WO2000063343A2

WO2000063343A2 - Bacterial culture

Info

Publication number: WO2000063343A2
Application number: PCT/EP2000/003254
Authority: WO
Inventors: Joseph Patrick Salanitro
Original assignee: Shell Internationale Research Maatschappij B.V.
Priority date: 1999-04-14
Filing date: 2000-04-10
Publication date: 2000-10-26
Also published as: AU4547600A; WO2000063343A3; EP1169431A2

Abstract

The present invention provides a pure bacterial culture isolable from a mixed bacterial culture ATCC No. 202057 and capable of degrading methyl t-butyl ether (MTBE) to carbon dioxide; a process for isolating such a pure bacterial culture, and a process for remediating a medium containing water and a branched alkyl ether (e.g. methyl t-butyl ether) and/or a branched alkyl alcohol (e.g. t-butyl alcohol (TBA)) using said pure bacterial culture.

Description

BACTERIAL CULTURE

Field of the Invention

The present invention relates to bacterial cultures, processes for their preparation and processes for remediating contaminated media using such bacterial cultures.

Background of the Invention

Alkyl-alkyl ethers such as methyl t-butyl ether (hereinafter "MTBE") are used as octane-enhancers in the reformulation of low volatility unleaded gasoline blends and for reducing the emission of volatile organic compounds from engines. In general, alkylethers, especially those alkylethers which have only one ether linkage and without other functional groups, are chemically stable compounds and there is little information on their biodegradability in soil, groundwater and activated sludge environments. The very slow rate of alkylether degradation by indigenous microbes in soils and biosludges may be attributed to the very stable and chemically unreactive ether linkage, the inability of these compounds to be transported into cells and/or the lack of inducible or existing enzyme activities (e.g. oxygenases, hydroxylases ) which can attack the ether bond. Furthermore, branched alcohols, such as t-butyl alcohol (hereinafter "TBA") , formed as metabolites in the degradation of branched ethers, such as MTBE, are also slow to degrade in the presence of indigenous microbes.

It is known that MTBE and/or TBA can persist in groundwater from accidental spills of unleaded gasoline from underground storage tanks. Certain mixed bacterial cultures have been used in attempts to degrade MTBE and/or TBA. However, it would be desirable to use a pure bacterial culture for degrading MTBE and/or TBA to effectively biotreat groundwater, wastewater, tank bottom wastes or soils containing branched carbon-containing ethers and alcohols. This is because relatively large quantities of mixed culture are typically needed for degrading or remediating MTBE and/or TBA because only minor portions of mixed cultures contain microbes which are able to degrade MTBE or its metabolites, and it is more costly to mass produce mixed cultures than pure cultures to obtain the same quantity of MTBE degradation activities. It is often necessary to use a mixed culture when attempting to biodegrade contaminants such as MTBE and/or TBA as a combination of different microbes may be effective in completely degrading a particular contaminant by virtue of different individual microbes being responsible for a respective one or a combination of steps in the metabolic pathway from contaminants through to carbon dioxide whilst no individual microbe is effective on its own.

Previous attempts to degrade MTBE and/or TBA using bacterial cultures have met with little success. One exception is described in U.S. Patent No. 5,750,364, which describes a process for preparing a mixed bacterial culture capable of degrading MTBE and/or TBA to carbon dioxide by adding a branched alkyl ether such as MTBE to an activated sludge, for example activated sludge obtained from a chemical plant, petrochemical plant or a refinery, or from a biotreater located in a wastewater treatment plant in a refinery or a petrochemical plant. A sample of a mixed bacterial culture prepared according to U.S. 5,750,364 has been deposited with the Americal Type Culture Collection (ATCC) , Patent Depository, 12301 Parklawn Drive, Rockville, MD. 20852, USA, with ATCC number 202057, under the Budapest Treaty (see also Column 2, line 64 to Column 3, line 4 of US Patent 5,902,734). Samples of this culture can be obtained from the permanent collection of the ATCC, Patent Depository (and Column 3, lines 1 to 4 of US Patent 5,902,734 indicate that all restrictions imposed by the depositor on the availability to the public were to be irrevocably removed upon granting of US Patent 5,750,364 (issued 12 May 1998) or of US Patent 5,902,734 itself). Mixed bacterial culture ATCC No. 202057 was prepared from activated sludge retrieved from the biotreater of the South Effluent Treater for treating wastewater from the Chemical Plant of Shell Deer Park Manufacturing Complex located at 5900 Highway 225, Deer Park Texas 77536, USA. The preparetion of mixed bacterial culture ATCC No.

202057 is described in U.S. Patent No. 5,750,364 at column 6, line 32, to column 7, line 8. At column 7, lines 22-31, of US Patent No. 5,750,364 it is stated that microscopic examination of phase-contrast and gram-stained smears of ATCC No. 202057 showed that it contains gram- positive filamentous species and several gram-negative smaller rod-shaped bacteria. Preliminary identification of colonies isolated on a minerals (Sturm solution) agar medium containing 200 ppm of MTBE indicate that ATCC No. 202057 contains at least 4-5 organisms including species of coryneforms. Pseudomonas and Achromobacter . All of these isolates utilize acetate, but none has been shown to grow on MTBE as a sole source of carbon. There is nothing in US Patent No. 5,750,364 to lead a person skilled in the art to conclude that he or she would isolate from ATCC No. 202057 a single organism which could metabolise MTBE through to carbon dioxide.

K. Mo, et al. Appl Microbiol Biotechnol (1997) 47:69-72 proposes isolating from activated sludge and fruit of the Gingko Tree three pure cultures, classified as belonging to the genuses Methylobacterium, Rhodococcus, and Arthrobacter, which are capable of degrading MTBE. However, the data presented by Mo proposes that only a minor portion of the MTBE was degraded by the cultures and very little if any, of MTBE degraded to carbon dioxide within the time frame of the experiment.

