Patent Publication Number: US-6210579-B1

Title: Bioremediation of pollutants with butane-utilizing bacteria

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. application Ser. No. 09/215,965 filed Dec. 18, 1998, now U.S. Pat. No. 6,051,130, which is a division of U.S. application Ser. No. 08/767,750 filed Dec. 17, 1996, now U.S. Pat. No. 5,888,396. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to the degradation of pollutants, and more particularly relates to bioremediation of pollutants such as chlorinated aliphatic hydrocarbons using butane-utilizing microorganisms. 
     BACKGROUND INFORMATION 
     Chlorinated, volatile, aliphatic hydrocarbons such as trichloroethene (TCE) are the most commonly reported contaminants of groundwater. Through releases of solvents, degreasers and other compounds, chlorinated compound contamination in surface and subsurface environments has reached high levels, and in many areas has seriously jeopardized drinking water aquifers and reservoirs. TCE is a suspected human carcinogen and remains the number one priority pollutant on the National Priority List of the U.S. Environmental Protection Agency. 
     When the discovery of the magnitude of chlorinated contamination in aquifer systems in the United States, and worldwide, came to light in the early 1980s, few approaches were developed to aggressively remediate the chlorinated contaminated sites. Available remediation methods for subsurface-environments include air sparging of the groundwater and the vacuum extraction of contaminants from the vadose zone. These remedial strategies transfer contamination from the subsurface environment to either the air or to activated carbon which must then be landfilled or incinerated. Landfilling contaminated activated carbon transfers the contamination from one source area to another while incineration is costly and requires considerable energy and costly equipment to completely volatilize organic compounds. Treatment strategies based on oxidation of contaminants that use ultraviolet radiation in combination with a chemical oxidant like hydrogen peroxide are also energy costly and require the injection of expensive chemicals. 
     Bioremediation is a method of harnessing the ability of microorganisms to degrade toxic pollutants. Anaerobic biodegradation of TCE usually results in the formation of harmful metabolites such as dichloroethylenes and the known carcinogen vinyl chloride. 
     The ability of aerobic methane-utilizing bacteria to degrade TCE cometabolically is known. However, the use of methane-utilizing bacteria is limited due to the toxic effects of chlorinated hydrocarbons like TCE in rather low concentrations. As disclosed by Broholm et al., “Toxicity of 1,1,1-Trichloroethane and Trichloroethene on a Mixed Culture of Methane-Oxidizing Bacteria”,  Applied and Environmental Microbiology , Aug. 1990, p. 2488-2493, the toxic effects of trichloroethene become substantial above 6 mg per liter (ppm) in water. In addition, trace amounts of copper have proven to inhibit methane monooxygenase. 
     The use of methane-utilizing bacteria to degrade TCE is disclosed in several patents. For example, U.S. Pat. No. 5,037,551 to Barkley and U.S. Pat. No. 5,057,221 to Bryant et al. disclose ex-situ bioreactors using a rigid substrate bed to support aerobic methanotrophic microorganisms which degrade halogenated organic compounds. The substrate bed may be made of manufactured solid material, such as activated carbon particles or contoured plastic spheres. In each of these patents, examples are provided wherein methane is supplied to an ex-situ bioreactor to degrade the halogenated organic compounds. In addition, U.S. Pat. No. 5,057,221 includes an example wherein propane is supplied to the bioreactor bed. 
     U.S. Pat. No. 5,384,048 to Hazen et al. discloses an in-situ groundwater bioremediation apparatus and method using a methane nutrient source. Bioremediation is carried out by periodically injecting nutrient fluid into the contaminant groundwater plume to stimulate the subsurface population of the microorganisms to increase. An oxygenated fluid is also injected into the plume to allow the aerobic microorganisms to break down the contaminants. The particular microorganisms disclosed are indigenous methanotrophs capable of biodegrading TCE by a series of enzymes including methane monooxygenase which are unique to this group of bacteria. 
     U.S. Pat. No. 5,326,703 to Hazen et al. discloses another in-situ method for biodegrading contaminants such as TCE. 
     U.S. Pat. No. 5,441,887 to Hanson et al. discloses an ex-situ method for biodegrading halogenated hydrocarbons by soluble methane monooxygenase. In the examples of this patent, methane is used as the food source for the methanotrophic bacteria. 
     U.S. Pat. No. 4,713,343 to Wilson Jr. et al. discloses a method for to biodegrading halogenated hydrocarbons such as TCE. The method may be performed either in-situ or ex-situ, and uses microorganisms such as methanotrophic bacteria. 
     U.S. Pat. No. 5,316,940 to Georgiou et al. discloses an ex-situ packed bed bioreactor utilizing a specific mutant methanotrophic bacteria to biodegrade TCE. Methane or methanol is used as the energy source. 
     U.S. Pat. No. 5,342,769 to Hunter et al. discloses an ex-situ bioremediation method for removing contaminants such as TCE from groundwater. A specific natural methanogenic bacteria is used in the process, along with methane as the food source. 
     Each of the above-noted patents is incorporated herein by reference. 
     Despite these bioremediation efforts, a need still exists for the effective degradation of pollutants such as chlorinated aliphatic hydrocarbons. The present invention has been developed in view of the foregoing, and to remedy other deficiencies of the prior art. 
     SUMMARY OF THE INVENTION 
     In accordance with the present invention, butane-utilizing organisms are used to degrade pollutants such as chlorinated aliphatic hydrocarbons. Degradation may occur cometabolically or by direct metabolism The butane-utilizing organisms of the present invention may be used for in-situ or ex-situ bioremediation of chlorinated hydrocarbon contaminants contained in air, soil and groundwater waste streams. In addition, salt- and acid-tolerant butane-utilizing bacteria may be used to restore saline and low pH groundwater systems impacted by chlorinated hydrocarbon contamination. 
     An object of the present invention is to provide an improved method of degrading hydrocarbon pollutants. 
     Another object of the present invention is to provide a bioremediation method of degrading TCE which utilizes bacteria demonstrating low TCE toxicity. 
     Another object of the present invention is to provide a method of degrading chlorinated aliphatic hydrocarbons with butane-utilizing bacteria by a cometabolic process. 
     Another object of the present invention is to provide a method of degrading chlorinated aliphatic hydrocarbons with butane-utilizing bacteria capable of directly metabolizing the hydrocarbon pollutants. 
     Another object of the present invention is to provide a method of degrading a hydrocarbon pollutant by treating the pollutant with butane-utilizing bacteria in the presence of oxygen for a sufficient time for the butane-utilizing bacteria to degrade the hydrocarbon pollutant. 
     Another object of the present invention is to provide a method of decontaminating water by treating the water with butane-utilizing bacteria to reduce or eliminate hydrocarbon pollutants contained in the water. 
     Another object of the present invention is to provide an ex-situ bioremediation apparatus that uses butane-utilizing bacteria and can be easily transported to various bioremediation sites. 
     These and other objects of the present invention will become more apparent from the following description. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIGS. 1 is a graph showing butane concentration at various times for experimental control samples. 
     FIG. 2 is a graph showing TCE concentration at various times for experimental control samples. 
     FIGS. 3-16 are graphs showing butane and TCE loss for various butane-utilizing bacteria in accordance with the present invention. 
     FIG. 17 is a partially schematic illustration of a bioreactor for use with butane-utilizing bacteria in accordance with the present invention. 
     FIG. 18 is a partially schematic illustration of multiple bioreactors connected in series using butane-utilizing bacteria in accordance with the present invention. 
     FIG. 19 is a graph showing butane consumption versus time during operation of a bioreactor in accordance with the present invention. 
     FIG. 20 is a graph showing TCE degradation versus time-during operation of a bioreactor in accordance with the present invention. 
     FIG. 21 is a graph showing butane consumption versus time during operation of a bioreactor in accordance with the present invention. 
     FIG. 22 is a graph showing TCE degradation versus time during operation of a bioreactor in accordance with the present invention. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present invention relates to a method and associated apparatus for the degradation of hydrocarbon pollutants. The hydrocarbon pollutants may include chlorinated aliphatics, chlorinated aromatics and non-chlorinated aromatics, with chlorinated aliphatic hydrocarbons being of particular interest. Specific hydrocarbon pollutants include methylene chloride, 1,1-dichloroethane, chloroform, 1,2-dichloropropane, dibromochloromethane, 1,1,2-trichloroethane, 2-chloroethylvinyl ether, tetrachloroethene (PCE), chlorobenzene, 1,2-dichloroethane, 1,1,1-trichloroethane, bromodichloromethane, trans-1,3-dichloropropene, cis-1,3-dichloropropene, bromoform, benzene, toluene, ethylbenzene, xylenes, chloromethane, bromomethane, vinyl chloride, chloroethane, 1,1-Idichloroethene, trans-1,2-dichloroethene, trichloroethene (TCE), dichlorobenzenes, cis-1,2-dichloroethene, dibromomethane, 1,4-dichlorobutane, 1,2,3-trichloropropane, bromochloromethane, 2,2-dichloropropane, 1,2-dibromoethane, 1,3-dichloropropane, bromobenzene, chlorotoluenes, trichlorobenzenes, trimethylbenzenes, trans-1,4-dichloro-2-butene and butylbenzenes. Trichloroethene is a particular hydrocarbon pollutant that may be degraded in accordance with the present invention. 
     The bioremediation process may be performed either in-situ or ex-situ to remove contaminants from various environments including aqueous systems. Aqueous systems suitable for treatment include drinking water, groundwater, industrial waste water and the like. 
     According to an embodiment of the present invention, it has been discovered that butane-utilizing bacteria are extremely effective at degrading low molecular weight halogenated aliphatic hydrocarbons such as TCE. The butane-utilizing bacteria may be used to aerobically degrade TCE by cometabolism and/or direct metabolism processes. 
     In contrast with conventional bioremediation techniques which typically use methane-utilizing bacteria to degrade TCE, the present invention provides a robust system using butane-utilizing bacteria having low TCE toxicity and improved TCE consumption capabilities. 
     The butane-utilizing bacteria of the present invention produce oxygenase enzymes and are capable of metabolizing butane. The operative enzymes may include extracellular enzymes, intracellular enzymes and cell-bound enzymes. The butane-utilizing bacteria typically produce butane monoxygenase and/or butane dioxygenase enzymes, and in some embodiments may also be capable of producing dehologenase enzymes which directly metabolize TCE. 
     The butane-utilizing bacteria of the present invention may contain gram negative and gram positive aerobic rods and cocci, facultative anaerobic gram negative rods, non-photosynthetic, non-fruiting gliding bacteria and irregular non-sporing gram positive rods. 
     Of the Pseudomomdaceae family comprising gram-negative aerobic rods and cocci, species of the following genera may be suitable: Pseudomonas; Variovorax; Chryseobacteriwn; Comamonas; Acidovorax; Stenotrophomonas; Sphingobacterium; Xanthomonas; Frateuria; Zoogloea; Alcaligenes; Flavobacterium; Derxia; Lampropedia; Brucella; Xanthobacter; Thermus; Thermomicrobium; Halomonas; Alteromonas; Serpens; Janthinobacterium; Bordetella; Paracoccus; Beijerinckia; and Francisella. 
     Of the  Nocardioform Actinomycetes  family comprising gram-positive Eubacteria and Actinomycetes, the following genera may be suitable: Nocardia; Rhodococcus; Gordona; Nocardioides; Saccharopolyspora; Micropotyspora; Promicromonospora; Intrasporangium; Pseudonocardia; and Oerskovia. 
     Of the Micrococcaceae family comprising gram-positive cocci, the following genera may be suitable: Micrococcus; Stomatococcus; Planococcus; Staphylococcus; Aerococcus; Peptococcus; Peptostreptococcus; Coprococcus; Gemella; Pediococcus; Leuconostoc; Ruminococcus; Sarcina; and Streptococcus. 
     Of the Vibrionaceae family comprising facultative anaerobic gram-negative rods, the following genera may be suitable: Aeromonas; Photobacterium; Vibrio; Plesiomonas; Zymomonas; Chromobacterium; Cardiobacterium; Calymmatobacterium; Streptobacillus; Eikenella; and Gardnerella. 
     Of the Rhizobiaceae family comprising gram-negative aerobic rods and cocci, the following genera may be suitable: Phyllobacterium; Rhizobium; Bradyrhizobium; and Agrobacterium. 
     Of the Cytophagaceae family comprising non-photosynthetic, gliding bacteria, non-fruiting, the following genera may be suitable: Cytophaga; Flexibacter; Saprospira; Flexithrix; Herpetosiphon; Capnocytophaga; and Sporocytophaga. 
     Of the Corynebacterium family comprising irregular, non-sporing gram-positive rods, the following genera may be suitable: Aureobacterium; Agromyces; Arachnia; Rothia; Acetobacterium; Actinomyces; Arthrobactera; Arcanobacterium; Lachnospira; Propionibacterium; Eubacterium; Butyrivibria; Brevibacterium; Bifidobacterium; Microbacterium; Caseobacter; and Thermoanaerobacter. 
     The following isolation techniques were used for obtaining pure and mixed cultures of various methane-, propane- and butane-utilizing bacteria. Enrichment procedures were used to increase bacterial population for a given growth substrate. Soil samples collected from a variety of sites underwent enrichment transfers weekly for a period of one year. The methods and materials used for the enrichment studies are described below. 
     Soil samples were collected with a stainless-steel hand auger at depths that varied between one to two feet. The soils samples were stored in dedicated glass containers and moistened with sterile deionized/distilled water for transport to the laboratory. The hand auger was decontaminated between sampling locations with three Alconox soap/distilled water rinses. Soil samples used as inocula were collected from the locations summarized in Table 1. 
     
