JP2016096764A - Butanol production method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of generating butanol with high efficiency in butanol fermentation.SOLUTION: A method for producing butanol comprises a step in which massive butanol fermentation microorganisms, or butanol fermentation microorganisms immobilized to a carrier are cultured in the presence of a substrate, and at the same time, the culture is subjected to solvent extraction using a C10-C12 alcohol.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a method for producing butanol by, for example, butanol fermentation.

  In butanol fermentation, acetone, n-butanol and ethanol are produced in a ratio of 3: 6: 1, respectively. Among these, n-butanol is expected as a fuel for drop-in automobiles from renewable resources, and is industrially useful as an alcohol for esters of various organic acids.

  However, of the products in butanol fermentation, n-butanol has the highest toxicity, and butanol fermentation usually stops growing with an accumulation of about 1%. Because of the high toxicity of butanol, the concentration of butanol accumulated in the medium is low. Conventionally, much energy is required to separate and recover n-butanol from the medium by distillation. Various methods have been developed to solve this problem, and examples include a solvent extraction method, a pervaporation method, a gas stripping method, and carrier adsorption.

  Among these, as a solvent extraction method, a method using a long-chain aliphatic alcohol having a large distribution ratio of n-butanol to water has been proposed. Patent Document 1 discloses a method using oleyl alcohol as an extractant. Oleyl alcohol is superior because of its relatively high partition coefficient of n-butanol and low toxicity to fermenting bacteria. However, since oleyl alcohol itself has a surfactant property, there is a problem that when it is mixed with a medium for extraction, an emulsion is formed and subsequent liquid-liquid separation cannot be performed.

  Patent Document 2 discloses performing fermentation while inoculating a fermented liquid with an extract containing an alcohol having 10 to 14 carbon atoms in a butanol production method by fermentation. Such aliphatic long-chain alcohols, which are shorter than oleyl alcohol, increase the distribution coefficient of n-butanol as the chain length decreases, making it difficult to form an emulsion, but on the other hand, the toxicity to fermenting bacteria increases. When an extraction operation using these solvents is performed using a culture solution containing fermenting bacteria, there is a problem that the fermenting bacteria are killed.

  In order to keep production costs low due to economic problems, it is desirable to use the broth after extracting n-butanol again for butanol fermentation, but the broth after extraction with a highly toxic long-chain alcohol solvent Because it is not suitable for the growth of fermenting bacteria, re-extract the long-chain alcohol from the culture solution with a second organic solvent that is harmless to the fermenting bacteria, and reduce the residual amount of long-chain alcohol remaining in the culture solution. A method of re-use has been devised. However, the method of extracting with two kinds of solvents is complicated and causes a cost increase.

  Furthermore, as an organic solvent with low toxicity suitable for extraction of n-butanol, fatty acid methyl ester and the like are listed in addition to oleyl alcohol. However, although fatty acid methyl ester has low toxicity to fermenting bacteria, it is not efficient because n-butanol has a small partition coefficient and a large amount of fatty acid methyl ester is required to extract n-butanol.

  On the other hand, Patent Document 3 discloses a method for converting an organic compound using a microorganism adhered and immobilized on a hydrophilic immobilization carrier. However, Patent Document 3 does not disclose that butanol production was performed using the immobilized microorganism.

  There is still a need for a method for efficiently producing butanol in butanol fermentation.

JP 60-172290 A JP 59-216591 Japanese Patent Laid-Open No. 5-91878

  In view of the above circumstances, an object of the present invention is to improve the yield of butanol in butanol fermentation.

  As a result of diligent research to solve the above-mentioned problems, in the case of butanol fermentation, a strain that becomes a lump or a strain immobilized on a carrier is used, and simultaneously with butanol fermentation, the number of carbon atoms such as dodecanol is 10 to 10 as an extraction solvent. It was found that the yield of butanol can be improved by extracting the produced butanol into the extraction solvent phase using 12 alcohols, and the present invention has been completed.

That is, the present invention includes the following.
(1) A step of culturing a lump of butanol-fermenting microorganisms or butanol-fermenting microorganisms immobilized on a carrier in the presence of a substrate, and simultaneously subjecting the culture to solvent extraction using an alcohol having 10 to 12 carbon atoms. And a method for producing butanol.
(2) The method according to (1), wherein the butanol-fermenting microorganism is a microorganism belonging to the genus Clostridium.
(3) the microorganism belonging to the genus Clostridium is selected from the group consisting of Clostridium saccharoperbutylacetonicum, Clostridium acetobutylicum and Clostridium beijerinckii (2) ) The method described.
(4) The method according to any one of (1) to (3), wherein the alcohol having 10 to 12 carbon atoms is selected from the group consisting of decanol, undecanol, and dodecanol.

