Elsevier

Journal of Biotechnology

Volume 325, 10 January 2021, Pages 312-324
Journal of Biotechnology

The effects of CO2-induced acidification on Tetraselmis biomass production, photophysiology and antioxidant activity: A comparison using batch and continuous culture

https://doi.org/10.1016/j.jbiotec.2020.10.005Get rights and content

Highlights

  • Tetraselmis has a higher antioxidant activity in balanced culture in acidic condition.

  • CO2-acidification enhances Tetraselmis growth in batch and continuous culture.

  • Culture mode and CO2-acidification have interactive effects on Tetraselmis physiology.

  • CO2-acidification with N-limitation induce a decrease of photosynthetic parameters.

  • CO2-acidification and culture mode are effective tools to optimize antioxidant activity.

Abstract

A Tetraselmis sp. was selected for its antioxidant activity owing to its high lipid peroxidation inhibition capacity. With the aim to monitor culture conditions to improve antioxidant activity, effects of CO2-induced acidification on Tetraselmis growth, elemental composition, photosynthetic parameters and antioxidant activity were determined. Two pH values were tested (6.5 and 8.5) in batch and continuous cultures in photobioreactors. Acidification enhanced cell growth under both culture methods. However, the microalgae physiological state was healthier at pH 8.5 than at pH 6.5. Indeed, photosynthetic parameters measured with pulse amplitude modulated (PAM) fluorometry showed a decrease in the photosystem II (PSII) efficiency at pH 6.5 in batch culture. Yet, with the exception of the PSII recovering capacity, photosynthetic parameters were similar in continuous culture at both pH. These results suggest that lowering pH through CO2-induced acidification may induce a lower conversion of light to chemical energy especially when coupled with N-limitation and/or under un-balanced culture conditions. The highest antioxidant activity was measured in continuous culture at pH 6.5 with an IC50 of 3.44 ± 0.6 μg mL−1, which is close to the IC50 of reference compounds (trolox and α-tocopherol). In addition, the principal component analysis revealed a strong link between the antioxidant activity and the culture method, the photophysiological state and the nitrogen cell quota and C:N ratio of Tetraselmis sp.. These results highlight Tetraselmis sp. as a species of interest for natural antioxidant production and the potential of PAM fluorometry to monitor culture for production of biomass with a high antioxidant activity.

Introduction

Many natural products isolated from microalgae have attracted attention owing to their broad spectrum of biological activities with health promoting effects. Microalgae produce specific metabolites that can be used in cosmetics and pharmaceuticals; they have bioactive compounds with anti-UV, antibacterial, antifungal, anticancer, anti-inflammatory and/or antioxidant activities (Abad et al., 2008; Assunção et al., 2016; Safafar et al., 2015; Sathasivam and Ki, 2018; Talero et al., 2015; Yuan et al., 2011). In addition, PUFAs (e.g., the omega-3 or -6 fatty acids), pigments (e.g., carotenoids), vitamins, sterols and polysaccharides from microalgae origin are key molecules to develop dietary supplements in human nutrition and to improve animal feed (Aklakur, 2016; Guedes et al., 2011a, b; Plaza et al., 2008).

Microalgae are currently studied as a potential source of natural antioxidant (Assunção et al., 2016; Goiris et al., 2012), as they could replace synthetic antioxidants in the food industry (Batista et al., 2019, 2017; Goiris et al., 2015a), and in the cosmetic, pharmaceutical and nutraceutical industries (Guedes et al., 2011a; Sansone and Brunet, 2019). In addition, microalgae biodiversity, productivity and controlled culture conditions offer great potentials to produce a sustainable source of natural antioxidants (Guedes et al., 2011a; Mimouni et al., 2012; Wijffels et al., 2010).

Recent screening showed that the antioxidant capacity of microalgae is species specific (Assunção et al., 2016; Coulombier et al., 2020; Custódio et al., 2012; Goiris et al., 2012). A Tetraselmis sp. isolated in New Caledonia was identified as a promising species for antioxidant production (Coulombier et al., 2020) owing to its high capacity to inhibit lipid peroxidation, growth rate, and ease of culture. In addition, Tetraselmis spp. were also identified for their antioxidant potentials in other screening (Assunção et al., 2016; Custódio et al., 2012; Goiris et al., 2012). This genus is cosmopolitan and can be found in different types of ecosystems, from oceanic to freshwater and hypersaline habitats (Fon-Sing and Borowitzka, 2016; Worden et al., 2004). Many Tetraselmis species have a high growth rate and dietary value of interest (e.g., polyunsaturated fatty acids, vitamins) for aquafeed formulation, fish, live feed, and shellfish nutrition (Cerezuela et al., 2012; Hemaiswarya et al., 2011; Ponis et al., 2003; Thinh et al., 1999), thus it is a genus exploited in aquaculture (e.g., T. chui, T. suecica). Concerning biotechnological applications, this genus is known to have a high content of bioactive compounds with antioxidant properties such as carotenoids (Ahmed et al., 2014), polyunsaturated fatty acids (Custódio et al., 2012), water soluble polysaccharides (Dogra et al., 2017), phenolic compounds (Farahin et al., 2016; Gam et al., 2020) and vitamins (Brown et al., 1999). Depending on abiotic stressors, microalgae can induce the production of antioxidant molecules to protect its organelles against ROS. This ability can then be used to enhance the production of specific metabolites of interest (Chen et al., 2017; Paliwal et al., 2017). Indeed, nutrient availability (Goiris et al., 2015b), light condition (Coulombier et al., 2020), pH and temperature (Guedes et al., 2011c) are abiotic factors that influence the antioxidant activity and productivity of microalgae. However, few studies assessed the effect of pH or CO2-induced acidification on microalgae antioxidant activity (Guedes et al., 2011c; Xia et al., 2018). Yet, pH and CO2-induced acidification affects dissolved inorganic carbon availability, intracellular acid base balance, structural rearrangement of pigment systems, and therefore, may influence growth, carbon assimilation, energy demand to maintain the membrane electrochemical potential and enzyme activity, and intracellular oxidative stress (Goss and Garab, 2001; Kramer et al., 2003; Milligan et al., 2009; Xia et al., 2018). Changes in carbon fixation will also modify the cellular concentration of ATP and NADPH, which in turn may modify photochemical processes and energy dissipation pathways (Takahashi and Murata, 2005). To monitor these photochemical processes (mainly PSII), PAM fluorometry is a commonly used technique based on chlorophyll fluorescence which offers the advantage to be non-invasive (Krause and Weis, 1991; Schreiber et al., 1995) and suitable in microalgal biotechnology to follow large scale culture fitness (Masojídek et al., 2010). This technique measure the light energy emitted from the light harvesting pigments associated with the process of photosynthesis. Briefly light energy absorbed by chlorophyll is either used by the photochemistry (photosynthesis), dissipated as heat (excess of energy) or re-emitted (fluorescence); consequently, by measuring the yield of fluorescence, information about the fitness of the PSII can be estimated (reviewed in Maxwell and Johnson, 2000; Consalvey et al., 2005).

