JP4565101B2 - Bioreactivity site bioactivity determination method - Google Patents

Description

  The present invention relates to a bioactivity determination method for a bioremediation site.

  Contamination of groundwater and soil with various organochlorine compounds, including tetrachlorethylene (PCE) and trichlorethylene (TCE), is a serious problem common to the world, and these substances are often taken up in the media on newspapers. There is a strong social demand for technological development to repair environmental pollution caused by

  Physicochemical methods and biological methods can be cited as the contaminated environment remediation techniques, but a bioenvironment remediation method (bioremediation) using microorganisms is particularly suitable for remediation of low concentration contamination. Bioremediation does not require soil excavation, is easy to repair the environment under the building, is low in cost and has a low environmental impact, and is expected to be put to practical use.

  Bioremediation is a method that enhances the ability of microorganisms to decompose and remove environmental pollutants by, for example, supplying various nutrients to microorganisms that naturally live in contaminated soil and groundwater. And a method of directly introducing a microorganism having the ability to decompose and remove environmental pollutants into the contaminated environment (bio augmentation: see, for example, Patent Document 1). In some cases, TCE-contaminated groundwater has been refurbished through biostimulation and bioaugmentation and has achieved excellent results.

When applying bioremediation, quickly determine whether the contaminated site can be biostimulated or whether bioaugmentation that introduces microorganisms capable of degrading contaminants from outside the system must be applied (For example, refer patent document 2).
JP 2003-154332 A Japanese Unexamined Patent Publication No. 2000-079000

  Therefore, an object of the present invention is to provide a method that can quickly determine the ability of microorganisms present in a contaminated environment to decompose pollutants.

  In order to achieve the above object, the environmental biological activity determination method of the present invention is a biological activity determination method for an environment contaminated with at least one of organochlorine compounds of tetrachlorethylene (PCE) and trichlorethylene (TCE), A nucleic acid extracted from a sample is amplified by a gene amplification method to be a target, and the target is hybridized with at least one DNA probe having a base sequence specific to a bacterium related to degradation of the organochlorine compound, thereby Bacteria are detected, and the removal ability of the organochlorine compound in the environment is determined based on the detected ability of the bacteria to decompose the organochlorine compound and its dechlorinated product.

  As for the bioremediation of polluted environments with organochlorine compounds such as PCE and TCE, the present inventors can determine whether biostimulation is possible if bacteria related to anaerobic organochlorine compound degradation can be rapidly detected from the polluted environment. We conducted extensive research focusing on the ease of judgment. As a result, for example, 18 kinds of bacteria can be detected at a time by using a DNA probe comprising a base sequence unique to each of the 18 anaerobic organochlorine compound degradation-related bacteria currently reported. And reached the present invention.

  According to the present invention, bacteria related to the degradation of organochlorine compounds in a contaminated environment can be quickly detected using a DNA probe, and the ability to remove PCE and its dechlorinated product in the contaminated environment can be determined. For example, bioremediation It is easy to determine whether or not biostimulation is possible, and it is possible to select an appropriate environmental restoration method. Furthermore, it is possible to make the environmental restoration inexpensive by selecting the environmental restoration method quickly and appropriately.

  Furthermore, since it is considered that the decomposition of PCE is difficult under aerobic conditions, and normally 50 cm or less from the ground surface is considered to be anaerobic, for example, a PCE contaminated environment or a contaminated environment having a surface surface of 50 cm or less. It may be an extra cost to repair a plant with an aerobic microorganism rather than an anaerobic microorganism that is originally available. However, according to the present invention, anaerobic organochlorine compound-degrading bacteria can be detected and the biological activity of the environment can be determined based on the decomposing activity of the bacteria, so that it is possible to avoid the use of excess energy. .

