WO2001059095A1 - Procede de purification ou de traitement de materiaux nuisibles pour l'environnement - Google Patents

Procede de purification ou de traitement de materiaux nuisibles pour l'environnement Download PDF

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Publication number
WO2001059095A1
WO2001059095A1 PCT/JP2000/009349 JP0009349W WO0159095A1 WO 2001059095 A1 WO2001059095 A1 WO 2001059095A1 JP 0009349 W JP0009349 W JP 0009349W WO 0159095 A1 WO0159095 A1 WO 0159095A1
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WO
WIPO (PCT)
Prior art keywords
microorganism
ability
transposon
microorganisms
environment
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Application number
PCT/JP2000/009349
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English (en)
Japanese (ja)
Inventor
Ginro Endo
Chien Chen Huang
Masaru Narita
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Japan Science And Technology Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Japan Science And Technology Corporation filed Critical Japan Science And Technology Corporation
Publication of WO2001059095A1 publication Critical patent/WO2001059095A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
    • B01D53/34—Chemical or biological purification of waste gases
    • B01D53/46—Removing components of defined structure
    • B01D53/64—Heavy metals or compounds thereof, e.g. mercury
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the invention of this application relates to a method for purifying an environment contaminated with harmful substances such as wastewater, exhaust gas or solid waste, and a method for treating harmful substances by mechanical equipment.
  • Heavy metals such as mercury and cadmium, and organic chlorine compounds such as PCB and trichloroethylene are widely used in industry, but they are extremely harmful to the human body. Therefore, not only acute poisoning due to these harmful substances, but also intake of agricultural products and marine products obtained from soil and water bodies contaminated with harmful substances, or groundwater, has a serious effect on the human body.
  • methyl mercury poisoning caused by ingestion of fish and crustaceans contaminated by industrial wastewater containing mercury compounds in Minamata Bay in Japan is a typical example.
  • microorganism capable of treating individual harmful substances has been isolated does not mean that the microorganism can be put to practical use as it is.
  • bio-regulation method when introducing microorganisms from outside into a contaminated environment (bio-regulation method), it is necessary to carefully evaluate the safety of the introduced microorganisms.
  • bio-simulation method a method of activating microorganisms already inhabiting the polluted environment.
  • the invention of this application has been made in view of the circumstances described above, and is intended to reduce the ability of various microorganisms living in a polluted environment and microorganisms applicable to a treatment apparatus to treat various harmful substances and to purify the environment.
  • the goal is to provide a method that can decompose and treat harmful substances safely and reliably. Disclosure of the invention
  • This application is an invention for solving the above-mentioned problem, and comprises the following steps:
  • the propagated microorganism A is introduced into a treatment device containing the microorganism B or a contaminated environment in which the microorganism B lives, and the transposon-like sequence region of the microorganism A is horizontally transmitted to the chromosome of the microorganism B, whereby the gene is transformed into the microorganism. Step of transferring to B
  • an environmental purification or harmful substance treatment method comprising purifying a contaminated environment or treating harmful substances with microorganisms B having an environmental purification ability or a toxic substance treatment ability obtained through these steps.
  • the gene related to the ability to purify the polluted environment or the ability to treat harmful substances is a gene derived from an organism other than the microorganism A.
  • the transposon-like sequence is a transposon-like sequence of the Gram-positive bacterium BaciIlus megateri urn MB1 strain.
  • TnNIER I 1 is another preferred embodiment.
  • the invention of this application provides for the establishment of a habitat for microorganisms A and Z or microorganisms B inside or outside the toxic substance treatment device or in the contaminated environment, and It is also preferred that the habitat be provided with a facility to which microorganisms can adhere and / or be entrapped.
  • FIG. 1 shows the results of gel electrophoresis of the PCR product of the CTB2 strain, which was cloned and cloned with Clostr idium bytur icum Mersaru having mero heron and Clostr idium bytur icum Mersaru having i_.
  • FIG. 2 is a schematic view illustrating a processing apparatus for performing the method of the present invention.
  • 10 is anaerobic tank
  • 11 is waste liquid
  • 12 is mercury vapor
  • 13 is microbial sludge
  • 14 is desorbed water
  • 20 is aerobic tank
  • 21 is storage tank
  • 22 is treated water
  • 23 is treated water Microbial sludge
  • 30 indicates an energy generating tank, 41, 42, 43, 44, 45, and 46 all indicate piping.
  • the inventors of the present application revealed that a gene group (mer operon) that acquires resistance by removing mercury is also encoded on the chromosome of anaerobic mercury-resistant bacteria. Moreover, the mer operon of this anaerobic bacterium almost completely matches the mer operon of a known aerobic bacterium (99.7% on a base sequence basis) (Gene 234: 361-369). , 1999).
  • the inventors have developed anaerobic water Finding that many strains of silver-resistant bacteria have the same mer operon, have a horizontal transposition of this gene in nature, and that this mer operon is coded for the transposon-like sequence TnMER M
  • TnMERM transposon-like sequence
  • the mer-operin is non-resistant by TnMERM. It was found that non-resistant bacteria acquired mercury resistance performance.
  • the method of the present invention based on the new findings as described above is composed of the following two steps.
  • the propagated microorganism A is introduced into a processing device containing the microorganism B or a contaminated environment in which the microorganism B lives, and the transposon-like sequence region of the microorganism A is horizontally transferred to the chromosome of the microorganism B. Transferring the offspring to microorganism B.
