Development Kraft pulping of bagasse by using

Egyptian Sugar Journal, June 2015 Vol.8 : 27 - 52 ‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬

Key Factors Affecting the Efficiency of Ethanol Fermentation Using Beet Molasses A. A. Zohri a, A. M. Ramadan b, M. M. El-TabakhC and K. Al-Tantawyc a)

Assiut University ,Faculty of Science, Egypt, Kafr El-Sheikh University, Faculty of Science, Egypt , c) Delta Sugar Company, Egypt. b)

Corresponding Author: Zohri, A.A., email: [email protected]

Abstract A number of factors affecting yeast fermentation performance have been investigated. These include temperature, pH and substrate concentration. Temperature, pH (physical factors) and substrate (sugar) concentration were observed to play a key role in productivity and fermentation efficiency of ethanol fermentation of beet molasses. The optima production conditions for bio-ethanol from beet molasses by the tested yeast strain were: 20% sugar concentration, pH at 5.0 for normal & high gravities and incubation temperature at 32˚C for normal gravity and 35˚C for high gravity conditions. Keywords:

bio-ethanol, fermentation efficiency, productivity, beet molasses, yeast, normal gravity and high gravity.

Introduction Beet molasses has high content of solids (approximately 80%), saccharose (51% in average), 1% rafinose, 0.25% glucose and fructose, 5% proteins, 6% betain, 1.5% nucleosides, 1.5% purine and primidine bases, organic acids and pectins (Šušić et al., 1989). Important constituents of molasses are minerals and vitamins. Minerals are nutrients necessary for growth and normal physiological functioning of yeast and are part of many enzymes and hormones (Bíró et al., 1988). In molasses, calcium, potassium and iron are present in substantial amounts although their contents vary over wide ranges. It is especially important to note that minerals in molasses are dissolved and that potassium is dominant with a share of 75% (by weight) of total cations (Šušić et al., 1989). Molasses also contains B group vitamins and does not contain fats and fibers. In addition, molasses shows humectancy, antioxidant and water

A. A. Zohri, et al (2015) Egyptian Sugar Journal, Vol.8 : 27 - 52 ‫ـــــــــــــــــــــــــــــــــــــــ ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬

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activity lowering properties which are important to the shelf-life of the products (Hickenbottom, 1996). Dodic et al. (2009) and El-Refai et al. (1992) reported that the increase in sugar concentration led to an increase in ethanol concentration and the highest ethanol yield was obtained at a sugar concentration of 20.8% (w.v -1). Roukas (1996) found that the maximum ethanol yield was obtained at a sugar concentration of 250 g L-1 and increasing the sugar concentration led to a decrease in ethanol yield which was also seen by Gőksungur and Zorlu (2001). Zayed and Foley (1987) found that the optimum pH for ethanol production was 4.5 in contrast to El-Refai et al. (1992) who found it to be 5. This was perhaps because two different yeast strains were investigated, each with their own optimum. Temperature extremes during fermentation can severely affect yeast growth and metabolism (Specht, 2003). Ethanol resistance is also influenced by temperature (Heard and Fleet, 1988; Bisson, 1999; Bisson and Butzke, 2000). At higher temperatures, the cell membrane fluidity increases and ethanol can enter the cell more readily, adversely affecting metabolism and cell viability. Cooler temperatures may enhance ethanol resistance by increasing sterol levels in yeast cell membranes (Suutari et al., 1990; Torija et al., 2003) resulting in decreasing accumulation of intracellular ethanol (Lucero et al., 2000). This study was designed to assess the effect of temperature, pH and sugar concentration on the ethanol fermentation efficiency of beet molasses with normal gravity (sugar level 190 gL-1) and high gravity (sugar level 220 gL-1)

Material and Methods Beet molasses The beet molasses used as a raw material for ethanol production after pretreated by hot hydrochloric acid and clarification was kindly supplied by the delta sugar company, Egypt. Organism and culture maintenance

Key factors affecting the efficiency of ethanol fermentation 37 ‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬

Saccharomyces cerevisiae GHM which used for brewing industry at Germany with codec numb (BG2). The strain of S.cerevisiae was maintained at 4˚C on agar slants. The composition of the agar medium was (g / l): yeast extract 3, malt extract 3, peptone 5, glucose 10 and agar 20. The cultures were maintained by sub-culturing every 20-days and the test tubes were then incubated at 30˚C for 36 h. Fermentation medium The fermentation beet molasses medium used in this study was treated by the method described by Rajagopalan and Krishnana (2008) with some modification. It was composed of (g L-1); mud-free, hot HCl-Treated beet molasses 200, Urea 1.08, MgSO4.7H2O 0.3 and H3PO4 0.3. The pH was adjusted at 5.0. Temperature Variations Temperature experiments were first carried out to discern what the optimal temperature for alcoholic fermentation at normal gravity (NG) and high gravity (HG). To optimize the fermentation temperature, fermentation was carried out at 25, 30, 32, 34, 36 and 38˚C for (NG), and 30, 32, 34, 35, 36 and 38˚C for (HG). Three liters of treated and sterilized beet molasses: 1.5L with sugar concentration 19% and another 1.5L with 22% was prepared. The pH was adjusted to 5.0. Fermentation was carried out in 500 ml sterilized conical flask which had 250 ml of working volume and inoculated with prepared inoculum of selected yeast at rate of 10%. Cultures were incubated at 120 rpm for 48 hours. The periodic samples were analyzed for dry yeast weight and ethanol content. pH Variations pH experiments were first carried out to discern what the optimal pH for alcoholic fermentation and yeast growth at normal gravity (NG) and high gravity (HG). Three liters of treated and sterilized beet molasses 1.5L with sugar concentration 19% (NG) and another 1.5L with 22% (HG) was prepared. The Temperature was adjusted to 32˚C for (NG) and 35˚C for (HG). Fermentation


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was carried out in 500ml sterilized conical flask which had 250 ml of working volume, and inoculated with prepared inoculum of selected yeast at rate of 10%. pH was controlled to 4.0, 4.5, 4.8, 5.0, 5.5, and 6.0 for both NG and HG by a controller that used NaCl and NaOH to keep the pH constant. Cultures were incubated at 120 rpm for 48 hours. The periodic samples were analyzed for dry yeast weight and ethanol content. Substrate Variation Different sugar concentrations of treated beet molasses (5%, 10%, 15%, 20%, 25%, and 30%) were prepared as fermentation medium. The pH and temperature were adjusted to 5.0 and 35˚C respectively. Fermentation was carried out in 500 ml sterilized conical flask which had 250 ml of working volume and inoculated with prepared inoculum of selected yeast at rate of 10%. Cultures were incubated at 120 rpm for 48 hours. The periodic samples were analyzed for dry yeast weight and ethanol content. Analytical method: Sugar Determination Sugar concentration was estimated using 3,5-dinitrosalicylic acid and glucose as standard (Miller, 1959). Determination of ethanol concentration and pH Ethanol concentrations were estimated by dichromate method described by Zohri and Eman Mostafa (2000). pH measured by using digital bench pHmeter, model pH-526/sentix – 20/AS- DIN/ SIN/ STH/ 650 according to procedure of Delta Sugar Company. Determination of biomass concentrations: Biomass concentration was measured by dry weight analysis. The carefully balanced samples (5ml each) were centrifuged at 4000 rpm for 5 minutes. The supernatant was poured out and 5 ml water was added to the remaining pellet. After dissolving with vortex they were centrifuged again. This washing step was carried out twice. The glass tubes containing pellets were put


in the oven at 105°C and were measured after 24 hours. The difference in the weights was calculated and the dry weight was expressed in gL-1 .

Results and Discussion Temperature Variation Temperature is one of the major constraints that determines the ethanol production. To know the optimum temperature for ethanol fermentation, the fermentation media were kept at 25, 30, 32, 34, 36 and 38˚C, and the growth, ethanol concentration and fermentation efficiency were detected. The maximum ethanol production 10.6%, biomass concentration 5.5 g.L-1 and fermentation efficiency 91.4%, (Table, 1 and Figure, 1) were obtained at 32˚C when used normal gravity pretreated beet molasses. On the other side, evident from Table 2 and Figure 2 that the optimum temperature for alcoholic fermentation at high gravity were 35˚C,

where ethanol concentration, growth and fermentation

efficiency recorded 12.3%, 6.2 g.L-1 and 91.8%, respectively. This result is in agreement with most previous studies on Saccharomyces cerevisiae. The effect of temperature has been investigated by Rogosa et al. (1947), Burgess and Kelly (1979), Demott et al. (1981), Chen and Zall (1982), Castillo et al. (1982), Marwaha and Kennedy (1984), Vienne and Von Stockar (1983), Tu et al. (1985), and Zayed and Foley (1987). Increasing in fermentation temperature was recorded as a successfully employed to increase the fermentation rate (Dragone et al., 2004). Zohri et al. (2014) studied ethanol production from treated cane molasses (sugar concentration 25%) using S. cerevisiae EC1118 strain and found that the fermentation efficiency recorded 99.97% at 35˚C. Lin et al. (2012) observed that when the temperature increased, the maximum fermentation time was shortened, but a much higher temperature, inhibit the growth of the cells then the fermentation significantly decline. Torija et al. (2003) found that alcohol fermentation increased as the temperature increased and recorded that the alcohol fermentation at 35˚C had no lag phase but they had a quick exponential phase and reached the maximal level earlier. Temperature tolerance was found


