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WO2024258308A1 - Appareil de mesure des paramètres d'une suspension de micro-organismes dans un photo-bioréacteur - Google Patents

Appareil de mesure des paramètres d'une suspension de micro-organismes dans un photo-bioréacteur Download PDF

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Publication number
WO2024258308A1
WO2024258308A1 PCT/RU2023/000183 RU2023000183W WO2024258308A1 WO 2024258308 A1 WO2024258308 A1 WO 2024258308A1 RU 2023000183 W RU2023000183 W RU 2023000183W WO 2024258308 A1 WO2024258308 A1 WO 2024258308A1
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WIPO (PCT)
Prior art keywords
photodetector
housing
emitter
fluorimeter
optical
Prior art date
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Pending
Application number
PCT/RU2023/000183
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English (en)
Russian (ru)
Inventor
Иван Владимирович КОНЮХОВ
Константин Викторович КОТЕЛЬНИК
Игорь Рашидович НАСИРОВ
Владимир Александрович КОВАЛЕВ
Тимофей Юльевич ПАН
Артем Сергеевич ПЕЧКИН
Антон Сергеевич ПОЛТОРАДНЕВ
Иван Дмитриевич ТОКАРЕВ
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"aa Plus Tech" LLC
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"aa Plus Tech" LLC
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Filing date
Publication date
Application filed by "aa Plus Tech" LLC filed Critical "aa Plus Tech" LLC
Priority to PCT/RU2023/000183 priority Critical patent/WO2024258308A1/fr
Publication of WO2024258308A1 publication Critical patent/WO2024258308A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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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

