Isolation and Identification of Bacillus species from soil for amylase production and phosphate solubilisation

Abstract

Soil microorganisms play a vital role in maintaining the ecological balance by active participation in Carbon, Nitrogen, Sulphur, Phosphorus and Potassium cycles in nature. Phosphate solubilising microbes plays an important role in plant nutrition through increase in phosphate uptake by plants and is used as bio fertilizers of agricultural crops. Phosphate is second most vital macronutrient required for the growth and development of plants. The potassium solubilising microorganisms (KSMs) are rhizospheric microorganism which solubilises the insoluble potassium (K) to soluble forms of K for plant growth and yield. K-solubilisation is carried out by a large number of saprophytic bacteria. Amylases are the enzymes that break down complex carbohydrates. Amylases are amongst the most widely used enzymes in industries such as food, fermentation, starch processing, textile and paper. Plants, animals and microbes can produce amylases. Microbial amylase have a broad spectrum of industrial applications as they are more stable with great genetic diversity, high enzymatic activity in a wide range of condition and (extreme pH, temperature, osmolarity, pressure etc) simple and cost effective production and easy manipulation to obtain enzymes of desired characteristics. The aim of this study was to isolate ten different Bacillus isolates from various soil samples and their identification on the basis of Gram staining, colony morphology and biochemical tests. Gram positive rod shaped bacteria showing large, circular, opaque colonies having white or greyish white appearance were preserved on Nutrient agar slants for further identification tests. All the isolates showed positive results for Catalase, Urease, Oxidase and motility tests. For other Biochemical tests mixed results were obtained. Phosphate solubilisation and Potassium solubilisation activities of different isolates was performed. Out of ten isolates, none of the isolates solubilised phosphate on Pikovskaya’s agar medium at 30°C after 15 days of incubation. Potassium solubilising activity was shown by two isolates (Sample code 3 and 9) out of ten. Among these two isolates, Sample code 3 showed highest potassium solubilisation and the clear zone measured on Aleksandrov agar medium was 9mm and that of Sample Code 9 was 5mm after 10 days of incubation at 30°C. 39 The isolates were screened for the production of amylase and its assay has been performed. Seven isolates displayed zone of clearance in starch hydrolysis test except Sample code 5, 7 and 8. Sample code 5, 7 and 8 were the only isolates that gave negative results for TSI tests among all ten isolates. The isolates were subjected to amylase activity test. Maximum amylase was produced by Sample code 9 i.e. 3.90 mg/ml after 24 hours of incubation. Sample code 10 showed least amylase production i.e. 0.86 mg/ml.