KR20220128722A - Composition for neutralizing acidic soil comprising microbial culture, and use thereof - Google Patents

Abstract

The present invention relates to a composition for neutralizing an acidic soil containing a microbial culture and use thereof. According to the present invention, mixed strain Y1 containing Serratia grimesii, Bacillus toyonensis, Pseudomonas protegens, and Atlantibacter hermannii has activity of growing microorganisms and increasing pH under acidic conditions of pH 4.5 and has ability to neutralize an acidic environment to a pH of 7 within 24 hours. Accordingly, the mixed strain Y1 is provided as a neutralizing agent for the acidic soil.

Description

Composition for neutralizing acidic soil comprising microbial culture, and use thereof

The present invention relates to a composition for neutralizing acidic soil comprising a microbial culture and a use thereof.

Soil acidity is the most basic factor to evaluate the geochemical properties of soil. The acidity of the soil plays a very important role in understanding the chemical properties of the soil as well as the biochemical and physical properties of the soil. In cultivation, soil acidity is regularly checked and proper management is required to maintain healthy soil and produce abundant crops. At the time of mining activities, the heavy metals and pH of the discharged mine are stable, but when the mine is closed or closed, pyrite is oxidized under the influence of oxygen and water flowing into the mine, and the mine drainage changes to a strongly acidic water with a pH of 4 or less. . As it passes through the surrounding rock layers, mine drainage dissolves heavy metals and is contaminated with various harmful substances. In addition, it is causing the destruction of the aquatic ecosystem due to the strong acidic water quality and the contamination of heavy metals in the surrounding agricultural land.

As a result of surveying soil contamination in the areas surrounding abandoned mines in Gangwon and Gyeongbuk in 2016, a total of 28 abandoned mines, including 17 abandoned metal mines, 7 abandoned coal mines, 3 mines containing asbestos substances, and 1 abandoned asbestos mine, were An area of 2,5757,120㎡ was found to be contaminated. In addition, according to a study by the Gangwon Research Institute, as of the end of 2018, there were 991 mines in Gangwon-do, including abandoned mines, and 462 of them, or 46.6%, were caused by leachate leakage, soil pollution, waste-rock loss, ground subsidence, and dust blowing. ) was worn. As such, abandoned mines scattered throughout Korea are not equipped with pollution prevention or treatment facilities, so mine drainage and waste-rock containing heavy metals have been exposed to the environment as they are, and continuous management and monitoring of environmental pollution are urgently needed.

Soil acidity and alkalinity are expressed as pH, and are important variables affecting the physicochemical and biological properties of soil. It is usually determined by the chemical equilibrium relationship between hydrogen ions and hydroxide ions, which is ultimately quantified as pH magnitude. Acidic soil means soil with a pH of less than 7, and can be subdivided into weakly acidic or strongly acidic depending on the pH. All soils in Korea except for Gangwon-do and part of the west coast are strongly acidic with a pH of 5.5 to 5.7 or less than 5.5. In Korea, more than 70% of bedrock is composed of acidic rocks such as rhyolite or granite, so the soil tends to acidify more easily. is indicating Considering that abandoned mines are distributed nationwide, it can be seen that acid soil in abandoned mine areas is not a problem limited to a specific area. Points with high pH are covered or neutralized, and points where tailings are directly exposed to the surrounding environment are acidic. Soil pollution is not easily visible, and it can cause more damage because it is only known after the damage has been done for a long period of time. Therefore, there is a need for an environmentally friendly acid soil neutralization technology that does not cause confusion in the soil ecosystem.

On the other hand, the process by which living organisms produce biological tissues into hard and stiff inorganic materials is called biomineralization. A schematic diagram of biomineralization occurring in the surrounding environment is shown in the following figure. The main functions of biomaterials formed through biomineralization are broad, such as support (bone), protective body (shell), ion storage (Ferritin), detector (Bacterial magnetite), crusher and crusher (teeth). More than 60 types of biomaterials are known so far, and despite the numerous types and diversity, most biomaterials are composed of calcium carbonate, calcium phosphate, calcium oxalate, and silicon dioxide. It is concentrated in minerals such as iron oxide.

