JP5305212B2 - Microbial community and dehalobacter genus bacteria that dechlorinate polychlorinated biphenyls and dioxins, and uses of the microbial community and dehalobacter bacteria - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new bacterial population for dechlorinating PCB and a bacterium. <P>SOLUTION: The bacterial population for dechlorinating polychlorinated biphenyls and dioxins has the following properties of (1) including a bacterium of the genus Dehalobacter as a bacterium having dechlorination ability, (2) carrying out dechlorination using hydrogen as an electron donor, (3) using acetic acid and carbon dioxide as a carbon source, (4) maintaining dechlorination activity in the presence of nitric acid or sulfuric acid having &le;10 mM and (5) inhibiting dechlorination activity by trivalent iron. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to novel microbial communities and Dehalobacter bacteria. More specifically, the present invention relates to a microbial community and dehalobacter genus bacteria that dechlorinate polychlorinated biphenyls and dioxins, and methods for obtaining and using them.

High-temperature incineration method, dechlorination decomposition method (chemical extraction decomposition method, organic alkali metal decomposition method, catalytic hydrodechlorination method) as treatment methods for polychlorinated biphenyl (hereinafter abbreviated as “PCB”), an environmental pollutant , Alkali catalyst decomposition method, metal sodium method, etc.), hydrothermal oxidative decomposition (supercritical hydration method, hydrothermal decomposition method, etc.), reductive thermochemical decomposition method (melting catalyst extraction method, gas phase hydrogen reduction method), etc. Has been. On the other hand, research on methods for biologically decomposing PCBs is also underway, and several reports have been made on microorganisms or microbial communities having PCB dechlorination ability (Non-Patent Documents 1 to 5). Specifically, a Chloroflexi genus Dehalococoides bacterium has been isolated as a microorganism that dechlorinates PCBs (Non-patent Document 1). From the marine environment, chloroflexigate o-17 and DF-1 have been obtained as accumulations (Non-Patent Documents 2 and 3). On the other hand, last year, it was reported that a Demicobacter genus of the Firmicutes genus, along with a Dehalococcideae bacterium, was suggested to dechlorinate PCBs in river sediment cultures. (Non-Patent Document 4). Moreover, it was shown that a dehalococcide genus bacterium dechlorinates alone (nonpatent literature 5).
Fennell, DE, I. Nijenhuis, SF Wilson, SH Zinder, and MM Haggblom. 2004. Dehalococcoides ethenogenes strain 195 reductively dechlorinates diverse chlorinated aromatic pollutants. Environ. Sci. Technol. 38: 2075? 2081. Cutter, LA, JEM Watts, KR Sowers, and HD May. 2001. Identification of a microorganism that links its growth to the reductive dechlorination of 2,3,5,6-chlorobiphenyl. Environ. Microbiol. 3: 699-709. Wu, Q., JEM Watts, KR Sowers, and HD May. 2002. Identification of a bacterium that specifically catalyzes the reductive dechlorination of polychlorinated biphenyls with doubly flanked chlorines. Appl. Environ. Microbiol. 68: 807-812. Yan, T., TM LaPara, and PJ Novak 2006. The effect of varying levels of sodium bicarbonate on polychlorinated biphenyl dechlorination in Hudson River sediment cultures.Environ. Microbiol. 8: 1288-1298. Bedard, D., L. Kirsti, M. Ritalahti, and Frank E. Loffler. (2007) Dehalococcoides in a Sediment-Free, Mixed Culture Metabolically Dechlorinate the Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 Appl. Envir. Microbiol. In press

As mentioned above, there are few reports of separation of microorganisms that dechlorinate PCBs. In addition, PCBs have various homologs, but the types of PCBs that can be dechlorinated by microorganisms or microbial communities reported so far are limited. For example, dehalococcides bacteria and DF-1 dechlorinate only the chlorine that is sandwiched between both sides. On the other hand, o-17 dechlorinates only at the ortho position of PCB. In complex cultures containing dehalococcidae bacteria, the PCB mixture is only dechlorinated up to 3-4 chlorides.
Accordingly, an object of the present invention is to provide a novel microbial community and microorganisms for dechlorinating PCBs and their uses. In particular, an object is to provide a novel microbial community and microorganisms that can dechlorinate PCBs up to 1 chlorinated product and their uses.

