CA2003100A1 - Rapid degradation of halogenated hydrocarbons by soluble methane monooxygenase or methanotrophic bacteria comprising same - Google Patents
Rapid degradation of halogenated hydrocarbons by soluble methane monooxygenase or methanotrophic bacteria comprising sameInfo
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- CA2003100A1 CA2003100A1 CA002003100A CA2003100A CA2003100A1 CA 2003100 A1 CA2003100 A1 CA 2003100A1 CA 002003100 A CA002003100 A CA 002003100A CA 2003100 A CA2003100 A CA 2003100A CA 2003100 A1 CA2003100 A1 CA 2003100A1
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- methane
- halogenated hydrocarbon
- bacterium
- hydrocarbon compound
- tce
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- C12N9/0004—Oxidoreductases (1.)
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- C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
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- C12Y114/13025—Methane monooxygenase (1.14.13.25)
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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Abstract
Abstract A method is disclosed for degradation of a halogenated hydrocarbon compound such as trichloroethylene (TCE) which utilizes a soluble methane monooxygenase or a bacterium comprising the monooxygenase. Methylosinus trichosporium OB3b is a soluble methane monooxygenase-producing bacterium which when cultivated by continuous culturing comprising exposing the bacterium to a continuous-flow gas mixture of air and methane in a ratio of about 25:1-1:20, respectively. Methylosinus trichosporium OB3b is capable of degrading TCE at rates from about 500-10,000 micromoles per hour per gram cells. The present method is useful to degrade halogenated hydrocarbon compounds such as TCE at initial concentrations up to 10,000 micromoles/l.
Description
3~
RAPID DE~.RADATION OF HALOGE~ATED H~DROCARBONS BY
SOLUBLE METHANE MONOOXYGENASE OR ~ET~IANOTROPHIC
BACTERIA COMPRISIN~ SAME
Field of the Invention This invention relates to methods of biologically degrading halogenated hydrocarbon compounds including trichloroethylene (TCE), wherein said methods utilize a soluble methane monooxygenase or the methane-oxidizing bacterium which comprises the soluble methane monooxygenase.
Backqrou~d of the Invention Halogenated hydrocarbon compounds are high-volume products of the chemical process industry; for example, more than 6 million metric tons of trichloroethylene (TCE), tetrachloroethylene (PCE), trichloroethane, carbon tetrachloride (CT), and chloroform (CF) are produced [in the United States] each year. Those halogenated hydrocarbon compounds most frequently found in groundwater are low molecular weight aliphatic halogenated hydrocarbons: TCE, dichloroethane (DCA), trichloroethane, and PCE. Many of these aliphatic halogenated hydrocarbon compounds, including TCE, have been listed as priority pollutants by the U.S.
Environmental Protection Agency, and are known or suspected carcinogens and mutagens. Haloforms (halogenated derivatives of methane) are al50 frequently detected in groundwaters and drinking waters. Some haloforms are produced during chlorination of water supplies, but ~ ~ , . . . .
~ . . , , ~ , . . .
-: . , ` :. . , , : -:
.
RAPID DE~.RADATION OF HALOGE~ATED H~DROCARBONS BY
SOLUBLE METHANE MONOOXYGENASE OR ~ET~IANOTROPHIC
BACTERIA COMPRISIN~ SAME
Field of the Invention This invention relates to methods of biologically degrading halogenated hydrocarbon compounds including trichloroethylene (TCE), wherein said methods utilize a soluble methane monooxygenase or the methane-oxidizing bacterium which comprises the soluble methane monooxygenase.
Backqrou~d of the Invention Halogenated hydrocarbon compounds are high-volume products of the chemical process industry; for example, more than 6 million metric tons of trichloroethylene (TCE), tetrachloroethylene (PCE), trichloroethane, carbon tetrachloride (CT), and chloroform (CF) are produced [in the United States] each year. Those halogenated hydrocarbon compounds most frequently found in groundwater are low molecular weight aliphatic halogenated hydrocarbons: TCE, dichloroethane (DCA), trichloroethane, and PCE. Many of these aliphatic halogenated hydrocarbon compounds, including TCE, have been listed as priority pollutants by the U.S.
Environmental Protection Agency, and are known or suspected carcinogens and mutagens. Haloforms (halogenated derivatives of methane) are al50 frequently detected in groundwaters and drinking waters. Some haloforms are produced during chlorination of water supplies, but ~ ~ , . . . .
~ . . , , ~ , . . .
-: . , ` :. . , , : -:
.
2~:33~
inadequate disposal techniques or accidental spillage may also be responsible for the presence of these haloforms.
Several of the halogenated hydrocarbon compounds mentioned a~ove are resistant to biodegradation in aerobic subsurrace environments, or their biological transformations are incomplete under anaerobic conditions. ~or example, under anaerobic conditions, TCE and PCE are known to undergo partial bioconversion to vinyl chloride, a compound which is as much or more of a problem as the original contaminants.
I0 Wilson and Wilson, A~ Rnv. Microbiol., ~9:242-243 (1985).
Current technology for reclaiming groundwater polluted with these halogenated hydrocarbon compounds involves pumping water to the surface and stxipping out the contaminants in aeration towers, or removing the pollutants on a sorbent. The former process is not permitted in some states, and the latter is expensive and involves the production of concentrated toxic materials tha~ may present future problems.
In an alternative reclamation method, acetate-degrading methanogenic bacteria have been reported todegrade halogenated hydrocarbon compounds. Chloroform (CE), bromodichloromethane (BDC~), dibromochloromethane (BDCM), ;
bromoform (BF), carbon tetrachloride (CT), 1,1,1-trichloroethane (1,1,1-TCA), 1,1,2,2-tetrachloroethane (1,1,2,2-TECE), and PCE ha~e all been substantially degraded . .
- . ~. . , - -2 ~
under methanogenlc conditions utilizing an anaerobic column with acetate employed as the primary substrate ln a medium seeded with a methanogenic mixed bacterial culture. A
continuous-flow, fixed-film laborato~y scale column operated under these conditions with a 2-day reten~ion time substantially removed these compounds present at column influent concentrations ranging from a~out 15-40 ~g/l. The acclimation period required for significant removal of CF, l,l,l-TCA, and 1,1,2,2- TECE was a~out 10 weeks. Bouwer and McCarty, A~ol. Env. Microbiol., 45:1286-1294 (1983).
Other anaerobic bacteria are also known to degrade halogenated hydrocarbon-containing compounds. For example, the anaerobic bacteria Methano~acterium thermoautotrophicum and D. autotrophicum have been shown to convert carbon lS tetrachloride to di- and trichloromethane, and to partially dehalogenate other chlorinated aliphatic compounds. EgLi et al., FEMS Nicrobiol. Letter, _ :257-261 (1987). The above results indicate that the use of methanogenic or other anaerobic bacteria to completely degrade all halogenated ~0 hydrocarbons is not commercially viable. These organisms exhibit slow rates of halogenated hydrocarbon destruction, even at low initial concentrations of the hydrocarbons, and are difficul~ to wor~ with given that anaerobic conditions are required.
~ , ,, ., . ,, , ~
Additionally, chloroform is oxidized at rates of 35 nano-moles per gram of cells per minute by the aerobe Methy.Zococcus capsu7at~s Bath. ~iggins et al., Nature, 286:561-564 (1980); ~aber e~ al., Science, 221:11~7-1153 5 (1983). SLmilar ra~es of degradation were observed for other haloforms except for carbon tetrachloride, which was not oxidized by Met~ylococcus capsulatus Bath. Higgins et al., su~ra; Haber et al., supra.
Certain methane-oxidizing bacteria are known to degrade chlorinated haloforms and halogenatad hydrocarbon compounds. For example, soil columns exposed to a surface mixture of 0.6% natural gas (primarily me~hane) in air for 3 weeks, and having water containing TCE at an average ~-concentration of 150 ~g/l added to the column influent at the end of the 3-week acclimation period, resulted in less than 5% of the applied TCE passing through the soil. Wilson a~d Wilson, suura. A me~hane-utilizing mixed culture isolated from a marsh has also recently been shown to completely oxidize TCE, vinyl chloride, vinylidene chloride, and dichloroethylene to carbon dioxide. Fogel et al., Appl.
Env. Microbiol., 51:720-724 (1986). However, the rate of TCE degradation reported by Fogel et al. was very ~low, approximately 2.5 ~moles per hour per gram of cells.
Additionally, tetrachloroethylene was not oxidized by the mixed culture.
~3~113QI
The above studies indicate that several chlorinated haloforms and halogenated hydrocarbon compounds are degradable by combined aerobic/anaerobic incubation under the proper conditions. ~owever, the real potential of methane-oxidizing bacteria, or methanotrophs, for ra~idly biodegrading halogenated hydrocarbon compounds such as TCE
has not yet been exploited. For example, when T OE was added to the soil column used by Wilson and Wilson, su~ra, the soil had previously been acclimated to the natural gas mixture for 3 weeks. Similarly, the acclimation period required for significant removal of 1,1,1-TCA and 1,1,2,2 TECE in the Bouwer and McCarty study was about 10 weeks.
It has been known for some time that obligate methanotrophs derive no energy from metabolism of compounds other than methane. Haber et al., supra; Higgins et al., su~ra. However, methanotrophs are able to deqrade numerous hydrocarbon compounds. The ability of methanotxophs to oxidize a wide range of compounds has been associated with the lack of specificity of methane monooxygenase (MMO), an enzyme produced by methanotrophs. Haber et al., supra;
Higgins et al., supra. The M~O system of methano~rophic bacteria catalyzes the cleavage of 2 and incorporation of one oxygen atom into methane to produce methanol.
The MMO system of methanotrophic bacterla can exist in either a soluble or a particulate (i.e., membrane-~ .
- ~
inadequate disposal techniques or accidental spillage may also be responsible for the presence of these haloforms.
Several of the halogenated hydrocarbon compounds mentioned a~ove are resistant to biodegradation in aerobic subsurrace environments, or their biological transformations are incomplete under anaerobic conditions. ~or example, under anaerobic conditions, TCE and PCE are known to undergo partial bioconversion to vinyl chloride, a compound which is as much or more of a problem as the original contaminants.
I0 Wilson and Wilson, A~ Rnv. Microbiol., ~9:242-243 (1985).
Current technology for reclaiming groundwater polluted with these halogenated hydrocarbon compounds involves pumping water to the surface and stxipping out the contaminants in aeration towers, or removing the pollutants on a sorbent. The former process is not permitted in some states, and the latter is expensive and involves the production of concentrated toxic materials tha~ may present future problems.
In an alternative reclamation method, acetate-degrading methanogenic bacteria have been reported todegrade halogenated hydrocarbon compounds. Chloroform (CE), bromodichloromethane (BDC~), dibromochloromethane (BDCM), ;
bromoform (BF), carbon tetrachloride (CT), 1,1,1-trichloroethane (1,1,1-TCA), 1,1,2,2-tetrachloroethane (1,1,2,2-TECE), and PCE ha~e all been substantially degraded . .
- . ~. . , - -2 ~
under methanogenlc conditions utilizing an anaerobic column with acetate employed as the primary substrate ln a medium seeded with a methanogenic mixed bacterial culture. A
continuous-flow, fixed-film laborato~y scale column operated under these conditions with a 2-day reten~ion time substantially removed these compounds present at column influent concentrations ranging from a~out 15-40 ~g/l. The acclimation period required for significant removal of CF, l,l,l-TCA, and 1,1,2,2- TECE was a~out 10 weeks. Bouwer and McCarty, A~ol. Env. Microbiol., 45:1286-1294 (1983).
Other anaerobic bacteria are also known to degrade halogenated hydrocarbon-containing compounds. For example, the anaerobic bacteria Methano~acterium thermoautotrophicum and D. autotrophicum have been shown to convert carbon lS tetrachloride to di- and trichloromethane, and to partially dehalogenate other chlorinated aliphatic compounds. EgLi et al., FEMS Nicrobiol. Letter, _ :257-261 (1987). The above results indicate that the use of methanogenic or other anaerobic bacteria to completely degrade all halogenated ~0 hydrocarbons is not commercially viable. These organisms exhibit slow rates of halogenated hydrocarbon destruction, even at low initial concentrations of the hydrocarbons, and are difficul~ to wor~ with given that anaerobic conditions are required.
~ , ,, ., . ,, , ~
Additionally, chloroform is oxidized at rates of 35 nano-moles per gram of cells per minute by the aerobe Methy.Zococcus capsu7at~s Bath. ~iggins et al., Nature, 286:561-564 (1980); ~aber e~ al., Science, 221:11~7-1153 5 (1983). SLmilar ra~es of degradation were observed for other haloforms except for carbon tetrachloride, which was not oxidized by Met~ylococcus capsulatus Bath. Higgins et al., su~ra; Haber et al., supra.
