Évaluation des microalgues sélectionnées en Nouvelle-Calédonie comme source potentielle d'anti-oxydants naturels. Identification des molécules anti-radicalaires et stimulation de leur biosynthèse par orientation métabolique

The demand has increased for natural antioxidant as an alternative to synthetic antioxidant for food, cosmetic and nutraceutical industry. Most natural antioxidant currently available on the market are derived from terrestrial plant. However, microalgae, with their chemodiversity, productivity and controlled cultivation possibilities, are a promising source of antioxidant. The objective of this thesis is to evaluate the potential of microalgae isolated in New Caledonia as a source of natural antioxidants. About thirty species of microalgae were isolated in the caledonian lagoon for their high growth rate by a dilution-exclusion method. A screening of antioxidant activity was then carried out on twelve species, grown at two light intensities (low and high), using four different antioxidant assays: DPPH (2,2-diphenyl-1-picrylhydrazyl), TEAC (Trolox equivalent antioxidant capacity), ORAC (Oxygen Radical Absorbance Capacity) and TBARS (Thiobarbituric Acid Reactive Substances). The results of the antioxidant screening showed the need to use several antioxidant assays and the significant impact of light intensity on antioxidant activity. The screening also highlighted two species of interest, Nephroselmis sp. and Tetraselmis sp.. The influence of some culture parameters on antioxidant activity was studied for these two species. Nephroselmis sp. has a strong capacity to scavenge peroxyl radicals owing to its high carotenoid content. In addition, this species has a carotenoid of interest that is still little studied, siphonaxanthin. It has been shown that the cultivation of Nephroselmis sp. should not be limited in nitrogen to promote the production of antioxidant carotenoids, in particular siphonaxanthin. Tetraselmis sp. has been shown to have a strong capacity to inhibit lipid peroxidation. The study of the influence of pH (6.5 and 8.5) and culture mode (batch vs. continuous) on the antioxidant activity and photophysiology of Tetraselmis sp. highlighted the importance of these two parameters on the antioxidant activity which is optimized when the microalgae is in a good physiological state. These results reveal the potential of microalgae isolated in New Caledonia as a source of natural antioxidants, and the high impact of culture conditions on antioxidant activity. Knowledge of the different mechanisms must now be deepened on cellular models and the origin of the molecules involved must be characterized by a metabolomic approach.Une demande croissante d’antioxydants naturels alternatifs aux antioxydants synthétiques a émergé dans l’industrie agroalimentaire, cosmétique et nutraceutique. La majorité des antioxydants naturels actuellement disponibles sur le marché sont issus des végétaux terrestres, cependant les microalgues par leur chimiodiversité, leur productivité et les possibilités de culture contrôlée offrent un grand potentiel. L’objectif de cette thèse est d’évaluer le potentiel de microalgues isolées en Nouvelle-Calédonie comme source d’antioxydants naturels. Une trentaine d’espèces de microalgues ont été isolées dans le lagon calédonien sur un critère de croissance par une méthode de dilution-exclusion. Un criblage de l’activité antioxydante a ensuite été réalisé sur douze de ces espèces, cultivées à deux intensités lumineuses (faible et forte) en utilisant quatre tests antioxydants différents : DPPH (2,2-diphenyl-1-picrylhydrazyl), TEAC (Trolox equivalent antioxidant capacity), ORAC (Oxygen Radical Absorbance Capacity) et TBARS (Thiobarbituric Acid Reactive Substances). Les résultats du criblage antioxydant ont montré l’importance d’utiliser plusieurs tests antioxydants et l’impact notable de l’intensité lumineuse sur l’activité antioxydante. Le criblage a également permis de mettre en évidence deux espèces d’intérêt Nephroselmis sp. et Tetraselmis sp. pour lesquelles l’influence de certains paramètres de culture sur l’activité antioxydante a été étudiée. Nephroselmis sp. a une forte capacité à neutraliser les radicaux peroxyles grâce à un contenu important en caroténoïdes, et contient un caroténoïde d’intérêt encore peu étudié, la siphonaxanthine. Il a été montré qu’il était nécessaire que la culture de Nephroselmis sp. ne soit pas limitée en azote pour favoriser la production de caroténoïdes antioxydants, en particulier la siphonaxanthine. Tetraselmis sp. a montré une forte capacité à inhiber la peroxydation des lipides. L’étude de l’influence du pH (6,5 et 8,5) et du mode de culture (batch vs continu) sur l’activité antioxydante et la photophysiologie de Tetraselmis sp. ont mis en évidence l’importance de ces deux paramètres sur l’activité antioxydante qui est optimisée lorsque la microalgue est dans un bon état physiologique. Ces résultats révèlent le potentiel des microalgues isolées en Nouvelle-Calédonie comme source d’antioxydants naturels, et l’impact important des conditions de culture sur l’activité antioxydante. La connaissance des différents mécanismes doit maintenant être approfondie sur des modèles cellulaires et l’origine des molécules impliquées doit être caractérisée par une approche métabolomique

