INTRODUCTION
Long-term economic and environmental concerns have resulted in a great amount
of research in past couple of decades on renewable source of liquid fuels to
replace fossil fuels. The utilization of cellulosic biomass continues to be
a subject of worldwide interest in view of fast depletion of our oil reserves
and food shortages (Kuhad et al., 1997). Important
distinguishing features of cellulose biomass among potential feeds for biological
processing include low purchasable price, potential for supply on very large
scale, recalcitrance to reaction and heterogeneous composition. Cellulosic waste
may be converted to products of commercial interest such as glucose, soluble
sugars, alcohol, single cell proteins (Ojumu et al.,
2002). The key element in bioconversion process of lignocellulosics to these
useful products is the hydrolytic enzymes mainly cellulases (Immanuel
et al., 2007). The saccharification process of cellulose waste relies
on participation of cellulolytic organisms and their cellulase enzymes (Lynd
et al., 2002). In addition, cellulolytic enzymes have been exploited
for commercial application in a variety of industries. However, the saccharification
process has not yet reached to the level of commercialization due to many factors-complexity
of cellulose structure, production of celluloses in low amounts by cellulolytic
organisms due to carbon repression, high cost of cellulase production and poor
yields of glucose (Gregg and Saddler, 1996). In order
to enhance the rate of saccharification, it has become necessary to search for
highly efficient cellulolytic organisms with secretion of higher titers of cellulase.
Members of fungal genus Trichoderma and Aspergillus have been
extensively studied, particularly due to their ability to secrete cellulose
degrading enzymes. The strains that have been mutagenized and genetically modified
to obtain an organism capable of producing high levels of cellulases (Vu
et al., 2009). The use of different mutagenic agents for strain improvement
was demonstrated by Parekh et al. (2000). Simultaneous
treatment with NTG and Ethidium bromide improved Fpase and CMCase activities
than wild type fungi (Chand et al., 2005).
The purpose of this study was to screen fungal culture for producing high levels
of cellulase enzyme by chemical mutations and depolymerization of lignocellulosics
by pretreatment with various chemical agents for cellulase. In this study pea
seed husk which is a cheap and locally available lignocellulosic waste was tested
as a novel substrate for cellulase production by Mutant Aspergillus niger.
MATERIALS AND METHODS
Microorganism: Aspergillus niger was isolated from the cotton
industry effluent soil collected from Nandyal, Andhra Pradesh, India (Narasimha
et al., 1999). This strain was cultivated on potato dextrose agar
medium at 28°C for 7 days.
Screening of Aspergillus niger for cellulase production: The cellulolytic nature of Aspergillus niger was confirmed first through the screening test. To this 1% of CMC was amended with Czapeck-Dox agar media and the pH was adjusted to 5. The media was poured into sterile Petri dishes, after solidification of media a small hole was made on the centre of Petri dish aseptically and the culture spores were added to this centre. The plates were incubated for 3 days at 30°C and 2 days at 50°C. After incubation the plates were stained with 1% Congo red solution for 15 min, after that the Congo red stain was neutralized with 1 M NaCl solution. The yellow color zone formation concern the ability of cellulose utilization and enzyme activity of fungal culture.
Mutation studies: The spore suspension of the Aspergillus niger was used for EMS chemical treatment. 4mL of spore suspension was added to 12 mL of EMS solution (4 mg mL-1) and the reaction was allowed to proceed. Two milliliter of this solution was taken at intervals of 30, 60, 90,120,150,180 and 210 min and centrifuged immediately for 10 min at 5000 rpm and the supernatant solution was decanted. Cells were washed three times with sterile water and resuspended in 10 mL of sterile phosphate buffer. The samples were serially diluted in the same buffer and plated over Czapeck-Dox agar medium. A total 10 colonies (designated as GNEM-1 to GNEM-10) were selected from the plates showing less than 1% survival rate (60 and 90 min EMS treated spore suspension) and tested for cellulase production.
Preparation of fungal spore inoculum for cellulase production: The mutant fungal culture was grown on Czapeck-Dox agar slants and they were incubated at room temperature for 7 days. After incubation 3 mL of sterile distilled water was added for each slant. Fungal spore concentration was determined by haemocytometer. Inoculum density was 2x106 spores were used for cellulase production.
Substrate: The substrate Pea seed husk was collected from local agricultural fields and it was pretreated with sodium hydroxide, hydrochloric acid and hydrogen peroxide and exposed to cellulolytic attack.
Preparation of substrates: Pea seed husk was sun dried for a period
of three weeks and subsequently oven dried slowly at 50°C for 2 days. The
dried substrate was chopped into bits, pulverized into coarse particle sizes
and then washed in several changes of hot water in order to remove the residual
sugars (Rezende et al., 2002).