Thus, there is a need for a pure bacterial culture capable of degrading branched alkyl ethers such as methyl t-butyl ether (MTBE) , and branched alcohols, such as t- butyl alcohol (TBA) . Summary of the Invention

The present invention provides a pure bacterial culture isolable from a mixed bacterial culture ATCC No. 202057 and capable of degrading methyl t-butyl ether

(MTBE) to carbon dioxide. The present invention further provides a process for isolating such pure bacterial culture and a process for remediating a medium containing water and a branched alkyl ether and/or a branched alkyl alcohol using said pure bacterial culture. Detailed Description of the Invention

A pure bacterial culture according to the present invention is isolable from a mixed bacterial culture ATCC No. 202057. Preferred pure bacterial cultures isolable from mixed bacterial culture ATCC No.202057 are aerobic organisms, more preferably aerobic organisms which are gram stain positive.

Whilst the pure bacterial culture of the invention is without limitation as to its morphology, preferred pure such cultures have been observed to be rod-shaped, such rod-shaped cultures preferably having a diameter of from 0.1 to 2 micron, more preferably from 0.5 to 1.5 micron. Preferred such cultures have also been observed to be appear coccal-shaped when cultured on solid media. The pure bacterial cultures of the present invention are preferably oxidase-negative and/or catalase- negative cultures, more preferably both oxidase-negative and catalase-negative cultures. Preferred pure bacterial cultures according to the present invention are bacteria which grow on one or more of the following substrates, Tweens ("TWEEN" is a trade mark - see "Encyclopedia of Surface-Active Agents", Vol. II, J.-P.Sisley, trans. P.J. Wood, Chemical Publishing Company Inc., New York, 1964), e.g. Tween 40 and Tween 80, dextrin, cellobiose, fructose, lactose, maltose trehalose, glucose, adonitol, arabinose, lactose, sorbitol, and the metabolic intermediates in the MTBE pathway, namely, isopropanol, acetone and acetate. More preferred are cultures which grow on all the aforementioned substrates.

Pure bacterial cultures which may be conveniently isolated from mixed culture having ATCC No.202057, and which are especially preferred are non-fermentative, gram positive, oxidase-negative, catalase-negative bacteria, preferably belonging to the family Actmomycetes, more preferably belonging to the genus Rhodococcus .

Preferred pure bacterial cultures according to the present invention are cultures capable of degrading a branched alkyl ether. Advantageously, the pure bacterial cultures are capable of aerobically degrading a branched alkyl ether, preferably a tertiary carbon atom-containing alkyl ether, more preferably MTBE, to carbon dioxide. Such preferred pure bacterial cultures are preferably capable of cleaving the ether linkage of methyl t-butyl ether (MTBE) with the transient formation of t-butyl alcohol (TBA) which is subsequently degraded substantially completely to carbon dioxide. As a preferred embodiment of the present invention, the pure bacterial culture can also metabolize other linear and branched ethers. Non- limiting and illustrative examples of the linear and branched ethers include diethyl ether (DEE) , dimethyl ether (DME) , methyl ethyl ether (MEE) , methyl n-propyl ether (MPE) , ethyl n-propyl ether, methyl isopropyl ether, ethyl isopropyl ether, diisopropyl ether (DIPE), ethyl t- butyl ether (ETBE) or methyl t-amyl ether.

A pure bacterial culture isolable from mixed culture ATCC No. 202057, as defined in the present invention, also includes any composition derivable from said pure bacterial culture. Illustrative examples of the compositions derivable from the pure bacterial, culture include, but are not limited to, member (s) of, fragment (s) of bacterial culture, membrane fragment (s) of bacterial culture, enzymes extracted and/or isolated from the bacterial culture, lyophilized and/or dried culture, lyophilized and/or dried fragments of culture, lyophilized and/or dried enzymes derivable from said culture, bacterial culture and/or fragment (s) thereof and/or enzymes derived therefrom bound to a carrier and/or binder and/or fixed bed, etc. Any method known to one skilled in the art for making composition derivable from the culture including but not limited to extraction or fragmentation to obtain active ingredients/fragments thereof is within the scope of the present invention. As one non-limiting example of the present invention, the pure culture can be first fragmented by sonification or lysing with lysozyme and/or a compound such as a chelating compound, followed by salting out the enzyme fractions using ammonium sulfate or NaCl.

Preferred pure bacterial cultures according to the present invention are cultures capable of degrading MTBE; preferably capable of degrading MTBE in 70 hours; more preferably capable of degrading at least 10% of the MTBE present in an MTBE-containing mixture from MTBE to carbon dioxide in 70 hours; even more preferably capable of degrading both MTBE and TBA, preferably to carbon dioxide in 70 hours; more preferably capable of degrading at least 10% of the MTBE and/or TBA added to the culture at a concentration of 0.01 to 500 ppm, to carbon dioxide within 70 hours; preferably capable of degrading to carbon dioxide MTBE and one or more of the following ether compounds: diisopropyl ether, ethyl t-butyl ether, di-t- butyl ether, diisobutyl ether, isopropyl isobutyl ether, isopropyl t-butyl ether, t-amylmethyl ether, t-amylethyl ether, t-amyl ethyl ether, t-amyl propyl ether, t- amylisopropyl ether, t-amyl-n-butyl ether, t-amyl isobutyl ether, and t-amyl methyl ether within 70 hours; more preferably capable of degrading to carbon dioxide MTBE and one or more of the following tertiary carbon-containing ether compounds: ethyl t-butyl ether, t-amyl-n-butyl ether, t-amylisobutyl ether, isopropyl t-butyl ether, t- amyl ethyl ether, t-amylpropyl ether, t-amylisopropyl ether, and t-amyl methyl ether within 70 hours.

