Photosynthetic organisms are exposed to a highly dynamic environment in nature and evolved multiple regulatory mechanisms of photosynthesis with different activation timescales, enabling responses to short- and long-term light dynamics (Roach and Krieger-Liszkay, 2019). The short-term mechanisms enable a quick response to changes in irradiance and they depend on either macromolecule already present in the cell or pools of molecules rapidly synthesized/degraded upon changes in irradiance. One of the fast responses to excess light is Non-Photochemical Quenching (NPQ), which drives the dissipation of a fraction of the absorbed energy as heat (Ruban, 2016), a mechanism that in microalgae mostly depends on the activity of antenna proteins belonging to the LHCSR/LHCX family (Roach et al., 2020; Blommaert et al., 2021). Another important mechanism triggered by excess light is the synthesis of the xanthophyll zeaxanthin from violaxanthin by Violaxanthin De-Epoxidase (VDE). Zeaxanthin enhances NPQ while also playing a direct role in scavenging reactive oxygen species (ROS) (Dall’Osto et al., 2012), generated from the interaction of excited chlorophyll states with molecular oxygen. When irradiance decreases, zeaxanthin is converted back to violaxanthin by zeaxanthin epoxidase (ZE), increasing light-harvesting ability. Whilst this cycle has been described in diverse microalgae species, from the green lineage (e.g., Chlamydomonas reinhardtii) to Eustigmatophytes (e.g., Nannochloropsis spp.), the analogous diadinoxanthin-diatoxanthin cycle is instead active in diatoms (Lacour et al., 2020).

The mechanisms described above are mainly active in protecting PSII, yet also PSI can experience radiation damage, when light fluctuations drive to over-reduction (Bellan et al., 2020). Two main mechanisms have been identified to be involved in protecting PSI. The first is called photosynthetic control and it is activated by the proton gradient across the thylakoids membrane generated in excess light. This regulation consists in the inhibition of the activity of the Cytochrome b₆f that consequently reduces the flux of electrons reaching PSI and thus the risk of over-reduction (Barbato et al., 2020). A second group of mechanisms, cyclic (CEF) and pseudo-cyclic (PCEF) electron flows, avoid PSI over-reduction by activating alternative pathways that recycle electrons back to the plastoquinone pool or Oxygen (Shikanai, 2014). Among the proteins that have been so far identified involved in CEF there are PGR5/PGRL1 (Munekage et al., 2002; DalCorso et al., 2008) and the chloroplast NADH-dehydrogenase-like (NDH-1) complex (Shikanai et al., 1998), whilst for PCEF two alternative pathways have been described so far, namely the Mehler reaction and Flavodiiron proteins (FDP or FLVs) (Shikanai and Yamamoto, 2017; Alboresi et al., 2019).

The impact of short-term mechanisms for the regulation of photosynthesis on biomass productivity in the outdoors environment has been demonstrated in plants, where NPQ, xanthophyll cycle and CEF (Hertle et al., 2013; Nawrocki et al., 2019) were shown to impact fitness (Kulheim et al., 2002). Also genetic optimization of the NPQ response was shown to enable significant improvement in biomass yield in tobacco plants cultivated in the field (Kromdijk et al., 2016). It can thus be expected that photosynthesis regulation is impacting productivity of microalgae cultivated in PBRs/ponds outdoors as well.

In the example reported in Figure 2, Nannochloropsis gaditana cultures were exposed to dynamic light changes with a highly different frequency of fluctuations, with the saturating light treatment lasting between 0.01 s and hours, while the limiting light being correspondingly six times longer to ensure that the total amount of photons received remained the same (Sforza et al., 2012; Bellan et al., 2020). The integrated photon flux corresponded to optimal light intensity for the species [i.e., 150 μmoles of photons m^–2 s^–1 (Sforza et al., 2012)] and nutrients and CO₂ were supplied in excess. These conditions thus enabled to assess the impact of illumination dynamics resembling those experienced in outdoor PBRs/ponds on microalgae photosynthetic productivity while maintaining all other parameters constant. Results showed that the frequency of light fluctuations alone has a major impact indeed, resulting in over a four-fold decrease in the growth rate and a reduction in the biomass produced by ≈ 85% (Figure 2). This is a clear exemplification that the impact of light dynamics must be strongly considered in all optimization efforts of outdoor productivity.

