GB2065699A - Ethanol production - Google Patents

Abstract

Continuous fermentation of fermentable sugars by use of a fermenting micro-organism is effected by a process in which an aqueous feedstock solution containing at least 140 g/l of one or more fermentable sugars is fermented by the micro- organism at a concentration in the fermentation vessel of at least 30 g/l of the cells of the micro-organism measured as a dry weight and through which vessel oxygen is passed as required to maintain the process, the fermentation being operated at a dilution rate of more than 0.07 h<-1> to give a fermented broth containing more than 70 g/l of ethanol, some cells, and residual fermentable sugar in an amount of 0 to 30 g/l.

Description

SPECIFICATION Ethanol production The present invention relates to ethanol production, and more particularly to the production of ethanol by fermentation of sugars.

It is well known that yeasts can be used to produce ethanol (that is, alcohol) by fermentation of glucose and other sugars. Such fermentations form the basis of the production of beer and other alcoholic drinks. There is now an increasing interest in the possibility of using fermentation to produce industrial alcohol.

On a world-wide basis, industrial alcohol is largely produced by the catalytic addition of water to ethylene. However it should be possible to produce industrial alcohol at an economically competitive price by fermentation. Particularly with the stimulus provided by the rising cost of oil, there has been a surge of interest in the use of ethanol as a fuel. This consideration applies especially in countries such as Brazil where the desire to save foreign exchange has stimulated a massive program to produce ethanol from sugar cane for use as a fuel.

A traditional batch fermentation can be used to produce industrial alcohol, but there would be considerable advantages if a continuous fermentation could be adopted. Continuous production potentially allows uniform operation in large-scale plants, simpler control, greater throughput, and savings in capital and operating costs. However, there have been difficulties in realising the potential of continuous fermentation.

In their article "Process Design and Economic Studies of Alternative Fermentation Methods for the Production of Ethanol" (Biotechnology and Bioengineering, XX, 1421(1978)), Cysewski and Wilke consider the limitations which apply to batch and continuous fermentations. They review some of their own previous experimental work, and develop some projected figures for a production of 78,000 gallons of 95% ethanol per day, using molasses as fermentable material.

From the article it will be seen that continuous fermentation is an appreciably better proposition on economic grounds than batch fermentation, but that ethanol inhibition and low cell-mass concentration are seen as limitations which apply equally to batch and to continuous procedures.

In order to overcome the cell-mass, that is, "biomass" limitation, a cell recycle system is adopted by Cysewski and Wilke, and in order to eliminate ethanol inhibition the ethanol is removed from the fermentation broth as the ethanol is formed. The removal of ethanol is seen as being particularly essential, since ordinarily the high ethanol concentrations inhibit years metabolism. Thus, by removing ethanol the concentration of fermentable sugar (glucose) can be raised from 10% to far higher levels, around 35%, thereby resulting in an increase in the maximum ethanol productivity to 82 g/litre-hour from the figure of 29 g/l-h for a continuous fermentation operated with cell recycle but without ethanol removal.

In an earlier article (Biotechnology and Bioengineering, XIX, 1125 (1977)), Cysewski and Wilke describe their technique for removal of ethanol during a continuous fermentation. The ethanol is removed by direct distillation from the fermenting broth, and in order to achieve distillation at temperatures compatible with the yeast it is necessary to apply a vacuum. In practice it was also found necessary to bleed liquid from the fermenter vessel to prevent the build-up of non-volatile material.

Particularly when coupled with the need to recycle cells from the bleed back to the fermentor vessel without breaking the vacuum, the proposals of Cysewski and Wilke present some appreciable demands on the equipment design if one is to achieve consistently high ethanol productivity at an economic cost.

Uhde GmbH, a member of the Hoechst group of companies, have recently put forward an improved continuous fermentation process for the production of alcohol. In their promotional literature they mention that difficulties have been encountered in developing a satisfactory continuous procedure, and offer a continuous process which employs a special adapted strain of yeast isolated as a result of a screening program.

The Uhde process employs a single-stage, carbon-limited fermentation to give a broth containing approximately 7% alcohol by volume, which can then be distilled.

More generally, there have been numerous proposals for continuous fermentation processes which can produce industrial alcohol.

