KR20130077296A - Clostridium sp. strain, a method for producing butyric acid using the clostridium sp. strain, and a method for isolating the clostridium sp. strain - Google Patents

Abstract

PURPOSE: A butyric acid producing method using a strain from Clostridium is provided to effectively produce butyric acid from galactan, a marine algae biomass, having a main carbohydrate component by producing butyric acid form galactose. CONSTITUTION: A strain from Clostridium uses galactose to produce butyric acid. A butyric acid producing method from galactose comprises treating the strain from the Clostridium on galactose and culturing to convert galactose to butyric acid. A separating method of the strain from the Clostridium comprises a step of removing a gram-negative bacteria by heat treating an anaerobic sludge, a step of inoculating the heat treated anaerobic sludge to the culture medium containing galactose and culturing, a step of selecting colonies with butyric acid resistancy by smearing the culturing solution obtained from the culturing to the culture medium containing butyric acid or the salt, and a step of separating the most butyric acid producing strain by measuring butyric acid of the selected colonies.

Description

Clostridium sp. Strain, method for producing butyric acid using the strain, and method for isolating the strain {Clostridium sp. strain, a method for producing butyric acid using Clostridium sp. strain, and a method for isolating the Clostridium sp. strain}

The present invention relates to a novel strain of Clostridium sp. On the other hand, a method for producing butyric acid from galactose. On the other hand, it relates to a novel method for isolating Clostridium sp. Strain which produces butyric acid from galactose.

Recently, fossil fuel depletion and global warming have attracted interest in sustainable and harmonious development, as well as the development of alternative energy sources for energy conservation and efficient environmental conservation.

Biomass refers to organic matter generated from renewable plants, including energy crops, trees, food, and feed. In recent years, interest in algae has increased as a new biomass. Algae produced in large scale in the oceans is a resource that does not trigger serious food supply problems like land plants or does not deplete resources like long-lived forest resources. In addition, seaweeds have a much faster growth rate than wood-based ones, and are easy to pretreat because they do not contain lignin.

Red algae contains the largest number of carbon sources (eg, 77.2%) in seaweeds, and the main carbohydrate constituents are galactan and cellulose. The galactan component is degraded to galactose by glycation, and the cellulose is decomposed into glucose.

Butyric acid is widely used in the chemical, food and pharmaceutical industries. Typical production strains include but are not limited to Clostridium tyrobutyricum) (Korea Patent Publication No. 10-2009-0103720). However, what is disclosed in Korean Patent Laid-Open No. 10-2009-0103720 is to produce butyric acid from glucose. Clostridium thyrobutiricum has a problem in that galactose contained in red algae is hardly utilized, and the yield of fermentation is low. To overcome this, it is urgently necessary to develop a new microorganism that produces galactose effectively and produces butyric acid.

Korean Patent Laid-Open No. 10-2009-0103720

Extractive Fermentation for Buryric Acid Production from Glucose by Clostridium tyrobutylicum (Biotech. And Bioeng., Vol. 82, No.1, P.93-102, Apr. 2003) J R Turvey, J Christison, "The hydrolysis of algal galactans by enzymes from a Cytophaga species", The Biochemical Journal (1967) Volume: 105, Issue: 1, Pages: 311-316 N J Kim et al., "Ethanol production from marine algal hydrolysates using Escherichia coli KO11" Bioresource technology (2011) Volume: 102, Issue: 16, Pages: 7466-7469

The technique disclosed herein is to provide a strain that produces butyric acid using galactose.

In another aspect, the technique disclosed herein provides a method for producing butyric acid using galactose.

In another aspect, the technique disclosed herein provides a strain that produces butyric acid from seaweed biomass with high efficiency.

In another aspect, the technique disclosed herein provides a method for efficiently producing butyric acid from seaweed biomass.

In another aspect, the technique disclosed herein provides a method for isolating strains that produce butyric acid using galactose.

The strain according to an embodiment of the present invention is a Clostridium strain producing butyric acid using galactose.

A method for producing butyric acid according to an embodiment of the present invention is a method for producing butyric acid from galactose, comprising treating a strain of Clostridium that produces butyric acid using galactose to convert galactose to butyric acid.

