AU602653B2 - Conversion of sucrose to ethanol and other products using zymomonas mobilis - Google Patents

Description

AU-AI 5 5 1 03 6 WORLD 1 L UA 'OP O N 3 PCT [8 [ernfjg~ INTERNATIONAL, APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 86/ 04925 C12P 007/06, C12N 001/20 Al (43) International Publication Date: 28 August 1986 (28.0&.86) (21) International Application Number: PCT/AU86/00042 (81) Designated States: AT (European patent), AU, BB, BE (European patent), BR, CH (European patent), DE (22) International Filing Date: 20 February 1986 (20.02.86) (European patent), FR (European patent), GB, GB (European patent), HU, IT (European patent), JP, KR, LK, LU (European patent), MW, NL (European (31) Priority Application Numbers: PG 9396 patent), SD, SE (European patent), SU, US.

PH 1811 (32) Priority Dates: 21 February 1985 (21.02.85) Published 6 August 1985 (06.08.85) With international search report.

(33) Priority Country: AU Thi. dcume nt c n.

amendrimnts made ire Co Scliun 49 and is correct tor (71) Applicant (for all designated States except US): UNI- I'inting VERSITY OF QUEENSLAND [AU/AU]; St. Lucia, QLD 4067 (AU).

(72) Inventor; and A P. 2 3 OC 1986 Inventor/Applicant (for US only) DOELLE, Horst, P.

Werner [AU/AU]; Belsize Street, Kenmore, QLD 4069 A TAA

AUSTRALIAN

(74) Agent: GRANT ADAMS COMPANY; 333 Adelaide 10 SEP 1986 Street, G.P.O. Box 1413, Brisbane, QLD 4000 (AU).

PATENT OFFICE (54) Title: CONVERSION OF SUCROSE TO ETHANOL AND OTHER PRODUCTS USING ZYMOMONAS MOB!-

LIS

(57) Abstract A single-stage fermentation process under micro-aerophilic conditions for the commercial production of ethanol with fructose and/or sorbitol from sucrose-based materials or glucose-fructose mixtures using strains of Zymomonas mobilis. Fructose utilization negative mutant strains of Zymomonas mobilis have been develnped which improve the fermentation efficiency.

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WO 86/04925 S86/4925 PCT/AU86/00042 -4i- .Title: "CONVERSION OF SUCROSE TO ETHANOL AND OTHER PRODUCTS USING ZYMOMONAS MOBILIS" BACKGROUND OF THE INVENTION Field of the Invention THIS INVENTION relates to a method for converting sucrose and/or glucose-fructose syrup mixtures to ethanol in combination with other products in a singlestage fermention process using pfl-en.tg mutant strains of Zymomonas mobilis in microaerophilia conditions.

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 sweetness, 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, vhich are very expensive and still do not completely remove glucose from the mixture.

The production of ethanol from sugar cane is SUBSTUTE: SHEET WO 86/04925 PCT/A U86/00042 -2well-known and in Brazil the ethanol is mixed with petrol to produce "gasohol", or is solely used as car fuel. In the ethanol is produced mainly from corn and to a lesser degree from sugar cane feedstocks whereby the etharol 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 0 preferred optimal temperature of fermentation is 300C and heat produced has to be controlled through the use of cooling equipment. The fermentation time for -t obtaining between 9 and 11% ethanol is 30 to 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.

L a i I WO 86104925 PCT/AU86/00042 -3- A second process for ethanol production is known, which utilizes the bacterium Zymomonas mobilis (see European Patent No. 0047641 George Weston Ltd.), This process is also a two-stage process as was described above for yeast, but the bacterium does nob require the addition of air for its growth stage (stage Instead, an adequate supply of nitrogen is required to keep conditions anaerobic. During the second stage of the process for the production of ethanol, the sugar concentration must never exceed 6% and thus the stage requires a stepwise or continuous addition of a concentrated sugar solution. The preferred temperature is 28 0 C to 33 0 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 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 fermentation system (see U.S. Patent No. 4,403,0 3 4 (Rogers et al and Australian Patent Application No. 78199/81 (Unisearch The second process is a continuous process using the glucose derived from starch hydrolysis for ethanol production. Both processes operate at 30 0

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L- 1 -4and pH 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 fructosesorbitol mixtures from sucrose-based materials (e.g.

sugar cane juice or syrup, sugar beet juice or syrup, molasses, palm sugar juices or syrup, raw or refined sugar) and/or glucose-fructose mixtures 10 high fructose corn syrup, artificial mixtures, high test molasses or invert sugar solutions).

