JPH0667314B2 - Method for microbial decomposition of aliphatic chlorine compound and its microorganism - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for decomposing a saturated and / or unsaturated aliphatic chlorine compound by a specific immobilized microorganism and a novel immobilized microorganism used in the method.

More specifically, it relates to a method for decomposing and removing a volatile aliphatic chlorine compound such as trichlorethylene contained in waste water or exhaust gas from a factory, or in soil by a microorganism.

[Prior Art] Various organic chlorine compounds are mixed in wastewater or exhaust gas from factories or soil, and due to problems such as environmental pollution recently, effective removal of these has become a focus of attention. There is.

In particular, trichlorethylene (TCE) is a persistent compound used in the IC industry and the like, has carcinogenicity, and has become a problem as a groundwater pollutant.

Conventionally, in order to remove organic chlorine compounds such as trichlorethylene from waste water or exhaust gas, an adsorption removal method using activated carbon has been performed, but these require a large amount of adsorbent and special equipment and facilities. However, it is not always an efficient and economical means of removal.

On the other hand, several methods using microorganisms have been attempted and reported as efficient and simple means for decomposing and removing organochlorine compounds.

For example, an example of decomposing and removing an organic chlorine compound such as polyphenyl polychlorinated by using a microorganism of the genus Rhodotorula, the genus Cladosporium, the genus Candida, the genus Saccharomyces, and the genus Streptomyces (JP-A-48-98085).
, JP-A-48-98086, JP-A-49-6186), and methanotrophic bacteria such as those of the genera Methylocinas, Methylocistis, Methylococcus and Methylobacterium. An example of decomposing a halogen-substituted benzene such as chlorotoluene (JP-A-55-127196) has been reported.

However, few microorganisms that effectively decompose and remove aliphatic chlorine compounds such as trichlorethylene and its related compounds have been reported, and only the proposal by the present applicant (Japanese Patent Application No. 63-239753) is present. .

[Problems to be Solved by the Invention] The present invention provides an improved method for industrially more advantageously utilizing the method for decomposing an aliphatic chlorine compound proposed in Japanese Patent Application No. 63-239753. The object is to provide a novel immobilized microorganism used in the method.

[Means for Solving the Problems] The present inventor has made extensive studies on immobilization of the microorganism disclosed in the above application, but found that a certain carrier is particularly effective for immobilization of the microorganism. The present invention has been reached.

That is, the present invention provides (1) a microorganism belonging to the genus Methylocinas and capable of degrading an aliphatic chlorine compound is immobilized on an agarose gel, a κ-carrageenan gel, and / or a calcium alginate gel, and the immobilized microorganism is treated with an aliphatic chlorine. A method for decomposing an aliphatic chlorine compound, which comprises contacting a compound or a substance containing the same, and (2) a methanotrophic bacterium that is a methane-utilizing bacterium that grows with methane and methanol as the sole carbon sources and decomposes trichlorethylene. Sporium ・ TSUKUB
It consists of an immobilized microorganism in which A is immobilized with an agarose gel, a κ-carrageenan gel and / or a calcium alginate gel.

The microorganism itself of the present invention is, as already described, Japanese Patent Application No. 63-23.
It is disclosed in No. 9753. It is widely distributed in various soils and can be collected from it. For example, the following method is used as a method for collecting the soil.

That is, the culture is performed by using a vial bottle sealed with a butyl rubber stopper and an aluminum seal and shaking at 30 ° C. Trichloroethylene amount takes a certain amount of gas phase from the headspace,
Quantify by gas chromatography and calculate the liquid phase concentration from Henry's law formula.

Using the above means, for example, the collected soil is repeatedly subjected to enrichment culture in the presence of 1 ppm trichlorethylene and methane to obtain a mixed microbial system that decomposes trichlorethylene well.
Since oxygen and methane are essential for the decomposition of trichlorethylene, methanotrophs will be isolated from mixed microbial systems.

The bacterium isolated in the present invention is a known methylosynase
Methylosinas, Trichosporium, TSUKUBA, which belongs to Trichosporium.