We have been attempting to find a novel process which can be used to produce ethanol at an economically attractive cost without requiring technically sophisticated equipment or a single, special strain of a micro-organism.

We have now found a process for continuous fermentation of fermentable sugars by use of a fermenting micro-organism in which process an aqueous feedstock solution containing at least 1 40 g/l of one or more fermentable sugars is fermented by the micro-organism at a concentration in the fermentation vessel of at least 30 g/l of the cells of the micro-organism measured as a dry weight and through which vessel oxygen is passed as required to maintain the process, the fermentation being operated at a dilution rate of more than 0.07 h ' to give a fermented broth containing more than 70 g/l of ethanol, some cells, and residual fermentable sugar in an amount of O to 30 g/l.

Any residual sugar can be allowed to ferment anaerobically prior to distillation or other purification of the broth.

By adoption of the present process it is readily possible on an industrial scale to produce fermented broths which contain high levels of ethanol, say 90 g/l of ethanol or more, or to produce with high productivity fermented broths which contain at least 70 g/l of ethanol. The process does not require vacuum distillation of ethanol from the primary fermentation vessel, nor is it founded upon use of a single special strain of a particular micro-organism.

We have found that the sugars, oxygen and ethanol concentration can be held at such a level that cell growth can be controlled. The cell growth is controlled so as to maintain the concentration of viable ethanol-producing cells at a substantially constant level, even over extended operating periods. Thus, in particular by regulation of the oxygen level it is possible to maintain the process by encouraging limited cell growth to replace dead cells and cells lost with the ethanol product.

In our process, we utilize to the full the potential of ethanol-tolerant micro-organisms (which are widely available) and to a large extent the only constraints are those imposed practically by the equipment which is employed.

The feedstock is one or more fermentable sugars comprising mono- and/or di-saccharides. It can be a solution prepared from a solid sugar by dissolution, for example, a dextrose solution, but on economic grounds it is greatly preferred to use an available carbohydrate raw material or waste product to prepare the feedstock.

Suitable raw materials or waste products include: a) Molasses. Molasses is a low-value by-product of sucrose processing. It typically contains from 30 to 40% sucrose, around 1 5% of other sugars, and lesser amounts of various impurities. Before it can be fermented it is normally diluted, for example to 160 g/l or more of sucrose, and supplemented as appropriate with nutrients.

Nutrients which can be added comprise assimilable sources of nitrogen eg ammonium sulphate, urea, or yeast autolysate, together with inorganic compounds providing trace elements such as zinc, copper, phosphorus etc and also organic compounds providing essential growth factors such as vitamins. Diluent water can also be added as required.

The diluted and supplemented molasses can then be pasteurized if necessary and cooled.

Other procedures can be used in preparing molasses for fermentation. For example, clarification procedures such as acidification or phosphatation (that is, in situ precipitation of calcium phosphate, suitably by adding phosphoric acid and lime) can be used. Where the resultant clarified molasses is not acidic, it may be appropriate to adjust the pH to be in the range 3 to 5.

b) Hydrolyzed starch. Starch from maize, wheat, cassava, potato or other materials represents an inexpensive source of fermentable sugars: after suitable preparation of the grain, root or other material, the starch can readily be hydrolyzed by known procedures to provide a suitable sugars solution. Acid or enzyme thinning and enzyme hydrolysis are examples of suitable procedures, with enzyme-enzyme procedures being preferred since acid-enzyme treatment is more likely to give unwanted reversion products.

The solutions obtained by the known procedures usually contain about 3545% by weight of mono- and di-saccharides of which 90 to 94% is glucose, with about 5% maltose, and other sugars forming the remainder. Before fermentation some preparation may be necessary to give a suitable medium, eg one or more of dilution, addition of nutrients, pasteurization or clarification.

c) Sugar juices. The processing of cane and beet sugar produces various streams which contain sucrose in differing degrees of concentrations and purity. Molasses is a particular by-product of sugar processing mentioned above, but it is a simple matter to utilize the other available streams. On economic grounds, a preferred available stream is raw cane juice in a sugar cane factory.

