1. INTRODUCTION:

Cellulosic waste material obtained from energy crops, food and chemicals could allow self-sustainable processes and products [1]. 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 [2]. The bioconversion of the agro waste material into fuel has received considerable interest during recent years. This lignocellulosic material was in abundant and available in free of cost. Enzymatic hydrolysis of cellulosic biomass is considered as the most efficient and least polluting methods for generating glucose from lignocellulosics, but the production economics of bioethanol is largely dependent on cost of cellulases [3]. Cellulases are comprise a complex system of three main enzymes viz. Endoglucanase, Exoglucanase and β glucosidase. These enzymes are involved in the natural degradation of cellulose, the major polysaccharide of plant cells [4]. The enzymatic complex can convert the cellulose too ligosaccharides and glucose [5]. Microorganisms such as fungi and bacteria are important producers of cellulases. Substrate costs account for a major fraction of the costs of cellulose production, and the use of cheap biomass resources as substrates can help to reduce cellulase prices [6].

Cellulase enzyme is industrially significant for starch processing, bioethanol production, malting and brewing, extraction of vegetables and fruits juice, pulp and paper making procedures and textile industries [7].

In this study some important agro bio-waste such as sugarcane bagasse and wheat bran have been used as substrate for production of enzyme by solid state fermentation using Aspergillus niger. Solid state fermentation (SSF) holds incredible prospective for production of commercially important enzymes because of several advantages, such as higher fermentation productivity, higher concentration of end product, higher product stability, lower catabolic repression and cultivation of microorganisms specialized for water insoluble substrates [8].This study reports the optimization of process parameters such as pH Value, substrate ratio, temperature and incubation time period to improve enzyme yield in SSF.

2. MATERIALS AND METHODS:

Sugarcane bagasse was procured from sugar Mill, Modinagar, U.P. wheat bran was used as co-substrate and was procured from local wheat Flour Mill. The media and media ingredients were obtained from Hi-Media Laboratories Pvt. Ltd., Mumbai and analytical grade chemicals obtained from E. Merck Ltd. Mumbai. The Aspergillus niger used for cellulase production was obtained from ATCC (American type culture collection), culture number- ATCC 16404^TM.

2.1 Fungal inoculum preparation:

Rose Bengal chloramphenicol agar medium was prepared for fungal inoculum preparation. This media was poured on petriplates. The solidified media was streaked with A. niger and incubated at 30^oC for 3 days for sporulation; the spores were harvested using sterilized water [9]. The spores concentration of 1-2x10⁸ spores/ml was used for inoculating on sugarcane bagasse and wheat bran, which was used as substrate for enzyme production.

2.2 Cellulase enzyme production:

The solid state fermentation using sugarcane bagasse and wheat bran in the ratio 3:1, 2:1 and 1:1 was mixed with culture media and used for the production of cellulase. Culture media was transferred uniformly on sterile 2000ml plastic trays containing 100gm of substrate; this uniformity was maintained to ensure proper aeration in the culture media. The effect of pH on cellulase production was determined at pH values of 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0 using either 1 M sodium hydroxide or 1 M hydrochloric acid. The trays were sterilised by autoclave at 121^oC for 15 min, cooled at room temperature, inoculated with 1 ml fungal spores suspension. The trays were covered with cellophane sheet, to prevent any contact with the external environment and incubated in incubator at 22.5^oC, 30^oC and 35^oC for 48hr, 72hr, 96hr and 120hr. The experiment was carried out at 70% moisture level for proper A. niger growth in the media by solid state fermentation (SSF) for the production of cellulase [10].

2.3 Extraction of enzyme:

1% Sodium benzoate (Sodium benzoate acts as an anti-fungal agent and kills the fungal spores in the sample) was prepared in distilled water. After that 2000ml of this solution was added to the tray. The entire setup was left for 24 hours at room temperature. All content was distributed to conical flasks (1000ml). These flasks were incubated at 30^oC for 30 minutes at 130 rpm in an orbital shaking BOD incubator (Narang Scientific Works Pvt. Ltd., New Delhi) to have the complete extraction of enzyme in the solution form and contents were then filtered through watt man filter paper (541). The filtrate was centrifuge at 5000xg at 4^oC for ten minutes [10]. Supernatant was collected and analysed for cellulase enzyme activity.

