JP2000262276A - New microorganism and biological treatment of marine life - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new microorganism consisting of Bacillus sp NA-3, Pseudomonas fluorescence NA-11, NA-12, etc., having a marine life decomposing activity, and useful for the decomposition treatment of shells, jelly fishes, etc., removed from a ship body or a water intake port. SOLUTION: This new microorganism is selected from a group consisting of Bacillus sp NA-3 (FERM P-17302), Pseudomonas fluorescens NA-11 (FERM P-17303), Pseudomonas fluorescens NA-12 (FERM P-17304), Enterobacter amnigeus NA-34 (FERM P-17305) and Ochrobactrum anthropi NA-43 (FERM P-17306), has a marine life decomposing activity and is useful for performing a decomposition treatment of shells attached to a ship body and a water intake port and marine life such as jelly fishes sucked at the water intake port of a water intake passage, without generating a bad smell and in a good efficiency.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel microorganism strain having the ability to degrade marine organisms and a method for biologically treating marine organisms using the same.

[0002]

2. Description of the Related Art Among sea creatures such as shellfish attached to a hull and an intake channel and jellyfish attracted to an intake of an intake channel,
For example, marine organisms adhering to a cooling intake channel of a factory, a power plant, or the like cause a reduction in heat exchange efficiency. In addition, sea creatures drawn into the intake of the intake channel cause a decrease in water intake to lower the water level at the intake,
This may cause the system to stop and the operating rate to decrease. Therefore, these marine organisms must be removed regularly or as needed. Heretofore, landfill treatment has generally been applied to the treatment of these removed marine organisms. However, marine organisms left unfilled have become a problem because they rot and emit intense odors.

[0003] The amount of marine organisms that adhere to the intake channels of factories or are drawn into the intake ports is enormous per year depending on the scale of the intake channels. This is a particularly big problem. Furthermore, it cannot be said that there is a limit to the landfill processing due to problems such as securing land for landfill.

[0004] As another treatment method, marine organisms removed and recovered from an intake channel or an intake port are incinerated. However, this method is not a desirable method as a method for treating waste, because the input energy is large and the treatment cost is high. Furthermore, considering the environmental issues surrounding us in the future, it is desirable to have a treatment method that not only disposes of marine life but also aims for effective utilization in some form. For this reason, technologies for efficiently processing marine products are being developed, and especially for marine organisms that regularly occur in large quantities, processing technologies are being developed including their effective use.

[0005] As a technique developed from the above-mentioned viewpoint, landed marine organisms are put into a treatment tank, and anaerobic microbial organisms that have been cultured in advance are injected into the tank. There is a technique of performing fermentation and decomposition treatment. This technique may be effective when treating small amounts of marine organisms. However, if a large amount of waste marine organisms are to be landed one after another, such as marine organisms that are removed from the intake channels of factories and power plants, a large-capacity treatment tank must be installed. There's a problem. In addition, when relatively large solids such as several millimeters to several centimeters are contained, such as marine organisms, the processing efficiency of the processing tank is low. further,
For example, in the case of shellfish, there are problems to be solved in order to achieve practical use, such as the separation of shells and shell meat takes about 5 days, and the liquefaction of shell meat requires a long period of about 1 to 2 months. Many.

[0006] Under such circumstances, it is necessary to develop a processing technique that can efficiently treat discarded marine organisms at low cost without generating a bad odor, and furthermore, with a view to effective use of shells and the like. It has been demanded.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a powerful marine biodegradation system for efficiently processing marine organisms without generating the offensive odor as described above. An object of the present invention is to provide a novel microorganism and a method for decomposing marine organisms using the microorganism.

[0008]

Means for Solving the Problems The present inventors have succeeded in isolating a marine biodegradable microorganism which can achieve the above object, and have accomplished the present invention.

The novel microorganism isolated according to the present invention is Bacillus sp. NA-
3: FERM P-17302), Pseudomonas fluorescens NA-11 (Pseudomonas fluolescens)
NA-11; FERM P-17303), Pseudomonas fluorescein NA-12 (Pseudomonas fluole)
scens NA-12; FERM P-17304), Enterobacter amnigeuus NA-34 (Enterobact
er amnigenus NA-34; FERM P-1730
5), and Ochrobactrum anthropi NA-43
(Ochrobactrum anthropi NA-43; FERMP
-17306).

The method for decomposing marine organisms according to the present invention comprises:
A first step of selecting a microorganism that can survive in an aqueous inorganic medium containing a marine organism from a microorganism source; and a marine biodegradability and 3% Na from the microorganism selected in the first step.
A second step of selecting a substance having a salt tolerance of not less than Cl, and a third step of decomposing the marine organism by bringing the microorganism obtained in the second step into contact with the marine organism to be treated. I do.

In a preferred method for decomposing marine organisms according to the present invention, microorganisms having marine organism resolution and salt tolerance of 3% NaCl or more are supported on a carrier in a culture tank and grown. Releasing the extracorporeal enzyme into the solution containing the extracorporeal enzyme obtained in the step,
The method includes a step of decomposing the marine organism by supplying the marine organism to a processing tank containing the marine organism, and a step of circulating the solution in the processing tank to the microorganism culturing tank.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The above-mentioned object is achieved by the present invention described below.

<Isolation method> As a result of searching for microorganisms that can efficiently degrade marine organisms, the present inventors have found from the soil in the Kanto region of Japan a strain that efficiently degrades marine organisms such as mussels and moon jellyfish. A method for decomposing the strain by contacting the strain with marine organisms was found. In the present invention, marine organisms may be shellfish, jellyfish, fish, seaweed, crustaceans, etc., but mainly include shellfish and jellyfish that cause problems when attached to an intake channel or sucked into an intake. I do.

In the actual treatment of marine organisms, if it is assumed that waste water is discharged or a carrier or the like in solid-phase cultivation is used as compost, a large amount of nutrients for the growth of degrading bacteria will be generated in the marine organism treatment field. It is easily assumed that it is difficult to add.

