JP3437377B2 - Novel microorganism and biodegradation method of dichloropropene using the microorganism, environmental purification / repair method - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel microbial strain, a method for biodegrading dichloropropene using the same, and further an aqueous medium such as wastewater and waste liquid containing dichloropropene, soil contaminated with dichloropropene, and air. Environmental purification and restoration methods.

[0002]

2. Description of the Related Art In recent years, environmental pollution caused by organochlorine compounds, which are harmful to living organisms and are difficult to decompose, such as soil and tap water source pollution problems caused by agricultural chemicals used in agriculture and golf courses, has become a serious problem. Is coming. Among them, in particular, 1,3-dichloropropene has been widely used as a main component of the nematicide DD, but it is widely suspected to be harmful to living bodies including humans, and the Ministry of Health and Welfare In both the “Water Quality Standards for Waterworks Law” revised in 1980 and the “Environmental Standards for Water Pollution” partially revised in 1993 by the Environment Agency, the standard values are 0.
It was determined to be 002 mg / l. Furthermore, in the Environmental Agency,
In the "Environmental Standards for Soil Contamination", 1,3-dichloropropene was cited as one of the additional items in 1975, and local governments and companies were urged to achieve the environmental standards.

From such a background, purification of an aqueous medium such as contaminated groundwater, which decomposes and decomposes 1,3-dichloropropene, and soil is an important issue from the viewpoint of environmental protection. Development is taking place.

For example, adsorption treatment with activated carbon and decomposition treatment with light or heat have been studied, but it is not always practical in terms of cost and operability.

On the other hand, it is relatively stable in the environment 1,3
-In recent years, decomposition of dichloropropene by microorganisms has been reported, and studies toward its practical use have begun.
That is, in the biodegradation treatment using microorganisms, it is possible to decompose organic chlorine compounds into harmless substances by selecting the microorganisms to be used, basically no special chemicals are required, and the labor and cost required for maintenance can be reduced. And so on.

However, there have been few reports of isolation in microorganisms capable of degrading 1,3-dichloropropene, for example, Xanthobacter strai, which decomposes 1,3-dichloropropene by alkene monooxygenase.
n Py2 ( Appl . Environ . Microb
iol . , 58, 3038 (1992)), which decomposes 1,3-dichloropropene by methane monooxygenase. Methylosinus trichospor
ium OB3b ( Appl . Environ , Mic
robiol . , 55, 2819 (1989)), and all of these microorganisms require an inducer (inducer) to decompose 1,3-dichloropropene.

[0007]

In addition to the above-mentioned degrading bacteria, it is considered that there are some microorganisms capable of degrading 1,3-dichloropropene due to their degrading mechanism and capable of inducing oxygenase which is an oxidase. However, in the above-mentioned degrading bacteria, hydrocarbons such as aromatic compounds and aliphatic compounds are inducers (inducers; indus) for degrading hydrocarbons.
cer), and it is highly undesirable to release such compounds into the environment with microorganisms.

Therefore, a method for microbially degrading 1,3-dichloropropene without the need for such an inducer has been strongly desired.

The object of the present invention is to obtain a novel microorganism capable of decomposing 1,3-dichloropropene strongly without the use of such an inducer, and to utilize the microorganism.
It is an object of the present invention to provide a method for decomposing 3-dichloropropene, and further a method for purifying and restoring the environment such as wastewater, waste liquid, an aqueous medium such as groundwater contaminated with 1,3-dichloropropene, soil and air.

[0010]

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

That is, the inventors of the present invention, Pseudomonas sp. ( Pseudomonas) , which is a microorganism that decomposes 1,3-dichloropropene without requiring an inducer
sp. ) Obtaining Q27 strain (Ministry of International Trade and Industry, Institute of Biotechnology, Industrial Technology deposit number: FERM P-15686), and contacting this strain with an aqueous medium containing this compound, soil, or gas phase causes pollution. We have found a way to decompose 1,3-dichloropropene.

