The invention relates to a process for producing microorganisms, Burkholderia cepacia (ex Pseudomonas cepacia), CIP I-2502, to the inoculum produced, and to its application in a process for degrading the alcohols produced, for example during degradation of ethers known as ether fuels when they are contained in aqueous effluents. The ethers are as follows: ethyl tert-butyl ether, hereinafter termed ETBE, methyl tert-butyl ether, hereinafter termed MTBE, and tert-amyl methyl ether, hereinafter termed TAME. The alcohols produced during their degradation are tert-butanol, hereinafter termed TBA or tert-amyl alcohol, hereinafter termed TAA.
Its particular industrial application is to water treatment.
The prior art is illustrated by the document: xe2x80x9cTransformation of carbon tetrachloride by Pseudomonas sp; strain KC under denitrification conditionsxe2x80x9d, Appl. Environ. Microbiol., vol 56, No. 11, Nov, 1990 (1990-11), p. 3240-3246, and by European patent EP-A-0 237 002 and French patents FR-A-2 735 497 and FR-A-2 766 478.
TBA and TAA are degradation products of the ethers MTBE and ETBE and of TAME respectively. Such ethers are currently used or can be used as additives to gasoline for which they increase the octane number. They are widely used since, for example, MTBE is added to gasoline in an amount of 10% to 15% (v/v). For this reason, the presence of such ethers in aquifers is being noted increasingly frequently (Andrews C., 1998, xe2x80x9cMTBExe2x80x94A Long Term Threat to Ground Water qualityxe2x80x9d, Ground Water, 36: 705-706)). Partial biodegradation of such ether fuels in aquifers due, for example, to limited available oxygen, can thus result in an accumulation of TBA and TAA as secondary contaminants.
Further, TBA and TAA can themselves be used as additives to gasoline to increase their octane number and in that case, their use may involve dispersing them in the environment as primary contaminants.
The presence of TBA or TAA in aquifer water for supplying drinking water or in residual water arriving at purification stations necessitates the use of specialised microorganisms which can degrade such alcohols, which latter are relatively resistant to biodegradation because of their branched structure.
According to United States patent U.S. Pat. No. 4,855,051, bacteria isolated from the earth, B. coagulans ATCC 53595, A. globiformis ATCC 53596 and P. stutzeri ATCC 53602, can degrade TBA added to a minimum mineral medium. According to U.S. Pat. No. 5,811,010, bacterial consortia are also capable of degrading TBA but the microorganisms constituting it have not yet been characterized. Further, in U.S. Pat. No. 5,814,514, different bacteria which are capable of growing on propane are capable of degrading TBA.
The Applicant has isolated an anerobic bacteria, Burkholderia cepacia CIP I-2052, for its ability to use TBA as a source of carbon and energy by degrading it to carbon dioxide (mineralisation). This bacteria was deposited by the Applicant as deposit number 1-2052 on Jul. 20, 1998 at the Institut Pasteur collection (CNCM [Collection Nationale de Cultures de Microorganisms, National Collection for Microorganism Cultures], 25 rue du Docteur-Roux, F-75724, Paris). This bacterium, according to the Applicant""s French patent application FR-A-2 766 478, can be used in mixed culture with bacteria that are capable of using ETBE as a carbon and energy source by degrading it to the TBA stage which then accumulates in the culture medium; for this reason, addition of an inoculum of Burkholderia cepacia CIP I-2052 with the ability of acting on the TBA produced, can produce total degradation of ETBE in effluents. In general, the incubation period for these microorganisms on the substrate is long; for example 400 h are required to degrade about 700 mg/l of TBA, and on the industrial scale, it turns out to be vital that a sufficient quantity of biomass is produced.
The aim of the present invention is to describe a modification to the Burkholderia cepacia CIP I-2052 bacteria culture which can very easily improve its growth in the presence of TBA or TAA provided as the only source of carbon and energy by adding a cobalt salt, used alone or as a mixture, to the culture medium.
More precisely, the invention concerns a process for producing a Burkholderia cepacia CIP 1-2052 bacterium in which said bacterium is cultured in the presence of air or oxygen and in the presence of a medium containing at least one source of nitrogen, tert-butanol (TBA) and/or tert-amyl alcohol (TAA), and an inoculum is recovered, characterized in that said medium contains at least one cobalt salt. The cobalt is preferably used in its divalent form.
In one embodiment of the process, the cobalt salt can be selected from the group formed by the chloride ion, the sulphate ion, the nitrate ion or a mixture thereof. It has been observed that hexahydrated cobalt chloride has a large effect on the biomass produced by the bacteria.
In a further embodiment of the invention, the bacterium Burkholderia cepacia CIP I-2052 can be seeded onto a saline vitamin-containing culture medium to which cobalt chloride has been added, used alone or as a mixture with other oligo-elements, in a final concentration in the medium of 0.01 to 4 mg/l, advantageously 0.03 to 2 mg/l and preferably 0.05 to 0.1 mg/l. Under these conditions, the carbon-containing substrates of Burkholderia cepacia CIP I-2052, i.e., TBA or TAA, can be added in to a concentration in the range 0.001 to 10 g/l of medium, preferably in the range 0.2 to 5 g/l.
Under these conditions, an inoculum with a higher cell density than that previously observed is produced wherein the final biomass concentration is, for example, of the order of 0.6 g dry weight/l of culture for an initial TBA concentration of 1 g/l. The inoculum thus prepared can be used alone to treat effluents containing TBA or TAA or it could also be used in a mixed culture with bacteria degrading ETBE or MTBE to TBA or TAME to TAA.
This inoculum produced in large quantities by the process of the invention can also be used directly to purify aqueous effluents containing TBA or TAA or the mixture of TBA and TAA. More precisely, the invention concerns a process for degrading TBA and/or TAA contained in aqueous effluents, in which an inoculum produced by the process for producing a Burkholderia cepacia CIP I-2052 bacterium is employed under aerobic conditions.
According to the Applicant""s French patent application FR-98/16520, the TBA can originate from degradation of MTBE or ETBE contained in aqueous effluents under aerobic conditions, by at least one bacterium which can be Gordona terrae CIP I-1889 or Rhodococcus equi CIP-2053. Gordona terrae CIP I-1889 was deposited under the terms of the Budapest Treaty by the Applicant as IFP-2001 on Jun. 25, 1997 and Rhodococcus equi CIP-2053 was deposited under the terms of the Budapest Treaty by the Applicant as IFP-2005 on Jul. 20, 1998. Both deposits were made at the Institut Pasteur collection (CNCM [Collection Nationale de Cultures Microorganisms, National Collection for Microorganism Cultures], 25 rue du Docteur-Roux, F-75724, Paris). These same bacteria can also degrade TAME or TAA under aerobic conditions.