JPS6018480B2 - freshwater purifier - Google Patents

Description

[Detailed description of the invention] The present invention relates to a freshwater purifying agent.

More specifically, the present invention relates to a novel freshwater purifying agent containing one or more non-pathogenic microorganisms. Water in swimming pools, water tanks, fire tanks, cooling tanks, reservoirs, fountain ponds, irrigation ponds, etc. is prone to spoilage, so the water must be changed at regular intervals, fresh water occasionally poured, and sterilized. Measures are being taken to add detergents and other cleaning agents.

However, the method of replacing or pouring water requires extremely heavy labor, and the method of using purification agents, although simple, is a harmful substance and causes pollution. In terms of health and safety,
There are a number of restrictions on its use. For this reason, the development of harmless purifying agents is eagerly awaited.

In addition, various freshwater fish and other seafood are raised in these ponds and tanks for both water purification and aquaculture purposes, but these fish and shellfish are harmless and have no purifying effect. At present, no purifying agent with a high
In addition, in aquariums, glass tanks, glass bowls, breeding containers, etc., large quantities or small numbers of fish and shellfish such as edible fish, ornamental fish, bred observation fish, bred experimental fish, and shellfish, shrimp, and crabs are bred. However, when kept in these artificial environments for long periods of time, a food chain is formed between the fish and shellfish, plants, and microorganisms in the water, and unlike in natural environments, where the fish and shellfish have a self-purifying effect, the fish and shellfish 8E Pond fish and feed residues accumulate and rot, polluting the water and producing toxic substances.If left untreated, fish and shellfish may become ill or their growth may be stunted. It may come to a halt or even lead to the foundation dying. Therefore, it is not only necessary to be careful about the amount of food fed, but also to change the water frequently within a short period of time. However, as mentioned above, even in the case of small-capacity tanks and pots, the task of replacing water is complicated. Furthermore, when you replace the water that the fish and shellfish have become accustomed to, the water temperature, amount of dissolved oxygen, and composition of ingredients in the water become slightly different, which can cause the fish and shellfish to stop growing.
It is undeniable that activity will slow down. As a result, the growth of fish and shellfish decreases, fish diseases occur due to a decline in physical strength, the number of crabs in farmed fish and shellfish decreases, and even large numbers of fish and shellfish die in bags. be hindered. Further, if dirty used water is disposed of, it will further cause secondary pollution, resulting in pollution problems. The present invention solves these shortcomings all at once and develops a new purifying agent that has an extremely excellent water purifying effect, does not have the expected effect on fish and shellfish, and whose effect lasts for a long period of time. It was made for the purpose of As a result of conducting research from various angles to achieve this goal, we obtained new knowledge that using chemical substances would not achieve the intended purpose, but that good results could be obtained by using microorganisms. Further research based on this basic idea led to the administration of one or a combination of two or more bacteria that decompose the respiratory effects of fish and shellfish and inorganic or organic nitrogen compounds such as ammonia and nitrite ions in wastewater. It was found that it has a remarkable purifying effect.

