WO2013137010A1

WO2013137010A1 - Novel bacillus microbes and use of same

Info

Publication number: WO2013137010A1
Application number: PCT/JP2013/055504
Authority: WO
Inventors: 世一大林; 良造栗田; 中山　浩二; 英夫橋本
Original assignee: 日之出産業株式会社
Priority date: 2012-03-12
Filing date: 2013-02-28
Publication date: 2013-09-19
Also published as: JP6105553B2; JPWO2013137010A1

Abstract

The purpose of the present invention is to discover microbes capable of treating oil-containing wastewater with good efficiency without spending a great deal of time reducing the oil content in the processing wastewater, and to provide a technology that uses those microbes to enable volume reduction of excess sludge in wastewater treatment and a reduction in wastewater oil content. Provided are a wastewater treatment device and a method comprising novel microbes belonging to the genus Bacillus and having the ability to decompose at least bacterial cell walls and lipids, and a wastewater probiotic that contains such microbes and is for preventing the generation or for volume reduction of excess sludge. The wastewater treatment device and method are characterized in that one or a mixture of two or more of the novel microbes are added to treated water of the wastewater treatment device.

Description

Novel Bacillus microorganism and use thereof

The present invention relates to a novel Bacillus microorganism and its use, and more specifically, suppresses excess sludge generated by waste water treatment by the activated sludge method, and efficiently treats oil and fat waste water by symbiosis with fungi. The present invention relates to a novel Bacillus microorganism, an aerobic sludge treatment method and a wastewater treatment apparatus using the microorganism, and the like.

According to the results in fiscal 2005, the amount of sludge generated from water and sewage in Japan accounted for 44.5% of the total industrial waste, generating approximately 1877,900,000 tons annually ). Of this, 48% (79.16 million tons) is sewage sludge generated from sewage treatment plants, and 21% (39.44 million tons) of animal manure and 31% (58.2 million for other industrial wastewater) T).

Typical sewage sludge treatment and disposal status is 9% regeneration rate, 86% intermediate treatment rate, 5% final disposal rate, intermediate treatment is mostly concentrated, dehydrated and incinerated, and much energy is added It will be. Since sludge often contains water with a water content of 60 to 90%, disposal of the treatment involves a large energy cost.

Some of the excess sludge from factory wastewater treatment is used as a soil conditioner and compost material, but most of it is pretreated by dehydration, incineration, etc. and then disposed of in landfills. This process consumes a lot of energy, the enterprise suffers from rising energy costs, and local governments and final disposal companies have difficulties in securing a disposal site and regulatory problems, which will continue to be a major issue in the future .

According to Non-Patent Document 1, techniques for sludge volume reduction include a chemical volume reduction method, a physicochemical volume reduction method, a biological volume reduction method and the like. Among them, chemical reduction methods include the ozone method of degrading cell walls by the oxidizing power of ozone, the enzyme method of solubilizing cell walls by enzymes such as cellulase, protease and amylase, and the alkali using protein denaturation by alkali There is a law etc. Among these, the ozone method has problems such as high initial cost and the enzymatic method has high running cost.

In the physicochemical volume reduction method, mechanical decomposition method that promotes solubilization by mechanically grinding sludge using high shear force by bead mill, high-speed rotating disk, etc., thermal decomposition It is generated by gasification pyrolysis method, hydrothermal reaction method in which all organic substances are hydrolyzed and decomposed by thermal decomposition using hydrothermal reaction of supercritical water or subcritical water, and solubilization, electrolysis of saline solution There are electrolytic oxidation methods that kill and solubilize microorganisms with chlorine and hypochlorous acid and biodegrade. These volume reduction methods have major problems with high initial investment and running costs.

In addition, biological volume reduction methods include auto-oxidation and food chain methods. Among them, in the self-oxidation method, other microorganisms are used as an organic energy source in the situation where microorganisms constituting sludge do not supply organic energy from the outside, and they are decomposed into water and carbon dioxide gas with the passage of time. The food chain method is a method of reducing the volume and creating a food chain system of bacteria → protozoan → metazoan. The self-oxidation method has problems with installing a volume-saving tank for one week's worth of sludge and running costs such as maintaining the temperature at 60 ° C, and the food chain method uses bacteria, protozoa and metazoans in an open air system. The issue is the accumulation of technical know-how to breed.

Furthermore, as a means for reducing the volume of sludge, a method of adding a hyperthermic microorganism as in Patent Document 1 or a biological treatment process by aeration in the presence of an aerobic microorganism under alkaline conditions as in Patent Document 2 A sludge treatment method is also proposed to be returned to Japan.

Patent Document 3 proposes a method of reducing the volume of sludge with a novel microorganism having sludge decomposability that can be grown under conditions of high alkalinity and high temperature. These techniques have high temperature and alkalinity, and the use of alkali agents and neutralizing agents, and high initial cost and running cost in the treatment of non-productivity sludge which use energy to make high temperature every day is a problem Become.

