JPH05308956A - Preservation of nitric acid bacteria and method for restoration - Google Patents

Abstract

PURPOSE:To enable preservation treatment of nitric acid bacteria without requiring operation such as concentration by freezing a solution of the bacteria and to extremely quicken restoration of nitrating ability in restoring the bacteria by culturing nitric acid bacteria in a medium containing a specific organic compound and freezing the bacterium solution. CONSTITUTION:First, nitric acid bacteria are cultured in a solution containing an organic compound selected from pyruvic acid (salt), oxaloacetic acid (salt), citric acid (salt), cisaconitic acid, (salt), isocitric acid, 2-oxoglutaric acid (salt), succinic acid (salt), fumaric acid (salt), malic acid (salt), glyoxylic acid (salt), acetic acid (salt), adenosine triphosphate (salt), D-glucose, D-fructose and a polymer polysaccharides obtained by polymerizing one molecule of alpha-L-rhamnose, one molecule of alpha-L-rhamnose, one molecule of D-glucuronic acid and two molecules of D-glucose as one unit in a straight-chain state. Then the solution of the bacteria is directly frozen and stored or mixed with silica gel and frozen and stored.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for preserving and restoring nitric acid bacteria among nitrifying bacteria contained in closed-system circulating water such as aquatic animal breeding / transportation facilities and aquariums, and wastewater, sewage and night soil treatment facilities, soil. It can also be used for the preservation and restoration of nitric acid bacteria widely distributed in nature such as seawater and river water.

[0002]

2. Description of the Related Art Nitrifying bacteria are autotrophic bacteria that oxidize to ammonia or nitrous acid or nitric acid, and synthesize bacterial cell components using carbon dioxide as the sole carbon source. It has been said that.

The nitrifying bacteria include ammonia-oxidizing bacteria that oxidize ammonia to nitrite (hereinafter referred to as "nitrite bacteria") and nitrite-oxidizing bacteria that oxidize nitrite to nitrate (hereinafter referred to as "nitrite bacteria"). It consists of two groups of bacteria, whose chemical reaction formulas are as follows.

[0004] (a) ammonia oxidation reaction of nitrifying bacteria ^{_{NH 4 + + 3 / 2O 2}} → NO 2 - + H 2 O + 39.5k
Nitrite oxidation reaction of cal (b) nitric acid bacteria ^{_{NO 2 - + 1 / 2O 2}} → NO 3 - + 21.6kcal

These bacteria are heterotrophic bacteria (proliferate by utilizing energy generated by oxidation reaction of organic matter)
The growth rate is slower than that of, and it takes a long time to reach the amount of bacteria sufficient for the nitrification reaction to appear (this period is referred to as the "acclimation period").

[0006] Therefore, in order to shorten the acclimatization period, it is most effective to add a large amount of separately prepared nitrifying bacteria as inoculum to a purification device or the like. For that purpose, a method for preserving nitrifying bacteria, which can be arbitrarily used in a timely manner and has excellent resilience, has been desired.

[0007] Conventionally, a subculture method has been used for the preservation of bacteria,
Although there are freeze-drying method and L-drying method, among them, the subculture method is the only effective preservation method for nitrite.

The subculture method means that nitrifying bacteria are inoculated in a medium and cultured at an optimum temperature for a certain period of time (in the case of nitrifying bacteria, it is usually 5
This is a method of preserving nitrifying bacteria by repeating the operation of immediately inoculating a part of the culture solution to another fresh medium after about 7 days).

[0009]

[Problems to be Solved by the Invention]

(1) The subculture method is complicated and requires a lot of labor.
In addition, mutation is likely to occur during repeated transplantation, resulting in a decrease in nitrification ability.

(2) When preserving the bacteria, the restoration efficiency is greatly improved by increasing the bacterial concentration in advance. However, the yield of nitric acid bacteria is 0.02-0.07g.
Nitrate bacteria / g-removed NO ₂ -N and extremely low compared to heterotrophic bacteria, and it takes a long time to obtain a high nitrate bacteria concentration. Therefore, conventionally, the nitric acid culture broth was collected by a centrifuge, concentrated, and then stored. This operation is complicated,
Easy to be contaminated by various bacteria. Further, it takes a long time to restore the nitrate bacteria that have been preserved, as compared with the heterotrophic bacteria.

