JP2002176972A - High-concentration nitrite-oxidizing bacterium, and method for high-concentration culture of nitrite- oxidizing bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for culturing in large quantities and in high concentrations nitrite-oxidizing bacteria in nitrifying bacteria contained in activated sludge. SOLUTION: The method for culturing in high concentrations nitrite-oxidizing bacteria in nitrifying bacteria slightly contained in activated sludge including sewage sludge and excrement sludge comprises the following process: activated sludge is subjected to nitrifying acclimatization by feeding an NH4-N-containing liquid in a dissolved oxygen concentration of >=2 mg/L at pH 7.0-9.0 and 20-40 deg.C for a specified period, and the pH of the system tending toward acidic side during the acclimatization step is maintained within the above-mentioned range by incorporation of a culture promoter consisting of a mixture of sodium carbonate and sodium bicarbonate to effect the acclimatizing accumulation of the ammonia-oxidizing bacteria and nitrite-oxidizing bacteria in the nitrifying bacteria contained in the activated sludge; thereafter, feed of the NH4-N-containing liquid is stopped and an NO2--containing liquid is fed instead to decrease the number of the ammonia-oxidizing bacteria, and the nitrite-oxidizing bacteria are further subjected to acclimatizing accumulation.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a high concentration nitrite-oxidizing bacterium,
And a high-concentration culture method for nitrite-oxidizing bacteria.

[0002]

2. Description of the Related Art Nitrifying bacteria (ammonia-oxidizing bacteria and nitrite-oxidizing bacteria) have a slow growth rate and have been confirmed for one hundred years due to the fact that they do not live as colonies. Until today, there have been no reports of successful industrial mass cultivation at high concentrations.

Furthermore, there is no report that the culture of only nitrite oxidizing bacteria at a high density (high concentration) was successful.

[0004] Nitrite oxidizing bacteria have a characteristic that they are susceptible to a high nitrite concentration in spite of the fact that they are nitric oxides that take over and oxidize nitrite produced by ammonia oxidizing bacteria. At most 2
At a nitrite concentration of 0 to 30 ppm, the oxidizing activity was lost.

[Object of the Invention] The present invention is to provide a freshwater nitrite oxidizing bacterium and a salt-tolerant nitrite oxidizing bacterium (for example, a nitrite oxidizing bacterium which can withstand a high salt concentration such as seawater) which has been considered impossible. It is an object of the present invention to provide a method for culturing a large amount and at a high density (high concentration), and culturing at a high density (high concentration) under a high nitrite concentration (for example, 50 ppm, 1 ppm).
(200 ppm, 200 ppm), it is an object of the present invention to provide a freshwater or salt-tolerant, high-density nitrite-oxidizing bacterium having excellent properties such as having the activity of oxidizing the nitrite.

[0006]

A nitrite according to claim 1
The high-concentration cultivation method of oxidizing bacteria is used for sewage sludge
Nitrite in nitrifying bacteria slightly contained in activated sludge
A method of culturing activated bacteria at a high concentration, wherein the activated sludge is
With NH₄-N containing solution to give the activity
Ammonia-oxidizing bacteria in nitrifying bacteria contained in sludge
After accumulating with nitrite oxidizing bacteria, the NH₄−
Stop application of N-containing liquid and NO ₂ ⁻With liquid
To reduce the number of ammonia oxidizing bacteria
And accumulate nitrite-oxidizing bacteria further.
It is a way to sign.

The method for culturing nitrite-oxidizing bacteria at a high concentration according to claim 2 is a method for culturing nitrite-oxidizing bacteria at a high concentration in nitrifying bacteria slightly contained in activated sludge such as sewage sludge and human waste sludge. And the activated sludge is dissolved in 2 m of dissolved oxygen.
g / liter or more, pH 7.0-9.0, temperature 20-4
Nitrogenization by applying an NH ₄ —N-containing liquid for a predetermined period under the condition of 0 ° C., and adjusting the pH leaning to the acidic side in the acclimation process to a culture promoting agent comprising a mixture of sodium carbonate and sodium hydrogen carbonate. By maintaining the concentration within the above-mentioned range, the ammonia-oxidizing bacteria and nitrite-oxidizing bacteria in the nitrifying bacteria contained in the activated sludge are acclimatized and accumulated, and then the application of the NH ₄ —N-containing liquid is stopped and NO ₂ is added. ^- by applying a solution containing, it allowed reducing the number of ammonia oxidizing bacteria, and a method characterized by further acclimatization integrated nitrite oxidizing bacteria.