When observed under a microscope, this fungus has a width of 0.6 to 1 μm and a length of 1
It is a short bacillus having a size of up to 5 μm and has the characteristics shown in the following table.

Chracteristics of methane−utilizing bacterium Gram stain Negative Cell shape Short rod Number of flagela 0 Motility − Growth on methane + ethane − propane − n-butane − dimethylether − methylamine − methanol + ethanol − nutrient broth − Growth at 30 ° C. + 37 ° C. + 45 ° C-Mol% G + C OF DNA 64.5 Major fatty acid C _{18: 1} (96.5%) Hydroxy fatty acid type 2-OH Quinone type Q _{8 Based on the} above mycological properties, the methylocinus strain of the present invention was identified. .

Methylosynus strains of the present invention, the morphology of the bacterium, microscopic findings such as Gram stain, physiological properties, etc., described the properties of the known bacterium Methylosinus trichosporium OP 3b (1. Journal of General Microbiology
61, 205-218 (1970), 2.Microbial Growth on C 1
Compounds p.123-133 (1984), 3.Journal of Ge
neral Applied Microbiology 33,135-165 (198
Based on the classification of Whittenbury et al. And Komagata et al. Described in [7)], it was identified as a strain closely related to Methylosinus trichosporium OB 3b.

However, it has no flagella, no saturated C ₁₉ fatty acids, and does not form rosettes, which clearly distinguishes it from Methylosynomyces trichosporium OB 3b and was identified as a new strain. It was named Rium TSUKUBA.

The bacterium of the present invention has been deposited in the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology as Microorganism Research Institute No. 10004.

The bacterium of the present invention is trichlorethylene and its various related compounds, namely, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, 1,1-dichloroethylene,
It has the property of decomposing 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane, 1,2-dichloroethane and chloroform, and has the ability to decompose 10ppm high concentration trichlorethylene to about half in 10 days. To have.

In the present invention, it is possible to repeatedly use the above-mentioned methylosynaceae trichosporium TSUKUBA, and to facilitate separation from the reaction system, and to utilize this bacterial cell industrially more advantageously, Immobilize

The cells used in the present invention have carrier specificity,
The choice of carrier for its immobilization is extremely important.
Polyurethanes, photocurable resins, polymer electrolytes and the like are also known as carriers for bacterial cells, but these carriers are not suitable for immobilizing the above bacterial cells. When these are used for immobilization, the characteristic inherent in the above-mentioned microbial cells for degrading aliphatic chlorine compounds such as trichlorethylene is significantly diminished.

According to the study of the present inventor, only when agarose gel, calcium alginate gel and κ-carrageenan gel were used as carriers for immobilization of the above-mentioned microbial cells, the microbial cells were immobilized without substantially impairing useful attributes be able to.

In carrying out the method of the present invention, the immobilized microorganism of the present invention is brought into contact with a waste water or exhaust gas containing trichlorethylene or the compound in a solution state.

[Examples] The present invention will be described in more detail with reference to Examples.

(Preparation method of immobilized cells) Put 30 ml of the following inorganic salt medium dissolved in distilled water into a vial (155 ml), and add Methylosynaceae trichosporium TS
After inoculating UKUBA, 1 ppm of trichlorethylene and methane were added, the mixture was completely sealed with a butyl rubber stopper and an aluminum seal, and shake culture was performed at 30 ° C.

_{KH 2 PO 4 0.45g / K 2} HPO 4 1.17g / NH 4 Cl 2.14g / Ca (NO 3) 2 · 2H 2 O 4.8mg / MgSO 4 · 7H 2 O 121mg / FeSO 4 · 7H 2 O 28mg / Trace metals DW pH7.2 CH ₄ 20 ml / bottle or CH ₃ OH 0.3 ml / bottle After collecting 60 ml of culture solution, 10 mM phosphate buffer solution (pH 7.
0) Suspended in 2.5 ml, 2.5 ml of 4% sodium alginate solution was added, and the mixture was added dropwise to a 5% calcium chloride solution to prepare calcium alginate gel-immobilized cells. In addition, 4% agarose and 2% κ-carrageenan gel-immobilized cells were prepared by adding agarose or κ-carrageenan to the cell suspensions and solidifying them. For comparison, a photocurable resin (ENT-340 manufactured by Kansai Paint Co., Ltd.
0), a polyelectrolyte (PDDA, KPVS) and a urethane polymer (PU6 manufactured by Toyo Tire & Rubber Co., Ltd.) were used as carriers for immobilization.

