AU603333B2 - Conversion of sucrose to ethanol using the bacterium zymomonas mobilis - Google Patents

Description

AU-AI 53970/ 86 6 PCT WORLD IN

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UA OP Y ANI\ I ,1 1 Ar J INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 86/ 04357 C12P 007/06, C12N 001/20 Al (43) International Publication Date: 31 July 1986 (31.07.86) (21) International Application Number: PCT/AU86/00015 (81) Designated States: AT (European patent), AU, BB, BE (European patent), BR, CH (European patent), DE (22) International Filing Date: 24 January 1986 (24.01.86) (European patent), FR (European patent), GB, GB (European patent), HU, IT (European patent), JP, KR, LK, LU (European patent), NL (European pa- (31) Priority Applicat un Numbers: PG 9046 tent), SD, SE (European patent), SU, US.

PG 9422 (32) Priority Dates: 25 January 1985 (25.01.85) Published 22 February 1985 (22.02.85) With international search report.

(33) Priority Country:.

AU

This doc.r,,. :n rin'; the (71) Applicant (for all designated States except US): UNI- ainC!' S *'dr VERSITY OF QUEENSLAND [AU/AU]; St. Lucia, Sction 4 ijr QLD 4067 printing.

(72) Inventor; and Inventor/Applicant ,(for US only) DOELLE, Horst, Werner [AU/AU]; Belsize Street, Kenmore, QLD 4069 A.Q. J P. 1 SEP 1986

(AU).

(74) Agent: GRANT ADAMS COMPANY; 333 Adelaide AUSTRALIAN Street, G.P.O. Box 1413, Brisbane, QLD 4000 1 3 AUG 1986 PATENT OFFICE (54) Title; CONVERSION OF SUCROSE TO ETHANOL USING THE BACTERIUM ZYMOMONAS MOBILIS (57) Abstract A new single-stage fermentation process for the commercial production of ethanol from refined sucrose, raw sugar, sugar cane juice, suar cane syrup, sucrose hydrolysates and invert sugars has been developed using Zymomonas mobilis.

The process gives a 94-98% sucrose hydrolysis efficiency and a 95-98% ethanol conversion efficiency. Within 24-30 hours; 200 g/L sucrose is converted to produce 95.5 g/L ethanol. Reinocu.':ion is carried out from the fermented broth without the need for centrifugation or membrane filtration.

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r r WO 86/04357 PCT/A U86/00015 -1- Title: "CONVERSION OF SUCROSE TO ETHANOL USING THE BACTERIUM ZYMOMONAS MOBILIS" BACKGROUND OF THE INVENTION Field of Invention This invention relates to a method for converting sucrose to ethanol in a single stage fermentation process using high efficiency strains of the bacterium Zymomonas mobilis in microaerophilic conditions.

Prior Art The traditional process of ethanol production is carried out in a two-stage batch process using yeast, whereby the first stage involves an aerobic propagation of the yeast referred to as the growth stage and the second stage involves the anaerob'ic 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 iermentation 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 300 C and heat produced has to be controlled through the use of cooling equipment. The fermentation time for 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 SUBSTITUTE SHE- I i 1 WO 86/04357 PCT/AU86/00015 -2increasing the inoculum density by 80-100 fold through cell recycling.

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 not 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 280 C to 330 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.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for producing ethanol from sucrose in a single-stage process using the bacterium Zymomonas mobilis.

It is a preferred object of the present invention to provide such a method using single-stage T ss ;.i-t~rdL batch fermentation, or if required, adjustments to this culturing method, e.g. fed-batch, continuous or multi-stage system, where the energy input is low.

It is a further preferred object to provide a method whereby the purity of the sucrose is not vital to the method.

In one aspect the present invention resides in a method for the production of ethanol from sucrose in a fermenter vessel characterized by: ee 10 fermenting the sucrose with a strain of the microorganism Zymomonas mobilis, the fermentation being carried out under microaerophilic conditions (as herein-before defined), in a single-stage process where the growth of the micro-organism and the 15 fermentation of the sucrose to ethanol by the microorganism occur in the same fermenter vessel, the sucrose concentration in the fermentation medium being in the range of 10-30% s A "single-stage process" is defined as a 20 process whereby growth and the ethanol production phase occur in the same fermenter vessel. Initiation of the process can be done either be a seed culture containing Zymomonas mobilis added to the fermenter vessel containing the fermentation medium or by adding the fermentation medium to the fermenter which contains a portion of the fermented medium

II

-ili i -iL i from a previous fermentation 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 atmosphere.

