KR20040019535A - The thermophilic microorganisms having the biological composting - Google Patents

Abstract

PURPOSE: Thermophilic microorganisms having improved organic matter decomposing activity are provided. The microorganisms can grow at high temperature of 60 deg. C or more and effectively decompose organic matter. Therefore, compost can be effectively prepared. CONSTITUTION: A microbial preparation for accelerating decomposition of organic matters comprises thermophilic microorganisms Bacillus stearothermophillus NIAST, Chaetomium thermophile NIAST and Thermoactinomyces NIAST. A method for culturing thermophilic microorganisms comprises the steps of: inserting a sterilized medium into a culture vessel; inoculating the thermophilic microorganisms into the medium; culturing the thermophilic microorganisms at an appropriate temperature with supplying air.

Description

The thermophilic microorganisms having the biological composting

The present invention relates to a thermophilic microorganism and a method for culturing the microorganisms that make up the organic material home-promoting microorganisms that can increase the home-combustion efficiency while decomposing various organic materials present in the organic by-products. More specifically, Bacillus stearothermophilus NIAST, Chaetomium thermophile NIAST and Thermoactinomyces NIAST as a basic strain using a thermophilic microorganism growing at a high temperature of 60 ℃ or more as an organic substance promoter and the organic material accelerating agent having excellent organic decomposition ability and a culture method of the microorganisms To provide.

Due to industrialization and urbanization, large quantities of organic by-products such as food processing by-products or food waste are generated in each factory and urban area.In the case of livestock farming, large quantities of organic by-products, such as manure, are emitted in large-scale breeding of livestock, which causes many problems in its treatment. This is causing. Of course, the treatment by methods such as landfill or incineration is also mobilized, but this method causes another secondary pollution and has many problems in installation because of local egoism.

On the other hand, according to the increase-oriented agricultural policy, excessive use of chemical fertilizers and pesticides has caused serious soil acidification and deterioration of farmland. Therefore, efforts have been made in many fields to improve intellect by supplying organic matter to farmland. In other words, while reducing chemical fertilizers or pesticides as possible, organic methods of improving organic power by supplying organic by-products to farmland are reducing the use of chemical fertilizers and pesticides.

However, when compost is prepared by the traditional method, there is a problem that a large amount of organic by-products cannot be processed quickly and odors are also involved. In other words, the conventional method is a method of stacking various compost raw materials and then fermenting them naturally, and it takes a long time to compost and generates a large amount of odor in the manufacturing process. Did. Therefore, in order to improve such a problem, various mechanical and biochemical methods have been devised and put into practical use, and among them, the use of home-promoting microorganisms, which select and formulate microorganisms involved in the composting process, has been gradually expanded.

However, among the many kinds of by-products used as organic resources, there are cases in which the housing is not sufficiently made due to the lack of microorganisms and the number of microorganisms associated with the housing, and various kinds of microbial homeopathic agents are used to promote the housing. In most cases, the effect is inadequate using mainly microorganisms such as mesophilic bacteria, yeast and filamentous fungi without considering the high temperature.

In addition, the aerobic home composting process used to manufacture organic compost requires a period of high temperature due to its biological characteristics, and is a very important period because the pathogenic organisms contained in the material to be composted are killed during the high temperature home composting period. to be. However, the composting method currently used, in particular, the composting process using livestock manure is not only relatively short of the high temperature maturation period, but also may not generate enough heat. The above phenomenon is particularly in winter when the thermophilic microorganisms do not reach the temperature at which the thermophilic microorganisms can grow, and the thermophilic bacteria, which play a key role in the housing process, do not fully grow, and the compost material does not have high-temperature microorganisms or is at an appropriate density. If present, it is a serious cause of remarkably reducing the quality of the home compost.

