JPH0760281A - Marine denitrifying bacterium immobilized material - Google Patents

Abstract

PURPOSE:To prevent marine denitrifying bacteria from flowing out and to secure a stable denitrification function through a long period of time by immobilizing microorganisms having a function to reduce nitrate or nitrite into gaseous nitrogen in sea water in a water vessel for culture, etc., on polymer. CONSTITUTION:A marine denitrifying bacteria immobilizing material for removing nitrogen in sea water which causes eutrophication is made by immobilizing microorganisms having a function to reduce nitrate or nitrite into gaseous nitrogen in natural or artificial sea water (hereafter called as marine denitrification bacteria) on polymer gel. As a polymer material used for the inclusively immobilized material is selected preferably from polyvinyl alcohol, polyethylene glycol, and polyacrylamide. The shape of the polymer gel is selected from sphere, cube, fiber, sheet, and tube; the gel is molded according to the purpose of application.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immobilized product of marine denitrifying bacteria capable of removing nitrogen in seawater which causes eutrophication.

[0002]

2. Description of the Related Art In a system for raising seafood in seawater such as an aquarium for appreciating fish, an aquarium for aquaculture, fish cages, aquariums, aquaculture farms and transportation of live fish, due to residual feed and excrement, ammoniacal Nitrogen is generated, which adversely affects seafood.
In order to remove this ammoniacal nitrogen, a method of adding water containing nitrifying bacteria, a method of waiting for the growth of nitrifying bacteria present in seawater, and a method of using a carrier on which nitrifying bacteria are entrapped and immobilized (JP-A-3-72996). No.) is known. However, ammoniacal nitrogen is only converted to nitrite nitrogen or nitrate nitrogen, and this alone does not result in nitrogen removal. Therefore, in order to remove this nitrite nitrogen or nitrate nitrogen, It is known to wait for the growth of the denitrifying bacteria present in. On the other hand, with respect to fresh water, a method using a carrier in which denitrifying bacteria are entrapped and immobilized is known (23rd Annual Meeting of Japan Society for Water Pollution, page 67 (1989)).

[0003]

The method of waiting for the growth of denitrifying bacteria is not effective because the nitrogen removing ability is slow to develop and the number of denitrifying bacteria cannot be increased. In addition, the denitrifying bacteria that have grown in the form of biofilms may drop out due to changes in the environment, and the nitrogen removal capacity is extremely unstable. On the other hand, denitrifying bacteria used in fresh water cannot be used for nitrogen removal in seawater because they grow in seawater and show no activity.

[0004]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, as a result, microorganisms capable of reducing nitrates or nitrites to nitrogen gas in seawater (natural seawater or artificial seawater) The present invention has been completed by discovering a marine denitrifying bacterium immobilization product obtained by immobilizing (in abbreviated form of denitrifying bacterium) on a polymer gel. By immobilizing marine denitrifying bacteria on polymer gel, it becomes possible to maintain the number of marine denitrifying bacteria at a high level. Nitrogen bacteria do not flow out and a stable nitrogen removal capacity can be obtained.

The present invention will be described in detail below. Marine denitrifying bacteria can be collected and separated from seawater.
It can also be collected from concrete, plastics, fibers, metals, ceramics, rocks, mud floors, animals and plants, etc., which have been immersed in seawater. The collected and separated marine denitrifying bacteria can be used as they are, or they can be cultured to enhance their ability. For the culture, natural seawater or known artificial seawater is used. The concentration of nitrate or nitrite during culture is preferably 0.1 ppm or more and 10% by weight or less, and particularly preferably 1 ppm or more and 1% by weight or less. Also,
Since marine denitrifying bacteria are heterotrophic bacteria, hydrogen donors such as organic carbons such as glucose and sodium acetate must be added during cultivation. There is no problem in changing the concentration of nitrate or nitrite during the culture, and it is also effective to make the concentration low at the beginning of the culture and gradually increase the concentration. The culture may be a closed system, or the water may be gradually replaced. Known nutrient salts may be added. The water temperature during culture is preferably 10 to 40 ° C,
Particularly, 15 to 35 ° C is preferable. The culture may be static culture, or may be agitated to the extent that air is not entrained. Also,
Even when the marine denitrifying bacteria are not added during the culture, a small number of the marine denitrifying bacteria that are resident in seawater grow. Propagation is faster and the culture time can be shortened.

