JP2902148B2 - Method for accelerating nitrification reaction of nitrate bacteria - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for accelerating the nitrification reaction of nitrifying bacteria among nitrifying bacteria (referred to as nitrifying bacteria) contained in closed-system circulating water for marine animal rearing and transportation facilities, aquariums, etc., and relates to wastewater or sewage treatment. The present invention relates to a method for accelerating the nitrification reaction of nitrate bacteria contained in equipment, soil, seawater, river water, and the like, and to a method that can be used as a means for measuring the number of bacteria.

[0002]

2. Description of the Related Art Nitrifying bacteria are autotrophic bacteria that oxidize ammonia or nitrite to nitrite or nitrate and synthesize cell components using carbon dioxide as the sole carbon source. Has been said to be inhibited.

[0003] Nitrifying bacteria include ammonia-oxidizing bacteria that oxidize ammonia to nitrite (hereinafter referred to as "nitrite").
And nitrite-oxidizing bacteria (hereinafter referred to as "nitrite bacteria") that oxidize nitrite to nitrate. Each chemical reaction formula is as follows. (A) ammonia oxidation reaction of nitrifying bacteria ^{_{NH 4 + + 3 / 2O 2}} → NO 2 - + H 2 O + 39.5kcal (b) nitrite-oxidizing reaction of nitric bacteria ^{_{NO 2 - + 1 / 2O 2}} → NO 3 - + 21.6kcal

[0004] These bacteria are heterotrophic bacteria (they grow using energy generated by the 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 sufficiently. The details will be described below by taking a conventional method for counting nitric acid bacteria as an example.

When measuring nitrate bacteria in sewage or soil,
For 1 liter of distilled water, 30m of sodium nitrite
g, potassium monophosphate 100mg, iron chelate 6m
g, magnesium sulfate heptahydrate 50 mg, calcium chloride dihydrate 20 mg, sodium bicarbonate 200 mg,
Add a small amount of calcium carbonate and a small amount of quartz sand, adjust the pH to 7.0, and sterilize by high pressure. Akira Kawakawa: Method for counting bacterial groups related to the metabolism of nitrogen compounds. 291-295 (196)
9)｝. 9 ml of this medium is dispensed into sterilized test tubes.

On the other hand, the sample is diluted 10-fold and 10-fold with a sterile diluent, and 1 ml of the sample in each dilution step is added to a test tube into which the above medium has been dispensed. Usually 3 or 5 at each stage
Contacted with the medium of the book, cultured at 20-30 ° C. for 60 days,
The remaining nitrite ion (NO ₂ ⁻ ) in each medium was detected, and from the results, the most probable number (M
PN) is calculated to estimate the number of bacteria in the sample.

[0007]

(1) In the conventional measuring method, the number of days required for culturing is usually extremely long, usually 60 days or more, and rapid measurement cannot be performed. For this reason, the operating conditions of the nitrification device and the like cannot be accurately determined.

(2) Use of three or five test tubes for each dilution step requires a lot of equipment, equipment and labor. In addition, since the number of days of culture is extremely long, more instruments, devices and labor are required to collectively measure a large number of samples, and it has been difficult in practice to measure efficiently.

(3) In a normal operation of the nitrification apparatus, the nitric acid bacterium may be damaged due to some cause in the system, the nitrification performance may be reduced, and nitrite ions may be accumulated. In this case, the time required for the performance recovery is longer than that required for an organic wastewater purification device or the like. This is because nitrate bacteria grow more slowly than heterotrophic bacteria, and it takes a long time to reach a bacterial amount sufficient to exhibit nitrification performance.

The present invention has been made in order to solve the above problems.
The present invention proposes a method for accelerating a nitrification reaction that can measure the number of nitrate bacteria among nitrifying bacteria.

[0011]

SUMMARY OF THE INVENTION The present invention is a method of promoting the nitrification reaction of nitric bacteria, (1) click enoic acid or salts thereof, cis-aconitic acid or a salt thereof, isocitric acid or a salt thereof, or a succinic acid Salt, fumaric acid or its salt, malic acid or its salt, glyoxylic acid or its salt, adenosine 3
Phosphoric acid (ATP) or a salt thereof, (2) L-glutamic acid or a salt thereof, L-aspartic acid or a salt thereof, L-serine or a salt thereof, (3) α-L-rhamnose, α-L-fucose, D -Glucuronic acid or a salt thereof, N-acetylglucosamine, N-acetyl-D-muramic acid, (4) one molecule of α-L-rhamnose, D-glucuronic acid 1
A nitric acid bacterium characterized in that a nitric acid bacterium is cultured in a liquid containing at least one or more organic compounds among high molecular polysaccharides in which the unit is composed of a molecule and two molecules of D-glucose. Is a method for accelerating the nitrification reaction.

[0012]

[Action] nitrification ability of nitric bacteria by specific organic compounds is promoted, a relatively short detectable concentration difference nitrite ions within the (NO ₂ ^-) are removed. The mechanism by which a specific organic compound promotes the nitrification ability of nitric acid bacteria is still unknown. Probably, these specific organic compounds are easily biosynthesized from other organic compounds in heterotrophic bacteria, but the biosynthesis in nitric acid bacteria may take considerable time due to differences in metabolic pathways. It is thought that there is not.

