KR100837876B1 - Functional microbial community fmc-ky7 being capable of degrading diesel oil - Google Patents

Abstract

A novel functional microbial community FMC-KY7 isolated from soil contaminated by diesel is provided to show excellent diesel oil bio-degrading efficiency. A functional microbial community FMC-KY7 with excellent diesel oil degrading capability is deposited as a deposition no. KCTC11077BP and comprises Sphingopyxis sp., Wautersia sp., Acinetobacter sp., Sphingomonas sp., Cupriavidus sp., Ralstonia sp., Pseudomonas sp., Alcaligenes sp. and Brucella sp..

Description

Functional microbial community FMC-KY7 being capable of degrading diesel oil}

1 is a analysis of dominant microorganisms of FMC-KY7 using terminal-Restriction Fragment Length Polymorphism (t-RFLP) [above; Using Hha I enzyme, below; Using Hae III enzyme].

2 shows microbial colonies isolated from FMC-KY7 using three types of solid media.

Figure 3 shows a schematic diagram of the distribution of microorganisms between the dominant microorganisms and isolated microorganisms of FMC-KY7.

Figure 4 shows the diesel degradation activity of FMC-KY7 and isolated microorganisms.

The present invention relates to a functional microbial community having a diesel oil decomposition activity, and more specifically, to identify the microorganisms isolated from FMC-KY7 [KCTC 11077BP] and FMC-KY7 functional microbial community secured from diesel contaminated soil New functional microbial community FMC-KY7 with diesel oil degradation activity by analyzing the biodegradation efficiency of diesel microorganisms of dominant microorganisms separated from the functional microbial community (FMC-KY7) and confirming the usefulness of the functional microbial community [KCTC 11077BP] It is about.

In natural environments, microorganisms rarely exist on their own and form a microbial community that interacts with microorganisms, other organisms, or the surrounding environment, which characterizes wastewater treatment and water purification systems, viability of pathogenic microorganisms, and antibiotic resistance. It is found in almost all microbial phenomena found around us, including the restoration of polluted environments and the corrosion of metals. Since the expressive characteristics of the microorganisms form a community are completely different from those of individual organisms, the microbial community is regarded as a functional expression unit rather than the individual microorganisms, and resources are secured. Information analysis is needed.

Therefore, rather than separating and applying microorganisms, application research through analysis of the dynamic characteristics of various microorganisms that make up the microbial community itself, microbial phenotype and activity according to environmental changes, and the interrelationship between microbial cells Is needed.

At present, the importance of microbial community as a research model of important biological resources and interactions between cells is gradually recognized, but full-scale microbial community acquisition and research have not been carried out yet. Through sequencing analysis, only the diversity of the microbial community is analyzed. The environmental restoration technology of pollutants mainly consists of the study of degradation metabolism using a single microbial strain on a single substance, and the research on the redesign of the degraded microbial cells and the development of probes for biomonitoring are mainly performed.

Katsivela et al. Investigated the changes in the indigenous microbial community before and after farmland in petroleum refinery Motor Oil Hellas in Greece by the t-RFLP (terminal-restriction fragment length polymorphism) method. Among the isolates that grew after the incubation, the well growing species were Enterobacter and Ochrobacterium based on the 16S rDNA sequence, and the same species were found in the t-RFLP results. In addition, they were found to have a significant effect on degradation activity [Wat. Air Soil Poll. 2003, 3: 103-115.

Laurie et al. Quantified the amount of nahAc and phnAc (phenanthrene dioxygenase) from two soils in Waikato, New Zealand, PAH-contaminated, uncontaminated Siberian and Antarctic islands [Appl. Environ. Microbiol. 2000, 66: 1814-1817.

Yeates et al. Conducted PCR and hive using primers made from known aromatic hydrocarbon degradation genes for soil samples from two aromatic hydrocarbon-contaminated and two non-contaminated soils in four of Wales, Australia. New gene search and their function were observed by the hybridization method [Environ. Microbiol. 2000, 2: 644-653.