Definitions

  • the claimed invention relates to the fields of electronics, optics and biotechnology, namely to measuring devices for continuous multiparameter monitoring of the physicochemical characteristics of biological suspensions (microorganisms and culture fluid) cultivated in a specialized device (photobioreactor) in an industrial manner, as well as to devices that control the development of phototrophic microorganisms in such areas as:
  • control over the stages of microbiological synthesis is carried out in two ways. The first is with the help of primary information sensors installed directly in the culturing device or on the communications adjacent to it, the second is with the help of laboratory analyses of suspension samples periodically taken from the photobioreactor. It is obvious that for an industrial installation the use of information sensors is a priority. This allows:
  • the main parameters of the suspension that need to be continuously monitored are the state of the photosynthetic apparatus of microorganisms and their concentration (see, for example, Babin M. Phytoplankton fluorescence: theory, current literature and in situ measurementZ/Real-time Coastal Observing Systems for Marine Ecosystem Dynamics and Harmful Algal Blooms: Theory, Instrumentation and Modelling I Ed. M. Babin, C. S. Roesler, J. J. Cullen. Paris UNESCO Publishing, 2008. URL: https://unesdoc.unesco.Org/ark:/48223/pf0000160000).
  • the so-called “variable” chlorophyll fluorescence (relative increase in fluorescence intensity during light saturation of photosynthesis processes) of microorganism cells is measured.
  • the concentration of cells the optical density of the suspension is measured, which directly depends on the concentration.
  • microorganisms are limited by cost factors (cost price).
  • cost factors is the cost of the photobioreactor equipment, an essential part of which are measuring devices, such as a fluorimeter and an optical density sensor. These devices are expensive, but they consist of similar elements: a power supply (voltage converter), a controller, an interface module, an emitter, a light filter, a detector, a signal amplifier, an analog-to-digital converter, and a housing.
  • a device for continuous multiparameter monitoring of the physicochemical characteristics of biological suspensions is known (RU Patent No. 2786399, published on 20.12.2022), which comprises a housing in which a cell with at least six measuring channels is located, allowing the parameters of biological suspensions to be determined based on the measured optical characteristics, wherein one of the channels is intended for measuring oxygen, a power source, a magnetic stirrer located in the cell, a heater of the analyzed substance and a temperature sensor located in the cell, an electronic control board, five optical radiation sources installed in the measuring channels and five optical radiation recorders, also installed in the measuring channels, wherein the optical radiation recorders are connected to the inputs of the amplifier and analog-to-digital converter unit, the output of which is connected to the input of the recording device, wherein at least the oxygen sensor is an assembly containing at least one Y-shaped fiber bundle with a composite core, composed of two physically mixed fiber bundles intended for separating the optical paths of excitation and recording of the optical signal, wherein the emitter of excitation radiation and
  • a photometer is known (RU Patent for Utility Model No. 75473, published on 10.08.2008), combining the capabilities of a fluorimeter and a photocolorimeter.
  • the device contains a source of excitation light energy, an optical system, a cuvette closed with a lid and a photodetector, equipped with an additional external optical cell with a lid and two flexible light guides made of fiber optics.
  • the light guides are fixed in the cuvette lid using guide metal tubes in such a way that their ends are strictly directed into the windows with the radiation source (primary light guide) and the photodetector (secondary light guide).
  • the opposite ends of the light guides are fixed in the external optical cell using removable light guide holders, with the possibility of installing them at both an angle of 90° and an angle of 180°.
  • the covers of the cuvette and the outer optical cell are provided with holes for the light guides, which are light-sealed with rubber seals. The following can be attributed to the disadvantages of the photometer:
  • the specified device does not measure the optical density of the suspension, its temperature and does not ensure the consistency of the obtained data.
  • a photometer is known (RU Patent for Invention No. 2176384, published on 27.11.2001), comprising an optically coupled light source, a light beam former, interference light filters, an optical distributor, a plate and a device for receiving radiation.
  • the photometer comprises a light flux switch with a rotor rotating inside a fixed housing, in which a flat mirror or light guide is fixed, and M fixed illuminating light guides are fixed in the housing, where M is the number of cells in a column or row of the plate.
  • the switch rotor may contain two light guides, and in the housing - additionally M measuring light guides and one common output light guide, optically coupled with one photodetector for alternately measured light fluxes. The following can be attributed to the disadvantages of the photometer:
  • the specified device does not measure the optical density of the suspension, its temperature and does not ensure the consistency of the obtained data
  • the specified device does not measure the optical density of the suspension, its temperature and does not ensure the consistency of the obtained data
  • a known spectrophotometer (RU Patent for Utility Model No. 147783, published on 20.11.2014) contains a light source in the form of a laser with distributed feedback, lenses, mirrors, cuvettes with the test and reference samples, condensers, a rotating device in the form of a bending piezoelectric plate, polarization attenuators, photocells, a two-channel amplifier, a sawtooth pulse generator, an analog-to-digital converter, a computer, a digital-to-analog converter, characterized in that the spectrophotometer is additionally equipped with an electronic switch, a photocell and a beam-splitting mirror by means of which part of the pump radiation is fed to the photocell, the signal from which is fed to the sawtooth pulse generator, and the electronic switch is connected to photocell via a computer.
  • the device is designed to determine the optical density, transmittance, fluorescence, scattering, concentration of ingredients of samples of different physical states.
  • the disadvantages of the device include the following:
  • a portable fluorometer/turbidimeter Aqua Fluor by Turner Designs is known (see http://docs.turnerdesiQns.com/t2/doc/manuals/998-0851.pdf, https://www.turnerdesiqns.com/aquafluor-handheld-fluorometer), which allows measuring fluorescence and optical density of a single sample.
  • the device can be configured with any combination of two channels: in vivo chlorophyll, extracted chlorophyll, cyanobacteria (phycocyanin or phycoerythrin), turbidity, rhodamine BT, fluorescein, ammonium, colored or fluorescent dissolved organic matter, optical brighteners or oil.
  • the disadvantages of the device include the following:
  • a high-speed spectroradiometry module based on the AvaSpec-2048-USB2 fiber-optic high-speed spectrometer is known (see https://www.avantes.ru/articles/up1/22.php), which can be used as a spectrophotometer, photocolorimeter, radiometer, nephelometer or turbidimeter, fluorimeter, fluoroscan or luminometer for analytical studies in the spectral range of 200-1150 nm with very high sensitivity.
  • the disadvantages of this device are:
  • the device itself does not have dust and moisture protection, which is necessary for placement in an industrial area, next to capacitive equipment;
  • the AquaPen-C AP 110-C fluorimeter is known (see httDs://biopik.ru/market/oborudovanie1/fluorimetry/Dsi photon systems instruments fluorime try/pribor aguapen ap110 c psi/, https://handheld.psi.cz/documents/AguaPen Manual- verze 02 2021.pdf) for working with suspensions of photosynthetic biological objects in field and laboratory conditions.
  • AquaPen-C is optimized for measuring the chlorophyll fluorescence of green algae and cyanobacteria.
  • the sensitivity of AquaPen is 500 ng chl / l and allows measurements in natural waters.
  • AquaPen-C has a built-in dual-wavelength LED turbidimeter (720 nm / 680 nm), allowing you to characterize the state of the suspensions under study.
  • the disadvantages of the fluorimeter include the following:
  • the closest device to the claimed device is a device for measuring the optical parameters of fluorescence intensity, optical density and refractive index in a test sample (patent application No. US20100182606A1, Apparatus and method for multi-parameter optical measurements, published on July 22, 2010), comprising: a) a housing with a detection area for receiving the test sample or a cuvette containing the test sample; b) one or more temperature sensors capable of measuring the temperature of one or more housings, detection areas, test samples or cuvettes; c) one or more temperature controllers for regulating the temperature of one or more housings, detection areas, test samples or cuvettes in a desired range; d) a probing light source in optical communication with the detection area for directing probing light onto the test sample; e) one or more sensors for detecting emitted light of the test sample and for converting the detected emitted light into an output signal; and f) a control system in operative communication with the probing light