Korean Patent No. 10-2125384 (2020. 06. 23. Announcement)

In order to solve the above problems, an object of the present invention is to provide a method for neutralizing acidic soils by selectively separating and identifying native microorganisms having neutralizing ability naturally present in acidic soils in Korea.

The present invention is Serratia grimesii ( Serratia grimesii ), Bacillus toyonensis ( Bacillus toyonensis ), Pseudomonas protegens ( Pseudomonas protegens ) and Atlantibacter hermannii ( Atlantibacter hermannii ) Acidic containing a microbial culture prepared by mixing A composition for neutralizing soil is provided.

In addition, the present invention Serratia grimesi ( Serratia grimesii ), Bacillus toyonensis ( Bacillus toyonensis ), Pseudomonas protegens ( Pseudomonas protegens ) and Atlantibacter hermani ( Atlantibacter hermannii ) A useful microorganism culture prepared by mixing Provided is a method for neutralizing acidic soil comprising mixing into acidic soil.

According to the present invention, Serratia grimesii ( Serratia grimesii ), Bacillus toyonensis ( Bacillus toyonensis ), Pseudomonas protegens ( Pseudomonas protegens ) and Atlantibacter hermannii ( Atlantibacter hermannii ) Mixed strain Y1 containing pH 4.5 As it was confirmed that it has the activity of increasing microbial growth and pH in phosphoric acidic conditions, and has the ability to neutralize an acidic environment of pH 7 within 24 hours, the mixed strain Y1 may be provided as a neutralizing agent for acidic soil.

1 is a graph showing the results of identification of microorganisms for the mixed strain Y1 using the next generation sequencing (NGS) technique.
Figure 2 is a graph showing the experimental results confirmed (yellow-red (pH < 5) is yellow (pH > 5)) for each acidic pH by the mixed strain Y1 of the agar plate.
3 is a graph showing the relationship between Serratia grimesii (YA-1) and Serratia spp.
4 is a graph showing the results of time series experiments on the neutralization ability of Y1 for acidic pH (a) pH, (b) OD ₆₀₀ , (c) NH ₄ ⁺ concentration (d) dehydrogenase activity.
5 is a photograph showing the results of colony formation of a single microbial species for Y1 after a neutralization experiment.
6 is a graph showing a comparison of time-series community changes for each single species of microorganisms during culture of Y1 according to acidic pH (a) pH 3.5, (b) pH 4, (c) pH 5, (d) pH 6.
7 is a graph showing the change in the concentration of microorganisms (OD ₆₀₀ ) and pH for each single strain in Y1 (A, B, C, D) (a).

The terms used in this specification have been selected as currently widely used general terms as possible while considering the functions in the present invention, which may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, and the like. In addition, in a specific case, there is a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than the name of a simple term.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Numerical ranges are inclusive of the values defined in that range. Every maximum numerical limitation given throughout this specification includes all lower numerical limitations as if the lower numerical limitation were expressly written. Every minimum numerical limitation given throughout this specification includes all higher numerical limitations as if the higher numerical limitation were expressly written. All numerical limitations given throughout this specification will include all numerical ranges that are better within the broader numerical limits, as if the narrower numerical limitations were expressly written.

Hereinafter, the present invention will be described in more detail.

The present inventors have completed the present invention by providing a method for neutralizing acidic soil by selectively isolating and identifying native microorganisms having neutralizing ability naturally present in acidic soils in Korea.

The present invention is Serratia grimesii ( Serratia grimesii ), Bacillus toyonensis ( Bacillus toyonensis ), Pseudomonas protegens ( Pseudomonas protegens ) and Atlantibacter hermannii ( Atlantibacter hermannii ) Acidic containing a microbial culture prepared by mixing A composition for neutralizing soil is provided.