Therefore, a search was made for microorganisms capable of growing using marine organisms as the sole carbon source and decomposing marine organisms efficiently without generating bad odors.

That is, first, as the first stage selection, 0.3 g of soil in the Kanto region, 100 mL of distilled water, and 1 g of marine organisms
(Fresh weight) was added to the container and culturing was repeated to perform enrichment culturing. The microorganisms growing at this point
It was isolated by single colony isolation by a plate dilution method using LB agar medium.

As described above, as a second stage of selection, microorganisms capable of growing only on marine organisms were further selected based on the resolution of marine organisms. That is, 100 mL of an inorganic salt medium suspension containing microorganisms that can grow only in these marine organisms
Was added with 1 g of marine organism, and the decomposition activity of the marine organism was verified.
As a result, microorganisms having high marine biodegradation activity were selected. In the actual treatment, NaCl contained in marine organisms
In addition, since NaCl in the seawater is present in the treatment environment, the salt tolerance of these microorganisms having a high resolution of marine organisms was verified for the purpose of selecting microorganisms for marine organism treatment that can be practically used. As a result, the NaCl concentration was 3% (% by weight).
Microorganisms having the above-mentioned resistance to NaCl were selected as marine biodegradable microorganisms.

The composition of the LB agar medium is shown below.

LB agar medium (in 1 L of medium: pH 7) NaOH: 10 g Tryptone: 10 g Yeast extract: 5 g Agar: 15 g Using the strain isolated by the above operation, degradation of marine organisms in the same manner as the above test The test was conducted to determine the presence or absence of decomposition and the growth of the test culture based on the increase in the absorbance of the test medium. Successfully obtained five strains with high levels.

<Mycological properties> The mycological properties of the five strains newly obtained in the present invention are shown below.

First, Bacillus species NA-3
(Bacillus sp. NA-3; FERMP-17302)
It shows about.

A) Morphological properties etc. (1) Morphology: Bacillus b) Physiological properties (1) Gram staining: + (2) Oxidase:-(3) Production of hydrogen sulfide:-(4) Reduction of nitrate: + (5) Formation of indole:-(6) VP test:-(7) ONPG:-(8) Liquefaction of gelatin: + (9) Or (decarboxylation from ornithine):-(10) Ly (decarboxylation from lysine): -(11) Ar (decarboxylation from arginine): + (12) Availability glucose: + fructose: + mannose: + maltose: + trehalose: + mannitol:-xylitol:-inositol:-sorbitol:-rhamnose:-sucrose: -Melviose:-amygdalin:-arabinose:-From the above properties, it is clear that this strain belongs to the genus Bacillus, and since the corresponding species is not known, Bacillus sp. Has been found to be suitable for belonging to Bacillus sp.

Next, Pseudomonas fluorescens NA-11;
FERM P-17303).

A) Morphological properties etc. (1) Morphology: Bacillus b) Physiological properties (1) Gram staining:-(2) Oxidase: + (3) Pigment-producing ability: + (4) Production of hydrogen sulfide:- (5) Reduction of nitrate:-(6) Formation of indole:-(7) Utilization of citric acid:-(8) VP test:-(9) ONPG:-(10) Liquefaction of gelatin: + (11) Or (Decarboxylation from ornithine):-(12) Ly (decarboxylation from lysine):-(13) Ar (decarboxylation from arginine): + (14) Availability glucose: + fructose: + maltose:-galactose: + xylose: + mannitol : + Sucrose:-lactose:-esculin:-From the above properties, it was recognized that this strain was appropriate to belong to Pseudomonas fluolescens.

Next, Pseudomonas fluorescens NA-12;
FERM P-17304).

A) Morphological properties etc. (1) Morphology: Bacillus b) Physiological properties (1) Gram staining:-(2) Oxidase: + (3) Pigment-producing ability: + (4) Production of hydrogen sulfide:- (5) Reduction of nitrate:-(6) Formation of indole:-(7) Utilization of citric acid:-(8) VP test: + (9) ONPG:-(10) Liquefaction of gelatin: + (11) Or (Decarboxylation from ornithine):-(12) Ly (decarboxylation from lysine):-(13) Ar (decarboxylation from arginine): + (14) Availability glucose: + fructose: + maltose:-galactose: + xylose: + mannitol : + Sucrose:-lactose:-esculin:-From the above properties, it was recognized that this strain was appropriate to belong to Pseudomonas fluolescens.

Next, Enterobacter amnigenus NA-34;
ERM P-17305).

A) Morphological properties etc. (1) Morphology: Bacillus b) Physiological properties (1) Gram staining:-(2) Oxidase:-(3) β-galactosidase: + (4) α-galactosidase: + ( 5) ONPG: + (6) urease:-(7) Or (decarboxylation from lysine): + (8) Ly (decarboxylation from lysine):-(9) Ar (decarboxylation from arginine): + (10) availability glucose : + Sucrose:-L-arabinose: + D-arabinose:-L-arabitol:-D-maltose: + D-mannitol: + L-rhamnose: + inositol:-D-setobiose: + D-sorbitol:-adinit :-Palatinose:-D-galacturonic acid: + L-ornithine monohydrochloride: + L-arginine monohydrochloride: + Lysine hydrochloride:-Potassium 5-ketogluconate:- Non-acid 5-bromoindoxyl:-Sodium pyruvate: + p-nitrophenyl-β-D-glucoside: + p-nitrophenyl-β-D-glucocolloid:-Sodium malonate: + L-tryptophan: -5 -Bromo-4-chloro-3-indoxyl-N-acetyl-D
-Glucosamide:-p-nitrophenyl-β-D-galactopyranoside: + p-nitrophenyl-α-D-glucoside:-p-nitrophenyl-α-D-galactopyranoside: + trehalose: +4 -Nitrophenyl-α-D-maltoside: -L-aspartic acid-4-nitroanilide:-From the above-mentioned properties, it is appropriate that this strain belongs to Enterobacter amnigenus. Admitted.