In addition, Jeon et al. Attempted to analyze the naphthalene-degrading microorganisms in situ using radioisotope techniques in coal tar-contaminated areas and to link the phylogenetic analysis and functional characteristics of the microorganisms [Jeon et al., 2003].

The diesel decomposition experiment of microorganisms thus far known has not been completely decomposed by the use of a single microorganism, and the problem of difficult environmental adaptation has been caused, and the microbial community is urgently required to solve these problems.

Therefore, the present inventors existed as a community rather than acting as a single microorganism in the natural world, but biodegradation of diesel oil in the absence of experiments related to the useful microbial community because research related to microorganisms has been concentrated on a single microorganism. The functional microbial community FMC-KY7 was secured to compare the degradable single microorganisms with the diesel oil degrading activity, confirming the superior diesel decomposing activity of FMC-KY7. The present invention has been completed by confirming that is reversed.

Therefore, the present invention is to investigate the microbial diversity and diesel oil degradation activity of the FMC-KY7 and the separated microorganisms excellent in the oil-degradable soil obtained from oil contaminated soil to confirm the superiority of the FMC-KY7 to confirm the usefulness of the functional microbial community There is a purpose.

The present invention is characterized by the functional microbial community FMC-KY7 [KCTC 11077BP] excellent in diesel oil resolution.

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

The present invention identifies the microorganisms isolated from FMC-KY7 [KCTC 11077BP] and FMC-KY7, which are functional microbial communities secured from diesel-contaminated soils, and systematically analyzes them and separates them from the functional microbial community (FMC-KY7). The present invention relates to a new functional microbial community FMC-KY7 [KCTC 11077BP] having diesel oil degradation activity by analyzing the biodegradation efficiency of diesel oil.

In the present invention, the medium used for pure separation from the functional microbial community was a Bushnell-Haas (BH) solid medium, an R2A solid medium, a 1/10 diluted nutrient solid medium (Nutrient agar, NA). Solid medium without diesel and a medium with l% (w / v) sterilized diesel were used. Smear cultures of functional microbial community samples in prepared media.

In order to confirm the distribution of microorganisms contained in FMC-KY7, DNA was extracted and amplified by PCR to confirm the community diversity of FMC-KY7 through t-RFLP. In order to confirm the distribution of microorganisms in the colony, a clone library was prepared by ligation to the T-vector and transformation. A total of 50 colonies were selected and examined for 16S rDNA partial sequencing to identify NCBI blasts. As a result, the predominant species are the genus Spingopyxis sp., Genus Wautersia sp., Genus Acinetobacter sp., Genus Sphingomonas sp., And genus Cupriaviders. ( Cupriavidus sp.), Ralstonia sp.

The FMC-KY7 was deposited with the Korea Biotechnology Research Institute Gene Bank on February 28, 2007, and was assigned the accession number KCTC 11077BP.

Microorganisms isolated from FMC-KY7 were purely separated into each medium according to the shape of the colonies. Also, in order to confirm the distribution of separable microorganisms, colonies grown in three types of media were collected, DNA was extracted, amplified by PCR, and transformed. The clone library was produced by the transformation. A total of 100 colonies were selected and examined for 16S rDNA partial sequencing to identify NCBI blasts. As a result, the predominant species were Brucella sp., Archangium sp., Sinorhizobium sp. And Gordonia sp.

Sterilized diesel was added to the Bushnell-Haas (BH) liquid medium and the colonies were suspended in saline and the same amount of microorganism was added to test the diesel degradation activity of the functional microbial community and the microorganisms purely isolated. Incubate aseptically. After 10 days of incubation, FMC-KY7 showed more than 95% diesel oil degradation activity, and Acinetobacter sp. Showed about 80% diesel oil degradation activity. However, the genus Brucella sp., Archangium sp., Sinorhizobium sp. And Gordonia sp. The oil degradation activity was found to be very low.

