CN102439161A - Continuous single-reaction kettle fermentation synthesis of butanol - Google Patents
Continuous single-reaction kettle fermentation synthesis of butanol Download PDFInfo
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- CN102439161A CN102439161A CN2009801588906A CN200980158890A CN102439161A CN 102439161 A CN102439161 A CN 102439161A CN 2009801588906 A CN2009801588906 A CN 2009801588906A CN 200980158890 A CN200980158890 A CN 200980158890A CN 102439161 A CN102439161 A CN 102439161A
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- butanols
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- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 title claims abstract description 167
- 238000006243 chemical reaction Methods 0.000 title claims description 47
- 238000000855 fermentation Methods 0.000 title abstract description 17
- 230000004151 fermentation Effects 0.000 title abstract description 17
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- 238000003786 synthesis reaction Methods 0.000 title abstract description 3
- 241000193403 Clostridium Species 0.000 claims abstract description 47
- 238000000034 method Methods 0.000 claims abstract description 33
- 238000004519 manufacturing process Methods 0.000 claims abstract description 30
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- 150000001720 carbohydrates Chemical class 0.000 claims abstract description 16
- 230000008569 process Effects 0.000 claims abstract description 6
- 238000005516 engineering process Methods 0.000 claims description 51
- 239000002904 solvent Substances 0.000 claims description 45
- 239000012071 phase Substances 0.000 claims description 30
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 25
- 239000007790 solid phase Substances 0.000 claims description 19
- 239000012634 fragment Substances 0.000 claims description 18
- 239000002253 acid Substances 0.000 claims description 17
- 235000014633 carbohydrates Nutrition 0.000 claims description 17
- 239000011159 matrix material Substances 0.000 claims description 17
- 235000010443 alginic acid Nutrition 0.000 claims description 13
- 229920000615 alginic acid Polymers 0.000 claims description 13
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- 239000000047 product Substances 0.000 claims description 13
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 12
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- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 8
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 8
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
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- WDMUXYQIMRDWRC-UHFFFAOYSA-N 2-hydroxy-3,4-dinitrobenzoic acid Chemical compound OC(=O)C1=CC=C([N+]([O-])=O)C([N+]([O-])=O)=C1O WDMUXYQIMRDWRC-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
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- 230000005526 G1 to G0 transition Effects 0.000 description 2
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- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000005862 Whey Substances 0.000 description 2
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- DNZWLJIKNWYXJP-UHFFFAOYSA-N butan-1-ol;propan-2-one Chemical compound CC(C)=O.CCCCO DNZWLJIKNWYXJP-UHFFFAOYSA-N 0.000 description 2
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- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
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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
- C12P7/16—Butanols
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/145—Clostridium
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Abstract
本发明公开了一种用梭状芽孢杆菌产酸相细胞和产溶剂相细胞的混合种群在单个反应釜中通过碳水化合物发酵生产丁醇的方法。所描述的本发明系统不需要间歇性调整pH或排出顶空气体。该方法提供了一种用于除去不可逆损害细胞的丁醇产物的工艺，并且描述了这类细胞可在相同的单个反应釜中重新开始合成丁醇的条件。如所公开的，本发明还描述了包含梭状芽孢杆菌细胞的组合物和生物纯培养物。
The present invention discloses a method for producing butanol by carbohydrate fermentation in a single reactor using a mixed population of acidogenic and solventogenic Clostridium cells. The described system of the present invention does not require intermittent pH adjustment or venting of headspace gas. The method provides a process for removing butanol products from irreversibly damaged cells and describes conditions under which such cells can resume butanol synthesis in the same single reactor. As disclosed, the present invention also describes compositions and biologically pure cultures comprising Clostridium cells.
Description
The cross reference of related application
The application is the part continuity of the USP 12/391,026 of submission on February 23rd, 2009, and this patent is discarded at present, and its disclosure is attached among this paper with its integral body by reference.
Technical field
The present invention relates generally to through the fermentative Production butanols, particularly relates to through producing microenvironment keeping single reaction still (vessel) production of butanol of producing acid and the subgroup of producing solvent phase cell symbiosis (coincident).
Background technology
Butanols (propyl carbinol (n-butanol) or just-butanols (n-butyl alcohol)) can be produced through the fermentation of glucide, said carbohydrate degradation become product as comprise five with the sugar (like glucose) of six carbon atom.During the World War I, developed this method charles's Wei graceful now (Charles Weizmann) and (seen that for example USP 1,315,585; 1,329,214; 1,437,697).Briefly, Wei graceful now method be included in the acetone-butanol fusobacterium (
Clostridium acetobutylicum) there is the fermentation of proper raw material down, the acetone-butanol fusobacterium becomes feedstock conversion the solvent mixture of acetone, butanols and ethanol (ABE).In solvent mixture, butanols: acetone: alcoholic acid ratio normally 6: 3: 1.
The ABE fermentation is two stages (biphasic) processes: during the stage, logarithmic growth is followed acetate and butyro-production first (producing acid), and this causes that also pH that follow and essential descends.Second (product solvent) mutually in, growth stops, and consumes aforesaid acid when producing solvent, comprises the raw material of further consumption input.Hydrogen and carbonic acid gas constantly produce during the fermentation.
Consequent solvent produces with rare aqs soln form, is generally about 1%-2% weight, makes its pure state recovery comprise itself and the separating of big water gaging.Isolating just expense makes and is less competitive than from the source production solvent that with the oil is the basis through these chemical preparationss of fermentative prodn.Yet, to the concern of global warming, realize the energy independently the increase of hope and petrochemical industry derived feed price people's interest is got back to can renewablely make with extra care raw material and convert on the technology of simple organic chemistry goods.
A problem with clostridium spp microbial fermentation ABE is that butanols is to culture poisonous (that is, under the situation of not considering sugared concentration, maximum tolerance butanol concentration is about 2%).Can avoid its toxicity through removing butanols in process of production continuously, be used for maximum and produce solvent.Various butanols are removed system, comprise absorption, pervaporation, infiltration extraction, r-o-, liquid-liquid extraction is gentle proposes all not exclusively successes.In addition, above-mentioned all methods have all increased production cost.Therefore, this area still needs a kind of improved technology of producing solvent from these organisms.
Summary of the invention
The present invention introduced a kind of through Clostridium (
ClostridiumGenus) cell carries out the method for fermentative prodn butanols to glucide, wherein produces sour phase (acidogenic-phase) cell and remains in the single reaction still with the mixture that produces solvent phase (solventogenic-phase) cell.System of the present invention does not need intermittent adjustment pH or discharges head space gas.Disclosed method provides a kind of technology that removes the butanols product, and said butanols product is not irreversibly to damage cell, and when the inhibition concentration of solvent no longer existed, this type cell can restart synthetic butanols.In addition, the present invention includes compsn (composition) and the biological pure culture that comprises the clostridium spp cell.
