KR101109120B1 - Functional microbial community being capable of degrading petroleum contaminated ocean environment - Google Patents

Abstract

The present invention relates to a functional microbial community having marine crude oil degrading activity, and more particularly, to identify microorganisms present in the functional microbial community FMC-CR9 secured in oil-contaminated tidal-flat soil, and functional microbial community (FMC-CR9) This study relates to a new functional microbial community FMC-CR9 [KCTC 11511BP] having marine crude oil degradation activity by analyzing the biodegradation efficiency of crude oil.

Marine Crude Degradation and Functional Microbial Communities

Description

Functional microbial community being capable of degrading petroleum contaminated ocean environment

The present invention relates to a functional microbial community having marine crude oil degradation activity.

Globally, marine pollution caused by oil spills is getting worse. According to the IPTOF's 2006 data, global spillages have decreased from 3 million tonnes in the 1970s, 1.17 million tonnes in the 1980s, and 1.13 million tonnes in the 1990s, but local and small spills are increasing year by year. to be. In Korea, according to the data of the Korea Coast Guard, there are 128 marine pollution accidents caused by oil spills occurring in coastal areas of Korea in 11979, 1854 cases in the 1980s, 3280 cases in the 1990s, and 1620 cases in 2000 ~ 2003. Pollution accidents are caused by fishing vessels, which account for 89% of the total accidents, but most of them are small-scale fishing boats that emit a small amount of waste oil. The number of cases is only 11%, but the amount of spillage is 98%, which is the most serious damage to the marine environment. In the future, damages caused by marine oil pollution are expected to increase further due to the increase of offshore transportation and development of waterfront industrial complexes, and marine oil spill accidents caused by human error will not only cause huge economic losses for fisheries, but also Other industries can be hit hard and are expected to have a serious impact on marine ecosystems.

Due to these problems, research on biological treatment of marine oil pollution continues, and microorganisms that decompose oil in the ocean are being separated, but field application is difficult. For this reason, microorganisms in the natural environment are rarely present on their own, and form a microbial community that interacts with microorganisms, other organisms, or the surrounding environment, and these characteristics can be attributed to wastewater treatment, water purification systems, and pathogenic microbial survival. It is found in almost all of the microbial phenomena found around us, including antibiotics, antibiotic resistance, environmental pollution, and metal corrosion. 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.

Currently, the importance of the microbial community as a research model of important biological resources and interactions between cells is gradually recognized, but the full-scale microbial community has not yet been secured and researched. Through its 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.

Wilfred et al. Investigated the microbial community changes through denaturing gradient gel electrophoresis (DGGE) during bioremediation of oil-contaminated tidal flats in Somerset, England. Dominant species during oil degradation resulted in 16S rDNA sequencing, Alcanivorax borkumensis and Pseudomonas. stutzeri ) [Appl. Environ. Microbiol. 2004, 70: 26032613.

Yakimov et al. Produced biological surfactants from the oceans and tidal flats of small islands close to the Arctic, and produced six surfactants and isolated six marine microorganisms that degraded n-alkanes. It confirmed that it was. As a result of the identification, it was confirmed that it is a new microorganism belonging to Alcanivorax borkumensis [Int. J. Syst. Bacteriol. 1998, 48: 339-348.

Katsivela et al. Investigated the change of indigenous microbial community before and after tillage in petroleum refinery Motor Oil Hellas in Greece by the t-RFLP (term-Restriction Fragment Length Polymorphism) method. . 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 contaminated with polycyclic aromatic hydrocarbons (PAH), uncontaminated Siberian soils and soils at Antarctose [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.

 The oil environment restoration experiment using the microorganisms known to date has been a problem of monitoring the decomposition process or separating the oil degradation microorganisms, and using a single microorganism, which does not completely decompose the decomposition substance and makes environmental adaptation difficult. Microbiological environmental purification of oil pollution is becoming more problematic for the method of microbial administration and adaptation. In order to solve this problem, there is an urgent need for a microbial community adapted from highly contaminated soil.

Therefore, the present inventors exist as a community rather than act as a single microorganism in the natural world, and in the marine state, the microbial community changes frequently depending on the pollutant component, so there is an urgent need for the study of a microbial community that decomposes pollutants. However, since researches related to microorganisms have been concentrated on single microorganisms, research on lack of useful microbial community has been conducted. As a result, research efforts have secured a functional microbial community, FMC-CR9 [KCTC 11511BP], which biodegrades marine crude oil. The present invention was completed by confirming the excellent crude oil cracking activity of FMC-CR9 by comparing the diesel cracking activity.

Accordingly, an object of the present invention is to investigate the microbial diversity and crude oil degradation activity of FMC-CR9 having excellent crude oil resolution obtained from oil-contaminated tidal-flat soil to confirm the superiority of FMC-CR9 to confirm the usefulness of the functional microbial community.

The present invention is characterized by a functional microbial community FMC-CR9 [KCTC 11511BP] with excellent crude oil resolution in a marine environment.

The functional microbial community building project according to the present invention will enable the resourceization of a new microbial group possessing excellent novelty (minimum unknown gene, 20%) and having marine crude oil degradation activity compared to other existing strains. The FMC samples, 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 the microorganisms present in FMC-CR9, a functional microbial community secured from oil-contaminated tidal-flat soil, and systematically analyzes them, and analyzes the crude biodegradation efficiency of the functional microbial community (FMC-CR9) to analyze the usefulness of the functional microbial community. The present invention relates to a new functional microbial community FMC-CR9 [KCTC 11511BP] with crude oil degradation activity in the marine environment.