Results reported in Figure 2 are consistent with similar experiments reported for other species (e.g., Chlamydomonas reinhardtii, Spirulina platensis, Chlorella spp.) where a broad range of light fluctuations was shown to have a strong impact on growth (Phillips and Myers, 1954; Nedbal et al., 1996; Vejrazka et al., 2011; Xue et al., 2011), clearly suggesting that this effect is largely similar in different photosynthetic microorganisms, from cyanobacteria to eukaryotic microalgae species. This observation also suggests that the phenomenon is intrinsic to the photosynthetic machinery and largely dependent on conserved functional mechanisms. Overall, literature data consistently show that very short light flashes do not cause significant damage and that the photons provided can be exploited with good efficiency even if illumination is very intense. This can be explained because intense light flashes are efficiently absorbed by pigments and can initiate photochemical reactions, but light is then switched off before electron flux becomes limited by diffusion-dependent transporters such as that mediated by the plastoquinone pool (Srivastava et al., 1995; Kirchhoff, 2014).

A second common observation is that cells can withstand even a very strong illumination, lasting for several minutes or hours and maintain a good growth rate. When exposed to intense light, regulatory mechanisms are activated, enabling the modulation of the photosynthetic efficiency and the protection from damage by dissipating light excesses as heat.

The most challenging conditions are thus those in which strong light exposure lasts long enough to cause damage but not long enough for the full activation of protection mechanisms. The fastest mechanism known is probably PCET, dependent on FDP whose activation is particularly fast and can provide significant protection in a few seconds after light is switched on (Gerotto et al., 2016). This mechanism, however, is not found in all eukaryotic microalgae, including all secondary endosymbionts like Nannochloropsis and diatoms (Bellan et al., 2020). NPQ, on the other hand, requires at least 60 s of intense light to be induced (Bellan et al., 2020). Xanthophyll cycle is instead slower and requires 1–3 min to be activated. Similar kinetics are also needed for the activation of the regulation of CET (Strand et al., 2015). Considering that microalgae are highly diverse and that this variability is also true for regulatory mechanisms of photosynthesis, different species may have slightly different kinetics. Anyhow, present knowledge is quite clear in suggesting that regulatory mechanisms of photosynthesis are not fully activated in the 1–60 s range, where still strong light lasts enough to saturate the electron transport chain and drive damage (Figure 2).

While the mechanisms discussed above are active in the seconds/minutes timescale, the prolonged exposition of cells to a specific irradiance induces the activation of long-term mechanisms for the regulation of the photosynthetic apparatus, such as acclimation, to maintain photosynthesis homeostasis. This phenomenon modulates the composition of the photosynthetic apparatus in response to irradiance intensity and includes the regulation of the content of proteins and other macromolecules (e.g., pigments) (Falkowski and Owens, 1980; Falkowski and LaRoche, 1991; Zou and Richmond, 2000; Walters, 2005). In most photosynthetic organisms, this long-term acclimation mechanism includes a reduction in the chlorophyll (Chl) content and an increase in photoprotective carotenoids (Car) and xanthophylls when exposed to saturating irradiance (Rodríguez et al., 2005). Another common feature of acclimation is the modulation of the photosynthetic capacity with, for example, cells acclimated to high light showing an increased photosynthetic electron transport, as observed in the diatom Phaeodactylum tricornutum and in the green microalga Chlamydomonas reinhardtii (Nymark et al., 2009; Bonente et al., 2012). This also correlates with an increase of sinks for photosynthetic electrons, such as the Calvin-Benson cycle enzymes, with the enzyme RuBisCO being the main target of the regulation (Fisher et al., 1989; Simionato et al., 2011).