The present invention provides a process for preparing a pure bacterial culture from a mixed bacterial culture ATCC No. 202057. The preparation of mixed culture ATCC No. 202057 is described in US Patent No. 5,750,364 at column 6, line 32 to column 7, line 8, wherein ATCC No. 202057 is prepared by adding MTBE to an activated sludge retrieved from the biotreater of the South Effluent Treater for treating waste-water from the Chemical Plant of Shell Deer Park Manufacturing Complex located at 5900 Highway 225, Deer Park, Tex. 77536, USA. Samples of mixed culture ATCC No. 202057, can be obtained from the permanent collection of the ATCC, Patent Depository, 12301 Parklawn Drive, Rockville, MD . 20852, USA. Any method known to one skilled in the art which is able to isolate a pure culture from a mixed culture may be used to isolate a pure culture according to the present invention. One process suitable for isolating the pure cultures of the present invention includes enhancing isolation of the pure microbes capable of degrading MTBE and/or TBA by first making dilution enrichments of the mixed culture ATCC No. 202057.

A process by which a pure bacterial culture may be conveniently isolated from mixed culture ATCC No. 202057 by way of making dilution enrichments, comprises the steps of:- a) adding sterile mineral dilution medium containing 0.01-1000 mg/L of MTBE, to a sample of mixed bacterial culture ATCC No. 202057, b) subsequently removing a proportion from the resulting culture volume and replacing that proportion with a corresponding volume of fresh dilution medium containing 0.01-1000 mg/L of MTBE, to yield a dilution enrichment culture, c) repeating step b) on resulting culture until a dilute suspension of dilution enrichment culture is found to consistently degrade MTBE, d) streaking the dilution enriched culture of step c) onto sterile plates containing a mineral medium and incubating said plates, e) observing the plates for the appearance of colonies and when colonies appear, picking the colonies from the plates and inoculating the separated colonies in sealed vessels containing mineral medium and MTBE, f) incubating the separated colonies and determining the loss of MTBE from headspace of the sealed vessels, and g) selecting and isolating a separated colony which substantially completely degrades MTBE to carbon dioxide.

Sterile mineral dilution medium, as used for example in step a) may conveniently comprise Bushnell-Haas minerals, hereinafter referred to as BH, containing in the range 0.01- 1000 mg/L, preferably in the range 0.1- 100 mg/L, more preferably in the range 1-10 mg/L of MTBE in a ratio with the amount of mixed culture of 5:1 to 0.2:1, preferably of 3:1 to 0.3: 1, more preferably of 1.5:1 to 1:1.5. Subsequently, a proportion of the resulting culture may be aseptically removed at suitable time intervals preferably weekly, biweekly or monthly most preferably weekly, and replaced with fresh sterile dilution medium which is added to the remaining culture. The dilution enrichment method, as described in step b) , is preferably continued for a period of time in the range 2-60 weeks, preferably in the range 4- 25 weeks, and more preferably in the range 7-14 weeks at a temperature in the range 10-40°C, preferably in the range 20-35°C and more preferably in the range 23°C to 32°C until a dilute suspension of bacteria is found to consistently degrade MTBE before each transfer interval. The resulting dilution enriched culture may subsequently be streaked onto sterile plates, for example Petri Plates, containing minerals and solidifying agent such as 1.5% "Difco" (Trade Name) Agar obtainable from B.D. Biosciences Inc.. The plates are then incubated at a temperature in the range 10-40°C, preferably in the range 20-35°C and more preferably in the range 23°C to 32°C and observed for the appearance of colonies after 1-70 days, preferably after 2-50 days, more preferably 2-10 days, most preferably after 3-5 days. The colonies are then individually picked from the plates, preferably with sterile needles, and inoculated in containers/vials containing mineral medium, preferably sterile mineral medium (such as BH medium) containing in the range 0.01- 1000 mg/L, preferably in the range 0.1-100 mg/L, more preferably in the range 1-10 mg/L of MTBE. The separated cultures are incubated at a temperature in the range 10-40°C, preferably in the range 20-35°C and more preferably in the range 23°C to 32°C. The loss of MTBE from the headspace of containers/vials is determined, and colonies which degrade MTBE substantially completely to carbon dioxide, preferably without the appearance of intermediates such as t-butyl alcohol are identified, selected and isolated. As a preferred embodiment of the present invention, the culture produced is capable of degrading alkyl ethers, preferably branched alkyl ethers, more preferably MTBE, to carbon dioxide. Preferably the culture prepared can also be used to degrade t-butyl alcohol, isopropyl alcohol and acetone, preferably to carbon dioxide.

In a preferred embodiment of the present invention, the MTBE and/or TBA degradation activity of the isolated culture is enhanced by h) growing the isolated pure culture in a sugar containing mineral solution and inducing enhanced MTBE and/or TBA degradation activity by adding MTBE and/or TBA to the resultant culture. Thus step (h) can, in a specific embodiment thereof, be regarded as growing the pure culture in a sugar- containing mineral solution and then inducing for higher MTBE and/or TBA degradation activity by spiking with MTBE and/or TBA. For example, the isolated pure bacterial culture can be grown to obtain a larger population of a larger quantity of the culture by growing on a sugar (such as glucose) containing BH mineral solution. Preferably, the isolated pure culture is grown in an MTBE and/or TBA-containing mineral media. More preferably, the culture is grown in a sugar containing mineral solution and the MTBE and/or TBA degrading activity is induced by adding MTBE and/or TBA to the culture. As a preferred aspect of this embodiment, the MTBE and/or TBA degrading activity, i.e. the capability of degrading MTBE and/or TBA (the concentration of the MTBE and/or TBA degraded within five hours, preferably 10 hours, more preferably 50 hours) is increased by at least 25%, preferably by at least 50%, more preferably by at least 100% after incubating with 1-1000 mg/L, preferably from 5-500 mg/L, more preferably from 20-200 mg/L of MTBE and/or TBA for less than 10 hours, preferably for less than 20 hours, and more preferably for less than 50 hours.

