Microbial growth and physiology: a call for better craftsmanship

Abstract

Virtually every microbiological experiment starts with the cultivation of microbes. Consequently, as originally pointed out by Monod (1949), handling microbial cultures is a fundamental methodology of microbiology and mastering different cultivation techniques should be part of every microbiologist's craftsmanship. This is particularly important for research in microbial physiology, as the composition and behavior of microbes is strongly dependent on their growth environment. It has been pointed out repeatedly by eminent microbiologists that we should give more attention to the media and culturing conditions. However, this is obviously not adhered to with sufficient rigor as mistakes in basic cultivation principles are frequently found in the published research literature. The most frequent mistakes are the use of inappropriate growth media and little or no control of the specific growth rate, and some examples will be discussed here in detail. Therefore, this is a call for better microbiological craftsmanship when cultivating microbial cultures for physiological experiments. This call is not only addressed to researchers but it is probably even more important for the teaching of our discipline.

Keywords: batch; continuous culture; cultivation; growth media; nutrient limitation; physiology.

FIGURE 1

Experimental confirmation of stoichiometric limitation of growth of different nutrients for a bacterial (Cometta et al., 1982) and a yeast culture (from Egli, 1980). Both microbial strains were cultivated in a chemostat culture at a fixed dilution rate. A distinct transition from limitation to excess is observed for carbon-, nitrogen, and phosphorus-limited growth, whereas the transition between limitation and excess is not as distinct in the case of magnesium and sulfur. (A,B) Rearranged from Cometta et al. (1982), and information kindly provided by B. Sonnleitner. (C) Rearranged from Egli (1980).

FIGURE 2

Patterns of growth, nutrient consumption, product formation and gross cellular composition during batch growth of Klebsiella pneumoniae in a defined mineral medium limited by different nutrients. The limiting nutrients were carbon (glucose), nitrogen (NH₄⁺), phosphorus (PO₄^3-), sulfur (SO₄^2-), and potassium (K⁺), respectively. Top panels show concentrations of dry biomass and limiting nutrient; middle panels show concentrations of residual glucose and acetate formed in the medium; the bottom panels show composition of biomass with respect to total protein and carbohydrates. Rearranged from Wanner and Egli (1990).

FIGURE 3

Zones of single- and dual-nutrient-limited growth (gray area) for a culture of Klebsiella pneumoniae cultivated in the chemostat as a function of dilution rate and C:N-ratio in the feed medium. (A) Conceptual scheme of residual steady-state concentrations for the nitrogen (ammonia) and the carbon source (glycerol) at a dilution rate (D) of 0.1 h^-1, as well as the steady-state biomass concentration in the culture and of an accumulated reserve material. In this experiment, the concentration of the nitrogen source in the feed medium is kept constant and the C:N-ratio of the medium is varied by changing the concentration of the carbon source). (B) The predicted boundaries for the three growth zones are shown, the boundaries were calculated from literature data reported (see Egli, 1991). The left hand border (full circles) was calculated from reported growth yields for N and C determined under glycerol-limited growth conditions; the right hand border (empty circles) was calculated from growth yields reported from cultures grown under ammonia-limited growth conditions. Adapted and extended from Egli (1991).

FIGURE 4

Batch growth curve of Escherichia coli K-12 MG1655 in complex medium (Luria-Bertani broth, 25% original strength) in vigorously shaken flask. Growth was measured as OD₅₄₆, the temperature was maintained at 37°C, and pO₂ was always >70% air saturation. The specific growth rate μ (h^-1) was calculated as the slope of five adjacent points (empty squares). The inset shows the first 2 h of the experiment with μ determined from the slope of three adjacent points. Adapted from Berney et al. (2006) and amended with the inset. For more details see Berney et al. (2006).