JP5066901B2 - Method for culturing vinyl chloride-degrading bacteria, groundwater and / or soil purification agent and purification method - Google Patents

Description

  The present invention relates to a method for culturing chlorinated ethylene-degrading bacteria, especially vinyl chloride-degrading bacteria. The present invention also relates to a purification agent and a purification method for groundwater and / or soil contaminated with chlorinated ethylene.

  With the development of science and technology in recent years, contamination of groundwater and soil has become a problem as various substances including harmful and hardly decomposable chemical substances are used. In particular, contamination by chlorinated ethylene such as tetrachloroethylene, trichlorethylene, cisdichloroethylene, and vinyl chloride is serious. That is, chlorinated ethylene has low water solubility and is chemically stable, so once released into the environment, it remains and accumulates in soil and groundwater for a long period of time.

  Against this background, there is an urgent need to purify groundwater and soil contaminated with chlorinated ethylene. In particular, bioremediation using anaerobic bacteria has attracted attention as an inexpensive purification method. Examples that have already been put to practical use have been reported in Europe and the United States, and efforts toward practical use are also active in Japan (see Non-Patent Document 1).

  In order to obtain microorganisms to be used for bioaugmentation, it is usually necessary to screen microorganisms having the intended function from the environment. In obtaining microorganisms with chlorinated ethylene degradability, enrichment culture is performed using a medium containing an organic substance as a carbon source and chlorinated ethylene as a respiratory substrate (electron acceptor), and further subculture is continued. To do. Examples of organic substances that can be generally used include organic acids such as lactic acid, acetic acid, propionic acid, and citric acid, alcohols such as methanol, saccharides, and proteins.

  By the way, there are many microorganisms that can assimilate such organic substances in the environment. For this reason, in the process of subculture after enrichment culture and the like, there is a concern that various microorganisms that grow quickly and become dominant in the system instead of bacteria having chlorinated ethylene decomposing ability. In such a case, it is difficult to accurately grasp the constituent species of the microbial group and maintain the proportions of the various groups, and thus to control the quality of the integrated culture used for bioaugmentation.

  Therefore, it is desirable that the microorganism used for bioaugmentation is an enriched culture in which bacteria having chlorinated ethylene degradability are purified or highly dominant. In fact, even in Non-Patent Document 1, it is desirable that the microorganisms used for bioaugmentation satisfy the condition that “they are composed of highly limited microorganisms and that the structure is continuously stable”. It is written.

  By the way, the degradation of chlorinated ethylene by anaerobic bacteria is generally performed reductively in the order of tetrachloroethylene, trichloroethylene, cis-1,2-dichloroethylene, vinyl chloride, and ethylene. Here, since vinyl chloride has been proven to have carcinogenicity, it is important to decompose it to ethylene. Only Dehalococcoides bacteria have been reported as anaerobic bacteria that can degrade vinyl chloride to ethylene.

However, among the bacteria belonging to the genus Dehalococcides, those having different degradation characteristics are mixed, such as those that do not have vinyl chloride resolution or those that have resolution but have a very slow degradation rate. . For this reason, establishment of the technique which can selectively accumulate dehalococcides genus bacteria which have the outstanding vinyl chloride resolution | decomposability is desired.
“Guidelines for the Use of Bioremediation by Microorganisms” Ministry of Economy, Trade and Industry and Ministry of the Environment, March 30, 2005

  However, as a method for preparing a microorganism used for bioaugmentation targeting chlorinated ethylene, a method that satisfies the above-mentioned conditions has not been established yet. In addition, there is no report on a technique capable of selectively accumulating microorganisms of the genus Dehalococcides having excellent vinyl chloride resolution.

  The present invention has been made in view of the above circumstances, a culture method capable of selectively accumulating bacteria belonging to the genus Dehalococcides having excellent vinyl chloride resolution, and contamination with chlorinated ethylene using this culture method. It is an object of the present invention to provide a purification agent and a purification method for groundwater and / or soil.

  The present inventors use a formic acid as a carbon source of a medium when culturing a bacterium belonging to the genus Dehalococcides using groundwater or soil contaminated with chlorinated ethylene as a planting source, thereby reducing vinyl chloride resolution. The present inventors have found that it is possible to easily and highly accumulate the dehalococcides bacteria that they have, and have completed the present invention. Specifically, the present invention provides the following.

