الفهرس 
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Abstract
The studies of ethanol fermentation reveal that nearly 90% of the energy cost during ethanol production comes from ethanol purification, since the fermented broth contains relatively low ethanol concentrations. Increasing the ethanol concentration in the broth via fermentation of media with high sugar concentration can significantly improve energy efficiency by reducing energy consumption in distillation. However, the high osmotic stress of the media related to the concentration of sugars and the inhibition effect of the produced ethanol are negative for ethanolic fermentation.
Therefore, the main goal of this work was to find a way to ferment artificial then natural media containing high sugar concentration in order to produce high amounts of ethanol. This goal was achieved through optimizing several factors that play the essential roles in the fermentation processes. Briefly these factors were: selection of a suitable yeast strain, optimizing the yeast nutrition (sugar concentration, nitrogen, growth factors and oxygen) and using an advanced fermentation technology. The choice of Saccharomyces cerevisiae used in this study was achieved according to the performances it displayed in fermentative activity, ethanol and temperature tolerance and flocculating ability compared to the other collected yeast strains. The strain showed marked fermentation activity, ethanol and temperature tolerance and good flocculation ability compared to the other strains, was selected for this work. The selected strain was originally a commercial pressed baker’s yeast strains (Saccharomyces cerevisiae) obtained from Sohaj Governorate in upper Egypt.
In a batch fermentation the used strain was able to ferment sucrose up to a concentration of 20% in media supplemented with (g/l): yeast extract 3, ammonium sulfate 0.5, magnesium sulfate 1.5, K(PHO4)2 1.5 and peptone 5 with a yeast inoculum level of 3%. A stuck fermentation had occurred at sucrose concentration of 25% under the above mentioned conditions and high amounts (12-13%) of residual sugars were left unutilized suggesting that the yeast cells suffered from product inhibition and/or osmotic stress.
A positive effect of increasing the yeast inoculum volume into 6% instead of 3% was observed on fermentation of media with 25% sucrose concentration. The data however showed that the fermentation was not complete and a considerable amounts of sugar were not utilized.
The ratio of consumed sucrose to added nitrogen (mg/g), which gave the best results (for the used yeast strain) was 5 mg nitrogen as ammonium sulfate/g of consumed sugar. It was concluded that this ratio should be nearly constant at any sugar concentration. Increasing the added ammonium sulfate level to 1250 mg/l of medium made this ratio right for fermentation of 25% sucrose (250 g/l). Addition of nitrogen at this ratio greatly enhanced the fermentation of 25% sucrose and produced 11.22 % ethanol with a residual sugar of only 3% (30 g/l). addition of nitrogen at level more than 5 mg ammonium sulfate/ g sugar had negative effects on the fermentation parameters.
Application of the above optimized factors on fermentation of 30% sucrose concentration medium did not led to a complete fermentation and the produced ethanol did not exceed 70% of it’s theoretical value.
Addition of yeast extract at level of 6 g/l together with thiamine at a level of 0.2 g/l markedly improved the fermentation pathway although the utilization of sugar was not complete and 42 g /l sucrose left without utilization. Under these conditions the ethanol yield from 30% sucrose concentration was 11.2. Addition of thiamine at concentration more than 0.2 g/l gave a negative effect concerning the ethanol production although the effect on yeast growth and sugar consumption was positive.
After several experiments the 2% alginate beads were selected for the immobilization experiments. Using these beads the fermentation was performed with sugar concentration of 30% using batch immobilized fermentation system. The results showed that concentration of sucrose was sharply decreased while the cell density and ethanol production were increased for a duration of 36 h. It seems that the lag phase was very short because the consumption of sucrose was high during the first 12 h of fermentation as compared with the free system data. The number of viable yeast increased very quickly and reached it’s maximum (150 x 106 cfu/g) after only 36 h of fermentation. At this short fermentation time the consumption of sugar was complete with ethanol production of 14.7, which equal to more than 95% of it’s theoretical value. By contrast, free yeast cells were not able to utilize the present sugar completely and their viability level was low with ethanol production of about 74 % of it’s theoretical value after a long fermentation time (60 h). The beads have been reutilized up to five times without observing any decrease in the yield or the rate of fermentation.
At this point it was decided to try fermenting the molasses medium with 30% fermentable sugars and to use some other factors such as the level of dissolved oxygen and the continuous fermentation to increase the fermentable sugar concentration as more as possible.
Alginate beads of S. cerevisiae were able to completely transform molasses with 30% fermentable sugar into ethanol within 48 h of fermentation. On the other hand the results showed that the fermentation was not complete and high residual sugar left with high rate of cell death if the free cell suspension was used as inoculum.
Both of the free and immobilized yeast cells were unsuccessful in the molasses medium contained 35% sugar concentration. This was a surprise result for the cell beads inoculum because the high biomass concentration of the immobilized system was expected to resist the high osmotic pressure of 35% sugar concentration.
Supplementation of the molasses medium containing 35% sugar concentration with sterilized air at rate of 175 ml/ min /liter of the production medium only during the first 12 h of fermentation showed a complete consumption of molasses sugar with alcohol yield near to the theoretical value although the fermentation time was slightly longer (60 h) than expected.
To ascertain whether the continuous cultivation could be suitable for fementation of molasses media using the immobilized yeast cells, set of experimnts were carried out at different flow rates.
When continuous ethanol fermentation was operated at flow rate of 2 ml/min, the sugar conversion was 100% with ethanol yield of 16.1%, equal to about 90% of it’s therotical value.
Continuous fermentation experiment with the immobilized Saccharomyces cerevisiae cells at the obtained optimum flow rate and all the previous optimized conditions was carried out. The steady state data for ethanol, sugar concentrations in the exit stream as well as the cell concentration in the beads were about 16% ethanol, 100% sugar consumption and approximately 150 cfu/g of beads.
The immobilized cell reactor was operated over a period of 35 days at a flow rate of 2m/min., without any loss in the immobilized cell activity. After 35 days the reactor was shut down and it was observed that there was no change in the physical appearance of the alginate beads.