Preferred pure bacterial cultures according to the present invention are cultures having a specific activity in the range 0.1 to 100, preferably in the range 1 to 50, more preferably in the range 5 to 30 mg MTBE/g cells/hr at 9°C. Although in laboratory experiments higher temperatures (e.g. 25°C) tend to be employed for evaluation of specific activity, in the field, ambient temperatures tend to be lower, e.g a temperature of 9°C. The present invention provides a process for degrading branched alkyl ethers such as MTBE, utilizing the above-mentioned novel pure culture by mixing or growing the aforementioned culture or composition derived therefrom with the branched alkyl ethers or a medium containing water and the branched alkyl ethers to be degraded. Conveniently, said process may be used for remediating a medium containing water and a branched alkyl ether and/or a branched alkyl alcohol, which process comprises growing in the presence of said medium a pure bacterial culture according to the present invention. The medium containing water to be remediated may be a liquid medium, for example groundwater or wastewater or it may be a mixed medium such as soil containing water contaminated with a branched ether and/or a branched alcohol. The remediation of the medium is preferably conducted under an oxygen-containing atmosphere, such as aerobic conditions. The remediation may be conveniently conducted at a temperature in the range 5°C to 80°C, preferably in the range 10°C to 60°C, more preferably in the range 15°C to 35°C, still more preferably at ambient temperature.

Branched alkyl ethers which may be degraded in said remediation process include methyl t-butyl ether (MTBE) diisopropyl ether (DIPE), ethyl t-butyl ether (ETBE) , di- t-butyl ether, diisobutyl ether, isopropyl isobutyl ether, isopropyl t-butyl ether, t-amylmethyl ether, t-amylethyl ether, t-amyl ethyl ether, t-amyl propyl ether, t- amylisopropyl ether, t-amyl-n-butyl ether, t-amyl isobutyl ether, and t-amyl methyl ether (TAME) . The process is very useful for remediating tertiary carbon-containing ethers and/or alcohols e.g. MTBE and/or TBA. Whilst application to degradation of aromatic ethers has not specifically been investigated, the possibility of such use is not excluded from consideration.

As an especially preferred embodiment of the present remediation process, a pure bacterial culture of the present invention is used to remediate a medium containing water and MTBE and/or TBA.

The present remediation process may very conveniently be used to remediate a medium containing water contaminated with branched alkyl ethers which are used in gasoline e.g. methyl t-butyl ether (MTBE) diisopropyl ether (DIPE), ethyl t-butyl ether (ETBE), and t-amyl methyl ether (TAME) . When branched alkyl ether contanining fuels are accidentally released into subsurface groundwater or soil, the water soluble ethers persist causing prolonged contamination. The present remediation process thus provides an effective means for remediating aqueous medium, e.g. groundwater, wastewater and soil contaminated with these ethers. In a preferred embodiment of the present invention, the ethers can be completely mineralized to carbon dioxide by a pure culture. Hence, the remediation process may be substantially free of environmentally undesirable end products.

The present pure culture is capable of degrading/ remediating a branched alkyl ether such as MTBE, more preferably a branched alkyl ether and/or a branched alkyl alcohol, such as MTBE and/or TBA in an aqueous medium containing in the range 0.001 ppmw to 5000 ppmw, preferably in the range 0.01 ppmw to 500 ppmw, more preferably in the range 0.05 ppmw to 100 ppmw of a branched alkyl ether and/or a branched alkyl alcohol; to reduce the content thereof by in the range 10% to 100%, preferably in the range 30% to 100%, more preferably in the range 50% to 100%, still more preferably in the range 80% to 100%, preferably within 70 hours, more preferably in 2 hours to 70 hours, still more preferably in 2 hours to 12 hours, and most preferably in 3 hours to 5 hours, by growing in an aqueous medium the culture of the present invention .

As a preferred embodiment of the present invention, the present pure culture is used in a concentration in the range 50 ppmw to 10,000 ppmw, preferably in the range 100 ppmw to 5000 ppmw, and more preferably in the range 1000 ppmw to 4000 ppmw, for example when used for remediating or degrading MTBE. Preferably, the present pure culture, when initially present at a concentration in the range 50 ppmw to 10,000 ppmw, preferably 50 ppmw to 5000 ppmw, more preferably in the range 300 ppmw to 4000 ppmw, in a medium containing .001 ppmw to 5000 ppmw of, for example MTBE, is capable of degrading the MTBE to carbon dioxide and water by 10 to 100 percent, preferably by 50 to 100 percent, more preferably by 80 to 100 percent, still more preferably by 90 to 100 percent, still more preferably by 95 to 100 percent in less than 70 hours, preferably in less than 50 hours, more preferably in less than 30 hours. As a non-limiting illustrative example, the pure culture is capable of degrading MTBE present initially at a concentration in the range 4 to 6 mg per liter down to about 0.5 to 0.001 mg/L, preferably to 0.1 to 0.01 mg/L, more preferably to 0.007 to 0.004 mg/L in less than 50 hours, more preferably in less than 30 hours at a temperature of 5 to 35°C.

