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WO2014067584A1 - Ensemble et procédé de dosage automatique répété d'une substance issue d'un échantillon contenant des cellules subissant une photosynthèse - Google Patents

Ensemble et procédé de dosage automatique répété d'une substance issue d'un échantillon contenant des cellules subissant une photosynthèse Download PDF

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Publication number
WO2014067584A1
WO2014067584A1 PCT/EP2012/071757 EP2012071757W WO2014067584A1 WO 2014067584 A1 WO2014067584 A1 WO 2014067584A1 EP 2012071757 W EP2012071757 W EP 2012071757W WO 2014067584 A1 WO2014067584 A1 WO 2014067584A1
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WO
WIPO (PCT)
Prior art keywords
sample
substance
assembly
headspace
temperature
Prior art date
Application number
PCT/EP2012/071757
Other languages
English (en)
Inventor
Thomas ABTS
Heike Enke
Anika HANS
Matthias Steffen
Ulf Duehring
Original Assignee
Algenol Biofuels Inc.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Algenol Biofuels Inc. filed Critical Algenol Biofuels Inc.
Priority to PCT/EP2012/071757 priority Critical patent/WO2014067584A1/fr
Publication of WO2014067584A1 publication Critical patent/WO2014067584A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/24—Automatic injection systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/22—Devices for withdrawing samples in the gaseous state
    • G01N1/2226—Sampling from a closed space, e.g. food package, head space
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/22—Devices for withdrawing samples in the gaseous state
    • G01N1/2226—Sampling from a closed space, e.g. food package, head space
    • G01N2001/2229—Headspace sampling, i.e. vapour over liquid
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
    • G01N2030/8854—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds involving hydrocarbons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • This invention is related to the field of analysis of chemical compounds of interest produced by photosynthetically active cells .
  • the laboratory routine generally involves cultivating candidate organisms under photosynthetic conditions and determining productivity by analyzing the amount of chemical compounds of interest in the sample after a given cultivation time.
  • US patent application 2011/039323 discloses a method for determining isoprene productivity of bacterial cultures, wherein transformed cells were grown in Luria Broth in sealed vials and the headspace over the cell cultures was assayed for isoprene by taking a 0.5-mL gas sample from the headspace air above the liquid culture with a syringe and analyzing the sample on an analytical gas chromatograph with a mass
  • Berry et al . disclose in international patent application 2010/062707 a method for determining ethanol produced by phototrophic microorganisms which includes drawing a cell-free sample from a main culture of organisms and then determining the liquid ethanol concentration by headspace gas chromatography.
  • US 2011/0151531 discloses a method for ethanol and acetaldehyde determination from a bacterial sample, wherein at given time points 1 mL of a main culture were withdrawn and spun down at 4 °C for 10 min at 21,000 RCF.
  • US 2011/0151531 further discloses a method for ethylene determination form a bacterial culture, wherein 1 ml of this culture was placed in a 10 ml headspace vial and incubated in a shaking incubator in the light for 1 h. The culture was then killed by incubating the sample at 80 °C for 5 min and analyzed for the presence of ethylene by headspace gas chromatography with flame ionization detector.
  • the invention described herein discloses an assembly configured for repeated automatic transferring and assaying of a volatile substance having in its pure form a vapour pressure at 20 "Celsius lower than 604 hPa from a sample comprising a liquid phase and a headspace, the liquid phase containing living cells undergoing photosynthesis, the cells thereby photosynthetically producing the substance.
  • the assembly comprises a sample holder with at least one sample holding
  • an illumination device for illuminating the sample in said sample holding position with photosynthetically usable radiant energy
  • a mixing device for mixing the sample in said sample holding position, and a temperature control device for controlling the temperature of the sample in said sample holding position,
  • an analytical unit comprising a gas chromatograph for assaying the substance
  • a transfer unit configured for repeated automatic transferring of the substance from the sample in said sample holding position from the headspace of said sample to the analytical unit.
  • the invention described herein also discloses a method for repeated automatic transferring and assaying of a volatile substance having in its pure form a vapour pressure at 20
  • the method comprises the steps: a) providing the sample in a sample container in the sample holder,
  • this invention provides an assembly
  • a sample holder configured for repeated automatic transferring and assaying of a volatile substance having in its pure form a vapour pressure at 20 "Celsius lower than 604 hPa from a sample comprising a liquid phase and a headspace, the liquid phase containing living cells undergoing photosynthesis, the cells thereby photosynthetically producing the substance, comprising a sample holder with at least one sample holding
  • an illumination device for illuminating the sample in said sample holding position with photosynthetically usable radiant energy
  • a mixing device for mixing the sample in said sample holding position
  • a temperature control device for controlling the temperature of the sample in said sample holding position
  • an analytical unit comprising a gas chromatograph for assaying the substance
  • a transfer unit configured for repeated automatic transferring of the substance from the same sample in said sample holding position from the headspace of said sample to the analytical unit.
  • the automatic transferring and assaying occurs at least at two different time points during incubation of the cells in the sample.
