United States Patent (19) (11) Patent Number: 5,055, 193 Hooper (45) Date of Patent: Oct. 8, 1991 54) BIODEGRADATION OF HALOGENATED Europaea with Alkanes, Alkenes and HYDROCARBONS UTILIZING Alkynes'. AMMONA-OXIDIZING BACTERIUM Wood, In , 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 , 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 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 -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

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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 , 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 of one molecule of hydrogen chloride from sentially all of a halogenated hydrocarbon compound, acetylene tetrachloride with alkali. preferably TCE, present at initial concentrations up to 15 300-400 micromoles/1 and more preferably at concen Nitrosomonas Bacteria trations of from trace amounts to about 100 mi The present invention utilizes ammonia-oxidizing cromoles/1 to undetectable amounts. These concentra bacteria to degrade the halogenated hydrocarbon-con tion values represent the initial concentration of the taining compounds described above. Preferably, bac halogenated hydrocarbon compound in a solution con teria of the strain Nitrosomonas europaea are utilized 20 prising the hydrocarbon compound, the bacterium, which produce the enzyme ammonia monoxygenase ammonia, and the aqueous media. It is to be understood (AMO). Other ammonia-oxidizing bacteria which pro that trace amounts refers to lower limits of detection by duce AMO may be useful in the present invention. assay techniques described in the Example herein. These other bacteria include, but are not limited to, Nitrosomonas, e.g., Nitrosospira species, Nitrosolubus 25 The invention will be further described with refer species, Nitrosococcus species, Nitrosovibrio species ence to the following detailed example. and Nitrosocystis species and other chemo-lithio Treatment of Contaminated Soil trophic ammonia oxidixing bacteria. Nitromonas europaea is an ubiquitous soil and water Solids dwelling ammonia oxidizing nitrifying bacteria which is 30 The TCE degrading capability of the present bacte an obligate aerobic autotroph which depends on the rium can be used to particular advantage in the treat oxidation of ammonia to nitrite for growth. Characteris ment of TCE contaminated solid material by the pro tics of Nitrosomonas europaea have been previously cess comprising contacting the TCE present in or on described. Hyman et al., Applied and Environmental 35 the solid material with water so as to extract the TCE, Microbiol. 54, 3187-3190 (1988); Wood, In Nitrification, which is then degraded with an effective amount of the p. 39 (J.J. Prosser Ed. 1986). Nitrosomonas europaea has present bacterium. Preferably the solid material will be been deposited with the American Type Culture Col slurried or leached with water (the leachate) in order to lection (ATCC 1978). remove the TCE therefrom. To increase the amounts of Degradation of Halogenated Hydrocarbon Compounds solid that can be detoxified with a given volume of The present invention provides a method of microbial leachate, the leachate preferably is intermittently or degradation of a halogenated hydrocarbon compound. continuously recycled through the solid, thus greatly The method comprises contacting a media containing increasing the TCE levels in the collected leachate to be the halogenated hydrocarbon compound, preferably 45 detoxified, and maximizing the detoxification efficiency TCE, with an amount of an autotrophic ammonia-oxi of the bacteria. dizing bacterium, preferably Nitrosomonas europaea, Specifically, TCE contaminated solids such as soil, or effective to completely degrade the halogenated hydro landfill can be detoxified by collecting the solids in a carbon compound. As used herein the halogenated hy holding area, such as a pit, and leaching the TCE from drocarbon containing media can be an aqueous solution 50 the contaminated material by passing pH-adjusted am or dispersion, a slurry containing contaminated soil or monia-containing water through the solids and separat halogenated hydrocarbon contaminated soil alone. In ing the leachate from the solids, preferably via a porous the method of the present invention the ammonia oxi solid layer positioned under the material. In the case of dizing bacterium is grown and harvested as described in intermittent leaching, the water will then be passed into Hooper and Nason, J. Biol. Chem., 240: 4044-4057 55 (1965), Hooper in Autotrophic Bacteria, Schlagel and a collection area such as a tank, pool or the like, where Bowein, eds. (1989) at pages 239-265, incorporated by it is held until reintroduced into the pit area. reference herein. As used herein, an effective amount of It is also envisioned that the method to degrade halo autotrophic ammonia oxidizing bacterium is an amount genated hydrocarbons described can be applied directly of the bacterium capable of degrading TCE to insignifi- 60 to soil containing halogenated hydrocarbon contami cant levels; preferably, at the rates stated herein. While nates, such as TCE together with ubiquitous Nitroso Nitrosomonas europaea can be used to degrade TCE monas present in the soil. In this case naturally present with little if any ammonia present, sustained growth of Nitrosomonas can be stimulated to degrade the haloge the bacteria for long-term use in the present invention is nated hydrocarbon compounds by adding an effective best accomplished when the organism is grown in an 65 amount of ammonia to the halogenated hydrocarbon aqueous media containing at least 10 umol/l ammonia contaminated soil. and preferably from about 1 mmol/l to about 10mmol/l The invention will be further described with refer ammonia. ence to the following detailed example. 