Gernot John - Academia.edu (original) (raw)
Papers by Gernot John
Chemie Ingenieur Technik
This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Shake flasks enjoy great popularity due to their ease-of-use and cost-effectiveness. Furthermore, using single-use shake flasks provides additional advantages over reusable glass versions, since they do not need to be cleaned or pre-sterilized. Despite their widespread use, there are, however, clear disadvantages associated with using shake flasks, which are primarily related to the lack of measurement and control capabilities. Therefore, this article discusses how optical sensors can be employed in single-use shake flasks to measure oxygen, carbon dioxide, pH, and biomass.
Bioengineering
Shake flasks remain one of the most widely used cultivation systems in biotechnology, especially ... more Shake flasks remain one of the most widely used cultivation systems in biotechnology, especially for process development (cell line and parameter screening). This can be justified by their ease of use as well as their low investment and running costs. A disadvantage, however, is that cultivations in shake flasks are black box processes with reduced possibilities for recording online data, resulting in a lack of control and time-consuming, manual data analysis. Although different measurement methods have been developed for shake flasks, they lack comparability, especially when changing production organisms. In this study, the use of online backscattered light, dissolved oxygen, and pH data for characterization of animal, plant, and microbial cell culture processes in shake flasks are evaluated and compared. The application of these different online measurement techniques allows key performance indicators (KPIs) to be determined based on online data. This paper evaluates a novel data ...
Shake flasks have been the work horse for microbial small-scale fermentations for decades. Recent... more Shake flasks have been the work horse for microbial small-scale fermentations for decades. Recently, single use versions are becoming more and more popular -for both microbial and cell cultivation. Typically, these vessels are still used as black boxes because no online measurement is integrated. The non-invasive measurement of oxygen and pH using chemical-optical sensors has already been commercially available for several years. With the recently added ability to measure biomass online, the metabolism is even more visible. This presentation discusses the use of a multitude of sensors in the small scale of shake flasks. It also presents a prototype sensor for the online measurement of CO2 that was developed recently and integrated into a multi-parameter platform. Applications are various: Although the CO2 sensor is only a prototype it is possible to follow a diauxie of E. coli cultivations online, while small changes in the growth curve detected by the biomass sensor indicate the exact time of limitations which was shown for different organisms. This presentation includes research supported by the Federal Ministry for Economic Affairs and Energy (BMWi) via AIF within the ZIM-Initiative #KF2516104FR2
Sensors, 2018
Buried explosive material, e.g., landmines, represent a severe issue for human safety all over th... more Buried explosive material, e.g., landmines, represent a severe issue for human safety all over the world. Most explosives consist of environmentally hazardous chemicals like 2,4,6-trinitrotoluene (TNT), carcinogenic 2,4-dinitrotoluene (2,4-DNT) and related compounds. Vapors leaking from buried landmines offer a detection marker for landmines, presenting an option to detect landmines without relying on metal detection. 2,4-Dinitrotoluene (DNT), an impurity and byproduct of common TNT synthesis, is a feasible detection marker since it is extremely volatile. We report on the construction of a wireless, handy and cost effective 2,4-dinitrotoluene biosensor combining recombinant bioluminescent bacterial cells and a compact, portable optical detection device. This biosensor could serve as a potential alternative to the current detection technique. The influence of temperature, oxygen and different immobilization procedures on bioluminescence were tested. Oxygen penetration depth in agaros...
BIOspektrum, 2015
Spinner flasks are often used for microcarrier-based cultivations of human mesenchymal stem cells... more Spinner flasks are often used for microcarrier-based cultivations of human mesenchymal stem cells (hMSCs). Normally, they are not equipped with pH and dissolved oxygen (DO) probes. This application note describes the cultivation of hMSCs in single-use spinner flasks equipped with optical pH (SP-HP8) and DO (SP-PSt3) sensors for the first time. While reaching peak cell numbers between 4.1 × 10 7 cells and 5.9 × 10 7 cells in two cultivation runs, reliable DO and pH data were delivered.
