JPH08141549A - Soil restoration method - Google Patents

Abstract

PURPOSE: To strengthen a decomposition treatment capacity for halogenated organic acids by inoculating soil polluted with the acids with a microorganism having dehalogenase which decomposes the acids hydrolysiswise. CONSTITUTION: In the decomposition process of halogenated organic acids which are a problem as byproducts in the sterilization and disinfection of water, especially the acids contained in soil, the soil is inoculated with a microorganism having dehalogenase which decomposes the acids hydrolysiswise. A microorganism of a renobacter group, preferably a renobacter species AC stock, is used as the microorganism. The halogenated organic acids include halogenoacetic acids and halognopropionic acids; the halogenoacetic acids include chloroacetic acid, dichloroacetic acid, and trichloroacetic acid; the halognopropionic acids include chloropropionic acid and dichloropropionic acid. The method is applicable to the soil restoration of both closed and open systems and can be implemented by a batch process, etc.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a soil remediation method using a novel microbial strain. More particularly, it relates to a method for remediating soil contaminated with halogen-substituted organic acids.

[0002]

2. Description of the Related Art Since a report by the United States EPA (Environmental Protection Agency) in the 1970s, sterilization and disinfection by-products have become a big problem in both sewage and tap water. Disinfection with chlorine is obligatory in Japan, but by-products such as trihalomethanes, haloacids, haloacetonitriles,
Substances such as haloketones have been confirmed, and their hepatotoxicity,
It is a very big problem due to its mutagenicity. Among them, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, which were taken up as environmental monitoring items in 1993,
Halogen-substituted organic acids such as bromoacetic acid (hereinafter referred to as halo acids) have been highlighted as a new problem. These are the 23rd Japan Society on Water Environment Seminar /
It is described in detail in the analytical method accompanying the revision of water quality environmental standards (Japanese Society for Water Environment) p55-p64 (November 1993).

Since such a halo acid has high water solubility, it remains in the soil through the environment in which water serves as a solvent.
It is spreading and becoming a huge problem.

Thus, the treatment of halo acids remaining in soil is very difficult by the conventional technique, and a very effective means for treating soil contaminated with volatile organochlorine compounds such as tetrachlorethylene and trichlorethylene. The vacuum extraction method and the like which are Degradation treatment is considered as an effective treatment method, but biodegradation treatment using microorganisms can be performed under extremely mild conditions, costs are relatively low, and harmful chemical substances can be introduced. Since it is not available, it is expected to be a very effective means.

For example, microorganisms that decompose halo acids include Trichoderma , Acrostalagmus , Penicillium , Clono.
Molds such as stachys , Pseudomonas , Arthrobacter ,
Rhizobium , Agrobacterium , Bacillus , Alcaligenes ,
Nocardia , Micrococcus , Achromobacter , Moraxella
(Above, protein nucleic acid enzyme, 29, 101-110 (198
Bacteria such as 4)) are being studied. In addition, Adachi showed that the unidentified bacterium OS-2 strain has an enzyme that decomposes chloroacetic acid, bromoacetic acid, and iodoacetic acid to almost the same degree, and dichloroacetic acid also decomposes although it has about half the activity. (Osaka Prefectural Public Health Institute, Public Health Edition, No. 30, 89)
(1992)). Also, Pseudomonas putida NCIMB 120
Studies have also been conducted to fix the dehalogenase extracted from 18 on carboxymethylcellulose or thioglycolic acid to decompose haloacids having 2 to 6 carbon atoms (European Patent No. 179603). Other than that, regarding the relationship between haloacid dehalogenase and genes, Pseudomonas putida AJ1
Strain (J. Gen. Microbiol., 138,675 (1992)), Pseudomona
s cepacia MBA4 strain (J. Biochem., 284,87 (1992)),
Pseudomonas sp. CBS3 strain (Biol. Chem. Hoppe-Seyl
er., 374,489 (1993)).

[0006] However, all of these are evaluations of the activity at the enzyme level, and it is the current situation that there is no knowledge of how these microorganisms actually behave in contaminated wastewater. Xant for haloacid decomposition in microorganisms themselves
hobacter autotrophicus GJ10 strain (Appl. Biochem. B
iotechnol., 40,158 (1993)) and 40,165 (1993)) have only been studied.

Regarding the decomposition of halopropionic acid, D
It has been reported that the dehalogenase extracted from Pseudomonas bacteria of type L and L-type chloropropionic acid decomposes to lactic acid (Japanese Patent Laid-Open No. 64-64544), but this case is also only an enzyme level study.

Moreover, from the viewpoint of satisfying various practical conditions and having sufficient decomposing ability when used in a method for treating haloacid-contaminated soil using microorganisms, the range of currently known bacterial species is Is not always sufficient. Therefore, at present, there is a strong demand for the acquisition of bacterial strains that satisfy the characteristics required in practical use.