The method of isolating Clostridium spp. According to an embodiment of the present invention comprises the steps of: heat treating anaerobic sludge to remove Gram-negative bacteria; Culturing the heat-treated anaerobic sludge in a culture medium containing galactose; Selecting a colony which is resistant to butyric acid by sprinkling the culture obtained from the culture on a medium containing butyric acid or a salt thereof; And isolating the strain having the highest amount of butyric acid production by measuring the butyric acid production amount of the selected colonies, wherein the Clostridium sp. Strain is a strain producing butyric acid using galactose.

By using the present invention, butyric acid can be effectively produced from galactose. Since butyric acid can be produced from galactose, it is possible to effectively produce butyric acid from seaweed biomass, a major carbohydrate ingredient, galactan. It can be widely used to produce chemical raw materials or bioenergy using seaweed biomass.

1 is a diagram showing a 16S rDNA nucleotide sequence of Clostridium tyrobutyricum S1 according to an embodiment of the present invention.
2 is a diagram showing a phylogenetic relationship based on the 16S rDNA region nucleotide sequence of Clostridium tyrobutyricum S1 according to an embodiment of the present invention.
Figure 3 is a graph showing the optimum growth temperature of Clostridium tyrobutyricum S1 according to an embodiment of the present invention.
Figure 4 is a graph showing the growth optimal pH of Clostridium tyrobutyricum S1 according to an embodiment of the present invention.
5 is a graph showing the growth characteristics and butyric acid production characteristics of Clostridium tyrobutyricum S1 according to an embodiment of the present invention in mMRS medium.
6A shows the result of GC-MS (Gas Chromatography-Mass Spectrometry) analysis of a standard sample of butyric acid.
Figure 6b shows the results of Gas Chromatography-Mass Spectrometry (GC-MS) of butyric acid obtained by using Clostridium tyrobutyricum S1 according to an embodiment of the present invention with lactic acid labeled ¹³ C .

The strain according to an embodiment of the present invention is a Clostridium strain producing butyric acid using galactose.

The Clostridium spp. Strain is an mMRS liquid including peptone, meat extract, yeast extract, galactose, polysorbate (Tween 80), dipotassium hydrogen phosphate, triammonium citrate, magnesium sulfate hepta hydrate and manganese sulfate tetrahydrate When cultured at 37 占 폚 for 3 days in a culture medium, has a property of converting at least 25% by weight, at least 30% by weight, at least 35% by weight, at least 40% by weight, at least 45% by weight or at least 50% by weight of galactose into butyric acid It may be Clostridium sp. The mMRS medium is specifically selected from the group consisting of 0.5-1.5% peptone, 0.5-1.5% meat extract, 0.2-0.8% yeast extract, 1.5-2.5% galactose, 0.05-0.15% polysorbate (Tween 80), 0.15-0.25% di Potassium hydrogen phosphate, 0.15-0.25% triammonium citrate, 0.005-0.015% magnesium sulfate heptahydrate, 0.002-0.008% manganese sulfate tetrahydrate, and optionally 0.2-0.8% sodium acetate. Specifically, the mMRS medium contained 1.0% peptone, 1.0% meat extract, 0.5% yeast extract, 2.0% galactose, 0.1% polysorbate (Tween 80), 0.2% dipotassium hydrogen phosphate, 0.2% triammonium citrate, 0.01 % Magnesium sulfate heptahydrate, 0.005% manganese sulfate tetrahydrate, and optionally 0.5% sodium acetate. The Clostridium spp. Strain may be a strain having an optimal growth temperature of 40 DEG C and an optimum growth pH of 5.5.

The Clostridial sp. Strain may be a Clostridial sp. Strain producing butyric acid using one or more of acetic acid or a salt thereof, and lactic acid or a salt thereof. When acetic acid or a salt thereof or lactic acid or a salt thereof is added to the medium, the production amount of butyric acid by Clostridium spp. Strain is further improved. The amount of acetic acid or its salt or lactic acid or its salt to be added may be 10 to 100 mM or 20 to 80 mM, 30 to 70 mM, 40 to 60 mM based on the total volume of the mMRS liquid medium. Where the salt may be a salt with all bases which form a salt through an ionic bond with the acid. For example, it may be a salt of a Group 1A metal. For example, a sodium salt or a potassium salt. The strain may have an increase in the production of butyric acid by 1.5 times, 1.8 times, 2.0 times, or 2.2 times by the addition of 50 mM acetic acid or a salt thereof. On the other hand, the strain may be a strain having an increase in butyric acid production by 1.8 times, 2.0 times, 2.2 times, or 2.4 times as much as the addition of 50 mM lactic acid or a salt thereof.