[0008] The method for culturing nitrite-oxidizing bacteria according to the third aspect of the present invention is the method for culturing nitrite-oxidizing bacteria according to the first or second aspect, wherein the nitrite oxidizing bacteria accumulate in the alkaline side. The method is characterized in that the pH that is inclined toward is maintained at a predetermined value by adding CO ₂ .

[0009] The high-concentration nitrite-oxidizing bacterium according to claim 4 is characterized in that it has a growth ability even in a freshwater environment, for example, in a high-salinity environment such as seawater even at a nitrite concentration of 50 ppm.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Activated sludge The activated sludge used in the present invention includes sewage sludge and human waste sludge. These may be those that have been subjected to fresh water dilution treatment, or may be those that have been subjected to sea water dilution treatment. Since a large amount of marine nitrite-oxidizing bacteria can be obtained, it is preferable to use activated sludge that has been subjected to a seawater dilution treatment.

[0011] To add a further explanation, natural seawater contains marine nitrite-oxidizing bacteria, which are considered to have higher salt tolerance than freshwater nitrite-oxidizing bacteria, but their abundance is very small. Due to the difficulty of pure isolation, its work has been delayed compared to freshwater nitrite-oxidizing bacteria. However, according to the culture method of the present invention, a high concentration of marine nitrite-oxidizing bacteria can be obtained by using the activated sludge subjected to the seawater dilution treatment as described above as a raw material. Marine nitrite-oxidizing bacteria have multi-layered cell walls and have strong resistance to various chemical substances that inhibit osmotic pressure change and growth of treated water.

Culture of nitrifying bacteria (cultivation) Culture of nitrifying bacteria contained in activated sludge is carried out by subjecting the activated sludge to sludge dewatered filtrate or (anaerobic) digestion for a predetermined period (for example, one month, two months or three months). Nitrogenation is performed by sludge treatment waste liquid such as a desorbed liquid. Since nitrification must be performed aerobically, the dissolved oxygen (DO) at this time must be 2 mg / liter or more. However, it was found for the first time in the present experiment that the growth speed tends to decrease when the dissolved oxygen concentration is too high. The details will be described below.

[0013] The nitrification speed by nitrifying bacteria is higher as the dissolved oxygen is higher. Therefore, it was thought that the higher the dissolved oxygen, the faster the nitrification accumulates. It was found that the acclimatization and accumulation of nitrifying bacteria using activated sludge as a raw material was slowed down from about 5 mg / liter of dissolved oxygen (DO). Note that the dissolved oxygen (DO) concentration is most preferably 2 to 4 mg / liter.

The pH must be 7.0-9.0 (especially when activated sludge diluted with seawater is used).
It is preferably from 5 to 8.5, more preferably from 7.5 to 7.8.

[0015] Regarding the cultivation temperature, the growth speed is high in the range of 20 to 40 ° C, and more preferably 25 to 35 ° C.

In the course of the culture, the alkalinity decreases as the pH decreases. That is, the oxidation of NH ₄ + to NO ₂ − by ammonia oxidizing bacteria and the oxidation of NO ₂ − to NO ₃ − by nitrite oxidizing bacteria are expressed by the following two equations (A) and (B). Expression (C) is an expression for the entire nitrifying bacteria.

[0017]

Embedded image

From these, 4.57 mg O ₂ / mg NH ₄ —N oxygen is required to oxidize NH ₄ —N to NO ₃ —N, and hydrogen ions are released as the nitrification reaction proceeds. It can be seen that the alkalinity decreases as the pH of the culture decreases. Since the cultivation rate decreases as the pH decreases, unless the pH is maintained at a predetermined value using a buffer solution or the like, the activity of the microorganism stops as in the conventional method.

Therefore, in the present invention, it is considered that a compound having a buffering action by mixing a non-hydride and a hydride with a pH which is inclined toward the acidic side during the culturing process is preferable. As a result of repeated trial and error with a large number of compounds, it has been found that it is most preferable to restore the pH by adding a culture promoting agent composed of a combination of sodium carbonate and sodium hydrogen carbonate.

It is generally known that the synthesis reaction of bacterial cells can be expressed by the following equation.

[0021]

Embedded image

When this is applied to the above-mentioned biochemical reaction formula of the mixed culture system (C), the following is obtained.

