(2) Prior Art

The sugar industry has become concerned following the announcements by many major food and drink manufacturing companies that they intend replacing sugar (sucrose) with fructose obtained from corn syrup, fructose/sorbitol or fructose/glucose (dextrose) mixtures to cater for the dietary, health and diabetics market. Fructose is nearly twice as sweet as sugar and so only half the amount is required for the same level of sweet- ηess, which reduces the calorific value, an important, aspect of this health-conscious world. In addition, sorbitol and fructose are safe sweeteners for diabetics in contrast to sucrose.

The conversion of corn syrup to fructose is energy-dependent as the corn mash must first be converted to glucose, which is normally carried out using the enzymes amylase and glucoamylase. The glucose must then be further converted to fructose using the enzyme glucose isomerase. This enzymic conversion results in approximately 50/50 mixtures of glucose and fructose. In order to obtain higher fructose values, glucose must be removed by chromatographical methods, which are very expensive and still do not completely remove glucose from the mixture. The production of ethanol from sugar cane is

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well-known and in Brazil the ethanol is mixed with petrol to produce "gasohol", or is solely used as car fuel. In U.S.A., the ethanol is produced mainly from corn and to a lesser degree from sugar cane feedstocks whereby the ethanol is primarily used as octane enhancer in unleaded petrol with the mix being referred to as "super unleaded gasoline".

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 optimize ethanol production the medium must either be supplemented with 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 30 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 30 to 70 hours with stage 2 batch fermentation. The time of this fermentation can be reduced to 10 hours by increasing the inoculum density by 80-100 fold through cell recycling.

i _•—-

A second process for ethanol production is known, which utilizes the bacterium Zymomonas mobilis (see European Patent No. 0047641 - George estori Ltd.). This process is also a two-stage process as was ^■ described above for yeast, 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 anaerobio. During the second stage of the process for the production of ethanol the sugar concentration must never exceed 6% (w/v) and thus the stage 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 about 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 in a two-stage process, each with a limited amount of sugar (i.e. 10% w/v) present (see British Patent No. 2055121 - Tanabe Sugaku Co. Ltd.).

In the case of yeast fermentation the examples for carbon source conversion are known to be sucrose, glucose, molasses and sugar cane juice, whereas in the case of the two-stage process utilizing Zymomonas the examples are limited to glucose, and in the case of the immobilized cells, to glucose and molasses.

Two processes are known to use glucose as substrate for ethanol production. One is a recycling process, whereby after fermentation the biomass is separated from the beer and recycled back to the fermen¬ tation system (see U.S. Patent No. -4,403,034 (Rogers et al _; and Australian Patent Application No. 78199/81 (Unisearch Ltd.)). The second process is a continuous process using the glucose derived from starch hydrolysis for ethanol production. Both processes operate at 30°C

and pH 5.0.

SUMMARY OF THE PRESENT INVENTION It is an object of the present invention to provide a method for producing ethanol in combination with fructose, sorbitol or fructose-sorbitol mixtures from sucrose-based materials (e.g. sugar cane juice or syrup, sugar beet juice or syrup, molasses, palm sugar juice or syrup, raw or refined sugar) and/or glucose-fructose mixtures (e.g. high fructose corn syrup, artificial mixtures, high test molasses or invert sugar solutions).

It is a preferred object of the present invent¬ ion to provide such a method using single-stage batch fermentation or, if required, adjustments to this culturing method, e.g. fed-batch, continuous or multi¬ stage systems, where the energy input is low.

It is a further preferred object to provide a method where the purity of the substrate is not vital to the success of the method. It is still a further preferred object .to provide a method where the ethanol produced can be used as an energy source to maintain the method in-train, and where the production of slime in the fermenter is eli¬ minated, or at least minimized. It is a still further preferred object to produce fructose utilization negative strains of Zymomonas mobilis suitable for the above method.

