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United States Patent (19) (11) Patent Number: 5,055, 193 Hooper (45) Date of Patent: Oct

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United States Patent (19) (11) Patent Number: 5,055, 193 Hooper (45) Date of Patent: Oct United States Patent (19) (11) Patent Number: 5,055, 193 Hooper (45) Date of Patent: Oct. 8, 1991 54) BIODEGRADATION OF HALOGENATED Nitrosomonas Europaea with Alkanes, Alkenes and HYDROCARBONS UTILIZING Alkynes'. AMMONA-OXIDIZING BACTERIUM Wood, In Nitrification, p. 39 (J. I. Prosser Ed. 1986). 75) Inventor: Alan Hooper, St. Paul, Minn. Hooper and Nason, J. Biol. Chem., 240: 4044-4057 (1965), "Characterization of Hydroxylamine-Cyto 73) Assignee: Regents of the University of chrome C Reductase from the Chemo Autotrophs Ni Minnesota, Minneapolis, Minn. trosomonas Europaea and Nitrosomonas Oceanus'. 21 Appl. No.: 321,498 Hooper, Autotrophic Bacteria, Schlagel and Bowein, 22 Filed: Mar. 9, 1989 eds. (1989) at pp. 239-265. Nelson et al., Appl. Envt. Microbiol. 53:949-954 (1987), 51) Int. Cl. ................................................ CO2F 3/00 "Biodegradation of Trichloroethylene and Involve 52 U.S. Cl. .................................... 210/601; 435/262: ment of an Aromatic Biodegrative Pathway'. 435/264 G. W. F. Wasser-Abwasser, vol. 129, No. 1, 1988, A. 58 Field of Search ................. 435/262, 264; 210/601 M. Cook, et al., "Mikrobieller Abbau von Halogeni 56) References Cited erten Aliphatischen Verbindungen', pp. 61-69. FOREIGN PATENT DOCUMENTS Hyman et al-Chem. Abst., vol. 103 (1985), p. 157,041g. 0300593 1/1990 European Pat. Off. Primary Examiner-Sam Rosen 0289350 1 1/1990 European Pat. Off. Attorney. Agent, or Firm-Merchant, Gould, Smith, OTHER PUBLICATIONS Edell, Welter & Schmidt Little et al., Appl. Envi. Microbiol. 54:951-956 (1989), (57) ABSTRACT "Trichloroethylene Biodegradation by a Methane Ox idiziny Bacterium.' A method is disclosed for degradation of a halogenated Wackett and Gibson, App. Envt. Microbiol. 54: hydrocarbon compound such as trichloroethylene 1703-1708 (1988), "Degradation of Trichloroethylene (TCE) which utilizes an ammonia-oxidizing bacterium by Toluene Dioxygenase in Whole-Cell Studies with of the genus Nitrosomonas, as well as other species of Pseudomonas Putida F." ammonia-oxidizing bacteria. Hyman et al., Appl. Envt. Microbiol. 54: .3187-3190 (1988), "Interaction of Ammonia Monooxygenase from 23 Claims, 1 Drawing Sheet U.S. Patent Oct. 8, 1991 5,055,193 Wu (ON) 3 is v V O N A N N O . N s TTTTTTon to O 5,055, 193 1. 2 BIODEGRADATION OF HALOGENATED SUMMARY OF THE INVENTION HYDROCARBONSUTELIZING We have discovered that certain autotrophic am AMMONIA-OXIDIZING BACTERIUM monia-oxidizing bacterium of the genus Nitrosomonas are capable of degrading halogenated hydrocarbon This invention was made with Government support compounds, such as TCE. Upon culturing these bac under USDA contract number 88-37120-3957. Govern teria in an aqueous medium with an amount of ammonia ment has certain rights in the invention. effective to sustain growth, the bacteria are then capa ble of essentially complete degradation of halogenated FIELD OF THE INVENTION 10 hydrocarbon compounds. This invention relates to methods of biologically Preferably, rates of halogenated hydrocarbon degra degrading halogenated hydrocarbon compounds in dation according to the present invention are from cluding trichloroethylene (TCE), wherein said methods about 1 to 100 umoles/hr./g of wet cells. In a preferred utilize an ammonia-oxidizing bacterium. embodiment we have achieved rates of TCE degrada 15 tion of about 2 to about 30 micromoles per hour per BACKGROUND OF THE INVENTION gram wet weight of Nitrosomonas europaea cells. The This invention relates to methods of biologically present invention provides for degradation of haloge degrading halogenated hydrocarbons including trichlo nated hydrocarbons present in initial concentrations of rethylene (TCE). up to about 300-400 micromoles/1 and preferably pro Halogenated hydrocarbon compounds are high vides for degradation of halogenated hydrocarbon com volume products of the chemical process industry; for pounds, such as TCE, at initial concentrations from trace amounts up to about 100 micromoles/l. Moreover, example, more than 6 million metric tons of trichloro the cultured cells are characterized by a membrane ethylene (TCE), tetrachloroethylene (PCE), trichloro bound ammonia monoxygenase (AMO). methane, carbon tetrachloride (CT), and chloroform 25 Further, in a preferred embodiment, Nitrosomonas (CF) are produced each year. Those halogenated hy europaea cells effect degradation of halogenated hydro drocarbon compounds most frequently found in carbons in a media when cells are employed in amounts groundwater are low molecular weight aliphatic halo of from about 10 mg to about 2000 mg wet wt. of cells/l. genated hydrocarbons: TCE, dichloroethane (DCA), In