Field of Invention
This invention relates to an improved process for production of alcohol using immobilized yeast beads and partitioned bioreactor. The present invention more particularly relates to utilization of partitioned batch reactor to improve the performance of the alcohol production with immobilized yeast. In addition, the invention also improved alcohol productivity due to reduction of fermentation time. Moreover, the present invention helps in elimination of separation step of catalyst from fermentation broth.
Background of the invention
Ethanol is one of the largest volume chemicals used after water and produced by biotechnological process since several decades using molasses as substrate. It has been widely used as a solvent, germicide, anti-freeze and as chemical raw material for the production of acetaldehyde, acetic acid, butadiene and ethylene. Recently its role as a valuable alternative source as clean fuel has renewed the interest worldwide. This situation created further interest in development of improved fermentation process to achieve higher ethanol yields.
Conventionally, ethanol production is performed by batch fermentation process using Sachhromyces cereviceae as a catalyst and molasses as substrate. This process of ethanol fermentation with yeast requires maintenance of yeast cultures and pre-fermentation for about 9-12 hours to develop sufficient quantity of catalyst for main fermentation. Hitherto several attempts have been made to improve the ethanol production either with free or immobilized yeast cells. The conventional free cell fermentation is generally performed using Sachharomyces cereviceae in diluted molasses solutions in a batch reactor for a period of 24-48 hours and requires pre-fermentation process for development of yeast for every batch [S.V. Ramakrishna, V.P. Sreedharan and P. Prema. In: Bioreactor Immobilized Enzymes and Cells: Fundamentals and Applications (Ed Mood Young), Elsevier Appl. Sci., Amsterdam, 1988, 251-260]. This process of alcohol production suffers from low productivity, longer fermentation time, high operating and capital investment [D. Das, R.G. Nandkishor, K. Murali and P.S. Gupta J.Ferment.Bioengg. 1993, 75, 132-137; D. Weuster-Botz Appl. Microbiol. Biotechnol, 1993, 39, 679-684]. In addition, one of the major problems associated with alcohol production is longer fermentation time due to low biomass concentration. Therefore, the maintenance of high cell densities in the fermentor is imperative, which was achieved by continuous mode of operation by cell recycle [C.W. Lee and H.N. Chang Biotehcnol Bioengg, 1987, 29, 1105-1112] but this system requires capital investment for procuring centrifuge and recurring expense for continuous operation. To over come this, use

