WO2014067584A1

WO2014067584A1 - Assembly and method for repeated automatic assaying of a substance from a sample containing cells undergoing photosynthesis

Info

Publication number: WO2014067584A1
Application number: PCT/EP2012/071757
Authority: WO
Inventors: Thomas ABTS; Heike Enke; Anika HANS; Matthias Steffen; Ulf Duehring
Original assignee: Algenol Biofuels Inc.
Priority date: 2012-11-02
Filing date: 2012-11-02
Publication date: 2014-05-08

Abstract

This invention provides an assembly configured for repeated automatic transferring and assaying of a volatile substance from a sample containing living cells undergoing photosynthesis, whereby the cells photosynthetically produce the substance, and a method for its use. Such an assembly and method generate a more time and cost efficient sample throughput and increased comprehensiveness and accuracy of acquired sample data compared to conventional technical set- ups and methods.

Description

Title of the invention

ASSEMBLY AND METHOD FOR REPEATED AUTOMATIC ASSAYING OF A SUBSTANCE FROM A SAMPLE CONTAINING CELLS UNDERGOING PHOTOSYNTHESIS

Field of the invention

This invention is related to the field of analysis of chemical compounds of interest produced by photosynthetically active cells .

Background of the Invention Chemical compounds, which derive from carbon fixation of carbon dioxide by photosynthetically active cells, are gaining importance in replacing products which have been

conventionally sourced from fossil fuels. A prominent task in this area is the identification and optimization of high performance phototrophic organisms which allow photosynthetic production of these chemical compounds in large scale. 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.

For instance, Anderson describes in US patent application US 2010/0196982 a method for isoprene assaying from bacterial cultures. Two ml of culture were incubated in sealed vials at an appropriate temperature with shaking for approximately 3 hours, after which the sample headspace is analyzed with a gas chromatography (GC) system that is highly sensitive to

isoprene. 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

sensitive detector. 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. The supernatant was then placed in a new tube and was analyzed for ethanol and acetaldehyde by use of a gas chromatograph equipped with a headspace analyzer and a flame ionization detector. 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. These methods are time and cost inefficient due to their labour intensiveness which impairs their suitability for assaying of a large number of samples with increased

throughput and economical use of sample resources. Therefore, there is a need for improved instrumentation and methods for assaying volatile compounds produced by

phototrophic cells which overcome or reduce some of the disadvantages of the above-mentioned methods. Summary of the Invention 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

position configured for holding the sample contained in a sample container in said sample holding position, 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, and

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

"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, with said

assembly. The method comprises the steps: a) providing the sample in a sample container in the sample holder,

b) incubating the sample by maintaining illumination and temperature required by the cells for undergoing

photosynthesis ,

c) maintaining a sample temperature suitable for providing the substance in the headspace of the sample and

automatically transferring a first aliquot containing the substance from the headspace of the sample to the analytical unit,

d) assaying the first aliquot in the analytical unit, e) continuing incubating said sample in the sample holder by maintaining illumination and temperature required by the cells for undergoing photosynthesis,

f) maintaining a sample temperature suitable for providing the substance in the headspace of the sample and

automatically transferring a second aliquot containing the substance from said sample to the analytical unit, g) assaying the second aliquot in the analytical unit.

Description of the Invention

In a first aspect, this invention provides 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, comprising a sample holder with at least one sample holding

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,

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.

As used herein, "repeated" automatic transferring and assaying shall have the meaning that, from a given sample, the

substance is transferred and assayed at least twice. In addition, the automatic transferring and assaying occurs at least at two different time points during incubation of the cells in the sample.

As used herein, "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. In

particular, the vapour pressure at 20 "Celsius shall be understood as a substance characteristic of the pure

substance, whereas the 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. A substance having in its pure form a vapour

pressure at 20 "Celsius lower than 604 hPa is essentially liquid at 20° Celsius. As used herein, the "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.

As used herein, "photosynthetically usable radiant energy" shall mean any spectral composition including wavelengths and intensities sufficient for the cells to undergo

photosynthesis .

