EP1638897A2 - Method of degrading tbp using a photosynthetic bacterial strain - Google Patents
Method of degrading tbp using a photosynthetic bacterial strainInfo
- Publication number
- EP1638897A2 EP1638897A2 EP04742718A EP04742718A EP1638897A2 EP 1638897 A2 EP1638897 A2 EP 1638897A2 EP 04742718 A EP04742718 A EP 04742718A EP 04742718 A EP04742718 A EP 04742718A EP 1638897 A2 EP1638897 A2 EP 1638897A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- tbp
- bacteria
- strain
- palustris
- degradation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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- RXPQRKFMDQNODS-UHFFFAOYSA-N tripropyl phosphate Chemical compound CCCOP(=O)(OCCC)OCCC RXPQRKFMDQNODS-UHFFFAOYSA-N 0.000 description 1
- 239000007102 tryptic soy broth medium Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
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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Abstract
The invention relates to: a method of treating liquid waste (liquid agricultural or industrial effluents or aquatic sites) which is loaded or polluted with tributyl phosphate (TBP), modified bacterial strains which can be used in the aforementioned treatment method, a method for monitoring changes in TBP pollution, and the device which is used to perform said treatment method. According to the invention, the liquid waste-treatment or -purification method essentially comprises: steps (1) consisting in bringing said liquid waste into contact with at least one non-sulphur purple photosynthetic bacterial strain which is resistant to TBP and which is selected from the group containing Rhodopseudomonas palustris (Rp. palustris), Rhodospirillum rubrum (Rs. rubrum), Rhodobacter capsulatus (Rb. capsulatus) or Rhodobacter sphaeroides (Rb. Sphaeroides) as well as the aforementioned modified bacterial strains in order to overexpress cytochrome P450 in conditions that enable the degradation of the TBP present in said waste, regardless of the initial TBP concentration; and (2) the recovery of the purified liquid effluents.
Description
PROCÉDÉ DEDÉGRADATIONDU TBP PARUNE SOUCHEBACTÉRIENNE PHOTOSYNTHÉTIQUE METHOD FOR DEGRADATION OF TBP BY A PHOTOSYNTHETIC BACTERIAL STRAIN
La présente invention est relative à un procédé de traitement de déchets liquides (effluents industriels ou agricoles liquides ou sites aquatiques) chargés ou pollués en phosphate de tributyle (TBP), à des souches bactériennes modifiées susceptibles d'être utilisées dans ledit procédé de traitement, à un procédé de surveillance de l'évolution de la pollution par le TBP ainsi qu'à un dispositif de mise en œuvre dudit procédé de traitement.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.
Le phosphate de tributyle (TBP) est un composé organophosphoré utilisé dans de nombreux domaines industriels et en particulier : comme solvant dans le recyclage des combustibles nucléaires et dans la purification des métaux rares ou dans la fabrication de plastifiants, de fluides hydrauliques, de pesticides, d'herbicides, d'agents anti-mousse ou d'agents anti-corrosion.Tributyl phosphate (TBP) 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.
Toutes ces applications génèrent des quantités importantes de déchets, qui sont très peu biodégradables, car le TBP est peu ou pas dégradé en milieu naturel : il est peu dégradé par les microorganismes indigènes et il est très peu sensible à la photolyse ou à l'hydrolyse naturelle.All of these applications generate significant quantities of waste, which are very little biodegradable, because TBP is little or not degraded in the natural environment: it is little degraded by indigenous microorganisms and it is very little sensitive to photolysis or hydrolysis. natural.
Bien que le TBP ne soit pas toxique pour l'organisme humain, il est néanmoins toxique envers divers organismes aquatiques (truites, crevettes, algues, bactéries) ce qui peut conduire à un déséquilibre écologique au niveau des sites contaminés.Although 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.
Il existe des documents qui décrivent des méthodes générales de dégradation de composés organiques dans des effluents liquides :There are documents which describe general methods of degradation of organic compounds in liquid effluents:
- le Brevet américain 6,472,198 décrit la dégradation de composés organiques chlorés, à l'aide de bactéries indigènes notamment de type méthanogène, acétogène, ou responsables de la deshalogénation ou de la dénitrification, ces bactéries fonctionnent soit en aérobiose, soit en anaérobiose. Un tel procédé n'est pas adapté à la dégradation du TBP.- American Patent 6,472,198 describes the degradation of chlorinated organic compounds, using indigenous bacteria in particular of the methanogenic, acetogenic type, or responsible for dehalogenation or denitrification, these bacteria function either aerobically or anaerobically. Such a process is not suitable for degrading TBP.
- le Brevet américain 6,106,719, décrit l'utilisation, pour le traite- ment des déchets liquides contenant à la fois des composés organiques et des composés inorganiques contenant de l'azote ou du phosphore, d'une combinaison de bactéries sous la forme de granules solides, en anaérobiose et en présence de lumière ;
ladite combinaison de bactéries comprend (a) un mélange de bactéries non-photosynthétiques : bactéries à fermentation acide et/ou bactéries productrices de méthane et (b) un mélange de bactéries photosynthétiques (bactéries pourpres non-sulfureuses, bactéries pourpres sulfureuses et bactéries vertes sulfureuses, telles que Chrorobium limicola, Chromatium vinosum, Rhodopseudomonas palustris ou Rhodobacter capsulatus). Un tel traitement permet la digestion de la matière organique, et celle des composés inorganiques comprenant de l'azote et/ou du phosphore. Dans le procédé décrit dans ce Brevet, la disparition du phosphore inorganique est liée à la croissance des bactéries photosynthétiques. Un tel procédé n'est donc pas adapté au traitement du TBP.- US Patent 6,106,719, describes the use, for the treatment of liquid waste containing both organic compounds and inorganic compounds containing nitrogen or phosphorus, of a combination of bacteria in the form of granules solids, anaerobically and in the presence of light; said combination of bacteria comprises (a) a mixture of non-photosynthetic bacteria: bacteria with acid fermentation and / or methane producing bacteria and (b) a mixture of photosynthetic bacteria (purple non-sulfurous bacteria, purple sulfurous bacteria and green sulfurous bacteria , such as Chrorobium limicola, Chromatium vinosum, Rhodopseudomonas palustris or Rhodobacter capsulatus). Such a treatment allows the digestion of organic matter, and that of inorganic compounds comprising nitrogen and / or phosphorus. In the process described in this patent, 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.
- Les Brevets américains 6,416,993 et 6,465,240 décrivent un procédé de dégradation de déchets organiques et inorganiques liquides, qui comprend deux étapes de traitement par des microorganismes photosynthétiques : un premier traitement par un ou plusieurs procaryote(s) photosynthétique(s) et de manière préfé- rée un consortium de bactéries photosynthétiques (bactéries pourpres non-sulfureuses : Rhodospirillum, Rhodopseudomonas, Rhodobacter, Chromatium, Rubrivivax ou cyanobactéries), puis un second traitement par des algues photosynthétiques. De manière plus précise, le procédé décrit permet de traiter des déchets contenant des concentrations élevées en carbone organique total (TOC), en demande d'oxygène biochimique (BOD), en azote (y compris l'ammoniaque) et en phosphore (P, y compris les phosphates, les polyphosphates, les phosphates organiques) ainsi que d'autres substances organiques ou inorganiques. Toutefois, ces procédés ne sont pas adaptés à la dégradation du TBP qui peut être présent à des concentrations élevées (de l'ordre de 100 mg/ml) ; en effet, les procédés décrits dans ces deux Brevets s'appliquent au traitement d'effluents agricoles, tels que le lisier, dans lequel on trouve peu ou pas de TBP. En outre, le TBP est réputé toxique pour les bactéries et les algues à de faibles concentrations (Nakamura A., 1991, International Program on Chemical Safety-Environmental Health Critiria 112- Tri-n-Butyl Phosphate QV 627. World Health Organisation). Les procédés de l'art antérieur visant au traitement de l'ensemble des produits organiques et inorganiques ne sont donc pas adaptés au traitement de
déchets comprenant du TBP, la croissance des algues et des bactéries non photosynthétiques mises en œuvre étant généralement inhibée par le 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. More precisely, 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. However, 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. In addition, 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.
La dégradation du TBP par les microorganismes a fait l'objet de peu d'études ; elle met généralement en œuvre des microorganismes qui agissent en aéro- biose :The degradation of TBP by microorganisms has been the subject of few studies; it generally implements microorganisms which act aerobically:
- Le Brevet américain 5,453,375 décrit un procédé de dégradation du TBP, qui met en œuvre la souche de bactérie Acinetobacter sp., en aérobiose.- The American patent 5,453,375 describes a process of degradation of TBP, which implements the strain of bacteria Acinetobacter sp., Aerobically.
- Dans d'autres études, l'équipe de R.A. THOMAS et al. [1, 2, 3, 4] a décrit l'utilisation d'un mélange de microorganismes du genre Pseudomonas spp., pour dégrader le TBP en aérobiose.- In other studies, the team of R.A. THOMAS et al. [1, 2, 3, 4] described the use of a mixture of microorganisms of the genus Pseudomonas spp., To degrade TBP aerobically.
- Dans l'article au nom de S. OWEN et al. [5], l'utilisation de bactéries Citrobacter sp. est préconisée en aérobiose pour dégrader le TBP.- In the article in the name of S. OWEN et al. [5], the use of Citrobacter sp. is recommended aerobically to degrade TBP.
Les conditions (aérobiose) exposées dans ces documents présentent les inconvénients suivants : - rendement de dégradation du TBP faible,The conditions (aerobic) exposed in these documents have the following drawbacks: - low degradation yield of TBP,
- reproductibilité des procédés aléatoire.- reproducibility of random processes.
En conséquence, l'ensemble des procédés de traitement des déchets organiques préconisés dans l'art antérieur ne sont donc pas adaptés au traitement du TBP, dans la mesure où aucun d'entre eux ne décrit un procédé à la fois stable, repro- ductible, efficace et à rendement élevé (> à 400 mg/1) pour dégrader le TBP, présent dans des fourchettes de concentrations variables.Consequently, all of the organic waste treatment methods recommended in the prior art are therefore not suitable for treating TBP, insofar as none of them describes a process that is both stable and reproducible. , effective and high yield (> 400 mg / 1) to degrade TBP, present in ranges of varying concentrations.
En conséquence, la Demanderesse s'est donné pour but de pourvoir à un procédé de traitement des déchets liquides contenant du TBP, qui réponde mieux aux besoins de la pratique que les procédés de l'art antérieur, notamment en ce qu'il permet effectivement de dégrader le TBP, avec un bon rendement et notamment, dans certaines conditions, un rendement supérieur à 400 mg/1, tout en étant reproductible.Consequently, 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.
La présente invention a pour objet un procédé de traitement ou d'épuration de déchets liquides chargés en TBP, caractérisé en ce qu'il comprend les étapes de : (1) mise en contact desdits déchets liquides avec au moins une souche de bactérie photosynthétique pourpre non-sulfureuse résistante au TBP sélectionnée dans le groupe constitué par Rhodopseudomonas palustris (Rp. palustris),
Rhodospirillum rubrum (Rs. rubrum), Rhodobacter capsulatus (Rb. capsulatus) ou Rhodobacter sphaeroides (Rb. sphaeroides) ainsi que lesdites souches de bactéries modifiées pour surexprimer le cytochrome P450, dans des conditions permettant la dégradation du TBP présent dans lesdits déchets, et ce, quelle que soit la concentration initiale en TBP ; etThe 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
(2) la récupération des effluents liquides purifiés.(2) recovery of purified liquid effluents.
Conformément à l'invention, pour une dégradation optimale, une source de carbone, soit endogène, soit exogène est nécessaire.According to the invention, for optimal degradation, a carbon source, either endogenous or exogenous, is necessary.
Selon un mode de mise en œuvre avantageux du procédé selon l'invention, l'étape (1) est réalisée en anaérobiose ou micro-anaérobiose.According to an advantageous embodiment of the method according to the invention, step (1) is carried out anaerobically or micro-anaerobically.
De manière générale, selon les besoins en oxygène, on définit différentes conditions : aérobiose, micro-anaérobiose (ou micro-aérobiose ou microaéro- philie) et anaérobiose stricte, de la manière suivante :Generally, depending on the oxygen requirements, different conditions are defined: aerobic, micro-anaerobic (or micro-aerobic or microaerobic) and strict anaerobic, as follows:
- aérobiose : un microorganisme aérobie est un microorganisme qui peut utiliser l'oxygène comme accepteur final d'électrons ;- aerobic: an aerobic microorganism is a microorganism which can use oxygen as final acceptor of electrons;
- micro-anaérobiose ou micro-aérobiose : un microorganisme capable de se développer en conditions dites de micro-aérobiose ou micro-anaérobiose est un microorganisme incapable de croître lorsque les concentrations en oxygène atteignent celles rencontrées dans l'air (20 %) mais qui est néanmoins capable de croître en présence de taux faibles en oxygène (2 à 10 %) ; de tels microorganismes présentent de meilleures conditions de croissance en présence de petites quantités d'oxygène libre ; en particulier, ils se développent sous la surface dans un tube, au niveau où les concentrations en oxygène sont optimales pour leur croissance (Microbiology. Principles and Applications, 1996, 3rd Edition, Black JG, Prentice Hall, Upper Saddle River, New Jersey, pages 144-148).- 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).
