A Process For The Preparation For Artificially Flocculated Yeast Particles Useful For Production Of Alcohol

Abstract

Flocculation is a useful property of industrial yeast (Saccharomyces) strains in fermentation, brewing, wine making and ethanol and biomass production. Preparation of biocatalyst particles containing yeast, for continuous production ofethanol, has a lot of economic feasibility due to increased microbial hold up, easeof separation of biomass from the fermentation media, possibilities for use in novel fermentation configuration. Recent advances in the design of continuous fermentation systems to shorten the fermentation time call for the use of flocculent strains for maintenance of cell population density. The degree of flocculation is a critical parameter since it severely affects the fermentation rate, time and production cost. It is essential that if high alcohol-yielding strains have to be used for fermentation, artificial flocculation methods have to be adopted. Flocculation by cationic polyelectrolyte (0.1-0.5 mg/mi) in the presence of Calcium ions (0.004-1.0 M/ml) was found to flocculate most of the yeast strains. Operation of a Tower fermentor for the production of ethanol with the flocculated /clustered yeast gave a productivity of 2- 4.5 gm/L/h which was higher than the batch production with the same flocculated yeast. This yeast can be used for various biotransformations as well as desugaring any solutions of importance. The use of artificially flocculated yeast will make the process cost effective. Compared to the gel entraped yeast particles , artificially flocculated cells have high cell density and have efficient conversions.

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The present invention relates to " A Process for the preparation of artificially flocculated yeast particles".
Flocculation is a useful property of industrial yeast (Saccharomyces) strains in fermentation, brewing, wine making and ethanol and biomass production. Flocculating yeast rapidly aggregate and settle in the later stage of fermentation.
And this phenomenon facilitates separation of yeast cells from the fermenting
liquor. Preparation of biocatalyst particles containing yeast, for continuous
production of ethanol, has a lot of economic feasibility due to increased microbial hold up, ease of separation of biomass from the fermentation media, possibilities for use in novel fermentation configuration. Recent advances in the design of continuous fermentation systems to shorten the fermentation time call for the use of flocculent strains for maintenance of cell population density. Flocculation is the process whereby destabilized particles are induced to come together, make contact and subsequently from larger agglomerates. Flocculation is confined to the reversible aggregation of cells. Significant flocculation of particles in suspension is brought about by inter particle collisions followed by their attachment. Being made up of thousands of cells, floes settle faster than cells with aggregation numbers below 10. Sedimentation can be measured as the amount of sediment at the end of a given time period or as the rate of sedimentation, expressed in volume, weight or cell number or as a fraction of biomass, the amount of sediment tells us the extent of flocculation.
For appreciable floe formation some form of induced turbulance or fluid velocity gradient is required to promote particle collisions and subsequent attachment. The
increased size of the floe enhanced the gravity settling. The rate of mixing is critical. Agitation (30 - 40 rpm) was required to overcome the like charge repulsion between yeast cells, thus enabling them to collect and form bonds, which is also strain dependent.
The degree of flocculation is a critical parameter since it severely affects the
fermentation rate, time and production cost. The flocculating phenomenon is
complex, but its genetic and environmental factors have been well investigated. The wine making industries use naturally flocculating strains of yeast (bottom yeast) for fermentation, which are very rare and yields very less alcohol (Rose, A. H and Harrison, J. S ' The Yeasts '(1987) Vol 2, pp 165). Flocculation occurs any where between pH 2 to 8, variable from strain to strain (Nichihara, H; Tiraya, T and Fukui,S (1964) J. Perm. Tech. 54, 351.). Being made up of thousands of cells, floes sediment faster than cells with aggregation number below 10. This occurs in the absence of cell division, under the conditions of cross bridging divalent ions (Miki, B.L.A.; Poon N.H.and Seligy V.I (1982) J. Bacteriology 150, 878.). The natural flocculance is a genetic trait in S.cerevisiae and is controlled by genes which are dominant ( FLO1, FLOS, FLOS ) recessive and /or suppressive (G.B.Calleja 'Cell aggregation in yeasts' Ed. Rose, A.M; and Harrison J.S.(1987) vol.2,p 131-161. Academic press.London.; Lima,N; Moreira, C; Texixeira, J.A; Mota M. Microbios (1995),S 1(328) 187-197). Several investigations have been made in the construction of flocculating ecological strains, which reveals the importance of selecting and constructing useful flocculent yeast (Russel, E., Stewart, G.G., Reader, H. P., Johnston, J. R. and Martin, P. A ( 1980 ) J. Institute
Brew. 86, 120). The walls of flocculent cells contain more mannan than those of free cells. The mannan - protein complex is the active site of cell-cell interaction. It is reported that ethanol and other alcohols, lipids, glycogen enhances the flocculation where as high temperature and anaerobic condition supress flocculance. Introduction of the flocculation property into wine yeast by hybridization by mating or protoplast fusion is also being carried out, with limited success.
Reference may also made to the yeast-ENCILIUM which is a self flocculating yeast strain developed by NCL pune.(Annual Report, NCL Poona 1992-1993.).

