CA1173381A - Ethanol production in a continuous process with cell recycle - Google Patents
Ethanol production in a continuous process with cell recycleInfo
- Publication number
- CA1173381A CA1173381A CA000371605A CA371605A CA1173381A CA 1173381 A CA1173381 A CA 1173381A CA 000371605 A CA000371605 A CA 000371605A CA 371605 A CA371605 A CA 371605A CA 1173381 A CA1173381 A CA 1173381A
- Authority
- CA
- Canada
- Prior art keywords
- culture medium
- ethanol
- cells
- zymomonas mobilis
- continuously
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/12—Bioreactors or fermenters specially adapted for specific uses for producing fuels or solvents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/18—External loop; Means for reintroduction of fermented biomass or liquid percolate
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/10—Separation or concentration of fermentation products
-
- 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
-
- 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
-
- 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
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/801—Anerobic cultivation
-
- 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
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/813—Continuous fermentation
-
- 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
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
Abstract
ABSTRACT
An improved method for the production of ethanol from a fermentable carbohydrate such as glucose, fructose or sucrose using the bacterium Zymomonas mobilis which method involves continuously anaerobically culturing the bacterium in a culture medium containing the fermentable carbohydrate, continuously or periodically drawing off a portion of the culture medium and replacing that portion with fresh culture medium, separating from the removed portion of the culture medium cells of Zymomonas mobilis contained therein, returning those cells to the culture medium, and recovering the ethanol contained in the portion of the culture medium from which the cells have been removed. It has been found that this method results in greatly improved ethanol production without the disadvantages which have been found when yeasts are continuously cultured.
An improved method for the production of ethanol from a fermentable carbohydrate such as glucose, fructose or sucrose using the bacterium Zymomonas mobilis which method involves continuously anaerobically culturing the bacterium in a culture medium containing the fermentable carbohydrate, continuously or periodically drawing off a portion of the culture medium and replacing that portion with fresh culture medium, separating from the removed portion of the culture medium cells of Zymomonas mobilis contained therein, returning those cells to the culture medium, and recovering the ethanol contained in the portion of the culture medium from which the cells have been removed. It has been found that this method results in greatly improved ethanol production without the disadvantages which have been found when yeasts are continuously cultured.
Description
~` ~1733~
The present invention relates to a method for the production of ethanol (alcohol) using the bacterium Z~momonas mobilis.
It is known to use the bacterium Zymomonas mobilis for the production of ethanol, and in fact this bacterium is used for the production of palm wine and other alcoholic beveraqes in tropical countries. It is also known that theoretically the production of ethanol using this bacterium has certain advantages as compared with yeasts which are conventionally used to produce alcohol by the fermentation of sugars, starches and other carbohydrate substrates. These advantages include the fact that Zymomonas mobilis produces less biomass than do yeasts due to the lower energy available for growth inherent in the Entner-Doudoroff pathway used by the bacterium which yields only one mole of ATP per mole of ~lucose me ~ olised as compared with the glycolytic pathway used by yeasts which yields two moles of ATP per mole of glucose. A further potential advantage is that Zymomonas ; mobilis grows anaerabically.
The present inventors have found that yreatly increased ethanol productivity can be achieved by continuously culturing Zymomonas mobilis with cell recycle.
It has been ~iscovered, and this could not have been predicted a priori, that continuous culture with cell recycle of zymomonas mobilis does not suffer from the difficulties encountered with continuous culture with cell recycle of conventional yeasts. These difficulties include the need to provide sterile oxygen to maintain yeast cell viabi~ity at high yeast concentrations, and the likelihood of contamination by unwanted bacteria during the extended period of continuous operation.
With Zymomonas mobilis fermentation using continuous -.. ~.
3 3 8 ~
culture with cell recycle as well as achieving higher ethanol productivities as compared with other techniques for Zymomonas mobilis fermentation and as compared with ;
yeast fermentation, it has been found that there is no requirement for oxygen to maintain viability at high cell concentrations. Furthermore due to the faster rates of sugar uptake and enhanced ethanol tolerance when compared to yeasts it has been found that no contamination occurs during extended periods of continuous operation.
The present invention consists in a process for the production of ethanol from a culture medium containing a fermentable carbohydrate which comprises continuously anaerobically culturing Zymomonas mobilis in the culture medium, continuously or periodically drawing off a `15 portion of the culture medium to give a withdrawn portion and replacing that portion with fresh culture medium, separating from the withdrawn portion of the culture medium cells of _ymomonas mobilis contained therein, ` returning the cells to the culture medium, and recovering the ethanol contained in the portion of the culture medium from which the cells have been removed.
