WO1987006615A1

WO1987006615A1 - Conversion of pre-treated and liquefied starch to ethanol using amyloglucosidase and zymomonas mobilis

Info

Publication number: WO1987006615A1
Application number: PCT/AU1987/000120
Authority: WO
Inventors: Horst Werner Doelle
Original assignee: University Of Queensland
Priority date: 1986-05-01
Filing date: 1987-04-30
Publication date: 1987-11-05
Also published as: JPS63503200A; BR8707684A; EP0307395A4; GB8824935D0; ES2005204A6; KR880701286A; GB2210384A; ZA873166B; EP0307395A1

Abstract

A method for the fermentation of pre-treated and liquefied starch to ethanol where the starch material is saccharified to glucose by the enzyme amyloglucosidase, and the glucose is fermented to ethanol by the micro-organism $i(Zymomonas mobilis) in a single fermentation vessel. The ratio of amyloglucosidase cells to $i(Zymomonas mobilis) cells can be varied so that saccharification and fermentation steps occur at the same rate. The starch concentration is in the range 10 % to 30 % (w/v).

Description

Title: "CONVERSION OF PRE-TREATED AND LIQUEFIED

STARCH TO ETHANOL USING AMYLOGLUCOSIDASE AND ZYMOMONAS MOBILIS" BACKGROUND OF THE INVENTION (1 ) Field of the Invention

THIS INVENTION relates to a method for con¬ verting pre-treated liquefied starch material to ethanol using high efficiency strains of the bacterium Zymomonas mobilis preferable under microaerophilic conditions.

(2 ) Prior Art

The traditional process of ethanol production is carried out in a two-stage batch process using yeast, whereby the first stage involves an aerobic propagation of the yeast referred to as the growth stage and the second stage involves the anaerobic process of ethanol production in the presence or absence of small amounts of oxygen. In order to further propagate yeast during the ethanol producing second stage, a slight addition of air or oxygen is required. The latter is required if the efficiency of the total process is to be increased using the occasional recycling of yeast cells by systems such as sedimentation or centrifugation. Since yeast fermentation is inherently dependent on coupling of growth with rate of ethanol production, to optimise ethanol production the medium must be supplemented with either growth enhancing substances or with finely controlled aeration.

The traditional yeast fermentation process (stage 2) is therefore dependent on large inoculum size of approximately 5 to 10 million cells per mL. The preferred optimal temperature of fermentation is between 30°C and 40°C and heat produced has to be controlled through the use of cooling equipment. The fermentation time for obtaining between 9% and 11% (v/v) ethanol is 40 to 60 hours with stage 2 batch or semi-continuous fermentation train process. The time of this fermen¬ tation can be reduced to 10 hours by increasing the inoculum density by 80-100 fold through cell recycling. Cost efficiencies in starch hydrolysate conversion processes to ethanol, however, prefer the use of a continuous addition of fresh yeast in fermentation train processes using between 3 to 5 fermenters for the stage 2 process to obtain maximal utilization efficiency and 11% (v/v) ethanol.

A second process for ethanol production is known, which utilizes the bacterium Zymomonas mobilis (see European Patent No. 0047641 - George eston Ltd.). This process is also a two-stage process as was des- cribed above for yeast batch fermentation, but the bacterium does not require the addition of air for its growth stage (stage 1), instead an adequate supply of nitrogen is required to keep conditions anaerobic. During the second stage of the process for the product- ion of ethanol, the sugar concentration must never exceed 6% (w/v) and thus requires a stepwise or contin¬ uous addition of a concentrated sugar solution. The preferred temperature is 28°C to 33°C and the preferred pH is 5.5. This process may also require a supply of nitrogen as well as additional nutrients . A third process for ethanol production has been described, which utilizes immobilized yeast or strains of Zymomonas mobilis in a two-stage process, each with a limited amount of sugar (10% (w/v) present (see British Patent No. 2,055,121 - Tanabe Sugaku Co. Ltd. ) .

A fourth process for ethanol production is known, which utilizes Zymomonas mobilis continuously with cell recycle (Australian Patent AU-B-67696/81 ) or utilizes a flocculent Zymomonas mobilis strain under semi-batch cultivation conditions" (Australian Patent AU-B-78199/81 ) . In both cases, the fermentation temper¬ ature was controlled at 30 C and pH at 5.0, the medium contained pure glucose with the addition of 5-10 g/L yeast extract.