In the present invention, the organochlorine compound degradation-related bacterium to be detected is preferably a bacterium comprising at least one selected from the group consisting of the following A to R bacteria.
A: Deharosupirirumu multi Tarnovo borane scan (Dehalospirillum multivorans)
B: Desulfitobacterium frappieri
C: Actinomycetales Sm-1 ( Rhodococcus sp. Sm-1)
D: Rhodococcus rhodococcus
E: key Santo Enterobacter flavus (Xanthobacter flavus)
F: Mycobacterium L1
G: Desulforobium norvegicum ( Desulfovibrio baculatus )
H: Desulfitobacterium dehalogenans
I: Desulfitobacterium hafniense
J: Clostridium formicoaceticum
K: Desulfuromonas chloroethenica
L: Acetobacterium woodii DSM 1030 ( Acetobacterium woodii DSM 1030)
M: Deharobakuta Resutorikutasu (Dehalobacter restrictus)
N: Desulfitobacterium sp. PCE1 ( Desulfitobacterium sp. Strain PCE1)
O: Desulfitobacterium frappieri TCE1
P: Acetobacterium woodii DSM 2396
Q: Desurufomoniru Tide Jay DCB-1 (Desulfomonile tiedjei DCB- 1)
R: Dehalococcoides ethenogenes 195

  In the environmental biological activity determination method of the present invention, when at least one of the bacteria J, L, and P is detected, it can be determined that the environment has the ability to decompose PCE into TCE.

  In the environmental biological activity determination method of the present invention, when at least one of the bacteria of A, G, and M is detected, it can be determined that the environment has the ability to decompose PCE into cis-type dichloroethylene (cisDCE). .

  In the biological activity determination method for an environment of the present invention, when at least one of the bacteria of B, I, H, N, O, and Q is detected, the environment has an ability to decompose PCE and TCE into cisDCE. Then it can be determined.

  In the environmental biological activity determination method of the present invention, when at least the K bacteria are detected, it can be determined that the environment has the ability to decompose PCE and TCE into DCE.

  In the environmental biological activity determination method of the present invention, when at least R bacteria are detected, it can be determined that the environment has the ability to decompose PCE, TCE, DCE and vinyl chloride (VC) into ethene.

  In the environmental biological activity determination method of the present invention, when at least C, D, and E bacteria are detected, it can be determined that the environment has the ability to decompose DCE and VC into carbon dioxide.

  In the environmental biological activity determination method of the present invention, when at least the F bacteria are detected, it can be determined that the environment has the ability to decompose VC into carbon dioxide.

In the environmental biological activity determination method of the present invention, examples of the DNA probe comprising a base sequence peculiar to the A to R bacteria include a DNA probe comprising any one of the following polynucleotides (1) to (4). .
(1) A polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOs: 1 to 17 and SEQ ID NOs: 19 to 105.
(2) A polynucleotide comprising a nucleotide sequence in which one to several bases of the polynucleotide of (1) are deleted, substituted or added, and complementary to the polynucleotide of (1) A polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a basic nucleotide sequence.
(3) A polynucleotide having a nucleotide sequence in which one to several bases of the polynucleotide of (1) are deleted, substituted or added, and the polynucleotide of (1) A polynucleotide having a homology of 90% or more.
(4) A polynucleotide comprising a base sequence complementary to the polynucleotide of any one of (1) to (3).

  Specifically, the DNA probe for detecting the bacterium A is preferably the DNA probe derived from any one of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NOs: 19 to 25. Here, the DNA probe derived from the base sequence of SEQ ID NO: 1 is a DNA probe comprising the polynucleotide of any one of (1) to (4), wherein the sequence number of (1) is SEQ ID NO: 1. means.

  As the DNA probe for detecting the bacterium B, the DNA probe derived from any one of the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NOs: 26 to 30 is preferable.

  As the DNA probe for detecting the bacterium C, the DNA probe derived from any one of the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NOs: 31 to 35 is preferable.

  The DNA probe for detecting the bacterium D is preferably the DNA probe derived from any one of the nucleotide sequences of SEQ ID NO: 4 and SEQ ID NOs: 36 to 40.

  As the DNA probe for detecting the bacterium E, the DNA probe derived from any one of the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NOs: 41 to 45 is preferable.

  As the DNA probe for detecting the bacterium F, the DNA probe derived from any one of the nucleotide sequences of SEQ ID NO: 6 and SEQ ID NOs: 46 to 48 is preferable.