Concentration or dilution may be appropriate, as also may be other pre-treatments such as addition of nutrients.

d) Other saccharide solutions. The present process is not restricted to the above sources of fermentable sugars. Examples of further feedstocks include sulphite waste liquor, whey, etc. A saccharide solution produced from bagasse or cellulosic material also represents a potentially useful source of fermentable sugars.

The concentration of the one or more sugars in the feedstock has to be at least 140 g/l, better still at least 180 g/l. The actual concentration which is used will depend on a variety of factors, including both operational and economic factors. In practice an upper limit is set by the particular micro-organism which is employed, with this limit being normally between 200 and 400 g/l, more usually at around 200 to 300 g/l.

We currently prefer to employ a solution containing between 200 and 250 g/l of sugars.

The micro-organism is a yeast which will tolerate the high ethanol level in the fermentation vessel.

Such micro-organisms are well-documented in the literature, and there is no difficulty in obtaining a strain which will continue to ferment at ethanol concentrations of above 70 g/l. Suitable strains are widely employed for instance in fermentations to produce wine and sake.

Suitable yeasts include for instance Saccharomyces spp such as S. uvarum, S. cerevisiae and Schizosaccharomyces spp such as Sz. poms6. Ethanol-tolerant strains of these yeasts are available to the public, for example as S. uvarum ATCC 26602, and S. cerevisiae ATCC 26603, NCYC 177 (ellipsoideus strain), NCYC 478 (sake strain), and NCYC 479 (sake strain).

The design of the fermentation vessel is not critical.

Preferably the vessel is generally tubular and constructed for aseptic fermentation. Suitably it has a volume of at least 100 I, with the total internal volume being about 1.2 times the intended liquid volume. In a preferred embodiment the vessel is generally tubular with a height to diameter ratio of about 2:1 to 2.5:1.

A stirrer is desirable to mix the contents of the vessel, and baffles may be employed to enhance mixing. Provision is made for inlet of oxygen or air, as well as of the feedstock, and an internal weir pipe or other means serves for withdrawal of fermented broth. Some means for cooling or heating liquid in the vessel is also desirable, the usual operating temperature being 30 to 350 C, as also is means for adding acid or alkali to adjust the pH of the liquid, the usual operating pH being 4 to 5.5, normally 4 to 4.5. Furthermore sensors can be provided to monitor the temperature, pH, dissolved oxygen, air flow rate, feedstock flow rate etc as appropriate or desired.

As is explained below means are also provided for maintaining in the vessel a cell concentration of more than 30 g/l, this weight being determined as a weight of dry matter present in the cells. It will be appreciated that the wet weight will be greater by a factor of several times.

The cell concentration in the primary fermentation vessel is maintained at 30 g dry cell matter/litre, or higher, and preferably at more than 35 g/l. The general objective is to maintain as high a cell concentration as is reasonably practicable, with the maximum often being determined by the limitations of the equipment. It is by the use of high cell concentrations that one can achieve high specific rates of fermentation.

Thus, we greatly prefer to maintain the liquid in the vessel as a slurry of the cells, especially a thick slurry containing 40 to 80 g dry cell matterllitre.

In the present process, the cell concentration is preferably maintained by recycling cells from the withdrawn fermented broth. Cells can be separated off from the withdrawn broth in a conventional manner, for example by centrifugation or sedimentation of the withdrawn broth, or by flotation of the cells, and recycled to the fermenter vessel.

Centrifugation of the withdrawn broth is widely employed elsewhere, for example in Brazil to process the product of batch fermentations, and can be used in the present continuous process. From the centrifugation there is obtained a solids-poor stream constituting the aqueous ethanol product of the process, and a solids-rich stream providing cells for recycle to the fermenter vessel.

Notwithstanding the proven worth of separation by centrifugation, it suffers from the disadvantage of high costs, both initially in equipping a production plant and also in maintaining the equipment.

Centrifuge equipment can often represent 10 or 1 5% of the total cost of a new fermentation plant.

Moreover, for a larger plant it is not simply a matter of designing a larger centrifuge: engineering and operating constraints dictate that a series of centrifuges is usually required in order to increase separating capacity.

In the development of our continuous process we have discovered an alternative to centrifugation.

Our alternative is based upon sedimentation or settling, and requires that the fermentation is effected with a flocculating yeast.

