WO2019162618A1 - Method for evaluating the biodegradable organic material content of an aqueous sample and kit for implementation thereof - Google Patents
Method for evaluating the biodegradable organic material content of an aqueous sample and kit for implementation thereof Download PDFInfo
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- WO2019162618A1 WO2019162618A1 PCT/FR2019/050394 FR2019050394W WO2019162618A1 WO 2019162618 A1 WO2019162618 A1 WO 2019162618A1 FR 2019050394 W FR2019050394 W FR 2019050394W WO 2019162618 A1 WO2019162618 A1 WO 2019162618A1
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- bacteria
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 9
- 229910052799 carbon Inorganic materials 0.000 claims description 9
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 7
- 229910052760 oxygen Inorganic materials 0.000 claims description 7
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- 244000063299 Bacillus subtilis Species 0.000 claims description 6
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 6
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- 241000589518 Comamonas testosteroni Species 0.000 claims description 6
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- 241000588724 Escherichia coli Species 0.000 claims description 6
- 241000588770 Proteus mirabilis Species 0.000 claims description 6
- 241000589776 Pseudomonas putida Species 0.000 claims description 6
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 claims description 6
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- 241001165358 Acinetobacter towneri Species 0.000 claims description 4
- 241000589155 Agrobacterium tumefaciens Species 0.000 claims description 4
- 241000977264 Bacillus funiculus Species 0.000 claims description 4
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- 241001631289 Bosea vestrisii Species 0.000 claims description 4
- 241000235863 Comamonas denitrificans Species 0.000 claims description 4
- 241001106040 Franconibacter helveticus Species 0.000 claims description 4
- 241000157908 Paenarthrobacter aurescens Species 0.000 claims description 4
- 241000589597 Paracoccus denitrificans Species 0.000 claims description 4
- 241001633977 Paracoccus pantotrophus Species 0.000 claims description 4
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- 241000736091 Sphingobium yanoikuyae Species 0.000 claims description 4
- 241001147687 Staphylococcus auricularis Species 0.000 claims description 4
- 241001147693 Staphylococcus sp. Species 0.000 claims description 4
- 241001288848 Thauera linaloolentis Species 0.000 claims description 4
- 241000123669 Zoogloea resiniphila Species 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 3
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- HSSLDCABUXLXKM-UHFFFAOYSA-N resorufin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3N=C21 HSSLDCABUXLXKM-UHFFFAOYSA-N 0.000 description 2
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- 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
- C02F3/341—Consortia of bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/1806—Biological oxygen demand [BOD] or chemical oxygen demand [COD]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/186—Water using one or more living organisms, e.g. a fish
- G01N33/1866—Water using one or more living organisms, e.g. a fish using microorganisms
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2209/00—Controlling or monitoring parameters in water treatment
- C02F2209/08—Chemical Oxygen Demand [COD]; Biological Oxygen Demand [BOD]
Definitions
- the present invention relates to a method for evaluating the biodegradable organic matter content of an aqueous sample, a kit for carrying out this evaluation and its application to effluents such as effluents from purification plants, industrial effluents or effluents. breeding.
- the measurement of oxygen is carried out by an iodometric method or by means of an electrochemical probe, the value of the DB05 being expressed in mg of 0 2 / L.
- This method is only suitable for values between 0 and 6 mg of 02 / L, which requires prior dilutions, and has a large variability (of the order of 20%).
- its duration (5 days) makes it impossible to follow up online: for example the monitoring of the quality of discharges from treatment plants.
- biosensors To obtain faster information and to enable an on-line measurement of the biodegradable carbonaceous material, biosensors have been developed. However, these devices require a heavy preliminary phase of calibration and parameterization on site.
- a first object of the invention is therefore to propose a method for evaluating the content of biodegradable organic matter in an aqueous sample which is rapid (preferably a few hours), allowing for example to pass on the information on environmental conditions. purification, precise and reproducible.
- Another aim of the invention is to propose a method for estimating the content of biodegradable organic matter over a large concentration, without requiring dilution of the sample and reducing the variability between measurements.
- Another object of the invention is to provide a method that is easy to implement and can be automated.
- Another object of the invention is to propose a method whose results can be converted into a value equivalent to the value of DBOs defined by the current standard.
- the present invention relates to a method for evaluating the content of biodegradable organic matter in an aqueous sample comprising the following successive steps:
- a marker of the metabolic activity of the bacteria such as a fluorescent and / or colored marker of the respiratory activity of said bacteria
- tracking is meant for example a measurement at regular intervals of the fluorescence of the sample.
- the final result giving information on the concentration of the biodegradable organic matter of the sample and the slope of the curve information on the rate of biodegradation by the bacterial strain in the presence.
- the marker of the metabolic activity of the bacteria is advantageously resazurin.
- the resazurin molecule is reduced to pink resorufin in the presence of a cellular metabolic activity, this reaction being quantitatively correlated with the bacterial respiration and therefore with the oxygen concentration induced by the biological degradation of the organic matter in the body. sample considered.
- the monitoring of the metabolic activity can be carried out by colorimetry (resazurin absorbance peak: 601 mm / resorufin absorbance peak: 571 mm) and / or by fluorescence spectrophotometry at a length of excitation wave of 540 nanometers and an emission wavelength of about 610 nanometers.
- the method according to the invention thus makes it possible to estimate the content of biodegradable organic matter over a large concentration, ranging from 0 to at least 300 mg 0 2 / L, without the need for dilution of the sample, which is a very advantageous important compared to methods using oxygen probes for example which are limited to about 6 mg O2 / L.
- This method can easily be automated.
- each measuring cell is prepared according to the steps following successive
- the culture step of the bacterial strain makes it possible in particular to have bacteria in an exponential growth phase with a homogeneous population.
- the protective agent is advantageously a substance that is not biodegraded by the bacterial strain in question.
- a diholoside such as sucrose or a triholoside such as raffinose may be used.
- the lyophilization step is preferably preceded by a freezing phase in a range of -20 ° C to -80 ° C, freeze drying occurring at a temperature below -40 ° C under a pressure of less than 0.10. mbar (for example about 0.05 mbar).
- Lyophilization under these conditions allows a conservation of the bacteria, without affecting their activity.
- the bacterial strains used in this method have been isolated from the natural environment. These are, in particular, microorganisms frequently encountered in urban wastewater.
- the bacterial strains are chosen from: Acinetobacter towneri, Paracoccus pantotrophus, Arthrobacter aurescens, Proteus mirabilis, Bacillus funiculus, Pseudomonas aeruginosa, Bacillus mycoides, Bacillus subtilis subsp. Subtilis, Pseudomonas putida, Bosea vestrisii, Budvicia aquatica, Rhizobium radiobacter, Comamonas denitrificans, Rhodococcus rhodochrous,
- the various fractions of the aqueous sample are distributed in at least 6, preferably at least 8, measuring cells each containing a bacterial strain belonging to one of the following groups:
- group A Bacillus funiculus, Bosea vestrisii, Zoogloea resiniphila, Paracoccus pantotrophus, Budvicia aquatica, or Acinetobacter towneri,
- group B Thauera linaloolentis, Comamonas denitrificans, Comamonas testosteroni, Bacillus mycoides, or Rhodococcus rhodochrous,
- group C Pseudomonas aeruginosa, Cupriavidus necator, or Pseudomonas putida,
- group D Escherichia coli, or Enterobacter helveticus
- group F Staphylococcus auricularis, or Corynebacterium pseudo diphtheriticum,
- Rhizobium radiobacter Bacillus subtilis subsp. Subtilis, Staphylococcus sp., Sphingobium yanoikuyae, or Arthrobacter aurescens.
