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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

Cultivations of E.coli BL21 expressing the recombinant enzyme ADH-A applying 2 temperature profiles in MTPs. (A) Cultivation and online monitoring of microbial growth (via scattered light) in complex auto-induction medium OnEx. (B) Temperature dependent volumetric activity at the end of the cultivation in the MTP. Reference values from additional cultivations in shake flasks at different temperatures. Culture conditions: 96well MTP, VL = 200 μL, n = 995 rpm, d0 = 3 mm, aeration with 100% oxygen. Temperature profile I for T = 32.2-46.3°C: RT = 37°C, Tset,low = 5°C, Tset,high = 60°C (comp. Figure 3D); Temperature profile II for T = 18.1-28,5°C: RT = 30°C, Tset,low = 5°C, Tset,high = 40°C (comp. Figure 3B). Reference cultivation: 250 ml shake flask, VL = 10 mL, n = 350 rpm, d0 = 50 mm, aeration with air. Online data of 10 exemplary wells.
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Figure 8: Cultivations of E.coli BL21 expressing the recombinant enzyme ADH-A applying 2 temperature profiles in MTPs. (A) Cultivation and online monitoring of microbial growth (via scattered light) in complex auto-induction medium OnEx. (B) Temperature dependent volumetric activity at the end of the cultivation in the MTP. Reference values from additional cultivations in shake flasks at different temperatures. Culture conditions: 96well MTP, VL = 200 μL, n = 995 rpm, d0 = 3 mm, aeration with 100% oxygen. Temperature profile I for T = 32.2-46.3°C: RT = 37°C, Tset,low = 5°C, Tset,high = 60°C (comp. Figure 3D); Temperature profile II for T = 18.1-28,5°C: RT = 30°C, Tset,low = 5°C, Tset,high = 40°C (comp. Figure 3B). Reference cultivation: 250 ml shake flask, VL = 10 mL, n = 350 rpm, d0 = 50 mm, aeration with air. Online data of 10 exemplary wells.

Mentions: As an additional expression system E. coli producing a recombinant alcohol dehydrogenase A from Rhodococcus ruber (ADH-A) was investigated regarding its temperature behavior. In order to look at a broader temperature range, two experimental sets were performed applying a low and a high temperature profile according to Figure 3B and D, respectively. In this way, temperatures of 18.1-46.3°C were realized. To avoid excess evaporation at higher temperatures the cultivation was aborted after 20 h when all cultures had entered the stationary phase. The low temperature profile was applied for 27 h. The biomass formation was followed on-line by scattered light measurement (Figure 8A). As discussed before, the typical growth behavior of E. coli in the auto-induction medium OnEx is observed at temperatures of 27.6-39.7°C. After a temperature dependent lag phase of 2–4.5 h a first exponential growth is observed. The subsequent growth inhibition indicated by decreasing slopes of the scattered light curves after 4–7 h was caused by the metabolic burden of recombinant ADH-A expression. As seen for E. coli expressing FbFP this growth inhibited production phase takes longer at lower temperatures, e.g. 2 h at 39.7°C and 4 h at 27.6°C. When cells recover from that metabolic load a second exponential increase occurs before the stationary phase is reached. At temperatures below 27.6°C the microbial growth becomes more linear so that product formation is not the prior growth inhibitor, but the temperature. At temperatures higher than 39.7°C almost no growth is detected.


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)

Cultivations of E.coli BL21 expressing the recombinant enzyme ADH-A applying 2 temperature profiles in MTPs. (A) Cultivation and online monitoring of microbial growth (via scattered light) in complex auto-induction medium OnEx. (B) Temperature dependent volumetric activity at the end of the cultivation in the MTP. Reference values from additional cultivations in shake flasks at different temperatures. Culture conditions: 96well MTP, VL = 200 μL, n = 995 rpm, d0 = 3 mm, aeration with 100% oxygen. Temperature profile I for T = 32.2-46.3°C: RT = 37°C, Tset,low = 5°C, Tset,high = 60°C (comp. Figure 3D); Temperature profile II for T = 18.1-28,5°C: RT = 30°C, Tset,low = 5°C, Tset,high = 40°C (comp. Figure 3B). Reference cultivation: 250 ml shake flask, VL = 10 mL, n = 350 rpm, d0 = 50 mm, aeration with air. Online data of 10 exemplary wells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Cultivations of E.coli BL21 expressing the recombinant enzyme ADH-A applying 2 temperature profiles in MTPs. (A) Cultivation and online monitoring of microbial growth (via scattered light) in complex auto-induction medium OnEx. (B) Temperature dependent volumetric activity at the end of the cultivation in the MTP. Reference values from additional cultivations in shake flasks at different temperatures. Culture conditions: 96well MTP, VL = 200 μL, n = 995 rpm, d0 = 3 mm, aeration with 100% oxygen. Temperature profile I for T = 32.2-46.3°C: RT = 37°C, Tset,low = 5°C, Tset,high = 60°C (comp. Figure 3D); Temperature profile II for T = 18.1-28,5°C: RT = 30°C, Tset,low = 5°C, Tset,high = 40°C (comp. Figure 3B). Reference cultivation: 250 ml shake flask, VL = 10 mL, n = 350 rpm, d0 = 50 mm, aeration with air. Online data of 10 exemplary wells.
Mentions: As an additional expression system E. coli producing a recombinant alcohol dehydrogenase A from Rhodococcus ruber (ADH-A) was investigated regarding its temperature behavior. In order to look at a broader temperature range, two experimental sets were performed applying a low and a high temperature profile according to Figure 3B and D, respectively. In this way, temperatures of 18.1-46.3°C were realized. To avoid excess evaporation at higher temperatures the cultivation was aborted after 20 h when all cultures had entered the stationary phase. The low temperature profile was applied for 27 h. The biomass formation was followed on-line by scattered light measurement (Figure 8A). As discussed before, the typical growth behavior of E. coli in the auto-induction medium OnEx is observed at temperatures of 27.6-39.7°C. After a temperature dependent lag phase of 2–4.5 h a first exponential growth is observed. The subsequent growth inhibition indicated by decreasing slopes of the scattered light curves after 4–7 h was caused by the metabolic burden of recombinant ADH-A expression. As seen for E. coli expressing FbFP this growth inhibited production phase takes longer at lower temperatures, e.g. 2 h at 39.7°C and 4 h at 27.6°C. When cells recover from that metabolic load a second exponential increase occurs before the stationary phase is reached. At temperatures below 27.6°C the microbial growth becomes more linear so that product formation is not the prior growth inhibitor, but the temperature. At temperatures higher than 39.7°C almost no growth is detected.

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