Flocculating yeasts are used in the brewing industry for making lager and related drinks, but have not been much used for the production of industrial or fuel alcohol. For non-potable alcohol, the accepted practice is to employ a top-fermenting yeast, such as a top-fermenting strain of Saccharomyces cerevisiae, for example the strain publicly available as strain NCYC 431.

In the present process it is possible to employ a sedimenter if instead of using a top-fermenting yeast, one uses a flocculating yeast, such as a bottom-fermenting strain of Saccharomyces uvarum (carlsbergensis). In order to achieve adequate separation of the yeast cells from the fermentation broths, it is usually necessary to employ the steps of feeding the broth to a sedimentation vessel, retaining the broth in the sedimenter vessel without turbulent agitation preventing the flocculation of the yeast, allowing the yeast cells to flocculate and to settle in the sedimenter vessel, passing a scraper around the bottom of the vessel in order gently to dislodge settled yeast cells without causing substantial vertical intermixing of the contents of the vessel, drawing off aqueous alcohol from towards the top of the sedimenter vessel, and drawing off an aqueous suspension of flocculated yeast cells from the bottom of the sedimenter vessel.

The sedimenter vessel will be sized in accordance with the designed output of the fermentation vessel being employed. For most purposes a sedimenter vessel with a broth capacity of from 0.1 to 0.5 that of the fermentation vessel is suitable, for example one with a broth capacity of from 0.2 to 0.33 that of the fermentation vessel. The residence time of broth in the sedimenter vessel is suitably from 20 to 300 minutes, though as with the capacity the actual value is not critical. The vessel is preferably constructed of polypropylene or other corrosion-resistant material in order to withstand corrosion by the broth, whose pH is usually around 4.

The preferred sedimenter vessel has a scraper arranged gently to dislodge settled yeast cells from around the bottom of the vessel. In order to minimize vertical intermixing of the contents of the vessel while the scraper is in operation, it is preferred to employ a rotating scraper rotating at less than 1 revolution per minute, preferably at less than 0.5 revolutions per minute. Furthermore, it is preferred that the inlet for the incoming broth is below the top of the level of liquid in the sedimenter vessel.Other steps can be taken to minimize intermixing vertically and between the incoming and outgoing streams: the broth inlet preferably includes a final horizontal section such that substantially no vertical motion is imparted to the broth as it enters the vessel, and the broth inlet is preferably encircled with a partition preventing horizontal intermixing between incoming broth from the broth inlet and outgoing aqueous ethanol being drawn off. Moreover, further improvements can be obtained by disentraining carbon dioxide from the broth as it is fed into the sedimenter vessel.

In general we prefer to recycle at least 85% of the cells back to the fermentation vessel. By this means, the cell mass in the fermentation vessel is effectively increased, resulting in increased fermentation rates per unit volume, while steady state operation is maintained.

As required, sufficient oxygen is passed through the primary fermentation vessel to maintain the process. The oxygen gives limited growth of the cells without promoting aerobic growth, thus serving to give regulated growth for cell replacement at the required level. The fermentation is effectively anaerobic and produces ethanol.

Very low levels of oxygen are sufficient for yeasts, to maintain the desired controlled growth rate, with an air flow rate of less than 0.5 vessel volumes per minute (vvm) usually being adequate. In our presently preferred process the air is introduced at around 0.07 vvm.

The oxygen level can also be assessed in terms of the oxygen uptake rate. For the present process, from 0.1 to 0.5 mg O2Ig celllhr is preferred, with from 0.2 to 0.4 mg Oz/g cell/hr being particularly appropriate, the cell weights being dry cell weights.

More generally, it is a simple matter to assess whether the oxygen level is suitable. Ethanol production shows a peak when the dissolved oxygen is at the optimum value. By varying the oxygen content and monitoring ethanol productivity, the optimum can easily be found. It is then greatly preferred to work in the region of this optimum though there is often some latitude before the oxygen level is too low or too high to maintain the necessary limited cell growth.

The dilution rate is more than 0.07 h-', which in turn means that the residence time of the liquid in the fermentation vessel has to be less than about 14.3 hours.

With increase in the dilution rate, the productivity increases but resulting overall in a lower ethanol concentration and a higher residual sugar level. With a decrease in the dilution the reverse applies; the productivity and residual sugar concentration fall and the ethanol concentration rises.

