GB1603183A

GB1603183A - Process and apparatus for continuous measurement of the enzymatic activity of the biomass in biological purification systems

Info

Publication number: GB1603183A
Application number: GB12472/78A
Authority: GB
Original assignee: Societe National Elf Aquitaine
Current assignee: Societe National Elf Aquitaine
Priority date: 1977-04-01
Filing date: 1978-03-30
Publication date: 1981-11-18
Also published as: IT7821746A0; CA1098431A; DE2813949A1; JPS541090A; FR2386034A1; CH634149A5; FR2386034B1; ES468425A1; BE865541A; NL7803479A; IT1096167B

Abstract

Process for the continuous measurement of the enzymatic activity of the biomass in biological purification systems for urban or industrial effluents. The process consists in applying the assay of dehydrogenases by a colorimetric method to the continuous measurement of the enzymatic activity of the biomass in biological purification systems, especially by the method employing the reduction of colourless 2,3,5-triphenyl- tetrazolium chloride to red triphenylformazan. The automatic device using this process has a sufficiently rapid response to enable poisoning of a biological purification station to be prevented. <IMAGE>

Description

(54) A PROCESS AND APPARATUS FOR CONTINUOUS MEASUREMENT OF THE ENZYMATIC ACTIVITY OF THE BIOMASS IN BIOLOGICAL PURIFICATION SYSTEMS (71) We, SOCIETE NATIONALE ELF AQUITAINE, a French Company, of Tour Aquitaine, 92400 Courbevoie, France, do hereby declare the invention, for which we pray that a Patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention concerns a process for continuous measurement of the enzymatic activity of installations to treat urban and industrial effluents by biological methods, and an apparatus to perform this process.

The efficiency of purification plants depends on preservation of a sufficient level of activity of the biomass in contact with the effluent during treatment. It is therefore important to be able to measure such activity at any moment, using a technique that can be performed rapidly. This is the only way of discovering variations in the level of activity quickly enough to allow suitable corrective action to be taken. It is vital to take such action as soon as the process of the reduction of activity begins, since the risk of inhibition of the biomass then becomes serious.

When such inhibition occurs, as the result of actual poisoning of the bacteria, prolonged stoppage of the plant becomes unavoidable.

Methods used have been based on measurement of matter in suspension or of volatile matter in suspension. This method includes not only living but also dead micro-organisms, as well as non-biological volatile matter in suspension. Such tests are thus incapable of providing a precise indication of the state of the biomass and its activity at a given moment in time.

Respirometric methods, which measure the oxygen consumption rate, provide direct information on the activity of living biomass fraction. However, they are difficult to apply directly, and are hard to plan for in the context of continuous measurements.

Quantitative analysis of the dehydrogenases in activated sludge was discussed in 1964 at the Second International Conference for Research into Water Pollution in Tokyo, by Misters G. Lenhard, L. Nourse and H.M. Schwartz.

Use of colourless triphenyl-tetrazolium chloride (TTC), which is reduced to red triphenyl formazan (TF) as a result of the action of dehydrogenases, has been recommended by various authors, such as Philip H. Jones and D. Prasad in Journal of Water Pollution Control Federation (Novembre 1969, pp. R 441/R 449; editor Peter J. Piecuch, 3900 Wisconsin Avenue, N.W., Washington D.C. 20016).

Until now, however, measurements have produced discordant results, which generally fail to give an accurate representation of oxidation reactions in the biomass.

In French patent 2 302 281 of 28th February 1975, Roger Ben-Aim and Jean-Pierre Legeron recommend the use of a limited proportion of TTC in a differential method, to prevent any interactions occurring between the TTC and TF and the biomass proper.

Measurement is done away from light and in the absence of substrates, which could affect the reaction medium. Under these operating conditions, with intermittent measurements, it is difficult to obtain results fast enough to allow quick corrective action to be taken.

This invention allows such problems to be overcome by defining the level of enzymatic activity by the quantity of TTC reducer under given conditions.

Since TF is insoluble in water, the quantity of TTC reduced is directly proportional to the concentration of dehydrogenases with a reducing effect on TTC, in other words dehydrogenases with a redox potential of less than -80 millivolts.

