WO1988000616A1 - Conversion of fermentable carbohydrates to ethanol using mixed cultures of zymomonas mobilis and yeast - Google Patents
Conversion of fermentable carbohydrates to ethanol using mixed cultures of zymomonas mobilis and yeast Download PDFInfo
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- WO1988000616A1 WO1988000616A1 PCT/AU1987/000224 AU8700224W WO8800616A1 WO 1988000616 A1 WO1988000616 A1 WO 1988000616A1 AU 8700224 W AU8700224 W AU 8700224W WO 8800616 A1 WO8800616 A1 WO 8800616A1
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- fermentation
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- ethanol
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- the present invention relates to a method for converting fermentable carbohydrates in a high salt medium by the use, under microaerophilic or anaerobic conditions, of a mixture consisting essentially of a Zymo onas bacterial species and a yeast, with or without cell recycling.
- yeast cells Most of the known methods for the conversion of fermentable carbohydrates to ethanol involve the use of a single strain of yeast or fermentative bacteria. Ethanol production utilizing yeast cells, for example, is often carried out in a two-stage batch process, where the term "batch" is us.ed to specify that the cells are not fixed to any surfaces or otherwise immobilized.
- the first "growth” or “propagation” stage involves propagation of yeast in an aerobic environment
- the second "fermentation” stage involves fermentation of fermentable substrates in a microaerophilic or an anaerobic environment, i.e., in the presence of a small amount of oxygen or in the total absence of oxygen.
- One disadvantage of the traditional process of yeast fermentation is the long fermentation time.
- “Fermentation time” is used here to denote the period of time required for complete conversion of substrate to end-products, or for the biocatalyst to reach the stage of maximum yield of end-products.
- a fermentation time of 30-70 hours has been found to be required for con ⁇ version of glucose to ethanol, using yeast cells.
- the rate-limiting factors in this process are the rates of glucose uptake and ethanol production by individual yeast cells. These rates are stringently regulated by the cell's enzyme control systems, e.g., by the enzymes invertase and hexokinase.
- One method devised to increase the yield of ethanol is to use a larger inoculum of yeast cells , i.e., a larger biomass density, on the order of 5-10 million cells per millilitre (mL) .
- This method still requires 30-50 hours of fermentation time. Reduction in fermentation time to the range of 10-30 hours has been achieved by using 80-100 times higher biomass density. This higher biomass density is achieved by recycling cells previously used in another fermentation. But the expediency of higher biomass density requires an adequate supply of nitrogen and phosphorus, and often sulfur and magnesium as well, which supplies are often limited.
- yeast fermentation process Another disadvantage of the yeast fermentation process, especially when cost is a major concern, is the diversion of fermentable substrate to the production of " by-products such as glycerol, amylalcohol and other fusel oils.
- by-products such as glycerol, amylalcohol and other fusel oils.
- the efficiency of conversion of substrate to end-product e.g., from glucose to ethanol, is lower than desirable, i.e., only between 85 and 91%.
- Substrate inhibition results from an osmotic effect of the substrate on the cells, and is reflected in reduced water activity inside the cell, plasmolysis, and decreased viability.
- End-product inhibition results from either ' a high solubility of cell membranes in ethanol, or the effect of ethanol on cells' internal regulatory mechanisms. In either case, there is inhibition of cell growth or inhibition of fermentation.
- Jones and Greenfield (1981) used a mixture of an "ethanol-tolerant " yeast and a "sugar- tolerant” yeast in a batch fermentation system which employed glucose as the fermentation substrate. They found that the performance of the mixture of cells was roughly equivalent to a weighted sum of the performance of the individual components, i.e., there was no effect over and above the sum of the individual performance of the two yeast types separately.
- a mixture of two types of yeasts was also used as biocatalysts by Fukushima and Hatakeyama (1987), in a study involving continuous fermentation of cane sugar.
- the mixture of cells was "immobilized", i.e., the cells were entrapped in aluminium alginate gel particles .
- a method for converting a fermentable carbohydrate to ethanol comprising the step of bringing a fermentable mixture into contact with a mixture of cells comprised of yeast cells and Zymomonas bacterial cells-, such that ethanol is produced by the action of the mixture of cells on the fermentable mixture, where the fermentable mixture comprises at least one fermentable carbohydrate and has a total salt content in excess of 3% .
- the bacterial cells used in the method are of the species Z. mobilis .
- a mixture of yeast and Zymomonas bacterial cells can, at a high conversion efficiency, convert fermentable carbo ⁇ hydrates, including sucrose and compounds comprising sucrose moieties, to ethanol in a high salt environment.
- the total content of salts e.g., salts of sodium, potassium and magnesium, ammonium salts, sulfates, chlorides, and phosphates, can be in excess of 3% (w/v), when both yeast and Zymomonas are inhibited.
- molasses is a complex fermentable mixture obtained as a residue after heating cane sugar juice and crystallizing from it a high quality sugar.
- the resulting mixture comprises the fermentable sugars sucrose, glucose and fructose.
- salts are concentrated in the residue, which detracts from the utility of the mixture in ethanol production based on yeast or Zymomonas alone .
- the same drawback has applied heretofore to the other complex fermentable mixtures, such a liquefied starch, high test molasses and, in certain instances, starch hydrolysates.
- yeasts that is capable of converting fer- mentable carbohydrates to ethanol can be used in the present invention.
- the choice of a particular strain of yeast depends on the fermentable substrate to be used in the fermentation.
- a yeast that can utilize those fer ⁇ mentable substrates not metabolized by the Zymomonas employed is preferred.
- the following yeasts are suit ⁇ able for use, according to the present invention: Saccharomyces species, Kluyveromyces species , Pachysolen tannopholus and Candida lusitaniae.
- Preferred strains of Saccharomyces uvarum and Saccharo- myces cerevisiae have been deposited in the Culture
- Any Zymomonas that is capable of converting fermentable carbohydrates to ethanol can be used in the present invention.
- Those suitable to be used according to the. present invention includes those currently classified taxonomically as Z. mobilis and Z. anaerobia, although there is, at this time, much uncertainty over the taxonomical classification of Zymomonas species.
- Particularly preferred are strains of the species Z.
- yeast cells and the bacterial cells to be used according to the present invention are propagated separately, in suitable growth media, to obtain the initial inocula for the use in the fermentation process.
- yeast and bacter ⁇ ial cells reach a logarithmic phase of growth, they are mixed in a fixed proportion, preferably in the ratio 1:1, 1:5, or 5:1, of yeast cells :Zymomonas mobilis cells (v:v ) .
- a complex fermentable mixture that is suitable for use in the present invention can contain any of the range of fermentable carbohydrates that can be used as substrates by yeast and Zymomonas, respectively.
- Substrates of this sort include, but are not limited to, sucrose, fructose, glucose and maltose. These and other sugars are found in . complex fermentable mixtures like molasses, high test molasses and liquefied starch. As previously mentioned, these mixtures have a high salt content, as do starch-hydrolysate mixtures when they are subjected to recycling.
