No abstract

Online monitoring of algal biotechnological processes still requires development to support economic sustainability. In this work, fluorescence spectroscopy coupled with chemometric modelling is studied to monitor simultaneously several compounds of interest, such as chlorophyll and fatty acids, but also the biomass as a whole (cell concentration). Fluorescence excitation-emission matrices (EEM) were acquired in experiments where different environmental growing parameters were tested, namely light regime, temperature and nitrogen (replete or deplete medium). The prediction models developed have a high R2 for the validation data set for all five parameters monitored, specifically cell concentration (0.66), chlorophyll (0.78), and fatty acid as total (0.78), saturated (0.81) and unsaturated (0.74). Regression coefficient maps of the models show the importance of the pigment region for all outputs studied, and the protein-like fluorescence region for the cell concentration. These results demonstrate for the first time the potential of fluorescence spectroscopy for in vivo and real-time monitoring of these key performance parameters during Nannochloropsis oceanica cultivation.

With the increase awareness for a healthier food regime and greener environmental processes, microalgae are being looked as a solution for a sustainable production of polyunsaturated fatty acids, such as omega-3 eicosapentaenoic acid (EPA). Nannochloropsis oceanica is an oleaginous microalga, well-known for the ability of EPA accumulation, although higher lipid productivities are still required to make the process competitive. Therefore, three cultivation parameters were tested in the present work (temperature, light cycles and nitrogen supply) in order to study the EPA profile in the polar and neutral fractions of the cells. In addition, an online monitoring tool based on a fluorescence spectroscopy technique was developed with the aim of increasing process knowledge at real time. The results of this work show that nitrogen depletion induces the highest variability in EPA accumulation in the neutral fraction (triacylglycerols). However, to increase the EPA content in the polar fraction a different strategy needs to be implemented, such as decreasing the cultivation temperature or the light available per cell. Chemometric models were developed through PCA (Principal Component Analysis) and PLS (Projection to Latent Structures), using only fluorescence spectra as inputs, enabling the monitoring of EPA in both fractions separately. High explained variance was observed (above 85%) in both fractions, with R 2 above 0.81 and slopes above 0.93 for both validation and training data sets. Lower values of cross-validation and prediction errors were observed (between 0.29 and 0.49% g/g DW ). The results obtained show that fluorescence spectroscopy is a powerful technique for online monitoring of non-fluorophore molecules, such as EPA, in complex process like microalgae cultivation.

The growth of the polyextremophile red microalga Galdieria sulphuraria ACUF 064 was evaluated under mixotrophy and heterotrophy in a 13-L lab-scale stirred photobioreactor, using buttermilk as a carbon source. Beforehand, G. sulphuraria ACUF 064 growth on glucose, galactose and lactose was evaluated. No significant differences were observed in terms of growth when lactose, glucose or galactose was used as a carbon source. Overall, the biomass yield on carbon was 70% higher in mixotrophy than heterotrophy for galactose (2.0 gx gC−1) and lactose (2.0 gx gC−1), while it was similar for glucose (1.5 gx gC−1). At the same time, the yield of biomass on nitrogen for cultures grown in lactose was the lowest in comparison to all the other tested substrates. This was also supported by a higher C-phycocyanin content, with 5.9% wC-PC/wx as the highest value in mixotrophy. A preliminary experiment in flasks under mixotrophic conditions with different buttermilk dilutions revealed that a dilution ratio of 40% v/v of buttermilk (corresponding to 2.0 gC L−1) was optimal for biomass production. Finally, G. sulphuraria ACUF 064 was grown in the optimal buttermilk dilution ratio in a 13 L photobioreactor. The highest biomass productivity was also obtained in mixotrophy (0.55 gx L−1 d−1), corresponding to a carbon removal of 61%. Overall, lactose-containing substrates such as buttermilk hold promise as a substrate for the growth of G. sulphuraria while revalorizing an industrial effluent.

After light, temperature is the most relevant environmental parameter in outdoors cultivation of microalgae. Suboptimal and supraoptimal temperatures negatively impact growth and photosynthetic performance with a subsequent effect on lipid accumulation. It is generally recognised that lower temperatures trigger an increase in fatty acid desaturation while higher temperatures trigger the opposite reaction. The effect of temperature on lipid classes has been less studied in microalgae and in certain cases, the effect of light cannot be completely excluded. In this research, the effect of temperature on growth, photosynthesis, and lipid class accumulation in Nannochloropsis oceanica was studied at a fixed light gradient with a constant incident light intensity (670 μmol m-2 s-1). A turbidostat approach was used to achieve temperature acclimated cultures of Nannochloropsis oceanica. Optimal growth was found at 25-29°C, while growth was completely arrested at temperatures higher than 31°C and lower than 9°C. Acclimation to low temperatures triggered a decrease in absorption cross section and photosynthesis rates with a tipping point at 17°C. Reduced light absorption was correlated with a decrease in content of the plastid lipids monogalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol. The increase of diacylglyceryltrimethylhomo-serine content at lower temperatures indicated a relevant role of this lipid class in temperature tolerance. Triacylglycerol content increased at 17°C and decreased at 9°C emphasising a metabolic switch in stress response. Total and polar eicosapentaenoic acid content remained constant at 3.5 and 2.4% w/w, despite the fluctuating lipid contents. Results show an extensive mobilisation of eicosapentaenoic acid between polar lipids classes at 9°C to ensure cell survival under critical conditions.