Engineering in Life Sciences, 2014
A novel online sensor system for noninvasive and continuous monitoring of cell growth in shake fl... more A novel online sensor system for noninvasive and continuous monitoring of cell growth in shake flasks is described. The measurement principle is based on turbidity measurement by detecting 180°-scattered light and correlation to OD by nonlinear calibration models. The sensor system was integrated into a commercial shaking tablar to read out turbidity from below the shake flasks bottom. The system was evaluated with two model microorganisms, Escherichic coli K12 as prokaryotic and Saccharomyces cerevisiae as eukaryotic model. The sensor allowed an accurate monitoring of turbidity and correlation with OD 600 ≤ 30. The determination of online OD showed relative errors of about 7.5 % for E. coli K12 and 12 % for S. cerevisiae. This matches the errors of the laborious offline OD and thus facilitates to overcome the drawbacks of the classical method as risk of contamination and decreasing volumes through sampling. One major challenge was to ensure a defined, nonvarying measurement zone as the rotating suspension in the shake flask forms a liquid sickle which circulates round the flasks inner bottom wall. The resulting alteration of liquid height above the sensor could be compensated by integration of an acceleration sensor into the tablar to synchronize the sensor triggering.
Chemie Ingenieur Technik, 2014
ABSTRACT No abstract is available for this article.
Sensors (Basel, Switzerland), 2014
In the context of this work we evaluated a multisensory, noninvasive prototype platform for shake... more In the context of this work we evaluated a multisensory, noninvasive prototype platform for shake flask cultivations by monitoring three basic parameters (pH, pO2 and biomass). The focus lies on the evaluation of the biomass sensor based on backward light scattering. The application spectrum was expanded to four new organisms in addition to E. coli K12 and S. cerevisiae [1]. It could be shown that the sensor is appropriate for a wide range of standard microorganisms, e.g., L. zeae, K. pastoris, A. niger and CHO-K1. The biomass sensor signal could successfully be correlated and calibrated with well-known measurement methods like OD600, cell dry weight (CDW) and cell concentration. Logarithmic and Bleasdale-Nelder derived functions were adequate for data fitting. Measurements at low cell concentrations proved to be critical in terms of a high signal to noise ratio, but the integration of a custom made light shade in the shake flask improved these measurements significantly. This senso...
Journal of laboratory automation, Jan 26, 2015
In this study, a slow-responding chemo-optical sensor for dissolved oxygen (DO) integrated into a... more In this study, a slow-responding chemo-optical sensor for dissolved oxygen (DO) integrated into a 96-well plate was developed. The slow response time ensures that the measured oxygen value does not change much during plate transport to the microplate reader. The sensor therefore permits at-line DO measurement of microbial cultures. Moreover, it eliminates the necessity of individual optical measurement systems for each culture plate, as many plates can be measured successively. Combined with the 96-well format, this increases the experimental throughput enormously. The novel sensor plate (Slow OxoPlate) consists of fluorophores suspended in a polymer matrix that were placed into u-bottom 96-well plates. Response time was measured using sodium sulfite, and a t90 value of 9.7 min was recorded. For application, DO values were then measured in Escherichia coli and Saccharomyces cerevisiae cultures grown under fed-batch-like conditions. Depending on the DO sensor's response time, dif...
On-line measurement of dissolved O 2 in shake-flasks was realized via immobilized sensor spots co... more On-line measurement of dissolved O 2 in shake-flasks was realized via immobilized sensor spots containing a fluorophore with an O 2-dependent luminescent decay time. An unaffected sensor signal during 80 autoclaving cycles suggests multi-usage of sensor equipped shake-flasks. The sensor had a response time of 6 s. Quantification of gas-liquid mass transfer revealed maximum k L a values of 150 h-1 , from which maximum O 2 transfer capacity of 33 mM h-1 was calculated. Liquid volume and shaking frequency have a strong influence on k L a. Exemplified by cultivations of Corynebacterium glutamicum the importance of shaking rate for O 2 supply of bacterial cultures is shown. Sampling of microbial cultures with intermittent shaking of a few minutes can cause O 2 limitation. Based on the results of this work a simple and straightforward tool is now available for accurate O 2 sensing in shake-flasks, which are widely used in microbial cultivations.