In order to achieve the above object, the present inventors first added a unique device to the microorganism accumulation method. That is, 4,5,6,7-tetrachlorophthalide (fusalide) was used as a PCB model compound, and microorganisms were accumulated using the dechlorination activity as an index. Specifically, first, paddy field soil was collected, water was added so as to be flooded, fusalide and an organic acid (lactic acid, formic acid, acetic acid, or butyric acid) were added, and culture was started at 30 ° C. The culture confirmed to be dechlorinated was subcultured using a medium supplemented with inorganic salts, fusalides and organic acids. The resulting enriched culture was subjected to a dechlorination test for chlorinated compounds such as PCB, and PCR-DGGE targeting the 16S ribosomal RNA (16SrRNA) gene and a base band analysis of the dominant band were performed. Microorganisms were identified. As a result, the accumulation (KLF culture) obtained under the condition of adding lactic acid was 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-tetrachlorobiphenyl) and 2,3,4-trichloro. Dechlorination of biphenyl (2,3,4-trichlorobiphenyl) to 2-monochlorobiphenyl or 4-monochlorobiphenyl, and 1,2,3-trichlorodibenzo-p-dioxin (1,2,3-Trichlorodibenzo-p-dioxin) was found to dechlorinate. Microbial community structure analysis based on the bacterial 16S rRNA gene was performed on KLF cultures. Eight dominant microbial species were identified, two were Dehalobacter bacteria, five were Clostridium bacteria, and one was a Sectientiactor ( Sedimentibacter) was closely related. Further investigation revealed that the bacteria (FTH1 and FTH2) that gave the DGGE bands that were most closely related to the genus Dehalobacter were actually involved in dechlorination, and these bacteria were dehalobacter restrictus ( Dehalobacter
restrictus) TEA strain was shown to be the closest relative. Moreover, it became clear that the KLF culture did not contain bacteria belonging to the genus Dehalococcides. On the other hand, as a result of various tests on the characteristics of the KLF culture, it was speculated that dechlorination was performed using hydrogen as an electron donor. Further characteristics of KLF culture include the availability of acetic acid and carbon dioxide as carbon sources, maintaining dechlorination activity in the presence of 10 mM nitric acid or sulfuric acid, and dechlorination activity with trivalent iron. Was found to be inhibited.
Subsequently, as a result of continuous subculture of the KLF culture under specific conditions, it was possible to increase the degree of accumulation to such an extent that three kinds of preferential microorganisms (two of these were FTH1 and FTH2) were recognized.
As described above, as a result of intensive studies by the present inventors, the present inventors have succeeded in obtaining a new microbial community that dechlorinates PCBs, and found new dehalobacter bacteria as microorganisms responsible for PCB dechlorination. Successful. Surprisingly, the microbial community has the ability to dechlorinate PCBs down to monochloride, and also has the ability to dechlorinate dioxins. On the other hand, since the microbial community having excellent PCB dechlorination ability was obtained in a relatively short period of time, the usefulness of the accumulation method adopted by the present inventor was confirmed.
The present invention is based on the above results, and provides the following microbial communities and the like.
[1] Microbial community having the following characteristics and dechlorinating polychlorinated biphenyls and dioxins:
(1) including bacteria belonging to the genus Dehalobacter as microorganisms having dechlorination ability;
(2) Dechlorination using hydrogen as an electron donor;
(3) Acetic acid and carbon dioxide can be used as a carbon source;
(4) maintain dechlorination activity even in the presence of 10 mM nitric acid or sulfuric acid;
(5) Dechlorination activity is inhibited by trivalent iron.
[2] 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-tetrachlorobiphenyl) and 2,3,4-trichlorobiphenyl (2,3,4-trichlorobiphenyl) to 2-monochlorobiphenyl (2 -monochlorobiphenyl) or 4-monochlorobiphenyl, and 1,2,3-trichlorodibenzo-p-dioxin The microbial community according to [1], which has an ability to
[3] The microbial community according to [1] or [2], comprising two types of bacteria belonging to the genus Dehalobacter as microorganisms having dechlorination ability.
[4] The base sequence of the 16S ribosomal RNA gene in the bacterium belonging to the genus Dehalobacter, and Dehalobacter
restrictus) The microbial community according to any one of [1] to [3], wherein the homology between the 16S ribosomal RNA gene in the TEA strain is 97% or more and less than 98%.
[5] The microbial community according to [3], wherein the 16S ribosomal RNA gene in one of the Dehalobacter bacteria has the base sequence of SEQ ID NO: 1, and the other 16S ribosomal RNA gene has the base sequence of SEQ ID NO: 2.
[6] The microbial community according to any one of [1] to [5], which does not contain bacteria belonging to the genus Dehalococcoides.
[7] The microbial community according to any one of [1] to [6], wherein the microorganism community is obtained by accumulating and cultivating medium coarse-grained strong clay soil.
[8] The enrichment culture includes a step of subculture in a nutrient medium containing 4,5,6,7-tetrachlorophthalide (4,5,6,7-tetrachlorophthalide) and lactic acid. The microbial community according to [7].
[9] The microbial community according to [1], wherein the accession number is NITE P-353 .
[10] Dehalobacter bacteria having the following characteristics and dechlorinating polychlorinated biphenyls and dioxins:
(1) Dechlorination using hydrogen as an electron donor;
(2) Acetic acid and carbon dioxide can be used as a carbon source;
(3) maintain dechlorination activity even in the presence of 10 mM nitric acid or sulfuric acid;
(4) Dechlorination activity is inhibited by trivalent iron.
[11] Ability to dechlorinate 2,3,4,5-tetrachlorobiphenyl and 2,3,4-trichlorobiphenyl to 2-monochlorobiphenyl or 4-monochlorobiphenyl, and 1,2,3-trichlorodibenzo- The Dehalobacter bacterium according to [10], which has an ability to dechlorinate p-dioxin.
[12] The base sequence of the 16S ribosomal RNA gene has a homology of 97% or more and less than 98% to the base sequence of the 16S ribosomal RNA gene in the Dehalobacter restrictus TEA strain, according to [10] or [11] Dehalobacter bacteria.
[13] The Dehalobacter genus bacterium according to [10] or [11], wherein the 16S ribosomal RNA gene has the nucleotide sequence of SEQ ID NO: 1 or 2.
[14] A polychlorinated biphenyl and dioxins comprising the step of agglomerating and culturing medium coarse-grained strong clay soil in the presence of 4,5,6,7-tetrachlorophthalide and lactic acid A method for obtaining a microbial community comprising dehalobacter bacteria to be dechlorinated.
[15] The method according to [14], wherein the enrichment culture comprises the following steps (1) and (2):
(1) adding 4,5,6,7-tetrachlorophthalide and lactic acid to a suspension of medium coarse-grained strong clay soil in paddy field and culturing;
(2) A step of subculturing using a nutrient medium containing 4,5,6,7-tetrachlorophthalide and lactic acid.
[16] The method according to [15], further comprising performing the following steps after step (2):
(3) A step of subculturing using a nutrient medium containing 4,5,6,7-tetrachlorophthalide and supplying hydrogen and carbon dioxide to the medium.
[17] The method according to [15], wherein the enrichment culture comprises the following steps (1) and (3):
(1) adding 4,5,6,7-tetrachlorophthalide and lactic acid to a suspension of medium coarse-grained strong clay soil in paddy field and culturing;
(3) A step of subculturing using a nutrient medium containing 4,5,6,7-tetrachlorophthalide and supplying hydrogen and carbon dioxide to the medium.
[18] Contamination containing the microbial community according to any one of [1] to [9] or the Dehalobacter genus bacteria according to any one of [10] to [13] containing polychlorinated biphenyl and / or dioxins A purification method characterized by acting on an object.
[19] The purification method according to [18], wherein the polychlorinated biphenyl is 2,3,4,5-tetrachlorobiphenyl or 2,3,4-trichlorobiphenyl.
[20] The purification method according to [18] or [19], wherein the dioxins are 1,2,3-trichlorodibenzo-p-dioxin.
[21] A method for dechlorinating a polychlorinated biphenyl to a monochlorinated product, which comprises allowing a Dehalobacter bacterium to act on the polychlorinated biphenyl.
[22] The polychlorinated biphenyl is 2,3,4,5-tetrachlorobiphenyl or 2,3,4-trichlorobiphenyl, and the monochlorinated product is 2-monochlorobiphenyl or 4-monochlorobiphenyl. The method according to [21], characterized by.
[23] The Dehalobacter genus bacterium described in any one of [1] to [9], or the Dehalobacter genus bacteria described in any of [10] to [13]. The method according to [21] or [22], wherein

  The first aspect of the present invention provides a microbial community that can be used for dechlorination of polychlorinated biphenyl (PCB) or dioxins. As shown in the Examples section described later, the present inventors have succeeded in obtaining a novel microbial community that dechlorinates PCBs and dioxins (hereinafter referred to as “microbial community of the present invention”). The “microbe community” refers to an aggregate of two or more microorganisms.

  The first feature of the microbial community of the present invention is to dechlorinate polychlorinated biphenyl (PCB) and dioxins. Here, “polychlorinated biphenyl (PCB)” is a general term for compounds in which a hydrogen atom of biphenyl is substituted with a chlorine atom. There are many isomers depending on the number and position of substituted chlorine. On the other hand, dioxins are a general term for polychlorinated dibenzopararadixin (PCDD) and polychlorinated dibenzofuran (PCDF). One of the PCBs, coplanar PCB (Co-PCB), is also classified as a dioxin.

In addition to the property of having dechlorination ability for PCBs and dioxins, the microbial community of the present invention is characterized by the following properties (1) to (5).
(1) A bacterium belonging to the genus Dehalobacter is included as a microorganism having dechlorination ability.
(2) Dechlorination is performed using hydrogen as an electron donor.
(3) Acetic acid and carbon dioxide can be used as a carbon source.
(4) Maintain dechlorination activity in the presence of 10 mM or less nitric acid or sulfuric acid.
(5) Dechlorination activity is inhibited by trivalent iron.

  The characteristic of (1) means that the microorganism responsible for dechlorination in the microbial community of the present invention is a Dehalobacter bacterium. For example, hydrogen produced from lactic acid can be used as the hydrogen in the characteristic (2). The characteristic of (3) means that the microbial community of the present invention can use not only acetic acid but also carbon dioxide as a carbon source. Carbon dioxide cannot be used as a carbon source in dehalococcides bacteria previously reported as PCB dechlorinated microorganisms.