  • a substance having in its pure form a vapour pressure at 20 “Celsius lower than 604 hPa” is meant to be a substance that, when pure, has a vapour pressure lower than 604 hPa, corresponding to approximately 453 mmHg.
  • vapour pressure at 20 "Celsius shall be understood as a substance characteristic of the pure
  • vapour pressure of the same substance when solute in a solvent, such as the sample liquid phase may deviate from that of the pure substance, as it depends on the concentration of the substance and the composition of the solution.
  • sample containing living cells undergoing photosynthesis shall mean that conditions are maintained in the sample which allow survival of the cells and continued photosynthetic production of the substance by the cells before, during and after at least one automatic transferring and assaying of the substance from the sample.
  • photosynthetically usable radiant energy shall mean any spectral composition including wavelengths and intensities sufficient for the cells to undergo
  • transferring or "assaying”, respectively, is considered “automatic” if it does not involve manual handling of either the sample or the substance, directly or indirectly, during the process of the substance being transferred from the headspace of the sample into the gas chromatograph .
  • automated is also applied to embodiments characterised in that no manual handling of the sample or the substance, directly or indirectly, is required once the sample has been introduced into the sample holding position of the assembly and the illumination, mixing and temperature have been set, at least until the final
  • the inventors of the present invention were the first to realise that a major drawback of conventional methods for headspace analysis of complex biological samples like
  • phototrophic bacterial cultures are that they typically require a separate specialised instrumentation for sample cultivation, headspace sample preparation, sample transfer and analysis and, as such, make multiple manual operations necessary.
  • the inventors discovered that configuring an assembly in such a way that illumination, mixing, temperature control and sampling work as a functional unit significantly reduces manual operations in headspace analysis of phototrophic bacterial samples. For example, the inventors accomplished adapting an illumination device in such a way that
  • the assembly of the present invention thus also furthers the economical use of samples and resources, since multiple measurements can be performed from a single sample of small volume, whereas end-point measurements generally require preparation and consumption of an extra sample for each measurement.
  • the accuracy of measurements is increased, since repeated measurements from the same sample obviate the need for different samples for multiple end-point measurements, thus avoiding sample variance arising from e.g. a different growth of the cells in the samples or introduced during preparation and processing of the samples.
  • the sample holder In one embodiment of the invention, the sample holder
  • sample holder is a device which avoids undesired moving, e.g. tilting or tipping over, of the sample container in the sample holding position.
  • the sample holder avoids moving of the sample container during mixing of the sample and automatic
  • the sample holder comprises a sample holding position which can be automatically spatially addressed in x and y coordinates, more preferably x, y and z coordinates, with the transfer unit.
  • a sample holder can, for instance, comprise holes or pockets for accommodating the sample.
  • a sample holder can comprise clamps for holding the sample.
  • a sample tray comprising a plurality of sample holding positions can be present.
  • a solid block with one or more drill-holes as sample holding positions can be used.
  • the plurality of sample holding positions is arranged in the sample holder in linear rows.
  • the sample holding positions can be arranged in two or more parallel rows of essentially rectangular arrangement.
  • sample holding positions is arranged in circular rows.
  • sample holding positions are arranged in concentric circles.
  • the sample holding positions can also be helically arranged.
  • the illumination device provides at least 10 ⁇ m ⁇ 2 s -1 photon flux of radiation usable by the cells for undergoing photosynthesis. With this configuration is achieved that the cells can undergo
  • the illumination device provides at least 50 ⁇ m ⁇ 2 s -1 photon flux of radiation usable by the cells for undergoing photosynthesis. Even more preferred is an illumination device that provides at least 100 ⁇ m ⁇ 2 s -1 photon flux up to at least 500 ⁇ m ⁇ 2 s -1 photon flux.
  • the illumination device comprises a dimmer for varying the intensity of the light output.
  • a dimmer for varying the intensity of the light output.
  • One example is a dimmer with which the
  • a dimmer configured for changing the illumination intensity in the sample holding position over time.
  • the dimmer is configured for automatically changing the illumination intensity in the sample holding position over time.
  • a dimmer configured for automatically performing light-dark-cycles, wherein a period of illumination is followed by a period without illumination in a recurring manner.
  • light-dark-cycles can comprise 12 hours of illumination followed by 12 hours of darkness.
  • a dimmer can be computer-controlled.
  • the computer can be adapted to control automatic changes to the light intensity output at given time points.
  • the illumination device can comprise a tubular lamp, for instance a fluorescence tube or a phosphorescence tube.
  • the tubular lamp is arranged to the side of the sample holding position to illuminate the sample at least partially from the side.
  • the tubular lamp is arranged above and set off to the side of the sample holding position to illuminate the sample at least partially from the top and at least partially from the side.
  • the sample holder comprises a plurality of sample holding positions arranged in the sample holder in at least two parallel linear rows and is configured for holding a plurality of samples in said sample holding positions.
  • the illumination device comprises at least two tubular lamps, for instance fluorescence or phosphorescence tubes, arranged above and set off to the opposite sides of the sample holding positions essentially parallel to said rows of sample holding positions to illuminate the samples in each row at least partially from the top and at least partially from the side.
