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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

Cultivation of E.coli BL21 expressing the fluorescent protein FbFP applying a temperature profile in a MTP. (A) Cultivation and online monitoring of microbial growth (via scattered light) in complex TB medium without induction. (B) Cultivation and online monitoring of microbial growth and (C) fluorescent protein formation in complex auto-induction OnEx. Culture conditions: 96well MTP, VL = 200 μL, n = 995 rpm, d0 = 3 mm, aeration with 100% oxygen. Temperature profile: RT = 37°C, Tset,low = 5°C, Tset,high = 60°C (comp. Figure 3D). Data of 12 (from 48) exemplary wells.
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Figure 4: Cultivation of E.coli BL21 expressing the fluorescent protein FbFP applying a temperature profile in a MTP. (A) Cultivation and online monitoring of microbial growth (via scattered light) in complex TB medium without induction. (B) Cultivation and online monitoring of microbial growth and (C) fluorescent protein formation in complex auto-induction OnEx. Culture conditions: 96well MTP, VL = 200 μL, n = 995 rpm, d0 = 3 mm, aeration with 100% oxygen. Temperature profile: RT = 37°C, Tset,low = 5°C, Tset,high = 60°C (comp. Figure 3D). Data of 12 (from 48) exemplary wells.

Mentions: An E. coli strain expressing a fluorescent model protein (FbFP) as product was investigated (Figure 4). Non-induced (MTP row A,C,E,G) and induced (MTP row B,D, F, H) cultivations were performed in parallel in one MTP. The temperature profile was essentially identical to Figure 3D. The results of 12 (out of 48) exemplary non-induced cultures in TB medium are shown in Figure 4A. All cultures begin immediately with their exponential growth without any lag phase. After 1 h the curves start spreading indicating different growth rates at different temperatures. The lowest rate is observed at the lowest temperature of 30.3°C. Higher temperatures lead to increased growth rates indicated by steeper curves. The maximum growth rate occurs at temperatures of 41.4-45.3°C. A further increase retarded the microbial growth again. Due to varied growth rate the time point for reaching the stationary phase differs as well. At 41.4°C the culture became stationary after 4.5 h, whereas it needed twice as long at 30.3°C. Comparing the final scattered light intensities, it can be seen that slightly more biomass was formed at lower temperatures. This might be explained by a higher energy demand for cell maintenance at higher temperatures which withdraws metabolic resources from growth [35]. It was already found that organisms growing at temperatures above their optimal growth temperature show lower cell yields. Thereby, it was postulated that biosynthetic reactions at high temperatures do not keep pace with catabolic reactions [36].


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)

Cultivation of E.coli BL21 expressing the fluorescent protein FbFP applying a temperature profile in a MTP. (A) Cultivation and online monitoring of microbial growth (via scattered light) in complex TB medium without induction. (B) Cultivation and online monitoring of microbial growth and (C) fluorescent protein formation in complex auto-induction OnEx. Culture conditions: 96well MTP, VL = 200 μL, n = 995 rpm, d0 = 3 mm, aeration with 100% oxygen. Temperature profile: RT = 37°C, Tset,low = 5°C, Tset,high = 60°C (comp. Figure 3D). Data of 12 (from 48) exemplary wells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Cultivation of E.coli BL21 expressing the fluorescent protein FbFP applying a temperature profile in a MTP. (A) Cultivation and online monitoring of microbial growth (via scattered light) in complex TB medium without induction. (B) Cultivation and online monitoring of microbial growth and (C) fluorescent protein formation in complex auto-induction OnEx. Culture conditions: 96well MTP, VL = 200 μL, n = 995 rpm, d0 = 3 mm, aeration with 100% oxygen. Temperature profile: RT = 37°C, Tset,low = 5°C, Tset,high = 60°C (comp. Figure 3D). Data of 12 (from 48) exemplary wells.
Mentions: An E. coli strain expressing a fluorescent model protein (FbFP) as product was investigated (Figure 4). Non-induced (MTP row A,C,E,G) and induced (MTP row B,D, F, H) cultivations were performed in parallel in one MTP. The temperature profile was essentially identical to Figure 3D. The results of 12 (out of 48) exemplary non-induced cultures in TB medium are shown in Figure 4A. All cultures begin immediately with their exponential growth without any lag phase. After 1 h the curves start spreading indicating different growth rates at different temperatures. The lowest rate is observed at the lowest temperature of 30.3°C. Higher temperatures lead to increased growth rates indicated by steeper curves. The maximum growth rate occurs at temperatures of 41.4-45.3°C. A further increase retarded the microbial growth again. Due to varied growth rate the time point for reaching the stationary phase differs as well. At 41.4°C the culture became stationary after 4.5 h, whereas it needed twice as long at 30.3°C. Comparing the final scattered light intensities, it can be seen that slightly more biomass was formed at lower temperatures. This might be explained by a higher energy demand for cell maintenance at higher temperatures which withdraws metabolic resources from growth [35]. It was already found that organisms growing at temperatures above their optimal growth temperature show lower cell yields. Thereby, it was postulated that biosynthetic reactions at high temperatures do not keep pace with catabolic reactions [36].

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