Ghaemghami, Jalal (234 Willow Avenue Somerville, MA, 02144, US)
Ghaemghami, Jalal (234 Willow Avenue Somerville, MA, 02144, US)
| 1. | A method of degrading a MTBE pollutant, the method comprising treating the MTBE pollutant with butaneutilizing bacteria in the presence of butane and oxygen for a treatment time sufficient for the butaneutilizing bacteria to degrade the MTBE pollutant. |
| 2. | The method of Claim 1, further comprising providing butane to the butaneutilizing bacteria during a portion of the treatment time. |
| 3. | The method of Claim 1, wherein the butane is provided at a substantially constant rate. |
| 4. | The method of Claim 3, wherein the butane is provided for substantially the entire treatment time. |
| 5. | The method of Claim 1, wherein the butane is provided in pulses. |
| 6. | The method of Claim 1, wherein the butane is provided as a butane substrate comprising butane as the most prevalent compound of the substrate. |
| 7. | The method of Claim 1, wherein the butane is provided as a butane substrate comprising at least about 10 weight percent butane. |
| 8. | The method of Claim 1, wherein the butane is provided as a butane substrate comprising at least about 50 weight percent butane. |
| 9. | The method of Claim 1, wherein the butane is provided as a butane substrate comprising at least about 90 weight percent butane. |
| 10. | The method of Claim 1, wherein the butane is provided as a butane substrate comprising at least about 99 weight percent nbutane. |
| 11. | The method of Claim 1, wherein the oxygen is provided to the butaneutilizing bacteria for substantially the entire treatment time. |
| 12. | The method of Claim 1, wherein the oxygen is provided to the butaneutilizing bacteria periodically. |
| 13. | The method of Claim 11, wherein the oxygen is provided as air. |
| 14. | The method of Claim 1, wherein the butaneutilizing bacteria comprises at least one bacterium selected from the group consisting of Pseudomonas, Variovorax, Nocardia, Chryseobacterium, Comamonas, Acidovorax, Rhodococcus, Aureobacterium, Micrococcus, Aeromonas, Stenotrophomonas, Sphingobacterium, Shewanella, Phyllobacterium, Clavibacter, Alcaligenes, Gordona, Corynebacterium and Cytophaga. |
| 15. | The method of Claim 1, wherein the butaneutilizing bacteria comprises at least one bacterium selected from the group consisting of putida, rubrisubalbicans, aeruginosa, paradoxus, asteroides, brasiliensis, restricta, globerula, indologenes, meningosepticum, acidovorans, delafieldii, rhodochrous, erythropolis, fascians, barkeri, esteroaromaticum, saperdae, varians, kristinae, caviae, maltophilia, <BR> <BR> <BR> thalpophilum, spiritivorum, putrefaciens B, myrsinacearum, michiganense, xylosoxydans, terrae, aquaticum B and johnsonae. |
| 16. | The method of Claim 1, wherein the butaneutilizing bacteria comprises at least one bacterium selected from the group consisting of Pseudomonas rubrisubalbicans, Pseudomonas aeruginosa, Variovorax paradoxus, Nocardia asteroides, Nocardia restricta, Chryseobacterium indologenes, Comamonas acidovorans, Acidovorax delafieldii, Rhodococcus rhodochrous, Rhodococcus erythropolis, Aureobacterium esteroaromaticum, Aureobacterium saperdae, Micrococcus varians, Micrococcus kristinae, Aeromonas caviae, Stenotrophomonas maltophilia, Sphingobacterium thalpophilum, Clavibacter michiganense, Alcaligenes xylosoxydans, Corynebacterium aquaticum B and Cytophaga johnsonae. |
| 17. | The method of Claim 1, wherein the MTBE pollutant is present within soil. |
| 18. | The method of Claim 1, wherein the MTBE pollutant is present in a gas. |
| 19. | The method of Claim 18, wherein the gas is air. |
| 20. | The method of Claim 1, wherein the MTBE pollutant is present in a liquid. |
| 21. | The method of Claim 20, wherein the liquid comprises water. |
| 22. | The method of Claim 1, wherein the MTBE pollutant is treated in situ at a contaminated site. |
| 23. | The method of Claim 22, wherein the contaminated site comprises groundwater. |
| 24. | The method of Claim 1, wherein the MTBE pollutant is treated ex situ in a bioreactor. |
| 25. | The method of Claim 1, wherein the MTBE pollutant is contained in water and the butaneutilizing bacteria degrades the MTBE pollutant at a rate of greater than about 0.1 mg/hr/liter. |
| 26. | The method of Claim 1, wherein the MTBE pollutant is contained in water and the butaneutilizing bacteria degrades the MTBE pollutant at a rate of greater than about 0.2 mg/hr/liter. |
