High cell density cultivation by anaerobic respiration.

Background: Oxygen provision is a bottleneck in conventional aerobic high cell density culturing (HCDC) of bacteria due to the low O₂ solubility in water. An alternative could be denitrification: anaerobic respiration using nitrogen oxides as terminal electron acceptors. Denitrification is attractive because NO₃^- is soluble in water, the end-product (N₂) is harmless, and denitrification is widespread among bacteria, hence suitable organisms for most purposes can be found. The pH must be controlled by injection of an inorganic acid to compensate for the pH increase by NO₃^--consumption, resulting in salt accumulation if feeding the bioreactor with NO₃^- salt. We avoid this with our novel pH-stat approach, where the reactor is supplied with 5 M HNO₃ to compensate for the alkalization, thus sustaining NO₃^--concentration at a level determined by the pH setpoint. Here we present the first feasibility study of this method, growing the model strain Paracoccus denitrificans anaerobically to high densities with glucose as the sole C-source and NO₃^- as the N-source and electron acceptor.

Results: Our fed-batch culture reached 20 g cell dry weight L^-1, albeit with slower growth rates than observed in low cell density batch cultures. We explored reasons for slow growth, and the measured trace element uptake indicates it is not a limiting factor. Bioassays with spent medium excluded accumulation of inhibitory compounds at high cell density as the reason for the slow growth. The most plausible reason is that high metabolic activity led to CO₂/H₂CO₃ accumulation, thus suppressing pH, leading to a paucity in HNO₃-feeding until N₂-sparging had removed sufficient CO₂. The three free intermediates in the denitrification pathway (NO₃^- → NO₂^- → NO → N₂O → N₂) can all reach toxic concentrations if the electron flow is unbalanced, and this did occur if cells were glucose-limited. On the other hand, accumulation of polyhydroxyalkanoates occurred if the cells were NO₃^--limited. Carefully balancing glucose provision according to the HNO₃ injected is thus crucial.

Conclusions: This work provides a proof of concept, while also identifying CO₂/H₂CO₃ accumulation as a hurdle that must be overcome for further development and optimization of the method.