The extent of this acclimation response is exemplified by merging data from Nannochloropsis gaditana cultivated at the lab scale in highly different conditions (Supplementary Table 1). These cultures differed for supports of cultivation (flasks, lab-scale photobioreactors with different designs), illumination intensities (10, 100, 400, 1000, 1200 μmol photons m^–2 s^–1), media (seawater media F/2, F/2 with additional/depletion of Nitrogen, Phosphorous), presence/absence of external CO₂ supply, cultivation strategies (batch or semi-continuous), as described in Supplementary Table 1. The comparative analysis of these data, obtained for the same microalgal strain and using a consistent set of analytical methods, highlights the response of microalgae to different growth conditions while minimizing effects due to other parameters, like species/strain and specific cultivation protocols. All data compared are obtained from cells under active growth and do not include cells under prolonged stresses.

The analysis of the pigment content highlights the activation of the acclimation response with a modulation of the Chl content of the cells (Figure 3). Under lower illumination, Chl content increases to maximize light-harvesting efficiency while, when light is intense, Chl content decreases to reduce absorption and avoid eventual photodamage. While this is a common behavior among different photosynthetic organisms (Li et al., 2009; Lepetit et al., 2012; Meneghesso et al., 2016), it is interesting to observe that this correlation emerges despite the differences in other cultivation parameters. Chl content in cells exposed to extremes illumination (10 or 1000 μmol photons m^–2 s^–1) is also more similar irrespective of other factors. This probably suggests the existence of a lower and higher limit for Chl accumulation per cell (Figure 3A), whose value is likely species-specific.

While the Chl content is largely influenced by the light intensity (Figure 3A), also other parameters like culture concentration, CO₂ and nutrient availability show an impact on acclimation, when their influence is specifically analyzed (Figure 3). Cells concentration plays a major effect indeed, since self-shading reduces the average light available to the culture, even if the incident light is equivalent (Figure 3B). A higher CO₂ availability, on the other hand, stimulates Chl accumulation (Figure 3C), likely because when the Calvin-Benson cycle is more active it re-generates acceptors for photosynthetic electron transport faster, thus enabling the use of photosynthetic excitation energy more efficiently. When the impact of nutrients is considered, Chl biosynthesis is highly dependent on Nitrogen availability and indeed cells with higher supply accumulate more pigments, while they accumulate far less in limiting conditions (Figure 3D). Phosphorous availability, on the other hand, has a smaller influence. In Nannocholopsis cultures Phoshoporus deficiency has no major impact on Chl content (Fattore et al., 2021), while significant effects were instead observed in cyanobacteria and other microalgae species (Kokabi et al., 2019, 2020; Solovchenko et al., 2020).

Overall, we observed that different parameters have a large impact on the acclimation of the photosynthetic apparatus (Alboresi et al., 2016; Meneghesso et al., 2016; Perin et al., 2017b; Fattore et al., 2021), highlighting the complexity of this phenomenon.

Carotenoids (Car) are the other major pigments in photosynthetic organisms, active in light-harvesting but also in protection from light excess and their content is also modulated during the acclimation response. Taking together the data of all experimental conditions reported in Supplementary Table 1, there is a clear linear correlation between Chl and Car content for Nannochloropsis cells, which is maintained independently from all the differences in growth parameters (Figure 4). This can be explained by the fact that pigment binding proteins in the photosynthetic apparatus bind Chl and Car with specific ratios and when their content changes both pigments are equally affected. It is interesting to observe that the tendency line trends to 0.01 pg Car/cell for a 0 Chl content, suggesting the presence of a carotenoid pool not bound to proteins of the photosynthetic apparatus. This pool might be free in membranes and active in scavenging ROS (Dall’Osto et al., 2010; Dell Aglio, 2021). Microalgae, including Nannochloropsis, can accumulate carotenoids in oil bodies, but this occurs especially under prolonged stress, and this is likely not influential here since all cultures considered were under active growth conditions.

