UNIT III

STERILIZATION KINETICS

 Thermal death kinetics of microorganisms,

 Batch and continuous heat sterilization of liquid media,

 Filter sterilization of liquid media,

 Air sterilization

 Design of sterilization equipment- batch and continuous.

Sterilization - Introduction:

 The overall biochemical processes should be carried out in a contamination-free environment.

 If fermentation is invaded by foreign organisms,

 Loss of productivity - medium would have to support the growth of both the production organism and the contaminant.

 Contaminant may 'outgrow' and displace the production organism – in continuous fermentation.

 Contamination in final product – Eg: SCP (product).

 Make subsequent recovery of the final product difficult – due to extra compounds produced by contaminants.

 The contaminant may degrade the desired product – Eg: Degradation of β-lactam antibiotics by β-lactamase-producing

bacteria.

 Sterilization is an important unit operation which differentiates biochemical and chemical process (do not require

sterilization).
 Fermentation is a biochemical process of producing metabolic products by the action of M.O. on a substrate, in the presence

of nutrients in the medium.

 So it involves = M.O., Medium, Fermenter, Nutrients/other additives, air (aerobic process).

 Sterile environment is required for all of the above.

Methods to avoid contamination in a fermentation process:

1. Using the pure inoculum to start the fermentation.

2. Sterilization of the medium.

3. Sterilization of the fermenter.

4. Sterilization of pipes, valves, bends that come in contact with fermentation process.

5. Sterilization of all materials to be added to the fermenter.

6. Sterilization of air.

7. Maintaining aseptic conditions during fermentation.

Sterilization of Media:

 Though various techniques can be used to sterilize the media, steam is used almost universally for the sterilization of

fermentation media.

 But animal cell culture – contains heat labile components – filtration is employed.

 Media should be sterilized to avoid the contaminating M.O.s which may,

 Use nutrients in the medium

 Change chemical structure of nutrients

 Change the pH

 Creates more foam that affects aeration

 Produce metabolic products

 Alter redox potential of medium

 Synthetic media do not require much sterilization (small heat) as compared to the crude media.

 Crude media contains more heat-resistant bacterial spores – require prolonged heating. .

 Utmost care must be taken that only adequate heating is done to sterilize thoroughly.

 Excessive heating may (a) denature the proteins (b) caramelize the sugars (c) thermal degradation of components by

inter-reactions.

 If pH is critical factor, adjust pH to neutral – Sterilize – bring to original pH (addition of pre-sterilized acid/alkali).

 If enzymes or vitamins are to be present – initially separated through biological filter – Sterilize - Added again

 Same method is followed for volatile components to prevent their loss during thermal sterilization.

 It should ensured that the thermo-sensitive components are present in the medium, at the same time the medium is

thoroughly sterilized and made free from contaminants.

 Therefore thermal death studies are to be performed to evaluate the sterilization time.
Thermal death kinetics of microorganisms:

 The rate of destruction of M.O.s by steam or moist heat is known as thermal death kinetics of micro-organisms.

 The destruction kinetics may be described as a first-order chemical reaction and is written as,

𝑑𝑁
 − 𝑑𝑡 = 𝑘𝑁 (1) N is the no. of live organisms present

𝑑𝑁 t is the sterilization time period

 − 𝑁 = 𝑘 𝑑𝑡
k is the first-order thermal kinetic rate constant
𝑁𝑡 1 𝑡
− 𝑁0 𝑁
𝑑𝑁 =𝑘 0
𝑑𝑡 The negative sign indicates that as ‘t’ increases, ‘N’ decreases.

𝑁
 −[ln 𝑁] 𝑡 = 𝑘 𝑡
𝑁0

 − ln 𝑁𝑡 + ln 𝑁0 = 𝑘𝑡 (2)

𝑁 𝑡
 𝑙𝑛 𝑁0 = 0
𝑘𝑑𝑡 (3)
𝑡
Equation (2) becomes,

𝑁𝑡
 = 𝑒 −𝑘𝑡
𝑁0

𝑁
 𝑙𝑛 𝑁𝑡 = −𝑘𝑡
0
(4)

𝑁
 ln 𝑁0 = 𝑘𝑡
𝑡
(5)
 Equation (4) is Survival factor and (5) is inactivation factor.