On the other hand, the most important thing when formulating microorganisms is how economically they can produce strains. In general, a method used in formulating microorganisms is used for shaking culture, fermenter culture, etc., but there are advantages and disadvantages in producing the cells by these cultures. That is, fermenter culture has the advantage of controlling the growth composition such as pH, temperature, oxygen concentration, agitation, but there is a problem that takes a lot of production costs. In addition, the shaking culture has a disadvantage of low cell growth rate.

Therefore, the present inventors have studied the thermophilic microorganisms that grow even at a high temperature of more than 60 ℃, found that the microorganisms of Bacillus stearothermophilus NIAST, Chaetomium thermophile NIAST and Thermoactinomyces NIAST has excellent resolution of organic matter and increased microbial activity in the compost It was.

Furthermore, as a result of extensive research by the present inventors, the present invention has been completed by finding that the microorganisms can propagate with a property using a political aeration method.

Accordingly, it is an object of the present invention to provide a thermophilic microorganism that grows even at a high temperature of 60 ° C or more, which forms an organic substance-promoting agent having excellent resolution of organic matter.

Another object of the present invention is to provide a culture method capable of propagating high temperature microorganisms as a property.

1 is a view showing a simple incubator device.

2 is a graph showing the growth rate according to the culture method.

3 is a graph showing the microbial change in pig manure according to the temperature.

4 is a graph showing the temperature change in pig manure compost.

5 is a graph showing the amount of ammonia gas released.

In order to achieve the above object, the present invention is characterized by promoting the maturation of organic material using high-temperature microorganisms, that is, Bacillus stearothermophilus NIAST, Chaetomium thermophile NIAST and Thermoactinomyces NIAST to grow at a high temperature of 60 ℃ or more.

A preferred form of the microorganism used in the present invention is a Bacillus (Bacillus) the genus Bacillus stearothermophilus NIAST, thermophilic actinomycetes (Thermoactinomyces) the genus Thermoactinomyces NIAST, fungi (Chaetomium) the genus Chaetomium thermophile NIAST.

Hereinafter, the present invention will be described in detail.

Bacillus ( Bacillus ) of the present invention forms spores and grows at 65 ℃ or more, and has the property of decomposing carbohydrates such as glucose (aracose), arabinose (xylose), mannitol (mannitol), etc. . It also hydrolyzes casein, gelatin and starch, and has catalase enzyme activity. General characteristics of Bacillus are shown in Table 1 below.

Characteristics reaction ㆍ cell diameter> 1.0㎛, spores are round, spore sac expansion, catalase, anaerobic growth, V-P test -++- ㆍ Productivity from sugar D-glucose L-arabinose D-xyloseD-mannitol ++++ ㆍ hydrolysis Casein gelatin starch +++ ㆍ growth by salt concentration 2% 5% 7% 10% ++- Growth temperature 30 ℃ 50 ℃ 65 ℃ +++

On the other hand, Chaetomium was selected from the genus Chaetomium of the family Chaetomiaceae , whose flora is olive- colored and has cellulase activity that degrades cellulose. General characteristics of filamentous fungi are shown in Table 2 below.

Characteristics Colony color Olive green Optimum growth temperature 50 ~ 60 ℃ Cellulose degrading enzyme activity positivity

On the other hand, thermophilic actinomycetes were isolated from Thermoactinomyces having the characteristics shown in Table 3 below, the mycelia of the isolates were white and did not secrete water-soluble pigments. In addition, it was confirmed that the isolate was a thermophilic bacterium grown at 55 ° C. without growing at 30 ° C., but grown in 25 μg / ml of novobiocin and 1.0% NaCl solution, but CYC agar containing 0.5% L-tyrosine. No melanin pigment was produced in the medium. In addition, isolates decompose casein, esculin, and gelatin, whereas chitin, hypoxantine, starch, tyrosine and xanthine There was no resolution for). On the other hand, the isolated strain was confirmed to use carbohydrates such as fructose (fructose), mannitol (mannitol), sucrose (sucrose), trehalose (trehalose) as a carbon source.

