Measurement method and apparatus for measuring gas concentration in a reaction vessel

Abstract

The present invention provides a method for measuring the concentration (Cz) of a target gas component (Z) in a gas mixture (12b) of known composition in a liquid solution within a reaction vessel (14), such as a bioreactor, in order to improve process control, particularly in cell culture and fermentation. The measurement method includes the steps of: a) preparing a measuring vessel (16) separated from the inside of the reaction vessel (14) by a gas-permeable separation membrane (18); b) measuring the gas density ρ, gas pressure p, and temperature T in the measuring vessel (16); and c) determining the concentration (Cz) of the target gas component (Z) from the values ​​measured in step b) and the molar masses of the gas components (X, Y, Z) of the gas mixture.

Description

Technical Field

[0001] The present invention relates to a method for measuring the concentration of a gas component to be measured in a gas mixture of a known composition (which changes during a reaction, for example, in a biological reaction (bioreaction) such as cell culture or fermentation), for example, in a reaction vessel designed as a bioreactor or the like. In particular, the present invention relates to a method for measuring the CO2 concentration in a reaction vessel in the food and beverage industry or a wastewater treatment facility, particularly in a bioreactor or reaction vessel. The present invention also relates to a reaction process carried out using this measurement method, particularly in a biological reaction process (bioreaction process) such as a cell culture process or a fermentation process, which is carried out in a reaction vessel such as a bioreactor. The present invention further relates to a measuring device for measuring the concentration of a gas component to be measured, particularly CO2, in a gas mixture of a known composition in a reaction vessel such as a bioreactor (which changes during a reaction, for example, in a biological reaction process). The present invention also relates to a biological reaction system particularly in a biological reaction including a reaction vessel in the form of a bioreactor, such a measuring device, and the use of the measuring device.

Background Art

[0002] For the technical background and the current state of the art, please refer to the following documents.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

[0004] Reference

[13] describes a biological reaction system for measuring CO2 concentration using known CO2 sensors, particularly the Sevelinghaus probe.

[0005] Reference

[14] describes a gas analysis system for analyzing the concentrations of multiple gas components in a gas mixture for energy supply, in which hydrogen is added to a component of natural gas or liquefied gas. For this purpose, infrared spectroscopy is used to determine (measure) the proportion of gas components that absorb infrared light, and gas density measurements are performed to determine the proportion of gas components that do not absorb infrared light from the results of infrared spectroscopy and gas density measurements. This method is implemented by energy suppliers to control the contamination of natural gas with hydrogen.

[0006] A preferred embodiment of the present invention relates to a method and apparatus for measuring gas concentration in a bioreactor or other reaction vessel for bioprocessing. In particular, the method and apparatus of the present invention are configured to determine the gas concentration of one or more gas components of a gas mixture contained in a liquid, in particular the gas concentration of dissolved gases.

[0007] Determining the gas concentration in a liquid medium is particularly important in bioreactions, and the CO2 content in bioreactors and especially in the liquids contained within them is of particular interest. A bioreaction is, for example, a process in which at least one component is transformed by cells, microorganisms, or their interactions. Other application areas of the methods and apparatus according to the embodiments of the present invention include chemical processes, processes in the food industry, particularly in beverage production (e.g., breweries), energy production processes, and food packaging processes.

[0008] According to reference [1], a bioreactor (often also called a fermenter) is a vessel in which specific microorganisms, cells, or small plants are cultured (fermented) under process conditions. Thus, the operation of a bioreactor is an application of biotechnology that utilizes or leverages biological processes (bioconversion, biocatalysis) in industrial facilities. Key factors that can be controlled or monitored in most bioreactors include the composition of the biocatalyst (also called the nutrient solution or substrate), the composition of the gas phase within the bioreactor, especially the oxygen supply, temperature, pH value, and sterility. The purpose of culturing in a bioreactor may be the extraction of cells or cellular components, or the extraction of metabolites. These are used, for example, as active ingredients in the pharmaceutical industry or as basic chemicals in the chemical or biochemical industries. The decomposition of chemical compounds may also occur in bioreactors, for example, during wastewater treatment at sewage treatment plants. A wide variety of organisms are cultured in bioreactors for various purposes. Examples of bioreactor use are shown in [1] and [3]. Bioreactors are often made of stainless steel or glass for multiple uses (in the biotechnology industry). However, recently, disposable plastic bioreactors have become more common, as shown in references [1], [2], and

[13] . In some embodiments of the invention, bioreactors can also be used in environmental and waste treatment technologies, for example, for biomass production or the decomposition of biodegradable waste, and can be formed from inexpensive materials such as concrete.

[0009] A bioreactor can contain up to three phases: a solid phase (biomass), a liquid phase (nutrient medium), and a gaseous phase (e.g., air, oxygen, carbon dioxide, nitrogen). Their distribution within the bioreactor is controlled by various means.

[0010] One factor affecting cell culture is the composition of the gases dissolved in the liquid phase within the bioreactor. These typically consist of oxygen, nitrogen, and carbon dioxide. To avoid ambiguity, the terms dissolved oxygen, dissolved nitrogen, and dissolved carbon dioxide (DO or DO2 or DCO2 or DCO2) are used. Generally, the composition of the target gas mixture within the bioreactor is known and is particularly influential in obtaining the desired results in cell culture, and is therefore adjusted or supplied.

[0011] In particular, the biotechnology industry relies on the precise control of CO2 concentration (level) within bioreactors such as incubators, fermenters, and other devices, which supports the growth and development of cells, tissues, and organisms.

[0012] The main reasons for measuring CO2 in a bioreactor during cell culture are as follows: High CO2 accumulation alters the quality and productivity of cells and products. Low CO2 concentrations also affect productivity. • A correlation has been established between biomass growth and CO2 emission rates (CER).

[0013] CO2 accumulation can be reduced by stripping. Dissolved CO2 easily passes through the cell membrane, affecting intracellular pH, which in turn directly impacts cellular mechanisms.

[0014] Therefore, online determination of CO2 levels in a bioreactor (process measurement) can significantly improve and / or optimize the process within the bioreactor. For this reason, it is desirable that the measurement results be available immediately and with a very short response time. Furthermore, the measurement should be economical, easy to perform, and as accurate as possible.

[0015] However, current CO2 sensors on the market are often expensive, prone to drift and inaccuracy, and require frequent calibration. This can lead to costly downtime and fluctuations in measurement results. In addition, known sensors are susceptible to reactive gases and have slow response times.

[0016] Methods for measuring CO2 in relation to biological reactions include, in particular, the Severinghaus method (see [4] and

[13] ), non-dispersive infrared (NDIR, see [5]), tunable diode laser (TDL, see [6]), photoacoustic (PAS, see [7]), thermal conductivity (TC), and blood gas analysis with Severinghaus (BGA, see also [8]). According to

[13] , it is also possible to use a sensor that can measure dissolved CO2 photochemically, in which case the sensor must have a chromophore that is sensitive to CO2.

[0017] Tunable diode laser (TDL) or tunable diode laser absorption spectroscopy (TDLAS) is a method used to estimate the concentration or density of a target gas or gas component from the measured absorbance. TDL has an accuracy of approximately 2%, requires a relatively long measurement path length (>50 mm), achieves a maximum reproducibility of 0.75%, and is relatively large and expensive. However, measurements can be performed very quickly (response time less than 10 seconds).

[0018] Another method involves exciting the gas or gaseous component CO2 in the infrared region. This generates measurable noise. However, this photo-acoustic measurement is considerably slower than TDL. A response time of 2 minutes is only achievable if a sterilizable separation membrane is unavailable. Especially in high humidity ranges, the measurement technique reaches its limits. A response time of 3 minutes is more realistic.