First, the mycological properties of the Pseudomonas species Q27 strain, which is a novel microorganism of the present invention, are shown below. β-galactosidase: Negative arginine dihydrolase: Positive lysine decarboxylase: Negative ornithine decarboxylase: Negative Citric acid availability: Positive H ₂ S production: Negative urease: Negative tryptophan deaminase: Negative indole production: Negative acetoin production: Positive gelatinase: Negative oxidase: Fermentation of positive sugar / oxidized glucose: Negative D-mannitol: Negative inosit: Negative D-sorbitol: Negative L-rhamnose: Negative sucrose: Negative D-melibiose: Negative D-Amygdalin: Negative L-arabinose: Negative Above Due to the above-mentioned characteristics, this strain is Pseudomonas ( Pse
It was determined to be a novel bacterial species belonging to the genus Udomonas ) and classified into Pseudomonas sp.

As described above, the strain of the present invention decomposes 1,3-dichloropropene of about 10 ppm almost completely in both cis-type and trans-type without the need for special chemical substances. Has resolution. Therefore, this strain was used as Pseudomonas species ( P
seudomonas sp. ) Named Q27 strain and deposited at the Institute of Biotechnology, Ministry of International Trade and Industry (deposit number: F
ERM P-15686).

The nutrient source of the medium that can be used for culturing the Q27 strain of the present invention is any carbon source or nitrogen source as long as it is a nutrient source necessary for the growth of ordinary microorganisms and that can be assimilated by this bacterium. Inorganic salts may also be used, and for example, it is also possible to culture with M9 medium supplemented with yeast extract or the like as a slight nutrient source.

The composition of the 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 1 liter of medium; pH 7.0) Cultivation can be carried out under aerobic conditions. It may be liquid culture or solid culture. The culture temperature is preferably around 30 ° C.

In the present invention, 1,3-dichloropropene is decomposed by treating 1,3-dichloropropene in an aqueous medium, soil or air.
It can be carried out by contacting 3-dichloropropene with the above strain Q27. The contact between the microorganism and 1,3-dichloropropene can be carried out by any method as long as the microorganism can exhibit a degrading activity, and can be carried out using various methods such as a batch method, a semi-continuous method and a continuous method. . The microorganism can be used in a semi-fixed state or immobilized on a suitable carrier. If necessary, various pretreatments may be applied to objects to be treated such as waste liquid, waste water, soil, and air.

The decomposition treatment of 1,3-dichloropropene in an aqueous medium in the present invention is carried out by
It can be carried out by contacting 3-dichloropropene with the above strain Q27. The main use forms are described below, but the present strain is not limited to these forms, and the strain can be used for purification treatment of 1,3-dichloropropene in an aqueous medium in any form.

For example, the simplest method is 1,3
There is a method of introducing strain Q27 directly into an aqueous medium contaminated with dichloropropene. In this case, it is desirable to adjust the pH, salt concentration, temperature, concentration of contaminants, etc. of the aqueous medium, but the Q27 strain maintains 1,3-dichloropropene-degrading activity unless it is extremely acidic or alkaline and has a high salt concentration. .

[0020] As another usage mode, there is a mode in which a culture tank is provided to cultivate the Q27 strain, and an aqueous medium contaminated with 1,3-dichloropropene is introduced into this culture tank at a predetermined flow rate to decompose it. . The introduction and drainage of the aqueous medium may be carried out continuously, or may be carried out intermittently or batchwise depending on the treatment capacity. It is advisable to optimize such control by controlling the system according to the concentration of 1,3-dichloropropene.

Further, there is a mode in which the Q27 strain is attached to a carrier such as soil particles, the reaction layer is filled with the same, and a 1,3-dichloropropene-contaminated aqueous medium is introduced into the reaction tank for decomposition treatment. . In this case, the carrier to be used is not limited to soil particles, and any carrier can be used, but it is more preferable that the carrier has excellent ability to retain microorganisms and maintains air permeability. For example, as a material that provides a habitat space for microorganisms, various microbial carriers that are commonly used in bioreactors conventionally used in the pharmaceutical industry, food industry, wastewater treatment system and the like can be used. More specifically, porous glass, ceramics, metal oxides, activated carbon, kaolinite, bentonite, zeolite, silica gel, alumina, inorganic particulate carriers such as anthracite, starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid. Gel carriers such as polyacrylamide, carrageenan, agarose and gelatin, ion exchangeable cellulose, ion exchange resins, cellulose derivatives,
Glutaraldehyde, polyacrylic acid, polyurethane,
Examples thereof include polyester. As a natural product, cotton,
Cellulose-based materials such as hemp and paper, and lignin-based materials such as wood flour and bark can also be used.

