M Bonnanfant

• H Marec

• B Jesus

J.-L Mouget

• J Pruvost

Investigation of the photosynthetic response of Chlorella vulgaris to light changes in a torus-shape photobioreactor

Keywords: Microalgae, Light response, Photosynthesis, Photoacclimation, Photoprotection

An efficient use of light is essential to achieve good performances in microalgae cultivation systems. This can be challenging particularly under solar conditions where light is highly dynamic (e.g., day/night cycles, rapid changes in wind and weather conditions). Microalgae display different mechanisms to optimize light use efficiency. In the short term, when high light is encountered, several processes of photoprotection can be involved to avoid cell damages (e.g., xanthophyll cycle). In the long term, when cells are exposed to a different light intensity, pigment content changes, i.e., photoacclimation. The purpose of this study is to investigate the photosynthetic response of Chlorella vulgaris cultures grown in closed lab-scale, torusshape photobioreactor under well-controlled light conditions, namely, constant and dynamic light transitions. Experiments were conducted in continuous mode with detailed characterization of the light attenuation conditions for each condition, as represented by the mean rate of photon absorption (MRPA), so as to characterize the time responses of the photosynthetic cells toward light changes. This enables to observe short-term and long-term responses with their own characteristic times. The mechanisms involved were found to be different between increasing and decreasing light transitions. Furthermore the MRPA was found a valuable parameter to relate the effect of light to biological responses (i.e., pigment changes) under constant light and dynamic light conditions.

Key points • MRPA proved valuable to relate C. vulgaris responses to light changes.

• A linear evolution was found between pigment content and MRPA in continuous light.

• A rising PFD step induced fast protection and acclimation mechanisms.

Introduction

An efficient use of light is essential to achieve good performances in microalgae production, particularly in mass cultivation systems under solar conditions where light is highly dynamic [START_REF] Richmond | Biological principles of mass cultivation of photoautotrophic microalgae[END_REF]. These light variations are mainly due to the diurnal cycles during which the incident photon flux density (PFD or q 0 ) is changing from null to very high values, greater than 1200 µmol photons m -2 s -1 in summer in Western France (Le [START_REF] Borgne | Développement d'un photobioréacteur solaire intensifié en vue de la production à grande échelle de biomasse microalgale[END_REF].

In order to avoid contaminations by bacteria, fungus, or other microalgal cells, microalgae are often cultivated in closed photobioreactors (PBRs). Controlling culture conditions prevents growth limitation due to pH, temperature, or culture medium variations [START_REF] Pruvost | Chapter fiveindustrial photobioreactors and scale-up concepts[END_REF]). As a result, light can become the main limiting parameter for the maximization of growth kinetics and biomass productivity. Light-use optimization is however complex, with the distribution of light in the culture medium being heterogeneous. Due to the cells in suspension and photosynthesis, light is absorbed, creating a gradient of light level along the culture depth [START_REF] Pruvost | Hydrodynamics influence on light conversion in photobioreactors: an energetically consistent analysis[END_REF]. Depending on external conditions such as PFD and dilution rate, even when the PFD applied onto the PBR is the same, the light attenuation profile, and thus light regimes into the culture medium, can be very different.

3

Microalgal cells absorb light energy through light harvesting complexes and use that energy for carbon fixation through photosynthesis (Crofts and Wraight 1983). However, if the light energy is excessive, the photosynthetic apparatus becomes overexcited and harmful reactive oxygen species (ROS) are produced [START_REF] Muller | Non-photochemical quenching. A response to excess light energy[END_REF]. In some cases, it results in cell photoinhibition leading to a loss of biomass productivity [START_REF] Kok | On the inhibition of photosynthesis by intense light[END_REF][START_REF] Nixon | Recent advances in understanding the assembly and repair of photosystem II[END_REF]). On the contrary, if light energy is too low, other mechanisms such as respiration occur in the PBR, also leading to productivity loss [START_REF] Bennoun | Evidence for a respiratory chain in the chloroplast[END_REF]Peltier and Cournac 2002).

To prevent these phenomena, microalgae have mechanisms to optimize the efficiency of their light utilization. In the short term, when excessive light is encountered, they can activate several photoprotection processes to avoid cell damages [START_REF] Prasil | Cyclic electron flow around photosystem II in vivo[END_REF][START_REF] Asada | The water-water cycle as alternative photon and electron sinks[END_REF][START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: mechanisms, evolution and ecophysiology[END_REF][START_REF] Kargul | Photosynthetic acclimation: structural reorganisation of light harvesting antenna -role of redox-dependent phosphorylation of major and minor chlorophyll a/b binding proteins[END_REF]. Among them, the non-photochemical fluorescence quenching (NPQ) is an important mechanism, which allows microalgae to quickly respond to a rapid increase in light received and avoid photo-damage. The major component of this process is due to the de-epoxidation of some xanthophylls, resulting in the dissipation as heat of the excess light energy, which cannot be used for photosynthesis [START_REF] Goss | Biodiversity of NPQ[END_REF].

In a longer time scale, when cells are exposed to a different light intensity, their pigment content is also known to change within a process named photoacclimation. When cells are exposed to low light, their pigment content per cell increases, which increases cell absorption capacity. On the contrary, when cells are exposed to high light, their pigment content decreases to reduce the production of ROS and the oversaturation effect [START_REF] Dubinsky | Photoacclimation processes in phytoplankton: mechanisms, consequences, and applications[END_REF].

In PBRs, the overall pigment concentration has a direct impact on the light transfer and therefore on the photosynthetic growth rate, and thus on the resulting PBR biomass productivity. So photoacclimation processes can influence the reactor performances [START_REF] Pruvost | Theoretical investigation of microalgae culture in the light changing conditions of solar photobioreactor production and comparison with cyanobacteria[END_REF].

The purpose of this study is to investigate the photoacclimation of Chlorella vulgaris cultures grown in a closed lab-scale PBR under well-controlled conditions of light received. First, the changes in the cellular pigment content due to photoacclimation will be studied in PBR operating at constant light. Then the dynamics of the acclimation processes will be examined during light step transitions.

Material and methods

Pre-culture protocol

The strain Chlorella vulgaris CCAP 21.119 was grown in a modified Bold's basal medium (BBM) in 250-mL Erlenmeyer flasks under 60 µmol photons m -2 s -1 [START_REF] Andersen | Algal culturing techniques -1st edition[END_REF].

The culture was continuously stirred. Algae were sub-cultured every 2 weeks; 1 mL from old culture was inoculated in 100 mL of new fresh BBM (Kazbar et al. 2019b).

Culture protocol

Cultivation system

Experiments were conducted in a laboratory-scale PBR of torus shape presenting a flat illuminated surface, a squarechannel (i.e., Cartesian geometry for light attenuation) and a depth of culture of 0.04 m (i.e., perpendicular to illuminated surface) for a total culture volume of 1.5 L (Fig. 1). A complete description can be found in [START_REF] Pruvost | Numerical investigation of hydrodynamic and mixing conditions in a torus photobioreactor[END_REF]. The interest of this system to control with a high accuracy all culture parameters influencing photosynthesis and growth has already been demonstrated in several works [START_REF] Perkins | A fully predictive model for one-dimensional light attenuation by Chlamydomonas reinhardtii in a torus photobioreactor[END_REF][START_REF] Degrenne | Investigation of the combined effects of acetate and photobioreactor illuminated fraction in the induction of anoxia for hydrogen production by Chlamydomonas reinhardtii[END_REF][START_REF] Takache | Investigation of light/dark cycles effects on the photosynthetic growth of Chlamydomonas reinhardtii in conditions representative of photobioreactor cultivation[END_REF][START_REF] Souliès | Investigation and modeling of the effects of light spectrum and incident angle on the growth of Chlorella vulgaris in photobioreactors[END_REF]Kazbar et al. 2019a).

To avoid any bias due to cell acclimation (as usually observed after inoculation), all cultures were carried out in continuous chemostat mode, with continuous addition of fresh culture medium. The flowrate was adjusted depending of the targeted dilution rate. Sufficient culture duration in constant conditions (i.e., usually several days) was applied to allow the culture to stabilize to the light conditions imposed.

The artificial incident PFD was provided by a panel of white light LEDs. The light from the LED panel was calibrated with a quantum sensor (Li-COR 250A, USA). Agitation was mainly provided by a marine impeller. During experiments, the temperature was maintained at 25 °C by automatic water cooling, the pH was monitored (InPro 3253i Mettler Toledo, USA) and maintained at 7.5 by automatic CO 2 injection, and the dissolved oxygen concentration was measured (InPro 6860i Mettler Toledo, USA) and controlled by automatic N 2 bubbling for degassing. Both pH and oxygen concentrations were regulated with a proportional integral derivative (PID) controller (Ifrim et al. 2014;[START_REF] Titica | Simultaneous control of pH and dissolved oxygen in closed photobioreactor[END_REF]. All regulations were done using Labview software (Laboratory Virtual Instrument Engineering Workbench, National Instrument, USA). Data were recorded in real time.

For these experiments, C. vulgaris cultures were carried out in Sueoka medium (i.e., with ammonium as the nitrogen source) supplemented with Hutner solution [START_REF] Sueoka | Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardii[END_REF][START_REF] Souliès | Investigation and modeling of the effects of light spectrum and incident angle on the growth of Chlorella vulgaris in photobioreactors[END_REF]. The concentration of nutrients was adjusted to be in excess, so that microalgae growth was only light-limited [START_REF] Souliès | Investigation and modeling of the effects of light spectrum and incident angle on the growth of Chlorella vulgaris in photobioreactors[END_REF]Kazbar et al. 2019a).

Culture conditions in constant light

In order to investigate C. vulgaris photoacclimation in constant light regime, several cultures were carried out in continuous mode (Table 1). The imposed PFD ranged between 50 and 800 µmol photons m -2 s -1 and the imposed dilution rate D ranged between 0.01 and 0.07 h -1 .

