NL8600431A - CONVERSION OF SUCROSE TO ETHANOL AND OTHER PRODUCTS USING ZYMOMONAS MOBILIS. - Google Patents

Description

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Conversion of sucrose to ethanol and other products using Zymomonas mobilis.

The present invention relates to a method for converting sucrose and / or glucose-fructose into ethanol mixtures in combination with other products in a single step fermentation process using basic or mutated strains of Zymomonas mobilis under micro aerophilic conditions.

The sugar industry has become concerned following announcements by many major food and beverage producing companies that they intend to replace sugar (sucrose) with fructose obtained from corn syrup, fructose / sorbitol or fructose / glucose (dextrose) mixtures to ensure the diet, health and diabetes market. Fructose is almost twice as sweet as sugar, so only half the amount is needed for the same level of sweetness, which reduces its caloric value, an important aspect in this health-conscious world. In addition, sorbitol and fructose are safe sweeteners for diabetics other than sucrose.

The production of ethanol from sugar cane is known, and in Russia ethanol is mixed with gasoline to produce "gasohol" or used as such as car fuel. In the USA, ethanol is mainly produced from corn and, to a lesser extent, 29 from sugar cane, the ethanol being used primarily as the octane booster in unleaded gasoline, the mixture being referred to as "super unleaded gasoline". The conversion of corn syrup to fructose is energy dependent, since the corn porridge must first be converted to glucose, which is normally done using the enzymes amylase and glucoamylase. The glucose then needs to be further converted to fructose using the enzyme glucose isomerase. This enzymatic conversion results in approximately 50/50 mixtures of glucose and fructose. In order to obtain higher levels of fructose, glucose must be removed by chromatographic techniques, which is particularly expensive and yet does not completely remove the glucose from the mixture. The traditional method of preparing ethanol is carried out in a two-stage batch process using yeast, the first stage comprising an aerobic propagation of the yeast, which is referred to as the growth stage, and the second stage the yeast anaerobic production of ethanol includes in the presence or absence of small amounts of oxygen. In order to allow the yeast to propagate further during the ethanol-providing second stage, a small addition of air or oxygen is required. The latter is required if the efficiency of the overall process is to be increased using incidental yeast cell recycling through systems such as sedimentation or centrifugation. Since yeast fermentation is basically dependent on a coupling of growth and the rate of ethanol production, in order to optimize ethanol production, either add growth enhancers or carefully controlled aeration to the medium.

The traditional yeast fermentation method (step 2) is therefore dependent on large inoculum amounts of about 5 to 10 million cells per ml. The preferred optimum temperature for the fermentation is 30 ° C and the heat produced should be controlled by the use of refrigeration equipment. The fermentation time to obtain between 9 and 11% (v / v) ethanol is 30 to 70 hours for the second stage batch fermentation. The time of this fermentation can be reduced to 10 hours by increasing the inoculum density by a factor of 80 to 100 by recycling cells.

A second method of ethanol preparation is known which uses the bacterium Zymomonas mobilis (European Patent No. 47 641 - George Weston Ltd.). This method also involves a two-step method, as described above for yeast, but the bacteria does not need the addition of air for the growth step (step 1). Instead, a sufficient supply of nitrogen is required to keep the conditions anaerobic. During the second step of the ethanol production process, the sugar concentration should never exceed 6% (w / v) and therefore this step requires a stepwise or continuous addition of a concentrated sugar solution.

The preferred temperature is from 28 to 33 ° C and the preferred pH is about 5.5. This process may also require the addition of oxygen as well as additional nutrients.

A third method of ethanol production has been described in which immobilized yeasts or strains of Zymomonas are used in a two-stage process, each with a limited amount of sugar (ie 10% w / v) (British Patent No. 2055121 - Tanabe Sugaku Co., Ltd.).

In the case of yeast fermentation, well-known examples for carbon sources are sucrose, glucose, molasses and sugarcane juice, while in the two-stage process using Zymomonas these examples are limited to glucose and in the case of immobilized cells, to glucose and molasses.

Two methods are known for the use of glucose as a substrate for ethanol preparation. One relates to a recycle process, where after fermentation the biomass is separated from the beer and returned to the fermentation system (US Patent 4,403,034, Rogers et al) and Australian Patent Application 78199/81, Unisearch Ltd.). The second method concerns a. continuous process using the glucose from starch hydrolysis for ethanol production. Both methods use a temperature of 30 ° C and a pH of 5.0.

