JP7032756B2 - Organic acid decomposition method, novel organic acid decomposition microorganism and compost production method - Google Patents

Description

NPMD NITE P-02597 NPMD NITE P-02598

The present invention relates to a method for decomposing an organic acid, a novel microorganism capable of decomposing an organic acid, and a method for producing compost using the microorganism.

The increase in waste has become a major problem due to the change in lifestyle to mass production, mass consumption, and mass disposal. In Japan, most of the waste is finally landfilled and treated, but there is a need to recycle the waste due to concerns about a shortage of landfill site. Among the wastes, food wastes discarded from homes, restaurants, food processing factories, etc. are organic substances that do not contain harmful substances such as heavy metals, so they are resources for agricultural use by composting. It is possible to change. Therefore, composting is attracting attention as an important technology for food waste recycling.

However, composting has been far from widespread until now. One of the reasons for this is that it takes a long time to manufacture compost. Compost consisting of food waste is obtained by decomposing food waste by aerobic fermentation of microorganisms, but it takes about 3 months to compost food waste by the usual natural fermentation method (see Patent Document 1). ). Therefore, in Patent Document 1, reconstituted compost containing microorganisms and water are added to food waste, and the mixture is stirred and granulated to a predetermined size to increase the decomposition rate of organic substances and efficiently produce compost. The method is described.

On the other hand, it has been confirmed that the influence of organic acids is the main reason why it takes a long time to compost food waste by the natural fermentation method (see Non-Patent Documents 1 to 3). In food waste, high concentrations of organic acids such as acetic acid and lactic acid are generated to lower the pH at the time of recovery or in the early stage of composting, and organic acids are generated and pH due to the decomposition of organic substances in composting. The activity of composting microorganisms is inhibited each time due to the phenomenon of decreasing pH, and therefore it takes a long time to compost. Therefore, for the purpose of shortening the time required for composting, various methods for suppressing the decrease in pH in the compost raw material have been tried.

Therefore, in Non-Patent Documents 1 to 3, a method of preventing a decrease in pH and promoting composting by adding alkaline substances such as calcium hydroxide, coal and lime or sodium acetate to the compost raw material, respectively. Is described. However, the method of adding an alkaline chemical substance is costly, and there is also a problem that the vegetables cultivated using the compost cannot meet the conditions for organic cultivation due to the use of the chemical substance at the time of compost production. be.

Therefore, a technique for adjusting the pH in the process of composting by utilizing a microorganism that decomposes an organic acid has been attracting attention. For example, Non-Patent Document 4 describes that in the composting of food residues, the organic acid in the compost raw material was decomposed and composting was promoted by inoculating the compost raw material with the Kluyveromyces marxianus Y60 strain of yeast. Has been done. Further, Non-Patent Document 5 describes that by inoculating the compost raw material with the yeast Pichia kudriavzevii RB1 strain, the organic acid in the compost raw material was decomposed, and the effect of promoting the decomposition of organic matter was confirmed accordingly. Has been done.

Japanese Patent No. 4782595

Process Biochem., 1971, Vol.6, p.32-36 Bioresour. Technol., 2009, Vol.100, p.3324-3331 Bioresour. Technol., 2009, Vol.100, p.2005-2011 Lett. Appl. Microbiol., 1998, Vol.26, p.175-178 Bioresour. Technol., 2013, Vol.144, p.521-528

However, the microorganisms that decompose organic acids disclosed in Non-Patent Document 4 and Non-Patent Document 5 belong to yeast having a growth temperature of about 30 ° C. and are not thermophilic microorganisms, and thus are in the process of composting. In the high temperature environment around 60 ° C. where organic matter decomposition is actively performed, it will die. Therefore, in the initial stage of composting (from the start of composting to before the temperature rise period), the above-mentioned organic acid-degrading microorganisms such as yeast decompose the organic acid, so that the organic acid originally present in the composting material is present. Although it is decomposed, in the high temperature period where composting is promoted and organic matter decomposition occurs actively, the organic acid generated by the organic matter decomposition is decomposed to suppress the decrease in pH, and the active organic matter decomposition in the high temperature period is carried out. It was difficult to maintain.

The present invention has been devised in view of the above points, and an object thereof is to provide a novel microorganism that actively decomposes an organic acid in a high temperature environment in the composting process, that is, under high temperature and aerobic conditions. It is an object of the present invention to provide a method for actively decomposing an organic acid using this novel microorganism and a method for promoting the decomposition of an organic substance to efficiently produce compost.

In order to solve the above problems, the method for decomposing an organic acid of the present invention is described in Geobacillus sp. (Geobacillus sp.) AD5 strain (NITE P-02597) or Air Bacillus sp. (Aeribacillus sp.) AD8 strain (NITE P-02598) is used to decompose organic acids.

Geobacillus sp. AD5 strain (NITE P-02597) and Air Bacillus sp. AD8 strain (NITE P-02598) is an aerobic, high-temperature resistant microorganism that does not inhibit growth even in a high-temperature environment of 50 ° C or higher and has the ability to decompose organic acids. Is. Therefore, even under aerobic and high temperature conditions, these microorganisms are actively active and can rapidly decompose organic acids.

Further, in the present invention, it is also preferable that the decomposition of the organic acid is carried out under a temperature condition of 50 ° C. or higher. As a result, the thermophilic bacterium Geobacillus sp. AD5 strain and Air Bacillus sp. A suitable culture temperature for the AD8 strain is selected.