Certain methane-oxidizing bacteria are known to degrade chlorinated haloforms and halogenatad hydrocarbon compounds. For example, soil columns exposed to a surface mixture of 0.6% natural gas (primarily me~hane) in air for 3 weeks, and having water containing TCE at an average ~-concentration of 150 ~g/l added to the column influent at the end of the 3-week acclimation period, resulted in less than 5% of the applied TCE passing through the soil. Wilson a~d Wilson, suura. A me~hane-utilizing mixed culture isolated from a marsh has also recently been shown to completely oxidize TCE, vinyl chloride, vinylidene chloride, and dichloroethylene to carbon dioxide. Fogel et al., Appl.
Env. Microbiol., 51:720-724 (1986). However, the rate of TCE degradation reported by Fogel et al. was very ~low, approximately 2.5 ~moles per hour per gram of cells.
Additionally, tetrachloroethylene was not oxidized by the mixed culture.
~3~113QI
The above studies indicate that several chlorinated haloforms and halogenated hydrocarbon compounds are degradable by combined aerobic/anaerobic incubation under the proper conditions. ~owever, the real potential of methane-oxidizing bacteria, or methanotrophs, for ra~idly biodegrading halogenated hydrocarbon compounds such as TCE
has not yet been exploited. For example, when T OE was added to the soil column used by Wilson and Wilson, su~ra, the soil had previously been acclimated to the natural gas mixture for 3 weeks. Similarly, the acclimation period required for significant removal of 1,1,1-TCA and 1,1,2,2 TECE in the Bouwer and McCarty study was about 10 weeks.
It has been known for some time that obligate methanotrophs derive no energy from metabolism of compounds other than methane. Haber et al., supra; Higgins et al., su~ra. However, methanotrophs are able to deqrade numerous hydrocarbon compounds. The ability of methanotxophs to oxidize a wide range of compounds has been associated with the lack of specificity of methane monooxygenase (MMO), an enzyme produced by methanotrophs. Haber et al., supra;
Higgins et al., supra. The M~O system of methano~rophic bacteria catalyzes the cleavage of 2 and incorporation of one oxygen atom into methane to produce methanol.
The MMO system of methanotrophic bacterla can exist in either a soluble or a particulate (i.e., membrane-~ .
- ~
3~
bound) form, depending on growth conditions. Burrows et al., J. Gen. Microbiol., 130:3327-3333 (1984), reported that copper availability during the growth of the methanotrophic bacterium Methylosinus trichosporium OB3b (Mt OB3b) determined the intracellular location of its MMO (i.e., wnether l~MO activity was located in the partlculate or the soluble fraction of the bacterium). ~owever, the tendency of methanotrophic bacteria cells to ela~orate only the membrane~bound (particulate) form of ~MO has been a recurring problem in the purification of soluble MMO in quantity. Fox and Lipscomb, Biochem. and Biophys. Res.
Comm., 154:165-170 (1988). Burrows et al., su~ra, reported that the particulate form of the MMO of Mt OB3b differed from the soluble form of the enzyme in that the particulate MMO was unable to oxidize aromatic or alicyclic hydrocarbon compounds. Both the particulate and soluble forms of the MMO of Mt OB3b were shown to oxidize methane, propene, and various n-alkanes.
To date, however, no one has fully exploited the degradation ability of methanotrophic bacteria, nor in particular the degradation ability of the soluble form of the NMO produced by these bacteria, in order to both rapidly and completely degrade halogenated hydrocarbon compounds.
For example, the rates of TCE degradation by methanotrophic bacteria reported thus far are unsatisfactorily slow and : . . .. .
. .
2~
thus impractical for commercial use. Rates of TC~
degradation reported under optimal conditions barely 2xcasd 100 ~moles per hour per gram of cells. Fogel et al., supra;
Nelson et al., ~. Env. Microbiol., 54: 604-606 (1988);
Nelson et al., AD~ . Env. Microbiol., 52: 383-384 (1986).
The time course of methanotrophic a~tac~ upon TCE reported in past studies suggests that TCE is in some way toxic to the bacteria cells, or to the enzymes functional in TC~
degradation.
Accordingly, there is a need for a method to rapidly and completely degrade halogenated hydrocarbon compounds such as TCE by employing the soluble fo~m of M~O, or by employing a methanotrophic bacterium which has been cultured in such a way as to produce the soluble MMO.
Su~marv of the In~ention The present invention provides a method of microbial degradation of a halogenated hydrocarbon compound.
The method comprises con~acting the halogenated hydrocarbon compound with an amount of a methane-Qxidizing ~acterium offective to completely degrade halogenated hydrocarbon compounds such as TCE at a rate from about 500 to about lO,000 micromoles per hour per gram of cells. The methane- - -oxidizing bacterium is cultured under continuous culture conditions in which the bacterium is exposed to a continuous-flow gas mixture of air and methane in a ratio of ~: .
3~
about 25~ 10, respectively. The continuously cultured bacterium produces a soluble methane monooxygenase (~O).
Pref~rably, rates of halogenated hydrocarbon degradation according to the present invention are from about 1,000 ~o about 9,000 micromoles per hour per gram of cells. In a preferred embodiment we have achieved rates of TCE degradation of 2000 to 4000 micromoles per hour per gram dry weight of Methylosinus trichospori~m OB3b cells. The present invention pxovides for degradation of halogenated hydrocarbons present in initial concentrations of up to 10,000 micromoles/l and preferably provides for degradation of halogenated hydrocarbon compounds such as TCE at initial concentrations from trace amounts of TCE up to about 1,000 micromoles/l. Moreover, the continuously cultured cells produce soluble NMO a~ cell densities well below the cell densities required in other studies.
Further, in a preferred embodiment, Methylosinus trichorsporium cells are employed in amounts of from about 0.10g/1 to about 20g/1, most preferably in amounts of from about 0.2 to 2.0 g/l. The air/methane mixture used for continuou~ culturing can vary. We have found that preferably, degradation of TCE is stimulated when methane is present in amounts from about 1 to about 20% of sa~uration~
The present method is ad~antageous in that it both rapidly and completely degrades halogenated hydrocarbon ' ' , .: ' ' ~ ': ' ~)3~
_9_ compounds such as TC~. The continuous culture conditions employed by the present method to culture the methane-oxidizing bacterium ensure that TCE will be completely degraded by the bacterium when the concentration of TCE is S significant. Additionally, the utili~ation of these continuous culture conditions provides for the generation of a methane-oxidizing bacterium which produces sufficient quantities of the soluble form of ~O. Using the continuous culture conditions of the present invention the amount of M~O in the c~altured cells is from about 5 to 30% of the weight of dry cells.
The present invention also provides a method of cultivating a methane-oxidizing bacterium capable of completeLy degrading a halogenated hydrocarbon compound.
The method comprises continuously culturing a methane-oxidizing bacterium so that the bacterium produces soluble ~MO in an amount effective to completely degrade the halogenated hydrocarbon compound. Continuous culture conditions include exposure to a gas mixture of air and methane in a ratio from about 25:1 to about 1:20 preferably from abou~ 10:1 to about 1:2, and most preferably about ;~
2.1:1. Preferably, the amount of MNO in the culture cells is from about 5% to about 30% of the weight of dry cells Further provided by the present invention is a method of degrading a halogenated hydrocarbon compound using ~' .
,., ~:~03~
methane monooxygenase, ~he method comprising continuously culturing 2 methane-oxidizing bacterium; separating a soluble methane monooxygenase therefrom; purifying the soluble methane monooxygenase to yield the purified S components reductase, component B, and hydroxylase; adding the purified components to an aqueous slurry of the halogenatad hydrocarbon compound to form a mixture; and reacting the mixture for a period of time sufficient to completely degrade the halogenated hydrocarbon compound.
LO Other features and advantages of the invention will be apparent from the following detailed description and appended claims.
Brief Description of_the Fiqures FIGURE 1 is a graphical representation o~ the time course of TCE degradation by continuously cultured Mt OB3b, determined at two different values of bacterial cell density.
FIGURE 2 is a graphical representation of the time course of TCE degradation by continuously cultured Nt Ob3b in the presence of different levels of methane.
FIGURE 3 shows a chemostat assembly of the type used to grow cultures in accordance with the present invention.
.
:, ' : : ' FIGURE 4 shows the head of a chemostat growth rlask oî the type used to grow cultures in accordance with the present invention.
FIGURE 5 shows the body of a chemos~at growth flask of the type used to grow cultures in accordance with the present invention.
Detailed Descriptian o~ the Xnvention Haloqenated HYdrocarbon-Containina ComPounds The present invention provides a method o~ rapidly and completely desrading a halogenated hydrocarbon compound.
Although the present invention preferably provides a method of degrading trichloroethylene (TCE), other halogenated hydrocarbons which may be degraded by the presen~ method include, but are no~ limited to, ~etrachloroethane, tetrachloroethylene (PCE), trichloroethane, dichloroethane (DCA), and chloroform.
The preferred halogenated hydrocarbon compound of the present invention, TCE (1,1,2 trichloroethene), is an aliphatic halogenated hydrocarbon with the chemical structure HClC=CCl2. TCE is primarily used in industry as a fire-resisting solvent. It can be produced by removal of one molecule of hydrogen chloride from ~ce~ylene tetrachloride with alkali.
- . . .
: : . , ~ ~ :
.: : . : .
2003~no Methanotro~hic Bacteria The present invention utilizes methane-oxidizing bacteria to degrade the halogena~ed hydIocarbon-containing compounds described above. Preferably, bacteria of the strain Methylosinus trichosporium OB3b (Mt OB3b) are utilized which produce the soluble fo~m of the enzyme methane monooxygenase (MMO). Other me~hane-oxidizing bacteria which produce M~O may be useful in the present ld invention. These other bacteria include, but are not limited to, Methylosinus sporium, Methylocytis parvus and other species of the genera Methylomonas, ~ethyl~acter and Methylococcus.
Nt OB3b is an obligate type II methanotrophic bacterium which is capable of growing on methane as its soLe -source of carbon and energy. This bacterium was discovered by Whittenbury et al., J. Gen. Mic _biol., 61:205-218 (1970). It is a gram-negative rod- or pear-shaped bacterium that forms exospores and which is typically found in rosette-shaped clusters of several cells. Mt OB3b colonies on methane media are white-yellow in color. Like other type II methanotrophs, Mt OB3b contains a complete tricarboxylic acid cycle, and utilizes the serine pathway for formaldehyde assimilation. The DNA of Mt OB3b has a G~C content of 62.5 mol-%. Mt OB3b grows at 3~C, but not at 45~C. Growth of 33~
~13-~t OB3b is not stimulated by yeast extract or ~y other multi-carbon compounds tssted by Whittenbury et al., ~u~ra.
~t OB3b is motile with polar tuffs of flagella. Capsules formed by ~t OB3b consist of short fibers radiating from the cell wall and do not respond to polysaccaride stain. The Mt OB3b strain used in the present invention was obtained from Professor R. Whittenbury, Narwick University, United Ringdom and has been deposited with the National Collection Qf Industrial Bacteria, Aberdeen, Scotland and assigned number NClB-11131.
Deqradation of Haloqenated ~drocarbon Com~ounds The present invention provides a method of microbial degradation of a halogenated hydrocarbon compound.
The method comprises contacting the halogenated hydrocarbon compound, preferably TC~, with an amount of a methane-oxidizing bacterium, preferably ~t OB3b, effective to completely degrade the halogenated hydrocarbon compound at a rate ~rom about 500 to about 10,000 ~micromoles per hour per gram of cells. In the method of the present invention the methane oxidizing bacterium is cultured under continuous culture conditions so as to produce soluble NMO. As used herein an effective amount of methane oxidizing bacterium is an amount of the bacterium capable of completely degrading T OE at the rates stated herein. Further. as used herein, 3~
the phrase "continuous culture conditions~' means that the methane-oxidizing bacterium has been cultured in a con~inuously replaced medium that is exposed to a continuous flow of a gas mix~ure comprising air and methane in a ratio of about 25:1-1:20, respectively; prererably about 10~ 2;
and most preferably about 2.1:1. Continuous culture and growth parameters are as described in Cornish, J. Gen.
~icro., 130: 2565-2575 (1984) with a dilution rate of continuous culture of about 0.1 volumes/volume culture per hour. The air-to-methane ratio of the gas mixture may be varied, and we have found that methane at concen~rations bet~een 1-20~ of saturation greatly stimulate TC~ oxidation.
We have determined that conventional (i.e. shake flask, non-continuous) methods of culturing the methane-oxidizing bacterium utilized in the present invention prove less than satisfactory in terms of the extent of remo~al of TCE from media comprising any significant initial concentration of TCE. Nore specifically, we have found that at initial TCE concentra~ions of one ppm or higher, oxidation of TCE ceased at about 50% removal when ~t OB3b grown by conventional methods (shake flask) was utilized.