The acclimation response impacts not only pigment composition but also photosynthetic functionality. This can be evidenced by analyzing the fluorescence parameter qL, which enables to estimate the photochemical activity. qL is 1 when all reaction centers are open and available for photochemical reactions and it decreases when cells are exposed to increasing illumination, trending to 0 when the photosynthetic capacity is saturated (Baker, 2008). In cultures acclimated to more intense illumination, qL values are generally higher, suggesting that these cells have a higher photochemical capacity and thus a stronger illumination is needed to reach saturation (Figure 5A). This common trend is observed under different culture conditions, but while the difference is strong in flask cultures, the acclimation of photosynthetic capacity is smaller in cultures cultivated at higher density in glass tubes with air bubbling (i.e., Multicultivator from Photon System Instruments, PSI, Supplementary Table 1), suggesting they are perceiving on average less light than the incident irradiation (Figure 5B).

Many of the parameters described above are affected when microalgae are cultivated outdoors, starting from the illumination intensity that changes continually during days, weeks, and seasons, but their impact on microalgae acclimation and photosynthetic functionality is still insufficiently investigated even if in situ probes for a continuous monitoring of photosynthetic efficiency were developed (Masojídek et al., 1999, 2004, 2010).

It is instead highly interesting and stratetic comparing the response of microalgae cultivated outdoors to lab-scale cultures and assessing whether the response to outdoor cultivation corresponds to the phenomena observed indoors, on the path to drive improvements of photosynthetic productivity in the intended environment of cultivation at industrial scale. Figure 6 shows data from Nannochloropsis gaditana cultivated outdoors in a pilot PBR (Figure 6A) in semicontinuous mode to maintain high cellular concentration and biomass productivity (Figure 6B) while providing nutrients and CO₂ in excess [Perin et al. (2015) as a reference on media composition]. These experiments were carried out in a greenhouse in February–April 2021 and while the temperature was controlled at 22°C, irradiance was natural.

Culture productivity was variable during different days, as expected, but it emerged a clear correlation with light intensity (Figure 6C). This suggests that, at least in the tested growth conditions, the culture was light-limited, and more available irradiation corresponded to higher productivity. Since productivity is limited from light availability, clearly any optimization of photon-to-biomass conversion efficiency would have a positive effect on yield.

The Chl content of cells in outdoor conditions showed large variability, going from 0.06 to 0.12 pg Chl/cell (Figure 6D). It is interesting to observe that this range of variability corresponds to the differences observed at the lab scale for cells exposed to different irradiances (Figures 3A, 6D). This suggests that microalgae cultivated outdoors activate an acclimation response as extensive as observed indoors and thus that this response is indeed largely activated when they are cultivated for an extended time at an industrial scale.

The pigment content measured outdoors also shows a correlation with the light intensity experienced by the culture, with higher average light intensities stimulating a decrease in Chl content. This is not true for external illumination below 100 μmoles of photons m^–2 s^–1, suggesting that below this value the maximal Chl content per cell was already reached. This is consistent with a similar conclusion obtained from lab-scale experiments, considering that, because of the culture density, the average illumination perceived by the cells is well below 10 μmoles of photons m^–2 s^–1 (Figure 3A).

Overall, these data highlight that acclimation is a phenomenon activated by microalgae also upon cultivation in PBR outdoors. In fact, while data at the lab scale are still significant to investigate the biological response in detail, parameters in outdoor conditions have other kinetics (e.g., light variability), whose impact should be investigated more thoughtfully. A better understanding of these responses in outdoors environments is strategic to drive microalgae genetic modifications effective in industrial conditions.