On the other hand, the culture method of the microorganism is a static aeration culture (static aeration culture), put the sterilized selective medium in a transparent incubator and inoculate the strain of the invention using a syringe through the rubber stopper in the center of the incubator lid. Thereafter, the incubator is placed in a thermostat adjusted to the incubation temperature of the invention strain and air is injected from the outside using a compressor. At this time, the injected air is controlled by the pressure regulator and the regulated air is introduced into the incubator after passing through the filter, and the discharged air blocks the spores from passing through the water and the filter.

In the stationary aeration method, oxygen is continuously supplied from the outside, and the inflowing air acts as a stirrer, so that the growth of microorganisms is made more smoothly than the conventional shaking culture in which oxygen is not supplied from the outside.

Hereinafter, the present invention will be described in more detail with reference to test examples, but the present invention is not limited only to these examples.

[Test Example 1]

Citrate buffer to examine the cellulose activity of the strains of the present invention 0.5 ml of the sample diluted with (210 g of citric acid monohydrate, 750 ml of distilled water, pH 4.3) was placed in a glass test tube, and 0.5 ml of substrate solution in which 2% carboxymethyl cellulose was dissolved in 0.05 M sodium citrate buffer (pH 4.8). Put and mix at 50 ℃. Next, 3.0 ml of DNS (3,5-Dinitrosalicylic acid) was added and mixed. Then, 10.6 g of DNS reagent and 19.8 g of NaOH were dissolved in 1416 ml of distilled water, 306 g of Rochelle salts (Na-K tartarate), 7.6 ml of phenol, And 8.3 g of Na metabisulfite were added. Next, the test tube containing the sample was put in a water tank, which was cut off, and allowed to stand for exactly 5 minutes, and immediately cooled in cold water. Thereafter, 20 ml of deionized or distilled water was added, and the test tube was inverted and mixed thoroughly. On the other hand, the sample, enzyme control, glucose standard solution and calibration control solution were also treated in the same manner as above.

Then, the color generated on the calibration curve control solution at 540 nm using a colorimeter, and the amount of glucose generated during the reaction using the Glucose standard curve is shown in Table 4 below.

Inventive strain CMCase (glucose mg / l) Bacillus genus 250 Thermoactinomyces genus 350 Chaetomium genus 380 Mixed strain 420

As can be seen in Table 4, were CMCase activity of the isolated strain is Bacillus genus 250 glucose ㎎ / ℓ, Thermoactinomyces genus 350 glucose ㎎ / ℓ and the Chaetomium genera 380 glucose ㎎ / ℓ, a mixed culture mixing three strains of the In case of 420 glucose ㎎ / ℓ it was confirmed that the relatively high activity compared to a single strain. This indicates that a mixture of the three strains can be formulated.

[Test Example 2]

When formulating microorganisms, it is important to improve the convenience of preservation and use of the microorganisms. Therefore, the survival rate in the inorganic (zeolite, pearlite) and organic (pitmos) extender was confirmed to confirm the survival rate of the strains of the present invention. First, the samples were diluted 10-fold step by step, and the diluted solution of appropriate concentration was inoculated into a curry containing a plate solid medium. Thermoactinomyces and Chaetomium kept the dilution intact, while Bacillus put the dilution bottle containing the diluent in the water bath at 80 ° C for 20 minutes to kill the cell body before inoculation and measured the remaining spore count.

At this time, Bacillus was used as Yeast Glucose agar medium, Thermoactinomyces genus CYC medium and Chaetomium genus oatmeal medium. Here, Yeast Glucose agar medium composition is yeast extract 3.0g, glucose 1.0g, K ₂ HPO ₄ 0.3g, KH ₂ PO ₄ 0.2g, MgSO ₄ ㆍ 7H ₂ O, agar 15.0g, distilled water 1000ml, pH 6.8, CYC medium was Sucrose 30.0g, Casamino acid vitamin-free 6.0g, NaNO ₃ 3.0g, Yeast extract 2.0g, K ₂ HPO ₄ 1.0g, MgSO ₄ 7H ₂ O, KCl 0.5g. 0.01 g of FeSO ₄ 7H ₂ O, 10.0 ml of antibiotic solution (novobiocin 25 µg- ¹ , cycloheximide 50 µml- ¹ containing solution), distilled water 1000 ml, pH 7.2.

