CA1337461C - Process for the quantification of methane gas bacteria - Google Patents
Process for the quantification of methane gas bacteriaInfo
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- CA1337461C CA1337461C CA000594621A CA594621A CA1337461C CA 1337461 C CA1337461 C CA 1337461C CA 000594621 A CA000594621 A CA 000594621A CA 594621 A CA594621 A CA 594621A CA 1337461 C CA1337461 C CA 1337461C
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- methane gas
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- 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
- C12Q1/06—Quantitative determination
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- 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
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analyzing Materials By The Use Of Fluid Adsorption Or Reactions (AREA)
- Heat Treatments In General, Especially Conveying And Cooling (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
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Abstract
The present invention provides a process for the quantification of methane gas bacteria and especially for monitoring the methane gas formation capacity of reactors containing methanogenic bacterial, wherein a representative sample of a medium containing the methane gas bacteria is subjected for at most one second to a brief irradiation by light with a wavelngth of 395 to 440 nm and the fluorescence thereby excited is deter-mined by flow cytometry.
Description
The present invention is concerned with a process for the quantification of methane gas bacteria.
Organic material is converted, with the exclusion of air and in the presence of an appropriate microflora, into methane gas. The material is thereby first hydrolysed in a multi-step decomposition (hydrolysis phase), then fermented to give organic acids (acidification phase), converted by acetogenic bacteria into molecular hydrogen and carbon dioxide (acetogenic phase) and then converted by methanogenic bacteria (methane gas bacteria) into methane gas (methanogenic phase). This reaction chain can only take place efficiently when, in the mixed bacterial population, sufficient methane gas bacteria are present and can multiply. Otherwise, the whole decomposition process is inhibited by the individual intermediate products.
This decomposition route takes place everywhere in nature where organic material is decomposed in the absence of air, i.e. in sumps and ponds, in eutrophied waters, on the beds of oceans and in the digestive tracts of humans and animals. In the so-called biogas reactors (fermentors), it is utilised for the conversion of organic waste from waste water clarification and from industrial and agricultural production into useful methane gas.
It is known that methane gas bacteria display an 3 1 337~61 autofluorescence in the case of brief irradiation.
This fluorescence is brought about by Factor F420 (flavine F420), a flavine mononucleotide analogue (cf. S.M. Stronach et al., Anaerobic Digestion Processes in Industrial Waste Water Treatment, pub.
Springer Verlag, Berlin, 1986). The characteristic light absorption and fluorescence spectrum of this substance makes it possible to detect these bacteria - under a fluorescence microscope. However, a micro-scopic quantification or counting in a counting chamber is not possible since the coloured material is destroyed by light absorption within seconds and the bacteria lose their fluorescence.
The concentration determination of F420 in sludge for the determination of the methanogenic activity is known. However, it is a prerequisite of the process that the~F420 is extracted from the sludge, pre-purified and then measured fluorimetrically. B.W.Reuter _ al. (J. Biotechn., 4, 325-332/1986) describe the in vivo measurement of the F fluorescence in cultures of Methanobacterium thermoautotrofikum by means of laser spectroscopy with a preceding time-consuming extraction process. However, because of their susceptibility to disturbance, these measure-ments are restricted to pure cultures of methanogenic bacteria and do not make possible a quantification of ~4~ l 337461 these bacteria in reactors used for the production of methane gas. For the relationship of methane form-ation and F420 content, reference is also made to J. Dolfing and J.-W.-Mulder in Appl. Environ. Micro-biol., 49, 1142-1145/1985.
It is an object of the present invention to provide a rapid, certain and simple method for the quantification of methane gas-producing bacteria in anaerobic decomposition processes, which method is especially also well suited for monitoring and controlling the methane gas formation capacity of biogas reactors. This object is solved by the process according to the present invention.
Thus, according to the present invention, there is provided a process for the quantification of methane gas bacteria and especially for monitoring the methane gas formation capacity of reactors con-taining ~ethanogenic bacteria, wherein a representative sample of a medium containing the methane gas bacteria is subjected for at most one second to a brief irradiation by light with a wavelength of from 395 to 440 nm and the fluorescence thereby excited is deter-mined by flow cytometry.
For a readily usable quantification and for the determination of the percentage of methane gas bacteria in the total bacteria, it is advantageous, besides the methane gas bacteria, also to determine the total ~5~ ~ 337461 amount of micro-organisms. This can take place simultaneously by measurerllent of the light scattering which, however, apart from the micro-organisms, also includes other fine particles or by colouring the DNA
of the micro-organisms, the two colour signals then being determined simultaneously. Scattered light deterrnination and coloration via the DNA can also be carried out in parallel, whereby the exactitude can be further increased.
