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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(cas:110-15-6), 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(cas:110-15-6), which comprises culturing a succinic acid(cas:110-15-6)-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(cas:110-15-6)-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(cas:110-15-6)-productivity can be improved, when a succinic acid(cas:110-15-6)-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, Msucciniciproducens 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(cas:110-15-6) 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(cas:110-15-6) 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(cas:110-15-6) 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(cas:110-15-6) 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(cas:110-15-6) 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.
FIG.1 is a schematic diagram showing the succinic acid(cas:110-15-6) production pathway using sucrose as a raw material in a succinic acid-producing microorganism.
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 -7O 0 C, 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 39 0 C for 8hr. Batch culture was performed by inoculating 250ml of the culture broth into a fermentor containing 2.25L of chemically defined medium under
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