Incubation period was 12 hours Bacillus , Thermoactinomyces and Chaetomium cultured for more than 4 days to examine the colonies appeared and the results are shown in Table 5 below.

Inventive strain Survival rate (%) Zeolite Pearlite Pete Moss Bacillus genus 90 95 87 Thermoactinomyces genus 87 80 85 Chaetomium genus 87 90 95 Mixed strain 86 93 87

As can be seen in Table 5, the genus Bacillus , pearlite, Thermoactinomyces genus zeolite, Chaetomium genus showed the highest survival rate in peatmoss, mixed strains showed high survival rate in pearlite.

[Test Example 3]

Proliferation amount of the genus Thermoactinomyces by the strain production method of the present invention was measured using a simple incubator (see Fig. 1) consisting of an air inlet (compressor), air volume regulator, bactericidal filter and transparent incubator. First, the sterilized selective medium was made in a transparent culture tank, and then the inoculated strain was inoculated using a syringe through a rubber stopper in the center portion of the incubator lid. Thereafter, the culture tank was placed in an incubator having a proper temperature, and air was injected from the outside using a compressor. At this time, the injected air is controlled by the pressure regulator and the regulated air is introduced into the incubator after passing through the filter, and the discharged air blocks the spores from passing through the water and the filter. Thereafter, the strain growth amount was collected by date by centrifugation and weighed by a balance. The results are shown in FIG.

As can be seen in Figure 2, the proliferation of thermoactinomyces is 10 mg / l shaking culture on the 10th day of culture, while 560 mg / l by static aeration method is about 5 times the recovery rate compared to the general shaking culture method It was confirmed that it can be raised.

[Test Example 4]

In order to utilize the strains of the present invention as a microbial agent, the maintenance period of survival rate was determined by investigating the density change of the strains treated with Bacillus , Thermoactinomyces and Chaetomium at 65 ° C. and strains treated at room temperature. At this time, the medium used for thermophilic Bacillus and mesophilic Bacillus is a yeast extract glucose agar medium, the culture was measured after incubation for 12 hours at 55 ℃, mesophilic Bacillus for 2 days at 28 ℃. At this time, Bacillus was measured by inoculating the sample diluent after heating at 80 ℃ for 20 minutes.

On the other hand, the thermophilic actinomycetes were used as CYC medium, and the mesophilic actinomycetes were used as starch casein medium. The composition of starch casein medium was 10.0 g of soluble starch, 0.3 g of Casein vitamin-free, 2.0 g of KNO _3, 2.0 g of NaCl, 2.0 g of K ₂ HPO ₄ , 0.05 g of MgSO ₄ 7H ₂ O, 0.03 g of CaCO ₃ , and FeSO _4. 0.01 g of 7H ₂ O, 75 μg ml of Cycloheximide- ¹ , 20.0 g of agar, pH 7.2, and 1000 ml of distilled water.

The culture conditions were 1 week at 28 ℃ for mesophilic actinomycetes, 4 days at 55 ℃ for the thermophilic actinomycetes was counted the number of colonies appeared on the surface of the medium. On the other hand, filamentous fungi were first counted after 3 days at 28 ° C. and second after 5 days using Rose bengal agar medium. The results are shown in Tables 6 to 7. In addition, microbial density change after 2 months is shown in Figure 3 below.