[0019] Examples of conventional range-oriented sensor systems include those based on NDIR (non-dispersive infrared). Tests with first-generation equipment have shown a response time of approximately 10 minutes (date: end of 2021).

[0020] Other suppliers of Bio Process Containers (BPC), which also offer sensors including SUBs, rely on fluorescent material-based technology. Similar to the “sevelinghaus” electrode described below, this actually measures the pH value of the electrolyte (bicarbonate). This means that CO2 first diffuses through a separation membrane, then “affects” the fluorophore, altering the buffer and ultimately enabling pH measurement. The instrument's accuracy is specified as 3% in the 0-10% CO2 range, possibly capable of measuring up to 15%. User reports indicate a response time of over 5 minutes. These instruments are based on measurements using PDMS (polydimethylsiloxane), and scientific papers also state a response time of 10 minutes.

[0021] The most commonly used measurement method is the Sevelinghaus electrode sensor technology. The Sevelinghaus principle is based on a pH probe immersed in a bicarbonate electrolyte surrounded by a CO2-permeable membrane. When CO2 reaches equilibrium with the bicarbonate buffer, carbonic acid is formed, lowering the pH, and this pH change correlates with the CO2 concentration. Probes based on the Sevelinghaus principle are used as inline sensors for real-time detection in the biopharmaceutical industry and have become the gold standard for analysis.

[0022] Reference

[13] aims to use commercially available oxygen and carbon dioxide (CO2) sensors in disposable bioreactors. These established systems are based on methods of eluting gaseous components present or dissolved in a medium (M), binding them to polymers for determination therein, or releasing them again into a specific fluid (e.g., bicarbonate buffer or specialized electrolyte).

[0023] Cost, handling, and maintenance are thought to be some of the reasons why carbon dioxide measurement has been slow to be adopted.

[0024] Blood gas analysis is also a form of Severinghaus's principle, and because it is always performed offline, it is outside the biological process.

[0025] The measurement of thermal conductivity (TC) has little practical importance and is mainly used in the food industry, specifically in processes where the gas or gas mixture is precisely known, such as packaging and beverage bottling under protective gas, and especially in beer.

[0026] Despite the critical importance of carbon dioxide content in culture media, this measurement has received little attention in the past. One reason for this is the lack of reliable sensors and measurement methods.

[0027] The present invention aims to provide a measurement method and apparatus for determining the concentration of an important gas in a process that can be carried out quickly and reliably using relatively simple means. [Means for solving the problem]

[0028] To solve this problem, the present invention provides a measurement method as described in claim 1 and a measurement apparatus as described in a further independent claim. Advantageous uses are covered in the further independent claims.

[0029] Advantageous embodiments of the present invention are subject to the dependent claims.

[0030] According to a first aspect, the present invention provides a method for measuring the concentration of a gas component to be measured in a gas mixture having a known composition in a liquid solution of a reaction vessel, comprising the following steps: a) Providing a measuring chamber separated by a separation membrane through which gas passes without passing liquid from the reaction vessel. b) Measuring the gas density ρ, gas pressure p, and temperature T in the measuring chamber. c) Determining the concentration of the gas component to be measured from the values measured in step b) and the molar mass of the gas components of the mixed gas.

[0031] The measuring chamber is preferably very small. Thereby, in particular, the reaction time is fast and the measurement time is short. The concentration change in the solution is transmitted very quickly to the very small volume of the measuring chamber.

[0032] In some embodiments, the volume V of the measuring chamber is less than 5000 mm , 3 ,

[0034] ,

[0033] , 3 , 3 , 3 , , , , , 3 , 3 In particular, V is less than 3000 mm 3 or less than 1000 mm 3 or significantly less than 100 mm 3 The lower limit of the volume of the measuring chamber depends on the degree of miniaturization of the sensors available for measuring the density, pressure, and temperature of the gas. For example, depending on the sensors used, the lower limit may be 0.5 mm 3 or 1 mm 3 or 2 mm 3 in some cases.

[0033] Particularly preferred is that step a) includes the following steps. a1) Providing a measuring chamber (16) with a measuring chamber volume V, where 0.5 mm 3 ≦ V ≦ 15 mm 3 [[ID=4,0]]In particular, 1 mm 3 ≦ V ≦ 10 mm 3 preferably 2 mm 3 ≦ V ≦ 5 mm 3 is.

[0034] As physical variables, gas density, pressure, and temperature are measured (determined). This means that no chemical conversion is required for measurement, which also contributes to reliability and rapid measurement. The sensors used are particularly miniaturizable. In particular, quartz oscillators or tuning fork type sensors are used for gas density measurement. Tuning fork type gas density sensors are known and are available on the market in other technological fields, particularly for monitoring insulating gases in electrical switchgear.

[0035] Step b) should ideally include the following steps: b1) A step of measuring the gas density using a tuning fork type sensor and / or a quartz crystal oscillator.

[0036] In particular, a quartz crystal oscillator tuning fork is used. This is especially designed to be as small as possible.

[0037] In some embodiments, a measurement method for measuring the concentration of a gas component to be measured (preferably one that changes during a bioreaction) in a gas mixture of known composition within a bioreactor includes the following steps: a) A step of providing a measuring chamber separated from the inside of a bioreactor by a gas permeable separation membrane. b) A step of measuring the gas density ρ, gas pressure p, and / or temperature T in the measuring chamber. c) A step in which the concentration of the gas component to be measured is determined from the measured value and / or the molar mass (known by the known composition) of the gas component of the gas mixture. In particular, the gas component being measured is CO2.

[0038] The motivation for developing embodiments of the invention lies in the interest in the concentration of a target dissolved gas component in a liquid phase. The measurement method according to the present invention is set to measure the concentration of a target gas component dissolved in a liquid phase. However, this measurement method also functions for measuring gas supply and exhaust gases from, for example, bioreactors, fermenters, or gas synthesis reactors.

[0039] In a particularly preferred embodiment, the concentration of a gas component to be measured is measured in a reaction vessel, especially a bioreactor, from a gas mixture having a known composition dissolved in a liquid phase. Preferably, the separation membrane is positioned in contact with the liquid phase of the reaction space, such as a bioreactor. Particularly preferably, the entire surface of the measuring membrane facing the inside of the reaction vessel is in the liquid phase. Particularly preferably, the measuring membrane is attached to or provided in the lower half or lower third of the reaction vessel.

[0040] Examples of reaction vessels include bioreactors, disposable bioreactors, bioreactors with a diameter of less than 10 mm, reaction vessels in the food industry, reaction vessels for beverage production, reaction vessels in breweries, reaction vessels in sewage treatment facilities, reaction vessels in wastewater treatment facilities, reaction vessels in CO2 extraction facilities, reaction vessels for process analysis, piping, and water supply piping.

[0041] A “gas-permeable separation membrane” means that the separation membrane is permeable to the gaseous components of a gas mixture. In the method according to the embodiments of the present invention, all analytes and gaseous components present in the medium are measured together, rather than measuring only one of the analytes or one of the gaseous components. At least two of the physical or chemical properties of the gas mixture (e.g., density, pressure, thermal conductivity, electrical conductivity and / or temperature, etc.) are measured (determined). In particular, density is measured together with one of the other physical properties.

[0042] In some embodiments, gaseous components in the medium contained within or dissolved in the bioreactor are eluted and then measured directly in a measurement chamber. The measurement is performed in the fluid phase. For this purpose, in some embodiments, the separation membrane is specified to be a composite material, including but not limited to polymers, and to be inflexible. The rigidity of this separation membrane ensures stable measurements even when the process pressure fluctuates (compared to current conventional membranes).

