JPH07108294A - Method for culturing denitrification bacteria, immobilized denitrification bacteria and denitrification method using the bacteria - Google Patents

Abstract

PURPOSE:To enable culture of high density in large quantity by a method wherein marine denitrification bacteria of specific genus Alkaligenes, wherein pH, temperature, and culture medium salt is in a specified optimum growth condition, are maintained in a predetermined condition and cultured until light absorbance becomes a predetermined value in a predetermined culture medium. CONSTITUTION:Marine denitrification bacteria of the genus Alkaligenes which has an optimum growth condition, wherein pH is 6-8, temperature 35 deg.C, and culture medium salt concentration is 0.85-3.4%, and has apparent metabolic pathway, wherein nitric acid is reduced directly to gaseous nitrogen, are selected. The bacteria are maintained in a condition similar to the optimum growth condition, such that salt concentration is 0.85-3.4%, pH 6-8, and temperature is 32-38 deg.C. And culture is continued until light absorbance of culture liquid becomes 2 or more at 610nm.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for culturing denitrifying bacteria, an immobilized denitrifying bacterium and a denitrifying method using the same. More specifically, it is applied as a purification microorganism to a water purification system in artificial fish breeding and the like. It is about denitrifying bacteria that can be made.

[0002]

2. Description of the Related Art Currently, the development of a circulating filtration fish-cultivating system that makes effective use of electricity and highly adjusts the breeding environment is currently underway for flounder and the like. In this system, which circulates and reuses the breeding water to control the temperature of the breeding water, fish excrement accumulates in the breeding water and causes water quality deterioration. Therefore, its purification is very important. Nitric acid, which is produced by the biological oxidation of ammonia, which is the main excrement of fish, inhibits the growth of fish at high concentrations and adversely affects the acidification of breeding water. In order to utilize it, it is necessary to reduce the nitric acid and perform denitrification to remove it as gaseous nitrogen.

For denitrification, it is known that a method using a purifying microorganism called a denitrifying bacterium is more practical than a physicochemical method. Denitrifying bacteria are bacteria that live widely in the natural world such as soil and ocean, and reduce nitrates and nitrites into gaseous nitrogen, which are released according to their apparent metabolic pathways. NO ₂ ^- → N ₂ B-type ^{_{NO 3 - → NO 2 - →}} N 2 C -type NO ₃ ^- → are known to be classified into N _2. The present inventor believes that C-type does not release toxic nitrous acid and is most useful for denitrification in fish rearing, and the marine denitrification having such a metabolic pathway and showing high denitrification activity. Bacteria were separated, and the removal of nitric acid from the breeding water by a biofilm method in which the bacteria were attached to the surface of the filter medium for purification was examined, and an effective filter medium was selected to confirm the denitrification effect to some extent. .

[0004]

However, in the denitrification by the biofilm method, in order to obtain an efficient denitrification effect, it is necessary to inoculate the cultivated bacteria in advance at a high density on the filter medium in the denitrification tank. It became clear that there is. For this reason,
This is not a problem in the case of application of the above-mentioned denitrifying bacteria to denitrification at the laboratory level, but when attempting to apply this biofilm method at the industrial level, the above-mentioned artificial inoculation of denitrification is performed. It is necessary to secure and stably supply bacteria, but a method for culturing the denitrifying bacteria in a large amount and at high density has not been found so far. Although the biofilm method has a high denitrification effect, its ability is limited, and there is a problem that the denitrification effect may become unstable in the case of long-term use. The present invention has been made to solve the above problems, a method for culturing marine denitrifying bacteria in a large amount and at high density, and immobilization of the denitrifying bacteria and the immobilized bacteria in a fluidized bed system. It is an object to provide a denitrification method to be used.

[0005]

The present inventors SUMMARY OF THE INVENTION The metabolic pathway NO apparent ₃ ^- → N perform various studies of the optimal growth conditions for utility marine denitrifying bacteria is _two, such a marine We found a high-density and large-scale culture method that had never been reported for sexually denitrifying bacteria, and by immobilizing the denitrifying bacteria and performing denitrification, the denitrification was performed more efficiently than the biofilm method. Find out that can be done,
Further intensive studies were conducted to complete the present invention.

That is, the present invention has a pH of 6 to 8 and a temperature of 3
Marine denitrifying bacterium of the genus Alcaligenes, which has an optimal growth condition of 5 ℃, culture medium salinity of 0.85-3.4%, and has an apparent metabolic pathway of reducing nitrate directly to gaseous nitrogen. Is cultivated in a culture medium having a salt concentration of 0.85 to 3.4% while maintaining the pH of the medium at 6 to 8 and the temperature at 32 to 38 ° C. until the absorbance of the culture solution at 610 nm becomes 2 or more. The present invention relates to a method for culturing denitrifying bacteria.

[0007] Alcaligenes marine denitrifying bacteria of the present invention, the following equation to reduce nitrous acid generated in the reduction of nitrate to rapidly gaseous nitrogen without release to the extracellular: NO ₃ ^- → N
_{It has two} apparent metabolic pathways. Such denitrifying bacteria are separated and purified by the nitric acid enrichment culture separation method using sea sand and bottom mud of various beaches and the filter sand of the filter tank of fish breeding equipment as samples, and their denitrification activity is confirmed. It has been classified and classified at the genus level. Since denitrifying bacteria are facultatively anaerobic and can grow aerobically, the above separation culture was performed under aerobic conditions, and denitrifying activity was confirmed by gas generation and increase in medium pH. In addition, the denitrification type is determined by the three states of inorganic nitrogen (N
O ₃ ⁻ , NO ₂ ⁻ , NH ₄ ⁺ ) was quantitatively changed, and classification at the genus level was performed by Gram staining, flagella observation, sugar fermentation test and the like.

In the present invention, a preferred marine denitrifying bacterium in terms of high denitrification ability, easy control of denitrification activity, and stable trait, is isolated from sand mud of Kamo Tidal Flat in Miyagi Prefecture.・ Separated from one strain named as purified Alcaligenes GA-2-1 strain and the filter sand of the fish breeding water filtration tank of the Central Research Institute of Electric Power Industry / Abiko Research Institute located in Abiko City, Chiba Prefecture. -Purified Alcaligenes Ab-A-
One strain and two strains named Alcaligenes genus Ab-A-2 strain. Among these three strains, from the viewpoint of high denitrification activity and wide range of culture conditions, the gene of Alcaligenes genus Ab-A-1
Strains are particularly preferred. In addition, these denitrifying bacteria were deposited at the Institute of Biotechnology, Institute of Industrial Technology, September 13, 1993, and have the following deposit numbers. Alcaligenes strain accession number G-A-2-1 FERM P-13861 Ab-A-1 FERM P-13862 Ab-A-2 FERM P-13860 Watanabe et al .: Central Research Institute of Electric Power Industry / Research Further details can be found in report U89035 (1989).