Example 1 The immobilized bacterial cells obtained above were molded into beads or cubes having a diameter of 3 mm and placed in one vial (Fig. 1) containing the inorganic salt medium, and a decomposition test of trichlorethylene was conducted. It was

In the decomposition experiment, resting cells were used without adding a growing carbon source to facilitate the analysis of the experimental results.

In the case of activating the immobilized resting cells, methane or methanol was added in the above amount and shake culture was carried out for a certain period of time.

The amount of trichlorethylene reduction was determined by gas chromatography using the headspace method.

First, the ability of various immobilized cells to decompose 1 ppm of trichlorethylene was measured comparatively. The results are shown in FIG.

The vertical axis shows the residual rate when the amount of trichlorethylene in the vial at the start of the reaction is 100%. As is clear from FIG. 2, trichloroethylene was hardly reduced in oriurethane and the photocurable resin, and even when immobilized with the polymer electrolyte, the reduction was only about 10%. On the other hand, about 60% of the free resting cells were decomposed after 14 hours, and when immobilized on agarose gel or κ-carrageenan gel, a similar decrease was observed.
When immobilized with a calcium alginate gel, the degradation rate was better than that of free bacteria, and a decrease of 90% or more was observed after 14 hours.

Example 2 Next, with respect to the immobilized bacterial cells by agarose gel, κ-carrageenan gel, and calcium alginate gel in which a remarkable decrease in trichlorethylene was observed, changes in the resolution due to repeated use and the durability of the gel were examined.

Here, repeated use was performed 5 times. First, Methylosinus trichosporium TSUKUBA was prepared by immobilizing methane as a carbon source, immobilized, and the first experiment was performed, and then methane or methanol was added to activate it, and the decomposition experiment was performed again as resting bacterial cells. The second and subsequent times were similarly activated every time and the decomposition experiment was repeated. The results are shown in Table 1. The numerical values in the table represent the decomposition rate, and the reduction amount of the first time is 100%. Regarding gel disintegration, + means normal gel, ± means disintegration of a part of gel, and-indicates complete disintegration.

When immobilized with a calcium alginate gel, higher resolution than free bacteria was observed at the first time in Fig. 2, but when repeatedly used, gel disintegration began at the third time. On the other hand, in the agarose gel, no gel disintegration was observed even after repeated use five times, and retention of constant resolution was observed. With κ-carrageenan gel, the gel began to break on the fourth occasion. In addition, when each immobilized cell was activated with methane or methanol, a higher decomposition rate was observed with the first activation with methanol than with the methane, but generally after the second activation It was recognized that activation with methane tended to be better.

Example 3 Next, the upper limit of the concentration of trichlorethylene capable of decomposing agarose-immobilized resting cells was examined. In FIG. 3, the vertical axis represents the relative residual rate with the initial amount of trichlorethylene in the vial as 100%.

From FIG. 3, it is clear that when the liquid phase concentration is 100 ppm, there is no reduction in trichlorethylene, but when the concentration is 35 ppm or less, a decrease is observed in each, and even at a considerably high concentration, decomposition is apparent. Although not shown here, almost the same results were obtained with free bacteria. Next, based on the results obtained here, the decomposition rate at each concentration was determined, and the relationship with the trichlorethylene concentration was investigated.

FIG. 4 is a diagram showing the relationship between the concentration of the substrate trichlorethylene and its decomposition rate. The vertical axis is the decomposition rate,
The horizontal axis shows the concentration of trichlorethylene. The top is the case of immobilized cells and the bottom is the case of free cells.