  As the DNA probe for detecting the bacterium G, the DNA probe derived from any one of the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NOs: 49 to 53 is preferable.

  As the DNA probe for detecting the bacterium H, the DNA probe derived from any one of the nucleotide sequences of SEQ ID NO: 8 and SEQ ID NOs: 54 to 57 is preferable.

  As the DNA probe for detecting the bacterium I, the DNA probe derived from any one of the nucleotide sequences of SEQ ID NO: 9 and SEQ ID NOs: 58 to 62 is preferable.

  The DNA probe for detecting the bacteria J is preferably the DNA probe derived from any one of the nucleotide sequences of SEQ ID NO: 10 and SEQ ID NOs: 63 to 68.

  As the DNA probe for detecting the bacterium K, the DNA probe derived from any one of the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NOs: 69 to 74 is preferable.

  As the DNA probe for detecting the bacterium L, the DNA probe derived from any one of the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NOs: 75 to 79 is preferable.

  As the DNA probe for detecting the bacterium M, the DNA probe derived from any one of the nucleotide sequences of SEQ ID NO: 13 and SEQ ID NOs: 80 to 86 is preferable.

  As the DNA probe for detecting the bacteria N, the DNA probe derived from any one of the nucleotide sequences of SEQ ID NO: 14 and SEQ ID NOs: 87 to 91 is preferable.

  As the DNA probe for detecting the bacteria O, the DNA probe derived from any one of the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NOs: 92 to 96 is preferable.

  The DNA probe for detecting the bacteria P is preferably the DNA probe derived from any one of the nucleotide sequences of SEQ ID NO: 16 and SEQ ID NOs: 97 to 99.

  The DNA probe for detecting the bacterium Q is preferably the DNA probe derived from any one of the nucleotide sequences of SEQ ID NO: 17 and SEQ ID NOs: 100 to 105.

  The environment to which the biological activity determination method of the present invention is applied includes at least one of soil, groundwater, pond, and seawater.

  The bioremediation method of the present invention is a method of bioremediation of an environment contaminated with at least one organochlorine compound of PCE and TCE, comprising the step of performing the bioactivity determination method of the environment of the present invention, and the organic chlorine A bioremediation method comprising a step of accelerating at least one of the growth and activity of the bacterium and accelerating the degradation of the organochlorine compound or a dechlorinated product thereof when a compound degradation-related bacterium is detected.

First, the organochlorine compound decomposition-related bacteria to be detected in the environmental biological activity determination method of the present invention will be described. Currently, 18 types of A to R are known as anaerobic bacteria related to the degradation of PCE. In the present invention, at least one of A to R is the detection target, and preferably all the bacteria of A to R are the detection target. This includes, but is not limited to, bacteria that will become apparent in the future related to the degradation of PCE. The bacteria from A to Q are given the deposit numbers of the following biological resource preservation organizations ATCC or DSMZ.
A: DSM 12446 B: DSM 13498 C: ATCC 51239 D: ATCC 21197 E: DSM 10330
F: DSM 6695 G: DSM 1741 H: DSM 9161 I: DSM 10644 J: ATCC 27076
K: DSM 12431 L: DSM 1030 M: DSM 9455 N: DSM 10344 O: DSM 12704
P: DSM 2396 Q: ATCC 49306

  For example, as shown in FIG. 5A, PCE is decomposed into PCE, TCE, dichloroethylene (DCE), and vinyl chloride (VC), and the VC is decomposed into ethene or carbon dioxide. FIG. 5B shows the degradation activity of the A to R bacteria against PCE and its dechlorinated product. As shown in FIG. 5B, the degradation activities of the A to R bacteria on PCE and its dechlorinated products vary. For example, the bacteria R dechlorinates PCE one by one up to ethene, but the bacteria M, A and G decompose PCE into cis-type DCE.