  • At least two of said sample holders are present, each of which comprises at least two tubular lamps arranged above a set of two opposite sides of the sample holding positions essentially parallel to said rows of sample holding positions to illuminate the samples in each row at least partially from the top and at least partially from the side.
  • the illumination device comprises a light-emitting diode (LED) .
  • the light-emitting diode can significantly reduce the space requirement of the illumination device so that a particularly compact design of the assembly can be achieved.
  • the LED can be arranged below the sample holding position to illuminate the sample at least partially from the bottom.
  • the sample holder can, for instance, comprise an opening or translucent material in the bottom of the sample holding position for providing a free optical light-path for the light of the LED to illuminate the bottom of the sample.
  • a particularly compact design of the assembly with good illumination of the sample is achieved.
  • an SMD-LED can be used.
  • a power LED or high-power LED can be used to obtain high
  • the sample holder comprises a plurality of sample holding positions in an arrangement comprising linear or circular rows and is configured for holding a plurality of samples in said sample holding
  • the illumination device comprises a
  • the mixing device is selected from a group consisting of a magnetic stirring system, an agitation system, and combinations thereof.
  • the magnetic stirring system can, for instance, employ a rotating magnetic field causing a stir bar in the sample to spin, thus stirring the sample.
  • the rotating magnetic field can, for instance, be created by a rotating magnet or a set of
  • the agitation system can, for instance, be configured for circular shaking, axial shaking, rocking or vibrating of the sample, and combinations thereof.
  • the inventors found that mixing of the sample in the sample holding position directly influenced the photosynthetic production of the substance in the sample as well as the accumulation of the produced substance in the headspace of the sample .
  • the temperature control device comprises a heating system for heating of the sample.
  • a heating system can, for instance, comprise a heating mat, a peltier unit, a water bath, a hot air unit, and combinations thereof.
  • the temperature control device can, for instance, comprise a temperature determining system for determining the temperature of the sample.
  • the temperature control device can also comprise a temperature feedback control, for example a proportional integral derivative (PID) controller.
  • PID proportional integral derivative
  • temperature control device can be further adapted for running a temperature program, which can, for instance, include automatically changing the incubation temperature of the sample, continuosly or step-wise, to a higher or lower incubation temperature at one or more time points during the sample cultivation.
  • a temperature program can, for instance, include automatically changing the incubation temperature of the sample, continuosly or step-wise, to a higher or lower incubation temperature at one or more time points during the sample cultivation.
  • the transfer unit comprises a sampling system configured for drawing an aliquot containing the substance from the headspace of the sample and introducing at least part of the aliquot into the analytical unit.
  • the sampling system can, for instance, comprise a gas- tight syringe, a sample loop, or combinations thereof.
  • the gas-tight syringe can be configured for drawing the aliquot from the headspace of the sample and directly injecting at least part of the aliquot into an injection port of the gas chromatograph .
  • the sample loop can be part of a balanced pressure sampling system. Alternatively, the sample loop can be part of a pressure loop sampling system. Such sampling systems can prevent substance carryover between two
  • the assembly is configured for transferring of the substance from the headspace of the sample at a sample temperature of 55 °C or lower.
  • a sample temperature of 55 °C or lower.
  • the assembly is configured for repeated automatic transferring and assaying of the volatile substance which has in its pure form a vapour pressure at 20 °C higher than 23.4 hPa but lower than 604 hPa.
  • the volatile substance can be a hydrocarbon-based compound.
  • the volatile substance can, for instance, be an alcohol.
  • the substance is ethanol.
  • the assembly comprises an
  • the autosampler including the sample holder with a plurality of said sample holding positions for holding a plurality of samples. Furthermore, the autosampler is configured for automatic transferring of the substance from each of the plurality of samples to the analytical unit.
  • the assembly further comprises a device adapted for controlling the repeated automatic transferring and assaying of the substance with the assembly.
  • the device can be a data processing unit, for example a computer.
  • the data processing unit can, for instance, be configured for assigning the sample with the sample holding position in the sample holder.
  • the processing unit can also be configured for addressing the sample in the sample holding position with the transfer unit. Moreover, the data processing unit can be configured for controlling and timing of the repeated automatic transferring and assaying of the substance from the sample. In certain embodiments, the data processing unit can be additionally configured for controlling the temperature control device, the illumination device, the mixing device, and combinations thereof .
  • the assembly is preferably configured for transferring of the substance from the headspace of the sample when the sample is contained in a gas-tight sample container.
  • the gas-tight sample container can be sealed with a septum comprising a self-sealing material.
  • the septum can, for instance, be included in a lid used for capping of the sample container.
  • the sample container can, for instance, be a sample vial, for example a gas-tight glass vial, such as a disposable glas vial for GC headspace analysis.
  • the sample container is preferably transparent, so that the photosynthetically usable radiant energy can pass through to the sample.
  • the septum can, for instance, comprise silicon.