       
         
           
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Sample Number/Matrix 
                 Sample Location 
               
               
                   
               
             
            
               
                 1/soil 
                 Landfill cell 
               
               
                 2/soil 
                 #2 fuel oil impacted soil 
               
               
                 3/soil 
                 Landfill cell 
               
               
                 4/soil 
                 Gasoline and waste oil impacted soils 
               
               
                 5/soil 
                 Shallow freshwater lagoon 
               
               
                 6/soil 
                 Salt marsh 
               
               
                 7/soil 
                 Industrial outfall 
               
               
                 8/soil 
                 #2 fuel oil impacted soil 
               
               
                   
               
            
           
         
       
     
     Cultures were transferred weekly for a period of one year in liquid media to increase the relative numbers of methane-, propane- and butane-utilizing bacteria. The liquid media was a mineral salts media (MSM) prepared from the following chemicals: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 MgSO 4 -7H 2 O 
                 1.0 
                 g; 
               
               
                   
                 CaCl 2   
                 0.2 
                 g; 
               
               
                   
                 NH 4 Cl 
                 0.5 
                 g; 
               
               
                   
                 FeCl 3 -6H 2 O 
                 4.0 
                 mg; 
               
               
                   
                 Trace elements solution 
                 0.5 
                 ml; and 
               
               
                   
                 Distilled water 
                 900 
                 ml. 
               
               
                   
                   
               
            
           
         
       
     
     A trace elements solution, which provides micronutrients for bacterial growth, was prepared comprising the following ingredients: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 ZnCl 2   
                 5.0 
                 mg; 
               
               
                   
                 MnCl 2 -4H 2 O 
                 3.0 
                 mg; 
               
               
                   
                 H 3 BO4 
                 30.0 
                 mg; 
               
               
                   
                 NiCl 2 -6H 2 O 
                 2.0 
                 mg; 
               
               
                   
                 (NH 4 ) 6 Mo 7 O 24 -4H 2 O 
                 2.25 
                 mg; and 
               
               
                   
                 Distilled water 
                 1000 
                 ml. 
               
               
                   
                   
               
            
           
         
       
     
     The pH of the MSM was adjusted to 6.8 before autoclaving (20 min at 121 degree C.) with 20.0 ml of a phosphate buffer solution comprising 3.6 g of Na 2 HPO 4  and 1.4 g of KH 2 PO 4  in 100 ml of distilled water. After autoclaving the MSM and the buffer solution, another 2.0 ml of the buffer solution was added to the MSM when the temperature of the media reached 60 degree C. The MSM cocktail was completed with the addition of 4.0 mg of casamino acids and 4.0 mg of yeast (Difco) dissolved in 100 ml of distilled water. The nutrient solution was filter sterilized by vacuum filtration through a 0.2 micron filter (Gelman) prior to addition to the MSM. 
     Prior to the first enrichment transfer, the inocula from the eight sampling locations summarized in Table 1 underwent a series of pre-treatments. The first two pre-treatments were conducted on the original soil materials used. as inocula. The last two treatments were applied as MSM alterations during the weekly transfers. The pre-treatments consisted of the following: (1) 30% ethanol saturation rinse followed by a sterile phosphate buffer rinse (ethanol); (2) 60° C. water bath for 15 minutes (heat); (3) no treatment (no-treat); (4) MSM containing 10% aqueous solution of sodium chloride (10% NaCl); and (5) MSM with pH of 2.0 (pH of 2). Treatment Nos. (4) and (5) were employed in an attempt to locate extreme halophiles and acidophiles capable of utilizing hydrocarbons as a growth substrate. 
     The first enrichment transfers for each sample series were conducted in 72 ml serum bottles (Wheaton) with 20 ml of MSM and 1.0 g of inocula. Subsequent culture transfers (5.0 ml) were conducted with sterilized plastic syringes (B&amp;D). The bottles were capped with red rubber plugs and crimped with aluminum seals (Wheaton). Each sample was handled aseptically and all glassware, materials and supplies were sterilized by autoclaving. Table 2 summarizes the enrichment transfer schedule and the concentration of methane or propane replaced in the headspace of each serum bottle using a dedicated gas tight syringe (Hamilton) with a Fisher Scientific inert sampling valve (on/off lever) to control gas loss from the needle tip between each transfer. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Sample No. 
                 Pre-Treatment 
                 Food Source 
                 Sample ID 
               
               
                   
               
             
            
               
                 1 
                 ethanol 
                 methane 
                 1EM 
               
               
                 1 
                 heat 
                 methane 
                 1HM 
               
               
                 1 
                 no-treat 
                 methane 
                 1NM 
               
               
                 1 
                 10% NaCl 
                 methane 
                 1SM 
               
               
                 1 
                 pH of 2.0 
                 methane 
                 1AM 
               
               
                 1 
                 ethanol 
                 propane 
                 1EP 
               
               
                 1 
                 heat 
                 propane 
                 1HP 
               
               
                 1 
                 no-treat 
                 propane 
                 1NP 
               
               
                 1 
                 10% NaCl 
                 propane 
                 1SP 
               
               
                 1 
                 pH of 2.0 
                 propane 
                 1AP 
               
               
                   
               
            
           
         
       
     
     The amount of oxygen required for mineralization of methane, propane and butane can be derived from the following equations.                  CH   4     +     2        O   2         =       CO   2     +     2        H   2        O               2:1                     C   3          H   8       +     5        O   2         =       3        CO   2       +     4        H   2        O               5:1                     C   4          H   10       +     6.5        O   2         =       4      CO2     +     5        H   2        O               6.5:1                         
     Table 2 summarizes the entire set of enrichment transfers prepared for Sample No. 1. The first sample series did not include a butane treatment. The remaining seven samples were prepared in identical fashion and, in addition, contained a butane treatment series, as shown in Tables 3 through 9. A control (serum bottle with sterilized MSM only) was maintained for each sample series. 
     All hydrocarbon gases described herein were research grade quality (Scott Specialty Gases). Methane was added at a concentration of 27% (vol/vol), propane at 10% and butane at 6%. After the first six months of enrichment transfers, the concentrations were reduced to 13% for methane and 9% for propane. The concentration of butane remained the same at 6%. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Sample No. 
                 Pre-Treatment 
                 Food Source 
                 Sample ID 
               
               
                   
               
             
            
               
                 2 
                 ethanol 
                 methane 
                 2EM 
               
               
                 2 
                 heat 
                 methane 
                 2HM 
               
               
                 2 
                 no-treat 
                 methane 
                 2NM 
               
               
                 2 
                 10% NaCl 
                 methane 
                 2SM 
               
               
                 2 
                 pH of 2.0 
                 methane 
                 2AM 
               
               
                 2 
                 ethanol 
                 propane 
                 2EP 
               
               
                 2 
                 heat 
                 propane 
                 2HP 
               
               
                 2 
                 no-treat 
                 propane 
                 2NP 
               
               
                 2 
                 10% NaCl 
                 propane 
                 2SP 
               
               
                 2 
                 pH of 2.0 
                 propane 
                 2AP 
               
               
                 2 
                 ethanol 
                 butane 
                 2EB 
               
               
                 2 
                 heat 
                 butane 
                 2HB 
               
               
                 2 
                 no-treat 
                 butane 
                 2NB 
               
               
                 2 
                 10% NaCl 
                 butane 
                 2SB 
               
               
                 2 
                 pH of 2.0 
                 butane 
                 2AB 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Sample No. 
                 Pre-Treatment 
                 Food Source 
                 Sample ID 
               
               
                   
               
             
            
               
                 3 
                 ethanol 
                 methane 
                 3EM 
               
               
                 3 
                 heat 
                 methane 
                 3HM 
               
               
                 3 
                 no-treat 
                 methane 
                 3NM 
               
               
                 3 
                 10% NaCl 
                 methane 
                 3SM 
               
               
                 3 
                 pH of 2.0 
                 methane 
                 3AM 
               
               
                 3 
                 ethanol 
                 propane 
                 3EP 
               
               
                 3 
                 heat 
                 propane 
                 3HP 
               
               
                 3 
                 no-treat 
                 propane 
                 3NP 
               
               
                 3 
                 10% NaCl 
                 propane 
                 3SP 
               
               
                 3 
                 pH of 2.0 
                 propane 
                 3AP 
               
               
                 3 
                 ethanol 
                 butane 
                 3EB 
               
               
                 3 
                 heat 
                 butane 
                 3HB 
               
               
                 3 
                 no-treat 
                 butane 
                 3NB 
               
               
                 3 
                 10% NaCl 
                 butane 
                 3SB 
               
               
                 3 
                 pH of 2.0 
                 butane 
                 3AB 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 Sample No. 
                 Pre-Treatment 
                 Food Source 
                 Sample ID 
               
               
                   
               
             
            
               
                 4 
                 ethanol 
                 methane 
                 4EM 
               
               
                 4 
                 heat 
                 methane 
                 4HM 
               
               
                 4 
                 no-treat 
                 methane 
                 4NM 
               
               
                 4 
                 10% NaCl 
                 methane 
                 4SM 
               
               
                 4 
                 pH of 2.0 
                 methane 
                 4AM 
               
               
                 4 
                 ethanol 
                 propane 
                 4EP 
               
               
                 4 
                 heat 
                 propane 
                 4HP 
               
               
                 4 
                 no-treat 
                 propane 
                 4NP 
               
               
                 4 
                 10% NaCl 
                 propane 
                 4SP 
               
               
                 4 
                 pH of 2.0 
                 propane 
                 4AP 
               
               
                 4 
                 ethanol 
                 butane 
                 4EB 
               
               
                 4 
                 heat 
                 butane 
                 4HB 
               
               
                 4 
                 no-treat 
                 butane 
                 4NB 
               
               
                 4 
                 10% NaCl 
                 butane 
                 4SB 
               
               
                 4 
                 pH of 2.0 
                 butane 
                 4AB 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 Sample No. 
                 Pre-Treatment 
                 Food Source 
                 Sample ID 
               
               
                   
               
             
            
               
