IL89766A - Process for the quantification of methane gas bacteria and especially for monitoring the methane gas formation capacity of reactors containing methanogenic bacteria - Google Patents
Process for the quantification of methane gas bacteria and especially for monitoring the methane gas formation capacity of reactors containing methanogenic bacteriaInfo
- Publication number
- IL89766A IL89766A IL8976689A IL8976689A IL89766A IL 89766 A IL89766 A IL 89766A IL 8976689 A IL8976689 A IL 8976689A IL 8976689 A IL8976689 A IL 8976689A IL 89766 A IL89766 A IL 89766A
- Authority
- IL
- Israel
- Prior art keywords
- bacteria
- methane gas
- monitoring
- process according
- quantification
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
-
- 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
Abstract
The method entails a representative sample of a medium containing the methane gas bacteria being exposed for a maximum of one second to brief irradiation with light of wavelengths from 395 to 440 nm, and the fluorescence excited thereby being determined by flow cytometry. This method makes it possible to determine methane gas bacteria in situ without previous extraction. The method is particularly suitable for use for monitoring and controlling reactors with methanogenic bacteria.
Description
/80/82 89766/2
m s η¾Ό jnp-Λ "Tm »3 ικηηη o »ρτ>>η n>m»o ny p^ "p^rm
o i¾iiioia ο»ρτ>»η ο>!? »η D 1?D>» ¾V ·)κη»η n
PROCESS FOR THE QUANTIFICATION OF METHANE GAS BACTERIA AND ESPECIALLY FOR MONITORING THE METHANE GAS FORMATION CAPACITY OF REACTORS CONTAINING METHANOGENIC BACTERIA
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
autofluorescence in the case of brief irradiation.
This fluorescence is brought about by Factor
(flavine
a flavi-ne mononucleotide analogue
(cf. S.M. Stronach e_t al_. , Anaerobic Digestion
Processes in Industrial Waste Water Treatment, pub.
Springer Verlag, Berlin, 1986). The characteristic light absorption and fluorescence spectrum of this substance (cf. Figs, la and lb of the accompanying drawings) makes it possible to detect these bacteria under a fluorescence microscope. However, a microscopic 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
for the determination of the methanogenic activity is known. However, it is a prerequisite of the process that the ^g is extracted from the sludge, pre-purified and then measured fluorimetrically. B.W.Reuter et al . (J. Biotechn. , 4, 325-332/1986) describe the in vivo measurement of the F^g 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 measurements are restricted to pure cultures of methanogenic bacteria and do not make possible a quantification of
e se acteria in reactors use or t e pro uct on o methane gas. For the relationship of methane formation and content, reference is also made to
J. Dolfing and J. -W. -Mulder in Appl . Environ. Micro- -biol., 49, 1142-1145/1985.
GB 2,155,631 discloses a method for quantitative determination of methane gas bacteria.. This publication does not teach the very short irradiation time of at most one second in accordance with the present invention.
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 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 from 395 to 440 nm and the fluorescence thereby excited is determined 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
amount of micro-organisms. This can take place
simultaneously by measurement 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
determination and coloration via the DNA can also be carried out in parallel, whereby the exactitude can be further increased.
Therefore, in an advantageous embodiment of the process according to the present invention, the total amount of micro-organisms present is also determined 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 accompanying materials 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.
Flow cytometry (laser flow cytometry; flow cyto-metry FCM) is a generally known and frequently used method for the analysis of cells of all kinds, whereby several parameters, such as DNA, RNA and protein
content, immunofluorescence, cell size and cell form can be simultaneously measured (cf., for example,
Biotechnology, 3, 337-356/1985; company brochure of
Orpegen, Heidelberg; company brochure of Skatron A.S., N-3401 Lier). Therefore, the choice of the process and apparatus embodiments used for the process according to the present invention depends especially on the specific sample to be investigated, upon the sample preparation and upon the process measures. For the simultaneous measurement of the light scattering and/or of the
coloration of the D A, there is very well suited, for example a Skatron flow cytometer. In our Federal
Republic of Germany Patent Application No. P 38 11 097.0, flow cytometry 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 mercury 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, which is filtered in the region of 470 nm, are measured and these signals analysed by a
computer. The bacteria are only exposed to the
excitation light for a fraction of a second so that the photolysis of the ""s excluded. This measurement arrangement makes it possible to measure all bacteria via the scattered light and to determine 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 simple quantification of methane gas bacteria via their own fluorescence in situ, for example in sludge, and thus without the 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 simple way, the decomposition potential of sludge mixtures in general and especially to assess the
operational efficiency of reactors with methanogenic 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 concentration of the methane gas bacteria. Such a monitoring
and control can also be carried out fully automatically. Therefore, the present invention is especially also concerned with the 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 determined and utilised as a regulation value for the control of the plant, for example via the regulation of pumps or heating elements.
By the term "reactors with methanogenic bacteria", in the scope of the present invention is to be understood all plant which are operated with methanogenic bacteria. These include, for example, 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.