- anaérobiose stricte : un microorganisme anaérobie n'est capable de se développer qu'en l'absence d'oxygène moléculaire.- strict anaerobic: an anaerobic microorganism is only able to develop in the absence of molecular oxygen.
Au sens de la présente invention, le terme anaérobiose, appliqué aux bactéries photosynthétiques non-sulfureuses est équivalent au terme microaérophilie ou micro-anaérobiose, plus communément utilisé pour ces bactéries ; une bactérie est dite microaérophile lorsqu'elle ne requiert qu'une concentration faible en oxygène (deWithin the meaning of the present invention, the term 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
2 à 10 %) pour sa croissance ; ce terme est également utilisé pour indiquer que les
bactéries concernées ont une activité métabolique en conditions aérobies mais croissent mieux dans des conditions anaérobies non strictes ([O2]>5 μM et < 70 μM) (Biology of Microorganisms, 9th Edition, 2000, MT Madigan, JM Martinko et J Parker, Prentice Hall, Upper Saddle River, New Jersey, pages 158-162). Pour ce qui concerne les bactéries photosynthétiques pourpres non-sulfureuses, l' anaérobiose (ou microaérophilie ou micro-anaérobiose ou micro-aérobiose), peut être obtenue soit en présence de lumière (lumière naturelle ou lumière artificielle, telle que lampe à incandescence ayant un spectre d'émission compris entre 350 et 1100 nm) et une énergie comprise dans une gamme de 1 à 2000 μmoles de photons/m2/s et dans un milieu ayant subi un cycle de dégazage pour obtenir une concentration résiduelle en oxygène comprise entre 5 μM et 70 μM, soit en l'absence de lumière, mais en présence d'un accepteur d'électrons couramment utilisé pour les bactéries photosynthétiques, tel que le triméthylamine-N-oxyde (TMAO), les nitrates et le diméthyl sulfoxyde (DMSO).2 to 10%) for its growth; this term is also used to indicate that bacteria concerned have metabolic activity under aerobic conditions but grow better under non-strict anaerobic conditions ([O 2 ]> 5 μM and <70 μM) (Biology of Microorganisms, 9 th Edition, 2000, MT Madigan, JM Martinko and J Parker , Prentice Hall, Upper Saddle River, New Jersey, pages 158-162). With regard to the non-sulfurous purple photosynthetic bacteria, 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 μmoles of photons / m 2 / s and in a medium having undergone a degassing cycle to obtain a residual oxygen concentration of between 5 μM and 70 μM, 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 ).
En général, dans les bassins de décantation utilisés, les bactéries pourpres non-sulfureuses mises en œuvre sont de préférence au fond dudit bassin (pourcentage en O2 de l'ordre de 2 à 10 %).In general, in the settling tanks used, 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%).
Selon un autre mode de mise en œuvre avantageux de l'invention, ladite souche de bactérie résistante au TBP mise en œuvre à l'étape (1) du procédé est de préférence sélectionnée dans le groupe constitué par Rp. palustris et Rs. rubrum, qui présentent une aptitude à dégrader le TBP, supérieure à 400 mg/1 et de préférence supérieure à 426 mg/1. En ce qui concerne Rp. palustris, sont également comprises dans l'invention, les souches dépourvues de plasmide endogène ; de telles souches sont dénommées ci-après souches ΔpRpal.According to another advantageous embodiment of the invention, 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. As regards Rp. Palustris, also included in the invention are the strains devoid of endogenous plasmid; such strains are hereinafter called ΔpRpal strains.
Selon un autre mode de mise en œuvre avantageux du procédé selon l'invention, l'étape (1) comprend la mise en œuvre d'un mélange de souches de bactéries pourpres non-sulfureuses résistantes au TBP comprenant au moins une souche sélectionnée dans le groupe constitué par Rhodospirillum rubrum (Rs. rubrum) et Rhodopseudomonas palustris (Rp. palustris) et au moins une autre souche sélectionnée dans le groupe constitué par Rhodobacter capsulatus (Rb. capsulatus) et Rhodo- bacter sphaeroides (Rb. sphaeroides).According to another advantageous embodiment of the method according to the invention, 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).
Conformément à l'invention, les souches de bactéries pourpres non- sulfureuses résistantes au TBP sont de préférence sélectionnées parmi les souches Rp.
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 et n°DSM 130, la souche Rs. rubrum SI n°ATCC 11170, la souche Rb. capsulatus Saint-Louis n°ATCC 23782 et la souche Rb. sphaeroides 2.4.1. n°ATCC 17023. De manière surprenante, les Inventeurs ont sélectionné des souches de bactéries pourpres non-sulfureuses résistantes au TBP, qui permettent d'obtenir des rendements de dégradation élevés, quelle que soit la concentration initiale en TBP et ce dans un procédé d'épuration en une seule étape ; le procédé selon l'invention est efficace et reproductible pour épurer et dépolluer l'environnement, et en particulier dégrader le TBP dans des déchets liquides et ce en raison de l'utilisation, de préférence en micro-anaérobiose, d'une ou plusieurs souches pures de bactéries pourpres non-sulfureuses résistantes au TBP, qui seules ou en mélange, permettent d'obtenir une dégradation du TBP au moins égal à 53 mg/1 et de préférence supérieur à 400 mg/I d'effluents liquides. Par « déchets liquides », on entend désigner les effluents et/ou les sites aquatiques pollués. Ces déchets liquides contiennent du TBP à dégrader mais peuvent aussi contenir d'autres composés organiques et inorganiques. Ils proviennent de l'industrie et notamment de l'industrie nucléaire.According to the invention, 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. Surprisingly, 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. By "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.
On entend, au sens de la présente invention par souche de bactérie pourpre non-sulfureuse résistante au TBP, une souche dont la croissance n'est pas inhibée en présence de concentrations de TBP au moins dans la fourchette de concentrations en TBP de 12-37 μM (Nakamura, précité) et bien entendu également à des concentrations supérieures en TBP.For the purposes of the present invention, the term “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 μM (Nakamura, cited above) and of course also at higher concentrations of TBP.
Selon encore un autre mode de mise en œuvre avantageux dudit pro- cédé, l'étape (1) est effectuée à une température comprise entre 10°C et 37°C, de préférence à 30°C, à un pH compris entre 5,5 et 8,5, de préférence à 6,9 et pendant au moins 15 jours, de préférence entre 15 et 21 jours.According to yet another advantageous embodiment of said method, 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.
Conformément à l'invention, l'étape (1) est de préférence réalisée dans un bassin de décantation. La période d'incubation ou de mise en contact doit être suffisante pour que la souche de bactérie ou le mélange de souches de bactéries puisse croître de façon exponentielle, pour permettre la dégradation du TBP avec un rendement élevé.
Cette période d'incubation est de préférence entre plusieurs jours et plusieurs semaines, en fonction de la température d'incubation utilisée ; par exemple, une période d'incubation de 21 jours à une température de 30°C, permet d'obtenir un rendement supérieur à 400 mg/1. Selon un autre mode de mise en œuvre avantageux dudit procédé, le taux de TBP dans lesdits déchets liquides avant traitement est compris entre 0,01 mM et 1 M ; cette gamme de valeurs est suffisamment large pour correspondre aux conditions habituellement rencontrées dans les cas de pollution ; en effet, les relevés effectués dans des conditions de pollution donnent des valeurs de rejet en TBP de l'ordre de 100 mg/1 soit 0,375 mM. En conséquence, il n'est généralement pas nécessaire de diluer ou de concentrer l'échantillon à traiter avant sa mise en contact avec l'inoculum de bactéries. En outre, dans cette gamme de valeurs, le rendement de dégradation est particulièrement élevé. En effet, le procédé selon l'invention permet la dégradation de concentrations en TBP de l'ordre de 0,1 à 2 mM ; aucune inhibition de la croissance des bactéries n'ayant été observée à ces concentrations. En particulier, lorsque la concentration initiale en TBP est supérieure à 1 mM, on obtient une dégradation complète du TBP, dans les conditions de l'invention. Plus précisément, en utilisant le procédé selon la présente invention, il est possible d'obtenir après 3 semaines d'incubation des rendements de dégradation du TBP compris entre 1 et 1000 mg par litre de milieu ; la valeur la plus élevée correspondant à la solubilité théorique maximale du TBP dans l'eau à 25°C.According to the invention, 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. According to another advantageous embodiment of said method, 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. In addition, in this range of values, 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. In particular, when the initial concentration of TBP is greater than 1 mM, a complete degradation of the TBP is obtained, under the conditions of the invention. More specifically, using the method according to the present invention, it is possible to obtain, after 3 weeks of incubation, degradation yields of TBP of between 1 and 1000 mg per liter of medium; the highest value corresponding to the maximum theoretical solubility of TBP in water at 25 ° C.
Pour pouvoir obtenir un procédé efficace et reproductible avec des rendements de dégradation de TBP élevés selon l'invention, il convient d'inoculer une souche pure ou un mélange de bactéries photosynthétiques pourpres non-sulfureuses résistantes au TBP. Selon l'invention, il est préférable d'utiliser une souche pure de bactéries photosynthétiques, dans un but d'optimisation des conditions de traitement et ainsi du rendement de dégradation du TBP.In order to be able to obtain an efficient and reproducible process with high TBP degradation yields according to the invention, a pure strain or a mixture of purple non-sulfurous photosynthetic bacteria resistant to TBP should be inoculated. According to the invention, it is preferable to use a pure strain of photosynthetic bacteria, for the purpose of optimizing the treatment conditions and thus the degradation yield of TBP.
Selon un autre mode de mise en œuvre avantageux de l'invention, les quantités de bactéries inoculées dans le milieu à traiter à l'étape (1), sont avanta- geusement comprises entre 104 et 1010 bactéries par ml de milieu à traiter. Toutefois, elles dépendent des conditions générales mises en œuvre (température, luminosité...). En conséquence, de manière générale, on entend par « quantité suffisante » de bacté-
ries à inoculer, la concentration d'ensemencement, c'est-à-dire le nombre initial de bactéries permettant, par croissance exponentielle, et dans des conditions adéquates, d'aboutir à une biomasse suffisante pour observer une dégradation du TBP.According to another advantageous embodiment of the invention, 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 . However, 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.
Les souches de bactéries photosynthétiques résistantes au TBP, telles que mises en œuvre dans la présente invention présentent comme avantage de fournir de bons rendements de biodégradation du TBP même après avoir été soumises à un traitement ultérieur (nombre important de sous-cultures, par exemple).The strains of photosynthetic bacteria resistant to TBP, as used in the present invention 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) .
Selon un autre mode de mise en œuvre avantageux du procédé selon l'invention, lorsque la source de carbone endogène n'est pas suffisante, on ajoute à l'étape (1), une source de carbone supplémentaire, dans le milieu à traiter ; cette source de carbone supplémentaire est avantageusement une solution tamponnée contenant un extrait de levure à une concentration comprise entre 0,1 et 10 g/1 (de préférence à 1 g/1), ou l'un des sels organiques suivants : succinate, glutamate, benzoate, malate ou fumarate à une concentration comprise entre 2 et 20 mM (de préférence à lO mM), et des facteurs de croissance dont au minimum la biotine et l'acide para amino benzoïque à une concentration comprise entre 2 et 40 μg/1 chacun.According to another advantageous embodiment of the method according to the invention, when the endogenous carbon source is not sufficient, in step (1), 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 μg / 1 each.
Préalablement à ladite mise en contact selon l'étape (1), les bactéries pourpres non-sulfureuses résistantes au TBP sont sélectionnées par culture sur milieu contenant au moins 12 μM et de préférence au moins 1 mM de TBP, puis sont culti- vées selon des méthodes connues de l'homme du métier comme par exemple celles décrites dans Bergey 's manual of systematic bacteriology ; Williams & Wilkins Edition ou dans « The photosynthetic bacteria » R.K. Clayton et W. R. Sistrom ; Plénum Press.Prior to said contacting according to step (1), the purple non-sulfur bacteria resistant to TBP are selected by culture on a medium containing at least 12 μM 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.
Dans le cadre de l'invention, le procédé est avantageusement mis en œuvre en lagunage non aéré.In the context of the invention, the method is advantageously implemented in non-aerated lagooning.
On entend par « lagunage non-aéré », un dispositif comprenant au minimum un bassin de décantation d'une profondeur allant de 0,5 à 1 mètre pour une surface en accord avec le débit des effluents, sachant que le temps de rétention doit être au minimum de 15-21 jours dans des conditions de température et d'éclairement optimales. La séparation ultérieure des bactéries et du milieu traité est favorisée par les conditions mises en œuvre (micro-anaérobiose, bassin de décantation).