The optimum growth conditions of the marine denitrifying bacterium belonging to the genus Alcaligenes of the present invention are pH 6-8, temperature 35 ° C., by comparing specific growth rates under various conditions of medium pH, temperature and salinity. Salt concentration 0.85-3.
It was found to be 4%. Further, by culturing the denitrifying bacteria while maintaining the above optimal growth conditions, it is possible to cultivate the bacteria at a high density until the absorbance (wavelength = 610 nm, turbidity) of the culture solution is about 2 to 6, and The culturing method was applicable to mass culture. The temperature is 3
It was also confirmed that a high growth rate was obtained in the range of 2 to 38 ° C. Therefore, the above-mentioned marine denitrifying bacteria should have a salinity of 0.8
To cultivate the denitrifying bacterium of the present invention in a high density in a large amount by culturing in a culture medium of 5 to 3.4% while maintaining the pH of the medium at 6 to 8 and the temperature at 32 to 38 ° C. Became possible. Application of the denitrifying bacterium of the present invention at an industrial level to a biofilm method, that is, a method of adhering bacteria at a high density on the surface of a filter medium to purify water by high-density and large-scale culture of the denitrifying bacterium of the present invention can do. Here, the filter medium is, for example, a sand filter medium (having a diameter of about 3 to 6 mm, etc.), a fibrous filter medium (such as ultra-fine chemical fibers woven into a ring into a string), or a plate-like filter medium (honeycomb-shaped plastic tube, etc.). Etc. are used, but sand filter media and fibrous filter media are preferred in terms of high denitrification activity after attachment of bacterial cells, and fibrous filter media are particularly preferred in terms of ease of handling.

The present invention also has a pH of 6 to 8 and a temperature of 35.
Immobilizing marine denitrifying bacteria of the genus Alcaligenes, which has the optimum growth conditions of 0.8 to 3.4% of the culture medium salt concentration and has an apparent metabolic pathway of reducing nitrate directly to gaseous nitrogen. Of immobilized denitrifying bacteria. In the present invention, the immobilization of the bacterial cells is carried out by encapsulating the bacterial cells in fine particles of a natural or synthetic polymer gel according to a conventional method, or by coating the bacterial cells with a semipermeable membrane, so-called It is preferably carried out by entrapping immobilization. Examples of the polymer gel include polyvinyl alcohol (PVA) gel, polyethylene glycol (PEG) gel, polyacrylamide gel, 2-hydroxymethylacrylamide gel, silica gel, alginic acid, urethane resin, and the like, which have a semipermeable property. Examples of the membrane include cellulose and its derivatives. Further, the immobilization of the bacterial cells in the present invention may be carried out by a carrier binding method or a cross-linking method which is carried out at the time of immobilizing the enzyme. In the case of application to water purification of marine fish, immobilization with PVA gel or PEG gel is preferable from the viewpoint of resistance to salt concentration of seawater.

Further, the present invention provides a denitrification method comprising contacting the immobilized denitrifying bacteria with inflow water in an anaerobic fluidized bed. In this denitrification method, it is preferable to carry out the treatment by setting the sugar concentration of the inflowing water to a carbon equivalent concentration of 120 to 360 mg-C / l and a residence time of 12 to 36 hours. In a particularly preferred embodiment of the denitrification method of the present invention, the sugar concentration of the inflow water is 240 mg-C / l in terms of carbon concentration.
And the residence time is 24 hours. In addition, in this specification, "residence time" means hydraulic retention time, and is the time when the inflow water contained in a certain system is replaced once. Further, the denitrification method of the present invention is effectively applied to a circulation filtration fish culture system in which the inflow water is breeding water in the circulation filtration fish culture system.

[0012]

EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited thereto. I. Selection of useful marine denitrifying bacteria A. Measurement of the number of general bacteria and denitrifying bacteria in marine sediments Atsumi Bay, Aichi Prefecture, Yokosuka Owada Bay, Kanagawa Prefecture, sea sand and sediment of Sendai Minami Gamo and Sendai Gamo, Miyagi Prefecture and Abiko City, Chiba Prefecture Five samples of the filtered sand from the fish breeding water filtration tanks of the Central Research Institute of Electric Power Industry and the Abiko Institute were used. About the number of general marine bacteria, 1/10 concentration ZoBe
ll 2216E agar medium and 1/10 concentration ORI agar medium were used in the dilution plate method. Regarding the number of denitrifying bacteria, a nitrate-added meat extract agar medium (hereinafter, abbreviated as meat extract medium) and 3% NaCl were added to seawater. Gi with added
It was calculated by the most probable value method (Most Probable Number method, hereinafter abbreviated as MPN method) using an ltay medium (hereinafter abbreviated as Giltay medium). In both cases, the culture was carried out at 25 ° C. for 14 days. The type and composition of the medium used are shown in Table 1.

[Table 1]

In the measurement of the number of denitrifying bacteria, the number of denitrifying bacteria in the Giltay medium was determined by gas generation (gas accumulation in the Durham tube put in the medium) and increase in the pH of the medium (blue coloration of the medium). It was determined to be positive, and denitrification was determined to be positive due to gas generation in the meat extract medium. The most probable number was converted to the number of bacteria. The number of general marine bacteria in the test sample is shown in Table 2 and the number of denitrifying bacteria is shown in Table 3.

[Table 2]

[Table 3] Thus, the density of general marine bacteria in the sample was at a level of 10 ⁴ to 10 ⁷ / g raw sample, which was almost the same as the density on a normal beach (Table 2). On the other hand, the density of the denitrifying bacteria was at a level of 10 ¹ to 10 ² / g raw sample, which was a very low ratio as compared with the number of general bacteria (Table 3), so it was determined that enrichment culture was necessary for separation.