On the other hand, the Clostridium sp is 2- (N- know Holly furnace) when working with ethane sulfonic acid (2- (N- morholino) ethanesulfonic acid, MES) buffer having the property of improving the acid producing Clostridium Lt; / RTI > The amount of MES added is 50 to 150 mM or 70 to 130 mM, 80 to 120 mM, 90 to 110 mM based on the total volume of the mMRS liquid medium. Specifically, the strain may be a strain having an increase in the production of butyric acid by 100 mM MES, 1.5 times, 1.8 times, 2.0 times, 2.2 times, or 2.3 times or more.

In addition, the Clostridial strain can be a strain having an increase in butyric acid production of 2.0 times or more, 2.2 times or more, 2.4 times or more, 2.6 times or 2.8 times or more when the acetic acid or its salt and 100 mM MES are simultaneously treated have.

 In addition, the Clostridial strain exhibits a property that the increase in butyric acid production is 2.2 times or more, 2.4 times, 2.6 times or more 2.8 times, 3.0 times or more or 3.1 times or more when the lactic acid or its salt and 100 mM MES are simultaneously treated Lt; / RTI > strain.

On the other hand, the strain of Clostridium sp. Is 2.2 times or more increased in butyric acid production by addition of acetic acid or its salt; The increase of butyric acid production by addition of lactic acid or its salt is more than 2.4 times; The addition of 100 mM MES increases butyric acid production by more than 2.3 times; The increase of butyric acid production by acetic acid or its salt and the addition of 100 mM MES is 2.8 times or more, and the increase of butyric acid production by lactic acid or its salt and 100 mM MES is 3.1 times or more.

The Clostridium spp. Strain may be a strain producing butyric acid using galactose derived from seaweed biomass.

The algae include green algae, brown algae and red algae. Green algae include blue, hearing, and blue light. Brown algae include seaweed, seaweed, kelp, rhubarb, and the like. Red algae include Kim, caranigin, and the like. Preferably, the algae are red algae. Red algae contains the most carbon sources of seaweed, and the main carbohydrate component is galactan. Galactan is degraded to galactose by glycation.

The strain of the genus Clostridium may be a strain belonging to Clostridium tyrobutyricum. The genus Clostridium strain may be Clostridium tyrobutyricum S1 strain. The Clostridium sp. Strain was deposited at KCTC 12103BP on Dec. 13, 2011 in the Korean Collection for Type Culture (KCTC) of the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Gt; Clostridium < / RTI > strain.

The method of producing butyric acid according to an embodiment of the present invention includes converting a galactose to a butyric acid by culturing and culturing a seaweed biomass in which galactose or galactan is decomposed into galactose by treating any one of the Clostridia sp. can do.

In the butyric acid production method, the cultivation may be carried out under the condition of pH 5 to 6. The culture can also be performed under the conditions of pH 5.2 to 5.8, pH 5.3 to 5.7, or pH 5.4 to 5.6. Since the optimum growth pH of the Clostridial sp. Strain is 5.5, adjusting the pH within the above range can increase the production efficiency of butyric acid.

In the butyric acid production method, the cultivation may be carried out at 38 to 42 ° C. In addition, the cultivation can be carried out at 35 to 45 캜, or 39 to 41 캜. Since the optimum growth temperature of the Clostridial sp. Strain is 40 캜, the production efficiency of butyric acid can be increased by performing the culture within the above temperature range.

The butyric acid production method is characterized in that at least one of acetic acid or a salt thereof and lactic acid or a salt thereof is added to a seaweed biomass in which galactose or galactan is degraded to galactose before or after the treatment of Clostridium spp. ≪ / RTI > Acetic acid or a salt thereof or lactic acid or a salt thereof may be added to further increase the amount of butyric acid produced in Clostridium spp. The amount of acetic acid or its salt or lactic acid or its salt to be added may be 10 to 100 mM or 20 to 80 mM, 30 to 70 mM, 40 to 60 mM based on the total volume of the mMRS liquid medium. Within this range, the production of butyric acid is effectively increased.

