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Sterilization

In fermentation industry it is mandatory to keepuncontaminated process from the preliminary culture to theproduction fermenter. The process employed to do so is calledsterilization.

Sterilization of fermentation media or equipment can beaccomplished by destroying all living organisms by means ofa) mechanical methods, for example, by filtration,

centrifugation, flotation, orb) Electrostatically.c) heat, chemical agents, or electromagnetic waves/radiation

(ultraviolet or X-rays)

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Mechanical abrasion can be used on a small scale, but thismethod is not satisfactory industrially. x-rays, beta rays, ultra-violet light, and sonic radiations, whileuseful for laboratory purposes are not applicable to thesterilization of large volume of fluids. Gamma rays, may prove useful, particularly in food industry.

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a) Mechanical Removal of Organisms Centrifugation, adsorption to ion exchange, adsorption toactivated carbon, or filtration are possible. Filtration is the only method in practical use. Filter sterilization is often used for components ofnutrient solutions which are heat -sensitive . Deep filters plate filters) are some time used to filtercomplex nutrient solutions.

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Filtration:

Sterilize solutions that may be damaged or denatured by high temperatures

or chemical agents.

The pore size for filtering bacteria, yeasts, and fungi is in the range of 0.22-0.45 m

The pore size for filtering viruses and some large proteins is in the range of0.01 mThe disadvantages of filtration are:1) certain components of the nutrient solution may beadsorbed on the filter material, and2) high pressures must be used up to 5 bar), which are undesirable inindustrial practice.

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Air sterilization:

- Aerobic fermentation require huge volumes of air (a 50,000 L fermenterrequires 7x106to 7x107L per day of air) which must be sterilized.

- Adiabatic compression of process air can increase air temperature (150oC to220oC). A temperature typically of 220oC for 30 s is required to kill spores.

- An air-filtration step is almost always used to ensure sterility of process air.

- Depth filters use glass wool, and rely on inertial impaction, interception,diffusion and electrostatic attraction (explained later).

- Surface filters using membrane cartridges use the sieving effect (explainedlater).

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Fig.1 Filtration mechanisms operating in depth filters

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Inertial impaction (or impingement) occurs when a particle travelling in theair stream and passing around a fibre, deviates from the air stream (due toparticle inertia) and collides with the fibre.

Impaction is the dominant collection mechanism for particles larger than 0.2m.

It is important in removing bacteria.

Fig.2Filtration mechanisms operating in depth filters

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R. Shanthini25 Nov 2011 9

Fig.3 Filtration mechanisms operating in depth filters

Interception occurs when a large particle, because of its size, collides with a fibrein the filter that the air stream is passing through.

Interception is the dominant collection mechanism for particles greater than 0.2m.

It is important in removing bacteria.

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Fig.4 Filtration mechanisms operating in depth filters

Diffusion occurs when the random (Brownian) motion of a particle causes thatparticle to contact a fibre.

Diffusion is dominant for particles less than 0.2 m.

It may be important for virus removal, but bacteria are sufficiently large thatdiffusion is relatively unimportant.

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Electrostatic attraction plays a very minor role in mechanical filtration. After fibrecontact is made, smaller particles are retained on the fibres by a weak electrostaticforce.

Fig.5 Filtration mechanisms operating in depth filters

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Sterilization of Culture Media

A number of means are available for sterilization, butin practice for large-scale installations, heat is themain mechanism used.

It is the most useful method for sterilization of

nutrient media.

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Theory of cell death kinetics and sterilization

operations utilizing moist heat Destroying a population generally follows first order

kinetics.

Initial number of cells No/ml, the number of destroyedorganisms N at time t (min), and the surviving cells N,the death rate can be calculated as follows:

(k is the specific death constant/min)

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When integrated between No at time t = 0 and N at time t = t, the followingequation is obtained:

No/N is the inactivation factor, N/No is the survival factor and

In No/N=V is the design criterion, a parameter which encompasses thecontamination level of the medium to be sterilized, No, and the desired sterilitylevel, N.

The temperature dependence of the specific death rate kd can be assumed

to follow the Arrhenius equation:

kd = kd0 exp (-- Ed/RT) (4) where Ed is activation energy, which can be obtained from the slope of

the In(kd) versus 1/T plot

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k is a constant which expresses the specific death rate.

It increases sharply with temperature Can be experimentally determined for an organism

using equation 3.

Plotting ln(N/N0

) against time (t) yields a straight linewith a slope equals k.

This kinetic description makes two predictions:

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An infinite time is required to achieve sterile

conditions (N=0).After certain time their will be less than one viable cell

present.

During fermentation the following points must beobserved to ensure sterility:

Sterility of the culture media

Sterility of incoming and outgoing air

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Factors influencing Heat Sterilization

the number and type of microorganisms present,

the composition of the culture medium,

the pH value,

the size of the suspended particles.

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Example Vegetative cells are rapidly eliminated at relatively low

temperatures. For destruction of spores, temperatures of 121C are

needed. Spores of Bacillus stearothermophilus are the most heat

resistant.

Therefore they are used as assay organisms for testing thevarious procedures used to sterilize equipment. Table 3.1 provides data on the relationship between

temperature, k value, and design criterion for this,organism.

A list of sterilization times and temperatures for variousorganisms is given in

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Sterilization of Fermentation Air Most industrial fermentations are operated under

vigorous aeration and the air supplied to the fermentermust be sterilized.