Finally, Okrobactrum Anthropi N
A-43 (Ochrobactrum anthropi NA-43; FER
MP-17306).

A) Morphological properties etc. (1) Morphology: Bacillus b) Physiological properties (1) Gram staining:-(2) Oxidase: + (3) Availability glucose: + rhamnose: + N-acetylglucosamine: + D-ribose: + inosit: + L-fucose: + sucrose: + sorbitol: + maltose: + L-arabinose: + glycogen:-D-melbiose:-salicin: + itaconic acid:-propionic acid: + suberic acid: -Sodium malonate:-sodium acetate: + lactic acid: + 5-ketogluconic acid:-L-alanine: + m-hydroxybenzoic acid:-L-serine: + D-mannitol: + n-capric acid: + n-k Herbic acid: + sodium citrate: + L-histidine hydrochloride: + 2-ketogluconic acid: + 3-hydroxybutyric acid:-p-hydrid Carboxymethyl acid: + L-Proline: from + above various characteristics, the present strain was recognized as that occupied Zokuse to Ochrobactrum anthropi (Ochrobactrumanthropi) is suitable.

The five strains of the present invention are capable of efficiently decomposing marine organisms such as mussels and moon jellyfish, as is clear from the examples described later. The present inventors attempted to degrade marine organisms using the five strains of the type strain, but did not always obtain as good a degrading activity as the present strain, and thus identified the present strain as a new strain. Each of the new strains was transformed into Bacillus species NA-3
Strain (Bacillus sp. NA-3: FERM P-1730)
2), Pseudomonas fluorescens NA-11 strain (FERM P
-17303), Pseudomonas fluorescens NA
-12 strain (Pseudomonas fluolescens NA-12: FE
RM P-17304), Enterobacter amnigenus NA-34 strain (Enterobacter amnigenus NA-3).
4: FERM P-17305), Ochrobactrum
Anthropi NA-43 strain (Ochrobactrum anthropi NA
-43: FERM P-17306) and deposited with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry. Less than,
The above five strains were used for NA-3, NA-11, NA-12, NA
-34, NA-43.

<Culture conditions> As a nutrient source of a medium used for culturing the five strains of the present invention, any nutrient source that is necessary for the growth of ordinary microorganisms and can be assimilated by each strain is used. A carbon source, a nitrogen source and inorganic salts may be used,
For example, it is possible to culture in an LB medium.

The composition of the LB medium is shown below.

LB medium (1 L of medium: pH 7) NaOH: 10 g Tryptone: 10 g Yeast extract: 5 g Culture can be performed under aerobic conditions, and either liquid culture or solid culture may be used.

The cultivation temperature may be within a temperature range in which each strain can grow well, and it is desirable to culture within this range. For example, NA-11, NA-12 and NA-3
4 is preferably 20 ° C to 30 ° C, particularly around 25 ° C,
3 ° C to 27 ° C is preferred. NA-3 and NA-43
Is preferably 35 ° C to 45 ° C, particularly around 37 ° C,
C. to 39 C. are preferred.

The pH at the time of culturing may be within a range in which each strain can grow well. For example, NA-3, NA-11,
NA-12, NA-34 and NA-43 have a pH of 5.5 to 5.5.
8.5 is preferable, and pH 6 to 8 is particularly preferable.

It is desirable to set the load of marine organisms within a range in which each strain can perform marine organism treatment well. For example,
In the culture tank, 10 ⁸ CFU / mL to 10 ⁹ CFU /
When about mL exists, 0.2 t to 0.5 t of marine organism is added per 1 m ^{3 of} the culture tank.

As described above, the treatment temperature, the treatment pH, and the marine organism load with the five strains of the present invention are desirably within the range in which each strain can perform good marine organism treatment. In the case where marine organism treatment is performed using an enzyme or the like produced by culturing the five strains, the treatment environment of the marine organism is adjusted to a temperature, pH, at which the enzyme or the like exerts a good treatment effect.
It is desirable to perform the processing as follows. It is also desirable to set the load on the marine organism within a range where the enzyme or the like can perform a satisfactory treatment.

<Enzyme activity> The body of marine organisms, particularly the bodies of shellfish and jellyfish, is mostly composed of protein (10%) except for water and ash (85%), followed by carbohydrate (10%). 3%) and lipids (2%).

Then, the protease, amylase and lipase activities of the five strains of the present invention were examined. As a result, it was found that all the strains released the protease outside the body (Examples 1 to 5). , Table 1).
In particular, the NA-11 strain showed high protease activity.
On the other hand, the amylase activity shows a low value for all strains,
No activity was observed for lipase activity.

From these facts, these five strains mainly degrade proteins which are components of marine organisms by the action of proteases released outside the cells, that is, soluble proteases, and degrade marine organisms with high efficiency. It is considered possible. However, there was not always a clear correlation between the protease activity of the five strains and the effect of sea biodegradation. Therefore, the present inventors
It is thought that protease activity is necessary for marine biodegradation, but it cannot be explained by soluble protease activity alone. That is, a strain having merely a high soluble protease activity is not always suitable for marine biodegradation. The reason for this is not clear at present, but the existence of soluble proteases that are effective in degrading marine organisms, and that factors other than protease activity may be important factors in the efficient degradation of marine organisms. Conceivable. Alternatively, even when the normal protease activity is low, the enzyme activity is improved by the induction of the protease by a substance derived from marine organisms,
It is also possible that the processing activity of marine organisms will be high.

As described above, when treating marine organisms that live in the sea, it is easily presumed that some seawater is mixed in with the marine organisms landed at the treatment site. In addition, NaCl contained in marine organisms will also be brought to the treatment site. For this reason, it was presumed that the marine organism-treated microorganisms required some degree of salt tolerance.

