KR101094447B1 - Biobutanoic acid and Preparing method thereof - Google Patents

Abstract

The present invention relates to a biobutanoic acid and a method for preparing the same, and in more detail, a fermentation broth obtained by fermenting carbohydrates into anaerobic bacteria is prepared from an aqueous butyrate salt solution by electrodialysis, and then acidified, After extraction with a solvent, n-butanoic acid is separated and purified from the extract to produce a biobutanoic acid and a biobutanoic acid prepared by the above method. Since the method of preparing the biobutanoic acid of the present invention hardly generates by-product salts of sulfate salts such as ammonium sulfate or calcium sulfate, it is not necessary to use a separate additional step for removing and discarding the biobutanoic acid with high yield. You can get it.

n-butanoic acid, electrodialysis, anaerobic bacteria, butyrate salt

Description

Biobutanoic acid and preparation method thereof

In the present invention, a fermentation broth obtained by fermenting carbohydrates into anaerobic bacteria is prepared from an aqueous solution of butyrate salt using electrodialysis, and then acidified, extracted with a specific solvent, and then separated and purified n-butanoic acid from the extract. It relates to a method for producing biobutanoic acid and to a biobutanoic acid produced by the above method.

The modern chemical and energy industry structure has been challenged by global dependence on excessive dependence on finite fossil fuels, rapidly depleting fossil fuels and increasing atmospheric concentrations of greenhouse gases resulting from overuse of fossil fuels. It is causing. However, biofuels are considered environmentally friendly fuels because they are made from biomass or biowaste that can be recycled periodically. Along with biofuels, biodiesel and bioethanol, biobutanol is attracting attention as a very promising energy source as a fuel that can be easily applied to future vehicles, and attention is being focused on the development of economical and environmentally friendly manufacturing methods.

Butanol has conventionally been produced via butyl aldehyde as a raw material of propylene produced in large quantities in naphtha crackers of petrochemical plants. Recently, n-butanoic acid is produced through a microbial fermentation process using organic waste and biomass as raw materials. After this, a method for producing butanol by esterification is also possible. However, when n-butanoic acid is prepared by using microorganisms, butanol is prepared using the same, the production yield may be higher than that of butanol using butyl aldehyde. In view of the additional separation and purification process for this purpose, there was a problem of low economic efficiency.

As a method of increasing the yield of n-butanoic acid, fermented co-extraction separation technology using a membrane contactor using a hollow fiber membrane module has been introduced in some documents. However, the membrane contactor is easily deteriorated or denatured by the oleyl alcohol, organic amine, and caustic soda solution used as the back extraction solvent in the extracting solvent mixture, so it is difficult to use it stably for a long time and actually contact the hollow fiber membrane material. Materials such as oleyl alcohol and organic amines are diffused and penetrated into the membrane material, causing swelling, solubilization, etc., thereby impairing the mechanical stability of the membrane and losing separation selectivity. In addition, since sulfuric acid or the like is used to acidify the compound obtained from the fermentation broth with n-butanoic acid, salt by-products such as ammonium sulphate or calcium sulphate are generated, which causes additional costs for mass disposal. , The method for producing n-butanoic acid using a membrane contactor has a problem of poor industrial performance.

Therefore, the present inventors have studied the method for producing biobutanoic acid with high economical efficiency, and have found the optimum fermentation conditions for fermenting carbohydrates into the genus Clostridium bacteria, and effectively separating butyrate salt from the fermentation broth. The introduction of an electrodialysis method that can be concentrated to realize that n-butanoic acid can be obtained in a high yield to complete the present invention. That is, an object of the present invention is to provide a biobutanoic acid and a method for producing the same.

The present invention for solving the above problems is the fermentation broth produced by fermenting the carbohydrate, the butyrate salt aqueous solution is separated and concentrated by electrodialysis, and then the acidified butyrate salt aqueous solution and n-butane using an organic solvent at the same time It is an object of the present invention to provide a method for producing biobutanoic acid with high yield of separating and purifying n-butanoic acid from the organic phase including n-butanoic acid after extracting an organic phase including an acid.

In addition, an object of the present invention is to provide a biobutanoic acid prepared by the above production method.

