Simultaneous photoautotrophic production of DHA and EPA by Tisochrysis lutea and Microchloropsis salina in co-culture

Abstract

Marine microalgae have received much attention as a sustainable source of the two health beneficial omega-3-fatty acids docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5). However, photoautotrophic monocultures of microalgae can only produce either DHA or EPA enriched biomass. An alternative may be the photoautotrophic co-cultivation of Tisochrysis lutea as DHA-producer with Microchloropsis salina for simultaneous EPA production to obtain EPA- and DHA-rich microalgae biomass in a nutritionally balanced ratio. Photoautotrophic co-cultivation processes of T. lutea and M. salina were studied, applying scalable and fully controlled lab-scale gas-lift flat-plate photobioreactors with LED illumination for dynamic climate simulation of a repeated sunny summer day in Australia [day-night cycles of incident light (PAR) and temperature]. Monocultures of both marine microalgae were used as reference batch processes. Differences in the autofluorescence of both microalgae enabled the individual measurement, of cell distributions in co-culture, by flow cytometry. The co-cultivation of T. lutea and M. salina in artificial sea water with an inoculation ratio of 1:3 resulted in a balanced biomass production of both microalgae simultaneously with a DHA:EPA ratio of almost 1:1 (26 mg_DHA g_CDW^-1, and 23 mg_EPA g_CDW^-1, respectively) at harvest after depletion of the initially added fertilizer. Surprisingly, more microalgae biomass was produced within 8 days in co-cultivation with an increase in the cell dry weight (CDW) concentration by 31%, compared to the monocultures with the same amount of light and fertilizer. What is more, DHA-content of the microalgae biomass was enhanced by 33% in the co-culture, whereas EPA-content remained unchanged compared to the monocultures.

Keywords: Microchloropsis salina; Tisochrysis lutea; Co-cultivation; Docosahexaenoic acid (DHA); Eicosapentaenoic acid (EPA); Photoautotrophic microalgae.

Fig. 1

Solar photosynthetic photon flux density (PPFD) and air temperature. Data recorded in Newcastle, Australia, on January 19, 2018 by BSRN station no: 52. Data were provided by Duck (2018) (BSRN). All batch processes in flat-plate gas-lift photobioreactors were performed under these repeatedly applied conditions

Fig. 2

Histograms of three samples with A 100% M. salina, B 100% T. lutea and C mixed sample containing both algal strains at an OD₇₅₀ of 0.2. X-axis shows the intensity of the fluorescence signal excited by a red laser at 640 nm and detected using the 660/10 nm bandpass filter. Y-axis shows the cell counts of the algae strains

Fig. 3

Phototrophic batch processes with T. lutea (right, red circle) and M. salina (left, green circle) in flat-plate gas-lift photobioreactors under simulated climate conditions. A Cell dry weight concentration, B nitrate concentration, C DHA (red circle) or EPA (green circle) concentration, and D DHA (red circle) or EPA (green circle) content of total cell dry weight. The batch processes were operated at a working volume of 1.8 L, pH 8.0 and an initial nitrate supply of 1.8 g L⁻¹. Monocultures were inoculated at an OD₇₅₀ of 0.2

Fig. 4

Variation of inoculation ratios in phototrophic batch processes with T. lutea (red circle) and M. salina (green circle) in co-culture at constant light irradiance of 83 ± 17 µmol m⁻² s⁻¹ and a constant temperature of 25 °C in 200 mL bubble column reactors operated in parallel. A Inoculation ratio 1:1, B inoculation ratio 1:2, C inoculation ratio 1:3, D inoculation ratio 1:4 (T. lutea:M. salina). The batch processes were operated at a working volume of 200 mL and pH 8.0

Fig. 5

Phototrophic batch process with T. lutea (red circle) and M. salina (green circle) in co-culture with an inoculation ratio of 1:3 in flat-plate gas-lift photobioreactors under simulated climate conditions. A cell dry weight concentration of T. lutea, B cell dry weight concentration of M. salina, C total cell dry weight concentration, D nitrate concentration, E DHA concentration, F EPA concentration, G DHA content of total cell dry weight, H EPA content of total cell dry weight. The batch process was operated at a working volume of 1.8 L, pH 8.0 and an initial nitrate supply of 1.8 g L⁻¹. Data are expressed as a mean ± SD (n = 3)

Fig. 6

A Absorption spectra of monocultures of M. salina (green dashed lines) and T. lutea (red dashed lines) and of a co-culture (green dashed lines) of both microalgae after acid acetone extraction. B Absorption spectra of the pigments Chlorophyll a (dashed dots) and Fucoxanthin (dashed line) after acid acetone extraction