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Friday, April 1, 2011



            To determine the holding time of reactor in batch kinetics.

            A fermentation product is produced by a culture of certain organisms in a nutrient medium. If a foreign microorganism invades the fermentation, the following may be the consequences:
·         There would be competition between the foreign microorganism and the production microorganism for the same nutrient medium, thus leading to loss of productivity.
·         If the fermentation is of continuous mode, the contaminant may outgrow the production microorganisms, and displace it from fermentation, thus leading to the loss of money, time and energy.
·         Final product may be contaminated.
·         Extraction of final product may be very difficult
·         Degradation of the desired product may take place due to metabolism of the contaminant. This is especially true in the case of the production of antibiotics. If the contaminant happens to be a bacterial strain, which is resistant to the action of the antibiotic, and this antibiotic also happens to be a part of the normal metabolic pathway of the contaminant, then it leads to a problem. Eg. Degradation of b- lactam antibiotics by b- lactamase producing bacteria.
·         Contamination of bacterial fermentation with bacteriophages could result in lysis of the bacterial cells. But this does not occur very often.
Avoidance of contamination may be achieved by:
·         Using a pure inoculum to start out with.
·         Sterilizing the medium, fermentor vessel and all the materials used in the process.
·         Maintaining aseptic conditions during the fermentation.

Liquid medium is most commonly sterilized in batch mode in the same fermentor where it will be eventually used. The liquid is heated to sterilization temperature by introducing steam into the coils or the jacket of the vessel. Other ways of doing this could be heating the vessel electrically or by bubbling steam into the medium. If direct steam injection is used, allowance must be made for dilution of the medium by condensate, which typically adds 10-20% to the liquid volume quality of the steam must also be high enough to avoid contamination of the medium by metal ions or organics.
            Depending upon the rate of heat transfer from the steam or electrical element, raising the temperature of the medium in large fermentors can take a significant period of time. Once the holding or sterilization temperature is reached, the temperature is held constant for the period of time thold. Cooling water in the coils or jacket of the fermentor is then used to maintain the temperature.
            For operation of batch sterilization systems, we must be able to estimate the holding time required to achieve the desired level of cell destruction. As well as destroying contaminants, heat sterilization may also destroy the nutrients in the medium. To minimize this loss, holding times at the sterilization temperature must be kept as short as possible.

            In a lethal environment, cells in a population do not all die at once. Deactivation of the culture occurs over a finite period of time depending on the initial number of viable cells and the severity of conditions imposed. Loss of cell viability can be described mathematically as follows.
     Cell death is assumed to be a first order process

            rd = -dN/dt = kd N                                                 (1)
Where rd is the cell death rate, N is the number of variable cells, and kd is the specific death rate.
Instead of N, we can also express the rate of cell death in terms of biomass (X) of variable cells.
If  kd is constant we can integrate eqn, (1) as follows to derive an expression for N as a function of time.
N                               t
(1/N) dN = kd t dt                                                         (2)
No                          o
To get
ln Nt = ln No-kdt                                                                (3)
Nt = No e (-kd t)                                                                    (4)
      Thus, if first order kinetics applies, a plot of ln Nt versus t gives a straight line with slope kd. However, first order kinetics does not always hold, particularly for bacterial spores immediately after exposure to heat. The value of kd is not only species dependent, but also dependent on the physiology of the cell itself. For eg. Bacillus stearothermophilus is the most heat resistant bacteria known.
Like other kinetic constants, the specific death constant also depends on temperature, T. this effect can be described using the Arrhenius relationship:

Kd = Ae(-Ea/RT)                                                                    (5)

Where A is the Arrhenius constant or frequency factor, Ea is the activation energy for thermal cell death and R is the universal gas constant.

Combining eqns 3 & 5, we get:
ln (No / Nt) = Ae (-Ea/RT)* t                                                       (8)
            This term, ln (No/Nt), has been described as Humphrey and Deindoerfer as the Del factor, Ñ, or the Nebla factor or the sterilization criterion.
            Thus, the Del factor is a measure of the fractional reduction in viable organism count by a certain heat and time regime.
 Rearranging (6), we get:
ln t = E/RT + ln (Ñ/A)                                                   (7)
            Thus, a plot of ln t versus T world yield a straight line of slope E/R and y intercept ln (Ñ/A).
Thus, the same degree of sterilization may be obtained by heating the substance at a high temperature for a short time, or a low temperature for a long time.

Two types of reactions contribute to loss of nutrient quality during sterilization.

A common reaction during sterilization is the Maillard type browning reaction. This 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 acids of proteins. Sterilizing the sugar separately and mixing it with the rest of the sterilized medium after cooling resolve this problem.