  Next, a DNA probe used for detection of PCE-degrading bacteria to be detected in the environmental biological activity determination method of the present invention will be described. The DNA probe in the present invention is not particularly limited as long as it is a sequence peculiar to the organochlorine compound-degrading-related bacteria. For example, sequences such as whole genome, tDNA, and rDNA can be used. ITS sequence. Prokaryotic ribosomal RNA (rRNA), 16SrRNA, 23SrRNA and 5SrRNA, are usually transcribed as one transcription unit (operon), so the 16SrRNA gene and the 23SrRNA gene are arranged side by side on the genome. The region between the 16SrRNA gene and the 23SrRNA gene is a region called 16S-23S Internal Transcribed Spacer (ITS). The present inventors first determined the sequence of the ITS in the A to Q bacteria (SEQ ID NOs: 1 to 17 respectively), and if this ITS sequence, a DNA probe specific to the A to Q bacteria was obtained. It was found for the first time that it could be produced.

  The genome sequence of the bacteria R was determined by Dr. Zinder of Cornell University. If the DNA probe comprising the ITS sequence of the bacterial R (SEQ ID NO: 18) is specific for the bacterial R and used in combination with a DNA probe from the ITS sequences of the 17 bacterial species, It was also found for the first time that a DNA probe capable of detecting all 18 types of bacteria of Q can be obtained.

Furthermore, the present inventors have found that a part of the ITS sequence (SEQ ID NOs: 1 to 18) can also be used as a DNA probe. Therefore, the DNA probe used in the environmental bioactivity determination method of the present invention includes a DNA probe comprising any one of the polynucleotides (1) to (4) below.
(1) A polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 18 or a part thereof.
(2) A polynucleotide comprising a nucleotide sequence in which one to several bases of the polynucleotide of (1) are deleted, substituted or added, and complementary to the polynucleotide of (1) A polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a basic nucleotide sequence.
(3) A polynucleotide having a nucleotide sequence in which one to several bases of the polynucleotide of (1) are deleted, substituted or added, and the polynucleotide of (1) A polynucleotide having a homology of 90% or more.
(4) A polynucleotide comprising a base sequence complementary to the polynucleotide of any one of (1) to (3).

  Here, the number of bases that can be deleted, substituted, or added is, for example, 1 to 6 by deletion / addition with respect to 40 bases, and preferably 1 to 3, more preferably 1 to 2 and 1 to 4 by substitution, preferably 1 to 2 and more preferably 1. Examples of the stringent conditions for hybridization include ± 10 ° C. of the Tm value of the base sequence represented by the SEQ ID NO. Moreover, as said homology, it is 90% or more, for example, 95% or more is preferable, More preferably, it is 97.5% or more.

  The DNA probe in the present invention is preferably a probe consisting of a part of the base sequence rather than the entire ITS sequence. This is because a DNA probe usually has a higher sequence specificity and a higher reliability as its length becomes shorter. On the other hand, the sequence itself needs to be unique to each of the bacteria. Therefore, the length of the DNA probe is not particularly limited. For example, the entire ITS sequence is from 10 bases to 40 to 80 bases.

  Specific examples of the base sequence that is a part of the ITS region of SEQ ID NOs: 1 to 18 are the base sequences of SEQ ID NOs: 19 to 115 having a length of 40 bases. SEQ ID NO: 19 to 25 correspond to a part of SEQ ID NO: 1, SEQ ID NO: 26 to 30 correspond to a part of SEQ ID NO: 2, SEQ ID NO: 31 to 35 correspond to a part of SEQ ID NO: 3, 36 to 40 correspond to part of SEQ ID NO: 4, SEQ ID NO: 41 to 45 correspond to part of SEQ ID NO: 5, SEQ ID NO: 46 to 48 correspond to part of SEQ ID NO: 6, and SEQ ID NO: 49 to 53 corresponds to a part of SEQ ID NO: 7, SEQ ID NOS: 54 to 57 correspond to a part of SEQ ID NO: 8, SEQ ID NOS: 58 to 62 correspond to a part of SEQ ID NO: 9, and SEQ ID NOS: 63 to 68 It corresponds to a part of SEQ ID NO: 10, SEQ ID NOS: 69 to 74 correspond to a part of SEQ ID NO: 11, SEQ ID NOS: 75 to 79 correspond to a part of SEQ ID NO: 12, and SEQ ID NOS: 80 to 86 represent SEQ ID NO: 13 corresponds to a part of SEQ ID NO: 14 of SEQ ID NO: 87 to 91 SEQ ID NO: 92 to 96 corresponds to a part of SEQ ID NO: 15, SEQ ID NO: 97 to 99 corresponds to a part of SEQ ID NO: 16, SEQ ID NO: 100 to 105 corresponds to a part of SEQ ID NO: 17 And SEQ ID NO: 106 to 115 correspond to a part of SEQ ID NO: 18.