- sugar cane juice or syrup, sugar beet juice or syrup, raw sugar and refined sugar can also have high salt contents when stillage of distillaries or steep liquor water is added to provide nutrient or diluting water .
- distilleries denotes a medium recycled from a prior fermentation in which there is no ethanol and little or no fermentable sugars.
- Step liquor water is a by-product of a wet-milling process that is used, in the starch manufacturing industry, to extract starch from plant materials.
- High test molasses denotes molasses to which an enzyme, invertase, has been added to convert some of the sucrose therein to glucose and fructose.
- “Liquefied starch” denotes a complex mixture of maltrin,, dextrin, starch, lipids and proteins obtained from wet or dry milling of starch-containing plant and root materials , the product of which is treated with physical agents (high temperature and pressure), with chemical agents (to effect acid hydro ⁇ lysis) or with alpha-amylase, to lower viscosity and thereby allow the use of a higher concentration in the fermentation mixture.
- Starch hydrolysates are complex mixtures obtained treating liquefied starch with amylo- glucosidase; they contain, as major components, glucose, maltrin, dextrin, maltose, lipids and proteins.
- the concentration of sucrose when it is used as a fermentable substrate according to the present invention, should be between 10 and 30% (w/v), preferably between 15 and 25%, and optimally at 18%.
- a suitable fermentation medium in this context is any suitable growth medium comprising at least one fermentable substrate and at least one of the following: yeast extract, peptone (i.e., casein hydrolysate) , dia monium phosphate, ammonium sulfate, magnesium sulfate, potassium dihydrogen phosphate, ammonium hydroxide, and urea.
- the concentration of the starch is such as to yield a glucose equivalent of between 10 and 25%, preferably, between 15 and 20%, and optimally 18%.
- the fermentation medium preferably com ⁇ prises at least one of the following components: peptone (casein hydrolysate), yeast extract, potassium dihydrogen phosphate, ammonium sulfate, ammonium hydroxide, urea, magnesium sulfate or dia monium phos ⁇ phate.
- peptone casein hydrolysate
- yeast extract potassium dihydrogen phosphate
- ammonium sulfate ammonium hydroxide
- urea magnesium sulfate or dia monium phos ⁇ phate.
- Each component is provided in a concentration between 0.01 and 0.05% (w/v), but preferably between 0.05 and 0.5% (w/v).
- Sugar cane juice, sugar beet juice, molasses or stillage from distilleries ⁇ known to comprise one or more of the above described components may be used as a-source of that component.
- Stillage from distilleries can be used in the present invention a concentration between 1.5 and 7.5% dry solids.
- the fermentation medium prefer ⁇ ably comprises at least one of the following components: yeast extract, peptone (casein hydrolysate), potassium dihydrogen phosphate, ammonium sulfate, urea, and magnesium sulfate.
- yeast extract peptone (casein hydrolysate)
- potassium dihydrogen phosphate potassium dihydrogen phosphate
- ammonium sulfate urea
- magnesium sulfate sulfate
- the above-described com ⁇ ponents are provided in a concentration between 0.01 and 0.5% each, the most optimum concentration being 0.2% each.
- Sugar cane juice, sugar beet juice or molasses known to comprise one or more of the above described components may be used as a source of those components.
- sugar cane juice or syrup, sugar beet juice or syrup, or molasses is used as a fermentable substrate, no supplementation of the fermentation medium is necessary.
- molasses is used as a fermentable substrate, on the other hand, stillage from distilleries may be used as a diluent or as source of nutrients .
- the fermentation medium may be supplemented with at least one of the following antibiotics to control secondary bacterial or yeast contamination: sorbic acid, penicillan V-acid, trimethoprim, strepto ⁇ mycin, neomycin, nalidixic acid, ampicillan, bacitracin, gentamicin, kanamycin, lincomycin, and polymyxin.
- the pH of the fermentation medium may be controlled or adjusted with sodium hydroxide, potassium hydroxide, ammonium hydroxide, ammonia or sulfuric acid, but preferably no pH control is performed. When a starch-based material is used as a fermentable sub ⁇ strate, the pH of the fermentation medium is maintained between 3.5 and 7.0.
- the pH of the fermentation medium initially is adjusted to between 4.. * 5 and 7.0, and preferably " between 3.9 and 5.0, except when starch .hydrolysate is used, as a fermentable sub ⁇ strate, in which case no pH adjustment is required initially.
- the pH of the medium may be adjusted to between 4.3 and 5.0 r and preferably between 4.3 and 4.8.
- the pH of the fermentation-medium may be adjusted by addition of sodium hydroxide or other suitable alkali. After the initial adjustment, fermentation can proceed without additional pH adjustment as the pH is maintained by the natural buffering action of the mixture.
- the pH of the fermentation medium is preferably maintained between 3.5 and 7.0, and optimally between 4.5 and 7.0. After 1-2 hours of fermentation the pH typically drops, and later stabi ⁇ lizes at between 3.7 and 4.7.
- the pH of the fermenta- tion medium may be adjusted by addition of sodium hydroxide or other suitable alkali .
- the fermentation within the present invention proceeds in the presence of a mixture of yeast cells and Zymomonas cells in either a batch system or an immobilized cell system.
- Suitable batch systems in this regard include a single-stage batch system, a fed-batch system, a continuous-fermenter train system and a multi ⁇ stage continuous batch system.
- a single-stage batch system involves a one- step process in which the biocatalysts are added to a fermentable mixture in a fermentation vessel and allowed to ferment, after which the ethanol is separated from the fermentable mixture and the biocatalysts are dis ⁇ carded.
- a fed-batch system an aliquot of the fer- mentative mixture in a fermentation vessel is removed at intervals during the fermentation process and is replaced by fresh fermentative mixture and ⁇ utrients, with or without the additional biocatalysts.
- Fermentation in a continuous-fermenter train system involves continuous addition of fresh fermentable mixture and nutrients, with or without additional bio ⁇ catalysts, to a fermentation vessel, which is serially connected to other vessels, such that overflow from the first vessel flows into the second vessel, and so forth.
- a multi-stage continuous batch system also has multiple vessels, but only nutrients, not fermentable substrates, are continuously added to one or more of the vessels.
- the biocatalysts are entrapped, e.g., in calcium or aluminium alginate gel particles.
- the cells used in the fermentation process according to the present invention can be recycled by centrifugation, filtration, flocculation, or coprecipitation with inert materials.
- the recycled cells can be added to the fermentation mixture at different points in the multi ⁇ stage batch system.
- Fermentation may be allowed to proceed .at a temperature of between 25 C and 40 C, with a constant temperature control between 32 C and 35 C being preferred.
- a sucrose-based material is used as substrate the temperature is maintained between 30°C and 40°C, preferably between 34°C and 37°C.
- amyloglucosidase is required when liquefied starch is used as a fermentable substrate.
- This enzyme is used at a concentration of between 0.01 and 1.0%, preferably between 0.1 and 0.5%, or at an activity of 0.32 and 1.0 GU/g starch, and preferably between 0.32 and 0.7 GU/g starch, where "GU" denotes glucose-units, and 1 G ⁇ denotes the release of 1 g of glucose per # hour from starch.