It is a preferred object of the present invention to provide a method whereby ethanol is produced from the glucose molecule only with fructose accumulating or being partly or completely transformed to sorbitol using a fructose utilization negative [equal to fructokinase (EC 2.7.1.4) negative] mutant of Zymomonas mobilis.

It is a further preferred object of the present 20 invention to provide such a method using single- Ij stage batch fermentation or, if required, adjustments to this culturing methods, e.g. fedbatch, continuous or multi-stage systems, where the energy input is low.

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It is still 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 from the glucose molecule can be used as an energy source to maintain the method in-train, and where the production of slime in the fermenter is eliminated, or at least minimized, reducing the cost for the recovery cf fructose, sorbitol or fructose-sorbitol mixtures.

It is a still further preferred object to produce fructose utilization negative [equal to fructokinase (EC 2.7.1.4) negative] strains of Zyinomonas mobills suitable for the above method.

Other preferred objects of the present invention will become apparent from the following description.

In a braod aspect the present invention resides in a method for the production of ethanol in combination with (simultaneously 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 with the microorganism Zymomnonas mobilis in a single-stage -6process under microaerophilic conditions wherein the glucose molecule from the sucrose-based material or glucose-fructose mixture contained in a fermentation medium as the substrate is selectively converted to ethanol and the fructose molecule is accumulated or partly or completely transformed selectively to sorbitol, characterized, in that the mutant strain is a fructose utilization negative [equal to fructokinase (EC 2.7.1.4) negative] mutant strain 10 deposited in the ATCC under Deposit No. 53431 or under Deposit No. 53432.

A "single-stage process" is defined as a process whereby growth and the production phase occur in the same fermenter vessel, whereby.the cultivation technique can be batch, fed->itch or continuous. Initiation of the process can be done w either by a seed culture containing Zymomonas mobilis 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 Zy7monas mobilis. "Microaerophilic conditions" are defined as conditions whereby no gas (oxygen, air, nitrogen etc.) is added to the fermenter and the surface of I'

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the fermentation medium is exposed to atmosphere.

The Zymomonas mjobilis organism does not require air or oxygen (aerobic) or nitrogen (anaerobic) for growth and production of ethanol plus fructose, sorbitol or sorbitol-fructose mixtures, but can tolerate the presence of air on the surface of the fermentation medium.

The fermentation may be carried out using free or immobilized form of the micro-organism. Sucrose- 10 based materials include sugar cane juice or syrup, sugar beet 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.

15 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 maedium are in the range of 0.01 to more preferably 0.2 to 0.5% Preferably the incubation stepos are carried out at approximately' 0 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 more preferably 1 to 2 *r i -6bil *e

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r One parent strain of the microorganism Zymomonas mjobilis 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 Zynomoonas 10 mobilis strain ATCC Deposit No. 29191 which is a second preferred parent strain suitable for use with the present invention. The pa.ent strain has also been deposited under Deposit No. NCIB 11199 at the National Collection of Industrial Bacteria, Torry 15 Research Station, Abbey Road, Aberdeen, United Kingdom AB9 8DG. 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 20 of sucrose conversion and these features are the only difference in the taxonomic description of the parent strain ATCC No. 29191 set out at pages 576- 580 of "Bergey's Manual of Determinative Bacteriology" (8th Edition) (1975).

The two fructose utilization negative (Fru-) strains of the microorganism Zymomonas mobilis (E -4 11 it 4 It "9 #0 9 a 0 1 89 064

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6 60 0 9 4 0 -6c- 977 and E 4381) have been deposited in -the Culture Collection of the University of Queenslan-d, Microbiology Department, St.Lucia, Queensland, Australia under Deposit No. UQM 2841 (;rE 977) and UQM 2864 4381) and deposited in the Amnerican Type Culture I /T T C WO 86/04925 PCT/AU86/00042 -7- Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852, U.S.A. on 17th January, 1986 under Deposit No. 53432 (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, respectively, 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 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: ppptone (caesin hydrolysate), yeast extract, calcium pantothenate, 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 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 in the range of 6.5 to 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 3 40C to 40 0 C. This temperature range appears to produce the best growth and product yields

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a 1 r .j WO086/ '04925 PCT/AU86/00042 and alleviates or markedly reduces the production of slime in the fermenter.

