JPH04501667A - Method for decomposing halogenated hydrocarbons using methane oxidizing bacteria that produce methane monooxygenase - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Halogenated hydrocarbons using methane oxidizing bacteria that produce methane monooxygenase Decomposition method of field of invention This invention relates to the use of halogenated hydrocarbon compounds including trichlorethylene (TCE). A biodegradation method using soluble methane monooxygenase or soluble methane monooxygenase. The present invention relates to a method using methane-oxidizing bacteria containing oxygenase.

Background of the invention Halogenated hydrocarbon compounds are bulk products of the chemical processing industry.

For example, over 6 million metric tons of trichlorethylene (TCE), Chloroethylene (PCE), dichloroethane, carbon tetrachloride (CT) and chlorine Roform (CF) is produced annually in the United States. most frequently from groundwater. The halogenated hydrocarbon compounds often found are low molecular weight aliphatic halogenated carbons. Hydrogen: TCE, dichloroethane (DCA), ) dichloroethane and PCE be. Many of these aliphatic halogenated hydrocarbon compounds, including TCE, are Listed as a priority pollutant by the United States Environmental Protection Agency and known or suspected carcinogen. substances and mutagens. In addition, haloform (halogenation inducer of methane) conductors) are often detected in groundwater and drinking water. Water supply to Haloform Some are produced during the chlorination process, but improper treatment techniques or accidental spills may also be responsible for the presence of these haloforms.

Some of the halogenated hydrocarbon compounds mentioned above are resistant to biodegradation in aerobic subaqueous environments. or their biological transformation is incomplete under anaerobic conditions. example For example, under anaerobic conditions, TCE and PCE have similar or is more problematic than the partial bioconversion of the compound to vinyl chloride. is known. Wilson and Wilson, Applied & Ember Ironmental Microbiology, 49:242-243 (1985) .

Latest recycling technology for groundwater contaminated with these halogenated hydrocarbon compounds Then, the water is pumped to the surface and the contaminants are removed in an aeration tower, or or remove contaminants with adsorbents. Regarding the previous method - some states do not allow C. Yes, and later methods are expensive and produce concentrated toxic substances that can cause future problems. Accompanied by formation.

In another recycling method, acetate-degrading methanogenic bacteria are It has been reported that it decomposes hydrogen compounds. Chloroform (CF), bromo Dichloromethane (BDCM), dibromochloromethane (BDCM), bromo Form (BF), carbon tetrachloride (CT), 1°1.1-1-IJ oo-1' 1,1,1-TCA), 1,1,2,2-atrachloroethane (1,1, 2,2-TECE) and PCE were all inoculated with methanogenic mixed bacterial cultures. method using an anaerobic column containing r-cetate as the next substrate in the medium. Substantially decomposed under tan-forming conditions. These conditions with a retention time of 2 days A continuous flow fixed film laboratory scale column operated at approximately 15-40 μg/ These compounds present at column injection concentrations in the range of 1 were substantially removed. C F, significantly removes 1.1.1-TCA and 1.1.2.2-TECE. The acclimatization period required was approximately 10 weeks.

Buware and Matsukaty, Applied & Environmental - Microbiology, 45:1286-1294 (1983).

Other anaerobic bacteria are also known to degrade halogenated hydrocarbon-containing compounds. It is. For example, the anaerobic bacterium Methanobacterium chir7autotrophicum and D autotrophicum convert carbon tetrachloride into di and trichloromethane. has been shown to partially dehalogenate other chlorinated aliphatic compounds. Ta. Egri et al., FEMS Microbiology Letters, 43:25 7-261 (1987). The above results support the use of methanogenic or other anaerobic bacteria. demonstrated that complete decomposition of all halogenated hydrocarbons by are doing. These microorganisms can survive even at low initial concentrations of halogenated hydrocarbons. Rogenated hydrocarbons have a slow rate of decay and do not work when anaerobic conditions are required. Peg.

Furthermore, chloroform is effective against the aerobic organism ``Methylococcus cubus Russ''. per gram of cells at a rate of 35 nanomoles/min. Higgins et al. , Nature, 286:561-564 (1980); Haber et al., Sci. Enns”, 221:1147-1153 (1983). ``Methylococcus capu Other haloforms that were not oxidized by the "Slatus" bath (but not carbon tetrachloride) A similar rate of decomposition was observed in the case of Higgins et al., supra, Harbor etc., supra.

Certain methane-oxidizing bacteria produce chlorinated haloforms and halogenated hydrocarbon compounds. It is known to break things down. For example, 0.6% natural gas (mainly At the end of the 3-week acclimatization period, the column inflow was exposed to a surface mixture of (as methane) Soil column with water containing TCE added to the water at an average concentration of 150 μg/l , less than 5% of the applied TCH passed through the soil. wilson and Wilson, supra. Additionally, methane-utilizing mixed cultures isolated from wetlands are , recently, TCE, vinyl chloride, vinylidene chloride and dichloroethylene dioxide It has been shown to completely oxidize to carbon.

7 Orgel et al., Appl, Env, Microbiol, 51 Near 2O- 724 (1986). However, the TCE degradation reported by Orgel et al. The rate was very slow, approximately 2.5 micromoles/hour per gram of cells.

Furthermore, tetrachlorethylene was not oxidized by the mixed culture.

The above test is suitable for some chlorinated haloforms and halogenated hydrocarbon compounds. can be degraded by combined aerobic/anaerobic incubation under Which indicates that. However, halogenated hydrocarbon compounds such as TCH methane-oxidizing bacteria or methanodroa troph)'s true potential has yet to be exploited. For example, TCE is When added to the soil column used by Ken and Wilson (ibid.), the soil , had previously been acclimated to the natural gas mixture for 3 weeks. Similarly, Bware and Matsuka Significance of 1.1.1-TCA and 1.1.2.2-TECE in Tee test The acclimatization period required for complete removal was approximately 10 weeks.

