EP3298157A1 - Method for quick measurement of biochemical oxygen demand - Google Patents

Abstract

The invention relates to a calibration method for measuring the BOD5 of samples by fluorescence, using samples with rising concentrations, referred to as reference samples or standard solutions (Std1 to Std8), said reference samples being prepared from a single stock solution comprising a mixture of oligo- and polysaccharides and of oligo- and polypeptides; and method for quick measurement of biochemical oxygen demand implementing said calibration method.

Description

 METHOD FOR RAPID MEASUREMENT OF BIOCHEMICAL DEMAND

 OXYGEN

The present invention relates to a calibration method for the quantification of BOD5, its use in a method for rapidly measuring the biochemical oxygen demand in an aqueous liquid medium containing biodegradable organic materials and a kit for the implementation of this process.

 The invention applies to the field of the analysis of the biodegradability of organic materials of various types, such as, for example, those of water and sludge treatment plant, agro-industrial effluents or livestock, industrial, agro-industrial and agricultural waste or compost.

 Organic substrates can be used by microorganisms as substrates from which they derive energy and the ability to grow. Many industrial applications use these biochemical degradation reactions, under aerobic or anaerobic conditions, including: wastewater treatment, production of high value-added compounds, energy production, and agro-food production. The sources of organic substrates available are very varied: crops and crop residues, production and agri-food residues, household and urban waste, biomass including algae.

There are different methods for measuring the biodegradability of organic substrates, that is, to measure the extent to which microorganisms will be able to use these substrates to transform them. Each field of application uses its own method very generally based on the measurement of the product formed during the reaction, sometimes on the measurement of the degraded substrate. These methods are long, complex to implement and require heavy equipment, cumbersome and expensive.

 One of the most commonly used industrial parameters for measuring the biodegradability of organic substrates is biochemical (or biological) oxygen demand (BOD). BOD is the amount of oxygen required by aerobic microorganisms in water to oxidize organic matter, dissolved or suspended in water. The most commonly performed measure is that of BOD5. BOD5 corresponds to the biochemical or (biological) oxygen demand after 5 days of incubation of the sample at a temperature of 20 ° C. Its standard measurement protocol is described in standard NF EN 1899-1 of May 1998. This measurement protocol is long and the results can vary mainly because of the fluctuations of the microbial diversity of the inoculum used.

 Many studies carried out over the last two decades have led to the development of different faster and more reliable technologies (Jouanneau S. et al., Water Research, (2014), 49, 62-82).

 Among these methods may also be mentioned the methods based on redox mediators such as that described by Dudal Y. et al. (Anal Bioanal Chem., (2006), 384, 175-179), which reproduces the contents of the international application WO 2006/079733 and describes a microplate fluorescence test for measuring the catabolic activity induced by the materials. dissolved organic matter (DOM). This technique allows, from glucose calibration curves, to calculate the mineralizable amount of a DOM and to classify DOMs according to their ability to react with bacteria.

Hudson et al. (Science of the total environment, 2007, 391, Nol, 149-158, Elsevier) and Heloise Garcia Knapil et al. (Environnmental Engineering Science, 2014, 31, nol2, 653-633) describe methods for measuring organic matter present in a sample, using the natural fluorescence of this organic material. This method aims to determine a BOD but without resorting to a degradation of organic matter by bacteria. Reshetilov A. et al. (State of the Art in Biosensors - Environmental and Medical Applications, 2013, 57-77, InTech) is a review of different methods for measuring BOD. Among these methods, the authors cite methods using biosensors using electron transport mediators, thus methods based on electrochemical measurements of BOD. The authors also report the possibility of using synthetic calibration solutions including rapidly degrading substrates, including those of the 2001 Test 303 mentioned in the OECD Guidelines (http://www.oecd-ilibrarv.org/ environment / test-no-303-simulation-test-aerobic-sewaqe-treatment-a-activated-sludqe -units-b-biofilms_9789264070424-en).

 Murakami Y et al. (Sensors and Actuators B: Chemical International Journal Devoted to Reasearch and Development of Physical and Chemical Transducers, 1998, 53, No. 3, 163-172, Elsevier) discloses an electrochemical system including immobilized bacteria, sensitive to pH changes resulting from the microbial activity.