  • the assembly can be configured for repeated automatic transferring and gas chromatographic assaying of oxygen from the headspace of the sample in addition to the volatile substance.
  • the assembly is configured for repeated automatic transferring and gas
  • Carbon dioxide and/or oxygen can, for instance, be determined from the same aliquot as the substance.
  • analytical unit can, for instance, be adapted for parallel gas chromatographic assaying of the substance, carbon dioxide and/or oxygen.
  • a direct correlation between the substance, carbon dioxide consumption and/or oxygen evolution in the sample headspace can be obtained to gain important insights into the growth characteristics and photosynthetic activity of cells.
  • the specific carbon conversion rate can be derived by directly correlating the carbon
  • the photon conversion rate can be derived for determining the proportion of photons that is used by the cells for production of the substance and biomass, respectively, and the proportion of photons that is not photosynthetically used.
  • the illumination device comprises a light source arranged at the inner side of a light-diffusing pane in an arrangement wherein the light emitted from the light source is scattered through the outer side of the light-diffusing pane, thereby acting as a diffuser by uniformly scattering the illumination from the light source over a surface area of the pane.
  • the terms “inner side” and “outer side” are to be understood as to describe the relative orientation of the light source and the light-diffusing pane in relation to each other only.
  • the invention provides for arrangements of the light source and the light-diffusing pane wherein the light source is arranged beneath the light-diffusing pane and the light is scattered through the top of the pane.
  • the light source can be arranged above the light-diffusing pane and the light is scattered through the bottom of the pane.
  • the sample holder preferably comprises a plurality of sample holding positions with an open and/or translucent bottom and is arranged above the light-diffusing pane, for illuminating the sample holding positions with photosynthetically usable radiant energy from the bottom.
  • a light table comprising the illumination device can be present.
  • the light source can, for instance, comprise one or more lamps, for example fluorescence or phosphorescence lamps.
  • the light source can comprise a plurality of LEDs, for instance SMD-LEDs or high-power LEDs.
  • the light-diffusing pane can comprise glass or transparent plastic material, such as acrylic glass.
  • the light-diffusing pane can comprise a light- scattering material or structure, for instance a frosting or a light-scattering grating.
  • the illumination device is configured to provide at least two areas of different illumination intensity simultaneously.
  • the illumination device and the sample holder are in an arrangement comprising at least one sample holding position in each of the at least two areas of different illumination intensity. In this way, parallel illumination of different sample holding positions with different illumination intensity is realised.
  • this invention provides a method for repeated automatic transferring and assaying of a volatile substance having in its pure form a vapour pressure at 20 "Celsius lower than 604 hPa from a sample comprising a liquid phase and a headspace, the liquid phase containing living cells undergoing photosynthesis, the cells thereby
  • the method comprises the steps a) providing the sample in a sample container in the sample holder,
  • method step b) , method step c) , method step e) or method step f) each independently further comprises mixing of the sample. In this way, a homogenous suspension of the cells and distribution of medium components in the sample can be achieved throughout the incubation.
  • the mixing can assist in achieving equilibrium of the substance between the liquid phase and the headspace of the sample, thus improving the accuracy of the assaying of the substance from the headspace of the sample.
  • carbon dioxide and/or a bicarbonate source can be added to the sample
  • the sample container of method step a) is a gas-tight sample container comprising a cap with a pierceable septum, said septum comprising a self-sealing material.
  • method step c) and method step f) can comprise puncturing said pierceable septum with a sampling needle and releasing the aliquot through the sampling needle into the transfer unit, for instance the gas-tight syringe or the pressure-loop of the sampling system.
  • the technical advantage is that the sample container remains gas-tight after an aliquot from the sample headspace has been taken, so that the substance can continue accumulating in the headspace for further sampling steps.
  • the above-described method is preferably carried out using a sample temperature in method c) and method step f) which essentially maintains the cells in the sample in a viable condition.
  • a viable condition as used herein shall mean that a temperature is maintained which does not have lethal effects on the cells.
  • temperature is adapted to allow continued growth of the cells and continued photosynthetic production of the substance.
  • At least 50% of the cells in the sample are maintained in a viable condition. More preferred are sample temperatures wherein at least 75% of the cells are maintained in a viable condition. Most preferred are sample temperatures that maintain at least 90% of the cells in a viable condition. The percentage of cells which are maintained in a viable condition can, for instance, be determined with a
  • fluorescence-based viability assay such as the fluorescein diacetate (FDA) assay or the chlorophyll auto-fluorescence assay.
  • FDA fluorescein diacetate
  • chlorophyll auto-fluorescence assay Another example is the 3- (4, 5-dimethylthiazol-2-yl) - 2 , 5-diphenyltetrazolium bromide (MTT) assay which can be used.
  • the sample temperature is 55 °C or lower.
  • sample temperature of 50 °C or lower. Even more preferred is a sample temperature of 45 °C or lower. The most preferred temperature is 40 °C or lower. Accordingly, the cells in the sample remain in a viable condition and are therefore available for further analyses requiring living cells, for instance the analysis of enzymatic activity, oxygen production or specific metabolite analyses.