For each condition, the culture was carried out in the PBR for at least 5 resident times until obtaining a steadystate (i.e., constant biomass and pigment concentrations). When steady-state was reached, 30 to 50 mL of culture was sampled for analyses. Samplings were done in triplicate in order to validate the steady-state regime.

Incident light transitions

To study C. vulgaris photoacclimation under dynamic light, two successive light step transitions in incident PFD were done (Fig. 2). Instead of light cycles, the choice of applying light transitions was made to allow the measurement of characteristic times after a single increasing or decreasing light change. A large amplitude between the two PFD levels was chosen to facilitate the observations.

A first, steady-state was reached for an incident PFD equal to 75 µmol photons m -2 s -1 , and then the light was changed to 765 µmol photons m -2 s -1 . When the second steadystate was reached, the incident PFD was reduced back to 75 µmol photons m -2 s -1 until the third steady-state was reached.

The dilution rate (D) was constant at 0.02 h -1 during the whole experiment.

Immediately after each PFD step, several culture samples were regularly taken and the sampling was less frequent over time thereafter. For each sample, about 30 mL of culture was taken. After each sampling, the harvest pump was stopped until the culture volume reached its pre-sampling value.

Analyses

PAM chlorophyll a fluorescence parameters

To measure photosynthetic parameters, off-line PAM chlorophyll a measurements were done using a PAM fluorometer (WaterPAM, Walz, Germany). Immediately after each sampling, cultures were diluted in Sueoka medium in a quartz cuvette so that the fluorescence signal was close to 200 units [START_REF] Walz | Dynamic pH model for autotrophic growth of microalgae in photobioreactor: a tool for monitoring and control purposes[END_REF]. The dilution mainly depended on the chlorophyll a concentration. The culture was left for 10 to15 min in darkness in order to allow the total oxidation of PSII reaction centers.

After dark acclimation, a rapid light curve (RLC) was carried on the samples [START_REF] Perkins | A fully predictive model for one-dimensional light attenuation by Chlamydomonas reinhardtii in a torus photobioreactor[END_REF]) with 8 incremental light levels, i.e., 0, 75, 109, 165, 245, 346, 479, 773, and 1127 µmol photons m -2 s -1 . After 1.5-min exposure to each light level, a saturating flash was applied for 0.8 s to close all open PSII reaction centers. After each light level, the following parameters were measured: F', F m ', F o (at the 1st measurement), F m (at the 1st measurement).

Maximum PSII quantum yield for photochemical energy conversion (F v /F m ) was calculated as follows [START_REF] Kolber | Use of active fluorescence to estimate phytoplankton photosynthesis in situ[END_REF]: PSII quantum yield of regulated non-photochemical energy loss (YNPQ) was calculated as follows (Klughammer and Schreiber 2008):

In Eq. 2, F m ' was the value measured for the actinic light equal to the average fluence rate of the culture (G mean , see next).

Biomass concentration

Microalgae biomass concentration was determined based on dry matter measurement (g L -1 ). Depending on the microalgae concentration, a known volume of culture (between 6 and 15 mL) was filtered on a pre-weighted dry glass microfiber filter with a pore size of 0.7 µm. Then the filters were dried at 105 °C. At least 24 h later, the filters were cooled in a desiccator and weighted again.

Pigment concentration

Depending on the culture concentration, a known volume (V samp ) of culture (between 0.1 to 2 mL) was centrifuged for 10 min at 13,400 g (MiniSpin Eppendorf, Germany). Supernatant was removed and the pellet was re-suspended in 1.5 mL of methanol. The tubes were left for 45 min at 45 °C to allow the extraction of pigments in methanol. After centrifugation, a white pellet was obtained, which proved the total extraction of the pigments. The absorbance was measured with a spectrophotometer (Jasco V-630, France) at 430 nm, 652 nm, 665 nm, and 750 nm. The following equations were used to estimate the concentrations of chlorophyll a, chlorophyll b, and total carotenoids [START_REF] Strickland | A practical handbook of seawater analysis[END_REF][START_REF] Ritchie | Universal chlorophyll equations for estimating chlorophylls a, b, c, and d and total chlorophylls in natural assemblages of photosynthetic organisms using acetone, methanol, or ethanol solvents[END_REF]):

with A 652 the absorbance at 652 nm, A 665 the absorbance at 665 nm, and A 480 the absorbance at 480 nm.

Total pigment concentration (C pig in g L -1 ) was calculated by adding these different concentrations together. The (1) total pigment (w pig ), chlorophyll a (w Chla ), chlorophyll b (w Chlb ), and total carotenoid (w car ) contents were calculated by dividing the pigment concentrations by the dry biomass concentration.

F v F m = F m -F o F m (2) YNPQ = F F m � - F F m (3) Chlorophyll a = 1.5 V samp -8.0962 × A 652 + 16.5169 × A 665 (4) Chlorophyll b = 1.5 V samp 27.4405 × A 652 + 12.1688 × A 665 (5) [Total carotenoids] = 1.5 V samp 4 × A 480

Inorganic carbon concentration

Inorganic carbon concentration was measured on some samples to check that the dissolved carbon concentration was not in a limiting concentration (i.e., inferior to 5 mM (Le [START_REF] Gouic | Investigation of growth limitation by CO 2 mass transfer and inorganic carbon source for the microalga Chlorella vulgaris in a dedicated photobioreactor[END_REF]). For that purpose, 15 mL of culture was centrifuged at 4150 g (Hettich Mikro 22R, Germany) for 10 min. Then, the supernatant was filtered and inorganic carbon concentration was measured with a carbon analyzer (TOC-L, Shimadzu, Japan).

Determination of the light attenuation conditions

Radiative transfer modeling

A two-flux model was used to determine the spectral value of the fluence rate G λ (z) (in µmol photons m -2 s -1 ) in the PBR [START_REF] Perkins | A fully predictive model for one-dimensional light attenuation by Chlamydomonas reinhardtii in a torus photobioreactor[END_REF]. Spectrally averaged fluence rate G mean was calculated by integrating G λ (z) over the photosynthetically active radiation (PAR) and over the culture volume.

The biomass specific rate of photon absorption A Cx (in µmol photons kg Cx -1 s -1 ) was obtained by integrating the product of G λ with the mass absorption coefficient Ea λ of C. vulgaris over the PAR [START_REF] Souliès | Investigation and modeling of the effects of light spectrum and incident angle on the growth of Chlorella vulgaris in photobioreactors[END_REF]:

By integrating A Cx over the culture volume, the mean biomass specific rate of photon absorption (MRPA Cx ) was determined. The radiative properties of C. vulgaris, necessary for this model, were obtained from [START_REF] Kandilian | Comparison of experimentally and theoretically determined radiation characteristics of photosynthetic microorganisms[END_REF] and are given as supplementary materials.

In the present study, the specific mean rate of photon absorption was also expressed per unit of total pigment (MRPA pig , in µmol photons kg pig -1 s -1 ). It was calculated by dividing MRPA Cx by the total pigment content w pig .

Light attenuation profile

For any microalgal species, a rate of photon absorption at which photosynthesis compensates for respiration can be defined (i.e., compensation point). This rate of photon absorption is named A C , and it was estimated equal to 2800 µmol photons kg Cx -1 s -1 in C. vulgaris [START_REF] Souliès | Investigation and modeling of the effects of light spectrum and incident angle on the growth of Chlorella vulgaris in photobioreactors[END_REF]). In the PBR used for the experiments, the light attenuation is one-dimensional [START_REF] Perkins | A fully predictive model for one-dimensional light attenuation by Chlamydomonas reinhardtii in a torus photobioreactor[END_REF]. In this case, an illuminated fraction can be defined and is equal to:

(6) A Cx = ∫ PAR Ea G d .
where V light is the light volume of the PBR (i.e., where A Cx > A C ), V PBR is the volume of the whole PBR, z(A C ) is the depth where A C is reached, and L the thickness of the PBR.

When γ is inferior to 1, there is a dark volume, and when γ is superior to 1, the culture volume is fully illuminated without dark volume [START_REF] Pruvost | Knowledge models for engineering and optimization of photobioreactors[END_REF]. For γ > 1 conditions, the transmittance T was also calculated, to estimate the fraction of the incident PFD passing through the reactor depth:

Results

Cultures in continuous light

Table 1 summarizes the values of biomass and pigment concentrations obtained at steady-states for the different cultures conditions. For each steady-state, light transfer conditions through radiative transfer modeling and corresponding MRPA values were calculated from Eq. 6. According to the PFD and dilution rate conditions, either diluted (γ > 1) or concentrated (γ < 1) cultures were obtained. The illuminated fraction γ was calculated for concentrated cultures and the transmittance T was calculated for diluted cultures. Figure 3 illustrates the pigment content obtained for each steady-state as a function of the MRPA Cx (Fig. 3a) and MRPA pig values (Fig. 3b). The illuminated fraction γ and the transmittance T are also given in Fig. 3.

The pigment content decreased with MRPA values (for both MRPA Cx and MRPA pig ). This can be approximated by two linear relations (minimum value of R 2 of 0.89), with a higher decrease for MRPA Cx values between 5 and 15 µmol photons g -1 s -1 , followed by a small evolution for values over 15 µmol photons g -1 s -1 . For the largest values of MRPA Cx , an asymptotic behavior was observed, as the pigment content never decreased under 4% Cx . This corresponded to an MRPA Cx about 25 µmol photons g -1 Cx s -1 (0.58 µmol photons mg pig -1 s -1 ), which was reached for an incident PFD equal to 400 µmol photons m -2 s -1 and a dilution rate equal to 0.07 h -1 . The resulting biomass concentration was 0.323 g L -1 .