Christian Guyard (Water, industry, nuisances, 2010, 334, 51-58) describes the method that is the subject of the application WO 2006/079733 which does not measure BOD5 but determines the biogeochemical reactivity of the organic matter contained in a sample. US Pat. No. 6,511,822 describes a method for the electrochemical detection of BDO5 using a calibration curve via a glutamic acid-glucose mixture which is the standard of the standard DBO5 NF EN 1899-1. The application US 2004/326617 relates to a determination method of the BOD5 based on the electrochemical detection of the oxygen consumption of an immobilized bacterial consortium for the degradation of the biodegradable MO of a sample. More recently, the inventors have developed a measurement of the biodegradability of organic substrates by the fluorescent and / or colorimetric detection of the microbial activity generated by the addition of organic substrates in a mixture of microorganisms making it possible to deduce therefrom calibration the expected value for each application domain and quantify the organic matter present. This method, described in the application EP2597461, is carried out on a format allowing the multiplicity of measurements with an automated data processing. It is based on the fact that during the incubation, the biodegradable organic material will be assimilated by the bacteria directly or after hydrolysis of higher molecular weight polymers (polysaccharides, polypeptides, ...). The different catabolic pathways lead to the production of pyruvate and / or acetyl-CoA which, under aerobic conditions, are oxidized within the Krebs cycle. During this oxidation, cofactors are reduced (NAD +, FAD, ...) and these reduced cofactors are subsequently re-oxidized at the level of the respiratory chain inducing the production of energy in the form of ATP via ATP. synthase and the process of oxidative phosphorylation. In aerobic conditions, the final acceptor of the electron at the level of the respiratory chain is the oxygen of the air which undergoes a reduction leading to the production of water. The molecular probe used will substitute for a small fraction of dioxygen as the final electron acceptor and be reduced to a highly fluorescent chemical form. In this way, the metabolic activity involved in the degradation of the organic matter can be measured by fluorescence and this measure is directly proportional to the consumption. of oxygen by the bacteria. This oxygen consumption is precisely what defines the biological oxygen demand; when this is measured over 5 days, it is referred to as BOD5. However, this method is not based on a calibration comprising a complex standard formed of different solutions, nor on a double calibration as a function of the incubation period. Jouanneau S et al. (Water Research, 2013, 49, 62-82, Elsevier) is a review of different BOD measurement methods which refers in particular to the inventors method of application EP2597461.

 Nevertheless, analytical laboratories involved in the monitoring of this parameter still need to be made available to reconcile their growing needs for profitability and responsiveness.

However, the inventors have discovered that they can use the derivative of the fluorescence intensity to measure the degradation of organic matter. The derivative of the fluorescence intensity shown in FIG. 1 represents the rate of consumption of O 2. The most easily assimilated organic molecules are rapidly degraded (<24 hours) while polymers requiring several hydrolysis steps are degraded after a longer incubation time (> 24 hours).

 Thus the inventors have developed, on the basis of these differences in assimilation, a specific complex standard of urban waste water to make the standard curves.

The subject of the invention is therefore a calibration method for the fluorescence measurement of the BOD5 of samples from samples of increasing concentrations of said reference samples or standard solutions (Std1 to Std8), said samples of reference being prepared from the same mother solution comprising:

 i) from 30 to 60 mg / L of a PI peptone,

 ii) from 20 to 50 mg / L of a polypeptide P2 of about 30 to 50 kDa comprising at most 400 amino acids iii) from 20 to 50 mg / L of a polypeptide P3 of about 50 to 70 kDa comprising at least maximum 600 amino acids

 iv) from 5 to 25 mg / L of a mixture of oligosaccharides Gl comprising between 15 and 25 units of glucose, advantageously 15 units of glucose.

 v) from 5 to 25 mg / L of a branched chain G2 polysaccharide comprising several hundred to several tens of thousands of glucose units

 vi) from 130 to 170 mg / L of a G3 polysaccharide with linear chains organized in fibers comprising several tens of thousands to several million units of glucose, the total of the proteins representing 35 to 45% by weight of the total organic matter present in the solution, advantageously 40% by weight and the total of carbohydrates representing 55 to 65% by weight of the total organic matter present in the solution, advantageously 60% .The PI and Gl components of the stock solution are of the material organic biodegradable in less than 24 hours of incubation at 30 ° C, the components P2, P3 and G2 of the stock solution are biodegradable organic material between 24 and 48 hours of incubation at 30 ° C and the G3 component of the solution mother is not or only slightly degraded in 48 hours of incubation at 30 ° C.

For the purposes of the present invention, peptone is understood to mean the product of the reaction of a protein hydrolysis, by a polypeptide a protein of animal or vegetable origin, by oligosaccharide a glucose polymer such as maltodextrin, branched chain polysaccharide a branched chain glucose polymer such as starch and linear chain polysaccharides a linear chain glucose polymer such as cellulose.

 In a particular embodiment of the invention, the ratios expressed in% relative to the total mass of organic materials) are:

 PI = 15%

 P2 and P3 = 12.5% each

 Gl and G2 = 5% each

 G3 = 50%

 In an advantageous embodiment of the invention, the peptone P1 is a slightly hydrolysed casein peptone comprising 20 to 30% by weight of free amino acids and 70 to 80% of low molecular weight oligopeptides and polypeptides. , less than 25 kDa, comprising not more than 200 amino acids.