  According to the present invention, the butanol yield in butanol fermentation can be improved.

It is a schematic diagram which shows the mode of (A) batch culture (bin) and (B) continuous extraction fermentation in this invention. 2 is a graph showing butanol production by dodecanol multi-layer culture in a massive butanol-fermenting strain in Example 1. FIG. 2 is a graph showing butanol production by dodecanol multi-layer culture in free cells in Comparative Example 1. FIG.

  A method for producing butanol according to the present invention (hereinafter referred to as “the present method”) is a method of culturing a butanol-fermenting microorganism in a bulk form or a butanol-fermenting microorganism immobilized on a carrier in the presence of a substrate, and simultaneously culturing the culture, It is a method including a step of subjecting to solvent extraction using an alcohol having 10 to 12 carbon atoms.

  This method is a method in which butanol extraction is performed simultaneously with cultivation using an alcohol solvent having a large butanol partition coefficient but high toxicity to butanol-fermenting microorganisms during butanol fermentation.

  When normal butanol-fermenting microorganisms are cultured by adding an amount of a highly toxic alcohol-based solvent that is two-phase separated from the medium, growth stops and butanol fermentation also stops. However, it has been found that butanol-fermenting microorganisms that become agglomerate exhibit growth and butanol-fermentability comparable to those in the case of not adding the two-phase separation amount of a highly toxic alcohol solvent. In addition, like butanol-fermenting microorganisms that become clumped, butanol-fermenting microorganisms immobilized on a carrier also have growth and butanol fermentability comparable to those without addition of a highly toxic alcohol solvent. Found to show.

  In this method, the butanol-fermenting microorganism is not particularly limited as long as it is a microorganism having a butanol-fermenting ability. Is mentioned. Moreover, as a strain of Clostridium saccharoper butylacetonicum, for example, ATCC27021 strain can be mentioned. Furthermore, examples of the strain of Clostridium saccharoperbutylacetonicum include, for example, gene mutant strains, specifically, butyric acid involved in the pathway for producing butyric acid from butyryl CoA described in JP-A-2014-207885. A strain of Clostridium saccharoperperbutylacetonicum in which a gene encoding enzyme (eg, ptb gene, buk gene) is deleted or destroyed; or acetic acid involved in a pathway for generating acetic acid from acetyl-CoA in addition to the butyric acid synthase gene Involved in genease genes (eg, pta gene, ack gene), acetone genease genes (eg, adc gene, ctfAB gene) involved in the pathway of acetone production from acetoacetyl-CoA and / or pathways of lactate production from pyruvate Clostridium saccharopurve with a deficient or destroyed lactic acid synthase gene (for example, ldh1 gene) High butanol yield strains such as the strains of Ruasetonikamu like.

  Examples of the lump (or aggregate) butanol-fermenting microorganisms include those obtained by culturing butanol-fermenting microorganisms for a long period of time by continuous culture or the like and adhering to a culture vessel. For example, microorganisms belonging to the genus Clostridium, such as Clostridium saccharoperbutylacetonicum, which are attached to the wall of the culture vessel by culturing TYA medium at a feed rate of 1 / day for 3 months are used for massive butanol fermentation It can be obtained as a microorganism. The attached bacterial cells are peeled off from the container and inoculated again into the culture container, so that the massive bacterial cells can be stably transplanted and cultured.

  Examples of butanol-fermenting microorganisms that have been agglomerated by immobilization on a carrier include butanol-fermenting microorganisms that are comprehensively immobilized using a gel such as carrageenan or calcium alginate as a carrier. For example, a butanol-fermenting microorganism can be added to a carrageenan aqueous solution, mixed, and dropped into a KCl solution to obtain a bead-shaped entrapping immobilization butanol-fermenting microorganism. Examples of other immobilization methods include entrapping immobilization with polyvinyl alcohol, agglomeration immobilization with polyethyleneimine, and the like, immobilization on porous urethane and inorganic carriers, and the like.

  On the other hand, in the present method, the alcohol used as the extraction solvent is an alcohol having 10 to 12 carbon atoms, and examples thereof include decanol, undecanol, and dodecanol.

  The toxicity of organic solvents is thought to have two effects. One is a solvent dissolved in an aqueous medium such as a medium, and the other is a solvent separated from an aqueous medium such as a medium.

