Claims:We claim:
1. A process for the preparation of ethanol comprising:
(a) providing an amount of yeast that is flocculating type;
(b) mixing said yeast with a desired amount of process water in a fermentor forming a first stream;
(c) treating said first stream with an acid at a desired pH and agitating it for a desired time forming a second stream;
(d) adding to said second stream a desired amount of sugary feedstock over a desired time and at a desired temperature forming a reaction mixture;
(e) allowing said reaction mixture to ferment further over a desired time and a desired temperature forming a third stream;
(f) keeping said third stream immobile for a desired time allowing said yeast to flocculate and settle at the bottom of said fermentor;
(g) removing between 80% to 90% of said third stream from said fermentor having only small amount of said yeast forming a forth stream ;
(h) subjecting said fourth stream to distillation to recover ethanol; and
(i) reusing remaining amount of said third stream having said yeast in step (a) for next round of said process in said fermentor.
2. The process of claim 1, wherein amount of said process water is between about 4 to 7 times of said amount of yeast.
3. The process of claim 1, wherein said acid is sulphuric acid or phosphoric acid.
4. The process of claim 1, wherein said acid treatment is performed for up to 1 hour under agitation.
5. The process of claim 1, wherein said pH is adjusted between about 2.5 to about 3.5 units.
6. The process of claim 1, wherein said sugary feedstock one of molasses, sugar syrup or sugar juice obtained from sugarcane or sugar beet.
7. The process of claim 1, wherein said desired time for continuously adding said sugary feedstock from about 6 to about 20 hours.
8. The process of claim 1, wherein said desired temperature is between about 30 ºC to about 35 ºC.
9. The process of claim 1, wherein said yeast is kept immobile for up to 1 hour.
10. The process of claim 1, wherein said reaction mixture is allowed to ferment further for about 10 hours.
11. The process of claim 1, wherein said yeast is a flocculating yeast.
12. The process of claim 1, wherein said yeast is a Saccharomyces sp.
, Description:FIELD OF THE INVENTION
The invention relates to a process for the preparation of ethanol by fermentation of a sugary feedstock by using a method of reusing the yeast used for said fermentation without removing it from the fermentor on completion of said fermentation. Further, an acidification in dilute suspension is performed to regenerate said yeast. Said process having advantages of requiring less capital expenditure and significantly less process time compared with the conventional method of yeast separation and recycling in the fermentation of ethanol.

BACKGROUND
The conversion of sugar to ethanol is rapidly expanding industry. Ethanol has widespread applications as an industrial chemical, a gasoline additive or a liquid fuel by itself, besides in potable ethanol and liquor industry.
Ethanol in India and other developing countries is mainly produced by fermentation of dilute molasses at temperature of about 25 ºC to about 35 ºC employing the yeast Saccharomyces cerevisiae. The sugarcane molasses is a complex mixture that varies in composition according to geographical sources, agricultural practices and sugar mill operations. The fermentable carbohydrates in molasses are sucrose, glucose and fructose. The non-sugars may consist of nitrogenous substances, gum polysaccharides, waxes, sterols, pigments and the salts of calcium, potassium or magnesium.
Ethanol production has been studied with different yeast strains to find out an effective method to increase the efficiency of ethanol production and reduce the capital expenditure on the equipment. The yeast flocculation is one of the well suited techniques for high density ethanol production. The use of flocculent cells, as a self immobilizing culture at the end of process is a very promising alternative since it reduces the expenses associated with the yeast separation units like the centrifuges and associated energy consumption. Further improves the ethanol productivity and reduces the fermentation time. So it has many advantages over conventional ethanol fermentation method using freely suspended yeast cells.
Several configurations of bioreactors, which use flocculating yeasts, have been suggested for continuous or batch mode of ethanol production. In conventional flocculating yeast technology continuous method requires more than one vessel like the yeast settling tank, treatment tank and activation tank, which operates in a tandem manner. Here, the fermented mash is continuously passed to the yeast settling tank and the overflow of it is taken to the distillation, while the bottom stream with settled yeast is taken to settling tank. Next, the yeast cream recovered from the settling tank is taken to treatment tank for acidification and further passed to activation tank for the yeast activation and then reused in fermentation. This process increasing the unit operations and is time consuming. Moreover; this technology involves high capital expenditure and space foot print due to several unit operations. Conversely the process of invention disclosed herein uses of a single vessel for the fermentation, the yeast settlement, the yeast treatment and for the activation of yeast cells which results in lower unit operation and low foot prints providing significant economic advantages.