 In another advantageous embodiment of the invention, the P2 polypeptide is hen egg white albumin.

 In another advantageous embodiment of the invention, the P3 polypeptide is bovine serum albumin.

 In another advantageous embodiment of the invention, the carbohydrate G1 is a weakly hydrolysed maltodextrin (equivalent dextrose = about 4-7%) resulting in a mixture of oligosaccharides having on average about fifteen glucose units.

 In another advantageous embodiment of the invention, the carbohydrate G2 is potato starch.

In another advantageous embodiment of the invention, the carbohydrate G3 is cotton fiber cellulose. The mother solution which makes it possible to produce the standard range is prepared by solubilizing the various constituents of the standard (G1 to G3 and P1 to P3) in a saline solution. The saline solution corresponds to a 1/50 dilution in distilled water of the Enverdi-DBO® buffer (reagent B). The Enverdi-DBO® buffer is strongly inspired by the buffer used in the French standard BOD5 (NF EN 1899-1 of May 1998) and includes:

Na ₂ HPO ₄ , 12H ₂ O = 33.4 g / L

MgSO ₄ , 7H ₂ O = 4.5 mg / L

FeCl, 6H ₂ O = 0.25 mg / L

CaCl ₂ = 5.5 mg / L

According to the invention, the mother solution also has the following characteristics:

 chemical oxygen demand determined using the closed tube method (ST-COD) is between 340-360 mg O2 / L (measured according to ISO 15705 of 2002),

 BOD5 between 120-160 mg O2 / L (measured according to standard NF EN 1899-1 of May 1998)

 total protein concentration between 100 and 130 mg / L, advantageously 115 mg / L, total carbohydrate concentration between 160 and 200 mg / L, advantageously 180 mg / L

pH = 7-7.5 (measured according to standard NF T90-008 2001), conductivity = 1.1 to 1.4 mS / cm (measured according to standard NF EN 27888 of 1994).

 Those skilled in the art will be able to choose, in the light of their general knowledge, the quantities of PI to P3 and G1 to G3 so that the parent solution has these characteristics.

 In an advantageous embodiment of the invention, the standard solutions are as follows:

 Standard 8 (Std 8) or stock solution (SM): useful BOD5 (without G3) = approx. 110 mgO2 / L

 Standard 7 (Std 7) or SM diluted 2/3: useful DBO5 (without G3) = approx. 73 mgO2 / L

 Standard 6 (Std 6) or SM diluted 1/3: useful BOD5 (without G3) = approx. 37 mgO2 / L

 Standard 5 (Std 5) or SM diluted 1/10: useful BOD5 (without G3) = approx. 11 mgO2 / L

 Standard 4 (Std 4) or SM diluted 1/15: useful BOD5 (without G3) = approx. 7.5 mgO2 / L

 Standard 3 (Std 3) or MS diluted 1/30: useful BOD5 (without G3) = approx. 3.5 mgO2 / L

 Standard 2 (Std 2) or SM diluted 1/75: useful BOD5 (without G3) = approx. 1.5 mgO2 / L

 Standard 1 (Std 1) or saline solution: DBO5

 0 mgO2 / L

For the purposes of the present invention, the term "useful BOD5" is understood to mean the BOD5 measured from the standard solutions as defined above and corresponding to the metabolizable material in 48 hours. It is therefore based on peptides PI to P3 and polysaccharides G1 and G2, G3 not being metabolized after 48 hours. Depending on their complexity, the molecules PI to P3 and G1 to G3, constituting the standard solutions, degrade more or less rapidly during the incubation. Similarly, the fluorescence intensity that results from their degradation varies according to the amount of oxygen required for their mineralization. Thus, the degradation profile is divided into 2 zones: zone 1 (ZI) from 0 to 24h and zone 2 (Z2) from 24 to 48h. Gl carbohydrate and PI protein are degraded to ZI, PI and P2 proteins, as well as carbohydrate G2 are degraded to Z2 (see Figure 2). G3 carbohydrate is not significantly degraded in 48 hours. The incubation time corresponding to the limit between zone 1 and 2 is indicative and may vary according to the inoculum considered.