Lorsque la croissance bactérienne a atteint une valeur importante, et avant de relarguer les effluents purifiés dans l'environnement, il peut être utile de récupérer les bactéries par filtration (en utilisant un filtre à diatomées par exemple) ou par centrifugation en continu avant de les réinjecter dans le système. La présente invention a également pour objet un procédé de suivi ou de surveillance de la dégradation du TBP dans des déchets liquides, lequel procédé comprend :“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). When 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:
- la mise en œuvre du procédé de traitement de déchets liquides chargés en TBP, tel que défini ci-dessus, puis - le prélèvement d'un échantillon de milieu traité au moins au temps t+15 jours et la mesure dans ledit échantillon prélevé de la concentration de TBP résiduel ; cette mesure peut être réalisée manuellement ou automatisée lorsqu'il existe un grand nombre d'échantillons à analyser. L'étape de mesure du TBP résiduel (n'ayant pas été dégradé par les bactéries) peut s'effectuer par toute technique connue en chimie analytique couramment utilisée, et plus particulièrement dans le domaine des composés organophosphorés. Pour une mesure fiable et précise, on utilise généralement une technique chromatographique, et plus précisément la chromatographie liquide à haute performance couplée à un réfractomètre. Tout le procédé peut être automatisé. La présente invention a également pour objet un coffret pour la mise en œuvre du procédé de traitement selon l'invention, caractérisé en ce qu'il comprend au moins une souche de bactérie photosynthétique pourpre non-sulfureuse résistante au TBP, choisie dans le groupe constitué par Rp. palustris, Rs. rubrum, Rb. capsulatus ou Rb. sphaeroides ainsi que lesdites souches de bactéries modifiées pour surexprimer le cytochrome P450, comme défini ci-dessus. Le mélange de souches comprend, de préférence, au moins la souche Rp. palustris ou la souche Rs. rubrum, comme précisé ci-dessus. En ce qui concerne Rp. palustris, sont également comprises dans le cadre de l'invention, les souches dépourvues de plasmide endogène ; de telles souches sont dénommées ci-après souches ΔpRpal. La présente invention a également pour objet des souches de bactéries photosynthétiques pourpres non-sulfureuses, caractérisées en ce qu'elles sont résistantes au TBP, en ce qu'elles sont sélectionnées dans le groupe constitué par Rp.
palustris, Rs. rubrum, Rb. capsulatus ou Rb. sphaeroides et en ce que leur ADN inclut au moins une copie supplémentaire du gène codant pour un cytochrome P450 homologue.- the implementation of the process for treating liquid waste loaded with TBP, as defined above, then - the taking of a sample of medium treated at least at time t + 15 days and the measurement in said sample taken of the concentration of residual TBP; this measurement can be carried out manually or automated when there is a large number of samples to be analyzed. The step of measuring the residual TBP (not having been degraded by bacteria) 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. With regard to Rp. Palustris, also included within the framework of the invention, the strains devoid of endogenous plasmid; such strains are hereinafter called ΔpRpal 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.
De telles souches, comprenant au moins une copie supplémentaire d'un gène homologue codant pour un cytochrome P450 permettent une surexpression dudit 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.
Selon un mode de réalisation avantageux desdites souches, elles sont constituées par une souche de Rp. palustris CGA009 comprenant un gène codant pour le cytochrome P450 de Rp. palustris, sous le contrôle du promoteur LHαβe, décrit dans Tadros M.H. et al. (10) (souche Rp. palustris LH5939). Le gène codant pour le cytochrome P450 de Rp. palustris est notamment décrit dans la base de données ά Oak Ridge National Laboratory (ORNL) sous le n° de gène 5939 (site : http ://genome .ornl . gov) ou sous le n° d'accès Genbank n°NZ_AAAF01000001.1.According to an advantageous embodiment of said strains, they 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 LHαβ 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 ά Oak Ridge National Laboratory (ORNL) under the gene number 5939 (site: http: // genome .ornl. Gov) or under the Genbank access number NZ_AAAF01000001.1.
La présente invention a également pour objet un dispositif d'épuration des déchets liquides chargés en TBP en mettant en œuvre le procédé de dégradation tel que décrit ci-dessus, caractérisé en ce qu'il comprend au moins un bassin de décantation adapté à la mise en contact des bactéries pourpres non-sulfureuses avec le milieu à traiter, de préférence en micro-anaérobiose. Un tel dispositif met notamment en œuvre la technique de lagunage non-aéré, qui consiste à faire pas- ser les effluents pollués, chargés en TBP dans une série de bassins à l'air libre dont le premier, appelé bassin de décantation, est spécifiquement ensemencé dans le cadre de l'invention par des bactéries photosynthétiques pourpres non-sulfureuses résistantes au TBP. Dans ce cas, la micro-anaérobiose se déroule au fond du bassin de tête où se dépose les boues. Ledit bassin de décantation est, de préférence, d'une profondeur allant de 0,5 à 1 mètre pour une surface en accord avec le débit des effluents, sachant que le temps de rétention doit être au minimum de 15-21 jours dans les conditions de température et d'éclairement optimales telles que définies ci-dessus.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. Such 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. In this case, 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.
Selon un mode de réalisation avantageux dudit dispositif, il comprend outre ledit bassin de décantation, des moyens d'introduction desdits déchets liquides contenant du TBP dans ledit bassin de décantation, des moyens d'introduction de Pinoculum de bactéries, des moyens d'évacuation des effluents épurés en TBP vers l'extérieur ainsi que des moyens de récupération des bactéries à recycler.
Selon un autre mode de réalisation avantageux dudit dispositif, ledit moyen de récupération des bactéries est avantageusement sélectionné dans le groupe constitué par les filtres à diatomées et les centrifugeuses en continu.According to an advantageous embodiment of said device, it 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. According to another advantageous embodiment of said device, said means for recovering bacteria is advantageously selected from the group consisting of diatom filters and continuous centrifuges.
Outre les dispositions qui précèdent, l'invention comprend encore d'autres dispositions qui ressortiront de la description qui va suivre, qui se réfère à des exemples de mise en œuvre de l'invention ainsi qu'aux dessins annexés, dans lesquels : la figure 1 représente la consommation de TBP (exprimée en pourcentage) en aérobiose (barres hachurées) et en anaérobiose (barres avec pointillés) pour chacune des souches de bactéries photosynthétiques pourpres non-sulfureuse testées (Rb. capsulatus Saint-Louis (ATCC 23782), Rb. sphaeroides 2.4.1 (ATCC 17023), Rs. rubrum S.l. (ATCC 11170), et Rp. palustris CGA009 (ATCC BAA-98)). Le milieu de culture de Hutner non inoculé est utilisé comme témoin ; la figure 2 représente la croissance de la souche Rp. palustris CGA009 (ATCC BAA-98) dans des conditions aérobies (A), photosynthétiques (micro-anaérobies) (B) et anaérobies (C) dans le milieu de culture de Hutner, en présence (ligne continue, (À.)) et en l'absence (ligne discontinue, (Δ)) de TBP 2 mM au cours de la croissance bactérienne. A la figure 2A, l'échelle des ordonnées de gauche correspond à la densité optique de la culture mesurée à 660 n . L'échelle des ordonnées de droite correspond à la concentration en TBP (ligne continue, (B)). En aérobiose, la courbe de dégradation peut être transcrite par la fonction a.eΛn2/ '2 + b avec a =28, è=0,55 et t1 2=l,l jour. En micro-anaérobiose, la courbe peut être transcrite par la fonction aj.eΛn2ln + a2. e"n2//2avec aj =23, a -ll, et t!=13,5 jours. Les échelles de la figure 2B sont identiques à celles de la figure 2A. - les figures 3A et 3B représentent respectivement la croissance de la souche Rp. palustris CGA009 (ATCC BAA-98) dans des conditions photosynthétiques (micro-anaérobiose) dans le milieu de culture de Hutner en présence de différentes concentrations de TBP, et une étude cinétique de la consommation de TBP pour la souche Rp. palustris CGA009 (ATCC BAA-98) dans les mêmes conditions. Les échelles de la figure 3 A sont identiques à celles de la figure 2. Celle de la figure 3B correspond à la concentration initiale en TBP exprimée en mM. Les concentrations
en TBP sont 0 mM (astérisques), 0,1 mM (croix), 0,5 mM (triangles), 1 mM (cercles), 1 ,5 mM (losanges), et 2 mM (carrés) ; la figure 4A représente la croissance de Serratia odorifera sp. dans du milieu Tsb en aérobiose, en présence (cercles) et en l'absence de monoxyde de carbone dans le milieu de culture (carrés) ; la figure 4B représente la croissance de la souche Rp. palustris CGA009 (ATCC BAA-98) exprimée en unités de densité optique à 660 nm dans des conditions photosynthétiques dans le milieu de culture de Hutner en présence (cercles) et en l'absence de monoxyde de carbone dans le milieu de culture (carrés). Toutes les bactéries sont incubées en présence (symboles remplis) et en l'absence (symboles vides) de TBP 2 mM ; la figure 5 représente la consommation de TBP exprimée en pourcentage de TBP résiduel par les souches Serratia odorifera sp. et Rp. palustris CGA009 (ATCC BAA-98) après 24 heures et 3 semaines respectivement, en l'absence (barres en pointillés) et en présence (barres hachurées) de monoxyde de carbone dans le milieu de culture ; la figure 6 représente le plasmide pCB07 comprenant le gène codant pour le cytochrome P450 de Rp. palustris n°5939 et le promoteur LHαβe. la figure 7 représente la consommation en TBP (en mM) par des extraits cellulaires de souches de Rp. palustris, sur une durée de 20 heures à 40°C, la concentration initiale en TBP étant de 2 mM. Les échantillons étudiés sont des protéines totales de : (1) : souche CGA009 non induite par du TBP en présence d'O2 ; (2) : souche CGA009 non induite par du TBP en l'absence d'O2 ; (3) souche de CGA009 induite par du TBP en présence d'O et (4) : souche ΔpRpal. Les contrôles sont les suivants : (5) : tampon de réaction sans protéine ; (6) extrait brut bouilli de Rp. palustris CGA009 et (7) : extrait brut d'E. coli DH5α. Les valeurs sont des moyennes ± les déviations standard, à partir de trois expériences indépendantes.In addition to the above arrangements, the invention also comprises other arrangements which will emerge from the description which follows, which refers to examples of implementation of the invention as well as to the appended drawings, in which: 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)). The uninoculated Hutner culture medium is used as a control; 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, (Δ)) of 2 mM TBP during bacterial growth. In FIG. 2A, the ordinate scale on the left corresponds to the optical density of the culture measured at 660 n. The ordinate scale on the right corresponds to the TBP concentration (solid line, (B)). In aerobic conditions, the degradation curve can be transcribed by the function ae Λn2 / ' 2 + b with a = 28, è = 0.55 and t 1 2 = l, l day. In micro-anaerobiosis, the curve can be transcribed by the function aj.e Λn2ln + a2. e " n2 // 2 with aj = 23, a -ll, and t ! = 13.5 days. 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. in TBP are 0 mM (asterisks), 0.1 mM (cross), 0.5 mM (triangles), 1 mM (circles), 1.5 mM (diamonds), and 2 mM (squares); Figure 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). All the bacteria are incubated in the presence (filled symbols) and in the absence (empty symbols) of 2 mM TBP; 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 LHαβ e . FIG. 7 represents the consumption of TBP (in mM) by cellular extracts of Rp. palustris strains, over a period of 20 hours at 40 ° C., the initial concentration of TBP being 2 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 ΔpRpal. The controls are: (5): protein-free reaction buffer; (6) raw boiled extract of Rp. Palustris CGA009 and (7): crude extract of E. coli DH5α. Values are means ± standard deviations, from three independent experiments.
La figure 8 représente (A) : la cinétique de consommation de TBP, par des extraits cellulaires à 40°C en présence de différentes concentrations initiales en TBP (en mM) ; les concentrations en TBP sont les suivantes : 0,05 mM (-), 0,1 mM (*), 0,25 mM (+), 0,5 mM (x), 0,75 mM (•), 1 mM (À), 1,5 mM (*) et 2 mM (•). Les valeurs sont des moyennes ± les déviations standard, à partir de trois expériences indépendantes. (B) : la vitesse de dégradation initiale vs la concentration ini-
tiale en TBP dans un extrait cellulaire brut (données obtenues en A). La ligne représente la meilleure correspondance de l'équation de Michaelis-Menten. Un diagramme de Lineweaver-Burk est illustré dans l'incrustation.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 (À), 1.5 mM (*) and 2 mM (•). Values are means ± standard deviations, from three independent experiments. (B): the initial degradation rate vs the initial concentration TBP in a crude cell extract (data obtained in A). The line represents the best match of the Michaelis-Menten equation. A Lineweaver-Burk diagram is illustrated in the overlay.
