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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

Determination of the optimal temperature for microbial growth and product formation of E.coli and K. lactis resulting from temperature profile experiments in MTPs. (A) Temperature dependent growth rate of E.coli (in rich and mineral medium) and K. lactis (in rich medium) under non-induced conditions. Temperature dependent maximum product formation (B) and maximum STY (C) of E.coli producing FbFP (in rich and mineral medium) and K. lactis producing GFP (in rich medium) under induced conditions. Dotted lines in A indicate Arrhenius fits due to Eq. 1.
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Figure 6: Determination of the optimal temperature for microbial growth and product formation of E.coli and K. lactis resulting from temperature profile experiments in MTPs. (A) Temperature dependent growth rate of E.coli (in rich and mineral medium) and K. lactis (in rich medium) under non-induced conditions. Temperature dependent maximum product formation (B) and maximum STY (C) of E.coli producing FbFP (in rich and mineral medium) and K. lactis producing GFP (in rich medium) under induced conditions. Dotted lines in A indicate Arrhenius fits due to Eq. 1.

Mentions: From the before described data sets (Figures 4 and 5) it is already possible to get an idea of the temperature optima for microbial growth and product formation in yeast and bacterial cells. But the high throughput of the MTP yields sufficient data for a more detailed characterization. For biomass formation the maximum growth rate under non-induced conditions was chosen as temperature dependent parameter (Figure 6A). As expected, the bacterial and the yeast system show a different behavior. E. coli in rich TB medium has a maximum growth rate of 0.41 h−1 at 43°C. The highest growth rate determined for K. lactis in rich YP medium is 0.16 h−1 at 32-33°C. In addition, the growth of E. coli in the mineral Wilms-MOPS medium was investigated. Compared to the growth in rich TB medium, a higher maximum growth rate of 0.67 h−1 was determined but at a lower temperature of 39.5°C. The observed values are in good agreement with earlier studies about E. coli[42-45] and Kluyveromyces strains [35,46,47]. For the product formation the maximum product fluorescence (indicator for product concentration) and the maximum space time yield were exemplary chosen as characteristic values (Figure 6B and C). Again differences between the microbial systems and cultivation media occur. K. lactis has the maximum product fluorescence and highest STY in the same range of 34-35°C which is slightly higher than the temperature for optimal growth. E. coli expressing FbFP in the rich auto-induction medium OnEx shows constant high fluorescence values over a relatively broad range from 35-42°C before it drops sharply at higher temperatures. The regarding maximum STY was determined at 41°C. For FbFP production in the mineral auto-induction medium the highest values for fluorescence and STY are observed at 44°C and 40-45°C, respectively.


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)

Determination of the optimal temperature for microbial growth and product formation of E.coli and K. lactis resulting from temperature profile experiments in MTPs. (A) Temperature dependent growth rate of E.coli (in rich and mineral medium) and K. lactis (in rich medium) under non-induced conditions. Temperature dependent maximum product formation (B) and maximum STY (C) of E.coli producing FbFP (in rich and mineral medium) and K. lactis producing GFP (in rich medium) under induced conditions. Dotted lines in A indicate Arrhenius fits due to Eq. 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Determination of the optimal temperature for microbial growth and product formation of E.coli and K. lactis resulting from temperature profile experiments in MTPs. (A) Temperature dependent growth rate of E.coli (in rich and mineral medium) and K. lactis (in rich medium) under non-induced conditions. Temperature dependent maximum product formation (B) and maximum STY (C) of E.coli producing FbFP (in rich and mineral medium) and K. lactis producing GFP (in rich medium) under induced conditions. Dotted lines in A indicate Arrhenius fits due to Eq. 1.
Mentions: From the before described data sets (Figures 4 and 5) it is already possible to get an idea of the temperature optima for microbial growth and product formation in yeast and bacterial cells. But the high throughput of the MTP yields sufficient data for a more detailed characterization. For biomass formation the maximum growth rate under non-induced conditions was chosen as temperature dependent parameter (Figure 6A). As expected, the bacterial and the yeast system show a different behavior. E. coli in rich TB medium has a maximum growth rate of 0.41 h−1 at 43°C. The highest growth rate determined for K. lactis in rich YP medium is 0.16 h−1 at 32-33°C. In addition, the growth of E. coli in the mineral Wilms-MOPS medium was investigated. Compared to the growth in rich TB medium, a higher maximum growth rate of 0.67 h−1 was determined but at a lower temperature of 39.5°C. The observed values are in good agreement with earlier studies about E. coli[42-45] and Kluyveromyces strains [35,46,47]. For the product formation the maximum product fluorescence (indicator for product concentration) and the maximum space time yield were exemplary chosen as characteristic values (Figure 6B and C). Again differences between the microbial systems and cultivation media occur. K. lactis has the maximum product fluorescence and highest STY in the same range of 34-35°C which is slightly higher than the temperature for optimal growth. E. coli expressing FbFP in the rich auto-induction medium OnEx shows constant high fluorescence values over a relatively broad range from 35-42°C before it drops sharply at higher temperatures. The regarding maximum STY was determined at 41°C. For FbFP production in the mineral auto-induction medium the highest values for fluorescence and STY are observed at 44°C and 40-45°C, respectively.

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