  • the microorganism A is a microorganism having a transposon-like sequence region on the genome, and a gene (hereinafter referred to as a gene related to the ability to purify the polluted environment or the ability to treat harmful substances) , And sometimes referred to as “resistance gene”).
  • the transposon-like sequence region is a nucleic acid sequence that can be transferred to the genome of another organism, for example, a Gram-positive bacterium Baci II us megaterium MB1 strain isolated from Minamata Bay The tumor can be exemplified by TnMERM of the anaerobic bacterium Clostr idium bytur i cum Mersaru.
  • this TnMERM (about 1 4 .5kb), genes mer operon involved in the far Ri mercury resolution of the (approximately 7. Okb) is coded. Therefore, by using these strains as microorganisms A in this step 1) in the next step 2), it becomes possible to purify the environment contaminated with heavy metals such as mercury and to treat heavy metals and their compounds. .
  • the resistance gene encoded in the transposon-like sequence region may be a gene derived from another organism.
  • a known or unknown gene encoding a substance that decomposes and volatilizes harmful substances such as mercury, cadmium, PCB, organochlorine compounds, and dioxins can be used.
  • unknown genes organisms having the ability to purify the environment or to treat harmful substances are separated from the polluted environment or harmful substance treatment processes including wastewater or exhaust gas treatment, and the environmental purification ability or Resistance genes related to toxic substance processing ability can be identified and used separately.
  • a gene recombination technique can be used.
  • step 2) the microorganism A is introduced into a harmful substance treatment device or an environment contaminated with harmful substances.
  • Microorganisms B suitable for survival are placed in this treatment device or in the contaminated environment, respectively.
  • the microorganism B may be an indigenous microorganism.
  • the resistance gene contained in the transposon-like sequence region of the microorganism A is transformed into the genome or plasmid of the microorganism B by the transposition function of the transposon-like sequence region.
  • Microbe B acquires the ability to purify the polluted environment or treat harmful substances.
  • this microorganism B is originally a microorganism that is suitable for survival in treatment equipment and contaminated environments, it inhabits well in each and By expression, harmful substances can be efficiently decomposed and volatilized. Furthermore, the treatment of harmful substances and the purification of the environment can be performed using both the microorganisms A and B. In this case, if microorganisms A and B have different properties (for example, anaerobic or aerobic), they can be separated after the mating to treat harmful substances under different conditions or to reduce environmental impact. Purification is possible at the same time.
  • a habitat for microorganisms A and / or B can be provided inside or outside the harmful substance treatment apparatus, or in the contaminated environment.
  • the habitat of the microorganisms A and B or the microorganisms B may be provided with a facility to which the microorganisms adhere and are fixed or entrapped.
  • the chromosomes DNA of the donor strains C. byturicum ersaru and CTB2 strain were isolated according to a conventional method, and whether or not the mer operon region had transferred from the donor strain to the CTB2 strain was examined by the PCR method.
  • PCR was performed using TP240 thermal cycler (Takara Shuzo) and LA PCR Kit Ver.2 (Takara Shuzo). 100 ng of longevity DNA, 0.4 l of forward primer (synthetic oligonucleotide of SEQ ID NO: 1) and reverse primer (synthetic oligonucleotide of SEQ ID NO: 2) were added in a volume of 0.4 l each. (Scale) Set in thermal cycler. After the set was completed, PCR was performed under the following conditions.
  • FIG. 1 shows the results of gel electrophoresis of the obtained PCR product. As is clear from FIG. 1, the presence of a mer operin of about 7. Okb was confirmed in the CTB2 strain derived from the bacterium B. subt iIs 168 having no mer operon.
  • FIG. 2 shows a heavy metal processing apparatus whose configuration is shown.
  • This processing device is equipped with an anaerobic tank (0), an aerobic tank (20), and an energy generating tank (30).
  • the anaerobic tank (10) is filled with the anaerobic microorganism A in which a gene for resistance to heavy metals such as mercury is coded in a transposon-like sequence, and the mercury in the waste liquid (11) is reduced under anaerobic conditions. Is decomposed and exhausted as steam (12).
  • the sludge (13) of microorganism A filled in the anaerobic tank (10) is collected as microbial fertilizer (compost) via the pipe ().
  • the desorbed water (14) of the anaerobic tank (10) is transported to the aerobic tank (20) via the pipe (42).
  • the aerobic tank (20) is filled with the aerobic microorganism B to which the resistance gene has been horizontally transferred from the microorganism A, and the heavy metals in the desorbed water ⁇ 4) are also contained in the aerobic tank (20). Decomposed.
  • the aerobic tank (20) is provided with a storage tank (21), and the treated water (22) is recovered via a pipe (43) and used as water resources or liquid fertilizer.
  • the storage tank (20 microbial sludges (23) are sent to an energy generation tank (30) via a pipe (44). In this energy generation tank, the microbial sludge (23) is fermented to produce hydrogen and sludge. Methane is generated and recovered via the pipe (45), and the anaerobic fermentation waste liquid generated in the energy-producing tank (30) is discharged through the pipe (46). Recirculated to (10).
  • the invention of this application allows microorganisms suitable for inhabiting the contaminated environment and treatment equipment to efficiently obtain the ability to treat harmful substances. Can be reliably purified.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Environmental & Geological Engineering (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Analytical Chemistry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Hydrology & Water Resources (AREA)
  • Water Supply & Treatment (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Processing Of Solid Wastes (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Treating Waste Gases (AREA)
  • Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)