                 5 
                 ethanol 
                 methane 
                 5EM 
               
               
                 5 
                 heat 
                 methane 
                 5HM 
               
               
                 5 
                 no-treat 
                 methane 
                 5NM 
               
               
                 5 
                 10% NaCl 
                 methane 
                 5SM 
               
               
                 5 
                 pH of 2.0 
                 methane 
                 5AM 
               
               
                 5 
                 ethanol 
                 propane 
                 5EP 
               
               
                 5 
                 heat 
                 propane 
                 5HP 
               
               
                 5 
                 no-treat 
                 propane 
                 5NP 
               
               
                 5 
                 10% NaCl 
                 propane 
                 5SP 
               
               
                 5 
                 pH of 2.0 
                 propane 
                 5AP 
               
               
                 5 
                 ethanol 
                 butane 
                 5EB 
               
               
                 5 
                 heat 
                 butane 
                 5HB 
               
               
                 5 
                 no-treat 
                 butane 
                 5NB 
               
               
                 5 
                 10% NaCl 
                 butane 
                 5SB 
               
               
                 5 
                 pH of 2.0 
                 butane 
                 5AB 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                 Sample No. 
                 Pre-Treatment 
                 Food Source 
                 Sample ID 
               
               
                   
               
             
            
               
                 6 
                 ethanol 
                 methane 
                 6EM 
               
               
                 6 
                 heat 
                 methane 
                 6HM 
               
               
                 6 
                 no-treat 
                 methane 
                 6NM 
               
               
                 6 
                 10% NaCl 
                 methane 
                 6SM 
               
               
                 6 
                 pH of 2.0 
                 methane 
                 6AM 
               
               
                 6 
                 ethanol 
                 propane 
                 6EP 
               
               
                 6 
                 heat 
                 propane 
                 6HP 
               
               
                 6 
                 no-treat 
                 propane 
                 6NP 
               
               
                 6 
                 10% NaCl 
                 propane 
                 6SP 
               
               
                 6 
                 pH of 2.0 
                 propane 
                 6AP 
               
               
                 6 
                 ethanol 
                 butane 
                 6EB 
               
               
                 6 
                 heat 
                 butane 
                 6HB 
               
               
                 6 
                 no-treat 
                 butane 
                 6NB 
               
               
                 6 
                 10% NaCl 
                 butane 
                 6SB 
               
               
                 6 
                 pH of 2.0 
                 butane 
                 6AB 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 Sample No. 
                 Pre-Treatment 
                 Food Source 
                 Sample ID 
               
               
                   
               
             
            
               
                 7 
                 ethanol 
                 methane 
                 7EM 
               
               
                 7 
                 heat 
                 methane 
                 7HM 
               
               
                 7 
                 no-treat 
                 methane 
                 7NM 
               
               
                 7 
                 10% NaCl 
                 methane 
                 7SM 
               
               
                 7 
                 pH of 2.0 
                 methane 
                 7AM 
               
               
                 7 
                 ethanol 
                 propane 
                 7EP 
               
               
                 7 
                 heat 
                 propane 
                 7HP 
               
               
                 7 
                 no-treat 
                 propane 
                 7NP 
               
               
                 7 
                 10% NaCl 
                 propane 
                 7SP 
               
               
                 7 
                 pH of 2.0 
                 propane 
                 7AP 
               
               
                 7 
                 ethanol 
                 butane 
                 7EB 
               
               
                 7 
                 heat 
                 butane 
                 7HB 
               
               
                 7 
                 no-treat 
                 butane 
                 7NB 
               
               
                 7 
                 10% NaCl 
                 butane 
                 7SB 
               
               
                 7 
                 pH of 2.0 
                 butane 
                 7AB 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 9 
               
               
                   
                   
               
               
                   
                 Sample No. 
                 Pre-Treatment 
                 Food Source 
                 Sample ID 
               
               
                   
                   
               
             
            
               
                   
                 8 
                 ethanol 
                 methane 
                 8EM 
               
               
                   
                 8 
                 heat 
                 methane 
                 8HM 
               
               
                   
                 8 
                 no-treat 
                 methane 
                 8NM 
               
               
                   
                 8 
                 10% NaCl 
                 methane 
                 8SM 
               
               
                   
                 8 
                 pH of 2.0 
                 methane 
                 8AM 
               
               
                   
                 8 
                 ethanol 
                 propane 
                 8EP 
               
               
                   
                 8 
                 heat 
                 propane 
                 8HP 
               
               
                   
                 8 
                 no-treat 
                 propane 
                 8NP 
               
               
                   
                 8 
                 10% NaCl 
                 propane 
                 8SP 
               
               
                   
                 8 
                 pH of 2.0 
                 propane 
                 8AP 
               
               
                   
                 8 
                 ethanol 
                 butane 
                 8EB 
               
               
                   
                 8 
                 heat 
                 butane 
                 8HB 
               
               
                   
                 8 
                 no-treat 
                 butane 
                 8NB 
               
               
                   
                 8 
                 10% NaCl 
                 butane 
                 8SB 
               
               
                   
                 8 
                 pH of 2.0 
                 butane 
                 8AB 
               
               
                   
                   
               
            
           
         
       
     
     After the first two weeks of enrichment transfers, all liquid suspensions, with the exception of the 10% NaCl treatments, the 2.0 pH treatments and the controls, demonstrated a significant increase in turbidity. 
     After conducting the enrichment transfers for 25 weeks, morphological descriptions and direct cell counts were compiled for all isolates. Morphological descriptions of the isolates were compiled using an Olympus BH-2 Phase Contrast Microscope. In addition, a Bright Line Hemacytometer (Fisher Scientific) was used to enumerate cell densities by the direct count method. Table 10 summarizes the descriptions and cell density enumerations. Serum bottles of sterilized MSM were maintained as controls. 
     
       
         
           
               
               
               
             
               
                 TABLE 10 
               
               
                   
               
               
                   
                   
                 Enumeration 
               
               
                 Sample ID 
                 Morphology 
                 (cells/ml) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 1EM 
                 
                   cocci 
                 
                 2.5E8 
               
               
                 1HM 
                 
                   cocci/bacilli 
                 
                 1.8E8 
               
               
                 1NM 
                 
                   bacilli 
                 
                 1.3E8 
               
               
                 1SM 
                 
                   cocci 
                 
                 5.8E6 
               
               
                 1AM 
                 
                   cocci 
                 
                 3.8E6 
               
               
                 1EP 
                 
                   bacilli 
                 
                 4.0E6 
               
               
                 1HP 
                 
                   cocci 
                 
                 1.3E7 
               
               
                 1NP 
                 
                   bacilli 
                 
                 9.8E6 
               
               
                 1SP 
                 
                   diplococci 
                 
                 4.0E6 
               
               
                 1AP 
                   bacilli  (variable) 
                 1.5E6 
               
               
                 2EM 
                 
                   cocci/bacilli 
                 
                 1.2E8 
               
               
                 2HM 
                 
                   cocci/bacilli 
                 
                 7.3E7 
               
               
                 2NM 
                 
                   streptococci/bacilli 
                 
                 1.1E8 
               
               
                 2SM 
                 comma-shaped 
                 6.6E7 
               
               
                 2AM 
                 comma-shaped 
                 8.3E6 
               
               
                 2EP 
                 
                   bacilli 
                 
                 1.2E8 
               
               
                 2HP 
                   bacilli /comma-shaped 
                 1.8E8 
               
               
                 2NP 
                   bacilli  (variable) 
                 1.1E8 
               
               
                 2SP 
                 
                   cocci 
                 
                 7.0E6 
               
               
                 2AP 
                 
                   cocci 
                 
                 3.3E6 
               
               
                 2EB 
                 
                   cocci/bacilli 
                 
                 2.1E8 
               
               
                 2HB 
                   bacilli  (variable) 
                 2.5E8 
               
               
                 2NB 
                   cocci /comma-shaped 
                 1.9E8 
               
               
                 2SB 
                 
                   bacilli 
                 
                 2.5E6 
               
               
                 2AB 
                 
                   cocci 
                 
                 3.0E6 
               
               
                 3EM 
                 
                   cocci/bacilli 
                 
                 1.4E8 
               
               
                 3HM 
                 
                   cocci 
                 
                 1.2E8 
               
               
                 3NM 
                 
                   cocci 
                 
                 5.8E7 
               
               
                 3SM 
                 
                   cocci 
                 
                 7.5E5 
               
               
                 3AM 
                 
                   cocci 
                 
                 7.5E5 
               
               
                 3EP 
                 
                   bacilli 
                 
                 7.8E7 
               
               
                 3HP 
                 
                   bacilli 
                 
                 3.0E7 
               
               
                 3NP 
                 
                   bacilli 
                 
                 7.1E7 
               
               
                 3SP 
                 
                   cocci 
                 
                 1.0E6 
               
               
                 3AP 
                 
                   bacilli 
                 
                 2.5E5 
               
               
                 3EB 
                   bacilli  (variable) 
                 1.5E8 
               
               
                 3HB 
                 
                   cocci/bacilli 
                 
                 3.1E7 
               
               
                 3NB 
                 
                   cocci 
                 
                 3.1E8 
               
               
                 3SB 
                   cocci  (irregular) 
                 1.7E7 
               
               
                 3AB 
                 
                   cocci/bacilli 
                 
                 2.5E5 
               
               
                 4EM 
                   cocci  (variable) 
                 1.6E8 
               
               
                 4HM 
                 
                   diplococci 
                 
                 3.1E8 
               
               
                 4NM 
                 
                   cocci 
                 
                 1.6E8 
               
               
                 4SM 
                 
                   cocci 
                 
                 1.3E6 
               
               
                 4AM 
                 
                   bacilli 
                 
                 2.5E5 
               
               
                 4EP 
                   bacilli  (variable) 
                 1.0E8 
               
               
                 4HP 
                   bacilli  (variable) 
                 2.2E8 
               
               
                 4NP 
                 
                   cocci 
                 
                 1.3E8 
               
               
                 4SP 
                 
                   cocci 
                 
                 1.5E6 
               
               
                 4AP 
                 
                   cocci/bacilli 
                 
                 6.5E6 
               
               
                 4EB 
                 
                   bacilli 
                 
                 3.6E8 
               
               
                 4HB 
                   bacilli  (variable) 
                 4.8E8 
               
               
                 4NB 
                 
                   bacilli 
                 
                 2.6E8 
               
               
                 4SB 
                 comma-shaped 
                 1.3E6 
               
               
                 4AB 
                 
                   cocci 
                 
                 1.0E6 
               
               
                 5EM 
                   cocci  (variable) 
                 1.3E8 
               
               
                 5HM 
                 
                   cocci 
                 
                 1.4E8 
               
               
                 5NM 
                 
                   cocci 
                 
                 2.4E8 
               
               
                 5SM 
                 no cells 
                 0.0 
               
               
                 5AM 
                 no cells 
                 0.0 
               
               
                 5EP 
                   cocci  (variable) 
                 5.1E7 
               
               
                 5HP 
                 
                   bacilli 
                 
                 3.2E7 
               
               
                 5NP 
                 
                   streptococci 
                 
                 2.1E8 
               
               
                 5SP 
                   cocci  (variable) 
                 2.8E6 
               
               
                 SAP 
                 
                   bacilli 
                 
                 5.0E5 
               
               
                 5EB 
                 
                   bacilli 
                 
                 3.1E8 
               
               
                 5HB 
                 
                   cocci 
                 
                 3.2E7 
               
               
                 5NB 
                 
                   cocci 
                 
                 1.6E8 
               
               
                 5SB 
                 
                   bacilli 
                 
                 1.0E6 
               
               
                 5AB 
                 
                   cocci 
                 
                 2.5E6 
               
               
                 6EM 
                   bacilli  (variable) 
                 1.7E8 
               
               
                 6HM 
                 
                   cocci 
                 
                 2.6E8 
               
               
                 6NM 
                 
                   cocci/spirochetes 
                 
                 1.3E8 
               
               
                 6SM 
                   cocci  (variable) 
                 1.3E6 
               
               
                 6AM 
                 
                   cocci (variable) 
                 
