KR20190122995A - Composition for microorganism fermentation comprising glycerol and method for producing butyric acid using the same - Google Patents

Abstract

The present specification provides a saccharification solution for microorganism fermentation comprising a carbon source and glycerol, wherein the carbon source includes one or more of glucose, pyruvic acid, acetic acid and xylose; and a method for producing butyric acid using the same. The saccharification solution can be used to produce butyric acid in a very good yield.

Description

Composition for microbial fermentation containing glycerol and a method for producing butyric acid using the same {COMPOSITION FOR MICROORGANISM FERMENTATION COMPRISING GLYCEROL AND METHOD FOR PRODUCING BUTYRIC ACID USING THE SAME}

The present specification describes a composition for microbial fermentation comprising glycerol and a method for producing butyric acid using the same.

In order to solve environmental pollution, greenhouse gas generation and exhaustion of petroleum resources due to the use of fossil fuels, research on the production of biofuels using biomass that can replace petroleum resources is being actively conducted. However, in Korea, due to limited arable land and limited resources, supply and demand of biomass is not easy, and if it is desired to produce biofuels and biochemicals using waste resources that are discarded, pretreatment and conversion to sugars that microorganisms can use As the saccharification process is difficult, it is necessary to obtain the maximum target product compared to the input biomass.

Biofuels and biochemicals are products obtained through fermentation of microorganisms, and as the microorganisms have various metabolic pathways, it is difficult to obtain a desired product in high yield. For example, Clostridium tyrobutyricum (CClostridium tyrobutyricum ATCC 25755) is a microorganism that is being studied a lot to produce butyric acid, but using a carbon source to produce not only the target butyric acid but also acetic acid (Fig. 1). Thus, in order to produce butyric acid in high yield, it is necessary to limit the metabolic flow of carbon to acetic acid.

Republic of Korea Patent Publication No. 10-0785997 Republic of Korea Patent Publication No. 10-1148255

In one aspect, the problem to be solved by the present invention is to provide a method for producing butyric acid in a high yield including glycerol in the composition for microbial fermentation.

In another aspect, the problem to be solved by the present invention is to provide a method for consuming glycerol by supplying a glycerol that can not be consumed as a sole carbon source in the production of butyric acid of microorganisms together with a fermentable carbon source and acetic acid (or acetate) will be.

In another aspect, the problem to be solved by the present invention is to increase the yield of butyric acid through the consumption of glycerol, fermentable carbon source and acetic acid (or acetate) as described above, and to convert the biodiesel byproduct glycerol to butyric acid To provide a way. In another aspect, the problem to be solved by the present invention is to provide a method for increasing the yield of butyric acid by increasing the NADH production in the process for fermentation of microorganisms.

In another aspect, the problem to be solved by the present invention is to use not only sugars as fermentable carbon sources included in the composition for fermentation of microorganisms but also acetic acid (or acetate) known as an inhibitor as an electron acceptor. It is to provide a method that can be converted to butyric acid.

In another aspect, the problem to be solved by the present invention is to provide a method for reducing or consuming all of the acetic acid produced as a byproduct in the production of butyric acid by fermentation of microorganisms.

In one aspect, the present invention provides a composition for fermentation of microorganisms comprising a fermentable carbon source and glycerol, wherein the fermentable carbon source comprises glucose.

In another aspect, the present invention provides a method for producing butyric acid, comprising culturing by adding glycerol and a fermentable carbon source to a medium containing Clostridium tyrobutyricum.

In one aspect, the present invention can provide a method for producing butyric acid in a high yield, including glycerol in the composition for microbial fermentation.

In another aspect, the problem to be solved by the present invention is to provide a method for consuming glycerol by supplying glycerol which cannot be consumed as the sole carbon source in the production of butyric acid of microorganisms together with the fermentable carbon source and acetic acid (or acetate). Can be.

In another aspect, the present invention is to provide a method for increasing the yield of butyric acid through the consumption of glycerol, fermentable carbon source and acetic acid (or acetate) as described above, and convert glycerol, a by-product of biodiesel, to butyric acid. .

In another aspect, the present invention may provide a method for increasing the yield of butyric acid by increasing the NADH production in the process for fermentation of microorganisms.

In another aspect, the present invention is not only a sugar as a fermentable carbon source contained in the microbial fermentation composition used for fermentation of microorganisms but also an acetic acid (or acetate) known as a fermentation inhibitor as an electron acceptor to convert into butyric acid Can produce biofuels and chemicals.

In another aspect, the present invention can use acetic acid contained in the saccharification liquid used for fermentation of microorganisms as an external electron acceptor, thereby alleviating the toxicity in the saccharification liquid at the same time as the production of biofuels and chemicals.

In another aspect, the present invention may provide a method for reducing the production of acetic acid as a by-product or producing no at all in the production of butyric acid by fermentation of microorganisms.