;~The process according to the present invention is preferably carried out at a temperature of from 20C
to 50C, most preferably at 25 to 40~C, and at a pH
between 3.7 and 8, most preferably 4.5 to 6.5. The medium preferably contains from 100 to 400 g/l of a fermentable carbohydrate, most preferably from 150 to 300 g/l.
The preferred carbohydrates for use as fermentable substrates in the culture medium include, in addition to glucose, simple sugars such as fructose, lactose and sucrose, starch and starch hydrolysates, and cellulosic raw materials. It will be recognised that any one strain oE Zymomonas mobilis will probably not ferment all of these substrates and therefore for any particular strain a suitable, fermentable, substrate ~17338 ~
should be selected.
It is preferred that the strain of _mombnas mobilis used in this process is a strain having a high specific ethanol , ' :
.
¦ !
~ ~733~ ~
'`' productivity, a large specific rate of glucose uptake and a high alcohol tolerance. A particularly suitable organism is CP4 from the type culture collection of:-Dr. J. De Ley Laboratory of Microbiology, Ledeganckstraat 35 Belgium Mutants of this strain have also been found to be particularly useful in carrying out the present process and may have a broader range of fermentable substrates than CP4 itself. The mutant strains may be produced by J, mutations of an existing strain as by the use O.e u.v.
~ radiation or nitrosoguanidine. Desirable properties may also ; be introduced into the zymomonas mobilis strains by plasmid transfex from other bacteria using, for example, membrane filter mating techniques.
The separation of the cells from the removed portion of the culture medium may be achieved in any suitable manner.
The ~ells may be removed by the use of a cros~-flow microfiltration membrane system, preferably using a membrane having a pore size of the order of 0.6 microns.
Alternatively the cells may be separated in a centrifuge or in a series of hydroclones. If flocculent Zymomonas mobilis cells are used these may be removed from the culture medium by allow~ them to ~ettle in a settling vessel or a series of hydroclones.
The strains of Zymomonas mobilis referred to in this specification have been deposited in the American Type Culture Collection , 12301 Park Lawn Drive Rockville, Maryland 20852, U.S.A. and have been assigned the following deposit numbers and dates:
- Specification Reference ATCC Deposit No. Deposit Date CP4 31821 Feb. 26, 1981 ZM481 31823 Feb. 26, 1981 ZM40l 31822 Feb. 26, 1931 '~' ' . .
11~13381 Examples In the following examples the bacterium Zymomonas mobilis was cultured in a fermentation medium comprising the following nutrient concentrations.-100-3~0 g~l glucose ; 5 g/l yeast extract 1 9/l (NH4)2 S4 9/ 1 }~H ;2 PO 4 0-5 9/l MgS04 7H2O
The cultures of the various strain of Z~momonas mobilis were maintained by transferring to fresh agar slants containing 20 g glucose, 10 g yeast extract and 20 9 agar at - pH 5.0 each week and storing at room temperature.
~ The continuous fermentation system was controlled at a temperature of 30C and pH 5.0 in a 1 litre fermentatlon vessel. The pH was controlled by the addition of 2 N NaOH.
In these experiments cell recycle was carried out using a cross-flow microfiltration membrane system including a "Millipore"*membrane BD having a nominal pore si2e of 0.6 microns. This membrane retained all cells and the permeate passing through the membrane was free from cells and contained the same ethanol concentration as the contents of the fermenter.
Figure 1 shows the arrangement of the fermenter and cell recycle system. In this Figure 10 is a fermentation vessel fitted with a stirrer 11. The culture medium is introduced into the fermenter 10 through line 12 and is drawn off through line 13 to a pump 14 and thence to a tangential flow microfilter 15. The permeate through the filter 15 is discharged through line 16 to storage prior to recovery of the ethanol therefrom while the retained cell concentrate is returned through line 17 to the fermenter 10. An overflow line 18 is provided.
.
* ~Trademark . . .
~. :
, ~7 338~
Table I shows the greatly improved maximum ethanol productivity of three strains of Zymomonas mobilis continuously cultured with cell recycle as compared with two strains of the yeast Saccharomyces Cerevisiae.