Other preferred objects of the present inven¬ tion will become apparent from the following description. In one aspect the present invention resides in a method for the production of ethanol in combina¬ tion with fructose and/or sorbitol from a sucrose- based material and/or a glucose-fructose mixture in a fermentation characterized by fermenting the sucrose- based material an /or glucose-fructose mixture with the

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micro-organism Zymomonas mobilis in a single-stage process under microaerophilic conditions wherein the sucrose-based material and/or glucose-fructose mixture is contained in a fermentation medium as the substrate. A "single-stage process" is defined as a process whereby growth and the production phase occur in the same fermentation vessel. Initiation of the process can be done either by a seed culture containing Zymomonas mobilis added to the fermenter vessel con- taining the fermentation medium or by adding the fermentation medium to the fermenter which contains a portion of the fermented medium from a previous fer¬ mentation run, the fermented medium containing Zymomonas mobilis. "Microaerophilic conditions" 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 at.mόsphere. 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 fermenta'tion medium.

The fermentation may be carried out using free or immobilized form of the micro-organism. Sucrose- based materials include sugar cane juice or syrup, sugar beet juice or syrup, molasses, palm sugar juice or syrup, raw or refined sugar. The glucose and/or fructose mixture include high fructose corn syrup, artificial mixtures, high test molasses or invert sugar solutions.

Preferably the base medium includes yeast extract, casein hydrolysate, ammonium sulfate, urea or magnesium sulfate, potassium dihydrogen phosphate and/or glucose. Preferably all the constituents in this base medium are

in the range of 0.01 to 1.0%, more preferably. 0.2 toO.5%

(w/v). Preferably the incubation steps are carried out at approximately 30°C for a period of e.g. 2 to 15 hours. Preferably the fructose and glucose are added to the medium in the range of 0.5 to 3%, more preferably 1 to 2% (w/v).

One preferred parent strain of the micro¬ organism Zymomonas mobilis has been deposited in the Culture Collection of the University of Queensland, Microbiology Department, St. Lucia, Queensland, Australia under Deposit No. UQM 2716 and deposited in the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852, U.S.A. on 24th April, 1984 under Deposit No. 39676. This strain has been developed from the Zymomonas mobilis strain ATCC Deposit No. 29191 which is a second preferred parent strain suitable for use with the present invention.The parentbstrain has also been deposited under Deposit No. NCIB 11199 at the National Collection of Industrial Bacteria, Torry Research Station, Abbey Road, Aberdeen, United Kingdom AB9 8-DG. The development of strain ATCC 39676 was carried out using the chemostat cultivation technique for increased substrate uptake i.e. improved performance and metabolic rate of sucrose conversion and these features are the only difference in the taxonomic des¬ cription of the parent strain ATCC No. 29191 set out at pages 576-580 of "Bergy's Manual of Determinative Bacteriology" (8th Edition) (1975).

Two preferred fructose utilization negative (Fru~) mutant strains of the micro-organism Zymomonas mobilis (E 977 and E 4381) have been deposited in the Culture Collection of the University of Queensland, Microbiology Department, St. Lucia, Queensland, Australia under Deposit No. UQM 2841 (=E 977) and UQM 2864 (=E 4381) and deposited in the American Type Culture

^* i ^■ '. ^' .

Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852, U.S.A. on 17th January, 1986 under Deposit No. 53 32 (UQM 2841) and Deposit No. 53431 (UQM 2864). The fructose utilization negative (Fru ^" ) mutant strains have been developed from the Zymomonas mobilis parent strains ATCC 29191 and 39676, respect¬ ively, by treatment using ethylmethane sulphonate (EMS). The taxonomic description for each strain is as for its parent with the exception of the fructose negative nature and each has a plump gram negative rod. The method of production of the fructose utilization negative strains is set out in Example I.

Preferably the sucrose concentration in the sucrose-based substrates is in the range of 10 to 40% (w/v) and the glucose and fructose concentration in the corresponding mixtures is in the range of 5 to 20% (w/v) of each.

Preferably the fermentation medium includes only one or more of the following components: peptone (caesin hydrolysate) , yeast extract, calcium panjsoth- enate, potassium dihydrogen phosphate, ammonium sulfate, urea, and magnesium sulfate. Preferably the components are provided in the concentration range of 0.01 to 0.5% each with approximately 0.2% being pre- ferred.