In addition, the drainage of ice cream for producing drainage with a high oil content, mayonnaise manufacturing plant, and the drainage of a restaurant using these or a shop for producing delicatess has a high oil content as well. Whether these fats or oils are vegetable or animal, it is difficult to treat the waste water with high fat content as it is, and waste water from which fats and oils have been physically and chemically removed in advance is waste water such as activated sludge method It must be sent to the treatment process.

As a method of removing fat and oil containing waste water, the fat and oil containing waste water of 200 to 500 mg / L as N-hexane extract is treated by installing a fat and oil separation tank, and when it exceeds 500 mg / L, a pressurized floatation tank is used. It is desirable to install to remove fats and oils and to process after reducing N-hexane extract. Treatment of fat and oil-containing wastewater involves running costs such as the initial cost of installation of such facilities and the disposal cost of fats and oils that are raised, and there is also an offensive odor for storage in summer, so microbial treatment is eagerly desired There is.

Then, the method of assimilation of animal and vegetable fats and oils is proposed using various yeast. Above all, the technology of Patent Document 4 is an invention of biological wastewater treatment which is easy to use because the range of growth pH of fat utilization yeast is wide and bacterial contamination does not have to be suppressed with a chlorinating agent. There was an issue that took time.

Japanese Patent Application Laid-Open No. 9-253699 Japanese Patent Application Laid-Open No. 9-136097 JP 2000-139449 Patent No. 4465332

The present invention was made from the above point of view, and improved the treatment of waste water, that is, found a microorganism capable of efficiently treating waste water, and using this, it was used to reduce excess sludge in waste water treatment and fats and oils in waste water. It is an object of the present invention to provide a technology that enables the reduction of the

In order to solve the above problems, the present inventor focused attention on microorganisms belonging to the genus Bacillus that secrete various enzymes, and searched for those that secrete decomposing enzymes effective for treating oil-containing waste water. We have found that wild strains of the genus Bacillus secrete lipids and enzymes that degrade bacterial cell walls. In addition, in order to strengthen the ability to secrete this enzyme, cells were made competent to secrete various enzymes, thereby completing the present invention.

That is, the present invention is a novel microorganism belonging to the genus Bacillus, which has at least the ability to degrade cell walls and lipids of bacteria.

Further, the present invention is a waste water treatment biocide for preventing excess sludge generation or reducing the volume, which contains the above-mentioned novel microorganism.

Furthermore, the present invention is a fat and oil containing waste water treatment viable bacterial composition comprising the above-mentioned novel microorganism and fat and oil utilization yeast.

Furthermore, the present invention is a method for reducing excess sludge in a wastewater treatment facility, characterized in that the above-mentioned new microorganisms are added singly or in combination of two or more to the water to be treated in the wastewater treatment facility. is there.

Furthermore, the present invention is characterized in that in the solid-liquid separation wastewater treatment method for removing water using a solid-liquid separation membrane, the above-mentioned novel microorganism is caused to act on the solid-liquid separation membrane singly or in combination of two or more kinds. , And a method of suppressing the decrease in flux through the membrane.

In addition, the present invention includes a treatment tank for storing and treating the water to be treated, an inflow pipe for letting the treatment water flow into the treatment tank, a discharge pipe for discharging the treated water, and aeration for the water to be treated. A waste water treatment apparatus having an aeration pipe, comprising a treatment agent feeding device for feeding a treatment agent.

The novel Bacillus microorganism of the present invention has at least the ability to degrade cell walls and lipids of bacteria, and also includes those having the action of degrading protein and starch, so this is the case in waste water treatment by conventional aerobic biological treatment. By using such a product, decomposition treatment of sludge and fats and oils can be easily performed, so that the amount of excess sludge and waste fat and oil can be reduced.

In particular, if the yeast fat and oil decomposition tank is provided during the wastewater treatment process and used in combination with fat and oil assimilation yeast here, the amount of excess sludge and waste fat and oil can be reduced or eliminated.

In addition, in the solid-liquid separation wastewater treatment method for removing water using a solid-liquid separation membrane, the novel Bacillus microorganism of the present invention is allowed to act on this solid-liquid separation membrane to prevent a decrease in flux passing through the membrane due to clogging of the membrane. I can do it.

It is drawing which showed typically the one aspect | mode of this invention waste water treatment apparatus.

The microorganism belonging to the genus Bacillus used in the present invention is at least capable of degrading bacterial cell walls and lipids. The microorganism having such ability can be obtained by screening from the natural world, or by performing transformation by means of genetic engineering based on the microorganism obtained by screening from the natural world.

As a specific example of the method for obtaining the microorganism belonging to the genus Bacillus according to the present invention, a microorganism isolated from plants, water, food or soil is selected using the degradability of bacterial cell wall and the degradability of lipid as an index. It can be obtained by In addition, these microorganisms are more preferable if they have the ability to degrade protein, starch and the like. The cell wall resolution is to destroy or dissolve all or part of the cell wall. By screening, strains HB-88 and HB-113 were obtained.

The microorganism thus obtained may be selected as it is from the resolution of the cell wall of the bacteria and the resolution of the lipids as described above, but for example, it is mutated by the usual method under conditions that grow by about 10% under ultraviolet irradiation. You may do, and then screen.