It is an object of the present invention to provide a process for the production of ethanol in combination with fructose, sorbitol or fructose-sorbitol mixtures from suctose-based materials (eg 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 (eg high fructose corn syrup, artificial mixtures, "high test" molasses or inverted sugar solutions).

It is a preferred object of the present invention to provide such a method using a single-stage batch fermentation, or if desired, adaptations to this culture method, eg, feed batch, continuous or multi-stage systems, in which the energy consumption is low.

It is a further preferred object to provide a method in which the purity of the substrate is not essential to the success of the method.

It is a still further object of the invention to provide a method in which the ethanol produced can be used as a source of energy to keep the process going, and in which the production of mucus in the fermentor is eliminated, or at least minimized .

It is an even further preferred goal to provide fructose-using negative strains of Zymomonas mobilis which are suitable for the above method.

Other preferred objects of the present invention will become apparent from the following description. In one aspect, the present invention can be viewed as a process for the production of ethanol in combination with fructose and / or sorbitol from a sucrose based material and / or a glucose-glucose-fructose mixture in a fermentation, characterized by fermenting the sucrose based material and / or glucose fructose mixture with the microorganism Zymomonas mobilis in a single stage method under microaerophilic conditions in which the sucrose based material and / or glucose fructose mixture is present in a fermentation medium as the substrate.

A single stage process is defined as a process in which growth and production phase take place in the same fermentation vessel. Initiation of the process can be obtained either by a seed culture containing Zymomonas mobilis and added to the fermentation vessel containing the fermentation medium, or by introducing the fermentation medium into the fermentor containing part of the fermented medium from a previous fermentation production containing fermented medium Zymomonas mobilis.

Microaerophilic conditions are defined as conditions in which no gas (oxygen, air, nitrogen, etc.) is added to the fermentor, while the surface of the fermentation medium is exposed to the atmosphere. The microorganism Zymomonas mobilis does not require air or oxygen (aerobic) or nitrogen (anaerobic) for the growth and production of ethanol, but it tolerates the presence of air on the oxygen of the fermentation broth.

The fermentation can be carried out using a free or immobilized form of the microorganism. Sucrose-based materials include sugarcane juice or syrup, sugar beet juice or syrup, molasses, palm sugar juice or syrup, raw or refined sugar.

The glucose and / or fructose mixtures include high fructose corn syrup, artificial mixtures, high test molasses or inert sugar solutions.

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Preferably, the base medium includes yeast extract, casein hydrolyzate, ammonium sulfate, urea or magnesium sulfate, potassium dihydrogen phosphate and / or glucose. Preferably, the amount of each of the ingredients in this base medium is 0.01 to 0.2%, preferably 0.2 to 1% (w / v). Preferably, incubation steps are performed at about 30 ° C for a period of, for example, 2 to 15 hours.

Preferably the fructose and glucose added to the medium in the amount of 0.5 to 3%, more especially 1 to 2% (w / v).

A preferred basic strain of the microorganism Zymomonas 10 mobilis is deposited in the Culture Collection of the University of Queensland, Microbiology Department, St. Lucia, Queensland, Australia. , under deposit no. UQM 2716 and registered with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852, United States of America on April 24, 1984 under Deposit No. 15 39676. This strain was developed from the Zymomonas mobilis strain ATCC no.

29191, which is a second preferred base strain suitable for use with the present invention. The base strain is also filed under No. NCI3 11199 with the National Collection of Industrial Bacteria, Torry Research Station, Abbey Road, Aberdeen, United Kingdom AB9 20 3 DG. Development of strain ATCC 39676 was performed using the chemostat cultivation technique for increased substrate uptake, i.e., improved activity and metabolic rate of sucrose conversion. and these properties are just some difference in the taxonomic description of the basic strain ATCC No. 29191 shown on pages 576-530 of "3ergy's Manual of Determinative Bacteriology" (3rd edition, 1975).

Two preferred fructose using negative (Fru) mutant states of the microorganism Zymomonas mobilis (E 977 and Ξ 4331) are deposited in the Culture Collection of the University of Queensland, Microbiology Department, St. Lucia, Queensland, Australia under deposit no. UQM 2S41 (E 977) and UQM 2864 (E 4281) and deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852, United States of America on January 17, 1936 under Deposit No. 53432 (UQM 2341) and Deposit No. 53431 (UQM 2364).

