We describe a method that enabled us to observe large numbers of individual bacterial cells during a long period of cell growth and proliferation. We designed a flow chamber in which the cells attached to a transparent solid surface. The flow chamber was mounted on a microscope equipped with a digital camera. The shear force of the flow removed the daughter cells, making it possible to monitor the consecutive divisions of a single cell. In this way, kinetic parameters and their distributions, as well as some physiological characteristics of the bacteria, could be analyzed based on more than 1,000 single-cell observations. The method which we developed enabled us to study the history effect on the distribution of the lag times of single cells.

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Methylotrophic yeast Pichia pastoris is an ideal host organism for recombinant protein production. However, adequate methanol feed is still a critical point of successful product formation in P. pastoris Mut 5 fermentations. Three methanol feed strategies were tested: an organic vapor sensor, a dissolved oxygen controlled methanol addition and a predetermined model, as well. The organic vapor sensor proved to be unsuitable for methanol concentration measurements when samples were taken from the head space of the bioreactor, but may have the potential to substitute expensive gas analyzers in methanol fed-batch with a suitable selector submerged into the fermentation media. Dissolved oxygen and substrate consumption did not show strict mathematical relation. However, drop of dissolved oxygen tension for periodic methanol addition may be applied for the determination of the substrate concentration in the media. The rate of methanol consumption shows peaks at 0.45 and 0.95% (v/v) substrate concentrations. The rate of the specific methanol consumption of our model organism was determined. Based on the value of 0.023 h`, which is significantly less than suggested by earlier experiments, a successful predetermined methanol feed strategy was set up.