| 27. | A method of decontaminating water comprising: providing contaminated water comprising a MTBE pollutant; and treating the MTBE pollutant with a butaneutilizing bacteria in the presence of butane and oxygen for a treatment time sufficient for the butaneutilizing bacteria to degrade the MTBE pollutant to produce decontaminated water having a lower concentration of the MTBE pollutant than the contaminated water. |
| 28. | The method of Claim 27, further comprising providing butane to the butaneutilizing bacteria during a portion of the treatment time. |
| 29. | The method of Claim 27, wherein the butane is provided at a substantially constant rate. |
| 30. | The method of Claim 29, wherein the butane is provided for substantially the entire treatment time. |
| 31. | The method of Claim 27, wherein the butane is provided in pulses. |
| 32. | The method of Claim 27, wherein the butane is provided as a butane substrate comprising butane as the most prevalent compound of the substrate. |
| 33. | The method of Claim 27, wherein the butane is provided as a butane substrate comprising at least about 10 weight percent butane. |
| 34. | The method of Claim 27, wherein the butane is provided as a butane substrate comprising at least about 50 weight percent butane. |
| 35. | The method of Claim 27, wherein the butane is provided as a butane substrate comprising at least about 90 weight percent butane. |
| 36. | The method of Claim 27, wherein the butane is provided as a butane substrate comprising at least about 99 weight percent nbutane. |
| 37. | The method of Claim 27, wherein the oxygen is provided to the butaneutilizing bacteria for substantially the entire treatment time. |
| 38. | The method of Claim 27, wherein the oxygen is provided to the butaneutilizing bacteria periodically. |
| 39. | The method of Claim 27, wherein the oxygen is provided as air. |
| 40. | The method of Claim 27, wherein the contaminated water comprises groundwater. |
| 41. | The method of Claim 27, wherein the contaminated water is treated insitu at a contaminated site. |
| 42. | The method of Claim 27, wherein the contaminated water is treated exsitu in a bioreactor. |
| 43. | The method of Claim 27, wherein the MTBE is degraded at a rate of greater than about 0.1 mg/hr/liter. |
| 44. | The method of Claim 27, wherein the MTBE is degraded at a rate of greater than about 0.2 mg/hr/liter. |
| 45. | The method of Claim 27, wherein the decontaminated water comprises less than about 70 ppb MTBE. |
| 46. | The method of Claim 27, wherein the decontaminated water comprises less than about 20 ppb MTBE. |
| 47. | The method of Claim 27, wherein the decontaminated water comprises less than about 5 ppb MTBE. |
| 48. | The method of Claim 27, wherein the decontaminated water comprises less than about 1 ppb MTBE. |
| 49. | A method of treating a site contaminated with a MTBE pollutant comprising: supplying a butane substrate to the contaminated site; and supplying an oxygencontaining gas to the contaminated site. |
| 50. | The method of Claim 49, wherein the butane substrate comprises at least about 10 weight percent butane. |
| 51. | The method of Claim 49, wherein the butane substrate comprises at least about 50 weight percent butane. |
| 52. | The method of Claim 49, wherein the butane substrate comprises at least about 90 weight percent butane. |
| 53. | 51 The method of Claim 49, wherein the butane substrate comprises at least about 99 weight percent nbutane. |
BACKGROUND INFORMATION Oxygenated gasoline additives such as MTBE have been in widespread use in the United States and other countries. MTBE exhibits very high mobility in soil and groundwater. Due to its solubility, groundwater contaminated with MTBE poses many problems. Many ethers such as MTBE are known to be resistant to biodegradation.
Exposure to MTBE has been shown to alter rat liver metabolic activities.
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. The ability of aerobic methane-utilizing bacteria to degrade
pollutants such as TCE cometabolically is known. However, bioremediation techniques using methane-utilizing bacteria are often not effective and are limited due to the toxic effects of certain chlorinated hydrocarbon pollutants in rather low concentrations.
Despite conventional remediation efforts, a need still exists for the effective degradation of MTBE pollutants. 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 MTBE pollutants. 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 MTBE contaminants contained in, for example, 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 MTBE contamination.
An aspect of the present invention is to provide an improved method of degrading MTBE pollutants.