Obligate methanodoroa derives no energy from the metabolism of compounds other than methane. It has been known for a long time that it does not. Haber et al., supra; Higgins et al., supra. . However, Methanodoroa can degrade many hydrocarbon compounds . The oxidizing ability of methanodroa for a wide range of compounds is In conjunction with the lack of specificity of the enzyme produced, methane monooxygenase (MMO) There is. Haber et al., supra; Higgins et al., supra. MMO of methanotrophic bacteria The system produces methanol by cleavage of 0□ and incorporation of monooxygen atom into methane. catalyze the growth of

The MMO system of methanotrophic bacteria can be either soluble or particulate (such as ie, membrane-bound). Preuss et al., “Journal of General Le Microbiology J, 30:3327-3333 (1984). methanotrophic bacterium Metylosinus trichosporium OB 3b (MtO B3b) The copper availability during growth depends on the intracellular location of its MMO (i.e. , whether MMO activity is located in bacterial particles or in soluble 7-lactones) I would like to report that. However, methanotrophic bacterial cells The tendency to assimilate only membrane-bound (particle) forms is a recurring trend in large-scale purification of soluble MMO. This is a recurring problem. Fox and Lipscomb, “Biochem. Cal and Biophysical Research Communications,” 154. :165-170 (1988). Preuss et al. (supra) found that the particle MMO was aromatic or The particle shape of MMO of MtOB3b is unique in that it cannot oxidize or alicyclic hydrocarbon compounds. reported that the state is different from the soluble form of the enzyme. MtOB3b MMO particles and soluble forms both contain methane, propene and various n-alkanes. It has been shown to oxidize.

However, to date, the degradative abilities of methanotrophic bacteria, especially these By fully developing the ability to degrade the soluble form of MMO produced by bacteria. However, no one has been able to rapidly and completely decompose halogenated hydrocarbon compounds. for example, The rates of TCE degradation by methanotrophic bacteria reported to date are satisfactory. It is so slow that it is impractical for commercial use. TC reported under optimal conditions The E-decomposition rate is slightly over 100 micromoles/hour per gram of cells. . Vogel et al., supra, Nelson et al., Applied and Environmental Men. Tal Microbiology, 54:604-606 (1988), Nelson et al. , Applied and Environmental Microbiology, 52 :383-384 (1986). Methanol against TCH reported in past studies The time course of the trophic attack indicates that TCE functions in bacterial cells or in TCE degradation. This suggests that it is toxic to enzymes in some way.

Therefore, using a soluble form of MMO or culturing in a manner that produces soluble MMO halogenated hydrocarbon compounds, e.g. There is a need for a method to rapidly and completely degrade TCE.

Summary of the invention The present invention provides a method for microbial degradation of halogenated hydrocarbon compounds.

This method involves applying a halogenated hydrocarbon compound, e.g. TCE, at t-per gram of cells. About 500 to about 10,000 micromoles, effective for complete degradation at a rate of 7 hours contacting a quantity of methane-oxidizing bacteria with the halogenated hydrocarbon compound. nothing. Methane-oxidizing bacteria are cultivated under continuous culture conditions, and the bacteria are separated from air and methane. exposed to a continuous flow of a gas mixture containing about 25:1-1:10 of each . Continuous culture bacteria produce soluble methane monooxygenase (MMO).

Preferably, the halogenated hydrocarbon decomposition rate according to the present invention is About 1000 to about 9000 micromoles/hour.

In a preferred embodiment, we use Metylocinus trichosporium 0B3b cells. 2000 to 4000 micromorph' hours of TCE per gram dry weight of Achieved solving speed. The present invention is 1ooo.

Provides a method for decomposing halogenated hydrocarbons present at initial concentrations below micromol/l. , preferably at initial concentrations from traces of TCE to about 1000 micromol/l. A method for decomposing halogenated hydrocarbon compounds, such as TCE, is provided. In addition, continuous culture Cultured cells were grown with soluble M at cell densities significantly lower than those required for other tests. Furthermore, in preferred embodiments, Methylocinus trichosborium does not produce MO. The cells are present in an amount of about 0.10 g/l to about 20 g/l, most preferably about 0.2 to 2.0 g/l. Used in an amount of 0 g/l. The air/methane mixture used for continuous cultivation is variable. obtain. We preferably decompose the TCE in an amount of about 1 to about 20% methane saturation. It was found that it is stimulated when it is present.

This method allows it to rapidly and completely remove halogenated hydrocarbon compounds such as TCE. It is advantageous in that it can be decomposed. Used in the present invention to culture methane-oxidizing bacteria Continuous culture conditions prevent complete degradation of TCE by bacteria when TCE concentrations are significant. Assure. Furthermore, by using these continuous culture conditions, a sufficient amount of MMO can be obtained. Methane-oxidizing bacteria are produced that produce the soluble form. Using the continuous culture conditions of the present invention When cultured, the amount of MMO in the cultured cells is about 5-30% of the dry cell weight.

The present invention also provides methane oxidizing particles that can completely decompose halogenated hydrocarbon compounds. A method for culturing bacteria is provided. This method involves continuously culturing methane-oxidizing bacteria. soluble amounts effective for complete degradation of halogenated hydrocarbon compounds by bacteria. to produce sexual MMO.

Continuous culture conditions include a ratio of air to methane of about 25:1 to about 1:20, preferably about 10 :1 to about 1:2, and most preferably about 2.1:1. Includes exposure to Preferably, the amount of MMO in the cultured cells is approximately 5% to about 30%.

Furthermore, the present invention provides halogenated hydrocarbon compounds using methane monooxygenase. A method of decomposing substances, in which methane-oxidizing bacteria are continuously cultivated and soluble methane is extracted from them. Isolating monooxygenase and purifying soluble methane monooxygenase The purified components reductase, component B and hydroxylase were obtained by by adding to an aqueous slurry of halogenated hydrocarbon compound to form a mixture. , allowing the mixture to react for a time sufficient for complete decomposition of the halogenated hydrocarbon compound. Provide a method for including.

Other features and advantages of the invention will be apparent from the following detailed description and claims. It should be white.

Brief description of the drawing Figure 1 shows the results of continuous culture MtOB3b measured at two different bacterial cell density values. FIG. 3 is a diagram showing the time course of TCE decomposition.

Figure 2 shows TCE by continuous culture MtOB3b in the presence of different levels of methane. FIG. 3 is a diagram showing the time course of decomposition.

FIG. 3 is an assembly diagram of a constant constituent culture tank of the type used for culturing the culture according to the invention. It is.

FIG. 4 shows a constant component fermenter growth flask of the type used for culturing cultures according to the invention. Showing Suko's head.

FIG. 5 shows a constant component fermenter growth flask of the type used for culturing cultures according to the invention. Indicates the main body of Sco.