  (1) A method for culturing vinyl chloride-degrading bacteria, comprising an accumulation procedure for culturing a bacterial group containing vinyl chloride-degrading bacteria in an accumulation medium containing formic acid and / or a salt thereof as a main carbon source.

  (2) The culture method according to (1), wherein the accumulation medium contains 1 mM or more and 5 mM or less of formic acid and / or a salt thereof.

  (3) The culture method according to (1) or (2), wherein the accumulation medium is a liquid medium in which hydrogen gas is added to a gas phase.

  (4) The bacterial group is prepared by culturing a seed source containing groundwater and / or soil contaminated with chlorinated ethylene under conditions to which neither formic acid or its salt is added. The culture method according to any one of (1) to (3), further comprising a preparation procedure before the accumulation procedure.

  (5) The culturing method according to (4), further comprising a filtration procedure for filtering gonococci from the bacterial group prepared in the preparation procedure.

  (6) A purification agent for groundwater and / or soil contaminated with chlorinated ethylene, comprising vinyl chloride-degrading bacteria obtained by the culture method according to any one of (1) to (5).

  (7) A method for purifying groundwater and / or soil contaminated with chlorinated ethylene, the method comprising purifying the groundwater and / or soil with the purifying agent according to (6).

  (8) Accumulating bacteria belonging to the genus Dehalococcides having vinyl chloride resolution by culturing a group of bacteria containing vinyl chloride-degrading bacteria under medium conditions using formic acid and / or a salt thereof as a main carbon source. Integration method.

  According to the present invention, a procedure for culturing a group of bacteria containing vinyl chloride-degrading bacteria in an accumulation medium containing formic acid and / or a salt thereof as a main carbon source is provided, so that microorganisms of a type having poor vinyl chloride resolution are eliminated. Is done. Therefore, it is possible to selectively accumulate dehalococcides bacteria having excellent vinyl chloride resolution.

  Hereinafter, although embodiment of this invention is described, this invention is not specifically limited to this.

<Culture method>
The culturing method of the present invention is characterized by having an accumulation procedure for culturing a bacterial group containing vinyl chloride-degrading bacteria in an accumulation medium containing formic acid and / or a salt thereof as a main carbon source.

  As the bacterial group, those already prepared may be used, or freshly prepared from a seed source including groundwater and / or soil contaminated with chlorinated ethylene. In the latter case, the culture method of the present invention has a preparation procedure for preparing a bacterial group before the accumulation procedure, and further includes a filtration procedure for filtering the koji mold from the bacterial group prepared in this preparation procedure. Also good.

  Hereinafter, the procedure of the culture method of the present invention will be described in time series.

[Preparation procedure]
The preparation procedure prepares bacterial groups by culturing a seed source containing groundwater and / or soil contaminated with chlorinated ethylene under conditions to which neither formic acid or its salt is added. It is a procedure.

  Here, “chlorinated ethylene” means a compound in which at least one of hydrogen atoms of ethylene is substituted with a chlorine atom, and includes tetrachloroethylene, trichloroethylene, cis-1,2-dichloroethylene, and vinyl chloride. .

  Groundwater and soil contaminated with chlorinated ethylene are preferably collected from contaminated sites where decontamination of bacteria belonging to the genus Dehalococcides is proliferating in the process of purification by biostimulation. .

  Dehalococcides bacteria are obligately anaerobic microorganisms and therefore die quickly in the presence of oxygen. Therefore, it is preferable to maintain anaerobic conditions in a series of operations such as collection and transfer of groundwater and soil, addition to culture vessels, and the like.

  First, groundwater and / or soil contaminated with chlorinated ethylene is put into a culture vessel. When groundwater is used as a seeding source, it may be put into a culture vessel as it is, or after diluting with water or the like. Moreover, when using soil as a seeding source, it may be put into a culture vessel as it is and suspended in a liquid medium, or it may be put into a culture vessel after being suspended in a suitable solvent (for example, water).