Further, the organic acid in the organic acid decomposition method of the present invention is preferably one or more compounds selected from the group consisting of acetic acid, propionic acid, butyric acid and lactic acid. Geobacillus sp. AD5 strain and Air Bacillus sp. A suitable organic acid for decomposing the AD8 strain is selected.

Further, the microorganism that decomposes the organic acid according to the present invention is Geobacillus sp. (Geobacillus sp.) AD5 strain (NITE P-02597) or Air Bacillus sp. (Aeribacillus sp.) AD8 strain (NITE P-02598).

In addition, the compost production method of the present invention uses Geobacillus sp. (Geobacillus sp.) AD5 strain (NITE P-02597) or Air Bacillus sp. It has a step of inoculating (Aeribacillus sp.) AD8 strain (NITE P-02598) and performing aerobic fermentation under a temperature condition of 50 ° C. or higher.

When organic substances are decomposed by composting microorganisms in the process of composting, organic acids are produced. Geobacillus sp. AD5 strain or Air Bacillus sp. By inoculating the AD8 strain into the organic matter that is the raw material for compost, the generated organic acid is promptly released into Geobacillus sp. AD5 strain or Air Bacillus sp. It is degraded by the AD8 strain. Therefore, the decrease in pH in the compost environment is suppressed, and the active activity of composting microorganisms is maintained. Geobacillus sp. AD5 strain and Air Bacillus sp. Since the AD8 strain is an aerobic thermophile, it can be actively decomposed even under temperature conditions in which organic matter is actively decomposed and high-temperature fermentation heat is generated.

According to the present invention, it is possible to provide an organic acid decomposition method, an organic acid decomposition microorganism and a compost production method having the following excellent effects.
(1) A novel microorganism having organic acid decomposing ability can efficiently decompose an organic acid under high temperature and aerobic conditions.
(2) Since organic acids such as acetic acid produced by decomposition of organic matter can be efficiently decomposed, a decrease in pH in the environment is suppressed, and the activity of composting microorganisms that decompose organic matter is maintained. Therefore, the decomposition of organic matter proceeds rapidly, the composting period is shortened, and composting can be efficiently performed.

It is a graph which shows the time-dependent change of the temperature in the apparatus of Run A (broken line) and Run B (solid line) in Example 1. FIG. It is a graph which shows the time-dependent change of pH of Run A (dashed line) and Run B (solid line) in Example 1. FIG. It is a graph which shows the time-dependent change of the organic acid concentration of Run A (dashed line) and Run B (solid line) in Example 1. FIG. The diamond-shaped marker indicates acetic acid (AA), the square marker indicates lactic acid (LA), the triangular marker indicates propionic acid (PA), and the round marker indicates butyric acid (BA). It is a graph which shows the time-dependent change of the microbial concentration of Run A (dashed line) and Run B (solid line) in Example 1. FIG. Triangular markers indicate yeast, round markers indicate room temperature bacteria, and square markers indicate thermophilic bacteria. It is a graph which shows the time-dependent change of the carbon dioxide gas generation rate of Run A (dashed line) and Run B (solid line) in Example 1. FIG. 2A and 2B are a graph showing changes in the microbial flora of Run A and a graph showing changes in the microbial flora of Run B in Example 2. 3 is a diagram showing (a) BLAST analysis results of AD5 (AD1 to AD7) strains and (b) a diagram showing BLAST analysis results of AD8 strains in Example 3.

The organic acid decomposition method of the present invention comprises Geobacillus sp., Which has organic acid decomposing ability. AD5 strain (NITE P-02597) or Air Bacillus sp. It is carried out by organic acid decomposition using AD8 strain (NITE P-02598). As shown in Examples described later, the present inventors conducted composting tests under various conditions, and found that the organic acid concentration in the composting raw material decreased in a high temperature environment of 50 ° C. to 70 ° C. to pH. It was discovered that there is a test plot (see Run B of Example 1 described later) in which the pH increases, the decomposition of organic matter is promoted, and the generation of carbon dioxide gas is actively performed. Geobacillus sp. Has a resolution of organic acids from compost samples in this test group under high temperature and aerobic conditions. AD5 strain and Air Bacillus sp. AD8 strain was isolated.

Geobacillus sp. AD5 strain (NITE P-02597) and Air Bacillus sp. The culture temperature of the AD8 strain (NITE P-02598) is preferably 50 ° C. or higher, more preferably 50 ° C. to 80 ° C., and particularly preferably 60 ° C. to 70 ° C. Under such culture conditions, Geobacillus sp. AD5 strain or Air Bacillus sp. By culturing the AD8 strain, these thermophilic microorganisms grow and the organic acids existing in the culture environment are decomposed.

In addition, in the decomposition of organic acids, a considerable amount of Geobacillus sp. AD5 strain or Air Bacillus sp. Cultures, spores, etc. of the AD8 strain may be added to the decomposition target containing an organic acid. Geobacillus sp. AD5 strain and Air Bacillus sp. All AD8 strains are microorganisms belonging to the family Bacillaceae, and are TS liquid media ((Trypticase peptone 17 g / L, Phyton peptone 3 g / L, Sodium chloride 5 g / L, K ₂ HPO ₄ 2.5 g / L, D). -Glucose 2.5 g / L, pH 7.3) or TS agar medium (TS liquid medium plus 20 g / L agar) is suitably cultured and propagated.