  • At least one reference sample containing a predetermined concentration of the substance to be assayed is incorporated into the method, such that the reference sample is treated in the same way as said sample during method steps a) through g) . In this way, assaying results from the sample and the reference sample can be compared to derive the concentration of the substance in the sample .
  • the method further comprises a plurality of reference samples containing a plurality of predetermined concentrations of the substance to be assayed. In this way, a temperature-dependent calibration curve for a given substance can be generated. The inventors found that this method greatly enhances the accurate determination of the substance
  • the substance is a hydrocarbon- based compound.
  • the substance is a hydrocarbon- based compound.
  • the substance is an alcohol.
  • the substance is ethanol.
  • the substance has in its pure form a vapour pressure at 20 °C which is higher than 23.4 hPa but lower than 604 hPa.
  • the aliquot of method steps c) and f) further contains carbon dioxide in addition to the substance, and the assaying in method steps d) and g) comprises detection of carbon dioxide.
  • the aliquot of method steps c) and f) further contains oxygen in addition to the substance, and the assaying in method steps d) and g) comprises detection of oxygen.
  • both carbon dioxide and oxygen are present in said aliquots, and the assaying in method steps d) and g) comprises detection of both carbon dioxide and oxygen. In this way, parallel determination of the substance, carbon dioxide and/or oxygen can be achieved. For instance, parallel time- courses of the photosynthetic production of the substance, the photosynthetic production of oxygen and/or the photosynthetic consumption of carbon dioxide by the cells can be obtained.
  • the conversion rate can be derived by directly correlating the carbon fixation rate and/or the oxygen production rate with the production rate of the substance.
  • the photon conversion rate can be derived for determining the proportion of photons that is used by the cells for production of the substance and biomass, respectively, and the proportion of photons that is not photosynthetically used.
  • Figure 1A and IB depict a schematic representation of an exemplary assembly in overview and in detail view of the sample holder.
  • Figure 2A and 2B schematically show an example of an LED-based light table.
  • Figure 3A and 3B show examples of ethanol calibration curves generated from GC headspace measurements of ethanol reference samples at 37 °C.
  • Figures 4A and 4B show examples of time courses of the
  • Figures 5A and 5B show examples of time courses of the
  • FIGS. 6A and 6B show examples of time courses of the
  • FIGS. 7A and 7B show examples of light saturation behaviour of the photosynthetic production of ethanol by a metabolically enhanced cyanobacterium under different illumination
  • FIG. 1A An example of an assembly that can be used in this experiment is schematically shown in overview in Figure 1A.
  • Figure IB shows, in more detail, the exemplary arrangement of sample holder, illumination device, mixing device, temperature control device and transfer unit within the assembly of Figure 1A.
  • the assembly can, as depicted, comprise an autosampler (AS) including the sample holder (SH) with a plurality of sample holding positions (SHP) .
  • the autosampler can, for example, include an XYZ-robot (PR) for automatic addressing of the sample holding positions and transferring of the substance from each of the plurality of samples to the analytical unit (AU) .
  • a gas-tight syringe (GTS) can be used for transferring of the substance from the sample in the sample holding
  • a Shimadzu PAL LHS2- SHIM/AOC-5000 autosampler can be used.
  • the autosampler can have more than one sample holder for increasing the sample holding capacity of the assembly.
  • two sample holders for holding a plurality of sample containers in the sample holding positions can be present.
  • Each sample holder can be illuminated with an illumination device comprising two tubular lamps (TFL) , for instance fluorescence or
  • NARVA fluorescence lamps BIO vital LT24WT5/958HQ
  • the lamps can be equipped with a dimmer for adjusting the illumination
  • the mixing device (MS) for mixing of the samples in the sample holder can, as in the given example, be arranged below the sample holders.
  • a magnetic stirrer can be used.
  • a suitable magnetic stirrer is for example the IKA R05 power.
  • the temperature control device (HM) can, for instance, be arranged between the sample holder and the mixing device.
  • the temperature control device can be equipped with a temperature regulator.
  • a heating mat coupled to a temperature regulator can be used.
  • a suitable combination is, for instance, the heating mat KM-SM3 of Mohr & Co in
  • the analytical unit comprises a gas chromatograph (GC) , which can, for instance, be equipped with a flame ionization
  • the gas chromatograph can be connected to a helium carrier gas. Hydrogen and artificial air can be
  • oxidizer gas can be connected as fuel gas and oxidizer gas, respectively, for the flame ionization detector.
  • a suitable gas chromatograph is the Shimadzu GC-2010 with FID.
  • the oxidizer air can be artificially generated.
  • the generator WGAZA50 from Science Support can be used.
  • chromatograph can be equipped with a capillary suitable for chromatographic separation of complex gaseous compositions from the headspace of the sample.
  • a capillary suitable for chromatographic separation of complex gaseous compositions from the headspace of the sample For instance, a medium bore capillary with a length of 30 m, internal diameter of 0.32 mm and film thickness of 1.8 ym can be used.
