Classifications

• • C—CHEMISTRY; METALLURGY
  • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F3/00—Biological treatment of water, waste water, or sewage
  • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
  • A01N25/08—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
  • A01N25/10—Macromolecular compounds
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
  • A01N37/46—N-acyl derivatives
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/795—Porphyrin- or corrin-ring-containing peptides
  • C07K14/80—Cytochromes

Definitions

• the present invention relates to a process for the treatment of liquid waste (liquid industrial or agricultural effluents or aquatic sites) loaded or polluted with tributyl phosphate (TBP), to modified bacterial strains capable of being used in said treatment process, a process for monitoring the evolution of pollution by TBP as well as a device for implementing said treatment process.
• liquid waste liquid industrial or agricultural effluents or aquatic sites
• TBP tributyl phosphate
• Tributyl phosphate is an organophosphorus compound used in many industrial fields and in particular: as a solvent in the recycling of nuclear fuels and in the purification of rare metals or in the manufacture of plasticizers, hydraulic fluids, pesticides, herbicides, anti-foaming agents, or anti-corrosion agents.
• TBP is not toxic to the human body, it is nonetheless toxic to various aquatic organisms (trout, shrimp, algae, bacteria) which can lead to an ecological imbalance in contaminated sites.
• Such a treatment allows the digestion of organic matter, and that of inorganic compounds comprising nitrogen and / or phosphorus.
• the disappearance of the inorganic phosphorus is linked to the growth of photosynthetic bacteria.
• Such a method is therefore not suitable for the treatment of TBP.
• the American Patents 6,416,993 and 6,465,240 describe a process for degrading liquid organic and inorganic waste, which comprises two stages of treatment with photosynthetic microorganisms: a first treatment with one or more photosynthetic prokaryote (s) and preferably created a consortium of photosynthetic bacteria (purple non-sulfurous bacteria: Rhodospirillum, Rhodopseudomonas, Rhodobacter, Chromatium, Rubrivivax or cyanobacteria), then a second treatment with photosynthetic algae.
• a first treatment with one or more photosynthetic prokaryote (s) and preferably created a consortium of photosynthetic bacteria (purple non-sulfurous bacteria: Rhodospirillum, Rhodopseudomonas, Rhodobacter, Chromatium, Rubrivivax or cyanobacteria) then a second treatment with photosynthetic algae.
• the process described makes it possible to treat waste containing high concentrations of total organic carbon (TOC), in demand of biochemical oxygen (BOD), nitrogen (including ammonia) and phosphorus (P, including phosphates, polyphosphates, organic phosphates) as well as other organic or inorganic substances.
• TOC total organic carbon
• BOD biochemical oxygen
• nitrogen including ammonia
• P phosphorus
• these methods are not suitable for degrading TBP, which can be present at high concentrations (of the order of 100 mg / ml); in fact, the methods described in these two patents apply to the treatment of agricultural effluents, such as liquid manure, in which there is little or no TBP.
• TBP is known to be toxic to bacteria and algae at low concentrations (Nakamura A., 1991, International Program on Chemical Safety-Environmental Health Critiria 112- Tri-n-Butyl Phosphate QV 627. World Health Organization).
• the processes of the prior art intended for the treatment of all organic and inorganic products are therefore not suitable for the treatment of wastes comprising TBP, the growth of algae and non-photosynthetic bacteria used being generally inhibited by TBP.
• TBP TBP
• the Applicant has set itself the goal of providing a method for treating liquid waste containing TBP, which better meets the needs of the practice than the methods of the prior art, in particular in that it effectively allows to degrade TBP, with a good yield and in particular, under certain conditions, a yield greater than 400 mg / 1, while being reproducible.
• the subject of the present invention is a method for treating or purifying liquid waste loaded with TBP, characterized in that it comprises the steps of: (1) bringing said liquid waste into contact with at least one strain of purple photosynthetic bacteria non-sulfurous resistant to TBP selected from the group consisting of Rhodopseudomonas palustris (Rp. palustris), Rhodospirillum rubrum (Rs. Rubrum), Rhodobacter capsulatus (Rb. Capsulatus) or Rhodobacter sphaeroides (Rb. Sphaeroides) as well as said strains of bacteria modified to overexpress cytochrome P450, under conditions allowing the degradation of TBP present in said waste, and regardless of the initial TBP concentration; and
• a carbon source either endogenous or exogenous, is necessary.
• step (1) is carried out anaerobically or micro-anaerobically.