Impact of Light Intensity on Antioxidant Activity of Tropical Microalgae

Twelve microalgae species isolated in tropical lagoons of New Caledonia were screened as a new source of antioxidants. Microalgae were cultivated at two light intensities to investigate their influence on antioxidant capacity. To assess antioxidant property of microalgae extracts, four assays with different modes of action were used: 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azino-bis (3-éthylbenzothiazoline-6-sulphonique) (ABTS), oxygen radical absorbance capacity (ORAC), and thiobabituric acid reactive substances (TBARS). This screening was coupled to pigment analysis to link antioxidant activity and carotenoid content. The results showed that none of the microalgae studied can scavenge DPPH and ABTS radicals, but Chaetoceros sp., Nephroselmis sp., and Nitzschia A sp. have the capacity to scavenge peroxyl radical (ORAC) and Tetraselmis sp., Nitzschia A sp., and Nephroselmis sp. can inhibit lipid peroxidation (TBARS). Carotenoid composition is typical of the studied microalgae and highlight the siphonaxanthin, detected in Nephroselmis sp., as a pigment of interest. It was found that xanthophylls were the major contributors to the peroxyl radical scavenging capacity measured with ORAC assay, but there was no link between carotenoids and inhibition of lipid peroxidation measured with TBARS assay. In addition, the results showed that light intensity has a strong influence on antioxidant capacity of microalgae: Overall, antioxidant activities measured with ORAC assay are better in high light intensity whereas antioxidant activities measured with TBARS assay are better in low light intensity. It suggests that different antioxidant compounds production is related to light intensity

Evaluation des microalgues sélectionnées en Nouvelle-Calédonie comme source potentielle d'anti-oxydants naturels : identification des molécules anti-radicalaires et stimulation de leur biosynthèse par orientation métabolique

