PRODUCTION OF LIGNOCELLULOSES FROM ASPERGILLUS NIGER USING ARECA NUT SPADE AS BIOMASS

Areca palm (ChrysalidoCarpus lutescenes) a widely used plant having feathery arching brands with 100 leaﬂets. All these plants produce much of waste in additions to greeny and nuts. This waste of spade is used for the production of various molecules that are used in industry and pharma sector. Fermentation techniques are used to generate economically important enzymes for industrial and pharmaceutical purposes. Cellulase enzyme degrades the cellulose in between β -1, 4 glucosidic link found in lignocellulosic complex which under physical treatment is slower to degrade. The present study of Aspergillus niger for cellulose production was carried in solid state (SS) and submerged (SM) fermentations for production of cellulase enzyme. Cellulase production in SSF after 72 h of fermentation was 8.02 and in SMF activity was 2.98 per ml of cultured broth at H 6 and temperature at 30°C. Both SMF and SSF were supplemented with lactose and lactobionic acid, which acted as cellulase P production inducers. The aim of the present work was to study the effect of Areca palm spade as substrate for Aspergillus niger and its cellulase production under SMF and SSF.


INTRODUCTION
The enzymes produced by fungi are broad array like endoglucanases or CMcases and β-glucosidases. This complex of cellulases combines and acts on celluloses. For example complete cellulose hydrolysis into glucose is brought about by endoglucanases and β-glucosidases, cellobiohydrolases degrade cellulose by removing cellulose from nonreducing ends of cyclodextrins. Finally, β-glucosidases hydrolyze cellobiose in to glucose and remove glucose units from non-reducing ends of cyclodextrins. These enzymes find multiple uses in production of food, animal feed, textiles, fuel, chemical, pharmaceuticals and in waste management.
Fungi are advantageous as the enzyme production rate is higher compared to other microorganisms. Aspergillus and Trichoderma species are very well known for cellulase production. Almost all fungi of the genus Aspergillus synthesize cellulase, therefore this genus has the potential to dominate enzyme industry. The enzymes of industrial importance have been produced in submerged fermentation [SMF] because of the ease of handling and good control of environmental factors such as temperature, aeration, agitation and pH (Singh et. al;. However, solid state fermentation (SSF) techniques are better adapted to enhance the yield (Ghildyal et al,1985) along with reducing the cost of enzyme production. Filamentous fungi such as Aspergillus niger are the most commonly used microorganisms in SSF due to their ability to grow on low water substrates such as lignocelluloses.

MATERIALS AND METHODS
Microorganism -Five soil samples were collected in an Areca farm of Tarikere Taluk in chickmagalur district of Karnataka state, India. To isolate and culture Aspergillus niger, the samples and were serially diluted and spread on plates as sterile triplicates of CMC agar [wood and bhat 1988]. The cultures were identified based on morphology of the colony and microscopic examination of four isolated colonies were further maintained on PDA agar slants, and later stored at 4°C until needed (Devanathan et al;. Spore counting was done by reading spore concentration in 1% triton x -100 and determined by using a haemocytometer with a phase contrast microscope.
Pre-treatment of Areca Spade -Areca spade acquired from areca farm was ground to a fine powder using a blender. The areca biomass was hydrolysed with 1% NaOH solution. The pH was brought to neutral by repeatedly washing the pre-treated biomass with distilled water and was filtered with a muslin cloth and air dried at room temperature.
Preparation of Aspergillus niger for Inoculation-Erlenmeyer flasks were used for growing the inoculums. 100 ml of purified water was taken and 2.4 grams of PD agar was added, mixed thoroughly. The above mixture was autoclaved at 121°C for 15 minutes. The broth was further supplemented with a g/litre of Sucrose, K HPO , Sodium 2 4 Nitrate, MgSO , KCl, FeSO (Trace) for minerals. 4 4 Substrate -Powdered Arecanut spade prepared as above was used in the study as the lignocellulosic substrate. Areca spade was procured from Tarikere Taluk in chickmagalur district of Karnataka state, India. The substrate was sterilized at 121°C. Solid state fermentation of Areca Spade Biomass -Solid state fermentation was carried out in 250 ml flasks that contained 10 grams of the Areca Spade powder which was moistened with mineral salt solution, the contents were sterilized, pH was maintained at 6. The 7day old slant was flooded with 2 ml of distilled water and inoculated, the contents were mixed well and incubated at 30°C in a humidified incubator for 7 days with a gentle shake . Post fermentation the contents of the flask were mixed with 100 ml of 0.2 M [pH 4.8] Sodium acetate buffer and the contents were shaken at 180 rpm on a shaker and the filtrates of the above were centrifuged at 6000 rpm for 10 minutes and further subjected to enzyme assessment. The reagent can be kept for 2 to 3 months. Reagent C consists of 1 volume Folin-Cio calteau reagent diluted with 15 volumes water. A series of dilutions were prepared of 0.3 mg/ml BSA in the same buffer containing the unknowns, to give concentrations of 30 to 150 micrograms/ml (0.03 to 0.15 mg/ml). Added 1.0 ml each dilution of standard, to 0.90 ml reagent A in separate test tubes and mixed. Tubes were incubated 10 min in a 50 degrees C bath, then cooled to room temperature. Added 0.1 ml of reagent B to the series of tubes, mixed, incubated for 10 min at room temperature. Rapidly added 3 ml of reagent C to each tube, mixed and incubated for 10 min at 50°degree in water bath, and cooled to room temperature. Final assay volume was 5 ml and absorbance was measured at 650nm in 1cm cuvettes. A standard curve of absorbance versus micrograms protein was plotted, and determined the amount of protein of extracellular enzymes using from this curve.