Detailed description of the invention Halogenated hydrocarbon-containing compounds The present invention provides a method for rapidly and completely decomposing halogenated hydrocarbon compounds. . The present invention preferably provides a method for decomposing trichlorethylene (TCE), Other halogenated hydrocarbons that can be decomposed by the method of the present invention include tetrachloro Ethane, tetrachloroethylene (PCE), l-dichloroethane, dichloroethane Examples include, but are not limited to, chloroform (DCA) and chloroform.

The preferred halogenated hydrocarbon compound of the present invention is TCE (1,1°2-tric Loloethene) is an aliphatic halogenated carbonized compound with the chemical structure HCIc-CC12. It is hydrogen. TCE is primarily used in industry as a refractory solvent. That is a In removing one molecule of hydrogen chloride from acetylene butrafluoride using acetylene butrafluoride can be manufactured from

methanotrophic bacteria In the present invention, the above-mentioned halogenated hydrocarbons are produced by utilizing methane-oxidizing bacteria. Decomposes the contained compounds. Preferably Metylocinus trichosporium OB 3 b (Mt OB 3b) strain of bacteria was used to produce the enzyme methane monooxygenase (M A soluble form of MO) is produced. Other methane-oxidizing bacteria that produce MMO are also present. may be useful in the invention. These other bacteria include Methylocinus subo Methylocinus bulbus and Methylomonas, Methylbacter and Methyromonas. There are other species of the genus Tyrococcus, including but not limited to.

MtOB3b utilizes methane as its sole source of carbon and energy It is an obligate type I+ methanotrophic bacterium that can grow by this bacterium Whittenbury et al. (Journal of General Microbiology J, 61:205-218 (1970)). That's outside forming viable spores, typically found in rosette-shaped clusters of several cells They are gram-negative rod or pear-shaped bacteria. MtOB in methane medium 3b colonies are white-yellow. Similar to other type II methanotrophs, Mt, O B3b contains a complete tricarboxylic acid cycle and is responsible for formaldehyde assimilation. It takes advantage of the serine pathway that MtOB3b DNA contains 62.5 mol% G+C It has a certain percentage. MtOB3b grows at 37°C instead of 45°C. Mt. Growth of OB3b was tested with yeast extract or Whittenbury et al. (supra). is not stimulated by other multi-carbon compounds. t formed by MtOB3b The membrane is composed of short fibers that radiate from the cell wall and can be stained with polysaccharides. does not respond to The MtOBSb strain used in the present invention is from Wau, UK. Obtained from Professor R. Whittenbury of the University of Chicago, National Collection of Industrial Bacteria (Avadi) NC], assigned number B-11131. It is being

Decomposition of halogenated hydrocarbon compounds The present invention provides a method for microbial degradation of halogenated hydrocarbon compounds.

This method produces about 500 to about 10,000 micromoles/hour per gram of cells. methanoic acid in an amount effective to completely decompose the halogenated hydrocarbon compound at a rate oxidizing bacteria, preferably MtOB3b, to a halogenated hydrocarbon compound, preferably T including contacting with CH. In the method of the present invention, methane-oxidizing bacteria are grown under continuous culture conditions. Soluble MrviO is produced by culturing under the following conditions. used here The effective amount of methane-oxidizing bacteria contained in the This is the amount of bacteria that can be broken down into Furthermore, as used in this specification, “continuous culture” The term "conditions" means that methane-oxidizing bacteria absorb air and methane at approximately 25:l-1: 20, preferably a ratio of about 10:1-1:2, and most preferably about 2.1+1 cultured in a continuous displacement medium that is exposed to a continuous flow of a gas mixture containing at a It means that. Continuous culture and growth parameters were described by Kornitsch, [Jer. Null Op Gen Microbiology J, 130:2565-257 5 (1984), and the dilution rate for continuous culture is 1 volume of culture. approximately 0.1 volume/hour. The air to methane ratio of the gas mixture can vary; We show that methane at concentrations of 1-20% saturation greatly stimulates TCE oxidation. I found it.

We used conventional (i.e., flask shaker) methane-oxidizing bacteria used in the present invention. (discontinuous) culture method is used to increase TCE from a medium containing a significant initial concentration of 'rCE. It was determined that the degree of removal was not very satisfactory. more specific For this purpose, we used conventional methods (flas When using MtOB3b grown by concussion), the oxidation of TCE is approximately 5 It was found that removal stopped at 0%.

The halogenated hydrocarbon compound decomposed by this method is preferably about 0.1 ( Methane-oxidizing bacteria in an aqueous medium containing methane-oxidizing bacteria at a rate of 1-20 g/l. more preferably in a proportion of about 0.2 to 2° Og/l. Use bacteria. The preferred aqueous medium used in the present invention is referred to as Higgins medium. Hi The method for producing Gins' medium is described in Cornish et al., "Journal of General Microbiology” (cited above). For manufacturing Higgins medium The method utilized in the present invention in this regard is illustrated in Example 1 below. higgins medium is classified as a minimal salts nitrate medium and contains approximately 250 μg/l Cu” . Other aqueous media suitable for use in the present invention include Batt, etc. ("International ・Journal of Systematic Bacteriology”, 26:226- 229 (1976)) and AMS and NMS minimal media. may include, but are not limited to. Huitztenberg et al. See "Journal of General Microbiology" (cited above).

This method provides an initial concentration of less than 10,000 micromoles/l, more preferably traces of halogenated hydrocarbon compounds present in concentrations ranging from 1000 micromol/l completely decompose the substance, preferably TCE. These concentration values are for hydrocarbon compounds , represents the initial concentration of a halogenated hydrocarbon compound in a solution containing bacteria and an aqueous medium. vinegar. Trace amount refers to the minimum detection limit by the verification technology described in the example below. It is understood that

The method includes at least 500 micromoles/hour of TCE per gram of cells. The TCE degradation rate range is approximately 5oo-ioo per gram of cells. oo micromol/hour. Preferably, this range is about 1000-9000 micromoles/hour. More preferably, the method , TCE degradation rate of approximately 2000-4000 micromoles/hour per gram of cells. achieve degree. However, “1 gram of cells” refers to the dry weight of methane-oxidizing bacterial cells. It is based on the amount of 1 gram.