In one embodiment; The continuous processing of production of butanol is disclosed; This technology comprises: the clostridium cell is contacted with the matrix phase that comprises glucide with buffer reagent; Culturing cell is until reaching the butanols inhibition concentration under optimum temps, reduces pressure in first reaction kettle up to violent agitation occurring, and the azeotrope that will comprise butanols is transferred to second from first reaction kettle and collected reaction kettle as condensation product (condensate); Wash reaction kettle with washing fluid, and constantly repeat above-mentioned steps.In related fields, flushing is the function of condensation and collection volume (condensation collection volume), when the condensation and collection volume accounts for about 4%-5% of culture volume, and the flushing beginning.
On the other hand, pressure drops to about 20mm Hg-30mm Hg from about 760mm Hg.On the other hand, when solvent production is about 35%-40% (wt/wt) of assimilable carbon hydrate in the matrix, replenish the matrix that comprises glucide.
In one aspect, these cells include but not limited to:
C. beijerinkii,
C. acetobutylicum,
C. aurantibutyricum,
C. tetranomorphum and C. thermocellumIn related fields, clostridium spp
BeijerinkiiNumbering be NRRL B-50244.
In another embodiment; The continuous processing of producing butanols is disclosed; It comprises: single culture of clostridium cell is contacted with the matrix phase that comprises glucide with buffer reagent; Culturing cell is until the inhibition concentration that reaches butanols under optimum temps; Rising culture temperature is to higher about 6 ℃-11 ℃ and the pressure of first reaction kettle is reduced to the generation violent agitation from about 760mm Hg than optimum temps; The azeotrope that will comprise butanols is transferred to second reaction kettle as condensation product from first reaction kettle, washes first reaction kettle and culture is cooled to the function of butanols synthetic optimum temps as the condensation and collection volume with washing fluid, and constantly repeat above-mentioned steps.
In one aspect, when solvent production is about 35%-40% (wt/wt) of assimilable carbon hydrate, replenish the matrix that comprises glucide.On the other hand, culture comprises the cell mixture that produces in acid and the product solvent phase.In related fields, the clostridium cell does
C. beijerinkiiOn the other hand, washing fluid is N
2Gas.
In one aspect, optimum temps is about 33 ℃-37 ℃.On the other hand, temperature rises to about 43 ℃-44 ℃.Aspect another, pressure drops to about 110mm Hg-100mm Hg.
In one aspect, the inhibition concentration of butanols is about 0.9%-2.0%.In related fields, the inhibition concentration of butanols is about 1.3%.
On the other hand, buffer reagent comprises the biogenic of lime carbonate, and it comprises Endoconcha Sepiae and oyster shells.In one aspect, this method also comprises cell is contacted with molecular skeleton.In related fields, molecular skeleton comprises the sponge fragment, wherein sponge-rubber pad carbonated calcium, silicon-dioxide spicule (silica spicules) or Mierocrystalline cellulose.
In one aspect, but the butanols output of this technology is about 35%-40% (w/w) of fermentable carbohydrates, comprises the butanols output of the about 20g/L of each circulation.On the other hand, this technology produces the head space gaseous mixture, and it comprises about 40-50%H
2, about 40-50%CO
2And 0-20%N
2In related fields, head space gas comprises 43%H
2, 43%CO
2And 14%N
2
In another embodiment, disclose a kind of compsn, it comprises the mixed population and the buffer reagent that comprises the biological calcium carbonate source that produces acid phase and product solvent phase Clostridium cell.In one aspect, the biological calcium carbonate source comprises Endoconcha Sepiae fragment and oyster shells fragment.On the other hand, buffer reagent is inorganic carbonate calcium source.
On the other hand, said composition also comprises cellulose biomass, comprises stem, leaf, chaff, wood chip, sawdust, withered tree, branch, household refuse, paper product, black liquid, grass or their combination.
In one aspect, cell is a clostridium spp
Beijerinkii(
Clostridium beijerinkii).In related fields, clostridium spp
BeijerinkiiNumbering be NRRL B-50244.On the other hand, clostridium spp
BeijerinkiiBe inserted in the solid phase, said solid phase comprises natural sponge, fiber sponge, alginate calcium microballon and SEPIGEL 305 sheet (sheet).
In one embodiment, clostridium spp is disclosed
BeijerinkiiThe biological pure culture of NRRL B-50244.In one aspect, this culture can continue to produce butanols under pH6.0.On the other hand, this culture is embedded in the solid phase.In related fields, solid phase comprises fragment, cuttlebone fragment and the SEPIGEL 305 sheet of natural sponge, fiber sponge, sponge gourd sponge, alginate calcium microballon, mollusk shell.
Description of drawings
Fig. 1 has shown the synoptic diagram of the equipment that can be used for continuous single reaction still production butanols;
Fig. 2 shown by
C. beijerinkiiThe chromatogram of the butanols that NRRL B-50244 culture is produced;
Fig. 3 illustrates the butanols generated time process of observed freedom and embedding cell.Filled squares representative comes from the culture freely
C. beijerinkiiThe data of cell.The solid diamond representative is from being embedded in the alginates microballon
C. beijerinkiiThe data of cell;
Fig. 4 illustrates OD
600nmThe cell growth rate of place's freedom and embedding cell.Butanols data point parallel acquisition among data point and Fig. 3.Filled squares representative from the culture freely
C. beijerinkiiThe data of cell.The solid diamond representative is from being embedded in the alginates microballon
C. beijerinkiiThe data of cell;
Fig. 5 (A)-(C) shown a series of show vacuum distilling after butanols restart the synthetic chromatogram.(A) shown the butanol concentration before the distillation.(B) shown the butanol concentration after the distillation.(C) shown the butanol concentration of cultivating again after (re-incubation).Each peak can be keyed among (keyed to) Fig. 2.All % values are vol/vol;
Fig. 6 has shown the synoptic diagram of the robotization embodiment of equipment shown in Figure 1.
Embodiment
Before describing compsn of the present invention, method and methodology, should be appreciated that, the invention is not restricted to described particular composition, method and experiment condition, because this based composition, method and condition can be different.Should be appreciated that also term as used herein is not intended restriction, because scope of the present invention will only be limited only from the purpose of describing particular in accompanying claims.
Used like this specification sheets and accompanying claims, " " of singulative, " one " and " be somebody's turn to do " comprise and quote plural form, indicate only if context has clearly in addition.Therefore, for example quote " cell " and comprise one or more cells, and/or the compsn of type that this paper describes, the compsn of this type will become after those skilled in the art read present disclosure obviously, and the rest may be inferred.
Only if definition is arranged in addition, otherwise all technology that this paper uses have the understanding identical implication common with one of ordinary skill in the art of the present invention with scientific terminology.Anyly describe method and material similar or that be equal to this paper and can be used for enforcement of the present invention and test, because of understanding, various improvement and modification are included in the spirit and scope of present disclosure.