; / \There are few patents, mainly on the periphery of the work, one is on Alcohol production. US patent No.4978618; and the other is on the Tower reactors for bioconversion of lignocellulosic material (US- Patent No. 5888806).
The drawbacks of the currently used flocculating yeast strains are the low yielders of alcohol and the cost of maintenance of the genetically engineered strains. It is essential that if high alcohol-yielding strains have to be used for fermentation, artificial flocculation methods have to be adopted ( Weir S, Ramsden D.K; Hugles J, and Lee Thomas, F. Biotechnol. Tech., 7 (1993) 199-204 ; Sousa, M.L, Texixeira, J.A ; J. Biotech.Letters (1996), 18,229-234). The yeast cells have a net negative charge, and hence ionic bonds, hydrogen bonds and hydrophobic interactions can also help stabilizing the floes. Adjusting the chemical environment of particle suspension induces flocculation. Application of cell flocculants and cross linking reagents requires that they have no detrimental effect on cell activity during fermentation. Flocculation involves the formation of an
open agglomeration of cells, the mechanisms of which depends upon molecules acting as bridges between cells., Usually Ca2+ ions was found to be the best to promote flocculation (Taylor, N.W and Orton, W.L (1975), J Institute Brew. 81, 53). Addition of metal ions reduced reverse charge repulsion and addition of surfactants to alter the hydrophobic, hydrophilic nature of the particle surface and so reduce the hydration stabilization. This technique is employed individually or in combination to achieve flocculation. There is not much work carried out to flocculate the yeast articicially with out affecting the fermentation ability.
The main object of the present invention 'A process for the preparation of articicially flocculated yeast particles' is to provide an efficient, flocculated yeast particles for the production of alcohol in the distillery industry, and in chemical biotransformations, which obviates the draw backs as detailed above.
Another objective of this invention is to provide a sturdy catalyst for the preparation of other biochemicals like high fructose syrup, vinegar, enzymes and many related chemicals.
Yet another object of the present inventions is to use the sturdy biocatalyst for many novel biotransformation in aqueous and organic media. Accordingly the present invention provides, a process for the preparation of artificially flocculated yeast particles useful for the production of alcohol, characterized in that coating yeast cell with non-ionic or cationic surfactant in the presence of a divalent metal ion, which comprises growing the yeast cells in a conventional medium as described herein , at a temperature ranging between 25°-30°C for a period ranging from 20-26 hrs, harvesting the cells and collecting the biomass by centrifugation at about 10,000 rpm at about 4°C for a period
ranging from 5-20 min., coating the above obtained wet yeast cells with a 0.1% to 3% non-ionic or cationic surfactant such as herein described , at a pH ranging between 8-10 , in the presence of a divalent metal ion selected from calcium, Barium, strontium and aluminium , under mild stirring of 20-40 rpm , for a period ranging from 0.5 - 4 hrs, clustering the above coated yeast cell by a 0.5 to 3% (v/v) conventional crosslinking reagent of the kind as described herein , in a phosphate buffer at a pH ranging 6-9 followed by centrifugation to obtain the desired flucculad yeast particles.
In another embodiment of the present invention, the medium used is aa conventional medium.
In another embodiment of the present invention, the surfactant used is selected from polyethylene imine, chiotosan and alumfloc.
In yet another embodiment of the present invention, the divalent metal ion used is selected from calcium, Barium, strontium and aluminium.
In yet another embodiment of the present invention the concentration of the cationic solution used for divalent metal ion is in the range of 0.1 to 5M.
In still another embodiment of the present invention, the yeast used is selected from any distillers or Brewers yeast, bakers yeast or any isolated yeast strain which is able to ferment sugar solutions.
In still another embodiment of the present invention, the crosslinking reagent used is selecte from glutaraldehyde, Dimethyl-3,3' dithiobisproponmidate, 2-iminothiolane, n-Succinimidyl-(4-azidophenyl)-1,3-dithiopropionate, Ethyl-4-azidophenyl-1,4-dithiobytryrimidate etc for 1 to 30 minutes.
In still another embodiment of the present invention the flocculated yeast
particles prepared is useful for the production of alcohol in the distillery industry, derusting of solutions and in chemical biotransformation in aqueous and organic media.
Any type of yeast cells, for example Distillers or Bakers yeast are taken, grown in the medium containing nutrients necessary for the growth which mainly
consists of glucose (0.1%) with malt extract, peptone and yeast extract for a