- the different fractions of the aqueous sample can be distributed in measuring cells each containing a single bacterial strain comprising at least the following eight bacterial strains: Bacillus subtilis subsp. Subtilis, Budvicia aquatica, Comamonas testosteroni, Corynebacterium pseudodiphtheriticum, Cupriavidus necator, Escherichia coli, Proteus mirabilis, Pseudomonas putida, Rhodococcus rhodochrous, Variovorax paradoxus.
- the present invention also relates to a kit for performing the evaluation of the biodegradable organic matter content of an aqueous sample, said kit comprising:
- measuring cells preferably six cells, preferably eight measuring cells, each containing a single, non-pathogenic bacterial strain, isolated from the natural medium, and lyophilized in the presence of a protective agent,
- a marker of the metabolic activity of said bacteria preferably a fluorescent and / or colored mark
- the measuring cells are advantageously transparent, for example glass, closed bottles, preferably in the form of individual tubes or arranged for example in microplates.
- the present invention also relates to the use of the evaluation method described above, for the quantification of the biodegradable organic matter content of an aqueous sample of treatment plants, industrial effluents or livestock effluents, or natural waters such as surface water, groundwater or marine waters.
- An advantageous use relates to the determination of the biological oxygen demand (BODs), by statistical processing of the data, obtained during the spectrophotometric monitoring of the bacterial activity in each sample fraction, using a multivariate method, such as a supervised learning algorithm of the neural network type, making it possible to correlate the results of said method with the corresponding value of BOD 5 of the ISO 5815 standard.
- BODs biological oxygen demand
- Figure 1 is a diagram showing the biodegradation capacity of different carbon sources by different bacterial strains, these strains can be classified into eight groups called A to H;
- Figure 2 in two parts, shows on the left the kinetics of biodegradation of the bacterial strains shown in Figure 1, and the diagram on the right the toxicological resistance index of these bacterial strains;
- Figure 3 presents the correlation (linear regression) obtained by an individual approach of each of the eight bacterial strains presented, the individual curves show the biological activity (in arbitrary unit u.a.) according to the BODs reference method;
- FIG. 4 presents different correlation tests between the value calculated by the method of the present invention expressed in mg BDO 5 / L according to the reference method, for samples of different origins (collective sanitation, autonomous sewerage, spillway). 'thunderstorm).
- Step 1 Culture of the strains before lyophilization
- the bacterial strain selected is isolated on a petri dish (LB medium agar: 5g / L NaCl, 5g / L Tryptone, 0.5g / L yeast extract, 12g / L agar) at 30 ° C for 24 to 48 hours . It is then inoculated and incubated in LB medium (5 g / L NaCl, 5 g / L Tryptone and 0.5 g / L yeast extract) at 30 ° C., with stirring at 250 rpm for 16 to 20 hours. The culture is monitored by optical density (cell density indicator) at 620 nm, until a homogeneous culture of bacteria in exponential growth phase. Step 2: Wash the stumps
- the pellet After centrifugation (6400 rpm / 4 min / 4 ° C.), the pellet is washed several times with a solution of MgSO 4 2 2 M, then conditioned in a lyophilization solution comprising: 50% of an amino acid solution IF-A (http://www.biolog.com) supplemented with nitrogen and phosphorus and 50% raffinose at 117.8 g / L.
- IF-A http://www.biolog.com
- the freeze-drying solution containing said suspended bacteria is distributed equally in different tubes, which are then subjected to freezing at -80 ° C for at least 3 hours, then to a lyophilization step at -50 ° C under 0, 05mbar for 36 hours. The whole can then be stored for several months at -20 ° C.
- the freeze-dried bacteria are "put back into activity" by adding a so-called regeneration solution composed of KH 2 PO 4 : 0.085 g / L, K 2 HPO 4 : 0.2175 g / L, Na 2 HPO 4 : 0.2665 g / L and NH 4 Cl: 0.02 g / L.
- the "regenerated" bacterial strain is then placed in an incubator for 20 hours at 30 ° C for a carbon depletion step, i.e. to allow the bacteria to consume the carbon resulting from the cell residues. dead bacteria in the medium during the previous steps (especially during the lyophilization step).
- the resazurin reagent and the test sample are added to the interfering carbon-free regenerated bacterial suspension in the following proportions: 10% of marker (resazurin), 23.3% of regenerated bacterial suspension and 66.7% of sample to test.
- the monitoring of the bacterial respiration is then followed by fluorescence (excitation wavelength of 540 nm and emission wavelength of 610 nm).
- Each selected strain gives partial information.
- the analyzes of the data are based on the set of data gathering the fluorescence values of each of the strains obtained. after three hours of exposure with the sample to be analyzed and the corresponding values of BODs measured according to ISO 5815.
- This database includes 208 conditions. Individually, the correlation between the strain responses and the BOD 5 reference values is low with an average correlation coefficient of 0.11 (see Figure 4). No strain is a relevant indicator of BOD 5 . However, each selected strain provides partial information. Consequently, the inventors' strategy is based on a multi-component analysis taking into account the information provided by the eight selected strains.
- the multi-varied approach proposed in the present patent application is based on the neuronal algorithm (neural network) for estimating the BOD of a bacterial panel after only three hours of measurement.
- biological provided by the bacterial panel in value of DBOs.
- new neural networks were generated with the learning panel.
- the choice was made from the correlation rate obtained between the estimated values (bacterial panel) and the BOD reference (see Figure 5) for several sample types: collective sanitation, non-collective sanitation or rainwater.
- the evaluation method according to the present invention is a robust, reliable method capable of providing information that can be correlated with standard BODs for broad ranges of biodegradable organic content concentrations of an aqueous sample.
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Abstract
A method for evaluating the biodegradable organic material content of an aqueous sample, comprising the following consecutive steps: - distributing various fractions of the sample into measurement cells each containing a single, non-pathogenic bacterial strain, isolated from the natural environment, said strains belonging to the following panel: Firmicutes, Actinobacteria and Proteobacteria; - adding, into each cell, a fluorescent or coloured marker of the respiratory activity of said bacteria in a thermostated enclosure; - monitoring, by spectrophotometry, the bacterial activity of each sample fraction for at least one hour, the final result and/or the kinematic results of the recordings relating to each fraction providing information on the metabolic activity of the bacteria, and thus an evaluation of the overall biodegradable organic material content of the aqueous sample. A correlation with the standard BOD5 method is presented. Use for effluents from purification or industrial plants, breeding stations or natural waters.
Description
Méthode d'évaluation de la teneur en matière organique biodégradable d'un échantillon aqueux et kit pour sa réalisation Method for evaluating the biodegradable organic matter content of an aqueous sample and kit for its production
DOMAINE DE L'INVENTION FIELD OF THE INVENTION
La présente invention concerne une méthode d'évaluation de la teneur en matière organique biodégradable d'un échantillon aqueux, un kit pour réaliser cette évaluation et son application à des effluents tels que des effluents de stations d'épuration, effluents industriels ou effluents d'élevage. The present invention relates to a method for evaluating the biodegradable organic matter content of an aqueous sample, a kit for carrying out this evaluation and its application to effluents such as effluents from purification plants, industrial effluents or effluents. breeding.
ART ANTERIEUR PRIOR ART
Les eaux usées urbaines, industrielles ou issues de l’agriculture sont généralement fortement chargées en matières organiques. Afin de déterminer les paramètres d’une épuration adaptée par voie biologique, ou pour vérifier la qualité des rejets en sortie d’une station d’épuration, il est nécessaire d’avoir une estimation de la charge en pollution organique biodégradable de ces effluents. La mesure de la DBOs (Demande Biochimique en Oxygène à 5 jours) est un test largement utilisé aujourd’hui. Cette méthode, qui fait l’objet d’une norme (ISO 5815) repose sur la mesure de la consommation en oxygène d’un échantillon aqueux par les microorganismes aérobies dudit échantillon dégradant les matières organiques présentes pendant une durée d’incubation de 5 jours à 20 °C à l’obscurité. La mesure de l’oxygène est réalisée par une méthode iodométrique ou au moyen d’une sonde électrochimique, la valeur de la DB05 étant exprimée en mg d’02/L. Cette méthode n’est adaptée qu’à des valeurs comprises entre 0 et 6 mg d’02/L, ce qui nécessite des dilutions préalables, et présente une importante variabilité (de l’ordre de 20 %). De plus, sa durée (5 jours) rend impossible un suivi en ligne : par exemple le suivi de la qualité de rejets de stations d’épuration.
