Development of Mini-Bioreactors for Evolution of Thermotolerance

In biotechnological processes thermotolerance is often a crucial issue. Especially in applications of yeast energy intensive cooling is required to maintain a constant temperature level when a process produces heat. While Saccharomyces cerevisiae is a rather thermosensitive organism, there are also tolerant and even well-established strains like Hansenula polymorpha und Kluyveromyces marxianus, which have been shown to grow at temperatures up to 50° C [1, 2]. Although a theoretical maximum temperature for eukaryotic growth is widely accepted to be 60° C, improvement of thermotolerance is desirable in any industrial organism. As the molecular mechanisms of thermotolerance are highly complex, a direct genetic manipulation is a very difficult task and has shown only limited success in previous approaches. Adaptive laboratory evolution seems to be more promising and might also be helpful for further understanding of the molecular mechanisms. Towards this goal we developed an array of mini-scale bioreactors, which are based on a previously described morbidostat design and assembled from low-cost customized materials and commonly available electric parts [3]. The focus on thermotolerance required the development of individually controllable heating units, which was realized with perforated ceramic elements connected to separate electric circuits with controllable voltage. Each reactor comprises a standard 20 mL glass vial (12 mL working volume), equipped with in-/outlets for feed, waste and gas alongside a probe for temperature measurement. Medium and air are pumped by piezo actuated micro membrane pumps. The vial is placed in a shell, in which an LED and a detector diode are integrated to approximate biomass formation inside the reactor. Stirring is achieved by a magnetic stirrer driven by a controllable motor connected to a magnet and embedded in a bottom construction beneath the reactor. A set of four reactors is operated via an Arduino® microcontroller board and controlled by a LabView based software, equipped with an intuitive user interface.

The morbidostat operation is based on maintaining a certain level of stress induced growth inhibition by raising the stressor once the evolving culture has recovered from the previous step of stressor increase. Apart from stressor increases, reactors are operated in a sequential batch mode in a narrow range of biomass concentration, where dilution steps are triggered by a maximum threshold of the biomass signal and terminated at a minimum threshold. The time interval required to trigger the next dilution step is used to estimate growth rate and thus growth inhibition.

Besides temperature stress, alternative modes of operation allow adaptive evolution to rising chemical stressor concentrations or evolution of growth rate at constant conditions. Several security loops have been integrated into the software to provide a fully automated operation of the device between manual inoculation and harvest of the evolved culture.

References:

[1]          Saraya, R. et al., FEMS Yeast Res., 12:271-8, 2012.

[2]          Fonseca, G. G. et al., Appl. Microbiol. Biotechnol., 79:339-354, 2008.

[3]          Toprak, E. et al., Nat. Protocols, vol. 8:555-567, 2013.