Enzymatic activity can accordingly be defined by the quantity of TTC reduced under certain conditions, and this is done by extracting the water-insoluble TTC with an organic solvent, the optical density of which can be measured.

In order to obtain a result proportional to the concentration of micro-organisms, namely dehydrogenases, standard incubation conditions have to be defined, in other words conditions that can be applied to sludge in the biological purification system regardless of the load involved: these include oxygen concentration, addition of reducing elements, pH value of the effluent.

Oxygen concentration must remain constant in the medium. The general layout of a respiratory chain shows that there is competition between the co-enzyme Q and TTC for oxidation of flavin adenine dinucleotide in reduced form (FADH2). In Science, pp. 107, 144, 1949, an article by E. Kun and L.G. Abood entitled "Colorimetric estimation of succinic dehydrogenases by triphenyl-tetrazolium chloride" shows this competition by studying, under aerobic and anoerobic conditions, the kinetics of the reduction of TTC by succinate dehydrogenase extracted from rat's liver. Estimation is therefore reproducible only insofar as the ratio of oxidized co-enzyme Q to TTC is constant.

This condition will be met if, by means of additives producing this result, the percentage of oxygen at the start of incubation is eliminated. During incubation, the percentage of TTC will remain very high in relation to the percentage of oxidized co-enzyme Q. TTC thereupon becomes the final electron acceptor.

Consequently, the estimation of activity can be defined as the measurement of a flow of electrons. This electron flow is proportional to the concentration of nictinamide adenine dinucleotide in reduced form (NADH2). Since the concentration of NADH2 depends on the concentration of enzymes (dehydrogenases) and assimilable substrate (e.g. lactose), it is only when there is an excess of this substrate that the electron flow is proportional to the dehydrogenase concentration.

When there is excess substrate, then, the activity potential which is measured reflects a concentrtion of enzyme in the sludge at a given moment, regardless of the substrate concentration at that same moment.

Enzymatic activity is known to increase in direct relation to the pH value, so that this value has to be fixed, if only to allow for the fact that reduction of TTC into TF is accompanied by acidification of the medium. A buffer solution of pH 7.5 corresponds to the pH value most commonly found in biological purification systems.

These are the three general, standard incubation conditions which characterize this new process for continuous measurement of the enzymatic activity of the biomass in the system.

Incubation time and the conditions for halting incubation are covered by special recommendations.

In this process for continuous measurement of the enzymatic activity of biological purification systems, the effluent is sampled continuously, and the sample placed under standard conditions.

An activated sludge represents a heterogeneous medium in which various species of bacteria live together, each being characterized by a minimum generation time, which can range from 16 minutes for Escherichia coli to 5- hours for Rhizobium japinicum; in most cases the generation time is approximately 20 minutes.

It is therefore necessary to choose a shorter incubation time, say 15 minutes, in order to measure the characteristic activity of a generation of bacteria.

The fact of replacing oxygen with TTC as final electron acceptor results in blocking the oxidative phosphorilation process which generates adenosine triphosphate (ATP), a product which stores energy at the level of the cell. This ATP is reused for the anabolism on which growth depends, and the choice of a longer incubation period would involve measuring activity resulting from disturbed growth.

In 15 minutes it is possible to measure an activity potential corresponding to a generation of bacteria and proportional to their concentration.

The process is performed at a temperature corresponding to maximum activity of the micro-organisms present in the medium.

In this novel process: - the sample is fractionated in the pipes by injecting an inert gas under optimum temperature conditions for the enzymatic activity of the bacterial content of the effluent involved, for a given period of time; - the sample is made to circulate under these optimum temperature conditions for the enzymatic activity for a given period, long enough for standard conditions of absence of oxygen and neutralized medium to prevail homogenously; - a substrate is added, such as lactose, which can be assimilated by the bacterial fauna, together with a measurable redox indicator which can be converted by reduction into a product that can be estimated, for example by colorimetry; - the sample circulates at the same optimum temperature for a given period, known as the incubation period.