Furthermore, using a transformation culture medium that regulates competent cells using Marburg strain of Bacillus subtilis having high natural transformation ability and other 160, 166, and 168-derived strains, transformation ability is enhanced and donors are used here. A strain may be introduced to have new properties. The Marburg strain and the strain derived therefrom can be purchased from RIKEN, NBRC, ATCC, etc.

Since the Marburg strain used had a starch-degrading enzyme and a lipolytic enzyme, transformation to add a gram-negative bacterial or gram-positive bacterial degrading enzyme was similarly performed. By this operation, the modified bacteria were modified to secrete lipids, bacterial cell walls, starches, and protein degrading enzymes. Among the 16 modified strains, among them, 1 strain which strongly secreted the enzyme was found. This strain was designated as HB-14.

As described above, three types of microorganisms, HB-14 strain, HB-88 strain and HB-113 strain, were obtained. As a result of comparing the bacteriological properties of this microorganism with "Bergey's manual of systematic biology Volume 2 (1984)", all the microorganisms belonged to the genus Bacillus.

Furthermore, as a result of performing the following DNA analysis, it was presumed that HB-14 strain was Bacillus subtilis (Bacillus subtilis (Bacillus subtilis), HB-88 strain was Bacillus methylotrophicus (Bacillus methylotrophicus) in 16s DNA gene elucidation ((strain ) Technosurga Lab). The HB-113 strain was determined to be Bacillus subtilis by API 50 CHB.

Next, the microbiological properties of HB-14 and HB-88 selected by the above screening are shown. The DNA analysis was carried out in Nutrient agar medium, and the culture was carried out in aerobic culture at a temperature of 30 ° C. for 40 hours, and used as a test fungus. For identification of bacterial species by 16S rDNA-Full, DNA is extracted with achromopeptidase (Wako Pure Chemical Industries, Ltd.), PCR of PrimeSTAR HS DNA polymerase (Takara Bio Inc.) is used, and BigDye Terminator v3 .1 Using the Cycle Sequence of Cycle Sequencing Kit (Applied Biosystems, CA, USA), the primers used are (PCR amplification: 9F, 1510R, sequence: 9F, 785F, 802R, 1510R), and the sequence is ABI PRISM 3130xl Genetic Analyzer System (Applied) ChromasPro 1.4 (For Biosystems, CA, USA) to determine the base sequence Apollo 2.0 as software for homology search and simplified phylogenetic analysis using Technelysium Pty Ltd., Tewantin, USA), Apollo DB-BA 7.0 as a database (Technosurga Labs Co., Ltd.), International Sequence Database (GenBank / DDBJ / Embl) was used. In addition, HB-113 was subjected to various searches according to the search method (Spore Experimental Manual p110 to 111, published by Shihobodou) and identified using the API system.

List of characteristics of new microorganisms I (30 ° C culture)

Characteristic list of new microorganisms II

These microorganisms are not included in the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (〒2-5-8, Kisarazu City, Chiba Prefecture, 〒 292-0818), HB-14 for NITE BP-1275, HB-88 NITE BP-1276 and HB-113 were internationally deposited on March 7, 2012 as NITE BP-1277.

The novel Bacillus microorganism of the present invention as described above can be used for all wastewater having BOD load in the aerobic wastewater treatment process, but can be preferably used for wastewater from sewage treatment and factories such as food factories.

When reducing the volume of sludge by utilizing the novel Bacillus microorganism of the present invention for the activated sludge method, microorganisms (10 ⁷ to 10 ⁸ / ml) cultured in a culture solution in the raw water tank or aeration tank of a wastewater treatment facility ) May be added in an amount of 1 to 1000 ppm, preferably 10 to 300 ppm. The addition of about once to 1 to 3 days is more preferable to continue the effect.

The novel Bacillus microorganism of the present invention is preferably used particularly in waste water treatment methods utilizing membrane treatment. That is, since the microorganism of the present invention has the resolution of the cell wall of bacteria as described above, it decomposes the bacteria and the viscous substances that grow in or around the pores of the treated membrane used in wastewater treatment. It is because sufficient sludge treatment capacity can be maintained without clogging the pores of the The treatment membrane may be a flat membrane or a hollow fiber membrane, and can be used similarly. The most appropriate type of membrane is the MF membrane from the size of the membrane's holes.

Moreover, the volume reduction rate of excess sludge can be raised more by utilizing the novel Bacillus-genus microbe of this invention for the process which combines the waste-water-treatment method of film | membrane use, and the activated sludge method. There are parallel processing method and serial processing method in combination of these two methods, but high concentration processing can be performed by using serial processing with this Bacillus which does not produce floc if the kind of organic matter in the drainage is constant. . On the other hand, in the case where the types of treated products are various and rich in variation, it is preferable to use a membrane to treat only high concentration drainage in parallel and then to flow it into an activated sludge tank.

Next, a waste water treatment apparatus that can be advantageously used in carrying out waste water treatment with the novel Bacillus microorganism of the present invention will be described.