[0043] In some embodiments, the membrane is reinforced and / or strengthened. In conventional bioreactor gas analysis sensors, the analyte is bound to a polymer or the like, but in embodiments of the present invention, all gas components move into the measurement chamber by diffusion.

[0044] In embodiments of the present invention, instead of measuring a single analyte, all dissolved gases in the measuring chamber are measured, and one component among them is identified. For example, in the measurement of CO2, it is not limited to measuring CO2 in an air mixture, but it is also possible to measure the CO2 content in a hydrogen-methane mixture. Other gases besides CO2 can also be measured.

[0045] The separation membrane should preferably contain a rigid material and / or a reinforcing material. The separation membrane should preferably be non-stretchable. The separation membrane should preferably be rigid and not flexible.

[0046] In some embodiments, the fluid is characterized within the measurement chamber. In some embodiments, the gas density of the reaction system is such that it prevents gas from escaping from the measurement chamber to the outside.

[0047] In some embodiments, the measurement chamber is airtight to the outside and connected to the inside of the bioreactor only through a gas-permeable separation membrane. In some embodiments, the measurement chamber is designed so that gas enters the measurement chamber only from the medium of the reaction vessel, through a gas-permeable membrane designed to be sealed from the environment.

[0048] The gas mixture should preferably include at least one additional gas component from the group consisting of air, industrial air, dry air, and a mixture of O2 and N2. In most cases, bioreactors and other reaction vessels operate in liquid phase and are introduced with a predetermined gas mixture. In particular, reaction vessels designed as bioreactors operate with, for example, air, or a specially introduced mixture of O2, N2, CO2, and other gases.

[0049] Preferably, the measurement method includes the following: A step to measure further physical parameters of the gas mixture.

[0050] In particular, other physical properties or other gas components are measured. For example, by further measuring other physical properties or gas components, other gas components can be accurately identified.

[0051] Preferably, the measurement method includes the following: Measure the relative humidity. In particular, the concentration of the target gas component, CO2, is determined as a function of the measured humidity value.

[0052] In the liquid phase, humidity and the resulting partial pressure are functions of temperature, but the gas component being measured in the measurement chamber on the opposite side of the separation membrane does not necessarily have to be 100%.

[0053] Preferably, the measurement method is configured as follows: This involves measuring the content of other gases in the gas mixture, such as O2. This allows for a more accurate determination of the average molar mass of the gas mixture, improving the measurement results. This applies to all components of the gas mixture.

[0054] It is desirable that the gas mixture be classified into a group of components containing the gas component to be measured and other gas components, that the weighted average molar mass be determined based on the component concentrations of the component groups, and that in step c), the concentration of the gas component to be measured be determined from the molar mass of the gas component to be measured and the weighted average molar mass. The corresponding step is known, for example, in a completely different technical field, such as measuring the withstand voltage of insulating gases present as a gas mixture in an electrical switchgear, as shown in [9].

[0055] In step b), it is desirable that the gas density be measured by a quartz crystal oscillator, more preferably by a tuning fork. For example, a quartz crystal oscillator known in

[12] can be used. Measuring gas density with a quartz crystal sensor is also known in the field of gas density measurement in electrical switching and power distribution systems (high voltage technology) of insulating gases, as described in [9] to

[11] .

[0056] From yet another perspective, the present invention provides a reaction method for carrying out a reaction in a reaction vessel, which includes the following steps. A step of measuring the concentration of the gas component of the gas mixture in the reaction vessel by performing a measurement method according to any of the embodiments described above, and performing further steps of the reaction method according to the measurement.

[0057] In some embodiments, the reaction method is a bioreaction method for carrying out a biological reaction (bioreaction) in a bioreactor, and this includes the following steps. A step of measuring the concentration of gaseous components in a gas mixture within a bioreactor by performing a measurement method according to any of the above-described embodiments, and further performing steps of a bioreaction method based on the measurement.

[0058] In particular, cell culture is carried out in a bioreactor, where it is especially preferably controlled by measuring the CO2 concentration by measuring the DCO2 concentration using the measurement method presented in the present invention.

[0059] Furthermore, the present invention provides a measuring device for measuring the concentration of a target gas component in a gas mixture of known compositions within a reaction vessel, the measuring device including: A measuring tank equipped with a gas-permeable separation membrane for separating the measuring tank from the inside of the reaction tank. A measuring device for measuring gas density ρ, gas pressure p, and temperature T in a measuring chamber. An evaluation device configured to determine the concentration of a gas component to be measured from the measured value obtained by the measuring device and the molar mass of the gas component of the gas mixture.

[0060] In some embodiments, the measuring device is configured to measure the concentration of a target gas component in a gas mixture of known composition within a bioreactor, preferably the concentration that changes during a biological reaction, and includes the following:

[0061] The measurement chamber is equipped with a gas-permeable separation membrane to separate it from the inside of the bioreactor.

[0062] The measuring device is used to measure the gas density ρ, gas pressure p, and temperature T inside the measuring chamber.

[0063] The evaluation device is configured to determine the concentration of the gas component to be measured from the measured value obtained by the measuring device and the molar mass of the gas component in the gas mixture.

[0064] A “gas-permeable separation membrane” means that the separation membrane is permeable to all gaseous components in a gas mixture. The separation membrane is configured to allow convection of the gas mixture within the measurement chamber. In some embodiments, the separation membrane has reinforcing or strengthening materials and is not flexible or stretchable.

[0065] In particular, when the volume of the measuring chamber is very small, very short reaction times can be achieved, such as less than 60 seconds in the liquid phase. Therefore, the volume of the measuring chamber is 5000 mm³. 3 Less than 3000mm, especially 3000mm 3 Less than 1000 mm, preferably 1000 mm 3 Less than 100mm 3 It is desirable that it be less than 0.5 mm. The lower limit of the measurement chamber volume depends particularly on the miniaturization capability of the gas density sensor, for example, 0.5 mm. 3 , 1mm 3 , or 2mm 3 That is the case.

[0066] In some embodiments, the measuring chamber has a measuring chamber volume V and 0.5 mm 3 ≤V ≤ 15mm 3 , especially 1mm 3 ≤V ≤ 10mm 3 , more preferably 2 mm 3 ≤V ≤ 5mm 3 It holds.

[0067] In some embodiments, the measuring device includes a quartz crystal oscillator and / or a tuning fork sensor for measuring gas density. In particular, a quartz crystal oscillator fork is used.

[0068] In some embodiments, the measuring device includes further measuring devices for measuring further physical properties or parameters of the gas mixture.

[0069] Preferably, the measuring device includes a humidity measuring device for measuring relative humidity. Preferably, the measuring device includes a gas content measuring device for measuring the content of at least one further gas in the gas mixture. Preferably, the evaluation device is designed to determine the concentration based on the measurement of further physical properties, the measurement from the humidity measuring device and / or the measurement from the gas content measuring device.

[0070] Preferably, the evaluation device is configured to determine the concentration of CO2 in the atmosphere, including O2 and N2, within a reaction vessel designed as a bioreactor.

[0071] Particularly preferably, the evaluation device is configured to estimate the partial pressure and concentration of CO2 dissolved in the liquid phase of the reaction vessel from the CO2 concentration in the atmosphere containing O2 and N2 in the measurement tank.

[0072] Preferably, a measuring device according to any of the embodiments described above is configured to carry out a measuring method according to any of the embodiments described above.

[0073] In another aspect, the present invention provides a reaction system for carrying out reactions including the following: The reaction system includes a measuring device and a reaction vessel according to any of the embodiments described above. Here, the measuring vessel is separated from the inside of the reaction vessel by a separation membrane.