Therefore, we screened and selected a large number of microorganisms,
Further examination of these combinations revealed that the above-mentioned inorganic or organic nitrogen compound-degrading bacteria are non-pathogenic saprophytes, cellulose-degrading bacteria and fat-degrading bacteria that decompose cellulose, fats and oils, etc. contained in feed and their residues. It was discovered that combining a large number of silks with silk has an even more surprising purifying effect, and that it is useful not only for purifying fresh water, but also for raising seafood and preventing diseases, leading to the present invention. That is, the gist of the present invention is one of the bacteria selected from the group consisting of Bacillus subtilis, Bacillus thuringiensis, Arthrobacter azilis, Asbergillus terreus, Bacteroides succinogenes, Bacteroides lipolyticum, and Brevibacterium lipolyticum. A freshwater purifying agent characterized by containing the above. The bacteria contained in the freshwater purifying agent of the present invention are classified into three groups: inorganic or organic nitrogen compound, cellulose-degrading bacteria, and fat-degrading bacteria, and the bacteria belonging to each group are as follows. Inorganic or organic nitrogen compound degrading bacteria {1) Pseudomonas denitrificans (Pseu
domonasdenitrmcans) (Commonwealth Research Institute No. 6121) '2} Bacillus subtilis (European cmuss costilis) (Commonwealth Research Institute No. 6114) '3} Bacillus thuringiensis (mother cmusthuringensis) 6115) ■ Arthrobacter azilis (~throcteragilis) (Central Research Institute No. 6113) {5} Kurthiazopfii (Central Research Institute No. 611) Cellulose-degrading bacteria [6' Bacteroides succinogenes (mother cteroides succlnogenes) of Asbergillus terreus (female)
Chokoken Bacteria No. 6117) Oil-degrading bacteria '8) / Bacteroides lipolyteicum
oideslipolytic mm)
No. 116) {91 Brevibacterium lipolypole ICU
m) (Choken Bacteria No. 6118) {10 Nocardia
Coralina (NMardiacoralljna) (Choken Bacteria Collection No. 612) Among the above-mentioned bacteria, ``1'' Pseumonas denitrificans, ``5'' Kurtia zophii, and {IQ Nocardia corallina are known bacteria.

・Ps. Denitrificans is a bacterium isolated from soil, and is an aerobic and aerobic bacterium.

B. Zuptilus and B. thuringiensis is a bacterium isolated from soil, is an aerobic bacterium, and has spore-forming ability. However, the latter fungus does not have the property of causing disease in insects. N. Coralina is an aerobic bacterium and has the ability to form chlamydospores. Many hydrocarbons, fats and oils, and phenols can be used as carbon sources. Hair ground squirrel is a polymorphic aerobic bacterium and has a wide temperature range. In addition to organic nitrogen compounds, a large number of hydrocarbons can be used as carbon sources. Bacteroides succinogenes is an anaerobic bacterium isolated from the rumen of cows. Bacteroides lipolyticum is a halophilic aerobic bacterium. Br
ev. Lipolyteicum is a bacterium isolated from a whey digestion plant, is an aerobic and facultative aerobe, and has a wide temperature range. And it can quickly break down fat. K. Zoffii was isolated from the decomposition product of chicken manure, and is an aerobic and aerobic bacterium.
Asp. Terreus is an aerobic filamentous fungus that has spore-forming ability. The above-mentioned m~[41 and blood bacteria can be cultured in a large capacity fermentation tank made of stainless steel.

The medium composition was 3% soybean meal, 1.0% sea flour, and 0.0% ammonium sulfate.
2%, calcium carbonate 0.3%, and the pH is adjusted to 7.0 before sterilizing the medium. Bacteria are grown by aeration culture at 30 qo.

The above-mentioned bacteria 'Kalpa', '7', and ■ can also be cultured in a stainless steel large-capacity fermentation tank.
The medium composition is 6% soybean meal, 2.0% starch, and 0.5% calcium carbonate.
Adjust H to 7.0. Cultivation is carried out at 30°C,
Aeration is not performed, nor is the forehead azusa done excessively; it is done slowly and under gentle conditions. The above-described {6} bacteria (ie, Asbergillus bacteria) can also be cultured in large quantities in a fermentation tank made of stainless steel. As for the culture medium, the culture medium used for the bacteria '1) to {4) is also used, but the pH' thereof is adjusted to 6.0. The bacteria are grown by aerobic stem culture in the same manner as for the bacteria m to (ii) above. [8
} (i.e. Bacteroides lipolyticum)
Added 3% salt to nutritional broth (Difco, USA),
Inoculate into a medium adjusted to pH 6.5 to 7.0 before sterilization,
Produced at 3530 in aerated vibrator or aerated lampshade culture.
In addition, in addition to the above-mentioned medium, other ingredients such as minerals, vitamins,
It is also possible to further add yeast extract, fish meal, plain heart infusion, NZ-amine, and other commonly used ingredients. In the present invention, although it is effective to purify fresh water even if each of the bacteria belonging to the above three groups is used alone, it is preferable to use one or two types of inorganic or organic nitrogen compound-degrading bacteria, cellulose-degrading bacteria, and oil and fat decomposing bacteria. It is ideal to combine these bacteria with degrading bacteria, and furthermore, to combine all of the bacteria listed in m to (ii) above.