The Serratia grimesii ( Serratia grimesii ) is 50 parts by weight to 99 parts by weight of 100 parts by weight of the total useful microorganism culture, Serratia grimesii ( Serratia grimesii ) It means that the strain is a dominant species of mixed strains for neutralizing acidic soil. .

The Serratia grimesii ( Serratia grimesii ) can survive at pH 4 to pH 8, and can neutralize acidic soil to neutral pH 7 within 24 hours.

The microbial culture means a microbial cell, or a culture solution containing the microbial cells, and the culture solution containing the microbial cells is a medium in which the microorganisms are grown by solid-state culture or liquid culture in a commonly known culture medium.

In addition, the present invention Serratia grimesi ( Serratia grimesii ), Bacillus toyonensis ( Bacillus toyonensis ), Pseudomonas protegens ( Pseudomonas protegens ) and Atlantibacter hermani ( Atlantibacter hermannii ) A useful microorganism culture prepared by mixing Provided is a method for neutralizing acidic soil comprising mixing into acidic soil.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

Example 1. Isolation of mixed strains for neutralizing acidic soil

To isolate and identify neutralizable native microorganisms from acidic soils in the mine area, 5 g of soil samples were placed in 25 mL of 0.85% (w/v) sodium chloride (NaCl) solution, and a funnel shaker (EYELA MMV-1000W, JAPAN) was used. and mixed at 200 rpm for 30 minutes. Thereafter, the solution was separated from the soil sample by standing for 30 minutes, and the supernatant was aliquoted and sterilized in Luria-Bertani (LB) broth (Bacto ^TM Tryptone 10 g/L; Bacto ^TM Yeast extract 5 g/L; NaCl 10 g). /L) was added to 100 mL and concentrated and cultured for 24 hours in a shaking incubator at 30 °C and 160 rpm. A microbial sample extracted from a soil sample and cultured separately was named a mixed strain. The separated supernatant and mixed strain culture were stored at 4° C., and microorganisms for neutralizing acidic soil were selectively separated from the mixed strain.

First, a neutralization-specific microorganism was identified among the mixed strains by using the next generation sequencing (NGS) technique. As shown in FIG. 1 , as a result of microbial identification for the mixed strain using the Next Generation Sequencing (NGS) method, Phodanobacter spp. (28.74%), Paraburkholderia spp. (24.17%), Natranearobacuium spp. (19.24%) was found to be dominant compared to other strains.

Experimental Example 2. Evaluation of neutralization ability of mixed strains for neutralizing acidic soil

In order to confirm whether the mixed strain exhibits acid resistance and neutralizing activity, the neutralization potential was evaluated by culturing the culture solution of the mixed strain on an acidic nutrient agar medium containing Methyl red. The nutrient agar medium is pH adjusted to 4, 4.5 and 5 with 4 MH ₂ SO ₄ solution, and then 0.03 g of Methyl red is added to 200 mL of 1.5X Nutrient Broth (NB, Difo TM Nutrient Broth, BD Biosciences, USA). and agar solution (4.5 g Micro agar/100 mL distilled water) were each autoclaved (121° C., 15 minutes), and then mixed to prepare a Petri dish (d 90 × h 15 mm).

As shown in Figure 2, under acidic conditions (pH < 5), the agar medium containing methyl red appeared red, but when the mixed strain was cultured, the pH increased and the color of the medium was discolored to yellow (pH > 5). appear. The above results demonstrate that the mixed strain contains a strain capable of neutralizing an acidic environment.

When Methyl red contained in the medium is pH 5.0 or higher, a microorganism with a potential to neutralize acidity was isolated by using the property of changing color from red to yellow. After completion of the culture, single colonies grown while discoloring the surrounding medium were separated and cultured again in nutrient broth (NB) under acidic conditions, and the selected strain was Glycerol stock [1 mL single strain culture solution + 250 μL 80% (v/v) Glycerol) ] and stored at -80°C.