Certain amino – acids and proteins may be degraded during a heat sterilization regime. Thermal destruction of nutrient components conforms with first order kinetics and may be degraded by a from similar to the Arrhenius equation:
    (Xt/Xo) = e (-kt)                                                         (8)
Values of activation energy Ea for thermal destruction of vitamins and amino acids are 84 to 92 kJ/gmol, for proteins it is about 165 kJ/gmol. As these values are a bit lower than that of microorganisms, raising the temperature has a greater effect on cell death than nutrient destruction. This means that sterilization at higher temperatures (and shorter times) has the advantage of killing cells with limited destruction of medium components.

            Let No denote the number of contaminants present in the raw medium . During the heating period, the holding period and at the end of the cooling period, the final number is reduced to N. Ideally N should be zero. But this is practically impossible to achieve, as absolute sterility would take infinitely long to reach. Normally the target level of contamination is expressed as a probability of contamination, and this value is fixed at 0.001 i.e. one batch in every 1000 is expected to be non-sterile. To apply this kinetics, we need to know some of the thermal death characteristics of the bacteria present in the medium. The assumption that Bacillus stearothermophilus  one of the most heat resistant bacteria known, is the only microbial contaminant present, is a safe one(we cannot practically calculate the average thermal death kinetic values of every known taxon that might constitute a contamination).Thus a considerable safety factor can be built into the design by adopting B.stearothermophilus as the design organism.
The death characteristics of this organism are:
Ea = 283000 J/mol                                                            (9)
A = 9.5 * 1037 / min
            We must keep in mind that these values will vary considerably depending on the type of medium used. This is particularly relevant when considering the sterilization of oils and fats (common fermentation substrates) where relative humidity may be relatively low (spores of B. stearothermophilus are ten times more resistant when dry, then when wet).


 If No and N known. We can determine the holding time required to reduce the number of cells by considering the kinetics of cell death.

Ñtotal = Ñheating + Ñholding +Ñ cooling                                                              (10)

Ñtotal = ln (No/N)                                                                                  from eqn (6)

We know that for the organism B.stearothermophilus, a good estimate of the number cells per unit volume of reactor would be                                             = 106 spores / mL
The reactor volume we had was                                             = 750mL
Thus, initial number of organisms we start out with               = No = 106 * 750 = 7.5 * 108

Ñheating and Ñcooling are collected as follows:
We know that Ñheating = k * t                                                      from eqns (5) & (6)
 k=Ae (-Ea/RT)                                                                                eqn (5)

For the organism B. stearothermophilus Ea and A are known. We also have a set of readings of T (temperature) vs. t (time) for heating and for cooling. Thus for each value of T, we can get the corresponding value of k (from eqn.5)
            If we now plotted k against t, we would obtain a graph whose AUC (or area under the curve) is = k* t, which in turn, is equal to Ñ. Depending on which set of readings we took of T vs. t (whether the heating or the cooling) we would get Ñheating or Ñcooling.
Thus, from eqn 10. we can calculate Ñholding. We know that the holding temperature is going to be 121°C. from this, we can calculate the corresponding value of k and t hence, the holding period for the sterilization.

            These design procedures apply to batch sterilization of medium when the temperature is uniform throught the vessel. However, if the liquid contains contaminant particles in form of flocs or pellets, temperature gradients may develop. Because heat transfer within solid particles is slower than in the liquid, temperature at the center of the solid will be lower than that in the liquid for some portion of the sterilizing time. As a result, cell death inside the particles is not as effective as in the liquid. Longer holding times are required to treat solid- phase substrates and media containing particles.


·         The experimental batch data is obtained using a 2L batch fermentor. It is filled to the 750mL mark with water.
·         The reactor is heated using a heating finger, and temperature was noted down regularly. A point to remember at this stage the safety valve has to be removed during the heating stage to prevent reactor bursting.
·         Once the temperature has reached 121°C, the heating element is disconnected, and cooling is begun. Once again, temperature is noted down regularly. We must remember to plug safety valve back in again at this cooling stage to prevent a vacuum from developing inside the reactor, which would induce it to suck in (non-sterile) air from outside.
·         This is the first part of the experiment. In the second part, 10ml culture was exposed to a temperature of 60 °C for 5 min, 10 min and 15 min. At the end of each time interval, 1 mL of culture was withdrawn and plated in LB plates at dilutions 1:102, 1:104, and 1:106. The same procedure was repeated for 80ºC.
·         The plates were incubated overnight and the colonies were counted the next day.


The holding time and thermal death rate constant (kd) of  batch heat sterilization   are determined

            Thus tholding can be considerably reduced by considering heating and cooling parts of cycles in the sterilization process.
The main aim of designing a process is to achieve the probability of obtaining sterility with minimum loss of nutritive quality. Hence, exposure of medium is kept to be minimum. An important observation is that the del factor does not include the absolute number of contaminant concentration and concentration of survival.
            Moreover , the holding time is kept as minimum as possible since heating sterilization may also destroy the medium nutrients. Thus by including heating and cooling cycles ,the holding times are kept minimum which minimizes the loss

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