Exoelectrogenesis response of Chlamydomonas reinhardtii in biophotovoltaic cells under iron and nitrogen deficiencies

The green microalga Chlamydomonas reinhardtii is a model organism for bioelectrogenic studies under nutrient stress, yet its exoelectrogenic behavior under iron and nitrogen deprivation remains underexplored. In this study, C. reinhardtii strain cw15 (cell wall deficient) was cultivated under nine defined media conditions with varying concentrations of FeCl₃ (0.02, 0.002, and 0 mM) and NH₄Cl (7.48, 3.74, and 0.374 mM). After four days of growth, cells were transferred to custom-built, single-chamber air-cathode biophotovoltaic (BPV) devices to evaluate voltage generation in the presence and absence of external electron mediators, including ferricyanide (FeCN), phenazine methosulfate (PMS), and their mixture. In the presence of FeCN the highest closed-circuit voltage (57 mV) was observed under iron starvation (0.0 mM FeCl₃) with full nitrogen supply. In contrast, the PMS-FeCN mixture yielded the highest output (121 mV) under combined Fe and N limitation. Polarization and mediator assays confirmed that the greatest electrogenesis rate (970 μW.m⁻²), 2.5 times that of the control, occurred in cells under Fe deficiency with moderate N supply, in the absence of any external mediators. These findings were verified by sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) and ferricyanide reduction assays, suggesting enhanced electron release capacity under nutrient stress.