stipulated time duration and then harvested and centrifuged to get a wet cake. This cake is dispersed in water containing either polyimine or a polysilane or any surfactant of cationic, anionic, amphoteric or neutral charge( 0.1 to 2 % ) stirred moderately ( ~ 100 rpm ) for Ito 4h. To this surfactant containing yeast slurry , any poly valent cation like CaCh (0.0004 to 1% ) is added to flocculate the yeast cells. Alternatively the cells are treated with 1% polyimine in the presence of sodium chloride at pH 8 for 2 hours and the washed cells are then treated 0.1 to 2 % v/v glutaraldehyde at pH 7.0 for a few minutes to get a cross-linked yeast particles. After washing repeatedly these clustered particles are used for fermentation of glucose , starch hydrolysates or molasses to alcohol. The clustered yeast particles are very sturdy and with good fermentation ability and can be used for the production of ethanol in a tower fermentor and also for biotransformations of different substrates in aqueous and organic media as well as for the desugaring of solutions which will make the processes economically feasible.
Flocculation by cationic polyelectrolyte (0.1- 0.5 mg/ml) in the presence of Calcium ions (0.004- 1.0 M/ml) was found to flocculate most of the yeast strains.
Chemical cross-linking can be used to cluster the yeast cells by first coating the yeast cells with a polyimine and then adding a bifuctional cross-linking agents like glutaraldehyde, Dimethyl-3,3'-dithiobispropionimidate, 2-iminothiolane, n-Succinimidyl-(4-azidophenyl)-1,3-dithiopropionate, Ethyl-4-azidophenyl-1,4-dithiobytryrimidateetc. The resultant floes are very stable and viable. To bring
about the monolayer coating salt is used in the medium. Too much of cross
• linking agent will kill the organism hence clustering parameters are standardized
to arrive at optimum condition.
Operation of a Tower fermentor for the production of ethanol with the flocculated yeast gave a productivity of 3.0 to 4.5 gm/L/h which was higher than the batch production with the same flocculated yeast. This yeast can be used for various biotransformations as well as desugaring any solutions of importance. The use of artificially flocculated yeast will make the process cost-effective. The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention.
Example 1
Strains of Saccharomyces cerevisiae a thermotolerant and high alcohol yielding, a distillery strain and baker's yeast from market were used for studying their flocculation ability. The cells were grown and maintained in MGYP medium with the following composition:
Malt extract - 3.0 g
Yeast extract - 3.0 g
Peptone - 5.0 g
Glucose - l.Og
Distilled water - 1000ml
pH was adjusted to 5.5
The cells were grown in shake flask at 120 rpm and 30+ 1°C for 24 hours, harvested and the biomass was collected by centrifugation at 10,000 rpm, 10 minutes, 4°C. These were used for flocculation and subsequent studies.
Preparation of flocculated yeast particles: 100 gms of fresh yeast is taken, made into a slurry and added enough CaCh to bring the concentration to 0.5 to 1.8% and then added cationic surfactant polyethylene imine or any similar reagent for example chiotosan, alum floe etc (0.1 to lg%).
Mild stirring ( 20 to 40 rpm ) is given to bring about the flocculation. After sufficient time ( 1 to 3 h) the floes are allowed to settle. The settling time is noted which correlates to the maximum flocculation. The floes are used for alcohol fermentation in batch mode. 5 % yeast floes are used in 10 % glucose solution and after 8 h about 95 % of glucose was converted to alcohol.
Comparison of flocculation characteristics of three different strains of S. cerevisiae at optimum conditions.
Yeast Strain Time taken for sedimentation hr
Termotolerant yeast 2 to 12
Distillary yeast 4 to 15
Bakers yeast 3 to 15
Example 2
Preparation of clustered yeast particles. 100 gm of* distiller's yeast which is previously grown in 5 % molasses is centrifuged to get the viable cells . The wet yeast cells were coated with 1 % of any non-ionic or cationic surfactants like polyethylene imine at a pH of 9.0 , and stirred at 20 to 50 rpm for 1 to 5 hrs in a rotary shaker . It was then centrifuged to recover the surfactant coated yeast cells . The pellets were treated with 0.1 to 2 % v /v glutaraldehyde in phosphate buffer at pH 7.0 to bring about the cross-linking between the yeast cells and centrifuged immediately. Over exposure to glutaraldehyde will reduce the viability of the yeast. The cross-linked yeast particles are washed thoroughly with distilled water. The sedimentation volume (SV) was measured by standard procedures . The clustered yeast particles are then collected and used for the preparation of alcohol both in batch and continuous fermentation with starch hydrolysate as substrate
Example 3
The chemically flocculated and clustered baker's yeast was used to study the fermentation of four different starch hydrolysates viz. Corn starch, Potato starch, Tapioca starch and Molasses in batch mode and the g % of sugar conversion was calculated. Flocculation by either method was found to be equally good in terms
of the percent sugar conversion. Molasses and cornstarch served better substrates for the selected strain.(Table Removed)

