CN102131936B - In synthesis gas sweat for multiple concentration of substrate reduce or in the absence of maintain the method for culture of microorganism - Google Patents
In synthesis gas sweat for multiple concentration of substrate reduce or in the absence of maintain the method for culture of microorganism Download PDFInfo
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- CN102131936B CN102131936B CN200980114615.4A CN200980114615A CN102131936B CN 102131936 B CN102131936 B CN 102131936B CN 200980114615 A CN200980114615 A CN 200980114615A CN 102131936 B CN102131936 B CN 102131936B
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- 238000012258 culturing Methods 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
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- 230000005611 electricity Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- JROGBPMEKVAPEH-GXGBFOEMSA-N emetine dihydrochloride Chemical compound Cl.Cl.N1CCC2=CC(OC)=C(OC)C=C2[C@H]1C[C@H]1C[C@H]2C3=CC(OC)=C(OC)C=C3CCN2C[C@@H]1CC JROGBPMEKVAPEH-GXGBFOEMSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
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- 238000011010 flushing procedure Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000003502 gasoline Substances 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
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- 239000002440 industrial waste Substances 0.000 description 1
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- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000012092 media component Substances 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- QMMRZOWCJAIUJA-UHFFFAOYSA-L nickel dichloride Chemical compound Cl[Ni]Cl QMMRZOWCJAIUJA-UHFFFAOYSA-L 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000032696 parturition Effects 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 235000015497 potassium bicarbonate Nutrition 0.000 description 1
- 229910000028 potassium bicarbonate Inorganic materials 0.000 description 1
- 239000011736 potassium bicarbonate Substances 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000007320 rich medium Substances 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- 239000011781 sodium selenite Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000000629 steam reforming Methods 0.000 description 1
- 230000033772 system development Effects 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 229960000344 thiamine hydrochloride Drugs 0.000 description 1
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 1
- 239000011747 thiamine hydrochloride Substances 0.000 description 1
- 235000021404 traditional food Nutrition 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 238000004804 winding Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
Classifications
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- 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
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- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
- C12M1/38—Temperature-responsive control
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- 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
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P7/00—Preparation of oxygen-containing organic compounds
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- 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
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- 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
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- 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
- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
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- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B3/00—Hydrogen; Gaseous mixtures containing hydrogen; Separation of hydrogen from mixtures containing it; Purification of hydrogen
- C01B3/02—Production of hydrogen or of gaseous mixtures containing a substantial proportion of hydrogen
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- 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
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- Y02E50/00—Technologies for the production of fuel of non-fossil origin
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- Y10S435/00—Chemistry: molecular biology and microbiology
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Abstract
The method that the present invention relates to maintain culture of microorganism in synthesis gas fermentation reactor in the case of reducing at multiple concentration of substrate or be non-existent, described method includes: adds carbon dioxide and is optionally added alcohol；Maintain free acetic acid concentration；And in special time, perform above-mentioned steps.
Description
Invention field
The present invention relates to comprise at least one produce acetic acid microorganism, for from the gaseous state containing at least one reducing gas
Substrate produces the improvement of the microbial fermentation processes of alcohol.
Background of invention
The conventional method maintaining culture of microorganism has many.But, these methods are perplexed by a large amount of poor efficiency behaviors.
For more having for other maintaining culture of microorganism in the case of multiple substrate is non-existent in synthesis gas sweat
The method of effect, there are still demand.
Have been described with three strain acetogens (Drake, 1994) for producing liquid fuel from synthesis gas: eat methylbutanoic acid
Bacillus (Butyribacterium methylotrophicum) (Grethlein etc., 1990；Jain etc., 1994b),
Clostridium autoethanogenum (Abrini etc., 1994), Clostridium ljungdahlii (Arora etc.,
1995；Barik etc., 1988；Barik etc., 1990；With Tanner etc., 1993).It is known that Clostridium
CO is transformed into ethanol by ljungdahlii and Clostridium autoethanogenum.
United States Patent (USP) No.5,173,429 of Gaddy etc. discloses Clostridium ljungdahlii ATCC
No.49587, a kind of CO and H from forming gas2O and/or CO2With H2Produce ethanol and the anaerobe of acetic acid.
United States Patent (USP) No.5,192,673 of Jain etc. discloses clostridium acetobutylicum (Clostridium
Acetobytylicum) mutant strain and use the method that this bacterial strain manufactures butanol.
United States Patent (USP) No.5,593,886 of Gaddy etc. discloses Clostridium ljungdahlii ATCC
No.55380.This microorganism can use waste gas (such as white carbon black waste gas) to produce acetic acid and ethanol as substrate anaerobism.
United States Patent (USP) No.5,807,722 of Gaddy etc. discloses use anaerobic bacteria such as Clostridium
Waste gas is transformed into the method and apparatus of useful products such as organic acid and alcohols by ljungdahlii ATCC No.55380.
United States Patent (USP) No.6,136,577 of Gaddy etc. discloses use anaerobic bacteria such as Clostridium
Waste gas is transformed into useful products such as organic acid and alcohols (particularly by ljungdahlii ATCC Nos.55988 and 55989
Ethanol) method and apparatus.
United States Patent (USP) No.6,136,577 of Gaddy etc. discloses use Clostridium ljungdahlii anaerobe
Waste gas is transformed into useful products such as organic acid and the method and apparatus of alcohols (particularly acetic acid) by strain.
United States Patent (USP) No.6,753,170 of Gaddy etc. discloses the anaerobe fermentation technology for producing acetic acid.
United States Patent (USP) No.7,285,402 of Gaddy etc. discloses the anaerobe fermentation technology for producing alcohol.
Have also been described for from other acetogen bacterial strains of synthesis gas production liquid fuel, such as: food methyl
Clostridium butylicum (Butyribacterium methylotrophicum) (Grethlein etc., 1990,
Appl.Biochem.Biotech.24/24:875-884) and Clostridium autoethanogenum (Abrini etc.,
1994, Arch.Microbiol.161:345-351).
In the art, for reducing or non-existent situation at multiple concentration of substrate in synthesis gas sweat
Lower holding culture, there are still demand.Cultivate for maintaining in the case of various interruptions in the industrial process that alcohol produces
Thing, also exists demand.Particularly, at CO, H2, or CO and H2Various concentration reduce in the case of maintain microorganism culturing
Thing, there are still demand.
Summary of the invention
The present invention relates in the case of multiple concentration of substrate reduces or be non-existent, remain micro-in synthesis gas fermentation reactor
The method of biological culture thing, described method includes: adds carbon dioxide and is optionally added into alcohol；Maintain free acetic acid concentration；With
And in special time, perform above-mentioned steps.
Present invention additionally contemplates that in the case of multiple concentration of substrate reduces or be non-existent, stoped synthesis gas fermentation reaction
The method of the culture of microorganism rapid loss in device, described method includes: adds carbon dioxide and is optionally added into alcohol；From fortune
Trip temperature landing low temperature；Maintain free acetic acid concentration；And in special time, perform above-mentioned steps.
Present invention also offers multiple concentration of substrate in feed gas supply thing to maintain in the case of reducing or being non-existent
The method of the culture of microorganism in synthesis gas fermentation reactor, described method includes: adds carbon dioxide and is optionally added into
Alcohol；From running temperature landing low temperature；Maintain free acetic acid concentration；And in special time, perform above-mentioned steps.
As embodiment of the present invention, alcohol can be as substrate utilization.Although having attempted several replacement growth substrate, but
Do not have one to behave like alcohol the best, do not have one to be readily available as alcohol yet.When forming gas supply recovers, micro-life
Thing culture easily returns to utilize synthesis gas.Additionally, as embodiment, not giving individually with acetic acid/alcohol approach may
Other competitive bacterias being present in culture fluid or process piping provide the chance of growth.And growth substrate such as glucose will
Their growth easily it is used for by the organism of any existence.
Prior art adjusts culture fluid to maintain low free acetic acid concentration by including.These technology will include raise pH and
Increase liquid to flow to wash out acetyl group.As embodiment, reduce temperature active to reduce culture, and use product ethanol
With carbon dioxide, energy is supplied back to culture to maintain viability.Concept in addition with new alternative substrate.
This is the improvement to technique because due to gasifier raw material supply, transmit equipment, drying equipment, gas purification or
The gas supply discontinuity caused along the interruption of any other device of gas supply line, will happen occasionally.The present invention's is another
One application includes from the three unities, inoculum is transported another place.In transit, culture may can not get synthesis
Gas is supplied, and therefore will need alternative substrate.Have and maintain the ability of more than 12 hours viability by being, technological ability to be changed
Enter.Therefore, there is the interruption during viable option will cause alcohol to produce in economic and technical and/or downturn plus work
The startup of factory and restarting is preferably minimized.
Accompanying drawing is sketched
Fig. 1 is schematic diagram, it is shown that the enforcement of the whole technological process estimated in the normal course of operation of the present invention
Scheme.Although being shown that ethanol in the drawings, but invention contemplates other alcohols.
Fig. 2 shows the schematic diagram of embodiment of the present invention, it is shown that carbon dioxide interpolation, alcohol consumption and culture
The trend recovered.
Fig. 3 shows the schematic diagram of the comparison of the present invention, it was demonstrated that shortage that alcohol consumes and the shortage that culture recovers.
Detailed Description Of The Invention
Definition
Except as otherwise noted, the following term otherwise used in whole this specification is defined as follows:
Term " about " refers to when modifying any amount (amount) that this amount is in the real world conditions maintaining culture of microorganism
Under, such as deviation encountered in laboratory, pilot plant or production facility.Such as, the composition used in the mixture
Amount when with " about " modify time, include under the experiment condition of factory or laboratory, the most typically used as deviation with
Degree of concern.The amount of such as product component, when modifying with " about ", includes batch in the many experiments of factory or laboratory
Between deviation and the intrinsic deviation of the method for analysis.Regardless of whether modify with " about ", amount all includes the equivalent amount with those amounts.
Any quantity (quantity) stated in this article and modify with " about " can also be used for as not having the amount modified with " about "
In the present invention.
Except as otherwise noted, otherwise term " acetic acid class thing (acetate) " is used for describing molecule present in fermentation liquid
Or the mixture of free acetic acid and acetate.The ratio of molecule acetic acid and acetic acid class thing depends on the pH of system, i.e. constant
Under " acetic acid class thing " concentration, pH is the lowest, and molecule acetic acid concentration is the highest relative to acetate.
Term " acetogen " or " producing acetic acid " refer to the antibacterial producing acetic acid class thing as anaerobic respiration product.This mistake
Journey is different from acetic fermentation, although both of which occurs and all to produce acetic acid class thing in the case of there is not oxygen.These are raw
Object also referred to as produces acetic acid bacteria, because all known acetogens are all antibacterials.In various habitats
Being found that acetogen, these habitats are usually (the shortage oxygen) of anaerobism.Acetogen can use various chemical combination
Thing is as the energy and carbon source；Research is the most clearly produced acetate metabolism form and is included using carbon dioxide as carbon source and hydrogen conduct
The energy.
Term " bioreactor ", " reactor " or " fermenting organism reactor " include by one or more containers and/or
Tower or pipeline arrange the installation for fermenting constituted, including CSTR (CSTR), immobilized cell reactor
(ICR), trickle bed reactor (TBR), bubble column, airlift fermentation device, static mixer or other be suitable for gas-liquids and connect
The device touched.For the method for the present invention, preferably fermenting organism reactor comprises to second fermenting organism reactor confession
The growth reactor of fermentation liquid, major part product ethanol is answered to produce wherein.
" cell concentration " in this specification is bacterial dry mass based on every liter of sample.Cell concentration is directly measured or passes through
The dependency of optical density is carried out calibration measurement.
Term used herein " continuation method " refers to a kind of fermentation process, and it is included in bioreactor and carries out even
Continuous nutrient feed, substrate charging, cell growth, and from bioreactor, remove (or removing) cell continuously and remove product
Thing.This continuous print charging, remove or cell growth can occur in identical or different stream.Continuation method causes giving birth to
Thing reactor reaches stable state." stable state " refers to that all these measurable variable (i.e. maintains in feed rate, bioreactor
Substrate and nutrient concentrations, bioreactor in cell concentration and the cell removed from bioreactor, from biological respinse
Product that device removes and conditional-variable such as temperature and pressure) keep constant in time.
" alcohol production rate " is the volume production rate of ethanol, is calculated as stable state concentration of alcohol and holds with liquid in continuous system
Stay the ratio of time (LRT), or in batch system, be calculated as the ratio of concentration of alcohol and the time produced needed for this concentration.Word
Group " high alcohol production rate " describes the every day of the volumetric ethanol productivity higher than 10g/L.
As H in feed gas2The twice of molal quantity and the molal quantity of the CO being converted and the CO that is converted2Molal quantity
Three times of sums ratio more than 1.0 time, " excess H2" can be used for alcohol production.If this ratio is less than 1.0, it is impossible to obtain
The H of excess2, ethanol can only be produced by different control mechanisms.
Term " ferments " and refers to that CO is fermented into alcohols and acetic acid class thing.Known many anaerobic bacterias are able to carry out CO to alcohols
Including butanol and ethanol and the fermentation of acetic acid, and it is suitable in the method for the present invention.Such it is suitable for the present invention's
The example of antibacterial includes the antibacterial that clostridium (Clostridium) belongs to, such as Clostridium ljungdahlii bacterial strain, including
At WO 00/68407, EP 117309, United States Patent (USP) Nos 5,173,429,5,593,886 and 6,368,819, WO 98/
Those described in 00558 and WO 02/08438, and Clostridium autoethanogenum (Aribini etc.,
Archives of Microbiology 161:pp 345-351).Other antibacterials being suitable for include Moore Salmonella (Moorella)
The antibacterial belonged to, including Moorella sp.HUC22-1 (Sakai etc., Biotechnology Letters 29:pp 1607-
, and carbonoxide Thermophilic Bacteria (Carboxydothermus) antibacterial (Svetlichny, V.A., Sokolova, the T.G. that belong to 1612)
Deng, (1991), Systematic and Applied Microbiology 14:254-260).The respective public affairs of these publications
Open content to be incorporated by reference in its entirety at this.Additionally, the professional of the art can screen other produce acetic acid anaerobism thin
Bacterium is in the method for the present invention.It is also acknowledged that the mixed culture of two or more antibacterial may be used for the present invention's
In method.A kind of microorganism being suitable for the present invention is the Clostridium can being purchased from DSMZ
Autoethanogenum, it has the diagnostic characteristics of DSMZ preserving number DSMZ 10061.Fermentation can be at any applicable biology
Reactor is carried out, such as CSTR (CSTR), bubble-column reactor (BCR) or trickle bed reactor
(TBR).Additionally, in the certain preferred embodiments of the present invention, bioreactor may be embodied in wherein cultivating microorganism
First growth reactor, and to its charging fermentation liquid and produce major part tunning (second from growth reactor wherein
Alcohol and acetic acid class thing) second fermentation reactor.
