BD Accuri News

Frank Delvigne Discusses Online Bioprocess Monitoring

Frank Delvigne, PhD, is associate professor in the Department of Bio-Industries at the University of Liège in Gembloux, Belgium. His research interests include bioreactor modeling, bioprocess scale-up and scale down, and microbial stress analysis. He spoke to us about designing and testing a low-cost interface to monitor a bioreactor using a BD Accuri™ C6 flow cytometer.

Q: Would you tell us about the research program in which you are using the BD Accuri C6?

A: I study microbial phenotypic heterogeneity in bioreactors and how it affects bioprocess optimization. I began to use a flow cytometer for cell analysis, but I missed a lot of information by sampling offline. Online flow cytometry systems are available, but the costs are very high. So we decided to build our own interface between the bioreactor and the flow cytometer, focusing on a low-cost benchtop flow cytometer—the BD Accuri C6 system. Its advantage is that it doesn’t require putting the tank under pressure. It works with peristaltic pumps, which make it very easy to connect the tubing of the bioreactor directly to the flow cytometer. We simply take the tubing and insert it directly in the sampling needles of the BD Accuri C6.

By using the BD Accuri C6 to work immediately with the cells coming directly from the bioreactor, we get very interesting results. When you have an online system, you can look at the dynamics of the response during the different phases of the cultivation.

Q: Could you describe the connections between the bioreactor and the BD Accuri C6?

A: Connections are made of silicone tubing and the fluid is transported by peristaltic pumps like those in the BD Accuri C6. All the fluid movement, staining, and cell withdrawal from the bioreactor are controlled by a microcontroller, which decides when the sample is taken from the bioreactor, how long the cells are in contact with the stain, and when the flow cytometry analysis occurs.

We also have a water reservoir for dilution when necessary, and the staining procedure occurs inline in a T-mixer—a T-junction between the fluid coming from the bioreactor and the water.

We are currently working to improve the design of the interface. The goal is to take the results of the flow cytometry experiment and insert them directly in the feedback control loops of the bioreactor. This new interface comprises a microreactor, for two reasons. First, the sample volume is very low in this kind of system, so we can work on mini-bioreactors without withdrawing a lot of sample. Second, in a microreactor, you can control the residence time of the stains very precisely. So we can improve the staining procedure and expand our work to use other stains that cost more money, which have to be used in lower amounts.

Q: Can you describe a typical study?

A: We are focused on E. coli bioprocessing. For that, we use dual marker systems: green fluorescent protein (GFP, measured in FL1), designed by engineering E. coli; and propidium iodide (PI, measured in FL3) to assess cell membrane permeability and cell viability. By combining these two biomarkers, we can assess the physiological states of E. coli during the bioprocess.

Q: Your recent paper1 discussed a phenomenon called segregation. Why is that important?

A: We know that even a clonal population of cells doesn’t exhibit the same phenotypes during a culture—even if the culture is carried out in homogeneous extracellular conditions. That’s why it’s very important to assess the distribution of these phenotypes. The segregation of the population can potentially impair the yield of a bioprocess. We found that, during a bioprocess with E. coli, there are three distinct subpopulations, according to the PI uptake of the cells.

Classically, you have two states or subpopulations, one that is not permeable to PI (healthy cells with a good membrane), and one that is permeable to PI, which takes up the PI (considered non-viable, damaged cells). But we found that, between these two subpopulations, there is an intermediate state, with cells taking up PI but not as much as the damaged subpopulation. This intermediate subpopulation is very important because it corresponds to viable cells that are potentially releasing proteins into the extracellular medium.

If we found a way during biopharmaceutical processes to release the proteins from the periplasm to the extracellular medium, we could lower the cost of the bioprocessing operation and improve the recovery of the recombinant proteins. That’s why we focus on this intermediate subpopulation.

Q: Which other features of the BD Accuri C6 are important to you?

A: It’s all about the cost. In a conventional flow cytometer, you have a lot of consumables, notably the sheath fluid, which can be very expensive. With the BD Accuri C6, you can use filtered, deionized water as sheath fluid, and you have to replace the tubing every 3–4 months—and that’s all. It’s very easy to maintain the equipment. And in bioreactor operation, we don’t have a lot of money, so if you want to perform repeated analyses of a bioreactor, low-cost analytical equipment is very important.

Q: Did anything surprise you about the BD Accuri C6 once you got it into your lab?

A: The quick setup. We took the flow cytometer out of the box, connected it to the electrical supply, and that’s it! We were able to do our first analysis immediately.

1 Brognaux A, Han S, Sørenson SJ, Lebeau F, Thonart P, Delvigne F. A low-cost, multiplexable, automated flow cytometry procedure for the characterization of microbial stress dynamics in bioreactors. Microb Cell Fact. 2013;12:100.