  In consideration of the fact that the culture medium contains chlorinated ethylene, the culture vessel is preferably made of glass. When the seed source is input, citric acid, yeast extract and the like are further added as organic nutrient sources. The concentration of citric acid is preferably 100 to 1000 mg / L, and the concentration of yeast extract is preferably 10 to 100 mg / L.

  In order to make the culture environment anaerobic, sodium sulfide and ferrous chloride are preferably added. The concentration of sodium sulfide and ferrous chloride in the medium is preferably 5 mg / L to 500 mg / L, respectively. In addition, anaerobicization of the internal environment of the culture vessel may be performed by deaeration or by filling the inside of the culture vessel with nitrogen gas or a mixed gas of nitrogen gas and carbon dioxide.

  After the culture vessel is sealed with a lid, the culture is started. The lid is preferably a butyl rubber stopper covered with polytetrafluoroethylene.

  The culture is performed by standing, and the temperature is preferably 15 to 30 ° C, more preferably 25 to 30 ° C. The culture is performed until all the cis-1,2-dichloroethylene contained in the medium is decomposed into ethylene, that is, until no cis-1,2-dichloroethylene and vinyl chloride are detected. The detection of cis-1,2-dichloroethylene and vinyl chloride is performed, for example, by collecting a small amount of the gas phase in the culture vessel and performing gas chromatography analysis.

  After completion of the culture, the culture solution is transplanted into another culture container in which a subculture medium is accommodated. This medium contains citric acid, yeast extract, sodium sulfide, and ferrous chloride as well as the above-mentioned additive components, and also contains predetermined inorganic salts.

  Alternatively, the medium contains organic acids (excluding formic acid) such as lactic acid, acetic acid, propionic acid, butyric acid, salts thereof (excluding formic acid salts), alcohols such as methanol, sugars such as sucrose, and the like. Also good. Further, cis-1,2-dichloroethylene is added to the culture vessel, and the concentration thereof is preferably 1 to 100 mg / L (10 μM to 1 mM), more preferably 1 to 10 mg / L (10 to 100 μM). .

  Resazurin, which is an indicator for examining anaerobic degree, may be added to the medium. The pH of the medium is preferably in a neutral range of about 6.0 to 8.5.

  In addition, the density | concentration of the culture medium component mentioned above is a density | concentration at the time of culture | cultivation. For this reason, it is preferable that the culture medium before adding groundwater has a higher concentration in consideration of the decrease in concentration due to dilution with the seed source.

  The subculture is performed under the same conditions as in the previous culture until neither cis-1,2-dichloroethylene nor vinyl chloride is detected. The detection of cis-1,2-dichloroethylene and vinyl chloride is performed, for example, by collecting a small amount of the gas phase in the culture vessel and performing gas chromatography analysis. By continuously performing such subculture, a bacterial group containing vinyl chloride-degrading bacteria is prepared.

[Filtering procedure]
The filtration procedure is a procedure for filtering the koji mold from the culture solution containing the bacterial group prepared in the preparation procedure. The filtrate obtained by filtration is one that contains a bacterial group from which gonococcus has been removed and includes vinyl chloride-degrading bacteria, and is used in the following accumulation procedure. The filtration is performed by ultrafiltration of the culture solution using a filter having a predetermined pore size. The pore diameter is not particularly limited, but is preferably about 0.45 μm, for example.

[Assembly procedure]
The accumulation procedure is a procedure in which a bacterial group including vinyl chloride-degrading bacteria is cultured in an accumulation medium containing formic acid and / or a salt thereof as a main carbon source.

  Here, the “main carbon source” means that a carbon source other than formic acid and / or a salt thereof is not contained to the extent that it affects the growth of microorganisms. That is, as long as it does not affect the growth of microorganisms, other carbon components may be contained. However, it is preferable that the carbon source consists only of formic acid and / or a salt thereof in terms of more effectively removing bacteria belonging to the genus Dehalococcides that are inferior in vinyl chloride resolution.

  One or both of formic acid and a formic acid salt may be added, but formic acid salt is preferred in order to maintain the pH of the accumulation medium in a neutral range. The salt of formic acid is not particularly limited as long as it does not inhibit the growth of the genus Dehalococcides, but may be, for example, sodium formate.

