Sources of Carbon Dioxide
Carbon dioxide inside bioreactors for animal cell culture originates from biological and non‑biological sources.
Biological sources refer to carbon dioxide produced by cellular respiration.
Non‑biological sources include bicarbonate salts present in culture media, as well as carbon dioxide, bicarbonates or carbonates fed into the bioreactor to maintain constant pH. In continuous culture, continuous medium feed leads to elevated CO₂ from non‑biological sources within the bioreactor. By contrast, in batch or fed‑batch culture, CO₂ is mainly generated via cellular respiration.
Causes of Carbon Dioxide Accumulation in Cultivation
Carbon dioxide accumulation is a prominent challenge in large‑scale high‑density animal cell culture, which can be summarized as follows:
First, elevated cell density increases the amount of CO₂ produced by cellular respiration per unit volume. Meanwhile, shifts in glucose and lactate metabolism occur alongside variations in cell viability throughout cultivation. Increased energy demand for product biosynthesis drives complete oxidation of glucose and lactate in the tricarboxylic acid (TCA) cycle, generating larger quantities of carbon dioxide.
Second, as bioreactor scale‑up proceeds, fluid shear stress increases under identical operating conditions. Cells are prone to damage caused by bubbles and foaming. Consequently, lower agitation speed and aeration rate are adopted, which reduce the CO₂ removal rate.
Third, microspargers are widely implemented to meet oxygen demand in large‑scale high‑density animal cell culture. Microspargers produce fine bubbles to expand gas‑liquid interfacial area and enhance oxygen mass‑transfer coefficient. Nevertheless, enhanced oxygen dissolution reduces bubble size, which in turn lowers the CO₂ stripping rate and results in CO₂ accumulation.
Effects of Carbon Dioxide Accumulation on Cells
Accumulated carbon dioxide readily transfers across gas‑liquid interfaces and cell membranes, and dissociates into HCO₃⁻ and H⁺ in aqueous solution. To stabilize pH, alkaline solution is dosed into the bioreactor, raising the osmotic pressure of the culture environment. Elevated osmotic pressure alters the exchange activity of cell membrane and increases intracellular ion concentration, which may exert adverse impacts on intracellular enzyme activities.
Research indicates that another mechanism of CO₂ action is intracellular pH reduction, which inhibits certain enzyme activities and impairs cell growth, metabolism and product formation. Experimental observations are listed below:
- Maximum specific product formation rate is achieved at pCO₂ of 30‑70 mmHg;
- Both cell growth rate and product titer decline when pCO₂ reaches 105 mmHg;
- When pCO₂ rises from 38 mmHg to 145 mmHg, the specific growth rate of CHO cells decreases by 50 %, and maximum viable cell density drops substantially;
- Incubator‑scale experiments show cells fail to grow at pCO₂ = 0 mmHg, whereas normal growth is observed at 30 mmHg.
Elevated pCO₂ reduces the specific consumption rates of glucose and glutamine as well as specific lactate production rate in CHO cell culture, while no significant change is observed in specific ammonia generation rate.
Apart from cell growth and metabolism, high pCO₂ compromises product expression. Kimura et al. reported that increased pCO₂ suppressed the specific t‑PA production rate of CHO cells. As pCO₂ increased from 36 mmHg to 140 mmHg and 190 mmHg, Qp decreased by 10 % and 33 %, respectively.
Carbon Dioxide Stripping in Bioreactors
For high‑density cultivation of CHO and BHK cells, CO₂ accumulation can be mitigated by minimizing non‑biological CO₂ input. Na₂CO₃ or NaOH can replace NaHCO₃ for pH control. Alternative buffering agents such as histidine and MOPS can substitute sodium bicarbonate buffer. For instance, a MOPS‑histidine buffer system with Na₂CO₃ for pH adjustment lowered pCO₂ from 230 mmHg to 68‑85 mmHg in BHK bioreactor cultures.
Bubble stripping is a conventional approach relying on gas‑liquid concentration gradient for CO₂ mass transfer. Stripping efficiency is governed by bubble size and aeration rate. Large bubbles favor CO₂ removal yet are disadvantageous for oxygen supply, while microbubbles (0.1‑0.25 mm) exhibit the opposite performance. Bubble diameter must be optimized to balance both requirements. For example, increasing bubble diameter from 2 μm to 2‑3 mm with aeration rate up to 0.006 vvm in a 500 L bioreactor, and replacing microspargers with open‑pipe spargers in a 1000 L bioreactor, have effectively decreased pCO₂.
Current mitigation strategies focus on agitation and aeration optimization. Trade‑offs must be made between shear‑sensitive cells and mass‑transfer requirements. Moreover, oxygen supply and CO₂ stripping have conflicting bubble‑size dependencies, adding complexity to mass‑transfer operations. Satisfying oxygen demand and preventing CO₂ build‑up without inflicting shear‑related cell damage remains a persistent technical challenge.