As used herein, "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 . Under certain circumstances, the term "automatic" 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

transferring and assaying of the substance has been completed.

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 is 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

illuminating the sample with photosynthetically usable energy directly at the site of sampling is realised. Therefore, widely automated processes are achieved which require less personnel attendance than conventional technical set-ups. The result is a higher sample throughput with increased time and cost efficiency and, ultimately, reduced overhead costs of the process . In addition, the inventor's achievement of configuring the assembly for repeated sampling of a substance from the same single sample in the presence of living cells, as opposed to conventional methods using end-point measurements after killing or removing of the cells, preserved the sample for further analyses requiring living cells, for instance the analysis of enzymatic activity or oxygen production. 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. In addition, 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.

In one embodiment of the invention, the sample holder

comprises a plurality of sample holding positions and is configured for holding a plurality of samples contained in a plurality of sample containers in said sample holding

positions. One example of a sample holder is a device which avoids undesired moving, e.g. tilting or tipping over, of the sample container in the sample holding position. In

particular, the sample holder avoids moving of the sample container during mixing of the sample and automatic

transferring of the substance from the sample. Preferably, 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. In one embodiment, the plurality of sample holding positions is arranged in the sample holder in linear rows. For instance, the sample holding positions can be arranged in two or more parallel rows of essentially rectangular arrangement. In another embodiment, the plurality of sample holding

positions is arranged in circular rows. For example, the sample holding positions are arranged in concentric circles. The sample holding positions can also be helically arranged.

In another embodiment of the invention, the illumination device provides at least 10 μΕ 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

photosynthesis and thereby produce the substance whilst the sample container is in the sample holding position. It is not necessary to remove the sample from the assembly for

illumination. Preferably, the illumination device provides at least 50 μΕ 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 μΕ m^~2 s^-1 photon flux up to at least 500 μΕ m^~2 s^-1 photon flux.

In yet another embodiment of the invention, the illumination device comprises a dimmer for varying the intensity of the light output. One example is a dimmer with which the

illumination can be specifically adjusted to the

photosynthetic needs of a particular cell type. Another example is a dimmer configured for changing the illumination intensity in the sample holding position over time. For instance, the dimmer is configured for automatically changing the illumination intensity in the sample holding position over time. One example is 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. For example, light-dark-cycles can comprise 12 hours of illumination followed by 12 hours of darkness. A dimmer can be computer-controlled. For instance, 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. In certain embodiments, the tubular lamp is arranged to the side of the sample holding position to illuminate the sample at least partially from the side. In preferred embodiments, 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. With these assemblies, particularly efficient and uniform illumination of one or more samples in the sample holder is accomplished .

In one preferred embodiment, 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. In this configuration, efficient and uniform

illumination of a plurality of samples is achieved, thus increasing the sample capacity of the assembly. In certain examples, 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.

Also provided is an assembly wherein 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. For example, 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. In this arrangement, a particularly compact design of the assembly with good illumination of the sample is achieved. For example, an SMD-LED can be used. A power LED or high-power LED can be used to obtain high

illumination intensity. The LED can, for instance, be combined with a material which assists in guiding the light from the LED to the sample. In yet another embodiment, 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

positions, wherein the illumination device comprises a

plurality of light-emitting diodes arranged below the sample holding positions, and wherein at least one light-emitting diode is assigned per one sample holding position to

illuminate the sample in said sample holding position at least partially from the bottom. This configuration realizes an energy and space efficient design with equally good

illumination of each of the plurality of samples. In certain embodiments of the invention, 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

stationary electromagnets. 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 .

In one embodiment, 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. The

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. With these assemblies, versatile and accurate thermostatisation of the sample is accomplished.

Also provided is an embodiment wherein 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

consecutive transfer operations during the repeated sampling and transferring.