  The concentration of formic acid and / or a salt thereof is usually preferably 1 mM or more and 5 mM or less in consideration of the removal effect of dehalococcides bacteria inferior in vinyl chloride resolution and cost. However, if hydrogen gas as an electron donor is added to the gas phase of the medium, the concentration of formic acid and / or its salt may be about 0.1 mM.

  About components other than a carbon source, you may be the same as the culture medium for the subculture mentioned above. Moreover, culture is performed under the same conditions, and planting is performed a predetermined number of times. The number of times of planting is not particularly limited, but if it is too small, there may be cases where Dehalococcide spp. Bacteria that are inferior in vinyl chloride resolution cannot be removed sufficiently, and if too large, the removal effect is saturated and economical. Not. Therefore, it is usually preferred to be about 3 to 10 times.

  The culture broth thus obtained is in a state in which a dehalococcide bacterium having excellent vinyl chloride resolution is highly dominant.

<Purifying agent>
The purification agent of the present invention is a purification agent for groundwater and / or soil contaminated with chlorinated ethylene, and is characterized by containing vinyl chloride-degrading bacteria obtained through an accumulation procedure. The vinyl chloride-degrading bacterium contained in the cleaning agent may be in the form of a culture solution obtained through an accumulation procedure, or may be in an appropriately processed form. However, it should be managed and used under anaerobic conditions in order to maintain the predominance of the genus Dehalococcides.

  This purification agent is in a highly prevalent state of the genus Dehalococcides with excellent vinyl chloride resolution, so that the quality of purifying groundwater and / or soil contaminated with chlorinated ethylene is raised to a high level. Can hold.

<Purification method>
The purification method of the present invention is a method for purifying groundwater and / or soil contaminated with chlorinated ethylene, characterized in that it has a procedure for introducing the above-mentioned purification agent into groundwater and / or soil. The groundwater and / or the soil may be in-situ, or may be pumped or excavated from the in-situ.

  The amount of the purification agent to be introduced into the groundwater and / or the soil, the input speed, and the like may be appropriately set according to the contamination status of the contaminated groundwater and / or the soil.

  Groundwater was collected from a site where the purification of soil and groundwater contaminated with chlorinated ethylene was performed by biostimulation. 100 mL of this groundwater was put into a glass vial having a volume of 155 mL, the medium components shown in Table 1 were added, and the gas phase was replaced with a nitrogen / carbon dioxide mixed (volume ratio 8/2) gas. Further, cDCE was added so that the concentration in the solution was about 6 mg / L, and then the vial was sealed with a butyl rubber stopper coated with polytetrafluoroethylene.

  Then, this vial was left still in a 30 degreeC thermostat, and culture | cultivation was started. 200 μL of the gas phase in the vial was periodically withdrawn, and the concentrations of cisdichloroethylene (cDCE), vinyl chloride (VC), and ethylene were measured using a gas chromatograph. After confirming that cisdichloroethylene and vinyl chloride were no longer detected, the aqueous phase of the medium was collected. 1 mL of the collected aqueous phase was transferred to another vial containing a synthetic medium having the composition shown in Table 2. In the same procedure, subculture was performed in which transplantation to the synthetic medium was performed a total of 5 times.

  Bacterial phase of the obtained culture broth was analyzed for terminal-restriction fragment length polymorphism (T-RFLP) analysis of 16S rDNA, 16S rDNA of dehalococcides genus bacteria and all bacteria Real-time PCR analysis and 16S rDNA clone analysis. As a result, it has been found that the proportion of the genus Dehalococcides in the total bacteria is very low, about 1%. Therefore, in order to accumulate the Dehalococcides bacteria more highly, the culture was continued by the following procedure.

  First, the culture solution after subculture was filtered with a filter having a pore size of 0.45 μm to remove most of the koji molds. It was confirmed that the filtrate retained the vinyl chloride decomposition activity. Moreover, when the composition of the filtrate was analyzed, the presence of a small amount of formic acid as well as acetic acid was confirmed as a decomposition product of citric acid. Therefore, it was decided to further subculture using a synthetic medium containing acetic acid and formic acid as organic substances. The medium components were basically the same except that sodium formate and sodium acetate were added to a concentration of 5 mM (corresponding to 295 mg / L and 225 mg / L, respectively) instead of sodium citrate and yeast extract. As shown in Table 2.