In the characteristic (4), “maintaining dechlorination activity” means that the dechlorination activity is reduced, but the dechlorination activity is still exhibited. Specifically, when evaluated using fusalide as a PCB model compound, when cultured in the presence of 10 mM nitric acid, the microbial community of the present invention is, for example, 47 to 67%, preferably 52 to 60%, more preferably about Shows 57% dechlorination activity. When cultured in the presence of 10 mM sulfuric acid, it exhibits a dechlorination activity of, for example, 59 to 79%, preferably 64 to 74%, more preferably about 69%. The dechlorination activity was determined by adding fusalide (100 μM) to the microbial community and culturing for about 10 days at 30 ° C., fusalide present in the culture, 4,6,7-trichlorophthalide (4,6 , 7-triCph), 4,7-dichlorophthalide (4,7-diCph), 4,5-dichlorophthalide (4,5-Cph), 4-monochlorophthalide (4-Cph) in% ) Based on the following formula:
Dechlorination activity (%) = (4,6,7-triCph (μM) + 4,7-diCph (μM) + 4,5-Cph (μM) + 4-Cph (μM)) / (fusalide (μM) +4, 6,7-triCph (μM) + 4,7-diCph (μM) + 4,5-Cph (μM) + 4-Cph (μM)) × 100

  In the characteristic (5), “dechlorination activity is inhibited” means that a significant decrease in dechlorination activity is observed. Specifically, when evaluated using fusalide as a PCB model compound, when cultured in the presence of 10 mM trivalent iron, for example, 30% or less, preferably 25% or less, more preferably 20% or less (specifically Shows 10-20% dechlorination activity.

  As shown in the examples described later, the microbial community successfully obtained by the present inventors was obtained by converting PCBs 2,3,4,5-tetrachlorobiphenyl and 2,3,4-trichlorobiphenyl into 2-monochlorobiphenyl. Or it was confirmed that it has the ability to dechlorinate to 4-monochlorobiphenyl. On the other hand, it was also confirmed that the microbial community has the ability to dechlorinate 1,2,3-trichlorodibenzo-p-dioxin, which is a dioxin. Based on this fact, it has the ability to dechlorinate 2,3,4,5-tetrachlorobiphenyl and 2,3,4-trichlorobiphenyl to 2-monochlorobiphenyl or 4-monochlorobiphenyl, and 1,2 The microbial community of the present invention can be further characterized by the property of having the ability to dechlorinate 3-trichlorodibenzo-p-dioxin. According to the microbial community of the present invention having such characteristics, unlike the PCB dechlorinated microorganisms (or microbial communities) reported so far, it is possible to dechlorinate up to 1 chlorinated product. Dioxins can also be dechlorinated. As described above, the microbial community of the present invention has a dechlorination ability that is clearly different from the previously reported PCB dechlorination microorganisms, and more diverse PCB isotope dechlorination and dioxin-contaminated environments. Application to purification is expected.

On the other hand, by the structural analysis based on the 16S rRNA gene, the microbial community successfully obtained by the present invention includes two new dehalobacter species (FTH1 and FTH2) that have the ability to dechlorinate PCBs and dioxins. It was found that Moreover, it became clear that the bacterium of the genus Dehalococcides was not included. Both FTH1 and FTH2 were closely related to the Dehalobacter restrictionus TEA strain isolated as a polychlorethylene (PCE) dechlorinated bacterium, and showed 97.5% and 97.3% homology with respect to the base sequence of the 16S rRNA gene, respectively. . The base sequence of the 16S rRNA gene of FTH1 is shown in SEQ ID NO: 1, the base sequence of FTH2 is shown in SEQ ID NO: 2, and the base sequence of the dehalobacter restrictus TEA strain is shown in SEQ ID NO: 3, respectively.
Based on the above findings, the microbial community of the present invention can be further characterized by the property of including two types of dehalobacter bacteria as microorganisms having dechlorination ability. In addition, the microbial community of the present invention is characterized by the fact that the homology between the base sequence of the 16S rRNA gene in the contained Dehalobacter genus bacteria and the base sequence of the 16S rRNA gene in the Dehalobacter restrictus TEA strain is 97% or more and less than 98%. It can also be characterized. Further, the microbial community of the present invention may be characterized by the characteristic that the 16S rRNA gene in one of the contained Dehalobacter bacteria has the base sequence of SEQ ID NO: 1, and the other 16S rRNA gene has the base sequence of SEQ ID NO: 2. it can. On the other hand, the microbial community of the present invention can be further characterized by the property of being free of dehalococcides bacteria.
In addition, the microbial community (KFC4HC) containing FTH1 and FTH2 is deposited with a predetermined depository as follows, and is easily available.
Depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary Center (2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan 292-0818)
Deposit date (Receipt date): April 3, 2007
Accession Number: NITE P-353
The morphological characteristics and physiological characteristics of KFC4HC are as described in the Examples section below.

  The second aspect of the present invention relates to a bacterium belonging to the genus Dehalobacter included in the microbial community of the first aspect. That is, this aspect provides new species of the genus Dehalobacter (FTH1 and FTH2) found in the microbial community as microorganisms having dechlorination ability for PCBs and dioxins. Here, since each of the above characteristics characterizing the microbial community provided by the first aspect of the present invention is caused by the new species of Dehalobacter genus, the new species of Dehalobacter genus itself is characterized by the above properties. Will be attached. The new genus Dehalobacter bacteria can be isolated from the microbial community of the present invention. As the isolation method, a limiting dilution culture method or an agar mixed culture method can be employed.