B. Separation of marine denitrifying bacteria and its denitrification type The separation of marine denitrifying bacteria was performed using the following two methods. First, a fixed amount of the liquid medium after culturing used in the MPN method was applied to Giltay agar medium and meat extract agar medium to separate denitrifying bacteria [Watahiki et al., Agric. Biol. B.
iochem. 47, 1991-1996 (1983)]. Since the denitrifying bacteria are facultatively anaerobic and can grow aerobically, the above separation culture was carried out under aerobic conditions. After the colonies that have grown are arbitrarily separated and purified, each of the isolated strains is Giltay.
Anaerobically cultivated in medium, denitrification activity is gas generation and medium pH
It was confirmed by the rise of. This series of separation operations was named separation method I. This separation method is a nitric acid accumulation culture separation method in which nitric acid is added as an oxidant (final electron acceptor) instead of oxygen to accumulate denitrifying bacteria. On the other hand, a series of separation operations shown below for separating denitrifying bacteria from the enriched culture solution by adding nitrite as a final electron acceptor and performing the enrichment culture was referred to as Separation Method II. From the point where the separation sample used in Separation Method I was taken, 1 g of the raw sample obtained in the same manner was added to a screw cap test tube in an amount of 20 ml of nitrous acid 1/10 concentration ZoBell.
In addition to the 2216E liquid medium, anaerobic culture was performed in an anaerobic glove box [internal gas phase N ₂ : H ₂ : CO ₂ = 17: 2: 1, oxygen concentration 0.1% (v / v) or less]. In addition,
The concentration of nitrous acid was 100 ppm-N, and it was added in the form of KNO ₂ . After subculturing in a medium of the same composition every 2 weeks, after 8 weeks, a certain amount of the culture solution was applied to an agar plate medium under anaerobic favorable conditions to isolate bacteria. The isolated strain was 1/10 concentration Zo containing 100 ppm-N equivalent of nitric acid or nitrous acid.
Bell 2216E liquid medium (20ml / tube)
1 week anaerobic culture at 22 ± 2 ° C. using, in the medium after culturing inorganic nitrogen three states ^{_{(NO 3 -, NO 2 -}} , NH 4 +) from the quantitative changes in the confirmation of the presence or absence of denitrification activity of Then, the mode of denitrification from nitric acid and nitrous acid (hereinafter referred to as denitrification type) was determined. The denitrifying bacteria separated by the separation method I were similarly judged as denitrifying type. In addition, Kikuchi et al .:
The method described in Research Report U87025 (1987) of the Central Research Institute of Electric Power Industry was followed.

The strains that could be isolated by the isolation method I were 235 strains, of which 81 strains showed denitrifying activity, and the ratio of the denitrifying strains to the total number of the isolated strains was 34%.
On the other hand, in the separation method II, 145 strains were separated and the denitrification activity was examined. As a result, 121 strains showed denitrification activity, and the ratio of the denitrification strain number to the number of the isolated strains was 83%. It was a high rate.

Next, the denitrification type judgment result will be described.
A typical result obtained by measuring the quantitative change of the three states of inorganic nitrogen in the medium after anaerobic culture is shown in FIG.
In addition, as shown in the footnotes of Table 5, the names of the strains shown below are derived from the sample of Atsumi Bay, where Y is represented by At, and similarly, Y is derived from the sample of Yokosuka Owada Bay,
G means that the sample is from Sendai Taiwan, and Ab is that from the Central Research Institute of Electric Power Industry, Abiko. Strain At-8
-R-2 produces nitrite in a nitric acid-containing medium,
The total amount of inorganic nitrogen did not change, and the amount of nitrite in the medium containing nitrite did not change. From this fact, this strain is a nitrate respiration type (NO ₃
^- → NO ₂ ^-) is determined to be. Strain At-8-
R-1 is a denitrifying bacterium because the amount of inorganic nitrogen is decreased in the nitric acid-added and nitrite-added media, but nitrite is detected in the nitric acid-added medium. Apparently, nitric acid is converted to gas through nitrite. Has been reduced to nitrogen. The strain At-B-4 is a denitrifying bacterium because the amount of inorganic nitrogen is decreased in the nitric acid-added medium and the nitrite-added medium, but in the nitric acid-added medium, At-8-
Unlike the R-1 strain, nitrite is not detected. This indicates that the nitrous acid produced by the reduction of nitric acid is rapidly reduced to gaseous nitrogen. The G-2-R-1 strain has a reduced amount of inorganic nitrogen only in the nitrite-added medium, and it is judged that the strain can denitrify only from nitrite. Therefore, denitrifying bacteria according to the metabolic pathway of its apparent as described above, A-type NO ₂ ^- → N ₂ B-type ^{_{NO 3 - → NO 2 - →}} N 2 C -type NO ₃ ^- → denitrification N ₂ It can be classified into types. Nitrous acid is harmful to fish, and since a large amount of nitric acid accumulates in breeding water, it is considered that C-type denitrifying bacteria are useful when considering application to a water purification system using a circulation filtration method. To be Table 4 shows the results of discrimination of the denitrification type of the strains judged to be positive for denitrification in the above experiment.

[Table 4] C-type denitrifying bacteria are 20% of the isolated denitrifying strains in the isolation method I.
(8 strains), and 23% of the strains isolated by isolation method II
(28 strains). This suggests that C-type denitrifying bacteria are rare among natural denitrifying bacteria.

C. Classification of isolated denitrifying bacteria For isolated bacterial strains that are considered to be useful from the denitrifying type, the stability of the strains was confirmed by further testing whether the activity was stable even after subculture, and the isolated strains that were stable were treated with fresh water. Method ["Marine Microbial Experiments" edited by Kadota and Taga, Academic Publishing Center, 2
28-233 (1985)].
Flagella observation, sugar fermentability test (OF test), etc. were performed to classify at the genus level.

The results are summarized in Table 5.

[Table 5] C-type denitrifying bacteria isolated by the isolation method I (A in the strain name is labeled), 7 strains out of 8 strains belong to the genus Alcaligenes,
All of the 8 strains of C-type denitrifying bacteria (B in the strain name) separated by Separation Method II were Pseudomonas spp.
was as).

D. Measurement of denitrification activity using acetylene inhibition method The denitrification bacteria of the above-mentioned useful denitrification bacteria were analyzed by O'hara et al., J. Gen. Micr.
obiol., 129, 2405-2412 (1983), and the acetylene inhibition method was used for the measurement. Acetylene inhibition method is acetylene denitrification process (NO ₃ ^- → NO
₂ ^- (NO) → N in ₂ O → N _2), it utilizes the property of inhibiting the reduction of the N ₂ O of N _2, the original N
_This is a method of calculating the denitrification activity by measuring the amount of ₂ and N ₂ O that should be generated as N ₂ O.