• aerobic, micro-anaerobic (or micro-aerobic or microaerobic) and strict anaerobic as follows:
• an aerobic microorganism is a microorganism which can use oxygen as final acceptor of electrons;
• micro- micro-anaerobic or micro-aerobic a microorganism capable of developing under conditions known as micro-aerobic or micro-anaerobic is a microorganism incapable of growing when the oxygen concentrations reach those encountered in the air (20%) but which is nevertheless able to grow in the presence of low oxygen levels (2 to 10%); such microorganisms have better growth conditions in the presence of small amounts of free oxygen; in particular, they develop beneath the surface in a tube, at the level where oxygen concentrations are optimal for their growth (Microbiology. Principles and Applications, 1996, 3 rd Edition, Black JG, Prentice Hall, Upper Saddle River, New Jersey , pages 144-148).
• an anaerobic microorganism is only able to develop in the absence of molecular oxygen.
• anaerobic, applied to non-sulfurous photosynthetic bacteria is equivalent to the term microaerophilic or micro-anaerobic, more commonly used for these bacteria; a bacterium is said to be microaerophilic when it requires only a low oxygen concentration (of
• anaerobiosis (or microaerophilia or micro-anaerobiosis or micro-aerobiosis), can be obtained either in the presence of light (natural light or artificial light, such as an incandescent lamp having a emission spectrum between 350 and 1100 nm) and an energy in a range from 1 to 2000 ⁇ moles of photons / m 2 / s and in a medium having undergone a degassing cycle to obtain a residual oxygen concentration of between 5 ⁇ M and 70 ⁇ M, either in the absence of light, but in the presence of an electron acceptor commonly used for photosynthetic bacteria, such as trimethylamine-N-oxide (TMAO), nitrates and dimethyl sulfoxide (DMSO ).
• TMAO trimethylamine-N-oxide
• DMSO dimethyl sulfoxide
• the purple non-sulfurous bacteria used are preferably at the bottom of said tank (percentage of O 2 of the order of 2 to 10%).
• said strain of bacteria resistant to TBP used in step (1) of the method is preferably selected from the group consisting of Rp. Palustris and Rs. Rubrum, which have an ability to degrade TBP, greater than 400 mg / 1 and preferably greater than 426 mg / 1.
• Rp. Palustris also included in the invention are the strains devoid of endogenous plasmid; such strains are hereinafter called ⁇ pRpal strains.
• step (1) comprises the implementation of a mixture of strains of purple non-sulfur bacteria resistant to TBP comprising at least one strain selected from the group consisting of Rhodospirillum rubrum (Rs. rubrum) and Rhodopseudomonas palustris (Rp. palustris) and at least one other strain selected from the group consisting of Rhodobacter capsulatus (Rb. capsulatus) and Rhodobacter sphaeroides (Rb. sphaeroides).
• the strains of purple non-sulfur bacteria resistant to TBP are preferably selected from the Rp strains. palustris CGA009 n ° ATCC BAA-98, n ° ATCC 17002, n ° ATCC 17007, n ° DSM 8283, n ° DSM 126, n ° DSM 7375, n ° DSM 131, n ° DSM 25, n ° DSM 124 and n ° DSM 130, the strain Rs. Rubrum SI n ° ATCC 11170, the strain Rb. capsulatus Saint-Louis n ° ATCC 23782 and the strain Rb. sphaeroides 2.4.1. n ° ATCC 17023.
• the inventors have selected strains of purple non-sulfurous bacteria resistant to TBP, which make it possible to obtain high degradation yields, whatever the initial concentration of TBP and this in a process of in one step; the method according to the invention is effective and reproducible for purifying and depolluting the environment, and in particular degrading TBP in liquid waste and this due to the use, preferably in micro-anaerobic, of one or more strains pure purple non-sulfur bacteria resistant to TBP, which alone or as a mixture, make it possible to obtain a degradation of TBP at least equal to 53 mg / 1 and preferably greater than 400 mg / I of liquid effluents.
• liquid waste is meant the effluents and / or polluted aquatic sites. This liquid waste contains TBP to be degraded but may also contain other organic and inorganic compounds. They come from industry and in particular from the nuclear industry.
• strain of purple sulfur-resistant bacteria resistant to TBP is understood to mean a strain whose growth is not inhibited in the presence of TBP concentrations at least in the TBP concentration range of 12-37 ⁇ M (Nakamura, cited above) and of course also at higher concentrations of TBP.
• step (1) is carried out at a temperature between 10 ° C and 37 ° C, preferably at 30 ° C, at a pH between 5, 5 and 8.5, preferably 6.9 and for at least 15 days, preferably between 15 and 21 days.
• step (1) is preferably carried out in a settling tank.
• the incubation or contacting period must be sufficient for the bacteria strain or the mixture of bacteria strains to grow exponentially, to allow degradation of TBP with high yield.
• This incubation period is preferably between several days and several weeks, depending on the incubation temperature used; for example, an incubation period of 21 days at a temperature of 30 ° C, allows a yield greater than 400 mg / 1 to be obtained.