[0023]

Embedded image

As is clear from the above formula, the cultivation of nitrifying bacteria requires a large amount of carbon source as compared with ammonium ion as an energy substrate.

As described above, by supplying a culture promoting agent comprising a combination of sodium carbonate and sodium bicarbonate, a carbon source for carbonic assimilation of nitrifying bacteria can be simultaneously supplied. Hereinafter, an explanation will be added.

When only sodium carbonate is used, the effect of increasing the decreasing pH is sufficiently recognized even when the sodium carbonate is a strong alkali, but the effect of increasing the pH is large, so that it can be used in a large amount. However, there is a point that it is not suitable to supply a sufficient carbon source. On the other hand, when only sodium hydrogen carbonate is used, there is no problem in terms of supply as an inorganic carbon source, but a large amount of supply is required in terms of maintaining pH, which is not preferable.

In view of such advantages and disadvantages, a mixture of sodium carbonate and sodium hydrogen carbonate can be suitably used.
By using an aqueous solution of the mixture, it has become possible to effectively supply an inorganic carbon source for assimilation of carbonic acid in a living body while keeping a gradually decreasing pH constant.

The mixing ratio of sodium carbonate and sodium hydrogencarbonate in the mixture is sodium carbonate:
It is preferable that the molar ratio of sodium hydrogen carbonate is 4 to 7: 4 to 8, and specifically, sodium carbonate is 0.4 to 0.4.
A mixed aqueous solution of 0.7 to 0.7 (mol / liter) and 0.4 to 0.8 (mol / liter) of sodium hydrogen carbonate is effective.

The monitoring of the pH of the culture system may be performed continuously or at predetermined intervals. pH
It is preferable to use a continuous pH monitoring device such as a controller, but not limited to this.
It can also be performed manually using an indicator.

The concentration of ammonia in the NH ₄ —N-containing liquid is preferably 100 mg / L or more and 300 mg / L or less, more preferably 200 mg / L or less. Ammonia is an energy source when ammonia-oxidizing bacteria, which are chemoautotrophic bacteria, grow by performing carbon assimilation.
It may inhibit growth. In addition, nitrite oxidizing bacteria contained in activated sludge, like ammonia oxidizing bacteria, are weak against high nitrite concentration, because they are bacteria that oxidize by taking over nitrite generated by ammonia oxidizing bacteria. The concentration cannot be set unnecessarily high. Therefore, the concentration of ammonia was 300 mg /
If it exceeds 1 liter, it is preferable to appropriately dilute it with seawater or fresh water.

As the NH ₄ —N-containing liquid, it is preferable to use a sludge treatment waste liquid such as a sludge dewatered filtrate or a digestion / desorption liquid generated in a water treatment plant.

Under the above culture conditions, activated sludge is nitrified by sludge treatment wastewater such as sludge dehydration filtrate and digestion / desorption solution, whereby the nitrifying bacteria contained in the activated sludge slightly can be cultured at a high concentration. Although it is possible, in addition to this, according to the present invention, activated sludge can be reduced to 1/3 to 2 months in 2 months.
Nitrified sludge which can be reduced in volume to 1/4 and has a large specific gravity can be obtained.

That is, it is said that activated sludge contains nitrifying bacteria at about 0.35% in the first place. By accumulating the activated sludge using the activated sludge as a raw material with an NH ₄ —N-containing liquid for about 2 months, the content of nitrifying bacteria in the activated sludge is increased by about 10 times (3.5%). To increase. In the process, other germs cannihilate and die because no external nutrients (feeds) are provided. As a result, the amount of activated sludge is reduced (volume is reduced).

When the germs almost die, they become hard-to-decompose organic substances having a large specific gravity called "granules", and nitrifying bacteria attach to the surroundings using these as nuclei. The hardly decomposable organic matter attached to the nitrifying bacteria sediments in the culture system due to its specific gravity. In order to culture nitrifying bacteria at a high concentration, this good sedimentation property is required. That is, nitrifying bacteria generally have a low specific gravity and float in pure culture. Therefore, nitrifying bacteria are likely to flow out of the culture system, and high-density culture cannot be expected. As a result, the production of nuclei (refractory organic substances) as described above is necessary for high-concentration culture.
It is not found in pure culture of nitrifying bacteria, but only when activated sludge is used as a raw material.