La figure 9 représente la dégradation en TBP par des extraits cellulaires dialyses ou non, sur une durée de 20 heures à 40°C, la concentration initiale en TBP étant de 2 mM. Les cofacteurs sont ajoutés à une concentration finale de 10 μM. Les valeurs sont des moyennes + les déviations standard, à partir de trois expériences indépendantes.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 μM. Values are means + standard deviations, from three independent experiments.
La figure 10 illustre la quantité de TBP dégradé (à partir d'une concentration initiale de 2 mM) sur une durée de 20 heures à 40°C, par différentes fractions d'extraits cellulaires : (1) : extrait brut ; (2) fraction soluble ; (3) fraction protéinique faiblement associée à la membrane et (4) fraction protéinique liée à la membrane. Les valeurs sont fonction du poids de protéine présent dans chaque cas et sont des moyennes + les déviations standard, à partir de cinq expériences indépen- dantes.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.
Il doit être bien entendu, toutefois, que ces exemples sont donnés uniquement à titre d'illustration de l'objet de l'invention, dont ils ne constituent en aucune manière une limitation.It should be understood, however, that these examples are given only by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation.
Exemple 1 : Comparaison du rendement de dégradation du TBP en aérobiose et en anaérobiose (micro-anaérobiose ou microaérophilie) par diverses bactéries photosynthétiques pourpres non-sulfureuses. A. Matériel et méthodesExample 1: Comparison of the degradation yield of TBP aerobically and anaerobically (micro-anaerobically or microaerophilically) by various purple non-sulfurous photosynthetic bacteria. A. Materials and methods
Au cours de cette expérience, on mesure la concentration du TBP résiduel en aérobiose et en micro-anaérobiose après 3 semaines de culture par les bacté- ries photosynthétiques suivantes : Rb. capsulatus Saint-Louis (ATCC 23782), Rb. sphaeroides 2.4.1 (ATCC 17023), Rs. rubrum S.l. (ATCC 11170) et Rp. palustris CGA009 (ATCC BAA-98).During this experiment, 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).
Les bactéries photosynthétiques sont incubées dans un milieu de Hutner [6] dans lequel le pH est compris entre 6,5 et 7,5, en présence de 2 mM de TBP, soit en aérobiose, soit en micro-anaérobiose. En aérobiose, les bactéries sont incubées dans l'obscurité à une température de 30°C ; le milieu de culture étant soumis à une agitation (150 rpm).
En micro-anaérobiose, les bactéries sont incubées à la lumièreThe 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
(lampe à incandescence ayant un spectre d'émission compris entre 400 et 900 nm) à(incandescent lamp having an emission spectrum between 400 and 900 nm) at
30°C. La micro-anaérobiose est obtenue par dégazage du milieu avant l'inoculation par mise sous dépression du flacon à l'aide d'une pompe à vide, pour atteindre une tension basse en oxygène de l'ordre de 70 μM.30 ° C. 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 μM.
Les milieux sont stérilisés par autoclavage à 120°C pendant 15 minutes. Le TBP est ajouté, après stérilisation, à une concentration finale de 2 mM, qui se situe au dessous du point de saturation du produit de solubilité du TBP dans l'eau qui est inférieur à lg/1 (3,7 mM) à 25°C. La croissance est suivie au spectrophotomètre à une densité optiqueThe 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
(DO) de 660 nm et les échantillons sont prélevés régulièrement et stockés à -20°C pour les mesures ultérieures du TBP résiduel.(OD) of 660 nm and the samples are taken regularly and stored at -20 ° C for subsequent measurements of the residual TBP.
On centrifuge la culture à 5000 g pendant 10 minutes pour séparer les bactéries du milieu de culture. On récupère alors le surnageant sur lequel on détermine la concentration du TBP résiduel. On ajoute au surnageant aqueux un volume égal de dichloroéthane et de phosphate de tripropyle (TPP). Le solvant permet d'extraire le TBP et les autres composés organiques hydrophobes, et le TPP est utilisé en tant que standard interne pour quantifier l'efficacité d'extraction et pour calibrer les résultats lors des analyses ultérieures par chromatographie liquide à haute perfor- mance (CLHP ou HPLC pour High Performance Liquid Chromatography). On mélange les échantillons par vortexage pendant 1 minute puis on laisse reposer pendant 30 minutes. Afin d'éliminer toute trace aqueuse, on filtre alors la phase organique (phase inférieure) sur un séparateur de phase (IPS, Whatman) suivie d'une évapora- tion pendant une nuit à 30°C, ou alternativement, on procède au passage d'un courant léger d'azote à température ambiante pendant 15 minutes. Les échantillons sont dilués dans du dodécane pour une analyse ultérieure par CLHP.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. In order to remove any aqueous trace, 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.
On procède alors à des mesures par analyses CLHP en utilisant une colonne Brownlee Spheri-5 aminée (Perkin Elmer) intégrée à une CLHP de modèle Waters 1525. On élue en utilisant un mélange d'heptane et d'acétate d'éthyle (75/25, vol/vol) avec un débit de 1 ml/min tel que décrit dans le brevet européen 0 578 579. On mesure le TBP en utilisant un réfractomètre de type Waters 2414 et on analyse les profils de chromatographie en utilisant le logiciel Breeze (commercialisé par la société
Waters). On définit la concentration en TBP par le rapport entre les aires de pics des composés TBP et TPP.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.
Les composés TBP et TPP, dont la pureté est estimée supérieure à 99 %, ainsi que tous les produits chimiques précédemment cités qui sont utilisés pour extraire le TBP et lors de l'analyse CLHP sont fournis par Sigma- Aldrich.The 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.
B. RésultatsB. Results
Les résultats sont présentés dans la figure 1.The results are shown in Figure 1.
En aérobiose, les rendements de dégradation du TBP atteignent 13 à 22 % après 21 jours d'incubation. Ces valeurs sont similaires à celles obtenues avec des isolats naturels tels que la souche Serratia odorifera sp..In aerobic conditions, the degradation yields of TBP reach 13 to 22% after 21 days of incubation. These values are similar to those obtained with natural isolates such as the Serratia odorifera sp. Strain.
Dans des conditions photosynthétiques (micro-anaérobiose), le rendement de dégradation est nettement amélioré chez certaines souches, atteignant presque 80 % pour Rp. palustris et Rs. rubrum après 3 semaines de culture. Ce rendement est reproductible, quel que soit le traitement ultérieur auquel sont soumises lesdites souches.Under photosynthetic conditions (micro-anaerobiosis), the degradation yield is clearly improved in certain strains, reaching almost 80% for Rp. Palustris and Rs. Rubrum after 3 weeks of culture. This yield is reproducible, regardless of the subsequent treatment to which said strains are subjected.
Exemple 2 : Croissance de la souche Rp. palustris CGA009 et cinétique de dégradation du TBP par la même souche soit en aérobiose, soit en micro-anaérobiose, soit en anaérobiose.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.
En raison des résultats obtenus à l'exemple 1, la souche Rp. palustris a plus particulièrement été étudiée.Due to the results obtained in Example 1, the strain Rp. Palustris was more particularly studied.
A. Matériel et méthodesA. Materials and methods
Souches et milieuxStrains and environments
Les souches et les milieux utilisés sont présentés dans le Tableau I ci-apres.
The strains and the media used are presented in Table I below.
Tableau I : Descriptif des différents plasmides et souches utilisésTable I: Description of the various plasmids and strains used
a Ap, ampicilline ; Km, kanamycine. a Ap, ampicillin; Km, kanamycin.
Rp. palustris a été cultivé dans le milieu de Hutner (6) ou dans un milieu PM: 12,5 mM Na2HPO4 ; 12,5 mM KH2PO4 ; 7,5 mM (NH4) 2SO4; 0,1 mM Na2S2O3, 5H2O 14,5 mM d'acide j aminobenzoïque ; 0,85 μM EDTA ; 3,8 nM ZnSO4 ; 7H2O; 2,5 nM FeSO4 ;7H2O, 0,9 nM MnSO4 ; 7H2O, 0,16 μM CuSO4 ; 7H2O, 86 nM Co(NO3) 2 ; 6 H2O, 46 nM Na2B4O7, 10 H2O ; 0,1 mM d'acide nitrilo- triacétique ; 0.24 mM MgSO4 anhydre; 45 μM CaCl2 ; 15 nM (NH )6Mo7O24 ; 4 H2O.Rp. 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 μM 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 μM 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 μM CaCl 2 ; 15 nM (NH) 6 Mo 7 O 24 ; 4 H 2 O.
Les milieux ont été stérilisés par autoclavage à 120°C pendant 15 minutes. Une source de carbone (acide succinique stérile) et de Na2CO3 (uniquement dans les conditions de photosynthèse) ont chacun été ajoutés au milieu PM à une concentration finale de 10 mM.The media were sterilized by autoclaving at 120 ° C for 15 minutes. A source of carbon (sterile succinic acid) and Na 2 CO 3 (only under photosynthetic conditions) were each added to the PM medium at a final concentration of 10 mM.
Pour cultiver ces bactéries sur des boîtes de milieu PM, une solution d'agar-agar 2X (34 g.1"1) a été mélangée, après autoclavage, avec du milieu PM 2X.
Pour les conditions de croissance photosynthétique, les boîtes ont été incubées à 30°C, dans un GENbag Anaer (BIOMERIEUX, France) sous exposition lumineuse.To grow these bacteria on plates of PM medium, a 2X agar-agar solution (34 g.1 "1 ) was mixed, after autoclaving, with PM 2X medium. For the photosynthetic growth conditions, the dishes were incubated at 30 ° C, in a GENbag Anaer (BIOMERIEUX, France) under light exposure.
Lorsque du TBP était requis, il a été ajouté directement au milieu de culture, après stérilisation, à une concentration finale de 2 mM (la solubilité du TBP dans l'eau est d'environ 2,5 mM). Les conditions de culture étaient soit celles de l'aérobiose (30°C, obscurité, sous agitation à 300 rpm), soit des conditions photosyn-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-
9 1 thétiques (30°C, lumière, 75 μmoles de photons.m" .s" ). Deux types de conditions photosynthétiques ont été utilisés conformément à la concentration en O2 : (1) La micro-anaérobiose a été obtenue par simple dégazage du milieu. La concentration résiduelle en O2 était d'environ 70 μM telle que mesurée par une électrode de Clark.9 1 thetics (30 ° C, light, 75 μmoles of photons.m " .s " ). Two types of photosynthetic conditions were used in accordance with the O 2 concentration: (1) Micro-anaerobiosis was obtained by simple degassing of the medium. The residual O 2 concentration was around 70 μM as measured by a Clark electrode.
(2) L' anaérobiose a été obtenue en soumettant le milieu à 5 cycles de dégazage suivis d'une étape de barbotage à l'argon suffisante pour obtenir une concentration en oxygène inférieure à 5 μM.(2) 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 μM.
La croissance a été suivie par densité optique à 660 nm (DOôeo). Pour les valeurs de DO660 > 0,8, les échantillons ont été dilués pour permettre une mesure précise. Des échantillons ont été prélevés périodiquement et stockés à -20°C pour d'autres tests TBP. Préparation des échantillons pour le dosage de TBPThe growth was followed by optical density at 660 nm (DOôeo). For OD 660 values> 0.8, the samples were diluted to allow precise measurement. Samples were taken periodically and stored at -20 ° C for other TBP tests. Preparation of samples for TBP assay
Les cultures ont été centrifugées pour éliminer les bactéries (10 minutes à 5 000 g) et le surnageant a été utilisé pour déterminer la concentration en TBP résiduel. Pour les extraits cellulaires, les dosages du TBP ont été effectués directement sur le mélange protéique. Le triphosphylphosphate (TPP) a été utilisé en tant que standard interne pour quantifier l'efficacité d'extraction et pour calibrer les résultats pour l'analyse de chromatographie en phase liquide haute performance (HPLC) subséquente. Le TBP a été extrait à partir des systèmes aqueux par l'addition d'un volume équivalent de dichloroéthane + TPP 2 mM aux surnageants aqueux. Les échantillons ont été mélangés vigoureusement au vortex pendant une minute. Après 30 minutes sans agitation, la phase organique (phase inférieure) a été filtrée sur un séparateur de phase (1PS, Whatman), pour éliminer toute trace de la phase aqueuse, et éva-
porée une nuit à 30°C. Les échantillons secs ont alors été dilués dans du dodécane pour l'analyse HPLC.The cultures were centrifuged to remove the bacteria (10 minutes at 5,000 g) and the supernatant was used to determine the concentration of residual TBP. For the cell extracts, the TBP assays were carried out directly on the protein mixture. Triphosphylphosphate (TPP) was used as an internal standard to quantify the extraction efficiency and to calibrate the results for the subsequent high performance liquid chromatography (HPLC) analysis. TBP was extracted from aqueous systems by the addition of an equivalent volume of 2 mM dichloroethane + TPP to the aqueous supernatants. The samples were vigorously mixed with the vortex for one minute. After 30 minutes without stirring, the organic phase (lower phase) was filtered through a phase separator (1PS, Whatman), to remove all traces of the aqueous phase, and evacuated. porée overnight at 30 ° C. The dry samples were then diluted in dodecane for HPLC analysis.
