JPH08256782A - Production of bacterium flocculant by fermentation method - Google Patents

Abstract

PURPOSE: To efficiently obtain a biodegradable bacterium flocculant causing a small sludge volume by culturing a bacterium belonging to the genus Rhodococcus by fermentation using a synthetic medium, useful for waste water treatment by activated sludge method, cell separation in fermentation engineering, etc. CONSTITUTION: A bacterium (Rhodococcus erythropolis KR-S-1 strain (FERM BP-4,913) belonging to the genus Rhodococcus is cultured by using a synthetic medium containing one or more saccharides such as glucose or fructose, one or more phosphates such as potassium phosphate and sodium phosphate, one or more nirogen sources such as urea or ammonium sulfate, one or more amino acids such as arginine, methionine, citrulline and glycine, one or more vitamins such as biotin, riboflavin, thiamine, vitamin B12 as active ingredients to give the objective bacterium flocculant effective for flocculation in waste water treatment and for recovery of useful substances from a fermented residue.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a microbial flocculant using a metabolite produced by a Rhodococcus bacterium. The microbial flocculant of the present invention is effective for flocculation in wastewater treatment, recovery of useful substances from fermentation residue, and the like, and is a flocculant that is safe for the human body without environmental pollution.

[0002]

Flocculants are synthetic polymeric flocculants (eg,
Polyacrylamide), inorganic flocculants (for example, sulfuric acid band), and microbial flocculants. Among these, the coagulants that are currently widely used are inorganic coagulants and synthetic polymer coagulants. These flocculants are used for wastewater treatment using activated sludge method, cleaning agents for civil engineering dredging work, etc. Furthermore, it covers a wide range of fields in various industrial processes and wastewater treatment such as application to the food industry. However, since the inorganic flocculant may be used in a large amount, the sludge volume generated is large, and the flocculant contains a metal. Sludge from synthetic polymer flocculants accumulates in soil and rivers without being decomposed if it is directly discharged to the outside. Therefore, most of them are incinerated, and all of them have major problems in terms of disposal.

Microbial flocculants have attracted great attention due to their excellent biodegradability and little accumulation in the environment, but they have not yet been put into practical use. The biggest problem is that the microbial flocculant has a higher production cost as compared with synthetic polymer flocculants and inorganic flocculants.
Various studies have been conducted so far on reduction of manufacturing cost, and many reports have been made. A method for producing a microbial flocculant using Rhodococcus spp., For example, a method using wastewater of a food factory for processing marine products containing a high concentration of protein (Japanese Patent Laid-Open No. 03-249909), malt generated in a beer factory Method of using root as a medium component (Japanese Patent Laid-Open No. 03-382)
03), a method of using a medium containing blood components (JP-A-02-92273), a method of using oil seeds as a medium component (JP-A-01-211492), and others (JP-A-52-148678). JP-A-54-32686
JP-A-58-183910, JP-A-49-004
685, JP-A-63-126596, JP-A-60-
71,099, JP-A-59-24649, JP-A-51
-86189, JP-A-62-250918, JP-A-62-176510, JP-A-02-215387,
JP-A-02-90903). All cultures of Rhodococcus spp. Found in these reports use natural medium components as nutrient sources. The natural medium contains many kinds of organic nutrients such as proteins, lipids and polysaccharides as well as inorganic salts as represented by yeast extract, and not only Rhodococcus spp.
It is an excellent medium that can meet the nutritional requirements of many types of microorganisms. However, on the contrary, certain essential nutrients in the medium are quickly depleted during culturing, the culturing is stopped in the middle, and growth is also inhibited by zinc atoms in natural products and metal atoms such as copper, In addition, it has a drawback that it secretes a growth inhibitor during growth and stops growing. From these reasons, the amount of culture products extracted from the culture broth is low, and the flocculation activity is low, which is one of the causes of low production efficiency as a microbial flocculant, which is a cause of high production costs. Has become.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides a method for improving such drawbacks of the conventional method and efficiently producing a microbial flocculant from a bacterium of the genus Rhodococcus.