Culture of methane-oxidizing bacteria Furthermore, the present invention provides for completely decomposing halogenated hydrocarbon compounds, preferably TCE. The present invention relates to a method for culturing methane-oxidizing bacteria. This method uses minimal salt media. Methane-oxidizing bacteria, preferably involves culturing MtOB3b. The above continuous culture conditions are such that air and methane are approximately 25:il:20, preferably about 1021-1:2, and most preferably about 2 ．． It involves exposing the bacteria to a continuous flow of a gas mixture contained in a 1:1 ratio. Like Alternatively, the gas mixture during TCE decomposition includes methane at a concentration of about 1-20% saturation. . This method allows continuous culture of methane-oxidizing bacteria to completely remove halogenated hydrocarbon compounds. It stipulates that it contains an amount of soluble MMO effective for decomposition. Preferably, continuous culture The amount of soluble MMO produced in cultivated bacterial cells is approximately 5-5% of the dry bacterial cell weight. It is 30%.

methane monooxygenase The methanotrophic bacterium MtOB3b produces the enzyme methane monooxygenase (MM can include both particulate (i.e., membrane-bound) and soluble forms of O). tongs etc. , FEBS Letters, 58:293-299 (1975), MMO is MtOB. 3b and that MMO is located in the particle fraction of phospholipase D. Soluble from particle 7 lactation by treatment, sonication or Triton X-100 treatment reported that it can be converted into Tong et al. also found that ascorbate was associated with MtOB3b. is an effective alternative to NADH, both in its crude extract or in its particle fraction. Although an electron donor surrogate, the related CO-bound cytochrome C is inactive for MMO activity. I reported what I thought was essential. However, sterling and dal Tong, “European Journal of Biochemistry J, 96:20. 5-2i 2 (1979) later reported that MM in cell-free extracts of MtOB3b O appears to be soluble and it serves as an electron donor for activity. reported that they require NAD(P)H. In addition, they found that ascorbic acid reported that salt is not an effective donor for MMO of MtOB3b. Also , Starling et al., Biochemical Journal, 177:361-364. (1979) contained its substrate specificity when present in the crude extract of MtOB3b. We tested the characteristics of MMO using several methods.

MMO catalyzes the first step in the oxidative metabolism of methane, where 0. is cleavage By inserting one oxygen atom into the C-H bond of methane, methane A rule is generated. 7 Otz et al., “Journal of Biological Chemistry” Street J, 263:10553-10556 (1988). Reaction at this stage The stoichiometry is shown below.

CH4+H(+)+O□+NADH-CH3OH+H,O+NAD Methane Nico Tinamide Methanol Nicotinamide Adenine Adenine dinucleotide, dinucleotide, reduction oxidation Fox and Lipscomb, “Biochemical and Biophysical ・Research Communications J, 154:165-170 (1988 (which is incorporated herein by reference) provides purified MMO from MtOB3b; The MMO system of MtOB3b was divided into three components. They believe that these three ingredients are all metabolites. was found to be required for in vitro reconstitution of oxidative activity. These ingredients are , Fox and Lipscomb respectively reductase, component B and hydro It is designated as xylase. Used in Fox and Lipscomb purification methods MtOB3b cells are described by Kornitsch et al., “Journal of General Le Microbiology J, 130:2565-2575 (1984). It was cultivated in continuous culture according to the following.

MtOB3b thus cultured reproducibly expresses the soluble form of MMO. and could be cultured with high yield.

The decomposition status of halogenated hydrocarbons by purified methane monooxygenase is as follows: It has been found that the MO component oxidizes TCE. Therefore, the present invention provides methane oxidizing Halogenated hydrocarbon formation by continuous cultivation of bacteria, preferably MtOB3b A method for decomposing a mixture of air and methane in a ratio of about 25:1 to 1:20, preferably about 25:1 to 1:20. a gas mixture comprised in a ratio of 10:1-1:2, and most preferably about 2.1=1. The above-mentioned bacteria were exposed to continuous culture conditions including a continuous flow of the compound, but the culture thus The bacteria containing soluble MMO in an amount of approximately 5-30% of the dry bacterial cell weight), Separating soluble MMO from subcultured bacterial cells and purifying soluble MMO A purified component containing reductase, component B and hydroxylase was obtained by , preferably in a weight ratio of about 110:13:250 of the three components, respectively. The ingredients are an aqueous slurry of a 7-logenated aliphatic hydrocarbon compound, preferably TCH. A mixture is formed by adding the above-mentioned norogenated aliphatic It is envisioned that the process would involve reacting for a sufficient time for complete decomposition of the hydrocarbon compound. It is.

The invention will now be further described by detailed examples.

Example 1 Manufacture of Higgins medium To provide a culture medium for methane-oxidizing bacteria suitable for use in the present invention, the following solutions were prepared. A solution was prepared and stored at 4°C.

100X Higgins Salt Solution Ingredients: Volume %: N　aN　Os　85 To, So, 17 MgSO4・7H,O3,7 CaC1z・2H! 0 04 100x Higgins phosphate solution Ingredients: Volume %: KH2P0. 53.0 Na2HPO486,0 This solution is adjusted to pH 7.0.

500x Higgins Trace Metal Solution Ingredients: Volume %: Zn5Oa・7H! OO, 287 Mn5 Oa” 7 H2O0-223H3BO30,062 NaMoO4・2Hto 0.048 CoC1z’ 6H200,048 KI　O,083 CuSOa@5HxOO-125 Add 1 ml of 1 mmol H2SO4 to 1 liter of trace metal solution.

1 The method for producing Higgins medium is described by Cornish et al. [Journal of Gene Ral Microbiology J, 130:2565-2575 (1984). It is shown.

1000x Higgins iron solution Ingredients: Concentration: FeSO4j 7HzO1,12g/100ml 1100ml Higgins iron melt Add 5 ml of 1 mmol HzSO* to the solution.

10 ml Higgins saline, 10 ml Higgins per liter of desired medium The phosphoric acid solution and 2 ml of Higgins trace metal solution were mixed together. Add distilled water The final capacity was determined by If agar plates have been prepared, 1 liter of liquid medium 17 g of purified agar was added per portion. Allow the mixture to drain slowly over the oven for 20 minutes. autoclaved. When the medium is cool enough to pour into the plate, or Or, before inoculation, add 1+al of Higgins iron solution by filter sterilization per liter. Well, I mixed it carefully. Mark the Higgins agar plate with a red stripe. did.

Example 2 Continuous culture of MtOB3b Approximately 10 hours per generation of yarn in Higgins medium prepared as described in Example 1. A continuous culture of MtOB3b was carried out in which the bacteria grew at a rate of . continuous culture, 0 ．． It was grown in a constant component growth chamber with a capacity of 185 liters. J , Devanfilis and R. Hanson, “Journal of Bacteriology J, 98:222-225 (1969), which is incorporated herein by reference.