Culture conditions are modulating factors to which microalgae cell will respond by adjusting their physiology inducing modifications in growth, photosynthetic parameters, biomass composition and consequently antioxidant activity (Coulombier et al., 2020). In this study, we focus on the effects of pH using CO2-induced acidification on antioxidant activity and physiological responses of a tropical strain of Tetraselmis sp. produced in stirred closed photobioreactor operated in batch and in continuous culture. The overall objectives were to assess the effect of pH and culture mode on growth, photosynthetic parameters, and elemental composition to suggest marker processes of antioxidant activity of Tetraselmis sp. and to optimize production of biomass with high antioxidant activity.

Section snippets

Microalgae culture

The microalgae Tetraselmis sp. was isolated from tropical coastal seawater (New Caledonia) (Coulombier et al., 2020). The inoculum was cultured in a 250 mL Erlenmeyer flask with filtered seawater (salinity 35; 0.2 μm) enriched in Conway medium (Walne, 1966). The cultures were exposed to a continuous light intensity of 190 μmol photons m−2 s-1, aerated, and gently homogenized daily for 11 days.

Experimental culture conditions

Continuous and batch cultures were carried out in six 2.5 L stirred closed PBRs made of transparent

Growth performances

Low pH had a significant influence on the growth (μmax, Table 1) of Tetraselmis sp. cultured in batch as μmax increased from 1.48 ± 0.16 day−1 at pH 8.5 to 2.45 ± 0.18 day−1 at pH 6.5, but the latency time (lambda) was slightly higher at low pH (0.44 ± 0.04 day) than at 8.5 (0.18 ± 0.03 day, Table 1). However, the maximum concentration (Cmax) was similar between the two conditions, 7.10 ± 0.02 × 106 cells mL−1 at pH 8.5 and 7.52 ± 0.02 × 106 cells mL−1 at pH 6.5 (Table 1 and Fig. 1A and C). In

Growth performances and fitness

Responses of microalgae to CO2-induced acidification are likely to be species specific, with potential “winners” and “losers” (Hinga, 2002). Tetraselmis F. Stein (1878) is a cosmopolitan genus known to live in a wide variety of habitats (Fon-Sing and Borowitzka, 2016; Worden et al., 2004). The species studied in this paper came from a coastal and transitional environment and is thus naturally exposed to sudden changes in pH (Wu et al., 2015). In culture, the effect of pH on a Tetraselmis sp.

Conclusions

This study provides evidence of the interactive effects of CO2-induce acidification and nutrient availability on growth, photophysiological state of the PSII and antioxidant activity of Tetraselmis sp.. It also highlights the great potential of Tetraselmis sp. as an alternative source of natural antioxidant, as well as on the role of pH and nutrients as effective tools to enhance the production of biomass with high antioxidant activity. In addition, it suggests that PAM fluorometry might be

Funding

The authors acknowledge the Province Nord, the Province Sud, the Government of New Caledonia and the Comité Interministériel de l'Outre-Mer (CIOM) for financial support through the AMICAL (Aquaculture of Microalgae in New CALedonie) 1 and 2 research programs, and The South Province of New Caledonia (n°26960, n°1546 and n°9705) and the North Province of New Caledonia (n°609011-55 and n°609011-54) delivered the sampling authorizations. We would like also to acknowledge Ifremer for financing the

CRediT authorship contribution statement

Noémie Coulombier: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - original draft, Writing - review & editing. Paul Blanchier: Conceptualization, Methodology, Formal analysis, Investigation, Writing - review & editing. Loïc Le Dean: Conceptualization, Methodology, Resources, Writing - review & editing, Project administration, Funding acquisition. Vanille Barthelemy: Investigation. Nicolas Lebouvier: Validation, Formal analysis, Writing - review & editing,

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgments

We would like to thanks Anne Desnues and Philippe Gérard from the LAMA (LAboratoire des Moyens Analytiques) (IRD, Nouméa) for the elemental and nutrient analyses.

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