The halogenated hydrocarbon compound to be degxaded by the present method i5 preferably contacted with the methane-oxidizing bacterium in an aqueous media which comprises about 0.10-20 g/l of the methane-oxidizi~g ' , ' 2~)3~
bact~rium, more preferably from about 0.2 to 2.Og/l o, the metnane-oxidizing bactsrium is used. ~he preferred aqueous media used in the present in~ention is referred to herein as Higgins media. The method of preparing Higgins media is S disclosed by Cornish et al., J. Gen. ~icro., su~ra. The reci~e utilized in the present invention for the preparation of Higgins media is given in Example 1 below. Higgins media is classified as a minimal salts nitrate media, and contains about 250 ~g/l of Cu2+. Other aqueous media suitable for use in the present invention may include, but are not limited to those described by Pat et al., Int. J. Svstematic Bacteriol., 25: 226-229 (1976) as well as AMS and ~MS
minimal media; see Whittenburg et al., J. Gen. Microiol., su~ra.
lS The present method provides for complete degradation of a halogenated hydrocarbon compound, pre~erably TCE, present at initial concentrations up to 10,000 micromoles/l and more preferably at concentrations of from trace amounts to 1000 micromoles/1. These concentration values represent the initial concentration of the halogenated hydrocarbon compound in a solution comprising the hydrocarbon compound, the bacterium, and the aqueous media. It is to be understood that trace amounts refers to lower limits of detection by assay techniques described in the Examples herein.
- .
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The presenl method achieves rates of TCE
degradation of at least 500 ~ moles per hour per gram of cells with a range or TCE degradation ra~e from about 500 -10,000 ~moles per hour per gram of cells; preferably the range is from about 1000 - 9000 ~moles per hour per gram of cells. ~ore preferably the present me~hod achieves rates oî
TCE degradation about 2000-4000 ~moles per hour per gram of cells, where "gram of cells~' means gram of methane-oxidizing bacterium cells on a dry weight basis.
Cultivation of Methane-OxidizLnq Bacteria The present invention is further directed to a method of cultivating a methane-oxidizing bacterium which is capable of completely degrading a halogenated hydrocarbon compound, preferably TOE . Th~ me~hod comprises culturing the methane-oxidizing bacterium, preferably Nt OB3b, under continuous culture conditions in a suitable minimal salts media, such as Higgins media. The continuous culture conditions comprise exposing the bacterium to a continuous-`~20 flow gas mixture of air and methane in a ratio of about 25:1-1:20, preferably about 10:1-1:2, and most preferably abo~t 2.1-:1. Preferably, the gas mixture during degradation of TCE comprises methane at a concentration of about 1-20% of saturation. The present method provides that the continuously cultured methane-oxidizing bacterium will : .
, -, . -, ., " ~
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comprise an amount of soluble l~O which is effective to completely degrade the halogenated hydrocaxbon compound.
Preferably, the amount of soluble ~O produced in the continuously cultured bacterial cells is from about 5-30% of the weight of the dry bacterial cells.
Methane NonooxYcenase The methanotrophic bacterium Mt OB3b can comprise both a particulate (i.e., membrane ~ound~ and a soluble form of the enzyme methane monooxygenase (NMO). Tonge et al., FEBS Letters, 58:293-2~9 (1975), reported that ~MO is located in the particulate fraction of Mt OB3b, and that MMO
could be solubilized from the particulate fraction by treatment with phospholipase D, sonication, or Triton X-100.
Tonge et al. also reported that ascorba~e was an effective electron donor substitute for NADH both in crude extracts of Nt OB3b or in its particulate fraction, but that associated CO-binding cytochrome c seemed to be essential for MMO
activity. However, Stirling and Dalton, Eur. J. Biochem., 96:205-212 (197~), later reported that the MMO in cell-free extracts of Nt OB3b appeared to be soluble, and that it required NAD(P)H as an electron donor for activity.
Further, they reported that ascorbate was not an effecti~e electron donor for the NNO of Nt OB3b. Stirling et al., Biochem J., 177:361-364 (1979), also characterized MMO in :: - . . . ... . .
2~3~
several ways including its substrate specificity when present in crude abstracts or Mt OB3b.
M~O catalyzes the first step in the oxidation metabolism of methane, in which O2 is cleaved and one oxygen atom is inserted into a C-H bond of methane to yield methanol. Fox et al., J. Biol._Chem., 263:10553-10556 (1988). ~he reaction stoichiometry of this step is shown below:
CH4 + H + O2 + NADH _____, C~30H + ~2O + NAD~
Nethane ~icotinamide Me~hanol Nicotinamide Adenine Adenine Dinucleotide, Dinucleotide, reduced oxidized Fox and Lipscomb, Biochem. & Bio~hys. Res. Comm., 154:165-170 (1988), incorporated by reference herein provided a purified NMO from Mt OB3b and resolved the MMO
system of Nt 0~3b into three components, all three of which they found were required for reconstituting methane-oxidizing activity in vitro. These components were denoted by Fox and Lipscomb as the reductase, component B, and the hydroxylase, respectively. The cells of Nt OB3b utilized in the purification process of Fox and Lipscomb were grown in continuous culture as described by Cornish et al., J. Gen.
Micro., 130:2565-2575 (1984). The Mt OB3b thus cultured reproducibly expressed the soluble form of MMO, and could b~
cultured in high yield.
.
:
:, - :
. . .
: ~ :
De~r~dation of Haloqenated HYdrocarbons by Purified Methane Monooxyaenase We have found that purified ~O components oxidize T G . ~ccordingly, the present invention envisions a method oî degrading a halogenated hydrocarbon compound by continuously culturing a methane-oxidizing bacterium, pre~arably Mt OB3b, by exposing the bacterium to continuous culture conditions including a continuous-flow gas mixture comorising air and methane in a ratio of about 25~ 20, prererably about 10~ 2, and most preferably about 2.1:1, and ~herein the bacterial thus cultured comprises soluble M~O in an amount from about 5-30% of the weight of the dry bacterium cells; separating the soluble ~O from the continuously cultured bacterium cells, purifying the soluble NMO to yield purified components comprising reductase, component B, and hydroxylase; dding an effective amount o~
the purified components, preferably in a ratio of about 110:13:250 by weight of the three components, respectively, .
to an aqueous slurry of the halogenated aliphatic hydrocarbon compound, preferably TCE, to form a mixture; and reacting the mixture for a period of time sufficient to completely degrade the halogenated aliphatic hydrocar~on compound.
The inven~ion will be further described by reference to the following detailed examples.
. - - - . . . ... ~ . . . . . . . . . .. . . .
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-2~-Example I - Pre~aratio~ of Hiq~i~s Medial In order to provide a medium for the cult~vation of the methane-oxidizing bacterium suitable for use in the present invention, the following solutions were prepared and S stored at 4C:
lOOx Hiqqins Salts Solution Inqredient: Volume ~:
NaNO3 85 ~SO4 17 ~gSO67H70 3.7 C~Cl7'2H2O 0.7 lOOx Hiqqins_Phos~hate Solution Inqredient: Volume ~:
KH7P04 53.0 ~0 Na7HPO4 86.0 Adjust this solu~ion to pH 7Ø
500x Hiqqins Trace Metals Solution Inqredient: Volume %~
znSO47H2O 0.287 MnSO47H2O 0.223 H3BO3 0.062 NaM006 2H20 o.n4s CoCl26H20 0.048 ~I 0.083 CuSO45H2O 0.125 Add l ml of l mM H7SO4 per liter of Trace Metals Solution.
.
I The method of preparation of Higgins media is disclosed by Cornish et al., J. Gen. Micro., 1 :2565-2575 ( 1sa4 ) .
:
3~
lOOOx Hiqains_Iron Solution Incredient: Concentration:
FeS0~7H70 1.12g/100 ml Add 5 ml of 1 mM H7S06 per 100 ml of Higgins Iron Solution.
Ten mL of Higgins Salts Solution, 10 ml of Hig~ins Phosphate Solution, and 2 ml of Higgins Trace MetaLs Solution were mixed together per liter of media desired.
Distilled water was added to make up the final volume. If agar plates were being made, 17 g of purified agar per liter of liquid media was added. The mixture was autoclaved for 20 minutes with slow exhaust. When the media had cooled sufficiently to pour plates, or prior to inoculation, 1 ml ~0 of Higgins Iron Solution was added by filter sterilization per liter and mixed carefully. Higgins media agar plates were marked with a red stripe.
. - , ~ .
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Ex~mple II - Co~tinuous C~lture of Mt OB3b A continuous cul~ure or M~ OB3b was perrormed in which tr.e bacterium grew at rate of approximately 10 hours S per generation on Higgins medium prepared as in Example I.
The continuous culture was grown in a chemostat growth chamber having a 0.185 li~er volume. J. Depamphillis and R.
Hanson, J. Bacterial., ~8: 222-225 (1969), incorporated by refarence herein.
The apparatus consisted of a water jacketed growth cham~er supplied with sterile, warm moist air and a constant supply of medium (See Pig. 3 in which arrows indicate the flow of medium air, warmed water and effluent: oxygen was supplied from the atmosphere). Referring to Figure 3, the alphabetic references describe: (A) chemostat growth flask;
(B) medium pump; (C) medium reservoir; (D) constant temperature bath; (E) constant temperature heater pump; (F) air humidifying chamber; (G) air sterilizing chamber, (H) air pump; and (I) culture collection flask.
~0 a. Growth Chamber This chamber is composed of two parts; a head section (Fig. 4) and a body section (Fig. 5). The head contains two parts, one for medium and one for air, and is fitted tightly onto the body section by a ground gLass joint. The body is water jacketed and has an overflow -~ ' : ': ' : :
., . : , : ~, :
~o~n~
device to maintain a constant volume in the cha~ber. The entrance to this overflow duct is shielded by a glass baffle which helps k~ep the volume in the chamber constant by preventing the slight amount of foam produced by sparging from leaving the overflow. The maximum volume of the growth chamber was 200 ml.
b. Constant Temperature Apparatus A. B. Braun Thermomix II (Melsungen, Germany) con~tant temperature water pump was used to circulate warmed water (~0 C) through the water jacket of ~he growth chamber. This warmed water also hea~s the air which is passed into the growth flask (See Fig. 3). The Thermomix II
is sensitive to changes of 0.1 C so the temperature variation is well within the limits of temperature control required.
c. Aeration System Bubbles of air were used to supply oxygen and aid in the mixing of the bacterial culture. Air was pumped rom the atmosphere by a B 2-F Model Aquarium Pump (Eugene G.
Danner Mfg. Co.). The air passed first into a wash bottle ~ontaining 1% HgCl2 Vid a sintered glass sparger and then through a warming bath (30 C) of sterile water contained in a wash bottle which was partially submerged in a water bath.
The air then passed into the growth chamber via a sintered glass gas dispersion tube.
.
-:
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- - , . .
, - . :
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-2~-25 ml of Mt OB3b cells were lnoculated into .185 1 of Higgins media and incubatod in the growth chamber at 30 C. New media was continuously added and expelled.
The continuous air feed was supplied wi~h an aquarium pump, and methane fram a pressurized tank. The gas mixture was applied in a volume ratio of approximately 1:1 (CE4:air).
The growth vessel was stirred vigorously. ~t OB3b cells ~ere grown to various turbidities and measured on a Spectronic 20 spectrometer l600 nm) at which time two-phase (Headspace) assays were performed.
Example III - Assayinc7 for Rate of TOE De~radation 1. Incubation Without Heads~ace2 General Protocol: ~ -~ Each culture of Mt OB3b was added to 1.8 ml serum bottle prewarmed to 30C and sealed with an 11 mm te~lon-lined rubber septa. For comparisons requiring similar initial dissolved oxygen levels, anaerobic make-up media (usually 0.6 ml) was added to the bottles first and then the bottles were sealed. Air-saturated 30C Higgins media prepared as in Example I was added via gas-tight syringe 2 A convenient liquid-liquid extraction method for the determination of halomethanes in water at the part$-per-billion level is disclosed by Henderson, J.E., G.R. Peyton and W.H. Glaze, in L.H. Reith, ed., Identification and Analysis of Orqanic Pollutants in Water, Ann Arbor Science Publishers, pp. 105-lll (1976~.
.
~:0~3~
(1.0 ml), while 1 atm pressure was maintained by allowing ai~ to bleed through a 25 gauge needle. Finally, culture was added (concentrated to give desir2d density) while remaining headspace was bled out through the 25 gauge needle. TC~ was added with a syringe at bottom of ~he sealed bottle, with a syringe at top of the bottle removing equivalent volume of culture. The assay time course was started with TCE (substrate) addition. Incubation was perfo~med at 30C with agita~ion at 200 rpm on a platform shaker.
Assays were terminated by extraction at desired time points. The liquid-liquid extraction technique used 0.6 ml of pentane con~aining 1,2-dibromoethane as an internal standard added via gas-tight syringe ~o inverted assay bottle, while a second syringe with a needle below first needle level collected displaced solution.
Partitioning was brought to equilibrium by centrifugation of bottles at S000 rpm for 10 min. The organic layer was ~emoved by gas-tight syringe and placed in a 1 ml serum bottle for chromatographic analysis. In some cases, dilution of sample or split injection was necessary.
