Chapter 5 Ft Edited

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CHAPTER 5

CHAPTER 5 STERILIZATION

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Introduction

A fermentation product is produced by the culture of a certain organism, or

organisms, in a nutrient medium. If the fermentation is invaded by a

foreign microorganism then the following consequences may occur:

i)

The m

The medi

edium wo

um would h

uld have t

ave to sup

o suppor

port the g

t the grow

rowth of b

th of both t

oth the pr

he produ

odu

cti

ction

on

organism & the contaminant, resulting in a

loss of productivity.

ii)

If the

ferm

fermentati

entati

on is a

contin

continuous o

uous one the

ne then the c

n the c

ontami

ontaminant

nant

may‘outgrow’

the production organism & displace it from the

fermentation.

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v) The contaminant may degrade the desired product; this is common in

bacterial contamination of antibiotic fermentations where the

contaminant would have to be resistant to the normal inhibitory effect of

the antibiotic & degradation of the antibiotic is a common resistance

mechanism, e.g. the degradation of

mechanism, e.g. the degradation of 

-lactam antibiotics by

-lactam antibiotics by



- lactamase-

-

lactamase-producing bacteria.

producing bacteria.

vi) Contamination of a bacterial fermentation with phage could result in the

lysis of the culture.

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•

• Avoidance of contamination may be achieved by:

Avoidance of contamination may be achieved by:

i)

Usi

Using a

ng a

pur

pure in

e inocu

oculum

lum

to s

tart

the

the fer

ferment

mentati

ation.

on.

ii)

Steril

Sterilizing

izing

the

the medium

medium

to be

employ

employed.

ed.

iii)

Steril

Sterilizing the ferment

izing the fermenter vessel.

er vessel.

iv)

Sterilizing all

Sterilizing all materials to be added to

materials to be added to the fermentation

the fermentation

during the process.

v)

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Medium Sterilization

•

• The first step in the sterilization of any medium is the

The first step in the sterilization of any medium is the

selection of an appropriate vessel in which to carry out the

sterilization process.

•

• This is obviously volume dependent and can range from

This is obviously volume dependent and can range from

something as simple as a screw cap bottle, through shake

flasks for inoculum development, to the complexicity of the

bioreactor itself.

•

• Sterilization is normally carried out by heat or filtration.

Sterilization is normally carried out by heat or filtration.

Although other methods exists, such as irradiation, chemical

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•

• Before the techniques which are used for the steam

Before the techniques which are used for the steam

sterilization for the culture medium are discussed, it is

necessary to discuss the kinetics of sterilization.

•

• The destruction of microorganisms by steam (moist heat) may

The destruction of microorganisms by steam (moist heat) may

be described as a first-order chemical reaction, & may be

represented by the following equation:

-dN/dt = kN

Where;

N is the number of viable organism present

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On integration of equation, give

N

_t

/N

_o

= e

– –

_kt

Where,

N

_o_o

is the number of viable organisms present at the start of the sterilization

treatment

N

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--

_{The relationship between T and}

_{the reaction rate constant is:}

d ln k/dT = E/RT

2

2 E

E

is

is the

the activation

activation energy

energy

R

is

is the

the gas

gas constant

constant

T

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Deindoerfer and Humphrey (1959) used the term ln No/Nt as a

design criterion for sterilization which has been variously called the

DEL FACTOR, Nabla factor & sterilization criterion represented by the

term



.

The Del factor is a measure of the fractional reduction in viable

org

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A risk factor of one batch in a thousand being contaminated is

frequently used in the fermentation industry.

Two basic types reaction contribute to the loss of nutrient

quality during sterilization:

i)

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ii)

Degradation of heat labile (heat sensitive) components such

as vitamins & amino acids.

•

• Reaction of this type may be minimized by using a suitable

Reaction of this type may be minimized by using a suitable

time-temperature sterilization regime.

The thermal destruction of essential media components

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•

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Advantages of continuous sterilization over batch

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Advantages of batch sterilization over continuous

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THE DESIGN OF

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The destruction of a nutrient may be considered a first-order

reaction & its rate will be affected by T

x

_t

/x

_o

= e

– –

kt

Where k is

Where k is the reaction rate constant

the reaction rate constant

Or

x

_o

/x

_t

= e

kt

Thus, taking natural logarithms,

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STERILIZATION OF THE FERMENTER

--

If t

If the m

he med

ediu

ium is s

m is ste

teri

rili

lize

zed in a s

d in a sep

epara

arate ba

te batc

tch co

h cook

oker, o

er, or is

r is

continuously sterilized, then the fermenter has to be

sterilized separately before the sterile medium is added to it.

--

Thi

This is

s is norm

norm

all

ally ac

y achie

hieved

ved by h

by heat

eating

ing the

the jac

jacket

ket or c

or coil

oils of

s of the

the

fermenter with steam & sparging steam into the vessel

through all entries.

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STERILIZATION OF AIR

--

Fi

Filt

lters

ers

fo

for t

r the r

he rem

emov

oval o

al of mi

f mi

cr

croo

oorga

rgani

nism f

sm fro

rom a

m an

n

environment may be divided into two larger group

i)

Tho

Those wh

se wh

ich t

ich the po

he pores i

res i

n the f

n the fil

ilter a

ter are sm

re small

aller tha

er than th

n the

e

particles which are to be removed.

ii)

Those w

hich the po

hich the pores are l

res are larger tha

arger tha

n the parti

n the particles w

cles which are

hich are

to be removed.

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