JPH0413655B2 - - Google Patents
Info
- Publication number
- JPH0413655B2 JPH0413655B2 JP59085533A JP8553384A JPH0413655B2 JP H0413655 B2 JPH0413655 B2 JP H0413655B2 JP 59085533 A JP59085533 A JP 59085533A JP 8553384 A JP8553384 A JP 8553384A JP H0413655 B2 JPH0413655 B2 JP H0413655B2
- Authority
- JP
- Japan
- Prior art keywords
- methane
- wavelength
- fluorescence
- bacteria
- light
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims description 172
- 241000894006 Bacteria Species 0.000 claims description 61
- 230000005284 excitation Effects 0.000 claims description 30
- 230000000694 effects Effects 0.000 claims description 22
- 238000012360 testing method Methods 0.000 claims description 7
- 230000001678 irradiating effect Effects 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 3
- 239000012491 analyte Substances 0.000 claims 1
- 230000001747 exhibiting effect Effects 0.000 claims 1
- 239000000126 substance Substances 0.000 description 25
- 230000004151 fermentation Effects 0.000 description 18
- 238000000855 fermentation Methods 0.000 description 18
- 244000005700 microbiome Species 0.000 description 17
- 238000000034 method Methods 0.000 description 15
- 238000002189 fluorescence spectrum Methods 0.000 description 14
- 238000005259 measurement Methods 0.000 description 12
- 238000012937 correction Methods 0.000 description 11
- 238000004519 manufacturing process Methods 0.000 description 11
- 238000010586 diagram Methods 0.000 description 10
- 238000001228 spectrum Methods 0.000 description 10
- 238000000695 excitation spectrum Methods 0.000 description 9
- 239000007788 liquid Substances 0.000 description 8
- 238000001514 detection method Methods 0.000 description 6
- 238000000691 measurement method Methods 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- 239000005515 coenzyme Substances 0.000 description 5
- 239000010802 sludge Substances 0.000 description 5
- 230000003595 spectral effect Effects 0.000 description 5
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 4
- 241001074903 Methanobacteria Species 0.000 description 4
- 230000000696 methanogenic effect Effects 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000037149 energy metabolism Effects 0.000 description 3
- 230000005855 radiation Effects 0.000 description 3
- 239000010865 sewage Substances 0.000 description 3
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 2
- 239000012670 alkaline solution Substances 0.000 description 2
- 230000002238 attenuated effect Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 229950006238 nadide Drugs 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 1
- 108091006149 Electron carriers Proteins 0.000 description 1
- 241000205275 Methanosarcina barkeri Species 0.000 description 1
- XJLXINKUBYWONI-NNYOXOHSSA-N NADP zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000027721 electron transport chain Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 230000033116 oxidation-reduction process Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000013214 routine measurement Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Sustainable Development (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Description
【発明の詳細な説明】
[産業上の利用分野]
この発明は、メタン菌を有する被検体中のメタ
ン菌の菌数またはメタン生成活性を測定する方法
に関し、下水処理システムにおけるメタン醗酵槽
内等のように、多数の微生物群および消化汚泥等
の種々雑多な異物と共存するメタン菌の菌数また
はメタンの生成活性の測定方法に関するものであ
る。Detailed Description of the Invention [Industrial Application Field] The present invention relates to a method for measuring the number of methane bacteria or methane production activity in a specimen containing methane bacteria, such as in a methane fermentation tank in a sewage treatment system. The present invention relates to a method for measuring the number of methane bacteria or methane production activity that coexists with a large number of microorganisms and various foreign substances such as digested sludge.
[従来の技術]
従来、微生物濃度から菌数または微生物の活性
を評価する方法として、微生物を含む溶液の可視
光の吸光度と微生物濃度との間に成立する一定の
相関関係を利用し、吸光度の測定により菌数等を
測定する方法が採られていた。[Prior art] Conventionally, as a method for evaluating the number of bacteria or the activity of microorganisms from the concentration of microorganisms, a certain correlation between the absorbance of visible light of a solution containing microorganisms and the concentration of microorganisms is used to calculate the absorbance. The method used was to measure the number of bacteria, etc. by measurement.
第2図は従来のこの種の測定方法を説明するた
めの装置系の要部ブロツク図である。図におい
て、1はメタン菌を有する被検体、2は光源、3
はこの光源2に電圧を印加する電源、4は光電子
増倍管、5はこの光電子増倍管4に電圧を印加す
る電源、6は光電子増倍管4の光電流を検出する
検出部である。 FIG. 2 is a block diagram of a main part of an apparatus system for explaining a conventional measuring method of this type. In the figure, 1 is a subject containing methane bacteria, 2 is a light source, and 3
4 is a power source that applies voltage to this light source 2; 4 is a photomultiplier tube; 5 is a power source that applies voltage to this photomultiplier tube 4; 6 is a detection unit that detects the photocurrent of photomultiplier tube 4. .