Next, the immobilization of marine denitrifying bacteria will be described. Polymer materials often used for entrapping immobilization include agar, alginate, carrageenan, polyacrylamide, polyvinyl alcohol, polyethylene glycol, and photocurable resin. Of these, polyvinyl alcohol (hereinafter abbreviated as PVA), polyethylene glycol (hereinafter abbreviated as PEG), polyacrylamide (hereinafter abbreviated as PAA) do not undergo microbial degradation,
It is preferable as an immobilization carrier because it has excellent habitability for microorganisms. The shape of the polymer gel is not particularly limited, but may be spherical, dice-shaped, fibrous, sheet-shaped, tubular, or the like, and the shape suitable for each use condition may be selected. Next, the polymer used in the polymer gel of the present invention will be described.

(1) In the case of PVA The average degree of polymerization of PVA is 1000 or more, preferably 170.
It is 0 or more, more preferably 1700 to 25,000, and the saponification degree is 98.5 mol% or more, preferably 99.
A fully saponified PVA of 85 mol% or more is preferable from the viewpoint of forming a PVA gel. Further, as the PVA in the present invention,
Various known modified PVA can be used as long as the effects of the present invention are not impaired. The concentration of the PVA aqueous solution can be from 0.1 to 40 wt% from the viewpoint of PVA gel formation. The higher the PVA concentration is, the stronger the gel is produced, but if the required gel strength is obtained, the PVA concentration is increased. A lower value is advantageous in terms of raw material cost. A water-soluble polymeric polysaccharide such as sodium alginate may be used to shape the PVA gel into a spherical shape. Also,
To the PVA aqueous solution, a microbial medium, a reinforcing agent for increasing the strength of the immobilization carrier, a filler for adjusting the specific gravity of the produced gel, and the like may be added within a range that does not inhibit the gelation of PVA. Marine denitrifying bacteria are mixed with this PVA aqueous solution. It is preferable to mix marine denitrifying bacteria that have been subjected to a concentration operation such as centrifugation because the concentration of microorganisms can be increased.

Various methods are known as gelation methods for PVA, but the following two methods are often used. The PVA aqueous solution is frozen at −5 ° C. or lower, preferably −10 ° C. or lower, and kept for at least 1 hour or longer, preferably 10 hours or longer, and then thawed. Freezing-thawing operation is performed at least once, preferably twice or more. The PVA aqueous solution is brought into contact with an aqueous solution containing a substance having a synergic action of PVA, for example, an aqueous sodium sulfate solution. The concentration of the sodium sulfate aqueous solution is preferably 100 mg / liter or more, and a saturated aqueous solution is particularly preferable. Immersion time is 1
0 minutes or more is preferable, and 30 minutes or more is more preferable. Further, the methods of and may be used in combination, or a drying operation and a crosslinking operation with formaldehyde or glutaraldehyde may be performed.

(2) In the case of PEG Marine denitrifying bacteria are mixed with an aqueous solution containing a polyethylene glycol prepolymer in an amount of 1 to 40% by weight, and a polymerization initiator and a polymerization accelerator are added thereto to cause gelation. As a prepolymer of polyethylene glycol, for example,
Examples thereof include polyethylene glycol diesters such as polyethylene glycol dimethacrylate, and methoxy polyethylene glycol monoesters such as methoxy polyethylene glycol methacrylate. Examples of the polymerization initiator include β-dimethylaminopropionitrile and NNN′N′-tetramethylethylenediamine. As the polymerization accelerator, potassium persulfate,
Examples thereof include ammonium persulfate.

(3) In the case of PAA Marine denitrifying bacteria are mixed with an aqueous solution containing acrylamide and methylenebisacrylamide, and a polymerization initiator and a polymerization accelerator are added thereto to cause gelation. As the polymerization initiator, β-dimethylaminopropionitrile, NNN ′
Examples thereof include N'-tetramethylethylenediamine. Examples of the polymerization accelerator include potassium persulfate and ammonium persulfate.