• the level of TBP in said liquid waste before treatment is between 0.01 mM and 1 M; this range of values is wide enough to correspond to the conditions usually encountered in the case of pollution; indeed, the surveys carried out under pollution conditions give TBP rejection values of the order of 100 mg / 1, ie 0.375 mM. Consequently, it is generally not necessary to dilute or concentrate the sample to be treated before it comes into contact with the inoculum of bacteria.
• the degradation yield is particularly high. Indeed, the method according to the invention allows the degradation of TBP concentrations of the order of 0.1 to 2 mM; no inhibition of the growth of bacteria was observed at these concentrations.
• the initial concentration of TBP is greater than 1 mM
• a complete degradation of the TBP is obtained, under the conditions of the invention.
• a pure strain or a mixture of purple non-sulfurous photosynthetic bacteria resistant to TBP should be inoculated.
• the amounts of bacteria inoculated into the medium to be treated in step (1) are advantageously between 10 4 and 10 10 bacteria per ml of medium to be treated .
• they depend on the general conditions implemented (temperature, brightness, etc.). Consequently, in general, the term "sufficient quantity" of bacteria ries to be inoculated, the seeding concentration, that is to say the initial number of bacteria allowing, by exponential growth, and under adequate conditions, to result in a biomass sufficient to observe a degradation of TBP.
• strains of photosynthetic bacteria resistant to TBP have the advantage of providing good yields of biodegradation of TBP even after being subjected to a subsequent treatment (large number of subcultures, for example) .
• an additional carbon source is added to the medium to be treated;
• this additional carbon source is advantageously a buffered solution containing a yeast extract at a concentration of between 0.1 and 10 g / 1 (preferably 1 g / 1), or one of the following organic salts: succinate, glutamate , benzoate, malate or fumarate at a concentration between 2 and 20 mM (preferably at 10 mM), and growth factors including at least biotin and para amino benzoic acid at a concentration between 2 and 40 ⁇ g / 1 each.
• the purple non-sulfur bacteria resistant to TBP are selected by culture on a medium containing at least 12 ⁇ M and preferably at least 1 mM TBP, then are cultured according to methods known to those skilled in the art such as, for example, those described in Bergey's manual of systematic bacteriology; Williams & Wilkins Edition or in "The photosynthetic bacteria” R.K. Clayton and W. R. Sistrom; Plenum Press.
• the method is advantageously implemented in non-aerated lagooning.
• Non-aerated lagoon is understood to mean a device comprising at least a settling tank with a depth ranging from 0.5 to 1 meter for a surface in agreement with the flow of effluents, knowing that the retention time must be at least 15-21 days under optimal temperature and lighting conditions.
• the subsequent separation of bacteria and the treated medium is favored by the conditions implemented (micro-anaerobic, settling tank).
• the bacterial growth has reached a significant value, and before releasing the purified effluents into the environment, it may be useful to recover the bacteria by filtration (using a diatom filter for example) or by continuous centrifugation before them feed back into the system.
• the present invention also relates to a process for monitoring or monitoring the degradation of TBP in liquid waste, which process comprises:
• the step of measuring the residual TBP can be carried out by any technique known in analytical chemistry commonly used, and more particularly in the field of organophosphorus compounds. For a reliable and precise measurement, a chromatographic technique is generally used, and more precisely high performance liquid chromatography coupled with a refractometer. The whole process can be automated.
• the present invention also relates to a kit for implementing the treatment method according to the invention, characterized in that it comprises at least one strain of non-sulfurous purple photosynthetic bacteria resistant to TBP, chosen from the group consisting by Rp. palustris, Rs. rubrum, Rb. capsulatus or Rb. sphaeroides as well as said strains of bacteria modified to overexpress cytochrome P450, as defined above.
• the mixture of strains preferably comprises at least the strain Rp. Palustris or the strain Rs. Rubrum, as specified above.
• the strains devoid of endogenous plasmid such strains are hereinafter called ⁇ pRpal strains.
• the present invention also relates to strains of non-sulfurous purple photosynthetic bacteria, characterized in that they are resistant to TBP, in that they are selected from the group consisting of Rp. palustris, Rs. rubrum, Rb. capsulatus or Rb. sphaeroides and in that their DNA includes at least one additional copy of the gene encoding a homologous cytochrome P450.
• Such strains comprising at least one additional copy of a homologous gene coding for a cytochrome P450, allow overexpression of said cytochrome P450.
• strains consist of a strain of Rp. Palustris CGA009 comprising a gene coding for the cytochrome P450 of Rp. Palustris, under the control of the promoter LH ⁇ e , described in Tadros MH et al. (10) (strain Rp. Palustris LH5939).
• the gene coding for the cytochrome P450 of Rp. Palustris is described in particular in the database ⁇ Oak Ridge National Laboratory (ORNL) under the gene number 5939 (site: http: // genome .ornl. Gov) or under the Genbank access number NZ_AAAF01000001.1.
