WO2009048202A1

WO2009048202A1 - Method for preparing succinic acid using glycerol as carbon source

Info

Publication number: WO2009048202A1
Application number: PCT/KR2008/000237
Authority: WO
Inventors: Sang Yup Lee; Jeong Wook Lee; Hyohak Song; Sol Choi; Ji Mahn Kim
Original assignee: Korea Advanced Institute of Science and Technology KAIST
Current assignee: Korea Advanced Institute of Science and Technology KAIST
Priority date: 2007-10-09
Filing date: 2008-01-15
Publication date: 2009-04-16
Anticipated expiration: 2010-04-09

Abstract

The present invention relates to a method for preparing succinic acid using glycerol as a carbon source, and more particularly, a method for preparing succinic acid with high purity and high yield, which comprises inoculating Mannheimia sp., which is a facultative anaerobic microorganism, into a culture medium containing glycerol as a carbon source and anaerobically culturing, then recovering succinic acid. According to the present invention, a significant amount of succinic acid can be produced compared with the methods using carbon sources such as glucose or sucrose, and formation of total by-products is inhibited or prevented upon microbial fermentation, thus making it possible to reduce costs required for separating and purifying succinic acid from final culture broth.

Description

Method for Preparing Succinic Acid Using Glycerol as Carbon Source

TECHNICAL FIELD

The present invention relates to a method for preparing succinic acid using glycerol as a carbon source, and more particularly, a method for preparing succinic acid with high purity and high yield, which comprises anaerobically culturing a succinic acid-producing microorganism such as Mannheimia sp., etc. in a culture medium containing glycerol as a carbon source, and then recovering succinic acid.

BACKGROUND ART

Succinic acid having two carboxylic groups can be used as a precursor of various chemical products which are industrially important, and thus the market for succinic acid is expected to grow dramatically (Zeikus et ah, Apph Microbiol. Biotechnoh, 51:545, 1999; Song et ah, Enzyme Microbial Technoh, 39: 352, 2006; Willke et al., Apph Microbiol. Biotechnoh, 66: 131 , 2004).

At present, most of succinic acid is produced by chemical synthesis using petroleum. However, the price of petroleum has been constantly rising and the pace of regulations for environmental pollution is increasing, under these circumstances there is an urgent need to develop a process for efficiently producing succinic acid using microorganisms, which can replace the current chemical synthesis based process. With recent development of culture technology, forecasting technology using a virtual cell model, strain improvement technology and metabolic engineering technology, technologies for succinic acid production using microorganisms have been rapidly developing throughout the world (Kim et ah, Biotech. Bioeng., 97:657, 2007; Song et al., Enzyme Microbial TechnoL, 39:352, 2006).

Recently, studies on succinic acid production from renewable raw materials through microbial fermentation are being conducted using various microorganisms such as Actinobacillus sp., Anaerobiospirillum sp., Bacteroides sp., Corynebacterium sp., Escherichia sp., Mannheimia sp., Succinimonas sp., Succinivibrio sp., which have the ability to produce succinic acid, and also E. colh by genetic manipulation.