The butyric acid production method is characterized in that the seaweed biomass in which galactose or galactan has been degraded to galactose is added to the biomass before or after treatment with Clostridium spp. Or simultaneously with 2- (N-morpholino) ethanesulfonic acid (MES) buffer May be added to galactose. Treatment of MES can further increase the production of butyric acid by Clostridium spp. The amount of MES added may be 50 to 150 mM or 70 to 130 mM, 80 to 120 mM, 90 to 110 mM based on the total volume of the mMRS liquid medium. Within this concentration range, butyric acid production can be further improved.

In the butyric acid production method, the galactose may be galactose derived from seaweed biomass.

The method for producing butyric acid may further include saccharifying the seaweed biomass to decompose galactan into galactose before treating the strain of Clostridium sp. As a method of saccharifying the seaweed biomass, a saccharification method using enzymes commonly used in the art can be used. For example, enzymes derived from Cytophaga species, dilute sulfuric acid and dilute hydrochloric acid can be used to hydrolyze galactan of seaweed biomass (JR Turvey, J Christison, "The hydrolysis of algal galactans by enzymes from "Ethanol production from marine algae hydrolysates using Escherichia coli KO11" Bioresource technology (2011) Volume: 102, Issue: 1, Pages: 311-316 , Issue: 16, Pages: 7466-7469).

Clostridium genus (Clostridium) separation method of the strain according to one embodiment of the present invention includes the steps of removing Gram-negative bacteria by heating the anaerobic sludge; Culturing the heat-treated anaerobic sludge in a culture medium containing galactose; Selecting a colony which is resistant to butyric acid by sprinkling the culture obtained from the culture on a medium containing butyric acid or a salt thereof; And isolating the strain having the highest production of butyric acid by measuring the production of butyric acid of the selected colonies. The Clostridium sp. Strain may be a strain producing butyric acid using galactose of seaweed biomass have.

In the method for isolating strains, the heat treatment may be a heat treatment at a temperature of 80-100 ° C for 10-60 minutes. Gram negative bacteria can be effectively removed if they are within the above temperature and time range.

In the strain isolation method, the medium containing the galactose may be MP2 liquid medium, which is a restricted medium. MP2 medium may be a medium comprising galactose, K ₂ HPO ₄ , KH ₂ PO ₄ , yeast extract, ammonium acetate, MgSO ₄ , MnSO ₄ and l-cysteine HCl. Specifically, the MP2 medium contained 15-25 g / L of galactose, 0.3-0.7 g / L of K ₂ HPO _4, 0.3-0.7 g / L of KH ₂ PO ₄ , 0.7-1.3 g / L of yeast extract, g / L, MgSO ₄ 0.1-0.3 g / L, MnSO ₄ 0.005-0.015 g / L and 1-cysteine HCl 0.3-0.7 g / L. Specifically, MP2 medium galactose _{20 g / L, K 2 HPO} 4 0.5 g / L, KH 2 PO 4 0.5 g / L, yeast extract 1 g / L, ammonium acetate, _{2.2 g / L, MgSO 4 0.2} g / L , 0.01 g / L MnSO ₄ and 0.5 g / L 1-cysteine HCl.

In the method for isolating strains, the step of inoculating and culturing the heat-treated anaerobic sludge in a culture medium containing galactose may be carried out at 37 ° C for 2-4 days.

The step of isolating the strain having the highest amount of butyric acid production in the method for isolating the strain may include selecting a strain having the highest amount of butyric acid production as a result of culturing in MP2 medium at 37 ° C for 2-4 days.

In the method for isolating Clostridium spp., Clostridium spp. May be any one of Clostridium spp. Mentioned above.

Hereinafter, the present invention will be described in more detail by way of examples, which are for illustrative purposes only and do not limit the scope of the patent.

<Example 1: Isolation of a strain producing butyric acid using galactose>

1 g of anaerobic sludge was collected from the anaerobic sludge of the wastewater treatment plant located in Jungnang-gu, Seoul, Korea to isolate butyric acid-producing microorganisms using galactose, and Gram-negative bacteria were removed by heat treatment at 90 ° C for 30 minutes. The heat-treated anaerobic sludge was treated with MP2 liquid medium (Galactose 20 g, K ₂ HPO ₄ 0.5 g, KH ₂ PO ₄ 0.5 g, Yeast extract 1 g, Ammonium acetate 2.2 g, MgSO ₄ 0.2 g, MnSO ₄ 0.01 g , l-cysteine HCl 0.5 g) and incubated at 37 ° C for 3 days.