PH Adjusting Agent in Nitrification Condition The mixture used both in the present invention and adjusting the pH and the carbon source may be used as a bacterial high-concentration culture promoting agent in the following embodiments. That is, 1. An autotrophic bacterium, which is an autotrophic bacterium high-concentration cultivation promoter that is a carbon source while maintaining the pH of the medium that tilts to the acidic side in the acclimatization process in a predetermined range when acclimating the autotrophic bacteria for a high concentration for a predetermined period, The two properties of a medium that exhibits basicity by dissociation and can maintain the pH of the medium inclined toward the acidic side within a predetermined range, and a property that can serve as a carbon source during growth of the autotrophic bacterium are as follows. If it can be provided by a kind of compound, at least one of the compounds
A species or, when the above two properties can be imparted by two or more kinds of compounds, a mixture of at least two kinds of the compounds is blended; .

2. 2. The autotrophic bacterium high concentration cultivation promoter according to claim 1, wherein the mixture is a mixture of the following components (A) and (B).

(A) A basic substance which is soluble in water and shows basicity when dissociated.

(B) Carbonates soluble in water.

3. The component (A) is an alkali metal carbonate or an alkaline earth metal carbonate, and the component (B) is an alkali metal hydrogencarbonate or an alkaline earth metal hydrogencarbonate. An autotrophic bacterial high concentration culture promoter.

4. The high-concentration autotrophic bacterium culture promoter according to claim 2 or 3, wherein a mixing ratio of the component (A): the component (B) is 4-7: 4-8 in a molar ratio.

5. The said (A) component is sodium carbonate, and the said (B) component is sodium hydrogen carbonate, The autotrophic bacterium high concentration culture promoter as described in any one of Claims 2-4 characterized by the above-mentioned.

(Specific Example) As a component of the above-mentioned accelerator, the pH of a medium which exhibits basicity by dissociation and leans toward the acidic side during acclimation is maintained in a predetermined range (7.0 to 9.0). The compound is not particularly limited as long as it has two properties, that is, a property that can be obtained and a property that can serve as a carbon source during the growth of the autotrophic bacterium.

When the above two properties can be imparted by one kind of compound, at least one kind of the compound becomes a component of the accelerator of the present invention.

As a specific example, trisodium bicarbonate (= sodium sesquicarbonate) (Na ₂ CO ₃ .Na
HCO ₃ .2H ₂ O) and compounds in which the alkali in this compound is partly or wholly substituted by potassium, magnesium, or the like.

When the above two properties can be imparted by two or more compounds, a mixture of at least two of the compounds is a component of the accelerator of the present invention. For example, a mixture of (A) an alkali which is soluble in water and shows basicity by dissociation and (B) a carbonate which is soluble in water can be mentioned. Specifically, as the component (A),
Hydroxides such as sodium hydroxide and potassium hydroxide,
Alkali metal salts such as sodium carbonate;
As the component (B), sodium hydrogen carbonate, magnesium hydroxide hydroxide (4MgCO ₃ .Mg (OH) ₂ .5H ₂
O) and the like.

Among them, the component (A) is an alkali metal carbonate or an alkaline earth metal salt which is soluble in water, such as sodium carbonate, sodium potassium carbonate and potassium carbonate, and the component (B) is It is preferable to use an alkali metal hydrogencarbonate or an alkaline earth metal hydrogencarbonate that is soluble in water, such as sodium hydrogencarbonate and potassium hydrogencarbonate, in that the two properties described above can be effectively imparted, Among them, a combination of sodium carbonate and sodium hydrogen carbonate is most preferable.

Cultivation (acclimation) of nitrite-oxidizing bacteria When the obtained nitrified sludge is further acclimated, supply of the NH ₄ —N-containing liquid (ammonia) is stopped, and NO ₂
^- give-containing liquid. As a result, among the ammonia-oxidizing bacteria and the nitrite-oxidizing bacteria which have been concentrated and cultured with each other, the ammonia-oxidizing bacteria stop growing and their numbers decrease. On the other hand, nitrite-oxidizing bacteria are given nitrite (NO
₂ ^-) continue to grow by.

Specific examples of nitrites that can be used include sodium nitrite, but are not limited thereto.

When the nitrite-oxidizing bacteria are acclimated, the pH of the medium tends to be alkaline.
To 8.5, preferably 7.4 to 8.0). In addition, since the bacterium is an autotrophic bacterium,
Since growth requires a carbon source, it is necessary to provide a carbon source during acclimation. It is the provision of carbon dioxide (CO ₂ ) that satisfies the two needs at one time.
It is not limited by this.

