1 Michigan Technological University David R. Shonnard 1 Chapter 10: Sterilization and Bioreactor Operation David Shonnard Department of Chemical Engineering Michigan Technological University Michigan Technological University David R. Shonnard 2 Sterilization Methods and Kinetics: 10.4 Sterility : the absence of detectable levels of viable organisms in a culture medium or in a gas Reasons for Sterilization 1. Economic penalty is high for loss of sterility 2. Many fermentations must be absolutely devoid of foreign organisms 3. Vaccines must have only killed viruses 4. Recombinant DNA fermentations - exit streams must be sterilized
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Michigan Technological UniversityDavid R. Shonnard1
Chapter 10: Sterilization and Bioreactor Operation
David ShonnardDepartment of Chemical Engineering
Michigan Technological University
Michigan Technological UniversityDavid R. Shonnard2
Sterilization Methods and Kinetics: 10.4
Sterility: the absence of detectable levels of viable organisms in a
culture medium or in a gas
Reasons for Sterilization
1. Economic penalty is high for loss of sterility
2. Many fermentations must be absolutely devoid of foreign
organisms
3. Vaccines must have only killed viruses
4. Recombinant DNA fermentations - exit streams must be
sterilized
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Michigan Technological UniversityDavid R. Shonnard3
Sterilization Agents
1. Thermal - preferred for economical large-scale sterilizations of
liquids and equipment.
2. Chemical - preferred for heat-sensitive equipment→ ethylene oxide (gas) for equipment→ 70% ethanol-water (pH=2) for equipment/surfaces→ 3% sodium hypochlorite for equipment
3. Radiation - uv for surfaces, x-rays for liquids (costly/safety)
4. Filtration→ membrane filters having uniform micropores→ depth filters of glass wool
Michigan Technological UniversityDavid R. Shonnard4
Kinetics of Thermal Sterilization (Death)
Practical considerations:
1. Not all organisms have identical death kinetics. → (increasing difficulty; vegetative cells < spores < virus)
2. Individuals within a population of the same organism may
respond differently
From Probability Theory:
p(t) = the probability that an individual cell is still viable at time t.
p(t) = e-k
dt (simplest form assuming 1st orde
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Michigan Technological UniversityDavid R. Shonnard5
Kinetics of Thermal Sterilization (cont.)
E[N(t)]= expected value (E) of the numb
of individual organis
at time t after sterilization starts.
= No p(t) = No e-k
dt
where No is the initial number of individuals
N(t)
N o
= e-kdt or ln
N(t)
N o
= -kdt "survival cur
Michigan Technological UniversityDavid R. Shonnard6
Temperature Effects on the Kinetics of Thermal Sterilization
Arrhenius Equation
kd = α e-Eod / RT
α = constant (time-1)
R = gas constant
T = absolute temperature
Eod = activation energy for death
(50 -150 kcal / g - mole) for spores
(2 - 20 kcal / g - mole) for vitamins / growth factors
ln
N(t
No
t
-kd
increasing
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Michigan Technological UniversityDavid R. Shonnard7
Population Effects on the Kinetics of Thermal Sterilization
Most Thermal Sterilizations are at 121˚C
Organism kd (min-1)
Vegetative cells >1010
Spores 0.5 to 5.0
Spores are the primary concern during thermal sterilization
Michigan Technological UniversityDavid R. Shonnard8
System Variables for Thermal Sterilization
Primary System Variables in Thermal Sterilization
1. Initial concentration of organisms
2. Temperature, T
3. Time (t) of exposure at temperature T.
Probability of an Unsuccessful Ferme [1-Po(t)
[1-Po(t)] = 1-[1-p(t)]N o
= 1-[1-e-k
dt]N o (for a homogeneous popul
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Michigan Technological UniversityDavid R. Shonnard9
Sterilization Chart
“Bioprocess Engineering: Basic Concepts”Shuler and Kargi, Prentice Hall, 2002
Michigan Technological UniversityDavid R. Shonnard10
System Variables for Thermal Sterilization
Use of Sterilization Charts:
1. Specify 1-Po(t) which is acceptable (e.g. 10-3)
2. Determine No in the system.
3. Read kdt from the chart.
4. Knowing kd for the spores (or cells), obtain the required time, t.
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Michigan Technological UniversityDavid R. Shonnard11
Scale-up of Sterilization
→ in batch sterilization, scale-up of small-scale sterilization data
to a much larger scale will result in unsuccessful sterilization
1-Liter Vessel 104 -L Vessel
no = 104 spores/L no = 10
4 spores/L
No = (1 L)(no) No = (104 L)(no)
[1-Po(t)] = 1-[1-e-k
dt]no [1-Po(t)] = 1-[1-e
-kdt]10
4no
= .003 = 1-5x10-14 ≈ 1
1515
Michigan Technological UniversityDavid R. Shonnard12
Batch vs. Continuous Sterilization
Batch
1. Longer heat-up/cool down time
2. Incomplete mixing
“Bioprocess Engineering: Basic Concepts”Shuler and Kargi, Prentice Hall, 2002
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Michigan Technological UniversityDavid R. Shonnard13
Batch vs. Continuous Sterilization
Continuous 1. Shorter time
2. Higher temperature
“Bioprocess Engineering: Basic Concepts”Shuler and Kargi, Prentice Hall, 2002
Michigan Technological UniversityDavid R. Shonnard14
Sterilization of Gases
→ aerobic fermentations require 0.1 to 1.0 (L air / (L liquid • min))→ 50,000 L fermenter requires 7x106 to 7x107 L air/day→ microorganism concentrations in air are about 1-10 / L air