The present invention separates the fermentation product (fermentation broth) from the fermenter's medium by simultaneously or intermittently operating the electrodialysis separator in parallel with the fermentation tank, so that the concentration of butyrate salt is suitable for the survival conditions of the fermentation bacteria in the fermentation tank. By overcoming the product inhibition, it is possible to increase the yield of the butyrate salt as the target substance and ultimately obtain high purity biobutanoic acid in high yield. In addition, the present invention can be used to separate, recycle and reuse the carbon source utilized from the fermentation broth using an electrodialysis separator, and in the present invention, since by-products such as sulfate salts such as ammonium sulfate or calcium sulfate are hardly generated, It is economical because no additional process is required to remove and discard it.

Hereinafter, the present invention will be described in more detail.

In the present invention, butyrate salts are defined as ammonium butyrate and calcium butyrate.

Biobutanoic acid production method of the present invention is a step of separating and concentrating an aqueous butyrate salt solution by electrodialysis from the fermentation broth produced by fermenting carbohydrate; Acidifying the concentrated butyrate salt aqueous solution with at least one selected from carbon dioxide, carbonic acid, and an inorganic acid, and simultaneously extracting an organic phase including n-butanoic acid using an organic solvent; And three steps of separating and purifying n-butanoic acid from the organic phase including n-butanoic acid.

In the first step, the fermentation broth is prepared by performing an anaerobic fermentation process using anaerobic bacteria with carbohydrates such as glucose and glucose. The anaerobes are Clostridium spp., Preferably Clostridium tyrobutyricum, Clostridium butryricum, Clostridium acetobutyricum, It may include one or two or more Clostridium bacteria selected from Clostridium beijerinckii, Clostridium populeti and Clostridium thermobutyricum.

And, in order to reduce the separation burden of hydrocarbons (carbohydrates) in the electrodialysis separation, it is preferable to adjust the hydrocarbon (carbohydrates) concentration of the fermentation tank below a certain concentration. Therefore, although the concentration of hydrocarbons (carbohydrates) such as glucose may be kept high in the state before electrodialysis separation, at least when the electrodialysis separation is performed, the concentration of hydrocarbons (carbohydrates) is 50 g / L or less, preferably 10 to It is preferable to maintain the concentration at 30 g / L, more preferably at a concentration of 10 to 20 g / L.

Maintaining anaerobic conditions in the fermentation tank immobilized inoculation of the anaerobes to provide a hydrocarbon feed, a fermentation broth containing butyrate salts such as ammonium butyrate, calcium butyrate and organic acids such as acetic acid. The fermentation is preferably maintained in the fermentation conditions of pH 5.8 ~ 6.5 and 35 ~ 40 ℃, more preferably maintaining the fermentation conditions of pH 5.9 ~ 6.4 and 37 ~ 39 ℃ in terms of the butyrate salt production. In addition, since the organic acid contained in the resulting fermentation broth causes an acidity drop of the fermentation broth, an acidity regulator containing ammonium ions or calcium cations that act as a pH buffer may be added. When the concentration of fermentation broth, especially the butyrate salt, increases, the limit of survival of the anaerobic bacteria is reached or the product inhibition concentration is reached, and the anaerobic bacteria no longer produce the material we want, and the activity is slowed. Since it is possible, it is necessary to remove the fermentation broth produced from the fermentation tank, and for this purpose, the present invention uses the following electrodialysis separation method to remove the fermentation broth from the fermentation tank.

The electrodialysis separation method may use an electrodialysis separator composed of two or three stages. Electric Dialysis separation applies DC voltage required for separation to a stacked structure in which unit membranes made of channels are stacked between spacers between the cation exchange type and the anion exchange type. This allows selective electrical separation between the electrolyte and the non-electrolyte material. Electrodialysis membranes can be manufactured in a more physicochemically stable form than the hollow fiber membranes used in conventional membrane contactors, and can also be regenerated during use by periodically changing the direction of current flow. Better. In addition, since the electrical conductivity changes according to the concentration of the material to be separated or the degree of separation, the amount of current is controlled to enable energy-efficient separation, and even when the stack is configured in a multi-stage separation type, the current flowing between the stacks is shared. Energy efficient separation can be achieved. Since water-soluble carbohydrates, such as glucose, are electrophilic membranes, which are basically hydrophilic membranes, they diffuse together with water to limit separation selectivity. This problem is controlled by lowering carbohydrate concentrations in the fermentor medium to fermenter in fed batch form. It can be solved by applying a method of driving, or using a multi-stage separation type electrodialysis module stack. In addition, it is preferable to operate the electrodialysis separator which is operated together with the fermentation tank continuously or intermittently simultaneously with the fermentation tank. That is, the electrodialysis can be carried out simultaneously with the fermentation or the electrodialysis can be performed intermittently, and more preferably, the leakage of the hydrocarbon raw material (carbohydrate) can be minimized by performing the electrodialysis separation intermittently. .

