JP3507151B2 - Biodegradation method of halogen-substituted organic acids and novel microorganisms used therefor - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel microbial strain and a method for biodegrading a halogen-substituted organic acid using the same, and more particularly to a biodegradation method useful for treating tap water, waste water and waste liquid containing the same.

[0002]

BACKGROUND OF THE INVENTION Since being reported by the US EPA in the 1970s, disinfection by-products have been a major problem in tap water. Although disinfection with chlorine is obligatory in Japan, substances such as trihalomethanes, haloacetic acids, haloacetonitriles, and haloketones have been confirmed as by-products, which are extremely large due to their hepatotoxicity and mutagenicity. It's a problem. Among them, haloacetic acids such as chloroacetic acid, dichloroacetic acid, trichloroacetic acid, and bromoacetic acid, which were taken up as environmental monitoring items in 1993, have been highlighted as a new problem. These are described in detail in the 23rd Japan Society for Water Environment Seminar / Analytical Method Accompanied by the Revision of Water Quality Environmental Standards (Japan Society for Water Environment) p55-p64 (November 1993). .

As described above, today, halogen-substituted organic acids (hereinafter referred to as halo acids) widely remain in environmental water as a disinfection by-product of tap water, and they are becoming a serious problem.

The treatment of halo acid in such an aqueous solution is impossible by a method such as aeration treatment, and it is necessary to perform decomposition treatment by some method. Although there are several possible methods to deal with this, biodegradation treatment such as bioreactor is a very useful method because it can be treated under mild conditions and is relatively low in cost. Conceivable.

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
(The above studies have been conducted on bacteria such as protein nucleic acid enzyme, 29, 101-110 (1984). Adachi is an unidentified bacterial OS.
-2 strain has an enzyme that decomposes chloroacetic acid, bromoacetic acid, and iodoacetic acid to about the same level, and dichloroacetic acid decomposes it with about half the activity (Osaka Prefectural Public Works Research Institute Public Health) Ed., No. 30, 89 (1992)). In addition, studies have also been conducted to decompose dehalogenase extracted from Pseudomonas putida NCIMB 12018 on carboxymethylcellulose or thioglycolic acid to decompose haloacids having 2 to 6 carbon atoms (European Patent No. 179603).
issue). In addition, regarding the relationship between haloacid dehalogenase and genes, Pseudomonas putida AJ1 strain (J. Gen. Micr
obiol., 138,675 (1992)), Pseudomonas cepacia MBA
4 strains (J. Biochem., 284,87 (1992)), Pseudomonas sp .
CBS3 strain (Biol. Chem. Hoppe-Seyler., 374,489 (19
93)) is being studied.

[0006] However, all of these are evaluations of activity at the enzyme level, and the actual behavior of these microorganisms in contaminated wastewater has not been studied at present. Xant for haloacid decomposition of microorganisms themselves
hobacter autotrophicus GJ10 strain (Appl. Biochem. B
iotechnol., 40,158 (1993)) and 40,165 (1993)) have only been studied.

Further, regarding the decomposition of halopropionic acid, it has been reported that dehalogenase extracted from D-type and L-type chloropropionic acid from a bacterium of the genus Pseudomonas decomposes to lactic acid (JP-A-4-64544). This case is only an enzyme-level study.

In addition, from the viewpoint of satisfying the practical conditions when applied to the method for decomposing halo acids using microorganisms and having sufficient resolution, 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 required for such a bacterial species, it is needless to say that it has a sufficient haloacid degrading property, but the growth conditions are different from those of the known bacterial species and its application range can be expanded, or its utilization. It is more preferable that the morphology is rich.

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 not only decomposing the dichloroacetic acid but also capable of growing under the bad environment of the waste liquid and maintaining the decomposition activity. .

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]

SUMMARY OF THE INVENTION It is an object of the present invention to develop a novel strong bacterium for decomposing halo acids, which is a problem as a sterilizing and disinfecting by-product, and a halo acid utilizing the microorganisms. It is an object of the present invention to provide a method for decomposing, particularly for decomposing halo acids contained in water such as wastewater / waste liquid, groundwater and water and sewerage.

[0013]

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

That is, as a result of searching for a bacterial species decomposing halo acid, the present inventors obtained a new bacterial species having a high concentration of halo acid decomposing ability from the Kanto loam soil of Japan, and obtained this bacterial species. By contacting the strain with an aqueous medium containing a halo acid,
We have found a way to decompose halo acids in aqueous media.

First, the mycological properties of the strain newly obtained in the present invention are shown below (identification criteria: Bergey's Manual (19
84)).

A. Morphological properties Gram stain: Size and shape of negative cells: C-shaped 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. Color: White to cream B. Growth status in various media BHIA: Good growth MacConkey: Bad growth C.I. Optimum temperature for growth: 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: Due to various properties above positive, 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 no haloacid-degrading bacteria are known among the strains belonging to Renovobacter, this strain was identified as a new strain, and the Renovobacter species AC strain ( Renobacter s
p . AC) and deposited at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science (accession number: FERM P-14641).

The AC strain has a very specific growth morphology as well as the morphology of the cell itself (S-shaped), and although the bacterium itself has not specified anything, it secretes a certain high molecular substance. And become a lump of bacteria to grow. Microscopically, such a property rapidly forms an AC strain's own habitat (habitat) and proliferates as a priority species, so that various miscellaneous bacteria are present in wastewater, waste liquid, and rivers. It is extremely advantageous when it is grown in a lake or lake and decomposed to treat halo acids.

The bacterium can be cultivated in a natural complete medium such as a usual 2YT medium or LB medium, but may 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; pH 7.0) Cultivation may be carried out under aerobic conditions. It may be liquid culture or solid culture. The culture temperature is preferably around 30 ° C.