Sensors and Actuators B: Chemical, 2006
ABSTRACT This paper describes properties of microplates (MTPs) with integrated, fluorescence-base... more ABSTRACT This paper describes properties of microplates (MTPs) with integrated, fluorescence-based sensors for pH and oxygen, and their application for enzyme screening and monitoring of bacterial respiratory activity. Thin, hydrophilic sensing films consisting of an analyte-sensitive indicator and a reference fluorophore, are deposited on the bottom of the MTP. This allows for calibration-free quantification of pH and pO2 with an acceptable accuracy and resolution for applications such as enzyme activity screening, respirometry, or toxicological assays. The sensor properties are mainly investigated with respect to their qualification for such applications. Specifically, enzyme activity screening is demonstrated for glucose oxidase using oxygen-sensitive plates, and for bacterial growth monitoring of Escherichia coli and Pseudomonas putida. Furthermore, a toxicological assay monitoring the respiration activity of P. putida was converted into a microplate format.
Journal of Dairy Research, 2003
A new method for characterization of acid production by dairy starter cultures is presented. Micr... more A new method for characterization of acid production by dairy starter cultures is presented. Microplates with integrated optical pH sensors are developed. Two fluorophores, a pH-sensitive and a pH-insensitive one are immobilised at the bottom of a polystyrene 96-well microtitre plate. The pH-insensitive fluorophore serves as an internal reference and makes calibration unnecessary. The sensor measures pH accurately in optically well-defined media. Particles and fluorophores contained in the bulk medium disturbed the measurements. Despite these disturbances it was possible to clearly sense differences in inoculum type and in inoculum sizes of cultures of Lactococcus lactis and of Streptococcus thermophilus at 30 and 37°C. Besides a pH-related signal there is information about other changes during milk fermentation. The cultivation results were compared with those from the established CINAC-method. From this comparison it can be concluded that the new method can be used reliably to cha...
Engineering in Life Sciences, 2010
While wave-mixed and stirred bag bioreactors are common devices for rapid, safe insect cell cultu... more While wave-mixed and stirred bag bioreactors are common devices for rapid, safe insect cell culturebased production at liter-scale, orbitally shaken disposable flasks are mainly used for screening studies at milliliter-scale. In contrast to the two aforementioned bag bioreactor types, which can be operated with standard or disposable sensors, shaker flasks have not been instrumented until recently. The combination of 250mL disposable shake flasks with PreSens's Shake Flask Reader enables both pH and dissolved oxygen to be measured, as well as allowing characterization of oxygen mass transfer. Volumetric oxygen transfer coefficients (k L a-values) for PreSens 250mL disposable shake flasks, which were determined for the first time in insect cell culture medium at varying culture volumes and shaker frequencies, ranged between 4.4 and 37.9/h. Moreover, it was demonstrated that online monitoring of dissolved oxygen in shake flasks is relevant for limitation-free growth of insect cells up to high cell densities in batch mode(1.6_10 7 cells/mL) and for the efficient expression of an intracellular model protein.
Current Microbiology, 2004
We have evaluated the OxoPlate OP96F for its usefulness in the characterization of antibacterial ... more We have evaluated the OxoPlate OP96F for its usefulness in the characterization of antibacterial compounds. Each well of the plate carries two different fluorescent dyes, an oxygen-sensitive indicator dye and a reference dye. Fluorescence intensity is proportional to the amount of dissolved oxygen in the medium. Growth of bacterial cells is measured indirectly via the gradual depletion of oxygen. In this study, Bacillus subtilis was treated with 14 different antibacterials of various modes of action. The OxoPlate was not only useful to deduce MIC values directly from the oxygen-depletion curves, but was also able to discriminate bactericidal from bacteriostatic compounds. Oxygen levels of cultures treated with bactericidal compounds dropped initially and then increased after the cells had died. In contrast, oxygen levels remained low when cultures were treated with bacteriostatic compounds. The system described herein gives valuable insight into the kinetics of growth inhibition and killing, and we conclude that the OxoPlate is highly useful for the initial characterization of antibacterials.