The demand has increased for natural antioxidant as an alternative to synthetic antioxidant for food, cosmetic and nutraceutical industry. Most natural antioxidant currently available on the market are derived from terrestrial plant. However, microalgae, with their chemodiversity, productivity and controlled cultivation possibilities, are a promising source of antioxidant. The objective of this thesis is to evaluate the potential of microalgae isolated in New Caledonia as a source of natural antioxidants. About thirty species of microalgae were isolated in the caledonian lagoon for their high growth rate by a dilution-exclusion method. A screening of antioxidant activity was then carried out on twelve species, grown at two light intensities (low and high), using four different antioxidant assays: DPPH (2,2-diphenyl-1-picrylhydrazyl), TEAC (Trolox equivalent antioxidant capacity), ORAC (Oxygen Radical Absorbance Capacity) and TBARS (Thiobarbituric Acid Reactive Substances). The results of the antioxidant screening showed the need to use several antioxidant assays and the significant impact of light intensity on antioxidant activity. The screening also highlighted two species of interest, Nephroselmis sp. and Tetraselmis sp.. The influence of some culture parameters on antioxidant activity was studied for these two species. Nephroselmis sp. has a strong capacity to scavenge peroxyl radicals owing to its high carotenoid content. In addition, this species has a carotenoid of interest that is still little studied, siphonaxanthin. It has been shown that the cultivation of Nephroselmis sp. should not be limited in nitrogen to promote the production of antioxidant carotenoids, in particular siphonaxanthin.Tetraselmis sp. has been shown to have a strong capacity to inhibit lipid peroxidation. The study of the influence of pH (6.5 and 8.5) and culture mode (batch vs. continuous) on the antioxidant activity and photophysiology of Tetraselmis sp. highlighted the importance of these two parameters on the antioxidant activity which is optimized when the microalgae is in a good physiological state. These results reveal the potential of microalgae isolated in New Caledonia as a source of natural antioxidants, and the high impact of culture conditions on antioxidant activity. Knowledge of the different mechanisms must now be deepened on cellular models and the origin of the molecules involved must be characterized by a metabolomic approach.Une demande croissante d’antioxydants naturels alternatifs aux antioxydants synthétiques a émergé dans l’industrie agroalimentaire, cosmétique et nutraceutique. La majorité des antioxydants naturels actuellement disponibles sur le marché sont issus des végétaux terrestres, cependant les microalgues par leur chimiodiversité, leur productivité et les possibilités de culture contrôlée offrent un grand potentiel. L’objectif de cette thèse est d’évaluer le potentiel de microalgues isolées en Nouvelle-Calédonie comme source d’antioxydants naturels. Une trentaine d’espèces de microalgues ont été isolées dans le lagon calédonien sur un critère de croissance par une méthode de dilution-exclusion. Un criblage de l’activité antioxydante a ensuite été réalisé sur douze de ces espèces, cultivées à deux intensités lumineuses (faible et forte) en utilisant quatre tests antioxydants différents : DPPH (2,2-diphenyl-1-picrylhydrazyl), TEAC (Trolox equivalent antioxidant capacity), ORAC (Oxygen Radical Absorbance Capacity) et TBARS (Thiobarbituric Acid Reactive Substances). Les résultats du criblage antioxydant ont montré l’importance d’utiliser plusieurs tests antioxydants et l’impact notable de l’intensité lumineuse sur l’activité antioxydante. Le criblage a également permis de mettre en évidence deux espèces d’intérêt Nephroselmis sp. et Tetraselmis sp. pour lesquelles l’influence de certains paramètres de culture sur l’activité antioxydante a été étudiée. Nephroselmis sp. a une forte capacité à neutraliser les radicaux peroxyles grâce à un contenu important en caroténoïdes, et contient un caroténoïde d’intérêt encore peu étudié, la siphonaxanthine. Il a été montré qu’il était nécessaire que la culture de Nephroselmis sp. ne soit pas limitée en azote pour favoriser la production de caroténoïdes antioxydants, en particulier la siphonaxanthine.Tetraselmis sp. a montré une forte capacité à inhiber la peroxydation des lipides. L’étude de l’influence du pH (6,5 et 8,5) et du mode de culture (batch vs continu) sur l’activité antioxydante et la photophysiologie de Tetraselmis sp. ont mis en évidence l’importance de ces deux paramètres sur l’activité antioxydante qui est optimisée lorsque la microalgue est dans un bon état physiologique. Ces résultats révèlent le potentiel des microalgues isolées en Nouvelle-Calédonie comme source d’antioxydants naturels, et l’impact important des conditions de culture sur l’activité antioxydante. La connaissance des différents mécanismes doit maintenant être approfondie sur des modèles cellulaires et l’origine des molécules impliquées doit être caractérisée par une approche métabolomique

Antioxidant Compounds from Microalgae: A Review

The demand for natural products isolated from microalgae has increased over the last decade and has drawn the attention from the food, cosmetic and nutraceutical industries. Among these natural products, the demand for natural antioxidants as an alternative to synthetic antioxidants has increased. In addition, microalgae combine several advantages for the development of biotechnological applications: high biodiversity, photosynthetic yield, growth, productivity and a metabolic plasticity that can be orientated using culture conditions. Regarding the wide diversity of antioxidant compounds and mode of action combined with the diversity of reactive oxygen species (ROS), this review covers a brief presentation of antioxidant molecules with their role and mode of action, to summarize and evaluate common and recent assays used to assess antioxidant activity of microalgae. The aim is to improve our ability to choose the right assay to assess microalgae antioxidant activity regarding the antioxidant molecules studied