• the present invention also relates to a device for purifying liquid waste loaded with TBP by implementing the degradation process as described above, characterized in that it comprises at least one settling tank suitable for placing in contact with the non-sulfurous purple bacteria with the medium to be treated, preferably in micro-anaerobiosis.
• a device notably implements the non-aerated lagooning technique, which consists in passing the polluted effluents, loaded with TBP in a series of basins in the open air, the first of which, called settling tank, is specifically seeded in the context of the invention by non-sulfurous purple photosynthetic bacteria resistant to TBP.
• micro-anaerobiosis takes place at the bottom of the head basin where the sludge is deposited.
• Said settling tank is preferably of a depth ranging from 0.5 to 1 meter for a surface in agreement with the flow rate of the effluents, knowing that the retention time must be at least 15-21 days under the conditions optimal temperature and lighting as defined above.
• said device comprises, in addition to said settling tank, means for introducing said liquid waste containing TBP into said settling tank, means for introducing pinoculum of bacteria, means for discharging effluents purified in TBP to the outside as well as means for recovering bacteria to be recycled.
• said means for recovering bacteria is advantageously selected from the group consisting of diatom filters and continuous centrifuges.
• FIG. 1 represents the consumption of TBP (expressed as a percentage) aerobically (hatched bars) and anaerobic (bars with dotted lines) for each of the non-sulfurous purple photosynthetic bacteria strains tested (Rb. Capsulatus Saint-Louis (ATCC 23782), Rb . sphaeroides 2.4.1 (ATCC 17023), Rs. rubrum Sl (ATCC 11170), and Rp. palustris CGA009 (ATCC BAA-98)).
• FIG. 2 represents the growth of the strain Rp. palustris CGA009 (ATCC BAA-98) under aerobic (A), photosynthetic (micro-anaerobic) (B) and anaerobic (C) conditions in Hutner's culture medium, in presence (solid line, (A.)) and absence (broken line, ( ⁇ )) of 2 mM TBP during bacterial growth.
• A aerobic
• B photosynthetic
• C anaerobic
• the curve can be transcribed by the function aj.e ⁇ n2ln + a2.
• the scales of figure 2B are identical to those of figure 2A.
• - figures 3A and 3B represent respectively the growth of the strain Rp.
• palustris CGA009 ATCC BAA-98 under photosynthetic conditions (micro-anaerobiosis) in the Hutner culture medium in the presence of different concentrations of TBP, and a kinetic study of the consumption of TBP for the strain Rp. palustris CGA009 (ATCC BAA-98) under the same conditions
• the scales in Figure 3A are identical to those in Figure 2.
• the scales in Figure 3B correspond to the initial TBP concentration expressed in mM.
• FIG. 4A shows the growth of Serratia odorifera sp. in aerobic Tsb medium, in the presence (circles) and in the absence of carbon monoxide in the culture medium (squares);
• FIG. 4B represents the growth of the strain Rp. palustris CGA009 (ATCC BAA-98) expressed in optical density units at 660 nm under photosynthetic conditions in the Hutner culture medium in the presence (circles) and in the absence of carbon monoxide in the culture medium (squares).
• FIG. 5 represents the consumption of TBP expressed as a percentage of residual TBP by the Serratia odorifera sp. and Rp. palustris CGA009 (ATCC BAA-98) after 24 hours and 3 weeks respectively, in the absence (dotted bars) and in the presence (hatched bars) of carbon monoxide in the culture medium;
• FIG. 6 represents the plasmid pCB07 comprising the gene coding for the cytochrome P450 of Rp. palustris n ° 5939 and the promoter LH ⁇ e .
• TBP in mM
• the samples studied are total proteins of: (1): strain CGA009 not induced by TBP in the presence of O 2 ; (2): strain CGA009 not induced by TBP in the absence of O 2 ; (3) strain of CGA009 induced by TBP in the presence of O and (4): strain ⁇ pRpal.
• the controls are: (5): protein-free reaction buffer; (6) raw boiled extract of Rp. Palustris CGA009 and (7): crude extract of E. coli DH5 ⁇ . Values are means ⁇ standard deviations, from three independent experiments.
• FIG. 8 represents (A): the kinetics of consumption of TBP, by cellular extracts at 40 ° C. in the presence of different initial concentrations of TBP (in mM); TBP concentrations are as follows: 0.05 mM (-), 0.1 mM (*), 0.25 mM (+), 0.5 mM (x), 0.75 mM (•), 1 mM ( ⁇ ), 1.5 mM (*) and 2 mM (•). Values are means ⁇ standard deviations, from three independent experiments.
• FIG. 9 represents the degradation of TBP by cell extracts, dialyzed or not, over a period of 20 hours at 40 ° C., the initial concentration of TBP being 2 mM.