Those linear evolutions were observed whatever the light attenuation condition (Fig. 3). For example, a concentrated culture with γ equal to 0.40 revealed the same MRPA Cx as a diluted culture with γ > 1 and T equal to 0.43. Both cultures exhibited same pigment content, about 6% Cx . Note that the diluted culture (T = 0.43) was obtained for an incident PFD

(7) = V light V PBR = z(A C ) L (8) T = G(z = L) q 0
equal to 100 µmol photons m -2 s -1 and a 0.05 h -1 dilution rate, resulting in a biomass concentration of 0.179 g L -1 . The concentrated culture (γ = 0.40) was obtained for an incident PFD equal to 800 µmol photons m -2 s -1 and a 0.02 h -2 dilution rate, resulting in a biomass concentration of 1.796 g L -1 . Very different biomass concentrations were then achieved in both cases. A similar observation can be made for the MRPA Cx values at 15 µmol photons g -1 Cx s -1 , with two different light attenuation conditions (T = 0.25 and γ = 0.40) resulting in the same pigment content (5.0% Cx ).

Culture in dynamic light conditions

Biomass concentration and pigment content

Figure 4a shows the changes in biomass concentration and total pigment content during the two successive light step transitions. After the first PFD change from 75 to 765 µmol photons m -2 s -1 , the biomass concentration needed between 150 and 200 h to almost stabilize around 1.45 (± 0.12, n = 12) g L -1 . Pigment content in biomass reached its lowest value of 4.5% Cx in the first 12 h and then it increased to reach 5.6 (± 0.2, n = 9) % Cx after 100 to 150 h. The culture at 765 µmol photons m -2 s -1 was highly unstable (i.e., biomass concentration and pigment content not constant), making difficult to define a precise time for the stabilization of biomass concentration. Nevertheless, it seemed that the increase in biomass concentration took more time than the decrease even if the rate for biomass increase during the first 12 h (0.026 g L -1 h -1 ) was higher than the decrease (0.013 g L -1 h -1 ). Instability of the culture at 765 µmol photons m -2 s -1 was probably due to both high value of dissolved oxygen concentration (around 250% of saturation) and strong agitation (50 to 100 mL (N2) min -1 ) to maintain this concentration. It must be noticed that a stronger agitation would have led to a risk of CO 2 limitation due to high degassing (Kazbar et al. 2019a).

When the PFD decreased from 765 to 75 µmol photons m -2 s -1 , pigment content took about 100 h to reach a stable value around 7.9 (± 0.1, n = 5) % Cx , which represents an increase of ca. 40%. The biomass concentration exhibited again a longer stabilization time, ca. 150 h, to reach a biomass concentration of 0.34 (± 0.01, n = 4) g L -1 . At the end of the experiment, at steady-state, biomass concentration and pigment content reached the same values as at the beginning of the experiment before the first light change.

Figure 4b shows the pigment composition (wt% of chlorophylls or carotenoids per total pigment) during the two successive light transitions. The composition was identical during the whole experiment, with chlorophyll a, chlorophyll b, and carotenoids representing respectively 67 ± 0.5 (n = 42) wt%, 16 ± 0.6 (n = 42) wt%, and 17 ± 0.4 wt% (n = 42) of the total pigment (the error is the standard deviation calculated on n measurements). We can thus conclude that even if the total pigment content changed a lot (from 5.6 to 7.9% Cx corresponding to a 40% increase) during the successive light Cx s -1 (a, black line), the equation is y = 8.6009 -0.2436x and R 2 = 0.89. For values of MRPA Cx between 14 and 25 µmol photons g -1 Cx s -1 (a, gray line), the equation is y = 6.089 -0.0679x and R 2 = 0.92. For values of MRPA pig between 0.04 and 0.29 µmol photons mg -1 pigments s -1 (b, black line), the equation is y = 8.1159 -11.321x and R 2 = 0.92. For values of MRPA pig between 0.29 and 0.58 µmol photons mg -1 pigments s -1 (b, gray line), the equation is y = 5.804 -2.488x and R 2 = 0.96 transitions, the pigment composition profile remained almost the same.

MRPA values and photosynthetic parameters

Figure 5a represents MRPA Cx as a function of time during the two successive light step transitions. When the PFD increased from 75 to 765 µmol photons m -2 , MRPA Cx values instantly increased from 6 to 63 µmol photons g -1 Cx s -1 . These values of MRPA Cx were much higher than those obtained at steady-state, i.e., tenfold increase. It took about 100 h for MRPA Cx values to decrease and stabilize around 13 (± 1.0, n = 12) µmol photons g -1 Cx s -1 . This decrease was the result of biomass concentration increase and cellular pigment content decrease. When the PFD decreased to 75 µmol photons m -2 s -1 , MRPA Cx values instantly decreased to 1.4 µmol photons g -1 Cx s -1 . Then MRPA Cx values increased again to reach, after about 150 h, the same values as at the beginning of the experiment. It should be noted that the stabilization time for MRPA Cx was the same as for biomass concentration or pigment concentration.

Microalgae are known to display different photoprotection and photoinhibition mechanisms to prevent damages caused by light stress. PAM chlorophyll a fluorescence was thus used to study the effect of PFD transitions on the dynamics of these mechanisms. Figure 5b gives the changes in F v /F m and YNPQ values during the two successive PFD steps. At the rising light step, F v /F m instantly decreased from 0.72 to 0.64 and stayed below 0.6 after 24 h. After about 150 h, F v /F m almost returned to its initial value. Then F v /F m progressively decreased over the next 300 h. When the light was set back to 75 µmol photons m -2 s -1 , F v /F m increased again after several hours to 0.72, and then slowly decreased over time to stabilize at 0.67.

During the whole experiment, YNPQ remained below 0.1. An increase was only observed at the beginning of the first light transition, corresponding to the largest MRPA values. All these results indicate a different behavior between rising and falling steps. Both were investigated in details in the following sections.

Increasing light step

Figure 6a shows the evolution with time of the biomass concentration and the pigment content over the first 24 h after the rising light change. The biomass concentration increased immediately but the rate was low during the first 2 h, from 0.27 to 0.33 g L -1 . Then the increase became a little higher for the next 8 h, and biomass concentration reached 0.58 g L -1 . During the first 4 h, pigment content strongly decreased, from 8.4 to 5.8% Cx . From 4 to 12 h, the rate was lower, from 5.8 to 4.5% Cx . From the 12th hour, it must be noted that pigment content slowly increased (0.020% Cx h -1 ).

Figure 6b shows the evolution with time of MRPA Cx values, resulting from biomass and pigment acclimation. As seen before, MRPA Cx instantly increased from 6 to 63 µmol photons g -1 Cx s -1 just after the rising PFD step. Then it quickly decreased during the first 4 h, from 63 to 42 µmol photons g -1 Cx s -1 and then the decrease slowed down to 30 µmol photons g -1 Cx s -1 during the following 10 h. Interestingly, if the pigment specific MRPA pig is observed (Fig. 6c), it can be noticed that at the beginning of the light transition, after increasing, MRPA pig did not decrease and stayed constant around 0.74 (± 0.01, n = 5) µmol photons mg -1 pigments s -1 for 4 h.

Figure 6d shows photosynthetic parameters (i.e., F v /F m and YNPQ values) along the first 24 h after the rising step. As seen before, F v /F m decreased from 0.72 to 0.64. But this decrease was not instantaneous and took about 2 h. Just after the PFD change, YNPQ decreased from 0.07 to 0.00 and after 4 h, it started to increase to reach 0.09 after 12 h. However, 24 h after the light increase, YNPQ was almost equal to 0.00. It can be noted that the first decreases in F v /F m and in YNPQ have the same characteristic times, around 5 h.

Decreasing light step

Figure 7a shows the changes in biomass concentration and pigment content over time after the decrease in light from 765 to 75 µmol photons m -2 s -1 . During the very first hours after the light decrease, no evolution was clearly observed. The biomass concentration remained at the same level, at 1.31 (± 0.01, n = 5) g L -1 during the first 4 h, and then it decreased linearly to reach 0.65 g L -1 after 50 h (R 2 = 0.9924). This decrease in biomass concentration corresponds to a loss of 0.013 g L -1 h -1 , equal to 1% of initial concentration lost per hour. After 50 h, the decrease became slower (loss of 0.0045 g L -1 h -1 ). Pigment content was almost constant during the first 12 h and stayed between 5.6 and 6.0% Cx . Then, pigment content started to increase and reached 7.8% Cx after 100 h.

Figure 7b shows the evolution of MRPA Cx with time. The rate of photon absorption at compensation point (A C ) is also shown (A C is equal to 2.8 µmol photons g -1 Cx s -1 (Souliès et al. 2016)). Below this value, respiration prevailed over photosynthesis, resulting in a negative growth. This condition was observed just after the PFD decrease, where MRPA Cx decreased from 14.4 to 1.4 µmol photons g -1 Cx s -1 . This value was found below A C , so at this time, respiration should be Figure 7c shows the evolution with time of F v /F m and YNPQ. When the incident light decreased, F v /F m took 8 h to reach a maximal value, from 0.62 to 0.72. Then it remained almost stable. YNPQ instantly decreased from 0.04 to 0.00 after the light change, and it stayed unchanged during the whole experiment.

Discussion

MRPA correlates changes in light received with biomass and pigment concentration evolutions

A decreasing relationship, with two successive linear relations, was found between total pigment content and MRPA for Chlorella vulgaris culture obtained in continuous light (Fig. 3). Those correlations were found valid whatever the light attenuation profile, as concentrated cultures (illumination fraction equal to 0.40) led to the same result as diluted cultures (transmittance equal to 0.43). Both light regimes inside the culture volumes were very different. Indeed a γ equal to 0.40 means that more than half of the culture volume was not illuminated. And a T equal to 0.43 means that all the culture volume was illuminated, and almost half of the incident PFD reached the bottom of the culture. This could be considered a surprising observation because having or not a dark volume in a PBR is well known to result in very different kinetic performances (e.g., averaged growth rate) of the microalgae culture [START_REF] Takache | Kinetic modeling of the photosynthetic growth of Chlamydomonas reinhardtii in a photobioreactor[END_REF][START_REF] Acien | Photobioreactors for the production of microalgae[END_REF]. In this study, the light attenuation profile (with or without dark volume) did not affect the pigment content in C. vulgaris. Only the averaged MRPA was found to have an influence.

Moreover, the fact that the pigment content almost remained stable at values of ca. 4% Cx suggested that this could be the minimal total pigment content that can be obtained in C. vulgaris when cultivated in nutrient-enriched conditions and continuous light.