The denitrification activity was measured as follows. The test strain was anaerobically cultured in a anaerobic glove box at 24 ± 1 ° C. for 5 days using a wide-mouth flask containing a ZoBell2216E agar medium containing nitric acid. The amount of nitric acid added to the medium was equivalent to 100 ppm-N, and was added in the form of KNO ₃ . After suspending the bacterial cells grown on the agar medium surface in sterile filtered seawater poured into a flask, centrifuge and discard the supernatant, suspend the pelleted bacterial cells in sterile filtered seawater again and centrifuge. The cells were washed. The cells were finally suspended in filtered seawater and used as a measurement sample. Cell suspension 1
1 ml of a glucose solution having a concentration of 180 mM and KNO ₃ having a concentration of 18 mM are placed in a test tube having a volume of 12.8 ml.
1 ml of the solution was added, the rubber stopper was applied, and immediately, 10% of acetylene in the internal gas phase (9.8 ml) was added using a gas tight syringe. All of the above operations were performed under anaerobic conditions. When acetylene is added, the denitrification activity measurement starts,
After culturing at 25 ° C for a certain period of time, the produced N ₂ O was charged with a PID type gas chromatograph (GC3000PI manufactured by Hitachi, Ltd.).
It was measured using D). The glucose concentration in the reaction solution was 60 mM, and the KNO ₃ concentration was 6 mM (84 ppm-
N). The bacterial cell suspension was subjected to ultrasonic treatment to disrupt the bacterial cells, and then subjected to measurement of protein content. The protein content was measured using a protein assay kit manufactured by Bio-Rad with albumin as a standard. The denitrification activity was expressed by the amount of N ₂ O produced per unit weight of protein.

First, when the N ₂ O production was measured for the Alcaligenes Ab-A-1 strain, it increased linearly up to 3 hours after the start of the measurement, as shown in FIG. Based on this result, the other test strains produced N ₂ produced 1 hour after the start of measurement.
O was measured and used as denitrification activity. The results are shown in Table 6.

[Table 6] Thus, the denitrification activity of each strain is 131-4330 nm
olN ₂ O / hour / mg protein, which varied depending on the strain. However, the average denitrifying activity is Smith et al., Soil.
Sci. Soc. Am. J. 50, 349-353 (1986), a representative denitrifying bacterium Pseudomonas isolated from soil.
Denitrification activity of Pseudomonas fluorescence (1350 nmol N ₂ O / hour / mg protein)
It was comparable to.

E. Relationship between Denitrification Activity and Kind of Organic Matter With respect to the 16 useful marine denitrifying bacteria strains, the relationship between denitrification activity and kind of organic matter was further investigated. Glucose, saccharose, peptone, glutamic acid and methanol were used as organic substances. Peptone manufactured by Difco was used, and other peptone manufactured by Wako Pure Chemical Industries, Ltd. was used.
The theoretical value of the amount of organic substances required for denitrification is 1 mg when glucose is used-1.07 mg-for denitrifying N NO _3.
Since C is required, the organic matter concentration is set to 120 here.
mg-C and nitrate ion concentration were 100 mg-N / l. The denitrification activity was measured by the acetylene inhibition method as in the previous section.

The results are shown in FIG. 3, and it can be seen that the relationship between organic matter and denitrification activity differs depending on the strain. The average denitrification activity of the 16 tested strains was 680 ± 429 nmol N ₂ O / hour / mg protein in the case of glucose. In addition, the denitrification rate in glucose is 1
When set to 00, sucrose 107 and peptone 14
5, glutamic acid 114, and methanol 87.8. Thus, peptone and glutamic acid tended to show high activity as added organic substances, but since these organic substances contain a little over 20% of organic nitrogen, the organic nitrogen is mineralized and released as ammonia. Considering the above, the pure denitrification activity is equivalent to 80% of the total denitrification activity. Taking this into account, the denitrification activity on glucose and saccharose is at a level comparable to peptone. Glucose and saccharose are cheaper than peptone and glutamic acid, and can be stored at room temperature in a high-concentration aqueous solution, and are easy to handle. From the above, it is judged that glucose and saccharose are suitable as the types of organic substances added as an energy source for denitrification. Furthermore, when comparing the test strains from the viewpoints of wide range of utilization to organic substances and high activity, strains G-A-2-1, G-A-2-2 and Ab-A belonging to the genus Alcaligenes −
The 1 strain and the Ab-A-2 strain are judged to be particularly effective strains. However, since the G-A-2-2 strain is more difficult to culture than the other three strains, it is expected that the actual application is difficult.

F. Relationship between Denitrification Activity and Dissolved Oxygen Concentration (DO) 3 strains belonging to the genus Alcaligenes, G-A-2-1 strain, Ab-A-1 strain and Ab-A
The relationship between the denitrification activity and DO was investigated for the -2 strain. In this case, the denitrification activity cannot be measured by the acetylene inhibition method, so that the experiment was carried out by a culture experiment. In the experiment, 5 constant temperature culture tanks kept at 25 ° C. were used, and DO was 0, 7, 15,
It was set to 30 and 60% oxygen saturation. The DO of the culture solution was adjusted by aeration and nitrogen gas, and the fluctuation range of the DO during the culture was about ± 10% of the set value.
For example, when the set value was 30% oxygen saturation, the actual DO fluctuation range was 27 to 33% oxygen saturation. The culture solution was stirred at 70 rpm using a screw-type stirring blade.
The denitrification activity depends on the culture solution in each culture tank (nitrate ion concentration 100
Calculated by measuring the decrease in inorganic nitrogen in the culture broth after 24 hours of culturing after inoculation of anaerobically grown cells into sterile filtered seawater (2 l) added with ppm-N and 300 ppm of glucose. . At the same time, the organic carbon concentration (TOC) in the culture solution was measured with a total organic carbon analyzer (TOC-500 manufactured by Shimadzu Corporation) to calculate the organic substance decomposition rate. The amount of inoculated cells in this experiment was G-
11.2-μg bacterial protein / ml culture solution for A-2-1 strain, 5.2 μg bacterial protein / ml for Ab-A-1 strain
The culture solution and Ab-A-2 strain contained 19.6 μg bacterial protein / ml culture solution.

The results are shown in FIG. 4. The denitrification activity here means the specific activity with respect to the denitrification rate at 0% oxygen saturation, and the organic matter decomposition rate is also shown. The denitrification activity of all strains decreased with the increase of DO, but the degree varied depending on the strain. Further, the specific activity of organic substance decomposition (TOC reduction amount) was 100% or more in any DO, and it was found that the organic substance decomposition was carried out rapidly. When the DO value showing 50% denitrification activity of 0% DO was estimated from the specific activity calculated from the amount of nitrogen reduction after 24 hours obtained in this experiment, it was 7 in G-A-2-1 strain.
% DO, 30-60% DO with Ab-A-1 strain, and A
It was 15% DO for the b-A-2 strain. From this result, it can be said that denitrification can be performed even when the DO is relatively high when the strain Alcaligenes genus Ab-A-1 is used. Thus, in order to obtain high denitrification activity, it is better to lower DO. However, if denitrification can be performed even under the condition that DO is relatively high,
There are many advantages from the perspective of recycling water for breeding fish. Therefore, it can be said that the above-mentioned strain Alcaligenes genus Ab-A-1 is a particularly preferable bacterial species.