1 is a diagram illustrating the metabolic pathway of Clostridium tyrobutyricum.
2 is a graph showing the organic acid production of Clostridium tyrobutyricum when glucose is used as a single carbon source in a medium without acetic acid.
Figure 3a is a graph showing the fermentation pattern of Clostridium tyrobutyricum when glucose is used as a single carbon source in the medium added with acetic acid.
Figure 3b is a graph showing the fermentation pattern of Clostridium tyrobutyricum when using glycerol as a single carbon source in the medium added with acetic acid.
4A is a graph showing the fermentation pattern of Clostridium tyrobutyricum when acetic acid is added when glucose and glycerol are simultaneously supplied to the medium as a carbon source.
Figure 4b is a graph showing the fermentation pattern of Clostridium tyrobutyricum when acetic acid is not added when glucose and glycerol are fed to the medium as a carbon source at the same time.
5a is a graph showing fermentation patterns of Clostridium tyrobutyricum when glucose and glycerol were used as the carbon source in a ratio of 7.5: 2.5 in an acetic acid-added medium.
Figure 5b is a graph showing the fermentation pattern of Clostridium tyrobutyricum when used as a carbon source in a 5: 5 ratio of glucose and glycerol in acetic acid added medium.
5C is a graph showing fermentation patterns of Clostridium tyrobutyricum when glucose and glycerol were used as carbon sources in a ratio of 2.5: 7.5 in acetic acid-added medium.
FIG. 6A is a graph showing butyric acid production of Clostridium tyrobutyricum in a medium containing about 3: 1 glucose and glycerol upon addition of 0 g / L acetic acid.
6B is a graph showing the butyric acid production of Clostridium tyrobutyricum in a medium containing about 3: 1 glucose and glycerol when 0.25 g / L acetic acid is added.
FIG. 6C is a graph showing butyric acid production of Clostridium tyrobutyricum in a medium containing about 3: 1 glucose and glycerol upon addition of 0.5 g / L acetic acid.
FIG. 6D is a graph showing the butyric acid production of Clostridium tyrobutyricum in a medium containing about 3: 1 glucose and glycerol upon addition of 1 g / L acetic acid.
FIG. 6E is a graph showing butyric acid production of Clostridium tyrobutyricum in a medium containing glucose and glycerol at about 3: 1 when 2 g / L acetic acid is added.
Figure 7 shows the calibration curve for the appropriate concentration of glycerol addition based on the acetic acid consumption concentration.
Figure 8a shows the butyric acid production pattern in the medium added 0g / L waste glycerol to the waste wood saccharification solution containing acetic acid.
Figure 8b shows the butyric acid production pattern in the medium in which 1.9 g / L of waste glycerol was added to the waste wood saccharification solution containing acetic acid.
Figure 8c shows the butyric acid production pattern in the medium of 2.3g / L waste glycerol to the waste wood saccharification solution containing acetic acid.
FIG. 8D shows a butyric acid production pattern in a medium in which 2.8 g / L of waste glycerol was added to a waste wood saccharification solution containing acetic acid.
FIG. 8E shows the butyric acid production pattern in a medium in which 3.7 g / L of waste glycerol was added to a waste wood saccharification solution containing acetic acid.
Figure 8f shows the butyric acid production pattern in a medium in which 5.4 g / L of waste glycerol was added to a waste wood saccharification solution containing acetic acid.
Figure 9 is a graph comparing the butyric acid production yield between the experimental group added waste glycerol to the model saccharification liquid (synthetic medium), waste wood saccharification liquid, waste wood saccharification liquid.
10a is a graph showing the growth of pH and Clostridium tyrobutyricum according to the culture time when no waste glycerol is added in butyric acid production using a model saccharification solution (synthetic medium) and a fermentation tank.
Figure 10b is a graph showing the concentration of glucose and xylose according to the incubation time in the absence of waste glycerol in the production of butyric acid using the model saccharification solution (synthetic medium) and the fermentation tank.
10c is a graph showing the concentrations of acetic acid and butyric acid according to the culture time when no waste glycerol is added in butyric acid production using a model saccharification solution (synthetic medium) and a fermentation tank.
FIG. 11A is a graph showing the growth of pH and Clostridium tyrobutyricum according to the culture time when waste glycerol is added in butyric acid production using a model glycosylated solution (synthetic medium) and a fermentor.
Figure 11b is a graph showing the concentration of glucose and xylose according to the culture time when the waste glycerol in the butyric acid production using the model saccharification solution (synthetic medium) and the fermentation tank.
11c is a graph showing the concentrations of acetic acid and butyric acid according to the culture time when waste glycerol is added in butyric acid production using a model saccharification solution (synthetic medium) and a fermentor.

Hereinafter, with reference to the accompanying drawings, it will be described embodiments of the present application in more detail. However, the technology disclosed in the present application is not limited to the embodiments described herein and may be embodied in other forms. It is merely to be understood that the embodiments introduced herein are provided so that the disclosure can be made thorough and complete, and the spirit of the present application to those skilled in the art will be fully communicated. In order to clearly express each component in the drawings, the size, such as the width or thickness of the component, is shown to be somewhat enlarged. In addition, although only a part of the components are shown for convenience of description, those skilled in the art will be able to easily understand the rest of the components. In addition, one of ordinary skill in the art may implement the spirit of the present application in various other forms without departing from the technical spirit of the present application.

In the present specification, "biomass" refers to organisms such as land plants, algae, and microalgae used as energy sources, and "biochemical" refers to pyrolysis / chemistry of the biomass. All chemicals that can be obtained by decomposition or fermentation. In addition, the term "biofuel" means a fuel obtained from the biomass, the broadest meaning including not only bioalcohol and bio oil but also metabolites that can be converted into fuel through chemical biological technology. to be.

