JP2553727B2 - Useful microorganisms and their use - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention is directed to Bacillus subtilis BN1001 (Bacillus sub).
tilis BN1001) and a method for reducing n-hexane extract substances in oil-containing wastewater, a method for decomposing scum accumulated in drain tanks such as kitchens, a method for removing waste odors and bad odors from toilets, a method for suppressing generation of bad odors, The present invention relates to a method for purifying water, a method for improving soil, a method for using organic fertilizer, and a method for improving feed efficiency and increasing body weight of animals.

[Prior Art] From the standpoint of environmental protection, stricter regulations on n-hexane extraction substances in oil-containing wastewater discharged from homes or various business establishments have been called for. Deodorizing treatment is required for wastewater discharged from homes or various business establishments, such as scum, which emits an unpleasant odor. , Various deodorants such as masking agents are commercially available. But n
There are few things that can be satisfied with the reduction of hexane extractables, decomposition of scum, deodorization, and prevention of bad odor. In particular, with the recent aging of the population, the number of people taking hypertension drugs, diabetes drugs, etc. has increased, and an odor different from the conventional manure odor controls the toilet and its surroundings, and there are many concerned persons who are having difficulty in countermeasures. Poultry odors of chicken farms, pig farms, quail farms and other animals are also a source of distress for workers and local residents. Further, in recent years, an increasing number of people are breeding ornamental fish and the like in an aquarium or a pond, but many people are worried about exchange of odor and water emitted from the aquarium or pond. There are also many people who love plants, and there is a desire to improve the soil and grow beautiful plants.
In addition, there are many demands for efficient economic animal breeding.

[Problems to be Solved by the Invention] A technical field in which it is desired to reduce n-hexane extractable substances in oil-containing wastewater discharged from homes or various establishments, disassembling scum accumulated in drainage tanks such as kitchens and generating from this scum. Technical fields that want to eliminate bad odors, technical fields that want to reduce or eliminate unpleasant odors generated from wastewater discharged from homes or various business establishments, and to prevent the occurrence of bad odors where it is predicted The technical field of wanting to reduce or eliminate the odor from excrement excreted by various animals including toilets or livestock, and the technical field of reusing raw garbage and excrement of various animals as organic fertilizer, Techniques for reducing odors emitted from aquariums or ponds for breeding ornamental fish, improving water purification technology, and soil It is an object of the present invention to provide a viable cultured bacterial cell and a viable bacterial agent which are excellent in safety and have an improving effect and which can be satisfied in the technical field and in the technical field demanding improvement in the efficiency of raising economical animals.

[Means for Solving the Problems] The present inventors conducted a screening of microorganisms from soils in each region in order to discover microorganisms that reduce n-hexane extract substances in oil-containing wastewater, and as a result, new bacteria belonging to the genus Bacillus. Was discovered and was named Bacillus subtilis BN1001.

While testing this live culture of Bacillus subtilis BN1001 with oil-containing wastewater, we discovered a deodorizing effect in addition to the oil-reducing effect, and the deodorizing effect from wastewater discharged from homes and various establishments, especially from places that emit unpleasant odors. The effect was repeatedly confirmed. In addition, the odor of toilet and various animal excrements should be reduced to the extent that it does not cause discomfort, and that foul odors should be suppressed where it is predicted that foul odors will be generated. What we could do, we also found a fish odor reduction and water purification effect in the ornamental fish tank and pond. Furthermore, after sowing in soil and mixing well, seeds of plants were sown and found to grow well, and as a result of being added to the feed of various economic animals, it is possible to improve feed utilization efficiency and increase body weight of animals. discovered. The present invention has been completed as a result of obtaining such new knowledge and further repeating the test.

That is, the present invention relates to live cultured bacterial cells of Bacillus subtilis BN1001 and a live bacterial agent.

The strain of the present invention is Bacillus subtilis BN1001 newly isolated from the soil by the present inventors, and the bacteriological properties of this bacterium are as follows.

