KR100879131B1 - Bacillus acidicola sh-1 strain and method for composting food garbage by using the same - Google Patents

Abstract

A Bacillus acidicola strain, and a method for making a compost from the food waste by using the strain are provided to improve the compost making velocity and to allow the acidic food waste to be made into a compost. A Bacillus acidicola strain is the Bacillus acidicola SH-1 KACC 91323P strain which has an optimum growth temperature of 50 °C, an optimum growth pH of 5.0 and can be proliferated even in the culture medium containing 7% NaCl. A method for making a compost from the food waste comprises the step of adding the Bacillus acidicola SH-1 KACC 91323P strain to the food waste.

Description

Bacillus acidicola (SHA strain) and the method for composting food garbage using the same {Bacillus acidicola SH-1 strain and method for composting food garbage by using the same}

The present invention relates to a microbial strain useful for composting food waste and a method for composting food waste using the strain.

Food waste is a major household waste with a high proportion of total household waste. In Korea, for example, the amount of food waste generated in 2004 was 11,463 tons per day, accounting for 23% of total household waste, and the amount of disposal was reported to be 15 trillion won. Especially in Korea, since 2005, a law prohibiting direct landfilling of food waste has come into force, and it is urgently needed to improve the recycling rate of waste generated.

Food waste consists of about 80% moisture, 17% flammable content, and 3% ash content. Excluding moisture, odor and leachate are generated due to high biodegradability. These odors and leachate are generated in all processes, such as collection, transfer, treatment, and the like, causing infestation of flies, insects, and pathogens. Food waste is expected to consume too much fuel due to high water content, low compression efficiency, and low calorific value when incinerated, and the high temperature of water causes the incineration temperature to be reduced, causing pollution of dioxin and other pollutants.

  In Korea, for example, in 2002, 52% of feed, 43.5% of compost, methanation and others accounted for 4.4%. Feeding is rapidly decreasing after mad cow disease or foot-and-mouth disease surge, and methanation is also lacking in related technologies. Therefore, the treatment rate of composting is expected to increase significantly in the future.

When using food waste for composting, the biggest problem is that first, bacteria are more likely to decay, and second, acidified food waste is difficult to compost.

Recently, high-temperature, high-speed composting has been used as a method for treating food waste with little environmental pollution. High-temperature fast composting is a technique that converts food waste into compost in a short period of time from one day to several days, mainly by the action of high temperature microorganisms. In the high temperature and high speed composting method, the food waste and the substrate having the high temperature microorganism are added to the composting container, followed by stirring, mixing, and warming to advance the conversion action.

However, the composting microorganisms known to date have a problem that the composting process is killed in an environment where the temperature is raised to 70 ℃ or more. In order to solve this problem, a method of keeping the temperature from rising to a high temperature during the composting process has been proposed. However, when the temperature is maintained at a moderate temperature, many mesophilic bacteria are prosperous. In addition, in order to be able to compost strong acid food waste, composting microorganisms must be able to grow under conditions of high acidity, and conventionally known composting microorganisms cannot be grown under acidic conditions of pH 5 or less. In addition, when the salinity of food waste is high, there is a problem in that the composting rate is lowered because the activity of the microorganism is lowered over time.

One embodiment of the present invention aims to solve the above problems.

One embodiment of the present invention is to provide a composting microorganism capable of growing at high temperature to improve the composting rate and prevent the breeding of mesophilic bacteria.

One embodiment of the present invention aims to effectively compost acidic food waste by providing a compostable microorganism resistant to acidity.

One embodiment of the present invention is to provide a composting microorganism capable of growing even at high salinity to effectively compost the high-salt food waste.

The strain according to the present invention for achieving the above object, the optimum growth temperature is 50 ℃, the optimum growth pH is 5.0, Bacillus acidicola ( Bacillus acidicola ) SH- which can be grown in the medium added with 7% NaCl 1 strain.

The strain according to the present invention is characterized in that it has 16s rDNA of SEQ ID NO: 1.

The strain according to the invention described above is characterized by having the bacteriological properties of Tables 1 and 2 herein.

The strain according to the present invention is characterized in that the accession number is KACC 91323P.

Food waste composting method according to the invention is characterized in that it comprises the step of adding the Bacillus acidicola ( Bcillus acidicola ) SH-1 strain to the food waste.

