WO2009014289A1

WO2009014289A1 - Method for preparing succinic acid using sucrose as a carbon source

Info

Publication number: WO2009014289A1
Application number: PCT/KR2008/000242
Authority: WO
Inventors: Sang Yup Lee; Hyohak Song; Jeong Wook Lee; Sol Choi; Ji Mahn Kim
Original assignee: Korea Advanced Institute Of Science And Technology
Priority date: 2007-07-25
Filing date: 2008-01-15
Publication date: 2009-01-29

Abstract

The present invention relates to a method for preparing succinic acid, which comprises culturing a succinic acid-producing microorganism in a medium containing sucrose as a carbon source, and more particularly, a method for preparing succinic acid, which comprises culturing a succinic acid-producing microorganism under batch or fed-batch culture conditions in a medium containing sucrose as a carbon source at a high concentration. According to the present invention, when inexpensive sucrose, whose price corresponds to 28.9% of glucose price, is used as a carbon source, microorganisms have improved resistance against organic acids including succinic acid compared to the case using other carbon sources such as glucose to significantly increase succinic acid productivity as well as final succinic acid concentration, thus making it possible to reduce costs required for separating and purifying succinic acid.

Description

METHOD FOR PREPARING SUCCINIC ACID USING SUCROSE AS A CARBON SOURCE

TECHNICAL FIELD

The present invention relates to a method for preparing succinic acid, which comprises culturing a succinic acid-producing microorganism in a medium containing sucrose as a carbon source, and more particularly, a method for preparing succinic acid, which comprises culturing a succinic acid-producing microorganism under batch or fed-batch culture conditions in a medium containing sucrose as a carbon source.

BACKGROUND ART

Succinic acid, which is widely known as an amber acid, has been produced by chemical synthesis and microbial fermentation, and can be used as a precursor of various chemical products and thus has high utility for economic and industrial applications (Zeikus et al, Appl. Microbiol. Biotechnol., 51 :545, 1999; Song et al, Enzyme Microbial Technol., 39: 352, 2006). Particularly, the demand for succinic acid is expected to be dramatically increased due to the finding that succinic acid can be used as a main source of biodegradable polymers (Willke et al., Appl. Microbiol. Biotechnol., 66:131, 2004). Most succinic acid for industrial use is currently produced using petroleum or liquefied natural gas as a raw material by chemical companies. When considering the latest sharp increase in petroleum prices, limited fossil resources and environmental concerns, there is an urgent need to develop a process for efficiently producing succinic acid by microbial fermentation, which can replace chemical synthesis process. With microbial culture technology, virtual cell technology and strain improvement through metabolic engineering, technology for succinic acid production has been remarkably progressed (Kim et al., Biotech. Bioeng., 97:657, 2007; Song et al., Enzyme Microbial Technol, 39:352, 2006).

Succinic acid production in a medium containing glucose as a culture material using Fusarium martii strain by Lockwood et al in 1938, triggered studies on succinic acid production using microorganisms. Since then, studies on the development of a process for effectively producing succinic acid through microbial fermentation, have been continuously carried out in various aspects, and various kinds of microorganisms (Actinobacillus, Anaerobiospirillum, Bacteroides, Mannheimia, Succinimonas, Succinivibrio etc.), which can efficiently produce succinic acid, were identified. Meanwhile, studies on the development of succinic acid-producing mutant strain through genetic manipulation, have been mainly carried out using Escherichia? coli, and various kinds of succinic acid-producing E. coli mutants such as strains capable of producing succinic acid in an aerobic condition, etc., have been developed (US 5,770,435; US 6,648,061; Hong et al, Biotechnol. Bioeng., 74:89, 2001; Vemuri et al., J. Ind. Microbiol. Biotechnol., 28:325, 2001; Lin et al, Metabol. Eng., 7:116, 2005; Lin et al, Biotechnol. Bioeng., 90:775, 2005).