従来の測定方法は第2図の構成において、光源
2からの可視光をメタン菌を有する被検体1に照
射して透過させ、この透過光を光電子増倍管4で
受光し、その強度を光電子増倍管4の光電流値と
して検出部6により測定していた。そして、測定
された吸光度から既知の吸光度と微生物濃度の相
関関係に基いて微生物濃度を評価し、それに関連
する菌数または微生物の活性を評価していた。 The conventional measurement method uses the configuration shown in FIG. 2, in which visible light from a light source 2 is irradiated onto a specimen 1 containing methane bacteria and transmitted, the transmitted light is received by a photomultiplier tube 4, and its intensity is measured by photoelectrons. The photocurrent value of the multiplier tube 4 was measured by the detection unit 6. Then, the microorganism concentration was evaluated based on the known correlation between the absorbance and the microorganism concentration from the measured absorbance, and the related number of bacteria or the activity of the microorganisms was evaluated.
他方、微生物の活性そのものを測定する方法と
して、微生物のエネルギー代謝サイクルにおける
アデノシン三リン酸ATP(Adenosine
Triphosphate)あるいはニコチンアミド アデ
ニンジヌクレオチドリン酸NAD(P)H
(Nicotinamide Adenine Dinucleotide
(phosphate))のような生体物質(主として補酵
素)の量を一定条件下で吸光度を光学的に測定す
る方法があつた。 On the other hand, as a method to measure the activity of microorganisms themselves, adenosine triphosphate ATP (ATP) in the energy metabolic cycle of microorganisms is used.
Triphosphate) or nicotinamide adenine dinucleotide phosphate NAD(P)H
(Nicotinamide Adenine Dinucleotide
There was a method to optically measure the amount of biological substances (mainly coenzymes) such as (phosphate) under certain conditions by measuring the absorbance.
[発明が解決しようとする課題]
上記のような例えば第2図に示した手段による
従来の測定方法は、被検体の吸光度を測定してい
るため、被検体に多種類の微生物あるいは異物が
含まれている場合は、全体の量が濃度として測定
されるため、メタン菌のみの濃度、それに関連す
る菌数または活性を選択的に測定することは不可
能であつた。他方、微生物の活性そのものを測定
する方法は、ATPやHAD(P)Hが生物のエネ
ルギー代謝系に係る生体物質であるため、全ての
微生物に存在し、メタン菌のみの生成活性および
菌数の測定は不可能であつた。[Problem to be Solved by the Invention] The conventional measurement method using the means shown in FIG. In the case of methane bacteria, the total amount is measured as the concentration, so it has been impossible to selectively measure the concentration of methane bacteria alone, the number of bacteria related thereto, or the activity. On the other hand, since ATP and HAD(P)H are biological substances related to the energy metabolism system of living organisms, methods that measure the activity of microorganisms exist in all microorganisms; Measurement was impossible.
この発明は上記のような課題を解決するために
なされたもので、被検体中の測定対象のメタン菌
に他のどのような微生物及び異物が共存していて
も、これらの共存物質による濁度の影響を受ける
ことなくメタン菌の菌数またはメタン生成活性を
測定することのできる測定方法を提供することを
目的とするものである。 This invention was made in order to solve the above-mentioned problem, and no matter what other microorganisms or foreign substances coexist with the methane bacteria to be measured in the sample, the turbidity caused by these coexisting substances can be reduced. It is an object of the present invention to provide a measurement method that can measure the number of methane bacteria or methane production activity without being influenced by methane bacteria.
[課題を解決するための手段]
この発明に係るメタン菌の菌数またはメタン生
成活性の測定方法は、希釈あるいはアルカリ処理
したメタン菌を有する被検体に励起光をその波長
を変化させて照射し、被検体が放射する放射螢光
を測定して、この放射螢光が、励起光の波長範囲
380〜440nmにおいて、ピーク強度を示す励起光
の波長を検出し、この波長の励起光を被検体に照
射して、被検体が放射する波長450〜490nmの範
囲における放射螢光のピーク強度を測定するとと
もに、励起光が被検体を透過した透過光量を測定
し、この透過光量から放射光の強度に対して被検
体の存在に由来する減弱効果の補正を行い、メタ
ン菌以外の共存物質による濁度の影響を除去して
上記メタン菌が放射する真の螢光強度を求め、メ
タン菌数と螢光強度の相関よりメタン菌数または
メタン生成活性を算出するものである。[Means for Solving the Problems] A method for measuring the number of methane bacteria or methane production activity according to the present invention involves irradiating a diluted or alkali-treated sample containing methane bacteria with excitation light while changing its wavelength. , the radiation fluorescence emitted by the subject is measured, and this radiation fluorescence falls within the wavelength range of the excitation light.
Detect the wavelength of the excitation light that shows the peak intensity in the range of 380 to 440 nm, irradiate the test object with the excitation light of this wavelength, and measure the peak intensity of the emitted fluorescent light in the wavelength range of 450 to 490 nm emitted by the test object. At the same time, the amount of transmitted light that the excitation light transmitted through the specimen is measured, and from this amount of transmitted light, the attenuation effect resulting from the presence of the specimen is corrected for the intensity of the emitted light, and the turbidity due to coexisting substances other than methane bacteria is corrected. The true fluorescence intensity emitted by the methane bacteria is determined by removing the influence of the methane bacteria, and the number of methane bacteria or the methane production activity is calculated from the correlation between the number of methane bacteria and the fluorescence intensity.
