Lipid production in association of filamentous fungi with genetically modified cyanobacterial cells

Abstract

Background: Numerous strategies have evolved recently for the generation of genetically modified or synthetic microalgae and cyanobacteria designed for production of ethanol, biodiesel and other fuels. In spite of their obvious attractiveness there are still a number of challenges that can affect their economic viability: the high costs associated with (1) harvesting, which can account for up to 50 % of the total biofuel's cost, (2) nutrients supply and (3) oil extraction. Fungal-assisted bio-flocculation of microalgae is gaining increasing attention due to its high efficiency, no need for added chemicals and low energy inputs. The implementation of renewable alternative carbon, nitrogen and phosphorus sources from agricultural wastes and wastewaters for growing algae and fungi makes this strategy economically attractive.

Results: This work demonstrates that the filamentous fungi, Aspergillus fumigatus can efficiently flocculate the unicellular cyanobacteria Synechocystis PCC 6803 and its genetically modified derivatives that have been altered to enable secretion of free fatty acids into growth media. Secreted free fatty acids are potentially used by fungal cells as a carbon source for growth and ex-novo production of lipids. For most of genetically modified strains the total lipid yields extracted from the fungal-cyanobacterial pellets were found to be higher than additive yields of lipids and total free fatty acids produced by fungal and Synechocystis components when grown in mono-cultures. The synergistic effect observed in fungal-Synechocystis associations was also found in bioremediation rates when animal husbandry wastewater was used an alternative source of nitrogen and phosphorus.

Conclusion: Fungal assisted flocculation can complement and assist in large scale biofuel production from wild-type and genetically modified Synechocystis PCC 6803 strains by (1) efficient harvesting of cyanobacterial cells and (2) producing of high yields of lipids accumulated in fungal-cyanobacterial pellets.

Keywords: Biofuel; Bioremediation; Flocculation; Fungi; Genetic modification; Renewable energy; Synechocystis PCC 6803; Wastewater treatment.

Fig. 1

Flocculation efficiency of Synechocystis PCC 6803 strains by A. fumigatus. Flocculation of Synechocystis cells by A. fumigatus/GLU (a), A. fumigatus/NEC (b) and A. fumigatus/TWS pellets (c). Flocculation efficiency of A. fumigatus with all SD strains showed significance levels, p < 0.01

Fig. 2

Flocculation of Synechocystis PCC 6803 cells by A. fumigatus. a SD100 culture mixed with A. fumigatus/TWS and A. fumigatus/GLU pellets, time = 0; Flocculation of SD100 (b), SD216 (c) and SD232 (d) cells with A. fumigatus/TWS and A. fumigatus/GLU pellets (t = 24 h); e Flocculation of SD277 with A. fumigatus/TWS and A. fumigatus/GLU pellets, t = 0; vertical bars show the levels of A. fumigatus pellets in SD277 culture; f same after 24 h; g, h A. fumigatus pellets before and after mixing with SD100 cells, respectively; i SD256 grown for 5 days under reduced CO₂ conditions and mixed with A. fumigatus/TWS and A. fumigatus/GLU pellets, t = 0; j same after 24 h. In a–f, i–j A. fumigatus/TWS pellets were shown on left and A. fumigatus/GLU on right. Scale 5 mm

Fig. 3

Microscopic analysis of A. fumigatus-Synechocystis associations. a, b A. fumigatus-SD100 pellets; c, d A. fumigatus-SD100 and A. fumigatus-SD216; e, f SD100 cells attached to A. fumigatus filaments; g, h A. fumigatus/GLU filaments stained with Nile red; i A. fumigatus/TWS filaments stained with Nile red; j FFA droplets secreted into growth media by SD277 growing in mono-culture; k–m FFA droplets attached to A. fumigatus filaments after mixing with SD232 (k) and SD277 (l, m) cultures at t = 0. Red colour is the autofluorescence of cyanobacterial phycobilisomes. Scales a, b = 1 mm; c–m = 20 µm

Fig. 4

Fatty acids composition of lipids in A. fumigatus, SD strains and A. fumigatus-SD pellets. 1 A. fumigatus/TWS; 2 A. fumigatus/GLU; 3 SD strains; 4 A. fumigatus/TWS-SD pellets; 5 A. fumigatus/GLU-SD pellets

Fig. 5

Composition of FFA secreted from A. fumigatus, SD strains and A. fumigatus-SD pellets. a SD strains; b A. fumigatus/TWS-SD pellets; c A. fumigatus/GLU-SD pellets

Fig. 6

Biomass and lipid production in A. fumigatus-SD100 pellets grown in 25 % swine wastewater. Af-SD100: A. fumigatus-SD100 pellets. Significance levels: *p < 0.05