Preferably when the fermentation has been completed, the micro-organism is separated from the fermentation products, the ethanol is distilled off and *Sv Lc -bose, sob cA L. of- z so Q\ co Ti-ot>re the product is concentrated or crystallized.

In a secondAaspect the present invention resides in a method for the production of a fructose utilization negative mutant strain of the micro-organism Zymomonas mobilis comprising mutating a fructose utilization negative strain of Zymomonas mobilis.

In a thirdjaspect 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 nitrosoguanidine to the first cell culture a.nd 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 generate the third cell culture.

A~ S 1. j LI r L I mF WO 86/04925 ,PCT/AU86/00042 -3 a- In a fourth aspect the present invention .resides in fructose ut ilization negativevstrains of Zymornonas mobilis produced by the above method.

IPTiieH BF THE PRE2FERED fttltttbt titstittt L" eef CDT i T

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-3 I WO 86/04925 PCT/AU86/00042 -9- To enable the invention to be fully understood, preferred examples of the invention will now be described. In all Examples, percentages are expressed as 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 the 10 mutant strains Zymomonas mobilis E 977 and E 4381).

The parent strain was grown in a medium containing 0.2% yeast extract, 0.2% casein hydrolysate, 0.2% ammonium sulfate, 0.2% MgSO4.

7H 2 0, 0.2% KH 2

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4 with 1% glucose to approximately 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 mi;tures were incubated for 2 hours at After centrifugation, the cells were washed in above 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% fructose and 1,000 U/mL penicillin G. After hours incubation at 30 0 C the cells were harvested and suspended in the above medium and left overnight at 300C. Surviving colonies were obtained on plating the culture in the above medium containing 1% glucose.

The isolated colonies were patched on the same medium containing either 1% glucose or 1% fructose.

Fructose utilization negative behaviour was confirmed using heavy inocula into above culture medium containing 1% and 10% fructose. If no growth occurred after 7 days, a fructose utilization negative mutant was obtained. (For mutant strain E 977, the penicillin step WO 86/04925 PCT/AU86/00042 may be omitted.) The parent strains ATCC 39676 and ATCC 29191 grew on both 1% and 10% sucrose, 18% (w/v) and 10% glucose, and 1% and 10% (w/v) fructose, but their mutant derivatives E 4381 and E 977, while being able to grow on 1% and 10% (w/v) sucrose and 1% and 10% glucose, did not grow on either 1% or 10% fructose.

Preliminary comparative investigations of sucrose fermentation were carried out using the Frumutants and their parent strains. U9ing 125 mL bottles and x medium containing 10% sucrose, the broth was inoculated with a seed culture grown on glucose and incubated statically for 48 hours at 37° 0 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% whereas the parent strains produced 66 to 71% EXAMPLE 2: Fermentation with Fru- Mutant Strains (Growth and production phases) 2,500 mL of a sucrose solution containing 192 g/L of sucrose are transferred into an open 3.

1 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 calcium pantothenate or the total medium can be replaced by 2,700 mL of sucrose containing sugar cane 0 m o a 1 t 2 WO 8'./04925 PCT/AU86/00042 -11msyrup, sugar beet syrup or a proportional addition of molasses.

Sbrou 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% sucrose, c0.2% yeast extract, 0.2% (w/v)casein hydrolysate (peptone), 0.2% potassium d.nn ydrogen phosphate, 0.2% magnesium sulfcate, hydrated, 0.2% (w/v) Sammonium sulfate at 37rC was added to the fermentation vessel. The initial pH of the fermentation medium was brought to 7.0. Over the first i to 2 hours, the pH Sdropped to 6.0 and was thereafter maintained at 6.0 by addition of 2 M NaOH (80 Cultivation was carried out at a temperature of 35°C with a stirring rate of 50 rpm.