- the mixture of yeast and bacterial cells are separated from the fermentation medium, and the ethanol is distilled.
- the cells may be separated e.g., by filtration or centrifugation, and recycled or reused with fresh fermentable substrate and medium. If the mixture of cells are not recycled, then 20% of the fermentation mixture is saved and used as inoculum for the next fermentation process .
- Zymomonas mobilis bacteria ATCC 39676
- Saccharomyces cells were propagated independently in two separate 50,000 L prefermenters .
- an inoculum consisting of 2,000 L of a Zymomonas culture was added to a growth medium compris ⁇ ing: (a) a mixture of B and C molasses in a ratio of 4:1, which exhibited a Brix reading of 20 , and (b) 80 kg of diammonium phosphate.
- B and C molasses are the residues remaining after a first and second cry ⁇ stallization of sugar, respectively, in the commercial production of sugar from cane sugar juice.
- the pH of this medium initially was 5.3.
- the yeast cells were propagated in the same growth medium as used for propagation of the bacteria, except, 250 kg of diammonium phosphate was used, instead of 80 kg, in the growth medium.
- the pH of this medium initially was 5.3, but was lowered to 4.0 by the addition of sulf ric acid. Agitation of the medium was accomplished by addition of air to the medium, and anti- foam was added to reduce foaming of the medium. After 12 hours of incubation, when the Brix reading was reduced to 16 , and the yeast cells are used in the same manner as the bacterial cells.
- the fermentation vessel to which the bacter- ial cells and yeast cells had been added, was filled up to a volume of 650,000 L with the same molasses mixture as was used in the growth medium but without addition of diammonium phosphate.
- The. fermentation mixture as constituted gave a Brix reading of 24.5° r a pH of 5.6, and was maintained at a temperature of 32°C. After 24 hours of fermentation r the fermentation mixture gave a Brix reading of 9 and an ethanol concentration of 9.24% (v/v) before distillation. Thus conversion efficiency of the sugars to ethanol was 91.0%.
- a parallel vessel containing only yeast cells required 36-45 hours for the same decrease in Brix reading. With yeast alone, the maximum substrate-to-ethanol conversion efficiency is in the range of 86%. Under the same conditions, Zymomonas would not sustain fermentation at all.
- EXAMPLE 2 EXAMPLE 2:
- Fermentation of molasses using a mixture of Zymomonas mobilis and yeast cells wherein the total sugar content of molasses and the concentration of sucrose, fructose and glucose are about 20%, 9.9%, 1.5% and 1.1%, respectively.
- sucrose Conversion of sucrose to ethanol by fermenta ⁇ tion using a mixture of Zymomonas mobilis and yeast cells, wherein the concentration of sucrose is about
- Zymomonas mobilis and Saccharomyces uvarum were separately propagated under microaerophilic con ⁇ ditions in a medium containing 5% glucose, 1.0% sucrose, 0.3% yeast extract, and 0.2% of each of the following: peptone, potassium dihydrogen phosphate, magnesium sulfate, and ammonium sulfate, at temperatures between 25°C and 37°C, After 12-24 hours, 240 L of the • Zymomonas culture, 50 mL of the Saccharomyces culture, and 2700 mL of a fermentation medium were added to a 3 L f rmentation vessel .
- the fermentation medium comprised 180 g/L sucrose, 3.0 g/L yeast extract, 3.0 g/L peptone, and 2.0 g/L each of potassium dihydrogen phosphate, ammonium sulfate and magnesium sulfate.
- the pH of the initial fermentatoin medium was adjusted to 6.0 by addition of a 2M sodium hydroxide solution. As fermentation progressed, the pH dropped to 4.5 and was thereafter maintained at 4.5 again by addition of 2M sodium hydroxide. Fermentation was carried out at a temperature of 35 C with stirring, at a rate of 60 rpm .
- sucrose Conversion of sucrose to ethanol by fermenta ⁇ tion using yeast cells alone, wherein the concentration of sucrose is about 18%.
- Saccharomyces uvarum was propagated under microaerophilic conditions in a medium as in Example 3 above. After 12-24 hours, 100 mL of the Saccharomyces culture, and 900 mL of a fermentation medium were added to a 1 L fermentation vessel.
- the fermentation medium comprised 180 g/L sucrose, 3.0 g/L yeast extract, 3.0 g/L peptone, and 2.0 g/L each of potassium dihydrogen phosphate, ammonium sulfate and magnesium sulfate.
- the pH of the initial fermentation medium was . adjusted to 6.0 by addition of a 2M sodium hydroxide solution. As fermentation progressed, the pH dropped to 4.5 and was thereafter maintained at 4.5. Fermentation was carried out at a temperature of 35°C without aeration, but with stirring, at a rate of 60 rpm.
- sucrose Conversion of sucrose to ethanol by fermenta ⁇ tion using Zymomonas mobilis alone, where the concen- tration of sucrose is about 18%.
- Zymomonas mobilis was propagated under micro ⁇ aerophilic conditions in a medium as in Example 3 above. After 12-24 hours, 300 mL consumof the Zymomonas culture, and 2700 mL of a fermentation medium were added to a 3 L fermentation vessel .
- the fermentation medium comprised 180 g/L sucrose, 3.0 g/L yeast extract, 3.0 g/L peptone, and 2.0 g/L each of potassium dihydrogen phosphate, ammonium sulfate and magnesium sulfate.
- the pH of the initial fermentation medium was adjusted to 6.0 by addition of a 2M sodium hydroxide solution. As fermentation progressed, the pH dropped to 4.5 and was thereafter maintained at 4.5. Fermenta ⁇ tion was carried out at a temperature of 35°C without aeration, but with stirring, at a rate of 60 rpm. After 24 hours, utilization of sugar was complete and ethanol yield was 80.67 g/L, or 10.22% (v/v), giving a conversion efficiency of 83.5%.
- EXAM LE 6 EXAM LE 6 :
- Zymomonas mobilis was propagated in a medium containing 10% sucrose or glucose, 0.2% each of yeast extract, peptone, potassium dihydrogen phosphate, magnesium sulfate, and ammonium sulfate, at 37 C for 12 hours.
- a volume of 200 mL of the Zymomonas culture, 200 mL of a Saccharomyces culture and a volume of the enzyme amyloglucosidase equivalent to 0.32 GU/g of starch (dry weight ) were added to a 10 L fermentation vessel, and the total volume of the fermentative mixture was made up to 4 L by the addition of a fermentation medium.
- the pH of the initial fermentation medium was adjusted to 4.5. Fermentation was carried out at a tteemmpperature of 32 C, with stirring, at a rate of 60 rpm.
- EXAMPLE 7 Conversion of liquefied starch to ethanol by fermentation using yeast cells, in the presence of amyloglucosidase, where the glucose equivalent is about 18.2% .
- a 50% dilution of a logarithmic phase culture of Saccharomyces, i.e. a 5% (v/v) cell suspension, and a volume of the enzyme, amyloglucosi ⁇ dase, equivalent to 0.32 GU/g of starch were added to a 10 L fermentation vessel, and the total volume of the fermentative mixture was made up to 4 L by the addition of a fermentation medium.