Preferably the pH of the fermentation process is maintained in the range of 4.0 to 7.0. Preferably the pH is initially set in the range of 6.5 to 7.0. As the fermentation process proceeds the pH drops and then after e.g. 1-2 hours, the pH is maintained in the range of 5.0 to 6.2. The pH range may be controlled by the addition of NaOH or other suitable alkali.

Preferably the temperature is maintained in the range of 34°C to 4θ°C. This temperature range appears to produce the best growth and product yields

and alleviates or markedly reduces the production of slime in the fermenter.

Preferably when the fermentation has been com¬ pleted, the micro-organism is separated from the fermentation products, the ethanol is distilled off and the product is concentrated or crystallized.

In a second aspect the present invention resides in a method for the production of a fructose utilizat¬ ion negative mutant strain of the micro-organism Zymomonas mobilis comprising mutating a fructose utiliz¬ ation negative strain of Zymomonas mobilis .

In a third aspect the present invention resides in a method for the production of a fructose utilization negative mutant strains of Zymomonas mobilis including the steps of: growing Zymomonas mobilis in a basic medium to form a first cell culture; adding ethylmethane sulphonate and/or nitrosog- uanidine to the first ceil culture and incubating the mixture; removing the cells from the first cell culture suspending the cells in the basic medium and incubating the mixture to generate a second cell culture; harvesting the cells from the second cell culture and suspending the cells in the base medium to generate a third cell culture; and obtaining the fructose utilization negative cells of Zymomonas mobilis by plating the cells of the third cell culture in the base medium combined with glucose and/or fructose.

Preferably at least a portion of the second cell culture is mixed with the basic medium combined with fructose and penicillin and the mixture is incubated to form a new cell culture which is the cell culture which is harvested and suspended in the base medium to gener¬ ate the third cell culture.

-8a-

In a fourth aspect, the present invention resides in fructose utilization negative strains of Zymomonas mobilis produced by the above method. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

i ^s<

-9-

To enable the invention to be fully understood, preferred examples of the invention will now be des¬ cribed. In all Examples, percentages are expressed as "% (w/v)" where 1% corresponds to 10 g/L. EXAMPLE 1: Production of Fructose Utilization Negative

(Fru~) Mutants The following procedure was used to obtain fructose utilization negative strain of Zymomonas mobilis parent strains ATCC 29191 and ATCC 39676 (e.g. the mutant strains Zymomonas mobjlis E 977 and E 4381).

The parent strain was grown in a medium contain¬ ing 0.2% (w/v) yeast extract, 0.2% (w/v) casein hydro- lysate, 0.2% (w/v) ammonium sulfate, 0.2% (w/v) MgSO- _j y 7H ₂ 0, 0.2% (w/v) KH ₂ P0 _jj with 1% (w/v) glucose to approxi- mately 109 cfu/mL, whereby cfu represents colony forming units. Into such a culture was directly added various volumes (0.1 to 0.5 mL) of ethylmethane sulphonate (EMS) and various amounts (60 to 1,000 mg) of nitrosoguanidine (NTG). The mixtures were incubated for 2 hours at 30°C. After centrifugation, the cells were washed in ab.ove basic medium and suspended in 10 mL of above medium. After 18 hours incubation at 30°C, 2 ML of the culture were transferred to 10 mL of the above medium containing 2% (w/v) fructose and 1,000 U/mL penicillin G. After 5 hours incubation at 30°C the cells were harvested and suspended in the above medium and left overnight at 30 C. Surviving colonies were obtained on plating the culture in the above medium containing 1% (w/v) glucose. The isolated colonies were patched on the same medium containing either 1% (w/v) glucose or 1% (w/v) fructose. Fructose utilization negative behaviour was confirmed using heavy inocula into above culture medium containing 1% and 10% (w/v) fructose. If no growth occurred after 7 days, a fructose utilization negative mutant was obtained. (For mutant strain E 977, the penicillin step

- 1 0-

may be omitted. )

The parent strains ATCC 39676 and ATCC 29191 grew on both 1% (w/v) and 10% (w/v) sucrose, 18% (w/v) and 10% (w/v) glucose, and 1% (w/v) and 10% (W/v) fructose, but their mutant derivatives E 4381 and E 977, while being able to grow on 1% (w/v) and 10% (w/v) sucrose and 1% (w/v) and 10% (w/v) glucose, did not grow on either 1% (w/v) or 10% (w/v) fructose.