[作用]
この発明においては、メタン菌を有する被検体
に波長を変化させて励起光を照射し、被検体から
放射される放射螢光が最大ピーク強度を示す波長
の励起光を照射して被検体が放射する放射螢光の
ピーク強度を測定し、同時に測定した励起光の透
過光量にもとづいて上記のピーク強度に対して減
弱効果の補正を行つて、メタン菌又はメタン生成
活性を求めている。ここで、その測定原理的作用
について説明をするとつぎのようになる。[Function] In the present invention, a specimen containing methane bacteria is irradiated with excitation light of varying wavelength, and the radiation fluorescence emitted from the specimen is irradiated with excitation light of a wavelength at which the maximum peak intensity is exhibited. Methanobacteria or methanogenic activity is determined by measuring the peak intensity of the emitted fluorescent light emitted by the specimen, and correcting the attenuation effect on the above peak intensity based on the amount of transmitted excitation light measured at the same time. . Here, the operation based on the measurement principle will be explained as follows.
メタン菌は通常の微生物と異なる生理的性質を
もち、メタン菌のエネルギー代謝に関与している
電子伝達系に関してはまだその全容は不明である
が、メタン菌に固有な補酵素によるものであるこ
とが知られている。すなわち、メタン菌のエネル
ギー代謝系に存在する電子伝達系の中には、F420
と命名される物質(補酵素)が電子キヤリアとし
て機能していることが知られており、これはメタ
ン菌に固有の物質であつて他の生物系には存在し
ていないものである。このように、メタン菌にの
み結合して電子伝達系(酸化還元系)に中心的に
係る物質(補酵素)F420が、メタン醗酵において
有機物から生成した各種の醗酵生産物からメタン
を生成する活性に機能するものであるとされてい
る。すなわちF420を中心としてメタン菌の電子伝
達系に関与する物質(以後F420物質という)は生
菌(生きた状態の菌)の状態で計測可能であると
共に、他の微生物あるいは異物と異なる特異的か
つ計測可能な物理化学的性質を持つため、メタン
菌の生理的機能から直接メタン生成機能と関連し
ており、メタン菌の菌数またはメタン生成活性の
測定のための有効な計測対象となる。 Methanobacteria have different physiological properties from normal microorganisms, and although the full details of the electron transport system involved in the energy metabolism of methanobacteria are still unknown, it is believed that this is due to coenzymes unique to methanobacteria. It has been known. In other words, in the electron transport chain that exists in the energy metabolism system of methane bacteria, F 420
It is known that a substance called a coenzyme (coenzyme) functions as an electron carrier, and this substance is unique to methane bacteria and does not exist in other biological systems. In this way, F 420 , a substance (coenzyme) that binds only to methane bacteria and is centrally involved in the electron transport system (oxidation-reduction system), produces methane from various fermentation products produced from organic matter in methane fermentation. It is said to be actively functional. In other words, substances involved in the electron transport system of methane bacteria, mainly F 420 (hereinafter referred to as F 420 substances), can be measured in the state of living bacteria (living bacteria) and have specific characteristics that are different from other microorganisms or foreign substances. Because it has physical and chemical properties that are both physical and measurable, it is directly related to the methane production function from the physiological function of methane bacteria, and is an effective measurement target for measuring the number of methane bacteria or methane production activity. .
実際に、特定の波長域の励起光をメタン菌に照
射すると、メタン菌のF420物質に起因して放射す
ると考えられる螢光特性が、消化汚泥中等のメタ
ン菌以外の微生物および異物に起因する螢光特性
と、生菌の状態で異なる挙動を示すことが明らか
となつている。したがつて、メタン菌のみの標準
試料による螢光特性と、被検体のメタン醗酵槽か
らの螢光特性との比較較正から、メタン醗酵槽内
の真のメタン菌の菌数またはメタン生成活性を求
め得ることとなる。 In fact, when methane bacteria are irradiated with excitation light in a specific wavelength range, the fluorescent properties thought to be emitted by the F 420 substance of methane bacteria are caused by microorganisms other than methane bacteria and foreign substances such as digested sludge. It has become clear that the behavior of bacteria differs depending on their fluorescent properties and the state of living bacteria. Therefore, it is possible to determine the true number of methane bacteria or methane production activity in the methane fermentation tank by comparing and calibrating the fluorescence properties of a standard sample containing only methane bacteria and the fluorescence properties from the methane fermentation tank to be tested. It is possible to ask for it.
以上はメタン菌のみに着目した測定原理である
が、メタン醗酵槽内の醗酵液等のメタン菌を有す
る被検体は、一般に他の物質の混入等によつて濁
度が高いために、原液または希釈した液であつて
も、螢光強度は濁度による減弱を受けている。こ
の対策として、励起光波長及び螢光波長における
被検体の濁度を励起光の透過光量から求めて上記
減弱効果を補正するので、被検体の濁度に影響さ
れることのないメタン菌の菌数またはメタン生成
活性の真の値が求められる。 The above is a measurement principle that focuses only on methane bacteria. However, samples containing methane bacteria, such as fermentation liquid in a methane fermentation tank, generally have high turbidity due to contamination with other substances. Even in diluted solutions, the fluorescence intensity is attenuated by turbidity. As a countermeasure against this, the turbidity of the specimen at the excitation light wavelength and fluorescence wavelength is determined from the amount of transmitted light of the excitation light and the above-mentioned attenuation effect is corrected, so that the methane bacteria are not affected by the turbidity of the specimen. The true value of the number or methanogenic activity is determined.