- the fermentation medium comprised liquefied starch and steep water liquor as in Example 6 above.
- the pH of the initial fermentation medium was adjusted to 4.5. Fermentation was carried out at a temperature of 32 C, with stirring, at a rate of 60 rpm.
- Fermentation was carried out as in Example 6 except that the inoculum of Zymomonas was 20% (v/v) of a logarithmic phase culture of the bacteria, and the inoculum of yeast was 1% (v/v) of a logarithmic phase culture of the yeast, and the liquefied starch contained a glucose equivalent of 173.9 g/L. After 36 hours, fermentation was complete and ethanol yield was 96.31 g/L or 12.19% (v/v), giving a conversion efficiency of 99.43%.
- the present invention provides, for the first time, a method for conversion of sucrose and other fermentable carbohydrates in high salt concentration, e.g., molasses, to ethanol by fermentation using a mixture of yeast and bacterial cells .
- the method generally operates at a higher temperature, and at a lower pH, and requires a much shorter fermentation time than that of yeast of Zymomonas alone.
- the resulting ethanol contains little or no fusel oils, thus making it an attractive candidate for use in automobiles which run on ethanol .
- the method of the present invention is parti ⁇ cularly suitable for fermentation of high test molasses because the yeast present in the yeast/Zymomonas cell mixture is able to ferment residuary sucrose, which is not utilized by Zymomonas .
- the present invention is also well-suited for fermentation of liquefied starch and high test molasses. It has been observed that the ratio of yeast to Zymomonas does not change during the course of fermentation. Therefore, the mixture of cells in the fermentative combination can be reused for subsequent fermentations.
- the fermentative mixture can be pumped off to harvest the ethanol produced, and the remaining 10-20% can act as inoculum for the next batch of fermentation.
- the yeast and Zymomonas can be separated from the fermentation medium, e.g., by centrifugation, and the cells can be recycled for use in the next batch of fermentation.
- Two or more strains of Zymomonas and two or more strains of yeast can be used in the fermentative mixture, according to the present invention, to maximise the yield of ethanol from a particular substrate.
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Abstract
A method for converting fermentable carbohydrates to ethanol in a high-salt environment employs a mixture of yeast cells and bacterial cells of the genus Zymomonas as biocatalysts.
Description
Title : "CONVERSION OF FERMENTABLE CARBOHYDRATES TO ETHANOL USING MIXED CULTURES OF ZYMOMONAS MOBILIS AND YEAST" BACKGROUND OF THE INVENTION The present invention relates to a method for converting fermentable carbohydrates in a high salt medium by the use, under microaerophilic or anaerobic conditions, of a mixture consisting essentially of a Zymo onas bacterial species and a yeast, with or without cell recycling.
Most of the known methods for the conversion of fermentable carbohydrates to ethanol involve the use of a single strain of yeast or fermentative bacteria. Ethanol production utilizing yeast cells, for example, is often carried out in a two-stage batch process, where the term "batch" is us.ed to specify that the cells are not fixed to any surfaces or otherwise immobilized. In this type of fermentation, the first "growth" or "propagation" stage involves propagation of yeast in an aerobic environment, and the second "fermentation" stage involves fermentation of fermentable substrates in a microaerophilic or an anaerobic environment, i.e., in the presence of a small amount of oxygen or in the total absence of oxygen. One disadvantage of the traditional process of yeast fermentation is the long fermentation time. "Fermentation time" is used here to denote the period of time required for complete conversion of substrate to end-products, or for the biocatalyst to reach the stage of maximum yield of end-products. A fermentation time of 30-70 hours has been found to be required for con¬ version of glucose to ethanol, using yeast cells. The rate-limiting factors in this process are the rates of glucose uptake and ethanol production by individual yeast cells. These rates are stringently regulated by
the cell's enzyme control systems, e.g., by the enzymes invertase and hexokinase.
One method devised to increase the yield of ethanol is to use a larger inoculum of yeast cells , i.e., a larger biomass density, on the order of 5-10 million cells per millilitre (mL) . This method still requires 30-50 hours of fermentation time. Reduction in fermentation time to the range of 10-30 hours has been achieved by using 80-100 times higher biomass density. This higher biomass density is achieved by recycling cells previously used in another fermentation. But the expediency of higher biomass density requires an adequate supply of nitrogen and phosphorus, and often sulfur and magnesium as well, which supplies are often limited.
Another disadvantage of the yeast fermentation process, especially when cost is a major concern, is the diversion of fermentable substrate to the production of" by-products such as glycerol, amylalcohol and other fusel oils. As a result, the efficiency of conversion of substrate to end-product, e.g., from glucose to ethanol, is lower than desirable, i.e., only between 85 and 91%.
Attempts to increase the yield of ethanol from sugar fermentation has also been limited by the sensi¬ tivity of yeast cells to high concentrations of both the sugar substrate ("substrate inhibition") and the ethanol end-product ("end-product inhibition"). Substrate inhibition results from an osmotic effect of the substrate on the cells, and is reflected in reduced water activity inside the cell, plasmolysis, and decreased viability. End-product inhibition results from either ' a high solubility of cell membranes in ethanol, or the effect of ethanol on cells' internal regulatory mechanisms. In either case, there is
inhibition of cell growth or inhibition of fermentation.
To overcome the problem of substrate and end- product inhibition, Jones and Greenfield (1981) used a mixture of an "ethanol-tolerant " yeast and a "sugar- tolerant" yeast in a batch fermentation system which employed glucose as the fermentation substrate. They found that the performance of the mixture of cells was roughly equivalent to a weighted sum of the performance of the individual components, i.e., there was no effect over and above the sum of the individual performance of the two yeast types separately.
A mixture of two types of yeasts was also used as biocatalysts by Fukushima and Hatakeyama (1987), in a study involving continuous fermentation of cane sugar. The mixture of cells was "immobilized", i.e., the cells were entrapped in aluminium alginate gel particles . The researchers found that the immobilized/mixed-cell system, comprising of wine yeast and alcohol yeast, produced 15% more ethanol than either immobilized yeast cells alone.
As an alternative to yeast, bacteria of the genus Zymomonas have been used in the conversion of fermentable carbohydrates to ethanol But several known advantages to employing Zymomonas in this regard -- the absence of an oxygen requirement in both the propagation and fermentation stages, a shorter fermentation time (typically, in the range of 15 to 24 hours), higher substrate-to-ethanol conversion efficiency -- are countered, at least in part, by a sensitivity of exist- ing Zymomonas strains to high salt environments, such as exist in molasses, as well as to high substrate and end-product concentrations .
Amin et al (1983) found that a higher concen¬ tration of ethanol could be produced, via fermentation of glucose in a simple medium, by using a combination
of yeast cells of the species S. bayanus and Zymomonas mobilis bacterial cells, relative to amounts produced by either yeast or bacteria alone. There .was no suggestion by Amin et al, however, that the more complex fermentable mixtures commonly employed in commercial ethanol production, which mixtures comprise a variety of different carbohydrates as well as salts in concen¬ trations potentially inhibitory to yeast and/or Z. mobilis bacteria, could be accommodated using a mixture of yeast and bacterial cells. SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a method for conversion of fer¬ mentable carbohydrates in a high salt medium to ethanol, using a mixture of Zymomonas cells and yeast cells .