[0013]

【Example】

(Example 1) The representative strains of nitric acid bacteria (Nitrobacter agilis
ATCC14123) will be described as an example to explain one embodiment of the present invention. The culture of nitric acid bacteria in this example was performed using a medium autoclaved as shown in Table 1 (the following medium was referred to. Lewis, RF and
D. Pramer, 1958, "Isolation of Nitrosomonas in p
ureculture ", 76, 524-528).

[Table 1]

10 ml of a nitric acid bacterium previously cultured in the medium of Table 1 for 10 days was added to 100 ml of fresh medium, and 2 ml of each Erlenmeyer flask (200 ml) was added.
It dispensed by 0 ml at a time. Then the addition concentration was 1 and 5 mM
Trisodium citrate / 2 water sterilized by filtration so that
Each of the hydrate solutions was added, and the temperature was 30 ° C.
The cells were rotated at 96 rpm for 96 hours. For these cultures, the nitrite concentration at the start and end of the culture was measured by the Grease-Romiin reagent method.

The additive material starve case of Suakonitto anhydride 5mM and 10 mM, isocitrate trisodium
5 mM and 10 mM for dihydrate, 1 mM and 5 mM for sodium succinate hexahydrate, 1 mM and 5 mM for monosodium fumarate, 1 mM and 10 mM for disodium L-malate, Glyoxylic acid monohydrate is added to the culture medium at 5 mM and 10 mM, and adenosine triphosphate (ATP) disodium trihydrate at 25 mg / l and 50 mg / l, respectively. Culture was performed.

For comparison, a nitrification test was also performed on a culture solution (control) containing only nitric acid bacteria containing no added substances in parallel. Table 2 shows the above results. From these results, it was found that the concentrations of the remaining nitrites in the other supplemented media except for 5 mM of succinic acid were lower than those in the control, and these supplemented substances had an effect of promoting the nitrification reaction of the nitrate bacteria.

Comparing the bacterial concentration at the end of the cultivation based on the simultaneous counting of the bacterial count, ATP is almost the same or lower than the control bacterial concentration (2.6 × 10 ⁸ cells / ml), and the growth promotion of nitrate bacteria is promoted. Had little effect.

[0018] One Hoku enoic acid, cis-aconitic acid, isocitric acid, succinic acid, fumaric acid, malic acid and Guruokishiru acids have different respective optimum addition concentration of growth-promoting and nitrification reaction promoting nitric acid bacteria, these substances Depending on the added concentration, there was an effect of promoting either the growth of nitric acid bacteria or the nitrification reaction.

[Table 2] Note) Additive names in the table are all abbreviations.

(Example 2) 1% sodium L-glutamate
96 hours of pre-cultivation of a medium supplemented with l-mg / l and 5 mg / l, sodium L-aspartate at 1 mg / l and 5 mg / l, and L-serine at 5 mg / l and 10 mg / l. The fungus was inoculated and cultured in the same manner as in Example 1. Table 3 shows the results.

From these results, it was found that in any of the added substances, nitrous acid was removed faster than the control, and had an effect of accelerating the nitrification reaction of nitrate bacteria.

However, a comparison of the bacterial concentration at the end of the culture based on the simultaneous counting of the bacterial count shows that these added substances are almost the same or lower than the control bacterial concentration (2.6 × 10 ⁸ cells / ml). It had no effect on the growth of nitrate bacteria.

[Table 3]

Example 3 α-L-rhamnose and α-L
-Fucose to 1 mM and 5 mM respectively, sodium D-glucuronate to 1 mM and 5 mM, N-acetyl-D-glucosamine to 1 mg / l and 5 mg / l
l, N-acetyl-D-muramic acid was added at 25 mg / l and 50 mg / l in a culture medium (Table 1), and a nitrate bacterium was inoculated for 96 hours in preculture, and cultured in the same manner as in Example 1. did. Table 4 shows the results.

From these results, it was found that nitrous acid was removed faster than the control with any of the added substances, and had an effect of accelerating the nitrification reaction of nitric acid bacteria.

When the cell concentrations at the end of the cultivation were compared based on the simultaneous counting of the number of cells, the optimal addition concentrations of L-rhamnose and L-fucose for promoting the growth of nitric acid bacteria and for promoting the nitrification reaction did not match. Depending on the added concentration, there was an effect of promoting either the growth of nitric acid bacteria or the nitrification reaction. However, D-glucuronic acid, N-acetyl-D-glucosamine and N-acetyl-D-muramic acid were almost the same or lower than the control bacterial concentration (2.6 × 10 ⁸ cells / ml). Had no effect.