To prevent contamination of either the fermentation by

airborne microorganisms or the environment by aerosolsgenerated within the fermenter, both air input and airexhaust ports have air filters attached.

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These filters are designed to trap and containmicroorganisms.

Filters are made of glass fibers, mineral fibers, poly tetra-fluoroethylene (PTFE) or polyvinyl chloride (PVC), and must

be steam steriliable and easily changed. Fermenter exhaust may also undergo dry heat sterilization

(incineration) as an additional safety measure and to avoidpathogens contamination.

AIR FILTERS

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Methods

Methods for sterilizing gases include filtration, gasinjection (ozone), gas scrubbing, radiation (UV), andheat.

Of these, filtration and heat are practical at an industrialscale.

Previously air was sterilized by passing it over electrically

heated elements. Due to high cost of electricity thismethod has become obsolete.

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b) H S ili i

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b) Heat Sterilization

moist heat is more effective than the dry heat because the intrinsic heatresistance of vegetative bacterial cells is greatly increased in a completely dry

state. As a result the death rate is much lower for the dry cells than for moistones. The heat conduction in dry air is also less rapid than in steam.

Therefore, dry heat is used only for the sterilization of glassware or heatable solidmaterials.

By pressurizing a vessel, the steam temperature can be increased significantlyabove the boiling point of water.

Laboratory autoclaves are commonly operated at a steam pressure of about 30psi, which corresponds to 121C. Even bacterial spores are rapidly killed at 121 C.

Advantages

most widely used can be employed for both liquid medium and heatable solid

object the most economical and efficient for the general sterilization

requirements of fermentation

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Figure 6: Installation of an air filter system in a fermenter

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Media and Fermenter Sterilization

Need of Sterilization For pilot-scale and industrial aseptic fermentations the

fermenter can be sterilized empty.

The vessel is then filled with sterile medium, prepared in a

batch or continuous medium cooker that may supply severalfermentations.

Alternatively, the fermenter is filled with formulated mediumand the two are sterilized together in one operation.

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However, some industrial fermentation are notaseptic, but microbial contamination is stillmaintained at a minimum level by boiling or

pasteurization of the media.

Otherwise, a fast-growing contaminant could outgrowthe industrial microorganism, or at least utilize somevaluable nutrients.

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Microbial contaminants may also metabolize thetarget product, produce toxic compounds or secreteproducts that may block filters and interfere with

downstream processing.

If the contaminant is a bacteriophage it may lyses theculture, as can occur in fermentations involving lacticacid bacteria.

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Process of Sterilization of fermenter Small laboratory-scale fermenters of 1-5L capacity are

usually filled with medium and then sterilized in asteam autoclave.

Here sterilization is normally performed usingpressurized steam to attain a temperature of 121C for15 min.

Care must be taken to avoid any pressure build-upinside the fermenter by venting withoutcontaminating the contents.

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For pilot-scale -and industrial fermenters more rigoroussterilization is necessary, involving increased sterilizationtime and/or higher temperature.

Normally, the aim is to provide sterilization conditions that

give an acceptable probability of contamination of 0.1 % (1 in1000).

Consequently, if the original number of microbial cells (No)is known, the Death factor (V =InNo/N,) can be calculated).(It is a measure of the rate of increase of the wave function)

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The valueN when a 0.1 % risk is adopted,will be 3-10 cells. Asterilization profile of a typical fermented is shown in Fig.7.

Destruction of cells occurs during heating (to 121C in this

case) and cooling (here from 121C to the fermentationoperating temperature).

Therefore the overall Death factor may be represented as

V total = V heating + V holding + V cooling

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Thus knowing both V heating and V cooling for aparticular fermentation system, the necessary holdingtime needed to achieve the required overall Death

factor can be calculated.

Alternatively, the Richards approximation can be used,which employs only that part of the curve above 100C(Fig. 7). This assumes that:

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1. heating and cooling between OC and 100C isunimportant for sterilization;

2. heating between 100C and 121 C; is at 1C perminute, i.e. 20 min; and

3. cooling from 121C to 100"C is at 1 C per minute, i.e.20 min.

Steps

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Figure 7: Sterilization Profile of a typical fermentation process

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In practice, steam sterilization involves passing the steamunder pressure into the vessel jacket and/or internalcoils.

Steam may also be injected directly into the headspaceabove the fermentation medium.

This aids sterilization, but can result in media volumechanges.

Steam sterilization is effective and cheaper than dry heatsterilization.

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Limitation of Steam Sterilization However, certain media constituents may be heat

labile and destroyed by excessive heat, e.g. glucose,some vitamins and components of animal cell culture

media. Such heat sensitive ingredients are often filter

sterilized before use; alternatively; some can be heatsterilized with minimal degradation using a hightemperature for a very short time, e.g. 140C for 50s.

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This is usually a continuous operation where theholding time is controlled by the flow rate through thesterilizer and the material is then rapidly cooled in a

heat exchanger.

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References

1. Miss Rahimah Othman .ERT 416/3 CHAPTER 7: UPSTREAM PROCESSING IN BIOPROCESS PLANT

(Email: rahimah@unimap.edu.my)

2. Bailey, J. E. and D. F. Ollis, Biochemical Engineering Fundamentals. New York,NY: McGraw-Hill Book Co., 1986.3.Industrial BiotechnologyCP504 Lecture 17 bhy R. Shanthini,25 Nov 20114. Fundamentals of Biochemical engg. By Rajiv Dutta 2008