Fermentation of Penaeus semisulcatus shrimp shell was performed by two bacteria to evaluate the degree of demineralization. Since the use of nutrient culture medium as the main culture medium increases costs, the rate of demineralization in commercial (nutrient medium) and non-commercial (distilled water) culture medium was compared. For this purpose, a successive two-step fermentation process was used. Thus, shrimp shells were first fermented by Bacillus licheniformis for 3 days and then by Lactobacillus plantarum for 4 days. During successive fermentation, the pH and demineralization efficiency of the two mediums were compared. In the first 3 days, the pH of the two mediums was not significantly different from each other, but in the next 4 days, a further decrease in pH was observed in the medium containing distilled water. At the end of 7 days of fermentation, the rate of demineralization in both commercial and non-commercial culture mediums was 87.29% and 98.09%, respectively. Therefore, the use of distilled water, in addition to the economic aspect, also showed the higher efficiency. Finally, the FTIR spectrum of chitin produced by distilled water was investigated.