It is preferred to employ as high a dilution rate as is possible, consistent with achieving the other requirements imposed on the process. A dilution rate of around 0.4 h-1 represents a convenient maximum towards which one can aim.

A growth rate of at least 0.003 h-' is usually required in order to maintain yeast cell viability at reasonable levels (say 80% viability). On the other hand, it is by maintaining a low growth rate that the inhibiting effect of high ethanol concentration is reduced. Hence, whereas a cell growth rate of 0.03 h-' represents a suitable maximum growth rate with yeast cells, we prefer to employ a growth rate of 0.003 h-' to 0.015 h-'.

The fermentation in the fermentation vessel results in an ethanol steady state concentration of more than 70 g/l, preferably 100 g/l or more.

It is a matter of practical expediency to balance the variable of the present process to produce a desired ethanol concentration. By using ethanol-tolerant strains as discussed above, there is little difficulty in obtaining the ethanol concentrations of more than 70 g/l. If the ethanol concentration is 70 g/i or less, then the dilution rate is lowered or the concentration of cells in the fermentation vessel or of the fermentable carbohydrate in the incoming solution is increased. A typicai maximum attainable ethanol concentration might be 150 to 200 g/l, with the actual value depending on the micro-organism employed.

By accepting some residual carbohydrate in the fermented broth from the fermentation vessel, it is appreciably easier to meet the constraints imposed by the present continuous process.

The level of residual carbohydrate has to be less than 30 g/l and will optimally be less than 2 g/l.

For a yeast-based fermentation the level will typically be from 0 to 1 5 g/l, usually from 0.1 to 3 g/l.

If desired, the residual carbohydrate can be fermented, using strictly anaerobic conditions.

Ordinarily there will be sufficient cells present in the aqueous ethanol product to achieve such secondary fermentation. For preference the secondary fermentation occurs in a hold tank for holding the fermented broth prior to subsequent purification.

The fermented broth is preferably purified by distillation, but other procedures may be appropriate, for example where a dilute aqueous solution of ethanol is desired.

Distillation normally involves an azeotropic distillation, and further stages can be included depending on the specification which the product is intended to meet. Further details regarding the conventional distillation of ethanol can be found in textbooks, eg in Biochemical Engineering (1964) by F. C. Webb, publisher van Nostrand, at pages 633 to 636.

The present invention is further illustrated by the following, small-scale and large-scale examples of processes embodying the invention. In the examples, reference is made to the accompanying drawings which comprise: Figure 1 is a schematic flow diagram of the apparatus employed in the small-scale example; Figure 2a, 2b and 2c together form a schematic flow diagram for the apparatus employed in the large-scale example; and Figure 3 is a chart showing results obtained using the apparatus of Figure 2.

EXAMPLE 1 Small-scale Fermentation A top-stirred 5 1 fermenter 10 was run at 2.5 1 working volume. Sterile medium was added by a calibrated peristaltic pump 11 and fermented broth displaced through an internal overflow weir 1 2.

The temperature was controlled to be at 300C and the pH to be at 4.5 by addition of 2 N NaOH (sensor 13). Oxygen requirements for yeast during fermentations were monitored by sensor 14 and met by diffusing air through silicone rubber coils (not shown) immersed in the broth, or by sparging air through a pipe 15 sited beneath the impeller, an outlet 21 with condenser 22 allowing for exit of air.

Agitation was varied according to experimental conditions.

Cell recycle was achieved by interposing two 750 ml yeast settling devices 16, 17 in series with the fermenter overflow (Figure 1). Sedimented yeast cream was pumped by pumps 1 8, 1 9 back into the fermenter and the product liquid taken from the overflow weir 20 of the second settling device 1 7. The product then passed to a secondary fermentation vessel (not shown).

The organism used was Saccharomyces uvarum ATCC 26602, a flocculant yeast. Cultures were maintained on wort agar slopes at 40C.

Using a ioop, culture was transferred from a slope to 50 ml malt extract medium (Table 1) and incubated for 24 h at 300C anaerobically in screw-capped bottles. 25 ml of this culture was used as inoculum for fermentation.