Therefore, in an advantageous embodinent of the process according to the present invention, the total amount of micro-organisms present is also detPr~ined by scattered light measurement and/or coloration of the DNA.
For the preparation for the flow cytometry, the samples are to be freed from disturbing accoinpanying rnaterials and possibly pre-treated in an appropriate way. This can take place in a manner generally known for this purpose. As a rule, a filtration suffices.
Thus, for example, sludge samples from a sludge tower of a conventionally constructed biogas plant for the purification of communal waste water are filtered through paper filters and the filtrate measured in a cytometer without further working up.
~low cytometry (laser flow cytom2try; flow cyto-r,letry FCM) is a generally known and frequently used method for the analysis of cells of all kinds, whereby several paraneters, such as DNA, RNA and protein --6- 1 33, 461 content, ir,mlunofluorescence, cell size and cell form can be simultaneously measured (cf., for example, Biotechnology, 3, 337-356/1985; com?any brochure of Orpegerl, Heidelberg; company brochure of Skatron A.S., N-3401 Lier). Therefore, the ~hoice of the process and a-pparatus embodiments used for the process according to the present invention depends especially on the specific sample to be investigated, upon the sarnple preparation and UpOtl the process .neasures. For the siMultaneous measurement of the light scattering and/or of the coloration of the DNA, there is very well suited, for exarnple a Skatron flow cytometer. In our Canadian Patent Application No. 594,620 flow cyto-netry is used for monitoring micro-organisms which frequently occur in the activated sludge of a clarification plant. Insofar as the process measures and constructions of apparatus of that process can be applied to the process of the present invention, they also constitute a part of the present description.
In one embodiment of the process according to the present invention, the bacteria are irradiated in a suspension by a blue excitation light which is emitted by a rnercury vapour lamp and is filtered in the range of from 395 to 440 nrn. Simultaneously, by means of two photomultipliers, scattered light and fluorescent light emitted by the bacteria, ~hich is filtered in the region of 470 nm, are measured and these signals analysed by a --7- l 337461 computer. The bacteria are only exposed to the excitation light for a fraction of a second so that the photolysis of the F420 is excluded. This measure-ment arrangement makes it possible to measure all bacteria via the scattered light and to deterrnine quantitatively the methane gas bacteria via the additional fluorescence light.
Thus, with the process according to the present invention, it is possible to carry out an exact, rapid and sirllple quantification of methane gas bacteria via their own fluorescence in situ, for exa~ple in sludge, and thus without t'ne disadvantages involved with the previously known processes, for example of extraction.
In particular, with the process according to the present invention, it is possible to assess, in a rapid and si;nple way, the decomposition potential of sludge r.lixtures in general and especially to assess the operational efficiency of reactors with ,~lethanogenic bacteria. Furthermore, it is possible, by means of a fully automatic and on-line connected measurement station, to monitor and control the biogas plant continuously.
Therefore, the present invention is also concerned with the use of the process according to the present invention for monitoring and controlling reactors with methanogenic bacteria which takes place via the concent-ration of the methane gas bact~ria. Such a ,-monitoring and control can also be carried out fully automaticall~.
Therefore, the present invention is especially also concerned with tlle use of the process according to the present invention for the automatic control of plant with methane gas bacteria in which, in a measurement value detection point based on flow cytometry, the concentration of the methane gas bacteria is deterrnined and utilised as a regulation value for the control of the plant, for exarnple via the regulation of pumps or heating elements.
~ y the term "reactors with methanogenic bacteria", in the scope of the present invention is to be under-stood all plant which are operated with methanogenic bacteria. Thes~ include, for exarnple, all biogas reactors, fermenters, sludge containers and sludge towers which serve for the removal of solid or liquid waste or which are provided for the production of methane gas.
Hcwever, in addition, the process according to the present invention is also suitable, for example, for the qualitative and quantitative determination (quantification) of methane gas bacteria and of their percentage proportion in the animal and human digestive tract, whereby it represents a valuable veterinary and hun~an medicinal method of investigation for diagnostic and also fo-r therapeutic purposes, and for monitoring and assessing naturally-occurring putrefaction processes `_ g of ecosystems, for example the degree of eutrophication of a lake.
Therefore, the present invention is also concerned with the use of the process according to the present invention for the determination of methane gas bacteria in the animal and human digestive tract and for monitor-ing and assessing naturally-occurring putrefaction proc-esses of ecosystems.