TABLE I
; Organism Glucose Ethanol Maximum Concn cOncn Productivity t /1) (q/l) (g/l/h) g, _ _ _ _ , _ _ _ S. Cerevisiae 100 43 29 (ATCC 4126) : S~ Cerevisiae 150 61 32 . ~NRRL Y-132) - z. ~obilis 100 45 120 (ATCC 10988) Z. Mobilis 140 65 200 (CP4) (ATCC 31821) z. Mobilis 1~0 . 85 85 (ZM 481)(ATCC 31823) - 20 From the above table it is clear that the continuous culture , ~
of zymomonas mobilis with cell recycle can giv~
productivities wbich are 200 to 300% higher than for yeasts fermentations carried out under similar conditions~ This means that the size of the fermentation vessels to produce ethanol may be reduced by an e~uivalent amount.
The organism Zymomonas mobilis ZM 481 was developed from strain CP4 as follows:-Strain CP4 was grown statically at 30C until it wasin ~he exponential growth phase. Nitrosoguanidine was then added to a final concentration of 50 ug/ml and the culture was incubated for 30 minutes at 30C. The cells were then washed twice in saline phosphate buffer and regrown overnight . at 30C. The culture was then plated on plates containing ,, , , , .
l 1~338 10%(v/v) ethanol. After several days incubation at 30C~
mutant colonies appeared at a frequency of approximately 10 7, and these were purified on similar medium and then tested for growth and ethanol production rates with 100, 200 and 250 g/l glucose, at 30C in tubes. The culture which produced the highest level of ethanol in the shortest time was numbered ZM48.
A second similar mutagenesis was clone with strain ZM48 but the cells were plated with 15% (v/v) ethanol. The mutant which produced the highest level of ethanol in the shortest time was numbered ZM481, and was kept as an ethanol tolerant strain in the type culture collection in the School of Biotechnology, University of New South Wales, Sydney, New South Wales, Australia.
~ The use of flocculent strains of Zymomonas mobilis in the present invention is advantageous. Table II records the use of a flocculent strain of Zymomonas mcbilis usmg a set~ing column as cc~ared with a flocculent strain of Sacc~aromyces cerevisiae.
In the following Table the results with a flocculent stra~n of Zymomonas mobilis are compared with the results ~or a flocculent strain o~ Saccharomyces cerevisiae.
TABLE II
Organism Glucose Ethanol Maximum Concn Concn Productivity (g/l) Ig/l) (g~l/h) _ S. Cerevisiae 100 43 29 (ATCC 4126) Z. Mobilis 100 45 51 (ZM 401) (ATCC 31822) The flocculent mutant ZM 401 was isolated as follows. A
culture of strain CP4 was grown in rich medium, RM, (20 g/l glucose, 10 g/l yeast extract, 2 9/1 KH2po~) statically at 30C until it was in the exponential phase of growth.
Then nitrosoguanidine (NTG) was added to a final . ' ' 3 3 8 ~
.
concentration of 50 ug/ml and the culture was further incubated for 1 hour. The cells were then washed twice in saline phosphate buffer, and incubated statically overnight in RM at 30 C. After this time, a few small granular flocs were visible at the bottom of the flask. The supernatant medium, with the majority of cells was carefully removed, and fresh medium was added. After repeatedly subculturing every 24 hours in this manner for 7 days, an apparently-pure culture of granular flocs was obtained. This culture was streaked on RM plates twice to further purify it by single colony isolation. A single colony was then retested in liquid medium, and was still flocculent. This was chosen as the flocculent strain, and was renumbered ZM 401.
From the Table it is clear that ZM 401 has considerable adva~ntages, in terms of enhanced productivities, over strains of flocculent yeast.
Table III shows the greatly increased ethanol productivity using a continuous culture with cell recycle of 7ymomonas mobilis (ATCC 10988) as compared with the batch :
cul~re and continuous culture without cell recycle of the same organism all in medium containing 100 g/l of glucose.
TABLE III
Cultivation Type Ethanol Productivity (g/l/h) Batch 5 Continuous 11 Continuous with cell recycle 120 It can be seen that there is a ten fold increase in the ethanol productivitY as between continuous culture of the organism and continuous culture with cell recycle.
. .
.
, . .
' .
~; ` .
`~
The present invention relates to a method for the production of ethanol (alcohol) using the bacterium Z~momonas mobilis.