The present inventors have reported that they isolated an excellent Mannheimia succiniciproducens MBEL55E (KCTC 0769BP) producing succinic acid with high efficiency from the rumen of Korean cow, and completed its full genome sequence and characterized the metabolic properties thereof (Hong et al, Nature Biotechnol., 22:1275, 2004). Also, the present inventors have constructed a mutant, M. succiniciproducens LPK (KCTC 10558BP) by disrupting a gene encoding lactate dehydrogenase(/d/k4) and a gene encoding pyruvate formate-lyase(/?/7) from M. succiniciproducens MBEL 55E and a mutant M. succiniciproducens LPK7 (KCTC 10626BP) by disrupting a phosphotransacetylase gene(pta) and an acetate kinase genc(ackA) 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 bacterial mutant, M. succiniciproducens PALK (KCTC 10973BP) (PCT/KR2007/003574) by disrupting a lactate dehydrogenase gva&ildhA), a phosphotransacetylase g&ne(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 the 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. Glycerol has advantages due to a low price and a high degree of reduction as a feedstock in microbial fermentation, compared with glucose, sucrose, xylose and the like, which are widely used as carbon sources in microbial fermentation. Meanwhile, microbial fermentation requires various chemical substances such as nitrogen, phosphorus, sulfur, etc. in addition to pure glycerol, and crude glycerol itself can be used as a good culture medium for microorganisms because it contains the above mentioned substances. Especially, succinic acid, ethanol and propanediol (1,2-propanediol, 1,3 -propanediol), which are produced through microbial fermentation, are more reduced metabolites than glucose, which causes oxidation/reduction inbalance in glucose fermentation via metabolic pathways, thus resulting in a lowered production yield with respect to the raw material, whereas theoredically, 1 mole of glycerol can provide all of 14 electrons required to produce 1 mole of succinic acid. Therefore, there is an advantage in that, when glycerol, which is a more reduced raw material than glucose, is used as a carbon source, it is possible to avoid a problem of oxidation/reduction inbalance, and thus, it is possible to increase production yield of more reduced chemicals than glucose, with respect to raw material.

Although some microorganisms cannot use glycerol as a carbon source, lots of microorganisms can use glycerol as a carbon source. Recently, there has been a study using E. coli, reporting that fermentation environment such as pH affects whether microorganisms can utilize glycerol, and more reduced chemicals, including ethanol, than glucose can be more efficiently produced using glycerol (Dharmadi et al, Biotehcnol. Bioeng., 94:822, 2006).

Also, an attempt to increase succinic acid production by culturing Anaerobiospirillum succiniciproducens using glycerol as a carbon source has been reported (Korean Patent Registration 10-0313134). In this case, succinic acid productivity could be increased to a certain extent by preventing the formation of acetic acid as a by-product, but no study succeeded in the production of homo- succinic acid using glycerol as a carbon source has been reported, including a study on the prevention of formation of formic acid, pyruvic acid, ethanol and the like, which are formed as by-products except acetic acid upon anaerobic fermentation of the above mentioned strain (US Patent 5, 143,834).

In a process for succinic acid production, costs required for separating and purifying succinic acid from the final culture broth completely depend on the amount of by-products (acetic acid, pyruvic acid, formic acid, lactic acid, ethanol etc.), the kind and number of by-products and various ingredients used as raw materials. According to the above mentioned study, a complex culture medium, which comprises excess amounts of expensive yeast extract and polypeptone whose chemical compositions are not known, was used, and succinic acid productivity was increased in the complex culture medium comprising large amounts of the above mentioned components. It has been known that it is difficult to apply the complex culture medium to industrial processes due to an increase in separation and purification costs caused by various unknown chemical components present in the medium, and high raw material costs. Specifically, it is generally known in the art that, since A. succiniciproducens is an obligate anaerobe, it is not suitable for producing succinic acid in a practical process. Thus, there is an urgent need to develop a process for producing homo-succinic acid at high yield, which comprises culturing a facultative anaerobic microorganism, not an obligate anaerobic microorganism in a chemically defined_f synthetic culture medium containing no expensive medium compounds such as yeast extract or polypeptone.

As a method for treating a large amount of glycerol produced as a by-product in the above-mentioned various processes, propylene glycol production is drawing much attention. Typical examples of propylene glycol compounds include 1,2- propanediol and 1,3 -propanediol, and they have been mainly produced by chemical synthesis till now. The annual production of propylene glycol compounds is about 4.5 billion kg and the production rate thereof increases by about 4% every year. However, there is a disadvantage in the production of propylene glycol through a chemical synthesis requires reaction conditions of high temperature and high pressure, which causes an increase in process initial capital costs. Therefore, there is a need to develop a technology for producing chemicals, which employs a low- temperature and low-pressure fermentation process using glycerol for culturing facultative anaerobic microorganisms that are relatively easy to culture.

