Microalgal extracts optimize lettuce quality through extraction-method-dependent modulation of primary and secondary metabolism

Microalgal extracts are rich in bioactive molecules capable of modulating crop quality and stress resilience. This study investigated the effects of crude and formulated extracts based on three extraction methods—acidic, alkaline, and enzymatic—from Nannochloropsis gaditana and Arthrospira maxima on the nutritional quality of lettuce (Lactuca sativa L.), focusing on primary and secondary metabolism. The extraction method significantly influenced the biochemical composition of the extracts. Alkaline extraction maximized protein yield (32.4% and 19.6% for N. gaditana and A. maxima, respectively), while enzymatic extraction enriched carbohydrate content (8.1–10.8%). Treatment with A. maxima enzymatic extracts significantly improved shoot (35.3%) and root (54.6%) biomass. Metabolomic analysis revealed species-specific metabolic reprogramming directly linked to nutritional quality. A. maxima functioned as a direct nutritional fortifier, significantly upregulating essential polyunsaturated fatty acids, including α-linolenic acid (an omega-3 fatty acid) by 47.2% and inducing the de novo accumulation of linoleic acid (an omega-6 fatty acid) at 10.99 µg.g⁻¹ FW. Crucially, A. maxima treatment uniquely induced the accumulation of γ-sitosterol at 10.07 µg.g⁻¹ FW, a phytosterol with documented cholesterol-lowering properties. This enhanced lipid and sterol profile was accompanied by a 31% increase in protein concentration and correlated with enhanced photosynthetic pigment content (chlorophyll a, chlorophyll b, and carotenoids). Conversely, N. gaditana induced a stress-adaptation profile, characterized by the accumulation of very-long-chain alkenes and alcohols, such as Nonacos-1-ene (5.84 µg.g⁻¹ FW) and 1-Pentacontanol (4.76 µg.g⁻¹ FW), associated with cuticular wax formation. This defensive shift occurred alongside a significant reduction in total sugars (-68%) and growth parameters, indicating a resource reallocation from growth and nutritional metabolism to structural defense. Our findings demonstrate that microalgal biostimulants optimize lettuce quality through extraction-method-dependent modulation of primary and secondary metabolism, with A. maxima enzymatic extracts showing the highest potential. This work provides a foundation for designing tailored algal biostimulants to address specific agronomic needs in sustainable horticulture.