Sterilization and Bioreactor Operation

Sterilization Methods and Kinetics Sterility: the absence of detectable levels of viable organisms in a culture medium or in a gas. It is an absolute concept. A system is never partially or almost sterile. Reasons for Sterilization 1. Economic penalty is high for loss of sterility 2. Many fermentations (such as antibiotic fermentation) must be absolutely devoid of foreign organisms. 3. Vaccines must have only killed viruses 4. Recombinant DNA fermentations (to make protein) -- exit streams must be sterilized

Sterilization Agents 1. Thermal - preferred for economical large-scale sterilizations of liquids and equipment. 2. Chemical - preferred for heat-sensitive equipment → ethylene oxide (gas) for equipment → 70% ethanol-water (pH=2) for equipment/surfaces → 3% sodium hypochlorite for equipment 3. Radiation – Ultraviolet (UV) radiation is effective to sterilize surfaces, but can not penetrate fluids easily. X-rays for liquids (costly/safety) because it can penetrate more deeply. 4. Filtration → membrane filters having uniform micropores (<0.2m). Not reliable as any defect in the membrane can lead to failure; virus can pass the filter. → depth filters of glass wool: rely on a combination of mechanisms (direct interception, electrostatic effects, diffusion and inertial effects) for the capture of particles.

Kinetics of Thermal Sterilization (Death) Practical considerations: 1. Not all organisms have identical death kinetics. → (increasing difficulty: vegetative cells < spores < virus) 2. Individuals within a population of the same organism may respond differently From Probability Theory: p(t) = the probability that an individual is still viable at time t. (simplest form assuming 1st order)

Kinetics of Thermal Sterilization (cont.) D – decimal reduction time. It is the time for the number of liable cells to decrease 10-fold. D=2.303/Kd

Temperature Effects on the Kinetics of Thermal Sterilization Increasing

Population Effects on the Kinetics of Thermal Sterilization

System Variables for Thermal Sterilization Primary System Variables in Thermal Sterilization 1. Initial concentration of organisms 2. Temperature, T 3. Time (t) of exposure at temperature T. N0 – the number of individuals in the reactor

System Variables for Thermal Sterilization (cont.) p(t) = the probability that an individual is still viable at time t. 1- p(t)= successful probability that an individual is killed at time t.

Sterilization Chart

System Variables for Thermal Sterilization Use of Sterilization Charts: Specify 1-P0(t) which is acceptable (e.g. 0.001) Determine N0 in the system. Read kdt from the chart. Knowing kd for the spores (or cells), obtain the required time, t.

Scale-up of Sterilization Let n0 be the concentration of particles. Now consider the probability of an unsuccessful sterilization in a 1 L and 10000 L reactor, where each contains the same identical condition (n0, kd and time of sterilization) in batch sterilization, scale-up of small-scale sterilization data to a much larger scale will result in unsuccessful sterilization

Sterilization Chart

Batch vs. Continuous Sterilization For spores, Kd falls very rapidly with temp. (10-fold drops from 121 C to 110 C ) Long heat-up and cool down time will damage to vitamins and proteins.

Batch vs. Continuous Sterilization Have less damage to medium (vitamin, protein and sugar)

Sterilization of Gases

Design and Operation of Bioreactors Types of Bioreactors Reactors with Mechanical Agitation see Fig. 10.1A a) disperse gas bubbles throughout tank b) increase residence time of bubbles c) shear large bubbles to smaller bubbles d) disk type or turbine type see Fig. 10.3A e) provide high kLa values f) baffles (4) augment mixing Bubble Column see Fig. 10.1B b) perforated plates enhance gas dispersion and mixing

Figure 10.1A Figure 10.1B a) disperse gas bubbles throughout tank b) increase residence time of bubbles c) shear large bubbles to smaller bubbles d) disk type or turbine type (figure 10.3A) e) provide high kLa values f) baffles (4) augment mixing a) disperse gas bubbles throughout tank b) perforated plates enhance gas dispersion and mixing

Figure 10.3A

Design and Operation of Bioreactors (cont.) 3. Loop Reactors

Figure 10.1C D E

Reactor Geometry and Layout

Reactor Types in Industry

Aeration and Heat Transfer

Oxygen Transfer Rate

kLa for Stirred Tanks

Pg Correlation

Pu Correlation (Figure 5.20 of Blanch and Clark)

NA Correlation (Figure 5.22 of Blanch and Clark)

Example Problem

Example Problem Solution

Example Problem Solution (cont.)

Example Problem Solution (cont.)

Example Problem Solution (cont.)

Example Problem Solution (cont.)

Example Problem Solution (cont.)

Measurement of OTR

Heat Generation Rate: Aerobic Growth

Heat Generation Rate: Agitation

Heat Balance