In one embodiment, the assembly is configured for transferring of the substance from the headspace of the sample at a sample temperature of 55 °C or lower. Preferred is a sample

temperature of 50 °C or lower. Even more preferred is a sample temperature of 45 °C or lower. The most preferred sample temperature is 40 °C or lower. The inventors found that an assembly configured for working at these temperatures provided unexpectedly accurate GC headspace analyses of the substance whilst at the same time circumventing thermal killing of the cells in the sample, so that repeated automatic transferring and assaying from the same sample could be realized. In a preferred embodiment, 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. In one preferred example, the substance is ethanol. The inventors found that unexpectedly accurate GC headspace analyses of ethanol can be performed with the assembly of the present invention at surprisingly low temperatures which circumvent thermal killing of the cells in the sample, whereas the applied art typically teaches ethanol headspace analysis at temperatures lethal for the cells in excess of 60 °C.

In yet another embodiment, the assembly comprises an

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.

In certain embodiments of the invention, the assembly further comprises a device adapted for controlling the repeated automatic transferring and assaying of the substance with the assembly. For instance, 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 data

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.

Also provided are embodiments, wherein the assembly is

configured for repeated automatic transferring and gas

chromatographic assaying of carbon dioxide from the headspace of the sample in addition to the volatile substance.

Furthermore, 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. In preferred embodiments, the assembly is configured for repeated automatic transferring and gas

chromatographic assaying of both carbon dioxide and oxygen from the headspace of the sample in addition to the volatile substance. Carbon dioxide and/or oxygen can, for instance, be determined from the same aliquot as the substance. The

analytical unit can, for instance, be adapted for parallel gas chromatographic assaying of the substance, carbon dioxide and/or oxygen. In this way, 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. For example, the specific carbon conversion rate can be derived by directly correlating the carbon

fixation rate, i.e. the time-dependent decrease of carbon dioxide content in the sample, and/or the oxygen production rate, i.e. the time-dependent oxygen increase, with the production rate of the substance. By further considering the illumination intensity, 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.

In certain variants of the invention, 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. In this context, 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. For instance, 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. In another example, the light source can be arranged above the light-diffusing pane and the light is scattered through the bottom of the pane. In some of these variants, 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. For example, 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. With these configurations,

particularly uniform illumination of a plurality of sample holding positions is achieved. This ensures comparability and reproducibility of assaying results obtained from samples accommodated in said plurality of sample holding positions.

In a further variant, the illumination device is configured to provide at least two areas of different illumination intensity simultaneously. In some of these variants, 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. For

example, dark filters with different light transmission characteristics can be arranged between the illumination device and the sample holding positions for providing areas of different illumination intensity. In some preferred variants, the illumination device comprises a plurality of areas of different illumination intensity and is in an arrangement with the sample holder comprising at least one sample holding position in each of the plurality of areas of different illumination intensity. In a second aspect, 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

photosynthetically producing the substance, using any of the assemblies described above. The method comprises the steps a) providing the sample in a sample container in the sample holder,

b) incubating the sample by maintaining illumination and temperature required by the cells for undergoing photosynthesis ,

c) maintaining a sample temperature suitable for providing the substance in the headspace of the sample and automatic transferring a first aliquot containing the substance from the headspace of the sample to the analytical unit,

f) maintaining a sample temperature suitable for providing the substance in the headspace of the sample and automatic transferring a second aliquot containing the substance from said sample to the analytical unit, and g) assaying the second aliquot in the analytical unit. The method steps f) and g) can be repeated by automatically transferring and assaying at least a third or further aliquot from the same sample. In this way, a time-course of the photosynthetic production of the substance by the cells contained in said sample can be obtained. The inventors realised that the single end-point measurements commonly used in methods of the applied art reflect the situation in the sample at a given instant only and therefore impaired both the accuracy of these methods as well as the economical use of samples and resources. In contrast, compiling time-resolved courses of the photosynthetic production of the substance with the methods of the present invention significantly increased the comprehensiveness of data obtained from a single sample. This increased information content improved the

characterisation of candidate organisms by providing valuable details about the cells producing the substance as well as the production course, which were previously inaccessible with conventional end-point measurements, such as determination of lag phases, local and global production maxima or minima, respectively, inhibitory product concentrations and specific production rates. The inventors found that the method steps f) and g) can be repeated over a duration of at least 72 hours.

In a preferred embodiment, 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.