Preliminary comparative investigations of sucrose fermentation were carried out using the Fru~ mutants and their parent strains. Using 125 mL bottles and x medium containing 10% (w/v) sucrose, the broth was inoculated with a seed culture grown on glucose and incubated statically for 48 hours at 37°C. Samples were taken after 15 minutes and at the end of incubation, centrifuged and analyised.

The results indicate that the mutant strains are able to accumulate up to 100% of the theoretically obtainable fructose, whereas the 'parent- strains exhibited minimal fructose accumulation of less than 18% of the theoretical value. Ethanol production by the mutants was 34 to 36% (w/v) whereas the parent strains produced 66 to 71% (w/v). EXAMPLE 2: Fermentation with Fru ^" Mutant Strains (Growth and production phases)

2,500 mL of a sucrose solution containing 192 g/L (w/v) of sucrose are transferred into an open 3.5 L fermentation vessel. If required, 200 mL of a medium is added aseptically containing any one or more of peptone, yeast extract, potassium dihydrogen phosphate, ammonium sulfate or urea, and magnesium sulfate, with each component having a concentration of 0.2% or less, whereby peptone and yeast extract can be replaced by 0.5% (w/v) calcium' pantothenate or the total medium can be replaced by 2,700 mL of sucrose containing sugar cane

- 1 1 -

syrup, sugar beet syrup or a proportional addition of molasses.

300 mL of a 12 to 24 hour seed culture of a fructose utilization negative mutant strain (E ^' 977 or E 4381) grown in a medium containing 10% (w/v) sucrose, 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 sulfate, hydrated, 0.2% (w/v) ammonium sulfate at 37°C was added to the fermentation vessel. The initial pH of the fermentation medium was brought to 7.0. Over the first 1 to 2 hours, the pH dropped to 6.0 and was thereafter maintained at 6.0 by addition of 2 M NaOH (80 g/L). Cultivation was carried out at a temperature of 35°C with a stirring rate of 50 rpm.

After 24 hours, maximal sucrose conversion has occurred giving an ethanol concentration of 46.7 g/L or 4.7% (w/v), a fructose concentration of 82.8 g/L (w/v) and a sorbitol concentration of 15.7 / (w/v). EXAMPLE 3; Fermentation with Fru ^" Mutant Strains

(Growth and production phases) 2,500 mL of a sucrose solution containing 217.1 g/L (w/v) of sucrose are transferred into an open 3-5 L fermentation vessel. If required, 200 mL of a medium is added aseptically containing any one or more of peptone, yeast extract, potassium dihydrogen phosphate, ammonium sulfate or urea, and magnesium sulfate, with each component having a concentration of 0.2% or less, whereby peptone and yeast extract can be replaced by 0.5% (w/v) calcium pantothenate or the total medium can be replaced by 2,700 mL of sucrose containing sugar cane syrup, sugar beet syrup or a proportional addition of molasses.

300 mL of a 12 to 24 hour seed culture of a fructose utilization negative mutant strain (E 977 or

_ϊ.-t _

- 12-

E 4381) grown in a medium containing 10% (w/v) sucrose, 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 sulfate, hydrated, 0.2% (w/v) ammonium sulfate at 37°C was added to the fermentation vessel. The initial pH was brought to 7.0 and pH was then maintained during the fermentation at 6.0 by addition of 2 M NaOH (80 g/L). Cultivation was carried out at a temperature of 35°C with a stirring rate of 50 rpm.