5,055, 193 5 6 EXAMPLE-TCE DEGRADATION than 1 uM in 24 hrs (Table I). With fresh cells the rate of TCE degradation was often not stimulated by the Materials and Methods addition of ammonia. With storage of the cells for sev Sources; TCE, Spectrophotometric grade, Aldrich eral days the rate of TCE degradation in the absence of Chem. Co., Milwaukee, Wis., aa'dipyridyl and calcium added ammonia decreased to as little as zero. With aged carbide, Fisher Chem, Fairlawn N.J.; tetrapyrin (2- cells, the addition of ammonia stimulated the rate of chloro-6-trichloromethylpyridine), Dow Chem. Co., TCE degradation. Midland, Mich. Saturated acetylene solutions were The disappearance of 11 uM TCE as catalyzed by a prepared by passing acetylene, generated from aqueous suspension of aged Nitrosomonas in the presence of 1 calcium carbide in one serum bottle, through buffer in a 10 mM ammonia occurred at an initial rate of at least 1.1 second serum bottle. Nitrosomonas europaea was grown nmoles (mg protein)- min-1 and continued at nearly and harvested as described in Hooper and Nason, J. that rate for many hours. Nearly all TCE had disap Biol. Chem., supra. Cells were stored at 4 C. as a 20% peared from the headspace by the end of the reaction. (wet weight) suspension. Oxygen utilization was as The amount of nitrite produced indicated that the sayed in a Gilson Medical Electronics, Middleton, Wis. 15 added and endogenous ammonia had been completely oxygraph with a Clark electrode. The chamber con utilized. At 3 hrs or 24 hrs the ratio of TCE degraded:- tained 1.5 mg ml wet weight cells and 1 mM ammo nitrite produced was approximately 1:100 (Table I). A nia. second and third addition of the same concentrations of Procedure 20 TCE and ammonia and subsequent incubation of 24 hrs For degradation of TCE, a 50 ml volume of reaction resulted in the further disappearance of 5 and 4 uM mixture containing, as indicated, 0.8 mg wet weight TCE and additional appearance of 430 and 63 uM ni (0.11 mg protein) ml of cells, 0.5 mM ammonium trite, respectively. The rate of degradation of TCE and sulfate, and TCE in 50 mM Na/K phosphate solution, production of nitrite progressively decreased during pH 7.5, was incubated at 20° C. in an inverted 125 ml 25 these long incubations. In cells lacking TCE, the loss of Wheaton (Millville, N.J. sorum bottle sealed with nitrite-producing ability occurred with approximately Pierce (Rockford, Ill.) "tufbond' teflon rubber lami the same time course. nated disc and hypo-vial aluminum clasp. A 5 ul vol Table I shows that the ammonia-dependent disap ume of 110 mM TCE in dimethylsulfoxide (DMSO) pearance of TCE was inhibited by aa'dipyridyl, nitra was added to start the reaction. The concentration of 30 pyrin or acetylene at concentrations where oxidation of TCE 11 uM is expressed as if it were all dissolved in 50 ammonia but not hydroxylamine is inhibited. ml, see Wackett and Gibson, Appl. Enyt, Microbio, 54: The above example demonstrates the disappearance 1703-1708 (1988). A sample of the head-space gas was of TCE from the headspace of bottles containing Nitro removed with a Hamilton (Reno, Nev.) 250 ul gas-tight somonas. Controls show that the disappearance is not syringe with 22S gauge blunt-tip (#3 point) needle and 35 due to leakage from the bottles or adsorption to glass, injected into a Hewlett-Packard 5890 Gas Chromoto The complete loss of activity in aged cells lacking am graph (HP#3396A integrator). Chromatography was monia and the sensitivity to specific inhibitors of oxida isothermal at 55 C. using He carrier gas (1.5 ml min) tion of ammonia indicates that TCE is being chemically on a 30 m, 0.25 mm ID DB-5 fused silica capillary col transformed rather than just absorbed by cells. Two umn (J&W Scientific, Folsom, Calif.). Analysis was by 40 observations indicate that degradation of TCE is cata electron capture detector at 250 C. with Ar:CH4 (95:5) lyzed by or strongly dependent on turnover of AMO: make up gas at 60 ml min-1. Under these conditions of (a) stimulation by ammonia of degradation of TCE (b) injection and assay and a split ratio of 1:40, 50 ul of 11 inhibition of TCE degradation by compounds which uM TCE had an Rf of 2.5 and gave an integrated peak are thought to specifically inhibit oxidation of ammonia. size of 160,000 area units. Three injections of single 45 TCE degradation continues for several days at the samples from the incubation vessel usually gave errors concentrations of Nitrosomonas, TCE and NH3 used in of 1% and no greater than 3%. Nitrite was assayed as the present study. The observed loss of TCE-degrading described in Hooper and Nasen, J. Biol. Chem. supra, activity seemed to be unrelated to the presence of TCE. Results Thus we have