Accordingly, the sea-biodegradable microorganism of the present invention was evaluated for salt tolerance at the selection stage. To have salt resistance,
In a medium containing a certain salt concentration (3% NaCl), Na
This means that the cells can be grown by drawing the same growth curve as in the absence of Cl. All five strains of the present invention were salt-tolerant in the presence of 3% NaCl (see Table 1). By having salt tolerance, the strain of the present invention can exhibit marine biodegradability under the same environment in which marine organisms grow. That is, the strain of the present invention can be directly introduced into seawater containing marine organisms to perform a decomposition treatment of marine organisms.

<Method of Decomposing Marine Organism> In the process of decomposing marine organisms in the present invention, the medium containing marine organisms may be, for example, one of the above-mentioned NA-3, NA-11, NA-12, NA-34, NA
It is characterized by being brought into contact with a microorganism having marine life resolution such as -43. Contact between the microorganism and the marine organism can be performed by a method of culturing the microorganism in a medium containing the marine organism, a method of adding the medium containing the marine organism to the culture system of the microorganism, or the like. It can be carried out using various methods such as a continuous method and a continuous method. The microorganism may be used in a semi-solid state or immobilized on a suitable carrier. Further, it is desirable to perform various pretreatments such as crushing treatment of marine organisms and to adjust and control the treatment environment, because treatment efficiency can be expected to be improved.
The adjustment of the treatment environment includes, for example,
By controlling the values of the pH in the treatment medium, the replenishment of various nutrients, and the like within a range in which the microorganisms to be used can most efficiently treat marine organisms, highly efficient treatment of marine organisms becomes possible.

The degradation treatment method of the present invention is not limited to the above five types of strains, and the same effect can be obtained by using other strains obtained by the above-mentioned isolation method. it can.

In order to explain the contents of the present invention, specific examples of the mode of decomposing marine organisms will be described below, but the present invention is not limited to these modes.

Example 1 As a method for treating a marine organism in the present invention, a microorganism having marine biodegradability, such as the five strains of the present invention, is cultured in a liquid medium by providing a culture tank. Next, a mode in which microorganisms and marine organisms are brought into contact with each other to be decomposed by introducing marine organisms into the culture tank is exemplified. Alternatively, there is a form in which a cultured microorganism having the ability to degrade a marine organism is added to a treatment tank to which a marine organism is added, and the microorganism is brought into contact with the marine organism to decompose the microorganism.

As a method for adding microorganisms having the ability to degrade marine organisms, any method can be used as long as microorganisms can be efficiently added. For example, various methods such as a method of adding a microorganism preparation such as a microorganism or a freeze-dried product of the microorganism to the reaction tank and a method of adding a culture solution of the microorganism can be appropriately selected and carried out.

There is no particular limitation on the method of introducing marine organisms, but from the viewpoint of increasing the efficiency of contact with microorganisms and enzymes produced by the microorganisms and improving the processing efficiency, marine organisms are finely crushed and then treated in a treatment tank. Alternatively, it is preferable to introduce into a culture tank. The introduction of marine organisms may be carried out continuously, but it is also possible to carry out the treatment intermittently or batchwise according to the treatment capacity. It is good to control such a control as a system according to the concentration of marine organisms, and to optimize it.

In order to carry out the above-mentioned marine organism treatment method, for example, a marine organism treatment apparatus shown in FIG. 1 can be used.

In the apparatus shown in FIG. 1, the microorganisms used are:
It is cultured in the marine biodegradable microorganism culture tank 3. The temperature of the microbial culture tank 3 is controlled by a heating device 8, and the culture solution in the culture tank is stirred by a stirring blade 5 driven by a stirring motor 6. On the other hand, the marine organisms crushed by the marine organism crusher 1 are transported to the marine organism treatment tank 2 by the marine organism transport belt conveyor 7. The marine biological treatment tank 2 also includes a heating device 8
The content in the processing tank is agitated by a stirring blade 5 driven by a stirring motor 6. By adding the culture solution in the microorganism culture tank 3 to the marine organism treatment tank 2 by the liquid sending pump 4, contact between microorganisms and marine organisms can be caused. Here, marine organisms are decomposed by microorganisms, and the liquid after the decomposition processing is discharged from the processing tank 2 as waste liquid.

Example 2 It is desirable that microorganisms be immobilized on a suitable carrier to prevent outflow from the reaction vessel. By holding microorganisms on a carrier, the concentration of microorganisms can be improved, and at the same time, survival of microorganisms derived from marine organisms and microorganisms derived from the outside world can be protected, and useful microorganisms can be protected from predators, etc. Becomes possible. As the carrier, any carrier can be used as long as it can hold microorganisms to be used, for example, inorganic materials such as ceramics, glass, calcium silicate, silica, alumina, and inorganic particles such as soil particles having an aggregated structure such as Kanuma soil, and the like. , Activated carbon, fiber activated carbon, urethane foam, photocurable resin, ion exchange resin,
Organic materials such as cellulose, lignin, chitin, chitosan, polyvinyl alcohol polymer, superabsorbent resin and superabsorbent resin, and porous bodies composed of them, and hydrophilic properties such as sodium alginate gel, carrageenan gel, agar, acrylamide gel A gel, a cloth such as a nonwoven fabric, a fibrous material, or the like can be used alone or in combination of two or more. The carrier is a microorganism used,
It is desirable to select materials, forms, and functions that exhibit good marine biodegradation efficiency.

In addition, when a component to be released from the microorganism such as an enzyme released by the microorganism used in the treatment is effective in decomposing the marine organism, a tank for culturing the microorganism and a tank for treating the marine organism are separately provided. It is also possible to install and circulate the liquid medium between them to perform the treatment. The above-mentioned treatment method can efficiently cope with the case where the culture condition of the microorganism and the treatment condition of the marine organism are significantly different.

In order to carry out the above-mentioned marine organism treatment method, for example, a marine organism treatment apparatus shown in FIG. 7 can be used.

