Glycerol production by anaerobic vacuum fermentation of molasses on pilot scale

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<ul><li><p>Glycerol Production by Anaerobic Vacuum Fermentation of Molasses on Pilot Scale </p><p>P. D. Virkar and M. S. Panesar Hindustan Lever Research Centre, Andheri (East), Bombay, 400 099 India </p><p>Accepted for publication March 6, 1986 </p><p>The use of sodium sulphite as a steering agent for enhancing the yield of glycerol during anaerobic ethanol fermentation is well established.' Several studies have been reported in the literature using free as well as immobilized cells of Saccharomyces cerevisiae.24 In these studies it was observed that a relatively high concentration of sulphite in the fermenting broth, typ- ically 40-100 g/L, was required to obtain a commer- cially significant yield of glycerol on sugar fermented. However, the dosing of large quantities of sulphite generally resulted in reduced viability of the micro- organisms and slow fermentations. The glycerol con- centration in the fermented broth was generally ob- served to be in the range 2040 g/L. The low productivity coupled with the high cost of sulphite rendered the process commercially unattractive. In order to reduce the sulphite requirement, whilst at the same time in- creasing the productivity, a modified vacuum fermen- tation was developed in our laborator ie~.~ The process was successfully established on a pilot scale and typical data obtained on scaleup are reported below. </p><p>EXPERIMENTAL </p><p>A proprietary strain of Saccharomyces cerevisiae, isolated from sugarcane molasses, was used in the fer- mentations reported here. The studies were carried out in an agitated stainless-steel fementer having a working volume of 0.25 m3. The temperature of the medium was maintained at 30 + 2"C, and the absolute gas pressure in the head space was kept at ca. 16 kPa by means of a water-ring vacuum pump. Sugarcane mo- lasses served as the sole source of C, N, P, nutrients, minerals, and growth factors. The typical composition of Indian canesugar molasses is given in Table I. The initial concentration of reducing sugars in the broth was adjusted to 30% (w/v) for all the fermentation. The initial pH of the medium was adjusted to ca. 7.0 and no control was exercised during the course of the fer- mentation. Sodium sulphite in powder form was dosed periodically such that the ratio of sugar fermented to sulphite added was maintained at ca. 13.5 (w/w). </p><p>Inoculum for the product fermenter was grown stagewise, initially in glass fermenters in the laboratory and subsequently in a 50-L stainless-steel fermenter in the pilot plant. An aqueous solution of 8-10% (w/v) molasses was used as the medium for inoculum growth, which was carried out aerobically (0.1 vvm) at a tem- perature of 30 k 2C. The growth time at each inoc- ulum stage was of the order of 24 h, and broth at a level of 1% (v/v) was used as seed material for the next growth stage. In the case of the product fermenter, however, an inoculum level of 10% (v/v) was used. </p><p>Inoculum growth fermenters were sterilized in situ at 120C for 20 min. The medium in the product fer- menter was only pasteurized by holding at 90C for 10 min. All inoculum transfers were carried out aseptic- ally. However, dosing of sodium sulphite was done nonaseptically. The concentration of glycerol in the fermenting broth was determined by the periodate ox- idation method after the separation from other polyols on silica gel TLC plates, developed using acetone as the solvent.6 The concentrations of reducing sugars and ethanol were determined by the procedures de- scribed by Patterson and co-workers' and Joslyn,8 re- spectively. The cell concentrations were obtained us- ing a Petroff Hawser chamber. </p><p>RESULTS AND DISCUSSION </p><p>The function of sodium sulphite in the fermentative production of glycerol is to chemically bind the acet- aldehyde formed during ethanol fermentation, and </p><p>Table I. Composition of Indian sugarcane molasses. </p><p>Number Item </p><p>I Total solids 70-75 2 Total sugar (as glucose)" 48-52 3 Nonsugar organicsb 8-12 4 Ash 9-1 1 </p><p>a Inverted sugar: 13-15%. Nitrogen: 0.3-0.5%. </p><p>Biotechnology and