# Screening for astaxanthin production by cultures of microalgae within integrated photobioreactors façade

Astrid de Luca, Catherine Dupré, Jeremy Pruvost, François Le Borgne, Olivier Lépine, Jack Legrand, Dominique M Grizeau

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Astrid de Luca, Catherine Dupré, Jeremy Pruvost, François Le Borgne, Olivier Lépine, et al.. Screening for astaxanthin production by cultures of microalgae within integrated photobioreactors façade. 3rd European Congress of Applied Biotechnology (ECCE10-ECAB3-EPIC5), Sep 2015, Nice, France. hal-02567648

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Submitted on 22 Aug 2023

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# SCREENING FOR ASTAXANTHIN PRODUCTION BY CULTURES OF MICROALGAE WITHIN INTEGRATED PHOTOBIOREACTORS FAÇADE

<figure><img src="image_3.png" /></figure>

Astrid De Luca<fnref n="1" />,², Catherine Dupré<fnref n="1" />, Jeremy Pruvost<fnref n="1" />,², François Leborgne², Olivier Lépine², Jack Legrand<fnref n="1" />,², Dominique Grizeau<fnref n="1" />

## Context

New closed photobioreactors designs for microalgae cultivation

* → sustainably producing high yields of value-added products astaxanthin 2000 $ / kg (Lorenz & Cysewski, 2000)

potent antioxidant

<figure><img src="image_4.png" /></figure>

prevention of age related macular degeneration (Yamashita, 2013)

* → Haematococcus pluvialis astaxanthin up to 3 % DW (Lorenz & Cysewski, 2000)

Symbio2 project :
Production of microalgae in biofacades photobioreactors (Pruvost et al., 2015)
Continuous-flow culture system
No biofouling or biomass sedimentation ; control of encystement
High astaxanthin productivities

## Objectives

Use of microplates to :

- screen microalgae species for astaxanthin accumulation.

- compare the efficiency of different factors for triggering astaxanthin synthesis

<table><thead><tr><td>Strain</td><td>Size (μm)</td><td>Cultivation in photobioreactors</td></tr></thead><tbody><tr><td>M1</td><td>13-27</td><td>X</td></tr><tr><td>M2</td><td>5-8</td><td>X</td></tr><tr><td>M3</td><td>15-30</td><td>✓</td></tr><tr><td>M4</td><td>18-23</td><td>X</td></tr></tbody></table>

<figure><img src="image_5.png" /></figure>

## Whole cells spectrophotometric characterization

<figure><img src="image_6.png" /></figure>

in situ measurements

## Miniaturization of end-point assays

<figure><img src="image_7.png" /></figure>

ex situ measurements

## Limits of quantitation (LOQ) and saturation (LOS)

Signals (inter)calibration

## Methodology

Cell incubation 96 and 24 wells microplates

Control of environmental parameters ; PFD, T, rpm

Inoculum pre-treatments for batch incubations

Well-controlled initial nutrients status

## Carotenoid induction

## Identification of potent factors

<figure><img src="image_8.png" /></figure>

## Data treatment

<figure><img src="image_9.png" /></figure>

Detection time (DT)

(Grizeau et al.)

## Microalgae incubations

* → Astaxanthin $C_{40}H_{52}O_4$

* → C-sufficient medium with $NaHCO_3$

* → Control of culture incubation :

<table><thead><tr><td>Parameter</td><td>Value</td></tr></thead><tbody><tr><td>Temperature (°C)</td><td>20</td></tr><tr><td>PFD (µmol photons m<sup>-2</sup> s<sup>-1</sup>)</td><td>100</td></tr><tr><td>Light path (mm)</td><td>96 wells : 5<br>24 wells : 4,5</td></tr></tbody></table>

* → incubation and reading

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>Multimode microplates reader</figcaption></figure>

## In situ measurements

<figure><img src="image_12.png" /></figure>

* A$_{750}$ to estimate residual growth

* Fluorescence $Ex_{440}/Em_{670} = f$ ([chlorophyll])

* A$_{480}$/ A$_{680}$ ration = f (time)

* Threshold of carotenogenesis = f ($\frac{1}{DT}$)

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>$[NaNO_3]_{opt}$ for carotenogenesis in M2 cells</figcaption></figure>

## Ex situ measurements

Exemple 24 wells microplate :

* 200 µL extracted with DMSO :

* spectrophotometry → [Total carotenoids]

* HPLC analysis → [Astaxanthin]

* carotenoid profile

<figure><img src="image_15.png" /></figure>

* 2 µL photosynthetic activities :

* Fv/Fm, NPQ and PI curves

* Microscopy / Image analysis

* cell numeration

Epifluorescence

* cell viability

Nitrate determination

* conversion yields

<figure><img src="image_16.png" /></figure>

## Conclusions - perspectives

* Efficiency of a microplates format utilization for comparative studies of microalgae carotenogenesis

* Combining *in situ* and *ex situ* measurements

<figure><img src="image_17.png" /></figure>

→ data acquisition for screening the efficiency of inducing factors on carotenogenesis

* Futur prospects ; improvements by using an automated workstation allowing semi-continuous cultures within microplates

<figure><img src="image_18.png" /></figure>

<footnote n="1">¹ GEPEA Laboratory, University Nantes, UMR-CNRS 6144, 44602 Saint-Nazaire Cedex, France, ² Algosource Technologies, Saint-Nazaire Cedex, France</footnote>