Other conditions, such as the dissolved oxygen concentration (D
O), temperature, etc. are the same as those described in the acclimation of nitrifying bacteria.

[0051]

The present invention will be described below with reference to one embodiment, but the present invention is not limited thereto.

High concentration culture of nitrifying bacteria (production of nitrifying activated sludge)
Construction) A fill and draw type culture tank (30 ) shown in FIG.
Liters) in a two-day batch culture. That is, urine sludge diluted with seawater, and anaerobic digestion and desorbent (NH ₄
-N is diluted with seawater so as to have a concentration of 100 mg / L) in a culture tank, and the temperature in the culture tank is adjusted to 27 ° C with a thermostat and a heater.
H controller and culture promoter (1N NaHCO
₃ and a buffer consisting of 0.5N Na ₂ CO ₃ ), and cultivation was carried out so as to be maintained at 7.5 to 8.5 (when the initial pH was 8.5 or more, diluted sulfuric acid was added. 8
5 or less). In addition, the dissolved oxygen (DO) concentration is 4
The amount of aeration was adjusted using a balloon to adjust the amount to mg / liter.

One day after the start of aeration, the digestion / elimination liquid was added again so that the final concentration became 100 mg / liter. Also,
On the second day, the aeration was stopped, the sludge was settled for 1 hour, the supernatant was removed, the digestion / desorption solution was added, and the operation of restarting the aeration was repeated.

Since the salt concentration of the original seawater diluted urine sludge corresponds to the seawater ratio of 80%, the habituation starts from the seawater ratio of 80%.
Raising the sea water ratio of 100% at _the stage _{of NH} 4 -N of 100mg / liter was to be nitrified completely _NO 3 -N after one day culture.

For several hours after the start of the aeration, the NH ₄ —N concentration decreases linearly.
The remaining NH ₄ —N concentration after 4 hours was measured, and the rate of change was determined from the slope of the section where the NH ₄ —N concentration changed linearly.
The value obtained by dividing this by the MLSS concentration was defined as the nitrification rate (see the following formula).

[0056]

(Equation 1)

SV30 of seawater acclimated nitrifying activated sludge (AMNS) which has been acclimatized after a seawater acclimation period of about 60 days,
The SVI was measured, sedimentation characteristics were examined, and the state of floc formation was observed using an optical microscope.

The graph of FIG. 2 shows the process of acclimating night soil sludge to seawater (FIG. 2 shows NH ₄ − at a concentration of 100 mg / liter).
N after 4 hours).

From the graph of FIG. 2, two months after the start of acclimation, 100 mg / liter of NH _4- at a seawater ratio of 100% was used.
It can be seen that AMNS capable of almost completely nitrifying N in 4 hours can be prepared. In order to prevent the loss of nitrification activated sludge due to the shortage of the inorganic carbon source, NaHCO ₃ and Na 2 C
Although pH adjustment using an inorganic carbon source combined with O ₃ was employed, the MLSS concentration of AMNS could be increased twice as much as before as shown in FIG.

The nitrification rate of nitrifying bacteria in AMNS is shown in the following table.

[0061]

[Table 1] It is reported that the abundance of nitrifying bacteria in the activated sludge is about 0.35%. From this calculation, it can be calculated that the nitrifying bacteria in seawater acclimated nitrifying activated sludge (AMNS) has a high concentration (about 2 to about 2%).
6%).

When the culture tank is allowed to stand, bacterial flocks can be confirmed, and most of the bacterial flocs precipitate because the specific gravity is higher than that of seawater. This is not seen in the pure culture of each of the ammonia-oxidizing bacteria and the nitrite-oxidizing bacteria, but appears in the mixed culture using activated sludge as a raw material.
As a result of observing the AMNS floc with a microscope, it was found that the sludge was composed of floc having a diameter of 50 to 100 µm. Further, as a result of observing the AMNS with a scanning electron microscope (SEM), it was found that the sludge flocs contained 20 to 100 μm granules made of filamentous fungi or sticky substances. Then, when SV30 and SVI of AMNS were determined, as shown in Table 1 above, 9%, 42.
It turned out to be 6, and it turned out that it is excellent in sedimentation. Thus, it was revealed that nitrification-activated activated sludge having high activity in seawater (of course, even in freshwater) can be mass-produced in a very short period of time from seawater-diluted human waste treatment plant sludge.

