KR20100087559A - The anaerobic functional microbial community being capable of vinyl chloride dechlorination - Google Patents

Abstract

PURPOSE: A functional microbial community is provided to ensure vinyl chloride dechlorination performance. CONSTITUTION: An anaerobic microbial community FMC-12T(KCTC 11435BP) has excellent vinyl chloride dechlorination performance. The microbial community includes Sulfurospillum sp., Dehalococcoides sp., Denitrovibrio acetophilus, and Dechlorospirillum sp. The anaerobic microbial community FMC-12T(KCTC 11435BP) is isolated from VC(vinyl chloride)-polluted river.

Description

The anaerobic functional microbial community being capable of vinyl chloride dechlorination}

The present invention relates to a functional microbial community excellent in vinyl chloride dechlorination.

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 are insignificant. The analysis mainly performs diversity analysis of the microbial community. 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.

Anaerobic and aerobic microorganisms are involved in the decomposition of ethylene chloride by microorganisms through various decomposition reactions. However, since ethylene chloride contaminated soil and groundwater are often deficient in oxygen or anaerobic, decomposition of ethylene chloride by microorganisms in the contaminated site is more effective in reducing dechlorination in anaerobic conditions than in aerobic decomposition. It is known to be rapidly progressed and dechlorinated stepwise by PCE (tetrachloroethene), TCE (trichloloethene), cis- DCE ( cis- 1,2-dichloroethene), VC (vinyl chloride), and ethene. It is known that the larger the number, the higher the reduction potential and the better the reductive dechlorination reaction. In dechlorination of PCE and TCE, the accumulation of the intermediate metabolites cis- DCE and VC was observed, and the conversion to ethene was reported to proceed very slowly. In addition, VC is a highly carcinogenic substance, a very toxic substance designated by the United States and Japan.

The conversion of VC to ethene, the last intermediate metabolite, is very important for the complete treatment of PCE.

However, although there are a variety of microorganisms in nature, there are very few anaerobic dechlorination microorganisms that have been found so far due to the difficulty of detecting and separating the microorganisms in the anaerobic state, and dechlorination by anaerobic mutual metabolism is the main cause of microorganisms decomposing chlorine compounds. It is considered a mechanism.

Therefore, it is important to secure a microbial community with high dechlorination efficiency from stream sloping of the river, dredged soil of the ocean, soil, and the like.

Therefore, the present inventors studied to confirm the usefulness of the anaerobic functional microbial community by confirming the diversity of the anaerobic functional microbial community and the microorganisms constituting the community, and the FMC-12T [KCTC] obtained from the VC contaminated sample. 11435BP] and FMC-12T [KCTC 11435BP] identified the dominant microorganisms and analyzed systematically, and anaerobic microbial community FMC-12T [KCTC 11435BP] and VC dechlorination rate of the dominant microorganisms identified to analyze the usefulness of the anaerobic functional microbial community By confirming that the present invention was completed.

Accordingly, an object of the present invention is to provide an anaerobic microbial community FMC-12T [KCTC 11435BP] excellent in VC dechlorination capacity obtained from VC-contaminated streams.

The present invention is characterized by the anaerobic microbial community FMC-12T [KCTC 11435BP] with excellent VC dechlorination ability.

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

The present invention is to identify the microorganisms in the anaerobic functional microbial community FMC-12T [KCTC 11435BP] with excellent VC dechlorination capacity obtained from VC-contaminated streams, and to examine the microbial diversity and VC dechlorination activity, and the usefulness of the anaerobic microbial community It was confirmed.

The present invention relates to FMC-12T [KCTC 11435BP] capable of biodegrading VC obtained from VC polluted streams collected in Incheon.

In order to secure the anaerobic microbial community in the present invention, the anaerobic politics culture is carried out in a mineral salt medium that is sufficiently purged with N ₂ -CO ₂ (80:20) gas in a serum bottle. do.

To confirm the distribution of microorganisms contained in FMC-12T [KCTC11435BP], DNA was extracted and amplified by PCR to confirm cluster diversity of FMC-12T with DGGE. In order to confirm the distribution of microorganisms in the colony, a clone library is prepared by ligation to the T-vector and transformation into E. coli. A total of 24 colonies were selected and examined for 16S rDNA partial sequencing to identify NCBI blasts. As a result, Sulfurospirillum sp. Was predominantly 75%.

The microbial community FMC-4P was deposited to the Korea Biotechnology Research Institute Gene Bank on December 1, 2008, and was given an accession number as KCTC 11435BP.