  Next, the method for detecting the organochlorine compound degradation-related bacteria using the DNA probe is not particularly limited. For example, a target is prepared from a nucleic acid extracted from a sample in a target environment, and the target, the DNA probe, It can detect by the method of utilizing hybridization. The target is a sequence of bacteria in a sample and is a complementary sequence of the sequence of the DNA probe, and can be detected by hybridization of the target labeled with a detection method and the DNA probe. Become. As the hybridization detection method, for example, a conventionally known gene detection technique such as Southern blot, DNA array, DNA microarray, or DNA chip can be used. Among these, it is preferable that DNA probes corresponding to all the bacteria from A to R are immobilized because bacteria in an environmental sample can be detected at a time.

  The target environment of the biological activity determination method of the present invention is not particularly limited, and examples thereof include contaminated soil, groundwater, pond, seawater and the like. The method for extracting the nucleic acid from the sample in the target environment is not particularly limited, and may be a conventionally known method. For example, a commercially available nucleic acid extraction kit can be used. The target can be prepared by amplifying a region corresponding to the DNA probe of the nucleic acid. The nucleic acid may be DNA or RNA, for example. The gene amplification method is also not particularly limited, and conventionally known amplification methods can be applied. It is also preferable to label the target simultaneously with gene amplification, depending on the hybridization detection method. The label is not particularly limited, and for example, a fluorescent label or an RI label can be used. When a target corresponding to a DNA probe using the ITS region is prepared by PCR, for example, a primer consisting of the base sequence represented by SEQ ID NO: 116 can be used as a sense primer, In addition to the bacterium R, a primer having a base sequence represented by SEQ ID NO: 117 can be used, and for the bacterium R, a primer having a base sequence represented by SEQ ID NO: 118 can be used.

  As described above, bacteria related to the decomposition of organochlorine compounds in the environment can be detected, but the determination of the removal ability of PCE and TCE in the environment is performed, for example, by comparing the detected bacteria with FIG. 5B. Can do.

  Next, the method of bioremediation of the present invention is a method of bioremediation of an environment contaminated with at least one organochlorine compound of PCE and TCE, and performing the method for determining biological activity of the environment of the present invention; A bioremediation method comprising the step of promoting the degradation of the organochlorine compound or its dechlorinated product by accelerating at least one of the growth and activity of the bacterium when the bacterium related to the degradation of the organochlorine compound is detected by the method. Is the method. In bioremediation, it is possible to select a more accurate and rapid bioremediation method by grasping in advance the ability to decompose and remove organochlorine compounds such as PCE in the environment using the biological activity determination method of the present invention. For example, if bacteria R is detected, PCE can be made into ethene by the bacteria R, so that biostimulation that introduces nutrients that enhance the growth and activity of the bacteria R into the environment can be selected. Further, for example, even when bacteria K and C are detected, PCE can be decomposed into carbon dioxide by the two kinds of bacteria. Therefore, a biostidy that introduces nutrients that enhance the growth and activity of the two kinds of bacteria into the environment. The simulation can be selected. In addition, when the organic compound degradation-related bacteria are not detected, it can be selected not to perform biostimulation.

  Examples of the present invention will be described below.