• the cofactors are added to a final concentration of 10 ⁇ M. Values are means + standard deviations, from three independent experiments.
• FIG. 10 illustrates the amount of degraded TBP (from an initial concentration of 2 mM) over a period of 20 hours at 40 ° C., by different fractions of cell extracts: (1): crude extract; (2) soluble fraction; (3) protein fraction weakly associated with the membrane and (4) protein fraction linked to the membrane. The values depend on the weight of protein present in each case and are averages + standard deviations, from five independent experiments.
• Example 1 Comparison of the degradation yield of TBP aerobically and anaerobically (micro-anaerobically or microaerophilically) by various purple non-sulfurous photosynthetic bacteria.
• the concentration of residual TBP in aerobic and microanaerobic conditions is measured after 3 weeks of culture with the following photosynthetic bacteria: Rb. capsulatus Saint-Louis (ATCC 23782), Rb. sphaeroides 2.4.1 (ATCC 17023), Rs. rubrum S.l. (ATCC 11170) and Rp. Palustris CGA009 (ATCC BAA-98).
• the photosynthetic bacteria are incubated in a Hutner medium [6] in which the pH is between 6.5 and 7.5, in the presence of 2 mM TBP, either aerobically or micro-anaerobically. Aerobically, the bacteria are incubated in the dark at a temperature of 30 ° C; the culture medium being subjected to stirring (150 rpm). In micro-anaerobiosis, bacteria are incubated in the light
• Micro-anaerobiosis is obtained by degassing the medium before inoculation by placing the vial under vacuum using a vacuum pump, to reach a low oxygen tension of the order of 70 ⁇ M.
• the media are sterilized by autoclaving at 120 ° C for 15 minutes.
• the TBP is added, after sterilization, to a final concentration of 2 mM, which is below the saturation point of the solubility product of TBP in water which is less than 1 g / 1 (3.7 mM) at 25 ° C. Growth is monitored with an optical density spectrophotometer
• the culture is centrifuged at 5000 g for 10 minutes to separate the bacteria from the culture medium. The supernatant is then recovered on which the concentration of residual TBP is determined. An equal volume of dichloroethane and tripropyl phosphate (TPP) is added to the aqueous supernatant. Solvent extracts TBP and other hydrophobic organic compounds, and TPP is used as an internal standard to quantify extraction efficiency and to calibrate results in subsequent analyzes by high performance liquid chromatography (HPLC or HPLC for High Performance Liquid Chromatography). The samples are mixed by vortexing for 1 minute and then left to stand for 30 minutes.
• HPLC or HPLC for High Performance Liquid Chromatography high performance liquid chromatography
• the organic phase is then filtered (lower phase) on a phase separator (IPS, Whatman) followed by evaporation overnight at 30 ° C, or alternatively, the process is carried out a light stream of nitrogen at room temperature for 15 minutes.
• the samples are diluted in dodecane for further analysis by HPLC.
• Measurements are then carried out by HPLC analyzes using an amine Brownlee Spheri-5 column (Perkin Elmer) integrated into a HPLC model Waters 1525. Elution is carried out using a mixture of heptane and ethyl acetate (75 / 25, vol / vol) with a flow rate of 1 ml / min as described in European patent 0 578 579.
• the TBP is measured using a Waters 2414 type refractometer and the chromatography profiles are analyzed using Breeze software ( marketed by the company Waters).
• the TBP concentration is defined by the ratio between the peak areas of the TBP and TPP compounds.
• TBP and TPP compounds the purity of which is estimated to be greater than 99%, as well as all the previously mentioned chemicals which are used to extract the TBP and during the HPLC analysis are supplied by Sigma-Aldrich.
• Example 2 Growth of the strain Rp. Palustris CGA009 and kinetics of degradation of TBP by the same strain either aerobically, or micro-anaerobically, or anaerobically.
• Example 1 Due to the results obtained in Example 1, the strain Rp. Palustris was more particularly studied.
• Palustris was cultured in Hutner's medium (6) or in PM medium: 12.5 mM Na 2 HPO 4 ; 12.5 mM KH 2 PO 4 ; 7.5 mM (NH 4 ) 2 SO 4 ; 0.1 mM Na 2 S 2 O 3 , 5H 2 O 14.5 mM aminobenzoic acid; 0.85 ⁇ M EDTA; 3.8 nM ZnSO 4 ; 7H 2 O; 2.5 nM FeSO 4 ; 7H 2 O, 0.9 nM MnSO 4 ; 7H 2 O, 0.16 ⁇ M CuSO 4 ; 7H 2 O, 86 nM Co (NO 3 ) 2 ; 6 H 2 O, 46 nM Na 2 B 4 O 7 , 10 H 2 O; 0.1 mM nitrilotriacetic acid; 0.24 mM MgSO 4 anhydrous; 45 ⁇ M CaCl 2 ; 15 nM (NH) 6 Mo 7 O 24 ; 4 H 2 O.
