Photosynthetic activity measured in-situ in microalgae cultures grown in large-scale raceway ponds

Photosynthetic activity measured in-situ in microalgae cultures grown in large-scale raceway ponds | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Photosynthetic activity measured in-situ in microalgae cultures grown in large-scale raceway ponds Jiří Masojídek, Karolína Štěrbová, Victor A. Robles Carnero, Giuseppe Torzillo, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4449619/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Two microalgae, Scenedesmus sp. and Chlorella sp. (Chlorophyceae), robust and well-growing species, with a potential for biostimulating activities, were cultured in raceway ponds (RWPs) placed in a greenhouse. The objective of this case study was to monitor the performance of microalgae cultures in-situ at various depths as concerns photosynthetic activity and physico-chemical variables (irradiance, temperature, dissolved oxygen concentration) including biostimulating activity. The data (photochemical yield and electron transport rate monitored by Chl fluorescence and photosynthetic oxygen production) both in-situ and ex-situ revealed that (i) even in thin cultures (0.5-1 g dry weight L -1 ), the active photic layer in the culture was only about 1 cm indicating that most of the culture was ˊphotosyntheticallyˋ in the dark and (ii) nevertheless, even at high dissolved oxygen concentrations of about 200 %sat and higher the cultures retained relatively high actual photochemical yield Y(II) of about 0.35 and higher when monitored in-situ . The presented work can be used as exemplary data to optimize the growth of microalgae cultures in large-scale raceway ponds by understanding the interplay between culture depth and cell concentration. Biological sciences/Biochemistry Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Microbiology Biostimulants Green microalgae In vivo Chl fluorescence Photosynthesis Raceway ponds Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Two basic approaches are used for microalgae production in the solar bioreactors: one possibility is the cultivation in open systems (with direct interaction of the microalgae culture with the environment), while the other employs closed or semi-closed bioreactors with no direct contact between the culture and the outside environment [ 1 , 2 ]. At present, the most frequently used ‘open’ cultivation systems for the commercial production of microalgae are various types of so-called raceway ponds (RWPs) due to their lower construction cost and easier maintenance compared to closed bioreactors. The early installations of RWPs were introduced in the 1950s-1960s by Oswald and co-workers [ 3 , 4 ] and their designs were advanced in the 1970s-1980s by the use of paddlewheel mixers which reduced the shearing forces on the cells and energy requirements [ 5 ]; then further improvements were made ever since [ 6 – 8 ]. The production RWPs from 100 to 5,000 m 2 were reported 1 . The key construction parameters are the depth of the culture and the total area occupied. The area is usually divided into two or more channels along which the culture is recirculated by paddle wheels or impellers (see Fig. 1 ) which maintain a culture velocity of about 0.3 m s − 1 . The recommended length-to-width (L/W) ratio of channels is about 15 [ 9 ]. The surface-to-total-volume ratio in these systems is about 5–10 m − 1 . A channel depth should be in the range of 0.2–0.4 m; it is recommended to operate at a lower culture depth to increase light penetration, biomass concentration and the stability of the cultures. The shorter the light path, the higher the biomass density can be maintained. However, the use of the RWPs is restricted – due to the limited control of cultivation conditions and contamination – to a few “robust” and fast-growing microalgae genera (e.g., Chlorella, Scenedesmus, Nannochloropsis ), or those that are cultured under selective conditions (e.g., Arthrospira or Dunaliella ) [ 2 ]. The cultures in the RWPs are usually grown at low biomass densities ranging between 0.5 and 1 g L − 1 depending on the pond depth. The cultures are usually operated in a semi-continuous regime collecting cells each 4–5 days depending on the season, and generally maintained at relatively high densities to facilitate harvesting. The available light is the main factor limiting the productivity of microalgae mass cultures outdoors [ 10 ]. However, the impact of irradiance on the productivity of outdoor microalgae cultures is rather complex due to the interaction with other environmental variables. The average amount of photon energy received by a single cell is a combination of several factors: light intensity, cell density, culture layer and the rate of mixing as well as the construction of the cultivation unit (for review see e.g. [ 11 – 13 ]. In any case, the irradiance regime should be optimised as light in excess can damage the photosynthetic apparatus, particularly in combination with temperature extremes or high oxygen concentration [ 14 , 15 ]. Microalgae, due to photosynthetic activity in outdoor mass cultures, can generate high concentrations of dissolved oxygen (DO). Though oxygen evolution is sometimes overlooked in large-scale units, high concentrations of DO in cultures occurring especially under high irradiance can result in photoinhibition and photorespiration which entail a reduction in photosynthetic activity and growth. In open units, the build-up of DO concentration as much as 3–4 times higher than the air saturation values can be observed during the day, which may partly decrease the photochemical yield of microalgae cultures [ 16 , 17 ]. The maintenance of DO levels below critical concentration (about 200% of air saturation) often requires degassing or efficient culture mixing. Optimizing culture productivity requires constant monitoring of physicochemical variables, such as pH, temperature, DO concentration, and nutrient levels, but most crucial is monitoring photosynthetic performance. The primary indications of adverse growth conditions can be detected as reduction of photosynthetic activity of a microalgae culture which subsequently slows down its growth and productivity [ 18 ]. Finding rapid, reliable and robust techniques to evaluate variations in microalgae activity has been one of the major tasks for the online monitoring of pilot and large-scale cultivation units [ 2 , 13 , 19 ]. Various monitoring methods have been used to adjust growth conditions for the production of biomass. The earlier reports showed that the photosynthetic variables (oxygen production, photochemical yield, electron transport rate) monitored in-situ/ex-situ well reflect the physiological status of the culture and provide primary information on photosynthetic activity which reflects the growth and biomass productivity [ 15 , 16 , 19 – 23 ]. Changes in oxygen production and in vivo chlorophyll (Chl) fluorescence are being used for monitoring microalgae culture, as they provide primary information on culture photosynthetic performance and consequently are reflected in culture growth [ 11 , 22 , 24 ]. The direct method of monitoring microalgae cultures is to follow the photosynthetic changes in-situ tracking the actual situation. The other possibility, but more time-consuming, is to assess the culture state ex-situ using microalgae samples withdrawn from a cultivation unit. Some variables, such as photosynthetic oxygen production, or PSII photochemical yield and electron transport rate are used to correlate with photosynthetic activity and growth [ 23 , 25 , 26 ]. In this case study, we aimed to survey the photosynthetic activity of outdoor microalgae cultures at various parts and depths of large RWPs using photosynthesis monitoring techniques. Particularly in this work, two strains of the same taxon Chlorophyta, Scenedesmus and Chlorella were examined which are characterized by their robustness and fast growth. Photosynthetic activity of microalgae cultures was measured in parallel with other variables (irradiance, temperature, dissolved oxygen concentration) and biostimulating activity. The data showed that even in not as dense cultures (0.5-1 g dry weight L − 1 ) the active photic layer in the culture is limited to about 1 cm. The presented work can be used as exemplary data to optimize the growth conditions of microalgae cultures in large-scale RWPs. Results Culture growth, irradiance and oxygen production Two 5-day trials were carried out in parallel using two RWP bioreactors during sunny and warm days in mid-summer (August). In trial 1, the culture of green microalga Scenedesmus sp. (further as Scenedesmus ) was grown in the RWP with an area of 76 m 2 in which 3 measuring sites were defined (1 – after the sump; 2 – at the first bend; 3 – close to the end of the second channel before deflectors) (Figs. 1 a,b). In trial 2, the culture of green microalga Chlorella sp. (further as Chlorella ) was grown in the RWP with an area of 730 m 2 in which 5 measuring positions were defined (1 – after the sump; 2 – middle of the first channel; 3 – at the first bend; 4 – middle of the second channel; 5 – close to the end of the channel before deflectors) (Figs. 1 c,d). The choice of measurement points included sites where the mixing was less efficient such as at the bends, or where it was expected to be considerably higher such as after the paddlewheels and the sump. In trial 1, the biomass density of the Scenedesmus culture was about 0.9 g L − 1 while in trial 2 ( Chlorella sp.) the biomass density was three times lower, about 0.3 g L − 1 . The chlorophyll (Chl) content was about 13.3 and about 5.6 mg L − 1 in trial 1 and trial 2, respectively. In both units, the culture depth was about 14 cm. As concerns the temperature and DO concentration changes in the Scenedesmus culture during trial 1, the data varied between 27.5–28°C in the morning (9:00 h) up to 35–36°C in the afternoon (17:00 h) (Fig. 2 a – values are a range of data recorded in 3 measuring positions). DO concentration values were found between 26–78%sat in the morning, increased to 171–258%sat at midday (13:00 h) and ranged between 178–258%sat at 17:00 h (Fig. 2 b – values represent a range of data recorded in 5 measuring positions). In trial 2 monitoring the Chlorella culture, the temperature varied between 27-27.5°C in the morning (9:00 h) up to 34–35°C in the afternoon (17:00 h) (Fig. 2 c). DO concentration values ranged between 92–102%sat in the morning, increased to 186 − 34 %sat at midday (13:00 h) and between 169 − 41 %sat at 17:00 h (Fig. 2 d). The typical daily course of irradiance measured in the greenhouse indicated the down at about 6:30 h and the dusk at about 21:00 hl; the range of the highest irradiance was seen between 14:00 h and 15:00 h (Fig. S1 a). The ambient irradiance maxima of about 1800 µmol photons m − 2 s − 1 measured at 13:00 h was usually 10–15% higher than those inside the greenhouse. Therefore, the irradiance intensities measured at the surface of RWPs during the trial were about 300, 1350, 1600 and 1100 µmol photons m − 2 s − 1 at 9:00, 11:00, 13:00 and 17:00 h, respectively. The irradiance intensities measured in-situ in the cultures at the depth of 1, 4 and 7 cm were between 85–250, 20–85 and between 6–21 µmol photons m − 2 s − 1 , respectively from 9:00 to 17:00 h (Fig. S1 b). The data showed that irradiance intensity at 7-cm depth is rather low and it is probably outside the photic zone of the culture. Photosynthesis monitoring in-situ Values of the actual photochemical yield Y(II) were measured in-situ in the culture at two depths – 0.6 and 4 cm during the 3-day trials in both trials (Fig. 3 ). The culture depths for measurements were selected to provide average light intensity (midday) 150–200 µmol photons m − 2 s − 1 at 0.6 cm depth (close to saturating irradiance for growth) and about 20 µmol photons m − 2 s − 1 at 4 cm (low irradiance close to the photosynthesis compensation point). It is important to note that data presented here are a mean of data measured at 3 (trial 1) or 5 positions (trial 2) in the RWPs (see Fig. 1 ) and there was not much variation among the values measured at various positions. In trial 1, the Y(II) values were measured at 9:00, 13:00 and 17:00 h on days 1, 2 and 3. In this trial, the highest values of Y(II) – between 0.49–0.71 were found at 9:00 h (Figs. 3 a,b,c); there was not much difference between the values measured at both depths. At 13:00 h and 17:00 h, the Y(II) values measured at 0.6-cm and 4-cm depths were lower – between 0.31–0.51 due to higher ambient irradiance penetrating the culture which caused higher PQ pool reduction. Nevertheless, there was little difference between Y(II) measured at 0.6-cm and 4-cm depths at 13:00 h and 17:00 h, which means that penetrating irradiance intensity was similar. In trial 2, the course of Y(II) changes was similar to that in trial 1, only the Y(II) values were mostly 15–20% lower compared to trial 1 suggesting lower photosynthetic activity of the culture. The highest values of Y(II) – between 0.42–0.62 were found at 9:00 h at both depths (Figs. 3 d,e,f). At 13:00 and 17:00 h, the measured Y(II) values were lower – between 0.33–0.38; and as in the case of Scenedesmus in trial 1 there were found small differences between the Y(II) values measured at 0.6-cm and 4-cm depths. In both trials, the ambient irradiance (PAR) penetrating inside the culture and actual PSII photochemical yield Y(II) were measured in-situ /online during the diurnal cycle using submerged sensors of irradiance and fluorescence placed close to each other which were connected to the Junior-PAM fluorimeter. Then, the relative electron transport rate rETR was estimated [Y(II) × E PAR ]. The irradiance intensity started to rise between 8:00 and 9:00 h and it was diminished at about 21:00 h which means that the culture recorded light for about 12–13 h (Fig. 4 ). As mentioned above, there was not much variation among the values of actual photochemical yield Y(II) measured at various positions (Fig. 3 ); thus only one position, point 1 downstream after the sump was selected for online measurements in both RWPs at two depths – 0.6 and 4 cm. In trial 1, the irradiance intensity of about 150–200 µmol photons m − 2 s − 1 was measured at the 0.6-cm depth between 14:00–17:00 h (Fig. 4 a). In this period, the maximum values of rETR were also estimated – between 115–160 µmol e − m − 2 s − 1 and these values followed the course corresponding to irradiance intensities measured inside the culture. The course of Y(II) values was antiparallel to irradiance due to the degree of PSII reduction – in light