  DNA was extracted from 250 mg of a soil sample provided by Matsushita Air Conditioning Environment Engineering Co., Ltd. using FastPrep bead-beater and soil DNA extraction kit (manufactured by Q Biogene) according to the instruction manual. About 1 μl of the DNA was mixed with 50 μl of standard Amplitaq Gold PCR mixture (Applied Bio) containing unlabeled sense primer 27F (SEQ ID NO: 116) and Cy3 labeled antisense primer 132R (SEQ ID NO: 117) or 341R (SEQ ID NO: 118). Added to Systems). After PCR was performed according to a standard protocol, the PCR amplification product was desalted using Autoseq G-50 (Pharmacia) and suction-dried using SpeedVac (Savant). The dried PCR amplification product was dissolved in a buffer having a final concentration of 5 × SSC, 0.2% SDS, 50% formamide, and the lysate was boiled at 94 ° C. for 3 minutes and ice-cooled for at least 2 minutes. Then, it applied on the DNA microarray. A cover glass was then placed over the microarray and the so-prepared one was placed in a hybridization chamber set at 42 ° C. for at least 4 hours. The DNA microarray was then washed with 0.2 × SSC, 0.2% SDS for 5 minutes, 0.2 × SSC for 5 minutes, and 0.05 × SSC for a few seconds and spin dried at 1,800 rpm. Thereafter, the DNA microarray was scanned with Scanarry version 5 (manufactured by PerkinElmer Japan).

  The DNA microarray was prepared by custom printing a DNA probe on a Takara Hubble Slide using Affymetrix 417 Arrayer. As for the probes on the DNA microarray used, the probes A1 to A7 of bacteria A have the base sequences of SEQ ID NOs: 19 to 25, respectively, and the probes B1 to B5 of bacteria B have the base sequences of SEQ ID NOs: 26 to 30, respectively. The probes C1 to C5 of bacteria C are the nucleotide sequences of SEQ ID NOs: 31 to 35, respectively, the probes D1 to D5 of bacteria D are the nucleotide sequences of SEQ ID NOs: 36 to 40, respectively, and Probes E1 to E5 are the nucleotide sequences of SEQ ID NOs: 41 to 45, bacteria F probes F1 to F3 are respectively the nucleotide sequences of SEQ ID NOs: 46 to 48, and bacteria G probes G1 to G5 are The nucleotide sequences of SEQ ID NOs: 49 to 53, respectively, H1 to H4 are the nucleotide sequences of SEQ ID NOs: 54 to 57, bacteria I probes I1 to I5 are the nucleotide sequences of SEQ ID NOs: 58 to 62, respectively, and bacteria J probes J1 to J6 are respectively , SEQ ID NOS: 63 to 68, the bacteria K probes K1 to K6 are the nucleotide sequences of SEQ ID NOS: 69 to 74, and the bacteria L probes L1 to L5 are the SEQ ID NOs: 75 to 79, respectively. The probes M1 to M7 of bacteria M are the nucleotide sequences of SEQ ID NOs: 80 to 86, respectively, and the probes N1 to N5 of bacteria N are the nucleotide sequences of SEQ ID NOs: 87 to 91, respectively. Bacteria O probes O1 to O5 are the nucleotide sequences of SEQ ID NOs: 92 to 96, respectively. The probes P1 to P3 of the bacteria P are the nucleotide sequences of SEQ ID NOs: 97 to 99, the probes Q1 to Q6 of the bacteria Q are the nucleotide sequences of the SEQ ID NOs: 100 to 105, respectively, and from the probes R1 of the bacteria R R10 is the base sequence of SEQ ID NOs: 106 to 115, respectively.

  A part of the result of scanning the DNA microarray is shown in FIG. FIG. 1A shows a scan image, and FIG. 1B shows a graph of quantified results. As shown in FIG. 1, significant hybridization to the probe of bacteria M (Dehalobacter restrictus DSM 945) was detected. Therefore, it was determined that the soil of the sample had the ability to decompose PCE into cisDCE (see FIG. 5B).

  It should be noted that the 97 types of DNA probes are specific to each bacterium and do not cross-hybridize, the targets are prepared from the A to R bacteria, and the targets are hybridized with the DNA microarray. Confirmed with.