As used herein, " produce acid mutually with the mixed population that produces the solvent phase cell " is meant the clostridium spp cell of number of combinations, and it comprises and is mainly butyric acid with the celliferous subgroup of acetate be mainly the celliferous subgroup of acetone/ethanol (ABE).This type bacterial strain comprises
C. beijerinkii,
C. acetobutylicum,
C. aurantibutyricum,
C. tetranomorphum,
C. thermocellumWith similar bacterium, said bacterium changes into solvent such as butanols, acetone, ethanol or Virahol with glucide, butyric acid and other acid.In some aspects, subgroup is from single strain or many bacterial strains.On the other hand, spendable cell can be naturally occurring or synthetical (that is, obtaining from the reorganization operation) in technology disclosed by the invention.
As used herein, " biological calcium carbonate source " is meant shell or the framework material (or its fragment) of carbonic acid Ca supply from animal.For example, belong to Mollusca (phylum
Mollusca) the shell of animal be the biological calcium carbonate source.
As used herein, " cellulose biomass " be meant have a small amount of protein, lipid (fat, wax and oil) and ash content, the material of forming by Mierocrystalline cellulose, semicellulose and xylogen.This biolobic material comprises assimilable glucide (that is carbohydrate matrix).Assimilable examples of carbohydrates includes but not limited to sugar as glucose, lactose, whey permeate, pentose, starch, liquefying starch, through liquefying starch, maltodextrin and the steeping water of enzyme processing.In one aspect, the dextrose equivalent (dextrose equivalents) that can analyze this type assimilable carbon hydrate is (for example, with using the YSI analyser after saccharifying enzyme and the AMS processing biomass; See that for example USP 5,192; 673; Or use the YSI analyser after handling biomass with dinitrosalicylic acid, see, for example the article delivered at " Biotech Bio-eng " 1970 the 12nd volumes 991-992 page or leaf of people such as Tasun).
As used herein, " solid phase " is meant that with resistance to deformation and volume change be the state of matter of characteristics.In one aspect, solid phase can be porous or atresia.In related fields, selected bacterium can form bacterium colony on the porous solid phase, and wherein the solid phase of bacterium colonyization is as the transplanting of new culture.For example, solid phase can be 3 dimension molecular skeletons, and wherein such skeleton is that the cell growth provides bigger surface-area (like sponge).In related fields, " embedding " is that phalangeal cell inserts in the solid phase space.
As used herein, " washing fluid " is meant liquid or the gas that is used to wash out other liquid or gas.For example, CO
2And N
2Gas is washing fluid.
Biogenic 1-butanols can use method well known in the art from fermention medium, to separate.For example, can from fermention medium, remove solid substance through centrifugal, filtration, decant or similar method.Then, method of use such as distillation, liquid-liquid extraction or be that the separation on basis comes from fermention medium, to separate the 1-butanols with the film.Because 1-butanols and water form the lower boiling azeotropic mixture, therefore distillation can only be used for mixture separation to its azeotropic is formed (azeotropic composition).Distillation combines to obtain to separate around azeotrope with another separation method usually.Can include but not limited to decant, liquid-liquid extraction, absorption and the technology that is the basis with the film with the method that distillation is used in combination to separate with purifying 1-butanols.
1-butanols-water mixture has formed heterogeneous azeotrope, makes distillation to combine to be used for separating and purifying 1-butanols with decant.The fermented liquid that in one approach, will contain the 1-butanols is distilled near azeotropic to be formed.Then, azeotropic mixture is condensed, and from fermention medium, separate the 1-butanols through decant.That inclines and contains water and can get back to first rectifying tower as backflow.The organic phase that is rich in the 1-butanols that of inclining can be further purified through distillation in second rectifying tower.
Also available liquid-liquid extraction combines from fermention medium, to separate the 1-butanols with distillation.In this method, use suitable solvent to come the 1-butanols in the liquid-liquid extraction fermented liquid.Then distillation contain the 1-butanols organic phase from solvent, to separate the 1-butanols.
Also available distillation combines from fermention medium, to separate the 1-butanols with absorption.In this method; Distillation contains the fermented liquid of 1-butanols to forming near azeotropic; Then through using sorbent material such as molecular sieve to remove remainder water (people's such as Aden that National Renewable Energy Laboratory is published in June, 2002 report NREL/TP-510-32438, diluted acid prehydrolysis and enzymic hydrolysis coupling to corn straw from technological design of lignocellulose biomass to alcoholic acid and economy).
In addition, distillation has been used to from fermention medium, separate the butanols with purifying 1-with the combination of pervaporation.In the method, the fermented liquid that distillation contains the 1-butanols is to forming near azeotropic, removes remainder water (article that people such as Guo delivers at " J. Membr. Sci. " 2004 the 245th volumes 199-210 page or leaf) with hydrophilic film through pervaporation then.
In all these distillating methods; Because the temperature that is commonly used to distill butanols-water azeotrope (for example; About 93 ℃) can irreversibly damage the cell of vegetative state (vegetative state), so must the fermenting organism body be separated from fermented liquid to be distilled.Therefore, compare with the inventive method, above said distil process can not be applied in the reaction kettle identical and not destroy cell with active cultures.
Use various technologies original position from the productivity culture and removed butanols.The technology of research comprises absorption, pervaporation, r-o-, liquid-liquid extraction is gentle carries.All methods of above-mentioned side all increase productive expense significantly, and none is best.To butanols demonstrate high selectivity with the film be the basis system's quilt
C. acetobutylicumWith
C. beijerinkiiStain and stop up.The model solution that the gas formulation is therefrom removed volatile constituent to hope goes on well (sees; For example U.S. Patent application 20050089979); But, when for example making culture keep being used to produce butanols imperturbably, be to forbid that rare gas element passes through solution to be cleaned and produces strong foamy.
In the present invention; A kind of production of butanol technology is disclosed; Comprise: in the first independent reaction kettle, single culture of clostridium cell is contacted with the matrix phase of carbohydrate containing with buffer reagent, culturing cell and reduces pressure in the same independent reaction kettle up to violent agitation occurring until reaching the butanols inhibition concentration under optimum temps; The azeotrope that will comprise butanols is transferred to the second collection reaction kettle as condensation product from first reaction kettle; Wash the function of first reaction kettle with washing fluid, wherein when the condensation product volume of collecting is about 4-5% of fermentation volume, begin flushing, and constantly repeat above-mentioned steps as the condensation and collection volume.Thereby the distillation of gained butanols-water azeotrope is carried out in the reaction kettle identical with active cultures.In addition, after the distillation, same culture can be used for restarting to produce butanols.
In one aspect, the every gram glucose that consumes in this technology or other carbohydrate (including but not limited to lactose) produce at least 0.4 gram butanols.On the other hand, but this technology can be reached the maximum butanols output of about 80%-90% by fermentable carbohydrates.In another related fields, the butanols output of this technology is about 20-40g/L.