Anaerobic fermentation microorganisms glycolysis for the production of ATP required for their growth, and removes the reducing power generated through fermentation. Clostridium tyrobutyricum (Clostridium tyrobutyricum) is an anaerobic fermentation microorganism that breaks down the glucose contained in the saccharified solution through glycolysis, and performs butyric acid fermentation using the reducing power produced by producing ATP. As shown in FIG. 1, the approximate metabolic pathways of butyric acid produced by Clostridium tyrobutyricum are converted into two molecules of pyruvate and two molecules of NADH. Is generated. Two molecules of NADH are used when two molecules of pyruvate are converted to butyric acid. Therefore, the present inventors further supply a carbon source in a reduced form than glucose and selectivity of butyric acid by using reducing power (NADH) generated when Clostridium tyrobutyricum metabolizes a high-reducing carbon source through glycolysis. ) To increase.

Glycerol is a highly reducing carbon source whose chemical structure is C3H8O3. The production rate of glycerol as a by-product of biodiesel production is quite high, at 10%. Since the production of biodiesel continues to increase due to depletion of petroleum resources and rising oil prices, the production of glycerol is also increasing. Therefore, using such glycerol as a carbon source with high reducing power may be advantageous to the environment and economics.

According to one embodiment of the present invention, it is possible to provide a composition for fermentation of microorganisms which can use glycerol as a carbon source.

The composition may allow glycerol, which cannot be used alone as a carbon source, in butyric acid production by microbial fermentation to be used for butyric acid production.

The composition may comprise a fermentable carbon source comprising glucose and glycerol.

The fermentable carbon source may include, without limitation, if Clostridium tyrobutyricum can be fermented to produce butyric acid, for example, it may include glucose (glucose).

The fermentable carbon source may be, in addition to glucose, monosaccharides such as galactose, mannose, mannose, rhamnose, fructose, xylose, arabinose and the like; Disaccharides such as cellobiose; And polysaccharides such as mannitol, alginic acid, laminaran and the like; It may further comprise one or more sugars selected from.

The composition may comprise acetic acid or acetate. "Acetic acid" is used herein to include acetic acid salt in a form that Clostridium tyrobutyricum can consume as acetic acid. The acetic acid may be converted to butyric acid by using a reducing power, that is, NADH generated during the fermentation process of the microorganism by acting as an external electron acceptor.

The acetic acid may be added separately to the composition, or may be in the form contained in the biomass saccharification liquid, and may be produced as a by-product in the butyric acid production process of Clostridium tyrobutyricum.

Comparing the glycolytic process between glycerol and glucose, two molecules of pyruvate produce two molecules of NADH for glucose while four times that of glycerol for four molecules of NADH. Therefore, Clostridium tyrobutyricum selectively produces NADH-consuming butyric acid to eliminate the high reducing force that occurs when glycolysis breaks down glycerol. In addition, glucose and xylose, as well as a large amount of acetic acid are present in the lignocellulosic biomass (wood, herbaceous) saccharified solution, which consumes glycerol to produce butyric acid and converts the remaining reducing power to acetic acid butyric acid. Can be removed

In one example, the fermentable carbon source may comprise a biomass saccharified liquid. The biomass saccharification liquid is a saccharification process in order to make biomass into a saccharide that microorganisms can use.

The biomass may include one or more of lignocellulosic biomass (woody, herbaceous, etc.) and seaweed biomass.

The lignocellulosic biomass (wood-based, herbaceous, etc.) generally includes lignocellulose, which is a complex composed of cellulose, hemicellulose, lignin, and the like. For example, the lignocellulosic biomass may contain 33 to 51% by weight of cellulose, 19 to 34% by weight of hemicellulose, 21 to 32% by weight of lignin, 0 to 2% by weight of ash, and other components. However, it is not limited thereto. The lignocellulosic biomass is not limited so long as it is a biological material including lignocellulosic, and may include, for example, wood-based, herbaceous, and the like, and is not limited to conifers and hardwoods, trees, and the like. For example, softwood from trees, softwood chips, slash or hog fuel from processing of softwood wood, forest residues. Straws such as rice straw, chaff, cereals, grasses, corn, corn husks, weeds, aquatic plants, hay and the like.

The lignocellulosic biomass saccharified solution means a product in which lignocellulosic biomass is degraded through a saccharification process. The saccharified solution may be prepared further including pretreatment before saccharification. The lignocellulosic biomass saccharified solution is, for example, glucose, galactose, mannose, rhamnose, xylose, and the like, in which the cellulose and the hemicellulose component are hydrolyzed. It may comprise pentose or hexose such as arabinose. In one example, the saccharified solution may further include a furan-based compound such as furan, hydroxymethylfurfural (HMF), furfural, acetic acid, and the like, which are generated due to sugar peroxidation. The lignin component of lignocellulosic biomass is hydrolyzed when ferulic acid, coumaric acid, benzoic acid, syringic acid, vanilic acid, (phenol), 4-hydroxybenzoic acid (4-hydroxybenzoic acid), 4-hydroxybenzaldehyde (4-hydroxybenzaldehyde), may be produced phenolic compounds such as syringaldehyde (syringaldehyde), the saccharified liquid is such It may further include a phenolic compound. In one embodiment, the lignocellulosic biomass saccharified solution is composed of glucose, galactose, mannose, rhamnose, xylose, and arabinose as glucose. One or more monosaccharides selected from the group; And disaccharide, cellobiose; It may include one or more selected from, but is not limited thereto.