2.4 Enzyme activity assay:

Filter paper assay was used to estimate total cellulase activity in the crude enzyme preparation [11]. For filter paper activity Wattman no. 1 filter paper strip of dimension 1.0× 6 cm (50 mg) was placed into each assay tube. The filter paper strip was saturated with 1.0 ml of sodium citrate buffer (0.05 M, pH 4.8) and was incubated for 10 min at 50°C, 0.5 ml of an appropriately diluted (in sodium citrate buffer) enzyme was added to the tube and incubated at 50°C for 60 min [12]. Also in case of endoglucanase activity half milliliter of 1 % carboxymethyl cellulose in 0.05M sodium citrate buffer, pH 4.8 was temperate for 10 min at 50 °C [13, 14]. After that half millilitre of an appropriately diluted enzyme was added to the tube and incubated at 50 °C for 30 min. Appropriate controls were also run along with the test. At the end of the incubation period, tubes were removed from the water bath, and the reaction was stopped by addition of 3 ml of 3,5-dinitrosalicylic acid reagent per tube. The tubes were incubated for 5 min in a boiling water bath for colour development and were cooled rapidly. The reaction mixture was diluted appropriately and was measured against a reagent blank at 540 nm in a UV-VIS spectrophotometer (Varian) [15]. The concentration of glucose released by enzyme was determined by comparing against a standard curve constructed similarly with known concentrations of glucose [16]. One unit of enzyme activity was defined as the amount of enzyme required for liberating 1 µM of glucose per milliliter per minute and was expressed as IU/ml (IU/gm).

3. RESULTS AND DISCUSSION:

The recent thrust in bioconversion of cellulosic biomass to chemical feedstock has led to extensive studies on cellulolytic enzymes produced by bacteria and fungi [17]. Though the growth period of bacteria is shorter than that of fungi, their half- backed cellulase system makes them less useful in the industrial production of cellulase. However, high cost of cellulases production hindered use of this enzyme in industry. For the utilization of cellulose and lignocellulose biomass, it is necessary step to enhance the cellulose production and reduces its production cost. The use of purified cellulosic as substrate is uneconomical for large scale production of cellulases. Therefore cheaply available agricultural cellulosic material viz. sugarcane bagasse and wheat bran were used for the production of cellulases by A. Niger under solid state fermentation. The production of cellulase enzyme was effected by many factors like pH, temperature, substrate ratio and incubation time. The production of cellulase enzyme was measured by enzyme activity assay in IU/gm.

3.1 Effect of Cellulase on Cellulose:

To test the cellulase activity 1% cellulase (12g) was prepared in 1200 ml of distilled water (mixed thoroughly and heated to a clear solution). This solution was then distributed in 6 different beakers with 200ml of 1% cellulose solution in each beaker. The amount of cellulase was added as tabulated below in Table-1and viscosity was then observed at different concentration of enzyme at 60rpm and 30^oC (Figure-1).

Table 1: Cellulase activity in presence of cellulose at 30°C and 60rpm

S.No.	Enzyme conc.(ml)	Viscosity(cP)
1	200ml CMC	110
2	200ml CMC+0.5 cellulase	30
3	200ml CMC+1.0 cellulase	21
4	200ml CMC+1.5 cellulase	19
5	200ml CMC+2.0 cellulase	16.5
6	200ml CMC+2.5 cellulase	15.5

Figure: 1 Cellulase activity with Cellulose

During catalysis some enzymes undergo large conformational changes as they progress through the catalytic cycle. It was observed that the viscosity decreases within increase in the concentration of crude cellulase, resulting in the free flowing nature of the solution and hence breaking down cellulose present in CMC (carboxymethyl cellulose) to a reducing sugars [18].Such similar studies were performed using different parameters such as pH, temperature and variant substrate ratios.