The invention is further explained and illustrated by reference to the accompanying drawings in which:
Figs. lA and lB show the characteristic light absorption and fluorescence spectrum of Factor F420;
Figs. 2A, 2B and 2C show measurements of a pure culture of Methanococcus vinelandii with a flow cytome-ter;
Fig. 2D is a graphical evaluation of the measure-ment of Fig. 2C;
Figs. 3A, 3B and 3C show measurements of a pure culture of bacteria of type Methanococcus vinelandii after photolysis of F420 with blue light;
Fig. 3D is a graphical evaluation of Fig. 3C;
Fig. 4 illustrates graphically the dependence of measured fluorescence impulse on the number of bacteria;
Fig. 5 illustrates graphically the dependence of the measured fluorescence impulse on the dilution of a Methanococcus pure culture;
-lo- 1 337461 Fig. 6A shows a cytogram of fluorescence v.
scatter light;
Figs. 6B, 6C, 6D and 6E are histograms of numbers of bacteria v. intensity of light scatter;
and Fig. ~F is a histogram similar to Fig. 6E.
The following Examples are given for the purpose of illustrating the present invention:
Example 1.
Measurement of a pure culture of methane gas bacteria.
: Methanogenic bacteria of the species Methanococcus vinelandii were cultured in a pure culture up to a density of 2.7 x ln8 per ml. and then measured in a flow cytometer.
Figs. 2A, 2B and 2C of the accompanying drawings show the measurernent of a pure culture of Methanococcus vinelandii with a flow cytometer.
Fig. 2A: Each point corresponds to a measured bacteriurîî;
after excitation with blue light in the range of from 395 to 440 nm, for each bacterium there is measured the total scattered light and the 470 nm fluorescent light.
Fig. 2B: Plotting of the linear scattered light a~ainst the rrequency of the rneasurements.
~ 1 337461 Fig. 2C: Plotting of the linear fluorescence signal against the frequency of the Measurements.
In these Figures, LIN SCT means the linear intensity of the scattered light and LIN FLl rneans the linear intensity of the fluorescent light.
Fig. 2D of the accompanying drawin~s shows the evaluation of Fig. 2C. The arrow marks the fluorescence intensity from which are graded particles as being fluorescent. From this, ~here is given a proportion of fluorescent bacteria of 94.22%.
As follows from the evaluation of the llistogram of the fluorescence intensity against the signal frequency from Fig. 2D, thus 94% of the 100,000 particles emitting scattered light show a clear fluorescence signal. For monitoring, the same bacteria suspension was exposed for 1 hour at a distance of 5 cm.
to the light of a lOOW mercury vapour lamp, the flavine thereby being photolysed. This suspension was again - measured in a cytometer. Figs. 3A, 3B and 3C of the accornpanying drawings show the measurement of a pure culture of bacteria of the Methanococcus vinelandii type after photolysis of the F420 wi~h blue ligl~t-Fig. 3D of the acco~,npanying drawings shows the evaluation of Fig. 3C.
It follo~s from Fig. 3 that the bacteria admittedly still scatter the excitation light but no longer emit any fluorescent light.
-12- l 33746 1 - Fig. 4 of the accompanying drawings shows the dependence of the measured fluorescence impulse upon the number of bacteria passed by the measurement beam and Fig. 5 of the accompanying drawings shows the dependence of the measured fluorescence impulse on the dilution of a Methanococcus pure culture.
Figs. 4 and 5 show that the methane gas bacteria ,can be detected quantitatively by the measurelnent arrangement via the fluoresc~nce light in a broad dilution and sample dosaging range. Only when the bacterial count passed by the measurement beam is less than 6.8 x 104/second is the measurement arrangement no longer able to d~tect all inethane gas bacteria.
Example 2.
Quantification of methane gas bacteria in the sludge of a biogas plant.
Sludge samples from the sludge tower of a convent-ionally constructed biogas plant for the purification of commurlal waste water were filtered over paper filters and the filtrate measured in a cytometer without further working up.
Fig. 6 of the accompanying drawings shows the measurement of a filter sludge sample from a sludge tower of a biogas plant. LIN SCT signifies the linear scattered light intensity and LIN rLi the linear fluorescence light intensity.
..
__ -13- 1337461 In particular Fig. 6 shows quantification of methane gas bacteria in fowling sludge, Fig. 6A
shows a cytogram of fluorescence (lin FLl) versus scatter light (lin SCT). Each point represents one registered bacterium. A total of 90,000 bacteria from fowling sludge were measured. The x- and y-axes were subdivided by channel numbers from 0 to 255.
