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Chapter 9 Steriliztion Process

The document discusses sterilization techniques for fermentation processes. It describes how contamination can negatively impact fermentation and outlines various sterilization methods for fermentation media, air, and fermenters. These include heating media to 121°C, filtering media and air, exposing materials to radiation or chemicals like ethylene oxide, and steaming fermenters in situ at 15 psig for 20 minutes. Proper sterilization of all materials, air, and the fermenter environment is necessary to maintain sterile and contamination-free conditions for microbial growth and product formation.

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Débora Costa Reis
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58 views16 pages

Chapter 9 Steriliztion Process

The document discusses sterilization techniques for fermentation processes. It describes how contamination can negatively impact fermentation and outlines various sterilization methods for fermentation media, air, and fermenters. These include heating media to 121°C, filtering media and air, exposing materials to radiation or chemicals like ethylene oxide, and steaming fermenters in situ at 15 psig for 20 minutes. Proper sterilization of all materials, air, and the fermenter environment is necessary to maintain sterile and contamination-free conditions for microbial growth and product formation.

Uploaded by

Débora Costa Reis
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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BIOREACTOR ENGINEERING

Chapter 9
Sterilization in Fermentation
by
Chew Few Ne
Faculty of Chemical & Natural Resources
Engineering
cfne@ump.edu.my

Sterilization in Fermentation by Chew Few Ne


Chapter Description

• Topic Outcomes
– Describe appropriate sterilization technique for medium,
air and fermenter.
– Perform sterilization calculation.

• References
– Doran, P.M. (2013) Bioprocess Engineering Principles.
Elsevier.
– Liu, S. (2013) Bioprocess Engineering: Kinetics,
Biosystem, Sustainability and Reactor Design. Elsevier.
– Rao, D.G. (2010) Introduction to Biochemical Engineering.
McGraw Hill.
Sterilization in Fermentation by Chew Few Ne
Topic Outline

• Introduction
• Medium Sterilization
• Air Sterilization
• Fermenter Sterilization

Sterilization in Fermentation by Chew Few Ne


Introduction

• Effect of contamination on fermentation:


– Medium is consumed unnecessarily  may affect the
growth of desired organism & outweigh the desired
product.
– Fermentation condition is affected  may affect the
growth of desired organism & outweigh the desired
product.
– Desired product is contaminated  interfere the
downstream process.
Foreign Foreign
produce
organisms products

Sterilization in Fermentation by Chew Few Ne


Introduction

• Fermentation involves:

Nutrients
Fermenter

- Microorganism
- Medium (thousands of liters )
Air (millions of liters )

• A sterile environment is needed for all the above.


Sterilization in Fermentation by Chew Few Ne
Introduction

• Methods to avoid contamination in a fermentation


process:
– Sterilization of the medium.
– Sterilization of the fermenter.
– Sterilization of all materials to be added to the fermenter.
– Sterilization of air.
– Employing pure inoculum.
– Sterilizing the pipes, valves, and bends. which come in
contact with the fermentation process.
– Maintaining aseptic conditions in the fermenters during
fermentation.
– Maintaining optimum or desired pH.
Sterilization in Fermentation by Chew Few Ne
Introduction

• Methods available for sterilization:


– Chemical - preferred for heat-sensitive equipment
• ethylene oxide (gas)
• 70% ethanol
• 3% sodium hypochlorite
– Exposure to radiation
• UV for surface
• X-ray for liquid
– Filtration
– Heating
Sterilization in Fermentation by Chew Few Ne
Medium Sterilization

• Heating method: The medium is heated to the


sterilization temperature (121ᵒC).
𝑑𝑁
– Thermal death kinetics of microorganisms: = −𝑘𝑑 𝑁
𝑑𝑡
where,
N = number of live cell
t = sterilization time
kd = specific death constant
𝑁𝑡
– For constant kd, integration: 𝑙𝑛 𝑁 = −𝑘𝑑 𝑡
𝑜
where,
No = initial number of live cell
Nt = number of live cell still present after a time period of t
Sterilization in Fermentation by Chew Few Ne
Medium Sterilization

• Exercise 1

Sterilization in Fermentation by Chew Few Ne


Medium Sterilization
𝐸
• kd is function of temperature: 𝑘𝑑 = − 𝑑
𝐴𝑒 𝑅𝑇
where,
A = Arrhenius constant
Ed = activation energy for thermal cell death
R = ideal gas constant
T = absolute temperature

– For Bacillus stearothermophilus, Ed is about 70 kcal/gmol.


– For E. coli, Ed is about 127 kcal/gmol.
– For vitamin in medium, Ed is about 2-20 kcal/gmol.
• EA > EB
– Increase in temperature will destroy more A compared to B.

Sterilization in Fermentation by Chew Few Ne


Medium Sterilization

• Exercise 2

Sterilization in Fermentation by Chew Few Ne


Medium Sterilization

• Filter method: Suitable for medium containing heat-


sensitive components.
– Membrane: made of cellulose esters or other polymers,
pore diameter between 0.2-0.45 μm.
– Membrane itself must be sterilized by steam or radiation
before use.
– Bacteria and other particles with dimensions greater than
the pore size are screened out and collected on the surface
of the membrane.
– Filtration is not as effective or reliable as heat sterilization.

Sterilization in Fermentation by Chew Few Ne


Air Sterilization

• The number of microbial cells in air = 103 – 104 /m3


• Method of air sterilization:
– Sterilization by heating (economically impractical).
– Radiation (UV rays).
– Use of germicidal sprays (e.g., phenol, ethylene oxide or
formalin).
– Sterilization by filtration.
• Filtration sterilization is commonly used.

Sterilization in Fermentation by Chew Few Ne


Air Sterilization

• Filter is also used to sterilize off-gases leaving the


fermenter.
– The concentration of cells in unfiltered fermenter off-gas is
several times greater than in air.
– Organisms (e.g., pathogens) are harmful to plant personnel
or environment.
• Two types of air filter:
– Depth filter (Fibrous filter): Filter with pores that are bigger
than the size of the microorganism to be removed.
– Surface filter (Absolute filter): Filter with pores that are
smaller than the size of the microorganism to be removed.
Sterilization in Fermentation by Chew Few Ne
Fermenter Sterilization

• Any joints, crevices, pits or flange joint are potential


hazardous points where the nutrients and microorganism
stay, hence contamination can take place.
• Fermenter should be free of crevices and stagnant areas,
have minimum number of joints, polished welded joints are
used, joint point should be as smooth as possible.
• Industrial fermenters are designed for in situ steam
sterilization under pressure (15 psig for 20 min).
• After sterilization, the fermenter should be flushed with
sterile air to keep under positive pressure.
• After sterilization, sterile medium and air entering
fermenter must be sterile. The transport line should be
maintained under aseptic conditions.
Sterilization in Fermentation by Chew Few Ne
CREDITS

Special thanks to
- Prof. Dr. Tey Beng Ti
- Prof. Madya Dr. Rosfarizan binti Mohamad
- Dr. Farhan binti Mohd Said

Thanks to Dr. Rozaimi bin Abu Samah for


proofreading the learning contents.

Sterilization in Fermentation by Chew Few Ne

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