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3.5.1.4 Sterilization

Sterilization is a process by which microorganisms are either killed or removed from the material or equipment. Sterilization is necessary to ensure that only the desired microorganism is present to carry out the fermentation, that products are made of predicted quality, that the environment is protected from undesirable contamination, and that deterioration (microbial spoilage) of products is prevented.

3.5.1.4.1 Medium Sterilization Techniques The sterilization techniques applicable for fermentation media sterilization include:

1. Sterilization by high temperature achieved by direct or indirect steam or electric heating, which is the most popular and efficient method.

2. Sterilization of the medium for animal cell culture by membrane filtration with absolute rating of 0.1-0.04 pm.

3. Microwave irradiation, which is used commonly in food industries and has been reported to cause cell death (92).

4 Newer techniques, like high voltage pulses and photosemiconductor powders (93), which involve the rupture of the cell membrane by increasing the transmembrane electric field strength beyond a certain threshold.

3.5.1.4.2 Sterilization by Heat When a medium containing microorganisms is heated above a certain temperature limit, the microorganisms are unable to survive. However, because endospores have higher heat tolerance, inactivation of spores is a good indication of the sterilization process efficiency. In the testing of a heat sterilization process, Bacillus stearothermophilus spores, which are the most temperature resistant, are widely used as test organisms (94).

Batch sterilization: Sterilization of medium in the fermentor can be carried out in batch mode by passing steam through the available heat transfer area (jacket or limpet coil) or through direct steam sparging or with electrical heaters. The highest temperature to be operated for batch sterilization of medium is 121°C. The batch heat sterilization is described by a first order kinetics resulting in the equation:

t o where N is the number of surviving microorganisms at t, N is the initial number of microorganisms, k is the specific death rate, and t is time.

Equation 3.1 can be rearranged as:

The reaction or sterilization rate increases with increase in temperature due to an increase in the reaction rate constant or specific death rate (k). The relationship between temperature (T) and specific death rate (k) can be expressed as:

where A is the Arrhenius constant, E is the activation energy, R is the gas constant and T is the absolute temperature.

Substituting k from Equation 3.3 into Equation 3.2:

The term "ln (No/Nt)" has been used as a design criterion for sterilization (95) and is represented as the Del factor (V), which is a measure of the fractional reduction in viable cell count produced in a certain temperature and time regime. Thus, we have:

where t is the time required to achieve a certain V value.

Rearranging Equation 3.5:

From a plot of ln (t) vs 1/T, the thermal death characteristics, namely activation energy (E) and Arrhenius constant (A), can be determined.

The batch sterilization cycle consists of heating, holding, and cooling cycles. The overall Del factor for destruction of cells during the sterilization period is represented as:

Voverall Vheating + Vholding + Vcooling (3.7)

By taking into account the Del factor during heating and cooling cycles in the design of a sterilization process, a minimum holding time is determined.

The advantages of batch sterilization are low capital equipment costs, low risk of contamination, easier manual control, and suitablity for media containing a high proportion of solids.

Continuous sterilization: Continuous sterilization has the advantages of relatively straightforward energy recovery, and lower nutrient degradation of heat labile substances due to shorter holding time. However, a disadvantage is that the presence of solids may give rise to inefficient sterilization, requiring a proper design.

Continuous heat sterilization essentially consists of a heating section using heat exchangers (typically spiral-type plates on an industrial scale), and a holding section where the temperature of the medium is maintained constant at the sterilization temperature. The inlet medium to the continuous sterilizer is preheated using hot fluid from the holding section in the heat exchanger. This process of energy transfer in the heat exchanger between the inlet raw medium and outlet sterilized medium not only cools the sterilized medium effectively, but also results in considerable energy savings. It is important to note that in the presence of solids, the rate at which medium can be heated is limited by the heat transfer rate from the liquid to the solid particles (96).

A continuous sterilizer has the advantage of being more economical in building area. It also offers greater possibility of automatic control, and reduction of sterilization process cycle time, resulting in minimum oversterilization and ease of scale up. Moreover, service requirements for steam and water are constant, compared with the fluctuating demand of batch sterilization. However, it may not be economical for small capacity batch fermentations.

3.5.1.4.3 Sterilization of Air Aerobic fermentation processes require significant quantities of sterile air. The sterilization of air in the strict bacteriological sense means the complete elimination of all viable microorganisms. The removal of bacteria by means of depth filters consisting of granular carbon or fibrous media has been almost universally adopted. In most fermentation systems, a prefilter usually made of cellulose, viscose, or glass wool is installed prior to the absolute filter to remove dust, oil droplets, and moisture from the process air. The most common material of construction of an absolute filter cartridge is pleated PTFE (Poly tetra fluoro ethylene, or Teflon™) membrane, which is hydrophobic and resists wetting. The membrane is intimately supported by pleated material, which prevents from distortion under high hydraulic or gas pressure. The absolute filters have a rating of 0.2-0.01 pm (for total removal of bacteria, viruses, bacteriophage, or spherical particles of that size) and can be sterilized using steam. After air filter sterilization, the filters are held under positive pressure until the end of fermentation. Pharmaceutical grade cartridges are processed with deionized, pyrogen free water and evaluated by bacterial challenge in accordance with international recommendations using standard test organisms (e.g., Pseudomonas diminuta, Acholeplasma laidlawii).

3.5.1.5 Inoculation

The process of inoculation is the transfer of seed material or inoculum into the fermentor. Inoculation of a laboratory fermentor is generally done using presterilized tubing and a peristaltic pump. However, on a larger scale, inoculum transfer is done by applying a positive pressure on the inoculum fermentor and connecting it aseptically to the production fermentor. The connecting lines are sterilized before being used for transfer of inoculum. Heat susceptible substances such as amino acids and some vitamins must be dissolved in small volumes of water, sterilized by filtration and added separately to the final medium aseptically.

The performance of a fermentation process is dependent largely upon the physiological status of the inoculum (97). It is necessary that the inoculum transfer time be determined experimentally at a laboratory scale and that a standard is set for cultural conditions needed for development of the inoculum. Inoculum transfer is commonly done with veg-etatively growing biomass determined by parameters such as turbidity, packed cell volume, dry weight, wet weight, or morphological characteristics (98). Online parameters which are considered for inoculum transfer include pH, dissolved oxygen, and oxygen and carbon dioxide concentration in the exit gas. The effect of inoculum age on the productivity of a secondary metabolite from Streptomyces species has been studied (99). The inoculum age at three time intervals, namely early log, log, and declining phases of growth determined online by the carbon dioxide production rates were carried out to determine the best inoculum for fermentation. It was observed that although there was a marginal influence on the biomass formation from the different inoculums, the log phase inoculum gave a significantly higher product concentration compared to the other two in the fermentor.

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