of whole cell immobilization by various techniques [S.V. Ramakrishna and R.S. Prakasham Current Science, 1999, 77, 87-100] has been attempted to enhance volumetric productivity of the system. Hitherto, several reactor configurations have been attempted to produce ethanol using entrapped cells [F. Godia, C. Casad, and C. Sola; Process Biochemistry, 1987, 43-48]. One of the major limitations associated with use of entrapped cells in various reactors is disintegration of immobilized beads, which cannot be used for long [S.V. Ramakrishna, V.P. Sreedharan and P. Prema. In: Bioreactor Immobilized Enzymes and Cells: Fundamentals and Applications (Ed MoodYoung), Elsevier Appl. Sci., Amsterdam, 1988, 251-260]. Research in this direction to enhance the stability of the entrapped cells resulted in development of stable immobilized yeast beads, which proved that they can be recycled several times without disintegration [S. V. Ramakrishna, R. S. Prakasham and P. Komariah, Indian Patent Application No. 186/DEL/2000; US Patent No. 6420146]. The major limitation in alcohol production with above immobilized yeast beads is that they tend to float on the surface of the fermentation broth during fermentation which results in decrease the rate of ethanol production. To overcome the above limitation, conventional reactor system was modified by incorporating the medium speed stirrer, which substantially improved the ethanol productivity [R S Prakasham, P N Sarma, S Venkata Mohan, K Krishna Prasad and K V Raghavan, India filing No. NF 358/02 Dt. 30.09.2002, US Patent filed]. Higher capital and operation cost were some of the major limitations associated with this modification. In addition, the above modifications in the fermentor reduce the life of immobilized yeast due to mechanical damage and the foam generated during fermentation carried away some immobilized yeast beads. This led to the reduction in effective utilization of the catalyst. The novelty in the present invention is the partitioning of batch reactor using perforated plates and recycling of fermentation broth through heat exchange system will provide mixing rate without using a mechanical stirrer and regulate the temperature of the fermentation broth. This enabled to improve the performance of the alcohol production with immobilized yeast beads. In addition, the present modification also enhanced alcohol productivity due to reduction in fermentation time. Utilization of perforated batch reactor for ethanol production using immobilized yeast beads also eliminates the solid liquid separation step, which is an essential step to retrieve the immobilized yeast beads for reuse in earlier patent (NF358/02). Further the present invention also reduces the operating cost and separation cost required for fermented broth and catalyst compared to earlier patent (NF358/02).
Objectives of the present invention
The main objective of the present invention is to provide an improved process for production of alcohol using immobilized yeast beads and partitioned bioreactor.
The other objective of the present invention is to produce the alcohol in a partitioned batch reactor. Yet another objective of the present invention is to enhance the rate of ethanol production.
Another objective of the present invention is the effective utilization of the catalyst for alcohol production.
Still other objective of the present invention is to provide cost effective improved alcohol production process.
Still another objective of the present invention is to eliminate the solid-liquid separation step required for retrieving the catalyst from the reaction mixture.
Yet another objective of the present invention is to reduce the fermentation time.
Further objective of the present invention is to reduce the operating cost of the fermentation process.
Summery of the invention
The novelty in the present invention is the reduction of fermentation time from 28-36 hours to 6-12 hours and considerable improvement in ethanol productivity with the use of a partitioned batch reactor compared to conventional process with activated stable yeast crystals. The present invention also enables to increase the output with the existing fermenter. In addition, the present invention also helps in effective utilization of catalyst to that of earlier inventions in this regard. Furthermore, the present invention edges in reduction of fermentation time and elimination of separation step of fermented broth and catalyst over the conventional system.
Detailed description of the invention
Accordingly, the present invention provides an improved process for production of alcohol using immobilized yeast beads and partitioned bioreactor comprising the steps of activating the immobilized yeast beads of Sachharomyces cerevisiae by innoculating in about 1.06 specific gravity
molasses solution for a period of about 8-12 hrs at a temperature ranging 28-38° C , inoculating about 0.25 to 2.0%w/v the activated immobilized yeast beads in sterilised dilute molasses medium aseptically having a specific gravity ranging between 1.060 to 1.120 of about 1.090 contained in partitioned bioreactor and the said bioreactor partitioned by incorporation of perforated sheets having 2mm diameter holes arranged 3-3.5 mm triangular pitch at the top and bottom of the conventional fermentor. incubating the inoculated molasses medium in partitioned bioreactor at a temperature 28-38°C for a period ranging 6-12 hrs to attain the specific gravity of fermentation broth ranging 1.01 to 1.05 and separating the immobilised yeast beads by draining out the fermented broth and recovering ethanol from the fermented broth by distillation