The multichannel pulse height analyser of the flow cytometer digitalizes the analogous signals from the photomultipliers by classifying them into those channels according to their size. As can be seen from this figure, two clusters of green fluorescent methanogenic bacteria of different scatter light can be separated from non-fluorescent part~icles.
Figs. 6B, C, D and E are histograms, where the numbers of bacteria were plotted versus the intensity of their scatter (lin SCT) and fluorescent light (lin FLl). In Figs. 6B and 6C a logarithmical and in Figs.
6D and 6E, a linear scale were used for the y-axes.
The x-axes were subdivided by channel numbers from 0 to 255. As can be seen from Figs. 6C and 6E, the green fluorescent methanogenic bacteria are separated from other particles of the size of bacteria in these dis-tributions.
Fig. 6F shows a histogram similar to that in Fig.
6E. The computer analysis reveals that all bacteria with fluorescent light above channel 40 can be considered as methanogenic bacteria. Of the 89,770 bacteria which had been analysed, 3,246 were methanogenic, which is 3.62%
_ -14- l 3 3 7 4 6 1 of the total bacterial population.
As call be seen from Fig. 6, the methane gas bacteria can be differentiated by their specific fluorescence from the other bacteria w'nich only produced scattered light. There can thereby be deter-mined not only their percentage proportion in the total bacterial population but also their absolute cell density in the suspension. The evaluation takes place by a histogram of the cell frequency against the fluorescence intensity, whereby the methane gas bacteria differ clearly from the other bacteria not only in the linear but also in the semi-logarithmic plotting. For monitoring, the sample was bleached as in Example l by the light of a mercury vapour lamp and then again measured. In this sample, there are only to be measured i particles with scattered light but without a fluorescence light part.
The same sample was then measured ten times successively and the percentage portion of methane gas bacteria in the total bacteria detennined in the case of each individual measurement. There was obtained an average value of 3. 71~o with a standard deviation of 0.186.
A monitoring and control of reactors witn methano-genic bacteria by way of the process according to thepresent invention can, for exal-nple, take place in the following way:
The concentration of the methane gas bacteria is --~ - 1 337461 determined according to the present invention and, by the following measures, their concentration influenced (kept constant) and thus an optimisation of the fermentation achieved:
1) Optimisation of the physico-chemical parameters of the reactor (pH value via the addition of lime, temper-ature via reactor heating, mixing up of the sludge mixture by means of appropriate circulating pumps);
Organic material is converted, with the exclusion of air and in the presence of an appropriate microflora, into methane gas. The material is thereby first hydrolysed in a multi-step decomposition (hydrolysis phase), then fermented to give organic acids (acidification phase), converted by acetogenic bacteria into molecular hydrogen and carbon dioxide (acetogenic phase) and then converted by methanogenic bacteria (methane gas bacteria) into methane gas (methanogenic phase). This reaction chain can only take place efficiently when, in the mixed bacterial population, sufficient methane gas bacteria are present and can multiply. Otherwise, the whole decomposition process is inhibited by the individual intermediate products.
This decomposition route takes place everywhere in nature where organic material is decomposed in the absence of air, i.e. in sumps and ponds, in eutrophied waters, on the beds of oceans and in the digestive tracts of humans and animals. In the so-called biogas reactors (fermentors), it is utilised for the conversion of organic waste from waste water clarification and from industrial and agricultural production into useful methane gas.
It is known that methane gas bacteria display an 3 1 337~61 autofluorescence in the case of brief irradiation.
This fluorescence is brought about by Factor F420 (flavine F420), a flavine mononucleotide analogue (cf. S.M. Stronach et al., Anaerobic Digestion Processes in Industrial Waste Water Treatment, pub.
Springer Verlag, Berlin, 1986). The characteristic light absorption and fluorescence spectrum of this substance makes it possible to detect these bacteria - under a fluorescence microscope. However, a micro-scopic quantification or counting in a counting chamber is not possible since the coloured material is destroyed by light absorption within seconds and the bacteria lose their fluorescence.
The concentration determination of F420 in sludge for the determination of the methanogenic activity is known. However, it is a prerequisite of the process that the~F420 is extracted from the sludge, pre-purified and then measured fluorimetrically. B.W.Reuter _ al. (J. Biotechn., 4, 325-332/1986) describe the in vivo measurement of the F fluorescence in cultures of Methanobacterium thermoautotrofikum by means of laser spectroscopy with a preceding time-consuming extraction process. However, because of their susceptibility to disturbance, these measure-ments are restricted to pure cultures of methanogenic bacteria and do not make possible a quantification of ~4~ l 337461 these bacteria in reactors used for the production of methane gas. For the relationship of methane form-ation and F420 content, reference is also made to J. Dolfing and J.-W.-Mulder in Appl. Environ. Micro-biol., 49, 1142-1145/1985.