After 6 weeks of the anaerobic standing culture, FMC-12T [KCTC 11435BP] showed 100% VC dechlorination activity. It showed a rapid decrease from the beginning of inoculation, and the added VC was found to be all degraded at 34 days. From day 4, when dechlorination of VC occurred rapidly, the concentration of ethene per unit time increased rapidly.

In the present invention, the microorganisms in the natural world exist as a community rather than act as a single microorganism, but since the researches related to microorganisms have been concentrated on a single microorganism, experiments related to the useful microorganism community have been lacking. Among them, the microbial community FMC-12T, which biodegrades VC, was confirmed to have excellent VC dechlorination activity, and the predominant microorganism of the microbial community was identified.

The microbial community building project carried out by the research team will enable the regeneration of new microbial populations that have outstanding novelty (minimum unknown gene, 79.2%) compared to other isolated strains, and will be provided through the microbial community bank to be built. The FMC sample is expected to accelerate the development of bioremediation processes for contaminated environments using microbial groups both domestically and internationally. In addition, it is expected to create economically significant added value as well as the technical achievement of the purification of polluted environment.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited by Examples.

Example 1: Microbial Diversity Analysis of FMC-12T

As an experiment on microbial diversity analysis of the functional microbial community FMC-12T with excellent VC dechlorination ability obtained through integrated culture, microbial diversity through denaturing gradient gel electrophoresis (DGGE) is shown in FIG. 1, and 16S rRNA gene clone library analysis Through microbial species corresponding to each band of the DGGE was identified.

Example 2: Culture for Maintenance of Dechlorination of FMC-12T

The medium and method used to obtain FMC-12T are as follows.

Mineral salt medium (NCl 1.0g, MgCl ₂ · 6H ₂ O 0.5g, KH ₂ fully purged with N ₂ -CO ₂ (80:20) gas in a 160 ml serum bottle PO ₄ 0.2 g, NH ₄ Cl 0.3 g, KCl 0.3 g, CaCl ₂ H ₂ O 0.015 g, trace element solution 1 ml, Na ₂ SeO ₃ -Na ₂ WO ₄ solution 1 ml, resazurin 10 mg / L) 50 ml 2 g of the sample was inoculated in an anaerobic chamber (Coy Lab., USA). At this time, Na ₂ S, L-cysteine is added to the final concentration of 0.2 mM as a reducing agent, and 0.5 ml of the vitamin solution is added. 1.4 mM VC was added as the electron acceptor, and 2 mM lactate was used as the electron donor. Anaerobic policing was carried out at 28 ° C. for 6 weeks.

After 6 weeks of the anaerobic standing culture, FMC-12T [KCTC 11435BP] showed 100% VC dechlorination activity. Dechlorination from VC to ethene proceeded rapidly from the first day of inoculation and the concentration of ethene per unit time increased rapidly. It was confirmed that the added VC was decomposed all on the 34th day as in FIG.

Example 3 DNA Extraction and PCR Conditions for Colony Library Construction

FMC-12T extracted DNA using FastDNA SPIN for soil kit (MP biomedicals) and PCR was performed as follows.

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

1541R: 5'-AAG GAG GTG ATC CAN CCR CA-3 '(SEQ ID NO: 2)

PCR Conditions for Colony Library Construction

Composition of the reaction mixture:

1 μl DNA

12.5 pmol 27F 2.5μl

12.5 pmol 1541R 2.5μl

2 μl 10 mM dNTP mixture

5 μl of 10 ㅧ 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 ℃, 5 minutes

Denaturation process-94 ℃, 1 minute

Annealing process-58 ° C, 1 minute

Polymerization process-72 ° C, 1 minute 30 seconds

Final extension process-72 ° C, 10 minutes

PCR reactions were identified on agarose gels.

Example 4: Cloning Conditions for Colony Library Construction

Clonal libraries were constructed using DNA extracted from FMC-12T.

PGEM-T easy (Promega) vector was used for TA-cloning and E. coli DH5α 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

PCR product 3µl

2 × buffer 5µl

Reaction condition:

ligation mixture room temp., 2 hr reaction

Thaw E. coli DH5α on ice

ligation mixture to E. coli Transfer to DH5α for 90 s at 42 ° C

30 min reaction on ice

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

Example 5: Identification of Clone Libraries of FMC-12T

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. A plasmid having an insert size of 1500 bp was selected and commissioned to Cosmojintech (Seoul) for sequencing. NCBI blasts were identified by searching. As a result of the identification of FMC-12T [KCTC 11435BP], the uncultured Sulfurospirillum sp. Showed the most predominance (Table 1). In addition, Dehalococcoides ethenogenus Strain 195 constituted other clusters.

