Production, Optimization and Partial purification of Cellulase by ...

Journal of Microbiology and Biotechnology Research

Scholars Research Library J. Microbiol. Biotech. Res., 2012, 2 (1):120-128

(http://scholarsresearchlibrary.com/archive.html) ISSN : 2231 –3168 CODEN (USA) : JMBRB4

Production, Optimization and Partial purification of Cellulase by Aspergillus niger fermented with paper and timber sawmill industrial wastes *M. Charitha Devi and M. Sunil Kumar Department of Virology, College of Sciences, Sri Venkateswara University, Tirupati, A.P, India _____________________________________________________________________________________________

ABSTRACT It was the goal to investigate the cellulase enzyme production ability of fungal strain Aspergillus niger against the lignocellulosic bio wastes like saw dust, paper cellulose at varying environmental parameters of pH (4-7), temperature (20-50°C) and incubation period (2-8 days). Production of cellulase was analysed by Dinitrosalicylic acid (DNS) and Filter paper assay methods. In the DNS method, maximum enzyme production of 3.9 IU was achieved at temperature of 45°C by Aspergillus niger in paper cellulose with pH of 5 on 7th day of growth. The partial purification of the cellulase enzyme produced by Aspergillus niger in the waste supplemented medium had two protein bands with the molecular weight of 33 and 24kDa respectively. Key words: Aspergillus niger, paper cellulose, DNS, FPA, Cellulase, Partial purification, Fermentation. _____________________________________________________________________________________________

INTRODUCTION The cellulose constitutes the major form of stocking of glucose obtained through photosynthesis and in the same time the major component of solar energy conversion to the biomass. The cellulose is also major constituent of all the Plant materials and that is why it is the most abundant organic material in nature, which is renewed every year. Because of its highly ordered structure, the cellulose is very hard to be degraded and that is why it is unusable and stocked in nature as waste. The capacity to degrade the natural cellulose implies the synthesis of the entire cellulolytic system. Cellulose has been used by man for centuries, however, its enormous potential as a renewable source of energy was recognized only after cellulose degrading enzymes or “cellulases” had been identified (Bhat & Bhat, 1997). A cellulosic enzyme system consists of three major components: endo-β-glucanase (EC 3.2.1.4), exo-β-glucanase (EC 3.2.1.91) and β120 Available online at www.scholarsresearchlibrary.com

M. Charitha Devi et al J. Microbiol. Biotech. Res., 2012, 2 (1):120-128 ______________________________________________________________________________ glucosidase (EC 3.2.1.21) (Knowles et al., 1987). These components act synergistically in the conversion of cellulose to glucose (Eveleigh, 1987). Ali and Akhand (1992) worked on cellulase production by mesophilic Trichoderma isolate during growth on water Hyacinth under optimized conditions. The cellulase complex of Aspergillus niger has been most thoroughly studied. It can convert native cellulose as well as derived celluloses to glucose (King & Nessal 1969). Ahmad et al., 2003 worked on Trichoderma harzianum for cellulase enzyme production by using different carbon sources and reported that Carboxy methyl cellulose is the best for substantial amount of enzyme production (Shazia shafique et al.,). Since growth of fungi as well as the enzyme production depends on the composition of the growth media, pH, temperature. The effect of environmental factors on the growth of fungi is generally less specific and restricted than the effect on secondary metabolite production. For example, the ranges of water activity, growth medium and pH within which formation of certain secondary metabolites occur, is narrower, than the range of conidial growth (Northolt & Bullerman, 1982). Several studies were carried out to produce cellulolytic enzymes from biowaste degradation process by many microorganisms including fungi such as Trichoderma, Penicillium, Aspergillus spp. etc. by Mandels and Reese (1985), Hoffman and Wood (1985), Brown et al. (1987), Lakshmikant and Mathur (1990) etc. Similarly celluloytic property of bacterial species like Pseudomonas, Cellulomonas, Bacillus, Micrococcus, Cellovibrio and Sporosphytophaga spp. were also reported (Nakamura and Kappamura, 1982; Immanuel et al., 2006). The specific cellulolytic activity shown by the bacterial species is found to be depending on the source of occurrence (Saxena et al., 1993). The work on cellulase enzyme production by Aspergillus niger has been conducted all over the world. But the physiological responses of same organism or species may vary with ecological variations. Therefore, the present research work was aimed to evaluate the potential cellulase production by native Aspergillus strains. MATERIALS AND METHODS Isolation and screening of Cellulase producing fungi: The samples were aseptically collected from local industrial wastes like paper, timber- saw mills etc., in and around Tirupati (Andhra Pradesh). Serially diluted sample prepared from the industrial wastes were spread on surface of potato dextrose agar and incubated for 7 days at 30ºC. Colonies were picked and sub-cultured to obtain pure culture. Stock cultures were maintained on potato dextrose agar at 4ºC. The isolated strains were carefully identified by morphological characteristics include color of the colony and growth pattern studies, as well as their vegetative and reproductive structures observed under the microscope. Cellulase producing fungi were screened on selective carboxymetyl cellulose agar containing (1%): NaNO3 2.0, KH2 PO4 1.0, MgSO4 .7H2O 0.5, KCl 0.5, carboxymethyl cellulose sodium salt 10.0, peptone 0.2, agar 17.0.Plates were spot inoculated with spore suspension of pure culture and incubated at 30ºC.After 3 days, plates were flooded with 1% Congo red solution for 15 minutes then de-stained with 1M NaCl solution for 15 minutes. The diameter of zone of decolorization around each colony was measured. The fungal colony showing largest zone of decolorization was selected for cellulase production.