The advantages of the mixed culture using activated sludge as a raw material as compared with the individual pure cultures of ammonia oxidizing bacteria and nitrite oxidizing bacteria are as follows.

In a pure culture system, ammonia and nitrite are separately required as energy substrates, whereas in a mixed culture system, only ammonia needs to be supplied as an energy substrate.

In a pure culture system, it is extremely difficult to grow the cells at a high concentration, but in a mixed culture system, it is easy to grow to the extent that the medium is suspended. This is presumed to be due to the ecology of ammonia-oxidizing bacteria and nitrite-oxidizing bacteria not forming colonies or flocs within their cognate and living in a floating life.

The marine nitrifying bacteria thus obtained have been deposited with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology (deposit number: FERM BP-7150, identification: B
ICOM Nitrifying Bacteria
SWAQ SP-78).

High concentration culture of nitrite-oxidizing bacteria (nitrite oxidation
Acclimatization of sludge) Figure 4 shows a schematic shows illustration of the culture device of nitrite-oxidizing bacteria.
Using this culture apparatus, high-density culture of nitrite-oxidizing bacteria (acclimation of nitrite-oxidized sludge) is performed.

That is, in the production of the acclimated seawater nitrification sludge as described above, the supply of the NH ₄ —N-containing liquid (ammonia) was stopped, and nitrite nitrogen was supplied instead.

Specifically, the seawater nitrifying sludge obtained in the above example was placed in a 10 L nitrite-oxidizing bacteria culture tank, and then subjected to ML.
SS was set to be 2000 mg / L, and acclimation of seawater nitrite oxidized sludge was performed in the medium for nitrite oxidizing bacteria described in [Table 2] by a one-day cycle repetitive half-batch restriction operation method (CF-ROM method). Was done.

[0070]

[Table 2]

Up to one month from the start of the culture, 50% of nitrite nitrogen was added.
mg / L (about 50 ppm) was added, and thereafter, 100 mg / L (about 100 ppm) of nitrite nitrogen was added.

During the culture period, the pH was constantly monitored by a pH controller, and carbon dioxide was appropriately supplied by a CO ₂ cylinder and a CO ₂ regulator so that the pH did not deviate from 7.5 (the carbon source was also supplied at the same time). ).
When pH adjustment was impossible with only carbon dioxide, a pH regulator was used. The cultivation temperature was maintained at 25 to 27 ° C. with a heater and a thermostat.

As a result, as shown in FIG. 5, among the ammonia-oxidizing bacteria and nitrite-oxidizing bacteria that have been enriched and cultured with each other, the ammonia-oxidizing bacteria stop growing, the number decreases, and the MLSS concentration decreases. Once dropped. on the other hand,
The nitrite-oxidizing bacteria continued to grow due to NO ₂ ⁻ in the given sodium nitrite, and the MLSS concentration was restored by continuing to adapt the nitrite-oxidizing bacteria.

In this nitrifying sludge, nitrite oxidizing bacteria were accumulated and cultured, and the number of ammonia oxidizing bacteria was reduced numerically, as shown in the graph of FIG. ) And the graph of the increase over time in the oxidation rate of nitrous acid (FIG. 5, bottom).

The obtained nitrite oxidized sludge (MLSS concentration =
The activity of nitrite-oxidizing bacteria in 4500 mg / L) was measured (when added to about 50ppm when the _NO 2 -N was about 100ppm added). The nitrite oxidation rate per hour up to 4 hours is shown in FIG.

[Results] Nitrite oxidation rate when about 100 ppm of NO ₂ -N was added: 4.99 mg / g · h (0 to 4 hr), 7.21
mg / g · h ( ₂ to 4 hr) Nitrite oxidation rate when about 50 ppm of NO ₂ -N is added: 4.45 mg / g · h (0 to ₂ hr).

Incidentally, nitrite-oxidizing bacteria have a characteristic that they are weak against a high nitrite concentration, though they are bacteria that take over and oxidize nitrite produced by ammonia-oxidizing bacteria, and usually have an activity of 20 to 30 ppm. Nitrite oxidizing bacteria obtained by the culturing method of the present invention can be reduced to 50 ppm, 100 ppm, 15 ppm in a freshwater environment, for example, in a high salinity environment such as seawater.
A microorganism that does not die even in the presence of extremely high nitrous acid, which cannot be considered with the common sense of 0 ppm or 200 ppm, in other words, a microorganism having an excellent ability to continue to grow as feed and have an activity of oxidizing the nitrite. It is.