It is also an object of the present invention to provide a method for production of ethanol, using a mixture of bacterial and yeast cells as biocatalysts, from complex mixtures of carbohydrates, including sucrose and sucrose-based compounds , in the presence of a high salt content .
In accomplishing these and other objects, there has been provided, in accordance with one aspect of the present invention, a method for converting a fermentable carbohydrate to ethanol, comprising the step of bringing a fermentable mixture into contact with a mixture of cells comprised of yeast cells and Zymomonas bacterial cells-, such that ethanol is produced by the action of the mixture of cells on the fermentable mixture, where the fermentable mixture comprises at least one fermentable carbohydrate and has a total salt content in excess of 3% . In a preferred embodiment, the bacterial cells used in the method are of the species Z. mobilis . Other objects, features and advantages of the
present invention will become apparent from the follow¬ ing detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS In a fermentation environment characterized by high salt and sucrose concentrations, both inhibitory of growth and fermentation by Zymomonas , the long fermen¬ tation time (at least 30 hours) of yeast cells would militate against a reduction of sucrose levels, by the action of yeast fermentation, to a level tolerable to the bacterial cells in a mixture comprising of yeast and Zymomonas cells. Therefore, the yeast/Zymomonas mixture would not have been expected to be more effic¬ ient in converting sucrose to ethanol than yeast alone. Moreover, the presence of a high concentration of salt in the fermentation medium would have been expected to inhibit bacterial-based fermentation activity irrespect¬ ive of sucrose levels in the medium.
It has been discovered, nevertheless, that a mixture of yeast and Zymomonas bacterial cells can, at a high conversion efficiency, convert fermentable carbo¬ hydrates, including sucrose and compounds comprising sucrose moieties, to ethanol in a high salt environment. In many of the complex fermentable mixtures used for the industrial production of ethanol, the total content of salts, e.g., salts of sodium, potassium and magnesium, ammonium salts, sulfates, chlorides, and phosphates, can be in excess of 3% (w/v), when both yeast and Zymomonas are inhibited. By a mechanism not completely under- stood, however, the high salt sensitivity of Zymomonas ,
as well as the above-mentioned sucrose intolerance, is largely obviated in a mixture consisting essentially of Zymomonas and yeas.t cells.
For example, molasses is a complex fermentable mixture obtained as a residue after heating cane sugar juice and crystallizing from it a high quality sugar. The resulting mixture comprises the fermentable sugars sucrose, glucose and fructose. By the process of con¬ centrating and crystallizing the high quality sugar, however, salts are concentrated in the residue, which detracts from the utility of the mixture in ethanol production based on yeast or Zymomonas alone . The same drawback has applied heretofore to the other complex fermentable mixtures, such a liquefied starch, high test molasses and, in certain instances, starch hydrolysates. But with the present invention, these high-salt, diverse-carbohydrate mixtures can provide the basis for high-efficiency conversion of carbohydrates to ethanol . Any yeast that is capable of converting fer- mentable carbohydrates to ethanol can be used in the present invention. The choice of a particular strain of yeast depends on the fermentable substrate to be used in the fermentation. A yeast that can utilize those fer¬ mentable substrates not metabolized by the Zymomonas employed is preferred. The following yeasts are suit¬ able for use, according to the present invention: Saccharomyces species, Kluyveromyces species , Pachysolen tannopholus and Candida lusitaniae. Preferred strains of Saccharomyces uvarum and Saccharo- myces cerevisiae have been deposited in the Culture
Collection of the University of Queensland, Department of Microbiology, St. Lucia, Queensland, 4067, Australia, under Deposit Nos . 61Y and 66Y for Saccharomyces uvarum, and Deposit No . 70Y for Saccharomyces cerevisiae. Any Zymomonas that is capable of converting
fermentable carbohydrates to ethanol can be used in the present invention. Those suitable to be used according to the. present invention includes those currently classified taxonomically as Z. mobilis and Z. anaerobia, although there is, at this time, much uncertainty over the taxonomical classification of Zymomonas species. Particularly preferred are strains of the species Z. mobilis , as exemplified by three strains that have been deposited in the Culture Collection of the University of Queensland, Microbiology Department, St. Lucia, Queensland, 4067, Australia, under Deposit Nos . UQM 2716, UQM 2841 and UQM 2864 and also in the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. on 24th April, 1984, under Deposit No. 39676, and on 17th January, 1986, under Deposit Nos. 53432 and 53431 respectively.
It is preferred that the yeast cells and the bacterial cells to be used according to the present invention are propagated separately, in suitable growth media, to obtain the initial inocula for the use in the fermentation process. As soon as both yeast and bacter¬ ial cells reach a logarithmic phase of growth, they are mixed in a fixed proportion, preferably in the ratio 1:1, 1:5, or 5:1, of yeast cells :Zymomonas mobilis cells (v:v ) .
A complex fermentable mixture that is suitable for use in the present invention can contain any of the range of fermentable carbohydrates that can be used as substrates by yeast and Zymomonas, respectively. Substrates of this sort include, but are not limited to, sucrose, fructose, glucose and maltose. These and other sugars are found in. complex fermentable mixtures like molasses, high test molasses and liquefied starch. As previously mentioned, these mixtures have a high salt content, as do starch-hydrolysate mixtures when they are
subjected to recycling. By the same token, sugar cane juice or syrup, sugar beet juice or syrup, raw sugar and refined sugar can also have high salt contents when stillage of distillaries or steep liquor water is added to provide nutrient or diluting water .
In the present description, the phrase "stillage from distilleries" denotes a medium recycled from a prior fermentation in which there is no ethanol and little or no fermentable sugars. "Steep liquor water" is a by-product of a wet-milling process that is used, in the starch manufacturing industry, to extract starch from plant materials. "High test molasses" denotes molasses to which an enzyme, invertase, has been added to convert some of the sucrose therein to glucose and fructose. "Liquefied starch" denotes a complex mixture of maltrin,, dextrin, starch, lipids and proteins obtained from wet or dry milling of starch-containing plant and root materials , the product of which is treated with physical agents (high temperature and pressure), with chemical agents (to effect acid hydro¬ lysis) or with alpha-amylase, to lower viscosity and thereby allow the use of a higher concentration in the fermentation mixture. "Starch hydrolysates" are complex mixtures obtained treating liquefied starch with amylo- glucosidase; they contain, as major components, glucose, maltrin, dextrin, maltose, lipids and proteins.
It is preferred that the concentration of sucrose, when it is used as a fermentable substrate according to the present invention, should be between 10 and 30% (w/v), preferably between 15 and 25%, and optimally at 18%.
A suitable fermentation medium in this context is any suitable growth medium comprising at least one fermentable substrate and at least one of the following: yeast extract, peptone (i.e., casein hydrolysate) ,
dia monium phosphate, ammonium sulfate, magnesium sulfate, potassium dihydrogen phosphate, ammonium hydroxide, and urea.