the case of solid material such as soil, decontamina trichloromethane, and PCE. Many of these aliphatic 30 tion may be conducted by an extractive process which halogeated hydrocarbon compounds, including TCE, preferably is operated so as to bring the bacteria of this have been listed as priority pollutants by the U.S. Envi invention into contact with solutions or slurries having ronmental Protection Agency, and are known or sus halogenated hydrocarbon concentrations of, e.g. from pected carcinogens and mutagens. Haloforms (haloge the lowest detectable concentration (trace announts) up nated derivatives of methane) are also frequently de 35 to about 300-400 uM. These aqueous solutions are ob tected in groundwaters and drinking waters. Some halo tained by continuously or intermittently leaching the forms are produced during chlorination of water solid material with pH-controlled water comprising the supplies, but inadequate disposal techniques or acciden halogenated hydrocarbon degrading bacterium and tal spillage may also be responsible for the release of ammonia. To degrade halogenated hydrocarbon com these haloforms. 40 pounds in the aqueous solution from about 10 umolar to Several of the halogenated hydrocarbon compounds about 40 mmolar ammonia is preferred. Surface and mentioned above are resistant to biodegradation in aer ground water may also be detoxified, by inoculating the obic subsurface environments, or their biological trans water with an effective amount of the present bacterium formations are incomplete under anaerobic conditions. 45 together with an ammonia source. For example, under anaerobic conditions, TCE and It is expected that methods known to the art could be PCE are known to undergo partial bioconversion to employed to produce mutations of the ammonia-oxidiz ing bacterium of the present invention which would vinyl chloride, a compound which is as much or more also possess useful properties with respect to the degra of a problem as the original contaminants. Wilson and dation of halogenated hydrocarbons such as TCE. Wilson, Appl. Eny. Microbiol, 49:242-243 (1985). 50 The present method is advantageous in that it both Current technology for reclaiming groundwater pol rapidly and completely degrades halogenated hydro luted with these halogenated hydrocarbon compounds carbon compounds such as TCE. Other features and involves pumping water to the surface and stripping out advantages of the invention will be apparent from the the contaminants in aeration towers, or removing the following detailed description and appended claims. pollutants on a sorbent. The former process is not per 55 mitted in some states, and the latter is expensive and BRIEF DESCRIPTION OF THE DRAWINGS involves the production of concentrated toxic materials FIG. 1 shows time course of production of nitrite that may present future problems. (dashed lines) and disappearance of TCE (solid lines) A number of methanotrophic bacteria have been catalyzed by Nitrosomonas. () cells, NH3, TCE, acet shown to degrade TCE. Little et al., Appl. Envit. Mi ylene (A) cells, TCS. (O) cells, NH3, TCE. (D) cells, crobiol. 54: 951-956 (1989); Wackett and Gibson, Appl. NH3. (O) cells, NH3, TCE. Enyi, Microbiol. 54: 1703-1708 (1988). However, to date DETAILED DESCRIPTION OF THE degradation of TCE by chemo-litho-autotrophic bac INVENTION teria has not been reported. In view of the environmen 65 tal problems associated with halogenated hydrocarbons Halogenated Hydrocarbon-Containing Compounds there is a need for a method to degrade halogenated The present invention provides a method of rapidly hydrocarbons utilizing ammonia-oxidizing bacterium. and completely degrading a halogenated hydrocarbon 5,055, 193 3 4 compound. Although the present invention preferably In one embodiment of the present invention the halo provides a method of degrading trichloroethylene genated hydrocarbon containing media to be degraded (TCE), other halogenated hydrocarbons which may be by the present method is contacted with the ammonia degraded by the present method include, but are not oxidizing bacterium in an aqueous media which com limited to, tetrachloroethane, tetrachloroethylene (PCE), trichloroethane, dichloroethane (DCA), and prises about 1 mg to about 2 g wet wit/l of the ammonia chloroform. oxidizing bacterium. The preferred aqueous media used The preferred halogenated hydrocarbon compound in the present invention is the nutrient medium de of the present invention, TCE (1,1,2-trichloroethene), is scribed in Hooper & Nason, J. Biol Chem., supra. Other an aliphatic halogenated hydrocarbon with the chemi 10 aqueous media suitable for use in the present invention cal structure HClC=CCl2. TCE is primarily used in can readily be identified by those of skill in the art. industry as a fire-resisting solvent. It can be produced The present method provides for degradation of es by removal
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