[0074] Examples of reaction vessels include bioreactors, disposable bioreactors, bioreactors with a diameter of less than 10 mm, reaction vessels for the food industry, reaction vessels for beverage production, reaction vessels for breweries, reaction vessels for wastewater treatment plants, reaction vessels for CO2 extraction plants, reaction vessels for process analysis, pipelines, and water supply piping.

[0075] In some embodiments, the reaction system is a biological reaction system for carrying out biological reactions such as cell culture and fermentation, and includes the following: Here, the measuring tank includes a measuring device and a bioreactor according to any of the embodiments described above. The measuring tank is separated from the inside of the bioreactor by a separation membrane.

[0076] The bioreactor is preferably a disposable reactor.

[0077] In a further aspect, the present invention provides the use of a measuring device having a measuring tank bounded by a gas-permeable separation membrane and a measuring device for measuring the gas density ρ, gas pressure p, and temperature T within the measuring tank, thereby enabling the measurement of the concentration of a target gas component in a gas mixture in a reaction vessel (e.g., a bioreactor) separated from the measuring tank by the separation membrane. Particularly preferably, the measuring device is used to measure the concentration of a target gas component in a gas mixture dissolved in the liquid medium of the reaction vessel (e.g., a bioreactor).

[0078] In embodiments of the method, apparatus, and system of the present invention, temperature measurement is also performed. Preferably, the measuring chamber is designed to raise its temperature faster than the temperature gradient of the reaction in the reaction vessel. This is also supported by the small volume of the measuring chamber.

[0079] A preferred embodiment of the invention relates to the measurement of gaseous component concentrations in a reaction vessel, particularly dissolved CO2 in the liquid phase, and is designed, for example, as a bioreactor, preferably using a composite sensor.

[0080] The relationships between the influencing variables density, pressure / partial pressure, and gas density, as well as their dependence on temperature and relative humidity, are known.

[0081] In a preferred embodiment of the invention, Henry's Law is applied using a gas-permeable separation membrane. This posits that the partial pressure of a gas on and in a liquid is equal to and directly proportional to the concentration of the gas in the liquid. The density, temperature, (optional) relative humidity, and absolute pressure of the gas mixture between the separation membrane and the measuring device are measured. If the determination is sufficiently accurate, it is possible to draw conclusions about the partial pressure of individual gas components, especially when they differ significantly in molar mass or / or can be divided into two groups. For example, even a difference of 12 g / mol is already significant, and reference [9] states that MA-MB is greater than 20 g / mol.

[0082] Preferred embodiments can be used to design factory-calibrated measuring devices that require no calibration for the end user. Some embodiments offer the possibility of coupling an electrical device (measuring device) with an oxygen sensor. This can improve accuracy by allowing for a more precise determination of the effective molar masses of the components of a gas mixture.

[0083] Preferably, the concentration of DCO2 is measured due to its high economic importance when cell culture or similar biological reactions are carried out in a bioreactor. The measurement of DCO2 concentration is also of interest in other reactions such as those in the food industry, beverage production, environmental technology, and wastewater treatment, and can be carried out using the methods and apparatus disclosed herein. Of course, other gas concentrations can also be measured using the same principle. For example, the measurement method can be applied to dissolved ammonia and acetone in the liquid phase, provided that a suitable separation membrane is available. Applications are also possible in the fields of biogas production (methane and carbon dioxide, including trace components of nitrogen, oxygen, hydrogen sulfide, hydrogen, and ammonia) and biofuel production (where CO2 and methane CH4 play a major role).

[0084] The principles and design will be explained in more detail below using an example of carbon dioxide measurement. A bioreactor is used as an example of a reaction vessel, but it is clear that the structure of the reaction vessel will differ in other embodiments. Examples of possible reaction vessels are shown above. [Brief explanation of the drawing]

[0085] Embodiments will be described in more detail below with reference to the attached drawings. [Figure 1] Figure 1 is a schematic block diagram of an embodiment of a measuring device for measuring the concentration of dissolved gas components in a reaction vessel designed, for example, as a bioreactor. [Figure 2] Figure 2 is a schematic diagram illustrating the grouping of gas components in a gas mixture. It shows the schematic principle of classifying the gas components to be measured into component groups during the process of evaluating the measurement signal from the sensor of the measuring device. [Figure 3]Figure 3 is a block diagram of a bioreactor system equipped with a bioreactor and measuring device. [Figure 4] Figure 4 is a schematic diagram of a disposable bioreactor equipped with a coupling for connecting a measuring device. [Figure 5] Figure 5 is a cross-sectional view of the coupling in Figure 4. [Figure 6] Figure 6 is a cross-sectional view showing both the coupling embodiment and the separation membrane module. [Figure 7] Figure 7 is a further schematic block diagram showing the basic configuration of the measurement principle according to a further embodiment of the invention. [Figure 8] Figure 8 is a micrograph of an embodiment of the composite sensor of the measuring device. [Modes for carrying out the invention]

[0086] Figure 1 shows an embodiment of the measuring device 10, with additional optional components of further possible embodiments of the measuring device 10 indicated by dotted lines. Figure 1 is a purely schematic block diagram illustrating the principle of the measuring device 10, and the dimensional ratios are not actual.

[0087] The measuring device 10 is configured to measure the concentration of dissolved gas component Z in dissolved gas mixtures 12, 12b of known composition, measured in a reaction vessel 14 designed as a bioreactor. For example, a predetermined or controlled atmosphere (gas phase - gas mixture 12a -) containing components X, Y, and Z is present in the reaction vessel 14. For example, air with a high CO2 content or a technically manufactured mixture of O2, N2, and CO2 is present in the reaction vessel designed as a bioreactor. Therefore, for example, a gas mixture 12b containing components X=O2, Y=N2, and Z=CO2 is present in the reaction vessel 14 in a dissolved state in the liquid phase. The measuring device 10 is configured to measure the concentration of any of the gas components in the liquid solution in the reaction vessel. In some embodiments, the measuring device 10 is designed to measure the concentration of CO2, in particular CO2, i.e., DCO2, dissolved in the liquid phase in the reaction vessel 14.

[0088] In the example shown, the measuring device 10 is designed as an electrical device equipped with a composite sensor 22 for determining the CO2 concentration.

[0089] The reaction vessel 14 is a container containing process media (liquid and gas). Examples of the reaction vessel 14 are described in detail below. In particular, biological reactions, i.e., cell culture and fermentation, take place in the reaction vessel 14, and in some embodiments it is designed as a bioreactor. In this case, the DCO2 content is measured and used to control and monitor the biological reactions.

[0090] The measuring device 10 has a measuring tank 16, which is equipped with a gas-permeable separation membrane 18 for separating the inside of the reaction vessel 14 from the measuring tank 16. During operation, the separation membrane 18 is in full contact with the liquid phase of the reaction vessel 14. The dissolved gas components of the gas mixture 12b dissolved in the liquid phase are allowed to pass through the separation membrane 18, and therefore the measuring tank 16 contains a gas mixture 12c corresponding to the gas mixture 12b dissolved in the liquid phase. The separation membrane 18 is permeable to all gas components of the gas mixture and allows convection. In some embodiments, the separation membrane 18 is neither flexible nor stretchable and is equipped with reinforcing and / or strengthening materials (not shown) for this purpose.

[0091] The measuring chamber 16 is designed with the smallest possible measuring chamber volume V. In particular, the measuring chamber volume V is 5000 mm³. 3 Less than 4000 mm 3 Less than 3000mm 3 Less than, and especially 500mm 3 Less than 100mm 3 It is less than this. By making the measuring chamber 16 particularly small in this way, it becomes possible to miniaturize the measuring device 20 described later. For example, the lower limit of the measuring chamber volume V is 0.5 mm depending on the degree of miniaturization. 3 2mm 3 That is the case.