II. Large-scale, high-density culture of marine denitrifying bacteria Next, 3 strains G-A-2-1 and Ab belonging to the genus Alcaligenes, which were particularly preferred in view of their high denitrification activity, etc.
-In order to search for conditions that enable large-scale cultivation of the A-1 and Ab-A-2 strains, a large-scale cultivation method is examined based on their optimal growth conditions, organic matter conditions and their results. did. A. Examination of Optimal Growth Conditions First, for the three strains of the genus Alcaligenes, using ZoBell2216E liquid medium as a basic medium, only one of the pH, seawater concentration, and temperature conditions of the medium was changed, and the respective conditions at that time were changed. The optimum conditions for each were clarified by measuring the specific growth rate (μ). When searching for the optimum medium pH, the medium pH is 5, 6,
The conditions were changed to 6 steps of 7, 8, 9 and 10, and the other conditions were that the seawater concentration was 100% and the temperature was constant at 30 ° C. The optimum seawater concentration is 100, 75,
It was changed to 5 steps of 50, 25 and 0%, and the other conditions were constant (pH: 7.8, temperature 30 ° C.). Then, in the case of searching for the optimum temperature, the temperature was changed in five stages of 20, 25, 30, 35 and 40 ° C., the pH was 7.8 and the seawater concentration was constant at 100%. The specific growth rate was calculated as follows. 10 for test tube (18mmφ × 18cm)
After adding ml of the medium and setting each condition, autoclave sterilization was performed. The pH was adjusted by adding 1N hydrochloric acid or sodium hydroxide solution to the medium, and the concentration of seawater was adjusted by adding distilled water before autoclave sterilization. A test tube containing the adjusted culture solution was inoculated with each strain in advance and then shake-cultured at 30 ° C for 3 days.
Bell 2216E culture solution (0.3 ml) was inoculated and shake-cultured by a reciprocal shaker under each set condition. A large number of test tubes for culture were prepared, and measurement samples were collected over time. The growth of the denitrifying bacteria depends on the turbidity (O.
D. 610 nm) to obtain a growth curve from the change in turbidity with time, and from the growth curve,
A specific growth rate was calculated, which indicates how many times the bacteria multiply per hour.

P on the growth of 3 strains of marine denitrifying bacteria
The effects of H, seawater concentration and temperature are shown in Figures 5, 6 and 7, respectively. (1) pH (see FIG. 5) The optimum p of the three strains of the genera Alcaligenes tested
H was 7 for all 3 strains. At pHs of 6 to 8, the specific growth rates of all strains were not significantly different from those at pH 7. At pH 5, 9 and 10, the specific growth rate tended to decrease, but the degree thereof varied depending on the strain. That is, G-A-2-1 strain and Ab
-The A-2 strain showed the lowest specific growth rate at pH 10
In the case of pH 7, the specific growth rate was reduced by about 20% compared to pH 7, but in the case of Ab-A-1 strain, at pH 10, it was markedly decreased by about 40%. At pH 5, GA-2-
One strain had a higher rate of decrease than the other two strains, but p
It was a 10% decrease compared to the case of H7.

(2) Seawater Concentration (See FIG. 6) Regarding the effect of seawater concentration on the growth of denitrifying bacteria, the following results were obtained. In all the strains, the specific growth rate was significantly reduced at 0% concentration (actual seawater concentration is about 3% seawater concentration due to the inclusion of salt from the inoculum) compared to other concentrations. Other concentrations, ie 25% to 100%
The strains showed almost the same level of specific growth rate among strains, although there were some differences depending on the strains. Seawater concentration is 25% to 1
The difference in specific growth rate between 00% was as follows. G
The -A-2-1 strain showed almost the same specific growth rate between 25% and 100%. In the Ab-A-2 strain, 25 and 50% had the highest specific growth rate, and at higher concentrations, the specific growth rate slightly decreased. In the case of the Ab-A-1 strain, the highest specific growth rate was observed at 50%, and the specific growth rates were slightly decreased at 25% and 75% and 100%. In the seawater concentration range of 25 to 100%, the difference in the specific growth rate is 16% at the maximum even with the largest Ab-A-1 strain.
(When the concentration is 50% and 100%). Generally, marine bacteria are low halophilic bacteria having an optimum salt concentration of 0.3 to 0.5 M (salt concentration of seawater is about 0.5 M), but from the above results, It was revealed that the three test strains also have the characteristics.

(3) Temperature (see FIG. 7) Regarding the effect of temperature on the growth of denitrifying bacteria, the following results were obtained. In the Ab-A-1 and Ab-A-2 strains, the specific growth rate increased linearly with the increase in temperature from 20 ° C to 35 ° C, and slightly decreased at 40 ° C. On the other hand, the strain G-A-2-1 had the highest specific growth rate at 35 ° C, which was almost the same as the value at 30 ° C and was remarkably low at 20 ° C and 40 ° C.
These three strains had an optimum temperature of 35 ° C. and were shown to be mesophilic bacteria. From the above examination results, 3
The optimum conditions for growing the strain are pH: 6 to 8, seawater concentration;
25% to 100% (that is, a salt concentration of 0.85 to 3.
4%), temperature; 35 ° C.

B. Effects of Organic Substances and Concentrations on the Growth of Marine Denitrifying Bacteria From the results of the examination in the previous section, the effects of adding organic substances to the basal medium were examined under the optimal growth conditions revealed for each strain. As a basic medium, ZoBel
2216E medium and Anderson medium (2.5 g peptone / liter medium, 2.5 yeast extract / liter)
g, FePO ₄ 0.1 g) pH of 7, seawater concentration of 50
% (Referred to as ZoBell modified medium and Anderson modified medium, respectively), and glucose, mannitol, saccharose, water-soluble starch or aspartic acid at two levels (1.0%).
And 0.5%) were added to compare specific growth rates. In addition, in order to investigate the effect of adding an inorganic nitrogen source, KNO
_{The group to which 3} was added was set to a group in which the concentration of the ZoBell modified medium was halved in order to investigate the influence when the organic matter concentration in the medium was low. The culture temperature was 35 ° C. in all cases.

The results are shown in FIG. The following results were obtained when using ZoBell modified medium as the basal medium. In the case of the GA-2-1 strain, the specific growth rate clearly decreased in the 1 / 2ZoBell modified medium as compared with the ZoBell modified medium group, and increased by 17% when 1.0% mannitol was added. However, there was no big difference in the other experimental plots. In the case of the Ab-A-1 strain, compared to the ZoBell modified medium group, there was no significant difference in the other experimental groups, but there was a tendency that the specific growth rate decreased except for the mannitol and sucrose addition groups. In the case of Ab-A-2 as well, compared to the ZoBell modified medium group, there was no big difference in the other experimental groups, but 1.0% starch added group,
It showed a tendency to decrease in the 1.0% sucrose addition group and the 1.0% glucose addition group. In each case, the maximum specific growth rate was observed in the ZoBell modified medium,
This was the case where 1.0% mannitol was added, and it was suggested that the addition of mannitol has a growth promoting effect on denitrifying bacteria. Further, in a similar study conducted using the Anderson modified medium as a basic medium, a specific growth rate equal to or inferior to that of the ZoBell modified medium was obtained. Therefore, when culturing a large amount of the test strain, Zo
It was considered effective to use Bell 2216E medium and to add mannitol. ZoBell modified medium, which had the highest specific growth rate for each strain, was added to ZoBell modified medium containing 1.0% mannitol.
The modified medium (II) was used for the next mass culture.