The cultures were cultured on MP2 medium containing 20 g of sodium butyrate, and colonies resistant to butyric acid were selected. Each of the selected colonies was inoculated into MP2 liquid medium and cultured at 37 ° C for 3 days, and the amount of butyric acid production was measured by gas chromatography (GC). As a result, microorganisms producing the largest amount of butyric acid were isolated. The isolated microorganism was deposited on December 13, 2011 in the Korean Collection for Type Culture (KCTC) of the Korea Research Institute of Bioscience and Biotechnology (KRIBB), a microorganism depository organization under the Budapest Treaty, KCTC 12103BP.

&Lt; Example 2: Identification and characteristics of strains >

The 16S rRNA gene of Clostridium sp. Isolate was identified by sequencing. Genomic DNA was extracted from the isolated strain using a genomic DNA preparation kit (Promega Co., USA), and then a universal primer 27F (5'-AGA GTT TGA TCC TGG CTC AG-3 16S rRNA gene was amplified by polymerase chain reaction (PCR) with 1492R (5'-TAC GGY TAC CTT GTT ACG ACT T-3 ': SEQ ID NO: 2) . The amplified products were purified using a PCR Purification kit, and 16S rDNA sequencing was performed on Macrogen.

The 16S rRNA gene sequence of the novel strain is shown in Fig. 1 and SEQ ID NO: 3. The 16S rDNA gene sequence of this isolate was searched in the blast (http://www.ncbi.nlm.nih.gov/BLAST) to analyze the phylogenetic relationship, and the Neighbor-Joining analysis The phylogenetic tree was established by -joining analysis method. As a result, it was confirmed that it was 95.7% homologous with Clostridium tyrobutyricum (GenBank accession no. M59113) (Fig. 2). Therefore, the inventor named the S1 strain "Clostridium tyrobutyricum S1".

Isolation strain confirmed as Clostridium tyrobutyricum S1 was confirmed carbohydrate availability using API 50 CHL (Bio-marieux, France). The separated samples were enriched by subculturing twice in MRS liquid medium and centrifuged at 10,000 xg for 10 minutes to collect the cells. The recovered cells were washed twice with 0.85% aqueous sodium chloride solution and the turbidity was adjusted to 4 Macfarland. 2 ml of the suspension was added to 5 ml of API 50CHL medium, and 100 쨉 l of each suspension was injected into each cuplet. The API 50CHL medium contained 1.0% peptone, 0.5% yeast extract, 0.1% polysorbate (Tween 80), 0.2% dipotassium hydrogen phosphate, 0.2% triammonium citrate, 0.02% magnesium sulfate heptahydrate, 0.005% manganese sulfate tetra Hydrate and 0.5% sodium acetate, and 0.017% bromocresol purple indicator. The completed kit was incubated at 37 ° C for 48 hours using an anaerobic incubator. The experimental results are shown in Table 1 below. As shown in the following Table 1, it was confirmed that the isolated strain produced an acid using D-xylose, galactose and mannose contained in the algae saccharification solution.

Carbohydrates Acid production D-xylose + ¹⁾ L-xylose - ²⁾ D-galactose + D-glucose + D-fructose + D-mannose + Potassium gluconate + Potassium 2-ketogluconate - Potassium 5-ketogluconate -

+ ¹⁾ : positive, - ²⁾ : negative

In order to confirm the optimum growth temperature and pH of Clostridium tyrobutyricum S1, the strain growth was observed according to each pH (5.5, 6.0, 6.5) and temperature (34, 37, 40, 43 ℃). It was.

As shown in Figures 3 to 4, it can be seen that the most suitable temperature and pH for growth of Clostridium tyrobutyricum S1 is 40 ℃, pH 5.5.

