Sterilization-1

Fermentation Sterilization

Introduction to Fermentation Contamination

  • Consequences of Contamination in Fermentation:

    • Medium may support growth of both producer and contaminant organisms, causing loss of productivity.

    • In continuous cultivation, contaminant may outgrow the producer organism.

    • Contamination leads to total loss of biomass as a product.

    • Contaminants may produce difficult-to-remove compounds in downstream processing.

    • Common in antibiotic fermentation such as β-Lactum antibiotics, where contaminants like β-Lactamase producing organisms degrade the product.

    • Contamination can lead to culture lysis due to phages.

Measures for Avoiding Contamination

  • Preventive Measures:

    1. Use pure inoculum to initiate fermentation.

    2. Sterilize the fermentation medium.

    3. Sterilize the fermenter.

    4. Sterilize all materials added during fermentation.

    5. Maintain aseptic conditions throughout the fermentation process.

Protected Fermentations

  • Some fermentations are described as protected:

    • Such as brewing beer, where hop resins inhibit growth of other organisms.

    • Protected fermentations may involve boiling the medium or using disinfectants without needing complete sterilization.

    • Most fermentations are not protected against contamination.

Sterilization Methods

  • Types of Medium Sterilization:

    • Filtration

    • Radiation

    • Ultrasonic treatment

    • Chemical treatment

    • Heat

UV Radiation

  • Many cellular materials absorb UV light, causing DNA damage and cell death.

  • Peak effectiveness at wavelengths around 265 nm, but poor penetration limits its use to clean chambers and operating theatres.

  • X-rays are lethal with good penetration but are expensive and pose safety concerns.

Chemical Sterilization

  • Common Chemical Agents:

    • Phenols and compounds: including cresol, orthophenylphenol.

    • Alcohols: such as ethyl and methyl.

    • Halogens: including iodine, hypochlorites, chloramines.

    • Detergents, acids, and alkalis.

    • Gaseous chemosterilizers: like ethylene oxide and formaldehyde.

Microorganism Destruction by Heat

  • Destruction of microorganisms by steam follows a first-order reaction:

    [-dN/dt = kN]

    • Where:

      • N = number of viable organisms

      • t = sterilization treatment time

      • k = rate constant or specific death rate

    • Importance placed on the number of organisms rather than concentration; even one contaminated organism can compromise the batch.

Integration of Death Rate Equation

  • Rearrange to get:

    • [-dN/N = kdt]

    • Integrating gives us:

    • [\ln(N_t/N_0) = -kt]

    • Where:

      • N_t = organisms present after sterilization

      • N_0 = organisms present before sterilization

Probability of Survival

  • As infinite time is required to achieve sterilization (N_t = 0), after a period there could be less than one viable cell present (N_t < 1).

  • The equation shows the probability of an organism surviving the treatment, e.g., N_t = 0.1 suggests a 10% chance of survival after ten treatments.

Temperature and Sterilization

  • The value of k is dependent on temperature, species, and physiological form of organisms.

  • Various microorganisms require different times and temperatures for effective sterilization, such as Bacillus stereothermophilus (B.st), known for its heat resistance.

Decimal Reduction Time (D)

  • Definition:

    • The time required to reduce cell population by a factor of ten:

    • [D = 2.303/k]

Problem Example: Thermal Death Kinetics

  • Testing Viable Spores of Bacillus subtilis:

    • Spore counts measured across various time intervals and temperatures (85°C, 90°C, 110°C, 120°C).

    • Analysis involves activation energy and specific death constants at given temperatures.

Steps for Activation Energy Calculation

  1. Plot (\ln(N_0/N_t)) against time to find slope, yielding k value.

  2. Plot (\ln k) against (1/T) to find E/R values and A.

  3. Calculate k for 100°C.

  4. Use k to calculate time needed to kill 99% of spores.

Batch Sterilization Techniques

  • Methods of Sterilization:

    • Direct steam sparging, electrical heaters, circulating steam for heating.

    • Cooling involves sparging steam through cooling coils.

Heating and Cooling Dynamics

  • Analyze overall sterilization time by combining heating and cooling periods to minimize contamination risks.

Decimal Reduction Challenges

  • If starting contaminants are at 10^17, a time of 17 times the decimal reduction time (D) needs to be calculated for adequate sterilization (e.g. 25.5 minutes at D=1.5 min).

Calculating Holdup Time

  • Given volumetric constraints, the contribution from heating, cooling, and overall time needs calculation for effective sterilization with B.st species.

Conclusion on Sterilization Strategies

  • The nature of microbial contamination necessitates establishing a suitable sterilization regime.

  • Considerations include nutrient interactions and potential degradation of heat-labile components.

  • Continuous sterilization methods may provide advantages such as improved maintenance of nutrient quality.