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to depend upon sugar concentration of the medium (Morimura et al., 1997). They observed that fermentation of molasses at 35˚C was possible when sugar concentration was 20%(w/v), while no fermentation when sugar concentration was 22%(w/v). Temperature extremes during fermentation can severely affect yeast growth and metabolism (Specht, 2003). Ethanol resistance is also influenced by temperature (Heard and Fleet, 1988; Bisson, 1999; Bisson and Butzke, 2000). At higher temperatures, the cell membrane fluidity increases and ethanol can enter the cell more readily, adversely affecting metabolism and cell viability. Cooler temperatures may enhance ethanol resistance by increasing sterol levels in yeast cell membranes (Suutari et al., 1990; Torija et al., 2003) resulting in lower accumulation of intracellular ethanol (Lucero et al., 2000). The general conclusion is that the optimum temperature is in the range 30 to 40 ˚C, with most reports identifying 35˚C as the peak for ethanol production. However, seems some variation depending on the strain of the yeast studied. pH Variation Table 3 and Figure 3 show clearly the effect of pH on the cell growth and fermentation efficiency at normal gravity and 32˚C in a medium containing a fixed amount of nitrogen source (NH4Cl, 1g/L) with an incubation time of 48 hrs. According to the experimental data, the pH range of 4.5-5.5 was selected for the cell growth and ethanol production. At pH 5, the maximum growth rate, ethanol concentration and fermentation efficiency were 4.1gL-1, 10.8%, 93%, respectively. On the other side, when the initial sugar concentration was increased at high gravity (Sugar concentration 220gL-1) and 35˚C with an incubation time of 48 hrs, the results appeared that at pH 5, the maximum growth rate, ethanol concentration and fermentation efficiency were 4.4gL-1, 12.2% and 90.8%, respectively (Table 4, Figure 4). These results are in agreement with that recorded by Zohri et al.( 2013 & 2013). On the other side; with a further increase in pH, ethanol production was decreased. This finding is in consistence with Mollison (1993) and Zohri et al. (2012). Control of pH


during ethanol fermentation is important for retarding the growth of harmful bacteria as well as improving the yeast growth by acidic solution (Mathewson, 1980). Also, with increase in pH, yeast produces acid rather than alcohol. Molasses has naturally alkaline pH and must be acidified prior to fermentation (Hodge and Hildebrandt, 1954). Yadav et al (1997) found that an increase in alcohol concentration, productivity as well as efficiency with an increase in pH from 4.0-5.0 and found that the optimum pH range for S.cerevisiae strain HAU-1 to be between pH 4.55.0. Zohri and Eman Mostafa (2000) found that the pH of 3.5 to 4.5 was the optimum for ethanol production by S. bayamus form date juice. Russell (2003) recorded that yeast prefers an acidic pH and its optimum pH is 5.0-5.2 but brewing and distilling strains are capable of good growth at the pH range of approximately 3.5 to 6.0. Narendranath and Power (2005) found that the optimum pH for yeast growth and ethanol production by S. cerevisiae was pH 4.9. But it doesn’t match results of Sivakumar et al, (2010), who found pH 4 optimum for ethanol production and this is due to difference in the tested strains. Substrate Variation Different sugar concentrations of treated beet molasses (5%, 10%, 15%, 20%, 25%, and 30%) were prepared as fermentation medium in 2000 ml Erlenmeyer flasks each contained 1000 ml molasses media and inoculated with 10 ml of 24 h, S. cerevisiae GHM yeast culture, and incubated in a shaking incubator at 35˚C and 200 rpm for 48 h. The optimum level of sugar was 20% (w/v) sugar in molasses medium (Table 5, Figure 5). Maximum amount of ethanol concentration, growth rate and fermentation efficiency (11.3%, 4.2 gL-1 and 92.5% respectively) were produced when the sugar concentration was 20% (w/v). Further increase in the sugar concentration, resulted in the decrease of its conversion to ethanol. So, sugar concentration plays an important role in ethanol fermentation by yeast. For economic reasons the residual sugar for maximum ethanol formation should be negligible at the end of fermentation.