FIG. 1 is a drawing schematically showing one aspect of the waste water treatment apparatus of the present invention. In the figure, 1 is a waste water treatment apparatus, 2 is a treatment tank, 3 is a treated water, 4 is an aeration pipe, 5 is an aeration hole, 6 is a treated water inflow pipe, 7 is a treatment agent feeding device, 8 is a treatment agent injection The pipe 9 is a fine air bubble generator, 10 is a submersible pump, and 11 is a water discharge pipe.

The waste water treatment apparatus shown in FIG. 1 contains a treatment tank 2 for storing and treating the water to be treated 3, an inflow pipe 6 for letting the water to be treated flow into the treatment tank, and a discharge for discharging the water to be treated. It includes the pipe 11, the aeration pipe 4 for aerating the water to be treated, and the treatment agent feeding device 7 for feeding the treatment agent.

The treatment tank 2 may be of any material and size as long as the treated water 3 can be stored, but in general, in the waste water treatment apparatus, one used as an adjustment tank is used It is preferable to do. Also, the aeration pipe 4 can be used without any problem as long as it is generally used for aeration in waste water treatment, as long as the aeration pipe 4 is provided with an appropriate number of aeration holes 5. The number and the diameter of the aeration holes 5 are preferably set so that a sufficient amount of air is blown in consideration of the volume and the depth of the treatment tank.

Furthermore, as long as the inflow piping 6 and the discharge piping 11 can also flow in and discharge the water to be treated, those which are conventionally used in the waste water treatment apparatus can be used as they are. The water to be treated that flows in from the inflow piping 6 is, for example, waste water including lipids, proteins, starch and the like discharged from a food manufacturing plant, a store that prepares and supplies food, etc., especially plant or animal It may be drainage having a high fat and oil content, and its flow rate may be greatly changed. The discharge pipe 11 may be provided in an overflow tank that naturally flows out when the water 3 to be treated reaches a predetermined amount or more, or it is a pipe connected to a pump, and it is electrically or continuously or intermittently. You may discharge it.

A feature of the waste water treatment apparatus is that a treatment agent feeding device 7 is provided, from which the treatment agent can be fed. That is, the waste water treatment apparatus not only aerates with air from the aeration pipe 4 but also a treatment agent such as a Bacillus microorganism having an ability to degrade at least bacterial cell walls and lipids in the aerated treated water. Hereinafter, by actively introducing a wastewater treatment living bacteria agent containing "the microorganism of the present invention" or a fat and oil containing wastewater treatment living bacteria composition containing the microorganism and a fat utilization yeast, a lipid is positively produced. , Protein, starch etc. can be degraded. Then, it is possible to charge all the treating agents at one time from the treating agent feeding device 7. However, quantitative, for example, by means of intermittent feeding, dropping, adding treating agent powder, etc. It is preferable to use this method because it can maintain a constant processing capacity. In addition, the treatment agent may be charged according to the amount of the water to be treated from the inflow pipe 6.

Further, in the waste water treatment apparatus, it is possible to further install the fine air bubble generator 9 having the submersible pump 10 in the treatment tank 2 to enable the circulation of the water 3 to be treated. By installing this micro air bubble generator 9, the amount of air in the treated water is increased, the treated water in the tank is circulated, the activity of the microorganism of the present invention becomes active, and more excellent lipid, protein, starch, etc. Disassembly can be expected.

The micro-bubble generator 9 of the present invention continuously releases micro-bubbles (1 to 1000 μm in diameter) and nano-bubbles (1 to 1000 nm in diameter), and the generation mechanism is crush, cavitation, turbulence or shear, Micropores, individual embedding, electrolysis, chemical reaction etc. These fine bubbles have the property of raising dissolved oxygen in water, and making the drainage in the tank flow and homogenize due to the release force and the difference in specific gravity of water.

The above-described waste water treatment apparatus 1 can greatly reduce the concentrations of BOD, COD and fats and oils as shown in the examples described later, so it can be used as an apparatus for directly discharging treated water to the sewerage However, it can also be used as an apparatus (primary biological treatment tank) for pre-stage treatment in a conventional wastewater treatment apparatus.

In the case of use of the latter, examples of treatment methods in the later stage (secondary biological treatment) include biological treatment such as activated sludge biological method, fluidized bed method, fixed bed method, aggregation treatment, membrane treatment and the like. Thus, the treated water treated in two stages can be discharged to the river in that state, and the waste water treatment apparatus can also significantly reduce the amount of excess sludge generated.

The microorganism used in the present invention described above is a microorganism of the genus Bacillus, which forms spores, and thus can be used in powder form using this. In this case, in order to facilitate dispersion in water, it can be used in the form of being mixed with minerals, grain components such as corn and organic substances such as glucose. Further, it can be kneaded with clay soil and used as a solid (including foamed concrete etc.), which may be dry or contain water. Furthermore, in order to control the growth, a bacteriostatic agent can be added, and it may be a liquid to which, for example, an alcohol, a salt, an emulsifier or the like is added or a component for lowering pH is added.