Electron-capture detection was preferred. The following gas chromatography operating parameters were used:
' .
3~
Table I - Gas Chromatoqra~hy Parameters GC: HPS79OA (with ECD) Column: Non-Pakd RS~-160 Thick Film Capillary (Alltech) Injection Temperature: 150C
Detection Temperature: 250C
Ramping: 35C (lst min), ramped to 120C at 15C/min Carrier Gas: X2 Carrier Gas Flow: 8 ml/min Injection Volume: l ml No-HeadsPace Assa~s of TCE Deqradatlon:
The following Tests 1, 2 and 3 were conducted according to the no-headspace assay procedure described generally above. A summary of the results of these 3 tests is given in Table II, below:
Table II - SummarY of Resul~s of No-HeadsPace Assa~s Rate of TCE-utilization Culture turbidity (~moles'hr~1'g cells~~) Test (Absorbance 600 nm) (no headspace assay) l 1.310 336 2 1.410 1070 3 1.460 ~400 -:
:. ' The exact assay protocol used and the detailed results o~t~ined in each test are given below.
Test 1 - ~3C'e~ 8 Protocol:
2 ml of Mt 0~3b cell suspension grown in a chemostat (procedure) with absorbance A6ao = 1.310 were added to 8 ml of 30C Higgins media prepared as in Example 1 in prewarmed 120 ml seruim bottles (l/S diLution A600 = 0.252).
The bottles were evacuated and refilled with air ha~ing 0%
- methane. One bottle was heat-killed and used as a control.
1.79 ml of Mt OB~b cell suspension was added to sealed 1.8 ml serum bottles. 11.25 ~l of 4mMi TCE stocX was added to start the assay with a nominal initial TCE
concentration of 25 ~M. Bottles were sacrificed at 2.5, 5.0, 10.0 and 15.0 minutes by displacing O.6 ml aqueous solution with pentane containing 1 ppm 1,2-dibromoethane as an internal standard.
. . .
The results of Test 1 ars given in Table III, below:
Table III - Test 1 Results R~n 1 Results:
Heat-killed control, 0% methane:
TLme rTCEl, ppm ~TC~ M
2.5 min 1.836 13.97 5~0 min 2.691 20.48 lS 15.0 min 2.883 21.94 Run 2 Results:
1/5 dilution, 0~ methane:
Time ~TCEl, ppm rTCEl, ~M
2.5 min 2.103 16.01 5.0 min 0.261 1.99 10.0 min 0.255 1.94 15.0 min 0.204 1.55 Test 2 - No-~eadsPace Assa~
Test 2 was performed follow mg the same procedures described above for Test 1, except that 1/20 and 1/50 dilutions were performed. The results of Test 2 are given .
in Table IV, below:
~:
Table IV - Test 2 Results A500 = 1.410 from chemostat 1/20 dilution A600 = 0.069 1/50 dilution Asoo = 0.026 Nominal initial TCE concentration = 25 ~M
Run 1 Results:
Heat-killed control, 0% methane:
Time rTC~l, PPm rTcEl~ ~M
2.5 min 2.749 20.92 5.0 min 2,861 21.77 10.0 min 2.901 22.08 15.0 min 3.132 23.84 Run 2 Results:
1/20 dilution, 0% methane:
Time rTcElr PPm rTCEl,_~M
2.5 min 4.227 32.17 5.0 min 3.475 26.45 10.0 min 2.843 21.64 15.0 min 2.742 20.87 Run 3 Results:
1/50 dilution, 0~ methane:
Time rTCEl, Ppm rTcEl~-~M
~0 2.5 min 2.924 22.25 5.0 min 1.843 14.03 10.0 min 2.717 20.68 15.0 min 3.183 24.22 "
: ~, , ' ~ ' . ~
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~es~ 3 - No-Heads~ace Assay In prewarmed (30C) 120 ml serum bottles~ 1 ml or ~t OB3b cell suspension grown ln a chemostat (A600 = 1.460) were mixed with 9 mls of 30C Higgins media prepared as in Example I. One bottle was prepared by adding "spent" media from the chemostat (decant media after pelleting cells with centrifugation - 10,000 rpm for 10 minutes) rather than fresh Higgins media. The bottles were sealed with 20 mm teflon-lined rubber septa, evacua~ed and refilled with air (0~ methane). One bottle was heat-killed and used as a control.
1.76 ml of Mt OB3b cell suspension was added to sealed 1.8 ml serum bottles (25 gauge needle used to equilibrate pressure). 45 ~1 of 4 mM TCE stock were added lS to start the assay. Bottles were inc~bated at 30C with agitation on a shaker bath. Bottles were sacrificed at 2.5, S.-0, 10.O, 15.O and 20.0 mins by displacing 0.6 ml aqueous solution with pentane containing 1 ppm 1,2-dibromoethane as an internal standard. Analysis was performed on a HP 5790A
GC using ECD.
The results of Test 3 are given in Table V
below:
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Table V - Test 3 Results Aso~ = 1.460 from chemostat 1/10 dilution ~0O = 0.140 ~ominal initial TCE concentration = 100 ~ (using 4mM TCE
stock in water) Dry weight = O.lOg.l~
Run 1 Results:
Heat-killed control, 0% methane:
Time, min rTCE1! ppm rTCE1L_~
lS 2.515.67~ llg~33 5.014.713 111.98 10.014.760 112.34 15.015.230 115.gl 20.014.691 111.81 Run 2 Results:
1/10 dilution in ~iggins media, 0% methane:
Rates Time, min rTcEl~ ~pm rTOE l, ~M mole_.h~l.q cells~;
2.5 14.119 107.46 :
5.0 14.651 111.41 10.0 ll.g81 gl.l9 15.0 8.406 63.98 3200 (lOmin-15min) 20.0 7.208 54.86 , -2~3~0 Run 3 Results:
1/10 dilution in spent Hig~ins media from chemostat, 0%
methane:
Rates Time, m_n L~5~ 2m rTcE~ moles.h~~
2.5 14.277 108.66 5.0 13.757 104.70 10.012.924 98.36 15.0 8.543 65.02 3g60 (10 min-lSmin) 20.0 7.650 58.22 _ .
The rate at those times in the reactions when most rapid oxidation of TCE occurred was approximately 3500 ~mol2s/h~'.g cells~l This rate was not stimulated by the addition of methane to the reaction mixture. The rate of TCE oxidation in the presence of methane over time is linear and TCE oxidation is more complete. TCE oxidation also occurs more rapidly in the presence of low concentrations of methane (Table VII). Therefore, it is believed that cells can oxidize TCE at rates of 10,000 ~moles.h~l.g cells~l under op~imal conditions.
- - . : ~ .
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2. Incubation ~ith Heads~ace Each culture of Mt OB3b was added in 2 ml quanti.ties ~o 10 ml serum bottles. Bottles were sealed with 20 mm teflon-lined ru~ber septa. TCE was added to start the S assay. Xeadspace accounted for 80~ of total volume within bottles, but TCE concentration was added based on 2 ml aqueous phase. TCE resided principally in the headspace.
All bottles were inverted in order to prevent possible TCE
loss by trapping the compound in the headspace between the liquid phase and glass. Samples were incubated inverted at 30C with agitation t200 rpm on a platfo~m shaker).
Pre~aration of GC Cali~ration Curves:
Headspace incubations were analyzed by direct injection of headspace into a gas chromatograph. This method may be performed without sacrificing a sample. It is important to prepare sound calibration curves for quantitation. The best method for external standardization is to prepare samples as if running an assay and heat-killing at 80C for 10 mins prior to TCE addition. Sampleswere incubated for 30 mins under test conditions ~o allow adequate time for TCE to partition among the numerous phases (air, water, cell material, and the like). Headspace samples were injected into a gas chromatograph using either FID or ECD. Numerous TCE concentrations were used to obtain . . . ~ ., ~ , , , :
. , : , a sound standardization curve.
Exampl~ IV - TCE De~radation with ~eadspace Assay Headspace assays of TCE degradation were conductad by adding bacterial cells from the continuous culture described in Example II or cells diluted with spent Xiggins medium. ( 2 mls) into assay vials (10 ml vials).
Dilution of cells in spent Higgins medium had no effect on the rate of TCE oxidation. Therefore, there do not seem to be protective compounds in the medium.
Heat-killed controls indica~ed that no TCE was lost from the vials.
The rates of TCE utilization in two-phase head space assays are shown in Table VI, below. Rates were calculated from peak heights of recorder tracings ~rom a gas chroma~ograph equipped with an electron capture detector.
The gas chromatograph parameters reported in Table I herein were utilized.
~0 - 2~3~
Table IV - TCE Deqradation in Two~Phase Assay Cell density Initial TCE Rate, mlcromoles in assay vials (g l~l) conc. TCE oxidized-(micromolar) g cells~lhr~
.6gS 22 281 .521 22 308 .347 22 465 .173 23 461 .070 22 ~08 Example V - Effect of Mt OB3b Çell De~sit~ o~ TCE
~ t~
In this example, the cell mass of the continuous culture (generated in Example II) increased to 0.825 g/l from O.6~5 g/l. As shown in FIGURE 1, the rate of TCE
degradation at low cell densities increased to 1200 ~0 micromoles TCE removed hr~lg cells~l at an initial ~CE
concentration of 80 ~N.
The rate of methane oxidation by these cells during culturing was 2860 ~moles hr~lg cells~l The curves at two different Mt OB3b cell densities ~5 shown in FIGURE 1 also illustrate that TCE oxidation was less complete at low cell densities than at high cell densities. Thls indicates that cells at high densities withstand toxic intermediates bscause there is more biomasq available to react with the reactive intermediate compounds.
Alternatively, the slower rates of oxidation per unit mass at high cell densities may have limited the rate of production of toxic intermediates to a rate at which they :~
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, % ~ ~3 were fur~her deqraded.
Exampl~ VI - TC~3 Dec~radation with Sin~le_Phase Assay In order to test the hypothesis that gas transfer limited oxidation rates at high cell densities, the rate of TC~ degradation was assayed in a single phase assay using techniques for no head space assay described in Example III.
The assay was performed at a ~t OB3b cell density of approximately 0.160 g/l, and an initial TCE concentration of 80 ~moles. The rate of TCE oxidation was 2400 micromoLes hr~lg cells~l or 315 mg hr~l.g cells~~. ;
, ' . .' .' :, : ~ .
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Example V~I - Effect of Methane Present Duri~c~
TCE Dearadation In this example the effect of methane present in the TCE oxidation vessel on the rate of oxidation of TCE was determined. A no head space assay of TCE degradation was performed according to the procedures of Example III in which the Mt OB3b cell density was approximately 0.16 g/1, and the nominal initial TCE concentration was 80 ~M. The results are presented in Table VII, below and Figure 2.
Table VII - Rate of TCE Deqradation in Presence of Methane Methane added to Rate of TCE oxidation, reaction system, ~moles-hr~~-g cells~
15(% sat~ration) none 660 5% 3350 10% 3G00 20% 875 50% 110 -We have subsequently observed that some cell batches oxidize TCE at rate of approx.imately 4000 micro-moles hr~l 'g cells~l without any methane present.
' '' .,, ' ~ :' ; . ~ .
. .
. . , ~ . .
., ,,, : , , Example VIII - Effect of Initial TCE Concentra~io~
on TCE Deqradation Rate Mt OB3b cells were continuously cultured under the following conditions:
Medium: Eiggins (prepared as in Example I) Gas flow rates: methane 45 ml/min air 135 ml/min Culture volume: 220 ml Culture temperature: 30C
A TOE no head space degradation assay of the type in Example III was performed using these cells. The results are shown in Table VIII, below:
~0 Table VIII - Effect of Initial TCE Concentration on Rate of TCE Oxidation Concentration micromoles TCE
of TCE, ~M removed-hr~'.g dry cells~L
82g These results and the results of other experiments led to the conclusion that the K, for TCE oxidation is approximately 5 ~M.
~03~no Example IX - Deqradation of TC2 at various Initial Concentrations The cell mass of the continuous culture described in Eæample II was grown to 0.64 g cells.l~l. A TCE no head space degradation assay using the procedure described in Example III was performed using these cells. The results are shown below in Table I~.
Table IX - TCE De~radation at Var-ous Initial TCE
Concentrations Concentration of Nicromoles TC~
TCE, ~m removed, ~moles/min.
S-10 min. 10-15 min.10-2S min.
.
2.96 5.69 3.03 320 15.54 10.2 640 ~0.48 .... .. . .. ..
. . .
- ~ . . .
