1. Ensure that the seed culture is at its exponential growth phase before transferring it in the fermenter.

2. Ensure that you operate the fermenter at the optimum conditions (i.e., pH, temperature, etc.) and the appropriate nutrients are added.

3. During the fermentation process, continuously check for contamination.

Thanks for the advice Bongo Mount-Batten Majeke

By applying suitable conditions for promoting the growth of the bacteria culture.

Thank you Obafemi Obajemihi

Hello Khrisna Putera Buddhiman ;

Thank you very much for posting your question in RG. First of all, I can see that the cell concentration that you are achieving is already high.. considering that some bacterial cultures will not grow higher than that. Can you elaborate on the method you used for CFU/mL determination?

In addition, your are reporting the wet biomass of your culture. It would be very helpful if you explain how you measured this. Regularly we report dry-basis biomass concentration, which for a regular bacterial culture it only goes up to around 1 g/L. So, to really have an idea of your cell yield it would be helfpul to know you the dry-weight biomass concentration of your culture. For this determination we use microfiltration membrane discs in our laboratory.

Overall, you should be aware that the maximum cell yield of a specific culture depends on many factors, among which the strain specificity is one of the most important. So I suggest that you check the literatures for cell yield reports for your specific strain, and compare your results with those of the literature. After this, you can proceed with other approaches related to bioprocesses to optimize the yield of your culture. If you can provide more details of your culture system as referred above, it could be possible for me to propose some ways to improve your cell yield.

Hope this was useful for your research.

Regards

Hello Rogelio Valadez-Blanco ,

Really appreciate your thoughtful inputs!

We use total plate count method for cfu/ml determination.

For the wet biomass determination, after the fermentation has finished, we centrifuge the culture and measure the weight of the pellet. By dividing with the original culture volume, the g/L wet biomass can be determined. Please let me know if there's any inputs on this method.

Regarding the culture system, currently we're still using batch fermentation supplemented with compressed air (not pure O2).

Thank you once again.

Hello Khrisna Putera Buddhiman;

Thanks for your reply. If you are sure of your CFU/mL determinations I can tell you that they are actually good compared with other bacterial reports. I don´t know why your goal is to reach a growth of 10^10 CFU/mL. Did you get that reference value from the literature or previous work for your specific strain?

The wet biomass measurement that you described is not a very reliable determination. I suggest to obtain the dry biomass measurement using microfiltration membranes as I described before. With this value, you can compare with the literature in which you may find this value for your specific bacteria.

I also recommend to measure the optical density at 600 nm. Depending on the color of your culturing medium, you may need to wash with RO (distilled) water the biomass using centrifugation cycles (one or two washing cycles are ok). This will serve you as another way to compare your celullar growth with literature values.

I think these steps are ok for ensuring the proper characterization of your cell growth. I would start obtaining a growth kinetics curve of your reactor batch. From this information you will obtain the growth rate. Then you need to do the same for different substrate concentrations to obtain the growth rate equation, and perhaps the Monod-kinetics parameters (if it behaves accordingly).

From these cell-growth kinetics experiments now you will be in position to apply different approaches to try to improve your current cell yield, which may include different experimental designs, such as factorial analysis considering temperature, pH, substrates, activators, etc; as well as different bioreactor configurations.

Hope this is useful for your research.

Kind regards