High cell density cultivation by anaerobic respiration

Area of Science:

• Biotechnology
• Microbial Culturing
• Anaerobic Respiration

Background:

• Oxygen limitation hinders conventional aerobic high cell density culturing (HCDC).
• Denitrification offers an alternative using nitrate (NO₃^-) as an electron acceptor.
• A novel pH-stat approach using nitric acid (HNO₃) avoids salt accumulation during denitrification.

Purpose of the Study:

• To assess the feasibility of a novel pH-stat denitrification method for bacterial HCDC.
• To cultivate the model strain Paracoccus denitrificans to high densities anaerobically.
• To identify challenges and optimize anaerobic high cell density culturing.

Main Methods:

• Fed-batch cultivation of Paracoccus denitrificans using glucose and nitrate.
• Employing a pH-stat system with nitric acid (HNO₃) for pH control.
• Monitoring cell dry weight, trace element uptake, and intermediate metabolite accumulation.

Main Results:

• Achieved 20 g cell dry weight L^-1 with slower growth rates compared to low-density cultures.
• Identified carbon dioxide (CO₂)/carbonic acid (H₂CO₃) accumulation as a primary cause for suppressed pH and limited HNO₃ feeding.
• Demonstrated that unbalanced electron flow can lead to toxic intermediate accumulation, while nutrient limitation causes polyhydroxyalkanoate accumulation.

Conclusions:

• The study presents a proof of concept for anaerobic HCDC via pH-stat denitrification.
• Carbon dioxide accumulation is a significant hurdle for method optimization.
• Careful balancing of glucose and nitric acid (HNO₃) is critical for efficient denitrification.