WO1986004357A1

WO1986004357A1 - Conversion of sucrose to ethanol using the bacterium zymomonas mobilis

Info

Publication number: WO1986004357A1
Application number: PCT/AU1986/000015
Authority: WO
Inventors: Horst Werner Doelle
Original assignee: University Of Queensland
Priority date: 1985-01-25
Filing date: 1986-01-24
Publication date: 1986-07-31
Also published as: AU5397086A; ZW1586A1; GB8716796D0; NL8600158A; MY101382A; AU603333B2; NZ214933A; EP0247044A4; GB2191503A; ES551206A0; ES8702327A1; KR870700097A; EP0247044A1; BR8606997A

Abstract

A new single-stage fermentation process for the commercial production of ethanol from refined sucrose, raw sugar, sugar cane juice, sugar cane syrup, sucrose hydrolysates and invert sugars has been developed using $i(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. Reinoculation is carried out from the fermented broth without the need for centrifugation or membrane filtration.

Description

Title: "CONVERSION OF SUCROSE TO ETHANOL USING THE BACTERIUM ZYMOMONAS MOBILIS" BACKGROUND OF THE INVENTION

(1) 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.

(2) Prior Art The traditional process of ethanol production is carried out in a two-stage batch process using yeast, whereby the first stage involves an aerobic propagation of the yeast referred to as the growth stage and the second stage involves the anaerobic process of ethanol production in the presence or absence of small amounts of oxygen. In order to further propagate yeast during the ethanol producing second stage, a slight addition of air or oxygen is required. The latter is required if the efficiency of the total process is to be increased using the occasional recycling of yeast cells by systems such as sedimentation or centrifugation. Since yeast fermentation is inherently dependent on coupling of growth with rate of ethanol production, to optimize ethanol production the medium must either be supplemented with growth enhancing substances or with finely controlled aeration.

The traditional yeast fermentation process (stage 2) is therefore dependent on large inoculum size of approximately 5 to 10 million cells per ml. The preferred optimal temperature of fermentation is 30° C and heat produced has to be controlled through the use of cooling equipment. The fermentation time for obtaining between 9 and 11% (v/v) ethanol is 30 to 70 hours with stage 2 batch fermentation. The time of this fermentation can be reduced to 10 hours by increasing the inoculum density by 80-100 fold through cell recycling.

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 1). 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% (w/v) and thus the stage requires a stepwise or continuous addition of a concentrated sugar solution. The preferred temperature is 28° C to 33° C and the pref.erred pH is about 5.5. This process may also require a supply of nitrogen as well as additional nutrients.

A third process for ethanol production has been described, which utilizes immobilized yeast or strains of Zymomonas in a two-stage process, each with a limited amount of sugar (i.e. 10% w/v) present (see British Patent No. 2055121 - Tanabe Sugaku Co. Ltd.).

In the case of yeast fermentation the examples for carbon source conversion are known to be sucrose, glucose, molasses and sugar cane juice, whereas in the case of the two-stage process utilizing Zymomonas the examples are limited to glucose, and in the case of the immobilized cells, to glucose and molasses. 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 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 (or its hydrolysis products or invert sugars) in a fermentation characterized by fermenting sucrose with the micro-organism Zymomonas mobilis in a single-stage process under microaerophilic conditions wherein the sucrose is contained in a fermentation medium.

Throughout the specification and claims the term "sucrose" shall be used to also include its hydrolysis products and invert sugars.

A "single-stage process" is defined as a process whereby growth and the production phase occur in the same fermenter vessel. Initiation of the process can be done either by a seed culture containing

Zymomonas mobilis added to the fermenter vejssel containing the fermentation medium or by adding the fermentation medium to the fermenter which contains a portion of the fermented medium from a previous fermentation run, the fermented medium containing Zymomonas mobilis.

"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 Zymomonas mobilis organism does not require air or oxygen (aerobic) or nitrogen (anaerobic) from growth and production of ethanol, but can tolerate the presence of air on the surface of the fermentation medium . The preferred strain of the micro-organism Zymomonas mobilis has been deposited in the culture collection of the University of Queensland, Microbiology Department, St. Lucia, Queensland, Australia under Deposit No. UQM 2716 and in the American Type Culture Collection (ATCC) 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. on the 24th April, 1984 under Deposit No. 39676.

This strain was derived by selection using continuous cultivation techniques from the strain deposited under Deposit No. NCIB 11199 at the National Collection of Industrial Bacteria, Torry Research Station, Abbey Road, Aberdeen, United Kingdom AB9 8DG and under Deposit No. 29191 at the ATCC. The selection was determined on improved performance and metabolic rate of sucrose conversion and these features are the only difference in the taxonom.ic description of the parent strain ATCC No. 29191 set out at pages 576-580 of "Bergy'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, crushed sugar cane juice, sugar beet juice, molasses (containing sucrose residues, sucrose hydrolysates and/or invert sugars) or the like or in combination of any other named substrates. Preferably the sucrose concentration is in the range of 10-30% with a concentration of 15-25% being more preferred for maximum ethanol yield, a concentration of 20% being most preferred.