3.2 Effect of pH on Cellulase activity on different days:

Effect of pH on cellulase activity was studied on different days viz on 4^th, 5^th and 6^th day according to the incubation period of A. niger at a pH of 3.5 to 6.0. The maximum activity of cellulase enzyme observed is 986.0IU/gm on day 5 at a pH of 5.0. The results are clearly visible in Table-2, Figure-2. pH is an important factor to judge cellulase activity. With increase in pH value there is a linear increase in cellulase activity till pH 4 beyond which the activity is not increasing linearly but is rather near to stable. The best activity was observed on day 5 at pH 5.

Table-2: Cellulase activity (IU/gm) at different pH and Time intervals

pH	Time (in Days)
pH	4	5	6
3.5	304.0	423.0	411.8
4.0	830.1	960.6	945.4
4.5	896.9	972.0	958.6
5.0	974.5	986.0	975.2
5.5	947.0	968.5	962.4
6.0	920.3	960.0	950.1

3.3 Effect of temperature on Cellulase activity (IU/gm) on different days:

The positive significance of temperature on cellulase enzyme production by A. niger was recorded with temperature optima between 22.5°C-35°C on different days viz on 4^th, 5^th and 6^th day. The maximum activity of cellulase enzyme observed is 990.0IU/gm on day 5 at 30°C. The results are clearly visible in Table-3 and Figure-3. With increase in temperature value there is an increase in cellulase activity till 3°C beyond which the activity starts decreasing. The best activity was observed on day 5 at 30°C.

Figure-2: Cellulase activity at different pH and Time intervals.

Table-3: Cellulase activity (IU/gm) at different Temperature and Time intervals

Temperature (^oC)	Time (in Days)
Temperature (^oC)	4	5	6
22.5	805.7	888.2	882.0
30	972.5	990.0	985.1
35	731.4	740.1	756.5

Figure-3: Cellulase activity at different Temperature and Time intervals.

3.4 Cellulase activity (IU/gm) at different substrate ratio:

In this study, different ratios of sugarcane bagasse and wheat bran at 3:1,2:1 and 1:1 were evaluated for maximum cellulase production. The maximum activity was observed when the substrates composed of sugarcane bagasse and wheat bran are taken in a ratio of 2:1 as shown in Table-4 and Figure-4. The activity observed is 998.5 IU/gm on day 5. Such similar types of studies were performed by using various cultures for the production of different metabolic enzymes as reported in the literature [19, 20].

Previous studies showed that supplementation of four parts of soyabean hulls with one part of wheat bran resulted in high titres of enzymes balanced proportion of various activities [20]. In this paper maximum amount of cellulase was obtained on 5^th day of incubation period at 30^oC and substrate ratio (2:1) until the end of the SSF.

Table-4: Cellulase activity (IU/gm) at different substrate ratio and Time intervals

Substrate Ratio	Time (in Days)
	4	5	6
3:1	902.0	945.4	936.2
2:1	960.0	988.5	978.0
1:1	736.1	804.7	780.0

Figure-4: Cellulase activity (IU/gm) at different substrate ratio

CONCLUSION:

In the present study the production of cellulases fromsugarcane bagasse and wheat bran under solid state fermentation was studied by Aspergillus niger. Optimal process parameters for enzyme production were found to be at pH 5.0, temperature 30°C, moisture content 70%. And a maximum 988.2IU/gm activity of enzyme was observed at a concentration of 2:1 (sugarcane bagasse: wheat bran) at 30°C and on 5^th day of incubation period. Sugarcane bagasse and wheat bran are cheap residues which can be used as a substrates for enzyme production and reduces the cost of enzyme production. The enzyme production does not require any specific environmental condition due to that this technology can be easily adopted for commercialization. Its manufacturing is very cheaper. It can be a milestone in enzyme industry to fulfill demand supply. Due to the easy handling, cheaper technology and excellent quality of this enzyme can be manufactured in any region of our country.

ACKNOWLEDGMENT:

We are thankful to Dabur India Ltd. Ghaziabad, for providing laboratory infrastructure to carry out this work.

REFERENCES:

1. Edward A Bayer, Henri Chanzy, Raphael Lamed, Yuval Shoham, (1998); Cellulose, cellulases and cellulosomes, Current Opinion in Structural Biology, Volume 8, Issue 5, Pages 548-557.