Example 4
Batch/continuous fermentations; Batch fermentation was carried out in 250 ml flasks for 96 hrs. with clustered yeast with glucose and starch hydrolysates. Samples were collected at regular intervals for the estimation of residual sugar. The alcohol content was also estimated. The same cells were used in a tower fermentor and compared with batch process.
Table
% alcohol by wt. 24h Glucose 10% Molasses-10% corn hydrolysate 10%
(Table Removed)Example 5
Different tower fermentors- Continuous production of ethanol
The first tower fermentor consists of a glass tube provided with an inlet at the bottom and an outlet at the top. The dimensions were 48 cm length, 34 mm internal diameter having a tower portion 5 cm x 41 mm to remove excess carbon dioxide( L/D ratio=14.1). A sintered plate was fixed at the bottom to prevent the yeast particles to escape through the inlet. Void volume was 375 ml. The column was filled with 5 g (Wet basis) of clustered yeast .The substrate was passed from the bottom and the fermented liquid was collected from the top outlet. The substrate used was glucose, molasses, and corn hydrolysate. Dilution rate is the ratio of flow rate by void volume. Productivity was calculated as the weight of alcohol produced/ L of reactor multiplied by dilution rate.
The second tower fermentor consists of a glass tube provided with an inlet at the bottom and an outlet at the top. The dimensions were 147cm length, 7.55mm having a tower portion 6cmx 10.29 mm. ( L/D ratio=196). The void volume was 450 ml. 100 ml of clustered yeast corresponding to 50 gm wet weight was used.
Table
Productivity of etanol in the fermentor at Retention Time: 16-20h(Table Removed)
The productivity was calculated by multiplying weight of alcohol produced per liter of reactor volume by the dilution rate. The dilution rate is calculated as the ratio of flow rate by void volume. The reactor having the higher L/D ratio gave better productivity.
The main advantages of the present invention are:
*
1.There is no need to search for a naturally flocculating strain or genetically manipulate the yeast strain to produce flocculent characteristics. The naturally flocculent strains, which are very rare, has poor fermentation ability, hence the choice of the best strains for artificial flocculation is very attractive.
2. A simple procedure makes any promising yeast strains to become flocculent.
This artificial flocculation has an advantage of utilizing the best strains for
fermentation; whereas, naturally flocculent strains are neither high yielding
nor thermotolerant for use in industrial situations.
3. The flocculated yeast has a better mechanical stability, hence different reactor
configurations can be used to suite a particular end product formation.
4. The flocculated yeast settles down faster and hence it is easier to separate from
the fermentation media. Avoiding filtration and centrifugation can reduce the operational and down stream processing cost.
5. The flocculated yeast is sturdy and the loss of cells during the reaction is
minimal and hence utilized for continuous operation.
6. The improved rheology at high biomass concentration is an added advantage.
*
7. Compared to the gel entrapped yeast particles, artificially flocculated cells
8. have high cell density and have efficient conversions.
9. Product inhibition can be avoided due to the easy recovery of the biocatalyst.
10. The product obtained is without much contamination and the costly separation
and purification steps can be reduced to minimum and hence the cost-
effectiveness of the process.

