3.1. Microorganisms and Media
Y. lipolytica DSM70562 was obtained from DSMZ collection culture. The growth medium for activation contained 100 g/L glucose and 10 g/L yeast extract. Yeast cultures maintained at 4°C and sub-cultured each 4 weeks. The production medium contained 200 g/L glucose, 10 g/L yeast extract, 10 mg/L MnSO4.4H2O, and 2 mg/L CuSO4.5H2O in flask culture.
3.2. Culture Conditions
A single colony Y. lipolytica was inoculated into a 100 mL Erlenmeyer flask containing 10 mL of production medium and incubated at 30°C, 180 rpm for 48 h. Two and half milliliters of the broth was transferred into a 250 mL Erlenmeyer flask containing 50 mL production medium and incubated at 30°C for seven days on a reciprocating shaker with 180 rpm. Initial pH of the production medium was adjusted at 5.5 (4, 10). Fermentation samples were run and analyses usually were performed at 24 h intervals or when the fermentation was judged as complete. Samples were analyzed for residual sugar, total polyol concentration, erythritol concentration, and biomass density.
3.3. Optimization of Erythritol Production
Effects of environmental factors on erythritol production were studied. The process was examined in the presence of various carbon sources as well as nitrogen source, temperature, and initial pH.
3.4. Choice of Carbon Source
To study the effect of carbon sources on erythritol production by Y. lipolytica, glucose from production medium was substituted by sucrose at the concentration of 100 g/L. Respective media were inoculated with two and half milliliters of seed cultures from the activated culture and incubated at 30°C, 180 rpm on rotary shaker. After one week, samples were analyzed for biomass, residual glucose, and polyol concentration.
3.5. Effect of Yeast Extract
The effect of the yeast extract concentration on erythritol production in production medium was examined by 20 % glucose as a carbon source. Two and half milliliters of seed cultures were inoculated in 250 mL Erlenmeyer flasks with 50 mL medium of varying yeast extract concentrations ranging from 0.5 to 2.5 % and incubated at 30°C, 180 rpm for seven days. Samples were analyzed as described earlier.
3.6. Effect of Initial pH
Effect of initial pH on erythritol production was studied in shake flasks with 50 mL production medium. The pH of a production medium plays a vital role in the production of various products. In this experiment a range of pH from 4 to 7 was studied and used for final production. Incubation temperature of fermentation medium was maintained at 30°C.
3.7. Analytical Methods
Yeast growth was measured by centrifuging a sample of the fermented liquor, washing the yeast twice with distilled water, and drying at 80 ºC in 12 h. Glucose was measured using dinitrosalicylic acid method (9) after cell removal by centrifugation. The supernatant liquid from the yeast centrifugation was used for further analyses. Erythritol was separated from the sugars and other polyols by thin layer chromatography (TLC). TLC was conducted using a solvent system composed of ethyl acetate: 2-butanol: water in the ratio of 6:3:1 by volume. Polyols were stained and detected by spraying 1 % NaIO4 on the samples followed by 1 % KMnO4 (11). Polyols create yellow spots on purple background. Total polyols were determined by colorimetric method of Bok and Demain (12); the same method was applied to elute and determine erythritol from the paper. Yield of erythritol production was calculated by this formula: yield (%) = (erythritol / carbon source consumption) ×100 %.
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