Therefore, the functional microbial community FMC-KY7 [KCTC 11077BP] according to the present invention was confirmed to have excellent diesel oil resolution, its usefulness was also verified, tidal flat, farmland oil pollution restoration, soil restoration during military relocation, waste gas station environmental purification It is expected to be useful for such purposes.

Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

Example 1: Microbial Diversity of Functional Microbial Community FMC-KY7

In experiments on the functional microbial community FMC-KY7 with 96% diesel decomposition efficiency obtained through integrated culture in KY7 soil collected from Gwangyang Works, the diversity through t-RFLP is shown in FIG. 1, and the 16S rDNA clone library The analysis identified microbial species corresponding to each peak of t-RFLP.

Example 2 Media Composition and Culture Conditions for Pure Microbial Separation from FMC-KY7

The medium used was BH medium (MgSO ₄ 0.2 g / L, CaCl ₂ 0.02 g / L, KH ₂ PO ₄ 1.0 g / L, K ₂ HPO ₄ 1.0 g / L, KNO ₃ 1.0 g / L, FeCl ₂ 0.05 g / L) was used as a solid medium, 1.5% agar powder (agar powder), R2A agar, 1/10 diluted nutrient solid medium (Nutrient agar, NA) was used, 1% (w / v) A solid medium containing diesel and sterilized zeolite (Sigma, USA) and a non-adding solid medium were prepared.

FMC-KY7 was plated on the prepared medium and sterilely cultured at 30 ° C. for 3 days to separate microorganisms as shown in FIG. 2. The separated microorganisms were visually separated and purified.

Example 3: DNA Extraction and PCR Conditions for Clonal Library Construction

FMC-KY7 extracted DNA using UltraClean soil DNA kit (Mobio), and microorganisms isolated from solid medium collected and mixed colonies and extracted DNA using UltraClean soil DNA kit (Mobio). PCR was performed in the following manner.

Primer used:

27F 5'-AGA GTT TGA TCC TGG CTC GA-3 '(SEQ ID NO: 1)

1542R; 5'-AGA AAG GAG GTG ATC CAG CC-3 '(SEQ ID NO: 2)

PCR Conditions for Clone Library Construction

Composition of reaction mixture:

1 μl DNA

12.5 pmol 27F 2.5 μl

12.5 pmol 1542R 2.5 μl

1 μl 10 mM dNTP mixture

5 μl 10 × PCR buffer

0.5 μl bobbin serum albumin (BSA)

0.5 μl of Taq DNA polymerase (TaKaRa, Japan)

36 μl dH ₂ O

Reaction conditions (30 repetitions except for initial denaturation and final extension):

Initial denaturation process-95 ℃, 3 minutes

Denaturation process-94 ℃, 45 seconds

Annealing process-58 ℃, 45 seconds

Polymerization process-72 ℃, 2 minutes

Final extension process-72 ° C, 7 minutes

PCR reactions were identified on agarose gels.

Example 4: Cloning Conditions for Clone Library Construction

A clone library was constructed using DNA extracted from FMC-KY7 and isolated microorganisms.

PGEM-T easy (Promega) vector was used for TA-cloning and DH5α (Real Biotech Corporation) was used for transformation. After transformation, white colonies were selected using X-gal + IPTG in LB + ampicillin solid medium.