Nitrogen in seawater can be removed by using the immobilized product of marine denitrifying bacteria obtained by the above method. As a method for removing nitrogen, seawater from which nitrogen should be removed may be contacted with a gel for immobilizing marine denitrifying bacteria. However, when removing ammoniacal nitrogen, it is difficult to convert the ammoniacal nitrogen directly into nitrogen gas.Therefore, a method of introducing seawater containing nitrifying bacteria, a method of multiplying nitrifying bacteria slightly present in seawater The method of using the carrier which entraps and fixes the nitrifying bacteria is used. Among them, it is preferable to use the one in which nitrifying bacteria are entrapped and immobilized. Alternatively, two or more of the above methods may be used in combination.
Thus, by immobilizing marine denitrifying bacteria in a polymer gel, it has become possible to remove nitrogen in seawater, which was difficult in the past.

[0012]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Example 1 0.1 g (dry weight conversion) of a deposit of porous concrete hanging on the quay of Izumisano Port in Osaka Prefecture was collected, and marine denitrifying bacteria purely separated therefrom were used. This was inoculated into 10 liters of the medium shown in Table 1 and cultured at about 20 ° C for 2 days. This was centrifuged at 9000 rpm to obtain about 1 g of cultured bacterial cells (dry weight conversion). Separately, PVA manufactured by Kuraray Co., Ltd. (average polymerization degree: 4000, saponification degree: 99.85 mol%) was washed with warm water at 40 ° C. for about 1 hour, and then the PVA concentration was 1
Add water to PVA so that the total amount becomes 500%.
g and treat at 110 ° C. for 2 hours to dissolve PVA, then
Cooled to 0 ° C. To this PVA aqueous solution, 250 g of a 4% by weight sodium alginate aqueous solution was added and mixed, and after cooling to room temperature, the above-mentioned cultured bacterial cells were added to bring the total amount to 1 liter, followed by sufficient stirring. Inner diameter of 4m
1 ml with a roller pump that uses one mφ vinyl tube
The solution was delivered at a flow rate of 1 / min, and was added dropwise to a 0.1 mol / liter calcium chloride (CaCl ₂ ) aqueous solution stirred with a stirrer from a height of 30 cm of the water surface. The dropped droplet is CaCl
₂ Immediately spheroidized and settled in aqueous solution. These spheroidized PVA mixed moldings were separated from the CaCl ₂ aqueous solution,
It was lightly washed with distilled water, frozen in a freezer at −20 ° C. for 12 hours, and then thawed. This gave an opaque brown flexible spherical PVA gel. This P
VA gel is formed into a spherical shape, has no tackiness, and has a particle size of 3
It was ~ 3.5 mmφ. To 5 liters of the medium for measuring denitrification activity shown in Table 2, 500 g of the obtained spherical PVA gel was added and left at 20 ° C. for 12 hours. As shown in Table 3,
The nitrate nitrogen concentration was significantly reduced, and denitrification activity was expressed (first time). Further, the PVA gel was washed with seawater and then the denitrification activity was measured, and the same activity was obtained (2
Second time).

Example 2 40% by weight of polyethylene glycol dimethacrylate
To 500 g of the aqueous solution, 1 g of the culture sludge (as dried weight) obtained in the same manner as in Example 1 was added and sufficiently stirred. To this, 5 g of NNN'N'-tetramethylethylenediamine as a polymerization accelerator and 2.5 g of potassium persulfate as a polymerization initiator were added, water was added so that the total amount was 1 liter, and the mixture was cast on a flat surface after stirring. Then, the mixture was polymerized at room temperature and formed into a sheet having a thickness of 3.0 to 3.2 mm. This was cut into 3.0 to 3.2 mm on a side to obtain a dice-like gel. To 5 liters of the solution for measuring denitrification activity shown in Table 2, 500 g of the obtained dice-shaped gel was added, and the mixture was allowed to stand at 20 ° C. for 12 hours. As shown in Table 3, the concentration of nitrate nitrogen was significantly decreased and the denitrification activity was expressed (first time). Further, the gel was washed with seawater and then the denitrification activity was measured, and the same activity was obtained (second time).