It is known to use the bacterium Zymomonas mobilis for the production of ethanol, and in fact this bacterium is used for the production of palm wine and other alcoholic beveraqes in tropical countries. It is also known that theoretically the production of ethanol using this bacterium has certain advantages as compared with yeasts which are conventionally used to produce alcohol by the fermentation of sugars, starches and other carbohydrate substrates. These advantages include the fact that Zymomonas mobilis produces less biomass than do yeasts due to the lower energy available for growth inherent in the Entner-Doudoroff pathway used by the bacterium which yields only one mole of ATP per mole of ~lucose me ~ olised as compared with the glycolytic pathway used by yeasts which yields two moles of ATP per mole of glucose. A further potential advantage is that Zymomonas ; mobilis grows anaerabically.
The present inventors have found that yreatly increased ethanol productivity can be achieved by continuously culturing Zymomonas mobilis with cell recycle.
It has been ~iscovered, and this could not have been predicted a priori, that continuous culture with cell recycle of zymomonas mobilis does not suffer from the difficulties encountered with continuous culture with cell recycle of conventional yeasts. These difficulties include the need to provide sterile oxygen to maintain yeast cell viabi~ity at high yeast concentrations, and the likelihood of contamination by unwanted bacteria during the extended period of continuous operation.
With Zymomonas mobilis fermentation using continuous -.. ~.
3 3 8 ~
culture with cell recycle as well as achieving higher ethanol productivities as compared with other techniques for Zymomonas mobilis fermentation and as compared with ;
yeast fermentation, it has been found that there is no requirement for oxygen to maintain viability at high cell concentrations. Furthermore due to the faster rates of sugar uptake and enhanced ethanol tolerance when compared to yeasts it has been found that no contamination occurs during extended periods of continuous operation.
The present invention consists in a process for the production of ethanol from a culture medium containing a fermentable carbohydrate which comprises continuously anaerobically culturing Zymomonas mobilis in the culture medium, continuously or periodically drawing off a `15 portion of the culture medium to give a withdrawn portion and replacing that portion with fresh culture medium, separating from the withdrawn portion of the culture medium cells of _ymomonas mobilis contained therein, ` returning the cells to the culture medium, and recovering the ethanol contained in the portion of the culture medium from which the cells have been removed.
;~The process according to the present invention is preferably carried out at a temperature of from 20C
to 50C, most preferably at 25 to 40~C, and at a pH
between 3.7 and 8, most preferably 4.5 to 6.5. The medium preferably contains from 100 to 400 g/l of a fermentable carbohydrate, most preferably from 150 to 300 g/l.
The preferred carbohydrates for use as fermentable substrates in the culture medium include, in addition to glucose, simple sugars such as fructose, lactose and sucrose, starch and starch hydrolysates, and cellulosic raw materials. It will be recognised that any one strain oE Zymomonas mobilis will probably not ferment all of these substrates and therefore for any particular strain a suitable, fermentable, substrate ~17338 ~
should be selected.
It is preferred that the strain of _mombnas mobilis used in this process is a strain having a high specific ethanol , ' :
.
¦ !
~ ~733~ ~
'`' productivity, a large specific rate of glucose uptake and a high alcohol tolerance. A particularly suitable organism is CP4 from the type culture collection of:-Dr. J. De Ley Laboratory of Microbiology, Ledeganckstraat 35 Belgium Mutants of this strain have also been found to be particularly useful in carrying out the present process and may have a broader range of fermentable substrates than CP4 itself. The mutant strains may be produced by J, mutations of an existing strain as by the use O.e u.v.
~ radiation or nitrosoguanidine. Desirable properties may also ; be introduced into the zymomonas mobilis strains by plasmid transfex from other bacteria using, for example, membrane filter mating techniques.
The separation of the cells from the removed portion of the culture medium may be achieved in any suitable manner.
The ~ells may be removed by the use of a cros~-flow microfiltration membrane system, preferably using a membrane having a pore size of the order of 0.6 microns.
Alternatively the cells may be separated in a centrifuge or in a series of hydroclones. If flocculent Zymomonas mobilis cells are used these may be removed from the culture medium by allow~ them to ~ettle in a settling vessel or a series of hydroclones.
The strains of Zymomonas mobilis referred to in this specification have been deposited in the American Type Culture Collection , 12301 Park Lawn Drive Rockville, Maryland 20852, U.S.A. and have been assigned the following deposit numbers and dates:
- Specification Reference ATCC Deposit No. Deposit Date CP4 31821 Feb. 26, 1981 ZM481 31823 Feb. 26, 1981 ZM40l 31822 Feb. 26, 1931 '~' ' . .