not observed reversible inactivation, by 50 TCE or its degradation product, of the TCE-degrading The rate of utilization of oxygen by cell suspensions capability of Nitrosomonas as has been observed with with 1 mM ammonia was inhibited by 98% in the pres organisms catalyzing the methane-or toluene-dependent ence of 1.1 mM TCE. When cells were incubated under degradation of TCE. Table I. Transformation of TCE these conditions for 15 min, washed by sedimentation by Suspensions of Nitrosomonas europaeal. and resuspended, complete ammonia-oxidizing activity 55 What is claimed is: was recovered. In contrast, the rate of oxygen utiliza 1. A method for microbial degradation of a haloge tion coupled to the oxidation of 2 mM hydroxylamine nated hydrocarbon compound comprising: was not inhibited by 1.1 mM TCE. The results suggest contacting an aqueous solution containing a concen that TCE may interact specifically with AMO and that, tration of ammonia effective to sustain growth and at least in short term incubations, cells are not inacti 60 said halogenated hydrocarbon with an amount of vated. The inhibitory effect of TCE on production of an autotrophic ammonia-oxidizing bacterium effec nitrate from ammonia is shown in FIG. 1 and Table I. tive to degrade essentially all of said halogenated Incubation of Nitrsomonas with 11 um TCE resulted in hydrocarbon compound. the progressive disappearance of TCE and very slow 2. The method of claim 1 wherein said halogenated production of nitrite (FIG. 1 and Table I). At the end of 65 hydrocarbon containing solution is contacted with an 24 hrs the ratio of TCE degraded: nitrite produced was aqueous media comprising from about 10 mg to about 2 approximately 1:10. In the absence of cells (in the pres g/of wet weight of cells/l of said ammonia-oxidizing ence or absence of NH3), TCE disappearance was less bacterium. 5,055,193 7 8 3. The method of claim 1 wherein said ammonia-oxi nated hydrocarbon compound at concentrations of at dizing bacterium is capable of degrading said haloge least about 300-400 micromoles/l. nated hydrocarbon compound at initial concentrations 12. The method of claim 8 wherein said ammonia-oxi of up to about 300-400 micromoles/1. dizing bacterium is Nitrosomonas europaea. 4. The method of claim 1 wherein said ammonia-oxi 13. The method of claim 8 wherein said halogenated dizing bacterium is Nitrosomonas europaea. hydrocarbon compound is an aliphatic halogenated 5. The method of claim 1 wherein said ammonia-oxi hydrocarbon compound. dizing bacterium is a species selected from the group 14. The method of claim 8 wherein said aliphatic consisting of Nitrosospira, Nitrosolabus, Nitrosovibrio halogenated hydrocarbon compound is trichloroethy and Nitrosocytis. O lene (TCE). 6. The method of claim 1 wherein said halogenated 15. A method of degrading a halogenated hydrocar hydrocarbon compound is an aliphatic halogenated bon compound comprising culturing an ammonia-oxi hydrocarbon compound. dizing bacterium of the genus Nitrosomonas in an aque 7. The method of claim 1 wherein said aliphatic halo ous medium comprising an amount of ammonia effec genated hydrocarbon compound is trichloroethylene 15 tive to sustain growth; and then contacting a haloge (TCE). nated hydrocarbon compound containing media with 8. A method for microbial degradation of a haloge an amount of said bacterium effective to degrade sub nated hydrocarbon compound, said method compris stantially all of said halogenated hydrocarbon. ling: 16. The method of claim 15 wherein said amount of contacting an aqueous solution containing a concen 20 ammonia is from trace amounts to about 40 mM. tration of ammonia effective to sustain growth and 17. The method of claim 15 wherein said bacteria is said halogenated hydrocarbon compound with an capable of degrading said halogenated hydrocarbon amount of a ammonia-oxidizing bacterium effec compound at a rate of about 1-100 micromoles per hour tive to degrade substantially all of said halogenated per gram wet cells. hydrocarbon compound at a rate of from about 1 to 25 18. The method of claim 15 wherein said bacterium is about 100 micromoles per hour per gram wet cells, capable of degrading TCE at initial concentrations of said ammonia-oxidizing bacterium producing an about 100 umoles/l. ammonia monoxygenase. 19. The method of claim 15 wherein from about 9. The method of claim 8 wherein said ammonia-oxi 10-2000 mg wet weight of said bacterium per liter are dizing bacterium is capable of degrading said haloge 30 added to said media. nated hydrocarbon compound at a rate of from about 2 20. The method of claim 16 wherein said media is an to about 30 umoles per hour per gram wet cells. aqueous solution containing TCE. 10. The method of claim 8 wherein said halogenated 21. The method of claim 15 wherein said media is an hydrocarbon containing solution is contacted with an aqueous slurry containing TCE. aqueous media comprising about 10 mg to about 2000 35 22. The method of claim 15 wherein said media is soil ng wet weight of said ammonia-oxidizing bacterium contaminated with TCE. per liter. 23. The method of claim 15 wherein said bacterium is 11. The method of claim 8 wherein said ammonia-oxi Nitrosomonas europaea. dizing bacterium is capable of degrading said haloge .x k k

