chapter 5 ft edited

8/4/2019 Chapter 5 Ft Edited

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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 medium would have to support the growth of both the production

organism & the contaminant, resulting in a loss of productivity.

ii) If the fermentation is a continuous one then the contaminant

mayoutgrow the production organism & displace it from the

fermentation.

iii) The foreign organism may contaminate the final product, e.g. single cellprotein where the cells, separated from the broth, constitute the product.

iv) The contaminant may produce compounds which make subsequent

extraction of the final product difficult


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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 resistancemechanism, e.g. the degradation of-lactam antibiotics by - lactamase-

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:

i) Using a pure inoculum to start the fermentation.

ii) Sterilizing the medium to be employed.

iii) Sterilizing the fermenter vessel.

iv) Sterilizing all materials to be added to the fermentation

during the process.v) Maintaining aseptic condition during the fermentation.


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Medium Sterilization

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

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.

Although other methods exists, such as irradiation, chemical

sterilization and ultrasonic treatment, however, steamis usedalmost universally for the sterilization of fermentation media.


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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

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

t is time of the sterilization treatment

k is the reaction rate constant of the reaction, or the specific

death rate.


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On integration of equation, give

Nt/No = ekt

Where,

No

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

Nt is the number of viable organism present after a treatment period, t

ln Nt/No = -kt

kt = ln Nt/No = ln No/Nt

As with the first-order reaction, the reaction rate increase with increase intemperature due to an increase in the reaction rate constant, which,in thecase of the destruction of microorganism, is the specific death rate (k).

Thus, the reaction-rate constant (k) is a true constant only under constanttemperature condition.


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- The relationship between T and the reaction rate constant is:

d ln k/dT = E/RT2

E is the activation energy

R is the gas constant

T is the absolute temperature

On integration :

k = Ae E/RT

ln K = ln A E/RT

(A is the Arrhenius constant)Expression that may be derived for the heat sterilization of a pure

culture at a constant temperature:

ln No/N

t= A. t. e E/RT


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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

organism count produced by a certain heat and time regime.

Therefore,

= ln No/Ntbut ln No/Nt = kt

and kt= A.t.e-(E/RT)

Thus, = A.t.e -(E/RT)

Rearranging eq.

ln t = E/RT + ln (/A)


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A risk factor of one batch in a thousand being contaminated isfrequently used in the fermentation industry.

Two basic types reaction contribute to the loss of nutrientquality during sterilization:

i) Interaction between nutrient components of the medium.

- A common occurrence during sterilization is the Maillard-typebrowning reaction which results in discolorization of themedium as well as a loss in nutrient quality.

- These reaction are normally caused by the reaction ofcarbonyl groups, usually from reducing sugars, with the amino

groups from amino acids & protein.- If browning reactions occur then it is usually necessary to

sterilized the carbohydrate component of the mediumseparately from the remainder of the medium & torecombine the two after cooling


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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

time-temperature sterilization regime.

The thermal destruction of essential media components

conforms approximately with first order reaction kinetic &

therefore, may be described by equations similar to thosederived for the destruction of bacterial.

xt/xo = e-kt

Where

Xt is the amount of nutrient after heat treatment period,t

Xo is the original amount of nutrient at the start of the heat

treatment


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in the consideration of Del factor it was evident that the sameDel factor could be achieved over a wide range oftemperature/time regime.

thus, it would be advantageous to employ a high temperaturefor a short time to achieve the desired probability of sterility,yet causing minimum nutrient degradation

thus, the ideal technique would be to heat the fermentation

medium to a high temp, at which it is held for a short period,before being cooled rapidly to the fermentation temperature

however, it is imposibble to heat a batch of many thousandsof litres of broth to a high temp, hold for a short period & coolw/out the heating & cooling periods contributing

considerably to the total sterilization time. the only practical method is to sterilize the medium in a

continuous stream.


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Advantages of continuous sterilization over batch

sterilization

i. Superior maintenance of medium quality

ii. Ease of scale-up

iii. Easier automatic control

iv. The reduction of surge capacity for steam

v. The reduction of sterilization cycle time

vi. Under certain circumstances, the reduction of fermenter

corrosion.


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Advantages of batch sterilization over continuous

sterilization

i. Lower capacity equipment costs

ii. Lower risk of contamination

iii. Easier manual control

iv. Easier to use the media containing a high proportion of solid

matter.


