Isolation and characterization of thermotolerant Gluconobacter strains catalyzing oxidative fermentation at higher temperatures

Thermotolerant acetic acid bacteria belonging to the genus Gluconobacter were isolated from various kinds of fruits and flowers from Thailand and Japan. The screening strategy was built up to exclude Acetobacter strains by adding gluconic acid to a culture medium in the presence of 1% D-sorbitol or 1% D-mannitol. Eight strains of thermotolerant Gluconobacter were isolated and screened for D-fructose and L-sorbose production. They grew at wide range of temperatures from 10 degreesC to 37 degreesC and had average optimum growth temperature between 30-33 degreesC. All strains were able to produce L-sorbose and D-fructose at higher temperatures such as 37 degreesC. The 16S rRNA sequences analysis showed that the isolated strains were almost identical to G. frateurii with scores of 99.36-99.79%. Among these eight strains, especially strains CHM16 and CHM54 had high oxidase activity for D-mannitol and D-sorbitol, converting it to D-fructose and L-sorbose at 37 degreesC, respectively. Sugar alcohols oxidation proceeded without a lag time, but Gluconobacter frateurii IFO 3264(T) was unable to do such fermentation at 37 degreesC. Fermentation efficiency and fermentation rate of the strains CHM16 and CHM54 were quite high and they rapidly oxidized D-mannitol and D-sorbitol to D-fructose and L-sorbose at almost 100% within 24 h at 30 degreesC. Even oxidative fermentation of D-fructose done at 37 degreesC, the strain CHM16 still accumulated D-fructose at 80% within 24 h. The efficiency of L-sorbose fermentation by the strain CHM54 at 37 degreesC was superior to that observed at 30 degreesC. Thus, the eight strains were finally classified as thermotolerant members of G. frateurii.