Applications: Growth dynamics

A true representation of growth dynamics

The removal of bubble interference makes UV-VIS spectroscopy a clean and reliable tool for monitoring microbial growth dynamics, even in highly aerated systems, such as aerobic yeast growth. Cytoprom’s UV-VIS systems provide an accurate measure of growth dynamics, whatever your aeration needs. The following data speak for themselves.

Figure 1: Fully aerated growth of S. cerevisiae W303-1a. Without bubble exclusion optical density values are inaccurate, as they either over or under represent the culture density, and imprecise, as bubbles cause significant scatter.

Figure 2: Growth of Y. lipolytica. Even when aeration is low, the level of scatter from bubble interference is significant because stirrer speed is the primary variable that effects bubble size distributions.

In each of Figures 1 and 2 the data were measured simultaneously by separate probes in the same fermentation run, one probe with and one probe without bubble exclusion. The results with bubble exclusion are accurate and reproducable, and it is clear that bubbles interfere with the measurements in different ways under all types of conditions – in fact the nature of the interference is a strong function of aeration, stirrer speed, and optical density (OD), as well as characteristics of the cells being cultured. For any particular growth curve, as shown above, even averaging the widely scattered data obtained without bubble exclusion gives an over-estimate of OD for some ranges of OD, and an under-estimate for other ranges, depending unpredictably on experimental conditions.

When reliable bubble exclusion is achieved, growth dynamics can be trusted and used to assess basic growth characteristics and kinetics with confidence. Figure 3 shows very nicely how even subtle differences in growth can be monitored. Here we compare two strains of a yeast expressing the mitochondrial ADP/ATP carrier. The only subtle difference in the strains is the introduction of three extra histidine residues on the expressed His-tagged protein. Even though this is a minor alteration, a small difference in the doubling times of the two strains can be observed.

Figure 3: Growth measurements in a fully aerated bioreactor of two strains of yeast expressing the mitochondrial ADP/ATP carrier with either an N-terminal six or nine-histidine tag. Continuous measurements were carried out with a fibre-optic probe fitted with an air bubble excluder. The results show the subtle effect on the doubling time, as a consequence of the introduction of three extra histidine residues on the expressed His-tagged protein.

Diauxic growth can also be accurately monitored and the change from one carbon substrate (glucose) to another (glycerol) can be clearly identified in Figure 4. The identification of limiting substrates can increase the yield of the fermentation significantly and thus growth monitoring is of great commerical value.

Figure 4. A strain of S. cerevisiae W303 was cultured with a mixed carbon substrate of 0.1 %w/v glucose and 3.0 %w/v glycerol. The glucose is more readily metabolised and becomes exhausted before a second phase of growth on glycerol.