Term used herein " gaseous state substrate " refers to single CO, CO and H2, CO2And H2, or CO, CO2And H2, optionally
Ground and other elements or compound include nitrogen and the methane blended of gaseous state.Such gaseous state substrate includes gas or air-flow, it
Typical case by directly or by burning release or be discharged in air.In some embodiment of the method, gaseous state substrate bag
Containing CO.In other embodiments of the method, gaseous state substrate comprises CO2And H2.In other embodiments, gaseous state substrate bag
Containing CO and H2.In particularly preferred embodiments, gaseous state substrate comprises CO, CO2And H2.Additionally, other substrates of the present invention can
To include those discussed above component and nitrogen, CO2, at least one gas in ethane and methane.Therefore, these substrate bags
Include and be commonly called " synthesis gas " or from the forming gas (including methane) of carbon product gasification and from various work
The gas of the waste gas of industry method.
Phrase " high concentration ethanol " refers to exceed in fermentation liquid the ethanol of about 10g/L, preferably greater than 15g/L, or ethanol with
The proportion of products of acetic acid class thing is 5: 1 or higher.
Term " restricted substrate " or " limiting nutrient thing " refer to the material in Nutrient medium or gaseous state substrate, its
Stable state or stable during cultured object is depleted to no longer biological support reactor during bacterial culture growth in bioreactor
The level of bacterial growth.Therefore, the every other material in Nutrient medium or gaseous state substrate is present in excess, and is " unrestricted
Property ".The evidence limited is to increase and adds the speed of restricted material to culture, i.e. have additional nutrients thing feed rate or gas
Feed rate, causes the corresponding increase of the gas uptake rate (mmol/min gas) caused due to the increase of cell density.
Term " microorganism " includes antibacterial, fungus, archeobacteria and protista；Microscopic plant (is referred to as chlorella)；With dynamic
Thing such as plankton, turbellarian worm and amebicide.Some people thinks and also includes virus, but other people think that they are without life
's.Microorganism is lived in all parts that there is aqueous water of biosphere, including soil, hot spring, seabed, atmosphere eminence and ground
Depths in rock in shell.Owing to playing the effect of decomposer, microorganism is to pass for the nutrient substance recirculation of ecosystem
Important.Microorganism also by human use in biotechnology, including traditional Food & Drink preparation and based on heredity work
The modern technologies of journey.It is contemplated that will utilize mixed microorganism bacterial strain in the present invention, it can comprise maybe can be without various
The bacterial strain of microorganism.In addition it is further contemplated that use the existing microbial strains selected, recombinant DNA technology can create micro-life
Thing.In certain embodiments of the invention, several exemplary bacterial strain of Clostridium ljungdahlii includes PETC strain
(United States Patent (USP) No.5,173,429), ERI2 strain (United States Patent (USP) No.5,593,886) and C-01 and O-52 strain (United States Patent (USP)
No.6,136,577).These bacterial strains are each deposited in American type culture collection (American Type Culture
Collection), 10801 University Boulevard, Manassas, Va.20110-2209, registration number is respectively
Nos.:55383 (in the past for ATCC No.49587) (preservation date be 1992 on December 22), 55380 (preservation date is
On June 28th, 1996), 55988 (on June 27th, 1997) and 55989 (preservation date is on June 27th, 1997).Each
Clostridium ljungdahlii bacterial strain is all the gram-positive bacterium of anaerobism, its guanine and cytosine (G+C) nucleoside
Acid content is about 22mole%.These antibacterials use various different substrate growth, but do not utilize methanol or lactic acid (salt).This
The difference of a little bacterial strains is its CO toleration, than gas uptake rate and specific production rate." wild " bacterium found in nature
In strain, only observe that minimal amount of ethanol produces.Clostridium ljungdahlii bacterial strain is preferable to be operated at 37 DEG C,
The ethanol typically produced under " wild " state and acetyl group (i.e. referring to free or molecule acetic acid and acetate) product ratio
Example is about 1: 20 (every 1 part of ethanol of 20 parts of acetyl group).Concentration of alcohol typical case is only 1-2g/L.Although the ability of this generation ethanol
It is useful, but due to low alcohol production rate, " wild " antibacterial cannot be used for producing economically ethanol on the basis of commercialization.Logical
Crossing manipulation micronutrient, Clostridium ljungdahlii bacterial strain above-mentioned has been used for producing ethanol and acetyl
Base, its proportion of products is 1: 1 (ethanol of equal parts and acetyl group), but concentration of alcohol is less than 10g/L, and this level causes low
Poor efficiency in 10g/L every day.Additionally, the stability of culture is a problem, mainly due to relatively high (8-10g/L)
The acetyl group (2.5-3g/L molecule acetic acid) of concentration is combined with the existence of ethanol.Additionally, attempting producing in more ethanol,
Along with gas velocity increases, culture is first subjected to molecule acetic acid and is then subjected to the suppression of CO.As a result, culture becomes unstable
Fixed, and gas can not be absorbed and produce additional product.Additionally, the Prior efforts carried out by the present inventor shows, in stable state
The ethanol producing more than 2: 1 in operation is difficult with acetyl group ratio.See for example Klasson etc., 1990 Applied
Biochemistry and Biotechnology, Proceedings of the 11th Symposium on
Biotechnology for Fuels and Chemicals (the 11st fuel and chemicals biotechnology seminar
Record), 24/25:857；Phillips etc., 1993 Applied Biochemistry and Biotechnology,
Proceedings of the 14thSymposium on Biotechnology for Fuels and Chemicals (the
14 fuel are recorded with chemicals biotechnology seminar), 39/40:559, etc..Lot of documents describes use
Anaerobic bacteria outside Clostridium ljungdahlii, is not consuming CO, CO2And H2Carbohydrate fermentation in produce solvent.
In the trial providing high alcohol yied, having changed many kinds of parameters, they include: nutrient type, and microorganism specifically adds
The reducing agent added, pH changes, and the interpolation of external source gas.See for example Rothstein etc., 1986 J.Bacteriol.,
165 (1): 319-320；Lovitt etc., 1988 J.Bacteriol., 170 (6): 2809；Taherzadeh etc., 1996
Appl.Microbiol.Biotechnol., 46:176.
When using term " hybrid bacterial strain ", it refers to the mixed culture of two or more microorganisms.The present invention's
Method make use of this " hybrid bacterial strain " of the microorganism herein above enumerated.
Term " native state " describes any compound, element or approach does not has the extra electronics of normal presence or matter
Son.On the contrary, term " reducing condition " describes any compound, element or approach and has one or more excess electron." reduction
State " by adding one or more electronics to " native state ", i.e. obtained by the oxidation-reduction potential reducing fermentation liquid.
" Nutrient medium " be generally used to illustrate containing vitamin and mineral be enough to allow the target bacteria selected raw
Long Conventional bacteria growth medium.Saccharide is not comprised in these culture medium.It is suitable for application various of the present invention
The component of Nutrient medium is known, and former publication, includes reporting in the publication of the present inventor.See example
As in international patent application No.WO08/00558, United States Patent (USP) No.5,807,722, United States Patent (USP) No.5,593,886 and the U.S.
Nutrient medium formula in patent No.5,821,111 and described in publication indicated above.According to the present invention, use
In from CO, CO2And H2The typical laboratory Nutrient medium producing acetic acid class thing comprises 0.9mg/L calcium pantothenate.But, for from
CO、CO2And H2The typical laboratory Nutrient medium producing ethanol comprises 0.02mg/L calcium pantothenate.
Term " reducibility gas " refers to CO and H2Any one or the two.Phrase is " more than the reproducibility needed for bacterial growth
The amount of gas " refer under the conditions of given nutrient medium ingredients, the amount of reducibility gas exceed antibacterial can be used in growth or
The amount of metabolism.Can be by increasing the net amount of reducibility gas or by reducing critical nutrients composition not increase gas
Just obtain excess air or by increasing the speed delivering gas to antibacterial, obtain this amount.When bacterial exposure is in than growth institute
When needing more reducibility gas, antibacterial responds by increasing the production of ethanol." target bacteria " be produce acetic acid anaerobism (or
Facultative) antibacterial, it is possible to by CO and water or H2And CO2It is transformed into ethanol and acetic acid product.According to the present invention, useful antibacterial includes
But it is not limited to triumphant 5 and produces vinegar bacterium (Acetogenium kivui), Wu Shi acetobacter (Acetobacterium woodii), tide
Wet anaerobism vinegar bacterium (Acetoanaerobium noterae), clostridium aceticum (Clostridium aceticum), food methylbutanoic acid
Bacillus (Butyribacterium methylotrophicum), clostridium acetobutylicum (C.acetobutylicum), hot acetic acid
Clostridium (C.thermoaceticum), mucus Eubacterium (Eubacterium limosum), Clostridium
ljungdahlii PETC、Clostridium ljungdahli iERI2、Clostridium ljungdahlii C-01、
Clostridium ljungdahlii O-52 and peptostreptococcus productus (Peptostreptococcus productus).
Other in these methods produce acetic acid anaerobic bacteria by professional's selection of the art.
Term " synthesis gas " refers to forming gas, and this title refers to the gas mixing containing the most commensurability carbon monoxide and hydrogen
Thing.The example of production method include the steam reforming of natural gas or liquid hydrocarbon with produce hydrogen, coal gasification and some
The refuse production capacity gasification facility of type.This title comes from them and is used as intermediate and use in producing synthetic natural gas (SNG)
In producing ammonia or methanol.Synthesis gas is also synthetically produced as fuel or the synthetic petroleum of lubricant through Fischer-Tropsch
Before and, the methanol of Mobil (Mobil) is used as intermediate in process of gasoline.Synthesis gas mainly by hydrogen, carbon monoxide and
The most common a certain amount of carbon dioxide composition, has the energy density less than natural gas half.Synthesis gas is flammable, generally
As fuels sources or as the intermediate producing other chemicals.
The detailed embodiment of the present invention
The present invention relates to tie up in the case of multiple concentration of substrate in synthesis gas fermentation reactor reduces or be non-existent
The method holding culture of microorganism, described method includes: adds carbon dioxide and is optionally added into alcohol；Maintain free acetic acid concentration
Below 5g/L free acetic acid；And in 0-30 minute, in 0-15 minute, in 15-30 minute, perform above-mentioned steps.
Present invention additionally contemplates that and reduced or non-existent feelings for multiple concentration of substrate in synthesis gas fermentation reactor
The method stoping culture of microorganism rapid loss under condition, described method includes: adds carbon dioxide and is optionally added into alcohol；Will
Temperature is reduced to maintain between 0-25 DEG C between 0-25 DEG C and by temperature from running temperature；Maintain free acetic acid concentration at 5g/L
Below free acetic acid；And in 0-30 minute, in 0-15 minute, in 15-30 minute, perform above-mentioned steps.
Present invention also offers in the case of in feed gas supply thing, multiple concentration of substrate reduces or be non-existent
The method maintaining culture of microorganism in synthesis gas fermentation reactor, described method includes: add carbon dioxide and optionally
Add alcohol；It is down to maintain between 0-25 DEG C between 0-25 DEG C and by temperature from running temperature by temperature；Maintenance free acetic acid is dense
Degree is below 5g/L free acetic acid；And in 0-30 minute, in 0-15 minute, in 15-30 minute, perform above-mentioned steps.
As embodiment, described maintenance culture of microorganism accounts for the time span of about 0-30 hour.As embodiment,
PH can maintain in the range of about 3.5-5.6.Further, it is contemplated that add bicarbonate solution to control pH.Bicarbonate
Solution can comprise: ammonium hydrogen carbonate, sodium bicarbonate and/or potassium bicarbonate.Embodiment of the present invention provide a method that, its
In optionally described carbon dioxide is moved in described reactor.Additionally, as embodiment, it is provided that have a high regard for described reaction
Selection of land adds nutrient.The invention provides and optionally add nutrient to described reactor.
Other embodiments of the present invention provides alcohol, including following one or more: ethanol, butanol, ethanol and butanol.
It is optionally possible to temperature is reduced between 0-25 DEG C from running temperature, temperature is maintained simultaneously 0-25 DEG C it
Between；It is optionally possible to add water to described reactor.This water can include fresh water, supplement water, recirculation water, distilled water, go from
Sub-water or a combination thereof.
The present invention take into account a kind of method, and wherein said culture of microorganism comprises at least one and produces acetic acid bacteria.Micro-
Biological culture thing can include selected from clostridium (Clostridium), Moore Salmonella (Moorella) and carbonoxide Thermophilic Bacteria
(Carboxydothermus) one or more bacterial strains or its genetic modification strain.
As embodiment, microorganism can include selected from PETC, ERI-2, O-52 and C-01 bacterial strain or a combination thereof
Clostridium ljungdahlii。
Present invention also offers a kind of method, before wherein culture of microorganism is returned to the suspension including adding synthesis gas
(pre suspension) condition.
Optionally, as embodiment, the present invention can provide: removes permeate；React with described in inert gas purge
Device；Or maintain low stirring to keep solid content to suspend.
Other aspects of the present invention and advantage will further describe in the following detailed description.
The product acetic acid autotrophic bacteria utilizing carbon monoxide and/or hydrogen and carbon dioxide (forming gas) to produce alcohol needs perseverance
Fixed gas supply produces alcohol.Intermediate product in alcohol production is acetic acid, and it can be in intracellular and extracellular.No
Enough forming gas supplies, beneficially acetic acid and produce limited ethanol.
Can be used for intermediate product acetic acid production forming gas reduce or do not have condition time, culture can exist
In the case of carbon dioxide, alcohol is converted back into acetic acid.In ethanol Already in culture fluid, and forming gas can be readily used for
Limited or in the case of not having.The alcohol separately added can also be supplied as required.Can be by by CO2Gas sparging is in culture
Add carbon dioxide, or it can be formed by adding bicarbonate in culture fluid.Sodium bicarbonate can be in fermentation
For maintaining required pH, therefore can readily use.In acid culture fluid, bicarbonate buffer reaction defines titanium dioxide
Carbon.Then antibacterial may utilize the carbon dioxide of formation and alcohol transformed back into acetic acid.