[0005]

[Means for Solving Problems] One of the major problems in producing a flocculant with a microorganism is that the concentration of the culture product is extremely low, about 0.1 to 1% in the culture solution after fermentation. It is in. As a result of various studies on the culture conditions of Rhodococcus bacteria, the present inventors increased the components suitable for the growth of Rhodococcus bacteria, and removed the medium constituents such as components that interfere with the growth of the bacteria. With clear control,
The present invention has been achieved by obtaining the knowledge that a culture product can be produced at a high concentration. That is, the present invention is a method for producing a microbial flocculant from a bacterium of the genus Rhodcoccus by a fermentation method using a synthetic medium. Among Rhodococcus bacteria, Rhodococcus erythropoles KR
-S-1 strain (FERM BP-4913) is most preferably used.

The synthetic medium in the present invention is composed of sugars, phosphates, nitrogen sources, amino acids and vitamins. Here, the synthetic medium is a medium in which the constituents are clearly controlled, and among the constituents, not only sugars and phosphates but also nitrogen sources, amino acids, and vitamins are chemically isolated. . Sugars are glucose, fructose,
One or more selected from sucrose and maltose, the phosphate is one or more selected from potassium phosphate and sodium phosphate, and specifically, sodium dihydrogen phosphate (NaH ₂ PO ₄ ), Potassium dihydrogen phosphate (KH ₂ PO ₄ ), disodium hydrogen phosphate (Na ₂ HP
O ₄ ), and dipotassium hydrogen phosphate (K ₂ HPO ₄ ).
The nitrogen source is one or more selected from urea, ammonium sulfate, ammonium nitrate, sodium nitrate, potassium nitrate, and the amino acids are arginine, methionine, citrulline, isoleucine, valine, phenylalanine,
One or more selected from tyrosine, tryptophan, histidine, glycine, glutamic acid, aspartic acid, proline, alanine, serine, ornithine, and vitamins include biotin, riboflamine, thiamine and vitamin B.
12. One or more selected from nicotinic acid, inositol, pantothenic acid, and ferroquinone.

The concentration of each component in the synthetic medium is 0.1 to 50 g of saccharide per 100 ml of water added to the medium,
Preferably 5 to 30 g, more preferably 10 to 20 g
Is. If the amount is less than 0.1 g, the characteristics of the present invention cannot be utilized, and if the amount is more than 50 g, the amount of aggregated active ingredients produced on the substrate decreases, which is inefficient. The phosphoric acid salt is potassium salt of phosphoric acid or sodium salt of phosphoric acid, and more preferably potassium phosphate. Its concentration is 0.01 to 10 g, preferably 0.5 to 3 g, and more preferably 1 to 2 g. If the amount is less than 0.01 g, the characteristics of the present invention cannot be utilized, and if the amount is more than 10 g, the amount of culture production on the substrate decreases, which is inefficient. The nitrogen source is 0.02 to 1 g, preferably 0.03 to
It is 0.5 g, more preferably 0.05 to 0.1 g. If the amount is less than 0.02 g, the characteristics of the present invention cannot be utilized, and if the amount is more than 1 g, the amount of culture produced on the substrate is reduced, which is inefficient. Amino acid concentration is 0.0
It is 1 to 10 g, preferably 0.05 to 1 g, and more preferably 0.08 to 0.5 g. If the amount is less than 0.01 g, the characteristics of the present invention cannot be utilized, and if the amount is more than 10 g, the amount of culture production on the substrate decreases, which is inefficient. The concentration of vitamins is 0.00001-0.01
g, preferably 0.0001 to 0.005 g, and more preferably 0.0005 to 0.001 g. 0.000
If it is less than 01 g, the characteristics of the present invention cannot be utilized, and if it is more than 0.01 g, the amount of culture production on the substrate is reduced, which is inefficient. In addition, if necessary, other amino acids, vitamins, sodium chloride, magnesium sulfate, or iron, calcium, manganese, zinc,
Copper sulfate, hydrochloride, nitrate, or elements of molybdenum, tungsten, or compounds may be added,
This does not limit the invention in any way.

