Vitamin C is industrially produced by the Reichstein method, which uses gluconobacters to oxidize sorbitol to sorbose then a chemical process to convert sorbose to 2-keto-L-gulonic acid (2-KLG). The establishment of a more extensive microbial process for 2-KLG production translates into a less expensive and more efficient production of vitamin C. I examined pure strains and mixed cultures for their ability to produce 2-KLG using thin layer and high performance liquid chromatography. The DSM 4027 mixed culture produced the highest yield, 25 g/L, of 2-KLG from 100 g/L of sorbose, while the gram-negative rods isolated from DSM 4027 produced 8.8 g/L, and B. megaterium isolated from DSM 4027 produced 1.4 g/L. Thus, the gram-negative rods in the mixed culture were the primary 2-KLG producer, but B. megaterium in the DSM 4027 mixture enhanced this synthesis. Authentic pure cultures of Gluconobacter oxydans IFO strain 3293 and ATCC strain 621 produced 3.4 g/L and 5.7 g/L, respectively. Attempts to co-culture the isolated B. megaterium with the isolated gram-negative rods and authentic Gluconobacter strains did not increase 2-KLG production, nor did growing the cultures on B. megaterium spent media. Bacillus megaterium produced an unidentified keto-compound detected on the TLC chromatograms, which suggested that B. megaterium converted sorbose to an intermediate that may then be converted by the gram-negative rods in DSM 4027 to 2-KLG. Limited phenotypic tests suggested that the gram-negative rods in the DSM 4027 mixture are not gluconobacters. / Master of Science