In the case of yeast fermentation the examples for carbon source conversion are known to be sucrose, glucose, molasses, sugar cane juice and starch hydro- lysates, whereas in the case of the Zymomonas mobilis fermentation the examples are limited to glucose; in the case of the immobilized cells to glucose and molasses, and for previous patent applications by the present inventor to sucrose, molasses, sugar cane syrup, sugar beet syrup and glucose/fructose mixtures. SUMMARY OF THE PRESENT INVENTION

It is an object of the present invention to provide a method for producing ethanol from starch material such as pre-treated liquefied starch from grain, corn and cassava, using the combined action of the enzyme amyloglucosidase (EC 3.2.1.3) and Zymomonas mobilis in free or immobilized form or a combination of both.

It is a preferred object that the method be effected in a single-stage fermentation process. It is another preferred object that the method be effected in the presence of a fermentation medium wherein the concentration of the starch component is greater than 10% (w/v).

It is a further preferred object of the present invention to provide such a method using single- stage batch fermentations or, if required, adjustments to this culturing method, e.g. fed-batch, semi- continuous fermenter trains, continuous or multi-stage systems, where the energy input is low. Other preferred objects of the present invention will become apparent from the following description.

In a broad aspect, the present invention resides in a method for the production of ethanol from pre-treated liquefied starch material in a fermenter characterized by the steps of:

(a) saccharification of the pre-treated liquefied starch material to glucose by the enzyme amyloglucosidase; and (b) fermenting the glucose to ethanol using the micro-organism zymomonas mobilis in the presence of a fermentation mediu .

"Pre-treated liquefied starch" is a complex mixture of maltrin, dextrin, starch, lipids and proteins obtained from wet or dry milling of starch containing plant materials, the product of which is treated by physical , chemical or enzymatic agents to lower the viscosity of starch and thus allows the use of higher concentrations in subsequent fermentation processes or food industries.

Preferably the saccharification and fermenta¬ tion steps are effected substantially simultaneously in a single-stage process in the same fermentation vessel and the level of amyloglucosidase added is preferably adjusted or controlled so that sufficient glucose is always produced by the saccharification prior or simult¬ aneous to the fermentation to meet the fermentation rate to ethanol demand by Zymomonas mobilis.

A "single-stage process" is defined as a process whereby growth and the production phase occur in the same fermenter vessel. Initiation of the process can be done either by a seed culture containing Zymomonas mobilis added to the fermenter vessel contain¬ ing the fermentation medium or by adding the fermenta- tion medium to the fermenter which contains a portion of the fermented medium from a previous fermentation run, the fermented medium containing Zymomonas mobilis. Preferably the fermentation is effected under microaerophilic conditions . "Microaerophilic conditions" are defined as conditions whereby no gas (oxygen, air nitrogen, etc.) is added to the fermenter and the surface of the fermen¬ tation medium is exposed to atmosphere. The Zymomonas mobilis organism does not require air or oxygen (aerobic) or nitrogen (anaerobic) for growth and pro¬ duction of ethanol, but can tolerate the presence of air on the surface of the fermentation medium.

The preferred strains of the micro-organism Zymomonas mobilis have been deposited in the culture collection of the University of Queensland, Micro¬ biology Department, St. Lucia, Queensland, 4067, Australia, under Deposit Nos . UQM 2716, UQM 2841 and UQM 2864, and in the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20952, U.S.A. on 24th April, 1984 and 17th January, 1986 under Deposit Nos. 39676, 53432 and 53431 respectively. The strain UQM 2716 was derived by selection using continuous cultivation techniques from the strain deposited under Deposit No. NCIB 11199 at the National Collection of Industrial Bacteria, Torrey Research

Station, Abbey Road, Aberdeen, AB9 8DG, United Kingdom and under ATCC Deposit 29191 and University of Queensland Deposit No. UQM 2007. The selection was determined on improved performance and metabolic rate of sucrose conversion relative to the parent strain UQM 2007.

