Microorganism strain using sphingobium yanoikuyae, and wastewater purification agent comprising same

Abstract

A microorganism strain using Sphingobium yanoikuyae, and a wastewater purification agent comprising same are disclosed. The microorganism strain (KCTC19105P) using Sphingobium yanoikuyae, according to an embodiment of the present invention, is produced by inducing a chemical mutation in Sphingobium yanoikuyae through ethyl methane sulfonate (EMS) treatment.

Description

Microbial strain using Sphingomonas yanoikuyae and wastewater purification agent containing the same

[0001] The present invention relates to a microbial strain using Sphingomonas yanoikuyae that has undergone chemical mutation by treatment with EMS (Eethyl Methane Sulfonate) and a wastewater purification agent containing the same.

[0002] As environmental issues become increasingly prominent worldwide, wastewater purification technologies utilizing microorganisms are gaining attention as a means of treating groundwater and wastewater. Microbial wastewater purification technologies involve wet treatment, decomposing or removing contaminants. These technologies are typically utilized in activated sludge systems or filtering systems in wastewater treatment facilities, utilizing contaminant-decomposing microorganisms.

[0003] Microorganisms added to wastewater undergo a microbial decomposition process over a period of time. Therefore, the purification effectiveness of pollutants varies depending on environmental conditions such as the type and amount of pollutants, sediment concentration, temperature, and oxygen supply. In particular, the types and strains of microorganisms used in the wastewater purification process significantly affect the types of pollutants removed and their effectiveness. Therefore, the development of wastewater purification agents optimized for efficient pollutant removal is urgently needed.

[0004] In order to solve the above-mentioned problems, the technical task of the present invention is to provide a microbial strain using Sphingomonas yanoikuyae, which provides a new, highly efficient Sphingomonas yanoikuyae strain by inducing a chemical mutation in Sphingomonas yanoikuyae through EMS treatment, and a wastewater purification agent containing the same.

[0005] As a means for solving the above-mentioned technical problem, a microbial strain (KCTC19105P) using Sphingomonas yanoikuyae according to an embodiment of the present invention is produced by inducing a chemical mutation in Sphingomonas yanoikuyae through treatment with EMS (Eethyl Methane Sulfonate).

[0006] The above Sphingomonas yanoikuya can be collected from wastewater or polluted areas.

[0007] Primary culture can be performed for 48 hours at a culture temperature of 28℃.

[0008] After distributing the above-mentioned cultured Sphingomonas yanoikuya strain, a predetermined amount of EMS can be treated and secondary culture can be performed at a culture temperature of 28°C for 48 hours.

[0009] It can be selected from among strains with a turbidity of 0.337 or less.

[0010] Meanwhile, a wastewater purification agent using Sphingomonas yanoikuyae according to another embodiment of the present invention includes a microbial strain (KCTC19105P) of any one of claims 1 to 5.

[0011] The above microbial strain (KCTC19105P) can be mixed with Bacillus subtilis KAC92451P and Bacillus licheniformis KACC92452P in a ratio of 1:1:1.

[0012] According to the present invention, there is an effect of providing a new Sphingomonas yanoikuyaesphingomonas strain by chemically mutagenesis of Sphingomonas yanoikuyaesphingomonas through EMS treatment, and providing a microbial strain using Sphingomonas yanoikuyaesphingomonas that is very effective in wastewater purification and a wastewater purification agent containing the same.

[0013] The effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0014] Figure 1 is a flow chart for explaining a method for producing a microbial strain using Sphingomonas yanoikuyae according to a preferred embodiment of the present invention.

[0015] Figure 2 is a genetic base sequence information of a microbial strain using Sphingomonas yanoikuyae according to a preferred embodiment of the present invention.

[0016] Figure 3 is a genetic relationship analysis diagram of a microbial strain using Sphingomonas yanoikuyae according to a preferred embodiment of the present invention.

[0017] Figure 4 is a flow chart for explaining a method for manufacturing a wastewater purifier according to a preferred embodiment of the present invention, and

[0018] Figures 5 to 10 are graphs showing the results of physicochemical analysis of a wastewater purification agent containing a microbial strain using Sphingomonas yanoikuyae according to the present invention.