Preferably, the concentration of the present pure culture used for remediating a medium containing water and a branched alkyl ether, preferably a branched alkyl ether and/or branched alkyl alcohol, is in the range 50 to 10,000, preferably in the range 100 to 3,000, and more preferably in the range 1,000 to 2500 mg of dry weight of cells per liter or Kg mixture, wastewater, groundwater or soil comprising in the range 3 ppb to 1000 ppm, preferably in the range 5 ppb to 500 ppm, more preferably in the range 10 ppb to 200 ppm of a branched alkyl ether and/or a branched alkyl alcohol; said pure culture being capable of degrading the branched alkyl ether and/or branched alkyl alcohol to carbon dioxide and water by 10 to 100 percent, preferably by 50 to 100 percent, more preferably by 80 to 100 percent, still more preferably by 90 to 100 percent, still more preferably by 95 to 100 percent in less than 70 hours, preferably in less than 50 hours, more preferably in less than 30 hours. As a preferred embodiment, the pure culture is capable of degrading a branched alkyl ether and/or a branched alkyl alcohol present at the above-mentioned concentration down to less than 100 ppb, preferably less than 40 ppb, more preferably less than 5 ppb, more preferably less than 1 ppb, in less than 70 hours, preferably in less than 50 hours, more preferably in less than 30 hours, still more preferably in less than 15 hours at a temperature in the range of 5 to about 35°C. The present invention further provides a process for aerobically degrading a branched alkyl ether, preferably a tertiary carbon-containing alkyl ether, in a branched alkyl ether-containing medium, preferably to carbon dioxide utilizing the present pure culture. Conveniently, such a process may be used for aerobically degrading a branched alkyl ether and/or a branched alkyl alcohol, preferably a tertiary carbon-containing alkyl ether and/or a tertiary carbon containing alcohol. The medium can be, but is not limited to, an aqueous medium, e.g. a soil medium, a mixture of soil and water, waste water, ground water, etc. In a preferred embodiment, the said process comprises growing in the presence of a branched alkyl ether and/or a branched alkyl alcohol-containing mixture the present pure culture, or its derivatives to reduce the concentration of the branched alkyl ether and/or the branched alkyl alcohol in the mixture. As one preferred embodiment, the branched alkyl ether and/or branched alkyl alcohol-containing medium comprises from 0.001 ppmw to 5000 ppmw, preferably from 0.01 ppmw to 500 ppmw, more preferably from 0.05 ppmw to 100 ppmw of ether and/or alcohol. The present process conveniently facilitates reduction of the content of branched alkyl ether and or branched alkyl alcohol, by 10% to 100%, preferably 30% to 100%, more preferably 50% to 100%, still more preferably 80% to 100% in 2 hours to 70 hours, preferably in 2 hours to 12 hours, more preferably in 3 hours to 5 hours. As a preferred embodiment, the process degrades a branched alkyl ether and/or a branched alkyl alcohol to carbon dioxide and water. Said process may be conveniently used to remediate a medium containing, as a branched alkyl ether, diisopropyl ether, ethyl t-butyl ether, di-t-butyl ether, diisobutyl ether, isopropyl isobutyl ether, isopropyl t-butyl ether, t-amylmethyl ether, t-amylethyl ether, t-amyl ethyl ether, t-amyl propyl ether, t- amylisopropyl ether, t-amyl-n-butyl ether, t-amyl isobutyl ether, and t-amyl methyl ether. The said process may very conveniently be used used to remediate a medium containing a tertiary carbon-containing ether and/or a tertiary carbon-containing alcohol, especially MTBE and/or TBA. The invention will be further understood from the following illustrative examples. In the examples, as elsewhere in this specification, parts and percentages are parts and percentages by weight, unless otherwise stated.

PART A: ISOLATION OF PURE CULTURE

Dilution enrichments of mixed culture ATCC No. 202057 were made to enhance isolation of a specific microbe capable of degrading MTBE. Mixed culture ATCC No. 202057 is prepared from an activated sludge retrieved from the biotreater of the South Effluent treater for treating wastewater from the Chemical Plant of Shell Deer Park Manufacturing complex located at 5900 Highway 225, Deer Park, Texas 77536, USA, as described in U.S. Patent No. 5,750,364 at column 6, line 32 to column 7, line 8. Samples of mixed culture ATCC No. 202057 are available from the permanent collection of the ATCC, Patent Depository, 12301, Parklawn Drive, Rockville, M.D. 20852, USA.

A pure culture was prepared by aαding 10 ml of the mixed culture ATCC No. 202057 to 10 ml sterile "Difco" (Trade Mark) Bushnell-Haas (hereinafter referred to as BH) (MgS0₄, 200 mg/L; CaCl₂, 20 mg/L; KH₂P0₄, 1000 mg/L; K₂HP0₄ 1000 mg/L; NH₄N0₃ 1000 mg/L; FeCl₃, 50 mg/L, pH 7.0) minerals medium (3.5 g/L) ; (obtained from BD. Biosciences Inc., 1 Becton Drive, Franklin Lakes, New Jersey, USA, formerly Becton Dickinson Microbiology Systems and Difco Laboratories) in stoppered serum bottles containing 1-5 mg/L MTBE. At weekly intervals, half of the culture volume (10 ml) was aseptically removed and 10 ml fresh sterile BH medium added to the remaining 10 ml of culture. The dilution enrichment method was continued for at least 2-3 months at 25°C until a dilute suspension of bacteria degrading MTBE consistently degraded MTBE before each transfer interval. This dilution enrichment culture was subsequently streaked onto sterile Petri plates containing BH minerals plus 1.5% "Difco" Agar, obtained from B.D. Biosciences, as solidifying agent. Plates were incubated at 25°C or 30°C and observed for the appearance of colonies after 3-5 days. Approximately 20 colonies were picked with sterile needles and inoculated into 20 serum vials containing sterile BH medium and 1-10 mg/L MTBE. These cultures were incubated at 25-30°C and the loss of MTBE from the headspace of the serum vials was determined. One of the pure isolates (hereinafter referred to as Culture A) was found to completely degrade MTBE without any significant appearance of intermediates such as t-butyl alcohol. PART B. DEGRADATION OF MTBE BY PURE CULTURE A