A. Morphological properties (1) The vegetative cells cultured in broth agar are 0.5 to 0.7 x 1.
5 to 3.0 μm bacillus (2) Gram stain is positive (3) Endospores are formed by culturing at 30 ° C for 2 to 3 days, 0.5 to
0.7 × 0.8-1.5μm oblong (4) Spores are located almost in the center of the sporangium, and almost no swelling of the sporangia is observed (5) Movement by the perilis (6) Negative acid-fast staining B .Cultural properties (1) Normal agar culture: Grows well in culture at 30 to 45 ° C for 1 to 3 days (2) Meat broth agar culture: Growth is good, the colony has a brown cream-like appearance, and diffusible pigment is produced Not (3) Meat juice liquid culture: White pellicle is formed on the liquid surface (4) Meat juice gelatin puncture culture: Layered liquefaction (5) Skim milk culture: Alkaline and peptoneized C. Physiological properties (1) Reducibility of nitrate: Positive (2) Denitrification reaction: Negative (3) VP reaction: Positive (4) MR test: Negative (5) Hydrolysis of starch: Positive (6) Utilizing nitrate and ammonium salts (7) Dye production: remarkable water solubility and water insolubility No pigment formation (8) Catalase: Positive (9) Utilization of citric acid: Positive (10) Arginine degradability: Negative (11) Indole formation: Negative (12) Hydrogen sulfide formation: Negative (13) Oxidase : Positive (14) Urease: Negative (15) Lysine decarboxylase: False positive (16) Salt tolerance: Grows on broth agar medium containing 10% salt (17) Growth temperature: Grows well in the temperature range of 25 to 45 ° C However, it does not grow at 55 ° C. (18) No growth under anaerobic conditions is observed (19) OF test: O type (20) Utilization of sugar in Hureyfson medium: D-glucose, L-arabinose, Generation of acid from D-xylose, D-mannitol, sorbitol, and sucrose is observed, but no gas generation is observed. From inositol, rhamnose, melibiose, and raffinose. Bagyizu Manual of systematic Chii' click Bact the BN1001 shares having a gas mycological properties of more than generation is not observed with
1986 Edition (Bergey's Manual of Systematic Bacteriol
ogy, 1986) compared with the known bacterial species described in Ogy, 1986), the strain was determined to belong to the genus Bacillus because it produced endospores with aerobic Gram-positive bacilli and was positive for catalase. It was identified as belonging to Bacillus subtilis based on its morphology and various physiological properties. However, it differs from the typical Bacillus subtilis in that it shows excellent utility in reducing the amount of n-hexane extract, decomposing scum, deodorizing, water purification, soil improvement, feed efficiency, and promotion of weight gain. Therefore, it is a new strain. This strain was produced by the Institute of Microbial Science and Technology, National Institute of Advanced Industrial Science and Technology, Microtechnical Research Institute No. 11132 (FERM P-1113).
2) has been deposited as

[Examples] Hereinafter, production examples of the present bacterium, and test examples using the obtained cultured viable cells and viable agent are shown, but the present invention is not limited thereto.

Production Example 1 In 15 L of tap water, dissolve 75 g of glucose, 105 g of meat extract, 150 g of peptone, 45 g of sodium chloride, put the medium adjusted to pH 7.2 in a 30 L jar fermenter, sterilize at 121 ° C for 30 minutes, and cool it. The culture solution of Bacillus subtilis BN1001 that had been precultured was inoculated into the inoculated plant, and aerated with stirring at 30 ° C. for 24 hours.

The thus obtained culture solution was used as Bacillus subtilis BN.
It can also be used as live cultured cells of 1001. However, perlite was added to this culture solution and mixed, and by further drying and pulverizing this, a viable agent 1. of Bacillus subtilis BN1001.
3kg is obtained. The number of viable bacteria contained in this probiotic agent is 1 × 10 ⁹
It was a piece / g.

Production Example 2 To 1 kg of commercially available soybean meal, 500 g of corn steep liquor (CSL) whose pH was adjusted to 7 and 300 g of tap water were added and mixed well, 1
It was sterilized at 21 ° C. for 60 minutes, inoculated with a culture solution of Bacillus subtilis BN1001 which had been precultured, and cultured at 30 ° C. for 120 hours. The culture thus obtained was mixed with calcium carbonate to obtain 1.6 kg of a viable agent of Bacillus subtilis BN1001. The viable cell count contained in this viable cell agent was 1 × 10 ⁹ cells / g.

