GB2200368A - Yeast flocculation - Google Patents

Abstract

A method of flocculating flocculent yeast in a yeast culture medium by agitating the medium above a certain degree of agitation after the stationary or decelerating phase of yeast growth has been reached. The method may be used in brewing and other biofermentation processes to speed the sedimentation of yeast from media where the media would otherwise have been left to stand to effect sedimentation. The agitation energy is preferably in the range of 0.25-1 joules hour<-1> ml<-1>

Description

IMPROVEMENTS IN YEAST FLOCCULATION PROCESSES This invention relates to an improved process for the flocculation of yeasts, particularly those which are used in the fermentation industry.

Yeasts are a well known class of micro-organism which belong to the class of fungi, and in which the unicellular form is conspicuous. Yeasts may be distinguished from bacteria and moulds inter alia by their size, shape, and cellular and cultivation characteristics. Many yeasts are industrially important in fermentation processes, particularly in the from duction of alcohol either for consumption in the form of beers, wines etc or for industrial use. In recent years yeasts have been genetically engineered to produce biosynthetic products such as drugs and proteins. Many strains of yeasts are known, and the prototype for the species commonly used in alcohol production is Saccharomyces cerevisiae.

Although a number of methods of classifying yeasts are known, one division that has been made is between "flocculent" and "nonflocculent" yeasts depending upon the tendency of the yeast cells to stay together in flocs or to remain separate. Flocculation may be defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended.

Although flocculation of simple materials such as inorganic precipitates has been well investigated, surprisingly little is known about the mechanism of flocculation of such complex biological systems as living yeasts. A number of hypotheses have been advanced for the mechanism, eg a lectin-like binding between a protein and cell-wall mannan, calcium bridging, and neutrilisation of the surface charge.

During a fermentation process, the yeast culture normally passes through a number

yeast cells increases with time, followed

during which the cell concentration remains essentially constant.

During the growth phase it is common to rouse" the yeast by blowing air through the medium, which usually causes agitation of the medium.

Termination of the fermentation process normally occurs during or after the stationary phase, either by stopping the process when the conçen- tration of the desired fermentation product, eg alcohol in beers, has reached a desired level, or when the yeast ceases to produce the product as a consequence of the concentration of the product which has been formed in the med Am.

After termination of the fermentation process it is usually necessary to separate as much as possible of the yeast from the fermentation medu m, so as to achieve for example a clear beer. This is normally achieved in practice by using a flocculent yeast in the fermentation process and causing the yeast to flocculate and consequently sediment from the supernatant fermentation medium. This flocculation is achieved by allowing the fermentation medium to stand as still as possible in conditions which minimise thermal convection currents and other forms of vibration, and only occurs during or after the stationary phase of the fermentation.

It is often necessary for the fermentation medium to be stood in this way for several days whilst flocculation takes place. The need to keep the fermentation medium standing in this manner for such long periods of time imposes considerable expense and is obviously otherwise inconvenient.

It is an object of the present invention to provide a method by means of which the flocculation time of yeast fermentation media may be significantly reduced.

According to the present invention, a method of initiating flocculation of a flocculent yeast (as defined above) in a yeast culture medium includes the step of agitating the medium, after the stationary orj

phase of yeast growth has been reached, above a certain degree of agitation.

The invention is based upon the discovery by the inventors that above a certain minimum degree of agitation, such agitation can initiate very rapid flocculation of flocculent yeasts during their stationary or late exponental growth stages. Thereafter continued agitation above this minimum degree can maintain a continued high rate of flocculation. This allows settling offlocculent yeasts to be achieved in a time that is very much less than the time for which fermentation tanks are normally allowed to stand still. In some cases settling may be achieved in minutes or hours rather than days. This discovery is contrary to the accepted belief in the fermen tat ion art that lengthy standing is desirable or necessary to achieve flocculation or settling.

The method is applicable to all flocculent yeasts, including the commercially useful S.cerevisiae and strains derived from this, such as S.cerevisiae var. ellipsoideus. Other suitable commercially important strains include S.carlsbergensis and S.fragilis.

The yeast culture medium may be any medium in which yeasts are cul tured, and includes in particular commercial fermentation media such as those used in the brewing (eg beer, ale, lager, stout manufacturing) or wine making industries, whether or not these fermentations are followed by distillation of the alcohol produced. The method may also be used in the course of other commercial fermentations, eg industrial alcohol production, or the fermentation of genetically modified yeasts to produce biosynthetic products.