𝑁0
 is the design criterion that indicate the contamination level of the medium.
𝑁𝑡

 The relationship of equation (1) is only with the sterilization of a pure culture in one physiological form, under ideal

sterilization conditions.

 The value of k is not only species dependent, but dependent on the physiological form of the cell;

 For example, the endospores of the genus Bacillus are far more heat resistant than the vegetative cells.
In the above equations,

 k is a constant which expresses the specific death rate.

 It increases sharply with temperature and

 can be experimentally determined for an organism using equation (4).

The graphical representations of equations are illustrated in the figure, from which it may be seen that viable
organism number declines exponentially over the treatment period.
𝑁0
A plot of the natural logarithm of against time yields a straight line, the slope of which equals - k.
𝑁𝑡
 This kinetic description makes two predictions which appear anomalous:

 An infinite time is required to achieve sterile conditions (i.e. Nt. = 0).

 After a certain time there will be less than one viable cell present.

 If the experimentally determined ‘ln k’ value from this equation is plotted against the reciprocal temperature (1/T) value, a

straight line should be obtained from which the ‘k’ value can be calculated for a desired temperature.

 The effect of the time of heat treatment on the survival of a population of bacterial endospores:

1)  The deviation from an immediate exponential decline in viable spore

number is due to the heat activation of the spores, that is the induction

of spore germination by the heat and moisture of the initial period of the

sterilization process.

 The activation of spores is significantly more than their destruction

during the early stages of the process and, therefore, viable numbers

increase before the observation of exponential decline.

2)  An initial stationary period observed during a sterilization treatment

due to the death of spores being completely compensated by the

heat activation of spores.

3)  Initial population decline at a sub-maximum rate during a

sterilization treatment due to the death of spores being compensated

by the heat activation of spores

 Sterilization of mixed cultures:

 Let us consider sterilization of mixed cultures (C) containing two species (A and B) with different heat sensitivities.

 It is clear that more sensitive organism will be getting destroyed in the early stages – Curve C follows line A (High

proportion of sensitive culture “A”); when more amount of A is destroyed, the curve follows the path of line B.

 Since the sensitive organism (A) is

less in number, it gets destroyed

C
C
quickly and the total no. of cells will
B
B always be equal to no. of resistant
A A
organisms (B) – Curve C follows

C C almost the line B (High proportion of

A A
B B resistant culture “B”).
Batch Sterilization:

 Two distinct methods of sterilization are : Batch and Continuous

 Batch Sterilization is the one in which all the contents are loaded in the sterilizer, steam is injected and later the contents

are discharged for processing or transferred directly to the fermenter.

 Advantages:

 The initial cost of investment is low

 The chances of contamination after sterilization is over are less – because can be performed in fermenter itself.

 Manual control, is usually done and so chances of mechanical failure is less.

 Media containing high proportion of solids can be handled easily.

 Disadvantages:

 Chances of risk of destruction of nutrients is more compared to continuous sterilization.

 Design Aspects:
 As with any first-order reaction, the reaction rate increases with increase in temperature due to an increase in the reaction rate

constant - in the case of the destruction of micro-organisms, is the specific death rate (k).

 Thus, k is a true constant only under constant temperature conditions.

 The relationship between temperature and the reaction rate constant was demonstrated by Arrhenius and may be represented by

the equation:

𝑑(ln 𝑘) 𝐸
 = 𝑅𝑇 2 (6) [E is the Activation energy; R is the gas constant and T is the absolute temperature]
𝑑𝑇

𝐸
 On integration, we get, 𝑘 = 𝐴𝑒 − 𝑅𝑇 (7) [A is the Arrhenius constant]

𝐸
 On taking natural logarithms, ln 𝑘 = ln 𝐴 − 𝑅𝑇

 This resembles equation of straight line (y = mx + c). So a plot of ‘ln k’ against the reciprocal of the absolute temperature (1/T)

will give a straight line.