periods it was between 0.2–0.45 while in dark periods increased up to 0.6. Then in trial 1, when the irradiance inside the culture was measured at the 4-cm depth, much lower maximum intensities – between 15–20 µmol photons m − 2 s − 1 were found which are probably close to the photosynthesis compensation point (Fig. 4 c). There was not much difference in the Y(II) values between the night and day periods as the penetrating irradiance was low, only the trend showed a slight decrease after sunset (Fig. 4 c). Thus, the estimated rETR showed maximum values between 10–20 µmol e − m − 2 s − 1 , which was less than one-tenth of that found at 0.6-cm depth. In trial 2, the maximum irradiance of 100–150 µmol photons m − 2 s − 1 intensity in the culture at the 0.6-cm depth was measured between 10:00–17:00 h (Fig. 4 b). In this period the rETR values were also estimated – between 30–50 µmol e − m − 2 s − 1 ; the course corresponded to that of the irradiance intensity inside the culture. The course of Y(II) values was the opposite of irradiance – in light periods it was between 0.1–0.2 while in dark periods it was between 0.2–0.3. Only the trend showed a slight decrease in Y(II) during the day and night periods. In this trial, when the irradiance was measured at the 4-cm depth, much lower maximum intensities of only up to 17 µmol photons m − 2 s − 1 were found which are probably below the photosynthesis-exciting level (Fig. 4 d). In this period, the rETR values were found only between 5–8 µmol e m − 2 s − 1 ; the course corresponded to that of the irradiance intensity inside the culture. Photosynthesis measurements ex-situ In both trials, the maximum photochemical yield F v /F m , maximum relative electron transport rate rETR max , maximal non-photochemical quenching NPQ max , the maximum rate of photosynthetic oxygen production P max , maximum rate of dark respiration Resp and the fraction of respiration in gross photosynthesis Resp/PS gross (in %; PS gross is the sum of P max + Resp) were measured ex-situ using culture samples taken from raceway ponds at particular daytimes – 9:00, 13:00 and 17:00 h (Figs. 5 and 6 ). Values of F v /F m , rETR max and NPQ max were calculated from light-response curves (LRC) of in vivo Chl fluorescence while P max , Resp and Resp/PS gross were determined from LRC of oxygen production/consumption. In trial 1 the values of F v /F m ranged between 0.63 and 0.79 and were relatively aligned (Fig. 5 a). The rETRmax values were found between 110–150 µmol e − m − 2 s − 1 (Fig. 5 b) while NPQ max was between 0.25–0.63 when the values were found decreasing from day 1 to day 3 and the lowest were found on day 3 (Fig. 5 c). The range of P max and Resp was found between 9-10.8 pmol O 2 m − 2 s − 1 and 0.48–1.6 pmol O 2 m − 2 s − 1 , respectively. (Figs. 5 d,e). The data of the rate between Resp/PS gross revealed the lowest ratio of Resp in PSgross was found in the morning – between 4.6–9.8% while the highest – between 17.8–28.9 pmol O 2 m − 2 s − 1 was in the afternoon (Fig. 5 f). These data also confirmed an increasing trend from the morning towards the afternoon and also from day toward day 3. The highest values between 11 and 15.4 were found on days 2 and 3 at 13:00 and 17:00 h. Considering the data of Pmax and Resp, it was obvious that the ratio of Resp/PSgross showed an increasing trend. In trial 2, the values of F v /F m ranged between 0.58 and 0.71 and were relatively comparable during the trial (Fig. 6 a). Compared to trial 1 they were about 10% lower. The rETR max values were found between 110–130 µmol e − m − 2 s − 1 (Fig. 6 b), which was about 15 below that recorded in trial 1, while NPQ was between 0.54–0.83 when the values were found lowest in the morning (Fig. 6 c). The range of Pmax was found mostly between 2.7–6.2 9-10.8 pmol O 2 m − 2 s − 1 , except the morning of day 1 when the value was 10.6 pmol O 2 m − 2 s − 1 . Compared to trial 1, Resp was found lower – between 0.27–0.70 pmol O 2 m − 2 s − 1 (Fig. 6 e). Similarly as in trial 1, the ratio between Resp/PS gross was low in the morning – between 2.5–6.7% while much higher – between 11.1–14.6 was found in the afternoon on days 2 and 3 (Fig. 6 f). Again, these data showed an increasing trend from the morning towards the afternoon and also from day 1 toward day 3. In both trials, culture samples were taken from the RWPs at 9:00, 13:00 and 17:00 h on days 1, 2 and 3. The samples were examined using the fast fluorescence induction kinetics (OJIP test) to estimate the redox status of quinone electron acceptors in the PSII complex. The examples of fluorescence induction curves were recorded at 13:00 h for all three days in trial 1 and trial 2 (Figs. 7 a, b). The curves were analyzed to evaluate the reduction status of the PSII electron acceptors. From the fluorescence levels at the J and I points, the variables Vj and Vi were calculated which express the redox status of quinone electron acceptors which is demonstrated by Vj and Vi variables (see Methods). The trends of Vj and Vi were similar in both Scenedesmus (Fig. 7 b) and Chlorella (Fig. 7 d) cultures. Nevertheless, in the Chlorella culture (trial 2) the values of Vj and Vi were about 22 and 14% higher, respectively compared to those in the Scenedesmus culture. Bioassays In the present trials, an important outcome was the biostimulating (auxin-like) activity of both Scenedesmus and Chlorella cultures. The biostimulating activities of the freeze-dried biomass samples were similar when detected by two bioassays. The mungbean rooting bioassay and the cucumber cotyledon rooting bioassay revealed that the sample of culture biomass taken in trial 2 had auxin-like activity equivalent to 0.3–0.5 mg IBA L − 1 , i.e. 125% of the control (Fig. 8 ). The biomass collected in trial 2 (Scenedesmus) showed similar bioactivity with the cucumber cotyledon rooting bioassay. Discussion In mass outdoor cultures, microalgae cells are subjected to changes in temperature and light intensity varying due to the diurnal and seasonal changes. Generally, the acceptable growth temperature for most microalgae species is between 15 and 35°C and temperature optima are about 30°C [ 19 ]. In the presented trials, the course of culture temperature was similar in both RWPs, on average about 31°C; only temporarily it was increased over 35°C (Fig. 2 a,c). The ambient irradiance maxima of about 1,800-2,000 µmol photons m − 2 s − 1 are usual on clear summer days which is about 10 times higher intensity than that required to saturate photosynthesis 19 . The measurement of light intensity in-situ in the 1-cm photic layer in microalgae cultures showed between 150–200 µmol photons m − 2 s − 1 (Figs. 3 and 5 ) which is considered optimal saturating irradiance for microalgae growth [ 23 , 27 ]. The presented data showed that only the photic layer at the culture surface in the relatively deep RWP can substantially contribute to photosynthetic productivity while lower layers are photo-limited, not photosynthesizing due to sub-saturating irradiance level (Fig. 4 ). These findings were supported by previous measurements in the green microalgae cultures in outdoor thin-layer cascades and RWPs [ 2 ]. Of course, we have to keep in mind that the culture is mixed and there is an exchange of the cell populations. Dissolved oxygen concentrations usually reflect the diurnal cycle of irradiance intensity showing the build-up from morning minima to maxima at midday and then the values decline through the afternoon. Under high photosynthesis rates, DO concentration can reach up to 25–30 mg L − 1 (200–400% of air saturation) at the top layer of microalgae cultures, even in open bioreactors [ 17 , 23 ]. High DO concentrations during the day might have a certain impact on growth due to the potential slowdown of photochemical yield [ 16 ]. In the present trials, a significant build-up of DO concentration (> 20 %sat) was observed which indicated that both cultures were photosynthetically active (Figs. 2 b,d). Later afternoon in trial 2, DO concentration increased up to 350 − 40 %sat which was probably caused by insufficient degassing (i.e. aeration in the sump) (Fig. 2 d). Nevertheless, the presented data show that in deep RWPs, the degassing is not as efficient enough to counteract oxygen accumulation in large culture volumes (Fig. 2 ). Dark respiration and also Mehler reaction related to PSI are contributing to reducing the photoxidation damages. Environmental constraints such as photo-stress, high temperatures, drought, or high salinity stimulate the activity of alternative PS I-driven electron transport pathways being an integral part of the energetic and regulatory functions of photosynthetic organisms and providing additional flexibility to protect against unfavorable conditions [ 28 ]. The ratio between the ETR and PS gross measured as oxygen production was close to the theoretical value of 5 below the light saturation point of photosynthesis whereas at higher irradiances the ratio was 7–15 indicating that the process of oxygen consumption in addition to respiration as photorespiration and Mehler reaction has an important role [ 20 ]. A higher ETR/PS gross ratio than 5 has been related to stressful conditions excess of light or nutrient limitations [ 29 ]. In this study, online measurements of the actual quantum yield Y(II) performed in the cultures of Scenedesmus and Chlorella at 4 cm depths showed a diurnal pattern similar to that observed close to the surface of the culture, i.e. antiparallel to light intensity as the minimum was recorded at midday (Fig. 4 ). Dueto the light extinction one might expect that the Y(II) yield would be higher in deeper layers (i.e. recovered via PSII reoxidation) but the cultures are mixed and cells can probably come from upper, more active (illuminated) layers. If we compare the course of Y(II) and DO concentration (Figs. 2 and 4 ), one may presume a certain decrease of Y(II) in trial 2 in the afternoon due to high DO concentrations of about 400%sat as it was found previously [ 16 ]. In the present trials, even when DO concentrations were quite high in trial 2 (Fig. 2 d), it did not have a considerable effect on photosynthetic activity (Fig. 3 f). It means that the culture in trial 2 was still photosynthesizing although its activity was markedly lower compared to that in trial 1 (Fig. 4 ). Further comparison of photosynthetic variables [Fv/Fm, Y(II), rETR max , P max , Resp] measured ex-situ in samples taken from the cultures revealed that the Chlorella culture in trial 2 was less photosynthetically active compared to that of Scenedesmus in trial 1 (Figs. 4 , 5 , 6 and 7 ). Higher photosynthesis rates P max in trial 1 were associated with higher respiration rates as in the Scenedesmus cultures the P max values and Resp rate were 2–3 times higher compared to those found in Chlorella (Figs. 6 d,e, vs. 7d,e). The Resp/PS gross ratios were not considerably different between the two species. In other words, cultures with similar Resp/PS gross ratios may have very different performances since both variables are substantially modified. The rETR max values measured in-situ and ex-situ were similar (115–160 µmol e-m − 2 s − 1 ) in trial 1 however in Chlorella, in-situ values (50–60 µmol e − m − 2 s − 1 ) were lower than those measured ex- situ (110–130 An in-situ study of photosynthetic oxygen exchange and electron transport rate in the marine macroalga Ulva lactuca (Chlorophyta)mol e − m − 2 s − 1 ). Higher in-situ values than those ex-situ have been reported in both green microalgae ( Chlorella fusca ) growing in layer cascades 26 or green macroalgae Ulva sp growing in outdoor tanks [ 30 , 31 ]. Generally, in-situ outdoor measurements are carried out under natural conditions (i.e. irradiance, temperature) which monitor the actual situation in microalgae cultures and usually show higher activities. The kinetics of fast fluorescence induction did not show any deep disturbance in the PSII complex in both cultures (Fig. 7 ). Nevertheless, the trends of Vj and Vi values in the Chlorella culture were about 22 and 14% higher, respectively compared to those in the Scenedesmus culture. It suggests that the electron transport on the acceptor side of the PSII complex might be slowed down, resulting in a decrease in electron transport activity in the Chlorella cultures. The changes of non-photochemical quenching NPQ max and Resp in both cultures (Fig. 5 c vs. Figure 6 c) showed that the values had an antiparallel course; these were either doubled or halved, respectively in Chlorella culture as compared with those of Scenedesmus . It indicates that the part of light energy harvested by the photosynthetic apparatus may be dissipated via non-photochemical quenching or respiration (Figs. 6 c, e vs. Figures 7 c, e) [ 13 , 32 ]. The relaxation mechanism of NPQ may not be fast enough to relax the photosynthetic apparatus 33 . This mechanism remained still partially active during the transition of the cells from the light-saturated layer on the surface of the culture to the light-limited zone. We may infer that cultures subjected to sufficient mixing can reduce the buildup of the NPQ on the surface and thus faster relax once the cells return to the light-limited part of the culture layer. An accelerated rate of NPQ relaxation might be related to the xanthophyll cycles during the transition from sun to shade which can be an important strategy for maintaining microalgae growth [ 33 , 34 ]. The loss of performance may happen in diluted cultures, such as in the case of Chlorella (Fig. 5 b,d). In this culture, NPQ very likely remains active even in deeper layers dissipating most of the light energy. Situations of intense light dissipation can occur frequently in mass cultures of microalgae due to excessive biomass harvesting (dilution) which exposes cells for some time to oversaturating irradiance [ 12 ]. The value NPQ max lower than 1 is an indicator that the thermal dissipation Y(NO) is dominating compared to the photoregulated energy dissipation mechanism Y(NPQ). Y(NO) is the fraction of energy passively dissipated as heat and fluorescence, mainly due to closed PSII reaction centers. High values indicate an inability of the alga to protect itself against photodamage by an excess of radiation [ 35 , 36 ]. Then, NPQ is calculated as the ratio of the two yield losses, i.e. Y(NPQ)/Y(NO) [ 37 ]. Conclusions Strains with similar features can behave differently depending on the set-up of cultivation units and growth conditions. The photosynthesis-measuring techniques using in vivo Chl fluorescence, both in-situ and ex-situ , were proven useful for culture monitoring as they reliably reflect the