The third aspect of the present invention provides a method for obtaining a microbial community of the present invention. The acquisition method of the present invention is characterized in that it comprises the step of accumulating and cultivating medium coarse-grained strong clay soil in the presence of fusalide and lactic acid. Hereinafter, this step will be described in detail.
In the acquisition method of the present invention, medium coarse-grained strong clay soil is used as a sample. The medium coarse-grained strong clay soil can be collected from, for example, 20 to 40 cm of the soil surface of the paddy field. It is preferable to store the soil after collection under a temperature condition of 10 to 25 ° C. until just before use.
The collected soil is subjected to enrichment culture. The enrichment culture is a culture method in which a target microorganism is purified (concentrated) by repeating a step of planting and culturing a part of a culture in which activity is recognized (that is, continuous subculture). The enrichment culture in the present invention is carried out in the presence of fusalide and lactic acid. The addition amount of a fusalide and lactic acid is not specifically limited. The amount of fusaride added is, for example, 10 to 600 μM, preferably 100 to 400 μM, with respect to 1 L of the culture. On the other hand, the addition amount of lactic acid is, for example, 10 mM to 30 mM, preferably 20 mM with respect to 1 L of the culture. It should be noted that the amount of fusaride and lactic acid need not be constant throughout the entire culture process. For example, the addition amount may increase as the number of passages increases, or conversely, the addition amount may decrease. Hereinafter, accumulation culture is demonstrated for every step. First, fusalide and lactic acid are added to a medium coarse-grained strong clay suspension and cultivated (step (1)). It is preferable to use distilled water for the preparation of the suspension. The culture temperature is, for example, 15 ° C to 30 ° C, preferably about 25 ° C. In addition, the culture period is approximately 10 days to 3 months, but it has been confirmed that the dechlorination of fusalides has progressed and the amount of fusalides in the culture solution has been reduced to 20% or less, preferably 5% or less. After that, it is a good idea to continue planting.
Next, subculture is performed using a nutrient medium containing fusalide and lactic acid (step (2)). That is, a part of the culture is transplanted and further cultured. The amount of culture used for transplanting is, for example, 1-20% (v / v) of the total culture, preferably 3-10% (v / v) of the total culture. The composition of the nutrient medium is not particularly limited. One suitable nutrient medium (FTH medium) is shown in the Examples column. The culture temperature and the culture period are the same as in step (1). The number of subcultures is not particularly limited, and is, for example, 1 to 30 times. In addition, the degree of accumulation is predicted by confirming the ability of dechlorination, the total number of bacteria, the form observed under the microscope, the PCR-DGGE band pattern and metabolic activity other than dechlorination such as methanogenesis. It is preferable to repeat the subculture until sufficient accumulation is observed.
For further accumulation, after step (2), subculture is performed using a nutrient medium containing fusalide and under conditions where hydrogen and carbon dioxide are supplied into the medium (step (3)). Also good. For example, if a sufficient amount of hydrogen and carbon dioxide is contained in the gas phase of the culture vessel, hydrogen and carbon dioxide can be supplied into the medium. The “sufficient amount” here is, for example, 30% to 90%, preferably 60% to 90% for hydrogen, and 5% to 40%, preferably 10% to 30%, for carbon dioxide. is there. An example of a suitable gas phase is a gas phase with 80% hydrogen and 20% carbon dioxide. Other conditions (temperature, culture period, etc.) in the subculture here are in accordance with step (2).
In one embodiment of the present invention, step (3) is performed instead of step (2). Thus, subculture in the presence of fusalide and lactic acid may be omitted.

The fourth aspect of the present invention relates to the use of the microbial community of the present invention and the Dehalobacter genus bacteria (hereinafter, these two are collectively referred to as “microorganisms of the present invention”). Specifically, a purification method using the microorganism of the present invention is provided. In the purification method of the present invention, the polychlorinated biphenyl and / or dioxins contained in the purification target are obtained by allowing the microorganisms of the present invention to act on the purification target (contaminant containing polychlorinated biphenyl and / or dioxins). Is dechlorinated. The purification target is not particularly limited as long as it contains polychlorinated biphenyls or dioxins. Typical examples of the purification target include industrial liquid waste, industrial waste water, industrial waste, and contaminated soil. As a method (application method) for causing the microorganisms or the like of the present invention to act, addition, spraying, mixing, etc. may be employed according to the form of the contamination target. The application amount can be arbitrarily set. An application amount suitable for the purification target can be set through a preliminary experiment. You may decide to apply the microorganisms of this invention, etc. in the state fix | immobilized thru | or supported by the support body.
You may decide to use together the microorganisms etc. of this invention, and other microorganism communities or microorganisms which dechlorinate PCB or dioxins. That is, PCBs or dioxins may be dechlorinated by combining the microorganisms of the present invention with other microorganisms. According to such a usage pattern, it is possible to improve the dechlorination rate, expand the types of compounds to be dechlorinated, and the like.

  By the way, as shown in the below-mentioned Example, as a result of examination by the present inventors, a dehalobacter genus bacterium that dechlorinates polychlorinated biphenyl to monochloride was found. In other words, it has been clarified that Dehalobacter bacteria are useful for dechlorinating polychlorinated biphenyl to monochloride. This finding cannot be predicted from previous reports, and its significance and value are extremely large. A further aspect of the present invention provides the following dechlorination method based on the above findings, that is, a method for dechlorinating polychlorinated biphenyl to monochlorinated product, which is characterized by allowing a Dehalobacter bacterium to act. The polychlorinated biphenyl here is preferably 2,3,4,5-tetrachlorobiphenyl or 2,3,4-trichlorobiphenyl. In this case, for example, 2-monochlorobiphenyl or 4-monochlorobiphenyl is produced as the monochloride. In addition, the dehalobacter genus bacteria of this invention can be used suitably as a dehalobacter genus bacteria. Moreover, you may make it the dehalobacter genus bacteria contained in the said microbial community act using the microbial community of this invention.

  We decided to adopt 4,5,6,7-tetrachlorophthalide (fusalide) as a model compound of PCB, and tried to acquire PCB dechlorinated microbial community by the following method. In addition, microbiological characteristics of the obtained microbial community were evaluated to confirm that PCB dechlorination was possible.

1. Sample and Method (1) Chlorine Compound Fusaride was purchased from Wako Pure Chemical Industries, Ltd. (Osaka). 4,5,6-trichlorophthalide, 4,5,6-trichlorophthalide, 4,7-dichlorophthalide, 4,6-dichlorophthalide and 4-monochlorophthalide are from Kureha Corporation (Tokyo) Received transfer. The following PCBs were purchased from AccuStandard Inc. (New Haven, Connecticut, USA). 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-TeCB); 2,3,4-trichlorobiphenyl (2,3,4-TriCB); 2,3-dichlorobiphenyl (2, 3-DiCB); 2,4-dichlorobiphenyl (2,4-DiCB); 2,5-dichlorobiphenyl (2,5-DiCB); 3,5-dichlorobiphenyl (3,5-DiCB); 2-monochloro Biphenyl (2-MCB); 3-monochlorobiphenyl (3-MCB) and 4-monochlorobiphenyl (4-MCB).

(2) Paddy field samples Paddy field samples were collected from a surface layer of 30 cm in a paddy field in Yatomi Town, Abe Prefecture. Distilled water was added to the soil to make a slurry. This slurry-like soil was passed through a 2.0 mm sieve, sealed in a plastic bag in a flooded state, and stored at 22 ° C. This soil had a water content of 35%, pH 6.8, total carbon content 0.99%, total nitrogen content 0.03%, and electric conductivity was 4.8 mS / m. Table 1 shows the other physicochemical properties of the soil.

(3) Accumulation of fusaride dechlorinated microbial communities
200 μl of 5 mM fusalide (dissolved in acetone) was added to a dry heat sterilized glass bottle (60 ml capacity), sealed, and then acetone was removed by vacuum suction. Once, 10 g [wet weight] of the above paddy soil was added thereto and filled with 10 ml of distilled water containing 1 mg L-1 resazurin. The soil suspension was adjusted to pH 7.0 with 1N hydrochloric acid and then subjected to nitrogen explosion for 15 minutes. The glass bottle was sealed with a fluororesin-coated butyl stopper, 20 mM lactic acid, formic acid, acetic acid, or butyric acid was added, and culture was started at 30 ° C. After culture for 1 month, about the culture in which dechlorination of fusaride was confirmed, 500 μl of the culture was transferred to FTH medium (Table 2) to which 10 ml of each organic acid was newly added and cultured. About the culture which confirmed dechlorination, 5% (v / v) was further transplanted, and the continuous subculture which culture | cultivates for 10 to 15 days was performed. Among these, lactic acid-added cultures that maintained dechlorination activity (hereinafter referred to as “KLF cultures”) were cultured in 600 ml glass bottles on a 1000 ml scale for the purpose of identifying the dechlorination pathway of fusalides and PCBs. went. KLF cultures were cultured with 20 mM pyruvic acid, 20 mM acetic acid, 100% hydrogen (gas phase) and 10 mM acetic acid, or 80% hydrogen (gas phase) and 20% carbon dioxide (gas phase). . In addition, experiments were conducted to evaluate the effects of electron acceptors and metabolic inhibitors.