The algae biomass includes cellulose (glucan) and may include polysaccharides such as alginic acid and laminaran and mannitol. Mannitol is a reduced form of sugar compared to glucose, which produces two reducing substances, NADH, which produces three NADHs per molecule when converted to pyruvic acid through glycolysis. It can be helpful if it can be used for production. In addition, since there is no lignin structurally, glycosylation is relatively easy, and since high toxic phenolic compounds derived from lignin are not present in the saccharified liquid, the toxicity may be lower than that of lignocellulosic biomass saccharified liquid.

The algae biomass includes, for example, red algae, brown algae, green algae, and the like, but is not limited thereto, and includes all obtained by directly harvesting or farming the ocean. Brown algae, which is known to have the highest yield in Korea, can produce large amounts of mannitol during saccharification, and seaweed (Laminaria japonica), Seaweed (Undaria pinnatifida), Hizikiafusiforme, Analipus japonicus, and Cordaria flagelliformis. , Ishige okamurai, Scytosiphon lomentaria, Seaweed (Endarachne binghamiae), Ecklonia cava, Ecklonia stolonifera, Eisenia bicyclis, Seaweed (Costaria costata), Sargasum fulvellum For example, Sargassum horneri, Sargassum thunbergii. The red alga is, for example, Gelidium amansii, Bangia atropurpurea, Porphyra suborbiculata, Radial laver (Porphyra yezoensis), Galaxala falcate, Scillaia japonica, Gelidium divaricatum, Gelidium pacificum, Lithophylum okamurae, Lithothammion cystocarpideum, Amphiroa anceps, Amphiroa beauvoisii (Coral of the lake) Corallina pilulifera, Marginisporum aberrans, Carpopeltis prolifera, Grateloupia filicina, Grateloupia elliptica, Grateloupia lanceolanta, Slippery silkworm Grateloupia turtuturu, Phacelocarpus japonicus, Glolopeltis furcata, Hypnea charoides, Hypnea japonitca, Hypnea saidu, Hypnea saidana Chondrus cripspus, Chondracanthus tenellus, Graotaria textorii, Lomentaria catenata, Hetrosiphonia japonica, Chondria crassicaulis, Cham-purple sacchara, Sylatius claa dia Seaweed (Porphyra yezoensis Ueda), Cotoni (Cottonii), Gambling (Grateloupia lanceolata), Prairie dog (Pterocladia tenuis), Acanthopeltis japonica, Gluiopeltis tenax, Irish moss, Gambling (Pachyptiopsi) ), Ceramium kondoi, Ceramium boydenii, Gigartina tenella, and Cactylaephora hypnaeoides. The green alga is, for example, Enteromorpha, Ulva lactuca, Spirogyra spp., Codium fragile, Codium minus, Caulerpa okamurai, Stone cloth (Nostoc commune) Etc.

The algae biomass saccharification liquid refers to a product in which algae biomass is degraded through a saccharification process. The saccharified solution may be prepared further including pretreatment before saccharification. The algae biomass saccharified solution may include, for example, one or more selected from the group consisting of mannitol, glucose, galactose, xylose and arabinose, mannitol, alginic acid, and laminaran. However, it is not limited thereto.

In one embodiment, the microorganism may be Clostridium tyrobutyricum. For example, the microorganism may be Clostridium tyrobutyricum ATCC 25755. The Clostridium tyrobutyricum can produce butyric acid from a carbon source.

In view of further improving the yield of butyric acid, the glycerol may include a weight ratio of 1:99 to 99: 1 of glucose to glycerol relative to glucose in the composition. For example, the weight ratio may be 1: 1 to 10: 1, for example, 3: 1.

In view of further improving the yield of butyric acid, the weight ratio of acetic acid and glycerol may be included in the range of 1:99 to 99: 1, for example, 1:10 to 1: 1, for example, 1: 2. have.

According to one embodiment of the present invention, it is possible to provide a method for enhancing butyric acid production of Clostridium tyrobutyricum using glycerol.

According to one embodiment of the present invention, it is possible to provide a method for producing butyric acid. The method may include culturing by adding glycerol to a medium containing Clostridium tyrobutyricum.

The culturing can be carried out without limitation as long as conditions for growth and butyric acid production of Clostridium tyrobutyricum (Clostridium tyrobutyricum) is possible. Strain growth conditions of Clostridium tyrobutyricum can be carried out as known in the art. In one embodiment the culturing may be carried out in anaerobic conditions. In one example, the culturing may be performed at 20 to 50 ° C., pH 4 to 7, for example, at 20 to 40 ° C., pH 4 to 7.

The medium is omitted for the fermentable carbon source contained in the microorganism fermentation composition according to an embodiment of the present invention described above.

In the culture of Clostridium tyrobutyricum, the glycerol may be added in the presence of acetic acid or acetate in the medium.

The acetic acid or acetate may be added to the medium separately, or included in the biomass saccharification liquid included as the fermentable carbon source, or as a by-product of the butyric acid production of Clostridium tyrobutyricum. May be produced.

The process uses acetic acid or acetate, a by-product from the butyric acid production of Clostridium tyrobutyricum, in production of butyric acid, which in turn reduces or eliminates the production of acetic acid as a byproduct in butyric acid production. Can be.

Clostridium tyrobutyricum is impossible to grow and produce butyric acid when using glycerol as the sole carbon source, glycerol, fermentable carbon source and a fermentable carbon source and acetic acid or acetic acid salt in accordance with an embodiment of the present invention and Both butyric acid or acetate can be consumed to produce butyric acid.