physiological status of microalgae cultures and can be used to adjust suitable growth regimes in large-scale bioreactors. Online measurements of oxygen production and fluorescence variables showed that the photic zone in RWPs contributing to growth is relatively thin, about 1 cm; in this case, less than 10% of the volume, meaning that most of the culture was ˊphotosyntheticallyˋ in the dark. The maximum photochemical yield of PSII, Fv/Fm is often used as an estimate of the photochemical yield of PSII and its decrease usually indicates that the cultures are exposed to unfavorable conditions. In the presented trials, the Fv/Fm values were found between 0.58-0.79 which indicated that microalgae cultures might be mildly constrained at certain daytimes. The similar biostimulating activity of the biomass samples harvested from the high-density Scenedesmus and low-density Chlorella culture approve that rather the species and its physiological status than the culture density determine the bioactivity. Results also revealed that the part of light energy harvested by the photosynthetic apparatus is dissipated via non-photochemical quenching or respiration in response to variable environmental conditions. The presented data can be used to optimize the growth of microalgae cultures in open large-scale bioreactors understanding the interplay between culture depth and cell concentration. Material and methods Plant material and cultivation The cultures of the green microalgae (class Chlorophyta) Scenedesmus sp. (in the form of four-cell coenobia; average size: 6 × 13 µm) and Chlorella sp. (solitary cells with 4 µm diameter) were grown phototrophically in an inorganic medium (0.9 g L − 1 NaNO 3 , 0.14 g L − 1 KH 2 PO 4 , 0.18 g L − 1 MgSO 4 and 0.015 g L − 1 commercially available mixture of micronutrients @karentol containing Fe, Cu, Mn, B, Zn, Mo and others in the form of chelate) in open cultivation systems – raceway ponds (RWPs) (Fig. 1 ). The two units – 75 and 730 m 2 were placed in a greenhouse which was located at the facilities of the company Biorizon Biotech in Alméria, Spain (GPS coordinates 36°49'59.7"N, 2°24'22.7"W). The average depth of bioreactors was about 14 cm (providing the surface-to-volume ratio of about 11) and a culture flow rate of about 0.3 m s − 1 was maintained by rotating paddle wheels. Carbon dioxide was supplied based on a pH-stat system to keep the value of about 8. Flow deflectors were placed at both ends of the RWPs and a sump was located downstream next to the paddle wheel where the culture can be CO 2 added, or aerated to remove oxygen [ 1 ]. Photosynthesis monitoring Photosynthetic activity of microalgae populations was monitored by Chl fluorescence and oxygen production measurements directly in the bioreactors in-situ as well as ex-situ in microalgae samples taken from outdoor cultures. The time in the figures corresponds to CEST (GMT + 1). In-situ measurements An underwater fluorimeter (Mini-PAM, H. Walz GmbH, Effeltrich, Germany) was used to measure fluorescence quantum yield Y(II) in-situ at two culture depths (about 0.6 cm and about 4 cm) at 3 positions (trial 1 - Fig. 1 a), or 5 positions trial 2 – Fig. 1 b) at three daytimes − 9:00, 13:00 and 17:00 h. Chl fluorescence data were recorded online in microalgae cultures during the diurnal cycle using four portable fluorimeters (Junior-PAM, H. Walz GmbH, Effeltrich) controlled by the WinControl-3 software via a USB interface which was also used for data acquisition [ 13 , 19 , 26 ]. The fluorimeter was fitted with blue light-emitting diodes (LED, 460 nm) to apply the measuring as well as saturating pulses. Ambient irradiance was used as actinic light. Two sets of a measuring light guide (plastic filament; 1.5 mm in diameter and length of 100 cm) and a mini-sensor were submerged at point 1 (downstream of the sump) into the culture at a depth of about 0.6 or 4 cm in both RWPs to measure photosynthesis variables. The incident photosynthetically active radiation E PAR (400–700 nm) and the actual quantum yield of PSII, Y(II) [= (Fm´- F´)/Fm´] were measured each 10 min where the variable F´ is the steady-state fluorescence level and Fm´ is the maximal fluorescence induced by a saturating light pulse which was measured at particular irradiance level (Masojidek et al. 2023). The relative electron transport rate rETR = [Y(II) × E PAR ] through PSII (µmol electrons m − 2 s − 1 ) was used to estimate photosynthetic activity where E PAR is the particular irradiance intensity measured in the culture (µmol photons m − 2 s − 1 ). This variable is easy to calculate and it is adequate to follow the diurnal changes in photosynthetic activity (and physiological conditions) in outdoor microalgae cultures. It is important to note that rETR represents a relative number and should be considered as a comparative variable. Irradiance, temperature, pH, and dissolved oxygen measurements Ambient irradiance (PAR) was measured as 10-s averaged values using a portable light meter (LI-250A with a flat quantum sensor LI-190SA, cosine-corrected up to 80° angle of incidence, Li-Cor, USA or with a spherical mini-sensor US-SQS, H. Walz GmbH, Germany). The culture temperature data were measured at several positions and the values were averaged (Fig. 2 a,c). The dissolved oxygen (DO) concentrations were recorded by a hand-held oximeter (model Oxi 330, WTW, Germany) with temperature compensation. The data were estimated in % of saturation (%sat). Ex-situ measurements For ex-situ measurements of Chl fluorescence and oxygen production, microalgae samples were taken from outdoor cultures at specified daytimes (9:00, 13:00 and 17:00 h) as described previously [ 13 , 17 ]. Photosynthesis light-response curves (LRC; photosynthesis vs. irradiance curve) of electron transport or oxygen production were measured in parallel using a pulse-amplitude-modulation fluorometer (PAM-2100, H. Walz, Germany) connected to a temperature-controlled chamber (DW2/2, Hansatech Instrument Ltd., Norfolk, UK). The curves were recorded in samples after 10–15 min of dark adaptation at 8 light intensities between 0 and 1800 µmol photons m − 2 s − 1 exposing them 2 min under each intensity at the temperature set according to the actual culture value. The minimum and maximum fluorescence levels F 0 and Fm (F 0 , basal fluorescence from fully oxidized reaction centers of PSII; Fm – maximum fluorescence from partially or fully reduced PSII reaction centers) were determined in the dark-adapted samples. The maximum PSII quantum yield was calculated as the ratio of variable and maximum fluorescence, Fv/Fm = (Fm -F 0 )/Fm, which expresses the maximum quantum efficiency of primary photochemistry [ 38 ]. The variable called the relative electron transport rate through PSII, rETR was calculated as the product of the actual photochemical efficiency Y(II) multiplied by the photosynthetically active radiation as mentioned above, see e.g. [ 39 , 40 ]. The values of rETRmax were calculated at the maxima of LRCs. Non-photochemical quenching NPQ [= (Fm – Fm’)/Fm’] was used to estimate non-photochemical energy dissipation [ 41 ]. Besides, rates of dark respiration Resp and photosynthetic oxygen evolution PS were measured as a function of irradiance in microalgae samples using a Clark-type oxygen electrode mounted in a temperature-controlled chamber (DW2/2 chamber, Hansatech Instrument Ltd., Norfolk, UK) connected to an oxygen monitoring system (Oxylab+, Hansatech Instr. Ltd., UK) and the programmable light source to adjust intensity increase in six 2-min steps between 0 and 950 µmol photons m − 2 s − 1 (saturating intensity for photosynthesis) [ 24 ]. The values of respiration and oxygen production are expressed in pmol O 2 cell − 1 s − 1 . Fast fluorescence induction kinetics (Kautsky curve or OJIP test) While the pulse-amplitude-modulation (PAM) technique gives information on the energy distribution between the photochemical and non-photochemical processes in photosynthesis, the fast fluorescence induction kinetics provides information on the redox status of the electron transport chain in the PSII complex. The fluorescence induction curves were measured ex-situ by a portable fluorimeter (AquaPen AP-100, P.S.I. Ltd. Brno, Czech Republic) in samples taken from outdoor cultures, dark-adapted for 5–10 min as described previously [ 22 ]. The curves were measured in the time range between 50 µs to 1 s when the signal rises rapidly from the origin (O) to the highest peak (P) via two infections – J and I [ 42 ]. The O point (50 µs) of the fluorescence induction curve represents a minimum value (designated as constant fluorescence yield F0) when PQ electron acceptors (Q A and Q B ) of the PSII complex are oxidized. The inflection J occurs after ~ 2–3 ms of illumination and reflects the dynamic equilibrium (quasi-steady-state) between Q A and Q B . The J–I phase (at 30–50 ms) is due to the closure of the remaining centers, and the I–P (ends at about 300–500 ms) corresponds to the full reduction of the plastoquinone pool (equivalent to the maximum fluorescence level Fm) [ 42 ]. From the fluorescence levels at the J and I points, the variables Vj and Vi were calculated as follows: Vj = (F 2ms - F 0 )/(Fm-F 0 ) and Vi = (F 30ms F 0 )/(Fm F 0 ) which showed redox status of quinone electron acceptors. Analytical measurements The measurement of biomass content was carried out as dry weight (DW) determination by filtering culture samples on pre-weighed glass microfiber filters (GC-50) as described previously [ 24 ]. The filters with the biomass were washed twice with deionized water and dried in an oven at 105°C for 8 h; then they were weighed (precision of ± 0.01 mg) and the biomass amount was calculated. Chl concentration was determined spectrophotometrically in methanol extracts. The cells were collected by centrifugation and the pellets were resuspended in 100% methanol, sea sand or glass beads were added and the tubes were put into the laboratory ultrasound bath heated to 40°C for 2 min, then cooled down in an ice bath and centrifuged. The absorbance of the supernatant was measured using a high-resolution spectrophotometer and the concentration of Chl was calculated according to Wellburn [ 43 ]. Bioassays The freeze-dried biomass of microalgae cultures harvested from the cultures at the end of cultivation was resuspended in distilled water (3 mg DW L − 1 ) and ultrasonicated (2 min) for testing biostimulating activities. The bioassays were carried out with biomass samples of 2 g L − 1 concentration. Two bioassays were used to detect plant biostimulating activities: the mung bean rooting test and the cucumber cotyledon root formation test. All bioassays were performed in triplicate. Mung bean rooting test (auxin-like activity) The plant biostimulating activity of the extracts was measured using the mung bean ( Vigna radiata ) rooting bioassay [ 44 , 45 ]. The beans were germinated in moist vermiculite at 26°C in a 16:8 h light:dark photoperiod and 120 µmol photons m − 2 s − 1 light intensity. On day 10, uniform mung bean cuttings with two leaves were placed in the growth chamber in the vials with the microalgae extracts (0.5, 1, 2 and 3 g L − 1 ) as the treatment for 6 h and transferred to vials containing water. Distilled water was used as the control. Then the plants were put back in the growth chamber for 8 days. After the incubation period, the number of roots (longer than 1 mm) was counted on each hypocotyl. The mean numbers of roots were compared to a standard curve prepared using indol-3-butyric acid (IBA – auxin equivalent) at concentrations of 0.3-1 mg L − 1 . Cucumber cotyledon rooting test (auxin-like activity) The cucumber ( Cucumis sativus L. ) cotyledon expansion bioassay was used to determine the auxin-like activity of microalgae [ 46 ]. For this bioassay, the freeze-dried biomass extracts were diluted to 2 g L − 1 concentration. Ten cotyledons were excised from 3-day-old seedlings and incubated on Petri dishes in a darkroom (26°C) for 5 days. The number of roots formed at the bases of the cotyledons was then counted and compared between the treatments with microalgae extract and the IBA solutions. Indol-3-butyric acid (IBA – auxin equivalent) was dissolved in 95% ethanol. Micro-algae extracts and IBA solutions were applied on individual 6-cm filter paper disks. To each treated disk, 3 mL of distilled water (control) was added to the bottom of each Petri dish; the IBA concentrations of 0.3-1 mg L − 1 were used as standard. Statistical analysis Most measurements were performed in triplicate ( n = 3); the means and standard deviations (SD) are reported in the figures. Sigma Plot 11.0 was used to determine significant differences between treatments. One-way analysis of variance (ANOVA) and the Holm-Sidac test were conducted for comparison of variables in the trials. For Figs. 2 , 3 , 5 , 6 , 7bd and 8, statistical differences between the samples collected at the same time and different sampling positions were studied. P values lower than 0.05 (P < 0.05) were considered to be significantly different. In graphs, the mean values designated by the same letter did not differ from each other. Declarations Acknowledgments The authors thank Ms. Soňa Pekařová for technical assistance and Mr. Eduard Pareis and Dr. Richard Lhotský for administrative support. Author contributions Conceptualization, J.M., F.L.F., G.T., C.G.-S., F.G.A.-F., J.P.D.; methodology, J.M., K.Š., F.L.F., G.T., C.G.-S., B.C., V.Ö., V.A.R.C., M.B-V., J.P.D., J.L.G.P., F.G.A.-F.; investigation, J.M., V.A.R.C., G.T., K.Š., C.G.-S., B.C., J.A.C.M., A.M.S.B., M.B.-V., V.Ö., J.L.G.P., F.L.F.; data curation, J.M., K.Š., V.A.R.C., G.T., C.G.-S., B.C., J.A.C.M., A.M.S.B., M.B-V., V.Ö., J.L.G.P., F.L.F.; writing—original draft preparation, J.M., G.T., F.L.F., K.Š.; writing—review and editing, J.M., G.T. F.L.F., K.Š., F.G.A-F.; visualization, K.Š., J.M.; G.T., F.L.F.; supervision, J.M., F.L.F., G.T., F.G.A.-F., J.P.D.; funding acquisition, F.G.A.-F., J.M., G.T., J.P.D., F.L.F. All authors have read and agreed to the published version of the manuscript. Funding This research was mainly funded by the EU Horizon 2020 Research and Innovation program, project SABANA (grant no. 727874), and in part by the MULTI-STR3AM project (grant No 887227) which received funding from Bio-based Industries Joint Undertaking. The financial support by the Alga Hub Project “Algae for More Sustainable and Healthy Functional Foods” (TED2021-131555B-C22) by the Ministry of Science and Innovation of the Spanish government has to be also acknowledged. Data availability: The datasets presented in this study are available from the corresponding authors upon reasonable request. The data are not publicly available without the permission of all co-authors. 