The Zvmomonas mobilis organism does not require air or oxygen (aerobic) or nitrogen (anaerobic) for growth and production of ethanol, but can tolerate 10 the presence of air on the surface of the fermentation medium.

The preferred strain of the micro-organism Zvmomonas mobilis has been deposited in the culture collection of the University of Queensland, Microbiology Department, St.Lucia, Queensland, 4067, Australia, under Deposit No. UQM 2716 and in the American Type Culture Collection (ATCC) 12301 Parklawn Drive, Rockville, Maryland, 20852, U.S.A.

on 24th April, 1984 under Deposit No. 39676.

20 This strain was derived by selection using continuous cultivation techniques from the strain o deposited under Deposit No. NCIB 11199 at the National Collection of Industrial Bacteria, Torrey Research Station, Abbey Road, Aberdeen, AB9 8DG, United Kingdom and under Deposit No. 29191 at the ATCC. The selection was determined on improved performance and metabolic rate of sucrose conversion

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4

I

-4aand 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 parent strain NCIB 11199/ATCC No. 29191 and other strains of Zymomonas mobilis may also be used.

Preferably the sucrose is obtained from sugar cane or sugar beet and may be supplied to the fermenter in the form of refined sugar, raw sugar, 10 crushed sugar cane juice, sugar beet juice, molasses or like or in combination of any other named substrates.

I. Preferably the sucrose concentration is in the range of 15-25% a concentration of 20% being most preferred for maximum ethanol yield.

In the case of refined or raw sugar, preferably the medium includes any one or more of the following components: yeast extract, peptone (casein hydrolysate); Se* :00 0o0 WO 86/04357 PCT/AU86/00015 potassium dihydrogen phosphate; ammonium sulphate ((NH 2

SO

4 or urea; and magnesium sulphate (MgSO 4 7 H 2 Preferably the components are provided in the concentration range of 0.01 to 0.5% each, with approximately 0.2% being preferred.

The abovenamed medium components may be replaced by the addition of sugar cane juice, sugar beet juice or molasses in appropriate concentrations as the medium components may be found in the juice or molasses.

In the case of any of the following; sugar cane juice or concentrate, sugar beet juice or concentrate and molasses as fermentation substrates, no additional supplements of any kind may be required.

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 6.0. The pH range may be controlled by the addition of NaOH or other suitable alkali.

Preferably the temperature in the fermenter is maintained in the range of 340 C to 400 C, with a constant temperature control at 350 C being preferred.

Preferably, when the fermentation has been completed, the micro-organism is separated from the fermentation medium, e.g. by filtration, centrifugation etc., and the ethanol is distilled off.

DETAILED DESCRIPTION OF THE PREFERRED EMBODI-

MENTS

To enable the invention to be fully understood, preferred examples of the method will now be described.

"In all Examples, percentages are expressed as where 1% corresponds to 10 g/L." EXAMPLE 1: (Initial growth and production phases SUBSTITUTE

SHEET

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WO 86/04357 PCT/AU86/00015 -6- Laboratory Scale) Sugar Cane Juice as Substrates: 1800 mL of sugar cane juice containing 12.1% sucrose was placed in a 2.0 L fermentation vessel.

200 mL of a 12-24 hours seed culture of Zymomonas mobilis (ATCC No. 39676) grown under microaerophilic condition in a medium containing 10% sucrose, 0.2% yeast extract, 0.2% casein hydrolysate (peptone), 0.2% KH 2 PO0, 0.2% MgSO 2 7 H 2 0, 0.2% ((NH 4 2

SO

4 at 370 was added to the fermenter vessel. The pH of the fermentation process was brought to 6.8 before initiation.

Over the first 1-2 hours, the pH dropped to 6.0 and was thereafter controlled at 6.0 by the addition of 2N NaOH (80 Cultivation was carried out at a temperature of 350 C with a stirring rate of 100 rpm.