Certaines améliorations ont été apportées à cette méthode de mesure : automatisation avec une mesure d’oxygène par sonde optique, méthode photométrique (suivi d’un colorant), ou méthode manométrique (modification de pression). Cependant, ces améliorations ne permettent pas d’avoir une estimation inférieure à 5 jours de la charge en pollution organique biodégradable des échantillons. Urban, industrial or agricultural wastewater is generally heavily loaded with organic matter. In order to determine the parameters of a biologically adapted treatment, or to check the quality of the discharges leaving a treatment plant, it is necessary to have an estimate of the biodegradable organic pollution load of these effluents. The measurement of BODs (Biochemical Oxygen Demand at 5 days) is a widely used test today. This method, which is the subject of a standard (ISO 5815), is based on the measurement of the oxygen consumption of an aqueous sample by the aerobic microorganisms of said sample degrading the organic materials present during an incubation period of 5 days. at 20 ° C in the dark. The measurement of oxygen is carried out by an iodometric method or by means of an electrochemical probe, the value of the DB05 being expressed in mg of 0 2 / L. This method is only suitable for values between 0 and 6 mg of 02 / L, which requires prior dilutions, and has a large variability (of the order of 20%). In addition, its duration (5 days) makes it impossible to follow up online: for example the monitoring of the quality of discharges from treatment plants. Some improvements have been made to this measurement method: automation with oxygen measurement by optical probe, photometric method (dye tracking), or manometric method (pressure modification). However, these improvements do not allow to have an estimate less than 5 days of the biodegradable organic pollution load of the samples.
Pour obtenir des informations plus rapides et permettre une mesure en ligne de la matière carbonée biodégradable, des biocapteurs ont été développés Cependant ces dispositifs nécessitent une phase préalable lourde de calibration et de paramétrage sur site. To obtain faster information and to enable an on-line measurement of the biodegradable carbonaceous material, biosensors have been developed. However, these devices require a heavy preliminary phase of calibration and parameterization on site.
Par ailleurs, des mesures alternatives à la mesure traditionnelle de la DB05 ont été proposées : par exemple les mesures réalisées par spectroscopie reposent sur une extrapolation de la teneur en matière organique biodégradable faite à partir de l’interprétation de spectres UV. De telles mesures sont peu précises. In addition, alternative measures to the traditional measurement of DB05 have been proposed: for example the measurements carried out by spectroscopy are based on an extrapolation of the content of biodegradable organic matter made from the interpretation of UV spectra. Such measures are not very precise.
Si les méthodes présentées ci-dessus améliorent partiellement la mise en œuvre de la DBOs ou permettent une estimation plus rapide de la charge en matière organique biodégradable d’un échantillon, elles sont soit incomplètes, soit imprécises et/ou peu adaptées à des mesures continues permettant de répondre aux besoins de gestion des effluents. BUTS DE L’INVENTION If the methods presented above partially improve the implementation of the BOD or allow a faster estimation of the biodegradable organic matter load of a sample, they are either incomplete or imprecise and / or unsuitable for continuous measurements. to meet effluent management needs. GOALS OF THE INVENTION
Un premier but de l’invention est donc de proposer une méthode d'évaluation de la teneur en matière organique biodégradable d'un échantillon aqueux qui soit rapide (de préférence quelques heures), permettant par exemple de répercuter l’information sur des conditions d’épuration, précise et reproductible. A first object of the invention is therefore to propose a method for evaluating the content of biodegradable organic matter in an aqueous sample which is rapid (preferably a few hours), allowing for example to pass on the information on environmental conditions. purification, precise and reproducible.
Un autre but de l’invention est de proposer une méthode permettant d’estimer la teneur en matière organique biodégradable sur une large concentration,
sans nécessiter de dilution de l’échantillon et tout en réduisant la variabilité entre les mesures. Another aim of the invention is to propose a method for estimating the content of biodegradable organic matter over a large concentration, without requiring dilution of the sample and reducing the variability between measurements.
Un autre but de l’invention est de proposer une méthode aisée à mettre en œuvre et pouvant être automatisée. Another object of the invention is to provide a method that is easy to implement and can be automated.
Enfin, un autre but de l’invention est de proposer une méthode dont les résultats puissent être convertis en valeur équivalente à la valeur de DBOs définie par la norme actuelle. Finally, another object of the invention is to propose a method whose results can be converted into a value equivalent to the value of DBOs defined by the current standard.
DESCRIPTION DE L’INVENTION DESCRIPTION OF THE INVENTION
A cet effet la présente invention concerne une méthode d'évaluation de la teneur en matière organique biodégradable d'un échantillon aqueux comprenant les étapes successives suivantes : For this purpose, the present invention relates to a method for evaluating the content of biodegradable organic matter in an aqueous sample comprising the following successive steps:
- répartition de différentes fractions de l'échantillon aqueux dans au moins quatre cellules de mesure renfermant chacune une souche bactérienne unique, non pathogène, isolée du milieu naturel, lesdites souches bactériennes appartenant au panel suivant : Firmicutes, Actinobacteria, et Proteobacteria, notamment Betaproteobacteria et Gammaproteobacteria ; - Distribution of different fractions of the aqueous sample in at least four measuring cells each containing a single bacterial strain, non-pathogenic, isolated from the natural environment, said bacterial strains belonging to the following panel: Firmicutes, Actinobacteria, and Proteobacteria, including Betaproteobacteria and Gammaproteobacteria;
- ajout dans chaque cellule d'un marqueur de l'activité métabolique des bactéries, tel qu’un marqueur fluorescent et/ou coloré de l’activité respiratoire des dites bactéries ; adding to each cell a marker of the metabolic activity of the bacteria, such as a fluorescent and / or colored marker of the respiratory activity of said bacteria;
- placement des cellules de mesure dans une enceinte thermostatée ; - placement of measuring cells in a thermostatically controlled chamber;
- suivi par spectrophotométrie de l'activité bactérienne de chaque fraction d'échantillon pendant au moins une, de préférence trois heures, le résultat final et/ou les résultats cinétiques (paramètres cinétiques de la biodégradation) relatifs à chaque fraction fournissant des informations sur l'activité métabolique des bactéries, et donc une évaluation de la teneur globale en matière organique biodégradable de l'échantillon aqueux. spectrophotometrically monitoring the bacterial activity of each sample fraction for at least one, preferably three hours, the final result and / or the kinetic results (kinetic parameters of the biodegradation) relating to each fraction providing information on the metabolic activity of the bacteria, and therefore an evaluation of the overall biodegradable organic matter content of the aqueous sample.
Il s’avère que cette méthode permet d’obtenir une estimation très rapide, en quelques heures (une à trois heures généralement) de la teneur en matière
organique biodégradable d'un échantillon, ce qui permet par exemple un suivi quasi en temps réel de la qualité d’épuration d’un traitement biologique d’une station. L’estimation de la réponse de chaque souche bactérienne est prise individuellement. It turns out that this method makes it possible to obtain a very rapid estimate, in a few hours (usually one to three hours) of the content content. organic biodegradable sample, which allows for example a near real-time monitoring of the quality of purification of a biological treatment of a station. The estimate of the response of each bacterial strain is taken individually.