[0092] The measuring device 10 includes a measuring device 20 for measuring the gas density ρ, gas pressure p, and temperature T in the measuring chamber. In particular, the measuring device 20 is designed as a composite sensor 22. The composite sensor 22 includes a known temperature sensor 24, a known pressure sensor 26, and a gas density sensor 28. The gas density sensor 28 is preferably a quartz crystal sensor designed as a tuning fork, which determines the gas density through the difference in quartz crystal vibration frequencies between a quartz crystal vibration fork exposed to the measuring medium and a quartz crystal vibration fork vibrating in a reference volume, as is known from reference

[12] , which will be referred to in detail therein. For further details of this measurement principle, see references [9] to

[12] . The different sensors 24, 26, and 28 may be supplied together in a single sensor housing or separately. They are designed to measure the temperature T, pressure p, and gas density ρ of the gas mixture 12c in the measuring chamber 16.

[0093] Furthermore, the measuring device 10 includes an evaluation device 30 configured to determine the concentration of the gas component Z to be measured from the values ​​measured by the measuring device 20 and the molar masses of gas components X, Y, and Z of the gas mixture 12c. The evaluation device 30 is, for example, part of an analysis and control unit 32 having a processor 34 and storage 36. The evaluation device 30 is programmed with a corresponding computer program for performing evaluations described later to determine the concentration of CO2 from the measured values ​​T, p, and ρ.

[0094] The measurement results are affected by the influencing variables, temperature and humidity. To mitigate this influence, some embodiments of the measuring device 10 optionally include one or more additional filters 38 and / or heaters 40. In some embodiments of the measuring device 10, a humidity sensor 42 is optionally provided to measure the relative humidity φ in the measuring chamber 16. Some embodiments of the measuring device 10 may include an optional external sensor 44 for detecting further gaseous components Y, particularly DO2, in the reaction chamber 14.

[0095] During the operation of the reaction vessel 14, a measurement method for measuring the concentration of the target gas component Z in a gas mixture 12b of known composition in a liquid solution is performed within the reaction vessel 14, and includes the following procedure. a) A step of supplying the measuring tank 16, which has been separated from the inside of the reaction tank 14 by the gas permeable separation membrane 18. b) A step of measuring the gas density ρ, gas pressure p, and temperature T in the measuring chamber 16. c) A step to determine the concentration of the gas component Z to be measured, based on the value measured in step b) and the molar masses of gas components X, Y, and Z of the gas mixture 12c (=12b).

[0096] The physical relationships of the measurements performed in a preferred embodiment are described in detail below.

[0097] Carbon dioxide typically exists as a gas. Therefore, measuring carbon dioxide in a process involves measuring the pressure generated by carbon dioxide in the gas or in the liquid in which it is dissolved.

[0098] The relevant physicochemical laws are as follows: • Dalton's Law (Law of Partial Pressures) Henry's Law • Law of Mass Balance (Law of Conservation of Mass)

[0099] Dalton's Law (Dalton's Law, Law of Partial Pressures) is: JPEG2026510994000002.jpg16170

[0100] Total pressure is the sum of the partial pressures of each gas component. The sum of all partial pressures gives the total pressure. Partial pressures correspond to the pressure exerted by each gas component if it were present individually within the given volume.

[0101] The extent to which a gas reacts with other substances or diffuses and dissolves in a liquid is determined not by the gas mixture or its concentration in the liquid, but by the partial pressure of the gas.

[0102] Therefore, dry air (p water For (=0), the following relationship holds: P total air =p nitrogen +p oxygen +p carbon dioxide +p argon +p trace gases (Total air pressure P) total ai = Partial pressure of nitrogen p nitrogen +Partial pressure of oxygen p oxygen +Partial pressure of carbon dioxide p carbon dioxide +Partial pressure of argon p argon +Partial pressure p of trace gas trace gases )

[0103] At atmospheric pressure of 1013 mbar (hPa), 0.038% of that pressure is accounted for by carbon dioxide. According to Dalton's law, p carbon dioxide This is 0.385 mbar.

[0104] Air is not recommended as a calibration gas because its volume percentage of carbon dioxide is low, and the expected carbon dioxide concentration in process applications increases. A gas mixture consisting of 5% carbon dioxide, 20% oxygen, and 75% nitrogen is more advantageous in both respects.

[0105] According to Dalton's Law, under normal conditions (10¹³ mbar, 25°C), a dry gas behaves as follows:

[0106] p carbon is 50.65 mbar, p oxyge is 202.60 mbar, p nitrogen It is 759.75 mbar.

[0107] Henry's Law: According to Henry's Law, each gas dissolves in a liquid according to its partial pressure in the gas phase. When distribution equilibrium is reached, each gas has the same partial pressure in both the gas and liquid phases.

[0108] Henry's Law is applied to the bioreactor 14 with the help of a gas-permeable separation membrane 18. According to this law, the partial pressure of gases above and in a liquid is equal and directly proportional to the concentration of gases in the liquid. The gas density ρ, temperature T, and, if necessary, relative humidity φ and absolute pressure p of the gas mixture 12c between the separation membrane 18 and the measuring device 20 are measured. If the measurements are sufficiently accurate, the partial pressure p of individual gas components can be measured only if the molar masses differ significantly. X , p Y , p Z This can be estimated. In particular, the gas components X, Y, and Z of the gas mixture can be divided into two groups A and B, as shown in Figure 2. For example, components X and Y are oxygen and nitrogen, respectively, and their partial pressures are p A =p X +p Y The components are classified into component group A, and at least one component Z, in this case carbon dioxide, is classified into component group B. For component group A, the weighted average molar mass is determined according to the concentrations of components X and Y. The molar mass of component Z is known. From this, the partial pressure of component group B and therefore the partial pressure and concentration of component Z (in this case carbon dioxide) can be determined, in more detail by the method described in reference [9].

[0109] The schematic diagram in Figure 1 shows a typical electrical device diagram for realizing the measuring device 10. The design of the container that holds the process medium (liquid or gas) is not specified. Therefore, it is also possible to determine the gas concentration of the gas component in a gas mixture in a liquid solution.

[0110] The electrical device is connected to a container (=bioreactor 14) containing the process medium via a process connection equipped with a gas-permeable separation membrane 18. The process connection with the gas-permeable separation membrane 18 constitutes a coupling 48 between the container containing the medium to be characterized and the measuring device 10.

[0111] The gas mixture 12b of the processing medium diffuses into the measuring chamber 16 through a gas-permeable separation membrane 18. Due to factors such as temperature and humidity, it may be advantageous or necessary in some applications to equip the measuring device 10 with additional filters 38 and heaters 40 to prevent condensation, particularly on the surfaces of the measuring chamber 16 and sensors 22, 24, 26, and 28.

[0112] For example, the measuring device 20, designed as a sensor package or composite sensor 22, measures the density ρ, absolute pressure p, temperature T, and, in this case, relative humidity φ of the gas mixture 12c. These signals are temporarily stored in the evaluation device 30 of the measuring device 10 and processed by the processor 34.

[0113] The components X, Y, and Z of the gas mixture, their proportions, and the accuracy of sensors 24, 26, 28, and 42 in the sensor package, are used to determine the partial pressure p of component B. B These are the influencing factors for accurately determining [the outcome].

[0114] In some embodiments, the measuring device 20 is designed as a Rho-pT sensor for measuring and analyzing the gaseous components of CO2, O2, and N2. The molar masses of the gaseous components are as follows: CO2: 44 g / mol O2: 32 g / mol N2: 28 g / mol

[0115] The three components are assigned to two defined component groups A and B. Molar mass M A and M B These are considered as weighted average molar masses with respect to their relative concentrations. This was done in [9] to determine the dielectric breakdown strengths of oxygen, nitrogen, and "C5". Bioreactor 14 has a higher concentration of CO2. Here, for example, CO2 is assigned to component group B, and N2 and O2 are assigned to component group A.