In the apparatus shown in FIG. 7, the microorganisms used are:
The cells are cultured in the immobilized microorganism packed tower 13. The temperature of the immobilized microorganism packed column 13 is controlled by a heating device 8. In the microorganism packed tower, microorganisms are immobilized on a carrier. The inside of the microorganism packed tower 13 is set to a culture condition preferable for the microorganism to release the extracorporeal enzyme, and the microorganism releases the extracorporeal enzyme efficiently. On the other hand, marine organisms crushed by the marine organism crusher 1 are transported to the marine organism treatment tank 2 by the marine organism transport belt conveyor 7. The liquid in the marine organism treatment tank 2 can be placed under conditions exposed to the outside temperature, but the temperature can be controlled by the heating device 8 as needed. The liquid in the marine organism treatment tank 2 is stirred by a stirring blade 5 driven by a stirring motor 6. By adding the culture solution in the immobilized microorganism packed tower 13 to the marine organism treatment tank 2 by the liquid sending pump 4,
Contact between marine organisms and microbial extracellular release enzymes can occur. Here, the marine organism is decomposed by the release enzyme, and the liquid after the decomposition is returned to the microorganism packed tower 13 again. As described above, by circulating the liquid between the microorganism packed tower 13 and the marine organism treatment tank 2, the marine organism is decomposed.

The waste liquid discharged by the marine organism treatment according to the present invention can be subjected to various treatments according to its properties, and can be reused and treated as reclaimed water or effluent water. As a method of treating the waste liquid, any method can be used as long as it can effectively treat the waste liquid, and examples thereof include a treatment with an anaerobic microorganism, a treatment with activated sludge, and a treatment with supercritical water. In addition, by controlling various conditions at the time of decomposition, it is possible not only to completely decompose marine organisms, but also to recover organic components derived from marine organisms contained in the treated medium as fertilizer etc. Becomes Furthermore, among shellfishes among marine organisms to be treated, shells and shell meat can be easily separated according to the present invention, so that shells can be recovered from the shellfish and reused as a Ca raw material. Become.

Example 3 Another form of the method for treating marine organisms according to the present invention is to mix marine organisms, a water conditioner, and a solid medium acting as a carrier for microorganisms in a reaction vessel, to which marine organisms can be decomposed. There is a form in which a decomposition treatment is performed by adding a microorganism and bringing the microorganism into solid phase contact with the marine organism. Decomposition efficiency can be improved by constantly or intermittently stirring the solid medium for aeration and fermentation heat dissipation. Also,
By controlling the oxygen concentration and water conditions in the reaction tank, the pH of the medium, and the like, the reaction can be efficiently advanced. It is desirable that these conditions be controlled within a range in which microorganisms to be used can treat marine organisms with high efficiency.

As a medium to be used, a medium having excellent water retention and having an ability to retain microorganisms is desirable. For example, sawdust, coffee grounds, vegetable medium such as okara, and, if somewhat less water-retaining, may have aggregate structures such as ceramics, glass, calcium silicate, silica, alumina, and Kanuma soil. Inorganic materials such as soil particles and their porous bodies, and activated carbon, fiber activated carbon, urethane foam, photocurable resin, ion exchange resin, cellulose, lignin, chitin, chitosan, polyvinyl alcohol polymer,
One or a combination of two or more of an organic material such as a superabsorbent resin and a porous body made of the same, and a cloth and a fibrous material such as a nonwoven fabric can be used. It is also effective to use a carrier made of a non-absorbable material as a spacer together with these carriers.

In order to carry out the above-mentioned marine life treatment method, for example, a marine life treatment apparatus shown in FIG. 13 can be used.

In the apparatus shown in FIG. 13, a solid medium 21,
Introduce marine organisms, microorganisms and water conditioning materials. The temperature in the processing tank is determined by a fan, a sensor 24, etc.
Is controlled by The microorganism to be used is added as a freeze-dried microbial preparation and brought into solid phase contact with a marine organism. The mixture in the processing tank is conveyed toward the medium outlet 25 while being stirred by the screw conveyor 22 in the processing tank. During the stirring and transport, the microorganism is subjected to a decomposition treatment of the marine organism. In addition, the agitation of the medium in the processing tank is promoted by this stirring, so that the microorganism treatment can be performed efficiently. While being decomposed, the marine organisms are sequentially sent to the medium outlet 25 and discharged.

When the carrier or the like is a biodegradable substance, the mixture in the reaction tank thus treated is not only treated as waste but also effectively used as compost or the like. It is also possible.

[0062]

EXAMPLES Hereinafter, preferred examples of the present invention will be described based on experimental results, but these do not limit the present invention in any way.

<Example 1> Bacillus species NA-3, a strain of the present invention, was evaluated for protease activity, amylase activity, and lipase activity. In addition, the salt resistance was evaluated.

After inoculating one loopful of NA-3 forming a colony on the LB agar medium into the LB liquid medium,
Cultured for days. Protease activity and amylase activity were evaluated by the azocasein method and the Bernfeld reducing sugar quantification method (dinitrile acetic acid method), respectively, using the medium after the cultivation as a sterile filtered enzyme solution. In addition, the lipase activity is NA-
3 was inoculated into a lipase activity measurement medium, and the activity was evaluated based on the presence or absence of a halo. The composition of the medium for measuring lipase activity is shown below.

A medium for measuring lipase activity Olive oil 1.0% Bile powder 1.0% Peptone 1.0% Yeast extract 0.5% NaCl 3.5% Agar 2.0% NA-3 was inoculated into the LB liquid medium adjusted to the concentration, and the wavelength 6 after 50 hours of culturing.
The observation was performed by observing the growth of the bacterial cells using the absorbance of light at 60 nm as an index. Table 1 shows the results of the enzyme activity and salt tolerance evaluation.

[0066]

[Table 1]

Example 2 The same test as in Example 1 was carried out for the strain of the present invention, Pseudomonas fluorescens NA-11. Table 1 shows the results.

Example 3 The same test as in Example 1 was carried out on the strain of the present invention, Pseudomonas fluorescens NA-12. Table 1 shows the results.

Example 4 The same test as in Example 1 was conducted for the strain of the present invention, Enterobacter amnigaeus NA-34. Table 1 shows the results.