Zones ZI and Z2 are independently calibrated because the relationship between fluorescence and oxygen consumed differs by the very nature of the molecules that constitute them and the physicochemical conditions of the medium at the incubation time considered. This calibration in two zones, allows a more accurate measurement since it takes into account that the bond uniting the fluorescence with the BOD5 differs according to the degradation time of the organic matter. The BOD5 portion of the degraded standard in ZI and that degraded in Z2 are calculated from the data resulting from the production of the standard and from standard BOD5 measurements made on the standard and the molecules constituting it using the same inoculum as that used with Enverdi-DBO®, either an urban wastewater treatment plant (WWTP) inlet or a commercial preparation using the following formulas: % DBO ZI =

 ([Gl] .DB05m-Gl + [Pl] .DB05m-Pl) / Σ [X] .DB05m-X

 % BOD Z2 =

 ([P2] DB05m-P2 + [P3] DB05m-P3 + [G2] .DB05m-G2) / Σ [X] .DB05m-X

 DBO ZI = DB05 Stdi x% BOD Zl

 BOD Z2 = DB05 Stdi x% BOD Z2

 with:

 X = Gl, G2, G3, PI, P2 or P3

 [X] = the concentration of the X molecule calculated from the weighing performed for the preparation of the stock solution - in mg / L

BOD5m-X = the specific BOD5 of the X molecule obtained by measurement according to the standard BOD5 method with the same inoculum as that used in the Enverdi-DBO® method - in mgO2 / mg of X Σ [X] .DB05m-X = the sum of the BOD5 provided by each of the molecules constituting the mother solution - in mgO2 / L

 DBO5 Stdi = the BOD5 of the standard solutions Stdl to Std8 calculated from the measurement made on the stock solution according to the standard BOD5 method with the same inoculum as that used in the Enverdi-DBO® method - in mgO2 / L

 Finally, four calibration curves are obtained:

 a calibration curve Zl high range (GH) obtained from standard solutions Stdl, Std5, Std6, Std7 and Std8;

 a calibration curve Z2 GH obtained from the same standard solutions, Stdl, Std5, Std6, Std7 and Std8;

a calibration curve Zl low range (GB) obtained from standard solutions Stdl, Std2, Std3, Std4 and Std5 and a calibration curve Z2 GB obtained from the same standard solutions Stdl, Std2, Std3, Std4 and Std5. The value plotted on the ordinate axis of the calibration lines is the area under the curve of the fluorescence derivatives (AUC), respectively, in ZI and Z2 for the zone 1 and zone 2 calibration curves. Mathematically, this amounts to taking the difference between the intensities of fluorescence obtained at 24 hours and Oh for ZI and those obtained between 48 and 24 hours for Z2.

 The concentrations of the different standard solutions therefore their BOD5 values vary according to the inoculum considered and the skilled person will be able to adapt them accordingly. To establish the calibration curves, the Stdl point corresponds to the measurement blank (buffered solution free of organic matter), the Std2 point corresponds to a DBO5 value of approximately 1.5 mg O2 / L, the Std5 point corresponds to a value BOD5 of about 11 mgO2 / L and the Std8 point corresponds to a BOD5 value of about 110 mgO2 / L. Moreover, for a given inoculum, the calibration of the Enverdi-DBO® method with respect to the standard BOD5 can be verified using the stock solution used to prepare the range and, if necessary, readjusted.

 In conclusion, the AUC is proportional to the amount of molecular probe reduced during the bacterial metabolism involved for the degradation of organic matter and therefore to the amount of oxygen required for it. The equivalence with BOD5 is obtained through a complex standard representative of urban waste water and whose BOD5 according to the standard method was measured.

For the quantification of the samples, the AUCs at ZI and Z2 are calculated from the fluorescence measurements made during the incubation and the BOD5 value is determined thanks to the calibration curves established by summing the value obtained in ZI and that obtained in Z2. Highly concentrated samples (treatment plant inlet) are quantified with GH calibration curves and those weakly concentrated (leaving the treatment plant) with GB calibration curves. The choice of the range suitable for quantifying a sample is performed automatically according to the value of the AUC obtained for the sample in comparison with those obtained for the standard solutions and independently between ZI and Z2. Thus, for example, the quantification of a sample which would present a lot of BIO5 in ZI and little in Z2, would use the GH range in ZI and the GB range in zone 2, this in order to guarantee a result as fair as possible.

 The invention also relates to a method for rapidly measuring the biochemical oxygen demand in samples containing biodegradable organic materials, said method comprising the following steps:

 a) incubation with a fluorescent bioreactant and an inoculum of microorganisms capable of degrading the sample to be tested, within a microplate with several wells:

 on the one hand at least one sample containing biodegradable organic materials, and

 on the other hand 8 samples of increasing concentrations called reference samples or standard solutions (Std l to Std8), prepared from the same mother solution comprising:

 i) from 30 to 60 mg / L of a PI peptone,

ii) from 20 to 50 mg / L of a polypeptide P2 of about 30 to 50 kDa comprising at most 400 amino acids, from 20 to 50 mg / L of a polypeptide P3 of about 50 to 70 kDa comprising at most 600 amino acids, iv) from 5 to 25 mg / L of a mixture of oligosaccharides Gl comprising between 15 and 25 units of glucose, advantageously 15 units of glucose, v) from 5 to 25 mg / L of a branched chain G2 polysaccharide comprising several hundred to several tens of thousands of glucose units,

vi) from 130 to 170 mg / L of a G3 polysaccharide with linear chains organized in fibers comprising several tens of thousands to several million units of glucose, the total of the proteins representing 35 to 45% by weight of the total organic matter present in the solution, advantageously 40% by weight and the total of carbohydrates representing 55 to 65% by weight of the total organic material present in the solution, advantageously 60%.