(3) In order to shorten the acclimatization period of the nitrification apparatus, the amount of nitrifying bacteria sufficient to sufficiently exert the nitrification performance may be prepared in advance and put into the apparatus at the time of startup.
However, as described above, nitrifying bacteria have a low cell yield and a slow growth rate, so that they have a drawback that only a low-concentration bacterial solution can be obtained even if they are cultured for a long time. Therefore, there was no choice but to transport a large amount of nitrifying bacteria solution.

(4) Even after the normal operation of the nitrification apparatus is completed, the nitrifying bacteria may be damaged due to some cause in the system and the nitrification performance may be deteriorated. In that case, the time required for performance recovery is longer than that of an organic wastewater purification device. This is also because the cell yield of nitrifying bacteria is low and the growth is slow. In order to shorten the performance recovery time, it is sufficient to re-supply the system with a high-concentration nitrifying bacterium that sufficiently restores the nitrifying performance, but it is difficult to achieve it because only a low-concentration nitrifying bacterium solution can be obtained. It was

Among the above problems, a method for obtaining a high-concentration bacterial solution in advance in a short time before preserving nitric acid bacteria has already been proposed (Japanese Patent Application No. 2-325613). The present invention further provides an effective method for preserving and restoring nitric acid bacteria in order to solve other problems.

[0014]

Means for Solving the Problems The present inventor has (1) a method for preserving nitric acid bacteria, wherein the nitric acid bacteria are pyruvic acid or a salt thereof, oxalacetic acid or a salt thereof, citric acid or a salt thereof, cisaconitic acid or Its salts, isocitric acid or its salts, 2-oxoglutaric acid or its salts, succinic acid or its salts, fumaric acid or its salts, malic acid or its salts, glyoxylic acid or its salts, acetic acid or its salts, adenosine 3
Phosphoric acid or a salt thereof, D-glucose, D-fructose, α-L-rhamnose, one molecule of α-L-rhamnose, one molecule of D-glucuronic acid and two molecules of D-glucose as one unit, and the unit is linear. A method for preserving nitric acid bacteria, characterized in that the nitric acid bacterium is cultivated in a liquid containing at least one kind of organic compound among the polymerized polysaccharides as described above, or the mixture is frozen and stored as it is or after mixing with silica gel.

(2) In the method for restoring frozen nitrite bacteria as described in 1 above, the organic compound of 1 above, lactic acid or a salt thereof, L-glutamic acid or a salt thereof, L-aspartic acid or a salt thereof, L-serine. Alternatively, a method for restoring a nitric acid bacterium, which comprises culturing in a medium containing one or more kinds of organic compounds among salts thereof. Is.

[0016]

[Operation] (1) It can be stored without damaging the nitric acid bacteria. (2) When the stored nitric acid bacteria are restored, specific organic compounds promote the growth or nitrification of nitric acid bacteria,
High-concentration nitric acid bacterium solution or restoration of nitrification capacity, which could not be obtained by conventional methods, can be obtained in a relatively short time.

The mechanism of the growth of nitric acid bacteria or the promotion of nitrification by a specific organic compound is still unknown. Probably, these specific organic compounds are easily biosynthesized from other organic compounds in heterotrophic bacteria, but in nitric acid bacteria, it is considered that the biosynthesis takes a considerable time due to the difference in metabolic pathway.

[0018]

【Example】

(Example 1) Below, representative strains of nitric acid bacteria (Nitrobacter ag
ilis ATCC 14123) will be described as an example of the present invention.

The culture of nitric acid bacteria in Examples was carried out by autoclaving the medium shown in Table 1 (Lewis, RF and D. Pramer, 1958,
"Isolation of Nitrosomonas in pure culture", J. Bac
teriol., 76, 524-528) (hereinafter referred to as "P medium"), and all were operated aseptically.