Example 5 The same test as in Example 1 was conducted for the strain of the present invention, Ochrobactrum anthropi NA-43. Table 1 shows the results.

Example 6 An apparatus as shown in FIG. 1 was prepared, and a test for decomposing marine organisms was performed.

A culture solution in the logarithmic growth phase of NA-3 (the medium is L
B medium) 3m^ThreeCrushed mussels as sea life
1.5 t was added, and the cells were cultured with stirring for 24 hours. Attachment
The crushed sea microorganisms added did not include shells and the like.
The cell concentration of the culture solution is about 10 ⁸Adjust to CFU / mL
Was.

At 12 hours and 24 hours after the start of the culture,
One liter of the culture solution was filtered through a 50 μm nylon mesh, and the weight of the residue on the mesh was measured. The result is shown in FIG.

FIG. 2 shows the weight (%) of the residue of the mussel after 12 hours and 24 hours, relative to the initial value (the weight at the time of introduction). The measurement of the initial value
The same operation as that for measuring the weight of the residue after 12 hours and 24 hours was performed by filtering the purple mussel at the time of feeding with a mesh and measuring the weight of the residue on the mesh.

The presence or absence of malodor during the test was determined by a sensory test at the time of sampling. Table 2 shows the results of the sensory test after 12 hours.

[0076]

[Table 2]

Sampling at 12 hours and 24 hours after the start of the culture was performed at n = 3, respectively, and the test was repeated three times.

Example 7 The same test as in Example 6 was performed on the strain NA-11 of the present invention. Table 2 shows the results.
And FIG.

Example 8 The same test as in Example 6 was performed on the strain NA-12 of the present invention. Table 2 shows the results.
And FIG.

Example 9 The same test as in Example 6 was performed on the strain NA-34 of the present invention. Table 2 shows the results.
And FIG.

Example 10 The same test as in Example 6 was performed on the strain NA-43 of the present invention. The results are shown in Table 2 and FIG.

Example 11 An apparatus as shown in FIG. 7 was prepared, and a test for decomposing marine organisms was performed.

The immobilized NA-3 on the chitosan porous carrier was filled in a microorganism packed tower 13, and water was circulated between the marine organism treatment tank 2 and the microorganism packed tower 13. The microorganism packed tower 13 was kept at about 37 ° C., and the seafood treatment tank 2 was treated under the outside air temperature condition.

To the marine organism treatment tank 2, 1.5 tons of crushed mussels as marine organisms were added for treatment. The crushed marine organism contained 20% of shells by wet weight. The water in the treatment tank was sampled periodically (every 4 hours until after 24 hours), filtered through a 50 μm nylon mesh, and the weight of the residue was measured. FIG. 8 shows the result.
The results were evaluated for the weight excluding the weight of shells and the like.
That is, the results in FIG. 8 show that both the initial value and the weight of the residue are the weight of the mussel shell (20% wet weight).
Is the value excluding.

The presence or absence of odor during the test was determined by a sensory test at the time of sampling. Table 2 shows the results of the sensory test after 12 hours.

The sampling was performed with n = 3, and the test was repeated three times.

<Example 12> The same test as in Example 11 was carried out on the strain NA-11 of the present invention except that the microorganism-filled tower was kept at 25 ° C. Table 2 and FIG.
Shown in

Example 13 The same test as in Example 11 was carried out on the strain NA-12 of the present invention except that the microorganism-filled tower was kept at 25 ° C. Table 2 and FIG.
0 is shown.

Example 14 The same test as in Example 11 was carried out on the strain NA-34 of the present invention except that the microorganism-filled tower was kept at 25 ° C. Table 2 and FIG.
It is shown in FIG.

Example 15 The same test as in Example 11 was performed on the strain NA-43 of the present invention. The results are shown in Table 2 and FIG.

Example 16 An apparatus as shown in FIG. 13 was prepared, and a test for decomposing marine organisms was performed. The inner volume of the processing tank is 2 m ³ .

As the solid medium 21, cedar sawdust 1.2
m ³ was used. NA-3 was used as a microbial preparation by culturing in an LB medium and freeze-drying in a conventional manner.
Three days prior to marine treatment, 10 ⁸ CFU of the above microbial preparation
/ G medium, and the inside of the processing tank is
The water content of the medium was controlled at 45 ± 10%, and the temperature was controlled at about 37 ° C. The stirring of the medium is performed by the screw conveyor 2 inside the processing tank.
2 and intermittently.

[0093] The introduction of marine organisms (mussels) was carried out at a rate of 120 kg per day. Temperature control after sea life input
The test was performed so that the temperature did not fall below 37 ° C. Twenty-four hours after the introduction of the marine organism, 1 L of the medium was sampled, and the presence or absence of decomposition of the marine organism was visually observed and evaluated. Table 3 shows the results.

[0094]

[Table 3]

Example 17 The same test as in Example 16 was carried out on the strain NA-11 of the present invention except that the temperature in the treatment tank was controlled at 25 ° C. Table 3 shows the results.

Example 18 The same test as in Example 16 was carried out on the strain NA-12 of the present invention except that the temperature in the treatment tank was controlled at 25 ° C. Table 3 shows the results.

Example 19 The same test as in Example 16 was carried out on the strain NA-34 of the present invention except that the temperature in the treatment tank was controlled at 25 ° C. Table 3 shows the results.

Example 20 The same test as in Example 16 was carried out on the strain NA-43 of the present invention. Table 3 shows the results.

Example 21 The same test as in Example 16 was conducted, except that a urethane foam cylindrical tip was used as a medium. Table 3 shows the results.

Example 22 The same test as in Example 16 was carried out, except that a hollow network-shaped molded product of polypropylene and coffee grounds were used as the medium. Table 3 shows the results.

<Example 23> A test similar to that in Example 6 was performed except that moon jellyfish was used as a marine organism, and the results are shown in Table 2 and Fig. 14.

Example 24 A test was conducted in the same manner as in Example 7 except that moon jellyfish was used as a marine organism. The results are shown in Table 2 and FIG.