 - Gl and PI being degraded between 0 and 24 hours of incubation (zone 1 or ZI), G2, P2 and P3 being degraded between 24 and 48 hours of incubation (zone 2 or Z2) and G3 not being degraded in 48 hours, said reference samples or standard solutions making it possible to construct four calibration curves according to the zone of degradation ZI or Z2 and function of the concentrations of the reference samples; b) analyzing the fluorescence emitted by said samples over time, said fluorescence analysis comprising three steps:

 measurement of a fluorescence intensity emitted following the degradation of the test sample and of the reference samples under the effect of the inoculum of microorganisms, during 45-55 hours, advantageously 48 hours,

using said measured fluorescence intensity for the sample to be tested to calculate a reference organic matter concentration, compared with the fluorescence intensity measured for the reference samples, and - conversion of this concentration of organic matter in mgO2.L ^{~ 1} values equivalent to the standard BOD5 (biological oxygen demand over 5 days), by applying a correspondence model obtained beforehand by comparison between measurements of the intensity of fluorescence carried out according to the same method on the reference samples and measurements made according to the BOD5 standard for said reference samples.

 In practice, the steps of using the fluorescence intensity and the conversion of the organic matter concentration can be carried out simultaneously, since one passes directly from the fluorescence to a value in mgO2 / L thanks to the calibration curves and the prior characterization of this standard according to the standard method.

The method according to the invention can be used routinely for the quantification of the BOD5 of urban waste water (raw, during or after treatment). In addition to the analysis of urban wastewater, this method can be used for any sample of industrial wastewater that has characteristics close to urban wastewater, ie where the biodegradable organic matter is predominantly composed of molecules biochemicals. The method thus proved valid for wastewater from the food and paper industries. The method can also be used for surface waters and natural waters provided that the limit of quantification is 0.5 mgO2 / L as required by the BOD5 standard for this type of sample (NF EN 1899-2 ); whatever the type of water analyzed, the pH of the samples must be between 6 and 8; if this is not the case, the pH must be adjusted first. According to the invention, samples containing organic material can be taken at any point in a wastewater treatment plant or at the factory outlet or at a waste generating site.

As for the standard method, the dilution of the samples before analysis is based on their total organic matter value measured by the COD and also expressed in mgO2 / L. If the estimated BOD5 from the COD is lower than the std8 BOD5 then the sample can be analyzed raw, otherwise the sample should be diluted appropriately in saline water (reagent B diluted 1: 50). ^th in deionized water). The automated data processing program provided by Envolure also allows for the determination of the appropriate dilution ratio of the samples from the measured COD as well as to define, before analysis, the plate plane, ie the position on the microplate of the different standard solutions (standard range) and samples. The analysis of the samples and the standard range must be carried out in duplicates or triplicates; a 96-well microplate makes it possible to simultaneously analyze 40 samples in duplicates or 24 samples in triplicates. The eventual pH adjustment of the samples, the determination of the dilution of the samples from their COD and the definition of the plate plan are thus the first steps of the analysis. The microplate reader / incubator should also be preheated to 30 ° C in order to be at the correct temperature when the time comes.

Once these first steps have been performed and the samples have been diluted, they must be pretreated. Pretreatment of the samples consists of partial sterilization to inhibit the activity of naturally occurring bacteria within them. This partial sterilization can be carried out by any conventional technique, in particular by microwave treatment or heat treatment; those skilled in the art will be able to adapt the conditions to be used according to the type and the quantity of samples to be treated. Thus, for a volume of 25 ml, the pretreatment can be carried out by microwaves for 3 minutes or, preferably, for a volume of 200 μl, by heating at 120 ° C. for 10 minutes and then cooling at 4 ° C. for 10 minutes. min.

 In this way, the samples and standards (which do not initially contain bacteria) are analyzed under the same conditions of inoculation which leads to comparable results. To do this, the previously diluted samples are transferred into the polypropylene microplate using a micropipette respecting the plate plane defined upstream. The volume transferred is 200 μΙ_. The microplate is covered with an aluminum film to prevent evaporation and placed in an oven at 120 ° C for 10 minutes. At the end of this time, the plate is placed at 4 ° C in a refrigerator for 10 min to return to room temperature and condense any vapors.