The pure culture isolate (Culture A) was grown in R₂A broth medium (yeast extract 0.5 g/L; peptone 0.5 g/L; casein acid hydrolyzate, 0.5 g/L; soluble starch, 0.5 g/L; glucose 0.5 g/L; KH₂P0₄, 0.3 g/L; MgS0_4/ 0.024 g/L; sodium pyruvate, 0.3 g/L; pH 7.0) for 24-48 hours at 25° C. The culture was then centrifuged (8000 rpm, 15 min.) and resuspended into 10 ml sterile phosphate-buffered saline (NaCl, 9 g/L; KH₂P0₄, 6.85 g/L; pH 7.0-7.2). The culture was transferred to a 30 ml serum vial. MTBE was added to a concentration of 5 mg/L and stoppered and sealed. The degradation of MTBE was followed at 25°C over several days. Table 1 shows an example of MTBE degraded by this pure culture from 5 mg/L to non-detectable concentrations (= 5μg/L) in 48 hours (Run #1) . This culture was respiked with 5 mg/L MTBE. MTBE was degraded 95% (0.24 mg/L) in 27 hours (Run #2 Table 2).

TABLE 1

Run #1: Degradation of MTBE by Pure Culture A

TABLE 2

Run #2: Degradation of MTBE by Pure Culture A

PART C: PHYSIOLOGICAL PROPERTIES OF PURE CULTURE A

Table 3 summarizes some physiological properties and substrates utilized by the MTBE-degrading isolate Culture A. The organism is an aerobic, morphologically irregular, gram-positive rod. The organism appears coccal-shaped when cultured on solid media. It grows on "Tween" (Trade Mark), surface active agents e.g. "Tween" 40 and "Tween" 80 dextrin, cellobiose, fructose, lactose, maltose, trehalose, glucose, adonitol, arabinose, lactose, sorbitol and acetate. Pure Culture A also grows well on the metabolic intermediates in the MTBE pathway, namely, isopropanol, acetone and acetate. The culture grows well on a variety of complex bacteriological media including Trypticase Soy Broth and Agar and Plate Count Agar (obtained from B.D., Bosciences, Inc). Based on physiological and biochemical features of Pure Culture A as a non-fermentative gram-positive, oxidase-negative, catalase-negative bacterium and substrate utilization patterns in the "Oxi/Ferm" (Trade Mark) and "Biolog" (Trade Mark) assays characteristics (see Table 3 for further details on the "Oxi/Ferm" and "Biolog" assays.) described in Bergey' s Manual of Systematic Bacteriology, pp 233-2339 (Holt, J.G. Ed. Williams and Williams. Baltimore MD, USA) , it is probable that this isolate belongs to the family of organisms known as actinomycetes . Further gene sequence and GC-Fatty Acid Methyl Ester (FAME) analyses confirmed that the pure culture belongs to the genus Rhodococcus .

TABLE 3

Physiological features & substrates utilized by pure Culture A

^a> Oxidase reaction negative when grown on most substrates . ^b) Biolog Identification System, .GN and GP plates (Biolog, Inc; Hayward, CA) a carbon source utilization method for identification of gram-negative and gram-positive bacteria, Miller and Rhoden (J. Clin. Microbiol .

29(6) .143-147, 1991) . ^c) Multimedia substrate utilization tubes for identification of oxidative and/or fermentative gam- negative rods (Hoffman-LaRoche, Inc.). Substrates tested in the Oxi/Ferm tube and Entertube include glucose, xylose, urea, citrate, arginine, lysine, lactose, sucrose, maltose, mannitol, dulcitol, phenylalanine and ornitine . ^d) Substrates were added to sterile minerals Bushnell Haas medium or the Rowbotham and Cross basal medium (J. Gen. Microbiol. 100:231-240, 1977) at concentrations of 100- 200 mg C/L (or 0.1-1% w/v for sugars, acids and hydrocarbons) and growth evaluated (visual) after inoculating media with 1 ml of acetate-grown (24 hour) and incubating cultures for 7-14 days at 30°C.

PART D: 16S rRNA GENE SEQUENCE ANALYSIS

The 16S rRNA gene of the pure Culture A was polymerase chain reaction (PCR) amplified from genomic DNA isolated from Culture A bacterial colonies. Primers used for the amplification correspond to E. coli positions 005 and 1540 (full length packages) and 005 and 531 (500 bp packages) . Amplification products were purified from excess primers and dNTPs using "Microcon 100" (Trade Name) concentrator from Amicon Inc., molecular weight cut-off membranes and checked for quality and quantity by running a portion of the products on an agarose gel.

Cycle sequencing of the 16S rRNA amplification products was carried out using "AmpliTaq" (Trade Mark) FS DNA polymerase and dRhodamine dye terminators. Excess dye-labeled terminators were removed from the sequencing reactions using "Sephadex" (Trade Mark) G-50 spin column. The products were collected by centrifugation, dried under vacuum and frozen at -20°C until ready to load.

Samples were resuspended in a solution of formamide/blue dextran/ethylenediamine triacetic acid (EDTA) and denatured prior to loading. The samples were electrophoresed on a ABI Prism 377 DNA Sequencer. Data was analyzed using PE/Applied Biosystem' s MicroSeq (Trade Mark) microbial analysis and DNA editing and assembly software and database.

The top ten alignment matches below are presented in a percent genetic distance format . In this format a low percent indicates a close match.

Alignment: 504 base pairs pure culture A

4.56 % 504 Rhodococcus coprophilus

4.57 % 503 Rodococcus rhodochrous 5.80 % 500 Mycobacterium tokaiense

6.40 % 500 Nocardia corynebacteroides

6.41 % 499 Mycobacterium brumae 6.97 % 502 Mycobacterium gadium

7.20 % 500 Tsukamurella wratislaviensis 7.37 % 502 Tsukamurella inchonensis 7.37 % 502 Tsukamurella pulmonis 7.37 % 502 Tsukamurella paurometabolum

The Neighbor joining (Saitou and Nei, Mol . Biol . Evol. 4 (4 ): 406-425, 1987) phylogenetic trees below are generated using the above top ten alignment matches.