The invention will be described in more detail with reference to the following examples, but the invention is not limited thereto.

Test example 1
Screening of useful bacteria Microorganisms belonging to the genus Bacillus were collected and separated from soil, dead leaves, drainage and food. Identification of bacteria used for the present invention was carried out according to a general separation / identification method of Bacillus genus in food (Spore experimental manual, p 110-111, published by Shiho Shiho).

Specifically, first, the collected sample is diluted or suspended with an appropriate amount of physiological saline, heated at 80 ° C. for about 10 minutes, streaked on a common agar medium (Eiken Chemical Co., Ltd.), and 37 ° C. It was cultured for 4 days. The number of germinated germs appeared at this time was over 3,000.

Gram-stained pure-cultured strains to confirm gram-positive spore-forming bacilli (Bacillus spp.) Were examined for biochemical characteristics according to the above-mentioned experimental manual to estimate the species. The presumed bacterial species is Bacillus licheniforms, B. coagulans, B. polymyxa, B. B. cereus, B. alvei, B. subtilis, B. pumilus, B .. stearothermophilus, B. macerans, B. et al. megaterium B. circulans B. firmus, B. laterosprus B. brevis, B. sphericus, B. et al. larvae B. popilliae, B. It was lentimorbus.

In the case of the gram-positive spore-forming bacilli, the test was carried out on catalase, VP test, viability on an anaerobic agar medium and starch hydrolyzability, respectively, and 180 strains were selected as Bacillus. The secretory properties of various enzymes were examined, the bacteria having two or more enzymes were stored, and 68 strains were selected except for harmful bacterial species due to the fermentability such as Apitest. These were subjected to the degradability test of Test Example 3 below, and finally Bacillus subtilis HB-88 and Bacillus HB-113 strains having high ability were further selected.

Test example 2
Acquisition of Transformed Bacteria Using Competence Method As a Marberg strain of Bacillus subtilis to be made competent, NBRC14144 strain obtained at NBRC was used. This strain has the ability to transform donor DNA into cells, but lacks strength and lack of secreted enzymes. As a donor strain, Bacillus subtilis strain HB-88 screened from among wild B. subtilis strains as a strain that strongly secretes the enzyme was used.

After suspending the NBRC 14144 strain in petri dish saline (0.85%), antibiotic resistant bacteria are prepared by ultraviolet irradiation, and the antibiotic gradient plate method (Manual of Methods for General Bacteriolyz p 230 1981) is used. Selected using chloralfenicol.

In natural transformation, competent cells were prepared using minimal medium according to the method of Microbial Genetic Experiment p96-101 (1982), and transformed by donating a donor strain. As a result, Bacillus strain HB-14 was obtained.

Test example 3
Selection of degrading enzyme-secreting Bacillus microorganism (1) The secretory enzyme productivity was determined according to the method described in “Research Methods of Natto”, p. The substrate to be examined was dissolved in a standard agar medium and sterilized according to a standard method, and the plate was made by pouring in a plastic dish having a diameter of 85 mm. The titer measured the clear zone of the colony outer side using a paper disc (Toyo Roshi Kogyo Co., Ltd.) of diameter 8 mm and thickness 1.5 mm. All bacterial solutions to be measured were streaked and cultured in a common broth medium for 20 hours with stirring culture, and the effective bacterial solutions were contained in 30 to 70 μl discs and attached to a measurement plate. In the determination of the titer, the clear zone was determined as (+) within 1 mm, 2-3 mm as (++), and 3 mm or more as (+++) from the colony outer edge. In addition, it is assumed that the clear zone is not created is (-).

(2) Confirmation of Degradability of Lipid A standard agar medium containing 3% of tributyrin was sterilized and then emulsified with a Polytron (POLYTRON) homogenizer (KINEMATICA. CH) 20000 rpm for 5 minutes, poured into a petri dish to prepare a lipolytic confirmation plate. As a first screening, the Bacillus microorganism previously separated on this plate was streaked to confirm the degradability of the lipid. 70 μl of positive bacterial solution was contained in a disc, placed on a plate, placed in a thermostat at 37 ° C. for 5 days, and the clear zone was measured. When the colony grew large, if the lower part was transparent, it was added to the clear zone. The HB-14 strain, the HB-88 strain and the HB-113 strain were all (+++).

(3) Confirmation of Degradability of Starch After sterilizing a standard agar medium containing 3% of soluble starch (Wako Pure Chemical Industries, Ltd.), it was poured into a petri dish to prepare a plate for confirming starch degradability. As a first screening, the Bacillus microorganism previously separated on this plate was streaked to confirm the degradability of starch. 30 μl of positive bacterial solution was contained in a disc, placed on a plate, placed in a thermostat at 37 ° C. for 8 hours, and a clear zone suitable for Lugol's solution was measured. The HB-14 strain, the HB-88 strain and the HB-113 strain were all (+++).