DETAILED DESCRIPTION OF THE INVENTION
In one embodiment of present invention, for a fermentative production of ethanol, a sugary feedstock [like molasses, sugar syrup or sugar juice obtained from sugarcane or sugar beet] is taken as a starting material for inoculum preparation of fermenting yeast. The feedstock is diluted with water and nitrogen source and other nutrients are added, if required, forming an inoculum preparation medium. The pH of said medium is adjusted between about 4 to about 5. Then said medium is inoculated with activated yeast culture [Saccharomyces sp.] and incubated at about 32 ºC for about 12 to about 20 hours. Next, it is added to a fermentor tank which is supplemented with nutrient like urea, diammonium phosphate and magnesium phosphate for robust growth of yeast. Further, desired quantity of water is added to form a medium which is agitated as per the requirement. Next, the desired quantity of feedstock having specific gravity about 1.5 is continuously added to said medium for about 6 hours to 20 hours [depending upon the quality of molasses] and simultaneously fermentation is carried out at about 30 ºC to about 35 ºC for about 14 hours to 18 hours. After this agitation in fermentor is stopped; and the fermented medium is kept immobile for about an hour. On the resting of agitation, said yeast flocculates and settles at the bottom of fermentor as the yeast cream and the fermented wash remaining as the supernatant. Then said fermented wash [the de-yeasted supernatant] is separated without disturbing said yeast cream and transferred to a collection vessel. Next, said fermented wash is subjected to distillation to recover ethanol. In the next round, said yeast cream in said fermentor is used as inoculum after the acid treatment. The desired quantity of process water [about 4 to 7 times] is again added to said yeast cream. Then it is acidified with concentrated sulfuric acid to get a pH between about 2.5 to about 3.5 and kept under agitation for about 1 hour. Then the nutrients are added to it as required. Next, the desired quantity of the feedstock having specific gravity about 1.5 is continuously added to said medium for about 6 to 20 hours and simultaneously the fermentation is allowed to proceed. In this process said yeast cream is re-used for several cycles in new batches of fermentation without any loss of efficacy in fermentation or appearance of contaminants over threshold during the serial fermentation. After each cycle the ethanol is recovered after distillation. After each cycle the ethanol concentration in fermented wash is about 8 to about 10 % by volume
In another embodiment of the invention, waste streams like process condensate or spent wash is used instead of process water during fermentation process.
In yet another embodiment of present invention, the fermentation process is designed to achieve about 8% to 12% ethanol by volume after each round of the batch fermentation. Herein the molasses or sugarcane syrup is used as sugary feed for fermentation having about 30% to about 80% sugars by weight.
In yet another embodiment of the invention, yeast culture is built up sufficiently in first fermentation batch which is used further for about 30 to 40 batches before the yeast biomass is replaced with fresh culture. Herein the pH of media after addition of molasses is self-maintained.
In yet another embodiment of the invention, said yeast remains in dispersed during fermentation stage and develops flocculation towards the end of fermentation and separates rapidly from fermentation broth and settles at the bottom of the fermentor. Further, the acidification step revives the yeast and inhibits the bacteria. The acidification of said yeast using concentrated sulfuric acid is carried out for about ½ to 1 hour under continuous agitation. Here more than two-log reduction in the viable bacteria takes place, while significant amount of the yeast cells remaining viable [see Example 2].
Embodiments provided above give wider utility of the invention without any limitations as to the variations that may be appreciated by a person skilled in the art. A non-limiting summary of various embodiments is given above, which demonstrate the advantages and novel aspects of the process disclosed herein.
ADVANTAGES:
1. Since the overall process of the ethanol production is carried out in a single vessel, operational parameter like cleaning, priming and operation of the fermentors are also substantially reduced.
2. Higher ethanol titer is achieved after every batch of fermentation with low effluent generation.
3. Low load on the ETP due to effective use of waste streams in fermentation process.
4. Easy unit operations and low foot print due to use of a single vessel for fermentation, yeast treatment and for the activation of yeast cells.
5. The yeast biomass produced in the process is very high which results in less bacterial contamination and higher fermentation efficiency.
6. Most of feedstock sugar is used for production of ethanol as the yeast biomass requires very small amount of sugar for growth.
7. Due to increase in the throughput the operational and capital expenses are substantially reduced relative to conventional process.
8. The process afforded about 1% more fermentation efficiency on sugary feed stocks compared with the conventional method.

EXAMPLE 1
About 485 mL diluted molasses having about 5% of sugar by weight was added in an inoculum preparation flask. Further about 500 PPM of urea as a nitrogen source was added to it. The pH of this medium was adjusted to about 4.5 and then inoculated with about 15 mL of active yeast culture and reaction was carried out at a temperature of about 32 ºC for about 12 hours to get the inoculum to initiate the process. Next, said inoculum was transferred into a 10-L fermentor and nutrients like urea (500 mg), diammonium phosphate (500 mg) and magnesium sulphate (250 mg) were supplemented for robust growth of said yeast. Further about 3440 mL of process water was added to it to form a liquid medium, which was agitated at about 100 RPM. Next, about 1557 g of molasses having specific gravity about 1.46 was continuously added to said medium for about 8 hours and simultaneously fermentation was carried out at about 34 0C for about 18 hours. Next the agitation in fermentor was stopped; and the fermented medium was kept immobile for about half an hour. On stoppage of agitation said yeast flocculated and settled at the bottom of fermentor as yeast cream (about 500 mL). Then the fermented wash (supernatant without yeast) of about 4500 mL was separated without disturbing said yeast cream and transferred to a collection vessel. Next, said fermented wash was subjected to distillation to recover ethanol. In the next round, said yeast cream in said fermentor was used as the inoculum after acid treatment for the next cycle of the process. First about 3440 mL of process water was added to said yeast cream. Then it was acidified with concentrated sulphuric acid to get a pH between about 2.8 to about 3.1 and kept under agitation for about 1 hour. The nutrients were added to it as above and fermentation was allowed to proceed for about 10 hours. In this process said yeast cream was re-used for up to 40 times without any loss of efficacy in fermentation or appearance of contaminants during the serial fermentation. After each cycle the ethanol concentration in fermented wash was about 9.2% by volume. This process afforded about 92% fermentation efficiency on molasses.

EXAMPLE 2
The effect of acid treatment on the yeast cream before and after the treatment was tested by measuring the viable yeast and bacterial cell numbers as shown below:
Acidification Yeast Bacteria
Before 2.8 x 108 6.2 x 105
After 1.1 x 108 1.6 x 103
Reduction in cell count
[% of log values] 4.8 44.73
The viability of the bacterial cells is about significantly reduced to more than two-log reduction, while more than 90% of the yeast cells remain viable after the acid treatment.