When composting food waste by using the strain according to the present invention, the composting action can be continued for a long time without additional exchange of microorganisms. It also has the advantage of easy maintenance of the composting process. In addition, it can contribute to improving the resource efficiency of food waste. In addition, since the composting proceeds at a high temperature of 50 ~ 70 ℃ can prevent the breeding of mesophilic bacteria, not only can improve the composting rate by the heat of oxidization generated by the microorganism, but also effectively compost the acidic food waste. Can be.

The microbial strain Bacillus acidicola SH-1 strain according to the present invention is a strain screened from nature. The primary screening for isolation of this strain was to isolate microorganisms that grow at high temperatures of 50 ° C. or higher and pH 5 or less. Enzyme activity of amylase and protease was measured in five isolates selected by primary screening. As a result, the highest activity strain was obtained. We named this strain Bacillus acidicola SH-1 strain. And the strain was deposited to Korea Agricultural Microorganism Support Center on July 13, 2007. The accession number is KACC 91323P.

Bacillus acidicola (SH) strain according to the present invention is a Gram-positive bacillus, aerobic spore-forming bacteria. Morphological and physiological characteristics of Bacillus acidicola SH-1 strain are shown in Table 1 below.

Characteristic Form Gram Dye Spore Formation Motility Voggs-Proskauer Test Starch and Casein Hydrolysis Nitrate Reduction Catalase Test Oxidase Test NaCl 3% NaCl 5 % NaCl 7% NaCl 10% On a rod + + + +--+ + + + + + + + +-

Bacillus acidicola SH-1 strain is extremely resistant to salt. Bacillus acidicola ( Bacillus acidicola ) SH-1 strain can be grown in the medium added with 7% NaCl. As a result of investigating the biochemical properties of 50 sugars using the API 50 CHB bacterial identification kit (Biomerieux, Vitek, Inc.), the strain according to the present invention was found to be a Bacillus strain. Bacillus axicola acidicola ) biochemical characteristics of SH-1 strain are shown in Table 2 below.

Characteristic Characteristic Glycerol － Esculin + Erythritol + Salincin + D-arabinose － Cellobinose + L-arabinose + Maltose － D-ribose + Lactose － D-xylose － Melibiose － L-xylose － Saccharose + Adonitol － Trehalose + β-xylopytanoside － Melezitose － Galactose － D-Raffinose － D-glucose + Amidon + D-fructose + Glycogen － D-mannose + Gentiobiose + L-sorbose － D-turanose + Rhamnose － D-lyxose － Dulcitol － D-tagatose － Inositol + D-fucose － Mannitol + L-fucose － Sorbitol + D-arabitol － Methy-α-D-mannopyranoside － L-arabitol － Methyl-α-D-glucopyranoside － Gluconate － N-acetylglucosamine － 2-ketogluconate － Amygdaline － 5-kerogluconate + Arbutine +

In addition, Bacillus acidicola (SH) strain according to the present invention has a 16s rDNA of SEQ ID NO: 1. This is 98% homology with the conventional Bacillus acidicola 105-2 strain.

The strain may continue to compost food waste for at least one year without exchanging the contents.

Hereinafter, one embodiment of the present invention will be described in more detail, but the scope of the present invention is not limited thereto.

<Example>

Example 1 Isolation and Identification of Strains

(1) Isolation of 5 strains

To isolate high-temperature and acid-resistant strains, 1 g of various fungal samples, such as soil, defoliation, and compost, in Gyeongsangnam-do, Korea, were suspended in 10 ml of 0.85% NaCl solution, diluted 10-fold, 100-fold, and 1000-fold to LB plate medium (bacto tryptone). 1%, yeast extract 0.5%, NaCl 0.5%, agar 1.8%, pH 5.0) and incubated at 50 ℃ for 24 hours. By observing the morphology of the resulting colonies, the colonies were divided into different colonies and the final four high temperature and acid resistant strains were selected.

(2) enzyme activity measurement

Amylase and protease activity were examined to assess substrate utilization of the five selected strains. Amylase activity was inoculated with the strain in LB plate medium to which 1% soluble starch was added and incubated overnight at 37 ℃ and then treated with iodine to observe the presence of a clear zone (clear zone). The protease activity was inoculated with the strain of LB plate medium to which 1% skim milk was added and cultured overnight at 37 ° C. The results were as shown in Table 3 below.