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This results are in agreement with that obtained by Zohri et al. (2012) who recorded that beet molasses with 20% sugar was suitable & best level for ethanol production by S. cerevisiae AU71 and found that the ethanol yield reached to 91% of the theoretical value. The decrease in fermentation efficiency by increasing the sugar level above 20% may be due to the substrate inhibition (Sedha and Verma, 2002). Monot et al. (1982) studied the effect of sugar in synthetic medium and found the yield of ethanol was maximum when sugar level ranged from 4.0 to 6.0% (w/v). The volume of ethanol production was 20 ml per 100 g of molasses. Zohri and Eman Mostafa (2000) studied the effect of different sugar concentration (13.5, 18 and 22.5%) in the date juice on the fermentation efficiency of two yeast strains named S. cerevisiae and S. bayamus at 28˚C and found that the date juice with 18% sugars was the more suitable and economic concentration. As general, increasing sugar concentration leads to increase the osmotic pressure and viscosity in fermentation medium and this had a highly inhibiting effect on yeast growth and their capability to ethanol production, (Table 5, Figure 5). Similar results were recorded previously in the fermentation medium with high gravity worts (Pratt-Marshall et al., 2002). They observed that fermentation of high gravity worts have a negative effect upon the yeast performance due to the elevated osmotic pressure. D Amore (1987) reported that as the initial wort gravity is increased, the rate of fermentation decrease and the amount of ethanol produced is lower than the one theoretically expected. Moaris et al. (1996) also studied viability of Saccharomyces sp. in 50% glucose and reported a viability of 10-98.8% in different strains of yeast. The detrimental effect of high sugar concentration on ethanol production was studied by Gough et al. (1996) in Kluyveromyces marxianus and a sucrose concentration more than 23% in molasses was found to affect ethanol production.


Incubation Time (h) Growth (gL-1) Alcohol % 25 Fermentation efficiency % Growth (gL-1) Alcohol % 30 Fermentation efficiency % Growth (gL-1) Alcohol % 32 Fermentation efficiency % Growth (gL-1) Alcohol % 34 Fermentation efficiency % Growth (gL-1) Alcohol % 36 Fermentation efficiency % Growth (gL-1) Alcohol % 38 Fermentation efficiency %

0 2.7 0.0 0.0 2.8 0.0 0.0 2.9 0.0 0.0 2.8 0.0 0.0 2.7 0.0 0.0 2.8 0.0 0.0

12 2.8 1.9 16.4 3.2 5.1 44.0 3.6 5.9 50.9 2.9 5.2 44.8 2.9 4.9 42.2 2.9 4.3 37.1

24 3.1 5.1 44.0 3.9 9.6 82.8 4.2 10.1 87.1 3.1 9.3 80.2 3.0 7.1 61.2 3.0 7.7 66.4

36 3.9 8.5 73.3 4.9 9.9 85.3 5.4 10.5 90.5 3.5 9.9 85.3 3.2 8.2 70.7 3.2 7.8 67.2

Growth (g/L‎) 25˚C






Efficiency‎%‎25˚C






5.6 5.2 4.8 4.4 4 3.6 3.2 2.8 2.4 2 1.6 1.2 0.8 0.4 0

48 4.1 8.9 76.7 4.9 10.4 89.7 5.5 10.6 91.4 3.8 10.2 87.9 3.3 8.5 73.3 3.3 7.9 68.1

95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 0

12

24

36

Fermentation efficiency %

Growth (g/L)

Temperature ˚C

Table(1): Effect of different temperature on both growth and fermentation efficiency by S. cerevisiae GHM at normal gravity (Sugar concentration 190 g.L-1), pH 5 and incubation time 48h.

48

Time

Figure (1):Saccharomyces cerevisiae GHM yeast growth, and fermentation efficiency% at different temperature, pH 5, incubation time 48 hrs and NG (Initial Sugar concentration 190 g.L-1).


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Table(2):Effect of different temperature on both growth and fermentation efficiency by S. cerevisiae GHM at high gravity (Sugar concentration 220 g.L-1), pH 5 and incubation time 48h.