[実施例]
第1図はこの発明による測定方法の一実施例を
示す測定装置の模式構成ブロツク図である。図に
おいて、7はメタン醗酵槽、8はメタン醗酵槽よ
りメタン菌を含む試料を採取するサンプリング管
であり、採取された試料はポンプ9により試料調
整器12に送液される。10は液溜であり、試料
調整器12に送液された試料を測定するのに適当
な被検体とするため、希釈あるいはアルカリ処理
するための溶液が溜められている。液溜10の溶
液は計量してポンプ11で試料調整器12に送液
され、試料と混合される。[Embodiment] FIG. 1 is a schematic block diagram of a measuring device showing an embodiment of the measuring method according to the present invention. In the figure, 7 is a methane fermentation tank, 8 is a sampling tube for collecting a sample containing methane bacteria from the methane fermentation tank, and the collected sample is sent to a sample regulator 12 by a pump 9. Reference numeral 10 denotes a liquid reservoir, which stores a solution for diluting or alkali-treating the sample sent to the sample conditioner 12 to make it a suitable specimen for measurement. The solution in the liquid reservoir 10 is measured and sent to the sample adjuster 12 by the pump 11, where it is mixed with the sample.
混合されて被検体として調整された溶液は、螢
光及び濁度を測定するための試料室13に移され
る。次いで試料室13の被検体に光源15の光を
分光器16を通して波長を測定した励起光が照射
される。被検体の濁度のため減弱した透過光は、
分光器23を通して光電子増倍管24で受光し、
光電流に変換されて検出器22で測定される。ま
た分光器16を通した励起光の照射により、被検
体のメタン菌固有の物質により放射する螢光は、
分光器20により螢光の波長を選定し、光電子増
倍管21で受光して光電流値に変換され、検出器
22で測定される。次いで、検出器22から検出
信号がマイクロプロセツサー26に送られる。マ
イクロプロセツサー26には、検出器22から螢
光の値を示す検出信号を濁度の値を示す検出信号
で補正をする計算機能を有しており、濁度を補正
した螢光の強度が得られる。又マイクロプロセツ
サー26はこのような計算機能のほかにサンプリ
ング管、送液等装置内の諸機能を自動制御する機
能をも備えている。マイクロプロセツサー26で
処理されたデーターは出力端子27で出力表示さ
れる。出力端子27は、処理データー及び制御シ
ーケンスの出力表示のみならず、マイクロプロセ
ツサー26への入力データーも出力端子27を通
じて行なわれる。なお測定の終了した被検体は、
ポンプ18により試料室から排液して廃液溜19
に棄てられる。 The mixed solution prepared as a sample is transferred to a sample chamber 13 for measuring fluorescence and turbidity. Next, the subject in the sample chamber 13 is irradiated with excitation light whose wavelength has been measured by passing the light from the light source 15 through the spectrometer 16 . The transmitted light, which is attenuated due to the turbidity of the sample, is
The light is received by a photomultiplier tube 24 through a spectrometer 23,
The photocurrent is converted into a photocurrent and measured by the detector 22. In addition, by irradiating the excitation light through the spectroscope 16, the fluorescence emitted by the substance specific to the methanogen bacteria in the specimen is
The wavelength of the fluorescent light is selected by a spectrometer 20, received by a photomultiplier tube 21, converted into a photocurrent value, and measured by a detector 22. A detection signal is then sent from the detector 22 to the microprocessor 26. The microprocessor 26 has a calculation function that corrects the detection signal indicating the fluorescence value from the detector 22 with the detection signal indicating the turbidity value, and calculates the fluorescence intensity with the turbidity corrected. is obtained. In addition to such calculation functions, the microprocessor 26 also has a function of automatically controlling various functions within the apparatus such as sampling tubes and liquid feeding. Data processed by the microprocessor 26 is output and displayed at an output terminal 27. The output terminal 27 not only outputs and displays processing data and control sequences, but also inputs data to the microprocessor 26 through the output terminal 27. In addition, for the subject whose measurement has been completed,
The liquid is drained from the sample chamber by the pump 18 to the waste liquid reservoir 19.
abandoned.
次に、前記の測定原理に基づき、第1図の測定
装置による測定方法の具体的手順について説明す
る。 Next, based on the measurement principle described above, the specific procedure of the measurement method using the measurement apparatus shown in FIG. 1 will be explained.