11~13381 Examples In the following examples the bacterium Zymomonas mobilis was cultured in a fermentation medium comprising the following nutrient concentrations.-100-3~0 g~l glucose ; 5 g/l yeast extract 1 9/l (NH4)2 S4 9/ 1 }~H ;2 PO 4 0-5 9/l MgS04 7H2O
The cultures of the various strain of Z~momonas mobilis were maintained by transferring to fresh agar slants containing 20 g glucose, 10 g yeast extract and 20 9 agar at - pH 5.0 each week and storing at room temperature.
~ The continuous fermentation system was controlled at a temperature of 30C and pH 5.0 in a 1 litre fermentatlon vessel. The pH was controlled by the addition of 2 N NaOH.
In these experiments cell recycle was carried out using a cross-flow microfiltration membrane system including a "Millipore"*membrane BD having a nominal pore si2e of 0.6 microns. This membrane retained all cells and the permeate passing through the membrane was free from cells and contained the same ethanol concentration as the contents of the fermenter.
Figure 1 shows the arrangement of the fermenter and cell recycle system. In this Figure 10 is a fermentation vessel fitted with a stirrer 11. The culture medium is introduced into the fermenter 10 through line 12 and is drawn off through line 13 to a pump 14 and thence to a tangential flow microfilter 15. The permeate through the filter 15 is discharged through line 16 to storage prior to recovery of the ethanol therefrom while the retained cell concentrate is returned through line 17 to the fermenter 10. An overflow line 18 is provided.
.
* ~Trademark . . .
~. :
, ~7 338~
Table I shows the greatly improved maximum ethanol productivity of three strains of Zymomonas mobilis continuously cultured with cell recycle as compared with two strains of the yeast Saccharomyces Cerevisiae.
TABLE I
; Organism Glucose Ethanol Maximum Concn cOncn Productivity t /1) (q/l) (g/l/h) g, _ _ _ _ , _ _ _ S. Cerevisiae 100 43 29 (ATCC 4126) : S~ Cerevisiae 150 61 32 . ~NRRL Y-132) - z. ~obilis 100 45 120 (ATCC 10988) Z. Mobilis 140 65 200 (CP4) (ATCC 31821) z. Mobilis 1~0 . 85 85 (ZM 481)(ATCC 31823) - 20 From the above table it is clear that the continuous culture , ~
of zymomonas mobilis with cell recycle can giv~
productivities wbich are 200 to 300% higher than for yeasts fermentations carried out under similar conditions~ This means that the size of the fermentation vessels to produce ethanol may be reduced by an e~uivalent amount.
The organism Zymomonas mobilis ZM 481 was developed from strain CP4 as follows:-Strain CP4 was grown statically at 30C until it wasin ~he exponential growth phase. Nitrosoguanidine was then added to a final concentration of 50 ug/ml and the culture was incubated for 30 minutes at 30C. The cells were then washed twice in saline phosphate buffer and regrown overnight . at 30C. The culture was then plated on plates containing ,, , , , .
l 1~338 10%(v/v) ethanol. After several days incubation at 30C~
mutant colonies appeared at a frequency of approximately 10 7, and these were purified on similar medium and then tested for growth and ethanol production rates with 100, 200 and 250 g/l glucose, at 30C in tubes. The culture which produced the highest level of ethanol in the shortest time was numbered ZM48.
A second similar mutagenesis was clone with strain ZM48 but the cells were plated with 15% (v/v) ethanol. The mutant which produced the highest level of ethanol in the shortest time was numbered ZM481, and was kept as an ethanol tolerant strain in the type culture collection in the School of Biotechnology, University of New South Wales, Sydney, New South Wales, Australia.
~ The use of flocculent strains of Zymomonas mobilis in the present invention is advantageous. Table II records the use of a flocculent strain of Zymomonas mcbilis usmg a set~ing column as cc~ared with a flocculent strain of Sacc~aromyces cerevisiae.
In the following Table the results with a flocculent stra~n of Zymomonas mobilis are compared with the results ~or a flocculent strain o~ Saccharomyces cerevisiae.
TABLE II
Organism Glucose Ethanol Maximum Concn Concn Productivity (g/l) Ig/l) (g~l/h) _ S. Cerevisiae 100 43 29 (ATCC 4126) Z. Mobilis 100 45 51 (ZM 401) (ATCC 31822) The flocculent mutant ZM 401 was isolated as follows. A
culture of strain CP4 was grown in rich medium, RM, (20 g/l glucose, 10 g/l yeast extract, 2 9/1 KH2po~) statically at 30C until it was in the exponential phase of growth.