In an embodiment of the present invention partitioned batch reactor comprises of a conventional reactor partitioned by incorporating perforated sheet having 2-millimeter diameter holes arranged 3.0 - 3.5 millimeter triangular pitch or a plate having slotted holes with 2 millimeter width and 6-8 millimeter length arranged in 5-9 millimeter triangular pitch at the top and bottom of the fermenter and an inlet and outlet being provided at the side of the fermentor and at the bottom of the reactor respectively to circulate the fermentation broth through heat exchanger and a distributor being fitted to the inlet of the reactor to have uniform distribution of the fermentation broth and the upper perforated plate in the fermentor being positioned below the inlet of the reactor and the distance between the outlet and upper perforated plate being about one fourth of the diameter of the fermentor and the lower perforated plate being positioned above the distributor and the the distance between the lower perforated plate and reactor inlet being kept at about one-fourth of the diameter of the fermenter (Figure 1).
In another embodiment of the present invention the molasses fermentation broth with specific gravity in the range of 1.060 - 1.120 is prepared by mixing known quantity of molasses and water in the conventional and partitioned batch reactor.
In an embodiment of the present invention the fermentation is carried out for a period ranging from 6 to 40 hours at 28 - 38 °C or till the specific gravity of the fermentation broth reached to 1.010- 1.050 in conventional and partitioned batch reactors.
In yet another embodiment of the present invention the temperature of the fermented broth in partitioned batch reactor is regulated by circulating the fermentation broth through a plate heat exchanger.
In yet another embodiment of the present invention the fermentation time is reduced from 24-40 hours to 6-12 hours without compromising on the ethanol production.
In yet another embodiment of the present invention ethanol production cost is reduced significantly as compared to conventional methodologies.
In yet another embodiment of the present the rate of ethanol production upto about 24.46 to 27.44 liters/h/tone as compared to that of conventional 6.65 to 8.07 liter/h/ton of molasses.
In yet another embodiment of the present invention the immobilized yeast beads used is recyclable upto about 60 batches for ethanol production.
The present invention of an improved ethanol production process using activated immobilized yeast beads in partitioned batch reactor is described with reference to the following examples which are explained by way of illustration only and should not therefore be construed to limit the scope of the present invention.
Example I
Ethanol production using immobilized yeast beads in conventional batch reactor
Sugar cane molasses was procured from local sugar industry and stored at 4°C till further use. The immobilized yeast beads were prepared as described in the earlier patent [Indian patent application No. 186/DEL/2000 and US Patent No. 6420146] and activated by incubating in the 1.060 specific gravity molasses solution for 8 hours at 28°C. Fermentation broth (10 liters) was prepared by diluting the molasses with water to get the specific gravity of
the solution of 1.090 and the initial sugar concentration in the fermentation broth was measured using conventional procedures. One percent of activated immobilized yeast beads were separated from activation solution and added to conventional fermentation vessel. The fermentation was continued at 30°C till the specific gravity of the fermentation broth reached to 1.024. During the fermentation, the temperature of the fermentation broth was controlled by circulating cooling water through cooling coil. The fermented broth and catalyst were separated using filter cloth and fermented broth was used for ethanol recovery and the results were indicated in the following table.
(Table Removed)
Example 2
Ethanol production using activated immobilized yeast beads in a partitioned batch reactor The same experiment was performed as example 1 in a partitioned batch reactor. The activated immobilized yeast beads of earlier experiments were reused in this experiment. Fermentation broth (10 liters) was prepared in partitioned fermentation vessel by diluting the molasses with water to get the specific gravity of the solution of 1.090 and the initial sugar concentration in the fermentation broth was measured using conventional procedures. One percent activated immobilized yeast beads were used in this experiment. The fermentation was continued at 30°C till the specific gravity of the broth reached to 1.026. A heat exchanger was used to control the temperature of the fermentation broth and have required mixing rate. At the end of the fermentation the fermented broth was drained out simply by opening drain value and used for ethanol recovery. The results were indicated in the following table.