Geobacillus sp. AD5 strain and Air Bacillus sp. The organic acid decomposed by the AD8 strain is not particularly limited, and examples thereof include formic acid, acetic acid, propionic acid, butyric acid, valeric acid, lactic acid, succinic acid, oxaloacetate, malonic acid, and fumaric acid. These organic acids are produced by decomposing compost raw materials such as food residues by composting microorganisms at the time of compost production. Therefore, Geobacillus sp. AD5 strain or Air Bacillus sp. The AD8 strain can be used for the decomposition of organic acids during compost production.

Next, Geobacillus sp. AD5 strain or Air Bacillus sp. A compost manufacturing method using the AD8 strain will be described. For compost raw materials mainly composed of food waste such as food residues, Geobacillus sp. AD5 strain or Air Bacillus sp. The process of inoculating the AD8 strain and performing aerobic fermentation will be described. For inoculation, Geobacillus sp. AD5 strain or Air Bacillus sp. Any one of AD8 strains or a combination thereof may be inoculated. A considerable amount of Geobacillus sp. AD5 strain or Air Bacillus sp. It can be easily inoculated by adding a culture of AD8 strain, spores, or the like to a compost raw material and mixing them. After inoculation, the composting temperature is preferably 50 ° C. or higher, more preferably 50 ° C. to 80 ° C., and particularly preferably 60 ° C. to 70 ° C. Under such culture conditions, Geobacillus sp. AD5 strain and Air Bacillus sp. By culturing the AD8 strain, these microorganisms grow in the compost environment.

When composting starts, the environmental temperature is about room temperature at the initial stage of composting, so it looks like the organic acid-degrading microorganisms of normal temperature bacteria originally present in the composting material, or the Pichia kudriavzevii RB1 strain separately inoculated into the composting material. Organic acid-degrading microorganisms (both of which cannot grow at high temperatures) decompose organic acids originally contained in compost raw materials. Along with this, composting microorganisms responsible for organic matter decomposition are activated and composted. Since the conversion temperature gradually rises and the temperature rises to the high temperature period, these organic acid-degrading microorganisms that cannot grow at high temperatures are killed. Further, under aerobic and high temperature conditions, which are suitable culture environments for composting microorganisms that decompose organic matter, active decomposition of organic matter occurs and a large amount of organic acid is produced at the same time. Geobacillus sp. AD5 strain or Air Bacillus sp. Since the AD8 strain is inoculated into the compost material, these microorganisms actively decompose the organic acid produced under high temperature conditions and prevent the accumulation of the organic acid. Therefore, the decrease in pH in the compost environment is suppressed, the active activity of microorganisms that decompose organic matter is maintained, and composting proceeds efficiently. The composting period is preferably about 3 to 21 days, and preferably about 5 to 14 days.

By the above-mentioned composting production method, a high temperature period in which microorganisms actively decompose organic matter is maintained, and composting of organic matter, which normally takes about 3 months, can be shortened to about 2 weeks or less. In addition, since the fermentation period at high temperature can be stably obtained, it is possible to sufficiently ferment and reduce the variation in compost products.

Geobacillus sp. With the organic acid decomposing ability of the present invention. AD5 strain (NITE P-02597) and Air Bacillus sp. The AD8 strain (NITE P-02598) can be used not only for composting as described above, but also for the decomposition of organic acids for any purpose. Although not particularly limited, it can be suitably used, for example, in the case of decomposing an organic acid contained in factory wastewater.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not particularly limited to these Examples.

[Example 1]
1. 1. Composting of compost mixed raw materials The compost raw materials and compost mixed raw materials used in this example are as follows.
(Compost raw material)
Generally, various food residues and food wastes are used as compost raw materials, but in this embodiment, in order to obtain reproducible data, a commercially available rabbit food (rabbit) is used as a model food residue. Food Timothy, a product of Easter Co., Ltd.) and rice (Sato rice, a product of Sato Foods Industry Co., Ltd.) were used. The elemental content of carbon contained in each material is 44.0% for rabbit food and 43.2% for cooked rice, and the elemental content of nitrogen is 2.4% for rabbit food and 0.89% for cooked grain. Met. The C / N ratio was 18.1 for rabbit food and 48.5 for cooked rice.

(Compost mixed raw material)
Rabbit food and cooked rice, which are the above-mentioned compost raw materials, were mixed at a dry weight ratio of 7: 3. A raw material consisting of this rabbit food and rice, sawdust, which is a breathable improving material, and a commercially available microbial material (Aures G, Matsumoto Microbial Research Institute Co., Ltd.) as an inoculum are mixed in a dry weight ratio of 10: 9: 1. Then, it was used as a compost mixed raw material. The concentrations of room temperature and thermophilic bacteria in the microbial material were 1.28 × 10 ⁶ CFU / g-ds, respectively, 3.98 × 10 ⁴ CFU / g-ds. This was inoculated with a yeast Pichia kudriavzevii RB1 strain capable of degrading organic acids (see Non-Patent Document ⁵ ) so as to be 105 CFU / g-ds in a compost mixed raw material. Normally, food residues discharged from homes, restaurants, food processing factories, etc. have a low pH because they are rancid and contain a large amount of organic acids before they are composted. In this example, four kinds of organic acids, specifically acetic acid, are used with reference to the description in the literature of Sundberg et al. (Bioresour. Technol., 2011, Vol.102, p.2859-2867). 90 g / kg-ds, propionic acid 3.02 g / kg-ds, butyric acid 2.43 g / kg-ds, and lactic acid 12.45 g / kg-ds were added to the compost mixed feedstock. After adding these organic acids, the pH of the compost mixed raw material was around 5.0, which was equivalent to the pH value of general food residues. Further, at the start of composting, distilled water was added to adjust the water content of the compost mixed raw material to 60%.