The present inventors isolated an excellent Mannheimia succiniciproducens MBEL55E (KCTC 0769BP) producing succinic acid with high efficiency from the rumen of Korean native cattle, and completed its full genome sequence and characterized metabolic properties thereof (Hong et al, Nature Biotechnol., 22:1275, 2004). Also, the present inventors have constructed its mutants, M. succiniciproducens LPK (KCTC 10558BP) by disrupting a gene encoding lactate dehydrogenase^/^) and a gene encoding pyruvate formate-lyase(#/7) from M. succiniciproducens MBEL 55E and M. succiniciproducens LPK7 (KCTC 10626BP) by disrupting a phosphotransacetylase gene(/??α) and an acetate kinase gene(αcfc4) in the mutant strain, M. succiniciproducens LPK (WO 2005/052135 Al; Lee et al, Appl Environ. Microbiol, 72:1939, 2006). In addition to that, the present inventors have constructed a mutant strain, M. succiniciproducens PALK (KCTC 10973BP) (PCT/KR2007/003574) by disrupting a lactate dehydrogenase gene(ldhA), a phosphotransacetylase gene (pta) and an acetate kinase gene (ackA) in the M. succiniciproducens MBEL55E strain, M. succiniciproducens ALKt (PCT/KR2008/000012) by overexpressing a phosphotransacetylase gene (pta) in PALK strain, and a mutant strain, M. succiniciproducens ALK (PCT/KR2008/000012) by disrupting a lactate dehydrogenase gene(ldhA) and an acetate kinase gene (ackA) in the M. succiniciproducens MBEL55E strain.

In fact, costs required for a succinic acid production process using microorganisms are higher than that required for a succinic acid production process using chemical synthesis. Thus, in order to reduce the costs required for the process of producing succinic acid using microbial fermentation, various studies on the development of culture processes such as a batch culture, a fed-batch culture, a continuous culture etc., have been conducted (Lee et al, Appl. Microbiol. Biotechnol., 54:23, 2000; Lee et al., BioProc. Biosystems Eng., 26:63, 2003; Urbance et al, Appl. Microbiol. Biotechnol., 65:664, 2004). Together with the culture process development, studies on the development of inexpensive raw materials such as wood hydrolysate, glycerol, whey, corn steep liquor etc., have been also carried out by many researchers (Samuelov et al., Appl Environ. Microbiol. 65:2260, 1999; Lee et al., Appl Microbiol Biotechnol., 54:23, 2000; Lee et al., Biotechnol Bioeng., 72:41, 2001; Lee et al, Biotechnol Lett., 25:111, 2003; Lee et al, BioProc. Biosystems Eng., 26:63, 2003). However, studies reported to date suggest that when the above mentioned materials were used as a raw material instead of glucose, succinic acid productivity was far lower than that when glucose was used as a raw material and thus cost-effective succinic acid production could not be achieved.

Sucrose consisting of glucose and fructose is a disaccharide which is produced by green plants in the process of undergoing photosynthesis and is very abundant in nature. Especially, it accounts for 15-20%(w/v) of sugar cane and sugar beet juice, and is industrially produced by a very simple process of repeating evaporation/concentration and thus production cost thereof is very low. According to a report by Koutinas et al. (Ind. Crops and Products. 20:75, 2004), in which prices of various raw materials, used in the production of chemical materials using microorganisms, are calculated based on glucose content, sucrose is 26.1 cents based on lkg glucose, which is a very low price corresponding to 77%, 50% and 28.9% of the prices of wheat, molasses and glucose, respectively.

With respect to studies on useful material production using sucrose, biodegradable polymers by cell culture at high concentration (polyhydroxybutyrate, Lee et al., Biotechnol Lett., 15:971, 1993; Lee et al., Biotechnol. Techniques, 1 :59, 1997), citric acid (Forster et al., App. Microbiol. Biotechnol., 75: 1409, 2007), acetone, butanol, ethanol, isopropanol (George et al., Appl. Environ. Microbiol., 45:1160, 1983; Durre, Appl. Microbiol. Biotechnol., 49:639, 1998), itaconic acid (Kautola et al., Biotechnol. Lett., 11 :313, 1989), xanthan gum (Letisse et al., Appl. Microbiol. Biotechnol., 55:417, 2001) and the like have been reported. However, in case of sucrose, only basic studies on whether succinic acid-producing microorganism can grow using sucrose as a carbon source, and the kinds of final metabolites produced by sucrose consumption, and the like, have been conducted (Lee et al., Proc. Biochem., 35:49, 1999; Lee et al., Appl. Microbiol. Biotechnol, 58:663, 2002). Namely, there is no report of whether succinic acid can be produced using sucrose, a method for preparing succinic acid using sucrose and conditions thereof, and in addition to that, the development of technologies that can improve succinic acid- productivity and the final succinic acid concentration, and the like by efficiently utilizing sucrose.