Another aspect of the present invention is to provide a method of degrading MTBE pollutants with butane-utilizing bacteria by a cometabolic process.
Another aspect of the present invention is to provide a method of degrading a MTBE pollutant by treating the pollutant with butane-utilizing bacteria in the presence of oxygen and butane for a sufficient time for the butane-utilizing bacteria to degrade the MTBE pollutant.
Another aspect of the present invention is to provide a method of decontaminating water by treating the water with butane-utilizing bacteria to reduce or eliminate MTBE pollutants contained in the water.
Another aspect of the present invention is to provide a method of treating a site contaminated with a MTBE pollutant. The method includes the steps of supplying a butane substrate to the contaminated site, and supplying an oxygen-containing gas to the contaminated site.
These and other aspects of the present invention will become more apparent from the following description.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing MTBE concentrations in aqueous samples at various times during treatment with butane utilizing bacteria in accordance with an embodiment of the present invention.
Fig. 2 is a graph showing butane concentrations in the aqueous samples of Fig. 1 at various times during treatment with the butane-utilizing bacteria.
Fig. 3 is a graph showing MTBE concentrations in aqueous samples at various times during treatment with butane-utilizing bacteria in accordance with another embodiment of the present invention.
Fig. 4 is a graph showing butane concentrations in the aqueous samples of Fig. 3 at various times during treatment with the butane-utilizing bacteria.
Fig. 5 is a graph showing MTBE concentrations in control samples at various times.
Fig. 6 is a graph showing butane concentrations in the control samples of Fig. 5.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to methods for the degradation of MTBE pollutants. As used herein, the term"MTBE pollutants"includes pollutants comprising methyl tertiary butyl ether, alone or in combination with other pollutants. In addition to MTBE, other gasoline oxygenates such as ethyl tert-butyl ether or tert-amyl methyl ether may be present in the pollutants, as well as chlorinated aliphatics, chlorinated aromatics and non-chlorinated aromatics. Such additional 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-
dichloroethene, 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, butylbenzenes, naphthalene, crude oil, refined oil, and Nos. 2,4 and 6 fuel oils.
In accordance with the present invention, butane is used to stimulate the growth of butane-utilizing bacteria which are effective in degrading MTBE pollutants.
The butane may be provided in the form of a butane substrate. The butane substrate includes liquids and/or gases in which butane is present in sufficient amounts to stimulate substantial growth of butane-utilizing bacteria. Butane is preferably the most prevalent compound of the butane substrate, on a weight percent basis. Butane typically comprises at least about 10 weight percent of the butane substrate. The other constituents of the butane substrate may include any suitable compounds, including inert gases and/or other alkanes such as methane, ethane and propane. Preferably, the butane substrate comprises at least about 50 weight percent butane, more preferably at least about 90 weight percent butane. In a particular embodiment, the butane substrate comprises at least about 99 weight percent n-butane.
The oxygen may be supplied in any suitable form, including air, pure oxygen and blends of oxygen with inert gases such as helium, argon, nitrogen, carbon monoxide and the like.
The bioremediation process of the present invention may be performed either in-situ or ex-situ to remove contaminants from various environments including aqueous systems such as groundwater, capillary fringe areas and vadose zones, and soil.
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 MTBE pollutants. The butane-utilizing bacteria may be used to aerobically degrade MTBE by cometabolism and/or direct metabolism processes.
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 enzymes which directly metabolize MTBE.
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 Pseudomonadaceae family comprising gram-negative aerobic rods and cocci, species of the following genera may be suitable: Pseudomonas; Variovorax; Chryseobacterium; Comamonas; Acidovorax; Stenotrophomonas; Sphingobacterium; Xanthomonas; Frateuria; Zoogloea; Alcaligenes ; Flavobacterium ; Derxia ; Lampropedia; Brucella; Xanthobacter;Thermus; Thermomicrobium; Halomonas; Serpens; Janthinobacterium;Janthinobacterium;Bordeteela; 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; Micropolyspora; 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 Location 1/soilLandfill cell fuel2/soil#2 oil impacted soil 3/soilLandfill cell 4/soil4/soilGasoline and waste oil impacted soils 5/soil5/soilShallow freshwater lagoon 6/soilSalt marsh Sample Number/MatrixSample Location 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: MgSO4-7H2O 1.0 g; CaCl2 0.2 g; NH4Cl 0.5 g; FeCl3-6H2O 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: ZnCl2 5.0 mg; MnCl2-4H20 3.0 mg; H3BO4 30.0 mg; <BR> <BR> <BR> <BR> NiCl2-6H20 2.0 mg;<BR> <BR> <BR> <BR> <BR> <BR> <BR> (N H4) 6M o7o24-4H2o 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 Na2HPO4 and 1.4 g of KH2PO4 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% NaCI); 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&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 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 1 EP 1heatpropane1HP 1 no-treat propane 1 NP Sample No. Pre-Treatment Food Source Sample ID 110% NaCIpropane1SP pH of 2. 0 propane lAP
The amount of oxygen required for mineralization of methane, propane and butane can be derived from the following equations.
