Microbial growth and kinetics

BY- Reddy Sailaja M (MSIWM030)

Microbial growth

Microbial growth is defined as the increase in cell number (rather than cell size) by asexual reproduction process called, binary fission. Cell division results in the growth of the cells in the representative population. Bacteria and archea undergo asexual reproduction, while fungi and higher organisms also exhibit sexual reproduction and increase in cell size is also considered as growth in higher organisms. 

Binary fission is the process of asexual reproduction method followed by most bacteria. In this process, initially a single cell starts doubling its machinery, including genetic material, enzymes and other essential components. Then a wall like structure, called septum forms in middle, dividing the cell into two equal halves, each half receives a set of genetic material and other essential components needed for life. A typical bacterium like Escherichia coli takes around 20 minutes to divide into two and is called a cell cycle. Multiple fission, budding and sporulation are some less frequent kinds of cell division shown by bacteria. The bacterial cell division by binary fission was shown in figure 1. 

Figure 1: Bacterial cell division – Binary fission

Bacterial growth curve

. When conditions favor (nutrients, environment, temperature etc), bacterial growth rate is rapid. When the bacteria undergo any change in their niche environment, growth rate is effected that might lead to dormancy or death. E.coli is taken as a model organism as it has very small life cycle of 20 minutes and easy to grow in the lab for variety of biological studies

E.coli can be grown in a glass test tube and its growth phase can be studied in a predictable approach. A typical bacterium under controlled conditions will exhibit four phases as follows:

i) Lag phase

ii) Log phase

iii) Stationary phase

iv) Decline/death phase

1. Lag phase: Bacteria in this phase will be in adaptive mode, trying to adjust to the surrounding new environment. If cells are in damaged state or transferred to entirely new environment, bacteria takes long time to adjust and the lag phase becomes lengthy. If the bacteria are exposed to familiar kind of environmental conditions with suitable nutrients, temperature etc, growth starts rapidly. In this case, lag phase takes less time.

In lag phase, cells will rejuvenate themselves; synthesize enzymes, metabolites, RNA etc that are required for the growth. Cells try to repair if there is any damage that was there in the cells. 

2.      Log phase: This phase is also called exponential phase. Cells will be in active mode and increase in number by multiplication, where 1 cell becomes 2, 2 becomes 4 etc. Rapid growth of the cells will result under ideal conditions, while showing slower growth is very rare depending upon the nutrient or other conditions. Cells in this phase are the healthiest and in the most active form. Exponential stage cells are the one that are being used in industry and research sectors for varied applications, like growth rate determination, metabolic activity studies, protein purification etc.

Log phase cells have steady growth rate, hence it is easy to estimate the generation time (g) of the cells. Generation time is calculated as the time taken for the bacterial cells to double in their number. The log of cell number can be plotted against time (hours or days) to generate a predictable slope. The number of cells after certain period of time can be calculated from the formula:

N=N₀2ⁿ

Where,

N = Final cell concentration (or number)

N₀ = Initial cell concentration

n = number of generations that happened over a period of time

When we apply log to the above formula,

Log N=Log N_o+n log10²

n = 3.3(Log10 N-Log10 No)

The above formula can be used to calculate number of generations during the bacterial growth.

Generation time (g) can also be represented as t/n, where ‘t’ is the specific period of time in minutes, hours, days etc. With the amount of ‘t’ used during the growth starting from the initial cell concentration, ‘g’ can be calculated.

3. Stationary phase: When the nutrients get deprived and waste products/secondary metabolites start accumulating in the medium, bacterial cells are unable to further grow and enter into stationary phase. During this phase, number of cells being died will be more than the number of cells being produced. As a result, flattening of the growth curve happens at this phase. The new cells being produced also vary in its shape – from bacilli to more circular, size – large to small, cells- individual to aggregated formation etc because of the new starvation conditions that started prevailing in the growth medium. 

All the physiological changes allow cells to survive the harsh environment rather than rapidly dividing. Cells also start producing secondary metabolites into the surrounding medium. Some bacteria like Bacillus form endospores and enter dormancy. 

Figure 2: Bacterial growth curve

4. Decline/Death phase: Cells start dying off rapidly in this phase, resulting in the steepness in the growth curve. Most cells are irreversibly damaged that when transferred to fresh growth medium, very few cells may respond and show further growth. Some cells that are not able to revive their growth are called as viable but nonculturable (VBNC). 

Bacterial growth kinetics

Optical density is a type of measure of Specific growth rate (µ). Spectrophotometer is used to measure optical density.

µ is calculated per hour as the generation time (T) at the time of lag phase as follows:

µ = (lnXn+1 – lnXn)/(tn+1 – t_n)

T = 0.69/ µ

Where,

InX = natural log of OD 

T =  time of OD measurement

Bacterial growth is an autocatalytic reaction i.e., the growth rate is directly associated to cell concentration.

The equation of bacterial growth kinetics is represented as follows:

ΣS + X → ΣP + nX

Substrates + cells → extracellular products a+ more cells.

Where,

S = substrate concentration measured in gram per litre (g/L)

X = Cell mass concentration (g/L)

P = Product concentration (g/L)

n = increased number of cells or biomass

Net specific growth rate (1/time) is represented as

μnet = 1/X. dX/dt

t = time

Monads equation: Monads equation describes the dependence of the bacterial growth rate on the concentration of the substrate, as follows:

μ = μ_ms / S +Ks

where,

μ =  specific growth rate of microorganism

μm =  maximal specific growth rate

S = concentration of the substrate that is limiting source for growth

Ks = “half-velocity constant” (is the value of S when μ/μmax = 0.5)

Measurement of bacterial growth: 

Following are the three major modes of measuring bacterial growth:

1. Microscopic count – counting the number of cells using a haemocytometer (originally used to count red and white blood cells). Haemocytometer is a special microscopic slide to count the cells in the gird present at the centre. However, it is not possible to distinguish live and dead cells unless cell permeable dyes like trypan blue is used.  

Figure 3: Bacterial cell count using haemocytometer

2. Plate count method – A bacterial culture is serially diluted and spreaded onto the plate. After incubation for growth, colonies are counted which are direct representation of growth.

Figure 4: Bacterial cell count using plate count

3. Turbidimetric method: Bacterial growth was measured based on the turbidity or cloudiness of the culture using a colorimeter. The number of cells grown is calculated by plotting the colorimetric values against standard graph generated using known cell numbers. 

Figure 5: Bacterial cell count using spectrophotometer (turbidity)

Apart from the above methods, bacterial growth can also be measured based on the dry weight and metabolic activity of the cells.