C. Large-scale culture of marine denitrifying bacteria For culturing marine denitrifying bacteria, a 2 liter Erlenmeyer flask and a 20 liter jar fermenter (manufactured by Mitsubishi Petrochemical Engineering Co., Ltd.) were used. First, 3 strains of denitrifying bacteria were cultured using an Erlenmeyer flask. 1 liter of ZoBell modified medium (II) was placed in a 2-liter Erlenmeyer flask and sterilized by an autoclave. After inoculating the medium with denitrifying bacteria, the medium was shake-cultured at 30 ° C. and 100 rpm. The culture solution was collected over time, and the turbidity (O.
D. 610 nm) was measured. At this time, when the bacterial density of the culture broth was high, the culture broth was diluted and measured so that the turbidity was 0.4 or less, and then converted. Next, culture was performed using a jar fermenter. The internal volume of the jar fermenter was 20 liters, the internal stirring was carried out with turbine type blades, and the supply of sterile air was carried out from the lower part of the culture tank.
The jar fermenter should be sterilized with hot water and 7
Performed with 0% alcohol solution. 18 liters of ZoBell modified medium (II) sterilized in a culture vessel container
After inoculation, 300 ml of the precultured culture solution was inoculated, and then the culture was performed under the conditions of 35 ° C., stirring speed of 100 rpm, and air flow rate of 0.5 liter air / liter culture solution / min. Under the experimental conditions, DO maintained 70% or more of the saturation during the culture period. The change in medium pH with the progress of culture was appropriately adjusted with 1N hydrochloric acid or sodium hydroxide solution. During the culture, a part of the culture solution was collected,
The growth rate was evaluated by measuring the turbidity (OD 610 nm) of the culture solution.

The results of culturing the 3 strains of denitrifying bacteria using a 2-liter Erlenmeyer flask are shown in FIG. Each strain entered logarithmic growth immediately after inoculation, but the culture time was about 12
O. in time. D. Entered the final logarithmic growth phase of about 0.6,
Even when the culture was continued for 2 hours, the final density was 0. D. (6
(10 nm) = 1 only. Figure 10
The results of mass culture with a 20 liter jar fermenter are shown in FIG. In the present experiment, as in the flask condition, logarithmic growth was started immediately after the start of the culture, and the final reached concentration in the flask condition was turbidity 1 (OD, 61).
0 nm) within 12 hours. After that, Ab-A-
The two strains started to grow slowly, entered the stationary phase, showed a maximum turbidity of 2 48 hours later, and then entered the death phase. G-A-2
-1 strain and Ab-A-1 strain continue to grow after 12 hours,
In the G-A-2-1 strain, the maximum turbidity was 5 at 36 hours,
In the Ab-A-1 strain, the maximum turbidity reached 6 at 48 hours. The transition of the medium pH is shown below each graph, and the arrow on the graph indicates that the pH was adjusted at that time. The number of bacteria at the maximum turbidity was counted by the dilution plate method. As a result, the GA-2-1 strain had 5.4 × 10 ¹⁵ cells / ml and the Ab-A-1 strain had 3.4 × 10 ¹⁵ cells. / Ml,
The Ab-A-2 strain reached 7.1 × 10 ¹⁵ cells / ml.
In addition, the dry matter weight of the bacterial cells obtained by centrifuging the culture solution under the maximum turbidity condition was measured to find that the strain G-A-2-1 had 1.
88 g dry matter / liter culture solution, 2.6 with Ab-A-1 strain
g dry matter / liter culture medium, 1.36 g with Ab-A-2 strain
It was a dry matter / liter culture solution. The bacterial cell production efficiency until reaching the maximum turbidity was 52 mg for the GA-2-1 strain.
Dry matter / liter culture solution / hour, 54 m with Ab-A-1 strain
g dry matter / liter culture solution / hour, 28 with Ab-A-2 strain
It was mg dry matter / liter culture solution. From the above examination results, it was shown that the Ab-A-1 strain among the three strains has the fastest growth and enables high-density culture. In addition, the pH of the culture solution changed with the progress of culture. In all three strains, the culture medium leaned toward the acid side at the early stage of the culture, but in the latter half of the culture, the medium leaned toward the alkali side. Ab out of 3 stocks
-A-1 strain had a low rate of pH fluctuation. Until now, few attempts have been made to cultivate and utilize marine denitrifying bacteria in a large amount and at high density, and there is no reported value to be referred to. Therefore, it can be said that the present invention reveals for the first time the high-density mass-culture conditions of denitrifying bacteria. Although all the cultures in this section were carried out under aerobic conditions, denitrifying bacteria induce denitrifying activity if induced under anaerobic conditions even when cultured under aerobic conditions. This is because it was judged that there is little need for the mass culture method under difficult anaerobic conditions.

III. Study on Efficiency Improvement of Denitrification System Applying Immobilized Denitrifying Bacteria Next, denitrification method applying entrapment and immobilization of denitrifying bacteria and applying fluidized bed denitrifying tank to which immobilized denitrifying bacteria are applied Was examined. A. Study on influence of substrate concentration and dissolved oxygen on denitrifying activity of immobilized denitrifying bacteria Preparation of immobilized carrier for denitrifying bacteria was performed by the method described in Uemoto et al .: Central Research Institute of Electric Power Industry, Research Report U90056 (1991). Obeyed. As the test strain, Alcaligenes sp. Ab-A-1 strain was used, and after culturing in ZoBell 2216E liquid medium, cells were recovered from the culture solution by centrifugation. As a polymer gel for entrapping fixation, polyvinyl alcohol (PV
A, Kuraray made Kuraray Poval PVA-HC) 10%
Used at a concentration of (w / v), the concentration of immobilized bacteria is 8 x 1
It was 0 ¹⁸ cells / liter PVA. The immobilized denitrifying bacteria thus prepared were added to ZoBell 2216E liquid medium in an amount of 500
The culture was conditioned with a culture medium supplemented with mg-N / l of KNO ₃ .
This acclimation was thoroughly performed, and the following studies were carried out using immobilized denitrifying bacteria in which expression of denitrification activity was observed.

