JPH062201B2 - Method for producing microbial flocculant - Google Patents

Description

TECHNICAL FIELD The present invention relates to a flocculant derived from a microorganism (referred to as a microorganism flocculant).
A method for easily and inexpensively manufacturing

The aggregating agent in the present specification means a substance having an ability to aggregate various other substances.

Conventional technology and its problems Conventionally, flocculants have been widely used as clarification treatment agents in wastewater treatment using the activated sludge method, civil engineering dredging work, and the like. In addition, the application of flocculants is advancing in a very wide range of fields, such as the water supply and waterworks water production fields, the downstream processing fields such as the separation of fermentation liquid and cultured cells in fermentation plants, and the food industry field. It's being done.

Currently, as coagulants, synthetic polymer coagulants (for example, polyacrylamide) and inorganic coagulants (for example, polyaluminum chloride, sulfuric acid band) are mainly used.
Is used. However, these flocculants
Although it is excellent in terms of aggregation ability and economical efficiency, it has a problem that it is inferior in safety and environmental hygiene and causes secondary pollution.

In order to solve such a problem, a microbial-derived flocculant that is biodegradable, safe, and free from the risk of secondary pollution is desired.

As a flocculant derived from microorganisms, Rhodococcu (Rhodococcu
s) A flocculant derived from a genus bacterium is known [Japanese Patent Publication Sho 56
No. 39633 (Patent No. 1096062)]. This flocculant can be produced by culturing Rhodococcus spp. In a medium containing glucose and / or fructose as a carbon source [Kurane et al., Agric. Biol.
m., 50 (9), 2301-2307, 1986, etc.]. However, since fructose used here is expensive, there is a problem that the culture cost increases. According to trial calculations,
When cultured under the conditions of 0.5% glucose and 0.5% fructose, glucose and fructose account for more than half of the cost of the medium. Further, when glucose is used alone, there is a problem that the amount of coagulant produced is small.

Means for Solving the Problems The present inventor has conducted extensive studies in view of the above problems of the prior art, and when culturing Rhodococcus in a medium containing alcohols as a carbon source, The inventors have found that a flocculant can be produced easily and inexpensively, and completed the present invention.

That is, the present invention, when producing a microbial flocculant by culturing a bacterium that belongs to the genus Rhodococcus and has the ability to produce a microbial flocculant, from the group consisting of methanol, ethanol, propanol, ethylene glycol and glycerol as a carbon source of the medium The present invention relates to a method for producing a microbial flocculant, which comprises using at least one selected.

The microorganism used in the present invention is not particularly limited as long as it belongs to the genus Rhodococcus and has the ability to produce a microbial flocculant. Specifically, bacteria belonging to Rhodococcus erythropolis, for example, Rhodococcus erythropolis KR-S-1 strain (hereinafter simply referred to as S-1 strain), Rhodococcus erythropolis KR-256- 2 strains (hereinafter simply referred to as 256-2 strains) and the like. Incidentally, Rhodococcus erythropolis (formerly Nocardia erythropolis) was rearranged and reclassified from the former name Nocardia erythropolis to Rhodococcus erythropolis by the International Commission for the Convention on Microbial Nomenclature in 1980. The above-mentioned S-1 strain and 256-2 strain are known strains and have been deposited at the Institute of Microbial Technology, Institute of Industrial Science. The deposit number is FERM P3530, 256 for S-1 strain.
-2 strain is FERM P3923.

In the present invention, when culturing a bacterium of the genus Rhodococcus,
Alcohols are used as the carbon source of the medium. The alcohol is not particularly limited, for example, methanol,
Examples thereof include primary alcohols such as ethanol and propanol, secondary alcohols such as ethylene glycol, and tertiary alcohols such as glycerol.
Among them, alcohols having 2 or more carbon atoms are preferable.
The alcohols can be used alone or in combination of two or more. The amount of alcohols added to the medium is not particularly limited, but is usually about 0.01 to 10% by weight, preferably 0.
It may be about 05 to 5% by weight.

As the medium components other than the carbon source, those conventionally used for culturing Rhodococcus bacteria can be used. Examples of the nitrogen source include yeast extract, urea, ammonium sulfate, ammonium chloride, peptone, soybean degradation product and the like. Among them, urea and yeast extract are particularly preferable. Examples of the inorganic salts include 1 potassium phosphate, 2 potassium phosphate, magnesium sulfate, sodium chloride and the like. In addition, vitamins, etc. may be included.

The culture may be liquid culture or solid culture. The culture conditions are
It may be similar to the conventional culture of Rhodococcus. For example, liquid culture is usually under aeration and stirring at about 10 to 50 ° C,
Preferably, it is carried out at a temperature of about 20 to 40 ° C. for usually about 1 to 30 days, preferably about 2 to 10 days.

The culture thus obtained can be used as it is as an aggregating agent. The flocculant component may be separated and purified from the culture by a known purification means such as centrifugation, ammonium sulfate precipitation method, gel filtration, high performance liquid chromatography, ion exchange chromatography, and ultrafiltration. Good.

Effects of the Invention According to the present invention, since inexpensive and easily available alcohols are used as the carbon source of the medium, the microbial flocculant can be easily and safely produced.

Examples Hereinafter, the present invention will be made clearer with reference to Examples and Comparative Examples.

In the examples, the amount of flocculant produced was expressed as the flocculating activity of the culture medium. The method for measuring agglutination activity is as follows.

In a test tube (φ14 mm), 9 ml of 8.9 mM glycylglycine (pH 7.0) containing 4444 ppm of kaolin is placed.

Next, add 50 μm of the sample and stir lightly.