And, as shown in B of FIGS. 1 to 4, the electrodialysis separator conveys unused hydrocarbon raw material remaining mostly in the first stage (retentate side) to the fermenter for recycling, thereby preventing microbial inhibition phenomenon and It is possible to maximize the conversion utilization rate of, and in the second stage (permeate side), by obtaining a concentrated solution of butyrate salt through the concentration process, it is possible to lower the cost of the separation and purification process proceeded to the subsequent process. As such, the present invention can be performed at the same time by fermentation and electrodialysis separation, overcoming the yield limitation of the product by the product inhibition phenomenon, it is possible to optimally maintain the butyrate salt production. In addition, the electrodialysis separator may be mainly composed of three stages to increase the separation removal rate of a hydrocarbon source such as glucose.

In the second step, the acidification is carried out by adding one or two or more selected from carbon dioxide, carbonic acid and inorganic acid to the concentrated aqueous butyrate salt in one step to perform an acidification reaction to acidify the butyrate salt with n-butanoic acid. The carbon dioxide can be recycled as shown in Figures 1 and 2 carbon dioxide generated in the fermentation process, and carbon dioxide may be separately added to the acidification reactor. Conventional processes have a problem of generating a large amount of by-products such as calcium sulfate and ammonium sulfate using inorganic acids such as sulfuric acid, but in the present invention, carbon dioxide can be recycled and recycled by decomposing carbonate generated using carbon dioxide and carbonic acid. By doing so, it is possible to reduce the inorganic salt by-products generated. Thus, the present invention does not completely exclude the use of inorganic acids. That is, the butyrate separated and concentrated by the electrodialysis process may be used as necessary or according to the environmental and economic evaluation of the process.

The two-step process will be described in more detail. The pKa values of carbonic acid are 6.4 and 10.3, respectively, with the former relating to the dissociation reaction (1) of the A and the dissociation reaction (2). On the other hand, the pKa value of n-butanoic acid is 4.82 and the pKa value of acetic acid that can be produced with n-butanoic acid during fermentation is 4.76. Below, their respective dissociation reactions are shown.

H⁺ + HCO₃ ^- pKa = 6.4 (1)H ₂ CO ₃

H ⁺ + HCO ₃ ^- pKa = 6.4 (1)

H⁺ + CO₃ ^2- pKa =10.3 (2)HCO ₃ ^-

H ⁺ + CO ₃ ^2- pKa = 10.3 (2)

H⁺ + C₃H₇COO^- pKa = 4.82 (3)C ₃ H ₇ COOH

H ⁺ + C ₃ H ₇ COO ^- pKa = 4.82 (3)

H⁺ + CH₃COO^- pKa = 4.76 (4)CH ₃ COOH

H ⁺ + CH ₃ COO ^- pKa = 4.76 (4)

If the pH value is equal to the pKa value of an acid, half the acid is dissociated from the relationship between pH and pKa obtained from the definition of pKa and pH. You can calculate the degree of dissociation accordingly. When the dissociation degree is calculated from the pKa value of the first dissociation reaction of the carbonic acid (1), the dissociation degree of the carbonic acid in the acidity range of 5.5 <pH <6 is in the lower range of about 10 to 30%. On the other hand, in the case of n-butanoic acid or acetic acid, the degree of dissociation in the corresponding pH range is about 82-95%, indicating high dissociation degree. That is, n-butanoic acid or acetic acid is present mainly in the acidity range butyrate or acetate, while carbon dioxide is mainly present in the form of non-ionized carbonic acid (H ₂ CO ₃ ) rather than bicarbonate ions or carbonate ions. do. Therefore, in order to acidify butyrate or acetate using carbon dioxide or carbonic acid to obtain an organic acid form, it is necessary to continuously separate and remove n-butanoic acid and acetic acid which are converted to the acid form from the process medium. Therefore, in the present invention, the converted organic acid is repeatedly extracted and separated by using an organic solvent.

