WO2002046370A1 - Promoter for the culture of autrotrophic bacterium at high density - Google Patents

Abstract

A promoter for the culture of an autotrophic bacterium at a high density having an effect of regulating the pH value of a medium, which varies during the acclimatization of the autotrophic bacterium in a definite period for the culture thereof at a high density, within a specific range and, at the same time, serving as a carbon source. This promoter comprises a mixture of sodium carbonate and sodium hydrogencarbonate and has a mixing ratio by mol of sodium carbonate : sodium hydrogen carbonate of 1: 4/7 to 17/7.

Description

Description: Promoter for culturing autotrophic bacteria at a high concentration [Technical Field] The present invention relates to an autotrophic bacterium high-concentration cultivator for culturing autotrophic bacteria at a high concentration (hereinafter simply referred to as “promoting”). It relates also referred) and agents ", more particularly, to accelerators for culturing autotrophic bacteria causing increasingly like medium _P H is changed in response to high concentrations in culture toothpick.

[Background Art] Autotrophic bacteria such as ammonia-oxidizing bacteria (nitrifying bacteria) and sulfur-oxidizing bacteria have been found to exist over 100 years ago because their growth rate is slow and they do not live as colonies. Until then, there have been no reports of successful high-density cultivation industrially.

 In other words, the conventional culture method is a small scale at the test tube level aiming at pure culture, and the medium in the flask does not suspend in about two months, and the culture method that can be applied industrially is I could not say.

 For example, pH decreases when nitrification starts in the culture of nitrifying bacteria, for example. Conventionally, there was no known way to increase this pH effectively (without affecting culture).

On the other hand, although the carbon source decreases with nitrification, carbon dioxide was conventionally used as a carbon source supply. Although the supply of carbon dioxide can surely prevent the depletion of the carbon source, the above-mentioned decline in PH This led to the cessation of nitrifying bacteria, which was the limit of bacterial growth.

 The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an accelerator for culturing autotrophic bacteria at a high concentration.

[Disclosure of the Invention]

The high-concentration autotrophic bacterium cultivation promoter according to claim 1 maintains the pH of a medium that is inclined to an acidic side in the acclimatization process in a predetermined range when the autotrophic bacterium is acclimated for a predetermined period to be cultured at a high concentration. An autotrophic bacterium high-concentration cultivation enhancer that serves as a carbon source together with the bacterium, which is capable of maintaining the pH of the culture medium that exhibits basicity by dissociation and tilts toward the acidic side within a predetermined range, and If two types of properties can be provided by one type of compound, a property that can be a carbon source during the growth of vegetative bacteria, and at least one type of the compound, or two or more types of the above properties can be provided by two or more types of compounds. In the case where the compound can be applied, a mixture of the compound is blended.

 The high-concentration cultivation agent for autotrophic bacteria according to claim 2 is the accelerator according to claim 1, wherein the mixture is a mixture of the following components (A) and (B). And

 (A) Alkali metal carbonate or alkaline earth metal carbonate.

 (B) Alkali metal bicarbonate or alkaline earth metal bicarbonate. The accelerator for autotrophic bacterium high concentration culture according to claim 3 is the accelerator according to claim 2, wherein the mixing ratio of the component (A): the component (B) is 1: 4 to 17 in a molar ratio. No.7.

The autotrophic bacterium high concentration culture promoter according to claim 4 is the promoter according to claim 2 or 3, wherein the component (A) is sodium carbonate. The component (B) is hydrogencarbonate. It is characterized by being sodium. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic explanatory view showing an example of a sludge nitrification acclimation apparatus.

 Figure 2 is a curve showing the concentration of NH 4 —N remaining 4 hours after injection of NH 4 — N (lO Omg / liter), showing the change in NH 4 —N concentration during the nitrification acclimation process of sludge. FIG. The mark in the figure indicates the introduction of NH4-N (100 mg Z liter).

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

 FIG. 4 is a graph showing changes in MLSS concentration after acclimation for 8 months by changing the composition ratio of the culture promoter.

 FIG. 5 is a graph showing changes in MLSS concentration after habituation by changing the type of culture promoter.

 FIG. 6 is a graph showing the daily change of the MLSS concentration in the process of acclimating sulfur-oxidizing bacteria in sludge.