Depositing, alcohol is transformed into free acetic acid in the case of carbon dioxide is relatively quick process.Microorganism is such as
Free acetic acid concentration in the culture fluid that Clostridium ljungdahlii can resist is limited.Forming gas reduce or
During forfeiture, need the concentration taking steps to control free acetic acid.A kind of such control method is to use temperature manipulation.?
Rise high-temperature in mesophilic scope and add culture activity rate.But temperature reduction makes this activity rate reduce in fermentation liquid.Cause
This, reduce temperature and be helpful to for the activity slowing down culture under conditions of reducing at gas or do not have, result in slower
Product acid.The another kind of method that free acetic acid controls is to change the pH of culture.The balance portion of acetyl group and acetic acid is controlled by pH
System.Raising pH during forming gas supply discontinuity, to make total acetyl group concentration, i.e. acetyl group add acetic acid higher, maintains relatively simultaneously
Low free acid concentration.
The third potential method controlling free acid concentration is to increase liquid and flows through system.When free acid concentration increases,
Increase liquid stream inflow system with increasing permeate removing, more free acids are washed out culture, prevents undesired simultaneously
Cell washes out.The additional liquid of entrance system can be that the flow of additional current or nutrient feed streams increases.
Technique detailed description under normal operating conditions
The present invention relates to the gaseous state group for the gaseous state substrate containing at least one reducibility gas, particularly industrial waste
Division and combination becomes gas (such as CO, CO2And H2) anaerobic fermentation produce ethanol method.By way biology of operation target bacteria
Footpath, these methods create the alcohol production rate higher than 10g/L every day.A kind of method of the present invention causes the abundance of NAD (P) H
More than NAD (P).NAD (P) H is oxidized to NAD (P) and causes the acetic acid produced by culture to be reduced into ethanol.Or, at this
In bright anaerobic fermentation, additive method for producing high concentration ethanol relates to the oxidation-reduction potential reducing fermentation liquid, and thus
Acetic acid is reduced into ethanol.The method of the present invention produces high concentration of alcohol (the most greater than about 10g/L, preferably above about 15g/L)
With low acetic acid class substrate concentration (i.e. in bioreactor below about 5g/L free acetic acid).These methods also maintain and control
System is for ethanol and acetic acid quantity-produced method condition, with help system fast quick-recovery from the disturbance of method.Additionally, this
Bright method helps to stop culture to adapt to low nutrition substrate concentration environment, and this adaptation has been probably for culture performance
Evil.The present invention is that alcohol production provides feasible commercial methods.
The biological pathway that the method for the present invention utilizes under normal operating conditions
Being not intended to be bound by theory, the present inventor advances a theory and thinks, from the increase second of method described herein
The method that alcohol anaerobism produces, is based on and relates to NAD (P) H in the Basic Ways circulation producing acetate pathway of autophyting growth
The biological pathway converted to NAD (P).The present invention includes operating those approach can produce continuously under the conditions of stable operation
With maintain high concentration of alcohol and low acetate class substrate concentration, thus provide for producing can be commercial use of ethanol from industrial gases
Method.In biological approach NAD (P) H to NAD (P) must participate in be described as follows: from gaseous component such as CO, CO2And H2Produce
Ethanol occurs in three step biological methods.In the first step, substrate CO and H2Oxidized, and discharge NAD in this case
(P) H:NAD (P) → NAD (P) H, CO+H2+H2O→CO2+4H+。
Then the product of step 1 is transformed into acetic acid, and this step needs NAD (P) H:NAD (P) H → NAD (P), CO+CO2+6H+
→CH3COOH+H2O.Finally, the reaction of the speed ratio step 2 carried out if as the reaction of step 1 faster causes excess NAD
(P) H can use, and acetic acid is reduced into ethanol.NAD (P) H → NAD (P), CH3COOH+4H+→C2H5OH+H2O.Therefore, from substrate
The availability of excessive NAD (P) H of oxidation, result in and produce ethanol from acetic acid.
There are two known Basic Ways circulations: (1) S-acetyl-coenzyme-A circulates, and (2) THF follows in producing acetate pathway
Ring, wherein CO2It is reduced into methyl.The order producing ethanol and acetic acid from which is described in J.R.Phillips etc., and 1994
Applied Biochemistry and Biotechnology, in 45/46:145.S-acetyl-coenzyme-A circulation has inner loop,
It is referred to herein as CO circulation.Because CO circulation reaction the most clockwise, therefore ferredoxin is reduced.Work as H2At hydrogenase
Time the most oxidized, ferredoxin also can be by H2Reduction.As a result, S-acetyl-coenzyme-A circulation is reacted the most clockwise, ferredoxin quilt
Oxidation.If internal CO circulation and S-acetyl-coenzyme-A circulate with same speed response, then ferredoxin is in oxidoreduction shape
Among state balance.But, if the two circulation occurs the most at the same rate, i.e. it is auxiliary that the speed of CO circular response is faster than acetyl
Enzyme A circulates, then the ferredoxin accumulation reduced.Additionally, work as H2During excess, it is also possible to produce the reduced iron oxygen also egg of excess
In vain.The reduction ferredoxin of this excess causes NAD (P) to be reproduced (reduction) one-tenth NAD (P) H, and this excess set up must quilt
Solution is divided by reaching balance, and in the process acetic acid is reduced into ethanol.
THF circulation cell growth works, and is required for cultivating continuously；Therefore it can not be stopped completely
Only.Reduce THF cycle rate to be also used for producing higher NAD (P) H Yu NAD (P) ratio.NAD (P) H in two places by oxygen
Change.By limit this oxidation, by keep total cellular NAD (P) H Yu NAD (P) ratio be in balance, NAD (P) H be used for by
Acetic acid is reduced into ethanol.
The second basic skills causing acetic acid to be reduced into ethanol is the oxidoreduction electricity by directly reducing fermentation liquid
Position.The reducing condition being sufficiently below culture native state causes NAD (P) H enrich and promote that acetic acid is reduced into ethanol.
Properly functioning method
The basic step of method includes following: describe continuous ferment process with reference to Fig. 1 and product reclaims.To thin containing target
The fermenting organism reactor 3 of bacterium, contains at least one reducibility gas such as CO or H with selected gas feed rate supply2
The continuous stream of gaseous state substrate 1, with the continuous stream of selected nutrient feed rate supply liquid phase Nutrient medium 2.At biology
In reactor 3, culture medium and gaseous state substrate are produced ethanol and acetic acid by bacterial fermentation.Reach stable the most under steady state conditions, a reactor
Cell concentration, just the component of operation continuous system is to reduce oxidation-reduction potential or to increase NAD (P) H and NAD in fermentation liquid
(P) ratio, keeps the free acetic acid concentration in bioreactor at below 5g/L simultaneously.The method of the present invention is designed to fair
Permitted and maintained ethanol and the production of acetic acid class thing in fermentation liquid so that the ethanol between 1: 1 to 20: 1 and acetic acid class thing ratio
Under, the productivity ratio of ethanol is more than 10g/L every day.In one embodiment, this ratio is more than 3: 1.In another embodiment
In, this ratio is more than 5: 1.In another embodiment, this ratio is more than 10: 1.In another embodiment, this ratio
More than 15: 1.The method of the present invention can also strengthen from CO, CO effectively2And H2With good method stability steady and continuous
(stable state) produce high concentration ethanol (15-35g/L ethanol) and low concentration of acetic acid class thing (0-5g/L acetic acid class thing), i.e. ethanol with
The proportion of products of acetic acid class thing is 3: 1 or higher.
During the method for the present invention, periodically take out broth samples, measure ratio by conventional method of analysis.Example
As, by such as centrifugal from sample separation cell, then cell-free sample is performed analysis method, such as preferably gas chromatogram
Method.But, the professional of the art can select other conventionals method of analysis.Additional optional step is increased to method
Rapid to reach and/or to maintain described ratio.
For operating system component and maintenance and/or the step realizing required alcohol production rate or ethanol and acetic acid ratio,
Including at least one the following step and the suitable combination of the following step: change Nutrient medium content, nutrient feed
Speed, water feed rate, operating pressure, operation pH, gaseous state substrate content, gas feed rate, fermentation broth agitation rate, keep away
Exempt from product inhibition step, reduce the cell density in bioreactor, or stop substrate suppression.Some preferably operate include to
Bioreactor provide liquid phase nutrient (pantothenic acid compound pantothenic acid compound or cobalt) limit, in feed gas slight excess of CO and
H2, minimize acetic acid concentration, it is to avoid culture is adapted to the environment of low liquid phase nutrient concentrations, will cultivate with relatively fast speed
Thing takes applicable cell concentration to, and the pH of culture is increased to more than 4.5, second present in the fermenting organism reactor meat soup
When the free acetic acid portion of acids thing is more than 2g/L, bacteria removal cell from bioreactor, make cell concentration be less than stable
Css to utilize all reducibility gas or nutritional substrate in bioreactor, and increase water feed rate, thus press down
Any undesired increase of free acetic acid concentration processed.All these steps will be discussed in more detail below.
Come autoreactor containing CO, CO2And H2Outside gas and unconverted CO, CO2And H2Waste gas 4 from instead
Device is answered to discharge, and for its fuel value.If the H2 of excess uses as control mechanism, then H in exit gas2Dividing potential drop and
Discharge H in gas2Dividing potential drop and CO2The ratio of dividing potential drop is used for the control identifying this step to ethanol Yu acetic acid class thing ratio.Cell
Recirculation is used for (but optional) increases the cell concentration within bioreactor, thus is CO, CO2And H2Conversion carries
Supply more biocatalyzer.Along with cell recirculation, the liquid efflunent carrying out autoreactor 5 is sent to cell separator 6,
Cell 7 is separated with permeate (cell free fluid) 8 there.Cell 7 is sent back to bioreactor, and permeate 8 is sent to product
Reclaim.
Cell separation by use continuous centrifuge, doughnut or spiral winding filtration system, ceramic filter system or
Other solid/liquid separator realize.Can be by various different technology, including distillation with absorb, from permeate (or such as
If fruit does not uses cell separation, instead carry out the effluent of autoreactor 5) reclaim ethanol.Permeate 8 divides in a distillation column
From, producing overhead 10 and the water 11 of 95% ethanol, water is used for being recycled back into reactor 3.Recirculation water 11 is containing sending out
Ferment does not has the supernutrition thing used, but any excess vitamin cracked from fermentation or cell is broken by thermal distillation
Bad.The overhead 10 of 95% ethanol is sent to molecular sieve 12, end-product dehydrated alcohol 13 required there and Diluted Alcohol
14 separately, and the latter is sent back to distillation column 9.
Growth, dead and cell clearance combination continuously maintain constant cell concentration so that produce ethanol (and
A small amount of acetic acid) the middle continuation method used can be by charging CO, CO2And H2And nutrient run many months and need not be another
Outer supplementary culture.The method of the present invention maintains and controls for ethanol and acetic acid quantity-produced condition, and stops or fast
The disturbance of speed bearing calibration.The method of the present invention also helps to prevent culture and is adapted to the environment of low nutrition substrate concentration, this suitable
Tackle be probably for culture performance harmful.In following description and embodiment, except as otherwise noted, otherwise use
Pressure be 1 atmospheric pressure, the temperature of use is between 36-41 DEG C.Preferably temperature and pressure can be by the art
Professional determines according to the selected microorganism used in bioreactor.
The various operations increased to basic step of the present invention described in detail below, it is allowed to strengthen the life of ethanol
Produce.Preferably, the liquid phase nutrient limitation (pantothenic acid compound pantothenic acid compound or cobalt) being described below in detail or excess H2Or the use of CO,
It is that the present invention is for obtaining and maintain required alcohol production rate and allowing to produce steady concentration and the ethanol of ratio in fermentation liquid
Method step with acetic acid class thing.These conditions permits produce ethanol and the acetic acid class thing of steady concentration in fermentation liquid.Excellent
Selecting in embodiment, the ethanol produced in fermentation liquid is more than 10: 1 with the Product ratios of acetic acid class thing, and concentration of alcohol is high
In 15g/L.
A. calcium pantothenate limits
In one embodiment of the invention, it is used for operating biological pathway and is beneficial to alcohol production and limits second
The method that acid produces, including the amount of calcium pantothenate in Nutrient medium being limited in less than antibacterial maintains fully utilized offer
Calcium pantothenate stable Css needed for amount.Pantothenic acid is the component of S-acetyl-coenzyme-A, therefore, cultivates by limiting nutrition
Calcium pantothenate in base, S-acetyl-coenzyme-A cycle rate reduces relative to CO cycle rate.This causes the long-pending of reproducibility ferredoxin
Tire out and NAD (P) is to the reduction of NAD (P) H, thus add the production of the ethanol as end-product.
For every gram (g) cell (dry weight) for producing in reactor, in reactor, the calcium pantothenate of charging is micro-
Time gram (μ g) number is in the range of 0.5 to 100, it was observed that pantothenic acid restriction.More preferably pantothenic acid limits relative in reactor
Every gram (g) dry cell weight produced, in the range of 2 to 75 μ g calcium pantothenates.Preferred pantothenic acid limits relative in reactor
Every gram (g) cell produced, in the range of 0.5 to 50 μ g calcium pantothenates.Another embodiment of this restriction is relative to instead
Answer every gram (g) cell produced in device, about 1-25 μ g calcium pantothenate.Another embodiment of this restriction is relative to reactor
Every gram (g) cell of middle generation, about 10-30 μ g calcium pantothenate.The amount of this nutrient maintains alcohol production to have precedence over acetic acid class thing
Produce.
At another aspect of this method, by regulation or adjustment fermentation parameter so that maintain calcium pantothenate concentration, adjust simultaneously
Whole gas feed rate, liquid feed rate, stir speed (S.S.) or H2At least one in dividing potential drop, parameter more than sometimes one,
The antibacterial in fermenting organism reactor is avoided to adapt to the environment of low restricted calcium pantothenate concentration.Avoid the bigger change of nutrient
Change, but maintain the nutrient feed concentration of relative constancy.In irreversible method, if allowing culture to adapt to low liquid
Phase limiting nutrient substance environment, will appear from the low Product ratios of 1.0g ethanol/g acetic acid class thing or lower.Therefore, reactor closes
Machine and renewed vaccination are required.Preferably, by first supply excess H in the feed gas of bioreactor2, then according to
Calcium pantothenate in restriction Nutrient medium described above, controls biological approach, raw with beneficially alcohol production and restriction acetic acid
Produce.