Example 3 Water was added to 200 g of acrylamide monomer and 10 g of methylenebisacrylamide to make 500 g. to this,
1 g of the cultured bacterial cell obtained in the same manner as in Example 1 (on a dry weight basis) was added and sufficiently stirred. To this, 5 g of NNN'N'-tetramethylethylenediamine as a polymerization accelerator,
Add 2.5 g of potassium persulfate as a polymerization initiator, add water so that the total amount becomes 1 liter, stir, cast on a flat surface and polymerize at room temperature to give a thickness of 2.9 to 3.1 mm. It was formed into a sheet. This was cut into a side of 2.9 to 3.1 mm to obtain a dice-like gel. The resulting dice-like gel 50 was added to 5 liters of the medium for measuring denitrification activity shown in Table 2.
0 g was added and the mixture was left at 20 ° C. for 12 hours. As shown in Table 3, the concentration of nitrate nitrogen was significantly decreased and the denitrification activity was expressed (first time). Also, after washing this gel with seawater,
When the denitrification activity was measured, similar activity was obtained (second time).

Comparative Example 1 500 g of a small piece of porous concrete suspended on the quay of Izumisano Port, Osaka Prefecture was placed in 10 liters of the medium shown in Table 1 and left at about 20 ° C. for 2 days. This porous concrete 50 was added to 5 liters of the solution for measuring denitrification activity shown in Table 2.
0 g was added and the mixture was allowed to stand at 20 ° C. for 12 hours. As shown in Table 3, the concentration of nitrate nitrogen decreased and the denitrification activity was expressed (first time). However, when the denitrification activity was measured after washing it with seawater, the denitrification activity was not observed (2
Second time).

Comparative Example 2 The same PVA as in Example 1 was washed with warm water at 40 ° C. for about 1 hour, and then water was added to PVA so that the concentration of PVA was 10% by weight to make the total amount 500 g, and the treatment was performed at 110 ° C. for 2 hours. PVA
Was dissolved and then cooled to 60 ° C. To this PVA aqueous solution, 250 g of a 4% by weight sodium alginate aqueous solution was added and mixed, and after cooling to room temperature, 1 g of denitrifying sludge having a denitrification activity in fresh water (on a dry weight basis) was added to bring the total amount to 1 liter, Stir well. Internal diameter 4 of these mixed liquids
1m with a roller pump that uses one mmφ vinyl tube
The solution was fed at a rate of 1 / min and added dropwise to a 0.1 mol / liter calcium chloride (CaCl ₂ ) aqueous solution stirred with a stirrer from a height of 30 cm of the water surface. The dropped droplet is CaC
Immediately spheroidized and settled in the ₁₂ aqueous solution. After separating all the spherical PVA-mixed molded products from the CaCl ₂ aqueous solution and lightly washing with distilled water, 1
It was frozen for 2 hours and then thawed. This gave an opaque brown flexible spherical PVA gel.
This PVA gel was formed into a spherical shape, had no tackiness, and had a particle size of 3 to 3.5 mmφ. Although this PVA gel had denitrification activity in fresh water, 500 g of spherical PVA gel was added to 5 liters of the solution for measuring denitrification activity shown in Table 2,
After standing at 20 ° C. for 12 hours, as shown in Table 3,
It was found that the concentration of nitrate nitrogen did not change (first time), and denitrification activity was not expressed in seawater (second time).

[0017]

[Table 1]

[0018]

[Table 2]

[0019]

[Table 3]

[0020]

According to the present invention, it is possible to denitrify seawater, which was difficult in the past. This is useful for removing nitrogen from the saltwater fish culture system and for preventing eutrophication of the ocean.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Takeshi Matsuda 1-2-1, Kaigan-dori, Okayama-shi Kuraray Co., Ltd. (72) Yuio Shiotani 1-2-12-39, Umeda, Kita-ku, Osaka Kuraray Co., Ltd. Within

Claims (1)

[Claims] 1. A marine denitrifying bacterium immobilization product in which a microorganism capable of reducing nitrate or nitrite to nitrogen gas in seawater is immobilized on a polymer gel.