(4) Analysis method Identification and quantification of fusalides, PCBs, and metabolites thereof were performed using a gas chromatograph mass spectrometer GC-MS (SHIMADZU GCMS-OP5050, hereinafter referred to as “GC-MS”).
1 mL was collected from the sample after completion of the culture, 1 ml of acetonitrile was added, and the mixture was stirred and mixed for 1 minute, 1 mL of ethyl acetate was added, and the mixture was mixed again for 1 minute. After allowing to stand for about 5 minutes, the organic solvent layer was recovered, and a small amount of anhydrous sodium sulfate was added thereto for dehydration. Of this, 2 μL was used for GC-MS analysis. GC-MS was analyzed using J & W DB-5MS (length 30 m x inner diameter 0.25 mm x film 0.25 mm) column with a vaporization chamber temperature of 320 ° C, an interface temperature of 280 ° C, and the following heating program: 140 ℃ (0.25min), 140 ℃ ～ 320 ℃ (10 ℃ ・ min ⁻¹ ), 320 ℃ (3min). It went on condition of. Metabolite identification was performed by comparing the results of all scan modes in the m / z 50-400 range with the residence time and mass spectrum of the standard sample. Quantification of fusaride and fusaride metabolites is in SIM mode detecting m / z 104, 139, 173, 207, and 243; PCB metabolite quantification is m / z 154, 188, 222, 222, 256, And in SIM mode to detect 290.
For the analysis of organic acids, high performance liquid chromatography (hereinafter referred to as “HPLC”; SHIMADZU SPD-10A was used) and ODS column (L-column, CERI) were used. From the sample after completion of the culture, 1 ml is collected using a syringe, filtered through a 0.2 μm filter (Millipore, Bedford, MA), 20 μl is injected into the HPLC, the flow rate is 1 ml · min ⁻¹ , and the column temperature is 40 ° C. , And detected by absorbance at UV 270 nm. As the mobile phase, a mixture of acetonitrile, water and acetic acid (0.1: 10: 89.9) was used.
Gas phase gas analysis was performed by gas chromatography (GC-14B, Shimadzu) and attached thermal conductivity detector (TCD). A stainless steel column packed with Molecular sieve 5A (GL-Science, Tokyo, Japan) was used for separation of hydrogen and methane, and activated carbon (GL-Science, Tokyo, Japan) was used for separation of carbon dioxide. The analysis was performed at a column temperature of 80 ° C., a vaporization chamber and detector temperature of 100 ° C. When detecting hydrogen and methane, 30 ml · min ⁻¹ nitrogen gas was used as the carrier gas, and when detecting carbon dioxide, 30 ml · min ⁻¹ helium gas was used as the carrier gas.

(5) Counting the total number of bacteria 1 ml was collected from the enrichment culture, suspended in 10 ml of 0.2 μm filter sterilized phosphate buffer (130 mM NaCl, 10 mM sodium phosphate buffer, 1 mM EDTA, pH 7.0) and stirred. Later, serial dilution was performed. For dilution of appropriate stage (10 ³ dilution) and filtered with suction over a membrane black filter (MILLIPORE). After filtration, spread the dried membrane on a glass slide, add approximately 5 μl of ProLong (registered trademark) Gold Antifade Reagent with DAPI Special Packaging (Molecular Probes, Eugene, Oregon, USA), cover the glass with a cover ballast, did. Observation was performed with an epi-illumination fluorescent microscope manufactured by Olympus, and images taken with an Olympus CCD camera (DP7, Olympus, Tokyo, Japan) were counted using image analysis software WINROOF (Flovel). Was measured.

(6) DNA extraction
Soil DNA extraction kit ISOIL (Nippon Gene, Toyama City, Japan) was used for DNA extraction. After completion of the culture, shake the serum bottle well, collect about 2 ml of the culture solution, centrifuge (20,630 × g, 5 min, 4 ° C), and use about 0.5 g of the precipitate, excluding the supernatant, for DNA extraction. It was. To this precipitate, 950 μl of Lysis Solution HE and 50 μl of Lysis Solution 20S were added, mixed thoroughly, and then incubated at 65 ° C. for 1 hour. This was centrifuged (12,000 × g, 1 min, 4 ° C.), 600 μl of the supernatant was transferred to a new tube, 400 μl of Purification Solution was added and mixed well. Add 600 μl of chloroform, stir and mix for 15 seconds, then centrifuge (12,000 × g, 10 min, room temperature), transfer 800 μl of aqueous layer to a new tube, and add 800 μl of Precipitation Solution. Mix and centrifuge (20,000 × g, 15 min, 4 ° C.). The supernatant was discarded, 1 ml of Wash Solution was added, mixed several times, and centrifuged (2,000 × g, 10 min, 4 ° C.). The supernatant was discarded, 1 ml of 70% ethanol and 2 μl of Ethachinmate were added, mixed several times, and centrifuged (20,000 × g, 5 min, 4 ° C.). After discarding the supernatant and air-drying, the precipitate was dissolved in 100 μl of TE solution (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

(7) PCR-DGGE (denaturing gradient gel electrophoresis)
Approximately 50 ng of extracted DNA was used as a template, and a 100 μl volume PCR reaction was performed. Among the 16S rRNA genes, the 341-534 variable region at the E. coli 16S rRNA gene position (Brosius et al. 1978. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci US A. 75 (10 ): 4801-5 (1978), GC357f (with 5 'end GC clamp) and 517r (Table 3) (Muyzer, G., EC de Waal, and AG Uitterlinden. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59: 695-700.). PCR was carried out for 35 cycles of hot start at 95 ° C for 2 minutes, heat denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1 minute, and extension at 72 ° C for 1.5 minutes.
* E.coli 16S rRNA gene position

DGGE has been reported (Muyzer, G., EC de Waal, and AG Uitterlinden. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59 : 695-700.) Using a denaturing gel electrophoresis apparatus (Bio-RAD Dcode). The gel for DGGE was 0.5 × TAE, 6% acrylamide, and 40-60% denaturant (100% denaturant solution; 7M urea, 40% formamide), DNA denaturant 0% acrylamide solution (Table 4) and DNA denaturation. An agent 80% acrylamide solution (Table 4) was mixed to prepare. Electrophoresis was performed with 0.5 × TAE buffer (40 mM Tris-base, 20 mM sodium acetate, 2 mM EDTA), 60 ° C., 100 V, and electrophoresis time of 16 hours. The gel stained with SYBR GREEN I (Molecular probe) was irradiated with a transilluminator (TOYOBO FAS-III mini + DS-30) to detect a PCR product. The main band was cut out and purified and then subcloned to determine the base sequence.