&Lt; Example 3: Comparison of galactose utilization characteristics of Clostridium Tyrobuticum strains with the strain of the present invention >

(Clostridium tyrobutyricum) strain, which is most widely used for butyric acid production and has the highest degree of similarity to the isolated strain, was fermented at 37 ° C for 3 days in MP2 medium containing galactose to produce butyric acid and strain Were compared with the isolates. The results are shown in Table 2 below.

As shown in Table 2, the strains of Clostridium Tyrobutilicum except for the isolate according to an embodiment of the present invention showed almost no use of galactose and no increase of butyric acid. However, In the case of the isolated strains according to the examples, it is observed that galactose is effectively used to produce butyric acid.

Strain OD ₆₀₀ Galactose consumption
(g / L) Butyric acid production
(g / L) C. tyrobutyricum ATCC 25755 1.34 ND ¹⁾ 0.67 C. tyrobutyricum NCIMB 9582 0.39 N.D. 0.5 C. tyrobutyricum NCIMB 9583 1.21 N.D. 0.135 C. tyrobutyricum NCIMB 9584 One N.D. 0.92 C. tyrobutyricum NCIMB 12573 1.11 N.D. 0.764 C. tyrobutyricum NCIMB 701715 0.89 N.D. 0.284 C. tyrobutyricum NCIMB 701753 0.67 N.D. 0.912 C. tyrobutyricum NCIMB 701755 1.12 N.D. 0.956 C. tyrobutyricum NCIMB 701756 0.87 N.D. 0.14 C. tyrobutyricum NCIMB 701757 1.17 N.D. 0.968 C. tyrobutyricum NCIMB 701790 1.51 N.D. 1.092 C. tyrobutyricum DSM 664 0.69 N.D. 0.56 C. tyrobutyricum DSM 1460 0.89 N.D. 0.964 C. tyrobutyricum S1 5.79 16.04 5.24

ND ¹⁾ : almost not used

<Example 4: Factors affecting the production of butyric acid by the isolated strain>

Cliprisdium tyrobutyricum S1,

1.0% peptone, 1.0% meat extract, 0.5% yeast extract, 2.0% galactose, 0.1% polysorbate (Tween 80), 0.2% dipotassium hydrogen phosphate, 0.2% triammonium citrate, 0.01% magnesium sulfate heptahydrate, The fermentation at 37 ° C for 3 days in the mMRS liquid medium containing 0.005% manganese sulfate tetrahydrate and 0.5% sodium acetate produced 6.99 g / L of butyric acid using 14.32 g / L of galactose, but acetic acid was only 4.1 g / L to 1.85 g / L (Fig. 5). In order to observe the increase of butyric acid production by addition of acetic acid and lactic acid, sodium acetate or sodium lactate was added to mMRS medium at 50 mM each and fermented at 37 ° C for 3 days. Since pH is also an important factor in the production of organic acids, the effect of butyric acid production and pH on the isolated strain was observed using 100 mM MES (2- (N-morholino) ethanesulfonic acid) buffer. The results are shown in Table 3 below. Run I is the result of incubation in mMRS medium lacking acetate and lactate. Run II is the result of culturing in mMRS medium supplemented with acetate. Run III is the result of culturing in mMRS medium supplemented with lactate. Run IV is the result of culturing in mMRS medium supplemented with 100 mM MES without acetate or lactate. Run V is the result of culturing in mMRS medium supplemented with acetate and 100 mM MES. Run VI was the result of incubation in mMRS medium with lactate and 100 mM MES.

As shown in Table 3, the production of butyric acid by the addition of sodium acetate of 50 mM was increased by 2.4-fold when sodium lactate of 2.2-fold and 50-mM was added, and increased 2.3-fold by addition of 100 mM of MES Respectively. In addition, it was observed that a simultaneous addition of 50 mM sodium lactate and 100 mM MES resulted in the production of 9.52 g / L butyric acid, 3.1 times higher (Table 3).