[0019] The above-described purposes, other purposes, features, and advantages of the present invention will be readily understood through the following preferred embodiments, illustrated in the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided to ensure that the disclosure is thorough and complete, and to sufficiently convey the spirit of the present invention to those skilled in the art.

[0020] Additionally, the terminology used herein is for the purpose of describing embodiments and is not intended to limit the present invention. In this specification, the singular also includes the plural unless specifically stated otherwise. As used herein, the terms "comprises" and / or "comprising" do not exclude the presence or addition of one or more other components.

[0021] Figure 1 is a flow chart illustrating a method for producing a microbial strain using Sphingomonas yanoikuyae according to a preferred embodiment of the present invention.

[0022] Sphingomonas yanoiculae is collected by collecting samples from a drainage ditch polluted with wastewater, nearby soil and floating matter, and isolating Sphingomonas yanoiculae from the collected samples (S100).

[0023] After collecting Sphingomonas yanoikuyae, strains with excellent growth activity are selected using a nutritional agar medium (NA) (S110), and the selected strains are subjected to primary culture (S120). The culture conditions for primary culture are culturing in liquid NA medium at a culture temperature of 28°C for 48 hours with stirring at 125 rpm.

[0024] After primary culture, the primary cultured Sphingomonas yanoikuyae strain is dispensed into 10 replicates of 0.9 ml each (S130), and EMS treatment is performed (S140). The EMS treatment condition is to treat with 100 μl / ml of EMS for 10 minutes.

[0025] Take 100 ㎕ of the suspension resulting from EMS treatment and spread it on NA medium, then perform secondary culture (S150). The culture conditions for secondary culture are 28℃ for 48 hours.

[0026] 100 strains of Sphingomonas yanoikuyae were purified and incubated in an incubator at 28℃ and 125 rpm while stirring for 48 hours. The turbidity was measured using an ultraviolet-visible spectrophotometer (UV / VIS spectrophotometer). Through this, the strain with a turbidity of 0.337 or less was selected as the final microbial strain (KCTC19105P) (S160).

[0027] FIG. 2 is a genetic base sequence information of a microbial strain using Sphingomonas yanoikuyae according to a preferred embodiment of the present invention, and FIG. 3 is a genetic relationship analysis diagram of a microbial strain using Sphingomonas yanoikuyae according to a preferred embodiment of the present invention.

[0028] As described in Figure 1, Sphingomonas yanoikuyae is subjected to EMS treatment to induce chemical mutations, and then the final strain is selected. More specifically, a single colony with vigorous in vitro growth is selected on NA medium, and this single colony strain is subjected to EMS mutations, after which any strains that die or exhibit poor in vitro growth are eliminated. One EMS mutant strain with vigorous in vitro growth on NA medium is selected as the final strain. The results of the genetic sequence analysis of the EMS mutant Sphingomonas yanoikuyae strain selected as the final strain are shown in Figure 2.

[0029] FIG. 3 shows a genetic relationship analysis of a microbial strain according to the present invention, and based on the genetic information shown in FIG. 2, a comparative analysis of the ITSl6s rRNA gene base sequence and a phylogenetic tree analysis with the existing Sphingomonas yanoikuyae registered in the National Center for Biotechnology Information in the United States, and a phylogenetic tree analysis, it is shown that the microbial strain according to the present invention was identified as a new Sphingomonas yanoikuyae bacterium compared to the Sphingomonas yanoikuyae of the same species discovered so far.

[0030] Figure 4 is a flow chart for explaining a method for manufacturing a wastewater purifier according to a preferred embodiment of the present invention.

[0031] A microbial strain according to the present invention was generated by the process illustrated in Fig. 1 (S200). This microbial strain was mixed with Bacillus subtilis and Bacillus licheniformis at a ratio of 1:1:1, respectively (S210).