                 2.0E6 
               
               
                 6EP 
                 
                   bacilli 
                 
                 2.8E7 
               
               
                 6HP 
                 
                   bacilli 
                 
                 1.3E8 
               
               
                 6NP 
                 
                   bacilli/spirochetes 
                 
                 2.0E8 
               
               
                 6SP 
                   cocci  (variable) 
                 3.5E6 
               
               
                 6AP 
                   cocci  (variable) 
                 5.0E5 
               
               
                 6EB 
                 
                   cocci 
                 
                 3.5E7 
               
               
                 6HB 
                 
                   bacilli 
                 
                 1.3E8 
               
               
                 6NB 
                 
                   bacilli 
                 
                 4.8E7 
               
               
                 6SB 
                 
                   cocci 
                 
                 2.3E6 
               
               
                 6AB 
                 
                   cocci 
                 
                 3.3E6 
               
               
                 7EM 
                 
                   streptococci 
                 
                 1.3E8 
               
               
                 7HM 
                 
                   staphylococci 
                 
                 3.2E7 
               
               
                 7NM 
                 
                   cocci/bacilli 
                 
                 3.1E8 
               
               
                 7SM 
                   cocci  (variable) 
                 3.0E6 
               
               
                 7AM 
                   cocci  (variable) 
                 4.0E6 
               
               
                 7EP 
                 
                   bacilli 
                 
                 1.4E8 
               
               
                 7HP 
                 
                   bacilli 
                 
                 4.1E8 
               
               
                 7NP 
                 
                   bacilli 
                 
                 3.5E8 
               
               
                 7SP 
                   cocci  (variable) 
                 1.2E7 
               
               
                 7AP 
                   cocci  (variable) 
                 1.5E6 
               
               
                 7EB 
                 
                   bacilli (variable) 
                 
                 1.6E8 
               
               
                 7HB 
                 
                   bacilli (variable) 
                 
                 3.9E8 
               
               
                 7NB 
                 
                   bacilli 
                 
                 4.2E8 
               
               
                 7SB 
                   cocci  (variable) 
                 4.3E6 
               
               
                 7AB 
                   cocci  (variable) 
                 2.8E6 
               
               
                 8EM 
                 
                   cocci 
                 
                 5.6E7 
               
               
                 8HM 
                 
                   cocci 
                 
                 6.1E7 
               
               
                 8NM 
                 
                   cocci 
                 
                 5.7E7 
               
               
                 8SM 
                   cocci  (variable) 
                 5.3E6 
               
               
                 8AM 
                 
                   bacilli 
                 
                 2.3E6 
               
               
                 8EP 
                 
                   bacilli 
                 
                 1.4E8 
               
               
                 8HP 
                 
                   cocci 
                 
                 3.8E8 
               
               
                 8NP 
                 
                   cocci 
                 
                 2.9E8 
               
               
                 8SP 
                 square-shaped 
                 6.5E6 
               
               
                 8AP 
                   cocci  (variable) 
                 3.8E6 
               
               
                 8EB 
                 
                   bacilli 
                 
                 1.3E6 
               
               
                 8HB 
                 
                   bacilli/streptococci 
                 
                 9.8E7 
               
               
                 8NB 
                   bacilli  (variable) 
                 1.2E8 
               
               
                 8SB 
                   bacilli  (variable) 
                 2.0E6 
               
               
                 8AB 
                   cocci  (variable) 
                 2.8E6 
               
               
                 Control-1 
                 no cells 
                 0.0 
               
               
                 Control-2 
                 no cells 
                 0.0 
               
               
                 Control-3 
                 no cells 
                 0.0 
               
               
                   
               
            
           
         
       
     
     Sample ID strains 3NB and 6NB were placed on deposit with the American Type Culture Collection (ATCC), Rockville, Md. on Aug. 22, 1996, under ATCC designation numbers 55808 and 55809, respectively. 
     A microcosm study using static headspace methods was conducted to evaluate methane, propane and butane consumption and TCE degradation rates for selected isolates. Static headspace involves a partitioning of volatile components between the aqueous and vapor phases enclosed in a gas tight vessel such as a serum bottle. 
     Each microcosm contained 25 ml of MSM and inoculum in. a 2 ml serum bottle, thus yielding an effective headspace of 47 ml. Low concentrations of research grade methane, propane or n-butane ranging from 200 to 1,200 parts per million (ppm) were added to the headspace in their respective microcosms, in contrast to concentrations used during enrichment transfers, in order to avoid saturating enzyme sites with the growth substrate which would reduce cometabolic rates. A TCE concentration in the range of 2 to 12 ppm was added to the headspace of each microcosm. According to Henry&#39;s Law, the TCE concentration in the headspace would yield a corresponding concentration of approximately 4 to 24 ppm in the aqueous phase. Hydrocarbon and TCE disappearance were evaluated at times 0, 48 and 96 hours for the methane and propane microcosms. TCE and n-butane disappearance was evaluated at times 0, 48, 96 and 144 hours. Serum bottles containing sterilized MSM and methane, MSM and propane, MSM and n-butane, and MSM and TCE were maintained as controls. The serum bottles were capped with gray butyl rubber plugs coated with Teflon and crimped with aluminum seals (Wheaton). The serum bottle caps were coated with paraffin wax and stored in an inverted position in a water bath. A 250microliter headspace sample from each serum bottle (after 2 min agitation of bottle) was analyzed using a Photovac 10S Plus gas chromatograph (GC) equipped with a photoionization detector (PID), an isothermal oven and a CP-Sil 5 CB capillary column. Duplicate analyses were conducted on each sample. Gas-tight Hamilton syringes with Fisher Scientific inert sampling valves comprising an on/off lever were used for on-column injections. The following GC parameters were employed for the microcosm evaluation: 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Oven temperature 
                 40 degrees C.; 
               
               
                   
                 Detector flow 
                 10 ml/min; 
               
               
                   
                 Backflush flow 
                 10 ml/min; 
               
               
                   
                 Carrier gas 
                 Ultra zero air (certified 
               
               
                   
                   
                 &lt;0.1 ppm total hydrocarbons); 
               
               
                   
                 TCE detection limit 
                 0.0588 parts per billion (ppb); 
               
               
                   
                 Methane detection limit 
                 100 ppb; 
               
               
                   
                 Propane detection limit 
                 75 ppb; and 
               
               
                   
                 N-butane detection limit 
                 50 ppb. 
               
               
                   
                   
               
            
           
         
       
     
     The results of the microcosm studies evaluating and comparing the TCE degradation rates of methane-, propane- and butane-utilizing bacteria are described below. 
     Table 11 summarizes the microcosm evaluation of methane consumption and TCE disappearance for selected isolates, given in ppm. Regression models were used to plot data to determine kinetic rates. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 11 
               
               
                   
                   
               
               
                   
                   
                 Zero Hour 
                 48 Hours 
                 96 Hours 
               
               
                   
                 Sample ID 
                 CH 4 /TCE 
                 CH 4 /TCE 
                 CH 4 /TCE 
               
               
                   
                   
               
             
            
               
                   
                 1EM 
                 1,144/5.909 
                 1,093/3.470 
                 909.5/2.854 
               
               
                   
                 1HM 
                 1,164/4.239 
                 708.5/2.538 
                  BDL/1.563 
               
               
                   
                 1NM 
                 1,043/4.215 
                 460.3/1.749 
                  BDL/1.107 
               
               
                   
                 2EM 
                 1,235/5.476 
                 806.2/3.498 
                 5.310/1.826 
               
               
                   
                 2HM 
                 1,260/5.741 
                 1,310/3.955 
                 972.1/2.530 
               
               
                   
                 2NM 
                 1,191/4.234 
                 1,130/3.360 
                  BDL/2.070 
               
               
                   
                 3EM 
                 1,191/5.525 
                 1,179/4.008 
                 559.7/2.212 
               
               
                   
                 3HM 
                 1,210/4.920 
                 1,204/3.586 
                 739.8/1.969 
               
               
                   
                 3NM 
                 1,147/5.678 
                 1,099/3.751 
                 581.9/1.815 
               
               
                   
                 4EM 
                   840/4.552 
                 352.7/0.767 
                  BDL/0.157 
               
               
                   
                 4HM 
                   431/3.799 
                 166.6/2.547 
                  BDL/1.520 
               
               
                   
                 4NM 
                 1,112/4.517 
                 460.5/3.447 
                 8.437/1.789 
               
               
                   
                 5EM 
                 1,068/9.672 
                 243.5/2.681 
                 8.786/1.386 
               
               
                   
                 5HM 
                 1,152/6.691 
                 967.6/1.570 
                  BDL/0.005 
               
               
                   
                 5NM 
                 1,108/7.775 
                 620.7/1.191 
                 4.856/0.074 
               
               
                   
                 6EM 
                   270/4.223 
                 151.6/2.956 
                  BDL/2.068 
               
               
                   
                 6HM 
                 1,145/4.638 
                 980.9/3.570 
                 123.9/1.599 
               
               
                   
                 6NM 
                 1,161/4.462 
                 1,034/4.100 
                 573.4/0.805 
               
               
                   
                 7EM 
                 1,218/4.687 
                 1,242/3.599 
                 925.3/1.640 
               
               
                   
                 7HM 
                 1,277/4.877 
                 1,309/3.860 
                 1,081/2.151 
               
               
                   
                 7NM 
                   464/4.337 
                   163/1.903 
                  BDL/1.004 
               
               
                   
                 8NM 
                 1,261/4.924 
                 1,260/3.608 
                 1,113/1.807 
               
               
                   
                 CH 4  control-1 
                 1,064/—   
                 998/—  
                 1,002/—   
               
               
                   
                 CH 4  control-2 
                 968/—  
                 8741/—  
                 8791/—  
               
               
                   
                 CH 4  control-3 
                 723/—  
                 7461/—  
                 721/—  
               
               
                   
                 TCE control-1 
                   —/7.830 
                   —/6.400 
                   —/6.200 
               
               
                   
                 TCE control-2 
                   —/4.874 
                   —/3.770 
                   —/4.100 
               
               
                   
                 TCE control-3 
                   —/11.69 
                   —/9.064 
                   —/10.20 
               
               
                   
                   
               
               
                   
                 BDL = below instrument detection limit  
               
            
           
         
       