Chemie Ingenieur Technik, 2012
Chemie Ingenieur Technik
This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Shake flasks enjoy great popularity due to their ease-of-use and cost-effectiveness. Furthermore, using single-use shake flasks provides additional advantages over reusable glass versions, since they do not need to be cleaned or pre-sterilized. Despite their widespread use, there are, however, clear disadvantages associated with using shake flasks, which are primarily related to the lack of measurement and control capabilities. Therefore, this article discusses how optical sensors can be employed in single-use shake flasks to measure oxygen, carbon dioxide, pH, and biomass.
Bioengineering
Shake flasks remain one of the most widely used cultivation systems in biotechnology, especially ... more Shake flasks remain one of the most widely used cultivation systems in biotechnology, especially for process development (cell line and parameter screening). This can be justified by their ease of use as well as their low investment and running costs. A disadvantage, however, is that cultivations in shake flasks are black box processes with reduced possibilities for recording online data, resulting in a lack of control and time-consuming, manual data analysis. Although different measurement methods have been developed for shake flasks, they lack comparability, especially when changing production organisms. In this study, the use of online backscattered light, dissolved oxygen, and pH data for characterization of animal, plant, and microbial cell culture processes in shake flasks are evaluated and compared. The application of these different online measurement techniques allows key performance indicators (KPIs) to be determined based on online data. This paper evaluates a novel data ...
Shake flasks have been the work horse for microbial small-scale fermentations for decades. Recent... more Shake flasks have been the work horse for microbial small-scale fermentations for decades. Recently, single use versions are becoming more and more popular -for both microbial and cell cultivation. Typically, these vessels are still used as black boxes because no online measurement is integrated. The non-invasive measurement of oxygen and pH using chemical-optical sensors has already been commercially available for several years. With the recently added ability to measure biomass online, the metabolism is even more visible. This presentation discusses the use of a multitude of sensors in the small scale of shake flasks. It also presents a prototype sensor for the online measurement of CO2 that was developed recently and integrated into a multi-parameter platform. Applications are various: Although the CO2 sensor is only a prototype it is possible to follow a diauxie of E. coli cultivations online, while small changes in the growth curve detected by the biomass sensor indicate the exact time of limitations which was shown for different organisms. This presentation includes research supported by the Federal Ministry for Economic Affairs and Energy (BMWi) via AIF within the ZIM-Initiative #KF2516104FR2
Sensors, 2018
Buried explosive material, e.g., landmines, represent a severe issue for human safety all over th... more Buried explosive material, e.g., landmines, represent a severe issue for human safety all over the world. Most explosives consist of environmentally hazardous chemicals like 2,4,6-trinitrotoluene (TNT), carcinogenic 2,4-dinitrotoluene (2,4-DNT) and related compounds. Vapors leaking from buried landmines offer a detection marker for landmines, presenting an option to detect landmines without relying on metal detection. 2,4-Dinitrotoluene (DNT), an impurity and byproduct of common TNT synthesis, is a feasible detection marker since it is extremely volatile. We report on the construction of a wireless, handy and cost effective 2,4-dinitrotoluene biosensor combining recombinant bioluminescent bacterial cells and a compact, portable optical detection device. This biosensor could serve as a potential alternative to the current detection technique. The influence of temperature, oxygen and different immobilization procedures on bioluminescence were tested. Oxygen penetration depth in agaros...
BIOspektrum, 2015
Spinner flasks are often used for microcarrier-based cultivations of human mesenchymal stem cells... more Spinner flasks are often used for microcarrier-based cultivations of human mesenchymal stem cells (hMSCs). Normally, they are not equipped with pH and dissolved oxygen (DO) probes. This application note describes the cultivation of hMSCs in single-use spinner flasks equipped with optical pH (SP-HP8) and DO (SP-PSt3) sensors for the first time. While reaching peak cell numbers between 4.1 × 10 7 cells and 5.9 × 10 7 cells in two cultivation runs, reliable DO and pH data were delivered.