Under dynamic light conditions, MRPA values had an amplitude larger than under continuous light conditions. Very high MRPA Cx values were then transitory observed (> 50 µmol photon g -1 Cx s -1 ) similar to those encountered in solar cultures due to high light fluctuations [START_REF] Pruvost | Development of a thin-film solar photobioreactor with high biomass volumetric productivity (AlgoFilm) based on process intensification principles[END_REF]. During the PFD light steps, time evolutions of biomass concentration, pigment content, and MRPA Cx were closely related. In both light transitions, the same stabilization times were observed for these parameters (around 150 h). Moreover, at the beginning of the rising light transition, a similar evolution of pigment content and MRPA Cx was observed, with a decrease of both parameters. This can be explained by the decrease in pigment content leading to a decrease in the capacity of light absorption by cells. As a consequence, less photons were absorbed for the same biomass concentration. Considering MRPA pig instead of the MRPA Cx , it remained unchanged for a few hours after the light increase. This illustrates the relation between MRPA and the pigment changes during photoacclimation, as already observed in continuous light with the linear evolution between MRPA and pigment content. Under the studied dynamic light conditions, a large variation of MRPA values was obtained, reflecting pigment content evolutions. When expressing MRPA values as a function of pigment content (MRPA pig ), it tends to reduce its variation in contrast to values determined as a function of biomass concentration.

All these results tend to indicate a universal relation between MRPA and pigment content values when C. vulgaris is cultivated in continuous light. And when a light step transition is applied, MRPA values are suddenly modified and the culture metabolism becomes unbalanced. From this point, both the biomass concentration and the pigment content evolve to reach a new steady-state, in which the relation between MRPA and pigment content achieved at steady-state in continuous light is again verified.

As already suggested in a previous study [START_REF] Pruvost | Development of a thin-film solar photobioreactor with high biomass volumetric productivity (AlgoFilm) based on process intensification principles[END_REF], MRPA thus reveals an interesting integrative parameter in microalgal culture systems as it enables to relate various quantities such as biomass concentration, pigment content (i.e., radiative properties), PFD, and PBR design (i.e., culture depth). Indeed, the present study shows that MRPA values both reflect the efficiency of light absorption due to the pigment content in C. vulgaris and the light attenuation conditions in the PBR. The former because it is a cell characteristic that changes with the microalga photoacclimation state, the latter because it is a consequence of this phenomenon and of cell density. In fact, this demonstrates the reliability of the MRPA parameter to pinpoint the tight coupling between PFD, biomass and pigment concentration, and metabolism.

Difference in dynamics between MRPA and microalgae biomass and pigment concentration

In both PFD transitions (high to low vs. low to high), MRPA values instantly changed after the light change. However, the resulting changes in biomass concentration and pigment content remained slower. In the rising light transition, the pigment content decreased very rapidly but this decrease was probably accentuated by the dilution rate and biomass increase was slow during the few first hours. After the PFD decrease, both biomass concentration and pigment content remained at the same values for a few hours. In both transitions, biomass concentration and pigment content took more than 100 h to stabilize.

Note that the increase in biomass concentration took longer than its decrease. This could be explained by the fact that in continuous chemostat cultures, a dilution rate is applied. As a consequence, to have a marked increase in the biomass concentration in such systems, a large increase in the biosynthesis rate is required and the energetic demand for this increase is high.

In both transitions, pigment content was found to stabilize faster than biomass concentration. In our case, the lowest value of pigment content was reached 10 h after the first light change, which was very fast compared to biomass evolution (more than 200 h to reach steady-state value). This is certainly explained by the global effect on light attenuation conditions: because biomass concentration increased with PFD, the light received into the PBR (i.e., the MRPA) decreased progressively and the pigment content increased again. When considering the PFD decrease, a progressive evolution toward steady-state values was also observed but, because of the need to synthetize de novo pigments, the dynamics was slower.

Contrary to biomass and pigment dynamics, parameters related to photosynthesis and MRPA responded quickly after both PFD transitions. Indeed, F v /F m and YNPQ decreased a few minutes just after the light increase. This transient slowdown of the photosynthesis efficiency is moderate and it could illustrate the capacity of C. vulgaris to rapidly quench energy in excess using state transition, a process different from photochemistry and other NPQ components (heat dissipation, photoinhibition). Indeed, green algae are known to adjust the absorption of light energy by both photosystems PSII and PSI, by transfer of mobile components of their light harvesting complexes (LHC) (e.g., [START_REF] Allen | Molecular recognition in thylakoid structure and function[END_REF][START_REF] Goldschmidt-Clermont | Sharing light between two photosystems: mechanism of state transitions[END_REF]. This process dissipates in minutes [START_REF] Muller | Non-photochemical quenching. A response to excess light energy[END_REF]). On the longer term, YNPQ increased again after a few hours. A few minutes after the opposite light step transition, YNPQ decreased and YPSII started to increase.

In summary, our study showed that a sudden transition in MRPA is associated to slow responses of biomass concentration and pigment content (characteristic times in the range of hours). But, on the other hand, it was observed a rapid time response of parameters related to photosynthesis. From this, it is deduced that rising and falling MRPA values dynamically reflect the direct impacts of light changes on the early photosynthetic events (PSII quantum yield and electron transport chain), and that it takes more time for the whole metabolism to respond and adapt to these light changes. This is in the line of previous observations on the diversity in short-term and a long-term photoacclimation phenomena [START_REF] Brunet | Pigments and photoacclimation processes. In: Phytoplankton pigments: characterization, chemotaxonomy and applications in oceanography Crofts AR, Wraight CA (1983) The electrochemical domain of photosynthesis[END_REF].

We can also note that the time to reach a steady-state was higher to 1 day after both transitions. As a consequence, in actual day-night cycles, microalgae will never have enough time to reach steady-state. In such conditions, cells and culture can be always considered in a transitory regime. This point deserves special consideration when designing and operating PBRs on the long term.

Different biological mechanisms involved when increasing and decreasing PFD transitions

Increasing and decreasing incident lights were shown to lead to different response times. This can be explained by the different photosynthetic mechanisms involved during these transitions.

When PFD increased, the main parameters that changed were F v /F m and YNPQ. A decrease in F v /F m was observed and it became below 0.7. It can be a sign of photoinhibition [START_REF] Malapascua | Photosynthesis monitoring to optimize growth of microalgal mass cultures: application of chlorophyll fluorescence techniques[END_REF] which corresponds to a damage of the photosynthetic apparatus leading to a decrease in the photosynthesis efficiency [START_REF] Kok | On the inhibition of photosynthesis by intense light[END_REF][START_REF] Nixon | Recent advances in understanding the assembly and repair of photosystem II[END_REF]. So as long as F v /F m was low, microalgae were not able to use light with the best efficiency to grow. F v /F m reached its initial value at the same time as pigment content stabilized. This supposes that photoacclimation processes allowed cells to recover from photoinhibition.

In parallel, after the light increase, when YNPQ values became a little higher, biomass concentration started to increase at a higher rate and the pigment content decrease at a lower rate. YNPQ was never high but in this experiment, considering light attenuation, the maximal mean fluence rate was about 300 µmol photons m -2 s -1 . At this level, photoprotection is known to remain low in C. vulgaris [START_REF] Malapascua | Photosynthesis and growth kinetics of Chlorella vulgaris R-117 cultured in an internally LED-illuminated photobioreactor[END_REF][START_REF] Bonnanfant | Photosynthetic electron transport transients in Chlorella vulgaris under fluctuating light[END_REF]. Thus, even being low, YNPQ might have played a role in photoprotection during the first few hours after the light increase.

Note that during the rising PFD transition, the chlorophyll a to carotenoids ratio was constant, indicating no marked light stress, which should have induced an increase in carotenoid relative concentration, a well-known effect of light stress response in C. vulgaris [START_REF] Dubinsky | Photoacclimation processes in phytoplankton: mechanisms, consequences, and applications[END_REF][START_REF] Sharma | Effects of culture conditions on growth and biochemical profile of Chlorella vulgaris[END_REF]). In our study, the light level applied was probably not high enough to induce the production of carotenoids known to protect the cells against the oxidative stress (Ortega- [START_REF] Ortega-Villasante | In vivo ROS and redox potential fluorescent detection in plants: present approaches and future perspectives[END_REF].

The mechanisms involved in the decreasing PFD transition were not the same as the ones observed in the opposite light transition. Here, the photoacclimation response was mainly due to the limitation of light in the PBR. Indeed, when the PFD decreased, MRPA value was below A C so the culture was light limited and respiration was predominant. As a consequence, biomass concentration decreased at a high rate, which, in turn, resulted in an increase in light availability for cells (increase in MRPA). From this point, F v /F m reached its maximum value, reflecting the fact that microalgae photoacclimated to the new light conditions, so they were able to use light with the best efficiency. As a consequence, the decrease in biomass concentration slowed down.

To conclude, for the two light acclimation phases (high to low vs. low to high), photosynthetic mechanisms involved were not the same. When incident light increased, YNPQ was transiently low, indicating that photoprotection only played a small role, whereas it remained close to zero when light decreased. F v /F m decreased instantly when light increased but it took several hours to increase again after PFD decreased. So under a rising light, photoinhibition occurred almost instantly and then photoprotection mechanisms were established for some hours; however, after decreasing light, no photoprotection was observed and the light received by the cells was probably too low for the photosystem to adjust quickly and to reach immediately an optimal photochemical yield.

Whatever the case, MRPA was found a very valuable parameter to relate light received by cells (as affected by changes in PFD, but also biomass and pigment concentrations) and corresponding biological responses. After each light change, several biological mechanisms took place and fine-tuned to let microalgae acclimate to the new conditions, both on the short term and on the long term. A rising PFD step to high irradiance trigger photoinhibition and photoprotection mechanisms whereas after a decreasing PFD step, cells do not rely on photoprotection to photoacclimate to low irradiance, and the culture was light limited for a few hours. Consequently, the response times for photoacclimation were not the same between these opposite transitions. However, the time for biomass concentration and pigment content to stabilize was always superior to 100 h. This again confirms that in the actual conditions of a solar production with day/night cycles, similarly to any photosynthetic organisms growing outdoor, C. vulgaris cultures will always be in a transitory regime with regards to their photosynthetic and metabolic responses.

