I suspect the problem is mechanical: bubbling gas in a liquid culture creates speed gradients and these disrupt cellular walls or somehow disturb the uptake of nutrients. I don't see why you need continuous sparging if your process is batch. Just flush once in order to eliminate O2. Some protocols recommend the volume of flush gas must be 5 times the headspace volume, in nearly 5 minutes. Once flushed, stir the reactor manually (shaking) or mechanically (motor stirrer) from time to time.

Thank you very much for your answer. I think that should be the problem. I will try your recommendation.

Hope it works. Rational criteria on how to adjust stirring, either mechanical or by gas bubbling, can be found in this book: https://www.crcpress.com/Managing-Biogas-Plants-A-Practical-Guide/Rosato/p/book/9781138626614. Good luck :-)

Ezgi, selection of the sparging mode (continuous or once) depends on your objectives. I use continuous sparging with pure N2 to monitor gases formed during anaerobic fermentation with mass-spectrometry (CO2, H2, CO, CH4 etc.). However the first day of cultivation, when you start the bioprocess with frozen cells as inoculum, it is advisable to stop N2 flow after initial purging and decrease the stirring/shaking degree. I prefer even to stop stirring completely until l see some signs of growth, e.g. release of CO2 or H2 or metabolic acidification (pH drop activating base titration). The reason is that anaerobic microorganisms are very sensitive to traces of oxygen (even the best quality ultra-pure N2 contain 100-300 ppm of O2) and ROS (reactive oxygen species) formed in bioreactor during autoclaving. Without stirring/purging, anaerobic cells adjust their micro-environment much easier, remove ROS and drop local Eh within few micrometers around cell clumps, while the mixing eliminates these favorable micro-zones.

If you do not plan to monitor gases and do not care about the physiological state of cells , do not use automated pH control and do not perform other quantitative assays (e.g. your objective is just to get some cells of Bifidobacterium), then I agree with Mario, your culture could be grown without sparging. Note that during such cultivation, there will be a build-up of pressure inside your flasks/reactors because of release of CO2.

Thank you Nicolai, Bifidobacteria do not produce gas, but I will be monitoring metabolites during growth. i have found some studies that shows pure N2 is diminishing the growth of bifidobacteria so my plan is to switch to gas mix (CO2, H2, N2) and try continuous sparging. I tried what Mario suggested but it took 3 days for them to grow and redox potential got high, which I am not sure if it is maintaining anaerobic conditions at some point. But you are right I will need the physiological state to be maintained. the only thing I do, I activate the cells from frozen state before bioreactor. So, the environment they are adjusting themselves is the stirring and gas. Therefore, I will switch to using gas mix. Thank you so much for your help!

Ezgi, biifdo ferment sugars into lactate, acetate, succinate and formate. Practically always, CO2 is one of products. It is resulted from decarboxylation. Therefore lack of visible gas bubbles does not mean that there is no gas production and there is no pressure build-up. Many anaerobic species are fastidious, so I am not surprised that they failed to grow for 3 days. Important point is to have active inoculum. Prepare seed material in small Hungate tubes, add complex medium, then transfer to bigger reactor. Yes, CO2 in the gas phase is good idea. But remember, that all metabolomic procedures are compromised by using complex media, therefore try to use minimal chemically defined media. Good luck.

Thanks for the answer. I need to make a correction, bifidobacteria that I study which is Bifidobacterium longum does not produce any gas in case anyone interested in studying this species. They ferment sugar , that is correct, but through a unique metabolic pathway, called fructose-6-phophatephosphoketolase, in other words bifid shunt, CO2 is not a product. yes lactate and acetate in some cases formate, ethanol and succinate get produced from sugar fermentation. Formate and ethanol are produced through conversion of pyruvate to acetylCoA to acetate. Succinate production might (!) occur through production of oxalaacetate from phosphenolpyruvate which requires CO2, not produce.

I activate bifidobacteria in anaerobic chamber. This is common practice that our lab do for several years. I activate bacteria from -80C on plate then media in anaerobic chamber and then transfer to reactor, as I said earlier.