121 Available online at www.scholarsresearchlibrary.com

M. Charitha Devi et al J. Microbiol. Biotech. Res., 2012, 2 (1):120-128 ______________________________________________________________________________ Carbon source: Fresh industrial wastes i.e., saw dust, paper cellulose were collected where the degraded samples were collected for isolation of fungi, they were washed and blended by a mixer and air dried. The blended materials were sieved and used as a carbon source for the production of cellulase. Cellulase production: The isolated Aspergillus niger pure culture harvested with sterile distilled water for inoculum preparation was used for cellulase production. The basal mediums supplemented with known volume of cellulosic substrates were used for production. 100ml of production medium (pH 5) in 250ml shake flask was inoculated with 2 ml of fungal spore suspension containing 105 spores per ml. The flask was incubated at 30oC with agitation speed of 200 rpm in rotary shaker incubator. After 7 days, culture filtrate was collected centrifuged at 6000 rpm for 15 min and supernatant was used as crude cellulase source. Cellulase assay: Total cellulase activity in the culture filtrate was determined according to the method of Mandels et al (1976).The reducing sugar released from filter paper per ml per min at 540nm was determined by Dinitro salicylic acid (DNS) method. One unit of total cellulase activity was defined as the amount of enzyme releasing 1µmole of reducing sugar per minute. Optimization of production of cellulase enzyme: Cellulase production depends upon the composition of the fermentation medium. Medium optimization for over production of the enzyme is an important step and involves a number of physico-chemical parameters such as the incubation period, pH, temperature and supplemented Substrate in submerged fermentation. For the initial optimization of the medium, the traditional method of “one variable at a time” approach was used by changing one component at a time while keeping the others at their original level. The selected cellulolytic strains were grown in selected media consisting of selected substrates for enzyme production. Studies were performed in shake flasks to optimize different fermentation conditions for hyper cellulase production. Carbon source: The basal medium supplemented with different volumes of Cellulose substrates as carbon source like paper cellulose and saw dust. The measured volume of Cellulose substrates was selected as optimum amount of carbon source required for cellulase production. Effect of Incubation period, temperature and pH: To select the suitable temperature, pH, incubation period for fermentative production of the enzyme the selected fungal strains were cultivated with varying temperatures of 30˚C-50˚C, pH range 3-8, incubation period range of 2-8 days, by keeping all other parameters constant for hours Partial purification of cellulose ◦ All procedures of the cellulase purification were carried out at 4 C. The culture supernatant was separated by centrifugation process, by using buffers like buffer A, 50mM Tris–HCl (pH 8.0); buffer B, buffer A containing 80% saturated ammonium sulphate (Wood 1988). Followed by Fractional ammonium sulphate precipitation by adding solid ammonium sulphate to the culture 122 Available online at www.scholarsresearchlibrary.com

M. Charitha Devi et al J. Microbiol. Biotech. Res., 2012, 2 (1):120-128 ______________________________________________________________________________ filtrate to 80% saturation. After 12 h the resulting precipitate was collected by centrifugation at 10,000 × g for 30 min and dissolved in buffer A and dialysed overnight against three changes of the same buffer. Insoluble material was removed by centrifugation at 10,000 × g for 10 min. The clear supernatant was filter sterilized and stored at 0oC. Partially purified Enzyme was confirmed by SDS- PAGE performed using the method of Laemmli (1970), with the stacking and separating gel concentrations were 4% and 12% of polyacrylamide, respectively. After the electrophoresis, the gels were stained with Coomassie brilliant blue R-250 (Sigma) for visualization of protein bands. Statistical analysis: Analysis of variance (ANOVA) was performed on all data using the SAS (1985) statistical package. The mean values were compared by the least significant difference (LSD) test at 5% level of confidence. RESULTS Isolation and screening of Cellulase producing fungi The fungi were isolated and screened from the samples collected from paper and timber- saw mills for cellulase production by congo red assay. The isolated strains were carefully identified by morphological Characteristics include color of the colony and growth pattern studies. Some of the microscopic characteristics examined under the microscope include spore formation and color (Fig.1). Isolated fungal strains were subjected to screening, the colony forming units per gram of sample (CFU/g) were calculated (Fig:2). The diameter of hydrolytic zone and colony diameter were measured (Fig:3). Isolate PIW-1 and TSMW-1 showed highest zone of hydrolysis as 12 mm out of 19 mm and 13mm out of 17mm of colony diameter.