(4) Confirmation of Degradability of Protein After sterilizing a standard agar medium containing 10% of soymilk (Future Co., Ltd.), it was poured into a petri dish to prepare a plate for confirming plant proteolysis. As a first screening, the Bacillus microorganism previously separated on this plate was streaked to confirm the protein degradation. 50 μl of positive bacterial solution was contained in a disc, placed on a plate, placed in a thermostat at 37 ° C. for 3 days, and the clear zone was measured. The HB-14 strain, the HB-88 strain and the HB-113 strain were all (+++).

After sterilizing a standard agar medium containing 5% non-fat milk (Taka pear milk industry), it was poured into a petri dish to prepare an animal protein degradation confirmation plate. As a first screening, the Bacillus microorganism previously separated on this plate was streaked to confirm the protein degradation. 70 μl of positive bacterial solution was contained in a disc, placed on a plate, placed in a thermostat at 37 ° C. for 10 hours, and the clear zone was measured. The HB-14 strain, the HB-88 strain and the HB-113 strain were all (+++).

(5) Confirmation of bacterial degradability (I)
E. coli (E. coli NBRC14237 strain) was cultured for 20 hours in a common broth medium, 1% was added to a sterile standard agar medium, mixed uniformly, and a plate for confirming bacterial degradation was prepared in a petri dish. As the first screening, the previously isolated Bacillus microorganism was streaked on this plate to confirm the degradability of the bacteria. 70 μl of positive bacterial solution was placed on a disc, placed on a plate, placed in a thermostat at 37 ° C. for 2 days, and the clear zone was measured. The HB-14 strain, the HB-88 strain and the HB-113 strain were all (+++).

Next, the lactic acid bacteria (Lactobacillus acidophilus (strain ATCC 53103)) are cultured in MRS synthetic medium for 48 hours, 1% is added to the sterilized standard agar medium, and mixed uniformly to prepare a bacterially degradable confirmation plate in a petri dish did. As the first screening, the previously isolated Bacillus microorganism was streaked on this plate to confirm the degradability of the bacteria. 70 μl of positive bacterial solution was placed on a disc, placed on a plate, placed in a thermostat at 37 ° C. for 1 day, and the clear zone was measured. The HB-14 strain, the HB-88 strain and the HB-113 strain were all (+++).

(6) Confirmation of bacterial degradability (II)
In order to clarify the degradation of the cell wall, E. coli and lactic acid bacteria were each cultured for 20 hours, and 160 ml of their respective culture broths were centrifuged at 5000 rpm for 15 minutes to separate the cells. The cells of E. coli and lactic acid bacteria were centrifuged and washed twice with 80 ml of sterile physiological saline (0.85%) to obtain 20 ml of bacterial solution. The bacterial solution was sterilized at 85 ° C. for 10 minutes and cooled with cold water, and then 5% (V / V) was added to a sterile standard agar medium, and mixed uniformly to prepare a cell wall degradable confirmation plate for E. coli and lactic acid bacteria. As the first screening, the previously isolated Bacillus microorganism was streaked on this plate to confirm the degradability of the bacteria. 70 μl of positive bacterial solution was contained in a disc, placed on each of the above plates, placed in a thermostat at 37 ° C. for 10 hours, and the clear zone was measured. The strains HB-14, HB-88 and HB-113 were both (+++) for E. coli and lactic acid bacteria.

(7) Viability from lysis The above-described E. coli was cultured in normal broth medium for 20 hours, and 40 ml of the culture solution was centrifuged at 5000 rpm (rpm) for 15 minutes. Similar centrifugation with physiological saline (0.85%) was repeated three times. Total amount of washed bacteria, 20%, minimum required inorganic salts (NH ₄ NO ₃ ; 1.0%, KH ₂ PO ₄ ; 0.25%, MgSO ₄ · 7H ₂ O; 0.25%, _{_{FeSO 4 · 7H 2 O; 0.0002}} %) and by adding agar to prepare a bacterial degradable check plate in a petri dish. The plate was streaked and cultured in a thermostat at 37 ° C. for 2 days to confirm its degradability. With HB-14 strain, HB-88 strain and HB-113 strain, their viability was confirmed and in the vicinity of the colony Medium became clear. In addition, it confirmed that it did not grow with the agar medium of only the said inorganic salt with respect to the positive strain.

From Test Example 3, the titers of the other degrading enzymes were confirmed in 68 strains having two degrading enzymes (lipid, starch) out of 180 strains. The results are shown in Table 3. All items were found 5 strains of degradable (+++) titer, and HB-13, HB-88, and HB-113 were representative of this.

Degradability titer of Bacillus decomposing enzymes

Example 1
General Analysis Value of Aerobic Treatment HB-113 (NITE BP-1277), which is rich in fat, cell wall, protein and amylolytic enzymes and can be used as a nutrient source, was used as a nutrient source. Take 200 L from the raw water conditioning tank into a 500 L tank and add 100 mg / L of the bacteria mentioned above from the dairy manufacturer's wastewater, and use the micro-bubble generator MAB (Japan Water Treatment Giken Co., Ltd.) The treatment was performed for 24 hours under aerobic conditions of L (25 ° C.) or higher. The analysis values when the wastewater is treated are shown in Tables 4 and 5 below. The bath treated with the addition of HB-113 cells showed very good results in terms of cut rate of BOD, COD, N-hexane, and SS compared to the case without addition.