The apparatus shown in FIG. 11 was used to measure the denitrification rate of the immobilized denitrifying bacteria. A culture tank 1 having a capacity of 2 liters is pre-filled with a fixed amount of immobilized denitrifying bacteria 2, and a reaction solution 3 is introduced therein (arrow a), a reaction solution introducing pipe 4, and a reaction solution 3 after treatment is discharged. (Arrow b) is provided with the reaction liquid discharge pipe 5. The stirring inside the culture tank 1 is performed using a stirrer 9 equipped with rotary blades (screw type blade 7 and turbine type blade 8) that rotate to the aroma of arrow c (rotation speed is 70 rpm). The pH of the reaction solution 3 is detected by the pH meter 13, and an appropriate acid or base is supplied into the reaction tank 1 if necessary. Similarly, the temperature of the reaction solution 3 is 1
4 and the temperature is higher than the set temperature (25 ° C.), the water jacket 10 that covers the periphery of the culture tank 1
Cooling water is injected from the water supply pipe 11 to fill the inside of the jacket, and then the water is drained from the drain pipe 12 to cool it to a set temperature. Conversely, when the temperature is lower than the set temperature, hot water or steam is Heat through jacket 10.
Further, the control of DO in the reaction solution 3 is detected by the DO sensor 15 provided in the culture tank 1, and the DO controller 16 connected to the sensor 15 operates the gas flow meter 17 and connects the line 18 as necessary. It is carried out by supplying a predetermined amount of nitrogen and air into the reaction liquid 3 from the air diffusing pipe 6 attached to the lower part of the stirrer 9 through. The DO was kept at an oxygen saturation of 1% or less.

In the examination of the reaction characteristics of the immobilized denitrifying bacteria, 360 ml of the immobilized denitrifying bacteria washed with seawater (which had been pumped up) was placed in a culture tank, 1440 ml of seawater was added, and the total volume was 1.8. The liter was 20% and the filling rate was 20%. To this, 200 mg-N / l KNO ₃ and 1000 mg / l glucose were added, and then anaerobic conditions were maintained while supplying nitrogen gas to induce denitrification activity. Then, the reaction liquid inside was replaced, and the denitrification activity under each condition was measured. The reaction conditions were set as follows. In order to examine the relationship between the amount of substrate nitric acid and the denitrification activity, the glucose concentration as a carbon source was set to 1000 mg / l reaction solution, and the nitric acid concentration was set to 100,
The denitrification activity was measured while changing it to 200, 500, 1000 mg / l. The denitrification activity was measured by measuring the three states of inorganic nitrogen in the reaction solution according to the above method and calculating the denitrification amount from the amount of decrease in inorganic nitrogen. To examine the relationship between the amount of substrate glucose and the denitrification activity, the nitric acid concentration was 200 mg-
Set the N / l reaction solution to a glucose concentration of 300, 60
The denitrification activity was measured by changing it to 0, 1000, 10000 mg / l.

Regarding the denitrification of the immobilized denitrifying bacterium, the result of studying the relationship with the amount of substrate nitric acid is shown in FIG. Initial nitric acid concentration from 100 mg-N / l to 1000 mg-N
Although it was changed within the range of 1 / l, there was no big difference in the denitrification amount up to the 24th hour, indicating that there was no big difference in the denitrification activity in the experimental section. After 24 hours, when the initial nitric acid concentration was 100 mg-N / l, the denitrification amount did not increase because nitric acid disappeared, but in the other sections, there was no significant difference in the experimental section. This indicates that the denitrification rate by the immobilized denitrifying bacteria is not significantly affected by the initial substrate nitric acid concentration range set in this experiment.

FIG. 13 shows the result of examination on the relationship between the substrate glucose and the denitrification activity. Although the initial glucose concentration was changed in the range of 300 mg / l to 10000 mg / l, there was a difference in the denitrification activity in the experimental section. That is, in the glucose concentration range of 300 to 1000 mg / l, there was no great difference in the denitrification amount between the experimental groups,
In the case of 10000 mg / l, the activity was remarkably reduced. Also, in this last experimental section, a scale-like substance, which is considered to be a filamentous fungus, proliferated in the reaction solution. It is considered that the sufficient presence of glucose substrate caused such a phenomenon, but it is expected that the immobilizing microorganism was affected by the filamentous fungus and the denitrification activity was lowered. The present inventor has confirmed that the Ab-A-1 strain does not significantly improve its denitrification activity even if the glucose concentration is increased under the condition of the bacterial cell suspension in the previous studies (Watanabe et al. : Central Research Institute of Electric Power Industry, Research Report U91002 (1
991), from the above results, the glucose concentration is 300 ~
In the range of 1000 mg / l, it is considered that the property does not change even when immobilized.

B. Examination of denitrification method by anaerobic fluidized bed to which immobilized denitrifying bacteria were applied Here, denitrification activity of immobilized denitrifying bacteria under continuous (Chemostat) conditions was examined. Using the apparatus shown in FIG. 11, a reaction solution was flowed in using a peristaltic pump to carry out a continuous reaction experiment. The experimental conditions are shown in Table 7. The capacity of the reaction tank was 1.8 liters, the temperature was 25 ° C., and the stirring speed was 70 rpm. DO was not controlled during the experiment. The three states of inorganic nitrogen and total organic carbon (TOC) contained in the inflow water and outflow water to the apparatus were measured according to the above-mentioned method.

[Table 7]

The time course of denitrification activity and TOC consumption of immobilized denitrifying bacteria under continuous reaction conditions is shown in FIG. The experiment was performed in duplicate, but the same transition was observed, so only the results in one series are shown. The three types of nitrogen and TOC of the inflow water of the reaction tank and the inflow water are also shown. In addition,
DO was kept below 5% of oxygen saturation throughout the experiment. During the first 7 days with a hydraulic retention time (HRT) of 48 hours, the denitrification rate was almost constant, about 60 mg-N /
1 was removed and most of the added TOC was consumed. Further, nitrite production was observed up to the 4th day, but it was not detected thereafter. In the denitrification by the biofilm method applying the Ab-A-1 strain, nitrite was accumulated for several days immediately after the start of operation of the denitrification tank, but no nitrite was accumulated thereafter. As described above, even when the immobilized denitrifying bacteria were applied, the same phenomenon as the biofilm method was observed. HRT
However, the denitrification amount increased during the next 7 days of 24 hours.
The amount of OC also increased. It is conceivable that the reason why the denitrification amount increased is that the residence time became shorter, the supply of the substrate became more active and the denitrification activity increased than when the HRT was 48 hours. Also, the doubled supply of carbon substrates may be the reason for the increase in unconsumed TOC. The denitrification amount is HRT24 for 7 days when HRT is 12 hours.
It decreased compared to the time, and the TOC amount of runoff water increased further. During 7 days when the substrate concentration of the inflow water was halved and the HRT was changed to 24 hours, the denitrification amount was almost the same and the TOC amount of the outflow water decreased compared to the HRT 12 hours.