45

50

55

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 5,055, 193 Page 1 of 3 DATED October 8, 1991

INVENTOR(S) : Alan Hooper it is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

At column 1 lines 19-20, for "trichlo-rethylene" read --trichloroethylene--

At column 2, line 61, for "TCS" read --TCE

At column 5, line 26, for "N.J. Sorum" read --N.J.) serum--

At column 5, line 27, after "teflon" insert --/--

At column 5, line 32, for "Microbio," read --Microbiol. , --

UNITED STATES PATENT ANDTRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 5,055, 193 Page 2 of 3

DATED October 8, 1991 INVENTOR(S) : Alan Hooper

it is certified that error appears in the above-indentified patent and that said Letters Patent is hereby corrected as shown below:

At column 6, line 54, after "europaea'." insert the following

table:

incubation - Mixture - Rate - Net - Change - ATCE +ANO ATCE +ANO ------(nmoles lin mg prot.') ( uM) TCE -0.05

cells, TCE 0.2 65

cells NH 73 400 cells TCENH 0.42 42 300

cells, NH., dipyridyl' wo l iOO cells, NH, dipyridyl, TCE 0.08 0.7 cells, NH, niträpyrin' 4. 60 cells, NH, nitrapyrin, TCE 0.024 0. 27

cells, NH, , acetylene' o -l. 4

cells, NH, acetylene, TCE 0.082 0.5 0.38 cells, acetylene, TCE -0.04 O 0.37 a---ee-ee------alm-m-m-m-ranaramurm-r-r-" "

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 5,055, 193 Page 3 of 3 DATED October 8, 1991 INVENTOR(S) : Alan Hooper

it is certified that error appears in the above-indentified patent and that said Letters Patent is hereby Corrected as shown below:

Assay conditions described in Materials and Methods. At 3 hrs. At 24 hours. "O. l. nM aa' dipyridyl (stock 50 mM in DMSO). See Wackett & Gibson, supra 0.05 mM nitrapyrin (stock 25 mM in DMSO). 'cells (5% w/w suspension) were preincubated for 30 min with stirring in the presence of lSO um acetylene (added from a 77 mM stock solution) in a sealed bottle with no head space.

Signed and Sealed this Eleventh Day of January, 1994 (a teen

BRUCELEHMAN Attesting Officer Commissioner of Patents and Trademarks