(Composting)
For composting, a laboratory-scale composting device (cylindrical, volume about 30 L) was used. The main body of the device that houses the compost mixed raw material is made of heat-resistant polyvinyl chloride, and a ribbon heater for adjusting the temperature inside the device is wrapped around the main body of the device and embedded in a heat insulating material made of styrofoam. .. About 3 kg of the compost mixed raw material was put into the apparatus by wet weight, and composting was performed while ventilating from the bottom. For composting, the test was conducted under two compost conditions, Run A and Run B. Run A performs composting without controlling the temperature rise process associated with the decomposition of organic matter, and Run B once controls the temperature inside the device to 40 ° C. during the temperature rise process, and yeast contained in the compost mixed raw material. After promoting the decomposition of organic acids in the Pichia kudriavzevii RB1 strain, the temperature is raised to 60 ° C. for composting.

Specifically, in Run A, the device temperature at the start of composting was set to room temperature (around 25 ° C.), and the aeration flow rate was adjusted to 45 L / h, which is the minimum required to maintain aerobic conditions. The temperature inside the compost increased due to self-heating due to the decomposition of organic matter by microorganisms, and after reaching 60 ° C, the temperature was controlled to 60 ° C by adjusting self-heating and external heating. The composting period was 10 days. On the other hand, in Run B, the device temperature at the start of composting was set to room temperature (around 25 ° C.), and the aeration flow rate was adjusted to 45 L / h. When the temperature inside the compost reaches 40 ° C in the process of raising the temperature, the aeration volume is increased to cool the inside of the compost while maintaining the temperature at 40 ° C for 24 hours. Promoted the decomposition of organic acids in the yeast. After that, the air volume was returned to 45 L / h, the temperature inside the compost was raised to 60 ° C., and the temperature was controlled to 60 ° C. by adjusting self-heating and external heating. The composting period was 10 days.

For uniform organic matter decomposition in the composting process, the lid of the device was opened once a day during the composting period, and the compost mixed raw material inside the device was mixed and agitated. At the time of turning back, distilled water was appropriately added so that the water content of the composted product did not decrease too much. At the time of turning back, about 15 g of compost sample was collected as an analysis sample.

In addition, the pH, water content, organic acid concentration, microbial concentration, and carbon dioxide generation rate in composting of the compost sample performed in this example were measured as follows.

(1) pH
The collected 2 g of compost sample was placed in a homogenize cup, 18 g of sterile water was added, and a homogenizer (Nissei Tokyo Office product, product name: AM-5) was used to make a uniform suspension at 10000 rpm for 10 minutes. .. The pH of the suspension was measured with a pH meter (HORIBA, Ltd. product, product name: D-51).

(2) Compost samples with a moisture content of about 1 g are collected in three weighing bottles, weighed, and then dried at 105 ° C for 24 hours in a constant temperature dryer (Yamato Kagaku Co., Ltd. product, product name; DS-600). rice field. The weight of the compost sample after drying was measured, and the value obtained by dividing the reduced weight after drying by the weight before drying was determined as the water content.

(3) Organic acid concentration The suspension of the sample prepared at the time of the above pH measurement was filtered using a 0.20 μm membrane filter to obtain a filtrate. The organic acid contained in this filtrate was analyzed by high performance liquid chromatography (HPLC; JASCO Corporation product, ChromNAV). SUGAR SH1011 (a product of Showa Denko KK) was used for the column, and the column temperature was set to 50 ° C. A degassed 5 mM sulfuric acid aqueous solution was used as the mobile phase of HPLC, and the flow rate was set to 1.0 mL / min. The HPLC detector used was a product of JASCO Corporation, model number; UV-2075, and the detection wavelength was 210 nm.

(4) Microbial concentration The concentrations of yeast, room temperature bacteria and thermophilic bacteria in the compost sample were determined by the dilution plate method using an agar medium. 9 times the amount of sterile water was added to 2 g of the collected compost sample, and the homogenized product at 10000 rpm for 10 minutes was appropriately diluted and smeared on the medium. A PD agar medium (a product of Eiken Chemical Co., Ltd.) was used to measure the yeast concentration, and the culture temperature was 30 ° C. and the culture period was 3 days. In addition, in order to suppress the growth of bacteria, 1 mL of chloramphenicol solution (100 mg of chloramphenicol dissolved in 1 mL of ethanol) was added to 1 L of PD agar medium. For measuring the concentrations of room temperature bacteria and thermophilic bacteria, TS agar medium (trypticase peptone 17 g / L, phyton peptone 3 g / L, sodium chloride 5 g / L, K ₂ HPO ₄ 2.5 g / L, D- Using 2.5 g / L of glucose, 20 g / L of agar, pH 7.3), the culture temperature was 30 ° C. for room temperature bacteria, 60 ° C. for thermophilic bacteria, and the culture period was 3 days. When measuring room temperature bacteria, 100 μL of amphotericin B solution (25 mg of amphotericin B dissolved in 1 mL of dimethyl sulfoxide) was added to 1 L of TS agar medium in order to suppress the growth of filamentous fungi. The cell concentration was expressed as CFU / g-ds, which is the number of colonies formed per 1 g of dry weight of the compost sample.