上記の原理に基づく測定方法の操作は次のよう
に行なう。試料をメタン醗酵槽7からサンプリン
グ管8で採取し試料調整器12で所定の被検体と
なるように希釈あるいはアルカリ処理して調整
し、試料室13の容器に入れて被検体とする。次
いで、分光器16でF420物質に螢光を発光させる
特定の波長の範囲の励起光を選択する。励起光の
波長として380〜440nmの波長範囲の光を使用す
る。この選択された特定波長の励起光は試料室1
3に照射され、被検体より生ずる螢光は分光器2
0を通して光電子増倍管21で受光され、また被
検体を透過した光は分光器24を通して光電子増
倍管24で受光する。螢光の強さを示す光電流及
び濁度を示す光電流は検出器22で時系列的に測
定され、検出信号としてマイクロプロセツサー2
6に入力される。マイクロプロセツサー26にお
いては、透過光量にもとづいて被検体の濁度によ
る螢光の強度の減弱を補正して真の螢光の強度を
演算する計算を行う。その結果は出力端子27に
表示される。 The measurement method based on the above principle is operated as follows. A sample is collected from the methane fermentation tank 7 using a sampling tube 8, diluted or treated with alkali to obtain a predetermined test sample using a sample conditioner 12, and placed in a container in a sample chamber 13 to be used as a test sample. Next, a spectroscope 16 selects excitation light in a specific wavelength range that causes the F 420 substance to emit fluorescence. Light in the wavelength range of 380 to 440 nm is used as the excitation light. This selected excitation light of a specific wavelength is applied to the sample chamber 1.
3, and the fluorescent light generated from the subject is sent to the spectrometer 2.
0 and is received by a photomultiplier tube 21, and the light transmitted through the subject is received by a photomultiplier tube 24 through a spectroscope 24. A photocurrent indicating the intensity of fluorescence and a photocurrent indicating turbidity are measured in time series by a detector 22, and sent to the microprocessor 2 as a detection signal.
6 is input. The microprocessor 26 performs calculations based on the amount of transmitted light to calculate the true intensity of the fluorescent light by correcting the attenuation of the fluorescent light intensity due to the turbidity of the subject. The result is displayed on the output terminal 27.
なお、第1図の測定装置においては、分光器2
0を特定波長(螢光波長)に固定し、分光器16
を特定波長域で走査してやればF420物質の励起ス
ペクトルが得られる。また、分光器16を特定波
長(励起波長)に固定し、分光器20を特定波長
域で走査することにより、F420物質の螢光スペク
トルが得られる。このようにすると、これらのス
ペクトルのいずれにおいてもF420物質に起因する
スペクトル強度のピークが得られ、F420物質の存
在量すなわち、メタン菌数またはメタン生成活性
に対応するスペクトル強度として同定することが
できる。ルーチン測定においては、励起及び螢光
スペクトルのうちのどちらかのスペクトルを測定
することで十分であることが、次図の実施例に示
すように確認されている。 In addition, in the measuring device shown in FIG. 1, the spectrometer 2
0 is fixed at a specific wavelength (fluorescence wavelength), and the spectrometer 16
By scanning in a specific wavelength range, the excitation spectrum of F420 material can be obtained. Further, by fixing the spectroscope 16 to a specific wavelength (excitation wavelength) and scanning the spectrometer 20 in a specific wavelength range, a fluorescence spectrum of the F 420 substance can be obtained. In this way, in any of these spectra, a peak of spectral intensity due to F 420 substances can be obtained, which can be identified as the spectral intensity corresponding to the abundance of F 420 substances, that is, the number of methanogens or methanogenic activity. I can do it. In routine measurements, it has been confirmed that it is sufficient to measure either the excitation or fluorescence spectra, as shown in the example shown in the next figure.
第3図はメタン醗酵槽から採取した被検体のメ
タン菌を含む消化汚泥の濁度補正前後の螢光特性
を示す特性線図である。図中、実線は補正前の励
起スペクトル、一点鎖線は補正前の螢光スペクト
ルを示している。図の縦軸はスペクトル強度であ
り、横軸は励起(実線)スペクトルに対しては励
起光の波長λEを、螢光(一点鎖線)スペクトルに
対しては螢光の波長λFを示す。なおA、Bで示す
領域は、それぞれ励起スペクトル、螢光スペクト
ルによる補正後のスペクトル強度を示す。 FIG. 3 is a characteristic diagram showing the fluorescence characteristics of digested sludge containing methane bacteria collected from a methane fermentation tank before and after turbidity correction. In the figure, the solid line shows the excitation spectrum before correction, and the dashed-dotted line shows the fluorescence spectrum before correction. The vertical axis of the figure is the spectral intensity, and the horizontal axis shows the wavelength λ E of the excitation light for the excitation (solid line) spectrum, and the wavelength λ F of the fluorescence for the fluorescence (dotted chain line) spectrum. Note that the regions indicated by A and B indicate the spectral intensities after correction by the excitation spectrum and fluorescence spectrum, respectively.
第3図の実線曲線は、分光器20を螢光波長λF
=470nmの螢光のみが通過するように固定し、分
光器16により励起波長λEを350〜440nmの範囲
内で連続走査して得られた励起スペクトルを示
す。この場合、約420nmの位置にピーク(a点)
を示している。さらに、一点鎖線曲線は、分光器
16を励起光波長λE=420nmに固定し、分光器
20を用いて螢光波長λFが380〜490nmの波長範
囲で連続走査して求めた螢光スペクトルである。
この場合、約470nmの螢光波長λFでピーク(b
点)を示している。a点とb点のスペクトル強度
がほぼ一致することは、両スペクトル最大値を互
にパラメータとして2つのスペクトルを求めたこ
とから当然の結果である。しかしこの特性は、は
じめにλFを任意に設定して励起スペクトルを求め
たのち、この励起スペクトルのピークを示すλEを
一定として螢光スペクトルを求め、もう一度、螢
光スペクトルの最大値を示すλFを一定として励起
スペクトルを求める操作により簡単に第3図の螢
光特性が求められる。 The solid curve in FIG. 3 indicates the fluorescence wavelength λ F
It shows an excitation spectrum obtained by fixing it so that only fluorescence of =470 nm passes through and continuously scanning the excitation wavelength λ E within the range of 350 to 440 nm using the spectrometer 16. In this case, the peak (point a) is at approximately 420 nm.