 Such a plot is termed an Arrhenius plot and enables the calculation of the activation energy and the prediction of the reaction

rate for any temperature.

 𝑁 𝐸
 Combining equation (5) and (7) ie., ln 𝑁0 = 𝑘𝑡 and 𝑘 = 𝐴𝑒 − 𝑅𝑇 , we get the following expression,
𝑡

𝑁 𝐸
 ln 𝑁0 = 𝐴. 𝑡. 𝑒 − 𝑅𝑇 . (8) This is the heat sterilization of a pure culture at a constant temperature.
𝑡

𝑁
 ln 𝑁0 is the design criterion for sterilization and is called as “Del factor” or “Nabla factor” and is represented as “Δ”.
𝑡

 Del factor is a measure of the fractional reduction in viable organism count produced by a certain heat and time regime.

Ln del=ln A+ln t-E/RT

Ln del-ln A+E/RT=ln t

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

𝐸 Δ
 On rearranging, ln t = 𝑅𝑇 + ln 9
𝐴

 The plot of ln (t) Vs (1/T) will give the del factor and the slope is dependent on the activation energy.
  A risk factor of one batch in a thousand being contaminated is frequently

used in the fermentation industry that is, the final microbial count in the

medium after sterilization should be 10-3 viable cells.

 To apply these kinetics it is necessary to know the thermal death

characteristics of all the taxa contaminating the fermenter and unsterile

medium which is impossible.

 Therefore, the assumption may be made that the only microbial

contaminants present are spores of Bacillus stearothermophilus - that is, one

of the most heat-resistant microbial types known.

 By adopting B. stearothermophilus as the design organism a considerable safety factor should be built into the

calculations.
 Activation energy = 283 kJ/mol.

 Arrhenius constant = A = 1*1036.2 s-1. These kinetic values will vary according to the medium.

 From this, it is clear that the usual assumption of a risk factor of one in thousand is justified, ie., Nt=10-3.

 However, fermentation medium is not an inert mixture of components and deleterious reactions may occur in medium

during the sterilization process, resulting in a loss of nutritive quality.

 This shows the deleterious effect of increasing medium

sterilization time on the yield of product of subsequent

fermentations.
 Loss of nutrient quality during sterilization:

Interaction between nutrient components:

 A common occurrence during sterilization is the Maillard-type browning reaction which results in discoloration of the

medium as well as loss of nutrient quality.

 These reactions are normally caused by the reaction of carbonyl groups, usually from reducing sugars, with the amino

groups of amino acids and proteins.

 Problems of this type are normally resolved by sterilizing the sugar separately from the rest of the medium and

recombining the two after cooling.

Degradation of heat labile compounds:

 Different compounds will have different sterilization periods and will be affected by the temperature of sterilization.

 Mostly by steam, animal cell culture is done by filtration.

 However, correct method of sterilization has to be chosen to avoid degradation of heat labile compounds in the media.
 Although a batch sterilization process involve destruction of nutrients, the objective in designing a batch process is still

to achieve the required probability of obtaining sterility with the minimum loss of nutritive quality.

 The highest temperature which appears to be feasible for batch sterilization is 121 °C so the procedure should be

designed such that exposure of the medium to this temperature is kept to a minimum.

 This is achieved by taking into account the contribution made to the sterilization by the heating and cooling periods of

the batch treatment.

 The following information must be available for the design of a batch sterilization process:

 A profile of the increase and decrease in the temperature of the fermentation medium during the heating and cooling

periods of the sterilization cycle.

 The number of micro-organisms originally present in the medium.

 The thermal death characteristics of the 'design‘ organism.

 We are considering the total number of organisms present in the medium and not the concentration.

