Bacterial Growth Curve Analysis and its Environmental Applications

Bacterial reproduction occurs via binary fission, in which one bacterial cell divides and becomes two cells (Figure 1). The time needed for cell division is known as the mean generation time, or doubling time, which is the time needed for the number of cells to double.

Figure 1. Exponential cell division. Each cell division results in a doubling of the cell number. At low cell numbers, the increase is not very large; however after a few generations, cell numbers increase explosively. After n divisions, there are 2ⁿ cells.

To understand and define the growth of particular microorganisms, they are placed in a flask, where the nutrient supply and environmental conditions are controlled. If the liquid medium supplies all the nutrients required for growth and environmental parameters conducive to growth, the increase in numbers can be measured as a function of time to obtain a growth curve. Several distinct growth phases can be observed within a growth curve (Figure 2). These include the lag phase, the exponential or log phase, the stationary phase, and the death phase, each of which are associated with specific physiological changes (Table 1).

Table 1. The four phases of bacterial growth.

Figure 2. A typical growth curve for a bacterial population. Compare the shape of the curves based on colony-forming units (CFUs) versus optical density, particularly in the death phase. The difference is due to the fact that dead cells still result in turbidity, but cannot form viable colonies in culture.

Overall, it is often critical to determine bacterial growth kinetics for a given bacterial isolate, in order to know the number of bacterial cells present in the liquid medium. There are different ways to measure growth in a liquid medium, including turbidity measurements using a colorimetric spectrophotometer, and serial dilution plating. Turbidity measurements rely on the fact that the more cells present in the liquid medium, the more turbid the liquid becomes. Serial dilution plating involves assaying the number of cells in the liquid medium that can form viable colonies on solid culture, a measurement known as the culture’s “colony-forming units”. Note, however, that such plating assays can only be used for bacteria that are, in fact, culturable.

Following a serial dilution plating experiment, the following data was obtained. At the beginning of exponential growth designated here as time t = 0, the initial concentration of bacterial cells is 1,000 CFU/mL. At time t = 6 h, the concentration of cells is 16,000 CFU/mL.

Now, X = 2ⁿ x X₀

Where: X₀ = initial concentration of cells = 1,000 CFU/mL
X = concentration of cells after time t = 16,000 CFU/mL
n = number of generations
16,000 = 2ⁿ x 1,000
2ⁿ = 16
log₁₀ 2ⁿ = log₁₀ 16
n x 0.301 = 1.204
n = 1.204 = 4
       0.301

Four generations elapsed in 6 h, so

Mean Generation Time = 6/4 = 1.5 h.

Figure 4. A root nodule that contains nitrogen-fixing bacteria.

Knowledge of bacterial growth kinetics and bacterial numbers in a culture medium is important from both a research and commercial point of view. In research, it is often critical to know the number of bacteria in a sample, so the experiment can be replicated, if need be, with the exact same numbers. For example, during experiments in which bacterial inoculants are added to a plot of soil, a minimum of 10⁴ CFU needs to be added per gram of soil to get the desired effect, such as enhanced biodegradation of toxic organic soil contaminants. Another example is the case of commercially produced rhizobial inoculants, where known numbers of rhizobia (bacteria that enter into symbiotic relationships with the roots of plants) are impregnated into a peat-based carbon medium (Figure 4). The medium is then used to inoculate legume seeds to enhance biological nitrogen fixation (i.e., the conversion of molecular nitrogen into organic forms that can be used by organisms as nutrients).

Growth kinetics is also useful for assessing whether particular strains of bacteria are adapted to metabolize certain substrates, such as industrial waste or oil pollution. Bacteria that are genetically engineered to clean up oil spills, for example, can be grown in the presence of complex hydrocarbons to ensure that their growth would not be repressed by the toxic effects of oil. Similarly, the slope and shape of growth curves produced from bacteria grown with mixtures of industrial waste products can inform scientists whether the bacteria can metabolize the particular substance, and how many potential energy sources for the bacteria can be found in the waste mixture.