Acid treatment (HCl): In this process the pea seed husk was soaked in 1N HCl in the ratio 1:10 (substrate: solution) for 60 min at room temperature and then washed with double distilled water for removal of chemicals and autoclaved at 121°C for one hour. The treated substrate was filtered for free of fibers and neutralized by washing with dilute aqueous sodium hydroxide. Then the treated substrate was washed with double distilled water until the filtrate becomes neutral. The substrates were dried at 60°C for 12h on hot air oven and used for enzyme assay.
Alkali treatment (NaOH): Pea seed husk was soaked in 1 N NaOH solution in the ratio 1:10 (substrate: solution) for 60 min at room temperature. After that, the substrate was subjected to washing with double distilled water for free of chemicals and autoclaved at 121°C for one hour. Then the treated substrate was washed with double distilled water until the wash water turned to become neutral pH and dried at 60°C for 12 h on hot air oven.
Hydrogen peroxide treatment (H2O2): Hydrogen peroxide (H2O2) at concentrations of 5 and 10% were used to pretreat the pea seed husk. The pH of the suspension was adjusted to 11.5 with 0.1 N NaOH and stirred gently at room temperature 25°C for 20 h. The contents were filtered and washed with distilled water until pH returns to neutral. The treated sample was dried at 110°C for overnight.
Filter paper saccharifying activity (Fpase): Filter paper sachharifying
activity in the culture filtrates was determined by the method of Ghose
(1987). It is a combined assay for endo β-1,4 glucanase and exo β-1,4glucanase.The
standard reaction mixture containing 50 mg of What man No.1 filter paper strips
(1x6 cm) as a substrate, suspended in a mixture containing 1 mL of 0.05 M sodium
citrate buffer (pH 4.8) and 0.5 mL of enzyme source. The enzyme control was
also prepared simultaneously by adding distilled water instead of enzyme. This
mixture was incubated for one hour at 50°C in water bath. The reducing sugar
was estimated by dinitrosalicylic acid method (Miller,
1959). After incubation; 3 mL of DNS reagent was added to each test tube
and boiled for 5 min in a boiling water bath. After boiling, transferred to
a cold water bath. Added 20 mL of distilled water. Mixed completely inverting
the tube several times so that the solution separates from the bottom of the
tube at each inversion. The color developed in the test tubes was read at 540
nm in a spectrophotometer. The enzyme activity was expressed in filter paper
units. Filter paper units were defined as the amount of enzyme releasing μ
moles of reducing sugar from filter paper per minute per mL.
Endoglucanase assay (CMCase): Endoglucanase activity of fungal culture
was quantified by Carboxy methyl cellulase method (Ghose,
1987). According to this method, 1 mL of 2% carboxy methyl cellulose as
a substrate was added to the mixture containing 1 mL of 0.05 M sodium citrate
buffer (pH 4.8) and 0.5 mL of enzyme. This mixture was incubated at 50°C
in a water bath for 30 min. The reducing sugar produced in the reaction was
estimated by DNS method. After incubation, 3 mL of DNS reagent was added to
each test tube and boiled for 5 min in a boiling water bath. After boiling,
transfer to a cold water bath and added 20 mL of distilled water. Mixed completely
inverting the tube several times so that the solution separates from the bottom
of the tube at each inversion. The color developed in the test tubes was read
at 540 nm in a spectrophotometer. The enzyme activity was expressed in terms
of CMC units. CMC units were defined as the amount of enzyme releasing μ
moles of reducing sugar from the substrate per minute per mL.
β-glucosidase assay: β-glucosidase activity in the culture
filtrates was determined by the method of Herr (1979).
According to this method, 0.2 mL of 5 mM ρ-nitro phenyl β-D-glucopyranoside
(PNPG) as a substrate was added to the mixture containing 1.6 mL of 0.05M sodium
citrate buffer (pH 4.8) and 0.2 mL of enzyme solution. After incubation, for
30 min at 50°C the reaction was stopped by the addition of 4 mL of 0.05
M NaOH-glycine buffer (pH-10.6) and the yellow colored para-nitro phenyl was
measured at 420 nm in spectrophotometer. One unit of β-glucosidase activity
is defined as that releasing μ mole of PNP from PNPG per minute per mL.
Protein estimation: Protein content in culture filtrate was determined
by Lowrys method (Lowry et al., 1951).
The culture filtrate with appropriate dilution was mixed with 5 mL of alkaline
solution. After 10 minu, 0.5 mL of folin-ciocalteau reagent was added. After
30 min of incubation in a dark place the colour developed was measured by spectrophotometer
at 660 nm.
RESULTS AND DISCUSSION
The fungal strain Aspergillus niger was screened for cellulase enzyme
production. The formation of clear yellow zone of hydrolysis concerns its ability
for cellulase production (Fig. 1).
Pea seed husk, a cheap and locally available lignocellulosic waste was tested
to find out whether it could support the production of cellulases by Aspergillus
niger under submerged fermentation. The Fpase, CMCase and β-glucosidase
activities of wild strain grown on pea seed husk were 3.2 IU, 2.8 and 0.4 per
mL per min, respectively (Fig. 2).