The fructose using negative (Fru) mutant strains are developed from the Zymomonas mobilis base strains ATCC 29191 and 39676, respectively ~ 4 St * 1 Λ * «v -Λ it j i; ij ί 'Λ j * ï * ni i * 4 «> w

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6 by treatment using ethyl methanesulfonate (EMS). The taxonomic description for each strain is similar to that of the base for it except for the fructose negative nature, and each has a thick gram negative bar. The method of production of the negative strains using fructose-5 is shown in Example I.

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

Preferably, the fermentation medium contains only one or more of the following components: peptone (casein hydrolyzate), yeast extract, calcium pentothenate, 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 about 0.2% being preferred.

Preferably, the pH of the fermentation process is maintained in the range of 4.0 to 7.0. Preferably, the pH is initially set at a value in the range of 6.0 to 7.0. During the course of the fermentation, the pH drops, and then after, for example, 1 to 2 hours, the pH is maintained in a range of 5.0 to 6.2. The pH range can be controlled by the addition of NaOH and other suitable alkali.

Preferably, the temperature is maintained in the range from 34 ° C to 40 ° C. This temperature range has been found to provide the best growth and product yields and alleviates or significantly reduces mucus production in the fermentor.

When the fermentation is complete, the organism is preferably separated from the fermentation products, the ethanol is distilled off and the product is concentrated or crystallized.

In a second aspect, the present invention is in a process for the production of a sucrose-using negative mutant strain of the microorganism Zymomonas mobilis comprising mutating a fructose-using negative strain of Zymomonas mobilis.

In a third aspect, the present invention is in a process for the production of fructose-employing negative mutant strains of Zymomonas mobilis comprising the steps of: 6600431 culturing Zymomonas mobilis in a basal medium to provide a first cell culture; adding ethyl methanesulfonate and / or nitroso-guanidine to the first cell culture and incubating the mixture; Removing the cells from the first cell culture, suspending the cells in the basal medium and incubating the mixture to obtain a second cell culture; harvesting the cells from the second cell culture and suspending the cells in the basal medium to generate a third cell culture; and obtaining the fructose-using negative cells of Zymomonas mobilis by plating the cells of the third cell culture in the basal medium combined with glucose or fructose.

Preferably, at least a portion of the second cell culture is mixed with the base 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 form the third cell culture. to deliver.

In a fourth aspect, the invention lies in fruit-using negative strains of Zymomonas mobilis produced by the method described above.

In order to provide a complete understanding of the invention, preferred examples of the invention will be described below. In all examples, percentages are expressed as (w / v) "25 where 1% corresponds to 10 g / l.

Example I

Production of fructose-applying negative (Fru) mutants

The following procedure was used to obtain fructose-using negative strain of Zymomonas mobilis base strains ATCC 30 29191 and ATCC 39576 (i.e. the mutant strains Zymomonas mobilis E 977 and W 4381:

The base strain was grown in a medium containing 0.2% w / v yeast extract, 0.2% w / v casein hydrolyzate, 0.2% w / v ammonium sulfate, 0.2% w / v MgSO ^ .7 ^ 0.2% w / v KS ^ PO ^ with 1% w / v glucose to about 109 cfu / mL, cfu denoting colony-forming units. To such culture was directly different volumes (0.1 to 0.5 mL) is is Ki Ü o 1 4; YOU

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8 ethyl methanesulfonate (EMS) and varying amounts (60 to 1000 mg) of nitrosoguanidine (NTG) were added. The mixtures were incubated at 30 ° C for 2 hours. After centrifugation, the cells were washed in the above-described basic medium and suspended in 10 mL of this medium.

After 18 hours of incubation at 30 ° C, 2 ml of the culture was transferred to 10 ml of this medium containing 2% w / v fructose and 1000 U / ml penicillin G. After incubation at 30 ° C for 5 hours, the cells were harvested and suspended in the medium described above and kept at 30 ° C overnight. Surviving colonies were obtained by plating the culture in the above medium containing 1% w / v glucose.

The isolated colonies were applied to the same medium containing either 1% w / v glucose or 1% w / v fructose. Fructose using negative behavior was confirmed using a heavy inocula in the above culture medium containing 1% and 10% w / v fructose. If no growth occurred after 7 days, a fructose using negative mutant was obtained (for mutant strain E 977, the penicillin step can be omitted).