During the pretreatment of the samples, the inoculum can be prepared. For this purpose, the used water used for this purpose (entry of wastewater treatment plant or WWTP) is either filtered at 1.2 μιτι using the syringe and syringe filters provided in the kit, or centrifuged at 5000 g for 30 s. The filtrate or the centrifugation supernatant is recovered and diluted in saline water (reagent B diluted ^1/50 in demineralized water).

Once the samples have been pretreated and the inoculum prepared, the polystyrene microplate can be filled in order to incubate the samples in the presence of the inoculum with fluorescence monitoring over time. For this, each well of the microplate that will be used for the analysis must be filled, in the following order, with: j- 20 μΙ_ Reagent A

| - 60 μΙ_ Reagent B

| - 170 μΙ_ of sample

 or solution

 standard

I (according to plate plan

defined)

20 μΙ of inoculum

 Stdl to Std8 correspond to Stdl to Std8 previously defined Ech = sample tested

These volumes must be introduced with micropipettes or with a pipette dispenser. With the microplate filled, the gas-permeable film is positioned thereon. In addition to allowing gas exchange to ensure aerobiosis, this film also limits the evaporation of the contents of wells. The microplate can then be introduced into the preheated reader / incubator at 30 ° C and the analysis sequence is started. The analysis sequence lasts 48 hours with a fluorescence measurement (Xex / Xem = 540/600 nm) every 30 min. Each fluorescence measurement is preceded by linear agitation of the microplate. When the analysis sequence is complete, the raw data can be processed automatically by a computer program provided by Envolure. The BOD5 of the samples is then calculated as previously described.

In an advantageous embodiment of the invention the fluorescent bioreactant is a derivative of Resazurin (CAS No. 550-82- 3) marketed by Invitrogen - Life Technologies under the name Prestoblue®.

 In an advantageous embodiment of the invention, the incubation temperature is between 25 and 35 ° C, preferably 30 to 35 ° C, more preferably 30 ° C. At these temperatures, the acceleration of the kinetics leads to a DBO5 equivalent after 48h only instead of 5 days under the conditions of the standard method (incubation at 20 ° C).

 In an advantageous embodiment of the invention, the incubation is carried out in a pH 7.2 buffer medium capable of supplying the essential nutrients to the bacteria: phosphorus, nitrogen and trace elements, advantageously in a phosphate buffer at pH 7.2 .

 According to the invention, the process can be applied to a purification plant effluent chosen from raw water, settled water, biologically treated water and discharges, surface water or groundwater, effluent or an effluent. waste from artisanal or industrial production.

 The invention also relates to a kit useful for implementing the method according to the invention and comprising: the fluorescent bioreactant, the reference samples and the buffer solution.

 Examples 1 and 2 and Figures 1 to 3 which follow illustrate the invention.

 FIG. 1 illustrates the derivative of the fluorescence intensity as a function of time for standard solutions Std1 to Std 8; the most easily assimilated organic molecules are rapidly degraded (<24h) polymers requiring several hydrolysis steps are degraded after a longer incubation time (> 24h).

Figure 2 shows the correlation between the substitution and reference DBO5 values for the three sampling sites (O Lab the Interdepartmental Union for the Sanitation of the Paris Urban Agglomeration (SIAAP); · Milwaukee Metropolitan Sewerage District Lab (MMSD) and ® Lab # 3). The lines Inc. sup. and Inc. inf. represent the generally accepted upper and lower limits of absolute uncertainty for the BOD5 values provided by the standard method.

 Figure 3 illustrates the degradation profile of PI, P2, Gl and G2; this profile is divided into 2 zones: zone 1 (ZI) from 0 to 24h and zone 2 (Z2) from 24 to 48h. Gl carbohydrate and PI protein are degraded to ZI, PI and P2 proteins, as well as G2 carbohydrate are degraded to Z2.

Example 1: Calibration protocol

As the BOD5 value of a sample according to the standard method may vary according to the inoculum used, it is necessary before routine use of the Enverdi-DBO® method to calibrate it according to the inoculum used in comparison with the inoculum used. standard BOD5 method. This step also allows, according to the characteristics of the inoculum, to specify its preparation protocol and in particular the dilution factor that must be applied before using it for analysis. Finally, for each inoculum, the incubation time is determined which exactly delimits the two calibration zones (ZI and Z2). For each method, the solutions indicated in the table below are used: Standard method

 (NF EN 1899-1 of May Enverdi-DBO® 1998)

 Std8 Stdl to Std 8

 Gl to 100 mg / L Gl to 100 mg / L

 G2 100 mg / L G2 100 mg / L

 G3 at 100 mg / L G3 at 100 mg / L

 PI at 100 mg / L PI at 100 mg / L

 P2 at 100 mg / L P2 at 100 mg / L

 P3 at 100 mg / L P3 at 100 mg / L

According to the standard method, the values DBO5 for StdO to Std7 are determined by calculation from the value of Std8; the DBO5 value of the standard solutions Std1 to Std8 is determined for the inoculum under consideration (DBO5-Stdi) and the fraction of degraded DBO5 in ZI and that degraded in Z2 are determined as% DBO ZI and% DBO Z2; these values can be integrated into an automated results processing program.