Since it has been found that enzymatic reactions are not completely blocked by the addition of the alcohol used for extraction, two apparatuses have been used successfully, one using the effect of a thermal shock, and the other a sudden change in pH, to halt the enzymatic activity of the biomass.

In this novel process: - the resulting product is extracted with a solvent, thereby localizing it in the liquid phase; - sludge is separated by decanting; - finally, the colorimetric intensity of the liquid phase is measured.

In such a process it is preferable to use 2,3,5-triphenyl tetrazolium chloride, which is reduced to triphenyl formazan, as redox indicator.

In one recommended embodiment, standard reducing-medium conditions, with zero oxygen content, are obtained by adding sodium sulphate and cobalt chloride.

In general, the sample circulates under optimum temperature conditions to suit the bacterial strains present.

The enzymatic activity of the biomass is halted either by raising the temperature above 65"C and keeping it there for a certain period of time, or by altering the pH value, by adding sulphuric acid, for example.

In some embodiments, an alcohol, such as ethyl alcohol, is used to extract and localize the resulting product in the liquid phase of the effluent.

Similarly, the substrate, which can be assimilated by the bacteria, and which is added to the sample before incubation, is a sugar, such as lactose.

The apparatus for carrying out the novel process for continuous measurement of the enzymatic activity of biological purification systems comprises a first tubular pipe with high heat-transmission coefficient, submerged in a bath of given temperature, and consisting of a first portion, the length, section and tortuosity of which are such that it forms a homogenizing mixer, supplied by a proportionating pump with micr-organisms, redox additives and inert fractionating gas, and also consisting of a second portion, the length, section and tortuosity of which are such that the time taken by the sample to pass through, related to the capacity of the proportionating pump, is between 15 and 20 minutes.

Said apparatus comprises, following the first tubular pipe, a second tubular pipe with high heat-transmission coefficient, immersed in a bath at a temperature of between 65 and 80"C, the beginning of this second passage containing an inlet for a flow of solvent, and being followed by a decanter containing two discharge outlets, one to remove extracted sludge and the other to allow the liquid phase to pass into another pipe leading to a colorimetric measuring apparatus.

In another embodiment, the apparatus for carrying out the novel process of continuous measurement of the enzymatic activity of biological purification systems comprises a first tubular pipe with high heat-transmission coefficient, immersed in a bath of given temperature, and consisting of a first portion, the length, section and tortuosity of which are such that it forms a homogenizing mixer, supplied by a proportionating pump with biomass, redox additives and inert fractionating gas, and also consisting of a second portion, the length, section and tortuosity of which are such that the time taken by the sample to pass through, related to the capacity of the proportionating pump, is between 15 and 20 minutes.

Said apparatus comprises, following the first tubular passage, and after a first inlet for a product halting all bacterial activity, a second inlet for solvent, this second inlet being followed by a decanter containing two discharge outlets, one to remove extracted sludge and the other to allow the liquid phase to pass into another pipe leading to a colorimetric measuring apparatus.

It will be easier to understand the invention from the following description of two of the possible industrial embodiments of the process and corresponding installations, illustrated by the accompanying Figures: Figure 1 is a diagram of a measuring apparatus in which enzymatic activity is stopped by means of a thermal shock.

Figure 2 is a diagram of a measuring apparatus in which enzymatic activity is stopped by altering the pH value.

Figure 1 shows a circuit 1, one end of which la forms a continuous sampling device, immersed in a channel (not shown here) containing a continuous flow of biomass and the other end 1b of which is a discharge outlet. The end of the pipe is connected directly to a proportionating pump 2, for example a peristaltic pump known in the previous art, through which the circuit 1 passes in the form of a component 1c made of an elastic material such as an elastomer. The proportionating pump 2 is connected by a component 1d to another component, consisting of two connected parts le and lf, with lf following le. Both these parts are immersed in a thermostatic bath 3.There are several inlets in the component ld, to admit various additives: in particular, an Na2SO3 inlet 4, a CoCl2 inlet 5 and an inlet 6 for an inert gas such as nitrogen.