• |
Filter paperase (FPase) is expressed in terms of filter paper
units. One unit is the amount of enzyme in the filtrate that releasing 1μmole
of reducing sugar from filter paper/mL/min |
• |
Carboxymethyl cellulose (CMCase) is expressed in terms of units. One unit
is the amount of enzyme releasing 1 μmole of reducing sugar from carboxymethyl
cellulose/mL/min |
• |
One unit of β-glucosidase activity is defined as the
amount of enzyme liberating 1 μmole of p-nitro phenol/mL/min |
|
Fig. 1: Aspergillus niger showed clear yellow zone
of hydrolysis which indicates CMC degradation |
|
Fig. 2: Cellulolytic activity of wild strain A. niger
grown on pea seed husk. Values represented are the Mean of two separately
conducted experiments |
The wild strain Aspergillus niger was subjected to ethyl methane sulfonate mutations and ten mutant strains from GNEM1 to GNEM10 were tested for total cellulase production (Fig. 3). Among the 10 EMS mutants GNEM7 showed maximum total cellulolytic activity (6.91 IU/mL/min) and this strain was selected for cellulase production on pre-treated pea seed husk.
In order to obtain a high amount of fermentable sugars various chemical pretreatments have been used in the present study. The combined effect of acid (1 N HCl), alkali (1 N NaOH) and oxidation (5 and 10% H2O2) pre-treatment methods was examined. The cellulolytic activities of GNEM7 on pea seed husk treated with different chemicals was determined (Fig. 4) and it showed maximum cellulase activities (FPase-19.73 IU, CMCase-18.09 IU and β-glucosidase-1.73 IU) on 10% H2O2 pre-treated pea seed husk than that of the remaining pre-treatments.
|
Fig. 3: Total cellulolytic activity of ethyl methane sulfonate
mutants grown on pea seed husk. Values represented are the Mean of two separately
conducted experiments |
|
Fig. 4: Cellulase activities of EMS mutant (GNEM7) on chemically
pretreated pea seed husk. Values represented are the Mean of two separately
conducted experiments |
Maximum reducing sugar concentration 1.6 mg mL-1 (Fig. 5) and protein concentration 2700 μg mL-1 (Fig. 6) were obtained when pea seed husk treated with 10% hydrogen peroxide.
Damisa et al. (2008) reported that the highest
cellulase activities on bagasse, corn cob and corn straw pre-treated with 2
M NaOH by Aspergillus niger AH3 were 0.067, 0.049 and 0.504
IU, respectively. Another study by Fatma et al.
(2010) reported that the maximum cellulase activity on rice straw treated
with 1% NaOH using Trichoderma reesei F418 was 11.17 IU g-1.
Similarly, in the present study, Aspergillus Niger GNEM7 showed
9.93 and 19.73 IU of cellulase activities on pea seed husk pre-treated with
1 N NaOH and 10% H2O2, respectively.
The total cellulolytic activity of Aspergillus niger GNEM7 on I N Hcl
pretreated pea seed husk was 9.13 IU/mL/min. Similar results were made with
Aspergillus fumigatus on Hcl pretreated wheat straw, values were found
to be 0.237 and 0.674 IU mL-1 (Dahot and Hanif,
1996). Similarly, the FPase, CMCase values found to be 0.089 and 1.023 U
mL-1, respectively when groundnut shells treated with 0.25 N HCl
(Vyas et al., 2005).
|
Fig. 5: Reducing sugar concentration in pea seed husk treated
with different chemicals. Values represented are the Mean of two separately
conductedexperiments |
|
Fig. 6: Total protein concentration in pea seed husk treated
with different chemicals. Values represented are the Mean of two separately
conducted experiments |
In the present study the cellulase activities, FPase (19.73 IU), CMCase (18.09
IU) activities were higher on 10% hydrogen peroxide pretreated pea seed husk.
A similar observation was made by Haq et al. (2008),
higher enzyme activities Fpase (5.82), CMCase (10.54), β-glucosidase (9.85
U mL-1) were obtained when sawdust treated with 5% hydrogen peroxide.
Singh et al. (2009) reported that the FPase
and CMCase activities on rice straw by Aspergillus niger were 0.96 and
0 .66 IU, respectively. The mutant Trichoderma when grown on wheat bran produced
FPase-6.2 IU and β-glucosidase 0.39 IU (Jun et
al., 2009). In the present study Aspergillus niger GNEM7 showed
higher Fpase-19.73 IU, CMCase-18.09 IU and β-glucosidase-1.73 IU activities
than the previous studies.
CONCLUSION
The production of cellulases on pea seed husk under submerged fermentation
was studied by mutant Aspergillus niger. Pea seed husk is a cheap and
locally available residue was used as a novel substrate for cellulase enzyme
production and reduces the cost of enzyme production. The enzyme activities
obtained on this substrate were maximum than the other lignocelluloses studied
earlier.
ACKNOWLEDGMENTS
We are grateful to University Grants Commission (UGC), New Delhi for providing
financial support to carry out this work.
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