Oleaginous yeasts have attracted increasing scientific interest as single cell oil (SCO) producers. SCO can be used as a fossil-free fuel substitute, but also as a source of rarely found odd-chain fatty acids (OCFAs), such as C15, C17, and C25 fatty acids which have a wide range of nutritional and biological applications. Volatile fatty acids (VFAs) have gained interest as sustainable carbon source for yeasts. This study aims to improve current knowledge on yeast species that yield high amounts of SCO using VFAs as a carbon source. Specifically, the growth of the promising yeasts Cryptococcus curvatus and Rhodotorula toruloides was evaluated on individual VFAs, such as acetic, propionic, and butyric acid. C. curvatus proved to be more tolerant in higher concentrations of VFAs (up to 60 g/L), while butyric acid favored biomass and lipid conversion (0.65 and 0.23 g/gsubstrate, respectively). For R. toruloides, butyric acid favored biomass conversion (0.48 g/gsubstrate), but lipid conversion was favored using acetic acid, instead (0.14 g/gsubstrate). Propionic acid induced the formation of OCFAs, which yielded higher amounts for C. curvatus (up to 2.17 g/L). VFAs derived from the anaerobic digestion of brewer’s spent grain were tested as a cost-competitive carbon source and illustrated the significance of the combination of different VFAs in the quality of the produced SCO, by improving the biodiesel properties and OCFAs production.

To address the issue of high organic carbon costs in heterotrophic cultivation of microalgae, we evaluated the hypotheses by employing microalgae as a biorefinery for proteins and advanced biofuels after cultivation on volatile fatty acids (VFAs) instead of pure glucose. To prevent the inhibitory effect of VFAs on lipid synthesis, strains capable of tolerating high levels of VFAs were selected. Growth and lipid synthesis by two freshwater microalgae, Auxenochlorella protothecoides and Chlorella sorokiniana, was optimized at different VFA concentrations. Maximum biomass and lipid content in A. protothecoides (10.66 g/L, 33.93%) and C. sorokiniana (7.98 g/L, 39.80%) were obtained by replacing glucose with 30 g/L acetate at C/N 60. The generated lipids were compliant with existing standards for biodiesel. Moreover, when grown on acetate, both microalgae contained the complete range of essential and non-essential amino acids. Finally, single-source commercial VFAs were replaced with VFAs mixture after acidogenic fermentation of waste lignocellulosic biomass from brewers’ spent grain. The mixture allowed successful mixotrophic and heterotrophic cultivation of both microalgae, demonstrating feasibility of this low-cost carbon source in fuel-grade biodiesel production.

The focus on sustainability and circular economy renders the microalgal biorefinery concept highly attractive. Although the diversity of microalgal composition makes them ideal feedstocks, their metabolic versatility challenges bioprocess optimization. To address this, an integrated, strain-specific approach was used to evaluate key cultivation parameters (nitrogen source, C/N ratio, and light intensity) as their interactions affect growth performance and biochemical composition. Heterotrophic cultivation of A. protothecoides (AP) and C. sorokiniana (CS) in glucose showed enhanced cell growth with organic N-sources. Biomass was consistently elevated across C/N ratios from 5 to 60 with corn steep liquor (CSL) (8.1 g L-1) and yeast extract (YE) (7.0 g L-1), while with urea it maximized at C/N 5 (6.2 g L-1). Protein synthesis increased at C/N 5, whereas lipid accumulation at C/N 60. Beechwood hydrolysate, a renewable glucose alternative, produced an average of 4.1 g L-1 protein (C/N 5) and 3.5 g L-1 lipids (C/N 60) between YE and CSL. Mixotrophic cultivation indicated better photosynthetic adaptation of AP at C/N 5, yielding 13.2 g L-1 biomass at 400 μmol m-2 s-1, whereas at C/N 60 growth was favored at 50 μmol m-2 s-1. The fatty acid profile of microalgal oil revealed de novo biosynthesis of odd-chain fatty acids at C/N 5 in both cultivation modes, while biodiesel-grade lipids produced in heterotrophic condition. These findings advance microalgal bioprocessing by emphasizing the importance of fine-tuning cultivation strategies and utilizing renewable nutrients to maximize resource efficiency and optimize the biosynthesis of valuable bioproducts, such as proteins, pigments, carbohydrates, and high-quality lipids.

Background: Biotransformation of waste oil into value-added nutraceuticals provides a sustainable strategy. Thraustochytrids are heterotrophic marine protists and promising producers of omega (ω) fatty acids. Although the metabolic routes for the assimilation of hydrophilic carbon substrates such as glucose are known for these microbes, the mechanisms employed for the conversion of hydrophobic substrates are not well established. Here, thraustochytrid Schizochytrium limacinum SR21 was investigated for its ability to convert oils (commercial oils with varying fatty acid composition and waste cooking oil) into ω-3 fatty acid; docosahexaenoic acid (DHA).

Results: Within 72 h SR21 consumed ~ 90% of the oils resulting in enhanced biomass (7.5 g L− 1) which was 2-fold higher as compared to glucose. Statistical analysis highlights C16 fatty acids as important precursors of DHA biosynthesis. Transcriptomic data indicated the upregulation of multiple lipases, predicted to possess signal peptides for secretory, membrane-anchored and cytoplasmic localization. Additionally, transcripts encoding for mitochondrial and peroxisomal β-oxidation along with acyl-carnitine transporters were abundant for oil substrates that allowed complete degradation of fatty acids to acetyl CoA. Further, low levels of oxidative biomarkers (H2O2, malondialdehyde) and antioxidants were determined for hydrophobic substrates, suggesting that SR21 efficiently mitigates the metabolic load and diverts the acetyl CoA towards energy generation and DHA accumulation.

Conclusions: The findings of this study contribute to uncovering the route of assimilation of oil substrates by SR21. The thraustochytrid employs an intricate crosstalk among the extracellular and intracellular molecular machinery favoring energy generation. The conversion of hydrophobic substrates to DHA can be further improved using synthetic biology tools, thereby providing a unique platform for the sustainable recycling of waste oil substrates.