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Example X - Dearadation of ~CE bv_Purified Methane Monooxvqe~ase Radiolabelled TCE was employed in order to demonstrate the oxidation of TC~ to products upon incubation wi~h soluble methane monooxygenase in the presence of reduced nicotinamide adenine dinucleotide ~NADH). Th~ three components of methane monooxygenase, reductase, component B, and hydroxylase, were purified by the method of Fox and Lipscomb, suPra, the disclosure of which is hereby incorporated by reference. Enzyme incubation mixtures were ~ -conducted in sealed 10 ml sept~m vi~ls, each ~ial containing the ingredienis shown in Table IX, below:
Table X - Enzyme Incubation Mixture ComPonents ::
Inqredient mount/
Concentration 3-[N-morpholino]propanesulfonic acid 1.5 ml/
(NOPS) bufferl at pH 7.5 25 mN
NADH2 0.1 mM
Reductase3 110 ~g Component B 13 ~g ~ydroxylase 250 ~g ____________________________________________ ___ .__________ . , 1 Sigma Chemical Company, St. Louis, Missouri.
2 Nicotinamide adenine dinucleotide, reduced form, Sigma Chemical Company, St. Louis, Missouri. ~.
3 Redùctase, component B, and hydro~ylase are the three -:
components of methane monooxygenase identified by Fox and Lipscomp, supra.
::~
31~0 105 nmol of uniformly labelled l4C-TCE was added to each vial by injection through the rubber septum above the reaction mixtur~ until a final TCE concentration or 70 ~M was reached. ~he reaction was allowed to proceed for 2 min at 25C and then quenched by the addition of sulluric acid (Sigma Chemical Company, St. Louis, Missouri) at pH
2Ø
Following centrifugation to remove precipitated protein, the reaction mixtures were analyzed by high pressure liquid chromatography (HPLC). The HPLC ooerating parameters employed are shown in Table ~I, below:
Table XI - HPLC O~eratinq Parameters Column: Aminex HPX-87H HPLC column (Bio- Rad) Nobile phase: 25% acetonitrile (Sigma Chemical Company, St. Louis, ~0 Missouri) in 0.01 N sulfuric acid (Sigma Chemical Company, St~ Louis, Missouri) Operating mode: Isocratic Rate: 0.3 ml per min Peak detaction: UItraviolet spectroscopy, 210 nm , This HPLC system resolved au~hentic standards of various potentLal organic acid products and ~633~
tri~hloroacetaldehyde (chloral). Under the enzyme incubation conditions described, 53% of the TCE was converted to HPLC-detectable products. These products were formic acid, glyoxylic acid, dichloroacetic acid, and S chloral.
In addition to the above incubations, control incubations were conducted in which each one of the methane monooxygenase componen~s or NADH were omitted. In all of these control experiment~, no products of TCE oxidation were detectable.
Thus, this experiment demonstrated that TCE
oxidation is catalyzed by the three-component methane monooxygenase enzyme system in the presence of NADH.
~ he invention has been described with reference to various specific and preferred embodiments and techniques.
However, it should be understood that many variations and ~;
modifications may be made while remaining within the spirit and scope of the invention. .
.. . . . . .
, :, . . '~ ,.. ... , :
, .
bound) form, depending on growth conditions. Burrows et al., J. Gen. Microbiol., 130:3327-3333 (1984), reported that copper availability during the growth of the methanotrophic bacterium Methylosinus trichosporium OB3b (Mt OB3b) determined the intracellular location of its MMO (i.e., wnether l~MO activity was located in the partlculate or the soluble fraction of the bacterium). ~owever, the tendency of methanotrophic bacteria cells to ela~orate only the membrane~bound (particulate) form of ~MO has been a recurring problem in the purification of soluble MMO in quantity. Fox and Lipscomb, Biochem. and Biophys. Res.
Comm., 154:165-170 (1988). Burrows et al., su~ra, reported that the particulate form of the MMO of Mt OB3b differed from the soluble form of the enzyme in that the particulate MMO was unable to oxidize aromatic or alicyclic hydrocarbon compounds. Both the particulate and soluble forms of the MMO of Mt OB3b were shown to oxidize methane, propene, and various n-alkanes.
To date, however, no one has fully exploited the degradation ability of methanotrophic bacteria, nor in particular the degradation ability of the soluble form of the NMO produced by these bacteria, in order to both rapidly and completely degrade halogenated hydrocarbon compounds.
For example, the rates of TCE degradation by methanotrophic bacteria reported thus far are unsatisfactorily slow and : . . .. .
. .
2~
thus impractical for commercial use. Rates of TC~
degradation reported under optimal conditions barely 2xcasd 100 ~moles per hour per gram of cells. Fogel et al., supra;
Nelson et al., ~. Env. Microbiol., 54: 604-606 (1988);
Nelson et al., AD~ . Env. Microbiol., 52: 383-384 (1986).
The time course of methanotrophic a~tac~ upon TCE reported in past studies suggests that TCE is in some way toxic to the bacteria cells, or to the enzymes functional in TC~
degradation.
Accordingly, there is a need for a method to rapidly and completely degrade halogenated hydrocarbon compounds such as TCE by employing the soluble fo~m of M~O, or by employing a methanotrophic bacterium which has been cultured in such a way as to produce the soluble MMO.
Su~marv of the In~ention The present invention provides a method of microbial degradation of a halogenated hydrocarbon compound.
The method comprises con~acting the halogenated hydrocarbon compound with an amount of a methane-Qxidizing ~acterium offective to completely degrade halogenated hydrocarbon compounds such as TCE at a rate from about 500 to about lO,000 micromoles per hour per gram of cells. The methane- - -oxidizing bacterium is cultured under continuous culture conditions in which the bacterium is exposed to a continuous-flow gas mixture of air and methane in a ratio of ~: .
3~
about 25~ 10, respectively. The continuously cultured bacterium produces a soluble methane monooxygenase (~O).
Pref~rably, rates of halogenated hydrocarbon degradation according to the present invention are from about 1,000 ~o about 9,000 micromoles per hour per gram of cells. In a preferred embodiment we have achieved rates of TCE degradation of 2000 to 4000 micromoles per hour per gram dry weight of Methylosinus trichospori~m OB3b cells. The present invention pxovides for degradation of halogenated hydrocarbons present in initial concentrations of up to 10,000 micromoles/l and preferably provides for degradation of halogenated hydrocarbon compounds such as TCE at initial concentrations from trace amounts of TCE up to about 1,000 micromoles/l. Moreover, the continuously cultured cells produce soluble NMO a~ cell densities well below the cell densities required in other studies.
Further, in a preferred embodiment, Methylosinus trichorsporium cells are employed in amounts of from about 0.10g/1 to about 20g/1, most preferably in amounts of from about 0.2 to 2.0 g/l. The air/methane mixture used for continuou~ culturing can vary. We have found that preferably, degradation of TCE is stimulated when methane is present in amounts from about 1 to about 20% of sa~uration~
The present method is ad~antageous in that it both rapidly and completely degrades halogenated hydrocarbon ' ' , .: ' ' ~ ': ' ~)3~
_9_ compounds such as TC~. The continuous culture conditions employed by the present method to culture the methane-oxidizing bacterium ensure that TCE will be completely degraded by the bacterium when the concentration of TCE is S significant. Additionally, the utili~ation of these continuous culture conditions provides for the generation of a methane-oxidizing bacterium which produces sufficient quantities of the soluble form of ~O. Using the continuous culture conditions of the present invention the amount of M~O in the c~altured cells is from about 5 to 30% of the weight of dry cells.
The present invention also provides a method of cultivating a methane-oxidizing bacterium capable of completeLy degrading a halogenated hydrocarbon compound.
The method comprises continuously culturing a methane-oxidizing bacterium so that the bacterium produces soluble ~MO in an amount effective to completely degrade the halogenated hydrocarbon compound. Continuous culture conditions include exposure to a gas mixture of air and methane in a ratio from about 25:1 to about 1:20 preferably from abou~ 10:1 to about 1:2, and most preferably about ;~
2.1:1. Preferably, the amount of MNO in the culture cells is from about 5% to about 30% of the weight of dry cells Further provided by the present invention is a method of degrading a halogenated hydrocarbon compound using ~' .
,., ~:~03~
methane monooxygenase, ~he method comprising continuously culturing 2 methane-oxidizing bacterium; separating a soluble methane monooxygenase therefrom; purifying the soluble methane monooxygenase to yield the purified S components reductase, component B, and hydroxylase; adding the purified components to an aqueous slurry of the halogenatad hydrocarbon compound to form a mixture; and reacting the mixture for a period of time sufficient to completely degrade the halogenated hydrocarbon compound.
LO Other features and advantages of the invention will be apparent from the following detailed description and appended claims.
Brief Description of_the Fiqures FIGURE 1 is a graphical representation o~ the time course of TCE degradation by continuously cultured Mt OB3b, determined at two different values of bacterial cell density.
FIGURE 2 is a graphical representation of the time course of TCE degradation by continuously cultured Nt Ob3b in the presence of different levels of methane.
FIGURE 3 shows a chemostat assembly of the type used to grow cultures in accordance with the present invention.
.
:, ' : : ' FIGURE 4 shows the head of a chemostat growth rlask oî the type used to grow cultures in accordance with the present invention.
FIGURE 5 shows the body of a chemos~at growth flask of the type used to grow cultures in accordance with the present invention.
Detailed Descriptian o~ the Xnvention Haloqenated HYdrocarbon-Containina ComPounds The present invention provides a method o~ rapidly and completely desrading a halogenated hydrocarbon compound.
Although the present invention preferably provides a method of degrading trichloroethylene (TCE), other halogenated hydrocarbons which may be degraded by the presen~ method include, but are no~ limited to, ~etrachloroethane, tetrachloroethylene (PCE), trichloroethane, dichloroethane (DCA), and chloroform.
The preferred halogenated hydrocarbon compound of the present invention, TCE (1,1,2 trichloroethene), is an aliphatic halogenated hydrocarbon with the chemical structure HClC=CCl2. TCE is primarily used in industry as a fire-resisting solvent. It can be produced by removal of one molecule of hydrogen chloride from ~ce~ylene tetrachloride with alkali.
- . . .
: : . , ~ ~ :
.: : . : .
2003~no Methanotro~hic Bacteria The present invention utilizes methane-oxidizing bacteria to degrade the halogena~ed hydIocarbon-containing compounds described above. Preferably, bacteria of the strain Methylosinus trichosporium OB3b (Mt OB3b) are utilized which produce the soluble fo~m of the enzyme methane monooxygenase (MMO). Other me~hane-oxidizing bacteria which produce M~O may be useful in the present ld invention. These other bacteria include, but are not limited to, Methylosinus sporium, Methylocytis parvus and other species of the genera Methylomonas, ~ethyl~acter and Methylococcus.
Nt OB3b is an obligate type II methanotrophic bacterium which is capable of growing on methane as its soLe -source of carbon and energy. This bacterium was discovered by Whittenbury et al., J. Gen. Mic _biol., 61:205-218 (1970). It is a gram-negative rod- or pear-shaped bacterium that forms exospores and which is typically found in rosette-shaped clusters of several cells. Mt OB3b colonies on methane media are white-yellow in color. Like other type II methanotrophs, Mt OB3b contains a complete tricarboxylic acid cycle, and utilizes the serine pathway for formaldehyde assimilation. The DNA of Mt OB3b has a G~C content of 62.5 mol-%. Mt OB3b grows at 3~C, but not at 45~C. Growth of 33~
~13-~t OB3b is not stimulated by yeast extract or ~y other multi-carbon compounds tssted by Whittenbury et al., ~u~ra.
~t OB3b is motile with polar tuffs of flagella. Capsules formed by ~t OB3b consist of short fibers radiating from the cell wall and do not respond to polysaccaride stain. The Mt OB3b strain used in the present invention was obtained from Professor R. Whittenbury, Narwick University, United Ringdom and has been deposited with the National Collection Qf Industrial Bacteria, Aberdeen, Scotland and assigned number NClB-11131.
Deqradation of Haloqenated ~drocarbon Com~ounds The present invention provides a method of microbial degradation of a halogenated hydrocarbon compound.
The method comprises contacting the halogenated hydrocarbon compound, preferably TC~, with an amount of a methane-oxidizing bacterium, preferably ~t OB3b, effective to completely degrade the halogenated hydrocarbon compound at a rate ~rom about 500 to about 10,000 ~micromoles per hour per gram of cells. In the method of the present invention the methane oxidizing bacterium is cultured under continuous culture conditions so as to produce soluble NMO. As used herein an effective amount of methane oxidizing bacterium is an amount of the bacterium capable of completely degrading T OE at the rates stated herein. Further. as used herein, 3~
the phrase "continuous culture conditions~' means that the methane-oxidizing bacterium has been cultured in a con~inuously replaced medium that is exposed to a continuous flow of a gas mix~ure comprising air and methane in a ratio of about 25:1-1:20, respectively; prererably about 10~ 2;
and most preferably about 2.1:1. Continuous culture and growth parameters are as described in Cornish, J. Gen.
~icro., 130: 2565-2575 (1984) with a dilution rate of continuous culture of about 0.1 volumes/volume culture per hour. The air-to-methane ratio of the gas mixture may be varied, and we have found that methane at concen~rations bet~een 1-20~ of saturation greatly stimulate TC~ oxidation.