Strain Growth at 50 ^1,2 Growth at pH 5.0 ^1,2 Enzyme activity ³ Amylase Protease SH-1 +++ +++ +++ +++ SH-5 +++ +++ - + SH-16 +++ ++ ++ - SH-24 ++ +++ +++ +

¹ The growth rate was measured after 12 hours on nutrient broth.

² +++ shows excellent growth

³ +++ shows good clear ring formation on screening medium; ++ good; + Fair; - none.

As shown in Table 3, the SH-1 strain and the SH-24 strain had both enzyme activities. Among them, the SH-1 strain showed higher activity than the SH-24 strain. Therefore, the SH-1 strain showing the highest enzyme activity was finally selected as a seed for aerobic high temperature fermentation of food waste.

Meanwhile, the 16s rRNA gene was amplified by PCR in order to analyze the 16s rDNA sequence of the isolated strain. Chromosomes were extracted using the GENEx ^™ genomic kit (General Biosystem, Korea). Two primers [16S-F (5'-AGAGTTTGATCATGGC-3 ') (SEQ ID NO: 2), 16S-R (5'-) designed based on Taq DNA polymerase (Takara Shuzo, Japan) and 16s rRNA gene of Escherichia coli AAGGAGGTGATCCAACC-3 ') (SEQ ID NO: 3)] was used to amplify the 16s rRNA gene. DNA sequencing was performed using an ABI PRISM 310 genetic analyzer (Perkin-Elmer Applied Biosystems, Foster City, CA, USA). Physiological and biochemical characteristics of the isolates were analyzed using API 50 CHB kit (Biomerieux, Vitek, Inc.), and the results were Bergy's Manual of Systematic Bacteriology [Sveath, PHA , et al., 1984. Bergy's Manual of Ayatematic Bacteriology. Vol. 2 , Williams and Wilkins, Baltimore.

As a result of investigating the characteristics of the common bacteria to identify the isolated bacterium D-15, the strain was Gram-positive bacilli and aerobic spore forming bacteria, and the other physiological characteristics were as shown in Table 1 above. In particular, since the resistance to salt was strong, growth was possible even in the medium to which 7% NaCl was added. The biochemical properties of 50 sugars were determined using the API 50 CHB bacterial identification kit (Biomerieux, Vitek, Inc.) and found to be Bacillus strains (see Table 2). In addition, 16s rDNA sequence was analyzed (SEQ ID NO: 1) to search for homology with other strains, showing a 98% homology with the Bacillus acidicola 105-2 strain. Therefore, this final selection strain is Bacillus acidicola ( Bacillus acidicola) It was identified and named Bacillus acidicola SH-1. And the strain was deposited to Korea Agricultural Microorganism Support Center on July 13, 2007. The accession number is KACC 91323P.

Example 2. Optimal Growth Temperature and pH of Strains

According to the growth conditions of Bacillus acidicola SH-1 strain according to the culture temperature, the strain was able to grow in the range of 25 ~ 65 ℃ and the growth of bacteria was less than 25 ℃ and above 65 ℃. Did not appear. In addition, the growth of the strain appeared to be the highest at 50 ℃, the optimum growth temperature was determined to 50 ℃ (Fig. 1). The growth of Bacillus acidicola SH-1 strain by culture pH was the highest. On the other hand, the growth of bacteria hardly occurred at less than pH 4.0 and more than pH 9.0 (FIG. 2).

Example 3. Composting of Food Waste

1. Food waste composition analysis

The samples used in this experiment were collected by removing the remaining foods discharged from the university cafeteria, and then uniformly mixed and frozen in a freezer (-20 ° C). In terms of food waste, there were many noodles and rice and boiled vegetables. This food waste was used after removing water because it was very watery. Bone and shellfish of animals that were difficult to decompose were used after direct fractionation. When used in the experiment, it was transferred to a refrigerator, thawed at 4 ° C. for 16 hours, and sterilized at 121 ° C. for 15 minutes to be used as a sample. The salinity and pH of the sample were eluted for 1 hour by adjusting the sample weight and the volume of distilled water to 1: 10, and then the salinity of the sample was measured using a salinity meter (Model SS-31A, Sekisui, Japan). (pH spear, Oaklon, USA). Items such as TS, VS and FS of food waste were measured according to Standard Methods [APHA 1995. Standard methods for the examination of wastewater, 19th Ed., Washington, DC, USA: American Public Health Association].