<Example 25> A test similar to that in Example 8 was carried out except that moon jellyfish was used as a marine organism, and the results are shown in Table 2 and FIG.

<Example 26> The same test as in Example 9 was carried out except that moon jellyfish was used as a marine organism, and the results are shown in Table 2 and Fig. 17.

<Example 27> The same test as in Example 10 was carried out except that moon jellyfish was used as the marine life, and the results are shown in Table 2 and FIG.

<Example 28> A test similar to that in Example 11 was carried out except that moon jellyfish was used as a marine organism, and the results are shown in Table 2 and Fig. 19.

<Example 29> A test similar to that in Example 12 was performed, except that moon jellyfish was used as a marine organism. The results are shown in Table 2 and FIG.

<Example 30> The same test as in Example 13 was carried out except that moon jellyfish was used as the marine life, and the results are shown in Table 2 and Fig. 21.

<Example 31> A test similar to that of Example 14 was carried out except that moon jellyfish was used as a marine organism, and the results are shown in Table 2 and FIG.

<Example 32> A test similar to that of Example 15 was performed except that moon jellyfish was used as a marine organism, and the results are shown in Table 2 and Fig. 23.

Example 33 A test was conducted in the same manner as in Example 16 except that moon jellyfish was used as a marine organism, and the results are shown in Table 3.

Example 34 A test was conducted in the same manner as in Example 17 except that moon jellyfish was used as a marine organism, and the results are shown in Table 3.

Example 35 A test was conducted in the same manner as in Example 18 except that moon jellyfish was used as a marine organism. Table 3 shows the results.

Example 36 A test was conducted in the same manner as in Example 19, except that moon jellyfish was used as a marine organism. Table 3 shows the results.

<Example 37> A test similar to that in Example 20 was carried out except that moon jellyfish was used as a marine organism, and the results are shown in Table 3.

The following is a comparative example in which marine organisms were degraded using the five strains of the present invention.

<Comparative Example 1> The same test as in Example 6 was carried out, except that Bacillus subtilis type strain (ATCC6051) was used as a microorganism to be used. The results are shown in Table 2 and FIG.

Comparative Example 2 Pseudomonas fluolescen was used as a microorganism.
The same tests as in Examples 7 and 8 were performed except that the type strain (ATCC13525) of s) was used. The results are shown in Table 2 and FIG.

Comparative Example 3 As a microorganism to be used, Enterobacter amnigenu
A test similar to that of Example 9 was performed, except that the type strain (s) (ATCC 33072) was used. The results are shown in Table 2 and FIG.

Comparative Example 4 As a microorganism to be used, Ochrobactrum anthropi was used.
The same test as in Example 10 was performed except that the type strain (ATCC49188) was used. The results are shown in Table 2 and FIG.

Comparative Example 5 Example 16 was repeated except that Bacillus subtilis type strain (ATCC6051) was used as the microorganism to be used.
The same test was performed. Table 3 shows the results.

Comparative Example 6 Pseudomonas fluolescen was used as a microorganism.
The same tests as in Examples 17 and 18 were performed except that the type strain (ATCC13525) of s) was used. Table 3 shows the results.

Comparative Example 7 As the microorganism to be used, Enterobacter amnigenu
The same test as in Example 19 was performed, except that the type strain (ATCC33072) of s) was used. Table 3 shows the results.

Comparative Example 8 As a microorganism to be used, Ochrobactrum anthropi was used.
The same test as in Example 20 was performed, except that the type strain (ATCC49188) was used. Table 3 shows the results.

<Comparative Example 9> The same test as in Comparative Example 1 was carried out except that moon jellyfish was used as a marine organism, and the results are shown in FIG.

<Comparative Example 10> The same test as in Comparative Example 2 was carried out except that moon jellyfish was used as a marine organism, and the results are shown in FIG.

<Comparative Example 11> The same test as in Comparative Example 3 was carried out except that moon jellyfish was used as a marine organism, and the results are shown in FIG.

<Comparative Example 12> The same test as in Comparative Example 4 was carried out except that moon jellyfish was used as a marine organism, and the results are shown in FIG.

<Comparative Example 13> The same test as in Comparative Example 5 was carried out except that moon jellyfish was used as a marine organism, and the results are shown in Table 3.

<Comparative Example 14> A test was conducted in the same manner as in Comparative Example 6 except that moon jellyfish was used as a marine organism. Table 3 shows the results.

<Comparative Example 15> A test similar to that of Comparative Example 7 was carried out except that moon jellyfish was used as a marine organism, and the results are shown in Table 3.

<Comparative Example 16> The same test as in Comparative Example 8 was carried out except that moon jellyfish was used as a marine organism, and the results are shown in Table 3.

<Experimental Results> From the results of Examples 1 to 5, it can be easily understood that the novel microorganism of the present invention has a high protease activity and has a tolerance at a salt concentration up to about seawater. However, the results of Examples 1 to 5 and Example 6
As is clear from the results of Comparative Examples 10 to 10 and Examples 23 to 27, the degradation treatment of marine organisms requires that the microorganism used has a certain level of protease activity, but only the protease activity necessarily has the processing ability. It can be understood that it is not determined uniquely. As described above, this is considered to be due to the presence of a protease effective for treating marine organisms or some other factor, or to a substance derived from marine organisms.

Further, Examples 6 to 15 and Examples 23 to
As is clear from the comparison between the results of Comparative Example 32 and the results of Comparative Examples 1 to 4 and Comparative Examples 9 to 12, the microorganism having the ability to degrade marine organisms of the present invention is very effective in treating marine organisms. Easy to understand. In addition, it can be easily understood that the use of the treatment method according to the present invention enables effective treatment of marine organisms in the liquid layer medium with little input energy and without generating offensive odor.

Further, Examples 16 to 22 and Example 33
To 37, Comparative Examples 5 to 8 and Comparative Examples 13 to 16
As is clear from the comparison of the results, it is easily understood that the treatment method of the present invention can efficiently treat marine organisms even in a solid phase medium.

