JPH0364103B2 - - Google Patents

Description

[Detailed description of the invention]

Industrial Application Field The present invention relates to a method for aggregating microbial cells by using chitosan of a specific molecular weight and polyacrylate in combination in order to efficiently separate microbial cells from a culture solution in the fermentation industry. . Prior Art There are many fermentation industries that cultivate microorganisms for the purpose of utilizing antibiotics, hormones, oxygen, etc. The culture method used in the fermentation industry is generally carried out by suspending microorganisms in a culture solution containing various nutrients. After the culture is completed, heating and pH adjustment are performed, and then the microbial cells and the culture solution are separated using a decanter, precoat filter, etc. Problems with the conventional technology Since the microorganisms in the culture solution mentioned above have poor permanence, a large amount of diatomaceous earth is used as a super-assistant or pre-coating agent, but the microorganisms contaminated with these diatomaceous earths cannot be incinerated. I had no choice but to dump it. When bacterial cells are needed, a decanter is used, but the sedimentation of microbial cells is poor, the treatment capacity is low, and the moisture content of the bacterial cake is high. How to solve the problem: By agglutinating the bacterial cells, the separation effect can be greatly increased. For this reason, various methods have been proposed for aggregating bacterial cells in a culture solution. In the field of water treatment, flocculants are well known and various types of flocculants are used. Among them, chitosan, which is produced by deacetylation of chitin, is a natural cationic polymer, and because it is highly safe, attempts have been made to apply it to the isolation of bacterial cells in culture solutions. For example, Japanese Patent Application Publication No. 1973-
71878 and JP-A-51-128474 disclose a method of flocculating microbial cells with chitosan, and JP-A-53-25027 discloses a method of aggregating microbial cells with chitosan by adding chitosan and polyanion to a bacterial culture solution and then adjusting the pH. A method of co-precipitation is disclosed. However, these methods are not put to practical use because of their weak cohesive force and lack of reproducibility. As a result of various studies based on aggregation theory, the present inventors succeeded in imparting a flocculating ability that is suitable for practical use by adding and mixing chitosan having an appropriate molecular weight to a culture solution, and then adding polyacrylate. The chitosan applied to the present invention has a viscosity of 1% chitosan solution in 1N saline at a temperature of 25°C PH.
It is necessary that it is in the range of 2 to 200 CP in the state of 4.0, and the range of 5 to 100 CP is particularly desirable. Commercially available chitosan only undergoes deacetylation treatment of chitin, and its viscosity is over 1000CP when measured under the same conditions. In order to obtain a desired molecular weight from such high-molecular-weight chitin or chitosan as a raw material, methods such as hydrolyzing it with an acid alkali and decomposing and cutting it with an oxidizing agent are known.
54-148890 and the method described in JP-A-62-184002 is applicable. These chitosan impart water solubility as a monobasic acid such as hydrochloric acid, acetic acid, or sulfamic acid, and are added as an aqueous solution with a concentration of 0.2 to 2%. The amount added is desirably 1 to 10% by weight based on the dry matter of the bacterial cells, and if the amount added is too large, re-decomposition will occur due to charge reversal. If commercially available high molecular weight chitosan is added under similar conditions, it will precipitate in a system and will not be uniformly dispersed in the culture solution. On the other hand, when chitosan in the present invention is added and stirred, fine flocs are formed due to coagulation. When a polyacrylate solution is added to the fine flocs and stirred, coarse flocs are formed due to cross-linking and adsorption, and both transient sedimentation properties are greatly improved. The polyacrylate used for this purpose is
It is a monovalent cation salt such as sodium, potassium, ammonium, etc., and the viscosity of a 1% solution of polyacrylic acid in 1N saline at a pH of 7.0 and a temperature of 25℃ is
Must be 10 PCs or more. Polyacrylate should be added in an amount of 0.2% or more based on the dry matter of the bacterial cells. When performing this agglutination operation, the pH of the culture solution is 4.5 ~
A range of 8.0 is desirable, and if the pH of the culture solution is below the isoelectric point of the bacterial cells or above the dissociation pH of chitosan, no aggregation will occur. The culture solution is usually subjected to various separation operations after heat treatment at 50 to 80°C, but the temperature of the culture solution at the time of adding the flocculant can be set arbitrarily. It is most preferable to add and mix the flocculant using a dedicated flocculation tank, but it is also possible to inject it into the suction of a pump or mix it by turbulent flow in piping. The aggregated bacterial cells are easily separated from the mother liquor using a known solid-liquid separator such as a decanter, filter press, belt filter, or belt press. It does not deviate from the present invention to use a super-aiding agent such as a pipe. Effect Although high-molecular-weight chitosan is used as a flocculant in wastewater treatment, the reason why high-molecular-weight chitosan is unsuitable for flocculating microbial cells in culture fluid is thought to be as follows. Biologically treated sludge in wastewater treatment is an aggregate of bacterial cells and the suspended particles have large particle sizes, so high-molecular-weight chitosan with strong cross-linking and adsorption properties is used. However, since the bacterial cells are individually dispersed in the culture solution, the particle size is small. Therefore, when an aqueous solution of high molecular weight chitosan is added to a culture solution, bacterial cells adhere to the surface of the aqueous chitosan solution and precipitate in the form of threads without being dispersed in the culture solution. On the other hand, in the case of a low-molecular-weight chitosan aqueous solution, it is uniformly dispersed in the culture solution and is effectively used for neutralizing charges on the surface of bacterial cells. As a result, the bacterial cells aggregate to form fine flocs, and cross-linking adsorption by polyacrylate works effectively to form coarse flocs with excellent transient sedimentation properties. In order to improve productivity, the culture solution is sent to the separation step at a bacterial cell concentration of usually 5% or more, at least 1% or more. Since the microbial cell concentration is thus high and is present in the form of fine particles, good dispersibility as in the product of the present invention is required in order to expect uniform distribution over the entire microbial cell surface. Sample Preparation The viscosity of the chitosan used in the test was adjusted according to the method described in Kyowa Yushi's Flownac N JP-A-54-148890. Table 1 shows the viscosity of each sample neutralized with hydrochloric acid and dissolved in 1% PH4 in 1N saline. Chitosan sample