The strain UQM 2864 is a fructose utilization negative mutant derived from the strain UQM 2716 and the third strain UQM 2841 is a fructose utilization negative mutant derived from the strain UQM 2007. The strains may be in free or immobilized forms and mutants or variants thereof may also be used.

Preferably the pre-treated liquefied starch is obtained from grain (e.g. wheat, barley, oats, rye, triticale, corn, cassava, arrow root etc.) and may be supplied to the fermenter in solubilized form, filtered or unfiltered, or in combination of any other named substrate. The pre-treatment method applied to the starch material will be dependent on that material. In a typical commercial realization of the present invention, a third party supplier provides the fermenter operator with the pre-treated liquefied starch in the form of a chemically indeterminate ("complex") mixture of products, which is produced by an enzymatic or a non-enzymatic method usually treated as a trade secret by the supplier.

Preferably the starch component should be in the concentration range of 10% to 30% (w/v) with a con¬ centration range of 15% to 20% (w/v) being more pre- ferred for maximum ethanol yield in single-batch fermentation or higher in a continuous feed system.

Preferably the fermentation medium includes any one or more of the following components : amyloglu¬ cosidase (EC 3.2.1.3), peptone (casein hydrolysate ) , yeast extract, potassium (or ammonium or sodium) di- hydrogen phosphate, ammonium sulphate, or ammonium hydroxide or urea, and magnesium sulphate.

Preferably the components, with the exception of amyloglucosidase, are provided in the concentration range of 0.01% to 0.5% each, with approximately 0.2% being preferred. The component amyloglucosidase is preferably provided in the concentration range of 10 to 100 mg/L or 0.32 to 1.0 GPU/g starch, whereby GPU expresses the enzyme activity in glucose producing units (one unit will liberate 1.0 mg glucose from soluble starch in three minutes at pH 4.5 at 55°C). The components yeast extract and peptone (casein hydrol- ysate) can be replaced by calcium pantothenate or beta- alanine. Preferably the abovenamed medium components, with the exception of amyloglucosidase, may be replaced by corn steep liquor, sugar cane juice or syrup or molasses, sugar beet juice or syrup or molasses in appropriate concentrations. Preferably the pH of the fermentation process is within the range of 3.5 to 7.0, with an initial pH of 4.5 to 7.0 and control between 3.9 and 5.0 being preferred. Alternatively, no pH control may be used with the initial pH of the pre-treated liquefied starch/ corn steep liquor mixture usually around pH 4.1 and slight adjustment c-f this natural pH to between 4.3 and 5.0 with the range 4.3 to 4.5 being preferred. Fermentation then proceeds and pH is maintained by the natural buffering action of the mixture. This gives the process a significant economic advantage.

Preferably the temperature in the fermenter is maintained in the range of 25 C to 40 C, with a constant temperature control between 30 C to 35 C being preferred. DETAILED DESCRIPTION OF THE PREFERRED

EMBODIMENTS

To enable the invention to be fully under¬ stood, preferred examples of the method will now be described. EXAMPLE 1

Six hundred (600) g maltrin (DE 10) was solu- bilized in 2 litres of distilled water. After the addition of 75 g corn steep liquor (50% solid content), the mixture was heated to 92 C for 5 to 10 minutes and transferred to a 4 L fermentatin vessel. After cooling, the volume was brought to 2,700 mL with dis¬ tilled water. To this fermentation medium was added an amount of amyloglucosidase equal to 0.64 GPU/g starch. Three hundred (300) mL of a 12 to 24 hour seed culture of Zymomonas mobilis grown in a medium containing 10% (w/v) glucose, 0.2% (w/v) yeast extract, 0.2% (w/v) casein hydrolysate (peptone), 0.2% (w/v) potassium dihydrogen phosphate, 0.2% (w/v) magnesium sulphate, hydrated, 0.2% (w/v) ammonium sulphate at 37°C was added to the fermenter.

