Candida Magnoliae Glycerol Yield

Candida Magnoliae Glycerol Yield Abstract Candida magnoliae, isolated from honey comb and Candida glycerinogenes, isolated from natural environment were compared for their potential to produce glycerol from glucose. The highest yield of glycerol was 55% for C. magnoliae and 64.5% for C. glycerinogenes. C. glycerinogenes yields 9% of higher concentration of glycerol than C. magnoliae. For C. magnoliae the optimum conditions were a temperature of 30 °C and a pH of 5. The optimum conditions for C. glycerinogenes were a temperature of 32 °C  and a pH of about 5. The required medium composition for glycerol production was 160 g/L of glucose, 3 g/L of yeast extract, 5 g/L of peptone for C. magnoliae and 230 g/L of glucose, 5 ml/L of corn step liquor, 5-6 g/L of phosphate for C. glycerinogenes. Introduction Glycerol, a simple alcohol which contains three hydroxyl group. Glycerol is also known as glycerine or 1,2,3-Propanetriol. Glycerol has many uses in pharmaceutical, food, paint, cosmetic industries. Glycerol can be used additional fuel in boilers due to its high calorific value. The physiochemical properties and chemical composition of glycerol varies from other fuels. Glycerol is a odourless, colourless, viscous liquid with sweet taste. Glycerol contains three hydrophilic hydroxyl groups which is responsible for its solubility in water. Glycerol has a melting point of 17.9 oC and boiling point of 290 oC. Molecular formula of glycerol is CH2OH-CHOH-CH2OH. Glycerol can be transformed to various value added chemicals such as dihydroxyacetone, succinic acid, citric acid, ethanol, hydrogen etc., Until now the fermentative metabolism of glycerol was being reported in species of bacteria like Citrobacter sp, Enterobacter sp, Lactobacillus sp, Propionibacterium sp, Clostridium and many fungi species. Glycerol has become an abundant carbon source and inexpensive. Glycerol is also produced by yeast fermentation process. Glycerol is obtained as a byproduct during the fermentation of sugar to ethanol using Saccharomyces cerevisiae. Increased glycerol production from monosaccharides can be obtained using yeast fermentation. The production of glycerol in the laboratory is possible by yeast Candida magnoliae and osmotolerant yeast Candida glycerinogenes. Using genetic information, there are new possibilities in the field of fermentation and metabolic engineering. The Overexpression or blocking of genes could potentially can increase yield or productivity. Triose phosphate isomerase is an important enzyme in glycolytic pathway that directs dihydroxyacetone phosphate to glyceraldehyde 3-phosphate. When this triose phosphate isomerase gene was deleted, the mutant is able to achieve higher yield of glycerol. Overexpression of GPD1 gene in yeast increases glycerol production simultaneously increases the accumulation of byproducts such as succinate, acetate, pyruvate etc., This work briefs about the comparison of glycerol production in Candida magnoliae and Candida glycerinogenes. For the design of fermentation process, culture media optimization is an essential step. Many parameters such as phosphate, sulfate, temperature and pH have been found to affect the productivity of glycerol by these microorganisms. Hence these were optimized in prior to other parameters. Materials and Methods Organisms and Media All fermentation procedures were carried out with both C. magnoliae and C. glycerinogenes. C. magnoliae cells were propagated in medium containing 3 g/L of yeast extract, 160 g/L of glucose, 3 g/L of malt extract, 5 g/L of peptone. C. glycerinogenes cells were propagated in medium containing 150 g/L of glucose, 2 g/L of urea and 7 ml/L of corn steep liquor. YEP medium is required to grow C. glycerinogenes and YM medium is required to grow C. magnoliae. The cells were grown until the density reaches 0.2 OD. Fermentation process Fermentations were carried out in 250 ml shake flasks with a working volume of 50 ml. To the working medium 5% (v/v) of C. glycerinogenes and C. magnoliae were inoculated in different flasks. Flask for C. glycerinogenes was incubated at 31 oC and flask for C. magnoliae was incubated at 30 oC for 48 hours. Magnetically stirred 3-l fermentor was used for fermentation with working volume of 1-l. The medium was agitated at 500 rpm and aerated at 2.0 l/min. Analytical methods Glucose concentration was determined using glucose analyzer and glycerol concentration was monitored and confirmed with HPLC using an Aminex HPX 87H column with differential refractive index detector. Other compounds like ethanol, organic acid were analyzed using gas chromatography. Results Candida magnoliae was isolated from honey comb. C. magnoliae can utilize glucose as a carbon source for growth. It strongly ferments glucose to glycerol. This yeast is able to grow in YM medium containing glucose, yeast extract, malt extract and peptone. C. glycerinogenes was isolated from natural environment of high osmotic pressure. Glucose can be used as carbon source by this organism for glycerol production. Optimization Effect of Glucose concentration