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Minireactor-based high-throughput temperature profiling for the optimization of microbial and enzymatic processes.

Kunze M, Lattermann C, Diederichs S, Kroutil W, Büchs J - J Biol Eng (2014)

Bottom Line: Microtiter plate-based high-throughput temperature profiling is a convenient tool for characterizing temperature dependent reaction processes.It allows the evaluation of numerous conditions, e.g. microorganisms, enzymes, media, and others, in a short time.The simple temperature control combined with a commercial on-line monitoring device makes it a user friendly system.

View Article: PubMed Central - HTML - PubMed

Affiliation: AVT-Chair for Biochemical Engineering, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.

ABSTRACT

Background: Bioprocesses depend on a number of different operating parameters and temperature is one of the most important ones. Unfortunately, systems for rapid determination of temperature dependent reaction kinetics are rare. Obviously, there is a need for a high-throughput screening procedure of temperature dependent process behavior. Even though, well equipped micro-bioreactors are a promising approach sufficient temperature control is quite challenging and rather complex.

Results: In this work a unique system is presented combining an optical on-line monitoring device with a customized temperature control unit for 96 well microtiter plates. By exposing microtiter plates to specific temperature profiles, high-throughput temperature optimization for microbial and enzymatic systems in a micro-scale of 200 μL is realized. For single well resolved temperature measurement fluorescence thermometry was used, combining the fluorescent dyes Rhodamin B and Rhodamin 110. The real time monitoring of the microbial and enzymatic reactions provides extensive data output. To evaluate this novel system the temperature optima for Escherichia coli and Kluyveromyces lactis regarding growth and recombinant protein production were determined. Furthermore, the commercial cellulase mixture Celluclast as a representative for enzymes was investigated applying a fluorescent activity assay.

Conclusion: Microtiter plate-based high-throughput temperature profiling is a convenient tool for characterizing temperature dependent reaction processes. It allows the evaluation of numerous conditions, e.g. microorganisms, enzymes, media, and others, in a short time. The simple temperature control combined with a commercial on-line monitoring device makes it a user friendly system.

No MeSH data available.


Related in: MedlinePlus

Optical temperature measurement in MTPs applying a fluorescent assay with the dyes Rhodamin B and Rhodamin 110. (A) Progress of the fluorescence signals of Rhodamin B (RhB) and Rhodamin 110 (Rh110) and their calculated ratio in a single well with varied thermostat set point temperature. Contrary to Figure 1C heating and cooling circulation systems were operated with one thermostat. (B) Calibration curve of the well temperature (measured via PT100 thermometer) vs. the fluorescence ratio RhB/Rh110. Experimental conditions: 96well MTP, VL = 200 μL, n = 995 rpm, d0 = 3 mm, RT = 37°C.
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Figure 2: Optical temperature measurement in MTPs applying a fluorescent assay with the dyes Rhodamin B and Rhodamin 110. (A) Progress of the fluorescence signals of Rhodamin B (RhB) and Rhodamin 110 (Rh110) and their calculated ratio in a single well with varied thermostat set point temperature. Contrary to Figure 1C heating and cooling circulation systems were operated with one thermostat. (B) Calibration curve of the well temperature (measured via PT100 thermometer) vs. the fluorescence ratio RhB/Rh110. Experimental conditions: 96well MTP, VL = 200 μL, n = 995 rpm, d0 = 3 mm, RT = 37°C.

Mentions: To characterize the behavior of microbial or enzymatic systems at different temperatures it is necessary to know the specific temperature in each single well. Equipping each well with a temperature sensor requires a very high degree of instrumentation. Just by using a temperature dependent fluorophore measurements can easily be done with the optical on-line monitoring system. In this work a combination of the fluorescent dyes Rhodamin B (RhB) and Rhodamin 110 (Rh110) was applied, where RhB is the temperature sensitive compound, whereas Rh110 acts as a reference. This measuring principle was described before [21]. In Figure 2A the fluorescence intensity of both dyes depending on the temperature is shown. Therefore, the thermostating block was tempered only by one thermostat to ensure a constant temperature distribution over the whole MTP. Thermostat set point temperatures from 5-95°C were adjusted. After each temperature shift (dotted vertical lines) the experimental conditions remained unchanged until both fluorescence signals showed constant values. The RhB signal decreases in a step like manner since its fluorescence intensity is decreasing with increasing temperature. After each temperature shift the fluorescence signal drops sharply before it remains constant when the temperature reaches its equilibrium. On the contrary, the fluorescence intensity of Rh110 is almost constant over the whole time. The slight decrease is due to a bleaching effect which is known to happen to RhB as well. To obtain a reliable measuring signal, the ratio of both fluorescence intensities was calculated [21].It must be considered that the thermostat set point temperatures in Figure 2A and the actual temperatures in the MTP’s wells are not identical since heat may be lost to the environment. For this reason, one well was equipped with an in-house constructed PT100 temperature sensor. In this way, the corresponding well temperatures for various RhB/Rh110 ratios were determined. In Figure 2B the resulting calibration curve is depicted. It is described by a polynomial equation of second degree applying MS Excel. The fluorescence ratios are average values of five measurements in one well. The maximum relative standard deviation was 0.4%. For further investigation of the measuring accuracy the heating block temperature, as well as the room temperature, was adjusted to 37°C. In this way, a constant temperature of 37°C in each well could be assumed. The regarding measurement of all 96 wells revealed an average value of 37°C with a maximum of 38.7°C and a minimum of 35.6°C. The standard deviation was 0.76 K. The reason for the deviation from well to well cannot be explained completely. Slight deviations in the properties of the transparent microtiter plate bottom are possible which may influence the optical signals. A systematic position effect could be excluded during the experiments.