[Table 1]

Sodium pyruvate 10 mM, oxalacetic acid 10 mM, trisodium citrate dihydrate 1
mM, cisaconitic acid hydrate 10 mM, trisodium isocitrate dihydrate 10 mM, 2-oxoglutarate 5 mM, sodium succinate hexahydrate 1 mM,
Fumarate monosodium is 10 mM, L-malate disodium is 10 mM, and gluoxylic acid monohydrate is 10 m.
M, sodium acetate 10 mM, adenosine triphosphate (ATP) disodium 5 mg / liter, D-glucose 5 mM, D-fructose 10 mM, α-L
-Rhamnose is 10 mM, 1 molecule of α-L-rhamnose, 1 molecule of D-glucuronic acid and 2 molecules of D-glucose are set as one unit, and the polymerized polysaccharide in which the unit is linearly polymerized (hereinafter referred to as "gellan gum"). Is 0.1%
Each of them was added to 30 ml of a separate medium (Table 1) so that 3 ml of nitric acid bacteria pre-cultured in the medium (Table 1) for 3 days was inoculated. This liquid at 30 ℃, 120 rp
The cells were cultivated at m.

In parallel with this, a culture medium containing no added substance (control) was similarly subjected to rotary culture for 5 days,
The numbers of bacteria in these cultures were counted directly. The results are shown in Table 2.

The final bacterial concentration was 7.0 × 10 ⁸ cells / ml or more in any of the supplemented media except pyruvic acid and glucose, but the final bacterial concentration of the non-supplemented medium (control) was 2.6 × 10 ⁸ Stayed at cells / ml.

[Table 2]

Bacterial fluid (2.0 × 10 ⁹ cells / ml) enriched with gellan gum-containing medium having the highest final bacterial concentration in Table 2,
The bacterial solution (1.0 × 10 ⁹ cells / ml) enriched with the glucose-containing medium having the lowest final bacterial concentration and the bacterial solution of the additive-free medium (control) were subjected to the following storage test.

The bacterial solution of the additive-free medium (control) was collected by centrifugation (9000 × 0.5 minutes) and then resuspended in the medium (Table 1) so as to be 5 times concentrated. The bacterial concentration was 2.0 × 10 ⁹ cells / ml).

First, colorless silica gel was washed with water and dried, and 0.4 g thereof was put into a test tube and sterilized by dry heat at 180 ° C. for 2 hours. While cooling with ice, 1 ml of the above-mentioned three kinds of bacterial solutions was added dropwise and frozen and stored in a deep freezer (-80 ° C).
In addition, 1 ml of the above-mentioned three kinds of bacterial solutions was directly frozen and stored in a deep freezer (-80 ° C). The control was also preserved in the same manner as the above operation.

After 24 hours, these preserved bacteria were suspended in 10 ml of a medium (Table 1) and cultured at 30 ° C. and 120 rpm to try to restore the nitrifying ability. The expression of nitrification ability was observed by quantifying the nitrite concentration in the culture. The results are shown in Table 3.

[Table 3]

From these results, the nitric acid bacteria consumed nitrite after 4 days at the latest after freezing or silica gel cryopreservation in any added or non-added medium.

Pyruvate, oxalacetic acid, citric acid, isocitric acid, 2-oxoglutaric acid, succinic acid,
Fumaric acid, malic acid, glyoxylic acid, acetic acid, ATP,
The same results as in Table 3 were obtained for the medium supplemented with glucose, fructose, rhamnose and gellan gum.

(Comparative Example 1) 1 ml of each of the three kinds of storage nitrite bacteria solution of Example 1 was placed in an ampoule, and water was removed by depressurizing at room temperature with a vacuum dryer (this method is called L-drying). Has been done).

Further, in parallel, three ml of each of the three types of nitrite bacteria solution for storage were placed in an ampoule, preliminarily frozen with dry ice-acetone, and then water was removed under reduced pressure by a vacuum dryer.

After 24 hours, these preserved bacteria were suspended in 10 ml of the medium (Table 1) and cultured at 30 ° C. and 120 rpm to try to restore the nitrification ability in the same manner as in Example 1. The expression of nitrification ability was observed by quantifying the nitrite concentration in the culture solution. The results are shown in Table 4.

[Table 4]

From these results, the nitrification reaction of nitric acid bacteria was developed after 5 days by L-drying in any of the added medium and the non-added medium, but the reconstitution was slow in freeze-drying.

The same results as in Table 4 were obtained when the product was suspended in a 3% L-sodium glutamate solution in L-drying.