2. Kuhad RC, Singh A, Eriksson KE, (1997); Microorganisms enzymes involved in the degradation of plant fiber cell walls. Adv. Biochem. Eng. Biotechnol. 57: 45-125.

3. Reith, J. H., den Uil, H., van Veen, H., de Laat WTAM, Niessen, J. J. and de Jong, E., (2002); Coproduction of bioethanol, electricity and heat from biomass residues. 12th European Conference and Technology Exhibition on Biomass from Energy, Industry and Climate Protection, Amsterdam, The Netherlands, 17–21 June.

4. Kuhad RC, Singh A, Eriksson KE, (1997); Microorganisms enzymes involved in the degradation of plant fiber cell walls. Adv. Biochem. Eng. Biotechnol. 57: 45-125.

5. Ander, P. Mishra, C. Farrell, R. Eriksson, K. E., (1990); Redox interations in lignin degradation: interaction between laccase. Different peroxidases and cellobiose: quinine oxidoreductase. J. Biotechnol. 13, 189-198.

6. Wen, Z., Liao, W., and Chen, S., (2005); Production of cellulase/ bglucosidase by the mixed fungi culture Trichodermareesei and Aspergillus phoenicis on dairy manure. Process Biochemistry, 40, 3087– 3094.

7. Bajpai, P., (1999); Application of enzymes in the pulp and paper industry. Biotechnol. Prog. 15, 147–157.

8. Benjamin, Sailas and Pandey, Ashok, (2001); Isolation and characterization of three distinct forms of lipases from Candida rugosa produced in solid state fermentation. Brazilian Archives of Biology and Technology, Vol. 44, no. 2, p213-221.

9. N. R. Kaminiet. at. (1998); Lipase production from Aspergillus niger by solid state fermentation using gingelly oil cake, Process Biochem. 33: 505-511.

10. Lavudi Saida, Harinder Singh Oberoi, M Laxmi vNarasu, (2013); Studies on cellulase production by solid state fermentation using Sweet sorgum bagasse; Helix Vol. 1: 261-266.

11. Ghose, T. K., (1987); Measurement of cellulase activities. Pure and Applied Chemistry 59: 257–268.

12. Mawadza, C., Rahni, H., Zvauya, R. and Bo, M., (2000); Purification and characterization of cellulases produced by two Bacillus strains. Journal of Biotechnology, 83:177-187.

13. Wood, T.M. and Bhat, K.M., (1998); Method for measuring cellulase activities. In Methods in Enzymology. Cellulose and Hemicellulose, W.A. Wood and J.A. Kellogg, Vol. 160, pp. 87-112. Academic Press, New York.

14. Coral, G., Arikan, B., Unaldi, M.N. and Guvenmez, H., (2002); Some properties of crude carboxymethyl cellulase of Aspergillus nigerZ10 wild-type strain. Turkey J. Biology, 26: 209-213.

15. Rashid SS, Alam MZ, Karim MI A, Salleh HM, (2009); Management of palm oil mill effluent through production of cellulases by filamentous fungi. World J. Microbiol. Biotechnol. 25: 2219-2226.

16. Wang, N.S. (2003) Glucose assay by dinitrosalicylic colorimetric method.

17. Kang, S.W., Park, Y.S., Lee, J.S. , Hong , S.I., Kim, S.W., (2004), Production of cellulases and hemicellulases by Aspergillus niger KK2from lignocellulosic biomass, Bioresource Technology, 91, 153–156.

18. Kansoh, A.L., Essam, S. A. and Zeinat A. N., (1999); Biodegradation and utilization of bagasse with Trichodermareesie. Polymer Degradation and Stability, 63(2): 273-278.

19. Sudhakar P., et. al., (2011); Production of Chitinase by solid state fermentation from Serratiamarcescens. Internation Journal of Chem Tech Research. Vol. 3, No. 2, pp 590-598.

20. Brijwani K., et. at., (2010); Production of Cellulolytic enzyme system in mixed culture solid state fermentation of soybean hulls supplemented with wheat bran. Process Biochem: 45: 120-128.

Received on 14.08.2013 Modified on 20.11.2013

Research J. Science and Tech. 6(1): Jan.-Mar. 2014; Page 16-19