a m After 24 h.urs, maximal sucrose conversion has occurred giaving an ethano concentration of 46.7 g/Ls or 4.7% a fructose concentration of 82.8 g/L and asorbitol conceentration of 15.7 g/L EXAMPLE 3: Fermentation with Fru Mutant Strains (Growth and production phases) S2,500 mL of a sucrose solution containing 217.1 g/L 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% 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 1 i^ 1 WO 86/04925 PCT/AU86/00042 -12- S E 4381) grown in a medium containing 10% sucrose, 0.2% yeast extract, 0,2% casein hydrolysate (peptone), 0.2% potassium dihydrogen phosphate, 0.2% 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 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 a fructose concentration of 109.7 g/L (w/v) and a sorbitol concentration of 4.1 g/L EXAMPLE 4: Fermentation with Fru- Mutant Strains (Growth and production-phases) 2,500 mL of a sucrose solution containing 390 g/L of sucrose- are transferred into a 3.5 L fermenter 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 sulfate, hydrated, with each component having a concentration of 0.2% 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% sucrose, 0.2% yeast extract, 0.2% casein hydrolysate (peptone), 0.2% potassium dihydrogen phosphate, 0.2% magnesium sulfate hydrated, 0.2% (w/v) ammonium sulfate at 370C was added to the fermentation "-4

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4 i i j1 ~.f*tr g-r i 86/04925 PCT/AU86/00042 -13vessel. The initial pH was brought to 7.0 and pH was then maintained at 6.0 by the addition of 2 M NaOH 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 a fructose concentration of 171.7 g/L and a sorbitol concentration of 34.5 g/L 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 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% whereby peptone and yeast extract can be replaced by 0.5% calcium pantothenate.

200 mL of a 12 to 24 hour seed culture of Zymomonas mobilis (ATCC No. 39676) grown in a medium containing 10% sucrose, 0.2% yeast extract, 0.2% casein hydrolysate (peptone), 0.2% (w/v) potassium dihyrdogen phosphate, 0.2% magnesium sulfate, hydrated and 0.2% ammonium sulfate at 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 Cultivation was carried out at a temperature of 35 C at a stiPring rate of 100 rpm.

After 72 hours, maximal sucrose conversion has occurred giving an ethanol concentration of 80 g/L sorbitol concentration of 86 g/L and fructose concentration of 50 g/L EXAMPLE 6: Fermentation with Parent Strain ATCC 39676 I. i. i i^- *LFPI~i53 WO 86/04925 PCT/AU86/00042 -14- (Growth and production phases) L of a sucrose solution containing 400 g/L 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, potassium dihydrogen phosphate, ammonium sulfate or urea, and magnesium sulfate, hydrated, with each component having a concentration of 0.2% whereby peptone and yeast extract can be replaced by 0.5% calcium pantothenate or the total mediumcan be replaced using 90 L of sucrose containing sugar cane syrup, sugar beet syrup or a proportional addition of molasses.

L of a 12 to 24 hour seed culture of Zymomonas mobilis (ATCC 39676) grown in a medium containing 10% sucrose, 0.2% yeast extract, 0.2% casein hydrolysate (peptone), 0.2% potassium dihydrogen phosphate, 0.2% magnesium sulfate, hydrated, 0.2% ammonium sulfate at 380C 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 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 a sorbitol concentration of 23.5 g/L and a fructose concentration of 88 g/L EXAMPLE 7: Fermentation with Parent Strain ATCC 39676 (Growth and production phases) 2,500 mL of a sucrose solution containing 430 g/L 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 ,^nJ c IIJ---YWL~~~ TLI*Il^l~--~ WO 86/04925 I ji PCT/AU86/00042

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dihydrogen phosphate, magnesium sulfate hydrated, ammonium sulfate or urea, with each component having a concentration of 0.2% whereby peptone and yeast extract can be replaced by 0.5% calcium panto- 5 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 containing 10% sucrose, 0.2% yeast extract, 0.2% casein hydrolysate (peptone), 0.2% potassium dihydrogen phosphate, 0.2% magnesium sulfate hydrated, 0.2% ammoniumi sulfate at 37 0 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 Cultivation was carried out at a temperature of 35 0 C with a stirring rate of 80 rpm.

After 72 hours, maximal sucrose conversion has occurred giving an ethanol concentration of 132 g/L and a fructose concentration of 104 g/L (wv).

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 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 dihydrogen phosphate, magnesium sulfate, hydrated, ammonium sulfate or urea, with each component having a concentration of 0.2% whereby peptone and yeast extract can be replaced by 0.5% calcium pantothenate.

