Physiological and Spectral Response of Broccoli Microgreens under Living and Dead Microalgae Biofertilizer Treatments in a Controlled Plant Factory

Abstract

Microalgae are promising biofertilizers for controlled plant factory systems, but the role of biomass viability in microgreen responses remains unclear. This study evaluated broccoli microgreens (Brassica oleracea) treated with three microalgae species (Nannochloropsis sp., Spirulina sp., and Chlorella sp.) applied as living suspensions or dead powders at 5% and 10% (v/v for living suspensions; w/v for dead powders), with or without AB Mix nutrient solution. Twenty-four treatment units were assessed at 14 days after sowing using proximal hyperspectral imaging (Specim IQ, 397-1004 nm), chlorophyll content (SPAD), and photosynthetic efficiency (Fv/Fm). Vegetation indices were extracted for statistical screening, and five primary indices (NDVI, GNDVI, PRI, CRI, and ARI) were reported in detail. Living and dead treatments were compared using Mann-Whitney U and Welch’s t-tests with Bonferroni correction. NDVI, PRI, and CRI significantly differentiated living from dead treatments (p < 0.006). PRI showed the largest effect size among the primary indices (|d| = 1.61) and strongly correlated with Fv/Fm (ρ = 0.832, p < 0.001). Living treatments maintained higher Fv/Fm than dead treatments (0.791 vs. 0.663), whereas SPAD differed minimally. Species-level analysis showed significant variation in four of five primary indices, with dead-form Nannochloropsis sp. showing the most suppressed spectral profile. These findings support PRI-based hyperspectral imaging as a non-destructive tool for monitoring microgreen physiological status in plant factory systems.