CHAPTER 5 STERILIZATION

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 cell protein 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

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 -lactam antibiotics by - lactamaseproducing bacteria.
vi) Contamination of a bacterial fermentation with phage could result in the lysis of the culture.

 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.

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, steam is used almost universally for the sterilization of fermentation media.

 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.

On integration of equation, give

Nt/No = e kt
Where, No is the number of viable organisms present at the start of the sterilization treatment 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 in temperature due to an increase in the reaction rate constant, which,in the case of the destruction of microorganism, is the specific death rate (k). Thus, the reaction-rate constant (k) is a true constant only under constant temperature condition.

- 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/Nt = A. t. e E/RT

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/Nt ln No/Nt = kt kt= A.t.e -(E/RT) = A.t.e -(E/RT) ln t = E/RT + ln (/A)

but and Thus,

Rearranging eq.

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) Interaction between nutrient components of the medium. - A common occurrence during sterilization is the Maillard-type browning reaction which results in discolorization of the medium as well as a loss in nutrient quality. - These reaction are normally caused by the reaction of carbonyl 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 medium separately from the remainder of the medium & to recombine the two after cooling

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 those derived 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

 in the consideration of Del factor it was evident that the same Del factor could be achieved over a wide range of temperature/time regime. thus, it would be advantageous to employ a high temperature for 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 thousands of litres of broth to a high temp, hold for a short period & cool w/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.

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.

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.

THE DESIGN OF BATCH STERILIZATION PROCESS Although a batch sterilization process is less successful in avoiding 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.

- 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 of Bacillus stearothermophilus iV) The risk of contamination considered acceptable for the process.

 The Del factor has already been defined as: = ln No/Nt Knowing the original number of organism present in the fermenter & the risk of contamination considered acceptable, the required Del factor may calculated. A frequently adopted risk of contamination is 1 in 1000 which indicates Nt should equal 10-3 of a viable cell. e.g: If unsterile broth contain 1011 viable organisms then Del factor may be calculated, thus; = ln 1011/10-3 = ln 1014 = 32.2 Therfore, overall Del factor required is 32.2

- 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 & time is 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 time increment can be calculated from the equation:

 1 = k1t 2 = k2t 3 = k3t Etc.

- 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 the cooling-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

- 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 of B. stearothermophilus spores at 121oC is 2.54 min-1. Threfore, t = /k or t = 12.3/2.54 = 4.84 min

- 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 sterilization process by the heating and cooling parts of the cycle a considerable reduction in exposure time is achieved.

Richards rapid method for the design of sterilization cycles.

- Richard (1968) proposed a rapid method for the design of sterilization cycles avoiding the time-consuming graphical integrations. - The method assumes that all spore destruction occurs at temperature above 100oC and that those parts of the heating and cooling cycle above 100oC are linear.

- 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 down from) 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 Del factors for heating and cooling may be read directly from the table. - If the temperature change deviates from 1o per minute, the Del factors may be altered by simple proportion.

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)
ToC 100 K min-1 0.019 -

101
102 103 104 105 106 107 108 109 110 111 112 113 114 115

0.025
0.032 0.040 0.051 0.065 0.083 0.105 0.133 0.168 0.212 0.267 0.336 0.423 0.531 0.666

0.044
0.076 0.116 0.168 0.233 0.316 0.420 0.553 0.720 0.932 1.199 1.535 1.957 2.488 3.154

116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

0.835 1.045 1.307 1.633 2.037 2.538 3.160 3.929 4.881 6.056 7.506 9.293 11.494 14.200 17.524

3.989 5.034 6.341 7.973 10.010 12.549 15.708 19.638 24.518 30.574 38.080 47.373 58.867 73.067 90.591

 E.g, if a fermentation broth were heated from 100o to 121oC in 30 minutes and cooled from 121o to 100o in 17 minutes, the Del factors for the heating & cooling cycles may be determined as follows:

From Table, if the changes in T had been 1o per minute the Del factor for both the heating & cooling cycles would be 12.549 but 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 17 minutes, therefore, Delcooling = 12.549 x 17 = 10.16 21

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.

Method of batch sterilization -The batch sterilization of the medium for a fermentation may be achieved either in the - fermentation vessel - separate mash cooker

The design of continuous sterilization process

- The design of continuous sterilization cycles may be approaches in exactly the same way as for batch sterilization system.

- The continuous system includes a time period during which the medium is heated to the sterilization temperature, a holding time at the desired T and cooling period at fermentation T.
- The major advantages of continuous process is that T may be used as a process variable, thus reducing the holding time & reducing the degree of nutrient degradation.

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

 Example A pilot sterilization were carried out in a 1000-dm3 vessel with a medium containing 106 organism cm-3 requiring a probability of contamination of 1 in 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.

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 cost 3) 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 that allowance be made for condense dilution & requires clean steam, free from anticorrosion additives

The destruction of a nutrient may be considered a first-order reaction & its rate will be affected by T

xt/xo = e kt
Where k is the reaction rate constant

Or

xo/xt = ekt

Thus, taking natural logarithms,

Ln xo/xt = kt

STERILIZATION OF THE FERMENTER - If the medium is sterilized in a separate batch cooker, or is continuously sterilized, 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 & 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 fermenter after the cycle is complete & positive pressure is maintained. Otherwise, a vacuum may develop & unsterile air may be drawn into the vessel.

STERILIZATION OF AIR - Filters for the removal of microorganism from an environment 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.

Filter made from fibrous material such as

- Cotton - Glass - Steel wool