The basic 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 20 and 10% w / v fructose, but their mutated derivative strains E 4381 and E 977, although they could 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 studies of sucrose fermentation were performed using the Fru mutants and their base strains. Using 120 mL vials and x medium with 10% w / v sucrose, the broth was seeded with a seed culture grown on glucose and incubated statically for 48 hours at 37 ° C. Samples were taken after 15 minutes and centrifuged and analyzed at the end of the incubation.

The results indicate that the mutant strains were able to absorb up to 100% of the theoretically available fructose, while the present strains showed minimal fructose accumulation of less than 18% of the theoretical value. Ethanol production in the mutants was 34 to 36% (w / v) while the base strains provided 66 to 71% 35 w / v.

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Example II

Fermentation with Fru mutant strains (growth and production phases) 2500 mL of a sucrose solution with 192 g / L (w / v) sucrose were transferred to an open 3.5 L fermentation vessel. If desired, 200 mL of a medium is added aseptically, which medium contains one or more of peptone, yeast extract, potassium dihydrogen phosphate, ammonium sulfate or urea, and magnesium sulfate, each component having a concentration of 0.2% or less, wherein peptone and yeast extract can be replaced with 0.5% w / v calcium pantothenate or the total medium can be replaced with 2700 mL of a 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 using negative mutant strain (Ξ 977 or S 4331) grown in a medium containing 10% w / v sucrose, 0.2% w / v yeast extract, 0.2% w / v casein-15 hydrolyzate (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 adjusted to 7.0. During the first 1 to 2 hours, the pH dropped to 6.0 and was then maintained at 6.0 by addition of 20 M NaQH (30 g / L ·). Cultivation was performed at a temperature of 35 ° C with a stirring speed of 50 rpm.

After 24 hours, maximum sucrose conversion had occurred, resulting in an ethanol concentration of 46.7 g / L, or 4.7% w / v, a fructose scan concentration of 82.3 g / L (w / v), and a sorbitol-25 concentration of 15.7 g / L (w / v).

Example III

Fermentation with Fru mutant strains (growth and production phases) 2500 mL of a sucrose solution containing 217.1 g / L w / v sucrose is placed in an open 3.5 L fermentation vessel. If desired, aseptically 200 mL of a medium containing one or more of peptone, yeast extract, potassium dihydrogen phosphate, ammonium sulfate or urea, and magnesium sulfate, each component having a concentration of 0.2% or less, replacing peptone and yeast extract can be replaced by 0.5% w / v calcium pantothenate or the total medium can be replaced with 2700 mL of a sucrose-containing sugar cane syrup, sugar beet syrup or a proportional addition of molasses.

Ώ ^ Λ Λ -7 d * «Λ yy ί_ί r J it * ^ · * V 'W Vs ai ί · ΐ 10 300 ML of a 12 to 24 seed culture of a fructose-using negative mutant strain (E 977 or E 4381) grown on a medium containing 10% w / v sucrose, 0.2% w / v yeast extract, 0.2% w / v casein hydrolyzate (peptone), 0.2% w (v potassium dihydrogen phosphate, 0.2% 5 w / v magnesium sulfate, hydrated, 0.2% w / v ammonium sulfate at 37 ° C was added to the fermentation vessel The initial pH was adjusted to 7.0 and then the pH was maintained at 6.0 during the fermentation by adding 2 M NaOH (80 g / L) Cultivation was performed at a temperature of 36 ° C using a stirring speed of 50 rpm.

After 24 hours, the maximum sucrose conversion was reached, 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) gave.

Example IV

Fermentation with Fru mutant strains (growth and production phases 2500 mL of a suerose solution with 390 g / L w / v sucrose is transferred to a 3m5 1 fermentation vessel. If desired, aseptically added 200 mL of a medium containing one or more 20 peptons (casein hydrolyzate), yeast extract, potassium dihydrogen phosphate, ammonium sulfate or urea, and magnesium sulfate, hydrated, each component having a concentration of 0.2% w / v, replacing peptone and yeast extract with 0.5% calcium pantothenate or the total media can be replaced using 2700 mL of sucrose-25 containing sugar black syrup, sugar beet syrup, or a proportional addition of molasses.

300 mL of a 12 to 24 hour seed culture of a fructose application 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 hydrolyzate (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 adjusted to 7.0 and the pH was then maintained at 6 by addition of 2 M NaOH (80 g / L). Cultivation was performed at a temperature of 35 ° C with a stirring speed of 50 rpm.

