(1 ) Field of the- Invention THIS INVENTION relates to a method for con¬ verting starch hydrolysates to ethanol in a single-stage process using high efficiency strains of the bacterium Zymomonas mobilis 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 requried. 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 con¬ trolled 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 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

fermentation can be reduced to 10 hours by increasing the inoculum density by 80-100 fold through cell recycling. Cost efficiencies in the conversion of starch hydrolysate to ethanol, however, prefer the use of a continuous addition of fresh yeast in fermentation train processes using between 3-5 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). This process may also require the supply of an organic or inorganic source of nitrogen for cell growth as well as additional nutrients and strict anaerobic conditions may require the slow bubbl¬ ing of nitrogen gas into the fermenter. During the second stage of the process for the production of ethanol , the sugar concentration must never exceed 6% (w/v) and thus requires a stepwise or continuous addition of a concentrated sugar solution. The preferred temperature is 28 C to 33 C and the preferred pH is 5.5. (It is not clear from this patent if the slow bubbling of nitrogen gas into the fermenter is also required in the second stage of the process.)

A third process for ethanol production has been described, which utilizes immobilized yeast or strains of Zymomonas 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 -

Unisearch Ltd.) or utilizes a flocculent Zymomonas mobilis strain under semi-batch cultivation conditions (Australian Patent AU-B-78199/81 - Unisearch Ltd.). In both cases, the fermentation temperature was controlled o

- 5 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- 10 lysates, whereas in the case of the Zymomonas fermenta¬ tion 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 15 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 based materials such as starch hydrolysates from grain 20 or root crops and where the purity of the substrate is not vital to the success of the method using the micro-organism Zymomonas mobilis as the fermenting agent .

It is a preferred object that the method be 25 effected in the presence of a fermentation medium where¬ in the concentration of the glucose component is greater than 10% (w/v) .

It is a further preferred object of the present invention to provide such a method using single- 30 stage batch fermentation 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.

¹¹ Other preferred objects of the present

35 invention will become apparent from the following

description.

In a broad aspect the present invention resides in a method for ethanol production by fermentation, characterized by the step of fermenting a starch hydrolysate to ethanol, in a single fermentation medium, using a Zymomonas mobilis strain in the fermentation medium.

The fermentation step may comprise:

(a) saccharification of a starch to glucose by amyloglucosidase action in the fermentation medium, and

(b) production of ethanol from the glucose in the fermentation medium by the action of the strain added to the fermentation medium after the saccharifi- cation.

Alternatively, the strain may act on a starch hydrolysate mixture added to the fermentation medium, whereby ethanol is produced.

"Starch hydrolysates" is a complex mixture obtained from liquified starch by physical, chemical or enzymatic treatment containing as the major component glucose with Maltrin, dextrin, maltose, lipids and protein as minor compounds .

A "single-stage process" is defined as a process whereby growth of the micro-organism and the ethanol 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 containing the fermentation medium or by adding the fermentation 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 condi-

tions" are defined as conditions whereby no gas (oxygen, air, nitrogen, etc.) is added to the fermenter and the surface of the fermentation medium is exposed to atmos¬ phere. The Zymomonas mobilis organism does not require air or oxygen (aerobic) or nitrogen (anaerobic) for growth and production 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, Microbiology 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, 20852, 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 No. 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 glucose is obtained from the starch hydrolysates obtained from grain (e.g.

wheat, barley, oats, rye, triticale, corn) or root crops (e.g. cassava, arrowroot, etc.) and may be supplied to the fermenter in the form of glucose syrup or glucose-fructose mixtures , either filtered or unfiltered, or in combination with any other named substrate .