Moreover, 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.

For efficient photosynthesis of the cells, carbon dioxide and/or a bicarbonate source can be added to the sample

container. Carbon dioxide and/or a bicarbonate source are preferably added in method step a) . For instance, CO2 gas can be injected into the gas phase of the sample container using a syringe. The bicarbonate source can, for instance, comprise sodium hydrogen carbonate NaHCC>3. In preferred embodiments, 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. Furthermore, 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. In particular, a viable condition as used herein shall mean that a temperature is maintained which does not have lethal effects on the cells. In particular, the

temperature is adapted to allow continued growth of the cells and continued photosynthetic production of the substance.

Preferably, 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. Another example is the 3- (4, 5-dimethylthiazol-2-yl) - 2 , 5-diphenyltetrazolium bromide (MTT) assay which can be used. In particular, the sample temperature is 55 °C or lower.

Preferred is a 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.

In a preferred embodiment, 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 .

In a variant, 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

concentration in the sample.

In a variant of the method, the substance is a hydrocarbon- based compound. In certain variants of the method, the

substance is an alcohol. In preferred variants, the substance is ethanol. Moreover, according to a further variant of the method, 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.

Given that, for volatile substances with these

characteristics, the state of the art commonly teaches

parameters for the headspace analysis which would entail thermal killing of cells in a bacterial culture, it is most surprising for those of skill in the art that highly accurate GC headspace analyses of these substances can be performed at temperatures which allow the presence of photosynthetically active, living cells in the sample. In particular, it is most unexpected to those of skill that ethanol can be accurately determined from the sample headspace of a photosynthetically active bacterial culture at a sample temperature of 55 °C or lower, and even more so at 40 °C or lower according to certain embodiments of the present invention.

In certain embodiments of the method, 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. In other certain embodiments, 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. In some embodiments, 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 inventors found that with these methods important insights into the growth characteristics and photosynthetic activity of cells can be gained. For example, the specific carbon

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. In addition, by further considering the illumination intensity, 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.

Detailed Description of Embodiments

In the following, certain embodiments of the invention will be explained in more detail with reference to figures and

experimental data. The figures and examples are not intended to be limiting with respect to specific details. Individual features can be identified with a reference numeral. This does not exclude that more than one of such feature can be present. Moreover, the features depicted in the figures are not

necessarily drawn to scale. 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

photosynthetic production of ethanol and acetaldehyde by a metabolically enhanced cyanobacterium without pre-induction of the plate-culture.

Figures 5A and 5B show examples of time courses of the

photosynthetic production of ethanol and acetaldehyde by the same metabolically enhanced cyanobacterium with pre-induction of the plate-culture. Figures 6A and 6B show examples of time courses of the

photosynthetic production of ethanol with cyanobacterial hybrid strains under simulated temperature stress conditions. Figures 7A and 7B show examples of light saturation behaviour of the photosynthetic production of ethanol by a metabolically enhanced cyanobacterium under different illumination

conditions . EXAMPLE 1

The following example describes the quantification of ethanol in the liquid phase of a cyanobacterial culture with an assembly and method according to the present invention.

Experimental setup:

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

position to the analytical unit, where it is injected into the injection port (IP) . For instance, 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. For example, 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

phosphorescence tubes, which are arranged above and set off to the side of the sample holding positions in each sample holder. For instance, NARVA fluorescence lamps (BIO vital LT24WT5/958HQ) of 24 Watt can be used. The lamps can be equipped with a dimmer for adjusting the illumination

intensity. 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. For instance, 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. For example, 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

combination with the JUMO dTRON 316 temperature regulator. The analytical unit (AU) comprises a gas chromatograph (GC) , which can, for instance, be equipped with a flame ionization

detector (FID) . The gas chromatograph can be connected to a helium carrier gas. Hydrogen and artificial air can be

connected as fuel gas and oxidizer gas, respectively, for the flame ionization detector. One example of a suitable gas chromatograph is the Shimadzu GC-2010 with FID. The oxidizer air can be artificially generated. For example, the generator WGAZA50 from Science Support can be used. The gas

chromatograph can be equipped with 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.