It shows. Furthermore, the dashed-dotted curve shows the fluorescence spectrum obtained by fixing the spectrometer 16 at the excitation light wavelength λ E =420 nm and continuously scanning the fluorescence wavelength λ F in the wavelength range of 380 to 490 nm using the spectrometer 20. It is.
In this case, the peak ( b
point). The fact that the spectral intensities at point a and point b almost match is a natural result since two spectra were obtained using the maximum values of both spectra as parameters. However, this characteristic requires first setting λ F arbitrarily to obtain the excitation spectrum, then determining the fluorescence spectrum by keeping λ E , which indicates the peak of this excitation spectrum, constant; The fluorescence characteristics shown in FIG. 3 can be easily obtained by determining the excitation spectrum with F constant.
一旦、第3図の特性が得られると、メタン菌を
測定対象とする未知の被検体に対しては、λF=
470nm一定で励起スペクトルの最大値を求める
か、λE=420nm一定で螢光スペクトルの最大値
を求めることのいずれか、又は両方(確認のた
め)のスペクトルを求めることで、メタン菌に対
する各スペクトルの補正前の最大値(a、b)が
容易に求められる。なお、試料調整器12におい
て処理されるアルカリ溶液の濃度等により各スペ
クトルの最大値(a、b)が左右にシフトするた
め、予め最大値の波長が特定できない場合、この
ような方法により最大値を示す波長を求めること
ができる。 Once the characteristics shown in Figure 3 are obtained, λ F =
By determining the maximum value of the excitation spectrum at a constant wavelength of 470 nm, or by determining the maximum value of the fluorescence spectrum at a constant λ E = 420 nm, or both (for confirmation), each spectrum for methanogens can be determined. The maximum value (a, b) before correction can be easily obtained. Note that the maximum value (a, b) of each spectrum shifts to the left or right depending on the concentration of the alkaline solution processed in the sample conditioner 12, so if the wavelength of the maximum value cannot be specified in advance, the maximum value can be determined using this method. It is possible to find the wavelength that shows .
濁度補正は、上記のスペクトル測定と同時に、
分光器23と光電子増倍管24によつて透過光量
を時系列的に求め、その減少度から濁度の補正率
を求め検出器22、マイクロプロセツサー26に
よつて、a、b点の補正を行い、それぞれ第3図
に示す補正後の強度A、Bを得ることができる。
なお、第3図におけるピーク(a、b点)は、試
料調整器12において処理されるアルカリ溶液の
濃度等によりシフトするものであり、励起光の波
長範囲が380〜440nm、放射螢光の波長範囲が
450〜490nmであれば、メタン菌の菌数またはメ
タン生成活性を測定することが可能である。 Turbidity correction is performed at the same time as the above spectrum measurement.
The amount of transmitted light is determined in time series by the spectroscope 23 and the photomultiplier tube 24, and the turbidity correction factor is determined from the degree of decrease. By performing the correction, it is possible to obtain the corrected intensities A and B shown in FIG. 3, respectively.
Note that the peaks (points a and b) in FIG. 3 shift depending on the concentration of the alkaline solution treated in the sample conditioner 12, etc., and the wavelength range of the excitation light is 380 to 440 nm, and the wavelength range of the emitted fluorescent light is 380 to 440 nm. The range is
If the wavelength is 450 to 490 nm, it is possible to measure the number of methane bacteria or methane production activity.