As a property of such a bacterial species, of course, it has a sufficient ability to decompose haloacids, but its growth range is different from that of known bacterial species and its application range can be expanded, or its utilization forms are abundant. Is more preferable.

For example, in the case of treating a waste liquid containing dichloroacetic acid, it is required that the microorganism to be applied is capable of growing in an adverse environment such as soil and maintaining its degrading activity, in addition to the ability to decompose dichloroacetic acid. .

Thus, it has sufficient haloacid degradability,
Further, there is a strong demand for bacterial species having practically advantageous properties over conventionally known bacterial species.

[0012]

DISCLOSURE OF THE INVENTION An object of the present invention is to decompose a halo acid using a powerful novel microorganism for decomposing the halo acid, which is a problem as a sterilization / disinfection by-product. Particularly, it is to provide a method for decomposing halo acids contained in soil.

[0013]

The above object can be achieved by the present invention described below.

That is, as a result of searching the bacterial species decomposing halo acids from the above viewpoints, the present inventors have found that new bacteria having a high concentration of halo acid degrading activity can be obtained from the soil of the Kanto loam layer in Japan. We obtained a seed, introduced this strain into soil, and found a method to decompose haloacids in the soil.

First, the mycological properties of the strain newly obtained by the present invention are shown below.

A. Morphological properties Gram staining: Negative cell size and shape: C- and / or S-shaped rods with a length of 1.0 to 2.0 μm and a width of 0.2 to 0.5 μm Motility: None Color: White to cream B. Growth status in various media BHIA: Good growth MacConkey: Bad growth C.I. Optimal growth temperature: 25 ° C to 35 ° C D. Physiological properties Distinction between aerobic and anaerobic: Aerobic TSI (slant / butt): Alkali / Alkali, H ₂ S
(−) Oxidase: Positive Catalase: Positive (identification standard: according to Bergey's Manual (1984)) From the above properties, this strain is a Renovobacter species.
( Renobacter sp .) Was found to be suitable.

Further, as is clear from the examples described later, this strain has excellent halo acid degrading ability. Since a haloacid-degrading bacterium of a strain belonging to Renovobacter has not been known so far, this bacterium was identified as a new strain, and a Renovobacter sp . AC strain ( Renobacter sp .
Strain AC), and deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology (accession number: FERM P-1464).
1).

The AC strain has a very specific growth morphology in addition to the morphology of the cell itself (S-shape), and the bacterium itself secretes some high molecular substance and grows as a lump of cells. Microscopically, such a property promptly forms a habitat (habitat) unique to the AC strain and grows as a priority species, so that it grows in soil where various other indigenous bacteria are present. Therefore, when the halo acid is decomposed, it is very advantageous.

The bacterium can be cultivated in a natural complete medium such as a usual 2YT medium or LB medium, but it can also be cultivated in an inorganic salt medium such as M9 medium to which yeast extract is added as a slight nutrient. It is possible.

The composition of M9 medium is shown below.

Na ₂ HPO ₄ : 6.2 g KH ₂ PO ₄ : 3.0 g NaCl: 0.5 g NH ₄ Cl: 1.0 g (in medium 11; p
H7.0) The culture can be performed under aerobic conditions, and may be liquid culture or solid culture. The culture temperature is preferably around 30 ° C.

Mutant strains obtained by mutating the bacterium naturally or by artificial means can be used in the present invention as long as they have good haloacid-degrading activity. Even if it exists, it shall be included in the present invention.

The halo acid decomposing treatment in the present invention can be carried out by bringing the halo acid in the soil into contact with the above-mentioned Renovobacter species AC strain. The contact between the microorganism and the halo acid can be carried out by a method such as culturing the microorganism in the soil containing the halo acid or mixing the contaminated soil into the culture system of the microorganism.

The method of the present invention can be applied to soil restoration of both closed and open systems, and can be carried out using various methods such as batch method, semi-continuous method and continuous method. The microorganism can be used in a semi-fixed state or immobilized on a suitable carrier. As described above, this strain is very simple and useful because the strain itself secretes a polymer to form a lump.

[0025]

EXAMPLES Examples will be described below.

The quantification of all halo acids was carried out by a high performance liquid chromatography (HPLC) method equipped with an ion exchange resin packed column (developing solvent: 0.01 N sulfuric acid aqueous solution / acetonitrile = 95/5, detected at 210 nm). went. (Example 1) Restoration of dichloroacetic acid-contaminated soil by Renovobacter species AC strain Colonies of the AC strain on an agar medium were treated with 100% M9 medium containing 0.1% yeast extract in a 200 ml Sakaguchi flask.
ml was inoculated and shake culture was carried out at 30 ° C. for 48 hours. Three
It was observed that the AC strain grew in a lump form until about 0 hours, and then detached.

