Analysis of Escherichia coli cell state by flow cytometry during whole cell catalyzed biotransformation for l-carnitine production
Flow cytometry was used to monitor Escherichia coli cellular state during the biotransformation of crotonobetaine into l-carnitine using growing and resting cells in batch and high-cell-recycle continuous membrane reactors. The cell physiological state and the DNA, RNA and protein cell content were analyzed during the bioprocess. The cell growth cycle was followed by reference to cellular DNA concentration and the entry in the stationary phase resulted in an increase in intracellular protein. The biochemical activity of resting cells was assessed for the first time at the molecular level, protein synthesis being observed despite the absence of nutrients. Freely suspended growing, both in batch and continuous cultures, and, more importantly, resting E. coli cells were seen to be made up of subpopulations differing in reproductive ability, metabolic activity and membrane integrity. In the case of growing cells, biotransformation was mostly performed by fully viable cells (68–75%), while in a resting cell system, also dead cells (1–5%) and cells with doubtful viability (60–70%) appeared to be involved in the process; in later stages, a population made up of phantom cells, containing little or no cellular DNA, was detected. In cell-recycle continuous reactors, the recording of DNA (40 to 60 fg), RNA (50 to 120 fg) and protein (100 to 220 fg) levels per unit of cell, and the evolution of cell population heterogeneity (three different populations of cells) threw light on the stress conditions imposed by high cell densities. The use of FCM allowed to follow the recovery of cell catalytic activity for resting biotransformation batch processes, thus showing its potential for the optimization of bioprocesses.
Journal: Process Biochemistry - Volume 42, Issue 1, January 2007, Pages 25–33