  Examples of alcohols having a longer chain than butanol include pentanol, hexanol, heptanol, octanol, nonanol, decanol, dodecanol, undecanol, and tridecanol. Become stronger. On the other hand, the longer the carbon chain, the lower the solubility in an aqueous medium such as a medium, and the lower the concentration of the alcoholic solvent in the medium when the medium is overlaid with the solvent. A long-chain alcohol substantially having decanol or higher has a solubility in water of 100 ppm or less, and even if a saturated alcohol solvent is added to the medium, the influence on butanol fermentation is small.

  On the other hand, the alcoholic solvent separated into two phases has an interface with an aqueous medium such as a medium, but the cell surface of the butanol-fermenting microorganism is considered to contain a hydrophobic portion, and has the property of adhering to this interface. Have. Ordinary butanol-fermenting microorganisms are killed when they are incorporated into this interface and the cell membrane of the fungus or the protein present in the cell membrane undergoes denaturation.

  However, in this method, even when the lump-like butanol-fermenting microorganisms adhere to the interface between the alcohol-based solvent and the aqueous medium such as a medium, only a part of the surface bacteria adhere to the organic solvent. Since the bacteria inside the mass do not come into direct contact with the alcoholic solvent, butanol and proteins present in the cell membrane are not denatured, butanol can be fermented without being killed.

  In this method, the above-mentioned massive butanol-fermenting microorganisms or butanol-fermenting microorganisms immobilized on a carrier are cultured in a medium containing a substrate (for example, glucose, xylose, sucrose, arabinose, etc.), and at the same time, the culture is carbonized. It is subjected to solvent extraction using several tens to twelve alcohols (hereinafter referred to as “alcohol solvent”), and the produced butanol is extracted into the alcohol solvent. For example, the above-mentioned massive butanol-fermenting microorganisms or butanol-fermenting microorganisms immobilized on a carrier are added to a medium containing 1 to 100 g / L of substrate and cultured. The temperature suitable for the cultivation is 20 to 40 ° C, preferably 25 to 35 ° C. Specifically, for example, when a microorganism belonging to the genus Clostridium is used, the culture is performed at 20 to 40 ° C. for 1 to 300 days under anaerobic conditions.

  FIG. 1 is a schematic diagram showing the mode of (A) batch culture (bottle) and (B) continuous extraction fermentation in the present invention.

  As shown in FIG. 1 (A), butanol can be extracted from the culture to the alcoholic solvent by overlaying the alcoholic solvent on the culture (medium). Alternatively, the alcohol-based solvent layered on the medium may be continuously taken out and a new alcohol-based solvent may be added. Alternatively, after removing butanol from the continuously extracted alcohol-based solvent by a method such as distillation, the alcohol A system solvent may be circulated through the medium.

  Furthermore, as shown in FIG. 1 (B), the medium is circulated externally, liquid-liquid extraction is performed between the alcoholic solvent, and then the two phases are separated into the medium and the alcoholic solvent. The medium is returned to the culture. As the alcohol solvent, a method in which butanol is removed and recovered by a method such as distillation and then reused for liquid-liquid extraction can be employed. In addition, sugar or medium components may be added when reused in the medium. Long-term continuous culture can be performed by adding sugar and medium components.

  In this method, butanol can be recovered by purifying butanol from an alcohol solvent by a method such as distillation.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

[Example 1] Butanol fermentation with massive butanol-fermenting strain and dodecanol overlay
1. Acquisition of massive cells The pta gene prepared by using Clostridium saccharoperbutylacetonicum ATCC27021 strain described in Example 1 of Japanese Patent Application Laid-Open No. 2014-207885 as a parent strain and a high butanol yield strain (ΔptaΔptb strain) in which the ptb gene was disrupted were The cells were continuously cultured for 3 months using an ABLE 100 ml microjar fermenter containing the medium. The feed rate of the medium was 100 ml / day. The composition of TYA medium is as follows: tryptone 6g, yeast extract 2g, glucose 40g, ammonium acetate 3g, magnesium sulfate heptahydrate 0.3g, potassium phosphate monobasic 0.5g, iron sulfate heptahydrate 10mg, distilled water 1L.

  The continuous culture for about 3 months resulted in bacterial deposits on the walls of the culture vessel and the stirring shaft. The adherent was peeled off and cultured in TYA medium to obtain microbial cells that maintained a clumped state.

2. Butanol fermentation using dodecanol overlay The butanol fermentation overlaying dodecanol was carried out using the massive cells obtained in section 1 above.