300 mL of a 12 to 24 hour seed culture of a fructose utilization negative mutant strain (E 977 or WO 86/04925 PCT/AU86/00042 E 4381) grewn in a medium containing 10% sucrose, 0.2% yeast extract, 0.2% casein hydrolysate (peptone), 0.2% potassium dihydrogen phosphate, 0.2% magnesium sulfate, hydrated, 0.2% (w/v) ammonium sulfate at 370C 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 'Cultivation was carried out at a temperature of 350C with a stirring rate of 80 rpm.

After 28 hours, maximal conversion has occurred giving an ethanol concentration of 42.2 g/L a fructose concentration of 70 g/L and a sorbitol concentration of 31.3, g/L 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 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 10,1 of the fermented medium from a previous fermentation run, the fermented medium containing Zymomonas mobilis. The fermented medium was centrifuged for 10 minutes at 44,000 rpm and was added to the fermentation medium of Examples 2 to 4 respectively, where the fermented medium contained the Frumutant 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 W0 86

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i /04925 PCT/AU86/00042 -17- Zymomonas mobilis was repeated several times and consistent results were achieved. It was observed that the Zymomonas mobilis cells grew rapidly in the fermentation 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, confectionery, 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 conditions, avoiding the need for aeration or addition of any other gas (and attendant equipment), the fermentation components and products requiring little mechanical 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 application's and the fermenter can be provided adjacent a sugar mill to reduce transport rrr L WO 86/04925 PCT/AU86/00042 -18costs. 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 applicable to the fermentation of invert sugar solutions as this comprises a mixture of 50% glucose and fructose 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 particularly 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.

*..nj WO 86/04925 PCT/AU86/00042 international Application No: PCTJ

MICROORGANISMS

Sline 2 to Optional Sheet In connection with the mlcroorgenlom reterred to on in.- of the doecriptiom A. IOEMTIFICATION OF OIPOSIT I Further depolit are Identifid on n tdditiorna sheet 01 Hems of drnoellary institution 4 AMERICAN TYPE CULTURE COLLECTION (ATCC) Address of deposltry Institution (Including postal code and ccuntry) 12301 Parkiawn Drive, Rockville, Maryland, 28852, U.S.A.

OteodrfeepI January, 1986 Accesslon Number A Januar 19865343 1 (17-01-86) 3 1. ADDITIONAL INDICATIONS Y (liove blaok I not applicblel. This Information is continued on a separale attached sheet C. 0EsNATIC STATIS FOR WHICH INOICATIONS ARK MADE I (If Ihe Indications are not for ait designated'Stls 0. SIPARATI FURNISHING OF INDICATIONS I (Iteen blank It not appilcable) The Indication lilatd t'tow wilt be submitted to Ite Internationai Burseu later I (Specify the generl naturr of the Indications e.g, "Acceslon Number O Depolit") 1. This sheot wit locsivod with the International application when flied (to be chocked by tie receiving Orcne (Authoiriod amour) 0 The de of receipt (from the applicant) by the Internationl Bureau It wee (Aulborlled Offeor) Form PCTIROII34 (January t11t) SUBSTITUTE

SHEET

F -1 WO 86/04925 PCT/AU86/00042 International Appllcatikn No: PCTI

MICROORGANISMS

Optional Sthoot In connection with the microorgcnnsm referred Is on of the description L A. ?OINTIFICATION OF 091-OSIT I Further deposits are IdentItled an an additional sheet O Nacme of depositary institution 4 AMERICAN TYPE CULTURE CENTRE (ATCC) Address ot dsnnsiltari Institution (includint; postal code anrd country) 12301 Parkiawn Drive, Rockville, Haryland, 28852, U.S.A.