TABLE 1 Composition of Inoculum Growth Medium Component Concentration (g/l) Malt Extract 50 Glucose 50 Sucrose 200 Fermentations were run batchwise initially until cells were in late log phase of growth at which time continuous medium feed was begun. After three fermenter throughputs, samples were analysed for centrifuged packed cell volume (CCV), cell dry weight concentrations, cell viability, ethanol and glucose.

Analysis of samples were continued until steady states were established in the fermenter.

The fermentation medium employed for the continuous fermentations was that shown in Table 2.

TABLE 2 Composition of Fermentation Medium Component Concentration (g/l) Glucose monohydrate 275 (NH4)2SO4 3.4 KH2PO4 5.6 MgSO4.7H2O 0.45 CaCI2. 6H2O 0.07 (NH4)2SO4. FeSO4. 6H2O 0.023 ZnSO4 7H2O 0.006 CuSO4.5H2O 0.0009 I nositol 0.06 Thiamin 0.013 Pyridoxin 0.0037 Ca++ pantothenate 0.0015 d-biotin 0.00009 All solutions were prepared in distilled water Glucose was sterilized separately from the saltsvitamins components and was subsequently mixed aseptically when cool.

A dilution rate of just greater than 0.11 h-1 was established. By varying between 99 and 80% the proportion of yeast cells recycled from the devices 1 6, 17 back to the fermenter 10, it was possible to achieve a cell concentration of 35 g/l for long periods to enable a steady state to be established. The steady state ethanol concentration in the fermenter 10 was from 80 to 100 g/l.

The product drawn from the second settling device 1 7 contained some residual glucose, and this was fermented out in the secondary fermentation vessel. Thereafter the aqueous ethanol was purified by filtration.

In two particular experimental runs, the following results were obtained for the steady state conditions in the fermenter 10.

TABLE 3 Experimental Results Dilution Rate Cell Concentration Ethanol Concentration Run No. (h-1) (ugli) (girl) 1 0.11 35.3 94 2 0.12 35 86 EXAMPLE 2 Large-scale Fermentation The feed from a storage tank 30 (Figure 2a) leads through a heat exchanger 31, with respective pipes 33, 34 being provided for the addition of water and a nutrient solution.

From the heat exchanger the feed is led through a pasteurization step 36, and cooled by passage back through the heat exchanger 31 and through a cooler 37 cooled by cold water passing from inlet 38 to outlet 39.

From the pasteurization step the feed is passed as shown in Figure 2b first to a seed fermenter 40, and thereafter to a main fermentation vessel 41.

The seed fermenter 40 is equipped with a stirrer 42 and inlets 43, 43', 44 and 45 respectively for sulphuric acid, sodium hydroxide solution, antifoaming agent (propylene glycol, MW 2025) and air (derived from an air blower 46 and passed through a sterile air filter 47). The seed fermenter 40 is further provided with a product outlet 48, and an air outlet 49, a jacket (not shown) and internal baffles (not shown).

The fermentation vessel 41 is around 1 50 1 capacity and made of stainless steel. It has internal baffles 52 and a stirrer 50 driven by a motor 51. A jacket 53 encircling the vessel 41 is linked to a supply of cooling water (not shown). An air inlet 55 extends to a ring sparge pipe 54 beneath the stirrer 50.

Inlets 57, 57', 58 are attached for feeding in of acid, alkali and antifoaming agent, respectively, and there are appropriate sensors and instrumentation for monitoring and/or control of temperature, pH, dissolved oxygen, pressure above liquid, air flow rate, feedstock, flow rate, etc.

A standpipe 59 with weir overflow is provided for removal of product from the fermentation vessel 41, and the product then passes through a sedimenter vessel 60. The sedimenter vessel 60 has an inlet 79 for introducing fermented broth from the fermentation vessel 41. Before entering the vessel 60 the broth is passed through a CO2-disentraining device 80 with a gas outlet. The point of discharge from the inlet 79 is below the surface of broth in the vessel 60 and at the end of a horizontal section. The sedimenter vessel has a scraper 81 fitted with scraper blades and with a motor (not shown) to rotate the scraper at about 6 rev/hr. Towards the top of the vessel is a circular channel forming a weir for offtake of liquid and feeding an outlet 83, while at the bottom of the vessel has an outlet 84. Encircling the discharge point of the broth inlet 79 is a partition.The vessel 60 has a gas vent (not shown) in its lid.