[0116] The following describes an example of determining the CO2 concentration (carbon dioxide concentration) in a gas mixture 12, which is a form of air.

[0117] Example 1: Dry air is filled into a container connected to a measuring device 10 equipped with a measuring tank 16 via a separation membrane 18. A gas mixer adds carbon dioxide until it contains 5% carbon dioxide by volume. The measurement is then performed.

[0118] The conditions are as follows: p Abs = 1013.25 mbar T=30℃ rH = φ = 0%

[0119] Ideally, the sensor provides the following signals: Density sensor: 1194.085354 g / m³ 3 Pressure sensor: 1013.25 mbar Temperature sensor: 30℃ Humidity sensor: 0%

[0120] The concentration is determined using the general law of state for gases. JPEG2026510994000003.jpg10170

[0121] Here, R is the universal gas constant (general gas constant), and M is the molar mass of the gas mixture. This equation can be solved based on the molar mass. JPEG2026510994000004.jpg8170

[0122] Therefore, the molar mass M within the processor is determined as follows: JPEG2026510994000005.jpg12170 As a result, a value of M = 29.70204501 g / mol is obtained.

[0123] Regarding the molar mass of CO2, the following holds true: ΔMCO2 is the molar mass of CO2. JPEG2026510994000006.jpg8170 Average molar mass M relative to dry air air The result was 28.949 g / mol.

[0124] As a result, the molar mass attributable to CO2 is ΔMCO2 = 0.753045011 g / mol.

[0125] From there, the CO2 concentration can be determined using a corresponding coefficient k that represents the rate at which the molar mass of the gas mixture increases for every 1% CO2 added.

[0126] This calibration coefficient k is determined by the following table.

[0127] [Table 1]

[0128] The molar mass of air is M air = 28.949 g / mol. If the volume ratio of CO2 increases to 10%, the total molar mass is M = 90% * M air +10%*MCO2 = 30.45509 g / mol, which is M air This shows a difference of 1.50609 g / mol. This means that for every 1% increase in the proportion of CO2, an increase of 0.150609 g / mol occurs.

[0129] Using the coefficient k = 0.150609 g / (mol*volt%), the concentration of CO2 can be calculated from the determined ratio (percentage) of molar masses. CCO2 = ΔMCO2: k = 0.753045011 g / mol: 0.150609 g / (mol * volume %) = 5.00000007 volume %

[0130] When expressing the partial pressure pCO2, it is as follows: pCO2=p*CCO2=1013.25mbar*0.05=50.6625007mbar

[0131] Given that the accuracy (relative value to the measured value) of the density sensor 28 is 0.50%, the pressure sensor 26 is 0.50%, the temperature sensor 24 is 0.15°C, and the humidity sensor 42 is 3%, the measuring device 10 provides values ​​in the range of 2.94% to 7.08% at a CO2 concentration of 5%, and the partial pressure can be estimated to be in the range of 29.8 mbar to 71.7 mbar.

[0132] Example 2: The gas measurement is performed in the same manner as in Example 1. The container is filled with dry air. CO2 is added to the gas mixer until it contains 20% by volume of CO2.

[0133] The conditions are as follows: pAbs = 1013.25 mbar T=30℃ Relative humidity rH = φ = 0%

[0134] Ideally, the sensor provides the following signals: Density sensor: 1284.90738 g / m³ 3 Pressure sensor: 1013.25 mbar Temperature sensor: 30℃ Humidity sensor: 0%

[0135] Within the processor, the molar mass M can again be calculated using the following formula. JPEG2026510994000008.jpg12170 This yields a value of M = 31.96118 g / mol.

[0136] The following applies to the proportion of molar mass attributable to CO2. JPEG2026510994000009.jpg8170 In the case of dry air, average molar mass M air = 28.949 g / mol

[0137] This allows us to determine the ratio of the molar mass generated to the CO2. ΔMCO2 = 3.01218 g / mol

[0138] Using the coefficient k = 0.150609 g / (mol * volume %), the measured concentration of CO2 can be determined from the ratio of molar masses. CCO2 = ΔMCO2: k = 3.01218 g / mol: 0.150609 g / (mol * volume %) = 20 volume %

[0139] If we are showing the partial pressure pCO2, it will look like this: pCO2=p*CCO2=1013.25mbar*0.2=202.65mbar

[0140] Considering the accuracy of the aforementioned sensors 24, 26, 28, and 42, the actual measured concentration is expected to be in the range of 17.78% to 22.24%, and the actual measured partial pressure is expected to be in the range of 180.2 mbar to 225.3 mbar.

[0141] Example 3: Example 3 shows how humidity sensor values ​​can be taken into consideration.

[0142] Similar to Example 1, the container is filled with moist air with rH=φ=50%, and gas measurements are performed. The gas mixer adds CO2 until the volume ratio reaches 5%.

[0143] The conditions are as follows: p=1034.34mbar T=35℃ rH=φ=50% Ideally, the sensor would output the following signal: Density sensor: 1174.710287 g / m³ 3 Pressure sensor: 1034.34 mbar Temperature sensor: 35℃ Humidity sensor: 50%

[0144] From known values ​​of relative humidity and temperature-dependent saturation pressure pm(T), the partial pressure pw of water vapor can be calculated using the formula pw = pm(T) * φ.

[0145] The processor first calculates the pressure of the dry air as follows: pAbs is calculated as p - pw = 1013.25 mbar. Next, the molar mass of the gas mixture is calculated as follows: JPEG2026510994000010.jpg11170

[0146] The following relationship holds true regarding the proportion of CO2 in the molar mass of CO2. JPEG2026510994000011.jpg8170 The average molar mass of dry air is M air The concentration was determined to be 28.949 g / mol.

[0147] As a result, the molar mass ratio of CO2, ΔMCO2, becomes 0.753045011 g / mol.

[0148] Using the coefficient k = 0.150609 g / (mol * volume %), the concentration of CO2 can be calculated from the determined molar mass ratio. CCO2 = ΔMCO2: k = 0.753045 g / mol: 0.150609 g / (mol * volume %) = 5.00000 volume %

[0149] If we are showing the partial pressure pCO2, it will be as follows: pCO2=p*CCO2=1034.34mbar*0.05=51.717mbar

[0150] If the accuracy of the density sensor 28 is 0.50%, the accuracy of the pressure sensor 26 is 0.50%, the accuracy of the temperature sensor 24 is 0.15°C, and the accuracy of the humidity sensor 42 is 3% for the measured values, then at a CO2 concentration of 5% and humidity of 50%, the measuring device 10 can provide values ​​ranging from 2.68% to 7.35%, and the partial pressure value can be estimated to be in the range of 27.7 mbar to 76.0 mbar.

[0151] The deviation from the actual value directly depends on the measurement accuracy of the gas density ρ, temperature T, arbitrary relative humidity φ, and absolute pressure p.

[0152] Figure 3 shows an example of a reaction system 50 designed, for example, as a bioreactor system, in which the measuring device 10 is used. The reaction system comprises a reaction vessel 14 (e.g., a bioreactor) and the measuring device 10. Inside the reaction vessel 14 are a stirrer 52 with a drive unit 54, a pH sensor 56, and a DO sensor 58 (an example of an external sensor 44) for detecting dissolved oxygen. Furthermore, there are valve-controlled inlets for steam 62, water 64, acidic medium 66, alkaline medium 68, nutrient solution 70, and air 72, as well as a valve-controlled exhaust port 74. Measurement signal lines transmit the measured values ​​to the transmitter and controller 78 and are shown as dotted lines. On the other hand, control signal lines sent from these transmitters and controllers 78 to corresponding valves and other actuators (e.g., drive unit 54) are shown as dashed lines.