As a result, the study of the dynamic mechanisms which intervene in the response of microalgae in variable light conditions is therefore essential for the good understanding and control of solar culture systems. Future studies will aim to extend the approaches developed here in real solar conditions, in particular taking into account UV damaging effects. Other biological models will also be studied to see how changes in quantities linked to the adaptive capacity of cells (as for example NPQ level and its kinetics of evolution toward light change) play on growth performance in solar culture conditions.

Fig. 1

 1 Fig. 1 Controlled photobioreactor used for the study of the photosynthetic response of C. vulgaris

Fig. 2

 2 Fig. 2 PFD step transitions applied during the investigation of photoacclimation of C. vulgaris in light changing conditions. Arrows show sampling times

Fig. 3

 3 Fig. 3 Pigment content obtained for each steady-state, as a function of the MRPA Cx values (a) or the MRPA pig values (b), in diluted (red circles) and concentrated (blue squares) cultures of C. vulgaris. Error bars show standard deviations (n = 3). Linear regressions were calculated (dashed line). For values of MRPA Cx between 3 and 14 µmol photons g -1 Cx s -1 (a, black line), the equation is y = 8.6009 -0.2436x and R 2 = 0.89. For values of MRPA Cx

Fig. 4 Fig. 5

 45 Fig. 4 Time response of C. vulgaris culture during a PFD step from 75 to 765 µmol photons m -2 s -1 , then from 765 to 75 µmol photons m -2 s -1 . D = 0.02 h -1 . a shows biomass concentration (blue circles, left axis) and pigment content (green squares, right axis). Black line represents 75 µmol photons m -2 s -1 and gray line presents 765 µmol photons m -2 s -1 . b shows pigment compositions (green squares correspond to chlorophyll a content, blue circles to chlorophyll b content and orange triangles to total carotenoids). The gray line (right axis) represents the PFD. Error bars represent standard deviations (n = 3)

Fig. 6

 6 Fig. 6 Time response of C. vulgaris culture over time during the first 24 h of the increase in PFD from 75 to 765 µmol photons m -2 s -1 . D = 0.02 h -1 . a shows biomass concentration (blue circles, left axis) and pigment content (green squares, right axis) changes with time. Black line represents 75 µmol photons m -2 s -1 and gray line represents 765 µmol photons m -2 s -1 . b shows the evolution of the biomass specific MRPA Cx with time (black circles, left axis). c shows the evolution of the pigment specific MRPA pig (black circles, left axis). d shows changes in F v /F m (blue squares) and YNPQ (black circles, left axis) with time. In b, c, and d, the gray line (right axis) represents the PFD. Error bars represent standard deviation (n = 3)

Fig. 7

 7 Fig. 7 Time response of C. vulgaris culture over time during the first 120 h after a PFD step from 765 to 75µmol photons m -2 s -1 . D = 0.02 h -1 . a shows the changes in biomass concentration (blue circles, left axis) and pigment content (green squares, right axis) with time. Black line represents 75 µmol photons m -2 s -1 and gray line represents 765 µmol photons m -2 s -1 . b shows the biomass specific MRPA Cx (black circles, left axis). c shows the changes in F v /F m (blue squares) and YNPQ (black circles, left axis) with time. In b and c, the gray line (right axis) represents the PFD. Error bars represent standard deviation (n = 3)

Table 1

 1

	Investigation of photoacclimation of C. vulgaris in constant light. Culture conditions (photon flux	Condition no	PFD (µmol photons m -2 s -1 )	Dilution rate, D (h -1 )	C x (g L -1 )	C pig (mg L -1 )	Illuminated fraction, γ	Trans-mit-tance, T
	densities, PFD and dilution rates, D) and results obtained at steady-states (biomass concentrations, C x ; pigment	1 2 3	50 50 100	0.01 0.02 0.02	0.40 ± 0.02 0.17 ± 0.00 0.41 ± 0.02	29.2 ± 0.8 13.7 ± 0.3 30.1 ± 0.8	0.31 0.84 0.52	0 0 0
	concentrations, C pig ; illuminated	4	100	0.05	0.18 ± 0.03	11.7 ± 2.0	> 1	0.43
	fractions, γ; and transmittances obtained, T). Errors are standard deviations (n = 3). A transmittance T equals to 0	5 6 7	250 250 400	0.02 0.05 0.02	0.67 ± 0.07 0.37 ± 0.02 1.21 ± 0.04	40.3 ± 4.8 18.4 ± 1.1 78.7 ± 1.0	0.60 > 1 0.41	0 0.25 0
	means that all light is absorbed	8	400	0.07	0.32 ± 0.04	14.0 ± 1.7	> 1	0.33
	by the culture and then no light pass through the culture	9 10	800 800	0.02 0.01	1.33 ± 0.08 1.80 ± 0.04	66.9 ± 7.1 105.6 ± 1.2	0.58 0.40	0 0
		11	800	0.04	1.01 ± 0.01	49.9 ± 1.9	0.80	0

Acknowledgements We are very grateful to the AMI (Atlantic MIcroalgae) scientific consortium and the Pays de la Loire region (France), who have supported our research. MB also benefitted from a training on fluorescence techniques in the laboratory of Dr. D. Campbell (Mount Allison University, Canada).

Funding The work was partly funded by the AMI (Atlantic MIcroalgae) project and the Pays de la Loire region (France). The European Commission Horizon 2020 Research and Innovation Program GHaNA, The Genus Haslea, New marine resources for blue biotechnology and Aquaculture, grant agreement No [734708/GHANA/H2020-MSCA-RISE-2016] funded the training of MB in the laboratory of Dr. D. Campbell (Mount Allison University, Canada).

Data availability

All data are available on simple request.

Code availability Not applicable.

Declarations

Consent to participate We state that no conflicts, informed consent, human or animal rights applicable.

Consent for publication

We hereby state that all authors mutually agree that it should be submitted for publication and that it is our original work. The manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.

Author contribution JP and MB conceived and designed research. MB conducted experiments. HM contributed to the conception and design of the experimental setups. JP and MB analyzed data. MB wrote the manuscript. All authors read, reviewed, and approved the manuscript.

Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Nesrine Gargouch

Raphaelle Touchard

Hélène Marec

Jean Luc Mouget

Jérémy Pruvost

Anthony Massé
email: anthony.masse@univ-nantes.fr

Submerged membrane photobioreactor for the cultivation of Haslea ostrearia and the continuous extraction of extracellular marennine

Keywords:

Successful implementation of membrane photobioreactor (MPBR) applied to H. Ostrearia. • Simultaneous H. Ostrearia cultivation and marennine continuous extraction. • No cell leaching even at high dilution rates. • High cell and marennine concentrations at low dilution rate.

Introduction

The cultivation and exploitation of marine microalgae have long been studied. Nevertheless, only few species whose physiology is well known are cultivated and exploited [START_REF] Morais Junior | Microalgae for biotechnological applications: Cultivation, harvesting and biomass processing[END_REF]. Among diatoms, Haslea ostrearia, an eukaryotic microalga of the class Bacillariophyceae remains difficult to cultivate at high productivities. Thus, the industrial exploitation of this microalga remains limited except for aquaculture. For the latter, the blue-green pigment "marennine" produced and excreted by the microalga is used for the oyster greening, giving them a better organoleptic quality. This biological activity has led to an increase in the market value of oysters by 20 to 30% for the French oyster industry [START_REF] Falaise | Harmful or harmless: Biological effects of marennine on marine organisms[END_REF]. This pigment could also be used for its antioxidant, antiproliferative, antibacterial, antiviral and antitumor properties [START_REF] Falaise | Antimicrobial Compounds from Eukaryotic Microalgae against Human Pathogens and Diseases in Aquaculture[END_REF][START_REF] Gastineau | Marennine-Like Pigments: Blue Diatom or Green Oyster Cult?[END_REF]Gastineau et al., , 2014aGastineau et al., , 2012b;;[START_REF] Hussein | Anticancer Compounds Derived from Marine Diatoms[END_REF]Prasetiya et al., 2020b).