On the other hand, the clostridium cell does
C. beijerinkii,
C. acetobutylicum,
C aurantibutyricum,
C. thermocellumOr
C. tetranomorphumIn related fields, cell does
C. beijerinkiiNRRL B-50244.
In general, the fermentation carry out at least about 36h, yet, fermentation can carry out 140h or more than.For the present invention, keep cell concn in logarithm later stage to stationary phase and be used for the continuous production solvent, when needing, change substratum and the cell used up.According to employed strains of clostridium, cell can be stirred or kept static.For example, work as use
C. beijerinkiiThe time, it is static to be used to produce butanols that culture will keep.
For the present invention, raw material can be that simple sugar is like glucose, lactose (for example, in whey permeate, containing), pentose; Compound carbohydrate such as starch, liquefying starch, liquefying starch, maltodextrin and the steeping water handled through enzyme; Maybe can comprise cellulose biomass.This type raw material adds as the aqueous solution and/or suspension-s.In one aspect, when when the raw material that comprises cellulose biomass obtains matrix solution, can this biolobic material be milled or micronize before the fermentation.Mill and reduced the size of carbohydrate containing feed composition, thereby make biomass be prone to the bacterial degradation that is chosen.Can adopt sterilization to kill end bacterium (background bacteria), selected bacterium is vigorously grown.Generally, glucide/carbohydrate is mixed as many fermentation systems are common, sterilize then (seeing that for example USP 5,753,474) with water.Once more; Can analyze constitutive material/carbohydrate glucide dextrose equivalent (for example; After handling biomass, (see that for example USP 5,192 with the analysis of YSI analyser with saccharifying enzyme and AMS; 673) handle after the biomass with the YSI analyser or through dinitrosalicylic acid and to analyze (seeing that for example people such as Tasun rolled up the article that the 991-992 page or leaf is delivered in 1970 the 12nd at " Biotech Bio-eng ")).
Except comprising the matrix of glucide, cell also contacts with nutritional medium.Useful nutritional medium comprises those nutritional mediums well known in the art, like P2 and peptone glucose yeast extract (TGY).Also can use other nutritional medium.Nutritional medium can be chosen wantonly and comprise additive for example salt and/or trace element.On the one hand, substratum is that lactose produces spore substratum (LSM), defines like table 1.
In general, if pH drop to 4.5 or below, the solvent production activity of producing solvent clostridium spp culture will stop.For the present invention, the initial pH of weak buffering growth medium is about pH6.5-7.0, to promote to produce acid phase (that is butyro-generation).Make the pH of solution drop to triggering then from producing acid phase to the transformation of producing solvent/butanols synthesis phase.In one aspect, the pH during butanols generates is about 4.8-5.0, about 5.0-5.3, about 5.3-5.5 or about 5.5-6.0.
Can use various salt (including but not limited to lime carbonate) to keep pH and be brought down below 4.8-6.0 with the pH that prevents culture.For the present invention, produce acid phase and the symbiosis of producing the solvent phase cell subsets in order in culture, to keep, using the buffering Yanyuan with big gradient buffer ability is useful (that is, showing putting than slow release of gegenion).In one aspect, replace the salt crystal with the biogenic that contains identical salt.For example, calcium carbonate crystal can use molluscan shell or framework material (including but not limited to Endoconcha Sepiae or oyster shells fragment) to replace.In addition, the available solid phase that contains 3 dimensional scaffolds provides the surface-area of increase to be used for bacterial growth.In one aspect, comprised sponge fragment (such as but not limited to natural sponge or fiber sponge), bacterium is bacterium colonyization above that.In related fields, by bacterium colonyization the sponge fragment as the transplanting of new culture to begin to produce solvent.
Well-known in the art; Concentration is that about 1.0% butanols (is seen most of clostridium spp culture is poisonous; For example; The article that people such as Hermann deliver at " App Env Microbiol " 1985 the 50th volumes the 5th phase 1238-1243 page or leaf), batch production solvent but its restricted passage is fermented.In one aspect, when butanol concentration reached about 0.9-1.3%, the temperature of culture can raise about 6 ℃-11 ℃ on the production of butanol optimum range, and can reduce pressure on the culture to begin to boil the azeotropic mixture of the fourth alcohol and water that obtains.The optimum temps of nourishing and growing in one aspect, is at about 33 ℃-35 ℃, about 35 ℃-36 ℃ or 36 ℃-37 ℃.In related fields, when temperature is elevated to about 41 ℃-42 ℃, about 42 ℃-43 ℃, about 43 ℃-44 ℃, about 44 ℃-46 ℃.In one aspect, temperature is 46.5 ℃.On the other hand, pressure can be reduced to about 110mm Hg-106mm Hg, about 106mm Hg-105mm Hg from about 760mm Hg, or about 105mm Hg-100mm Hg.Make the vapour pressure of azeotrope equal the pressure on the culture through being combined on the vegetative cell growth optimum temps elevated temperature and reducing pressure, can in cultivating reaction kettle, realize vacuum distilling and can damage cell with recovering.
On the other hand, temperature can maintain the optimum temps of production of butanol, and pressure can reduce to about 50mm Hg-40mm Hg, about 40mm Hg-30mm Hg from about 760mm Hg, or about 30mm Hg-20mm Hg.Therefore,, make the vapour pressure of azeotrope equal the pressure on the culture, can be implemented in once more to cultivate and carry out vacuum distilling in the reaction kettle and can damage cell with recovering through reducing pressure fully.
For the solvent that generates with disclosed technology, butanols can separate with active cultures through vacuum distilling, and wherein gained butanols-water azeotrope is captured as condensation product.In addition, because cell is not irretrievably to be damaged by disclosed technology, so butanols can generate, separates and restart to produce solvent in the single reaction still.
Fig. 1 has shown the synoptic diagram of technology described herein.In this embodiment, the source of containing glucide can be sent into first (cultivation) reaction kettle (20) from batch can (10).First reaction kettle (20) can comprise P2 or other nutritional medium and suitable carbohydrate source (such as but not limited to, glucose, lactose, pentose; Compound carbohydrate such as starch, liquefying starch, liquefying starch, maltodextrin and the steeping water handled through enzyme; Or the glucide that obtains from cellulose biomass etc.), it is with selected clostridium cell inoculation.As disclosed, the steam that vacuum distilling produced can obtain circulation through the system that uses suitable pump (30), and available cooler (40) and condensing surface (50) condensation, and wherein solvent collection is in reaction kettle (60) independently.The condensation product that comprises solvent can be transferred to and is used for further operation or storage in one or more independent reaction kettles (61).The extra assembly of this technology can include but not limited to: one or more froth breaking reaction kettles (70), one or more leak-off pipe (80), one or more input tube (90), one or more valve (100), one or more monitoring equipment (110) are (for example; Be used for monitoring cell concn, butanol concentration, set(ting)value, temperature etc.) and one or more instrument (120) is (for example; Pressure/vacuumometer), be used for continuous or batchwise operation.In one aspect, one or more pipe (80,90) and one or more valve (100) can electricity consumption, machinery or manually controlled, or controls with their combination.