     
     The average loss of methane and TCE in the control bottles was 51.0 and 1.30 ppm, respectively. Methane was completely consumed within 96 hours by the following isolates: 1HM, 1NM, 2NM, 4EM, 4HM, 5HM, 6EM and 7NM. However, the initial methane concentrations varied. A lag phase of methane consumption was observed in several isolates between zero and 48 hours, for example, 1EM, 3EM, 3HM, 3NM, 7EM, 7HM and 8NM. This lag phase apparently represents an acclimation period for the isolates and not a manifestation of the toxic effects of TCE since methane consumption resumed between hours 48 and 96. 
     Isolates in 4EM, cell density 1.6E8 (Table 10), had initial CH 4 /TCE concentrations of 840/4.552 ppm, respectively, and final CH 4 /TCE concentrations of 0/0.157 ppm. The methane consumption rate (apparent zero order kinetics) for isolate 4EM was calculated to be 8.75 mg h −1  L −1 , and the TCE degradation rate (first order kinetics) was 0.04 mg h −1  L −1 . Therefore, 219 ppm of methane was required to degrade 1.0 ppm TCE. 
     Isolates in 5HM, cell density 1.4E8, had initial CH 4 /TCE concentrations of 1,152/6.691 ppm, respectively, and final CH 4 /TCE concentrations of 0/0.005 ppm. The methane consumption rate (zero order kinetics) for isolate 5HM was calculated to be 12.0 mg h −1  L −1 , and the TCE degradation rate (zero order kinetics) was 0.07 mg h −1  L −1 . Therefore, 171 ppm of methane was required to degrade 1.0 ppm TCE. 
     Isolates in 5NM, cell density 2.4E8, had initial CH 4 /TCE concentrations of 1,108/7.775 ppm, respectively, and final CH 4 /TCE concentrations of 4.856/0.074 ppm. The methane consumption rate (apparent zero order kinetics) for isolate 5NM was calculated to be 11.5 mg h −1  L −1 , and the TCE degradation rate (first order kinetics) was 0.05 mg h −1  L −1 . Therefore, 230 ppm of methane was required to degrade 1.0 ppm TCE. 
     Isolates in 6NM, cell density 2.6E8, had initial CH 4 /TCE concentrations of 1,161/4.462 ppm, respectively, and final CH 4 /TCE concentrations of 573.4/0.805 ppm. The methane consumption rate (apparent zero order kinetics) for isolate 6NM was calculated to be 6.12 mg h −1  L −1 , and the TCE degradation rate (zero order kinetics) was 0.04 mg h −1  L −1 . Therefore, 153 ppm of methane was required to degrade 1.0 ppm TCE. 
     Review of the results indicate that some isolates produce enzymes that are effective at mineralizing TCE while others harvested on the same growth substrate produce enzymes that are ineffectual. 
     Table 12 summarizes the microcosm evaluation of propane consumption and TCE disappearance for selected isolates, given in ppm. Regression models were used to plot data to determine kinetic rates. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 12 
               
               
                   
                   
               
               
                   
                   
                 Zero Hour 
                 48 Hours 
                 96 Hours 
               
               
                   
                 Sample ID 
                 C 3 H 8 /TCE 
                 C 3 H 8 /TCE 
                 C 3 H 8 /TCE 
               
               
                   
                   
               
             
            
               
                   
                 1HP 
                 1,653/4.437 
                 1,055/&gt;4.0  
                 1,414/2.760 
               
               
                   
                 2EP 
                 1,099/4.226 
                   ***/1.496 
                 1,024/0.152 
               
               
                   
                 2HP 
                 1,787/2.609 
                 1,421/2.166 
                 494.4/1.796 
               
               
                   
                 2NP 
                 1,730/&gt;4.0  
                 1,424/5.075 
                 267.6/2.536 
               
               
                   
                 3HP 
                 1,555/1.922 
                 800.9/1.812 
                 17.92/1.130 
               
               
                   
                 3NP 
                 1,693/2.292 
                 1,211/&gt;2.0  
                 8.612/1.328 
               
               
                   
                 4EP 
                 1,869/1.922 
                 985/*** 
                 525.3/1.991 
               
               
                   
                 4HP 
                 1,071/1.972 
                 975.6/0.724 
                 646.1/0.062 
               
               
                   
                 4NP 
                 1,128/2.401 
                 1,070/&gt;2.0  
                 925.4/2.536 
               
               
                   
                 5HP 
                 1,731/1.896 
                 955.3/***   
                 678.2/1.383 
               
               
                   
                 5NP 
                 1,837/2.468 
                 865.4/&gt;2.01 
                 247.5/1.919 
               
               
                   
                 6EP 
                 1,760/***   
                 1,639/12.50 
                 1,402/10.0  
               
               
                   
                 6HP 
                 1,738/7.392 
                   ***/2.317 
                 1,133/0.625 
               
               
                   
                 6NP 
                 3,458/8.061 
                 1,062/5.196 
                 910.1/2.562 
               
               
                   
                 7HP 
                 1,737/5.327 
                  BDL/3.299 
                 20.70/4.583 
               
               
                   
                 7NP 
                 1,733/5.024 
                 BDL/***  
                 15.30/4.017 
               
               
                   
                 8EP 
                 1,985/5.260 
                 779.6/5.771 
                 15.91/3.193 
               
               
                   
                 8HP 
                 1,806/4.772 
                 1,315/&gt;4.5  
                 553.1/2.723 
               
               
                   
                 8NP 
                 1,868/4.768 
                  BDL/2.616 
                 15.02/1.984 
               
               
                   
                 C 3 H 8  control-1 
                 1,586/—   
                 1,618/—   
                 1,332/—   
               
               
                   
                 C 3 H 8  control-2 
                 1,732/—   
                 1,781/—   
                 1,448/—   
               
               
                   
                 C 3 H 8  control-3 
                 1,777/—   
                 1,879/—   
                 1,518/—   
               
               
                   
                 TCE control-1 
                   —/2.638 
                   —/5.416 
                   —/5.022 
               
               
                   
                 TCE control-2 
                   —/5.280 
                   —/5.089 
                   —/7.760 
               
               
                   
                 TCE control-3 
                   —/6.844 
                   —/10.08 
                   —/9.269 
               
               
                   
                   
               
               
                   
                 BDL = below instrument detection limit  
               
               
                   
                 ****= high standard deviation  
               
            
           
         
       
     
     The average loss of propane in the control bottles did not exceed 15 percent of total added over a period of 96 hours. Propane was completely consumed within 48 hours by the isolates 7HP, 7NP and 8NP. The average initial propane concentration for these three isolates was 1,770 ppm. In contrast, no selected methane-utilizing isolates achieved complete methane oxidation in less than 96 hours. The lag phase observed in the methane microcosm study between zero and 48 hours for several isolates was not apparent in the propane microcosm study. Of particular note are the isolates 4HP and 6NP. Isolates 7HP and 7NP demonstrated minimal TCE degradation averaging 20% of the total added. 
     Isolates in 4HP, cell density 2.2E8 (Table 10), had initial C 3 H 8 /TCE concentrations of 1,071/1.972 ppm, respectively, and final C 3 H 8 /TCE concentrations of 646.1/0.062 ppm. The propane consumption rate (apparent zero order kinetics) was calculated to be 4.43 mg h −1  L −1  and the TCE degradation rate (zero order kinetics) was 0.02 mg h −1  L −1 . Therefore, 222 ppm of propane was required to degrade 1.0 ppm TCE. 
     Isolates in 6NP, cell density 2.6E8, had initial C 4 H 8 /TCE concentrations of 1,161/4.462 ppm, respectively, and final C 4 H 8 /TCE concentrations of 573.4/0.805 ppm. The propane consumption rate (apparent zero order kinetics) was calculated to be 26.5 mg h −1  L −1 , and the TCE degradation rate (zero order kinetics) was 0.06 mg h −1  L −1 . Therefore, 442 ppm of propane are required to degrade 1.0 ppm TCE. There was a nearly equivalent correlation for the linear (r=0.8914) and exponential (r=0.9140) data plots for the propane consumption rate for isolate 6NP. In addition, the data plots for the TCE degradation rate showed the same linear (r=0.9997) and exponential (r=0.9910) trends. 
     Review of the results indicate that some isolates produce enzymes, as observed for the methane microcosm evaluation, that are effective at mineralizing TCE while others produce enzymes that are apparently ineffectual. 
     Table 13 summarizes the microcosm evaluation of n-butane consumption and TCE disappearance for selected isolates in accordance with the present invention, given in ppm. Corresponding graphs illustrating the butane microcosm results are included in FIGS. 1-16. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 13 
               
               
                   
               
               
                 Sample 
                 Zero Hour 
                 48 Hours 
                 96 Hours 
                 144 Hours 
               
               
                 ID 
                 C 4 H 10 /TCE 
                 C 4 H 10 /TCE 
                 C 4 H 10 /TCE 
                 C 4 H 10 /TCE 
               
               
                   
               
             
            
               
                 2EB 
                 694.6/8.321 
                 584.5/3.740 
                   500/1.768 
                 BDL/0.945 
               
               
                 2HB 
                 659.4/4.110 
                 488.7/2.615 
                  BDL/1.274 
                 BDL/0.477 
               
               
                 2NB 
                 701 9/5.415 
                 616.6/≧5.0  
                 452.7/2.405 
                 BDL/1.760 
               
               
                 3EB 
                  1077/9.399 
                 551 0/7.330 
                 476.8/2.641 
                 BDL/1.260 
               
               
                 3NB 
                 657.3/11.42 
                 564.8/10.12 
                  ≧564/3.827 
                 559.8/2.368  
               
               
                 4EB 
                 670.3/7.065 
                 544.1/1.758 
                 473.2/0.774 
                 BDL/0.158 
               
               
                 4HB 
                 709.2/6.752 
                 333.7/3.006 
                  BDL/2.900 
                 BDL/1.825 
               
               
                 4NB 
                 710.6/6.817 
                  BDL/2.750 
                  BDL/1.383 
                 BDL/1.199 
               
               
                 5EB 
                 659.8/5.873 
                 517.5/2.737 
                  BDL/2.253 
                 BDL/1.113 
               
               
                 6HB 
                 625.9/5.508 
                 576.3/1.046 
                 542.4/0.142 
                 397.4/BDL   
               
               
                 6NB 
                 675.8/5.996 
                 622.0/0.836 
                 620.9/0.268 
                 BDL/BDL  
               
               
                 7EB 
                 726.0/2.357 
                 619.8/2.331 
                   BDL/≧2.300 
                 BDL/2.100 
               
               
                 7HB 
                 656.3/5.055 
                 355.0/2.703 
                   BDL/≧2.700 
                 BDL/2.648 
               
               
                 7NB 
                 687.5/6.232 
                 297.2/3.064 
                  BDL/2.440 
                 BDL/1.721 
               
               
                 8EB 
                 651.5/2.934 
                 448.5/***   
                  BDL/2.684 
                 BDL/2.446 
               
               
                 8HB 
                 718.4/2.599 
                 576.2/1.967 
                 BDL/***  
                 BDL/1.953 
               
               
                 Butane 
                 632.0/—   
                 681.9/—   
                 762.5/—   
                 986.6/—   
               
               
                 Control- 
               
               
                 1 
               
               
                 Butane 
                 884.5/—   
                 889.0/—   
                 942.7/—   
                 1075/—   
               
               
                 Control- 
               
               
                 2 
               
               
                 Butane 
                 966.9/—   
                 1012/—  
                 1053/—  
                 1168/—   
               
               
                 Control- 
               
               
                 3 
               
               
                 TCE 
                   —/8.149 
                   —/7.840 
                   —/7.717 
                  —/7.392 
               
               
                 Control- 
               
               
                 1 
               
               
                 TCE 
                    —/11.720 
                   —/9.831 
                   —/8.949 
                  —/8.922 
               
               
                 Control- 
               
               
                 2 
               
               
                 TCE 
                    —/11.835 
                    —/11.605 
                    —/10.438 
                  —/9.954 
               
               
                 Control- 
               
               
                 3 
               
               
                   
               
               
                 BDL = below instrument detection limit  
               
               
                 ***= high standard deviation  
               
            
           
         
       