[0153] For example, a specific cell culture based on predetermined process parameters is carried out in a reactor 14 designed as a bioreactor.

[0154] As shown in Figure 3, the CO2 concentration value provided by the measuring device 20 can be used directly to control, for example, the addition of nutrients, such as the nutrient solution 70.

[0155] The values ​​from the oxygen sensor 58 may be supplied to the evaluation device 30 to take into account the change in the molar mass of component group A (in this case, air with correspondingly changing oxygen content).

[0156] Examples of reaction vessels include bioreactors, disposable bioreactors 80, bioreactors with a diameter of less than 10 mm, reaction vessels for the food industry, reaction vessels for beverage manufacturing, reaction vessels for breweries, reaction vessels for sewage treatment plants, reaction vessels for wastewater treatment facilities, reaction vessels for CO2 extraction plants, reaction vessels for process analysis, pipelines, and water pipes.

[0157] The transition to disposable bioreactors (SUBs) and bioprocess containers (BPCs) is described below.

[0158] In conventional biopharmaceutical and biotechnology processes, bioreactors made primarily of stainless steel or glass, such as the one shown in Figure 3, have long been used for large-batch production.

[0159] However, the use of disposable bioreactors (SUBs) is increasing, mainly due to their advantages in scalability, flexibility, and cost. While the use of SUBs offers several significant advantages for specific batch production, it also presents unique challenges, particularly in terms of measuring the environment within the SUB during production.

[0160] One parameter that is particularly affected is the measurement of CO2. A search of the catalogs of major process analysis manufacturers (Who's Who, prominent figures) reveals that while solutions exist for measuring CO2 levels in conventional processes, none exist for sub-processes (SUBs).

[0161] Since SUBs are typically shipped to pharmaceutical and biotech companies in a sterile, production-ready state, the sensors must either be installed in the bioreactor 80 before shipment or be able to be inserted into the bioreactor without affecting the sterile internal environment.

[0162] To date, there are no practical solutions that can be installed before the delivery of the SUB80. Installing measurement technology on-site without affecting the sterile internal environment is currently extremely expensive in terms of both cost and measurement performance.

[0163] Preferred embodiments of the measurement method and measuring apparatus 10 address both of these aspects, particularly the possibility of installing the separation membrane 18 in the SUB80 before the SUB80 is delivered to the end user. Figure 4 shows a highly schematic diagram of a disposable bioreactor 80 with the coupling 48 already attached, Figure 5 shows a cross-sectional view of the coupling 48, and Figure 6 shows a diagram of the coupling with the separation membrane module 82 attached.

[0164] The two most important parameters in a bioreactor, DO and pH, can be complemented by the use of CO2 measurement technology, just as in conventional processes.

[0165] The shown embodiment of the cup-long 48 with the separation membrane 18 is by no means the only possible embodiment. In some embodiments, a needle-size embodiment is sufficient.

[0166] As described above, in a preferred embodiment, components X, Y, and Z of gas 12c are grouped into groups A and B. From this, it can be inferred from the description of the measurement principle that knowledge of the individual components X, Y, and Z of gases 12a, 12b, and 12c has a very significant impact on the accuracy of the measurement under certain circumstances. For example, there are niche applications involving pure oxygen or high oxygen concentrations. Signals from the DO sensor 58 or other external sensors 44, which are measurement techniques for measuring and monitoring oxygen concentration, can be input to the composite sensor 22 via an interface. This improves accuracy.

[0167] A similar scenario is interesting when heavy volatile organic compounds (VOCs) are present in the process and need to be monitored.

[0168] In particular to improve process control of a bioreactor (14) in cell culture and fermentation, several embodiments of the present invention provide a method for measuring the concentration (Cz) of a target gas component (Z) from a gas mixture (12, 12a, 12b, 12c) having a known composition in a solution within the bioreactor (14). a) A step of providing a measuring chamber (16) separated from the inside of the bioreactor (14) by a gas-permeable separation membrane (18). b) A step of measuring the gas density ρ, gas pressure p, and temperature T in the measuring chamber (16), and c) A step in which the concentration (Cz) of the gas component to be measured (Z) is determined from the values ​​and molar masses of the gas components (X, Y, Z) of the gas mixture measured in step b).

[0169] Dissolved CO2 (dCO2) is a crucial parameter for understanding the progress of any biotechnology process. Therefore, solutions for determining the proportion of CO2 in a liquid solution have been specifically proposed. However, this method and apparatus are also suitable for measuring the concentrations of other gaseous components, and some embodiments include configurations for this purpose.

[0170] Measuring dissolved gas components in liquid samples has been particularly challenging. Conventional measurements were typically performed indirectly, for example, via pH values. Sensors often required calibration, were relatively inaccurate, had very long response times, and were susceptible to interference from other gases that affected pH values.

[0171] Figure 7 shows the measurement principle of several preferred embodiments of the measuring apparatus. The liquid sample is separated from the measuring tank 16 by a liquid-impermeable but gas-permeable separation membrane 18, where the density of the gaseous component in the gas phase is measured. The gas density GD is measured by quartz crystal microbalance density measurement (QCM). As shown in the graph on the right side of Figure 7, the obtained frequency difference (difference frequency) df is proportional to the gas density, and in this case, proportional to the amount of CO2 in the solution. The measurement principle does not require any intermediate chemical steps, and the measurement is performed only in the gas phase in equilibrium with the solution via the separation membrane 18. This results in a very small measurement error (e.g., ±5 mbar in an unoptimized system). Experiments showed a response time of only 4 seconds in addition to the diffusion time through the separation membrane 18.

[0172] The measuring device 20 can be miniaturized to a very small size as a composite sensor 22. Figure 8 shows a microscopic image of one embodiment of the measuring device designed as a composite sensor 22 with a quartz crystal oscillator (tuning fork type) as a gas density sensor 28, composite pressure / temperature sensors 24, 26 and an optional humidity sensor 42 on its side. The diameter of the sensor head can be made less than 1 mm, which allows the volume V of the measuring chamber to be made very small. The sensors 28, 24, 26, and 42 are arranged on a plate (PCB) 84, on the back side of which a chip with the evaluation device 30 or a part thereof can be placed. The sensor head shown in Figure 8 is mounted on a holder, which allows the composite sensor 22 to be placed in a disposable bioreactor 80 or other reaction vessel with a separation membrane 18 sandwiched in between.

[0173] The measurement requires no further calibration after initial calibration at the manufacturing plant. The design is simple and can be assembled using commercially available parts. The separation membrane 18 is removable. The measurement is purely physical and does not require any wet chemical processes. The signal is linear and has a fast response time in the range of a few seconds. Other gas components exhibiting acidic or basic reactions do not affect the measurement.

[0174] The gas density sensor 28 is equipped with a resonant quartz oscillator fork. As the gas density increases, the oscillation frequency decreases.