As is clear from this figure, the decomposition pattern of trichlorethylene when immobilized on agarose is Michaelis-M.
You can see that it is an enten type. So Lineweaver-Burk
The maximum decomposition rate Vmax and the saturation constant Ks were obtained by the method of.

FIG. 5 is a Lineweaver-Burk plot.

When Vmax and ks are calculated from this, Vmax of the immobilized cells is 3.15.
μgTCE / mg cell / hr was calculated to be 2.62 for free cells, while ks was 100 μM for immobilized cells and 66 μ for free cells.
It was M. From these values, even if immobilized, no significant decrease in resolution was observed as compared to free bacteria.
Tends to be slightly higher when fixed. It is also understood that ks is larger in the immobilized cells and has a weaker affinity for the substrate trichlorethylene. The weak affinity with the substrate is thought to be because the agarose gel, which is the immobilization carrier, inhibits the diffusion of the substrate into the cells, and especially in the case of high concentration trichlorethylene, the gel protects the cells and is effective. Seems to be.

Then, the influence of temperature on the decomposition rate was examined. The result is shown in FIG. The decomposition rate on the vertical axis is shown from 0.10.

The decomposition rate drops sharply at 15 ℃ and 35 ℃, but at 20 ℃
Showed the highest decomposition rate. It was also found to be relatively stable from 20 ℃ to 30 ℃.

FIG. 7 shows the results of examining the effect of pH on the decomposition rate. It can be seen that the decomposition rate is almost constant over a wide range of pH 6 to 7.5, but that at pH 8 the decomposition rate drops sharply. Also, the reason that the decomposition rate increased slightly at pH 6 is considered to be that the strength of the agarose gel was weakened because it was an acidic solution, and the bacterial cells leaked out to some extent, which promoted the decomposition of trichlorethylene by the free bacteria. Therefore, it can be said that in this immobilized cell, the degrading activity decreases on the alkaline side, but the activity does not change so much at neutral.

[Effects of the Invention] As described above, by selecting a specific carrier according to the present invention, methylosynaceous trichosporium / TSUKUB is selected.
It can be immobilized without substantially degrading the decomposition characteristics of the aliphatic chlorine compound possessed by A. By using this novel immobilized microorganism, it is possible to efficiently decompose persistent pollutants. .

[Brief description of drawings]

FIG. 1 is an explanatory view of a test apparatus used in an example of the present invention, and FIG.
Figure shows the decomposition characteristics of trichlorethylene when immobilized on various carriers. Figure 3 shows the change in the decomposition characteristics of trichlorethylene by cells fixed to agarose depending on its concentration. Figure 4 shows the same cells. For explaining the effect of the concentration of trichlorethylene on the decomposition rate of trichlorethylene, Fig. 5 shows the decomposition of trichlorethylene by the fungus body.
The figure showing a Lineweaver-Burk plot, FIG. 6 is a figure explaining the influence of the temperature which acts on the trichlorethylene decomposition characteristic by the same microbial cell, and FIG. 7 is a figure explaining the influence of pH which acts on the trichlorethylene decomposition characteristic by the same microbial cell.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location C02F 3/34 ZAB Z C12N 11/10 // (C12N 1/20 C12R 1:01)

Claims (4)

[Claims] 1. A member of the genus Methylosinus,
A microorganism having a capability of degrading an aliphatic chlorine compound is immobilized on an agarose gel, a carrageenan gel, and / or an alginic acid gel, and the immobilized microorganism is brought into contact with the aliphatic chlorine compound or a substance containing the aliphatic chlorine compound. Disassembly method. 2. The method according to claim 1, wherein the microorganism is a methane-utilizing bacterium that decomposes trichlorethylene. 3. The microorganism is methylosynaceus trichosporium TSUKUBA (Microtechnology Research Institute No. 10004) (1)
Alternatively, the method according to (2). 4. Methylosynus trichosporium TSUKUBA which is methane-utilizing and decomposes trichlorethylene.
An immobilized microorganism obtained by immobilizing (Microtechnology Research Institute No. 10004) with agarose gel, κ-carrageenan gel and / or calcium alginate gel.