The decomposition treatment of 1,3-dichloropropene in soil in the present invention can be carried out by bringing 1,3-dichloropropene existing in soil into contact with the above strain Q27. The main usage patterns are described below.
Without being limited to these forms, the strain of the present invention can be used for purification treatment of 1,3-dichloropropene in soil in any form.

For example, the simplest method is 1,3
-Q directly in the soil contaminated by dichloropropene
There is a method of introducing 27 strains. The method of introduction includes not only the method of spraying on the soil surface, but also the method of introduction from a well inserted into the ground in the case of treatment in a relatively deep formation. Furthermore, when pressure is applied by air, water, etc., the Q27 strain spreads over a wide area, which is more effective. In this case, various conditions in the soil are adjusted so as to be suitable for the Q27 strain, but the Q27 strain grows faster in the presence of a carrier such as soil particles, and in that sense, the condition in the soil is convenient. .

Further, the Q27 strain is attached to a carrier, the carrier is filled in a reaction tank, and the reaction tank is introduced into a soil tank contaminated with 1,3-dichloropropene, mainly into an aquarium tank for decomposition treatment. There is a form to do. It is desirable that the form of the reaction tank is one that can cover a wide range in the soil, such as a fence or a film. In this case, any carrier can be used, but it is more preferable that the carrier has excellent ability to retain microorganisms and maintains air permeability. For example, as a material that provides a habitat space for microorganisms, various microbial carriers that are commonly used in bioreactors conventionally used in the pharmaceutical industry, food industry, wastewater treatment system and the like can be used. More specifically, porous glass, ceramics, metal oxides, activated carbon, kaolinite, bentonite, zeolite, silica gel, alumina, inorganic particulate carrier such as anthracite, starch, agar, chitin,
Examples thereof include gel carriers such as chitosan, polyvinyl alcohol, alginic acid, polyacrylamide, carrageenan, agarose and gelatin, ion exchangeable cellulose, ion exchange resins, cellulose derivatives, glutaraldehyde, polyacrylic acid, polyurethane and polyester.
As natural products, cellulose-based products such as cotton, hemp, and paper, and lignin-based products such as wood flour and bark can also be used.

The decomposition treatment of 1,3-dichloropropene in the air according to the present invention can be carried out by bringing 1,3-dichloropropene existing in the air into contact with the strain Q27. The main use forms are described below, but the present strain is not limited to these forms, and the strain can be used for purification treatment of 1,3-dichloropropene-contaminated air in any form.

For example, a culture tank is provided to culture the Q27 strain,
There is a mode in which a gas contaminated with 1,3-dichloropropene is introduced into this culture tank at a predetermined flow rate and decomposed. There is no limitation on the method of introducing the gas, but a form in which the culture solution is agitated by the introduction of the gas to promote aeration is more preferable. The introduction and exhaust of the gas may be performed continuously, but it is also possible to perform the treatment intermittently or in a batch manner depending on the treatment capacity. This kind of control
It is advisable to optimize the system by controlling the system according to the concentration of 3-dichloropropene.

As another utilization form, the strain Q27 is adhered to a carrier such as soil particles, the reaction layer is filled with the same, and 1,3-dichloropropene polluted gas is introduced into the reaction tank for decomposition treatment. There is a form to do. In this case, the carrier to be used is not limited to soil particles, and any carrier can be used, but it is more preferable that the carrier has excellent ability to retain microorganisms and maintains air permeability. For example, as a material that provides habitat for microorganisms,
Various microbial carriers widely used in bioreactors used in the food industry, wastewater treatment systems, etc. can be used. More specifically, porous glass, ceramics,
Inorganic particulate carriers such as metal oxides, activated carbon, kaolinite, bentonite, zeolite, silica gel, alumina, anthracite, starch, agar, chitin, chitosan,
Examples thereof include gel-like carriers such as polyvinyl alcohol, alginic acid, polyacrylamide, carrageenan, agarose and gelatin, ion-exchangeable cellulose, ion-exchange resins, cellulose derivatives, glutaraldehyde, polyacrylic acid, polyurethane and polyester. As natural products, cellulose-based products such as cotton, hemp, and paper, and lignin-based products such as wood flour and bark can also be used.