(A) Purification of main band The excised gel was washed by pipetting with 1 ml of distilled water, and PCR was performed again using the gel fragment (about 0.5 mm square) as template DNA. The reaction was carried out for 35 cycles with a hot start at 95 ° C for 2 minutes, heat denaturation at 94 ° C for 1 minute, annealing at 60 ° C for 1 minute, and extension at 72 ° C for 1.5 minutes. The obtained PCR product was DGGE to confirm the isolation of the band. The non-isolated band was cut out again, the annealing temperature and the number of cycles were changed, and the same operation was repeated until it was isolated. The PCR product in which band isolation was confirmed was purified after ethanol precipitation, and electrophoresed with 1 × TAE buffer using a 4% agarose gel. After electrophoresis, the target DNA was excised from the gel, purified using GENECLEAN II Kit (BIO101), and the nucleotide sequence was determined. The band that could not be isolated was subcloned and the nucleotide sequence was determined.

(B) Subcloning
PCR products were subcloned using pTBlue Perfectly Blunt cloning kit (Novagen) according to the accompanying instructions. After blunt end reaction (22 ° C., 15 minutes), ligation (16 ° C., 14-16 hours) was performed, and transformation by heat shock was performed. 50 mM IPTG and 40 mg ml in LBA agar medium (10 gL ^-1 tryptone / peptone, 5 g L ^-1 yeast extract, 10 g L ^-1 NaCl, 20 g L ^-1 agar, 100 mg L ^-1 ampicillin) ^The transformed bacterial solution was applied to a medium coated with 50 μl of ⁻¹ X-gal and cultured at 37 ° C. for 12 to 16 hours. After culture, white colonies were collected and the plasmid was amplified using Templiphi ^™ plasmid amplification kit (Amersham Biosciences, Sunnyvale, CA, USA) and used as a template for subsequent sequencing and PCR reactions.

(8) Construction of 16S rRNA gene clone library and RFLP analysis
The 16S rRNA gene clone library uses approximately 50 ng of extracted DNA as a template, and primer sets 27f and 1492r (Table 5) targeting bacteria (Weisburg, WG, SM Barns, DA Pelletier, and DJ Lane. 1991. 16S The PCR product amplified by ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173: 697-703.) was cloned and constructed. PCR was performed at 95 ° C for 2 minutes, followed by 30 cycles of heat denaturation at 94 ° C for 1 minute, annealing at 50 ° C for 1 minute, and extension at 72 ° C for 1.5 minutes. An extension reaction was performed for 5 minutes. The PCR product was purified by 1% agarose electrophoresis and cloned as described above. The obtained clone was subjected to PCR again using a plasmid as a template, and the PCR product was subjected to a restriction enzyme digestion reaction with HaeIII, HhaI and SspI. The digested product was run on a 3% agarose gel, and operational taxonomic units (OTUs) were determined from this migration pattern.
* E.coli 16S rRNA gene position

(9) Determination of base sequence and systematic analysis
The DNA base sequence was determined using BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems). The sequencing reaction was performed by heat denaturation at 96 ° C. for 1 minute, followed by 25 cycles of heat denaturation at 96 ° C. for 10 seconds, annealing at 50 ° C. for 5 seconds, and extension at 72 ° C. for 4 minutes. The sequence primer of DGGE band clone was subjected to cycle sequence reaction using sequence primers pT7-F and pT7-R (Table 6) targeting the insert upstream and downstream sites of the pT7 vector. For the clones amplified with the 27f and 1492r primers, a cycle sequence reaction was performed using the primers shown in Table 7. The sequence reaction was analyzed with 3100-Avant genetic analyzer (Applied Biosystems). The obtained base sequence was edited with the GENETIX ver 7 software development program. About the edited nucleotide sequence, the homology search program (BLAST Homology Search) in the database on the Internet (DDBJ: http // www.ddbj.nig.ac.jp/E-mail/homology.html) (Altschul, SF , Gish, W., Miller, W., Myers, EW and Lipman, DJ (1990) "Basic local alignment search tool." J. Mol. Biol. 215: 403-410.) It was.

* E.coli 16S rRNA gene position

2. Results (1) Fusaride dechlorinated microbial community As a result of adding various organic acids and fusarides to paddy soil and culturing for one month, anaerobic dechlorination of all organochlorine compounds was confirmed. Among these, the lactic acid and formic acid-added aggregates maintained the dechlorination activity even after 10 passages (FIG. 1). As a result of dechlorination test of other chlorinated compounds on both cultures, only KLF culture (lactate supplemented with lactate) was found to be 2,3,4,5-tetrachlorobiphenyl, 2,3,4-trichloro. Since biphenyl was dechlorinated, it was decided to use a KLF culture in subsequent experiments.

(2) KLF culture
The KLF culture was examined for the dechlorination pathway of fusaride. All KLF cultures were dechlorinated with 100 μM fusaride to 4-monochlorophthalide in approximately 5 days. The state of desulfurization of the KLF culture is shown in FIG.

(3) Effect of electron donor on fusalide dechlorination in KLF culture Table 8 shows the results of investigating the influence of various electron donors on the KLF culture. KLF cultures maintain dechlorination activity up to 10 ^-5 dilution when 20 mM lactic acid or hydrogen is added, and maintain dechlorination activity in 20 mM acetic acid, pyruvate, propionic acid or 5 mM lactic acid addition systems. There wasn't. Also, only when 0.01% peptone was added, the KLF culture used hydrogen produced from lactic acid as an electron donor because it showed dechlorination activity in a ^10-6 diluted culture in a hydrogenated system. Thus, it was speculated that dechlorination of fusalide was performed.
“Nt” in the table indicates “not tested”. *: Desulfurization of fusalide was confirmed under the condition of 0.01% peptone addition. **: Gas concentration in the gas phase v / v (%).

(5) Effects of metabolic inhibitors on fusaride dechlorination in KLF cultures
The KLF culture was performed chloramphenicol 50 mg L ^-1, vancomycin 50 mg L ^-1, 5 mM bromo ethanesulfonic acid (BES), or the heat treatment of the case and 80 ° C. for 30 minutes was added 10mM molybdate As a result of examining the fusalide dechlorination activity in each case and evaluating the presence or absence of the inhibition tendency with respect to the untreated culture, it was shown that all the metabolic inhibition treatments inhibit the dechlorination of fusalide (Table 9). In addition, as a result of culturing with addition of 10 mM nitric acid, sulfuric acid, and trivalent iron and evaluating the effect of these electron acceptors on the desulfurization of nitric acid, the nitric acid and sulfuric acid addition system showed a slight decrease in the speed, but the desulfurization of the fusaride was slightly reduced. While chlorination occurred, the dechlorination of fusalide was completely inhibited in the iron addition system (Table 9). From these results, it was found that this microorganism group can maintain the dechlorination ability of fusalide in the presence of 10 mM or less sulfuric acid or nitric acid, but its dechlorination activity is greatly inhibited by trivalent iron.