For the economical production of succinic acid for industrial use by microbial fermentation, the following requirements should be satisfied; use of inexpensive raw materials such as sucrose, and an increase in succinic acid productivity and the final succinic acid concentration. Particularly, when considering the fact that the costs of raw materials account for about 50% of overall production cost of chemicals by microbial fermentation, there is an urgent need to develop a method for preparing succinic acid using inexpensive raw materials.

Accordingly, the present inventors have made extensive efforts to develop an effective technology for producing succinic acid at a high concentration using sucrose, and as a result, developed a method for producing succinic acid using inexpensive sucrose instead of glucose, and confirmed that when succinic acid is produced in a medium containing sucrose as a carbon source, final succinic acid concentration is much higher than that in the conventional succinic acid production process, particularly, sucrose-containing medium prevents succinic acid from inhibiting cell growth resulted in the increase of final cell concentration, so that overall succinic acid productivity is significantly increased, thereby completing the present invention.

SUMMARY OF INVENTION

It is an object of the present invention to provide a method for preparing succinic acid at high concentration using sucrose, which comprises effectively producing succinic acid by various culture processes using sucrose as a carbon source.

To achieve the above objects, the present invention provides a method for preparing succinic acid, which comprises culturing a succinic acid-producing microorganism in a medium containing sucrose as a carbon source.

In addition, the present invention provides a method for preparing succinic acid, which comprises culturing a succinic acid-producing microorganism Mannheimia sp. in a medium containing sucrose as a carbon source.

Other features and aspects of the present invention will be apparent from the following detailed description and the appended claims. BRIEF DESCRIPTION OF DRAWINGS

FIG.1 is a schematic diagram showing the succinic acid production pathway using sucrose as a raw material in a succinic acid-producing microorganism.

FIG.2 is a graph showing succinic acid production characteristics of a M succiniciproducens PALK strain in a batch culture using sucrose as a carbon source.

FIG.3 is a graph showing succinic acid production characteristics of a M succiniciproducens PALK strain in a batch culture using glucose as a carbon source.

FIG.4 is a graph showing succinic acid production characteristics of a M succiniciproducens PALK strain in a fed-batch culture using sucrose as a carbon source.

FIG.5 is a graph showing succinic acid production characteristics of a M succiniciproducens PALK strain in a fed-batch culture using glucose as a carbon source.

DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS

The present invention intends to examine whether succinic acid-productivity can be improved, when a succinic acid-producing microorganism is cultured in a medium containing sucrose, not glucose, as a carbon source. Sucrose, which is a disaccharide consisting of glucose and fructose, is abundant in nature, and is inexpensive compared with glucose.

In examples of the present invention, a succinic acid-producing microorganism, M succiniciproducens PALK was cultured in a medium containing sucrose as a carbon source under batch and fed-batch culture conditions, and as a result, it was confirmed that overall succinic acid-productivity, maximum cell concentration and the like were much higher compared with the case when glucose is used as a carbon source.

Therefore, in one aspect, the present invention relates to a method for preparing succinic acid using sucrose, which comprises culturing a succinic acid-producing microorganism in a medium containing sucrose as a carbon source.

The method for preparing succinic acid using sucrose according to the present invention comprises the steps of inoculating a succinic acid-producing microorganism into a medium containing sucrose as a culture material to culture in batch or fed-batch mode, and recovering succinic acid from the culture broth.

In the method for preparing succinic acid using sucrose according to the present invention, sucrose can completely replace all other carbon sources including glucose, so that there is no need to add a separate carbon source except sucrose to a medium.