  A summary of the results is shown in FIG. FIG. 2 is a graph showing whether or not the ITS sequence target of 18 bacteria shown on the vertical axis and the 97 DNA probes shown on the horizontal axis were hybridized. This indicates that the DNA probe and the target are hybridized. The phylogenetic tree on the horizontal axis is prepared from the alignment of ITS sequences. As shown in FIG. 2, it was shown that the target prepared from each of the bacteria from A to R significantly hybridized only with the DNA probe of the bacteria from A to R without cross-hybridization.

  The groundwater sample was obtained in the same manner as in Example 1 except that a 300 ml groundwater sample provided by Matsushita Air Conditioning Environment Engineering Co., Ltd. was used instead of the soil sample, and DNA was extracted from debris centrifuged at 7,000 rpm. Bacteria inside were detected using the DNA microarray.

  A part of the result is shown in FIG. FIG. 3A shows a scan image, and FIG. 3B shows a graph of the quantified results. As shown in FIG. 3, significant hybridization to a probe of bacteria J (Clostridium formicoaceticum ATCC 27076) was detected. Therefore, it was determined that the soil of the sample had the ability to decompose PCE into TCE (see FIG. 5B).

  The bacteria in the sample were detected in the same manner as in Example 1 except that an anaerobic enrichment culture sample provided by Dr. T.H. Lee (Korea) was used instead of the soil sample.

  The result is shown in FIG. As shown in FIG. 4, strong signals were detected for some probes of bacteria A, J, M, N, and O, and weak signals were also detected for bacteria B and I probes. Therefore, it was determined that the sample had the ability to convert PCE to cisDCE (see FIG. 5B). This determination result was consistent with the PCE / cisDCE analysis data of the enrichment culture by Dr. T.H. Lee.

  As described above, the environmental biological activity determination method of the present invention is useful in a method for repairing a contaminated environment, particularly in the field of bioremediation.

FIG. 1 is a diagram showing the detection result of bacteria in an example of the present invention. FIG. 2 is a diagram showing a detection result by a DNA microarray in an example of the present invention. FIG. 3 is a diagram showing the result of detection of bacteria in another example of the present invention. FIG. 4 is a diagram showing a result of detecting bacteria in still another example of the present invention. FIG. 5 is a diagram illustrating the degradation activity of anaerobic organochlorine compound degradation-related bacteria according to the present invention.

SEQ ID NO: 116 sense primer 27F for PCR
SEQ ID NO: 117 antisense primer 132R for PCR
SEQ ID NO: 118 antisense primer 341R for PCR

Claims (9)