According to the Enverdi-DBO® method, the incubation time delimiting ZI and Z2 (ZI must contain the degradation peaks of G1 and PI, Z2 must contain the degradation peaks of P2, P3 and G2) is determined and the dilution factor of the inoculum: the fluorescence obtained for StdI should be as low as possible and the Std8 analysis should not lead to a limitation by the probe of the fluorescence produced (saturation). At the same time, the inoculum must be able to degrade P2, P3 in less than 48h. The optimal dilution of the inoculum therefore corresponds to the best compromise leading to the verification of these two criteria. After the Enverdi-DBO® method has been calibrated against the normed BOD5 for a given inoculum and determined the optimal dilution factor of the inoculum, this calibration and inoculum dilution should be validated by comparing the results. obtained by the two methods on a varied panel of samples very weakly to highly concentrated in BOD5, as well as on the control recommended in the standard BOD5. If necessary, calibration or dilution of the inoculum can be adjusted at this stage to obtain statistically comparable results for both methods for the same samples and the same inoculum. For this calibration, 20 samples of low to very low (<20 mgO2 / L) DBO5 and 20 medium to high (> 20 mgO2 / L) DBO5 samples were used. A glutamic acid-glucose (AGG) control at 210 mgO 2 / L is used as a control of the BOD5 standard.

 Calibration can also be performed using standard mixed solutions: Std ZI (mixture of a solution of G1 and a solution of PI at 15 and 45 mg / L respectively), Std Z2 (mixture of a solution of P2, a solution of P3 and a solution of G2 at concentrations of 35, 35 and 15 mg / L respectively) and Std8 corresponding to the standard solution itself which contains Gl, P1, P2, P3, G2 and G3 at respective concentrations of 15, 45, 35, 35, 15 and 150 mg / L).

Example 2 Comparison of the Results Obtained with the Method According to the Invention and Standard Methods (US Standard and EU Standard)

 1. Operating procedure

A total of 109 samples averaged 24 h of raw and treated wastewater was collected over a 20-day period from the various WWTPs of the two MMSDs (Milwaukee, USA) and the SIAAP (Paris, France). These samples were stored at 4 ° C and analyzed within 48 hours. Just prior to analysis, 25 ml of the samples were partially sterilized for 3-7 min using a microwave oven set to a minimum power (<200 W) to reduce its native bacterial load. The BOD5 and COD measurements taken before and after sterilization showed no impact of this procedure on the content and biodegradability of organic matter.

 The inoculum was prepared by centrifugation of raw wastewater from one of the treatment plants (30s, 2000 x g) to remove particulates.

 The ENVERDI® kit (Envolure, France and Envolus, USA) was used on all samples following the procedure given in the kit, by mixing the appropriate volumes of fluorescent reagent, buffer solution, and inoculum with samples or specimens. standards in a 96-well microplate. Except for inocula, all reagents and standards are provided in the kit. Fluorescence (λεχ = 540 nm, λειτι = 600 nm) was recorded every 15 minutes using a fluorescence microplate reader (FLx800, Biotek, USA) also used for incubation at 30 ° C and to agitate the microplate for 48 h.

 The fluorescence data was then translated into BOD5, in mg O2.L-1, using a standard-based model provided with the kit. These standards are treated exactly like the samples and their response is used to derive the BOD5.

 Standard BOD5 was performed for all samples using standard methods (Standard Method 5210 at MMSD and NF EN 1899-1 at SIAAP).

The equivalence between values measured by ENVERDI® DBO5 and standard methods was tested by the three most commonly used statistical tests: the linear regression of least squares, the Bland and Altman method and the Wilcoxon test. These tests were performed using the XLSTAT software (Addinsoft, France). 2. Results

 More than 80% of the BOD5 values measured with the substitution method are within the absolute uncertainty range of the standard method (see Figure 2). The substitution results are in accordance with the standard BOD5 measurements: the coefficients of determination (R2) are all greater than 0.9 and the guiding coefficients are very close to 1.

 The results obtained with Enverdi-DBO® are statistically equivalent to those obtained by the standard methods US-EPA 5210 (http://www.polyseed.com/misc/bod- std% 20methods-8.04.pdf) and NF EN 1899-1 of May 1998 (study carried out on 3 different sites in France and in the United States on a panel of 133 samples).