As the growth material for the bacterium, a medium for generally culturing microorganisms as described above can be used. For example, broth medium, M9 medium, 2xYT medium, L medium, or a medium in which polypeptone, yeast extract or the like and a carbon source such as glucose are arbitrarily mixed is effective. In addition, these media can be used either in liquid form or in gel form by adding agarose.

[0029] Further, as a material capable of both holding bacteria and supplying nutrients, many examples can be given to compost used in the agriculture, forestry and fisheries industries. That is, grain-derived straws and sawdust such as straw, plant-derived dried products such as rice bran, snow flowers, sugar cane dregs, and marine wastes such as crab and shrimp shells can be used.

The purification of the air contaminated with dichloropropene may be carried out by pre-filling it with a substance to be a carrier and then introducing the bacteria, or by pre-culturing. In order to proceed the decomposition reaction more efficiently, the nutrients and water content mentioned above,
It is advisable to adjust the oxygen concentration. Further, the ratio of the carrier to the water content in the reaction tank may be appropriately selected depending on the growth and air permeability of the microorganisms and the form of the reaction tank depending on the amount and concentration of the gas to be treated. For example, columns, tubes, tanks, and box-shaped ones can be used in consideration of facilitating contact with. Furthermore, such a shape may be unitized with an exhaust duct, a filter, or the like, or some of them may be connected in accordance with the ability.

The polluted air may be adsorbed to the carrier material at first, and the effect of microbial utilization is not often observed immediately after the start, but after a certain period of time, the pollutant adhered to the carrier material is decomposed. Then, the contaminants are again adsorbed on the surface of the decomposed material, so that the adsorptivity to the carrier material is regenerated. In this way, 1,3-dichloropropene removing ability is not saturated, and constant decomposition can always be expected.

The environmental purification / restoration method of the present invention is a closed system,
It can be applied to any open system wastewater / wastewater treatment and soil / groundwater treatment methods, and microorganisms may be immobilized on a carrier or the like, or various methods for promoting growth may be used in combination.

[0033]

[Examples] Example 1 Method for obtaining strain Q27, 1 g was added to 3 g of soil collected from the Kanto Loam Formation in the east Kanto region.
M9 medium 1 containing 10 ppm of 3-dichloropropene
0 ml was added, and the mixture was cultured at 22 ° C. for 10 days with shaking. 1 ml of the suspended culture solution was added to 10 ml of a medium having the same composition, and the mixture was similarly cultured with shaking for 7 days. This operation was repeated 3 times, and when the culture solution, which had been cultured for one month for convenience, was applied to the M9 agar medium containing 0.2% of yeast extract, colony formation was observed. M9 containing 10 ppm 1,3-dichloropropene and 0.2% yeast extract for this colony
The medium was inoculated and examined for its ability to decompose 1,3-dichloropropene, and Pseudomonas species Q27 strain having 1,3-dichloropropene decomposing ability was isolated. Example 2 Degradation of 1,3-dichloropropene by strain Q27 (liquid culture system) The colonies on the agar medium of strain Q27 obtained as in Example 1 contain 0.2% of yeast extract in a Sakaguchi flask. 200 ml of M9 medium was inoculated and shake culture was performed at 22 ° C. for 20 hours.

Next, 1,3-dichloropropene (cis (c
(is) type, trans type mixed product; manufactured by Kanto Chemical Co., Ltd.) and 5 ml of M9 medium containing 0.1% yeast extract were injected into a vial, and 0.1 ml of the bacterial solution cultured as described above was added. After inoculation, it was completely sealed with a butyl rubber stopper and an aluminum cap, and cultured with shaking at 22 ° C. The amount of cis-type and trans-type 1,3-dichloropropene in the sample was quantified by gas chromatography by a headspace method, and the cis-type and trans-type were sequentially measured.
The type 1,3-dichloropropene reduction was measured. As a control, in the same experimental system, the amount of cis-type and trans-type 1,3-dichloropropene was also quantified in a system in which the strain Q27 was not added, and the residual rate relative to the amount of the control was obtained. The results are shown in Fig. 1.

