CHEMICAL ENGINEERING DEPARTMENT

INTRODUCTION TO BIOTECHNOLOGY
MWF 1400 – 1500
Engr. Jeremiah Emier C. Villanueva

BATCH MICROBIAL GROWTH

Prepared by: Credo, Allen

De Padua, Princess Faye
Dela Cruz, Khuert Alfred
Gusto, Mae Deshiela
Tamoria, Patrice Pauline
Microbial Growth
- Increase in number of cells, not
cell size
- “One cell becomes colony of
millions of cells”
- Control of growth is important for
a.) infection control and b.) growth
of industrial and biotechnical
organisms
Factors Regulating Microbial Growth
Energy Sources
Sunlight = phototroph
Organic = chemotroph
Chemotroph – derive both carbon and
energy from organic compounds
Chemoorganic autotroph – derives
energy from organic compounds and
carbon source from inorganic
compounds
Litoautotroph – neither sunlight nor
organics are used, relies only on
inorganics
Saprobe – lives on organic matter of
dead organisms

- Nutrients Parasite – lives on organic matter of

- Environmental conditions living host = pathogens
o Temperature
o pH Environmental Factors Influencing
o osmotic pressure Growth
- Generation time
1. Temperature
Chemical Requirements 2. O2
3. pH
1. Water 4. Osmotic Pressure
2. CHONPS 5. Others (radiation, atmospheric
- C is 50% of cell’s dry weight pressure, etc.)
- Trace elements
3. Organic compounds Temperature Optima
- Glucose (source of energy)
- Vitamins (Coenzymes) Psychrophiles: cold-loving
- Amino acids, purines, pyrimidines Mesophiles: moderate temperature-
loving
Nutritional Categories Thermophiles: heat-loving
Each has a minimum, optimum, and
Carbon Sources maximum growth temperature
CO2 = autotroph
Organic = heterotroph
1|Batch Microbial Growth (04102019 )
 CHEMICAL ENGINEERING DEPARTMENT
INTRODUCTION TO BIOTECHNOLOGY
MWF 1400 – 1500
Engr. Jeremiah Emier C. Villanueva

BATCH MICROBIAL GROWTH

• Helicobacter pylori lives in

stomach under mucus layer

Measuring Bacterial Growth

Bacterial Division
• Bacteria divide by binary fission
• Alternative means
• Budding
• Conidiospores
(filamentous bacteria)
• Fragmentation

- Optimum growth temperature is usually

near the top of the growth range
- Death above the maximum temp.
comes from enzyme inactivation
- Mesophiles most common group of
organisms
- 40ºF (5°C) slows or stops growth of
most microbes

Oxygen Requirements

• Obligate aerobes – require O2

• Facultative anaerobes – can use
O2 but also grow without it
• Obligate anaerobes – die in the
presence of O2

pH

• Most bacteria grow between pH

6.5 and 7.5
• Acid (below pH 4) good
preservative for pickles,
sauerkraut, cheeses
• Acidophiles can live at low pH
• Many bacteria and viruses survive
low pH of stomach to infect
intestines

2|Batch Microbial Growth (04102019 )

 CHEMICAL ENGINEERING DEPARTMENT
INTRODUCTION TO BIOTECHNOLOGY
MWF 1400 – 1500
Engr. Jeremiah Emier C. Villanueva

BATCH MICROBIAL GROWTH

Generation Time

• Time required for cell to divide/for

population to double
• Average for bacteria is 1-3 hours
• E. coli generation time = 20 min
• 20 generations (7 hours), 1
cell becomes 1 million cells
Plotting growth on graphs