そして、これらのスペクトルからメタン菌及び
メタンの生成活性を同定する。同定するにはあら
かじめ螢光スペクトルの強度のピーク及びスペク
トルの強度の変化に対応したメタン菌の菌数ある
いはメタン生成活性が既知である必要がある。そ
こで標準試料としてのメタン菌について、それら
の関係を求める測定をしておく。それにはメタン
醗酵槽の消化汚泥からメタン菌を単離する。その
単離したメタン菌、例えばメタノザルチナ バル
ケリ(Methanosarcina barkeri)を他の生体物
質を含まない培地に懸濁し、そのメタン菌を標準
試料として励起光の波長を変化した場合の螢光ス
ペクトルの強度のピークが表われる点を求め、前
記の濁度補正によつて補正後の螢光強度を算出す
る。一方、この培地上に生育するメタン菌のコロ
ニーを数えることによりメタン菌の菌数を求める
ことができ、また、このメタン菌が発生するメタ
ン量を測定することにより標準試料におけるメタ
ン発生量を求めることができる。その結果螢光ス
ペクトルとメタン菌数及びメタン生成活性との相
関関係が明らかとなる。第4図、第5図はそのよ
うにして求められた相関関係の一例を示す校正線
図であり、励起光の波長を420nmとした場合の
螢光スペクトルの強度と、メタン菌数およびメタ
ン発生量の関係を示したものである。第4図の横
軸は螢光スペクトル強度、縦軸はメタン菌数であ
り、第5図の横軸は第4図と同じであるが、縦軸
はメタン(ガス)の発生量を示す。いずれの校正
曲線もよい直線性が得られ、十分に使用可能であ
ることが認められる。 Then, from these spectra, methane bacteria and methane production activity are identified. For identification, it is necessary to know in advance the number of methane bacteria or the methane-producing activity corresponding to the intensity peak of the fluorescence spectrum and the change in the intensity of the spectrum. Therefore, we will conduct measurements to determine the relationship between the two using methane bacteria as a standard sample. To do this, methane bacteria are isolated from the digested sludge of the methane fermentation tank. The peak of the intensity of the fluorescence spectrum when the isolated methane bacteria, such as Methanosarcina barkeri, is suspended in a medium containing no other biological substances and the wavelength of the excitation light is changed using the methane bacteria as a standard sample. The point where appears is determined, and the fluorescence intensity after correction is calculated by the turbidity correction described above. On the other hand, the number of methane bacteria can be determined by counting the colonies of methane bacteria growing on this medium, and the amount of methane generated in the standard sample can be determined by measuring the amount of methane generated by these methane bacteria. be able to. As a result, the correlation between the fluorescence spectrum, the number of methanogens, and the methanogenic activity was revealed. Figures 4 and 5 are calibration diagrams showing an example of the correlation obtained in this way, and show the intensity of the fluorescence spectrum when the wavelength of the excitation light is 420 nm, the number of methane bacteria, and the methane generation. This shows the relationship between quantities. The horizontal axis of FIG. 4 is the fluorescence spectrum intensity, and the vertical axis is the number of methane bacteria. The horizontal axis of FIG. 5 is the same as that of FIG. 4, but the vertical axis shows the amount of methane (gas) generated. It is recognized that both calibration curves have good linearity and are fully usable.
本発明の測定方法を実施する装置を第1図に示
したが、励起光を照射する光源15に印加される
電圧または光電子増培管21,24に導入される
光の強度に応じて印加する電圧を変化させ、光電
子増培管20,24に流れる光電流値をその光電
子増培管21,24に適した範囲内に保つと共
に、放射する螢光の強度または各印加電圧に対す
る光電流値を一定螢光強度または一定印加電圧に
対する光電流値に換算するという動作を、システ
ムコントローラーにより自動的に行うこともでき
る。また、メタン醗酵槽7に直接に検出子を導入
して、別々に螢光及び濁度を検出しても同様に本
発明の目的を達成できる。 The apparatus for carrying out the measurement method of the present invention is shown in FIG. By changing the voltage, the value of the photocurrent flowing through the photomultiplier tubes 20 and 24 is kept within a range suitable for the photomultiplier tubes 21 and 24, and the intensity of the emitted fluorescence or the photocurrent value for each applied voltage is adjusted. The operation of converting into a photocurrent value for a constant fluorescence intensity or a constant applied voltage can also be automatically performed by the system controller. Alternatively, the object of the present invention can be similarly achieved by introducing a detector directly into the methane fermentation tank 7 and detecting fluorescence and turbidity separately.
なお、被検体について下水道処理システムなど
のメタン醗酵槽における他の微生物群及び異物の
共存する系について説明したが、それ以外の微生
物群及び異物が共存しても同様にして測定するこ
とができる。 Although a system in which other microbial groups and foreign substances coexist in a methane fermentation tank such as a sewage treatment system has been described as a test subject, measurements can be made in the same manner even if other microbial groups and foreign substances coexist.
[発明の効果]
この発明は以上説明したとおり、メタン菌に他
のどのような微生物および異物が共存していて
も、濁度の影響を受けることなくメタン菌の菌数
またはメタン生成活性を測定することが可能とな
る。そのためメタン醗酵槽が効率よく運転でき、
下水道処理システム等メタン醗酵槽を有するプロ
セスの運転及び管理システムの最適化を容易に達
成しうる効果がある。[Effects of the Invention] As explained above, the present invention is capable of measuring the number of methane bacteria or methane production activity without being affected by turbidity, regardless of what other microorganisms and foreign substances coexist with methane bacteria. It becomes possible to do so. Therefore, the methane fermentation tank can operate efficiently,
This has the effect of easily optimizing the operation and management system of a process having a methane fermentation tank, such as a sewage treatment system.