(5) Carbon dioxide generation rate The rate of carbon dioxide decomposition in composting is the rate of carbon dioxide generation (unit:) by measuring the amount of carbon dioxide generated by composting because carbon dioxide is generated when organic matter is decomposed. Time and unit The amount of carbon dioxide generated per dry weight of the compost sample; mol / h / g-ds) was quantified. For the carbon dioxide generation rate, the carbon dioxide concentration in the exhaust gas is measured with an infrared gas analyzer (Riken Keiki Co., Ltd. product, model; RI-555), and the ventilation speed is measured with a gas meter (Shinagawa product, model; DC-2). ) Was continuously measured and calculated. The amount of organic matter decomposed was quantified by the carbon change rate defined as the ratio of the amount of carbon lost as carbon dioxide to the amount of carbon in the food residue.

After the composting started, the temperature inside the device was measured. In addition, the pH, water content, organic acid concentration and microbial concentration were measured using the compost sample collected as an analysis sample. In addition, the amount of carbon dioxide gas generated by composting was measured, and the carbon dioxide gas generation rate per unit time was determined. These results are shown in FIGS. 1 to 5, respectively. The horizontal axis shows the number of composting days (days) after the start of composting. Run A (dashed line) shows the results under the test conditions where composting was performed without controlling the temperature rise process associated with organic matter decomposition, and Run B (solid line) shows the temperature inside the device once during the temperature rise process. The results are shown under the test conditions in which the temperature was maintained at 40 ° C. for 24 hours, the temperature was raised to 60 ° C. after promoting the decomposition of organic acids in the yeast Pichia kudriavzevii RB1 strain contained in the compost mixed raw material, and then composting was performed. ..

FIG. 1 shows the time course of the temperature inside the device. In Run A, the temperature first rose to 30 ° C. or higher, dropped once, and then reached around 53 ° C. After that, the temperature dropped to room temperature again, but the temperature reached 60 ° C. 5 days after the start of the test, and thereafter maintained at 60 ° C. by external heating. On the other hand, in Run B, after reaching 40 ° C. in the temperature rising period, the state of 40 ° C. was maintained for 24 hours by increasing the aeration flow rate and cooling. After that, when the aeration flow rate was returned to 45 L / h, the temperature reached 60 ° C. by 4 days from the start of the test, and thereafter, the temperature was maintained at 60 ° C. by combining self-heating and external heating.

FIG. 2 shows the change over time in pH. In composting under any of the test conditions of Run A and Run B, the pH rapidly increased from 5 to around 6.5 and maintained at that value until 2 days after the start of the test. After that, in Run A, the pH dropped to about 4.5, and although it rose slightly around 5 days, it dropped again and maintained a low pH around 5 until the end of composting. On the other hand, in Run B, the pH decreased to around 5.3 3 days after the start of the test, then gradually increased from around 6 days, and finally showed a high value of about 8.5. According to this result, since the pH of Run B is finally weakly alkaline, not only the organic acid is decomposed, but also the decomposition of the protein produces ammonia, which is an alkaline substance, that is, the decomposition of the protein. It is believed that it was actively carried out. As a result of the above, there was a large difference in pH change between Run B with temperature control of 40 ° C during the temperature rise period and Run A without temperature control, and Proteolysis was more active in Run B. I understood. In both Run A and Run B composting, the water content of the composted product was maintained at around 60%, which satisfied the condition that active organic matter decomposition occurred in composting.

FIG. 3 shows the change over time in the organic acid concentration. In composting under any of the test conditions of Run A and Run B, acetic acid (AA), lactic acid (LA), propionic acid (PA) and butyric acid (BA) in the compost mixed raw material were decomposed immediately after the start of composting. .. However, in Run A, 24 g / kg-ds acetic acid accumulated 3 days after the start of the test. It is considered that this is because not only carbon dioxide gas is generated by the decomposition of organic matter, but also a part of acetic acid, which is a decomposition intermediate, is accumulated without being decomposed into carbon dioxide gas. After that, the acetic acid contained in Run A decreased once on the 5th day from the start of the test, but after that, a high concentration of acetic acid of 20 to 25 g / kg-ds was accumulated again. On the other hand, in Run B, the acetic acid concentration was maintained at a low value until 4 days after the start of the test as compared with Run A. It is considered that this is because the RB1 strain decomposed acetic acid and the acetic acid concentration was suppressed to a low level as a result of maintaining the temperature inside the apparatus at around 40 ° C. suitable for the activity of the RB1 strain in the temperature raising process. After that, in Run B, when the temperature inside the device reaches 60 ° C., the yeast RB1 strain loses its activity, so acetic acid gradually accumulates and the acetic acid concentration rises to about 15 g / kg-ds. After the day passed, active decomposition of acetic acid began again, and finally all organic acids including acetic acid were decomposed, and no organic acid remained.

FIG. 4 shows the time course of the concentration of microorganisms (yeast, room temperature bacteria, thermophilic bacteria). ^In both the test conditions of Run A and Run B, yeast proliferated rapidly and the yeast concentration reached 109 CFU / g-ds or more. The medium used for counting yeast was a medium for fungi, but it was confirmed that most of the colonies that appeared were Pichia kudriavzevii RB1 strains. After that, in Run A, the concentration of the RB1 strain changed as it decreased and increased, but since it could not survive at high temperature, the concentration decreased to below the detection limit after 6 days when the temperature was maintained at 60 ° C. On the other hand, in Run B, the temperature inside the device during the temperature rise period was maintained at 40 ° C., so after maintaining the high concentration for several days, the concentration dropped to below the detection limit 4 days after the start of the test when the temperature reached 60 ° C. .. As for room temperature bacteria, Run A grew to ¹⁰⁸ CFU / g-ds and then kept a constant value, but Run B grew to around ¹⁰⁹ CFU / g-ds, which is higher than that of Run A. The final concentration is shown. As for thermophilic bacteria, the concentration of Run B with temperature control at 40 ° C was an order of magnitude higher than that of Run A without temperature control. These differences in microbial concentration correspond to the fact that Run B promoted organic matter decomposition compared to Run A. In addition, in both Run A and Run B, the change in the concentration of the room temperature bacterium and the thermophilic bacterium was small in the latter half of the test, and the microbial concentration became substantially constant.