So far H. ostrearia has been mainly cultivated in open culture systems of 200 L and 10 m 3 or in photobioreactors (PBRs, less than7 L) (Gastineau et al., 2014b;Rossignol et al., 2000a;[START_REF] Turpin | Oyster greening by outdoor mass culture of the diatom Haslea ostrearia Simonsen in enriched seawater[END_REF]. Whatever the system, high volumes of culture often have to be set-up in order to produce a consistent amount of biomass or marennine, as their concentrations are usually low [START_REF] Bélanger | Extraction Improvement of the Bioactive Blue-Green Pigment "Marennine" from Diatom Haslea ostrearia's Blue Water: A Solid-Phase Method Based on Graphitic Matrices[END_REF][START_REF] Francezon | Electrochromic Properties and Electrochemical Behavior of Marennine, a Bioactive Blue-Green Pigment Produced by the Marine Diatom Haslea ostrearia[END_REF]. Thus, cultivation process intensification remains a challenge, notably to increase marennine or biomass concentration but also the productivities. Currently, the photobioreactors (PBR) used for Haslea ostrearia cultivation are mostly developed for growing microalgae in suspension [START_REF] Nghiem Xuan | Optimization of the growth and marennine production by the diatom Haslea ostrearia in photobioreactor[END_REF]Rossignol et al., 2000a, Rossignol et al., 2000b) whereas this diatom settles and forms biofilms in natural environments. Moreover, the nutrient requirements of this species have long been questioned, as well as the culture parameters (temperatures, pH and light). In general, enrichment solutions such as those proposed by [START_REF] Provasoli | The development of artificial media for marine algae[END_REF] and containing nitrates, phosphorus, sulfur, vitamins and trace metals, are added to artificial or natural filtered seawater to feed microalgae cultures. Culture mediums have also been developed specifically for H. ostrearia such as the diatom artificial medium (DAM) [START_REF] Gagneux-Moreaux | Diatom artificial medium (DAM): a new artificial medium for the diatom Haslea ostrearia and other marine microalgae[END_REF], a modified version of the [START_REF] Harrison | A broad spectrum artificial seawater medium for coastal and open ocean phytoplankton[END_REF] artificial seawater (ASW) [START_REF] Mouget | Light is a key factor in triggering sexual reproduction in the pennate diatom Haslea ostrearia[END_REF] and the NX medium [START_REF] Nghiem Xuan | Design of an artificial culture medium to optimize Haslea ostrearia biomass and marennine production[END_REF]. Techniques of immobilized cultures (active and/or passive) have also been developed such as culture in tanks for the greening of oysters [START_REF] Turpin | Oyster greening by outdoor mass culture of the diatom Haslea ostrearia Simonsen in enriched seawater[END_REF] and culture on alginate beads [START_REF] Lebeau | Marennine production by agar-entrapped Haslea ostrearia Simonsen[END_REF]. An immersed membrane PBR (MPBR) was also implemented for the continuous extraction of extracellular marennine (EMn) while maintaining algal cells within the photobioreactor (Rossignol et al., 2000a). This kind of photobioreactor is promising because the biomass and hydraulic residence times can be decoupled [START_REF] Castaing | Immersed hollow fibres microfiltration (MF) for removing undesirable micro-algae and protecting semi-closed aquaculture basins[END_REF][START_REF] Castaing | Investigating submerged ultrafiltration (UF) and microfiltration (MF) membranes for seawater pre-treatment dedicated to total removal of undesirable micro-algae[END_REF][START_REF] Massé | Comparison of sludge characteristics and performance of a submerged membrane bioreactor and an activated sludge process at high solids retention time[END_REF]. Furthermore, the cells are not submitted to any shear stress due to pumping or circulation. With this cultivation system, Rossignol et al. (2000a) improved the production of biomass and marennine with marennine concentration, productivity and specific productivity equal to 25 mg L -1 , 3 mg L -1 d -1 and 33 × 10 -9 mg cells -1 d -1 respectively. Moreover, this system induces low energetic costs compared to tangential filtration [START_REF] Humeau | Estimated costs of implementation of membrane processes for onsite greywater recycling[END_REF][START_REF] Massé | Dead-end and tangential ultrafiltration of natural salted water: Influence of operating parameters on specific energy consumption[END_REF][START_REF] Mo | Application of membrane dewatering for algal biofuel[END_REF][START_REF] Zhang | Membrane technologies for microalgal cultivation and dewatering: Recent progress and challenges[END_REF].

Recently, Nghiem [START_REF] Nghiem Xuan | Design of an artificial culture medium to optimize Haslea ostrearia biomass and marennine production[END_REF][START_REF] Nghiem Xuan | Optimization of the growth and marennine production by the diatom Haslea ostrearia in photobioreactor[END_REF] showed that growing Haslea in a stirred flat panel PBR allows better access of the cells to nutrients and light. Till now, they reached a better productivity of extracellular marennine of 4.5 ± 0.2 mg L -1 d -1 (Nghiem Xuan et al., 2020) against 0.7 ± 0.1 mg L -1 d -1 for immobilized cell culture [START_REF] Nghiem Xuan | Optimization of the growth and marennine production by the diatom Haslea ostrearia in photobioreactor[END_REF].

On the basis of a literature review, it appears that air-lift as well as submerged membrane photobioreactor could be of interest for Haslea ostrearia cultivation. Therefore, in this study, an integrated air-lift membrane photobioreactor was implemented and compared to a conventional air-lift photobioreactor. This work aims at studying the versatility of the membrane process facing different conditions of dilution rates (0.1, 0.3, 0.5 d -1 ) as well as its performances (productivities) compared to the same air-lift photobioreactor without membrane.

Material and methods

Microalgae and culture maintenance

A diatom strain Haslea ostrearia (NCC 529), isolated from oysterponds in the Bay of Bourgneuf, France and provided by the Nantes culture collection of the Mer-Molécules-Santé (MMS) laboratory, was used in this study.

Provalosi medium (ES 1/3) was used as culture media. It was prepared in 1 L of natural seawater (Le Croisic Ocearium, Loire-Atlantique, France) filtered using 0.7 µm then 0.45 µm filters (Whatman GF/F). pH and salinity were adjusted at 7.8 and 28 g L -1 respectively. The medium was sterilized by filtration at 0.2 μm.

The microalgae were maintained in 500 mL flasks filled with 250 mL of ES 1/3 medium, at 20 • C . Light was supplied by white led light for the day/night cycle 14/10 at 150 µmol photons m -2 s -1 . 500 mL of these starter cultures were used to inoculate a 1 L PBR.

Photobioreactor system

All experiments, with or without immersed membrane, were sequentially carried out in a lab-scale airlift PBR with a volume of 1 L, as described in [START_REF] Nghiem Xuan | Optimization of the growth and marennine production by the diatom Haslea ostrearia in photobioreactor[END_REF]. Mixing resulted from gas injection at the bottom of the PBR and the pH, fixed at 7.8, was controlled by CO 2 injection, supplied by an automated regulating valve. The PBR or MPBR was placed in an air-conditioned room with temperature adjusted to 20 • C. Cultures were conducted with modified Provasoli ES 1/3 medium, enriched in macronutrients with a NaNO 3 concentration of 0.15 g L -1 , NaHCO 3 equal to 0.42 g L -1 and Na 2 SiO 3 , 5H 2 O of 0.03 g L -1 , needed for cell metabolism. In order to prevent any precipitation phenomenon and to have a chemically stable growth medium, carbon (NaHCO 3 ) and silica (Na 2 SiO 3 , 5H 2 O) were directly and separately added in "fed-batch" to the photobioreactor. Light was supplied by a led panel across the day/ night cycle 14 h/10 h at 200 µmol photons m -2 s -1 .

Two kinds of cultivation experiments were carried out using one airlift PBR without membrane (thereafter named PBR, as control) and one with membrane of filtration (MPBR) (Fig. 1). The latter was equipped with an immersed ultrafiltration membrane. For the present work, PolyVinyliDene Fluoride (PVDF) hollow fiber membranes were chosen with an average pore diameter equal to 0.05 µm and a total membrane surface area equal to 0.0373 m 2 . Culture medium containing extracellular marennine was sucked through the membrane, from the outside to the inside of the fibers, while biomass was retained. An equivalent volume of nutrient medium was supplied from the top of MPBR through a peristaltic pump. No backwashes were implemented.

For both photobioreactors, air-lift alone or with a membrane, three dilution rates (D) equal to 0.1, 0.3 and 0.5 d -1 , were fixed (renewal rate of the culture medium equal to 10%, 30% and 50%). Experiments at different dilution rates were performed separately.

The dilution rate was calculated as follows:

D ( d -1 ) = Q/V r (1)
With Q the flow rate (mL d -1 ) and V r the reaction volume (mL).

Analytical methods

Before each sample, stirring with a magnetic bar was carried out to push over the cells remaining stuck to the walls of the photobioreactor and around the fiber of the filtration membrane. The cell number in suspension was determined using a Nageotte counting chamber (Marienfeld, AQRA, India). The cell concentration (N) was calculated using the following formula:

N ( cells L -1 ) = n/V (2)
With n: the number of cells, V: represents 5 µL for 4 bands of 1.25 µL. All measurements were carried out in triplicates for a minimum of 300 cells.

Extracellular marennine (EMn) concentration was determined by measuring the optical density (A 669 ) at 669 nm, as described by [START_REF] Pouvreau | Preliminary characterisation of the blue-green pigment "marennine" from the marine tychopelagic diatom Haslea ostrearia (Gaillon/Bory) Simonsen[END_REF]. The pigment concentration (mg L -1 ) is then calculated by the following equation, using the Beer-Lambert law:

[EMn] ( mg L -1 ) = (A 669 /ε) × M (3)
With A: the absorbance of the sample at 669 nm, ε: the molar extinction coefficient (ε = 12 × 10 4 L mol -1 cm -1 for the extracellular marennine), l: the optical path of the cell in cm and M: the molar mass of the EMn which is equal to 9893x10 3 mg mol -1 according to [START_REF] Pouvreau | Preliminary characterisation of the blue-green pigment "marennine" from the marine tychopelagic diatom Haslea ostrearia (Gaillon/Bory) Simonsen[END_REF].

The marennine volumetric (P v ) and specific (P s ) productivities were calculated from the dilution rate, according to the following equations Eq. ( 4) and Eq. ( 5) respectively:

P v ( mg L -1 d -1 ) = C m × D (4) P s ( mg cells -1 d -1 ) = P v /C (5)
With C m : the average concentration of EMn at the steady state (mg L - 1 ), D: dilution rate (d -1 ), C: the average cell number in suspension at the pseudo-steady state (Cells L -1 )

Retention rate

The retention rate (RR) of marennine by the membranes was calculated as follows:

RR(%) = ( 1 - [EMn] p [EMn] r ) × 100 (6)
With [EMn] p : the concentration of EMn in the permeate, [EMn] r : the concentration of EMn in the PBR (retentate).

