Plasma discharge intensity modulates cell death pathways in pulsed electric field-treated Chlorella vulgaris

Sequential physical treatments can produce cellular responses qualitatively different from individual applications, yet the principles governing such interactions remain poorly understood in microalgae. We investigated Chlorella vulgaris subjected to gliding arc discharge (GAD) plasma (supply voltage 130-250 V) followed by a fixed pulsed electric field (PEF; 25 kV/cm, 7 μs). Rather than dose-dependent enhancement, plasma intensity produced a bifurcation in the inactivation phenotype. At low plasma intensity (130 V), GAD+PEF was indistinguishable from PEF alone, yielding extensive macromolecule release (protein 20-24% of dry biomass; DNA >20 mg/L), complete metabolic inactivation, and cell disintegration consistent with PEF-mediated programmed cell death. At high plasma intensity (≥210 V), GAD+PEF caused high membrane permeabilization (>70%) but strongly suppressed macromolecule release (<6% protein; <5% DNA), reduced caspase-3-like activity, and preserved cellular architecture for 72 h, while still fully inactivating metabolism. High-intensity treatment increased free amino acid release, suggesting non-caspase proteolysis. Control experiments showed that secondary plasma effects (increased temperature, conductivity, and accumulation of long-lived H₂O₂/NO₂^-/NO₃^-) did not account for the bifurcation, implicating direct plasma-cell interactions or short-lived species. These findings show that plasma preconditioning intensity can redirect PEF-triggered cell-death responses in microalgae, enabling membrane permeabilization without structural collapse.