Figure.1: Microscopic features of Aspergillus sp. stained with lactophenol cotton blue at magnifications of 40X

Table.1: Colony forming units of isolated fungi per gram of sample (CFU/g). S. No.

Nature of sample

1.

Paper industrial waste

2.

Timber saw mill waste

Dilution 10-3 10-4 10-5 10-3 10-4 10-5

Number of Fungal population (CFU) 45 14 04 24 03 -


M. Charitha Devi et al J. Microbiol. Biotech. Res., 2012, 2 (1):120-128 ______________________________________________________________________________ Table.2: The diameter of hydrolytic zone and colony diameter S. No. 1. 2. 3. 4. 5. 6.

Colony Microscopic Colony diameter Hydrolytic Zone colour observation (mm) (mm) Black Aspergillus sp. PIW-1 19 12 Black Aspergillus sp. 12 5 PIW-2 Black Aspergillus sp. 10 PIW-2 Black Aspergillus sp. TSMW-1 17 13 Black Aspergillus sp. 9 TSMW-2 8 4 Black Aspergillus sp. TSMW-3 PIW, Paper industrial waste; TSMW, Timber saw mill waste. (-) indicates diameter of hydrolytic zone is less than 2mm.

Strain No

Figure.3: Isolated fungal primary colonies (A, B, C and D) and CMC-Congo red plate assay of Aspgergillus sp.

Fig.4. Effect of incubation period on cellulase production


M. Charitha Devi et al J. Microbiol. Biotech. Res., 2012, 2 (1):120-128 ______________________________________________________________________________

Fig.5. Effect of pH on cellulase production

Fig.6. Effect of temperature on cellulase production

Fig.7. SDS-PAGE analysis of partially purified endo and exo glucanase. Lane 1- Marker proteins lanes 2 partially purified enzyme stained with Comassie brilliant blue.


M. Charitha Devi et al J. Microbiol. Biotech. Res., 2012, 2 (1):120-128 ______________________________________________________________________________ Optimization of production of cellulase enzyme: Maximum activity was achieved at the cultural optimized conditions - recorded by Aspergillus niger PIW-1 and TSMW-1 in Papercellulose with temperature of 45°C, pH of 5 at the growth of 7th day. The activity calculated was 3.9 IU/min/ml and 3.8 IU/min/ml respectively. Partial purification of cellulose Two protein bands with the molecular weight of 33 and 24kDa respectively were identified in SDS-PAGE analysis of partially purified endo and exo glucanase. DISCUSSION This study could establish that paper industrial waste, timber saw mill waste which does not find any significant commercial use especially in developing countries like India and is disposed of in municipal bins for rotting could serve as an ideal substrate for production of cellulases. Hence, the technology using these cheap and readily available substrates for production of cellulases in optimum quantities in about 96 h holds promise for the future. This study could also demonstrate that a single enzyme, if partially purified, could be used for saccharification of lignocellulosic biomass rather than using a concoction of cellulases. The results are significant for the study on cellulase production and provide a potential approach for the industry. Several fungal species were isolated from diverse environments for the production of cellulase enzyme, the two fungal isolates of Aspergillus sp. possessing the potential cellulolytic activity were selected for the production of cellulase enzyme by carboxy methyl cellulose clearance plate assay by measuring the clearing zone. Czapak dox broth was selected as viable media for the production of cellulase. The production of cellulase in the medium containing papercellulose and saw dust as substrates were suitable in case of Aspergillus sp. submerged fermentation and gave highest production of total cellulase activity. The cultural conditions were optimized for higher yield of cellulase enzyme. For the initial optimization of the medium, the traditional method of “one variable at a time” approach was used by changing one component at a time while keeping the others at their original level. The optimized cultural conditions resulted in increased total cellulase production of 3.9 IU and 3.8 IU for Aspergillus sp. PIW-1, TSMW-1 respectively. The results obtained in this study are better or comparable with the results obtained by Krishna (1999) or Ananda Muniswaran et al. (1994) who used banana stalk and coconut coir for production of cellulases, respectively. However, Kang et al. (2004) have reported higher enzyme yields using different ratios of rice straw and wheat bran using Aspergillus sp. Thus, this study could reveal that if the crude filtrate prepared using above treatments could be partially purified using ammonium sulphate precipitation followed by membrane filtration, it could alone be sufficient for efficient saccharification of pretreated lignocellulosic material. Two bands showing cellulolytic activity were detected on gel during electrophoresis of the partially purified enzyme. The molecular weights of these bands were estimated to be 24 and 33 kD. These bands (proteins) may be isoenzymes or the different subunits of the same enzyme protein on electrophoresis gel (Coral et al., 2002). CONCLUSION The fungi as enzyme sources have many advantages that, the enzymes produced are normally extracellular, making easier for downstream process. The development of economically feasible 126 Available online at www.scholarsresearchlibrary.com