After 24 hours the utilization of the sugar was complete giving an ethanol concentration of 60.8 g/L or 6.08% EXAMPLE 2: (Growth and production phases Laboratory Scale) Sugar Cane Juice as Substrate: 90 L of sugar cane juice containing 15.2% sucrose was placed into a 100 L fermentation vessel.

L of a 12-24 hours seed culture of Zymomonas mobilis (ATCC 39676) grown in the same medium as in Example 1 was added to the fermentation vessel.

The initial pH was brought to 6.8 and pH was then maintained during the fermentation at 6.0 or 5.0 by the addition of 8 N NaOH. Cultivation was carried out at a temperature of 350 C with a stirring rate of 250 rpm.

After 26 hours the utilization of the sugar was complete giving an ethanol concentration of 85 g/L or EXAMPLE 3: (Growth and production phases Laboratory Scale) B T T TE- S WO 86/04357 PCT/AU86/00015 -7- Refined Sugar as Substrate: 1800 mL of a fermenation medium containing 9.69% (96.94 g/L) refined sucrose, 0.2% yeast extract, 0.2% casein hydrolysate (peptone), 0.2% (NH 4 2 S0 4 0.2% MgSO 4 7 H20 and

KH

2

PO

4 were placed into a 2.0 L fermentation vessel.

200 mL of a 12-24 hours seed culture of Zymomonas mobilis (ATCC 39676) grown in the same medium as described in'Example 1 was added to the fermenter vessel. The initial pH was brought to 6.8 and pH was then maintained at 5.0 or 6.0 by the addition of 2 N NaOH. Cultivation was carried out at a temperature of 350 C with a stirring rate of 100 rpm.

After 10 hours the fermentation was complete giving an ethanol concentration of 4.7% or 47.22 g/L.

EXAMPLE 4: (Growth and production phases Laboratory Scale) Refined Sugar as Substrate: 1800 ml of a fermentation medium containing 18.25% (182.54 g/L) refined sucrose plus the media components described in Example 3 were placed into a 2.0 L fermentation vessel.

200 mL of a 12-25 hours seed culture Zymomonas mobilis (ATCC 39676) grown in the s ,me medium as described in Example 1 was added to the fermentation vessel. The initial pH was brought to 6.8 and pH was then maintained at 5.0 or 6.0 by the addition of 2 N NaOH. Cultivation was carried out at a temperature of 350 C with a stirring rate of 100 rpm.

After 19 hours the fermentation was complete giving an ethanol concentration of 8.94% or 89.44 g/L.

EXAMPLE 5: (Production phase Laboratory Scale) Refined Sugar as Substrate: This example is concerned with the continuous use of bacterial SUBSTITUTE

SHEET

WO 86/435 P- CT/A;- U86/O00 WO 86/04357 PCT/A U86/00015 -8fermentation broth from one fermenter to be used for the subsequent batch process as seed culture.

200 mL of fermentation broth from Example 4 was used as seed culture for 1800 mL fermentation medium of the same composition. Under identical cultivation conditions, the fermentation was complete after 19 hours giving the same ethanol concentration.

This reuse does not require any filtration, concentration or centrifugation and has been carried out successfully up to 6 times.

EXAMPLE 6: (Production phase Laboratory Scale) Mill Raw Sugar (Sucrose) a3 Substrate: The fermentation was carried out using mill raw sugar and the fermentation broth of Example 4 and the only supplementation needed was 0.05% calcium pantothenate, 0.02% MgSO 4 7 H 2 0 and 0.02% KH 2

PO

4 .(No supplementation is .required for sugar cane juice or concentrate as the substrate.) EXAMPLE 7: (Production phase Laboratory Scale) Refined Sugar as Substrate: 1800 mL of a fermentation medium containing 15% (150.0 g/L) refined sucrose, 0.2% yeast extract, 0.2% casein hydrolysate (peptone), 0.2% (NH 4 2

SO

4 0.2% MgSO 4 7 H20, and 0.2%

KH

2 PO4 were placed into a 3.0 L fermentation vessel.

200 mL of a 12-24 hours seed culture of Zymomonas mobilis (NCIB 11199/ATCC 29191) grown in the same medium was added to the fermenter vessel. The pH was brought to 6.8 at the start of the fermentation, and after the culture decreased the pH to 5.0 or the pH was maintained at 5.0 or 6.0 by the addition of 2 N NaOH. Cultivation was carried out at a temperature of 350 C with a stirring rate of 100 rpm.