These inlets 4, 5 and 6 are connected by passages 4', 5' and 6' to storage or supply facilities (not shown here) for the various additives, by means of pipes each passing through sections of the proportionating pump 2. These sections comprise suitable means (not shown here) of imparting a given flow velocity to each additive, and accordingly a given flow-rate.

The parts le and if of the passage consist of a tube made of a material with a high heat-transmission coefficient, such as a metal alloy selected for its very good resistance to corrosion.

The length, section and tortuosity of the part le of the circuit are such that the mixture, when it leaves this part, may be regarded as homogeneous in temperature, chemical composition and biological state.

At the junction between le and lf, there is an inlet 7 to admit a compound which can be assimilated by the bacteria in the sample and a redox indicator. This inlet 7 is connected by a pipe 7', which passes through a section of the proportionating pump 2, to separate containers for the assimilable compound and the indicator.

The length, section and tortuosity of part if of the circuit are such that the effluent, moving at the velocity imparted to it by the proportionating pump and at the flow-rates also imparted by the proportionating pump to the various additives, takes a given length of time to pass through this part lf.

The end of part lf is connected by a component 1g to a component lh, immersed in a thermostatic bath 8, the temperature of which is significantly higher than in the bath 3, preventing any enzymatic activity in the bacterial content of the effluent.

Component 1g contains an inlet 9 to admit a solvent of the coloured product into which the redox indicator has been converted. This inlet 9 is connected by a pipe 9', which passes through a section of proportionating pump 2, to a solvent container (not shown here). The solvents used are selected from alcohols such as C2H5OH.

The length, section and tortuosity of component lh, which is made of the same material as parts le and lf, are such that the effluent takes a given length of time to pass through it.

Component 1h is connected by another component li to a defrothing device 10 of a type known per se, from which emerge a pipe 11 for the gas phase, leading to a discharge outlet, and a pipe lj, leading to a decanter 12 of a type known per se.

From the decanter emerge a pipe 13 to extract and convey sludge to a discharge outlet, and a component 1m which conveys the liquid phase to the inlet of a recording colorimeter 14 of a type known in the previous art, as regards both the actual colorimeter 14 and the recording device 14', which may be separate.

Pipe component 1m extends beyond the colorimeter outlet in the form of another component in which leads to the discharge outlet lb.

Pipe 12 and components 1m and ln each pass each through a convenient member of sections of the proportionating pump 2, in order to ensure continuous circulation of the effluents.

Figure 2 shows the same components designated by the same references, except for the component 1h and the thermostatic tank 8, which are replaced by a mixer device 8' containing an inlet 15 to admit a product such as sulphuric acid, to inhibit the enzymatic activity of the micro-organism fauna; it also contains the solvent inlet 9.

This apparatus functions as follows. The proportionating pump 2 contains a sufficient number of separate sections to provide a flow of sample and of various additives, and to collect the liquid phase and sludge following the decanter 11, in order to dispatch the liquid phase to the colorimeter 13 and sludge to the discharge outlet.

Before reaching the recording colorimeter inlet, the sample undergoes the following phases of treatment: - the sample receives the additives Na2SO3 and CoCl2 in proportions such that free oxygen is completely eliminated and the pH value is adjusted, to 7.5 for example (neutralized medium).

- the effluent passes through the part le of the circuit submerged in a thermostatic tank, which is maintained at the optimum temperature for enzymatic activity, generally 37"C; on leaving this part of the circuit, the sample is homogeneous in temperature, chemical composition and biological state; - the sample then receives, through the inlet 7, a carbohydrate compound which can be assimilated by the bacteria, such as lactose, and a redox indicator, in this case 2,3,5-triphenyl-tetrazolium chloride (TTC); - the sample then passes through a part if of the circuit for a given period, for example 15 minutes, at the temperature chosen, under conditions such that the pH value of the medium remains constant (neutralized medium) and the oxygen concentration nil; under such conditions TTC is a final electron acceptor, which makes quantitative analysis reproducible.