The initial pH was brought to 4.8 and pH was controlled at 4.5 by addition of 2N alkaline (e.g. 80 g/L NaOH) . Cultivation was carried out at a temperature of 35 C with a stirring rate of 150 rpm. After 15 hours maximal ethanol production has occurred giving an ethanol concentration of 78 g/L or 9.87% (v/v). EXAMPLE 2

Five hundred (500) g maltrin M-100 was solu- bilized in 2 litres of distilled water, heated at 92 C for 5 to 10 minutes and transferred to a 4 L fermenter vessel. Two hundred (200) mL of a medium is added aseptically containing any one or more of peptone (case¬ in hydrolyste), yeast extract, potassium dihydrogen phosphate, ammonium sulphate or urea or ammonia or ammonium dihydrogen phosphate, and magnesium sulphate, hydrated with each component having a concentration of 0.2% (w/v), whereby peptone and yeast extract can be replaced by calcium pantothenate or the total medium can be replaced by the addition of appropriate amounts of corn steep liquor, sugar cane syrup, molasses or sugar beet syrup.

Three hundred (300) mL of a 12 to 24 hour seed culture of Zymomonas mobilis grown as outlined in Example 2 was added to the fermenter together with an amount of amyloglucosidase equal to 0.64 GPU/g starch. The initial pH was brought to 5.0 and pH was controlled at 4.5 by addition of 2N alkaline (e.g. 80 g/L NaOH) . Cultivation was carried out at a temperature of 35°C with a stirring rate of 150 rpm.

After 15 hours maximal ethanol production has occurred, giving an ethanol concentration of 74 g/L or 9.36% (v/v). EXAMPLE 3 Five hundred and forty (540) g of altrin

M-100 was dissolved in 2 litres of distilled water and heated to 92°C for 10 minutes. In 700 mL, 9 g of yeast extract, 9 g of peptone, and 6 g each of potassium dihydrogen phosphate, magnesium sulphate, hydrated and ammonium sulphate are dissolved. The total medium may be replaced by addition of appropriate amounts of corn steep liquor (1% to 5% by volume) plus starch hydro- lysate, sugar cane syrup, juice or molasses, or sugar beet syrup or molasses. The 2 L of maltrin plus 700 mL of nutrient solution is loaded into a 3 L fermenter and the tempera¬ ture adjusted to 25°C to 38°C, preferably 30°C to 35°C most preferred. Prior to addition of inoculum, the pH is to be within the range 4.5 to 6.5 with 5.0 to 5.5 most preferred.

Three hundred (300) mL of a 12 to 24 hour seed culture of Zymomonas mobilis grown in a medium contain¬ ing 5% to 10% (w/v) glucose with 3 g/L yeast extract, 3 g/L peptone and 2 g/L each of potassium dihydrogen phosphate, hydrated magnesium sulphate and ammonium sulphate, and cultivated at 30 C to 37 C was added to the fermenter (3L) together with 400 mg/L amylogluco¬ sidase (12,000 U/g enzyme).

The initial pH was brought to the range 4.5 to 6.5 with pH 6.0 selected. During the fermentation the pH was controlled at 4.5 by the addition of 2N alkali (e.g. 80 g/L NaOH) . Cultivation was carried out at 35°C with a minimal agitation rate of 60 rpm. After 22 hours maximal ethanol production has occurred giving an ethanol concentration of 89.7 g/L or 11.4% (v/v). The temperature of the fermenter is preferably maintained in the range 28°C to 40°C with 35°C most preferred. EXAMPLE 4 Example 4 was repeated where 300 mg/L amylo¬ glucosidase was added to the fermenter and fermentation was carried out for 18 hours.

The resultant ethanol concentration was 88.6 g/L or 11.2% (v/v) . EXAMPLE 5

Example 4 was repeated using 20 mg/L amylo¬ glucosidase and a fermentation time of 43 hours. The resultant ethanol concentration ws 86.4 g/L or 11.0% (v/v). The ethanol produced has commercial value as a component for gasoline or as a base product in the chemical industry, e.g. for the production of ethylene, while the other by-product, carbon dioxide, may be used for dry ice or as a carbon source for the growth of algae biomass.

The fermentation process required only a low energy input as the micro-organism produces a fair amount of heat during the fermentation process. In addition, the fermentation is carried out in micro- aerophilic conditions, avoiding the need for aerating or addition of nitrogen pumps, the fermentation compo¬ nents and products only requiring little mechanical stirring and pH control.

When the fermentation is completed, the Zymomonas mobilis cells may be separated from the fermentation medium and the ethanol distilled off. Alternatively, a portion of the fermented medium from a preceding fermentation may be added to the fermenter as the inoculum of the Zymomonas mobilis cells for the succeeding fermentation.