The culture medium may conveniently be at a temperature which is normally employed in the fermentation process, which will vary with the strain of yeast employed, but will generally be between 5-300C. Similarly the pH of the medium may be in the range normally encountered in yeast fermentation, eg in brewing generally between pH 2-9, especially 3-7. A preferred pH is the isoelectric point of the yeas, ie the point at which surface charges are seutrilised eg around pM 2-3, and the medium may have acid or base added to it to adjust the pH to a desired value. It is common 2+ for Ca ions to be added to a yeast fermentation medium, in view of the 2+ 2+ belief that flocculation may be Ca dependent, and the addition of Ca in media used with the method of the invention may be advantageous in encoura ging rapid flocculation.

With the method of the invention the initial rate of flocculation and the subsequent rate if agitation is maintained is generally found to be cell concentration dependent, in many cases directly proportional to the square of the initial cell concentration.

The point at which the stationary phase of yeast growth in the medium is reached may be determined by measuring the yeast cell density in the medium. This increases with time from the start of fermentation, then begins to level off as the stationary phase is approached. The stationary phase is marked by an essentially constant cell density with increasing time, or at the very least by a marked drop in the rate of increase.

With yeasts that do not show a.marked drop in the rate of increase of cell density, the late

rate of increase of cell density has decreased ideally to zero or to less than ca 5% of the maximum rate of increase shown in the growth phase.

During the stationary or

phase an FLO gene appears to be activated in flocculent yeasts, which is not activated during the growth phase. Whilst this gene is activated, flocculation may occur.

Agitation of the medium to initiate or maintain flocculation by the method of the invention may be by mechanical means such as a stirrer, eg of propellor shape, or by means of a cycling pump in which case agitation may advantageously also occur in the pump cavity, or simply py agitation of the

also be by means such as' bubbling air through the medium. Other means will be apparent to those skilled in the art.

bove a certain minimum level. Whereas in known processes eg water treatment where flocculation in the presence of chemical flocculating agents is encouraged by gentle agitation followed by lengthy standing, in the present process the efficiency of flocculation as expressed in terms of the settling rate appears to be directly proportioned to the degree of agitation above the minimum. The upper limit is set by practical considerations, not by any characteristics of the yeast or medium.

The minimum degree of agitation required in each case to initiate and/ or maintain flocculation depends upon the strain of yeast, and is generally found to increase with increasing pH for any strain, at least between pH 2.0 and 9.0. As a corollary, for any degree of agitation, the initial flocculation rate generally declines with increasing pH. This decline is less conspicuous at higher degrees'of agitation.

The initial flocculation rate of the yeast generally increases with increasing degrees of agitation above the minimum level necessary to initiate flocculation. Continued agitation of the yeast culture is generally found to lead to progressive and continuous flocculation. Cessation of agitation when flocculation has been initiated is found to lead to cessation of flocculation, and consequent cessation of precipitation of suspended yeast. This phenomenon provides a further advantage of the invention in that agitation may be applied to initiate and maintain flocculation and precipitation, and when a required amount of yeast has precipitated, agitation and hence flocculation may be stopped, leaving a required quantity of yeast still in suspension. This may be achieved only with difficulty in prior art processes.

The size of the flocs formed is also found to be dependent upon the degree of agitation of the medium, more violent agitation leading to smaller flocs.

The degree of agitation is normally measured by the violence of the agitation, and is directly related to the amount of agitation energy applied to the vessel containing the medium and transferred to the medium. The experimental data presented below are at present limited to laboratory scale volumes of medium, but there is no reason to doubt that the principal could be applied to industrial scale fprmenters. The amount of agitation energy transferred to the medium in the experiments described below

be broadly determined by calorimetry, erg by measuring the temperature increase

methods.The degree of agitation produced in the laboratory scale experiments described below could be achieved in an industrial scale process by the agitation methods described above, and relatively simple experimental work is necessary to determine the mechanical requirements to achieve a suitable degree of agitation necessary to initiate flocculation of the yeast.

When agitation of the culture medium at a degree of agitation greater than the minimum necessary to initiate flocculation is maintained, flocculation generally continues until an equilibrium is reached between the proportion of floccerlated and suspended yeast cells, which may be as high as more than 99% flocculated.

The invention will now be described by way of example only with reference to the following drawings, in which:

Fig ) > Is a graph of shaking speed against flocculation rate for a yeast strain.

Fig P3 3 Is a graph of shaking speed against flocculation rate for other yeasts Fig 14 Is a graph showing the effect of shaking a yeast suspension and then ceasing, and vice versa.

Fig 5 Is a graph of flocculation rate against yeast cell concentra tion.

Fig 6 Is a graph of flocculation rate against (yeast cll concentra tion) 2 Fig 81 Is a graph of Log (velocity constant) against 1/shaking speed.

Fig 3 Is a graph of flocculation rate against shaking speed at vary ing pH.

Fig so Is a graph of flocculation rate against pH at constant shaking speed.

Fiq %\o Is a graph of'activation energy'against DH.