After 45 hours the fermentation was complete giving an ethanol concentration of 6.1% or 61.21 g/L.

SUBSTITUTE SHEET WO 86/04357 PCT/AU86/00015 -9- EXAMPLE 8: (Growth and production phases Commercial Scale) 100 L of the fermentation broth of Example 4 was used as a seed culture for 3,000 L of a fermentation vessel. The initial pH was brought to 6.8, and after the culture decreased the pH to 5.0, it was maintained at 5.0 by the addition of 2 NaOH.

After 12-24 hours, 27,000 L of the fermentation medium was added to the vessel to fill the vessel and the fermentation conditions above were repeated.

After 24-30 hours, 27,000 L of the fermented medium was pumped from the vessel with an ethanol concentration of 9.5% To the 3,000 L of the fermented medium remaining in the vessel was added 27,000 L of the fermentation medium and the fermentation process was repeated.

The fermentation process, using fermented medium from a preceding process as an inoculum for the Zymomonas mobilis, was repeated several times and the ethanol concentration remained in the range of 9.5 to 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 addition of the fresh fermentation medium.

EXAMPLE 9: (Growth and production phases Laboratory Scale) L of a fermentation medium containing 1600 g of sucrose, 500 g molasses, 5.0 g (NH4) 2 S0 4 5 g MgSO 4 7 H 2 0 (total of 1800 g sucrose) were added to a 14.0 L fermentation vessel.

L of a seed culture of Zymomonas mobilis (ATCC 39676) grown in a medium as described in earlier examples was added. The pH was adjusted to 7.0 and the pH control was maintained at 6.0. The temperature of SU) POITITurF SH WO- 8 PC/A86001 Wo 86/04357 PCT/A U86/QO015 incubation was 350 C and the stirring rate 100 rpm.

After 36 hours, the sucrose was utilized and 89.6 g/L (8.96% (or 11.3% ethanol was produced.

Four litres of the culture medium was centrifuged for 10 minutes at 4,000 rpm and used as seed culture for the next fermentation run.

L of a fermentation medium containing 1500 g sucrose, 500 g molasses, 5 g (NH4) 2 SO4, 5 g MgS0 4 7 H20 (total of 1600 g sucrose) were added to the 14.0 L fermentation vessel.

L of the resuspended culture from 4.0 L of the previous run was added. The pH was adjusted to and the pH control was maintained at 5.0. The temperature of incubation was 350 C and the stirring rate 100 rpm.

After 17 hours the fermentation was complete and 76.69 g/L (7.67% (or 9.7% ethanol was produced.

TABLE 1 gives the results of fermentation experiments with the 2.0 L bench-top and 100 L pilot plant fermenters using a variety of refined sugar (sucrose) concentrations.

The fermentation process can be carried out using other strains of Zymomonas mobilis, including parent strain NCIB 11199/ATCC 29191 but the best results are produced using the ATCC 39676 strain.

The ethanol produced has commercial value as a component for gasohol or as a base product in the chemical industry e.g. for the production of ethylene, while the other by-product, carbon dioxide, may be used for dry ice or as a carbon source for the growth of algae biomass.

The fermentation process requires only a low energy input as the micro-organism produces a fair amount ISU 83st!TUTF. .43HkEr WO 86045 P; CT/A- U86/000 WO 86/04357 PCT/A U8/00015 -11of heat during the fermentation process. In addition, the fermentation is carried out in microaerophilic conditions, avoiding the need for aerating or addition of nitrogen pumps, the fermentation components and products only requiring little mechanical stirring and pH control.

Experiments have shown that the success of the fermentation process is not wholly dependent on the quality of the sucrose as substrate. Successful tests have been carried out even on rotting cane which indicates that the process is particularly suited for industrial application and the fermenter can be adjacent a sugar mill to reduce transport costs. As the sugar cane juice does not have to be sterilized, the energy input can be kept low.