When mixing these bacteria, they are usually mixed in equal amounts, but in this case, the ratio is not limited. When these bacteria are mixed, they are individually cultured using the method described above, and then mixed. Culture is from 12 to
After continuing the culture for 4 pods, the growths were collected by centrifugation, and the thus obtained bacterial cell paste was freeze-dried in an acetone dry ice bath, and then mixed with each other. It is. It is also possible to purely separate only the bacterial cells from the bacterial cell paste and dry them, or to leave them wet without drying. However, if it remains in a wet state such as a bacterial cell paste, it is not only inconvenient to handle and store, but also the bacteria may become inactivated or become excessively active.

Therefore, in order to solve these problems, it is preferable to mix the bacterial cell paste with an inert carrier. As inert carriers, any material can be used as long as it is inert towards these fungi, for example clays or clay minerals such as diatomite, montmorillonite, chirolin, /miculite, bentonite. Alumina, silica, calcium carbonate, aluminum carbonate, apatite, attapulgite, etc. can be widely used. These inert carriers and microbial cells are mixed at the most suitable ratio depending on the carrier used, the amount of microbial cells, and its moisture content. The inert carrier acts not only as a drying agent for the bacterial cells, but also as a protective agent for the bacterial cells, and therefore, the ratio of the bacterial cells and the inert carrier to the ratio of, for example, 1 to 9:9 to 9 is more preferable. is 1:1
If mixed at a ratio of ~1:5, the lifespan of the bacterial cells will be extended, and germination and proliferation of the bacterial cells will also be inhibited. There will no longer be a need for special packaging.

There are no special precautions to be taken when storing it, just store it in a dry storage area.

Although microbial products usually require very delicate storage and preservation precautions, the simple operation of simply mixing the wet cake with the carrier without forming a lyophile is sufficient for long-term storage. Can be stored and preserved. Of course, it is of course possible to use the lionfish as is. To use the freshwater purifying agent according to the present invention, it is sufficient to dissolve it in a small amount of water and sprinkle it on the water surface, or it can be simply added in a small amount directly into the water. It is very convenient and does not require complicated usage.

It can also be used by mixing it with feed, if necessary. There are no particular restrictions on when to use it, but it is most efficient to use it when the water becomes cloudy. However, it is of course also possible to use it from the beginning or at any other suitable time. If you are raising seafood, it is best to use it after changing the water, but there is no problem if you use it without changing the water. The amount used can be very small, but as a rough guideline, if you are raising fish and using it in a tank or aquarium, about 15 to 80 fish should be used as a mixture of diatoms and a bacterial mixture. Preferably 25-5 kg of fence, when used for ponds, about 20-8 blood, preferably 25-5 ■ blood, and for maintenance use, 2-3 times a year, 3-3 times as necessary. It is only necessary to immerse yourself at a level of 18, preferably 5 to IQ, and maintenance is extremely simple. When the purpose is to purify water when no seafood is being raised, the desired purpose can be sufficiently achieved with a smaller amount than the above. Of course, the above usage amount can be adjusted as necessary. The purifying agent of the present invention has extremely high safety, and when handling it, there is no irritation to the eyes, no permeation, no oral poison, and no irritation to the skin.

Moreover, since the amount used is very small and the bacteria themselves are not harmful, there is no problem with pollution. No toxicity is observed for seafood. Furthermore, there is no problem in replacing the water in a small-capacity tank or pot, for example, and adding it to tap water that has a faint chlorine odor, or to something that has been neutralized with chlorine or the like. By using the purifying agent of the present invention, the water purification effect can be maintained for a long period of 90 to 140 days, which is a remarkable effect.Furthermore, it is possible to raise freshwater fish at high density, which is very effective. High economic efficiency can be obtained. If necessary, it is of course possible to use clean water from which bacteria have been removed using a bacterial filter or the like. Hereinafter, the present invention will be explained based on examples.