Moreover, for the medium for culturing a succinic acid-producing microorganism, any medium can be used without limitations as long as it is a medium capable of culturing anaerobic microorganisms. In addition, the succinic acid-producing microorganism is preferably selected from the group consisting of Actinobacillus sp., Anaerobiospirillum sp., Bacteroides sp., Mannheimia sp., Succinimonas sp._s Succinivibrio sp. and recombinant E. coli, more preferably a microorganism Mannheimia sp., but is not limited thereto as long as it can produce succinic acid.

In the present invention, the microorganism Mannheimia sp. is preferably selected from the group consisting of M. succiniciproducens LPK, M. succiniciproducens LPK7, M. succiniciproducens PALK, M. succiniciproducens ALKt and M. succiniciproducens ALK.

The inventive method for preparing succinic acid using sucrose uses sucrose as a carbon source, and is effective to produce succinic acid at a high concentration by efficient use of sucrose by a succinic acid-producing microorganism.

When sucrose is used as a carbon source, the final succinic acid concentration is much higher than that resulted from culture processes using various kinds of carbon sources including glucose, and resistance against various kinds of organic acids including succinic acid, which inhibit cell growth, is significantly improved, thus making it possible to achieve excellent succinic acid-productivity. Specifically, in the method for producing succinic acid using sucrose according to the present invention, succinic acid productivity can be increased by more than 19% compared to a method using glucose as a carbon source.

The study results that sucrose functions to protect microorganisms from an adverse external environment, was reported recently. Kiliman et al have performed an experiment in which Lactococcus lactis strain was exposed to a medium containing sucrose and a medium containing no sucrose at the lethal temperature for a certain period of time to examine its viability and protein structure, and as a result, found that viability of the strain, which was exposed to the medium containing sucrose, was much higher than that of the strain, which was exposed to the medium containing no sucrose, and secondary structures of proteins present inside a cell were preserved much better in the strain exposed to the medium with sucrose than they were in the strain exposed to the medium without sucrose (Kilimann et al, Biochimica et Biophysica Acta, 1764:1188, 2006). The above result indicates that sucrose functions to protect cells from the harmful environments to cells.

In the present invention, the culture of the succinic acid producing microorganisms and recovery process of succinic acid can be performed by the culture methods known in the conventional fermentation process and methods for separating and purifying succinic acid.

Examples

Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention.

Particularly, the following examples illustrate only Mannheimia sp. which is a succinic acid-producing microorganism, as a host cell, but, it is obvious to a person skilled in the art that other kinds of succinic acid producing microorganisms can also be used as a host cell.

Example 1: Batch culture using sucrose as a culture material in a chemically defined medium

ImL of M. succiniciproducens PALK (KCTC 10973BP), which was kept in a 15% glycerol solution at -7O⁰C, was inoculated into 19ml of complex medium containing 50 mM of glucose, and cultured in anaerobic conditions at 39°C for 8hr, and then 2.5mL of the culture broth was again moved to 250ml of complex medium containing 5OmM of sucrose and cultured at 39⁰C for 8hr. Batch culture was performed by inoculating 250ml of the culture broth into a fermentor containing 2.25L of chemically defined medium under conditions of a first sucrose concentration of 13OmM at 39⁰C and 200rpm. In order to keep anaerobic conditions during the fermentation, carbon dioxide was continuously supplied at a flow rate of 0.2wm(500mL/min). pH during the fermentation was adjusted to 6.5 by using 28%(w/v) ammonia solution, and 50μ g/L of spectinomycin was added to the medium as an antibiotic. The complex medium consisted of 5.0g/L of yeast extract, 5.0g/L of polypeptone, l.Og/L of NaCl, 8.708g/L Of K₂HPO₄, 9.996g/L of NaHCO₃, 0.02g/L of CaCl₂-2H₂O and 0.2g/L of MgCl₂-OH₂O. The chemically defined medium consisted of l.Og/L of NaCl, l.Og/L of (NH₄)₂SO₄, 8.708g/L of K₂HPO₄, 9.996g/L of NaHCO₃, 0.02g/L of CaCl₂-2H₂O, 0.2g/L of MgCl₂-OH₂O, 5mL/L of trace metal solution (Lee et ai, J. Environ. Polymer Degrad, 4:131, 1996), O.5g/L of cysteine, O.5g/L of methionine, O.5g/L of alanine, O.5g/L of asparagine, 0.5g/L of aspartic acid, O.5g/L of proline, O.5g/L of serine, 0.005g/L of nicotinic acid, 0.005g/L of Ca-pantothenate, 0.005g/L of pyridoxine-HCl, 0.005g/L of thiamine, 0.005g/L of ascorbic acid and 0.005g/L of biotin.

