Production Of Amylase From Eichhornia Crassipes And Pistia Stratiotes As Substrates Using Solid State Fermentation

T. Godbole, C. Velhal and C. S. Kulkarni
Mitcon Biotechnology and Pharmaceutical Center, Shivajinagar, Pune – 411005.

ABSTRACT:
B. Subtilis (NCIM 2063) and A. Niger (NCIM 620) were used for amylase production from E. Crassipes and P. Stratiotes. The fermentation batches were set up in minimal basal media. Maximum amylase activity was seen by Aspergillus niger on Pistia stratiotes (Day 2) and Eicchornia crassipes (Day 4) at 32°C. A. niger with a spore count of 6.4×109 spores/ml after partial purification produced 6.8 units/ml enzyme in P. stratiotes and 7.3units/ml enzyme in E. crassipes with a spore count of 3.2×109 spores/ml. B. subtilis with CFU of 4.1×109 after partial purification produced 6.239 nits/ml enzyme in P. stratiotes and 6.08 units/ml enzyme in E. crassipes with CFU of 4.6×109. The results obtained are significant as there have been no reports of production of amylase from these two plants using solid state fermentation (SSF).

Keywords: Aspergillus niger, Bacillus subtilis, Eichhornia crassipes, Pistia stratiotes, Amylase and Solid substrate

INTRODUCTION

Amylases are a group of enzymes which consists of α- amylase, β-amylase and glucoamylase which hydrolyses starch and sugars. These are industrially important enzymes which are obtained from various microbial sources usually through submerged fermentation method. These enzymes capture about 25-30% of the worlds industrially important enzymes market1. Among these enzymes the most widely used and
important enzyme is α-amylase also called as 1, 4-α-Dglucan glucanohydrolase ([EC 3.2.1.1]) which find a wide array of application in the food industry, fermentation industry paper industry and textile industry.

These enzymes have been produced traditionally by submerged fermentation method owing to its ease in controlling various environmental parameters and handling. Glancing at the history we see that the submerged fermentation had become a role model technique since the development of penicillin due to which the ancient technique of solid state fermentation had got neglected2. Solid state fermentation is a
technique involving the absence of free water in the moist substrate3. This method of fermentation is an interesting alternative to the other techniques which eases the downstream processing of the metabolites4. The beginning of fermentation technique in nature is thought to begin on the moist surfaces of solids in ancient times (solid state fermentation). This method involves a number of advantages like it involves less
energy requirements, less waste water as no free water is involved in it, less downstream processing, more concentrated products after downstream processing and utilisation of natural waste substrates thus taking care of the waste disposal making this method ecofriendly5. Also the environmental conditions that are provided to the organism are more similar to its habitat.

Thus there is a renewed interest in harnessing this method in the industrial or large scale production of the enzymes and has been often used for the same3. It has been reported that by using solid state  fermentation the cost for the production media and downstream processing of the enzyme can be reduced by 50-60% as compared to the traditional submerge fermentation method thus making it a cost effective and cheaper method5. Eichhornia crassipes which belongs to the family Pontederiaceae, commonly known as water hyacinth is a perennial free floating aquatic plant bearing long, pendant roots, rhizomes, stolons, leaves, inflorescences and fruit clusters and usually 40 cm in height which may grow upto 1m also6. This plant is also considered as a noxious weed as it grows very fast and depletes the nutrients thereby lowering the BOD of the water body adversely affecting the flora and fauna. They can achieve a growth rate of 17.5 metric tons per hectare per day7. They reproduce sexually and asexually through seeds and stolons respectively. These seeds remain viable for up to 20 years making it difficult to control. If not controlled in time there have been instances when they have caused blockage of the water body making recreational activities like fishing very difficult also making them inhabitable and inaccessible. They also out-complete the other species in its vicinity outgrowing them and decreasing biodiversity. These plants also act as breeding grounds for insects and pests and also facilitate the water evaporation from the water bodies7. They also make the ecosystem less fertile by absorbing the nutrients in the water bodies surrounding it6, 8. Pistia stratiotes commonly called as water lettuce is a free floating aquatic weed which resembles an open head of lettuce. It forms dense mats restricting the water flow causing an increased evaporation in the water bodies, reduces infiltration of sunlight affecting the submerged aquatic plants and also acts as a breeding ground for mosquitoes9. Similar to Eicchornia crassipes this plant also acts as a noxious element and harms the environment adversely9. As both these plants have been causing a nuisance worldwide the need to control them is the necessity at the present date as they grow and spread at an alarming rate. These plants cause enormous blockages and facilitate the breeding and spread of various diseases, insects as well as they themselves grow and proliferate in such conditions8. A substantial amount is spent annually worldwide to control them by various physical, chemical, and biological means. These plants have been reported to act as substrates for the production of various industrially important enzymes. Here in this study we are going to use these nuisance causing plants as substrates for the production of amylase and check for its feasibility for the same. These plants will be used as carbon sources for Aspergillus niger, which is known to produce amylase in large quantities with other substrates10.

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