Par "suivi", on entend par exemple une mesure à intervalles réguliers de la fluorescence de l’échantillon. Le résultat final donnant une information sur la concentration de la matière organique biodégradable de l’échantillon et la pente de la courbe une information sur la vitesse de biodégradation par la souche bactérienne en présence. By "tracking" is meant for example a measurement at regular intervals of the fluorescence of the sample. The final result giving information on the concentration of the biodegradable organic matter of the sample and the slope of the curve information on the rate of biodegradation by the bacterial strain in the presence.
Le marqueur de l'activité métabolique des bactéries est avantageusement la résazurine. The marker of the metabolic activity of the bacteria is advantageously resazurin.
En effet, la molécule de résazurine est réduite en résorufine de couleur rose en présence d’une activité métabolique cellulaire, cette réaction étant quantitativement corrélée à la respiration bactérienne et donc à la concentration en oxygène induite par la dégradation biologique de la matière organique dans l’échantillon considéré. Avec ce marqueur, le suivi de l'activité métabolique peut être réalisé par colorimétrie (pic d'absorbance de la résazurine : 601 mm / pic d'absorbance de la résorufine : 571 mm) et/ou par spectrophotométrie de fluorescence à une longueur d'onde d'excitation de 540 nanomètres et une longueur d'onde d'émission de 610 nanomètres environ. Indeed, the resazurin molecule is reduced to pink resorufin in the presence of a cellular metabolic activity, this reaction being quantitatively correlated with the bacterial respiration and therefore with the oxygen concentration induced by the biological degradation of the organic matter in the body. sample considered. With this marker, the monitoring of the metabolic activity can be carried out by colorimetry (resazurin absorbance peak: 601 mm / resorufin absorbance peak: 571 mm) and / or by fluorescence spectrophotometry at a length of excitation wave of 540 nanometers and an emission wavelength of about 610 nanometers.
La méthode selon l’invention permet ainsi d’estimer la teneur en matière organique biodégradable sur une large concentration, allant de 0 à au moins 300 mg 02/L, sans nécessiter de dilution de l’échantillon, ce qui présente un avantage très important par rapport aux méthodes mettant en œuvre des sondes à oxygène par exemple qui sont limitées à 6 mg O2/L environ. The method according to the invention thus makes it possible to estimate the content of biodegradable organic matter over a large concentration, ranging from 0 to at least 300 mg 0 2 / L, without the need for dilution of the sample, which is a very advantageous important compared to methods using oxygen probes for example which are limited to about 6 mg O2 / L.
La présente méthode peut aisément être automatisée. This method can easily be automated.
De manière préférée, chaque cellule de mesure est préparée selon les étapes
successives suivantes : Preferably, each measuring cell is prepared according to the steps following successive
- isolement et culture de la souche bactérienne sélectionnée, - isolation and culture of the selected bacterial strain,
- lavage pour éliminer toute impureté carbonée, washing to eliminate any carbon impurity,
- puis conditionnement dans ladite cellule avec ajout d'un agent protecteur de la souche bactérienne vis-à-vis de la lyophilisation, - conditioning in said cell with addition of a protective agent of the bacterial strain vis-à-vis lyophilization,
- lyophilisation de la souche bactérienne pour sa conservation, lyophilization of the bacterial strain for its preservation,
- puis, avant son utilisation pour l'évaluation de la teneur en matière organique dégradable d'un échantillon aqueux, réhydratation et régénération au moyen d’une solution aqueuse de régénération de type tampon NPK, et maintien de la souche bactérienne dans un incubateur pendant environ vingt heures permettant aux bactéries de consommer le carbone résultant des résidus cellulaires de bactéries mortes pendant les étapes précédentes (dénommée étape d’épuisement de la partie carbonée). L’étape de culture de la souche bactérienne permet notamment d’avoir des bactéries en phase exponentielle de croissance avec une population homogène. - then, before its use for the evaluation of the degradable organic matter content of an aqueous sample, rehydration and regeneration by means of an NPK buffer-type aqueous regeneration solution, and maintenance of the bacterial strain in an incubator during approximately twenty hours allowing the bacteria to consume the carbon resulting from cell residues of dead bacteria during the previous steps (called the stage of depletion of the carbon part). The culture step of the bacterial strain makes it possible in particular to have bacteria in an exponential growth phase with a homogeneous population.
L’agent de protection est avantageusement une substance non biodégradée par la souche bactérienne considérée. On peut utiliser par exemple un diholoside tel que le saccharose ou un triholoside tel que le raffinose. The protective agent is advantageously a substance that is not biodegraded by the bacterial strain in question. For example, a diholoside such as sucrose or a triholoside such as raffinose may be used.
L’étape de lyophilisation est de préférence précédée d'une phase de congélation dans une plage de -20°C à -80°C, la lyophilisation ayant lieu à une température inférieure à -40 °C sous une pression inférieure à 0,10 mbar (par exemple environ 0,05 mbar). The lyophilization step is preferably preceded by a freezing phase in a range of -20 ° C to -80 ° C, freeze drying occurring at a temperature below -40 ° C under a pressure of less than 0.10. mbar (for example about 0.05 mbar).
La lyophilisation dans ces conditions permet une conservation des bactéries, sans affecter leur activité. Lyophilization under these conditions allows a conservation of the bacteria, without affecting their activity.
Les souches bactériennes utilisées dans la présente méthode ont été isolées du milieu naturel. Ce sont, en particulier, des microorganismes fréquemment
rencontrés dans les eaux usées urbaines. The bacterial strains used in this method have been isolated from the natural environment. These are, in particular, microorganisms frequently encountered in urban wastewater.
Selon un mode de réalisation avantageux de l’invention les souches bactériennes sont choisies parmi : Acinetobacter towneri, Paracoccus pantotrophus, Arthrobacter aurescens, Proteus mirabilis, Bacillus funiculus, Pseudomonas aeruginosa, Bacillus mycoides, Bacillus subtilis subsp. Subtilis, Pseudomonas putida, Bosea vestrisii, Budvicia aquatica, Rhizobium radiobacter, Comamonas denitrificans, Rhodococcus rhodochrous,According to an advantageous embodiment of the invention, the bacterial strains are chosen from: Acinetobacter towneri, Paracoccus pantotrophus, Arthrobacter aurescens, Proteus mirabilis, Bacillus funiculus, Pseudomonas aeruginosa, Bacillus mycoides, Bacillus subtilis subsp. Subtilis, Pseudomonas putida, Bosea vestrisii, Budvicia aquatica, Rhizobium radiobacter, Comamonas denitrificans, Rhodococcus rhodochrous,
Comamonas testosteroni, Sphingobium yanoikuyae, Corynebacterium pseudodiphtheriticum, Staphylococcus auricularis, Cupriavidus necator, Staphylococcus sp., Enterobacter helveticus, Thauera linaloolentis, Escherichia coli, Variovorax paradoxus, Paracoccus denitrificans, Zoogloea resiniphila. Comamonas testosteroni, Sphingobium yanoikuyae, Corynebacterium pseudodiphtheriticum, Staphylococcus auricularis, Cupriavidus necator, Staphylococcus sp., Enterobacter helveticus, Thauera linaloolentis, Escherichia coli, Variovorax paradoxus, Paracoccus denitrificans, Zoogloea resiniphila.