• the media were sterilized by autoclaving at 120 ° C for 15 minutes.
• a source of carbon sterile succinic acid
• Na 2 CO 3 only under photosynthetic conditions
• TBP When TBP was required, it was added directly to the culture medium, after sterilization, to a final concentration of 2 mM (the solubility of TBP in water is approximately 2.5 mM).
• the culture conditions were either those of aerobiosis (30 ° C., darkness, with stirring at 300 rpm), or photosyn-
• the anaerobiosis was obtained by subjecting the medium to 5 degassing cycles followed by a step of bubbling with argon sufficient to obtain an oxygen concentration of less than 5 ⁇ M.
• TBP Triphosphylphosphate
• HPLC high performance liquid chromatography
• the HPLC analyzes were carried out using a Spheri-5 amino column (Perkin Elmer) connected to an HPLC of Waters 1525 model, as specified in Example 1.
• a mixture of heptane and acetate d ethyl 75/25, v / v was used at a flow rate of 1 ml.min "1 as described in European Patent 0 578 579.
• TBP and TPP were detected by refractometry (Waters Refractometer 2414) and the refraction chromatography profiles were analyzed using the Waters Breeze software.
• the TBP concentration was estimated from the ratio between the areas of the TBP and TPP peaks. Under these conditions, the detection threshold for TBP was about 5 ⁇ M (see also example 1).
• the crude extracts and fractionated proteins were used for the degradation studies of TBP.
• sterile buffer containing 2 mM TBP, boiled crude extracts (10 minutes at 95 ° C) and crude extracts of E. coli DH5 ⁇ were tested to estimate the abiotic disappearance of TBP.
• the crude extracts were dialyzed with the Ultrafree Biomax 10,000 D system (Millipore) so as to lower the concentration of small molecules.
• FADH 2 and FMNH 2 were obtained by reduction of a solution of FMN and F AD with a small excess of sodium dithionite. All the cofactors used were added to the cell extracts at a final concentration of 10 ⁇ M.
• the assays were carried out by testing the degradation of TBP by the proteins in 1 ml of 400 mM Tris-HCl pH 6.5 at 40 ° C. The optimum temperature and pH have been determined. Residual TBP was assayed by HPLC.
• TBP TBP (2 M) or in the absence of TBP.
• Figure 2A aerobically
• Figure 2B micro-anaerobic
• the growth of the strain begins after a delay, it is shorter in aerobic, then the growth kinetics accelerates to reach that of witness (absence of TBP).
• the kinetics of degradation is monophasic; it can result in an exponential curve with a tj / of 1.1 days (28% amplitude).
• the initial consumption rate of TBP is 13 ⁇ mol / l / h ( Figure 2A).
• the growth kinetics is biphasic, it can result in a series of two exponential curves with a t ⁇ / 2 of 0.94 day (23% amplitude) and a tj / of 13.5 days ( 77% amplitude).
• the initial consumption rate of TBP is 15 ⁇ mol / l / h ( Figure 2B).
• Example 3 Effect of different concentrations of TBP on the growth of the strain Rp. Palustris CGA009 and the kinetics of degradation of TBP by the same strain in micro-anaerobiosis.
• the culture and analysis conditions for TBP are identical to those used in Example 2.
• the results are illustrated in FIG. 3.
• the strain Rp. Palustris CGA009 is cultured in the presence of various concentrations of TBP: 2; 1.5; 1, 0.5 and 0.1 mM in micro-anaerobiosis. According to these results, there is a shortening of the growth time when the concentration of TBP is reduced.
• the growth of the strain is similar to that observed in the medium devoid of TBP (FIG. 3A).
• TBP degradation yields improve when the TBP concentration is less than 1 mM; TBP is then almost entirely degraded in 21 days (Figure 3B).
• the presence of CO in the medium does not affect the growth of each of the two strains when the TBP is absent from the medium, and also for the strain S. odorifera when it is put in the presence of TBP.
• the growth of the strain Rp. Palustris is greatly reduced in the presence of CO ( Figure 4B).
• the growth of R. palustris is actually reduced by the inhibition of cytochrome P450 when the TBP is present.
• the assay of the concentration of residual TBP after 21 days of culture shows a significant decrease in the degradation of TBP ( Figure 5). This suggests the existence of a correlation between the inhibition of cytochrome P450 and the low degradation yield of TBP. This correlation does not exist with the S. odorifera strain for which CO treatment does not significantly modify the yield of degradation in TBP.
• Example 5 Construction of a strain of Rp. Palustris overexpressing cytochrome P450 in a constitutive manner.
• a transcriptional fusion is carried out of the promoter of the gene cluster of the light recovery complex II (LH) ⁇ e and of the gene 5939 so as to obtain a constitutive expression of the gene in question.