Thus, in the butyric acid production method, the glycerol, fermentable carbon source, acetic acid or acetate in culture can maintain the condition that all three are present to improve the yield of butyric acid. For example, if there is a component that is exhausted of the three components may include additionally adding the component.

In one example, in view of further improving the yield of butyric acid, the medium may include glucose and glycerol in a weight ratio of glucose: glycerol in a range of 1:99 to 99: 1. For example, the weight ratio may be 1: 1 to 10: 1, for example, 3: 1.

In one example, in view of further improving the yield of butyric acid, the medium may include acetic acid and glycerol in a weight ratio of 1:99 to 99: 1, for example 1:10 to 1: 1. For example 1: 2.

The method allows the fermentation metabolic flow of Clostridium tyrobutyricum to selectively flow to butyric acid, not acetic acid, due to the high reducing power of glycerol to reduce acetic acid production. In addition, acetic acid produced as a by-product is converted back to butyric acid due to high reducing power, and as a result, production can be reduced or eliminated.

Hereinafter, the present invention will be described in detail with reference to Examples, Comparative Examples and Test Examples. These are only presented by way of example only to more specifically describe the present invention, it is to those skilled in the art that the scope of the present invention is not limited by these Examples, Comparative Examples and Test Examples. Will be self explanatory.

[Experimental Conditions and Analysis Methods]

In the following experiment was carried out according to the following strains and experimental conditions.

(1) use strain

Clostridium tyrobutyricum ATCC 25755 was used as a microorganism to proceed with fermentation.

(2) medium composition and culture conditions

The basal medium used for strain culture was 5 g yeast extract, 0.2 g magnesium sulfate, 0.01 g manganese sulfate, 0.01 g iron sulfate, 0.01 g sodium chloride, 0.5 g potassium phosphate (KH2PO4) per liter of total volume. A medium containing 0.5 g of dibasic potassium phosphate (K 2 HPO 4) and 2.2 g of ammonium acetate was used. For the medium without acetic acid, 2.2 g of ammonium acetate was removed and 2.2 g of ammonium sulfate was added. In addition, the rapid decrease in pH due to the production of acetic acid and butyric acid in the culture inhibits the growth of microorganisms, so 2- (N-morholino) ethanesulfonic acid (MES) buffer 100 mM was added. The initial pH of the medium was adjusted to 6.5 with 4 N potassium hydroxide (KOH). In the case of batch culture, 20 mL of medium was added to a 60 mL serum bottle, oxygen was removed with argon gas, sealed with a rubber stopper and aluminum crimp seal, and sterilized to make anaerobic conditions. Thereafter, 10% inoculation of the microorganisms incubated in the base medium for 24 hours and strain growth and metabolite analysis were performed while culturing at a temperature of 37 ° C. and a rotational speed of 200 rpm in a shaking incubator.

(3) analysis method

As the incubation progressed, the concentrations of sugar, glycerol, acetic acid and butyric acid were measured using an Agilent model 1200 liquid chromatograph. Microbial growth was determined at 600 nm with a spectrophotometer (UVmini-1240, SHIMAZU). Absorbance was measured.

Test Example 1 Organic Acid Production of Microorganisms Using Glucose as a Carbon Source in a Medium Without Acetic Acid

Clostridium tyrobutyricum ATCC 25755 (Clostridium tyrobutyricum ATCC 25755) was cultured by adding 10 g / L of glucose to the basal medium without acetic acid and observed the growth of the strain, the results are shown in FIG.

In the results of FIG. 2, when about 10 g / L of glucose was consumed, 0.77 g / L of acetic acid and 3.39 g / L of butyric acid were produced, and the production ratio of acetic acid and butyric acid was 1: 4.4 among the total organic acids. The proportion of acetic acid was found to be quite high, about 20%. Butyric acid yield was 0.35 g / g glucose based sugar.

Test Example 2 Comparison of Fermentation Patterns of Microorganisms Using Glucose and Glycerol as Carbon Sources in Acetic Acid-Added Medium

Glycerol is a highly reduced form of carbon source and most absolute anaerobic strains do not use it as a single carbon source. Thus, the following experiment was conducted to determine whether Clostridium tyrobutyricum uses glycerol as a single carbon source.

Clostridium tyrobutyricum ATCC 25755 (Clostridium tyrobutyricum ATCC 25755) was incubated in a medium in which 10 g / L glucose and 10 g / L glucose were added to the medium to which acetic acid was added, respectively, and the growth and metabolites of the strains were compared. The results are shown in FIGS. 3A and 3B.

In the results of FIGS. 3A and 3B, when only glucose was added, 9.65 g / L of glucose was consumed in 14 hours to produce 0.77 g / L of acetic acid and 3.39 g / L of butyric acid, and the yield of butyric acid was 0.35 g / g. (FIG. 3A). However, when glycerol was added as a single carbon source, the growth and butyric acid production of the strains were very small (Fig. 3b). This is a result of Clostridium tyrobutyricum not using glycerol as a single carbon source, which can not remove high reducing power when metabolized.