CH4 =CO2+2H2O2:12O2 C3H8 + 502 3CO2 + 4H20 5: 1 C4H, o + 6.502 = 4C02 + 5H2O 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. SourceSampleIDFood methane2EM2ethanol methane2HM2heat methane2NM2no-treat NaClmethane2SM210% of2.0methane2AM2pH propane2EP2ethanol propane2HP2heat propane2NP2no-treat NaClpropane2SP210% of2.0propane2AP2pH butane2EB2ethanol butane2HB2heat butane2NB2no-treat NaClbutane2SB210% 22pH of 2ABbutane 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 propane3NP3no-treat NaClpropane3SP310% 3 pH of 2. 0 propane 3AP 3 ethanol butane 3EB 3 heat butane 3HB butane3NB3no-treat NaClbutane3SB310% 3 pH of 2. 0 butane 3AB Table 5 Sample No. SourceSampleIDFood 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 NaClbutane4SB410% 4 pH of 2. 0 butane 4AB Table 6 Sample No. Pre-Treatment Food Source Sample ID 5 ethanol methane 5EM methane5HM5heat methane5NM5no-treat NaClmethane5SM510% of2.0methane5AM5pH propane5EP5ethanol propane5HP5heat 5 no-treat propane 5NP 5 10% NaCl propane 5SP of2.0propane5AP5pH butane5EB5ethanol butane5HB5heat 5 no-treat butane 5NB 510% NaCIbutane5SB 5 pH of 2. 0 butane 5AB Table 7 Sample No. SourceSampleIDFood 6 ethanol methane 6EM methane6HM6heat methane6NM6no-treat 6 10% NaCl methane 6SM 6 pH of 2. 0 methane 6AM propane6EP6ethanol propane6HP6heat propane6NP6no-treat 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 NaClbutane7SB710% 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 NaClmethane8SM810% 88pH of 8AMmethane 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) 2.5E81EMcocci 1.8E81HMcocci/bacilli 1.3E81NMbacilli 5.8E61SMcocci 3.8E61AMcocci IEP bacilli 4. OE6 1.3E71HPcocci 9.8E61NPbacilli ISP diplococci 4. OE6 lAP bacilli (variable) 1.5E6 1.2E82EMcocci/bacilli 7.3E72HMcocci/bacilli 1.1E82NMstreptococci/bacilli 2SM comma-shaped 6.6E7 2AM comma-shaped 8.3E6 2EP bacilli 1. 2E8 2HP bacilli/comma-shaped 1.8E8 2NP bacilli (variable) l. lE8 2SP cocci 7. OE6 3.3E62APcocci 2.1E82EBcocci/bacilli 2HB2HBbacilli (variable) 2.5E8 1.9E82NBcocci/comma-shaped 2.5E62SBbacilli 3.0E62ABcocci 1.4E83EMcocci/bacilli 3HM cocci 1.2E8 3NM cocci 5.8E7 7.5E53SMcocci 7.5E53AMcocci Enumeration Sample ID Morphology (cells/ml) 3EP bacilli 7. 8E7 3HP bacilli 3. OE7 7.1E73NPbacilli 3SP cocci I. OE6 3AP bacilli 2. 5E5 3EB bacilli (variable) 1.5E8 3HB cocci/baciili 3. 1E7 3NB cocci 3. 1E8 (irregular)1.7E73SBcocci 2.5E53ABcocci/bacilli 4EM cocci (variable) 1.6E8 3.1E84HMdiplococci 4NM cocci 1.6E8 1.3E64SMcocci 4AM bacilli 2. 5E5 4EP bacilli (variable) 1. OE8 4HP bacilli (variable) 2.2E8 1.3E84NPcocci 4SP cocci 1. SE6 6.5E64APcocci/bacilli 4EB bacilli 3.6E8 4HB bacilli (variable) 4.8E8 2.6E84NBbacilli 1.3E64SBcomma-shaped 1.0E64ABcocci SEM cocci (variable) 1.3E8 1.4E85HMcocci 2.4E85NMcocci cells0.05SMno SAM no cells 0.0 SEP cocci (variable) 5. 1E7 3.2E75HPbacilli Enumeration Sample ID Morphology (cells/ml) 2.1E85NPstreptococci (variable)5SPcocci 2.8E6 5.0E5SAPbacilli 5EB bacilli 3. 1E8 5HB cocci 3.2E7 1.6E85NBcocci 1.0E65SBbacilli 2.5E65ABcocci 6EM bacilli (variable) 1.7E8 6HM cocci 2.6E8 1.3E86NMcocci/spirochetes 6SM cocci (variable) 1.3E6 6AM cocci (variable) 2. 0E6 6EP bacilli 2.8E7 6HP bacilli 1.3E8 6NP bacilli/spirochetes 2. OE8 6SP cocci (variable) 3.5E6 6AP cocci (variable) 5. 