It is an object of the present invention to provide a rapid, certain and simple method for the quantification of methane gas-producing bacteria in anaerobic decomposition processes, which method is especially also well suited for monitoring and controlling the methane gas formation capacity of biogas reactors. This object is solved by the process according to the present invention.
Thus, according to the present invention, there is provided a process for the quantification of methane gas bacteria and especially for monitoring the methane gas formation capacity of reactors con-taining ~ethanogenic bacteria, wherein a representative sample of a medium containing the methane gas bacteria is subjected for at most one second to a brief irradiation by light with a wavelength of from 395 to 440 nm and the fluorescence thereby excited is deter-mined by flow cytometry.
For a readily usable quantification and for the determination of the percentage of methane gas bacteria in the total bacteria, it is advantageous, besides the methane gas bacteria, also to determine the total ~5~ ~ 337461 amount of micro-organisms. This can take place simultaneously by measurerllent of the light scattering which, however, apart from the micro-organisms, also includes other fine particles or by colouring the DNA
of the micro-organisms, the two colour signals then being determined simultaneously. Scattered light deterrnination and coloration via the DNA can also be carried out in parallel, whereby the exactitude can be further increased.
Therefore, in an advantageous embodinent of the process according to the present invention, the total amount of micro-organisms present is also detPr~ined by scattered light measurement and/or coloration of the DNA.
For the preparation for the flow cytometry, the samples are to be freed from disturbing accoinpanying rnaterials and possibly pre-treated in an appropriate way. This can take place in a manner generally known for this purpose. As a rule, a filtration suffices.
Thus, for example, sludge samples from a sludge tower of a conventionally constructed biogas plant for the purification of communal waste water are filtered through paper filters and the filtrate measured in a cytometer without further working up.
~low cytometry (laser flow cytom2try; flow cyto-r,letry FCM) is a generally known and frequently used method for the analysis of cells of all kinds, whereby several paraneters, such as DNA, RNA and protein --6- 1 33, 461 content, ir,mlunofluorescence, cell size and cell form can be simultaneously measured (cf., for example, Biotechnology, 3, 337-356/1985; com?any brochure of Orpegerl, Heidelberg; company brochure of Skatron A.S., N-3401 Lier). Therefore, the ~hoice of the process and a-pparatus embodiments used for the process according to the present invention depends especially on the specific sample to be investigated, upon the sarnple preparation and UpOtl the process .neasures. For the siMultaneous measurement of the light scattering and/or of the coloration of the DNA, there is very well suited, for exarnple a Skatron flow cytometer. In our Canadian Patent Application No. 594,620 flow cyto-netry is used for monitoring micro-organisms which frequently occur in the activated sludge of a clarification plant. Insofar as the process measures and constructions of apparatus of that process can be applied to the process of the present invention, they also constitute a part of the present description.
In one embodiment of the process according to the present invention, the bacteria are irradiated in a suspension by a blue excitation light which is emitted by a rnercury vapour lamp and is filtered in the range of from 395 to 440 nrn. Simultaneously, by means of two photomultipliers, scattered light and fluorescent light emitted by the bacteria, ~hich is filtered in the region of 470 nm, are measured and these signals analysed by a --7- l 337461 computer. The bacteria are only exposed to the excitation light for a fraction of a second so that the photolysis of the F420 is excluded. This measure-ment arrangement makes it possible to measure all bacteria via the scattered light and to deterrnine quantitatively the methane gas bacteria via the additional fluorescence light.
Thus, with the process according to the present invention, it is possible to carry out an exact, rapid and sirllple quantification of methane gas bacteria via their own fluorescence in situ, for exa~ple in sludge, and thus without t'ne disadvantages involved with the previously known processes, for example of extraction.
In particular, with the process according to the present invention, it is possible to assess, in a rapid and si;nple way, the decomposition potential of sludge r.lixtures in general and especially to assess the operational efficiency of reactors with ,~lethanogenic bacteria. Furthermore, it is possible, by means of a fully automatic and on-line connected measurement station, to monitor and control the biogas plant continuously.
Therefore, the present invention is also concerned with the use of the process according to the present invention for monitoring and controlling reactors with methanogenic bacteria which takes place via the concent-ration of the methane gas bact~ria. Such a ,-monitoring and control can also be carried out fully automaticall~.