Engineering in Life Sciences, 2014
A novel online sensor system for noninvasive and continuous monitoring of cell growth in shake fl... more A novel online sensor system for noninvasive and continuous monitoring of cell growth in shake flasks is described. The measurement principle is based on turbidity measurement by detecting 180°-scattered light and correlation to OD by nonlinear calibration models. The sensor system was integrated into a commercial shaking tablar to read out turbidity from below the shake flasks bottom. The system was evaluated with two model microorganisms, Escherichic coli K12 as prokaryotic and Saccharomyces cerevisiae as eukaryotic model. The sensor allowed an accurate monitoring of turbidity and correlation with OD 600 ≤ 30. The determination of online OD showed relative errors of about 7.5 % for E. coli K12 and 12 % for S. cerevisiae. This matches the errors of the laborious offline OD and thus facilitates to overcome the drawbacks of the classical method as risk of contamination and decreasing volumes through sampling. One major challenge was to ensure a defined, nonvarying measurement zone as the rotating suspension in the shake flask forms a liquid sickle which circulates round the flasks inner bottom wall. The resulting alteration of liquid height above the sensor could be compensated by integration of an acceleration sensor into the tablar to synchronize the sensor triggering.
Chemie Ingenieur Technik, 2014
ABSTRACT No abstract is available for this article.
Sensors (Basel, Switzerland), 2014
In the context of this work we evaluated a multisensory, noninvasive prototype platform for shake... more In the context of this work we evaluated a multisensory, noninvasive prototype platform for shake flask cultivations by monitoring three basic parameters (pH, pO2 and biomass). The focus lies on the evaluation of the biomass sensor based on backward light scattering. The application spectrum was expanded to four new organisms in addition to E. coli K12 and S. cerevisiae [1]. It could be shown that the sensor is appropriate for a wide range of standard microorganisms, e.g., L. zeae, K. pastoris, A. niger and CHO-K1. The biomass sensor signal could successfully be correlated and calibrated with well-known measurement methods like OD600, cell dry weight (CDW) and cell concentration. Logarithmic and Bleasdale-Nelder derived functions were adequate for data fitting. Measurements at low cell concentrations proved to be critical in terms of a high signal to noise ratio, but the integration of a custom made light shade in the shake flask improved these measurements significantly. This senso...
Journal of laboratory automation, Jan 26, 2015
In this study, a slow-responding chemo-optical sensor for dissolved oxygen (DO) integrated into a... more In this study, a slow-responding chemo-optical sensor for dissolved oxygen (DO) integrated into a 96-well plate was developed. The slow response time ensures that the measured oxygen value does not change much during plate transport to the microplate reader. The sensor therefore permits at-line DO measurement of microbial cultures. Moreover, it eliminates the necessity of individual optical measurement systems for each culture plate, as many plates can be measured successively. Combined with the 96-well format, this increases the experimental throughput enormously. The novel sensor plate (Slow OxoPlate) consists of fluorophores suspended in a polymer matrix that were placed into u-bottom 96-well plates. Response time was measured using sodium sulfite, and a t90 value of 9.7 min was recorded. For application, DO values were then measured in Escherichia coli and Saccharomyces cerevisiae cultures grown under fed-batch-like conditions. Depending on the DO sensor's response time, dif...
On-line measurement of dissolved O 2 in shake-flasks was realized via immobilized sensor spots co... more On-line measurement of dissolved O 2 in shake-flasks was realized via immobilized sensor spots containing a fluorophore with an O 2-dependent luminescent decay time. An unaffected sensor signal during 80 autoclaving cycles suggests multi-usage of sensor equipped shake-flasks. The sensor had a response time of 6 s. Quantification of gas-liquid mass transfer revealed maximum k L a values of 150 h-1 , from which maximum O 2 transfer capacity of 33 mM h-1 was calculated. Liquid volume and shaking frequency have a strong influence on k L a. Exemplified by cultivations of Corynebacterium glutamicum the importance of shaking rate for O 2 supply of bacterial cultures is shown. Sampling of microbial cultures with intermittent shaking of a few minutes can cause O 2 limitation. Based on the results of this work a simple and straightforward tool is now available for accurate O 2 sensing in shake-flasks, which are widely used in microbial cultivations.