Impact of Light Intensity on Antioxidant Activity of Tropical Microalgae

Twelve microalgae species isolated in tropical lagoons of New Caledonia were screened as a new source of antioxidants. Microalgae were cultivated at two light intensities to investigate their influence on antioxidant capacity. To assess antioxidant property of microalgae extracts, four assays with different modes of action were used: 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azinobis (3-éthylbenzothiazoline-6-sulphonique) (ABTS), oxygen radical absorbance capacity (ORAC), and thiobabituric acid reactive substances (TBARS). This screening was coupled to pigment analysis to link antioxidant activity and carotenoid content. The results showed that none of the microalgae studied can scavenge DPPH and ABTS radicals, but Chaetoceros sp., Nephroselmis sp., and Nitzschia A sp. have the capacity to scavenge peroxyl radical (ORAC) and Tetraselmis sp., Nitzschia A sp., and Nephroselmis sp. can inhibit lipid peroxidation (TBARS). Carotenoid composition is typical of the studied microalgae and highlight the siphonaxanthin, detected in Nephroselmis sp., as a pigment of interest. It was found that xanthophylls were the major contributors to the peroxyl radical scavenging capacity measured with ORAC assay, but there was no link between carotenoids and inhibition of lipid peroxidation measured with TBARS assay. In addition, the results showed that light intensity has a strong influence on antioxidant capacity of microalgae: Overall, antioxidant activities measured with ORAC assay are better in high light intensity whereas antioxidant activities measured with TBARS assay are better in low light intensity. It suggests that different antioxidant compounds production is related to light intensity

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

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

Full-scale validation of an algal productivity model including nitrogen limitation

International audienceAccurate predictions of algal productivity under nutrient-limiting conditions are needed to assess the economics of full-scale algal cultivation for the developing markets of food, feed, and at longer term, green chemistry and biofuel. In particular, predicting pigments production from micro-algae is a critical milestone in the assessments of high-value chemicals production from micro-algae. This study validates a mathematical model predicting algal biomass productivity in outdoor raceway ponds under nitrogen-limiting conditions. The model was first validated from experimental data collected during Dunaliella salina cultivation in indoor photobioreactors and accounts for the impact of light, temperature, and nitrogen concentration on algal productivity (overall accuracy on algal concentration of ±2.7 mg L−1, N = 48). The model was then validated against data collected in outdoor raceway ponds over a period of 2 years, representing a total of 111 days of cultivation. Biomass and extracellular nitrogen concentrations predictions were accurate within ±0.055 g L−1 (N = 69) and ±0.0024 g L−1 (N = 26), respectively. Model inaccuracies were mostly due to measurement errors and uncertainties on model inputs. Measured carotenoids concentrations were found proportional to the biomass concentrations in the outdoor raceway ponds. By coupling this linear correlation to the productivity model, predicted carotenoids concentrations were in good agreement with experimental data (accuracy within ±0.0046 g L−1, N = 55). The mathematical model developed in this study has therefore the potential to refine previous assessments of algal cultivation for biofuels and pigments production

Targeted and non-targeted metabolomic analyses of Heterocapsa cf. bohaiensis under nickel and iron high concentration and its photophysiology

