Current study was focused on the development of a non-fastidious lactic acid producing strain having better growth rate, low pH tolerance and good productivity by genome shuffling of a mutant strain of Lactobacillus delbrueckii NCIM 2025 and an amylase producing non-fastidious Bacillus amyloliquefaciens ATCC 23842. After the third cycle of the protoplast fusion, lactic acid production by few fusants was monitored and the best fusant was selected for further studies. Optimization of the important process parameters for lactic acid production was conducted using Plackett-Burman design and response Surface methodology. Selected fusant could utilize the liquefied cassava bagasse starch directly with minimum nutrient supplementation for lactic acid production. During validation, 40 g/L of lactic acid was obtained (similar to 96% conversion of starch to lactic acid) by using fusant inoculum (3%, v/v) from 83 g/L cassava bagasse (starch content 50% w/w) supplemented with yeast extract and peptone (0.2% each, w/v) and the buffering agent (2% CaCO3, w/v). (C) 2008 Elsevier Ltd. All rights reserved.