The method according to the invention, because:

the use of a unique standard specifically developed by the inventors in order to mimic the biodegradable organic matter of real samples,

 an incubation time of between 45 to 50 hours, advantageously equal to 48 hours at a temperature of between 25 and 35 ° C., advantageously equal to 30 ° C.,

 - a calibration in 2 zones 0-24h and 24-48h and

 - pre-treatment of real samples before analysis

allows precise quantification of the BOD5 of real samples. This simple and fast process shows performances identical to those obtained with the standardized method NF EN 1899-1 of May 1998.

Claims

1. Calibration method for the fluorescence measurement of the BOD5 of samples from samples of increasing concentrations of said reference samples or standard solutions (Std1 to Std8), said reference samples being prepared from the same solution mother comprising:  i) from 30 to 60 mg / L of a PI peptone,  ii) from 20 to 50 mg / L of a polypeptide P2 of about 30 to 50 kDa comprising at most 400 amino acids iii) from 20 to 50 mg / L of a polypeptide P3 of about 50 to 70 kDa comprising at least maximum 600 amino acids  iv) from 5 to 25 mg / L of a mixture of oligosaccharides Gl comprising between 15 and 25 units of glucose, advantageously 15 units of glucose.  v) from 5 to 25 mg / L of a branched chain G2 polysaccharide comprising several hundred to several tens of thousands of glucose units  vi) from 130 to 170 mg / L of a G3 polysaccharide with linear chains organized in fibers comprising several tens of thousands to several million units of glucose, the total of the proteins representing 35 to 45% by weight of the total organic matter present in the solution, advantageously 40% by weight and the total of carbohydrates representing 55 to 65% by weight of the total organic material present in the solution, advantageously 60%. A method for rapidly measuring the biochemical oxygen demand in samples containing biodegradable organic materials, said method comprising the steps of: a) incubation, with a fluorescent bioreactant and an inoculum of microorganisms capable of degrading the sample to be tested, in a microplate with several wells:  on the one hand at least one sample containing organic matter, and  on the other hand 8 samples of increasing concentrations called reference samples (Std1 to Std8), prepared from the same stock solution comprising: comprising:  i) from 30 to 60 mg / L of a PI peptone,  ii) from 20 to 50 mg / L of a polypeptide P2 of about 30 to 50 kDa comprising at most 400 amino acids, iii) from 20 to 50 mg / L of a polypeptide P3 of about 50 to 70 kDa comprising not more than 600 amino acids,  iv) from 5 to 25 mg / L of a mixture of oligosaccharides Gl comprising between 15 and 25 units of glucose, advantageously 15 units of glucose,  v) from 5 to 25 mg / L of a branched chain G2 polysaccharide comprising several hundred to several tens of thousands of glucose units,  vi) from 130 to 170 mg / L of a G3 polysaccharide with linear chains organized in fibers comprising several tens of thousands to several million units of glucose, the total of the proteins representing 35 to 45% by weight of the total organic matter present in the solution, advantageously 40% by weight and the total of carbohydrates representing 55 to 65% by weight of the total organic material present in the solution, advantageously 60%. - G1 and P1 being degraded between 0 and 24 hours of incubation (zone 1 or ZI), G2, P2 and P3 being degraded between 24 and 48 incubation hours (zone 2 or Z2) and G3 not being degraded in 48 hours, said reference samples or standard solutions making it possible to construct four calibration curves according to the zone of degradation ZI or Z2 and depending on the concentrations of the samples of reference; b) analyzing the fluorescence emitted by said samples over time, said fluorescence analysis comprising three steps:  measuring a fluorescence intensity emitted following the degradation of the test sample and the reference samples under the effect of the inoculum of microorganisms, for 45 to 50 hours, advantageously 48 hours, using said measured fluorescence intensity for the sample to be tested to calculate a reference organic matter concentration, compared with the fluorescence intensity measured for the reference samples, and conversion of this concentration of organic matter to mgO.L ^"1 values equivalent to the standard BOD5 (biological oxygen demand over 5 days), by applying a correspondence model obtained beforehand by comparison between measurements of the fluorescence intensity performed according to the same method on reference samples and measurements made according to the BOD5 standard for said reference samples. 3. Process according to claim 2, characterized in that the concentration of biodegradable organic material of the reference samples Std1 to Std8 corresponds to a value of BOD between 0 and approximately 110 mgO .L ^-1 . 4. Method according to any one of claims 2 or 3 characterized in that the incubation is carried out in a phosphate buffer medium at pH 7.2 at a temperature between 25 and 35 ° C. 5. Method according to any one of claims 2 to 4 characterized in that the organic sample is a sewage treatment plant effluent selected from raw water, settled water, biologically treated water and discharges, a water of surface or groundwater, effluent or waste from artisanal or industrial production. 6. Kit useful for the implementation of the method according to claim 2 characterized in that it comprises: the biofluorescent reagent, the reference samples and the buffer solution.