During the operate continuously of the open technology of the present invention; When the condensation product volume of collecting reaches about 4%-5% of initial culture volume; Cultivate the available washing fluid of reaction kettle (20) (like nitrogen) flushing, and under suitable situation, temperature can be returned to optimum temps.In addition; For operate continuously; When solvent production is that (this shows when (by weight) for about 35%-40% of assimilable carbon hydrate; Culture has consumed most of available carbohydrate matrix), add more matrix solution at this time point and replace from cultivating the liquid volume that reaction kettle (20) distills out.
In order from disclosed condensation product, to produce>99% butanols, can divide dried up (for example adsorb, absorption, pervaporation, infiltration extraction, r-o-, liquid-liquid extraction is gentle carries) through method well known in the art from solvent.Solvent from fermentation can be analyzed through method well known in the art, includes but not limited to gc.
In another embodiment, disclose a kind of compsn, it comprises mixing subgroup of producing acid phase and the Clostridium cell that produces solvent phase and the buffer reagent that comprises the biological calcium carbonate source.In one aspect, the biological calcium carbonate source comprises Endoconcha Sepiae fragment or oyster shells fragment.
In one aspect, the present invention can use
C. beijerinkiiThis culture can be inoculated in the LSM substratum and in about 33 ℃ of-37 ℃ of following anaerobism and cultivate 36h at least.
In related fields,
C. beijerinkiiNumbering be NRRL B-50244.According to the clause of budapest treaty,
C. beijerinkiiThe farming research culture collection (Agricultural Research Culture Collection) that NRRL B-50244 is deposited in the Illinois State peoria on February 12nd, 2009 (NRRL) in.In another related fields,
C. beijerinkiiNRRL B-50244 produces butanols in the pH of about 5.0-5.5 scope.In addition, the bacterial strain of preservation can continue to produce butanols under pH6.0.On the other hand,
C. beijerinkiiBe embedded in the solid phase, said solid phase comprises natural sponge, fiber sponge, sponge gourd sponge, alginate calcium microballon and SEPIGEL 305 sheet.
In one embodiment, clostridium spp is disclosed
BeijerinkiiThe biological pure culture of NRRL B-50244.In one aspect, this culture is embedded in the solid phase.In related fields, solid phase comprises natural sponge, fiber sponge, alginate calcium microballon and SEPIGEL 305 sheet.
As is known to the person skilled in the art, suitable reactor drum is big or small, condensing surface is big or small and the change of miscellaneous equipment through selecting, and technology described herein can be amplified to fairly large easily.Utilize the explanation of this paper and those skilled in the art's knowledge, concentration, glucide concentration and other parameter of regulating the clostridium cell are to provide successive technology.
Technological operation described herein for example can be used, and microprocessor system realizes robotization.For example, this system can monitor total solvent concentration, total solvent concentration and preset value are compared, and removes a part of culture during greater than preset value when concentration, or when concentration is lower than preset value, adds inoculum.This system also can add water or other additive, in fermentor tank/cultivation reaction kettle, to keep pre-determined volume.This system can monitor glucide concentration, the concentration and the preset value of glucide compared, and if glucide concentration be lower than preset value, add carbohydrate solutions.Perhaps, this system can monitor fermented liquid butanol concentration, butanol concentration and preset value are compared, and if butanol concentration during greater than preset value, add carbohydrate solutions.In related fields, if butanol concentration greater than preset value, system can raise culture temperature and reduce on the culture pressure with from fermented liquid the distillation and separating butanol.
This system can monitor the condensation product collection rate from fermented liquid, and collection rate and preset value are compared, and if collection rate be lower than preset value, available N
2Purge of gas is cultivated reaction kettle and cooling temperature (suitable time) to restart to produce butanols.
This automation system is as shown in Figure 6.In this embodiment; Primary clustering (10), (20), (30), (40), (50), (60), (61), (70), (80), (90), (100), (110) and (120) are identical with shown in Figure 1 those, and can further comprise microprocessor (130) and exhaust passage/storage (vent/reservoir) (140).Exhaust passage/storage (140) is electrically coupled to valve (101), and this valve (101) is equipped with to carry out fluid with exhaust passage/storage (140), leak-off pipe (80) and input tube (90) and exchanges with mechanical, and carries out function with microprocessor (130) and exchange.In related fields, exhaust passage/storage (140) can comprise washing fluid (for example, N
2).On the other hand, exhaust passage/storage (140) but the entering of convection cell and go out and carry out Electromechanical Control.In addition, electric coupling be equipped with exhaust passage/storage (140) and carry out function with microprocessor (130) and exchange.On the other hand, one or more pumps (30), one or more valve (100), one or more monitoring equipment (110) and one or more instrument (120) but electric coupling and be equipped with to carry out function and exchange with microprocessor (130).
Following embodiment is intended to explanation, does not limit the present invention.
Embodiment
Species specificity PCR.
According to the record that people such as Klijn rolled up in the 8th phase 2919-2924 page or leaf in " Appl Env Microbiology " nineteen ninety-five the 61st, carry out the specific amplification in 16S rRNA gene V6 district with PCR.Be reflected in the aseptic 0.5ml pipe and carry out, this pipe contains the following damping fluid of 50 μ L: 10mM Tris-HCl (pH8.8), 3.0mM MgCl
2, 50mM NaCl, 2.5mM (every kind) deoxynucleoside triphosphate, 1U
TaqPolysaccharase and 15ng (every kind) primer P1 5'-GCG GCG TGC CTA ATA CAT GC-3' (SEQ ID NO:1) and P2 5'-GGG TTG CGC TCG TTG CGG GA-3' (SEQ ID NO:2).After being heated to 95 ℃ of elimination protease activities, add 5 μ L template DNAs (that is the DNA that, from culture, extracts).Make the DNA 1min that unwinds at 94 ℃, 55 ℃ of annealing 1.5min also extend 2.5min at 72 ℃, circulates to accomplish for 30 times and increase.