Result: HB-113 bacteria addition treatment

Result: no added bacteria

Example 2
Sludge volume reduction test Water containing 7% milk is used as a raw water model for drainage, and 200 ml of the strain is a strain of Bacillus HB-14 (NITE BP-1275) cultured in ordinary broth medium (Eiken Chemical Co., Ltd.) 2 ml was added and culture was carried out with stirring at 30 ° C. for 24 hours to obtain pre-culture. This pre-culture solution is added to the sludge solution (test sludge solution; COD = 31) of the waste water treatment of a food company producing 800 ml of confectionery, and the strength of circulating sludge through air stone used for tropical fish (1. The air was fed at 2 to 2.0 mg oxygen / L), and aeration culture was performed. One day later, 200 ml samples were taken and the preculture was added as on the first day. This was continued for one week, and the value of MLSS of the amount of sludge was measured.

In addition, as a comparison, instead of the HB-14 strain, a Bacillus microorganism (not soluble in bacteria, proteolytic (+), lipolytic (-), starch-degradable (+++)) isolated from natto is used. It was. These results are shown in Table 6.

Result:

Example 3
Clogging prevention test in waste water treatment using a membrane (1) 1000 ml of 10% soymilk (component unadjusted soymilk; Inc., Inc.) was used as a drainage stock solution model. To this solution, 20 ml of Bacillus subtilis HB-88 strain (NITE BP-1276) cultured for 20 hours in ordinary broth medium was added, and the strength of circulating sludge through the air stone used for stock tropical fish (1.2 to 2.). Air was sent at 0 mg oxygen / L), and aeration culture was performed all day. After 1 day to 4 days, 200 ml of each sample was taken, and 10% soy milk dewatered stock solution model was added each time to make 1000 ml, and aeration culture was continued. Each sample was centrifuged (3000 rpm) after measuring MLSS, and drainage was possible for 5 days by using a 0.45 micron sterile membrane.

(2) On the other hand, the same procedure as in (1) above is used except that the activated activated sludge liquid (obtained at a food factory; COD = 31) is used instead of Bacillus subtilis HB-88 strain cultured in ordinary broth medium. It was processed. In this case, the 0.45 micron sterile membrane was clogged and could hardly be filtered.

Example 4
Fat and oil reduction test in waste water (1) Inorganic salt ((NH ₄ ) ₂ SO ₄ : 5.0 g, Na ₂ HPO ₄ : 0.5 g, MgSO ₄ · 7H ₂ O: 0.25 g, CaCO ₃ : 5. A solution of 500 ml of the aqueous solution in which 0) was dissolved was sterilized, and 100 ml of skimmed milk (Takahana non-fat milk) made 500 ml of water was added thereto to make a total of 1000 ml as a pre-culture liquid. Two sets of this preculture solution were prepared.

(2) In this pre-culture solution, 20 ml of a yeast (YH-01 strain Hinode Sangyo Co., Ltd.) culture solution, which was cultured with stirring for 2 days at 30 ° C. in normal bouillon medium, and a Bacillus HB-113 strain (NITE BP) -1277) Add 20 ml of the culture solution, keep the container at 30 ° C, send air at a strength (1.2 to 2.0 mg oxygen / L) at which the solution circulates through an air stone, and culture for 1 day. 2 sets).

Water was added to 60 ml of milk (Taka pear unadjusted milk, milk fat percentage: 3.6%) to make the total amount 1000 ml, and used as a model raw water of oil-containing drainage. The initial culture solution was added to this model raw water to make a total of 2000 ml of oil-containing (1080 mg / L) waste water. The oil-containing waste water (2 sets) was kept at 30 ° C. in a container, and air was sent at the strength at which the solution circulated through the air stone in the same manner as above, and was cultured for 3 days. 1000 ml per day was taken as a sample, and the amount of n-hexane extract (oil amount) in treated water was measured.

(3) As a comparative example, only 20 ml of the yeast (YH-01 strain Hinode Sangyo Co., Ltd.) culture solution which was cultured with stirring in a medium of 30 ° C. for 2 days was added to the two sets of precultures prepared in (1). In addition, the culture was conducted in the same manner as in (2) below, and the amount of n-hexane extract in the treatment solution was measured.

(4) As a comparative example, only 20 ml of HB-113 culture solution cultured with stirring at 30 ° C. for 2 days in ordinary bouillon medium is added to the two sets of pre-culture solutions prepared in (1) above. And the amount of n-hexane extract in the treatment solution was measured. The results of (2), (3) and (4) are shown in Table 7.