Cloning Conditions for Clone Libraries

Composition of reaction mixture:

1 μl vector (pGEM-Teasy)

1 μl T4 ligase

3 μl PCR product

5 μl 2 × buffer

Reaction condition:

ligation mixture 4 ℃, reaction for 2 hours

Comp. thaw cells on ice

ligation mixture to comp. Transfer to cell for 90 seconds at 42 ℃

30 minutes reaction on ice

Smear 37 ° C o / n on LB-amp (X-gal + IPTG)

Example 5: Identification of clone libraries of FMC-KY7 and isolated microorganisms

Selected white colonies were extracted from the plasmid using a Qiagen spin miniprep kit (Qiagen), and the presence or absence of an insert was determined using the restriction enzyme EcoRI. The plasmid showing the size of the insert of 1500 bp was selected and asked for solgent (charge) to analyze the sequencing. NCBI blast was identified by searching. Identification results of the FMC-KY7, Scotland pinggo Pixies in (Spingopyxis sp.), Water Asia in (Wautersia sp.) And Acinetobacter in was shown the most dominant two (Acinetobacter sp.), Alkali jeneseu in (Alcaligenes sp.) And Brucella sp. Found only one colony (Table 2).

As a result of the identification of clone libraries of isolated microorganisms, 94 clone libraries were produced by microorganisms cultured and isolated under 12 conditions in a total of three media, and the predominant species were Brucella sp. And Archangium sp. .), Genus Sinorhizobium sp., Genus Gordonia sp. (Table 3).

The microorganisms identified were Brucella sp., Spingopyxis sp., Cupriavidus sp., Acinetobacter sp., And Wautersia sp. Pseudomonas sp. Was found in FMC-KY7 and isolated microorganisms, but Brucella sp. Showed the highest predominance in isolated microorganisms but only 3% in FMC-KY7. The remaining common microorganisms were predominant in FMC-KY7 but their distribution was very low in isolated microorganisms (FIG. 3).

Example 6 Investigation of Diesel Degradation Activity of Major Microorganisms

FMC-KY7 and the observed common to separate the microorganism's pinggo Pixies in (Spingopyxis sp.), Water Asia in (Wautersia sp.), Coupe Ria bideoseu in (Cupriavidus sp.), Acinetobacter genus (Acinetobacter sp.) FMC-KY7 and diesel degrading activity were analyzed in Pseudomonas sp. And Brucella sp. Each microorganism was inoculated by diluting with saline to 10 ⁶ . The medium used was BH medium (MgSO ₄ 0.2 g / L, CaCl ₂ 0.02 g / L, KH ₂ PO ₄ 1.0 g / L, K ₂ HPO ₄ 1.0 g / L, KNO ₃ 1.0 g / L, FeCl ₂ 0.05 g / L) inoculated 400 μl of microorganisms in a 20 ml BH medium, 0.2 g diesel, 0.2 g zeolite (Sigma, USA) culture in a 100 ml Erlenmeyer flask and shaken for 10 days at 120 rpm in a 30 ℃ incubator It was. As a result, as shown in FIG. 4, the decomposition rate of FMC-KY7 was 95% or more, and the microorganism showing the best diesel decomposition activity showed diesel degradation rate of about 80% in the genus Acinetobacter sp. Dominant in the paper had Acinetobacter the FMC-KY7 (Acinetobacter sp.) , Scotland pinggo Pixies in (Spingopyxis sp.), And water Shia in (Wautersia sp.) Occupies a dominant species in the separated microorganisms eoteuna demonstrate the diesel-degrading activity Brucella sp., Archangium sp., Sinorhizobium sp. And Gordonia sp. Were found to have little diesel degradation activity.

The functional microbial community building project according to the present invention will enable the resourceization of a new microbial group having excellent novelty (minimum unknown gene, 60%) compared to other conventionally strained strains, and will be provided through the microbial community bank to be built. FMC samples will accelerate the development of bioremediation processes for contaminated environments using microbial populations both domestically and internationally, thereby creating economically significant added value as well as technical achievements in the purification of contaminated environments. It is expected to be.

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Claims (1)

Spingopyxis sp., Wautersia sp., Acinetobacter sp., Spingomonas sp., Cupriavidus sp., Lal Functional microorganism community FMC- with excellent resolution for diesel oil, characterized by containing the genus Ralstonia sp., Pseudomonas sp., Alcaligenes sp. And Brucella sp. KY7 [KCTC 11077BP].

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