゛　The above device consists of a water jacket growth chamber supplied with sterile warm and humid air and Consisting of a constant supply of medium (see Figure 3, arrows indicate medium air, hot water and Showing the flow of effluent water: oxygen was supplied from the air). Regarding Figure 3, The alphabetical representations are (A) constant component culture tank growth 7 lasco, (B) medium pump, (C) Medium reservoir, (D) Constant temperature bath, (E) Constant temperature heater pump, (F) Air humidification chamber, (G) air sterilization chamber, (H) air pump and (1) culture medium. A nutrient collection flask is shown.

a. Growth chamber This chamber consists of two parts: the head (Fig. 4) and the main body (Fig. 5). be done. The head contains two parts, one for the medium and one for the air, and has a ground glass joint. It fits firmly into the main body part.

The main body is water-jacketed to maintain a constant volume in the chamber. It has over 70-devices. The entrance to this over 70-duct is a gas It is shielded by a lath buckle, which prevents traces of foam from spreading. Helps maintain constant volume in the chamber by preventing flow.

The maximum capacity of the growth chamber was 200ml.

b. Constant temperature device A, B, Braun Thermomix II (Melsungen, Germany) constant temperature water pump By using circulated. This hot water also heats the air, which is passed through the growth flask (see Figure 3). . Temperature changes are not required as the Thermomix II is sensitive to changes of 0.1°C. well within the temperature control limits.

CO aeration system Air bubbles were used to provide oxygen and promote mixing of the bacterial culture. B2 -F model aquarium pump (Niegene G.

Danner Manufacturing Company) to extract air from the atmosphere. injected with a pump. First, a wash bottle containing 1% HgCl, via a sintered glass sparger. Pass air through the chamber and then through a warm bath (30°C) of sterile water contained in a wash bottle. Partially submerged in a water bath. Then into the growth chamber via a sintered glass gas sparge tube. Let the air pass through.

25 ml of MtOB3b cells were inoculated into 1851 Higgins medium and incubated at 30"C. Incubated in a growth chamber. Continuously add and drain new medium I let it happen. Continuous air supply is provided by an aquarium pump, and methane is supplied by a pressurized tank. Supplied from. The gas mixture is applied in a volume ratio of approximately 1:1 (CH,:methane). Ta. The growth container was agitated vigorously. Growing MtOB3b cells to various turbidities , measured on a Spectroconitsuta 20 spectrometer (600 nm), at which point two-phase (head) test was performed.

Example 3 Testing of TCE decomposition rate 1. Incubation without head 2 General protocol: Add each culture of MtOB3b to a 1.8 ml serum bottle preheated to 30°C. It was sealed with a 11+nn+ Teflon-reinforced rubber partition. Similar initial dissolved oxygen levels To make comparisons that require a large amount of anaerobic media, we first prepare an anaerobic make-up medium (usually 0.0 , 6 ml) was added to the bottle and the bottle was then sealed. Empty manufactured according to Example 1 At the same time, add air-saturated Higgins medium at 30°C using an airtight syringe (1.0ml). , 1 atm was maintained by removing air through a 25 gauge needle. Finally, the rest The culture was added (concentrated to the desired density) while removing the head with a 25 gauge needle.

TCE is added with a syringe at the bottom of the sealed bottle, and an equal volume is added with a syringe at the top of the bottle. Cultures were removed. Assay time course was started by adding TCE (substrate) . Incubate on a platform shaker with stirring at 200 rpm. The test was carried out at 30°C.

Conventional liquid-liquid extraction method for the determination of halomethanes in water at the 21 billion part level The law is reviewed by Henderson, J.E., G.R. Payton and W.H. Glaze. “Identification and Analysis of Organic Polytan in Water J (ed. L. H. Case, Ann Arbor SA) Jens Publishers), pp. 105-111 (1976). Ru.

Assays were terminated by extraction at the desired time points. In liquid-liquid extraction techniques, internal standards 005m1(7)pentane containing 1,2-dibromoethane was used as into the inverted assay bottle with an airtight syringe, with the needle lower than the first needle level. Collect the drainage solution with a syringe. By centrifuging the bottle at 5000 rpm for 10 minutes The distribution was allowed to reach equilibrium. The organic layer was removed using an airtight syringe and chromatographed. It was placed in a 1 ml serum bottle for graphical analysis. However, sample dilution or In some cases, split injections were required. Electron capture detection methods were preferred. the below described gas chromatography operating parameters were used.

Table 1 Gas chromatography parameters cc: HP5790A (by ECD) ) Column: Non-packed R3L-160 thick film capillary (all-tic ) Injection temperature: 150℃ Detection temperature = 250°C Ramping = 35°C (1st minute), ramping to 120°C at 15°C/min Carrier gas: ■(2 Carrier gas flow rate: 8 ml/l /Dispensing volume: 1m1 Headless test for TCE decomposition: Tests 1, 2, and 3 below were conducted generally in accordance with the headless test method described above. this A summary of the results of the three tests is shown in Table 2 below.

Table 2 = Summary of results of headless space test Test culture turbidity (absorbance 600 nm) TCE utilization rate (many per gram of cells) icromol/hour) (headless assay) 1 1.310 336 2 1.410 1070 3 1.460 2400 The exact assay protocols used and detailed results obtained for each test are provided below. vinegar.

Test l - headless head determination protocol: Absorbance A. . MtOB grown in a constant component culture tank (method) according to -1,310 3b Cell suspension The cell suspension was prepared according to Example 1 in a 120 ml bottle of serum. (115 dilution rate A,...-0- 252). The bottle was emptied and refilled with air with 0% methane content. add one bottle It was heat sterilized and used as a control.

1. Add 79 ml MtOB3b cell suspension and add 1.8 ml (7) cell suspension. The tor was sealed. 11. By adding 25 μl of 4 mmol TCE stock The assay was started at a nominal TCE initial concentration of 25 micromolar. 0.6ml aqueous solution was used as an internal standard and replaced with pentane containing i ppm of 1,2-dibromoethane. The bottle was discarded at 2.5.5.0.10.08 and 15.0 minutes by .

The results of Test 1 are shown in Table 3 below.