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THE DESIGN OF BATCH STERILIZATION PROCESS

Although a batch sterilization process is less successful inavoiding the destruction of nutrients than a continuous , the

objective of designing a batch process is still to achieve the

required probability of obtaining sterility with the minimum

loss of nutrient quality.

The highest T for batch sterilization is 121oC. Heating &

cooling periods of the batch treatment must be considered.


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- The following information must be available for the design of

a batch sterilization process:

i) A profile of the increase & decrease in temperature of the

fermentation medium during heating & cooling periods of the

sterilization cycle.

ii) The number of microorganism originally present in the

medium

iii) The thermal death characteristics of the design organism.,

it is being assumed that all the organism detected are spores

ofBacillus stearothermophilus

iV) The risk of contamination considered acceptable for the

process.


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The Del factor has already been defined as:

= ln No/Nt

Knowing the original number of organism present in thefermenter & the risk of contamination considered acceptable,the required Del factor may calculated.

A frequently adopted risk of contamination is 1 in 1000 whichindicates Nt should equal 10

-3 of a viablecell.

e.g: If unsterile broth contain 1011 viable organisms then Delfactor may be calculated, thus;

= ln 1011/10-3

= ln 1014

= 32.2

Therfore, overall Del factor required is 32.2


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- However the destruction of cells occurs during the heating &cooling of the broth as well as during the period at 121oC,thus the overall Del factor may be represented as:

overall = heating + holding + cooling

Calculation of the Del factor during heating & cooling

- The relationship between Del factor, the temperature & timeis given by the equation:

= A.t.e-(E/RT)

- However, during this periods the temperature is not constant.

- The value of the Del factor corresponding to each timeincrement can be calculated from the equation:


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1 = k1t

2 = k2t

3 = k3tEtc.

- The sum of the Del factors for all the increments will then equal

the Del factor for the heating-up period. The Del factor for thecooling-down period may be calculation in a similar fashion.

- Calculation of the holding time at constant temperature (121oC)

- Del factor to be achieved during the holding time may

calculated using eq.

holding = overall - heating - cooling


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- The temperature of the holding period is normally 121oC so the

holding time, at that temperature, to achieve the desired Del

factor should be calculated.

- Using the example where the overall Del factor was shown to

be 32.2, if is taken that the heating Del factor was 9.8 and the

cooling Del factor 10.1, the holding Del factor may be

calculated

- holding = 32.2 9.8 10.1

holding = 12.3

But = kt

e.g the specific death rate ofB. stearothermophilus spores at121oC is 2.54 min-1.

Threfore, t = /k or t = 12.3/2.54 = 4.84 min


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- If the contribution made by the heating and cooling parts of

the cycle were ignored then the holding time would be given

by the equation:

t = overall/k = 32.2/2.54 = 12.68 min

- Thus by considering the contribution made to the sterilizationprocess by the heating and cooling parts of the cycle a

considerable reduction in exposure time is achieved.


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Richards rapid method for the design of sterilization cycles.

- Richard (1968) proposed a rapid method for the design ofsterilization cycles avoiding the time-consuming graphical

integrations.

- The method assumes that all spore destruction occurs attemperature above 100oC and that those parts of the heating

and cooling cycle above 100oC are linear.


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- Based on these assumptions, Richards has presented a table

of Del factors for B. stearothermophilus spores which would

be obtained in heating and cooling a broth up to (and downfrom) holding temperature of 101-130oC, based on a T

changes of 1oC per minute.

- If the rate of temperature changes is 1oC per minute, the Delfactors for heating and cooling may be read directly from the

table.

- If the temperature change deviates from 1o per minute, theDel factors may be altered by simple proportion.