Standard Growth Curve

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BATCH MICROBIAL GROWTH
Growth Phases
• 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
Measuring Growth
• Direct methods – measure the
count individual cells
• Indirect Methods – intracellular
components of cells are
measured (e.g., RNA, DNA,
protein)
- Cell Number Density -
Petroff – Hausser Slide
- also known as hemocytometer
- a calibrated grid is placed over the
culture chamber, and the number of cells
per grid square is counted using a
microscope
-To be statistically reliable, at least 20
grid squares must be counted and
averaged.
4|Batch Microbial Growth (04102019 )
CHEMICAL ENGINEERING DEPARTMENT
INTRODUCTION TO BIOTECHNOLOGY
MWF 1400 – 1500
Engr. Jeremiah Emier C. Villanueva
Plate Count Method
- Culture samples are diluted and spread
on the agar surface and the plates are
incubated. Colonies are counted on the
agar surface following the incubation
period
Ring Mounted Slides
- Agar-gel medium is placed in a small
ring mounted on a slide
- Cells are spread on dish, incubated and
examined in a microscope
Particle Counters
- 2 electrodes (1 in tube at vacuum and 1
in solution) and 1 electrolyte solution
- Vacuum is applied at tube to let solution
pass through orifice
- Electrical resistance causes pulses in
voltage
- No. of pulses = no. of cells
- Height of the pulse = measure of cell
size
Turbidity
- employs the use of a spectrometer and
correlates the amount of cell in a sample
with the percentage of light transmitted
- Cell Mass Concentration -
Dry Weight
- Used as cell mass determining
technique for cells in solid-free
medium
- Culture is washed then dried
- Mass of filter with and without the
culture is obtained
 CHEMICAL ENGINEERING DEPARTMENT
INTRODUCTION TO BIOTECHNOLOGY
MWF 1400 – 1500
Engr. Jeremiah Emier C. Villanueva

BATCH MICROBIAL GROWTH

Advantage: Easy and Quick If cell response is fast compared

Disadvantage: No distinction between to external changes and if the magnitude
live and dead cells of these changes is not too large (e.g., a
10% or 20% variation from initial
Packed Cell Volume conditions), then the use of unstructured
models can be justified, since the
- used for volume determination of cells deviation from balanced growth may be
- parallel test in a hemocytometer and small.
packed cell volume is done for cell count
density determination Structure – Component of an individual
cell
Considerations for Modelling Segregation – Setting apart of individual
Microbial Growth cells in a colony

Cellular composition and biosynthetic Unstructured Non-segregated Models

capabilities change in response to new
growth conditions (unbalanced growth). Substrate-limited growth
Although these kinetic ideas are evident - a single chemical species, S, is
in batch culture, they are equally evident growth-rate limiting (i.e., an
and important in other modes of culture increase in S influences growth
(e.g., continuous culture). rate, while changes in other
nutrient concentrations have no
The complete description of the effect)
growth kinetics of a culture would involve
recognition of the structured nature of
each cell and the segregation of the
culture into individual units (cells) that
may differ from each other. If the ratio of
Monod Equation
these components can change in
response to perturbations in the
Monod Equation - describes substrate-
extracellular environment, then the
limited growth only when growth is slow
model is behaving analogously to a cell
and population density is low
changing its composition in response to
environmental changes.
If the consumption of a carbon–
Unstructured model – assumes fixed cell energy substrate is rapid, then the
release of toxic waste products is more
composition or balanced growth
likely.
Balanced growth assumption - valid
primarily in single-stage, steady-state At high population levels, the buildup
continuous culture and the exponential of toxic metabolic by-products becomes
phase of batch culture more important.

5|Batch Microbial Growth (04102019 )

 CHEMICAL ENGINEERING DEPARTMENT
INTRODUCTION TO BIOTECHNOLOGY
MWF 1400 – 1500
Engr. Jeremiah Emier C. Villanueva

BATCH MICROBIAL GROWTH

Inhibition by toxic compounds

The following rate expressions are

used for competitive, noncompetitive,
and uncompetitive inhibition of growth in
analogy to enzyme inhibition.

In some cases, the presence of toxic

compounds in the medium results in the
inactivation of cells or death.

Other models are based on

enzyme inhibition.

Substrate Inhibition

At high substrate concentrations,

microbial growth rate is inhibited by the
substrate. As in enzyme kinetics,
substrate inhibition of growth may be
competitive or noncompetitive. If a
single-substrate enzyme-catalyzed
reaction is the rate limiting step in
microbial growth, then inhibition of
enzyme activity results in inhibition of
microbial growth by the same pattern.

Product inhibition

High concentrations of product

can be inhibitory for microbial growth.
Product inhibition may be competitive or
noncompetitive, and in some cases when
the underlying mechanism is not known,
the inhibited growth rate is approximated
to exponential or linear decay
expressions.

6|Batch Microbial Growth (04102019 )