Both the cis type and the trans type were decomposed almost completely in 3 days. Example 3 Decomposition treatment of 1,3-dichloropropene in soil by strain Q27 (brown forest soil) The same 1,3-dichloropropene 2 used in Example 2
1 ml of M9 medium containing 0 ppm and 0.2% yeast extract
Was added to a vial, 4 g of brown forest soil was added, and 0.1 ml of the bacterial solution cultured as in Example 2 was further inoculated, followed by complete sealing with a butyl rubber stopper and an aluminum cap.
Static culture was performed at 2 ° C. Cis type and tra in sample
The amount of ns-type 1,3-dichloropropene was quantified by gas chromatography by the headspace method, and the decrease in cis-type and trans-type 1,3-dichloropropene was measured over time. As a control, cis-type and trans in a system without addition of strain Q27 in a similar experimental system
The amount of 1,3-dichloropropene in the mold is also quantified,
The residual rate with respect to the amount of 1,3-dichloropropene of the control was determined. The results are shown in Figure 2.

Both the cis type and the trans type were decomposed almost completely in 5 days. Example 4 Separation treatment of 1,3-dichloropropene in soil by strain Q27 (loam soil) A cis-type and trans-type 1,3 chronological process was performed in the same manner as in Example 3 except that the soil sample was loam soil. -The reduction of dichloropropene was measured. The results are shown in Fig. 3.

Both the cis type and the trans type were decomposed almost completely in 5 days. Example 5 Decomposition treatment of 1,3-dichloropropene in soil by strain Q27 (fine sand soil) The same method as in Example 3 except that the soil sample was fine sand soil (silt content: about 10%) Cis type and t
The reduction of trans type 1,3-dichloropropene was measured. The results are shown in Fig. 4.

Both the cis type and the trans type were decomposed almost completely in 4 days. Example 6 Decomposition treatment of 1,3-dichloropropene in the gas phase by aeration of the culture solution using the Q27 strain 0.1 ml of the bacterial solution of the Q27 strain cultured as in Example 2
30 ml in a vial containing 0.1% yeast extract
Of M9 medium. Air aerated in a saturated aqueous solution of 1,3-dichloropropene was allowed to flow into the solution for 30 minutes at a flow rate of 60 ml / minute, then completely sealed with a butyl rubber stopper and an aluminum seal, and shake culture was performed at 22 ° C. The amount of 1,3-dichloropropene was quantified by gas chromatography by the headspace method, and the cis-type and tran values were calculated daily.
S-type 1,3-dichloropropene was measured.

As a control, Q27 in a similar experimental system
Cis-type and trans-type 1,3 in a strain-free system
-The amount of dichloropropene was also quantified, and the
The residual ratio with respect to the amounts of s-type and trans-type 1,3-dichloropropene was determined. Results are shown in FIG.

95 days after 4 days for both cis type and trans type
It was decomposed by more than%. Example 7 Decomposition treatment of 1,3-dichloropropene in the gas phase by soil aeration using the Q27 strain 0.1 m of the bacterial solution of the Q27 strain cultured in the same manner as in Example 2.
30 ml in a vial containing 0.2% yeast extract
In addition to 1 M9 medium, sterilized brown forest soil was added to the surface of the water. After sealing with a butyl rubber stopper and leaving it at 22 ° C. overnight, the excess culture solution was decanted and removed. Air aerated in a saturated aqueous solution of 1,3-dichloropropene was allowed to flow into the soil for 30 minutes at a flow rate of 60 ml / minute, then completely sealed with a butyl rubber stopper and an aluminum seal, and shake cultured at 22 ° C. The amounts of cis-type and trans-type 1,3-dichloropropene in the sample were quantified by gas chromatography by the headspace method, and the amounts of cis-type and trans-type 1,3-dichloropropene were measured daily.

As a control, Q27 in a similar experimental system
Quantification of 1,3-dichloropropene was also performed in a system without addition of strain, and cis-type and trans-type 1,3
-The residual rate with respect to the amount of dichloropropene was determined. Results are shown in FIG.

Both the cis type and the trans type are 95 in 4 days.
It was decomposed by more than%.