0E5 3.5E76EBcocci 6HB bacilli 1.3E8 6NB bacilli 4.8E7 6SB cocci 2.3E6 3.3E66ABcocci 7EM streptococci 1.3E8 7HM staphylococci 3.2E7 7NM cocci/bacilli 3. lE8 7SM cocci (variable) 3. 0E6 7AM cocci (variable) 4. 0E6 7EP bacilli 1.4E8 4.1E87HPbacilli 7NP bacilli 3. 5E8 Enumeration Sample ID Morphology (cells/ml) 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. lE7 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. SE6 8AP cocci (variable) 3.8E6 8EB bacilli 1.3E8 8HB bacilli/streptococci 9.8E7 8NB bacilli (variable) 1.2E8 8SB bacilli (variable) 2.0E6 SAB cocci (variable) 2.8E6 Control-l 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 August 22,1996, under ATCC designation numbers 55808 and 55809, respectively.
Methyl tertiary butyl ether (MTBE) cometabolism was evaluated using consortia of butane-utilizing bacteria suspended in 45 ml of sterile mineral salts (MS)
medium. The consortia consisted of groups of cells from the following genera: Pseudomonas, Variovorax, Nocardia, Chrvseobacterium, Comamonas and Micrococcus.
Bacterial inocula used for this study were isolated from gasoline- contaminated soil and groundwater, enumerated using the Most Probable Number Method and characterized using Fatty Acid Analysis by gas chromatography. Negative stain preparations of selected isolates were examined using transmission electron microscopy.
Growth was monitored in 120 ml serum bottles under a butane, MTBE and air mixture.
Butane consumption rates between 1.5 and 6.0 mgh-'L-'were measured for selected consortia using a gas chromatograph equipped with a photoionization detector (GC-PID).
MTBE degradation from starting headspace concentrations as high as 10 mgL-'to sub-part per billion concentration was observed within a 48-hour period and quantified using the GC-PID. Byproducts such as carbon dioxide and dissolved oxygen concentrations were measured using Chemetrics titration kits. The data collected demonstrate the potential for the bioremediation of MTBE using a cometabolic substrate.
Table 11 illustrates the MTBE degradation and butane consumption results for Sample IDs 6NB and 3NB, as well as the control sample.
Table 11 Sample ID 6NB Butane Concentration MTBE Concentration (ppm) (ppm) 0 hours 956 10 12 hours 807 7.5 24 hours 664 6.6 36 hours 346 3.3 48 hours 221 0 Sample ID 3NB Butane Concentration MTBE Concentration (ppm) (ppm) 0 hours 879 10 12 hours 807 8.4 24 hours 562 5.5 36 hours 231 2.9 48 hours 112 0 Control Sample Butane Concentration MTBE Concentration (ppm) (ppm) 0 hours 1186 8 12 hours 1046 7. 5 24 hours 1032 7.3 36 hours 1017 7 48 hours 1010 6.7
Fig. 1 illustrates MTBE co-metabolism for butane-utilizing bacteria Sample ID 6NB of the present invention. As shown in Fig. 1, MTBE concentration decreases from 10 parts per million to 0 parts per million after a 48 hour treatment time. During the same treatment period, the butane concentration decreases from 956 ppm to 221 ppm, as shown in Fig. 2.
Fig. 3 illustrates MTBE co-metabolism for butane-utilizing bacteria Sample ID 3NB of the present invention. As shown in Fig. 3, MTBE concentration decreases from 10 parts per million to 0 parts per million after a 48 hour treatment time. During the same treatment period, the butane concentration decreases from 879 ppm to 112 ppm, as shown in Fig. 4.