[0136]

As described above, the novel marine biodegradable bacterium Bacillus sp. NA-3 (Bacillus sp.) Of the present invention is described.
sp. NA-3: FERM P-17302), Pseudomonas fluorescens NA-11 (Pseudomonas fl
uolescens NA-11: FERM P-17303),
Pseudomonas fluorescens NA-12 (Pseudomo
nas fluolescens NA-11: FERM P-1730
4), Enterobacter amnigeus (Enterobacter
amnigenus NA-34: FERM P-1730
5), and Ochrobac anthropi (Ochrobac
trum anthropi NA-43: FERM P-1730
If 6) is used, marine organisms can be efficiently decomposed. In addition, the method for treating marine organisms according to the present invention does not generate a bad odor, is extremely economical, and has an extremely large industrial value.

[Brief description of the drawings]

FIG. 1 is a schematic view of an apparatus used in Examples 6 to 10 and Examples 23 to 27.

FIG. 2 is a view showing preferable results in Example 6.

FIG. 3 is a view showing preferable results in Example 7.

FIG. 4 is a view showing preferable results in Example 8.

FIG. 5 is a view showing preferable results in Example 9.

FIG. 6 is a view showing preferable results in Example 10.

FIG. 7 is a schematic view of an apparatus used in Examples 11 to 15 and Examples 28 to 32.

FIG. 8 is a view showing preferable results in Example 11.

FIG. 9 is a view showing preferable results in Example 12.

FIG. 10 is a view showing preferable results in Example 13.

FIG. 11 is a view showing preferable results in Example 14.

FIG. 12 is a view showing preferable results in Example 15.

FIG. 13 is a schematic view of an apparatus used in Examples 16 to 22 and Examples 33 to 37.

FIG. 14 is a view showing preferable results in Example 23.

FIG. 15 is a view showing preferable results in Example 24.

FIG. 16 is a view showing preferable results in Example 25.

FIG. 17 is a view showing preferable results in Example 26.

FIG. 18 is a view showing preferable results in Example 27.

FIG. 19 is a view showing preferable results in Example 28.

FIG. 20 is a view showing preferable results in Example 29.

FIG. 21 is a view showing preferable results in Example 30.

FIG. 22 is a view showing preferable results in Example 31.

FIG. 23 is a view showing preferable results in Example 32.

FIG. 24 is a view showing preferable results in Comparative Example 1.

FIG. 25 is a view showing preferable results in Comparative Example 2.

FIG. 26 is a view showing preferable results in Comparative Example 3.

FIG. 27 is a view showing preferable results in Comparative Example 4.

FIG. 28 is a view showing preferable results in Comparative Example 9.

FIG. 29 is a view showing preferable results in Comparative Example 10.

FIG. 30 is a view showing preferable results in Comparative Example 11.

FIG. 31 is a view showing preferable results in Comparative Example 12.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Sea creature crusher 2 ... Sea creature treatment tank 3 ... Sea creature decomposing microorganism cultivation tub 4 ... Liquid feed pump 5 ... Stirring blade 6 ... Stirring motor 7 ... Sea creature conveyor belt 8 ... Heating device 13 ... Fixed Packing tower for immobilized microorganisms 21 ... Solid medium 22 ... Screw conveyor 23 ... Medium, sea organism input / sampling port 24 ... Fan, sensor, etc. 25 ... Media outlet 26 ... Heating device

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:07) (C12N 1/20 C12R 1:39) (C12N 1/20 C12R 1:01) F term (Reference) 4B029 AA02 AA21 BB01 BB02 BB16 CC01 CC02 CC03 CC10 CC13 DA04 DA06 DA07 DA10 DB01 DD02 DF01 DF05 DG06 DG08 4B033 NA01 NA12 NB12 NB22 NB32 NB49 NB68 NC04 ND04 ND15 ND20 4B031A22 BC15 AB12ACB BCX BC42 BD42 BD43 BD50 CA27 CA31 CA32 CA33 CA41 CA49 CA55

Claims (4)

[Claims] 1. Bacillus species NA-3 (Baci
llus sp. NA-3; FERM P-17302), Pseudomonas fluorescens NA-11 (Pseudomona
s fluolescens NA-11; FERM P-1730
3), Pseudomonas fluorescens NA-12 (Ps
eudomonas fluolescens NA-12; FERM P-
17304), Enterobacter amnigueus NA-
34 (Enterobacter amnigenus NA-34; FERM
P-17305), Ochrobactrum anthropi N
A-43 (Ochrobactrum anthropi NA-43; FE
RM P-17306). A novel microorganism selected from the group consisting of: 2. A first step of selecting a microorganism that can survive in an aqueous inorganic medium containing a marine organism from a source of microorganisms; A second step of selecting a substance having a salt tolerance of not less than% NaCl, and a third step of contacting the microorganism obtained in the second step with a marine organism to be treated to decompose the marine organism. A method for decomposing marine organisms, comprising: 3. The microorganism obtained in the second step is Bacillus sp. NA-3 (Bacillus sp.
NA-3; FERM P-17302), Pseudomonas fluorescens NA-11 (Pseudomonas fluolesc)
ens NA-11; FERM P-17303), Pseudomonas fluorescens NA-12 (Pseudomonas
fluolescens NA-12; FERM P-1730
4), Enterobacter amnigueus NA-34 (En
terobacter amnigenus NA-34; FERM P-1
7305), Ochrobactrum anthropi NA-43
(Ochrobactrum anthropi NA-43; FERMP
The method according to claim 2, wherein the microorganism is selected from the group consisting of: (-17306). 4. A process in which the microorganism having the ability to degrade marine organisms is supported on a carrier in a culture vessel and grown, and an extracorporeal enzyme is released from the microorganism into the culture vessel. And supplying a solution containing the marine organism to a treatment tank containing the marine organism to decompose the marine organism; and circulating the solution in the treatment tank to the microorganism culturing tank. Item 4. The method for decomposing marine organisms according to Item 3.