It is true that when starting, usual restricted liquid phase nutrient calcium pantothenate keeps excess dense to avoid low nutrition thing
The adaptation of degree environment, if not using excess H2, this adaptive state may cause excessively poor performance, and make culture lose
Carry out producing to obtain the ability of the high alcohol production rate exceeding 10g/L every day.
B. cobalt limits
In another embodiment of the present invention, it is used for operating biological approach be beneficial to alcohol production and limit acetic acid life
The method produced, including the amount of cobalt in Nutrient medium being limited in less than antibacterial maintains the steady of fully utilized provided cobalt
The fixed amount needed for Css.When for every gram (g) cell (dry weight) produced in bioreactor, to reactor
When microgram (μ g) number of the cobalt of middle charging is in the range of 5 to 100, it was observed that cobalt restriction.Preferably, cobalt limit include relative to
The every gram of cell produced in reactor provides about 20 to 50 μ g cobalts to reactor.This cobalt amount maintains the alcohol production in technique preferential
In acetic acid production.
Cobalt in restricted fermentation liquid can also reduce S-acetyl-coenzyme-A cycle rate.Because cobalt is used for following methyl from THF
Ring transfers to S-acetyl-coenzyme-A circulation, and therefore the cobalt amount in restricted fermentation liquid also reduces, by not allowing to shift, the merit that THF circulates
Energy.Cobalt limits and reduces THF cycle rate, and this also causes higher NAD (P) H Yu NAD (P) ratio, thus produces ethanol.
By stoping the adaptation to low restricted cobalt concentration environment, method is carried out further manipulation.With with avoid
Adapt to the mode that low pantothenic acid concentration environment is almost identical, maintain constant cobalt concentration, adjust one or more fermentation ginseng simultaneously
Number (gas velocity, liquid rate, stir speed (S.S.), CO2Content and H2Partial pressure).Avoid the large change of nutrient, but tie up
Hold the nutrient feed concentration of relative constancy.
Preferably, by first to reactor feed excess H2, the cobalt in Nutrient medium the most limited as described above is controlled
Biological pathway processed, in order to be conducive to alcohol production and limit acetic acid production.When starting, restrictive liquid phase nutrient cobalt
Keeping excess to avoid the adaptation to low nutrition substrate concentration environment, this adaptive state may cause excessively poor cultivation physical property
Can, and make culture lose the ability of the generation Product ratios more than 1: 1.
C. excess supply hydrogen
In another embodiment, for operating biological approach so that beneficially alcohol production limit acetic acid production
Method, is included in feed gas charging excess H2, or limit gaseous carbon thus cause excess H2, then used by biological approach.
Preferably, H2Reducibility gas is relative to CO excess, the H of excess2Antibacterial is caused to produce high ethanol and acetic acid class thing in fermentation liquid
Ratio.If H2The twice of the CO (number of moles of gas) of (number of moles of gas of charging) and conversion and the CO of conversion2(gas molar
Number) the ratio of summation of three times more than 1, then fermentation tank is limit carbon.Discharge H present in gas2Dividing potential drop is preferably greater than
0.4atm.Finally, H2Dividing potential drop and CO2The ratio of dividing potential drop have to be larger than 3.0, to guarantee to obtain enough H2Use all of
CO2.If CO2Dividing potential drop is more than 0.1atm, it is possible to grow the most limited.
When starting, use excess H2It is better than nutrient limitation, is primarily due to it and is easier to control.Use excess H2's
Have an advantage that it avoids acetic acid excess and produces, this excess produce can cause the poor and potential acetic acid of Product ratios suppress with
And the adaptation to low nutrition substrate concentration environment.
D. excess supply carbon monoxide
The another way of the component of operational approach is included in for excess supply reproducibility in the gaseous state substrate of approach
Gas CO, it can be used for the oxidation-reduction potential directly reducing in fermentation liquid.Therefore, according to this embodiment, to biological respinse
Device provides containing the gaseous state substrate of CO, wherein present in bioreactor the amount of CO higher than antibacterial being maintained fully utilized institute
Amount needed for the stable Css of the CO provided.Specific speed (every gram of cell (dry weight) in bioreactor when CO picked-up
MM number of the CO of picked-up per minute, or mmol/g cell minute) more than 0.3 time, CO excess supply becomes makes alcohol production
The method being better than acetic acid production.More particularly, this step includes that the specific speed that CO absorbs is more than 0.5.This means average every
Individual cell with at least 0.3mmol/gmin or more preferably utilizes CO with the speed of at least 0.5mmol/gmin in its metabolism.Excellent
Choosing, CO provide speed make CO picked-up be 0.3 to CO/ gram of bacterial cell of 2mmol (dry weight)/minute.Another embodiment party
In case, CO with 0.5 to 1.5mmolCO/ gram of bacterial cell (dry weight)/minute speed provide.In another embodiment, CO
With CO/ gram of bacterial cell of about 1mmol (dry weight)/minute speed provide.
This CO uptake rate maintains alcohol production to have precedence over acetic acid class thing and produces.If the supply of CO makes in fermentation liquid
The CO dissolved is notable owing to gas pressure or fabulous material transmit, and fermentation liquid will become more to be reduced.Excess supply CO
There are two additional benefits.The CO of excess can cause CO circulation to operate with speed faster, and if S-acetyl-coenzyme-A circulation
Otherwise it is restricted and can not catch up with CO circulation, will build up on the ferredoxin of reduction.CO also is able to pass through substrate
Suppression is slowed down the step 2 (producing intermediate product acetic acid) in three one step process altogether.This of step 2 reduces speed relative to step 1
Rate causes NAD (P) H excess, and this causes alcohol production to be better than acetic acid.
Although excess CO can cause alcohol production to increase by the oxidation-reduction potential directly reducing fermentation liquid, but excessive
The existence of CO is also by suppression CO dehydrogenase and therefore suppresses H2Picked-up, thus Developing restraint.There is excess CO the most unfortunately to lead
The H caused2It is poor to convert, and this is not likely to be favourable economically.Under substrate suppresses, long playing consequence is H2Picked-up is not
Good.This ultimately results in cell and cracks and need to reset reactor.When during the initial growth of culture or thereafter, should
When method has undesired CO substrate suppression result (there is too many CO for available cell), reduce gas feed rate
And/or stir speed (S.S.), until substrate suppression is alleviated.
E. additional operating step
In addition to main method described above strengthens step, method ethanol production comprises several method step
It is preferable.
1. increase material transmission
A kind of such additional embodiment includes ensuring that CO or H2Transmit from gas feed to the material of liquid fermentation liquid
It is faster than antibacterial and utilizes the ability of dissolved gas.Such as, if containing the bioreactor of Clostridium ljungdahlii
By charging CO, CO2 and H2, and limit or excess H at nonnutritive thing (such as calcium pantothenate or cobalt)2Run under conditions of existence, then
Cell growth is by the gas quantitative limitation being delivered in liquid phase, and system produces acetic acid as product.If fed to culture
The desired amount of CO or H is grown only slight beyond culture2, it produces ethanol.But, if too much gas is delivered in liquid phase as training
Supporting used by thing, substrate suppression will occur, this can cause culture disturbance and cell death.Therefore, the behaviour of excess material transmission
Make scope the narrowest.
Circulating with reference to S-acetyl-coenzyme-A, in order to produce the reduction ferredoxin of excess, CO circulation or ferredoxin pass through
The reduction of hydrogenase must carry out circulating faster than S-acetyl-coenzyme-A.Method described herein can be used for antibacterial by restriction
Required nutrient such as calcium pantothenate or cobalt or other substrates such as CO2The speed of gas, or by providing the excess end to culture
Thing H2Or CO, limit organism and can utilize the speed of dissolved gas.
The Theoretical Rate of material transmission can be calculated, even if it can utilize substrate than antibacterial under not having other to limit
Speed is faster.This speed when reached, is limited by organism nature growth rate.Therefore, there is most the enforcement of production efficiency
Scheme is that wherein material transmits (gas flow rate or stir speed (S.S.)) than the cell of highest possible concentration in the feelings not having any restriction
The faster embodiment of speed of substrate can be utilized under condition.Because substrate suppression can quickly cause cell death, and institute
The by-product concentration generated is poisonous to culture, and therefore opereating specification is the narrowest.
2. supply excess CO and H2
In another embodiment of the inventive method, limiting cobalt or calcium pantothenate or the H enriched is being provided2Or CO
In method, it is achieved that the stability of high concentration of alcohol/limited acetic acid production.According to this step, when culture uses gaseous state substrate
CO、H2And CO2During as carbon source and the energy, the most excessively supply CO and H2.By reaching steady-state operation, it is then gradually increased
Gas feed rate and/or stir speed (S.S.) (being incremented by with 10% or lower) are until CO and H2Convert and just start to reduce, obtain
Slight excess of CO and H2.This is that the material transmission avoiding beneficially acetic acid production limits and provides the reduced iron oxygen of excess
Also albumen to be reduced into NAD (P) H and producing a kind of means of ethanol by NAD (P).If CO and H2Do not carry with slightly excess
Confession, will occur material transmission to limit, and described approach reaches balance.This causes ethanol and acetic acid poor (the high acetic acid of class thing Product ratios
Class substrate concentration).High acetic acid class substrate concentration may finally cause acetic acid to suppress, and which has limited antibacterial picked-up H2Ability and may be
Culture is caused to lose efficacy eventually.
The step avoiding material transmission to limit includes increasing stir speed (S.S.) or gas velocity to be passed by more CO and H2
It is delivered in liquid phase, thus is returned to there is slight excess of CO and H2.If owing to material transmission limits and there occurs that product presses down
System, it is necessary to increase liquid feed rate, removes acetic acid suppression by being diluted to relatively low acetic acid class thing generation concentration.Because increasing
Adding medium feed speed and will increase pantothenic acid compound pantothenic acid compound or the μ g number of cobalt of the every gram of cell produced, therefore this must be only
Of short duration can carry out, maybe must eliminate excessive pantothenic acid compound pantothenic acid by adjusting culture medium concentration or increase water feed rate
Thing or cobalt.
3. regulation acetic acid product suppression
In method described above, if bioreactor accumulates too many molecule acetic acid, i.e. ＞ 2g/L, thus do not permit
Permitted cell growth and further alcohol production, then it may happen that acetic acid product suppresses.Employ another operating procedure to avoid
Culture lost efficacy.A kind of amendment includes of short duration increase liquid or water feed rate, is reduced to by the liquid concentration of inhibition acetic acid
Less than 2g/L.
4. water recirculation step
In the method for the invention, for the stable training maintaining generation ethanol not have clean acetic acid to produce as exclusive product
Support another selectable method step of thing, including increasing the water recirculation returning to fermentation reactor from distillation.As relatively
Mentioning time early, water (containing the highest 5g/L acetic acid class thing) recirculation has an advantage that and is recycled back into by the acetic acid class thing of generation
In reactor, so that not having clean acetic acid to produce.Therefore, balance is set up between ethanol and acetic acid class thing in the reactor.Knot
Really, all CO, CO being fed to reactor and being transformed into product2And H2, in addition to maintaining for culture, result in ethanol
Produce.
5. reduce cell density
Can be used for another operating procedure of method is to start the most clear from bioreactor of bacterial cell
Remove, to reduce the cell concentration in bioreactor.This operation utilizes bioreactor for being fallen below by cell concentration
In all reducibility gas or the stable steady state cell concentration of nutritional substrate.Therefore, by changing cell density, anti-at biology
The production of ethanol in device is answered to be better than the production of acetic acid class thing.
6. two benches CSTR
The problem limiting lower alcohol production with culture medium relevant is that culture finally adapts to limit after running some months
Property condition processed and do not continue to produce the ability of ethanol or tendency.Instead acetic acid class thing ultimately becomes predominant product.This right
The adaptation of low limiting nutrient substrate concentration, causes culture to produce (ethanol and acetic acid class produce thing more more than ethanol acetic acid
Ratio is 1.0 or lower), and produce low concentration of alcohol (sometimes as little as 1g/L).When comparing ethanol production rate in growth rate
During prior starting not when culture provides enough nutrients, it is most likely to occur adaptation.Additionally, in steady-state operation
Cheng Zhong, particularly when lower limiting nutrient substrate concentration so that response system breaks away from acetic acid class thing time, there is also culture can
The danger that low limiting nutrient substrate concentration can be adapted to.
When using above-mentioned pantothenic acid or cobalt conditioning step rather than allowing culture to use the growth of obtainable nutrient,
In order to avoid this adaptation and danger above-mentioned, it is possible to use the another kind of amendment of method.The another kind of amendment of method
Being two-stage type CSTR system, wherein to occur mainly in limiting nutrient thing in the first order slight excess of in the good growth of culture
In the case of (may be attended by acetic acid produce), followed by produce level, wherein from culture the being restricted property now of the first order
The restriction of nutrient also is used for producing high concentration ethanol.This amending method is able to maintain that not to the pantothenic acid compound pantothenic acid reduced
Compound or cobalt concentration produce the stable culture adapted to.This amending method includes running two-stage type CSTR, wherein growth response
Device (1 grade) is to producing reactor (2 grades) charging, and the production of a large amount of ethanol occurs in producing reactor.Growth reactor is not
Nutrient limitation step described above is used to run, so culture is not easy to adapt to restrictive condition.
According to the embodiment of two-stage type CSTR, growth level runs the liquid residence time (LRT) of about 24 hours.Growth level
CSTR 1 charging culture medium 2 containing enough pantothenic acid compound pantothenic acid compounds or cobalt (and also may be used to produce healthy culture
Some acetic acid can be produced).Therefore, create excessive acetic acid in the reactor, but stability increases.This pantothenic acid compound pantothenic acid
Compound or cobalt concentration exceed the concentration being normally fed in single-stage CSTR for producing ethanol.It is fed to the gas in this reactor
Body is that liquid feedstock is fresh culture 2 from the unconverted gas 3 producing level 4.The operation of growth level CSTR does not use cell
Recirculation.The purpose of this growth stage reactor is to provide not to low pantothenic acid compound pantothenic acid compound concentration for alcohol production subsequently
Produce the healthy culture adapted to.