on glycerol production The effect of different concentration of glucose and glycerol production by C. magnoliae and C. glycerinogenes was determined in 250 ml flask containing 50 ml of medium. For C. magnoliae, concentration of glycerol yield increases when the concentration of glucose was increased from 100 g/l to 200 g/l and for C. glycerinogenes, concentration of glycerol increases when the concentration of glucose ranges between 150 g/L to 250 g/L . Further increase in the concentration of glucose causes a remarkable decrease in the yield. The optimum concentration of glucose for the growth using C. magnoliae was found to be 160 g/L and for C. glycerinogenes, glucose concentration was found to be 230 g/L. Table 1 Effect of different concentration of sulfate on glycerol productivity Glucose (g/L) Glycerol for C. magnoliae (g/L) Glycerol for C. glycerinogenes (g/L) 0 40.9 99.8 100 45.3 104.1 130 49.1 110.3 160 52.6 116.7 190 45.0 120.9 210 41.2 123.2 240 38.5 117.4 270 34.6 106.8 Fig. 1 Effect of initial concentration of glucose in the medium on the production of glycerol by C.magnoliae and C. glycerinogenes based on the amount of glucose consumed. Effect of Phosphate concentration on glycerol production It was found that phosphate is also an important factor in determining the glycerol productivity. A concentration of phosphate between 0 and 2 g/l increases the glycerol yield. Beyond 2 g/l of phosphate the yield of glycerol decreases gradually for C. magnoliae simultaneously the glycerol concentration for C. glycerinogenes decreases beyond 6 g/l of phosphate. Table 2 Effect of different concentration of phosphate on glycerol productivity Phosphate (g/L) Glycerol for C. magnoliae (g/L) Glycerol for C. glycerinogenes (g/L) 0 45.8 76.2 2 46.2 84.1 4 43.8 119.6 6 39.5 123.3 8 37.9 81.3 10 30.3 47.2 Fig. 2 Effect of initial concentration of phosphate on the production of glycerol in the medium by C.magnoliae and C. glycerinogenes based on the amount of consumed glucose. Effect of Temperature on glycerol production Using shake-flask culture, the optimum temperature was determined for glycerol production. The concentration of glycerol varies with temperature from 26 oC to 34 oC. The yield of glycerol increases till 30 oC for C.magnoliae and beyond this temperature the yield decreases. Similarly for C.glycerinogenes concentration of glycerol decreases beyond 32 oC. Table 3 Effect of temperature on glycerol productivity Temperature (oC) Glycerol for C. magnoliae (g/L) Glycerol for C. glycerinogenes (g/L) 26 68.6 79.6 28 73.2 101.1 30 77.3 114.2 32 74.1 130.4 34 70.5 125.7 Fig. 3 The temperature significantly affected the production of glycerol by à ¢-  C.magnoliae and à ¢-   C.glycerinogenes based on the amount of glucose consumed. Effect of pH on glycerol production Batch experiments were done to determine the effect of pH on glycerol production from C.magnoliae and C.glycerinogenes. At acidic pH the yield was found to be low. Between the pH 4 and 6, the production of glycerol was not significantly affected. At pH 5 there was a significant increase in the growth rate and glycerol production. Finally the optimum pH was found to be 5 for both the organisms. Table 4 Effect of pH on glycerol productivity pH Glycerol for C. magnoliae (g/L) Glycerol for C. glycerinogenes (g/L) 3.0 113.4 3.5 4.0 60.3 120.2 4.5 75.9 5.0 80.1 134.7 5.5 76.0 6.0 66.2 127.1 6.5 7.0 103.5 Fig. 4 Effect of initial pH of medium on production of glycerol by à ¢-  C. magnoliae and à ¢-   C. glycerinogenes based on the amount of glucose consumed. Discussion Glycerol yield by microbial fermentation of glucose using S. cerevisiae is less than 50%(w/w). But newly discovered C. magnoliae and osmotolerent yeast C. glycerinogenes produced glycerol in higher concentration compared with S. cerevisiae. 64.5% of glycerol obtained from C. glycerinogenes and C. magnoliae yields 55% of glycerol after recovery. Therefore C. glycerinogenes gives 9% more yield when compared to C. magnoliae. Increase in glycerol by C. glycerinogenes is due to overexpression of GPD1 gene. Also glycerol concentration increases in C. magnoliae when GPD1 gene overexpressed. But it also accumulated higher amount of byproducts such as acetate, succinate, pyruvate and acetoin. Many parameters such as temperature, pH, phosphate and glucose were found to affect the glycerol productivity by C. magnoliae and C. glycerinogenes. The optimum concentration of glucose for C. magnoliae was found to be 160 g/L and for C. glycerinogenes it was found to be 230 g/L, beyond these concentration range the yield of glycerol decreases. 2 g/L of phosphate was estimated as optimum concentration for glycerol production by C. magnoliae and similarly for C. glycerinogenes, maximum glycerol was obtained at 6 g/L of phosphate. 28 oC – 32 oC of temperature favors the good growth of cells and better glycerol production. Thus the optimum temperatures was declared to be 30 oC for C. magnoliae and 32 oC for C. glycerinogenes. Finding different species other than S. cerevisiae will give new knowledge, beyond physiological effect of glycerol production.