Among the medium compositions in the prior art, the addition amount of saccharides such as glucose and fructose can be accurately adjusted as a single composition, and phosphates such as potassium salts and sodium salts and nitrogen components such as urea and ammonium sulfate. Can be added exactly as well. But the amino acids,
Proteins or vitamins are so-called natural mediums to which natural products such as yeast extract and peptone are added. Amino acids, proteins in the natural products,
It is not possible to accurately control the content and content of vitamins, metals, and other ingredients. Glucose, sugar and the like are also separated from nature, but they are single substances and the amount added in the medium can be controlled, so they are not natural products in the concept of the present invention.

[0009] In the case of a natural medium, it is possible to increase the amount of nutrient components, particularly sugars added to the medium in order to promote the growth of bacteria, but a specific amino acid in the natural product or other specific essential nutrients. The components are quickly depleted and the culture stops halfway. In addition, growth may be hindered by metal atoms such as zinc and copper in natural products.
Furthermore, Rhodococcus bacteria secrete growth inhibitory substances as they grow. This component is mainly generated during the digestion of amino acids and vitamins, and when the concentration of natural product components is increased in order to increase the amount of the culture product that is an agglutinating active component, many growth inhibitory substances are also produced. As a result, the culture stops halfway, and it is not possible to obtain the desired culture output. As described above, it has been substantially difficult to increase the concentration of the medium components and increase the production amount of the flocculation-active culture product by the conventional techniques.

The conditions for culturing the Rhodococcus bacterium in the present invention are not particularly limited, but are usually 1 to 1 under aerobic conditions such as shaking culture or aeration-agitation deep culture.
Do it on day 0. The culture temperature is preferably in the range of 15 to 40 ° C,
The pH is preferably 4 to 10, and more preferably 7 to 9. The microbial flocculant used in the present invention is a culture product of a bacterium of the genus Rhodococcus, and its isolation method is not particularly limited. Usually, a large amount of a water-soluble organic solvent such as acetone or ethanol is added to the culture solution, and the precipitate can be filtered and taken out. However, in many cases, the purpose can be achieved sufficiently by using the culture medium as it is.

[0011]

[Function] The amount of nutrient components in the medium, especially sugars, has the greatest influence on the production amount of culture products. However, simply increasing the added amount of sugar is not enough. Other nutrients such as coexisting amino acids are also important.
In the case of a natural product medium, nutrient components, which is the process of culturing, are depleted quickly and the culturing stops halfway. Also,
Rhodococcus bacteria secrete growth inhibitory substances as they grow. This component is mainly generated during the digestion of amino acids and vitamins. Increasing the concentration of natural product components to increase the amount of culture products produced produces not only the desired culture products but also growth inhibitory substances. Will be done. And after all, the culture stopped halfway,
Only the component having the expected agglutinating activity does not have a high concentration.
In addition, natural products originally contain growth inhibitory substances for Rhodococcus bacteria such as zinc and copper when their concentrations increase, and the concentration added to the medium is naturally limited. By using the synthetic medium of the present invention, it became possible to culture under the condition that the components necessary for the growth of Rhodococcus were sufficiently contained and the inhibitory components were not contained.

[0012]

EXAMPLES The present invention will be described in more detail by way of examples.
The present invention is not limited to the examples below. [Measurement of production amount of culture product]
A double volume of acetone was added, and the precipitate was collected by filtration using No6 filter paper, dried at 105 ° C. for 2 hours, and then weighed.

[Measurement of Aggregation Activity] The measurement was carried out by the following procedure.
The absorbance used here is the absorbance with respect to this transmittance, which is obtained by measuring, as the transmittance, the sum of the scattered light to the front of the optical path when the light passes through the suspension and the partially attenuated transmitted light. (1) 0.5 g of kaolin (particle size 62 to 74 μm specified by JIS-K8746) was put in 90 mL of distilled water to form a suspension, and the suspension was adjusted to pH = 7.0. (2) Culture medium containing cells during or after culture.
5 mL was taken, distilled water was added thereto to make 10 mL in total, and the mixture was added to the above kaolin suspension. For comparison, the culture solution was not added, and 10 mL of distilled water was added to the kaolin suspension. (3) Put the mixed solution in a 100 mL volumetric flask, and
After performing the inverted stirring 5 times, the mixture was left standing for 5 minutes. (4) After collecting 50 mL of the supernatant of the volumetric flask, the absorbance (550 nm) was measured with a spectrophotometer. (5) The aggregation activity was calculated based on the following formula. Aggregation activity = (1 / absorbance of sample)-(1 / absorbance of comparative solution)