The results are shown in Table 4 below. The salinity of food waste was 0.7 ~ 1.5%, the water content was 74.5 ~ 82.5%, pH was 4.2 ~ 5.4, and it was slightly acidic, and the content of nonvolatile solids was 5.5 ~ 7.6%.

Properties pH Salinity (%) MC (%) TS (%) VS (%) FS (%) Measures 4.2-5.7 0.7-1.5 74.5-82.5 17.5-21.4 92.4-94.5 5.5-7.6

2. Composting of food waste

Using Bacillus acidicola SH-1 (Accession No.:KACC 91323P), which grows well under high temperature (above 50 ℃) and weakly acid (below pH 5) conditions and degrades starch and protein, High temperature and acid composting was tested. For the purpose of composting the food waste, the apparatus used in this experiment used a 10 L capacity food waste composting device (manufactured by Freshwater Hyclean). The temperature inside the reactor was always maintained at 50 ° C. by a temperature sensor mounted on the bottom, and three stirring shafts were installed at right angles to the horizontally arranged rotating shaft. As the components of the medium for culturing the strain, 1% Bakto tryptone, 0.5% yeast extract, 0.5% NaCl was prepared. 1L of distilled water was added to the prepared media components, and the pH was adjusted to 5 with a pH adjusting solution. The pH-adjusted culture was sterilized at 121 ° C. for 15 minutes in an autoclave, and then the seed solution of Bacillus acidicola SH-1 was added to obtain a final concentration of 1% to obtain a seed culture. In the composting device, 500 ml of spawn culture solution was evenly poured into 1 kg of cedar sawdust serving as a microbial carrier, stirred for 1 hour, and then administered food waste.

(1) determination of viable cell count during composting

After administering food waste, the number of viable cells in the reactor was measured for 24 hours. As a result, the number of viable cells increased to 6.1 × 10 ¹⁰ / ml after 8 hours of fermentation, and the rapid growth rate was shown, and the number of viable cells increased to 9.2 × 10 ¹⁰ / ml in 16 hours and maintained up to 24 hours (FIG. 3). As a result, Bacillus axicola acidicola ) SH-1 was found to be compostable food waste in a short time under high temperature and acidic conditions.

(2) acid composting experiment

It is common for the contents of a general food waste disposal device to be acidic at first, but later to weakly alkaline at pH 7-9. In addition, composting under acidic conditions has not been performed effectively. The present inventors put 1kg of food waste into the composting device for 30 days a day and proceed acidic composting in order to test whether acidic composting of food is possible using Bacillus acidicola SH-1 according to the present invention. The compost weight, temperature, pH in the apparatus were measured. As a result, the temperature of the composting during the experiment period was 40 ~ 62 ℃ it was confirmed that the pH is maintained at around 4.5 (C, D of Figure 4). The composting function was not reduced and the food waste was continuously reduced. After 30 days of the experiment, the cumulative input amount of the food waste was about 31 kg and about 4 kg of compost was obtained, indicating a food waste reduction rate of about 87%. A, B).

(3) fertilizer analysis of compost

On the other hand, organic matter, C / N ratio, arsenic, cadmium, mercury, lead, chromium, copper, nickel, zinc, salts from compost generated from food waste fertilizer analysis method [Ministry of environment. 2002. Methods for soil pollution process test, pp. 118-194].

The results are shown in Table 5 below.

Item (unit) Reference value Measures Organic matter (Wt%) More than 25 62.7 Ratio of organic matter / nitrogen Less than 50 25.4 Arsenic (mg / kg) Less than 50 0 Cadmium (mg / kg) Less than 5 0 Mercury (mg / kg) Less than 2 0 Lead (mg / kg) Less than 150 0 Chromium (mg / kg) Less than 300 8.9 Copper (mg / kg) Less than 300 5.0 Nickel (mg / kg) Less than 50 3.8 Zinc (mg / kg) Less than 900 18.3 Salinity (Wt%) Less than 1 1.4