• the promoter LH ⁇ e is amplified by PCR from 300 bp upstream of the starting codon to 68 bp downstream of it using the genomic DNA of Rp.
• the PCR amplified fragment is cloned into the cloning vector pGEM-T TM (Proméga) to give the plasmid pCB04.
• the entire 5939 gene is then amplified using the following primers: 5'-CCCAAGCTTGGGTGAACAACAACGAGGGAGTG-3 '(SEQ ID NO:
• the PCR amplified fragment is digested with Hindlll and S ⁇ cl and ligated into the plasmid pBBRlMCS2 (Kovach et al, 1994) at the same sites, which makes it possible to obtain the plasmid pCB06.
• the plasmid pCB04 is digested with Kpnl and Hindlll and cloned into the plasmid pCB06 digested with these same restriction enzymes, to obtain the plasmid pCB07.
• the map of plasmid pCB07 is shown in Figure 6.
• the plasmid pCB07 containing the gene 5939 under the control of the LH ⁇ e promoter is transferred by tri-parental conjugation from the E. coli OY15aphe strains (Eraso, et al. 1994) and HB101 (Boyer et al., 1969) in the strain Rp. Palustris CG009 to obtain the strain Rp. Palustris H5939 overexpressing cytochrome P450 (positive clones). These positive clones are selected in a medium containing kanamycin at a concentration of between 20 and 100 ⁇ g / ml.
• Example 6 Comparative study of the degradation of TBP by different cell extracts.
• Centrifugation for 90 minutes at 200,000 g at 4 ° C was carried out to sediment the membranes.
• the supernatant made up the fraction of soluble proteins.
• the pellet was resuspended in 2 ml of 2M NaBr buffer, 200 mM sucrose, 50 mM glycylglycine, at pH 6 and added with a cocktail of protease inhibitors, then extracted by gentle stirring for 30 minutes at 4 ° C; the supernatant constituted the fraction enriched in proteins weakly associated with the membrane.
• the pellet containing the proteins strongly associated with the membrane, was resuspended in 2 ml of 50 mM Tris-HCl buffer pH 8, lauryl diaminooxide (LDAO) 0.5% (vol / lvol) and added with a cocktail of inhibitors proteases.
• the proteins were dissolved by gentle agitation for 60 minutes at 4 ° C.
• the resulting extract was centrifuged for 90 minutes at 200,000 g at 4 ° C to pellet the insoluble materials. The supernatant constitutes the fraction of membrane proteins.
• the protein concentration in each extract was determined by the biuret method according to the BCA Protein Assay Reagent kit (Uptima) using bovine serum albumin as standard.
• TBP degradation tests were carried out with proteins extracted from Rp. Palustris CGA009.
• the optimal test conditions are as follows: pH 6.5 and 40 ° C.
• the cell extracts are obtained under the conditions set out above in the chapter Materials and methods; they are in particular obtained by extraction of the proteins from the strain Rp. palustris cultivated under micro-anaerobic conditions, in Hutner's medium in the presence (cultures induced) or in the absence (cultures not induced) of 2 mM from TBP.
• Figure 7 shows that 70% of TBP is degraded by cellular extracts at 40 ° C under aerobic conditions for 20 hours, independently of the possible presence of TBP in the culture medium (Figure 7: (1) and (3) ). An experiment was also carried out under anaerobic conditions. In this case, no degradation is observed (Figure 7: (2)); this implies that oxygen is necessary to degrade TBP, and this, as for whole cells.
• TBP degradation tests were compared with the results obtained with various controls: protein-free reaction buffer, boiled crude extract of Rp. Palustris CGA009 and crude extract of E. coli DH5 ⁇ ( Figure 7: (5), (6) and (7)).
• the initial optimal O 2 concentration is approximately 70 ⁇ M, in the selected micro-anaerobic medium.
• the degradation of TBP is a constitutive mechanism of the cell, insofar as the cellular extracts of bacteria cultured in the presence or in the absence of TBP have the same effectiveness; in addition, a subculture obtained from a strain previously cultivated in a medium containing TBP has the same profile as a subculture obtained from a non-induced culture (growth in a medium in l absence of TBP): the degradation kinetics are identical.
• Example 7 Comparative study of the kinetics of degradation of TBP in the presence of different concentrations of TBP and by different cell extracts.
• FIG. 8A shows the kinetics of degradation of TBP by cell extracts as a function of different concentrations of TBP: 2, 1.5, 1, 0.75, 0.5, 0.25, 0.1 and 0.05 mM .
• FIG. 8B shows the best correspondence of the Michaelis-Menten equation with values of Vm ap and Km ap of 0.48 + 0.04 mM.h-1 and 2.6 + 0.4 mM respectively.