Table 3 - Results of test l Operation result: Heat sterilization control, 0% methane: Time [TCE], ppm [TCE], μM2.5 min 1.836 13 ．． 97 5.0 minutes 2.691 20.48 15.0 minutes 2.883 21.94 Operation 2 result: 115 dilution, 0% methane: Time [TCE], ppm [TCE], μM 2.5 minutes 2.103 16.0 1 5.0 minutes 0.261 1.99 10.0 minutes 0.255 1.94 15.0 minutes 0-204 1.55 Test 2 - Headless test Test 2 was conducted according to the same method as in Test 1 above, except that 1/20 and A 1150 dilution was made. The results of Test 2 are shown in Table 4 below.

Table 4 - Results of test 2 A. . - 1.4101/20 dilution A6° from color component culture tank. -0,069 1150 dilution A. . -0,026 Nominal TCE initial concentration - 25 micromolar Operation l result 2 Heat sterilization control, 0% methane: Time [TCE], ppm [TCE], μM 2.5 minutes 2.749 20.9 2 5.0 minutes 2.861 21.77 IO0θ min 2.901 22.08 15.0 minutes 3.132 23.84 Operation 2 result: I/20 dilution rate, 0% methane: Time [TCE], ppm [TCE], μM 2.5 minutes 4.227 32.1 7 5.0 minutes 3.475 26.45 1090 minutes 2.843 21.64 15.0 minutes 2.742 20.87 Operation 3 result: 1150 dilution rate, 0% methane: Time [TCE], ppm [TCE], μM 2.5 minutes 2.924 22.2 5 5.0 minutes 1.843 14.03 1010 minutes 2.717 20.68 15.0 minutes 3.183 24.22 Test 3 - Headless test In a preheated (30'C) 120ml serum bottle, add a color component culture tank (A aoo- 1 ml of MtOB3b cell suspension grown in 1-460) as described in Example 1. The mixture was mixed with 9 ml of 30° C. Higgins medium prepared according to the method described above. fresh higgins broth By adding "spent" medium from the color component culture tank (for 10 min. Decanted medium after sedimentation of cells by centrifugation at 110,000 rp), one bottles were prepared. Seal the bottle with a 20 mm Teflon-reinforced rubber septum, empty it, and release air. (0% methane). One bottle was heat sterilized and used as a control.

Added 1.76 ml of MtOB3b cell suspension and sealed the 1.8 ml serum bottle. (Pressure was equilibrated using a 25 gauge needle). 4 mmol TC of 45 μm The assay was started by adding E stock. While stirring on a shaking bath The bottles were incubated at 30°C. 0.6 ml of aqueous solution was used as an internal standard. By replacing it with pentane containing 1 ppm of 1,2-dibromoethane The bottles were discarded at , 2.5, 5.0, 1080, 15.0 and 20.0 minutes. E Analysis was performed on an HP5790A GC using CD.

The results of Test 3 are shown in Table 4 below.

Table 4 - Results of test 3 A　a　　　o　- 1.4601/l O dilution from color component culture tank A6. ,=0. 140 Nominal TCE initial concentration==lQO micromolar (4 mmol TCE concentration in water ) Dry weight -〇-10g/l Operation result: Heat sterilization control, 0% methane: Hour, minute [TCE], ppm [TCE], μM2.5 15.679 1 i9.335.0 14.713 111.98 10.0 14.760 112.3415.0 15.230 115.91 20.0 14.691 111.81 Operation 2 result: 1/1O dilution in Higgins medium, θ% methane: hours, minutes [TCtJ, ppm [ TCE], μM rate, μM/hour/g2.5 cells 14.119 107. 46 5.0 14-651 111.41 10.0 11.981 91.19 15.9 8.406, 63.98 3200 (10 minutes - 15 minutes) 20.0 7.208 54.86 Operation 3 result: l/10 dilution in spent Higgins medium from a constant-nutrient culture tank, 0% methane: Hour, minute [TCE], ppm [TCE], pM speed, ttM/hour Between/cell 1g2.5 14.277 108.66 5.0 13.757 104.70 10.0 12.924 98.36 1.5.0 8.543 65.02 3960 (10 minutes - 15 minutes) 20.0 7.650 58.22 If the most rapid oxidation of TCH were to take place, the rate at this point in the reaction would be: It was approximately 3500 micromoles/hour per gram of cells. This rate reduces methane No irritation occurred when added to the reaction mixture.

The rate of TCE oxidation in the presence of methane during the above time is linear, and TCE oxidation is Even more complete. Additionally, TCE oxidation is even more rapid in the presence of low concentrations of methane. (Table 7). Therefore, under optimal conditions the cells can contain 1000 It is believed that TCE can be oxidized at a rate of 0 micromoles/hour.

2. Incubation with heads Each culture of MtOB3b was added in a 2 ml volume. Added to 10ml serum bottle. Seal the bottle with a 20m1 Teflon-reinforced rubber partition. Sealed. The assay was started by adding TCE. The head is 8 of the total capacity in the bottle. 0%, but the TCE concentration was added based on 2 ml aqueous phase. TCE is the main It was present in the head. By doing so, the combination occurs in the liquid phase and the head between the glasses. To prevent TCE loss that could occur due to trapping, all bottles was reversed. While stirring the sample at 30°C (on a platform shaker 2 00 rpm) and incubated in reverse direction.

Creation of GC calibration curve Analyze head incubations by direct injection of heads into a gas chromatograph did. This method can be performed without sacrificing the sample. Normal calibration curve for quantification It is important to create The best external standardization method is to conduct a test and By preparing the sample assuming that it will be heat sterilized at 80°C for 10 minutes before adding be. TCE by incubating the sample under test conditions for 30 minutes. left for an appropriate amount of time to distribute among many phases (air, water, cellular matter, etc.) . Head samples were injected into a gas chromatograph using FID or ECD. Many A normal calibration curve was obtained by using multiple TCE concentrations.

Example 4 TCE decomposition by head test Bacterial cells from the continuous culture described in Example 2 or cells diluted in spent Higgins medium. TC cells (2+nl) were added to the assay vial (10 ml vial). A head test for E-decomposition was performed.

The dilution rate of cells in spent Higgins medium has no effect on the rate of TCE oxidation. It was. Therefore, it is unlikely that protective compounds are present in the medium.

The heat sterilization control showed that no TCE was lost from the vial.

The TCE utilization rate in the two-phase headspace test is shown in Table 6 below. Equipped with electronic capture detector t; Calculate the velocity from the peak height of the recorder record from the gas chromatograph. Ta. The gas chromatographic parameters recorded in Table 1 are used here. ;.