As shown in Table 5, the organic matter and organic matter / nitrogen ratio (C / N ratio) were 62.7% and 25.4, respectively, all of which satisfied the reference values. The effect of C / N ratio on composting can be divided into the reaction rate and the quality of composting. If the C / N ratio is high (generally 80 or higher), the composting period is long due to nitrogen deficiency and the temperature rise is not good. If the C / N ratio is too low (typically 10 or less), odor is more likely to occur and the composting reaction will be slower. In general, as the compost progresses, the C / N ratio gradually decreases, and the stable range is known to be 10-20. Therefore, it is considered that the compost produced in this composting process has an appropriate C / N ratio. In addition, no harmful arsenic, cadmium, mercury, and lead were detected, and chromium, copper, nickel, and zinc all met the standard. The compost thus obtained was used as land or neutralized with lime, mixed with soil, left for a while, and then used as compost after secondary fermentation.

1 is a graph showing the growth rate change according to the temperature change of the strain according to the present invention.

Figure 2 is a graph showing the growth rate change according to the pH change of the strain according to the present invention.

3 is a graph showing a change in the number of strains according to the present invention during the composting process of food waste according to an embodiment of the present invention.

Figure 4 shows the result of monitoring the composting process of food waste according to an embodiment of the present invention. A is the change in cumulative food waste input, B is the weight of compost, C is the change in temperature, and D is the change in pH.

<110> DONG-EUI EDUCATIONAL, FOUNDATION          CHUNG SU HIGH CLEAN CO., LTD <120> Bacillus acidicola SH-1 strain and method for composting <130> 07P405IND <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 1387 <212> DNA <213> Bacillus acidicola <400> 1 tgctcaggac gaacgctggc ggcgtgcnta atacatgcaa gtcgagcgga cttttaaaag 60 cttgctttta aagttagcgg cggacgggtg agtaacacgt gggtaacctg cctgtaagac 120 tgggataact ccgggaancc ggggctaata ccggataact tcttttcctc gcatgaggaa 180 aagattgaaa gatggcttcg gctatcactt acagatggac ccgcggcgca ttagctagtt 240 ggtgaggtaa cggctcacca aggcgacgat gcgtagccga cctgagaggg tgatcggcca 300 cactgggact gagacacggc ccagactcct acgggaggca gcagtaggga atcttccgca 360 atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaaa 420 ctctgttgtt agggaagaac aagtatcgtt cgaatagggc ggtaccttga cggtacctaa 480 ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt 540 gtccggaatt attgggcgta aagcgcgcgc aggcggtttc ttaagtctga tgtgaaagcc 600 cacggctcaa ccgtggaggg tcattggaaa ctgggagact tgagtgcaga agaggagagt 660 ggaattccac gtgtagcggt gaaatgcgta gagatgtgga ggaacaccag tggcgaaggc 720 gactctctgg tctgtaactg acgctgaggc gcgaaagcgt ggggagcgaa caggattaga 780 taccctggta gtccacgccg taaacgatga gtgctaagtg ttagagggtt tccgcccttt 840 agtgctgcag ctaacgcatt aagcactccg cctggggagt acggccgcaa ggctgaaact 900 caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg 960 cgaagaacct taccaggtct tgacatcctc tgacaaccct agagataggg cgttcccctt 1020 cgggggacag agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg 1080 ttaagtcccg caacgagcgc aacccttgac cttagttgcc agcattcagt tgggcactct 1140 aaggtgactg ccggtgacaa ccggaagagg tggggatgac gtcaatcatc atgcccttta 1200 tgactgggct cacacgtgct acaatggatg gtacaagggc tgcgagacgc gaggttaagc 1260 aatcccataa accattctca gttcgattgc angctgcact ctgctgcatg agccggaatc 1320 gctagtaatc gcggatcgca tgccgcggtg aatacgttcc cggccctgta cacaccgccg 1380 tcacaca 1387 <210> 2 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 agagtttgat catggc 16 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 aaggaggtga tccaacc 17  

Claims (5)

Bacillus acidicola SH-1 KACC 91323P strain with an optimal growth temperature of 50 ° C., an optimal growth pH of 5.0, and growth in medium containing 7% NaCl. The Bacillus acidicola SH-1 KACC 91323P strain according to claim 1, wherein the strain has a 16s rDNA of SEQ ID NO: 1. delete delete As a way to compost food waste, A method of composting food waste, comprising adding Bacillus acidicola SH-1 KACC 91323P strain according to claim 1 to food waste.

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