1. Materials and Methods Strains of S.cerevisiae S646-lB (FLO l/FL0l adel/adel) and JDX-7-176 (his 3 trpl ura 3 FLO1) were obtained from the National Research Council of Canada and the University of Strathclyde, Glasgow, respectively.

Strains were maintained on YEPD slopes, containing per litre of yeast; yeast extract (Oxoid-Trade Mark), 10g; bacteriological peptone (Oxoid -Trade Mark), 20g; glucose 20g; Agar (Difco-Trade Mark), 20g. Routinely cells were grown at 250C in YEPD broth (1.25L) in 2L conical flasks on a LH fermenta tion Mk V orbital shaker (}linche . iE mm orbital radius) at 150 orbits minute -.

Growth of yeast cells was monitored spectrophotometrically (Unicam Trade Mark SP600) (E600nm - 1cm) following addition of 50mM EDTA to dis perse flocs, and compared to a calibration curve.

Stationary phase cultures (Fig.1) achieved after 48 hours of culture were harvested by centrifugation (2000g x 5 min) and washed twice with vigorous mixing in 50 mM EDTA, then finally resuspended in 5 mM EDTA.

ii. Measurement of Flocculation 16 ml of harvested cell suspension (at a determined cell density) in 5 mM EDTA with 16 ml citrate/phosphate buyer (50 mM pH 4.5) or buer as specified, were placed in 100 ml capacity conical flasks and shaken at pre determined rates (0-180 orbits per minute (rpm)) on the flinch (35 mm) orbital shaken referred to above, at 250C.

Calcium chloride solution (8 ml 50 mM) was added and the reaction was followed by removing samples samples from immediately below the surface with a wide-bored pipette, diluting with EDTA (50 mM) and measuring the cell density spectrophotometrically (E600 nm - 1 cm). Absorbance of unfloccu lated control flasks was measured following addition of water 8 ml) in place of calcium chloride. The populations of cells in flocs were then determined.

ili A. Results A number of experiments were carried out investigating the effect of increased orbital shaken speed, pH, cell concentration on flocculation rate, and the use of the method on different strains of yeasts.

(Qh M Shaker Speed The shaker speed in rpm is the best measure of the degree of 1.

agitation. Fig / shows the effect of increasing shaker speed on the initial rate of flocculation of S.cerevisiae S646-1B. Below 50 rpm (of a cell concentration of 6 mg dry weight per ml, pH 4.5) no floccu lation was observed, with cells sedimenting at an identical rate to that of non-flocculent cells.

Fig Z shows results under identical conditions for five other strains of S.cerevisiae. In each case there is a definite minimum degree of agitation necessary to initiate flocculation, for four of the five being in the range 50-100 rpm, and for strain NCYC 1224 being about 20 rpm. In the case of NCYC 1119 the onset of flocculation was very sharply defined. Strain X is a laboratory strain derived from an Allied Breweries (Trade Mark) strain.

Fig X shows the effect of shaking a suspension of S.cerevisiae S646-1B for a period of time (curve A) followed by ceasing to shake the suspension ie leaving it to stand (Al), and the converse effect of allowing the culture to stand for a period (curve B) followed by a period of shaking (curve B1). The effect of shaking is clearly shown to initiate rapid flocculation and sedimentation of the yeast, compared with the effect of leaving the suspension standing still. Curves A2 and B2 respectively show the effect of not ceasing to shake the suspen sion, and of simply allowing the suspension to stand still on identical initial suspended cell concentrations.

Fig 6 shows the dependence upon shaking speed of a value known as the velocity constant K. Log K was found to be inversely propor tional to the reciprocal of the shaking speed for S.cerevisiae S646-1B. The velocity constant K was determined by the expression: - de dt = KC2 where C is cell concentration, K is the velocity constant and t the time. From the slope of Fig 5, K could be determined, and at pH 4.5 and 100 rpm K = 0.5.

like Cell Concentration Fig 6 and 7 respectively show the dependence of the initial rate of flocculation at 100 rpm of S.cerevisiae S646-1B upon the initial cell concentration and the square of the initial cell concentration pH being constant'at 4.5. The initial rate of flocculation being shown to be directly proportional to the square. Each point on Figs 8 and 2 represents the mean of at least 4 independent determina tions.

(razz Effect of PH a Figs / and ss show the effect of pH on flocculation whilst shak ing of S.cerevisiae S646-1B. From pH2 to pH9 the effect of increase of pH is to increase the minimum shaking speed necessary to initiate flocculation, and at constant shaking speed to decrease the initial rate of flocculation.

Table 1 below lists the initial rates of flocculation for various shaking speeds and differing pH values.