  • a suitable capillary is the FS-CS-624 from the GC supplier Chromatographie Service GmbH.
  • Hybrid clones are raised on BG11 plates containing 5 ⁇ of the inducing agent and on BG11 plates without supplementation of the inducing agent. Twelve individual samples are prepared by scratching six individual clones from the BG11 plates with inducing agent and six individual clones from the BG11 plates without inducing agent, respectively. An individual sample is prepared from each of the clones by resuspending the corresponding clone in marine BG11 liquid medium (mBGll) .
  • mBGll marine BG11 liquid medium
  • the cell density in the samples is then adjusted to an optical density at 750 nm of approximately 1.0.
  • Two millilitres of each sample are then filled into gas-tight GC vials for headspace autosampling with a nominal volume of 20 millilitres.
  • the sample headspace is supplemented with 3 millilitres CO2 ⁇
  • the vials are tightly closed with caps with self-sealing silicone septa and placed into the autosampler rack which is temperature controlled at 37°C.
  • Reference samples are prepared as 2 millilitre aliquots with 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5 and 10 mg/ml ethanol in 35 psu sodium cloride. Reference samples are placed into the same 20 ml sample containers with self-sealing silicon septum caps for headspace autosampling . For each reference sample at least six measurements are applied. After the measurements, the resulting peak areas of the reference samples are used for generating two calibration curves, the first in the concentration range from 0.005 to 0.5 mg/ml ethanol and the second one for the concentration range from 0.5 to 10 mg/ml ethanol. The calibration curves have to fulfil linearity .
  • the sample incubation temperature in the autosampler is adjusted to 37 °C.
  • the illumination is set at 100 ⁇ .
  • the magnetic stirrer is configured for interval mixing of the samples, with cycles of 2 minutes mixing at 400 rpm, followed by 90 minutes without mixing.
  • An automated process follows, wherein after approximately 0, 8.5, 17, 25.5, 34 and 42.5 hours aliquots of 500 ⁇ of the headspace of the samples are automatically drawn with the gas-tight headspace syringe and injected via the injection port into the gas chromatograph for analysis. Before each headspace autosampling, the mixing is changed for 10 min to continuous mixing with 750 rpm at 37°C incubation temperature.
  • the syringe temperature is set at 70 °C.
  • the fill speed is 250 ⁇ per second, following an initial lag time of 1 second after the septum of the samples has been pierced by the syringe needle.
  • the injection of the aliquot into the gas chromatograph happens with an injection speed of 500 ⁇ per second. Afterwards, the syringe flushes for 3 minutes with air to prevent sample carryover between two injections.
  • the gas chromatograph runtime is 4 minutes and 30 seconds.
  • the injection temperature on the gas chromatograph is 230 °C.
  • the column temperature is 60 °C. Detection is
  • the flame ionization detector at 250 °C process temperature.
  • the makeup gas is nitrogen at 30 ml per minute, the fuel gas is hydrogen at 35 ml per minute and the oxidizer gas is artificial air at 400 ml per minute.
  • the final optical density at 750 nm of the samples is measured and an average cell density for each sample is determined by calculating the arithmetic mean of the optical density at the starting point and the optical density at the end point of the process divided by two.
  • FIG. 2A shows an alternative embodiment of the illumination device in combination with a sample holder for a plurality of samples.
  • a light table (LT) is used.
  • the light table can for example be assembled by arranging an array of light emitting diodes (LED) around the edges beneath a light- diffusing pane, for instance a translucent glass plate (GP) , which scatters the light emitted from the LEDs uniformly through its top plane.
  • the LEDs can, for instance, be SMD- LEDs.
  • the LEDs can be power LEDs or high-power LEDs.
  • 24 power LEDs can be arranged below the edge of the long side, and 18 power LEDs below the edge of the short side of a rectangular glass plate measuring 24 x 17 cm.
  • the glass plate can, for instance, comprise acrylic glass.
  • the glass plate can, for instance, comprise a grating (LSG) which assists in scattering the light emitted from the LEDs
  • the grating can, for
  • the grating can, for example, comprise a polymer material.
  • the sample holder (SH) can, for example, have an open bottom in the sample holding positions (SHP) and is placed on top of the light table, so that the light emitted through the top of the light table can illuminate the samples from the bottom.
  • Figure 2B shows in more detail an exemplary schematic arrangement incorporating the light table (LT) and sample holder (SH) loaded with sample containers (SC) in some of the sample holding positions (SHP) within an autosampler.
  • the mixing device can, for instance, be arranged between below the light table (MS) .
  • the temperature control device (HM) can, for instance, be arranged between the light table and the mixing device.
  • a heating mat can be used as the temperature control device with the light table.
  • a magnetic stirrer can for example be used as the mixing device with the light table.
  • Figure 4A and 4B show the analysis results of the six
  • Synechococcus PCC7002 which have been raised on the BGll plates without inducing agent.
  • the samples were simultaneously present in the autosampler and processed in parallel by assaying the headspace of each of the samples after 0, 8.5, 17, 25.5, 34 and 42.5 hours.