After 31 hours, the maximum sucrose conversion was reached 8 S 0 0 4 3 1 11 giving an ethanol concentration of 69.4 g / L w / v, a fructose concentration of 171.7 g / L w / v and sorbitol concentration of 34.5 g / L w / v.

Example V

Fermentation with basic step ATCC 39676 (growth and production phases) 1500 ml of a sucrose solution containing 400 g / L sucrose are transferred to a 2 L fermentation vessel. If desired, aseptic 390 raL of a medium is added which medium contains one or more components selected from peptone, yeast extract, potassium hydrogen phosphate, ammonium sulfate or urea and magnesium sulfate, each component having a concentration of 0.2% w / v, and wherein peptone and yeast extract can be replaced by 0.5% w / v calcium pantothenate.

200 mL of a 12 to 24 hour seed culture of Zymomonas mobiiis (ATCC 39676) grown in a medium containing 10% w / v sucrose, 15 '3.2% w / v yeast extract, 9.2% w / v casein hydrolysafc (peptone ), 0.2% w / v potassium dihydrogen phosphate, 0.2% w / v magnesium sulfate, hydrated and 0.2% w / v ammonium sulfate at 35 or 37 ° C was added to the fermentation vessel. The initial pK was adjusted to 6.5 and then maintained at 5.5 by the addition of 2 M NaOH (30 g / L). Cultivation was performed at a temperature of 35 ° C at a stirring speed of 100 rpm.

After 72 hours, the maximum sucrose conversion was reached giving an ethanol concentration of 80 g / L w / v, sorbitol concentration of 36 g / L w / v and a fructose concentration of 50 g / L w / v.

Example VI

Fermentation with basic strain ATCC 39676 (growth and production phases) 85 l of a sucrose solution with 400 g / L w / v sucrose are transferred to a 100 l semi-technical fermentation vessel. If desired, 5 liters of a medium are added, which medium contains one or more components of peptone (case hydrolyzate), yeast extract, potassium dihydrogen phosphate, ammonium sulfate or urea, and magnesium sulfate, hydrated. each component having a concentration of 9.2% w / v, where peptone and yeast extract can be replaced with 0.5% calcium pantothenate or the total medium can be replaced using 90 L of a sucrose-containing sugar cane syrup, 35 sugar beet or a proportional addition of molasses.

19 1 of a 12 to 24 hour seed culture Zymomonas mobiiis (ATCC 39676) grown in a medium containing 10% w / v sucrose, 0.2% w / v bo ύ U τ ó i si w <·· 12 yeast extract, 0 2% w / v casein hydrolyzate (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 adjusted to 6.5 and the pH was then maintained at 6.2 by the addition of 2 M NaOH (80 g / L. Cultivation was performed at a temperature of 35 ° C with a stirring speed of 250 rpm. .

After 72 hours the maximum sucrose conversion was reached 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 .

10 Example 7

Fermentation with basic strain ATCC 39676 (growth and production phases) 2500 mL of a sucrose solution with 400 g / L w / v sucrose are transferred to a 3.5 L fermentation vessel. If desired, aseptic 200 mL of a medium containing one or more of the following components is added: peptone (case hydrolyzate), yeast extract, potassium dihydrogen phosphate, magnesium sulfate, hydrated, ammonium sulfate or urea, each component having a concentration of 0 , 2% w / v, where peptone and yeast extract can be replaced with 0.5% w / v calcium pantothenate or the total medium can be replaced with 2700 20 mL of a 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 containing 10% w / v sucrose, 0.2% w / v yeast extract, 0.2% w / v casein hydrolyzate (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 adjusted to 7.0 and the 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 speed of 80 rpm.

After 72 hours, the meximal sucrose conversion was reached to give 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)

2500 mL of a solution containing 102 g / L glucose and 105 g / L

8600431? ? 13 containing fructose is transferred to a 3.5 L fermentation vessel. if desired, aseptically 200 mL of one. medium containing one or more components of peptone (casex hydrolyzate), yeast extract, potassium dihydrogen phosphate, magnesium sulfate, hydrated, ammonium sulfate or urea, each component having a concentration of 0.2% w / v, and wherein peptone and yeast extract can be replaced by 0.5% w / v calciun pantothenate.