[0032] In this way, the microbial strains according to the present invention, Bacillus subtilis and Bacillus licheniformis, are mixed in a ratio of 1:1:1 to manufacture a wastewater purifier (S220).

[0033] Sphingomonas yanoikuyae is an aerobic, gram-negative, non-motile, non-spore-forming bacterium that forms yellow or grayish-white colonies. The genome size is approximately 5,353,044 base pairs, with a genome content of 64.3%. Sphingomonas yanoikuyae can survive at a pH of 6.0 to 9.0, with an optimum pH of 6.8. Its growth temperature is 13 to 30°C, with an optimum temperature of 28°C. Sphingomonas yanoikuyae is slightly halophilic (2 to 5% NaCl) and is found in a variety of environments from terrestrial to aquatic. Recently, Sphingomonas yanoikuyae has been attracting attention as a potential source-remediation microorganism, following its discovery in areas contaminated with toxic compounds such as pentachlorophenol (PCP), polychlorinated biphenyl (PCB), and herbicides. The potential of Sphingomonas yanoikuyae to biodegrade various aromatic hydrocarbons (C6H5)2, such as PCBs, naphthalene, phenylthrene, toluene, and xylene, has been revealed in the fields of biological remediation and biotechnology.

[0034] Bacillus subtilis (B. subtilis) is a facultatively anaerobic, Gram-positive, weakly white, rod-shaped bacterium with flagella that allows it to move rapidly in liquids. Therefore, it is primarily found in the upper layers of soil and is known as a commensal microorganism in the gut of many animals, including humans. Furthermore, it forms endospores, demonstrating superior survival rates compared to other microorganisms, even in extremely harsh environments. Therefore, it is attracting attention as a microorganism ideally suited to decomposing various contaminants under extreme conditions.

[0035] Bacillus licheniformis is a facultatively anaerobic, Gram-positive, mesophilic bacterium. However, the optimal temperature for the production of enzymes such as serine protease is 37°C. It is also resistant to high temperatures, surviving above 50°C, and thus can withstand exothermic reactions during the decomposition of pollutants. Bacillus licheniformis is an effective microorganism for serine protease synthesis and probiotics. Recently, its ability to decompose recalcitrant proteins has also been confirmed, drawing attention as a bioremediation microorganism.

[0036] The effects of a wastewater purification agent containing a microbial strain, Bacillus subtilis, and a mixture of Bacillus subtilis, having these characteristics are examined in FIGS. 5 to 10 described below.

[0037] Figures 5 to 10 are graphs showing the results of physicochemical analysis of a wastewater purification agent containing a microbial strain using Sphingomonas yanoikuyae according to the present invention.

[0038] A wastewater purifier was manufactured by mixing the microbial strain using Sphingomonas yanoikuyae according to the present invention, Bacillus subtilis (B. subtilis) and Bacillus subtilis (B. subtilis) in a ratio of 1:1:1.

[0039] After adding the wastewater purifier according to the present invention to the wastewater of equal volume at a ratio of 1:9, the physicochemical properties were analyzed by time period, and the results were presented in graphs in Figs. 5 to 10. Here, Figs. 5 to 10 are analysis results according to the standard analysis method of AOAC (Association of Official Analytical Chemists), and the untreated area corresponds to the raw wastewater, and the treated area corresponds to the state in which the wastewater purifier according to the present invention was added to the raw wastewater.

[0040] The physicochemical analysis of the raw wastewater collected for mixing with the wastewater purifier according to the present invention is shown in Table 1 below.

[0041] COD(mg / L)BOD(mg / L)TN(mg / L)TP(mg / L)SS(mg / L)TOC(mg / L)Raw water content372.2540.639.815.150.2241.8

[0042] FIG. 5 shows the change in chemical oxygen demand (COD) when a wastewater purifier according to the present invention is added to wastewater. The COD of the raw wastewater was 372.1 mg / L, and in the untreated group, it stabilized at 20 mg / L after 150 hours, but in the treated group, it reached 21 mg / L in just 110 hours after treatment and stabilized at 15 mg / L after 170 hours, showing an efficiency of 1.4 times and a COD reduction effect of 1.3 times compared to the untreated group.