From the sedimenter a solids-rich fraction, mainly cells, is cycled through a balance tank 62 and then either back to the fermentation vessel 41 or onwards to a pre-still tank 63.

The solids-poor fraction, mainly aqueous ethanol, is fed onwards to the tank 63, which is equipped with a stirrer 64 and also acts as a secondary fermentation vessel.

From the tank 63 a pipe extends (Figure 2c) to an inlet 65 of a steam stripping still 66 fitted with a steam inlet 67. A product outlet 68 then leads from the stripping still 66 to a heat exchanger 70 and thereafter to the centre plate column 71. Outlets 72, 73, 74 and 75 allow four fractions to be taken off, and the fraction from towards the bottom of the column is taken from outlet 74, through heat exchanger 76 and into a rectifying column 77. Input to the column 77 is at just below the middleplate, and the product outlet 78 is near the top.

The process was set in operation by culturing an inoculum of the desired organism, Saccharomyces uvarum ATCC 26602 in this instance, in the seed fermenter 40.

In this instance the apparatus was used for fermentation of glucose derived from corn starch.

Sufficient water was added through pipe 33 to achieve a final glucose concentration of around 230 g/l.

Nutrient solution of the composition given in Table 2 but lacking the glucose monohydrate was added through pipe 34 to the glucose solution. The supplemented glucose 'solution was fed at about 300C into the seed fermenter and air, alkali, and antifoaming agent supplied as required.

The cultured cells were then fed to the main fermentation vessel 41, together with the pre-treated glucose, and air, alkali and antifoaming agent as required. The stirrer 50 maintained homogeneity of the contents of the vessel, with the baffles 52 serving to prevent dead spaces. Product was taken by the standpipe 59 to the sedimenter 60. The fermented broth was fed in through the inlet 79 at a rate faster than liquid drained out through the outlet 84. The broth level gradually built up to just above the horizontal section of the inlet, and liquid then ran over the weir into the channel 82 and thence out through the outlet 83. The motor was started, thus setting in motion the scraper 81 at a speed of 6 rev/hr. By virtue of the slow rotational speed of the scraper, the broth was retained temporarily in the vessel without turbulent agitation.

The cells of the yeast then flocculated and settled at the bottom of the vessel 60. The scraper blades 81 gently dislodged the settled cells without causing them to migrate vertically and mix with broth towards the top of the vessel. In this way, aqueous ethanol containing at most 5 g/l of yeast cells was obtained as the liquid drawn off through the outlet 83 while an aqueous suspension of floccuiated yeast cells (up to 85 g/l) was drawn off from the bottom of the sedimenter vessel.

95% of the cells were recycled back to the fermenter vessel 41, and in this way a cell concentration of about 40 to 50 g/l was built up in the vessel 41.

The dilution rate was initially set to be 0.11 h-', and steady state conditions were approached.

After about 180 hours the dilution rate was altered to 0.1 h-', and then at about 320 hours to 0.08 h-'.

At the dilution rate of 0.08 h-', air was being introduced through inlet 50 at the rate of 111/m, while 2.7 N sodium hydroxide had to be added through inlet 43' at the rate of 0.07 I/h in order to maintain the pH at 4.5. A fermented broth containing more than 92 g/l of ethanol was easily obtained.

The aqueous ethanol separated in the sedimenter 60 contained residual glucose, about 1.4 girl, and was passed at 0.74 kg/min to the pre-still tank 63 which also acted as a secondary fermentation vessel. Anaerobic fermentation occurred in tank 63, thereby slightly raising the ethanol concentration.

From the vessel 63 the ethanol solution was fed in to the stripping still 66 and gave at the outlet 68 a dilute ethanol still containing part of the unwanted volatile components. In the column 71 four fractions were produced, namely volatile esters and aldehydes from outlet 72, fusel oil from outlet 73, 25% ethanol from outlet 74 and water and other involatile matter from outlet 75. The 25% ethanol was then azeotropically distilled in the column 78, giving ethanol containing 4.4% water. If desired this product could then have been further purified by a ternary azeotropic distillation using a third component which forms an azeotrope with the water and only a small part of ethanol.