Produce stage reactor 4 to run with the specified LRT less than 20 hours.Should feed fresh with the CSTR of cell recirculation
Gas feed 5, and be likely to be of low conversion.It is fed fresh culture charging 6 and the culture from growth level
Charging 7.Because there is the excess quantity from growth level to use, so to this reactor feed minimal amount of pantothenic acid compound or cobalt.?
This reactor employs cell recirculation 8, in order to the cell produced by great majority sends back to reactor 9.Product liquid
Discharge concentration of alcohol in 10 should be higher than 20g/L.The feature of two-stage CSTR system includes seldom changing over the pantothenic acid to low
Thing or cobalt concentration adapt to, and total LRT is less equal than 30 hours, pre-in respect of higher compared with single-stage CSTR of same size
Alcohol production rate and higher concentration of alcohol.
7. start amendment
In the practice of the invention preferably by additive method step, be included in the initial start of fermentation culture is thin
Born of the same parents produce.Start bioreactor charging CO, CO2And H2It is by inoculating from stock culture in batches to produce ethanol and acetic acid
Or by using the vaccinization thing from pre-existing reactors to realize as culture charging.As avoiding training time relatively early
Support thing and low pantothenic acid compound or cobalt concentration occurred in the discussion adapted to pointed, but optimum limit nutrient to
Culture supply excess H2Before, culture is made to reach high-cell density.This quick starting avoids culture and adapts to also
Create good proportion of products (high ethanol and low acetate class substrate concentration).If not using fast starting, then it is likely to occur product
Thing ratio is poor, and low liquid phase nutrient concentrations can be produced and adapt to by culture and it needs to reactor renewed vaccination.
Reactor uses batch liquid phase (fluid medium is the most discontinuously fed in reactor), with low stirring speed
Rate is (at use for laboratory New Brunswick Scientific BiofloTMMay be for 400-600rpm in reactor) and in institute
Need to start under pH.Therefore, the liquid phase in reactor is made up of a certain amount of Nutrient medium containing vitamin and salt, has specified
The limiting nutrient thing calcium pantothenate of concentration or cobalt (20 μ g/L pantothenic acid compound and 75ppb cobalt).If used from pre-existing reactors
Vaccinization thing, batch liquid-phase operation is not likely to be required.In this case, during initial start with at a slow speed to
Reactor continuous feed gas.Ideally, gas during starting mutually will be without CO2, rich in H2, and gas velocity and stirring
Mix speed and will remain in low-level to avoid CO substrate to suppress.
For from CO, CO2And H2The exemplary universal start-up program of the concentration of alcohol of production and maintenance viable commercial is by three
Individual different phase is constituted: (a) initial start, and the most crucially cell produces；B () starts, wherein throughput rate becomes crucial；
And (c) steady-state operation.Essentially, the feature of initial start is that inoculation contains choosing under required pH (being typically 4.5-5.5)
From cobalt (75ppb) or the batch liquid of the specified limiting nutrient thing of calcium pantothenate (20 μ g/L).For the ease of starting, preferably keep
Low gas feed rate and stir speed (S.S.), simultaneously overfeeding H2.In starting process, the reason of alcohol production is excess H2；
Nutrition limits later generation.Therefore, starting process there are in fact the liquid nutritional thing of excess, to avoid culture to low
There is undesired adaptation in nutrient.Along with carrying out fermentation in several hours after inoculation, create CO2And consume H2.These speed
Change shows that stir speed (S.S.) should specified be slowly increased (in laboratory reactor, may increase 200-within 2-3 days time
300rpm), to avoid material transmission to limit.
In the system using vaccinization, inoculating contrary with the batch from stock culture, what CO2 produced starts appearance
Much faster.But, if stir speed (S.S.) increase is too fast, the suppression of CO substrate will occur.This observation H2Convert (or CO2Produce)
The program of the most specified increase stir speed (S.S.) is carried out with relatively fast speed, until reaching target stir speed (S.S.).Train at batch liquid
In Yanging in the time course of this increase stir speed (S.S.), most importantly cell produces rather than product is formed.
Once reach target stir speed (S.S.) (at use for laboratory New Brunswick Scientific BiofloTMReactor
Middle 800-1000rpm) after, it is allowed to culture reaches stable to confirm H2Picked-up.Starting becomes wherein throughput rate and becomes weight
The pattern wanted.Convert it is desirable that have the CO more than 80% and discharge H high in gas2Dividing potential drop (at least 0.55atm) is with really
Protect alcohol production, with limit acetic acid class thing and free molecule acetic acid concentration.Then start fluid medium feed rate (for
From stock culture batch inoculation system for) with start continuous liquid charging, and with 10% increment by gas velocity court
Increase to target flow velocity.Keep H2Excess is to avoid excessive acetic acid to produce.When gas velocity increases, limit liquid phase nutrient (general
Acid calcium or cobalt), the effect of this restriction is to reduce H on a small quantity in target production department2Convert.
In steady-state operation, 15-35g/L ethanol and the production of 0-5g/L acetic acid class thing are reached.In this stage, need
A small amount of adjustment limiting nutrient thing, liquid feed rate and gas feed rate, by the professional of the art by this
The existing knowledge of technical field and the teachings of the present invention select.If to add cell in method ethanol production to follow again
Ring, it adds along with gas flow rate (increasing) and the adjustment of nutrient concentrations (reduction) the most at this moment.
Described above produce continuously under the conditions of stable operation and maintain high concentration ethanol and by-product acetic acid class thing
The method that concentration is low, adds described target bacteria on a commercial scale for the purposes of alcohol production.In the above methods
The step of general introduction overcomes and utilizes described target bacteria from CO, CO2And H2Carry out the restriction of commercialization alcohol production.Preferably described
Method uses continuous bioreactor, although batch and fed-batch type fermentation process can also use, but on a large scale
It is unlikely to be economically feasible for alcohol production.
The following examples shall illustrate some specific embodiments of invention disclosed herein.But, these are implemented
Example should not be construed as limited to neoteric scope, because as the professional of the art is recognized, and can
The present invention to be carried out many amendments spirit without departing from disclosed invention.
Embodiment
Having carried out initial experiment uses ethanol and carbon dioxide to maintain Clostridium as the energy using research
The viability of ljungdahlii.In this experiment, carbon dioxide provides as by the gas of culture bubbling.By by temperature
Degree is reduced to 25 DEG C and controls free acid concentration by increasing pH set point.Close forming gas and delay in order to about 30ml/min
Slowly the carbon dioxide blasted replaces.Stirring is reduced to just provides enough mixing so that heat and liquid additive are distributed to
Low-level in reactor.PH is brought up to 4.7 from 4.5.Reactor has the cell recirculation loop using hollow-fibre membrane,
It allows to utilize permeate to be carried out the loss cell stoped in experimentation.The cultivation of entrance system it is equal to through liquid stream
Base flow.Liquid residence time is constant, is maintained at 30 hours.
After supplying 12 hours without forming gas, as expected, ethanol and the total acetyl group concentration measured have changed
Become.Levels of ethanol is reduced to 12.8g/L from 24.0, and total acetyl level increases to 10Ag/L from 4.2.Temperature set-point returns
To 38 DEG C.When culture heats, the phase same level used before stirring is increased to experiment；It is the front stream used of experiment with flow velocity
The forming gas stream of the 50% of speed replaces carbon dioxide.
Stop permeate cleaning.Culture is maintained under this condition 14 hours.In this time course, carbon monoxide is taken the photograph
Going bail for, it is fixed to keep steady, and hydrogen picked-up is stable to be increased.After once hydrogen picked-up fully increases, it is stepped up gas flow rate to arrive
Flow velocity before experiment.Forming gas flow velocity returned to test front speed in 47.5 hours.When feed gas stream increases, total acetyl group
Concentration reduces, and concentration of alcohol increases.Total acetyl group concentration fell back to test front level in 32 hours.Concentration of alcohol is little 70.5
Time interior reach close to experiment before level.
In this experiment, carbon dioxide is provided by 7.7% sodium bicarbonate solution is continuously flowed into culture.By temperature
It is reduced to 25 DEG C.By temperature being reduced to 25 DEG C, increasing pH and control to dissociate by increasing the liquid stream through culture
Acetic acid concentration.Close forming gas and flow replacement continuously with 7.7% sodium bicarbonate.In the presence of sour environment, sodium bicarbonate
Resolve into sodium ion, water and carbon dioxide, thus provide for ethanol conversion being become carbon dioxide necessary to free acid.Will
Stirring is reduced to just to provide and enough mixes so that the low-level that is distributed in reactor of heat and liquid additive.Do not control pH
Set point, but be because sodium bicarbonate and be added in culture, in whole experimentation, pH is slowly increased, and this contributes to control
The concentration of free acid processed.Reactor has the cell recirculation loop using hollow-fibre membrane, and it allows to utilize permeate to carry out
Clean to stop the loss cell in experimentation.Additional bicarbonate is added equal to the base flow of cultivating of entrance system through liquid stream
Sodium stream.Liquid residence time was reduced to 21 hours from 29 hours by extra bicarbonate stream.
In experimentation, along with total acetyl group concentration stably raises, concentration of alcohol reduces.In 5.5 hours, ethanol
Concentration is reduced to 14.1g/L from 21.0, and total acetyl level increases to 9.1g/L from 4.4.Measure the pH arrived also
Increase to 4.84 from 4.48.In order to attempt to control the concentration of acid, after experiment starts 5.6 hours, the flow velocity of nutrition logistics
2.81mL/min is increased to from 1.33mL/min.It is undesired to stop that permeate cleaning also increases to 3.48mL/min from 1.86
Cell washout.Liquid residence time was reduced to 12 hours from 21 hours by these changes.This has and keeps low required of acid concentration
Effect.Changing after fluid flow 2 hours, total acetyl group concentration is only only increased to 9.4g/L.But concentration of alcohol is with faster
Speed is reduced to 10.7g/L from 14.1.Behind do not have that forming gas supplies 8 hours, measure the ethanol arrived and total acetyl group is dense
Degree changes as expected.Levels of ethanol is reduced to 10.7g/L from 21.0, and total acetyl level increases to 9.4g/L from 4.4.Temperature
Set point liter returns to 38 DEG C.When culture heats, stirring is added to and the phase same level testing front use；Stop adding carbon
Acid hydrogen sodium, 50% beginning forming gas stream of the flow velocity to use before experiment；And stop permeate cleaning.Culture is in this state
Under only maintain 50 minutes.It is stepped up gas flow rate to reach former flow velocity.In 29.2 hours, forming gas flow velocity is
Return to test front speed.When feed gas stream increases, in 43.2 hours, total acetyl group concentration reduces and concentration of alcohol
Increase to concentration before experiment.The most Already in the ethanol in fermentation tank can be at forming gas together with carbon dioxide
Culture viability is maintained during interruption.
Embodiment 1
Use ethanol and CO2Convert the bacterial gas bulk diffusion research of production capacity
The purpose of Experiment on Microbiology determines that and loses feed gas at feed gas forfeiture long period (＞ 30 minutes)
In the case of maintain the method for culture.In the present embodiment, interpolation is focused on for the CO that ethanol is transformed into free acid2Make
During forming gas is lost, a kind of mode of energy is obtained for culture.
Since significant period of time, have appreciated that some produces acetic acid microorganism and can use CO2Ethanol is transformed back into
Acetic acid, but the most do not carry out overtesting to determine whether this process can maintain for long-time when not having forming gas can use
Culture.Embodiment of the present invention provide for the solution that survives when losing feed gas, this be due to
When normal bio reactor runs, ethanol and CO2(with the form of sodium bicarbonate) be readily available for.Except adding CO2With
Outside ethanol is transformed into acid, some experimentation reduces the temperature one as the culture activity that slows down of culture
Mode.Slower cytoactive will reduce the requirement of energy, required CO2With amount of alcohol and the amount of the acid of generation.
For these are tested, straight as with cell recirculation and culture cooling coil loop of bioreactor
Logical CSTR runs.Use permeate to clean to stop undesired loss cell in experimentation, but wash liquid stream is turned
It is not recycled back in bioreactor to waste liquid.In normal bio reactor running, culture temperature keeps
At 38 DEG C；Stirring is 400rpm；Culture approximate volume is about 2.4L；Culture pH set point is 4.5.7.7%NaHCO3Molten
Liquid controls for pH.Feed gas is containing 15%H2, 45%N2, 30%CO and 10%CO2Forming gas.Synthesis gas feeds
Speed is about 475mL/min.Culture medium is fed in reactor with about 1.30-1.35mL/min or about 1870-1940mL/ days.
Liquid and cell residence time average out to 25-30 hour.The culture medium used is to be conventionally used for the 1x EtOH cultivation that C-01 cultivates
Base.Medium component and concentration thereof are listed in table 1 below.
In normal course of operation, CL antibacterial uses gas composition CO, H2And CO2As carbon source and the energy or electron source.
Therefore must carefully stop to maintain culture.But, if feed gas supply thing interrupts, then utilize ethanol and CO2 to produce
Acetic acid, as shown in equation below (1).
Equation (1)
By this reaction, cell advantageously can obtain electronics for survival from alcohol to the oxidation of carboxylic acid form.If entered
Material gas halts, culture typically obtains electronics and does not obtain carbon, and therefore cell growth reduces.Therefore, it is believed that this process
Provide the means of culture survival, although not being optimal production.During losing feed gas, it should avoid thin
Born of the same parents wash out or remove any cell from system, to maintain cell density.
This process causes the accumulation of acid.Must take steps to ensure that free acetic acid level maintains below inhibition concentration
The concentration level of (≤5g/L).Realization to this can increase by being by pH set-point brings up to the scope of 5.1-5.3
The liquid stream of system thus LRT is reduced to 15-20 hour, and by limiting available CO2 and/or ethanol to limit the life of acid
Produce, or by reducing temperature to reduce culture metabolism.
The generation of acid consumes the concentration of alcohol in reactor.Because culture shows the second of reduction under these conditions
Alcohol produces, it is therefore necessary to monitoring concentration of alcohol is to guarantee that it will not excessively consume.When feed gas does not increases, may
Need add to system or supplement ethanol.In these experimentations, the concentration of alcohol in bioreactor has been depleted to
As little as 4g/L, and culture is not had adverse effect.