Plus particulièrement les différentes fractions de l'échantillon aqueux sont réparties dans au moins 6, de préférence au moins 8, cellules de mesure renfermant chacune une souche bactérienne appartenant à l’un des groupes suivants : More particularly, the various fractions of the aqueous sample are distributed in at least 6, preferably at least 8, measuring cells each containing a bacterial strain belonging to one of the following groups:
- groupe A : Bacillus funiculus, Bosea vestrisii, Zoogloea resiniphila, Paracoccus pantotrophus, Budvicia aquatica, ou Acinetobacter towneri, group A: Bacillus funiculus, Bosea vestrisii, Zoogloea resiniphila, Paracoccus pantotrophus, Budvicia aquatica, or Acinetobacter towneri,
- groupe B : Thauera linaloolentis, Comamonas denitrificans, Comamonas testosteroni, Bacillus mycoides, ou Rhodococcus rhodochrous, group B: Thauera linaloolentis, Comamonas denitrificans, Comamonas testosteroni, Bacillus mycoides, or Rhodococcus rhodochrous,
- groupe C : Pseudomonas aeruginosa, Cupriavidus necator, ou Pseudomonas putida, group C: Pseudomonas aeruginosa, Cupriavidus necator, or Pseudomonas putida,
- groupe D : Escherichia coli, ou Enterobacter helveticus, group D: Escherichia coli, or Enterobacter helveticus,
- groupe E : Variovorax paradoxus, - group E: Variovorax paradoxus,
- groupe F : Staphylococcus auricularis, ou Corynebacterium pseudo diphtheriticum, group F: Staphylococcus auricularis, or Corynebacterium pseudo diphtheriticum,
- groupe G : Paracoccus denitrificans, ou Proteus mirabilis, - group G: Paracoccus denitrificans, or Proteus mirabilis,
- groupe H : Rhizobium radiobacter, Bacillus subtilis subsp. Subtilis, Staphylococcus sp., Sphingobium yanoikuyae, ou Arthrobacter aurescens. - group H: Rhizobium radiobacter, Bacillus subtilis subsp. Subtilis, Staphylococcus sp., Sphingobium yanoikuyae, or Arthrobacter aurescens.
Il est en effet apparu que les souches considérées pouvaient être classées en
groupes de profils métaboliques de biodégradation similaires, les souches bactériennes d’un même groupe étant capables de dégrader les mêmes composés organiques. Ainsi, à titre d’exemple, les différentes fractions de l'échantillon aqueux peuvent être réparties dans des cellules de mesure renfermant chacune une souche bactérienne unique comprenant au moins les huit souches bactériennes suivantes : Bacillus subtilis subsp. Subtilis, Budvicia aquatica, Comamonas testosteroni, Corynebacterium pseudodiphtheriticum, Cupriavidus necator, Escherichia coli, Proteus mirabilis, Pseudomonas putida, Rhodococcus rhodochrous, Variovorax paradoxus. It appeared in fact that the strains in question could be classified as groups of similar biodegradation metabolic profiles, the bacterial strains of the same group being able to degrade the same organic compounds. Thus, by way of example, the different fractions of the aqueous sample can be distributed in measuring cells each containing a single bacterial strain comprising at least the following eight bacterial strains: Bacillus subtilis subsp. Subtilis, Budvicia aquatica, Comamonas testosteroni, Corynebacterium pseudodiphtheriticum, Cupriavidus necator, Escherichia coli, Proteus mirabilis, Pseudomonas putida, Rhodococcus rhodochrous, Variovorax paradoxus.
La présente invention concerne également un kit pour réaliser l'évaluation de la teneur en matière organique biodégradable d'un échantillon aqueux, ledit kit comprenant : The present invention also relates to a kit for performing the evaluation of the biodegradable organic matter content of an aqueous sample, said kit comprising:
- au moins quatre cellules de mesure, de préférence six cellules, de préférence encore huit cellules de mesure, chacune renfermant une souche bactérienne unique, non pathogène, isolée du milieu naturel, et lyophilisée en présence d'un agent de protection, at least four measuring cells, preferably six cells, preferably eight measuring cells, each containing a single, non-pathogenic bacterial strain, isolated from the natural medium, and lyophilized in the presence of a protective agent,
- un marqueur d'activité métabolique desdites bactéries, de préférence, un marque fluorescent et/ou coloré, a marker of the metabolic activity of said bacteria, preferably a fluorescent and / or colored mark,
- une notice détaillée de mise en œuvre de la méthode telle que décrite ci- dessus. Les cellules de mesure sont avantageusement des flacons transparents, par exemple en verre, fermés, de préférence sous la forme de tubes, individuels ou agencés par exemple en microplaques. - a detailed record of implementation of the method as described above. The measuring cells are advantageously transparent, for example glass, closed bottles, preferably in the form of individual tubes or arranged for example in microplates.
La présente invention concerne également l’utilisation de la méthode d’évaluation décrite ci-dessus, pour la quantification de la teneur en matière organique biodégradable d'un échantillon aqueux de stations d'épuration,
d'effluents industriels ou d'effluents d'élevage, ou encore des eaux naturelles telles que des eaux de surface, des eaux souterraines ou des eaux marines. The present invention also relates to the use of the evaluation method described above, for the quantification of the biodegradable organic matter content of an aqueous sample of treatment plants, industrial effluents or livestock effluents, or natural waters such as surface water, groundwater or marine waters.
Une utilisation avantageuse porte sur la détermination de la demande biologique en oxygène (DBOs), par traitement statistique des données, obtenues lors du suivi spectrophotométrique de l'activité bactérienne dans chaque fraction d'échantillon, mettant en œuvre une méthode multivariée, tel qu’un algorithme par apprentissage supervisé de type réseau de neurones, permettant de corréler les résultats de ladite méthode à la valeur correspondante de DBO5 de la norme ISO 5815. An advantageous use relates to the determination of the biological oxygen demand (BODs), by statistical processing of the data, obtained during the spectrophotometric monitoring of the bacterial activity in each sample fraction, using a multivariate method, such as a supervised learning algorithm of the neural network type, making it possible to correlate the results of said method with the corresponding value of BOD 5 of the ISO 5815 standard.
BREVE DESCRIPTION DES DESSINS BRIEF DESCRIPTION OF THE DRAWINGS
L'invention sera bien comprise à la lecture de la description suivante d'exemples de réalisation, en référence aux dessins annexés dans lesquels : The invention will be better understood on reading the following description of exemplary embodiments, with reference to the appended drawings in which:
La figure 1 est un schéma présentant la capacité de biodégradation de différentes sources de carbone par différentes souches bactériennes, ces souches pouvant être classées en huit groupes dénommés de A à H ; La figure 2, en deux parties, présente à gauche la cinétique de biodégradation des souches bactériennes présentées la figure 1 , et le schéma de droite l’indice de résistance toxicologique de ces souches bactériennes ; Figure 1 is a diagram showing the biodegradation capacity of different carbon sources by different bacterial strains, these strains can be classified into eight groups called A to H; Figure 2, in two parts, shows on the left the kinetics of biodegradation of the bacterial strains shown in Figure 1, and the diagram on the right the toxicological resistance index of these bacterial strains;
La figure 3 présente la corrélation (régression linéaire) obtenue par une approche individuelle de chacune des huit souches bactériennes présentées, les courbes individuelles montrent l'activité biologique (en unité arbitraire u.a.) en fonction de la méthode de référence DBOs ; Figure 3 presents the correlation (linear regression) obtained by an individual approach of each of the eight bacterial strains presented, the individual curves show the biological activity (in arbitrary unit u.a.) according to the BODs reference method;
La figure 4 présente différents essais de corrélation entre la valeur calculée par la méthode de la présente invention exprimée en mg BDO5/L en fonction de la méthode de référence, pour des échantillons d’origines différentes (assainissement collectif, assainissement autonome, déversoir d’orage).