After 24 hours, maximal sucrose conversion has occurred giving an ethanol concentration of 51.7 g/L (w/v), a fructose concentration of 109.7 g/L (w/v) and a sorbitol concentration of 4.1 g/L (w/v). EXAMPLE : Fermentation with Fru ^" Mutant Strains

(Growth and production ^* phases) 2,500 mL of a sucrose solution ^* containing 390 g/L (w/v) of sucrose are transferred into a 3.5 L fer¬ menter vessel. If required, 200 mL of a medium is added aseptically containing one or more of peptone

(casein hydrolysate), yeast extract, potassium dihydrogen phosphate, ammonium sulfate or urea, and magnesium sul¬ fate, hydrated, with each component having a concen¬ tration of 0.2% (w/v), whereby peptone and yeast extract can be replaced by 0.5% calcium pantothenate or the total medium can be replaced using 2,700 mL of sucrose containing sugar cane syrup, sugar beet syrup or a proportional addition of molasses.

300 mL of a 12 to 24 hour seed culture of a fructose utilization negative mutant strain (E 977 or E 4381) grown in a medium containing 10% (w/v) sucrose, 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 sulfate hydrated, 0.2% (w/v) ammonium sulfate at 37°C was added to the fermentation

- 13-

vessel. The initial pH was brought to 7.0 and pH was then maintained at 6.0 by the addition of 2 M NaOH (80 g/L). Cultivation was carried out at ^' a temperature of 35°C with a stirring rate of 50 rpm. After 31 hours, maximal sucrose conversion has occurred giving an ethanol concentration of 69.4 g/L (w/v), a fructose concentration of 171.7 g/L (w/v) and a sorbitol concentration of 3*4.5 g/L (w/v). EXAMPLE 5: Fermentation with Parent Strain ATCC 39676 (Growth and production phases)

1,500 mL of sucrose solution containing 400 g/L of sucrose are transferred into a 2 L fermentation vessel. If required, 300 mL of a medium is added aseptiσally containing any one or more of peptone, yeast extract, potassium dihydrogen phosphate, ammonium sulfate or urea and magnesium sulfate, with each component having a concentration of 0.2%> (w/v), whereby peptone and yeast extract can be replaced by 0.5% (w/v) calcium pantothe¬ nate. 200 mL of a 12 to 24 hour seed culture of

Zymomonas mobilis (ATCC No. 39676) grown in a medium containing 10% (w/v) sucrose, 0.2% (w/v) yeast extract, 0.2% (w/v) casein hydrolysate (peptone), 0.2% (w/v) potassium dihyrdogen phosphate, 0.2% (w/v) magnesium sulfate, hydrated and 0.2% (w/v) ammonium sulfate at

35 C or 37°C was added to the fermentation vessel. The initial pH was brought to 6.5 and was then maintained at 5.5 by the addition of 2 M NaOH (80 g/L). Cultivation was carried out at a temperature of 35°C at a stirring rate of 100 rpm.

After 72 hours, maximal sucrose conversion has occurred giving an ethanol concentration of 80 g/L (w/v), sorbitol concentration of 86 g/L (w/v) and fructose con¬ centration of 50 g/L (w/v). EXAMPLE 6: Fermentation with Parent Strain ATCC 39676

- 1 4 -

(Growth and production phases) 85 L of a sucrose solution containing 400 g/L (w/v) of sucrose are transferred into a 100 L pilot plant fermentation vessel. If required, 5 L of a medium is added aseptically containing any one or more of peptone (casein hydrolysate), yeast extract, potas¬ sium dihydrogen phosphate, ammonium sulfate or urea, and magnesium sulfate, hydrated, with each component having a concentration of 0.2% (w/v), whereby peptone and yeast extract can be replaced by 0.5% calcium pan¬ tothenate or the total mediumcan be replaced using 90 L of sucrose containing sugar cane syrup, sugar beet syrup or a proportional addition of molasses.