However, not much progress has been made in studies relating to succinic acid production through microbial fermentation using glycerol until now, and the importance of studies on the understanding of metabolic pathways involving glycerol and the application thereof and studies on the production of various more reduced metabolites than glucose using glycerol as a carbon source, will increase as biodiesel demand increases.

In order for the succinic acid production process through microbial fermentation to be competitive in succinic acid market where most of succinic acid is at present produced from petroleum, it is necessary to develop inexpensive, reduced raw materials such as glycerol together with continuous technology development. Moreover, when considering raw material prices, and separation and purification costs account for most of succinic acid production costs in the production of chemicals through microbial fermentation, there is an urgent need to develop a technology for producing succinic acid, which uses a chemically defined synthetic culture medium and provides a high production yield with respect to a raw material and, at the same time, can minimize the total amount of by-products generated during fermentation or produce homo-succinic acid.

Accordingly, the present inventors have made extensive efforts to develop a technology to produce homo-succinic acid using glycerol as a carbon source, and as a result, confirmed that homo-succinic acid can be produced at a high yield while remarkably decreasing the amount of total by-products by culturing Mannheimia sp. microorganisms in a culture medium containing glycerol, which is inexpensive, abundant in nature and more reduced than glucose, as a carbon source and recovering succinic acid from the culture broth, thereby completing the present invention.

SUMMARY OF INVENTION

It is an object of the present invention to provide a method for preparing homo- succinic acid with high yield using glycerol as a carbon source

To achieve the above object, the present invention provides a method for preparing succinic acid using glycerol as a carbon source, which comprises the steps of culturing a succinic acid-producing microorganism in a glycerol-containing culture medium; and recovering succinic acid from the culture broth.

In addition, the present invention provides a method for preparing succinic acid using glycerol as a carbon source, which comprises the steps of culturing M. succiniciproducens PALK in a glycerol-containing chemically defined culture medium; and recovering succinic acid from the culture broth.

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 a metabolic pathway in which succinic acid is synthesized using glycerol as a raw material in succinic acid-producing rumen bacteria. FIG.2 is a graph showing succinic acid production characteristics of M. succiniciproducens PALK in a batch culture using glycerol as a carbon source.

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

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

FIG.5 is a graph showing succinic acid production characteristics of M. succiniciproducens PALK in a batch culture using glycerol together with glucose as carbon sources.

FIG.6 is a graph showing succinic acid production characteristics of M. succiniciproducens PALK in a batch culture using glycerol together with sucrose as carbon sources.

DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS

In one aspect, the present invention relates to a method for preparing succinic acid using glycerol as a carbon source, which comprises the steps of culturing a succinic acid-producing microorganism in a glycerol-containing culture medium; and recovering succinic acid from the culture broth.

Specifically, the method for preparing succinic acid according to the present invention is preferably performed by inoculating Mannheimia sp., which is a facultative anaerobic microorganism, into a culture medium containing glycerol as a carbon source, and then anaerobically culturing. In the present invention, glycerol enables succinic acid production with high yield by properly providing reducing equivalents required for succinic acid production, and effectively prevents the formation of by-products during fermentation in a culture medium, thus making it possible to reduce raw material prices needed for succinic acid production and costs required for separating and purifying succinic acid from final culture broth.

In the present invention, glycerol is preferably contained in a culture medium, either alone or together with other carbon sources as a carbon source and the other carbon sources except glycerol is preferably selected from the group consisting of glucose, sucrose and xylose.

In the present invention, the culture medium is preferably a chemically defined culture medium, and preferably contains NaCl, (NH₄)₂SO₄, K₂HPO₄, NaHCO₃, CaCl₂-2H₂O, MgCl₂-OH₂O, cysteine, methionine, alanine, asparagine, aspartic acid, proline, serine, nicotinic acid, Ca-pantothenate, pyridoxine-HCl, thiamine, ascorbic acid and biotin.