With 10 times of gained PCR product dilutions, be used for pcr amplification for the second time with 5 these diluents of μ l as template then, its primer is one of P5 5'-GGA ATC TTC CAC AAT GGG CG-3' (SEQ ID NO:3) and following Auele Specific Primer:
Pac-A?GGA?CTT?CAT?CCA?TTA?CGG?ACT?AAC?(SEQ?ID?NO:4)；
Pbe-A?CTT?CCC?CGA?TTA?AGG?GTA?ATT?CAG?(SEQ?ID?NO:5)；
Pbu-A?GTG?GCT?TGC?TCC?ATT?ACA?GAG?TAA?(SEQ?ID?NO:6)；
Pty-A?CGC?CTA?TCT?CTA?GGT?TAT?TCA?G?(SEQ?ID?NO:7)；
Psp-A?CAC?CTA?TCT?CTA?GGC?TAT?GCA?A?(SEQ?ID?NO:8)。
In second step, revise the condition of annealing temperature (63 ℃ or 72 ℃) and cycle index (20 or 25).When annealing temperature during, reduce cycle index near elongating temperature (that is, 72 ℃).
Culture condition.
C. beijerinkiiNeed anaerobic condition.Yet, with being not to be fatal in the of short duration oxygen that is exposed to atmospheric concentration of culture.Table 1 has provided the component of the substratum that uses.
Resazurin is the indicator of oxygen contamination, and it is colourless under the situation of oxygen not having.To be incorporated in 121 ℃ of autoclaving 15min except that all components the dense sugar storage liquid (concentrated sugar stocks) mixes.Sugar soln carries out autoclaving separately, avoiding forming deleterious compound, and when solution temperature is cooled to about 65 ℃, just adds.Trace element solution is sterilized through micro-filtration (0.2 micron).Initial sugar substrate concentration is 50g/L.In case mix the component of all substratum, through solution is placed-vacuum of 60mm Hg gets off and reduce the oxygen that has infiltrated through solution, and is colourless up to resazurin.Use nitrogen wash reaction kettle head space then.With the active inoculum inoculation anaerobic substratum of packing, and, do not stir 33 ℃ of-37 ℃ of cultivations.In cultivating reaction kettle, produce data with the 400ml working volume.
Product analysis
Analyze tunning with gc (GC).The single-column of filling with Chromosorb-101 is enough to differentiate all relevant tunnings.Detect through flame ion from the material of post wash-out.Be sent to from the signal of flame ionization detector and be used for drawing and calculating peak area in the integrator.Fig. 2 has shown the typical chromatogram of the active cultures of 1 microlitre packing.As can be seen from the figure, the primary product of fermentation is a butanols.
Production of butanol and growth
In order to confirm sane growth of cell and butanols synthetic condition, culture is grown in above-mentioned substratum, and pass the generation of getting equivalent sample analysis butanols in time.For the data that generate in Fig. 3 and 4, at " freedom " cell (that is the cell in the suspension-s) be embedded in thinner intracellular growth/production of butanol between the cell in the alginates microballon." gel " of the cell suspension through will being added with alginates drops in CaCl
2Carry out the embedding of alginates microballon in the solution (100mM).Contain alginates microballon culture inoculation medium and begin to cultivate with the logarithm later stage of packing the spending the night of " freedom " culture or packing of spending the night.The cell culture of packing is analyzed with GC as stated.5.0 * 10
6The butanol concentration of the GC peak area of unit corresponding about 1% (w/w).
As can beappreciated from fig. 3, for " freedom " cell, production of butanol increased sharply at the 1st day to the 4th day, and production trend to the 7 days downwards.For the cell of embedding, after about 1 day lag-phase, speed ratio " freedom " cell that production of butanol increases is slow a little, and still, in inoculation back 7 days, the production of embedding cell was significantly higher.
" microenvironment " control pH is provided
Solvent is sane production under pH5.0-5.5.If pH drop to 4.5 or below, the activity of culture significantly reduces.The initial pH of weak buffering growth medium is pH6.5-7.0, to promote to produce sour phase (that is, butyric acid generates).The pH of solution must drop to triggering to the transformation of producing the synthetic phase of solvent/butanols.
Add lime carbonate and be lower than 4.8-5.0 to prevent culture pH.Perhaps, achieve this end, not as using biological calcium carbonate source (for example, about 10 milligrams every milliliter Endoconcha Sepiae or oyster shells fragment) with its use calcium carbonate crystal.In the single reaction still, use these sources that provide to realize " microenvironment " and bigger " surge capability gradient ", to promote to keep product acid and the subgroup of producing the solvent cell.
In addition, in view of the result shows that the cell that is embedded in stationary phase has advantage, the sponge fragment is put into " freedom " cell culture (natural sponge or fiber sponge), bacterium forms bacterium colony on fragment.Sponge fragment through embedding is found to be fabulous culture inoculum, and said culture begins to produce butanols immediately; That is, the cell of " sponge binding " has realized that the butanols synthetic begins fast, and not observed hysteresis on the alginates microballon of embedding.
Because the summary of pertinent literature shows that the disclosed system of Ramey (that is, adopts clostridium spp
AcetobutylicumWith
C. tyrobutyricumTwo stage fixed-bed reactor) than adopting clostridium spp
AcetobutylicumSystem obviously have more yield-power, so production of butanol be in the original plan the expansion Ramey work (seeing that for example USP 5,753,474).Screening
C. acetobutylicumBacterial strain is to differentiate the bacterial strain of tool yield-power; Observe NRRL "
C. acetobutylicum" bacterial strain B597 has Ideal Characteristics.Yet, according to showing that the B597 bacterial strain is
C. beijerinkiiA strain (through above-mentioned with the dna sequence dna be the basis test implement).From B597, separate mutant strain, the B597 bacterial strain saved as NRRL B-50244 on February 12nd, 2009.According to showing that the two mutants of this purifying has new phenotype, shows
C .beijerinkiiNRRL B-50244 can produce butanols outside normal pH scope, said normal pH scope is to producing the typical pH scope of solvent clostridium spp kind; That is pH4.8-5.0.In fact,
C. beijerinkiiNRRL B-50244 can produce butanols under pH6.0.Therefore, the two-phase method of Ramey is no longer suitable.Therefore, for present embodiment, production of butanol is used
C beijerinkiiNRRL B-50244 carries out.The butanols output of the technology of announcing compares favourably with the technology (seeing table 2) of Ramey, but the complicacy in design and execution is wanted much less.
Use
C. acetobutylicumOr
C. beijerinkiiA problem of fermentative prodn butanols is that the butanols pair cell is poisonous.Do not consider under the situation of sugared concentration that the maximum butanol concentration that can tolerate is about 2% (wt/v).
Attempted a kind of possible method that addresses this problem, that is, and vacuum distilling butanol/water azeotrope.The boiling point of butanols is about 118 ℃, and the boiling point of water is 100 ℃.Yet if the mixture of fourth alcohol and water is heated under barometric point, the lower boiling azeotropic mixture of fourth alcohol and water will be 93 ℃ of evaporations.The steam of azeotrope consists of about 55.5wt% butanols and 45.5wt% water.The solubleness of butanols in water has only 7.7 weight %, so condensation product will form two-layer.The upper strata is the butanols of 58 moles of % and the water of 42 moles of %: promptly, by weight, 80% butanols/20% water.Lower floor comprises the butanols of 7.7% weight.
