Using jackfruit (Artocarpus heterophyllus) pulp as substrate for cellulase production by Rhizopus stolonifer

T. Kavitha¹, Dr. R. Nelson², D. Ramesh Babu¹

¹PG and Research Department of Microbiology, J.J. College of Arts and Science, Pudukkottai, Tamil Nadu India.

²Assistant Professor and Head Department of Botany, Government Arts College, Ariyalur, Tamil Nadu. India

ABSTRACT:

In this present investigation three fungal species namely Rhizopus stolonifer, Penicilium sp Verticillium verticillate were isolated from the ripened Jack fruit (Artocarpus heterophyllus) pulp waste and their cellulase producing ability was screened on Carboxy methyl cellulase agar. Of the three isolates, Rhizopus stolonifer showed maximum cellulase activity. The enzyme production by Rhizopus stolonifer was assayed and the optimum pH, incubation period, temperature, nitrogen source, carbon source were standardized. The fermentation experiments were studied in solid state fermentation (SSF).

KEYWORDS: Jack fruit pulp waste, solid state fermentation, cellulase enzyme, Rhizopus stolonifer.

INTRODUCTION:

Cellulases are industrially important enzymes that are sold in large volumes for use in different industrial applications such as starch processing, animal feed production, grain alcohol fermentation, malting and brewing, extraction of fruit and vegetable juices, pulp and paper industry and textile industry (Roopesh et al., 2006). There are growing markets for cellulases in the field of detergent industry and saccharifaication of agriculture wastes for bioethanol technology (Sukumaran et al., 2009). Lignocellulosic wastes are the largest group of wastes present on this planet causing environmental pollution (Rani and Nand 2000) .It has been reported that solid state fermentation (SSF) was an attractive process to produce cellulases .which is economical due to its lower capital investment and lower operating expenses (Singhania et al., 2009). Production of cellulases by fungi in SSF using agriculture wastes has been reported (Dogaris et al., 2009).Cellulose is the most abundant and renewable bio-polymer on earth. It is the dominating waste material from agriculture and constitutes the waste generated from both natural and man – made activities.

Artocarpus heterophyllus lam is known as jackfruit in Vernacular and Pala in Tamil, is a medium-sized evergreen tree with heavy fruiting near the root or trunk. It is native to Asia and known to have high nutritional value and rich in fibers, minerals and vitamins. The peels, which are around the edible part of jack fruit are highly fibrous but not edible, are thrown away as wastes. This can be dried and powdered as cheap raw materials for the production of cellulase by fungi. The main objectives of this study were to formulate the medium for cellulase enzyme production using jackfruit waste and to optimize the growth parameter.

MATERIALS AND METHODS:

Sample collection and Media preparation

Ripened Jack fruit pulp waste was collected from market at Pudukkottai, Tamil Nadu. The collected sample was allowed to air dry and ground to powder. The powder was kept in a clean bottle separately and used for further studies.

Fungi Isolation.

Fungal species were isolated and maintained on potato Dextrose Agar (PDA) medium, in slant culture throughout the study at 4^oC.

Preparation of Inoculums:

10 ml of distilled water containing 0.1% Tween 80 was transferred to a fully speculated (7 days) PDA slant culture. The old spores were dislodged using the inoculation needle under aseptic conditions and the suspensions were used as inoculums.

Screening for cellulase enzyme activity.

The isolated fungal species were inoculated in the CMC agar plates and incubated at 28^oC for 5 days. After incubation the plates were treated with 1% congo red and 5% of NaCl were added. The fungal growth was stopped by flooding with HCl (Hydrochloric acid) which chances the dye color to blue violet.

Media formulation for Solid State Fermentation - SSF

Ripened Jack fruit pulp powder was used as a substrate. The solid substrate (5g) was weighed in 250 ml Erlenmyer flasks and mixed with a mineral salt solution containing (g/l)2 (NH₄)₂ So₄.10g; KH₂ Po₄, 3.0g; MgSo₄-7H₂o, 0.5g; CaCl₂.H₂o, 0.5g and distilled water was added to obtain the desired initial moisture content. The contents of the flask were mixed and autoclaved at 120^oC for 20 minutes.

Optimization of Cellulase Production

The effect of various parameters on cellulase production such as incubation period, temperature, pH, carbon sources and nitrogen sources were studied.

Effect of incubation period on cellulase production

The effect of incubation period on enzyme activity was studied by inoculating 1 ml of the spore suspension in to the production media. The flasks were incubated at 30^oC for various incubation periods such as 24 hrs, 48 hrs, 72 hrs, 94 hrs, and 120 h.