[Test Example 3] Comparison of fermentation pattern of microorganisms depending on the presence or absence of acetic acid when glucose and glycerol were fed to the medium as a carbon source

As confirmed in Test Example 2, Clostridium tyrobutyricum cannot use glycerol as a single carbon source. Thus, this experiment was carried out to check whether glycerol is consumed when glycerol and glucose are simultaneously supplied to the medium. Clostridium tyrobutyricum ATCC 25755 (Clostridium tyrobutyricum ATCC 25755) was incubated in a medium containing 15 g / L of glucose and 3 g / L of glycerol in the basal medium containing acetic acid. The results are shown in FIG. 4A.

In the results of FIG. 4A, 8.17 g / L butyric acid was produced using both 15.18 g / L glucose and 2.64 g / L glycerol in 18 hours with acetic acid. Glycerol was consumed within 12 hours, and added acetic acid was consumed up to 12 hours using glycerol. In addition, it was confirmed that after 12 hours of exhaustion of glycerol, acetic acid was produced again. Glycerol consumption is an unexpected result, especially when acetic acid is not produced and consumed while glycerol is consumed. The butyric acid production yield is 0.538 g / g glucose, which is higher than the theoretical butyric acid production yield of 0.489 g / g glucose, from which it can be inferred that glycerol and acetic acid are consumed to increase butyric acid production.

From the results of FIG. 4a, it was considered that glycerol consumption and acetic acid consumption were related, and thus culture was performed in a medium without acetic acid. The results are shown in Figure 4b. No additional acetic acid was added to the medium, but a small amount of acetic acid was transferred from the seed culture at seeding but very little (0.22 g / L or less). When acetic acid was not added, only 6.28 g / L and 1.5 g / L of glycerol were used until 48 hours of fermentation, and it was confirmed that only 2.35 g / L of butyric acid was produced (FIG. 4B). The concentration of acetic acid does not increase or decrease significantly compared to the concentration produced at the beginning. Therefore, the production of butyric acid using glucose, together with the production of acetic acid and the large amount of NADH generated during the glycerol process, It is assumed that it is used to convert acetic acid to butyric acid and removed.

From the above results, it was confirmed that in order to simultaneously use glucose and glycerol as a carbon source, an external electron acceptor is required to remove the reducing power generated during metabolism.

Test Example 4 Comparison of Fermentation Patterns of Microorganisms According to Glucose and Glycerol Concentration Ratios in Acetate Added Medium

As confirmed in Test Example 3, when Clostridium tyrobutyricum ATCC 25755 (Glostridium tyrobutyricum ATCC 25755) supplies glucose and glycerol as a carbon source to the medium at the same time, it can be seen that acetic acid is required to consume glycerol. . Thus, the total concentration of glucose and glycerol in the basal medium was about 10 g / L, and cultured in a medium added at various ratios (glucose: glycerol = 7.5: 2.5, 5: 5, 2.5: 7.5) and growth of the strain was performed. Was compared. The results are shown in FIGS. 5A-5C.

In the results of FIG. 5A, all the carbon sources (7.31 g / L glucose and 2.32 g / L glycerol) were used for the experimental group to which glucose and glycerol were added in a ratio of 7.5: 2.5, and 4.72 g / L butyric acid. Was produced (FIG. 5A). It was observed that 0.95 g / L acetic acid was consumed to remove NADH occurring during the corresponding process of glycerol and converted to butyric acid to increase sugar-based butyric acid yield (yield 0.64 g / g glucose). This is higher than 0.35 g / g glucose, the yield in Test Example 1, and 0.489 g / g, the theoretical yield of glucose based butyric acid production. It can be seen that butyric acid production increased by consuming glycerol and acetic acid. In the experimental group in which glucose and glycerol were added at a ratio of 5: 5, the added glucose (4.93 g / L) was consumed (FIG. 5B). However, it was observed that glycerol consumed 2.64 g / L until 11 hours, when all the added glucose was consumed, and then was not used. The yield is 0.89 g / g glucose, higher than 0.489 g / g, the theoretical yield of glucose based butyric acid production. In the experimental group in which glucose and glycerol were added in a ratio of 2.5: 7.5, it can be seen that the use of glycerol stopped from 8 hours when the added glucose (2.64 g / L) was used up (FIG. 5C). Butyric acid produced 2.7 g / L, yield 1.02 g / g glucose.

Clostridium tyrobutyricum requires other carbohydrates, such as glucose, to produce butyric acid using glycerol, and has been found to convert acetic acid to butyric acid using the high reducing power generated when using glycerol.

[Test Example 5] Comparison of microorganism fermentation pattern according to acetic acid concentration in a medium containing about 3: 1

Clostridium tyrobutyricum ATCC 25755 (Clostridium tyrobutyricum ATCC 25755) was subtracted from ammonium acetate in the base medium composition, 2.2 g of ammonium sulfate was added, 7.5 g / L of glucose and 2.5 g / L of glycerol was added. Sodium acetate (CH3COONa) was added and cultured so that the acetic acid concentration was 0, 0.25, 0.5, 1, 2 g / L, respectively, and the fermentation pattern was confirmed.