We Claim:
1. A process for the preparation of artificially flocculated yeast particles
useful for the production of alcohol, characterized in that coating yeast cell
with non-ionic or cationic surfactant in the presence of a divalent metal ion
, which comprises growing the yeast cells in a conventional medium as
described herein , at a temperature ranging between 25°-30°C for a period
ranging from 20-26 hrs, harvesting the cells and collecting the biomass by
centrifugation at about 10,000 rpm at about 4°C for a period ranging from
5-20 min, coating the above obtained wet yeast cells with a 0.1% to 3%
non-ionic or cationic surfactant such as herein described , at a pH ranging
between 8-10 , in the presence of a divalent metal ion selected from
calcium, Barium, strontium and aluminium , under mild stirring of 20-40
rpm , for a period ranging from 0.5 - 4 hrs, clustering the above coated
yeast cell by a 0.5 to 3% (v/v) conventional crosslinking reagent of the
kind as described herein , in a phosphate buffer at a pH ranging 6-9
followed by centrifugation to obtain the desired flucculad yeast particles.
2. A process as claimed in claims 1 , wherein the surfactant used is selected
from polyethylene imine, chiotosan and alumfloc.
3. A process as claimed in claims 1-2, wherein the concentration of the
cationic solution used for divalent metal ion is in the range of 0.1 to 5M.
4. A process as claimed in claims 1 - 3, wherein the yeast used is selected
from any distillers, Brewers yeast, bakers yeast and any isolated yeast
strain which is able to ferment sugar solutions.

5. A process as claimed in claims 1-4, wherein the crosslinking reagent
used is selected from glutaraldehyde, Dimethyl-3,3'-dithioispropionimidate,
2-iminothiolane, n-Succinimidyl-(4-azidophenyl)-1,3-dithiopropionate,
Ethyl-4-azidophenyl-1,4-dithiobytryrimidate.
6. A process as claimed in claims 1 - 5 , wherein the flocculated yeast
particles prepared is useful for the production of alcohol in the distillery
industry, derusting of solutions and in chemical biotransformation in
aqueous and organic media.
7. A process for the preparation for artificially flocculated yeast particles
useful for production of alcohol substantially as herein described with
reference to the examples accompanying this specification.

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