Run I Run II Run III Run IV Run V Run VI Galactose consumption (g / L) 11.9 14.32 15.46 21.31 24.05 21.95 Acetic acid consumption (g / L) - 2.25 - - 0.44 - Lactic acid consumption (g / L) - - 5.79 - - 5.1 Butyric acid production (g / L) 3.12 6.99 7.45 7.20 8.84 9.52 Final pH 4.62 4.99 4.88 4.99 5.11 5.37 Yield (g / g) 0.26 0.49 0.48 0.34 0.37 0.43

Run I: mMRS without acetate and lactate

Run II: mMRS with acetate

Run III: mMRS with lactate

Run IV: mMRS without acetate and lactate (100 mM MES)

Run V: mMRS with acetate (100 mM MES)

Run VI: mMRS with lactate (100 mM MES)

By Clostridium tie as shown in Table 3 butynyl rikum (Clostridium tyrobutyricum) is S1, and effective use of lactic acid, lactic acid increase in the drive acid production, so to see if the lactic acid is converted to acid l- [3- ¹³ C] Metabolic pathways were observed using lactate. The results are shown in Figs. 6A to 6B.

As shown in FIGS. 6A and 6B, 90.1, 91.1 and 92.1 mass / charge peaks from ¹³ C-lactate which are not observed in GC-MS analysis (FIG. 6A) of the standard sample butyric acid are observed, Can be confirmed to be converted to butyric acid.

In conclusion, the strain according to an embodiment of the present invention can produce butyric acid by effectively utilizing galactose present in algae biomass that is not available from existing strains of Clostridium tyrobutyricum, product selectivity) is almost 100%, it can be used as a good candidate strain for production of butyric acid by seaweed biomass.

Korea Biotechnology Research Institute KCTC12103BP 20111213

<110> Korea Institute of Science and Technology <120> Clostridium sp. strain, a method for producing butyric acid using          the Clostridium sp. strain, and a method for isolating the          Clostridium sp. strain <130> 11p572 / ind <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tacggytacc ttgttacgac tt 22 <210> 3 <211> 1392 <212> DNA <213> Clostridium tyrobutyricum <400> 3 ggacgaacgc tggcggcgtg cctaacacat gcaagtcgag cgagagagtc ccttcgggga 60 caatctagcg gcggacgggt gagtaacacg tgggtaacct gcctcaaaga gggggatagc 120 ctcccgaaag ggagattaat accgcataag acctggattc acatggatcc gggatgaaag 180 gagaaatccg ctttgagatg gacccgcggc gcattagcta gttggtgggg taaaggccta 240 ccaaggcagc gatgcgtagc cgacctgaga gggtgatcgg ccacattgga actgagagac 300 ggtccagact cctacgggag gcagcagtgg ggaatattgc acaatgggcg aaagcctgat 360 gcagcaacgc cgcgtgagtg atgaaggttt tcggatcgta aagctctgtc ttctgggaag 420 ataatgacgg taccagagga ggaagccacg gctaactacg tgccagcagc cgcggtaata 480 cgtaggtggc gagcgttgtc cggatttact gggcgtaaag ggtgcgtagg cggatgttta 540 agtgagatgt gaaatacccg ggcttaaccc gggggctgca tttcaaactg gacatctgga 600 gtgcaggaga ggagaatgga attcctagtg tagcggtgaa atgcgtagag attaggaaga 660 acaccagtgg cgaaggcgat tctctggact gtaactgacg ctgaggcacg aaagcgtggg 720 tagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgagta ctaggtgtag 780 gaggtatcga ccccttctgt gccgcagtta acacattaag tactccgcct gggaagtacg 840 atcgcaagat taaaactcaa aggaattgac gggggcccgc acaagcagcg gagcatgtgg 900 tttaattcga agcaacgcga agaaccttac ctggacttga catcccctgc atagcctgga 960 gacaggcgaa gcccttcggg gcagggagac aggtggtgca tggttgtcgt cagctcgtgt 1020 cgtgagatgt taggttaagt cctgcaacga gcgcaaccct tgttgttagt tgctaacatg 1080 taaagatgag cactctaacg agactgccgc ggttaacgcg gaggaaggtg gggatgacgt 1140 caaatcatca tgccccttat gtccagggcg acacacgtgc tacaatgggc agaacagaga 1200 gatgcgaaac cgcgaggtgg agccaaacta gaaaactgcc ctcagttcgg attgcaggct 1260 gaaacccgcc tgcatgaagt tggagttgct agtaatcgcg aatcagaatg tcgcggtgaa 1320 tacgttcccg ggccttgtac acaccgcccg tcacaccatg agagctggca acacccgaag 1380 tccgcagtct aa 1392

Claims (22)