300 mL of a 12 to 24 hour seed culture of a fructose-used negative mutant strain S 977 or E 4381, grown in 13 medium containing 10% w / v sucrose, 0.2% w / v yeast extract, 0.2% w / v Caecne hydrolyzate (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 pEE was adjusted to 7.0 and the pH was then maintained at 6.0 or 5.5 by addition of 2 M NaOH (33 g / L). Cultivation was performed at a temperature of 35 ° C with a stirring speed of 30 rpn.

At 23 hours the maximum conversion was reached giving an ethanol concentration of 42.2 g / lw / v, a fructose concentration of 70 g / L w / v and a sorbitol concentration of 31.1 g / L w / v.

Experiments have been conducted with a number of giucose-fructose mixtures containing glucose and fructose at a level of 53 to 23 C g / L w / v using the seed culture of Example VIII under the same conditions and equivalent ethanol, fructose and sorbitol concentrations reached.

Experiments have also been conducted in which the fermentation medium is added to the fermentor containing about 10% w / v of the fermented medium from a previous fermentation, which cemented medium contains Zymomonas mobilis. The demented medium was centrifuged for 10 minutes at 4000 rpm and was added to the fermentation medium of Examples II to IV, respectively, the fermented medium containing the Fru mutant strains, and to the fermentation medium of Examples V and VI, where the irmentatxamsdxun da base strain ATCC 39676 contained. The pH and temperature conaxis of the examples were maintained and in all cases -5 rarmeatatia occurred with results corresponding to those described in each of our respective examples.

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14

The fermentation process, using fermented medium from a previous fermentation as an inoculum for the Zymomonas mobilis, was repeated several times and consistent results were obtained. The Zymomonas mobilis cells were observed to grow rapidly in the fermentation medium and both growth and production phases occurred simultaneously after the initial growth phase upon addition of the fresh fermentation medium to the fermentor containing the fermented medium.

In Examples V, VI and VII, the base strain ATCC 39676 can be replaced by other strains, including the second strain ATCC 29191, but the best results are achieved using ATCC 39676.

The ethanol produced has commercial value as a component in gasoline or as octane improvers in unleaded gasoline or as a base product in the chemical industry, carbon dioxide can be used for dry ice or as a carbon source for the growth or algal biomass . Fructose, sorbitol and mannitol are very valuable nutritive sweeteners with different commercial value in diet food, health food, diabetic food such as soft drinks, confectionery, and related industries.

The fermentation process requires only a low amount of energy, since the organism produces a reasonable amount of heat during the fermentation. In addition, the fermentation is carried out under micro-aerophilic conditions, which avoids the need for aeration or addition of another gas (and associated equipment), while the fermentation components and products require little mechanical stirring and pH control.

In the case of sucrose, tests have shown that the success of the fermentation process does not depend entirely on the quality of the substrate. Preliminary experiments with sugarcane juice and sugarcane syrup demonstrate that the method is particularly suitable for industrial applications, and the fermenter can be located near a sugar factory to reduce transportation costs. Since the sugarcane juice does not need to be sterilized, energy consumption 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 lie in. the bersik from 1: 4 to 4: 1 glucose: fructose. The method is clearly applicable to the fermentation of inert sugar solutions as it comprises a mixture of 50% glucose and 50% fructose 5 (i.e. 1: 1 ratio) obtained by the hydrolysis of sucrose; invert sugar is commercially prepared by inversion of 96% cane sugar solution. This indicates that the process is particularly suitable for industrial applications if the fermentor can be placed near a fructose corn syrup or isomerization plant providing such mixtures to reduce transports.

It will be apparent to those skilled in the art that various changes and modifications can be made to the described examples without departing from the scope of the present invention as defined in the following claims.

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Claims (28)