In a typical commercial realization of the present invention, a third party supplier provides the fermenter operator with the starch hydrolysates in the for of a chemically indeterminate ("complex") mixture of starch hydrolysis products, which mixture is produced by an enzymatic or a non-enzymatic method usually treated as a trade secret by the supplier. (Hitherto, it has not been considered possible to ferment a range of complex mixtures of starch hydrolysates , as distin¬ guished from chemically well-defined "starch material", using Zymomonas mobilis. )

Preferably the glucose 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 preferred for maximum ethanol yield in single-batch fermentation or higher, in a continuous feed system.

In the case of glucose syrup and the sacchari¬ fied starch, the medium may include any one or more of the following components: peptone (casein hydrolysate) yeast extract, potassium dihydrogen phosphate, (KH-PO. ), ammonium sulphate C(NH. )-SO^] or ammonium hydroxide (NH^OH) or urea, and magnesium sulphate (MgS0 ₄ .7H~0) and diammonium phosphate [ (NH. HPO.] . Preferably the components, are provided in the concentration range of 0.01 to 0.5% (w/v) each, with approximately 0.05 to 0.5% being preferred. The components yeast extract and peptone (casein hydrolysate) can be replaced by calcium pantothenate or ^_?-alanine. The abovenamed medium components may be

replaced by the addition of sugar cane juice, sugar beet juice or molasses in appropriate concentrations, or by corn steep liquor in the final concentration range of 1.5 to 7.5% (dry solids). (Steep liquor water is a by-product of the wet-milling processes used in the starch manufacturing industry to force the starch from the plant materials. Depending on the usage, the total dry solid content may vary from 9 to 13% or may be concentrated to 35 to 50% (animal feed 30 to 35% (fermentation nutrient). It contains a mixtu protein, amino acids, carbohydrates, phytic acid, and a number of different minerals in inorganic and organic form. )

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 starch hydrolysate/corn steep liquor mixture usually around pH 4.1 and slight adjustment of 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 Cassava roots were mashed and saccharified

using amylase enzyme (i.e. amyloglucosidase at 60 C. The hydrolyzed mash (2,500 mL) containing 75 g/L glucose was transferred into a 3L fermenter vessel. 200 mL of a medium is added aseptically containing any one or more

5 of peptone (casein hydrolysate), yeast extract potassium dihydrogen phosphate, ammonium sulphate or urea and magnesium sulphate, with each component having a_ concentration of 0.2% whereby peptone and yeast extract can be replaced by calcium pantothenate or the

10 ^" to:tal medium can be replaced by the addition of. appropriate amounts of sugar cane syrup, molasses or sugar beet syrup.

300 mL of a 12-24 hour seed culture of Zymomonas mobilis (ATCC 39676) grown in a medium con-

15 taining 10% (w/v) sucrose or glucose, 0.2% (w/v) yeast extract, 0.2% (w/v) casein hydrolysate, 0.2% KH-PO. , or

0.2% (w/v) MgSo. ^H^O, 0.2 % (w/v) (NH. ) S0„ at 37°C 4 Δ 4 2 4 was added to the fermenter vessel . The initial pH was brought to 7.0 and pH was controlled at 5.5 by

20 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 80 rpm.

After 12 hours maximal ethanol production has occurred giving an ethanol concentration of 36.31 g/L

25. αf. 92% conversion effiency.

If the cassava hydrolysate was centrifuged after saccharification, the conversion efficiency could be increased to at least 95%. EXAMPLE 2

30 330 g of glucose was dissolved in 1.0 L of distilled water and heated to 60 ^~ C for 10 minutes. In 500 mL, 100 g of corn steep liquor (48 to 50% dry sub¬ stance) was dissolved and made up to 800 mL then mixed with the 1.0 L glucose solution. The resulting 1.8L

35 solution was loaded into a 3L fermenter and the

temperature adjusted to 25 to 38 C with 32 C being most preferred. Prior to addition of inoculum the pH is to be within the range 3.9 to 5.5 with 4.2 to 5.0 most preferred. 200 mL of a 12-24 hour seed culture of

Zymomonas mobilis grown in a medium containing 5 to 10% (w/v) glucose with 3 g/L yeast extract, 3 g/L peptone and 2 g/L each of KH PO (NH ) ₂ SO and MgS0 ₄ .7H ₂ 0, and cultivated at 22 to 37°C, was added to the fermenter. Glucose may be replaced by starch hydrolysate; yeast extract, peptone, KfLPO^ (NH SO and MgS0..7_L0 may be replaced by 1.5 to 7.5% (dry substance) corn steep liquor (48 to 50% dry substance corn steep liquor solution) in both the inoculum and main fermentation stages .