  Further, as a carbon source in the culture solution, a medium added with citric acid and yeast extract instead of sodium formate and sodium acetate and a medium added with xylose were used as comparative examples.

  Each culture medium was subcultured 5 times after planting, and then analyzed by denaturing gradient gel electrophoresis (DGGE) targeting 16SrDNA. The main band observed on the gel is cut out, and the result of determining the base sequence is also shown in FIG. Lane A in FIG. 1 represents an example in which citric acid and yeast extract were added, Lane B represents an example in which xylose was added, and Lane C represents an example in which sodium formate and sodium acetate were added.

  As shown in FIG. 1, many main bands were observed in Lane A, but the number of main bands was reduced in Lane B and Lane C. That is, it was found that a small number of microorganisms are selectively accumulated by using xylose or sodium formate and sodium acetate as a carbon source.

  As a result of the nucleotide sequence determination, each band indicated by an arrow is a Dehalococcides sp. GT strain (Appl. Environ. Microbiol., Vol. 72, 1980-1987, 2006); It had 99% or more homology with the ethenegenes 195 strain (Science, vol. 276, 1568-71, 1997). Therefore, focusing on these bands, only 195 strains were observed in lane B, but both GT strain and 195 strain were observed in lane C.

  The GT strain has been reported to have vinyl chloride resolution and the 195 strain has no (or very low) vinyl chloride resolution. In view of this, when xylose is used as a carbon source, 195 strain-type dehalococcides bacteria having no vinyl chloride resolution are accumulated, while sodium formate and sodium acetate are used as a carbon source. It was found that the bacteria can be accumulated including dehalococcides bacteria having excellent vinyl chloride resolution.

  Next, it was decided to conduct further studies with the aim of more selectively accumulating GT strain type dehalococcides bacteria.

  First, the enrichment culture obtained by the culture using the above-mentioned sodium formate and sodium acetate is used as a seed source, and as a carbon source, sodium acetate alone (5 mM), sodium formate alone (5 mM), sodium formate and sodium acetate Culturing was carried out in three types of synthetic media containing both (5 mM each). DGGE analysis was performed on the culture solution obtained 5 times after transplanting. The main band observed on the gel is cut out, and the results of determining the base sequence are shown together in FIG. In FIG. 2, lane A is an example in which only acetic acid is added, lane B is an example in which only formic acid is added, and lane C is an example in which formic acid and acetic acid are added.

  As shown in FIG. 2, in lane A and lane C, not only the GT strain but also the band of 195 strain is observed with almost the same density, while in lane B, the band of the GT strain is much darker. At the same time, the band of 195 shares was extremely thin.

  Furthermore, DNA was extracted from the culture solution obtained by the culture using sodium formate, and quantitative PCR analysis for 16SrDNA was performed. Then, the ratio of 16S rDNA of the genus Dehalococcides to the 16S rDNA derived from all bacteria is about 24%, which is much higher than the ratio obtained by culturing with citric acid and yeast extract (about 1% as described above). It was confirmed that the value was high.

  From the above results, it was found that by culturing in an accumulation medium containing sodium formate as the main carbon source, the GT strain type dehalococcides genus bacteria having excellent vinyl chloride resolution can be selectively accumulated.

  There have been no reports of pathogenicity, carcinogenicity, toxin production, etc. for Dehalococcides bacteria. For this reason, it is expected that the purifying agent in which dehalococcides bacteria having excellent vinyl chloride resolution are selectively accumulated can be safely used in bioaugmentation.

It is a figure which shows the result of one test example of this invention. It is a figure which shows the result of one Example of this invention.

Claims (1)

The bacteria containing and vinyl chloride degrading bacteria obtained using the inoculum containing contaminated groundwater and / or soil by chlorinated ethylene, formic acid and / or media conditions to a salt thereof as a main carbon source An accumulation method for accumulating bacteria belonging to the genus Dehalococcides having vinyl chloride resolution by culturing them originally.