Figs. 5 and 6 illustrate MTBE and butane concentrations for control samples. In Fig. 5, the concentration of MTBE in the control sample decreases a negligible amount during the 48 hour treatment period. In Fig. 6, the butane
concentration in the control sample likewise decreases a negligible amount during the 48 hour treatment time.
The results listed in Table 11 and shown in Figs. 1-6 demonstrate that a consortium of butane-utilizing bacteria can cometabolize MTBE under butane and air tension.
The butane-utilizing bacteria of the present invention are preferably capable of degrading MTBE at a rate of greater than about 0.1 mg/hr/liter in water, more preferably at a rate of greater than about 0.2 mg/hr/liter. After treatment in accordance with the present invention, MTBE levels are reduced significantly. For example, the concentration of MTBE in water is typically reduced to levels of less than about 70 ppb, preferably less than about 20 ppb. More preferably, the concentration of MTBE is reduced to less than about 5 ppb, most preferably less than about 1 ppb.
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 first order kinetics. Another cause of the higher MTBE cometabolic rates for the butane-utilizers in comparison with the methane-or propane-utilizers may be the molecular structure of the compounds and enzymes. MTBE is a five carbon, oxygen-containing molecule. 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 MTBE degraders in comparison with the methane and propane operative enzymes. 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 Similarity 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, 4HP-4HB, 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 may have the ability to degrade MTBE are identified in Table 12.
Table 12 SampleID Genus Species 2HB* Pseudomonas putida 2EB Pseudomonas rubrisubalbicans 3EB Pseudomonas rubrisubalbicans 5EB Pseudomonas aeruginosa 6NB Pseudomonas aeruginosa paradoxus2EBVariovorax paradoxus2HBVariovorax 3EB Variovorax paradoxus paradoxus3NBVariovorax paradoxus4HBVariovorax 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 GenusSpeciesSampleID indologenes4HBChryseobacterium 7NB indologenes 5EB Chryseobacterium meningosepticum acidovorans2EBComamonas acidovorans3NBComamonas acidovorans6HBComamonas acidovorans6NBComamonas delafieldii4EBAcidovorax delafieldii4NBAcidovorax delafieldii6NBAcidovorax 4NB rhodochrous rhodochrous7NBRhodococcus 2EB erythropolis 3EB erythropolis erythropolis6HBRhodococcus fascians4EB*Rhodococcus fascians5EB*Rhodococcus barkeri4NBAureobacterium esteroaromaticum4HBAureobacterium esteroaromaticum4NBAureobacterium saperdae6HBAureobacterium varians5EBMicrococcus varians7NBMicrococcus kristinae7NBMicrococcus caviae6HBAeromonas 6NB caviae maltophilia2EBStenotrophomonas maltophilia3EBStenotrophomonas SampleID Genus Species maltophilia4EBStenotrophomonas 5EB maltophilia 6HB Stenotrophomonas maltophilia 6NB maltophilia 4EB Sphingobacterium thalpophilum 4NB* Sphingobacterium spiritivorum 4NB Shewanella putrefaciens B ll myrsinacearum3NB*Phyllobacterium michiganense6HBClavibacter 6HB Clavibacter michiganense**** 6NB Alcaligenes xylosoxydans 7HB* 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-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 is enhanced through the addition of butane, oxygen and, optionally, bacterial nutrients in any subsurface environment in which MTBE, chlorohydrocarbons or other pollutants have been introduced, thereby creating an
effective treatment zone. Butane, oxygen and optional bacterial nutrients such as inorganic and organic nitrogen-containing compounds and phosphorous-containing compounds 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. The butane-utilizing organisms of the present invention may be applied in-situ in saline or low pH environments as well.
A preferred system and method of in-situ bioremediation which may be used to degrade MTBE pollutants are described in U. S. Patent Application entitled "System and Method of In-Situ Bioremediation with Butane-Utilizing Bacteria"filed March 24,1999, which is incorporated herein by reference.
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 that the contaminants are not merely transferred but destroyed.
MTBE-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 MTBE which could be treated as an air waste stream described above. Another type of soil reactor may degrade MTBE pollutants 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 MTBE pollutants 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 as described in U. S. Patent Application Serial No. 08/767,750 may be used to restore surface water or groundwater impacted with MTBE pollutants, 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 MTBE 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.
In addition to batch-type processes, the bioreactors may also operate by continuous flow techniques. MTBE 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.