The medicinal and food industries have long used vitamin B12, a vital nutrient. Numerous bacterial strains, including Propionibacterium freudenreichii, Propionibacterium shermani, and Pseudomonas denitrificans, have been effectively exploited in the industrial production of vitamin B12 because chemical synthesis is too expensive, labor-intensive, and time-consuming. The major goal of this work was to identify microbial strains that produce vitamin B12 and to synthesize vitamin B12 from industrial wastewater, particularly dairy waste water. Propionibacterium species were isolated, identified, and characterized using molecular techniques, particularly polymerase chain reaction (PCR). The purpose of this study was to investigate how to increase vitamin B12 yield from Propionibacterium species utilizing RSM approach. Recent developments in biotechnology have produced techniques that limit modification, deliver results faster, and are less expensive.