Sample preparation:

Cyanobacterial hybrid strains have been generated by

metabolically enhancing Synechococcus PCC7002 for ethanol production by introducing a recombinant pyruvate decarboxylase gene and a recombinant synAdh alcohol dehydrogenase gene under the control of an inducible promoter. Hybrid clones are raised on BG11 plates containing 5μΜ 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) . Addition of 10 μΜ inducing agent, 50 mM TES pH 7.3 and 20 mM NaHC0₃ triggers ethanol production in the samples by induction of the inducible promoter driving over-expression of the recombinant pyruvate decarboxylase and alcohol dehydrogenase gene. 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 .

Procedure :

The sample incubation temperature in the autosampler is adjusted to 37 °C. The illumination is set at 100 μΕ . 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 μΐ 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 μΐ 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 μΐ 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

accomplished with 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.

After the final measurement, 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.

Afterwards, the average ethanol production rate per cell density is calculated.

Figure 2A shows an alternative embodiment of the illumination device in combination with a sample holder for a plurality of samples. In this case, 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. As an example, 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

uniformly through its top plane. The grating can, for

instance, be generated with laser ablation. 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. As an example, 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.

Results :

The results of the measurements of ethanol reference samples at 37 °C are summarised as ethanol calibration curves shown in Figure 3. Figures 3A shows the calibration curve generated for the concentration range from 0.001 to 0.02 mg/ml ethanol and Fig. 3B shows the calibration curve generated for the

concentration range of 0.02 to 1 mg/ml ethanol. Both

calibrations correspond to a linear function with Revalues >0.99, confirming the accuracy of ethanol headspace analysis in this temperature range.

Figure 4A and 4B show the analysis results of the six

individual clones of the cyanobacterial hybrid strain

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

cultivation. Figure 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

individual clones. Figure 5A and 5B show the analysis results of the

corresponding six individual clones of the cyanobacterial hybrid strain Synechococcus PCC7002 which have been raised on the BG11 plates supplemented with 5 μΜ of the inducing agent to establish inducing conditions already during the plate- culture stage. 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 the strain in each of the individual clones over the monitored 42.5 hours of cultivation. Figure 5A displays the ethanol productϊοπ 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 μΜ 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

consolidated time-courses of the photosynthetic ethanol production by the pre-induced clones. The acetaldehyde level is rapidly increasing in the early stages of the liquid cultures and remains afterwards almost constant over the monitored cultivation time. Under these conditions, the averaged slope of the ethanol concentration time-course roughly corresponds to an ethanol production rate between 0.013% and 0.016% (v/v) per OD and day for the individual clones. This corresponds to a 20-30% increased ethanol

production rate compared to the average production rate obtained with the cells that were not pre-induced (Figure 4A) . Thus, the lag-phase in ethanol production observed with these hybrid strains of Synechococcus PCC7002 in the liquid culture stage can be compensated by establishing pre-induction

conditions with inducing agent already during the plate- culture stage. The compilation of the accurate time-course of photosynthetic ethanol production according to the present invention therefore allowed identification of a delayed response of the inducible promoter system in the Synechococcus PCC7002 hybrid strains, which provides economically important insights into the ethanol production with these hybrid

strains .

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.

Experimental setup:

Essentially as described in example 1.

Sample preparation Three different cyanobacterial hybrid strains HS1-HS3 have been generated by metabolically enhancing Synechocystis 6803 for ethanol production by introducing a recombinant pyruvate decarboxylase gene under transcriptional control of an

inducible promoter and a recombinant synAdh alcohol

dehydrogenase gene under transcriptional control of a

constitutive promoter. 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 CO₂. The vials are tightly closed with caps with self-sealing silicone septa and placed into the autosampler rack.

Procedure : Essentially as described in example 1, but with the

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 productϊοπ 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,

obtaining a time-course of the ethanol production from

ethanologenic hybrid strains directly during simulated heat stress conditions in the sample can be a useful means for the screening and optimization of thermo-tolerant production strains . EXAMPLE 3

The following example describes the determination of the light saturation characteristics and the optimum illumination intensity for photosynthetic ethanol production with a

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. In addition, 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 μΕ are generated.