  • the objective of the present invention is to create a combined device that combines the capabilities of a fluorimeter, turbidimeter and thermometer, creating conditions for continuous automatic multiparameter monitoring of the physicochemical characteristics of suspensions of phototrophic microorganisms cultivated in a specialized device (photobioreactor) in an industrial manner.
  • the technical result which the claimed invention is aimed at achieving, consists in automating the measurement process, increasing the accuracy of measurements and the reliability of the obtained data while simultaneously reducing the cost of manufacturing the device.
  • a combined device for measuring the parameters of a suspension of phototrophic microorganisms from inside a photobioreactor consisting of a housing, inside which are located: a temperature sensor, an emitter directing radiation to the measured medium, a fluorimeter photodetector, a turbidimeter photodetector and an electronic control unit connected to an external information system and a power source, containing an analog-to-digital converter and a signal amplifier connected via communication channels to the emitter, the fluorimeter photodetector and the turbidimeter photodetector.
  • the housing is made hermetically sealed and has a U-shape, the vertical parts of which form a groove intended for moving the measured medium inside it.
  • Parallel walls of the groove contain openings in which sealing glasses are installed on one optical axis.
  • a frame of optical devices consisting of two parts, in one part of which the mentioned photodetector of the fluorimeter is installed, and in the other part the mentioned emitter is installed so that the conjugation of the optical axes of the emitter and the photodetector of the fluorimeter is ensured at the boundary of the sealing glass and the measured medium.
  • light filters In front of the photodetector of the fluorimeter and the emitter there are light filters that transmit only radiation of the chlorophyll fluorescence spectrum.
  • the photodetector of the turbidimeter is placed inside the other vertical part of the housing on the same optical axis with the sealing glasses and the emitter.
  • the electronic unit and the temperature sensor are placed in the horizontal part of the housing.
  • At least one mounting bracket is installed on the outer walls of the housing.
  • the frame of the optical devices can be designed with the possibility of adjusting the angle of conjugation of the optical axes of the photodetector of the fluorimeter and the emitter.
  • One of the outer U-shaped parts of the body can be made in the form of a cover, the edges of which are covered with a sealing gasket along the perimeter.
  • the electronic unit can be connected to an external information system via a sealed cable connector.
  • the bit depth of the analog-to-digital converter of the electronic unit can be more than twelve.
  • the combined device consists of a U-shaped housing 1 with a lid (not shown in the figure), the edges of which are covered along the perimeter with a sealing gasket, ensuring the tightness of the internal volume of the housing 1.
  • the vertical parts of the housing form a groove 2 between themselves, inside which the measured medium - the photobioreactor suspension - moves. Inside the walls of the housing 1, which are parallel to the walls of the groove 2, there are openings in which sealing glasses 3 and 4 are installed. All optical elements of the device are placed inside the vertical parts of the housing 1.
  • Emitter 6 is in immediate proximity to fluorimeter photodetector 8 so that the radiation of the fluorescence-inducing spectrum created by emitter 6 for rapid saturation of chlorophyll molecules does not fall on fluorimeter photodetector 8.
  • Emitter light filter 7 is placed after emitter 6, and fluorimeter photodetector light filter 9 is placed in front of fluorimeter photodetector 8. relative to their optical axes in the direction of the sealing glass 3.
  • the photodetector of the turbidimeter 10 is placed and fixed on the sealing glass 4.
  • the optical elements 6-9 are fixed inside the frame of the optical elements 5 using screw connections 11, which provide the ability to adjust the location and direction of the optical axes of the emitter 6 and the photodetector of the fluorimeter 8 so that their optical axes intersect at a point located immediately after the sealing glass 3 (at the boundary of the sealing glass 3 and the measured medium), and the emitter 6 and the photodetector of the turbidimeter 10 are on the same optical axis.
  • An electronic unit 12 is placed inside the horizontal part of the housing 1.
  • the electronic unit 12 consists of a temperature sensor 13, an analog-to-digital converter, a signal amplifier connected via communication channels to the emitter 6, the photodetector of the fluorimeter 8 and the photodetector of the turbidimeter 10.
  • the capacity of the analog-to-digital converter of the electronic unit 12 is more than twelve (for example, sixteen) in order to increase the accuracy of determining the fluorescence intensity.
  • the photodetector of the turbidimeter 10, the photodetector of the fluorimeter 8, the emitter 6 and the temperature sensor 13 are connected to the electronic unit 12 via communication channels.
  • the lines that connect the photodetector of the turbidimeter 10 and the photodetector of the fluorimeter 8 with the electronic unit 12 must be protected from external electromagnetic fields, since the signals created by them have a low level of electrical voltage. In addition, the power supply lines of the signal amplifiers must also be protected. Such protection is ensured by the device being inside the suspension volume, therefore shielding of the electronic components of the device is not required.
  • the device is connected to the external information system using a sealed cable connector 14.
  • Mounting brackets 15 are installed on the outer surface of the walls of the housing 1 to fix the device on the inner wall of the photobioreactor or any other device cultivating the biological environment. The most preferable number of mounting brackets 15 for reliable fixation of the combined device is 2.
  • the operation of the combined device is carried out as follows. Before carrying out measurements of the parameters of the suspension, the device is prepared for operation: open the cover of the housing 1, using screw connections 11 set the required location of the optical elements 6-9 and the direction of the optical axes of the emitter 6 and the photodetector of the fluorimeter 8, close the cover of the housing 1 back.
  • the electronic unit 12 Upon a signal from the external information system to request the parameter of the optical density of the suspension, the electronic unit 12 generates a series of pulses (consisting of at least one pulse) of rectangular electric current, supplied to the emitter 6.
  • the emitter 6 creates light radiation of the required intensity and duration.
  • the light filter of the emitter 7 provides the specified width of the radiation spectrum of the emitter 6.
  • the electrical voltage created by the photodetector of the turbidimeter 10 depends on the intensity of the received radiation, which in turn depends on the optical density of the suspension.
  • the electronic unit 9, having received the signal from the photodetector of the turbidimeter 10, amplifies and digitizes it. After processing the received signal, the electronic unit 12 transmits the received value to the information system.
  • the electronic unit 12 Based on a signal from an external information system to request the parameter of fluorescence intensity of microorganism cells, the electronic unit 12 generates a series of pulses (consisting of at least one pulse) of rectangular electric current supplied to the emitter 6.
  • the emitter 6 creates light radiation of the required intensity and duration.
  • the light filter of the emitter 7 provides a specified width of the spectrum of radiation of the emitter 6.
  • the generated radiation is supplied to the cells of microorganisms moving through the groove 2, next to the sealing glass 3 and is absorbed by the chlorophyll molecules located in them, transferring them to an excited state. When chlorophyll molecules return from the excited state to the ground state, molecule glow (i.e.
  • the photodetector of the fluorimeter 8 captures the glow of the chlorophyll molecules of the cells of the suspension and forms an electric voltage signal transmitted to the input of the electronic unit 12.
  • the electronic unit 12 amplifies the signal received from the photodetector of the fluorimeter 8 and digitizes it.
  • the electronic unit 12 After processing the received signals, the electronic unit 12 transmits the value of the ratio of the average fluorescence intensity at the beginning to the average fluorescence intensity at the end of the light pulse of the entire series of pulses created by the emitter 6 to the information system.
  • the electronic unit Block 12 Upon a signal from the external information system to a request for a suspension parameter, the electronic unit Block 12 digitizes the signal received from temperature sensor 13 and transmits the received value to the information system.
  • the claimed device allows:

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  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne des dispositifs de mesure pour contrôler les caractéristiques de suspensions biologiques. Cet instrument combiné comprend un corps dans lequel sont disposés un capteur de température, un émetteur, un photodétecteur, un fluorimètre, un photo-détecteur de turbidimètre et une unité de commande électronique. Le corps est étanche et possède une forme de U dont les parties supérieures définissent une gorge. Les parois parallèles de la gorge comprennent des ouvertures dans lesquelles sont disposés des verres d'étanchéité. Dans une des parties verticales du corps se trouve un cadre d'instruments optiques dans lequel sont disposés un photodétecteur de fluorimètre et un émetteur de manière à assurer le couplage des axes optiques de l'émetteur et du photodétecteur du fluorimètre. Des filtres lumineux sont disposés en amont du photodétecteur du fluorimètre et de l'émetteur. Le photodétecteur du fluorimètre est disposé dans l'autre partie verticale du corps. L'unité électronique et le capteur de température sont disposés dans la partie horizontale du corps. Sur les parois externes du corps se trouve au moins une console de fixation. Le résultat technique de l'invention consiste en une automatisation du processus de mesure, une augmentation de leur précision et de la fiabilité des données obtenues tout en réduisant les coûts intrinsèques de production de l'instrument.
PCT/RU2023/000183 2023-06-15 2023-06-15 Appareil de mesure des paramètres d'une suspension de micro-organismes dans un photo-bioréacteur Pending WO2024258308A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/RU2023/000183 WO2024258308A1 (fr) 2023-06-15 2023-06-15 Appareil de mesure des paramètres d'une suspension de micro-organismes dans un photo-bioréacteur