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Zhang, T.-Y., Wu, Y.-H., Zhu, S., Li, F.-M. & Hu, H.-Y. Isolation and heterotrophic cultivation of mixotrophic microalgae strains for domestic wastewater treatment and lipid production under dark condition. Bioresour. Technol. 149 , 586–589 (2013). Additional Declarations No competing interests reported. Supplementary Files FigureS1.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4449619","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":309429230,"identity":"4ad869af-d842-453d-8305-2a76dd871bd8","order_by":0,"name":"Jiří Masojídek","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIie3QMQrCMBSA4VcCnQqurxT0BEKkUAQRrxJxcHHwBlYcXDyALj1G5sADu1RcOzgoBV0cCi4OHWwE16ibYP4hU74kLwA226+GnAECqa/JVnx1jyYe/2xve5mejt1pr+nPs1tZVodWO2ZFaSJRNgk58nEYwE76a+/ckcrlxvsiNXEROQ2TmjAPyZHKA2Ek+8tZk1kCWXGrOA00UUaSi0gTEUAGAQga1sSJzeT6nKWzibeRv1I0kuSGJlE/bHwqsOq1MKeivFfUl+nC/GM6hnrF1wDs3f4653lowziAzWaz/XMPDYNPVnigNLcAAAAASUVORK5CYII=","orcid":"","institution":"Centre ALGATECH, Institute of Microbiology","correspondingAuthor":true,"prefix":"","firstName":"Jiří","middleName":"","lastName":"Masojídek","suffix":""},{"id":309429231,"identity":"a01f5846-0567-4623-a34f-3151413e74d8","order_by":1,"name":"Karolína Štěrbová","email":"","orcid":"","institution":"Centre ALGATECH, Institute of Microbiology","correspondingAuthor":false,"prefix":"","firstName":"Karolína","middleName":"","lastName":"Štěrbová","suffix":""},{"id":309429232,"identity":"5e9ff1ca-f216-4c47-a9d6-3d1cc21450ee","order_by":2,"name":"Victor A. Robles Carnero","email":"","orcid":"","institution":"Malaga University","correspondingAuthor":false,"prefix":"","firstName":"Victor","middleName":"A. Robles","lastName":"Carnero","suffix":""},{"id":309429233,"identity":"ed29d4c3-733d-4642-af96-af06bdd3480d","order_by":3,"name":"Giuseppe Torzillo","email":"","orcid":"","institution":"CNR - Institute of Bioeconomy","correspondingAuthor":false,"prefix":"","firstName":"Giuseppe","middleName":"","lastName":"Torzillo","suffix":""},{"id":309429234,"identity":"6d45d98a-34c3-4bdc-8012-81ec87d78835","order_by":4,"name":"Cintia Gómez-Serrano","email":"","orcid":"","institution":"University of Almería","correspondingAuthor":false,"prefix":"","firstName":"Cintia","middleName":"","lastName":"Gómez-Serrano","suffix":""},{"id":309429235,"identity":"06633fba-0992-4856-add3-18ad142f4e21","order_by":5,"name":"Bernardo Cicchi","email":"","orcid":"","institution":"CNR - Institute of Bioeconomy","correspondingAuthor":false,"prefix":"","firstName":"Bernardo","middleName":"","lastName":"Cicchi","suffix":""},{"id":309429236,"identity":"7d1aa50c-9057-4e0f-9866-8e3329ea83dd","order_by":6,"name":"João Artur Câmara Manoel","email":"","orcid":"","institution":"Centre ALGATECH, Institute of Microbiology","correspondingAuthor":false,"prefix":"","firstName":"João","middleName":"Artur Câmara","lastName":"Manoel","suffix":""},{"id":309429237,"identity":"44749671-798e-4764-888b-23ce610a59e0","order_by":7,"name":"Ana Margarita Silva Benavides","email":"","orcid":"","institution":"CIMAR, Universidad de Costa Rica","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Margarita Silva","lastName":"Benavides","suffix":""},{"id":309429238,"identity":"f9551028-a90b-48ce-9dc6-6736c3d9214f","order_by":8,"name":"Marta Barceló-Villalobos","email":"","orcid":"","institution":"CHLYDRO","correspondingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Barceló-Villalobos","suffix":""},{"id":309429239,"identity":"fe94f6f4-53f7-4ea8-8913-06b2f2381813","order_by":9,"name":"Joaquín Pozo Dengra","email":"","orcid":"","institution":"BIORIZON BIOTECH, Parque Científico Tecnológico de Almería","correspondingAuthor":false,"prefix":"","firstName":"Joaquín","middleName":"Pozo","lastName":"Dengra","suffix":""},{"id":309429240,"identity":"a76ccc98-683b-44f0-8f70-9f2e0bd4fbc0","order_by":10,"name":"Vince Ördög","email":"","orcid":"","institution":"Széchenyi István University","correspondingAuthor":false,"prefix":"","firstName":"Vince","middleName":"","lastName":"Ördög","suffix":""},{"id":309429241,"identity":"c2566e39-2bd5-4eb2-a091-1ecb7e8ac938","order_by":11,"name":"Juan Luis Gómez Pinchetti","email":"","orcid":"","institution":"Universidad de Las Palmas de Gran Canaria","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"Luis Gómez","lastName":"Pinchetti","suffix":""},{"id":309429242,"identity":"f95f86ce-b874-4f2e-987d-7e92e9c8e008","order_by":12,"name":"Francisco Gabriel Acién Fernándéz","email":"","orcid":"","institution":"University of Almería","correspondingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"Gabriel Acién","lastName":"Fernándéz","suffix":""},{"id":309429243,"identity":"3132e397-1c67-4a49-858e-e81ea8ecb9cf","order_by":13,"name":"Félix López Figueroa","email":"","orcid":"","institution":"Malaga University","correspondingAuthor":false,"prefix":"","firstName":"Félix","middleName":"López","lastName":"Figueroa","suffix":""}],"badges":[],"createdAt":"2024-05-20 14:07:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4449619/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4449619/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58071073,"identity":"70549914-75b0-44bf-8d39-7bdfe8abd9dd","added_by":"auto","created_at":"2024-06-10 18:41:18","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1615788,"visible":true,"origin":"","legend":"\u003cp\u003ePictures and schematic diagrams of two raceway ponds of 76 and 730 m\u003csup\u003e2\u003c/sup\u003e used in trials 1 and 2. (a, c) The bioreactors were placed in a greenhouse which was located at the facilities of the company Biorizon Biotech in Alméria, Spain (GPS coordinates 36°49'59.7\"N, 2°24'22.7\"W). (b, d) Schematic diagram of raceway ponds with numbered positions where culture variables were monitored. In trial 1, three measuring positions were defined (1 – after the paddlewheel and sump; 2 – at the first bend; 3 – close to the end of the second channel before deflectors and paddlewheel)m while in trial 2, five measuring positions were defined (1 – after the paddlewheel and sump; 2 – middle of the first channel; 3 – at the first bend; 4 – middle of the second channel; 5 – close to the end of the channel before deflectors)\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4449619/v1/df3c0d314c478d458c3e6081.jpg"},{"id":58070543,"identity":"13725905-eea1-4927-ba35-e01cc3b095fb","added_by":"auto","created_at":"2024-06-10 18:33:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":797367,"visible":true,"origin":"","legend":"\u003cp\u003e(a, c) Daily changes in the temperature (°C) and (b, d) dissolved oxygen concentration (saturation in %) measured in the cultures during trials 1 and 2. Data were measured at 9:00, 13:00 and 17:00 h; values are presented as a mean of figures measured at 3 or 5 positions in the RWPs (see Fig. 1). Values are presented as a mean ±SE.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4449619/v1/25c0cf9e7bae02669f5e136e.jpg"},{"id":58070546,"identity":"d5033292-2a43-4b90-af87-d658a2bf878c","added_by":"auto","created_at":"2024-06-10 18:33:18","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":941158,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of the actual photochemical yield of PSII, Y(II) measured at 9:00, 13:00 and 17:00 h during day 1 (a, d) day 2 (b, e) and day 3 (c, f) during trial 1 (a, b, c) and trial 2 (d, e, f). Data were measured \u003cem\u003ein-situ\u003c/em\u003e, 0.6 and 4 cm deep in the culture using a portable fluorimeter Mini-PAM (for details see Material and Methods). Values are averaged throughout 3 (trial 1) or 5 measuring points (trial 2) in the RWPs (see Fig. 1).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4449619/v1/d2d17548aba4014b3f012ec6.jpg"},{"id":58071074,"identity":"c98e69d7-0ee0-4708-a7c6-8e649ec02ce0","added_by":"auto","created_at":"2024-06-10 18:41:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3106244,"visible":true,"origin":"","legend":"\u003cp\u003eDiurnal changes in irradiance intensity (µmol photons m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e), the actual photochemical yield of PSII, Y(II) and relative electron transport rate rETR (µmol e\u003csup\u003e-\u003c/sup\u003e m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e) measured \u003cem\u003ein-situ\u003c/em\u003e in the culture during trial 1 (a, c) and trial 2 (b, d). Values were measured by four portable fluorimeters Junior-PAM (for details see Material and Methods) in 10-min intervals. Fibreoptics and a light minisensor were placed next to each other at position 1 in both RWPs (Fig. 1) and submerged at the depth of 0.6-cm (a, b) or 4-cm (c, d). The bars at the upper edge of the panels indicate a diurnal cycle.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4449619/v1/2d989633125bdfdd5a9fd44b.jpg"},{"id":58070548,"identity":"6f90ef9f-c209-4945-8919-c356a6cabc68","added_by":"auto","created_at":"2024-06-10 18:33:19","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1130773,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the maximum photochemical yield of PSII, Y(II) (a), rETR\u003csub\u003emax\u003c/sub\u003e (b), non‑photochemical quenching NPQ (c), photosynthetic oxygen evolution Pmax (d), dark respiration Resp (e) and the ratio of Resp/PS\u003csub\u003egross\u003c/sub\u003e (f). The data were measured at 9:00, 13:00 and 17:00 h on day 1 (a, d) day 2 (b, e) and day 3 (c, f) during trial 1. Values were estimated from light-response curves of Chl fluorescence (portable fluorimeter PAM-2100) and oxygen production (an oxygen monitoring system Oxylab+) which were measured \u003cem\u003eex-situ\u003c/em\u003e using the culture samples taken from RWP (for details see Material and Methods). The values are presented as a mean (n = 3) ±SE and those designated by the same letter did not differ significantly from each other.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePS\u003csub\u003egross\u0026nbsp;\u003c/sub\u003e=\u0026nbsp;Resp\u0026nbsp;+\u0026nbsp;P\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4449619/v1/552dd872487c88be76f63176.jpg"},{"id":58070549,"identity":"41419fea-e732-4dd7-8e70-d3e6baa13b28","added_by":"auto","created_at":"2024-06-10 18:33:19","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1070841,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the maximum photochemical yield of PSII, Y(II) (a), rETR (b), non-photochemical quenching NPQ (c), photosynthetic oxygen evolution Pmax (d), dark respiration Resp (e) and the ratio of Pmax/Resp (f). The data were measured at 9:00, 13:00 and 17:00 h on day 1 (a, d) day 2 (b, e) and day 3 (c, f) during trial 2 (\u003cem\u003eChlorella\u003c/em\u003e). Values were estimated from light-response curves of Chl fluorescence and oxygen production which were measured \u003cem\u003eex-situ\u003c/em\u003eusing the culture samples taken from outdoor cultures grown RWPs (for details see Material and Methods). The values are presented as a mean (n = 3) ±SE and those designated by the same letter did not differ significantly from each other.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4449619/v1/b7503fdf17cf6f5d214212e8.jpg"},{"id":58070547,"identity":"61d07fb7-c32b-4bcb-892b-e6cee3a72a2c","added_by":"auto","created_at":"2024-06-10 18:33:18","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1515556,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Exemplary fast fluorescence induction kinetics measured \u003cem\u003eex-situ\u003c/em\u003e at 13:00 h on days 1, 2 and 3 during trials 1 and 2 using a portable fluorimeter Aquapen AP-100. (b) Daily changes in the variables Vj and Vi in trials 1 and 2 were measured on days 1, 2 and 3 at 9:00, 13:00 and 17:00 h using samples taken from cultures at particular daytimes. Values were calculated from fluorescence induction curves (for more detail see Material and Methods) and are presented as a mean (n = 3) ±SE and those designated by the same letter did not differ significantly from each other.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4449619/v1/22d93d471b77ab72754a9222.jpg"},{"id":58070551,"identity":"05445d6c-5f6b-493b-b463-3d80da632952","added_by":"auto","created_at":"2024-06-10 18:33:19","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":628602,"visible":true,"origin":"","legend":"\u003cp\u003eThe auxin-like activity was detected (a) by mungbean rooting test or (b) by cucumber cotyledon rooting test using water extracts of freeze-dried microalgae samples (2 mg L\u003csup\u003e-1\u003c/sup\u003e) taken from trial 1 (\u003cem\u003eScenedesmus\u003c/em\u003e) and trial 2 (\u003cem\u003eChlorella\u003c/em\u003e) on day 2. Indol-3-butyric acid (IBA) was used as the standard. Dashed lines represent the level of the control (distilled water). The values are presented as a mean (n = 3) ±SE and those designated by the same letter did not differ significantly from each other.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4449619/v1/49dd9790326924dd13a5a535.jpg"},{"id":59547251,"identity":"fa242759-9835-4103-969c-13cc0b05605f","added_by":"auto","created_at":"2024-07-03 05:26:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11606430,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4449619/v1/b3626c7e-2c35-476e-a0bd-a8c689eac425.pdf"},{"id":58070552,"identity":"f4b6759f-b3f4-47b6-bf93-74d0b8aaf178","added_by":"auto","created_at":"2024-06-10 18:33:19","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":223534,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4449619/v1/cf62e54fe3cc99e3372ca3cf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Photosynthetic activity measured in-situ in microalgae cultures grown in large-scale raceway ponds","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTwo basic approaches are used for microalgae production in the solar bioreactors: one possibility is the cultivation in open systems (with direct interaction of the microalgae culture with the environment), while the other employs closed or semi-closed bioreactors with no direct contact between the culture and the outside environment [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. At present, the most frequently used \u0026lsquo;open\u0026rsquo; cultivation systems for the commercial production of microalgae are various types of so-called raceway ponds (RWPs) due to their lower construction cost and easier maintenance compared to closed bioreactors. The early installations of RWPs were introduced in the 1950s-1960s by Oswald and co-workers [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and their designs were advanced in the 1970s-1980s by the use of paddlewheel mixers which reduced the shearing forces on the cells and energy requirements [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]; then further improvements were made ever since [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe production RWPs from 100 to 5,000 m\u003csup\u003e2\u003c/sup\u003e were reported\u003csup\u003e1\u003c/sup\u003e. The key construction parameters are the depth of the culture and the total area occupied. The area is usually divided into two or more channels along which the culture is recirculated by paddle wheels or impellers (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) which maintain a culture velocity of about 0.3 m s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The recommended length-to-width (L/W) ratio of channels is about 15 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The surface-to-total-volume ratio in these systems is about 5\u0026ndash;10 m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. A channel depth should be in the range of 0.2\u0026ndash;0.4 m; it is recommended to operate at a lower culture depth to increase light penetration, biomass concentration and the stability of the cultures. The shorter the light path, the higher the biomass density can be maintained.