First, the following microorganisms were cultured according to their respective methods. [11Ps. Denitrificanth■B. Subtilis '3' B. thuringiensis '4'A Agilis ■ K. Zofiii [6} ASp. Tereus'7} Bact. Succinogenes■ Bact. Lipolyticum side Brev. Lipolyticum '10 N. A medium with a composition of 3% Coralina soybean meal, 1% starch, 0.2% ammonium sulfate, and 0.3% charcoal was sterilized after adjusting the pH to 7. Inoculate with seed culture.

Each culture was carried out in a stainless steel fermenter, and the temperature was maintained at 30°C, with aeration and concentration.
Cultivation continued for a period of time. The bacteria '5}, '7}, and '9} are cultured in a stainless steel fermenter in the same manner as above, but the culture is carried out using 6% soybean meal, 2.0% starch, and 0.0% charcoal. In a medium with a composition of 5%, incubation was performed slowly and without aeration at 30°C.
Each culture was cultured for two years. For the {61 bacteria, culture was carried out in the same way as in cases '1} to {4-, but the pH of the medium was 6.0 instead of 7.0.
．． Adjusted to 0. For the bacteria on the shelves, add 3% salt to nutrient broth (Difco, USA) and adjust the pH to 6.5-7.0 before sterilization.
The cells are inoculated into a medium adjusted to 35° C. and grown at 35° C. by aerated shaking or aerated aerobic culture. After the cultivation was completed, the culture was centrifuged to obtain a bacterial paste. This paste was freeze-dried in acetone-dry ice. The thus-prepared overnight culture of the fungus was mixed with an equal amount (weight) of diatoms to obtain a mixture of the fungus and the carrier.

Next, the mixtures of these bacteria and carriers were combined to prepare freshwater purifying agents having the formulations shown in Examples 1 to 9 below.

In each example, the numbers represent the number of bacteria (unit: 100 million) per 1 g of powdered formulation. The powder formulation of Example 9, which is a combination of all of the flea bacteria described in 1 above, is a light pink to brown color and is a free-flowing powder with a slightly sour odor (this formulation is referred to as "USC-600 }).Example 1 Ps. Denitrificans 2.5
ASp. Tereus 1
．． 082ev. lipolyte cum
1.0 Example 2B. subtilis
2.5 ct. succinogenes
0.5N. Coralina
0.5 Example 3A Agilis
2.5Bact. succinogenes
0.5B2ev. lipolyte cum
0.5 Example 4Ps. Denitrificans 2.0K. Zofiii
0.4Bact. Succinogenes 0.4N. Coralina 0.4B26v.
Lipolytecum 0.4 Example
5A. Agilis 2.
0B. subtilis 0
．． 4B. Thuringiensis 0.
4ASp. Tereus
0.4N. Coralina
0.4 Example 6Ps. Denitrificanth
2.0A Agilis
1.0ASp. Tereus
0.5Bact. succinogenes
0.5Bact. lipolyte cum
0.5 Example 7Ps. Denitrificanth
1.0B. subtilis
1.0B. thuringiensis
0.5ASp. Tereus
0.5 mother ct. succinogenes
0.5 Brev. lipolyte cum
0.5N. Coralina
0.1 Example 8 haired ground squirrel
0.5K. Zofiii
0.2Ps. Denitrificans 2.0Bact. Succinogenes 0.5 Bact. Lipolytecum 0.5 Brev
．． Lipolytecum 0.5N.
Coralina 0.5 Example 9 Hairy Ground Squirrel 0
．． 5B. subtilis 1
B. thuringiensis 0.5K.
Zofiii 0.1Ps.
Denitrificans IASp. Tereus 0.582c
t. Succinogenes 0.5B
2ct. Lipolytecum 0.
5Brev. lipolyte cum
0.5N. Coralina
0.1 The effects of each of the formulations of Examples 1 to 9 of the present invention were compared with untreated formulations in which no formulation was administered, and evaluated according to Test Examples 1 to 3 below.

