**3.4. Optimisation of raw starch degrading thermostable amylase and lactic acid production**

The amylase and lactic acid production was optimized by studying the effect of cultural and environmental variables (carbohydrate and nitrogen sources, metal salts and surfactants) individually and simultaneously. The effect of carbohydrate sources was studied by replacing soluble starch in basal medium with different sugars, gelatinized and raw natural crude starch sources (glucose, fructose, maltose, amylose, amylopectine, cassava, corn, rice tapioca, and sorghum flours at final concentration of 1% (w/v)). Nitrogen sources were tested by replacing yeast extract with various nitrogen sources (peptone, tryptone, beef extract, soyabean meal, ammonium sulphate, and urea at final concentration of 1.5% (w/v)). The effect of metal salts was studied by adding individually various metal salts (CaCl2.2H2O, MgSO4.7H2O, FeSO4.7H2O, FeCl3, NaCl at concentration of 0.1% (w/v)). Similarly the effect of surfactants was studied by supplementing the culture medium with Tween 80 and Tween 40 at concentration of 1.5% (v/v).

All media containing gelatinized starch sources were autoclaved at 121 °C for 20 min, while for the media containing raw starch flour, starch powder was sterilized by washing in ethanol and added to sterile nutrient broth.