Comparing this Comparative Example 1 with Example 1, it can be seen that the preservation of the nitric acid bacterium is superior to the cryopreservation or the silica gel cryopreservation.

(Example 2) In the same manner as in Example 1, 1 part of the nitrite bacterium solution enriched with a medium supplemented with rhamnose and glucose was used.
It was stored frozen for 0 months on silica gel. 1 ml of this preserved bacterial solution
Sodium pyruvate 10 mM, oxalacetic acid 10 mM, trisodium citrate dihydrate 1 m
M, cisaconitic acid hydrate 10 mM, trisodium isocitrate dihydrate 10 mM, 2-oxoglutarate 5 mM, sodium succinate hexahydrate 1 mM, monosodium fumarate 10 mM, L-malic acid 10 mM disodium, 10 mM gluoxylic acid monohydrate,
Sodium acetate 10 mM, adenosine triphosphate (AT
P) Disodium 5 mg / liter, D-glucose 5 mM, D-fructose 10 mM, α-L-rhamnose 10 mM, gellan gum 0.1%, sodium lactate 1 mM, sodium L-glutamate 1 mg /
l, L-sodium aspartate 5 mg / l, L-
The cells were suspended in 10 ml of a medium (Table 1) added with serine at 10 mg / l and cultured at 30 ° C. and 120 rpm to restore the nitrification ability. The expression of nitrification ability was observed by quantifying the nitrite concentration in the culture solution as in Example 1. The results are shown in Table 5.

[Table 5]

From this, it can be seen that the nitrification performance of nitric acid bacteria is expressed faster and the restoration is faster in the supplemented medium than in the non-supplemented medium.

[0037]

【The invention's effect】

(1) In the present invention, it is possible to increase the concentration of nitric acid bacteria by culturing for a relatively short period of time, and therefore, the bacterial solution can be preserved as it is when it is preserved, and operations such as concentration are unnecessary.

(2) Since the storage period can be significantly extended by cryopreserving silica gel, it does not require much labor as in the subculture method, and furthermore, the occurrence of mutations associated with transplantation can be minimized.

(3) When restoring the nitrite bacteria that have been frozen and stored on silica gel, the restoration of the nitrification ability is remarkably accelerated as compared with the conventional culture with an inorganic compound (nitrite).

(4) In addition to the above effects, as an inoculum for acclimatizing the nitrification apparatus or for troubleshooting, a nitrifying performance is recovered or recovered within a short time with a smaller volume of nitrifying bacterium solution compared to the conventional one. To be made.

[Procedure amendment]

[Submission date] April 30, 1991

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 2

[Correction method] Change

[Correction content]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0002

[Correction method] Change

[Correction content]

[0002]

2. Description of the Related Art Nitrifying bacteria use ammonia or nitrous acid
It is an autotrophic bacterium that oxidizes to nitric acid or nitric acid and synthesizes bacterial components using carbon dioxide as the sole carbon source, and it has been said that the nitrification reaction of nitrifying bacteria is inhibited when organic substances coexist.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0021

[Correction method] Change

[Correction content]

In parallel with this, a culture medium (control) containing no added substance was similarly subjected to rotary culture for 5 days, and the number of bacteria in these culture solutions was directly counted. The results are shown in Table 2.

[Procedure amendment 4]

[Document name to be amended] Statement

[Name of items to be corrected] Table 2

[Correction method] Change

[Correction content]

[Table 2]

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0024

[Correction method] Change

[Correction content]

In addition, the bacterial solution of the additive-free medium (control) was resuspended in the medium (Table 1) so as to be concentrated 5 times after collecting the cells by centrifugation (9000 × G, 5 minutes). (The bacterial concentration at this time was 2.0 × 10 ⁹ cells / m 2.
l) was used.

[Procedure Amendment 6]

[Document name to be amended] Statement

[Correction target item name] 0026

[Correction method] Change

[Correction content]

After 24 hours, these preserved bacteria were suspended in 10 ml of a medium (Table 1) and cultured at 30 ° C. and 120 rpm to try to restore the nitrifying ability. The expression of nitrification ability was observed by quantifying the nitrite concentration in the culture. The results are shown in Table 3.