Effect of temperature on cellulase production

The effect of temperature on enzyme activity was studied by inoculating the organisms in to the production media and incubated at different temperature 20^oC, 25^oC, 30^oC, 35^oC.

Effect of pH on cellulase production

The effect of PH on enzyme activity was studied by inoculating the organisms in to the production media have different PH 4, 5, 6 and incubated at 35^oC.

Effect of carbon source on cellulase production

Effect of carbon sources in enzyme activity was studied by inoculating the organisms in to the production media that containing different carbon source such as glucose, lactose, sucrose and CMC (Carboxyl methyl cellulose).

Effect of nitrogen source on cellulase production

The effect of nitrogen source on enzyme activity was studied by inoculating the organism into the production media containing different nitrogen sources such as yeast extract, beef extract, peptone and casein and incubated at 35^oC.

Enzyme extraction

To the culture flask phosphate buffer (0.2m, pH 7) was added to the fermented substrate to a total volume of 100 ml and mixed for one hour on rotary shaker. The suspension was filtered and centrifuged and the supernatant was used as the crude enzyme preparation for assay of enzyme activity.

Enzyme extraction

To the culture flask phosphate buffer (0.2m, pH 7) was added to the fermented substrate to a total volume of 100 ml and mixed for one hour on rotary shaker. The suspension was filtered and centrifuged and the supernatant was used as the crude enzyme preparation for assay of enzyme activity.

Enzyme assay by DNS method

The enzyme activity was determined by DNS method (Miller 1959) using glucose as standard.

RESULTS AND DISCUSSION:

From the Ripened Jack fruit pulp waste Rhizopus stolonifer, Penicilium sp, Verticillium verticillate were isolated. Among the isolated fungus Rhizopus stolonifer was found to be more potential cellulase producer. The result obtained in this study showed that Ripened Jack fruit pulp waste served as good substrate enabling the growth of all the three isolates however Rhizopus stolonifer exhibited more cllulase activity on CMC agar which produced a considerable amount of the cellulase enzyme under different physicochemical parameters.

When the incubation period extended for 120hr the maximum activity was detected as 3.75 IU/ml. In the present investigation the cellulase activity increased steadily and reached maximum at 96 and 120 hours of incubation (Figure1). Pothiraj et al., (2006) reported Maximum activities of cellulase on the eighth day by A. niger and A. terreus fermentation, tenth day by R. stolonifer on cassava waste. When the culture was incubated at different temperature, the maximum cellulase activity was detected as2.03 IU/ml at 35^oC (Figure -2). Similar observations have been expressed (Ali A. Juwaied et al., 2011) using sugar cane waste.

Figure -3 shows the enzyme activity at various pH values. At pH 4.5, A. niger produced maximum enzyme (2.7 IU/ml) (Ali A. Juwaied et al.,2011), strongly support our report (2.75 IU/ml) at pH 5. Among the carbon sources used CMC was found to be the effective carbon source (3.75IU/ml) followed by Lactose (2.75 IU/ml.) While sucrose and glucose produced almost similar results (Figure- 4). Municipal solid waste residue (4-5% (w/v)) and peptone and yeast extract (1.0% (w/v)) were found to be the best combination of carbon and nitrogen sources for the production of cellulase by A. niger and Trichoderma sp. (Gautam et al., 2011). Beef extract served as ideal nitrogen source (3.25 IU/ml) which is followed by yeast extract, milk Casein and peptone as shown in Figure -5.

The media optimization is an essential aspect in the development of enzyme production technology. The maximum productivity of cellulase was achieved by utilization of Ripened Jackfruit pulp waste as solid substrate. Further it was recorded that the optimum incubation period was120 hrs, temperature 35^oC, pH -5 with CMC (1%) and beef extract as carbon and nitrogen sources respectively.

Figure-1

Figure-2

Figure-3

Figure-4

Figure -5

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Gautam S.P, Bundela P.S, Pandey A K, Jamaluddin Khan, Awasthi M K, and Sarsaiya S. Optimization for the Production of Cellulase Enzyme from Municipal Solid Waste Residue by Two Novel Cellulolytic Fungi Biotechnology Research international vol: 2011.

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Pothiraj C, Balaji P, and Eyini M Enhanced production of cellulase by various fungal cultures in solid fermentation of cassava waste. African Journal of biotechnology 5: (2006). 1882 – 1885.

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Received on 03.04.2012

Modified on 12.04.2012

Accepted on 22.04.2012

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Research J. Science and Tech. 4(2): March-April 2012: 67-69