EXEMPLES FIG. 4 presents different correlation tests between the value calculated by the method of the present invention expressed in mg BDO 5 / L according to the reference method, for samples of different origins (collective sanitation, autonomous sewerage, spillway). 'thunderstorm). EXAMPLES
Les différentes étapes de préparation des souches bactériennes avant leur introduction dans les cellules de mesure sont présentées ci-après : The various stages of preparation of the bacterial strains before their introduction into the measuring cells are presented below:
Etape 1 : Culture des souches avant lyophilisation Step 1: Culture of the strains before lyophilization
La souche bactérienne sélectionnée est isolée sur boite de pétri (milieu LB gélosé : 5g/L NaCI, 5g/L Tryptone, 0,5g/L Extrait de levure, 12g/L d’agar) à 30°C pendant 24 à 48 heures. Elle est ensuite inoculée et incubée en milieu LB (5g/L NaCI, 5g/L Tryptone et 0,5g/L Extrait de levure) à 30°C, sous agitation à 250 tours/min pendant 16 à 20 heures. La culture est suivie par densité optique (indicateur de densité cellulaire) à 620 nm, jusqu’à obtenir une culture homogène de bactéries en phase exponentielle de croissance. Etape 2 : Lavage des souches The bacterial strain selected is isolated on a petri dish (LB medium agar: 5g / L NaCl, 5g / L Tryptone, 0.5g / L yeast extract, 12g / L agar) at 30 ° C for 24 to 48 hours . It is then inoculated and incubated in LB medium (5 g / L NaCl, 5 g / L Tryptone and 0.5 g / L yeast extract) at 30 ° C., with stirring at 250 rpm for 16 to 20 hours. The culture is monitored by optical density (cell density indicator) at 620 nm, until a homogeneous culture of bacteria in exponential growth phase. Step 2: Wash the stumps
Après centrifugation (6400 tours par min/ 4min/ 4°C), le culot est lavé plusieurs fois par une solution de MgS04 102 M, puis conditionné dans une solution de lyophilisation comprenant : 50% d’une solution d’acides aminés IF-A (http://www.biolog.com) complémentée en azote et phosphore et 50% de raffinose à 117,8 g/L. After centrifugation (6400 rpm / 4 min / 4 ° C.), the pellet is washed several times with a solution of MgSO 4 2 2 M, then conditioned in a lyophilization solution comprising: 50% of an amino acid solution IF-A (http://www.biolog.com) supplemented with nitrogen and phosphorus and 50% raffinose at 117.8 g / L.
Etape 3 : Lyophilisation des souches Step 3: Lyophilization of the strains
La solution de lyophilisation renfermant lesdites bactéries en suspension sont réparties de manière égale dans différents tubes, qui sont ensuite soumis à une congélation à -80°C pendant au moins 3 heures, puis à une étape de lyophilisation à -50°C sous 0,05mbar pendant 36 heures. L’ensemble peut alors être conservé pendant plusieurs mois à -20 °C. The freeze-drying solution containing said suspended bacteria is distributed equally in different tubes, which are then subjected to freezing at -80 ° C for at least 3 hours, then to a lyophilization step at -50 ° C under 0, 05mbar for 36 hours. The whole can then be stored for several months at -20 ° C.
Etape 4 : Préparation de l’analyse Step 4: Prepare the analysis
Les bactéries lyophilisées sont « remises en activité » par ajout d’une solution dite de régénération composée de KH2P04 : 0,085 g/L, K2HP04 : 0,2175 g/L, Na2HP04 : 0,2665 g/L et NH4CI : 0,02 g/L.
La souche bactérienne « régénérée » est ensuite placée dans un incubateur pendant 20 heures à 30°C pour une étape d’épuisement du carbone, c’est-à- dire de manière à permettre aux bactéries de consommer le carbone résultant des résidus cellulaires de bactéries mortes dans le milieu pendant les étapes précédentes (notamment pendant l’étape de lyophilisation). The freeze-dried bacteria are "put back into activity" by adding a so-called regeneration solution composed of KH 2 PO 4 : 0.085 g / L, K 2 HPO 4 : 0.2175 g / L, Na 2 HPO 4 : 0.2665 g / L and NH 4 Cl: 0.02 g / L. The "regenerated" bacterial strain is then placed in an incubator for 20 hours at 30 ° C for a carbon depletion step, i.e. to allow the bacteria to consume the carbon resulting from the cell residues. dead bacteria in the medium during the previous steps (especially during the lyophilization step).
Chaque tube renfermant une souche bactérienne unique est alors prêt pour l’analyse proprement dite. Each tube containing a single bacterial strain is then ready for the actual analysis.
Etape 5 : Analyse Step 5: Analysis
A la suspension bactérienne régénérée et exempte de carbone interfèrent est ajouté le réactif résazurine puis l’échantillon à tester dans les proportions suivantes : 10% de marqueur (résazurine), 23,3 % de suspension bactérienne régénérée et 66,7 % d’échantillon à tester. The resazurin reagent and the test sample are added to the interfering carbon-free regenerated bacterial suspension in the following proportions: 10% of marker (resazurin), 23.3% of regenerated bacterial suspension and 66.7% of sample to test.
Le suivi de la respiration bactérienne est alors suivi par fluorescence (longueur d'onde d'excitation de 540 nm et longueur d'onde d'émission de 610 nm). The monitoring of the bacterial respiration is then followed by fluorescence (excitation wavelength of 540 nm and emission wavelength of 610 nm).
Chaque souche sélectionnée donne une information partielle. Each selected strain gives partial information.
Cependant pour obtenir une estimation plus globale de la teneur en matière organique biodégradable d'un échantillon, il est nécessaire de combiner les informations provenant de plusieurs souches bactériennes. However, to obtain a more global estimate of the biodegradable organic matter content of a sample, it is necessary to combine the information from several bacterial strains.
A cet effet, les inventeurs ont sélectionné 28 souches bactériennes (voir tableau 1 ) issues du milieu naturel et les ont confrontées à différentes sources organiques carbonées : les résultats sont présentés en figure 1.
For this purpose, the inventors selected 28 bacterial strains (see Table 1) from the natural environment and confronted them with different organic carbon sources: the results are presented in FIG.
DSM: Collection Allemande de microorganismes DSM: German Collection of Microorganisms
ATCC: American Type Culture Collection ATCC: American Type Culture Collection
CIP: Collection de l’Institut Pasteur CIP: Institut Pasteur Collection
Tableau 1 Table 1
Ceci a permis notamment de classer des différentes souches bactériennes en 8 groupes principaux (notés de A à H sur la figure 1 ), dans lesquels les bactéries présentaient un profil de biodégradation comparable. This made it possible, in particular, to classify different bacterial strains into 8 main groups (denoted from A to H in FIG. 1), in which the bacteria had a comparable biodegradation profile.
Dans chacun des groupes, a été retenue la souche bactérienne ayant notamment la meilleure cinétique de biodégradation (voir figure 2 schéma de gauche) et/ou la meilleure résistance à des tests toxicologiques (réalisés par la méthode selon la demande de brevet français du même déposant déposée le même jour) (voir figure 3 : index de résistance toxicologique). In each of the groups, the bacterial strain having the best kinetics of biodegradation (see figure 2 on the left) and / or the best resistance to toxicological tests (made by the method according to the French patent application of the same applicant filed the same day) (see Figure 3: toxicological resistance index).
Les informations partielles (rapportées en valeur de DBOs) données par chaque souche individuelle sélectionnée sont regroupées à la figure 4. The partial information (reported in value of DBOs) given by each individual strain selected are grouped together in FIG.
Corrélation avec la DBO5 Correlation with BOD 5
Les analyses des données sont basées sur l'ensemble des données regroupant les valeurs de fluorescence de chacune des souches obtenues
après trois heures d'exposition avec l'échantillon à analyser et les valeurs correspondantes de DBOs mesurées selon la norme ISO 5815. The analyzes of the data are based on the set of data gathering the fluorescence values of each of the strains obtained. after three hours of exposure with the sample to be analyzed and the corresponding values of BODs measured according to ISO 5815.