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ToC K min-1 100 0.019 -

101 0.025 0.044

102 0.032 0.076

103 0.040 0.116

104 0.051 0.168

105 0.065 0.233

106 0.083 0.316

107 0.105 0.420

108 0.133 0.553

109 0.168 0.720

110 0.212 0.932

111 0.267 1.199

112 0.336 1.535

113 0.423 1.957

114 0.531 2.488

115 0.666 3.154

Del value for B. stearothermophilus spores for the heating-up period over a T range of 100 to

130oC, assuming a rate of T change of 1o min-1 and negligible spore destruction at

temperature below 100o (Richard, 1968)


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116 0.835 3.989

117 1.045 5.034

118 1.307 6.341

119 1.633 7.973

120 2.037 10.010

121 2.538 12.549

122 3.160 15.708

123 3.929 19.638

124 4.881 24.518

125 6.056 30.574

126 7.506 38.080

127 9.293 47.373

128 11.494 58.867

129 14.200 73.067

130 17.524 90.591


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E.g, if a fermentation broth were heated from 100o to 121oC in30 minutes and cooled from 121o to 100o in 17 minutes, the Delfactors for the heating & cooling cycles may be determined asfollows:

From Table, if the changes in T had been 1o per minute the Delfactor for both the heating & cooling cycles would be 12.549but the temperature changes in the heating cycle was 21o in 30

minutes, therefore

Delheating = 12.549 x 30 = 17.93

21

And the temperature changes in the cooling cycle was 21o

in 17minutes, therefore,

Delcooling = 12.549 x 17 = 10.16

21


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The scale up of batch sterilization process

-The use of the Del factor in the scale up of batch sterilization process

has been discussed by Banks (1979).

-In this stage the Del factor does not include a volume term, i.e.

absolute numbers of contamination & survivors are considered, NOT

their concentration.

-If the size of fermenter is increased, the initial number of spores in

the medium will also be increased but if the same probability of

achieving sterility is required the final spore number should remain

the same, resulting in an increase in the Del factor.


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Method of batch sterilization

-The batch sterilization of the medium for a fermentation may beachieved either in the

- fermentation vessel

- separate mash cooker


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The design of continuous sterilization process

- The design of continuous sterilization cycles may be

approaches in exactly the same way as for batchsterilization system.

- The continuous system includes a time period during whichthe medium is heated to the sterilization temperature, aholding time at the desired T and cooling period atfermentation T.

- The major advantages of continuous process is that T may beused as a process variable, thus reducing the holding time &reducing the degree of nutrient degradation.

- The required Del factor may be achieved by the combinationof T & holding time which gives an acceptably small degree ofnutrient decay.


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Example

A pilot sterilization were carried out in a 1000-dm3 vessel with a mediumcontaining 106 organism cm-3 requiring a probability of contamination of 1in 1000. The Del factor would be:

= ln 106 x 103 x 103 = ln 1012

10-3 10-3

= ln 1015 = 34.5

- If the same probability of contamination were required in a 10,000-dm3

vessel using the same medium the Del factor would be:

= ln 106 x 103 x 104 = ln 1013

10-3

10-3

= ln1016 = 36.8

Del factor increase with an increase in the size of the fermenter volume.


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Types of continuous sterilizer

1) Continuous-plate heat exchanger.

2) Continuous injector-flash cooler. (steam injector)

Advantages and disadvantages of the steam injection system

Advantages:

1) It may be used for media containing suspended solids

2) Low capital cost3) Easy cleaning & maintenance

4) Shorter heating & cooling periods

5) Higher steam utilization efficiency

Disadvantages

1) Foaming may occur during both heating & cooling

2) Direct contact of the medium with steam requires thatallowance be made for condense dilution & requires clean

steam, free from anticorrosion additives


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

reaction & its rate will be affected by T

xt/xo = ekt

Where k is the reaction rate constant

Or xo/xt = ekt

Thus, taking natural logarithms,

Ln xo/x

t= kt


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STERILIZATION OF THE FERMENTER

- If the medium is sterilized in a separate batch cooker, or is

continuously sterilized, then the fermenter has to besterilized separately before the sterile medium is added to it.

- This is normally achieved by heating the jacket or coils of thefermenter with steam & sparging steam into the vessel

through all entries.

- Steam pressure is held 15 psi in the vessel for 20 min

- It is essential that sterile air is sparged into the fermenterafter the cycle is complete & positive pressure is maintained.Otherwise, a vacuum may develop & unsterile air may bedrawn into the vessel.


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STERILIZATION OF AIR

- Filters for the removal of microorganism from anenvironment may be divided into two larger group

i) Those which the pores in the filter are smaller than the

particles which are to be removed.

ii) Those which the pores are larger than the particles which are

to be removed.


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Filter made from fibrous material such as

- Cotton- Glass

- Steel wool

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