Sensors and Actuators B: Chemical, 2006
ABSTRACT This paper describes properties of microplates (MTPs) with integrated, fluorescence-base... more ABSTRACT This paper describes properties of microplates (MTPs) with integrated, fluorescence-based sensors for pH and oxygen, and their application for enzyme screening and monitoring of bacterial respiratory activity. Thin, hydrophilic sensing films consisting of an analyte-sensitive indicator and a reference fluorophore, are deposited on the bottom of the MTP. This allows for calibration-free quantification of pH and pO2 with an acceptable accuracy and resolution for applications such as enzyme activity screening, respirometry, or toxicological assays. The sensor properties are mainly investigated with respect to their qualification for such applications. Specifically, enzyme activity screening is demonstrated for glucose oxidase using oxygen-sensitive plates, and for bacterial growth monitoring of Escherichia coli and Pseudomonas putida. Furthermore, a toxicological assay monitoring the respiration activity of P. putida was converted into a microplate format.
Journal of Dairy Research, 2003
A new method for characterization of acid production by dairy starter cultures is presented. Micr... more A new method for characterization of acid production by dairy starter cultures is presented. Microplates with integrated optical pH sensors are developed. Two fluorophores, a pH-sensitive and a pH-insensitive one are immobilised at the bottom of a polystyrene 96-well microtitre plate. The pH-insensitive fluorophore serves as an internal reference and makes calibration unnecessary. The sensor measures pH accurately in optically well-defined media. Particles and fluorophores contained in the bulk medium disturbed the measurements. Despite these disturbances it was possible to clearly sense differences in inoculum type and in inoculum sizes of cultures of Lactococcus lactis and of Streptococcus thermophilus at 30 and 37°C. Besides a pH-related signal there is information about other changes during milk fermentation. The cultivation results were compared with those from the established CINAC-method. From this comparison it can be concluded that the new method can be used reliably to cha...
Engineering in Life Sciences, 2010
While wave-mixed and stirred bag bioreactors are common devices for rapid, safe insect cell cultu... more While wave-mixed and stirred bag bioreactors are common devices for rapid, safe insect cell culturebased production at liter-scale, orbitally shaken disposable flasks are mainly used for screening studies at milliliter-scale. In contrast to the two aforementioned bag bioreactor types, which can be operated with standard or disposable sensors, shaker flasks have not been instrumented until recently. The combination of 250mL disposable shake flasks with PreSens's Shake Flask Reader enables both pH and dissolved oxygen to be measured, as well as allowing characterization of oxygen mass transfer. Volumetric oxygen transfer coefficients (k L a-values) for PreSens 250mL disposable shake flasks, which were determined for the first time in insect cell culture medium at varying culture volumes and shaker frequencies, ranged between 4.4 and 37.9/h. Moreover, it was demonstrated that online monitoring of dissolved oxygen in shake flasks is relevant for limitation-free growth of insect cells up to high cell densities in batch mode(1.6_10 7 cells/mL) and for the efficient expression of an intracellular model protein.
Current Microbiology, 2004
We have evaluated the OxoPlate OP96F for its usefulness in the characterization of antibacterial ... more We have evaluated the OxoPlate OP96F for its usefulness in the characterization of antibacterial compounds. Each well of the plate carries two different fluorescent dyes, an oxygen-sensitive indicator dye and a reference dye. Fluorescence intensity is proportional to the amount of dissolved oxygen in the medium. Growth of bacterial cells is measured indirectly via the gradual depletion of oxygen. In this study, Bacillus subtilis was treated with 14 different antibacterials of various modes of action. The OxoPlate was not only useful to deduce MIC values directly from the oxygen-depletion curves, but was also able to discriminate bactericidal from bacteriostatic compounds. Oxygen levels of cultures treated with bactericidal compounds dropped initially and then increased after the cells had died. In contrast, oxygen levels remained low when cultures were treated with bacteriostatic compounds. The system described herein gives valuable insight into the kinetics of growth inhibition and killing, and we conclude that the OxoPlate is highly useful for the initial characterization of antibacterials.
Chemie Ingenieur Technik, 2012