The upper strata can be used as directly burning and need not to be further purified of fuel, and lower floor comprises enough butanols (about 1.0M butanols) and need not further concentrated in " methanol fuel cell ", to produce available current.Perhaps, but lower floor's vapor enrichment to obtain 80% extra butanols.Can be through as obtaining with " molecular sieve " doing for obtaining 100% ethanol>butanols of 99% concentration.
The low-pressure distillation of production of butanol culture
Confirmed under low-temperature reduced-pressure, in the butanol/water mixture of fermented liquid, to set up the condition of azeotropic behavior (azeotropic behavior).If observe the condition that produces this behavior; The technology of available simple and inexpensive realizes two targets: 1) results butanols and its concentration in substratum is reduced to suppresses below horizontal; And 2) if cell be not killed; After top condition rebulids, will restart synthetic butanols, wherein this technology is fit to carry out continuous production butanols from single fermentation reaction still.
The temperature of culture is elevated to about 43 ℃, and reaches vacuum (seeing, for example Fig. 1 (40) and (50)) through condensate trap (condensation trap).When vacuum reached about 110mm Hg, culture solution began violent agitation.When the condensation product collection rate begins to descend, cultivate reaction kettle and make temperature retrieval to 35 ℃ with nitrogen wash.When solvent production shows most available sugar of cell consumption or raw material (butanols has small amount of acetone, sees, for example Fig. 5 (A)), add more raw materials in this time, and replace the liquid volume of distillation loss with the raw material volume.
Can find out that from Fig. 5 (C) when top condition rebulid, the butanols that between about 43 ℃ and 110mm Hg-100mm Hg, carries out vacuum distilling suppressed culture and restarts synthetic butanols.
Though invention has been described with reference to top embodiment, should be understood that various improvement and distortion include within the spirit and scope of the present invention.
All reference of listing all are attached among this paper with its integral body by reference.
< 110>Eugene T Butler three generations (Butler, Eugene T. III)
< 120>butanols is synthesized in the fermentation of single reaction still continuously
<130> JQIP111126US
<150> US09/35327
<151> 2009-02-26
<160> 8
<170> PatentIn?version?3.5
<210> 1
<211> 20
<212> DNA
< 213>artificial sequence
<220>
< 223>primer, clostridium spp kind (Clostridium Species) 16S rRNA conservative region 41-60
<400> 1
gcggcgtgcc?taatacatgc 20
<210> 2
<211> 20
<212> DNA
< 213>artificial sequence
<220>
< 223>primer, clostridium spp kind (Clostridium Species) 16S rRNA conservative region 1094-1114
<400> 2
gggttgcgct?cgttgcggga 20
<210> 3
<211> 20
<212> DNA
< 213>artificial sequence
<220>
< 223>primer, clostridium spp kind (Clostridium Species) 16S rRNA conservative region 361-380
<400> 3
ggaatcttcc?acaatgggcg 20
<210> 4
<211> 24
<212> DNA
< 213>artificial sequence
<220>
< 223>primer, clostridium spp acetobutylicum (Clostridium acetobutylicum) 1000-1050 V6 zone
<400> 4
ggacttcatc?cattacggac?taac 24
<210> 5
<211> 24
<212> DNA
< 213>artificial sequence
<220>
< 223>primer, clostridium spp beijerinkii (Clostridium beijerinkii) 1000-1050 V6 zone
<400> 5
cttccccgat?taagggtaat?tcag 24
<210> 6
<211> 24
<212> DNA
< 213>artificial sequence
<220>
< 223>primer, clostridium spp butyricum (Clostridium butyricum) 1000-1050 V6 zone
<400> 6
gtggcttgct?ccattacaga?gtaa 24
<210> 7
<211> 22
<212> DNA
< 213>artificial sequence
<220>
< 223>primer, clostridium spp tyrobutyricum (Clostridium tyrobutyricum) 1000-1050 V6 zone
<400> 7
cgcctatctc?taggttattc?ag 22
<210> 8
<211> 22
<212> DNA
< 213>artificial sequence
<220>
< 223>primer, clostridium spp sporogenes (Clostridium sporogenes) 1000-1050 V6 zone
<400> 8
cacctatctc?taggctatgc?aa 22
Claims (29)
1. continuous processing that is used to produce butanols, it comprises:
(a) liquid culture of clostridium cell is contacted with the matrix phase that comprises glucide with buffer reagent;
(b) under the optimum temps of solvent production culturing cell up to reaching the butanols inhibition concentration;
(c) pressure in reduction by first reaction kettle is up to violent agitation occurring;
The azeotrope that (d) will comprise butanols is transferred to the second collection reaction kettle as condensation product from first reaction kettle;
(e) wash first reaction kettle with washing fluid; And
(f) continuous repeating step (b)-(e).
2. technology according to claim 1, wherein flushing is the function of condensation and collection volume, and wherein said flushing begins when the condensation and collection volume is the 4%-5% of culture volume.
3. technology according to claim 1 also comprises when solvent production is about 35%-40% (wt/wt) of assimilable carbon hydrate in the matrix, replenishes the matrix that contains glucide.
4. technology according to claim 1, wherein culture comprises the mixture that produces acid and product solvent phase cell.
5. technology according to claim 1, wherein the clostridium cell is selected from:
C. beijerinkii,
C. acetobutylicum,
C. aurantibutyricum,
C. tetranomorphumWith
C. thermocellum
6. technology according to claim 1, wherein pressure drops to about 20mm Hg-30mm Hg from about 760mm Hg.
7. technology according to claim 1, wherein the inhibition concentration of butanols is about 0.9%-2.0%.
8. technology that is used to produce butanols, it comprises:
(a) liquid culture of clostridium cell is contacted with the matrix phase that comprises glucide with buffer reagent;
(b) under optimum temps culturing cell until reaching the butanols inhibition concentration;
(c) temperature of culture is elevated on the solvent production optimum temps about 6 ℃-11 ℃ and reduce pressure in first reaction kettle up to the beginning violent agitation;
The azeotrope that (d) will comprise butanols is transferred to the second collection reaction kettle as condensation product from first reaction kettle;
(e) wash first reaction kettle and the temperature of culture is cooled to optimum temps with washing fluid; And
(f) continuous repeating step (b)-(e).
9. technology according to claim 8, wherein optimum temps is about 33 ℃-37 ℃.
10. technology according to claim 9, wherein temperature is elevated to about 43 ℃-44 ℃ in the step (c).
11. the function of condensation and collection volume is wherein washed and be cooled to technology according to claim 8, wherein when the condensation and collection volume is about 4%-5% of culture volume, and flushing and cooling beginning.
12. technology according to claim 8, wherein pressure drops to about 110mm Hg-100mm Hg from about 760mm Hg.
13. technology according to claim 8, wherein inhibition concentration is about 0.9%-2.0%.