[Best Mode for Carrying Out the Invention] Autotrophic Bacteria

 The autotrophic bacterium according to the present invention refers to any bacterium that causes a reaction such that the pH of the medium decreases during cultivation. Specifically, ammonia-oxidizing bacteria (nitrifying bacteria), sulfur-oxidizing bacteria, Examples thereof include carbon monoxide oxidizing bacteria, and a mixed bacterium of two or more of these.

These bacteria are contained, for example, in activated sludge and seawater. However, it is more preferable to use activated sludge because it contains more bacteria. . Accelerator component

 The components of the accelerator of the present invention have the property of exhibiting basicity by dissociation and having the property of maintaining the pH of the culture medium inclined toward the acidic side during acclimation within a predetermined range (7.0 to 9.0). The compound is not particularly limited as long as it has two properties, namely, 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 is a component of the accelerator of the present invention.

 Specific examples include trisodium hydrogen bicarbonate (== sodium sesquicarbonate) (Na 2 CO 3 -NaHCO 3 · 2H 2 O) and sodium in this compound is potassium magnesium. And the like, a compound partially or wholly substituted for.

 Further, when the above two properties can be imparted by two or more compounds, a mixture of the compounds is a component of the accelerator of the present invention. For example, a mixture of (A) a carbonate which is soluble in water and exhibits basicity by dissociation and (B) a bicarbonate which is soluble in water can be mentioned. Examples include hydroxides such as sodium hydroxide and potassium hydroxide, and alkali metal salts such as sodium carbonate. As the component (B), sodium hydrogen carbonate, magnesium carbonate (4Mg C03 ' Carbonates such as Mg (OH) 2-5H2O).

Among them, the component (A) is an alkali metal carbonate or an alkaline earth metal carbonate which is soluble in water, such as sodium carbonate, sodium carbonate, potassium carbonate, etc .; The two properties described above are effective when the component is a water-soluble alkaline metal bicarbonate or an alkaline earth metal bicarbonate that is soluble in water, such as sodium bicarbonate or bicarbonate. And sodium carbonate and carbonic acid. Most preferred is a combination of sodium hydrogen.

 Activated sludge

 The activated sludge used in the present invention includes sewage sludge and night soil sludge. These may be freshwater-diluted or seawater-diluted, but if the autotrophic bacteria are cultured using seawater-diluted sludge as a raw material, they are rare. Since a large amount of valuable marine bacteria (salt-resistant bacteria) can be obtained, it is preferable to use activated sludge that has been subjected to seawater dilution.

 To explain, although natural seawater contains marine bacteria that are considered to have higher salt tolerance than freshwater bacteria, their abundance is very small and pure separation is difficult. Its research has lagged behind freshwater bacteria. However, according to the culture method of the present invention, a large amount of marine bacteria having a high concentration can be obtained by using activated sludge that has been subjected to seawater dilution treatment as described above as a raw material. Marine bacteria have multi-layered cell walls and have strong resistance to various osmotic pressures of treated water and various chemicals that inhibit growth.

 Culture (acclimation) conditions for nitrifying bacteria

 The culture of nitrifying bacteria contained in the activated sludge is carried out by

(For example, 1 month, 2 months or 3 months), sludge dewatered filtrate ゃ (anaerobic) Nitrification by sludge treatment wastewater such as digestion and desorption solution is required. Therefore, the dissolved oxygen (DO) at this time must be 2 mg Z liter or more. However, for the first time in this experiment, it was found that if the dissolved oxygen concentration was too high, the growth rate would tend to decrease. The details will be described below.

The rate of nitrification by nitrifying bacteria is higher as the dissolved oxygen is higher. However, the acclimation of nitrifying bacteria from activated sludge as a raw material is likely to be slowed down by more than 5 mg / liter dissolved oxygen (DO). I understood. In addition, dissolved oxygen (DO) concentration S mgZ liter is most preferable.

 Further, the pH must be 7.0 to 9.0, particularly when using activated sludge diluted with seawater, preferably 7.5 to 8.5, more preferably 7.5 to 7.8. preferable.

 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 cultivation, the pH decreases and the alkalinity decreases. That is, the oxidation of NH4 + to NO 2− by ammonia oxidizing bacteria and the oxidation of NO 2 − to NO 3 − by nitrite oxidizing bacteria are as follows:

 (A) and (B). Equation (C) is an equation for the entire nitrifying bacteria.

