocelluloses were milled and fractionated into two particle sizes of
210μ and 500μ for testing cellulase activity. Portions of unscreened ground crystalline
lignocellulosics were pretreated in two concentrations (0.5M and 2M) of both acid
(sulphuric acid) and alkali (sodium hydroxide) independently and were left for varying
residence time of one hour or three hours in the digester. Incubation temperature, pH,
inoculum concentration, soluble protein and proximate constituents were all investigated.
Component cellulase enzymes: exoglucanase, endoglucanase and β glucosidase were
evaluated and compared with Trichoderma reesei cellulase preparation (ECONASE CEP)
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as control. Aseptic conditions were used in all the growth studies. Supernatants of crude
enzyme were taken and assayed at 24 hours interval. The optimum pH of 4.8 was found to
have strong influence on enzyme production. Maximum enzyme activity was at 96 and 120
hours for the mutant and wild strains respectively. Optimum spore concentration for
cellulase expression was 1.0 x 106 spores/ml. Higher spore concentration was not
advantageous while lower concentration resulted in prolonged lag. Optimum incubation
temperature of the crude cellulase from the mutant was 500C. Soluble protein recovery
correlated with cellulase production and cellulose material utilization by the organism.
Particle size and compositional variance of the lignocellulosics were found to have a
substantial influence on the enzyme expression by the organism. Larger particle size
(500μ) gave low yield of cellulase whereas the smaller size fraction (210μ) gave a
corresponding high yield of cellulase. Generally, enzyme expression in the lignocellulosics
was in the order: bagasse > corn cob > corn strain. Alkali pretreated residues gave
significantly higher cellulase yield than acid treated residues. Non-pretreated residues gave
only low enzyme titers. Maximal activity of exoglucanase and endoglucanase was reached
at 96 hours. Mutant strain showed improved production of the cellulase in the pretreated
lignocellulosics by 51%, 40% and 16% for bagasse, corncob, and corn straw respectively.
Exoglucanase, endoglucanase and β–glucosidase activities of the mutant increased by
48%, 35% and 64% respectively. The mutant was found to be an efficient producer of the
β-glucosidase; 25% higher than the ECONASE. Maximal cellulose yield, 0.068IU/ml/min,
was obtained from bagasse digested in 2M NaOH for one hour and fermented with the
mutant. This translated to 39% increase in enzyme expression when compared with nontreated
bagasse of 0.049IU/ml/min. Mutations using sub-lethal concentrations of
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hydroxylamine for repeated bouts produced an elite strain of Aspergillus Niger with
improved rate of cellulose production. Sugarcane gasses therefore has a great potential as
a raw material for commercial production of cellulose.
Description
BEING A DISSERTATION SUBMITTED TO THE POSTGRADUATE
SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF DOCTOR OF
PHILOSOPHY (Ph.D) IN MICROBIOLOGY
DEPARTMENT OF MICROBIOLOGY
FACULTY OF SCIENCES
AHMADU BELLO UNIVERSITY
ZARIA
JULY, 2007
Keywords
EFFECTS,, MUTAGENIC,, TREATMENT,, RATE,, CELLULASE,, PRODUCTION,, ASPERGILLUS,, NIGER,, ISOLATED,, SOIL