     
     As shown in Table 13, n-butane was completely consumed within 48 hours by 4NB and 96 hours by 2HB, 4HB, 4NB, 5EB, 7EB, 7HB and 7NB. A lag phase was not observed for growth between zero and 48 hours. According to the graphs of FIGS. 3,  5 ,  7 ,  9 ,  11 ,  13  and  15 , depicting n-butane consumption versus time for selected isolates, n-butane consumption appears to follow apparent zero order kinetics, indicating that the rate of reaction may be independent of the concentration of the isolates. However, according to the graphs of FIGS. 4,  6 ,  8 ,  10 ,  12 ,  14  and  16 , depicting TCE loss over time, zero and first order kinetics were observed. The occurrence of first order kinetics indicates that the TCE degradation rate may be dependent on the concentration of the isolates such that there is a linear relationship between the natural log of concentration at a given time over the initial concentration. Of particular note are the isolates 2EB, 2HB, 2NB, 3EB, 3NB, 4EB, 4HB, 4NB, 5EB, 6HB, 6NB and 7NB. The average loss of TCE in the control bottles (1-3) ranged from 9 to 23 percent. 
     Isolates in 2EB, cell density 2.1E8 (Table 10), had initial C 4 H 10 /TCE concentrations of 694.6/8.321 ppm, respectively, and final C 4 H 10 /TCE concentrations of 0/0.945 ppm (see FIGS.  3  and  4 ). The n-butane consumption rate (apparent zero order kinetics) for isolate 2EB was calculated to be 4.52 mg h −1  L −1 , and the TCE degradation rate (first order kinetics) was 0.02 mg h −1  L −1 . Therefore, 226 ppm of n-butane was required to degrade 1.0 ppm TCE. Since the 2EB-containing microcosm showed no correlation between TCE and n-butane degradation, TCE apparently serves as a carbon source. 
     Isolates in 2HB, cell density 2.5E8, had initial C 4 H 10 /TCE concentrations of 659.4/4. 110 ppm, respectively, and final C 4 H 10 /TCE concentrations of 0/0.477 ppm (see FIGS.  3  and  4 ). In comparison with the propane-utilizing microcosm, 2HP, that degraded 31 percent of the total TCE added, 2HB degraded 88 percent of the total added. The n-butane consumption rate (apparent zero order kinetics) for 2HB was calculated to be 5.06 mg h −1  L −1 , and the TCE degradation rate (zero order kinetics) was 0.03 mg h −1  L −1 . Therefore, 169 ppm of n-butane was required to degrade 1.0 ppm TCE. The 2HB-ontaining microcosm showed a good correlation between TCE and n-butane degradation characteristic of cometabolic degradation. 
     Isolates in 2NB, cell density 1.9E8, had initial C 4 H 10 /TCE concentrations of 701.9/5.415 ppm, respectively, and final C 4 H 10 /TCE concentrations of 0.0/1.760 ppm (see FIGS.  3  and  4 ). Therefore, 473 ppm of n-butane was required to degrade 1.0 ppm TCE. The propane-utilizing microcosm, 2NP, degraded 25 percent less of the total added TCE than the 2NB-containing microcosm. The n-butane consumption rate (apparent zero order kinetics) for 2HB was calculated to be 4.73 mg h −1  L −1 , and the TCE degradation rate (first order kinetics) was 0.01 mg h −1  L −1 . Since the 2NB-containing microcosm showed no correlation between TCE and n-butane degradation, it is apparent that this isolate uses TCE as a carbon source. 
     Isolates in 3EB, cell density 1.5E8, had initial C 4 H 10 /TCE concentrations of 1077/9.399 ppm, respectively, and final C 4 H 10 /TCE concentrations of 0/1.260 ppm (see FIGS.  5  and  6 ). Inoculum 3EB degraded 87 percent of the total TCE added. The n-butane consumption rate (apparent zero order kinetics) for 3EB was 6.89 mg h −1  L −1 , and the TCE degradation rate (data correlated with zero and first order kinetics) was 0.06 mg h −1  L −1 . Therefore, 115 ppm of n-butane was required to degrade 1.0 ppm TCE. Since the 3EB-containing microcosm showed little correlation between TCE and n-butane degradation, this isolate apparently uses TCE as a carbon source. 
     Isolates in 3NB, cell density 3. 1E8, had initial C 4 H 10 /TCE concentrations of 657.3/11.42 ppm, respectively, and final C 4 H 10 /TCE concentrations of 559.8/2.368 ppm (see FIGS.  5  and  6 ). In contrast, the propane-utilizing microcosm, 3NP, degraded 42 percent of the total TCE added whereas 3NB degraded 79 percent of the total added. The TCE degradation rate was 0.01 mg h −1  L −1 . Therefore, 10.77 ppm of n-butane was required to degrade 1.0 ppm TCE. 
     Isolates in-4EB, cell density 3.6E8, had initial C 4 H 10 /TCE concentrations of 670.3/7.065 ppm, respectively, and final C 4 H 10 /TCE concentrations of 0/0.158 ppm (see FIGS.  7  and  8 ). The propane-utilizing microcosm, 4EP, showed no TCE degradation. The inoculum 4EB degraded 98 percent of the total TCE added. The n-butane consumption rate (apparent zero order kinetics) for 4EB was calculated to be 4.34 mg h −1  L −1 , and the TCE degradation rate (first order kinetics) was 0.03 mg h −1  L −1 . Since the 4EB-containing microcosm showed no correlation between TCE and n-butane degradation, it is apparent that this isolate uses TCE as a carbon source. 
     Isolates in 4HB, cell density 4.8E8, had initial C 4 H 10 /TCE concentrations of 709.2/6.752 ppm, respectively, and final C 4 H 10 /TCE concentrations of 0/1.825 ppm (see FIGS.  7  and  8 ). The inoculum 4HB degraded 73 percent of the total TCE added. The n-butane consumption rate (apparent zero order kinetics) for 4HB was calculated to be 5.13 mg h −1  L −1 , and the TCE degradation rate (first order kinetics) was 0.01 mg h −1  L −1 . Since the 4HB-containing microcosm showed no correlation between TCE and n-butane degradation, it is apparent that this isolate uses TCE as a carbon source. 
     Isolates in 4NB, cell density 2.6E8, had initial C 4 H 10 /TCE concentrations of 709.2/6.752 ppm, respectively, and final C 4 H 10 /TCE concentrations of 0/1.825 ppm (see FIGS.  7  and  8 ). The propane-utilizing microcosm, 4NP, showed no TCE degradation whereas 4NB degraded 82 percent of the total TCE added. The n-butane consumption rate (apparent zero order kinetics) for 4NB was calculated to be 4.44 mg h −1  L −1 , and the TCE degradation rate (first order kinetics) was 0.01 mg h −1  L −1 . Since the 4NB-containing microcosm showed no correlation between TCE and n-butane degradation, it is apparent that this isolate uses TCE as a carbon source. 
     Isolates in 5EB, cell density 3.1E8, had initial C 4 H 10 /TCE concentrations of 659.8/5.873 ppm, respectively, and final C 4 H 10 /TCE concentrations of 0/1.113 ppm (see FIGS.  9  and  10 ). The inoculum 5EB degraded 81 percent of the total TCE added. The n-butane consumption rate (apparent zero order kinetics) for 5EB was 5.20 mg h −1  L −1 , and the TCE degradation rate (first order kinetics) was 0.01 mg h −1  L −1 . Therefore, 520 ppm of n-butane was required to degrade 1.0 ppm TCE. Since the SEB-containing microcosm showed no correlation between TCE and n-butane degradation, it is apparent that this isolate uses TCE as a carbon source. 
     Isolates in 6HB, cell density 1.3E8, had initial C 4 H 10 /TCE concentrations of 625.9/5.508 ppm, respectively, and final C 4 H 10 /TCE concentrations of 397.4/0.0 ppm (see FIGS.  11  and  12 ). The inoculum 6HB degraded 100 percent of the total TCE added. The n-butane consumption rate was 1.50 mg h −1  L −1 , and the TCE degradation rate (zero order kinetics) was 0.04 mg h −1  L −1 . Therefore, 37.50 ppm of n-butane was required to degrade 1.0 ppm TCE, a very low butane requirement. These isolates may degrade TCE in the absence of butane. 
     Isolates in 6NB, cell density 4.8E7, had initial C 4 H 10 /TCE concentrations of 675.8/5.996 ppm, respectively, and final C 4 H 10 /TCE concentrations of 0.0/0.0 ppm (see FIGS.  11  and  12 ). The inoculum 6NB degraded 100 percent of the total TCE added. The n-butane consumption rate (apparent zero order kinetics) was 4.23 mg h −1  L −1 , and the TCE degradation rate (zero order kinetics) was 0.04 mg h −1  L −1 . Therefore, 106 ppm of n-butane was required to degrade 1.0 ppm TCE. The 6NB-containing microcosm showed a good correlation between TCE and n-butane consumption. Thus, degradation is apparently a cometabolic process. 
     Isolates in 7EB, cell density 1.6E8, had initial C 4 H 10 /TCE concentrations of 726.0/2.357 ppm, respectively, and final C 4 H 10 /TCE concentrations of 0.0/2.100 ppm (see FIGS.  13  and  14 ). The inoculum 7EB degraded only 11% of the total TCE added. The n-butane consumption rate (apparent zero order kinetics) was 5.0 mg h −1  L −1 , and the TCE degradation rate (first order kinetics) was 0.002 mg h −1  L −1 . 
     Isolates in 7HB, cell density 3.9E8, had initial C 4 H 10 /TCE concentrations of 656.3/5.05 ppm, respectively, and final C 4 H 10 /TCE concentrations of 0.0/2.700 ppm (see FIGS.  13  and  14 ). The inoculum 7HP degraded 14 percent of the total TCE added, while the inoculum 7HB degraded 47% of the total TCE. added. The n-butane consumption rate for 7HB (apparent zero order kinetics) was 4.6 mg h −1  L −1 , and the TCE degradation rate (first order kinetics) was 0.02 mg h −1  L −1 . The 7HB-containing microcosm showed a good correlation between TCE and n-butane consumption. Thus, degradation is apparently a cometabolic process. 
     Isolates in 7NB, cell density 4.2E8, had initial C 4 H 10 /TCE concentrations of 687.5/6.232 ppm, respectively, and final C 4 H 10 /TCE concentrations of 0.0/1.721 ppm (see FIGS.  13  and  14 ). The inoculum 7NP degraded 20 percent of the total TCE added, while the inoculum 7NB degraded 72 percent of the total TCE added. The n-butane consumption rate (apparent zero order kinetics) was 4.92 mg h −1  L −1 , and the TCE degradation rate (first order kinetics) was 0.01 mg h −1  L −1 . Since the 7NB-containing microcosm showed no correlation between TCE and n-butane degradation, it is apparent that this isolate uses TCE as a carbon source. 
     TCE degradation in a few microcosms was independent of butane consumption. For example, the isolates in 4NB and 7NB degraded TCE continuously in the absence of butane. Other microcosms such as 4EB demonstrate cometabolic degradation of TCE with butane. 
     From the above-noted results, it appears that TCE toxicity for the butane-utilizing bacteria is minimal. The butane-utilizing bacteria are preferably capable of surviving TCE levels above about 6 mg/l, more preferably above about 10 mg/l, and most preferably above about 20 mg/l aqueous phase. As set forth in U.S. Pat. No. 4,713,343 to Wilson Jr. et al., cited previously, the growth of methane-utilizing bacteria are inhibited by the presence of a chlorinated epoxide that forms during the cometabolic degradation of TCE mediated by the enzyme methane monooxygenase. According to Broholm et al., 1990, cited previously, TCE is toxic to methane-utilizing bacteria at concentrations above 6 mg/l in water. A quantitative comparison of TCE toxicity between methane-, propane- and butane-utilizing bacteria showed that the butane-utilizing bacteria of the present invention were substantially less susceptible to the effects of TCE toxicity. Butane-utilizing bacteria survived at an average of 1.4 times higher concentrations of TCE than propane oxidizers. Moreover, the butane-utilizing bacteria are capable of surviving at several orders of magnitude higher TCE concentrations than methane oxidizers. 
     In accordance with a preferred embodiment of the present invention, TCE is degraded at higher rates with butane-utilizing bacteria in comparison with methane- or propane-utilizing bacteria. Over 50% of the butane-utilizing bacteria degraded TCE to half of its initial concentration within 48 hours after incubation. This high rate of degradation was not observed in over 80 percent of the microcosms containing methane- or propane-utilizing bacteria. The butane-utilizing bacteria of the present invention are preferably capable of degrading TCE at a rate of greater than about 1 mg/hr/liter in water, more preferably at a rate of greater than about 1.5 or 2 mg/hr/liter. As discussed more fully below, processing parameters may be controlled during the bioremediation process to achieve high TCE degradation rates of 10 to 20 mg/hr/liter or higher. 
     As a food source for microbial consumption, butane has been found to be a superior substrate to methane or propane due to its solubility factor. Methane and propane are characterized as slightly soluble in water while butane is characterized as very soluble in water. At 17 degrees centigrade, 3.5 ml of methane and 6.5 ml of propane dissolves in 100 ml of water. In contrast, 15 ml of butane dissolves in 100 ml of water. Higher solubility increases microbial access to the growth substrate for metabolism, and may produce reaction rates demonstrating zero order kinetics. Another cause of the higher TCE cometabolic rates for the butane-utilizers in comparison with the methane- or propane-utilizers may be the molecular structure of the compounds and enzymes. TCE is a large planar molecule composed of two carbon atoms and three chlorine atoms. Methane is a small single tetrahedral carbon molecule, while propane is a three carbon molecule. On the other hand, butane is a large non-planar four carbon molecule. While not intending to be bound by any particular theory, molecular structure, reactive surface area and size may play a role in causing the operative enzymes of the butane oxidizers to be superior TCE degraders in comparison with the methane and propane operative enzymes. The occurrence of TCE degradation in absence of any other growth substrate clearly provides highly improved TCE degradation capability. Furthermore, while methane-utilizing bacteria are typically sensitive to normal oxygen tension of an air atmosphere and require decreased oxygen levels for growth, the butane-utilizing bacteria of the present invention are not sensitive to ambient oxygen tension and can be used with normal atmospheres. In addition, the butane-utilizers do not exhibit copper toxicity, and do not require carbon dioxide as a supplementary carbon source. 
     Various propane-utilizing and butane-utilizing bacteria were characterized as follows. Microorganism identification is based on the Similaritv Index. The Similarity Index in the Microbial Identification System (MIS) is a numerical value which expresses how closely the fatty acid composition of an unknown sample compares with the mean fatty acid methyl ester composition of the strains used to create the library entry listed as its match. The database search presents the best matches and associated similarity indices. An exact match of the fatty acid make-up of the unknown sample to the mean of a library entry results in a similarity index of 1.000. The similarity index will decrease as each fatty acid varies from the mean percentage. Strains with a similarity of 0.500 or higher and with a separation of 0.100 between first and second choice are good matches (good or excellent). A similarity index between 0.300 and 0.500 may be a good match but would indicate an atypical strain (OK). Values lower than 0.300 suggest that the species is not in the database but those listed provide the most closely related species (weak or poor). 
     In the cases where a strain remained unidentified after fatty acid analysis, the Biolog system was employed where microorganisms are identified by comparing substrate utilization characteristics of the unknown isolate to the Biolog database. 
     The following isolates were chosen for identification at two independent laboratories: propane-utilizers 2EP, 3EP, 4HP, 6HP, 6NP and 8NP; and butane-utilizers 2EB, 2HB, 3EB, 3NB, 4EB, 4HB, 4NB, 5EB, 6HB, 6NB and 7NB. 
     The majority of the propane-utilizers and butane-utilizers were characterized as different genera/species by both laboratories for the comparison-pair isolates 2EP-2EB, 3EP-3EB, 4HP4HB, 6HP-6HB, and 6NP-6NB, thus indicating that the butane-utilizers are a distinct class of microorganism from the propane degraders. Since methane-utilizing bacteria are obligate methane oxidizers, no isolates from the methane microcosms were submitted for laboratory analysis. Most isolates from the microcosms were mixed. Between both laboratories, 59 genus/specie were identified with “good or excellent” precision, 14 with “OK” precision (atypical strains) and 22 with “weak” precision (species not in database and remain as unknowns). A summary of the butane-utilizers that have demonstrated the ability to degrade TCE are identified in Table 14. 
     