[Table 4]

Example 4 Pseudomonas elodea (ATCC31461)
A nitrification test of nitric acid bacteria was performed using a high-molecular-weight polysaccharide (product name: gellan gum manufactured by Wako Pure Chemical Industries, Ltd.) obtained by deacetylating and producing a mucous substance produced by Nitto. This substance is α-L
-One molecule of rhamnose, one molecule of D-glucuronic acid and D
-Two units of glucose are defined as one unit, and the unit is a linearly polymerized high molecular weight polysaccharide (average weight molecular weight is 650,000). This substance was added to a concentration of 0.1, 0.2, 0.5, 1.0 and 5.0%, and a medium (Table 1) sterilized by high pressure was inoculated with nitrate bacteria for 6 days of preculture. The culture was carried out for 96 hours in the same manner as in Example 1. Table 5 shows the results.

From these results, it was found that, regardless of the concentration of gellan gum, the concentration of residual nitrite was lower than that of the control, and the nitrification reaction of nitric acid bacteria was promoted.

Comparing the bacterial concentration at the end of the cultivation based on the simultaneous counting of the number of bacteria, the optimum concentration of this additive differs in the promotion of the growth of nitric acid bacteria and the promotion of the nitrification reaction. Had the effect of promoting either the proliferation or the nitrification reaction.

[Table 5]

[0028] (Example 5) click enoic acid sodium dihydrate is 1 mM, cis-aconitic anhydride 5 mM, isocitric trisodium dihydrate is 5 mM, sodium succinate hexahydrate is 1 mM,
Monosodium fumarate is 5 mM, disodium L-malate 1 mM, glyoxylic acid monohydrate 5 mM, adenosine triphosphate disodium trihydrate (ATP) 25
mg / l, 1 L-sodium glutamate monohydrate
mg / l, sodium L-aspartate monohydrate 1 mg / l, L-serine 5 mg / l, α-L-rhamnose 1 mM, α-L-fucose 1 mM, sodium D-glucuronate 1 The medium in Table 6 (Engel, MS and M) was adjusted so that the concentration of hydrate was 1 mM, N-acetylglucosamine was 1 mg / l, N-acetyl-D-muramic acid was 25 mg / l, and gellan gum was 0.5%. Alexander,
1951. "Growth and autotrophic metabolism of Nitoro
somonas europaea ", J. Bacteriol., 76, 217-222), and supplemented media of each substance were prepared. Each of the supplemented media was dispensed into three test tubes in an amount of 5 ml.

On the other hand, a nitrate bacterium purely separated from sewage sludge was cultured in a medium (Table 6) for 7 days (at this time, the bacterial concentration was 1.2 × 10 ⁸ cells / ml by direct counting). It was diluted 10 ⁸ to 10 ¹⁰ times with physiological saline.
1 ml of each serial dilution was added to each of the three test tubes prepared for each of the above-mentioned supplemented media. These test tubes were shake-cultured at 30 ° C. for 180 strokes per minute.

In parallel with this, three test tubes (control) into which a medium (Table 6) to which no additive was added were dispensed.
1 ml of each serially diluted solution was added thereto, and the cells were cultured under the same conditions as above.

The residual nitrite concentration of these test tubes was measured every 5 days. Table 7 shows the number of days required for the nitrite concentration in the medium to decrease by 20 mg / l or more, and the estimated bacterial concentration based on the most probable number (MPN) value obtained from these results.

From this, the most probable number (MPN) and the bacterial concentration determined by direct counting agreed relatively well. It was also found that the required culture days of the supplemented medium were significantly shorter than those of the control.

[Table 6]

[Table 7] Note) Additive names in the table are all abbreviations.

[0033]

(1) In the present invention, the nitrification ability (nitrite removal ability) is improved 3 to 6 times as compared with the conventional culture of nitric acid bacteria using an inorganic compound (nitrite). (2) At the time of starting or at the time of occurrence of a trouble, the nitrification device has a low nitrification ability. Even in such a case, according to the present invention, the nitrification ability can be rapidly improved or restored so as not to be compared with the related art. (3) With the conventional method, the required number of culture days is extremely long, and the measurement of the number of nitrate bacteria, which could not be performed quickly, can be performed in a relatively short number of days. It is possible to make a judgment. (4) Since the counting of nitrate bacteria can be performed relatively easily and in a short number of days, it is possible to reduce many instruments, devices and labor required by the conventional method.

Claims (1)

(57) [Claims] 1. A method for promoting nitrification reaction of nitric bacteria, (1) click enoic acid or salts thereof, cis-aconitic acid or a salt thereof, isocitric acid or salts thereof, succinic acid or salts thereof, fumaric acid or a salt thereof , Malic acid or a salt thereof, glyoxylic acid or a salt thereof, adenosine 3
Phosphoric acid (ATP) or a salt thereof, (2) L-glutamic acid or a salt thereof, L-aspartic acid or a salt thereof, L-serine or a salt thereof, (3) α-L-rhamnose, α-L-fucose, D -Glucuronic acid or a salt thereof, N-acetylglucosamine, N-acetyl-D-muramic acid, (4) one molecule of α-L-rhamnose, D-glucuronic acid 1
A nitric acid bacterium characterized in that a nitric acid bacterium is cultured in a liquid containing at least one or more organic compounds among high molecular polysaccharides in which the unit is composed of a molecule and two molecules of D-glucose. A method for accelerating the nitrification reaction.