Experiments have shown that the success of the fermentation process is not wholly dependent on the quality of the substrate .

The invention is not limited to the specific examples described and various changes and modifications may be made to the Examples without departing from the scope of the present invention defined in the appended claims .

Claims

1. A method for the production of ethanol from starch material in a fermenter characterized by the steps of:

(a) saccharification of the starch material to glucose by the enzyme amyloglucosidase; and

(b) fermenting the glucose to ethanol using the micro-organism Zymomonas mobilis in the presence of a fermentation medium.

2. A method according to Claim 1 characterized in that: the Zymomonas mobilis is a strain deposited in the ATCC under Deposit No. 39676 or a mutant or variant of ATCC 39676.

3. A method according to Claim 1 characterized in that: the Zymomonas mobilis is a strain deposited in the ATCC under Deposit No. 29191 or a mutant or variant of ATCC 29191.

4. A method according to Claim 1 characterized in that: the Zymomonas mobilis is a strain deposited in the ATCC under Deposit No. 53431 or a mutant or variant of ATCC 53431.

5. A method according to Claim 1 characterized in that: the Zymomonas mobilis is a strain deposited in the ATCC under Deposit No. 53432 or a mutant or variant of ATCC 53432.

6. A method according to any one of Claims 1 to 5 characterized in that: the saccharification and fermentation steps are effected simultaneously in a single-stage process in the same fermentation vessel and the number of Zymomonas mobilis cells is adjusted or controlled so that the glucose produced by the saccharification step is immediately fermented to ethanol .

7. A method according to any one of Claims 1 to 6 characterized in that: the fermentation is effected under micro¬ aerophilic conditions .

8. A method according to any one of Claims 1 to 7 characterized in that: the starch hydrolysates are obtained from grain, corn, cassava or arrow root.

9. A method according to Claim 8 characterized in that: the grain comprises wheat, barley, oats, rye or triticale.

10. A method according to Claim 8 or Claim 9 characterized in that: the starch is supplied to the fementer in filtered or unfiltered solubilized form.

11. A method according to any one of Claims 1 to 10 characterized in that: the starch concentration is in the range of 10% to 30% (w/v) .

12. A method according to Claim 11 characterized in that : the starch concentration is in the range of 15% to 20% (w/v) .

13. A method according to any one of Claims 1 to 12 characterized in that: the fermentation medium includes any one or more of the following components: amyloglucosidase (EC 3.2.1.3), peptone (casein hydrolysate ) , yeast extract, potassium (or ammonium or sodium) dihydrogen phosphate, ammonium sulphate, or ammonium hydroxide, or urea and magnesium sulphate.

14. A method according to Claim 13 characterized in that : the concentration of each component, with the exception of amyloglucosidase, is in the range of 0.01% to 8.5% (w/v) .

15. A method according to Claim 14 characterized in that: the concentration of each component, with the exception of amyloglucosidase, is 0.2% (w/v).

16. A method according to Claim 13 characterized in that: the concentration of amyloglucosidase is in the range of 10 to 100 mg/L or 0.32 to 1.0 GPU/g starch.

17. A method according to any one of Claims 13 to

16 characterized in that: yeast extract and peptone are replaced by calcium pantothenate or beta-alanine.

18. A method according to any one of Claims 1 to

17 characterized in that the pH of the fermentation medium is main¬ tained within the range of 3.5 and 7.0.

19. A method according to Claim 18 characterized in that: the pH is in the range of 4.5 to 5.0.

20. A method according to any one of Claims 1 to 19 characterized in that: the temperature is maintained in the range of 34° to 40°C.

21. A method according to Claim 20 characterized in that : the temperature is maintained at 35 C.

22. A method according to any one of Claims 1 to 21 characterized in that: when the fermentation is completed, the Zymomonas mobilis cells are separated from the fermentation medium and the ethanol is distilled off.

23. A method according to any one of Claims 1 to 21 characterized in that: a portion of the fermented m'edium from a preceding fermentation is added to the fermenter as an inoculum for the Zymomonas mobilis cells for the succeeding fermentation.

24. Ethanol produced from starch material by the method according to any one of Claims 1 to 23.