[0043]

INDUSTRIAL APPLICABILITY The novel 1 brought about by the present invention
3-Dichloropropene-degrading bacterium Pseudononas
sp. Efficient biodegradation of an aqueous medium containing 1,3-dichloropropene, soil and air by the Q27 strain, and efficient purification of the environment such as an aqueous medium contaminated with 1,3-dichloropropene, soil or air.・ Repair is possible.

[Brief description of drawings]

FIG. 1 shows the decomposition of 1,3-dichloropropene by a liquid culture system using strain Q27.

FIG. 2 is a diagram showing decomposition of 1,3-dichloropropene by brown forest soil using Q27 strain.

FIG. 3 is a view showing decomposition of 1,3-dichloropropene by loam soil using Q27 strain.

FIG. 4 is a diagram showing the decomposition of 1,3-dichloropropene by fine sand soil using the Q27 strain.

FIG. 5 is a view showing decomposition of 1,3-dichloropropene by aeration of a culture medium using Q27 strain.

FIG. 6 is a diagram showing decomposition of 1,3-dichloropropene by soil aeration using strain Q27.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI C02F 3/34 C12R 1:38 // (C12N 1/20 B01D 53/34 120D C12R 1:38) B09B 3/00 E (56 ) References Appl Environ Micro obiol, 1992, Vol. 58, No. 9, p. 3038-3046 Soil Biol Biochem, 1995, Vol. 27, No. 12, p. 1547-1557 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 1/20 BIOSIS / WPI (DIALOG) PubMed

Claims (22)

(57) [Claims] 1. A novel microorganism Pseudomonas sp. With dichloropropene resolution (Pseudomon
as sp. ) Q27 strain. 2. A medium containing dichloropropene, wherein Pseudomonas s ( Pseudomonas s)
p. ) A biodegradation method of dichloropropene, which comprises contacting strain Q27 to decompose dichloropropene. 3. The method of claim 2, wherein the medium is an aqueous medium. 4. The method according to claim 3, wherein the contacting comprises contacting the carrier supporting the microorganism with an aqueous medium containing dichloropropene. 5. The method according to claim 4, wherein the contact is performed by accommodating the carrier carrying the microorganism in a container, and introducing the aqueous medium containing dichloropropene from one of the containers and discharging it from the other. the method of. 6. The method according to claim 2, wherein the medium is soil. 7. The method of claim 2 wherein the medium is air. 8. The dichloropropene is cis-1,3-
The method according to claim 2, which is one or more of dichloropropene and trans-1,3-dichloropropene. 9. A medium contaminated with dichloropropene,
An environmental purification / restoration method comprising contacting the microorganism to decompose dichloropropene. 10. The method of claim 9, wherein the contaminated medium is an aqueous medium. 11. The method according to claim 10, wherein the contacting comprises contacting the carrier carrying the microorganism with an aqueous medium containing dichloropropene. 12. The method according to claim 11, wherein the contact is performed by accommodating the carrier carrying the microorganism in a container, and introducing an aqueous medium containing dichloropropene from one of the containers and discharging it from the other. the method of. 13. The method according to claim 9, wherein the polluting medium is soil. 14. The method according to claim 13, wherein the microorganism is grown in the soil by introducing an aqueous medium containing the microorganism into a contaminated soil and supplying nutrients and / or oxygen. Method. 15. The method according to claim 14, wherein the introduction of the microorganism into the soil is carried out by pressure from an injection well provided in the soil. 16. The method according to claim 13, wherein the contacting introduces a soil containing dichloropropene into a liquid phase containing the microorganism. 17. The method according to claim 13, wherein the contacting is carried out by contacting the carrier carrying the microorganism with the soil containing dichloropropene. 18. The method of claim 9, wherein the contaminated medium is air. 19. The method of claim 18, wherein the contacting introduces contaminated air into the liquid phase containing the microorganisms. 20. The method according to claim 18, wherein the contacting comprises contacting polluted air with a carrier carrying the microorganisms. 21. A carrier is placed in a container, the contacting of which carries the microorganism, and polluted air is introduced from one of the containers,
21. The method according to claim 20, characterized in that it is discharged from the other. 22. The dichloropropene is cis-1,3.
22. The method according to claims 9 to 21, which is one or more of dichloropropene and trans-1,3-dichloropropene.