M. Charitha Devi et al J. Microbiol. Biotech. Res., 2012, 2 (1):120-128 ______________________________________________________________________________ technologies for cellulase production and for the enzymatic hydrolysis of cellulosic materials will enable to utilize the large quantities of biomass such as the residues of both food industries and agriculture. Thus the present investigation was selected to conduct an extensive study on cellulases from A. niger. Present study was aimed at isolation of promising cellulase producing Aspergillus sp. and its identification, optimization of cultural conditions for production of cellulolytic enzymes. Fungal cultures were initially identified as species of the genera of Aspergillus based on cultural, morphological, microscopic characterstics. The cellulololytic activity of these cultures was studied by standard CM-cellulose and congo red plate assay method. The process development is the key step in fermentation processes. The study related to process development involves optimization of different fermentation conditions (physical and nutritional) towards enhancement of cellulolytic enzymes production. Shakeflask cultural conditions (physical and nutritional factors) for cellulolytic enzymes production by the isolated promising Aspergillus niger were optimized. Rice straw was selected as a best substrate for cellulase production using Aspergillus niger. The cultural conditions were optimized for higher yield of cellulase enzyme. One factor at a time (OFAT) strategy was used for the optimization of medium components. The selected cellulolytic strains were grown in selected media for enzyme production and the studies were performed in shake flasks. Cellulase production with Aspergillus niger was highest at temperature 45 ˚C, pH-5.0, incubation time (7 days) and in presence of substrates (rice straw). the partial purification of cellololytic enzymes from the culture filtrate was performed by ammonium sulphate salt precipitation followed by desalting through dialysis. The partial purification of cellololytic enzymes from the culture filtrate was performed by ammonium sulphate salt precipitation followed by desalting through dialysis. Two protein bands with the molecular weight of 33 and 24kDa respectively Acknowledgement This work was supported by DBT (Department of Biotechnology) Ministry of Sciences & Technology, New Delhi, We are very thankful for providing the financial support and my colleagues for their moral support. REFERENCES [1]. Ananda Muniswaran, P. K., Selvakumar, P., and Narasimha Charyulu, N. C. L. (1994). Journal of Chemical Technology and Biotechnology, 60, 147–151. [2]. Badal, C.S. (2004). Process Biochemistry. 39, 1871–1876. [3]. Bhat, M. and Bhat, S. (1997) Biotechnology Advances 15, 583- 620. [4]. Coral G, Arikan B, Unaldi MN, Guvenmes H (2002). Turk J. Biol. 26:209-213. [5]. Ghose, T.K. (1969) Biotechnology and Bioengineering 11, 239-261. [6]. Han, Y.W. and Callihan, C.D. (1974) Applied Microbiology 27, 159-165. [7]. Jahangeer S, Khan N, Jahangeer S, Sohail M, Shahzad S, Ahmad A, Ahmed Khan S(2005). Pak.J. Bot. [8]. Kang, S. W., Park, Y. S., Lee, J. S., Hong, S. I., & Kim, S. W. (2004). Bioresource Technology, 91, 153–156. [9]. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall (1951) J. Biology Chem. 48: 17-25. [10]. Miller, G. L. (1959) Anal. Chem. 31: 426-428. [11]. Nakamura K, Kppamura K (1982) J. Ferment. Technol. 60 (4): 343 - 348. 127 Available online at www.scholarsresearchlibrary.com

M. Charitha Devi et al J. Microbiol. Biotech. Res., 2012, 2 (1):120-128 ______________________________________________________________________________ [12]. Reese, E.T. and Mandels, M. (1984) Rolling with the time: production and applications of Trichoderma reesei cellulase. Annual Report of Fermentation Processes. 7, 1–20. [13]. Shamala, T. R. and Srikantaiah, K. R. (1986) Enzyme Microbial Technology 8, 178-182. [14]. Wood, T.M. and Bhat, M.K. (1988), Methods in Enzymology, 160, 87-112.