  Specifically, 50 ml of dodecanol was added to a 100 ml medium bottle containing 50 ml of TYA medium, and 1 ml of massive cells was added thereto and cultured at 30 ° C. under anaerobic conditions while stirring with a stirrer. Note that the glucose concentration of the TYA medium was 83.6 g / L.

Sampling was performed every day from the second day of culture, and the butanol content in the medium and dodecanol was analyzed.
The results are shown in Table 1 and FIG.

  As shown in Table 1 and FIG. 2, butanol fermentation proceeds normally in bulk cells, butanol is extracted normally, butanol is extracted into the organic phase (dodecanol phase), and the concentration of butanol in the medium (aqueous phase) is low. Maintained. The total amount of butanol contained in the medium and dodecanol was 27.5 g per liter of medium.

[Comparative Example 1] Dodecanol Multilayer Culture with Free Bacteria In the same manner as in Example 1 except that the high butanol yield strain (ΔptaΔptb strain) described in Example 1 that grows in a free state was used, The culture was overlaid with dodecanol. Note that the glucose concentration of the TYA medium was 83.2 g / L.
The results are shown in Table 2 and FIG.

  As shown in Table 2 and FIG. 3, the culture was continued until the fifth day of culture, but the turbidity of the culture broth did not increase, and neither glucose consumption nor butanol production was observed.

[Example 2] Incubation with alcohol of each chain length
50 ml of each alcohol having 7 to 14 carbon atoms is layered on 50 ml of TYA medium, and the massive cells described in Example 1 or the free cells described in Comparative Example 1 are inoculated and stirred at 30 ° C. with a stirrer. Incubated under anaerobic conditions. Note that the glucose concentration of the TYA medium was 40 g / L.

  The results are shown in Table 3. In Table 3, the total amount of butanol contained in the alcohol layered on the culture medium after 7 days of culture is shown per notation per liter of the medium.

  As shown in Table 3, free microbial cells that grow uniformly dispersed did not grow when long-chain alcohols having up to 12 carbon atoms were added. Tridecanol and tetradecanol were solidified at a culture temperature of 30 ° C. and grew, but the amount of butanol produced was the same as when no long-chain alcohol was added.

  On the other hand, massive cells grew under the condition of adding a long-chain alcohol having 10 or more carbon atoms, and the amount of butanol produced increased to 27 g / L when a long-chain alcohol having 10 to 12 carbon atoms was added. In the case of long chain alcohols having 13 and 14 carbon atoms, the long chain alcohol was solidified, and the amount of butanol produced was the same as when no long chain alcohol was added. However, even when the culture was carried out using tridecanol or tetradecanol in which the solvent was solidified, the massive cells produced more butanol than the free cells.

  In this way, free cells did not grow in the presence of liquid long-chain alcohols, whereas massive cells grew in the presence of liquid long-chain alcohols having 10 to 12 carbon atoms to produce butanol. We were able to.

[Example 3] Bacterial immobilized butanol fermentation with carrageenan 8 ml of 2% aqueous solution of κ-carrageenan (manufactured by Wako Pure Chemical Industries, Ltd.) was heated to 100 ° C to completely dissolve and then cooled to 42 ° C or lower. did. To this carrageenan aqueous solution, 3 ml of the cell suspension of the high butanol yield strain (ΔptaΔptb strain) described in Example 1 was added and mixed, and the solution was added dropwise to a 2% KCl solution to form the bead-shaped immobilized cells. Prepared.

  The immobilized cells were separated by filtration, inoculated into 50 ml of 50 ml of Tdecane medium containing 90 g / L glucose, and cultured under anaerobic conditions at 30 ° C. while stirring with a stirrer.

  When analyzed after 4 days of culture, glucose was completely consumed, and the medium and dodecanol were combined and contained 27.3 g of butanol per liter of the medium.

Claims (4)

  A process for culturing butanol-fermenting microorganisms immobilized on a bulk butanol-fering microorganism or a carrier in the presence of a substrate, and simultaneously subjecting the culture to solvent extraction using an alcohol having 10 to 12 carbon atoms. Production method.   The method according to claim 1, wherein the butanol-fermenting microorganism is a microorganism belonging to the genus Clostridium.   The microorganism belonging to the genus Clostridium is selected from the group consisting of Clostridium saccharoperbutylacetonicum, Clostridium acetobutylicum and Clostridium beijerinckii. Method.   The method according to any one of claims 1 to 3, wherein the alcohol having 10 to 12 carbon atoms is selected from the group consisting of decanol, undecanol and dodecanol.

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