However, 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 human medicinal method of investigation for diagnostic and also for therapeutic purposes, and for monitoring and assessing naturally-occurring putrefaction processes
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
monitoring and assessing naturally-occurring putrefaction processes of ecosystems.
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
o
density of 2.7 x 10 per ml. and then measured in a flow cytometer.
Figs. 2A, 2B and 2C of the accompanying drawings show the measurement of a pure culture of Methanococcus vinelandii with a flow cytometer.
Fig. 2A: Each point corresponds to a measured bacterium; 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 against the frequency of the measurements.
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 FL1 means the linear intensity of the fluorescent light.
Fig. 2D of the accompanying drawings shows the evaluation of Fig. 2C. The arrow marks the fluorescence intensity from which are graded particles as being fluorescent. From this, there is given a proportion of fluorescent bacteria of 94.22%.
As follows from the evaluation of the histogram 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 100W mercury vapour lamp, the flavine thereby being photolysed. This suspension was again measured in a cytometer. Figs. 3A, 3B and 3C of the accompanying drawings show the measurement of a pure culture of bacteria of the Methanococcus vinelandii type after photolysis of the F^ 0 ^1-1 blue light.
Fig. 3D of the accompanying drawings shows the
evaluation of Fig. 3C.
It follows from Fig. 3 that the bacteria
admittedly still scatter the excitation light but no longer emit any fluorescent light.
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 measurement arrangement via the fluorescence 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 10^/second is the measurement arrangement no longer able to detect all methane gas bacteria.
Exam le 2.
Quantification of methane gas bacteria in the sludge of a biogas plant.
Sludge samples from the sludge tower of a conventionally constructed biogas plant for the purification of communal 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 FLi the linear
fluorescence light intensity. Fig. 6F shows the
evaluation of Fig. 6E.
As can be seen from Fig. 6, the methane gas bacteria can be differentiated by their specific
fluorescence from the other bacteria which 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 1 by the light of a mercury vapour lamp and then again
measured. In this sample, there are only to be measured 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 determined in the case of each individual measurement. There was obtained an average value of 3.71% with a standard deviation of 0.186.
A monitoring and control of reactors with methano-genic bacteria by way of the process according to the present invention can, for example, take place in the following way:
The concentration of the methane gas bacteria is
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, temperature via reactor heating, mixing up of the sludge mixture by means of appropriate circulating pumps);
2) Synchronisation of the mixing ratio of inoculation sludge to introduced waste to give an optimum concentration 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 determined 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 optimisation of the reactor temperature).
Claims (6)
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 microorganisms present by scattered light measurement and/or coloration of the DNA.
3. Process according to claim 1 for the quantific-ation of methane gas bacteria, substantially as hereinbefore described and exemplified and with reference to the accompanying drawings.
4. Use of the process according to any of the preceding claims for monitoring and controlling reactors with methane gas bacteria.
5. Use of the process according to any of claims 1 to 3 for the determination of methane gas bacteria in the animal or human digestive tract.
6. Use of the process according to any of claims 1 to 3 for monitoring and assessing naturally-occurring putrefaction processes and ecosystems.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3811098A DE3811098A1 (en) | 1988-03-31 | 1988-03-31 | METHOD FOR QUANTIFYING METHANE GAS BACTERIA |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| IL89766A0 IL89766A0 (en) | 1989-09-28 |
| IL89766A true IL89766A (en) | 1994-01-25 |
Family
ID=6351230
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| IL8976689A IL89766A (en) | 1988-03-31 | 1989-03-27 | Process for the quantification of methane gas bacteria and especially for monitoring the methane gas formation capacity of reactors containing methanogenic 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 RU SU894613724A patent/RU2028383C1/en active
- 1989-03-23 CA CA000594621A patent/CA1337461C/en not_active Expired - Fee Related
- 1989-03-27 IL IL8976689A patent/IL89766A/en not_active IP Right Cessation
- 1989-03-29 EP EP89105556A patent/EP0337189B1/en not_active Expired - Lifetime
- 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
- 1989-03-30 NO NO89891354A patent/NO891354L/en unknown
- 1989-03-31 JP JP1078751A patent/JPH01300900A/en active Pending
- 1989-03-31 DK DK157589A patent/DK157589A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| AU3164889A (en) | 1989-10-05 |
| IL89766A0 (en) | 1989-09-28 |
| AU621594B2 (en) | 1992-03-19 |
| EP0337189A1 (en) | 1989-10-18 |
| CA1337461C (en) | 1995-10-31 |
| ES2058370T3 (en) | 1994-11-01 |
| DE3811098A1 (en) | 1989-10-12 |
| DK157589A (en) | 1989-10-01 |
| EP0337189B1 (en) | 1993-06-30 |
| RU2028383C1 (en) | 1995-02-09 |
| DE58904824D1 (en) | 1993-08-05 |
| DK157589D0 (en) | 1989-03-31 |
| ATE91153T1 (en) | 1993-07-15 |
| JPH01300900A (en) | 1989-12-05 |
| NO891354L (en) | 1989-10-02 |
| NO891354D0 (en) | 1989-03-30 |
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