𝑁
 Δ = ln 𝑁0 and if we consider that unsterile medium contains initially 1011 viable cells.
𝑡

 Δ = ln (1011/10-3) = ln (1014)

 Hence, Δ = 32.2

 This is the overall del factor.

 Normally, destruction of cell takes place at 121 °C. This period is called “holding period”.

 Some number of organisms would be destroyed during the temperature building up to 121 °C. This is “heating period”.

 Some cells would be destroyed during “cooling period” from 121 °C to room temperature. Hence the overall period can

be given as,

Δoverall = Δheating + Δholding + Δcooling (10)

Calculation of Del factor during Heating and Cooling:

 The relationship between Del factor, the temperature and time is given by equation

𝐸
 Δ = 𝐴. 𝑡. 𝑒 − 𝑅𝑇 (a)

 However, during the heating and cooling periods the temperature is not constant and, therefore, the calculation of Δ would

require the integration for the time-temperature regime observed.

𝑁 𝑡
 Δ = ln 𝑁0 = 𝑘 0
𝑑𝑡
𝑡

 This is possible if the functionality of time and temperature are known.

 Deindoerfer and Humphrey (1959) – analytical expressions to integrate functions of t and T by linear, hyperbolic or

exponential - practically very difficult.

 Richards (1968) – graphical integration is possible by experimentally noting the data.

Graphical integration Method:

 The time – temperature profile can be experimentally recorded, and drawn in the form of plot.

 X-axis is divided into large number of small equal intervals like t1, t2, t3, …t.

 The temperature T1 during t1 period is noted as the average temperature at the beginning of t1 and ending of t1 ie., T1 is the

midpoint of t1.

 If the total time is divided into 30 equal parts, the method would be accurate.

 Having known the temperature T and time t, Δ can be calculated with equation (a) for B.stearothermophilus using the values of

activation energy and Arrhenius constant as follows:

 E = 283 kJ/mol; A = 1*1036.2 s-1

Δ1 = k1t; Δ2 = k2t; Δ3 = k3t etc., and the overall Δ is a summation of individual Δ’s

 Hence Δ = Δ 1+ Δ 2+ Δ 3+….

𝑛
 Δheating= 𝑖=1 Δ𝑖
 Similarly, Δcooling can be calculated by having the time-temperature profile during cooling process of sterilization.

Calculation of the Holding time with constant temperature:

 From the previous calculations the overall Del factor, as well as the Del factors of the heating and cooling parts of the

cycle, have been determined.

 Therefore, the Del factor to be achieved during the holding time may be calculated by difference:

Δholding = Δoverall - Δheating - Δcooling

 Using our example where the overall Del factor is 32.2 and if it is taken that the heating Del factor was 9.8 and the

cooling Del factor 10.1, the holding Del factor may be calculated:

Δholding = 32.2 - 9.8 - 10.1

Δholding = 12.3

But the specific death rate (k) of B. stearothermophilus at 121 °C is 2.54 min-1.

t = Δ/k = 32.2/2.54 = 4.84 min.

Methods to calculate Del factors:

 There are two methods of calculation of del factors during heating and cooling.

 Graphical integration (previously discussed)

 Richard’s rapid calculation method for heat sterilization.

 Richard’s rapid calculation method:

 Proposed by Richards in 1968 to design sterilization cycles by avoiding the time consuming graphical integrations.

 Assumptions: 1. spore destruction occurs at temperatures > 100 °C (ie., destruction occurs only between 100 – 121 °C).

2. heating and cooling cycles above 100 °C are linear.

 These assumptions were reasonable and the method is accurate and simple.

 Richards has presented a table of Del factors for B. stearothermophilus spores - obtained in heating and cooling a broth up

to (and down from) holding temperatures 101-130 °C, based on a temperature change of 1 °C per minute.
 If the rate of temperature change is 1°C per minute, the Del factors for heating

and cooling may be read directly from table; if the temperature change deviates

from 1°C per minute, the Del factors may be altered by simple proportion.

