Astaxanthin and eicosapentaenoic acid production by S4, a new mutant strain of Nannochloropsis gaditana

Abstract

Background: Astaxanthin is a ketocarotenoid with high antioxidant power used in different fields as healthcare, food/feed supplementation and as pigmenting agent in aquaculture. Primary producers of astaxanthin are some species of microalgae, unicellular photosynthetic organisms, as Haematococcus lacustris. Astaxanthin production by cultivation of Haematococcus lacustris is costly due to low biomass productivity, high risk of contamination and the requirement of downstream extraction processes, causing an extremely high price on the market. Some microalgae species are also primary producers of omega-3 fatty acids, essential nutrients for humans, being related to cardiovascular wellness, and required for visual and cognitive development. One of the main well-known producers of omega-3 fatty eicosapentaenoic acid (EPA) is the marine microalga Nannochloropsis gaditana (named also Microchloropsis gaditana): this species has been already approved by the Food and Drug Administration (FDA) for human consumption and it is characterized by a fast grow phenotype.

Results: Here we obtained by chemical mutagenesis a Nannochloropsis gaditana mutant strain, called S4, characterized by increased carotenoid to chlorophyll ratio. S4 strain showed improved photosynthetic activity, increased lipid productivity and increased ketocarotenoids accumulation, producing not only canthaxanthin but also astaxanthin, usually found only in traces in the WT strain. Ketocarotenoids produced in S4 strain were extractible in different organic solvents, with the highest efficiency observed upon microwaves pre-treatment followed by methanol extraction. By cultivation of S4 strain at different irradiances it was possible to produce up to 1.3 and 5.2 mgL^-1 day^-1 of ketocarotenoids and EPA respectively, in a single cultivation phase, even in absence of stressing conditions. Genome sequencing of S4 strain allowed to identify 199 single nucleotide polymorphisms (SNP): among the mutated genes, mutations in a carotenoid oxygenase gene and in a glutamate synthase gene could explain the different carotenoids content and the lower chlorophylls content, respectively.

Conclusions: By chemical mutagenesis and selection of strain with increased carotenoids to chlorophyll ratio it was possible to isolate a new Nannochloropsis gaditana strain, called S4 strain, characterized by increased lipids and ketocarotenoids accumulation. S4 strain can thus be considered as novel platform for ketocarotenoids and EPA production for different industrial applications.

Keywords: Astaxanthin; Carotenoids; Eicosapentaenoic acid; Microalgae; Omega-3 fatty acids.

Fig. 1

Screening of Nannochloropsis gaditana strains with increased carotenoids per chlorophyll content. a 500/680 nm absorption ratio of strains previoulsy selected for having a visible orange phenotype. The 500/680 nm absoprtion ratio meaured were normalized as a percentage to average 500/680 nm value calculated considering all strains analyzed. The strains with 500/680 nm absorption ratio increased by at least 10% compared to the average were further analyzed and are indicated with arrows. b Carotenoids per chlorophylls content of selected mutant strains compared to WT analyzed by HPLC. c Microscopy images of WT and S4 cells. d Average diameter and area of WT and S4 strains Error bars indicate standard deviation (n = 3 for panel b, n = 75 for panel d. Significant different values are marked with an asterisk as determined by unpaired two-sample t-test (p < 0.05)

Fig. 2

ECS, de-epoxidation index, NPQ and chlorophylls bleaching of S4 strain compared to WT. a Proton motive force (pmf) obtained by electrochromic shift measurement (ECS) on whole cells at 1000 µmol photons m⁻² s⁻¹ and normalized to chlorophylls content. b De-epoxidation index of WT and S4 strains upon exposure to high light (2500 µmol photons m⁻² s⁻¹) for 1 h. Pigment composition was evaluated at different time points by HPLC analysis and de-epoxidation index was calculated as (zeaxanthin + antheraxanthin/2) /(antheraxanthin + violaxanthin + zeaxanthin). c Non-photochemical quenching (NPQ) induction of WT and S4 mutant strain at actinic light of 1500 µmol photons m⁻² s⁻¹. After 10 min of illumination actinic light was turned off to induce NPQ relaxation. d Chlorophylls bleaching kinetics: absorption of chlorophylls was measured for about 14 h upon exposure of cells to 2500 µmol photons m⁻² s⁻¹. Experimental data were fitted with linear function. Error bars are reported as standard deviation (n = 3). Significant different values are marked with ** as determined by unpaired two-sample t-test (n = 3, p < 0.01)

Fig. 3

a Net oxygen evolution rate at different light intensities for WT and S4 mutant strains normalized on a chlorophyll basis. Experimental data were fitted with hyperbolic function. b Net oxygen evolution rate normalized on a cell basis. c Dark respiration rate normalized on a cell basis for WT and S4 mutant strains. Error bars showed the standard deviation (n = 3). Significant different values are marked with ** as determined by unpaired two-sample t-test (n = 3, p < 0.01)

Fig. 4

Growth curve, biomass, and lipid productivity of WT and S4 mutant strain. a Growth curve obtained measuring absorption at 720 nm. b Daily biomass productivity expressed as g L⁻¹ day⁻¹. c Lipid accumulation per dry weight measured by Nile red staining in cells at the end of the growth curve. Error bars are reported as the standard deviation (n = 3). Significant different values are marked with ** as determined by unpaired two-sample t-test (n = 3, p < 0.01)

Fig. 5

FAME composition of N. gaditana S4 strain. FAME composition of S4 strain is reported as percentage of total FAME for cells grown in absence (a) or presence (b) of 10% glucose (Glu) in the growth medium. The different irradiances of growth, from 100 to 2000 µmol m⁻² s⁻¹, are reported

Fig. 6

Correlation between EPA and ketocarotenoids content. The dark squares refer to a growth medium without glucose, instead grey dot refer to a growth medium with 10 g L⁻¹ of glucose. Error bars showed the standard deviation (n = 4)