In the present invention, in order to secure a functional microbial community from oil-contaminated tidal-flat soils, liquid medium containing 5,000 to 10,000 ppm of Bshernell-Haas (BH) medium and crude oil was used in artificial seawater. The functional microbial community was secured through integrated culture in the prepared liquid medium. Changes in the microbial community until the acquisition of FMC-CR9 were confirmed by the DNA extraction, PCR amplification of DNA at each culture stage, and the change of 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, predominant species were identified as uncultured bacterium, Alcanivorax sp. And Idiomarina sp.

The FMC-CR9 was deposited with the Korea Biotechnology Research Institute Gene Bank on May 14, 2009, and was assigned the accession number KCTC 11511BP.

The experiments on the change of diesel oil degradation activity in the integrated culture of the functional microbial community were carried out by gas chromatography after liquid culture in which crude oil and Bushnell-Haas (BH) were added to sterilized artificial seawater for 10 days. . FMC-CR9 showed a crude oil resolution of 91% at a concentration of 10,000 ppm of crude oil.

Therefore, the functional microbial community FMC-CR9 according to the present invention was confirmed to have excellent crude oil resolution, and its usefulness was also verified, and it is expected to be useful for marine oil accidents caused by ships and purification of oil-contaminated tidal flats.

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: functional microbial community FMC - CR9 Microbial Diversity

Samples used were oil-contaminated tidal-flat soil near Gomso Port oil depot. In the artificial seawater prepared according to the composition of the following Table 1 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 / 0.2 g of crude oil (10,000 ppm) was added to a 20 ml BH liquid medium prepared by adding 3.27 g of L and FeCl ₂ , and then inoculated with 0.4 g of the mud flat soil sample. 10 days of incubation. As a result, the diesel decomposition efficiency was improved during the four integrated cultures, thereby securing a functional microbial community FMC-CR9 having a diesel decomposition efficiency of 91% in the fourth integrated culture [FIG. 1]. As a result of DGGE, which showed the change of community during agglutination, 13 bands were observed in soil, but as bandage progressed, community became simpler and 7 bands were observed in 4th aggression. The major bands identified in the 4th integrated culture were Alcanivorax, respectively. borkumensis ) , uncultured bacterium, Sphingomonas paucimobilis and uncultured Fulvimonas sp.

Example  2: for creating clone libraries DNA  Extraction PCR  Condition

FMC-CR9 extracted DNA using Power soil DNA kit (Mobio, USA), and microorganisms separated from solid medium collected and mixed colonies and extracted DNA using Power 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 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: for creating clone libraries Cloning  Condition

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.

For clone libraries Cloning  Condition

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  4: FMC - CR9 Identification of clone libraries

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 (charge), and the NCBI blasts were identified by searching. As a result of the identification of FMC-T9, uncultured bacterium, Alcanivorax sp. And Idiomarina sp. Predominantly (Table 2).

Example  5: FMC - CR9 Investigation of Crude Oil Degradation Activity in Rats and Oil-Degrading Microorganisms

Oil-Bacterial Microbial Microbacterium oxydans [KCTC11366BP], purely isolated from tidal-flats, Rhodobacteraceae bacterium [KCTC11367BP], Pseudomonas Pseudomonas marincola ) and [KCTC11368BP] Sphingomonas , known as an oil-degrading microorganism xenophga ) [KCTC2978BP], Acinetobacter calcoaceticus [KCTC2702], and Burkholderia sp. were compared with FMC-CR9 for crude oil degradation activity. 0.2 g of crude oil (10,000 ppm) was added to 20 ml of BH liquid medium made by adding 3.27 g of BH medium to artificial seawater, and then inoculated with 400 μl of microorganisms in 1 × 10 ⁷ cell / ml. Shake culture was performed for 10 days at 120 rpm in a 20 ℃ incubator.

As a result, as shown in FIG. 3, the decomposition rate of FMC-CR9 reached 91%, whereas the pure bacterium Microbacterium oxydans ( Microbacterium) oxydans ) , Rhodobacteraceae bacterium , Pseudomonas marincola showed degradation rates of 72%, 58% and 68%, respectively. Sphingomonas sphingomonas xenotopa xenophga ) , Acinetobacter calcoaceticus ) and Burkholderia sp. showed very low degradation rates of 5%, 3% and 0%, respectively.

Figure 1 shows the marine crude oil degradation activity of microorganisms in the integrated culture of FMC-CR9 [1: primary integrated culture, 2: secondary integrated culture, 3: tertiary integrated culture, 4: fourth integrated culture].

Figure 2 shows the change in diversity during the incubation of FMC-CR9 using denaturing gradient gel electrophoresis (DGGE) [1: primary integration culture, 2: secondary integration culture, 3: tertiary integration culture, 4: fourth Integrated culture].

Figure 3 shows the marine crude decomposition activity of FMC-CR9 and purely isolated microorganisms.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Functional microbial community being capable of degrading          petroleum contaminated ocean environment <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)

Alcanivorax sp., Idiomarina sp., Oleiphilus sp. And Sphingomonas sp. Functional microbial community FMC-CR9 [KCTC 11511BP]. A method for purifying seawater contaminated with crude oil, comprising inoculating the seawater contaminated with crude oil with the functional microbial community FMC-CR9 [KCTC 11511BP] of claim 1.

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