JP3227488B2 - Purification method for mercury pollutants - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for purifying water or soil contaminated with mercury using a recombinant microorganism.

[0002]

2. Description of the Related Art Mercury has been found to cause environmental pollution in various parts of the country, such as Minamata Bay, and methods for purifying it have been studied.
In order to purify the environment, decomposing and removing mercury compounds that cause pollution is essential for a fundamental solution. Conventionally,
The restoration of the environment contaminated by mercury compounds has been carried out by excavating contaminated soil and cultivating uncontaminated soil, or reclaiming land like Minamata Bay. However, excavation or landfill methods are very expensive, and they are not a radical solution because they do not decompose and remove mercury compounds.

On the other hand, it is known that a mercury compound is decomposed and reduced to metallic mercury by the action of microorganisms.

[0004]

However, the ability of such microorganisms to reduce mercury compounds is insufficient for efficiently decomposing and removing mercury compounds in mercury-contaminated water and soil. For this reason, there is a demand for a treatment method with a higher ability to decompose and remove mercury compounds.

An object of the present invention is to provide a method for purifying mercury contaminants by using microorganisms efficiently under such circumstances.

[0006]

Means for Solving the Problems As a result of intensive studies, the present inventor has found that it is effective to utilize a microorganism incorporating a mercury compound reductase gene group derived from Escherichia coli together with a thiol compound. Reached.

[0007] The present invention provides: (1) the mercury contaminants, a method for purifying a recombinant microorganism obtained by introducing mercury compounds reductase genes, the
The recombinant microorganism is Pseudomonas putida PpY101
/ PSR134 and a method for purifying mercury contaminants, wherein a thiol compound coexists in the system. (2) The method according to the above (1), wherein the thiol compound is thioglycolic acid, a salt thereof or mercaptoethanol. (3) The method for purifying mercury contaminants according to (1) or (2), wherein the mercury contaminants are mercury-contaminated soil.

[0008] The microorganism used in the present invention is a recombinant plasmid pSR obtained by incorporating a mercury compound reductase gene group into a broad host range vector pSUP104.
Putida This is introduced into microorganisms belonging to the genus Pseudomonas such as the PpY101 strain. This recombination can be performed, for example, by the following method. That is, Escherichia coli JE51 in which the mer operon of the mercury compound degrading enzyme gene group is encoded.
The plasmid NR1 derived from Escherichia coli is isolated and partially digested with a restriction enzyme EcoRI to purify a sample containing a DNA fragment encoding the mer operon. Then, this is inserted into the EcoRI site of the broad host range vector pSUP104 to prepare a recombinant plasmid pSR134. This recombinant plasmid is transformed into a microorganism belonging to the genus Pseudomonas, for example, Pseudomonastida PpY101.
Introduced into the strain by the electric pulse method. Thus 100
It is possible to prepare a recombinant microorganism having a remarkably high ability to decompose mercury compounds, which can be proliferated even on a plate medium containing mg / l mercuric chloride. And this recombinant microorganism is 10
It is capable of almost completely decomposing mercuric chloride at a high concentration of 0 ppm in one day. The recombinant microorganism Pseudomonas putida PpY101 / pSR134 is described in Biosci. Biotech. Bioche
m. , 57 (8), 1264-1269, 1993.

Similarly, Pseudomonas putida P
RS 2000, P.S. Fluorescence (fluor)
escens) LB303 strain, P. Arginosa (ae
ruginosa) PA01 strain, Escherichia coli HB101 strain, etc., and the above-mentioned pSR134 for microorganisms belonging to the genus Pseudomonas.
Can be introduced by an electric pulse method or the like. In fact, the present inventors have confirmed introduction into these hosts by the method shown below.

[0010] The recombinant plasmid pSR134 has the ability to take in mercuric chloride existing outside the cell into the cell and decompose it into mercury metal. The metallic mercury generated at this time becomes vapor and is removed out of the system. Whether various recombinants have this ability can be qualitatively confirmed by an x-ray film method.

[0011] The microorganism to be tested is placed in a microplate and covered with an X-ray film from above. When the metallic mercury vapor is generated, it reacts with the emulsion (silver) of the x-ray film and is exposed. By this method, the ability of various recombinants and their hosts to convert mercury was compared. As a result, in the strains into which the recombinant plasmid pSR134 had been introduced, exposure to x-ray film was clearly observed, and it was confirmed that these strains had the ability to convert mercury. But,
No photosensitization was observed in the non-recombinant.

In order to confirm the change in the properties of the obtained recombinants, the specific growth rate, the resistance to mercury chloride and the resolution of mercury chloride between the recombinant and its host were also compared.
Table 1 shows the results. It was found that the growth rate of the recombinant was slightly slower in all strains. Also,
The results of examining the resistance to mercury chloride and tetracycline derived from the recombinant plasmid pSR134 showed that all the recombinants had improved resistance to these.

The present inventors have inferred the mercury reduction mechanism using the microorganism of the present invention as follows. That is, first, the SH compound binds to divalent mercury ions generated from an inorganic mercury compound such as mercuric chloride in an aqueous solution,
This mercury-SH compound complex is metabolized by the action of the mercury reductase in the cells and is decomposed into metallic mercury. Accordingly, the thiol compound is essential for the mercury reduction reaction by the microorganism used here. On the other hand, mercuric chloride in the soil is adsorbed on the soil particles, and in order to be purified by microorganisms, mercury ions must be eliminated. The above thiol compound not only promotes the decomposition reaction of the mercury compound by the microorganism, but also has an action of effectively releasing mercury from the soil.

