KR20100123513A - Functional microbial community being capable of degrading high level diesel oil - Google Patents

Abstract

PURPOSE: A functional microorganism community having activity of decomposing diesel of high concentration is provided to purify contaminated soil. CONSTITUTION: A microorganism community FMC-T9(KCTC 11340BP) has an ability of decomposing diesel. The microorganism community includes Pseudomonas sp., Burkholderia sp. and Rhodanobacter sp. A Bushnell-Haas liquid medium containing 10,000 ppm sterilized diesel is used to obtain the functional microorganism community from a contaminated soil.

Description

Functional microbial community being capable of degrading high level diesel oil}

The present invention relates to a functional microbial community having a high concentration of diesel oil decomposition activity.

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. When microorganisms form a community, the characteristics of expression are completely different from those of individual organisms. Therefore, the microbial community is considered as a functional expression unit rather than individual microorganisms, and resources are collected. Analysis is necessary.

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 tillage in petroleum refinery Motor Oil Hellas in Greece by t-RFLP (term-Restriction Fragment Length Polymorphism). The dominant species among the isolates after the incubation were Enterobacter and Ochrobacterium as a result of 16S rDNA sequencing, 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.

Jeon et al. Analyzed naphthalene-degrading microorganisms in situ using radioisotope technology in coal tar-contaminated regions, and analyzed the systematic and functional characteristics of the microorganisms [Proc. Natl. Acad. Sci. 2003, 100: 13591-13596.

 The diesel decomposition experiment of microorganisms known to date has a problem that complete decomposition of decomposition products is not achieved by using a single microorganism, and that it is difficult to adapt to the environment. It is becoming. In order to solve this problem, there is an urgent need for a microbial community adapted from highly contaminated soil.

Therefore, the present inventors existed as a community rather than acting as a single microorganism in the natural world, but since the research related to microorganisms has been concentrated on a single microorganism, a high concentration of diesel oil is lacking in the experiments related to the useful microbial community. The biodegradable functional microbial community FMC-T9 was secured to compare the degradable activity of single microorganisms with diesel oil, confirming the superiority of the diesel oil degradation activity of FMC-T9.

Therefore, the present invention confirms the superiority of FMC-T9 by examining the microbial diversity and diesel oil degrading activity of diesel microorganisms with excellent degradability of diesel oil obtained from high concentration of oil contaminated soil and confirming the usefulness of functional microbial community. Its purpose is to.

The present invention is characterized by the functional microbial community FMC-T9 [KCTC 11340BP] excellent in diesel oil resolution.

Functional microbial community building project according to the present invention will enable the regeneration of new microbial group possessing excellent novelty (at least unknown gene, 28%) and high concentration of diesel decomposition activity compared to other isolated strains, and construction The FMC sample, which will be provided through the Microbial Community Bank, will accelerate the development of bioremediation processes for contaminated environments using microbial populations both domestically and internationally. It is also expected to create great added value.

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

The present invention identifies and systematically analyzes microorganisms present in the functional microbial community FMC-T9 obtained from high concentration diesel contaminated soil, and analyzes the diesel biodegradation efficiency of the functional microbial community (FMC-T9). A new functional microbial community, FMC-T9, having high concentration (10,000-30,000 rpm) diesel oil degrading activity by identifying its usefulness.

In order to secure a functional microbial community from soil contaminated with high concentration diesel oil, a Büschnell-Haas (BH) liquid medium added with 10,000 ppm of sterile diesel was used. The functional microbial community was secured through integrated culture in the prepared medium. Changes in the microbial community until the FMC-T9 was obtained, the DNA was extracted at each culture stage, amplified DNA by PCR and confirmed the change of the community through DGGE.

In order to confirm the distribution of microorganisms in the colony, the PCR amplification product was ligation to the T-vector and transformed to produce a clone library. A total of 80 colonies were selected and examined for 16S rDNA partial sequencing to identify NCBI blasts. As a result, the predominant species were identified as uncultured bacterium, Pseudomonas sp., And fenthion-degrading bacterium.

The FMC-T9 was deposited with the Korea Biotechnology Research Institute Gene Bank on May 30, 2008 and was assigned the accession number KCTC 11340BP.

Experiments on the change of diesel oil degradation activity during the incubation of the functional microbial community were analyzed by gas chromatography after 10 days of cultivation of the Bshernell-Haas (BH) liquid medium to which sterilized diesel was added. FMC-T9 decomposed 93% of 10,000 ppm of diesel. In addition, the decomposition activity experiment with 30,000 ppm high-concentration diesel showed 53% decomposition activity at 14,000 ppm after 7 days.

Therefore, the functional microbial community FMC-T9 according to the present invention has been found to have excellent diesel oil resolution, and its usefulness has been verified, and thus, it will be useful for high-contaminated soils such as soil restoration during military relocation and purification of waste gas station environment. It is expected.