A method for judging the biological activity of an environment contaminated with at least one organochlorine compound of tetrachlorethylene (PCE) and trichlorethylene (TCE), wherein a nucleic acid extracted from an environmental sample is amplified by a gene amplification method to obtain a target, The organochlorine compound and its dechlorinated product of the bacteria detected by detecting the bacteria in the environment by hybridizing with a combination of DNA probes comprising a base sequence unique to the bacteria related to the degradation of the organochlorine compounds Determining the ability of the environment to remove the organochlorine compound based on its ability to decompose
The combination of the DNA probes is
DNA for detecting bacteria A Dehalospirillum multivorans, comprising a polynucleotide represented by any of the nucleotide sequences of SEQ ID NOS: 19 to 25 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence At least one of the probes;
Bacteria B for detecting Desulfitobacterium frappieri, comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOs: 26 to 30 and a nucleotide sequence complementary to the nucleotide sequence At least one of the DNA probes;
Bacterial C actinomycetales Sm-1 (Rhodococcus sp. Sm-1), comprising a polynucleotide represented by any of the nucleotide sequences of SEQ ID NOs: 31 to 35 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence At least one DNA probe for detecting (Actinomycetales Sm-1 (Rhodococcussp. Sm-1)),
At least a DNA probe for detecting bacteria D Rhodococcus rhodococcus, comprising a polynucleotide represented by any of the nucleotide sequences of SEQ ID NOS: 36 to 40 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence With one
DNA probe for detecting Bacterium E Xanthobacter flavus, comprising a polynucleotide represented by any one of the nucleotide sequences SEQ ID NO: 41 to 45 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence At least one of
DNA probe for detecting bacteria F Mycobacterium L1 (Mycobacterium L1), comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOs: 46 to 48 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence At least one of
Bacterial G desulfomicrobium norbegicam (Desulfomicrobium baculatas) (Desulfomicrobium) comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOs: 49 to 53 and a nucleotide sequence complementary to the nucleotide sequence at least one DNA probe for detecting norvegicum (Desulfovibrio baculatus),
Detecting bacteria H Desulfitobacterium dehalogenans, comprising a polynucleotide represented by any of the nucleotide sequences of SEQ ID NOS: 54 to 57 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence At least one of the DNA probes;
A method for detecting bacteria I Desulfitobacterium hafniense, comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOs: 58 to 62 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence. At least one of the DNA probes;
DNA for detecting bacterial J Clostridium formicoaceticum, comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOS: 63 to 68 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence At least one of the probes;
At least a DNA probe for detecting bacterial K Desulfuromonas chloroethenica, which comprises a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOS: 69 to 74 and a nucleotide sequence complementary to the nucleotide sequence; With one
Bacteria L acetobacterium woodii DSM 1030 (Acetobacterium woodii DSM 1030) comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOS: 75 to 79 and a nucleotide sequence complementary to the nucleotide sequence is detected. At least one of DNA probes for
At least a DNA probe for detecting bacteria M Dehalobacter restrictus, comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOs: 80 to 86 and a nucleotide sequence complementary to the nucleotide sequence With one
Bacteria N Desulfitobacterium sp. PCE1 (Desulfitobacterium sp. Strain PCE1) comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOs: 87 to 91 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence A DNA probe for detecting
Bacterium O Desulfitobacterium frappieri TCE1 (Desulfitobacterium frappieri TCE1) comprising a polynucleotide represented by any of the nucleotide sequences SEQ ID NO: 92 to 96 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence is detected At least one of DNA probes for
Bacteria P acetobacterium woodi DSM 2396 (Acetobacterium woodii DSM 2396) comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOS: 97 to 99 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence is detected. At least one of DNA probes for
Detects bacterial Q desulfomonyl tiedjei DCB-1 comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOs: 100 to 105 and the nucleotide sequence complementary to the nucleotide sequence At least one of the DNA probes for
An environmental biological activity determination method , which is a combination of DNA probes containing Wherein J, if it is detected L and at least one bacterium of P, the environment, biological activity determination method of determining according to claim 1, wherein as having ability to decompose the TCE and PCE. The biological activity determination according to claim 1 or 2 , wherein when at least one kind of bacteria of A, G and M is detected, the environment is determined to have an ability to decompose PCE into cis-type dichloroethylene (cisDCE). Method. Wherein B, I, H, N, when it is detected O and Q at least one of bacteria, the environment, both the PCE and TCE from determining claims 1 to have a decomposing capability CisDCE 3 of The biological activity determination method according to claim 1. When the bacteria of the K has been detected, the environment, biological activity determination method according to any one of claims 1 determines to have the ability to decompose PCE and TCE to DCE 4. The organism according to any one of claims 1 to 5 , wherein the environment is determined to have an ability to decompose DCE and VC into carbon dioxide when at least one bacterium of C, D and E is detected. Activity determination method. When the bacteria of the F is detected, the environment, biological activity determination method according to any of VC from determining claims 1 to have the ability to resolve carbon dioxide 6. The biological activity determination method according to claim 1, wherein the contaminated environment is at least one of soil, groundwater, pond, and seawater. A method of bioremediation of an environment contaminated with at least one organochlorine compound of PCE and TCE, comprising the step of performing the biological activity determination method of the environment by the method according to any one of claims 1 to 8 , A method of bioremediation, comprising the step of promoting the degradation of the organochlorine compound or a dechlorinated product thereof by promoting at least one of the growth and activity of the bacterium when an organochlorine compound-degrading bacterium is detected.

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