 For example, if a fermentation broth were heated from 100° to 121°C in 30

minutes and cooled from 121° to 100° in 17 minutes, the Del factors for the

heating and cooling cycles may be determined as follows:

 From table, Δ = 12.549 (if 1°/min; But the change in the heating cycle was 21° in

30 minutes), therefore,

 Δheating = 12.549*(30/21) = 17.93

 Temperature change in the cooling cycle was 21° in 17 minutes, therefore,

 Δcooling= 12.549*(17/21) = 10.16

 Δholding at constant temperature can be found using previous method.

 Scale up of batch sterilization process:

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

 The holding time in the large vessel may be calculated by the graphical integration method or by the rapid method of

Richards (1968), based on the temperature-time profile of the sterilization cycle in the large vessel.

 However, extending the holding time on the larger scale will result in increased nutrient degradation.

 The decrease in the yield of a fermentation when it is scaled up is often due to problems of nutrient degradation during batch

sterilization – can be overcome only by sterilizing the medium continuously.

 Methods of batch sterilization process:

In-situ (sterilization is inside the

fermenter itself)
Batch Sterilization
Ex-situ (sterilization is in a separate
vessel)
Advantages of separate vessel:

 One vessel may be used to serve several fermenters and the medium may be sterilized as the fermenters are being cleaned

and prepared for the next fermentation, thus saving time between fermentations.

 The medium may be sterilized in a cooker in a more concentrated form than would be used in the fermentation and then

diluted in the fermenter with sterile water prior to inoculation.

 In some fermentations, the medium is at its most viscous during sterilization and therefore would require agitation.

 The fermenter can be prevented from the corrosion which may occur with medium at high temperature.

Disadvantages of separate vessel:

 The cost of constructing a batch medium sterilizer is much the same as that for the fermenter.

 Pipework would be necessary to transport the sterile medium – chances of contamination.

 Mechanical failure in a cooker supplying medium to several fermenters would render all the fermenters temporarily

redundant.
Continuous Sterilization:

 In this sterilization process, there will be continuous inflow and outflow of material.

 The continuous system includes a time period during which the medium is heated to the sterilization temperature (using heat

exchanger), a holding time at the desired temperature (using insulated holding coil) and a cooling period to restore the

medium to the fermentation temperature (using two heat exchangers).

 The length of holding period is dictated by the length of the coil and the flow rate of the medium.

Advantages:

 The throughputs (capacity) can be higher.

 The medium quality can be maintained better.

 The system can be automated for control; avoids human error during operation.

 Running costs are less.

 Sterilization times are shorter

 Ease in scaling-up of the process.

Disadvantages:

 The initial capital investment is more.

 Require aseptic transfer system.

 However, HTST (High Temperature Short Time) is possible only in continuous mode of sterilization.

Methods of continuous sterilization:

Plate heat exchange sterilization

Continuous
Sterilization
Steam injection and flash cooling
sterilization
Plate heat exchange sterilization:

 Incoming unsterile medium is preheated by heat exchange with the outgoing sterile medium.

 Then again heated with steam in a heat exchanger.

 Then passed through the holding section.

 Heating time and cooling time are considerable.

Steam injection flash cooling sterilization:

 Steam is directly injected along with the medium

continuously.

 Heating time and heating section are negligible.

 Holding time is based on the length of the holding pipe

(sterilization occurs).

 Steam and sterilized medium under pressure are passed

through the expansion valve into the vacuum chamber.

 Steam is removed out under vacuum.

 Sterile medium then passes through the cooling zone

(considerable); but temperature is much < 100 °C, so

neglected.
Sterilization of the fermenter:

 If the medium is sterilized in a separate batch cooker or is sterilized continuously, then the fermenter has to be

sterilized separately before the sterile medium is added to it.

 This is normally achieved by heating the jacket or coils of the fermenter with steam and sparging steam into the

vessel through all entries.