When liquefied starch is used as a fermentable substrate, the concentration of the starch is such as to yield a glucose equivalent of between 10 and 25%, preferably, between 15 and 20%, and optimally 18%. In such a case, the fermentation medium preferably com¬ prises at least one of the following components: peptone (casein hydrolysate), yeast extract, potassium dihydrogen phosphate, ammonium sulfate, ammonium hydroxide, urea, magnesium sulfate or dia monium phos¬ phate. Each component is provided in a concentration between 0.01 and 0.05% (w/v), but preferably between 0.05 and 0.5% (w/v). Sugar cane juice, sugar beet juice, molasses or stillage from distilleries^ known to comprise one or more of the above described components may be used as a-source of that component. Stillage from distilleries can be used in the present invention a concentration between 1.5 and 7.5% dry solids.
When refined sugar or raw sugar is used as fermentable substrate, the fermentation medium prefer¬ ably comprises at least one of the following components: yeast extract, peptone (casein hydrolysate), potassium dihydrogen phosphate, ammonium sulfate, urea, and magnesium sulfate. Preferably the above-described com¬ ponents are provided in a concentration between 0.01 and 0.5% each, the most optimum concentration being 0.2% each. Sugar cane juice, sugar beet juice or molasses known to comprise one or more of the above described components may be used as a source of those components.
When sugar cane juice or syrup, sugar beet juice or syrup, or molasses is used as a fermentable substrate, no supplementation of the fermentation medium is necessary. When molasses is used as a fermentable
substrate, on the other hand, stillage from distilleries may be used as a diluent or as source of nutrients .
The fermentation medium may be supplemented with at least one of the following antibiotics to control secondary bacterial or yeast contamination: sorbic acid, penicillan V-acid, trimethoprim, strepto¬ mycin, neomycin, nalidixic acid, ampicillan, bacitracin, gentamicin, kanamycin, lincomycin, and polymyxin. The pH of the fermentation medium may be controlled or adjusted with sodium hydroxide, potassium hydroxide, ammonium hydroxide, ammonia or sulfuric acid, but preferably no pH control is performed. When a starch-based material is used as a fermentable sub¬ strate, the pH of the fermentation medium is maintained between 3.5 and 7.0. In particular, the pH of the fermentation medium initially is adjusted to between 4..*5 and 7.0, and preferably "between 3.9 and 5.0, except when starch .hydrolysate is used, as a fermentable sub¬ strate, in which case no pH adjustment is required initially.
When steep liquor water is used as a fermen¬ table substrate, the pH of the medium may be adjusted to between 4.3 and 5.0r and preferably between 4.3 and 4.8. The pH of the fermentation-medium may be adjusted by addition of sodium hydroxide or other suitable alkali. After the initial adjustment, fermentation can proceed without additional pH adjustment as the pH is maintained by the natural buffering action of the mixture.
When a sucrose-based material is used as a fermentable substrate, the pH of the fermentation medium is preferably maintained between 3.5 and 7.0, and optimally between 4.5 and 7.0. After 1-2 hours of fermentation the pH typically drops, and later stabi¬ lizes at between 3.7 and 4.7. The pH of the fermenta- tion medium may be adjusted by addition of sodium
hydroxide or other suitable alkali .
The fermentation within the present invention proceeds in the presence of a mixture of yeast cells and Zymomonas cells in either a batch system or an immobilized cell system. Suitable batch systems in this regard include a single-stage batch system, a fed-batch system, a continuous-fermenter train system and a multi¬ stage continuous batch system.
A single-stage batch system involves a one- step process in which the biocatalysts are added to a fermentable mixture in a fermentation vessel and allowed to ferment, after which the ethanol is separated from the fermentable mixture and the biocatalysts are dis¬ carded. In a fed-batch system, an aliquot of the fer- mentative mixture in a fermentation vessel is removed at intervals during the fermentation process and is replaced by fresh fermentative mixture and αutrients, with or without the additional biocatalysts. Fermentation in a continuous-fermenter train system involves continuous addition of fresh fermentable mixture and nutrients, with or without additional bio¬ catalysts, to a fermentation vessel, which is serially connected to other vessels, such that overflow from the first vessel flows into the second vessel, and so forth. A multi-stage continuous batch system also has multiple vessels, but only nutrients, not fermentable substrates, are continuously added to one or more of the vessels. In an immobilized cell system, the biocatalysts are entrapped, e.g., in calcium or aluminium alginate gel particles.
In each of these systems, the cells used in the fermentation process according to the present invention can be recycled by centrifugation, filtration, flocculation, or coprecipitation with inert materials. For example, the recycled cells can be added to the
fermentation mixture at different points in the multi¬ stage batch system.
Fermentation may be allowed to proceed .at a temperature of between 25 C and 40 C, with a constant temperature control between 32 C and 35 C being preferred. When a sucrose-based material is used as substrate the temperature is maintained between 30°C and 40°C, preferably between 34°C and 37°C.
Addition of the enzyme amyloglucosidase is required when liquefied starch is used as a fermentable substrate. This enzyme is used at a concentration of between 0.01 and 1.0%, preferably between 0.1 and 0.5%, or at an activity of 0.32 and 1.0 GU/g starch, and preferably between 0.32 and 0.7 GU/g starch, where "GU" denotes glucose-units, and 1 Gϋ denotes the release of 1 g of glucose per #hour from starch.
Preferably when fermentation has been com¬ pleted, the mixture of yeast and bacterial cells are separated from the fermentation medium, and the ethanol is distilled. The cells may be separated e.g., by filtration or centrifugation, and recycled or reused with fresh fermentable substrate and medium. If the mixture of cells are not recycled, then 20% of the fermentation mixture is saved and used as inoculum for the next fermentation process .
Preferred embodiments of the invention are further described by reference to the following Examples, which are illustrative only and in no sense limiting. Except where indicated otherwise, reported percentages are expressed in weight per volume (w/v), where 1% corresponds to 1 gram per 100 mL, or 10 g/L. EXAMPLE 1 :
Fermentation of molasses using a mixture of Zymomonas mobilis and yeast cells, in 1:1 ratio (v:v), wherein the total sugar content of molasses and the
concentrations of sucrose, fructose and glucose are about 24.5%, 10.7%, 2.7% and 1.9%, respectively.
Zymomonas mobilis bacteria (ATCC 39676) and Saccharomyces cells were propagated independently in two separate 50,000 L prefermenters . For propagation of the bacteria, an inoculum consisting of 2,000 L of a Zymomonas culture was added to a growth medium compris¬ ing: (a) a mixture of B and C molasses in a ratio of 4:1, which exhibited a Brix reading of 20 , and (b) 80 kg of diammonium phosphate. (B and C molasses are the residues remaining after a first and second cry¬ stallization of sugar, respectively, in the commercial production of sugar from cane sugar juice.) The pH of this medium initially was 5.3. After 12 hours of incubation, when the Brix reading was reduced to 12 , the cells were ready for the fermentation stage, and, approximately 45 ,'000 L of this Zymomonas culture was inoculated into a 750,000 L fermentation vessel which had a working volume of 650,000 L. (The remaining 5,000 L of Zymomonas cells could be used as an inoculum for the next prefermenter . ) Propagation of the bacter¬ ial cells was carried out without any agitation.
