Growth and Cultivation of micro-organisms by E. Börje Lindström This learning object has been funded by the European Commissions FP6 BioMinE project

Growth Definition: Growth implies that all building blocks of the cell increases with the following consequences: Growth Normally   Increase in cell number Multi cellular Uunicellular organisms organisms increases size increase number of organism of organisms

Mathematical representation The bacteria divide binary usually perpendicular to the length axis and thereby two new cells are produced For a unicellular bacterium the cell number increase exponentially with base 2 as seen in the table below: Cell no. Exponential expression n = no. of doublings 20 21 time for each doubling = g (min, hr) 22 23 Etc. 2n The following mathematical expression is then obtained:

Math. cont. Nt = N0 x 2n (1) Where: Nt = the cell number at time t N0 = the cell number at t = 0 and n represents the number of doublings (generations) if g = time for a generation and t = total time, then Nt = N0 x 2n = N0 x 2t/g (2) set m = 1/g where m is the specific growth rate constant; inserted in (2) gives

Math. cont. Nt = N0 x 2tm (3) take the logarithm of equation (3), which gives: log Nt = log N0 + t x m x log 2 (4) in a semi-logarithmic graph this is a strait line log Nt slope = m x log 2 t

Cultivation Batch cultivation Cultivation is normally performed batch-wise or continuously. Batch cultivation - the growth medium and the bacteria (inoculum) are added to the growth vessel once at the start of the experiment! - any growth vessel can be used: shake flasks, stirred tank reactors etc. - batch-wise cultivation is chosen to rapidly obtain growth data During batch cultivation of a bacterial culture you can have four (4) growth phases as shown below:

Growth curve during batch cultivation log Nt t Stationary phase Death phase Lag-phase Log-phase

Properties of the growth phases Lag-phase: - the cell devision is delayed due to how the inoculum has been treated - the previous medium - the temperature, etc. Log-phase: - exponential growth - as fast as the soluble nutrients permit - the doubling time, g, can be determined here - in bioleaching there is often no exponential phase due to that the energy source is a particle

Properties, cont. Stationary phase: Death phase: - growth is stopped due to changes in the medium - an essential nutrient has ceased - pH-changes due to end products - dissolved oxygen for aerobic organisms Death phase: - an exponential curve due to some toxic substance excreted from the bacteria

Quantitative methods for measuring bacterial growth The growth of the bacterial population can be followed either by the changes in number of cells or weight of cell mass. In the following table a comparison of a few methods are found.   Sensitiveness (cells/ml) Parameter Method Note Gravimeter 108 Direct method Cell mass (dry weight/ml) Turbid meter (O.D.) 107 Indirect method (depending on the compound) Indirect method Chemical analysis Cell number, total Microscopy 106 Direct method  Cell number, viable Viable count (V.C.) 1-10 Indirect method

Comments to the quantitative methods Some of the methods are noted as direct or indirect. The direct methods show the cell mass or cell number directly in the sample. In the indirect methods you need a standard curve comparing a direct and an indirect method. If e.g. during growth the same sample is measured by the direct method gravimeter and the indirect method turbid meter and those values are plotted in a diagram you will have a standard curve for use in later experiments. (dry weight/ml) Cell mass Turbid meter, (O.D.)

Comments, cont. The method gravimeter uses ordinary balances after removal of the water content of the sample. Given a sample size of one ml and assuming that an average dry bacterium is weighing 10-12 g and that a ordinary balance can detect 10-4 g this means that you must have >108 bacteria per ml in the sample to be able to use weighing. The sensitivity given for any turbid meter e.g.ordinary spectrophotometers measuring optical density, is arbitrary. For microscopy the sensitivity value means that you have on average one cell in the smallest square on the special object glass used. Depth Grid in the bottom

Comments, cont. In viable count you usually pore out 0.1 ml of the sample onto the surface of a nutrient agar plate. If you get one colony after incubation then you have had 10 bacteria per ml in the sample. 0.1 ml 1 ml Nutrient agar plate 10 bact/ml 1 bact/ml

Balanced vs. Unbalanced growth The growth of a bacterial culture is related to the composition of the medium. In a minimal medium the growth is slower than in a complex medium. If all the essential nutrients are freely available the growth is balanced, which means that all the building bocks are synthezised with the same speed (see figure below). However, if the synthesis of one of the building blocks is stopped, the growth is terminated due to unbalanced growth, which often leads to death of the culture (see figure below). Time Amount Protein RNA DNA Time Amount Protein RNA DNA Balanced Unbalanced

Enrichment and isolation When a special bacterial species is nutritional favoured during cultivation that species will be enriched in the culture. A small sample of this culture is then transferred to new fresh medium of the same type and the cultivtion is continued. This procedure is continued several times. Isolation: A small sample of the last enrichment culture is then spread on top of a agar plate with the same nutrient media as in the enrichment. Among those colonies appearing on the plate after proper incubation the wanted bacterial species will be. Testing these colonies will evetually result in the isolation.