KR20010002820A - Method for Manufacturing Succinic Acid Using Glycerol - Google Patents

Abstract

PURPOSE: A method for producing succinic acid using glycerol is provided, thereby acetic acid, which is generally produced as a byproduct in production processes of succinic acid using glucose, can be remarkably inhibited, so that the production costs can be lowered. CONSTITUTION: The succinic acid is produced by batch culturing microorganism selected from Anaerobiospirillum succiniciproducens, Succinivibrio dextrinosolvens, Fibrobacter succinogenes, or Ruminococcus flavefaciens, preferably Anaerobiospirillum succiniciproducens in a medium. The medium comprises 5-30g of glycerol and 1-15g of yeast extract per 1L thereof, and is suitable for culturing anaerobic microorganisms. The culture is carried out at the temperature of 35 to 42 deg.C, preferably 37 to 40 deg.C, and most preferably 39 deg.C, and at pH 6.0 to 7.5, with supplying carbonic acid gas at a flow rate of 0.25 to 0.5 vvm.

Description

Method for manufacturing succinic acid using glycerol {Method for Manufacturing Succinic Acid Using Glycerol}

The present invention relates to a method for producing succinic acid using glycerol. More specifically, the present invention is inoculated succinic acid fermentation microorganisms in a culture medium containing glycerol (glycerol) which is a waste from the oil industry as a culture material, and cultured through a batch culture process, high yield of succinic acid And it relates to a production method of succinic acid using glycerol that can be produced with high productivity.

Recently, due to the strengthening of environmental regulations, the production cost, utilization and processing cost of petrochemical products have risen sharply, but biochemicals by biological methods are supported not only by strengthening environmental regulations but also by the breakthrough development of microbial culture technology and genetic engineering technology. The production of biochemicals is of increasing interest. This method is recognized for its economic superiority over chemical synthesis in the sense that the raw materials are cheap renewable resources (Landucci, et al., Appl. Biochem. Biotechnol., L45: 678-696 (1994)). As it is an environmentally friendly process, research is gradually expanding to reduce costs by mass production. In addition, the price of biochemicals has recently decreased a lot, and thus the marketability of such biochemicals has been very high. For this reason, lactic acid, acetic acid or succinic acid from biomass using microorganisms is used. Efforts to produce organic acids, such as succinic acid, have been actively made (Lim Euk-kyu, Bioindustry, 5: 60-75 (1992)).

The study of the biological production of succinic acid began in 1938 when Lockwood et al. Announced the production of succinic acid from sugar by 18% yield using the microorganism Fusarium martii. Thereafter, various types of anaerobic microorganisms (Succinivibrio dextrinosolvens), Fibrobacter succinogenes or Ruminococcus flavefaciens, etc. (see Zeikus, Annu. Rev. Microbiol., 34: 423-464 (1980)) has been reported to produce succinic acid as a final product through glucose metabolism, but no industrially useful strains producing high yields of succinic acid have been reported. However, unlike recent succinic acid-producing anaerobic microorganisms, Anaerobiospirillum succinici producens, which produces succinic acid in high concentrations and yields from glucose in the presence of excess carbonic acid, directly produces succinic acid. It turns out to be one of the most promising strains to produce.

On the other hand, glycerol is a by-product from the oil industry, and is a renewable resource that can be usefully reused through processing, and can use this to produce useful biochemicals by microorganisms. Glycerol is currently produced in excess in soap manufacturing, fatty acid manufacturing, wax and surfactant production manufacturing, etc. To date, compounds studied or produced using glycerol are reuterin (Talarico, et al., Antimicrob). Agents Chemother., 32: 1854-1858 (1988)), 2,3-butanediol (2,3-butanediol, see Biebl, et al., Appl. Microbiol.Biotechnol., 50: 24-29 (1998) ), 1,3-propanediol (1,3-propanediol, see Menzel, et al., Enzyme Microb. Technol., 20: 82-86 (1997)). However, despite efforts to produce useful biochemicals using glycerol as a starting material, studies on succinic acid production method using glycerol have not been conducted at home and abroad.