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

SUBSTITUTE SHEET TABLE 1 Sucrose initial Sucrose residual gIL Sucrose hydr'olysis efficiency Ethanol gIL Ethanol theoret g/L Conversion efficiency (Hydrolysed sucrose to ethanol Fermentation time h gIL 96.9 41.85 95.0 47.0 49,52 94I.9 121.0* 3.63 97.0 60.8. 63.1~4 96.2 29 152.0 6.94 95.)4 75.0 78.1 96.1 2)4 179.7 11.26 93.7 83*.7 90.62 92.41 2)4 182.5 7.3 96.0 89.41 9)4.25 9)4.8 2)4 200.0 11.25 9)4.0 95.5 101.54 9)4.1 32 229.2 7.63 96.6 96.3 119.20 80.8 32 250.0 7.64 96.9 96.5 130.)4 741.0 32 '166.2 25.414 90.41 87.0 12.5 67232 268.9 25.75 90.41 105.41 130.81 80.57 32 Pilot plant fermentation with totaLl volume of 100 litre>.

Ethanol production from sucrose as substrate- Zymomonas mobilis using refined sugar (sucrose) 0 -7 WO 86/04357 PCT/AU86/00015 -13- International Application No: PCT/

MICROORGANISMS

Optional Sheet In connection with the micrororganlem referred io on page lne-_ of the description S A. IDOINTIFICATION OF DIPOSIT I Further depoolts are identllfed on an additional shoeelt Name of depository Intiltution 4 AMERICAN TYPE CULTURE CENTRE (ATCC) Addressa f depositary inaltituten (Including postal ode and country) 4 12301 Parklawn Drive, Rockville, Maryland, 28852, U.S.A.

Daoe of depoit 24 April, 1984 Accession Number (24-04-84) ATCC No. 39676 U. ADDITIONAL INDICATIONS I (leave blank Ii not appollcable). This Information Ia continued on a separate attached she t C. DAoIONATED TATES FOR WHICH INDICATIONS ARE MADA I (it the Indications are not for all designated Slates) D. SEPARATE FURNISHING OF INDICATIONS I (leave blank If not applicable) The Indicatione listed below will be submitted o the Internallonal Bureau liater (Specify Ihe general nature of the Indications e.g., Accession Number of Deposit") I. This sheet was received with the International appllction when filed (to be checked by the recelving Offlce) (Authorized Ofcer) U The dete of receipt (from the applicant) by the Internallonal Bureau Is was (Authorled Ofrcer) Form PCT/RO/134 (January till) SUBSTITUTE SHE-

Claims (13)

1. A method for the production of ethanol from sucrose in a fermentation medium in a fermenter vessel characterized by: fermenting the sucrose with a strain of the micro-organism Zymomonas mobilis, the fermentation being carried out under microaerophilic conditions (as herein- before defined), in a single-stage process where the growth of the micro-organism and the fermentation of the sucrose to ethanol by the micro-organism occur in the same fermenter vessel, the sucrose concentration in the S* fermentation medium being in the range of 10-30%

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

3. A methodaccording to Claim 1 characterized in that the Zymomonas mobilis is a strain deposited in the ATCC under Deposit No. 29191.

4. A method according to any one of Claims 1 to 3 characterized in that the sucrose is added to the fermentation medium in the form of refined sugar, raw sugar, crushed sugar cane juice, sugar beet juice, molasses or a combination of two or more of these. A method according to any of Claims 1 to 4 characterized in that the sucrose concentration is in the range 15-25% I i Y is -ts-

6. A method according to Claim 5 characterized in that the sucrose concentration is 20%

7. A method according to any one of Claims 1 to 6 characterized in that the fermentation medium contains one or more of the following components: peptone, yeast extract, potassium dihydrogenphosphate, ammonium sulphate, urea or magnesium sulphate, the concentration of each component being in the range 0.01 to 0.5%

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

9. A method according to any one of Claims 1 to 8 00 g 00* characterized in that the pH of the fermentation medium S. is maintained in the range 4.0 to A method according to Claim 10 characterized in that the pH is initially in the rang, 6.5 to 7.0, allowed to fall as fermentation commences and then maintained in the range 5.0 to

11. A method according to any one of Claims 1 to characterized in that the temperature in the fermenter is see. maintained in the range 340 to 40 0 C.