Analyse HPLCHPLC analysis
Les analyses HPLC ont été effectuées en utilisant une colonne Spheri-5 amino (Perkin Elmer) connectée à une HPLC de modèle Waters 1525, comme précisé à l'exemple 1. Pour l'élution, un mélange d'heptane et d'acétate d'éthyle (75/25, v/v) a été utilisé à un débit de 1 ml.min"1 tel que décrit dans le Brevet européen 0 578 579. Le TBP et le TPP ont été détectés par réfractométrie (Waters Refractometer 2414) et les profils de chromatographie de réfraction ont été analysés en utilisant le Software Waters Breeze. La concentration en TBP a été estimée à partir du rapport entre les aires des pics de TBP et de TPP. Dans ces conditions, le seuil de détection de TBP était d'environ 5 μM (voir également exemple 1).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. For the elution, 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 μM (see also example 1).
Oosae.es enzvmatiquesEnzmatic Oosae.es
Les extraits bruts et les protéines fractionnées ont été utilisés pour les études de dégradation du TBP. En tant que contrôle, du tampon stérile contenant 2 mM de TBP, des extraits bruts bouillis (10 minutes à 95°C) et des extraits bruts d'E. coli DH5α ont été testés pour estimer la disparition abiotique du TBP. Les extraits bruts ont été dialyses avec le système Ultrafree Biomax 10 000 D (Millipore) de manière à abaisser la concentration en molécules de faible taille. FADH2 and FMNH2 ont été obtenus par réduction d'une solution de FMN et de F AD avec un faible excès de dithionite de sodium. Tous les cofacteurs utilisés ont été ajoutés aux extraits cellulaires à une concentration finale de 10 μM.The crude extracts and fractionated proteins were used for the degradation studies of TBP. As a control, sterile buffer containing 2 mM TBP, boiled crude extracts (10 minutes at 95 ° C) and crude extracts of E. coli DH5α 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 μM.
Les dosages ont été effectués en testant la dégradation du TBP par les protéines dans 1 ml de Tris-HCl 400 mM pH 6,5 à 40°C. Les optimums de tempé- rature et de pH ont été déterminés. Le TBP résiduel a été dosé par HPLC.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.
B. RésultatsB. Results
Les résultats sont présentés dans la figure 2.The results are shown in Figure 2.
On mesure la croissance de la souche Rp. palustris en présence deThe growth of the strain Rp. Palustris is measured in the presence of
TBP (2 M) ou en l'absence de TBP. Aussi bien en aérobiose (figure 2A) qu'en micro-anaérobiose (figure 2B), la croissance de la souche commence après un délai, celui-ci étant plus court en aérobiose, puis la cinétique de croissance s'accélère pour atteindre celle du témoin (absence de TBP). En aérobiose, la cinétique de dégradation
est monophasique ; elle peut se traduire par une courbe exponentielle avec un tj/ de 1,1 jours (28 % d'amplitude). Le taux de consommation initiale de TBP est de 13 μmol/l/h (Figure 2A).TBP (2 M) or in the absence of TBP. Both aerobically (Figure 2A) and micro-anaerobic (Figure 2B), 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). In aerobic conditions, 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 μmol / l / h (Figure 2A).
En micro-anaérobiose, la cinétique de croissance est biphasique, elle peut se traduire par une série de deux courbes exponentielles avec un tι/2 de 0,94 jour (23 % d'amplitude) et un tj/ de 13,5 jours (77 % d'amplitude). Le taux de consommation initial du TBP est de 15 μmol/l/h (Figure 2B).In micro-anaerobiosis, the growth kinetics is biphasic, it can result in a series of two exponential curves with a tι / 2 of 0.94 day (23% amplitude) and a tj / of 13.5 days ( 77% amplitude). The initial consumption rate of TBP is 15 μmol / l / h (Figure 2B).
Ces résultats démontrent l'existence de deux mécanismes différents : un mécanisme rapide fonctionnant en aérobiose et en micro-anaérobiose, et un autre plus lent qui fonctionne seulement en micro-anaérobiose. Il est à noter que dans les deux conditions de culture, les phases rapides correspondent presque à la même courbe exponentielle suggérant la survenue de deux mécanismes similaires indépendant des conditions de culture, alors que la phase lente semble être liée à la micro- anaérobiose. Il est remarquable qu'en micro-anaérobiose, la phase rapide de dégrada- tion du TBP apparaisse de façon concomitante pendant le délai de croissance mentionnée précédemment, alors que l'accélération nette de la croissance de la souche est accompagnée par un ralentissement de la cinétique de dégradation du TBP.These results demonstrate the existence of two different mechanisms: a rapid mechanism operating in aerobic and micro-anaerobic conditions, and a slower one which works only in micro-anaerobic conditions. It should be noted that in the two culture conditions, the rapid phases correspond almost to the same exponential curve suggesting the occurrence of two similar mechanisms independent of the culture conditions, while the slow phase seems to be linked to microanaerobiosis. It is remarkable that in micro-anaerobiosis, the rapid phase of degradation of TBP appears concomitantly during the growth period mentioned previously, while the marked acceleration of the growth of the strain is accompanied by a slowing down of the kinetics of TBP degradation.
Par ailleurs, dans des conditions d'anaérobiose stricte, on n'observe aucune croissance en présence de TBP 2mM, tandis qu'en l'absence de TBP, on observe une croissance du même type que celle observée dans les conditions de micro-anaérobiose (figure 2C).Furthermore, under strict anaerobic conditions, no growth is observed in the presence of 2 mM TBP, while in the absence of TBP, a growth of the same type as that observed under micro-anaerobic conditions is observed. (Figure 2C).
Exemple 3 : Effet de différentes concentrations de TBP sur la croissance de la souche Rp. palustris CGA009 et la cinétique de dégradation du TBP par la même souche en micro-anaérobiose. A. Matériel et méthodesExample 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. A. Materials and methods
Les conditions de culture et d'analyse du TBP sont identiques à celles utilisées dans l'exemple 2.The culture and analysis conditions for TBP are identical to those used in Example 2.
B. RésultatsB. Results
Les résultats sont illustrés par la figure 3. La souche Rp. palustris CGA009 est mise en culture en présence de concentrations variées de TBP : 2 ; 1,5 ; 1, 0,5 et 0,1 mM en micro-anaérobiose.
D'après ces résultats, on note un raccourcissement du délai de croissance lorsque la concentration du TBP est réduite. De plus, à une concentration inférieure à 1 mM, la croissance de la souche est similaire à celle observée dans le milieu dépourvu de TBP (Figure 3 A). Les rendements de dégradation en TBP s'améliorent lorsque la concentration en TBP est inférieure à 1 mM ; le TBP est alors presque entièrement dégradé en 21 jours (Figure 3B).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. In addition, at a concentration of less than 1 mM, 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).
On n'observe aucune inhibition de la dégradation du TBP par un métabolite secondaire avec la souche Rp. palustris CGA009, en conditions de micro- anaérobiose. Exemple 4 : Rôle du cytochrome P450 par l'étude de l'effet d'un inhibiteur du cytochrome P450, le monoxyde de carbone (CO) sur la croissance de la souche Rp. palustris en présence ou en l'absence de TBP.No inhibition of degradation of TBP by a secondary metabolite with the strain Rp. Palustris CGA009 is observed, under microanaerobic conditions. Example 4 Role of Cytochrome P450 by Studying the Effect of an Inhibitor of Cytochrome P450, Carbon Monoxide (CO) on the Growth of the Rp. Palustris Strain in the Presence or in the Absence of TBP.
Afin de démontrer qu'un ou plusieurs cytochrome(s) participent activement à la dégradation du TBP par la souche Rp. palustris CGA009, on incube cette souche de bactéries en présence de monoxyde de carbone (CO), qui est un inhibiteur du cytochrome P450, en présence (2 mM) ou en l'absence de TBP. Il est possible de procéder au traitement par le CO, préalablement à l'inoculation de la culture, de deux manières différentes: soit par bullage léger, soit par addition d'un milieu de culture déjà saturé en CO. Une souche S. odorifera, qui ne peut croître qu'en condition aérobie dans un milieu Tryptic soy broth (Tsb ; Sigma-Aldrich), est utilisée à titre de comparaison ; cette souche étant capable de dégrader le TBP mais avec un rendement global plus faible.In order to demonstrate that one or more cytochrome (s) actively participate in the degradation of TBP by the strain Rp. Palustris CGA009, this strain of bacteria is incubated in the presence of carbon monoxide (CO), which is an inhibitor of cytochrome P450 , in the presence (2 mM) or in the absence of TBP. It is possible to carry out the treatment with CO, before the inoculation of the culture, in two different ways: either by light bubbling, or by adding a culture medium already saturated with CO. A strain S. odorifera, which can only grow in aerobic condition in a Tryptic soy broth medium (Tsb; Sigma-Aldrich), is used for comparison; this strain being capable of degrading TBP but with a lower overall yield.
Les conditions de culture et de mesure du TBP sont identiques à celles utilisées dans l'exemple 2.The conditions for culturing and measuring TBP are identical to those used in Example 2.
Les résultats sont illustrés par la figure 4.The results are illustrated in Figure 4.
Comme le montrent les figures 4A et 4B, respectivement, la présence du CO dans le milieu n'affecte pas la croissance de chacune des deux souches lorsque le TBP est absent du milieu, et également pour la souche S. odorifera lorsqu'elle est mise en présence de TBP. De plus, la croissance de la souche Rp. palustris est fortement réduite en présence de CO (Figure 4B). Ainsi, la croissance de R. palustris est réellement réduite par l'inhibition des cytochromes P450 lorsque le
TBP est présent. Le dosage de la concentration du TBP résiduel après 21 jours de culture montre une diminution importante de la dégradation du TBP (Figure 5). Ceci suggère l'existence d'une corrélation entre l'inhibition des cytochromes P450 et le rendement faible de dégradation du TBP. Cette corrélation n'existe pas avec la souche S. odorifera pour laquelle le traitement au CO ne modifie pas d'une façon significative le rendement de la dégradation en TBP.As shown in FIGS. 4A and 4B, respectively, 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. In addition, the growth of the strain Rp. Palustris is greatly reduced in the presence of CO (Figure 4B). Thus, 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.
Exemple 5 : Construction d'une souche de Rp. palustris surexprimant le cytochrome P450 d'une façon constitutive.Example 5: Construction of a strain of Rp. Palustris overexpressing cytochrome P450 in a constitutive manner.
Le gène 5939 de Rp. palustris CG009 (toutes les informations concernant ce gène peuvent être trouvées sur le site http://genome.ornl.gov) codant pour le cytochrome P450 a été introduit dans la même souche.The gene 5939 of Rp. Palustris CG009 (all the information concerning this gene can be found on the site http://genome.ornl.gov) coding for cytochrome P450 was introduced into the same strain.
Pour ce faire, on réalise une fusion transcriptionnelle du promoteur du cluster génique du complexe de récupération de la lumière II (LH) αβe et du gène 5939 de façon à obtenir une expression constitutive du gène en question. Le promo- teur LHαβe est amplifié par PCR à partir de 300 pb en amont du codon de départ jusqu'à 68 pb en aval de celui-ci en utilisant l'ADN génomique de Rp. palustris CG009 comme matrice (10), avec les amorces suivantes :To do this, a transcriptional fusion is carried out of the promoter of the gene cluster of the light recovery complex II (LH) αβ e and of the gene 5939 so as to obtain a constitutive expression of the gene in question. The promoter LHαβ 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. Palustris CG009 as template (10), with the following primers:
5'-GGGGTACCCCTGGGATGTCCGGTATGACA-3' (SEQ ID NO:l) contenant un site de restriction Kpnl (indiqué dans la séquence en gras), et 5'-CCCAAGCTTGGGTTGTGGAGCTCTTCCGTTC-3' (SEQ ID5'-GGGGTACCCCTGGGATGTCCGGTATGACA-3 '(SEQ ID NO: l) containing a restriction site Kpnl (indicated in the bold sequence), and 5'-CCCAAGCTTGGGTTGTGGAGCTCTTCCGTTC-3' (SEQ ID
NO:2) contenant un site de restriction Hindlll (indiqué dans la séquence en gras).NO: 2) containing a HindIII restriction site (indicated in the sequence in bold).