Date of deposit 2 p il, 1 8 Accession Nurmber (24-0 4 Api, 9 ATCC No. 39676 S. ADDITIONAL INDICATIONS I toan btank if net aptticable). This tntormatton ts coninued en a separate attached shoet C. ISIGNATED STATIES FOR WHICH tNDICATIONS Ang MAO*E the Indications are not tor sil desitnated Stalst 0. SIPARATE FURNISHING OF INDICATIONS I (teens blsnkr It not applicable) Ti,. Indications listsd bato w nitt be submitted to the Internattonsl Bureau later I (Specify the general nature of the Indications e-q., -Accession Number of Deposit This sheot was received with the International aptlication wrhen filied (to be chocked by the receiving Omien) (Authorized Officsr) IZThe dats of receipt (frontr the applicant) by the Internatioas Bureau It Fornm PCTIRO/t34 iti1) (Authorized 011Ir CbS C WO 86/04925 PCT/AU86/00042 Internatlonal ApplIcation No: PCT/ *1

MICROORRGANISMS

A line-? to Optional Shooet In connection with the mnicroorvaniarm ratfrad io am pe*7- flna.J 4. the description a A. IDENTIFICATION OF ONPOSuarI Further deprosts era Idantitlad orm on addlitonal ah@et Nanm* of depositary Institution, 4 AMERICAN TYPE CULTURE COLLECTION (ATCC) Address of depositary Institution (including posial coda and country)4 12301 Par'klawn Drive, Rockville, Maryland, 28852, U.S.A.

Oat. 0 January, 1986 Accession Numrber a (17-01-86) 153432 B. ADDITIONAL INDICATIONS t (l~ea btank It not applicable), This Iniormaion (a continuad on a searate attached shoot

U:

Ii 14

U:

4 C. ORSIONATIO STATES FORl WHICH INDICATIONS Ant MAOCS 1(if iht indications are not lt all deaignatod States) 0. St1'AiATII FURNISHING OF IN131CATIONS I (leavea biankr Ifnot applicablol The Indicaions Ilaud bolow wiil ha iubmiliad to tha iniarnillonal Bureau lae (Specify the ganeral nature of th10 Indtcations a.

"Acctuion Number oi Daposit", 1. Th~ia os witma received woiih the Iniarnational appicutloul ,hon ilad (to be chocked by the recaiving Ot11ca)f (Authofixad Officer) LIThe dioto racaini (from, the applicant) by ihe Iniernaional fluraau (Authorliad Officar) rtm PCTIRO/i34 (January 1961) suBssoTtr S14 ET

Claims (13)

1. A method for the production of ethanol in conjunction with fructose, or sorbitol or sorbitol- fructose mixtures 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 with the micro-organism Zymomonas mobilis in a single-stage process under microaerophilic conditions selectively converting the glucose molecule only to ethanol and accumulating the fructose molecule or transforming the fructose molecule partly or completely to sorbitol, characterized in that the Zympomonas mobilis strain is a mutant strain unable to utilize fructose to ethanol and that this fructose utilization negative [equal to fructokinase (EC2.7.1.4) negative) mutant strain is deposited in the ATCC under Deposit No. 53431 or under Deposit No. 53432.

2. A method according to Claim 1 characterized in that the sucrose-based material comprises raw sugar, refined sugar, sugar cane juice or syrup, sugar beet juice or syrup, palm juice or syrup or a combination of two or more of these.

3. A method according to Claim 2 characterized in that the sucrose concentration in the sucrose-based material is in the range of 5 to 20%

4. A method according to any of Claim 1 to 3 characterized in that the glucose-fructose mixture *1- u -e -23- comprises high fructose corn syrup, artificial glucose- fructose mixtures, high t st molasses or invert sugar solutions or a combination of two or more of these. A method according to Claim 4 characterized in that the concentration of both glucose and fructose in the glucose-fructose mixture is in the range of 5 to

6. A method according to any one of Claims 1 to characterized in that the fermentation medium contains 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 of 0.01 to 0.5%

7. A method according to Claim 6 characterized in that the concentration of each component is 0.2%

8. A method according to any one of Claims 1 to 7 characterized in that the pH of the fermentation medium 0o is maintained in the range of 4.0 to

9. A method according to Claim 8 characterized in that the pH is initially in the range of 6.5 to allowed to fall as fermentaion commences and then maintained in the range of 5.0 to 6.2. A method according to any one of Claims 1 to 9 characterized in that the temperature during fermentation is maintained in the range 340 to 40 0 C. c -e 1 I ii 9 I. i :1? j* eq 4. 4 I 11 A method according to Claim 10 characterized in that the temperature is maintained at

12. A method according to any one of Claims 1I to 11 characterized in that, when the fermentation is completed, the microorganism Zymomons mobilLs is separated from the fermentation medium and the ethanol is destilled off.