Table 4 - TCE decomposition in two-phase test Initial TCE concentration rate in assay glass bottle, cell density of oxidized TCE (g/l) (Micromole) μM/cell 1 samurai, 521 22' 308 ．． 347 22 465 ．． 173 23 461 ．． 070 22 808 Example 5 Effect of MtOB3b cell density on TCE degradation rate In this example, continuous culture The cell density of (produced in Example 2) was increased from 0.825 g/l to 0.695 g/l. Ta. As shown in Figure 1, the TCE degradation rate at low cell density was The initial concentration of CE increased to 1200 micromoles TCE removed per g of cells/hour. Addition.

The rate of methane oxidation by these cells in culture was 2860 m/g of cells. chromol/hour.

Additionally, the curves shown in Figure 1 at two different MtOB3b cell densities are At high cell densities, TCE oxidation was more incomplete than at high cell densities. child This means that there is more biomass available for reaction with reactive intermediate compounds. This shows that cells at high density are resistant to toxic intermediates.

Alternatively, if the oxidation rate per unit dose at high cell densities is slow, It is possible that the rate of formation of intermediates is limited by the rate of their further decomposition. .

Example 6 TCE decomposition by single-phase test To test the hypothesis that gas transfer limits the oxidation rate at high cell densities, a practical TCE degradation rate was tested in a single-phase assay using the headless assay technique described in Example 3. Established. This assay requires an MtOB3b cell density of approximately 0.160 g/l and a An initial TCE concentration of icromolar was performed. The TCE oxidation rate is 2/g of cells. 400 micromoles/hour or 315 mg/hour.

Example 7 Effect of methane present during TCE decomposition In this example, the effect of methane present in the TCE oxidation vessel on the TCE oxidation rate is Measure f:. The MtOB3b cell density was approximately 0.16 g/l and the nominal TCE A headless assay of TCE degradation was carried out according to the method of Example 3 with an initial concentration of 80 micromolar. I made a decision. The results are shown in Table 7 below and FIG. 2.

Table 7 - TCE decomposition rate in the presence of methane TCE oxidation rate added to the reaction system Every time, Methane (% saturation) micromoles per gram of cells/h without 660 5% 3350 10% 3000 20% 875 50% 110 In total, we determined that several batches of cells (batches) were was observed to oxidize TCE at a rate of approximately 4000 micromol/hour.

Example 8 Effect of initial TCE concentration on TCE degradation rate MtOB3b cells were cultured under the following conditions. Continued cultivation.

Medium: Higgins (manufactured as described in Example 1) Gas flow rate: Methane 45ml /min, air 135ml/min Culture capacity: 220ml Culture temperature = 30° Using these cells, a TCE headless space head assay of the type described in Example 3 was performed.

The results are shown in Table 8 below.

Table 8 - Effect of “I’ G E initial concentration on TCE oxidation rate TCE concentration (μM) Micromoles of TCE removed per gram of dry cells/hour From these and other experimental results, K for TCE oxidation.

was concluded to be approximately 5 micromolar.

Example 9 Degradation of TCE at various initial concentrations The continuous culture cell mass described in Example 2 was grown to 0.64 g cells/l. fruit The f:T CE headless decomposition assay was performed using these cells using the method described in Example 3. It was. The results are shown in Table 9 below.

Table 9 - TCE decomposition at various initial TCE concentrations (μM) removed Micromoles of TCE, micromoles 7 minutes 5-10 minutes 10-15 minutes 10-25 minutes 40:196 80 5.69 3.03 320 15.54 10.2 640 10.48 Example 1O Degradation of TCH by purified methane monooxygenase using radiolabeled TCE By reducing nicotinamide adenine dinucleotide (NADH), After incubation with soluble methane monooxygenase in the presence of The oxidation of TCH to product was demonstrated. Three components of methane monooxygenase, Leda 7 ox and Lipscomb (formerly (the disclosures of which are incorporated herein by reference). enzyme The incubation mixture was introduced into a sealed 10 ml septum glass bottle. Each moth The glass bottle contained the ingredients shown in Table 9 below.

Table 10 - Enzyme Incubation Mixture Components Components Amount/Concentration 3-[N-Morphopropanesulfonic acid 1.5ml/(MOPS) buffer’ , pH 7, 525mM NADH” 0.1mM Reductase 3 110Pg Ingredient 8 13μg Hydroxylase 250Itg l Sigma Chemical Company, St. Louis, Missouri.

2 Nicotinamide adenine dinucleotide, reduced form, Sigma Chemical - Company, St. Louis, Missouri.

Coreductase, component B and hydroxylase are These are the three components of methane monooxygenase identified by Comb (supra).

105 nmoles of homogeneously labeled 14C-TCE to a final TCE concentration of 70 μM. Add to each vial by injecting through the rubber septum above the reaction mixture until the I got it. The reaction was allowed to proceed for 3 minutes at 25°C and then treated with sulfuric acid (Sigma) at pH 2-0. Chemical Company, St. Louis, Missouri).

After removing precipitated proteins by centrifugation, the reaction mixture was subjected to high performance liquid chromatography. It was analyzed by HPLC. The HPLC operating parameters used are listed below. It is shown in Table 11.

Table 1l - HPLC operating parameters Column: Aminex HPX-87HHPLC column (Bio-Rad) Mobile phase: 0.01N sulfuric acid (Sigma Chemical Company, St. Louis, MI) Zuri) in 25% acetonitrile (Sigma Chemical Company, Centrol) (Issue, Missouri) Operation mode: Isocratic Speed: 0.3ml per minute Peak detection: Ultraviolet spectroscopy, 210nm This HPLC system is capable of various Authentic standard for organic acid products and trichloroacetaldehyde (chloral) Divided the substance.

Under the enzyme incubation conditions described, 53% of the TCH was analyzed by HPLC. Converted to a detectable product. These products include formic acid, glyoxalic acid, These were roloacetic acid and chloral.

In addition to the above incubations, each of the methane monooxygenase components or performed control incubations omitting NADH.

In all of these control experiments, no TCE oxidation products could be detected. .

That is, this experiment demonstrated that TCE oxidation is a ternary methane monomer in the presence of NADH. It was demonstrated that it is catalyzed by the xygenase enzyme system.