An attempt was made to deposit the high-density nitrite-oxidizing bacterium thus obtained at the Patent Microorganisms Depositary Center of the National Institute of Bioscience and Biotechnology, which was rejected. Oxidizing bacteria [Identification of microorganisms: BICOM Nitrifying Bacter
ia SWAQ NB] is stored by the following corporations and has a system for receiving applications for sale from third parties. However, it is necessary to conclude a contract with the microorganism custodian prior to the sale request. Requests for microbial distribution contract and application for microbial distribution are also below. "Bicom, Inc., 1-4-2 Shinsenri-Higashicho, Toyonaka-shi, Osaka, Senri Life Science Center (560-0082), TEL: 06-486
3-7500 (age) ".

[0079]

According to the present invention, nitrite-oxidizing bacteria, which have been considered impossible so far, can be grown and cultured at a high concentration (high density).

[Brief description of the drawings]

FIG. 1 is a schematic explanatory view showing an example of a sludge nitrification acclimation apparatus.

FIG. 2 is a graph showing a change in NH ₄ —N concentration in a nitrification acclimation process of sludge.

FIG. 3 is a graph showing the change over time of the MLSS concentration during the nitrification acclimation process of sludge.

FIG. 4 is a schematic explanatory view showing an example of an apparatus for culturing nitrite-oxidizing bacteria.

FIG. 5 is a graph showing the transition of the MLSS concentration over time in the enrichment culture of nitrite-oxidizing bacteria (top), and a graph showing the transition of the ammonia oxidation rate during the acclimatization period (middle). FIG. 10 is a graph (middle) showing a change in nitrite oxidation rate during the acclimatization period.

FIG. 6 is a graph showing the activity of nitrite-oxidizing bacteria in the obtained nitrite-oxidized sludge.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B029 AA02 BB02 CC01 DF01 DF02 DF04 4B065 AA01X BB03 BB38 BB40 BC02 BC14 CA55 CA56 4D040 BB01

Claims (4)

[Claims] 1. A method for culturing nitrite-oxidizing bacteria in nitrifying bacteria, which is slightly contained in activated sludge such as sewage sludge or human waste sludge, at a high concentration, wherein the activated sludge is provided with an NH ₄ —N-containing liquid. In this way, ammonia-oxidizing bacteria and nitrite-oxidizing bacteria in the nitrifying bacteria contained in the activated sludge are acclimatized and accumulated, and then the application of the NH ₄ —N-containing liquid is stopped and NO ₂
^- by applying a solution containing a high concentration culture method nitrite oxidizing bacteria, characterized in that allowed reducing the number of ammonia oxidizing bacteria, and further acclimatized integrated nitrite oxidizing bacteria. 2. A method for culturing nitrite oxidizing bacteria in nitrifying bacteria contained in activated sludge such as sewage sludge or night soil sludge at a high concentration, wherein the activated sludge is dissolved in dissolved oxygen at a concentration of 2 mg / liter or more at a pH of at least 2.
Under the conditions of 7.0 to 9.0 and a temperature of 20 to 40 ° C., the NH ₄ —N-containing solution is applied for a predetermined period of time to acclimate the nitrification, and at the same time acclimate to the acidic side in the acclimation process.
Is maintained within the above-mentioned range by adding a culture promoting agent comprising a mixture of sodium carbonate and sodium hydrogencarbonate, thereby accumulating ammonia-oxidizing bacteria and nitrite-oxidizing bacteria in the nitrifying bacteria contained in the activated sludge. , NO ₂ stops the application of later the NH 4 _-N containing liquid ^- by applying a solution containing, allowed reducing the number of ammonia oxidizing bacteria, and characterized in that it further acclimatization integrated nitrite oxidizing bacteria High concentration culture method for nitrite oxidizing bacteria. 3. The method according to claim 1, wherein the pH inclined toward the alkali side in the process of accumulating nitrite-oxidizing bacteria is maintained at a predetermined value by adding CO _2. For high concentration cultivation of nitrite-oxidizing bacteria 4. A high-concentration nitrite-oxidizing bacterium having the ability to oxidize nitrite even in a freshwater environment, for example, in a high-salinity environment such as seawater even at a nitrite concentration of 50 ppm.