[Procedure Amendment 7]

[Document name to be amended] Statement

[Name of item to be corrected] 0030

[Correction method] Change

[Correction content]

In addition, in parallel, 1 ml of each of the three types of storage nitrite bacteria solution for preservation was placed in an ampoule, preliminarily frozen with dry ice-acetone, and then water was removed under reduced pressure by a vacuum dryer.

[Procedure Amendment 8]

[Document name to be amended] Statement

[Correction target item name] 0031

[Correction method] Change

[Correction content]

After 24 hours, these preserved bacteria were suspended in 10 ml of a medium (Table 1), cultured at 30 ° C. and 120 rpm, and an attempt was made to restore their nitrification ability in the same manner as in Example 1. The expression of nitrification ability was observed by quantifying the nitrite concentration in the culture solution. The results are shown in Table 4.

[Procedure Amendment 9]

[Document name to be amended] Statement

[Name of item to be corrected] 0032

[Correction method] Change

[Correction content]

From these results, nitrification reaction of nitric acid bacteria appeared after 5 days by L-drying in any addition medium or non-additional medium, but by lyophilization, reconstitution was slow.

[Procedure Amendment 10]

[Document name to be amended] Statement

[Correction target item name] 0035

[Correction method] Change

[Correction content]

Example 2 As in Example 1, a nitric acid solution supplemented with a medium containing rhamnose and glucose was cryopreserved for 10 months on silica gel. This preserved bacterial solution 1m
1m each, sodium pyruvate 10 mM, oxalacetic acid 10 mM, trisodium citrate dihydrate 1 m
M, cisaconitic acid hydrate 10 mM, trisodium isocitrate dihydrate 10 mM, 2-oxoglutarate 5 mM, sodium succinate hexahydrate 1 mM, fumaric acid-sodium 10 mM, L-malic acid 10 mM disodium, 10 mM glyoxylic acid hydrate,
Sodium acetate 10 mM, adenosine triphosphate (AT
P) disodium 5 mg / l , D-glucose 5 m
M, D-fructose 10 mM, α-L-rhamnose 10 mM, gellan gum 0.1%, sodium lactate 1 mM, sodium L-glutamate 1 mg / l, L
-Suspension in 10 ml of a medium (Table 1) supplemented with sodium aspartate at 5 mg / l and L-serine at 10 mg / l, and the suspension was cultured at 30 ° C and 120 rpm to restore its nitrification ability. The expression of nitrification ability was observed by quantifying the nitrite concentration in the culture solution as in Example 1. The results are shown in Table 5.

[Procedure Amendment 11]

[Document name to be amended] Statement

[Correction target item name] 0039

[Correction method] Change

[Correction content]

(3) Nitric acid stored in a frozen state on silica gel
When the bacteria are restored, the restoration of nitrification ability is remarkably accelerated as compared with the conventional culture using an inorganic compound (nitrite).

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Claims (2)

[Claims] 1. A method for preserving nitric acid bacteria, wherein the nitric acid bacteria are pyruvic acid or a salt thereof, oxalacetic acid or a salt thereof, citric acid or a salt thereof, cisaconic acid or a salt thereof, isocitric acid or a salt thereof, and 2-oxoglutar. Acid or its salt, succinic acid or its salt,
Fumaric acid or its salt, malic acid or its salt, glyoxylic acid or its salt, acetic acid or its salt, adenosine triphosphate or its salt, D-glucose, D-fructose, α-L-rhamnose, α-L-rhamnose. 1
Molecule and D-glucuronic acid molecule and D-glucose 2
A nitric acid bacterium is cultivated in a liquid containing at least one organic compound of a high molecular polysaccharide in which the molecule is used as one unit and the unit is linearly polymerized, and the bacterium liquid is stored as it is or after mixing with silica gel and then frozen. A method for preserving nitric acid bacteria, which is characterized in that 2. The method for restoring frozen nitrite bacteria according to claim 1, wherein the organic compound according to claim 1, lactic acid or a salt thereof, L-glutamic acid or a salt thereof,
A method for restoring a nitric acid bacterium, which comprises culturing in a medium containing at least one organic compound selected from L-aspartic acid or a salt thereof and L-serine or a salt thereof.