In the present invention, a facultative anaerobic microorganism Mannheimia sp. is cultured under anaerobic conditions in a chemically defined culture medium whose exact chemical composition is known, without using a complex culture medium comprising excess amounts of yeast extract and polypeptone, thus producing homo- succinic acid with high yield. There is a method in the prior art that enables synthesis of succinic acid using glycerol as a carbon source in succinic acid- producing rumen bacteria (FIG.1).

In the present invention, the culture is preferably performed using a method selected from the group consisting of batch culture, fed-batch culture and continuous culture. In the present invention, the succinic acid-producing microorganism is preferably a bacterium selected from the group consisting of Actinobacillus sp., Anaerobiospirillum sp., Bacteroides sp., Corynebacterium sp., Escherichia sp., Mannheimia sp., Succinimonas sp. and Succinivibrio sp., and the microorganism Mannheimia sp. is preferably selected from the group consisting of M. succiniciproducens LPK, M. succiniciproducens LPK7, M. succiniciproducens PALK and M. succiniciproducens ALKt.

The inventive process of culturing a succinic acid-producing microorganism Mannheimia sp. and recovering succinic acid can be performed using a conventional culture method and a method for separating and purifying succinic acid, which are generally known in prior art fermentation.

In the present invention, the succinic acid yield is preferably 20-90% based on the amount of added carbon sources including glycerol.

In the present invention, succinic acid can be produced with much less by-product formation upon microbial fermentation, and the production ratio of succinic acid to by-products is preferably more than 70%.

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 M. succiniciproducens PALK, which is a microorganism Mannheimia sp., but, it is obvious to a person skilled in the art that other microorganisms can also be used. Moreover, the following examples illustrate only batch culture, but fed-batch and continuous culture methods are intended to be included within the scope of the present invention.

Example 1: Batch culture using glycerol as a carbon source in a chemically defined culture medium

1 ml of M succiniciproducens PALK, which was kept in 15% glycerol solution of - 70°C, was inoculated into 19 ml of complex medium containing 50 mM of glucose (Table 1), and cultured in anaerobic conditions at 39°C for 8 hr, and then 2.5 ml of the culture broth was again transferred to 250 ml of complex medium containing 25 mM of glycerol and cultured at 39⁰C for 8 hr.

250 ml of the culture broth was inoculated into a bioreactor containing 2.25 L of chemically defined medium (Table 2), and batch culture was performed under conditions of an initial glycerol concentration of 50 mM, a culturing temperature of 39°C and a stirring rate of 200rpm. In order to keep anaerobic conditions during the culture, carbon dioxide was continuously supplied with a flow rate of 0.2 wm (500 ml/min), and pH during the culture was adjusted to 6.5 using 28% (w/v) ammonia solution. 50 μg/L of spectinomycin as an antibiotic was added.

Cell concentration in the culture broth during the culture was estimated with a spectrophotometer using the previously measured optical density at 600 nm (OD₆Qo) and the verification test for dried-cell weight. During the culture, samples for the analysis of organic acids including succinic acid, and ethanol produced as metabolites, and glycerol used as a carbon source were collected from the bioreactor regularly, and centrifuged at 13,000 rpm at 4⁰C for 10 minutes, followed by analyzing the supernatants using a High-Performance Liquid Chromatography, thus calculating concentrations thereof. Table 1 : Composition of a complex medium