FIG. 5 shows the change over time in the carbon dioxide gas generation rate. For both Run A and Run B, first, the peak of the carbon dioxide gas generation rate, which is considered to correspond to the decomposition of the organic acid contained in the compost mixed raw material, was observed. In Run A, a second peak of carbon dioxide generation rate was subsequently observed 2 days after the start of the test, but the peak shape was sharp and carbon dioxide generation did not continue for a long period of time. At this time, in Run A, a peak of temperature rise corresponding to this second peak was observed (see FIG. 1). After that, a third peak of the carbon dioxide generation rate was observed around 5 days after the start of the test, and this third peak also corresponded to the rapid rise in the temperature of Run A on the 5th day shown in FIG. It turned out that. This result is considered to be due to the accumulation of metabolic heat generated when organic matter is actively decomposed and the temperature rise. On the other hand, in Run B, the second peak is broad. This means that the decomposition of organic acids is continued by maintaining 40 ° C., which is suitable for the activity of the yeast RB1 strain, during the temperature rise period, the activity of microorganisms other than the RB1 strain in the composted product is also enhanced, and the decomposition of organic matter proceeds. It shows that. After that, the carbon dioxide generation rate decreased, but in Run B, the carbon dioxide generation rate increased again 7 days after the start of the test, and a third peak was observed. This result indicates that the environment for active decomposition of organic matter by thermophilic microorganisms was set up before 7 days.

Comprehensive analysis of the data shown in FIGS. 1 to 5 shows that in Run A, first, the organic acid in the compost mixed raw material is decomposed by the yeast RB1 strain, so that the organic acid concentration decreases, the pH increases, and carbonic acid. Gas is being generated. As the pH rises, the activity of microorganisms other than the RB1 strain contained in the composted product also increases, so that organic matter decomposition progresses. Two days after the start of the test, the carbonic acid gas generation rate increases and the organic matter decomposition intermediate Organic acid is produced. Here, when the organic acid is accumulated, the pH is lowered and the activity of the microorganism (yeast RB1 strain and other microorganisms) is also lowered, so that the carbon dioxide gas generation rate is also lowered. This reduces calorific value and lowers the temperature, allowing the surviving RB1 strain to grow again, thus decomposing the accumulated organic acids and slightly increasing the pH. It is considered to be. When the pH rises, carbon dioxide gas is also generated again, so that heat of metabolism is generated, and the temperature rises to reach 60 ° C. As the decomposition of organic matter progresses, the production of organic acid and carbonic acid gas progresses, but the RB1 strain is dead due to the high temperature condition of 60 ° C., and the pH drops due to the accumulation of organic acid, so that the whole microorganism The activity has also decreased, and carbon dioxide is no longer generated. At this time, almost no heat of metabolism is generated, but the temperature is maintained at 60 ° C. because it is maintained by external heating.

On the other hand, in Run B, first, since the organic acid in the compost mixed raw material is decomposed by the yeast RB1 strain, the organic acid concentration decreases, the pH increases, and carbonic acid gas is generated. After the second day from the start of the test, organic acid, which is an intermediate for decomposition of organic matter, is also produced, but since the yeast RB1 strain decomposes the organic acid while the temperature inside the device is maintained at 40 ° C. It occurs more often than Run A. When the temperature control at 40 ° C in the device is completed, the temperature inside the device gradually rises due to the accumulation of metabolic heat and reaches 60 ° C on the 4th day after the start of the test, but at 60 ° C, the yeast RB1 strain is killed. , Organic acids are gradually accumulated, the pH is lowered, the activity of the whole microorganism is inhibited, and the carbonic acid gas generation rate is extremely low. However, the decomposition of the organic acid started 6 days after the start of the test in which the temperature inside the apparatus was maintained at 60 ° C., the pH also increased, and a large amount of carbon dioxide gas was generated. The results of this series of Run B show that the accumulation of organic acids can be reduced and the decomposition of organic substances can be promoted by controlling the temperature at 40 ° C. More importantly, the decomposition of organic acids was carried out in composting, which was maintained at a high temperature of 60 ° C. under aerobic conditions. This means that there are microorganisms that decompose organic acids under high temperature and aerobic conditions. The existence of microorganisms that decompose organic acids under normal temperature conditions, such as yeast RB1 strain, has been confirmed, and there are microorganisms that decompose fatty acids to produce methane in methane fermentation under high temperature and anaerobic conditions. Is also known. However, until now, the existence of microorganisms capable of actively decomposing organic acids in the process of composting under high temperature and aerobic conditions has not been known. Therefore, in Examples 2 and 3 below, an attempt was made to analyze and isolate a microorganism that decomposes an organic acid confirmed by Run B.