It is obvious that even mutant strains obtained by mutating the bacterium naturally or by artificial means can be applied to the present invention as long as they have good haloacid-degrading activity. Even in such cases, these are all included in the scope of the present invention.

The decomposition treatment of the haloacid in the present invention can be carried out by bringing the haloacid in an aqueous medium such as a waste liquid 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 an aqueous medium containing the halo acid, or adding the aqueous medium to a culture system of the microorganism, a batch method, a semi-continuous method. It can be carried out by using various methods such as a continuous method and a continuous method. The microorganism can be used in a semi-fixed state or immobilized on a suitable carrier. As described above, the strain itself is very simple and useful because the strain itself secretes a high-molecular substance to form a lump.

[0024]

EXAMPLES Examples will be described below. In addition, the quantification of all halo acids was carried out by a high performance liquid chromatography (HPLC) method (developing solvent: 0.
01N sulfuric acid aqueous solution / acetonitrile = 95/5, 210
nm detection). (Example 1) Degradation of dichloroacetic acid by Renovobacter species AC strain A colony of the AC strain on an agar medium was 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, 50 ml of the same medium containing 200 ppm of dichloroacetic acid was inoculated with 1 ml of the above-mentioned culture solution and 30 ° C.
The culture was performed with shaking. After that, 1 ml of the reaction solution was collected every 8 hours, the cells were removed by centrifugation, the pH was adjusted to 2 or less with diluted sulfuric acid, and the mixture was introduced into HPLC, and the decrease in dichloroacetic acid was measured daily. The result is shown in FIG.

Decomposition started after 8 hours, and after 48 hours, 200 ppm of dichloroacetic acid was completely decomposed.
Also, glyoxylic acid is detected as a decomposition intermediate,
It was confirmed that the decomposition was carried out by the action of dehalogenase. The glyoxylic acid was completely decomposed into carbon dioxide and water. (Example 2) Degradation of other haloacetic acid by Renovobacter species AC strain In the same manner as in Example 1, chloroacetic acid (200 ppm), trichloroacetic acid (50 ppm), and bromoacetic acid (50 ppm) were used as AC to decompose AC Attempted decomposition by strain. The incubation time and the residual concentration of each compound are shown in FIG.
This is shown in FIGS.

All the compounds were completely decomposed by 72 hours. Glycolic acid was detected as an intermediate product of decomposition, confirming that the decomposition was carried out by the action of dehalogenase. The glycolic acid was completely decomposed into carbon dioxide and water. (Example 3) Degradation of chloropropionic acid by Lenobacter species AC strain In the same manner as in Example 1, 2-chloropropionic acid and 3-chloropropionic acid were used as decomposition target substances, and AC
Attempted decomposition by strain. The concentration was 1000 ppm.
The incubation time and the residual concentration of each compound are shown in FIG.

All the compounds were completely decomposed by 48 hours. Lactic acid was detected as a decomposition intermediate product, confirming that the decomposition was performed by the action of dehalogenase. The lactic acid was completely decomposed into carbon dioxide and water. (Example 4) Degradation of 2,2-dichloropropionic acid by the Renovobacter species AC strain In the same manner as in Example 1, 2,2-dichloropropionic acid was used as a substance to be degraded, and degradation by the AC strain was tried. The concentration was 100 ppm. The culture time and the residual concentration are shown in FIG.

By the 48th hour, 100 ppm of 2,2
-Dichloropropionic acid was completely decomposed. Further, pyruvic acid was detected as an intermediate product of decomposition, and it was confirmed that the decomposition was carried out by the action of dehalogenase. The pyruvic acid was completely decomposed into carbon dioxide and water.

[0030]

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 waste liquids containing haloacids.

[Brief description of drawings]

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

FIG. 2 is a diagram showing decomposition of chloroacetic acid by AC strain.

FIG. 3 is a diagram showing decomposition of trichloroacetic acid by an AC strain.

FIG. 4 is a diagram showing decomposition of bromoacetic acid by an AC strain.

FIG. 5 is a diagram showing degradation of chloropropionic acid by AC strain.

FIG. 6 is a diagram showing the decomposition of 2,2-dichloropropionic acid by an AC strain.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:01) C12R 1:01 (56) References JP-A-5-260964 (JP, A) JP-A-3-80085 ( JP, A) JP 8-141586 (JP, A) JP 8-141549 (JP, A) JP 5-130879 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 1/00 C12N 9/00 C02F 1/00 BIOSIS / WPI (DIALOG)

Claims (6)

(57) [Claims] 1. A method for biodegrading a halogen-substituted organic acid.
And a novel microorganism Renovobacter species AC having a dehalogenase for degrading the halogen-substituted organic acid.
(Biotechnology Institute of Technology Contract number: FERM P -1464
No. 1 ), and culturing the microorganism together with the halogen-substituted organic acid under the condition that the dehalogenase acts.
And a step of decomposing the halogen-substituted organic acid. Wherein said halogen-substituted organic acids, also haloacetic
The method according to claim 1 is a halopropionyl. 3. The method according to claim 2, wherein the haloacetic acid is any one of chloroacetic acid, dichloroacetic acid, trichloroacetic acid and bromoacetic acid. 4. The method according to claim 2, wherein the halopropionic acid is chloropropionic acid or dichloropropionic acid. 5. A novel microbial Renovobacter spp. Having a dehalogenase for degrading halogen-substituted organic acids.
AC (Biotechnology Institute of Technology Contract number: FERM
P- 14641 ) . 6. A wastewater characterized in that an aqueous waste solution containing a halogen-substituted organic acid is contacted with Renovobacter Species AC (Institute of Industrial Science and Technology, Accession No .: FERM P-14641) or a culture secretion thereof. Biological purification method.