       
         
           
               
               
               
             
               
                 TABLE 14 
               
               
                   
               
               
                 Sample ID 
                 Genus 
                 Species 
               
               
                   
               
             
            
               
                  2HB* 
                 Pseudomonas 
                 
                   putida 
                 
               
               
                 2EB 
                 Pseudomonas 
                 
                   rubrisubalbicans 
                 
               
               
                 3EB 
                 Pseudomonas 
                 
                   rubrisubalbicans 
                 
               
               
                 5EB 
                 Pseudomonas 
                 
                   aeruginosa 
                 
               
               
                 6NB 
                 Pseudomonas 
                 
                   aeruginosa 
                 
               
               
                 2EB 
                 Variovorax 
                 
                   paradoxus 
                 
               
               
                 2HB 
                 Variovorax 
                 
                   paradoxus 
                 
               
               
                 3EB 
                 Variovorax 
                 
                   paradoxus 
                 
               
               
                 3NB 
                 Variovorax 
                 
                   paradoxus 
                 
               
               
                 4HB 
                 Variovorax 
                 
                   paradoxus 
                 
               
               
                 4NB 
                 Variovorax 
                 
                   paradoxus 
                 
               
               
                  5EB* 
                 Variovorax 
                 
                   paradoxus 
                 
               
               
                 6HB 
                 Variovorax 
                 
                   paradoxus 
                 
               
               
                 2EB 
                 Variovorax 
                   paradoxus ** 
               
               
                 6NB 
                 Variovorax 
                   paradoxus *** 
               
               
                 7NB 
                 Nocardia 
                 
                   asteroides 
                 
               
               
                 2HB 
                 Nocardia 
                   asteroides *** 
               
               
                 3EB 
                 Nocardia 
                   asteroides *** 
               
               
                  4HB* 
                 Nocardia 
                   asteroides *** 
               
               
                 4NB 
                 Nocardia 
                   asteroides *** 
               
               
                 7NB 
                 Nocardia 
                   asteroides *** 
               
               
                  5EB* 
                 Nocardia 
                 
                   brasiliensis 
                 
               
               
                 2EB 
                 Nocardia 
                 
                   restricta 
                 
               
               
                 2HB 
                 Nocardia 
                 
                   globerula 
                 
               
               
                 2HB 
                 Chryseobacterium 
                 
                   indologenes 
                 
               
               
                 4HB 
                 Chryseobacterium 
                 
                   indologenes 
                 
               
               
                 7NB 
                 Chryseobacterium 
                 
                   indologenes 
                 
               
               
                 5EB 
                 Chryseobacterium 
                 
                   meningosepticum 
                 
               
               
                 2EB 
                 Comamonas 
                 
                   acidovorans 
                 
               
               
                 3NB 
                 Comamonas 
                 
                   acidovorans 
                 
               
               
                 6HB 
                 Comamonas 
                 
                   acidovorans 
                 
               
               
                 6NB 
                 Comamonas 
                 
                   acidovorans 
                 
               
               
                 4EB 
                 Acidovorax 
                 
                   delafieldii 
                 
               
               
                 4NB 
                 Acidovorax 
                 
                   delafieldii 
                 
               
               
                 6NB 
                 Acidovorax 
                 
                   delafieldii 
                 
               
               
                 4NB 
                 Rhodococcus 
                 
                   rhodochrous 
                 
               
               
                 7NB 
                 Rhodococcus 
                 
                   rhodochrous 
                 
               
               
                 2EB 
                 Rhodococcus 
                 
                   erythropolis 
                 
               
               
                 3EB 
                 Rhodococcus 
                 
                   erythropolis 
                 
               
               
                 6HB 
                 Rhodococcus 
                 
                   erythropolis 
                 
               
               
                  4EB* 
                 Rhodococcus 
                 
                   fascians 
                 
               
               
                  5EB* 
                 Rhodococcus 
                 
                   fascians 
                 
               
               
                 4NB 
                 Aureobacterium 
                 
                   barkeri 
                 
               
               
                 4HB 
                 Aureobacterium 
                 
                   esteroaromaticum 
                 
               
               
                 4NB 
                 Aureobacterium 
                 
                   esteroaromaticum 
                 
               
               
                 6HB 
                 Aureobacterium 
                 
                   saperdae 
                 
               
               
                 5EB 
                 Micrococcus 
                 
                   varians 
                 
               
               
                 7NB 
                 Micrococcus 
                 
                   varians 
                 
               
               
                 7NB 
                 Micrococcus 
                 
                   kristinae 
                 
               
               
                 6HB 
                 Aeromonas 
                 
                   caviae 
                 
               
               
                 6NB 
                 Aeromonas 
                 
                   caviae 
                 
               
               
                 2EB 
                 Stenotrophomonas 
                 
                   maltophilia 
                 
               
               
                 3EB 
                 Stenotrophomonas 
                 
                   maltophilia 
                 
               
               
                 4EB 
                 Stenotrophomonas 
                 
                   maltophilia 
                 
               
               
                 5EB 
                 Stenotrophomonas 
                 
                   maltophilia 
                 
               
               
                 6HB 
                 Stenotrophomonas 
                 
                   maltophilia 
                 
               
               
                 6NB 
                 Stenotrophomonas 
                 
                   maltophilia 
                 
               
               
                 4EB 
                 Sphingobacterium 
                 
                   thalpophilum 
                 
               
               
                  4NB* 
                 Sphingobacterium 
                 
                   spiritivorum 
                 
               
               
                 4NB 
                 Shewanella 
                   putrefaciens  B 
               
               
                  3NB* 
                 Phyllobacterium 
                 
                   myrsinacearum 
                 
               
               
                 6HB 
                 Clavibacter 
                 
                   michiganense 
                 
               
               
                 6HB 
                 Clavibacter 
                   michiganense **** 
               
               
                 6NB 
                 Alcaligenes 
                 
                   xylosoxydans 
                 
               
               
                  7NB* 
                 Gordona 
                 
                   terrae 
                 
               
               
                 7NB 
                 Corynebacterium 
                   aquaticum  B 
               
               
                 7NB 
                 Cytophaga 
                 
                   johnsonae 
                 
               
               
                   
               
               
                 *low similarity index indicating a poor match with the fatty-acid database. In these cases, the species in the consortia listed was matched to a database testing substrate utilization and remained unidentified. The (*) best describes an unknown genera/species.  
               
               
                 **GC Subgroup A subspecies  
               
               
                 ***GC Subgroup B subspecies  
               
               
                 ****tessellarius subspecies  
               
            
           
         
       