[Example 2]
2. 2. Analysis of the microbial flora in the compost sample The changes in the microbial flora in the process of composting the compost mixed raw material in Example 1 were analyzed by the method of Next Generation Sequencing (NGS). In Example 1, when the compost mixed raw material was cut back, DNA was extracted from 0.2 g of the compost sample collected from the inside of the apparatus as an analysis sample using a DNA extraction kit (ISOIL for Beads Beating; Nippon Gene Co., Ltd.). For the extracted DNA, a part of the 16S rRNA gene (gene region containing V3 and V4 regions) was amplified by PCR. For PCR, a DNA polymerase for hot start PCR (TaKaRa EX Taq (registered trademark) Hot Start Version, RR006A; Takara Bio Inc.) is used, and a PCR device (TaKaRa PCR Thermal Cycler Dice (registered trademark) Standard, TP600; Takara; Bio Co., Ltd.). The composition of the PCR reagent, the sequence of the primers and the amplification conditions are shown in Tables 1 and 2, respectively. The underlined part of each primer sequence in Table 2 is a sequence for indexing to identify each sample, and the sequence other than the underlined part corresponds to the gene region containing the V3 and V4 regions of prokaryotic 16S rRNA. ing. For each PCR product, 1.0 × Tris-borate-EDTA (TBE) agarose gel (agar concentration 2%) electrophoresis was performed, and the gene region containing the V3 and V4 regions of the prokaryotic 16S rRNA, which is the target sequence, was amplified. I confirmed that.

Each PCR product was purified using a DNA purification kit (Agencourt AMPure XP; Beckman Coulter, Inc.). Purified PCR products were indexed for multisample analysis using the Nextera XT Index kit (Illumina, Inc.) and DNA polymerase (TaKaRa EX Taq® Hot Start Version; Takara Bio Inc.). The composition of the reagent for indexing is shown in Table 3. The PCR conditions for indexing were the same as the PCR conditions shown in Table 2. Then, the indexed DNA fragment was purified and the base sequence was determined.

The analysis of the obtained nucleotide sequence data is performed by a data analysis tool, Quantitative Insights Into Microbial Ecology (QIIME) ver 1.9.1 (Caporaso et al., Nature Methods, 2010, Vol.7, p.335-336). ) Was used. The adapter sequence was removed, and the sequence of about 300 bases read from the forward and reverse primers was aligned, and then the sequence was complemented to obtain a sequence of about 465 bases. Then, 16S rRNA sequence data was analyzed by OTU and distributed to a bacterial taxon to obtain a microbial flora distribution. Table 4 below shows a list of analyzed microbial taxa.

In addition, FIG. 6 shows the results of NGS analysis of the microbial flora that changes in the process of composting in Example 1. First, the change in the microbial flora in Run A is shown in FIG. 6 (a). Throughout the composting process, NO. 113 Bacillus microorganisms are overwhelmingly predominant. However, in Run A, the organic acid was accumulated in the composted product and the pH remained low. The 113 Bacillus microorganisms were considered to have extremely low organic acid degrading activity under aerobic and high temperature composting conditions.

Then, the change of the microbial flora in Run B in which the presence of the microorganism that decomposes the organic acid was confirmed is shown in FIG. 6 (b). One day after the start of composting, NO. The abundance of 110 Bacillaceae microorganisms is high, but NO. 113 Bacillus microorganisms are overwhelmingly predominant, especially on the 4th day. The relative abundance of 113 microorganisms was 80%. After that, after 6 days from the start of composting, NO. The abundance of 113 microorganisms was reduced to 20-30%, but the abundance was maintained higher than that of other microorganisms. In addition, after 6 days from the start of composting, NO. 114 Bacillaceae microorganisms, NO. 202 Microorganisms of the genus Symbiobacterium, NO. 108 Bacillales microbes and NO. The abundance of microorganisms of the genus Ureibacillus such as 137 is remarkably increased. Of these, NO. The abundance of 114 Bacillaceae microorganisms reached a maximum of 20% on the 6th day of composting, and then gradually decreased with the progress of composting. According to the results of Run B of Example 1, active decomposition of organic matter was observed at a high temperature of 60 ° C. after the 6th day from the start of composting. Microorganisms other than 113, which showed a high abundance on the 6th day and after the 6th day of composting, that is, NO. 114 (Bacillaceae), NO. 202 (Genus Symbiobacterium), NO. 108 (Bacillales) and NO. It was considered highly likely that any or more of the 137 (Ureibacillus) microorganisms were degrading organic acids.

[Example 3]
3. 3. Isolation and identification of organic acid-degrading microorganisms (isolation of microorganisms)
Among the Run B compost samples collected from the inside of the apparatus as an analysis sample when the compost mixed raw material was cut back in Example 1, the organic acid-degrading microorganisms were found in the compost sample on the 6th day when active organic acid decomposition occurred. An attempt was made to isolate. After diluting the compost sample to 1/100 with sterile water, inoculate it into the inorganic salt acetic acid culture solution shown in Tables 5 and 6 below, which contains acetic acid as the only carbon source, and inoculate the culture at a culture temperature of 60 ° C. for 3 days. Enrichment culture was performed. 100 μL of this culture solution was smeared on an agar medium of an inorganic salt acetic acid culture solution and cultured at a culture temperature of 60 ° C. and a culture period of 5 days. Eight colonies were observed on the agar medium after culturing. The AD1 to AD8 strains of these eight colonies were recovered and isolated as organic acid-degrading microorganisms.

(Identification of microorganisms)
DNA was extracted from eight isolated microbial strains AD1 to AD8 using a DNA extraction kit (ISOIL for Beads Beating; Nippon Gene Co., Ltd.). The 16S rRNA region of DNA extracted from microorganisms was amplified by PCR. For PCR, a DNA polymerase for hot start PCR (TaKaRa EX Taq (registered trademark) Hot Start Version, RR006A; Takara Bio Inc.) is used, and a PCR device (TaKaRa PCR Thermal Cycler Dice (registered trademark) Standard, TP600; Takara; Bio Co., Ltd.). The composition of the PCR reagent, the sequence of the primers and the amplification conditions are shown in Tables 7 and 8, respectively. For each PCR product, 1.0 × Tris-borate-EDTA (TBE) agarose gel (agar concentration 2%) electrophoresis was performed, and it was confirmed that the 16S rRNA region, which was the target sequence, was amplified.