[0043] Figure 6 shows the change in biological oxygen demand (BOD) when a wastewater purifier according to the present invention is added to wastewater.

[0044] The BOD of the raw wastewater was 540.2 mg / L, and the untreated group stabilized at 25 mg / L after 190 hours, but the treated group reached 21 mg / L after 160 hours and stabilized at 15 mg / L after 180 hours, showing an efficiency of 1.2 times and a BOD reduction effect of 1.6 times compared to the untreated group.

[0045] Figure 7 shows the change in ammonia nitrogen (T-NH4), that is, the change in total nitrogen (TN), when a wastewater purifier according to the present invention is added to wastewater.

[0046] The ammonia nitrogen content of the raw wastewater was 39.8 mg / L, and in the untreated group, it stabilized at 5 mg / L after 280 hours, but in the treated group, it reached 3 mg / L after 130 hours and stabilized at 3 mg / L after 170 hours, showing an efficiency of 2.1 times and a TN reduction effect of 1.6 times compared to the untreated group.

[0047] Figure 8 shows the change in total phosphorus (TP) when a wastewater purifier according to the present invention is added to wastewater.

[0048] The total phosphorus content of the raw wastewater was 15.1 mg / L, and in the untreated group, it stabilized to less than 1.2 mg / L after 290 hours, but in the treated group, it reached this level only 210 hours after treatment and stabilized to 0.3 mg / L after 280 hours, showing an efficiency 1.4 times higher and a TP reduction effect 4 times higher than in the untreated group.

[0049] Figure 9 shows the change in suspended solids (SS) when a wastewater purifier according to the present invention is added to wastewater.

[0050] The suspended solids (SS) content of the raw wastewater was 50.2 mg / L, and although it fluctuated in the untreated group, it remained within the 30 mg / L range after 60 hours, while the treated group reached it 20 hours after treatment and stabilized at 25 mg / L after 65 hours, showing an efficiency of 3.0 times and an SS reduction effect of 1.2 times compared to the untreated group.

[0051] Figure 10 shows the change in total organic carbon (TOC) when a wastewater purifier according to the present invention is added to wastewater.

[0052] The total organic carbon (TOC) content of the raw wastewater was 241.8 mg / L. Although the untreated group fluctuated, it remained within the 10 mg / L range after 160 hours, and the treated group reached it 120 hours after treatment and stabilized at 2 mg / L after 160 hours, showing an efficiency of 1.3 times and a TOC reduction effect of 5.0 times compared to the untreated group.

[0053] As shown in FIGS. 5 to 10, when a physicochemical analysis was performed on wastewater and the wastewater purifier according to the present invention was added to the wastewater, it was confirmed that all characteristics of COD, BOD, TN, TP, SS, and TOC were excellent.

[0054] Those skilled in the art will appreciate that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims below rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims

1. A microbial strain (KCTC19105P) created by chemically mutagenesis of Sphingobium yanoikuyae by treatment with EMS (Eethyl Methane Sulfonate).

2. In paragraph 1, The above Sphingomonas yanoikuya is a microbial strain (KCTC19105P) characterized by being collected from wastewater or a polluted area.

3. In paragraph 1, A microbial strain (KCTC19105P) characterized by primary culture at a culture temperature of 28℃ for 48 hours.

4. In paragraph 3, A microbial strain (KCTC19105P) characterized in that the strain is divided into the cultured Sphingomonas yanoikuya, a predetermined amount of EMS is treated, and secondary culture is performed at a culture temperature of 28°C for 48 hours.

5. In paragraph 1, A microbial strain (KCTC19105P) characterized by being selected from among strains having a turbidity of 0.337 or less.

6. A wastewater purification agent comprising a microbial strain (KCTC19105P) of any one of clauses 1 to 5.

7. In paragraph 6, A wastewater purifier comprising the above microbial strain (KCTC19105P) mixed with Bacillus subtilis KAC92451P and Bacillus licheniformis KACC92452P in a ratio of 1:1:1.

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