On the other hand, due to the steady development of the oil industry, the problem of overproduction of glycerol has been continuously raised, and in consideration of environmental issues, research on biochemicals, especially succinic acid, using glycerol as a starting material is being conducted. In addition, since succinic acid is one of the few alternative biochemicals that are competitive in price compared to the current chemical process, many studies are expected to be conducted in the future, but the research on the production process has not been carried out yet. Therefore, not only the production of succinic acid by biological method is economical, but also in consideration of environmental aspects, the development of a culture process that can efficiently use glycerol, in particular, the establishment of culture processes and conditions that can produce succinic acid using glycerol Therefore, there is a constant need to develop a technology that can effectively utilize glycerol.

Therefore, the present inventors have made efforts to establish a technology that can effectively produce succinic acid by the culturing process using glycerol efficiently, instead of glucose used in the existing culturing process, succinic acid using glycerol as a by-product and waste of the oil industry As a result of the development of a batch culture process capable of producing succinic acid from a culture medium containing glycerol through the microbial culture technology, it was possible to produce succinic acid with a higher yield than previously reported succinic acid yield. It was confirmed that the production of acetic acid, which is a by-product produced, can be significantly suppressed, thereby completing the present invention.

After all, the main object of the present invention is to provide a method for producing succinic acid using glycerol to effectively produce succinic acid by a batch culture process using glycerol as a culture raw material.

1 is a graph showing the fermentation characteristics of succinic acid in a batch culture using glycerol.

2 is a graph showing the fermentation characteristics of succinic acid in the batch culture using glycerol according to the addition of yeast extract.

The production method of succinic acid using glycerol of the present invention comprises the step of inoculating succinic acid fermentation microorganisms in a culture medium containing glycerol as a culture raw material and culturing in batch, and obtaining succinic acid from the electric culture.

In this case, the glycerol may include 5 to 30 g per liter of culture medium, and may be used by mixing glucose and glycerol in an appropriate ratio, but when using the production method of the present invention, glycerol may be used to completely replace glucose which is a carbon source of the culture medium. Therefore, other carbon sources other than glucose or glycerol need not be included in the culture medium. In addition, the culture medium for the culture of succinic acid fermentation microorganisms can be used as long as the medium for culturing anaerobic microorganisms, but the amount of yeast extract is important for the optimization of cell growth and succinic acid production of succinic acid fermentation microorganisms, yeast extract It is desirable to include 5 to 15 g of the ingredient per liter of culture medium. In addition, the succinic acid fermentation microorganisms are Anaerobiospirillum succiniciproducens, Succinivibrio dextrinosolvens, Fibrobacter succinogens Luminox or Fibrobacter uccino Ruminococcus flavefaciens and the like can be used, but most preferably Anaerobiospirillum succiniciproducens (David, et al., Int. J. Syst. Bacteriol) , 26: 498-504 (1976). In addition, the cultivation is a batch cultivation, it is a 35 to 42 ℃, preferably 37 to 40 ℃, most preferably at a temperature of 39 ℃ and pH range of 6.0 to 7.5, carbon dioxide gas flow rate of 0.25 to 0.5vvm The succinic acid can be produced with high concentration and high productivity only when the culture is performed while feeding.

In the method for producing succinic acid using glycerol of the present invention, succinic acid is produced while glycerol is efficiently consumed by succinic acid fermentation microorganisms. At this time, the use of glycerol as a raw material for the production of succinic acid that exceeds the succinic acid productivity obtained in the conventional glucose cultivation process, as well as, in particular, overproduction of acetic acid that causes inhibition of the growth of microorganisms is remarkable It is accompanied by the effect of suppressing the succinic acid, which enables high efficiency and high economic efficiency.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1: Batch Culture with Glycerol