Further, 1 ml of a 10 mM glycylglycine solution (pH 7.0) containing 500 mM CaCl ₂ is added, and the mixture is stirred with a vortex mixer.

Let stand for 5 minutes.

From the position 1 cm from the liquid surface, collect 1 ml of the reaction solution.

The absorbance (turbidity, OD ₅₅₀ ) of the collected reaction solution at 550 nm is measured, and the reciprocal thereof (1 / OD ₅₅₀ ) is determined.

Using distilled water instead of the sample, a control value (1 / OD ₅₅₀ ) c is determined in the same manner as in the above.

(1 / OD ₅₅₀ ) − (1 / OD ₅₅₀ ) c is defined as aggregation activity and is represented as Δ (1 / OD ₅₅₀ ).

In the examples, "%" means "% by weight".

Example 1 A 500 ml Erlenmeyer flask was charged with a medium (pH 8.
0) 150 ml was put and autoclaved for autoclaving.
This was inoculated with S-1 strain and shake-cultured at 30 ° C. for 9 days, and the amount of coagulant produced [Δ (1 / OD ₅₅₀ )] was examined daily. The results are shown in Table 1. In Table 1, the degree of bacterial growth (cell concentration) is also shown as the turbidity at 660 nm (OD ₆₆₀ ) of the culture solution.

_{K 2 HPO 4 0.5% KH 2} PO 4 0.2% MgSO 4 · 7H 2 O 0.02% NaCl 0.01% Yeast extract 0.05% Ethanol 1% urea 0.05% Comparative Example 1 Ethanol The S-1 strain was cultured in the same manner as in Example 1 except that 0.5% of glucose and 0.5% of fructose were used instead of 1%, and the production amount of the aggregating agent was examined daily. The results are shown in Table 1.

From Table 1, it can be seen that in the method of the present invention, the concentration of cells (the degree of growth with bacteria) was lower than in the conventional method, but the aggregating activity was almost equivalent.

Further, the cost of the medium of Example 1 was about 70% of the cost of the medium of Comparative Example 1.

Example 2 S-1 strain was cultured for 13 days in the same manner as in Example 1 using various alcohols (concentration 1%) as carbon sources. Cell concentration (OD ₆₆₀ ) and coagulant production [Δ (1 / OD
₅₅₀ )]. The results are shown in Tables 2 and 3.

Also, when ethanol is used as a carbon source,
Table 4 shows the daily changes in the amount (concentration) of ethanol. The amount of ethanol was determined from the amount of increase in NADH (increase in absorbance at 340 nm) according to the following measurement principle.

Comparative Example 2 The S-1 strain was cultured in the same manner as in Example 2 except that 0.5% glucose and 0.5% fructose were used instead of 1% alcohol, and the bacterial cell concentration and the amount of coagulant production were obtained. Was investigated daily. The results are shown in Tables 2 and 3.

Example 3 The culture solution of the S-1 strain obtained in the same manner as in Example 1 was used as it was as a flocculant, and the purifying effect of the flocculant on various suspended waters was examined.

(1) Preparation of suspended water 1) River polluted water 40g of soil collected from the river coast is replaced with 100ml of 10m
The supernatant was used after being suspended in M glycylglycine-NaOH solution (pH 7.0) and allowed to stand for 15 minutes. PH of supernatant
Was adjusted to 7.0 with NaOH.

2) Contaminated water from thermal power plant tank yard 20g soil from thermal power plant tank yard
The supernatant (pH 7.36) after suspending in 0 mM glycylglycine-NaOH (pH 7.0) and standing for 15 minutes was used.

3) India ink 60 μl of commercially available India ink stock solution was suspended in 100 ml of 10 mM glycylglycine-NaOH (pH 7.0) and used (pH 6.94).

4) Kaolin Commercially available kaolin was suspended with 8.9 mM glycylglycine so that the concentration was 4444 ppm, and the pH of the suspension was adjusted to 7.0 with NaOH.

(2) Method To 9 ml of the suspension, 100 μ of culture solution and 500 mM Ca
10 mM glycylglycine-NaOH (p containing Cl ₂
H7.0) 1 ml was added. This was stirred with a vortex mixer for 20 seconds, allowed to stand, and the turbidity (absorbance at 550 nm) of the supernatant was measured with time. For comparison, the turbidity was also measured for the sample to which the culture solution was not added. The results are shown in Table 5.

From Table 5, it can be seen that the microbial flocculant obtained by the method of the present invention has an excellent substance flocculating ability.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Masaki Hirano 3-3-22 Nakanoshima, Kita-ku, Osaka City, Osaka Prefecture 3-22 Kansai Electric Power Co., Inc. (72) Masaki Sugimoto 3--3 Nakanoshima, Kita-ku, Osaka City, Osaka Prefecture No. 22 In Kansai Electric Power Co., Inc. (72) Inventor Masahiko Hirano 1111 Tehiro, Kamakura-shi, Kanagawa Toray Research Center Co., Ltd. (72) Inventor Yoshitaka Taniguchi 1111 Tehiro, Kamakura-shi, Kanagawa Toray Research Center Co., Ltd. Secretary Kawakami Mihide

Claims (2)

[Claims] 1. A method for producing a microbial flocculant by culturing a bacterium that belongs to the genus Rhodococcus and has the ability to produce a microbial flocculant, which is selected from the group consisting of methanol, ethanol, propanol, ethylene glycol and glycerol as a carbon source of a medium A method for producing a microbial flocculant, which comprises using at least one selected. 2. The method according to claim 1, wherein the bacterium belonging to the genus Rhodococcus and having the ability to produce a microbial flocculant is Rhodococcus erythropolis.