13. A method according to Claim 1 characterized in that the Zy7noomonas inobilis comprises Zymomonas mobilis bacterial cells suspended in the fermentation medium.

14. A method according to Claim 1 characterized in that a portion of the fermented medium from a preceding fermentation is provided as an inoculum of the Zyomonas moblis bacterial cells for the succeeding fermentation. Ethanol and fructose and/or sorbitol produced from sucrose-based material by the method according to any one of Claims 1 to 14.

16. Ethanol and fructose and/or sorbitol produced from a glucose-fructose mixture by the method according to any one of Claims 1 to 14. .4 4* 4. I -1$ F- j r i -iA A INTERNATIONAL SEARCH REPORT International Application No PCT/AU 86/00042 I. CLASSIFICATION OF SUBJECT MATTER (iI several classticlion symools aooly, .noicate allt According to International Patent Classificailon (IPC) or to both National Classification and IPC Int. Cl. C12P 007/06, C12N 001/20 II. FIELDS SEARCHED Minimum Documentation Searched Classification System Classification Symbols IPC C12G 003/02, C12P 007/06, 7/08, 7/10, 7/18, C12N 001/20, Chemical Abstracts; Keyword Zymomonas mobilis Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched I III. DOCUMENTS CONSIDERED TO ME RELEVANT' Category Citation of Document, wit h indication, where approprlate, of the relevant passages it2 Relevant to Claim No, X,P AU,A, 29530/84 (UNIVERSITY OF QUEENSLAND) 4 April 1,2,4,6,8, 1985 (04.04.85) 10-20 AU,B, 78199/81 (540186) (UNISEARCH LTD) 17 June 1982 1,6-10,12, (17.06.82) 17-20 WO,A, 82/01563 MATTIASSON, B. HAGERDAL, 1,7,10,12, P. ALBERTSSON) 13 May 1982 (.13.05.82) 19,20 Xi AU,B, 67697/81 (537029) (UNISEARCH LTD) 19 November 1,6-10,12, 1981 (19.11.81) 17-20 X AU,B, 59181/80 (531176) (TANABE SEIYAKU CO. LTD) 1,7,10,14, S 18 December 1980 (18.12.80) 19,20 Advances in Biochemical Engineering, Volume 23, 1,4,6-12, issued 1982 (Heidelberg, West Germany) P.L. Rogers, 17-20 K.J. Lee, M.L. Skotnicki and D.E. Tribe, 'Ethanol Production by Zymomonas mobilis', see pages 34-84 Continued SSpecial categories of cited documents: to later document ouolished after the international filing date or priority date and not in conflict with the apolication out document defining the general state of the art which is not cited to understand the princlole or theory underlying the considered to be of particular relevance invention earlier document but published on or after the international document of particular relevance: the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or Involve an inventive step which is cited to establish the publication date of another document of particular relevance; the claimed invention citation or other special reason (as soecified) cannot be considered to involve In inventive steo when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination eling obvious to a person skilled document published prior to the international filing date but in the art, later than the priority date claimed document member of the same patent family IV, CERTIFICATION Date of the Actual Completion of the International Search ate of Miling of t ti International Search Report 22 April 1986 (22.04.86) iQ_. i l t >Y /91 ii I: *I- International Searching Authority Australian Patent Office Signature of horle fric J.W. ASHMAN Form PCT/ISAI210 (second sheet) (January 19S) i' -r I- -L-I-Cs;LU;r~PI -~-nT~LlrXI( International ApIlication No, PCT/AU 86/00042 III DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) C3aegory Ctain of Document, winl indication, where approprate. of me relevant passages Relevant to Claim No European Journal of Applied Biotechnology, Volume 11, issued 1981 (Heidelberg, West Germany) S. Cromie and H.W.Doelle, 'Nutritional Effects on the Kinetics of Ethanol Production from Glucose by Zymomonas mobilis' see pages 116-119 S Biotechnology Letters, Volume 4 No. 4, issued April 1982 (Surrey, England) H.J.J. Van Vuuren and L. Meyer, 'Production of Ethanol from Sugar Cane Molasses', see pages 253-256 y Biotechnology Letters, Volume 3 No. 5, issued Mayri981 (Surrey, England) E. Lyness and H.W. Doelle, 'Ethanol Production from Cane Juice by Zymomonas mobilis', see pages 257-260 X x /1 Biotechnology Letters, Volume 2 No. 12, issued December 1980 (Surrey, England) E. Lyness and H.W. Doelle, 'Effect of Temperature on Sucrose to Ethanol Conversion by Zymomonas mobilis Strains', see pages 549-554 Applied Microbiology and Biotechnology, Volume 20, issued 1984 (Heidelberg, West Germany) K.D. Barrow, J.G. Collins, D.A. Leigh, Rogers and R.G. Warr, 'Sorbitol Production by Zymomonas mobilis', see pages 225-232 Applied Microbiology and Biotechnology, Volume 20, issued 1984 (Heidelberg, West Germany) L. Viikara, 'Formation of Sorbitol by Zymomonas mobilis', see pages 118-123 Applied Microbiology and Biotechnology, Volume 19, issued 1984 (Heidelberg, West Germany) L. Viikari, 'Formation of Levan and Sorbitol from Sucrose by Zymomonas mobilis', see pages 252-255 Biotechnology Letters, Volume No. 10, issued October 1979 (Surrey, England) K.J. Lee, D.E. Tribe, P.L. Rogers, 'Ethanol Production by Zymomonas mobilis in Continuous Culture at High Glucose Concentrations' see pages 421-426 1,6,8-15,