The type of the strain was determined through 16S rDNA sequencing of the single stored strain. At this time, 16S rDNA sequencing was performed by isolating strains that form colonies differently depending on the type of nutrient agar medium (NA, LB agar, TSA, R2A agar). Genomic DNA of each strain was extracted using HiGene ^TM Genomic DNA Prep Kit (BIOFACT, Korea), and 16S universal primers 27F (5'-AGA GTT TGA TCM TGG CTC AG-3'; SEQ ID NO: 1) and 1492R ( Poly chain reaction (PCR) was performed using 5`-TAC GCY TAC CTT GTT ACG ACT T-3`, SEQ ID NO: 2, Bioneer, Korea) and BioFact ^TM Taq DNA Polymerase kit (BIOFACT, Korea). amplified through The amplified DNA was purified by HiGene ^TM Gel&PCR Purification System (BIOFACT, Korea), and the nucleotide sequence of 16S rDNA was analyzed using ABI 3730XL DNA Analyzer (Applied Biosystems, USA). Thereafter, the nucleotide sequence analysis result was compared with the nucleotide sequence described in the Gene Bank of NCBI (National Center for Biotechnology Information)-BLAST (Basic Local Alignment Search Tools) to identify the microbial species, and a microorganism specific for neutralization was finally selected. As a result of separating and culturing strains capable of neutralizing acidic environments at pH 4.5 and 5, the results are shown in Table 1 below, and among them, Serratia spp. The relationship between the two is shown in FIG. 3 .

Indigenous soil microbes pH Phylogenetically similar species Similarities Differences Y1 4.5 Burkholderia stabilis 1452/1454 (99%) 0/1454 (0%) 5 Serratia grimesii. (YA-1) 1413/1415
(99%) 0/1415 (0%) 5 Atlantibacter hermannii 1429/1444 (98%) 2/1444 (0%)

Example 3. Characterization of mixed strains for neutralizing acidic soil

To analyze the characteristics of the mixed strain, the microbial cell mass (OD ₆₀₀ ), microbial activity, pH and ammonium ion (NH ₄ ⁺ ) concentration were measured. The mixed strain isolated from the soil sample collected from the soil site was inoculated into 300 mL of NB medium and cultured for 10 days in a shaking incubator at 30°C and 160 rpm. The characteristics of the mixed strain were analyzed by culturing in the same way as a control group that was inoculated with microorganisms in a medium without pH adjustment.

After centrifuging the sample (3,000 rpm, 15 minutes), the pH was measured with a pH meter (BP3001, Trans Instruments, Singapore) by taking the supernatant. The amount of microorganisms (OD ₆₀₀ ) was measured by measuring the absorbance of 1 mL of the microorganism suspension at a wavelength of 600 nm using a UV-Vis spectrophotometer (HS-3300, Humas, Korea). Ammonium ions (NH ₄ ⁺ ) were obtained using the Nessler method. After centrifuging 1 mL of the sample (3,000 rpm, 15 minutes) to take the supernatant, it was diluted 50-fold with distilled water. After adding 3 drops each of polyvinyl alcohol dispersing agent and mineral stabilizer solution (HACH, USA) to the diluted solution, add 1 mL of Nessler reagent (HACH, USA) and react for 30 seconds. The absorbance of the reaction solution was measured at 425 nm using a UV-Vis spectrophotometer (HS-3300, HUMAS, Korea). The concentration of the sample was converted through the calibration curve obtained using the NH ₄ Cl standard solution. Microbial activity was measured by INT assay. After centrifuging 0.5 mL of the sample at 13,000 rpm for 10 minutes, the precipitated cells are infused with 0.2% (w/v) INT (Indonitrotetrazolium chloride, TCI, JAPAN) solution (in 200 mM Phosphate buffer, pH 7.6) 0.5 mL and 0.1 mL of 1% (w/v) Surcose solution, and then at 30°C for 30 minutes. Incubated at 160 rpm. Then, 50 μL of HCl (35%) and 1 mL of 1,4-Dioxane were added to extract INT formazan, and absorbance was measured at 481 nm.