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); potassium dihydrogen phosphate; ammonium sulphate

( (NH₄ )₂SO₄) or urea; and magnesium sulphate

(MgSO₄. 7 H₂O). 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 7.0. As the fermentation process proceeds the pH drops and then after e.g. 1-2 hours, the pH is maintained in the range of 5.0 to 6.0. 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 34° C to 40° C, with a constant temperature control at 35° 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 EMBODIMENTS

To enable the invention to be fully understood, preferred examples of the method will now be described. "In all Examples, percentages are expressed as "% (w/v)" where 1% corresponds to 10 g/L." EXAMPLE 1 : (Initial growth and production phases - 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% KH₂PO₄, 0.2% MgSO₂. 7 H₂O, 0.2% ((NH₄)₂SO₄) at 37° 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 g/L). Cultivation was carried out at a temperature of 35° 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% (w/v). 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.

10 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 35° 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 8.5% (w/v). EXAMPLE 3 : (Growth and production phases - Laboratory Scale) 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% (NH₄)₂SO₄, 0.2% MgSO₄. 7 H₂O and 0.2% KH₂PO₄ 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 35° C with a stirring rate of 100 rpm.

After 10 hours the fermentation was complete giving an ethanol concentration of 4.7% (w/v) 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 same 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 35° C with a stirring rate of 100 rpm. After 19 hours the fermentation was complete giving an ethanol concentration of 8.94% (w/v) 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 fermentation 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) as 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% MgSO₄. 7 H₂O and 0.02% KH₂PO₄. (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% (NH₄ )₂SO₄, 0.2% MgSO₄. 7 H₂O, and 0.2% KH₂PO₄ 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 6.0, 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 35º C with a stirring rate of 100 rpm.

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

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 10% (w/v). 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) 9-0 L of a fermentation medium containing 1600 g of sucrose, 500 g molasses, 5.0 g (NH₄)₂SO₄, 5 g MgSO₄. 7 H₂O (total of 1800 g sucrose) were added to a 14.0 L fermentation vessel.

1.0 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 incubation was 35° C and the stirring rate 100 rpm.

After 36 hours, the sucrose was utilized and

89.6 g/L (8.96% w/v)) (or 11.3% v/v)) ethanol was produced. Four (4.0) litres of the culture medium was centrifuged for 10 minutes at 4,000 rpm and used as seed culture for the next fermentation run.

9.0 L of a fermentation medium containing 1500 g sucrose, 500 g molasses, 5 g (NH₄)₂SO₄, 5 g MgSO₄. 7 H₂O (total of 1600 g sucrose) were added to the 14.0 L fermentation vessel.

1.0 L of the resuspended culture from 4.0 L of the previous run was added. The pH was adjusted to

7.0 and the pH control was maintained at 5.0. The temperature of incubation was 35° C and the stirring rate

100 rpm.

After 17 hours the fermentation was complete and 76.69 g/L (7.67% (w/v)) (or 9.7% (v/v)) 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 of 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.

Claims

1. A method for the production of ethanol from sucrose (or its hydrolysis products or invert sugars) in a fermenter characterized by fermenting sucrose with the micro-organism Zymomonas mobilis in a single-stage process under microaerophilic conditions wherein the sucrose is contained in a fermentation medium.

2. A method according to Claim 1 characterized in that the Zymomonas mobilis is a strain deposited in the ATCC under Deposit No. 396?6 or a mutant or variant of ATCC 39676.

3. A method according 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 (which may contain sucrose hydrolysis products or invert sugars).

5. A method according to any one of Claims 1 to 4 characterized in that the sucrose concentration is in the range 10-30% (w/v).

6. A method according to Claim 5 characterized in that the sucrose concentration is in the range 15-25% (w/v).

7. A method according to Claim 6 characterized in that the sucrose concentration is 20% (w/v).

8. A method according to any one of Claims 1 to 7 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% (w/v).

9. A method according to Claim 8 characterized in that the concentration of each component is 0.2% (w/v).

10. A method according to any one of Claims 1 to 9 characterized in that the pH of the fermentation medium is maintained in the range 4.0 to 7.0.

11. A method according to Claim 11 characterized in that the pH is initially in the range 6.5 to 7.0, allowed to fall as fermentation commences and then maintained in the range 5.0 to 6.0.

12. A method according to any one of Claims 1 to 11 characterized in that the temperature in the fermenter is maintained in the range 34º C to 40° C.

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

14. A method according to any one of Claims 1 to 13 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.

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

16. A method according to Claim 15 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.

17. A strain of Zymomonas mobilis deposited in the ATCC under Deposit No. 39676.

18. A mutant or variant Zymomonas mobilis strain of ATCC 39676.

19. Ethanol produced from sucrose (or its hydrolysis products or invert sugars) by the method according to any one of Claims 1 to 16.