The specific growth rate, mercury chloride tolerance and mercury chloride degradability of the various recombinants obtained and their hosts were compared. It was found that the growth rate of the recombinant was slightly slower in all strains. Recombinant plasmid pSR1
As a result of examining the resistance to mercury chloride and tetracycline derived from No. 34, it was shown that all the recombinants had improved resistance to these.

[0015]

[Table 1]

In the present invention, when purifying a mercury contaminant using the above-mentioned microorganism having a reducing ability of a mercury compound, it is important to coexist a thiol compound in order to enhance the reducing ability.

The thiol compound is not particularly limited as long as it has a thiol group, but is preferably a water-soluble thiol compound. Particularly preferred are water-soluble thioglycolic acid, alkali metal salts of thioglycolic acid, and sulfur-containing amino acids such as mercaptoethanol and cysteine. The present inventors infer the mercury reduction mechanism using the microorganism of the present invention as follows. That is, first, a thiol compound binds to divalent mercury ions generated from an inorganic mercury compound such as mercuric chloride in an aqueous solution, and this mercury-thiol compound complex is metabolized by the action of mercury reductase in the cells, Decomposes into metallic mercury. Accordingly, the thiol compound is essential for the mercury reduction reaction by the microorganism used here. In addition,
The mechanism of action of thiol compounds is unknown in detail. On the other hand, mercuric chloride in the soil is adsorbed on the soil particles, and in order to be purified by microorganisms, mercury ions must be desorbed. In addition to promoting the action, it has the effect of effectively desorbing mercury ions from the soil.

Table 2 below shows the measured values of the Hg + ² concentration when a predetermined concentration of a thiol compound was added to a slurry in which 10 g of soil (solid content) containing 54 mg of mercuric chloride was dispersed in 100 g (ml) of water. It is. Table 2 shows that mercuric chloride adsorbed on the soil particles is desorbed by the addition of the thiol compound to release mercury ions.

[0019]

[Table 2]

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION As described above, by introducing a broad host range vector incorporating a mercury compound reductase gene group as described above, the microorganisms to be treated from various microorganisms can be used. It has the feature that it is possible to select microorganisms suitable for ecosystems, and it can be applied to various soil environments.

The method of the present invention can of course be applied to purification of mercury-contaminated water, but is particularly suitable for purification of mercury-contaminated soil.

[0022]

The present invention will be described in more detail with reference to the following examples.

Preparation of Recombinant Escherichia coli JE encoding the mer operon of mercury degrading enzyme gene group
The plasmid NR1 from 51 is isolated and the mer operon is purified. This is then transformed into the broad host range vector pSUP1.
04 to produce the recombinant plasmid pSR134. This recombinant plasmid was introduced into Pseudomonas putida PpY101 strain by the electric pulse method. Culture method of bacterial cells 100 ml of L nutrient medium was added to a 500 ml culture device,
Mercuric chloride is added at a concentration of 20 ppm. A recombinant microorganism Pseudomonas grown on the same plate medium
Inoculate with Putida PpY101 / pSR134. 30
Shake culture at 20 ° C for 20 hours. After the culture, the cells are collected by centrifugation and used for removing mercury compounds.

Example 1 Decomposition and Removal of Contaminated Soil (Example 1) Recombinant microorganism Pseudomonas putida PpY101 / pSR134 cultured in a phosphate buffer solution containing 40 ppm of mercuric chloride as described above [Accession No. FERM P-16495] Was sprayed at 5 × 10 ⁶ per ml of buffer solution, and 1 mM of thioglycolic acid was added as a thiol compound. After 24 hours, mercuric chloride in the buffer solution was completely removed. On the other hand, no removal was observed in the system to which thioglycolic acid was not added.

(Example 2) A recombinant microorganism Pseudomonas putida PpY101 / p cultured in a paddy soil slurry contaminated with 40 ppm of mercuric chloride as described above.
When 5 × 10 ⁶ SR134 and 50 μM thioglycolic acid were applied simultaneously per 1 g of soil, about 90% of mercuric chloride in the solution was removed after 4 hours. On the other hand, no removal was observed in the system to which the recombinant microorganism was not added.

[0026]

As described above, according to the present invention, soil and water contaminated with a mercury compound such as an inorganic mercury compound can be efficiently purified, and the microorganism to be used is a mercury compound reductase. It is more advantageous to introduce a broad host range vector into which the gene group has been incorporated, since it is possible to select a microorganism suitable for the ecosystem of the soil to be treated.

Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI // (C12N 1/21 B09B 3/00 ZABE C12R 1:40) C12N 15/00 A (56) References JP-A-10-229873 (JP) , A) PLASMID 27 p. 4-16 (1992) Biosci. Biotechnol l. Biochem. 57 (8) p. 1264-1269 (1993) (58) Fields investigated (Int. Cl. ⁷ , DB name) B09B 3/00 C12N 15/00 C12N 1/21 MEDLINE (DIALOG)

Claims (3)

(57) [Claims] 1. A method for purifying mercury contaminants using a recombinant microorganism into which a mercury compound reductase gene group has been introduced, wherein the recombinant microorganism is Pseudomonas putida Pp.
A method for purifying mercury contaminants , which is Y101 / pSR134, and wherein a thiol compound coexists in the system. 2. The method for purifying mercury contaminants according to claim 1, wherein the thiol compound is thioglycolic acid, a salt thereof or mercaptoethanol. 3. The method for purifying mercury contaminants according to claim 1, wherein the mercury contaminants are mercury contaminated soil.