Minireactor-based high-throughput temperature profiling for the optimization of microbial and enzymatic processes.

Kunze M, Lattermann C, Diederichs S, Kroutil W, Büchs J - J Biol Eng (2014)

Optical temperature measurement in MTPs applying a fluorescent assay with the dyes Rhodamin B and Rhodamin 110. (A) Progress of the fluorescence signals of Rhodamin B (RhB) and Rhodamin 110 (Rh110) and their calculated ratio in a single well with varied thermostat set point temperature. Contrary to Figure 1C heating and cooling circulation systems were operated with one thermostat. (B) Calibration curve of the well temperature (measured via PT100 thermometer) vs. the fluorescence ratio RhB/Rh110. Experimental conditions: 96well MTP, VL = 200 μL, n = 995 rpm, d0 = 3 mm, RT = 37°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4128537&req=5

Figure 2: Optical temperature measurement in MTPs applying a fluorescent assay with the dyes Rhodamin B and Rhodamin 110. (A) Progress of the fluorescence signals of Rhodamin B (RhB) and Rhodamin 110 (Rh110) and their calculated ratio in a single well with varied thermostat set point temperature. Contrary to Figure 1C heating and cooling circulation systems were operated with one thermostat. (B) Calibration curve of the well temperature (measured via PT100 thermometer) vs. the fluorescence ratio RhB/Rh110. Experimental conditions: 96well MTP, VL = 200 μL, n = 995 rpm, d0 = 3 mm, RT = 37°C.
Mentions: To characterize the behavior of microbial or enzymatic systems at different temperatures it is necessary to know the specific temperature in each single well. Equipping each well with a temperature sensor requires a very high degree of instrumentation. Just by using a temperature dependent fluorophore measurements can easily be done with the optical on-line monitoring system. In this work a combination of the fluorescent dyes Rhodamin B (RhB) and Rhodamin 110 (Rh110) was applied, where RhB is the temperature sensitive compound, whereas Rh110 acts as a reference. This measuring principle was described before [21]. In Figure 2A the fluorescence intensity of both dyes depending on the temperature is shown. Therefore, the thermostating block was tempered only by one thermostat to ensure a constant temperature distribution over the whole MTP. Thermostat set point temperatures from 5-95°C were adjusted. After each temperature shift (dotted vertical lines) the experimental conditions remained unchanged until both fluorescence signals showed constant values. The RhB signal decreases in a step like manner since its fluorescence intensity is decreasing with increasing temperature. After each temperature shift the fluorescence signal drops sharply before it remains constant when the temperature reaches its equilibrium. On the contrary, the fluorescence intensity of Rh110 is almost constant over the whole time. The slight decrease is due to a bleaching effect which is known to happen to RhB as well. To obtain a reliable measuring signal, the ratio of both fluorescence intensities was calculated [21].It must be considered that the thermostat set point temperatures in Figure 2A and the actual temperatures in the MTP’s wells are not identical since heat may be lost to the environment. For this reason, one well was equipped with an in-house constructed PT100 temperature sensor. In this way, the corresponding well temperatures for various RhB/Rh110 ratios were determined. In Figure 2B the resulting calibration curve is depicted. It is described by a polynomial equation of second degree applying MS Excel. The fluorescence ratios are average values of five measurements in one well. The maximum relative standard deviation was 0.4%. For further investigation of the measuring accuracy the heating block temperature, as well as the room temperature, was adjusted to 37°C. In this way, a constant temperature of 37°C in each well could be assumed. The regarding measurement of all 96 wells revealed an average value of 37°C with a maximum of 38.7°C and a minimum of 35.6°C. The standard deviation was 0.76 K. The reason for the deviation from well to well cannot be explained completely. Slight deviations in the properties of the transparent microtiter plate bottom are possible which may influence the optical signals. A systematic position effect could be excluded during the experiments.

Bottom Line: Microtiter plate-based high-throughput temperature profiling is a convenient tool for characterizing temperature dependent reaction processes.It allows the evaluation of numerous conditions, e.g. microorganisms, enzymes, media, and others, in a short time.The simple temperature control combined with a commercial on-line monitoring device makes it a user friendly system.

View Article: PubMed Central - HTML - PubMed

Affiliation: AVT-Chair for Biochemical Engineering, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.

ABSTRACT

Background: Bioprocesses depend on a number of different operating parameters and temperature is one of the most important ones. Unfortunately, systems for rapid determination of temperature dependent reaction kinetics are rare. Obviously, there is a need for a high-throughput screening procedure of temperature dependent process behavior. Even though, well equipped micro-bioreactors are a promising approach sufficient temperature control is quite challenging and rather complex.

Results: In this work a unique system is presented combining an optical on-line monitoring device with a customized temperature control unit for 96 well microtiter plates. By exposing microtiter plates to specific temperature profiles, high-throughput temperature optimization for microbial and enzymatic systems in a micro-scale of 200 μL is realized. For single well resolved temperature measurement fluorescence thermometry was used, combining the fluorescent dyes Rhodamin B and Rhodamin 110. The real time monitoring of the microbial and enzymatic reactions provides extensive data output. To evaluate this novel system the temperature optima for Escherichia coli and Kluyveromyces lactis regarding growth and recombinant protein production were determined. Furthermore, the commercial cellulase mixture Celluclast as a representative for enzymes was investigated applying a fluorescent activity assay.

Conclusion: Microtiter plate-based high-throughput temperature profiling is a convenient tool for characterizing temperature dependent reaction processes. It allows the evaluation of numerous conditions, e.g. microorganisms, enzymes, media, and others, in a short time. The simple temperature control combined with a commercial on-line monitoring device makes it a user friendly system.

No MeSH data available.


Related in: MedlinePlus