Table 2: Composition of a chemically defined medium

As a result, as shown in FIG. 2 and Table 3, after M succiniciproducens PALK was cultured in a chemically defined medium, 3.9623 g/L of glycerol was consumed, and 4.8124 g/L of succinic acid was produced as a final product. Succinic acid yield based on glycerol consumed was 1.2146 g/g (1.8944 mol/mol, per mole of glucose), which is a 72.33% increase in the weight yield of succinic acid (79.17%, per mole of glucose) compared with Comparative example 1 in which glucose was used as a carbon source. In addition, the weight yield of succinic acid was increased by 76.26% (89.69%, per mole of glucose) compared with Comparative example 2 in which sucrose was used as a carbon source. Especially, when M. succiniciproducdens PALK was cultured in a chemically defined medium whose exact culture composition is known, using only glycerol as a carbon source, only homo-succinic acid was only produced without production of acetic acid and pyruvic acid, which are produced as by-products during fermentation in a culture medium containing other carbon sources, as shown in Comparative examples 1 and 2, and Examples 2 and 3.

Table 3 : Succinic acid yield and production ratio of succinic acid to by-products according to carbon sources

Comparative example 1: Batch culture using glucose as a carbon source in a chemically defined culture medium A batch culture of M. succiniciproducens PALK in a chemically defined medium using glucose instead of glycerol as a single carbon source, and analysis for the culture broth were performed under the same conditions, using the same method as described in Example 1. Glucose used as a raw material was also analyzed under the same conditions, with the same method as described in Example 1, using a High-Performance Liquid Chromatography.

As a result, as shown in FIG. 3 and Table 3, after M. succiniciproducdens PALK was cultured in the chemically defined medium, 18.45 g/L of glucose was consumed, and 13.00 g/L of succinic acid was produced as a final product. At this time, succinic acid yield based on glucose consumed was 0.7048 g/g (1.0573 mol/mol), and particularly, 0.69 g/L of acetic acid and 4.83 g/L of pyruvic acid were produced, suggesting that the production ratio of succinic acid to by-products is 2.3587 g/g (1.6623 mol/mol).

Comparative example 2: Batch culture using sucrose as a carbon source in a chemically defined culture medium

A batch culture of M. succiniciproducens PALK in a chemically defined medium using sucrose instead of glycerol as a single carbon source, and analysis for the culture broth were performed under the same conditions, using the same method as described in Example 1. Sucrose used as a raw material was also analyzed under the same conditions, with the same method as described in Example 1, using a High-Performance Liquid Chromatography.

As a result, as shown in FIG. 4 and Table 3, after M. succiniciproducdens PALK was cultured in the chemically defined medium, 22.46 g/L of sucrose was consumed, and 15.48 g/L of succinic acid was produced as a final product. At this time, succinic acid yield based on sucrose consumed was 0.6891 g/g (0.9987 mol/mol, per mole of glucose), and additionally, 3.96 g/L of pyruvic acid was produced as a by-product, suggesting that the production ratio of succinic acid to by-product is 3.9083 g/g (2.9144 mol/mol).

Example 2: Batch culture using glycerol and glucose as carbon sources in a chemically defined culture medium

A batch culture of M. succiniciproducens PALK was performed in a chemically defined medium containing 50 mM of glycerol and 100 mM of glucose. The batch culture and analysis for the culture broth were performed under the same conditions, with the same method as described in Example 1. Glycerol and glucose used as raw materials were also analyzed under the same condition, with the same method as described in Example 1, using a High-Performance Liquid Chromatography.

As a result, as shown in FIG. 5 and Table 3, after M. succiniciproducdens PALK was cultured in the chemically defined medium, 18.63 g/L of glucose and 3.04 g/L of glycerol were consumed, and 18.87 g/L of succinic acid was produced as a final product. At this time, succinic acid yield based on the sum of glucose and glycerol used as carbon sources was 0.8706 g/g (1.3323 mol/mol, per mole of glucose), which is a 23.52% increase in the weight yield of succinic acid (26.01%, per mole of glucose), compared with Comparative example 1 in which glucose alone was used as a carbon source. And 0.47g/L of acetic acid and 1.59 g/L of pyruvic acid were produced as by-products, suggesting that the production ratio of succinic acid to by-products was 9.1700 g/g (6.1824 mol/mol), which is a 289% increase (272% mol/mol) based on weight, compared with Comparative example 1 in which glucose alone was used as a carbon source.