Cette base de données comprend 208 conditions. De manière individuelle, la corrélation entre les réponses des souches et les valeurs de références de DBO5 est faible avec un coefficient de corrélation moyen de 0,11 (voir figure 4). Aucune souche n'est un indicateur pertinent de la DBO5. Cependant, chaque souche sélectionnée fournit une information partielle. En conséquence, la stratégie des inventeurs est fondée sur une analyse multi- composants prenant en compte l'information fournie par les huit souches sélectionnées. This database includes 208 conditions. Individually, the correlation between the strain responses and the BOD 5 reference values is low with an average correlation coefficient of 0.11 (see Figure 4). No strain is a relevant indicator of BOD 5 . However, each selected strain provides partial information. Consequently, the inventors' strategy is based on a multi-component analysis taking into account the information provided by the eight selected strains.
L'approche multi-variée proposée dans la présente demande de brevet est basée sur l'algorithme neuronal (réseau de neurones) pour estimer la DBOs d'un panel bactérien après seulement trois heures de mesure. The multi-varied approach proposed in the present patent application is based on the neuronal algorithm (neural network) for estimating the BOD of a bacterial panel after only three hours of measurement.
Dans un premier temps, la base de données générale (n=208) a été introduite dans le logiciel Neuro One (version 6.13.0.5, Nétral, France) pour réaliser des modèles (réseaux de neurone-perceptron) permettant de convertir l'information biologique fournie par le panel bactérien en valeur de DBOs. As a first step, the general database (n = 208) was introduced in the Neuro One software (version 6.13.0.5, Nétral, France) to create models (neuron-perceptron networks) for converting information. biological provided by the bacterial panel in value of DBOs.
Plusieurs architectures neuronales ont été comparées (nombre de neurones cachés, mode d'activation, standardisation des données). Plus de 800 modèles ont été générés afin de sélectionner les meilleures architectures. Several neural architectures have been compared (number of hidden neurons, activation mode, data standardization). More than 800 models have been generated to select the best architectures.
A partir des architectures sélectionnées, de nouveaux réseaux de neurones ont été générés avec le panel d'apprentissage. Le choix a été réalisé à partir du taux de corrélation obtenu entre les valeurs estimées (panel bactérien) et la référence de DBOs (voir figure 5) pour plusieurs types d'échantillon : assainissement collectif, assainissement non collectif ou eau de pluie.
Le modèle sélectionné consiste en une couche cachée de trois neurones activés par une fonction tangente hyperbolique. Ce modèle de réseaux de neurones a été capable de fournir une estimation de la DBOs (R2 = 0,851 après seulement trois heures en comparaison des cinq jours requis par la méthode de référence). From the selected architectures, new neural networks were generated with the learning panel. The choice was made from the correlation rate obtained between the estimated values (bacterial panel) and the BOD reference (see Figure 5) for several sample types: collective sanitation, non-collective sanitation or rainwater. The selected model consists of a hidden layer of three neurons activated by a hyperbolic tangent function. This model of neural networks was able to provide an estimate of BODs (R 2 = 0.851 after only three hours compared to the five days required by the reference method).
En conséquence, la méthode d'évaluation selon la présente invention est une méthode robuste, fiable, apte à fournir des informations pouvant être corrélées avec la DBOs standard pour de larges gammes de concentrations de teneurs en matière organique biodégradable d'un échantillon aqueux.
Accordingly, the evaluation method according to the present invention is a robust, reliable method capable of providing information that can be correlated with standard BODs for broad ranges of biodegradable organic content concentrations of an aqueous sample.
Claims
1. Méthode d'évaluation de la teneur en matière organique biodégradable d'un échantillon aqueux comprenant les étapes successives suivantes : 1. A method of evaluating the biodegradable organic matter content of an aqueous sample comprising the following successive steps:
- répartition de différentes fractions de l'échantillon aqueux dans au moins quatre cellules de mesure renfermant chacune une souche bactérienne unique, non pathogène, isolée du milieu naturel, lesdites souches bactériennes appartenant au panel suivant : Firmicutes, Actinobacteria, et Proteobacteria, notamment Betaproteobacteria et Gammaproteobacteria ; - Distribution of different fractions of the aqueous sample in at least four measuring cells each containing a single bacterial strain, non-pathogenic, isolated from the natural environment, said bacterial strains belonging to the following panel: Firmicutes, Actinobacteria, and Proteobacteria, including Betaproteobacteria and Gammaproteobacteria;
- ajout dans chaque cellule d'un marqueur de l'activité métabolique des bactéries, tel qu’un marqueur fluorescent et/ou coloré de l’activité respiratoire des dites bactéries ; adding to each cell a marker of the metabolic activity of the bacteria, such as a fluorescent and / or colored marker of the respiratory activity of said bacteria;
- placement des cellules de mesure dans une enceinte thermostatée ; - placement of measuring cells in a thermostatically controlled chamber;
- suivi par spectrophotométrie de l'activité bactérienne de chaque fraction d'échantillon pendant au moins une, de préférence trois heures, le résultat final et/ou les résultats cinétiques relatifs à chaque fraction fournissant des informations sur l'activité métabolique des bactéries, et donc une évaluation de la teneur globale en matière organique biodégradable de l'échantillon aqueux. spectrophotometrically monitoring the bacterial activity of each sample fraction for at least one, preferably three hours, the final result and / or the kinetic results relating to each fraction providing information on the metabolic activity of the bacteria, and therefore an evaluation of the overall organic biodegradable content of the aqueous sample.
2. Méthode selon la revendication 1 , caractérisée en ce que chaque cellule de mesure est préparée selon les étapes successives suivantes : 2. Method according to claim 1, characterized in that each measuring cell is prepared according to the following successive steps:
- isolement et culture de la souche bactérienne sélectionnée, - isolation and culture of the selected bacterial strain,
- lavage pour éliminer toute impureté carbonée, washing to eliminate any carbon impurity,
- puis conditionnement dans ladite cellule avec ajout d'un agent protecteur de la souche bactérienne vis-à-vis de la lyophilisation, - conditioning in said cell with addition of a protective agent of the bacterial strain vis-à-vis lyophilization,
- lyophilisation de la souche bactérienne pour sa conservation, lyophilization of the bacterial strain for its preservation,
- puis, avant son utilisation pour l'évaluation de la teneur en matière organique dégradable d'un échantillon aqueux, réhydratation et régénération au moyen d’une solution aqueuse de régénération de type tampon NPK, et maintien de la souche bactérienne dans un incubateur pendant environ vingt heures permettant aux bactéries de consommer le carbone résultant des résidus
cellulaires de bactéries mortes pendant les étapes précédentes (dénommée étape d’épuisement de la partie carbonée). - then, before its use for the evaluation of the degradable organic matter content of an aqueous sample, rehydration and regeneration by means of an NPK buffer-type aqueous regeneration solution, and maintenance of the bacterial strain in an incubator during about twenty hours allowing the bacteria to consume the carbon resulting from the residues bacterial cell dead during the previous steps (called the stage of depletion of the carbonaceous part).
3. Méthode selon la revendication 2, caractérisée en ce que la phase de lyophilisation est précédée d'une phase de congélation dans une plage de -20°C à -80°C, la lyophilisation ayant lieu à une température inférieure à - 40 °C sous une pression inférieure à 0,10 mbar. 3. Method according to claim 2, characterized in that the lyophilization phase is preceded by a freezing phase in a range of -20 ° C to -80 ° C, freeze drying taking place at a temperature below -40 ° C under a pressure of less than 0.10 mbar.
4. Méthode selon l'une quelconque des revendications précédentes, caractérisée en ce que le marqueur de l'activité métabolique des bactéries est la résazurine, le suivi de l'activité métabolique des bactéries étant réalisé par colorimétrie et/ou par spectrophotométrie de fluorescence. 4. Method according to any one of the preceding claims, characterized in that the marker of the metabolic activity of the bacteria is resazurin, the monitoring of the metabolic activity of the bacteria being carried out by colorimetry and / or fluorescence spectrophotometry.