  • the assaying results were used for compiling a time course of the photosynthetic production of ethanol and acetaldehyde by each of the individual clones of the hybrid strain over the monitored 42.5 hours of
  • FIG. 4A displays the ethanol production in ⁇ 6 ethanol (v/v) over the cultivation time for each of the six clones.
  • Figure 4B shows the results of the corresponding acetaldehyde production in % (v/v) over the cultivation time.
  • This strain exhibits a lag-phase in the ethanol production of approximately 10 hours, after which the production rate significantly increases.
  • the lag phase results from the pre- cultivation of the clones on the plates in the absence of inducer, leading to complete repression of recombinant pdc and adh activity in the early stages of the liquid culture.
  • the lag-phase correlates well with the delayed accumulation of acetaldehyde in the samples during the first 10 hours of cultivation.
  • the averaged slope of the ethanol concentration time-course roughly corresponds to an ethanol production rate between 0.011% and 0.012% (v/v) per OD and day for the
  • Figure 5A and 5B show the analysis results of the
  • Figure 5A displays the ethanol product ⁇ in "6 ethanol (v/v) over the cultivation time for each of the six pre-induced clones.
  • Figure 5B shows the corresponding results of the acetaldehyde production in % (v/v) over the cultivation time. Due to the pre-induction of the plate-cultures by supplementation of 5 ⁇ inducing agent, almost no lag-phase in the ethanol production is present. A more constant ethanol production is achieved, as can be derived from the almost linear slope of the
  • EXAMPLE 2 The following example describes the screening of heat-tolerant ethanologenic cyanobacterial hybrid strains by quantification of ethanol in the liquid phase of cyanobacterial cultures under simulated heat stress conditions with an assembly and method according to the present invention.
  • Clones of the hybrid strains are raised on BG11 plates. Two individual clones are picked from each hybrid strain. Each clone is used to prepare an individual sample by resuspending the corresponding clone in mBGll liquid medium. Ethanol production in the samples is triggered by induction of the inducible promoter driving over-expression of the recombinant pyruvate decarboxylase gene. The cell density in the samples is then adjusted to an optical density at 750 nm of approximately 1.3. Two millilitres of each sample are then filled into gas-tight GC vials for headspace autosampling with a nominal volume of 20 millilitres. The sample headspace is supplemented with 3 millilitres CO 2 . The vials are tightly closed with caps with self-sealing silicone septa and placed into the autosampler rack.
  • temperature control device adapted to run a temperature profile with a base incubation temperature of approximately 36 °C interspersed with temperature stress peaks of approximately 46 °C after 10 hours and 35 hours cultivation time using a heating rate of approximately 2 °C/hour.
  • the headspace of each of the samples is assayed after approximately 1, 4, 17, 28, 39, 51, 59 and 65 hours.
  • the assaying results were used for compiling a time-course of the photosynthetic production of ethanol by the individual clones of each of the three hybrid strains under heat-stress conditions. Results :
  • Figure 6A shows the analysis results of the six individual clones. Displayed is the ethanol product ⁇ in "6 ethanol (v/v) per sample OD over the cultivation time. Graphs with square and diamond marker points represent the results from the hybrid strain HS1, graphs with triangular and cross marker points represent the results from the hybrid strain HS2 and graphs with circle and plus marker points mark the results from the third hybrid strain HS3.
  • Figure 6B shows the recorded temperature profile of the temperature control device. All three strains exhibit a similar ethanol production during the first 15 hours of cultivation. In the course of the following heat stress conditions, however, higher ethanol production is achieved with hybrid strains HS1 and HS2 than with hybrid strain HS3. The highest thermo-tolerance of ethanologenesis is observed in the two clones of hybrid strain HS1. Thus,
  • cyanobacterial hybrid strain by quantification of ethanol in the liquid phase of cyanobacterial cultures under different illumination conditions with an assembly and method according to the present invention.
  • Experimental setup Essentially as described in example 1, but wherein the light table shown in figures 2A and 2B is used as the illumination device.
  • the light table is subdivided into eight distinct zones of different sample illumination intensity by inserting dark filters with different light transmission properties between the top of the light table and the sample holding positions in the sample rack. In this way, distinct sample holding positions with an illumination intensity of 2, 33, 34, 75, 81, 165, 324 and 400 ⁇ are generated.
  • a cyanobacterial hybrid strain has been generated by
  • the cell density in the sample is then adjusted to an optical density at 750 nm of approximately 1.0.
  • Eight individual samples are then prepared from the master sample by aliquoting two millilitres each of the master sample into eight individual gas-tight GC vials for headspace autosampling with a nominal volume of 20 millilitres.
  • the sample headspace is supplemented with 3 millilitres CO 2 .
  • the vials are tightly closed with caps with self-sealing silicone septa and placed into the different illumination zones of the adapted light table and autosampler rack so that every sample is illuminated with a different illumination intensity.
  • Figure 7A shows the specific ethanol production in % (v/v) per OD of the eight differently illuminated samples over the cultivation time.