Test Example 1 Corn oil 1000 ppm, yeast extract 0.3%, ammonium dihydrogen phosphate 0.1%, potassium chloride 0.02%, magnesium sulfate 0.02%, viable bacterium of Bacillus subtilis BN1001 prepared in Production Example 1 in an oil medium of pH 7.8 The agent was added, and the mixture was shaken at 30 ° C. for 22 hours to measure the amount of n-hexane extract. The results are shown in Table 1.

Test Example 2 Various fats and oils were tested by the method described in Test Example 1,
The amount of decrease is divided by the amount of n-hexane extract at the start of the test, and the ratio is shown in Table 2 as the amount of fat and oil decomposition.

Test Example 3 Wastewater (raw water) from a confectionery factory was sampled, and Production Example 1
The viable agent of Bacillus subtilis BN1001 prepared in 1. was added, and the mixture was reacted by shaking at 30 ° C. for 22 hours, and the amount of n-hexane extract was measured. The results are shown in Table 3.

Test Example 4 In a wastewater treatment tank (6 tons capacity) of a Chinese restaurant, the probiotic agent obtained in Production Example 1 was 50 ppm for the first time and 50 ppm every two weeks thereafter.
Then, 5 g each was added daily from the sink in the kitchen to see the change in the solidified scum. As a result, after 2 weeks, the liquid surface was darkened and slightly softened, and after 4 weeks, the liquid surface was clearly softened and the liquid surface became visible when pressed with a stick from above.

It should be noted that, before the addition of the probiotic agent, a bad odor was emitted from around the scum, but it was deodorized after 2 weeks. After three months, the scum was no longer solidified.

Test Example 5 The probiotic agent prepared in Production Example 1 was added to the wastewater treatment tank (combined tank having a capacity of 12 tons) of a family restaurant in the same manner as in Test Example 4. As in Test Example 4, the scum was solidified and did not crack even if it was poke with a stick from the top, and it was difficult to soften even if it was sprinkled with water. The odor was even worse, and there were more complaints from the surrounding area, and the people involved in the restaurant were in trouble.

After the introduction of the probiotic agent was started, changes in the state of scum in the wastewater treatment tank, the visibility of the discharged water, and the odor of the treatment tank and its surroundings were observed. A fluorometer was used for water transparency, a portable odor sensor XP-329 (manufactured by New Cosmos Electric Co., Ltd.) was used for odor, and the state of scum was visually observed. Prior to the on-site test, wastewater having a strong unpleasant odor was sampled from the wastewater treatment tank, placed in an Erlenmeyer flask, and the live bacterial agent obtained in Production Example 1 was not added.
The amount of hexane extract and the odor were measured. The live bacterial agent was added only at the start of the test, and the reaction was performed at 30 ° C with shaking. The results of the test in the flask are shown in Table 4, and the results of the in-situ test are shown in Table 5.

Measurement of odor The odor sensor measured the odor of the wastewater treatment tank and its vicinity by adjusting the odor at the windward side of the wastewater treatment tank 5 to 10 meters away from the place where no unpleasant odor is felt to 100. The higher this value is, the stronger the unpleasant odor is, and there is no unit.

Numerical level of 130 to 150: Somewhat odorous level of 200 to 250: Anyone recognizes it as an odor Numerical level of 300 or more: Odor that makes anyone feel uncomfortable As shown in Table 4, the odor,
The amount of n-hexane extract is decreasing. The field test results in Table 5 also support the flask test results. That is, by adding the probiotic agent, the transparency can be improved, the malodor can be suppressed, and the amount of the n-hexane extractable substance can be reduced, so that it can be used in a restaurant kitchen facility such as a family restaurant or a hotel.

Test Example 6 5 g of probiotic agent from Production Example 1 per 1 kg of feces discharged from Uzura Farm
As a result of measuring the odor after 1 month by the method shown in Test Example 1, the odor that was 450 at the start of the test was reduced to 130.

In addition, 5 g per ton of feces was added daily at the Uzara site, and 250 g was added every week, and the odor was measured after 2 months. As a result, the odor at the start of the test was reduced to 130 and decreased. Due to the odor effect, it can be used in porridge and similar pig farms, poultry farms, etc.