Le fragment amplifié par PCR est clone dans le vecteur de clonage pGEM-T™ (Proméga) pour donner le plasmide pCB04. On amplifie ensuite le gène 5939 entier en utilisant les amorces suivantes : 5'-CCCAAGCTTGGGTGAACAACAACGAGGGAGTG-3' (SEQThe PCR amplified fragment is cloned into the cloning vector pGEM-T ™ (Proméga) to give the plasmid pCB04. The entire 5939 gene is then amplified using the following primers: 5'-CCCAAGCTTGGGTGAACAACAACGAGGGAGTG-3 '(SEQ
ID NO:3) contenant un site de restriction Hindlll (indiqué dans la séquence en gras), etID NO: 3) containing a HindIII restriction site (indicated in the sequence in bold), and
5'-CCCGAGCTCGGGCTATGGCGTGGAAATGGA-3' (SEQ ID NO:4) contenant un site de restriction Sαcl (indiqué dans la séquence en gras). Le fragment amplifié par PCR est digéré par Hindlll et Sαcl et ligaturé dans le plasmide pBBRlMCS2 (Kovach et al, 1994) aux mêmes sites, ce qui permet d'obtenir le plasmide pCB06.
Le plasmide pCB04 est digéré par Kpnl et Hindlll et clone dans le plasmide pCB06 digéré par ces mêmes enzymes de restriction, pour obtenir le plasmide pCB07. La carte du plasmide pCB07 est indiquée dans la figure 6.5'-CCCGAGCTCGGGCTATGGCGTGGAAATGGA-3 '(SEQ ID NO: 4) containing a Sαcl restriction site (indicated in the sequence in bold). The PCR amplified fragment is digested with Hindlll and Sαcl 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.
Afin de surexprimer le cytochrome P450 dans la souche Rp. palus- tris CG009, le plasmide pCB07 contenant le gène 5939 sous contrôle du promoteur LHαβe est transféré par conjugaison tri-parentale à partir des souches E.coli OYl5aphe (Eraso, et al, 1994) et de HB101 (Boyer et al., 1969) dans la souche Rp. palustris CG009 pour obtenir la souche Rp. palustris H5939 surexprimant le cytochrome P450 (clones positifs). Ces clones positifs sont sélectionnés dans un milieu contenant de la kanamycine à une concentration comprise entre 20 et 100 μg/ml.In order to overexpress cytochrome P450 in the Rp. Palustris CG009 strain, the plasmid pCB07 containing the gene 5939 under the control of the LHαβ 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 μg / ml.
Exemple 6 : Etude comparative de la dégradation du TBP par différents extraits cellulaires.Example 6: Comparative study of the degradation of TBP by different cell extracts.
A. Matériels et méthodesA. Materials and methods
Extraction et fractionnement des protéines La souche de Rp. palustris, cultivée sur du milieu de Hutner en présence ou en l'absence de 2 mM de TBP, a été récoltée à la fin de la phase exponentielle de croissance par centrifugation pendant 10 minutes à 8 000 g à 4°C. Les culots ont été lavés deux fois dans du tampon phosphate pH 7,2 et remis en suspension dans 20 ml du même tampon contenant un cocktail d'inhibiteurs de protéases (P8849, Sigma-Aldrich). Les cellules ont été cassées mécaniquement par trois passages à travers une presse cellulaire à 16000 psi et le lysat bactérien a été centrifugé pendant 20 minutes à 10 000 g pour enlever les débris cellulaires et les cellules intactes. L'extrait brut a été divisé en 3 fractions. Une centrifugation de 90 minutes à 200 000 g à 4°C a été effectuée pour sédimenter les membranes. Le surnageant a constitué la fraction des protéines solubles. De façon à dissocier les protéines faiblement associées à la membrane, le culot a été remis en suspension dans 2 ml de tampon 2M NaBr, 200 mM saccharose, 50 mM glycylglycine, à pH 6 et additionné d'un cocktail d'inhibiteurs de protéases, puis extrait par une agitation douce durant 30 minutes à 4°C ; le surnageant a constitué la fraction enrichie en protéines faiblement associées à la membrane. Le culot, contenant les protéines fortement associées à la membrane, a été remis en suspension dans 2 ml de tampon 50 mM Tris-HCl pH 8, lauryl- diaminooxyde (LDAO) 0,5% (vol/lvol) et additionné d'un cocktail d'inhibiteurs de
protéases. Les protéines ont été solubilisées par une agitation douce pendant 60 minutes à 4°C. L'extrait résultant a été centrifugé pendant 90 minutes à 200 000 g à 4°C pour culotter les matériaux insolubles. Le surnageant constitue la fraction des protéines membranaires. La concentration en protéines dans chaque extrait a été déterminée par la méthode du biuret selon le kit BCA Protein Assay Reagent (Uptima) en utilisant l'albumine sérique bovine comme standard. B. RésultatsProtein extraction and fractionation The Rp. Palustris strain, cultured on Hutner's medium in the presence or absence of 2 mM TBP, was harvested at the end of the exponential growth phase by centrifugation for 10 minutes at 8 000 g at 4 ° C. The pellets were washed twice in phosphate buffer pH 7.2 and resuspended in 20 ml of the same buffer containing a cocktail of protease inhibitors (P8849, Sigma-Aldrich). The cells were mechanically broken by three passes through a cell press at 16,000 psi and the bacterial lysate was centrifuged for 20 minutes at 10,000 g to remove cell debris and intact cells. The crude extract was divided into 3 fractions. 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. In order to dissociate the proteins weakly associated with the membrane, 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. B. Results
Les tests de dégradation du TBP ont été réalisés avec des protéines extraites de Rp. palustris CGA009. Les conditions optimales du test sont les suivan- tes : pH 6,5 et 40°C.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.
Les extraits cellulaires sont obtenus dans les conditions exposées ci- dessus au chapitre Matériel et méthodes ; ils sont en particulier obtenus par extraction des protéines à partir de la souche Rp. palustris cultivée en conditions micro-anaéro- bies, dans du milieu de Hutner en présence (cultures induites) ou en l'absence (cultures non induites) de 2 mM de TBP.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.
La figure 7 montre que 70 % de TBP est dégradé par des extraits cellulaires à 40°C dans des conditions aérobies pendant 20 heures, indépendamment de la présence éventuelle de TBP dans le milieu de culture (figure 7 : (1) et (3)). Une expérience a également été réalisée dans de conditions anaérobies. Dans ce cas, on n'observe aucune dégradation (figure 7 : (2)) ; ceci implique que l'oxygène est nécessaire pour dégrader le TBP, et ce, comme pour les cellules entières.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.
Les essais de dégradation du TBP ont été comparés aux résultats obtenus avec différents contrôles : tampon de réaction sans protéine, extrait brut bouilli de Rp. palustris CGA009 et extrait brut d'E. coli DH5α (figure 7 : (5), (6) et (7)).The 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 DH5α (Figure 7: (5), (6) and (7)).
Ces résultats montrent :These results show:
- que la dégradation se fait par un processus enzymatique ; en effet, on n'observe aucune dégradation dans un certain nombre de tests (voir figure 7) ainsi qu'aucune accumulation significative de TBP dans les cellules pendant une période de culture de 21 jours : il n'y a donc pas de séquestration du composé non dégradé.- that the degradation takes place by an enzymatic process; indeed, no degradation is observed in a certain number of tests (see FIG. 7) as well as no significant accumulation of TBP in the cells during a culture period of 21 days: there is therefore no sequestration of the undegraded compound.
- le processus de dégradation est dépendant de l'oxygène à la fois in vitro et in vivo. Cependant, il n'existe clairement aucune corrélation stoechiométrique
entre la quantité d'oxygène et la quantité de TBP dégradé. La concentration initiale en O2 optimale est d'environ 70 μM, dans le milieu micro-anaérobie sélectionné.- the degradation process is dependent on oxygen both in vitro and in vivo. However, there is clearly no stoichiometric correlation between the amount of oxygen and the amount of degraded TBP. The initial optimal O 2 concentration is approximately 70 μM, in the selected micro-anaerobic medium.
- la dégradation de TBP est un mécanisme constitutif de la cellule, dans la mesure où les extraits cellulaires de bactéries mis en culture en présence ou en l'absence de TBP ont la même efficacité ; en outre, une sous-culture obtenue à partir d'une souche préalablement mise en culture dans un milieu contenant du TBP présente le même profil qu'une sous-culture obtenue à partir d'une culture non induite (croissance dans un milieu en l'absence de TBP) : les cinétiques de dégradation sont identiques. Exemple 7 : Etude comparative de la cinétique de dégradation du TBP en présence de différentes concentrations en TBP et par différents extraits cellulaires.- 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.
A. Matériel et méthodesA. Materials and methods
Analyses cinétiques Les paramètres cinétiques de la dégradation du TBP ont été calculés à partir de l'équation de Michaelis-Menten, V= 'Km+[S] ou ^ correspond à la vitesse de dégradation du TBP et S à la concentration en TBP. Cette équation a été utilisée pour ajuster les différentes données expérimentales de V= [S]) en utilisant une méthode d'ajustement non-linéaire (SigmaPlot). Cette approche est plus précise que l'ajustement linéaire du point double inverse (Lineweaver-Burk), du fait de la taille variable des barres d'erreurs dans cette dernière. Comme l'activité enzymatique a été dosée dans un système d'extrait brut, les paramètres cinétiques ont été exprimés en tant que concentration apparente de TBP pour la moitié de l'activité maximale (Kmap) et pour la vitesse maximale apparente (Vmap). B. RésultatsKinetic analyzes The kinetic parameters of the degradation of TBP were calculated from the Michaelis-Menten equation, V = 'Km + [S] where ^ corresponds to the rate of degradation of TBP and S to the TBP concentration. This equation was used to adjust the different experimental data of V = [S]) using a non-linear adjustment method (SigmaPlot). This approach is more precise than the linear adjustment of the double inverse point (Lineweaver-Burk), due to the variable size of the error bars in the latter. As the enzymatic activity was measured in a crude extract system, the kinetic parameters were expressed as the apparent concentration of TBP for half of the maximum activity (Km ap ) and for the maximum apparent speed (Vm ap ). B. Results
La figure 8A montre la cinétique de dégradation du TBP par des extraits cellulaires en fonction de différentes concentrations en TBP : 2, 1,5, 1, 0,75, 0,5, 0,25, 0,1 et 0,05 mM.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 .
La figure 8B montre la meilleure correspondance de l'équation de Michaelis-Menten avec des valeurs de Vmap et de Kmap respectivement de : 0,48+0,04 mM.h-1 et 2,6+0,4 mM.
L'analyse de la cinétique de dégradation réalisée à l'aide d'extraits cellulaires est consistante avec l'équation de Michaelis-Menten. Ceci permet d'estimer le Kmap et la Vmap, bien que la vitesse maximale ne puisse pas être atteinte dans les conditions d'expérimentations (solubilité faible du TBP dans l'eau). Un Kmap de 2,6 mM indique une affinité apparente modérée du système enzymatique pour le TBP. La Vmap était de 0,48 mM.li'1.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 .
Pour des concentrations en TBP comprises entre 2 et 0,5 mM, la cinétique présente un ralentissement significatif, au bout d'un moment, ce qui est probablement dû à une inhibition par le produit (courbe biphasique). Pour des concentra- tions en TBP inférieures à 0,5 mM, la cinétique suit une courbe monophasique (voir également exemple 2).For TBP concentrations between 2 and 0.5 mM, the kinetics show a significant slowing down, after a while, which is probably due to inhibition by the product (biphasic curve). For TBP concentrations less than 0.5 mM, the kinetics follow a single-phase curve (see also example 2).
Exemple 8 : Influence de cofacteurs sur la dégradation du TBP par des extraits cellulaires.Example 8 Influence of cofactors on the degradation of TBP by cell extracts.
Un extrait brut, obtenu dans les conditions décrites à l'exemple 6 est dialyse de manière à réduire la concentration en molécules de bas poids moléculaire. Après la dialyse, le dégradation du TBP est diminuée d'un facteur 3, en comparaison avec le contrôle non dialyse (figure 9, (1) et (2)). Dans ces conditions, l'effet de différents cofacteurs a été testé : NADH, NADPH, FMNH2 et FADH2. Sur des extraits cellulaires non dialyses, l'addition de ces cofacteurs n'améliore pas de manière signi- ficative la dégradation du TBP. Par contre, la dégradation par des échantillons dialyses est augmentée par l'addition de NADPH ou de FMNH2 (figure 9, (4) et (6)). La combinaison de ces deux cofacteurs restaure complètement l'activité de dégradation observée avec les échantillons non dialyses (figure 9, (11)). Exemple 9 : Dégradation du TBP par des fractions de protéines Pour être en mesure de localiser l'activité enzymatique impliquée dans la dégradation du TBP par Rp. palustris, un extrait brut est fractionné en trois fractions de protéines : fraction soluble, fraction faiblement associée à la membrane et fraction liée à la membrane ; ces différentes fractions sont obtenues dans les conditions exposées à l'exemple 6. Les tests de dégradation du TBP sont effectués dans les conditions exposées à l'exemple 2.A crude extract obtained under the conditions described in Example 6 is dialyzed so as to reduce the concentration of low molecular weight molecules. After dialysis, the degradation of TBP is reduced by a factor of 3, in comparison with the non-dialysis control (Figure 9, (1) and (2)). Under these conditions, the effect of different cofactors was tested: NADH, NADPH, FMNH 2 and FADH 2 . On non-dialyzed cell extracts, the addition of these cofactors does not significantly improve the degradation of TBP. On the other hand, 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.