10 L of a 12 to 24 hour seed culture of Zymomonas mobilis (ATCC 39676) grown in a medium con¬ taining 10% (w/v) sucrose, 0.2% (w/v) yeast extract, 0.2% (w/v) casein hydrolysate (peptone), 0.2% potassium dihydrogen phosphate, 0.2% (w/v) magnesium sulfate, hydrated, 0. ^" 2% (w/v) ammonium sulfate at 38°C was added to the fermentation vessel. The initial pH was .brought to 6.5 and pH was then maintained at 6.2 by the addition of 2 M NaOH (80 g/L). Cultivation was carried out at a temperature of 35 C with a stirring rate of 250 rpm. After 72 hours, maximal sucrose conversion has occurred giving an ethanol concentration of 75 g/L (w/v), a sorbitol concentration of 23.5 g/L (w/v) and a fructose concentration of 88 g/L (w/v). EXAMPLE 7: Fermentation with Parent Strain ATCC 39676 (Growth and production phases)

2,500 mL of a sucrose solution containing 400 g/L (w/v) of sucrose are transferred into a 3.5 L fermentation vessel. If required, 200 mL of a medium is added aseptically containing any one or more of peptone (casein hydrolysate), yeast extract, potassium

- 15-

dihydrogen phosphate, magnesium sulfate hydrated, ammonium sulfate or urea, with each component having a concentration of 0.2% (w/v), whereby peptone and yeast extract can be replaced by 0.5% (w/v) calcium panto- thenate or the total medium can be replaced by 2,700 mL of sucrose containing sugar cane syrup, sugar beet syrup or a proportional addition of molasses.

300 mL of a 12 to 24 hour seed culture of Zymomonas mobilis (ATCC 39676) grown in a medium contain- ing 10% (w/v) sucrose, 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 sulfate hydrated, 0.2% (w/v) ammonium sulfate at 37 C was added to the fermentation vessel. The initial pH was brought to 7.0 and pH was then maintained at 6.0 by addition of 2 M NaOH (80 g/L). Cultivation was carried out at a temperature of 35 C with a stirring rate of 80 rpm.

After 72 hours, maximal sucrose conversion has occurred giving an ethanol concentration, of 132 g/L (w/v) and a fructose concentration of 104 g/L (w/v). EXAMPLE 8: Fermentation of Glucose-Fructose Mixtures with Fru ^" Mutants (Growth and production phases) 2,500 mL of a solution containing 102 g/L glucose and 105 g/L fructose are transferred into a 3-5 L fermentation vessel. If required, 200 mL of a medium is added aseptically containing any one or more of peptone (casein hydrolysate), yeast extract, potas¬ sium dihydrogen phosphate, magnesium sulfate, hydrated, ammonium sulfate or urea, with each component having a concentration of 0.2% (w/v), whereby peptone and yeast extract can be replaced by 0.5% (w/v) calcium panto¬ thenate.

300 mL of a 12 to 24 hour seed culture of a fructose utilization negative mutant strain (E 977 or

- 16-

E 4381) grown in a medium containing 10% (w/v) sucrose, 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 sulfate, hydrated, 0.2% (w/v) ammonium sulfate at 37°C was added to the fermentation vessel. The initial pH was brought to 7.0 and pH was then maintained at 6.0 or 5.5 by addition of 2 M NaOH (80 g/L). ^' Cultivation was carried out at a temperature of 35°C with a stirring rate of 80 rpm. After 28 hours, maximal conversion has occurred giving an ethanol concentration of 42.2 g/L (w/v), a fructose concentration of 70 g/L (w/v) and a sorbitol concentration of 31.1 g/L (w/v).

Experiments have been carried out with a number of glucose-fructose mixtures containing glucose and fructose in the range of 50 to 200 g/L (w/v) using the seed culture of Example 8 under the same conditions and • equivalent ethanol, fructose and sorbitol concentrations have been achieved. Experiments have also been carried out where the fermentation medium is added to the fermenter which contains approximately 10% (w/v) of the fermented medium from a previous fermentation run, the fermented medium containing Zymomonas mobilis. The fermented medium was centrifuged for 10 minutes at 4,000 rpm and was added to the fermentation medium of Examples 2 to 4 respect¬ ively, where the fermented medium contained the Fru~ mutant strains, and to the fermentation medium of Examples 5 and 6 where the fermentation medium contained the parent strain ATCC 39676. The pH and temperature conditions' of the Examples were followed and in all cases fermentation occurred with results corresponding to those described for each respective Example.