We have determined that conventional (i.e. shake flask, non-continuous) methods of culturing the methane-oxidizing bacterium utilized in the present invention prove less than satisfactory in terms of the extent of remo~al of TCE from media comprising any significant initial concentration of TCE. Nore specifically, we have found that at initial TCE concentra~ions of one ppm or higher, oxidation of TCE ceased at about 50% removal when ~t OB3b grown by conventional methods (shake flask) was utilized.
The halogenated hydrocarbon compound to be degxaded by the present method i5 preferably contacted with the methane-oxidizing bacterium in an aqueous media which comprises about 0.10-20 g/l of the methane-oxidizi~g ' , ' 2~)3~
bact~rium, more preferably from about 0.2 to 2.Og/l o, the metnane-oxidizing bactsrium is used. ~he preferred aqueous media used in the present in~ention is referred to herein as Higgins media. The method of preparing Higgins media is S disclosed by Cornish et al., J. Gen. ~icro., su~ra. The reci~e utilized in the present invention for the preparation of Higgins media is given in Example 1 below. Higgins media is classified as a minimal salts nitrate media, and contains about 250 ~g/l of Cu2+. Other aqueous media suitable for use in the present invention may include, but are not limited to those described by Pat et al., Int. J. Svstematic Bacteriol., 25: 226-229 (1976) as well as AMS and ~MS
minimal media; see Whittenburg et al., J. Gen. Microiol., su~ra.
lS The present method provides for complete degradation of a halogenated hydrocarbon compound, pre~erably TCE, present at initial concentrations up to 10,000 micromoles/l and more preferably at concentrations of from trace amounts to 1000 micromoles/1. These concentration values represent the initial concentration of the halogenated hydrocarbon compound in a solution comprising the hydrocarbon compound, the bacterium, and the aqueous media. It is to be understood that trace amounts refers to lower limits of detection by assay techniques described in the Examples herein.
- .
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The presenl method achieves rates of TCE
degradation of at least 500 ~ moles per hour per gram of cells with a range or TCE degradation ra~e from about 500 -10,000 ~moles per hour per gram of cells; preferably the range is from about 1000 - 9000 ~moles per hour per gram of cells. ~ore preferably the present me~hod achieves rates oî
TCE degradation about 2000-4000 ~moles per hour per gram of cells, where "gram of cells~' means gram of methane-oxidizing bacterium cells on a dry weight basis.
Cultivation of Methane-OxidizLnq Bacteria The present invention is further directed to a method of cultivating a methane-oxidizing bacterium which is capable of completely degrading a halogenated hydrocarbon compound, preferably TOE . Th~ me~hod comprises culturing the methane-oxidizing bacterium, preferably Nt OB3b, under continuous culture conditions in a suitable minimal salts media, such as Higgins media. The continuous culture conditions comprise exposing the bacterium to a continuous-`~20 flow gas mixture of air and methane in a ratio of about 25:1-1:20, preferably about 10:1-1:2, and most preferably abo~t 2.1-:1. Preferably, the gas mixture during degradation of TCE comprises methane at a concentration of about 1-20% of saturation. The present method provides that the continuously cultured methane-oxidizing bacterium will : .
, -, . -, ., " ~
, . , , :
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comprise an amount of soluble l~O which is effective to completely degrade the halogenated hydrocaxbon compound.
Preferably, the amount of soluble ~O produced in the continuously cultured bacterial cells is from about 5-30% of the weight of the dry bacterial cells.
Methane NonooxYcenase The methanotrophic bacterium Mt OB3b can comprise both a particulate (i.e., membrane ~ound~ and a soluble form of the enzyme methane monooxygenase (NMO). Tonge et al., FEBS Letters, 58:293-2~9 (1975), reported that ~MO is located in the particulate fraction of Mt OB3b, and that MMO
could be solubilized from the particulate fraction by treatment with phospholipase D, sonication, or Triton X-100.
Tonge et al. also reported that ascorba~e was an effective electron donor substitute for NADH both in crude extracts of Nt OB3b or in its particulate fraction, but that associated CO-binding cytochrome c seemed to be essential for MMO
activity. However, Stirling and Dalton, Eur. J. Biochem., 96:205-212 (197~), later reported that the MMO in cell-free extracts of Nt OB3b appeared to be soluble, and that it required NAD(P)H as an electron donor for activity.
Further, they reported that ascorbate was not an effecti~e electron donor for the NNO of Nt OB3b. Stirling et al., Biochem J., 177:361-364 (1979), also characterized MMO in :: - . . . ... . .
2~3~
several ways including its substrate specificity when present in crude abstracts or Mt OB3b.
M~O catalyzes the first step in the oxidation metabolism of methane, in which O2 is cleaved and one oxygen atom is inserted into a C-H bond of methane to yield methanol. Fox et al., J. Biol._Chem., 263:10553-10556 (1988). ~he reaction stoichiometry of this step is shown below:
CH4 + H + O2 + NADH _____, C~30H + ~2O + NAD~
Nethane ~icotinamide Me~hanol Nicotinamide Adenine Adenine Dinucleotide, Dinucleotide, reduced oxidized Fox and Lipscomb, Biochem. & Bio~hys. Res. Comm., 154:165-170 (1988), incorporated by reference herein provided a purified NMO from Mt OB3b and resolved the MMO
system of Nt 0~3b into three components, all three of which they found were required for reconstituting methane-oxidizing activity in vitro. These components were denoted by Fox and Lipscomb as the reductase, component B, and the hydroxylase, respectively. The cells of Nt OB3b utilized in the purification process of Fox and Lipscomb were grown in continuous culture as described by Cornish et al., J. Gen.
Micro., 130:2565-2575 (1984). The Mt OB3b thus cultured reproducibly expressed the soluble form of MMO, and could b~
cultured in high yield.
.
:
:, - :
. . .
: ~ :
De~r~dation of Haloqenated HYdrocarbons by Purified Methane Monooxyaenase We have found that purified ~O components oxidize T G . ~ccordingly, the present invention envisions a method oî degrading a halogenated hydrocarbon compound by continuously culturing a methane-oxidizing bacterium, pre~arably Mt OB3b, by exposing the bacterium to continuous culture conditions including a continuous-flow gas mixture comorising air and methane in a ratio of about 25~ 20, prererably about 10~ 2, and most preferably about 2.1:1, and ~herein the bacterial thus cultured comprises soluble M~O in an amount from about 5-30% of the weight of the dry bacterium cells; separating the soluble ~O from the continuously cultured bacterium cells, purifying the soluble NMO to yield purified components comprising reductase, component B, and hydroxylase; dding an effective amount o~
the purified components, preferably in a ratio of about 110:13:250 by weight of the three components, respectively, .
to an aqueous slurry of the halogenated aliphatic hydrocarbon compound, preferably TCE, to form a mixture; and reacting the mixture for a period of time sufficient to completely degrade the halogenated aliphatic hydrocar~on compound.
The inven~ion will be further described by reference to the following detailed examples.
. - - - . . . ... ~ . . . . . . . . . .. . . .
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-2~-Example I - Pre~aratio~ of Hiq~i~s Medial In order to provide a medium for the cult~vation of the methane-oxidizing bacterium suitable for use in the present invention, the following solutions were prepared and S stored at 4C:
lOOx Hiqqins Salts Solution Inqredient: Volume ~:
NaNO3 85 ~SO4 17 ~gSO67H70 3.7 C~Cl7'2H2O 0.7 lOOx Hiqqins_Phos~hate Solution Inqredient: Volume ~:
KH7P04 53.0 ~0 Na7HPO4 86.0 Adjust this solu~ion to pH 7Ø
500x Hiqqins Trace Metals Solution Inqredient: Volume %~
znSO47H2O 0.287 MnSO47H2O 0.223 H3BO3 0.062 NaM006 2H20 o.n4s CoCl26H20 0.048 ~I 0.083 CuSO45H2O 0.125 Add l ml of l mM H7SO4 per liter of Trace Metals Solution.
.
I The method of preparation of Higgins media is disclosed by Cornish et al., J. Gen. Micro., 1 :2565-2575 ( 1sa4 ) .
:
3~
lOOOx Hiqains_Iron Solution Incredient: Concentration:
FeS0~7H70 1.12g/100 ml Add 5 ml of 1 mM H7S06 per 100 ml of Higgins Iron Solution.
Ten mL of Higgins Salts Solution, 10 ml of Hig~ins Phosphate Solution, and 2 ml of Higgins Trace MetaLs Solution were mixed together per liter of media desired.
Distilled water was added to make up the final volume. If agar plates were being made, 17 g of purified agar per liter of liquid media was added. The mixture was autoclaved for 20 minutes with slow exhaust. When the media had cooled sufficiently to pour plates, or prior to inoculation, 1 ml ~0 of Higgins Iron Solution was added by filter sterilization per liter and mixed carefully. Higgins media agar plates were marked with a red stripe.
. - , ~ .
, . . . . .
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Ex~mple II - Co~tinuous C~lture of Mt OB3b A continuous cul~ure or M~ OB3b was perrormed in which tr.e bacterium grew at rate of approximately 10 hours S per generation on Higgins medium prepared as in Example I.
The continuous culture was grown in a chemostat growth chamber having a 0.185 li~er volume. J. Depamphillis and R.
Hanson, J. Bacterial., ~8: 222-225 (1969), incorporated by refarence herein.
The apparatus consisted of a water jacketed growth cham~er supplied with sterile, warm moist air and a constant supply of medium (See Pig. 3 in which arrows indicate the flow of medium air, warmed water and effluent: oxygen was supplied from the atmosphere). Referring to Figure 3, the alphabetic references describe: (A) chemostat growth flask;
(B) medium pump; (C) medium reservoir; (D) constant temperature bath; (E) constant temperature heater pump; (F) air humidifying chamber; (G) air sterilizing chamber, (H) air pump; and (I) culture collection flask.
~0 a. Growth Chamber This chamber is composed of two parts; a head section (Fig. 4) and a body section (Fig. 5). The head contains two parts, one for medium and one for air, and is fitted tightly onto the body section by a ground gLass joint. The body is water jacketed and has an overflow -~ ' : ': ' : :
., . : , : ~, :
~o~n~
device to maintain a constant volume in the cha~ber. The entrance to this overflow duct is shielded by a glass baffle which helps k~ep the volume in the chamber constant by preventing the slight amount of foam produced by sparging from leaving the overflow. The maximum volume of the growth chamber was 200 ml.
b. Constant Temperature Apparatus A. B. Braun Thermomix II (Melsungen, Germany) con~tant temperature water pump was used to circulate warmed water (~0 C) through the water jacket of ~he growth chamber. This warmed water also hea~s the air which is passed into the growth flask (See Fig. 3). The Thermomix II
is sensitive to changes of 0.1 C so the temperature variation is well within the limits of temperature control required.
c. Aeration System Bubbles of air were used to supply oxygen and aid in the mixing of the bacterial culture. Air was pumped rom the atmosphere by a B 2-F Model Aquarium Pump (Eugene G.
Danner Mfg. Co.). The air passed first into a wash bottle ~ontaining 1% HgCl2 Vid a sintered glass sparger and then through a warming bath (30 C) of sterile water contained in a wash bottle which was partially submerged in a water bath.
The air then passed into the growth chamber via a sintered glass gas dispersion tube.
.
-:
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- - , . .
, - . :
, -2~
-2~-25 ml of Mt OB3b cells were lnoculated into .185 1 of Higgins media and incubatod in the growth chamber at 30 C. New media was continuously added and expelled.
The continuous air feed was supplied wi~h an aquarium pump, and methane fram a pressurized tank. The gas mixture was applied in a volume ratio of approximately 1:1 (CE4:air).
The growth vessel was stirred vigorously. ~t OB3b cells ~ere grown to various turbidities and measured on a Spectronic 20 spectrometer l600 nm) at which time two-phase (Headspace) assays were performed.
Example III - Assayinc7 for Rate of TOE De~radation 1. Incubation Without Heads~ace2 General Protocol: ~ -~ Each culture of Mt OB3b was added to 1.8 ml serum bottle prewarmed to 30C and sealed with an 11 mm te~lon-lined rubber septa. For comparisons requiring similar initial dissolved oxygen levels, anaerobic make-up media (usually 0.6 ml) was added to the bottles first and then the bottles were sealed. Air-saturated 30C Higgins media prepared as in Example I was added via gas-tight syringe 2 A convenient liquid-liquid extraction method for the determination of halomethanes in water at the part$-per-billion level is disclosed by Henderson, J.E., G.R. Peyton and W.H. Glaze, in L.H. Reith, ed., Identification and Analysis of Orqanic Pollutants in Water, Ann Arbor Science Publishers, pp. 105-lll (1976~.
.
~:0~3~
(1.0 ml), while 1 atm pressure was maintained by allowing ai~ to bleed through a 25 gauge needle. Finally, culture was added (concentrated to give desir2d density) while remaining headspace was bled out through the 25 gauge needle. TC~ was added with a syringe at bottom of ~he sealed bottle, with a syringe at top of the bottle removing equivalent volume of culture. The assay time course was started with TCE (substrate) addition. Incubation was perfo~med at 30C with agita~ion at 200 rpm on a platform shaker.