Optionally, in this process, cultivation temperature has played vital as the mode controlling cell metabolic rates
Effect.When the temperature decreases, the metabolic rate of cell slows down.Turn, this reduces sour generation and ethanol and CO2Utilization.When instead
When answering device there is no feed gas, reduce temperature and extend the time span that culture can be survived.On the contrary, if temperature is maintained at
38 DEG C, acid produces speed at its peak and it needs to carefully the monitoring gentle levels of ethanol of sour water is to keep culture healthy.Experiment
Culture temperature is reduced to about 25 DEG C, successfully cell viability is maintained under not having feed gas about 30 hours.
Embodiment of the present invention provide controlled quatity CO2Delivering method, including NaHCO3Add.When sodium bicarbonate quilt
When importing in sour environment such as fermentation liquid, as shown in following equation 2, create CO2.It is believed that this to system interpolation CO2
Method with by CO2It is ejected in culture that to compare be favourable, because sodium bicarbonate not only adds CO2, and by culture
PH increases to about 5.1, helps compensate for, by balance free acid level, the acid that system produces.
Equation 2
After feed gas is lost, ethanol or started to occur the most immediately to the conversion of acid within the several seconds.At feed gas
When losing beginning, by using the high N of about 400-450mL/min2CO is dissolved present in stream elution culture2, it is possible to stop acid
Quickly and a large amount of accumulation.Nitrogen should spray about 0-15 by culture after losing feed gas stream as quickly as possible
Minute, this step is proceeded more quickly, the most favourable to the present invention.Using nitrogen stream in first 5 minutes is embodiment of the present invention.
The CO once dissolved2Stock be removed, it is possible to use controlled feed speed starts to add NaHCO3。
Use NaHCO3Add and CO is provided2The pH of culture can be increased.If cell keeps utilizing all available carbon
The activity of acid hydrogen sodium, pH will be added to about 5.1 and then remains there.This is required the second effect, should not be prevented from.
Slow and stable pH increases the product acid that antagonism will be assisted to raise by constraint free acid level.But, if cell is lived
Property impaired, pH will be added to about more than 5.3, show culture can variability or otherwise function reduce.
When feed gas can use, should stop adding sodium bicarbonate, and the thing of feed gas should be increased as quickly as possible
Matter is transmitted, but the most too gives cell.Within the time of about 10-15 minute, it should stirring is increased to return and loses
Go the same setting used before feed gas, and feed gas flow velocity is increased to return to about the 50% of initial feed rate.Cause
For utilizability and high total acetyl level of substrate, culture stably will transform back into ethanol acid.This will be reflected as pH
Increase, and be intended.At this moment should convert the feed gas flow velocity to fermentation tank according to gas to be changed, as
In any normal reaction device operates like that.
If in terms of preserving cell viability, all are carried out well, and once feed gas stream is subsequently restarted, and enters
Material gas flow rate can reach properly functioning setting in about 20-26 hour.When the concentration of ethanol and acid may spend longer
Between reach normal operating level, about 26-72 hour.
Need the stirring of minimum to maintain the Temperature Distribution in whole bioreactor and to maintain culture pH.Minimum
The stirring of amount will be defined as mixing just to be enough to keep liquid distribution.With the two-forty of properly functioning middle use such as
400rpm compares, and this can be about 50rpm, or 40-60rpm.This stirring the most effectively keeps cell suspension.It is believed that cell should
This is suspended, in order to provide and CO2Constant contact with ethanol performs required reaction.
Produce acetic acid microorganism and need CO or H2And CO2To obtain the required electronics for cell growth and carbon.Do not entering
In the time course of material gas, CO can not be obtained and can not obtain H2For cell growth process.It is believed that at feed gas
In those times lost, cell growth stops.In the feed gas supply thing possible limited time, relatively low feed gas
Supply thing can be used in culture survival, and this is probably reasonably.The feeding gas scale of construction reducing supply provides culture to product
The return of Radix rumicis acetosae formula.When substrate feed rate reduces, culture is sour to the conversion of ethanol by being automatically stopped, and causes ethanol and acid
The gradual decline of ratio.Once this gas forfeiture process is well understood, then may be able to advise producing at feed gas being stranded
During the difficult time, best course of action is to stop feed gas supply completely rather than provide the substrate of relatively low rate.As
Really determine and preferably reduce substrate feed rate, then must take action to deal with the increase of acid.Such action will include
Increasing by the liquid stream of system to remove acid, culture pH is to maintain tolerable free acid level in increase, and/or from system
Middle removing most cells is to maintain the ratio of the healthy gas picked-up needed for low yield acid and cell.
Nutrient media components in table 1. 1x EtOH culture medium and concentration thereof
| Component/ion | Additive | 1x EtOH |
| Concentration (ppm) in culture medium | ||
| NH4 + | NH4Cl/(NH4)2HPO4 | 838 |
| Fe | FeCl2·4H2O | 16.8 |
| Ni | NiCl2·6H2O | 0.198 |
| Co | CoCl2·6H2O | 0.991 |
| Se | Na2SeO3 | 0.0913 |
| Zn | ZnSO4·7H2O | 0.455 |
| Mo | Na2MoO4·2H2O | 0.238 |
| Mn | MnCl2·4H2O | 0.167 |
| B | H3BO3 | 1.05 |
| Cu | CuCl2·2H2O | 0.149 |
| W | Na2WO4·2H2O | 1.12 |
| K | KCl | 78.6 |
| Mg | MgCl2·6H2O | 59.8 |
| Na | NaCl | 78.7* |
| Ca | CaCl2·2H2O | 54.5 |
| Cysteine hydrochloride | Cysteine hydrochloride | 250 |
| PO4 -2 | H3PO4/(NH4)2HPO4 | 816 |
| Pantothenic acid | Pantothenic acid | 0.025 |
| Biotin | Biotin | 0.020 |
| Thiamine | Thiamine | 0.050 |
*Na+Concentration is only from NaCl.It does not include from other compositions such as Na2WO4·2H2The Na of O+。
**Ca+2Concentration does not comprise the calcium from pantothenic acid calcium salt.
Table 2 describes the culture parameter before and after experiment, such as pH, oxidoreduction, ethanol and acetic acid in detail.One
For as, when culture uses ethanol and CO2During survival, levels of ethanol reduces, and acid concentration and culture pH increase.Table 2 is also
List the high free acid level measured in those experimentations and experiment terminate after return to initial gas feed rate
Hourage.It should be remembered that the key element of culture survival is to maintain free acid concentration ＜ during losing feed gas
5.0g/L.When acid produces, higher culture pH or acid faster is needed to remove speed to stop acid suppression.When acid produces
Time, need higher culture pH or acid faster to remove speed to stop acid suppression.
The CO added to culture described in detail by table 32, unit is the mmol/min of every gram of cell in culture.Calculate
It is according to the feed rate of sodium bicarbonate in bioreactor and total cellular score.At 25 DEG C, the CO of 0.014mmol/min g2
Feed rate be enough under conditions of not having feed gas maintain culture 12 hours.When the time span of experiment increases to 24
Little constantly, healthy culture survival needs the average CO of 0.034mmol/min g2Feed rate.It is interesting that work as culture
When temperature increases to 38 DEG C, culture needs the minimum CO of 0.114mmol/min g2Feed rate maintains healthy culture.
At 38 DEG C, the metabolism of cell is higher, needs more energy for survival, and therefore more ethanol conversion becomes acid.
Embodiment 2
Culture is survived 17 and 24 hours in the case of not having feed gas
Experiment condition:
16.9hr without feed gas
Temperature is reduced to 25 DEG C
In experiment, culture medium interpolation is not changed in
The CO of every gram of cell 0.030mmol/min2Feed rate
Permeate is used to clean to keep cell
From culture broth, CO is not removed when experiment starts2
Before experiment starts, culture cell density be 3.7g/L, pH be 4.44, oxidoreduction be-440mV, CO and
H2Picked-up respectively 5.0 and 1.2mmol/min, CO and H2Converting and be respectively 86 and 40%, ethanol is 23.5g/L, and acid is 3.9g/
L。
Little constantly at t=9511.6, feed gas flow velocity is reduced to 53mL/min from 474mL/min.Stirring is from about 400
Being reduced to about 50rpm, the temperature set points on reactor was reduced to 25 DEG C from 38 in 12 minutes.Once cool down, with
The 38.5g/L sodium bicarbonate that 0.57mL/min starts, it is provided that the CO of every gram of cell 0.030mmol/min2Feed rate；Stop into
Material gas stream；Start permeate with 1.95mL/min to clean, and cultivation base flow is maintained at 1.37mL/min.To reactor head
Spatially slowly add nitrogen and form vacuum in the reactor to stop.Culture is retained under this condition 16.9 hours.
In experimentation, within the most every 2 hours, take out fluid sample, to monitor the pH of culture, cell density, product and thin
Born of the same parents' form.In whole experiment, the pH of culture stably increases, and reaches 5.07 at the end of experiment.Concentration of alcohol is stable from 23.5
Be reduced to experiment at the end of 7.0g/L.Total acetyl group concentration stably increases to 8.2g/L from 3.9.The most about 12 is little
Time, culture morphology shows that the cell of only 5-10% is granular or hollow body.Cell length is meansigma methods, has light
Spend or there is no warpage or bending.
Little constantly at t=9528.5, the temperature set-point increase on reactor is returned to about 38 DEG C；Feed gas is with about
53mL/min restarts, and culture medium B and permeate clean and stop；Enter the N of reactor head space2Stream stops.Work as temperature
When reaching about 28.0 DEG C, feed gas flow velocity is increased to 143mL/min.When about 30 DEG C, feed gas stream is increased again
To 236mL/min, or the 50% of initial gas flow velocity.When about 32 DEG C, stirring is increased to 200rmp.When about 34 DEG C, will
Stirring increases to about 400rpm.
After gas, stirring and temperature increase about 40 minutes, initial conversion was good, H2It is 47% and CO to be 88%.About 15
After minute, convert the best, H2It is 47% and CO to be 87%.Immediately begin to increase gas flow rate.Take about 18.3
Hour reach the maximum gas stream used before experiment starts.When gas flow rate increases, pH continues to reduce, at about 18.3 hours
Inside reach about 4.60.After experiment terminates 40.6 hours, ethanol increase returned to 20.0g/L, and after 24.9 hours, acid rolls back
3.4g/L。
Embodiment 3
Experiment condition:
Without feed gas about 24hr
Temperature is reduced to about 25 DEG C
In experiment, culture medium interpolation is not changed in
The CO of every gram of cell 0.035mmol/min2Feed rate
Permeate is used to clean to keep cell
From culture broth, CO is not removed when experiment starts2
Before experiment starts, it is 4.50 that culture cell density is about 3.2g/L, pH, and oxidoreduction is about-425mV,
CO and H2Picked-up is respectively 4.7 and 1.5mmol/min, and ethanol is about 17.7g/L, and acid is about 2.93g/L.
Little constantly at t=1888, feed gas flow velocity is reduced to 53mL/min from about 474mL/min.Stirring is from about 400
Being reduced to about 50rpm, the temperature set points on reactor was reduced to about 25 DEG C from about 38 in about 14 minutes.Once cool down
Become, use the NaHCO of about 38.5g/L3Stream starts to add sodium bicarbonate with 0.58mL/min, it is provided that every gram of cell 0.035mmol/
The CO of min2Feed rate；Stop feed gas stream；Start permeate with 1.81mL/min to clean, and cultivation base flow is maintained at
1.30mL/min.It is slowly added to nitrogen to reactor head space and forms vacuum in the reactor to stop.By culture at this
Under the conditions of retain about 24 hours.
The most about 15.5 hours, reactor condition provided: the cell density of about 2.4g/L；The pH of about 4.96；About
6.06g/L EtOH；The acid of about 7.87g/L.Morphocytology shows that the cell of about 5-10% is granular or the most granular
's.Owing to staying concentration of alcohol in the reactor low, little constantly at t=1904,9L culture medium A adds 115mL Gem
Clear grain alcohol, creates the concentration of alcohol of about 10g/L.Medium feed speed keeps identical, it is provided that every gram of cell
The ethanol feed rate of 0.037mmol/min.
At the end of 24 hours, condition of culture provides: the cell density of about 2.9g/L；The pH of about 5.04；About 4.10g/l
EtOH；The acid of about 8.68g/L.It is granular or the most granular that morphocytology shows that the cell of about 10-15% is converted to.
Little constantly at t=1912, the temperature set-point increase on reactor is returned to about 38 DEG C；Feed gas is with about
53mL/min restarts, and culture medium B and permeate is cleaned and stops；The N of reactor head space will be entered2Stream stops.Will
Culture medium changes over the normal 1xEtOH culture medium without ethanol.Along with being stepped up of temperature, increase at regular intervals
Feed gas and stirring.When temperature reaches about 28.0 DEG C, feed gas flow velocity is increased to about 179mL/min.About 30.0
DEG C time, feed gas stream is increased again to about 248mL/min, or about the 50% of initial gas flow velocity.When about 32.0 DEG C, will
Stirring increases to about 200rmp.When about 34 DEG C, stirring is increased to about 400rpm.
As embodiment, after gas, stirring and temperature increase about 35 minutes, initial conversion was H2It is 60% and CO
It is 84%.As embodiment, after about 15 minutes, it is provided that convert: H2It is 62% and CO to be 91%.Carry out gas stream immediately
The increase of speed.In this case, the maximum gas flow rate reaching to use before experiment starts is taken about 19.5 hours.
Embodiment 4
Experiment condition:
23.5hr without feed gas
Temperature is reduced to 25 DEG C
Culture medium is added the half being reduced to proper flow；Semicystinol concentration in culture medium doubles
The CO of every gram of cell 0.039mmol/min2Feed rate
Permeate is used to clean to keep cell
From culture broth, CO is not removed when experiment starts2
In embodiments, lose the gas experiment of 24 hours and demonstrate in the case of without feed gas, when providing every gram
When cell 0.035mmol/min CO2 adds, culture can very well be survived about 24 hours.In experimentation culture medium and
Sodium bicarbonate flows into reactor, needs to remove about 2.6L permeate to stop cell to lose owing to washing out.This is slightly higher than
The culture volume of 2.4L.On a laboratory scale, required liquid stream can tolerate well with the ratio of culture volume.But
Being at industrial scale, wastewater flow rate is required monitored, and the need to, it is necessary to reduce.In this experiment, except by culture medium
Flow velocity reduces outside half permeate flushing dose needed for reducing, and all parameters are all identical with previous experiments holding.In the past
Experiment in had some sign to show the reduction of cysteine feed rate may interfere with experiment, so at this experimentation
Middle double to keep cysteine feed rate by the concentration of cysteine in culture medium.