     
     In accordance with the present invention, butane-utilizing bacteria have been found to withstand TCE concentrations that have proven fatal to methanotrophic bacteria. The butane-utilizing bacteria also demonstrate higher TCE degradation than propane-utilizing bacteria. In addition, analysis of TCE degradation by selected isolates indicates that certain butane-utilizing bacteria use TCE as a carbon source. 
     In-situ bioremedial processes that may be used in accordance with the present invention include the injection of non-indigenous butane-utilizing microorganisms into the surface or subsurface and/or the use of indigenous butane-utilizing microorganisms. Indigenous microorganisms can be stimulated to flourish by the addition of nutrients and a growth substrate that may be limited in the ecosystem under scrutiny. For aerobic metabolism, oxygen is usually in limited concentrations. The growth of butane-utilizing bacteria may be enhanced through the addition of oxygen, nutrients and butane in any subsurface environment in which chlorohydrocarbons have been introduced, thereby creating an effective treatment zone. Oxygen, nutrients such as inorganic and organic nitrogen-containing compounds and butane gas can be delivered into the subsurface through injection or diffusion wells or some other type of delivery system. Alternatively, non-indigenous strains of butane-utilizing organisms may be injected into a subsurface environment. For TCE-utilizing bacteria, the introduction of the aliphatic hydrocarbon carbon source may not be necessary. The butane-utilizing organisms of the present invention may be applied in-situ in saline or low pH environments as well. 
     Furthermore, butane-utilizing organisms of the present invention may be provided in an ex-situ bioreactor capable of treating air, soil or groundwater (freshwater, saline or low pH) waste streams. The ex-situ bioreactor may be used in a batch-type process and/or in a continuous flow process. 
     For air or gas treatment, butane-utilizing bacteria may be grown in a bioreactor on any suitable type of packing material or substrate capable of withstanding turbulent gas streams. The gas stream laden with chlorinated volatile organic compounds may be extracted from the subsurface or other environment with a vacuum blower and treated in a bioreactor. In this embodiment, treatment consists of passing the chlorinated air waste stream through the bioreactor in much the same fashion as conventional activated carbon systems, with the exception Lthat the contaminants are not merely transferred but destroyed. 
     Chlorohydrocarbon-impacted soils may be bioremediated in accordance with the present invention with butane-utilizing organisms in an ex-situ bioreactor. This apparatus may agitate soil through mixing or fluidizing, thereby accelerating the volatilization of chlorohydrocarbons which could be treated as an air waste stream described above. Another type of soil reactor may degrade chlorohydrocarbons in a bioreactor capable of treating a soil slurry matrix through either the introduction of non-indigenous butane-utilizing bacteria, or the stimulation of indigenous butane-utilizing bacteria. Oxygen, nutrients including alternate limited carbon and nitrogen sources such as casamino acids and yeast and butane may be introduced into this type of bioreactor. The use of surfactants may accelerate the removal of the chlorinated compounds from the soil matrix thereby lower treatment time and increasing bioreactor performance. 
     In accordance with an embodiment of the present invention, an ex-situ bioreactor may be used to restore surface water or groundwater impacted with chlorohydrocarbons such as TCE, by employing butane-utilizing bacteria. The impacted water may comprise fresh water, salt water, low pH water or the like. The ex-situ bioreactor may comprise one or multiple chambers, each housing a substrate such as biofilm fabric or packing material seeded with specific strains or a consortia of butane-utilizing bacteria. Each bioreactor chamber preferably comprises an oxygen, nutrient and butane gas delivery system. Bioreactor systems employing butane-utilizing organisms that demonstrate the ability to use TCE as a direct food source may not require the introduction of butane. However, in a cometabolic system, timers are preferably included to regulate the introduction of the butane, thereby reducing the likelihood of saturating the enzyme sites which would result in a lower contaminant destruction rate. 
     FIG. 17 illustrates an ex-situ bioreactor for use in accordance with an embodiment of the present invention. The bioreactor  10  includes a generally cylindrical container  12  having a cap  14 . The cap  14  is sealed to the container  12  by clamps  15  and  16 , along with an O-ring gasket  17 . A support member  18  secures a microorganism substrate  20  at the desired position within the container  12 . 
     The substrate  20  may comprise any suitable material such as a bioscreen made of aluminum, steel, stainless steel or any substantially non-reactive, non-absorbent support material, preferably coated with trichlorofluoroethylene or other suitable coating for growth/colonization of bacterial cells through biofilm formation. In a preferred embodiment, the substrate  20  comprises a bioscreen made of aluminum screen with pore size 0.0039 square inch, rolled in concentric circles to increase available surface area for biofilm formation. 
     A fluid inlet  22  communicates with the interior of the bioreactor  10 . In accordance with the present invention, the term “fluid” includes flowable materials such as liquids, gases, slurries, fluidized solids and the like. A fluid outlet  24  communicates with the interior of the bioreactor  10  to transport fluid therefrom. During operation, the fluid fills the bioreactor  10  to the desired level  25 . 
     As shown in FIG. 17, a gas inlet  32  passes through the bioreactor cap  14 . A tube  33  connects with a sparger  34  to deliver the gas to the interior of the bioreactor  10 . A gas outlet  39  allows gas to exit the bioreactor. A pressure relief valve  38  prevents excessive buildup of pressure within the bioreactor. while a gas sampling port  36  allows for testing of headspace gas within the bioreactor. 
     Single or multiple bioreactor chambers may be used in accordance with the present invention. For example, as shown in FIG. 18, multiple bioreactors  10  may be connected in series. In FIG. 18, influent enters the leftmost bioreactor  10  via the fluid inlet  22 . After treatment in the first bioreactor, fluid travels to the middle bioreactor by a connecting tube  23 . Similarly, after treatment in middle bioreactor, fluid travels to the rightward bioreactor by a connecting tube  23 . After treatment in the multiple bioreactors, the effluent exists the system. 
     In accordance with a preferred embodiment of the present invention, the ex-situ bioreactor is provided as a portable unit that can easily be transported to various bioremediation sites. For example, single or multiple chamber bioreactors are preferably transportable by truck or other suitable means, and may be small enough to permit hand carrying. The bioreactor may be placed adjacent to a surface or subsurface water contamination site. Alternatively, for surface water bioremediation, the bioreactor may be constructed to float on or in the contaminated water. 
     The following examples illustrate the batch-type operation of a bioreactor similar to that shown in FIG.  17 . 
     EXAMPLE 1 
     A biofilm was formed on an aluminum screen substrate  20  as shown in FIG.  17 . Growth was established by operating the bioreactor shown in FIG. 17 as a growth chamber in which butane and oxygen were provided by injection into the bioreactor headspace through port  36 . Butane concentrations were kept low after biofilm formation in order to limit competitive inhibition during operation of the bioreactor. One 6-liter batch bioreactor was used for the TCE cometabolic degradation test run. The bioreactor contained 4.2 liters of autoclaved deionized water with a MSM amendment and an aluminum screen with pore size 0.0039 square inches. The isolates in Sample ID 3NB were chosen to seed the bioscreen. The bioreactor headspace consisted of 2-liters of volume within the reactor and a maximum of 3-liters total headspace throughout the gas recirculation lines. The recirculation system was driven by a hermetically-sealed bellows vacuum pump that operated at 0.22 cfm and 10 psi, producing 622 ml/min of air flow. The pump was scheduled to run 30 minutes every 4 hours to produce a pulsing effect within the bioreactor between the two phases (liquid and gaseous). At the time of circulation, both TCE and butane concentrations were increased within the aqueous phase, thereby increasing the bioavaillbility of both compounds. At the time the pump was off, TCE volatilization and butane evolution increased in the headspace concentrations. Once the initial parameters, i.e., dissolved oxygen concentration, pH, chloride ion concentration, temperature and pressure were established using Chemetrics Titration kits and hand-held meters, headspace samples were collected using gas tight syringes at a sampling port to determine the butane and TCE concentrations using a Photovac IOS Plus gas chromatograph. The parameters are set forth in Table 15. 
     
       
         
           
               
               
               
             
               
                 TABLE 15 
               
               
                   
               
               
                   
                 Initial Concentrations 
                 Final Concentrations 
               
               
                 Parameters 
                 at 14:28 hrs. 
                 at 23:30 hrs. 
               
               
                   
               
             
            
               
                 Dissolved Oxygen 
                 8 ppm 
                 7 ppm 
               
               
                 pH 
                 7.0 
                 6.5 
               
               
                 Chloride Ions 
                 300 ppm* 
                 300 ppm* 
               
               
                 Temperature 
                 25 degrees C. 
                 25 degrees C. 
               
               
                 Log (Cell Counts) 
                 12 
                 N/A 
               
               
                 Pressure 
                 0 psi 
                 0 psi 
               
               
                   
               
               
                 *differentiation from the initial concentration of the chloride ions in the solution was not detected due to low sensitivity of the titration kit.  
               
            
           
         
       
     
     FIGS. 19 and 20 demonstrate the degradation of TCE and butane in the bioreactor of FIG. 17 while it was running in batch mode. Butane was provided 24 hours prior to the introduction of TCE. TCE disappearance from its initial concentration, exceedingly higher than its toxic concentration to methanotrophs and propane oxidizers, was measured utilizing headspace analysis. The TCE was degraded at a rate of about 1.6 mg/hr/liter. The recorded concentration in headspace is half of its dissolved concentration as describen by Henry&#39;s constant. Therefore, the data indicates that the butane oxidizing bacteria within this bioreactor survives at a TCE concentration of 13 mg/l, in comparison with the TCE toxic level for methanotrophs reported at concentrations above 6 mg/l by Broholm et al., 1990. 
     EXAMPLE 2 
     Two 6liter bioreactors were operated simultaneously for six and a half hours. The first bioreactor was seeded with isolates from 3NB and the second bioreactor with isolates from 4EB. A biofilm was formed on an aluminum screen substrate as shown in FIG.  17 . Each bioreactor contained 4.2 liters of autoclaved deionized water with a MSM amendment and an aluminum screen with pore size 0.0039 square inches. The two bioreactors contained 4-liters of headspace total volume and a maximum of 6liters total headspace throughout the gas recirculation lines. The recirculation system was driven by a hermetically-sealed bellows vacuum pump that operated at 0.22 cfm and 10 psi, producing 622 ml/min of air flow. The pump simultaneously recirculated the headspace volumes in both bioreactors. The butane and TCE loss over time is shown in Table 16. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 16 
               
               
                   
                   
               
               
                   
                 Time 
                   
                 Butane (ppm) 
                 TCE (ppm) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0 
                 hours 
                 371.9 
                 11.01 
               
               
                   
                 1.0 
                 hours 
                 387.7 
                 8.471 
               
               
                   
                 1.2 
                 hours 
                 209.5 
                 4.878 
               
               
                   
                 2.0 
                 hours 
                 205.1 
                 3.974 
               
               
                   
                 2.2 
                 hours 
                 121.7 
                 3.731 
               
               
                   
                 3.0 
                 hours* 
                 127.6 
                 3.028 
               
               
                   
                 3.2 
                 hours 
                 74.98 
                 2.593 
               
               
                   
                 4.8 
                 hours 
                 82.92 
                 2.580 
               
               
                   
                 5.0 
                 hours 
                 49.45 
                 2.173 
               
               
                   
                 5.8 
                 hours 
                 58.76 
                 2.432 
               
               
                   
                 6.1 
                 hours** 
                 42.58 
                 1.875 
               
               
                   
                 6.7 
                 hours 
                 38.93 
                 1.273 
               
               
                   
                   
               
               
                   
                 *80 ml of oxygen added to system  
               
               
                   
                 **pump ran continuously for 30 minutes  
               
            
           
         
       
     
     FIGS. 21 and 22 demonstrate the degradation of TCE and butane in the dual-bioreactors while running in batch mode. TCE disappearance from an initial aqueous-phase concentration of 22 mg/liter (ppm), exceedingly higher than its toxic concentration to methanotrophs and propane oxidizers, was measured utilizing headspace analysis. TCE was degraded at a rate of about 2.7 mg/hr/liter for the first 3 hours of operation, and then at a rate of 0.5 mg/hr/liter for the final 3.7 hours of operation. 
     In addition to batch-type processes, the bioreactors of the present invention may also operate by continuous flow techniques. For example, process, scale-up and operation in continuous flow mode in three tandem bioreactors similar to those shown in FIG. 18 may provide a degradation rate of 120 mg of TCE per hour at a flow rate of 36 liters per hour. TCE removal efficiency may be increased substantially by controlling process parameters such as increasing biofilm surface area with the medium, improving butane and oxygen delivery systems and adjusting adequate conditions for optimum bacterial growth. Various other support media, i.e., non-metallic screens, pellets, beads, etc., for the biofilm in the bioreactors listed above may provide a larger surface area for biofilm formation prior to the treatment phase. Other types of support media may also optimize bacterial growth and surface to volume ratio in the bioreactor thus improving biodegradation conditions, and effectively reducing the required residence times within the bioreactor. Greater performance may be achieved by utilizing effective oxygen and growth substrate delivery systems such as sparging. This can be accomplished by reducing bubble size during sparging which would increase the availability of the compounds to the microorganism inside the bioreactor. In certain cases, it may be desirable to reduce the negative effects of extremely stressed influent streams to the bioreactor by pre-adjusting pH, temperature and other related physico-chemical parameters. 
     Whereas particular embodiments of this invention have been described above for purposes of illustration, it will be evident to those skilled in the art that numerous variations of the details of the present invention may be made without departing from the invention as defined in the appended claims.