Estimation of Microbial Growth

Estimation of Microbial Growth
Standard Growth Curve

Growth in Batch Culture
1. Bacteria growing in batch culture produce a growth curve with up to four distinct phases.
2. Batch cultures are grown in tubes or flasks and are closed systems where no fresh nutrients
are added or waste products removed.
3. Lag phase occurs when bacteria are adjusting to them medium. For example, with a
nutritionally poor medium, several anabolic pathways need to be turned on, resulting in a
lag before active growth begins.
4. In log or exponential phase, the cells are growing as fast as they can, limited only by
growth conditions and genetic potential. During this phase, almost all cells are alive, they
are most nearly identical, and they are most affected by outside influences like
disinfectants.
5. Due to nutrient depletion and/or accumulation of toxic end products, replication stops and
cells enter a stationary phase where there is no net change in cell number.
6. Death phase occurs when cells can no longer maintain viability and numbers decrease as a
proportion.
Phases of Growth
• Lag phase – making new enzymes in response to new medium
• Log phase – exponential growth
• Desired for production of products
• Most sensitive to drugs and radiation during this period
• Stationary phase –
• nutrients becoming limiting or waste products becoming toxic
• death rate = division rate
• Death phase – death exceeds division
Bacterial growth
• The time taken for a microbial population to double in
number is called the doubling time. The time taken for a
single cell to divide is called the generation time
2N
1N
N DT
time
• The mean generation time of a population is equal to the

doubling time.
• Doubling time is a measure of growth rate

a short doubling time implies a fast growth rate.
Mean Generation Time and Growth Rate
• The mean generation time (doubling time) is the amount of time

required for the concentration of cells to double during the log
phase. It is expressed in units of minutes.
1
• Growth rate (min-1) =
mean generation time
• Mean generation time can be determined directly from a

semilog plot of bacterial concentration vs time after inoculation
Mean Generation Time and Growth Rate
As growth increases biomass increases: during log phase
dx = μx x = cell conc (biomass) (mg/m3)

dt t = incubation time (h)
μ = specific growth rate (h-1)
dx. 1 =μ
dt x
dx
dt μ =slope
x
Beginning of log phase t=0 biomass X0
On integration of equation 1
∫dx = ∫ μ dt
x
ln X = μ t + K (integration constant)
when t=0
ln X0 = K
ln X = μ t + ln X0
ln X –ln X0 = μ t
X X
ln =𝜇t or 2.303 log10 =𝜇t
X0 X0
ln X . 1 = td
X0 μ
𝑊ℎ𝑒𝑛 𝑡 = 𝑡𝑑
X = 2𝑋0
2𝑋0 1
𝑙𝑛 . = 𝑡𝑑
𝑋0 𝜇
ln 2
= 𝑡𝑑
μ
0.693
= 𝑡𝑑
μ
0.693
μ=
𝑡𝑑
μ is inversely proportional to 𝑡𝑑
If 𝑡𝑑 is high μ is low and vice versa
Concept check
• Starting with four bacterial cells per milliliter in a rich nutrient
medium, with a 1 h lag phase and a 20 min generation time, how
many cells will there be in 1 liter of this culture after 1 h? After 2 h?
After 2 h if one of the initial four cells was dead?
Answer
All cells are viable: After one hour, there would still be only the four
original cells since the lag time is one hour.
After two hours, three divisions would have occurred, since the
generation time is 20 minutes; the population would be 4 x 23 = 32
cells/ml.
One of the initial cells is dead: After two hours the population would be
3 x 23 = 24 cells/ml.
Constitutive Rate Expressions for Biological Processes
• Model of Microbial Growth

• Structured and Segregated- cells are different from one another
• Unstructured, Non-segregated models lump the population into one biophase
interacting with external environment and is one species in solution; they are
mathematically simple
• External environment influences cell response and can confer new growth characteristics
on the cell
13
Unstructured Growth Models
• Simple relationships that describe exponential growth
• Kinetics of cell growth are described using cell and nutrient concentration
profiles
• Malthus’s simple model: rx = µX where rx is the increase in dry cell weight
and µ(hr-1) is a constant
14
Growth Kinetics
Introduction
Net specific growth rate (1/time):
net   g  kd
g : Gross specific growth rate (1/time)
kd : The rate of loss of cell mass due to cell death

or endogenous metabolism
Endogenous metabolism: during the stationary phase,

the cell catabolizes cellular reserves for new building blocks and
for energy-producing monomers.
15
Growth Kinetics
Introduction
Net specific replication rate (1/time):
1 dN
R 
N dt
R
R   '  kd
R
N : Cell number concentration (cell number /L)