Assays were terminated by extraction at desired time points. The liquid-liquid extraction technique used 0.6 ml of pentane con~aining 1,2-dibromoethane as an internal standard added via gas-tight syringe ~o inverted assay bottle, while a second syringe with a needle below first needle level collected displaced solution.
Partitioning was brought to equilibrium by centrifugation of bottles at S000 rpm for 10 min. The organic layer was ~emoved by gas-tight syringe and placed in a 1 ml serum bottle for chromatographic analysis. In some cases, dilution of sample or split injection was necessary.
Electron-capture detection was preferred. The following gas chromatography operating parameters were used:
' .
3~
Table I - Gas Chromatoqra~hy Parameters GC: HPS79OA (with ECD) Column: Non-Pakd RS~-160 Thick Film Capillary (Alltech) Injection Temperature: 150C
Detection Temperature: 250C
Ramping: 35C (lst min), ramped to 120C at 15C/min Carrier Gas: X2 Carrier Gas Flow: 8 ml/min Injection Volume: l ml No-HeadsPace Assa~s of TCE Deqradatlon:
The following Tests 1, 2 and 3 were conducted according to the no-headspace assay procedure described generally above. A summary of the results of these 3 tests is given in Table II, below:
Table II - SummarY of Resul~s of No-HeadsPace Assa~s Rate of TCE-utilization Culture turbidity (~moles'hr~1'g cells~~) Test (Absorbance 600 nm) (no headspace assay) l 1.310 336 2 1.410 1070 3 1.460 ~400 -:
:. ' The exact assay protocol used and the detailed results o~t~ined in each test are given below.
Test 1 - ~3C'e~ 8 Protocol:
2 ml of Mt 0~3b cell suspension grown in a chemostat (procedure) with absorbance A6ao = 1.310 were added to 8 ml of 30C Higgins media prepared as in Example 1 in prewarmed 120 ml seruim bottles (l/S diLution A600 = 0.252).
The bottles were evacuated and refilled with air ha~ing 0%
- methane. One bottle was heat-killed and used as a control.
1.79 ml of Mt OB~b cell suspension was added to sealed 1.8 ml serum bottles. 11.25 ~l of 4mMi TCE stocX was added to start the assay with a nominal initial TCE
concentration of 25 ~M. Bottles were sacrificed at 2.5, 5.0, 10.0 and 15.0 minutes by displacing O.6 ml aqueous solution with pentane containing 1 ppm 1,2-dibromoethane as an internal standard.
. . .
The results of Test 1 ars given in Table III, below:
Table III - Test 1 Results R~n 1 Results:
Heat-killed control, 0% methane:
TLme rTCEl, ppm ~TC~ M
2.5 min 1.836 13.97 5~0 min 2.691 20.48 lS 15.0 min 2.883 21.94 Run 2 Results:
1/5 dilution, 0~ methane:
Time ~TCEl, ppm rTCEl, ~M
2.5 min 2.103 16.01 5.0 min 0.261 1.99 10.0 min 0.255 1.94 15.0 min 0.204 1.55 Test 2 - No-~eadsPace Assa~
Test 2 was performed follow mg the same procedures described above for Test 1, except that 1/20 and 1/50 dilutions were performed. The results of Test 2 are given .
in Table IV, below:
~:
Table IV - Test 2 Results A500 = 1.410 from chemostat 1/20 dilution A600 = 0.069 1/50 dilution Asoo = 0.026 Nominal initial TCE concentration = 25 ~M
Run 1 Results:
Heat-killed control, 0% methane:
Time rTC~l, PPm rTcEl~ ~M
2.5 min 2.749 20.92 5.0 min 2,861 21.77 10.0 min 2.901 22.08 15.0 min 3.132 23.84 Run 2 Results:
1/20 dilution, 0% methane:
Time rTcElr PPm rTCEl,_~M
2.5 min 4.227 32.17 5.0 min 3.475 26.45 10.0 min 2.843 21.64 15.0 min 2.742 20.87 Run 3 Results:
1/50 dilution, 0~ methane:
Time rTCEl, Ppm rTcEl~-~M
~0 2.5 min 2.924 22.25 5.0 min 1.843 14.03 10.0 min 2.717 20.68 15.0 min 3.183 24.22 "
: ~, , ' ~ ' . ~
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~es~ 3 - No-Heads~ace Assay In prewarmed (30C) 120 ml serum bottles~ 1 ml or ~t OB3b cell suspension grown ln a chemostat (A600 = 1.460) were mixed with 9 mls of 30C Higgins media prepared as in Example I. One bottle was prepared by adding "spent" media from the chemostat (decant media after pelleting cells with centrifugation - 10,000 rpm for 10 minutes) rather than fresh Higgins media. The bottles were sealed with 20 mm teflon-lined rubber septa, evacua~ed and refilled with air (0~ methane). One bottle was heat-killed and used as a control.
1.76 ml of Mt OB3b cell suspension was added to sealed 1.8 ml serum bottles (25 gauge needle used to equilibrate pressure). 45 ~1 of 4 mM TCE stock were added lS to start the assay. Bottles were inc~bated at 30C with agitation on a shaker bath. Bottles were sacrificed at 2.5, S.-0, 10.O, 15.O and 20.0 mins by displacing 0.6 ml aqueous solution with pentane containing 1 ppm 1,2-dibromoethane as an internal standard. Analysis was performed on a HP 5790A
GC using ECD.
The results of Test 3 are given in Table V
below:
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2~3~
Table V - Test 3 Results Aso~ = 1.460 from chemostat 1/10 dilution ~0O = 0.140 ~ominal initial TCE concentration = 100 ~ (using 4mM TCE
stock in water) Dry weight = O.lOg.l~
Run 1 Results:
Heat-killed control, 0% methane:
Time, min rTCE1! ppm rTCE1L_~
lS 2.515.67~ llg~33 5.014.713 111.98 10.014.760 112.34 15.015.230 115.gl 20.014.691 111.81 Run 2 Results:
1/10 dilution in ~iggins media, 0% methane:
Rates Time, min rTcEl~ ~pm rTOE l, ~M mole_.h~l.q cells~;
2.5 14.119 107.46 :
5.0 14.651 111.41 10.0 ll.g81 gl.l9 15.0 8.406 63.98 3200 (lOmin-15min) 20.0 7.208 54.86 , -2~3~0 Run 3 Results:
1/10 dilution in spent Hig~ins media from chemostat, 0%
methane:
Rates Time, m_n L~5~ 2m rTcE~ moles.h~~
2.5 14.277 108.66 5.0 13.757 104.70 10.012.924 98.36 15.0 8.543 65.02 3g60 (10 min-lSmin) 20.0 7.650 58.22 _ .
The rate at those times in the reactions when most rapid oxidation of TCE occurred was approximately 3500 ~mol2s/h~'.g cells~l This rate was not stimulated by the addition of methane to the reaction mixture. The rate of TCE oxidation in the presence of methane over time is linear and TCE oxidation is more complete. TCE oxidation also occurs more rapidly in the presence of low concentrations of methane (Table VII). Therefore, it is believed that cells can oxidize TCE at rates of 10,000 ~moles.h~l.g cells~l under op~imal conditions.
- - . : ~ .
, . . . : ~ ..
~20~3~
2. Incubation ~ith Heads~ace Each culture of Mt OB3b was added in 2 ml quanti.ties ~o 10 ml serum bottles. Bottles were sealed with 20 mm teflon-lined ru~ber septa. TCE was added to start the S assay. Xeadspace accounted for 80~ of total volume within bottles, but TCE concentration was added based on 2 ml aqueous phase. TCE resided principally in the headspace.
All bottles were inverted in order to prevent possible TCE
loss by trapping the compound in the headspace between the liquid phase and glass. Samples were incubated inverted at 30C with agitation t200 rpm on a platfo~m shaker).
Pre~aration of GC Cali~ration Curves:
Headspace incubations were analyzed by direct injection of headspace into a gas chromatograph. This method may be performed without sacrificing a sample. It is important to prepare sound calibration curves for quantitation. The best method for external standardization is to prepare samples as if running an assay and heat-killing at 80C for 10 mins prior to TCE addition. Sampleswere incubated for 30 mins under test conditions ~o allow adequate time for TCE to partition among the numerous phases (air, water, cell material, and the like). Headspace samples were injected into a gas chromatograph using either FID or ECD. Numerous TCE concentrations were used to obtain . . . ~ ., ~ , , , :
. , : , a sound standardization curve.
Exampl~ IV - TCE De~radation with ~eadspace Assay Headspace assays of TCE degradation were conductad by adding bacterial cells from the continuous culture described in Example II or cells diluted with spent Xiggins medium. ( 2 mls) into assay vials (10 ml vials).
Dilution of cells in spent Higgins medium had no effect on the rate of TCE oxidation. Therefore, there do not seem to be protective compounds in the medium.
Heat-killed controls indica~ed that no TCE was lost from the vials.
The rates of TCE utilization in two-phase head space assays are shown in Table VI, below. Rates were calculated from peak heights of recorder tracings ~rom a gas chroma~ograph equipped with an electron capture detector.
The gas chromatograph parameters reported in Table I herein were utilized.
~0 - 2~3~
Table IV - TCE Deqradation in Two~Phase Assay Cell density Initial TCE Rate, mlcromoles in assay vials (g l~l) conc. TCE oxidized-(micromolar) g cells~lhr~
.6gS 22 281 .521 22 308 .347 22 465 .173 23 461 .070 22 ~08 Example V - Effect of Mt OB3b Çell De~sit~ o~ TCE
~ t~
In this example, the cell mass of the continuous culture (generated in Example II) increased to 0.825 g/l from O.6~5 g/l. As shown in FIGURE 1, the rate of TCE
degradation at low cell densities increased to 1200 ~0 micromoles TCE removed hr~lg cells~l at an initial ~CE
concentration of 80 ~N.
The rate of methane oxidation by these cells during culturing was 2860 ~moles hr~lg cells~l The curves at two different Mt OB3b cell densities ~5 shown in FIGURE 1 also illustrate that TCE oxidation was less complete at low cell densities than at high cell densities. Thls indicates that cells at high densities withstand toxic intermediates bscause there is more biomasq available to react with the reactive intermediate compounds.
Alternatively, the slower rates of oxidation per unit mass at high cell densities may have limited the rate of production of toxic intermediates to a rate at which they :~
~ :: ~ : :
,.: - : ,:~ : :.
'", ': ~ ~: ' ' ' .
, % ~ ~3 were fur~her deqraded.
Exampl~ VI - TC~3 Dec~radation with Sin~le_Phase Assay In order to test the hypothesis that gas transfer limited oxidation rates at high cell densities, the rate of TC~ degradation was assayed in a single phase assay using techniques for no head space assay described in Example III.
The assay was performed at a ~t OB3b cell density of approximately 0.160 g/l, and an initial TCE concentration of 80 ~moles. The rate of TCE oxidation was 2400 micromoLes hr~lg cells~l or 315 mg hr~l.g cells~~. ;
, ' . .' .' :, : ~ .
)3~
Example V~I - Effect of Methane Present Duri~c~
TCE Dearadation In this example the effect of methane present in the TCE oxidation vessel on the rate of oxidation of TCE was determined. A no head space assay of TCE degradation was performed according to the procedures of Example III in which the Mt OB3b cell density was approximately 0.16 g/1, and the nominal initial TCE concentration was 80 ~M. The results are presented in Table VII, below and Figure 2.
Table VII - Rate of TCE Deqradation in Presence of Methane Methane added to Rate of TCE oxidation, reaction system, ~moles-hr~~-g cells~
15(% sat~ration) none 660 5% 3350 10% 3G00 20% 875 50% 110 -We have subsequently observed that some cell batches oxidize TCE at rate of approx.imately 4000 micro-moles hr~l 'g cells~l without any methane present.
' '' .,, ' ~ :' ; . ~ .
. .
. . , ~ . .
., ,,, : , , Example VIII - Effect of Initial TCE Concentra~io~
on TCE Deqradation Rate Mt OB3b cells were continuously cultured under the following conditions:
Medium: Eiggins (prepared as in Example I) Gas flow rates: methane 45 ml/min air 135 ml/min Culture volume: 220 ml Culture temperature: 30C
A TOE no head space degradation assay of the type in Example III was performed using these cells. The results are shown in Table VIII, below:
~0 Table VIII - Effect of Initial TCE Concentration on Rate of TCE Oxidation Concentration micromoles TCE
of TCE, ~M removed-hr~'.g dry cells~L
82g These results and the results of other experiments led to the conclusion that the K, for TCE oxidation is approximately 5 ~M.
~03~no Example IX - Deqradation of TC2 at various Initial Concentrations The cell mass of the continuous culture described in Eæample II was grown to 0.64 g cells.l~l. A TCE no head space degradation assay using the procedure described in Example III was performed using these cells. The results are shown below in Table I~.