The concentration of cells in the culture broth was measured with a spectrophotometer, and then calculated using the previously measured optical density at 600 nm (OD₆₀o) and the verification test for dried-cell weight. During the fermentation, samples were collected from the fermentor regularly. The collected samples were centrifuged at 13,000 rpm at 4°C for 10 minutes, and then the supernatants were used to analyze the concentrations of various kinds of organic acids including succinic acid and ethanol, produced as metabolites, and sucrose used as a carbon source by using a High-Performance Liquid Chromatography.

As shown in FIG. 2 and Table 1, M. succiniciproducens PALK strain completely consumed 22.26g/L of sucrose in the chemically defined medium after 8hr culture, and produced 17.76g/L of succinic acid as a final product. At this time, succinic acid-productivity was 2.22g/L, which is a 25% increase in succinic acid productivity compared with Comparative example 1 in which glucose was used as a carbon source. Unlike Comparative example 1 in which glucose was used as a carbon source, when sucrose was used as a carbon source, cell growth was not inhibited by organic acids including succinic acid until sucrose was completely consumed. Comparative example 1; Batch culture using glucose as a culture material in a chemically defined medium

Batch culture and analysis for culture broth in a composition medium using glucose instead of sucrose as a carbon source for M. succiniciproducens PALK strain were performed under the same conditions, using the same methods as described in Example 1. The concentration of glucose used as a raw material was also analyzed under the same conditions, using a High-Performance Liquid Chromatography as described in Example 1.

As shown in FIG. 3 and Table 1, M. succiniciproducens PALK strain completely consumed 23.43g/L of glucose in the chemically defined medium after lOhr culture, and produced 17.81g/L of succinic acid as a final product. At this time, succinic acid-productivity was 1.781g/L/h. Most of all, when the concentration of succinic acid was more than 2.11g/L during the fermentation, cell growth rate was sequentially and rapidly decreased.

Table 1 : Comparison between succinic acid-productivities in a medium containing glucose and a medium containing sucrose as a raw material in a batch culture using M. succiniciproducens PALK

Example 2: Fed-batch culture using sucrose as a culture material in a chemically defined medium

M. succiniciproducens PALK strain was cultured using sucrose as a raw material in a chemically defined medium under fed-batch culture conditions. At this time, initial culture conditions were the same as those in Example 1, and 5OmM of glycerol was added to an initial medium for reduction. When sucrose concentration in the medium became lower than 10g/L, 700g/L of concentrated sucrose solution was intermittently supplied to a fermentor to maintain sucrose concentration in the medium at 5~10g/L, thus attempting to produce succinic acid at a high concentration.

As a result, as shown in FIG. 4 and Table 2, M. succiniciproducens PALK strain produced 73.5g/L of succinic acid in the medium containing sucrose after 20hr culture, and at this time, succinic acid-productivity was 3.68g/L/h, which indicates that the final succinic acid concentration was increased by 19% and overall succinic acid-productivity was also increased by 19% compared with the case where glucose is used as a raw material. In addition, succinic acid-productivity in the medium containing sucrose showed a maximum value of 4.6g/L/h, producing 55.19g/L of succinic acid at lhr after the culture, which is 22% higher than that of the case where glucose is used as a raw material. Moreover, the maximum volumetric productivity of succinic acid in the medium using sucrose as a raw material was 7.94g/L/h at 7.5-9 hr after the culture, and this is a more than 62% increase compared with the case using glucose, which suggests that, if sucrose is used as a raw material in an actual fermentation process, it is possible to increase succinic acid-productivity by more than 62% compared with the case using glucose. Specifically, cell growth rate was remarkably increased since cell growth was not inhibited by organic acids including succinic acid until succinic acid concentration in the medium reached 24.5g/L, and the cell concentration showed a maximum value of 4.49g/L 9hr after the culture. That is, maximum cell concentration in the medium using sucrose as a raw material was increased by more than 40% compared with the medium using glucose as a raw material. Although, unlike the case using glucose as a raw material, succinic acid production was observed even after 20hr culture in the case of using sucrose as a raw material, the culture was performed only until 20hr in order to compare with the case using glucose.