Before experiment starts, culture cell density is about 2.5g/L, pH and is about 4.50, oxidoreduction be about-
440mV, CO and H2Picked-up is about 4.8 and about 1.2mmol/min respectively, and ethanol is about 21.3g/L, and acid is about 2.96g/L.
Little constantly at t=2008.5, feed gas flow velocity is reduced to 53mL/min from 474mL/min.Stirring is from about 400
Being reduced to about 50rpm, the temperature set points on reactor was reduced to 25 DEG C from 38 in about 13 minutes.Once cool down, made
With the NaHCO of 38.5g/L3Solution starts to add sodium bicarbonate with 0.57mL/min；Stop feed gas stream；With 1.20mL/min
Start permeate to clean, and cultivation base flow is reduced to 0.68mL/min.It is slowly added to nitrogen with resistance to reactor head space
Only form vacuum in the reactor.In culture medium A, semicystinol concentration is increased to 5g/L.CO2With every gram of cell
The offer of 0.039mmol/min.Culture is maintained under this condition 24 hours.
In experimentation, within the most every 2 hours, take out fluid sample, to monitor the pH of culture, cell density, product and thin
Born of the same parents' form.As it is expected that in whole experiment pH stably increase, to experiment at the end of reach 5.14.Concentration of alcohol is steady from 21.3
Surely the 6.03g/L at the end of experiment it is reduced to.Total acetyl group concentration stably increases to 10.38g/L from 2.96.Little about 24
Shi Hou, culture morphology shows that the cell of about 10-20% is granular or the most granular.
Little constantly at t=2032, the temperature set-point increase on reactor is returned to about 38 DEG C；Feed gas is with about
53mL/min restarts；Stop sodium bicarbonate adding and permeate cleaning；And the N of reactor head space will be entered2Stream stops
Only.Culture medium flow velocity is increased back 1.37ml/min.When culture medium is heated, feed gas flow velocity increase is returned to 248mL/
Min, and stirring is stepped up about 400rpm.
Gas, stirring and temperature increase after about 30 minutes, initial conversion is H2It is 50% and CO to be 87%.Immediately begin to
The increase of gas flow rate.Take the maximum gas stream reaching to use before experiment starts about 24 hours.
Embodiment 5
25 DEG C, the survival of 12 hours cultures time minimize CO2Feed rate
Experiment condition:
12hr without feed gas
Temperature is reduced to 25 DEG C
In experiment, culture medium interpolation is not changed in
The CO of every gram of cell 0.014mmol/min2Feed rate
Permeate is used to clean to keep cell
From culture broth, CO is not removed when experiment starts2
This experimental evaluation maintains culture 12 hours required minimum CO at 25 DEG C2Adding rate.As embodiment party
Case, reduces the NaHCO as culture medium B3The concentration of solution, keeps every other experiment parameter identical simultaneously.In this experiment
In, NaHCO3Concentration is lowered to about 19.3g/L, provides every gram of cell about 0.014mmol/min CO in the reactor2's
CO2Feed rate.This is low feed rate for culture is survived.
Before experiment starts, culture cell density is about 4.0g/L, pH and is about 4.43, oxidoreduction be about-
430mV, CO and H2Picked-up is about 4.9 and about 1.3mmol/min, CO and H respectively2Converting and be about 86 and about 44% respectively, ethanol is
About 18.9g/L, acid is about 3.8g/L.
2015, t=9580.7 little constantly, feed gas flow velocity is reduced to about 53mL/min from about 474mL/min.Stir
Mixing and be reduced to about 50rpm from about 400, the temperature set points on reactor was reduced to about 25 DEG C from about 38 in about 12 minutes.One
Denier has cooled down, and starts sodium bicarbonate stream with 0.56mL/min；Stop feed gas stream；Permeate is started clear with 1.96mL/min
Wash, and cultivation base flow is maintained at 1.36mL/min.It is slowly added to nitrogen to stop in the reactor to reactor head space
Form vacuum.Culture is maintained under this condition about 12 hours.
In experimentation, within the most every 2 hours, take out fluid sample, to monitor the pH of culture, cell density, product and thin
Born of the same parents' form.In whole experiment, pH all-the-time stable in an experiment increases, and reaches about 4.72 at the end of experiment.Concentration of alcohol from
About 18.9 are stably reduced to the about 10.7g/L at the end of experiment.Total acetyl group concentration stably increases to about from about 3.8
6.0g/L.Cell density is reduced to about 2.8g/L from about 4.0.After about 12 hours, morphocytology provides the thin of about 95+%
Born of the same parents are averaged, and obtain length slightly longer, only have very small amount warpage or bending, granular cell or hollow body the most accidentally occur.
Little constantly at t=9592.7, the temperature set-point increase on reactor is returned to about 38 DEG C；Feed gas is with about
53mL/min restarts；Culture medium B and permeate are cleaned and stops；The N of reactor head space will be entered2Stream stops.When
When culture medium being heated in following 15 minutes, it is stepped up feed gas flow velocity and stirring.When temperature reaches about 28.0 DEG C
Time, feed gas flow velocity is increased to about 178mL/min.The pH of culture slowly reduces, and shows the activity of culture.About
When 30.0 DEG C, feed gas stream is increased again to 236mL/min, or the 50% of initial gas flow velocity.When about 32.0 DEG C, will
Stirring increases to about 200rmp.When about 34 DEG C, stirring is increased to about 400rpm.
Increasing latter 50 minutes in gas, stirring and temperature, initial conversion provides about 53%H2About 89%CO.Open immediately
The increase of beginning gas flow rate.Take the maximum airflow reaching to use before experiment starts 14.6 hours.When gas flow rate increases
Time, concentration of alcohol increases and returns to about 23.7g/L for 49.4 hours after experiment terminates, and after experiment terminates 9.3 hours, acid rolled back
About 3.5g/L.
Embodiment 6
38 DEG C, the survival of 6 hours cultures time minimize CO2Feed rate
Experiment condition:
6hr without feed gas
Temperature is maintained at 38 DEG C
In experiment, culture medium interpolation is not changed in
The CO of every gram of cell 0.114mmol/min2Feed rate
Permeate is used to clean to keep cell
N is started with in experiment2CO is removed from culture broth2
Before experiment starts, culture cell density is about 2.76g/L, pH and is about 4.60, oxidoreduction be about-
440mV, CO and H2Picked-up is about 4.5 and about 1.2mmol/min respectively, and ethanol is about 19.0g/L, and acid is about 2.43g/L.
Little constantly at t=3080.5, feed gas flow velocity is reduced to about 53mL/min from about 475mL/min, then closes
Close.By high N2Stream begins through feed gas ejector, still stirring is kept 3 minutes with by CO at about 400rpm simultaneously2From training
Support in thing and remove.Removing CO2Time, control pH to close to add any sodium bicarbonate to stop.After about 3 minutes, by N2Stream fall
Low, by N2Entrance changes to headroom rather than ejector.Stirring is reduced to about 50rpm from about 400.Use about 77g/L
NaHCO3Start to add CO with about 0.82mL/min2, every gram of cell about 0.114mmol/min CO is provided in the reactor2.Will training
Support base flow speed and be maintained at about 1.34mL/min, start permeate with 2.25ml/min and clean stream.
Due to a large amount of acid, experiment stops constantly t=3086.5 is little after 6 hours.By sodium bicarbonate add, permeate clear
Wash and add to the N of headroom2Stop.Feed gas stream restarts with 53mL/min.Increase feed gas and stirring
Interval and afore-mentioned test increase when culture is warmed to about 38 DEG C gas and to stir the interval that used about the same.?
Experiment stops latter 2 minutes, and feed gas stream increases to about 170mL/min.Experiment stop after about 4 minutes, by feed gas stream
Increase to about 248mL/min (the 50% of initial gas flow velocity).Experiment stop after about 6 minutes, stirring is increased to 200rpm.Real
Test after stopping about 8 minutes, stirring is increased to about 400rpm.
Add CO2After using low stir about 6 hours, pH is about 5.08.Fluid analysis display product is about 10.4g/L second
Alcohol and about 8.21g/L acid.The morphology of culture demonstrates that the cell of about 3% is granular, and additionally having about 22% is almost grain
Shape.
After experiment stops, the initial conversion of about 30 minutes is about 47%H2About 87%CO.Immediately begin to increase gas stream
Speed.T=3108 hour or experiment terminate after about 21.5 little constantly, reached original feed gases flow velocity.
Table 2, at about 25 DEG C and about 38 DEG C, lose the culture parameter in gas experiment, wherein cultivate and use stirring and extremely
The initial GFR of few 50% restarts.
Table 3, for lose feed gas experiment add bicarbonate time, use known CO2It is anti-that adding rate calculates
Answer every gram of cell CO in device2Feed rate (mmol/min g)
Table 4, the CO2 feed rate lost in feed gas experimentation, EtOH uptake rate and rate of producing acid.
Embodiment 7
Comparative example: the illustrative methods of the present invention
By containing CO and/or the forming gas of carbon dioxide/Gaseous Hydrogen or waste gas, be continuously introduced into containing
Clostridium ljungdahlii bacterial strain and comprise the stirring of conventional liq culture medium of vitamin, trace meter and salt
In autoclave bioreactor.
During using the starting method of culture inoculum of less than 10%, reactor uses batch liquid phase fortune
OK, the most not to reactor continuous feed fluid medium.Therefore, liquid phase in reactor is had nominal concentrations by a collection of
The Nutrient medium of limiting nutrient thing calcium pantothenate or cobalt is constituted.Or, it is possible to use containing yeast extract, tryptone
Or the rich medium of other complicated nutrient substance.
It is desirable that gas when starting is mutually without CO2And containing excess H2.Gas velocity and stir speed (S.S.) are maintained at low
Level is (at New Brunswick Scientific BiofloTMLess than 500rpm in fermenting organism reactor), to cause CO
And H2Slightly excess, but avoid CO substrate to suppress at the same time.Such as, at 1 liter of laboratory New Brunswick Scientifc
BiofloTMIn fermenting organism reactor, feed gas composition is 63%H2, 32%CO and 5%CH4, the stirring speed of initial start
Rate is 400rpm, and gas velocity is 20ml/min.The reason producing ethanol in starting process is the H of excess2；Nutrient substance
Occur when being limited in later.Therefore, starting process in fact exists the liquid nutritional thing (pantothenic acid compound, cobalt) of excess, with
Avoid the adaptation to low nutrition substance environment of the undesired culture.
After inoculation, along with fermentation has carried out the time of several hours, convert from CO and create CO2, H2With CO2Together by
Consuming, this is the signal that specified increase stir speed (S.S.) limits to avoid gaseous matter transmission.At New Brunswick
Scientific BiofloTMIn CSTR, discharge gas and contain 25%CO, 67%H2, 2%CO2And 6%CH4.If stirring speed
Rate increase is too fast, the suppression of CO substrate will occur, and after increasing as stirring, methane concentration reduction is confirmed.Therefore, typical case
Lower stir speed (S.S.) can increase 200rpm in 24 hours.This monitoring CO2Produce (or H2Convert) the most specified increase stir speed (S.S.)
Program carry out with relatively fast speed, until reach target stir speed (S.S.).At New Brunswick Scientific
BiofloTMIn fermenting organism reactor, typical target stir speed (S.S.) is 900rpm.This increase stirring speed in batch liquid culture
During the time of rate, it is most important that cell produces rather than product is formed.Therefore, reached the cell concentration of about 1.5g/L,
And be 10g/L ethanol and 2g/L acetic acid class thing from the typical production concentration of batch experiments.
Once reach target stir speed (S.S.), it is allowed to system development to maximum H2Picked-up.There is the highest H2Discharge concentration (allusion quotation
Type ground ＞ 60%) it is preferable, to guarantee that alcohol production is with limit acetic acid production.Then fluid medium charging is started (right
For the system from stock culture batch inoculum) to start continuous liquid charging, and by gas feed rate towards target
Flow velocity increases.At use for laboratory New Brunswick Scientific BiofloTMIn fermenting organism reactor, liquid feedstock
Speed is typically 0.5mL/min, and the every 24 hours target speed towards 125mL/min of gas flow rate increases by 10 to 15%.
It is important that provide excess H in feed gas2To avoid the acetic acid production of excess.Increasing along with gas flow rate
Adding, cell produces to be increased, until reactor is ultimately limited by liquid phase nutrient (calcium pantothenate or cobalt), as under target productivity ratio
H2The a small amount of reduction converted is confirmed.At New Brunswick Scientific BiofloTMIn CSTR, this is realized
It is H under the target productivity ratio of 20g/L days2Convert reduction by 10%.
Then production method and reactor assembly maintain production 15 make to 35g/L ethanol and 0 to 5g/L acetic acid class thing
For under the steady statue of product, adjust limiting nutrient thing, liquid rate and gas velocity the most on a small quantity.Thin not having
The use for laboratory New Brunswick Scientific Bioflo of born of the same parents' recirculationTMIn fermenting organism reactor, typical stable state
Condition be gas residence time (gas flow rate/liquid reactor volume) be 20 minutes, liquid residence time (flow rate of liquid/anti-
Answer device liquid volume) it is 30 hours, stir speed (S.S.) is 900rpm, uses pantothenic acid compound to limit the CO conversion and 60% producing 92%
H2Convert.
Increase in the embodiment of cell recirculation in this method to reactor assembly, add adjustment the most together
Gas velocity (increasing) and nutrient concentrations (reduction).At New Brunswick Scientific BiofloTMCSTR makes
During with cell recirculation, gas residence time is typically 8 minutes, and liquid residence time is 12 hours, and cell residence time is 40
Hour, stir speed (S.S.) is 900rpm.These conditions use pantothenic acid compound limit time typically produce 92% CO convert and
The H of 50%2Convert.
Embodiment 8
Comparative example: recover from serious method upsets
Due to unpredictalbe change of method condition, the such as mechanical problem of reactor, use cell recirculation, contain
C.ljungdahliiC-01 bacterial strain, continuous feed gas and liquid nutritional thing also produce 15-35g/L ethanol and 0-5g/L acetic acid
The CETR being in steady statue, receive upset.Upset to reactor assembly can be slight, the ofest short duration increase gas
Body speed causes short-term substrate to suppress, or great, and such as longer-term increases gas velocity, and this ultimately results in acetic acid production and increases
Add and more serious molecule acetic acid product suppresses.
