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Bioprocess optimization, Cyanobacteria, Small-scale cultivation, Squalene, Illumination intensity

Abstract

Cyanobacteria pose as an interesting host for biotechnological production processes with regard to closing industrial carbon cycles and thus minimizing industry greenhouse gas emissions. This is the case since cyanobacteria possess the ability to fix CO2 directly through photosynthesis. Compared with land plants, cyanobacteria exhibit higher photosynthetic efficiency (Branco Dos Santos et al., 2014), are more accessible to genetic engineering techniques and usually their cultivation sites do not compete with arable land (Dietsch et al., 2021).

Nonetheless, production processes involving cyanobacteria or microalgae are still scarce in the biotechnological industry. This is most often associated with high production costs that stem from growth media (Ashokkumar et al., 2019) or the application of artificial lighting (Blanken et al., 2013). Especially, the optimization of lighting conditions can substantially lower the cost of a cyanobacterial cultivation by increasing the cell density through optimization of the incident illumination intensity or optical path length of the photobioreactor (Pfaffinger et al., 2019). Moreover, the composition of the applied light source with regard to different wavelength regimes can influence growth and product synthesis (Nur et al., 2023). Additionally, limitations of cyanobacterial growth by media components are still not fully understood (van Alphen et al., 2018). Thus, it is of great importance to understand and optimize these factors in a cyanobacterial production process starting from early process development for these processes to become viable alternatives. In our work, we employ two novel photobioreactor systems in microtiter and shake flask scale to cultivate a genetically engineered Synechocystis sp. PCC6803 strain, with the ability to produce squalene, an interesting terpene with applications in the vaccine and cosmetics industry (German et al., 2023). By employing small-scale cultivation systems, we enable high throughput experiments towards optimizing the illumination conditions and the media composition. The set-up in microtiter scale comes equipped with a custom-made LED module that can illuminate the wells of a microtiter plate with 48 individually dimmable white light LEDs and thus facilitates the execution of up to 48 parallel illumination intensity experiments (Loogen et al., 2021). The system in shake flask scale on the other hand allows the combination of LEDs with different emission spectrums at different individual illumination intensities (Beuel et al., 2021). Both systems are combined with the Respiratory Activity Monitoring System (RAMOS) (Anderlei & Büchs, 2001) or μRAMOS (Flitsch et al., 2016) respectively. While RAMOS devices enable the continuous measurement of oxygen and carbon dioxide transfer rates throughout cultivations, μRAMOS devices solely measure the oxygen transfer rate continuously.

By measuring the oxygen transfer rate, limitations of the cultivation caused by nutrients or light can be identified (Anderlei et al., 2004). Moreover, the overall net oxygen transfer is a measure of photosynthetic activity which in turn is directly correlated with carbon dioxide fixation in cyanobacteria. Thus, by comparing the integral of net oxygen transfer with the integral of incident illumination, it is possible to determine and subsequently maximize the effective quantum yield and carbon dioxide fixation during cyanobacterial cultivations. In our experiments, we could uncover limitations induced by media components as well as lighting by online monitoring the oxygen transfer rate. Furthermore, our measurements revealed that the oxygen transfer rate reaches a constant maximum value regardless of media concentration and incident illumination intensity in the photo saturated regime. We believe that this value constitutes an intrinsic characteristic of the photobioreactor system, being the maximum biomass fraction that is photosynthetically active. Thus, this parameter will prove crucial in the optimization and scale-up of cyanobacterial production processes as well as in understanding limitations induced by lighting. Overall, these results showcase the unexploited potential of utilizing cyanobacteria for future bioeconomic processes.

Acknowledgement

We thank the Fraunhofer IME Aachen for providing a LEDitSHAKE module to the Chair of Biochemical Engineering and acknowledge funding by the Ministry of Culture and Science of the State of North Rhine-Westphalia within the framework of the NRW Strategieprojekt Bioeconomy Science Center (BioSC) in the project LEDCyans.

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