The fermentation process, using fermented medium from a preceding process as an inoculum for the

- 17-

Zymomonas mobilis was repeated several times and con¬ sistent results were achieved. It was observed that the Zymomonas mobilis cells grew rapidly in the fermen¬ tation medium and both growth and production phases occurred simultaneously after the initial growth phase on the addition of the fresh fermentation medium to the fermenter containing the fermented medium.

In Examples 5, 6 and 7, the parent strain ATCC 39676 may be replaced by other strains, including the second strain ATCC 29191 but. the best results are achieved using ATCC 39676.

Ethanol produced has commercial value as a component in gasoline or octane boosters in lead-free petrol or as a base product in the chemical industry, carbon dioxide may be used for dry ice or as a carbon source for the growth of algal biomass. Fructose, sorbitol and mannitol are highly valuable nutritive sweeteners of different commercial value in dietary, health food, diabetic foods such as soft drinks, con- fectionery, and related industries.

The fermentation process requires only a low energy input as the organism produces a fair amount of heat during the fermentation process. In addition, the fermentation is carried out in microaerophilic con- ditions, avoiding the need for aeration or addition of any other gas (and attendant equipment), the fermen¬ tation components and products requiring little mechani¬ cal stirring and pH control.

In the case of sucrose, experiments have shown that the success of the fermentation process is not wholly dependent on the quality of the substrate. Preliminary experiments with sugar cane juice and sugar cane syrup indicate that the process is particularly suited for industrial applications and the fermenter can be provided adjacent a sugar mill to reduce transport

- 1 8- costs. As the sugar cane juice does not have to be sterilized, the energy input can be kept low.

In the case of glucose-fructose mixtures, experiments have shown that the ratio of both sugars play an important role. It is preferred that the glucose-fructose mixtures be in the range of 1:4 to 4:1 glucose: fructose. The method is clearly applic¬ able to the fermentation of invert sugar solutions as this comprises a mixture of 50% glucose and 50% fructose (i.e. a ratio of 1:1) obtained by the hydrolysis of sucrose; invert sugar being prepared commercially from the inversion of 96% cane sugar solution. This indicates that the process is partic¬ ularly suited for industrial applications when the fermenter can be provided adjacent to a fructose corn syrup or isomerization plant producing such ^' mixtures to reduce transport costs.

It would be readily apparent to the skilled addressee that various changes and modifications may be made to the examples described without departing from the scope of the present invention defined -in the appended claims.

A iNfΛ »3

International Application No: PCT/

Form PCT' .0'13. (January 1981)

SUBSTITUTE SHEET

International Application No: PCT/

MICROORGANISMS

Optional Sheet In connection with the mlcroorgnπltm referred to on pege -7 , tine !__ -- — of Iha deierlptlon >

A. IDENTIFICATION OF DEPOSIT •

Further depαalta are Identified on an additional ahaet | [ '

Name ol depoaltary Inatltutlon <

AMERICAN TYPE CULTURE CENTRE ( ATCC )

Addraaa of dapoaltary tπiUtullon {Including pot I coda and country) «

1 2301 Parklawn Drive , Rockville , Maryland , 28852 , U . S .

Oat.of .po.,.. _{24 April j 1 g84} A eaaalon Number *

( 24-04-84 ) ATCC No . 39676

ADDITIONAL INDICATIONS ' (leeve blank II not applicable). Thla Information la continued on a aaparata attached iha.t Q

C. DESIβNATEO STATES FOR WHICH INDICATIONS ARE MAOS > (If the Indication! are not for all deelgnetad Statea)

D. SEPARATE FURNISHING OF INDICATIONS • (leav. blink II not applicable)

The Indlcatlona liitad below will be lubmltted to the Internetlonel Bureau later * (Specify the general nature of tha Indlcatlona e.g., " Acceialon Number of Oepo.lt ")

E. HI Thli theel wai recalved with the Intarπelloπal application whan filed (to be check. by the r.e.lvlng Office)

(Authorized Officer) j I The data of receipt (from the applicant) by the Internetlonel Bureau '• CO <_r>

wee . . r _c_J

(Authorized Officer)

Form PCTmo/134 (January tttl)

* .... - _t _ t - S 'H - -ΈZT

International Application No: PCT/

SUBSTIITOT'E SB ET