Test Example 7 20-60 mg of the probiotic agent of Production Example 1 in an ornamental fish tank (60 L capacity)
As a result of daily addition, the fish odor disappeared shortly after addition, and the water exchange in the aquarium could be delayed for about 1 to 2 weeks.
It should be noted that the fish did not die due to the addition of the protozoal agent. That is, it can be used at home or in a fish farm.

Test Example 8 3 to 5 g of the probiotic agent of Production Example 1 on raw garbage discharged from home
As a result of putting it in before bed, the next morning, it was deodorized so that no one could notice it. The same phenomenon was confirmed to be effective in the sink, which emits an unpleasant odor, in the bathroom, in the washing machine room, and in the toilet. Moreover, the slimy condition in the sink or the bathroom disappears, and it can be used in a place where a bad odor has already been generated at home or a place where a bad odor is expected to occur in the future.

Test Example 9 3-5 g of the probiotic agent of Production Example 1 was mixed with 500 g of soil and seeds of morning glory were sown, and the growth was compared. Compared with this, it grew about 6 to 10% longer, and the number of seeds obtained was about 10% larger.

In addition, as a result of mixing the probiotic agent of Production Example 1 in soil with strong odor, the odor disappeared in 1 to 2 days. As a result of planting leeks on this soil and comparing it with general soil, the growth of leeks was similar to the morning glory. A difference of about 5% was recognized, and the result that the yield could be increased was obtained.

Test Example 10 50 mg of the probiotic agent of Production Example 2 was added to or not added to the feed for commercial broilers for the first half (combination feed), and the growth test was performed by feeding the broilers 2 to 3 days after birth. . The results are shown in Table 6.

As shown in Table 6, by feeding the compounded feed to which the probiotic agent of the present invention was added, a significant weight gain effect was observed and the feed conversion rate could be improved.

EFFECTS OF THE INVENTION The present invention provides a newly isolated Bacillus subtilis BN.
It is related to 1001 and when it is put into oil-containing wastewater and wastewater that gives off an unpleasant odor, it has an oil-reducing effect, a deodorizing effect, and a water-purifying effect. By further administering to animals, the effect of improving edema is exerted as a multi-functional, wide-ranging and outstanding effect such as a weight gain effect and an effect of improving feed efficiency.

Further, the present invention plays an important role in each technical field such as environmental improvement, wastewater treatment, agriculture, animal husbandry, and fishery as a safe wastewater treatment agent without side effects or as a feed additive.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12R 1: 125) (56) Reference JP-A-1-144971 (JP, A) JP-A-49 -18671 (JP, A) JP 62-232343 (JP, A) JP 64-86842 (JP, A) Actual opening 63-209580 (JP, U) Actual opening 57-42591 (JP, U) )

Claims (2)

(57) [Claims] 1. Bacillus subtilis BN1001 having the following mycological properties (1) Bacillus (2) Gram stain: Positive (3) Endospore formation, oval (4) Motility Yes (5) Anti-acid stain: Negative (6) Normal agar culture: Well grown (7) Meat agar culture: Good growth, colony is brown cream-like (8) Meat juice liquid culture: White pellicle is formed on the liquid surface (9) Nitrate reducibility: positive (10) VP reaction: positive (11) Starch hydrolysis: positive (12) Catalase: positive (13) Citric acid availability: positive (14) Arginine degradability: negative (15) Indole formation: Negative (16) Hydrogen sulfide formation: Negative (17) Oxidase: Positive (18) Urease: Negative (19) Lysine decarboxylase: False positive (20) Salt tolerance: Meat agar with 10% salt Grow on medium (21) Growth temperature: 25-45 ℃ (22) OF test: O type (23) Sugar utilization by Hureyfson medium: Acids from D-glucose, L-arabinose, D-xylose, D-mannitol, sorbitol, sucrose However, the generation of gas is not recognized. The generation of acid and gas from inositol, rhamnose, melibiose, and raffinose is not recognized. (24) Reduce the amount of n-hexane extracted substances in oil-containing wastewater to reduce Decomposes scum that accumulates in the drainage tank, and has the action of removing and purifying odors from wastewater and toilets. 2. A method for decomposing oil by introducing cultured viable cells of Bacillus subtilis BN1001 and a probiotic agent into oil-containing wastewater to reduce the amount of n-hexane extractable substance.