Growth (gL-1) Alcohol %

Temperature ˚C

32



34



35



36



38


Growth (g/L)

Growth‎(g/L)‎30˚C Growth‎(g/L)‎36˚C Efficiency‎%‎34˚C

Growth‎(g/L)‎32˚C Growth‎(g/L)‎38˚C Efficiency‎%‎35˚C

0 3.7

12 4.1

24 4.8

36 5.1

48 5.2

0.0 0.0 3.8 0.0 0.0 3.9 0.0 0.0 3.8 0.0 0.0 3.7 0.0 0.0 3.8 0.0 0.0

5.1 38.1 4.3 5.8 43.3 4.7 5.8 43.3 4.5 6.1 45.5 4.6 5.9 44.0 4.2 6.0 44.8

9.9 73.9 5.0 10.3 76.9 5.2 10.7 79.9 5.6 11.9 88.8 5.7 11.2 83.6 4.9 10.9 81.3

10.5 78.4 5.3 11.7 87.3 5.7 11.5 85.8 6.1 12.1 90.3 5.9 11.9 88.8 5.5 11.4 85.1

10.8 80.6 5.5 11.9 88.8 5.8 12.1 90.3 6.2 12.3 91.8 6.0 12.2 91.0 5.5 12.0 89.6

Growth‎(g/L)‎34˚C Efficiency‎%‎30˚C Efficiency‎%‎36˚C

Growth‎(g/L)‎35˚C Efficiency‎%‎32˚C Efficiency‎%‎38˚C

6.4 6 5.6 5.2 4.8 4.4 4 3.6 3.2 2.8 2.4 2 1.6 1.2 0.8 0.4 0

95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 0

12

24 Time (h)

36


Incubation Time (h) Growth (gL-1) 30 Alcohol %

48

Figure (2): Saccharomyces cerevisiae GHM yeast growth, and fermentation efficiency% at different temperature, pH 5, incubation time 48 hrs and HG ( Initial Sugar concentration 220 g.L-1).


pH

Effect of different pH on both growth and fermentation efficiency by S. cerevisiae GHM at normal gravity (Sugar concentration 190 g.L-1), 32˚C and incubation time 48h.

Incubation Time (h) Growth (gL-1) 4.0 Alcohol % Fermentation efficiency % Growth (gL-1) 4.5 Alcohol % Fermentation efficiency % Growth (gL-1) 4.8 Alcohol % Fermentation efficiency % Growth (gL-1) 5.0 Alcohol % Fermentation efficiency % Growth (gL-1) 5.5 Alcohol % Fermentation efficiency % Growth (gL-1) 6.0 Alcohol % Fermentation efficiency %

Growth g/L

Growth g/L pH4 Growth g/L pH5 Fermentation efficiency % pH4 Fermentation efficiency % pH5

0 2.6 0.0 0.0 2.5 0.0 0.0 2.6 0.0 0.0 2.5 0.0 0.0 2.7 0.0 0.0 2.6 0.0 0.0

12 2.8 6.9 59.4 2.8 7.6 65.5 2.9 7.7 66.3 2.9 7.8 67.2 3.1 7.5 64.6 2.9 6.6 56.9

Growth g/L pH4.5 Growth g/L pH5.5 Fermentation efficiency % pH4.5 Fermentation efficiency % pH5.5

24 3.1 9.8 84.4 3.2 10.2 87.9 3.3 10.2 87.9 3.2 10.5 90.4 3.5 10.2 87.9 3.0 9.7 83.6

36 3.5 10.0 86.1 3.8 10.4 89.6 3.9 10.6 91.3 3.8 10.8 93.0 3.9 10.6 91.3 3.5 9.9 85.3

48 3.5 10.1 87.0 3.9 10.6 91.3 4.0 10.7 92.2 4.1 10.8 93.0 4.1 10.6 91.3 3.6 10.0 86.1

Growth g/L pH4.8 Growth g/L pH6 Fermentation efficiency % pH4.8 Fermentation efficiency % pH6

4.2 4 3.8 3.6 3.4 3.2 3 2.8 2.6 2.4 2.2 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 0

12

24

36

Fermentation efficiency%

Table(3):

48

Time hus

Figure (3): Saccharomyces cerevisiae GHM yeast growth, and fermentation efficiency% at different pH, temperature 32˚C, incubation time 48 hrs and NG ( Initial Sugar concentration 190 gL-1).


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Table (4): Effect of different pH on both growth and fermentation efficiency by S. cerevisiae GHM at high gravity (Sugar concentration 220 g.L-1), 35˚C and incubation time 48h.