The genus of sulforopylium is known as a microorganism that degrades PCE to cis- DCE. Dihalococoid etenogenus strain 195 was isolated as the only microorganism that degrades PCE to ethene. However, it has been reported that dihalocoid etenogenus strain 195 rapidly degrades PCE to VC, but does not always dechlorinate VC to ethene due to unknown factors. Other dihalocoid strains are frequently found in cultures that dechlorinate under anaerobic conditions. Their microorganisms include strains FL2, CBDB1 (AJ965256) and BAV1 (CP000068). In the dechlorination process of PCE, TCE, cis- DCE and VC, the dihalocoid genus is known as an important microorganism.

A clone of dihalocoide etenogenus strain 195 (8%) was present in the FMC-12T culture, but the genus Sulphorospyrilium (75%) was predominant. This may be inferred from the fact that the genus of sulfospiryliumium plays a greater role in the dechlorination of VC. Other microorganisms included 4.2% of Dechlorospirillum sp. And 8% of Denitrovibrio acetophilus in the clone library. Microorganisms appearing in both DGGE and clone libraries were found in the genus Sulphorospyrilium, Dinitrovibrio acetophyllus, and Dichlorospiryllium genus. The genus of sulfospirylium is known as a dechlorinating microorganism, and two other microorganisms have been reported as microorganisms present in dechlorination culture. However, the dihalocoid genus isolated from microorganisms that degrade PCE to ethene in the clone library was not detected in DGGE.

FMC-12T sample sikimyeo disassemble the VC to ethene, which is the dominant microbial prisoners stand in RY Lilium reported as microbial dechlorination (Sulfurospillum sp.) Have appeared, other microorganisms dihalo rococo id in (Dehalococcoides sp.), Denitrovibrio ( Denitrovibrio acetophilus ) and Dechlorospirillum sp. Other microorganisms are clustered together with uncultured bacterium.

TABLE 1

Microbial Distribution Identified in Clone Library of FMC-12T

Accession Species % AY530551 Dechlorospirillum sp. DB 16S ribosomal RNA gene 4.2 CP000027 Dehalococcoides ethenogenes 195 8.3 AF146526 Denitrovibrio acetophilus 16S ribosomal RNA gene 8.3 AJ009499 uncultured bacterium SJA-168 16S rRNA gene 4.2 DQ234133 Uncultured Sulfurospirillum sp. clone DS049 16S rRNA gene 75.0

Example 6: PCR reaction conditions for DGGE of FMC-12T

Primary PCR Conditions for DGGE

Composition of the reaction mixture:

1 μl DNA

12.5 pmol 27F 2.5μl

12.5 pmol 1541R 2.5μl

2 μl 10 mM dNTP mixture

5 μl of 10 ㅧ 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 ℃, 5 minutes

Denaturation process-94 ℃, 1 minute

Annealing process-58 ° C, 1 minute

Polymerization process-72 ° C, 1 minute 30 seconds

Final extension process-72 ° C, 10 minutes

PCR reactions were identified on agarose gels.

Secondary PCR Conditions for DGGE

341F-GC: 5'-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG

             AGG CAG CAGCCT ACG GGA GGC AGC AG-3 '(SEQ ID NO: 3)

786R: 5'-CTA CCA GGG TAT CTA ATC-3 '(SEQ ID NO: 4)

Composition of the reaction mixture:

1 μl primary PCR product

12.5 pmol 341F-GC 2.5 μl

12.5 pmol 786R 2.5μl

2 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 ℃, 5 minutes

Denaturation process-94 ℃, 45 seconds

Annealing process-60 ° C, 45 seconds (-20 ° C decrease per cycle) 20 cycles

Polymerization process-72 ° C, 45 seconds

Denaturation process-94 ℃, 45 seconds

Annealing process-50 ° C, 45 seconds (15 cycles)

Polymerization process-72 ° C, 45 seconds

Final extension process-72 ° C, 10 minutes

PCR reactions were identified on agarose gels.

Example 7: DGGE of FMC-12T

D-Code 16 / 16-cm gel system (Bio-Rad, Hercules, USA) was used, the denaturation gel concentration was 40-70%, acrylamide concentration 10% to make a denaturation gel. Electrophoresis conditions were performed at 1 × TAE (20 mM Tris acetate, 0.5 mM EDTA [pH 8.0]), 60 V, 17 hours maintained at 60 ° C. After the electrophoresis gel was stained with ethidium bromide for 30 minutes was observed. The image was converted using a Kodak 1.0 software package (Kodak), as shown in FIG. 1 (eubacteria).