(Ammonia oxidizing bacteria)

 NH4 + + 1.5 θ2 → Ν02— + Η2θ + 2Η + …… (A) (Nitrite oxidizing bacteria)

 N02- + 0.5 02 → Νθ3-"…-(B)

(Mixed system)

NH4 + + 2.0 θ2 → Ν03— + Η2θ + 2Η + …… (C) From these, to oxidize ΝΗ4—4 to NO 3— 4., 4.57 mg O 2 / mg NH4—N oxygen Since hydrogen ions are released as the nitrification reaction progresses, it can be seen that the pH of the culture system decreases and the strength decreases. As the culture speed decreases with decreasing pH If the pH is not maintained at a predetermined value using a buffer or the like, the activity of microorganisms stops as in the conventional method.

 Therefore, in the present invention, it is considered that a buffer which has a pH which is inclined toward the acidic side in the culture process and which has a buffering action as a mixture of a normal salt and an acid salt is preferable. As a result of repeated trial-and-error tests, it was found that it is most preferable to restore pH by introducing a culture promoter composed of a combination of sodium carbonate and sodium bicarbonate.

 In general, it is known that the synthesis reaction of bacterial cells can be expressed by the following formula <

4C02 + HC03 + NH4 + + Η2θ → C5H7NO2 + 50

If this is applied to the biochemical reaction formula of the mixed culture system (C) described above, it is roughly as follows.

NH4 + 4- 1.86 02 + 1.98 HC〇3-→

 0.021 C5H end N02 + 0.98 Νθ3-+ 1.04 Η2θ + 1.88 H2C03

As is clear from the above equation, culturing nitrifying bacteria requires a large amount of carbon source even when compared to the energy substrate, ammonium ion.

 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 supplied at the same time. The explanation is added below.

If only sodium carbonate is used, the sodium carbonate is In some cases, the effect of increasing the pH can be sufficiently recognized.However, the effect of increasing the pH is so large that it cannot be used in a large amount and is not suitable for supplying a sufficient carbon source. is there. On the other hand, when using only sodium hydrogen carbonate, 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 became possible to effectively supply an inorganic carbon source for assimilation of carbonic acid in a living body while maintaining a gradually decreasing pH.

 The mixture ratio of sodium carbonate and sodium hydrogencarbonate in the mixture is preferably 1: 4Z7 to 17/7 in a molar ratio of sodium carbonate: sodium hydrogencarbonate. For 1 mo 1 Z liter of sodium, sodium bicarbonate is 4Z 7 (0.571-) mo 1 Z liter ~ 17/7 (2.4 8 8) mo 1 liter An aqueous solution of the mixture is effective.

 The monitoring of the pH of the culture system may be performed continuously or at predetermined time intervals. It is preferable to use a continuous pH monitoring device such as a pH controller, but the present invention is not limited to this. It is also possible to perform the measurement manually using a pH indicator such as phenol red.

The concentration of ammonia in the NH4-N-containing liquid is preferably 100 mg / liter or more and 300 mg / liter or less, and more preferably 200 mg / liter or less. Ammonia is an energy source for the growth of ammonium oxidizing bacteria, which are chemolithotrophic bacteria, by assimilating carbon dioxide. However, excessive amounts of ammonia may inhibit growth and growth. Nitrite oxidizing bacteria contained in activated sludge, like ammonia oxidizing bacteria, are bacteria that take over and oxidize nitrite generated by ammonia oxidizing bacteria. In this case, the initial concentration of ammonia cannot be set unnecessarily high because it is weak to high nitrite concentration. Therefore, the concentration of ammonia

If it exceeds 300 mg / liter, it is preferable to appropriately dilute with seawater or fresh water.

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

 Under the above culture conditions, activated sludge is nitrified by sludge treatment wastewater such as sludge dewatered filtrate and digestion desorbent, so that nitrifying bacteria slightly contained in the activated sludge can be cultured at a high concentration. However, in addition to this, according to the present invention, the activated sludge can be reduced to 1/3 to 1/4 in two months, and nitrified sludge having a large specific gravity can be obtained.

In other words, it is said that activated sludge originally contains about 0.35% of nitrifying bacteria. By accumulating activated sludge using such activated sludge as a raw material in an NH4-N-containing liquid for about two months, the content of nitrifying bacteria in the activated sludge can be increased by about 10 times (3.5%). ). In the process, other germs cannihilate and die because no external nutrients (feeds) are provided. As a result, the amount of activated pollutants is reduced. When the germs almost die, they become hard-to-decompose organic matter called "granules", which are attached to nitric bacteria around the nucleus. Persistent organic matter attached to 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. In other words, 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-concentration culture cannot be expected. As a result, the production of nuclei (refractory organic substances) as described above is required for high-concentration culture, but nucleation is not seen in pure culture of nitrifying bacteria, and is not active. Only seen when sludge is used as raw material.