[0175] Potential applications include biochemical processes, particularly CO2 measurement in disposable bioreactors. Further applications include the food and beverage industry, water management (wastewater treatment and drinking water supply), CO2 capture and storage, and direct measurement of CO2 emissions in industry. Applications in the energy supply sector are also possible, such as producing methane and other hydrocarbon gases from surplus renewable energy for later energy generation, or fuel production from algae cultivation. [Explanation of symbols]

[0176] 10 Measuring device 12 Gas mixtures 12a Gas mixture in the gas phase of a bioreactor 12b Gas mixture liquefied in the liquid phase of a bioreactor 12c Gas mixture in the measuring chamber 14. Reaction vessel (e.g., bioreactor) 16 Measuring tank 18 Separation membrane 20 Measuring devices 22. Combined Sensors 24 Temperature Sensors 26 Pressure Sensor 28 Gas density sensor 30 Evaluation device 32 Analysis and Control Units 34 processors 36 storage 38 filters 40 Heater 42 Humidity Sensor 44 External sensors 48 Couplings 50. Reaction systems (e.g., bioreactor systems) 52 Stirrer 54 Drive unit 56 pH sensor 58. Dissolved Oxygen (DO) Sensor 60 steam 64 water 66 Acidic media 68 Alkaline media 70 Nutritional Solution 72 Air 74 Exhaust gas 78 Transmitters and Controllers 80 disposable bioreactors 82 Separation Membrane Module 84 plates X, Y, Z gas components A and B gas component groups Concentration of gas component X (CX) Concentration of gas component Y in CY Concentration of CZ gas component Z Concentration of CA gas component group A Concentration of gas component group B (CB) Partial pressure of gas component group B in PB GD Gas Density df frequency difference QCM

Claims

1. A method for measuring the concentration (Cz) of a target gas component (Z) in a gas mixture (12b) of known composition in a liquid solution in a reaction vessel (14), a) A step of providing a measuring tank (16) separated from the reaction tank (14) by a separation membrane (18) that is impermeable to liquids but permeable to gases, b) A step of measuring the gas density ρ, gas pressure p, and temperature T in the measuring chamber (16), c) A measurement method comprising the step of determining the concentration (Cz) of the gas component (Z) to be measured from the value measured in step b) and the molar mass of the gas components (X, Y, Z) of the gas mixture.

2. The measurement method according to claim 1, Step a) is a1) Set the volume V of the measuring chamber (16) to 5000 mm ３ Less than 1000 mm, preferably 1000 mm ３ Less than 5 mm, preferably 5 mm ３ ≤V ≤ 15mm ３ , especially 1 mm ３ ≤V ≤ 10mm ３ Preferably 2 mm ３ ≤V ≤ 5mm ３ A measurement method that includes a step of setting [something].

3. A measurement method according to claim 1 or 2, Step b) is b1) A measurement method comprising the step of measuring the gas density using a tuning fork type gas density sensor.

4. A measurement method according to any one of claims 1 to 3, The reaction tank is selected from the group consisting of a bioreactor, a disposable bioreactor, a bioreactor with a diameter of less than 10 mm, a reaction tank in the food industry, a reaction tank for beverage production, a reaction tank in a brewery, a reaction tank in a wastewater treatment facility, CO ２ A measuring method selected from the group including an extraction plant reaction tank, a process classification reaction tank, piping, and water pipes.

5. A measurement method according to any one of claims 1 to 4, The gas component (Z) to be measured is, 5.1 The gaseous components of the gas mixture (12b) that change during the reaction in the reaction vessel (14), in particular the gaseous components of the biological reaction in the reaction vessel (14) designed as a bioreactor, and / or 5.2 CO ２ The measurement method.

6. A measurement method according to any one of claims 1 to 5, The gas mixture (12b) is air, industrial air, dry air, and / or O ２ and N ２ A measurement method characterized by comprising at least one further gaseous component (X, Y) from the group consisting of a mixture of the above.

7. A measurement method according to any one of claims 1 to 6, 7.1 Steps for measuring relative humidity, and / or 7.2 The step of measuring the content of other gases in the gas mixture, comprising at least one of the steps, Here, step c) is a measurement method determined based on the value measured in step 7.1 or 7.

2.

8. A measurement method according to any one of claims 1 to 7, A measurement method comprising: the gas mixture (12b) being divided into the gas component (Z) to be measured and a group of components (A) consisting of further components (X, Y) of the gas mixture (12b); the weighted average molar mass of the group of components (A) being determined based on the concentrations of the components (X, Y); and in step c), the concentration of the gas component (Z) to be measured being determined from the molar mass and weighted average molar mass of the gas component (Z) to be measured.

9. A measurement method according to any one of claims 1 to 8, A measurement method in which the gas density is measured using a quartz crystal oscillator in step b).

10. A reaction method for carrying out a reaction in a reaction vessel (14), The steps include: measuring the concentration of the gas component of the gas mixture (12b) in the reaction vessel (14) according to the measurement method described in any one of claims 1 to 9; A reaction method comprising the step of performing a further step of the reaction method in accordance with the measurement.

11. The reaction method according to claim 10, A reaction method wherein the reaction method is a bioreaction process for carrying out a biological reaction in a reaction vessel designed as a bioreactor.

12. The reaction method according to claim 11, A reaction method in which the biological reaction includes cell culture in a bioreactor (14).

13. A measuring device (10) for measuring the concentration (CZ) of a target gas component (Z) of a gas mixture (12b) having a known composition in a reaction vessel (14), The measuring tank (16) is equipped with a gas-permeable separation membrane (18) for separating the measuring tank (16) from the inside of the reaction tank (14), A measuring device (20) for measuring the gas density ρ, gas pressure p, and temperature T in the measuring chamber (16), and A measuring device comprising: an evaluation device (30) configured to determine the concentration of a gas component to be measured from the value measured by the measuring device (20) and the molar mass of the gas component in the gas mixture (12b).

14. A measuring device according to claim 13, The measuring chamber (16) has a measuring chamber volume V, where 0.5 mm ３ ≤V ≤ 15mm ３ , especially 1 mm ３ ≤V ≤ 10mm ３ Preferably 2 mm ３ ≤V ≤ 5mm ３ It is a measuring device.

15. A measuring device according to claim 13 or 14, The measuring device (20) includes a quartz crystal oscillator and / or a tuning fork type sensor for measuring the gas density.

16. A measuring device (10) according to any one of claims 13 to 15, 16.1 Humidity measuring device (42) and / or for measuring relative humidity 16.2 Includes a gas content measuring device (44, 58) for measuring the content of at least one other gas in the gas mixture, The evaluation device (30) is a measuring device configured to determine the concentration based on the measurements of the humidity measuring device and / or the gas content measuring device.

17. A measuring device (10) according to any one of claims 13 to 16, The evaluation device (30) is used to determine the O in the reaction vessel (14). ２ and N ２ CO2 in the atmosphere ２ A measuring device configured to determine concentration.

18. A measuring device (10) according to any one of claims 13 to 17, which is designed to perform the measuring method according to any one of claims 1 to 10.

19. A reaction system (50) for carrying out a reaction, The system includes a reaction vessel (14) and a measuring device (20) according to any one of claims 13 to 18. A reaction system in which the measuring tank (16) is separated from the inside of the reaction tank (14) by a separation membrane (18).

20. The reaction system (50) according to claim 19, The aforementioned reaction vessel is a bioreactor, a disposable bioreactor (80), a bioreactor with a diameter of less than 10 mm, a reaction vessel for the food industry, a reaction vessel for beverage manufacturing, a reaction vessel for a brewery, a reaction vessel for a wastewater treatment plant, CO ２ A reaction system (50) selected from a group consisting of reaction vessels for extraction plants, reaction vessels for process analysis, pipelines, and water pipes.

21. The measuring device (10) comprises a measuring tank (16) partitioned by a gas-permeable separation membrane (18), and a measuring device (10) for measuring the gas density ρ, gas pressure p, and temperature T within the measuring tank (16), and is used to measure the concentration (Cz) of the gas component (Z) of the gas mixture (12b) to be measured in a reaction tank (14) separated from the measuring tank (16) by the separation membrane (18).

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