Airlift bioreactor-based strategies for prolonged semi-continuous cultivation of edible Agaricomycetes

Abstract

Submerged cultivation of edible filamentous fungi (Agaricomycetes) in bioreactors enables maximum mass transfer of nutrients and has the potential to increase the volumetric productivity of fungal biomass compared to solid state cultivation. These aspects are paramount if one wants to increase the range of bioactives (e.g. glucans) in convenient time frames. In this study, Trametes versicolor (M9911) outperformed four other Agaricomycetes tested strains (during batch cultivations in an airlift bioreactor). This strain was therefore further tested in semi-continuous cultivation. Continuous and semi-continuous cultivations (driven by the dilution rate, D) are the preferred bioprocess strategies for biomass production. We examined the semi-continuous cultivation of T. versicolor at dilution rates between 0.02 and 0.1 h^-1. A maximum volumetric productivity of 0.87 g/L/h was obtained with a D of 0.1 h^-1 but with a lower total biomass production (cell dry weight, CDW 8.7 g/L) than the one obtained at lower dilution rates (12.3 g/L at D of 0.04 and vs 13.4 g/L, at a D of 0.02 h^-1). However, growth at a D of 0.1 h^-1 resulted in a very short fermentation (18 h) which terminated due to washout (the specific D exceeded the maximum growth rate of the fungal biomass). At a D of 0.04 h^-1, a CDW of 12.3 g/L was achieved without compromising the total residence time (184 h) of the fermentation. While the D of 0.04 h^-1 and 0.07 h^-1 achieved comparable volumetric productivities (0.5 g/L/h), the total duration of the fermentation at D of 0.07 h^-1 was only 85 h. The highest glucan content of cells (27.8 as percentage of CDW) was obtained at a D of 0.07 h^-1, while the lowest glucan content was observed in T. versicolor cells grown at a D of 0.02 h^-1. KEY POINTS: • The highest reported volumetric productivity for fungal biomass was 0.87 g/L/h. • Semi-continuous fermentation at D of 0.02 h-1 resulted in 13.4 g/L of fungal biomass. • Semi-continuous fermentation at D of 0.07 h-1 resulted in fungal biomass with 28% of total glucans.

Keywords: Airlift fermentation; Bioactives; Filamentous fungi; Volumetric productivity.

Fig. 1

Diagram picture of the Bionet® airlift bioreactor (Fuente Alamo de Murcia, Spain) equipped with an internal draught tube and a bottom stainless-steel ring sparger

Fig. 2

A Selected Agaricomycetes strains growth curves in YSMG media in shaken flask experiments at 25 °C and 150 RPM. B Selected Agaricomycetes strains growth curves in CSL media in shaken flask experiments at 25 °C and 150 RPM. Each set of cultures was grown in triplicate experiments

Fig. 3

A Fungal biomass in CSL media reductions. B β-glucans (as percentage of CDW) produced by fungal biomass in CSL media reductions. Baseline: glucose, G 10 g/L; yeast extract, YE 3 g/L; corn steep liquor, CSL 15 mL/L. G0 = same as baseline but no glucose; Y0 = same as baseline but no yeast extract; GY0 = same as baseline but no glucose and yeast extract; G/2 = same as baseline but glucose = 5 g/L); Y/2 = same as baseline but YE 1.5 g/L; GY/2 = same as baseline but halved glucose and yeast extract (5 and 1.5 g/L respectively); C0 = same as baseline but no CSL; C/2 = same as baseline but halved CSL (7.5 mL/L); CG/2 = same as baseline but halved CSL and glucose (7.5 mL and 5 g/L respectively). Each media condition was tested in triplicate experiments

Fig. 4

Glucose consumption and dissolved oxygen trend by the four selected fungal strains (A. blazei, H erinaceus, L. edodes and T. versicolor) in the airlift bioreactor in batch mode. Each plotted batch is an average of triplicate experiments

Fig. 5

(A1–3) Microscopic mycelial pellet of A. blazei, pelletised aggregation of A. blazei in the seeding flask, pelletised aggregation of A. blazei in the airlift bioreactor. (B1–3) Microscopic mycelial pellet of H. erinaceus, pelletised aggregation of H. erinaceus in the seeding flask, pelletised aggregation of H. erinaceus in the airlift bioreactor. (C1–3) Microscopic mycelial pellet of L. edodes, pelletised aggregation of L. edodes in the seeding flask, pelletised aggregation of L. edodes in the airlift bioreactor. (D1–3) Microscopic mycelial pellet of T. versicolor, pelletised aggregation of T. versicolor in the seeding flask, pelletised aggregation of T. versicolor in the airlift bioreactor. The bar size in Figure A1 is 100 μm; in Figure B1, it is 50 μm; and in Figures C1 and D1, it is 200 μm respectively.

Fig. 6

Average plot of triplicate semi-continuous fermentations targeting a dilution rate (D) of 0.02 h⁻¹ for the growth of T. versicolor after a batch phase of 48 h. A red vertical dotted line indicates the end of the batch phase and therefore the start of the semi-continuous feeding regime in the semi-continuous fermentations. A vertical black dashed line identifies the onset of macroaggregate pellet formation

Fig. 7

A Average plot of triplicate semi-continuous fermentations targeting a dilution rate (D) of 0.04 h⁻¹ for the growth of T. versicolor after a batch phase of 48 h. A vertical red dotted line identifies the start of the feeding regime, while the vertical black dashed line identifies the onset of macroaggregates pellets formation. B Evidence of mycelia aggregates in airlift bioreactor after a semi-continuous fermentation with a D of 0.04 h⁻¹

Fig. 8

Average plot of triplicate semi-continuous fermentations targeting a dilution rate (D) of 0.07 h⁻¹ for the growth of T. versicolor after a batch phase of 68 h. A vertical red dotted line identifies the start of the feeding regime. A vertical black dashed line identifies the onset of macroaggregate pellet formation