12. A method according to Claim 11 characterized in that the temperature is maintained at 35 0 C.

13. A method according to any one of Claims 1 to 12 characterized in that, when the fermentation is completed, the micro-organism Zymomonas mobilis is separated from the fermentation medium and the ethanol is distilled off. I L-31~--g-~CI---~I1IIICI UIIIIP I LI DUI DYPLI-L~- I

14. A method according to Claim 1 characterized in that the Zymomonas mobilis comprises Zymomonas mobilis bacterial cells suspended in the fermentation medium. A method according to Claim 14 characterized in that a portion of the fermented medium from a preceding fermentation is added to the fermenter as the inoculum of the Zymomonas mobilis bacterial cells for the succeeding fermentation.

16. Ethanol produced from sucrose by the method according to any one of Claims 1 to S S.. @0 0 OSS g m. 0 *0 0 gO go 0 S em S gem goof *0 0 .me, e *0 0 0000 0OGS S m INTERNATIONAL SEARCH REPORT 1.i-.C18 Ati.i,ia(' No. PCT/AU 86/00015 IC L ASSitIC ATION Ofif igtC I MATIP i1 1*.r1 a. At CDrI0nq 1ii Iner r.01-r.i w C 16110 ii. (IP C tD. Iii toii, NIol.0 a clasi Cl-o iP'C Int. Cl. C12P 007/06, C12N 001/20 I FIELLDS StAPICNIO Molr.,uu DoLumentaoo Stoic thei,' Cloe I Ciees-f-cei-on Symbol% IPC C12G 003/02, C12P 007/06, 7/08, 7/10; Chemical Abstracts Keyword Zymomonas mobilis Documvinlalion Sea ochord otner tham M,nsrrium Documt~r'il,cn to the Ell Mri lha, such Docufmtni are Im~ctoe r, In# foC Sesrcpec a III DOCUMENTS CONSIDERED TO BE RELEVANT' CAtlego, Citaiof a Document. 1, .win iindicaiorn, wriere obcproprif, of thre ftrea~ paaa;ts It Relenenil to Cie. X,P AU,A, 29530/84 (UNIVERSITY OF QUEENSLAND) 4 April 1985 1-4,8-19 (04.04.85) X AU,B, 78199/81 (540186) (UNISEARCH LTD) 17 June 1982 1,4-6,8,i5, (17.06 .82) 16,19 If X WO,A, 82/01563 MATTIASSON, B. HAGERDAL, 1,5-7,10,19 P. ALBERTSSON) 13 May 1982 (13.05.82) X AU,B, 67697/81 (537029) (UNISEARCH LTD) 19 November 1,4,5-8,10, 1981 (19.11.81) 15,16,19 X AU,B, 59181/80 (531176) (TANABE SEIYAKU CO. LTD) 1,5,6,10,19 18 December 1980 (18.12.80) X Advances in Biochemical Engineering, Volume 23, issued 1,3,5-11,15 1982 (Heidelberg, West Germany) P.L. Rogers, K.J. Lee, 16,19 M.L. Skotnicki and D.E. Tribe, 'Ethanol Production by Zymomonas mobilis', see pages 34-84 Conti nued *Special cattgoris o ci ctedi Documenits "ST" Ists, ocmei1 :V.1~hroc aft.' tre Internrational Al-; dale of pricilt l a niot or conflict wii tie goo, CaSC'o t. "Adocument doef-ring lire general stale 0 o te aml wohich is not 0.1Ct to urrnitro tire principle or nlit tha O ttl nbr' 1 Ire rtDriiteo to be of cparlrcila, iele6ernct munneiror, so, genidocunrment but published on or sti tire ifite'fi1tn1flal "It document of pa-icull' froloence, tire clirnme i.eo"o fl'In file cantor be co"1110Oa'ed ncooel or cannot be come:iCofIC iC document which may Ithrow doubter oft 01oi'le CIA-MI111 Of rIntolio ein Inrooeilie ase winch is Cited to sisillnish the yOUbotaiom Osle of another document Of Dar.Culal r~eeace, the Claimed ino@^-Cl Cntatrloo or other special lesson (as scooacifect Camrotcobe cottace'ao tcrnnoinofe an rnvnieton lo -hon theo 0cCUmeri refailing to an oral disclosure, uso. exhibtion or documnent .6 CIDDMSc with onie of more 01i"0 sci Do,,, Dther ?meant mania such Cominationi berng obvious to a peison abIlleC Pdocument publihed proof to tire intotarnhlonat fibme dote but in the cit totar them thre pricifiir data Climed documntr mainbe' of the same patent family IV CERTIFICATION Dale of the Actual Completion af the InternatIional Searchr D tIct Mahlig of tis Internatiornal Search Report 22 April 1986 (22.04.86) o j 9 inrternationlal %sarhng Authorilif SgiI1aa fjerrred Offi.e Australian Patent Office J.W. ASHMAN pornt PCTjtIArjlO reacond 111%940 IJefuP '111S 417 d International Aplication No. PCT/AU 86/00015 ill. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) Category Ciatoan of Document. with indication, where appropriate, of the relevant passages Relevant to Clam No X i European Journal of Applied Biotechnology, Volume 11, 1,8-13,15, Sissued 1981 (Heidelberg, West Germany) S. Cromie and 16,19 H.W. Doelle, 'Nutritional Effects on the Kinetics of Ethanol Production from Glucose by Zymomonas mobilil;', see pages 116-119 X Biotechnology Letters, Volume 4 No. 4, issued i 1,4,10,12,15 April 1982 (Surrey, England) H.J.J. Van Vuuren and 16,19 L. Meyer, 'Production of Ethanol from Sugar Cane Molasses', see pages 253-256 X Biotechnology Letters, Volume 3 No. 5, issued 1,3,4,10,12, May 1981 (Surrey, England) E. Lyness and H.W. Doelle, 13,15,16,19 'Ethanol Production from Cane Juice by Zymomonas mobilis', see pages 257-260 X Biotechnology Letters, Volume 2 No. 12, issued 1,3,5-8,10, December 1980 (Surrey, England) E. Lyness and 12,13,15,16, H.W. Doelle, 'Effect'of Temperature on Sucrose to 19 Ethanol Conversion by Zymomonas mobilis Strains', see pages 549-554 X Biotechnology Letters, Volume 1 No. 10, issued 1,5-7,10,15, October 1979 (Heidelberg, West Germany) K.J. Lee, 16,19 D.E. Tribe, P.L. Rogers, 'Ethanol Production by Zymomonas mobilis in' Cntinuous Culture at High Glucose Concentrations', see pages 421-426 Form PCT ISA 210 (extra heetl) (January 195) (I ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL APPLICATION NO. PCT/AU 86/00015 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 for these particulars which are merely given for the purpose of information. 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 W .8201563 EP 63146 .AU 77269/81 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 lne-to~l oi a'o toPCT/AU86/OOO FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET VR, OIBSERVATM~NS WHERE CERTAIN Z;LAIAS WERE FOUND UNSEARCHAULE This Intsrriacional silarch report has not been astaolished in respect of certain claims under Article 17(2) for the following reasons: IL Claim numbtrst. 7 .8 because they relate to subject matter not required to be searched by this Authority, namely: Plant or animal varieties 2. Claim numbiers because they relate to opans of the international application that do not Comply with the prescribed require- merits to such en extent that no meaningful international searcn can be carries olut, specifically 3Claim numoers...... becausisthey are deendentt claims ari are riot crafled in accoroance Vvith the sit,)nd arid third saenrces of PCT Rule 6 Vi.i_- ONSERVATIONS WHERtE UNITY OF INVENTION IS LACKING I This International Searching Authority found multiple Inventions In this International Application am follows: 1,71 As all required additional search lssa were timely paid by the applicant, this International search report Covers all searchable claims of the international application. 2.rI As only some of the required additional search fees were timely Paid by the aoplicant, this international search report covers only those claims of the International application for which fees were paid, specifically claims: No required additional search fees were timely paid by the mpolicont. Consequently, this international search report Is restricted to the Invention first mentioned In the claims;t It Is covered by claim numbers, 4 DAs all searchable claims could be searched without e#Mort justifying an additional fee, the International Searching Authority did not invite payment of any additional fee. Remark on Protet CDThe additional search fees were accomponled by applicant's Protest, HD o protest accompanied the Payment of additional search fees. Form PCTjISA2SO (supplemental street IJen~uarY 1965)