Therefore, the present invention is especially also concerned with tlle use of the process according to the present invention for the automatic control of plant with methane gas bacteria in which, in a measurement value detection point based on flow cytometry, the concentration of the methane gas bacteria is deterrnined and utilised as a regulation value for the control of the plant, for exarnple via the regulation of pumps or heating elements.
~ y the term "reactors with methanogenic bacteria", in the scope of the present invention is to be under-stood all plant which are operated with methanogenic bacteria. Thes~ include, for exarnple, all biogas reactors, fermenters, sludge containers and sludge towers which serve for the removal of solid or liquid waste or which are provided for the production of methane gas.
Hcwever, in addition, the process according to the present invention is also suitable, for example, for the qualitative and quantitative determination (quantification) of methane gas bacteria and of their percentage proportion in the animal and human digestive tract, whereby it represents a valuable veterinary and hun~an medicinal method of investigation for diagnostic and also fo-r therapeutic purposes, and for monitoring and assessing naturally-occurring putrefaction processes `_ g of ecosystems, for example the degree of eutrophication of a lake.
Therefore, the present invention is also concerned with the use of the process according to the present invention for the determination of methane gas bacteria in the animal and human digestive tract and for monitor-ing and assessing naturally-occurring putrefaction proc-esses of ecosystems.
The invention is further explained and illustrated by reference to the accompanying drawings in which:
Figs. lA and lB show the characteristic light absorption and fluorescence spectrum of Factor F420;
Figs. 2A, 2B and 2C show measurements of a pure culture of Methanococcus vinelandii with a flow cytome-ter;
Fig. 2D is a graphical evaluation of the measure-ment of Fig. 2C;
Figs. 3A, 3B and 3C show measurements of a pure culture of bacteria of type Methanococcus vinelandii after photolysis of F420 with blue light;
Fig. 3D is a graphical evaluation of Fig. 3C;
Fig. 4 illustrates graphically the dependence of measured fluorescence impulse on the number of bacteria;
Fig. 5 illustrates graphically the dependence of the measured fluorescence impulse on the dilution of a Methanococcus pure culture;
-lo- 1 337461 Fig. 6A shows a cytogram of fluorescence v.
scatter light;
Figs. 6B, 6C, 6D and 6E are histograms of numbers of bacteria v. intensity of light scatter;
and Fig. ~F is a histogram similar to Fig. 6E.
The following Examples are given for the purpose of illustrating the present invention:
Example 1.
Measurement of a pure culture of methane gas bacteria.
: Methanogenic bacteria of the species Methanococcus vinelandii were cultured in a pure culture up to a density of 2.7 x ln8 per ml. and then measured in a flow cytometer.
Figs. 2A, 2B and 2C of the accompanying drawings show the measurernent of a pure culture of Methanococcus vinelandii with a flow cytometer.
Fig. 2A: Each point corresponds to a measured bacteriurîî;
after excitation with blue light in the range of from 395 to 440 nm, for each bacterium there is measured the total scattered light and the 470 nm fluorescent light.
Fig. 2B: Plotting of the linear scattered light a~ainst the rrequency of the rneasurements.
~ 1 337461 Fig. 2C: Plotting of the linear fluorescence signal against the frequency of the Measurements.
In these Figures, LIN SCT means the linear intensity of the scattered light and LIN FLl rneans the linear intensity of the fluorescent light.
Fig. 2D of the accompanying drawin~s shows the evaluation of Fig. 2C. The arrow marks the fluorescence intensity from which are graded particles as being fluorescent. From this, ~here is given a proportion of fluorescent bacteria of 94.22%.
As follows from the evaluation of the llistogram of the fluorescence intensity against the signal frequency from Fig. 2D, thus 94% of the 100,000 particles emitting scattered light show a clear fluorescence signal. For monitoring, the same bacteria suspension was exposed for 1 hour at a distance of 5 cm.
to the light of a lOOW mercury vapour lamp, the flavine thereby being photolysed. This suspension was again - measured in a cytometer. Figs. 3A, 3B and 3C of the accornpanying drawings show the measurement of a pure culture of bacteria of the Methanococcus vinelandii type after photolysis of the F420 wi~h blue ligl~t-Fig. 3D of the acco~,npanying drawings shows the evaluation of Fig. 3C.
It follo~s from Fig. 3 that the bacteria admittedly still scatter the excitation light but no longer emit any fluorescent light.
-12- l 33746 1 - Fig. 4 of the accompanying drawings shows the dependence of the measured fluorescence impulse upon the number of bacteria passed by the measurement beam and Fig. 5 of the accompanying drawings shows the dependence of the measured fluorescence impulse on the dilution of a Methanococcus pure culture.