 The promoter of the present invention enables cultivation of autotrophic bacteria, which has been impossible so far, in a large amount and at a high concentration.

 Hereinafter, an example of the present invention will be described, but the present invention is not limited thereto.

 Example 1 (High concentration culture of nitrifying bacteria [Production of nitrifying activated sludge])

 Batch culture was performed in a two-day cycle in a fililand draw type culture tank (30 liters) shown in FIG. That is, urine sludge diluted with seawater and anaerobic digestion / elimination liquid (diluted in seawater so that the concentration of NH4-N is 100 mgZ liter) are put into a culture tank, and the temperature in the culture tank is thermostated. So that the temperature becomes 27 ° C with a heater and a heater, and adjust the pH with a pH controller and a culture promoter (0.5 mol / liter Na 2 CO 3 and 1 mol 1 Z liter). The culture was performed by setting the pH to 7.5 to 8.5 with a buffer consisting of NaHCO 3 (when the initial pH was 8.5 or more, dilute sulfuric acid was added. 8.5 or less). The amount of aeration was adjusted with a balloon to adjust the dissolved oxygen (DO) concentration to 4 mg / litre.

 One day after the start of the aeration, the replenisher / desorbent was added to a final concentration of 10 Omg / liter. 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 aeration was restarted.

 Since the salt concentration of the original seawater diluted urine sludge reaches 80% of seawater ratio, habituation starts from 80% of seawater ratio, and 10 mg of NH4-N is completely nitrified to NO3-N after 1 day of culture. At that point, the seawater ratio was raised to 100%.

Since NH 4 — N concentration decreases linearly for several hours after the start of aeration, the remaining NH 4 — N concentration at 0, 1, 2, 3, and 4 hours after the start of aeration is measured. The rate of change was determined from the slope of the section where the NH4-N concentration changes linearly, and this was divided by the MLSS concentration to obtain the nitrification rate (see the following equation).

R dNH4-N 1

 NH4- N

 d t S

RNH4-N NH4-N reduction rate (mg—Nh—N / g—MLSS · hr)

 S sludge MLS S concentration (g liter) Measure the SV30 and SVI of seawater acclimated nitrifying activated sludge (AMN S), which has been acclimated after a seawater acclimation period of about 60 days, and examine sedimentation characteristics At the same time, the state of floc formation was observed using an optical microscope.

 The graph in Fig. 2 shows the process of acclimating night sludge to seawater (the figure shows the concentration of 100 mg / liter of NH4-N after 4 hours).

From the graph in Fig. 2, two months after the start of acclimation, it is possible to prepare AMN S that can almost completely nitrify 100 mg / liter of NH4-N at a seawater ratio of 100% in 4 hours. I understand. In order to prevent nitrification-activated sludge loss due to a shortage of inorganic carbon sources, ί> Η adjustment using an inorganic carbon source combining NaHC03 and Na2C03 was adopted. As shown in the figure, the MLSS concentration of AMN S could be doubled compared to that before acclimation. The nitrification rate of nitrifying bacteria in AMN S is shown in the following table _c

 [table 1 ]

* Unit of nitrification rate: mg—NH4—NZg—MLSS · hr It is reported that the abundance of nitrifying bacteria in activated sludge is about 0.35%. From this calculation, it was found that the nitrifying bacteria in seawater acclimated nitrifying activated sludge (AMN S) had a high concentration (about 3. 5%). When the culture tank is allowed to stand, bacterial flocs can be confirmed, and most of the bacterial flocs precipitate because the specific gravity is heavier than 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. Microscopic observation of the AMNS floc revealed that the sludge consisted of flocs of 50 to 100 µm in diameter. In addition, observation of AMNS with a scanning electron microscope (SEM) revealed that the sludge flocs contained granules consisting of 20 to 100 m of filamentous fungi and sticky substances. Then, when SV30 and SV-I of AMNS were determined, they were 9% and 42.6 as shown in Table 1 above, indicating that the sedimentation was excellent. From this, it was clarified that nitrification-activated sludge having high activity in seawater (and 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 mixed cultivation using activated sludge as compared with the pure cultivation 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, but 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 habitat of the ammonia-oxidizing bacteria and nitrite-oxidizing bacteria not forming colonies and flocks within their cognate and living in a floating life. The marine nitrifying bacteria obtained in this manner have been deposited with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology (Deposit No .: F ERM BP—710, Identification: BI COM Nitrifying Bacteria S WAQ SP—7 8).