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHowever, the use of the RWPs is restricted \u0026ndash; due to the limited control of cultivation conditions and contamination \u0026ndash; to a few \u0026ldquo;robust\u0026rdquo; and fast-growing microalgae genera (e.g., \u003cem\u003eChlorella, Scenedesmus, Nannochloropsis\u003c/em\u003e), or those that are cultured under selective conditions (e.g., \u003cem\u003eArthrospira\u003c/em\u003e or \u003cem\u003eDunaliella\u003c/em\u003e) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The cultures in the RWPs are usually grown at low biomass densities ranging between 0.5 and 1 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e depending on the pond depth. The cultures are usually operated in a semi-continuous regime collecting cells each 4\u0026ndash;5 days depending on the season, and generally maintained at relatively high densities to facilitate harvesting.\u003c/p\u003e \u003cp\u003eThe available light is the main factor limiting the productivity of microalgae mass cultures outdoors [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the impact of irradiance on the productivity of outdoor microalgae cultures is rather complex due to the interaction with other environmental variables. The average amount of photon energy received by a single cell is a combination of several factors: light intensity, cell density, culture layer and the rate of mixing as well as the construction of the cultivation unit (for review see e.g. [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In any case, the irradiance regime should be optimised as light in excess can damage the photosynthetic apparatus, particularly in combination with temperature extremes or high oxygen concentration [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMicroalgae, due to photosynthetic activity in outdoor mass cultures, can generate high concentrations of dissolved oxygen (DO). Though oxygen evolution is sometimes overlooked in large-scale units, high concentrations of DO in cultures occurring especially under high irradiance can result in photoinhibition and photorespiration which entail a reduction in photosynthetic activity and growth. In open units, the build-up of DO concentration as much as 3\u0026ndash;4 times higher than the air saturation values can be observed during the day, which may partly decrease the photochemical yield of microalgae cultures [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The maintenance of DO levels below critical concentration (about 200% of air saturation) often requires degassing or efficient culture mixing.\u003c/p\u003e \u003cp\u003eOptimizing culture productivity requires constant monitoring of physicochemical variables, such as pH, temperature, DO concentration, and nutrient levels, but most crucial is monitoring photosynthetic performance. The primary indications of adverse growth conditions can be detected as reduction of photosynthetic activity of a microalgae culture which subsequently slows down its growth and productivity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Finding rapid, reliable and robust techniques to evaluate variations in microalgae activity has been one of the major tasks for the online monitoring of pilot and large-scale cultivation units [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Various monitoring methods have been used to adjust growth conditions for the production of biomass. The earlier reports showed that the photosynthetic variables (oxygen production, photochemical yield, electron transport rate) monitored \u003cem\u003ein-situ/ex-situ\u003c/em\u003e well reflect the physiological status of the culture and provide primary information on photosynthetic activity which reflects the growth and biomass productivity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eChanges in oxygen production and \u003cem\u003ein vivo\u003c/em\u003e chlorophyll (Chl) fluorescence are being used for monitoring microalgae culture, as they provide primary information on culture photosynthetic performance and consequently are reflected in culture growth [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The direct method of monitoring microalgae cultures is to follow the photosynthetic changes \u003cem\u003ein-situ\u003c/em\u003e tracking the actual situation. The other possibility, but more time-consuming, is to assess the culture state \u003cem\u003eex-situ\u003c/em\u003e using microalgae samples withdrawn from a cultivation unit. Some variables, such as photosynthetic oxygen production, or PSII photochemical yield and electron transport rate are used to correlate with photosynthetic activity and growth [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this case study, we aimed to survey the photosynthetic activity of outdoor microalgae cultures at various parts and depths of large RWPs using photosynthesis monitoring techniques. Particularly in this work, two strains of the same taxon Chlorophyta, \u003cem\u003eScenedesmus\u003c/em\u003e and \u003cem\u003eChlorella\u003c/em\u003e were examined which are characterized by their robustness and fast growth. Photosynthetic activity of microalgae cultures was measured in parallel with other variables (irradiance, temperature, dissolved oxygen concentration) and biostimulating activity. The data showed that even in not as dense cultures (0.5-1 g dry weight L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) the active photic layer in the culture is limited to about 1 cm. The presented work can be used as exemplary data to optimize the growth conditions of microalgae cultures in large-scale RWPs.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCulture growth, irradiance and oxygen production\u003c/h2\u003e \u003cp\u003eTwo 5-day trials were carried out in parallel using two RWP bioreactors during sunny and warm days in mid-summer (August). In trial 1, the culture of green microalga \u003cem\u003eScenedesmus\u003c/em\u003e sp. (further as \u003cem\u003eScenedesmus\u003c/em\u003e) was grown in the RWP with an area of 76 m\u003csup\u003e2\u003c/sup\u003e in which 3 measuring sites were defined (1 \u0026ndash; after the sump; 2 \u0026ndash; at the first bend; 3 \u0026ndash; close to the end of the second channel before deflectors) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea,b). In trial 2, the culture of green microalga \u003cem\u003eChlorella\u003c/em\u003e sp. (further as \u003cem\u003eChlorella\u003c/em\u003e) was grown in the RWP with an area of 730 m\u003csup\u003e2\u003c/sup\u003e in which 5 measuring positions were defined (1 \u0026ndash; after the sump; 2 \u0026ndash; middle of the first channel; 3 \u0026ndash; at the first bend; 4 \u0026ndash; middle of the second channel; 5 \u0026ndash; close to the end of the channel before deflectors) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec,d). The choice of measurement points included sites where the mixing was less efficient such as at the bends, or where it was expected to be considerably higher such as after the paddlewheels and the sump.\u003c/p\u003e \u003cp\u003eIn trial 1, the biomass density of the \u003cem\u003eScenedesmus\u003c/em\u003e culture was about 0.9 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e while in trial 2 (\u003cem\u003eChlorella\u003c/em\u003e sp.) the biomass density was three times lower, about 0.3 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The chlorophyll (Chl) content was about 13.3 and about 5.6 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in trial 1 and trial 2, respectively. In both units, the culture depth was about 14 cm. As concerns the temperature and DO concentration changes in the \u003cem\u003eScenedesmus\u003c/em\u003e culture during trial 1, the data varied between 27.5\u0026ndash;28\u0026deg;C in the morning (9:00 h) up to 35\u0026ndash;36\u0026deg;C in the afternoon (17:00 h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea \u0026ndash; values are a range of data recorded in 3 measuring positions). DO concentration values were found between 26\u0026ndash;78%sat in the morning, increased to 171\u0026ndash;258%sat at midday (13:00 h) and ranged between 178\u0026ndash;258%sat at 17:00 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb \u0026ndash; values represent a range of data recorded in 5 measuring positions). In trial 2 monitoring the \u003cem\u003eChlorella\u003c/em\u003e culture, the temperature varied between 27-27.5\u0026deg;C in the morning (9:00 h) up to 34\u0026ndash;35\u0026deg;C in the afternoon (17:00 h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). DO concentration values ranged between 92\u0026ndash;102%sat in the morning, increased to 186\u0026thinsp;\u0026minus;\u0026thinsp;34 %sat at midday (13:00 h) and between 169\u0026thinsp;\u0026minus;\u0026thinsp;41 %sat at 17:00 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe typical daily course of irradiance measured in the greenhouse indicated the down at about 6:30 h and the dusk at about 21:00 hl; the range of the highest irradiance was seen between 14:00 h and 15:00 h (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea). The ambient irradiance maxima of about 1800 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e measured at 13:00 h was usually 10\u0026ndash;15% higher than those inside the greenhouse. Therefore, the irradiance intensities measured at the surface of RWPs during the trial were about 300, 1350, 1600 and 1100 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 9:00, 11:00, 13:00 and 17:00 h, respectively. The irradiance intensities measured \u003cem\u003ein-situ\u003c/em\u003e in the cultures at the depth of 1, 4 and 7 cm were between 85\u0026ndash;250, 20\u0026ndash;85 and between 6\u0026ndash;21 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively from 9:00 to 17:00 h (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb). The data showed that irradiance intensity at 7-cm depth is rather low and it is probably outside the photic zone of the culture.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhotosynthesis monitoring\u003c/b\u003e \u003cb\u003ein-situ\u003c/b\u003e\u003c/p\u003e \u003cp\u003eValues of the actual photochemical yield Y(II) were measured \u003cem\u003ein-situ\u003c/em\u003e in the culture at two depths \u0026ndash; 0.6 and 4 cm during the 3-day trials in both trials (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The culture depths for measurements were selected to provide average light intensity (midday) 150\u0026ndash;200 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 0.6 cm depth (close to saturating irradiance for growth) and about 20 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 4 cm (low irradiance close to the photosynthesis compensation point). It is important to note that data presented here are a mean of data measured at 3 (trial 1) or 5 positions (trial 2) in the RWPs (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and there was not much variation among the values measured at various positions. In trial 1, the Y(II) values were measured at 9:00, 13:00 and 17:00 h on days 1, 2 and 3. In this trial, the highest values of Y(II) \u0026ndash; between 0.49\u0026ndash;0.71 were found at 9:00 h (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,b,c); there was not much difference between the values measured at both depths. At 13:00 h and 17:00 h, the Y(II) values measured at 0.6-cm and 4-cm depths were lower \u0026ndash; between 0.31\u0026ndash;0.51 due to higher ambient irradiance penetrating the culture which caused higher PQ pool reduction. Nevertheless, there was little difference between Y(II) measured at 0.6-cm and 4-cm depths at 13:00 h and 17:00 h, which means that penetrating irradiance intensity was similar. In trial 2, the course of Y(II) changes was similar to that in trial 1, only the Y(II) values were mostly 15\u0026ndash;20% lower compared to trial 1 suggesting lower photosynthetic activity of the culture. The highest values of Y(II) \u0026ndash; between 0.42\u0026ndash;0.62 were found at 9:00 h at both depths (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed,e,f). At 13:00 and 17:00 h, the measured Y(II) values were lower \u0026ndash; between 0.33\u0026ndash;0.38; and as in the case of \u003cem\u003eScenedesmus\u003c/em\u003e in trial 1 there were found small differences between the Y(II) values measured at 0.6-cm and 4-cm depths.