第1図はこの発明の測定方法の一実施例を示す
測定装置の模式構成ブロツク図、第2図は従来の
測定方法を説明する装置系を含む要部ブロツク
図、第3図はこの発明によるメタン醗酵槽から採
取した被検体のメタン菌を含む消化汚泥の濁度補
正前後の螢光特性を示す特性図、第4図はメタン
菌数と螢光スペクトル強度との関係を示す校正線
図、第5図はメタン発生量と螢光スペクトル強度
との関係を示す校正線図である。
図において、7はメタン醗酵槽、8はサンプリ
ング管、9はポンプ、10は液溜、11はポン
プ、12は試料調整器、13は試料室、14は電
源、15は光源、16は分光器、17は排液管、
18はポンプ、19は廃液溜、20は分光器、2
1は光電子増培管、22は検出器、23は分光
器、24は光電子増培管、25は電源、26はマ
イクロプロセツサー、27は出力端子である。
なお、図中同一符号は同一または相当部分を示
す。
Fig. 1 is a schematic structural block diagram of a measuring device showing an embodiment of the measuring method of the present invention, Fig. 2 is a block diagram of the main parts including the device system explaining the conventional measuring method, and Fig. 3 is a block diagram of a measuring device according to the present invention. A characteristic diagram showing the fluorescence characteristics before and after turbidity correction of digested sludge containing methane bacteria collected from a methane fermentation tank, and Figure 4 is a calibration diagram showing the relationship between the number of methane bacteria and fluorescence spectrum intensity. FIG. 5 is a calibration diagram showing the relationship between the amount of methane generated and the fluorescence spectrum intensity. In the figure, 7 is a methane fermentation tank, 8 is a sampling tube, 9 is a pump, 10 is a liquid reservoir, 11 is a pump, 12 is a sample conditioner, 13 is a sample chamber, 14 is a power source, 15 is a light source, and 16 is a spectrometer. , 17 is a drain pipe,
18 is a pump, 19 is a waste liquid reservoir, 20 is a spectrometer, 2
1 is a photomultiplier tube, 22 is a detector, 23 is a spectrometer, 24 is a photomultiplier tube, 25 is a power source, 26 is a microprocessor, and 27 is an output terminal.
Note that the same reference numerals in the figures indicate the same or corresponding parts.
Claims (1)
菌を有する被検体に励起光をその波長を変化さ
せて照射し、 (b) 上記被検体が放射する放射螢光を測定して、
該放射螢光が、励起光の波長範囲380〜440nm
において、ピーク強度を示す励起光の波長を検
出し、 (c) 次いで、該波長の励起光を上記被検体に照射
して、上記被検体が放射する波長範囲450〜
490nmにおける放射螢光のピーク強度を測定す
るとともに、 (d) 上記励起光が上記被検体を通過する透過光量
を測定し、 (e) 該透過光量に基づいて上記放射螢光のピーク
強度に対して上記被検体の存在に由来する減弱
効果の補正を行い、 (f) 上記メタン菌が放射する真の螢光強度を求め
る ことを特徴とするメタン菌の菌数またはメタン生
成活性の測定方法。[Claims] 1. (a) irradiating a diluted or alkali-treated specimen containing methane bacteria with excitation light while changing its wavelength, and (b) measuring the emitted fluorescence emitted by the specimen. hand,
The wavelength range of the excitation light is 380 to 440 nm.
(c) Detecting the wavelength of the excitation light exhibiting the peak intensity; (c) Next, irradiating the test subject with the excitation light at the wavelength to obtain a wavelength range of 450 to 450 nm emitted by the test subject.
While measuring the peak intensity of the emitted fluorescent light at 490 nm, (d) measuring the amount of transmitted light through which the excitation light passes through the subject, (e) calculating the peak intensity of the emitted fluorescent light based on the amount of transmitted light. (f) correcting the attenuation effect resulting from the presence of the analyte, and (f) determining the true fluorescence intensity emitted by the methane bacteria.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP59085533A JPS60230040A (en) | 1984-04-27 | 1984-04-27 | Method for measuring number of mathane bacteria and methane forming activity |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP59085533A JPS60230040A (en) | 1984-04-27 | 1984-04-27 | Method for measuring number of mathane bacteria and methane forming activity |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS60230040A JPS60230040A (en) | 1985-11-15 |
JPH0413655B2 true JPH0413655B2 (en) | 1992-03-10 |
Family
ID=13861520
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP59085533A Granted JPS60230040A (en) | 1984-04-27 | 1984-04-27 | Method for measuring number of mathane bacteria and methane forming activity |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS60230040A (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0762652B2 (en) * | 1986-08-22 | 1995-07-05 | 株式会社日立製作所 | Fluorescence analysis method and apparatus |
JPS63247643A (en) * | 1987-04-02 | 1988-10-14 | Kondo Kogyo Kk | Method for measuring suspended bacteria |
JP5148387B2 (en) * | 2008-06-30 | 2013-02-20 | 浜松ホトニクス株式会社 | Spectrometer, spectroscopic method, and spectroscopic program |
US20130078730A1 (en) * | 2011-09-23 | 2013-03-28 | Michael J. Murcia | Method for monitoring and control of a wastewater process stream |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5194886A (en) * | 1975-02-19 | 1976-08-19 | ||
JPS5543461A (en) * | 1978-09-22 | 1980-03-27 | Gunze Ltd | Corrected turbidity measuring method for treating agent concentration containing in suspension |
-
1984
- 1984-04-27 JP JP59085533A patent/JPS60230040A/en active Granted
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5194886A (en) * | 1975-02-19 | 1976-08-19 | ||
JPS5543461A (en) * | 1978-09-22 | 1980-03-27 | Gunze Ltd | Corrected turbidity measuring method for treating agent concentration containing in suspension |
Also Published As
Publication number | Publication date |
---|---|
JPS60230040A (en) | 1985-11-15 |
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