Based on the above results, the denitrification activity and TOC consumption rate of the immobilized denitrifying bacteria under continuous reaction conditions were evaluated, and the results are shown in Table 8. The denitrification activity and the TOC consumption rate were evaluated by using the measured values on the last day of each experimental condition, assuming that this time is a steady state under each condition. The measured value is an average value of duplicates.

[Table 8] The following are clear from the results summarized in Table 8. H
If the RT is 48 hours, 1 in a 1.8 liter reaction tank
88.2 mg-N is removed per day, which is 49 mg-N when converted per liter of the reaction tank. The denitrification capacity by the biofilm method established by the present inventor is about 40 m.
g-N / l denitrification tank / day (HRT is 48 hours,
Under a glucose concentration of 300 mg / l), the value according to the present invention is 1.2 times that, which is slightly improved as compared with the biofilm method. For HRT 24 hours, 1.
202 mg-N was removed per day in an 8-liter reaction tank (112 mg-N per 1-liter reaction tank),
It was 2.8 times more potent than the biofilm method and was much more active than the biofilm method. 220 mg per day in a 1.8 liter reaction tank when HRT is 12 hours
-N has been removed and is three times more potent than the biofilm method. However, 77 of the TOC added to the denitrification tank
There is a problem that% is leaked. Therefore, if TOC is considered to affect fish, it may be necessary to combine a fluidized bed denitrification tank using immobilized denitrifying bacteria with a TOC treatment tank that flows out. Also, H
When the substrate concentration was halved under the condition of RT 24 hours, both the denitrification amount and the TOC consumption rate decreased to about half, but the denitrification efficiency was slightly improved as compared with the biofilm method. Based on the above examination results, the denitrification method using an anaerobic fluidized bed to which immobilized denitrifying bacteria are applied generally improves the denitrification efficiency as compared with the biofilm method. At 240m
It was clarified that the efficiency of denitrification was increased to about 2.8 times when the GRT was increased to twice or more and the HRT was accelerated to 24 hours for 24 hours.

[0042]

The present invention has the following effects by adopting the above configuration. First, by enabling large-scale cultivation of useful marine denitrifying bacteria at high density, artificial inoculation of filter media is possible, and the marine denitrifying bacteria can be used not only at the laboratory level but also at the industrial level. It became applicable to denitrification by the biofilm method. Furthermore, the present invention makes it possible to perform denitrification with higher efficiency by comprehensively immobilizing the denitrifying bacteria and using an anaerobic fluidized bed denitrifying tank to which the immobilized denitrifying bacteria are applied. . Further, the marine denitrifying bacterium of the present invention is a type whose apparent metabolism is a direct reduction from nitric acid to gaseous nitrogen, and therefore, as a purification microorganism for water purification in a circulating filtration fish culture system or the like in which nitrite generation is not desirable, It can preferably be used in the form of biofilms or more efficiently immobilized bacteria.

[Brief description of drawings]

FIG. 1 is a graph showing the quantitative changes of three states of inorganic nitrogen in a medium after anaerobic culture of various denitrifying bacterial strains.

FIG. 2 is a graph showing changes over time in the amount of N ₂ O produced by the denitrifying bacterium Alcaligenes Ab-A-1 strain under acetylene-added anaerobic conditions.

FIG. 3 is a graph showing the relationship between the denitrification activity of denitrifying bacteria and the type of added organic matter.

FIG. 4 is a graph showing the effect of dissolved oxygen concentration on the denitrification activity of denitrifying bacteria and the decomposition rate of organic matter.

FIG. 5 is a graph showing the effect of pH on the growth of three useful marine denitrifying bacteria.

FIG. 6 is a graph showing the influence of the concentration of Mediterranean water on the growth of three useful marine denitrifying bacteria.

FIG. 7 is a graph showing the effect of temperature on the growth of three useful marine denitrifying bacteria.

FIG. 8 is a graph showing the effect of adding an organic substance on the growth of three useful marine denitrifying bacteria.

FIG. 9 is a graph showing the time course of growth of three useful marine denitrifying bacteria under shaking culture conditions.

FIG. 10 is a graph showing the time course of growth and medium pH adjustment of three useful marine denitrifying bacteria strains under large-scale culture conditions.

FIG. 11 is a cross-sectional view showing an apparatus used for measuring reaction characteristics of immobilized denitrifying bacteria in a batch system and a continuous system.

FIG. 12 is a graph showing the relationship between the amount of denitrification by immobilized denitrifying bacteria and the concentration of substrate nitric acid.

FIG. 13 is a graph showing the relationship between the amount of denitrification by immobilized denitrifying bacteria and the concentration of substrate glucose.

FIG. 14 is a graph showing the time course of denitrification activity and total organic carbon (TOC) consumption of immobilized denitrifying bacteria under continuous reaction conditions.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display area // (C12N 1/20 C12R 1:02)

Claims (6)

[Claims] 1. Alkaligenes, which has an optimum growth condition of pH 6 to 8, temperature 35 ° C., culture medium salt concentration of 0.85 to 3.4%, and has an apparent metabolic pathway for direct reduction of nitric acid to gaseous nitrogen. Marine denitrifying bacteria of the genus 0.85 in salinity
A denitrifying bacterium which comprises culturing in a culture medium of up to 3.4% while maintaining the pH of the medium at 6 to 8 and the temperature at 32 to 38 ° C until the absorbance of the culture solution at 610 nm becomes 2 or more. Culture method. 2. Alkaligenes, which has an optimal growth condition of pH 6 to 8, temperature of 35 ° C., culture medium salt concentration of 0.85 to 3.4% and an apparent metabolic pathway for direct reduction of nitric acid to gaseous nitrogen. An immobilized denitrifying bacterium obtained by immobilizing a marine denitrifying bacterium of the genus. 3. The marine denitrifying bacterium is Alcaligenes genus G-
A-2-1 strain (FERM P-13861), Alcaligenes genus Ab-A-1 strain (FERM P-13862)
No.) or Alcaligenes genus Ab-A-2 strain (FERM
P-13860), The immobilized denitrifying bacterium according to claim 2. 4. A denitrification method comprising contacting the immobilized denitrifying bacteria according to claim 2 or 3 with inflow water in an anaerobic fluidized bed. 5. The sugar concentration of the inflow water is converted to a carbon concentration of 12
The denitrification method according to claim 4, wherein the treatment is carried out with 0 to 360 mg-C / l and a residence time of 12 to 36 hours. 6. The denitrification method according to claim 4, wherein the inflow water is breeding water in a circulating filtration fish culture system.