Then nitrosoguanidine (NTG) was added to a final . ' ' 3 3 8 ~
.
concentration of 50 ug/ml and the culture was further incubated for 1 hour. The cells were then washed twice in saline phosphate buffer, and incubated statically overnight in RM at 30 C. After this time, a few small granular flocs were visible at the bottom of the flask. The supernatant medium, with the majority of cells was carefully removed, and fresh medium was added. After repeatedly subculturing every 24 hours in this manner for 7 days, an apparently-pure culture of granular flocs was obtained. This culture was streaked on RM plates twice to further purify it by single colony isolation. A single colony was then retested in liquid medium, and was still flocculent. This was chosen as the flocculent strain, and was renumbered ZM 401.
From the Table it is clear that ZM 401 has considerable adva~ntages, in terms of enhanced productivities, over strains of flocculent yeast.
Table III shows the greatly increased ethanol productivity using a continuous culture with cell recycle of 7ymomonas mobilis (ATCC 10988) as compared with the batch :
cul~re and continuous culture without cell recycle of the same organism all in medium containing 100 g/l of glucose.
TABLE III
Cultivation Type Ethanol Productivity (g/l/h) Batch 5 Continuous 11 Continuous with cell recycle 120 It can be seen that there is a ten fold increase in the ethanol productivitY as between continuous culture of the organism and continuous culture with cell recycle.
. .
.
, . .
' .
~; ` .
`~
Claims (7)
1. A process for the production of ethanol from a culture medium containing a fermentable carbohydrate, said process comprising continuously anaerobically culturing Zymomonas mobilis in the culture medium, continuously or periodically drawing off a portion of the culture medium to give a withdrawn portion and replacing that portion with fresh culture medium, separating from the withdrawn portion of the culture medium cells of Zymomonas mobilis contained therein, returning those cells to the culture medium, and recovering the ethanol contained in the portion of the culture medium from which the cells have been removed.
2. A process as claimed in claim 1 in which the fermentable carbohydrate is selected from the group consisting of glucose, fructose and sucrose.
3. A process as claimed in claim 1 in which the Zymomonas mobilis is of a strain selected from the group consisting of CP4, ZM 481 and ZM 401.
4. A process as claimed in any one of claims 1 to 3 in which the cells are recovered from the withdrawn portion of the culture medium by filtration.
5. A process as claimed in any one of claims 1 to 3 in which the cells are flocculent and are recovered from the withdrawn portion of the culture medium in a settling vessel.
6. A process as claimed in any one of claims 1-3 in which the culture medium is maintained at a temperature between 20°C and 50°C.
7. A process as claimed in any one of claims 1 to 3 in which the culture medium is maintained at a pH between 3.7 and 8.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AUPE265580 | 1980-03-05 | ||
| AU2655/80 | 1980-03-05 | ||
| AU3561/80 | 1980-05-15 | ||
| AUPE356180 | 1980-05-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1173381A true CA1173381A (en) | 1984-08-28 |
Family
ID=25642373
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000371605A Expired CA1173381A (en) | 1980-03-05 | 1981-02-24 | Ethanol production in a continuous process with cell recycle |
| CA000371592A Expired CA1174191A (en) | 1980-03-05 | 1981-02-24 | Ethanol production |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000371592A Expired CA1174191A (en) | 1980-03-05 | 1981-02-24 | Ethanol production |
Country Status (9)
| Country | Link |
|---|---|
| US (2) | US4403034A (en) |
| JP (1) | JPS56164790A (en) |
