Abstract
The marine protist Schizochytrium limacinum SR21 is a promising producer of docosahexaenoic acid (DHA) from short-chain carboxylic acids (SCCAs). Dark fermentation effluent (DFE) is an SCCA- and nutrient-rich substrate derived from biogenic residues and has previously been evaluated for microbial lipid production. However, its low carbon concentration limits volumetric productivity and causes substantial culture dilution when larger feed volumes are required. Membrane-based cell retention is an established process intensification strategy for low-concentration feed solutions, as it decouples hydraulic residence time from biomass residence time. However, the combination of DFE utilisation and cell retention for DHA production by S. limacinum has received little attention. In this study, repeated fed-batch and cell retention strategies were evaluated to overcome dilution limitations and enable intensified biomass and lipid production from DFE. Additional cultivations with defined SCCA mixtures showed preferential uptake of acetate and butyrate over propionate and lactate, which was also observed during DFE conversion. In repeated fed-batch operation, biomass output reached 7.5 g after 95 h. In contrast, a final biomass output of 33 g was achieved in cell retention mode, representing a 4.4-fold increase. This improvement was not associated with enhanced substrate conversion efficiency but resulted from retaining biomass while increasing DFE throughput. While applying cell retention, the specific palmitic acid concentration increased to 116 mg g⁻¹, while DHA remained comparatively stable at 57 mg g⁻¹. This resulted in a DHA titre of 1.9 g L⁻¹ compared with 0.24 g L⁻¹ in repeated fed-batch. Nitrogen and phosphate removal reached 58% and 87%, respectively. However, the relatively high nutrient content of DFE limited the establishment of strong nitrogen limitation. Overall, cell retention enabled process intensification with a total lipid space-time yield of 0.07 glipids L⁻¹ h⁻¹ and a DHA space-time yield of 19.7 mg L⁻¹ h⁻¹. The results demonstrate that cell retention is an effective strategy for valorising low-concentration DFE while operational boundaries for DFE-based DHA production are identified.