(Table Removed)
Example 3
Comparative evaluation of alcohol fermentation process performed in conventional and partitioned batch reactor using activated immobilized yeast beads The ethanol fermentation performed in example 1 and 2 using activated immobilized yeast beads in conventional and partitioned batch reactor were compared to evaluate the benefits of the present invention.

(Table Removed)
Example 4
Ethanol production using activated immobilized yeast beads in conventional batch reactor The activated yeast beads of the above experiments were reused in this experiment also. Fifteen liters of molasses based fermentation broth having specific gravity of 1.098 was prepared by diluting the molasses with water. The initial sugar concentration in the fermentation broth was measured using conventional procedures. Fermentation was initiated by pouring the fermentation broth into the reactor. The temperature of the fermentation broth was maintained by circulating cold water through a cooling coil. The fermentation was continued till the specific gravity of the fermentation broth reached to 1.038. The fermented broth and catalyst were separated using filter cloth and fermented broth was used for ethanol recovery and the results were indicated in the following table.

(Table Removed)
Example 5
Ethanol production using activated yeast beads in a partitioned batch reactor
The above experiment was conducted in a partitioned batch reactor by reusing yeast
beads of above experiment. Fifty liters of high gravity fermentation broth (1.098 specific
gravity) having initial sugar concentration of 16.81 (%) was prepared and poured in the above
partitioned reactor. The fermentation was continued at 30°C till the specific gravity of the

fermentation broth reached to 1.036. A heat exchanger was used to control the temperature of the fermentation broth and have required mixing rate. The activated immobilized yeast beads were then separated by draining the fermented broth and used for ethanol recovery. The observations are listed in the following table.

(Table Removed)
Example 6
Comparative evaluation of alcohol fermentation process performed in conventional and partitioned batch reactor using activated immobilized yeast beads The ethanol fermentation performed in example 4 and 5 using activated immobilized yeast beads in conventional and partitioned batch reactor were compared to evaluate the benefits of the present invention.

(Table Removed)
Example 7
Recycling of the immobilized yeast for ethanol production in partitioned batch reactor
The prepared immobilized yeast was used in the reactor employing the procedure mentioned in the above examples. Two liters of fermentation broth having specific gravity of 1.098) was prepared and poured in the above partitioned reactor. The fermentation was continued at 30°C till the specific gravity of the fermentation broth reached to 1.036. A heat exchanger was used to control the temperature of the fermentation broth and have required mixing rate. The activated immobilized yeast beads were then separated by draining the fermented broth and used for ethanol recovery. This process was continued to elucidate the recycling capability of the immobilized yeast beads for ethanol production in the reactor for

more than 60 batches. The ethanol production was measured after each batch and found to be varying between 3-5%.
Tentative economic benefits
Basis: - 30 KL Distillery
Alcohol yield = 6% (Total fermentation broth to be fermented is = 5.0 lakh liters)
Yeast cake required per batch for 5.0 lakh liters fermentation broth is 50 Kg @ Rs 40/-
= 50 X 40 = Rs 2,000/- per batch Immobilized yeast required 1% i.e. for 5.0 lakh liters is 5 tonnes Cost of immobilized yeast @ Rs 125/-kg = 5,000 x 125 = 6,25,000/-
Sixty batches of operation
Cost of immobilized yeast = Rs 6,25,000
- Cost of the yeast cake required = 2000 x 60 = Rs 1,20,000
Extra cost in put through immobilized yeast = Rs 5,05,000
Benefit due to use of immobilized yeast
a) By elimination of prefermentation (when immobilized yeast is used)
Basis:
Molasses used per batch for 30 KL distillery is 150 tones
10% of this is used for prefermentation, i.e., 15 tones per batch
Tone of molasses cost ranges from Rs. 1,800 to 2,800/- (average Rs.
2,300/-)
Cost of molasses per batch of prefermentation is = 15 x 2,300 = Rs
34,500/-
For 60 batches of operation =34,500x60 = Rs. 20,70,000/-
Extra expenditure due to immobilized yeast 5,05,000/-
Benefit with immobilized yeast = Rs. 15,65,000/-
b) In conventional fermentation, per batch operation requires 24 to 30 hours while with immobilized yeast in partitioned batch reactor requires 9-15 hours.
The advantages of the present invention:
1. The main advantage of the present invention is reduction of batch fermentation time from 28-44 hours to 6-14 hours depending on the initial specific gravity of the fermentation broth.
2. The other advantage of the present invention is improvement of alcohol yield per tone of molasses used during batch fermentation.
3. Another advantage of the present invention is improvement in rate of alcohol production.
4. Yet other advantage of the present invention is increasing of the plant capacity substantially with minor modification of present basic fermentor structure.
5. Yet another advantage of the present invention is the elimination of solid-liquid separation step of fermented broth and catalyst.