Neighbor Joining Tree

Rhodococcus rhodochrou Culture A

Rhodococcus coprophilus Tsukamurella pulmonis

Tsukamurella inchonensis

^■"T- Tsukamurella paurometabolu Nocardia corynebacteroides

- Tsukamurella wratislaviensis Mycobacterium brumae

- Mycobacterium tokaiense

- Mycobacterium gadium Concise alignments are also included below. These illustrate positions that differ between pure culture A and the first match in the database. The position of the mismatch is read vertically from top to bottom and the sequences are read horizontally from left to right.

Concise Alignment - 500 bp

111111222223444444444

68777999012567333456668

95389135913613578960130 Culture A ATGTCTCGTCGCCCTCCGGGGAC

Rhodococcus coprophilus TCTATCTTATATTACGGATCCGT

Data from the partial sequencing of 16S rRNA have enabled the suprageneric relationship of actinomycetes to be established and this places Rhodococcus beside Nocardia and Mycobacterium among the nocardioform actinomycetes (Goodfellow, M 1989 Suprageneric classification of Actinomycetes, In: Bergey' s Manual of Systematic Bacteriology. Pp. 2333-2339. Holt, J.G. Ed. Williams & Williams, Baltimore, MD) . PART E:GC-FAME ANALYSIS

The strain was streaked onto trypticase soy agar [TSA] . The TSA plates were prepared for use in the GC- FAME analyses after 24 hour incubation. The strain was examined against both the Aerobe (TSBA [rev. 3.90]) and the Clinical Aerobe (CLIN [rev. 3.90]) databases. The strain was subsequently prepared for "Biolog" (Trade Mark) assay analysis by suspending it in sterile saline and loading the solution into the appropriate microtiter plates (Gram positive) . The plates were incubated for 24 hours and then examined against . version 3.5 of the "Biolog" (Trade Mark) database using an automated microplate reader. TABLE 4

Summary of Results by GC-FAME and Biolog

Part F: Degradation of MTBE with Pure Culture A

Aqueous solutions containing various concentrations of MTBE: 5.7, 11.7, 20.9, 44.5, 90.2, 165, 350 ppm of MTBE were mixed with the present pure Culture A (2.76 g/L Total Suspended Solids (TSS) at 25°C) and incubated at 25°C for various lengths of time and the concentrations of MTBE were measured. The results of the experiment is listed in Table 5 below.

Table 5

MTBE Cone

Part G: Degradation of MTBE with Pure Culture A

Aqueous solutions containing various concentrations of MTBE: 6.5, 14, 19.2, 40, and 100 ppm of MTBE were mixed with the present pure Culture A (330 mg/L TSS at 9°C) and incubated at 25°C for various lengths of time and the concentrations of MTBE were measured. The results of the experiment is listed in Table 6 below. Table 6 MTBE Cone (ppm)

Part H : Degradation of TBA with Pure Culture

Aqueous solutions containing various concentrations of t-butyl alcohol (TBA) were mixed with the present pure Culture A (820 mg/L TSS at 9°C) and incubated at 25°C for various lengths of time and the concentrations of TBA were measured. The results of the experiment is listed in Table 7 below.

Table 7

PART I : EFFECT OF STORAGE CONDITIONS (TEMPERATURE) ON Culture A FOR MTBE DEGRADATION

A culture Medium (BHCιo)was prepared with BH + lOg/L "Cerelose" (Trade Mark of a product obtainable from Corn Products International Inc.), (1000 ml) at pH 7.2 - 7.4. BHCio was inoculated with 10 - 20 ml of pure Culture A grown on BHCi (BH + lg/L "Cerelose" ) for 2-4 days at room temperature. The effect of storage on the activity of the resulting culture was investigated by storing samples of the culture at a temperature of 25°C, 4°C and -70°C.

The culture was assayed for MTBE degradation prior to set up at 25°C, 4°C and -70°C. After being stored at the aforementioned temperatures for a period of 24h, 48h and 72 hours, samples of the culture were assayed for MTBE activity in a die-away test system conducted using the following procedure:- (Analysis was conducted on MTBE in vials using a calibrated Photovac (photonionisation detector) Gas Chromatograph in aliquots of 0.1 ml sample . ) a) 10 ml of culture was added to a 30 ml serum vial, sealed, and 5-8 ppm of MTBE (0.5 ml of 200 ppm sterile soln) was subsequently introduced, b) after a designated period of time, the culture sample was assayed for MTBE concentration, wherein for each analysis a packed cell (pellet) aliquot was removed at the designated time and washed by adding 40 ml BH minerals solution and vortexing well to suspend cells, and 10 ml of washed solution was removed for die away test.

The results of the study are summarized in Tables 8-11 below. Table 8 illustrates the importance of washing the samples before analysis. TABLE 8

MTBE DIE-AWAY (WASHED AND UNWASHED CULTURE)

MTBE ppm

^Washed culture was centrifuged and resuspended in BH minerals 2x before running die-way assay.

TABLE 9

MTBE DIE-AWAY (WASHED CELLS)

24 HR STORED SAMPLES:

MTBE ppm

TABLE 10 MTBE DIE-AWAY (WASHED CELLS) 48 HR STORED SAMPLES: MTBE PPM

TABLE 11

MTBE DIE-AWAY (WASHED CELLS)

72 HR STORED SAMPLES:

MTBE ppm

Therefore, the above data show that the pure culture did not show any significant deterioration in MTBE activities after 72 hours of storage.