 Steam pressure is held at 15 psi in the vessel for approximately 20 minutes.

 It is essential that sterile air is sparged into the fermenter after the cycle is complete and a positive pressure is

maintained.

 Otherwise a vacuum may develop and unsterile air be drawn into the vessel.
Filter sterilization of liquid media:

 Media for animal-cell culture cannot be sterilized by steam because they contain heat-labile proteins.

 Thus, filtration is the method of choice and fixed pore or absolute filtration is the better system to use.

 An ideal filtration system for the sterilization of animal cell culture media must fulfil the following criteria:

 The filtered medium must be free of fungal, bacterial and mycoplasma contamination.

 There should be minimal adsorption of protein to the filter surface.

 The filtered medium should be free of viruses.

 The filtered medium should be free of endotoxins.

 Nowadays, absolute filtration systems are used for the sterilization of animal cell culture medium (specific with pore

sizes smaller than the particles to be removed).

 Such systems consist of membrane cartridges which are fitted into stainless steel, steam sterilizable modules.

 The membranes for media filtration are constructed from steam sterilizable hydrophilic material and are treated to

produce a filtrate of particular quality.

 For example, if minimal protein adsorption is a major criterion then a specially coated filter membrane is used.

 It would be very difficult to construct a single filtration membrane which would fulfil all four criteria.

 Thus, a series of filters are used to achieve the desired result.

 The figure illustrates a system to produce sterile, mycoplasma free serum and consists of four filters arranged in sequence.
 The first filter is a positively (+) charged polypropylene pre-filter with an absolute rating of 5 μm for the removal of

coarse precipitates, clot-like material and other gross contaminants.

 The second filter is also positively (+) charged polypropylene but with an absolute rating of 0.5 μm for bulk microbial

removal, deformable gels, lipid-based materials and endotoxin reduction.

 The third filter is a single layered, nylon/polyester positively (+) charged filter with a 0.1 μm absolute rating for further

microbial and endotoxin removal and optimum protection of the final filter.

 The fourth filter is similar to the third and has the same rating, but is double layered and removes mycoplasmas, gives

absolute sterility and final endotoxin control.

 Thus, the combination of four filters gives a sequential removal of decreasingly small particles and prolongs the life of

the final filter.

 To remove viral contamination then a final 0.04 μm nylon / polyester filter would be added.
Sterilization of Air:

 Aerobic fermentations require the continuous addition of considerable quantities of sterile air.

 Achieving sterility for air is stupendous task for biochemical engineers.

 1 cubic meter of air contains – 4*103 to 20*103 particles (average – 103 to 104 particles/m3; avg. size of particles is 0.6

micron).
Methods of Air Sterilization:

 Sterilization by heating

 Use of UV rays and other electromagnetic waves

 Use of germicidal sprays

 Sterilization by filtration

 Although it is possible to sterilize air by heat treatment, it is not economical due to lower heat transfer coefficient.

 UV rays are effective in killing air-borne microbes, but it is not reliable to sterilize large volumes of air in factories.

 Spraying small amounts of germicides like phenol, ethylene oxide or formalin (formaldehyde) can be an effective

method, but it cannot ensure 100 % sterility of air.

 Due to many drawbacks of all other methods in one way or the other, filtration can be an effective method to sterilize air.
 Fixed pore filters (which have an absolute rating) are very widely used in the fermentation industry and several

manufacturers produce filtration systems for air sterilization.

 These systems, like those for the sterilization of liquids, consist of pleated membrane cartridges designed to be

accommodated in stainless steel modules.

 Types of air filters:

The air filters used for the removal of M.O.s or spores from the air are of two types:

 Filters whose pores are smaller than the size of M.O.s to be removed. – virtually all M.Os are filtered off

(absolute filters)

 Filters whose pore size is bigger than the size of M.O.s – fibrous materials are used as filter beds – fibrous type

air filters – cannot assure 100 % sterility.

Thank You !!!!