In the results of Figure 6a, it was confirmed once again that the fermentation does not proceed in the absence of acetic acid (Figure 6a). When 0.25 g / L acetic acid was added, fermentation proceeded smoothly until only 8 hours of acetic acid was exhausted. Butyric acid production was stopped after acetic acid was exhausted (Fig. 6b), 0.5 g / L, 1 g. Similar behavior is also seen when / L acetic acid is added (FIGS. 6C and 6D). When all acetic acid was consumed and glycerol remained, it was confirmed that Clostridium tyrobutyricum rarely used glucose and glycerol. When 2 g / L acetic acid was added, a small amount of acetic acid remained until all glycerol (3.16 g / L) was used, so glucose and glycerol were the highest at 7.35 g / L and 3.16 g / L, and the production of butyric acid was 6.06. g / L confirmed more than other experimental groups (FIG. 6E). The yield is 0.824 g / g glucose, which is higher than the theoretical yield of 0.489 g / g.

Based on the results when the glucose concentration is 7-8 g / L and the ratio of glucose to glycerol is about 3: 1, a reliable value is obtained when a calibration curve for the consumption concentration of glycerol is compared to the consumption concentration of acetic acid (R2 = 0.998) was observed (FIG. 7). Knowing the acetic acid concentration in the medium or saccharification through this calibration curve will help to predict the appropriate amount of glycerol added proportionally.However, if the sugar, glycerol and acetic acid concentrations are outside the ranges of the experimental group of Figs. The ratio is different. In particular, since the acetic acid produced when the sugar concentration is high, the glycerol consumption is higher than the calibration curve of FIG. An example can be seen in Test Example 6 below.

[Test Example 6] Promoting butyric acid production using waste wood saccharification liquid and waste glycerol

Since waste wood saccharified solution contains high concentration of sugar, all sugars are not available through batch cultivation. After diluting 1/4 of the waste wood saccharification process with concentrated sulfuric acid with distilled water, per liter of volume, 5 g yeast extract, 0.2 g magnesium sulfate, 0.01 g manganese sulfate, 0.01 g iron sulfate, 0.01 g Sodium chloride, 0.5 g of potassium phosphate (KH 2 PO 4), 0.5 g of potassium phosphate (K 2 HPO 4), 2.2 g of ammonium sulfate are added and 0, 1.9, 2.3, 2.8, 3.7, 5.4 g / L Pretreated waste glycerol was added and used as a fermentation medium for Clostridium tyrobutyricum ATCC 25755. The initial pH of the medium was adjusted to 6.5 with 4 N potassium hydroxide (KOH). In the case of batch culture, 20 mL of medium was added to a 60 mL serum bottle, and the microorganisms were inoculated, followed by incubation for 24 hours at a temperature of 37 ° C. and a rotational speed of 150 rpm in a shake incubator.

Waste glycerol was mixed in the saccharified solution after pretreatment to remove the substances that inhibit the microbial growth. Specifically, waste glycerol: distilled water is mixed at a volume of 1: 3, adjusted to pH 4 with 2.6 N HCl, and then left to stand. After centrifugation, the supernatant (including fatty acid) containing the inhibitor is discarded and the remaining lower layer is collected. Was used in the experiment as waste glycerol pretreated. Pre-treated waste glycerol was added at various concentrations to confirm the fermentation pattern during fermentation of the waste saccharification solution (FIGS. 8A to 8F).

After adding various concentrations of pretreated waste glycerol, the strain growth (OD600) at 12 hours was OD600 6.39, 6.13, 6.11, 6.47, 6.5 compared to the control group OD600 6.05, respectively. Even if it was observed that the growth of the strain was not inhibited.

As in the waste wood saccharification solution, the acetic acid contained in the saccharification solution also acts as an external electron acceptor, as in the synthetic medium. As a result, the consumption of glucose from 24 hours after the fermentation was due to the lack of acetic acid in the saccharification solution from the addition of 3.7 g / L And it was confirmed that the production of butyric acid does not proceed smoothly (Fig. 8e). At this time, the consumption of acetate was 1.05 g / L and the consumption of glycerol was 3.5 g / L. This is more than 2.12 g / L of glycerol consumption predicted when 1.05 g / L of acetate is consumed in FIG. 9, which is higher than the sugar concentration (7-8 g / L) in FIG. 9 (about 25 g / L) is higher.

When 2.8 g / L spent glycerol was added, it was observed that all glucose contained in saccharified solution was consumed without additional acetic acid production, and 11.19 g using 17.99 g / L glucose and 4.44 g / L xylose. / L of butyric acid was produced (FIG. 8D). The added glycerol was also consumed, and 0.38 g / L of acetic acid contained in saccharified solution was further confirmed.

The experimental group to which only the waste wood saccharified liquid was added, the experimental group to which the waste wood saccharified liquid was added pretreated glycerol of 2.8 g / L, and the model saccharified liquid (80 g of glucose, 30 g of xylose, and 6 g of acetic acid) The yield of butyric acid of the contained saccharified solution) is compared and shown in FIG. 9. Production yield was calculated according to the following formula.

<Equation 1>

Butyric Acid Production Yield = (Butyric Acid Production Weight (g)) / (Waste Glucose + Xylose + Glycerol Weight (g))

In the case of the model saccharification liquid and the waste wood saccharification liquid addition group, the yield was confirmed to be almost the same as 0.36 and 0.37. However, when waste glycerol was added to the saccharified solution, a high yield of butyric acid was obtained at 0.45 g / g.

