Imperatives for anaerobic high cell density culturing by denitrification: technical and physiological perspectives

Single-cell protein (SCP) is gaining attention as a source of food and feed, offering a lower environmental footprint than traditional agriculture. Efficient SCP production requires high cell density culturing (HCDC; >20 g cell dry weight L− 1), but O2 supply can become limiting in conventional aerobic systems. To circumvent this bottleneck, we recently proposed an anaerobic strategy using nitrate as an electron acceptor, exploiting denitrification-driven alkalinization in a pH-stat system to regulate substrate provision. Using the model organisms Paracoccus denitrificans and Paracoccus pantotrophus, we achieved biomass concentrations of up to 60 g dry weight L− 1 and protein contents up to 75% (75 ± 5%) in a 3 L fed-batch bioreactor. However, growth rates at high cell density were markedly lower than µmax observed in low-density cultures. In vivo and in silico experiments revealed three interacting constraints: (i) a CO2-pH lag where CO2 accumulation delayed alkalization by denitrification and thus substrate injection; (ii) mixing limitations, leading to poor substrate distribution; and (iii) physiological stress from nitrite accumulating during imbalanced denitrification. The CO2-pH lag emerged as the dominant barrier, resulting in long starvation periods. Lowering the pH setpoint of the pH-stat accelerated CO2 removal and thus substrate provision, but intensified nitrite toxicity. Insufficient mixing compounded the growth limitations as nitrate was only briefly available to a fraction of the population. Small-batch bioassays ruled out accumulation of inhibiting compounds other than nitrite. However, cells grown at high density in the reactor displayed reduced respiration rates, suggesting chronic stress under these conditions. Anaerobic HCDC by denitrification is feasible and yields high-quality biomass, but barriers remain to achieving competitive production rates. The CO2-pH lag appears to be the primary constraint, amplified by incomplete mixing and nitrite toxicity. These factors interact, e.g. mitigating the CO2-pH lag by lowering the process pH exacerbates nitrite toxicity. Future work should integrate reactor engineering to improve mixing and gas removal and strain selection for tolerance to low pH and nitrite, supported by omics and metabolic modelling to understand denitrifier physiology at high cell density.