Phenotyping Nannochloropsis gaditana under different conditions in controlle photobioreactor in laboratory and upscaled photobioreactors in greenhouse

Since resources of fossil fuels are limited, alternatives for energy production need to be explored. Besides plants as biomass and energy crops, the interest in microalgae has been increasing, as they can synthesize many valuable compounds with a large application range, including transport fuels or fish and animal feed, without competing with agricultural food production for arable land. Moreover, microalgae can utilize flue gas from industrial emissions and municipal wastewater as a nutrient source. For economically viable production of algae, however, it is necessary to explore the maximal potential of algae by optimized cultivation conditions and targeted genetic modifications based on the knowledge about their physiology as well as regulatory mechanisms of growth and metabolic processes, in addition to technical improvement of photobioreactors (PBRs) and downstream processes. Circadian clocks synchronize certain physiological, metabolic and developmental processes of organisms with specific phases of recurring changes in their environment, e.g. day and night or seasons. In this study it was investigated whether the circadian clock plays a role in regulation of growth and chlorophyll accumulation in Nannochloropsis gaditana, an oleaginous marine microalga that is considered as a potential feedstock for biofuels and for which a draft genome sequence has been published. Optical density (OD) of N. gadinata culture was monitored at 680 and 735 nm under 12 h/12 h or 18 h/6 h light-dark (LD) cycles and after switching to continuous illumination (LL) in controlled PBRs in the laboratory. In parallel, chlorophyll fluorescence was measured to assess the quantum yield of photosystem (PS) II. Furthermore, to test if redor blue-light photoreceptors are involved in clock entrainment in N. gaditana, some of the experiments were conducted by using only red or blue light. Growth and chlorophyll accumulation were confined to light periods in the LD cycles, increasing more strongly in the first half than in the second half of the light periods. After switching to continuous light, rhythmic oscillations persisted (especially for OD680) at least in the first 24 h, with a 50% decrease in the capacity to grow and accumulate chlorophyll during the first subjective night. Pronounced free-running oscillations were induced by blue light, but not by red light. In contrast, the PS II quantum yield was determined by light conditions. Continuous blue light also enhanced accumulation of vaucheriaxanthin. The results indicate interactions between circadian and light regulation of growth and chlorophyll accumulation in N. gaditana. Mutants with reduced pigment contents and lower capacities of thermal energy dissipation, allowing better penetration of light into PBRs with a smaller loss of absorbed light energy, are considered as a good strategy to improve the yield of biomass or other high-value products under light-limited conditions. Therefore, two EMS-mutants of N. gaditana, npq3 and npq21 previously selected for their pale appearance and low capacities for thermal dissipation, were examined for their growth and photosynthetic properties in comparison with the wild type (WT) under different light regimes with fluctuating or constant light in LD cycles or LL as well as temperature cycles of 30◦C/23◦C and 23◦C/15◦C under controlled laboratory conditions. The OD680 and OD735 as well as PS II quantum yield were monitored during cultivation and pigment composition was analyzed after several days of acclimation to the different conditions. In addition, biomass production was measured for LD cycle experiments. The npq21 showed higher OD680 and OD735 values compared to WT and npq3 under LD and LL with fluctuating or constant light. No differences for PS II quantum yield were found between the genotypes or treatments under LD, but lower PS II quantum yields were found for npq3 under LL. In particular, higher biomass production was found for npq21 under fluctuating LD. The presence of antheraxanthin, an intermediate xanthophyll of the violaxanthin cycle, was found in both mutants under all conditions tested, whereas WT did not accumulate antheraxanthin under non-stressful conditions, such as constant LD or LD with temperature cycle 23◦C/15◦C. Higher carotenoid to chlorophyll ratios under fluctuating light conditions found in npq21 seem to facilitate this mutant to better cope with photooxidative stress than WT, while npq3 always showed the lowest performance of the three, presumably due to negative effects of the mutations. Cultivation under temperature cycles of 23◦C/15◦C and 30◦C/23◦C resulted in similar increase in OD680 and OD735 but lower carotenoid to chlorophyll ratios under 30◦C/23◦C for npq21. In contrast, WT showed enhanced OD680 and OD735 increase under 30◦C/23◦C compared to 23◦C/15◦C without changes in carotenoid to chlorophyll ratios but retention of antheraxanthin under 30◦C/23◦C, indicating stress. The results show improved growth for npq21 under fluctuating light and varying temperature regimes which resemble the conditions in large-scale cultivation in PBRs in greenhouses. Four replicate PBR systems designed for industrial-scale production of microalgae were established in greenhouses to monitor growth of N. gaditana under more natural and realistic conditions. The comparability of the four systems was confirmed by cultivating N. gaditana WT. Based on the results obtained under the laboratory conditions, npq21 was selected for the scale-up trial to compare with the WT under the greenhouse conditions. The algae were harvested whenever the culture density exceeded a threshold during the experimental periods. The results showed higher biomass and higher chlorophyll a production for npq21 especially during the initial phase until the cell density reached the threshold value. The addition of water, salt and nutrient solution per produced biomass was nearly the same for both genotypes. Thus, npq21 outcompeted WT under most of the laboratory and greenhouse conditions tested in this study, even though the originally reported phenotypes of low pigmentation and low capacity for thermal energy dissipation had been lost during cultivation of the stock culture in the growth cabinet, suggesting that mutations in other physiological or metabolic processes may have contributed to the increased stress tolerance and higher biomass production of npq21. Whatever the genetic explanation for better performance may be, npq21 seems to be a promising candidate for further investigations.