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn both trials, the ambient irradiance (PAR) penetrating inside the culture and actual PSII photochemical yield Y(II) were measured \u003cem\u003ein-situ\u003c/em\u003e/online during the diurnal cycle using submerged sensors of irradiance and fluorescence placed close to each other which were connected to the Junior-PAM fluorimeter. Then, the relative electron transport rate rETR was estimated [Y(II) \u0026times; E\u003csub\u003ePAR\u003c/sub\u003e]. The irradiance intensity started to rise between 8:00 and 9:00 h and it was diminished at about 21:00 h which means that the culture recorded light for about 12\u0026ndash;13 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). As mentioned above, there was not much variation among the values of actual photochemical yield Y(II) measured at various positions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e); thus only one position, point 1 downstream after the sump was selected for online measurements in both RWPs at two depths \u0026ndash; 0.6 and 4 cm. In trial 1, the irradiance intensity of about 150\u0026ndash;200 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was measured at the 0.6-cm depth between 14:00\u0026ndash;17:00 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In this period, the maximum values of rETR were also estimated \u0026ndash; between 115\u0026ndash;160 \u0026micro;mol e\u003csup\u003e\u0026minus;\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and these values followed the course corresponding to irradiance intensities measured inside the culture. The course of Y(II) values was antiparallel to irradiance due to the degree of PSII reduction \u0026ndash; in light periods it was between 0.2\u0026ndash;0.45 while in dark periods increased up to 0.6. Then in trial 1, when the irradiance inside the culture was measured at the 4-cm depth, much lower maximum intensities \u0026ndash; between 15\u0026ndash;20 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were found which are probably close to the photosynthesis compensation point (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). There was not much difference in the Y(II) values between the night and day periods as the penetrating irradiance was low, only the trend showed a slight decrease after sunset (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Thus, the estimated rETR showed maximum values between 10\u0026ndash;20 \u0026micro;mol e\u003csup\u003e\u0026minus;\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which was less than one-tenth of that found at 0.6-cm depth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn trial 2, the maximum irradiance of 100\u0026ndash;150 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e intensity in the culture at the 0.6-cm depth was measured between 10:00\u0026ndash;17:00 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In this period the rETR values were also estimated \u0026ndash; between 30\u0026ndash;50 \u0026micro;mol e\u003csup\u003e\u0026minus;\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; the course corresponded to that of the irradiance intensity inside the culture. The course of Y(II) values was the opposite of irradiance \u0026ndash; in light periods it was between 0.1\u0026ndash;0.2 while in dark periods it was between 0.2\u0026ndash;0.3. Only the trend showed a slight decrease in Y(II) during the day and night periods. In this trial, when the irradiance was measured at the 4-cm depth, much lower maximum intensities of only up to 17 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were found which are probably below the photosynthesis-exciting level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). In this period, the rETR values were found only between 5\u0026ndash;8 \u0026micro;mol e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; the course corresponded to that of the irradiance intensity inside the culture.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhotosynthesis measurements\u003c/b\u003e \u003cb\u003eex-situ\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn both trials, the maximum photochemical yield F\u003csub\u003ev\u003c/sub\u003e/F\u003csub\u003em\u003c/sub\u003e, maximum relative electron transport rate rETR\u003csub\u003emax\u003c/sub\u003e, maximal non-photochemical quenching NPQ\u003csub\u003emax\u003c/sub\u003e, the maximum rate of photosynthetic oxygen production P\u003csub\u003emax\u003c/sub\u003e, maximum rate of dark respiration Resp and the fraction of respiration in gross photosynthesis Resp/PS\u003csub\u003egross\u003c/sub\u003e (in %; PS\u003csub\u003egross\u003c/sub\u003e is the sum of P\u003csub\u003emax\u003c/sub\u003e + Resp) were measured \u003cem\u003eex-situ\u003c/em\u003e using culture samples taken from raceway ponds at particular daytimes \u0026ndash; 9:00, 13:00 and 17:00 h (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Values of F\u003csub\u003ev\u003c/sub\u003e/F\u003csub\u003em\u003c/sub\u003e, rETR\u003csub\u003emax\u003c/sub\u003e and NPQ\u003csub\u003emax\u003c/sub\u003e were calculated from light-response curves (LRC) of \u003cem\u003ein vivo\u003c/em\u003e Chl fluorescence while P\u003csub\u003emax\u003c/sub\u003e, Resp and Resp/PS\u003csub\u003egross\u003c/sub\u003e were determined from LRC of oxygen production/consumption.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn trial 1 the values of F\u003csub\u003ev\u003c/sub\u003e/F\u003csub\u003em\u003c/sub\u003e ranged between 0.63 and 0.79 and were relatively aligned (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The rETRmax values were found between 110\u0026ndash;150 \u0026micro;mol e\u003csup\u003e\u0026minus;\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) while NPQ\u003csub\u003emax\u003c/sub\u003e was between 0.25\u0026ndash;0.63 when the values were found decreasing from day 1 to day 3 and the lowest were found on day 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The range of P\u003csub\u003emax\u003c/sub\u003e and Resp was found between 9-10.8 pmol O\u003csub\u003e2\u003c/sub\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 0.48\u0026ndash;1.6 pmol O\u003csub\u003e2\u003c/sub\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed,e). The data of the rate between Resp/PS\u003csub\u003egross\u003c/sub\u003e revealed the lowest ratio of Resp in PSgross was found in the morning \u0026ndash; between 4.6\u0026ndash;9.8% while the highest \u0026ndash; between 17.8\u0026ndash;28.9 pmol O\u003csub\u003e2\u003c/sub\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was in the afternoon (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). These data also confirmed an increasing trend from the morning towards the afternoon and also from day toward day 3. The highest values between 11 and 15.4 were found on days 2 and 3 at 13:00 and 17:00 h. Considering the data of Pmax and Resp, it was obvious that the ratio of Resp/PSgross showed an increasing trend.\u003c/p\u003e \u003cp\u003eIn trial 2, the values of F\u003csub\u003ev\u003c/sub\u003e/F\u003csub\u003em\u003c/sub\u003e ranged between 0.58 and 0.71 and were relatively comparable during the trial (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Compared to trial 1 they were about 10% lower. The rETR\u003csub\u003emax\u003c/sub\u003e values were found between 110\u0026ndash;130 \u0026micro;mol e\u003csup\u003e\u0026minus;\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), which was about 15 below that recorded in trial 1, while NPQ was between 0.54\u0026ndash;0.83 when the values were found lowest in the morning (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). The range of Pmax was found mostly between 2.7\u0026ndash;6.2 9-10.8 pmol O\u003csub\u003e2\u003c/sub\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, except the morning of day 1 when the value was 10.6 pmol O\u003csub\u003e2\u003c/sub\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Compared to trial 1, Resp was found lower \u0026ndash; between 0.27\u0026ndash;0.70 pmol O\u003csub\u003e2\u003c/sub\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Similarly as in trial 1, the ratio between Resp/PS\u003csub\u003egross\u003c/sub\u003e was low in the morning \u0026ndash; between 2.5\u0026ndash;6.7% while much higher \u0026ndash; between 11.1\u0026ndash;14.6 was found in the afternoon on days 2 and 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). Again, these data showed an increasing trend from the morning towards the afternoon and also from day 1 toward day 3.\u003c/p\u003e \u003cp\u003eIn both trials, culture samples were taken from the RWPs at 9:00, 13:00 and 17:00 h on days 1, 2 and 3. The samples were examined using the fast fluorescence induction kinetics (OJIP test) to estimate the redox status of quinone electron acceptors in the PSII complex. The examples of fluorescence induction curves were recorded at 13:00 h for all three days in trial 1 and trial 2 (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b). The curves were analyzed to evaluate the reduction status of the PSII electron acceptors. From the fluorescence levels at the J and I points, the variables Vj and Vi were calculated which express the redox status of quinone electron acceptors which is demonstrated by Vj and Vi variables (see Methods). The trends of Vj and Vi were similar in both \u003cem\u003eScenedesmus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb) and \u003cem\u003eChlorella\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed) cultures. Nevertheless, in the \u003cem\u003eChlorella\u003c/em\u003e culture (trial 2) the values of Vj and Vi were about 22 and 14% higher, respectively compared to those in the \u003cem\u003eScenedesmus\u003c/em\u003e culture.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBioassays\u003c/h2\u003e \u003cp\u003eIn the present trials, an important outcome was the biostimulating (auxin-like) activity of both \u003cem\u003eScenedesmus\u003c/em\u003e and \u003cem\u003eChlorella\u003c/em\u003e cultures. The biostimulating activities of the freeze-dried biomass samples were similar when detected by two bioassays. The mungbean rooting bioassay and the cucumber cotyledon rooting bioassay revealed that the sample of culture biomass taken in trial 2 had auxin-like activity equivalent to 0.3\u0026ndash;0.5 mg IBA L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, i.e. 125% of the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The biomass collected in trial 2 (Scenedesmus) showed similar bioactivity with the cucumber cotyledon rooting bioassay.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn mass outdoor cultures, microalgae cells are subjected to changes in temperature and light intensity varying due to the diurnal and seasonal changes. Generally, the acceptable growth temperature for most microalgae species is between 15 and 35\u0026deg;C and temperature optima are about 30\u0026deg;C [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In the presented trials, the course of culture temperature was similar in both RWPs, on average about 31\u0026deg;C; only temporarily it was increased over 35\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,c). The ambient irradiance maxima of about 1,800-2,000 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are usual on clear summer days which is about 10 times higher intensity than that required to saturate photosynthesis\u003csup\u003e19\u003c/sup\u003e. The measurement of light intensity \u003cem\u003ein-situ\u003c/em\u003e in the 1-cm photic layer in microalgae cultures showed between 150\u0026ndash;200 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) which is considered optimal saturating irradiance for microalgae growth [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The presented data showed that only the photic layer at the culture surface in the relatively deep RWP can substantially contribute to photosynthetic productivity while lower layers are photo-limited, not photosynthesizing due to sub-saturating irradiance level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These findings were supported by previous measurements in the green microalgae cultures in outdoor thin-layer cascades and RWPs [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Of course, we have to keep in mind that the culture is mixed and there is an exchange of the cell populations.\u003c/p\u003e \u003cp\u003eDissolved oxygen concentrations usually reflect the diurnal cycle of irradiance intensity showing the build-up from morning minima to maxima at midday and then the values decline through the afternoon. Under high photosynthesis rates, DO concentration can reach up to 25\u0026ndash;30 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (200\u0026ndash;400% of air saturation) at the top layer of microalgae cultures, even in open bioreactors [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. High DO concentrations during the day might have a certain impact on growth due to the potential slowdown of photochemical yield [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the present trials, a significant build-up of DO concentration (\u0026gt;\u0026thinsp;20 %sat) was observed which indicated that both cultures were photosynthetically active (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,d). Later afternoon in trial 2, DO concentration increased up to 350\u0026thinsp;\u0026minus;\u0026thinsp;40 %sat which was probably caused by insufficient degassing (i.e. aeration in the sump) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Nevertheless, the presented data show that in deep RWPs, the degassing is not as efficient enough to counteract oxygen accumulation in large culture volumes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Dark respiration and also Mehler reaction related to PSI are contributing to reducing the photoxidation damages. Environmental constraints such as photo-stress, high temperatures, drought, or high salinity stimulate the activity of alternative PS I-driven electron transport pathways being an integral part of the energetic and regulatory functions of photosynthetic organisms and providing additional flexibility to protect against unfavorable conditions [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The ratio between the ETR and PS\u003csub\u003egross\u003c/sub\u003e measured as oxygen production was close to the theoretical value of 5 below the light saturation point of photosynthesis whereas at higher irradiances the ratio was 7\u0026ndash;15 indicating that the process of oxygen consumption in addition to respiration as photorespiration and Mehler reaction has an important role [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. A higher ETR/PS\u003csub\u003egross\u003c/sub\u003e ratio than 5 has been related to stressful conditions excess of light or nutrient limitations [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, online measurements of the actual quantum yield Y(II) performed in the cultures of \u003cem\u003eScenedesmus\u003c/em\u003e and \u003cem\u003eChlorella\u003c/em\u003e at 4 cm depths showed a diurnal pattern similar to that observed close to the surface of the culture, i.e. antiparallel to light intensity as the minimum was recorded at midday (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Dueto the light extinction one might expect that the Y(II) yield would be higher in deeper layers (i.e. recovered via PSII reoxidation) but the cultures are mixed and cells can probably come from upper, more active (illuminated) layers. If we compare the course of Y(II) and DO concentration (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), one may presume a certain decrease of Y(II) in trial 2 in the afternoon due to high DO concentrations of about 400%sat as it was found previously [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the present trials, even when DO concentrations were quite high in trial 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), it did not have a considerable effect on photosynthetic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). It means that the culture in trial 2 was still photosynthesizing although its activity was markedly lower compared to that in trial 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurther comparison of photosynthetic variables [Fv/Fm, Y(II), rETR\u003csub\u003emax\u003c/sub\u003e, P\u003csub\u003emax\u003c/sub\u003e, Resp] measured ex-situ in samples taken from the cultures revealed that the \u003cem\u003eChlorella\u003c/em\u003e culture in trial 2 was less photosynthetically active compared to that of \u003cem\u003eScenedesmus\u003c/em\u003e in trial 1 (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Higher photosynthesis rates P\u003csub\u003emax\u003c/sub\u003e in trial 1 were associated with higher respiration rates as in the \u003cem\u003eScenedesmus\u003c/em\u003e cultures the P\u003csub\u003emax\u003c/sub\u003e values and Resp rate were 2\u0026ndash;3 times higher compared to those found in \u003cem\u003eChlorella\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed,e, vs. 7d,e). The Resp/PS\u003csub\u003egross\u003c/sub\u003e ratios were not considerably different between the two species. In other words, cultures with similar Resp/PS\u003csub\u003egross\u003c/sub\u003e ratios may have very different performances since both variables are substantially modified.