Test Example 1 Nishikigoi Breeding Test Two 75 x 55 skin x 35 arc glass aquariums were each filled with sterilized river sand at a thickness of 5 cm on the bottom, and tap water that had been stagnant all day and night was poured in for 100 water each to determine the average weight. 20 Nishikigoi each weighing about 75g were put in each.

The same inner-layer type bran strainer was used in both areas, and aeration was performed using an aerator. Nosan Inn Nishikigoi No. 7 was used for feeding, and care was taken to leave as little uneaten food as possible. After the release, the microbial preparation of the present invention US was placed in one aquarium.
Melt C-600 with powder so that it has a bait level of 5,
The rope was quietly prayed so that it spread well throughout the tank, and this was designated as a treatment area. The water temperature was 15 qo to 24 qo and the breeding test period was 60 days. The following results were obtained by not performing any water changes, replenishing only the naturally evaporated water, and not cleaning the filter or replacing the filter. (Table 1). Table 1 01 The filter in the control area was almost clogged, the water was suspended, and the odor was quite strong.

The treated area had no suspension, was transparent, and had a very weak odor. '2} After the test was completed, the ammonia concentration and nitrite ion in both tanks were examined and the following results were obtained (Table 2).

Table 2 {3i When the contents of the filter tube were taken from the treated and control areas into test tubes and left at room temperature, the control group showed further decomposition and fermentation, but the treated area showed no change. In addition to the fact that the amount of contents in the strainer in the treated area was overwhelmingly lower than that in the control area, it was inferred that the application of the microbial preparation of the present invention had changed it into a difficult-to-decompose substance.

Test example 2 High-density rearing test of eel Prepare 12 glass aquariums of 75 arc x 55 x 35 arc.
No. 1 corresponding to Examples 1 to 9 and two comparative examples. 1
~shame. Number the aquarium up to 11 and enter the aquarium No. 1~N
o. No. 9 to No. 9 were treated as the microorganism preparation treatment area of the present invention. 10
and No. No. 11 was used as a control group (untreated group) (Table 3).

Each aquarium was filled with sand 5 skins deep and filled with 100 bottles of tap water that had been brewed overnight. For free-raised eels, unless the size variation is as small as possible, the feed will be consumed evenly and effectively, resulting in a large loss of feed, which will make it difficult to judge the effectiveness of microbial preparations. Choose fish of the same size as much as possible, and choose fish with a weight of 45. Female~47. Twenty fish within the female range were placed in each tank. Relaxation aft. 1~No.
In the aquarium of treatment area No. 9, a microorganism preparation was prepared according to the preparation example in the attached sheet, and the preparation No. 91 was added to the snake. Into the water tank 1, mix the microbial preparation powders prepared in accordance with the table below, such as the preparation example 1' (5
Willow level). The same type of activated carbon sieve was installed in both the control and treatment areas, and aeration was performed with air stones to reduce the dissolved oxygen level to 7. I was careful not to go below Norimisaki. In addition, in order to reproduce the high temperature conditions where water tends to change, we use an IkW heater with an automatic temperature regulator, and the water temperature is 27.
The temperature was adjusted to 0 (soil 0.5°C). The exam period is 18
Day 0 was set as 0, and the water was not replaced during that time, but only natural evaporation amount was replenished at timely replenishment, and efforts were made to maintain a constant water amount. Only in the treated area, a microbial preparation with a weight ratio of 5 cm was added to the amount of water added during water replenishment. Showa Sangyo Eel Marnir B was used as the feed, and initially the same amount was fed to all groups, but after about three weeks, the control group's feed intake decreased, while the treated group had a strong appetite. Although the amount of food was increased, care was taken to ensure that there was as little leftover food as possible in all tanks after feeding. The results are shown in Table 4. Table 3 Table 4 Table 1 In the treated area, the water was very clear and there was little odor, and when the eels burrow into the sand at the bottom after ingesting food, they become suspended, but there is no difference. Even if it is, it will settle in about 5 to 1 minute,
Suspension is gone.

2. In the control plot, about three weeks after being released, the water became noticeably cloudy, there was a foul odor, the animals began to move slowly, and the roots began to die.