Test Example 7 Confirmation of Butyric Acid Production Enhancement Effect of Glycerol Using Fermentor

In a model saccharification solution containing 80 g glucose, 30 g xylose, and 6 g acetic acid per liter of P2 medium components (25 g yeast extract per liter, 0.2 g magnesium sulfate, 0.01 g manganese sulfate, 0.01 g Clostridium tyrobuti in a medium containing iron sulfate, 0.01 g sodium chloride, 0.5 g potassium phosphate (KH 2 PO 4), 0.5 g potassium diphosphate (K 2 HPO 4), and 2 g ammonium sulfate Changes in butyric acid production with and without waste glycerol were observed using fermentation medium of Lycum ATCC 25755 (Clostridium tyrobutyricum ATCC 25755). Waste glycerol was added 10g per liter and added when exhausted during the fermentation process.

The pH of the medium was fermented by fixing to 6.0 with 6 N potassium hydroxide (KOH). In the case of batch culture, 1 L of medium was added to a fermenter of 3 L and 5% of the microorganism was inoculated, followed by incubation for 56 hours at a temperature of 37 ° C. and a rotational speed of 200 rpm in a shaker.

The fermenter was used to determine the growth and butyric acid production characteristics of the strain according to the presence / absence of waste glycerol in the model saccharified solution containing high concentration of sugar (Figs. 10a to 10c and 11a to 11c).

In case of strain growth, there was no significant difference between the two experimental groups (FIGS. 10A and 11A), but in the production of butyric acid at 49.9 g / L when glycerol was added (FIG. 11C), no glycerol-added experimental group (35.5 g / L; FIG. 10C) There was an increase in production of 14.4 g / L compared to Calculation of fermentation yield (g butyric acid production g / consumed sugar g) shows 0.42 for the addition of glycerol and 0.34 for the glycerol-free experimental group, a significant improvement in yield. In the experimental group to which glycerol was added, the concentration of acetic acid was decreased at the beginning of fermentation, and the concentration of acetic acid increased after about 15 hours, which is 'acetic acid consumption rate of sugar consumption' rather than 'acetic acid consumption rate of glycerol consumption'. It's happening because it's faster. After 20 hours after the addition of glycerol again, the rate of acetic acid consumption was faster than that of acetic acid production, and the concentration of acetic acid was continuously reduced and almost exhausted. On the other hand, in the experimental group without addition of glycerol (Fig. 10c) it was observed that more than 9.3 g / L acetic acid was produced than the initial concentration. Based on the above results, when glycerol is added, the flow of fermentable carbon sources (glucose and xylose) in the saccharified solution can be concentrated to butyric acid, thereby significantly increasing the yield of butyric acid production while reducing the generation of by-products (acetic acid). At the same time, it can be seen that the non-fermentable carbon source (acetic acid) is also converted to butyric acid.

Claims (20)

A composition for fermentation of microorganisms comprising a fermentable carbon source and glycerol,
The fermentable carbon source comprises glucose, microbial fermentation composition. The method of claim 1,
The microorganism is Clostridium tyrobutyricum (Clostridium tyrobutyricum), microbial fermentation composition. The method of claim 1,
The fermentable carbon source comprises at least one of lignocellulosic biomass saccharification liquid and seaweed biomass saccharification liquid, microbial fermentation composition. The method of claim 1,
Further comprising acetic acid or acetate salts, microbial fermentation composition. The method of claim 1,
The fermentable carbon source, in addition to glucose, further comprises a sugar selected from monosaccharides, disaccharides and polysaccharides, microbial fermentation composition. The method of claim 5,
The sugars are galactose, mannose, mannose, rhamnose, fructose, xylose, arabinose, cellobiose, mannitol, and alginic acid. alginic acid) and laminaran (laminaran), microbial fermentation composition. The method of claim 1,
The weight ratio of glucose to glycerol is 1:99 to 99: 1, microbial fermentation composition. The method of claim 1,
The composition is a composition for fermentation microorganisms for butyric acid production. A method for producing butyric acid, comprising culturing by adding glycerol and a fermentable carbon source to a medium containing Clostridium tyrobutyricum. The method of claim 9,
The fermentable carbon source comprises glucose, method of producing butyric acid. The method of claim 9,
The fermentable carbon source comprises at least one of lignocellulosic biomass saccharification liquid and seaweed biomass saccharification liquid, butyric acid production method. The method of claim 9,
A method for producing butyric acid, which further comprises adding acetic acid or acetate to the medium. The method of claim 9,
The medium comprises acetic acid or acetate produced through the culture of Clostridium tyrobutyricum, butyric acid production method. The method according to claim 12 or 13,
The Clostridium tyrobutyricum is the fermentable carbon source; Acetic acid or acetates; And fermentation of glycerol to produce butyric acid. The method according to claim 12 or 13,
The Clostridium tyrobutyricum is the fermentable carbon source; Acetic acid or acetates; And acetic acid or acetate in the presence of glycerol; And fermentation of glycerol to produce butyric acid. The method of claim 9,
The medium comprises glucose,
The glycerol is added in a weight ratio of 1:99 to 99: 1 of glucose: glycerol, butyric acid production method. The method of claim 10,
The fermentable carbon source further comprises a sugar selected from monosaccharides, disaccharides and polysaccharides in addition to glucose, butyric acid production method. The method of claim 17,
The sugars are galactose, mannose, mannose, rhamnose, fructose, xylose, arabinose, cellobiose, mannitol, and alginic acid. alginic acid) and laminaran (laminaran) is at least one, butyric acid production method. The method of claim 9,
The method is to enhance butyric acid production. The method of claim 9,
The method produces butyric acid by consuming acetic acid or acetate to produce butyric acid.

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