Sample preparation:

A cyanobacterial hybrid strain has been generated by

metabolically enhancing Synechococcus PCC7002 for ethanol production by introducing a recombinant pyruvate decarboxylase gene and a recombinant synAdh alcohol dehydrogenase gene under transcriptional control of an inducible promoter. Clones of the hybrid strain are raised on BG11 plates. A master sample is prepared from a single clone by scratching an isolated clone from the plate and resuspending the clone in mBGll liquid medium. Ethanol production in the sample is triggered by addition of the inducing agent to the medium, driving over- expression of the recombinant pyruvate decarboxylase and alcohol dehydrogenase gene. 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 CO₂. 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. Procedure :

Essentially as described in example 1, with automatic GC headspace analysis of the samples after approximately 1, 4, 7, 10, 14 and 14 hours.

Results : 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

corresponding ethanol production rate in % (v/v) per OD and day over the sample illumination intensity. The ethanol production increases with increased illumination intensity, until in a range of about 75-100 μΕ illumination intensity an ethanol production maximum is reached with this clone. A decreased ethanol production is again observed for the highest of the tested illumination intensities. This example illustrates how obtaining a time-course of the ethanol production directly from samples incubated under different light conditions can be a useful means for determining the light saturation behaviour of ethanologenesis in hybrid strains, which ultimately allows optimising the cultivation conditions in the production scale and economising the production process.

Reference numeral

AS autosampler

AU analytical unit

PR XYZ-robot

GTS gas-tight syringe

GC gas chromatograph

FID flame ionisation detector

IP injection port

SH sample holder

SHP sample holding position

SC sample container

HM temperature control device

MS mixing device

TFL tubular lamp

LT light table

GP glass plate

LSG light scattering grating

LED light emitting diode

Claims

1. 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, comprising

- a sample holder with at least one sample holding

position configured for holding the sample contained in a sample container in said sample holding position,

- 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, and

- 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.

2. The assembly of the previous claim, wherein the sample holder comprises a plurality of sample holding positions and is configured for holding a plurality of samples contained in a plurality of sample containers in said sample holding positions .

3. The assembly of the previous claim, wherein the sample holding positions are arranged in the sample holder in linear or circular rows.

4. The assembly of any of the preceding claims, wherein the illumination device provides at least 10 μΕ m^~2 s^-1 photon flux of radiation usable by the cells for undergoing

photosynthesis .

5. The assembly of any of the preceding claims, wherein the illumination device comprises a dimmer for varying the

intensity of the light output.

6. The assembly of any of the preceding claims, wherein the illumination device comprises a tubular lamp.

7. The assembly of the previous claim, wherein the tubular lamp is arranged to the side of the sample holding position to illuminate the sample at least partially from the side, preferably wherein 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.

8. The assembly of any of the preceding claims,

- wherein 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, and

- wherein the illumination device comprises at least two tubular lamps arranged above and set off to 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.

9. The assembly of any of the preceding claims, wherein the illumination device comprises a light emitting diode (LED) .

10. The assembly of the previous claim, wherein the light emitting diode (LED) is arranged below the sample holding position to illuminate the sample at least partially from the bottom.

11. The assembly of any of the preceding claims,

- wherein 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 positions,

- wherein the illumination device comprises a plurality of light emitting diodes arranged below the sample holding positions, and

- wherein at least one light emitting diode is assigned per one sample holding position to illuminate the sample in said sample holding position at least partially from the bottom.

12. The assembly of any of the preceding claims, wherein the mixing device is selected from a group consisting of: a magnetic stirring system, an agitation system, or combinations thereof .

13. The assembly of the previous claim, wherein the agitation system is configured for circular shaking, axial shaking, rocking or vibrating of the sample, or combinations thereof.

14. The assembly of any of the preceding claims, wherein the temperature control device comprises a heating system for heating of the sample.

15. The assembly of the previous claim, wherein the heating system comprises a heating mat, a peltier unit, a water bath, a hot air unit, or combinations thereof.