Abstract

L'invention concerne un procédé qui confère à un micro-organisme la possibilité de traiter efficacement des matériaux nuisibles, en agissant de manière appropriée dans un environnement pollué ou un dispositif de traitement à l'intérieur duquel il se trouve, ce qui assure un traitement des matériaux nuisibles et une purification de l'environnement selon les étapes suivantes: 1) prolifération d'un micro-organisme A porteur de gène capable de purifier un environnement pollué ou de traiter des matériaux nuisibles dans le domaine de séquence de type transposon, par transfert dans le génome d'autres organismes ou plasmides; et 2) transfert du micro-organisme A ayant ainsi proliféré dans le dispositif de traitement renfermant un autre micro-organisme B ou dans l'environnement pollué contenant ce micro-organisme B, puis transfert horizontal du domaine de séquence de type transposon du micro-organisme A dans le chromosome du micro-organisme B visant à transférer le gène susmentionné dans le micro-organisme B. On purifie ainsi l'environnement pollué ou bien on traite les matériaux nuisibles par le biais du micro-organisme B, lequel est désormais capable de purifier l'environnement ou de traiter les matériaux nuisibles en question.
PCT/JP2000/009349 2000-02-14 2000-12-27 Procede de purification ou de traitement de materiaux nuisibles pour l'environnement WO2001059095A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2000035902A JP2001225096A (ja) 2000-02-14 2000-02-14 環境浄化または有害物質処理方法
JP2000-35902 2000-02-14

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992019738A1 (fr) * 1991-05-02 1992-11-12 Sbp Technologies, Inc. Decomposition microbienne de trichlorethylene, de dichlorethylenes et de polluants aromatiques
JPH10327850A (ja) * 1997-05-29 1998-12-15 Res Dev Corp Of Japan 微量要素・無機栄養塩類拡散型菌体培養用担体
JPH11341976A (ja) * 1998-06-01 1999-12-14 Japan Science & Technology Corp 微量要素を抱括した菌体増殖用担体

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992019738A1 (fr) * 1991-05-02 1992-11-12 Sbp Technologies, Inc. Decomposition microbienne de trichlorethylene, de dichlorethylenes et de polluants aromatiques
JPH10327850A (ja) * 1997-05-29 1998-12-15 Res Dev Corp Of Japan 微量要素・無機栄養塩類拡散型菌体培養用担体
JPH11341976A (ja) * 1998-06-01 1999-12-14 Japan Science & Technology Corp 微量要素を抱括した菌体増殖用担体

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"Structure analysis of a class II transposon encoding the mercury resistance of the Gram-positive bacterium Bacillus megaterium MB1, a strain isolated from Minamata Bay, Japan", Gene, Vol. 234, pages 361-369 (1999). *

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