The initial jp was brought to 4.5. During the fermentation the pH was controlled at 4.0 to 4.1 by the addition of 1 to 2N alkali (for example 80 g/L sodium hydroxide) with an agitation rate of 60 to 80 rpm. After initial mixing agitation is not required. After 17 hours maximal ethanol production has occurred giving an ethanol concentration of 83.0 g/L or 10.54% (v/v). The temperature of the fermenter is preferably maintained in the range 25 to 39 C with 32 C most preferred. Less than 0.1 g/L glucose remains in the fermenter at maximum ethanol production. EXAMPLE 3

Example 2 was repeated using 165 g glucose and 50 g corn steep liquor (48 to 50% dry substance) in a volume of 1.0 L. A seed culture of 100 mL as described in Example 2 was used to start the fermentation. After 17 hours maximal ethanol production occurred giving an ethanol concentration of 83.7 g/L or 10.63% (v/v). The temperature was maintained at 32 C and pH controlled at 4.1. Less than 0.1 g/L glucose remains at maximal

ethanol production. EXAMPLE 4

Example 2 was repeated using 180 g glucose and 50 g corn steep liquor (48 to 50% dry substance) in a

5 volume of 1.0 L. A seed culture of 100 mL as described in Example 2 was used to start the fermentation. After 23.6 hours maximal ethanol production occurred giving an ethanol concentration of 93.1 g/L or 11.8% (v/v) with 0.6. g/L glucose remaining in the fermenter at maximal

10 ethanol production. EXAMPLE 5

1,500 mL _. of culture medium containing 255 g glucose and 75 g of corn steep liquor (48.8% dry substance) was loaded into a 1.5L fermenter. 300 mL of

15 a 12-24 hour seed culture of Zymomonas mobilis grown at 22 to 30°C in a medium containing 5% (w/v) glucose, 5 g/L yeast extract, 3 g/L peptone and 2 g/L each * KH ₂ P0 ₄ , MgSO 7H ₂ 0 and ( H ₄ ) SO was centrifuged at 2,500 x g, at ambient temperature for 25 minutes. The

20 resulting cell pellet was retained and a portion

(approximately 200 L) of the glucose-corn steep liquor mixture used to resuspend the cells. The cell suspen¬ sion was then added to the 1.5L culture vessel.

The incubation temperature was set to 32°C

25. with agitation at 60 rpm. A temperature range of 25 to 37 C with 30 to 33 C most preferred is suitable. Agitation is not essential. pH control was not required. After 15 'hours the utilization of glucose was complete giving an

30 ethanol concentration of 82.9 g/L or 10.5% (v/v).

In all examples, only a small seed culture of the Zymomonas mobilis was added to the fermenter vessel. Because of the specific conditions in the fermenter vessel , growth of the Zymomonas mobilis and

35 ethanol production occurred simultaneously in a true

single-stage process. This avoided the requirement to first grow the Zymomonas mobilis cells in a fermentation medium (below an ethanol concentration of 6%) to produce a cell suspension, and then transferring the cell sus- pension to a fermenter vessel for the ethanol production stage e.g. as described in European Patent No. 0047641.

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 mobjlis cells for the succeeding fermentation.

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, the fermentation compo- nents and products only requiring little mechanical stirring and pH control. (In some cases, no pH control is required. )

Experiments have shown that the success of the fermentation process is not wholly dependent on the quality of the substrate- since unfiltered hydrolysates have been used.

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.