Sample collection and processing. Soil samples were collected in a sterile beaker using a sterile spatula at a depth of 2-3 cm from a garden in Ramna Park, Dhaka, Bangladesh. One gram of soil sample was dispensed into 99 ml of sterile distilled water and homogenized. One ml of homogenized soil sample was transferred into 9 ml sterile distilled water and serial dilution was carried out up to 10-8 dilution.
Isolation and screening of amylolytic bacteria. Serially diluted bacterial cultures (100 µl) were spread on nutrient agar media and incubated at 37oC for 24 h. Their colony morphology of isolated colonies were noted and streaked on starch agar media containing starch as sole carbon source for starch hydrolysis test to detect their amylolytic activity. The plates were incubated at 37oC for 24-48 h. Following incubation, plates were flooded with Gram’s iodine solution (Gram’s iodine- 250 mg iodine crystals added to 2.5 g potassium iodide solution and 125 ml of water) to identify zone of clearance around the colony. Deep blue color around the growth indicates negative result that is no amylolytic activity where zone of clearance was produced by amylase producers. The pure cultures showing clear zones were subcultured at regular interval and maintained on to nutrient agar slants at 4oC.
Identification of amylase producing bacteria. Isolated amylase producing bacteria were presumptively identified by Gram staining, cultural and biochemical characteristics. The biochemical tests included methyl red, voges-proskauer, citrate utilization, indole production, H2S production, motility, gelatine hydrolysis, sugar hydrolysis, oxidase, catalase and carbohydrate fermentation. Mannitol egg yolk polymyxin (MYP) media (Egg yolk emulsion and Polymyxin B sulphate were added in MYP agar base) was used to selectively isolate only Bacillus cereus which was purchased as dehydrated media from Oxoid Ltd (Basingstoke, UK), prepared according to the manufacturer’s instructions. Only those isolates that are presumptively isolated as Bacillus spp. were further analyzed by different experiments.
Crude enzyme preparation and extraction. To extract amylase enzyme, a loopful of pure culture was inoculated in production media containing starch (10 g/l), peptone (5 g/l), (NH4)2SO4 (2 g/l), KH2PO4 (1 g/l), K2HPO4 (2 g/l), MgCl2 (0.01 g/l) at pH 7 and incubated at 37oC in a shaking water bath for 24 h. Following incubation, 10 ml of 24 h old culture was centrifuged at 5,000 rpm for 15 min. Cells were discarded and supernatant was decanted for crude enzyme that was used for optimization of assay condition for amylase activity.
Amylase assay. Amylase activity was assayed by employing 3, 5 dinitrosalicylic acid (DNS) method as described previously with few modifications (11). In brief, 1% starch solution was prepared freshly by dissolving 1 g of soluble starch in 100 ml of 0.02 M sodium phosphate buffer (pH, 7). To prepare assay condition, 1 ml of 1% starch solution and 0.5 ml of crude enzyme extract was incubated at 50oC for 30 min. The assay was stopped by adding 3 ml of DNS reagent and heated the solution in a boiling water bath for 10 min. Then, with running tap water the solution was cooled. The solution volume was brought up to 10 ml by adding distilled H2O and the absorbance was recorded using spectrophotometer. A blank was always prepared without the enzyme. Enzyme activity was measured by preparing a standard graph with known concentrations of standard (glucose) and plotted. Here, one unit (U/ml) of amylase activity is defined as the amount of amylase required to catalyze 1 µmol of reducing sugar (glucose) from starch per minute under the assay condition.
Production of ammonia. Isolates with the ability to release simple ammonia from complex proteins are considered as potential for plant growth. To detect ammonia excretion, freshly grown bacterial cultures were inoculated in 5 ml sterilized peptone water containing test tubes and incubated at 37oC for 48 h. Following incubation, 1 ml of Nessler’s reagent was added to peptone water. The tubes were thoroughly shaken and centrifuged at 12,000 rpm for 15 min. The supernatants were taken to measure absorbance at 450 nm in a spectrophotometer.
Denitrification. Denitrification is a step where nitrates are reduced to nitrites, ammonia, nitrous oxide and finally to elemental nitrogen in the form of nitrogen gas. To detect nitrification, freshly grown bacterial isolates were inoculated in nitrate broth containing Durham tube. Following 48 h of incubation at 37oC, the test cultures were examined for the presence or absence of the air bubble in the Durham tubeo.
Nitrogen fixation capability. To detect whether the isolates can fix atmospheric nitrogen, freshly grown bacterial cultures were streaked in nitrogen-free Jensen’s media (sucrose 10 g, dipotassium hydrogen phosphate 1 g, magnesium sulphate 0.5 g, sodium chloride 0.5 g, ferrous sulfate 0.1 g, sodium molybdate 0.005 g, agar 20 g, for 1 liter, pH 7.0-7.2). The media is devoid of any nitrogen source. The media were incubated at 37oC and checked each day for any growth till 15 days.