[Test 1] 100 mL of medium (pH = 8.5) prepared with the composition shown in Table 1 was sampled and
After putting it in a 00 mL Erlenmeyer flask, it was sterilized by autoclave at 121 ° C. for 15 minutes. Next, Rhodococcus erythropoles KR-S-1 strain (FERM
BP-4913) 1 platinum loop inoculation, then at 7
Culture was performed for one day (Examples 1 to 8). The amount of culture products produced from each culture and the agglutination activity were measured. In the synthetic medium of the present invention, even if the carbon source glucose is concentrated up to 12% by weight, the bacterial cells grow, and finally 3.2 g / 1 in the culture solution.
00 mL of culture output was obtained and the aggregation activity was also high. In the medium (Comparative Examples 1 and 2) using the yeast extract, which is a natural product, when the glucose concentration was 10 g / 100 mL or more, the cells did not grow, and the desired culture product could not be obtained.

[0015]

[Table 1] [Test 2] Medium 290 prepared with the composition shown in Table 2
0 mL (pH = 8.5) was sampled, put in a 5 L fermenter, and then sterilized by an autoclave. After the medium has cooled down, Rhodococcus erythraceus KR-S-
1 strain (FERM BP-4913), Rhodococcus erythraceus KR-256-2 strain (FERM P3
No. 923). 500 rpm
Aeration was performed at 3 L / min while culturing at 30 ° C., and culture was performed at 30 ° C. for 8 days (Examples 9 to 11). After culturing, the production amount of the culture product and the aggregating activity were measured.

As a result, with the synthetic medium of the present invention, it is possible to obtain a culture product even if the saccharide concentration in the medium is as high as 10 g / 100 mL or more, and it is recognized that it is very efficient as a microbial flocculant. Was given.

[0017]

[Table 2]

[0018]

By using the synthetic medium of the present invention,
Contains enough components necessary for the growth of Rhodococcus bacteria,
Cultivation can be performed under conditions that do not contain inhibitory components. Used as a microbial flocculant is a culture product of Rhodococcus spp. The isolation method is not particularly limited, and usually a large amount of a water-soluble organic solvent such as acetone or ethanol is added to the culture solution, and the deposited material can be filtered and taken out. However, in many cases, the use of the culture solution as it is can sufficiently achieve the purpose.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1:01) (C12N 1/20 C12R 1:01) (72) Inventor Ryuichiro Kurane Higashiichi Tsukuba, Ibaraki Prefecture 1 to 3 Industrial Technology Institute of Biotechnology, Industrial Technology

Claims (5)

[Claims] 1. A method for producing a microbial flocculant from a bacterium of the genus Rhodcoccus by a fermentation method using a synthetic medium. 2. The genus Rhodococcus is Rhodococcus
Erythropoles KR-S-1 strain (FERM BP-4
913). The method for producing a microbial flocculant according to claim 1. 3. The synthetic medium comprises at least one sugar selected from the group consisting of glucose, fructose, sucrose and maltose, and at least one selected from the group consisting of K salt of phosphoric acid and Na salt. At least one nitrogen source selected from the group consisting of phosphate, urea, ammonium sulfate, ammonium nitrate, sodium nitrate, and potassium nitrate, arginine, methionine,
At least one or more amino acids selected from the group consisting of citrulline, isoleucine, valine, phenylalanine, tyrosine, tryptophan, histidine, glycine, glutamic acid, aspartic acid, proline, alanine, serine, ornithine, biotin, riboflamine,
The method for producing a microbial flocculant according to claim 1 or 2, which is an active ingredient comprising at least one vitamin selected from the group consisting of thiamine, vitamin B12, nicotinic acid, inositol, pantothenic acid and ferroquinone. 4. The amino acid is arginine, citrulline,
Glycine, glutamic acid, aspartic acid, alanine,
The method for producing a microbial flocculant according to claims 1 and 2, wherein the synthetic medium according to claim 3 is at least one selected from the group consisting of: 5. The method for producing a microbial flocculant according to claim 1 or 2, wherein the weight of the saccharide is at least 10 g per 100 ml of water in the medium.