La figure 10 montre que l'activité de dégradation est plus particulièrement localisée dans la fraction de protéines faiblement associées à la membrane,
avec 3,5 μM de TBP dégradé.mg de protéines"1, comparé aux quantités dégradées respectivement par la fraction de protéines totales (1,2 μM de TBP dégradé.mg de protéines"1), la fraction soluble (2 μM de TBP dégradé.mg de protéines"1) et la fraction liée à la membrane (1,65 μM de TBP dégradé.mg de protéines"1). Les protéines impliquées dans la dégradation du TBP sont faiblement associées à la membrane. L'activité est dépendante à la fois du NADPH et du FMNH2 et confirme le rôle du cytochrome P450 dans l'activité de dégradation du TBP.FIG. 10 shows that the degradation activity is more particularly localized in the fraction of proteins weakly associated with the membrane, with 3.5 μM of degraded TBP.mg of protein "1 , compared to the amounts degraded respectively by the total protein fraction (1.2 μM of degraded TBP.mg of protein " 1 ), the soluble fraction (2 μM of TBP degraded.mg of protein "1 ) and the membrane-bound fraction (1.65 μM 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.
Exemple 10 : Activité d'une souche de Rp. palustris dépourvue du plasmide en- dogène pRpalExample 10 Activity of a strain of Rp. Palustris lacking the endogene plasmid pRpal
A. Matériels et méthodes Techniques moléculairesA. Materials and methods Molecular techniques
L'isolement des plasmides a été réalisé à l'aide du kit NucleoSpin Plasmid (Macherey-Nagel) en suivant les instructions du fabricant. L'isolement de l'ADN génomique a été effectué à l'aide du kit NucleoSpin Tissue (Macherey-Nagel) en suivant les instructions du fabricant.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.
Durant les processus de conjugaison, les plasmides ont été obtenus par croisement tri-parental à partir des souches d'E. coli DH5α phe (donneur), contenant le plasmide pMG105, et HB101 (helper) vers la souche Rp. palustris (accepteur). Les cellules d'E. coli ont été cultivées dans du milieu LB + Km jusqu'à ce que la valeur de DO660 atteigne 1,5, et les cellules de Rp. palustris ont été cultivées sur le milieu de Hutner jusqu'à ce que la valeur de DO660 atteigne 1 (cultures pendant une nuit). Les bactéries « donneur, helper et accepteur » (100 μl, 10 μl et 1 ml respectivement) ont été lavées et regroupées sur une boîte de milieu de Hutner. Cette boîte a été incubée pendant \6 heures à 30°C. Les bactéries ont été remises en suspension dans 1 ml de milieu PM, étalées sur des boîtes PM + Km et incubées 4 jours, soit dans des conditions aérobies, soit dans des conditions de micro-aérobiose. Expériences d'élimination de plasmidesDuring the conjugation processes, the plasmids were obtained by tri-parental crossing from E. strains. coli DH5α 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 μl, 10 μl 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
Pour éliminer le plasmide pRpal, le plasmide pMG105 (11), un vecteur de clonage présentant un gène de résistance à Km, qui possède la même origine de réplication que pRpal a été introduit par croisement chez R. palustris. Les transconjugants porteur de pMG105 ont été sélectionnés sur des boîtes PM + Km.
Généralement, les bactéries Km résistantes ont perdu pRpal. Les souches porteuses de pMG105 ont été cultivées sur un milieu sans Km pour expulser ce plasmide après plusieurs sous-cultures et la souche résultante sans plasmide est conservée en tant que Rp. palustris ΔpRpal. Chaque étape de l'expérience d'élimination est contrôlée par un isolement du plasmide. Les bactéries R. palustris ΔpRpal sont testées pour leur capacité à dégrader le TBP.To eliminate the plasmid pRpal, 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 ΔpRpal. Each step of the elimination experiment is monitored by isolation of the plasmid. R. palustris ΔpRpal bacteria are tested for their ability to degrade TBP.
B. RésultatsB. Results
Une extraction de plasmide a montré que Rp. palustris comprend un plasmide endogène (pRpal, 9,8 kb). Cette caractéristique a été confirmée par le séquençage du génome de Rp. palustris (12).Plasmid extraction has shown that Rp. Palustris comprises an endogenous plasmid (pRpal, 9.8 kb). This characteristic was confirmed by the sequencing of the genome of Rp. Palustris (12).
La croissance, la forme morphologique ainsi que la pigmentation des colonies sont identique dans la souche ΔpRpal et dans la souche sauvage. Rp. palustris est naturellement résistant à la gentamycine (50 μg.ml"1) et la souche sans plasmide croit en présence de cet antibiotique, comme la souche sauvage. La souche sans plas- mide croit également dans un milieu contenant du TBP, comme la souche sauvage, et présente une cinétique de dégradation du TBP similaire à celle de la souche sauvage. Ceci indique que les enzymes impliquées dans la dégradation du TBP par Rp.palustris ne sont pas codées par des gènes portés par le plasmide endogène mais plutôt par l'ADN chromosomique. Rp. palustris est efficace dans le processus de dépollution et la souche sans plasmide également ; cette dernière peut en outre constituer un outil pour déterminer la localisation plasmidique ou chromosomique de gènes impliqués dans d'autres voies de dégradation. REFERENCES BIBLIOGRAPHIQUES 1. Thomas R. A. et al.,. Appl Microbiol Biotechnol., 1998, 49, 202-209.The growth, the morphological form as well as the pigmentation of the colonies are identical in the ΔpRpal strain and in the wild strain. Rp. Palustris is naturally resistant to gentamycin (50 μg.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. Rp. 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. 1. Thomas RA et al., Appl Microbiol Biotechnol., 1998, 49, 202-209.
2. Thomas R. A. P. et al., Biotechnol Techn., 1997, 11, 781-785.2. Thomas R. A. P. et al., Biotechnol Techn., 1997, 11, 781-785.
3. Thomas, R. A. P. et al., Environ. Sci. and Biotechnol, 1996, 30, 2371-2375.3. Thomas, R. A. P. et al., Environ. Sci. and Biotechnol, 1996, 30, 2371-2375.
4. Thomas R. A. P. et al., FEMS Microbiology Lett, 1997,155, 155-159.4. Thomas R. A. P. et al., FEMS Microbiology Lett, 1997, 155, 155-159.
5. Owen S. et al., Appl Biochem Biotechnol, 1992, 34/35, 693-707. 6. Clayton R., K., Biochim. Biophys. Acta. , 1960, 37, 503-512.5. Owen S. et al., Appl Biochem Biotechnol, 1992, 34/35, 693-707. 6. Clayton R., K., Biochim. Biophys. Acta. , 1960, 37, 503-512.
7. Boyer H.W. et al., J Mol. Biol, 1969, 41, 459-72.7. Boyer H.W. et al., J Mol. Biol, 1969, 41, 459-72.
8. Eraso J.M. et al., J Bacteriol, 1994, 176, 32-43.
9. Kovach M.E. et al., Biotechniques, 1994, 16, 800-2.8. Eraso JM et al., J Bacteriol, 1994, 176, 32-43. 9. Kovach ME et al., Biotechniques, 1994, 16, 800-2.
10. Tadros M.H. et al., Eur. J. Biochem., 1993, 217, 867-75.10. Tadros M.H. et al., Eur. J. Biochem., 1993, 217, 867-75.
11. Inui M et al., Appl. Environ. Microbiol, 2000, 66, 54-6311. Inui M et al., Appl. About. Microbiol, 2000, 66, 54-63
12. Larimer F. et al., Nat. Biotechnol., 2004, 22, 55-61
12. Larimer F. et al., Nat. Biotechnol., 2004, 22, 55-61
Claims
REVENDICATIONS
1°) Procédé de traitement ou d'épuration de déchets liquides chargés en phosphate de tributyle (TBP), caractérisé en ce qu'il comprend essentiellement les étapes de : (1) mise en contact desdits déchets liquides avec au moins une souche de bactérie photosynthétique pourpre non-sulfureuse résistante au TBP sélectionnée dans le groupe constitué par Rhodopseudomonas palustris (Rp. palustris), Rhodospirillum rubrum (Rs. rubrum), Rhodobacter capsulatus (Rb. capsulatus) ou Rhodobacter sphaeroides (Rb. sphaeroides) ainsi que lesdites souches de bactéries modifiées pour surexprimer le cytochrome P450, dans des conditions permettant la dégradation du TBP présent dans lesdits déchets et ce quelle que soit la concentration initiale en TBP ; et1°) Process for treating or purifying liquid waste loaded with tributyl phosphate (TBP), characterized in that it essentially comprises the steps of: (1) bringing said liquid waste into contact with at least one strain of bacteria non-sulfurous purple photosynthetic 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 the TBP present in said waste, regardless of the initial TBP concentration; And
(2) la récupération des effluents liquides purifiés.(2) recovery of purified liquid effluents.
2°) Procédé selon la revendication 1, caractérisé en ce que l'étape (1) de mise en contact est mise œuvre en micro-anaérobiose.2°) Method according to claim 1, characterized in that the contacting step (1) is carried out in micro-anaerobiosis.
3°) Procédé selon la revendication 1 ou la revendication 2, caractérisé en ce que ladite souche de bactérie résistante au TBP mise en œuvre à l'étape (1) du procédé est de préférence sélectionnée dans le groupe constitué par Rp. palustris et Rs. rubrum. 4°) Procédé selon l'une quelconque des revendications 1 à 3, caractérisé en ce que l'étape (1) comprend la mise en œuvre d'un mélange de souches de bactéries pourpres non-sulfureuses résistantes au TBP comprenant au moins une souche sélectionnée dans le groupe constitué par Rhodospirillum rubrum (Rs. rubrum) et Rhodopseudomonas palustris (Rp. palustris ) et au moins une autre souche sélec- tionnée dans le groupe constitué par Rhodobacter capsulatus (Rb. capsulatus) et Rhodobacter sphaeroides (Rb. sphaeroides).3°) Method according to claim 1 or claim 2, characterized in that said strain of bacteria resistant to TBP implemented in step (1) of the method is preferably selected from the group consisting of Rp. palustris and Rs . rubrum. 4°) Method according to any one of claims 1 to 3, characterized in that step (1) comprises the implementation of a mixture of strains of non-sulfurous purple 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) .
5°) Procédé selon l'une quelconque des revendications 1 à 4, caractérisé en ce que les souches de bactéries pourpres non-sulfureuses résistantes au TBP sont sélectionnées dans le groupe constitué par les souches de Rp. 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 et n°DSM 130, la souche Rs.
rubrum SI n°ATCC 11170, la souche Rb. capsulatus Saint-Louis n°ATCC 23782 et la souche Rb. sphaeroides 2.4.1. n°ATCC 17023.5°) Method according to any one of claims 1 to 4, characterized in that the strains of non-sulfurous purple bacteria resistant to TBP are selected from the group consisting of the strains of Rp. palustris CGA009 n°ATCC BAA-98 , No. ATCC 17002, No. ATCC 17007, No. DSM 8283, No. DSM 126, No. DSM 7375, No. DSM 131, No. DSM 25, No. DSM 124 and No. DSM 130, strain Rs. rubrum SI n°ATCC 11170, the Rb strain. capsulatus Saint-Louis n°ATCC 23782 and the Rb strain. sphaeroides 2.4.1. n°ATCC 17023.
6°) Procédé selon l'une quelconque des revendications 1 à 5, caractérisé en ce que l'étape (1) de mise en contact est effectuée à une température com- prise entre 10°C et 37°C, de préférence à 30°C, à un pH compris entre 5,5 et 8,5, de préférence à 6,9 et pendant au moins 15 jours, de préférence entre 15 et 21 jours.6°) Method according to any one of claims 1 to 5, characterized in that the contacting step (1) is carried out at a temperature between 10°C and 37°C, preferably at 30°C. °C, at a pH between 5.5 and 8.5, preferably at 6.9 and for at least 15 days, preferably between 15 and 21 days.
7°) Procédé selon l'une quelconque des revendications 1 à 6, caractérisé en ce que le taux de TBP dans lesdits déchets liquides avant traitement est compris entre 0,01 mM et 1 M. 8°) Procédé selon l'une quelconque des revendications 1 à 7, caractérisé en ce que les quantités de bactéries inoculées dans le milieu à traiter à l'étape (1) sont avantageusement comprises entre 104 et 1010 bactéries par ml de milieu à traiter.7°) Method according to any one of claims 1 to 6, characterized in that the level of TBP in said liquid waste before treatment is between 0.01 mM and 1 M. 8°) Method according to any one of claims 1 to 7, characterized in that the quantities 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.