PART J: COMPARISON OF DEGRADING ACTIVITIES IN Culture A AND ATCC 15998

The degrading activities of the Culture A of the present invention, which belongs to Rhodococcus sp . , was compared with the pure culture with ATCC No. 15998 which is a Rhodococcus rhodochrous "ruber strain".

Table 12

CULTURE MEDIUM/GROWTH CONDITIONS INCUBATION/DAYS

1. A UGA + .01% Tyrosine 30°C, 5d

2. A UGA + .1% Tyrosine 30°C, 5d

3. ATCC15998 BHNPC10 (Cerelose lOg/L) 30°C, 4d

4. ATCC15998 UGAC10 (Cerelose lOg/L) 30°C, 6d

UGA - (Universal Growth Agar)

One hundred mililiters of each of the cultures were centrifuged at 8000 rpm for 15 minutes and washed twice with sterile BH Mineral Solution. Each culture was then resuspended in 10 ml BH and then sparged 20 -30 seconds with 100% oxygen. 10 ml of the solution was then dispensed to 30 ml serium vials. The vials were sealed and about 10 ppm MTBE was added. MTBE die away test system was followed using the growth medium and conditions specified in Table 12 above. The results were analyzed with Photovac Gas Chromatography .

TABLE 13

MTBE CONDITIONS (DIE-AWAY) IN CULTURES MTBE ppm

The results of the tests propose that the pure culture Rhodococcus rhodochrous "ruber strain" ATCC No. 15998 does not show the ability to degrade MTBE as demonstrated by the present pure Culture A.

PART K: Induction of MTBE Biodegradation in Culture A

The pure culture A was grown on a medium (100 ml) containing BH minerals (BH) , lg/L ((NH₄)₂ S0₄, lg/L K₂HP0₄ and 10 g/L "Cerelose" (Trade Mark) (glucose) substrate. The culture was incubated on a shaker (200 rpm) at 30°C for 48-72 hrs and then centrifuged at 8000 rpm for 15-20 minutes. The supernatant was decanted and the collected cells were washed twice in BH by the same centrifugation decanting procedure. After the final wash, the cell pellet is resuspended in 10 ml BH (w/o cerelose) and transferred first to a 30 ml serum bottle. The 20 ml headspace was fluidized with 100% oxygen and sealed with a butyl rubber stopper. MTBE was added in consecutive spikes at 10, 20, 40, 80 & 160 ppm and the biodegradation of MTBE by the culture was followed by taking a headspace sample (10-50 microliters) and determining the amount of MTBE using the Photovac GC (Model 10S Plus) . In the induction method, doses of MTBE were added (from stock concentrated solutions of ether) in increasing concentrations after each previous dose was degraded e.g., after the 10 ppm dose degrades (e.g. 10-24 hr) then 20 ppm was added and the decline of MTBE in headspace was followed. This induction method is a method to induce the enzyme pathway in Culture A that is responsible for biodegradation of the ether.

Table 14

MTBE Biodegradation in Culture A Consecutive Spiking

Experiment

From the results of the above experiment, it can be seen that the MTBE degrading activity of the pure bacterial culture were induced to a higher level after the culture was exposed to MTBE for an extended period of time .

Claims

C L A I M S

1. A pure bacterial culture isolable from a mixed bacterial culture ATCC No. 202057 and capable of degrading methyl t-butyl ether (MTBE) to carbon dioxide.

2. A pure bacterial culture according to claim 1 which pure culture is an aerobic, gram stain positive culture.

3. A pure bacterial culture according to claim 1 or claim 2, belonging to the genus Rhodococcus.

4. A pure bacterial culture according to any one of claims 1-3, having a specific activity of from 0.1 to 100 mg MTBE/g cells/hour at 9°C.

5. A pure bacterial culture according to any one of claims 1-4, which culture is capable of aerobically degrading at least 10% of the methyl t-butyl ether (MTBE) and/or t-butyl alcohol (TBA) added to the culture at a concentration in the range 0.01 to 500 ppm, to carbon dioxide within 70 hours.

6. A process for isolating a pure bacterial culture according to any one of claims 1-5, which process comprises a) adding sterile mineral dilution medium containing

0.01-1000 mg/L of MTBE, to a sample of mixed bacterial culture ATCC No. 202057, b) subsequently removing a proportion from the resulting culture volume and replacing that proportion with a corresponding volume of fresh dilution medium containing 0.01-1000 mg/L of MTBE, to yield a dilution enrichment culture, c) repeating step b) on resulting culture until a dilute suspension of dilution enrichment culture is found to consistently degrade MTBE, d) streaking the dilution enriched culture of step c) onto sterile plates containing a mineral medium and incubating said plates, e) observing the plates for the appearance of colonies and when colonies appear, picking the colonies from the plates and inoculating the separated colonies in sealed vessels containing mineral medium and MTBE, f) incubating the separated colonies and determining the loss of MTBE from headspace of the sealed vessels, and g) selecting and isolating a separated colony which substantially completely degrades MTBE to carbon dioxide .

7. A process according to claim 6, which further comprises h) growing the isolated pure culture in a sugar- containing mineral solution and inducing enhanced MTBE and/or TBA degradation activity by adding MTBE and/or TBA to the resulting culture.

8. A process for remediating a medium containing water and a branched alkyl ether and/or a branched alkyl alcohol, which process comprises growing in the presence of said medium under aerobic conditions a pure bacterial culture according to any one of claims 1-5, or a pure bacterial culture prepared by a process according to claim 6 or 7.

9. A process according to claim 8, wherein the branched alkyl ether and/or a branched alkyl alcohol is present in the medium at a concentration in the range 0.001 to 5000 ppmw.

10. A process according to claim 8 or claim 9, wherein the medium contains MTBE and /or TBA.