Growth g/L pH4 Growth g/L pH5 Fermentation efficiency % pH4 Fermentation efficiency % pH5

0 2.5 0.0 0.0 2.5 0.0 0.0 2.6 0.0 0.0 2.5 0.0 0.0 2.6 0.0 0.0 2.6 0.0 0.0

12 2.8 7.4 55.1 2.7 7.9 58.8 2.7 8.0 59.5 2.8 8.0 59.5 2.7 7.9 58.8 2.7 7.3 54.3

Growth g/L pH4.5 Growth g/L pH5.5 Fermentation efficiency % pH4.5 Fermentation efficiency % pH5.5

24 3.2 10.9 81.1 3.2 11.1 82.6 3.8 11.3 84.1 3.7 11.4 84.8 3.5 11.3 84.1 3.0 10.8 80.3

36 3.8 11.7 87.0 3.7 11.7 87.0 4.0 12.1 90.0 4.1 12.1 90.0 3.9 11.9 88.5 3.8 11.4 84.8

Growth g/L pH4.8 Growth g/L pH6 Fermentation efficiency % pH4.8 Fermentation efficiency % pH6

4.8

95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0

4.4 4 3.6 3.2 Growth (g/L)

48 3.9 11.8 87.8 4.1 11.9 88.5 4.3 12.1 90.0 4.4 12.2 90.8 4.1 12.0 89.3 4.0 11.7 87.0

2.8 2.4 2 1.6 1.2 0.8 0.4 0 0

12

24 Time hus

36


pH

Incubation Time (h) Growth (gL-1) 4.0 Alcohol % Fermentation efficiency % Growth (gL-1) 4.5 Alcohol % Fermentation efficiency % Growth (gL-1) 4.8 Alcohol % Fermentation efficiency % Growth (gL-1) 5.0 Alcohol % Fermentation efficiency % Growth (gL-1) 5.5 Alcohol % Fermentation efficiency % Growth (gL-1) 6.0 Alcohol % Fermentation efficiency %

48

Figure (4): Saccharomyces cerevisiae GHM yeast growth, and fermentation efficiency% at different pH, temperature 35˚C, incubation time 48 hrs and HG ( Initial Sugar concentration 220 gL-1).


Table (5): Effect of different sugar concentration on both growth and fermentation efficiency by S. cerevisiae GHM at pH 5, 35˚C and incubation time 48 (h)

Growth (g/L) Sugar 5% Growth (g/L) Sugar 20% Fermentation efficiency% Sugar 5% Fermentation efficiency% Sugar 20%

0 2.2 0.0 0.0

12 2.5 1.9 62.2

24 2.8 2.5 81.8

36 3.1 2.5 81.8

48 3.5 2.5 81.8

2.2 0.0 0.0

2.7 4.1 67.1

3.2 5.2 85.1

3.5 5.2 85.1

3.8 5.2 85.1

2.3 0.0 0.0

2.7 5.1 55.6

3.5 7.8 85.1

3.8 8.2 89.5

4.0 8.2 89.5

2.2 0.0 0.0

2.9 6.2 50.7

3.7 10.5 85.9

3.9 11.1 90.8

4.2 11.3 92.5

2.3 0.0 0.0

2.7 5.1 33.4

3.3 9.9 64.8

3.6 10.6 69.4

3.8 10.9 71.4

2.3 0.0 0.0

2.3 0.0 0.0

2.5 0.0 0.0

2.8 0.0 0.0

2.9 0.0 0.0



4.4

95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0

4 3.6 Growth (g/L)

3.2 2.8 2.4 2 1.6 1.2 0.8 0.4 0 0

Figure (5):

12

24 Time hus

36


Total sugar concentration gL-1

Incubation Time (hrs.) Growth (gL-1) 5.0 Alcohol % Fermentation efficiency % Growth (gL-1) 10.0 Alcohol % Fermentation efficiency % Growth (gL-1) 15.0 Alcohol % Fermentation efficiency % Growth (gL-1) 20.0 Alcohol % Fermentation efficiency % Growth (gL-1) 25.0 Alcohol % Fermentation efficiency % Growth (gL-1) 30.0 Alcohol % Fermentation efficiency %

48

Saccharomyces cerevisiae GHM yeast growth and fermentation efficiency at different sugar concentration, pH 5, 35˚C and incubation time 48 (h)


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Conclusion The optimum production conditions for ethanol from beet molasses by the tested yeast strain were: 20% sugar concentration, pH at 5.0 for normal & high gravity and incubation temperature at 32˚C for normal gravity and 35˚C for high gravity.

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