The 2,3,5-triphenyl-tetrazolium chloride is reduced by the dehydrogenases into triphenyl formazan (TF), in accordance with the following reaction:

,N-N - C6H5 + 2H N-NH-C6H5 +H + + C,H, CN=N - C6H5 + 2H C6H5-C N=N-C6H5 Cl (colourless) (red) On completion of its passage, part if lf receives an amount of a triphenyl formazan solvent, such as C2H5OH.

The sample is then directed towards a device which blocks the enzymatic reaction. This phenomenon can be obtained either by a thermal shock (Figure 1), or by the addition of an inhibiting compound such as sulphuric acid (Figure 2).

After having the froth removed, the sample is sent to a decanter 12 in which the sludge, freed of the red triphenyl formazan which had attached itself to the solid particles, is separated and sent to the discharge outlet, while the liquid phase containing all the triphenyl formazan resulting from enzymatic activity during the incubation period in the part if of the circuit is directed to the recording colorimeter inlet.

This quantitative analysis process and the apparatus for its performance can be used to measure the enzymatic activity of any fermentation system, aerobic and anaerobic.

The process is particularly suitable for determining the possibility of treatment of industrial effluents in a laboratory pilot unit.

When the apparatus is equipped with a device for crushing non-biological matter in suspension, it can be used for continuous recording of the activity of the biomass of an aeration plant.

If the apparatus is positioned in front of a station and supplied with effluent to be treated and with the receiving micro-organism fauna, it can be used to detect possible inhibition of the fauna, for example by a toxic substance, and to prevent it.

Naturally, this invention is in no way confined to the embodiment described above: many variant forms are possible for someone skilled in the art, depending on applications, and without any departure from the invention as defined in the claims.

WHAT WE CLAIM IS: 1. A process for the continuous measurement of the enzymatic activity of the biomass in biological purification systems, wherein: - a continuous sample of the biomass fauna is taken; - this sample is placed under standard conditions, comprising neutralized reducing medium with zero oxygen content; - the sample is made to flow under these optimum temperature conditions for the enzymatic activity for a given period, long enough for standard conditions of absence of oxygen and neutralized medium to prevail homogeneously; - a compound which can be assimilated by the bacteria present is added, together with a measurable redox indicator which can be converted by reduction into a product that can be estimated; - the sample is made to flow at the same optimum temperature for a given period, known as the incubation period;; - the enzymatic activity of the bacterial fauna is halted; - the reduced redox indicator is extracted with a solvent, thereby localizing it in the liquid phase of the sample.

- sludge is separated out by decanting; - the resulting product is measured in the liquid phase.

2. A process as defined in claim 1, wherein the redox indicator is 2,3,5-triphenyltetrazolium chloride, which after reduction forms triphenyl formazan.

3. A process as defined in claim 2, wherein the sample is placed under standard conditions, comprising a reducing medium with zero oxygen content, by the addition of sodium sulphate and cobalt chloride.

4. A process as defined in claim 2, wherein: - the sample is made to flow and fractionated by means of an inert gas, under selected optimum temperature conditions; - the enzymatic activity of the bacterial fauna is halted by means of a thermal shock, by raising the temperature to a given level above 65"C and keeping it at this level for a given period of time.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. The 2,3,5-triphenyl-tetrazolium chloride is reduced by the dehydrogenases into triphenyl formazan (TF), in accordance with the following reaction: ,N-N - C6H5 + 2H N-NH-C6H5 +H + + C,H, CN=N - C6H5 + 2H C6H5-C N=N-C6H5 Cl (colourless) (red) On completion of its passage, part if lf receives an amount of a triphenyl formazan solvent, such as C2H5OH. The sample is then directed towards a device which blocks the enzymatic reaction. This phenomenon can be obtained either by a thermal shock (Figure 1), or by the addition of an inhibiting compound such as sulphuric acid (Figure 2). After having the froth removed, the sample is sent to a decanter 12 in which the sludge, freed of the red triphenyl formazan which had attached itself to the solid particles, is separated and sent to the discharge outlet, while the liquid phase containing all the triphenyl formazan resulting from enzymatic activity during the incubation period in the part if of the circuit is directed to the recording colorimeter inlet. This quantitative analysis process and the apparatus for its performance can be used to measure the enzymatic activity of any fermentation system, aerobic and anaerobic. The process is particularly suitable for determining the possibility of treatment of industrial effluents in a laboratory pilot unit. When the apparatus is equipped with a device for crushing non-biological matter in suspension, it can be used for continuous recording of the activity of the biomass of an aeration plant. If the apparatus is positioned in front of a station and supplied with effluent to be treated and with the receiving micro-organism fauna, it can be used to detect possible inhibition of the fauna, for example by a toxic substance, and to prevent it. Naturally, this invention is in no way confined to the embodiment described above: many variant forms are possible for someone skilled in the art, depending on applications, and without any departure from the invention as defined in the claims. WHAT WE CLAIM IS:

1. A process for the continuous measurement of the enzymatic activity of the biomass in biological purification systems, wherein: - a continuous sample of the biomass fauna is taken; - this sample is placed under standard conditions, comprising neutralized reducing medium with zero oxygen content; - the sample is made to flow under these optimum temperature conditions for the enzymatic activity for a given period, long enough for standard conditions of absence of oxygen and neutralized medium to prevail homogeneously; - a compound which can be assimilated by the bacteria present is added, together with a measurable redox indicator which can be converted by reduction into a product that can be estimated; - the sample is made to flow at the same optimum temperature for a given period, known as the incubation period;; - the enzymatic activity of the bacterial fauna is halted; - the reduced redox indicator is extracted with a solvent, thereby localizing it in the liquid phase of the sample.

5. A process as defined in Claim 2, wherein:

- the sample is made to flow and fractionated by means of an inert gas, under selected optimum temperature conditions; - the enzymatic activity of the bacterial fauna is halted by altering the pH value.

6. A process as defined in claim 1, wherein the resulting product is extracted and localized in the liquid phase of the effluent using an alcohol.

7. A process as defined in claim 1, wherein the compound which can be assimilated by the bacteria is a sugar.

8. A process as defined in claim 7, wherein the sugar which can be assimilated by the bacteria is lactose.

9. A process as defined in claim 1, wherein the measurable product is measured in the liquid phase by colorimetry.

10. A process as defined in claim 1, wherein the measurable product is measured in the liquid phase by an electrochemical process of the redox reagent.

11. An apparatus for continuous measurement of the enzymatic activity of the biomass in biological purification systems, comprising a first tubular pipe with high heattransmission coefficient, immersed in a bath at a temperature selected to suit the bacterial fauna present, and consisting of a first portion, the length, section and tortuosity of which are such that it forms a homogenizing mixer, supplied by a proportionating pump with the micro-organism sample, redox additives and inert gas, and also consisting of a second portion, the length, section and tortuosity of which are such that the time taken by the sample to pass through, related to the capacity of the proportionating pump, corresponds to the generation time of the bacterial fauna involved, said apparatus further comprising, following the first tubular pipe, a second tubular pipe with high heat-transmission coefficient, immersed in a bath at a temperature of above 65"C, the beginning of this second tubular pipe containing an inlet for a flux of solvent, and being followed by a decanter containing two discharge outlets, one to remove extracted sludge and the other to allow the liquid phase of the sample to pass into another pipe leading to a measuring apparatus.

12.An apparatus for continuous measurement of the enzymatic activity of the biomass in biological purification systems, comprising a first tubular pipe with high heat-transmission coefficient, immersed in a bath at a temperature selected to suit the bacterial fauna present, and consisting of a first portion, the length, section and tortuosity of which are such that it forms a homogenizing mixer, supplied by a proportionating pump with the micro-organism sample, redox additives and inert gas, and also consisting of a second portion, the length, section and tortuosity of which are such that the time taken by the sample to pass through, related to the capacity of the proportionating pump, corresponds to the generation time of the bacterial fauna involved, said apparatus further comprising, following the first tubular pipe, a system of two inlets, one for a flux of solvent, and the other for a product halting all bacterial activity in the sample, this inlet system being followed by a decanter containing two discharge outlets, one to remove extracted sludge and the other to allow the liquid phase of the sample to pass through into another pipe leading to a colorimetric measuring apparatus.