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
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...
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...
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...
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.
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...
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
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...
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...
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...
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.
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...
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