Comparative example 2: Fed-batch culture using glucose as a culture material in a chemically defined medium

M. succiniciproducens PALK strain was cultured using glucose as a raw material in a chemically defined medium under fed-batch culture conditions. At this time, initial culture conditions were the same as those in Example 2, and 5OmM of glycerol was added to an initial medium for reduction. In order to produce succinic acid at a high concentration, when glucose concentration in the medium became lower than 10g/L, 700g/L of concentrated glucose solution was intermittently supplied to a fermentor to maintain glucose concentration in the medium at 5~10g/L.

As a result, as shown in FIG. 5 and Table 2, M. succiniciproducens PALK strain produced 61.9g/L of sucrose in the medium containing glucose after 20hr culture, and at this time, succinic acid-productivity was 3.09g/L/h. Succinic acid- productivity showed a maximum value of 3.77g/L/h, producing 47.12g/L of succinic acid at 12.5hr after the culture. In addition, the maximum volumetric productivity of succinic acid (the amount of succinic acid produced per unit culture time and unit culture volume) was 4.88g/L/h, which was observed at 6.5~8hr after the culture. The maximum volumetric productivity of succinic acid represents the productivity at which succinic acid can be produced at the maximum level through fermentation, which is industrially very important. Cell growth rate was gradually decreased starting 1.33hr after the culture, at which succinic acid concentration was 3.5g/L, and cell concentration showed a maximum value of 3.19g/L at 9.5 hr after the culture, and then started to decrease thereafter. After 20hr culture, succinic acid was not more produced.

Table 2: Comparison between succinic acid-productivities in a medium containing glucose and a medium containing sucrose under fed-batch culture conditions using M. succiniciproducens PALK

INDUSTRIAL APPLICABILITY

As described in detail above, the method for preparing succinic acid according to the present invention is effective for increasing the final succinic acid concentration since sucrose decreases cell growth inhibition by succinic acid and various kinds of organic acid, and thus, costs required for separating and purifying succinic acid from the final culture broth can be significantly reduced. Especially, sucrose as a raw material is very inexpensive compared with the existing carbon sources including glucose, and thus, can outstandingly contribute to the replacement of chemical process for succinic acid production by biological process therefor. Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

THE CLAIMS

What is Claimed is:

L A method for preparing succinic acid using sucrose, the method comprises culturing a succinic acid-producing microorganism in a medium containing sucrose as a carbon source.

2. The method for preparing succinic acid using sucrose according to claim 1, wherein the culture is batch culture or fed-batch culture.

3. The method for preparing succinic acid using sucrose according to claim 1, wherein the succinic acid-producing microorganism is selected from the group consisting of Actinobacillus sp., Anaerobiospirillum sp., Bacteroides sp., Mannheimia sp., Succinimonas sp., Succinivibrio sp. and recombinant Escherichia? coli.

4. The method for preparing succinic acid using sucrose according to any one claim among claims 1-3, wherein succinic acid-productivity is increased by more than 19% compared to a method using glucose as a carbon source.

5. A method for preparing succinic acid using sucrose, the method comprising culturing a succinic acid-producing microorganism Mannheimia sp. in a medium containing sucrose as a carbon source.

6. The method for preparing succinic acid using sucrose according to claim 5, wherein the culture is batch or fed-batch culture.

7. The method for preparing succinic acid using sucrose according to claim 5, wherein the succinic acid-producing microorganism Mannheimia sp. is selected from the group consisting of M. succiniciproducens LPK, M. succiniciproducens LPK7, M. succiniciproducens PALK, M. succiniciproducens ALKt and M. succiniciproducens ALK

8. The method for preparing succinic acid using sucrose according to any one claim among claims 5-7, wherein succinic acid productivity is increased by more than 19% compared to a method using glucose as a carbon source.