Table 1 Initial Rates of Flocculation (mg.dry.wt.ml s ) (means of at least 3 separate determinations)

Shaking speed (rev.min1) p11 2.0 pH 5.8 pH 9.0 60 0.028 + 0.008 0.008 + 0.006 0 80 0.108 + 0.012 0.072 + 0.010 0.005 + 0.003 120 1.33 + 0.19 1.17 + 0.17 1.15 + 0.11 Fig 9 shows the dependence upon pH of the "activiation" energy in terms of shaking speed to initiate flocculation (arbitrary units) determined from Arrhenius-like plots of K and shaking speed for S.cerevisiae S646-1B.The method of derivation of the activiation energy was analagous that used for velocity constant - temperature plots in chemistry for second-order reactions. A clear minimum is seen at the yeast isoelectric point (pH 2-3).

disk) Control Experiment To prove that flocculation on agitation was not so'imply an effect caused by redistribution of the Ca by agitation but was indeed due to the input of mechanical energy a series of parallel experiments were performed in which blue dye was added in place of or in addition to CA 2+ . Dispersion of the dye to form an even distribution was seen to occur within 5-10 seconds. Similarly, shaking of a yeast cell Ca 2+ suspension before addition of Ca ions did not result in floccula- tion.

All the above experiments were repeated with the yeast strain S.cerevisiae JDX1-7-176 and the results were found to be substantially identical.

In all cases therefore the absolute necessity of agitation of suspensions of flocculent yeasts above a certain minimum degree of agitation is demonstrated both to initiate flocculation and to cause continued flocculation at a significant useful rate.

Claims (1)

1. UK Claims

   1. A method of initiating flocculation of a flocculent yeast (as defined above) in a veast culture medium includes the step of agitating the medium, after the stationary or

   phase of yeast growth has been reached1 above a certain degree of agitation.

   2. A method of initiating flocculation of a flocculent yeast as claimed in claim 1 wherein the yeast is selected from S.cerevisiae, S.cerevisiae var, ellipsoideus, S.carlsbergensis and S.fragilis.

   3. A method of initiating flocculation of a flocculent yeast as claimed in claims 1 or 2 wherein the yeast culture medium is commercial fermentation media.

   4. A method of initiating flocculation of a flocculent yeast as claimed in any one of the preceding claims wherein the yeast culture medium is selected from those used for brewing beer, ale, larger, stout, making wine or distilled alcohol.

   5. A method of initiating flocculation of a flocculent yeast as claimed in any one of claims 1 to 4 wherein the method is for industrial alcohol production, or the fermentation of genetically modified yeasts to produce biosynthetic products.

   6. A method of initiating flocculation of a flocculent yeast as claimed in any one of the preceding claims wherein the temperature of the culture medium is generally between 5-300C.

   7. A method of initiating flocculation of a flocculent yeast as claimed in any one of the preceding claims wherein the pH of the culture medium is between pH 2-9.

   8. A method of initiating flocculation of a flocculent yeast as claimed in any one of the preceding claims wherein the pH of the culture medium is between pH 3-7.

   9. A method of initiating flocculation of a flocculent yeast as claimed in any one of the preceding claims wherein the pH of the culture medium is around pH 2-3.

   10. A method of initiating flocculation of a flocculent yeast as claimed in any one of the preceding claims wherein the pH of the culture medium is the pH at which the yeast cells have their isoelectric point.

   11. A method of initiating flocculation of a flocculent yeast as claimed in any one of the preceding claims wherein Ca2+ ions are added to the culture medium.

   12. A method of initiating flocculation of a flocculent yeast as claimed in any one of the preceding claims wherein agitation of the medium is commenced when the rate of increase of cell density has decreased to between zero and less than about 5% of the maximum rate of increase shown in the growth phase.

   13. A method of initiating flocculation of a flocculent yeast as claimed in any one of the preceding claims wherein agitation is achieved by mechanical means.

   14. A method of initiating flocculation of a flocculent yeast as claimed in any one of the preceding claims wherein the agitation means is a stirrer, in the shape of a propeller, a cycling pump, mechanical shaker or vibrator.

   6. A method of initiating flocculation of a flocculent yeast as claimed in any one of claims 1-12 wherein the agitation means is air bubbled through the medium.

   \8 15. A method of initiating flocculation of a flocculent yeast as claimed in any one of the preceding claims wherein the initial flocculation rate of the yeast generally increases with increasing degrees of agitation above the minimum, level necessary to initiate flocculation.

   I s Y7. A method of initiating flocculation of a flocculent yeast as claimed in any one of the preceding claims wherein continuous agitation of the yeast culture generally leads to progressive and continuous flocculation.

   tq A. A method of initiating flocculation of a flocculent yeast as claimed in any one of the preceding claims wherein cessation of agitation when flocculation has been initiated leads to cessation of flocculation and cessation of precipitationtof suspended yeast.

   22. 22. . A method of initiating flocculation of a flocculent yeast substantially as herein described with reference to the accompanying figures.