\u003c/p\u003e \u003cp\u003eThe rETR\u003csub\u003emax\u003c/sub\u003e values measured \u003cem\u003ein-situ\u003c/em\u003e and \u003cem\u003eex-situ\u003c/em\u003e were similar (115\u0026ndash;160 \u0026micro;mol e-m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in trial 1 however in \u003cem\u003eChlorella, in-situ\u003c/em\u003e values (50\u0026ndash;60 \u0026micro;mol e\u003csup\u003e\u0026minus;\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were lower than those measured ex-\u003cem\u003esitu\u003c/em\u003e (110\u0026ndash;130 An \u003cem\u003ein-situ\u003c/em\u003e study of photosynthetic oxygen exchange and electron transport rate in the marine macroalga \u003cem\u003eUlva lactuca\u003c/em\u003e (Chlorophyta)mol e\u003csup\u003e\u0026minus;\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Higher \u003cem\u003ein-situ\u003c/em\u003e values than those \u003cem\u003eex-situ\u003c/em\u003e have been reported in both green microalgae (\u003cem\u003eChlorella fusca\u003c/em\u003e) growing in layer cascades\u003csup\u003e26\u003c/sup\u003e or green macroalgae \u003cem\u003eUlva\u003c/em\u003e sp growing in outdoor tanks [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Generally, \u003cem\u003ein-situ\u003c/em\u003e outdoor measurements are carried out under natural conditions (i.e. irradiance, temperature) which monitor the actual situation in microalgae cultures and usually show higher activities.\u003c/p\u003e \u003cp\u003eThe kinetics of fast fluorescence induction did not show any deep disturbance in the PSII complex in both cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Nevertheless, the trends of Vj and Vi values in the \u003cem\u003eChlorella\u003c/em\u003e culture were about 22 and 14% higher, respectively compared to those in the \u003cem\u003eScenedesmus\u003c/em\u003e culture. It suggests that the electron transport on the acceptor side of the PSII complex might be slowed down, resulting in a decrease in electron transport activity in the \u003cem\u003eChlorella\u003c/em\u003e cultures.\u003c/p\u003e \u003cp\u003eThe changes of non-photochemical quenching NPQ\u003csub\u003emax\u003c/sub\u003e and Resp in both cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec vs. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec) showed that the values had an antiparallel course; these were either doubled or halved, respectively in \u003cem\u003eChlorella\u003c/em\u003e culture as compared with those of \u003cem\u003eScenedesmus\u003c/em\u003e. It indicates that the part of light energy harvested by the photosynthetic apparatus may be dissipated via non-photochemical quenching or respiration (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, e vs. Figures\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, e) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The relaxation mechanism of NPQ may not be fast enough to relax the photosynthetic apparatus\u003csup\u003e33\u003c/sup\u003e. This mechanism remained still partially active during the transition of the cells from the light-saturated layer on the surface of the culture to the light-limited zone. We may infer that cultures subjected to sufficient mixing can reduce the buildup of the NPQ on the surface and thus faster relax once the cells return to the light-limited part of the culture layer. An accelerated rate of NPQ relaxation might be related to the xanthophyll cycles during the transition from sun to shade which can be an important strategy for maintaining microalgae growth [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The loss of performance may happen in diluted cultures, such as in the case of \u003cem\u003eChlorella\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb,d). In this culture, NPQ very likely remains active even in deeper layers dissipating most of the light energy. Situations of intense light dissipation can occur frequently in mass cultures of microalgae due to excessive biomass harvesting (dilution) which exposes cells for some time to oversaturating irradiance [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe value NPQ\u003csub\u003emax\u003c/sub\u003e lower than 1 is an indicator that the thermal dissipation Y(NO) is dominating compared to the photoregulated energy dissipation mechanism Y(NPQ). Y(NO) is the fraction of energy passively dissipated as heat and fluorescence, mainly due to closed PSII reaction centers. High values indicate an inability of the alga to protect itself against photodamage by an excess of radiation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Then, NPQ is calculated as the ratio of the two yield losses, i.e. Y(NPQ)/Y(NO) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cul\u003e\n \u003cli\u003eStrains with similar features can behave differently depending on the set-up of cultivation units and growth conditions.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eThe photosynthesis-measuring techniques using \u003cem\u003ein vivo\u003c/em\u003e Chl fluorescence, both \u003cem\u003ein-situ\u0026nbsp;\u003c/em\u003eand \u003cem\u003eex-situ\u003c/em\u003e, were proven useful for culture monitoring as they reliably reflect the physiological status of microalgae cultures and can be used to adjust suitable growth regimes in large-scale bioreactors.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eOnline measurements of oxygen production and fluorescence variables showed that the photic zone in RWPs contributing to growth is relatively thin, about 1 cm; in this case, less than 10% of the volume, meaning that\u0026nbsp;most of the culture was ˊphotosyntheticallyˋ in the dark.\u003c/li\u003e\n \u003cli\u003eThe maximum photochemical yield of PSII, Fv/Fm is often used as an estimate of the photochemical yield of PSII and its decrease usually indicates that the cultures are exposed to unfavorable conditions. In the presented trials, the Fv/Fm values were found between 0.58-0.79 which indicated that microalgae cultures might be mildly constrained at certain daytimes.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eThe similar biostimulating activity of the biomass samples harvested from the high-density \u003cem\u003eScenedesmus\u003c/em\u003e and low-density \u003cem\u003eChlorella\u003c/em\u003e culture approve that rather the species and its physiological status than the culture density determine the bioactivity.\u003c/li\u003e\n \u003cli\u003eResults also revealed that the part of light energy harvested by the photosynthetic apparatus is dissipated via non-photochemical quenching or respiration\u0026nbsp;in response to variable environmental conditions.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eThe presented data can be used to optimize the growth of microalgae cultures in open large-scale bioreactors understanding the interplay between culture depth and cell concentration.\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePlant material and cultivation\u003c/h2\u003e \u003cp\u003eThe cultures of the green microalgae (class Chlorophyta) \u003cem\u003eScenedesmus\u003c/em\u003e sp. (in the form of four-cell coenobia; average size: 6 \u0026times; 13 \u0026micro;m) and \u003cem\u003eChlorella\u003c/em\u003e sp. (solitary cells with 4 \u0026micro;m diameter) were grown phototrophically in an inorganic medium (0.9 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaNO\u003csub\u003e3\u003c/sub\u003e, 0.14 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.18 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MgSO\u003csub\u003e4\u003c/sub\u003e and 0.015 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e commercially available mixture of micronutrients @karentol containing Fe, Cu, Mn, B, Zn, Mo and others in the form of chelate) in open cultivation systems \u0026ndash; raceway ponds (RWPs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The two units \u0026ndash; 75 and 730 m\u003csup\u003e2\u003c/sup\u003e were placed in a greenhouse which was located at the facilities of the company Biorizon Biotech in Alm\u0026eacute;ria, Spain (GPS coordinates 36\u0026deg;49'59.7\"N, 2\u0026deg;24'22.7\"W). The average depth of bioreactors was about 14 cm (providing the surface-to-volume ratio of about 11) and a culture flow rate of about 0.3 m s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was maintained by rotating paddle wheels. Carbon dioxide was supplied based on a pH-stat system to keep the value of about 8. Flow deflectors were placed at both ends of the RWPs and a sump was located downstream next to the paddle wheel where the culture can be CO\u003csub\u003e2\u003c/sub\u003e added, or aerated to remove oxygen [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePhotosynthesis monitoring\u003c/h2\u003e \u003cp\u003ePhotosynthetic activity of microalgae populations was monitored by Chl fluorescence and oxygen production measurements directly in the bioreactors \u003cem\u003ein-situ\u003c/em\u003e as well as \u003cem\u003eex-situ\u003c/em\u003e in microalgae samples taken from outdoor cultures. The time in the figures corresponds to CEST (GMT\u0026thinsp;+\u0026thinsp;1).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn-situ\u003c/b\u003e \u003cb\u003emeasurements\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAn underwater fluorimeter (Mini-PAM, H. Walz GmbH, Effeltrich, Germany) was used to measure fluorescence quantum yield Y(II) \u003cem\u003ein-situ\u003c/em\u003e at two culture depths (about 0.6 cm and about 4 cm) at 3 positions (trial 1 - Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), or 5 positions trial 2 \u0026ndash; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) at three daytimes \u0026minus;\u0026thinsp;9:00, 13:00 and 17:00 h. Chl fluorescence data were recorded online in microalgae cultures during the diurnal cycle using four portable fluorimeters (Junior-PAM, H. Walz GmbH, Effeltrich) controlled by the WinControl-3 software via a USB interface which was also used for data acquisition [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The fluorimeter was fitted with blue light-emitting diodes (LED, 460 nm) to apply the measuring as well as saturating pulses. Ambient irradiance was used as actinic light. Two sets of a measuring light guide (plastic filament; 1.5 mm in diameter and length of 100 cm) and a mini-sensor were submerged at point 1 (downstream of the sump) into the culture at a depth of about 0.6 or 4 cm in both RWPs to measure photosynthesis variables. The incident photosynthetically active radiation E\u003csub\u003ePAR\u003c/sub\u003e (400\u0026ndash;700 nm) and the actual quantum yield of PSII, Y(II) [= (Fm\u0026acute;- F\u0026acute;)/Fm\u0026acute;] were measured each 10 min where the variable F\u0026acute; is the steady-state fluorescence level and Fm\u0026acute; is the maximal fluorescence induced by a saturating light pulse which was measured at particular irradiance level (Masojidek et al. 2023). The relative electron transport rate rETR = [Y(II) \u0026times; E\u003csub\u003ePAR\u003c/sub\u003e] through PSII (\u0026micro;mol electrons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was used to estimate photosynthetic activity where E\u003csub\u003ePAR\u003c/sub\u003e is the particular irradiance intensity measured in the culture (\u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). This variable is easy to calculate and it is adequate to follow the diurnal changes in photosynthetic activity (and physiological conditions) in outdoor microalgae cultures. It is important to note that rETR represents a relative number and should be considered as a comparative variable.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eIrradiance, temperature, pH, and dissolved oxygen measurements\u003c/h2\u003e \u003cp\u003eAmbient irradiance (PAR) was measured as 10-s averaged values using a portable light meter (LI-250A with a flat quantum sensor LI-190SA, cosine-corrected up to 80\u0026deg; angle of incidence, Li-Cor, USA or with a spherical mini-sensor US-SQS, H. Walz GmbH, Germany). The culture temperature data were measured at several positions and the values were averaged (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,c).\u003c/p\u003e \u003cp\u003eThe dissolved oxygen (DO) concentrations were recorded by a hand-held oximeter (model Oxi 330, WTW, Germany) with temperature compensation. The data were estimated in % of saturation (%sat).