We Claim:
1 An improved process for production of alcohol using immobilized yeast beads
and partitioned bioreactor comprising:
a) activating the immobilized yeast beads of Sachharomyces cerevisiae by innoculating in about 1.06 specific gravity molasses solution for a period of about 8-12 hrs at a temperature ranging 28-38° C
b) inoculating about 0.25 to 2.0%w/v the activated immobilized yeast beads as obtained in step a) in sterilised dilute molasses medium aseptically having a specific gravity ranging 1.060 to 1.120 contained in partitioned bioreactor and the said bioreactor partitioned by incorporation of perforated sheets having 2mm diameter holes arranged 3-3.5 mm triangular pitch at the top and bottom of the conventional fermentor.
c) incubating the inoculated molasses medium in partitioned bioreactor at a temperature 28-38°C for a period ranging 6-12 hrs to attain the specific gravity of fermentation broth ranging 1.01 to 1.05
d) separating the immobilised yeast beads by draining out the fermented broth and recovering ethanol from the fermented broth by distillation
2 An improved process for production of alcohol as claimed in claims 1 wherein the
partitioned batch reactor comprises of a conventional reactor partitioned by incorporating
perforated sheet having 2-millimeter diameter holes arranged 3.0 - 3.5 millimeter triangular pitch
or a plate having slotted holes with 2 millimeter width and 6-8 millimeter length arranged in 5-9
millimeter triangular pitch at the top and bottom of the fermenter and an inlet and outlet being
provided at the side of the fermentor and at the bottom of the reactor respectively to circulate the
fermentation broth through heat exchanger and a distributor being fitted to the inlet of the reactor
to have uniform distribution of the fermentation broth and the upper perforated plate in the
fermentor being positioned below the inlet of the reactor and the distance between the outlet and
upper perforated plate being about one fourth of the diameter of the fermentor and the lower
perforated plate being positioned above the distributor and the the.distance between the lower
perforated plate and reactor inlet being kept at about one-fourth of the diameter of the fermenter (Figure 1).
3 An improved process for production of alcohol as claimed in claims 1-2 wherein the molasses fermentation broth with specific gravity in the range of 1.060 - 1.120 is prepared by mixing known quantity of molasses and water in the conventional and partitioned batch reactor.
4 An unproved process for production of alcohol as claimed in claims 1-3 wherein the fermentation is carried out for a period ranging from 6 to 40 hours at 28 - 38 °C or till the specific gravity of the fermentation broth reached to 1.010 - 1.050 in conventional and partitioned batch reactors.
5 An improved process for production of alcohol as claimed in claims 1-4 wherein the temperature of the fermented broth in partitioned batch reactor is regulated by circulating the fermentation broth through a plate heat exchanger.
6 An improved process for production of alcohol as claimed in claims 1-5 wherein the fermentation time is reduced from 24-40 hours to 6-12 hours without compromising on the ethanol production.
7 An improved process for production of alcohol as claimed in claims 1-6 wherein ethanol production cost is reduced significantly as compared to conventional methodologies.
8 An improved process for production of alcohol as claimed in claims 1-7 improves the rate of ethanol production upto about 24.46 to 27.44 liters/h/tone as compared to that of conventional 6.65 to 8.07 liter/h/ton of molasses.
9 An improved process for production of alcohol as claimed in claims 1-8 wherein the immobilized yeast beads used is recyclable upto about 60 batches for ethanol production.
10 An improved process for production of alcohol as substantially herein described with reference to the examples accompanying the specification.