The yeast cells were propagated in the same growth medium as used for propagation of the bacteria, except, 250 kg of diammonium phosphate was used, instead of 80 kg, in the growth medium. The pH of this medium initially was 5.3, but was lowered to 4.0 by the addition of sulf ric acid. Agitation of the medium was accomplished by addition of air to the medium, and anti- foam was added to reduce foaming of the medium. After 12 hours of incubation, when the Brix reading was reduced to 16 , and the yeast cells are used in the same manner as the bacterial cells.
The fermentation vessel, to which the bacter- ial cells and yeast cells had been added, was filled up
to a volume of 650,000 L with the same molasses mixture as was used in the growth medium but without addition of diammonium phosphate. The. fermentation mixture as constituted gave a Brix reading of 24.5°r a pH of 5.6, and was maintained at a temperature of 32°C. After 24 hours of fermentation r the fermentation mixture gave a Brix reading of 9 and an ethanol concentration of 9.24% (v/v) before distillation. Thus conversion efficiency of the sugars to ethanol was 91.0%. At the same time, a parallel vessel containing only yeast cells required 36-45 hours for the same decrease in Brix reading. With yeast alone, the maximum substrate-to-ethanol conversion efficiency is in the range of 86%. Under the same conditions, Zymomonas would not sustain fermentation at all. EXAMPLE 2:
Fermentation of molasses using a mixture of Zymomonas mobilis and yeast cells, wherein the total sugar content of molasses and the concentration of sucrose, fructose and glucose are about 20%, 9.9%, 1.5% and 1.1%, respectively.
The growth and fermentation conditions in this Example were the same as in Example 1, except that the initial Brix reading of the fermentation mixture was 20 instead of 24.5 . After 24 hours, ethanol yield was 8.24% and conversion efficiency was 98.4%. EXAMPLE 3 :
Conversion of sucrose to ethanol by fermenta¬ tion using a mixture of Zymomonas mobilis and yeast cells, wherein the concentration of sucrose is about
Zymomonas mobilis and Saccharomyces uvarum were separately propagated under microaerophilic con¬ ditions in a medium containing 5% glucose, 1.0% sucrose, 0.3% yeast extract, and 0.2% of each of the following:
peptone, potassium dihydrogen phosphate, magnesium sulfate, and ammonium sulfate, at temperatures between 25°C and 37°C, After 12-24 hours, 240 L of the • Zymomonas culture, 50 mL of the Saccharomyces culture, and 2700 mL of a fermentation medium were added to a 3 L f rmentation vessel . The fermentation medium comprised 180 g/L sucrose, 3.0 g/L yeast extract, 3.0 g/L peptone, and 2.0 g/L each of potassium dihydrogen phosphate, ammonium sulfate and magnesium sulfate. The pH of the initial fermentatoin medium was adjusted to 6.0 by addition of a 2M sodium hydroxide solution. As fermentation progressed, the pH dropped to 4.5 and was thereafter maintained at 4.5 again by addition of 2M sodium hydroxide. Fermentation was carried out at a temperature of 35 C with stirring, at a rate of 60 rpm .
After 24 hours, utilization of sugar was complete and ethanol yield was 90.14 g/L, or 11.42% (v/v), giving a conversion efficiency of 93.2%. EXAMPLE 4:
Conversion of sucrose to ethanol by fermenta¬ tion using yeast cells alone, wherein the concentration of sucrose is about 18%.
Saccharomyces uvarum was propagated under microaerophilic conditions in a medium as in Example 3 above. After 12-24 hours, 100 mL of the Saccharomyces culture, and 900 mL of a fermentation medium were added to a 1 L fermentation vessel. The fermentation medium comprised 180 g/L sucrose, 3.0 g/L yeast extract, 3.0 g/L peptone, and 2.0 g/L each of potassium dihydrogen phosphate, ammonium sulfate and magnesium sulfate.
The pH of the initial fermentation medium was . adjusted to 6.0 by addition of a 2M sodium hydroxide solution. As fermentation progressed, the pH dropped to 4.5 and was thereafter maintained at 4.5.
Fermentation was carried out at a temperature of 35°C without aeration, but with stirring, at a rate of 60 rpm.
After 47-55 hours, utilization of sugar was complete and ethanol yield was 75.8 g/L, or 9.61% (v/v), giving an conversion efficiency of 78.4%. EXAMPLE 5 :
Conversion of sucrose to ethanol by fermenta¬ tion using Zymomonas mobilis alone, where the concen- tration of sucrose is about 18%.
Zymomonas mobilis was propagated under micro¬ aerophilic conditions in a medium as in Example 3 above. After 12-24 hours, 300 mL„of the Zymomonas culture, and 2700 mL of a fermentation medium were added to a 3 L fermentation vessel . The fermentation medium comprised 180 g/L sucrose, 3.0 g/L yeast extract, 3.0 g/L peptone, and 2.0 g/L each of potassium dihydrogen phosphate, ammonium sulfate and magnesium sulfate.
The pH of the initial fermentation medium was adjusted to 6.0 by addition of a 2M sodium hydroxide solution. As fermentation progressed, the pH dropped to 4.5 and was thereafter maintained at 4.5. Fermenta¬ tion was carried out at a temperature of 35°C without aeration, but with stirring, at a rate of 60 rpm. After 24 hours, utilization of sugar was complete and ethanol yield was 80.67 g/L, or 10.22% (v/v), giving a conversion efficiency of 83.5%. EXAM LE 6 :
Conversion of liquefied starch to ethanol by fermentation using a mixture of Zymomonas mobilis and yeast cells, in 1:1 ratio (v:v), in the presence of amyloglucosidase, wherein the glucose equivalent is about 19.6%.
Zymomonas mobilis was propagated in a medium containing 10% sucrose or glucose, 0.2% each of yeast
extract, peptone, potassium dihydrogen phosphate, magnesium sulfate, and ammonium sulfate, at 37 C for 12 hours. A volume of 200 mL of the Zymomonas culture, 200 mL of a Saccharomyces culture and a volume of the enzyme amyloglucosidase equivalent to 0.32 GU/g of starch (dry weight ) were added to a 10 L fermentation vessel, and the total volume of the fermentative mixture was made up to 4 L by the addition of a fermentation medium. The fermentation medium comprised (a) liquefied starch, originally containing 38% dry solids, but diluted to a concentration containing 18% dry solids, which thus yield a glucose equivalent content of 180 x 1.09 = 196.2 g/L = 19.62%, and (b) stillage from distilleries, originally containing 12.5% dry solids, diluted to a concentration containing 2.5 - 3.0% dry solids .
The pH of the initial fermentation medium was adjusted to 4.5. Fermentation was carried out at a tteemmpperature of 32 C, with stirring, at a rate of 60 rpm.