• the analysis of the degradation kinetics carried out using cell extracts is consistent with the Michaelis-Menten equation. This makes it possible to estimate the Km ap and the Vm ap , although the maximum speed cannot be reached under the experimental conditions (low solubility of TBP in water).
• a Km ap of 2.6 mM indicates a moderate apparent affinity of the enzymatic system for TBP.
• the Vm ap was 0.48 mM.li '1 .
• Example 8 Influence of cofactors on the degradation of TBP by cell extracts.
• a crude extract obtained under the conditions described in Example 6 is dialyzed so as to reduce the concentration of low molecular weight molecules.
• the degradation of TBP is reduced by a factor of 3, in comparison with the non-dialysis control ( Figure 9, (1) and (2)).
• the effect of different cofactors was tested: NADH, NADPH, FMNH 2 and FADH 2 .
• the addition of these cofactors does not significantly improve the degradation of TBP.
• the degradation by dialysis samples is increased by the addition of NADPH or FMNH 2 (FIG. 9, (4) and (6)). The combination of these two cofactors completely restores the degradation activity observed with the non-dialyzed samples ( Figure 9, (11)).
• EXAMPLE 9 Degradation of TBP by Protein Fractions To be able to localize the enzymatic activity involved in the degradation of TBP by Rp. Palustris, a crude extract is fractionated into three protein fractions: soluble fraction, fraction weakly associated with the membrane and membrane-bound fraction; these different fractions are obtained under the conditions set out in Example 6. The degradation tests for TBP are carried out under the conditions set out in Example 2.
• FIG. 10 shows that the degradation activity is more particularly localized in the fraction of proteins weakly associated with the membrane, with 3.5 ⁇ M of degraded TBP.mg of protein "1 , compared to the amounts degraded respectively by the total protein fraction (1.2 ⁇ M of degraded TBP.mg of protein " 1 ), the soluble fraction (2 ⁇ M of TBP degraded.mg of protein "1 ) and the membrane-bound fraction (1.65 ⁇ M of TBP degraded.mg of protein " 1 ).
• the proteins involved in the degradation of TBP are weakly associated with the membrane. The activity is dependent on both NADPH and FMNH 2 and confirms the role of cytochrome P450 in the degradation activity of TBP.
• the isolation of the plasmids was carried out using the NucleoSpin Plasmid kit (Macherey-Nagel) following the manufacturer's instructions.
• the isolation of genomic DNA was carried out using the NucleoSpin Tissue kit (Macherey-Nagel) following the manufacturer's instructions.
• the plasmids were obtained by tri-parental crossing from E. strains. coli DH5 ⁇ phe (donor), containing the plasmid pMG105, and HB101 (helper) to the strain Rp. palustris (acceptor).
• the cells of E. coli were grown in LB + Km medium until the DO 660 value reached 1.5, and Rp. palustris cells were grown in Hutner's medium until the DO 660 value reaches 1 (overnight cultures).
• the "donor, helper and acceptor” bacteria 100 ⁇ l, 10 ⁇ l and 1 ml respectively) were washed and pooled on a box of Hutner medium.
• This dish was incubated for 6 hours at 30 ° C.
• the bacteria were resuspended in 1 ml of PM medium, spread on PM + Km dishes and incubated for 4 days, either under aerobic conditions or under micro-aerobic conditions. Plasmid elimination experiments
• the plasmid pMG105 (11), a cloning vector exhibiting a resistance gene to Km, which has the same origin of replication as pRpal was introduced by crossing in R. palustris.
• the transconjugants carrying pMG105 were selected on PM + Km boxes. Generally, resistant Km bacteria have lost pRpal.
• the strains carrying pMG105 were cultivated on a medium without Km to expel this plasmid after several subcultures and the resulting strain without plasmid is preserved as Rp. Palustris ⁇ pRpal. Each step of the elimination experiment is monitored by isolation of the plasmid.
• R. palustris ⁇ pRpal bacteria are tested for their ability to degrade TBP.
• Rp. Palustris comprises an endogenous plasmid (pRpal, 9.8 kb). This characteristic was confirmed by the sequencing of the genome of Rp. Palustris (12).
• the growth, the morphological form as well as the pigmentation of the colonies are identical in the ⁇ pRpal strain and in the wild strain.
• Palustris is naturally resistant to gentamycin (50 ⁇ g.ml "1 ) and the plasmid-free strain believes in the presence of this antibiotic, like the wild strain.
• the plasmid-free strain also grows in a medium containing TBP, like the wild strain, and has a degradation kinetics of TBP similar to that of the wild strain. This indicates that the enzymes involved in the degradation of TBP by Rp.palustris are not encoded by genes carried by the endogenous plasmid but rather by chromosomal DNA.
• palustris is effective in the depollution process, as is the plasmid-free strain, which may also be a tool for determining the plasmid or chromosomal location of genes involved in other pathways of degradation.

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