Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
  • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• 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.
• stage 2 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.
• 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.).
• 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.
• 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.
• 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.
• 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.
• the medium includes any one or more of the following components: yeast extract; peptone (casein hydrolysate); potassium dihydrogen phosphate; ammonium sulphate
• 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.
• sugar cane juice or concentrate, sugar beet juice or concentrate and molasses as fermentation substrates no additional supplements of any kind may be required.
• the pH of the fermentation process is maintained in the range of 4.0 to 7.0.
• 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.
• 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.
• the micro-organism is separated from the fermentation medium, e.g. by filtration, centrifugation etc., and the ethanol is distilled off.
• 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 PO 4 , 0.2% MgSO 2 . 7 H 2 O, 0.2% ((NH 4 ) 2 SO 4 ) 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.
• 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% (NH 4 ) 2 SO 4 , 0.2% MgSO 4 . 7 H 2 O and 0.2% KH 2 PO 4 were placed into a 2.0 L fermentation vessel.
• 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.
• This reuse does not require any filtration, concentration or centrifugation and has been carried out successfully up to 6 times.
• 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% MgSO 4 . 7 H 2 O 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 H 2 O, and 0.2% KH 2 PO 4 were placed into a 3.0 L fermentation vessel. 200 mL of a 12-24 hours seed, culture of
• 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.
• 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 (NH 4 ) 2 SO 4 , 5 g MgSO 4 . 7 H 2 O (total of 1800 g sucrose) were added to a 14.0 L fermentation vessel.
• sucrose was utilized and
• 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.
• 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.

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• 1986
  • 1986-01-21 ZW ZW15/86A patent/ZW1586A1/xx unknown
  • 1986-01-24 WO PCT/AU1986/000015 patent/WO1986004357A1/fr not_active Application Discontinuation
  • 1986-01-24 NL NL8600158A patent/NL8600158A/nl not_active Application Discontinuation
  • 1986-01-24 GB GB08716796A patent/GB2191503A/en not_active Withdrawn
  • 1986-01-24 ES ES86551206A patent/ES8702327A1/es not_active Expired
  • 1986-01-24 EP EP19860901009 patent/EP0247044A4/fr not_active Withdrawn
  • 1986-01-24 BR BR8606997A patent/BR8606997A/pt unknown
  • 1986-01-24 AU AU53970/86A patent/AU603333B2/en not_active Expired - Fee Related
  • 1986-01-24 NZ NZ214933A patent/NZ214933A/xx unknown
  • 1986-01-24 KR KR860700660A patent/KR870700097A/ko not_active Application Discontinuation
• 1987
  • 1987-09-30 MY MYPI87002630A patent/MY101382A/en unknown

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