The organic solvent used in the present invention is tri (C ₃ ~ C ₁₂ ) alkylamine; And low boiling point organic solvents; One or two selected from among them can be used in combination. In addition, the tri (C ₃ ~ C ₁₂ alkyl) amine is preferably used one or two or more selected from tripentylamine, trihexylamine, trioctylamine and tridecylamine, the low boiling point organic solvent Preference is given to using one or two or more selected from dichloromethane, chloroform and benzene. As an organic solvent, the use of tri (C ₃ -C ₁₂ ) alkylamines or low-boiling organic solvents in combination with each other, rather than discontinuous use, significantly increases the extraction efficiency even when the concentration of n-butanoic acid is low. You can.

Step 3 is a process for separating and purifying n-butanoic acid from an organic phase containing n-butanoic acid, and the separation and purification may be performed using a method used in the art, but is not particularly limited in the present invention, but the vacuum distillation method Preference is given to using. In addition, the tri (C ₃ ~ C ₁₂ ) alkylamine or the low boiling point organic solvent may be recovered in three steps, and returned and reused in two steps.

In Figures 1 to 4 A shows a schematic diagram of the manufacturing process of n-butanoic acid according to the present invention as a specific example, in Figures 1 to 4 B specific method for realizing the schematic diagram of A using a chemical apparatus Illustrated by way of example.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited by the following examples.

Example 1

(1) fermentation

Bacteria Clostridium tyrobutyricum ATCC 25755 cells were inoculated with glucose as a carbohydrate (nutrition), and the fermentation tank was formed in an anaerobic atmosphere while flowing 100 ml of nitrogen gas per minute. In addition, pH = 6.0 was maintained using 14% ammonia water to adjust the acidity of the fermentor, and stirred at a speed of 200 rpm at a temperature of 37 ° C. The seed culture after inoculation was 100 ml, and the glucose concentration at this time was 50 g / L. To add depleted glucose, 200 ml of 500 g / L of glucose solution were added over a total of four times (once every 17 hours, 28 hours, 41 hours and 48 hours). And fermentation time was a total of 72 hours.

The fermentation broth was centrifuged and ultrafiltration separated to obtain analytical samples. The analytical samples were quantitatively analyzed using a high speed liquid chromatography (HPLC) analysis system in which an ultraviolet (UV) detector and a refractive index (RI) detector were connected in series. . Ammonium butyrate and ammonium acetate were quantified using an ultraviolet detector and glucose was quantified using a refractive index detector. In addition, an OA-1000 organic acid column was used as a separation column, and a mobile phase used an aqueous 0.01 N-H 2 SO 4 solution.

As a result, a fermentation broth containing 56.1 g / L ammonium butyrate and 14.1 g / L ammonium acetate and 19.5 g / L glucose was obtained, and the fermentation yield of glucose was about 14 (w / w)%. It was.

(2) electrodialysis separation

A two-stage (two stage) electrodialysis separator of laboratory scale was used to carry out electrodialysis simultaneously with a fermenter to separate and concentrate an aqueous butyrate salt solution. As a result of electrodialysis of one stage, the concentrations of ammonium butyrate and ammonium acetate decreased to 0 within the measurement limits within one hour at one stage (retentate side) of the membrane, and separation of two stages (permeate side) began. It was isolated at a concentration similar to the previous concentration (56 g / L ammonium butyrate, 14 g / L ammonium acetate). However, in the case of glucose, after the separation, the concentration on the first side decreased to about 15 g / L, and the concentration on the second side increased to about 4.5 g / L, which caused the ion exchange membrane under the electrodialysis separation condition, not the electrical separation. It is due to the effect of the movement of water through. In the case of two-stage electrodialysis, the water circulating in the two-stage side was reduced to half the amount of one-stage side, and within 60 minutes both butyrate and acetate migrated to the two-stage side and concentrated to about twice the initial concentration. In addition, in the case of glucose, it was confirmed that the concentration on the two-stage side under the separation conditions was about 0.1 g / L or less diffused.