Example 8: Determination of the base sequence of the DGGE band of FMC-12T

The band was cut out, 50 μl of TE buffer was added, frozen at a deep freezer of −70 ° C., and dissolved in a 50 ° C. water bath three times, followed by centrifugation (6,300 × g, 1 min) to obtain a supernatant. PCR was performed in the same manner as above using DNA recovered from each band using 341F-GC and 786R primers as templates. PCR products were purified using a PCR purification kit (Qiagen, Hilden, Germany). The purified product was determined by sequencing using an automatic DNA sequencer (Applied Biosystem, Foster, USA). The determined nucleotide sequences were analyzed through the BLAST search program using the GenBank database of NCBI (www.ncbi.nlm.nih.gov). The results identified by analyzing the DGGE bands are shown in Table 2 below.

TABLE 2

Microorganisms Identified in DGGE of FMC-12T

No. Accession Species Max. ident. One DQ234133 Uncultured Sulfurospirillum sp. 16S rRNA gene 98% 2 DQ234133 Uncultured Sulfurospirillum sp. 16S rRNA gene 97% 3 AF146526 Denitrovibrio acetophilus 16S rRNA gene 90% 4 DQ234133 Uncultured Sulfurospirillum sp. 16S rRNA gene 98% 5 AY530551 Dechlorospillum sp. DB 16S rRNA gene 97% 6 DQ234133 Uncultured Sulfurospirillum sp. 16S rRNA gene 97%

In case of band 5 ( Dechlorospillum sp. DB 16S rRNA gene), the intensity of the band was strong. On the other hand, bands 1, 2, 4, and 6 (Uncultured Sulfurospirillum sp. 16S rRNA gene) and band 3 ( Denitrovibrio acetophilus 16S rRNA gene) showed weak band intensities (FIG. 1, Table 2). The most predominant microorganisms through DGGE were analyzed in the 5th band, dichlorospirilium. However, it has been shown that various microorganisms in various sulfospirylium species exist. Sulfurospillum sp. Has been reported to degrade PCE to cis -DCE. Dechlorospirillum sp. And Denitrovibrio acetophilus are present in dechlorinated cultures. It is reported.

Example 9 Dechlorination Analysis for Securing Anaerobic Functional Microbial Community FMC-12T

VC dechlorination secured through integrated culture around the river in Incheon city experiments with excellent functional microbial community FMC-12T, dechlorination rate through GC analysis is shown in FIG. FMC-12T [KCTC11435BP] showed 100% VC dechlorination activity. The decrease in VC showed a very rapid decrease from the beginning of inoculation, and it was confirmed by GC analysis that the added VC was all degraded and converted to ethene at 34 days. Ethene, a decomposition product of VC, was found to rapidly increase the concentration of ethene per unit time as VC decreased. Dechlorination of VC progressed rapidly and decreased slowly from day 21 until day 21 of inoculation.

1 is a dominant microorganism analysis of FMC-12T [KCTC11435BP] using DGGE [1, 2, 4, 6: Uncultured Sulfurospirillum sp., 3: dinitrovibrio acetofiler Denitrovibrio acetophilus , 5: Dechlorospillum sp.

Figure 2 is a measure of the dechlorination rate of VC through GC analysis of FMC-12T [-◆-: vinyl chlorolide,-■-: ethene].

<110> Korea Research Institute of Bioscience and Biotechnology <120> The anaerobic functional microbial community being capable of          vinyl chloride dechlorination <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F primer <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 1541R primer <400> 2 aaggaggtga tccanccrca 20 <210> 3 <211> 74 <212> DNA <213> Artificial Sequence <220> 3223F-GC primer <400> 3 cgcccgccgc gcgcggcggg cggggcgggg gcacgggggg cctacgggag gcagcagcct 60 acgggaggca gcag 74 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> 223 786R primer <400> 4 ctaccagggt atctaatc 18  

Claims (2)

Vinyl chloride Anaerobic microbial community with excellent dechlorination capacity FMC-12T [KCTC 11435BP]. According to claim 1, Sulfurospillum sp., Dehalococcoides sp.), Microbial community FMC-12T [KCTC 11435BP], which includes Denitrovibrio acetophilus and Dechlorospirillum sp.

Images