Results and discussion

PBR and MPBR performances

Biomass cultivation follow-up

Fig. 2 shows the evolution of the suspension cell concentration of Haslea ostrearia in the PBR and MPBR operating at different dilution rates. For both photobioreactors, cell concentrations first increased then stabilized. The stabilization of cell concentration occurred sooner for the PBR and reached lower values. As shown in Table 1, whatever the µ max : growth rate, N t cell number at time (t), N t-1 cell number at time (t-1). tg: generation time (day).

dilution rate adopted for the MPBR, the specific growth rates were almost similar (from 0.28 to 0.32 d -1 ). These values depend on the hydraulic and biomass residence times as well as the assimilation level of photons and nutrients. In the case of PBR, the biomass and hydraulic residence times are similar whereas they are equal to infinite and 10, 3.3 or 2 days respectively for the MPBR. As a consequence, the PBR did not allow maintaining a high cell density. Cell washout was observed with the PBR from a dilution rate equal to 0.3 d -1 . From this value, no cell concentration stabilization was observed (Figs. 2 and3). Therefore, for the PBR, the maximum stabilized cell concentration reached 211 ± 5 × 10 6 cells L -1 after 4 days of operation (Fig. 2). This value of cell density at low dilution rates (0.1 d -1 ) seems to be congruent with previously published results. Indeed, a maximum cell density of 150 ± 0.1 × 10 6 cells L -1 was reached at day 5 when Haslea ostrearia was cultured under mixed conditions in a crystal PBR (i.e. airlift PBR) with a dilution rate equal to 0.1 d -1 and under a light intensity of 300 µmole photons m -2 s -1 with a periodicity of 14 h light/10 h dark [START_REF] Nghiem Xuan | Optimization of the growth and marennine production by the diatom Haslea ostrearia in photobioreactor[END_REF].

Nowadays, different types of microalgae cultivation systems exist such as open raceway ponds and closed PBR like vertical column, flat panel and tubular PBR. When classically operated in continuous mode, biomass concentration and then productivity depend on the dilution rate [START_REF] Bilad | Membrane technology in microalgae cultivation and harvesting: A review[END_REF]. In 2012, [START_REF] Tang | Continuous microalgae cultivation in a photobioreactor[END_REF] demonstrated that by increasing the dilution rate of a continuous PBR up to 8-folds (from 0.076 to 0.64 d -1 ), the cell concentration of the microalga Chlorella minutissima decreased from 14.7 × 10 7 to 2.1 × 10 7 cells mL -1 . In addition to these classic PBRs mentioned above, closed or semi-closed PBR with an integrated membrane have also been studied for the cultivation and harvesting of microalgae and mainly for the waste water treatment [START_REF] Bilad | Membrane technology in microalgae cultivation and harvesting: A review[END_REF][START_REF] Zhang | Membrane technologies for microalgal cultivation and dewatering: Recent progress and challenges[END_REF]. Whatever the type of membrane bioreactors, for microalgae or bacteria, the membrane often prevents cell washout and allows increasing the concentration of biomass while keeping a renewal of the growth medium [START_REF] Bilad | Membrane technology in microalgae cultivation and harvesting: A review[END_REF][START_REF] Marbelia | Membrane photobioreactors for integrated microalgae cultivation and nutrient remediation of membrane bioreactors effluent[END_REF][START_REF] Mo | Application of membrane dewatering for algal biofuel[END_REF][START_REF] Zhang | Membrane technologies for microalgal cultivation and dewatering: Recent progress and challenges[END_REF]. Moreover, MPBR allow to reduce energy costs [START_REF] Akkoyunlu | Membrane bioreactors for the production of value-added products: Recent developments, challenges and perspectives[END_REF]. Nevertheless, few works deal with the interest of MPBR for the simultaneous extraction of exometabolites, which is of interest if those exometabolites can be recovered from the membrane while cells are kept in the culture vessel. Thus, for the present study, a self-designed airlift PBR was used in order to maintain a better access of cells to nutrients and light through aeration and thus better metabolite production [START_REF] Nghiem Xuan | Optimization of the growth and marennine production by the diatom Haslea ostrearia in photobioreactor[END_REF][START_REF] Pruvost | Industrial Photobioreactors and Scale-Up Concepts[END_REF]. In addition, this PBR was equipped with a submerged membrane in order to increase Haslea ostrearia cell density and simultaneously to extract the marennine exometabolite. As a consequence, the specific MPBR made it possible to cultivate the Haslea strain at high concentrations never reported in the literature with a maximum suspension cell concentration of 555 ± 25 × 10 6 cells L -1 obtained at a dilution rate equal to 0.1 d -1 (Fig. 2), a value 3 times higher than the PBR device (Fig. 3). It was also able to maintain relatively higher cell concentration over a wider range of dilution rates (at least up to 0.5 d -1 with a cell concentration of 399 ± 11 × 10 6 Cells L - 1 ) (Figs. 2 and3). In fact, high cell density in the photobioreactor will facilitate the downstream processing of biomass in the event of refining. Moreover, at the end of a culture period, the biomass could be more concentrated thanks to a last suction of the medium through the membrane. This method could then also be an effective approach to reduce harvest costs as demonstrated by [START_REF] Bilad | Membrane technology in microalgae cultivation and harvesting: A review[END_REF]. Then, the recovered biomass can be exploited for the extraction of intracellular marennine as well as the siliceous exoskeleton called frustule which could change the ways of manufacturing devices for energy storage, optoelectronics, solar cells and batteries [START_REF] Abdusatorov | FEM exploration of the potential of silica diatom frustules for vibrational MEMS applications[END_REF].

In this study and as shown in Fig. 2, the pseudo-stabilization phase depends on the dilution rate applied. The higher dilution rate is, the faster the stabilization of the culture (10, 6 and 5 days for 0.1 d -1 , 0.3 d -1 and 0.5 d -1 respectively, Table 2). Despite the total retention of cells within the MPBR, without the presence of a purge and/or a biomass outlet, a pseudo-steady state was reached for all the experiments performed with the MPBR. However, our results are consistent with the study of Rossignol et al. (2000a) where they show that during a threeweek experiment at a dilution rate equal to 0.1 d -1 , the viable biomass of H. ostrearia and the marennine productivity reached a plateau phase at the end of the first week. This behavior may be due to the nutritional composition of the culture medium used in this study which may no longer be sufficient for their multiplication but only to keep them alive. This state can be explained by a cell renewal in which the number of living cells makes up for the number of dead cells. Moreover, it's noted that the values of the pseudo-stabilized cell concentrations are different between the dilution rates 0.1 d -1 (555 ± 25 × 10 6 Cells L -1 ) and the couple 0.3 d -1 and 0.5 d -1 (390.4 ± 6 × 10 6 Cells L -1 and 399 ± 11 × 10 6 Cells L -1 respectively). In fact, researchers suggest that EMn is synthesized as a light shielding molecule (Gastineau et al., 2014a;[START_REF] Pouvreau | Antioxidant and Free Radical Scavenging Properties of Marennine, a Blue-Green Polyphenolic Pigment from the Diatom Haslea ostrearia (Gaillon/Bory) Simonsen Responsible for the Natural Greening of Cultured Oysters[END_REF]Rossignol et al., 2000b). Consequently, the more the medium is renewed, the more EMn concentration in the PBR decreases and therefore the cells will be more exposed to light which stresses them and affects their growth.

Extracellular marennine production

For the MPBR device, marennine concentrations increased then stabilized at higher values for the lowest dilution rates (Fig. 4.A, 4.B, 4. C). Thus, at 0.1 d -1 , a maximum concentration of EMn equal to 36 ± 0.02 mg L -1 was obtained for the MPBR versus 14 ± 0.01 mg L -1 for the PBR (Fig. 4.A). As presented in Fig. 4.A and 4.B, the stabilized EMn concentrations were always lower for the PBR compared to the MPBR. By plotting the stabilized concentration of EMn as a function of pseudostabilized cell concentration (Fig. 4.D), it appears that EMn concentration increased with cell density for both photobioreactors. The stabilization was always faster for the highest dilution rate.

In addition to the continuous agitation provided by gas injection, manual agitation with a magnetic stirred, already present in the bioreactor, was carried out every day and before each sample. This manipulation allowed to push over the cells which remain stuck on the photobioreactor walls and around the fibers of the filtration membrane. Thus, the two devices (PBR and MPBR) were compared on the basis of the cells in suspension which are the easiest to recover for an industrial application. It should be noted that very few cells remain attached to the filtration membranes (barely visible to the naked eye), comparing to those in suspension. The MPBR achieved the highest volume productivity of EMn (Fig. 5.A). Indeed, its maximum productivity was equal to 7.2 ± 0.01 mg L -1 d -1 (D = 0.5 d -1 ), that means 2 to 5 times higher than the productivity of the PBR (Fig. 5.A and 5.B), but also higher than cell culture systems used so far. For example, for agar/alginate immobilized cells under a light intensity of 3 x10 16 quanta cm -2 s -1 with a 14/10 h light/dark cycle, [START_REF] Lebeau | Continuous marennin production by agar-entrapped Haslea ostrearia using a tubular photobioreactor with internal illumination[END_REF]; Lebeau et al. (2002) found a maximum marennine productivity of 0.5-0.6 mg L -1 d -1 at a dilution rate equal to 0.25 d -1 . Passive cell immobilization at the bottom of open ponds of 10 m 3 resulted in the same range of EMn concentration (3.4 mg L -1 ) and productivity (0.4 mg L -1 d -1 ) [START_REF] Turpin | Oyster greening by outdoor mass culture of the diatom Haslea ostrearia Simonsen in enriched seawater[END_REF]. Nghiem Xuan et al. ( 2021) also found a lower productivity equal to 4.5 ± 0.16 mg EMn L -1 d -1 during the cultivation within a conventional mixed PBR fed with optimized culture medium and with an irradiance equal to 300 µmole photons m -2 s -1 with a photoperiod 14/10 h light/dark. Finally, the volume productivities of marennine from the study presented here, were 0.7 to 2 times higher than the productivity obtained by Rossignol et al. (2000a) who used a cylindrical photobioreactor (L = 40 cm and d = 15 cm) equipped with a submerged membrane. The cylindrical PBR is known to be one of the most practical PBR due to its low energy consumption. Nevertheless, it presents a small specific illumination (a s ) area of 26.66 m -1 (a s cylindrical PBR = 2/R, with R is the internal radius of the cylinder) which may result in a high dark fraction inside the reactors. This value is below the specific illumination area of our air-lift PBR which is equal to 33.33 m -1 (a s flat plate PBR = 1/L, with L is the thickness of the PBR). In addition, Airlift PBR (also called gas/liquid reactor) allows a good constant mixing, by gas injection, which improves light access to cells. This was not the case with the study of Rossignol et al., (2000a) who only mixed with magnetic stirrer for 5 min when they injected the culture medium. With the MPBR configuration, the stabilized concentration and volume productivity of extracellular marennine are inversely proportional (Fig. 5.A and 5.C). A dilution rate of 0.1 d -1 , resulted in the highest EMn concentration (36.00 ± 0.02 mg L - 1 ) and the lowest productivity (3.60 ± 0.01 mg L -1 d -1 ) whereas it is the opposite for 0.5 d -1 ([EMn] = 14.00 ± 0.03 mg L -1 , P v = 7.20 ± 0.01 mg L -1 d -1 ). This is in agreement with the study of Rossignol et al. (2000b) who increased the dilution rate by 10 times (0.025 and 0.25 d -1 ) to improve the volume productivity of marennine from 0.17 to 2.7 mg L -1

