STERILISATION (PAGE 371, Doran e-book)

Introduction Commercial fermentations typically require thousands of litres of liquid medium and millions of litres of air. For processes Operated with axenic cultures, these raw materials must be provided free from contaminating organisms. Methods available for sterilisation including chemical treatment, exposure to ultraviolet, gamma and X-ray radiation,sonication, filtration and heating. Only the last two (filtration and heating) are used in large-scale operations.

Batch Heat Sterilisation of Liquids Liquid medium is most commonly sterilised in batch in the vessel where it will be used. The liquid is heated to sterilisation temperature by: introducing steam into the coils or jacket of the vessel steam is bubbled directly into the medium, or the vessel is heated electrically. 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 sufficiently high to avoid contamination of the medium by metal ions or organics. A typical temperature-time profile for batch sterilisation is shown in Figure 13.35(a). Once the holding or sterilisation temperature is reached, the temperature is held constant for a period of time thd. Cooling water in the coils or jacket of the fermenter is then used to reduce the medium temperature to the required value.

Normally, cell death below about 100°C is minimal; However,when heating and cooling are relatively slow, temperatures remain close to the maximum for considerable periods of time and, as indicated in Figure 13.35(b), cell numbers can be reduced significantly outside of the holding period. Usually, holding periods are of the order of minutes whereas heating and cooling of large liquid volumes take hours.

For operation of batch sterilisation systems, we must be able to estimate the holding time required to achieve the desired level of cell destruction. As well as destroying contaminant organisms, heat sterilisation also destroys nutrients in the medium. How to minimise this loss?(relates with holding time) Cell death occurs at all times during batch sterilisation, including the heating-up and cooling-down periods. The holding time thd can be minimised by taking into account cell destruction during these periods.

The final number of contaminant after cooling is Nf The final number of contaminant after cooling is Nf. Ideally, Nf is zero; at the end of the sterilisation cycle we want to have no contaminants present. However, it is theoretically impossible to achieve (require an infinitely-long sterilisation time.) Normally, the target level of contamination is expressed as a fraction of a cell, which is related to the probability of contamination. For example, we could aim for an Nf value of 10-3; this means we accept the risk that one batch in 1000 will not be sterile at the end of the process. If No and Nf are known, the holding time required to reduce the number of cells from N 1 to N 2 can be determined by considering the kinetics of cell death.

Rate of heat sterilisation is governed by the equations for thermal death. For first-order death kinetics, in a batch vessel where cell death is the only process affecting the number of viable cells: where N is number of viable cells, t is time and kd is the specific death constant. Eq. (13.95) applies to each stage of the batch sterilisation cycle: heating, holding and cooling. However, because kd is a strong function of temperature, direct integration of Eq. (13.95) is valid only when the temperature is constant, i.e. during the holding period. The result is:

Important points…. The design procedures outlined in this section apply to batch sterilisation of medium when the temperature is uniform throughout the vessel. However, if the liquid contains contaminant particles in the form of flocs or pellets, temperature gradients may develop. WHY?? Because heat transfer within solid particles is slower than in liquid, the temperature at the centre of the solid will be lower than that in the liquid. As a result, cell death inside the particles is not as effective as in the liquid. How does this affect the holding time?

When heat sterilisation is scaled up to larger volumes, longer treatment times are needed to achieve the same sterilisation results at the same holding temperature. Sustained elevated temperatures during heating and cooling effect the media. WHY??

Continuous Heat Sterilisation of Liquids Continuous sterilisation, particularly a high T, short-exposure-time process: significantly reduce damage to medium ingredients while achieving high levels of cell destruction. improved steam economy and more reliable scale-up. (20-25% that used in batch processes) the time required is also significantly reduced (Because heating and cooling are virtually instantaneous) Typical equipment configurations for continuous sterilisation are: Continuous steam sterilization with flash cooling Heat transfer using heat exchangers

(a) CONTINUOUS STEAM INJECTION WITH FLASH COOLING

(a) CONTINUOUS STEAM INJECTION WITH FLASH COOLING: Descriptions Raw medium entering the system is first pre-heated by hot, sterile medium in a heat exchanger. This economises on steam requirements for heating and cools the sterile medium. Steam is then injected directly into the medium as it flows through a pipe; the liquid temperature rises almost instantaneously to the desired sterilisation temperature. The time of exposure to this temperature depends on the length of pipe in the holding section of the steriliser. After sterilisation, the medium is cooled instantly by passing it through an expansion valve into a vacuum chamber (flash cooler) Further cooling takes place in the heat exchanger where residual heat is used to pre-heat incoming medium.

(b) HEAT TRANSFER USING HEAT EXCHANGERS.

(b) HEAT TRANSFER USING HEAT EXCHANGERS: Descriptions Raw medium is pre-heated with hot, sterile medium in a heat exchanger then brought to the sterilisation temperature by further heat exchange with steam. The sterilisation temperature is maintained in the holding section Temperature is reduced to the fermentation temperature by heat exchange with incoming medium.

Disadvantages Heat-exchange systems : Steam injection: are more expensive to construct than injection devices; fouling of the internal surfaces also reduces the efficiency of heat transfer between cleanings. Steam injection: dilution of the medium by condensate foaming from direct steam injection can also cause problems with operation of the flash cooler.

From Figure 13.38 above, rates of heating and cooling in continuous sterilisation are much more rapid than in batch.