After 36 hours, fermentation was complete and ethanol yield was 12.42% (v/v), giving a conversion efficiency of 97.8%. EXAMPLE 7: Conversion of liquefied starch to ethanol by fermentation using yeast cells, in the presence of amyloglucosidase, where the glucose equivalent is about 18.2% .
200 mL of a 50% dilution of a logarithmic phase culture of Saccharomyces, i.e. a 5% (v/v) cell suspension, and a volume of the enzyme, amyloglucosi¬ dase, equivalent to 0.32 GU/g of starch were added to a 10 L fermentation vessel, and the total volume of the fermentative mixture was made up to 4 L by the addition of a fermentation medium. The fermentation medium
comprised liquefied starch and steep water liquor as in Example 6 above.
The pH of the initial fermentation medium was adjusted to 4.5. Fermentation was carried out at a temperature of 32 C, with stirring, at a rate of 60 rpm.
After 41 hours, fermentation was complete and ethanol yield is 92.18 g/L or 11.61% (v/v), giving a conversion efficiency of 91.17%. EXAMPLE 8:
Conversion of liquefied starch to ethanol by fermentation using a mixture of Zymomonas mobilis and yeast cells, in 20:1 ratio (v:v), in the presence of amyloglucosidase, wherein the glucose equivalent is about 17.4%.
Fermentation was carried out as in Example 6 except that the inoculum of Zymomonas was 20% (v/v) of a logarithmic phase culture of the bacteria, and the inoculum of yeast was 1% (v/v) of a logarithmic phase culture of the yeast, and the liquefied starch contained a glucose equivalent of 173.9 g/L. After 36 hours, fermentation was complete and ethanol yield was 96.31 g/L or 12.19% (v/v), giving a conversion efficiency of 99.43%. The present invention provides, for the first time, a method for conversion of sucrose and other fermentable carbohydrates in high salt concentration, e.g., molasses, to ethanol by fermentation using a mixture of yeast and bacterial cells . The method generally operates at a higher temperature, and at a lower pH, and requires a much shorter fermentation time than that of yeast of Zymomonas alone. As a result of the high efficiency of conversion of substrate to ethanol obtained by this method, the resulting ethanol contains little or no fusel oils, thus making it an
attractive candidate for use in automobiles which run on ethanol .
The method of the present invention is parti¬ cularly suitable for fermentation of high test molasses because the yeast present in the yeast/Zymomonas cell mixture is able to ferment residuary sucrose, which is not utilized by Zymomonas . The present invention is also well-suited for fermentation of liquefied starch and high test molasses. It has been observed that the ratio of yeast to Zymomonas does not change during the course of fermentation. Therefore, the mixture of cells in the fermentative combination can be reused for subsequent fermentations. For example, at the end of a fermenta- tion step, 80-90% of the fermentative mixture can be pumped off to harvest the ethanol produced, and the remaining 10-20% can act as inoculum for the next batch of fermentation. Alternatively, the yeast and Zymomonas can be separated from the fermentation medium, e.g., by centrifugation, and the cells can be recycled for use in the next batch of fermentation.
Two or more strains of Zymomonas and two or more strains of yeast can be used in the fermentative mixture, according to the present invention, to maximise the yield of ethanol from a particular substrate.
REFERENCΞS
(1) Amin et al , "Determination of by-products formed during the ethanolic fermentation using batch and immobilized cell systems of Zymomonas mobilis and Saccharomyces bayanus" , Eur. J. Appl . Microbiol . biotechnol. 18: 1-5 (1983)
(2) Fukushima & Hatakeyama, "Rapid continuous alcohol fermentation of cane juice in a novel bioreactor packed with immobilized mixed cells", in INTERNATIONAL CONFERENCE ON BIOMASS (Applied Science Publ. 1982).
(3) Jones & Greenfield, "Batch ethanol production with dual organisms", Biotech. Letters 3: 225-30 (1981)
Claims
1. A method for converting a fermentable carbo¬ hydrate to ethanol, comprising the step of bringing a fermentable mixture into contact with a mixture of cells comprised of yeast cells and Zymomonas bacterial cells, such that ethanol is produced by the action of said mixture of cells on said fermentable mixture, wherein said fermentable mixture comprises at least one fermentable carbohydrate and has a total salt content in excess of 3% .
2. A method according to Claim 1, wherein said fermentable carbohdyrate comprises at least one of sucrose, fructose, glucose and maltose.
3. A method according to Claim 1 , wherein said fermentable mixture comprises liquefied starch.
4. A method according to Claim 1, wherein said fermentable mixture comprises molasses.
5. A method according to Claim 1, wherein said fermentable mixture comprises high test molasses .
6. A method according to Claim 3, wherein said fermentable mixture further comprises amyloglucosidase.
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
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AUPH6974 | 1986-07-17 | ||
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AU730586 | 1986-08-06 | ||
AUPH7305 | 1986-08-06 | ||
AU918986 | 1986-11-27 | ||
AUPH9189 | 1986-11-27 |
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WO1988000616A1 true WO1988000616A1 (en) | 1988-01-28 |
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PATENT ABSTRACTS OF JAPAN, C-192, page 27; & JP,A,58 129 984 (HITACHI ZOSEN K.K.) 3 August 1983 (03.08.83). * |
PATENT ABSTRACTS OF JAPAN, C-192, page 27; & JP,A,58 129 985 (HITACHI ZOSEN K.K.) 3 August 1983 (03.08.83). * |
PATENT ABSTRACTS OF JAPAN, C-192, page 28; & JP,A,58 129 986 (HITACHI ZOSEN K.K.) 3 August 1983 (03.08.83). * |
PATENT ABSTRACTS OF JAPAN, C-192, page 28; & JP,A,58 129 987 (HITACHI ZOSEN K.K.) 3 August 1983 (03.08.83). * |
PATENT ABSTRACTS OF JAPAN, C-192, page 29; & JP,A,58 129 989 (HITACHI ZOSEN K.K.) 3 August 1983 (03.08.83). * |
PATENT ABSTRACTS OF JAPAN, C-281, page 136; & JP,A,60 006 195 (SHINNENRIYOUYU KAIHATSU-GIJUTSU KENNKIYUU KUMIAI) 12 January 1985 (12.01.85). * |
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US4885241A (en) * | 1988-10-13 | 1989-12-05 | University Of Queensland | Ethanol production by zymomonas cultured in yeast-conditioned media |
WO1991007511A1 (en) * | 1988-10-13 | 1991-05-30 | The University Of Queensland | Ethanol production by zymomonas cultured in yeast-conditioned media |
WO2000070072A1 (en) * | 1999-05-17 | 2000-11-23 | Aivars Upenieks | Method of sugar beet complex processing |
WO2002070642A1 (en) * | 2001-03-02 | 2002-09-12 | Natu-Licores, S.L. | Method for obtaining a fermented beverage from fruits and/or other agricultural produce |
CN114540146A (en) * | 2022-03-18 | 2022-05-27 | 宁夏红枸杞产业有限公司 | Medlar fermented wine and brewing method thereof |
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