Topic: Sterilization of air, media and fermenter

By: Ms Sonia Chauhan Asst. Prof. Department of Biotechnology

Sterilization
In fermentation industry it is mandatory to keep

uncontaminated process from the preliminary culture to the production fermenter. The process employed to do so is called sterilization. Sterilization of fermentation media or equipment can be accomplished by destroying all living organisms by means of
a) mechanical methods, for example, by filtration,

centrifugation, flotation, or b) Electrostatically. c) heat, chemical agents, or electromagnetic waves/radiation (ultraviolet or X-rays)

 Mechanical abrasion can be used on a small scale, but this

method is not satisfactory industrially. x-rays, beta rays, ultra-violet light, and sonic radiations, while useful for laboratory purposes are not applicable to the sterilization of large volume of fluids. Gamma rays, may prove useful, particularly in food industry.

a) Mechanical Removal of Organisms

Centrifugation, adsorption to ion exchange, adsorption to

activated carbon, or filtration are possible. Filtration is the only method in practical use. Filter sterilization is often used for components of nutrient solutions which are heat -sensitive . Deep filters (plate filters) are some time used to filter complex nutrient solutions.

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 0.220.45 m The pore size for filtering viruses and some large proteins is in the range of 0.01 m The disadvantages of filtration are: 1) certain components of the nutrient solution may be adsorbed on the filter material, and 2) high pressures must be used (up to 5 bar), which are undesirable in industrial practice.

Air sterilization:
-

Aerobic fermentation require huge volumes of air (a 50,000 L fermenter requires 7x106 to 7x107 L per day of air) which must be sterilized.
Adiabatic compression of process air can increase air temperature (150oC to 220oC). 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 (explained later).

Fig.1 Filtration mechanisms operating in depth filters

Fig.2Filtration mechanisms operating in depth filters

Inertial impaction (or impingement) occurs when a particle travelling in the air stream and passing around a fibre, deviates from the air stream (due to particle inertia) and collides with the fibre. Impaction is the dominant collection mechanism for particles larger than 0.2 m. It is important in removing bacteria.

Fig.3 Filtration mechanisms operating in depth filters

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

Interception is the dominant collection mechanism for particles greater than 0.2 m. It is important in removing bacteria.

R. Shanthini 25 Nov 2011

Fig.4 Filtration mechanisms operating in depth filters

Diffusion occurs when the random (Brownian) motion of a particle causes that particle 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 that diffusion is relatively unimportant.

Fig.5 Filtration mechanisms operating in depth filters

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

Sterilization of Culture Media

A number of means are available for sterilization, but

in practice for large-scale installations, heat is the main mechanism used. It is the most useful method for sterilization of nutrient media.

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 destroyed organisms 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)

 When integrated between No at time t = 0 and N at time t = t, the following

equation 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 the

contamination level of the medium to be sterilized, No, and the desired sterility level, N. The temperature dependence of the specific death rate k d can be assumed to follow the Arrhenius equation:
k d = k d 0 exp (-- Ed /RT) (4) where Ed is activation energy, which can be obtained from the slope of the In(kd) versus 1/T plot

 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 line with a slope equals k. This kinetic description makes two predictions:

 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 be observed to ensure sterility: Sterility of the culture media Sterility of incoming and outgoing air

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.

 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 the various 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 various organisms is given in

Heat Sterilization

Sterilization of Fermentation Air

Most industrial fermentations are operated under

vigorous aeration and the air supplied to the fermenter must be sterilized. To prevent contamination of either the fermentation by airborne microorganisms or the environment by aerosols generated within the fermenter, both air input and air exhaust ports have air filters attached.

AIR FILTERS
These filters are designed to trap and contain

microorganisms. Filters are made of glass fibers, mineral fibers, poly tetrafluoroethylene (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 avoid pathogens contamination.

Methods
Methods for sterilizing gases include filtration, gas

injection (ozone), gas scrubbing, radiation (UV), and heat. Of these, filtration and heat are practical at an industrial scale. Previously air was sterilized by passing it over electrically heated elements. Due to high cost of electricity this method has become obsolete.

Sterilization of Fermentation Air by Filter

Filter sterilization by glass wool filters are used. Particles are trapped by a combination of physical effect

which include inertial effects, blocking effect, diffusion, gravity separation. and electrostatic attraction. The disadvantages are shrinkage and solidification during steam sterilization. Glass Fiber filter cartridges has replaced glass wool filter as these do not have the drawback mentioned.

b) Heat Sterilization
moist heat is more effective than the dry heat because the intrinsic heat resistance 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 moist ones. 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 solid materials. By pressurizing a vessel, the steam temperature can be increased significantly above the boiling point of water. Laboratory autoclaves are commonly operated at a steam pressure of about 30 psi, 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

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

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 several fermentations. Alternatively, the fermenter is filled with formulated medium and the two are sterilized together in one operation.

 However, some industrial fermentation are not

aseptic, but microbial contamination is still maintained at a minimum level by boiling or pasteurization of the media. Otherwise, a fast-growing contaminant could outgrow the industrial microorganism, or at least utilize some valuable nutrients.

 Microbial contaminants may also metabolize the

target product, produce toxic compounds or secrete products that may block filters and interfere with downstream processing. If the contaminant is a bacteriophage it may lyses the culture, as can occur in fermentations involving lactic acid bacteria.

Process of Sterilization of fermenter

Small laboratory-scale fermenters of 1-5L capacity are

usually filled with medium and then sterilized in a steam autoclave. Here sterilization is normally performed using pressurized steam to attain a temperature of 121C for 15 min. Care must be taken to avoid any pressure build-up inside the fermenter by venting without contaminating the contents.

 For pilot-scale -and industrial fermenters more rigorous

sterilization is necessary, involving increased sterilization time and/or higher temperature. Normally, the aim is to provide sterilization conditions that give an acceptable probability of contamination of 0.1 % (1 in 1000). Consequently, if the original number of microbial cells (No) is known, the Death factor (V =In No/N,) can be calculated).
(It is a measure of the rate of increase of the wave function)

 The value N when a 0.1 % risk is adopted, will be 3-10 cells. A

sterilization 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 121 C to the fermentation operating temperature). Therefore the overall Death factor may be represented as V total = V heating + V holding + V cooling

 Thus knowing both V heating and V cooling for a

particular fermentation system, the necessary holding time 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:

Steps
1. heating and cooling between OC and 100C is

unimportant for sterilization; 2. heating between 100C and 121 C; is at 1C per minute, i.e. 20 min; and 3. cooling from 121C to 100"C is at 1 C per minute, i.e. 20 min.

Figure 7: Sterilization Profile of a typical fermentation process

 In practice, steam sterilization involves passing the steam

under pressure into the vessel jacket and/or internal coils. Steam may also be injected directly into the headspace above the fermentation medium. This aids sterilization, but can result in media volume changes. Steam sterilization is effective and cheaper than dry heat sterilization.

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 heat sterilized with minimal degradation using a high temperature for a very short time, e.g. 140C for 50s.

 This is usually a continuous operation where the

holding time is controlled by the flow rate through the sterilizer and the material is then rapidly cooled in a heat exchanger.

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 Biotechnology CP504 Lecture 17 bhy R. Shanthini,25 Nov 2011 4. Fundamentals of Biochemical engg. By Rajiv Dutta 2008