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. 

  1.      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 No+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.

  1. 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. 

E:\Sailaja\CONTENT WRITING - MICROSCOPIA IWM\microbial growh and kinetics\bacterila growth curve.jpg
Figure 2: Bacterial growth curve
  1. 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 – tn)

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 = μms / 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.  
Direct Microscopic Count of Bacteria- Lab 13 - YouTube
Figure 3: Bacterial cell count using haemocytometer
  1. 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
  1. 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.

BACTERIA-REPRODUCTION & GENE TRANSFER

bacteria: reproduction and gene transfer

Content:

  • Binary fission
  • Transformation
  • Transduction
  • Conjugation

Reproduction and growth:

  • In unicellular organism cell growth and reproduction are two tightly linked processes unlike multicellular organism.
  • After gaining a fixed size bacteria reproduce through binary fission, budding and fragmentation.
  • Bacteria in optimum condition grow and divide rapidly and double its population in every 9.8 minutes.

Binary fission:

  • It is the most common mode of cell division and growth cycle of bacterial population.
  • In binary fission single cell divides into two identical cells with development of transverse septum (cross wall).
  • Two daughter cells contains nucleus of its own which is identical to the parent cell.
  • Cytoplasm divides leads to production of two equal sized cells.

Process of binary fission:

  • Before the division DNA in the bacterial cell is tightly coiled
  • DNA is then uncoiled and duplicated.
  • Each copy of the DNA is pulled to the separate poles.
  • Synthesis of new cell wall begins
  • Once the new cell wall is synthesised fully it results in complete split of bacterium.
  • New daughter cells now have tightly coiled DNA, plasmids and ribosomes.
Types of binary fissionExample
TransverseParamecium
ObliqueCeratium
LongitudinalEuglena
Irregularamoeba

Gene transfer:

  • Gene transfer means movement of genetic information in organisms.
  • There are two types of gene transfer method one is vertical in which gene is transferred from parents to offspring and another one is horizontal in which gene is transferred in between two organisms.
  • In prokaryotes vertical gene transfer is by the means of binary fission and horizontal gene transfer method consist of three process i.e. transformation, transduction and conjugation.

Transformation:

  • In 1928 Fred Griffith discovered this method of horizontal gene transfer.
  • In this process naked DNA molecule or fragment from surrounding environment is uptake by the recipient and incorporated in its chromosome.
  • It is of two types natural and artificial, natural transformation is very rare event and observed in both gram negative and gram positive bacteria.
  • Ability of bacteria to uptake DNA fragment and get transformed is known as competence.

Process of transformation:

  • Competent bacteria naturally pull DNA fragment into their cell from the environment.
  • These DNA fragment naturally released in the environment after a bacterial cell die.
  • Ds DNA once crosses the membrane in cytoplasm the 3’ end is leading.
  • The translocated strand interested in the chromosome of recipient bacteria by homologous recombination.
  • Now the recipient bacteria undergoes replication and the cells acquired new phenotype are said to be transformed.

Transduction:

  • In transduction, DNA is transfer from donor bacteria to recipient bacteria by bacteriophage (functions as vector).
  • It was discovered by Lederberg and Zinder in 1951.
  • Bacteriophage due to high specificity of surface receptors has narrowest host range.
  • Transduction has one advantage over conjugation is that it doesn’t require physical contact of donor to recipient cell.
  • Transduction process is resistant to the DNase enzyme.

Steps:

  • The phage infects the host and inserts its phage DNA into the cytoplasm of the host.
  • During lytic cycle the phage DNA along with the bacterial chromosome is broken down into pieces
  • Bacterial chromosome packed into the viral capsid is released by the lysis of the bacterium.
  • Now the transducing phage with bacterial chromosome is ready to infect another bacterium in this way donor’s DNA enters into the cytoplasm of second bacterium.
  • Host recombinase recA is present in the cell due to which donor DNA recombines with homologous bacterial DNA and produces transductants.

Conjugation:

  • The process of transfer of plasmid or other transmissible DNA element from donor to recipient via sex pilus or conjugation tube.
  • Recipient of conjugation is known as transconjugants.
  • Is can transfer DNA regions of hundreds to thousands of kilobases and has board host range fro DNA transfer.
  • Occur in between many species of gram negative and gram positive bacteria even occurs between plants and bacteria.
  • Conjugation involves F plasmid is most common.

Steps:

  • F+ structure contains tra locus which has pilin gene with some regulatory proteins responsible for the formation of pili on surface.
  • Proteins present on pili attach to the F- cell surface and responsible for making contact between them but doesn’t transfer plasmid.
  • The traD enzyme on the base of the pili makes the membrane to fuse.
  • After the conjugation initiated the enzyme relaxes attached to the conjugative plasmid and make  a nick at oriT.
  • The nicked strand is now transferred to the recipient cell
  • F+ cell carry such integrated F element is known as Hfr cell.
  • The F element of Hfr cell is replicated along with the bacterial chromosome and in this way transmitted from one to next generation.