9°) Procédé selon l'une quelconque des revendications 1 à 8, caractérisé en ce qu'au cours de l'étape (1), on ajoute une source de carbone supplémen- taire, dans le milieu à traiter.9°) Method according to any one of claims 1 to 8, characterized in that during step (1), an additional carbon source is added to the medium to be treated.
10°) Procédé selon la revendication 9, caractérisé en ce que ladite source de carbone supplémentaire est avantageusement une solution tamponnée contenant un extrait de levure à une concentration comprise entre 0,1 et 10 g/1 ou l'un des sels organiques suivants : succinate, glutamate, benzoate, malate ou fumarate à une concentration comprise entre 2 et 20 mM (de préférence à 10 mM), et des facteurs de croissance dont au minimum la biotine et l'acide para amino benzoïque à une concentration comprise entre 2 et 40 μg/1 chacun.10°) Method according to claim 9, characterized in that said additional carbon source is advantageously a buffered solution containing a yeast extract at a concentration of between 0.1 and 10 g/1 or one of the following organic salts: succinate, glutamate, benzoate, malate or fumarate at a concentration between 2 and 20 mM (preferably 10 mM), and growth factors including at least biotin and para amino benzoic acid at a concentration between 2 and 40 μg/1 each.
11°) Procédé selon l'une quelconque des revendications 1 à 10, caractérisé en ce que préalablement à ladite mise en contact selon l'étape (1), les bacté- ries pourpres non-sulfureuses résistantes au TBP sont sélectionnées par culture sur milieu contenant au moins 12 μM et de préférence au moins 1 mM de TBP.11°) Method according to any one of claims 1 to 10, characterized in that prior to said contacting according to step (1), the non-sulfurous purple bacteria resistant to TBP are selected by culture on medium containing at least 12 μM and preferably at least 1 mM of TBP.
12°) Procédé de suivi ou de surveillance de la dégradation du TBP dans des déchets liquides, lequel procédé comprend :12°) Process for monitoring or monitoring the degradation of TBP in liquid waste, which process comprises:
- la mise en œuvre du procédé de traitement de déchets liquides chargés en TBP selon l'une quelconque des revendications 1 à 1 1, puis
- le prélèvement d'un échantillon de milieu traité au moins au temps t+15 jours et la mesure dans ledit échantillon prélevé de la concentration de TBP résiduel.- the implementation of the process for treating liquid waste loaded with TBP according to any one of claims 1 to 1 1, then - taking a sample of treated medium at least at time t+15 days and measuring the concentration of residual TBP in said sample taken.
13°) Coffret pour la mise en œuvre du procédé de traitement selon l'une quelconque des revendications 1 à 1 1, caractérisé en ce qu'il comprend au moins une souche de bactérie photosynthétique pourpre non-sulfureuse résistante au TBP, choisie dans le groupe constitué par Rp. palustris, Rs. rubrum, Rb. capsulatus ou Rb. sphaeroides ainsi que lesdites souches de bactéries modifiées pour surexprimer le cytochrome P450, telles que définies aux revendications 1, 3, 4 ou 5. 14°) Souches de bactéries photosynthétiques pourpres non- sulfureuses, caractérisées en ce qu'elles sont résistantes au TBP, en ce qu'elles sont sélectionnées dans le groupe constitué par Rp. palustris, Rs. rubrum, Rb. capsulatus et Rb. sphaeroides, et en ce que leur ADN inclut au moins une copie supplémentaire du gène codant pour un cytochrome P450 homologue. 15°) Souche de bactéries selon la revendication 14, caractérisée en ce qu'il s'agit d'une souche de Rp. palustris CGA009 n°ATCC BAA-98 comprenant un gène codant pour le cytochrome P450 de Rp. palustris, sous le contrôle du promoteur LHαβe.13°) Box for implementing the treatment process according to any one of claims 1 to 1 1, characterized in that it comprises at least one strain of non-sulfurous purple photosynthetic bacteria resistant to TBP, chosen from the group consisting of Rp. palustris, Rs. rubrum, Rb. capsulatus or Rb. sphaeroides as well as said strains of bacteria modified to overexpress cytochrome P450, as defined in claims 1, 3, 4 or 5. 14°) Strains of non-sulphurous 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 and Rb. sphaeroides, and in that their DNA includes at least one additional copy of the gene coding for a homologous cytochrome P450. 15°) Strain of bacteria according to claim 14, characterized in that it is a strain of Rp. palustris CGA009 n°ATCC BAA-98 comprising a gene coding for cytochrome P450 of Rp. palustris, under the control of the LHαβ e promoter.
16°) Souche de bactérie photosynthétique pourpre non-sulfureuse, caractérisée en ce qu'elle est résistante au TBP et en ce qu'elle est constituée par une souche de Rp. palustris dépourvue de plasmide endogène.16°) Strain of non-sulfurous purple photosynthetic bacteria, characterized in that it is resistant to TBP and in that it consists of a strain of Rp. palustris devoid of endogenous plasmid.
17°) Dispositif d'épuration des déchets liquides chargés en TBP, caractérisé en ce qu'il comprend au moins un bassin de décantation adapté à la mise en contact des bactéries pourpres non-sulfureuses avec le milieu à traiter, de préfé- rence en anaérobiose ou micro-anaérobiose.17°) Device for purifying liquid waste loaded with TBP, characterized in that it comprises at least one settling basin adapted to bringing non-sulfurous purple bacteria into contact with the medium to be treated, preferably in anaerobiosis or micro-anaerobiosis.
18°) Dispositif selon la revendication 17, caractérisé en ce que ledit bassin de décantation est, de préférence, d'une profondeur allant de 0,5 à 1 mètre pour une surface en accord avec le débit des effluents, sachant que le temps de rétention doit être au minimum de 15-21 jours. 19°) Dispositif selon la revendication 17 ou la revendication 18, caractérisé en ce qu'il comprend outre ledit bassin de décantation, des moyens d'introduction desdits déchets liquides contenant du TBP dans ledit bassin de décan-
tation, des moyens d'introduction de l'inoculum de bactéries, des moyens d'évacuation des effluents épurés en TBP vers l'extérieur ainsi que des moyens de récupération des bactéries à recycler.18°) Device according to claim 17, characterized in that said settling basin is, preferably, of a depth ranging from 0.5 to 1 meter for a surface in accordance with the flow rate of the effluents, knowing that the time of retention should be a minimum of 15-21 days. 19°) Device according to claim 17 or claim 18, characterized in that it comprises, in addition to said settling basin, means for introducing said liquid waste containing TBP into said settling basin. tation, means of introducing the bacterial inoculum, means of evacuating the effluent purified in TBP to the outside as well as means of recovering the bacteria to be recycled.
20°) Dispositif selon l'une quelconque des revendications 17 à 19, caractérisé en ce que ledit moyen de récupération des bactéries est avantageusement sélectionné dans le groupe constitué par les filtres à diatomées et les centrifugeuses en continu.
20°) Device according to any one of claims 17 to 19, characterized in that said bacteria recovery means is advantageously selected from the group consisting of diatomaceous earth filters and continuous centrifuges.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR0305762A FR2854886B1 (en) | 2003-05-14 | 2003-05-14 | METHOD FOR DEGRADING TBP BY A BACTERIAL PHOTOSYNTHETIC STRAIN |
| PCT/FR2004/001165 WO2004101449A2 (en) | 2003-05-14 | 2004-05-13 | Method of degrading tbp using a photosynthetic bacterial strain |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP1638897A2 true EP1638897A2 (en) | 2006-03-29 |
Family
ID=33306335
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP04742718A Withdrawn EP1638897A2 (en) | 2003-05-14 | 2004-05-13 | Method of degrading tbp using a photosynthetic bacterial strain |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US9029127B2 (en) |
| EP (1) | EP1638897A2 (en) |
| CA (1) | CA2525314C (en) |
| FR (1) | FR2854886B1 (en) |
| WO (1) | WO2004101449A2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101269227B (en) * | 2007-03-21 | 2011-07-27 | 北京世纪星伊埃姆生物技术研究所 | Biological agent for purifying air, preparation method and uses thereof |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100934601B1 (en) * | 2005-04-21 | 2009-12-31 | 이비덴 가부시키가이샤 | Method of treating wastewater containing organic compound |
| US8658411B2 (en) | 2005-04-21 | 2014-02-25 | Ibiden Co., Ltd. | Method of treating wastewater containing organic compound |
| CN103043804A (en) * | 2012-12-26 | 2013-04-17 | 中国农业大学 | Method for treating soybean processing wastewater by utilizing photosynthetic bacteria and recycling wastewater |
| CN103058387B (en) * | 2012-12-26 | 2014-12-10 | 中国农业大学 | Method of treating and recycling food processing waste water by using photosynthetic bacterium |
| CN105174492B (en) * | 2015-10-09 | 2019-07-09 | 天津大学 | A kind of nitrifier fast quick-recovery promotor after being inhibited by heavy metal |
| CN108611360B (en) * | 2018-05-14 | 2022-04-05 | 陕西师范大学 | Lycopene-producing rhodobacter sphaeroides engineering strain and construction method thereof |
| CN110076193B (en) * | 2018-08-24 | 2021-06-11 | 马莹 | Pseudomonas libanoides MY and application thereof in heavy metal polluted saline soil remediation |
| CN109554298A (en) * | 2019-01-10 | 2019-04-02 | 三峡大学 | The enrichment method of a kind of microbial flora and its in the processing heavy metal-polluted application caught of Cr |
| CN110261267B (en) * | 2019-05-30 | 2023-06-13 | 中国水产科学研究院南海水产研究所 | Detection method of photosynthetic bacteria preparation product for fishing |
| CN110627224A (en) * | 2019-09-29 | 2019-12-31 | 海南卓越生物有限公司 | Formula and preparation method of water quality purifying agent for bog red pseudo single-package flora |
| CN113862186B (en) * | 2021-10-12 | 2024-04-16 | 南通华宇化工科技有限公司 | Microbial composite microbial agent and preparation method and application thereof |
| CN116161799B (en) * | 2022-01-07 | 2024-04-26 | 南通聚益成广生物科技有限公司 | Method for treating food waste water by utilizing photosynthetic bacteria |
| CN114956352B (en) * | 2022-05-31 | 2023-06-02 | 广西民族大学 | Rhodopseudomonas palustris-nano TiO 2 Composite material, preparation method and application thereof |
| CN116337791B (en) * | 2023-05-31 | 2023-08-15 | 北京挑战生物技术有限公司 | In-vitro detection method for release rate of phosphorus phytate in feed raw material |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59173197A (en) * | 1983-03-24 | 1984-10-01 | Chiyoda Chem Eng & Constr Co Ltd | Fermentating method of organic waste liquid |
| US5453375A (en) * | 1993-08-18 | 1995-09-26 | Lockheed Idaho Technologies Company | Method and compositions for the degradation of tributyl phosphate in chemical waste mixtures |
| FR2712604B1 (en) * | 1993-11-15 | 1996-02-02 | Commissariat Energie Atomique | Cloning and expression cassette, expression vectors and transformed bacteria comprising the Frup promoter from R. Capsulatus; their applications. |
| US6106719A (en) * | 1998-09-14 | 2000-08-22 | Director-General Of Agency Of Industrial Science And Technology | Process for treating liquid waste using light under anaerobic conditions |
| JP2003089997A (en) * | 2001-09-14 | 2003-03-28 | Toru Oki | Biodegradable paper product |
-
2003
- 2003-05-14 FR FR0305762A patent/FR2854886B1/en not_active Expired - Fee Related
-
2004
- 2004-05-13 EP EP04742718A patent/EP1638897A2/en not_active Withdrawn
- 2004-05-13 US US10/556,324 patent/US9029127B2/en not_active Expired - Fee Related
- 2004-05-13 WO PCT/FR2004/001165 patent/WO2004101449A2/en active Application Filing
- 2004-05-13 CA CA2525314A patent/CA2525314C/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| See references of WO2004101449A2 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101269227B (en) * | 2007-03-21 | 2011-07-27 | 北京世纪星伊埃姆生物技术研究所 | Biological agent for purifying air, preparation method and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2004101449A8 (en) | 2005-04-07 |
| FR2854886A1 (en) | 2004-11-19 |
| CA2525314C (en) | 2015-01-13 |
| US20080145917A1 (en) | 2008-06-19 |
| FR2854886B1 (en) | 2007-07-20 |
| WO2004101449A2 (en) | 2004-11-25 |
| US9029127B2 (en) | 2015-05-12 |
| CA2525314A1 (en) | 2004-11-25 |
| WO2004101449A3 (en) | 2005-02-03 |
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