The present invention has been described with reference to various embodiments and preferred aspects and techniques. I posted it. However, many modifications and variations may be made without departing from the spirit and scope of the invention. It should be understood that changes and modifications may be made.

Time (minutes) Time (minutes) Constant component culture tank assembly FIG, 4 FIG, 5 international search report --^--” PCT/US 89105177 International Search Report PCT/US 89105177

Claims (31)

[Claims] (1) A method for microbial decomposition of halogenated hydrocarbon compounds, comprising: Completion of hydrogen compounds at a rate of about 500 to about 10,000 micromoles/hour per gram of bacteria. An amount of methane-oxidizing bacteria effective to completely degrade the halogenated hydrocarbon is brought into contact with the halogenated hydrocarbon. The above-mentioned methane-oxidizing bacteria are cultured under continuous culture conditions, and the above-mentioned continuous For subsequent cultivation, the above bacteria are mixed with air and methane in a ratio of approximately 25:1-1:10, respectively. said continuously cultured methane oxidizing cell. A method in which the bacterium produces soluble methane monooxygenase. (2) Methane-oxidizing bacteria absorb halogenated hydrocarbons at a rate of about 1000 to about 90 per gram of bacteria. 2. The composition according to claim 1, which can be completely decomposed at a rate of 0.00 micromol/hour. Method. (3) Methane-oxidizing bacteria absorb halogenated hydrocarbons at a rate of about 2,000 to about 40 per gram of bacteria. 2. The composition according to claim 1, which can be completely decomposed at a rate of 0.00 micromol/hour. Method. (4) the gas mixture comprises air and methane in a ratio of about 10:1 to 1:2, respectively; The method according to claim 1. (5) Claim in which the gas mixture comprises air and methane in a ratio of about 2.1:1, respectively. The method described in 1. (6) Methane-oxidizing bacteria absorb approximately 0.10-20g/halogenated hydrocarbon compound. 2. The method of claim 1, wherein the method is contacted with an aqueous medium containing μ. (7) Methane-oxidizing bacteria absorb approximately 0.2-2.0 g of halogenated hydrocarbon compounds/ 2. The method of claim 1, wherein the method comprises contacting an aqueous medium containing l. (8) Halogenation by methane-oxidizing bacteria at an initial concentration of 10,000 micromol/l or less 2. The method of claim 1, wherein the method is capable of cracking hydrocarbons. (9) Methane-oxidizing bacteria are exposed to trace amounts of halogenated hydrocarbons at an initial concentration of 1000 m 2. The process as claimed in claim 1, which is capable of decomposing up to 1 mol/l of halogenated hydrocarbons. (10) The method according to claim 1, wherein the methane-oxidizing bacterium is a species of the genus Metylocinus. . (11) The methane-oxidizing bacterium is Metylocinus trichosporium OB3b, The method according to claim 10. (12) Halogenated hydrocarbons containing methane at a concentration of approximately 1-20% saturation. 2. The method of claim 1, wherein the decomposition is carried out in the presence of a gas mixture comprising: (13) the halogenated hydrocarbon compound is an aliphatic halogenated hydrocarbon compound, The method according to claim 1. (14) The aliphatic halogenated hydrocarbon compound is trichlorethylene (TCE). 2. The method according to claim 1. (15) Bacteria are exposed to soluble methane monooxygenate in an amount of about 5-30% of the dry bacterial cell weight. 2. The method of claim 1, wherein the method produces shigenase. (16) Cultivation of methane-oxidizing bacteria that can completely decompose halogenated hydrocarbon compounds A method in which the methane-oxidizing bacteria is separated from air and methane in a ratio of about 25:1-1, respectively. A continuous culture that involves exposing the bacteria to a continuous flow of a gas mixture containing a ratio of: It consists of culturing under nutrient conditions, and the methane oxidizing bacteria continuously cultured above are Approximately 500 to 10,000 micromoles of the above halogenated hydrocarbon compound per 1 g of bacteria. soluble methane monooxygenase in an amount effective to completely degrade it at a rate of A method of producing. (17) The gas mixture contains air and methane in a ratio of about 10:1-1:2, respectively. 17. The method of claim 16. (18) A claim in which the gas mixture contains air and methane in a ratio of approximately 2.1:1, respectively. The method according to item 16. (19) The method according to claim 16, wherein the bacterium is a species of the genus Metylosinus. (20) Claim 16, wherein the bacterium is Methylocinus trichosporium OB3b. How to put it on. (21) Bacteria absorb soluble methane monooxygen in an amount of about 5-30% of the dry bacterial cell weight. 17. The method of claim 16, wherein the method produces a cygenase. (22) the halogenated hydrocarbon compound is an aliphatic halogenated hydrocarbon compound, 17. The method according to claim 16. (23) The aliphatic halogenated hydrocarbon compound is trichlorethylene (TCE). 23. The method of claim 22. (24) A method for decomposing a halogenated hydrocarbon compound, the method comprising: (a) methane-oxidizing bacteria; is a gas mixture containing air and methane in a ratio of approximately 25:1 to 1:20, respectively. The continuous culture conditions described above include exposing the bacteria to a continuous flow of the compound. The methane-oxidizing bacteria that have been (b) the above-mentioned continuous culture methane oxidizing Isolating the above soluble methane monooxygenase from bacteria, (c) Purify the soluble methane monooxygenase separated above to obtain reductase. , obtain a purified component containing component B and hydroxylase, and (d) obtain a purified component containing the purified component. Adding an effective amount to an aqueous slurry of the halogenated hydrocarbon compound to form a mixture. , (e) the mixture is heated for a time sufficient to completely decompose the halogenated hydrocarbon. A method comprising reacting for a period of time. (25) The gas mixture contains air and methane in a ratio of about 10:1-1:2, respectively. 25. The method of claim 24. (26) A claim in which the gas mixture contains air and methane in a ratio of approximately 2.1:1, respectively. The method according to item 25. (27) The method according to claim 24, wherein the bacterium is a species of the genus Metylosinus. (28) Claim 27, wherein the bacterium is Methylosinus trichosporium OB3b. How to put it on. (29) Purified component reductase, component B and hydroxylase each in approximately 1 25. The method of claim 24, wherein the method is added to the aqueous slurry in a weight ratio of 10:13:250. (30) the halogenated hydrocarbon compound is a halogenated aliphatic hydrocarbon compound, 25. The method according to claim 24. (31) The halogenated aliphatic hydrocarbon compound is trichlorethylene (TCE) 31. The method of claim 30.