Each PCR product confirmed to be amplified in the target region was purified using a DNA purification kit (Wizard® SV Gel and PCR Clean-Up System; Promega Co., Ltd.). A portion of 16S rRNA containing the V3 and V4 regions was amplified using Primer 357F and Primer 907R on the purified PCR product. Table 9 shows the primer sequences and amplification conditions.

The amplified PCR product was sequenced using a DNA sequencing reaction kit (BigDye® Terminator v3.1 Cycle Sequencing Kit; Applied Biosystems, Inc.). The base sequence was analyzed by a DNA sequencer (ABI Prism (registered trademark) 310 Genetic Analyzer; Applied Biosystems Co., Ltd.). For the determined nucleotide sequence, BLAST analysis was performed on the sequence registered in NCBI (http://www.ncbi.nlm.nih.gov/) to identify the most closely related microorganism.

When the DNAs of AD1 to AD8 isolated as organic matter-degrading microorganisms were analyzed, it was found that the seven microbial strains of AD1 to AD7 had the same base sequence, and that AD1 to AD7 were all the same microorganism. On the other hand, the base sequence of AD8 is different from that of AD1 to AD7, and it was found that AD8 and AD1 to AD7 are different microorganisms. FIG. 7 shows the BLAST analysis results of the AD5 (AD1 to AD7) strains and the AD8 strains. According to this, the AD5 (AD1 to AD7) strains are bacteria belonging to the genus Geobacillus of the family Bacillaceae, and have the highest homology with Geobacillus thermogenitrificans (homology 98%). On the other hand, the AD8 strain was found to be a bacterium belonging to the genus Aeribacillus of the family Bacillaceae and closely related to Aeribacillus pallidus (homology 99%). The "AD5" and "AD8" were deposited at the Patent Microorganisms Depositary Center as AD5: NITE P-02597 and AD8: NITE P-02598.

In order to reflect the microbial identification results of the AD5 (AD1 to AD7) strains and the AD8 strains in the NGS analysis results in Example 2, the AD5 (AD1 to AD7) strains and the AD8 strains were prepared using the base sequence information of AD5 and AD8. It was confirmed which microorganism classification group shown in Table 4 and FIG. 6 in Example 2 was classified. As a result, the AD5 (AD1 to AD7) strains identified as microorganisms belonging to the genus Geobacillus of the family Bacillaceae in this example were designated as NO. It was found to be classified in 114 Bacillaceae microbial taxa. From this result, in Run B of Example 1, NO. It was revealed that 114 microorganisms, that is, AD5 strain, were abundantly present on the 6th day of composting, and the organic acid decomposition was actively carried out under aerobic and high temperature conditions. On the other hand, the AD8 strain identified as the genus Aeribacillus of the family Bacillaceae in this example is the NO. It was classified into 137 microbial taxa of the family Plananococasee. The AD8 strain is closely related to Aeribacillus pallidus, which was reclassified from the genus Geobacillus in 2010. Therefore, the AD8 strain was expected to be classified into at least the microbial taxon belonging to the Bacillaceae family, but unlike the expectation, it was classified into a different microbial taxon (at the Order level, the AD8 strain was also classified. Since both microorganisms of No. 137 are classified in the order Bacillales, they are in agreement). The reason for this is considered to be that the microbial databases used for BLAST search and NGS analysis are different. NO., Which is a microbial taxon corresponding to this AD8 strain. 137 was a microorganism that was abundant in Run B of Example 1 on the 6th day of composting and was presumed to be involved in organic acid decomposition. Therefore, in Run B of Example 1, NO. It was speculated that 137 microorganisms, that is, AD8 strain, were abundantly present on the 6th day of composting, and that organic acid decomposition was actively carried out under aerobic and high temperature conditions. From these results, it was clarified that the AD5 (AD1 to AD7) strains and the AD8 strains proliferate under aerobic and high temperature conditions in composting and actively decompose organic acids.

The present invention is not limited to the above-described embodiment or embodiment, and various design-modified forms within the scope of the invention described in the claims are also included in the technical scope. It is a thing.

NITE P-02597
NITE P-02598

Claims (5)

Geobacillus sp. (Geobacillus sp.) AD5 strain (NITE P-02597) or Air Bacillus sp. (Aeribacillus sp.) A method for decomposing an organic acid, which comprises decomposing an organic acid using an AD8 strain (NITE P-02598). The organic acid decomposition method according to claim 1, wherein the decomposition of the organic acid is carried out under a temperature condition of 50 ° C. or higher. The organic acid decomposition method according to claim 1 or 2, wherein the organic acid is one or more compounds selected from the group consisting of acetic acid, propionic acid, butyric acid and lactic acid. Geobacillus sp. (Geobacillus sp.) AD5 strain (NITE P-02597) or Air Bacillus sp. (Aeribacillus sp.) AD8 strain (NITE P-02598). Geobacillus sp. (Geobacillus sp.) AD5 strain (NITE P-02597) or Air Bacillus sp. (Aeribacillus sp.) A compost production method comprising a step of inoculating AD8 strain (NITE P-02598) and performing aerobic fermentation under a temperature condition of 50 ° C. or higher.

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