Batch cultivation was carried out using glycerol as a culture raw material: the strain was used for anaerobic microorganisms, Anaerobiospirillum succiniciproducens (ATCC 29305), and 5 g / L glucose, 10 g / L polypeptone, 5 g / L yeast extract, 3 g / L K ₂ HPO ₄ , 1 g / L NaCl, 1 g / L (NH ₄ ) ₂ SO ₄ , 0.2 g / L Pre-culture medium consisting of CaCl ₂ · 2H ₂ O, 0.2g / L MgCl ₂ · 6H ₂ O and 1g / L Na ₂ CO ₃ was prepared, and then in a 100 ml medium injected with carbon gas for anaerobic culture. The bacteria were inoculated and precultured at 39 ° C. for 14 hours. Next, 6.5 g / L glycerol, 10 g / L polypeptone, 5 g / L yeast extract, 3 g / L K ₂ HPO ₄ , 1 g / L NaCl, 5 g / L (NH ₄ ) ₂ SO ₄ , Culture medium consisting of 0.2 g / L CaCl ₂ · 2H ₂ O, 0.2 g / L MgCl ₂ · 6H ₂ O, 5 mg / L FeSO ₄ · 7H ₂ O, and 5 g / L Na ₂ CO ₃ The mixture was inoculated with 0.9 L of medium into a 2.5 L culture tank, and then the temperature and pH were adjusted to 39 ° C. and 6.5, respectively, inoculated with 100 ml of pre-cultured succinic acid fermentation microorganism, and carbon dioxide gas was maintained to maintain anaerobic condition. The microorganisms were cultured while feeding at a flow rate of 0.25 vvm. While the culture was performed for a total of 37 hours, the medium was taken from the incubator hourly, and the amount of succinic acid, glycerol and acetic acid in the culture medium was analyzed using an Aminex HPX-87H column (Bio-Rad, USA). It was measured (see FIG. 1). In Figure 1, (●) is the cell concentration; (○) is succinic acid; (■) is glycerol; And (◇) represents the concentration according to the incubation time of acetic acid. As shown in FIG. 1, the concentrations of glycerol and succinic acid consumed after 37 hours of incubation were 3.7 and 4.9 g / L, respectively, and the yield of succinic acid (amount of succinic acid produced / amount of glycerol consumed) was 135%. . In particular, the production of by-product acetic acid was significantly suppressed, and the ratio of acetic acid to succinic acid (amount of succinic acid produced / amount of acetic acid produced) was 25.8: 1, which is about 6.5 times higher than the ratio of 4: 1 obtained using glucose. I could confirm it.

Example 2 Culture Characteristics According to the Ratio of Glycerol and Glucose

In batch cultivation, in order to optimize the medium composition capable of producing succinic acid efficiently, the batch cultivation was carried out under the culture conditions shown in Example 1 while varying the ratio of glycerol and glucose: while incubating for a total of 36 hours, The amount of succinic acid, glycerol, acetic acid and glucose in the culture medium was measured using an Aminex HPX-87H column, and the maximum cell concentration, maximum specific growth rate, succinic acid concentration, and succinic acid yield in this medium. , The ratio of acetic acid to succinic acid and the overall productivity was compared (see Table 1).

As shown in Table 1 below, when only glycerol was included as the carbon source than when only glucose was included as the carbon source, the cell concentration and the succinic acid concentration of the succinic acid fermentation microorganisms were lower, whereas by showing a high succinic acid yield and the ratio of acetic acid to succinic acid, Glycerol could be effectively substituted for glucose, it was found that the yield of succinic acid and the ratio of acetic acid to succinic acid increased by glycerol. In addition, productivity also increases as the ratio of glucose increases, but the ratio of acetic acid to succinic acid tends to decrease. Therefore, in order to prevent production of unnecessary acetic acid, it is preferable to include more glycerol in the culture medium instead of glucose. Was evaluated.

Example 3: Culture characteristics according to complex nitrogen source

In general, since it is reported that nutrients contained in the complex nitrogen source have an important effect on cell growth, in this embodiment, in order to observe the effect of the complex nitrogen source during glycerol production, complex nitrogen known as an important factor for succinic acid production and cell growth Batch culture was carried out under the culture conditions shown in Example 1 with varying concentrations of causative polypeptone and yeast extract (Lee, PC et al., Process Biochem., In press (1999)). The culture was carried out for 36 hours and the medium was collected from the flask for each hour. The amount of succinic acid, glycerol, and acetic acid in the culture was measured using an Aminex HPX-87H column, and the maximum cell concentration, succinic acid concentration, and succinic acid according to each condition were measured. The yield and the ratio of acetic acid to succinic acid were compared (see Table 2).