5. Méthode selon l'une quelconque des revendications précédentes, caractérisée en ce que les souches bactériennes sont choisies parmi : 5. Method according to any one of the preceding claims, characterized in that the bacterial strains are chosen from:
Acinetobacter towneri, Paracoccus pantotrophus, Arthrobacter aurescens, Proteus mirabilis, Bacillus funiculus, Pseudomonas aeruginosa, Bacillus mycoides, Bacillus subtilis subsp. Subtilis, Pseudomonas putida, Bosea vestrisii, Budvicia aquatica, Rhizobium radiobacter, Comamonas denitrificans, Rhodococcus rhodochrous, Comamonas testosteroni, Sphingobium yanoikuyae, Corynebacterium pseudodiphtheriticum, Staphylococcus auricularis, Cupriavidus necator, Staphylococcus sp., Enterobacter helveticus, Thauera linaloolentis, Escherichia coli, Variovorax paradoxus, Paracoccus denitrificans, Zoogloea resiniphila. Acinetobacter towneri, Paracoccus pantotrophus, Arthrobacter aurescens, Proteus mirabilis, Bacillus funiculus, Pseudomonas aeruginosa, Bacillus mycoides, Bacillus subtilis subsp. Subtilis, Pseudomonas putida, Bosea vestrisii, Budvicia aquatica, Rhizobium radiobacter, Comamonas denitrificans, Rhodococcus rhodochrous, Comamonas testosteroni, Sphingobium yanoikuyae, Corynebacterium pseudodiphtheriticum, Staphylococcus auricularis, Cupriavidus necator, Staphylococcus sp., Enterobacter helveticus, Thauera linaloolentis, Escherichia coli, Variovorax paradoxus , Paracoccus denitrificans, Zoogloea resiniphila.
6. Méthode selon l'une quelconque des revendications précédentes, caractérisée en ce que les différentes fractions de l'échantillon aqueux sont réparties dans au moins 6, de préférence au moins 8, cellules de mesure renfermant chacune une souche bactérienne appartenant à l’un des groupes suivants : 6. Method according to any one of the preceding claims, characterized in that the different fractions of the aqueous sample are distributed in at least 6, preferably at least 8, measurement cells each containing a bacterial strain belonging to one following groups:
- groupe A : Bacillus funiculus, Bosea vestrisii, Zoogloea resiniphila, Paracoccus pantotrophus, Budvicia aquatica, ou Acinetobacter towneri,
- groupe B : Thauera linaloolentis, Comamonas denitrificans, Comamonas testosteroni, Bacillus mycoides, ou Rhodococcus rhodochrous, group A: Bacillus funiculus, Bosea vestrisii, Zoogloea resiniphila, Paracoccus pantotrophus, Budvicia aquatica, or Acinetobacter towneri, group B: Thauera linaloolentis, Comamonas denitrificans, Comamonas testosteroni, Bacillus mycoides, or Rhodococcus rhodochrous,
- groupe C : Pseudomonas aeruginosa, Cupriavidus necator, ou Pseudomonas putida, group C: Pseudomonas aeruginosa, Cupriavidus necator, or Pseudomonas putida,
- groupe D : Escherichia coli, ou Enterobacter helveticus, group D: Escherichia coli, or Enterobacter helveticus,
- groupe E : Variovorax paradoxus, - group E: Variovorax paradoxus,
- groupe F : Staphylococcus auricularis, ou Corynebacterium pseudo- diphtheriticum, group F: Staphylococcus auricularis, or Corynebacterium pseudo-diphtheriticum,
- groupe G : Paracoccus denitrificans, ou Proteus mirabilis, - group G: Paracoccus denitrificans, or Proteus mirabilis,
- groupe H : Rhizobium radiobacter, Bacillus subtilis subsp. Subtilis, Staphylococcus sp., Sphingobium yanoikuyae, ou Arthrobacter aurescens. - group H: Rhizobium radiobacter, Bacillus subtilis subsp. Subtilis, Staphylococcus sp., Sphingobium yanoikuyae, or Arthrobacter aurescens.
7. Méthode selon l'une quelconque des revendications précédentes, caractérisée en ce que les différentes fractions de l'échantillon aqueux sont réparties dans des cellules de mesure renfermant chacune une souche bactérienne unique comprenant au moins les huit souches bactériennes suivantes : Bacillus subtilis subsp. Subtilis, Budvicia aquatica, Comamonas testosteroni, Corynebacterium pseudodiphtheriticum, Cupriavidus necator, Escherichia coli, Proteus mirabilis, Pseudomonas putida, Rhodococcus rhodochrous, Variovorax paradoxus. 7. Method according to any one of the preceding claims, characterized in that the various fractions of the aqueous sample are distributed in measuring cells each containing a single bacterial strain comprising at least the following eight bacterial strains: Bacillus subtilis subsp. Subtilis, Budvicia aquatica, Comamonas testosteroni, Corynebacterium pseudodiphtheriticum, Cupriavidus necator, Escherichia coli, Proteus mirabilis, Pseudomonas putida, Rhodococcus rhodochrous, Variovorax paradoxus.
8. Kit pour réaliser l'évaluation de la teneur en matière organique biodégradable d'un échantillon aqueux, ledit kit comprenant : Kit for performing the evaluation of the biodegradable organic matter content of an aqueous sample, said kit comprising:
- au moins quatre cellules de mesure, de préférence six cellules, de préférence encore huit cellules de mesure, chacune renfermant une souche bactérienne unique, non pathogène, isolée du milieu naturel, et lyophilisée en présence d'un agent de protection, at least four measuring cells, preferably six cells, preferably eight measuring cells, each containing a single, non-pathogenic bacterial strain, isolated from the natural medium, and lyophilized in the presence of a protective agent,
- un marqueur d'activité métabolique desdites bactéries, a marker of metabolic activity of said bacteria,
- une notice détaillée de mise en œuvre de la méthode conforme à l'une des revendications précédentes.
- Detailed instructions for implementing the method according to one of the preceding claims.
9. Kit selon la revendication 8, caractérisé en ce que les cellules de mesure sont des flacons transparents, fermés, de préférence sous la forme de tubes individuels ou agencés en microplaques. 9. Kit according to claim 8, characterized in that the measuring cells are transparent, closed bottles, preferably in the form of individual tubes or arranged in microplates.
10. Utilisation de la méthode d'évaluation selon l'une quelconque des revendications 1 à 7, pour la quantification de la teneur en matière organique biodégradable d'un échantillon aqueux de stations d'épuration, d'effluents industriels ou d'effluents d'élevage ou d’eaux naturelles telles que les eaux de surface, les eaux souterraines ou les eaux marines. 10. Use of the evaluation method according to any one of claims 1 to 7, for the quantification of the biodegradable organic matter content of an aqueous sample of treatment plants, industrial effluents or effluents d or natural waters such as surface water, groundwater or marine waters.
11. Utilisation de la méthode d'évaluation selon l'une quelconque des revendications 1 à 7, pour la détermination de la demande biologique en oxygène (DBOs), par traitement statistique des données, obtenues lors du suivi spectrophotométrique de l'activité bactérienne dans chaque fraction d'échantillon, mettant en œuvre une méthode multivariée, tel qu’ un algorithme par apprentissage supervisé de type réseau de neurones, permettant de corréler les résultats de ladite méthode à la valeur correspondante de DBOs de la norme ISO 5815.
11. Use of the evaluation method according to any one of claims 1 to 7, for the determination of the biological oxygen demand (BODs), by statistical treatment of the data, obtained during the spectrophotometric monitoring of the bacterial activity in each sample fraction, implementing a multivariate method, such as a supervised learning algorithm of the neural network type, making it possible to correlate the results of said method to the corresponding value of DBOs of the ISO 5815 standard.
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