Example 5
E. coli inhibitory action Inorganic salt ((NH ₄ ) ₂ SO ₄ : 5.0 g, Na ₂ HPO ₄ : 0.5, MgSO ₄ · 7 H ₂ O: 0.25 g, CaCO ₃ : 5.0) in 970 ml water It melt | dissolved, 30 ml of milk (fat percentage 3.6%) was added to this, and it was set as the oil-and-fat containing drainage model of 1000 ml. 10 ml of yeast (YH-01 strain) culture broth cultured at 30 ° C. for 2 days in ordinary bouillon medium and 10 ml of HB-88 strain culture broth (NITE BP-1276) cultured at 35 ° C. for 1 day in ordinary bouillon medium Then, 10 ml of E. coli (NBRC 14237 strain) which was statically cultured at 35 ° C. in a normal broth medium for 1 day was added, and gently culture was carried out at 32 ° C. for 3 days. An analysis sample was collected after one day and three days, and the oil content and the number of bacteria added were counted. The method of measuring the number of bacteria is appropriately diluted with sterile saline according to a standard method, and the detection of yeast (YH-01) is carried out using a subrow-agar medium (Eiken) for 5 days at 30 ° C., Bacillus (HB-88) Were cultured at 37 ° C. for 2 days using a standard agar medium. For E. coli, 0.1 ml of a stock solution was applied to a plate of deoxycholate medium and cultured at 37 ° C. for 2 days. The results are shown in Table 8. As a result, both after 1 day and after 3 days, E. coli decreased, and the degradability of the oil was also sufficiently degraded as in Example 4.

The novel Bacillus microorganism of the present invention has the property of degrading the cell wall, assimilating and growing the degraded cells, secreting an enzyme that degrades lipid, and further secretes an enzyme that degrades protein and starch. It is.

And it becomes possible to improve the volume reduction of the excess sludge of an activated sludge method, the reduction of the fats and oils in waste water, and solid-liquid separation using the characteristic of the said Bacillus-genus microbe. That is, the sludge contains protein viscous substance, polysaccharide mucilage, fat agglomerate, bacteria lump, mineral and the like as a component, and these are aggregated, but the Bacillus microorganism of the present invention is not mineral among these Since it is possible to secrete an enzyme capable of decomposing and to grow with the lysed components, volume reduction of excess sludge can be advantageously promoted.

Moreover, to the waste water containing fats and oils, more excellent fats-and-oils decomposition action can be obtained by making it coexist with the yeast which assimilate fats and oils.

1 ... ... Waste water treatment device 2 ... ... Treatment tank 3 ... ... Water to be treated 4 ... ... Aeration pipe 5 ... ... Air aeration 6 ... ... Water inflow piping to be treated 7 ... ... Treatment agent feeding device 8 ... ... Treatment agent injection tube 9 ... ... Fine air bubble generator 10 ... ... Submersible pump 11 ... ... Processed water discharge piping

Claims

A novel microorganism belonging to the genus Bacillus, which has the ability to degrade at least bacterial cell walls and lipids.
The novel microorganism according to claim 1, further having the ability to degrade protein and starch.
The novel microorganism according to claim 1 or 2, which kills gram-negative bacteria and can be grown using the components of the bacteria as a nutrient source.
The method according to any one of claims 1 to 3, which is designated as Bacillus HB-14 strain (NITE BP-1275), Bacillus HB-88 strain (NITE BP-1276) and Bacillus HB-113 strain (NITE BP-1277). Novel microorganism described in paragraph.
An agent for treating waste water, comprising the novel microorganism according to any one of claims 1 to 4, for preventing excess sludge generation or reducing the volume.
An oil- and fat-containing wastewater treatment composition comprising the novel microorganism according to any one of claims 1 to 4 and an oil-utilizing yeast.
A method for reducing excess sludge in a wastewater treatment facility, characterized in that the novel microorganism according to any one of claims 1 to 4 is added singly or in combination of two or more to the water to be treated in the wastewater treatment facility. Method of packaging.
In the solid-liquid separation wastewater treatment method of removing water using a solid-liquid separation membrane, the action of the novel microorganism according to any one of claims 1 to 4 in the solid-liquid separation membrane is exerted alone or in combination. A method of suppressing flux reduction through a membrane, characterized in that
A treatment tank for containing and treating treated water, an inflow pipe for introducing treated water into the treated tank, a discharge pipe for discharging treated water, and an aeration pipe for aerating treated water An apparatus, comprising: a treatment agent input device for injecting a treatment agent.
10. The waste water treatment apparatus according to claim 9, wherein the treatment agent feeding device feeds a treatment agent in a fixed amount.
The waste water treatment apparatus according to claim 9 or 10, wherein the treatment agent is the waste water treatment biocide according to claim 5.
The waste water treatment apparatus according to claim 9 or 10, wherein the treatment agent is the fat and oil containing waste water treatment living microbe composition according to claim 6.
The wastewater treatment device according to any one of claims 9 to 12, wherein the discharge pipe is provided in an overflow tank provided in the treatment tank.
The waste water treatment apparatus according to any one of claims 9 to 12, wherein the discharge pipe is provided to pump treated water from the treatment tank with a pump.
The waste water treatment apparatus according to any one of claims 9 to 12, further comprising a microbubble generator installed in the treatment tank to enable circulation of the water to be treated.