| BR (2) | BR8101334A (en) |
| CA (2) | CA1173381A (en) |
| DE (2) | DE3108386A1 (en) |
| FR (2) | FR2477572A1 (en) |
| GB (1) | GB2074188B (en) |
| NZ (1) | NZ196399A (en) |
| ZA (2) | ZA811434B (en) |
Families Citing this family (38)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58129979A (en) * | 1982-01-26 | 1983-08-03 | Hitachi Zosen Corp | Continuous preparation of alcohol by fluidizing immobilized microbial cell |
| JPS58152491A (en) * | 1982-03-05 | 1983-09-10 | Hitachi Zosen Corp | Production of alcohol through fermentation |
| JPS5959195A (en) * | 1982-09-27 | 1984-04-04 | Shinenerugii Sogo Kaihatsu Kiko | Continuous preparation of alcohol by immobilized mold of microorganism |
| GB2134540A (en) * | 1983-02-08 | 1984-08-15 | Us Energy | Biological conversion system |
| US5135853A (en) * | 1983-07-22 | 1992-08-04 | Rensselaer Polytechnic Institute | Three compartment bioreactor and method of use |
| PH19835A (en) * | 1983-09-27 | 1986-07-16 | Univ Queensland | Conversion of sucrose to fructose and ethanol |
| US4833078A (en) * | 1984-06-22 | 1989-05-23 | Celgene Corporation | Semi-continuous fermentation process for aromatic hydrocarbon bioconversion |
| AT385282B (en) * | 1984-10-18 | 1988-03-10 | Vogelbusch Gmbh | METHOD FOR THE CONTINUOUS PRODUCTION OF AETHANOL |
| US4808526A (en) * | 1985-04-12 | 1989-02-28 | George Weston Limited | Continuous process for ethanol production by bacterial fermentation |
| US4816399A (en) * | 1985-04-12 | 1989-03-28 | George Weston Limited | Continuous process for ethanol production by bacterial fermentation |
| DE3673054D1 (en) * | 1985-04-12 | 1990-09-06 | Weston George Ltd | CONTINUOUS PROCESS FOR PRODUCING ETHANOL BY BACTERIAL FERMENTATION. |
| US4808527A (en) * | 1985-04-12 | 1989-02-28 | George Weston Limited | Continuous process for ethanol production by bacterial fermentation |
| US4812410A (en) * | 1985-04-12 | 1989-03-14 | George Weston Limited | Continuous process for ethanol production by bacterial fermentation |
| US4755467A (en) * | 1985-06-03 | 1988-07-05 | Unisearch Limited | Method for the production of sorbitol and gluconate |
| FR2583060B1 (en) * | 1985-06-06 | 1987-09-18 | Inst Francais Du Petrole | PRODUCTION OF BUTANOL AND ACETONE BY FERMENTATION WITH RECYCLING OF BIOMASS BY ULTRAFILTRATION |
| DE3528933A1 (en) * | 1985-08-13 | 1987-02-19 | Kernforschungsanlage Juelich | FERMENTATION METHOD FOR FRUCTOSE GAIN OR ENRICHMENT AGAINST GLUCOSE AND THEREFORE USEABLE ZYMOMONAS MOBILIS MUTANTS |
| FR2608170B1 (en) * | 1986-12-12 | 1989-10-20 | Speichim Equip Ind Chimiq | NEW PROCESS FOR THE CONTINUOUS FERMENTATION OF AN AQUEOUS MUST WITH A VIEW TO PRODUCING ETHANOL AND / OR YEAST BIOMASS |
| US4840902A (en) * | 1987-05-04 | 1989-06-20 | George Weston Limited | Continuous process for ethanol production by bacterial fermentation using pH control |
| JPH0247140U (en) * | 1988-09-26 | 1990-03-30 | ||
| US4885241A (en) * | 1988-10-13 | 1989-12-05 | University Of Queensland | Ethanol production by zymomonas cultured in yeast-conditioned media |
| US5258293A (en) * | 1991-05-03 | 1993-11-02 | Trustees Of Dartmouth College | Continuous process for ethanol production from lignocellulosic materials without mechanical agitation |
| AU6875298A (en) * | 1997-04-07 | 1998-10-30 | University Of Florida Research Foundation, Inc. | Development of high-ethanol resistant (escherichia coli) |
| US6566107B1 (en) | 2000-11-01 | 2003-05-20 | Midwest Research Institute | Recombinant Zymomonas mobilis with improved xylose utilization |
| US7015039B1 (en) * | 2002-08-08 | 2006-03-21 | Massachusetts Eye & Ear Infirmary | Separating epithelium from stroma in LASEK surgery |
| GB0511602D0 (en) | 2005-06-07 | 2005-07-13 | Tmo Biotec Ltd | Microorganisms |
| CN100429303C (en) * | 2005-09-05 | 2008-10-29 | 福建农林大学 | Motion fermentation single cell bacterium acid-resistant strain |