17-20 1,6,8,12,14, 17-20 1,4,6,12,14, 17-20 1,4,6-10,12, 14,15,17-20 1,6-10,12, 17-20 1,6-10,12, 17-20 1,6-10,12, 17-20 1,6-10,12, 17-20 Form PCT ISA 210 (extra sheet) (January 1985) c u I l-- ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL APPLICATION NO. PCT/AU 86/00042 This Annex lists the known publication level patent family members relating to the patent documents cited in the above-mentioned international search report. The Australian Patent Office is in no way liable tion. for these particulars which are merely given for the purpose of informal Patent Document Cited in Search Patent Family Members Report AU 29530/84 BR 8404428 EP 142230 ES 531928 JP 60214887 ZA 8402294 AU 78199/81 BR 8107912 DE 3148329 FR 2495637 US 4443543 WO 8201563 AU 77269/81 EP 63146 AU 67697/81 BR 8101334 BR 8101335 CA 1173381 CA 1174191 DE 3108384 DE 3108386 FR 2477571 FR 2477572 GB 2074188 JP 56164790 NZ 196399 US 4403034 US 4443544 ZA 8101434 ZA 8101435 PH 17444 AU 59181/80 BR 8003540 DE 3022063 ES 492372 FI 801829 FR 2458586 GB 2055121 IN 154144 JP 55165796 PH 16533 SE 8004386 SU 1181555 US 4350765 ZA 8003474 CA 1143307 JP 56048887 END OF ANNEX i International Application No. PCT/AU 86/00042 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET VE 1 OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABSLE This International search reort has not been established in resoect of certain claims under Artilte 17(2) lOr the following reasons: iCtCaim numbers because they (etate to subtect metter not required to be searched by this Authority, namely; Ctaim numbers because they relate to oaris of the internationat soolication that do not compty with the prescribed require- m~ents to such an eatent that no meaning~ful international search can be carried out, soeciticaily claim numemers.......because they are dedrt cisir.4 and are rot drafiedI in accordlance withl trie sairtd and third seintences of PCT Rule 6.44a). OBSERVATIONS WHERE UNITY OF INVENTION tS LACKIPNG This International Searching Authority found multipie Inventions in. this International application as fotlowi: 1,7 As aii required additional search fees were timnoly paid by the applicant, this International search report covers all saarchaoie claims of the international application. 27As oniy some of the required additional search fees were fimely paid by the epolicant, this international search report covers only those claims of the International application for which fae were paid. spelcifidally clims. H.7 o required additional search feea were timely paid by the applicant. Consequently, this international search report Ia restricted to the invention first mentioned In the claims; It Is covered by claim numbers; 4,JAs ail searchsble claims could be searched without effort justifying an additional fee, the lnternaticnal Searching Authority did not Invite payment of any additionai fee. Remarki on Proteat E The additional search fees were accompanied by applicant's protest. C] No protest accompanied the payment of additional search fees. Form PCTIISA1210 (supplemental areat l(tanuary 19ssi