Bioengineering, Vol. XXIX, Pp. 773-774 (1987) 0 1987 John Wiley &amp; Sons, Inc. CCC 0006-3592/87/060773-02$04.00 </p></li><li><p>300 </p><p>- </p><p>- </p><p>- 200 4 </p><p>\ 01 - </p><p>d </p><p>E \ U </p><p>01 u d 4 </p><p>0.0 y - z i x </p><p>U z 0 U </p><p>2 3 </p><p>0.4 u </p><p>\ </p><p>100 </p><p>80 - \ 01 - </p><p>60 2 U z 0 U </p><p>40 g a </p><p>3 20 * </p><p>W U </p><p>0 0 24 48 72 </p><p>T I M E (hrs) </p><p>Figure 1. Time-course of the fermentation for glycerol production. Data points shown are averages of three batches carried out under identical conditions: (A) concentration of sugar in broth (g/L), (0) concentration of glycerol in broth (g/L), (0) cell concentration in broth (cells/mL), (X) cumulative amount of sulphite dosed (g/L), and (0) broth pH. </p><p>thereby enhance the yield of glyceroL3 In the present work, the sulphite requirement was reduced by the application of vacuum in the fermenter head space thus achieving the continuous removal of acetaldehyde, to- gether with ethanol and carbon dioxide, from the fer- menting broth. This also enabled the use of high initial sugar concentrations without concomitant accumula- tion of high levels of ethanol in the fermented broth, and consequent loss of vitality of the yeast. Under the conditions employed the peak value of ethanol con- centration was of the order of 30 g/L. </p><p>Typical data obtained in the course of our work are shown in Figure 1. Each data point shown is the av- erage for three fermentations carried out under iden- tical conditions. The sulphite concentration in the fer- menting broth was noted to be an important parameter requiring careful control. In practice this was achieved by following the sulphite dosing protocol stated above. The batch time was observed to be of the order of 72 h. The average yield of glycerol on sugar fermented was computed to be 26.3% (w/w). At the end of the fermentation, ca. 10% of the total reducing sugars re- mained unfermented. The average concentration of glycerol in the fermented broth was 71 g/L. </p><p>Independent mass balance studies on the laboratory scale had shown that the yield of ethanol under the conditions employed was also of the order of 25% (w/w). The yields of acetaldehyde and carbon dioxide were not measured experimentally. However, calculations based on the reported stoichiometry of sugar metabolism indicated yields of ca. 1 1% (w/w) and 36% (w/w), respectively. On the commercial scale, stripping of the volatiles from the fermented broth can also be </p><p>achieved by sparging an inert gas, e.g. carbon dioxide, through the medium. Theoretical computations, as- suming the outlet gas to be in equilibrium with a well- mixed broth, indicated that a gas flow rate of ca. 0.5 vvm would give results similar to those obtained with vacuum fermentation. This has been confirmed in bench- scale experiments using carbon dioxide as the inert gas.5 In practice, the carbon dioxide exiting the fer- menter could be recirculated after scrubbing the vol- atiles from the gas in an absorption column. </p><p>The pH of the fermenting broth has a significant effect on glycerol yield. In fermentations where the pH of the broth was maintained at ca. 5.0, average yields of glycerol were observed to be somewhat lower, of the order of 20% (w/w) rather than 26% (w/v). </p><p>References </p><p>1. S. C. Prescott and C. G. Dunn, Industrial Microbiology, 3rd ed. </p><p>2. G . G. Freeman and G. M . S. Donald, Appl. Microbiol., 5, 197 </p><p>3 . J. H. Harris and G . J. Hajny, J . Biochem. Micrubiol. Technol. </p><p>4. B. Bisping and H. J . Rehm, Eur. J . Appl . Microbiol. Biotechnol., </p><p>5 . G . P. Kalle, S. C. Naik and B. 2. Lashkari, J . Ferment. Technol., </p><p>6 . C . F. Smullin, L. Hartman, and R. S. Stetzler, JAOCS, 35, 179 </p><p>7. G . Patterson, E. B. Cowling, and J . Porath, J . Biochem. Biophys. </p><p>8. M . A. Joslyn, Methods in Food Analysis, 2nd ed. (Academic </p><p>(McGraw-Hill, New York, 1959), p. 208. </p><p>( 1957). </p><p>Eng., 2, 9 (1960). </p><p>14, 136 (1982). </p><p>63, 231 (1985). </p><p>( 1953). </p><p>Acta. , 67, 1 (1963). </p><p>Press, New York, 1970). p. 457. </p><p>774 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 29, APRIL 1987 </p></li></ul>