Effect of fermentation parameters on SmF and SSF -The
Aspergillus niger and the areca spade powder were used for the production of fermentation product under various conditions.

RESULTS AND DISCUSSION
Tarikere is abundant in areca nut farms and also Aspergillus niger in these farms. The local strain of Aspergillus niger has natural capacity to degrade the lignocellulosic components by secreting lignocellulytic enzymes. These enzymes were studied and analysed by using enzyme assay and to confirm the presence of cellulases like Fpase, βglucosidase and Endoglucanase. These enzymes convert the substrate into monomers which are used as sources of industry and pharmacy.
Aspergillus niger was isolated by serial dilution of soil sample cultured from the areca farm. The pure culture of fungal colonies of white colony morphology were collected from PD agar plates and then turned black during sporulation, the sporulated organism was microscopically examined stained with lacto phenol cotton blue dye using for morphological confirmation. The identification feature like hyaline septate hyphae, conidial head, conidiophores, globose shaped vesicles, sterigmata confirmed Aspergillus niger.
Cellulases Secreted by Aspergillus niger -Cellulolytic enzymes were secreted in to the broth and substrate of areca biomass by Aspergillus niger. After 7days of incubation the filtrate of SmF and SSF contained Fpase, CMCase and β-glucosidase. CMCase as the most prominent enzyme forming the larger moiety than Fpase and β-glucosidase. Table: 01: Production of hydrolases from aspergillus niger in the presence of areca biomass.°M aximum enzyme production was recorded around 30 C both under SMF and SSF. Around 80% of Cellulase production was observed °°°a round 35 C of temperature whereas 50% of cellulase around 20 C, 25 C °a nd 40 C. Maximum enzyme concentration was recorded at 30-35°C. According to a study by (Devanathan et al. 2007), 37°C was said to be the optimum temperature for cellulose production by Aspergillus niger. H P change also affected the product stability, growth of the fungi and H caused morphological changes in microorganisms. The P effect can be H described by Michaelis constant in the P range 3 to 8. The maximum H cellulose was produced at P 6.0 Short incubation period for enzyme production offers the potential for inexpensive production of enzyme. Incubation time necessary for optimal production varied between different enzymes produced from the substrate (Smith et al;1996). In this study the enzyme production steadily increased along with incubation and reached the maximum at 96h (4days incubation) then started declining after, the lowest enzyme concentration being recorded at 24 hours due to no stimulating compound on the substrate. Cellobiose was produced during the early stages of incubation. The formation of specific enzyme at specific incubation period greatly varies with the substrate also. Fermenting some organisms, there were examples of obtaining maximum yield only at 7 days of incubation, with Trichoderma reesei cultivated on rice straw and 66 hrs with Trichoderma harzianum. Cellulase production also differed with the carbon supplement used in the culture media. Among the different carbon sources tested Lactose promoted maximum yield and acted as enzyme production promoter.
The early availability of carbon source induces a good growth in the microorganism. The enzyme production is influenced by different sources and different concentrations of organic source of nitrogen in the medium. The enzyme production is sensitive to different levels of nitrogen present in the medium. Biosynthesis and secretion of enzyme is also influenced by the moisture content in SSF. The ratio of 1:2 is found to be ideal for high enzyme production lower than optimal moisture content results in no solubility of nutrients from the substrate and therefore decreased enzyme production , Higher moisture levels will also reduce the yield by interfering the oxygen transfer and by increasing porosity i.e. inter particle spaces, thus hindering the growth there by affecting enzyme synthesis. Table.03: Production of enzyme in smf process with the dilution of and biomass with h o in the presence of aspergillus niger. 2 µ Moles of enzymes produced in a minimum per mg protein the substrate present in Areca nut spade biomass under different dilution conditions. So 1:10 of w/v of moisture content can be allowed for synthesis of cellulases by Aspergillus niger. Inoculum size also should be optimum so as to facilitate proper usage of substrate by microorganisms 5 to 15% of inoculums size is optimum for SmF and 10% for SSF.
In this study Aspergillus niger was comparatively cultured under SSF and SMF. The SSF condition provided proper utilisation of the substrate for enzyme production; comparatively minimum inoculation was needed for SSF. SSF has less liquid wastage, proper usage of substrate was achieved, cellulases produced in SSF were greater than enzymes produced by SmF.