Next, the concentration of dichloroacetic acid was 50 mg / g w in 50 g of air-dried soil of brown forest soil collected in Atsugi City, Kanagawa Prefecture.
Add 10 ml of dichloroacetic acid aqueous solution so that it becomes the et soil, inoculate 10 ml of the above culture solution, stir well, and mix.
Static culture was carried out at 30 ° C. in a 0 ml Erlenmeyer flask. 1g of soil is collected every 24 hours thereafter, and 5ml of 0.01N
A sulfuric acid aqueous solution was added and the mixture was stirred for 1 hour, the soil was removed by centrifugation and filtration, the pH was adjusted to 2 or less with dilute sulfuric acid, the solution was introduced into HPLC, and the decrease in dichloroacetic acid was measured daily. The result is shown in FIG. As a control experiment, an aseptic medium was added instead of the bacterial solution to perform the experiment in the system, and the residual rate was expressed by comparison with the control.

Decomposition started after 2 days, and after 4 days, 50 ppm of dichloroacetic acid in soil was completely decomposed. (Example 2) Restoration of other haloacid-contaminated soil by Renovobacter species AC strain In the same manner as in Example 1, chloroacetic acid (50 ppm), trichloroacetic acid (10 ppm), and bromoacetic acid (10 ppm) were used as decomposition target substances. , AC strain was attempted. The number of culture days and the residual rate of each compound are shown in FIG.

Chloroacetic acid on the third day, trichloroacetic acid,
Bromoacetic acid was also completely decomposed by the 5th day. (Example 3) Degradation of chloropropionic acid by Renovobacter species AC strain In the same manner as in Example 1, 2-chloropropionic acid and 3-chloropropionic acid were used as the substances to be degraded, and AC was used.
Attempted decomposition by strain. The concentration was 200 ppm. The number of culture days and the residual rate of each compound are shown in FIG.

All the compounds were completely decomposed by the 3rd day. (Example 4) Decomposition of 2,2-dichloropropionic acid by Lenobacter species AC strain Using the same method as in Example 1, 2,2-dichloropropionic acid was used as a substance to be decomposed, and decomposition by the AC strain was tried. The concentration was 50 ppm. The number of culture days and the residual rate of each compound are shown in FIG.

All the compounds were completely decomposed by the 3rd day. (Example 5) Degradation mode of each halo acid by Renovobacter species AC strain The culture solution used in Example 1 was treated with dichloroacetic acid, chloroacetic acid,
50 ml of the same medium containing trichloroacetic acid, bromoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid and 2,2-dichloropropionic acid separately (concentration is the same as in each example) was inoculated into the medium at 1 ml at 30 ° C. Shake culture was performed. After that, 1 ml of the reaction solution was sampled at a time when it was considered to be decomposed by about half, and the cells were removed by centrifugation. Then, the pH was adjusted to 2 or less with dilute sulfuric acid, and the mixture was introduced into HPLC to analyze the decomposition intermediate products. As a result, glyoxylic acid was detected from dichloroacetic acid, glycolic acid was detected from chloroacetic acid, trichloroacetic acid, and bromoacetic acid, lactic acid was detected from 2-chloropropionic acid, and pyruvic acid was detected from 2,2-dichloropropionic acid. (Not detected from 3-chloropropionic acid), Renovobacter Species A
It was revealed that the decomposition of each haloacid by strain C was due to the action of dehalogenase, which is a hydrolytic dehalogenase. It was confirmed that these intermediate products were completely decomposed thereafter.

[0032]

EFFECTS OF THE INVENTION The novel haloacid-degrading bacterium provided by the present invention enables the biodegradation of haloacids, which is currently becoming a problem, and enables efficient biological treatment of soil contaminated with haloacids.

[Brief description of drawings]

FIG. 1 is a diagram showing decomposition of dichloroacetic acid in soil by an AC strain.

FIG. 2 is a diagram showing decomposition of haloacetic acid in soil by AC strain.

FIG. 3 is a diagram showing decomposition of chloropropionic acid in soil by an AC strain.

FIG. 4 is a diagram showing decomposition of 2,2-dichloropropionic acid in soil by an AC strain.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication C12R 1:01)

Claims (6)

[Claims] 1. A soil characterized by inoculating a soil contaminated with a halogen-substituted organic acid with a microorganism having a dehalogenase that hydrolytically decomposes the halogen-substituted organic acid to decompose the halogen-substituted organic acid. How to repair. 2. The soil remediation method according to claim 1, wherein the microorganism is of the genus Renovobacter. 3. The microorganism is Renovobacter species A.
The soil restoration method according to claim 2, which is a C strain. 4. The halogen-substituted organic acid is at least one of haloacetic acid and halopropionic acid.
The soil remediation method described in. 5. The soil remediation method according to claim 4, wherein the haloacetic acid is at least one selected from chloroacetic acid, dichloroacetic acid, trichloroacetic acid, and bromoacetic acid. 6. The soil remediation method according to claim 4, wherein the halopropionic acid is at least one of chloropropionic acid and dichloropropionic acid.