 Example 2 (acclimation experiment in which the composition ratio of the accelerator was changed)

 The acclimatization experiment of Example 1 described above was further continued for 6 months (8 months in total), and the MLSS concentration of AMN S after acclimation was measured. Figure 4 shows the results.

 The same habituation experiment was performed for 8 months by changing the mixing ratio of the culture promoter (Na 2 CO 3 (0.5 mol 1 liter), Na HCO 3 (Imol Z liter)), and ML The increase in SS concentration was measured. The results are shown in Figure 4.

 Example 3 (Accustomed experiment in which the composition of the accelerator was changed)

 Type of culture promoter (composition) High-density cultivation of nitrifying bacteria (production of nitrifying activated sludge) was performed in the same manner as in Example 2, except that [NaHC03, Na2CO3] was changed. . Figure 5 shows the MLSS concentration of AMN S after habituation.

 Example 4 (High concentration culture of sulfur oxidizing bacteria)

 Basically, it is sufficient to follow the same culture method as that for the high-density cultivation of nitrifying bacteria described above.

Put the sludge returned from the sewage sludge treatment plant in a 5-liter beaker so that the MLSS concentration becomes 130 mg gZL, and use the sulfur-oxidizing bacterial medium shown in [Table 2] below to limit the amount of the repetitive half-day cycle. The oxidized sludge was acclimated by the operation method (CF-ROM method). The incubation temperature is 25-27 with heater and thermostat. C, and the pH is 7.0 to 7.8 with a mixture of NaHC03 and Na2C〇3 (Mono ratio: NaHC03: Na2C03 = 1.0: 0.5). Adjusted within the range. [Table 2]

Na 2 S203-5H20 1 g K2HP04 4 g ivrl 2 Jr U 4 4 g ΝΗ4 C 10.5 g g: ¾ ¾ ¾ ¾ 属 属 属 属 η ^ U η U m JL

 IT ノ 十 ソ 1 1 IT ”k / k Trace metal mixture *

 7 n SO 70 Ω mJL Π

 NaMo 2-2Η2θ 10 Omg KC 1 10 Omg.

Mn S04 100 Omg KA1 (S04) 2 2H20 10 Om g

CuS 04 · 7H20 5 Omg EDTA 975 mg deionized water 1 liter Figure 6 shows the MLSS concentration that increases over time.

[Reference to deposited biological material]

① Name of the depositary institution:

 National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary

 (Name of former depositary institution:

 Ministry of International Trade and Industry, Institute of Industrial Technology, Institute of Biotechnology and Industrial Technology)

 Address: Zip code 30 5— 8 5 6 6

 1-chome, 1-chome, Tsukuba, Ibaraki, Japan 1 Chuo No. 6

 (Old address: Tsukuba East 1-chome, Ibaraki, Japan 3) TE L: 02 9 8-6 1-60 2 9

 ② Deposit date: April 27, 2000

 ③ Accession number: F ERM B P— 7 1 5 0

Claims

The scope of the claims 1. When acclimating autotrophic bacteria for a specified period of time to culture them at a high concentration, maintain the pH of the medium that tilts to the acidic side in the acclimation process within a predetermined range, and use an autotrophic bacteria high-concentration culture promoter that serves as a carbon source. So,  The following two properties are considered: a property that can maintain the pH of the medium that exhibits basicity by dissociation and leans toward the acidic side within a predetermined range; and a property that can serve as a carbon source during growth of the autotrophic bacterium. When it can be provided by one kind of compound, at least one kind of the compound, or when two or more kinds of the above properties can be provided by two or more kinds of compounds, a mixture of such compounds is blended. Autotrophic bacteria high concentration culture promoter. 2. The autotrophic bacterium high-concentration culture promoter according to claim 1, wherein the mixture is a mixture of the following components (A) and (B). (A): Alkali carbonate metal salt or Alkali carbonate earth metal salt. (B): alkali metal bicarbonate or alkaline earth metal bicarbonate _c 3. The high concentration of autotrophic bacterium according to claim 2, wherein the mixing ratio of the component (A) and the component (B) is 1: 4/7 to 17/7 in molar ratio. . '  4. The autotrophic bacterium high concentration cultivation promoter according to claim 2, wherein the component (A) is sodium carbonate, and the component (B) is sodium hydrogen carbonate.