(6) Microbial community structure analysis based on 16S rRNA gene of KLF culture In each KLF culture in each subculture process, PCR-DGGE of about 150 bp was performed using a primer having a 16S rRNA gene sequence specific to bacteria. The results are shown in FIG. From FIG. 3, the microbial composition in the KLF culture changed greatly until the 4th subculture, and then changed to a relatively stable microbial composition up to the 12th culture. As a result of performing DGGE on the culture (Fig. 3-F) in which the 10th generation culture was transplanted twice to the culture without addition of fusalide, two bands A and B which were the main bands in the culture with addition of fusalide were found. The disappearance (FIG. 3) suggested that these two microorganisms are responsible for the dechlorination of fusalides. This DGGE band A is closely related to Dehalobacter restricus PER-K23 strain known as a tetrachlorethylene (PCE) dechlorination bacterium and shows 94.9% homology (Holliger, C., D. Hahn, H. Harmsen, W. Ludwig, W. Schumacher, B. Tindall, F. Vazquez, N. Weiss, and JA Zehnder. 1998. Dehalobacter restrictus gen. Nov. And sp. Nov., A strictly anaerobic bacterium that reductively dechlorinates tetra- and trichloroethene in An anaerobic respiration. Arch. Microbiol., 169: 313-321.), B also showed 95.9% homology to the D. restricus TEA strain, also a PCE dechlorinating bacterium (Wild, A., R Hermann, and T. Leisinger. 1996. Isolation of an anaerobic bacterium which reductively dechlorinates tetrachloroethene and trichloroethene. Biodegradation 7: 507-511).
For the purpose of phylogenetic analysis based on the almost full length of 16SrRNA gene of each dominant microorganism in the culture with and without addition of fusalide, PCR using primers that amplify almost the full length of 16SrRNA gene was performed, and a 16SrRNA gene clone library was constructed did. 50 clones (100 clones in total) were obtained for the cultures with and without fusaride. As a result of comparing restriction enzyme fragment patterns by HaeIII, HhaI, and SspI for each clone, a total of 8 OTUs were obtained, and base sequence analysis was performed for each clone from 8OTUs. Of the 8 OTUs obtained from the KLF cultures with and without the addition of fusaride, OTU1 and OTU2 belonged to the genus Dehalobacter, 5 OTUs belonged to the genus Clostridium, and 1 belonged to the genus Sedimentbacter (Table 10). Of these, OTU1 and OTU2 appeared only in the fusalide-added culture, and corresponded to bands A and B shown by PCR-DGGE, respectively. Since OTU1 and OTU2 were thus shown to be microorganisms responsible for fusalide dechlorination in KLF cultures, they are referred to as FTH1 and FTH2 for the purpose of distinguishing them from other clones. FTH1 and FTH2 were isolated as PCE dechlorinated bacteria, Dehalobacter restrictus TEA strain (Wild, A., R. Hermann, and T. Leisinger. 1996. Isolation of an anaerobic bacterium which reductively dechlorinates tetrachloroethene and trichloroethene. Biodegradation 7: 507-511), showing 97.5% and 97.3% homology, respectively. FIG. 4 shows a phylogenetic tree created by the neighborhood joining method for the FTH1 and FTH2 sequences and their related sequences. FTH1 and FTH2 are related clones (von Wintzingerode, F., B. Selent, W. Hegemann, and UB Gobel. 1999. Phylogenetic analysis of anion) obtained from previously isolated bacteria of the genus Dehalobacter and the environment related to chlorinated compounds. anaerobic, trichlorobenzene-transforming microbial consortium. Appl. Environ. Microbiol. 65: 283-286., Schlotelburg, C., F. von Wintzingerode, R. Hauck, W. Hegemann, and UB Gobell. 2000. Bacteria of an anaerobic 1 , 2-dichloropropane-dechlorinating mixed culture are phylogenetically related to those of other anaerobic dechlorinating consortia.Int.J. Syst.Evol.Microbiol. 50: 1505-1511., Lowe, M., EL Madsen, K. Schindler, C. Smith, S. Emrich, F. Robb, and RU Halden. 2002. Geochemistry and microbial diversity of a trichloroethene-contaminated Superfund site undergoing intrinsic in situ reductive dechlorination.FEMS Microbiol. Ecol. 40: 123-134., Van Doesburg, W ., MHA van Eekert, PJM Middeldorp, M. Balk, G. Schraa, and AJM Stams. 2005. Reductive dec hlorination of [β] -hexachlorocyclohexane ([β] -HCH) by a Dehalobacter species in coculture with a Sedimentibacter sp. FEMS Microbiol. Ecol. 54: 87_95 .; Yan, T., TM LaPara, and PJ Novak 2006. The effect of varying levels of sodium bicarbonate on polychlorinated biphenyl dechlorination in Hudson River sediment cultures.Environ. Microbiol. 8: 1288-1298.), a new cluster at least at the species level, even from 16S rRNA gene homology. It was shown to be a new bacterium. The 16S rRNA gene sequences of FTH1 and FTH2 are accession numbers AB294742 (SEQ ID NO: 1) and AB294743 (SEQ ID NO: 1) in DDBJ / EMBL / GenBank (http://www.ddbj.nig.ac.jp/Welcome-j.html). Registered with number 2).

(7) Evaluation of dechlorination ability of PCBs and PCDDs in KLF culture
For KLF culture, the gas phase was replaced with 80% hydrogen and 20% carbon dioxide, and 53,4 volume of KLF 12th culture was added as inoculum, 2,3,4-triCB, 2,3,4, The dechlorination ability of 5-teCB, 1,2,3-triCDD or 1,2,3,4-teCDD was evaluated. The transition of the chlorine compound concentration in the culture solution of each culture is shown in FIG. In cultures supplemented with 2,3,4-triCB, 2,3,4,5-teCB, and 1,2,3-triCDD, the decrease in added chlorine compounds and the accompanying dechlorination over time Product production was confirmed (FIGS. 5A, B, C). On the other hand, such changes suggesting dechlorination were not observed in the 1,2,3,4-TeCD-added culture (FIG. 5D). From this, it was shown that the KLF culture has the ability to dechlorinate 2,3,4-TriCB, 2,3,4,5-TeCB, and 1,2,3-TriCDD.
As shown in FIG. 5, in the PCB-added KLF culture, the amount of dechlorinated product produced with respect to the added PCB is very small, so that the dechlorinated product is vaporized in the gas phase. Expected. Therefore, when 20 μl of gas in the gas phase was analyzed by GC-MS, it was observed that PCB was vaporized mainly in 1-2 chlorinated biphenyl.

  FIG. 6 shows the total amount of PCBs or PCDDs present in the gas phase and culture medium on day 0 and day 60 of the KLF culture. The total amount on day 0 in this figure is the amount of each added PCB and PCDD detected in the culture medium on day 0 and the presence of the liquid phase / gas phase of the added compound in the detection results on day 60 It is the total of gas phase abundance estimated from the ratio. In PCDD cultures, the amount of PCBD increased by dissolution into the culture medium, and in PCB cultures, it was suggested that PCBs disappeared slightly due to vaporization, but approximately the same amount of PCB or PCDD was obtained. Therefore, in KLF cultures, 2,3,4-TriCB, 2,3,4,5-TeCB and 1,2,3-TriCDD are reduced by dechlorination and 1,2,3,4 -TeCDD was shown not to be dechlorinated.

(8) Dechlorination pathway of fusarides, PCBs and PCDDs by KLF culture
Produced by dechlorination of fusaride (A), 2,3,4-triCB (B), 2,3,4,5-teCB (C), and 1,2,3-triCDD (D) by KLF culture The dechlorination pathway of these compounds was estimated from the dechlorination product (FIG. 7).