process Housing period (days) 0 3 7 14 21 35 No vaccination Thermophilic Actinomycetes 0.2 0.3 2.6 9.1 15.7 19.0 Mesophilic actinomycetes 2.0 1.0 9.0 63.7 47.5 50.0 Thermophilic Bacillus 3.1 1.2 32.0 45.3 50.0 24.9 Mesophilic Bacillus 46.0 368.0 234.0 2895.0 2125.0 2873.0 Filamentous fungi 2.5 1.2 300.0 1590.0 3040.0 1335.0 Average 10.7 74.3 115.5 915.2 1055.6 860.3 inoculation Pyogenic actinomycetes 2985.4 3385.4 4485.7 4539.3 3957.6 3816.2 Mesophilic actinomycetes 60.5 31.1 272.3 1112.8 147.5 1513.2 Thermophilic Bacillus 156.4 1605.7 2115.3 2286.7 2523.9 2256.9 Mesophilic Bacillus 277.3 2218.9 1410.9 17456.0 17813.1 17323.4 Filamentous fungi 2.2 1.1 264.2 1400.3 2677.4 1175.7 Average 696.4 1448.4 1709.7 5359.0 5681.9 5217.1

process Microbial mass (Biomass C mg / kg) No vaccination 151 Inoculation 1,005

As can be seen in Table 6, it was confirmed that the thermophilic actinomycetes and the thermophilic Bacillus genus do not have a significant difference in density change. This indicates that the activity of the thermophilic microorganism, which is the basic strain of the present invention, remains very stable. In addition, the average density of the total microorganisms in the inoculation was maintained very high, as can be seen in Table 7, it was confirmed that the body weight of the microorganism is also significantly increased compared to the inoculation.

[Test Example 5]

In order to measure the temperature change during the critical maturation period as an indicator of compost maturation, microorganisms were inoculated and mixed with microbial agent: compost in a volume ratio of 1: 100, and then put into a pilot. The conditions were the same as in the natural state, and the air volume was allowed to flow 1 liter of air per 1 kg of sample per minute (in 1 liter min ^-1 kg building), and was periodically measured using a thermometer installed in the pilot. The results are shown in FIG. 4.

As can be seen in Figure 4, it can be seen that the initial calorific value due to the inoculation of microorganisms is higher than the control group and maintains the temperature around 50 ℃ continuously in the second half of the house. In addition, it was confirmed that there is a temperature difference of more than 20 ℃ as compared to the control as a whole, which means that the activity of the thermophilic microorganism of the present invention is continuously maintained.

[Test Example 6]

Generally, odorous gases generated during composting of animal waste are known as ammonia, sulfur compounds such as methylmercaptan, dimethylsulfide and dimethyldisulfide, and volatile fatty acids. The most emitted during the composting process is ammonia gas. Therefore, the amount of ammonia gas generated by using the glass detector tube is described in the instruction manual of the detector tube and the air inhaler.The both ends of the detector tube are cut off and inserted into the tip of the air inhaler. The discoloration length of the reactant contained in the detection tube was measured based on the scale marked on the surface of the detection tube. The results are shown in FIG. 5.

As can be seen in Figure 5, most of the ammonia is released during the early maturity, it was confirmed that the amount of ammonia gas released in the case of the microorganism according to the present invention compared to the control.

As described above, it was confirmed that the high temperature development strain of organic matter decomposition was effective in organic matter decomposition ability, survival rate, temperature increase rate of house temperature, ammonia reduction rate of pig manure and microbial body weight in compost. This indicates that inoculation of strains using high temperature microorganisms, especially mixed strains, is more efficient than the conventional general process in organic material ripening process requiring metabolic mechanism of microorganisms.

Claims (2)

A Bacillus stearothermophilus NIAST, a strain of the genus Bacillus , Chaetomium thermophile NIAST, a strain of the genus Streptococcus sp. Place the sterilized selective medium into a transparent incubator and inoculate the strain of the invention using a syringe through the rubber stopper in the center of the incubator lid. Method for culturing high-temperature microorganisms, characterized in that the microorganisms are cultured by injecting.