Clostridium strain producing butyric acid using galactose. The method according to claim 1, wherein the Clostridia spp.
Culturing for 3 days at 37 ° C in mMRS liquid medium containing peptone, meat extract, yeast extract, galactose, polysorbate (Tween 80), dipotassium hydrogen phosphate, triammonium citrate, magnesium sulfate hepta hydrate and manganese sulfate tetrahydrate , Clostridium sp. Strain having a property of converting 25 wt% or more of galactose into butyric acid. 2. The method according to claim 1, wherein the Clostridial subsp.
Clostridium sp. Strain having an optimal growth temperature of 40 ° C and an optimal growth pH of 5.5. The Clostridial sp. Strain according to claim 1, wherein the Clostridial sp. Strain produces butyric acid by further using at least one of acetic acid or a salt thereof and lactic acid or a salt thereof. 2. The method according to claim 1, wherein the Clostridial sp. Strain has a property of improving butyric acid production when 2- ( N- morholino) ethanesulfonic acid (MES) Clostridial &lt; / RTI &gt; [Claim 2] The method according to claim 1, wherein the galactose is galactose derived from seaweed biomass, Clostridium sp. The strain of the genus Clostridium according to claim 1, wherein the strain of the genus Clostridium is a Clostridium tyrobutyricum strain. The strain of the genus Clostridium according to claim 7, wherein the strain of the genus Clostridium is Clostridium tyrobutyricum S1 strain. The strain of the genus Clostridium according to claim 8, wherein the strain of the genus Clostridium is Clostridium tyrobutyricum S1 strain (KCTC 12103BP). As a method for producing butyric acid from galactose,
A method for producing butyric acid from galactose, comprising converting galactose to butyric acid by treating galactose with the strain of the genus Clostridium according to any one of claims 1 to 9. The method of claim 10,
Before or after treatment of Clostridium spp., Or simultaneously with acetic acid or a salt thereof; Lactic acid or a salt thereof; A method for producing butyric acid from galactose, wherein at least one of 2- (N-morpholino) ethanesulfonic acid (MES) buffer is added to galactose. 11. The method for producing butyric acid according to claim 10, wherein the cultivation is performed under the condition of pH 5 to 6. 11. The method of producing butyric acid according to claim 10, wherein the cultivation is carried out at 38 to 42 DEG C. 14. The method according to any one of claims 10 to 13,
Wherein said galactose is galactose derived from seaweed biomass. 15. The method of claim 14,
A method for producing butyric acid from galactose, which further comprises saccharifying the seaweed biomass to decompose the galactan to galactose before treating the strain of Clostridium sp. A method for producing butyric acid from seaweed biomass,
Hydrolyzing the seaweed biomass to decompose galactan into galactose; And
Butyric acid from algae biomass comprising converting galactose to butyric acid by treating and incubating a strain of Clostridium sp. Production method. 17. The method of claim 16,
Before or after treatment of Clostridium spp., Or simultaneously with acetic acid or a salt thereof; Lactic acid or a salt thereof; And 2- (N-morpholino) ethanesulfonic acid (MES) buffer to the seaweed biomass, to produce butyric acid from the seaweed biomass. 17. The method of producing butyric acid according to claim 16, wherein the cultivation is performed under the condition of pH 5 to 6. 17. The method according to claim 16, wherein the cultivation is carried out under the condition of 38 to 42 DEG C from the seaweed biomass. As a method of obtaining a Clostridium strain,
The Clostridium sp. Strain is a strain that produces butyric acid using galactose,
The method comprises:
Treating the anaerobic sludge with heat to remove Gram-negative bacteria;
Culturing the heat-treated anaerobic sludge in a culture medium containing galactose;
Selecting a colony which is resistant to butyric acid by sprinkling the culture obtained from the culture on a medium containing butyric acid or a salt thereof; And
And isolating the strain having the highest production of butyric acid by measuring the butyric acid production of the selected colonies. The method of claim 20, wherein the strain of the genus Clostridium is a strain of the genus Clostridium according to any one of claims 1 to 9. 21. The method according to claim 20, wherein the culture medium is a culture of Clostridium sp. Strain MP2 medium containing galactose, K ₂ HPO ₄ , KH ₂ PO ₄ , yeast extract, ammonium acetate, MgSO ₄ , MnSO ₄ and 1-cysteine HCl Way.

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