【table】

【table】 Sample sodium polyacrylate

[Table] Example 1 Candida utilis was cultured in a culture solution consisting of glucose, acid mother extract, and nutrient salts at pH 6.6.
A solution with a bacterial cell concentration of 5% was used for the test. Transfer 200 ml of the main culture solution to a 300 ml glass beaker, and mix the flocculant with a jar tester at a rotation speed of 150 rpm. After adding chitosan and stirring for 2 minutes, sodium polyacrylate was added and stirred for 1 minute. Chitosan is
Dissolve in 0.1N acetic acid solution to a concentration of 1%, and use a 0.2% aqueous solution of sodium polyacrylate. Aggregated bacterial bodies were placed in a Buchner funnel with a diameter of 7 cm No.2.
The time required for the paper to be filtered under reduced pressure of 700 mmHg was measured. The results are shown in Table-3. Overtest results

【table】

[Table] Example 2 Streptomyces griseus was cultured in a culture solution consisting of glucose, yeast extract, and nutrients.
A solution with a pH of 6.8 and a bacterial cell concentration of 6% was used for the test. The results of an overtest similar to Example 1 are shown in the table below.
Shown in 2. Overtest results

【table】

[Table] Example 3 Aspergillus niger was cultured in a culture solution containing glucose, yeast extract, and nutrients at pH 6.5.
A solution with a bacterial cell concentration of 5.5% was used for the test. The results of an overtest similar to Example 1 are shown in the table below.
5. Overtest results

【table】

【table】

Claims (1)

[Claims] 1 Chitosan, which has a viscosity of 2 to 200 centipoise of a 1% polymer solution in 1N saline at pH 4.0, was added to the culture solution and mixed, and then sodium polyacrylate was added and mixed to aggregate the microbial cells. A method for promoting the separation of microbial cells and culture mother liquor in the fermentation industry, which is characterized by mulching.