A method for the preparation of ethanol in combination 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 mixture and / or glucose-fructose mixture 5 with the micro-organism Zymomonas mobilis in a single-stage process under micro-aerophilic conditions wherein the sucrose-based material and / or glucose-fructose mixture is included in a fermentation medium as the substrate, 2. A method according to claim 1, characterized in that the Zymomonas mobilis is a strain deposited with ATCC under Deposit No. 39676 or a mutant or variant of ATCC 39676, The method according to claim 2, characterized in that the mutant strain is a fructose using negative mutant strain deposited under No. ATCC 53431 or a fructose using negative mutant or variant of ATCC 52431. A method according to claim 1, characterized in that the Zymomonas mobilis is a strain deposited with the ATCC under deposit no. 29191 or a mutant or variant of ATCC 29191. Method according to claim 4, characterized in that the mutant strain is a fructose-using negative mutant deposited with the ATCC under Deposit No. 53432 or a fructose using negative mutant or variant of ATCC 53432. 6. A method according to any one of claims 1 to 5, characterized in that the sucrose-based material comprises raw sugar, refined sugar, sugarcane juice or syrup, sugar beet juice or syrup, palm sugar juice or syrup or a combination of two or more of these. A method according to claim 6, characterized in that the sucrose concentration in the sucrose-based material is between 5 and 20% w / v. A method according to any one of claims 1 to 5, characterized in that the glucose-fructose mixture comprises high fructose corn syrup, artificial glucose fructose mixtures, high test molasses or inert sugar solutions or a combination of two or more of these . ö -Sï ft ft f ”7 4 v) U V U 4 Ó 1 A method according to claim 8, characterized in that the concentration of both glucose and fructose in the glucose-fructose mixture is in the range from 5 to 20% w / v. 10. A method according to any one of claims 1 to 9, characterized in that the fermentation medium comprises one or more of the following components, peptone, yeast extract, potassium dihydrogen phosphate, ammonium sulfate, urea or magnesium sulfate, the concentration of each component being in the range from 0.01 to 0.5% w / v. 11. A method according to claim 10, characterized in that the concentration of each component is 0.2% w / v. A method according to any one of claims 1 to 11, characterized in that the pH of the fermentation medium is maintained in the range from 4.0 to 7.0. Process according to claim 12, characterized in that 15 da pH is initially in the range of 6.5 to 7.0, then may drop as soon as fermentation begins and is then maintained in the range of 5.0 to 6.2. 14. Process according to any one of claims 1-13, characterized in that the temperature in the fermenter is maintained in the range from 34 to 40 ° C. A method according to claim 14, characterized in that the tsp nutature is maintained at 353C. Method according to any one of claims 1-15, characterized in that when the fermentation is complete, the microorganism Zymomonas 25 mobills is separated from the fermentation medium and the ethanol is distilled off. Method according to claim 1, characterized in that the Zvmcmonas mobills comprise Zymomonas mobiiis baeterial cells suspended in the fermentation broth. Process according to claim 1, characterized in that part of the fermented medium from a previous fermentation is added to the fermentor as the inoculum of the Zymomonas mobills baeterial cells for the subsequent fermentation. 19. Ethanol and fructose and / or sorbitol prepared from sucrose-based material using the process according to any one of claims 1-13. 'pi fi .Λ "* ~ *' J L« * 7 v * i Τ ·· 20. Ethanol and fructose and / or sorbitol prepared from a glucose-fructose mixture using the method according to any one of claims 1-18. 21. A method for the production of a fructose using negative mutant strain of the microorganism Zymomonas mobilis comprising mutating a fructose using positive strain of Zymomonas mobilis to form a fructose using negative strain of Zymomonas mobilis. 22. A method of producing a fructose utilize the negative mutant strain of the microorganism Zymomonas mobilis, characterized by the following steps: culturing Zymomonas mobilis in a basal medium to form a first cell culture; adding ethyl methanesulfonate and / or nitrosoguanidin-15 to the first cell culture and incubating the mixture; removing the cells from the first cell culture, suspending cells in the basal 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 basal medium to generate a third cell culture; and obtaining the fructose-using negative cells of Zymomonas mobil's by plating the cells of the third cell culture in the basal medium in combination with glucose and / or fructose. The method according to claim, characterized in that after generating the second cell culture, at least a portion of the second cell culture is mixed with the base medium in combination with fructose and penicillin and the mixture is incubated to form a new cell culture, wherein the cells are harvested from the cell culture and suspended in the basal medium to yield the third cell culture. A method according to claim 22, characterized in that the strain of Zymomonas mobilis in the base medium is the strain deposited with the ATCC under Deposit No. 39676. 25. A method according to claim 22, characterized in that the strain of Zymomonas mobilis in the base medium is the strain deposited at 8600431 x —-—-- ψ ---- -4. The ATCC under deposit-no. 29191. 26. Sen. fructose-using negative mutant strain of Zyncmonas rcbiiis obtained using the method of claim 22. 27. A strain of Zymomanas mobilis deposited with the ATCC under Deposit No. 53431. 23. A strain of Zymomonas mobilis deposited with the ATCC under Deposit No. 53432. ^ η λ K ï - - * »* V ν '- ^