International audienceMicroalgae are able to adapt and to synthesize valuable compounds in response to abiotic stresses such as temperature, UV-radiation or metallic trace elements. We studied this faculty of adaptation by assessing the impact of high metal concentrations on Heterocapsa cf. bohaiensis, a newly isolated dinoflagellate from the New Caledonian coastal water rich in metals. We cultivated H. cf. bohaiensis in continuous culture mode using 10L photobioreactors and exposed the algae to high concentrations (10-3M) of nickel (Ni2+) and/or iron (Fe2+). We then followed its photosynthetic efficiency using Pulse Amplitude Modulated chlorophyll fluorometry and its metabolome with targeted (HPLC-UV-DAD, GC-MS) and non-targeted (LC-MS2 and NMR) approaches. The photosynthetic measurements indicate that H. cf. bohaiensis is tolerant to Ni2+ but sensitive to Fe2+ high concentrations. In presence of Fe2+, Fv/Fm and rETRm decreased from 0.62 to 0.47 and from 156 to 102, respectively. The JIP-tests (i.e. the fast chlorophyll fluorescence transient) suggest that the reduction of photosynthesis in response to Fe2+ is due to a disruption in the electron transport chain rather than a defect in the PSII light absorption and trapping which are on the contrary enhanced by Fe2+. The metabolomics analyses highlight the variation of the main dinoflagellate pigments, Chlorophyll c and a, peridinin, diadinoxanthin and diatoxanthin in response to time and metals and give a first glimpse on the complex metabolomic response to metal stress. These results bring new knowledge on this species and on the impact of nickel and iron on the microalgae photosynthetic pathway and metabolomic responses

A method to assess algicidal activity of microalgal extracts coupling microalgae produced in stirred closed photobioreactor operating in continuous with pulse amplitude modulated (PAM) fluorometry

We describe in the present study a quick and reliable method based on chlorophyll a fluorescence to assess putative algicidal effect of different microalgal extracts. We couple microalgal production under chemostat cultivation mode to continuously produce a given microalgae species (e.g. Dunaliella salina in this study) at a stable physiological state to ease comparison between extracts tested; with a non-destructive method based on chlorophyll a fluorescence. Pulse amplitude modulated (PAM) fluorometry was used to assess over time the effect of different microalgal crude extracts on the efficiency of the photosystem II (PSII) of a tested microalgae (Dunaliella salina). • Microalgal production at stationary phase in stirred closed photobioreactor (PBR) operating in continuous have stable photophysiological parameters, which is a prerequisite to compare the impact of different algicidal compounds. • The combination of both methods, allows to quickly assess the algicidal effect of diverse microalgal (crude) extracts on the PSII efficiency of a tested microalgae. • The method may be used to identify and isolate algicidal molecules affecting algal PSII using a bio-guided isolation protocol

Targeted and non-targeted metabolomic analyses of Heterocapsa cf. bohaiensis under nickel and iron high concentration and its photophysiology

International audienceMicroalgae are able to adapt and to synthesize valuable compounds in response to abiotic stresses such as temperature, UV-radiation or metallic trace elements. We studied this faculty of adaptation by assessing the impact of high metal concentrations on Heterocapsa cf. bohaiensis, a newly isolated dinoflagellate from the New Caledonian coastal water rich in metals. We cultivated H. cf. bohaiensis in continuous culture mode using 10L photobioreactors and exposed the algae to high concentrations (10-3M) of nickel (Ni2+) and/or iron (Fe2+). We then followed its photosynthetic efficiency using Pulse Amplitude Modulated chlorophyll fluorometry and its metabolome with targeted (HPLC-UV-DAD, GC-MS) and non-targeted (LC-MS2 and NMR) approaches. The photosynthetic measurements indicate that H. cf. bohaiensis is tolerant to Ni2+ but sensitive to Fe2+ high concentrations. In presence of Fe2+, Fv/Fm and rETRm decreased from 0.62 to 0.47 and from 156 to 102, respectively. The JIP-tests (i.e. the fast chlorophyll fluorescence transient) suggest that the reduction of photosynthesis in response to Fe2+ is due to a disruption in the electron transport chain rather than a defect in the PSII light absorption and trapping which are on the contrary enhanced by Fe2+. The metabolomics analyses highlight the variation of the main dinoflagellate pigments, Chlorophyll c and a, peridinin, diadinoxanthin and diatoxanthin in response to time and metals and give a first glimpse on the complex metabolomic response to metal stress. These results bring new knowledge on this species and on the impact of nickel and iron on the microalgae photosynthetic pathway and metabolomic responses