Table 2

Cell or marennine concentrations as well as marennine productivity following operating conditions of the MPBR of this study. d -1 in free-cells bioreactor associated with an external flat membrane module and from 0.12 to 0.7 mg L -1 d -1 in immobilized-cells bioreactor, under light intensity equal to 80 µmole photons m 2 s -1 with the following cycles: 14 h light/ 10 h dark. Moreover, Fig. 5.C shows that the specific productivity of marennine, calculated from the number of cells in suspension, increases by increasing the dilution rate. The highest EMn specific productivity was equal to 0.018 × 10 -6 mg cells -1 d -1 at D = 0.5 d -1 , 3 times higher than the specific productivity obtained at D = 0.1 d -1 . In fact, an increase of medium renewal rate would allow to stimulate the synthesis of EMn by increasing the accessibility of light and nutrient to microalgal cells. Besides, marennine, which presents a significant bioactivity, could have inhibited the growth of H. ostrearia cells at high concentrations [START_REF] Nghiem Xuan | Design of an artificial culture medium to optimize Haslea ostrearia biomass and marennine production[END_REF]. This phenomenon was not observed during this study. In fact, at high concentration of 36.00 ± 0.02 mg L -1 , a high suspension cell concentration of 555 ± 25 × 10 6 cells L -1 was still reached, higher than the literature values (Gastineau et al., 2012a;[START_REF] Gaudin | Microalgal cell immobilization for the longterm storage of the marine diatom Haslea ostrearia[END_REF][START_REF] Lebeau | Diatom cultivation and biotechnologically relevant products. Part I: Cultivation at various scales[END_REF][START_REF] Nghiem Xuan | Design of an artificial culture medium to optimize Haslea ostrearia biomass and marennine production[END_REF], 2021;Prasetiya et al., 2020b;Rossignol et al., 2000a). All these results show that membrane technology can play a crucial role in microalgae biorefinery processes and depending on the biotechnological application envisaged. As shown above, low dilution rate operations (0.1 d -1 ) induced a high concentration of marennine in the photobioreactor. In this case, the entire culture (cells plus the culture medium) could be harvested just after the stabilization of the concentrations. The cells would then be separated from marennine by centrifugation. This operating mode, which resembles a batch operation, would allow to recover a small volume of marennine-rich solution as well as pellet of cells.

On the other hand, high dilution rate operations (0.5 d -1 ) may also have an interest. Indeed, even if the concentration of marennine into the MPBR is lower, but already high compared to classical photobioreactors, this makes it possible to continuously produce a solution of marennine. In that case, the marennine solutions will already be pre-purified since the membrane will have retained all the biomass. This type of operation could be exploited in oyster farming in order to continuously supply ponds with marennine solutions at concentrations below the toxicity level. The process of greening oysters during the final farming stage before marketing could be improved [START_REF] Prasetiya | Does culture supernatant of Haslea ostrearia containing marennine affect short-term physiological traits in the adult blue mussel Mytilus edulis? Aquac[END_REF]. In addition, this process can be integrated into aquaculture industries where marennine could serve as a source of antimicrobial substances to control pathogens affecting certain larval, juvenile or adult stages of farmed animals (bivalves, fish) (Prasetiya et al., 2020a).

Finally, marennine is known for its multiple biological activities (Gastineau et al., 2014b). One of the scientific and technological issues related to its extraction concerns the preservation of its properties and activities. For the present study, EMn is recovered in its native configuration, in a medium with the same composition of soluble compounds, pH and ionic strength. The use of the filtration membrane enables to prepurify EMn without adding chemicals and under relatively mild operating conditions. In contrast to cross-flow filtration, the molecules do not pass at high speeds through pumps, which makes them less susceptible to be damaged.

However, the performance of the submerged membrane photobioreactor strongly depends on several parameters including light, carbonization of CO 2 and nutrient concentrations which can be affected by the presence of a membrane in MPBR systems. It would therefore be interesting to study the influence of these parameters. In addition, membrane fouling could constitute a major challenge for extended application in the culture of microalgae notably for long term filtrations where a biofilm could settle. Backwashes with permeates could be used to alleviate severe fouling [START_REF] Massé | Performances of dead-end ultrafiltration of seawater: From the filtration and backwash efficiencies to the membrane fouling mechanisms[END_REF]. Moreover, the molecular weight cut-off of the membrane as well as the conditions of its use (shear stress at the surface of the membrane) could be changed in order to enhance the hydraulic performances and the transmission rates [START_REF] Balti | Fractionation of Arthrospira platensis (Spirulina) water soluble proteins by membrane diafiltration[END_REF][START_REF] Gifuni | Recovery of microalgal protein by three-steps membrane filtration: Advancements and feasibility[END_REF]. Nevertheless, for industrial applications, larger-scale operations will be needed even if the process of EMn recovery using membranes is optimized. This scale-up remains a challenge, because the behavior of the culture of microalgae will change.

Membrane performance

The membrane retention rate was estimated in this study at each dilution rates after stabilization. As shown in Fig. 6, the membrane allowed marennine to pass relatively easily since retention rates were less than 40%. Furthermore, an increase of the dilution rate induced a decrease of the retention rate (Fig. 6). Put another way, the higher the renewal rate of the culture medium, the more marennine passes freely through the membranes. In fact, at a dilution rate of 0.1 d -1 , 35% of EMn were retained by the filtration membranes against 10% at 0.5 d -1 . The highest suspension cell concentration at 0.1 d -1 (555 ± 25 × 10 6 cells L -1 against 399 ± 11 x10 6 cells L -1 at 0.5 d -1 ) as well as EMn concentration (36 ± 0.02 mg L -1 against 14 ± 0.03 mg L -1 at 0.5 d -1 ) could induce a higher membrane fouling or polarization concentration at the membrane surface. In addition, since the retention rates were calculated at the end of the stabilization period, it should be noted that the membrane was submerged for the longest time in the case of the 0.1 d -1 before evaluation of the retention rate. Thus, a different membrane fouling could occur due to different residence time of membrane. In order to mitigate this mass transfer decrease, a backwash could be implemented with a fresh growth medium.

Conclusion

This work demonstrates the high potential of air-lift membrane photobioreactors for simultaneous cultivation of the diatom H. ostrearia and extraction of its valuable exometabolite marennine. High cell concentrations in the photobioreactor were achieved, depending on the dilution rate. Productivities and concentrations of extracellular marennine are significantly increased compared to previous works using classical photobioreactor. Thus, the operation of MPBRs at high dilution rates is a promizing field for continuous extraction of pre-purified algal exometabolites.

Fig. 1 .

 1 Fig. 1. The schematic illustration of the microalga cultivation process using MPBR and PBR. A) PBR without membrane filtration, B) PBR with membrane filtration. Both of them are operated at different dilution rate (D) equal to 0.1, 0.3 and 0.5 d -1 . Before sampling stirring with a magnetic bar on the photobioreactor walls as well as manual stirring of fiber bundle was carried out.

Fig. 2 .

 2 Fig. 2. Growth kinetics of Haslea ostrearia in PBR (◆) and MPBR (•) operating at different dilution rates of 0.1, 0.3 and 0.5 d -1 . Washout occurred in the PBR, indicated by the continuous decrease of the cell concentration. Results were expressed as mean ± standard deviations of triplicate determinations.

Fig. 3 .

 3 Fig. 3. Stabilized cell density of Haslea ostrearia in both PBRs operating at different dilution rates. Washout in PBR (◆) at dilution rate D equal to 0.3 d -1 . MPBR (■) was able to prevent washout up to D = 0.5 d -1 allowing higher cell concentration. Results were expressed as mean ± standard deviations of triplicate determinations.

Fig. 4 .

 4 PhotobioreactorsFree cells/ immobilized cells

Fig. 5 .

 5 Fig. 5. Concentration (◆) and volume productivity (•) of extracellular marennine at different dilution rates 0.1 d -1 , 0.3 d -1 and 0.5 d -1 . A, B) EMn concentration and productivity in the MPBR and the PBR. The stabilized concentration and volume productivity of extracellular marennine are inversely proportional. C) Evolution of volume (•) and specific (■) productivity of EMn in the MPBR depending on the marennine concentration. Results were expressed as mean ± standard deviations of triplicate determinations.

Fig. 6 .

 6 Fig. 6. Membrane retention rate with the MPBR operating at different dilution rates.

Table 1

 1 Growth parameter of Haslea ostrearia in MPRB at different dilution rate.

		MPBR
	Dilution rate (d -1 )	0.1	0.3	0.5
	µ 0.31	0.32	0.28
	t g = 2/ µ max	2.25	2.19	2.49

max = ln(Nt) -ln(Nt-1 ) tt (t-1)

Funding

This work received funding from .

CRediT authorship contribution statement

Nesrine Gargouch: Conceptualization, Methodology, Validation, Investigation, Writingoriginal draft. Raphaelle Touchard: Resources, Investigation. Hélène Marec: Resources, Investigation. Jean Luc Mouget: Funding acquisition, Writingreview & editing. Jérémy Pruvost: Conceptualization, Supervision, Writingreview & editing. Anthony Masse: Conceptualization, Methodology, Validation, Supervision, Writingoriginal draft.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.