  • Figure 7B shows the plot of the

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Abstract

Cette invention concerne un ensemble conçu pour un transfert et un dosage automatique répété d'une substance volatile issue d'un échantillon contenant des cellules vivantes subissant une photosynthèse, les cellules produisant de manière photosynthétique la substance, et un procédé d'utilisation de cet ensemble. Un tel ensemble et un tel procédé génèrent un débit d'échantillons plus économique en termes de temps et de coût, et une exhaustivité et une précision accrues des données d'échantillons acquises en comparaison des installations et des procédés techniques traditionnels.
PCT/EP2012/071757 2012-11-02 2012-11-02 Ensemble et procédé de dosage automatique répété d'une substance issue d'un échantillon contenant des cellules subissant une photosynthèse WO2014067584A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106872622A (zh) * 2017-02-06 2017-06-20 国网山东省电力公司招远市供电公司 一种带有防尘塞的油样色谱分析定量进样装置

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020006360A1 (en) * 2000-03-10 2002-01-17 Neal David M. Three stage needle for use with an autosampler
US20070184553A1 (en) * 2006-02-06 2007-08-09 Hartlein Thomas M Method of automatically regulating and measuring pressure during sampling and analysis of headspace gas
US20100062707A1 (en) 2008-09-05 2010-03-11 Huawei Technologies Co., Ltd. Frequency conversion device, method and system
US20100196982A1 (en) 2007-05-01 2010-08-05 Stephen Anderson Methods for The Direct Conversion of Carbon Dioxide Into a Hydrocarbon Using a Metabolically Engineered Photosynthetic Microorganism
US20110039323A1 (en) 2009-08-14 2011-02-17 Eric Lawrence Singsaas Isoprene Production
US20110151531A1 (en) 2008-03-03 2011-06-23 Joule Unlimited, Inc. Ethanol production by microorganisms
WO2011111050A2 (fr) * 2010-03-11 2011-09-15 Jacob Edrei Procédés de génération d'hydrogène
US20120238002A1 (en) * 2011-03-16 2012-09-20 Arizona Board Of Regents For And On Behalf Of Arizona State University Photobioreactors Comprising Membrane Carbonation Modules and Uses Thereof
WO2012145692A2 (fr) * 2011-04-21 2012-10-26 The Regents Of The University Of California Procédé basé sur la diffusion pour obtenir des hydrocarbures volatils produits par des microorganismes photosynthétiques dans des réacteurs à deux phases

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020006360A1 (en) * 2000-03-10 2002-01-17 Neal David M. Three stage needle for use with an autosampler
US20070184553A1 (en) * 2006-02-06 2007-08-09 Hartlein Thomas M Method of automatically regulating and measuring pressure during sampling and analysis of headspace gas
US20100196982A1 (en) 2007-05-01 2010-08-05 Stephen Anderson Methods for The Direct Conversion of Carbon Dioxide Into a Hydrocarbon Using a Metabolically Engineered Photosynthetic Microorganism
US20110151531A1 (en) 2008-03-03 2011-06-23 Joule Unlimited, Inc. Ethanol production by microorganisms
US20100062707A1 (en) 2008-09-05 2010-03-11 Huawei Technologies Co., Ltd. Frequency conversion device, method and system
US20110039323A1 (en) 2009-08-14 2011-02-17 Eric Lawrence Singsaas Isoprene Production
WO2011111050A2 (fr) * 2010-03-11 2011-09-15 Jacob Edrei Procédés de génération d'hydrogène
US20120238002A1 (en) * 2011-03-16 2012-09-20 Arizona Board Of Regents For And On Behalf Of Arizona State University Photobioreactors Comprising Membrane Carbonation Modules and Uses Thereof
WO2012145692A2 (fr) * 2011-04-21 2012-10-26 The Regents Of The University Of California Procédé basé sur la diffusion pour obtenir des hydrocarbures volatils produits par des microorganismes photosynthétiques dans des réacteurs à deux phases

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
K HANCKE ET AL: "Temperature effects on respiration and photosynthesis in three diatom-dominated benthic communities", AQUATIC MICROBIAL ECOLOGY, vol. 37, no. 3, 15 December 2004 (2004-12-15), pages 265 - 281, XP055071433, ISSN: 0948-3055, DOI: 10.3354/ame037265 *
NEDBAL L ET AL: "A Photobioreactor System for Precision Cultivation of Photoautotrophic Microorganisms and for High-Content Analysis of Suspension Dynamics", BIOTECHNOLOGY AND BIOENGINEERING, WILEY & SONS, HOBOKEN, NJ, US, vol. 100, no. 5, 1 August 2008 (2008-08-01), pages 902 - 910, XP002608893, ISSN: 0006-3592, [retrieved on 20080212], DOI: 10.1002/BIT.21833 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106872622A (zh) * 2017-02-06 2017-06-20 国网山东省电力公司招远市供电公司 一种带有防尘塞的油样色谱分析定量进样装置
CN106872622B (zh) * 2017-02-06 2020-12-08 嘉兴市大明实业有限公司 一种带有防尘塞的油样色谱分析定量进样装置

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