As shown in Table 2, it was confirmed that cell growth and succinic acid production increased as the yeast extract was added more, and in the case of polypeptone, cell growth and succinic acid production increased only when the yeast extract was added together and added alone. It was found that there was no effect on cell growth or succinic acid production.

Example 4: Culture Characteristics according to Yeast Extract

In order to confirm the effect of the yeast extract observed in Example 3, a certain amount of yeast extract was intermittently added to the culture of succinic acid fermentation, and the culture characteristics of cell growth and succinic acid production were examined: 8 g / L as the initial condition. Glycerol and yeast extract of 5g / L was added, and the other culture conditions were carried out by the method described in Example 1, while the culture was carried out for a total of 120 hours, the medium was taken from the culture tank by the hour and in the culture medium The amount of succinic acid, glycerol and acetic acid was measured using an Aminex HPX-87H column (see Figure 2). In Figure 2, (●) is the cell concentration; (○) is succinic acid; (■) is glycerol; And (◇) represents the concentration according to the incubation time of acetic acid. As shown in Figure 2, after 48 hours glycerol was consumed 3.3g / L, cell growth increased to OD ₆₆₀ = 0.85. In addition, when yeast extract was added when no more glycerol was consumed, glycerol was consumed again and cell growth was increased. In order to further confirm the dependence of glycerol on yeast extract, the addition of glycerol and yeast extract was confirmed that the added glycerol was also consumed, and the concentration and yield of the produced succinic acid were 19 g / L and 160%, respectively. It was confirmed that the ratio of acetic acid to succinic acid increased to 31.7: 1 (g / g). The ratio of succinic acid and acetic acid reported so far was about 4: 1 (g / g) when using glucose. According to the production method of succinic acid using the glycerol of the present invention, it was found that the production ratio was increased by about 8 times. .

These results indicate that the production of succinic acid in a culture medium containing glycerol as a raw material can inhibit excess acetic acid production which inhibits the growth of microorganisms. It can be a great help in producing this. In addition, these results are a groundbreaking process that can replace the glucose used to produce succinic acid with glycerol, a by-product produced in the oil industry, and can be positively evaluated in terms of environment.

As described above in detail specific parts of the present invention, it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

As described and demonstrated in detail above, the present invention uses a glycerol which is a by-product of the oil industry to produce succinic acid in high yield under anaerobic conditions, and at the same time, a method for producing succinic acid in batch culture that significantly suppresses the production of by-product acetic acid. to provide. According to the production method of succinic acid using the glycerol of the present invention, it is possible to produce succinic acid from glycerol in high yield, it is possible to significantly suppress the production of acetic acid, which is a by-product produced during the production of succinic acid using glucose, of course. In the case of succinic acid purification, cost reduction can be made. In addition, glycerol is a by-product produced in excess in the oil industry, and the necessity of reuse thereof is increasing. The present invention is more valuable because it can produce succinic acid currently produced by a chemical process using such glycerol. Can be said to be high.

Claims (6)

A method of producing succinic acid using glycerol comprising the step of inoculating succinic acid fermentation microorganisms in a culture medium containing glycerol as a culture raw material and culturing in batchwise to obtain succinic acid from the electric culture. The method of claim 1, Glycerol is characterized in that it contains 5 to 30g per liter of culture medium Method for producing succinic acid using glycerol. The method of claim 1, The culture medium is a medium capable of culturing anaerobic microorganisms, characterized in that Method for producing succinic acid using glycerol. The method of claim 1, Yeast extract in the culture medium is characterized in that it contains 5 to 15g per liter culture medium Method for producing succinic acid using glycerol. The method of claim 1, Succinic fermentation microorganisms include Anaerobiospirillum succiniciproducens, Succinivibrio dextrinosolvens, Fibrobacter succinogenes or Luminococcus flaviencus flavicoccus Ruminococcus flavefaciens) characterized in that Method for producing succinic acid using glycerol. The method of claim 1, Cultivation is carried out while supplying carbon dioxide gas at a flow rate of 0.25 to 0.5vvm at a temperature of 35 to 42 ℃ and pH 6.0 to 7.5 range Method for producing succinic acid using glycerol.