Variable in continuous sterilisers An important variable affecting performance of continuous sterilisers is the nature of fluid flow in the system. Ideally, all fluid entering the equipment at a particular instant should spend the same time in the steriliser and exit the system at the same time. No mixing should occur in the tubes; if fluid nearer the entrance of the pipe mixes with fluid ahead of it, there is a risk that contaminants will be transferred to the outlet of the steriliser. The type of flow in pipes where there is neither mixing nor variation in fluid velocity is called plugflow.

Plug flow is an ideal flow pattern (in reality, fluid elements in pipes have a range of different velocities.) As illustrated in Figure 13.39, flow tends to be faster through the centre of the tube than near the walls. However, plug flow is approached in pipes at turbulent Reynolds numbers above about 2 x 104; operation at high Reynolds numbers minimises fluid mixing and velocity variation. Deviation from plug-flow behaviour is characterised by the degree of axial dispersion in the system, i.e. the degree to which mixing occurs along the length or axis of the pipe. Axial dispersion is a critical factor affecting design of continuous sterilisers.

FIGURE 13.39

For perfect plug flow, Dz is zero and Pe is infinitely large; The relative importance of axial dispersion and bulk flow in transfer of material through the pipe is represented by a dimensionless variable called the Peclet number: where Pe is the Peclet number, u is the average linear fluid velocity, L is the pipe length and Dz is the axial-dispersion coefficient. For perfect plug flow, Dz is zero and Pe is infinitely large; In practice, Peclet numbers between 3 - 600 are typical. The value of Dz for a particular system depends on the Reynolds number and pipe geometry (Figure 13.40)

Da is another dimensionless number called the Damkiihler number: where kd is the specific death constant, L is the length of the holding pipe and u is the average linear liquid velocity. Figure 13.41 shows that, at any given sterilisation temperature defining the value of kd and Da, performance of the steriliser declines significantly as the Peclet number decreases. The lower the value of N2/N l, the greater is the level of cell destruction.

Example 13.8 Holding temperature in a continuous steriliser

Take note that…. Heating and cooling in continuous sterilisers are so rapid that in design calculations they are considered instantaneous. While reducing nutrient deterioration, this feature of the process can cause problems if there are solids present in the medium. During heating, the temperature at the core of solid particles remains lower than in the medium. Because of the extremely short contact times in continuous sterilisers compared with batch systems, there is a greater risk that particles will not be properly sterilised. It is important therefore that raw medium be clarified as much as possible before it enters a continuous steriliser.

Filter Sterilisation of Liquids Media containing heat-labile components such as enzymes and serum are easily destroyed by heat and must be sterilised by other means. Typically, membranes used for filter sterilisation of liquids are made of cellulose esters or other polymers have pores between 0.2 and 0.45 pm in diameter. The membranes themselves must be sterilised before use, usually by steam. As medium is passed through the filter, bacteria and other particles with dimensions greater than the pore size are screened out and collect on the surface of the membrane. The small pore sizes used in liquid filtration mean that the membranes are readily blocked unless the medium is pre-filtered to remove any large particles. To achieve high flow rates, large surface areas are required.

Liquid filtration is generally not as effective or reliable as heat sterilisation. Viruses and mycoplasma are able to pass through membrane filters. Care must also be taken to prevent holes or tears in the membrane. Usually, filter-sterilised medium is incubated for a period of time before use to test its sterility.

Sterilisation of Air The number of microbial cells in air is of the order 103-104 m-3. Filtration is the most common method for sterilising air in large-scale bioprocesses; heat sterilisation of gases is economically impractical. Depth filters consisting of compacted beds or pads of fibrous material such as glass wool have been used widely in the fermentation industry. Distances between the fibres in depth filters are typically 2-10 μm, about 10 times greater than the dimensions of the bacteria and spores to be removed. Air-borne particles penetrate the bed to various depths before their passage through the filter is arrested; the depth of the filter medium required to produce air of sufficient quality depends on the operating flow rate and the incoming level of contamination.

Cells are collected in depth filters by a combination of impaction, interception, electrostatic effects, and for particles smaller than about 1.0 μm, diffusion to the fibres. Depth filters do not perform well if there are large fluctuations in flow rate or if the air is wet; liquid condensing in the filter increases the pressure drop, causes channelling of the gas flow, and provides a pathway for organisms to grow through the bed.

Membrane cartridge filters Increasingly, depth filters are being replaced for industrial applications by membrane cartridge filters. These filters use steam-sterilisable polymeric membranes which act as surface filters trapping contaminants as on a sieve. Membrane filter cartridges typically contain a pleated configuration (allows a high filtration area to be packed into a small cartridge volume) and hydrophobic filter (minimises problems with filter wetting) small and uniformly-sized pores 0.45 m or less in diameter. Pre-filters built into the cartridge or up-stream reduce fouling of the membrane by removing large particles, oil, water droplets and foam from the incoming gas.

Filters are also used to sterilise effluent gases leaving fermenters. The objective is to prevent release into the atmosphere of any microorganisms entrained in aerosols in the headspace of the reactor. The concentration of cells in fermenter off-gas is several times greater than in air. Containment is particularly important when organisms used in fermentation are potentially harmful to plant personnel or the environment Companies operating fermentations with pathogenic or recombinant strains are required by regulatory authorities to prevent escape of the cells.

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