| GB0520344D0 (en) * | 2005-10-06 | 2005-11-16 | Tmo Biotec Ltd | Microoganisms |
| KR101367942B1 (en) | 2006-07-21 | 2014-02-27 | 질레코 인코포레이티드 | Conversion systems for biomass |
| MX2009002296A (en) | 2006-09-28 | 2009-04-16 | Tmo Renewables Ltd | Thermophilic microorganisms for ethanol production. |
| US7815741B2 (en) | 2006-11-03 | 2010-10-19 | Olson David A | Reactor pump for catalyzed hydrolytic splitting of cellulose |
| US7815876B2 (en) | 2006-11-03 | 2010-10-19 | Olson David A | Reactor pump for catalyzed hydrolytic splitting of cellulose |
| US20080299624A1 (en) * | 2007-06-01 | 2008-12-04 | Edward Heslop | Continuous fermentation apparatus and method |
| GB0715751D0 (en) | 2007-08-13 | 2007-09-19 | Tmo Renewables Ltd | Thermophilic micro-organisms for ethanol production |
| US8252566B2 (en) * | 2008-05-20 | 2012-08-28 | Jj Florida Properties Llc | Ethanol production from citrus waste through limonene reduction |
| US9255280B2 (en) * | 2008-05-20 | 2016-02-09 | Jj Florida Properties Llc | Removal of fermentation inhibiting compounds from citrus waste using solvent extraction and production of ethanol from citrus waste |
| GB0820262D0 (en) | 2008-11-05 | 2008-12-10 | Tmo Renewables Ltd | Microorganisms |
| MX2014008376A (en) * | 2012-01-10 | 2015-04-09 | Archer Daniels Midland Co | Process for making hmf and hmf derivatives from sugars, with recovery of unreacted sugars suitable for direct fermentation to ethanol. |
| WO2015130883A1 (en) * | 2014-02-27 | 2015-09-03 | E. I. Du Pont De Nemours And Company | Enzymatic hydrolysis of disaccharides and oligosaccharides using alpha-glucosidase enzymes |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE430171B (en) * | 1978-01-31 | 1983-10-24 | Alfa Laval Ab | CONTINUOUS PROCEDURE FOR THE PRODUCTION OF ETHANOL IN A FERMENTOR ADDED TO A SUBSTRATE WITH HIGH CARBOHYDRATE CONCENTRATION, WHICH DISPOSES FERMENTATION LIQUID AFTER COMPOUNDING A FRENCH PREPARED FLUID ... |
-
1981
- 1981-02-24 CA CA000371605A patent/CA1173381A/en not_active Expired
- 1981-02-24 CA CA000371592A patent/CA1174191A/en not_active Expired
- 1981-03-03 US US06/240,099 patent/US4403034A/en not_active Expired - Fee Related
- 1981-03-03 US US06/240,140 patent/US4443544A/en not_active Expired - Lifetime
- 1981-03-03 GB GB8106694A patent/GB2074188B/en not_active Expired
- 1981-03-04 FR FR8104338A patent/FR2477572A1/en active Granted
- 1981-03-04 ZA ZA00811434A patent/ZA811434B/en unknown
- 1981-03-04 ZA ZA00811435A patent/ZA811435B/en unknown
- 1981-03-04 FR FR8104337A patent/FR2477571A1/en active Granted
- 1981-03-05 DE DE19813108386 patent/DE3108386A1/en active Granted
- 1981-03-05 BR BR8101334A patent/BR8101334A/en unknown
- 1981-03-05 NZ NZ196399A patent/NZ196399A/en unknown
- 1981-03-05 BR BR8101335A patent/BR8101335A/en unknown
- 1981-03-05 DE DE19813108384 patent/DE3108384A1/en not_active Ceased
- 1981-03-05 JP JP3190081A patent/JPS56164790A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| ZA811435B (en) | 1982-03-31 |
| FR2477571A1 (en) | 1981-09-11 |
| GB2074188B (en) | 1984-04-18 |
| JPS56164790A (en) | 1981-12-17 |
| ZA811434B (en) | 1982-03-31 |
| DE3108386C2 (en) | 1989-02-02 |
| NZ196399A (en) | 1984-03-30 |
| BR8101335A (en) | 1981-09-08 |
| DE3108386A1 (en) | 1982-01-28 |
| JPS6357039B2 (en) | 1988-11-10 |
| CA1174191A (en) | 1984-09-11 |
| US4403034A (en) | 1983-09-06 |
| US4443544A (en) | 1984-04-17 |
| FR2477572A1 (en) | 1981-09-11 |
| FR2477572B1 (en) | 1984-04-20 |
| BR8101334A (en) | 1981-09-08 |
| FR2477571B1 (en) | 1984-07-13 |
| GB2074188A (en) | 1981-10-28 |
| DE3108384A1 (en) | 1981-12-24 |
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| Date | Code | Title | Description |
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| MKEX | Expiry |