Short-term upsets and (such as gas velocity is reduced to it initial easily by the parameter only readjusting multilated
Level) and monitor the progress of reactor and also correct without result in longer problem to guarantee to upset.
But, acetic acid product suppression is more serious problem.If as long-term substrate suppression, nutrient excess add,
CO2Accumulate the result of the most eurypalynous mechanical problem, culture create excess molecular acetic acid, it is necessary to first correction causes
The problem of excessive acetic acid.Then by increase liquid rate to wash out acetic acid (washing out ethanol with unfortunate) from system, from system
Middle removing quickly results in the excessive acetic acid of Product inhibiton.Once acetic acid class thing level is less than 3-5g/L, then reset liquid rate, and
Reactor is put back into excess H2Under conditions of charging or vitamin or cobalt limit (with or without cell recirculation).Will
Reactor restores to the original state to be included in before cell is washed out and cracks and reduces gas velocity to avoid substrate suppression and/or to stir
Mix speed.Then stir speed (S.S.) or gas velocity are increased.
In a specific embodiment, with cell recirculation, containing Clostridium ljungdahlii C-01 bacterium
Strain is for from CO, CO2And H2Produce ethanol and the CSTR of acetic acid, mechanical problem is responded and starts to produce acetic acid.2100ml's
Reactor feeds containing 62%H with the gas residence time of 15 minutes2, 31%CO and 7%C2H6Gas, and use 600rpm
Stir speed (S.S.) and 4.86 pH run.Liquid residence time is 23 hours, and cell residence time is 68 hours.Containing salt and
The liquid nutrient media of vitamin exists B-vitamin solution (50.5mg/l calcium pantothenate, 20.6mg/L D-biotin and
The aqueous mixture of 50.6mg/L thiamine hydrochloride), its concentration is every liter of culture medium 0.4ml vitamin solution (seeing table 2).Second
Determining alcohol drops to 7g/L, and acetic acid class substrate concentration is increased to 7g/L, the situation operation for reactor or the warp of alcohol production
Ji is all unstable for learning.Cell density is 2.4g/L, and it is 85% that CO converts, H2Conversion is 25%.
The strategy used in recovering reactor is by first drastically reducing to the gas feed rate of reactor, then depositing
At excess H2In the case of gradually recover reactor constitute.Because acetic acid class substrate concentration is not undue height, do not have in the present embodiment
Have and reduce the liquid rate flowing to reactor, to remove Product inhibiton.Instead, the reduction and subsequently of gas flow rate is used
At excess H2In the presence of run, it is allowed to acetic acid class substrate concentration is more gently reduced to non-inhibited level.In reactor recovers
Specific procedure is discussed below.
Being closed by cell recirculation, gas velocity drastically reducing 70% to gas residence time is 62 minutes, will simultaneously
Liquid residence time was the most slightly adjusted to 30 hours (t=0) from 23 hours.Vitamine concentration in culture medium is constant.Along with this
The change of gas velocity, CO converts increases to 98%, H2Conversion increases to 80%.The more important thing is, system has the H of excess2Deposit
, it is reduced to 5% from 19% is confirmed as discharging CO2 in gas.Along with excess H2Appearance, under acetic acid class substrate concentration
Fall, concentration of alcohol increases simultaneously.Such as, in t=66 little constantly (after closing cell recirculation 66 hours), acetic acid class substrate concentration is
Through dropping to 4g/L, concentration of alcohol has risen slightly to 7.5g/L.
Excess H2Existence (and reduce acetic acid class substrate concentration) can increase the most lentamente in speed subsequently, so
After with faster rate increase.Little constantly to t=215, gas is held and stayed is 29 minutes, and concentration of alcohol is 12g/L, acetic acid class substrate concentration
It is 3g/L.Alcohol production rate is 8g/L days.CO present in exit gas2Being 6%, it is 98% that CO converts, H2Conversion is 80%.
Little constantly to t=315, concentration of alcohol is 16g/L, and acetic acid class substrate concentration is 4g/L, and gas converts same good, and gas is held when staying
Between be 20 minutes.Alcohol production rate is 11g/L days.Little constantly to t=465, concentration of alcohol has reached 20g/L, there is also
3.5-4g/L acetic acid class thing.Alcohol production rate is 16g/L days.Gas residence time has already decreased to 16 minutes, CO and H2 converts
It is respectively 95% and 73%.These conditions maintain the continuous operation close to 200 hours, it was demonstrated that reactor assembly is the most recovered
It produces the ability of ethanol, and the operation conditions before substantially maintaining.
All published document are incorporated herein by reference.The many modifications and changes of the present invention are included in the explanation being determined above
In book, and suspect for the professional of the art it is obvious.Repairing of this composition to the present invention and method
Change and change is considered to contain within the scope of the appended claims.
Claims (16)
1. one kind is used for maintaining in the case of synthetic gas density reduces or be non-existent in synthesis gas fermentation reactor producing acetic acid
The method of anaerobe culture, described method includes:
Add carbon dioxide；Free acetic acid concentration is maintained below 5g/L free acetic acid；Consume alcohol；Described in 30 minutes
Synthetic gas density performs above-mentioned steps in the case of reducing or being non-existent；And subsequently from the permeate of cell separation or from instead
The effluent answering device reclaims alcohol；
Wherein said culture of microorganism comprises selected from clostridium (Clostridium), Moore Salmonella (Moorella) and carbonoxide
One or more bacterial strains of Thermophilic Bacteria (Carboxydothermus) or its genetic modification strain.
2. the process of claim 1 wherein that described maintenance culture of microorganism includes 30 hours interior persistent period.
3. the process of claim 1 wherein that pH is maintained in the range of 3.5-5.6.
4. the method for claim 3, wherein adds bicarbonate solution and controls pH.
5. the process of claim 1 wherein and described carbon dioxide is moved in described reactor.
6. the method for claim 1, adds nutrient to described reactor.
7. the process of claim 1 wherein that described alcohol includes ethanol, butanol or ethanol and butanol.
8. the method for claim 1, is reduced to temperature between 0-25 DEG C from running temperature, temperature is maintained 0-25 DEG C simultaneously
Between.
9. the method for claim 1, adds water to described reactor.
10. the method for claim 1, adds water to described reactor, and described water includes: fresh water, supplementary water, recirculation water, steaming
Distilled water, deionized water or a combination thereof.
11. the process of claim 1 wherein that culture of microorganism returns to the suspension precondition including adding synthesis gas.
12. the process of claim 1 wherein carry out remove permeate step.
13. the process of claim 1 wherein with reactor described in inert gas purge.
14. the process of claim 1 wherein that the low stirring of maintenance is to keep solid content at suspended state.
15. 1 kinds stop product acetic acid anaerobism in synthesis gas fermentation reactor in the case of multiple concentration of substrate reduces or be non-existent
The method of culture of microorganism rapid loss, described method includes:
Temperature is reduced between 0-25 DEG C from running temperature, temperature is maintained between 0-25 DEG C simultaneously；Maintain free acetic acid
Concentration is below 5g/L free acetic acid；And in 30 minutes, perform above-mentioned steps,
Wherein said culture of microorganism comprises selected from clostridium (Clostridium), Moore Salmonella (Moorella) and carbonoxide
One or more bacterial strains of Thermophilic Bacteria (Carboxydothermus) or its genetic modification strain.
16. 1 kinds in feed gas supply thing multiple concentration of substrate maintain synthesis gas fermentation anti-in the case of reducing or being non-existent
Answering the method producing acetic acid anaerobe culture in device, described method includes:
Temperature is reduced between 0-25 DEG C from running temperature, temperature is maintained between 0-25 DEG C simultaneously；Maintain free acetic acid
Concentration is below 5g/L free acetic acid；And in 30 minutes, perform above-mentioned steps,
Wherein said culture of microorganism comprises selected from clostridium (Clostridium), Moore Salmonella (Moorella) and carbonoxide
One or more bacterial strains of Thermophilic Bacteria (Carboxydothermus) or its genetic modification strain.
Applications Claiming Priority (5)
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| US6450608P | 2008-03-10 | 2008-03-10 | |
| US61/064,506 | 2008-03-10 | ||
| US12/381,193 | 2009-03-09 | ||
| US12/381,193 US9034618B2 (en) | 2009-03-09 | 2009-03-09 | Method for sustaining microorganism culture in syngas fermentation process in decreased concentration or absence of various substrates |
| PCT/US2009/001522 WO2009114127A1 (en) | 2008-03-10 | 2009-03-10 | Method for sustaining microorganism culture in syngas fermentation process in decreased concentration or absence of various substrates |
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| CN102131936A CN102131936A (en) | 2011-07-20 |
| CN102131936B true CN102131936B (en) | 2016-08-10 |
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| KR (1) | KR20110002029A (en) |
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| HUE055340T2 (en) | 2009-02-26 | 2021-11-29 | Lanzatech New Zealand Ltd | Methods of sustaining culture viability |
| US7927513B1 (en) * | 2009-10-27 | 2011-04-19 | Coskata, Inc. | Method of treating a hot syngas stream for conversion to chemical products by removing ammonia and COS |
| US8795995B2 (en) * | 2010-06-30 | 2014-08-05 | Coskata, Inc. | Method for injecting a feed gas stream into a vertically extended column of liquid |
| US10100336B2 (en) * | 2012-05-22 | 2018-10-16 | Ineos Bio S.A. | Syngas fermentation process and medium |
| US9193947B2 (en) | 2012-05-22 | 2015-11-24 | Ineos Bio Sa | Process for culturing microorganisms on a selected substrate |
| US10100338B2 (en) * | 2012-05-22 | 2018-10-16 | Ineos Bio S.A. | Method of operation of a syngas fermentation process |
| US10233478B2 (en) * | 2012-09-19 | 2019-03-19 | Ineos Bio Sa | Process for reducing CO2 emissions and increasing alcohol productivity in syngas fermentation |
| US10100337B2 (en) * | 2013-02-14 | 2018-10-16 | Ineos Bio Sa | Process for fermenting co-containing gaseous substrates |
| US9850503B2 (en) * | 2013-06-10 | 2017-12-26 | Ineos Bio Sa | Control of conductivity in anaerobic fermentation |
| US9885063B2 (en) * | 2013-06-10 | 2018-02-06 | Ineos Bio Sa | Process for fermenting co-containing gaseous substrates in a low phosphate medium effective for reducing water usage |
| WO2015002552A1 (en) * | 2013-07-04 | 2015-01-08 | Lanzatech New Zealand Limited | Multiple reactor system and process for continuous gas fermentation |
| JP6309304B2 (en) * | 2014-02-24 | 2018-04-11 | 積水化学工業株式会社 | Recombinant cells and method for producing 1,4-butanediol |
| US9701987B2 (en) * | 2014-05-21 | 2017-07-11 | Lanzatech New Zealand Limited | Fermentation process for the production and control of pyruvate-derived products |
| US10370685B2 (en) * | 2014-07-09 | 2019-08-06 | Synata Bio, Inc. | Processes for controlling the concentration of co-produced oxygenated organics in anaerobic fermentation broths for the bioconversion of syngas to product oxygenated organic compound |
| JP6789116B2 (en) * | 2014-07-30 | 2020-11-25 | 積水化学工業株式会社 | Equipment for producing organic matter from waste and methods for producing organic matter from waste |
| EP3199619A4 (en) * | 2014-09-19 | 2018-06-13 | Sekisui Chemical Co., Ltd. | Microorganism culture method and culture device |
| WO2017015022A1 (en) * | 2015-07-17 | 2017-01-26 | Synata Bio, Inc. | Methods for sustaining the viability of microorganisms during a cessation of syngas flow and processes for storage and reactivation of microorganisms |
| CA3071542A1 (en) * | 2017-07-31 | 2019-02-07 | Synata Bio, Inc. | System and method for concentrating suspended solids prior to removal |
| WO2019191767A1 (en) * | 2018-03-30 | 2019-10-03 | Invista Textiles (U.K.) Limited | High hydrogen utilization and gas recycle |
| US11104877B2 (en) * | 2018-05-21 | 2021-08-31 | Jupeng Bio, Inc. | Composition for obtaining protein-rich nutrient supplements from bacterial fermentation process |
| EP3833771A1 (en) * | 2018-08-08 | 2021-06-16 | Jupeng Bio, Inc. | Carbon dioxide bioconversion process |
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| US7285402B2 (en) * | 2000-07-25 | 2007-10-23 | Emmaus Foundation, Inc. | Methods for increasing the production of ethanol from microbial fermentation |
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| JPS62214399A (en) * | 1986-03-17 | 1987-09-21 | 株式会社東芝 | Method of processing radioactive waste organic solvent |
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2009
- 2009-03-10 NZ NZ588464A patent/NZ588464A/en not_active IP Right Cessation
- 2009-03-10 KR KR1020107022608A patent/KR20110002029A/en not_active Application Discontinuation
- 2009-03-10 AU AU2009223801A patent/AU2009223801B2/en not_active Ceased
- 2009-03-10 EP EP09721160A patent/EP2268824A1/en not_active Withdrawn
- 2009-03-10 CN CN200980114615.4A patent/CN102131936B/en active Active
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- 2009-03-10 WO PCT/US2009/001522 patent/WO2009114127A1/en active Application Filing
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| US7285402B2 (en) * | 2000-07-25 | 2007-10-23 | Emmaus Foundation, Inc. | Methods for increasing the production of ethanol from microbial fermentation |
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Also Published As
| Publication number | Publication date |
|---|---|
| KR20110002029A (en) | 2011-01-06 |
| JP5777889B2 (en) | 2015-09-09 |
| BRPI0909841B1 (en) | 2019-02-05 |
| MX2010009983A (en) | 2010-11-26 |
| BRPI0909841A2 (en) | 2016-06-21 |
| JP2011512870A (en) | 2011-04-28 |
| NZ588464A (en) | 2012-01-12 |
| WO2009114127A1 (en) | 2009-09-17 |
| EP2268824A1 (en) | 2011-01-05 |
| AU2009223801B2 (en) | 2014-09-25 |
| CN102131936A (en) | 2011-07-20 |
| CA2718132A1 (en) | 2009-09-17 |
| AU2009223801A1 (en) | 2009-09-17 |
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