14. process according to claim 8 also comprises when solvent production is about 35%-40% (wt/wt) of assimilable carbon hydrate in the matrix, replenishes the matrix that contains glucide.
15. technology according to claim 8, wherein buffer reagent comprises the biological calcium carbonate source.
16. technology according to claim 15, wherein biogenic is Endoconcha Sepiae or oyster shells.
17. technology according to claim 8 also comprises cell is contacted with the sponge fragment.
18. technology according to claim 17, wherein sponge-rubber pad carbonated calcium, silicon-dioxide spicule or Mierocrystalline cellulose.
19. technology according to claim 8, but wherein this technology reaches the butanols output between about 80%-90% by fermentable carbohydrates.
20. technology according to claim 8, wherein the butanols output of this technology is about 20-40g/L.
21. technology according to claim 8, wherein this technology produces the head space gaseous mixture, and it comprises about 40-50%H
2, 40-50%CO
2And 0-20%N
2
22. technology according to claim 21, wherein this technology produces the head space gaseous mixture, and it comprises about 43%H
2, 43%CO
2And 14%N
2
23. a compsn, it comprises product acid phase and mixed population that produces solvent phase clostridium spp cell and the buffer reagent that contains the biological calcium carbonate source.
24. compsn according to claim 23, wherein the biological calcium carbonate source is Endoconcha Sepiae fragment or oyster shells fragment.
25. compsn according to claim 23, wherein cell is a clostridium spp
Beijerinkii
26. compsn according to claim 25, wherein
C. beijerinkiiBe NRRL B-50244.
27. compsn according to claim 25, wherein clostridium spp
BeijerinkiiBe embedded in the solid phase, said solid phase is selected from: natural sponge, fiber sponge, sponge gourd sponge, alginate calcium microballon and SEPIGEL 305 sheet.
28. clostridium spp
BeijerinkiiThe biological pure culture of NRRL B-50244.
29. biological pure culture according to claim 28, wherein this culture is embedded in the solid phase, and said solid phase is selected from: natural sponge, fiber sponge, alginate calcium microballon and SEPIGEL 305 sheet.
Applications Claiming Priority (3)
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| US39102609A | 2009-02-23 | 2009-02-23 | |
| US12/391,026 | 2009-02-23 | ||
| PCT/US2009/035327 WO2010096065A1 (en) | 2009-02-23 | 2009-02-26 | Continuous single vessel butanol synthesis by fermentation |
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| CN102439161B CN102439161B (en) | 2015-11-25 |
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| EP (1) | EP2398906A4 (en) |
| CN (1) | CN102439161B (en) |
| AU (1) | AU2009340524B2 (en) |
| BR (1) | BRPI0924257A2 (en) |
| WO (1) | WO2010096065A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108690853A (en) * | 2017-04-05 | 2018-10-23 | 中国石油化工股份有限公司 | A kind of method of fermenting and producing butanol |
| CN109154007A (en) * | 2016-07-22 | 2019-01-04 | 佛兰芒技术研究所有限公司 | Methods and systems for producing products by fermentation |
| CN109486868A (en) * | 2017-09-09 | 2019-03-19 | 中国石油化工股份有限公司 | A method of isopropanol and butanol are produced by fermenting raw materials of lignocellulosic |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948737B (en) * | 2010-08-31 | 2013-06-05 | 天津理工大学 | Acetone-butanol in-situ extraction continuous fermentation device and technology |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4359533A (en) * | 1980-11-26 | 1982-11-16 | The United States Of America As Represented By The Department Of Energy | Fermentative alcohol production |
| WO2008076749A1 (en) * | 2006-12-15 | 2008-06-26 | Dow Global Technologies Inc. | Recovery of volatile products from fermentation broth |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63254986A (en) * | 1987-04-10 | 1988-10-21 | Res Assoc Petroleum Alternat Dev<Rapad> | Production of alcohol |
| AU7384098A (en) * | 1997-05-14 | 1998-12-08 | Board Of Trustees Of The University Of Illinois, The | A method of producing butanol using a mutant strain of (clostridium beijerinckii) |
| US8426173B2 (en) * | 2007-05-02 | 2013-04-23 | Butamax (Tm) Advanced Biofuels Llc | Method for the production of 1-butanol |
| RU2375454C1 (en) * | 2008-08-28 | 2009-12-10 | Открытое акционерное общество "Корпорация Биотехнологии" | Method of producing organic solvents, mainly butanol |
-
2009
- 2009-02-26 EP EP09840537.6A patent/EP2398906A4/en not_active Withdrawn
- 2009-02-26 AU AU2009340524A patent/AU2009340524B2/en not_active Ceased
- 2009-02-26 CN CN200980158890.6A patent/CN102439161B/en not_active Expired - Fee Related
- 2009-02-26 WO PCT/US2009/035327 patent/WO2010096065A1/en active Application Filing
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4359533A (en) * | 1980-11-26 | 1982-11-16 | The United States Of America As Represented By The Department Of Energy | Fermentative alcohol production |
| WO2008076749A1 (en) * | 2006-12-15 | 2008-06-26 | Dow Global Technologies Inc. | Recovery of volatile products from fermentation broth |
Non-Patent Citations (2)
| Title |
|---|
| 王鑫昕: "原位萃取发酵耦合工艺高产丁醇的初步研究", 《河北农业大学学报》 * |
| 顾阳等: "生物丁醇制造技术现状和展望", 《生物工程学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109154007A (en) * | 2016-07-22 | 2019-01-04 | 佛兰芒技术研究所有限公司 | Methods and systems for producing products by fermentation |
| CN109154007B (en) * | 2016-07-22 | 2023-01-03 | 佛兰芒技术研究所有限公司 | Methods and systems for producing products by fermentation |
| CN108690853A (en) * | 2017-04-05 | 2018-10-23 | 中国石油化工股份有限公司 | A kind of method of fermenting and producing butanol |
| CN108690853B (en) * | 2017-04-05 | 2021-10-08 | 中国石油化工股份有限公司 | Method for producing butanol by fermentation |
| CN109486868A (en) * | 2017-09-09 | 2019-03-19 | 中国石油化工股份有限公司 | A method of isopropanol and butanol are produced by fermenting raw materials of lignocellulosic |
| CN109486868B (en) * | 2017-09-09 | 2022-04-08 | 中国石油化工股份有限公司 | Method for producing isopropanol and butanol by fermenting lignocellulose serving as raw material |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2009340524A1 (en) | 2011-10-20 |
| CN102439161B (en) | 2015-11-25 |
| EP2398906A1 (en) | 2011-12-28 |
| BRPI0924257A2 (en) | 2016-07-19 |
| EP2398906A4 (en) | 2013-11-06 |
| AU2009340524B2 (en) | 2015-09-17 |
| WO2010096065A1 (en) | 2010-08-26 |
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