As shown in FIG. 4 , at pH 4 or lower, the growth of microorganisms and increase in pH and NH ₄ ⁺ concentration did not appear, but at pH 4.5, the growth rate of microorganisms and the increase in pH increased after 2 hours. At pH 5 and pH 6, growth and acidity were neutralized immediately after inoculation, and it took 24 hours and 8 hours, respectively, for the mixed strain under the conditions of pH 4.5 and pH 5, until the neutralization reaction was completed up to pH 7 or higher. . The pH increased to above pH 8 within 24 hours of incubation time.

Example 4. Identification and Characterization of Acidic Soil Neutralizing Single Microorganisms

Using the mixed strain, some of the samples collected for evaluation of acid neutralization ability in the liquid medium were diluted and spread on a nutrient agar medium. community composition) and changes in the total number of viable cells were observed (FIG. 6).

As shown in Figure 5, as a result of identifying strains representing different colonies, A is Bacillus toyonensis (1.27%), B is Serratia grimesii (YA-1) (97.47%), C is Pseudomonas protegens (0.84%) ), D were identified as Atlantibacter hermannii (0.42%). A mixed strain comprising the four identified strains was named YA1 mixed strain.

As a result of comparing the changes in bacterial community composition and live cell concentration at acidic pHs of pH 3.5 (a), pH 4 (b), pH 5 (c), and pH 6 (d) of each of the YA1 mixed strains, the results shown in FIG. As can be seen, at pH 4.5 or lower, only the B strain ( Serratia grimesii (YA-1)) was detected at a very low concentration, and the number of viable cells was generally reduced and inactivated within 12 hours. In the case of an initial pH of 5 or higher, strains of other species other than Serratia grimesii (YA-1) were also observed within 24 hours after inoculation, but after that, strain B appeared to dominate. That is, in neutralizing the acidic liquid medium among the Y1 mixed strains, it was found that the B strain had the highest acid resistance, and made a great contribution to neutralizing the acidity.

In addition, as a result of comparing the growth rate and pH change of each of the strains A to D, as shown in FIG. 7 , the OD ₆₀₀ of strains B and C after 3 days of culture was 0.565 and 0.417, respectively, and the pH was 6.8 and 6.59 increased to In particular, strain B grew rapidly within the same incubation time, and the pH was also increased to a high value, so it was found to be the most suitable strain for preparing a soil conditioner for neutralizing acid soil.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Composition for neutralizing acidic soil comprising microbial culture, and use thereof <130> ADP-2021-0084 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 1492R <400> 2 tacgcytacc ttgttacgac tt 22

Claims (7)

Serratia grimesii ( Serratia grimesii ), Bacillus toyonensis ( Bacillus toyonensis ), Pseudomonas protegens ( Pseudomonas protegens ) and Atlantibacter hermannii ( Atlantibacter hermannii ) For neutralizing acidic soil containing a microbial culture prepared by mixing composition. According to claim 1, wherein the Serratia grimesi ( Serratia grimesii ) Acidic soil neutralization composition, characterized in that 50 parts by weight to 99 parts by weight in 100 parts by weight of the total useful microorganism culture. The composition for neutralizing acidic soil according to claim 1, wherein the Serratia grimesi ( Serratia grimesii ) survives at pH 4 to pH 8. The composition for neutralizing acidic soil according to claim 1, wherein the microbial culture is a microbial cell or a culture solution containing the microbial cell. The composition for neutralizing acidic soil according to claim 1, wherein the microbial culture further comprises a Burkholderia stabilis strain. Serratia grimesii ( Serratia grimesii ), Bacillus toyonensis ( Bacillus toyonensis ), Pseudomonas protegens ( Pseudomonas protegens ) and Atlantibacter hermannii ( Atlantibacter hermannii ) Mixing a microbial culture prepared by mixing in acidic soil A method of neutralizing acidic soil comprising a. The method according to claim 6, wherein the microbial culture further comprises a Burkholderia stabilis strain.

Images