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEx-situ\u003c/b\u003e \u003cb\u003emeasurements\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eex-situ\u003c/em\u003e measurements of Chl fluorescence and oxygen production, microalgae samples were taken from outdoor cultures at specified daytimes (9:00, 13:00 and 17:00 h) as described previously [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePhotosynthesis light-response curves (LRC; photosynthesis vs. irradiance curve) of electron transport or oxygen production were measured in parallel using a pulse-amplitude-modulation fluorometer (PAM-2100, H. Walz, Germany) connected to a temperature-controlled chamber (DW2/2, Hansatech Instrument Ltd., Norfolk, UK). The curves were recorded in samples after 10\u0026ndash;15 min of dark adaptation at 8 light intensities between 0 and 1800 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e exposing them 2 min under each intensity at the temperature set according to the actual culture value. The minimum and maximum fluorescence levels F\u003csub\u003e0\u003c/sub\u003e and Fm (F\u003csub\u003e0\u003c/sub\u003e, basal fluorescence from fully oxidized reaction centers of PSII; Fm \u0026ndash; maximum fluorescence from partially or fully reduced PSII reaction centers) were determined in the dark-adapted samples. The maximum PSII quantum yield was calculated as the ratio of variable and maximum fluorescence, Fv/Fm = (Fm -F\u003csub\u003e0\u003c/sub\u003e)/Fm, which expresses the maximum quantum efficiency of primary photochemistry [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The variable called the relative electron transport rate through PSII, rETR was calculated as the product of the actual photochemical efficiency Y(II) multiplied by the photosynthetically active radiation as mentioned above, see e.g. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The values of rETRmax were calculated at the maxima of LRCs. Non-photochemical quenching NPQ [= (Fm \u0026ndash; Fm\u0026rsquo;)/Fm\u0026rsquo;] was used to estimate non-photochemical energy dissipation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBesides, rates of dark respiration Resp and photosynthetic oxygen evolution PS were measured as a function of irradiance in microalgae samples using a Clark-type oxygen electrode mounted in a temperature-controlled chamber (DW2/2 chamber, Hansatech Instrument Ltd., Norfolk, UK) connected to an oxygen monitoring system (Oxylab+, Hansatech Instr. Ltd., UK) and the programmable light source to adjust intensity increase in six 2-min steps between 0 and 950 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (saturating intensity for photosynthesis) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The values of respiration and oxygen production are expressed in pmol O\u003csub\u003e2\u003c/sub\u003e cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFast fluorescence induction kinetics (Kautsky curve or OJIP test)\u003c/h2\u003e \u003cp\u003eWhile the pulse-amplitude-modulation (PAM) technique gives information on the energy distribution between the photochemical and non-photochemical processes in photosynthesis, the fast fluorescence induction kinetics provides information on the redox status of the electron transport chain in the PSII complex. The fluorescence induction curves were measured \u003cem\u003eex-situ\u003c/em\u003e by a portable fluorimeter (AquaPen AP-100, P.S.I. Ltd. Brno, Czech Republic) in samples taken from outdoor cultures, dark-adapted for 5\u0026ndash;10 min as described previously [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The curves were measured in the time range between 50 \u0026micro;s to 1 s when the signal rises rapidly from the origin (O) to the highest peak (P) via two infections \u0026ndash; J and I [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The O point (50 \u0026micro;s) of the fluorescence induction curve represents a minimum value (designated as constant fluorescence yield F0) when PQ electron acceptors (Q\u003csub\u003eA\u003c/sub\u003e and Q\u003csub\u003eB\u003c/sub\u003e) of the PSII complex are oxidized. The inflection J occurs after ~\u0026thinsp;2\u0026ndash;3 ms of illumination and reflects the dynamic equilibrium (quasi-steady-state) between Q\u003csub\u003eA\u003c/sub\u003e and Q\u003csub\u003eB\u003c/sub\u003e. The J\u0026ndash;I phase (at 30\u0026ndash;50 ms) is due to the closure of the remaining centers, and the I\u0026ndash;P (ends at about 300\u0026ndash;500 ms) corresponds to the full reduction of the plastoquinone pool (equivalent to the maximum fluorescence level Fm) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. From the fluorescence levels at the J and I points, the variables Vj and Vi were calculated as follows: Vj = (F\u003csub\u003e2ms\u003c/sub\u003e - F\u003csub\u003e0\u003c/sub\u003e)/(Fm-F\u003csub\u003e0\u003c/sub\u003e) and Vi = (F\u003csub\u003e30ms\u003c/sub\u003e F\u003csub\u003e0\u003c/sub\u003e)/(Fm F\u003csub\u003e0\u003c/sub\u003e) which showed redox status of quinone electron acceptors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnalytical measurements\u003c/h2\u003e \u003cp\u003eThe measurement of biomass content was carried out as dry weight (DW) determination by filtering culture samples on pre-weighed glass microfiber filters (GC-50) as described previously [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The filters with the biomass were washed twice with deionized water and dried in an oven at 105\u0026deg;C for 8 h; then they were weighed (precision of \u0026plusmn;\u0026thinsp;0.01 mg) and the biomass amount was calculated.\u003c/p\u003e \u003cp\u003eChl concentration was determined spectrophotometrically in methanol extracts. The cells were collected by centrifugation and the pellets were resuspended in 100% methanol, sea sand or glass beads were added and the tubes were put into the laboratory ultrasound bath heated to 40\u0026deg;C for 2 min, then cooled down in an ice bath and centrifuged. The absorbance of the supernatant was measured using a high-resolution spectrophotometer and the concentration of Chl was calculated according to Wellburn [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBioassays\u003c/h2\u003e \u003cp\u003eThe freeze-dried biomass of microalgae cultures harvested from the cultures at the end of cultivation was resuspended in distilled water (3 mg DW L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and ultrasonicated (2 min) for testing biostimulating activities. The bioassays were carried out with biomass samples of 2 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e concentration. Two bioassays were used to detect plant biostimulating activities: the mung bean rooting test and the cucumber cotyledon root formation test. All bioassays were performed in triplicate.\u003c/p\u003e \u003cp\u003eMung bean rooting test (auxin-like activity)\u003c/p\u003e \u003cp\u003eThe plant biostimulating activity of the extracts was measured using the mung bean (\u003cem\u003eVigna radiata\u003c/em\u003e) rooting bioassay [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The beans were germinated in moist vermiculite at 26\u0026deg;C in a 16:8 h light:dark photoperiod and 120 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e light intensity. On day 10, uniform mung bean cuttings with two leaves were placed in the growth chamber in the vials with the microalgae extracts (0.5, 1, 2 and 3 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) as the treatment for 6 h and transferred to vials containing water. Distilled water was used as the control. Then the plants were put back in the growth chamber for 8 days. After the incubation period, the number of roots (longer than 1 mm) was counted on each hypocotyl. The mean numbers of roots were compared to a standard curve prepared using indol-3-butyric acid (IBA \u0026ndash; auxin equivalent) at concentrations of 0.3-1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCucumber cotyledon rooting test (auxin-like activity)\u003c/p\u003e \u003cp\u003eThe cucumber (\u003cem\u003eCucumis sativus L.\u003c/em\u003e) cotyledon expansion bioassay was used to determine the auxin-like activity of microalgae [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. For this bioassay, the freeze-dried biomass extracts were diluted to 2 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e concentration. Ten cotyledons were excised from 3-day-old seedlings and incubated on Petri dishes in a darkroom (26\u0026deg;C) for 5 days. The number of roots formed at the bases of the cotyledons was then counted and compared between the treatments with microalgae extract and the IBA solutions. Indol-3-butyric acid (IBA \u0026ndash; auxin equivalent) was dissolved in 95% ethanol. Micro-algae extracts and IBA solutions were applied on individual 6-cm filter paper disks. To each treated disk, 3 mL of distilled water (control) was added to the bottom of each Petri dish; the IBA concentrations of 0.3-1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were used as standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eMost measurements were performed in triplicate (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3); the means and standard deviations (SD) are reported in the figures. Sigma Plot 11.0 was used to determine significant differences between treatments. One-way analysis of variance (ANOVA) and the Holm-Sidac test were conducted for comparison of variables in the trials. For Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, 7bd and 8, statistical differences between the samples collected at the same time and different sampling positions were studied. P values lower than 0.05 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were considered to be significantly different. In graphs, the mean values designated by the same letter did not differ from each other.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Ms. Soňa Pekařov\u0026aacute; for technical assistance and Mr. Eduard Pareis and Dr. Richard Lhotsk\u0026yacute; for administrative support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, J.M., F.L.F., G.T., C.G.-S., F.G.A.-F., J.P.D.; methodology, J.M., K.\u0026Scaron;., F.L.F., G.T., C.G.-S., B.C., V.\u0026Ouml;., V.A.R.C., M.B-V., J.P.D., J.L.G.P., F.G.A.-F.; investigation, J.M., V.A.R.C., G.T., K.\u0026Scaron;., C.G.-S., B.C., J.A.C.M., A.M.S.B., M.B.-V., V.\u0026Ouml;., J.L.G.P., F.L.F.; data curation, J.M., K.\u0026Scaron;., V.A.R.C., G.T., C.G.-S., B.C., J.A.C.M., A.M.S.B., M.B-V., V.\u0026Ouml;., J.L.G.P., F.L.F.; writing\u0026mdash;original draft preparation, J.M., G.T., F.L.F., K.\u0026Scaron;.; writing\u0026mdash;review and editing, J.M., G.T. F.L.F., K.\u0026Scaron;., F.G.A-F.; visualization, K.\u0026Scaron;., J.M.; G.T., F.L.F.; supervision, J.M., F.L.F., G.T., F.G.A.-F., J.P.D.; funding acquisition, F.G.A.-F., J.M., G.T., J.P.D., F.L.F. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was mainly funded by the EU Horizon 2020 Research and Innovation program, project SABANA (grant no. 727874), and in part by the MULTI-STR3AM project (grant No 887227) which received funding from Bio-based Industries Joint Undertaking. The financial support by the Alga Hub Project \u0026ldquo;Algae for More Sustainable and Healthy Functional Foods\u0026rdquo; (TED2021-131555B-C22) by the Ministry of Science and Innovation of the Spanish government has to be also acknowledged.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003eThe datasets presented in this study are available from the corresponding authors upon reasonable request. The data are not publicly available without the permission of all co-authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration:\u003c/strong\u003e The authors declare that the experiment with microalgae species complied with relevant institutional, national, and international guidelines and legislation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAci\u0026eacute;n, F. G. \u003cem\u003eet al.\u003c/em\u003e Photobioreactors for the production of microalgae. \u003cem\u003eMicroalgae-Based Biofuels Bioprod. From Feed. Cultiv. to End-Products\u003c/em\u003e 1\u0026ndash;44 (2017) doi:10.1016/B978-0-08-101023-5.00001-7.\u003c/li\u003e\n\u003cli\u003eMasoj\u0026iacute;dek, J., Lhotsk\u0026yacute;, R., \u0026Scaron;těrbov\u0026aacute;, K., Zittelli, G. C. \u0026amp; Torzillo, G. 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Technol.\u003c/em\u003e \u003cstrong\u003e149\u003c/strong\u003e, 586\u0026ndash;589 (2013).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biostimulants, Green microalgae, In vivo Chl fluorescence, Photosynthesis, Raceway ponds","lastPublishedDoi":"10.21203/rs.3.rs-4449619/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4449619/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTwo microalgae, \u003cem\u003eScenedesmus\u003c/em\u003e sp. and \u003cem\u003eChlorella\u003c/em\u003e sp. (Chlorophyceae), robust and well-growing species, with a potential for biostimulating activities, were cultured in raceway ponds (RWPs) placed in a greenhouse. The objective of this case study was to monitor the performance of microalgae cultures \u003cem\u003ein-situ \u003c/em\u003eat various depths as concerns photosynthetic activity and physico-chemical variables (irradiance, temperature, dissolved oxygen concentration) including biostimulating activity. The data (photochemical yield and electron transport rate monitored by Chl fluorescence and photosynthetic oxygen production) both \u003cem\u003ein-situ \u003c/em\u003eand \u003cem\u003eex-situ\u003c/em\u003e revealed that (i) even in thin cultures (0.5-1 g dry weight L\u003csup\u003e-1\u003c/sup\u003e), the active photic layer in the culture was only about 1 cm indicating that most of the culture was ˊphotosyntheticallyˋ in the dark and (ii) nevertheless, even at high dissolved oxygen concentrations of about 200 %sat and higher the cultures retained relatively high actual photochemical yield Y(II) of about 0.35 and higher when monitored \u003cem\u003ein-situ\u003c/em\u003e. The presented work can be used as exemplary data to optimize the growth of microalgae cultures in large-scale raceway ponds by understanding the interplay between culture depth and cell concentration.\u003c/p\u003e","manuscriptTitle":"Photosynthetic activity measured in-situ in microalgae cultures grown in large-scale raceway ponds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-10 18:33:13","doi":"10.21203/rs.3.rs-4449619/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dc37d825-673b-4c48-96f8-a8281fd7c45a","owner":[],"postedDate":"June 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":32695941,"name":"Biological sciences/Biochemistry"},{"id":32695942,"name":"Biological sciences/Biological techniques"},{"id":32695943,"name":"Biological sciences/Biotechnology"},{"id":32695944,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2024-07-03T05:10:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-10 18:33:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4449619","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4449619","identity":"rs-4449619","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}