After the eel burrowed into the bottom sand, it became cloudy and the suspension did not subside for more than three strokes, and the floating matter remained suspended for a long time. The foul odor and suspension progressed until the end of the test, and death continued to be the No. 1 problem. 10
A total of 13 fish in the tank, No. A total of 16 fish died in 11 aquariums. 3 The filters in the control area were completely drained and the filters were replaced in both aquariums, and the filters were replaced frequently every 20 to 30 days after that, but the filters in the treatment area were completely drained by the water tank. Although there were some differences, the air flow became thinner around the 90th day, but it was not completely clogged, and the cartridges were replaced only once for each aquarium around the 113th to 129th day.

4. Observation of the contents of the sieve in the treated area revealed that it was smooth and had little odor, and the sieve was easy to clean when replacing activated carbon.

On the other hand, the contents in the control area were slimy and had a strong odor of putrefaction, and even though the sieve filter had to be scrubbed a little, the stains did not come off and it took a long time to clean it. The breeding density at the time of pick-up in the treatment area was 20732XQ/100k ~ 30716K9/1
00If you convert that to an eel pond with a water depth of 2 hours, it will be 41.4k.
9/M2 to 61.4k9/M, and in the case of the forced oxidation method, the density is about 12kg/M2.Compared to this, the treated area has a density of 3.45 to 5.9kg/M2.
An 11 times higher density is achieved.

Test Example 3 High Density Rearing Test for Rainbow Trout Twelve glass aquariums of 75 skins x 55 skins x 35 skins (water volume 100) were prepared and No. 1 fish tanks were prepared corresponding to Examples 1 to 9 and two comparative examples. 1~No. Dial up to 11 and enter the aquarium no.
1~Side. No. 9 to No. 9 were treated as the microorganism preparation treatment area of the present invention.
10 and no. No. 11 was used as a control group (untreated group) (Table 5).

Each aquarium had a sterilized stream at a depth of three bays, and the same type of strainer and aerator were used. The water temperature was set at 18oo±1°C using a heater. Rainbow trout of the same weight as possible were selected in each tank, and 10 trout were placed in each tank. (Average weight is 67g) After adding fish, No. 1~No. In the aquarium of treatment area 9, N
o. In the aquarium of 1, {1- formulation example, No. 2 is [2}
Similarly, 5 g of each microbial preparation powder was poured into the corresponding mold as shown in the example of preparation. The compound feed used was Maruka Inryo Complete Feed for Trout P6, with a 70%
It was kept for days. In addition, water was not replaced at all in all areas, and water was replenished only from natural evaporation in a timely manner.

Treatment area no. 1~No. At the time of water replenishment, microorganism preparation powder was added to the water tank No. 9 at a weight ratio of 5 to the amount of water refilled. The results are shown in Table 6. Table 5 Table 6 Table 1 The water in the treated area was clearer and the odor was much weaker than in the control area, but the odor in the control area was strong and the water was significantly cloudy.

2. When we examined the rainbow trout that died in the control area, we observed adhesion of secondary sea bream valves and dilation of sea bream capillaries in all of them, and it was observed that the hair died due to ammonia poisoning. It was presumed that when the amount of food was 1/2 compared to the treated area, activity decreased due to blood loss anemia caused by ammonia, and body weight did not increase as much as in the treated area.

3. The filter filter clogged much faster in the control plot than in the control plot, and it completely clogged on the 24th and 27th day.Although the filter was replaced, the filter in the treated plot did not clog until the end of the experiment.

The contents of the transfer vessel were placed in test tubes for both the control and treated areas and left for two weeks.The contents of the control area produced fine bubbles coming out of the bottom, fermentation, and decomposition progressed, but the contents of the treated area did not. Things did not rot, but were transformed into somewhat decomposed substances.

Claims (1)

[Claims] 1 Bacillus subtilis, Bacillus thuringiensis, Arthrobacter agilis, Aspergillus
A freshwater purifier characterized by containing one or more bacteria selected from the group consisting of Brevibacterium terreus, Bacteroides succinogenes, Bacteroides lipolyteicum, and Brevibacterium lipolyteicum.