Applications Claiming Priority (1)

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PCT/RU2023/000183 WO2024258308A1 (fr) 2023-06-15 2023-06-15 Appareil de mesure des paramètres d'une suspension de micro-organismes dans un photo-bioréacteur

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5483080A (en) * 1992-09-18 1996-01-09 Tam; Lisa A. Method and device for measuring and controlling cell density in microbiological culture
US20110162979A1 (en) * 2010-01-07 2011-07-07 Pharmaco-Kinesis Corporation Method and Apparatus for Forming of an Automated Sampling Device for the Detection of Salmonella Enterica Utilizing an Electrochemical Aptamer Biosensor
US20180187234A1 (en) * 2015-06-29 2018-07-05 The Arizona Board Of Regents On Behalf Of The University Of Arizona Optical device for in-line and real-time monitoring of microorganisms

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5483080A (en) * 1992-09-18 1996-01-09 Tam; Lisa A. Method and device for measuring and controlling cell density in microbiological culture
US20110162979A1 (en) * 2010-01-07 2011-07-07 Pharmaco-Kinesis Corporation Method and Apparatus for Forming of an Automated Sampling Device for the Detection of Salmonella Enterica Utilizing an Electrochemical Aptamer Biosensor
US20180187234A1 (en) * 2015-06-29 2018-07-05 The Arizona Board Of Regents On Behalf Of The University Of Arizona Optical device for in-line and real-time monitoring of microorganisms

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LADISLAV NEDBAL ET AL.: "Photobioreactor System for Precision Cultivation of Photoautotrophic Microorganisms and for High-Content Analysis of Suspension Dynamics", BIOTECHNOLOGY AND BIOENGINEERING, vol. 100, no. 5, August 2008 (2008-08-01), pages 902 - 910, XP002608893, DOI: 10.1002/BIT.21833 *

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