(3) Acidification and Preparation of n-butanoic Acid

Trioctylamine was added to the concentrated butyrate salt solution at room temperature, followed by injecting carbon dioxide, and then pressurized and stirred at about 10 bar to perform acidification. Next, the mixture was stagnant to extract an organic phase including n-butanoic acid from the lower organic layer. The extracted organic phase containing n-butanoic acid was subjected to vacuum distillation in a batch while controlling a vacuum degree of 250 mmHg in a 20-stage Sieve Tray distillation column. As a result, n-butanoic acid with 98% purity was obtained on the column. Then, the organic solvent recovered from the column bottom was reextracted, and the above process was repeated three times. As a result, the final recovery rate of n-butanoic acid reached 81%.

Example 2

(1) Fermentation and Electrodialysis Separation

Fermentation and electrodialysis were performed in the same manner as in Example 1. However, the fermentation conditions were maintained at an acidity of pH = 6.4, and the electrodialysis was performed intermittently. As a result, the fermentation yield of glucose to ammonium butyrate reached about 19 (w / w)%.

(2) Acidification and Preparation of n-butanoic Acid

The acidification reaction was performed in the same manner as in Example 1, but an organic solvent in which trioctylamine and dichloromethane were mixed at a volume ratio of 90:10 was used. As a result, the purity of n-butanoic acid was 96% and the final recovery of n-butanoic acid was 83%.

Example 3

In the same manner as in Example 2, only dichloromethane was used as the organic solvent. As a result, the purity of n-butanoic acid was 98% and the final recovery of n-butanoic acid was 82%.

Through the above examples, it was confirmed that the butyrate salt can be obtained in high yield by operating the electrodialysis separator simultaneously or intermittently with the fermentation tank, and as a result, it was confirmed that high purity biobutanoic acid can be obtained in high yield.

1 to 4 shows a specific example of a schematic diagram of a method for producing biobutanoic acid according to the present invention, and B of FIGS. 1 to 4 shows a schematic view of A as a chemical apparatus.

<Explanation of symbols for the main parts of the drawings>

       ROF: Organic carbon source returned for recycling from electrodialysis to fermenter

       AQ: aqueous phase ORG: organic phase

       CD: Carbon Dioxide AA: Acetic Acid

       BA: n-butanoic acid MC: low boiling organic solvent

       TAA: Trialkylamine

Claims (13)

A step of separating and concentrating an aqueous butyrate salt solution from the fermentation broth produced by fermenting the carbohydrate by electrodialysis; Acidifying the concentrated butyrate salt aqueous solution with at least one selected from carbon dioxide, carbonic acid, and an inorganic acid, and simultaneously extracting an organic phase including n-butanoic acid using an organic solvent; And Separating and purifying n-butanoic acid from the organic phase including n-butanoic acid; Biobutanoic acid production method characterized in that consisting of. The method of claim 1, wherein the electrodialysis separation method is characterized by using an electrodialysis separation device composed of two or three stages of separation stages. According to claim 1, wherein the fermentation broth is Clostridium tyrobutyricum (Clostridium tyrobutyricum), Clostridium butyricum, Clostridium acetobutyricum (Clostridium acetobutyricum), Clostridium baizerinkai beijerinckii), Clostridium populeti (Clostridium populeti) and Clostridium thermobutyricum (Clostridium thermobutyricum), characterized in that the carbohydrate is fermented with one or two or more species of the genus Clostitridium. The method according to claim 1, wherein the fermentation is performed at a pH of 5.8 to 6.5 and 35 to 40 ° C. The method of claim 4, wherein the pH is controlled by ammonium salt or calcium salt buffer. The process according to claim 1, wherein the step of electrodialysis is carried out continuously or intermittently simultaneously with the fermentation, and the unfermented hydrocarbon separated from the fermentation broth is reused. The method of claim 1, wherein the organic solvent Tri (C ₃ -C ₁₂ ) alkylamines; And One or two or more low boiling organic solvents selected from dichloromethane, chloroform and benzene; Production method characterized in that one or two selected from. 8. The method according to claim 7, wherein the tri (C ₃ -C ₁₂ alkyl) amine is at least one selected from tripentylamine, trihexylamine, trioctylamine and tridecylamine. The method of claim 1, wherein some or all of the carbon dioxide is produced when the hydrocarbon is fermented in one step. The method of claim 1, wherein the separation and purification using a vacuum distillation method. The method of claim 1, wherein the organic solvent is recovered from the organic phase of the three steps and reused in the second step. The method according to claim 1, wherein the butyrate salt comprises at least one selected from ammonium butyrate and calcium butyrate. delete

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