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

Example 1: Microbial Diversity of Functional Microbial Community FMC-T9

Samples used were soil collected from oil tanks contaminated with 93,836 ppm diesel. 20 ml BH medium (0.2 g / L MgSO ₄ , 0.02 g / L CaCl ₂ , 1.0 g / L, KH ₂ PO ₄ ) with 0.2 g diesel (10,000 ppm) and 0.2 g zeolite (Sigma, USA) ₂ HPO ₄ 1.0 g / L, KNO ₃ 1.0 g / L, FeCl ₂ 0.05 g / L) was inoculated with 0.4 g of soil sample was incubated for 10 days at 120 rpm in a 24 ℃ incubator. As a result, the diesel decomposition efficiency was improved during the four times of the incubation culture to secure a functional microbial community FMC-T9 having a diesel decomposition efficiency of 93% in the fourth integration culture (FIG. 1). As a result of the DGGE showing the change of clustering in the culture, more than 30 bands were observed in the soil, but as the culture progressed, the community became simpler and 6 bands were observed in the 4th integration culture (FIG. 2).

Example 2: DNA Extraction and PCR Conditions for Clonal Library Construction

FMC-T9 extracted DNA using UltraClean soil DNA kit (Mobio, USA), and microorganisms isolated from solid medium collected and mixed colonies and extracted DNA using UltraClean soil DNA kit. 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 the 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 of 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 3: Cloning Conditions for Clone Library Construction

Clonal libraries were constructed using DNA extracted from FMC-T9 and isolated microorganisms. PGEM-T easy (Promega, USA) vector was used for TA-cloning and DH5α (RBC Bioscience, Taiwan) 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 the 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 4 Identification of Clone Library of FMC-T9

Selected white colonies were extracted from the plasmid using a Qiagen spin miniprep kit (Qiagen, Germany), and the presence or absence of an insert was determined using the restriction enzyme EcoR I. Plasmids with an insert size of 1500 bp were selected and quenched by Solent (Daejeon). The sequences were analyzed and NCBI blasts were identified. As a result of the identification of FMC-T9, uncultured bacterium, Pseudomonas sp. And penthion-degrading bacterium were predominant (Table 1).

Distribution of Microorganisms Identified in FMC-T9 Configuration (%) uncultured bacterium 27 Pseudomonas sp. 25 fenthion-degrading bacterium FP1-6 12 Burkholderia sp. 6 Rhodanobacter sp. 6 Achromobacter sp. 4 Alcaligenes sp. 4 Bordetella sp. 4 Mesorhizobium ap. 4 Rhizobium sp. 4 Diaphorobacter sp. 2 Dyella sp. 2

Example 5 Investigation of Diesel Degradation Activity of FMC-T9 and Oil-Degrading Microorganisms

The diesel cracking activity of FMC-T9 was analyzed. Dilute microorganisms in saline solution at 1 × 10 ⁶ cell / ml in 20 ml BH medium containing 0.2 g of diesel (10,000 ppm) and 0.2 g of zeolite (Sigma, USA), inoculate 400 μl, and then incubate at 24 ℃. Shaking culture at 120 rpm for 10 days. As a result, as shown in FIG. 3, the degradation rate of FMC-T9 was 93%, whereas the single strains of Sphingopyxis sp., Acinetobacter sp. And Pseudomonas sp. The degradation rates were 52%, 75% and 16%, respectively.

Example 6: Investigation of Diesel Degradation Activity of FMC-T9 at High Concentration

Diesel degradation activity of FMC-T9 at high concentration was analyzed. Inoculate 400 μl of FMC-T9 (2%) in 20 ml of BH medium containing three different concentrations of diesel (10,000, 20,000 and 30,000 ppm) and 0.2 g of zeolite (Sigma, USA). Shake culture was performed for 7 days at rpm. As a result, it was confirmed that diesel decomposition proceeded in the experimental group inoculated with FMC-T9, while the experimental group not inoculated with microorganisms at a high concentration of diesel concentration of 30,000 ppm decreased by 4%. It was confirmed that the 53% decrease in ppm (Table 2).

Figure 1 shows the diesel degradation activity of microorganisms in the integrated culture of FMC-T9.

Figure 2 shows the change in diversity during the integrated culture of FMC-T9 using denaturing gradient gel electrophoresis (DGGE) [soil: soil sample, 1: primary integrated culture, 2: secondary integrated culture, 3: tertiary integrated culture , 4: 4th integrated culture].

Figure 3 shows the diesel degradation activity of FMC-T9 and lyolytic microorganisms.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Functional microbial community being capable of degrading high          level diesel oil <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F primer <400> 1 agagtttgat cctggctcga 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 1542R primer <400> 2 agaaaggagg tgatccagcc 20  

Claims (2)

Microbial community FMC-T9 with good diesel oil resolution [KCTC 11340BP]. The microbial community FMC-T9 [KCTC 11340BP] according to claim 1, comprising Pseudomonas sp., Burkholderia sp. And Rhodanobacter sp.

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