Table IX - TCE De~radation at Var-ous Initial TCE
Concentrations Concentration of Nicromoles TC~
TCE, ~m removed, ~moles/min.
S-10 min. 10-15 min.10-2S min.
.
2.96 5.69 3.03 320 15.54 10.2 640 ~0.48 .... .. . .. ..
. . .
- ~ . . .
. . ~ , , ,, :
3~
Example X - Dearadation of ~CE bv_Purified Methane Monooxvqe~ase Radiolabelled TCE was employed in order to demonstrate the oxidation of TC~ to products upon incubation wi~h soluble methane monooxygenase in the presence of reduced nicotinamide adenine dinucleotide ~NADH). Th~ three components of methane monooxygenase, reductase, component B, and hydroxylase, were purified by the method of Fox and Lipscomb, suPra, the disclosure of which is hereby incorporated by reference. Enzyme incubation mixtures were ~ -conducted in sealed 10 ml sept~m vi~ls, each ~ial containing the ingredienis shown in Table IX, below:
Table X - Enzyme Incubation Mixture ComPonents ::
Inqredient mount/
Concentration 3-[N-morpholino]propanesulfonic acid 1.5 ml/
(NOPS) bufferl at pH 7.5 25 mN
NADH2 0.1 mM
Reductase3 110 ~g Component B 13 ~g ~ydroxylase 250 ~g ____________________________________________ ___ .__________ . , 1 Sigma Chemical Company, St. Louis, Missouri.
2 Nicotinamide adenine dinucleotide, reduced form, Sigma Chemical Company, St. Louis, Missouri. ~.
3 Redùctase, component B, and hydro~ylase are the three -:
components of methane monooxygenase identified by Fox and Lipscomp, supra.
::~
31~0 105 nmol of uniformly labelled l4C-TCE was added to each vial by injection through the rubber septum above the reaction mixtur~ until a final TCE concentration or 70 ~M was reached. ~he reaction was allowed to proceed for 2 min at 25C and then quenched by the addition of sulluric acid (Sigma Chemical Company, St. Louis, Missouri) at pH
2Ø
Following centrifugation to remove precipitated protein, the reaction mixtures were analyzed by high pressure liquid chromatography (HPLC). The HPLC ooerating parameters employed are shown in Table ~I, below:
Table XI - HPLC O~eratinq Parameters Column: Aminex HPX-87H HPLC column (Bio- Rad) Nobile phase: 25% acetonitrile (Sigma Chemical Company, St. Louis, ~0 Missouri) in 0.01 N sulfuric acid (Sigma Chemical Company, St~ Louis, Missouri) Operating mode: Isocratic Rate: 0.3 ml per min Peak detaction: UItraviolet spectroscopy, 210 nm , This HPLC system resolved au~hentic standards of various potentLal organic acid products and ~633~
tri~hloroacetaldehyde (chloral). Under the enzyme incubation conditions described, 53% of the TCE was converted to HPLC-detectable products. These products were formic acid, glyoxylic acid, dichloroacetic acid, and S chloral.
In addition to the above incubations, control incubations were conducted in which each one of the methane monooxygenase componen~s or NADH were omitted. In all of these control experiment~, no products of TCE oxidation were detectable.
Thus, this experiment demonstrated that TCE
oxidation is catalyzed by the three-component methane monooxygenase enzyme system in the presence of NADH.
~ he invention has been described with reference to various specific and preferred embodiments and techniques.
However, it should be understood that many variations and ~;
modifications may be made while remaining within the spirit and scope of the invention. .
.. . . . . .
, :, . . '~ ,.. ... , :
, .
Claims (31)
1. A method for microbial degradation of a halogenated hydrocarbon compound, said method comprising:
contacting said halogenated hydrocarbon compound with an amount of a methane-oxidizing bacterium effective to completely degrade said halogenated hydrocarbon compound at a rate from about 500 to about 10,000 micromoles per hour per gram cells, said methane oxidizing bacterium being cultured under continuous culture conditions, said continuous culture conditions comprising exposing said bacterium to a continuous-flow gas mixture comprising air and methane in a ratio of about 25:1-1:10, respectively, said continuously cultured methane-oxidizing bacterium producing a soluble methane monooxygenase.
contacting said halogenated hydrocarbon compound with an amount of a methane-oxidizing bacterium effective to completely degrade said halogenated hydrocarbon compound at a rate from about 500 to about 10,000 micromoles per hour per gram cells, said methane oxidizing bacterium being cultured under continuous culture conditions, said continuous culture conditions comprising exposing said bacterium to a continuous-flow gas mixture comprising air and methane in a ratio of about 25:1-1:10, respectively, said continuously cultured methane-oxidizing bacterium producing a soluble methane monooxygenase.
2. The method of claim 1 wherein said methane-oxidizing bacterium is capable of completely degrading said halogenated hydrocarbon compound at a rate of from about 1000 - 9000 µmoles per hour per gram cells.
3. The method of claim l wherein said methane-oxidizing bacterium is capable of completely degrading said halogenated hydrocarbon compound at a rate of about 2000-4000 µmoles per hour per gram cells.
4. The method of claim 1 wherein said gas mixture comprises air and methane in a ratio of about 10:1-1:2, respectively.
5. The method of claim 1 wherein said gas mixture comprises air and methane in a ratio of about 2.1:1.
6. The method of claim 1 wherein said halogenated hydrocarbon compound is contacted with an aqueous media comprising about 0.10-20 g/1 of said methane-oxidizing bacterium.
7. The method of claim l wherein said halogenated hydrocarbon compound is contacted with an aqueous media comprising about 0.2 to about 2.0 g/l of said methane-oxidizing bacterium.
8. The method of claim 1 wherein said methane-oxidizing bacterium is capable of degrading said halogenated hydrocarbon compound at initial concentrations up to 10,000 micromoles/l.
9. The method of claim 1 wherein said methane oxidizing bacterium is capable of degrading said halogenated hydrocarbon compound at initial concentrations from trace amounts of said halogenated hydrocarbon compound up to 1000 micromoles/l.
10. The method of claim 1 wherein said methane-oxidizing bacterium is a member of the genus Methylosinus.
11. The method of claim 10 wherein said methane-oxidizing bacterium is Methylosinus trichosporium OB3b.
12. The method of claim 1 wherein said halogenated hydrocarbon is degraded in the presence of a gas mixture comprising methane at a concentration of about 1-20% of saturation.
13. The method of claim 1 wherein said halogenated hydrocarbon compound is an aliphatic halogenated hydrocarbon compound.
14. The method of claim 13 wherein said aliphatic halogenated hydrocarbon compound is trichloroethylene (TCE).
15. The method of claim 1 wherein said bacterium produce said soluble methane monoxygenase in an amount from about 5-30% of the weight of dry bacterium cells.
16. A method of cultivating a methane-oxidizing bacterium capable of completely degrading a halogenated hydrocarbon compound, said method comprising:
culturing said methane-oxidizing bacterium under continuous culture conditions comprising exposing said bacterium to a continuous-flow gas mixture comprising air and methane in a ratio of about 25:1-1:20, respectively, said continuously cultured methane-oxidizing bacterium producing an amount of a soluble methane monooxygenase effective to completely degrade said halogenated hydrocarbon compound at a rate from about 500 to about 10,000 micromoles per hour per gram cells.
culturing said methane-oxidizing bacterium under continuous culture conditions comprising exposing said bacterium to a continuous-flow gas mixture comprising air and methane in a ratio of about 25:1-1:20, respectively, said continuously cultured methane-oxidizing bacterium producing an amount of a soluble methane monooxygenase effective to completely degrade said halogenated hydrocarbon compound at a rate from about 500 to about 10,000 micromoles per hour per gram cells.
17. The method of claim 16 wherein said gas mixture comprises air and methane in a ratio of about 10:1-1:2, respectively.
18, The method of claim 16 wherein said gas mixture comprises air and methane in a ratio of about 2.1:1.
19. The method of claim 16 wherein said bacterium is a member of the genus Methylosinus.
20. The method of claim 19 wherein said bacterium is Methylosinus trichosporium OB3b.
21. The method of claim 16 wherein said bacterium produces said soluble methane monooxygenase in an amount from about 5-30% of the weight of dry bacterium cells.
22. The method of claim 16 wherein said halogenated hydrocarbon compound is an aliphatic halogenated hydrocarbon compound.
23. The method of claim 22 wherein said aliphatic halogenated hydrocarbon compound is trichloroethylene (TCE).
24. A method of degrading a halogenated hydrocarbon compound, said method comprising:
(a) culturing said methane-oxidizing bacterium under continuous culture conditions comprising exposing said bacterium to a continuous-flow gas mixture comprising air and methane in a ratio of about 25:1-1:20, respectively, said continuously cultured methane-oxidizing bacterium producing a soluble methane monooxygenase in an amount from about 5-30% of the weight of dry bacterium cells;
(b) separating said soluble methane monooxygenase from said continuously cultured methane-oxidizing bacterium;
(c) purifying said separated soluble methane monooxygenase to yield purified components comprising reductase, component B, and hydroxylase;
(d) adding an effective amount of said purified components to an aqueous slurry of said halogenated hydrocarbon compound to form a mixture; and (e) reacting said mixture for a period of time sufficient to completely degrade said halogenated hydrocarbon compound.
(a) culturing said methane-oxidizing bacterium under continuous culture conditions comprising exposing said bacterium to a continuous-flow gas mixture comprising air and methane in a ratio of about 25:1-1:20, respectively, said continuously cultured methane-oxidizing bacterium producing a soluble methane monooxygenase in an amount from about 5-30% of the weight of dry bacterium cells;
(b) separating said soluble methane monooxygenase from said continuously cultured methane-oxidizing bacterium;
(c) purifying said separated soluble methane monooxygenase to yield purified components comprising reductase, component B, and hydroxylase;
(d) adding an effective amount of said purified components to an aqueous slurry of said halogenated hydrocarbon compound to form a mixture; and (e) reacting said mixture for a period of time sufficient to completely degrade said halogenated hydrocarbon compound.
25. The method of claim 24 wherein said gas mixture comprises air and methane in a ratio of about 10:1-1:2, respectively.
26. The method of claim 25 wherein said gas mixture comprises air and methane in a ratio of about 2.1:1.
27. The method of claim 24 wherein said bacterium is a member of the genus Methylosinus.
28. The method of claim 27 wherein said bacterium is Methylosinus trichosporium OB3b.
29. The method of claim 24 wherein said purified components reductase, component B, and hydroxylase are added to said aqueous slurry in a ratio or about 110:13:250, by weight, respectively.
30. The method of claim 24 wherein said halogenated hydrocarbon compound is a halogenated aliphatic hydrocarbon compound.
31. The method of claim 30 wherein said halogenated aliphatic hydrocarbon compound is Trichloroethylene (TCE).
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US27253888A | 1988-11-17 | 1988-11-17 | |
| US272,538 | 1988-11-17 | ||
| US38485989A | 1989-07-21 | 1989-07-21 | |
| US384,859 | 1989-07-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2003100A1 true CA2003100A1 (en) | 1990-05-17 |
Family
ID=26955588
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002003100A Abandoned CA2003100A1 (en) | 1988-11-17 | 1989-11-16 | Rapid degradation of halogenated hydrocarbons by soluble methane monooxygenase or methanotrophic bacteria comprising same |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0445224A1 (en) |
| JP (1) | JPH04501667A (en) |
| CA (1) | CA2003100A1 (en) |
| WO (1) | WO1990005708A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6171844B1 (en) | 1996-08-19 | 2001-01-09 | Toyota Jidosha Kabushiki Kaisha | Microorganism and method for environmental purification using the same |
| KR100363209B1 (en) * | 2000-02-16 | 2002-12-05 | 장덕진 | Dechlorination method of chlorinated organic compounds sewage and a device thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4323649A (en) * | 1979-03-09 | 1982-04-06 | Imperial Chemical Industries Limited | Biotransformations using methane-utilizing bacteria |
| US4713343A (en) * | 1985-08-29 | 1987-12-15 | The United States Of America As Represented By The Administrator Of The U.S. Environmental Protection Agency | Biodegradation of halogenated aliphatic hydrocarbons |
| US4959315A (en) * | 1987-04-30 | 1990-09-25 | The United States Of America As Represented By The Administrator Of The Environmental Protection Agency | Biodegradation of chloroethylene compounds |
-
1989
- 1989-11-13 WO PCT/US1989/005177 patent/WO1990005708A1/en not_active Ceased
- 1989-11-13 EP EP90901207A patent/EP0445224A1/en not_active Withdrawn
- 1989-11-13 JP JP2501257A patent/JPH04501667A/en active Pending
- 1989-11-16 CA CA002003100A patent/CA2003100A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
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| WO1990005708A1 (en) | 1990-05-31 |
| JPH04501667A (en) | 1992-03-26 |
| EP0445224A1 (en) | 1991-09-11 |
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