Figs. 4 and 5 show that the methane gas bacteria ,can be detected quantitatively by the measurelnent arrangement via the fluoresc~nce light in a broad dilution and sample dosaging range. Only when the bacterial count passed by the measurement beam is less than 6.8 x 104/second is the measurement arrangement no longer able to d~tect all inethane gas bacteria.
Example 2.
Quantification of methane gas bacteria in the sludge of a biogas plant.
Sludge samples from the sludge tower of a convent-ionally constructed biogas plant for the purification of commurlal waste water were filtered over paper filters and the filtrate measured in a cytometer without further working up.
Fig. 6 of the accompanying drawings shows the measurement of a filter sludge sample from a sludge tower of a biogas plant. LIN SCT signifies the linear scattered light intensity and LIN rLi the linear fluorescence light intensity.
..
__ -13- 1337461 In particular Fig. 6 shows quantification of methane gas bacteria in fowling sludge, Fig. 6A
shows a cytogram of fluorescence (lin FLl) versus scatter light (lin SCT). Each point represents one registered bacterium. A total of 90,000 bacteria from fowling sludge were measured. The x- and y-axes were subdivided by channel numbers from 0 to 255.
The multichannel pulse height analyser of the flow cytometer digitalizes the analogous signals from the photomultipliers by classifying them into those channels according to their size. As can be seen from this figure, two clusters of green fluorescent methanogenic bacteria of different scatter light can be separated from non-fluorescent part~icles.
Figs. 6B, C, D and E are histograms, where the numbers of bacteria were plotted versus the intensity of their scatter (lin SCT) and fluorescent light (lin FLl). In Figs. 6B and 6C a logarithmical and in Figs.
6D and 6E, a linear scale were used for the y-axes.
The x-axes were subdivided by channel numbers from 0 to 255. As can be seen from Figs. 6C and 6E, the green fluorescent methanogenic bacteria are separated from other particles of the size of bacteria in these dis-tributions.
Fig. 6F shows a histogram similar to that in Fig.
6E. The computer analysis reveals that all bacteria with fluorescent light above channel 40 can be considered as methanogenic bacteria. Of the 89,770 bacteria which had been analysed, 3,246 were methanogenic, which is 3.62%
_ -14- l 3 3 7 4 6 1 of the total bacterial population.
As call be seen from Fig. 6, the methane gas bacteria can be differentiated by their specific fluorescence from the other bacteria w'nich only produced scattered light. There can thereby be deter-mined not only their percentage proportion in the total bacterial population but also their absolute cell density in the suspension. The evaluation takes place by a histogram of the cell frequency against the fluorescence intensity, whereby the methane gas bacteria differ clearly from the other bacteria not only in the linear but also in the semi-logarithmic plotting. For monitoring, the sample was bleached as in Example l by the light of a mercury vapour lamp and then again measured. In this sample, there are only to be measured i particles with scattered light but without a fluorescence light part.
The same sample was then measured ten times successively and the percentage portion of methane gas bacteria in the total bacteria detennined in the case of each individual measurement. There was obtained an average value of 3. 71~o with a standard deviation of 0.186.
A monitoring and control of reactors witn methano-genic bacteria by way of the process according to thepresent invention can, for exal-nple, take place in the following way:
The concentration of the methane gas bacteria is --~ - 1 337461 determined according to the present invention and, by the following measures, their concentration influenced (kept constant) and thus an optimisation of the fermentation achieved:
1) Optimisation of the physico-chemical parameters of the reactor (pH value via the addition of lime, temper-ature via reactor heating, mixing up of the sludge mixture by means of appropriate circulating pumps);
2) Synchronisation of tlle mixing ratio of inoculation sludge to introduced waste to give an optimum concent-ration of the methane gas bacteria and/or 3) Dosing in of activators, for example commercially ~ available polyelectrolytes (for example Bio-Klar Algin, Methane-active) or of dry bacteria of the hydrolytic and acid-forming stage of the fermentation.
The monitoring and control of plants with methane gas bacteria can also be carried out fully automatically, the concentration of the methanogenic bacteria being det~-nined in a measurement value detection point depending upon flow cytometry and being used as a regulating value for controlling pumps (for example for the dosing in of bacteria, inoculation sludge, substrate or activators) or of heating elements (for the opti-.nisation of the reactor ternperature).
The monitoring and control of plants with methane gas bacteria can also be carried out fully automatically, the concentration of the methanogenic bacteria being det~-nined in a measurement value detection point depending upon flow cytometry and being used as a regulating value for controlling pumps (for example for the dosing in of bacteria, inoculation sludge, substrate or activators) or of heating elements (for the opti-.nisation of the reactor ternperature).