Example 3: Batch culture using glycerol and sucrose as carbon sources in a chemically defined culture medium A batch culture of M. succiniciproducens PALK was performed in a chemically defined medium containing 5OmM of glycerol and 5OmM of sucrose. The batch culture and analysis for the culture broth was performed under the same conditions, with the same method as described in Example 1. Glycerol and sucrose used as raw materials were also analyzed under the same condition, with the same method as described in Example 1 using a High-Performance Liquid Chromatography.

As a result, as shown in FIG. 6 and Table 1, after M. succiniciproducdens PALK was cultured in the chemically defined medium, 19.06 g/L of sucrose and 1.66g/L of glycerol were consumed, and 17.17g/L of succinic acid was produced as a final product. At this time, succinic acid yield based on the sum of glucose and glycerol used as carbon sources was 0.8286 g/g (1.2077 mol/mol, per mole of glucose), which is a 20.24% increase in the weight yield of succinic acid (20.93%, per mole of glucose), compared with Comparative example 2 in which sucrose alone was used as a carbon source. And 0.61 g/L of acetic acid and 1.65g/L of pyruvic acid were produced as by-products, suggesting that the production ratio of succinic acid to by-products was 7.6081 g/g (5.0416 mol/mol), which is a 94.67% increase (72.99% mol/mol) based on weight, compared with Comparative example 2 in which sucrose alone was used as a carbon source.

INDUSTRIAL APPLICABILITY

As described in detail above, according to the present invention, a significant amount of succinic acid can be produced compared with the conventional methods using carbon sources such as glucose or sucrose, particularly, formation of total byproducts is inhibited or prevented, thus reducing costs required for separating and purifying succinic acid from the final culture broth. In addition, Mannheimia sp., which is a facultative anaerobic microorganism, not an obligate anaerobic microorganism, is cultured in a chemically defined medium whose exact chemical composition is known, and thus homo-succinic acid can be produced at high yield. 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 glycerol as a carbon source, which comprises the steps of culturing a succinic acid-producing microorganism in a glycerol-containing culture medium; and recovering succinic acid from the culture broth.

2. The method according to claim 1, wherein the succinic acid-producing microorganism is a bacterium selected from the group consisting of Actinobacillus sp., Anaerobiospirillum sp., Bacteroides sp., Coryne bacterium sp_v Escherichia sp., Mannheimia sp., Succinimonas sp. and Succinivibrio sp.

3. The method according to claim 1, wherein the glycerol is contained as a sole carbon source in a culture medium.

4. The method according to claim 1, wherein the glycerol is contained together with other carbon sources except glycerol in a culture medium.

5. The method according to claim 4, wherein the other carbon source except glycerol is selected from the group consisting of glucose, sucrose and xylose.

6. The method according to claim 1, wherein the culture medium is a chemically defined culture medium.

7. The method according to claim 6, wherein the chemically defined culture medium contains NaCl, (NH₄)₂SO₄, K₂HPO₄, NaHCO₃, CaCl₂-2H₂O, MgCl₂-OH₂O, cysteine, methionine, alanine, asparagine, aspartic acid, proline, serine, nicotinic acid, Ca-pantothenate, pyridoxine-HCl, thiamine, ascorbic acid and biotin.

8. The method according to claim I₅ wherein the culture is performed using a method selected from the group consisting of batch culture, fed-batch culture and continuous culture.

9. The method according to claim 2, wherein the microorganism is selected from the group consisting of M. succiniciproducens LPK, M. succiniciproducens LPK7, M. succiniciproducens PALK, and M. succiniciproducens ALKt.

10. The method according to any one claim among claims 1-9, wherein by-products are not formed upon succinic acid production by a microbial fermentation.