16. The assembly of any of the preceding claims, wherein the temperature control device comprises a temperature determining system for determining the temperature of the sample.

17. The assembly of any of the preceding claims, wherein the temperature control device comprises a temperature feedback control.

18. The assembly of the previous claim, wherein the

temperature feedback control comprises a proportional- integral-derivative (PID) controller.

19. The assembly of any of the preceding claims, wherein 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.

20. The assembly of the previous claim, wherein the sampling system comprises a gas-tight syringe, a sample loop, or combinations thereof.

21. The assembly of any of the preceding claims, configured for transferring of the substance from the headspace of the sample at a sample temperature of 55 "Celsius or lower, preferably at a sample temperature of 50 "Celsius or lower 50°C, more preferred at a sample temperature of 45 "Celsius or lower, most preferred at a sample temperature of 40 "Celsius or lower.

22. The assembly of any of the preceding claims, configured for repeated automatic transferring and assaying of the volatile substance, wherein the substance is a hydrocarbon- based compound.

23. The assembly of any of the preceding claims, wherein an autosampler is present including the sample holder with a plurality of said sample holding positions for holding a plurality of samples configured for automatic transferring of the substance from each of the plurality of samples to the analytical unit.

24. The assembly of any of the preceding claims, configured for transferring of the substance from the headspace of the sample, wherein said sample is contained in a gas-tight sample container with a septum comprising a self-sealing material.

25. The assembly of any of the preceding claims, configured for repeated automatic transferring and assaying of carbon dioxide from the headspace of the sample in addition to the volatile substance.

26. The assembly of any of the preceding claims, configured for repeated automatic transferring and assaying of oxygen from the headspace of the sample in addition to the volatile substance .

27. The assembly of any of the preceding claims, wherein 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.

28. The assembly of any of the preceding claims, wherein the illumination device is configured to provide at least two areas of different illumination intensity simultaneously.

29. 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 photosynthetically producing the substance, using an assembly according to any of the preceding claims, comprising the method steps

a) providing the sample in a sample container in the sample holder,

photosynthesis ,

30. The method of the previous claim, comprising repeating method steps f) and g) by automatically transferring and assaying at least a third or further aliquot from the same sample for obtaining a time course of the photosynthetic production of the substance by the cells contained in said sample .

31. The method of claim 29 or 30, wherein method step b) , method step c) , method step e) or method step f) each

independently further comprises mixing of the sample.

32. The method of one of the claims 29-31, wherein during method step a) CO₂ and/or a bicarbonate source is added to the sample container.

33. The method of one of the claims 29-32, wherein the sample container is gas-tight and comprises a cap with a pierceable septum, said septum comprising a self-sealing material, and wherein method step c) and method step f) comprises puncturing said pierceable septum with a sampling needle and releasing the aliquot through the sampling needle into the transfer unit.

34. The method of one of the claims 29-33, wherein the sample temperature of method step c) and method step f) essentially maintains the cells in the sample in a viable condition.

35. The method of the previous claim, wherein the sample temperature is 55 "Celsius or lower, preferably 50 "Celsius or lower, more preferably 45 "Celsius or lower, most preferred 40 "Celsius or lower.

36. The method of one of the claims 29-35, further comprising at least one reference sample containing a predetermined concentration of the substance to be assayed, the reference sample being treated in the same way as said sample during method steps a) through g) .

37. The method of the previous claim, further comprising a plurality of reference samples containing a plurality of predetermined concentrations of the substance to be assayed.

38. The method of one of the claims 29-37, wherein the

substance is a hydrocarbon-based compound.

39. The method of one of the claims 29-38, wherein the

substance is ethanol.

40. The method of one of the claims 29-39, wherein the aliquot of method steps c) and f) further contains carbon dioxide in addition to the substance, and wherein the assaying in method steps d) and g) comprises detection of carbon dioxide.

41. The method of one of the claims 29-40, wherein the aliquot of method steps c) and f) further contains oxygen in addition to the substance, and wherein the assaying in method steps d) and g) comprises detection of oxygen.