Continuous operation of the present process was achieved for the period of 532 hours, the process then being voluntarily terminated. Throughout the entire period of 532 hours the process yielded a broth containing between 90 and 100 g/l ethanol, and there was no difficulty in maintaining the process.

The consistent performance and long-term stability of the process is illustrated by the analytical results shown on the chart forming Figure 3 of the accompanying drawings. Plotted against time are the readings taken at regular intervals for dilution rate, cell viability, glucose concentration, ethanol concentration and fermenter cell concentration. With the dilution rate remaining between 0.11 to 0.08 h-', the cell viability varied between 52 and 99%, with 75 to 80% being a typical value. The glucose concentration in the feed was kept at between 200 and 230 g/l, and gave a product containing between 70 and 100 g/l of ethanol and 0 to 20 g/l of residual glucose. The conversion efficiency was typically 85%. The concentration of cells in the fermenter was held at between 30 and 60 g/l, the growth rate being about 0.01 h-' and the recycle ratio about 9:1.

Claims (20)

1. A process for continuous fermentation of fermentable sugars by use of a fermenting microorganism in which process an aqueous feedstock solution containing at least 140 g/l of one or more fermentable sugars is fermented by the micro-organism at a concentration in the fermentation vessel of at least 30 gil of the cells of the micro-organism measured as a dry weight and through which vessel oxygen is passed as required to maintain the process, the fermentation being operated at a dilution rate of more than 0.07 h-' to give a fermented broth containing more than 70 g/l of ethanol, some cells, and residual fermentable sugar in an amount of O to 30 g/l.

2. A process according to claim 1, wherein the cell concentration is maintained by recycling at least 85% of the cells from the withdrawn fermented broth.

3. A process according to claim 2, wherein cells for recycling are separated off from the withdrawn broth by centrifugation.

4. A process according to any of claims 1 to 3, wherein the fermentation is effected with a flocculating yeast.

5. A process according to claim 4 wherein cells for recycling are separated from the withdrawn fermented broth by sedimentation or settling.

6. A process according to claim 5, wherein separation of the yeast cells from the fermentation broth is effected by the steps of feeding the broth to a sedimentation vessel, retaining the broth in the sedimenter vessel without turbulent agitation preventing the flocculation of the yeast, allowing the yeast cells to flocculate and to settle in the sedimenter vessel, passing a scraper around the bottom of the vessel in order gently to dislodge settled yeast cells without causing substantial vertical intermixing of the contents of the vessel, drawing off aqueous alcohol from towards the top of the sedimenter vessel, and drawing off an aqueous suspension of flocculated yeast cells from the bottom of the sedimenter vessel.

7. A process according to any preceding claim, wherein the feedstock solution is prepared from an available carbohydrate raw material or waste product.

8. A process according to any of claims 1 to 6, wherein the feedstock solution is prepared from molasses.

9. A process according to any of claims 1 to 6, wherein the feedstock solution is prepared from maize, wheat, cassava, potato or other starch-containing material.

10. A process according to any of claims 1 to 6, wherein the feedstock solution is prepared from raw sugar can juice.

11. A process according to any preceding claim, wherein the concentration of the one or more sugars in the feedstock solution is at least 1 80 g/l.

12. A process according to claim 11, wherein the feedstock solution contains between 200 and 250 g/l of fermentable sugars.

13. A process according to any preceding claim, wherein the cell concentration in the fermentation vessel is maintained at more than 35 g/l.

14. A process according to claim 13, wherein the cell concentration is 40 to 80 g/litre.

1 5. A process according to any preceding claim, wherein oxygen is passed through the fermentation vessel using an air flow rate of less than 0.5 vessel volumes per minute.

1 6. A process according to any preceding claim, wherein the fermentation results in an ethanol steady state concentration of 100 g/l or more.

1 7. A process according to any preceding claim, wherein the fermented broth contains from 0 to 1 5 gll of residual sugars.

1 8. A process according to any preceding claim, wherein residual carbohydrate is fermented under anaerobic conditions.

1 9. A process according to any preceding claim, wherein the fermented broth is purified by distillation.

20. A process for continuous fermentation of fermentable sugars substantially as hereinbefore described with reference to the Figures 2 and 3 of the accompanying drawings.