(9) Accumulation of KLF culture
A portion of the KLF culture was subjected to continuous subculture. First, 5% (v / v) KLF culture was subcultured and cultured in FTH medium supplemented with 0.01% (w / v) peptone. The gas phase conditions were 80% hydrogen and 20% carbon dioxide, and the culture temperature was 30 ° C. After 14 days of culture, an additional 5% of the culture was inoculated and cultured. This operation was repeated to increase the degree of integration. After confirming that FTH1 and FTH2 were contained and that dechlorination activity was observed, the finally obtained culture (KFC4HC) was deposited at a predetermined depository as follows.
Depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary Center (2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan 292-0818)
Deposit date (Receipt date): April 3, 2007
Accession Number: NITE P-353

KFC4HC is a mixed microorganism containing a total of three dominant microbial species. Two species (FTH1, FTH2) are novel microorganisms at the dehalobacter species level. The two microbial species dechlorinate fusarides. In order to confirm whether or not the two microbial species are alive, it is only necessary to observe whether dechlorination of fusalide occurs in the cultured culture. Moreover, when PCR-DGGE is performed and the same band pattern as FIG. 9 is obtained, it can confirm that each microbial species which comprises KFC4HC is maintained including the said 2 types of microbial species. Other characteristics of KFC4HC are shown below.
(Form)
Neisseria gonorrhoeae. FIG. 8 shows a DFC (4 ′, 6-diamino-2-phenylindole) stained image of KFC4HC.
(Physiological features)
(1) Even after growing, the turbidity of the medium cannot be confirmed visually.
(2) Died at 40 ° C or higher.
(3) Does not grow when 0.5mM or more of hydrogen sulfide is added.
(4) Grows fusalide as an electron acceptor.
(PCR-DGGE)
The result of analyzing KFC4HC by PCR-DGGE is shown in FIG.

4). Consideration
Using fusalide as a model compound instead of PCB, we obtained a KLF aggregate that dechlorinates fusalide and PCB, and showed that KLF cultures dechlorinate fusalide even under conditions of reduced iron addition. The microorganisms responsible for dechlorination of fusalides in KLF cultures are the two Dehalobacter bacteria FTH1 and FTH2, which are novel microorganisms at least at the species level. FTH1 and FTH2 can dechlorinate 2,3,4,5-tetrachlorobiphenyl and 2,3,4-trichlorobiphenyl to monochlorinated compounds such as 2- or 4-monochlorobiphenyl.
In addition, we succeeded in obtaining a highly concentrated microbial community by subculturing KLF cultures. It should be noted that such a high degree of accumulation was obtained despite a relatively short period of accumulation of 16 months in total.

  The microbial community and microorganisms of the present invention are capable of dechlorinating PCB alone up to 1 chlorinated product, and can be expected to be applied and applied to the dechlorination of various PCB congeners. Since the microbial community and microorganisms of the present invention also have a dechlorination activity for dioxins, application to purification of dioxin-contaminated environments is also expected. The microbial community or microorganism of the present invention can be used in combination with other PCB dechlorinated microbial groups or microorganisms to enhance the dechlorination ability.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Dechlorination of fusaride in each organic acid-added paddy field culture. ace: acetic acid-added culture, for: formic acid-added culture, lac: lactic acid-added culture, but: butyric acid-added culture. Dechlorination of fusarides in KLF cultures. The mean and standard deviation of the results of three experiments performed in parallel are shown. ●: Fusaride, ▲: 4,6,7-trichlorophthalide, □: 4,4-dichlorophthalide, ■: 4,5-dichlorophthalide, ○: 4-monochlorophthalide PCR-DGGE analysis results of microbial community structure in KLF culture. Each lane number indicates the number of passages of the KLF subculture. “-F” indicates a culture in which the 10th generation KLF culture was subcultured in the fusalide-free medium for 2 generations. For bands A and B, sequence analysis was performed. A phylogenetic tree created by the neighbor-joining method for FTH1 and FTH2 sequences and their related sequences. The clones indicated by * are all clones acquired from the environment related to chlorine compounds, and the characters displayed beside them indicate the names of chlorine compounds. 2,3,4-triCB (A), 2,3,4,5-teCB (B), 1,2,3-triCDD (C) and 1,2,3,4-teCDD (D ) Dechlorination ability. Total amount of PCBs or PCDDs present in the gas phase and culture medium at day 0 and day 60 of KLF culture. Dechlorination route of each chlorinated compound by KLF culture: Fusaride (A), 2,3,4-triCB (B), 2,3,4,5-teCB (C), 1,2,3-triCDD ( D). DAPI (4 ', 6-diamino-2-phenylindole) stained image of the finally obtained microbial community (KFC4HC). Results of KFC4HC PCR-DGGE analysis. Lane A: KFC4HC (deposited microorganism culture), Lane B: KLF culture (original culture of KFC4HC), Lane C: Fusaride dechlorinated bacterial clone FTH2, Lane D: Fusaride dechlorinated bacterial clone FTH1. The band indicated by a in lane A is derived from clone FTH1, and the band indicated by b is derived from clone FTH2.

Claims (7)

The following characteristics:
(1) including bacteria belonging to the genus Dehalobacter as microorganisms having dechlorination ability;
(2) Dechlorination using hydrogen as an electron donor;
(3) Acetic acid and carbon dioxide can be used as a carbon source;
(4) maintain dechlorination activity even in the presence of 10 mM nitric acid or sulfuric acid;
(5) Dechlorination activity is inhibited by trivalent iron.
A microbial community that dechlorinates polychlorinated biphenyls and dioxins,
Contains two dehalobacter bacteria as microorganisms with dechlorination ability,
The 16S ribosomal RNA gene in one of Deharobakuta bacteria has a nucleotide sequence of SEQ ID NO: 1, 16S ribosomal RNA gene in the other will have a nucleotide sequence of SEQ ID NO: 2,
A microbial community whose accession number is NITE P-353 . The following characteristics:
(1) Dechlorination using hydrogen as an electron donor;
(2) Acetic acid and carbon dioxide can be used as a carbon source;
(3) maintain dechlorination activity even in the presence of 10 mM nitric acid or sulfuric acid;
(4) Dechlorination activity is inhibited by trivalent iron.
A dehalobacter bacterium that dechlorinates polychlorinated biphenyls and dioxins,
16S ribosomal RNA gene have a nucleotide sequence of SEQ ID NO: 1 or 2,
Included in the microbial community whose deposit number is NITE P-353 ,
Dehalobacter bacteria. Microbial community according to claim 1, or a Deharobakuta bacterium according to claim 2, purification method which comprises causing to act on contaminants including polychlorinated biphenyls and / or dioxins. The purification method according to claim 3 , wherein the polychlorinated biphenyl is 2,3,4,5-tetrachlorobiphenyl or 2,3,4-trichlorobiphenyl. The purification method according to claim 3 or 4 , wherein the dioxins are 1,2,3-trichlorodibenzo-p-dioxin. A method for dechlorinating polychlorinated biphenyl to monochlorinated product, characterized by allowing dehalobacter bacteria to act on polychlorinated biphenyl,
Wherein said Deharobakuta bacterium, characterized in that a Deharobakuta bacterium according to Deharobakuta bacterium, or claim 2 included in the microbial community of claim 1. The polychlorinated biphenyl is 2,3,4,5-tetrachlorobiphenyl or 2,3,4-trichlorobiphenyl, and the monochlorinated product is 2-monochlorobiphenyl or 4-monochlorobiphenyl. The method according to claim 6 .