 ' : Gross specific replication rate (1/time)
R
kd : The rate of cell death (1/time) 16

Monod Model
• Developed by Jaques Monod, exemplifies the effect of
nutrient concentration based on E. coli growth are
various sucrose concentrations and assumes only the
limiting substrate is important in determining the rate of
cell proliferation
17
For the Monod Model
• Cell growth might follow the form
rx = µX = µmaxSX/(Ks + S)
and batch growth at constant volume
dX/dt = µmaxSX/Ks + S
µmax is max specific growth rate of cells
Ks is value of the limiting nutrient concentration; substrate affinity
Two limiting forms:
• At high substrate concentrations S>>Ks, µ= µmax
• At low substrate concentrations S<<Ks, µ= µmax/KsS
18
 = μ max S/(Ks +S)
μ = specific growth rate

μ
μ max
1/2 μ max
Ks = substrate concentration
Residual substrate conc. [S]
19
ks
• Bacteria with a high affinity for substrate has a low Ks and vice versa
• The higher the affinity the less growth is affected until substrate levels
are very low
20
Models of Growth and Non-growth Associated
Product Formation
• Primary metabolites- growth associated; rate of production parallels
growth of cell population; Ex: gluconic acid
• Secondary metabolites- non-growth associated; kinetics do not
depend on culture growth rate; Ex: antibiotics, vitamins
• Intermediate products- partially growth associated; Ex: amino acids,
lactic acid
21
Kinetic Pattern of Growth and Product Formation
Growth-associated Mixed-growth-associated Non growth-associated

22
Kinetic Pattern of Growth and Product Formation
1 𝑑𝑃
𝑞𝑃 = , specific rate of product formation
𝑋 𝑑𝑡
Growth-associated
𝑞𝑃 = 𝑌𝑃/𝑋 𝜇𝑔
Mixed-growth-associated
𝑞𝑃 = 𝛼 𝜇𝑔 + 𝛽 𝛼, 𝛽 = 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡
Non growth-associated
𝑞𝑃 = 𝛽
23
Example 1
A strain of mold was grown in a batch culture on glucose and the following data were obtained.
Time (h) Cell conc. (g/L) Glucose conc. (g/L)

0 1.25 100 1. Calculate the maximum net specific growth
rate.
9 2.45 97
16 5.1 90.4 2. Calculate the apparent growth yield.
23 10.5 76.9
3. What maximum cell conc. could one expect if
30 22 48.1
150 g of glucose were used with the same size
34 33 20.6 of inoculum ?
36 37.5 9.38
40 41 0.63
24
Solution
ln 𝑋2 −ln 𝑋1 ln 37.5 −ln 5.1

1. 𝜇𝑛𝑒𝑡 = = ≅ 0.1 ℎ−1
𝑡2 −𝑡1 36 −16
41 −1.25
2. 𝑌𝑋/𝑆 = ≅ 0.4 𝑔 𝑐𝑒𝑙𝑙𝑠/𝑔 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒
0.63 −100
3. 𝑋𝑚𝑎𝑥 = 𝑋0 + 𝑌𝑋/𝑆 𝑆0 = 1.25 + 0.4 150 = 60.25 𝑔 𝑐𝑒𝑙𝑙𝑠/𝐿
25

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Estimation of Microbial Growth

Standard Growth Curve

• The mean generation time of a population is equal to the

• Doubling time is a measure of growth rate

• The mean generation time (doubling time) is the amount of time

• Mean generation time can be determined directly from a

dx = μx x = cell conc (biomass) (mg/m3)

• Model of Microbial Growth

kd : The rate of loss of cell mass due to cell death

Endogenous metabolism: during the stationary phase,

N : Cell number concentration (cell number /L)

kd : The rate of cell death (1/time) 16

μ = specific growth rate

Residual substrate conc. [S]

Growth-associated Mixed-growth-associated Non growth-associated

Time (h) Cell conc. (g/L) Glucose conc. (g/L)

ln 𝑋2 −ln 𝑋1 ln 37.5 −ln 5.1

3. 𝑋𝑚𝑎𝑥 = 𝑋0 + 𝑌𝑋/𝑆 𝑆0 = 1.25 + 0.4 150 = 60.25 𝑔 𝑐𝑒𝑙𝑙𝑠/𝐿

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