Claims (5)
1. Process for the quantification of methane gas bacteria and especially for monitoring the methane gas formation capacity of reactors containing methanogenic bacteria, wherein a representative sample of a medium containing the methane gas bacteria is subjected for at most one second to a brief irradiation by light with a wavelength of 395 to 440 nm and the fluorescence thereby excited is determined by flow cytometry.
2. Process according to claim 1, wherein, at the same time, there is determined the total amount of micro-organisms present by scattered light measurement and/or coloration of the DNA.
3. Use of the process according to claim 1 or 2 for monitoring and controlling reactors with methane gas bacteria.
4. Use of the process according to claim 1 or 2 for the determination of methane gas bacteria in the animal or human digestive tract.
5. Use of the process according to claim 1 or 2 for monitoring and assessing naturally-occurring putrefaction processes and ecosystems.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3811098.9 | 1988-03-31 | ||
| DE3811098A DE3811098A1 (en) | 1988-03-31 | 1988-03-31 | METHOD FOR QUANTIFYING METHANE GAS BACTERIA |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1337461C true CA1337461C (en) | 1995-10-31 |
Family
ID=6351230
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000594621A Expired - Fee Related CA1337461C (en) | 1988-03-31 | 1989-03-23 | Process for the quantification of methane gas bacteria |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP0337189B1 (en) |
| JP (1) | JPH01300900A (en) |
| AT (1) | ATE91153T1 (en) |
| AU (1) | AU621594B2 (en) |
| CA (1) | CA1337461C (en) |
| DE (2) | DE3811098A1 (en) |
| DK (1) | DK157589A (en) |
| ES (1) | ES2058370T3 (en) |
| IL (1) | IL89766A (en) |
| NO (1) | NO891354L (en) |
| RU (1) | RU2028383C1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10106312B4 (en) * | 2001-02-12 | 2008-10-09 | Uwe Heinemann | Regulatory procedure for methane gas production |
| DE102010023486A1 (en) * | 2010-06-11 | 2011-12-15 | B. Braun Avitum Ag | Detection device and method |
| CN110146497B (en) * | 2019-05-29 | 2020-12-29 | 哈尔滨商业大学 | Copper ion detection method based on methane-oxidizing rhzomorph functionalized nanogold |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60181637A (en) * | 1984-02-28 | 1985-09-17 | Mitsubishi Electric Corp | Measurement of number of methanobacterium or methane generation activity |
-
1988
- 1988-03-31 DE DE3811098A patent/DE3811098A1/en not_active Withdrawn
-
1989
- 1989-03-22 AU AU31648/89A patent/AU621594B2/en not_active Ceased
- 1989-03-23 CA CA000594621A patent/CA1337461C/en not_active Expired - Fee Related
- 1989-03-23 RU SU894613724A patent/RU2028383C1/en active
- 1989-03-27 IL IL8976689A patent/IL89766A/en not_active IP Right Cessation
- 1989-03-29 AT AT89105556T patent/ATE91153T1/en not_active IP Right Cessation
- 1989-03-29 ES ES89105556T patent/ES2058370T3/en not_active Expired - Lifetime
- 1989-03-29 DE DE8989105556T patent/DE58904824D1/en not_active Expired - Fee Related
- 1989-03-29 EP EP89105556A patent/EP0337189B1/en not_active Expired - Lifetime
- 1989-03-30 NO NO89891354A patent/NO891354L/en unknown
- 1989-03-31 DK DK157589A patent/DK157589A/en not_active Application Discontinuation
- 1989-03-31 JP JP1078751A patent/JPH01300900A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| NO891354D0 (en) | 1989-03-30 |
| ES2058370T3 (en) | 1994-11-01 |
| NO891354L (en) | 1989-10-02 |
| AU621594B2 (en) | 1992-03-19 |
| DK157589D0 (en) | 1989-03-31 |
| IL89766A0 (en) | 1989-09-28 |
| RU2028383C1 (en) | 1995-02-09 |
| EP0337189A1 (en) | 1989-10-18 |
| EP0337189B1 (en) | 1993-06-30 |
| DE58904824D1 (en) | 1993-08-05 |
| DE3811098A1 (en) | 1989-10-12 |
| JPH01300900A (en) | 1989-12-05 |
| DK157589A (en) | 1989-10-01 |
| ATE91153T1 (en) | 1993-07-15 |
| AU3164889A (en) | 1989-10-05 |
| IL89766A (en) | 1994-01-25 |
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