Scale-Up to Pilot of a Non-Axenic Culture of Thraustochytrids Using Digestate from Methanization as Nitrogen Source

Abstract

The production of non-fish based docosahexaenoic acid (DHA) for feed and food has become a critical need in our global context of over-fishing. The industrial-scale production of DHA-rich Thraustochytrids could be an alternative, if costs turned out to be competitive. In order to reduce production costs, this study addresses the feasibility of the non-axenic (non-sterile) cultivation of Aurantiochytrium mangrovei on industrial substrates (as nitrogen and mineral sources and glucose syrup as carbon and energy sources), and its scale-up from laboratory (250 mL) to 500 L cultures. Pilot-scale reactors were airlift cylinders. Batch and fed-batch cultures were tested. Cultures over 38 to 62 h achieved a dry cell weight productivity of 3.3 to 5.5 g.L^-1.day^-1, and a substrate to biomass yield of up to 0.3. DHA productivity ranged from 10 to 0.18 mg.L^-1.day^-1. Biomass productivity appears linearly related to oxygen transfer rate. Bacterial contamination of cultures was low enough to avoid impacts on fatty acid composition of the biomass. A specific work on microbial risks assessment (in supplementary files) showed that the biomass can be securely used as feed. However, to date, there is a law void in EU legislation regarding the recycling of nitrogen from digestate from animal waste for microalgae biomass and its usage in animal feed. Overall, the proposed process appears similar to the industrial yeast production process (non-axenic heterotrophic process, dissolved oxygen supply limiting growth, similar cell size). Such similarity could help in further industrial developments.

Keywords: Aurantiochytrium; DHA; PUFA; Thraustochytrids; digestate; oxygen; pilot; scale-up.

Figure 1

Biomass production kinetics of A. mangrovei cultivation in a modified YEP medium, with air bubbling 0.4 VVM. Volume of culture was 8 L before day 3, and 10 L from day 3 to day 10, due to addition of 2 L of a 500 g.L⁻¹ glucose solution on day 3. Three replicates, error bars represent S.D.

Figure 2

Cultures in bubbling and shaking 800 mL supplemented with digestates from three different sources and with 0.297 g.L⁻¹ NH₄Cl (final concentration) as control source of nitrogen (see Section 4.1.2 for detailed description of culture medium). Mean values (n = 3, error bars = S.D.). (A): biomass production kinetics; (B): glucose concentration in cultures; (C): relative lipid content in cells as measured by BODIPY-stained cell fluorescence; (D): NH₄ concentrations in cultures.

Figure 3

Biomass growth kinetics of A. mangrovei in 150 mL cultures in 500 mL shaken flasks. Digestate as % v/v. Mean values (n = 3, error bars = S.D.). Cell concentrations were estimated using O.D. measurement at 620 nm. Relationship between cell concentration and O.D. at 620 nm was previously established by the following equation: cell concentration = 1 × 10⁸ × (O.D. at 620 nm) − 1 × 10⁷ (R² = 0.98).

Figure 4

Effect of air bubbling systems on cultures (6 L) in non-axenic airlift cylinders and in an axenic carboy with air bubbling and 100 rpm shaking as control. (a): biomass production kinetics; (b): Cell concentration kinetics; (c): Dissolved oxygen concentration; (d): relative lipid content in cells as measured by BODIPY stained cell fluorescence.

Figure 5

Effect of medium supplementation on biomass production in 500 L reactor cultures (2 replicates). 1X-1, 1X-2: simple batch (no feeding); 1.3X-1, 1.3X-2: feeding with 30% of initial medium organic concentrate after 16 h; 2X-1, 2X-2: feeding with 100% of initial medium organic concentrate after 16 h.

Figure 6

Relative distribution of the main fatty acids from 14 cultures of 450–500 L (black bars are an average of 3 axenic cultures in carboys on modified YEP medium. Error bars = S.D). January A and January B are the fed batch cultures (+30% of the initial media; see Section 2.5 for details).

Figure 7

Biomass (DCW g.L⁻¹), glucose (g.L⁻¹) and nitrogen (g.L⁻¹) concentrations in 500L airlift fed-batch culture; 100% of initial organic substrate was added at 16 h. Mean (n = 2, error bars = S.D.).

Figure 8

Cellular protein (a) and total fatty acid (b) concentrations versus biomass concentration, from a data set of 14 kinetics in 450 and 500 L cultures and 3 kinetics in 260 L cultures, over a 6 month period.

Figure 9

Mean concentration (n = 2, error bars = S.D.) over a 2X-fed-batch kinetic in a 500 L culture, of neutral and polar fatty acids in biomass, and percentage (w/w) of neutral lipids over total lipids.